WO2002034903A2 - Acide nucleique regulateur du gene abca7, molecules modulant son activite et applications therapeutiques - Google Patents

Acide nucleique regulateur du gene abca7, molecules modulant son activite et applications therapeutiques Download PDF

Info

Publication number
WO2002034903A2
WO2002034903A2 PCT/FR2001/003219 FR0103219W WO0234903A2 WO 2002034903 A2 WO2002034903 A2 WO 2002034903A2 FR 0103219 W FR0103219 W FR 0103219W WO 0234903 A2 WO0234903 A2 WO 0234903A2
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
transcription
abca7
polynucleotide
gene
Prior art date
Application number
PCT/FR2001/003219
Other languages
English (en)
French (fr)
Other versions
WO2002034903A3 (fr
Inventor
Patrice Denefle
Marie-Françoise ROSIER
Catherine Prades
Isabelle Arnould-Reguigne
José OSORIO Y FORTEA
Nicolas Duverger
Giovanna Chimini
Original Assignee
Aventis Pharma S.A.
Inserm
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from FR0013649A external-priority patent/FR2822165B1/fr
Application filed by Aventis Pharma S.A., Inserm filed Critical Aventis Pharma S.A.
Priority to JP2002537874A priority Critical patent/JP2004512045A/ja
Priority to EP01978557A priority patent/EP1334186A2/de
Priority to AU2002210661A priority patent/AU2002210661A1/en
Priority to CA002426294A priority patent/CA2426294A1/fr
Publication of WO2002034903A2 publication Critical patent/WO2002034903A2/fr
Publication of WO2002034903A3 publication Critical patent/WO2002034903A3/fr

Links

Classifications

    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • 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
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • 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
    • 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

