EP1299539A1 - Herzmuskel-spezifischer promoter - Google Patents

Herzmuskel-spezifischer promoter

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Publication number
EP1299539A1
EP1299539A1 EP01955410A EP01955410A EP1299539A1 EP 1299539 A1 EP1299539 A1 EP 1299539A1 EP 01955410 A EP01955410 A EP 01955410A EP 01955410 A EP01955410 A EP 01955410A EP 1299539 A1 EP1299539 A1 EP 1299539A1
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European Patent Office
Prior art keywords
gene
nucleic acid
acid molecule
transcription
sites
Prior art date
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EP01955410A
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English (en)
French (fr)
Inventor
Marc Fiszman
Bernard Prudhon
Yves Fromes
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Institut National de la Sante et de la Recherche Medicale INSERM
Association Francaise Contre les Myopathies
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Institut National de la Sante et de la Recherche Medicale INSERM
Association Francaise Contre les Myopathies
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Withdrawn legal-status Critical Current

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    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • 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
    • 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 a promoter active in the myocardium and to its application for expressing genes of interest in this tissue, in particular for the treatment of myocardial diseases.
  • the impairment of myocardial functions can induce serious pathologies, for example familial hypertrophic cardiomyopathy, myocardial infarction and heart rhythm disturbances.
  • the possibility of modulating the responses of cardiac muscle cells (cardiomyocytes) by directing the expression of a gene of interest in these cells can find many applications such as for example: in the case of cardiomyopathies of genetic origin, the synthesis functional non-mutated cardiac protein such as cardiac protein C (familial hypertrophic cardiomyopathy), in the case of acquired pathologies:
  • control of cell division either to stimulate it, for example to regenerate a functional muscle after a heart attack, or to slow it down, for example to inhibit the growth of a heart tumor.
  • This can be achieved, for example, by the manufacture of dominant negative mutants for growth factor receptors or by the use of antisense RNA,
  • cardiomyocytes the study of the in vivo functions of cardiomyocytes, as well as the modification of these functions for therapeutic purposes requires the use of animal models making it possible to individually assess the role of each of the proteins expressed by cardiomyocytes, which '' it involves over-expressing or under-expressing a protein produced naturally by these cells, evaluating the activity of new potentially therapeutic molecules, or studying the functional consequences of the expression of a heterologous protein .
  • MLCs myosin light chains
  • ⁇ -MHC heavy chain of myosin ⁇
  • Some of the promoters regulating the expression of the above genes have been cloned and used in vivo to direct the expression of heterologous genes in transgenic mice, in particular those of the MLC2v genes (HUNTER and al. , J. Biol. Chem., 270, 23173-23178, 1995), Ca (BIBEN et al., Cited above), ANF, (FIELD et al., Science, 239, 1029-1033, 1988), ⁇ - and ⁇ -MHC (RINDT and al., Transgenic Res., 4, 397-405, 1995; KNOTTS et al., Dev. Dyn., 206, 182-192, 1996) and cTnT (ZHU et al., Dev. Biol., 169, 487- 503, 1995).
  • the promoter of the ⁇ -MHC gene is not adapted to transgenic expression in adults; its expression is restricted to the ventricles from the ninth day of development in mice, and drops a few days before birth (NG et al., Cire. Res., 68, 1742-1745, 1991),
  • the promoter of the MLC2v gene is restricted to the ventricles (HUNTER et al., supra) (O'BRIEN et al., supra), the promoter of the ANF gene is restricted to the left ventricle during embryonic development before becoming specific for the atria in adults (ZELLER et al., Genes Dev., 1, 693-698, 1987; FIELD et al., cited above),
  • the promoter of the ⁇ -MHC gene is expressed in the atria during embryonic development and its expression in the ventricles increases a few days before birth and persists after birth (PALERMO et al., Cell., Mol. Biol. Res. , 41, 501-509, 1995; NG et al., Cited above),
  • the promoter of the ⁇ -cA gene (GUNNING et al., Mol. Cell. Biol., 3, 1985-1995, 1983) and the promoter of the cTNT gene (MAR et al., J. Cell. Biol., 107, 573-585, 1988; MAR et al., Symp. Soc. Exp. Biol., ⁇ ,, 237-249, 1992) are expressed both in the myocardium and in the skeletal muscle in the embryo and in l 'adult. Studies carried out in different species have made it possible to isolate a certain number of transcription factors involved in cardiac differentiation and cardiogenesis.
