JP2004512045A - Nucleic acids that regulate the ABCA7 gene, molecules that modulate its activity, and therapeutic uses - Google Patents

Abstract

The present invention relates to nucleic acids that can regulate the transcription of the ABCA7 gene, which encodes a carrier protein that can mediate lipid metabolism and / or processes including the immune system and inflammation. In addition, depending on the location of the gene at q13 on chromosome 19, ABCA7 may be involved in other pathologies genetically linked to the locus. The present invention also relates to a nucleotide construct comprising a polynucleotide encoding a predetermined polypeptide or nucleic acid placed under the control of a nucleic acid that regulates the ABCA7 gene. Further, the present invention relates to a recombinant vector, a transformed host cell and a non-human transgenic mammal comprising a nucleic acid that regulates the transcription of the ABCA7 gene and / or the nucleotide construct described above, and the ABCA7 gene. The present invention relates to a method for screening a molecule or substance capable of modulating the activity of a nucleic acid to be regulated.

Description

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【図面の簡単な説明】
【図１】
ヒトおよびマウスで見出されたＡＢＣＡ７遺伝子のプロモーター領域における転写因子部位を示す図式的な説明である。
【図２Ａ】
配列番号１の配列を示し、様々な転写因子に結合する各特徴的なユニットの位置は太字で示し、対応する配列に特異的な転写因子の記号表示は、そのヌクレオチド配列の上に示す。
【図２Ｂ】
図２Ａの続きである。
【図３Ａ】
配列番号４の配列を示し、様々な転写因子に結合する各特徴的なユニットの位置は太字で示し、対応する配列に特異的な転写因子の記号表示は、そのヌクレオチド配列の上に示す。
【図３Ｂ】
図３Ａの続きである。
【図４】
配列番号９および１０のプライマーを用いて作製されたアンプライマー（表４）とハイブリダイズした、様々な成体および胎児組織のノーザンブロット（クロンテック社）上のヒトＡＢＣＡ７遺伝子の発現パターンを示す。
【図５】
マウス転写物に特異的なプライマーを用いて作製されたアンプライマーとハイブリダイズした様々な成体組織のノーザンブロット上のマウスＡＢＣＡ７遺伝子の発現パターンを示す。
【図６】
炎症を起こした動脈の断面図における、ＡＢＣＡ７タンパク質をコードする遺伝子の発現のプロフィールを、ＡＢＣＡ７アンチセンスプローブとのインサイチュハイブリダイゼーションにより示す。
【図７】
喘息患者の気管支チューブの断面図における、ＡＢＣＡ７タンパク質をコードする遺伝子の発現のプロフィールを、ＡＢＣＡ７アンチセンスプローブとのインサイチュハイブリダイゼーションにより示す。
【図８】
クローン病に罹った患者の結腸の断面図における、ＡＢＣＡ７タンパク質をコードする遺伝子の発現のプロフィールを、ＡＢＣＡ７アンチセンスプローブとのインサイチュハイブリダイゼーションにより示す。
【図９】
リンパ節の断面図における、ＡＢＣＡ７タンパク質をコードする遺伝子の発現のプロフィールを、ＡＢＣＡ７アンチセンスプローブとのインサイチュハイブリダイゼーションにより示す。
【図１０】
リウマチ様関節炎に罹った患者の滑膜の断面図における、ＡＢＣＡ７タンパク質をコードする遺伝子の発現のプロフィールを、ＡＢＣＡ７アンチセンスプローブとのインサイチュハイブリダイゼーションにより示す。
【図１１】
乾癬に罹った患者の皮膚の断面図における、ＡＢＣＡ７タンパク質をコードする遺伝子の発現のプロフィールを、ＡＢＣＡ７アンチセンスプローブとのインサイチュハイブリダイゼーションにより示す。[0001]
The present invention relates to nucleic acids capable of regulating ABCA7 gene transcription, wherein the ABCA7 gene is a gene involved in lipid metabolism of hematopoietic tissues, as well as in cell signaling mechanisms relating to immune response and inflammation.
The invention also describes polypeptides and polynucleotides that may affect diseases associated with the chromosome 19 locus q13 by a sequence or expression defect.
