WO1997035974A1 - Sequences en amont du gene sm22, vecteurs les contenant et leurs utilisations therapeutiques, notamment dans le traitement des maladies vasculaires - Google Patents
Sequences en amont du gene sm22, vecteurs les contenant et leurs utilisations therapeutiques, notamment dans le traitement des maladies vasculaires Download PDFInfo
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- WO1997035974A1 WO1997035974A1 PCT/FR1997/000543 FR9700543W WO9735974A1 WO 1997035974 A1 WO1997035974 A1 WO 1997035974A1 FR 9700543 W FR9700543 W FR 9700543W WO 9735974 A1 WO9735974 A1 WO 9735974A1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4716—Muscle proteins, e.g. myosin, actin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2799/00—Uses of viruses
- C12N2799/02—Uses of viruses as vector
- C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
- C12N2799/022—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from an adenovirus
Definitions
- the present invention relates to sequences upstream of a gene expressed in smooth muscle cells such as SM 22, as well as to vectors containing them.
- vectors and sequences in therapy, for example for the production of locally or systemically active polypeptides, in particular immunogenic polypeptides, endogenous regulatory polypeptides such as cytokmes, or also for the production RNA of therapeutic interest, for example an antisense RNA
- the invention relates to the use of vectors and nucleotide sequences in the treatment of vascular diseases.
- Atherosclerosis is a degenerative disease of the arteries associating lesions of arteriosclerosis and atheroma, and thus combining a hardening of the arteries and a fatty degeneration of their internal coat.
- This coronary insufficiency is generally remedied by percutaneous coronary angioplasty consisting of introduce into the coronary artery network a catheter provided at its end with a balloon, advance the catheter to the level of the stenosis and inflate the balloon in order to crush the lesion against the wall and thus restore a coronary caliber allowing sufficient myocardial perfusion.
- This myocardial revascularization technique is very widely used (50,000 procedures per year in France and 500,000 procedures per year in the United States) but is however limited by the reappearance, in the months following the operation, of a new lesion in the dilated site, or restenosis, in approximately 30% of cases
- intimal hyperplasia is due to the proliferation of smooth muscle cells in the intima, which results in the synthesis of a bulky extra cellular matrix This activation would be induced by the local release of growth factors, and cytokines, as well as by the suppression of the synthesis of certain antiproliferative endothéaux peptides
- arterial reshaping consists in a reduction of the caliber of the artery without modification of the thickness of the wall, and therefore without proliferation of cells.
- the proteins constituting smooth muscles have been the subject of plot studies.
- the protein SM 22 which constitutes one of the first markers to appear during the differentiation of smooth muscle cells, has been studied and the coding part of its gene has been sequenced in mice (SOLWAY and AL, 1995, J Biol Chem, 270 22, 13460-13469) and in rats (KEMP and AL 1995, BIOCHEM J 310, 1037-1043).
- the gene coding for the protein SM 22 alpha in mice is composed of five exons and comprises 6.2 kb II is present in a single copy in the genome SOLWAY et al, have shown that the 441 base pairs upstream of the region coding gene were necessary and sufficient to induce a high level of transcription of a reporter gene in primary cell cultures of rat aortas and in A 7r5 lines. They also demonstrated that the messenger RNAs of this gene were expressed at high levels in the aorta, in the uterus, in the lungs and in the intestine.
- OSBOURN and AL (1995 Gene 154, 249-253) have also studied the 5 'region upstream of the rat SM 22 gene. They have demonstrated the presence of two introns in this region.
- the applicant set out to identify in vivo, on adult individuals, the activity of the various regions constituting the region upstream of the expressed coding sequence of the gene for the SM 22 II protein, on the other hand, endeavored to determine whether changes in this sequence induced changes in gene expression in different tissues.
- the invention also relates to any nucleotide sequence or oligonucleotide of natural origin or obtained by chemical synthesis having a homology with the sequence SEQID No. 1 of at least 70%.
- nucleotide sequence of natural origin is intended to mean a fragment of complementary DNA obtained by reverse transcription of cellular messenger RNA or alternatively a fragment of genomic DNA obtained after cleavage of cellular DNA using enzymes. restriction.
- nucleotide sequence obtained by chemical synthesis means a DNA fragment of known sequence generated by automatic synthesis of polynucleotides, for example using a suitable automatic device.
- protein or RNA of therapeutic interest means a molecule capable of inhibiting the growth of smooth muscle cells, activating the growth of endothelial cells, consolidating the arteries and / or capable of inducing a cellular or humoral immune response .
