JP2000507819A - Sequences upstream of the gene SM22, vectors containing these sequences, and their therapeutic use, especially for the treatment of vascular diseases - Google Patents

Abstract

  (57) [Summary] A fragment of a sequence preceding the coding portion of the gene for protein SM22 or a sequence that hybridizes under high stringency conditions to the preceding sequence, wherein the fragment is susceptible to elicit specific expression of this gene in eukaryotic cells; Also disclosed are coding sequences for proteins of therapeutic interest, or RNA. The sequences or vectors containing them can be used to treat coronary artery disease, especially restenosis.

Description

DETAILED DESCRIPTION OF THE INVENTION Sequences upstream of the gene SM22, vectors containing these sequences, and Their therapeutic use for treatment   The present invention relates to a sequence upstream of a gene expressed in smooth muscle such as SM22, and A vector comprising the same.   Another object is the use of these vectors and sequences for therapy. example For example, locally or systemically active polypeptides, especially immunogenic polypeptides, To produce endogenous regulatory polypeptides such as itokine or Use to produce targeted RNA, eg, anti-sense RNA. In particular, A clear objective is to use nucleotide vectors and sequences in the treatment of vascular diseases. is there.   Atherosclerosis is a disease of the arteries that combines arteriosclerosis and atherosclerotic lesions. It is a sexual disease and is thus accompanied by hardening of the arteries and steatosis of the intima of the arteries. This crown Arterial insufficiency is generally caused by connecting a catheter with a balloon at the tip to the arterial network. The catheter, advance the catheter until it reaches the stenosis, and inflate the balloon. To restore the coronary artery inner diameter to adequate myocardial perfusion. Treated by percutaneous coronary dilatation consisting of Is done.   This myocardial revascularization technique is very widely used (in France, 50 000 surgeries in the United States and 500 000 surgeries per year in the United States), about 30% of all cases Several months after surgery, new lesions reappear or re-stenosis in the dilated area There is a limit from that.   Various treatments proposed to treat this type of lesion include FELDMAN and STE "Perspective de la th by G ummary, 12, 47-55).   Restenosis involves two mechanisms: intimal thickening and arterial remodeling . The first of these mechanisms, intimal hyperplasia, By proliferation of intimal smooth muscle cells leading to synthesis. This activation involves growth factors and By local release of cytokinins and by combining several antiproliferative endothelial peptides Triggered by the exclusion of the adult. The second mechanism, arterial remodeling, is to reduce wall thickness It is to reduce the inner diameter of the artery without changing it. Therefore, accompanying cell growth I don't know.   The authors described various attempts to inhibit intimal hyperplasia by gene therapy. But the results may vary for various reasons. Not very satisfying. Poor metastasis is one way to explain these results. This is one of the reasons generally shown. This problem is caused by the use of adenovirus vectors. But another problem was the uncontrolled recombination by adenovirus. Risk of spreading these vectors into the systemic circulatory system It appears to be related to the immunogenicity of these viruses.   These authors also report that preproendothelin or adenovirus-ligand Specific promoter sequences that vascular cells have, such as complexes, Comment on using it as a system of great interest for research without occurring. Has been reached.   Application WO 96 / 05.321 (Rhone Poulenc Rorer S.A.) also claims suicide for the treatment of restenosis. A defective recombinant adenovirus that makes up the gene is described. Suicide gene Can be placed under the control of an active promoter in vascular smooth muscle cells. Smooth muscle Only the tin alpha promoter is mentioned for the purposes of this embodiment. This Thailand None of the examples of the use of loop constructs illustrate this application.   Piecewise studies were performed on proteins that make up smooth muscle. For example, flat Study of protein SM22, one of the first markers to appear during differentiation of smooth muscle cells And The coding portion of the gene is a mouse (SOLWAY et al., 1995, J, Biol. Chem, 270, 22, 13460-13469) and rats (KEMP et al., 1995, BIOCHEM. J. 310, 1037-1043). Sequenced.   The sequences of the chicken and human genes were also determined (for chicken genes). And Nishida et al., 1991, Biochem. Int., 23, 663-668; Th for human genes weatt et al., 1992, Biochem. Biophyse. Res. Comm., 187, 1-7).   Is the coding gene for protein SM22α in mouse 5 exons? It comprises 6.2 kb. That single copy is present in the genome. SOLWAY et al. Indicates that 441 base pairs upstream of the coding region of the gene Primary cell culture and A₇r_FiveInduces a high transcription rate of the reporter gene Has been shown to be necessary and sufficient to guide. They also have a large amount of It has been demonstrated that sussenger RNA is expressed in the aorta, uterus, lung and intestine.   KEMP et al. (1995, BIOCHEM, J. 310, 1037-1043) reported on the coding of the rat gene SM22. The 1.9 kb fragment upstream of the signaling sequence was cloned and sequenced. Hula Fragment from the promoter region between nucleotides +65 and -303 Region contains most of the untranslated region of the gene. Was shown to be more active than the 1.5 kb fragment. This suggests the presence of a regulatory sequence at the 5 'end of the gene.   OSBOURN et al. (1995, Gene, 154, 249-253) also proposed a 5 'upstream of rat gene SM22. The area was studied. They have demonstrated the presence of two introns in this region.   These studies were performed in vitro using primary aortic cell cultures. Note that it was not in vivo. However, these experimental conditions Does not indicate the activity of the gene in the cells of the organism.   In addition, they are very limited in the non-coding parts of the gene located at the 5 'end. Region, in other words, the portion downstream of nucleotide -441.   More recently, Li et al. (1996, J. Cell Biol, 132, 849-859) The regulation of the gene SM22 was studied using mice.   However, this document does not cover the vector containing a fragment of this gene. No application is mentioned and there is obviously no therapeutic application.   In addition, a limited number of constructs have been produced, and generally only effects on embryos have been tested. It has not been performed on adults.   The authors have shown from these studies that the sequence between nucleotides -2735 and -445 Speculation does not include any essential muscle regulatory elements.   Applicants believe that the upstream of the expressed coding sequence of the gene for protein SM22 The activities of various regions that make up the region And tried. He also noted that modifications in this sequence could alter gene expression in various tissues. An attempt was made to determine whether to induce a change.   