Definitions

  • the present invention relates to a nucleic acid capable of regulating the transcription of the ABCA7 gene, which is a gene capable of integrating into lipid metabolism in the hematopoietic tissues, as well as in the cellular signaling mechanisms linked to the immune reaction. and inflammation.
  • the present invention also describes polypeptides and polynucleotides whose alteration of the sequence or expression is potentially implicated in diseases associated with the genetic locus q13 of chromosome 19.
  • the present invention also relates to nucleotide constructs comprising a polynucleotide coding for a polypeptide or producing a nucleic acid of interest, placed under the control of a nucleic acid regulating the human or murine gene ABCA7.
  • the invention also relates to recombinant vectors, transformed host cells, and non-human transgenic mammals, comprising a nucleic acid regulating the transcription of the human and mouse ABCA7 gene or a abovementioned nucleotide construct, as well as methods for the production. screening of molecules or substances capable of modulating the activity of the nucleic acid regulating the ABCA7 gene.
  • the invention further relates to methods for detecting an alteration in the transcription of the ABCA7 gene and thus for diagnosing a possible dysfunction in lipid metabolism in hematopoietic tissues and in cellular signaling mechanisms of immunity. It also relates to substances or molecules modulating the activity of the nucleic acid regulating the transcription of the ABCA7 gene as well as pharmaceutical compositions containing such substances or such molecules.
  • ABC transporter proteins (ATP-Binding Cassette) constitute a superfamily which is extremely conserved during evolution, from bacteria to humans. These proteins are involved in the membrane transport of various substrates, for example ions, amino acids, peptides, sugars, vitamins or even steroid hormones (Higgins et al., Annu Rev. Cell Biol, 8, (1992 ) 67-1 13).
  • ABC transporter proteins have been identified in humans and a number of them have been associated with various diseases.
  • cystic fibrosis is caused by mutations in the CFTR (cystic fibrosis transmembrane conductance regulator) gene, also known as ABCC7.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • ABC transporter PFIC2 or ABCB11
  • PFIC2 PFIC2
  • ABCB11 another ABC transporter, designated PFIC2 or ABCB11, seems to be involved in a form of progressive familial intrahepatic cholestasis, this protein being potentially responsible, in humans, for the export of bile salts.
  • ABCA ABC transporters
  • the members of this subfamily are also highly conserved during the evolution of multicellular eukaryotes.
  • the ABCA1 and ABCA4 transporters which are the best known have an identity of 95% and 88% respectively with their murine orthologs.
  • the members of this subfamily are also strongly related, since for example the transporters ABCA1 and ABCA4 have a protein sequence identity of 50.9%, as well as a very similar genomic organization (Allikmets et al., Nat. Genêt. (1997) ) 15, 236-2446; Broccardo et al., Biochim. Biophys.
  • ABCA1 there is an abnormal distribution of membrane phospholipids within the layers of the plasma membrane, which results more precisely in the presence in greater amount of phosphatidylserine in the outer layer, and a disruption in the concentration of Ca 2+ .
  • the transporters ABCA1 and ABCA4 have been particularly studied.
  • ABCA1 indeed seems to be involved in pathologies linked to a dysfunction of the cholesterol metabolism inducing diseases like atherosclerosis, or family deficiencies in HDL (FHD) like Tangier disease (FR 99/7684000; Rust et al., Nat.Genet, 22 (1999) 352-355; Brooks-Wilson et al., Nat. Genêt, 22 (1999) 336-345; Bodzioch et al., Nat.Genet. 22 (1999) 347-351; Orso and al., Nat. Genet, 24 (2000) 192-196). Tangier's disease seems to be linked to a cellular deficit in the translocation of cellular cholesterol which leads to a degradation of HDLs, and thereby a disturbance of lipoprotein metabolism.
  • HDL particles which do not incorporate cholesterol from peripheral cells, are not metabolized correctly, but are instead eliminated quickly from the body.
  • the HDL plasma concentration of these patients is therefore extremely reduced and HDLs no longer ensure the return of cholesterol to the liver.
  • This cholesterol accumulates in these peripheral cells and causes characteristic clinical manifestations such as the formation of orange tonsils.
  • other lipoprotein disturbances such as overproduction of triglycerides as well as increased synthesis and intracellular catabolism of phospholipids are observed.
  • the transporter ABCA4 has also been associated with degenerative and inflammatory diseases of the eye such as recessive Stargardt's disease (Allikmets et al., 1997) and degeneration of the macular region of the retina linked to age (AMD) (Allikmets and al., Nat. Genet. 15 (1997) 236-246; Allikmets et al., Science, 277 (1997) 1805-1807; Cremers et al., Hum. Mol. Genêt. (1998), 7 (3), 355-62; Martinez-Mir et al., Nat. Genêt. 18 (1998) 1 1 -12; Weng et al., Ce // (1999) 98 (1), 13-23).
  • degenerative and inflammatory diseases of the eye such as recessive Stargardt's disease (Allikmets et al., 1997) and degeneration of the macular region of the retina linked to age (AMD) (Allikmets and al., Nat. Genet. 15 (1997) 236-246
  • ABCA7 a cDNA comprising the entire open reading phase of a new member of the A family of ABC transporters (“ATP-Binding Cassette”) has recently been cloned from RNA of human macrophages, and is designated ABCA7 (Kaminski et al., BBR, 273 (2000), 532-538).
  • the characterization of the complete amino acid sequence of ABCA7 indicates that the protein product has the general structure characteristic of ABCA transporters, in that it comprises the symmetrical structure comprising the two transmembrane domains and two NBF motifs.
  • the ABCA7 protein has other motifs which have been recently
  • the transporter protein ABCA7 seems to have a regulatory profile dependent on sterol fluxes, similar to that of the other members of the subfamily A, and in particular of the transporter ABCA1 (Langman et al., BBR Corn; 257 (1999), 29-33; Laucken et al., PNAS, 97 (2000) 817-822). I! was indeed observed by Kaminski et al. (supra) an increase in the expression of ABCA7 after incubation of human macrophages in the presence of low density acetylated lipoproteins (AcLDL) which induce a sterol load, as well as a decrease in expression in the presence of the acceptor of HDL3 cholesterol which causes a decrease in the sterol load.
  • AcLDL low density acetylated lipoproteins
  • ABCA7 presents like the other ABCA members a certain specialization of its tissue expression, the messenger of ABCA7 being present predominantly in the hematopoietic tissues constituted by lymphocytes, granulocytes, thymus, spleen, bone marrow , or fetal tissues, whereas the expression of ABCA1 is predominant in macrophages and the placenta, and that of ABCA4 is restricted in the retina (Rust et al., Nat. Genêt, 22, (1999) 352-355 ).
  • the characterization of the regulatory sequences of the human ABCA7 gene would make it possible to detect mutations in patients, in particular to diagnose individuals belonging to family groups at risk. Furthermore, the isolation of these regulatory sequences would make it possible to complement the mutated sequence with a functional sequence capable of overcoming the metabolic dysfunctions induced by the mutation or mutations diagnosed, by means of the construction of targeted therapeutic means, such as means intended to gene therapy. b) The characterization of the regulatory sequences of the ABCA7 gene would make available to those skilled in the art means capable of allowing construction by genetic engineering and then the expression of genes determined in the cell types in which the ABCA7 gene is preferentially expressed.
  • the inventors have now isolated and then analyzed a human genomic DNA of 33.5 kb comprising the 46 exons of the open reading frame of the ABCA7 gene as well as the non-transcribed region of approximately 1.1 kb located on the 5 'side of exon 1 , upstream of the +1 transcription site, and comprising regulatory signals of the human ABCA7 gene.
  • the inventors also isolated and then analyzed a murine genomic DNA of 20Kb comprising the 45 exons of the open reading frame of the ABCA7 gene as well as the non-transcribed region of approximately 1.2Kb in the mouse located on the 5 'side of exon 1 , upstream of the +1 transcription site, and comprising signals for regulating the murine ABCA7 gene.
  • isolated in the sense of the present invention designates a biological material (nucleic acid or protein) which has been removed from its original environment (the environment in which it is naturally located).
  • a polynucleotide naturally occurring in a plant or animal is not isolated.
  • the same polynucleotide separated from adjacent nucleic acids within which it is naturally inserted into the genome of the plant or animal is considered to be "isolated”.
  • Such a polynucleotide may be included in a vector and / or such a polynucleotide may be included in a composition and nevertheless remain in an isolated state since the vector or the composition does not constitute its natural environment.
  • purified does not require that the material be present in a form of absolute purity, exclusive of the presence of other compounds. Rather, it is a relative definition.
  • a polynucleotide is in the "purified" state after purification of the starting material or of the natural material of at least one order of magnitude, preferably 2 or 3 and preferably 4 or 5 orders of magnitude.
  • nucleotide sequence can be used to denote either a polynucleotide or a nucleic acid.
  • nucleotide sequence encompasses the genetic material itself and is therefore not limited to information regarding its sequence.
  • nucleic acid includes RNA, DNA, cDNA or even RNA / DNA hybrid sequences of more than one nucleotide, in single chain form or in duplex form.
  • nucleotide designates both natural nucleotides (A, T, G, C) as well as modified nucleotides which comprise at least one modification such as (1) a purine analog, (2) a d analog '' a pyrimidine, or (3) a sugar analogous, examples of such modified nucleotides being described for example in PCT application No. WO 95/04064.
  • a first polynucleotide is considered to be "complementary" to a second polynucleotide when each base of the first nucleotide is paired with the base complementary to the second polynucleotide whose orientation is reversed.
  • the complementary bases are A and T (or A and U), or
  • variant of a nucleic acid is meant a nucleic acid which differs by one or more bases with respect to the reference polynucleotide.
  • a variant nucleic acid may be of natural origin, such as an allelic variant found naturally, or may also be an unnatural variant obtained for example by mutagenesis techniques.
  • the differences between the reference nucleic acid and the variant nucleic acid are reduced so that the nucleotide sequences of the reference nucleic acid and the variant nucleic acid are very close and, in many regions , identical.
  • the nucleotide modifications present in a variant nucleic acid can be silent, which means that they do not alter the amino acid sequences encoded by said variant nucleic acid.
  • nucleotide changes in a variant nucleic acid can also produce substitutions, additions, deletions in the polypeptide encoded by the variant nucleic acid with respect to the peptides encoded by the reference nucleic acid.
  • modifications of nucleotides in coding regions can produce substitutions, conservative or non-conservative in the amino acid sequence.
  • the variant nucleic acids according to the invention code for polypeptides which retain substantially the same biological function or activity as the polypeptide of the reference nucleic acid or the ability to be recognized by antibodies directed against the polypeptides coded by the initial nucleic acid.
  • nucleic acids will thus code for mutated forms of polypeptides whose systematic study will make it possible to deduce structure activity relationships from the proteins in question. Knowledge of these variants in relation to the studied disease is fundamental since it allows to understand the molecular cause of the pathology.
  • fragment will be understood to mean a reference nucleic acid according to the invention, a nucleotide sequence of reduced length compared to the reference nucleic acid and comprising, on the common part, a nucleotide sequence identical to the nucleic acid of reference.
  • Such a “fragment” of nucleic acid according to the invention may, where appropriate, be included in a larger polynucleotide of which it is constitutive.
  • Such fragments comprise, or alternatively consist of, oligonucleotides of length ranging from 20 to 25, 30, 40, 50, 70, 80, 100, 200, 500, 1000 or 1500 consecutive nucleotides of a nucleic acid according to the invention .
  • biologically active fragment of a transcription regulating acid according to the invention is meant a nucleic acid capable of modulating the transcription of a DNA sequence placed under its control.
  • a biologically active fragment comprises a basic promoter and / or a regulatory element, as defined in the present description.
  • regulatory nucleic acid is meant a nucleic acid which activates and / or regulates the expression of a DNA sequence selected and placed under its control.
  • promoter is meant a DNA sequence recognized by the proteins of the cell involved in the initiation of transcription of a gene.
  • the basic promoter is the minimal regulatory nucleic acid capable of initiating the transcription of a determined DNA sequence which is placed under its control.
  • the basic promoter consists of a region of genomic DNA upstream of the transcription initiation site where there is very often a CAAT sequence (where one or more protein transcription factors bind) as well as, except in rare cases such as in certain domestic genes, the sequence TATA or "TATA box” or a related box. It is at the level of this box that an RNA polymerase is fixed as well as one or more transcription factors, such as the proteins which bind to the "TATA box” (TATA box Binding Proteins or TBPs).
  • a nucleotide sequence is "placed under the control" of a regulatory nucleic acid when this regulatory nucleic acid is located, relative to the nucleotide sequence, so as to control the initiation of transcription of the nucleotide sequence by an RNA polymerase.
  • regulatory element or “regulatory sequence” within the meaning of the invention is meant a nucleic acid comprising elements capable of modulating the transcription initiated by a basic promoter, such as sites for binding of various transcription factors, “enhancer” sequences for increasing transcription or “silencer” sequences for inhibiting transcription.
  • enhancer sequence is meant a DNA sequence included in a regulatory nucleic acid capable of increasing or stimulating the transcription initiated by a basic promoter.
  • silica is meant a DNA sequence included in a regulatory acid capable of decreasing or inhibiting the transcription initiated by a basic promoter.
  • Regulatory elements may be present outside the sequence located on the 5 ′ side of the transcription initiation site, for example in introns and exons, including in coding sequences.
  • the basic promoter and the regulatory element can be "specific for one or more tissues", if they allow transcription of a determined DNA sequence, placed under their control, preferably in certain cells (for example cells specific of a tissue), that is to say either exclusively in the cells of certain tissues, or at different levels of transcription depending on the tissue.
  • transcription factor proteins which preferentially interact with regulatory elements of a regulatory nucleic acid according to the invention, and which stimulate or on the contrary suppress transcription. Certain transcription factors are active in the form of monomers, others being active in the form of homo- or heterodimers.
  • the term “modulation” is aimed either at positive regulation (increase, stimulation) of transcription, or negative regulation (decrease, inhibition, blockage) of transcription.
  • the "percentage of identity" between two nucleotide or amino acid sequences can be determined by comparing two optimally aligned sequences, through a comparison window.
  • the part of the nucleotide or polypeptide sequence in the comparison window can thus include additions or deletions (for example "gaps") with respect to the reference sequence (which does not include these additions or these deletions) so as to obtain an optimal alignment of the two sequences.
  • the percentage is calculated by determining the number of positions at which an identical nucleic base or amino acid residue is observed for the two sequences (nucleic or peptide) compared, then by dividing the number of positions at which there is identity between the two bases or amino acid residues by the total number of positions in the comparison window, then multiplying the result by 100 to obtain the percentage of sequence identity.
  • the optimal alignment of the sequences for the comparison can be achieved by computer using known algorithms contained in the package of the company WISCONSIN GENETICS SOFTWARE PACKAGE, GENETICS COMPUTER GROUP (GCG), 575 Science Doctor, Madison, WISCONSIN.
  • the percentage of sequence identity may be carried out using the BLAST software (BLAST versions 1.4.9 of March 1996, BLAST 2.0.4 of February 1998 and BLAST 2.0.6 of September 1998), using only the default parameters (AltschuI et al, J. Mol. BioL, (1990) 215: 403-410; AltschuI et al, Nucleic Acids Res. (1997) 25: 3389-3402).
  • the query sequence and the databases used can be peptide or nucleic, any combination being possible.
  • hybridization conditions described above are suitable for hybridization under high stringency conditions, of a nucleic acid molecule of variable length from 20 nucleotides to several hundred nucleotides.
  • hybridization conditions described above can be adapted as a function of the length of the nucleic acid for which hybridization is sought or of the type of labeling chosen, according to techniques known to those skilled in the art.
  • the suitable hybridization conditions can for example be adapted according to the teaching contained in the work of HAMES and HIGGINS (1985) (Nucleic acid Hybridization iapractical Approach, Hames and Higgins Ed., IRL Press, Oxford) or even in the work by F. AUSUBEL et al (1999) (Currents Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, NY).
  • transformation within the meaning of the invention means the introduction of a nucleic acid (or a recombinant vector) into a host cell.
