Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2830/00—Vector systems having a special element relevant for transcription
  • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
  • C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2830/00—Vector systems having a special element relevant for transcription
  • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2830/00—Vector systems having a special element relevant for transcription
  • C12N2830/42—Vector systems having a special element relevant for transcription being an intron or intervening sequence for splicing and/or stability of RNA
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2830/00—Vector systems having a special element relevant for transcription
  • C12N2830/80—Vector systems having a special element relevant for transcription from vertebrates
  • C12N2830/85—Vector systems having a special element relevant for transcription from vertebrates mammalian
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2840/00—Vectors comprising a special translation-regulating system
  • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron

Definitions

• the present invention relates to the field of biology. It relates in particular to the field of regulation of gene expression and, more particularly, it describes the development and development of a new system for pharmacological regulation of the expression of transgenes.
• the invention is based in particular on the use of constructs of human origin to activate the transcription of the transgene.
• the invention thus describes new compositions, constructions and methods allowing the efficient regulation of the expression of a nucleic acid in vitro, ex vivo or in vivo, for example in muscle cells.
• the applications which flow from the present invention are numerous, in the experimental, clinical, therapeutic or diagnostic fields, for example.
• control of the level and duration of expression of the transgenes is necessary for many applications.
• the success of the therapy may require a specific assay of the protein synthesized from the transgene.
• the production of recombinant proteins in vitro can be improved by using inducible expression systems, making it possible for example to decouple the growth and production phases. Building transgenic animals, studying the effects of a gene or the bioavailability of a protein, etc. are all situations in which appropriate control of gene expression can be implemented and make improvements.
• a first illustration of these regulators was constructed by fusion of the Lac repressor of E. coli with the transactivating domain of VP16 of the herpes simplex virus (HSV).
• IPTG isopropyl bD-thiogalactoside
• Another system was constructed by fusing the Tet repressor from E. coli with the VP16 transactivator domain from HSV.
• Another system was built by fusion of the DNA binding domain of the GAL4 protein of S. cerevisiae with the ligand binding domain of the human progesterone receptor and the transactivating domain of HSV VP16, this version is activated. by a progesterone analog such as RU486 (Wang Y. et al., Proc NatI Acad Sci USA, 91 (1994) 8180-8184).
• RU486 Wang Y. et al., Proc NatI Acad Sci USA, 91 (1994) 8180-8184.
• a fusion of the Drosophila ecdysone receptor with the HS16 VP16 transactivating domain has also been described, activated by ecdysone and analogues of this steroid hormone (No D. et al., Proc NatI Acad Sci USA, 93 (1996) 3346-3351).
• a transcriptional regulator then consists of two protein subunits, the first can be formed by the fusion of a binding domain to chimeric DNA and three copies of the human protein FKBP and the second by the fusion of the domain of binding to rapamycin of the human protein FRAP and of the transactivating domain of the p65 subunit of human NFkB.
• 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).
• these transcription regulators are xenogenic proteins in humans. They are in fact made up of protein fragments originating from bacteria, viruses, yeasts or insects or, when the protein domains are of human origin, their junction creates sequences foreign to man.
• the present invention provides a solution to these problems.
• the present invention indeed relates to a regulatory system using an activator of human origin. This should make it possible to avoid repeated administrations of the therapeutic gene.
• the present invention describes in particular an improved system of expression mductible using nuclear receptors PPAR (Peroxisome proiiferator-activated receptors) as transc ⁇ ptional regulators.
• PPAR Peroxisome proiiferator-activated receptors
• the use of PPRE in a hepatospecific expression system was described in application WO 98/21349
• the improved system according to the invention now makes it possible to produce the transcriptional regulator (a PPAR protein of human origin, and therefore essentially not xenogenic in humans), and the mductible promoter, 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).
• PPRE PPAR response element
• the system of the invention can be activated, in vitro and also in vivo, in particular in muscle, by the specific ligands of PPARs.
• 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 therefore has many advantages, both in terms of significant induction, tolerance (especially for use in vivo), strength and conditions of use
• the invention therefore describes new constructs for the production and implementation of this system, in particular promoter regions, expression cassettes and plasmids.
• the invention also describes new constructs of PPAR allowing improved control of the expression of genes, as well as combinations of these different constructs.
• the invention further shows that these methods and compositions allow significant control and regulation of expression in vitro and in vivo
• the invention also relates to cells comprising constructs of the invention, as well as methods for screening for PPAR ligand compounds, for example.
• a first subject of the invention resides in a composition comprising:
• a first element comprising a nucleic acid of interest under the control of a mductible promoter comprising a response element to a PPAR and a minimal transcriptional promoter
• a second element comprising a nucleic acid encoding a PPAR under the control of a transcriptional promoter, with a view to their simultaneous, separate or spaced-apart use over time.
• compositions of the invention further comprise (c) a PPAR gand, also for simultaneous, separate or time-spaced use.
• elements (d) comprising a nucleic acid encoding a retmoide X receptor (RXR) under the control of a transcriptional promoter
• RXR retmoide X receptor
• elements (a), (b), if appropriate (c), and / or (d) can be prepared separately, packaged separately , and used sequentially, to allow control of the expression of the nucleic acid of interest
• elements (a) and (b), and optionally (d) are prepared and packaged together, while compound c) is packaged separately and used spaced over time with (a) and (b), and possibly (d), the combination of these different elements in a cell, tissue, organ, etc. leading to the regulatory effect expression sought
• the elements (a) and (b) and possibly (d) are carried by separate genetic constructions.
• the elements (a) and (b) and optionally (d) are assembled in the same genetic construction.
• the present invention thus describes complex genetic constructions allowing the expression of a product of interest and a PPAR. These constructions are particularly advantageous since they alone contain all of the genetic elements necessary for the regulated expression of the nucleic acid of interest.
• the genetic construct (s) may be of varied nature and / or origin, in particular plasmid, episomal, chromosomal, viral, phage, etc.
• the genetic construct is a plasmid or viral vector
• plasmids carrying the elements separately ( a) or (b) mention may be made, for example, of the plasmids JxnS-TK-pGL3, JxnAS-TK-pGL3, DRI xnS- TK-pGL3, DR1xnAS-TK-pGL3, JxnAS-CMV-pGL3, pSG5-hPPARg2g2, or Jx10AS -CMV-EF-pGL3, which will be described in detail later.