Surprisingly, he modifies the 5 'region of the mouse protein SM22 by modifying it. Thus, the specific expression of the reporter gene in arteries, especially in the aortic smooth muscle, is Demonstrated that it is possible in adults.   He was also identified by LI et al. As containing no essential regulatory sequences Since the sequence (1996, supra) actually lacks the specific expression of the SM22 gene in adults, It has been demonstrated that it contains a kinase sequence.   Finally, surprisingly, he found that intron sequences induce specific expression in arteries. It is necessary to do it.   Therefore, the object of the present invention is to     -Sequence or high upstream of the coding part of the gene for protein SM22 Fragment of a sequence that hybridizes with the upstream sequence under stringent conditions G Fragmentation is likely to induce specific in vivo expression of this gene in arterial cells), and And     -Coding sequence or RNA of a protein of therapeutic interest   And a DNA sequence comprising:   The present invention also relates to naturally occurring or at least 70% similarities to SEQ ID NO: 1. Is any nucleotide or oligonucleotide sequence obtained by chemical synthesis. About the columns.   Naturally occurring nucleotide sequences are derived by reverse transcription of cellular messenger RNA. Complementary fragments of the resulting DNA, or after cleavage of cellular DNA with restriction enzymes It means a fragment of the genomic DNA obtained.   Nucleotide sequences obtained by chemical synthesis, for example, Using a DN having a known sequence produced by automated synthesis of a polynucleotide Refers to the A fragment.   Proteins or RNAs of therapeutic interest are those that inhibit the growth of smooth muscle cells, May activate endothelial cell proliferation and strengthen arteries, and / or A molecule capable of eliciting a humoral immune response.   For the purposes of the present invention, "highly stringent articles The term “case” is defined in Maniatis et al., 1982 (Molecular cloning, A Laboratory Manual , Cold Spring Harbor Lab. CSH, N.Y. USA) or one of the more recent reprints Used in the sense that it is. Preferably, the hybridization conditions are Ma niatis (1982 edition). Fragments that did not hybridize 3 washes are required to remove the cells, in the presence of 0.2 SSC and 0.1% SDS At 65 ° C. in the absence of formamide.   Advantageously, the sequence corresponds to nucleotide -2126 shown in the Appendix as SEQ ID NO: 1 And +4135, nucleotides -2126 shown in the Appendix as SEQ ID NO: 2 +65, or nucleotide -2126 shown as SEQ ID NO: 3 in the Appendix And -445.   It may also include the fragment of SEQ ID NO: 4.   In particular, fragments of this type have a coding between nucleotides -2126 and -1340. Less of the sequence of SEQ ID NO: 5 corresponding to the portion upstream of the 5 'end of the And a portion. One of the objects of the invention is that the sequence is given in the appendix Is the sequence of SEQ ID NO: 5 of this type.   The sequences of SEQ ID NO: 2 and SEQ ID NO: 1 are based on the plasmid p2 126nlz and p2126INTnlz, respectively, and CNCM (Na National Collection of Microorganism Cultures) on March 25, 1996 Included in strains deposited under numbers I-1685 and I-1686.   It is another object of the present invention to provide a method for treating SEQ ID NO: 1, It is a sequence that hybridizes with the sequences of SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 5.   The sequence containing the first intron sequence of gene SM22, in particular the sequence of SEQ ID NO: 1 By an adult of a protein or RNA of therapeutic interest of specific artery Which form a particularly advantageous embodiment of the present invention.   However, the invention is limited to sequences containing fragments of the mouse gene. To induce gene expression in arterial cells. It relates to any other sequence of any other kind that has the property of emitting. Therefore, Expert reveals sequence upstream of rat gene in protein SM22KE It is useful to refer to the literature of MP et al. (1995, supra). The invention also relates to proteins Any sequence, including fragments of sequences upstream of the gene in SM22, e.g., complete Deletion of several structures with the same or similar function as the function in the entire sequence And a sequence modified by   A protein of therapeutic interest is thymidine kinase in herpes virus. Such proteins may induce the formation of such cytotoxic compounds. This protein Has ganciclovir, which has special features and resembles guanosine, in cells with DN Transform A into a derivative that interferes with replication synthesis (Merck Index, reference 4262). For this, the coding gene of this protein is introduced into the sequence according to the invention. And block cell growth in the case of intimal thickening. Thymidine kinase The sequences to be loaded are described in particular by Caruso and Klatzmann (1992, Proc. Natl. . Acad. Sci., USA, 89, 182-186).   The protein of therapeutic interest may also be encoded by the Rb gene (Chang et al., 1995, Science, 267, 518-522) or encoded by the eNOS gene (Hamon et al., 19 94, Circulation, 90, 1357-1362) has a cytostatic effect, such as protein It may be a protein.   It is also a protein with lipolytic activity, such as lipoprotein lipase. It can be quality. This type of protein is available from Reyner (1995, Nature Genetics). , 10, 28) and Ming Sun Liu (1994, J. Biol. Chem, 269-11417). Coded into an array.   It may also be a growth factor for endothelial cells. In particular, interleukins One such protein.   It can also be a muscle protein such as myosin or actin, or It may be a protein having a tissue structure like lagen or elastin.   It is a protein that elicits an immune response as described in application WO 90 11092 Especially the glycoprotein gp120 or the HIV Nef protein (human immunodeficiency virus) Virus antigen.   Generally, the protein will be therapeutically active, such as a protein of immunotherapeutic interest. Or proteins of interest as vaccines, such as interleukins, growth factors Or, for example, fibroblast growth factor (FGF) or nerve growth factor (NGF); A protein capable of eliciting an immune response for cytotoxic or cellular immunity; or Can normally complement the activity of a gene, but either mutate or delete its sequence Depending on one or more of the proteins that are no longer expressed in the treated individual. It can be any kind of protein.   This sequence may also encode an RNA of therapeutic interest. This type of RNA Antisense RNA of protein p53 Or as described in application WO 9011092 deposited by the VICAL Company Such RNA may be used.   Another object of the invention is characterized in that it comprises an arrangement according to the invention as described above. Vector. This type of vector is useful for proteins of therapeutic interest or It may include any other DNA sequence required for expression of the RNA in the target tissue, particularly, arterial cells, In particular, it may contain an efficient origin of replication for smooth muscle cells.   