[0002]
The present invention also relates to a polynucleotide comprising a polynucleotide encoding a predetermined polypeptide or a polynucleotide producing a predetermined nucleic acid, which is placed under the control of a nucleic acid that regulates the human or mouse ABCA7 gene.
The present invention also relates to recombinant vectors, transformed host cells, and non-human transgenic mammals comprising the human and mouse ABCA7 genes or nucleic acids that regulate the transcription of the above-described nucleotide constructs. The present invention relates to a method of screening for a molecule or a substance capable of modulating the activity of a nucleic acid to be expressed.
[0003]
In addition, the present invention makes it possible to detect defects in ABCA7 gene transcription, thereby diagnosing lipid metabolism at the level of hematopoietic tissue and possible dysfunction in immune cell signaling mechanisms. On how to make it.
Also, an object of the present invention is a substance or molecule that modulates the activity of a nucleic acid that regulates ABCA7 gene transcription, as well as a pharmaceutical composition containing such a substance or molecule.
[0004]
ABC (ATP binding cassette) transport proteins constitute a very well conserved superfamily during evolution from bacteria to humans. These proteins are involved in the membrane transport of various substances (eg ions, amino acids, peptides, sugars, vitamins or steroid hormones) (Higgins et al., Annu Rev. Cell Biol, 8, (1992) 67-113). .
[0005]
By characterizing the complete amino acid sequence of some ABC transporters, a common general structure can be defined, which specifically describes two nucleotide binding folds (NBFs) containing Walker type A and B units. ) And two transmembrane domains (transmembrane domains each consisting of six helices) (Klein et al., BBA, 1461 (1999), 237-262). The specificity of the ABC transporter for various transported molecules appears to be determined by the structure of the transmembrane domain, while the energy required for transport activity is provided by ATP degradation at the NBF fold level (Dean et al., Curr. Opin. Genet. Dev. 5 (1995) 779-785).
[0006]
Several ABC transport proteins have been identified in humans, many of which are involved in various diseases.
For example, cystic fibrosis is caused by mutations in the CFTR (cystic fibrous transmembrane regulator) gene, which is also called ABCC7, as well as some multidrug resistance in tumor cells. Is associated with a mutation in the gene encoding the MDR (multidrug resistance) protein, which is also called ABCB, which also has an ABC transporter structure.
Other ABC transporters are associated with neuronal and tumor conditions (US Pat. No. 5,858,719) or may be involved in diseases due to defects in metal homeostasis, ABC-3 protein.
Similarly, another ABC transporter, called PFIC2 or ABCB11, appears to be involved in the formation of progressive familial intrahepatic cholestasis, a protein involved in bile salt transport in humans. there's a possibility that.
[0007]
Also, a subfamily A of ABC transporters called ABCA has been identified. This is characterized by the presence of a highly hydrophobic segment (HH1: highly hydrophobic) between the two transmembrane domains bound to the two NBF units (Broccardo et al., BBA 1461 (1999) 395). -404). So far, four members of this subfamily have been characterized. They include transporters ABCA1 and ABCA2 (both located at loci 9q22-9q31 and 9q34, respectively, on chromosome 9), as well as transporter ABCA3 (located at 16pl3.3 on the chromosome). Transporters ABCA4 or ABCR (located at 1p22 of the chromosome) (Broccardo et al., 1999). Also, members of this subfamily are highly conserved during the evolution of multicellular eukaryotes. As an example, the best known transporters ABCA1 and ABCA4 show 95% and 88% identity with their mouse orthologs, respectively. In addition, members of this subfamily are closely related, for example, because the transporters ABCAl and ABCA4 show 50.9% protein sequence identity and very similar genomic organization (Allikmets et al. (1997) 15, 236-246; Broccardo et al., Biochim. Biophys. Acta (1999) 1461, 395-404; Luciani et al., Genomics (1994) 21, 150- (1), 150-. 9; Remalley et al., Proc. Natl. Acad. Sci. USA (1999) 96 (22), 12685-90).