  • MEF-2 proteins Myocyte Enhancer Factor-2
  • homeo box factors Myocyte Enhancer Factor-2
  • Biol., 13, 4432-4444, 1993 also influences the differential expression of muscle genes in the heart and could represent an additional mechanism for controlling the transcription of genes specifically in the heart (SARTORELLI et al., Cire. Res., 72, 925-931, 1993; LYONS et al., Curr. Opin. Genêt. Dev., 6, 454-460, 1996; OLSON et al., Supra; MABLY et al., Supra; FIRULLI et al., Trends Genêt., 13, 364-369 , 1997; DUROCHER et al., 1998, cited above).
  • the cDNA and the MYBPC3 gene encoding human cardiac protein C were isolated (KASAHARA et al., J. Clin. Invest., 94, 1026-1036, 1995).
  • the MYBPC3 gene is at least 21 kb in size, it is composed of 35 exons (CARRIER et al., Cire. Res., 80, 427-434, 1997) and is located on the fold 2 band of chromosome 11 (llpll.2), (GAUTEL et al., Cire. Res., 82, 124-129, 1998).
  • CARRIER et al., Cire. Res., 80, 427-434
  • SVpll.2 fold 2 band of chromosome 11
  • GAUTEL et al. Cire. Res., 82, 124-129, 1998.
  • no sequence controlling the transcription of this gene has been precisely identified.
  • protein C is an abundant protein of the striated muscles which represents 2 to 4% of the fibrillar proteins (OFFER et al., J. Mol. Biol., 74, 653-676, 1973) . It is part of a set of proteins having structural and / or regulatory functions associated with the heavy chain of myosin in the thick filament (EPSTEIN et al., Science, 251, 1039-1044, 1991).
  • cardiac protein C is restricted exclusively to the heart muscle and cannot be never found in skeletal muscle throughout development in humans and mice, and transcripts of the MYBPC3 gene (Myosin Binding Protein-Cardiac 3) encoding cardiac protein-C are present only in the heart and in a uniform manner , in mouse or human embryos (GAUTEL et al., cited above; FOUGEROUSSE et al., Cire. Res., 82, 130-133, 1998).
  • MHC is a familial pathology which is transmitted in an autosomal dominant Mendelian fashion, characterized by an unexplained enlargement of the left ventricle, predominant in the interventricular septum and associated with large cellular and tissue disorganization (HENGSTENBERG et al., J. Mol. Cell. Cardiol., 26, 3-10, 1994).
  • the present invention relates to a nucleic acid molecule derived from a mammalian MYBPC gene, which comprises the cis-regulatory elements necessary and sufficient to direct the specific expression of a gene in the myocardium.
  • the nucleic acid molecule according to the invention essentially consists of the sequences necessary for controlling the initiation of transcription of the MYBPC3 gene (basal promoter), as well as by the sequences involved in the cis regulation of the initiation of the transcription, and this by mechanisms similar to the natural mechanisms regulating the transcription of the endogenous MYBPC3 gene.
  • nucleic acid molecules according to the invention can advantageously be used to direct the expression of a heterologous gene, in particular a gene of therapeutic interest.
  • heterologous relative to a sequence of a given gene means any nucleic acid sequence other than those which, in nature, are immediately adjacent to said sequence.
  • RNA polymerase a canonical motifs recognized by RNA polymerase such as the sequences of TATA boxes and CAT boxes.
