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  • C12N15/09—Recombinant DNA-technology
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  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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  • C12N2799/00—Uses of viruses
  • C12N2799/02—Uses of viruses as vector
  • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid

Definitions

• the present invention relates to a new system for gene expression. It also relates to the use of this system in gene or cell therapy, to increase the expression of genes of interest, or for the production of recombinant proteins.
• Gene and cell therapies consist in correcting a deficiency or an anomaly (mutation, aberrant expression, etc.) or in ensuring the expression of a protein of therapeutic interest by introducing genetic information into the affected cell or organ. .
• This genetic information can be introduced either ex vivo in a cell extracted from the organ, the modified cell then being reintroduced into the body (cell therapy), or directly in vivo in the appropriate tissue (gene therapy).
• Different techniques exist for performing gene transfer including various transfection techniques involving chemical or biochemical, natural or synthetic vectors such as DNA and DEAE-dextran complexes (Pagano et al., J. Virol.
• viruses as vectors for gene transfer.
• various viruses have been tested for their capacity to infect certain cell populations and in particular, retroviruses (RSV, HMS, MMS, etc.), HSV virus, adeno-associated viruses and adenoviruses.
• the present invention now describes a particularly efficient new gene expression system.
• the present invention stems in particular from the identification of regions endowed with transcription enhancer activity, making it possible to increase the strength of promoters, and thus the levels of gene expression.
• the present invention more particularly results from the identification of regions of the first intron of the tyrosine hydroxylase gene capable of increasing the activity of transcriptional promoters. These regions are located more precisely at the level of the HUMTH01 microsatellite present in the first intron of this gene.
• Microsatellites represent an abundant class of repeat DNA sequences which exhibit a large polymorphism linked to variations in the number of repeat motifs and / or in the sequence of these motifs. These microsatellites have therefore been used as genetic markers for the construction of genetic maps and for the identification of loci involved in pathologies.
• the size of the different alleles of a microsatellite depends on variations in the number of repeated patterns. Sequencing experiments on repeated dimers have thus made it possible to show a variation in the number of repeating units and in their sequence. These variations may correspond in particular to "perfect” repetitions, that is to say without interruption in the base sequence, or to "imperfect” repetitions, containing one or more interruptions in the sequence of patterns, whether deletion (s) or insertion (s).
• the HUMTH01 rpicrosatellite is located in the first intron of the tyrosine hydroxylase (TH) gene, which is the limiting enzyme in the catecholamine biosynthetic pathway. Part of the sequence of this intron is shown SEQ ID N. 1 (from nucleotide 871). This sequence includes the HUMTH01 microsatellite (allele (TCAT) 9, shown in bold), which is located at position 1170 (GenBank, accession # D00269). The HUMTH01 microsatellite is made up of repeated TCAT tetrameric patterns. It has a certain polymorphism, different alleles having been described having variable numbers of repeating units. The most frequently encountered allele (allele Ei) comprises 10 repeating units and a deletion of a base pair in the fifth repeating unit, which comprises the sequence CAT (SEQ ID N. 2). Other alleles have been identified carrying from 5 to 10 TCAT motifs.
• a first subject of the invention therefore relates to a DNA fragment having an activity of transcriptional enhancement consisting essentially of a portion of the first intron of the tyrosine hydroxylase gene.
• the fragment of the invention comprises less than 200 bp and includes an allele of the HUMTH01 microsatellite. More preferably, the fragment comprises less than 100 bp.
• the fragment consists essentially of an allele of the HUMTHOI microsateliite.
• the HUMTH01 microsatellite is located at position 1170 approximately of the human TH gene (GenBank accession # D00269), and consists mainly of repeated TCAT tetrameric motifs. Different alleles of this microsateilite have been described having variable numbers of repeating units (from 5 to 10) and variations in sequences, certain alleles having base mutations. Thus, the most frequently encountered allele (allele Ei) comprises 10 repeated motifs and a deletion of a base pair in the fifth repeated motif, which comprises the sequence CAT (SEQ ID N. 2). Other alleles have been identified carrying from 5 to 10 TCAT motifs (SEQ ID N. 9-15).