• plasmids in which the elements (a) and (b) have been assembled mention may, for example, be made of the plasmids Jx5AS-TK-Luc-hPPARg2, SV-g2-J10-C-pGL3. hPPARg2-CMV-Jx5AS-TK-pGL3 or hPPARg2-CMV-Jx10AS-CMV-pGL3, which will be described in detail later.
• a viral vector there may be mentioned in particular a recombinant adenovirus, a recombinant retrovirus, an AAV, a herpes virus, a vaccinia virus, etc., the preparation of which can be carried out according to methods known to those skilled in the art. .
• element (a) comprises an inducible promoter comprising at least:
• a PPAR response element (PPRE, "Peroxysome Proliferator Response Element”) is a region of nucleic acid capable of binding a PPAR, the binding of PPAR can then mediate a signal to neighboring nucleic regions.
• a response element to a PPAR is therefore a region of nucleic acid capable of binding PPARs.
• the response element to a PPAR more particularly comprises one or more binding sites of the PPAR. Such binding sites have been described in the prior art, such as, for example, in various human promoters (apolipoprotein Ail gene, for example). Such sites can also be constructed artificially, and tested for their PPRE properties, as described below.
• the response element to a PPAR comprises one or more sites of sequence TCAACCTTTACCCTGGTAG (SEQ ID NO: 1) or of functional variants of this sequence.
• the sequence SEQ ID NO: 1 corresponds to the J region of the human apoAII promoter (nucleotides -734 to -716).
• the response element to a PPAR comprises one or more sites of sequence AGGTCAAAGGTCA (SEQ ID NO: 5) or functional variants of this sequence.
• the sequence SEQ ID NO: 5 corresponds to the consensus region DR1.
• the term functional variant designates any modified sequence retaining the properties of PPRE as mentioned above, that is to say in particular the capacity to bind a PPAR.
• the modifications may include one or more additions, mutations, deletions and / or substitutions of nucleotides in the sequence considered. These modifications can be introduced by conventional methods of molecular biology, such as in particular site-directed mutagenesis or, more practically, by artificial synthesis of the sequence in a synthesizer.
• the variants retain at least 50% of the residues of the initial sequence indicated. More preferably, the variants have modifications affecting less than 5 nucleotides in the sequence considered.
• the variants thus obtained are then tested for their PPRE activity. This property can be checked in different ways, including:
• a variant is considered to be functional within the meaning of the present invention when the activity measured, for example in (ii) above, is preferably at least 50% equal to that measured with a site of sequence SEQ ID NO: 1 or 5, more preferably at least equal to 75%.
• Functional variants of PPAR binding sites within the meaning of the invention are described for example in Juge-Aubry et al (J Biol. Chem 272 (1997) 25252) and in Nakshat ⁇ et al. (NAR 26 (1998) 2491), incorporated herein by reference
• Retmoid X receptors are coded by three genes RXR ⁇ , RXR ⁇ , and RXR ⁇ , whose isolation and sequence have been described (Mangelsdorf DJ et al (1990) Nature 345, 224-229, Mangelsdorf DJ et al. ( 1992), Genes Dev 6, 329-344)
• element (d) codes for human RXR ⁇
• the PPAR response element can comprise several sites for binding to a PPAR. It can 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 from 3 to 20, more preferably from 5 to 15.
• a preferred embodiment of the invention is a construction comprising from 10 to 15 binding sites , the results present in the examples indeed show the advantageous properties of such constructions in terms of induction and expression levels, in particular in muscle cells
• the PPAR response element is associated with a minimal transcriptional promoter.
• the minimal promoter is a transcriptional promoter having a weak or nonexistent basal activity, and capable of being increased in the presence of a transcriptional activator (the interaction of an active PPAR with the element PPRE).
• a minimal promoter can therefore be a naturally weak promoter in mammalian cells, i.e. producing a non-toxic and / or insufficient expression to obtain a pronounced biological effect.
• a minimal promoter is a construct prepared from a native promoter, by deletion of region (s) not essential (s) to transcriptional activity.
• it is preferably a promoter essentially comprising a TATA box, generally of a size less than 160 nucleotides, centered around the codon for initiating transcription.
• a minimal promoter can thus be prepared from viral, cellular, strong or weak promoters, such as for example the promoter of the thymidine kinase (TK) gene of the herpes virus, the immediate CMV promoter, the PGK promoter.
• TK thymidine kinase
• the promoter of the muscle creatine kinase (MCK) gene the promoters of the genes of the various actin isoforms of skeletal muscle, the promoter of the desmin gene, the promoter of the vimentin gene, the promoters of the chain genes light or heavy chain of myosin, etc.
• MCK muscle creatine kinase
• Specific examples of minimum promoters are represented by nucleotides -54 to +48 of CMV or
• the minimal promoter (Pmin), the PPAR response element (PPRE) and the nucleic acid of interest (AN) are functionally arranged in element (a), that is to say so that the minimal promoter controls the expression of the nucleic acid of interest and that its activity is regulated by the element PPRE.
• element (a) Generally, these regions are therefore arranged in the following order, in the 5 '-> 3' orientation: PPRE-Pmin-AN.
• any other functional arrangement can be envisaged by a person skilled in the art without departing from the present invention.
• the different functional domains above can be linked directly to each other, or separated by nucleotides which do not significantly affect the regulated nature of the promoter of element (a).
• nucleotides can be functionally neutral residues, resulting for example from cloning steps (PCR ends, restriction sites, etc.).
• These nucleotides can also have biological properties, making it possible to confer improved characteristics or performances to the system of the invention (amplifier of household genes, amplifier of specific tissues, silencer, intron, splicing site, etc.).
• the inducible promoter further comprises an amplifying region.
• Such a region advantageously makes it possible to increase the expression levels of the nucleic acid of interest.
• an amplifying region (E) is preferably positioned 3 'to the minimum promoter, between the latter and the nucleic acid of interest, according to the following scheme (5'-> 3 '): PPRE-Pmin-E-AN.
• the minimal promoter and the response element to a PPAR can be present either in the same orientation (that is to say in the direction of transcription), or in reverse orientation (that is to say that the response element to a PPAR is in the antisense orientation with respect to transcription by the Pmin promoter).
• these two embodiments allow effective control of the regulation of expression in vitro as well as in vivo.
• element (b) of the compositions according to the invention comprises at least: - a nucleic acid encoding a PPAR,
• PPARs belong to the nuclear hormone receptor superfamily, and are grouped into three distinct groups, PPAR ⁇ , PPAR ⁇ (also called NUC-1 or PPAR ⁇ ) and PPAR ⁇ .
