NZ516678A

NZ516678A - Regulation system of expression using nuclear PPAR receptors

Info

Publication number: NZ516678A
Application number: NZ516678A
Authority: NZ
Inventors: Raphael Darteil; Joel Crouzet; Bart Staels; Abderrahim Mahfoudi
Original assignee: Aventis Pharma Sa
Priority date: 1999-06-22
Filing date: 2000-06-22
Publication date: 2004-04-30
Also published as: NO20016347L; CZ20014609A3; BR0011897A; JP2003503034A; WO2000078986B1; CN1370240A; WO2000078986A1; KR20020028909A; MXPA01013227A; HUP0203584A2; IL147104A0; AU6164500A; NO20016347D0; EP1190084A1; CA2375869A1

Abstract

Methods and compositions for the pharmacological regulation of the expression of transgenes. A composition comprising: (a) a first element comprising a nucleic acid of interest under the control of an inducible promoter comprising an element containing a response element to a Peroxisome Proliferator-Activated Receptor (PPAR) and a minimal transcriptional receptor; and (b) a second element comprising a nucleic acid coding for a PPAR under the control of a transcriptional promoter, for simultaneous, separate or prolonged use And the use of said compositions and methods in the experimental, clinical, therapeutic or diagnostic fields.

Description

<div class="application article clearfix" id="description"> 516678 WO 00/78986 PCT/FR00/01744 1 SYSTEM FOR REGULATION OF EXPRESSION USING PPAR NUCLEAR RECEPTORS The present invention relates to the field of biology. It relates in particular to the field of the regulation of the expression of genes and, more particularly, it describes the design and development of a new system for the pharmacological regulation of the expression of transgenes. The invention is based in particular on the use of constructs of human origin for activating the transcription of the transgene. The invention thus describes new compositions, constructs and methods allowing the effective regulation of the expression of a nucleic acid in vitro, ex vivo or in vivo, for example in muscle cells. The applications which result from the present invention are many, in the experimental, clinical, therapeutic or diagnostic fields, for example. Controlling the level and the duration of the expression of transgenes is necessary for many applications. Thus, in gene therapy, the success of the therapy may require a specific assay of the protein synthesized from the transgene. Likewise, the production of recombinant proteins in vitro may be r improved using inducible expression systems, allowing for example uncoupling of the growth and production phases. The construction of transgenic animals, the study of the effects of a gene and of the INTELLECTUAL PROPERTY OFFICE OF N.Z. -9 FEB m RECEIVED WO 00/78986 2 PCT/FROO/01744 bioavailability of a protein, and the like, are so many situations in which an appropriate control of genetic expression may be used and may provide improvement. Various artificial regulators of 5 transcription have been designed in the prior art, which are activated by a xenobiotic molecule which bind to the promoter sequences for transcription of the transgene. A first illustration of these regulators was 10 constructed by fusion of the E. coli Lac repressor with the herpes simplex virus (HSV) VP16 transactivator domain. Two versions of these regulators exist, one capable of being activated by isopropyl (3-D-thiogalactoside (IPTG) and the other inactivated by 15 IPTG (Baim S. et al. , Proc Natl Acad Sci USA, 88 (1991) 5072-5076; Labow M. et al., Mol. Cell. Biol., 10 (1990) 3343-3356). Another system was constructed by fusion of the E. coli Tet repressor with the HSV VP16 20 transactivator domain. Two versions of these regulators also exist, one capable of being activated by tetracycline or its derivatives and the other inactivated by these same molecules (Gossen M. and Bujard H., Proc Natl Acad Sci USA, 89 (1992) 5547-5551; 25 Gossen M. et al., Science, 286 (1995) 1766-1769). Another system was constructed by fusion of the DNA-binding domain of the S. cerevisiae GAL4 WO 00/78986 3 PCT/FRO0/01744 protein with the ligand-binding domain of the human progesterone receptor and the HSV VP16 transactivator domain; this version is activated by a progesterone analogue such as RU486 (Wang Y. et al., Proc Natl Acad 5 Sci USA, 91 (1994) 8180-8184). A fusion of the drosophila ecdysone receptor with the HSV VP16 transactivator domain has also been described, activated by ecdysone and the analogues of this steroid hormone (No D et al., Proc Natl Acad Sci USA, 93 (1996) 10 3346-3351). Another system takes advantage of the capacity of certain immunosuppresive molecules (cyclosporin A, rapamycin and its derivatives) to promote the combination of certain cellular proteins. A transcriptional regulator then consists of two protein 15 subunits; the first may be formed by the fusion of a chimeric DNA-binding domain and of three copies of the human FKBP protein and the second by the fusion of the rapamycin-binding domain of the human FRAP protein and of the transactivator domain of the human NFkB p65 20 subunit. This transcriptional regulator is activated by rapamycin which allows the dimerization of the two subunits (Rivera V. et al., Nat. Med., 2 (1996) 1028-1032) . Although these systems make it possible to 25 obtain satisfactory levels of regulation in some tissues, they exhibit, nevertheless, certain disadvantages which limit their conditions of use. WO 00/78986 4 PCT/FRO0/01744 Thus, these transcriptional regulators are xenogenic proteins in humans. They indeed consist of protein fragments obtained from bacteria, viruses, yeasts or insects or, when the protein domains are of human 5 origin, their joining creates sequences which are foreign to humans. These protein domains may therefore induce a cytotoxic immune reaction, causing the destruction of the cells which express the gene of interest under the control of the xenogenic 10 transcriptional regulator, and thus the termination of the expression of the transgene. This situation may necessitate the use of repeated administrations of the therapeutic gene, which constitutes a major disadvantage, in particular when this involves a 15 traumatic surgical act, and which is not always effective, in particular when the vector of the therapeutic gene is a virus whose first injection causes an immune reaction. In addition, the levels of expression observed with the prior art systems of 20 regulation are not always satisfactory. The need therefore exists for an improved system of regulating expression, compatible with use in vivo, which can be used in various tissues, and which ensures high levels of expression in the activated 2 5 state. The present invention provides a solution to these problems. WO 00/78986 5 PCT/FR00/01744 The present invention indeed relates to a system of regulation using an activator of human origin. This should make it possible to avoid the repeated administrations of the therapeutic gene. 5 The present invention describes in particular an improved system of inducible expression using the PPAR (Peroxisome proliferator-activated receptors) nuclear receptors as transcriptional regulators. The use of PPRE in a hepatospecific expression system has 10 been described in application WO 98/21349. The improved system according to the invention currently makes it possible to produce the transcriptional regulator (a PPAR protein of human origin, and therefore essentially nonxenogenic in humans) and the inducible promoter, 15 which controls the expression of the transgene, is composed, on the one hand, of a minimum promoter and, on the other hand, of a PPAR response element (PPRE). The system of the invention is activable, in vitro and also in vivo, in particular in the muscle, by the 20 ligands specific for the PPARs. Furthermore, the level of expression of the transgene, obtained after activation, is comparable to that of a strong promoter such as the hCMV-IE promoter. The system according to the present invention 25 therefore exhibits numerous advantages, simultaneously in terms of substantial induction, of tolerance (in WO 00/78986 6 PCT/FR00/01744 particular for an in vivo use), of strength and of conditions of use. The invention therefore describes new constructs for the preparation and the use of this 5 system, in particular promoter regions, expression cassettes and plasmids. The invention also describes new PPAR constructs allowing an improved control of the expression of genes, as well as combinations of these different constructs. The invention shows, in addition, 10 that these methods and compositions allow substantial control and regulation of the expression in vitro and in vivo. The invention also relates to cells comprising constructs of the invention, as well as methods for screening compounds which are ligands for PPARs, for 15 example. More particularly, a first subject of the invention consists in a composition comprising: (a) a first element comprising a nucleic acid of interest under the control of an inducible promoter 20 comprising a PPAR response element and a minimal transcriptional promoter, and (b) a second element comprising a nucleic acid encoding a PPAR under the control of a transcriptional promoter, 25 for their use simultaneously, separately or sequentially. ii'siTc*5 ACTUAL F~0?c?,"fY OFFICE OF N.Z. ;WO 00/78986 ;7 ;PCT/FRO0/01744 ;In a more specific mode, the compositions of the invention comprise in addition: ;(c) a ligand for PPAR, ;also for a use simultaneously, separately or sequentially. ;5 ;Advantageously, they comprise, in addition, an element (d) comprising a nucleic acid encoding a retinoid X recepetor (RXR) under the control of a transcriptional promoter. ;10 The expression for a use simultaneously, ;separately or spaced out over time indicates that the elements (a), (b) and, where appropriate, (c) and/or (d) may be prepared separately, packaged separately, ;and used sequentially, to allow control of the 15 expression of the nucleic acid of interest. Typically, the elements (a) and (b), and optionally (d) -are prepared and packaged together, whereas the compound (c) is packaged separately and used spaced out over time with (a) and (b), and optionally (d), the 20 combination of these different elements in a cell, a tissue, an organ and the like leading to the desired effect of regulation of expression. ;In this regard, in a specific embodiment of a r ;composition of the invention, the elements (a), (b) and 25 optionally (d) are carried by distinct genetic constructs. ;INTELLECTUAL PXO'rHRTY OFFICE OF N.Z. ;- 3 DEC 2003 ;RECEIVED. ;WO 00/78986 ;8 ;PCT/FRO0/01744 ;In another specific and preferred embodiment of a composition of the invention, the elements (a), (b) and optionally (d) are assembled in the same genetic construct. The present invention thus describes 5 complex genetic constructs allowing the expression of a product of interest and of a PPAR. These constructs are particularly advantageous since they contain, on their own, all the genetic elements necessary for the regulated expression of the nucleic acid of interest. 10 The genetic construct(s) may be of a varied, ;in particular plasmid, episomal, chromosomal, viral or phage, nature and/or origin, and the like. Preferably, the genetic construct is a plasmid or viral vector. ;By way of illustration of plasmids separately 15 carrying the elements (a) or (b), there may be mentioned for example the plasmids JxnS-TK-pGL3, JxnAS-TK-PGL3, DRlxnS-TK-pGL3, DRlxnAS-TK-pGL3, JxnAS-CMV-pGL3, pSG5-hPPARg2g2 or JxlOAS-CMV-EF-pGL3, which will be described in detail later. ;20 By way of illustration of plasmids in which the elements (a) and (b) have been assembled, there may be mentioned for example the plasmids Jx5AS-TK-Luc-hPPARg2, SV-g2-J10-C-pGL3, hPPARg2-CMV-Jx5AS-TK-pGL3 or hPPARg2-CMV-JxlOAS-CMV-pGL3, which will be described in 25 detail later. ;As an example of a viral vector, there may be mentioned in particular a recombinant adenovirus, a ;WO 00/78986 ;9 ;PCT/FR00/01744 ;recombinant retrovirus, an AAV, a herpesvirus, a vaccinia virus and the like, whose preparation may be carried out according to methods known to persons skilled in the art. ;5 The arrangement and the structure of the genetic constructs will be described in greater detail in the text which follows. ;In this regard, as indicated above, the element (a) comprises an inducible promoter comprising 10 at least: ;- a PPAR response element, and ;- a minimal transcriptional promoter. ;A PPAR response element (PPRE, "Peroxysome Proliferator Response Element") is a nucleic acid 15 region capable of binding a PPAR, it being possible for the binding of the PPAR to then mediate a signal around neighbouring nucleic regions. A PPAR response element is therefore a nucleic acid region capable of binding PPARs. For carrying out the invention, the PPAR 2 0 response element comprises more particularly one or more PPAR-binding sites. Such binding sites have been described in the prior art, such as for example, in different human promoters (gene for apolipoprotein All, for example). Such sites may also be artificially 25 constructed, and tested for their PPRE properties, as is described below. ;WO 00/78986 PCT/FR00/01744 ;10 ;In a specific embodiment, the PPAR response element comprises one or more sites having the sequence TCAACCTTTACCCTGGTAG (SEQ ID NO:l) or functional variants of this sequence. The sequence SEQ ID N0:1 5 corresponds to the J region of the human apoAII promoter (nucleotides -734 to -716). ;In another specific embodiment, the PPAR response element comprises one or more sites having the sequence AGGTCAAAGGTCA (SEQ ID NO:5) or functional 10 variants of this sequence. The sequence SEQ ID NO:5 corresponds to the consensus region DRl. ;The term functional variant designates any-modified sequence conserving the properties of