• a DNA fragment carrying 3 TCAT motifs can be synthesized and tested as a transcriptional enhancer, according to the invention.
• the invention also relates to an isolated DNA fragment characterized in that it has a transcriptional enhancer activity and in that it has the sequence (TCAT) n - (CAT) 0 - ( TCAT) p , in which n is between 1 and 50; o is between 0 and 20 and p is between 0 and 50.
• the transcriptional enhancer has a sequence (TCAT) fl - (CAT) 0 - (TCAT) p in which n is between 2 and 20 inclusive, and o and p are 0.
• the transcriptional enhancer has a sequence (TCAT) n - (CAT) 0 - (TCAT) p in which n is between 1 and 10, o is between 1 and 5 and p is included between 1 and 10.
• transcriptional enhancers there may be mentioned the fragments of sequence SEQ ID N.1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15.
• Another object of the present invention resides in an expression cassette comprising a coding sequence, a promoter allowing its expression and a transcription enhancer as defined above.
• the cassette also comprises a signal for termination of the transcription.
• the promoter is advantageously chosen from functional promoters in mammalian cells, preferably human. It may especially be a promoter allowing the expression of a nucleic acid in a hyperproliferative cell (cancerous, restenosis, etc.).
• various promoters can be used, such as the promoter of the p53 gene. They can also be regions of different origin (responsible for the expression of other proteins, or even synthetic). It can thus be any promoter or derived sequence stimulating or repressing the transcription of a gene in a specific way or not, inducible or not, strong or weak. Mention may in particular be made of the promoter sequences of eukaryotic or viral genes.
• promoters may be promoter sequences originating from the genome of the target cell.
• ubiquitous promoters can be used in particular (promoter of the HPRT, PGK genes, a-actin, tubulin, etc.), promoters of intermediate filaments (promoter of the GFAP genes, desmin, vimentin, neurofilaments, keratin, etc. ), promoters of therapeutic genes (for example the promoter of the MDR, CFTR, Factor VIII, ApoAl, ApoAII, Albumin, Thymidine kinase, etc.
• tissue-specific promoters promoter of the pyruvate kinase gene, of villin, of intestinal fatty acid binding protein, smooth muscle a-actin, promoter of neuronal specific enolase (Forss-Petter et al., Neuron 5 (1990) 187); etc), of the promoter generating the form VAChT PARNm V1 (acetylcholine transcporter: Cervini et al., J. Biol. Chem. 270 (1995) 24654) or promoters responding to a stimulus (steroid hormone receptor, retinoic acid receptor , etc).
• promoter sequences originating from the genome of a virus such as for example the promoters of the E1A and MLP genes of adenovirus, the early promoter of CMV, or also the promoter of the LTR of RSV or of the MMTV, the promoter of the TK gene of a herpes virus, etc.
• these promoter regions can be modified by addition or deletion of sequences.
• the promoter used may be a "minimal" promoter, that is to say a reduced promoter whose activity essentially depends on the presence of a transactivator such as a enhancer of the invention. Therefore, in the absence of the enhancer, the activity of the promoter is reduced and the gene is little or not expressed.
• the minimum promoter generally consists of a TATA or INR box. These elements are in fact the minimum elements necessary for the expression of a gene in the presence of a transactivator. It advantageously comprises less than 200 bp, including the TATA or INR region.
• the minimal promoter can be prepared from any promoter by genetic modification. As a specific example of a candidate promoter, mention may be made of the promoter of the thymidine kinase gene.
• the minimal promoter can also be derived from human CMV. In particular, it may consist of the fragment between the nucleotides -53 to +75 or -31 to +75 of the CMV (+1 corresponding to the ATG codon). Any conventional promoter can however be used such as for example the promoter of the genes coding for chloramphenicol acetylated transferase, ⁇ -galactosidase or even luciferase.
• the transcriptional promoter is a functional promoter in mammalian cells, and in particular a ubiquitous viral (TK, CMV) (PGK) or specific promoter (promoter of neuronal specific enolase, promoter generating the V1 form of mRNA of VAChT.
• TK ubiquitous viral
• CMV CMV
• PGK specific promoter
• promoter of neuronal specific enolase promoter generating the V1 form of mRNA of VAChT.
