KR20010075338A - Use of specific hybrid promoters for controlling tissue expression - Google Patents

Abstract

The present invention provides a hybrid promoter comprising all or part of an enhancer region of a strong ubiquitous promoter / enhancer; It relates to a promoter region that enables specific expression in smooth muscle cells and any vectors or cells containing such promoters and uses thereof.

Description

Use of specific hybrid promoters to control tissue expression {USE OF SPECIFIC HYBRID PROMOTERS FOR CONTROLLING TISSUE EXPRESSION}

Possible ways of controlling and directing gene expression are an important area of development with biotechnology. In vitro, certain cell populations can be used to improve the conditions for producing recombinant proteins. It also enables in vitro to detect or confirm the presence of a particular cell population in a sample, and to test the properties or gene regulation of the product in a particular cell population. Gene expression control is very important for ex vivo or in vivo therapies in view of the ability to selectively regulate the production of therapeutic molecules. Indeed, depending on the application, depending on the gene to be transferred, it is important to be able to target only certain tissues or specific parts of the organism in order to focus the therapeutic effect and limit metastasis and side effects.

Targeting of a given nucleic acid expression can be accomplished according to various methods. The first approach is to use, for example, delivery agents or vectors that exhibit defined cell specificity. However, the specificity conferred by this kind of vector is generally very messy and does not allow precise targeting of the cell population. Another method is to use expression signals specific to specific cell types. In this respect, so-called "specific" promoters, for example, promoters of genes encoding pyruvate kinases, borrowers, GFAP, promoters of fatty acid-binding intestinal proteins, α-actin promoters of smooth muscle cells, SM22 promoters or human albumin genes The promoter of is described in the literature. However, while such promoters exhibit tissue specificity, they exhibit relatively low strength. Accordingly, the majority of such promoters generally have activity levels below the activity level of the so-called "strong" promoter at least 10 to 100 magnifications. In addition, it is generally believed that the specificity of the promoter is inversely proportional to the intensity and the higher the intensity, the higher the level of nonspecific activity.

It is particularly advantageous to have a strong promoter that is specific to a particular tissue. It is an object of the present invention to precisely provide new constructs that allow for high and targeted expression of genes.

The present invention relates to the field of biology, in particular the field of controlling the expression of genes. The present invention describes in particular new constructs and new vectors that allow for the targeting and high expression of genes. The invention can be used in a number of areas, in particular for the production of recombinant proteins, the creation of transgenic animal models, the creation of cell lines, the development of screening tests, or gene and cell therapies.

Table I : Hybrid promoters evaluated in transient transfection with rabbit smooth muscle cells (rabbit SMC), ECV304 cells, myoblast cells C2C12, HeLa cells, NIH 3T3 cells, sarcoma cells TU182, and kidney cells 293 in primary culture in vitro ( hSMα-actin) relative activity. The relative activity of each promoter is expressed as a percentage of luciferase activity obtained using the plasmid pCMV-leadTK. Enh-X: The enhancer sequence of hCMV-IE is cloned upstream of the X promoter in a normal orientation. HnE-X: The enhancer sequence of hCMV-IE is cloned upstream of the X promoter in the opposite orientation.

Table II : Hybrid promoters evaluated in transient transfection in rabbit smooth muscle cells (rabbit SMC), ECV304 cells, myocytes C2C12, HeLa cells, NIH 3T3 cells, sarcoma cells TU182, and renal cells 293 in primary culture in vitro ( relative activity of mSM22). The relative activity of each promoter is expressed as a percentage of luciferase activity obtained using the plasmid pCMV-leadTK. Enh-X: The enhancer sequence of hCMV-IE is cloned upstream of the X promoter in normal culture. HnE-X: The enhancer sequence of hCMV-IE is cloned upstream of the X promoter in the opposite orientation.

1 : Schematic diagram of a plasmid in which the expression cassette contains a hSMα-actin hybrid promoter.

2 : Schematic diagram of the plasmid in which the expression cassette contains the hybrid promoter mSM22α.

Figure 3 : Endothelial cells derived from rabbit smooth muscle cells (rabbit SMC), human navel spinal cord sarcoma (ECV304) induced endothelial cells, mouse myoblasts (C2C12) and human cervical sarcoma (HeLa) in primary culture in vitro Activity of hybrid promoters evaluated in transient transfections. The relative activity of each promoter is expressed as a percentage of luciferase activity obtained using the plasmid pCMV-leadTK. Enh-X: The enhancer sequence of hCMV-IE is cloned upstream of the X promoter in a normal orientation. hnE-X: The enhancer sequence of hCMV-IE is cloned upstream of the X promoter in the opposite orientation.

4 : Activity of hybrid promoters evaluated in transient transfection into mouse embryonic fibroblasts (NIH 3T3), human ENT sarcoma (TU182), and transformed human embryonic kidney cells 293 in vitro. The relative activity of each promoter is expressed as a percentage of luciferase activity obtained using the plasmid pCMV-leadTK. Enh-X: The enhancer sequence of hCMV-IE is cloned upstream of the X promoter in a normal orientation. hnE-X: The enhancer sequence of hCMV-IE is cloned upstream of the X promoter in the opposite orientation.

Figure 5 : Activity of hybrid promoters assessed in gene delivery to C57BL6 mouse cranial tibia in vivo. The relative activity of each promoter is expressed as a percentage of luciferase activity obtained using the plasmid pCMV-leadTK.