• PPAR ⁇ also called NUC-1 or PPAR ⁇
• PPAR ⁇ also called NUC-1 or PPAR ⁇
• PPAR ⁇ nuclear hormone receptor superfamily
• the isolation and sequence of many human PPARs have been described in the literature (see especially 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. Biol. Chem., 272 (1997) 18779-18789; Mukherjee R. et al., J. Biol.
• the nucleic acid encoding a PPAR encodes a human PPAR, in particular a PPAR ⁇ or a PPAR ⁇ .
• it is a PPAR ⁇ or a PPAR ⁇ in its native form, that is to say without modification of primary structure with respect to the natural molecule.
• it is a modified PPAR comprising several ligand binding sites.
• the present invention describes and also relates to any modified PPAR comprising several ligand binding sites. More particularly, it is a PPAR ⁇ or a PPAR ⁇ , even more preferably a PPAR ⁇ .
• the PPARs modified according to the invention comprise from 2 to 5 ligand binding sites, more preferably from 2 to 4 binding sites. It is more particularly PPAR comprising 2 to 5 copies of the E and F domains involved in ligand binding.
• the PPAR proteins contain different domains: the N-terminal A / B domain which contains a non-ligand-dependent transactivating region, the C domain which is the DNA binding domain (DBD), the D domain which is a hinge region , and the E / F domains which contain a ligand-dependent transactivating region.
• 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).
• LBD ligand binding domain
• the boundaries of the E / F domains vary from one PPAR to another.
• the E / F domain extends from amino acid 284 to amino acid 505.
• modified PPARs comprising several repeating E and F domains, and that these modified PPARs are functional and have improved properties of inducibility by the ligands of the PPARs.
• Such constructions therefore represent an embodiment and a particular object of the present invention.
• a typical example of PPAR modified according to the invention is a PPAR ⁇ comprising 2 ligand binding sites (that is to say two domains E and F).
• the protein sequence of PPAR ⁇ 2 ⁇ 2 is represented in the sequence SEQ ID NO: 24.
• the invention also relates to any variant of the sequence SEQ ID NO: 24 retaining a PPAR type activity (the ability to activate, in the presence of a PPAR ligand such as BRL49653, a promoter comprising a PPRE sequence).
• the variants are understood to mean any mutant, dei réelle, and / or polypeptide comprising one or more additional residues.
• the invention also relates to any nucleic acid coding for such a modified PPAR.
• It may be a DNA (in particular a cDNA or a synthetic or semi-synthetic DNA) or an RNA.
• This DNA can be constructed according to conventional molecular biology methods known to those skilled in the art (synthesis, ligations, screening of libraries, etc.). It is advantageously any nucleic acid comprising a sequence encoding a polypeptide of sequence SEQ ID NO: 24, or hybridizing with a sequence encoding a polypeptide of SEQ ID NO: 24, and encoding a polypeptide with activity of PPAR type.
• this DNA may include a promoter and / or a transcriptional terminator, for example.
• the second transcriptional promoter controlling the expression of the nucleic acid encoding PPAR, may be any strong or weak, ubiquitous or selective, constitutive or regulative promoter, functional in mammalian cells, in particular in human cells. It can be a domestic cellular promoter (ie, a mammalian gene, in particular a human gene), a viral, bacterial, insect, plant promoter, natural or synthetic, simple or complex, etc.
• suitable promoters for this element (b) are in particular viral promoters (immediate promoter of the SV40 virus, immediate promoter of the CMV virus, retrovirus LTR, promoter TK of the herpes virus) or cellular (PGK promoter, albumin, EF1 ⁇ , or of genes strongly expressed in the muscle as: promoter of the muscle creatin kinase (MCK) gene, promoters of the genes of the various actin isoforms of skeletal muscle, promoter of the desmin gene, promoter of the vimentme gene , promoters of the light chain or heavy chain genes of myosin).
• viral promoters immediate promoter of the SV40 virus, immediate promoter of the CMV virus, retrovirus LTR, promoter TK of the herpes virus
• PGK promoter cellular
• albumin albumin
• EF1 ⁇ EF1 ⁇
• genes strongly expressed in the muscle as: promoter of the muscle creatin kinase (MCK) gene, promoters of the genes
• the promoter can be modified by the introduction of one or more enhancer regions, such as the enhancer region of intron 2 of the beta globule gene, enhancer of the very early gene of the CMV virus, enhancer of EF1 ⁇ , of region (s) silencer, of regions conferring tissue specificity (for example regions isolated from specific tissue promoters such as: promoter of the muscle creatin kinase (MCK) gene, promoters of the genes of the various actin isoforms of skeletal muscle, promoter of the desmin gene, promoter of the vimentin gene, promoters of the light chain or heavy chain genes of myosin) or a controllable character, or by deletion of regions not essential to activity, for example.
• tissue specificity for example regions isolated from specific tissue promoters such as: promoter of the muscle creatin kinase (MCK) gene, promoters of the genes of the various actin isoforms of skeletal muscle, promoter of the desmin gene, promoter of the vimentin gene, promoter
• the second promoter are the viral promoters, in particular the early promoter of the SV40 virus and the immediate promoter of the CMV, or derivatives thereof. Furthermore, in a particular mode of implementation, when the elements (a) and (b), and possibly (d), are assembled in the same genetic construct, the second transcriptional promoter (of element (b)) and the inducible promoter of element (a), and optionally the promoter of element (d) can be grouped to form only one promoter region common, in particular bidirectional, as will be explained in detail later in the text.
• Another object of the present invention resides in a vector comprising an element (a) and an element (b), and optionally an element (d), as defined above.
• the elements (a) and (b), and possibly (d), are in the same orientation in the vector.
• Such a variant is illustrated for example by the plasmid SV-g2-J10-C-pGL3 (FIG. 17).
• the elements (a) and (b), and possibly (d), are in opposite orientation in the vector.
• Such a variant is illustrated for example by the plasmids represented in FIGS. 16, 18 and 19.
• the inducible promoter of element (a) and the transcriptional promoter of element (b) are assembled into the vector to form a regulatable bidirectional promoter.
• Such a mode of implementation is illustrated for example by the plasmids represented in FIGS. 18 and 19.
• a particular object of the invention resides in a vector characterized in that it comprises, in the 5 ′ direction -> 3 ', a first nucleic acid encoding a PPAR, a first minimal transcriptional promoter controlling the expression of said first nucleic acid, one or more response element (s) to a PPAR, a second minimal transcriptional promoter and, under the control said second minimal transcriptional promoter, a second nucleic acid encoding a product of interest.