  • transformation also encompasses a situation " in which the genotype of a cell has been modified by an exogenous nucleic acid, and this cell thus transformed expresses said exogenous nucleic acid, for example in the form of a recombinant polypeptide or still in the form of a sense or antisense nucleic acid.
  • transgenic animal within the meaning of the invention is meant a non-human animal, preferably a mammal, in which one or more cells contain a heterologous nucleic acid introduced by human intervention, such as by transgenic techniques. well known to those skilled in the art.
  • the heterologous nucleic acid is introduced directly or indirectly into the cell or the precursor of the cell, by genetic manipulation such as micro-injection or infection with a recombinant virus.
  • the heterologous nucleic acid may be integrated into the chromosome, or may be in the form of DNA replicating extra-chromosomally.
  • NUCLEIC ACID REGULATOR OF THE ABCA7 GENE From libraries of BAC type vectors prepared from human and murine genomic material, the inventors have succeeded in isolating a nucleic acid regulating the human and murine ABCA7 genes.
  • the inventors have determined, by a comparative analysis of human and murine genomic sequences, a regulatory nucleic acid comprising in particular two regulatory modules conserved in humans and mice.
  • the inventors have therefore determined that the nucleic acid regulating the transcription of the ABCA7 gene, when it is defined in the broadest way, consists of a polynucleotide comprising, from the 5 ′ end to the 3 ′ end : a non-transcribed region of approximately 1.2 kb located upstream of the site for initiating transcription of the ABCA7 gene, and
  • a first object of the invention consists of a nucleic acid comprising a polynucleotide having at least 20 consecutive nucleotides of the nucleotide sequence SEQ ID No. 1, or a nucleic acid of complementary sequence.
  • the region of approximately 1.1 kb located upstream from the site of initiation of transcription of the ABCA7 gene, and comprising the basic promoter and multiple regulatory elements for transcription is also included in the sequence identified as SEQ ID N ° 2 according to the invention.
  • nucleotide at position 1 of the sequence SEQ ID No. 2 is the nucleotide at position -1111, with respect to the site of initiation of transcription of the ABCA7 gene.
  • the invention relates to a nucleic acid comprising a polynucleotide having at least 20 consecutive nucleotides of the nucleotide sequence SEQ ID No. 2, or a nucleic acid of complementary sequence.
  • the nucleic acid regulating the transcription of the ABCA7 gene of sequence SEQ ID No. 1 comprises, in addition to a 5 ′ regulatory region not transcribed, also the 5 ′ part of the first exon of the human ABCA7 gene.
  • the partial sequence of the first exon of the ABCA7 gene is defined as the sequence SEQ ID No. 3.
  • the invention relates to a nucleic acid comprising a polynucleotide having at least 20 consecutive nucleotides of nucleotide sequence SEQ ID No. 3 or a nucleic acid of complementary sequence.
  • nucleic acid according to the invention will be in an isolated and / or purified form.
  • nucleic acid having at least 80% nucleotide identity with a nucleic acid as defined above.
  • this nucleic acid can be of murine origin, and consists of a polynucleotide of nucleotide sequence SEQ ID NO: 4 comprising from the 5 ′ to 3 ′ end:
  • the region of approximately 1.2Kb located upstream from the site of initiation of transcription of the ABCA7 gene, and comprising the basic promoter and multiple regulatory elements for transcription is also included in the sequence identified as SEQ ID NO: 5 according to the invention.
  • the invention also encompasses a nucleic acid characterized in that it hybridizes, under conditions of high stringency, with any one of the nucleic acids according to the invention.
  • the invention also relates to a nucleic acid having at least 80%, advantageously 90%, preferably 95% and very preferably 98% of nucleotide identity with a nucleic acid comprising at least 20 consecutive nucleotides of a polynucleotide chosen from the group consisting of the nucleotide sequences SEQ ID No. 1 to SEQ ID No. 5.
  • the nucleic acid of sequence SEQ ID No. 2 included in the nucleic acid regulating the human gene ABCA7 of sequence SEQ ID No. 1, comprises the elements constituting a basic promoter, respectively a box Degenerated "TATA" (TTAAG) located 30 bp upstream from the point of initiation of transcription.
  • TTAAG Degenerated "TATA"
  • TTAAA degenerate "TATA" box
  • the regulatory sequences SEQ ID No. 2 and SEQ ID No. 5 also include numerous sites for binding various transcription factors capable of positively or negatively regulating the activity of the base promoter. Thus, the different sequences characteristic of the binding sites of various transcription factors in the sequences SEQ ID No. 2 and SEQ ID No. 5 were identified by the inventors in the following detail.
  • sequences SEQ ID No. 2 and SEQ ID No. 5 were used as reference sequences and processed according to the algorithms of the Matlnspector software (Quandt et al., Nucl Acid Res (1995) 23 (23), 4878-4884) and compared with the data indexed in several databases such as Transfac and the presence as well as the localization of the various characteristic sites of the sequences SEQ ID N ° 2 and 5, and in particular the sites of binding of the transcription factors were determined according to well methods known to those skilled in the art.
  • the positions of the start nucleotides of each of the transcription factor binding sites are designated with reference to the numbering of the nucleotides of the sequences SEQ ID No. 2 and NO: 5 with respect to the +1 point of initiation of transcription, content in the sequences SEQ ID No. 1 and No. 4, as shown in FIG. 1.
  • Figure 2 shows the sequence SEQ ID NO: 1, which contains the sequence SEQ ID No 2.
  • the first nucleotide at position 5 'of the sequence of FIG. 2 is also the first nucleotide at position 5' of one of the nucleic sequences SEQ ID No. 1 and SEQ ID No. 2.
  • the sites of attachment to transcription factors are illustrated in bold type which delimit their respective positions, and their respective designations are indicated above each of the corresponding boxes.
  • the nucleotides of the sequence represented in FIG. 2 have been numbered with respect to the transcription initiation site, numbered "+1", the nucleotide 5 ′ of nucleotide +1 being itself numbered "-1" .
  • Figure 3 shows the sequence SEQ ID NO: 4, which contains the sequence
  • the first nucleotide at position 5 'of the sequence of FIG. 3 is also the first nucleotide at position 5' of one of the nucleic sequences SEQ ID No. 4 and SEQ ID No. 5.
  • the sites of attachment to transcription factors are illustrated in bold type which delimit their respective positions, and their respective designations are indicated above each of the corresponding boxes.
  • the nucleotides of the sequence represented in FIG. 3 have been numbered with respect to the transcription initiation site, numbered "+1", the nucleotide 5 ′ of nucleotide +1 being itself numbered "-1" .
  • the two regulatory modules stored in mice and humans also include sites for binding transcription factors such as GFI1 and NFkappaB (NFkB), which are mainly present in the lymphatic organs.
  • GFI1 and NFkappaB NFkB
  • NF1 factor The binding characteristics of the NF1 factor can be found in particular in the following entries in the Medline database: 88319941, 91219459, 861401 12, 87237877, 90174951, 89282387, 90151633, " 892618136, 86274639, 87064414, 89263791.
  • factor NF1 recognizes the following palindromic sequence: "TGGCANNNTGCCA (NNTTGGCNNNNNNNCCCCN)" which is present in viral and cellular promoters and at the level of the origin of replication of adenovirus type 2. These proteins are capable of activating transcription and replication They bind to DNA in the form of a homodimer.
  • NFY factor The NFY factor is described in particular in entry No. P25.208 of the Swissprot database. It is a factor which recognizes a "CCAAT" motif in promoter sequences such as those of the gene coding for type 1 collagen, albumin and ⁇ -actin. It is a stimulator of transcription.
  • the factor AP4 has a DNA binding domain of the "helix loop helix” type (bHLH) as well as two dimerization domains.
  • the consensus site for factor AP4 is the following "CWCAGCTGGN", and this generally overlaps with a binding site for factor AP1.
  • the CEBP factor binding characteristics can be found in particular in the following entries in the Medline database: 93315489, 91248826, 94193722, 93211931, 92390404, 90258863, 94088523, 90269225 and 96133958. It is an activator of the important transcription in the regulation of genes involved in immune and inflammatory responses. It specifically binds to an IL-1 response element in the IL-6 gene. It probably plays a role in regulating the acute phase of inflammation and in hemopoiesis. The consensus recognition site is as follows: "T (T / G) NNGNAAfJVG)".
  • HNF3B factor HNF3B factor
  • OVERDIER et al. (1994, Mol. Cell Biol. VoU4: 2755-2766), as well as the following Medline database entries: 91352065, 91032994, 92345837, 89160814, 91187609, 91160974, 91029477, 94301798 and 94218249.
  • This transcription factor acts as an activator of many liver genes such as AFT genes, albumin, tyrosine aminotransferase and interacts with cis-acting regulatory regions of these genes.
  • the FM G factor binding characteristics can be found in particular in the following entries in the Medline database: 10762661, 9931446, 9571157, 9285685, 9070650, and 7789186.
  • the GFI1 gene codes for a zinc finger protein involved in the transcriptional regulation and more particularly in the interleukin 2 signaling pathway.
  • the consensus recognition site is as follows: "NNNNNNAAATCANNGNNNNNNN” NFkappa-B factor:
  • the factor NFkappa-B is a heterodimer consisting of a first 50 kDa subunit and a second 65 kDa subunit. Two heterodimers can form a labile tetramer. Its attachment to DNA depends on the presence of zinc (Zn ++ ). It can be induced by many agents such as TNF, PKA or even PKC. It is a key regulator of genes involved in responses to infection, inflammation, and stress.
  • An essential characteristic of the regulatory nucleic acid according to the invention, and more particularly of the sequence located upstream of the transcription initiation site included both in the sequence SEQ ID No. 2 and in the sequence SEQ ID N ° 5 is the presence of motifs characteristic of putative sites of binding to transcription factors involved in the gene expression of T lymphocytes, such as the transcription factors CEBP, NFKB, and GFI1
  • GFI1 is a proto-oncogene which codes for a zinc finger nuclear protein involved in the signaling pathway by cytokines and in the clonal amplification of T cells (Zweidler-McKay, et al., Mol. Cell. Biol. ( 1996), 16 (8), 4024-4034).
  • the transcription factor GFI1 which acts as a transcriptional repressor of genes which inhibit T cell activation and oncogenesis. It is specifically present in the thymus, spleen and T lymphocytes.
  • CEBP and NFkappaB transcription factors which are expressed in the thymus, spleen and T lymphocytes, are well known to those skilled in the art and act in cooperation in mediating the induction of expression of the genes of T lymphocytes. (Runch et al., 1994) and HepB3 cells (Shimizu et al., Gene, (1994) 149, 305-310).
  • the invention relates to a nucleic acid comprising a polynucleotide having at least 20 consecutive nucleotides of one of the nucleotide sequences SEQ ID No. 1 or 2, and SEQ ID No. 4 or 5, as well as an acid nucleic acid of complementary sequence.
  • nucleic acids comprising one or more "biologically active" fragments of one of the sequences SEQ ID No. 1 or 2, and SEQ ID No. 4 or 5.
  • Those skilled in the art can easily obtaining biologically active fragments of these sequences, with particular reference to Table 3 above as well as to Figures 2 and 3 in which the different motifs characteristic of the regulatory sequence of the ABCA7 gene are presented.
  • Those skilled in the art will thus be able to obtain such biologically active fragments by total or partial chemical synthesis of the corresponding polynucleotides or even by causing restriction endonucleases to act in order to obtain the desired DNA fragments, the restriction sites present on the sequences.
  • SEQ ID N ° 1 to SEQ ID N ° 5 can be easily found from the sequence information, using current restriction mapping software such as GCG version 9J module map.
  • nucleic acid fragments determined using restriction endonucleases is for example described in the work by SAMBROOK et al., (Molecular cloning: a laboratory manual, 2ed. Cold Spring Harbor Laboratory, Cold spring Harbor, New York (1989)
  • the invention therefore also relates to a nucleic acid as defined above, capable of modulating the transcription of a polynucleotide placed under its control.
  • a biologically active fragment of a transcription-regulating nucleic acid comprises a first conserved module (module 2) which comprises the basic promoter (TATA box) going from the nucleotide in position - 1 at the nucleotide at position -390, relative to the site of initiation of transcription, the first nucleotide transcribed being the nucleotide at position 1 1 12 of the nucleotide sequence SEQ ID No. 1, or the nucleotide at position 1221 of the sequence nucleotide SE ID NO: 4.
  • module 2 which comprises the basic promoter (TATA box) going from the nucleotide in position - 1 at the nucleotide at position -390, relative to the site of initiation of transcription, the first nucleotide transcribed being the nucleotide at position 1 1 12 of the nucleotide sequence SEQ ID No. 1, or the nucleotide at position 1221 of the sequence nucleotide SE ID NO: 4.
  • a biologically active fragment of a transcription regulating nucleic acid comprises the conserved modules 1 and 2 (FIG. 1) from the nucleotide in position -1 to the nucleotide in position -860, relative at the transcription initiation site, the first nucleotide transcribed being the nucleotide in position 1 1 12 of the nucleotide sequence SEQ ID No. 1, or the nucleotide in position 1221 of the nucleotide sequence SE ID NO: 4.
  • such a biologically active fragment of a transcriptional acid comprises, in addition to the basic promoter (core promoter) and the proximal regulatory elements, also other regulatory elements such as different GFI1, HNF3B, CEBPB, NF1 sites and extends from the nucleotide at position -1 to the nucleotide at position -1 1 1 1, relative to the transcription initiation site, the first nucleotide transcribed being the nucleotide at position 1 1 12 of the nucleotide sequence SEQ ID N ° 1, and at the nucleotide in position - 1220, with respect to the transcription initiation site, the first nucleotide transcribed being the nucleotide in position 1221 of the nucleotide sequence SEQ ID N ° 4 .
  • EXON 1 ANALYSIS The applicant has also identified the nucleotide sequences located downstream of the transcription initiation site and corresponding to the 5 'end of the exon
  • exon 1 begins at the nucleotide at position 1121 of the sequence SEQ ID No 1 and ends at the nucleotide at position 2322 of the sequence SEQ ID # 1.
  • the 5 'end of exon 1 is identified as the sequence SEQ ID No. 3 and the complete sequence of exon 1 is identified as the sequence SEQ ID No. 6.
  • Exon 1 contains the start of the open reading phase of the human ABCA7 gene, nucleotide A of the ATG codon being located at position 1208 of the sequences SEQ ID Nos. 3 and 6.
  • Exon 1 codes for the polypeptide of sequence SEQ ID # 7.
  • Exon 1 is likely to contain elements for regulating the expression of the ABCA7 gene, in particular elements of the enhancer enhancer type and / or elements of the silencer or repressor type.
  • a transcription regulating nucleic acid according to the invention may also contain, in addition to biologically active fragments of the sequence SEQ ID No. 1, also nucleotide fragments, or even all of the sequences SEQ ID No. 2 to SEQ ID N ° 3 and 6.
  • nucleotide sequences SEQ ID N ° 1 to SEQ ID N ° 3, and 6, as well as their fragments can in particular be used as nucleotide probes or primers to detect the presence of at least one copy of the ABCA7 gene in a sample, or further to amplify a target sequence determined within the regulatory sequence of the ABCA7 gene.
  • a further subject of the invention is therefore a nucleic acid having at least 80% nucleotide identity with a nucleic acid as defined above, in particular originating from one of the sequences SEQ ID N ° 1 to SEQ ID N ° 3 and 6.
  • the invention also relates to a nucleic acid hybridizing, under conditions of high stringency, with any of the nucleic acids according to the invention, in particular a nucleic acid originating from a sequence chosen from the sequences SEQ ID N ° 1 to SEQ ID N ° 3 and 6.
  • the invention also relates to a nucleic acid as defined above and further characterized in that it is capable of modulating the transcription of a polynucleotide of interest placed under its control.
  • such a nucleic acid is capable of activating the transcription of the polynucleotide of interest placed under its control.
  • a regulatory nucleic acid according to the invention can be characterized in that it is capable of inhibiting the transcription of the polynucleotide of interest placed under the control thereof.
  • a transcription regulating nucleic acid according to the invention when it is suitably located with respect to a polynucleotide of interest whose expression is sought, will allow the transcription of said polynucleotide of interest, either constitutively or inducibly.
  • the inducible nature of the transcription initiated by a regulatory nucleic acid according to the invention can be conferred by one or more of the regulatory elements which it contains, for example the presence of one or more sites as previously defined in the sequence SEQ ID N ° 1 or SEQ ID N ° 2.
  • tissue-specific expression of the polynucleotide of interest can be sought by placing this polynucleotide of interest under the control of a regulatory nucleic acid according to the invention capable, for example, of initiating the transcription of this polynucleotide d interest specifically in certain categories of cells, for example cells of hematopoietic tissue, such as peripheral leukocytes, thymus cells, spleen cells, and bone marrow.
  • a regulatory nucleic acid according to the invention capable, for example, of initiating the transcription of this polynucleotide d interest specifically in certain categories of cells, for example cells of hematopoietic tissue, such as peripheral leukocytes, thymus cells, spleen cells, and bone marrow.