This type of vector allows for the corresponding recombination in the organism to be treated. A sequence specific for the gene to be replaced, said sequence being upstream of the sequence of the invention. Or is substituted downstream. Due to the presence of these sequences, they are present in the treated organism. Undesired genes can be expressed in vectors or vectors required to be expressed in organisms. It is replaced by the gene contained in the column.   A corresponding recombinant method of this type is described by Le Mouellic et al. (1990, Proc, Nat. Acad. S. ci. USA, 87, 4712-4716) or in application PCT WO 91 / 06.667. Type.   This type of vector can be a vector derived from an adenovirus. Book Adenoviruses particularly adapted for embodiments of the invention include FELDMAN and STEG (1996, Above) (1994, Sciences, 265, 781-784) or application FR 94.03.151 (Pasteur I nstitute, Inserm). These viruses usually have their excess Rejected by individuals due to poor immunogenicity. Nevertheless, these will Strains were selected to reduce their immunogenic properties. In any case However, even though these vectors are highly immunogenic, the arteries are not affected by the host immune system. In a relatively short period of the order of a few weeks to accommodate the adenovirus rejection period As treated, it can be used within the framework of the present invention.   Nevertheless, it is not possible to introduce the sequences according to the invention into the above-mentioned vectors. Not indispensable, arterial cells are directly transduced by the DNA that makes up these sequences. It should be noted that it can be transfected.   The nucleic acid sequence according to the invention may also be introduced after the nucleic acid has been covalently linked to the compound. it can. The compounds promote their penetration into cells or their transport to the nucleus And the resulting conjugate is to Meidsorb Technologies International As described in the international application WO 94/27238 filed, Can be putselled.   According to another embodiment, the nucleic acid sequence is Seikagaku Corp. their permeation into cells, as in international application WO95 / 10534 filed by oration Included in the transfection system containing the polypeptide obtain.   These vectors or sequences can be prepared in situ by any means known to those of skill in the art. Can be administered. For example, they are described in FELDMAN and STEG (1996, supra). Using a catheter with a balloon covered by a channel, It can be delivered in situ.   They can also be administered during surgery or by removing the aorta. Other methods of administration are described in FELDMAN et al. (1995, J. Clin. Invest., 95, 2662-2671). Screen with a small mesh impregnated with DNA containing these sequences And the introduction along the aorta.   These sequences or vectors may be in the form of compositions containing them, for example, Gels that facilitate their transfection in the vesicles and can be advantageously administered . This gel was applied to MIDOUX (1993, Nucleic Acid Research, 21, No. 4, 871-878). Complexes of poly-L-lysine and lactose or PASTORE (1994, Circ ulation, 90, I-517). They are Can be placed in a solution in a buffer solution, or It can be combined with liposomes.   The invention also relates to an agent comprising these sequences, vectors or compositions, as well as To a pharmaceutical composition comprising a pharmaceutically effective amount and a pharmaceutically compatible excipient. You.   These types of sequences, vectors or compositions are particularly useful for treating heart disease, and DNA capable of expressing a gene encoding a protein of particular interest in the treatment of restenosis ( (cDNA or genomic DNA) can be advantageously used in the manufacture of a medicament for delivering arteries . Nevertheless, they are also drugs for the treatment of mutations that weaken blood vessels Can also be used for the production of For example, this type of drug is Expression of coding genes for various proteins or CAM-type muscle adhesion proteins Could contain a sequence or vector of the invention.   Another object of the present invention is to express sequences and vectors expressed in the present invention. To determine RNA, especially messenger RNA.   Another object of the present invention relates to the regulatory sequences of the gene encoding the protein SM22. Methods for screening molecules in vitro to test the activity of a molecule is there. For example, this method uses the promoter of the gene encoding the protein SM22. -Under the control of all or part of the array Transfecting a cell with a sequence or vector of the invention containing the reporter gene And a first step of performing the process. In the second step, the transfected Incubated cells in the presence of the molecule to be tested, followed by a reporter -Expression of the gene is quantified.   The cells used for transfection are advantageously in primary culture or Smooth muscle cells, especially aortic cells, which are any of the forms of stable cell lines.   Cells are transfected with constructs such as p2126nlz or p2126INTnlz When produced, the β-galactosidase produced is, for example, recovered by Boehringer. Detection kit sold in Reference 1 669 893 (Boehringer-Mannheim "Biochemi cals "catalog, 1996, p. 236, beta-Gal Reporter Gene Assay, Chemilumin escent) and is advantageously quantified by optical readings.   In another embodiment of the in vitro screening method according to the present invention, The target gene is a luciferase coding gene. In this case, Lucif Quantification of the expression of cellulase is performed, for example, by the method of Boehringer in Reference 1 Diagnostic kit sold in 1 (Boehringer-Mannheim "Biochemicals" catalogue, 199 6, Luciferase Reporter Gene Assay) Will be displayed.   The present invention also relates to a transgenic vector comprising a sequence or vector as defined above. The present invention relates to cook animals, particularly mice. A protein of therapeutic interest in said animal Has been replaced by a reporter gene.   Finally, the present invention relates to a sequence upstream of the coding part of the protein SM22 gene. A method for detecting a mutation in a column. These mutations The expression of the protein SM22 coding gene can be modified, especially in smooth muscle. Could be reduced or even eliminated. Protein SM22 regulates calcium fixation It is related to a family of knot-related calponin-type proteins, Mutations of type will cause functional modifications to smooth muscle (Ayme-Southg ate et al., 1989, J. Mol. Cell. Biol.). This type of mutation detection method advantageously comprises It may be the method described in French patent application FR-93 10821.   In a complementary manner, the present invention also modifies to allow for increased expression of the SM22 gene. Including the decorated sequences of the present invention.   