  • a fragment of the MYBPC3 gene comprising the TATA box and two GATA-4 sites is capable of activating the expression of a heterologous gene, only in cardiomyocytes in vitro and in vivo.
  • said nucleic acid molecule comprises two binding sequences for the transcription factor GATA-4 (canonical motif 5 '- (A / G) GATA (A / G) -3).
  • said GATA-4 sites are located between positions -60 and -70 and between positions -1010 and -1020, relative to the site of initiation of transcription.
  • the GATA factors belong to a family of nuclear proteins which have a specific DNA binding domain consisting of two adjacent zinc fingers of the C2 / C2 family (HYATT, EMBO J., 12, 4993-5005, 1993).
  • the C-terminal zinc finger associated with an adjacent basic region constitutes the minimal domain for recognition of the canonical motif 5 '- (A / G) GATA (A / G) -3' (KO et al., Mol. Cell. Biol ., 13, 4011-4022, 1993; MERIKA et al., Mol. Cell. Biol., 13, 3999-4010, 1993).
  • GATA-4/5/6 factors Six GATA factors have been characterized in vertebrates including the GATA-4/5/6 factors which are involved on the one hand in cardiogenesis and cardiac differentiation (JIANG et al., Dev. Biol., 174, 258-270, 1996; GOVE et al., EMBO J., 16, 355-368, 1997; GREPIN, Development, 124, 2387-2395, 1997), and on the other hand the differentiation of intestinal epithelial cells (GAO, Mol. Cell. Biol., 18, 2901-2911, 1998).
  • the GATA-4/5/6 factors are capable of transactivating numerous cardiac genes expressed in cardiomyocytes ('LAVERRIERE et al., J. Biol.
  • factor GATA-4 activates the genes for sarcomeric proteins such as troponin C.
  • said nucleic acid molecule also comprises one or more site (s) for binding a factor involved in the restriction of transcription of the MYBPC3 gene in cardiomyocytes.
  • said binding site (s) are selected from the group consisting of: factor NFAT-3 binding sequences, Ets protein binding sequences and RSRF factor binding sequences.
  • the inventors have further observed that the sequence which extends from the position -1500 to -800 relative to the site of initiation of transcription of the MYBPC3 gene is involved in the restriction of the transcription of the gene in cardiomyocytes.
  • the inventors analyzed said sequence and located potential binding sites:
  • - RSRFs factors canonical motif of CarG type: 5 '-CC (A / T) GG-3'.
  • the factor NFAT-3 which is capable of interacting with the factor GATA-4 is expressed in the heart, it plays a role in cardiogenesis (DE LA POMPA et al., Nature, 392, 182-186, 1998; RANGER and al., Nature, 392, 186-190, 1998) and is also involved in cardiac hypertrophy (MOLKENTIN et al., Cell, 93, 215-226, 1998).
  • Ets proteins regulate tissue-specific expression via activation and repression mechanisms CONRAD et al., Mol. Cell. Biol., 14, 1553-1565, 1994; ROSEN et al., J. Biol. Chem ., 269, 15652-15660, 1994; UMEZAWA et al., Mol. Cell. Biol., 17, 4885-4894, 1997), among these, the ERP / Net protein which is expressed in many tissues is also present in the heart (LOPEZ et al., Mol. Cell.
  • binding of these factors to elements of this sequence could be responsible for the restriction of the transcription of the MYBPC3 gene in cardiomyocytes and represents an additional mechanism for controlling the activity of the endogenous MYBPC3 promoter in cardiomyocytes.
  • the GATA-4 sites as well as the NFAT-3, Ets and RSRFs sites, if they are present, are located respectively at the following positions, relative to the transcription initiation site:
  • - NFAT-3 sites -822 / -815; -850 / -844; - Ets sites: -935 / -932; -1102 / -1099;
  • a nucleic acid molecule according to the invention is represented for example by:
  • nucleic acid molecules specified above which can be obtained from the murine MYBPC3 gene, only constitute an illustration of the object of the invention. This also includes in particular nucleic acid molecules reproducing homologous sequences existing in humans, or in other mammals, and which can be obtained by those skilled in the art by conventional techniques of molecular biology.