PPRE as mentioned above, that is to say in particular the 15 capacity to bind a PPAR. The modifications may comprise one or more additions, mutations, deletions and/or substitutions of nucleotides in the sequence considered. These modifications may be introduced by conventional molecular biology methods, such as in 20 particular site-directed mutagenesis or, more practically, by artifical synthesis of the sequence in a synthesizer. Generally, the variants conserve at least 50% of the residues of the initial sequence indicated. More preferably, the variants possess 25 modifications affecting less than 5 nucleotides in the sequence considered. The variants thus obtained are ;WO 00/78986 ;11 ;PCT/FR00/01744 ;then tested for their PPRE activity. This property may be checked in various ways, and in particular: ;(i) by bringing the test sequence into contact with a PPAR and a retinoid X receptor (RXR), ;5 preferably in an acellular test, and detecting the formation of a complex (for example by gel migration retardation); ;(ii) by inserting the test sequence into an expression cassette comprising a minimal promoter and a ;10 reporter gene, introducing the cassette into a cell, and detecting (where appropriate assaying) the expression of the reporter gene in the presence and in the absence of a PPAR and of a ligand for a PPAR; ;(iii) by any other technique known to a ;15 person skilled in the art, making it possible to detect the interaction between a nucleic acid and a protein, for example. ;A variant is considered to be functional for the purpose of the present invention when the activity 2 0 measured, for example in (ii) above, is preferably at least equal to 50% of that measured with a site having the sequence SEQ ID NO:l or 5, more preferably at least equal to 75%. Functional variants of PPAR-binding sites for the purpose of the invention are described, for 25 example, in Juge-Aubry et al., (J. Biol. Chem. 272 (1997) 25252) and in Nakshatri et al. (NAR 26 (1998) ;WO 00/78986 ;12 ;PCT/FRO0/01744 ;2491), which are incorporated into the present application by way of reference. ;The retinoid X receptors (RXR) are encoded by three genes RXRa, RXR(3 and RXRy, whose isolation and 5 sequence have been described (Mangelsdorf DJ et al. (1990), Nature 345, 224-229; Mangelsdorf DJ et al. (1992), Genes Dev 6, 329-344). Preferably, the element (d) encodes the human RXRa. ;As regards the PPAR/RXR heterodimerization, 10 two reviews may be consulted: Mangelsdorf DJ and Evans RM (1995), Cell 83, 841-850 and Wilson TM and Wahli W (1997), Current Opinion in Chemical Biology 1, 235-241. The article by Schulman IG et al. (1989), Molecular and Cellular Biology 18, 3483-3494 describes 15 transactivation by the PPARy/RXRa heterodimer. ;The use of the element (d) is capable of synergizing the activity of the element (b). ;As indicated above, in the compositions according to the invention, the PPAR response element 20 may comprise several sites for binding to a PPAR. This may be a repetition of the same site, or combinations of different sites, the repetition of identical sites being preferred. More particularly, the response element comprises up to 30 binding sites, preferably 2 5 from 3 to 20, more preferably from 5 to 15. A preferred embodiment of the invention is a construct comprising from 10 to 15 binding sites, the results presented in ;WO 00/78986 ;13 ;PCT/FROO/01744 ;the examples indeed show the advantageous properties of such constructs in terms of induction and of level of expression, in particular in the muscle cells. ;For the preparation of an inducible promoter 5 according to element (a) of the compositions of the invention, the PPAR response element is combined with a transcriptional minimal promoter. The minimal promoter is a transcriptional promoter having a basal activity which is low or inexistant, and capable of being 10 increased in the presence of a transcriptional activator (interaction of a PPAR activated with the PPRE element). A minimal promoter may therefore be a promoter which is naturally weak in mammalian cells, that is to say which produces a nontoxic and/or an 15 insufficient expression in order to obtain a pronounced biological effect. Advantageously, a minimal promoter is a construct prepared from a native promoter, by deletion of a region or regions not essential for the transcriptional activity. Thus, this is preferably a 20 promoter comprising essentially a TATA box, generally of less than 160 nucleotides in size, centred around the codon for initiation of transcription. A minimal promoter can thus be prepared from strong or weak viral or cellular promoters such as for example the promoter 25 of the herpesvirus thymidine kinase (TK) gene, the CMV immediate promoter, the PGK promoter, the promoter of the muscle creatine kinase (MCK) gene, the promoters of ;WO 00/78986 ;14 ;PCT/FROO/01744 ;the genes for the various skeletal muscle actin isoforms, the promoter of the desmin gene, the promoter of the vimentin gene, the promoters of the myosin light chain or heavy chain genes, and the like. Specific 5 examples of minimum promoters are represented by nucleotides -54 to +48 of the CMV or -105 to +56 of the TK promoter, for example. It is understood that any variant of these promoters or similar constructs from other promoters may be constructed by persons skilled 10 in the art and used within the framework of the present invention. ;The minimal promoter (Pmin), the PPAR response element (PPRE) and the nucleic acid of interest (NA) are arranged functionally in the element 15 (a), that is to say such that the minimal promoter controls the expression of the nucleic acid of interest and that its activity is regulated by the PPRE element. Generally, these regions are therefore arranged in the following order, in the 5'—>3' orientation: PPRE-Pmin-20 NA. However, any other functional arrangement can be envisaged by persons skilled in the art without departing from the present invention. ;In addition, the various functional domains above may be directly linked to each other, or 25 separated by nucleotides which do not significantly affect the regulated character of the promoter of the element (a). Such nucleotides may be neutral residues ;WO 00/78986 ;15 ;PCT/FROO/01744 ;from the functional point of view, resulting, for example, from cloning steps (PCR ends, restriction sites, and the like). These nucleotides may also possess biological properties which make it possible to 5 confer improved characteristics or performances to the system of the invention (enhancer of housekeeping genes, tissue specific enhancer, silencer, intron, splicing site, and the l.ike>. In this regard, in a specific embodiment of the invention, the inducible 10 promoter comprises, in addition, an enhancer region. Such a region advantageously makes it possible to increase the levels of expression of the nucleic acid of interest. Such an enhancer (E) region is preferably positioned in 3' of the minimal promoter, between the 15 latter and the nucleic acid of interest, according to the following scheme (5'—>3')*- PPRE-Pmin-E-NA. Moreover, in the constructs of the invention, the minimal promoter and the PPAR response element may be present either in the same orientation (that is to 20 say in the direction of transcription), or in the opposite orientation (that is to say that the PPAR response element is in the antisense orientation relative to transcription by the Pmin promoter). As illustrated in the examples, these two embodiments 25 allow an effective control of the regulation of the expression in vitro and in vivo. WO 00/78986 16 PCT/FR00/01744 As indicated above, the element (b) of the compositions according to the invention comprises at least: - a nucleic acid encoding a PPAR, 5 - under the control of a second transcriptional promoter. The PPARs belong to the superfamily of nuclear hormone receptors, and are grouped into three distinct groups, PPARa, PPAR8 (also called NUC-1 or 10 PPAR(3) and PPARy. The isolation and the sequence of many human PPARs have been described in the literature (see in particular Sher T. et al., Biochemistry, 32 (1993) 5598-5604; Mukherjee R. et al., J. Steroid Biochem. Molec. Biol., 51 (1994) 157-166; Fajas L. et al., J. 15 Biol. Chem., 272 (1997) 18779-18789; Mukherjee R. et al., J. Biol. Chem., 272 (1997) 8071-8076; Schmidt A. et al., Mol. Endocrinol. 6 (1992) 1634-1641). The PPARy promoter has, in addition, been recently cloned, as described by application WO 99/05161. 2 0 In a preferred embodiment of the invention, the nucleic acid encoding a PPAR encodes a human PPAR, in particular a PPARa or a PPARy. The results presented in the examples indeed show that the use of these molecules ensures for the system of the invention high 25 levels of regulation and expression, in particular in the muscle cells. WO 00/78986 17 PCT/FROO/01744 According to a first embodiment, this is a PPARa or a PPARy in its native form, that is to say without modification of primary structure relative to the natural molecule. 5 According to another embodiment, this is a modified PPAR comprising several ligand-binding sites. In this regard, the present invention describes and also has as subject any modified PPAR comprising several ligand-binding sites. More 10 particularly, this is a PPARa or a PPARy, still more preferably a PPARy. Preferably, the modified PPARs according to the invention comprise from 2 to 5 ligand-binding sites, more preferably from 2 to 4 binding sites. This is more particularly PPAR containing 2 to 5 15 copies of the E and F domains involved in the binding to the ligand. The PPAR proteins contain different domains: the N-terminal A/B domain which contains a transactivating region not dependent on the ligand, the C domain which is the DNA-binding domain (DBD) and the 20 D domain which is a hinge region, and the E/F domains which contain a transactivating region dependent on the ligand. The E/F domains are also called ligand-binding domain (LBD) (see in particular Schoonjans K. et al., Biochim. Biophys. Acta, 1302 (1996) 93-109) . The limits 25 of the E/F domains vary from one PPAR to another. By way of example, for the human PPARy2 isoform used, the E/F domain extends from amino acid 284 to amino acid WO 00/78986 18 PCT/FR00/01744 505. The present invention now shows that it is possible to construct modified PPARs comprising several repeated E and F domains, and that these modified PPARs are functional and possess improved properties of inducibility by the ligands for the PPARs. Such constructs therefore represent an embodiment and a specific subject of the present invention. A typical example of modified PPAR according to the invention is a PFARy containing 2 ligand-binding sites (that is to say two E and F domains). The complete protein sequence of PPARy2y2 is represented on the sequence SEQ ID NO:24. SEQ ID NO:24 mgetlgdspidpesdsftdtlsanisqemtmvdtempfwptnfgissvdlsvmedhshsfdi kpfttvdfssistphyedipftrtdpwadykydlklqeyqsaikvepasppyysektqlyn kpheepsnslmaiecrvcgdkasgfhygvhacegckgffrrtirlkliydrcdlncrihkks rnkcqycrfqkclavgmshnairfgrmpqaekekllaeissdidqlnpesadlralakhlyd syiksfpltkakarailtgkttdkspfviydmnslmmgedkxkfkhitplqeqskevairif qgcqfrsveavqeiteyaksipgfvnldlndqvtllkygvheiiytmlaslmnkdgvliseg qgfmtreflkslrkpfgdfmepkfefavkfnalelddsdlaifiaviilsgdrpgllnvkpi ediqdnllqalelqlklnhpessqlfakllqkmtdlrqivtehvqllqvikktetdmslhpl lqeiykdlyawailtgkttdkspfviydmnslmmgedkikfkhitplqeqskevairifqgc qfrsveavqeiteyaksipgfvnldlndqvtllkygvheiiytmlaslmnkdgvlisegqgf mtreflkslrkpfgdfmepkfefavkfnalelddsdlaifiaviilsgdrpgllnvkpiedi qdnllqalelqlklnhpessqlfakllqkmtdlrqivtehvqllqvikktetdmslhpllqe iykdly The invention also relates to any variant of the sequence SEQ ID NO:24 conserving a PPAR-type activity (the capacity to activate, in the presence of a PPAR ligand such as BRL49653, a promoter containing a PPRE sequence). The variants are understood to mean any mutant, deletant and/or polypeptide containing one or more additional residues. Preferably a variant WO 00/78986 19 PCT/FROO/01744 conserving at least 80% of the residues of the sequence ID NO:24. In addition, the invention also relates to any nucleic acid encoding such a modified PPAR. This 5 may be a DNA (in particular a cDNA or a synthetic or semisynthetic DNA) or an RNA. This DNA may be constructed according to conventional molecular biology methods known to persons skilled in the art (synthesis, ligations, screening of libraries and the like). It is 10 advantageously any nucleic acid comprising a sequence encoding a polypeptide having the sequence SEQ ID NO:24, or hybridizing with a sequence encoding a polypeptide of SEQ ID NO-.24, and encoding a polypeptide with a PPAR-type activity. In addition, this DNA may 15 comprise a transcriptional promoter and/or terminator, for example. The second transcriptional promoter, controlling the expression of the nucleic acid encoding the PPAR, may be any strong or weak, ubiquitous or 20 selective, constitutive or regulated promoter which is functional in mammalian cells, in particular in human cells. This may be a domestic cellular promoter (i.e., a mammalian, in particular a human, gene), a natural or synthetic, simple or complex, viral, bacterial, insect 25 or plant promoter, and the like. Examples of appropriate promoters for this element (b) are in particular viral promoters (SV40 virus immediate WO 00/78986 20 PCT/FR00/01744 promoter, CMV virus immediate promoter, retrovirus LTR, herpesvirus TK promoter) or cellular promoters (PGK, albumin or EFla promoter, or promoter of genes which are highly expressed in the muscle such as: promoter of 5 the muscle creatine kinase (MCK) gene, promoters of the genes of the various skeletal muscle actin isoforms, promoter of the desmin gene, promoter of the vimentin gene, promoters of the myosin light chain or heavy chain genes). Moreover, the promoter may be modified by 10 introduction of one or more enhancer regions, such as the enhancer region of intron 2 of the beta-globin gene, enhancer of the CMV virus very early gene, EFla enhancer, silencer region(s), regions conferring a tissue specificity (for example regions isolated from 15 tissue-specific promoters such as: promoter of the muscle creatine kinase (MCK) gene, promoters of the genes of the various skeletal muscle actin isoforms, promoter of the desmin gene, promoter of the vimentin gene, promoters of the myosin light chain or heavy 20 chain genes) or a regulable character, or by deletion of regions not essential to the activity, for example. Such promoters may be used to express the RXR, contained in the element (d). Preferred examples of a second promoter are 25 the viral promoters, in particular the SV40 virus early promoter and the CMV immediate promoter, or derivatives thereof. WO 00/78986 21 PCT/FROO/01744 Moreover, in a specific embodiment, when the elements (a) and (b), and optionally (d), are assembled in the same genetic construct, the second transcriptional promoter (of the element (b)) and the 5 inducible promoter of the element (a), and optionally the promoter of the element (d), may be grouped so as to form only one common, in particular bidirectional, promoter region, as will be explained in detail in the text which follows. 