This type of transgenic mouse has a molecular Quality SM22 coding gene It can be used to screen for activity against regulatory sequences. The molecule is Mice can be administered and subsequently killed before being colored by a reporter gene. Histological sections are made to show the affected tissue.   For embodiments of the present invention, one of skill in the art will advantageously recognize the manual "SAMBROK et al. (Molec ular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989) "or one of its latest reprints.   The present invention is illustrated by, but not limited to, the following examples:   Figure 1 shows the region between -2474 and +6030 (measured for transcription start site) Figure 2 shows a map of the restriction and exon / intron structure of the SM22 site, including Exo Are indicated by open squares, and arrows indicate the position of the repeating B1 sequence.   FIG. 2 shows the sequences of three types of repetitive B1 sequences, showing the basic changes therein.   FIG. 3 is an autoradiogram showing the identification of the transcription start site. Well P Corresponds to the primer used.   FIG. 4A shows the sequence of the 5 ′ region upstream of the transcription start site and exons of the SM22 gene. Is shown. Shows fixation sites for various factors with various consensus sequences . The dashed line indicates the position of the repeating B1 sequence.   FIG. 4B is a schematic restriction map of the SM22 gene.   FIG. 4C is a detailed restriction map of the SM22 gene.   FIG. 5 shows the construction of plasmid p352nlz.   FIG. 6 shows the construction of plasmid p2126nlz.   FIG. 7 shows the construction of plasmid p2126INTnlz.   8A and 8B show the expression of the gene lacZ in transgenic mouse embryos. You. Figures 8A and 8B show embryos at the 12.5 and 15.5 day stages, respectively.   9A-9D show histological embryo sections stained with lacZ. FIG. 9A Shows a cross-section of the heart at the 12.5 day stage of the embryo, showing the myocardium and arterial wall of the right ventricle (RV). Only a strong staining (abbreviation -AA = flexion of the fourth aortic arch artery; CA = carotid artery; E = esophagus; lv = Left ventricle; PA = proximal aorta; PT = pulmonary trunk; RA = right atrium; RV = right ventricle; T = trachea; V = right and left Main vein).   FIG. 9B shows a cross-sectional view of the abdominal region of the embryo at the 14.5 day stage, showing staining of the zen artery (UA). And lack of visceral markers (B = bladder; D = duodenum; H = large intestine; L = liver; M = small intestine; ST = stomach) .   FIG. 9D shows a cutaway view of the tail at the 12.5 day stage of the embryo, showing the myocardium (my) and the tail artery (a ) Indicates a label.   FIG. 9C is an enlargement of the portion of the photograph corresponding to the instant artery at the 12.5 day stage. m) only, and endothelium (e) Indicates lack of knowledge.   FIGS. 10A and 10C show transgene expression in adult mice.   FIG. 10A shows dense markers for aorta (a) and pulmonary artery (pa) and markers for vena cava (cv). 1 is an upper image of the entire heart showing a lack of insight. FIGS. 10B and 10C show the adult colon. Sections of the layer of smooth muscle (sm) and adjacent mesenteric artery (ma). Labeling of lacZ (FIG. 10B) Indicates that the expression of the transgene is restricted to the arteries, but is immunized by the SM22 antibody. Epidemiological fluorescence (FIG. 10C) shows the inheritance of endogenous smooth muscle cells in both arteries and colon. 3 shows expression of offspring SM22.   FIG. 11 shows the regulatory region of SM22 gene and thymidine kinase of herpes virus. 1 shows a vector containing a gene encoding   12A-12D show embryos 15.5 days after mating (FIGS. 12A-12C) and 2 months of age. 12 shows expression of the SM445nlz construct in adults (FIG. 12D).Example Apparatus and method   1.Cloning and characterization of mouse gene SM22   Delta-EMBL 3 phage (provided by Dr. D. Plachov, Münster, Germany) About 10 of the genomic mouse bank c57 / bl constructed in⁶Recombinant phages Of the mouse protein SM22 (Almendral et al., 1989; Exp. Cell Res. 181, 518-530). Screening was performed using the 890 bp BALI / Eco RV fragment of the additional DNA as a probe. Learning. Probes are radiolabeled by random initiation, DN A was placed on a filter left overnight at 65 ° C in Church buffer. It was hybridized. The filters are then washed at 65 ° C in 0.2xSSC / 0.1% SDS. The positive plate was purified by three successive rescreens under the same conditions and homogenized. I knew it. One of the clones isolated was shown to contain the entire SM22 locus. Subsequently, it was mapped using restriction enzymes and subcloned. Clonin Mouse genomic DNA and genomic DNA to avoid re-arrangement during the Southern blot hybridization analysis was performed on Segment comparison charts.   The SM22 gene sequence was sequenced using the Sequence kit V2.0 (United States Biochemical, Brand, Ohio) using two strands using the chain termination method Were determined.   2.Northern type hybridization, primer extension, 5'RA Analysis of protection by CE and RNAse   Total RNA from cell lines and mature mouse tissues was obtained from Chirgwin et al. (1979, Biochemistry, 18 , 5294-5299). Produce messenger RNA Oligotex kit (Quiagen Inc, Chatsworth, CA) (Near State).   Analysis by Northern type hybridization Using an agarose gel containing Hybond-N membrane (Amersham) using a method known to those skilled in the art. Life Science, Little Chalfont, UK) Performed by zation.   For analysis by primer extension, the phase of the SM22 gene A 30 nucleotide synthetic oligonucleotide that is complementary to the complementary DNA sequence +36 to +65 ( P27N1) was prepared.   (5'-AAGGCTTGGTCGTTTGTGGACTGGAAGGAG-3) to polynucleotide kinase T4 Therefore, it was labeled. 5 μg of mouse uterine messenger RNA was added to 1-2 × 10^Fivecpm purification After hybridization with the probe at 55 ° C, the primer extension The test procedure was used. Results are 6% denatured It was analyzed by electrophoresis on a polyacrylamide gel. Direct determination of transcription start site Load the reaction with the same primers into adjacent wells to allow Was.   Bases +112 to +129 (primer 2) and +162 to +179 (primer of SM22 complementary DNA) Using two 18 bp antisense synthetic oligonucleotides corresponding to 1), 5′R The ACE method is described in FROHMAN et al. (1988, Proc. Natl. Acad. Sci, 85, 8998-9002). It was performed as follows.   Based on the results of RNase protection, the -352 to +65 region of the SM22 gene obtained by PCR And a 417 bp fragment containing Cloned using the terminating method. Sequence of the fragment obtained by PCR Revealed. To generate a radiolabeled antisense RNA probe, use Sph  Using I to linearize the construct at position -203 of the SM22 gene,³²Polymer in the presence of P-CTP Rahse RNA T₇Was used for transcription. Probe (3 × 10^Fivecpm) with 5 μg of mouse uterus Hybridized overnight at 42 ° C. with ssenger RNA. Hybridization Later, samples were incubated with RNases A and T (1 hour at 37 ° C) and allowed to settle. And analyzed as described for primer extension.   