- the term "strongly stringent conditions" is used in the sense given by Maniatis et al., 1982 (Molecular cloning, A Laboratory Manual, Cold Spring Harbor Lab. CSH, NY USA or one of its recent reissues
- the hybridization conditions are as described by Maniatis (edition of 1982) Three washes intended to remove the non-hybrid fragments are necessary and are carried out at 65 ° C. in the presence of 0.2 SSC and 0.1 % SDS, in the absence of formamide.
- said sequence comprises a fragment of the sequence between nucleotides -2126 and +4135, whose sequence SEQ ID No. 1 is given in the appendix, of the sequence between nucleotides
- Such a fragment may in particular comprise at least part of the sequence SEQ ID No. 5 which corresponds to the part upstream of the 5 'end of the coding part comprised between the nucleotides -2126 and -1340.
- sequence SEQ ID No. 5 the sequence of which is given in the appendix constitutes an object of the present invention.
- sequences SEQ ID No. 2 and SEQ ID No. 1 are included respectively in the plasmids p2126nlz and p2126INTnlz carried by the strains deposited on March 25, 1996 with the Collection
- the subject of the invention is also the sequences hybndant under conditions of high stringency with the sequences SEQ ID No. 1, SEQ ID No. 2. SEQ ID No. 3 and SEQ ID No. 5.
- sequences including the sequence of the first intron of the SM gene
- sequence SEQ ID No. 1 constitute particularly advantageous modes of implementation of the present invention, because they allow expression of proteins or RNA of therapeutic interest, in adults, specific for arteries.
- the present invention is however not restricted to sequences containing fragments of the mouse gene, but relates to any other sequence of any other species having the same properties, namely to specifically induce expression of a gene in artery cells .
- a person skilled in the art may advantageously refer to the article by KEMP et AL (1995, previously cited) which describes the sequence upstream of the rat gene of the SM 22 protein.
- the present invention also covers any sequence comprising fragments sequences upstream of genes of the SM 22 protein, modified for example by deletion of certain structures which retain identical or similar functions to those of the complete sequence.
- the protein of therapeutic interest can be a protein inducing the formation of a cytotoxic compound, such as the thymidine kmase of the herpes virus.
- This protein has the particularity of transforming ganciclovir (Merck Index, reference 4262), an analogue of the guanosine, a derivative which blocks the synthesis of DNA in replicating cells
- transforming ganciclovir Merck Index, reference 4262
- an analogue of the guanosine a derivative which blocks the synthesis of DNA in replicating cells
- the introduction of a gene coding for this protein in the sequences according to the present invention therefore makes it possible to block the proliferation of cells in the case of intimal hyperplasia.
- a sequence encoding thymidine kinase is described in particular by Caruso and Klatzmann (1992, Proc Natl Acad Sci., USA, 89, 182-186).
- Said protein of therapeutic interest can also be a protein exhibiting a cytostatic effect, such as the protein encoded by the Rb gene (Chang et al., 1995 Science, 267, 518-522), or by the eNOS gene (Hamon et al ., 1994, Circulation, 90, 1357-1362).
- a lipoprotein lipase Such proteins are coded by the sequences described by Reyner (1995, Nature Genetics, 10, 28) and Ming Sun Liu (1994, J. Biol. Chem, 269-11417).
- Such a protein can in particular be a mterleukin.
- It can be a muscle protein, such as myosin, or organism, or a tissue structure protein such as collagen or elastin.
- It can be a protein inducing an immune response as described in application WO 90 11092, and in particular a viral antigen such as the glycoprotein gp120 or the protein Nef of HIV (human immunodeficiency virus).
- a viral antigen such as the glycoprotein gp120 or the protein Nef of HIV (human immunodeficiency virus).
- said protein can be one or more proteins of therapeutic or vaccine interest, such as proteins of immunotherapeutic interest, for example mterleukins, growth factors, for example fibroblast growth factors (FGF) or NGF (Nerve Growth Factor), proteins which can induce an immune response, whether it is humoral, cytotoxic or cellular immunity, or proteins making it possible to complement the activity of a gene normally expressed in the individual to be treated but which no longer is, either by mutation or deletion of its sequence.
- FGF fibroblast growth factors
- NGF Neve Growth Factor
- This sequence can also code for an RNA of therapeutic interest.
- Such an RNA can be the antisense RNA of the protein p 53 or an RNA as described in application WO 90 11092 filed by the company VICAL.