10 To this effect, another subject of the present invention consists in a vector comprising an element (a) and an element (b), and optionally an element (d), as defined above. According to a first variant of the 15 invention, the elements (a) and (b), and optionally (d) , are in the same orientation in the vector. Such a variant is illustrated for example by the plasmid SV-g2-J10-C-pGL3 (Figure 17). According to another variant of the 20 invention, the elements (a) and (b), and optionally (d), are in the opposite orientation in the vector. Such a variant is illustrated for example by the plasmids represented in Figures 16, 18 and 19. More preferably, in this variant embodiment, the inducible 25 promoter of the element (a) and the transcriptional promoter of the element (b) are assembled in the vector to form a regulable bidirectional promoter. Such an WO 00/78986 22 PCT/FROO/01744 embodiment is illustrated for example by the plasmids represented in Figures 18 and 19. In this regard, a specific subject of the invention consists in a vector characterized in that it 5 comprises, in the 5'—>3' direction, a first nucleic acid encoding a PPAR, a first minimal transcriptional promoter controlling the expression of the said first nucleic acid, one or more PPAR response element(s), a second minimal transcriptional promoter and, under the 10 control of the said second minimal transcriptional promoter, a second nucleic acid encoding a product of interest. This type of construct is advantageous since it allows the co-expression of the two nucleic acids in 15 the same plasmid, and the amplification of this expression by the regulation of the two nucleic acids by the PPARs and their ligands. The expression of the nucleic acid of interest in the compositions of the invention is 20 generally activated in the presence of a PPAR ligand (element (c)). In this regard, according to the PPAR used, various types of ligands, natural or synthetic, may be used. Thus, the PPARa-activating ligands are for 2 5 example the fibrates such as fibric acid and its analogues. As analogues of fibric acid, there may be mentioned in particular gemfibrozyl (Atherosclerosis WO 00/78986 23 PCT/FROO/01744 114(1) (1995) 61), bezafibrate (Hepatology 21 (1995) 1025), ciprofibrate (BCE&M 9(4) (1995) 825), clofibrate (Drug Safety 11 (1994) 301), fenofibrate (Fenofibrate Monograph, Oxford Clinical Communications, 1995), 5 clinofibrate (Kidney International. 44(6) (1993) 1352), pirinixic acid (Wy-14,643) or 5,8,11,14-eicosatetranoic acid (ETYA). These various compounds are compatible with a biological and/or pharmacological use in vitro or in vivo. 10 The PPARy-activating ligands may be chosen from natural and synthetic ligands. As natural ligands, there may be mentioned fatty acids and eicosanoids (for example linoleic acid, linolenic acid, 9-HODE, 5-HODE) and as synthetic ligands, there may be mentioned 15 thiazolidinediones, such as in particular rosiglitazone (BRL49653), pioglitazone or troglitazone (see for example Krey G. et al., Mol. Endocrinol., 11 (1997) 779-791 or Kliewer S. and Willson T., Curr. Opin. in Gen. Dev. 8 (1998) 576-581) or the compound RG12525. 20 Moreover, the compositions according to the invention may contain several PPAR activators in combination, and in particular a fibrate or a fibrate analogue combined with a retinoid. The subject of the invention is also the use 25 of a composition or of a vector as defined above for expressing a nucleic acid of interest in a cell ex vivo or in vitro. WO 00/78986 PCT/FR00/01744 24 In this regard, the nucleic acid may be any nucleic acid (DNA, RNA) encoding a product of interest (RNA, protein, polypeptide, peptide and the like). It may be a product of interest in the food, therapeutic 5 or vaccine sector, a marker, and the like. The invention also relates to the use of a composition or of a vector as defined above for the preparation of a product^ intended for expressing a nucleic acid of interest in a cell in vivo. 10 The subject of the invention is also a method for the regulated expression of a nucleic acid in a cell, comprising bringing the said cell into contact with a composition or a vector as defined above. For a use in vitro or ex vivo, the cells may 15 be brought into contact with the compositions or vectors of the invention according to various protocols. Thus, the cells in culture may be incubated directly with the elements (a), (b) and (c), and optionally (d), of the invention, for example with a 20 vector containing the elements (a) and (b) and in the presence of the ligand (c). Alternatively, the cells may be incubated in a first instance with the elements (a) and (b) and optionally (d) (in particular assembled in the same vector) and then, in a second instance 25 (after culture and optionally selection of the modified cells), the element (c) may be added. This latter type of protocol makes it possible, for example, to uncouple WO 00/78986 25 PCT/FR00/01744 the culture phase (or the cell expansion phase) from the nucleic acid expression phase. These experiments may be carried out in any appropriate medium and device, preferably in a plate, dish, flask, in the 5 sterile condition. The quantities of cells, vector and ligand can be easily adapted by persons skilled in the art, on the basis of the information provided in the examples and of their general knowledge. For a use in vivo, the cells (or organs, 10 tissue, and the like) are brought into contact, by administration of the elements (a), (b) and (c), and optionally (d), in vivo, simultaneously, separately or spaced out over time. To this effect, the elements (a) and (b), and optionally (d), optionally in the form of 15 a single genetic construct, are generally administered by the parenteral, in particular intramuscular, intravenous, subcutaneous, intradermal, intratumoral or stereotaxic route. The choice of the mode of adminstration may be guided by the application 2 0 envisaged, the tissue targeted and/or the type of product of interest encoded by the transgene. For this administration, the compositions of the invention may comprise any agent promoting cellular transfection (cationic polymer, lipid and the like). In a specific 25 mode, the compositions are administered by the intramuscular route, and the genetic constructs are WO 00/78986 26 PCT/FROO/01744 used in the form of a "naked" nucleic acid, that is to say without added transfection agent. Likewise, when the elements (a) and (b), and optionally (d), are introduced by means of viral 5 vectors, no additional transfection agent is necessary. As illustrated in the examples, the ligand (c) may be administered before, simultaneously or after the elements (a) and (b), and optionally (d). In this regard, the administration of the 10 ligand may be carried out by the oral, anal, intravenous, intraperitoneal or intramuscular route, for example. The doses used may be adapted by persons skilled in the art, on the basis of the in vivo data 15 published in the literature. Thus for example, for a form not soluble in water, typical doses of ligand such as BRL49653 are between 5 and 50 mg/kg, for example 30 mg/kg, which make it possible to obtain a plasma concentration close to about 15 |iig/ml at least. For a 20 water-soluble form of ligand, whose bioavailability is greater (for example a maleate salt of BRL49653), the typical doses are lower, generally less than 5 mg/kg, for example from 0.01 to 1 mg/kg. These doses can be quite obviously adapted by persons skilled in the art 25 as a function of the constructs used, the ligands used, and the desired applications and effects. In general, the results presented in the examples advantageously WO 00/78986 PCT/FROO/01744 27 show that the compositions of the invention make it possible to obtain in vivo a high and regulated expression, at ligand doses less than those normally-used. In addition, although repeated administrations of 5 ligand may be carried out, the results presented also show that the expression is high after a single dose of ligand. Generally, the vector doses used may vary between 0.01 and 1000 jltg, or more, depending on the 10 desired applications. The invention may be used for expressing a gene in various types of cells, tissues or organs, in vitro, ex vivo or in vivo. In particular, this may be a mammalian, preferably a human, cell, tissue or organ. 15 By way of illustration, there may be mentioned muscle cells (or a muscle), hepatic cells (or the liver), cardiac cells (or the heart, the arterial or vascular wall), nerve cells (or the brain, the marrow and the like) or tumour cells (or a tumour). 20 Preferably, the constructs, compositions and method of the invention are used for the regulated expression of a nucleic acid in a muscle cell (or a muscle) in vitro, ex vivo or in vivo. The results presented in the examples illustrate more particularly 25 the advantages of the invention in vivo or in vitro in this type of cells. WO 00/78986 28 PCT/FR00/01744 The invention also relates to any cell modified by bringing into contact with a composition or a vector as defined above. The invention also relates to the use of a 5 composition, of a vector or of a cell as defined above, in which the nucleic acid of interest is a reporter gene (such as for example secreted alkaline phosphatase or luciferase) for the screening in vitro, ex vivo or in vivo (in particular in the muscle cells or a muscle) 10 for PPAR ligands. In this regard, the invention also describes a method for identifying PPAR ligands comprising the bringing into contact of a cell as defined above with a test molecule (or composition), and the detection of the expression of the nucleic acid 15 of interest (the latter being preferably a reporter gene). The expression may, in addition, be compared with that observed in the absence of test compound or in the presence of a reference ligand, in order to evaluate the activity of the compound tested. 20 The invention also relates to the use of a composition or of a vector as defined above, for the construction of transgenic animals, in particular of nonhuman mammals, useful for preclinical studies, or for studies of bioavailability, or labelling, and the 25 like. The present invention will be described in greater detail with the aid of the examples which WO 00/78986 29 PCT/FROO/01744 follow and which should be considered as illustrative and non1imi t ing. LEGEND TO THE FIGURES 5 Figure 1 : Schematic representation of the plasmid FTKpGL3. Figure 2 : Schematic representation of the plasmid 10 Jx3S-TK-pGL3. Figure 3 : Schematic representation of the plasmid Jx3AS-TK-pGL3. 15 Figure 4 s Schematic representation of the plasmid DRlx3S-TK-pGL3. Figure 5 : Schematic representation of the plasmid DRlx3AS-TK-pGL3. 20 Figure 6 : Activities of the inducible promoters evaluated in transient transfections in vitro in mouse myoblasts (C2C12). The cells are cotransfected with : (i) 10 ng of plasmid FTKpGL3 (a), or Jx3S-TK-pGL3 (b), 25 or Jx3AS-TK-pGL3 (c), or DRlx3S-TK-pGL3 (d) , or DRlx3AS-TK-pGL3 (e), (ii) increasing quantities of plasmid pSG5-hPPARg2, and (iii) 20 ng of plasmid pRL- WO 00/78986 30 PCT/FROO/01744 null. The activity of each inducible promoter represents the luciferase activity of Photinus pyralis normalized using the activity of Renilla reniformis luciferase. 5 Figure 7 : Activities of the inducible promoters evaluated in transient transfections in vitro in mouse myoblasts (C2C12). The cells are cotransfected with : (i) 10 ng of plasmid FTKpGL3 (a), or Jx3S-TK-pGL3 (b), 10 or Jx3AS-TK-pGL3 (c), or DRlx3S-TK-pGL3 (d), or DRlx3AS-TK-pGL3 (e), (ii) increasing quantities of plasmid pSG5-hPPARa(Koz), and (iii) 20 ng of plasmid pRL-null. The activity of each inducible promoter represents the luciferase activity of Photinus pyralis 15 normalized using the activity of Renilla reniformis luciferase. Figure 8 : Schematic representation of the plasmid Jx5AS-CMV-pGL3. 