[0008]
Moreover, members of subfamily A appear to show similar functional specificities at the level of membrane lipid and phospholipid transport. Indeed, loss of function of these transporters has been shown to affect the regeneration of phospholipids in bilayers of cell membranes. In the case of ABCA4, first, normal regeneration of phosphatidyl-ethanolamine (PE) in the membrane part in rod cells is observed, which leads to blindness via a series of events (Weng et al., Cell (1999) 98). (1), 13-23). In the case of ABCA1, an abnormal distribution of membrane phospholipids in the cytoplasmic membrane layer is observed, which more precisely results in the presence of a larger amount of phosphatidylserine in the outer layer and Ca²⁺Disturb the concentration.
[0009]
Transporters ABCA1 and ABCA4 are of particular interest. The ABCA1 gene does indeed appear to be involved in pathologies relating to cholesterol metabolic dysfunction, these being diseases such as, for example, atherosclerosis or familial HDL deficiency (FHD) (eg Tangier disease). (FR99 / 7684000; Rust et al., Nat. Genet., 22 (1999) 352-355; Brooks-Wilson et al., Nat. Genet., 22 (1999) 336-345; Bodzioch et al. 22 (1999) 347-351; Orso et al., Nat. Genet., 24 (2000) 192-196). Tangier's disease appears to be associated with a cellular defect in the translocation of cellular cholesterol, which causes HDL degradation, thereby disrupting lipoprotein metabolism. That is, HDL particles that do not take up cholesterol from surrounding cells are not properly metabolized, but, on the contrary, are thought to be rapidly eliminated outside the body. Therefore, the HDL concentration in the plasma of such patients is drastically reduced and HDL no longer ensures the return of cholesterol to the liver. This cholesterol accumulates in cells around it, causing the development of characteristic clinical symptoms, such as the formation of orange palatine tonsils. Moreover, perturbations of other lipoproteins are observed, for example overproduction of triglycerides and increased intracellular synthesis and catabolism of phospholipids.
[0010]
Moreover, ABCA4 transporters are associated with debilitating and inflammatory eye diseases such as recessive Stargardt's disease (Allikmets et al., 1997) and age-related degradation of the area of the retinal macula (AMD). (Allikmets et al., Nat. Genet. 15 (1997) 236-246; Allikmets et al., Science, 277 (1997) 1805-1807; Cremers et al., Hum. Mol. Genet. (1998), 7 (). Martinez-Mir et al., Nat. Genet. 18 (1998) 11-12; Weng et al., Cell (1999) 98 (1), 13-23).
[0011]
In humans, a cDNA containing the entire open reading frame of a new member of the A subfamily of ABC (ATP binding cassette) transporters has recently been cloned from human macrophage RNA in humans and is termed ABCA7 (Kaminski et al., BBR, 273 (2000), 532-538).
[0012]
Characterization of the complete amino acid sequence of ABCA7 indicates that the protein product has the general structural features of an ABCA transporter, ie, a symmetrical protein comprising two transmembrane domains and two NBF units. Having a structure. In addition to these characteristic units, the ABCA7 protein has other units recently identified as characteristic of the ABCA transporter, namely the HH1 region and the hot spot region (Broccardo et al. , Biochim. Biophys. Acta (1999) 1461, 395-404).
[0013]
Like other members of the A subfamily of ABC transporters, the sequence of the ABCA7 protein is highly conserved in mouse and human, with 79% interspecies identity. Moreover, the ABCA7 protein characterizes the intron-exon organization of ABCA subfamily members, as well as high sequence homology, especially at 54% and 49% for the human transporters ABCAl and ABCA4, respectively. .
[0014]
Moreover, the protein transporter ABCA7 appears to exhibit sterol flow-dependent regulatory properties and is similar to other members of the A subfamily, particularly the ABCAl transporter (Langman et al., BBR Com; 257 (1999), 29-33; Laucken et al., PNAS, 97 (2000) 817-822). In fact, Kaminski et al. (Supra) have shown that incubation of human macrophages in the presence of acetylated low-density lipoprotein (AcLDL), which induces sterol uptake, increases ABCA7 expression, as well. It has been found that expression is reduced in the presence of HDL3 cholesterol receptor, which causes decreased sterol uptake.