Materials and methods

Methods commonly used in molecular biology, such as preliminary extraction of plasmid DNA, centrifugation of plasmid DNA in cesium chloride gradient, agarose gel electrophoresis, purification of DNA fractions by electroelution, saline medium using ethanol or isopropanol Precipitation of plasmid DNA and transformation in Escherichia coli are well known to those skilled in the art and are described abundantly 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 cloning various promoter regions, is commercially available (Promega Corporation). Plasmids pCMVβ (Clontech Laboratories Inc.) and pUC18 (Boehringer Mannheim) are also commercially available.

Enzymatic amplification of DNA fractions by PCR (polymerase chain reaction) technology is performed using DNA thermal cycler ^™ (Perkin Elmer Cetus) according to the manufacturer's recommendations.

Electroporation of plasmid DNA into E. coli can be performed by an electroporator (Bio-Rad) according to the manufacturer's recommendations.

Identification of the nucleotide sequences can be performed using a method developed by Sanger et al. (Proc. Natl. Acad. Sci. USA, 74 (1977) 5463-5467) using a kit distributed by Applied Biosystems according to the manufacturer's recommendations. Can be.

The present invention particularly describes novel chimeric promoters that allow for high and specific expression of smooth muscle cells. The present invention also describes vectors containing such promoters and their use for the delivery of genes to cells in vitro, ex vivo and in vivo. Constructs of the present invention may first have opposing properties, ie high selectivity and high transcriptional activity. The present invention provides particularly effective means for targeting gene expression in smooth muscle cells in vivo or in vitro, and for regulating such expression.

The invention is more specifically based on the construction of chimeric (or hybrid) promoters comprising different origins and functional regions. More specifically, the first subject matter of the present invention

All or part of an enhancer region of a strong ubiquitous promoter / enhancer, and

Promoter region allowing specific expression in smooth muscle cells

There is a hybrid promoter comprising a.

Combinations of enhancer regions and promoters have already been described in the prior art. Thus, for example, a method of coupling the CMV enhancer region with a nonspecific promoter such as the promoter of the chicken β-actin gene (WO96 / 13597) to increase the intensity is known. However, such constructs have never been described or implied for the purpose of obtaining high expression specific for smooth muscle cells. The present invention is based, in part, on the selection and combination of particular "enhancer" elements and particular "promoter" elements. The present invention is based on the demonstration that such a combination of elements can achieve high levels of expression without affecting the selectivity of the promoter for target cells of smooth muscle. The present invention provides constructs that are particularly advantageous because they allow targeted production with high levels of molecules in smooth muscle cells. In addition, the present application shows that such constructs can be used both in vitro and in vivo.

In the hybrid promoter according to the present invention, the enhancer region and the promoter region are functionally combined, i.e., the enhancer region is functionally combined such that it promotes promoting activity on the activity of the promoter region. Generally, these two regions are genetically linked and the enhancer regions are close enough to each other to activate the promoter region. Preferably, the distance between the enhancer region and the promoter region is 1 kb or less, more preferably 500 bp or less. In a particularly preferred construct according to the invention, these two regions are 400 bp or less apart, more preferably 200 bp or less. In addition, as can be seen from the examples, the orientation of each of the two regions does not have a significant impact on the activity of the hybrid promoter of the present invention. As a result, the enhancer region can be located in the same orientation or in the opposite orientation with respect to the transfer direction of the promoter region.

In a preferred embodiment, the enhancer region is selected from the enhancer region of the cytomegalovirus immediate-early (CMV-IE) gene, the enhancer region of the Raus sarcoma virus LTR (RSV-LTR), the enhancer region of the SV40 virus, and the enhancer region of the EF1α gene. do. More preferably, in the hybrid promoter of the present invention, the enhancer region is an enhancer region of cytomegalovirus (CMV-IE), preferably the immediate-early gene of human cytomegalovirus (hCMV-IE). A particular enhancer region consists of any fraction that includes, for example, fractions -522 to -63, or at least a portion thereof, of the hCMV-IE gene and exhibits enhancer activity.

In the case of a promoter region, advantageously a region comprising all or part of a promoter encoding a-actin of smooth muscle cells (SMact) or a promoter of the SM22 gene is used to carry out the invention. Promoters of such genes have been described for smooth muscle cell-specific properties (especially Ueyama H. et al., Mol. Cell. Biol., 4 (1984) 1073-1078; Solway J. et al., J. Biol) Chem., 270 (1995) 13460-13469).

The first particularly advantageous variant of the invention

All or part of an enhancer region of a human cytomegalovirus immediate-early (hCMV-IE) gene, and

A gene encoding SM-actin of smooth muscle cells (SMact), preferably a hybrid promoter comprising all or part of a promoter of the human Smact gene.

A second particularly advantageous variant of the invention

All or part of an enhancer region of a human cytomegalovirus immediate-early (hCMV-IE) gene, and

A hybrid promoter comprising all or part of a promoter of the SM22 gene, preferably the mouse SM22 alpha gene.

In addition, in certain embodiments of the invention, the promoter region used is a chimeric region comprising a base promoter and tissue specificity consensus sequence, which sequence is derived from a SMact promoter or SM22 promoter, or a combination of both. In such embodiments, the base promoter may be a "minimal" promoter, ie a promoter comprising only sequences necessary for transcriptional promoter activity (eg, TATA boxes). Such base promoters may be SMact or SM22 genes, or actual base promoters of heterologous origin (eg, β-globin, HSV-TK, SV40 or EF1-α). Sequences that confer tissue specificity are advantageously SMact promoters (RT Shimizu et al., J. Biol. Chem. 270 (1995) 7634-7643) and / or SM22 promoters (L. Li et al., J. Cell. Part of the sequence of 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). It includes.

Certain forms of hybrid promoters in accordance with the present invention

All or part of an enhancer region of a strong ubiquitous promoter / enhancer,

The primary promoter, and

Sequences conferring tissue specificity, including all or part of the SMact promoter and / or SM22 promoter

It includes.

