EP1299539A1

EP1299539A1 - Myocardium-specific promoter

Info

Publication number: EP1299539A1
Application number: EP01955410A
Authority: EP
Inventors: Marc Fiszman; Bernard Prudhon; Yves Fromes
Original assignee: Institut National de la Sante et de la Recherche Medicale INSERM; Association Francaise Contre les Myopathies
Current assignee: Institut National de la Sante et de la Recherche Medicale INSERM; Association Francaise Contre les Myopathies
Priority date: 2000-07-12
Filing date: 2001-07-12
Publication date: 2003-04-09
Also published as: AU2001277581A1; FR2811678B1; FR2811678A1; US20040175699A1; WO2002004637A1

Abstract

The invention concerns a myocardium-specific promoter, derived for the <i>MYBPC</i> gene of a mammal and comprising regulating <i>cis</i>-elements necessary and sufficient for directing specific expression of a gene in the myocardium. The invention is applicable to treatment of diseases affecting the myocardium.

Description

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, whether genetic or acquired, 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,

* control of the force of contraction or the rhythm of contractions of the heart muscle.

On the other hand, 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 . To be able to effectively use the potentials of a transfer of genes into the heart muscle, it is necessary to have vectors making it possible to obtain a stable expression of a gene of interest in the 5 cardiomyocytes, in vi tro and also in vivo as well as efficient and reliable techniques for transferring therapeutic genes into the tissues concerned.

This expression must also, in some cases, be specific to this fabric, in order to avoid problems

10 which could result from ubiquitous expression insofar as certain viral vectors such as adenovirus are capable of penetrating into numerous tissues.

Currently a number of proteins are known which are expressed more or less specifically

1. in the heart muscle, and whose promoters constitute potential candidates for the specific tissue expression of a heterologous gene; myosin light chains (MLCs), (O'BRIEN et al., Proc. Nat. Acad. Sci., 90, 5157-5161, 1993; LEE et al., J. Biol. Chem., 267, 15875- 15885, 0 1992; KELLY et al., J. Cell Biol. 129, 383-396, 1995), the heavy chain of myosin α (α-MHC), (SUBRAMANI7AM et al., J. Biol. Chem. , 268, 4331-4336, 1993), the myosin β heavy chain (β-MHC), (RINDT et al., J. Biol. Chem., 268, 5332-5338, 1993), cardiac troponin T ( cTnT), (IANNELLO et 5 al., J. Biol. Chem., 266, 3309-3313, 1991; CHRISTENSEN et al., Mol. Cell. Biol., 13, 6752-6765, 1993), muscle creatinine kinase (MCK), (AMACHER et al., Mol. Cell. Biol., 13, 2753-2764, 1993), atrial natriuretic factor (ANF), (SEIDMAN et al., Can. J. Physiol. Phar acol., 69, 1486-1492, 0 1991; ARGENTIN et al., Mol. Cell. Biol., 14, 777-790, 1994), cardiac α-actin (α-cA), (BIBEN et al., Dev. Biol., 173, 200-212, 1996), type-A (ANP) and -B (BNP) natriuretic peptides, (DUROCHER et al. , Dev. Genet., 22, 250-262, 1998). 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), α- and β-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).

Even if these sequences make it possible to express genes at the cardiac level, their expression profile is either restricted to a compartment of the heart, or nonspecific of the heart, at stages of embryonic development or in adults (LYONS et al. , J. Cell. Biol., 111, 2427-2436, 1990 and J. Cell. Biol., 111, 1465-1476, 1990).

For exemple :

- the promoter of the β-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 α-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 α-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., Ζ,, 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. These factors bind to specific sequence motifs in the regulatory regions of these genes, called transcriptional cis-regulatory elements, and are capable of transactivating many cardiac genes expressed specifically in cardiomyocytes. Among these factors, the best known are: MEF-2 proteins (Myocyte Enhancer Factor-2), homeo box factors

Csx / Nkx2.5 and Mhox (Muscle Hox), GATA factors, ehand and dhand proteins, and TEF-1 factors

(Transcription Enhancer factor-1), (OLSON et al., Science,

272, 671-676, 1996; MABLY et al. , Wax. Res., 79, 4-13, 1996).