- the present invention further relates to vectors characterized in that they contain a sequence according to the invention, as described above.
- a vector may contain any other DNA sequence necessary for the expression of the protein, or RNA, of therapeutic interest in the target tissues, and in particular may contain an origin of replication which is effective in artery cells, in particular in smooth muscle cells.
- Such a vector may comprise sequences allowing homologous recombination in the organism treated, specific for the gene to be replaced, said sequences being placed upstream and downstream of the sequence according to the invention. Due to the presence of such sequences, the gene unwanted, present in the organism treated will be replaced by the gene carried by the vector or the sequence and which one wishes to see expressed in the organism.
- Such a homologous recombination method can be of the type described by Le Mouellic et al, (1990, Proc. Nat Acad Sci USA, 87, 4712-4716) or in PCT application WO 91 / 06.667.
- Such a vector can be a vector derived from an adenovirus
- Adenoviruses suitable for the implementation of the present invention are in particular those described by FELDMAN and STEG (1996, previously cited) or OHNO et al (1994, Sciences, 265, 781-784) or in application FR 94 03.151 (Institut Pasteur, Inserm) These viruses are generally rejected by the individual, due to their too strong immunogenicity Nevertheless, strains of these viruses have been selected in order to reduce their character immunogenic.
- these vectors even having a strong immunogenicity, can be used in the context of the present invention, since the arteries are treated for relatively short periods of time of the order of a few weeks, compatible with the times of rejection of adenoviruses by the host's immune system. It should nevertheless be noted that the introduction of the sequences according to the present invention into the vectors described above is not essential, and that the cells of the arteries can be directly transfected with DNA comprising these sequences.
- nucleic acid sequences according to the present invention can be introduced after covalent coupling of the nucleic acid with compounds promoting their penetration into cells or their transport to the nucleus, the resulting conjugates being optionally encapsulated in polymeric microparticles, as in international application WO 94/27238 from Meidsorb Technologies International.
- the nucleic sequences can be included in a transfection system comprising polypeptides promoting their penetration into cells, as in international application WO 95/10534 from Seikagaku Corporation.
- vectors or sequences can be administered in situ by any means known to a person skilled in the art.
- they can be delivered in situ using a balloon catheter covered with channels, as described by FELDMAN and STEG (1996). , previously cited).
- aorta can also be administered during an operation, or by stripping the aorta.
- Another mode of administration consists in introducing a small mesh grid impregnated with DNA comprising these sequences, along the aorta, as described. by Feldman et al. (1995, J. Clin. Invest 95 2662-2671).
- sequences or vectors can advantageously be administered in the form of a composition containing them, for example in a gel facilitating their transfection into artery cells.
- a gel can be a complex of poly-L lysine and lactose, as described by MIDOUX (1993, Nucleic Acid Research, 21, n ° 4, 871 -878) or poloxamer 407 as described by PASTORE (1994, Circulation, 90, 1-517). They can also be dissolved in a buffered solution or be combined with liposomes.
- the present invention further relates to medicaments containing such sequences, vectors or compositions, as well as to pharmaceutical compositions containing them in amounts pharmaceutically effective as well as pharmaceutically compatible excipients.
- Such sequences, vectors or compositions can be advantageously used for the manufacture of medicaments for the delivery to the arteries of a DNA (cDNA or genomic DNA) which can express in particular a gene coding for a protein of interest, for the treatment of coronary heart disease, and in particular for the treatment of restenosis. They can nevertheless also be used for the manufacture of a medicament for the treatment of mutations which weaken the vessels.
- a medicament could, for example, comprise a sequence or a vector according to the invention capable of expressing a gene coding for a normal protein of desmin or a muscle adhesion protein of the CAM type.
- the present invention also relates to RNAs expressed from the sequences and vectors which are the subject of the present invention, and in particular messenger RNAs.
- the present invention also relates to a method for screening molecules in vitro, with a view to testing their activity on the regulatory sequences of the gene coding for the SM protein 22.
- a method for screening molecules in vitro will comprise, for example, a first step according to which one transfects cells with a sequence or a vector according to the invention, comprising a reporter gene placed under the control of all or part of the promoter sequence of the gene coding for the SM 22 protein.
- the cells thus transfected are incubated in the presence of the molecule to be tested, then the expression of the reporter gene is quantified.
- the cells used for the transfection are advantageously smooth muscle cells, in particular aortic cells, either in the form of a primary culture, or in the form of a stable cell line.