• the transcriptional promoter is a minimal promoter, essentially composed of a region of less than 200 bp comprising a TATA or INR box.
• the coding sequence advantageously comprises one or more sequences coding for proteins. It may especially be a sequence coding for a therapeutic product, whether it is a peptide, polypeptide, protein, ribonucleic acid, etc. More particularly, the coding sequence is a DNA sequence (cDNA, gDNA, synthetic DNA, human.animal, vegetable, etc.) coding for a protein product such as enzymes, blood derivatives, hormones, lymphokines: interleukins, interferons, TNF, etc. (FR 9203120), growth factors, neurotransmitters or their synthetic precursors or enzymes, trophic factors: BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NT5, etc.
• apolipoproteins ApoAl, ApoAIV, ApoE, etc (FR 93 05125), dystrophin or a minidystrophin (FR 9111947), tumor suppressor genes: p53, Rb, RaplA, DCC, k-rev, etc (FR 93 04745) , genes coding for factors involved in coagulation: Factors VII, VIII, IX, etc., or all or part of a natural or artificial immunoglobulin (Fab, ScFv, etc.), a ligand RNA (WO91 / 19813), etc. .
• the coding sequence can also be an antisense sequence, the expression of which in the target cell makes it possible to control the expression of genes or the transcription of cellular mRNAs.
• Such sequences can, for example, be transcribed, in the target cell, into RNAs complementary to cellular mRNAs and thus block their translation into protein, according to the technique described in patent EP 140 308.
• the present invention is also suitable for the expression of sequences coding for toxic factors. It can in particular be poisons for cells (diphtheria toxin, pseudomonas toxin, ricin A, etc.) of product inducing sensitivity to an external agent (suicide genes Thymidine kinase, cytosine deaminase, etc.) or even capable killer genes to induce cell death (Grb3-3 (PCT / FR94 / 00542), anti-ras ScFv (W094 / 29446), etc).
• This system is also particularly interesting for the expression of cytokines, interferons, TNF or TGF for example.
• the expression cassette advantageously consists of the following elements: - As enhancer region, a sequence (TCAT) n - (CAT) 0 - (TCAT) p , in which is not between 1 and 50; o is between 0 and 20 and p is between 0 and 50,
• the promoter consists of the region -109 to +52, or -37 to +19 of the promoter of the HSV-1 thymidine kinase gene.
• the enhancer region is chosen from the fragments of sequence SEQ ID N.1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15.
• a a particularly remarkable property of the enhancer regions of the invention is that they seem to be active both in the sense orientation (normal orientation of the microsatellite in the TH gene) and in the antisense orientation (orientation opposite to that of the microsatellite in the gene TH). Therefore, in the expression cassettes of the invention, the enhancer region can be positioned in the two orientations (5 '-> 3' or 3 '- * 5').
• Another aspect of the invention also resides in an expression vector comprising an expression cassette as defined above.
• the vector can be plasmid or viral in nature.
• the viral vectors there may be mentioned more preferably the adenoviruses, the retroviruses, the herpes viruses or even the associated adeno viruses.
• the viruses according to the present invention are defective, that is to say incapable of replicating autonomously in the target cell.
• the genome of the defective viruses used in the context of the present invention is therefore devoid of at least the sequences necessary for the replication of said virus in the infected cell. These regions can be either eliminated (in whole or in part), or made non-functional, or substituted by other sequences and in particular by the sequences of the invention.
• the defective virus nevertheless retains the sequences of its genome which are necessary for the packaging of the viral particles.
• adenoviruses of type 2 or 5 Ad 2 or Ad 5
• Ad 2 or Ad 5 adenoviruses of animal origin
• adenoviruses of animal origin mention may be made of adenoviruses of canine, bovine, murine origin,
• the adenovirus of animal origin is a canine adenovirus, more preferably an adenovirus
• CAV2 Manhattan strain or A26 / 61 (ATCC VR-800) for example.
• adenoviruses of human or canine or mixed origin are used.