For the construction of the hybrid promoter of the present invention, molecular biology techniques known to those skilled in the art can be used. Thus, enhancer regions and promoter regions (including sequences that confer basic promoter and tissue specificity) can be isolated from nucleic acid libraries or total cell DNA by conventional techniques, such as by amplification by specific probes. These fractions can be artificially synthesized using sequence information available as prior art. When these fractions are obtained, they can be easily combined with each other by ligase and other restriction enzymes, resulting in the hybrid promoter of the present invention. In addition, these fractions can be modified by cleavage, mutation, insertion or addition of base pairs to facilitate cloning or to change functionality. In addition, as noted above, the fractions can be combined directly with each other, while separated by base pairs that do not significantly affect the activity of the hybrid promoter.

The hybrid promoter of the present invention retains the ability to specifically express related nucleic acids in smooth muscle cells. "Specific" of expression means that the activity of the promoter is significantly higher in smooth muscle cells. Although nonspecific expression may be present in other cells, the corresponding activity level is generally very low (to negligible) as observed in smooth muscle cells, and is generally at least 10 magnifications lower. The results shown in the Examples show a difference in expression, which may correspond to 140 magnifications in this respect, reflecting the high selectivity of the promoters of the invention. In this regard, the results shown also show high specificity for smooth muscle cells because no expression is detected in endothelial cells present in the vicinity, blood vessels, especially arteries. The results shown in the examples show that the strength of the promoter of the present invention is higher than the non-hybrid specific promoter, the difference may exceed 100 magnifications. These properties explain the advantageous and unexpected properties of the hybrid promoters of the present invention in terms of the strength and specificity of expression of related nucleic acids in smooth muscle cells.

In this regard, another subject of the invention relates to an expression cassette comprising a nucleic acid encoding a relevant RNA or polypeptide, which is under the control of the hybrid promoter described above. Advantageously, the cassette of the present invention additionally comprises a signal for termination of transcription placed on nucleic acid 3 '.

Given the population of cells targeted by the cassette of the present invention, the nucleic acid may encode, for example, a protein selected from:

Proteins involved in the cell cycle, such as p21 or any other protein that inhibits cyclin-dependent kinase (cdk), the product of the retinoblastoma (Rb) gene, GAX, GAS-1, GAS-3, GAS-6, Gadd 45, Gadd 153, cyclin A, B and D

Apoptosis inducing proteins such as p53, apoptosis inducers such as Bas, Bcl-X _s , Bad or members of any other antagonist lineage of Bcl ₂ and Bcl-X ₁

Inhibit the activity of receptors on proteins capable of modifying the proliferation of smooth muscle cells, such as intracellular antibodies or proteins involving cell proliferation, such as Ras proteins, map-kinases, or tyrosine-kinases or growth factors ScFv

Proteins for labeling (LacZ, GFP, Luc, secreted alkaline phosphatase (SeAP), growth hormone (GH), etc.) for the purpose of conducting proliferation studies or diagnostic studies

Angiogenesis inducing proteins, for example members of the VEGF strain, members of the FGF strain, more particularly FGF1, FGF2, FGF4, FGF5, angiogenin, EGF, TGFα, TGFβ, TNFα, scatter factor / HGF Synthesis of members of the opioetine line, cytokines, in particular interleukin, including IL-1, IL-2, IL-8, angiotensin-2, plasminogen activator (TPA), urokinase (uPA), active lipids Molecules involved (prostaglandins, Cox-1).

Natural or chimeric receptors including transcription factors such as DNA-binding domains, ligand-binding domains, transcriptional activity or inhibition domains, for example tetR-NLS-VP16 fusion proteins, fusion proteins derived from estrogen receptors, steroids Fusion proteins derived from hormone receptors, fusion proteins derived from progesterone receptors, proteins of the CID (chimeric inducer of dimerization) system described by Rivera et al. (Rivera et al. (1996), A humanized system for pharmacologic control of gene expression , Nature Medicine, 2: 1028-1032).

Although the invention is not limited to specific examples of proteins or RNAs, one of ordinary skill in the art can use simple routine experimental procedures for the expression of any nucleic acid in smooth muscle cells.

Another subject of the invention additionally relates to any vector comprising a hybrid promoter or cassette as described above. Vectors of the invention can be, for example, plasmids, cosmids or any DNA not encapsidated by viruses, phages, artificial chromosomes, recombinant viruses and the like. Preferably plasmid or recombinant virus.

Among the plasmid vectors, all cloning and / or expression plasmids which are known to those skilled in the art and generally contain origins of replication may be mentioned. Mention may be made of neoplasmic plasmids carrying improved origins of replication and / or markers as described, for example, in the applications WO96 / 26270 and PCT / FR96 / 01414.

Among the vectors of the recombinant virus type, mention may be made of recombinant adenoviruses, retroviruses, huff viruses or adeno-associated viruses, preferably. The construction of this type of replication-defective recombinant virus, and the infectivity of these vectors, has been extensively described in the literature (especially in S. Baeck and KLMarch (1998), Circul. Research vol. 82, pp. 295-305, T. Shenk, BN Fields, DM Knipe, PM 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).

Particularly preferred recombinant viruses for carrying out the invention are defective recombinant adenoviruses.

Adenoviruses are linear double stranded DNA viruses of about 36 kb (kilobase) in size. There are a variety of serotypes that differ somewhat in structure and properties but exhibit significant genetic structure. More specifically, the recombinant adenovirus may be of human or animal origin. For adenoviruses of human origin, mention may be made of those classified as group C, in particular of type 2 (Ad2), 5 (Ad5), 7 (Ad7) or 12 (Ad12). Among the various adenoviruses of animal origin, preferably all of the adenoviruses of dog origin, in particular CAV2 adenovirus strains, can be mentioned (eg Manhattan or A26 / 61 strains (ATCC VR-800)). Other adenoviruses of animal origin are in particular cited in application WO94 / 26914, which is incorporated herein by reference.