20 Figure 9 : Activities of the inducible promoters evaluated in transient transfections in vitro in mouse myoblasts (C2C12). The cells are cotransfected with : (i) 10 ng of plasmid Jx5AS-TK-pGL3 (a), or Jx5AS-CMV-25 pGL3 (b), (ii) increasing quantities of plasmid pSG5-hPPARg2, and (iii) 20 ng of plasmid pRL-null. The activity of each inducible promoter represents the: WO 00/78986 31 PCT/FROO/01744 luciferase activity of Photinus pyralis normalized using the activity of Renilla reniformis luciferase. Figure 10 : Activities of the inducible promoters 5 evaluated in transient transfections in vitro in mouse myoblasts (C2C12). The cells are cotransfected with : (i) 10 ng of plasmid JxnAS-TK-pGL3, (ii) 10 ng of plasmid pSG5-hPPARg2, and (iii) 20 ng of plasmid pRL-null. The activity of each inducible promoter 10 represents the luciferase activity of Photinus pyralis normalized using the activity of Renilla reniformis luciferase. Figure 11 : Activities of the inducible promoters 15 evaluated in transient transfections in vitro in mouse myoblasts (C2C12). The cells are cotransfected with : (i) 10 ng of plasmid JxnAS-CMV-pGL3, (ii) 10 ng (a) or 50 ng (b) of plasmid pSG5-hPPARg2, and (iii) 20 ng of plasmid pRL-null. The activity of each inducible 20 promoter represents the luciferase activity of Photinus pyralis normalized using the activity of Renilla reniformis luciferase. 25 Figure 12 : Schematic representation of the plasmid pSG5-hPPARg2g2. WO 00/78986 32 PCT/FROO/01744 Figure 13 : Comparison of the transcriptional regulators hPPARg2 and hPPARg2g2. Mouse myoblasts (C2C12) are contransfected with: (i) 10 ng of plasmid JxlOAS-CMV-pGL3, (ii) increasing quantities of plasmid 5 pSG5-hPPARg2 (a) or pSG5-hPPARg2g2 (b), and (iii) 20 ng of plasmid pRL-null. The activity of the inducible promoter represents the luciferase activity of Photinus pyralis normalized using the activity of Renilla reniformis luciferase. (c) : factors for induction by 10 BRL49653 obtained with the plasmid pSG5-hPPARg2 or the plasmid pSG5-hPPARg2g2. This induction factor is calculated by dividing the activity in the presence of BRL49653 by the activity in the presence of DMSO. 15 Figure 14 Schematic representation of the plasmid JxlOAS-CMV-EF-pGL3. Figure 15 : Activities of the inducible promoters evaluated in transient transfections in vitro in mouse 20 myoblasts (C2C12). The cells are cotransfected with : (i) 10 ng of plasmid JxlOAS-CMV-pGL3 (a), or JxlOAS-CMV-EF-pGL3 (b), (ii) increasing quantities of plasmid pSG5-hPPARg2g2, and (iii) 20 ng of plasmid pRL-null. The activity of each inducible promoter represents the 25 luciferase activity of Photinus pyralis normalized using the activity of Renilla reniformis luciferase. WO 00/78986 33 PCT/FROO/01744 Figure 16 : Schematic representation of the plasmid Jx5AS-TK-luc-hPPARg2. Figure 17 : Schematic representation of the plasmid SV-5 g2-JlO-C-pGL3. Figure 18 : Schematic representation of the plasmid hPPARg2-CMV-Jx5AS-TK-pGL3. 10 Figure 19 : Schematic representation of the plasmid hPPARg2-CMV-JxlOAS-CMV-pGL3. Figure 20 : Comparison of the different versions of the system inducible in vitro. Mouse myoblasts (C2C12) are 15 transfected with, for each version of the system, the same number of moles of inducible expression cassettes. The results are expressed as a percentage of the activity of the hCMV-IE promoter obtained using the plasmid pCMV-leadTK. The factors for induction with 20 BRL49653 are calculated by dividing the activity in the presence of BRL49653 with the activity in the presence of DMSO. 1 = pSG5-hPPARg2 + Jx5AS-TK-pGL3; 2 = Jx5AS-TK-luc-hPPARg2; 3 = pSG5-hPPARg2g2 + Jxl0AS-CMV-pGL3; 4 = pSG5-hPPARg2 + JxlOAS-CMV-pGL3; 5 = SV-g2-J10-C-pGL3; 25 6 = hPPARg2-CMV-Jx5AS-TK-pGL3; 7 = hPPARg2-CMV-JxlOAS-CMV-pGL3; 8 = hPPARg2-CMV-Jxl5AS-CMV-pGL3; 9 = hPPARg2-CMV-Jx20AS-CMV-pGL3. WO 00/78986 34 PCT/FR00/01744 Figure 21 : Comparison in vitro of the ligands BRL49653 and RG12525. Mouse myoblasts (C2C12) are transfected with: (i) 10 ng of plasmid hPPARg2-CMV-JxlOAS-CMV-pGL3 5 whose expression cassette is presented in (a) and (ii) 10 ng of plasmid pRL-null. (b) The activity of the inducible promoter represents the luciferase activity of Photinus pyralis normalized using the activity of Renilla reniformis luciferase. 10 Figure 22 : Comparison of the different versions of the system inducible in vivo. C57BI/6 mice (6 mice per group) are injected bilaterally, in their cranial tibial, with, for each version of the system, the same 15 number of moles of inducible expression cassettes. An electrotransfer is then applied on each muscle. The treated animals receive each day, by force-feeding, 30 mg/kg of BRL49653. Four days after the injection of DNA, the animals are sacrificed and the muscles are 2 0 removed in order to measure the luciferase activity. 1 = pCMV-1eadTK; 2 = pSG5-hPPARg2 + JxlOAS-CMV-pGL3; 3 = pSG5-hPPARg2g2 + JxlOAS-CMV-pGL3; 4 = hPPARg2-CMV-JxlOAS-CMV-pGL3. 25 Figure 23 : Comparison, in vivo, of various protocols for induction with BRL49653. C57BI/6 mice (6 mice per group) are injected bilaterally, in the cranial tibial, WO 00/78986 35 PCT/FROO/01744 with 10 |Xg of DNA containing 1 jig of plasmid hPPARg2-CMV-JxlOAS-CMV-pGL3 whose expression cassette is presented in (a). The activities obtained with the various induction protocols are assembled in the panel 5 (b) . Figure 24 : Schematic representation of the plasmid pRDA02. 10 Figure 25 : Kinetics of induction obtained in vivo with the inducible system. (A) Ten C57BI/6 mice are injected bilaterally, in the cranial tibial, with a DNA mixture containing 3 mg of plasmid pRDA02 and 3 mg of plasmid pSG5-hPPARg2. An electrotransfer is then applied on 15 each muscle. Four days, and then 39 days after the injection of DNA, the animals are treated, by force-feeding, with 30 mg/kg of BRL49653. At various times, blood samples are collected over heparin and the enzymatic activity of the secreted alkaline phosphatase 20 (hSeAP) is measured in the plasma, using the Phospha-Light™ kit (Tropix, PE Biosystems, Foster City, CA). (B) C57BI/6 mice (2 groups of 10 mice) are injected bilaterally, in the cranial tibial, with a DNA mixture containing 3 mg of plasmid pRDA02 and 3 mg of plasmid 25 pSG5-hPPARg2. An electrotransfer is then applied on each muscle. Four days after the injection of DNA, the animals receive, by force-feeding, either a single dose WO 00/78986 36 PCT/FROO/01744 of BRL49653 (30 mg/kg), or one dose per day (30 mg/kg) for 5 days. At various times, blood samples are collected over heparin, and the enzymatic activity of hSeAP is measured in the plasma, using the "Phospha-Light" kit (Tropix). The results presented (induction factors) correspond to the ratio between the hSeAP activity measured on the day of interest and that obtained on D4. Figure 26: Comparison, in vivo, of different PPARg ligands, and study of the dose effect of one of them. C57BI/6 mice (5 mice per group) are injected bilaterally, in the cranial tibial, with a DNA mixture containing 5 mg of plasmid pRDA02 and 5 mg of plasmid pSG5-hPPARg2. An electrotransfer is then applied on each muscle. Six days (A) or 10 days (B) after the injection of DNA, the animals are treated, by force-feeding, either with different PPARg ligands (A; BRL49653, Actos™ (Takeda Pharmaceuticals) and Avandia™ (SmithKline Beecham)), or with various doses of BRL49653 (B). At various times, blood samples are collected over heparin, and the enzymatic activity of hSeAP is measured in the plasma, using the "Phospha-Light" kit (Tropix). The results presented (induction factors) correspond to the ratio between the hSeAP activity measured on the day of interest and that obtained on D6 (A) or D10 (B). WO 00/78986 37 PCT/FROO/01744 Figure 27: Schematic representation of the plasmid Jxl OAS - CMV-VEGFa1 6 5 . MATERIALS AND METHODS 5 The methods conventionally used in molecular biology, such as preparative extractions of plasmid DNA, caesium chloride gradient centrifugation of plasmid DNA, electrophoresis on agarose gels, 10 purification of DNA fragments by electroelution, precipitation of plasmid DNA in saline medium with ethanol or isopropanol, transformation in Escherichia coli are well known to persons skilled in the art and are abundantly described in the literature (Sambrook et 15 al., "Molecular Cloning, a Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989) . The plasmid pGL3-Basic, used for the clonings of the various promoter regions, as well as the plasmid 20 pRL-null, are of commercial origin (Promega Corporation). The plasmids pSG5 (Stratagene), pBluescript II SK+ (Stratagene) and pSL301 (Invitrogen Corporation) are also of commercial origin. The constructions of the expression plasmids pSG5-hPPARg2 25 (Fajas L. et al., J. Biol. Chem., 272 (1997) 18779-18789) and pSG5-hPPARa(Koz) (Gervois P. et al., Mol. WO 00/78986 38 PCT/FR00/01744 Endocrinol., 13 (1999) 400-409) have previously been described. The construction of the plasmid pCMV-leadTK has also previously been described in patent 5 application FR 98/120000 of 25/09/98, and in patent application US SN 60/123,298 (provisional application). It is recalled that this plasmid is constructed in the following manner. The expression vector pCGN previously described by Tanaka et al. 10 (Cell, 60 (1990) 375-386) contains the CMV promoter (-522/+72) fused with the "leader" of the HSV tk gene (+51/+101) upstream of a sequence encoding the haemagglutinin epitope. The plasmid pCGN (10 ng) was used as template for a PCR amplification. The primers 15 which were .used are the following: - Primer 6718 (5'CCCGTTACATAACTTACGGTAAATGGCCCG3') (SEQ ID NO : 26), this primer hydridizes with the CMV promoter at position -522 (8 nucleotides downstream of the EcoRI 20 site of pCGN). - primer 6719 (5'GGGACGCGCTTCTACAAGGCGCTGGCCGAA3') (SEQ ID NO : 27), this primer hybridizes up to position 101 of the tk "leader". The first nucleotide G in bold is intended to 25 restore the Ncol site of pGL3-Basic as will be explained below. WO 00/78986 PCT/FR00/01744 39 The PCR fragment thus obtained is purified and then phosphorylated with the aid of the T4 phage polynucleotide kinase (New England Biolabs). In parallel, the vector pGL3-Basic (Promega) was 5 linearized with Ncol, purified and then treated with Klenow DNA polymerase (Boehringer Mannheim) so as to fill the Ncol site. This vector is then dephosphorylated with the aid of alkaline phosphatase (Boehringer Mannheim) and then used for the insertion 10 of the phosphorylated PCR fragment. Thus, the guanosine (G) of the primer 6719 makes it possible to restore only the Ncol site when the CMV-tk leader fragment is oriented with the 5' part (primer 6718, position -522 of the CMV) downstream of the Hindlll site of pGL3-15 Basic and its 3' end (primer 6719, tk leader) is ligated to the Ncol site of pGL3-Basic (first ATG of luciferase). The plasmid thus obtained is designated pCMV-leadTK. The enzymatic amplification of DNA fragments 20 by the PCR (polymerase chain reaction) technique may be carried out using a DNA thermal cycler™ (Perkin Elmer Cetus) according to the manufacturer's recommendations. The electroporation of plasmid DNA into Escherichia coli cells may be carried out with the aid 25 of an electroporator (Bio-Rad) according to the manufacturer's recommendations. WO 00/78986 PCT/FR00/01744 40 The verification of the nucleotide sequences may be carried out by the method developed by Sanger et al., [Proc. Natl. Acad. Sci. USA, 74 (1977) 5463-5467) using the kit distributed by Applied Biosystems 5 according to the manufacturer's recommendations. The murine myoblasts C2C12 are cultured in DMEM™ medium (Life Technologies Inc.) supplemented with 10% foetal calf serum (FCS). The cultures are carried out in an oven at 37°C, under a humid atmosphere and at 10 a C02 partial pressure of 5%. The tranfections are carried out in 24-well plates and each transfection is carried out three times. Twenty-four hours after the transfection, the cells are inoculated at 3xl04 cells per well in DMEM™ 15 medium. For each well, 500 ng of plasmid DNA (plasmids of interest and pBluescript II SK+ in order to adjust to 500 ng) are mixed with the cationic lipid RPR120535 B (WO 97/18185) in an amount of 6 nmol of lipid per (Xg of DNA in DMEM™ medium (20 ^.1 final) comprising 150 mM 20 NaCl and 50 iriM bicarbonate. After 20 minutes at room temperature, the 2 0 |Xl of the DNA/lipid mixture are brought into contact with the cells, in the absence of FCS, for 2 hours, The culture medium is then supplemented with FCS or with ULTROSER™ (BioSepra Inc.) 25 so as to obtain a final concentration of 10% or 2% respectively. The PPAR ligands, dissolved in DMSO, are added to the culture medium at the same time as the FCS WO 00/78986 41 PCT/FROO/01744 or the ULTROSER™. Forty-eight hours after the transfection, the culture medium is removed and the cells are rinsed twice with PBS (Life Technologies Inc.). The activity of the Photinus pyralis luciferase 5 and the activity of the Renilla reniformis luciferase are then determined with the aid of the Dual-Luciferase Reporter Assay System™ kit (Promega Corporation) according to the supplier's recommendations. The in vivo gene transfer experiments are 10 carried out on 6-week-old C57BI/6 female mice. The animals are anaesthetized with 250 jil of a ketamine (Rhone Merieux, 10 mg/ml final)/Xylazine (Bayer Pharma, 0.3 mg/ml final) mixture by the intraperitoneal route. An injection of a total quantity of 10 jig of DNA is 15 then carried out into each cranial tibial muscle. Each leg is then subjected to an electric field (frequency of 1 Hz; 4 pulses of 20 ms at 250 V/cm). During the entire duration of the experiment, the animals receive each morning, by force-feeding, either 30 mg/kg of 20 BRL49653 (SmithKline Beecham) in 1% carboxycellulose (weight/volume), or 1% carboxycellulose alone. Four days after the gene transfer, the animals are sacrificed and the muscles collected in PLB™ lysis buffer (Promega Corporation) in Lysing Matrix™ tubes 25 (BIO 101, Inc.). The grinding of the muscles, which makes it possible to extract luciferase, is carried out with the aid of the FastPrep™ apparatus (BIO 101, Inc.) WO 00/78986 42 PCT/FR00/01744 for 25 seconds at 6.5 m/s. The activity of the Photinus pyralis luciferase is then determined with the aid of the Luciferase Assay System™ kit (Promega Corporation) according to the supplier's recommendations. 