3.Cell culture and transfection   Cell lines NIH 3T3, 10T1 / 2 and 8/47 were transformed with 10% fetal calf serum (Hyclone Inc., In DMEN (Life Technologies, Inc, Vienna, Austria) 7% CO at 37 ° C_TwoCultured in an atmosphere.   For transfection experiments, seed confluent cells in 6 cm Petri dishes. And grow to approximately 75% confluence before adding the transfection mixture I let it.   Using a Lipofectamine reagent sold by Life Technologies Inc. The transfection was performed.   In summary, 6 μg of plasmid containing the supercoiled reporter gene and internal As a part control, 24 μg of Lipofetamine was added to 2 μg of PCMβgal. 3ml After adding DMEM, the transfection cocktail was added to the cells. 6 hours later This mixture was replaced with growth medium containing 10% serum and changed again after 12 hours. The expression of the reporter gene product was analyzed after 24 hours. Simulated by serum Transfected cells can be collected for 24 hours Previously, they were placed in DMEM containing 20% serum.   4.CAT Of β and galactosidase   Cells are removed from the Petri dishes using a cell scraper, centrifuged and 150 μl of Z buffer (100 mM sodium phosphate PH 7.5, 1mM MgCl_Two, 10nM KCI, 50nM β-mercaptoethanol) Thawed in three freeze / thaw cycles. Centrifuge at low speed to remove debris Using 2-20 μl of suspended material using processes known to those skilled in the art, β-galacto Sidase activity was measured. GORMAN et al. (1982 Mol. Cell. Biol., 2, 1044-1051). Therefore, 10-100 μl of lysate was used for the determination of CAT activity. Transfer CAT activity was normalized by βgal activity to take into account the variation in the collection efficiency.   5.Production of transgenic mouse   Plasmid pnlz2126 deposited with CNCM under numbers I-1685 and I-1686, respectively And the insert of p2126INTnlz were isolated by digestion with SalI / PmeI and subsequently Electrophoresis, then use Geneclean kit (Bio 101, La Jolla, CA) And then resuspended in 10 mM Tris HCI pH 7.5, 0.25 mM EDTA. LI et al. ( 1993, Development, 177/3, 947-959). Sgenic animals were produced.   Mice containing the transgene were transferred to Southern Type Hi using PCR and DNA probes. Identified by hybridization. The pnlz2126 construct out of 17 mice Three positive animals were obtained, two of which transmitted this construct, Express.   For the construct p2126INTnlz, 2 of the 28 tested positive animals were obtained. Again, only one animal expresses and transmits this construct.   Transgenic mice were backcrossed with C57 / bl mice to Histochemical staining was performed with hemizygous mice.   6.Histochemical staining   Whole embryos or adult tissues are treated with Buffer A (100 mM sodium phosphate) for 15-30 minutes at 4 ° C. Um, pH7.3, 2mM MgCl_Two, 0.1% sodium deoxycholate, and 0.2% NP-4 Fixed in 1% formaldehyde in 0). After washing, the specimens were collected at 30 ml X-gal (Sigma, St. Louis, Mo.), 5 mM potassium ferrocyanide, Stained in buffer A containing 5 mM potassium ferricyanide, 20 mM Tris-Cl pH 7.3 Was. Next, the stained sample is washed and destained with increasing concentrations of ethanol. Water, clarified in xylene and coated with paraffin. Histological section thickness 7 ~ 10 μm and stained again with Ehrlich's hematoxylin and eosin. Was. Next, the embryos are again clarified using benzyl alcohol and benzyl benzoate. It has become.   For immuno-histological staining, mouse tissues were treated with 3% paraformaldehyde in PBS buffer. The cells were fixed at 4 ° C. for 3 hours in a hide, followed by Tissue-Tek medium (Reichert-Jung, Wis.). ) And 5-10 μm thick on a cryo-microtome Thinned. The selected sections were fixed on a microscopic plate, and a monoclonal antibody against SM22 was used. Body (Duband et al., 1993, Differentiations, 55, 1-11) at a dilution of 1/30, and Staining was performed using a labeled second antibody Cy3 (Biological Detection Systems, USA). Off The sections were examined using a fluorescence microscope.     Example 1 SM22 Characterize the zodiac   Complete screening from genomic mouse bank hybridization Three overlapping clones containing the entire SM22 gene were isolated. Fig. 1 partially reproduced A restriction map of the exon, It was determined by the hybridization used.   The region of interest was subcloned and sequenced.   The sequence was determined between -2474 and +110 downstream of the polyadenylation site and Data banks. Po Limitations in which the rear denaturation site and coding sequence are common to the five exons The gene covers 5923 bp starting from the transcription start site (FIG. 1). All Eku The son-intron boundary corresponds to the consensus sequence.   The first exon contains only one leader sequence at the 5 'and has a transcription initiation codon. Therefore, the second, more than 4 kbp intron sequence from the transcription start site Localized in Exxon.   Two potential polyadenylation signals are localized at positions 5905 and 5913 However, some TGT regions placed after the polyadenylation site are probably transcribed. Involved in the suspension.   Sequence analysis also reveals three repeats with significant similarity to the repeat B1 element. The presence of the return sequence (Figure 2) was clearly demonstrated, some of which were Transcribed and encodes 5S RNA. All repeats have the opposite arrangement compared to the SM22 locus. It is noted that   The transcription start site was mapped by analysis by primer extension (FIG. 3). At the 3 'end of exon 1, a complementary antisense oligonucleotide comprises four Produce extension products, which differ by only one base pair (bp) in length, the longest of them It is 65bp. Therefore, the transcript Starts with G and the upstream region of the transcription initiation codon is 77 bp.   The shortest extension product is probably a modification at the 5 'end of the messenger RNA (capping ) Caused by incomplete termination of primer extension.   These results were confirmed by analysis with RNAse protection, which revealed that 65b Protected fragment of p and antisense located in the second exon of the gene And sequencing of extension products derived from independent reactions of primers with primers Brought a decision.   These results are consistent with the results obtained by Solway et al. (1995 supra). this The minor sequence differences between the two results are probably due to the different mouse strains. Due to an allelic mutation.   The -28 bp TTTAAA sequence (FIG. 4) is closely related to the TATAAA sequence and is probably Work as a box. Computer analysis of the sequence at 5 'regulates transcription of muscle genes Some of the factors and elements that are known to contribute to (Fig. 4). 11 E-type boxes (CA NNTG), 4 Mef-2 / rSRF-type patterns (YTAWAAATAR), 4 possible SRF linkers Element (CC (A / T) 6GG), 5 AP-2 link sites (CCCMNSSS) and 5 SP1 All of the patterns (GGGCGG) The position was determined. Finally, similar to the TGT3-3 pattern, recently the nuclear muscle Identified as actor link sites and regulates transcription of smooth muscle alpha-actin The position of the element contributing to was determined (FIG. 4).     