The adenovirus genome includes at each end a reverse repeating sequence (ITR), a capsidizing sequence (Psi), an early gene and a late gene. Basic early genes are contained in the E1, E2, E3 and E4 regions. Of these, genes contained in the El region are particularly essential for virus propagation. Basic late genes are contained in the L1 to L5 region. The genome of adenovirus Ad5 has been fully sequenced and available as a database (see in particular Genebank M73260). In addition, some or all of the other adenovirus genomes (Ad2, Ad7, Ad12, etc.) were sequenced.

For use as recombinant vectors, various constructs derived from adenoviruses were prepared by introducing various therapeutic genes. In each of these constructs, the adenovirus was modified to be unable to replicate in infected cells. Thus, the constructs described in the prior art are adenoviruses that lack the E1 region essential for viral replication, at which the heterologous DNA sequence is inserted (Levrero et al., Gene 101 (1991) 195; Gosh-Choudhury et al. , Gene 50 (1986) 161). It is also proposed to make other deletions or modifications in the genome of adenoviruses to improve the properties of the vector. Accordingly, heat-sensitive point mutations can be introduced into the ts125 mutant to inactivate the 72 kDa DNA-binding protein (DBP) (Van der Vliet et al., 1975). Other vectors include deletion of another region, the E4 region, which is essential for replication and / or viral propagation. The E4 region is actually involved in regulating expression of late genes, stability of late nuclear RNA, killing of host cell protein expression and replication efficiency of viral DNA. Adenovirus vectors that have deleted the E1 and E4 regions possess transcriptional background noise and highly reduced expression of viral genes. Such vectors are described in applications WO94 / 28152, WO95 / 02697, WO96 / 22378. Additionally, vectors that carry modifications 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 group C human adenovirus. More preferably, it is adenovirus Ad2 or Ad5.

Advantageously, the recombinant adenovirus used within the structure of the present invention comprises a deletion in the El region of its genome. More specifically, this includes deletions in the El and El regions. Specifically, for example, nucleotides 454-3328; Deletions affecting 382-3446 or 357-4020 (based on the Ad5 genome) may be mentioned.

According to a preferred variant, the recombinant adenovirus used within the structure of the present invention additionally comprises a deletion in the E4 region of the genome. More specifically, deletions in the E4 region affect all open decoding frames. Specifically, for example, deletions 33466-35535 or 33093-35535 may be mentioned. Other forms of deletion in the E4 region are described in the applications WO95 / 02697 and WO96 / 22378 referenced herein.

Expression cassettes can be inserted at various sites in the recombinant genome. It can be inserted as a replacement or supplement to a sequence deleted at the level of the El, E3 or E4 region. It may also be inserted at other sites outside the sequence required in cis state for virus production (ITR sequence and capsidation sequence).

Recombinant adenoviruses are produced in capsidized lines, ie, cell lines capable of trans-complementing one or more functions deficient in the recombinant adenovirus genome. Among capsidizing strains known to those skilled in the art, mention may be made of, for example, 293 strains into which a portion of the adenovirus genome is integrated. More precisely, the 293 lineage comprises adenovirus genome serotype 5 (Ad5) comprising the left ITR, the encapsidation region, the E1 region (including E1a and E1b), the region encoding protein pIX and the region encoding protein pIVa2. Human embryonic kidney cell line containing the left extremity (about 11-12%). This lineage may be trans-complementary to recombinant adenoviruses lacking the El region, ie, all or a portion of the El region, and may produce viral titers with high titers. This line can also produce viral stock further comprising a heat-sensitive E2 mutation at an acceptable temperature (32 ° C.). Other cell lineages that may be complementary to the El region are described based on human lung sarcoma cells A549 (WO 94/28152) or human retinoblastoma (Hum. Gen. Ther. (1996) 215). Also described are strains capable of trans-complementing the various functions of adenoviruses. In particular, lines complementary to the E1 and E4 regions (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 lines complementary to the E1 and E2 regions (WO94 / 28152, WO95 / 02697, WO95 / 27071) may be mentioned.

Recombinant adenoviruses are usually produced by introducing viral DNA into the encapsidation line and then lysing the cells after about 2 or 3 days (the kinetics of the adenovirus cycle is 24 to 36 hours). To carry out the method, the introduced viral DNA may be a fully recombinant viral genome, optionally constructed in bacteria (WO96 / 25506) or yeast (WO95 / 03400) and transfected into cells. It may be a recombinant virus used to infect the encapsidated lineage. Viral DNA can be introduced in the form of fractions, each containing a portion of the recombinant viral genome and a homologous region (which can be reconstituted by homologous recombination between the various fractions after introduction into the capsidized cells).

After cell lysis, the recombinant virus particles are separated by cesium chloride gradient centrifugation. Another method is described in patent FR96: 08164, incorporated herein by reference.

The invention also relates to a composition comprising a vector as described above and a chemical or biochemical delivery agent. The term “chemical or biochemical delivery agent” is interpreted to mean any compound that promotes penetration of a nucleic acid into a cell (ie, except for a recombinant virus). These include cationic nonviral agents such as cationic lipids, peptides, polymers (polyethylene imine, polylysine), nanoparticles; Or noncationic nonviral agents, such as noncationic liposomes, noncationic nanoparticles or polymers. Such formulations are well known to those skilled in the art.

The invention also relates to a composition comprising the recombinant virus described above and a physiologically acceptable vehicle.