[0015]
Moreover, ABCA7, like other ABCA members, shows some differentiation with respect to its tissue expression, and ABCA7 messengers are mainly involved in hematopoietic tissue consisting of lymphocytes, granulocytes, thymus, spleen, bone marrow or fetal tissue. In contrast, ABCA1 expression is predominant in macrophages and placenta, and ABCA4 expression is restricted to the retina (Rust et al., Nat. Genet, 22, (1999) 352-355).
[0016]
With all the above data on protein sequence identity, regulatory mechanisms, and specificity of expression, the ABCA7 gene constitutes another transporter of the A subfamily and functions of other transporters, especially the ABCA1 transporter It is shown to have similar or more abundant functions.
[0017]
Therefore, this transporter is likely to act as a mediator of lipid metabolism and, like the ABCAl transporter, is highly likely to be involved in certain metabolic dysfunctions or deficiencies. Moreover, the differentiation of ABCA7 transporter expression is likely to be due to the latter, as demonstrated by Kaminski et al. (Supra), probably in the case of, for example, the pathogenesis of atherosclerosis. It has a role in transport (export) and may also play a role in immune signaling mechanisms of lymphocytes.
[0018]
Although the expression of the human ABCA7 gene appears to be regulated according to the cell type or the metabolic state of a particular cell type, it is not known whether its sequence allows for regulation of this gene.
However, in the context of the art, it is necessary to identify these regulatory sequences for the following reasons.
[0019]
a) These sequences can be mutated in patients suffering from a condition related to defective transport of lipids or possible ABCA7 protein substrates, or in patients who are likely to develop such a condition.
Characterization of the regulatory sequences of the human ABCA7 gene makes it possible to detect patient mutations and in particular to diagnose individuals belonging to potentially dangerous families. In addition, the separation of these regulatory sequences will result in a functional sequence that can compensate for the metabolic dysfunction induced by the diagnosed mutation, based on the composition of the target therapeutic means (eg, a means for gene therapy). It can complement the mutated sequence it has.
[0020]
b) The characterization of the regulatory sequence of the ABCA7 gene allows the skilled person to take advantage of the tools which can be constructed by genetic engineering, whereby the defined gene, preferably the cell type in which the ABCA7 gene is expressed, Can be expressed in
c) Moreover, some parts of the regulatory sequences of the ABCA7 gene can construct high-level constitutive promoter sequences which, in cells, provide a novel means of expressing the defined sequences. It is of the type that can be built and complement any existing means.
[0021]
However, it must be noted that despite the efforts made to date, the regulatory sequences of the ABCA7 gene have hitherto remained completely unknown.
The inventors have now reported that 33.5 kb of human genomic DNA (including 46 exons of the open reading frame of the ABCA7 gene), similarly located +1 upstream from the transcription site on the 5 'side of exon 1, An approximately 1.1 kb non-transcribed (non-transcribed) region containing the signal that regulates the ABCA7 gene was isolated and subsequently analyzed.
[0022]
In addition, the present inventors have found that a 20 kb mouse genomic DNA containing 45 exons of the open reading frame of the ABCA7 gene and a mouse ABCA7 gene which is similarly located +1 upstream from the transcription site on the 5 'side of exon 1 The approximately 1.2 kb untranscribed region of the mouse containing the regulatory signals was isolated and subsequently analyzed.
[0023]
General definition
The term "isolated" for the purposes of the present invention refers to a biological material (nucleic acid or protein) that has been removed from its natural environment (the environment in which it naturally occurs).
For example, a polynucleotide that occurs naturally in a plant or animal is not isolated.
A polynucleotide that is naturally inserted into the genome of a plant or animal is "isolated" if it is separated from adjacent nucleic acids.
Such a polynucleotide can be included in a vector, and / or such a polynucleotide can be included in a composition, but since the vector or composition does not constitute its natural environment, It remains isolated.
[0024]
In the term "purified", the material need not be in completely pure form, where the presence of other compounds has been ruled out. The terms are rather relative definitions.
A polynucleotide is in a "purified" state after purifying the starting material or natural material on a scale of at least one order of magnitude, preferably two or three orders of magnitude, more preferably four or five orders of magnitude.
For the purpose of describing the invention, the expression "nucleotide sequence" can be used to indicate either a polynucleotide or a nucleic acid.