5 EXAMPLES EXAMPLE 1 : Construction of promoters inducible by the PPARs and of expression plasmids containing them. 10 1.1. Plasmid FTKpGL3. A DNA fragment, corresponding to part of the promoter of the TK gene of the type 1 herpes simplex 15 virus (HSV-1), between positions -105 and +5 6 relative to the site of initiation of transcription, was amplified by PCR using the plasmid pBLCAT2 (Luckow B. and Schutz G., Nucleic Acids Res., 15 (1987) 5490) as template and the oligonucleotides 5' CGA CTC TAG AAG 20 ATC TTG CCC CGC CCA GCG 3' (SEQ ID NO:28) and 5' TCG CCA AGC TTC TCG TGA TCT GCG GCA 3' (SEQ ID NO:2) as primers. This fragment was digested with Bglll and Hindlll and was then cloned into the plasmid pGL3-Basic previously digested with Bglll and Hindlll in order to 25 obtain the plasmid FTKpGL3. A schematic representation of this plasmid is presented in Figure 1. WO 00/78986 43 PCT/FROO/01744 1.2 Plasmids JxnS-TK-pGL3. A DNA fragment, containing one or more (n) J sites of the promoter of the human ApoA-II gene, was 5 amplified by PCR using the plasmid J3TKpGL3 (Vu-Dac N. et al., J. Clin. Invest., 96 (1995) 741-750) as template and the oligonucleotides 3RDA37 (5' ACG TGT CGA CAC TAG TGG CTA GAG GAT CTC TAC CAG G 3'; SEQ ID NO:3) and 4RDA48 (5' CGA TGG TAC CCT CGA GCA ATG TGC 10 TAG CGA GAT CCT TCA ACC TTT ACC 3'; SEQ ID NO:4) as primers. This fragment was digested with Xhol and Spel and was then cloned into the plasmid FTKpGL3 previously digested with Xhol and Nhel, in the direction of transcription of the minimal TK promoter (S), in order 15 to obtain the plasmids JxlS-TK-pGL3, Jx2S-TK-pGL3 and Jx3S-TK-pGL3, depending on the number of J sites present. A schematic representation of the plasmid Jx3S-TK-pGL3 is presented in Figure 2. The DNA fragment, amplified by PCR using the 20 plasmid J3TKpGL3 and the oligonucleotides 3RDA37 and 4RDA48 as primers, digested with Xhol and Spel, was also cloned into the plasmid Jx3S-TK-pGL3 previously digested with Xhol and Nhel, in order to obtain the plasmids Jx4S-TK-pGL3, Jx5S-TK-pGL3 and Jx6S-TK-pGL3, 25 depending on the number of J sites present. WO 00/78986 44 PCT/FROO/01744 1.3. Plasmids JxnAS-TK-pGL3. The plasmids JxnAS-TK-pGL3 differ from the plasmids JxnS-TK-pGL3 by the orientation of the J sites 5 present in the inducible promotor. A DNA fragment containing one or more J sites of the promoter of the human ApoA-II gene, was amplified by PCR using the plasmid J3TKpGL3 as template and the oligonucleotides 3RDA37 and 4RDA48 as primers. This fragment was 10 digested with Sail and Nhel and was then cloned, in the opposite direction of transcription of the minimal TK promotor (AS), into the plasmid FTKpGL3 previously digested with Xhol and Nhel in order to obtain the plasmids JxlAS-TK-pGL3, Jx2AS-TK-pGL3 and Jx3AS-TK-15 pGL3, depending on the number of J sites present. A schematic representation of the plasmid Jx3AS-TK-pGL3 is presented in Figure 3. The DNA fragment, amplified by PCR using the plasmid J3TKpGL3 and the oligonucleotides 3RDA37 and 2 0 4RDA48 as primers, digested with Kpnl and Spel, was also cloned in the antisense (AS) orientation into the plasmid Jx3AS-TK-pGL3 previously digested with Kpnl and Nhel in order to obtain the plasmids Jx4AS-TK-pGL3 and Jx5AS-TK-pGL3 depending on the number of J sites 25 present. WO 00/78986 45 PCT/FROO/01744 1.4 Plasmids DRlxnS-TK-pGL3. These plasmids contain, as PPAR response element (PPRE), a consensus sequence (AGGTCA A AGGTCA, 5 SEQ ID NO:5) called consensus DR1. A DNA fragment, containing one or more consensus DR1 sites, was amplified by PCR using the oligonucleotides 1RDA69 (5' ACG TGT CGA CAC TAG TCA AAA CTA GGT CAA AGG TCA CGG AAA ACT AGG TCA AAG GTC ACG GAG AAC TAG 3'; SEQ ID NO:6) 10 and 2RDA64 (5' CGA TGG TAC CCT CGA GCA ATG TGC TAG CCG TGA CCT TTG ACC TAG TTT TCC GTG ACC TTT GAC C 3'; SEQ ID NO:7) as primers. This fragment was digested with digested with Xhol and Spel and was then cloned, in the sense orientation, into the plasmid FTKpGL3 previously 15 digested with Xhol and Nhel in order to obtain the plasmids DRlx2S-TK-pGL3 and DRlx3S-TK-pGL3, depending on the number of consensus DR1 sites present. A schematic representation of the plasmid DRlx3S-TK-pGL3 is presented in Figure 4. 20 The DNA fragment, amplified by PCR using the oligonucleotides 1RDA69 and 2RDA64 as primers, digested with Xhol and Spel, was also cloned into the plasmid DRlx3S-TK-pGL3 previously digested with Xhol and Nhel in order to obtain the plasmids DRlx5S-TK-pGL3, DRlx6S-2 5 TK-pGL3 and DRlx7S-TK-pGL3, depending on the number of consensus DR1 sites present. WO 00/78986 46 PCT/FROO/01744 1.5. Plasmids DRlxnAS-TK-pGL3. The plasmids DRlxnAS-TK-pGL3 differ from the plasmids DRlxnS-TK-pGL3 by the orientation of the 5 consensus DRl sites present in the inducible promoter. A DNA fragment, containing one or more consensus DRl sequences, was amplified by PCR using the oligonucleotides 1RDA69 and 2RDA64 as primers. This fragment was digested with Sail and Nhel, and was then 10 cloned, in the antisense orientation, into the plasmid FTKpGL3 previously digested with Xhol and Nhel in order to obtain the plasmids DRlx2AS-TK-pGL3 and DRlx3AS-TK-pGL3, depending on the number of consensus DRl sites present. A schematic representation of the plasmid 15 DRlx3AS-TK-pGL3 is presented in Figure 5. The DNA fragment, amplified by PCR using oligonucleotides 1RDA69 and 2RDA64 as primers, digested with Kpnl and SPel, was also cloned into the plasmid DRlx3AS-TK-pGL3 previously digested with Kpnl and Nhel 2 0 in order to obtain the plasmids DRlx5AS-TK-pGL3 and DRlx6AS-TK-pGL3 depending on the number of consensus DRl sites present. EXAMPLE 2 s Specificity of the PPARs for different 25 response elements. 2.1. System using hPPARg2. WO 00/78986 PCT/FR00/01744 47 The activity of the inducible promotors, using hPPARg2 as transcriptional regulator, was evaluated in transient tranfection in mouse myoblasts 5 (Figure 6). The results show that, depending on the response element (PPRE) used, the induction by the hPPARg2 ligand (BRL49653) and the final activity after activation vary. The best results were obtained using J sites as PPRE. Furthermore, the orientation of the PPRE 10 is also important. In the case of the J site, the AS orientation is more favourable (Panel c). 2.2. System using hPPARa. 15 The results obtained with hPPARa as transcriptional regulator are assembled in Figure 7. Unlike hPPARg2, it is the consensus DRl which is the best PPRE for hPPARa (Panels d and e). These results therefore show (1) the 20 functionality of the plasmids of the invention and (2) that depending on the PPAR chosen in the inducible system, it is important to select the PPRE most appropriate for the transcriptional regulator. This choice may influence the induction factor due to the 25 presence of the ligand but also the level of activity reached after induction. It is understood that other PPREs can be used in the system of the invention. WO 00/78986 48 PCT/FROO/01744 EXAMPLE 3: Construction of promoters inducible by the PPARs containing a minintum promoter other than that of HSV1-TK such as for example the hCMV-IE minimum 5 promoter. 3.1. Construction of the plasiriids containing the hCMV-IE minimum promoter. 10 A DNA fragment, containing the hCMV-IE minimum promoter (from position -54 to position +48 relative to the site of initiation of transcription), was amplified by PCR using the plasmid pCMVfi (Clontech) as template and the oligonucleotides 5RDA32 (5' ACG TAG 15 ATC TCG GTA GGC GTG TAC GGT GGG AG 3'; SEQ ID NO:8) and 6RDA29 (5' ACG TAA GCT TCT ATG GAG GTC AAA ACA GC 3'; SEQ ID NO:9) as primers. This fragment was digested with Hindlll and Bglll and was then cloned into the plasmid FTKpGL3 previously digested with Hindlll and 2 0 Bglll in order to obtain the plasmid FCMVpGL3. The plasmid Jx5AS-TK-pGL3 was digested with Bglll and Nhel in order to isolate the Bglll-Nhel fragment of 179 bp containing 5 copies of the J site. This fragment was inserted into the plasmid FCMVpGL3 25 previously digested with Bglll and Nhel in order to give the plasmid Jx5AS-CMV-pGL3. A schematic WO 00/78986 49 PCT/FROO/01744 representation of the plasmid Jx5AS-CMV-pGL3 is presented in Figure 8. The plasmid Jx5AS-CMV-pGL3 was digested with SphI and Nhel in order to isolate the SphI-Nhel 5 fragment of 982 bp containing 5 copies of the J site, the hCMV-IE minimum promoter and the 5' part of the gene encoding luciferase. This fragment was inserted into the plasmid Jx5AS-CMV-pGL3 previously digested with SphI and Spel in order to give the plasmid JxlOAS-10 CMV-pGL3. The plasmids Jxl5AS-CMV-pGL3 and Jx20AS-CMV-pGL3 were also obtained by following the same strategy. 3.2. Activity of plasmids containing the hCMV-IE minimum promoter. 15 A comparison of the minimum promoters which can be used in the inducible system was made in transient transfection. The results, which are assembled in Figure 9, show that depending on the 2 0 minimum promoter, the final activity after induction may vary by a factor of 2. These results show in particular that, under the conditions tested, the CMV promoter appears to give a higher activity. Of course other minimum promoters, such as promoters not 25 containing a TATA box, may be used. WO 00/78986 50 PCT/FROO/01744 EXAMPLE 4 : Importance of the number of response elements present in the inducible promoters. The optimization of the number of PPREs 5 present in the inducible promoter was studied in transient transfection. The results, presented in Figure 10, show that the higher the number of copies of the PPRE, the greater the induction factor for the ligand and the activity induced. On the other hand, if 10 this number is too high, both the induction factor and the induced activity decrease, this being regardless of the quantity of hPPARg2 present in the assay (Figure 11). The optimum number of PPRE appears to be between 10 and 15. 15 EXAMPLE 5: Construction of a transcriptional regulator highly inducible by the PPAR ligands. 5.1. Construction of a transcriptional 2 0 regulator comprising two copies of the ligand-binding domain. Construction of the plasmid pSG5-hPPARg2g2. A DNA fragment, noted A, containing the DNA region complementary to hPPARg2 encoding the C-terminal part of the F domain, was amplified by PCR using the 25 plasmid pSG5-hPPARg2 as template and the oligonucleotides 20RDA21 (5' GGT TTG CTG AAT GTG AAG CCC 3'; SEQ ID NO:10) and 21RDA42 (5' AGT CTC TAG AGC WO 00/78986 51 PCT/FROO/01744 TAC GCG TAC AAG TCC TTG TAG ATC TCC TGC 3'; SEQ ID NO:11) as primers. A DNA fragment, noted B, containing the DNA region complementary to hPPARg2 encoding the E and F domains, was amplified by PCR using the plasmid 5 pSG5-hPPARg2 as template and the oligonucleotides 22RDA32 (5' AGT CAC GCG TGG GCG ATC TTG ACA GGA AAG AC 3'; SEQ ID NO:12) and 23RDA21 (5' GCC TTT GAG TGA GCT GAT ACC 3'; SEQ ID NO:13) as primers. The A fragment, digested with SacI and Mlul and the B fragment, 10 digested with Mlul and Xbal, were cloned together into the plasmid pSG5-hPPARg2 previously digested with SacI and Xbal in order to obtain the plasmid pSG5-hPPARg2g2. This plasmid, whose schematic representation is presented in Figure 12, contains a complementary DNA 15 which encodes a transcriptional regulator (noted hPPARg2g2) comprising two copies of the E and F domains, that is to say two ligand-binding domains. The complete sequence of PPARy2v2 is represented below (SEQ ID NO:24): mgetlgdspidpesdsftdtlsanisqemtmvdtempfwptnfgissvdlsvmedhshsfdi kpfttvdfssistphyedipftrtdpwadykydlklqeyqsaikvepasppyysektqlyn kpheepsnslmaiecrvcgdkasgfhygvhacegckgffrrtirlkliydrcdlncrihkks rnkcqycrfqkclavgmshnairfgrmpqaekekllaeissdidqlnpesadlralakhlyd syiksfpltkakarailtgkttdkspfviydmnslmmgedkikfkhitplqeqskevairif qgcqfrsveavqeiteyaksipgfvnldlndqvtllkygvheiiytmlaslmnkdgvliseg qgfmtreflkslrkpfgdfmepkfefavkfnalelddsdlaifiaviilsgdrpgllnvkpi ediqdnllqalelqlklnhpessqlfakllqkmtdlrqivtehvqllqvikktetdmslhpl lqeiykdliyawailtgkttdkspfviydmnslmmgedkikfkhitplqeqskevairifqgc qfrsveavqeiteyaksipgfvnldlndqvtllkygvheiiytmlaslmnkdgvlisegqgf mtreflkslrkpfgdfmepkfefavkfnalelddsdlaifiaviilsgdrpgllnvkpiedi qdnllqalelqlklnhpessqlfakllqkmtdlrqivtehvqllqvikktetdmslhpllqe 20 IYKDLY WO 00/78986 52 PCT/FROO/01744 The sequence of the C-terminal part of PPARY2y2, comprising the E and F domains, is the following sequence SEQ ID NO:25: MMGEDKIKFKHITPLQEQSKEVAIRIFQGCQFRSVEAVQEITEYAKSIPGFVNLDLNDQVTL LKYGVHEIIYTMLASLMNKDGVLISEGQGFMTREFLKSLRKPFGDFMEPKFEFAVKFNALEL DDSDLAIFIAVIILSGDRPGLLNVKPIEDIQDNLLQALELQLKLNHPESSQLFAKLLQKMTD LRQIVTEHVQLLQVIKKTETDMSLHPLLQEIYKDLYAWAILTGKTTDKSPFVIYDMNSLMMG EDKIKFKHITPLQEQSKEVAIRIFQGCQFRSVEAVQEITEYAKSIPGFVNLDLNDQVTLLKY GVHEIIYTMLASLMNKDGVLISEGQGFMTREFLKSLRKPFGDFMEPKFEFAVKFNALELDDS DLAIFIAVIILSGDRPGLLNVKPIEDIQDNLLQALELQLKLNHPESSQLFAKLLQKMTDLRQ IVTEHVQLLQVIKKTETDMSLHPLLQEIYKDLY 5.2. Activity of the plasmid pSG5-hPPARg2g2. The results presented in Figure 13 show that 10 if the induced activity is lower using hPPARg2g2 as transcriptional regulator (Figure 13 a and b), the induction factor for the ligand (Figure 13 c) is much higher with this regulator. The difference between the two transcriptional regulators is explained by the fact 15 that for hPPARg2g2, the background noise of the system in the absence of ligand is low and remains low, regardless of the quantity of regulator present. On the other hand, the higher the increase in hPPARg2g2, the higher the induced activity, which is not the case for 2 0 the system using hPPARg2 which appears to saturate. The presence of a second ligand-binding domain (hPPARg2g2) therefore confers on the transcriptional regulator greater inducibility by the ligand. WO 00/78986 53 PCT/FROO/01744 EXAMPLE 6 : Increase in the final activity of the inducible promoters. 