- the quantification of the beta-galactosidase produced will advantageously be carried out by optical reading, for example using the detection kit sold by Boehringer under the reference 1 669 893 ( Boehringer catalog Mannheim - "Biochemicals”. 1996, page 236, beta-Gal Reporter Gène Assay, Chemilummescent).
- the reporter gene is the gene coding for luciferase
- the quantification of the expression of luciferase is advantageously carried out by chemiluminescence, for example by means of the diagnostic kit marketed by Boehringer under the reference 1 758 241 (Boehringer-Mannheim catalog "Biochemicals", 1996, Luciférase Reporter Gène Assay).
- the present invention also relates to transgenic animals and in particular mice carrying a sequence or a vector as defined above in which the gene coding for the protein of therapeutic interest is replaced by a reporter gene
- the present invention finally relates to a method for detecting mutations in the sequence upstream of the coding part of the gene for the protein SM22.
- Such mutations are indeed capable of modifying the expression of the gene coding for the protein SM22, in particular reducing or even abolishing the expression of this gene in smooth muscles.
- Such mutations would be likely to cause a modification of the functioning of smooth muscle, the SM22 protein being related to a family of proteins involved in the regulation of calcium binding, of the calponine type (Ayme-Southgate et al. 1989, J Cell . Biol.).
- Such a mutation detection method can advantageously be the method which is the subject of French patent application FR-93 10821.
- sequences according to the invention modified so that they allow an increase in the expression of the SM22 gene are also part of the invention.
- transgenic mice can be used to screen molecules for their activity on the regulatory sequences gene coding for the SM 22 protein. Molecules can be administered to mice, then after sacrifice, histological sections are carried out in order to highlight the tissues stained by the reporter gene.
- a person skilled in the art can advantageously refer to the following manual "SAMBROK et al (Molecular cloning, A Laboratory Manual Cold Spring Harbor
- FIG. 1 represents the restriction map and the exon / intron structure of the SM 22 site, comprising the region -2474 to +6030 (relative to the site of initiation of transcription)
- the exons are represented by white squares while the arrows indicate the position of repeated B1 type sequences.
- FIG. 2 represents the alignment of three repetitive sequences of type B1, in which the basic exchanges are indicated
- FIG. 3 is an autoradiogram illustrating the identification of the site of initiation of transcription.
- the well P corresponds to the primer used.
- FIG. 4A represents the sequence of the 5 ′ region upstream of the transcription initiation site, and of the exon of the SM 22 gene. The sites for the attachment of the different factors are indicated, as well as the various concensus sequences. The broken lines. indicate the position of repeated B1 type sequences.
- Figure 4B is a schematic restriction map of the SM 22 gene.
- Figure 4C is a detailed restriction map of the SM 22 gene.
- Figure 5 illustrates the manufacture of the plasmid p352nlz.
- FIG. 6 illustrates the manufacture of the plasmid p2126 nlz.
- Figure 7 illustrates the manufacture of the plasmid p2126INTnlz.
- FIGS. 8A and 8B illustrate the expression of the lacZ gene in embryos of transgenic mice.
- FIGS. 8A, 8B represent embryos at the 12.5 and 15.5 day stages, respectively.
- FIGS. 9A to 9D represent histological sections of embryos stained using lacZ
- FIG. 9A represents a section of an embryo at the 12.5 day stage in the heart, showing an intense coloration exclusively in the myocardium of the ventricle right (RV) and in the artery walls (abbreviations: AA: elbow of the fourth aortic artery; CA: carotid artery; E: esophagus; Iv: left ventricle; PA: proximal part of the aorta; PT: pulmonary trunk; RA: right atrium; RV: right ventricle; T: trachea; V: right and left cardinal vein).
- AA elbow of the fourth aortic artery
- CA carotid artery
- E esophagus
- Iv left ventricle
- PA proximal part of the aorta
- PT pulmonary trunk
- RA right atrium
- RV right ventricle
- T trachea
- FIG. 9B represents a section through the abdominal region of an embryo at the 14.5 day stage, showing a coloration of the umbilical arteries (AU) and the absence of labeling of the viscera (B: bladder; D: duodenum; H: large intestine; L: liver; M: small intestine; ST: stomach).
- AU umbilical arteries
- Figure 9D is a section through the tail segments at the 12.5 day stage showing the labeling in the myotome (my) and the tail artery (a).