The expression "nucleotide sequence" includes the genetic material itself and is therefore not limited to information about the sequence.
[0025]
The term “nucleic acid”, “polynucleotide”, “oligonucleotide” or “nucleotide sequence” includes an RNA, DNA or cDNA sequence or an RNA / DNA hybrid sequence of two or more nucleotides and is single-stranded or double-stranded. Any of chain forms may be used.
The term "nucleotide" refers to both natural nucleotides (A, T, G, C) and modified nucleotides, where the modified nucleotides are at least one modification, such as (1) a purine analog, Examples of such modified nucleotides comprising 2) a pyrimidine analog, or (3) a similar sugar, are described, for example, in PCT application WO 95/04064.
[0026]
For the purposes of the present invention, a first polynucleotide is considered to be `` complementary '' to a second polynucleotide, wherein each base of the first nucleotide is a second polynucleotide in the opposite direction. Is paired with the complementary base of The complementary bases are A and T (or A and U) or C and G.
[0027]
A “variant” of a nucleic acid according to the invention is understood to mean a nucleic acid which differs by one or more bases from the reference polynucleotide. A variant nucleic acid may be a variant of a naturally occurring, eg, naturally occurring allele, or a non-natural variant, eg, obtained by mutagenesis techniques.
[0028]
In general, the differences between a reference nucleic acid and a variant nucleic acid are such that the nucleotide sequences of the reference nucleic acid and the variant nucleic acid are so similar that the nucleotide sequences are low and identical in many regions. The modification of the nucleotide present in the mutant nucleic acid may be silent, which means that the amino acid sequence encoded by the mutant nucleic acid is not changed.
However, changes in the nucleotides in the mutant nucleic acid also result in substitutions, additions or deletions in the polypeptide encoded by the mutant nucleic acid with respect to the peptide encoded by the reference nucleic acid. In addition, nucleotide modifications in such coding regions can result in conservative or non-conservative substitutions in the amino acid sequence.
[0029]
Preferably, the mutant nucleic acid according to the present invention has substantially the same function or biological activity as the polypeptide of the reference nucleic acid, or the ability to be recognized by an antibody directed against the polypeptide encoded by the original nucleic acid. Encodes a conserved polypeptide.
Thus, some mutant nucleic acids encode mutant forms of the polypeptide, the systematic study of which allows one to infer the structure-activity relationship of the protein in question. Knowledge of these variants about the disease being studied is important because it allows an understanding of the molecular causes of the condition.
[0030]
"Fragment" is understood to mean a reference nucleic acid according to the invention, a nucleotide sequence of a shorter length compared to the reference nucleic acid and a nucleotide sequence encompassing the same common part as the reference nucleic acid over the common part. .
Such a "fragment" of the nucleic acid according to the present invention may optionally be included in a larger polynucleotide of which it is a component.
Such fragments comprise oligonucleotides of 20 to 25, 30, 40, 50, 70, 80, 100, 200, 500, 1000 or 1500 contiguous lengths of nucleotides of the nucleic acids according to the invention, or , Consisting of the oligonucleotide.
[0031]
A “biologically active fragment” of a transcription-regulating acid according to the invention is understood to mean a nucleic acid which is able to modulate the transcription of a DNA sequence placed under its control. Such a biologically active fragment contains the core promoter and / or regulatory elements as defined in the description of the present invention.
A "regulatory nucleic acid" according to the present invention is understood to mean a nucleic acid which activates and / or regulates the expression of a DNA sequence selected and placed under its control.
[0032]
"Promoter" is understood to mean a DNA sequence recognized by a cellular protein involved in the initiation of transcription of a gene. A core promoter is the minimum regulatory nucleic acid capable of initiating transcription of a defined DNA sequence placed under its control. In general, the core promoter consists of a region of the genomic DNA upstream of the transcription start site, where the CAAT sequence (where one or more transcription protein factors bind) is frequently present, as well as several Except in rare cases, such as the housekeeping gene of TATA, there is a TATA or "TATA box" sequence or related box. It is at this box level that RNA polymerase, as well as one or more transcription factors, eg, “TATA” box binding protein (TBP), bind.