• This type of construction is advantageous since it allows the co-expression of the two nucleic acids in 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 generally activated in the presence of a PPAR ligand (element (c)).
• a PPAR ligand (element (c)
• different types of ligands can be used, natural or synthetic.
• the activating ligands of PPAR ⁇ are for example fibrates such as fibric acid and its analogs.
• gemfibrozyl As analogs of fibric acid, mention may be made in particular of gemfibrozyl (Atherosclerosis 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), ciinofibrate (Kidney International. 44 (6) (1993) 1352), pirinixic acid (Wy-14,643) or 5,8,11,14-eicosatetranoic acid (ETYA). These different compounds are compatible with biological and / or pharmacological use in vitro or in vivo.
• the PPAR ⁇ activating ligands can be chosen from natural and synthetic ligands.
• 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 thiazolidinediones, such as especially rosiglitazone (BRL49653), pioglitazone or troglitazone (see for example Krey G. et al., Mol. Endochnol., 11 (1997) 779-791 or Kliewer S. and Willson T., Curr. Opin. in Gen. Dev 8 (1998) 576-581) or the compound RG12525.
• compositions according to the invention may comprise several activators of PPAR in combination, and in particular a fibrate or a fibrate analog associated with a retinoid.
• a subject of the invention is also the use of a composition or a vector as defined above for expressing a nucleic acid of interest in a cell ex vivo or in vitro.
• the nucleic acid can be any nucleic acid (DNA, RNA) coding for a product of interest (RNA, protein, polypeptide, peptide, etc.). It can be a product of agrifood, therapeutic, vaccine interest, a marker, etc.
• the invention also relates to the use of a composition or a vector as defined above for the preparation of a product intended for expressing a nucleic acid of interest in a cell in vivo
• the invention also relates to a method for the regulated expression of a nucleic acid in a cell, comprising bringing said cell into contact with a composition or a vector as defined above.
• the cells can be brought into contact with the compositions or vectors of the invention according to different protocols.
• the cells in culture can be incubated directly with the elements (a), (b) and (c), and optionally (d), of the invention, for example with a vector comprising the elements (a) and (b ) and in the presence of ligand (c).
• the cells can be incubated first with elements (a) and (b), and optionally (d) (in particular assembled in the same vector) then, secondly (after culture and possibly selection of modified cells), item (c) can be added.
• This latter type of protocol makes it possible, for example, to decouple the culture (or cell expansion) phase from the expression phase of the nucleic acid.
• the cells are brought into contact by administration of elements (a), (b) and (c), and optionally (d), vivo, simultaneously, separately or spaced over time.
• elements (a) and (b), and optionally (d), possibly in the form of a single genetic construction are generally administered parenterally, in particular intramuscular, intravenous, subcutaneous, intradermal, intratumoral or stereotaxic
• the compositions of the invention may include any agent promoting cell transfection (cationic polymer, lipid, etc.).
• the compositions are administered intramuscularly, and the genetic constructs are used in the form of "naked" nucleic acid, that is to say without added transfection agent.
• the ligand (c) can be administered before, simultaneously, or after elements (a) and (b), and optionally (d).
• the administration of the ligand can be carried out by oral, anal, intravenous, intraperitoneal or intramuscular route, for example.
• typical doses of ligand such as BRL 49653 are between 5 and 50 mg / kg, for example 30 mg / kg, making it possible to obtain a plasma concentration close to 15 ⁇ g. / ml at least.
• typical doses are lower, generally less than 5 mg / kg, for example from 0.01 to 1 mg / kg.
• the results presented in the examples advantageously show that the compositions of the invention make it possible to obtain in vivo a strong and regulated expression, at doses of ligand lower than those usually used.
• the results presented also show that the expression is strong after a single intake of ligand.
• the vector doses used can vary between
• the invention can be used to express a gene in different types of cells, tissues or organs, in vitro, ex vivo or in vivo.
• it may be a cell, a tissue or a mammalian organ, preferably a human.
• muscle or a muscle
• hepatic or liver
• cardiac or heart, arterial or vascular wall
• nerve or brain, marrow, etc.
• tumor cells or a tumor).
• 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 the advantages of the invention in vivo or in vitro in this type of cells.
• the invention also relates to any cell modified by contacting with a composition or a vector as defined above.
• the invention also relates to the use of a composition, a vector or a cell as defined above, in which the nucleic acid of interest is a reporter gene (such as, for example, luciferase or secreted alkaline phosphatase) for screening in vitro, ex vivo or in vivo (in particular in muscle cells or a muscle) of PPAR ligands.
• the invention also describes a method for identifying PPAR ligands comprising bringing a cell as defined above into contact with a test molecule (or composition), and demonstrating a expression of the nucleic acid of interest (this preferably being a reporter gene). The expression can also 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 test compound.
• the invention also relates to the use of a composition or a vector as defined above, for the construction of transgenic animals, in particular of non-human mammals, useful for preclinical studies, or for studies of bioavailability, labeling, etc.
• a composition or a vector as defined above for the construction of transgenic animals, in particular of non-human mammals, useful for preclinical studies, or for studies of bioavailability, labeling, etc.
• the present invention will be described in more detail with the aid of the following examples which should be considered as illustrative and not limiting.
• Figure 1 Schematic representation of the plasmid FTKpGL3.
• Figure 2 Schematic representation of the plasmid Jx3S-TK-pGL3.
• Figure 3 Schematic representation of the plasmid Jx3AS-TK-pGL3.
• Figure 4 Schematic representation of the DR1x3S-TK-pGL3 plasmid.
• Figure 5 Schematic representation of the DR1x3AS-TK-pGL3 plasmid.
• Figure 6 Activities of 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), or Jx3AS-TK-pGL3 (c), or DR1x3S-TK-pGL3 (d), or DR1x3AS -TK-pGL3 (e), (ii) increasing amounts of plasmid pSG5-hPPARg2, and (iii) 20 ng of plasmid pRL-null.
• the activity of each inducible promoter represents the luciferase activity of Photinus pyralis normalized by the luciferase activity of Renilla reniformis.
• Figure 7 Activities of 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), or Jx3AS-TK-pGL3 (c), or DR1x3S-TK-pGL3 (d), or DR1x3AS -TK-pGL3 (e), (ii) increasing amounts of plasmid pSG ⁇ -hPPARa (Koz), and (iii) 20 ng of plasmid pRL-null.