  • a regulatory nucleic acid according to the invention may comprise one or more "discrete" regulatory elements, such as enhancer and silent elements.
  • a regulatory nucleic acid may comprise one or more sites for potential attachment to the transcription factors as defined in FIG. 2.
  • a regulatory acid according to the invention also includes a sequence not comprising the basic promoter, that is to say the sequence going from the nucleotide in position -1 to the nucleotide in position -25, with respect to the initiation site of the transcription.
  • Such a regulatory nucleic acid will then preferably comprise a basic promoter called “heterologous”, that is to say a polynucleotide comprising a "TATA” box and a “homebox” not originating from the nucleic acid regulating the gene ABCA7.
  • transcription regulating nucleic acid comprising all or part of the sequence SEQ ID No. 1 which has been modified, for example by addition, deletion or substitution of one or more nucleotides. Such modifications can modulate the transcriptional activity by causing an increase or on the contrary a decrease in the activity of the promoter or of the regulatory element.
  • Such a modification can also affect the tissue specificity of the promoter or of the regulatory element. So, for example, a nucleic acid regulator according to the invention can be modified in order to stimulate transcription in only one of the tissues in which it is naturally expressed.
  • a transcription regulating acid according to the invention can also be modified and be made inducible by a particular compound, for example by creating in the sequence a site inducible by a given therapeutic compound.
  • Modifications in a sequence comprising all or part of the sequence SEQ ID No. 1 and comprising the promoter or a regulatory element can be carried out with methods well known to those skilled in the art, such as mutagenesis.
  • the activity of the promoter or of the modified regulatory element can then be tested, for example by cloning the modified promoter upstream of a reporter gene, by transfecting the resulting DNA construct in a host cell and by measuring the level of expression of the reporter gene in the transfected host cell.
  • the activity of the modified promoter can also be analyzed in vivo in transgenic animals. It is also possible to construct libraries of modified fragments which can be screened using functional tests in which, for example, only promoters or regulatory elements having the desired activity will be selected.
  • Such tests may be based, for example, on the use of reporter genes conferring resistance to specific compounds, for example antibiotics.
  • the selection of cells having a regulatory nucleic acid / reporter gene construction and containing a promoter or a regulatory element having the desired modification can then be isolated by culturing host cells transformed with such a construction in the presence of the determined compound, for example the antibiotic determined.
  • the reporter gene can also code for any easily detectable protein, for example an optically detectable protein such as luciferase.
  • the subject of the invention is also a nucleic acid comprising: a) a transcription regulating nucleic acid as defined above; and b) a polynucleotide of interest encoding a polypeptide or a nucleic acid of interest.
  • the polynucleotide of interest whose transcription is sought encodes a protein or a peptide.
  • the protein can be of any kind, for example a protein of therapeutic interest, including cytokines, structural proteins, receptors or even transcription factors.
  • the nucleic acid transcription regulator will advantageously comprise a nucleic acid going from the nucleotide in position -1 to the nucleotide in position -1111, relative to the site of initiation of transcription of the sequence SEQ ID No. 1 or 2, and going from the nucleotide in position -1 at the nucleotide in position -1220 SEQ ID N ° 4 or 5.
  • the polynucleotide of interest will code for a gene involved in the fight against inflammation, such as a cytokine receptor or even for a superoxide dismutase. If an antitumor effect is sought, we will then seek to stimulate the number and activation of cytotoxic T lymphocytes specific for a given tumor antigen.
  • a regulatory nucleic acid according to the invention will be used in combination with a polynucleotide of interest coding for the protein ABCA7.
  • the polynucleotide of interest can also be a sense type oligonucleotide.
  • the polynucleotide of interest can also produce a nucleic acid, such as an antisense nucleic acid specific for a gene whose translation is sought to be inhibited.
  • the polynucleotide of interest whose transcription is regulated by the regulatory nucleic acid is a reporter gene, such as any gene coding for a detectable protein.
  • reporter genes there may be mentioned in particular the gene for luciferase, ⁇ -galactosidase (LacZ), chloramphenicol acetyl transferase (CAT) or any gene coding for a protein conferring resistance to a particular compound, particularly to an antibiotic.
  • LacZ ⁇ -galactosidase
  • CAT chloramphenicol acetyl transferase
  • RECOMBINANT VECTORS any gene coding for a protein conferring resistance to a particular compound, particularly to an antibiotic.
  • vector within the meaning of the present invention is meant a circular or linear DNA or RNA molecule which is either in the form of single strand or double strand.
  • a recombinant vector according to the invention is used in order to amplify the regulatory nucleic acid according to the invention which is inserted therein after transformation or transfection of the desired cellular host.
  • these are expression vectors comprising, in addition to a regulatory nucleic acid according to the invention, sequences the expression of which is sought in a host cell or in a determined multicellular organism.
  • a recombinant vector according to the invention will notably comprise the following elements:
  • a regulatory nucleic acid (1) a regulatory nucleic acid according to the invention; (2) a polynucleotide of interest comprising a coding sequence included in the nucleic acid to be inserted into such a vector, said coding sequence being placed in phase with the regulatory signals described in (1); and
  • the recombinant vectors according to the invention may include one or more origins of replication in cellular hosts in which their amplification or expression is sought, markers or selection markers.
  • the bacterial promoters could be the Lacl, LacZ promoters, the RNA polymerase promoters of bacteriophage T3 or T7, the PR or PL promoters of phage lambda.
  • Promoters for eukaryotic cells will include the HSV virus thymidine kinase promoter or the mouse metallothionein-L promoter.
  • bacteriophage vectors such as bacteriophage P1 vectors such as the vector p158 or even the vector p158 / neo8 described by Sternberg (Trends Genet., (1992) 8: 1-16) are preferably used. ; Mamm. Genome (1994) 5: 397-404).
  • the preferred bacterial vectors according to the invention are for example the vectors pBR322 (ATCC37017) or also vectors such as pAA223-3 (Pharmacia, Uppsala, Sweden), and pGEM1 (Promega Biotech, Madison, Wl, USA). Mention may also be made of other commercially available vectors such as the vectors pQE70, pQE60, pQE9 (Qiagen), psiX174, pBluescript SA, pNH8A, pNH16A, pNH18A, pNH46A, pWLNEO, pSV2CAT, pOG44, pXTI, pSG (Stratagene).
  • a recombinant vector according to the invention can also be a retroviral vector or also an adeno-associated vector (AAV).
  • AAV adeno-associated vector
  • Such adeno-associated vectors are for example described by Flotte et al., Am. J. Respir., Cell Mol. Biol. (1992) 7: 349-356; Samulski et al., J. Virol. (1989) 63: 3822-3828; MacLaughlin et al., Am. J. Hum. Broom. (1996) 59: 561-569).
  • the polynucleotide construct comprising the regulatory sequence and the coding sequence must be introduced into a host cell.
  • the introduction of such a polynucleotide construct according to the invention into a host cell can be carried out in vitro, according to techniques well known to those skilled in the art to transform or transfect cells, either in primary culture or in the form cell lines.
  • a person skilled in the art may advantageously refer to various techniques, such as the calcium phosphate precipitation technique (Graham et al., Virology (1973) 52: 456-457; Chen et al., Mol. Cell. Biol. (1987) 7: 2745-2752), DEAE Dextran (Gopal et al., Mol. Cell. Biol., (1985) 5: 1188-1190), electroporation (Tur-Kaspa et al., Mol Cel. Biol, (1986) 6: 716-718 .; Potter et al., Proc. Natl. Acad. Sci.
  • the polynucleotide Once the polynucleotide has been introduced into the host cell, it can be stably integrated into the genome of the cell. Integration can be carried out at a specific location in the genome, by homologous recombination, or can be integrated at random. In certain embodiments, the polynucleotide can be stably maintained in the host cell in the form of an episome fragment, the episome comprising sequences permitting maintenance and replication thereof, either independently, either synchronized with the cell cycle.
  • a method for introducing a polynucleotide according to the invention into a host cell comprises a step during which a preparation comprising a vector is introduced of compatible pharmaceutical expression and a “naked” polynucleotide according to the invention, placed under the control of appropriate regulatory sequences, by local injection into the chosen tissue, for example a smooth muscle tissue, the “naked” polynucleotide being absorbed by cells in this tissue.
  • compositions for in vitro and in vivo use comprising "naked" polynucleotides are for example described in PCT application No. WO 95/11307 as well as in the articles by Tacson et al. (Nature Med. (1996) 2 (8), 888-892) and de Huygen et al., (Nat. Med. (1996) 2 (8), 893-898).
  • compositions for the in vivo production of a protein of interest comprising a polynucleotide coding for the polypeptide of interest placed under the control of a regulatory sequence according to the invention, in solution in a physiologically acceptable vector.
  • the amount of vector that is injected into the chosen host organism varies depending on the site of the injection. As an indication, it can be injected between approximately 0.1 and approximately 100 ⁇ g of the polynucleotide construct regulatory sequence / coding sequence into the body of an animal.
  • the vector is preferably injected in the body of a patient who is likely to develop a disease related to a protein deficiency ABCA7.
  • the invention also relates to a pharmaceutical composition intended for the prevention or treatment of subjects affected by a dysfunction of the ABCA7 protein, comprising a regulatory nucleic acid according to the invention and a polynucleotide of interest coding for the ABCA7 protein. , in combination with one or more physiologically compatible excipients.
  • such a composition will comprise the regulatory nucleic acid defined by one of the sequences SEQ ID No. 1 or 2, and SEQ ID No. 4 or 5, or a biologically active fragment of this regulatory nucleic acid.
  • the invention further relates to a pharmaceutical composition intended for the prevention or treatment of subjects affected by a dysfunction in lipid metabolism, comprising a recombinant vector as defined above, in association with one or more physiologically excipients compatible.
  • a pharmaceutical composition intended for the prevention or treatment of subjects affected by a dysfunction of the processes involving the immune system and inflammation comprising a recombinant vector as defined above, in association with a or more physiologically compatible excipients.
  • the invention also relates to the use of a polynucleotide construct in accordance with the invention and comprising a nucleic acid regulating the ABCA7 gene as well as a sequence coding for the protein ABCA7, for the manufacture of a medicament intended for the prevention or treatment of affected subjects a dysfunction of lipid metabolism or a problem of immunological or inflammatory origin.
  • the invention also relates to the use of a recombinant vector according to the invention, comprising, in addition to a regulatory nucleic acid of the invention, a nucleic acid coding for the protein ABCA7, for the manufacture of a medicament intended for preventing or treating subjects affected by dysfunction of the processes involving the immune system and inflammation.
  • Vectors useful in somatic gene therapy methods and compositions containing such vectors are provided.
  • the present invention also relates to a new therapeutic approach for the treatment and / or prevention of pathologies linked to lipid metabolism as well as for the treatment and / or prevention of pathologies linked to dysfunction of the lymphocytic mediation mechanisms of inflammation. It offers an advantageous solution to the drawbacks of the prior art, by demonstrating the possibility of treating pathologies, in particular pathologies linked to a dysfunction of lipid metabolism in myelo-lymphatic tissues, by gene therapy, by transfer and in vivo expression of a polynucleotide construct comprising, in addition to a regulatory nucleic acid according to the invention, a sequence coding for an ABCA7 protein which is strongly presumed to be involved in the transport and / or metabolism of lipids.
  • the invention thus provides a simple means allowing a specific and effective treatment of subjects affected by a dysfunction of the processes involving the immune system and inflammation.
  • Gene therapy consists of correcting a deficiency or an anomaly
  • viruses as vectors for gene transfer has emerged as a promising alternative to these physical transfection techniques.
  • retroviruses RSV, HMS, MMS, etc.
  • the HSV virus adeno-associated viruses
  • adenoviruses adenoviruses
  • the present invention therefore also relates to a new therapeutic approach for the treatment of pathologies linked to the transport of lipids, consisting in transferring and expressing in vivo genes coding for ABCA7 placed under the control of a regulatory acid according to the invention. It is particularly advantageous to construct recombinant viruses containing a DNA sequence comprising a regulatory nucleic acid according to the invention and a sequence coding for an ABCA7 protein involved in lipid metabolism, to administer these recombinant viruses in vivo, and that this administration allows stable and efficient expression of a biologically active ABCA7 protein in vivo, and without cytopathological effect.
  • Adenoviruses are particularly effective vectors for the transfer and expression of the ABCA7 gene.
  • the use of recombinant adenoviruses as vectors makes it possible to obtain sufficiently high expression levels of the gene of interest to produce the desired therapeutic effect.
  • Other viral vectors such as retroviruses or adeno-associated viruses (AAV), allowing stable expression of the gene are also claimed.
  • the present invention thus offers a new approach for the treatment and prevention of pathologies linked to dysfunctions in lipid metabolism and in the signaling pathways of inflammation by lymphocytes.
  • the invention therefore also relates to a defective recombinant virus comprising a regulatory nucleic acid according to the invention and a nucleic sequence coding for an ABCA7 protein involved in lipid metabolism or in processes involving the immune system and inflammation.
  • the invention also relates to the use of such a defective recombinant virus for the preparation of a pharmaceutical composition intended for the treatment and / or the prevention of dysfunctions in the signaling of inflammation by lymphocytes.
  • the present invention also relates to the use of cells genetically modified ex vivo by a virus as described above, or of cells producing such viruses, implanted in the organism, allowing a prolonged and efficient expression in vivo of a protein.
  • ABCA7 biologically active.
  • the present invention shows that it is possible to incorporate a DNA sequence coding for ABCA7 in the control of a regulatory nucleic acid as defined above in a viral vector, and that these vectors make it possible to efficiently express a mature, biologically active form. More particularly, the invention shows that the in vivo expression of ABCA7 can be obtained by direct administration of an adenovirus or by implantation of a producer or genetically modified cell by an adenovirus or by a retrovirus incorporating such a DNA.
  • the present invention is particularly advantageous because it makes it possible to induce a controlled expression and without harmful effect of ABCA7 in organs which are not normally concerned with the expression of this protein.
  • a significant release of the ABCA7 protein is obtained by implantation of cells producing vectors of the invention, or infected ex vivo with vectors of the invention.
  • the mediator activity in the lipid metabolism produced in the context of the present invention can be of the human or animal ABCA7 type.
  • the nucleic sequence used in the context of the present invention can be a cDNA, a genomic DNA (gDNA), a RNA (in the case of retroviruses) or a hybrid construct consisting for example of a cDNA in which one or more introns would be inserted. .
  • a cDNA or a gDNA is used.
  • the use of a gDNA allows better expression in human cells.
  • these sequences are advantageously modified, for example by site-directed mutagenesis, in particular for the insertion of suitable restriction sites.
  • the sequences described in the prior art are in fact not constructed for use according to the invention, and prior adaptations may prove to be necessary, in order to obtain important expressions.
  • a construct coding for a derivative of these ABCA7 proteins are also possible to use.
  • a derivative of these ABCA7 proteins comprises for example any sequence obtained by mutation, deletion and / or addition with respect to the native sequence, and coding for a product retaining the activity of mediator of lipid metabolism. These modifications can be carried out by techniques known to a person skilled in the art (see general molecular biology techniques below). The biological activity of the derivatives thus obtained can then be easily determined, as indicated in particular in the examples describing the measurement of the influx of lipids from the cells.
  • the derivatives within the meaning of the invention can also be obtained by hybridization from nucleic acid libraries, using as probe the native sequence or a fragment thereof.