- FIG. 9C is an enlargement of the part of the photograph corresponding to the umbilical artery at the 12.5 day stage, showing an exclusive marking of the muscular layer (m) and an absence of marking of the endothelium (e).
- FIGS 10A to 10C illustrate the expression of the transgene in adult mice
- FIG. 10A represents a top view of the whole of the heart showing an intense marking of the aorta (a) and of the pulmonary artery (pa) and an absence of marking of the vena cava (vc).
- Figures 10B and 10C are sections through the smooth muscle layer (sm) of the adult colon and an adjacent mesentery artery (ma).
- the lacZ labeling shows that the expression of the transgene is restricted to the artery while the immunofluorescence with SM 22 antibodies (FIG. 10C) highlights the expression of the endogenous SM 22 gene in the cells of the smooth muscle of both the artery and the colon.
- Figure 11 represents a vector comprising the regulatory regions of the SM 22 gene and the gene encoding the thymidin kinase of the herpes virus.
- FIGS. 12A to 12D illustrate the expression of the SM 445 nlz construction in embryos of 15.5 pc days (FIGS. 12A to 12C) and in an adult of 2 months (FIG. 12D).
- phage delta-EMBL 3 Approximately 10 6 recombinant phages from a c57 / bl mouse genomic library constructed in phage delta-EMBL 3 (supplied by Dr D. Plachov Munster, Germany) were screened using the BAL 1 / Eco RV fragment of 890 bp from the complementary DNA of the mouse protein SM22 (Almendral et al, 1989; Exp. Cell Res 181, 518-530), as probe The probe was radioactively labeled by random initiation and the DNA was hybridized on filters left overnight at 65 ° C in Church buffer.
- the sequence of the SM 22 gene was determined on both strands by the chain termination method (Sanger et al, 1977, Proc. Natl. Acad Sci. 74, 5463) using the Sequenase V2.0 kit (United States Biochemical , Cleveland, Ohio).
- RNA from cell lines and adult mouse tissue was isolated by the method of Chirgwin et al, (1979, Biochemistry, 18.
- the 5'RACE method was carried out using two synthetic 18 bp antisense oligonucleotides corresponding to the bases +112 to +129 (primer 2) and +162 to +179 (primer 1) of the complementary DNA SM 22, as described in substance by FROHMAN et al, (1988, Proc. Natl. Acad Sci, 85, 8998-9002).
- RNA polymerase T 7 RNA polymerase T 7 in the presence of 32 P-CTP.
- the probe (3 X 10 5 cpm) was hybridized with 5 ⁇ g of messenger RNA from mouse uterus at 42 ° C. overnight After hybridization, the samples were incubated with RNases A and T (1 hour at 37 ° C), precipitated, and analyzed as described for primer extension analysis.
- the NIH 3T3, 10T1 / 2 and 8/47 cell lines were cultivated in DMEM (Life Technologies, Inc, Vienna, Austria) supplemented with 10% fetal calf serum (Hyclone Inc., United States) at 37 ° C, in an atmosphere of Co2 at 7%.
- DMEM Life Technologies, Inc, Vienna, Austria
- 10% fetal calf serum Hyclone Inc., United States
- Co2 Co2
- the cells were removed from petri dishes with a cell scraper, centrifuged, resuspended in 150 ⁇ l of buffer Z
- mice carrying the transgene were identified by PCR and Southern hybridization using a DNA probe.
- the construction pnlz2126 made it possible to obtain three positive animals out of 17 mice, two of them transmitting and expressing this construction.
- transgenic mice were backcrossed with c57 / bl mice and the histochemical stains were carried out with hemizygous mice for the transgene.
- the histological sections were made with a thickness of 7 to 10 ⁇ m and again stained with Ehrhch hematoxline and eosin On the other hand, the embryos could be clarified again using a mixture of benzyl alcohol and benzyl benzoate.
- mice were fixed in 3% of paraformaldehyde in PBS buffer at 4 ° C for 3 hours, then coated in Tissue-Tek medium (Reichert-Jung, Vienna) and sectioned on a cryopreservation. microtome at thicknesses of 5 to 10 ⁇ m
- the selected sections were mounted and stained using a monoclonal antibody directed against SM 22 (Duband et al, 1993, Differentiations, 55, 1-11), at a dilution 1 / 30 as well as using a secondary antibody labeled Cy3 (Biological Detection Systems, USA). The sections were examined using a fluorescence microscope.
- the regions of interest have been subcloned and sequenced.
- the sequence was determined between positions -2474 and +1 10 downstream of the polyadenylation site and was compared in the EMBL database.