• the genome of the recombinant adenoviruses of the invention comprises at least the ITRs and the packaging region of an adenovirus, and the nucleic acid sequence coding for a chimeric molecule and an expression cassette as defined above. before. More preferably, in the genome of the adenoviruses of the invention, the E1 region at least is non-functional.
• the viral gene considered can be made non-functional by any technique known to a person skilled in the art, and in particular by total suppression, substitution (for example by sequences of the invention), partial deletion, or addition of one or more bases in the gene or genes considered.
• the adenovirus according to the invention comprises a deletion in the E1 and E4 regions. According to another preferred embodiment, it comprises a deletion in region E1 at the level of which the region E4 and the sequences of the invention are inserted (Cf FR94 13355).
• the defective recombinant adenoviruses according to the invention can be prepared by any technique known to those skilled in the art (Levrero et al., Gene 101 (1991) 195, EP 185 573; Graham, EMBO J. 3 (1984) 2917). In particular, they can be prepared by homologous recombination between an adenovirus and a plasmid carrying, inter alia, the expression cassette of the invention and sequences approved for adenovirus. Approved recombination occurs after co-transfection of said adenovirus and plasmid in an appropriate cell line.
• the adenoviral genome can also be prepared in vitro, in a bacterium (FR95 01632) or in a yeast (WO95 / 03400).
• the cell line used for the production of adenoviruses must preferably (i) be transformable by said elements, and (ii), contain the sequences capable of complementing the part of the genome of the defective adenovirus, preferably in integrated form to avoid risks of recombination.
• a line mention may be made of the human embryonic kidney line 293 (Graham et al., J. Gen. Virol.
• Ad5 adenovirus (12%) or lines capable of complementing the E1 and E4 functions as described in particular in applications No. WO 94/26914 and WO95 / 02697. Then, the adenoviruses which have multiplied are recovered and purified according to conventional techniques of molecular biology, as illustrated in the examples.
• AAV adeno-associated viruses
• the defective recombinant AAVs according to the invention can be prepared by co-transfection, in a cell line infected with a human helper virus (for example an adenovirus), of a plasmid containing the expression cassette of the invention bordered by two inverted repeat regions (ITR) of AAV, and of a plasmid carrying the packaging genes (rep and cap genes) of AAV.
• a human helper virus for example an adenovirus
• ITR inverted repeat regions
• rep and cap genes packaging genes
• retroviruses are integrative viruses, selectively infecting dividing cells. They therefore constitute vectors of interest for cancer applications.
• the genome of retroviruses essentially comprises two LTRs, an encapsidation sequence and three coding regions (gag, pol and env).
• the gag, pol and env genes are generally deleted, in whole or in part, and replaced by a heterologous nucleic acid sequence of interest.
• These vectors can be produced from different types of retroviruses such as in particular MoMuLV ("murine moloney leukemia virus”; also designated MoMLV), MSV ("murine moloney sarcoma virus”), HaSV ("harvey sarcoma virus”) ; SNV ("spleen necrosis virus”); RSV ("rous sarcoma virus”) or the Friend virus.
• MoMuLV murine moloney leukemia virus
• MSV murine moloney sarcoma virus
• HaSV human moloney sarcoma virus
• SNV spleen necrosis virus
• RSV rous sarcoma virus
• Friend virus Friend virus
• a plasmid comprising in particular the LTRs, the packaging sequence and the sequences of the invention (expression cassette) is generally constructed, then used to transfect a cell line called packaging. , capable of providing deficient retroviral functions in the plasmid in trans.
• packaging lines are therefore capable of expressing the gag, pol and env genes.
• packaging lines have been described in the prior art, and in particular the line PA317 (US4,861, 719); the PsiCRIP line (WO90 / 02806) and the GP + envAm-12 line (WO89 / 07150).
• the recombinant retroviruses may include modifications at the level of the LTRs to suppress transcriptional activity, as well as extended packaging sequences comprising a part of the gag gene. O 97/40172 PC17FR97 / 00636
• the expression cassette can also be inserted into a plasmid vector (FR95 02117).
• the gene expression system of the invention is particularly suitable for use in gene or cell therapy, or for the production of recombinant proteins.