Example 2 Cloning of recombinant plasmids to create transgenic mice And build     1.pnlz Production of   Plasmid pGEM7 (in other words, Promega, Madison, WI, USA) Obtained at the plasmid pGEM-72F (+/-)-Gen Bank sold by Corp. (SEQ ID NOs: X65310 and X65311) are the ampicillin resistance gene, lac Z gene. Children, and the origin of replication. It contains 3000 base pairs. Nae I / Bsp 12 Digested with 01. The extensible terminal Bsp1201 of the vector was ligated to the blunt The ends were cut off and the plasmid was recircularized in the presence of a 10 pb Sal I linker. Then, pG The lacZ fragment of EM 7 was deleted, a Sal I site was introduced, and a Bsp 1201 site Was restored.   Constructed by inserting a PmeI linker with single-stranded extensible terminal NsiI A Pme I site was introduced at the product Nsi I site.   Finally, Hind III and Sac yielding two fragments of the reporter gene Using pDes2.2nlz (LI et al., 1993, supra) with I, coupled with nuclear localization signals The isolated lacZ gene was isolated.   The two fragments were inserted between the modified pGEM7 vector between Hind III and Sac I. And their orientations were clarified. This procedure allows pn without promoter This produces the lz vector.     2.p352nlz Production of   FIG. 5 shows the construction of this plasmid.   SEQ ID NO: 1, comprising the strand from nucleotide -2126 to +4135, the SM22 fragment Produce a 417 pb PCR fragment covering the region -352 to +65 of the gene The tSK + vector was used to insert into the blunt-ended Hinc II site. PCR flag The sequence of the statements was revealed. Fragment recovery with Xba I and Xho I sites And inserted into the corresponding site of plasmid pBLCAT3, resulting in recombinant p352CAT. p35 Insert the 2CAT Xba1 / Xhol fragment into the corresponding pnlz site to create p352nlz did.     3.p2126nlz Production of   To create p2126nlz, a 1.9 kb Bs covering the -2126 to -206 region of the SM22 locus p1201 / Sph I fragment ( Is contained in SEQ ID NO: 1 which includes the strand from nucleotides -2126 to +4145). It was inserted into each site of 352nlz. This plasmid was transferred to CNCM on March 25, 1996. Deposit No. I-1685. The production is shown in FIG.     4.p2126INTnlz Production of   The construction of this plasmid is shown in FIG.   Bsp 1201 / Hind III 5.8 kb flag covering the region (2126 to +3651 at the SM22 locus) Was inserted into the corresponding site of pnlz to create plNt-nlz. Then, SM22 seat The +3648 to +4143 bases 496 pb PCR fragment was added to the Hind III site at positions 4135-4140. Using front and rear primers designed to introduce Generated. This PCR product was digested with Hind III and ligated into the pINT-nlz Hind III site. To generate p2126INT-nlz. Thus, the p2126INT-nlz construct is 5 ′ from the start of transcription. 2126 base pairs from the region, exon 1, entire intron 1 and translation initiation The last four base pairs are removed from CATG to remove the codon and introduce the above Hind III site. It contains a 15 base pair second exon mutated to GCTT. Plasmid p2126 INTnlz The part of the insert derived from the SM22 promoter contained in Become. This plasmid was deposited with the CNCM on March 25, 1996 under the number I-1686. The production As shown in FIG.     Example 3 Expression of constructs p2126 nlz and p2126INTnlz in transgenic mice   SV₄₀Using the lacZ reporter gene with the viral nuclear localization signal, Two constructs were made as described in Example 2.   The first construct, p2l26nlz, contains the upstream region 2126 bp and the first exon (65 bp) . The second p2126INTnlz construct contains the first 12 bp of the first intron and exon. Is the same as the first, except for the addition of   As described in the apparatus and method sections, each of these constructs Generate at least one transgenic mouse expressing the porter gene did.   Embryos obtained on days 12.5-15.5 after mating (dpc) were stained to demonstrate βgal activity. Was colored. Expression of the two constructs is identical in all conditions observed, but p212 6INTnlz shows slightly stronger staining.   Expression was first detected at 8.5 days in regions of the embryonic heart and in blood vessels in the pericardium. 9 At day 5, lacZ expression in the heart increased and was localized to the right ventricle and arteries. ing.   Between day 12.5 and day 15.5, as well as the function of the area, The segregation of the label becomes clear and the label is localized to the right ventricle (Fig. (8B). The cut plane shows that expression occurs in the right ventricular myocardium and minor in the left ventricle and right atrium. Or no labeling (FIG. 9A).   This expression in the heart is only temporary and does not appear in adults. It probably It may be inhibited at a later stage of fetal development. Early onset from day 9.5 Observed in the area of the starting snout, it spreads to the area of the tail at day 10.5 and at day 11.5 Reach high levels.   Expression began to decrease in the rostral zone from day 12.5 (FIG. 8B) and was no longer detected at day 14.5 Not performed (FIG. 10E).   Cross sections show that transgene expression is restricted to the area of the muscle plate (FIG. 9D). .   The largest portion of expression occurs in the vasculature of the developing embryo. Starting on day 9.5 , Expression is detected in the dorsal aorta. On day 10.5, dorsal aorta and aortic flexion Increased coloration, 1acZ expression on day 11.5, dorsal aorta, aortic flexion, iliac artery It is clearly seen in the umbilical, carotid and main arteries of the head.   Transgene expression in blood vessels begins at day 12.5, when intercostal vessels can be easily seen Increase (FIG. 8A).   On day 15.5 (FIG. 8B), the primary including limb and tail vessels LacZ is present in the blood vessels and the pulmonary trunk becomes colored. Expression in the vascular system , Sustained in the adult, aorta, pulmonary trunk and right pulmonary artery (FIG. 10A), intestine (FIG. 1). OB), clearly seen in the bladder and uterine vessels.   Histological sections reveal differences in transgene expression in arteries and veins Show. Sections from 12.5 day old embryos (FIG. 9A) were obtained from the aortic flexure, proximal pulmonary trunk and From the fourth artery from the proximal part in the ascending aorta, very high levels of carotid muscularis Although showing expression, the left and right portions of the anterior main vein remain uncolored. In the muscle vein Lack of transgene expression is evident in venous ducts, and in portal and umbilical veins . The same condition was observed in adults, for which the expression was in vena cava (FIG. 10A) or pulmonary Pulses can never detect it.   These observations have never been confirmed between layers of arterial and venous smooth muscle. Identify any differences that were not made. Other findings in the artery are that expression is restricted to the muscle layer and Indicates that it does not exist (FIG. 9C).   