  • derivatives are in particular molecules having a greater affinity for their binding sites, molecules exhibiting greater resistance to proteases, molecules having greater therapeutic efficacy or less side effects, or possibly having new biological properties.
  • the derivatives also include the modified DNA sequences allowing improved expression in vivo.
  • the present invention relates to a defective recombinant virus comprising a regulatory nucleic acid according to the invention and a cDNA sequence coding for an ABCA7 protein involved in the transport and metabolism of cholesterol.
  • the DNA sequence is a gDNA sequence.
  • the cDNA sequence coding for the protein ABCA7, and usable in a vector according to the invention is advantageously the sequence SEQ ID No. 8.
  • the vectors of the invention can be prepared from different types of virus.
  • vectors derived from adenoviruses, adeno-associated viruses (AAV), herpes viruses (HSV) or retroviruses are used. It is very particularly advantageous to use an adenovirus, for direct administration or for the ex vivo modification of cells intended to be implanted, or a retrovirus, for the implantation of producer cells.
  • the viruses according to the invention are defective, that is to say that they are unable to replicate autonomously in the target cell.
  • the genome of 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 nucleic sequence coding for the protein ABCA7.
  • the defective virus nevertheless retains the sequences of its genome which are necessary for the packaging of the viral particles.
  • adenoviruses various serotypes, whose structure and properties vary somewhat, have been characterized.
  • serotypes it is preferred to use, in the context of the present invention, human adenoviruses of type 2 or 5 (Ad 2 or Ad 5) or adenoviruses of animal origin (see application WO 94/26914).
  • adenoviruses of animal origin which can be used in the context of the present invention, mention may be made of adenoviruses of canine, bovine, murine origin (example: Mav1, Beard et al., Virology 75 (1990) 81), ovine, porcine , avian or even simian (example: after-sales service).
  • the adenovirus of animal origin is a canine adenovirus, more preferably a CAV2 adenovirus [Manhattan or A26 / 61 strain (ATCC VR-800) for example].
  • a frame of the invention are of human origin adenovirus or canine or mixed.
  • the defective adenoviruses of the invention comprise ITRs, a sequence allowing the packaging and the sequence coding for the protein ABCA7 placed under the control of a nucleic acid according to the invention.
  • the E1 region at least is made non-functional.
  • the E1 gene and at least one of the E2, E4, L1-L5 genes are non-functional.
  • the viral gene considered can be made non-functional by any technique known to those skilled in the art, and in particular by total suppression, substitution, 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 and the sequence coding for ABCA7 is inserted at the level of the inactivated E1 region.
  • the defective recombinant adenoviruses according to the invention can be prepared by any technique known to a person skilled in the art (Levrero et al., Gene (1991) 101: 195, EP 185 573; Graham, EMBO J. (1984) 3: 2917 ). In particular, they can be prepared by homologous recombination between an adenovirus and a plasmid carrying inter alia the DNA sequence coding for the protein ABCA7.
  • the cell line used 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 the risks of recombination.
  • a line mention may be made of the human embryonic kidney line 293 (Graham et al., J. Gen. Virol. (1977) 36: 59) which contains in particular, integrated into its genome, the left part of the genome of an Ad5 adenovirus (12%) or lines capable of complementing the E1 and E4 functions as described in particular in applications No. WO 94/26914 and WO 95/02697. 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 rest of the genome is divided into 2 essential regions carrying the packaging functions: the left part of the genome, which contains the rep gene involved in viral replication and the expression of viral genes; the right part of the genome, which contains the cap gene coding for the capsid proteins of the virus.
  • the use of vectors derived from AAVs for gene transfer in vitro and in vivo has been described in the literature (see in particular WO 91/18088; WO 93/09239; US 4,797,368, US 5,139,941, EP 488,528).
  • 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 sequence coding for the ABCA7 protein bordered by two repeated regions inverted (ITR) from AAV, and a plasmid carrying the encapsidation genes (rep and cap genes) from AAV.
  • a human helper virus for example an adenovirus
  • ITR inverted
  • rep and cap genes encapsidation genes
  • retroviruses are integrative viruses, infecting dividing cells.
  • 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 said coding sequence is generally constructed and then used to transfect a cell line called packaging, capable of providing trans functions deficient retrovirals in the plasmid.
  • 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 (US 4,861, 719); the PsiCRIP line (WO 90/02806) and the GP + envAm-12 line (WO 89/07150).
  • 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 (Bender et al., J. Virol. 61 (1987) 1639).
  • the recombinant retroviruses produced are then purified by conventional techniques.
  • the results given below indeed demonstrate the particularly advantageous properties of adenoviruses for the expression in vivo of a protein having an activity of mediator of lipid metabolism.
  • the adenoviral vectors according to the invention are particularly advantageous for direct administration in vivo of a purified suspension, or for the ex vivo transformation of cells, in particular autologous, with a view to their implantation.
  • the adenoviral vectors according to the invention also have significant advantages, such as in particular their very high infection efficiency, making it possible to carry out infections from small volumes of viral suspension.
  • a line producing retroviral vectors containing a regulatory nucleic acid according to the invention and the sequence coding for the protein ABCA7 is used for implantation in vivo.
  • the lines which can be used for this purpose are in particular the cells PA317 (US4,861,719), PsiCrip (WO 90/02806) and GP + envAm-12 (US 5,278,056), modified to allow the production of a retrovirus containing a nucleic sequence coding for an ABCA7 protein according to the invention.
  • a retrovirus containing a nucleic sequence coding for an ABCA7 protein for example totipotent stem cells, precursors of blood cell lines, can be removed and isolated from the subject. These cultured cells can then be transfected with the retroviral vector containing the sequence coding for the ABCA7 protein under the control of its own promoter. These cells are then reintroduced into the subject.
  • the differentiation of these cells will be at the origin of cells of the hematopoietic tissue expressing the ABCA7 protein, in particular T lymphocytes which participate in the signaling of inflammation.
  • the sequence coding for the ABCA7 protein is placed under the control of a regulatory acid according to the invention comprising the regulatory elements allowing its expression in the infected cells, and very particularly the regulatory elements of NFkappaB, CEBP, and GFH type.
  • the present invention also relates to any use of a virus as described above for the preparation of a pharmaceutical composition intended for the treatment and / or prevention of pathologies linked to lipid metabolism or else to dysfunction related to processes involving the immune system and inflammation.
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising one or more defective recombinant viruses as described above.
  • These pharmaceutical compositions can be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal, etc. administration.
  • the pharmaceutical compositions of the invention contain a pharmaceutically "acceptable vehicle for an injectable formulation, in particular for intravenous injection such as, for example into the portal vein of the patient.
  • This may be in particular isotonic sterile solutions , or dry compositions, in particular lyophilized, which, by addition as the case may be of sterilized water or physiological saline, allow the constitution of injectable solutes
  • Direct injection into the portal vein of the patient is advantageous because it makes it possible to target the infection in the liver and thus, to focus the therapeutic effect on this organ.
  • the doses of defective recombinant virus used for injection can be adapted as a function of various parameters, and in particular as a function of the viral vector, of the mode of administration used, of the pathology concerned or also of the duration of the treatment sought.
  • the recombinant adenoviruses according to the invention are formulated and administered in the form of doses of between 10 4 and 10 14 pfu / ml, and preferably 10 6 to 10 10 pfu / ml.
  • the term pfu (“plaque forming unit”) corresponds to the infectious power of a virus solution, 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.
  • the compositions according to the invention can directly comprise the producer cells, with a view to their implantation.
  • another subject of the invention relates to any mammalian cell infected with one or more defective recombinant viruses as described above. More particularly, the invention relates to any population of human cells infected with these viruses. They may in particular be cells of blood origin (totipotent stem cells or precursors), fibroblasts, myoblasts, hepatocytes, keratinocytes, smooth and endothelial muscle cells, glial cells, etc.
  • the cells according to the invention can come from primary cultures. These can be removed by any technique known to those skilled in the art, then cultured under conditions allowing their proliferation. As regards more particularly fibroblasts, these can be easily obtained from biopsies, for example according to the technique described by Ham (Methods Cell Biol (1980) 21a: 255). These cells can be used directly for infection by viruses, or stored, for example by freezing, for the establishment of autologous libraries, for later use.
  • the cells according to the invention can also be secondary cultures, obtained for example from pre-established banks (see for example EP 228458, EP 289034, EP 400047, EP 456640).
  • the cultured cells are then infected with the recombinant viruses, to give them the capacity to produce a biologically active ABCA7 protein.
  • the infection is carried out in vitro according to techniques known to those skilled in the art. In particular, according to the type of cells used and the number of copies of virus per cell desired, a person skilled in the art can adapt the multiplicity of infections and possibly the number of cycles of infections carried out. It is understood that these steps must be carried out under conditions of appropriate sterility when the cells are intended for administration in vivo.
  • the doses of recombinant virus used for infection of the cells can be adapted by a person skilled in the art according to the aim sought.
  • the conditions described above for administration in vivo can be applied to infection in vitro.
  • retroviruses For infection with retroviruses, it is also possible to co-cultivate the cells which it is desired to infect with cells producing the recombinant retroviruses according to the invention. This makes it possible to dispense with the purification of retroviruses.
  • Another subject of the invention relates to an implant comprising mammalian cells infected with one or more defective recombinant viruses as described above or cells producing recombinant viruses, and an extracellular matrix.
  • the implants according to the invention comprise 10 ⁇ to 10 1 ° cells. More preferably, they include 10 6 to 10 8 .
  • the extracellular matrix comprises a gelling compound and optionally a support allowing the anchoring of the cells.
  • gelling agents are used for the inclusion of cells in a matrix having the constitution of a gel, and to promote the anchoring of the cells on the support, if necessary.
  • Different cell adhesion agents can therefore be used as gelling agents, such as in particular collagen, gelatin, glycosaminoglycans, fibronectin, lectins, etc.
  • collagen is used. It can be collagen of human, bovine or murine origin. More preferably, type I collagen is used.
  • compositions according to the invention advantageously comprise a support allowing the anchoring of the cells.
  • anchoring designates any form of biological and / or chemical and / or physical interaction resulting in the adhesion and / or fixing of the cells on the support.
  • the cells can either cover the support used, or penetrate inside this support, or both. It is preferred to use, within the framework of the invention, a solid, non-toxic and / or biocompatible support.
  • PTFE polytetrafluoroethylene
  • the present invention thus provides a very effective means for the treatment or prevention of pathologies linked to the transport of cholesterol, in particular obesity, hypertriglyceridemia, or, in the field of cardiovascular diseases, myocardial infarction, angina, sudden death, heart decompensation and stroke.
  • this treatment can concern both humans and any animal such as sheep, cattle, domestic animals (dogs, cats, etc.), horses, fish, etc.
  • the invention also relates to a recombinant host cell comprising any of the nucleic acids of the invention of sequence SEQ ID No. 1 to SEQ ID No. 6, and more particularly a nucleic acid of sequence SEQ ID NO 1 to SEQ ID # 3.
  • the invention also relates to a recombinant host cell comprising a recombinant vector as described above.
  • the preferred host cells according to the invention are for example the following: a) prokaryotic host cells: strains of Escherichia coli (strain DH5- ⁇ ), of
  • Bacillus subtilis from Salmonella typhimurium, or strains of species such as Pseudomonas, Streptomyces and Staphylococus; b) eukaryotic host cells: HeLa cells (ATCC No. CCL2), Cv 1 cells (ATCC No. CCL70), COS cells (ATCC No. CRL 1650), Sf-9 cells (ATCC No. CRL 1711), CHO cells ( ATCC N ° CCL-61) or 3T3 cells (ATCC N ° CRL-6361), or cells of the Hepa1-6 line referenced in the American Type Culture Collection (ATCC, R ⁇ ckville, MD, United States of America).
  • HeLa cells ATCC No. CCL2
  • Cv 1 cells ATCC No. CCL70
  • COS cells ATCC No. CRL 1650
  • Sf-9 cells ATCC No. CRL 1711
  • CHO cells ATCC N ° CCL-61
  • 3T3 cells ATCC N ° CRL-6361
  • the invention provides methods for treating a subject with a pathology related to the level of expression of the ABCA7 protein.
  • a treatment method consists in administering to the subject a compound modulating the expression of the gene.
  • ABCA7 which can be identified by various in vitro screening methods as defined below.
  • a first method consists in identifying compounds modulating the expression of the ABCA7 gene. According to such a method, cells expressing the ABCA7 gene are incubated with a candidate substance or molecule to be tested and the level of expression of the ABCA7 messenger RNA or the level of production of the ABCA7 protein is then determined.
  • the messenger RNA levels of ABCA7 can be determined by hybridization on a Northern type gel, well known to those skilled in the art.
  • the messenger RNA levels of ABCA7 can also be determined by methods using PCR or the technique described by WEBB et al. (Journal of
  • Production levels of the ABCA7 protein can be determined by immunoprecipitation or immunochemistry using an antibody that specifically recognizes the ABCA7 protein.
  • a nucleotide construct as defined above, comprising a regulatory nucleic acid according to the invention as well as a reporter polynucleotide placed under the control of the regulatory nucleic acid is used, said regulatory nucleic acid comprising at least one basic promoter and at least one regulatory element from one of the sequences SEQ ID No. 1 to SEQ ID No. 3.
  • the reporter polynucleotide can be a gene encoding a detectable protein, such as a gene encoding a luciferase.
  • the cells are transfected with the polynucleotide construct containing the regulatory nucleic acid according to the invention and the reporter polynucleotide, in a stable or transient manner.
  • the transformed cells are then incubated in the presence or in the absence of the molecule or of the candidate substance to be tested for a sufficient time, then the level of expression of the reporter gene is determined.
  • the compounds which induce a statistically significant change in the expression of the reporter gene are then identified and, where appropriate, selected.
  • the subject of the invention is also a method for the in vitro screening of a molecule or of a substance modulating the activity of a regulatory nucleic acid according to the invention, in particular modulating the transcription of the reporter polynucleotide constituting a polynucleotide construct according to the invention, characterized in that it comprises the steps consisting in: a) culturing a recombinant host cell comprising a polynucleotide of interest placed under the control of a regulatory nucleic acid according to the invention; b) incubating the recombinant host cell with the substance or molecule to be tested; c) detecting the expression of the polynucleotide of interest; d) compare the results obtained in step c) with the results obtained when the recombinant host cell is cultured in the absence of the candidate molecule or substance to be tested.
  • the invention also relates to a kit or set for the in vitro screening of a candidate molecule or substance capable of modulating the activity of an acid nucleic regulator according to the invention, comprising: a) a host cell transformed with a polynucleotide construct as defined above, comprising a reporter polynucleotide of interest placed under the control of a regulatory nucleic acid according to the invention; and b) if appropriate, means for detecting the expression of the reporter polynucleotide of interest.
  • the reporter polynucleotide of interest is the coding sequence of a luciferase.
  • the regulatory nucleic acid according to the invention is inserted into a vector, upstream of the sequence coding for luciferase. It may for example be the vector pGL3-basic (pGL3-b) sold by the company PROMEGA
  • the recombinant vector comprising the coding sequence of luciferase placed under the control of a regulatory nucleic acid according to the invention is transfected into recombinant host cells, the luciferase activity of which is then determined after culturing in the presence or not of the candidate substance or molecule to be tested.
  • pGL3-b vectors containing either the cytomegalovirus (CMV) promoter, the ApoAl promoter or even no promoter can be used as controls.