6.1. Construction of an inducible expression 5 cassette comprising the intron of hEFla. Construction of the plasmid JxlOAS-CMV-EF-pGL3. A DNA fragment, containing the first intron of the gene encoding hEFla (from position +16 to 10 position +984 relative to the site of initiation of transcription; Genbank accession number: E02627), was amplified by PCR using the oligonucleotides 2 5RDA35 (5' AGT CAC TAG TAA GCT TTT TGC CGC CAG AAC ACA GG 3'; SEQ ID NO:14) and 2 6RDA36 (5' AGT CAC TAG TCC ATG GCT GCC 15 CAG TGC CTC ACG ACC 3'; SEQ ID NO:15) as primers. This fragment was digested with Hindlll and Ncol and was then cloned into the plasmid JxlOAS-CMV-pGL3 previously digested with Hindlll and Ncol in order to obtain the plasmid JxlOAS-CMV-EF-pGL3. A schematic representation 2 0 of the plasmid JxlOAS-CMV-EF-pGL3 is presented in Figure 14. 6.2. Activity of the plasmid JxlOAS-CMV-EF- pGL3 . 25 With the aim of increasing the final activity of the system, an enhancer sequence, situated in the WO 00/78986 54 PCT/FR00/01744 first intron of the hEFla gene, was cloned in the vicinity of the inducible promoter. The results presented in Figure 15 show that the presence of the enhancer region increases the induced activity of the 5 system, this being regardless of the quantity of transcriptional regulator used. EXAMPLE 7 : Construction of plasmids comprising both a cassette for expression of the transcriptional 10 regulator and an inducible expression cassette. 7.1. Plasmid Jx5AS-TK-luc-hPPARg2. The plasmid pSG5-hPPARa(Koz) was digested 15 with Mlul and Seal in order to isolate the Mlul-Scal fragment of 1229 bp containing the 3' region of the DNA complementary to hPPARa. This fragment was inserted into the plasmid pSL301 previously digested with Mlul and Smal in order to give the plasmid pSL-3'hPPARa. 2 0 The plasmid pSG5-hPPARa(Koz) was digested with Sail and Mlul in order to isolate the Sall-Mlul fragment of 1406 bp containing the SV40 virus early promoter and the 5' region of the DNA complementary to hPPARa. This fragment was inserted into the plasmid 25 pSL-3'hPPARa previously digested with Xhol and Mlul in order to give the plasmid pSL-hPPARa. WO 00/78986 55 PCT/FROO/01744 The plasmid pSL-hPPARa was digested with Spel and Sail in order to isolate the Spel-Sail fragment of 2664 bp containing the SV40 virus early promoter and the DNA complementary to hPPARa. This fragment was 5 inserted into the plasmid pBluescript II SK+ previously digested with Spel and Sail in order to give the plasmid pBS-hPPARa. The plasmid pSG5-hPPARg2 was digested with Avrll and SacI in order to isolate the Avrll-SacI 10 fragment of 2070 bp, noted C, containing the 5' region of the DNA complementary to hPPARg2. A DNA fragment, noted D, containing the 3' region of the DNA complementary to hPPARg2, was amplified by PCR using the plasmid pSG5-hPPARg2 as template and the 15 oligonucleotides 10RDA21 (5' CAG GTT TGC TGA ATG TGA AGC 3'; SEQ ID NO:16) and 11RDA40 (5' TGA CGT GTC GAC CTA GTA CAA GTC CTT GTA GAT CTC CTG C 3'; SEQ ID NO:17) as primers. The C fragment and the D fragment, digested with SacI and Sail, were cloned together into the 2 0 plasmid pBS-hPPARa previously digested with Avrll and Sail in order to obtain the plasmid pBS-hPPARg2. The plasmid Jx5AS-TK-pGL3 was digested with Kpnl and Sail in order to isolate the Kpnl-SalI fragment of 2324 bp containing the luc+ gene under the 25 control of an inducible promoter. This fragment was inserted into the plasmid pBS-hPPARg2 previously digested with Kpnl and Sail in order to give the WO 00/78986 56 PCT/FROO/01744 plasmid Jx5AS-TK-luc-hPPARg2. A schematic representation of the plasmid Jx5AS-TK-luc-hPPARg2 is presented in Figure 16. 5 7.2. Plasmid SV-g2-JlO-C-pGL3. The plasmid pBS-hPPARg2 was digested with NotI and Sail in order to isolate the Notl-Sall fragment of 2622 bp, noted E, containing the DNA 10 complementary to hPPARg2 under the control of the SV40 early promoter. A DNA fragment, noted F, containing the SV40 virus polyadenylation site, was amplified by PCR using the plasmid FTK-pGL3 as template and the oligonucleotides 18RDA31 (5' AGT CGT CGA CGC TTC GAG 15 CAG ACA TGA TAA G 3'; SEQ ID NO:18) and 19RDA35 (5' AGT CGC TAG CGA CGG ATC CTT ATC GAT TTT ACC AC 3'; SEQ ID NO:19) as primers. The E fragment and the F fragment, digested with Sail and Nhel, were cloned together into the plasmid JxlOAS-CMV-pGL3 previously digested with 20 NotI and Nhel in order to obtain the plasmid SV-g2-J10-C-pGL3. A schematic representation of the plasmid SV-g2-JlO-C-pGL3 is presented in Figure 17. 7.3. Plasmid hPPARg2-CMV-Jx5AS-TK-pGL3. 25 A DNA fragment, noted G, containing the DNA complementary to hPPARg2, was amplified by PCR using WO 00/78986 57 PCT/FROO/01744 the plasmid Jx5AS-TK-luc-hPPARg2 as template and the oligonucleotides 12RDA50 (5' GTC AGC TAG CCT ACT CGA GCC ACC ATG GGT GAA ACT CTG GGA GAT TCT CC 3'; SEQ ID NO:20) and 13RDA42 (5' TAC GGG GTA CCC AGA CAT GAT AAG ATA CAT TGA TGA GTT TGG 3'; SEQ ID NO:21) as primers. A DNA fragment, noted H, containing the hCMV-IE minimum promoter (from position -54 to position +48 relative to the site of initiation of transcription), was amplified by PCR using the plasmid pCMVP as template and the oligonucleotides 14RDA3 3 (5' GTC AGC TAG CCG GTA GGC GTG TAC GGT GGG AGG 3'; SEQ ID NO:22) and 15RDA33 (5' TAC GCT CGA GCT TCT ATG GAG GTC AAA ACA GCG 3'; SEQ ID NO:23) as primers. The G fragment, digested with Kpnl and Xhol and the H fragment, digested with Xhol and Nhel, were cloned together into the plasmid Jx5AS-TK-pGL3 previously digested with Kpnl and Nhel in order to obtain the plasmid hPPARg2-CMV-Jx5AS-TK-pGL3. A schematic representation of the plasmid hPPARg2-CMV-Jx5AS-TK-pGL3 is presented in Figure 18. 7.4. Plasmids hPPARg2-CMV-JxnAS-CMV-pGL3. The plasmid Jx5AS-CMV-pGL3 was digested with Nhel and SphI in order to isolate the Nhel-SphI fragment of 982 bp containing the 5' region of the luc+ gene under the control of an inducible promoter. This fragment was inserted into the plasmid hPPARg2-CMV- WO 00/78986 58 PCT/FROO/01744 Jx5AS-TK-pGL3 previously digested with Spel and SphI in order to give the plasmid hPPARg2-CMV-JxlOAS-CMV-pGL3. A schematic representation of the plasmid hPPARg2-CMV-JxlOAS-CMV-pGL3 is presented in Figure 19. 5 The plasmid Jx5AS-CMV-pGL3 was digested with Nhel and SphI in order to isolate the Nhel-SphI fragment of 982 bp containing the 5' region of the luc+ gene under the control of an inducible promoter. This fragment was inserted into the plasmid hPPARg2-CMV-10 JxlOAS-CMV-pGL3 previously digested with Spel and SphI in order to give the plasmid hPPARg2-CMV-Jxl5AS-CMV-pGL3. The plasmid JxlOAS-CMV-pGL3 was digested with Nhel and SphI in order to isolate the Nhel-SphI 15 fragment of 1151 bp containing the 5' region of the luc+ gene under the control of an inducible promoter. This fragment was inserted into the plasmid hPPARg2-CMV-Jxl0AS-CMV-pGL3 previously digested with Spel and SphI in order to give the plasmid hPPARg2-CMV-Jx20AS-20 CMV-pGL3. EXAMPLE 8 : Comparison of the different versions of the inducible system in vitro. 25 Figure 20 assembles the results obtained in vitro with various versions of the inducible system. These results show that the systems using two plasmids WO 00/78986 59 PCT/FROO/01744 (Figure 20, lines 1, 3 and 4) like the systems with only one plasmid (Figure 20, lines 2 and 5 to 9) are functional; that is to say that the presence of a PPARg ligand (here BRL49653) greatly increases the expression 5 of the gene placed under the control of the inducible promoter. It is also observed that for some systems (Figure 20, lines 3 and 7 to 9), the induction factor for the ligand is greater than 30, and that for the system presented in Figure 20, line 4, the activity 10 after induction is equal to that of a strong promoter such as that of the hCMV-IE promoter. EXAMPLE 9 : Various PPAR ligands can activate the inducible system 15 9.1. System using hPPARg2. The results presented in Figure 21 show that hPPARg ligands other than BRL49653, here RG12525 (RPR 20 ligand for hPPARg), may be used to activate the inducible system. At a concentration of 100 |Jm, a treatment with RG12525 even leads to a higher induction than that obtained with BRL49653. Any other PPARg ligand may therefore be used as inducer of the system. 25 WO 00/78986 60 PCT/FROO/01744 9.2. System using hPPARa. In the same manner as for the system using hPPARg, a system using hPPARa as transcriptional 5 regulator may be activated with the fibrates or WY-14,643 for example or any other hPPARa ligand. EXAMPLE 10 : The inducible system may be activated in vivo, in the muscle. 10 Figure 22 assembles the results obtained in vivo, in the muscle, with different versions of the inducible system. The results show that for the three versions tested (Figure 22, lines 2 to 4), a treatment 15 by force-feeding with a hPPARg ligand is capable of greatly increasing, in the muscle, the activity of the inducible promoters. The induction factors are: Xl4 for the Figure 22 line 2 version, x8 for the Figure 22 line 3 version, and X24 for the Figure 22 line 4 version. 20 Furthermore, for one of the versions (Figure 22, line 2), the activity obtained in the animals treated with BRL49653 is of the order of that of a strong promoter such as the hCMV-IE promoter. The results, presented in Figure 23, also 25 show that a single dose of ligand can induce the system, whether this dose is taken before or after the gene transfer. This experiment also shows that a dose WO 00/78986 61 PCT/FROO/01744 which is two times smaller than that normally used makes it possible to obtain the same induction factor. The system, using a PPAR nuclear receptor as transcriptional regulator, is therefore functional in 5 vivo and may be induced by the oral administration of a PPAR ligand. EXAMPLE 11: Construction of a plasmid allowing the inducible expression of a gene whose product is 10 secreted. 11.1 Construction of the plasmid pRDA02 The plasmid JxlOAS-CMV-pGL3 was digested with 15 Hindlll and Mlul in order to isolate the Hindlll-Mlul fragment of 459 bp. This fragment was inserted into the plasmid pXL3010 (Bettan M. et al., Anal. Biochem., 271 (1999) 187-189) previously digested with Hindlll and Mlul in order to give the plasmic pRDA02. This plasmid 20 contains the DNA complementary to the gene encoding the secreted form of human placental alkaline phosphatase (hSeAP) whose expression is under the control of a promoter inducible by the system using the PPARs as transcriptional regulator. A schematic representation 25 of the plasmid pRDA02 is presented in Figure 24. WO 00/78986 62 PCT/FROO/01744 EXAMPLE 12: The inducible system makes it possible to regulate, in vivo, the plasma concentration of a secreted protein. 5 The results presented in Figure 25 show that, by using the inducible system, it is possible to regulate, over time, the plasma concentration of a protein secreted from the muscle, this being with a simple oral administration of a PPAR ligand. The plasma 10 concentration of hSeAP is increased by a factor of 18 (Figure 25A) two days after the administration of ligand, and then returns to its base level one week later. Between the 21st and 39th day, an immune response directed against hSeAP of human origin is 15 observed and results in a decrease in the plasma concentration of this protein. Despite this immune response, it is possible to carry out a second induction cycle (Figure 25A). As shown in Figure 25B, the inducible system 20 also makes it possible, by daily administrations of ligand, to maintain the plasma level of hSeAP at a high level for a period equal to the duration of the treatment. WO 00/78986 63 PCT/FROO/01744 EXAMPLE 13: Various PPAR ligands can activate the inducible system in vivo, this being in a dose-dependent manner. 5 BRL49653, in its commercial form for the treatment of type II diabetes (Avandia™, SmithKline Beecham) and pioglitazone, in its commercial form for this same treatment (Actos™, Takeda Pharmaceuticals) can also activate the inducible system (Figure 2 6A). 10 Figure 2 6B also shows that the induction factor is directly correlated with the dose of ligand used. The system, using a PPAR nuclear receptor as transcriptional regulator, therefore makes it possible to control, very precisely, the plasma level of a 15 secreted protein. Furthermore, this regulation may be obtained using various PPAR ligands. EXAMPLE 14: Construction of a plasmid allowing the inducible expression of a gene whose product is an 2 0 angiogenic factor. 14.1 Construction of the plasmid JxlOAS-CMV- VEGFA165. 2 5 The human VEGF165 reading frame was cloned by reverse transcription and PCR from total RNA of human placenta (Clontech) (Houck et al. Mol. Endocrinol. 12 WO 00/78986 64 PCT/FROO/01744 (1991) 1806-1814) and then inserted into a plasmid pBluescript (Stratagene) containing the CMV E/P promoter from position -522 to +72 and the SV40 late polyA, in order to give the plasmid pXL3218. The latter 5 was then digested with Hindlll and BsrGI in order to isolate the Hindlll-BsrGI fragment A of 482 bp. The plasmid pXL3218 was also digested with BsrGI and BamHI in order to isolate the BsrGI-BamHI fragment B of 390 bp. Fragments A and B were inserted into the 10 plasmid JxlOAS-CMV-pGL3 previously digested with Hindlll and BamHI in order to give the plasmid JxlOAS-CMV-VEGFA165. This plasmid contains the DNA complementary to the gene encoding VEGFA165 whose expression is under the control of a promoter inducible 15 by the system using PPARs as transcriptional regulator. A schematic representation of the plasmid JxlOAS-CMV-VEGFA165 is presented in Figure 27. This plasmid can be used, for example, to control, over time, the angiogenic activity of VEGF for 2 0 therapeu tic purpo s e s. -65- </div>