The invention also relates to a pharmaceutical composition comprising the vector described above. The pharmaceutical compositions of the present invention may be formulated for administration by topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular or transdermal routes and the like.

Preferably, the pharmaceutical composition contains a pharmaceutically acceptable vehicle for injection, in particular intravenous injection or injection into smooth muscle tissue. These are particularly isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride, etc., or mixtures of these salts), or, optionally, the injection solution upon addition of sterile water or physiological saline. Dry, in particular lyophilized, composition. Other excipients such as hydrogels can be used. Such hydrogels can be prepared from biocompatible and nontoxic (single- or hetero-) polymers. Such polymers are described, for example, in application WO93 / 08845. Some of these, especially those obtained from ethylene and / or propylene oxide, are useful. The use of hydrogels is particularly advantageous when delivering nucleic acids to the vascular wall, in particular smooth muscle cells of the vascular wall. The dosage used for injection can be adjusted as a function of various parameters, in particular the dosage form used, the purpose (labeling, pathology, screening, etc.), the gene to be expressed, or the desired expression duration. In general, the recombinant virus according to the invention is formulated and administered at a dosage of 10 ⁴ to 10 ¹⁴ pfu, preferably 10 ⁶ to 10 ¹⁰ pfu. The term pfu (“plaque forming unit”) corresponds to the degree of infection of a viral solution and is measured by counting the plaque number of infected cells by infecting the appropriate cell culture. Techniques for measuring pfu titers of viral solutions are well described in the literature. For in vitro or in vitro use, the cassettes, vectors or compositions of the invention can be cultured at conventional dosages in the presence of selected cell populations. Such incubation may be carried out in a culture dish, flask, fermenter or any other optional equipment.

The invention also relates to any cell modified by the cassette or vector (especially adenovirus) described above. "Modified" cell means any cell containing a construct according to the invention. These cells can be used for the production of recombinant proteins in vitro. It can be transplanted into an organism according to the method described in application WO95 / 14785. Such cells are preferably human smooth muscle cells.

The invention also relates to the use of a hybrid promoter as defined above for the specific expression of nucleic acids in smooth muscle cells in vitro, ex vivo or in vivo.

The invention also relates to the use of the hybrid promoter described above for the preparation of a composition for expressing a nucleic acid in smooth muscle cells (except endothelial cells present adjacent to an artery) in vivo.

Because of the smooth muscle cell-specificity, the constructs according to the invention can be used to create animal models of vascular pathology or to carry out methods for detecting or selecting the presence of smooth muscle cells in labeling studies or samples.

Subject of the invention is also a method of producing a recombinant protein comprising introducing a culture of the recombinant cell population into a cell population of a vector as described above and recovering the protein thus produced. Advantageously, smooth muscle cells are used to carry out the method according to the invention. It may be an established line or primary culture.

The present application will be described in more detail by the following examples (specific and without limitation).

Example 1 Construction of Hybrid Promoter and Expression Plasmid Containing the Same

1.1. Hybrid promoter hSMα-actin

Plasmid phSMact. High molecular weight genomic DNA was prepared from primary cultures of human aortic smooth muscle cells (Clonetics) according to methods described by Sambrook et al. ("Molecular Cloning, a Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989). did.

This DNA was used as template for initial amplification of PCR using the following primers:

Primer 6417 (starting at position −1034 (promoter region) of human specific smooth muscle α-actin gene (Ueyama H. et al., Mol. Cell. Biol., 4 (1984) 1073-1078. Access Genbank D00618) 5 'GATGGTCCCTACTTATGCTGCTA 3') (SEQ ID NO: 1).

primer 6418 (5 'CTTCCATCATACCAAACTACATA 3') at position 1974 of the D00618 sequence located inside the first intron of the hsMact gene (SEQ ID NO: 2). The reaction mixture is 1 mg genomic DNA, 10 pmol each of two primers (6417 and 6418), 100 mM each deoxyribonucleotide (dATP, dCTP, dGTP, dTTP), 2 mM MgCl ₂ and 5 units of Taq DNA polymerase (PerkinElmer) It includes. The reaction volume is 50 ml to fit the optimal PCR buffer concentration recommended by Perkin Elmer.

PCR amplification is performed in a Micoamp ^™ tube (Perkin Elmer) using a thermocycler PTC-100 ^™ (MJ Research, Inc.). This amplification consists of 30 cycles including a denaturation step at 95 ° C. for 2 minutes followed by a denaturation step at 95 ° C. for 15 seconds, an annealing step at 60 ° C. for 30 seconds and an extension step at 72 ° C. for 1 minute. These 30 cycles are followed by an additional 5 minutes and then the PCR reaction is stored at 100 ° C.

One microliter of this reaction is collected from the initial reaction and then diluted in 10 ml of water. Next, a second PCR was performed using 1 ml of dilution under the same conditions as the initial (above) except for the following different primer pairs:

Primer 6453 (5 'CTGCTAAATTG ctcgag GACAAATTAGACAAA 3') (SEQ ID NO: 3), which introduces the XhoI site (lower case, lowercase) upstream of the hSMact promoter (position -680).

Primer 6456 (5 'CCCTGACA aagctt GGCTGGGCTGCTCCACTGG 3') (SEQ ID NO: 4), which introduces the HindIII site at position +30 of hsMact.

After analysis on agarose gel and purification, the DNA fractions amplified by PCR were cleaved with XhoI and HindIII for 3 hours at 37 ° C. and then into the previously cut vector pGL3-Basic (Promega) using the same restriction enzyme. Cloning yields plasmid phSMact (FIG. 1).