• the cassette or plasmid vectors can be used in the form of naked DNA, or complex to inert transfer vectors. Different types of inert vectors capable of promoting the transfer and expression of nucleic acids in eukaryotic cells have indeed been described.
• Chemical or biochemical vectors represent an alternative to viruses, in particular for reasons of convenience, security and also by the absence of a theoretical limit as regards the size of the DNA to be transfected.
• These synthetic vectors have two main functions, to compact the nucleic acid to be transfected and to promote its cell fixation as well as its passage through the piasmic membrane and, where appropriate, the two nuclear membranes. To compensate for the polyanionic nature of nucleic acids, the non-viral vectors all have polycationic charges.
• cationic polymers of polylysine type (LKLK) n, (LKKL) n, immine polyethylene (WO96 / 02655) and DEAE dextran or else cationic lipids or lipofectants are the most advantageous. They have the property of condensing DNA and promoting its association with the cell membrane. Among the latter, mention may be made of lipopolyamines (lipofectamine, transfectam, WO95 / 18863) and various cationic or neutral lipids (DOTMA, DOGS, DOPE, etc.).
• the present invention also relates to any pharmaceutical composition comprising a vector as defined above.
• These compositions can be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, etc. administration.
• the composition according to the invention contains pharmaceutically acceptable vehicles for an injectable formulation.
• They can be in particular saline solutions (monosodium phosphate, disodium, sodium chloride, potassium, calcium or magnesium, etc., or mixtures of such salts), sterile, isotonic, or dry compositions, in particular lyophilized, which, by addition, as appropriate, of sterilized water or physiological saline, allow the constitution of injectable solutes.
• retroviruses they may be advantageous to directly use packaging cells or cells infected ex vivo with a view to their reimplantation in vivo, possibly in the form of neo ⁇ organs (WO94 / 24298).
• the vector doses used for the injection can be adapted according to different parameters, and in particular according to the mode of administration used, the pathology concerned or the duration of the treatment sought.
• the recombinant viruses according to the invention are formulated and administered in the form of doses of between 10 4 and 10 14 pfu / ml.
• doses of 10 6 to January 10 0 pfu / ml may also be used.
• pfu plaque forming unit
• plaque forming unit corresponds to the infectious power of a suspension of virions, and is determined by infection of an appropriate cell culture, and measures, generally after 48 hours, the number of plaques of infected cells. The techniques for determining the pfu titer of a viral solution are well documented in the literature.
• Another subject of the invention consists of a cell comprising a DNA fragment, an expression cassette or a vector as defined above.
• These cells are obtained by any technique known to those skilled in the art allowing the introduction of DNA into a given cell. It may especially be transformation, transfection, infection, electroporation, conjugation, fusion of protoplasts or any other technique known to those skilled in the art. With regard to in vitro or ex vivo transformation, various protocols have been described in the prior art. In particular, the transformation of cells can be carried out by treating the whole cells in the presence of lithium acetate and of polyethylene glycol according to the technique described by Ito et al. (J. Bacteriol.
• the cells according to the invention can be of different origins.
• it may be bacteria or eukaryotic cells (yeasts, animal cells, plant cells) etc.
• eukaryotic cells yeasts, animal cells, plant cells
• the bacteria there may be mentioned more preferably JELfioJi, ⁇ . subtilis. Stre p tomvces. Pseudomonas (fL pjjtjdâ, £. Aemginosa ) .
• Rhizobium jnejMi Agrobacterium tumefaciens
• Staphylococcus aureus Streptomyces pristi ⁇ ae ⁇ PiraliS, Enterococcus faecium or Clostridium. etc.
• bacteria it is preferred to use E. coli.
• yeasts there may be mentioned Kluweromvces. Saccharomvces. Pichia. Hansenula. etc.
• mammalian animal cells mention may be made of CHO, COS, NIH3T3, PC12 cells, etc.
• hematopoietic stem cells WO88 / 08450, WO93 / 20195, WO93 / 09815, WO93 / 11230), endothelial cells (WO89 / 05345, WO90 / 06757, WO92 / 09222 ), myoblasts (WO93 / 03768, W093 / 24151, WO94 / 01129), fibroblasts (US 5,219,740, WO89 / 02468, WO93 / 07906), hepatocytes (WO89 / 07Î36, W092 / 12242, WO93 / 03142), astrocytes (WO94 / 01135), neuroblasts (WO94 / 10292, WO94 / 16059), keratinocytes (WO94 / 11011), macrophages (FR93 / 10222).