[0033]
The nucleotide sequence is "under control" of the regulatory nucleic acid, where the regulatory nucleic acid is positioned in association with the nucleotide sequence in such a way that transcription initiation of the nucleotide sequence is controlled by the RNA polymerase.
[0034]
"Regulatory element" or "regulatory sequence" for the purposes of the present invention is understood to mean a nucleic acid comprising an element capable of modulating transcription initiated by a core promoter, such as, for example, There are binding sites for various transcription factors, "enhancer" sequences to increase transcription, or "silencer" sequences to suppress transcription.
An “enhancer” sequence is understood to mean a DNA sequence contained in a regulatory nucleic acid which can increase or stimulate transcription initiated by the core promoter.
[0035]
A “silencer” sequence is understood to mean a DNA sequence contained in a regulatory nucleic acid which can reduce or repress transcription initiated by the core promoter.
Regulatory elements may be outside of the sequence located 5 'to the transcription start site, for example, in introns and exons contained in the coding sequence.
Under their control, preferably in particular cells (eg, tissue-specific cells), ie, in cells of a particular tissue, or at transcription levels that vary from tissue to tissue, from the core promoter and regulatory elements. The core promoter and regulatory elements can be "one or more tissue-specific" if the restricted DNA sequence located in is transcribed.
[0036]
"Transcription factors" are understood to mean proteins which preferably interact with the elements regulating the regulatory nucleic acids according to the invention, and proteins which stimulate and, in contrast, suppress transcription. Some transcription factors are active in monomeric form, and others are active in homodimeric or heterodimeric form.
The term "modulating" means either positively regulating (increase, stimulate) transcription or negatively regulating (decreasing, suppressing, preventing) transcription.
[0037]
For the purposes of the present invention, the "percent identity" between two nucleotide or amino acid sequences can be determined by comparing the two optimally aligned sequences via a comparison window.
Thus, a portion of the nucleotide or polypeptide sequence in the comparison window is compared to the reference sequence (not including these additions or deletions) so that an optimal alignment of the two sequences is obtained. It may include deletions (eg, "gaps").
[0038]
The above ratio determines the number of positions where the same nucleobase or the same amino acid residue is observed in the two compared sequences (nucleic acid or peptide) in order to determine the ratio of sequence identity, Calculated by dividing the number of identical positions between two base or amino acid residues by the total number of positions in the comparison window, and then multiplying the result by 100.
Optimal comparative sequence alignments can be achieved using computers assisted by known algorithms, such as the WISCONSIN GENETICS SOFTWARE PACKAGE, GENETICS COMPUTER GROUP (GCG) (575 Science Doctor (MDISON, W. NDISON, Wisconsin). ) Included in the package.
[0039]
By way of example, with the assistance of BLAST software (versions of BLAST 1.4.9, March 1996, BLAST 2.0.4, February 1998, and BLAST 2.0.6, September 1998), exclusively default parameters The sequence identity ratio can be obtained using only the sequence identity (Altschul et al, J. Mol. Biol. (1990) 215: 403-410; Altschul et al., Nucleic Acids Res. (1997) 25: 3389. -3402). BLAST searches for sequences similar / homologous to the referenced "request" sequence, with the aid of the algorithm of Altschul et al. (Supra).
The request sequence and database used are of the peptide or nucleic acid type and can be in any combination.
“Highly stringent hybridization conditions” for the purposes of the present invention is understood to mean the following conditions.
[0040]
1-Membrane competition and prehybridization:
Mix 40 μl salmon sperm DNA (10 mg / ml) +40 μl human placental DNA (10 mg / ml),
Denature at 96 ° C. for 5 minutes, then immerse the mixture in ice,
Remove 2 × SSC buffer and pour 4 ml of formamide mix into hybridization tube containing membrane,
Adding a mixture of the two denatured DNAs,
Incubate at 42 ° C. with rotation for 5-6 hours.
[0041]
2-label probe competition;
To the labeled purified probe, add 10 to 50 μl of CotI DNA, depending on the amount of non-specific hybridization.
Denature at 95 ° C for 7-10 minutes,
Incubate at 65 ° C for 2-5 hours.