Plasmids pXL3130 and pXL3131. DNA fraction corresponding to the enhancer region of the promoter of the human cytomegalovirus IE (hCMV-IE) gene between positions -522 and -63 relative to the transcription initiation site using plasmid pCMVβ as a template and oligonucleotide 8557 (5 ') as a primer. PCR amplification using ATC GAC GCG TGC CCG TTA CAT AAC TTA CGG 3 ') (SEQ ID NO: 5) and 8558 (5' ATC GAC GCG TCC GCT CGA GCG TCA ATG GGG CGG AGT TG 3 ') (SEQ ID NO: 6) . This fraction was cleaved with MluI and then cloned into plasmid phSMact previously cleaved with MluI and treated with alkaline phosphatase. Depending on the direction of insertion of the fractions, two different plasmids were obtained: pXL3130 and pXL3131. A schematic of this plasmid is shown in FIG. 1 (FIG. 1). These plasmids contain a hybrid promoter consisting of a promoter of the hCMV-IE gene and an enhancer of the promoter of the hSMα-actin gene in the form of MluI-NcoI fractions.

1.2. Hybrid promoter mSM22

Plasmid pmSM22. High molecular weight genomic DNA was prepared from Balbc mouse liver according to the method described by Sambrook et al. ("Molecualr Cloning, a Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989).

This DNA was used as template for PCR amplification using the following primers:

Primer 6517: (5 'CCAGGCTGCA ctcgag ACTAGTTCCCACCAACTCGA 3') (SEQ ID NO: 7), which introduces an XhoI site (underlined lowercase) at position -436 of the promoter of the mouse SM22 alpha gene (Solway J. et al. J. Biol. Chem., 270 (1995) 13460-13469; Access Genbank L41161).

Primer 6518: (5 'TCGTTTG aagctt GGAAGGAGAGTAGCTTCGGTGTC 3') (SEQ ID NO: 8), which introduces the HindIII site at position +43 of mSM22 alpha.

Using the same reagents and concentrations as for hSMact, a reaction mixture was prepared comprising 1 mg of mouse genomic DNA and 10 pmol of each of the two primers (6517 and 6518), followed by PCR amplification under the same conditions (see Example 1.1). Was done.

After analysis on agarose gels and after purification, the DNA fractions amplified by PCR were cleaved with XhoI and HindIII at 37 ° C. for 3 hours and then cloned into the vector pGL3-Basic (Promega), previously cut with the same restriction enzyme. . The resulting plasmid was called pmSM22 (FIG. 2).

Plasmids pXL3152 and pXL3153. DNA fraction corresponding to the enhancer region of the promoter of the hCMV-IE gene at positions -522 to -63 relative to the transcription initiation site using plasmid pCMVβ as a template and oligonucleotide 8557 (5 'ATC GAC GCG TGC CCG TTA CAT) as a primer PCR amplification was performed using AAC TTA CGG 3 ') (SEQ ID NO: 5) and 8558 (5' ATC GAC GCG TCC GCT CGA GCG TCA ATG GGG CGG AGT TG 3 ') (SEQ ID NO: 6). This fraction was cleaved with MluI and cloned into plasmid pmSM22 previously cleaved with MluI and treated with alkaline phosphatase. Depending on the direction of insertion of the fractions, two different plasmids were obtained: pXL3152 and pXL3153. Schematic diagrams of these plasmids are shown in FIG. 2. These plasmids, in the form of MluI-NcoI fractions, contain a hybrid promoter consisting of an enhancer of a promoter of the hCMV-IE gene and a promoter of the mSM22 gene.

1.3 control plasmid pCMV-leadTK

The expression vector pCGN described previously by Tanaka et al. (Cell, 60 (1990) 375-386) was fused with a "leader" of the HSV tk gene (+ 51 / + 101) upstream of the sequence encoding the hemagglutinin epitope. CMV promoter (-522 / + 72). Plasmid pCGN (10 ng) was used as template for PCR amplification. PCR reactions and amplifications were performed under the same conditions as used for hSMact and mSM22 (Examples 1.1 and 1.2). The primers used are as follows:

Primer 6718 (5 'CCCGTTACATAACTTACGGTAAATGGCCCG 3') (SEQ ID NO: 9), this primer hybridizes with the CMV promoter at position -522 (8 nucleotides downstream of the EcoRI site of pCGN).

- primer 6719 (5 'g GACGCGCTTCTACAAGGCGCTGGCCGAA 3' ) ( SEQ ID NO: 10), this primer hybridized to the location 101 or less of the tk "reader". Dark initial nucleotide G restores the NcoI site of pGL3-Basic, as clearly described below.

The PCR fraction thus obtained is purified and then phosphorylated by T4 phage polynucleotide kinase (New England Biolabs). At the same time, the vector pGL3-Basic (Promega) was linearized with NcoI, purified and treated with Klenow DNA polymerase (Boehringer Mannheim) to fill the NcoI site. This vector is dephosphorylated by alkaline phosphatase (Boehringer Mannheim) and then used to insert phosphorylated PCR fractions. Accordingly, guanosine (G) of primer 6719 is such that the CMV-tk leader fraction is oriented along the 5 'site downstream of the HindIII site of pGL3-Basic (primer 6718, position -522 of CMV) and its 3' end (primer). 6719, tk leader) can only recover the NcoI site when linked into the NcoI site of pGL3-Basic (initial ATG of luciferase). The plasmid thus obtained is named pCMV-leadTK.

Example 2: Specificity of an In Vitro Hybrid Promoter

This example illustrates the tissue specificity of the hybrid promoter of the present invention in vitro.