• These cells generally have the capacity to produce a product of therapeutic interest and can be implanted in vivo.
• blood cells mention may be made of erythrocytes, neutrophilic, basophilic and eosinophilic granulocytes, B and T lymphocytes, in particular CD4 lymphocytes, cytotoxic lymphocytes (CD8 CTL), tumor infiltrating lymphocytes (TIL) and LAK, monocytes and macrophages, dendritic cells, megakaryocytes and platelets.
• Figure 1 Representation of the plasmid pTK-Luc; minimal promoter Thymidine kinase; Perfect repetition, sense of TH intron; Perfect repetition, antisense; Imperfect repeat, sense of TH intron; Imperfect repetition, antisense.
• the enzymes were provided by New-England Biolabs (Beverly, MA).
• the DNA fragments are separated according to their size on 0.8 to 1.5% agarose gels, purified by GeneClean (BIO101,
• Amplification by PCR (Polymerase Chain Reaction), was also carried out according to Maniatis ⁇ î aJ., 1989, with the following specifications:
• oligonucleotides are synthesized using the chemistry of phosphoramidites protected in ⁇ by a cyanoethyl group, (Sinha e! SJ., 1984, Giles 1985), with the automatic DNA synthesizer Applied.
• the sequencing was carried out on double-stranded templates by the chain termination method using fluorescent primers.
• HUMTH01 microsatellite in the regulation of gene expression has been studied by gel retardation experiments (electrophoretic mobility test, EMSA). For this, nuclear extracts of Hela cells were prepared, and incubated in the presence of oligonucleotides corresponding to sequences of the HUMTH01 microsatellite.
• the nuclear extracts of Hela cells were prepared according to the Dignam method (NAR 11 (1983) 1474), and the protein concentrations according to the Bradford technique (Anal. Biochem. 72 (1976) 248).
• the electrophoretic mobility test was carried out in a final volume of 10 ⁇ l, in the presence of 10 to 15 ⁇ g of nuclear extract incubated for 5 min in the presence of 1 ⁇ g of poly (dl-dC) - (dl-dC), 4 mM HEPES buffer, pH 7.6, 100 mM KCI, 12% glycerol.
• oligonucleotides labeled with ⁇ ⁇ P-ATP by means of T4 polynucleotide kinase (Amersham) were then incubated for 5 min with the proteins, at room temperature.
• the oligonucleotides used are probes corresponding to the Ei and Ep alleles of the HUMTH01 microsatellite.
• unlabeled oligonucleotides were added 5 min before the labeled probes.
• the sequence of the oligonucleotides is as follows:
• Ep-sens TCATTCATTC ATTCATTCAT (SEQ ID N. 5)
• Ep-as ATGAATGAAT GAATGAATGA (SEQ ID N. 6)
• the DNA-protein complexes formed were then resolved by non-denaturing gel electrophoresis, 6% acrylamide in 0.5X TBE 4C buffer. After drying, the gels are autoradiographed overnight at room temperature.
• a series of competition experiments with different oligonucleotides was carried out.
• the oligonucleotides used correspond to the TRE sequence for binding of factor AP1 (TGACTCA).
• TGACTCA TRE sequence for binding of factor AP1
• competition experiments were also carried out with oligonucleotides containing other protein binding sequences: CRE-TH; CRE; Pou; SP1; AP4 and E-TH. None of these sequences, in molar excess of 100 times, made it possible to shift the binding of the nuclear extracts of the Hela cells to the oligonucleotides of the microsatellite, confirming the specificity of the interaction.
• oligonucleotides were synthesized on a Beckman oligo 1000 synthesizer, according to the manufacturer's recommendations.
• the enhancers produced are:
• This DNA fragment includes the imperfect allele of the HUMTH01 microsatellite, in the sense orientation (normal orientation in the TH intron)
• This DNA fragment includes the imperfect allele of the HUMTH01 microsatellite, in the antisense orientation (reverse orientation of the TH intron)
• This DNA fragment comprises the perfect allele of the HUMTH01 microsatellite ((TCAT) 10 ).