• each inducible promoter represents the luciferase activity of Photinus pyralis normalized by the luciferase activity of Renilla reniformis.
• Figure 8 Schematic representation of the plasmid Jx5AS-CMV-pGL3
• Figure 9 Activities of inducible promoters evaluated in transient m vitro transfections in mouse myoblasts (C2C12)
• the cells are cotransfected with (i) 10 ng of plasmid Jx5AS-TK-pGL3 (a), or Jx5AS- CMV-pGL3 (b) , (u) increasing amounts of plasmid pSG5-hPPARg2, and (m) 20 ng of plasmid pRL-null
• the activity of each inducible promoter represents the luciferase activity of Photinus pyralis normalized by the activity of the luciferase of Renilla reniformis
• FIG. 10 Activities of inducible promoters evaluated in transient transfections in vitro in mouse myoblasts (C2C12).
• the cells are cotransfected with (i) 10 ng of plasmid JxnAS-TK-pGL3, (n) 10 ng of plasmid pSG5-hPPARg2, and (m) 20 ng of plasmid pRL-null
• the activity of each inducible promoter represents the luciferase activity of Photinus pyralis normalized by the activity of renilla reniformis luciferase
• FIG. 11 Activities of inducible promoters evaluated in transient m vitro transfections in mouse myoblasts (C2C12)
• the cells are cotransfected with (i) 10 ng of plasmid JxnAS-CMV-pGL3, (M) 10 ng (a) or 50 ng ( b) of plasmid pSG5-hPPARg2, and (ni) 20 ng of plasmid pRL-null
• the activity of each inducible promoter represents the luciferase activity of Photinus pyralis normalized by the activity of the luciferase of Renilla reniformis
• FIG. 12 Schematic representation of the plasmid pSG5-hPPARg2g2
• FIG. 13 Comparison of hPPARg2 and hPPARg2g2 transcriptional regulators.
• Mouse myoblasts (C2C12) are cotransfected with (i) 10 ng of plasmid Jx10AS-CMV-pGL3, (n) increasing amounts of plasmid pSG5-hPPARg2 (a) or pSG5-hPPARg2g2 (b), and (m) 20 ng of plasmid pRL-null
• the activity of the mductible promoter represents, the luciferase activity of Photinus pyralis normalized by the activity of Renilla reniformis luciferase.
• Figure 14 Schematic representation of the plasmid Jx10AS-CMV-EF-pGL3.
• Figure 15 Activities of inducible promoters evaluated in transient transfections in vitro in mouse myoblasts (C2C12).
• the cells are cotransfected with: (i) 10 ng of plasmid Jx10AS-CMV-pGL3 (a), or JxIOAS- CMV-EF-pGL3 (b), (ii) increasing amounts of plasmid pSG5- hPPARg2g2, and (iii) 20 ng of plasmid pRL-null.
• the activity of each inducible promoter represents the luciferase activity of Photinus pyralis normalized by the luciferase activity of Renilla reniformis.
• Figure 16 Schematic representation of the plasmid Jx5AS-TK-luc-hPPARg2.
• Figure 17 Schematic representation of the plasmid SV-g2-J10-C-pGL3.
• Figure 18 Schematic representation of the plasmid hPPARg2-CMV-Jx5AS-TK-pGL3.
• Figure 19 Schematic representation of the plasmid hPPARg2-CMV-Jx10AS-CMV-pGL3.
• Figure 20 Comparison of the different versions of the inducible system in vitro.
• Mouse myoblast of (C2C12) are transfected with, for each version of the system, the same number of moles of expression cassettes inducible. The results are expressed as a percentage of the activity of the hCMV-IE promoter, obtained using the plasmid pCMV-leadTK.
• Induction factors by BRL49653 are calculated by dividing the activity in the presence of BRL49653 by the activity in the presence of DMSO.
• Figure 21 In vitro comparison of ligands BRL49653 and RG12525.
• Mouse myoblasts C2C12 are transfected with: (i) 10 ng of plasmid hPPARg2-CMV-Jx10AS-CMV-pGL3, the expression cassette of which is presented in (a) and (ii) 10 ng of plasmid pRL-null .
• the activity of the inducible promoter represents the luciferase activity of Photinus pyralis normalized by the activity of the luciferase of Renilla reniformis.
• Figure 22 Comparison of different versions of the inducible system in vivo.
• C57BI / 6 mice (6 mice per group) are injected bilaterally into the cranial tibial with, for each version of the system, the same number of moles of inducible expression cassettes.
• An electrotransfer is then applied to each muscle.
• the treated animals receive, daily, by gavage, 30 mg / kg of BRL49653.
• Four days after the DNA injection the animals are sacrificed and the muscles are removed to measure the luciferase activity.
• Figure 23 Comparison, in vivo, of different induction protocols with BRL49653.
• C57BI / 6 mice (6 mice per group) are injected bilaterally into the cranial tibial with 10 ⁇ g of DNA containing 1 ⁇ g of the plasmid hPPARg2- CMV-Jx10AS-CMV-pGL3, the expression cassette of which is presented in (at).
• the activities obtained with the different induction protocols are collated in panel (b).
• Figure 24 Schematic representation of the plasmid pRDA02.
• Figure 25 Induction kinetics obtained in vivo with the inducible system.
• mice Ten C57BI / 6 mice are injected bilaterally into the cranial tibial with a DNA mixture containing 3 mg of plasmid pRDA02 and 3 mg of piasmid pSG5-hPPARg2. An electrotransfer is then applied to each muscle. Four days, then 39 days after the DNA injection, the animals are treated, by gavage, with 30 mg / kg of BRL49653. At different times, blood samples are taken on heparin, and the enzymatic activity of secreted alkaline phosphatase (hSeAP) is measured in the plasma, using the Phospha-Light TM Kit (Tropix, PE Biosystems, Foster City, CA ).
• hSeAP secreted alkaline phosphatase
• mice C57BI / 6 mice (2 groups of 10 mice) are injected bilaterally, into 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 to each muscle.
• the animals receive, by gavage, either a single dose of BRL49653 (30 mg / kg), or one dose per day (30 mg / kg) for 5 days.
• blood tests on heparin are carried out, and the enzymatic activity of hSeAP is measured in the plasma, using the "Phospha-Light" Kit (Tropix).
• the results presented correspond to the ratio between the activity of hSeAP 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 into 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 to each muscle.
• Figure 27 Schematic representation of the plasmid Jx10AS-CMV-VEGF A 165.