However, while the participation of each of the above factors in regulating the expression of endogenous muscle genes is clearly established, the exact combination of these tissue-specific factors necessary and sufficient to express heterologous genes of therapeutic interest, specifically in the heart has not been determined.

To this first degree of complexity in the control of tissue-specific expression, is added an additional degree of complexity insofar as it has been reported that the particular combination of these tissue-specific factors with ubiquitous factors such as factors related to SRF (Serum Response Factor) called RSRF (Rela ted Sérum Response Factor) (PARI et al., Mol. Cell. Biol., 11, 4796-4803, 1991; KARNS et al., J. Biol Chem., 270, 480-487, 1995; PARKER et al., J. Biol. Chem., 267, 3343-3450, 1992; CHEN et al., Mol. Cell. Biol., 16, 6372- 6384, 1996), Spl (SAFFER et al., Mol. Cell. Biol., 11, 2189-2199, 1991) and Egr-1 (MUTERO et al., J. Biol. Chem., 270, 1866-1872, 1995; THOMPSON et al. , J. Biol. Chem., 266, 22678-22688, 1991; GUPTA et al., J. Biol. Chem., 266, 12813-12816, 1991; PARMACECK et al., Mol. Cell. Biol., 14, 1870-1885, 1994; ZHU et al., Mol. Cell. 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).

Consequently, no promoter allowing the expression of a gene of interest in all the compartments of the heart, early in the embryo and in a lasting way in the adult has not yet been characterized. In addition, the combination of cis-elements, necessary and sufficient to ensure expression of a gene, specifically in the heart muscle has not been identified.

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). However, no sequence controlling the transcription of this gene has been precisely identified.

Among the proteins appearing expressed specifically in the myocardium, 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).

There are three isoforms of protein C, each encoded by a gene and expressed specifically in a type of muscle: rapid skeletal protein C, slow skeletal protein and cardiac protein C (cMyBPC), (YAMAMOTO et al., J. Biol. Chem., 258, 8395-8401, 1983).

The expression of 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).

In humans, the MYBPC3 gene encoding the cardiac isoform is one of the eight genes involved in cardiac disorders of the MHC type (TOWBIN et al., Curr. Opin. Cell. Biol., 10, 131-139, 1998 ; MOGENSEN et al., J. Clin. Invest., 103, 39-43, 1999). 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).

From a mouse genomic clone comprising a fragment of the MYBPC3 gene encoding the cardiac protein C, the inventors have now identified and cloned the promoter sequence of the MYBPC3 gene, and have also located the regulatory sequences necessary and sufficient to tissue-specific activity of this promoter. This work enabled them to isolate fragments of this promoter capable of activating the expression of a gene specifically and exclusively in the myocardium at all stages of development, from the embryo to the adult.

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.

The 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.

For the purposes of the present invention, the term "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.

Among the sequences necessary for controlling the initiation of transcription, mention may in particular be made of the canonical motifs recognized by RNA polymerase such as the sequences of TATA boxes and CAT boxes.

Among the potential sequences involved in cis in the regulation of transcription in the heart muscle, mention may be made of the sequences of binding of the transcription factors, ubiquitous or specific of the heart muscle, such as the sequences of binding of the factors described above.

Unexpectedly, the inventors have shown that 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.

According to an advantageous embodiment of the invention, said nucleic acid molecule comprises two binding sequences for the transcription factor GATA-4 (canonical motif 5 '- (A / G) GATA (A / G) -3).

Advantageously, 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). 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. Chem., 269, 23177-23184, 1994; KELLEY et al., Development, 118, 817-827, 1993; JIANG et al., Cited above; MORRISEY et al., Dev. Biol., 177, 309-322, 1996; 183, 21-36, 1997; PARMACECK et al., Mol. Cell. Biol., 12, 1967-1976, 1992). For example, factor GATA-4 activates the genes for sarcomeric proteins such as troponin C.

Advantageously, 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. Advantageously, 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:

- factor NFAT-3 ("Nuclear Factor of Activa ted T cells-3"; canonical motif 5 '- (A / T) GGAAAAT-3'), Ets proteins (canonical motif Ets 5'- GGA (A / T) -3 ', and

- 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. Biol., 14, 3292-3309, 1994; PRICE et al., EMBO J., 14, 2589-2601, 1995) and it seems that it can interact with the RSRFs binding site (PRICE et al., supra; LOPEZ et al., supra; ASYLYK et al., Eur. J. Biochem., 211, 7-18, 1993).