• a vector comprising a heterologous gene (luciferase) under the control of a TK promoter, as well as an enhancer according to the invention was constructed.
• the plasmid pTK-Luc (DeThe et al., Nature 343 (1990) 177) was used.
• This vector comprises a modified (minimal) TK promoter, comprising nucleotides -109 to +52.
• a similar construction is carried out with a smaller fragment (nucleotides -37 to +19).
• Different enhancer regions were introduced into this plasmid, upstream of the TK promoter. More particularly, the 54 mer enhancers described in Example 2.1.
• Vector including another promoter The vector described above can easily be modified to replace the TK promoter with any other transcriptional promoter desired, such as the early CMV promoter or any minimal promoter derived therefrom such as a promoter consisting of the fragment comprised between nucleotides -53 to +75 or -31 to +75 of the CMV. It can also be the promoter of the murine or human PGK gene, the neuronal specific penolase promoter, or the promoter promoter generating the V1 form of the VAChT mRNA. Furthermore, different vectors or cassettes carrying these different promoters have been described in the literature.
• the plasmid RSV-CAT was used in the experiments to determine the efficiency of cell transfection. This plasmid was therefore cotransfected with the vectors of the invention. This plasmid includes the gene
• This example describes a study of the activity of the sequences of the invention on the expression levels of genes in cells of a carcinoma.
• the cells studied are the Hela cells. These are cells from a human epithelial carcinoma. These cells were deposited at PATCC under the reference ATCC CCL-2.
• Hela cells were cultured in Dulbecco medium supplemented with 7% Supreme serum. 10 B cells were taken up in 0.15 ml of serum-free medium, and transfected by electroporation using the "Gene Extract "from Biorad, with 1 pmol of each vector to be tested, 5 pmol of vehicle DNA (Bluescript II), and 0.5 pmol of RSV-CAT plasmid making it possible to evaluate the transfection efficiency.
• the cells were harvested 24 and 48 hours after transfection, and tested in triplicate for luciferase activity
• the LUMAT LB9501 luminometer (Berthold) was used, with a reaction volume of 150 ⁇ l (luciferin 0.08 mM; ATP 0.1 mM; Tris- Phosphate 25 mM pH 7.8; MgCI2 8 mM; dithiothreitol 2mM; EDTA 1mM; Triton 1%; Glyceral 15%)
• the luciferase activity of each vector was then normalized to the CAT activity resulting from the vector RSV-CAT co-transfected.
• CAT activity was measured by liquid scintillation counting. Normalized luciferase activity is expressed in RLU (Relative Light Units). The results given are the means of three experiments.
• results obtained are presented in FIG. 2. They show that the T10i and T10p sequences both induce a strong increase in the expression of the gene.
• the T10i sequence induces an increase in luciferase activity by a factor of 90 and the T1 Op sequence by a factor of 160.
• the increase observed is by a factor of approximately 250 for the two sequences, when these are arranged in the reverse orientation.
• the increase in expression levels observed is approximately 300 to 350 times, for the two sequences, in the 2 orientations.
• This example describes a study of the activity of the sequences of the invention on the expression levels of genes in cells of a rat pheochromocytoma.
• the cells studied are the PC12 cells, deposited at ATCC under the reference ATCC CRL-1721.
• PC12 cells were cultured in RPMI medium supplemented with 10% horse serum and 5% fetal calf serum.
• the protocol transfection and activity measurement is identical to that used in Example 3 for Hela cells.
• results obtained are presented in FIG. 3. They show that the sequences T10i and T10p induce, whatever their orientation, an increase in the expression of the gene by a factor 25 after 24 hours and by a factor 50 after 48 hours.
• CCCTTCCCAG GCTCTAGCAG CAGCTCATGG TGGGGGGTCC TGGGCAAATA GGGGGCAAAA 240 TTCAAAGGGT ATCTGGGCTC TGGGGTGATT CCCATTGGCC TGTTCCTCCC TTATTTCCCT 300

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