  • the transfected cells are washed with PBS buffer and lysed with 500 ⁇ l of lysis buffer (50 mM tris, 150 mM NaCl, 0.02% sodium azide, 1% NP- 40, 100 ⁇ g / ml of AEBSF and 5 ⁇ g / ml of leupeptin).
  • lysis buffer 50 mM tris, 150 mM NaCl, 0.02% sodium azide, 1% NP- 40, 100 ⁇ g / ml of AEBSF and 5 ⁇ g / ml of leupeptin.
  • the polynucleotide constructs producing high levels of luciferase activity in the transfected cells are those which contain a regulatory nucleic acid according to the invention included in the sequence SEQ ID No. 1 which is capable of stimulating transcription.
  • first probes specific to the messenger RNA of the reporter polynucleotide are first prepared, for example using the multiprime labeling kit (sold by the Amersham Life Sciences Company, Cleveland, Ohio, United States).
  • compositions modulating the activity of a regulatory nucleic acid according to the invention can be identified in vivo, in non-human transgenic animals.
  • a non-human transgenic animal for example a mouse
  • a candidate molecule or substance to be tested for example a candidate substance or molecule previously selected by an in vitro screening method as defined above.
  • the activity level of the regulatory nucleic acid according to the invention is determined and compared with the activity of an identical non-human transgenic animal, for example an identical transgenic mouse, which has not received the candidate molecule or substance.
  • the activity of the regulatory nucleic acid according to the invention which is functional in the transgenic animal can be determined by various methods, for example the measurement of the levels of messenger RNA corresponding to the reporter polynucleotides of interest placed under the control of said nucleic acid. regulator by Northern hybridization, or by in situ hybridization, or by non-invasive biophotonic imaging (Xenogen Corporation).
  • the activity of the regulatory nucleic acid according to the invention can be determined by measuring the expression levels of protein encoded by the reporter polynucleotides of interest, for example by immunohistochemistry, in the case where the reporter polynucleotide of interest includes an open reading frame encoding a protein detectable by such a technique.
  • non-human mammals such as mice, rats or guinea pigs or rabbits which have their genome modified by the insertion of a polynucleotide construct comprising a reporter polynucleotide of interest placed under the control of a regulatory nucleic acid according to the invention.
  • transgenic animals according to the invention comprise the transgene, that is to say the abovementioned polynucleotide construct, in a plurality of their somatic and / or germ cells.
  • transgenic animals can be carried out according to conventional techniques well known to those skilled in the art.
  • a person skilled in the art may in particular refer to the production of transgenic animals, and particularly to the production of transgenic mice, as described in US patents No. 4,873,191 (issued October 10, 1989), US No. 5,464,764 ( issued November 7, 1995) and US 5,789,215 (issued August 4, 1998) the content of these documents being incorporated herein by reference.
  • a polynucleotide construct comprising a regulatory nucleic acid according to the invention and a reporter polynucleotide of interest placed under the control of the latter is inserted into an ES type stem cell line.
  • the insertion of the polynucleotide construct is preferably carried out by electroporation, as described by Thomas et al. (1987, Cell, Vol. 51: 503-512).
  • the cells which have undergone the electroporation step are then screened for the presence of the polynucleotide construct (for example by selection using markers, or by PCR or by analysis on Southern DNA-type electrophoresis gel. ) in order to select the positive cells which have integrated the exogenous polynucleotide construct into their genome, if appropriate following a homologous recombination event.
  • Such a technique is for example described by MANSOUR et al. (Nature (1988) 336: 348-352).
  • ES type cells are brought into contact with 2.5-day embryos at an 8-16 cell stage (moruiae) as described by WOOD et al. (1993, Proc. Natl. Acad. Sci. USA, vol.90: 4582-4585) or by NAGY et al. (1993, Proc. Natl. Acad. Sci. USA, vol. 90: 8424-8428), the ES cells being internalized in order to colonize the blastocyst extensively, including the cells giving rise to the germ line. The descendants are then tested to determine those who have integrated the polynucleotide construct (the transgene).
  • the invention therefore also relates to a non-human transgenic animal whose somatic and / or germ cells have been transformed by a nucleic acid or a polynucleotide construct according to the invention.
  • the invention also relates to recombinant host cells obtained from a transgenic animal as described above.
  • Recombinant cell lines from a transgenic animal according to the invention can be established in long-term culture from any tissue of such a transgenic animal, for example by transfection of primary cell cultures with vectors expressing oncogenes such as the large T antigen of SV40, as described for example by CHOU (1989, Mol. Endocrinol. Vol. 3: 1511-1514) and SCHAY et al. (1991, Biochem. Biophys.
  • the invention also relates to a method for the in vivo screening of a molecule or of a candidate substance modulating the activity of a regulatory nucleic acid according to the invention, comprising the steps consisting in a) administering the substance or the candidate molecule for a transgenic animal as defined above; b) detecting the expression level of a reporter polynucleotide of interest placed under the control of the regulatory nucleic acid; c) compare the results obtained in b) with the results obtained in a transgenic animal which has not received the candidate substance or molecule.
  • the invention further relates to a method for in vivo screening of a substance or molecule modulating the transcription of a polynucleotide of interest constituting a nucleic acid according to the invention, characterized in that it comprises the steps consisting in (a) administering the candidate substance or molecule to a non-human transgenic mammal as defined above; (b) detecting the expression of the polynucleotide of interest in the transgenic mammal as treated in step a); and (c) comparing the detection results of step (b) with the results observed in a non-human transgenic mammal as defined above who have not received the administration of the candidate substance or molecule.
  • the invention also relates to a kit or kit for the in vivo screening of a candidate molecule or substance modulating the activity of a regulatory nucleic acid according to the invention, comprising (a) a transgenic animal as defined above above ; (b) if appropriate, the means for detecting the level of expression of the reporter polynucleotide of interest.
  • the invention also relates to pharmaceutical compositions intended for the prevention or treatment of a deficit in lipid metabolism, or of a dysfunction in the processes involving the immune system and inflammation.
  • the subject of the invention is also a substance or a candidate molecule modulating the activity of a regulatory nucleic acid according to the invention.
  • the invention also relates to a candidate substance or molecule characterized in that it increases the activity of a regulatory nucleic acid according to the invention, and very particularly of a regulatory nucleic acid comprising sequence SEQ ID N ° 1, 2, 4 or 5.
  • a substance or molecule capable of modulating the activity of a regulatory nucleic acid according to the invention was selected according to one of the screening methods in vitro or in vivo defined above.
  • a subject affected in lipid metabolism or in the signaling of immunity is treated by the administration on this subject of an effective amount of a compound modulating the activity of a regulatory nucleic acid according to the invention .
  • a patient with low activity of the ABCA7 promoter can be treated with an aforementioned molecule or substance in order to increase the activity of the ABCA7 promoter.
  • a patient with abnormally high ABCA7 promoter activity may be treated with a compound capable of decreasing or blocking the activity of the ABCA7 promoter.
  • the present invention also relates to a substance or molecule used as an active principle of a medicament.
  • a compound can be a compound which modulates the interaction of at least one transcription factor with the ABCA7 promoter or a regulatory element of a regulatory nucleic acid according to the invention.
  • the compound can inhibit the interaction of one of the transcription factors listed in Table 1 with a regulatory nucleic acid according to the invention.
  • the compound can also be a compound which modulates the activity of a transcription factor which binds to the promoter of ABCA7 or of a regulatory element present on the latter.
  • a compound of therapeutic interest according to the invention can also be a compound which modulates the interaction of a first transcription factor with a second transcription factor.
  • a compound of therapeutic interest according to the invention is preferably chosen from nucleic acids, peptides and small molecules.
  • such a compound can be an antisense nucleic acid which binds specifically on a region of the ABCA7 promoter or on a regulatory element of an ABCA7 regulatory nucleic acid and inhibiting or decreasing the activity of the promoter.
  • This compound of therapeutic interest can also be an antisense nucleic acid which interacts specifically with a gene coding for a transcription factor modulating the activity of the promoter ABCA7, such that the interaction of the antisense nucleic acid with the coding gene for the transcription factor which binds to the promoter ABCA7 decreases the production of this transcription factor, resulting in an increase or a decrease in the activity of the promoter ABCA7, depending on whether the transcription factor increases or on the contrary reduces the activity of promoter ABCA7.
  • the toxicity and the therapeutic efficacy of the therapeutic compounds according to the invention can be determined according to standard pharmaceutical protocols in cells in culture or in experimental animals, for example to determine the lethal dose LD 50 (i.e. the lethal dose for 50% of the population tested) as well as the effective dose ED 5 o (that is to say the therapeutically effective dose in 50% of the population tested).
  • LD 50 i.e. the lethal dose for 50% of the population tested
  • ED 5 o that is to say the therapeutically effective dose in 50% of the population tested.
  • the therapeutically effective dose can be estimated initially from tests carried out in cell cultures in vitro.
  • a subject of the invention is also pharmaceutical compositions comprising a therapeutically effective amount of a substance or molecule of therapeutic interest according to the invention.
  • compositions according to the invention are more particularly intended for the treatment and / or prevention of deficiencies in the metabolism of lipids, or in the mechanisms involving the immune system and inflammation.
  • compositions can be formulated in conventional manner using one or more physiologically acceptable vectors or excipients.
  • the compounds of therapeutic interest according to the invention can be formulated for administration by injection, inhalation or also by oral, buccal, parenteral or rectal administration.
  • Techniques for preparing pharmaceutical compositions according to the invention can be easily found by those skilled in the art trade, for example in the book Remmington's Pharmaceutical Sciences, Mead Publishing Co., Easton, PA, USA.
  • compositions according to the invention can be formulated in the form of liquid solutions, preferably in physiologically compatible solutions or buffers.
  • the invention further relates to methods for determining whether a subject presents a risk for the development of a pathology linked to a deficit in lipid metabolism, or in processes involving the immune system and inflammation.
  • Such methods include detecting, in cells of a biological sample from the test subject, the presence or absence of a genetic alteration characterized by an alteration in the expression of a gene whose expression is regulated by the promoter of ABCA7.
  • a genetic alteration characterized by an alteration in the expression of a gene whose expression is regulated by the promoter of ABCA7.
  • such genetic alterations can be detected in order to determine the existence of a deletion of one or more nucleotides in the sequence of an ABCA7 regulatory nucleic acid, of the addition of one or more nucleotides or alternatively the substitution of one or more nucleotides in said sequence SEQ ID No. 1, 2, 3 or 6.
  • the genetic alteration is identified according to a method comprising the sequencing of all or part of the sequence SEQ ID N ° 1, or alternatively of all or part of at least the sequence SEQ ID N ° 2.
  • Sequencing primers can be constructed in order to hybridize with a determined region of the sequence SEQ ID No. 1. Such sequencing primers are preferably constructed so as to amplify fragments of approximately 300 about 500 nucleotides from the sequence SEQ ID No. 1 or from a complementary sequence.
  • the amplified fragments for example by the PCR method, are then sequenced and the sequence obtained is compared with the reference sequence SEQ ID No. 1 in order to determine whether one or more deletions, additions or substitutions of nucleotides are found in the amplified sequence from the DNA contained in the biological sample from the test subject.
  • the invention therefore also relates to a method for detecting an alteration in the transcription of the ABCA7 gene in a subject, comprising the following steps: a) sequencing of an amplifiable nucleic acid fragment using at least one nucleotide primer hybridizing with the sequence SEQ ID No. 1 or SEQ ID No. 2 according to the invention; b) align the sequence obtained in a) with the sequence SEQ ID No. 1 or SEQ ID No. 2; c) determining the presence of one or more deletions, additions or substitutions of at least one nucleotide in the sequence of the nucleic acid fragment, relative to the reference sequence SEQ ID No. 1 or SEQ ID No. 2.
  • the invention also relates to a kit or kit for the detection of an alteration in the transcription of the ABCA7 gene in a subject, comprising one or more sequencing primers capable of hybridizing with a region of the sequence SEQ ID No: 1, and thus allow the sequencing of a polynucleotide located upstream of the site of initiation of transcription of the ABCA7 gene in the test subject.
  • part of the invention also includes oligonucleotide probes hybridizing with a region of the sequence SEQ ID No. 1 or of the sequence SEQ ID No. 2 in which an alteration in the sequence was determined during implementation of the detection method described above.
  • oligonucleotide probes hybridizing specifically with a corresponding region of the sequence SEQ ID No 1 or of the sequence SEQ ID No 2, for which one or more deletions, additions or substitutions of at least one nucleotide has been determined in a subject.
  • Such oligonucleotide probes constitute means for detecting alterations in the regulatory sequence of the ABCA7 gene and therefore also means for detecting a predisposition to the development of a pathology linked to a deficit in lipid metabolism or to dysfunction at the level processes involving the immune system and inflammation.
  • a further subject of the invention is therefore a kit or kit for detecting an alteration in the transcription of the ABCA7 gene in a subject, comprising: a) one or more primers hybridizing with a region of the sequence SEQ ID No. 1 or the sequence SEQ ID No. 2; b) if necessary, the means necessary for carrying out an amplification reaction.
  • the subject of the invention is also a kit or kit for the detection of an alteration in the transcription of the ABCA7 gene in a subject, comprising: a) one or more oligonucleotide probes as defined above; b) where appropriate, the reagents necessary for carrying out a hybridization reaction.
  • the subject of the invention is also a kit or kit for the detection of an alteration in the transcription of the ABCA7 gene in a subject, comprising one or more probes hybridizing with a region of the sequence SEQ ID No: 1 or of the sequence SEQ ID No: 2 used to quantify the messenger RNA of ABCA7 in biological material originating from said test subject.
  • nucleic acid fragments derived from any one of the nucleotide sequences SEQ ID No. 1 -6 are therefore useful for the detection of the presence of at least one copy of a nucleotide sequence regulating the ABCA7 gene or else of a fragment or variant (containing a mutation or polymorphism) of the latter in a sample.
  • nucleotide probes or primers according to the invention comprise at least 8 consecutive nucleotides of a nucleic acid chosen from the group consisting of sequences SEQ ID NO 1 -5, or of a nucleic acid of complementary sequence.
  • nucleotide probes or primers according to the invention will have a length of 10, 12, 15, 18 or 20 to 25, 35, 40, 50, 70, 80, 100, 200, 500, 1000, 1500 consecutive nucleotides d 'a nucleic acid according to the invention, in particular a nucleic acid of nucleotide sequence chosen from sequences SEQ ID NO 1- 5.
  • a probe or a nucleotide primer according to the invention will consist and / or include fragments with a length of 12, 15, 18, 20, 25, 35, 40, 50, 100, 200, 500, 1000, 1500 nucleotides sequences of a nucleic acid according to the invention, more particularly of a nucleic acid chosen from the sequences SEQ
  • the definition of a probe and of a nucleotide primer according to the invention therefore includes oligonucleotides which hybridize, under the conditions of high stringency hybridization defined above, with a nucleic acid chosen from the sequences SEQ ID NO 1 - 5, 6 or 8 or with a sequence complementary to these.
  • a primer or a nucleotide probe according to the invention can be prepared by any suitable method well known to those skilled in the art, including by cloning and action of restriction enzymes or also by direct chemical synthesis according to techniques such as the method to the phosphodiester of Narang et al., (Methods Enzymol (1979) 68: 90-98) or of Brown et al. (Methods Enzymol (1979) 68: 109-151), the diethylphosphoramidite method of Beaucage et al. (Tetrahedron Lett (1980) 22: 1859-1862) or the solid support technique described in patent EP 0 707 592.