The 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.

Advantageously, 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:

- GATA-4 sites: -63 / -58; -1015 / -1010;

- NFAT-3 sites: -822 / -815; -850 / -844; - Ets sites: -935 / -932; -1102 / -1099;

- RSRF sites of the CarG type: -861 / -853; -870 / -865; -1231 / -1221. A nucleic acid molecule according to the invention is represented for example by:

- the sequence which extends from position -1535 to position +28 relative to the site of initiation of transcription of the murine MYBPC3 gene; this sequence is represented in the annexed sequence list under the number SEQ ID NO: 1, and the sequence represented in the annexed sequence list under the number SEQ ID NO: 2 which extends from position 464 to position 1563 of the sequence SEQ ID NO: 1 above.

It should be understood that the 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.

Likewise, from 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.

The 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

MYBPC3, or of a chimeric sequence, comprising at least the cis-regulatory elements of the MYBPC3 gene, as defined above, and a heterologous sequence placed under transcriptional control of said nucleic acid molecule. b) recombinant vectors, comprising an insert consisting of a nucleic acid molecule according to the invention. Advantageously, these expression vectors comprise at least one expression cassette as defined above.

Many vectors into which a 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. Preferably, 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. For example, in the case of animal cells, viral vectors such as adenoviruses, retroviruses, lentiviruses and AAVs, in which the sequence of interest has been inserted, may be used, among others. one can also associate said sequence (isolated or inserted into a plasmid vector) with a substance enabling it to cross the membrane of host cells, for example a preparation of liposomes, lipids or cationic polymers, or else inject it directly into the host cell, in the form of naked DNA.

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.

These 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. 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.

The present invention will be better understood using the additional description which follows, which refers to examples describing the obtaining and characterization of the promoter of the MYBPC3 gene and fragments thereof allowing specific expression in muscle. cardiac, as well as their use for the expression of heterologous genes specifically in cardiac muscle cells in cell cultures and in transgenic animals, as well as in the appended drawings in which:

- Figure 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. 2 illustrates the comparison of the proximal sequence of the promoter of the human and murine MYBPC3 gene. (a) the sequence which extends from position -238 to position +83, relative to the site of initiation of transcription (+1) of the mouse MYBPC3 gene is presented and the potential factor binding sites transcription are framed, (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.

- Figure 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.- - Figure 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 λFIXII library of mouse genomic DNA using a probe of 215 bp corresponding to the 5 'end of the coding sequence for cardiac protein C.

Three fragments of 2 kb, 4 kb and 6 kb, obtained by restriction of the 15 kb fragment derived from the genomic clone with the enzyme Sac1, were subcloned at the SacI site of the plasmid pBluescript (STRATAGENE). The plasmids obtained, named pSac2, pSac4 and pSacδ respectively, were sequenced and the sequence of the above fragments was compared with the databases.

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.

In humans, the chromosomal organization of the Spi -1 genes (MOREAU-GACHELIN, Oncogene, 4, 1449-1456, 1989) and MyBPC3 (GAUTEL et al., Cited above) is similar to that observed in mice: they are both located in the fold 2 region of chromosome 11.

The 2.3 kb intergenic region comprising the promoter of the MYBPC3 gene, derived from the plasmid pSac4, was then analyzed.

Example 2 Analysis of the promoter of the human and murine MYBPC3 gene

2.1. Determination of the Murine MYBPC3 Gene Transcription Initiation Site 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.

2.2. Investigation of specific patterns of transcription factor binding in human and mouse MYBPC3 genes

The alignment of the 2.3 kb sequence with the sequence of the human gene (accession number Y10129) shows a homology of almost 80% between the positions -200pb and +1 (Figure 2b). The 2.3 kb sequence was analyzed using the Ma tlnspector software which allows the search for binding sites of factors involved in transcription (QUANDT et al., Nucleic Acid Res., 23, 4878-4884, 1995 ; http // www. gsf. de / biodv / matinspector .html). Sequence elements described as having importance in the regulation of genes encoding muscle proteins have also been sought directly on these sequences. The potential binding sites of ubiquitous or specific transcription factors of the heart identified in the proximal sequence -238 to +83 of the murine promoter and the conservation of these sequences in the human promoter are presented respectively in FIGS. 2a and 2b.