Claims

<div class="application article clearfix printTableText" id="claims"> CLAIMS

1. Composition comprising: (a) a first element comprising a nucleic acid of interest under the control of an inducible promoter comprising a PPAR response element and a minimal transcriptional promoter, and (b) a second element comprising a nucleic acid encoding a PPAR under the control of a transcriptional promoter, for their use simultaneously, separately or sequentially.

2. Composition according to claim 1, characterized in that it comprises in addition: (c) a ligand for PPAR, for a use simultaneously, separately or sequentially.

3. Composition according to claim 1 or 2, characterized in that the elements (a) and (b) are carried by distinct genetic constructs.

4. Composition according to claim 1 or 2, characterized in that the elements (a) and (b) are assembled in the same genetic construct.

5. Composition according to claim 3 or 4, characterized in that the genetic construct(s) is/are a plasmid or viral vector.

6. Composition according to claim 5, characterized in that the viral vector is an adeno-associated virus (AAV).

7. Composition according to any one of claims 1 to 6, characterized in that the PPAR response element comprises one or more PPAR-binding sites.

8. Composition according to claim 7, characterized in that the PPAR response element comprises one or more sites having the sequence SEQ ID NO:1.

9. Composition according to claim 7, characterized in that the PPAR response element Comprises one or more sites having the sequence SEQ ID NO:5

10. Composition according to any one of claims 7 to 9, f—— — / --ACTUAL PROPERTY OFFICE OF N.Z. - 9 FES 2004 DECEIVED -66- characterized in that the response element comprises up to 30 binding sites, preferably from 3 to 20, more preferably from 5 to 15.

11. Composition according to any one of claims 1 to 10, characterized in that the minimal promoter is a promoter of a cellular or viral gene deleted for the region(s) not essential for transcriptional activity.

12. Composition according to any one of claims 1 to 11, characterized in that the inducible promoter comprises, in addition, an enhancer region.

13. Composition according to any one of claims 1 to 12, characterized in that the minimal promoter and the PPAR response element are in the same orientation.

14. Composition according to any one of claims 1 to 12, characterized in that the minimal promoter and the PPAE response element are in the opposite orientation.

15. Composition according to any one of claims 1 to 14, characterized in that the nucleic acid encoding a PPAR encodes a PPARa or a PPARy.

16. Composition according to any one of claims 1 to 15, characterized in that the nucleic acid encoding a PPAR encodes a modified PPAR comprising several ligand-binding sites.

17. Composition according to any one of claims 1 to 16, characterized in that it comprises, in addition, an element (d) comprising a nucleic acid encoding an RXR under the control of a transcriptional promoter.

18. Vector comprising an element (a) and an element (b) according to claim 1.

19. Vector according to claim 18, characterized in that the elements (a) and (b) are in the opposite orientation.

20. Vector according to claim 18 or 19, characterized in that the inducible promoter of the element (a) and the transcriptional promoter of the element (b) are assembled in the vector to form a regulable bidirectional promoter.

21. Vector according to claim 20, characterized in that it comprises, ii\TELL^CVUAL PROPERTY OFFICE OF N.Z. - 9 FES 2004 RECEIVED INTELLECTUAL PROPERTY OFFICE OF N.Z. .67- -9 FEB 200* RECEIVED in the 5'->3' direction, a first nucleic acid encoding a PPAR, a first mirrtmat- "———- transcriptional promoter controlling the expression of the said first nucleic acid, one or more PPAR response elements, a second minimal transcriptional promoter and , under the control of the said second minimal transcriptional promoter, a second nucleic acid encoding a product of interest.

22. Vector according to any one of claims 18 to 21, characterized in that it comprises, in addition, an element (d) according to claim 17.

23. Use of a composition according to any one of claims 1 to 17 or of a vector afccording to any one of claims 18 to 22 for expressing a nucleic acid of interest in a cell ex vivo or in vitro.

24. Use of a composition according to any one of claims 1 to 17 or of a vector according to any one of claims 18 to 22 for the preparation of a product intended for expressing a nucleic acid of interest in a cell in vivo.

25. Method for the regulated expression of a nucleic acid in a cell, in vitro or ex vivo comprising bringing the said cell into contact with a composition according to any one of claims 1 to 17 or a vector according to any one of claims 18 to 22.

26. Method according to claim 25, characterized in that it is a mammalian, preferably human cell.

27. Method according to claim 26, characterized in that it is a muscle cell.

28. Use of a first element and a second element as defined in any one bf claims 1 to 17 in the ; preparation of a product for regulating the expression of a nucleic acid in vivo. i

29. Use of a first element and a second element as defined in any one of claims 1 to 17 in the preparation of a medicament for regulating the expression of a nucleic acid in vivo in a patient in need thereof.

30. Use of a first element as defined in any one of claims 1 to 17 in the preparation of a product formulated for coadministration with a second element as defined in any one of claims 1 to 17 for regulating the expression of a nucleic acid in vivo,

31. Use of a second element as defined in any one of claims 1 to 17 in the preparation of a product formulated for coadministration with a first element as defined in any one of claims 1 to 17 for regulating the expression of a nucleic acid in vivo. -68-

32. Use of a first element as defined in any one of claims 1 to 17 in the preparation of a product formulated for sequential administration with a second element as defined in any one of claims 1 to 17 for regulating the expression of a nucleic acid in vivo.

33. Use of a second element as defined in any one of claims 1 to 17 in the preparation of a product formulated for sequential administration with a first element as defined in any one of claims 1 to 17 for regulating the expression of a nucleic acid in vivo.

34. Use of a first element as defined in any one of claims 1 to 17 in the preparation of a medicament formulated for coadministration with a second element as defined in any one of claims 1 to 17 for regulating the expression of a nucleic acid in vivo in a patient in need thereof.

35. Use of a second element as defined in any one of claims 1 to 17 in the preparation of a medicament formulated for coadministration with a first element as defined in any one of claims 1 to 17 for regulating the expression of a nucleic acid in vivo in a patient in need thereof.

36. Use of a first element as defined in any one of claims 1 to 17 in the preparation of a medicament formulated for sequential administration with a second element as defined in any one of claims 1 to 17 for regulating the expression of a nucleic add in vivo in a patient in need thereof.

37. Use of a second element as defined in any one of claims 1 to 17 in the preparation of a medicament formulated for sequential administration with a first element as defined in any one of claims 1 to 17 for regulating the expression of a nucleic acid in vivo in a patient in need thereof.

38. Cell modified by bringing into contact with a composition according to any one of claims 1 to 17 or a vector according to any one of claims 18 to 22.

39. Modified PPAR comprising several ligand-binding sites.

40. Nucleic acid encoding a PPAR according to claim 39.

41. Method for identifying PPAR ligands, comprising the bringing of a cell according to claim 38 into contact with a test molecule and the detection of the expression of the nucleic acid of interest.

42. Method for identifying PPAR ligands in vivo, characterized in that there is administered a composition according to any one of claims 1 to 17 or a vector according to any one of claims 18 to 22 as well as a test molecule, and in that the expression of the nucleic acid of interest Is detected.

43. Composition according to claim 1 substantially as hereinbefore described in any one of the Examples. Wlectual reoraJT OFFICE OF N.Z. - 9 FEB 2004 s RECEIVED -69-

44. Vector according to claim 18 substantially as hereinbefore described in any one of the Examples.

45. Use according to any one of claims 23, 24 or 28 to 37 substantially as hereinbefore described in any one of the Examples.