• the plasmid pGL3-Basic, used for cloning the different promoter regions, as well as the plasmid 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 construction of the plasmid pCMV-leadTK was also described previously in the patent application FR 98/120000 of 25/1598 and in the 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 and coli contains the CMV promoter (-522 / + 72) merges with " leader ”of the HSV tk gene (+ 51 / + 101) upstream of a sequence coding for the hemagglutinin epitope
• the plasmid pGCN (10ng) was used as template for PCR amplification
• the primers which have been used are the following
• Primer 6718 (5 'CCCGTTACATAACTTACGGTAAATGGCCCG 3') (SEQ ID NO 26), this primer is hybridized with the CMV promoter in position - 522 (8 nucleotides downstream of the EcoRI site of pCGN) - Primer 6719 (5 'GGGACGCGCTTCTACAAGGCGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGA )
• this primer hybridizes to position 101 of the "leader" tk
• the first nucleotide G in bold is intended to restore the Ncol site of pGL3-Bas ⁇ c as will be explained below
• the PCR fragment thus obtained is purified and then phosphorylated using the phage T4 polynucleotide kinase (New Engiands Biolabs).
• the vector pGL3-Bas ⁇ c (Promega) was linearized by Ncol, purified and then treated with Klenow DNA.
• the guanosine (G) of the primer 6719 restores the Ncol site only when the CMV-leader tk fragment is oriented with the 5 ′ part (primer 6718, position -522 of CMV) downstream of the HindIII site of pGL3-Bas ⁇ c and its 3 ′ end (primer 6719, leader tk) is ligated to the Ncol site of pGL3-Basic (first luciferase ATG)
• the plasmid obtained is designated pCMV-leadTK
• the enzymatic amplification of DNA fragments by the PCR technique can be carried out using a DNA thermal cycler TM (Perkm Elmer Cetus) according to the manufacturer's recommendations
• the electroporation of plasmid DNA in Escherichia coli cells can be carried out using an electroporator (Bio-Rad) according to the manufacturer's recommendations.
• Verification of the nucleotide sequences can be carried out by the method developed by Sanger et al. (Proc. NatI. Acad. Sci. USA, 74 (1977) 5463-5467) using the kit distributed by Applied Biosystems according to the manufacturer's recommendations.
• the C2C12 murine myoblasts are cultured in DMEM TM medium (Life Technologies Inc.) supplemented with 10% fetal calf serum (SVF).
• DMEM TM medium Life Technologies Inc.
• SVF fetal calf serum
• the transfections are carried out in 24-well plates and each transfection is carried out three times. Twenty four hours before transfection, the cells are seeded at 3 ⁇ 10 4 cells per well in DMEM TM medium.
• 500 ng of plasmid DNA (plasmids of interest and pBluescript II SK + to make up to 500 ng) are mixed with the cationic lipid RPR120535 B (W097 / 18185) at the rate of 6 nmol of lipid per ⁇ g of DNA in DMEM TM medium (20 ⁇ l final) comprising 150 mM NaCl and 50 mM bicarbonate.
• the 20 ⁇ l 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 SVF or ULTROSER TM (BioSepra Inc.) 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 SVF or ULTROSER TM. Forty-eight hours after transfection, the culture medium is removed and the cells are rinsed twice with PBS (Life Technologies Inc.).
• the activity of Photinus pyralis luciferase and the activity of Renilla reniformis luciferase are then determined using the Dual-Luciferase Reporter Assay System TM kit (Promega Corporation) according to the supplier's recommendations.
• the in vivo gene transfer experiments are carried out on 6 week old female C57BI / 6 mice. Animals are anesthetized with 250 ⁇ l of a ketamine (Rhône Mérieux, 10 mg / ml final) / Xylazine (Bayer Pharma, 0.3 mg / ml final) mixture intraperitoneally. An injection of a total amount of 10 ⁇ g of DNA is then performed in each cranial tibial muscle.
• Each leg is then subjected to an electric field (frequency of 1 Hz; 4 20 ms dots at 250 V / cm).
• the animals receive each morning, by gavage, either 30 mg / kg of BRL49653 (SmithKIine Beecham) in 1% carboxycellulose (weight / volume), or 1% carboxycellulose alone.
• BRL49653 SmithKIine Beecham
• the animals are sacrificed and the muscles removed in PLB TM lysis buffer (Promega Corporation) in Lysing Matrix TM tubes (BIO 101, Inc.).
• the crushing of the muscles, which makes it possible to extract the luciferase, is carried out using the FastPrep TM device (BIO 101, Inc.) for 25 seconds at 6.5 m / s.
• the activity of Photinus pyralis luciferase is then determined using the Luciferase Assay System TM kit (Promega Corporation) according to the supplier's recommendations.
• EXAMPLE 1 Construction of promoters inducible by PPARs and expression plasmids containing them.
• the JxnAS-TK-pGL3 plasmids differ from the JxnS-TK-pGL3 plasmids by the orientation of the J sites present in the inducible promoter.
• a schematic representation of the plasmid Jx3AS-TK-pGL3 is presented in Figure
• AS antisense orientation
• plasmids contain, as an element of response to PPAR (PPRE), a consensus sequence (AGGTCA A AGGTCA, SEQ ID NO: 5) called DR1 consensus.
• the DR1xnAS-TK-pGL3 plasmids differ from the DR1xnS-TK-pGL3 plasmids in the orientation of the consensus DR1 sites present in the inducible promoter.
• a DNA fragment, containing one or more consensus DR1 sequences, was amplified by PCR using the oligonucleotides 1 RDA69 and 2RDA64 as primers.
• This fragment was digested with Sali and Nhel and then was cloned, in the antisense orientation, into the plasmid FTKpGL3 previously digested with Xhol and Nhel to obtain the plasmids DR1x2AS-TK-pGL3 and DR1x3AS-TK-pGL3 DR1 consensus sites present.
• a schematic representation of the plasmid DR1x3AS-TK-pGL3 is presented in FIG. 5.
• EXAMPLE 2 Specificity of PPARs for different response elements.
• EXAMPLE 3 Construction of promoters inducible by PPARs containing a minimum promoter other than that of HSV1 -TK such as for example the minimum promoter of hCMV-IE.