Analysis of the murine promoter shows that 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. In addition, 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. However, it is possible that the 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 following transcription factor binding sites have been identified in the murine promoter at the specified positions, those identical in the human promoter are indicated in bold. :

• if your are rich in GC of binding of the factors Spl and Spl - like:

-47 / -41; -192 / -187; -282 / -278

• if your are rich in GC for the binding of Egr-1 factors: -169 / -161; -227 / '-222; -293 / -287; -503 / -490;

-669 / -662

• if your are rich in MEF-2 factor binding AT:

-32 / -24; -92 / -83 The first MEF-2 binding site covers the TATA box and the second is adjacent to another cis-element for transcription regulation (box E).

• if your rich in AT binding proteins to homeodomains such as MHox:

-260 / -252; -758 / -752; -813 / -806; -1071 / -1063; -1710 / -1697

• canonical motifs GATA-4 ((5 '- (A / G) GATA (A / G) -3'):

-63 / -58; -1015 / -1010; -2239 / -2234 • GATA-4 like pattern

-117 / -112; -244 / -239

• canonical motif Nkx2. 5 (5 '-TNAAGTG-3'):

-981 / -975; -1078 / -1072; -1631 / -1625, (on the anti-sense strand)

• if your GTIIC, SphI and SpHII of binding of factors TEF-1

-105 / -100; -640 / -646; -812 / -804; -2132 / -2124, The sites in position -105 / -100 and -640 / -646 are juxtaposed to an E box.

• thyroid hormone binding site (TRE: Thyroid Responsive Element):

-70 / -64; -161 / -143; -205 / -198; -967 / -959; -1591 / -1585; -1611 / -1604,

• canonical motif 5 ^r -CA (G / C) (C / G) TG-3 '(box E):

-82 / -11, -109 / -104,

• canonical motif (5 '^' -GGA (A / T) -3 '^' ) of Ets factors:

-101 / -95; -935 / -932; -1102 / -1099,

• palindromic patterns with a strong affinity for Ets factors:

-116 / -128

• if you are rich in AT of the CarG or GArC type for binding RSFR factors:

-820 / -814; -848 / -843; -870 / -865; -861 / -853; -1231 / -1221;

-1721 / -1710,

• canonical motif (5 '- (A / T) GGAAAAT-3') for binding of factor NFAT-3 (Nuclear Factor of Activa ted cells):

-822 / -815; -850 / -844; -2200 / -2193. These results show on the one hand that many transcription factors are capable of binding to the promoter of the murine and human MYBPC3 gene, and on the other hand that most of the binding sites of these factors are conserved between the human and murine promoters . However, the presence of these sites even if they are canonical does not mean that they are directly involved in the regulation of the endogenous promoter. Consequently, only the functional analysis of the role of these different sites in the activity of the promoter of the MYBPC3 gene makes it possible to determine the minimum combination of these sequences, capable of ensuring the tissue-specific expression of the MYBPC3 gene in the heart. Example 3: Determination of a minimum promoter fragment specific for cardiac cells

3.1 Promotional activity of the 2.3 kb intergene sequence and of fragments derived from this sequence

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). In the 3 'primer, 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 β-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 μF) for 5 to 7 ms in a volume of 800 μl of phosphate buffer pH 7.4, containing 25 μg of plasmid. Then, the cells are distributed in culture dishes containing 0.1% gelatin supplemented with proliferation medium.

2 days after electroporation, 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 μl (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 expression of EGFP is determined from a set of 15 embryonic bodies observed under an inverted fluorescence microscope (OLYMPUS IX50 / IX70 + IX = FLA) and evaluated by the percentage of embryonic bodies emitting fluorescence at the level of swinging areas.

The results of this experiment are as follows: 1) No expression of EGFP is observed in differentiated or undifferentiated P19 cells, electroporated with the plasmids in their antisense version, which demonstrates that the fluorescence observed is specific for the activity of the promoter.