Looking at all the observations (FIGS. 8A and 8B), the expression of the transgene was found to be It is clear that it has disappeared. Endogenous SM22 gene is found in vascular smooth muscle tissue This is unexpected, as expression is expected in the gut. Muscle layers of the stomach, intestine and developing bladder were histologically sectioned in the abdominal region of the embryo at day 14.5 It is easily recognized (FIG. 9C). Transgene expression in this region is evident in the umbilical artery In the stomach, duodenum, small and large intestine, visceral muscle layer and bladder muscle No detectable in the layer, where no lacZ expression is observed. In addition, the esophagus and No expression is observed in the trachea (FIG. 9A) or in the lung bronchi.   Staining of adult muscle tissue yields primarily the same results. FIG. 10B shows translocation inheritance. Mesenteric vasculature is very potent by lacZ, although offspring expression is absent in colonic muscle Indicates that it is stained. However, sections of similar intestinal sections by immunofluorescence were Staining shows that the endogenous protein SM22 is present in both tissues (FIG. 10C). Lack of transgene expression in adult visceral smooth muscle is also associated with esophagus, trachea, bladder , Confirmed by staining for βgal in the vas deferens and uterus. βgal expression is It is never detected in the muscle layers of these tissues.   Oddly, significant staining was seen in the cytoplasm of the epithelium of the vas deferens and of the renal tract. Be guessed. Staining is also seen in non-transgenic control mice This is probably due to endogenous galactosidase activity.Example 4 SM22 Encodes the regulatory region of the gene and the herpesvirus thymidine kinase SM22 INT-TK production of constructs containing genes   By Hind III digestion, HIV-LTR fragment (-167, +80) was converted to pLTR-TK plasmid. And its ends were blunted by the Klenow enzyme followed by XhoI digestion. The plasmid was placed 5 ′ with an Xho1 site, 5 ′ → 3 ′ in the order of HindIII and Bsp1201 sites. And closed by inserting a linker containing a blunt end 3 'to give the p-TK vector I got it. 5.8 kb Bsp 1201 / Hind flag covering the region -2126 to +3648 of the SM22 locus Inserts the p-TK into SM22 INT-TK. This fragment has the sequence number Contained in one nucleotide chain.   This construction is shown schematically in FIG.     Example 5 lacZ Plasmid containing the -445 to +65 fragment of the SM22 gene fused to the gene Of D445 nlz and acquisition of transgene Apparatus and method p445nlz Building   Plasmid p2126nlz was cut with XbaI to give SM22 It has a unique site in the 5 'upstream "polylinker". Protruding from the fragment The 5 'end was filled in with dNTPs using Klenow polymerase.   After precipitation, the transcription start site was set to +1 nucleotide by Pst I, and the The fragment with the neak site was cut.   The protruding 3 'end obtained from Pst I digestion uses Klenow polymerase without dNTPs And hydrolyzed. -445 to +65 fused to the lacZ gene in addition to the plasmid sequence The fragment containing the SM22 sequence from was purified using an agarose gel and ligated. Ring closed with ze T4.   For transgene experiments, use the PstI / NsiI double digestion of plasmid p2126nlz The fragments injected into mouse eggs were purified.   The results obtained with this construct as well as the comparison between p2126 INTlacZ and p2126lacZ The results obtained are shown in the table below.   Transcriptional regulatory properties of the SM22 gene sequence between nucleotides -445 and +65 Sgenic mice were analyzed in vivo using two methods. Temporary analysis and call The first method involves expressing the lacZ gene directly at a given stage in the developing embryo FO. It analyzes patterns. The second approach is to start with a stable FO Creating a system It consists of     1. Temporary analysis   Temporary analysis revealed that the p445nlz transgene was integrated into the genome for a total of 13 embryos. Four integrated embryos were produced. 4 positive embryos analyzed 12.5 days after development One did not express the transgene. Two embryos were p2126INTnlz at the same stage Has the same expression pattern as that of the system, in other words, a specific expression pattern of arterial smooth muscle cells. The last third embryo has the same area of arterial expression and has spread primarily to the embryonic mesenchyme. It had ectopic expression of various sites scattered.     2．Stable system   Two stable independent systems are male (system No. 1) and female (system No. 9) start trees Erect.   The expression of the transgene was in embryos at day 15.5 for line 1 and for line 9 for line 9. Analyzed on embryos at 17.5 days of development. The two systems have the same type of expression in the arteries Transgenes in the heart are expressed in the dorsal aorta and pulmonary trunk.   Unlike the system obtained with the transgenes p2126nlz and p2126INTnlz, two p445n lz is, at this stage, the esophageal wall and respiratory trachea composed of smooth muscle, 1a Expresses cZ very strongly.   The two systems are not identical to each other in some regions of the embryo. System No. In 1, the trachea In the subsequent bronchi, the transgene is strongly expressed on day 15.5, but lacZ is present in the trachea. It is not seen in strain No. 9 on day 17.5, which is only present. This difference is the difference between the two systems. Altered regulation due to different integration sites or lacZ expression between day 15.5 and 17.5 May be due to a difference in stages, meaning that it can be suppressed between the eyes.   Therefore, the two systems are based on transgenes in organs that contain a strong component of smooth muscle cells. Expression. It consists of at least bronchial-type smooth muscle cells, trachea, and Several visceral types of smooth muscle cells, associated with the esophagus, are interesting and noteworthy .   Finally, sites with different ectopic expression, in other words proteins or endogenous Sites where the presence of SM22 RNA was not transferred were found in two systems. Corollary 1 is The two posterior fingers of each limb contain very strong localized expression. System 9 is before the brain LacZ is strongly expressed in some regions.   Expression of this construct at two stages of development is shown in FIGS. Those Embryos (12A), 12B, 12C) at 15.5 days after mating (p.c.) and 2 month old adults. Figure 5 shows staining for β-galactosidase activity of SM445 nlz in the body (12D). 12A, B and C arteries, esophagus and Bronchial smooth muscle tissue, ectopic site such as spinal cord (12B) or after two of each limb Note the expression on the finger (12A). In the adult thoracic aorta or pulmonary trunk Note also the lack of current (12D). Only tracheal expression is maintained.   The results obtained indicate that the 450 base pair sequence maintains the same expression in embryos and fetuses. Is sufficient, but not enough for adults.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 48/00 A61K 48/00 C07K 14/475 C07K 14/475 14/54 14/54 C12N 5/10 C12N 9/12 9/12 9/20 9/20 C12Q 1/68 A C12Q 1/68 C12N 5/00 B (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB , GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (GH, KE, LS, MW, SD, SZ, UG), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AU, AZ, BA, BB , BG, R, BY, CA, CN, CU, CZ, EE, GE, HU, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LV, MD, MG , MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SG, SI, SK, TJ, TM, TR, TT, UA, UG, US, UZ, VN (72) Inventor Small Victor Austria A-5411 Oberalm Schulstraße 870 (72) Inventor Mosler Herbel Austria A-5201 Siekirchen Weinbergstrasse 23 (72) Inventor Lee Zellin France E-75006 Paris Rue Mazarin 33 ( 72) Inventor Merix Cay Mathias France France 94700 Maison-Alfort Avenue de General de Gaulle 9