2.1. Cell culture

Rabbit smooth muscle cells (SMC) are cultured in DMEM ^™ medium (Life Technologies Inc.) supplemented with 20% fetal calf serum (FCS). ECV304 cells are cultured in 199 ^™ medium (Life Technologies Inc.) supplemented with 10% FCS. C2C12 myoblasts, HeLa cells, NTH 3T3 cells and TU182 cells are cultured in DMEM ^™ medium supplemented with 10% FCS. 293 cells are cultured in MEM ^™ medium (LifeTechnologies Inc.) supplemented with pyruvate, non-essential amino acids and 10% FCS. All incubations are performed in an incubator at 37 ° C. under a humid atmosphere and a CO ₂ partial pressure of 5%.

2.2. In vitro transfection

Transfection is carried out in 24-well plates and each transfection is performed three times. 24 hours prior to transfection, cells were (i) at 5 × 10 ⁴ cells / well for rabbit smooth muscle cells, ECV304, NIH 3T3 and HeLa cells, (ii) at 10 ⁵ cells / well for TU182 cells, (iii) Culture at 3 × 10 ⁴ cells / well for C2C12 cells and (iv) at 2 × 10 ⁵ cells / well for 293 cells.

For each well, 500 ng of plasmid DNA (250 ng of related plasmid and 250 ng of pUC18) was added in an amount of 6 nmol of lipid per μg of DNA in DMEM ^™ medium (final 20 μl) containing 150 mM NaCl and 50 mM bicarbonate. Mix with cationic lipid RPR120535 B (WO 97/18185). After 20 minutes at room temperature, 20 μl of the DNA / lipid mixture is contacted with the cells for 2 hours in the absence of FCS. Supplement the FCS to the culture medium to obtain the percentage of FCS required for the culture of each cell type.

48 hours after transfection, the culture medium is transferred and the cells are washed twice with PBS (Life Technologies Inc.). Luciferase activity is measured by the Luciferase Assay System ^TM Kit (Promega Corporation) according to the manufacturer's recommendations.

2.3. Specific Activity of Hybrid Promoter

The relative luciferase activity (for the pCMV-lead TK promoter) of the hybrid promoters measured in vitro in seven different cell types is shown in FIGS. 3 and 4 and Tables I and II. The results show that for promoters hSMact (Table I) and mSM22 (Table II), the relative activity is a value reflecting the specificity of these promoters for smooth muscle cells; Specificity is 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 SMC is at least 5 times higher than that observed in other cell types: (i) 5 to 20 times higher for promoter hSMact (Table I) and (ii) 5 to 25 times higher for promoter mSM22. (Table II).

The results shown in Tables I and II show that in smooth muscle cells, the activity of the four hybrid promoters (Enh-hSMact, hnE-hSMact, Enh-mSM22 and hnE-mSM22) according to the present invention is comparable to that of the CMV promoter in terms of intensity. Shows. In addition, the relative activity of each of these promoters in another cell type is at least 10 times less for the hSMact hybrid promoter and at least 4 times less for the mSM22 hybrid promoter than that observed for smooth muscle cells: (i) of the hSMact hybrid promoter. 10-140 times for and (ii) 4-55 times for the mSM22 hybrid promoter. These hybrid promoters preserve the same tissue specificity as observed for the specific promoters hSMact and mSM22.

These results additionally show that the orientation of the enhancer region in the hybrid promoter of the present invention does not significantly affect the activity.

The four hybrid promoters retain activity in smooth muscle cells comparable to the promoters hSMact and mSM22 in vitro, or as high as CMV promoters (known as strong promoters), with higher tissue specificity.

Example 3: Specificity of Hybrid Promoters in Vivo

This example illustrates the tissue specificity of the hybrid promoter of the present invention in vivo.

3.1. Gene Delivery to Skeletal Muscle

Various plasmids were injected into the two tibialis muscles of female C57BL6 mice grown for 5 weeks via the intramuscular route. Each plasmid diluted with the final 150 mM NaCl solution is injected in an amount of 10 μg per muscle. Three days after injection, the muscles are collected in 2 ml of Cell Culture Lysis Reagent ^™ buffer (Promega Corporation) and ground in a Diax grinder (Heidolph). The milled product is centrifuged at 4000 g for 15 minutes and luciferase activity is assessed using Luciferase Assay System ^™ kit (Promega Corporation) according to the manufacturer's recommendations.

3.2. Activity of Hybrid Promoter in Skeletal Muscle in Vivo

The relative activity of the two specific promoters (hSMact and mSM22) and the relative activity of the two hybrid promoters (Enh-hSMact and Enh-mSM22) of the present invention were evaluated after transferring naked DNA to the cranial tibialis muscle in vivo. The results shown in FIG. 5 show that the activity of the Enh-hSMact promoter is 100 times lower than the CMV promoter. In addition, the activity of the Enh-mSM22 promoter is 17 times lower than the CMV promoter. Thus, the tissue specificity observed in C2C12 cells that make up the model closest to those used in vitro, particularly in vivo, is preserved in vivo.

Example 4 Construction of Recombinant Adenovirus Expressing GAX Proteins Under the Control of Specific Hybrid Promoters

This example will describe an adenovirus vector carrying a gene encoding a GAX protein operably linked to a hybrid promoter of the invention consisting of a CMV enhancer and an SMα-actin promoter (enh-hSMact).

The human gax gene contains 912 base pairs and encodes a transcription factor of 303 amino acids involved in the inhibition of cell growth (growth-inhibition-specific homeobox) and responsible for the proliferation of human smooth muscle cells. These homeodomain-containing genes were initially isolated from the aorta and specifically expressed in adult cardiovascular tissue (Gorski et al., 1993).