- the gene covers 5923 bp from the start of transcription site to the polyadenylation site and the coding sequence is divided into 5 exons ( Figure 1). All exon-intron boundaries correspond to consensus sequences.
- the first exon contains only a 5 'leader sequence and the transcription start codon is therefore located in the second exon separated from the transcription start site by more than 4 kbp of intron sequence.
- Two potential polyadenylation signals have been located at positions 5905 and 5913 and several TGT regions placed downstream of the polyadenylation site, presumably take part in the termination of transcription.
- the transcription start site was mapped by primer extension analysis ( Figure 3).
- An antisense oligonucleotide complementary to the 3 ′ end of exon 1 leads to the production of four extension products which differ only in a pair of bases (bp) in length, the longest of them being 65 bp.
- the transcription product therefore starts with a G, 77bp upstream of the transcription initiation codon.
- the shortest extension products were thought to be caused by premature termination of primer extension due to capping at the 5 'end of the messenger RNA.
- the TTTAAA sequence in position -28bp ( Figure 4) is closely related to the TATAAA sequence and probably has the function of a TATA box.
- Computer analysis of the 5 'sequence reveals the presence of several potential binding sites for factors as well as for elements which are known to contribute to the regulation of muscle gene transcription (Figure 4).
- a total of 11 E-type boxes (CANNTG), four Mef-2 / rSRF-type patterns (YTAWAAATAR), four potential SRF linkers (CC (A / T) 6GG), five AP-2 link sites (CCCMNSSS) and five SP1 motifs (GGGCGG) have been located.
- CCCMNSSS AP-2 link sites
- GGGCGG five SP1 motifs
- the plasmid pGEM7 (that is to say the plasmid pGEM-72F (+/-), marketed by Promega Corp., Madison, Wi, USA. Sequences available in Gen Bank, under the following numbers X65310 and X 65311) comprises an ampiciliin resistance gene, a lac Z gene and an origin of replication II comprises 3000 base pairs. It was digested with Nae 1 / Bsp 120I. The outgoing Bsp 120I end of the vector was made "blunt ends" with the Klenow enzyme, and the plasmid was closed in the presence of a 10 bp Sal I linker. The lacZ fragment of pGEM 7 was therefore deleted. a Sal I site introduced, and a Bsp 120I site restored.
- a Pme I site was introduced by insertion of a Pme I linker with Nsi I outgoing single-stranded ends, into the Nsi I site of the construction.
- lacZ gene coupled to the nuclear localization signal was isolated from pDes2.2nlz (Ll et al, 1993, cited above) with Hind III and Sac I giving two fragments of the reporter gene.
- the two fragments were inserted between the Hind III and Sac I sites of the modified pGEM7 vector and checked for their orientation. This procedure gives the vector pnlz without promoter.
- Figure 5 illustrates the manufacture of this plasmid.
- the first construction, p2126nlz contains 2126 bp of the upstream region and the first exon (65 bp)
- the second construction p2126INTnlz is identical to the first except for the addition of the first mtron and the first 12bp of the second exon.
- each of the constructs gave rise to at least one transgenic mouse expressing the reporter gene.
- the embryos obtained from 12.5 to 15.5 days after coitus (dpc) were stained so as to highlight the ⁇ gal activity.
- the expressions of the two constructions are identical, at all stages observe although p2126INTnlz gives a little more intense coloring.
- the sections show that the expression of the transgene is restricted to the myotomal region of the segments (FIG. 9D).
- the expression takes place in the vascular system of the developing embryo. From 9.5 days, the expression is detected in the dorsal aorta. At 10.5 days, the coloration of the dorsal aorta and aortic elbows increases and at 11.5 days, the lacZ expression is clearly visible in the dorsal aorta, the aortic elbows the iliac arteries, the umbilical arteries, the carotid arteries and in the major vessels of the head.
- lacZ is present in the major vessels including those of the limbs and of the tail, and the coloration of the pulmonary trunk becomes visible
- Expression in the system vascular disease persists in adults and is clearly visible in the aorta. in the pulmonary trunk and in the right pulmonary artery ( Figure 10A), as well as in the vessels of the intestines ( Figure 10B), in the bladder and in the uterus.
- FIG. 10B shows that the expression of the transgene is absent in the muscles of the colon whereas the mesentery vessel is very strongly colored by lacZ
- FIG. 10B shows that the expression of the transgene is absent in the muscles of the colon whereas the mesentery vessel is very strongly colored by lacZ
- the coloring by immunofluorescence of the section of a similar intestinal region shows that the endogenous SM 22 protein is present in both tissues (FIG.