  • Each of the nucleic acids according to the invention can be marked, if desired, by incorporating a marker detectable by spectroscopic, photochemical, biochemical, immunochemical or even chemical means.
  • markers can consist of radioactive isotopes (32P, 33P, 3H, 35S), fluorescent molecules (5-bromodeoxyuridine, fluorescein, acetylaminofluorene, digoxigenin) or also ligands such as biotin.
  • the labeling of the probes is preferably carried out by incorporation of labeled molecules within the polynucleotides by extension of primers, or by addition on the 5 ′ or 3 ′ ends or also by “nick translation”.
  • the probes according to the invention may have structural characteristics such as to allow amplification of the signal, such as the probes described by Urdea et al. (Mol. Cell. Biol., (1991) 6: 716-718), or also in European patent n ° EP-0 225 807 (CHIRON).
  • the oligonucleotide probes according to the invention can be used in particular in hybridizations of the Southern type with genomic DNA or else of the northern type with RNA.
  • the probes according to the invention can also be used for the detection of PCR amplification products or even for the detection of mismatches.
  • Nucleotide probes or primers according to the invention can be immobilized on a solid support.
  • solid supports are well known to those skilled in the art and include surfaces of the wells of microtiter plates, polystyrene beds, magnetic beds, nitrocellulose strips, or even microparticles such as latex particles.
  • the present invention also relates to a method for detecting the presence of a nucleic acid as described above in a sample, said method comprising the steps consisting in: 1) bringing one or more nucleotide probes according to l invention with the test sample;
  • the oligonucleotide probe (s) are immobilized on a support.
  • the oligonucleotide probes include a detectable marker.
  • the invention further relates to a kit or kit for detecting the presence of a nucleic acid according to the invention in a sample, said kit comprising: a) one or more nucleotide probes as described above; b) where appropriate, the reagents necessary for the hybridization reaction.
  • the detection kit or kit is characterized in that the probe or probes are immobilized on a support.
  • the detection kit or kit is characterized in that the oligonucleotide probes comprise a detectable marker.
  • the oligonucleotide probes comprise a detectable marker.
  • such a kit will comprise a plurality of oligonucleotide probes in accordance with the invention which can be used to detect target sequences of interest or alternatively to detect mutations in the coding regions or the non-coding regions of the nucleic acids according to the invention, more particularly nucleic acids of sequences SEQ ID NO 1-5, 6 and 8 or the nucleic acids of complementary sequence.
  • the probes according to the invention immobilized on a support can be ordered in matrices such as "DNA chips".
  • matrices such as "DNA chips”.
  • nucleotide primers according to the invention can be used to amplify any of the nucleic acids according to the invention, and more particularly all or part of a nucleic acid of sequences SEQ ID NO 1-5, or alternatively a variant of that -this.
  • Another subject of the invention relates to a method for the amplification of a nucleic acid according to the invention, and more particularly a nucleic acid of sequences SEQ ID NO 1-5 or a fragment or a variant thereof contained in a sample, said method comprising the steps consisting in: a) bringing the sample, in which the presence of the target nucleic acid is suspected, into contact with a pair of nucleotide primers whose position hybridization is located respectively on the 5 ′ side and on the 3 ′ side of the region of the target nucleic acid whose amplification is sought, in the presence of the reagents necessary for the amplification reaction; and b) detecting amplified nucleic acids.
  • amplification method as defined above, one will advantageously have recourse to any one of the nucleotide primers described above.
  • the subject of the invention is also a kit or kit for the amplification of a nucleic acid according to the invention, and more particularly all or part of a nucleic acid of sequences SEQ ID NO 1-5, said kit or kit comprising a) a pair of nucleotide primers in accordance with the invention, the hybridization position of which is located respectively on the 5 'side and on the 3' side of the target nucleic acid whose amplification is sought; b) where appropriate, the reagents necessary for the amplification reaction.
  • Such an amplification kit or kit will advantageously comprise at least one pair of nucleotide primers as described above.
  • FIG. 1 is a schematic representation of the sites of transcription factors found in humans and mice within the promoter region of the ABCA7 genes.
  • FIG. 2 illustrates the sequence SEQ ID No. 1 and the position of each of the characteristic motifs for attachment to different transcription factors is shown in bold type, the designation of the specific transcription factor of the corresponding sequence being indicated above the nucleotide sequence.
  • FIG. 3 illustrates the sequence SEQ ID No. 4 and the position of each of the characteristic motifs for attachment to different transcription factors is shown in bold type, the designation of the specific transcription factor of the corresponding sequence being indicated above the nucleotide sequence.
  • FIG. 4 illustrates the pattern of expression of the human ABCA7 gene on northern blots of different adult and fetal tissues (Clontech) hybridized with an amplimer produced with the primers SEQ ID Nos. 9 and 10 (table 4).
  • FIG. 5 illustrates the pattern of expression of the murine ABCA7 gene on a northern blot of different adult tissues hybridized with an amplimer produced with primers specific for the murine transcript.
  • FIG. 6 represents the expression profile of the gene coding for the protein ABCA7 on a cross section of an inflamed artery, by in situ hybridization with an antisense probe ABCA7.
  • FIG. 7 represents the expression profile of the gene coding for the protein ABCA7 on a section of the bronchi of an asthmatic patient, by in situ hybridization with an antisense probe ABCA7.
  • FIG. 8 represents the expression profile of the gene coding for the protein
  • ABCA7 on a colon section of a patient with Crohn's disease, by in situ hybridization with an ABCA7 antisense probe.
  • FIG. 9 represents the expression profile of the gene coding for the protein ABCA7 on a section of the lymph node, by in situ hybridization with an antisense probe ABCA7.
  • FIG. 10 represents the expression profile of the gene coding for the ABCA7 protein of a synovial section of a patient suffering from rheumatoid arthritis, by in situ hybridization with an ABCA7 antisense probe.
  • FIG. 11 represents the expression profile of the gene coding for the protein ABCA7 on a section of skin of a patient suffering from psoriasis by in situ hybridization with an antisense probe ABCA7.
  • RACE RT-PCR
  • MRNAs polyA
  • the first amplification primers and the internal primers were chosen from the cDNA sequence.
  • the amplifications carried out with the internal PCR amplification primers were cloned. Specific clones were then amplified using primers whose sequences are respectively (CAGGAAACAGCTATGAC) and (GCCAGTGTGATGGATAT) and sequences on both strands.
  • the transcription initiation site was located on the promoters of the human and murine genes of ABCA7 using the following three software: TSSG and TSSW (Solovyev et al., Ismb (1997) 5, 294-302) and NNPP ( Reese MG, et al., 1999).
  • a prediction of the binding sites for human and murine transcription factors was made using the Matlnspector motif search program (Quandt et al., Nucl Acid Research (1995) 23 (23) 4878-84).
  • the calculation of the scores for each transcription factor binding site is carried out using the following formula: (Of- Tf) / (Tf) 1/2 , in which Of is the frequency of observation of a pattern and Tf is the calculated frequency of a consensus pattern.
  • a first filtration step was carried out by adjusting the “matrix similarity” score of the Matlnspector program above 0.85 and the “core similarity” score above 0.99.
  • a comparative analysis of the interspecies promoters was carried out according to what is described by Werner T (Models for prediction and recognition of eukaryotic promoters, Mammalian Génome (1999) 10: 168-175) in order to define transcription modules comprising sites with a similar motif and present both on the human and murine sequences of the sequence upstream of the ABCA7 gene.
  • the expression profile of the polynucleotides according to the present invention is determined according to the northern blot analysis and reverse transcription protocols coupled to the PCR described in particular by Sambrook et al (ref. CSH Sambrook, J., Fritsch, EF, and Maniatis, T. (1989). "Molecular Cloning: A Laboratory Manual,” 2 ⁇ ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.).
  • PCR polymerase chain reaction
  • Reverse transcription into cDNA is carried out with the enzyme SUPERSCRIPT II (GibcoBRL, Life Technologies) according to the conditions described by the manufacturer.
  • the polymerase chain reaction is carried out according to standard conditions, in 20 ⁇ l of reaction mixture with 25 ng of the cDNA preparation.
  • the reaction mixture is composed of 400 ⁇ M of each of the dNTPs, of 2 units of Thermus aquaticus (Taq) DNA polymerase (Ampli Taq Gold; Perkin Elmer), of 0.5 ⁇ M of each primer, of 2.5 mM MgCI2, and of PCR buffer.
  • Taq Thermus aquaticus
  • PCR cycles (denaturation 30 s at 94 ° C, 30 s hybridization broken down as follows during the 30 cycles: 64 ° C 2 cycles, 61 ° C 2 cycles, 58 ° C 2 cycles and 55 ° C 28 cycles and one elongation of one minute per kilobase at 72 ° C) are carried out after a first denaturation step at 94 ° C for 10 min in a Perkin Elmer 9700 thermocycler.
  • the PCR reactions are visualized on agarose gel by electrophoresis.
  • the cDNA fragments obtained can be used as probes for analysis by Northern blot and can also be used for the exact determination of the polynucleotide sequence.
  • a cDNA probe produced as described above is labeled with 32 P using the High Prime DNA labeling system (Boehringer) according to the instructions indicated by the manufacturer. After labeling, the probe is purified on a micro-column of Sephadex G50 (Pharmacia) according to the instructions indicated by the manufacturer. The labeled and purified probe is then used for the detection of the expression of mRNAs in different tissues.
  • the blot is analyzed after a night of exposure on contact with a phosphor screen revealed using the Storm (Molecular Dynamics, Sunnyvale, CA).
  • the results presented in FIG. 5 show that the mouse ABCA7 gene is expressed in adult tissues. A higher amount of murine ABCA7 mRNA is detected in hematopoietic tissues such as the spleen and thymus, which is consistent with the expression of ABCA7 which has been observed in myelomonocytic and lymphocytic lines. No expression of the ABCA7 gene was detected in the fibroblastic cell lines.
  • Figure 4 shows a similar expression pattern of the human ABCA7 gene with, however, a strong signal of hybridization in the fetal liver.
  • RNAs are obtained from hematopoietic tissues of normal subjects or those affected by the guanidine isothiocyanate method (Chomczynski et al., Anal Biochem (1987) 162: 156-159).
  • Poly (A) + mRNAs are obtained by affinity chromatography on oligo (dT) -cellulose columns (Sambrook et al., (1989) Molecular cloning: a laboratory manual. 2 ed.
  • cDNAs used as probes are obtained by RT-PCR (DeRisi et al., Science (1997) 278: 680-686) with oligonucleotides labeled with a fluorescent product (Amersham Pharmacia Biotech; CyDye TM).
  • the glass slides containing the sequences according to the present invention corresponding to the ABCA7 gene are hydridized with the nucleotide probes prepared from the messenger RNA of the cell to be analyzed.
  • the use of the Amersham / molecular Dynamics system (Avalanche Microscanner TM) allows the differential quantification of the expressions of the sequence products on the healthy or affected cell type.
  • EXAMPLE 5 Test intended for the screening of molecules activating or inhibiting the expression of the ABCA7 gene.
  • the screening test makes it possible to identify a substance capable of modulating the activity of synthesis of the ABCA7 protein.
  • the region of the regulatory acid of the human ABCA7 gene going from the nucleotide at position -1111 to the nucleotide at position -1, relative to the site of initiation of transcription, can be amplified by the PCR technique using the pair of primers specific for the region described above from human genomic DNA present in a BAC vector from a collection of human BAC vectors.
  • the amplified DNA fragment is digested with the restriction endonuclease Sal 1, then inserted into the vector PXP1 described by Nordeen et al. (Bio Techniques, (1988) 6: 454-457), at the Sal 1 restriction site of this vector. The insert was then sequenced.
  • E-MEM medium Minimun Essential Medium with Earle's Salts
  • 10% (v / v) fetal calf serum BioWhittaker, Walkersville, MD.
  • Approximately 1.5 X 10 5 cells were distributed into each of the wells of a 12-well culture plate (2.5 cm), and were grown to about 50-70% confluence, then co-transformed with 1 ⁇ g of the plasmid Sal-Lucif and 0.5 ⁇ g of the control vector pBetagal (CloneTech Laboratories Inc., Palo Alto, CA, USA) using the necessary Superfectin Reagent Kit (QIAGEN Inc., Valencia, CA, USA). Two hours after the addition of the DNA, the culture medium is removed and replaced with complete AMEM medium (Minimum Esential Medium Eagle's Alpha Modification).
  • AMEM medium Minimum Esential Medium Eagle's Alpha Modification
  • the cells are placed in fresh medium of the DMEM type (Dulbecco's Minimum Essential Medium) supplemented with 2 ⁇ g / ml of glutamine, 100 units / ml of streptomycin and 0.1% bovine serum albumin (BSA , Fraction V), in the presence or absence of molecules at different concentrations.
  • DMEM Dulbecco's Minimum Essential Medium
  • BSA bovine serum albumin
  • the cells are recovered 16 hours after the last change of medium using a lysis solution (Lysis Solution) from the Tropix Luciferase Assay Kit (Tropix Inc., Bedford, MA, USA).
  • the cell lysate is divided into aliquots which are used to quantify the proteins using the MicroBCA Kit (Pierce, Rockford, IL, USA) as well as to quantify the production of luciferase and beta-galactosidase respectively.
  • required Tropix Luciférase Assay Kit and Galacto-Light Plus Kit The tests are carried out according to the manufacturer's recommendations.
  • the molecules active on the ABCA7 promoter are then selected according to the ratio "luciferase activity / beta-galactosidase activity.
  • RNA probes Tissue samples involved in paraffin were hybridized with complementary radioactive labeled RNA probes. More specifically, a fragment of the ABCA7 gene corresponding to the nucleotide sequence going from nucleotide 594 to nucleotide 1055 of the GenBank sequence designated NM-0191 12 was subcloned in the plasmid pCRI1 (Invitrogen).
  • RNA probes labeled with Uridin triphosphate 35 S were then generated with the RNA polymerases SP6 and T7, then hybridized to the different tissue sections.
  • the different sections of tissue were digested with proteinase K and hybridized with the previously described probes at a concentration equal to approximately 3.5 ⁇ 10 7 dpm / ml for 18 hours at 65 ° C.
  • the slides were then treated with RNAase A and washed in SSC, 0.1 X at 70 ° C for 2 hours, and were covered with a Kodak NTB-2 photographic emulsion, exposed for 7 days to 4 ° C, then revealed using a Kodak D-19 solution.
  • Figure 6 is a section through the artery of a 92-year-old man who underwent an amputation below the knee, with arteriosclerosis and acute inflammation. There is a weak specific marking of the macrophages in the thrombi and in the site of inflammatory infiltration in the adventitious coat.
  • Figure 7 which is a section of bronchi taken during the autopsy of a 63 year old asthmatic woman, shows a weak marking of lymphocytes and macrophages in the inflammatory submucosal infiltrate.
  • Figure 8 is a section of colon collected during the operation of an 81-year-old woman with a clinical diagnosis of Crohn's disease. We observes a labeling of macrophages and a subfamily of lymphocytes in the lamina propria.
  • Figure 9 corresponds to a section of a lymph node removed during the operation of a 48-year-old man.
  • the ganglion cells are weakly labeled, and isolated macrophages are also labeled in the lymph node.
  • FIG. 10 which represents a section of the synovium of a 25 year old woman presenting a clinical diagnosis of rheumatoid arthritis, shows a strong marking of the sub synovial histiocytes and the macrophages.
  • FIG. 11 which represents a section of skin obtained following the biopsy of a 55 year old woman suffering from psoriasis, shows an average marking of the macrophages in the perivascular inflammatory infiltrate. Isolated perivascular lymphocytes are also labeled.
  • Table 1 Sites, scores, consensus, and positions relative to the transcription initiation point (TSS) predicted by NNPP, TSSG and TSSW software in humans
  • mice Sites, scores, consensus, and positions relative to the point of initiation of transciription (TSS) predicted by NNPP, TSSG and TSSW software in mice
  • Oligonucleotides specific for the human ABC-A7 gene are Oligonucleotides specific for the human ABC-A7 gene.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Zoology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Wood Science & Technology (AREA)
  • Biophysics (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Plant Pathology (AREA)
  • Pain & Pain Management (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Rheumatology (AREA)
  • Physics & Mathematics (AREA)
  • Diabetes (AREA)
  • Hematology (AREA)
  • Obesity (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)
PCT/FR2001/003219 2000-10-24 2001-10-17 Acide nucleique regulateur du gene abca7, molecules modulant son activite et applications therapeutiques WO2002034903A2 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
JP2002537874A JP2004512045A (ja) 2000-10-24 2001-10-17 Ａｂｃａ７遺伝子を調節する核酸、その活性をモジュレートする分子および治療用途
EP01978557A EP1334186A2 (de) 2000-10-24 2001-10-17 Nukleinsäure der regulierungsequenzen von abca7, deren modulatoren und therapeutischen anwendungen
AU2002210661A AU2002210661A1 (en) 2000-10-24 2001-10-17 Nucleic acid generating the ABCA7 gene, molecules modulating its activity and therapeutic applications
CA002426294A CA2426294A1 (fr) 2000-10-24 2001-10-17 Acide nucleique regulateur du gene abca7, molecules modulant son activite et applications therapeutiques