[0042]
3-Hybridization:
Remove the prehybridization mix,
Mix 40 μl salmon sperm DNA + 40 μl human placenta DNA; denature at 96 ° C. for 5 minutes, then immerse in ice
To the hybridization tube, add 4 ml of the formamide mix, a mixture of the two DNAs and the denatured labeled probe / CotI DNA,
Incubate at 42 ° C with rotation for 15-20 hours.
[0043]
4-wash:
Rinse by washing once with 2 × SSC at room temperature,
Wash twice with 2 × SSC and 0.1% SDS at room temperature for 5 minutes,
Wash twice with 0.1 × SSC and 0.1% SDS at 65 ° C. for 15 minutes,
Wrap the membrane in Saran and expose.
[0044]
The above hybridization conditions are compatible with the hybridization of nucleic acid molecules of various lengths ranging from 20 nucleotides to hundreds of nucleotides under conditions of high stringency.
It goes without saying that the above hybridization conditions can be adjusted according to the function of the length of the nucleic acid for which hybridization is sought and the function of the type of labeling selected, according to techniques known to those skilled in the art.
Appropriate hybridization conditions are described, for example, in the manuals of HAMES and HIGINS (1985) (Nucleic Acid Hybridization practical Approach, Hames and Higgins Ed., IRL Press, Oxford). AUSUBEL et al. (1999) manual (Currents Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, NY).
[0045]
"Transformation" for the purposes of the present invention is understood to mean introducing a nucleic acid (or a recombinant vector) into a host cell. The term "transformation" also includes the state in which a cell has been genotyped by an exogenous nucleic acid, such that the transformed cell has the exogenous nucleic acid, for example, a recombinant polypeptide. Or in the form of a sense or antisense nucleic acid.
A “transgenic animal” for the purposes of the present invention is understood to mean a non-human animal, preferably a mammal, in which case one or more of the cells is transfected by gene transfer techniques well known to those skilled in the art. And heterologous nucleic acids introduced by human intervention such as Heterologous nucleic acids are introduced directly or indirectly into cells or cell precursors by, for example, microinjection or genetic engineering, such as infection with a recombinant virus. The heterologous nucleic acid may be integrated into the chromosome or may be provided in the form of a DNA that is replicated extrachromosomally.
[0046]
Nucleic acid regulating ABCA7 gene
Using BAC-type vector libraries prepared from human and mouse genomic sources, the inventors have successfully isolated nucleic acids that regulate the human and mouse ABCA7 genes.
The inventors have determined, by comparative analysis of human and mouse genomic sequences, regulatory nucleic acids comprising two regulatory modules that are conserved, especially in humans and mice. Thereby, we believe that the nucleic acid that regulates the transcription of the ABCA7 gene, in its broadest definition, is between the 5 'and 3' ends.
A non-transcribed region of about 1.2 kb located upstream of the transcription start site of ABCA7 gene;
A partial sequence of the first exon of the ABCA7 gene,
It was determined to be composed of a polynucleotide containing
[0047]
In its broadest definition, a nucleic acid that regulates the transcription of the ABCA7 gene comprises all the nucleotide regions defined above and is considered to be identical to SEQ ID NO: 1 according to the present invention.
Accordingly, a first object of the present invention consists of a nucleic acid comprising a polynucleotide having at least 20 contiguous nucleotides (having the nucleotide sequence of SEQ ID NO: 1) or a nucleic acid having a complementary sequence.
[0048]
In addition, a region of about 1.1 kb, which is located upstream of the transcription start site of the ABCA7 gene and contains a core promoter and a plurality of elements that regulate transcription, is included in the sequence identified as SEQ ID NO: 2 according to the present invention.
More precisely, the nucleotide at position 1 of the sequence SEQ ID NO: 2 is the nucleotide at position -1111 with respect to the transcription start site of the ABCA7 gene.
According to a second aspect, the present invention relates to a nucleic acid comprising a polynucleotide having at least 20 contiguous nucleotides (having the nucleotide sequence of SEQ ID NO: 2) or a nucleic acid having a complementary sequence.
[0049]
As already specified above, the nucleic acid that regulates ABCA7 gene transcription having the sequence of SEQ ID NO: 1 also contains, in addition to the non-transcribed 5 'regulatory region, the 5' portion of the first exon of the human ABCA7 gene.