2) With the plasmids pEGFP10 or pEGFP12, - a very weak expression of EGFP is observed in non-differentiated P19 cells, and in P19 cells differentiated into cardiac cells, a majority expression of EGFP is observed in the zones with a weak and variable expression in cells outside these beating zones.

3) With the plasmids pEGFP4, pEGFP6 and pEGFP8, there is an absence of expression of EGFP in undifferentiated P19 cells and expression of EGFP only in the beating zones in P19 cells differentiated into cardiac cells.

These results make it possible to establish that only the fragments of 2.5 kb, 1.5 kb and 1.1 kb contain all of the promoter sequences of the murine MYBPC3 gene allowing specific expression of cardiac muscle cells.

3.2. Determination of the elements of sequences involved in the cardiac specificity of the promoter. 3.2.1. Mutagenesis of GATA-4 sites

It was previously shown (Example 2) that 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

-1015 / -1010 bp is included only in the fragment of

1.5 kb (Figure 3).

These reasons being important in the specific expression of cardiac genes, the sequences TGATAA (site GATA.l) and AGATAA (site GATA.2) were mutated independently in restriction site Xbal (TCTAGA) in the plasmid pEGFP6.

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 pEGFPβ.l, pEGFP6.2, pEGFP6.1.2, pEGFPlO.l and pEGFPl2.1.

3.2.2 Transfection of the mutated plasmids into non-cardiac cells. In order to verify that the mutations effectively prevent the binding of the transcription factor GATA-4, the constructs pEGFP6 and pEGFPδ doubly mutated (pEGFP6.1.2) 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 lμg of mutated or non-mutated pEGFPδ plasmid, 1 μg of plasmid GATA-4 and- 3 μl of non-liposomal lipid Fugeneβ (BOEHRINGER ), according to the manufacturer's recommendations. The cells transfected with the mutated plasmids pEGFPδ and pEGFP6 alone are used as a negative control. 48 hours after transfection, the emission of fluorescence is observed under an inverted fluorescence microscope.

It is observed that only the mutated construct on the two sites is not transactivated by the expression of factor GATA-4. The other constructs are to varying degrees, the most significant transactivation being obtained with the non-mutated plasmid pEGFP6. These results therefore establish that only the two canonical sites in position -63 / -58 and -1015 / -1010 allow good transactivation of the 1.5 kb fragment of the promoter, by the factor GATA-.

3.2.3 Transfection of mutated plasmids into heart cells.

The mutated or non-mutated plasmids are electroporous in P19 cells differentiated into cardiac cells as described in Example 3.1.

The results of 2 series of independent experiments are presented in Table I below. Board

* ND = not determined

The results observed make it possible to establish that in cardiac cells: 1) the 1.5 kb fragment mutated at the two sites

GATA has no activity,

2) 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.

The results confirm that the fragment of

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.

On the other hand, the results observed with the plasmids pEGFP10.l and pEGFP12.1 compared to those observed for the plasmid pEGFPδ.1.2 make it possible to establish that there is a negative regulatory mechanism involving the sequences

-15,007 to 800.

Analysis of the 700 bp (see example 3) reveals consensus reasons for binding to the transcription factor NF-AT3 [-822 / -815 bp (5 '-TGGAAAAT-3'), -850 / -844 bp (5 ' -TGGAAAT- 3 ')].

In addition, the Ets sites (-935 / -932, -1102 / -1099) and the SRFR sites (-1231 / -1221; -861 / -853; -870 / -865) are potential targets of the ERP / Net factor, which is expressed in the heart.

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).

The Ets proteins which recognize the 5'-GGA (A / T) -3 'sequence (TREISMAN et al., Curr. Opin. Dev. Genêt., 4, 96-101, 1994) 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 including the heart (LOPEZ et al., Mol. Cell. Biol., 14-3292-3309, 1994; PRICE et al., EMBO J., 14, 2589-2601, 1995) interacts with a single Ets binding site (LOPEZ et al., Cited above) and it seems that it can also interact with the SRFR binding site (PRICE et al., EMBO J., 14, 2589-2601, 1995; LOPEZ et al., Cited above; WASYLYK et al., Eur. J. Biochem., 211, 7-18, 1993).