46. Method according to claim 25, 41 or 42 substantially as hereinbefore described in any one of the Examples.

47. Cell according to claim 38 substantially as hereinbefore described in any one of the Examples.

48. Modified PPAR according to claim 39 substantially as hereinbefore described in any one of the Examples.

49. Nucleic acid according to claim 40 substantially as hereinbefore described in any one of the Examples. INTELLECTUAL PRODCRTY OFFICE Or '7. - 9 FEB m RECEIVED I08660J.DOC WO 00/78986 PCT/FROO/01744 SEQUENCE LISTING <110> AVENTIS PHARMA S.A. <120> System for the pharmacological regulation of expression using PPAR nuclear receptors and their ligands <130> SEQUENCES <140> <141> <160> 28 <170> Patentln Ver. 2.1 <210> 1 <211> 19 <212? DNA <213> Homo sapiens <400> 1 tcaaccttta ccctggtag <210> 2 <211> 27 <212> DNA <213? Homo sapiens <400> 2 tcgccaagct tctcgtgatc tgcggca <210> 3 <211> 37 <212> DNA <213> Homo sapiens <400> 3 acgtgtcgac actagtggct agaggatctc taccagg <210> 4 <211> 48 <212> DNA <213> Homo sapiens 4400> 4 cgatggtacc ctcgagcaat gtgctagcga gatccttcaa cctttacc WO 00/78986 PCT/FROO/01744 1 <210> 5 <211> 13 <212 > DNA <213> Homo sapiens <400> 5 aggtcaaagg tea 13 <210> 6 <211> 69 <2X2> DNA <213> Homo sapiens <400> 6 acgtgtcgac actagtcaaa actaggtcaa aggtcacgga aaactaggtc aaaggtcacg 60 gaaaactag 6 9 <210> 7 <211?. 64 <212> DNA <213> Homo sapiens <4 00> 7 cgatggtacc ctcgagcaat gtgctagccg tgacctttga cctagttttc cgtgaccttt 60 gacc 64 <210? 8 <211> 32 <212> DNA <213> Homo sapiens <400> 8 aegtagatet eggtaggegt gtacggtggg ag 32 <210> 9 <211? 29 <212> DNA <213> Hotno sapiens <400> 9 aegtaagett ctatggaggt caaaacagc 29 <210> 10 <211> 21 <212> DNA <213> Homo sapiens <4 00> 10 WO 00/78986 PCT/FROO/01744 ggtttgctga atgtgaagcc c 21 <210> 11 <211? 42 <212> DNA <213> Homo sapiens <400> 11 agtctctaga gctacgcgta caagtccttg tagatctcct gc <210> 12 <211> 32 <212> DNA <213> Homo sapiens <400> 12 agtcacgcgt gggcgatctt gacaggaaag ac <210> 13 <211> 21 <212? DNA <213> Homo sapiens <400> 13 gcctttgagt gagctgatac c <210> 14 <211> 35 <212> DNA <213 > Homo sapiens <4 00> 14 agtcactagt aagctttttg ccgccagaac acagg <210> 15 <211> 36 <212> DNA <213> Homo sapiens <400> 15 agtcactagt ccatggctgc ccagtgcctc acgacc <210> 16 <211> 21 <212> DNA <213 > Homo sapiens <400? 16 caggtttgct aaatgtgaag c 21 WO 00/78986 PCT/FR00/01744 4 <210> 17 <211> 40 <212> DNA <213 > Homo sapiens <400> 17 tgacgtgtcg acctagtaca agtccttgta gatctcctgc 40 <210> 18 <211> 31 <212> DNA <213> Homo sapiens <210> 19 <211> 35 <212> DNA <213> Homo sapiens <400> 19 agtcgctagc gacggatcct tatcgatttt accac 35 <2l0s. 20 <211> 50 <212> DNA <213 :> Homo sapiens <210> 21 <211> 42 <212> DNA <213> Homo sapiens <400> 21 tacggggtac ccagacatga taagatacat tgatgagttt gg 42 <210> 22 <211> 33 <212> DNA <213> Homo sapiens <4 00> 18 agtcgtcgac gcttcgagca gacatgataa g 31 <400> 20 gtcagctagc ctactcgaac caccatgggt gaaactctgg gagattctcc SO <400> 22 gtcagctagc cggtaggcgt gtacggtggg agg 33 WO 00/78986 PCT/FR00/01744 <210> 23 <211> 33 <212> DNA <213> Homo sapiens <400> 23 tacgctcgag cttctatgga ggtcaaaaca gcg 3 3 <210> 24 <211> 750 <212> PRT <213> Homo sapiens <400> 24 Met Gly Glu Thr Leu Gly Asp Ser Pro lie Asp Pro Glu Ser Asp Ser 15 10 15 Pile Thr Asp Thr Leu Ser Ala Asn lie Ser Gin Glu Met Thr Met Val 20 25 30 Asp Thr Glu Met Pro Phe Trp Pro Thr Asn Phe Gly lie Ser Ser Val 35 40 45 Asp Leu Ser Val Met Glu Asp His Ser His Ser Phe Asp lie Lys Pro 50 55 60 Phe Thr Thr Val Asp Phe Ser Ser lie Ser Thr Pro His Tyr Glu Asp 65 70 75 80 lie Pro Phe Thr Arg Thr Asp Pro Val Val Ala Asp Tyr Lys Tyr Asp 85 90 95 Leu Lys Leu Gin Glu Tyr Gin Ser Ala lie Lys val Glu Pro Ala Ser 100 105 110 Pro Pro Tyr Tyr Ser Glu Lys Thr Gin Leu Tyr Asn Lys. Pro His Glu 115 120 125 Glu Pro Ser Asn Ser Leu Met Ala He Glu Cys Arg Val Cys Gly Asp 130 135 140 Lys Ala Ser Gly Phe His Tyr Gly Val His Ala Cys Glu Gly Cys Lys 145 150 155 160 Gly Phe Phe Arg Arg Thr lie Arg Leu Lys Leu lie Tyr Asp Arg Cys 165 170 175 Asp Leu Asn Cys Arg lie His Lys Lys Ser Arg Asn Lys Cys Gin Tyr 180 185 190 Cys Arg Phe Gin Lys Cys Leu Ala Val Gly Met Ser His Asn Ala lie 195 200 205 WO 00/78986 PCT/FR00/01744 Arg Phe Gly Arg Met Pro Gin Ala Glu Lys Glu Lys Leu Leu Ala Glu 210 215 220 lie Ser Ser Asp lie Asp Gin Leu Asn Pro Glu Ser Ala Asp Leu Arg 225 230 235 240 Ala Leu Ala Lys His Leu Tyr Aep Ser Tyr He Lys Ser Phe Pro Leu 245 250 255 Thr Lys Ala Lys Ala Arg Ala lie Leu Thr Gly Lys Thr Thr Asp Lys 260 265 270 Ser Pro Phe Val He Tyr Asp Met Asn Ser Leu Met Met Gly Glu Asp 275 280 285 Lys lie Lys Phe Lys His lie Thr Pro Leu Gin Glu Gin Ser Lys Glu 230 255 300 Val Ala lie Arg lie Phe Gin Gly Cys Gin Phe Arg Ser Val Glu Ala 305 310 315 320 Val Gin Glu lie Thr Glu Tyr Ala Lys Ser lie Pro Gly Phe Val Asn 325 330 33$ Leu Asp Leu Asn Asp Gin Val Thr Leu Leu Lys Tyr Gly Val Hie Glu 340 34S 350 He lie Tyr Thr Met Leu Ala Ser Leu Met Asn Lys Asp Gly Val Leu 355 360 365 lie Ser Glu Gly Gin Gly Phe Met Thr Arg Glu Phe Leu Lys Ser Leu 370 375 380 Arg Lys Pro Phe Gly Asp Phe Met Glu Pro Lys Phe Glu Phe Ala Val 385 390 355 400 Lys Phe Asn Ala Leu Glu Leu Asp Asp Ser Asp Leu Ala He Phe lie 405 410 415 Ala Val lie lie Leu Ser Gly Asp Arg Pro Gly Leu Leu Asn Val Lys 420 425 430 Pro lie Glu Asp lie Gin Asp Asn Leu Leu Gin Ala Leu Glu Leu Gin 435 440 445 Leu Lys Leu Asn His Pro Glu Ser Ser Gin Leu Phe Ala Lys Leu Leu 450 455 460 Gin Lys Met Thr Asp Leu Arg Gin lie Val Thr Glu His Val Gin Leu 465 470 475 480 Leu Gin Val He Lys Lys Thr Glu Thr Asp Met Ser Leu His Pro Leu 485 490 495 f Leu Gin Glu lie Tyr Lys Asp Leu Tyr Ala Trp Ala lie Leu Thr Gly WO 00/78986 PCT/FR00/01744 7 500 505 510 Lys Thr Thr Asp Lys Ser Pro Phe Val lie Tyr Asp Met Asn Ser Leu 515 520 525 Met Met Gly Glu Asp Lys lie Lys Phe Lys His lie Thr Pro Leu Gin 530 535 540 Glu Gin Ser Lys Glu Val Ala lie Arg lie Phe Gin Gly Cys Gin Phe 545 550 555 560 Arg Ser Val Glu Ala Val Gin Glu lie Thr Glu Tyr Ala Lys Ser lie 565 570 575 Pro Gly Phe Val Asn Leu Asp Leu Asn Asp Gin Val Thr Leu Leu Lys 580 585 590 Tyr Gly Val His Glu lie lie Tyr Thr Met Leu Ala Ser Leu Met Asn 595 600 605 Lys Asp Gly Val Leu lie Ser Glu Gly Gin Gly Phe Met Thr Arg Glu 610 615 620 Phe Leu Lys Ser Leu Arg Lys Pro Phe Gly Asp Phe Met Glu Pro Lys 625 630 635 640 Phe Glu Phe Ala Val Lys Phe Asn Ala Leu Glu Leu Asp Asp Ser Asp 645 650 655 Leu Ala lie Phe lie Ala Val lie lie Leu Ser Gly Asp Arg Pro Gly 660 665 670 Leu Leu Asn Val Lys Pro lie Glu Asp lie Gin Asp Asn Leu Leu Gin 675 680 685 Ala Leu Glu Leu Gin Leu Lys Leu Asn His Pro Glu Ser Ser Gin Leu 690 695 700 Phe Ala Lys Leu Leu Gin Lys Met Thr Asp Leu Arg Gin'lie Val Thr 705 710 715 720 Glu Kis Val Gin Leu Leu Gin Val lie Lys Lys Thr Glu Thr Asp Met 725 730 735 Ser Leu His Pro Leu Leu Gin Glu He Tyr Lys Asp Leu Tyr 740 745 750 <210> 25 <211> 467 <212> PRT <213> Homo sapiens <:400> 25 WO 00/78986 PCT/FROO/01744 Met Met Gly Glu Asp Lys lie Lys Phe Lys His lie Thr Pro Leu Gin 15 10 15 Glu Gin Ser Lys Glu Val Ala lie Arg lie Phe Gin Gly Cys Gin Phe 20 25 30 Arg Ser Val Glu Ala Val Gin Glu lie Thr Glu Tyr Ala Lys Ser lie 35 40 45 Pro Gly Phe Val Asn Leu Asp Leu Asn Asp Gin Val Thr Leu Leu Lys 50 55 60 Tyr Gly Val His Glu lie lie Tyr Thr Met Leu Ala Ser Leu Met Asn 65 70 75 80 Lys Asp Gly Val Leu lie Ser Glu Gly Gin Gly Phe Met Thr Arg Glu 85 90 95 Phe Leu Lys Ser Leu Arg Lys Pro Phe Gly Asp Phe Met Glu Pro Lys 100 105 110 Phe Glu Phe Ala Val Lys Phe Asn Ala Leu Glu Leu Asp Asp Ser Asp 115 120 125 Leu Ala lie Phe lie Ala Val lie lie Leu Ser Gly Asp Arg Pro Gly 130 135 140 Leu Leu Asn Val Lys Pro lie Glu Asp lie Gin Asp Asn Leu Leu Gin 145 150 1S5 160 Ala Leu Glu Leu Gin Leu Lys Leu Asn His Pro Glu Ser Ser Gin Leu 165 170 175 Phe Ala Lys Leu Leu Gin Lys Met Thr Asp Leu Arg Gin lie Val Thr 180 185 190 Glu His Val Gin Leu Leu Gin Val He Lys Lys Thr Glu Thr Asp Met 195 200 205. Ser I^eu His Pro Leu Leu Gin Glu lie Tyr Lys Asp Leu Tyr Ala Trp 210 215 220 Ala lie Leu Thr Gly Lys Thr Thr Asp Lys Ser Pro Phe Val He Tyr 225 230 235 240 Asp Met Asn Ser Leu Met Met Gly Glu Asp Lys lie Lys Phe Lys His 245 250 255 lie Thr Pro Leu Gin Glu Gin Ser Lys Glu Val Ala lie Arg lie Phe 260 265 270 Gin Gly Cys Gin Phe Arg Ser Val Glu Ala Val Gin Glu lie Thr Glu 275 280 285 / Tyr Ala Lys Ser He Pro Gly Phe Val Asn Leu Asp Leu Asn Asp Gin WO 00/78986 PCT/FROO/01744 9 290 295 300 Val Thr Leu Leu Lys Tyr Gly Val His Glu lie lie Tyr Thr Met Leu 305 310 315 320 Ala Ser Leu Met Asn Lys Asp Gly Val Leu He Ser Glu Gly Gin Gly 325 330 335 Phe Met Thr Arg Glu Phe Leu Lys Ser Leu Arg Lys Pro Phe Gly Asp 340 345 350 Phe Met Glu Pro Lys Phe Glu Phe Ala Val Lys Phe Asn Ala Leu Glu 355 360 365 Leu Asp Asp Ser Asp Leu Ala He Phe lie Ala Val lie lie Leu Ser 370 375 380 Gly Asp Arg Pro Gly Leu Leu Asn Val Lys Pro lie Glu Asp lie Gin 385 390 395 400 Asp Asn Leu Leu Gin Ala Leu Glu Leu Gin Leu Lys Leu Asn His Pro 405 410 415 Glu Ser Ser Gin Leu Phe Ala Lys Leu Leu Gin Lys Met Thr Asp Leu 420 425 430 Arg Gin lie Val Thr Glu His Val Gin Leu Leu Gin Val lie Lys Lys 435 440 445 Thr Glu Thr Asp Met Ser Leu His Pro Leu Leu Gin Glu He Tyr Lys 450 455 460 Asp Leu Tyr 465 <210> 26 <2ll> 30 <212> DNA <213> Homo sapiens <400> 26 cccgttacat aacttacggt aaatggcccg 30 <210> 27 <211> 30 <212> DNA <213> Homo sapiens <400> 27 gggacgcgct tctacaaggc gctggccgaa 30 WO 00/78986 PCT/FR00/01744 10 <210> 2B <211> 30 <212> DNA <213> Homo sapiens <400> 28 cgactctaga agatcttgcc ccgcccagcg 30 </div>