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
FR0013649A FR2822165B1 (fr) 2000-10-24 2000-10-24 Acide nucleique regulateur du gene abca7, molecules modulant son activite et applications therapeutiques
FR00/13649 2000-10-24
US25314100P 2000-11-28 2000-11-28
US60/253,141 2000-11-28

Publications (2)

Publication Number Publication Date
WO2002034903A2 true WO2002034903A2 (fr) 2002-05-02
WO2002034903A3 WO2002034903A3 (fr) 2002-08-08

Family

ID=26212696

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/FR2001/003219 WO2002034903A2 (fr) 2000-10-24 2001-10-17 Acide nucleique regulateur du gene abca7, molecules modulant son activite et applications therapeutiques

Country Status (5)

Country Link
EP (1) EP1334186A2 (de)
JP (1) JP2004512045A (de)
AU (1) AU2002210661A1 (de)
CA (1) CA2426294A1 (de)
WO (1) WO2002034903A2 (de)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005056796A1 (en) * 2003-12-15 2005-06-23 Noab Biodiscoveries Inc. Materials and methods for analysis of atp-binding cassette transporter gene expression

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000058473A2 (en) * 1999-03-31 2000-10-05 Curagen Corporation Nucleic acids including open reading frames encoding polypeptides; 'orfx'
WO2001009314A1 (fr) * 1999-07-30 2001-02-08 Institut National De La Sante Et De La Recherche Medicale-Inserm Nouvelles applications des transporteurs de type abca

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000058473A2 (en) * 1999-03-31 2000-10-05 Curagen Corporation Nucleic acids including open reading frames encoding polypeptides; 'orfx'
WO2001009314A1 (fr) * 1999-07-30 2001-02-08 Institut National De La Sante Et De La Recherche Medicale-Inserm Nouvelles applications des transporteurs de type abca

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
BROCCARDO C ET AL: "Comparative analysis of the promoter structure and genomic organization of the human and mouse ABCA7 gene encoding a novel ABCA transporter." CYTOGENETICS AND CELL GENETICS, vol. 92, no. 3-4, 2001, pages 264-270, XP001028244 ISSN: 0301-0171 *
DATABASE EMBL [en ligne] EMBL; 3 mars 2000 (2000-03-03) DOE JOINT GENOME INSTITUTE: "H.sapiens chromosome 19 clone CTD-2621i17, 51 unordered pieces." retrieved from WWW.EBI.AC.UK Database accession no. AC024580 XP002178168 *
DATABASE GENESEQ [en ligne] EMBL; 21 juillet 2000 (2000-07-21) WEI Y. ET AL.: "EST" retrieved from WWW.EBI.AC.UK Database accession no. BE247495 XP002178167 *
KAMINSKI W E ET AL: "COMPLETE CODING SEQUENCE, PROMOTER REGION, AND GENOMIC STRUCTURE OFTHE HUMAN ABCA2 GENE AND EVIDENCE FOR STEROL-DEPENDENT REGULATION IN MACROPHAGES" BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, ACADEMIC PRESS INC. ORLANDO, FL, US, vol. 281, no. 1, 16 février 2001 (2001-02-16), pages 249-258, XP000990418 ISSN: 0006-291X *
KAMINSKI WOLFGANG E ET AL: "Genomic organization of the human cholesterol-responsive ABC transporter ABCA7: Tandem linkage with the minor histocompatibility antigen HA-1 gene." BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 278, no. 3, 30 novembre 2000 (2000-11-30), pages 782-789, XP000990417 ISSN: 0006-291X *
KAMINSKI WOLFGANG E ET AL: "Identification of a novel human sterol-sensitive ATP-binding cassette transporter (ABCA7)." BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 273, no. 2, 5 juillet 2000 (2000-07-05), pages 532-538, XP002178649 ISSN: 0006-291X cité dans la demande *
See also references of EP1334186A2 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005056796A1 (en) * 2003-12-15 2005-06-23 Noab Biodiscoveries Inc. Materials and methods for analysis of atp-binding cassette transporter gene expression

Also Published As

Publication number Publication date
EP1334186A2 (de) 2003-08-13
CA2426294A1 (fr) 2002-05-02
WO2002034903A3 (fr) 2002-08-08
AU2002210661A1 (en) 2002-05-06
JP2004512045A (ja) 2004-04-22

Similar Documents

Publication Publication Date Title
Garg et al. Tbx1, a DiGeorge syndrome candidate gene, is regulated by sonic hedgehog during pharyngeal arch development
Webster et al. Requirement for both Shc and phosphatidylinositol 3′ kinase signaling pathways in polyomavirus middle T-mediated mammary tumorigenesis
EP1471926B1 (de) Zusammensetzungen und verfahren zur therapeutischen anwendung einer mit dem gen atonal assoziierten sequenz
FR2809414A1 (fr) Acide nucleique regulateur du gene abc1, molecules modulant son activite et applications therapeutiques
WO2002034903A2 (fr) Acide nucleique regulateur du gene abca7, molecules modulant son activite et applications therapeutiques
JP2003523177A (ja) ヒトａｂｃ１遺伝子の核酸、並びに治療及び予防へのそれらの適用
US20030044895A1 (en) Nucleic acids of the human ABCA5, ABCA6, ABCA9, AND ABCA10 Genes, vectors containing such nucleic acids, and uses thereof
WO2000078970A1 (fr) Acides nucleiques et proteines correspondant au gene abc1 humain
WO2002004637A1 (fr) Promoteur specifique du myocarde
FR2746815A1 (fr) Sequences en amont du gene sm 22, vecteurs les contenant et leurs utilisations therapeutiques, notamment dans le traitement des maladies vasculaires
WO1997035974A9 (fr) Sequences en amont du gene sm22, vecteurs les contenant et leurs utilisations therapeutiques, notamment dans le traitement des maladies vasculaires
FR2822165A1 (fr) Acide nucleique regulateur du gene abca7, molecules modulant son activite et applications therapeutiques
JP2004531241A (ja) ヒトａｂｃｃ１１遺伝子の核酸、このような核酸を含むベクター、およびこれらの使用
CA2272994A1 (fr) Promoteur de la ve-cadherine et ses utilisations
US20030077591A1 (en) Nucleic acid for regulating the ABCA7 gene, molecules modulating its activity and therapeutic applications
WO2000063402A1 (fr) Cassette de regulation de l'expression d'un acide nucleique heterologue dans une cellule eucaryote, en particulier musculaire
AU2007240161A1 (en) Nucleic acid generating the ABCA7 gene, molecular modulating its activity and therapeutic applications
CN114787361A (zh) 微生物胆固醇催化基因的靶向表达降低过量脂质
WO2002090545A2 (fr) Acide nucleique codant le polypeptide cgl1 et application de cet acide nucleique et du polypeptide cgl1 au diagnostic et en therapeutique
JP2004537983A (ja) ヒトａｂｃｃ１２遺伝子の核酸、このような核酸を含むベクター、およびこれらの使用

Legal Events

Date Code Title Description
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
AK Designated states

Kind code of ref document: A3

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PH PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A3

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 2426294

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 2002537874

Country of ref document: JP

WWE Wipo information: entry into national phase

Ref document number: 2001978557

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 2002210661

Country of ref document: AU

WWP Wipo information: published in national office

Ref document number: 2001978557

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWR Wipo information: refused in national office

Ref document number: 2001978557

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 2001978557

Country of ref document: EP