- SM 22 INT-TK herpes virus thymidine kinase
- the HIV-LTR fragment (-167, + 80) is deleted from the plasmid pLTR-TK by Hind III digestion, the ends of which are made blunt with the Klenow enzyme, followed by Xho I digestion.
- the plasmid closed by inserting a linker containing the Xhol sites in 5 ', the Hind III and Bsp 120I sites in the order 5' -> 3 'and a 3' blunt end to give the vector p-TK
- a Bsp fragment 120I / H ⁇ nd III of 5 8 kb covering the region -2126 to + 3648 of the SM 22 locus is inserted into p- TK to obtain SM 22 INT-TK This fragment is included in the nucleotide sequence of SEQ ID N ° 1 .
- the plasmid p2126nlz was cut by Xba I which has a unique site in the "polylinker" 5 'upstream of the sequence SM 22.
- the protruding ends of the fragment were filled using the Klenow polymerase with dNTP.
- the fragment injected into the eggs of mice was purified from a double digestion Pst 1 / Ns ⁇ I of the plasmid p2126nlz.
- transient analysis consists in analyzing the expression pattern of the lacZ gene directly in founder FO embryos at a given stage
- second method consists in establishing stable lines from adult FO founders.
- the transient analysis made it possible to obtain four embryos having integrated the transgene p445nlz into their genome out of a total of 13 embryos.
- the four positive embryos analyzed at 12.5 days of development one embryo did not express the transgene
- the two lines p445nlz express lacZ very strongly in the walls of the esophagus and the respiratory trachea which are composed of smooth muscle at this stage.
- the two lines show differences between them in certain regions of the embryo
- the transgene is strongly expressed in the bronchioles in continuity of the trachea at 15.5 days which is not observed in line n ° 9 to 17.5 days when lacZ is found only in the trachea
- This difference can be due either to a modification of the regulation due to different integration sites in the two lines, or to the difference in stage which makes that the expression of lacZ can be repressed between day 15.5 and 17.5
- FIGS. 12A to 12D represent 15.5 day pc embryos (12A. 12B 12C) and a 2 month old adult (12 D) SM 445 nlz colored for ⁇ -galactosidase activity
- FIGS. 12A to 12D represent 15.5 day pc embryos (12A. 12B 12C) and a 2 month old adult (12 D) SM 445 nlz colored for ⁇ -galactosidase activity
- Myosin light chain 3F regulatory sequences confer regionalized cardiae and skeletal muscle expression in transgenic mice. J. of Cell Biol., 129/2, 383-396.
- the 5'-flanking region of the mouse vascular smooth muscle ⁇ -ac tin gene contains evolutionarily co ns erv ed sequence motifs with in a functional promoter. J. Biol. Chem., 265/27, 16667-16675.
- T ransgelin A transfomation and shape change sensitive actin-gelling protein. J. Cell Biol., 121/5, 1065-1073.
- Thweatt R., Lumpkin, C. K. and Goldrtein, S. (1992).
- a novel gene encoding a smooth muscle protein is overexpressed in senescent human fibroblasts. Biochem. Biopkys. Res.