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 ^Λ superlinker phagemid "(PHARMACIA) was inserted between the SalI and NotI sites of the plasmid pEGFP-1 (CLONTECH) to give the plasmid pΔEGFP, deleted from the promoter and from the coding part of the EGFP cDNA,

2) the Xhol-Smal fragment of the plasmid pCMVβ (CLONTECH) containing an intron as well as a donor site and a splice acceptor site (SD / SA) was inserted between the SalI and Smal sites of the plasmid pΔEGFP,

3) a Pmel site was introduced by PCR at the ends of the Eco47III-Afl-II fragment of the plasmid pΔEGFP (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

5) the SalI-HindIII fragment of the plasmid pGEM-T6 containing the fragment of 1.5 kb of the promoter of the MYBPC3 gene, obtained as described in Example 3, was inserted between the Xhol and HindIII sites of the Pmel fragment of the plasmid pPmel for give the plasmid pU523.

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.

The plasmid pEGFP6, obtained as described in Example 3, was digested with the endonucleases Sac1 and AflII and the restriction fragment obtained, which comprises the 1.5 kb fragment of the promoter of the MYBPC3 gene linked to the EGFP gene has been purified. This fragment was injected into the eggs of fertilized mice which were then reimplanted in pseudo-pregnant mice. The expression of EGFP in the 10 mouse lines obtained was analyzed by direct observation of different organs under an inverted fluorescence microscope.

7 of 10 lines strongly express EGFP, this expression is detected only in the heart

These results confirm that the 1.5 kb fragment is active in vivo and makes it possible to express a heterologous gene at high levels, exclusively in the heart. The expression cassette as defined above therefore makes it possible to evaluate the activity of new therapeutic molecules for cardiac or cardiovascular purposes and to study the functional consequences of the expression of a heterologous protein in the heart muscle. 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 β-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 pCMVβ (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. Adult male Wistar rats were injected with 50 to 500 μg of the recombinant plasmid obtained, containing the β-galactosidase gene under the transcriptional control of the 1.5 kb fragment of the gene promoter MYBPC3. 2 series of experiments were carried out on batches of 3 animals:

1) intramyocardial injection, at the level of the wall of the left ventricle, of 50 to 250 μg of recombinant plasmid in 5% W / V glucose solution in an injection volume of 100 μl,

2) intramuscular injection, at the level of the Tibialis anterior skeletal muscle, of 50 to 500 μg of recombinant plasmid in 5% W / V glucose solution in an injection volume of 100 μl.

The plasmid containing no promoter was used as a negative control and the plasmid pCMVβ (CLONTECH) as a positive control for the expression of β-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.

After incubation in the presence of the X-gal substrate (5-bromo-4-chloro-3-indolyl-β-galactoside), the sections were observed under a microscope in order to analyze the cells expressing β-galactosidase.

After injection of the control plasmid without promoter into the myocardium or else into the skeletal muscle, no cell expressing β-galactosidase is detected in the two types of muscle.

After injection into the myocardium, numerous cells expressing β-galactosidase are detected both in animals having received the plasmid pCMVβ and in animals having received the plasmid pShMYBPC3-LacZ.

On the other hand, after injection into the skeletal muscle, no cell expressing β-galactosidase is detected in animals having received the plasmid pShMYBPC3-LacZ while many cells expressing β-galactosidase are detected in animals having received the plasmid pCMV.beta.

These results make it possible to affirm that the in vivo administration of an expression vector plasmid containing a heterologous gene under the control of the 1.5 kb fragment of the MYBPC3 promoter makes it possible to express this heterologous gene, only in the myocardium. 6.2 Injection of a recombinant adenovirus A recombinant adenovirus, called AdMYBPC3-βgal containing the β-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 ΔE1, was obtained by homologous recombination from the shuttle plasmid of the adenovirus pShMYBPC3.

The recombinant adenovirus described by DAVIDSON et al. (Nature Genetics, 3, 219-223, 1993), containing the βgalactosidase gene under the transcriptional control of the ubiquitous cytomegalovirus promoter (AdCMV-βgal) was used as a control.