  • nucleic acid molecules comprising at least the cis-regulatory elements of the MYBPC3 gene specified above
  • a person skilled in the art can construct different nucleic acid molecules in accordance with the invention, for example by carrying out the mutation or the deletion of sequences located outside the cis-regulatory elements, and / or possibly their substitution by other sequences.
  • nucleic acid molecules comprising the cis-regulatory elements of the MYBPC3 gene can be combined with regulatory cis-elements or else with a basal promoter originating from genes other than the MYBPC3 gene, according to different combinations, to obtain molecules chimeric nucleic acids which differ from each other by their level of activity and their degree of specificity.
  • the nucleic acid molecules according to the invention can be used to control expression of a heterologous gene in mammalian cells, and advantageously, to obtain specific expression in cardiac muscle cells.
  • the object of the present invention also encompasses recombinant nucleic acid molecules comprising at least one nucleic acid molecule according to the invention linked to at least one heterologous sequence.
  • the object of the present invention includes in particular: a) expression cassettes comprising: a nucleic acid molecule according to the invention; it may be a sequence from the gene
  • these expression vectors comprise at least one expression cassette as defined above.
  • nucleic acid molecule of interest can be inserted in order to introduce and maintain it in a eukaryotic or prokaryotic host cell, are known in themselves; the choice of an appropriate vector depends on the use envisaged for this vector (for example replication of the sequence of interest, expression of this sequence, maintenance of this sequence in extrachromosomal form, or else integration into the chromosomal material of the host), as well as the nature of the host cell.
  • the invention further relates to prokaryotic or eukaryotic cells transformed with at least one nucleic acid molecule according to the invention.
  • these cells are animal cells, in particular mammalian cells. It could be cardiac muscle cells or totipotent embryonic cells capable of differentiating into cardiac muscle cells.
  • Transformed cells according to the invention can be obtained by any means, known in themselves, making it possible to introduce a nucleic acid molecule into a host cell.
  • viral vectors such as adenoviruses, retroviruses, lentiviruses and AAVs, in which the sequence of interest has been inserted, may be used, among others.
  • the transfer of genes into the heart muscle can for example be carried out using recombinant adenovirus, recombinant virus associated with adenovirus (AAV), liposomes, lipids or cationic polymers, or by direct injection of molecule nucleic acid.
  • AAV recombinant virus associated with adenovirus
  • viral or non-viral vectors can be administered either by direct injection into the myocardium or by coronary perfusion (BARR et al., Gene Therapy, 1, 51-58, 1994; BUDKER et al., Gene Therapy, 5, 272- 276, 1998) or even by injection into the pericardial envelope (LIM et al., Circulation, 83, 2007-2011, 1991; MUHIHAUSER et al., Gène Therapy, 3, 145-153, 1996; ROTHMANN et al. , Gene Therapy, 3, 919-926, 1996).
  • the inventors also obtained transgenic animals, in which a heterologous gene was placed under transcriptional control of a nucleic acid molecule comprising the cis-regulatory elements of the MYBPC3 gene, according to the invention and thus found that the properties of this nucleic acid molecule, and in particular the specificity of expression in cardiac muscle cells was manifested not only ex vivo, but also in vivo at all stages of development, from the embryo to the adult.
  • the subject of the present invention is animals and in particular non-human transgenic mammals, characterized in that all or part of their cells are transformed by a nucleic acid molecule according to the invention.
  • animals and in particular non-human transgenic mammals characterized in that all or part of their cells are transformed by a nucleic acid molecule according to the invention.
  • These are for example animals into which a gene of interest has been introduced under the control of cis-regulatory elements of the MYBPC3 gene, or a chimeric promoter constructed from cis-regulatory elements of this ci which confer specificity of expression in cardiac muscle cells; the gene of interest is then expressed specifically in cardiac muscle cells.