• Plasmid Jx5AS-CMV-pGL3 was digested with Sphl and Nhel to isolate the Sphl-Nhel fragment of 982 bp containing 5 copies of site J, the minimum promoter hCMV-IE and the 5 'part of the gene coding for luciferase. This fragment was inserted into the plasmid Jx5AS-CMV-pGL3 previously digested with Sphl and Spel to give the plasmid Jx10AS-CMV-pGL3. The plasmids Jx15AS-CMV-pGL3 and Jx20AS-CMV-pGL3 were also obtained by following the same strategy.
• EXAMPLE 5 Construction of a transcriptional regulator highly inducible by PPAR ligands.
• the fragment A, digested with Sacl and Mlul and fragment B, digested with Mlul and Xbal were cloned together in the plasmid pSG5-hPPARg2 previously digested with Sacl and Xbal to obtain the plasmid pSG5-hPPARg2g2.
• This plasmid contains complementary DNA which codes for a transcriptional regulator (denoted hPPARg2g2) comprising two copies of the E and F domains, ie two ligand binding domains.
• PPAR ⁇ 2 ⁇ 2 The complete sequence of PPAR ⁇ 2 ⁇ 2 is shown below (SEQ ID NO: 24): MGETLGDSPIDPESDSFTDTLSANISQEMTMVDTEMPFWPTNFGISSVDLSVMEDHSHSFDI KPFTTVDFSSISTPHYEDIPFTRTDPWADYKYDLKLQEYQSAIKVEPASPPYYSEKTQ YN KPHEEPSNS MAIECRVCGDKASGFHYGVHACEGCKGFFRRTIRLKLIYDRCDLNCRIHKKS RNKCQYCRFQKCLAVGMSH AIRFGRMPQAEKEKLI_AEISSDIDQLNPESADLRAAKHLYD SYIKSFPLTKAKARAILTGKTTDKSPFVIYDMNSI ⁇ VIMGEDKIKFKHITPLQEQSKEVAIRIF QGCQFRSVEAVQEITEYAKSIPGFVNLDLNDQVTLLKYGVHE1IYTMLASLMNKDGVLISEG QGF TREFLKS
• sequence of the C-terminal part of PPAR ⁇ 2 ⁇ 2, comprising the domains E and F, is the following sequence SEQ ID NO: 25 -
• hPPARg2g2 The presence of a second ligand-binding domain (hPPARg2g2) therefore gives the transcriptional regulator greater inducibility by the ligand.
• EXAMPLE 6 Increase in the final activity of inducible promoters.
• EXAMPLE 7 Construction of plasmids comprising both an expression cassette for the transcriptional regulator and an inducible expression cassette.
• the plasmid pSG5-hPPARa (Koz) was digested with Mlul and Seal 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 SmaI to give the plasmid pSL-
• the plasmid pSG5-hPPARa (Koz) was digested with SalI and Mlul to isolate the Sall-MIul 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 pSL-3'hPPARa previously digested with Xhol and Mlul to give the plasmid pSL-hPPARa.
• the plasmid pSL-hPPARa was digested with Spel and SalI to isolate the Spel-SalI fragment of 2664 bp containing the early promoter of the SV40 virus and the complementary DNA of hPPARa. This fragment was inserted into the plasmid pBluescript II SK + previously digested with Spel and SalI to give the plasmid pBS-hPPARa.
• the plasmid pSG5-hPPARg2 was digested with Ayril and SacI to isolate the Ayrll-SacI fragment of 2070 bp, noted C, containing the 5 ′ region of the DNA complementary to hPPARg2.
• Fragment C and fragment D digested with Sac1 and SalI, were cloned together in the plasmid pBS-hPPARa previously digested with Ayril and Sali to obtain the plasmid pBS-hPPARg2.
• Plasmid Jx5AS-TK-pGL3 was digested with Kpnl and SalI to isolate the Kpnl-SalI fragment of 2324 bp containing the luc + gene under the control of an inducible promoter. This fragment was inserted into the plasmid pBS-hPPARg2 previously digested with Kpnl and SaN to give the plasmid Jx5AS-TK-luc-hPPARg2.
• a schematic representation of the plasmid Jx5AS-TK-luc-hPPARg2 is presented in FIG. 16.
• the plasmid pBS-hPPARg2 was digested with NotI and SalI to isolate the NotI-SalI fragment of 2622 bp, noted E, containing the complementary DNA of hPPARg2 under the control of the SV40 early promoter.
• HPPARg2-CMV-Jx5AS-TK-pGL3 plasmid A DNA fragment, denoted G, containing the DNA complementary to hPPARg2, was amplified by PCR using 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.
• G A DNA fragment, denoted Jx5AS-TK-luc- hPPARg2
• 12RDA50 5 'GTC AGC TAG CCT ACT CGA GCC ACC ATG GGT GAA ACT CTG GGA GAT TCT CC 3
• Plasmid Jx5AS-CMV-pGL3 was digested with Nhel and Sphl 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-Jx5AS-TK-pGL3 previously digested with Spel and Sphl to give the plasmid hPPARg2-CMV-Jx10AS-CMV-pGL3.
• a schematic representation of the plasmid hPPARg2-CMV-Jx10AS-CMV-pGL3 is presented in Figure 19.
• Plasmid Jx5AS-CMV-pGL3 was digested with Nhel and Sphl 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-Jx10AS-CMV-pGL3 previously digested with Spel and Sphl to give the plasmid hPPARg2-CMV-Jx15AS-CMV-pGL3.
• the plasmid Jx10AS-CMV-pGL3 was digested with Nhel and Sphl to isolate the Nhel-SphI 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-Jx10AS-CMV-pGL3 previously digested with Spel and Sphl to give the plasmid hPPARg2-CMV-Jx20AS-CMV-pGL3.
• Figure 20 combines the results obtained in vitro with several versions of the inducible system. These results show that the systems using two plasmids (Figure 20 lines 1, 3 and 4) like the systems with a single 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 of the gene placed under the control of the inducible promoter.
• a PPARg ligand here BRL49653
• the factor of induction by the ligand is greater than 30, and that for the system presented on figure 20 line 4, the activity after induction is equal to that of a strong promoter like that of the hCMV-IE promoter.
• ligands of hPPARg other than BRL49653, here RG12525 ligand RPR of hPPARg
• RG12525 ligand RPR of hPPARg
• treatment with RG12525 even leads to a stronger induction • than that obtained with BRL49653.
• Any other PPARg ligand can therefore be used as a system inducer.
• a system using hPPARa as a transcriptional regulator can be activated with the fibrates or WY-14,643 for example or any other ligand of hPPARa.
• the inducible system can be activated in vivo, in the muscle.
• FIG. 22 brings together the results obtained in vivo, in the muscle, with different versions of the inducible system.