Claims (1)

[Claims] 1. A fragment of the sequence upstream of the coding part of the gene of the protein SM22 or of a sequence which hybridizes under high stringent conditions with said sequence, said fragment being liable to induce specific expression of this gene in eukaryotic cells, And-a DNA sequence which comprises a protein coding sequence or RNA of therapeutic interest. 2. A fragment of the sequence upstream of the coding part of the gene of the protein SM22 or of a sequence which hybridizes under high stringent conditions with said sequence, said fragment being liable to induce the specific expression of this gene in vivo in arterial cells And-a DNA sequence characterized by containing a protein coding sequence or RNA of therapeutic interest. 3. 3. The method according to claim 1, wherein the protein or the RNA is capable of inhibiting smooth muscle cell growth, activating endothelial cell proliferation, stiffening the arterial wall and / or eliciting an immune response. The sequence according to any of the above. 4. The fragment according to any one of claims 1 to 3, characterized in that the fragment is included in the sequence between nucleotides -2126 and +4135 of the mouse gene of protein SM22 (SEQ ID NO: 1). Array. 5. The fragment according to any one of claims 1 to 4, characterized in that said fragment is contained in the sequence between nucleotides -2126 and +65 of the mouse gene of protein SM22 (SEQ ID NO: 2). Array. 6. The sequence according to any one of claims 1 to 5, characterized in that it comprises at least a part of the sequence between nucleotides -2126 and -445 of the mouse gene of the protein SM22 (SEQ ID NO: 3). 7. The sequence according to any one of claims 1 to 6, wherein said protein of therapeutic interest is a protein that induces the formation of a cytotoxic compound. 8. The sequence according to claim 7, characterized in that the protein is a herpes virus thymidine kinase. 9. The sequence according to claim 1, wherein the protein of therapeutic interest has a cytostatic effect. 10. The sequence according to any one of claims 1 to 6, wherein said protein of therapeutic interest has lipolytic activity. 11. 11. The sequence according to claim 10, wherein said protein is lipoprotein lipase. 12. 7. The sequence according to claim 1, wherein the protein of therapeutic interest is an endothelial cell growth factor. 13. The sequence according to claim 11, wherein the factor is an interleukin. 14． 7. The sequence according to claim 1, wherein the protein of therapeutic interest is a muscle protein or a tissue structure. 15. The sequence according to any one of claims 1 to 6, wherein the RNA of therapeutic interest is an antisense RNA of the protein p53. 16. A DNA sequence upstream of the coding part of the mouse gene for the protein SM22, characterized in that it comprises at least part of the sequence SEQ ID No. 5 or a sequence which hybridizes therewith under stringent conditions. 17． A DNA sequence upstream of the coding part of the mouse gene for the protein SM22, characterized in that it comprises at least part of the sequence SEQ ID No. 1 given below, or a sequence which hybridizes therewith under stringent conditions. 18. A DNA sequence upstream of the coding part of the mouse gene for the protein SM22, characterized in that it comprises at least part of the sequence SEQ ID No. 2 shown below or a sequence which hybridizes therewith under stringent conditions. 19. A strain deposited with the CNCM on March 25, 1996 as No. I-1685, comprising the plasmid p2126nlz containing the sequence of claim 18. 20. A strain deposited with the CNCM on March 25, 1996 as No. I-1686, comprising the plasmid p2126INTnlz containing the sequence of claim 17. 21. A vector comprising the sequence according to any one of claims 1 to 16. 22. 21. The vector according to claim 20, characterized in that it contains an effective origin of replication in arterial cells. 23. The vector according to any one of claims 21 or 22, wherein the vector is a derivative of an adenovirus. 24. An RNA that can be expressed by the sequence or vector according to any one of claims 1 to 23. 25. A composition comprising a sequence or a vector according to any one of claims 1 to 16 and 21 to 23. 26. 26. The composition of claim 25, wherein the sequence or vector is included in a composition that facilitates its transfection in a cell. 27. 26. The composition according to claim 25, comprising a gel that is a complex of poly-L-lysine and lactose. 28. A drug comprising the sequence or vector according to any one of claims 1 to 16 and 21 to 23. 29. A pharmaceutical composition comprising a pharmaceutically effective amount of the nucleic acid sequence or vector according to any one of claims 1 to 16 and 21 to 23, and a pharmaceutically compatible excipient. 30. Use of a vector or sequence according to any one of claims 1 to 16 and 21 to 23 for the manufacture of a medicament for treating coronary artery disease. 31. Use of a vector or sequence according to any one of claims 1 to 16 and 21 to 23 for the manufacture of a medicament for treating restenosis. 32. 24. Use of a vector or sequence according to any one of claims 1 to 16 and 21 to 23 for the manufacture of a medicament for treating mutations that make blood vessels brittle. 33. A transgenic mouse comprising the sequence or the vector according to any one of claims 1 to 16 and 21 to 23, wherein the coding gene of the protein of therapeutic interest is replaced by a reporter gene. . 34. A method for in vitro screening of the activity of a molecule against the regulatory sequence of the coding gene of the protein SM22, comprising the following steps:-by means of the sequence or vector according to any one of claims 1 to 16 and 21 to 23 Transfection of cells (where the coding gene for the protein of therapeutic interest is replaced by a reporter gene),-incubation of cells transfected with the molecule to be tested, and-quantification of reporter gene expression A method for screening a molecule comprising: 35. A method for detecting a mutation in a region containing the sequence according to any one of claims 1 to 16 or the vector according to any one of claims 21 to 23, wherein the gene is located after the sequence. A method characterized by altered expression of a. 36. A method for expressing a protein of therapeutic interest which comprises using the product according to any one of claims 1 to 24.