  • the transformed cells and the transgenic animals in accordance with the invention can in particular be used as models for studying and / or modifying the expression of different genes in cardiac muscle cells.
  • the invention also relates to the use of nucleic acid molecules in accordance with the invention for obtaining medicaments, in particular medicaments intended for the treatment of myocardial pathologies.
  • FIG. 1 illustrates the restriction map and the organization of introns and exons of the 15 kb fragment of the MYBPC3 gene isolated from a mouse genomic library.
  • This fragment comprises a 2.3 kb sequence between the Spi-1 gene and the MYBPC3 gene (intergenic region), containing the promoter of the MYBPC3 gene.
  • S Sacl; Sp: SphI; Ac: AccI; Xb: Xbal; H: HindIII; E: EcoRI; K: Kpnl.
  • the exons of the Spi-1 gene are shown in black and the exons of the MYBPC3 gene are shown in gray.
  • FIG. 1 illustrates the restriction map and the organization of introns and exons of the 15 kb fragment of the MYBPC3 gene isolated from a mouse genomic library.
  • This fragment comprises a 2.3 kb sequence between the Spi-1 gene and the MYBPC3 gene (intergenic region), containing the promoter of the MYBPC3
  • FIG. 2 illustrates the comparison of the proximal sequence of the promoter of the human and murine MYBPC3 gene.
  • (b) the sequence extending from position -255 to position +1 relative to the start site of transcription of the mouse MYBPC3 gene sequence is aligned with the corresponding of human MYBPC3 gene (accession number Y10129) and potential transcription factor binding sites are boxed.
  • FIG. 3 illustrates the structure of the four fragments of 2.5, respectively; 1.5; 0.8 and 0.35 kb of the promoter of the MYBPC3 gene coupled to the reporter gene EGFP in the vector pEGFP.
  • FIG. 4 illustrates the restriction map of an expression vector containing the fragment of 1.5 kb of the promoter of the MYBPC3 gene.
  • Example 1 Molecular cloning and chromosomal location of the promoter of the murine MYBP-C3 gene A 15 kb fragment of the MYBPC3 gene was isolated by screening a ⁇ FIXII library of mouse genomic DNA using a probe of 215 bp corresponding to the 5 'end of the coding sequence for cardiac protein C.
  • the restriction map of the 15 kb fragment is represented in FIG. 1: it contains exons 1 to 20 of the cardiac protein C gene, the last two exons of the Spi-1 oncogene (accession number: X17463) and 2.3 kb of intergenic sequence.
  • the 2.3 kb intergenic region comprising the promoter of the MYBPC3 gene, derived from the plasmid pSac4, was then analyzed.
  • the transcription initiation site was determined by the primer extension technique and by cloning the 5 'end of the protein messenger RNA. C cardiac, using respectively the AMV Reverse Transcriptase Primer Extension System kit (PROMEGA) and the 5 'RACE kit (Rapid Amplification System of cDNA ends), according to the manufacturer's instructions. The results show that the initiation site of the transcription of the MYBPC3 gene is located 47 bp upstream of the ATG.
  • PROMEGA AMV Reverse Transcriptase Primer Extension System kit
  • 5 'RACE kit Rapid Amplification System of cDNA ends
  • the transcription initiation site is located 30 bp downstream of the first nucleotide of a TATA box (5 '-TAAATA-3') recognized by RNA polymerase II which is also present in the human promoter.
  • TATA box 5 '-TAAATA-3'
  • CAT box no canonical motif of the :: AAT type (CAT box) was identified in the first 200 nucleotides upstream of the site for initiating transcription of the murine or human promoter.
  • CAAGT sequence of the murine promoter or the CAAT sequence of the human promoter, located between -137 / -141pb can play the role of a CAT box.
  • the first MEF-2 binding site covers the TATA box and the second is adjacent to another cis-element for transcription regulation (box E).