• the results show that for the three versions tested (FIG. 22 lines 2 to 4), a treatment by gavage with a ligand of hPPARg is capable of strongly increasing, in the muscle, the activity of the inducible promoters.
• the induction factors are: x14 for the version figure 22 line 2, x8 for the version figure 22 line 3, and x24 for the version figure 22 line 4.
• the activity obtained in animals treated with BRL49653 is of the order of that of a strong promoter such as the hCMV-IE promoter.
• results, presented in FIG. 23, also show that a single take of ligand can induce the system, whether this take place before or after the gene transfer. This experience also shows that a dose twice less than that usually used, allows to obtain the same induction factor.
• the system, using a PPAR nuclear receptor as a transcriptional regulator, is therefore functional in vivo and can be induced by the oral intake of a PPAR ligand.
• EXAMPLE 11 Construction of a plasmid allowing the inducible expression of a gene whose product is secreted.
• Plasmid Jx10AS-CMV-pGL3 was digested with HindIII and Mlul to isolate the HindIII-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 HindIII and Mlul to give the plasmid pRDA02.
• This plasmid contains DNA complementary to the gene coding for the secreted form of human placental alkaline phosphatase (hSeAP), the expression of which is under the control of a promoter inducible by the system using PPARs as transcriptional regulator.
• hSeAP human placental alkaline phosphatase
• EXAMPLE 12 The inducible system makes it possible to regulate, in vivo, the plasma concentration of a secreted protein.
• FIG. 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, and this with a simple oral intake of a ligand of PPAR .
• the plasma concentration of hSeAP is increased by a factor of 18 (Figure 25A) two days after taking the ligand, then returns to its basic level a week later. Between 2 and 39 ⁇ eme ee day, an immune response directed against the hSeAP of human origin is observed and results in a decrease in plasma concentration this protein. Despite this immune response, it is possible to perform a second induction cycle (FIG. 25A).
• the inducible system also makes it possible, by taking daily ligands, to maintain the plasma level of hSeAP at a high level, for a period equal to the duration of the treatment.
• EXAMPLE 13 Different ligands of PPARs can activate the inducible system in vivo, and this in a dose-dependent manner.
• BRL49653 in its form marketed for the treatment of type II diabetes (Avandia TM, SmithKiine Beecham) and pioglitazone, in its form marketed for the same treatment (Actos TM, Takeda Pharmaceuticals) can also activate the inducible system (Figure 26A ).
• Figure 26B also shows that the induction factor is directly correlated to the dose of ligand used.
• the system using a nuclear PPAR receptor as a transcriptional regulator, therefore makes it possible to very precisely control the plasma level of a secreted protein.
• this regulation can be achieved by using various PPAR ligands.
• EXAMPLE 14 Construction of a plasmid allowing the inducible expression of a gene whose product is an angiogenic factor.
• 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 (1991) 1806-1814) and then inserted into a plasmid pBluescript
• plasmid pXL3218 (Stratagene) containing the CMV E / P promoter from position -522 to +72 and the SV40 late polyA, to give the plasmid pXL3218.
• the latter was then digested with HindIII and BsrGI to isolate the HindIII-BsrGI fragment A of 482 bp.
• the plasmid pXL3218 was also digested with BsrGI and BamHI to isolate the B fragment BsrGI-BamHI of 390 bp. Fragments A and B were inserted into the plasmid Jx10AS-CMV-pGL3 previously digested with HindIII and BamHI to give the plasmid Jx10AS-CMV-VEGF A 165.
• This plasmid contains the DNA complementary to the gene coding for VEGF A 165 of which expression is under the control of a promoter inducible by the system using PPARs as a transcriptional regulator.
• a schematic representation of the plasmid Jx10AS-CMV-VEGF A 165 is presented in FIG. 27.
• This piasmid can be used, for example, to control over time the angiogenic activity of VEGF for therapeutic purposes.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Genetics & Genomics (AREA)
• Chemical & Material Sciences (AREA)
• Engineering & Computer Science (AREA)
• Organic Chemistry (AREA)
• Zoology (AREA)
• Biotechnology (AREA)
• Molecular Biology (AREA)
• General Engineering & Computer Science (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Wood Science & Technology (AREA)
• Biomedical Technology (AREA)
• General Health & Medical Sciences (AREA)
• Biophysics (AREA)
• Biochemistry (AREA)
• Plant Pathology (AREA)
• Microbiology (AREA)
• Physics & Mathematics (AREA)
• Medicinal Chemistry (AREA)
• Toxicology (AREA)
• Animal Behavior & Ethology (AREA)
• Gastroenterology & Hepatology (AREA)
• Cell Biology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Pharmacology & Pharmacy (AREA)
• Epidemiology (AREA)
• Immunology (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
• Medicines Containing Material From Animals Or Micro-Organisms (AREA)
• Peptides Or Proteins (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Applications Claiming Priority (5)

Publications (1)

Family

ID=26234999

Family Applications (1)

Country Status (14)

Families Citing this family (5)

Family Cites Families (2)

• 2000
  • 2000-06-22 NZ NZ516678A patent/NZ516678A/en unknown
  • 2000-06-22 IL IL14710400A patent/IL147104A0/xx unknown
  • 2000-06-22 KR KR1020017016447A patent/KR20020028909A/ko not_active Application Discontinuation
  • 2000-06-22 CA CA002375869A patent/CA2375869A1/fr not_active Abandoned
  • 2000-06-22 HU HU0203584A patent/HUP0203584A2/hu unknown
  • 2000-06-22 CN CN00811928A patent/CN1370240A/zh active Pending
  • 2000-06-22 WO PCT/FR2000/001744 patent/WO2000078986A1/fr not_active Application Discontinuation
  • 2000-06-22 AU AU61645/00A patent/AU6164500A/en not_active Abandoned
  • 2000-06-22 EP EP00948058A patent/EP1190084A1/fr not_active Withdrawn
  • 2000-06-22 BR BR0011897-4A patent/BR0011897A/pt not_active IP Right Cessation
  • 2000-06-22 CZ CZ20014609A patent/CZ20014609A3/cs unknown
  • 2000-06-22 MX MXPA01013227A patent/MXPA01013227A/es unknown
  • 2000-06-22 JP JP2001505726A patent/JP2003503034A/ja not_active Withdrawn
• 2001
  • 2001-12-21 NO NO20016347A patent/NO20016347L/no not_active Application Discontinuation

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events