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Application Number | Priority Date | Filing Date | Title |
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EP97919424A EP0914426A1 (fr) | 1996-03-26 | 1997-03-26 | Sequences en amont du gene sm22, vecteurs les contenant et leurs utilisations therapeutiques, notamment dans le traitement des maladies vasculaires |
JP9534091A JP2000507819A (ja) | 1996-03-26 | 1997-03-26 | 遺伝子sm22の上流の配列、これらの配列を含むベクター、および特に血管疾患の処置のためのそれらの治療的使用 |
AU23907/97A AU2390797A (en) | 1996-03-26 | 1997-03-26 | Sequences ahead of gene sm22, vectors containing same, and therapeutical uses thereof, particularly for treating vascular diseases |
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FR96/03750 | 1996-03-26 | ||
FR9603750A FR2746815A1 (fr) | 1996-03-26 | 1996-03-26 | Sequences en amont du gene sm 22, vecteurs les contenant et leurs utilisations therapeutiques, notamment dans le traitement des maladies vasculaires |
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WO1997035974A9 WO1997035974A9 (fr) | 1997-12-24 |
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JP (1) | JP2000507819A (fr) |
AU (1) | AU2390797A (fr) |
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Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998015575A1 (fr) * | 1996-10-07 | 1998-04-16 | Arch Development Corporation | Promoteur pour l'expression de cellule musculaire lisse |
WO2003083105A1 (fr) * | 2002-03-28 | 2003-10-09 | Medicalseed Co., Ltd. | Traitement et prevention de l'angiostenose |
US7169764B1 (en) | 1995-10-05 | 2007-01-30 | Arch Development Corporation | Promoter for smooth muscle cell expression |
EP1925626A1 (fr) | 2003-07-21 | 2008-05-28 | Transgene S.A. | Nouvelles cytokines multifonctionnelles |
EP2476703A1 (fr) * | 2011-01-14 | 2012-07-18 | Bracco Imaging S.p.A | Anticorps humains à réaction croisée avec des antigènes bactériens et propres de plaques athérosclérotiques |
WO2014066443A1 (fr) | 2012-10-23 | 2014-05-01 | Emory University | Conjugués de gm-csf et d'il-4, compositions et procédés associés |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2010143860A (ja) | 2008-12-19 | 2010-07-01 | Chisso Corp | タンパク質の安定化剤 |
WO2012095516A1 (fr) | 2011-01-14 | 2012-07-19 | Bracco Imaging Spa | Anticorps humain à réaction croisée avec un antigène bactérien et un auto-antigène de plaques athéroscléreuses |
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- 1997-03-26 EP EP97919424A patent/EP0914426A1/fr not_active Withdrawn
- 1997-03-26 JP JP9534091A patent/JP2000507819A/ja active Pending
- 1997-03-26 AU AU23907/97A patent/AU2390797A/en not_active Abandoned
- 1997-03-26 CA CA002250099A patent/CA2250099A1/fr not_active Abandoned
- 1997-03-26 WO PCT/FR1997/000543 patent/WO1997035974A1/fr not_active Application Discontinuation
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US6331527B1 (en) | 1995-10-05 | 2001-12-18 | Arch Development Corporation | Promoter smooth muscle cell expression |
US7169764B1 (en) | 1995-10-05 | 2007-01-30 | Arch Development Corporation | Promoter for smooth muscle cell expression |
US6114311A (en) * | 1995-10-05 | 2000-09-05 | Arch Development Corporation | Method for modulating smooth muscle cell proliferation |
US6284743B1 (en) | 1995-10-05 | 2001-09-04 | Arch Development Corporation | Method for modulating smooth muscle cell proliferation |
US6291211B1 (en) | 1995-10-05 | 2001-09-18 | Arch Development Corporation | Promoter for smooth muscle cell expression |
US6297221B1 (en) | 1995-10-05 | 2001-10-02 | Arch Development Corporation | Method for promoting angiogenesis with a nucleic acid construct comprising an SM22α0 promoter |
US6090618A (en) * | 1996-10-07 | 2000-07-18 | Arch Development Corporation | DNA constructs and viral vectors comprising a smooth muscle promoter |
WO1998015575A1 (fr) * | 1996-10-07 | 1998-04-16 | Arch Development Corporation | Promoteur pour l'expression de cellule musculaire lisse |
WO2003083105A1 (fr) * | 2002-03-28 | 2003-10-09 | Medicalseed Co., Ltd. | Traitement et prevention de l'angiostenose |
EP1944318A1 (fr) | 2003-07-21 | 2008-07-16 | Transgene S.A. | Nouvelles cytokines multifonctionnelles |
EP1925626A1 (fr) | 2003-07-21 | 2008-05-28 | Transgene S.A. | Nouvelles cytokines multifonctionnelles |
EP2476703A1 (fr) * | 2011-01-14 | 2012-07-18 | Bracco Imaging S.p.A | Anticorps humains à réaction croisée avec des antigènes bactériens et propres de plaques athérosclérotiques |
WO2014066443A1 (fr) | 2012-10-23 | 2014-05-01 | Emory University | Conjugués de gm-csf et d'il-4, compositions et procédés associés |
EP3587455A1 (fr) | 2012-10-23 | 2020-01-01 | Emory University | Conjugués de gm-csf et d'il-4, compositions et procédés associés |
Also Published As
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AU2390797A (en) | 1997-10-17 |
FR2746815A1 (fr) | 1997-10-03 |
CA2250099A1 (fr) | 1997-10-02 |
JP2000507819A (ja) | 2000-06-27 |
EP0914426A1 (fr) | 1999-05-12 |
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