  • TRE thyroid hormone binding site
  • the intergenic region was amplified by PCR from the plasmid pSac4 so as to generate fragments of 2.5 kb, 1.5 kb respectively (SEQ ID NO: 1), 1.1 kb (SEQ ID NO: 2), 0.8 kb and 0.35 kb, having an identical 3 'end corresponding to the position +28 relative to the site of initiation of transcription and a variable 5' end (FIG. 3).
  • SEQ ID NO: 1 fragment of 2.5 kb, 1.5 kb respectively
  • SEQ ID NO: 2 1.1 kb
  • 0.8 kb and 0.35 kb having an identical 3 'end corresponding to the position +28 relative to the site of initiation of transcription and a variable 5' end (FIG. 3).
  • the ATG in position +24 to +26 of the MYBPC3 gene which could disturb the initiation of translation at the level of the first ATG of a heterologous gene cloned behind these sequences is replaced by a SacI site.
  • the amplification products obtained are cloned at the SacI site of the plasmid "pGEM-T easy vector" (PROMEGA) and then subcloned in both orientations (sense and antisense) at the SacI site of the plasmid pEGFP-1 (CLONTECH) which contains the Eucaryotic Green Fluorescent Protein (EGFP) cDNA.
  • the plasmids obtained are called pEGFP4 (2.5 kb), pEGF 6 (1.5 kb), pEGFP8 (1.1 kb), pEGF 10 (0.8 kb) and pEGFP12 (0.35 kb).
  • the plasmids containing the insert in its antisense orientation are used as negative controls and the plasmid containing the 2.5 kb fragment is used as a positive control.
  • the plasmids containing the different promoter fragments are electropores in embryonic carcinoma cells of mouse C3H / He strain (P19 cells; Me BURNEY et al., Nature, 299, 165-167, 1982). Stable transfectants having integrated copies of these plasmids are selected in the presence of geneticin, then maintained in the undifferentiated state or induced in differentiation.
  • P19 cells are totipotent embryonic cells capable of differentiating into cardiac cells in the presence of dimethyl sulfoxide, at the final concentration of 1% v / v.
  • Embryonic bodies made up of cardiac cells in contact with each other, are recognizable by rhythmic beating zones, a phenotype characteristic of the heart developing in vivo (DOETSCHMAN, J. Embryol. Exp. Morphol. 87, 27-45, 1985 ,; RUDNIKI, Dev. Biol., 138, 348-358, 1990; MALTSEV, Mech. Dev., 44, 41-50, 1993).
  • the P19 cells are maintained in the undifferentiated state in a proliferation medium (DMEM (GIBCO), 15% fetal calf serum, 0.1 mM ⁇ -mercaptoethanol). After trypsination, 2.10 6 to 5.10 6 cells in suspension are electroporated (GENE PULSER, BIO-RAD, set to 250 V and 500 ⁇ F) for 5 to 7 ms in a volume of 800 ⁇ l of phosphate buffer pH 7.4, containing 25 ⁇ g of plasmid. Then, the cells are distributed in culture dishes containing 0.1% gelatin supplemented with proliferation medium.
  • DMEM fetal calf serum
  • 0.1 mM ⁇ -mercaptoethanol ⁇ -mercaptoethanol
  • the selection is carried out in a proliferation medium containing 0.7 mg / ml of geneticin.
  • the differentiation is carried out in DMEM medium containing 20% fetal calf serum and 1% DMSO, by the technique of pendant drops; the cells in suspension (25,000 cells / cm 3 ) are deposited in drops of 20 ⁇ l (500 cells) on the lid of a cell culture dish and incubated for 3 days (the cells aggregate to form embryonic bodies), then the embryonic bodies are detached and cultivated in suspension for 4 days, and on the seventh day of differentiation they are transferred to a gelatin culture medium allowing their adhesion to the support.
  • the swinging zones appear between the fourteenth and the twentieth day of differentiation.