Lots of 3 adult male Wistar rats were injected with 10 ⁹ pfu (plaque forming units) of the adenovirus AdMYBPC3-bgal or AdCMV-βgal. 4 series of experiments were carried out: 1) intramyocardial injection, at the level of the wall of the left ventricle, of the preparation of adenovirus in Ringer-lactate solute in an injection volume of -100 μl,

2) intramuscular injection, at the level of the Tibialis anterior skeletal muscle of the preparation of adenovirus in Ringer-lactate solute in an injection volume of 100 μl,

3) intrapericardial injection, according to the method described in FROMES et al. (Gene Therapy, 6, 683-688, 1999), of the preparation of adenovirus in Ringer-lactate solute in an injection volume of 750 μl,

4) intra-arterial injection of the adenovirus preparation in Ringer-lactate solution, in an injection volume of 100 μl 7 days after the injection, the animals were sacrificed and the heart, the muscle and the other organs ( lung, diaphragm, liver, kidney, spleen) were removed. of the serial transverse and / or longitudinal cryocuts of these organs were performed.

After injection into the skeletal muscle, no cell expressing β-galactosidase is detected in the animals having received AdMYBPC3-βgal whereas numerous positive cells are observed in animals having received AdCMV-βgal.

After intrapericardial injection, the expression of β-galactosidase is limited to myocardial cells as well as to spleen cells in animals which have received AdMYBPC3-βgal while cells expressing β-galactosidase are observed in numerous organs (liver, heart, spleen, lung, kidney) of animals having received AdCMV-βgal. After intraarterial injection, no cells expressing β-galactosidase are observed in animals having received AdMYBPC3-βgal while cells expressing β-galactosidase are observed in many organs, in particular in the liver, but never in the heart, animals having received AdCMV-βgal.

These results make it possible to affirm that the administration in vivo, directly in the heart or else systemically, of a recombinant adenovirus containing a heterologous gene under the control of the 1.5 kb fragment of the promoter MYBPC3 makes it possible to express this heterologous gene, at high levels, only in the myocardium.

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.

Claims

CLAIMS 1) Nucleic acid molecule capable of directing the specific expression of a gene in the myocardium, characterized in that it comprises the cis-elements for regulating the transcription of the promoter of a mammalian MYBPC3 gene, which transcriptional regulatory elements comprise at least two binding sequences of a GATA-4 transcription factor.

2 °) nucleic acid molecule according to claim 1, characterized in that it comprises a site

GATA-4 between positions -60 and -70 and between positions

-1010 and -1020, relative to the site of initiation of transcription.

3 °) nucleic acid molecule according to claim 2, characterized in that it also comprises one or more binding sites of a transcription factor involved in the restriction of the transcription of the MYBPC3 gene in cardiomyocytes, said one or more binding sites being chosen from the NFAT-3 factor binding sequences, the Ets protein binding sequences and the RSRF factor binding sequences.

4 °) nucleic acid molecule according to claim 3, characterized in that said binding sites are located at the following positions with respect to the transcription initiation site:

- GATA-4 sites: -63 / -58; -1015 / -1010;

- NFAT-3 sites: -822 / -815; -850 / -844;

- Ets sites: -935 / -932; -1102 / -1099;

- RSRF sites of the CarG type: -861 / -853; -870 / -865; -1231 / -1221.

5 °) nucleic acid molecule according to any one of claims 1 to 4, characterized in that its sequence is selected from the group consisting of: the sequence represented in the sequence list in the appendix under the number SEQ ID NO: 1, and the sequence shown in the sequence list in the appendix under the number SEQ ID NO: 2. 6 °) Recombinant vector comprising an insert consisting of a nucleic acid molecule according to any one of claims 1 to 5.

7 °) cell transformed with at least one nucleic acid molecule according to any one of claims 1 to 5.

8 °) transformed cell according to claim 7, characterized in that said cell is a _cell of mammal. 9 °) non-human transgenic animal, characterized in that all or part of its cells are transformed by a nucleic acid molecule according to any one of claims 1 to 5.

10 °) transgenic animal according to claim 9, characterized in that it is a mammal.

11 °) Use of a nucleic acid molecule according to any one of claims 1 to 5 for obtaining medicaments.