Human gax gene sequences were cloned from skeletal muscle cDNA libraries by PCR (polymerase chain reaction) using sequences derived from the human gax gene as primers and published by Walsh et al. (Genomics (1994), 24, p535). The sequence was cloned into the expression vector pXL3297. Such plasmids were found in plasmid bluescript (Stratagene) containing human CMV IE enhancer / promoter (-522 / + 72) (Cell (1985), 41, p521) and SV40 poly A (2538-2759) (GenBank SV4CG position). Induced.

The construct was made using the plasmid pXL3130 described in Example 1 (FIG. 1), and the SMα-actin promoter was introduced in advance. Plasmid pXL3297 is an expression vector containing human gax gene. This was cleaved using the enzymes HindIII and AvrII and introduced into the human gax gene into the plasmid pXL3282 previously cleaved with the enzymes HindIII and AvrIII to make plasmid pXL3300. As indicated in FIG. 6 where various steps relating to the construction of the plasmids described above are depicted, the final plasmid, pXL3310, contains an expression cassette consisting of a CMV-IE enhancer, a SMα-actin promoter (pSMA), which are combined according to the present invention. And operably linked to genes encoding human GAX protein and SV40 poly A termination signal.

The human gax gene expression cassette is a recombinant human adeno of serotype 5 (Ad5) that lacks the E1 and E3 regions by co-transfection and homologous recombination between adenovirus and a plasmid carrying a cassette expressing the gax gene in capsidated cells. It is introduced into the virus. These cells are preferably 293 lineages. The production of adenovirus stocks containing cassettes expressing the human gax gene is due to infection and lysis of capsidated cells 2 or 3 days after separation of recombinant virus particles by cesium chloride gradient centrifugation.

Viral particles are used to study the expression of the human gax gene in smooth muscle cells under the control of the promoter of the present invention.

Expression of GAX protein is checked 24 hours after infection of primary smooth muscle cells by immunoblotting or western blotting with rabbit anti-gax polyclonal antibodies.

Expression of messenger RNA is analyzed 24 hours after infection of smooth muscle cells by dot blot and northern blotting using oligonucleotides of the sequence present in the gax gene.

Biological activity analysis of adenoviruses encoding gax genes is carried out in the following manner:

Smooth muscle cells of the logarithmic proliferative phase are infected with adenoviruses containing genes encoding GAX proteins under the control of the enh-hSMact promoter in the absence and presence of 125 ng of lipofectin (48-well plate). Adenovirus (variably diluted) and lipofectamine are incubated for 30 minutes in serum-free medium at room temperature. The mixture or virus alone is contacted with the cells at 37 ° C. for 1 hour. At the end of infection, the virus containing medium is transferred and the cells are incubated in DMEM medium containing 0.5% SVF. Within 24 hours after infection, the culture medium is replaced with a growth medium equivalent to 1/2 of the culture and continued incubation for 48 hours to allow cells to enter S phase. For the other half of the culture, weakly mitotic medium is added to maintain the cells. The Alamar protocol is used to count live cell numbers 72 hours after infection.

Claims (18)

All or part of an enhancer region of a strong ubiquitous promoter / enhancer, and A hybrid promoter comprising a promoter region that allows specific expression in smooth muscle cells. The method of claim 1, wherein the enhancer region is selected from the group consisting of an enhancer region of cytomegalovirus immediate-early (CMV-IE) gene, an enhancer region of Rau's sarcoma virus LTR (RSV-LTR), an enhancer region of SV40 virus, and an enhancer region of EF1α gene. Hybrid promoter, characterized in that selected. 3. The hybrid promoter according to claim 2, wherein the enhancer region is an enhancer region of the immediate-early gene of cytomegalovirus (CMV-IE), preferably human cytomegalovirus (hCMV-IE). The hybrid promoter according to claim 1, wherein the promoter region comprises all or part of a gene encoding SM-actin of smooth muscle cells or SM22 gene. All or part of an enhancer region of a human cytomegalovirus immediate-early (hCMV-IE) gene, and A hybrid promoter comprising all or part of a promoter of a gene encoding α-actin (SMact) of smooth muscle cells. All or part of an enhancer region of a human cytomegalovirus immediate-early (hCMV-IE) gene, and A hybrid promoter comprising all or part of a promoter of the SM22 gene. The hybrid promoter of claim 1, wherein the promoter region comprises a basic promoter and a tissue specific transport sequence, said sequence derived from an SMact promoter and / or SM22 promoter. An expression cassette under the control of a hybrid promoter according to any one of claims 1 to 7 and comprising a nucleic acid encoding a relevant RNA or polypeptide. 10. The cassette of claim 8, further comprising a signal for transcription termination. 10. The method according to claim 8 or 9, wherein the nucleic acid encodes a protein selected from among the proteins involved in the cell cycle, apoptosis inducing protein, a protein capable of modifying the proliferation of smooth muscle cells, angiogenesis inducing protein and a transcription factor. Characterized by a cassette. A vector comprising a hybrid promoter according to claim 1 or a cassette according to claim 8. The vector of claim 11, wherein the vector is any DNA that is not encapsidated by a plasmid, cosmid or virus. 12. The vector according to claim 11, which is preferably a recombinant virus derived from an adenovirus, retrovirus, huff virus or adeno-associated virus. A composition comprising a vector according to claim 12 and a chemical or biochemical delivery agent. A composition comprising the recombinant virus according to claim 13 and a physiologically acceptable vehicle. A cell modified by the cassette according to claim 8 or the vector according to claim 11. Use of a hybrid promoter according to any one of claims 1 to 7 in preparing a composition intended for the selective expression of nucleic acids in smooth muscle cells. Use of a hybrid promoter according to any one of claims 1 to 7 in the preparation of a composition intended for the expression of nucleic acid in smooth muscle cells that are not endothelial cells found near blood vessels.