  • the promoter of the MYBPC3 gene contains 2 canonical GATA-4 motifs, the most proximal of which is between -63 / -58 bp (GATA.l site) is included both in the fragments of 1 , 5 kb, 0.8 kb and 0.35 kb, and the most distal (GATA.2 site) located between
  • TGATAA site GATA.l
  • AGATAA site GATA.2
  • TCTAGA restriction site Xbal
  • the GATA.l site present in the plasmids pEGFP10 and pEGFP12 was also mutated into the Xbal site.
  • the plasmids obtained comprising these mutations are called pEGFP ⁇ .l, pEGFP6.2, pEGFP6.1.2, pEGFPlO.l and pEGFPl2.1.
  • the constructs pEGFP6 and pEGFP ⁇ doubly mutated are transiently transfected in non-cardiac cells (quail fibroblast line QT6; MOSCOVICI et al., Cell, 11, 95-103, 1977), in the presence or absence of a plasmid vector for the expression of factor GATA-4.
  • 500,000 QT6 cells cultured in DMEM medium containing 10% fetal calf serum are transfected with a mixture containing l ⁇ g of mutated or non-mutated pEGFP ⁇ plasmid, 1 ⁇ g of plasmid GATA-4 and- 3 ⁇ l of non-liposomal lipid Fugene ⁇ (BOEHRINGER ), according to the manufacturer's recommendations.
  • the cells transfected with the mutated plasmids pEGFP ⁇ and pEGFP6 alone are used as a negative control. 48 hours after transfection, the emission of fluorescence is observed under an inverted fluorescence microscope.
  • the mutated or non-mutated plasmids are electroporous in P19 cells differentiated into cardiac cells as described in Example 3.1.
  • GATA has no activity
  • the 1.5 kb fragment mutated on one or other of the GATA sites has weaker activities than that of the non-mutated fragment, 3) the shorter fragments (0.8 kb or 0.5 kb) which do not have any of the GATA sites by deletion and / or mutation still retain an activity.
  • 1.5 kb constitutes a strong minimum promoter of the MYBPC3 gene best regulated in cardiac cells and makes it possible to establish that the two GATA-4 sites (-63 / -58 and -1015 / -1010) are essential for activity of this promoter.
  • Ets sites 935 / -932, -1102 / -1099
  • SRFR sites 1231 / -1221; -861 / -853; -870 / -865
  • the factor NFAT-3 which binds to the canonical motif 5 '- (A / T) GGAAAAT-3' (HOEY et al., I munity, 2, 461-472, 1995) is part of a multigenic family whose members are mainly expressed in lymphocytes (RAO et al., Annu. Rev. Immunol., 15, 707-747, 1997).
  • NFAT-3 is expressed in the heart and plays a role in cardiogenesis (DE LA POMPA et ai., Nature, 392, 182-186, 1998; RANGER et al., Nature, 392, 186-190, 1998), it It has also been reported that NFAT-3 interacts with GATA-4 to activate transcription of the BNP gene (type B Natriuretic Peptide), involved in cardiac hypertrophy (MOLKENTIN et al., cited above).
  • Ets proteins which recognize the 5'-GGA (A / T) -3 'sequence regulate tissue-specific expression via activation and repression mechanisms (CONRAD et al., Mol. Cell. Biol., 14, 1553-1565, 1994; ROSEN et al., J. Biol. Chem., 269, 15652-15660, 1994; UMEZAWA et al., Mol. Cell. Biol., 17, 4885-4894, 1997).
  • the ERP / Net protein which is expressed in many tissues including the heart (LOPEZ et al., Mol. Cell.
  • This negative regulatory mechanism represents an additional mode of control of specific expression in the heart for the 1.5 kb fragment of the promoter of the MYBPC3 gene.
  • Example 4 Construction of an Expression Vector According to the Invention An expression vector, called pU523, allowing the expression of a heterologous gene under the transcriptional control of the 1.5 kb fragment of the promoter of the MYBPC3 a gene was built in the following stages: 1) the Sall-NotI fragment of the multiple cloning site of the plasmid pSL1190 ⁇ superlinker phagemid "(PHARMACIA) was inserted between the SalI and NotI sites of the plasmid pEGFP-1 (CLONTECH) to give the plasmid p ⁇ EGFP, deleted from the promoter and from the coding part of the EGFP cDNA,
  • a Pmel site was introduced by PCR at the ends of the Eco47III-Afl-II fragment of the plasmid p ⁇ EGFP (700 bp), containing the intron, the SD / SA and the polyadenylation sequence of SV40. 4) the amplification product obtained was cloned into the vector pGEM-T easy plasmid (PROMEGA) to give the plasmid pPmel, and
  • the vector map pU523 is illustrated in FIG. 4: it comprises the 1.5 kb fragment of the promoter of the MYBPC3 gene, a small intron, a multiple cloning site and a polyadenylation sequence, bordered by a rare restriction site ( Pmel site) which allows the expression cassette to be isolated and cloned into another vector.
  • Example 5 Activity of the minimum promoter of 1.5 kb ⁇ n vivo in transgenic mice.
  • Example 6 Activity of the minimum promoter of 1.5 kb in vivo in the heart muscle after injection of a recombinant vector containing the promoter linked to the reporter gene for ⁇ -galactosidase 6.1. Injection of a recombinant plasmid
  • a recombinant plasmid called pShMYBPC3-LacZ, containing the coding sequence of the lacZ gene, under the control of the 1.5 kb fragment of the murine MYBPC3 gene promoter was constructed by following the following steps.
  • the Xhol-Sall fragment of the plasmid pCMV ⁇ (CLONTECH) containing the coding sequence of the lacZ gene was inserted at the SalI site of the adenovirus shuttle plasmid pAdeasy (HE et al., Proc. Nat. Acad. Sci., 95, 2509 -2514, 1998).
  • the plasmid obtained contains the coding sequence of the lacZ gene, flanked 5 ′ by a splice acceptor site, an intron and a splice donor site originating from the late region of SV40 and in 3 ′ a polyadenylation site.
  • This plasmid was then digested with the endonucleases Xhol and HindIII and the Xhol-HindIII fragment of the plasmid pEGFP6 containing the promoter of the MYBPC3 gene was inserted between these two sites to give the plasmid pShMYBPC3-LacZ.
  • the plasmid containing no promoter was used as a negative control and the plasmid pCMV ⁇ (CLONTECH) as a positive control for the expression of ⁇ -galactosidase in the two types of muscle tissue 7 days after the injection, the animals were sacrificed and the heart and muscle were removed. Serial transverse or longitudinal cryocuts of these two organs were performed.
  • AdMYBPC3- ⁇ gal containing the ⁇ -galactosidase gene under the transcriptional control of the 1.5 kb fragment of the promoter of the MYBPC3 gene, in place of the El region of a defective adenovirus ⁇ E1, was obtained by homologous recombination from the shuttle plasmid of the adenovirus pShMYBPC3.
  • adenovirus described by DAVIDSON et al. (Nature Genetics, 3, 219-223, 1993), containing the ⁇ galactosidase gene under the transcriptional control of the ubiquitous cytomegalovirus promoter (AdCMV- ⁇ gal) was used as a control.
  • ⁇ -galactosidase After intrapericardial injection, the expression of ⁇ -galactosidase is limited to myocardial cells as well as to spleen cells in animals which have received AdMYBPC3- ⁇ gal while cells expressing ⁇ -galactosidase are observed in numerous organs (liver, heart, spleen, lung, kidney) of animals having received AdCMV- ⁇ gal.
  • AdMYBPC3- ⁇ gal no cells expressing ⁇ -galactosidase are observed in animals having received AdMYBPC3- ⁇ gal while cells expressing ⁇ -galactosidase are observed in many organs, in particular in the liver, but never in the heart, animals having received AdCMV- ⁇ gal.
  • This recombinant vector therefore makes it possible to limit the undesirable effects linked to the expression of a heterologous gene in a tissue other than the myocardium.

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