The partial sequence of the first exon of the ABCA7 gene is defined as SEQ ID NO: 3.
According to a third aspect, the invention relates to a nucleic acid comprising a polynucleotide having at least 20 contiguous nucleotides (having the nucleotide sequence of SEQ ID NO: 3), or a nucleic acid having a complementary sequence.
[0050]
Preferably, the nucleic acids according to the invention are in an isolated and / or purified form.
Also, a component of the present invention is any "biologically active" fragment of a nucleic acid as defined above.
According to yet another aspect, the invention relates to a nucleic acid having at least 80% nucleotide identity with a nucleic acid as defined above.
[0051]
In particular, the nucleic acid may be from a mouse,
A polynucleotide having the nucleotide sequence of SEQ ID NO: 4 including the 5 'to 3' ends:
A non-transcribed region of about 1.2 kb located upstream of the transcription start site of mouse ABCA7 gene;
A partial sequence of the first exon of the ABCA7 gene,
including.
[0052]
Further, a region of about 1.2 kb, which is located upstream of the transcription start site of the ABCA7 gene and contains a core promoter and a plurality of elements that regulate transcription, is also included in the sequence identical to SEQ ID NO: 5 according to the present invention.
The present invention also includes a nucleic acid characterized in that it hybridizes with any of the nucleic acids according to the present invention under high stringency conditions.
The invention also relates to a nucleic acid comprising at least 20 contiguous nucleotides of a polynucleotide selected from the group consisting of nucleotide SEQ ID NO: 1 to SEQ ID NO: 5, comprising at least 80%, advantageously 90%, preferably 95%, Most preferably, it relates to nucleic acids having 98% nucleotide identity.
[0053]
Detailed analysis of SEQ ID NO: 2 and SEQ ID NO: 5
According to the main characteristics, the nucleic acid having the sequence of SEQ ID NO: 2 (included in the nucleic acid that regulates the human ABCA7 gene having the sequence of SEQ ID NO: 1) contains the components of the core promoter and each is 30 bp upstream of the transcription start site. The degenerate "TATA" box (TTAAG) is located. Similarly, the degenerate "TATA" box (TTAAA) is located 30 bp upstream of the transcription start site of the mouse nucleic acid having the sequence of SEQ ID NO: 5 and is included in nucleic acids that regulate the mouse ABCA7 gene having the sequence of SEQ ID NO: 4. It is. The location of the "TATA" box on the promoter of the human and mouse ABCA7 gene, as well as the transcription start site, is shown in FIG.
[0054]
In addition, the regulatory sequences of SEQ ID NO: 2 and SEQ ID NO: 5 contain many binding sites for various transcription factors that can positively or negatively regulate the activity of the core promoter.
Accordingly, the various sequence features of the various transcription factor binding sites in the sequences of SEQ ID NO: 2 and SEQ ID NO: 5 have been identified by the inventors in the following detailed manner.
Using the sequences of SEQ ID NO: 2 and SEQ ID NO: 5 as reference sequences, processing according to the algorithm of the MatInspector software package (Quantt et al., Nucl Acid Res (1995) 23 (23), 4878-4884), for example Transfac Compared to data stored in several databases-the presence and location of various sites characteristic of the sequences of SEQ ID NOs: 2 and 5, especially the presence and location of binding sites for transcription factors, are well known to those skilled in the art. Was determined according to the method described above.
[0055]
More specifically, the software package NNPP (Reese et al. J. Comput. Biol. (1997) 4 (3) 311-23), TSSG and TSSW (Soloriev et al., Ismb (1995), 5, 294-302) are described. And detailed analysis was performed on 1.1 kb and 1.2 kb upstream of the start site of the sequences of SEQ ID NOs: 2 and 5, respectively, so that in the first step of the search, 193 and 233 transcription factors in humans and mice A putative site that binds to was identified. This is summarized in Tables 1 and 2. By collecting, filtering, and comparing human and mouse regulatory sequences as described above, it was determined that they contained two modules common to human and mouse regulatory sequences and could bind to various transcription factors. Five and three sites were selected in modules 1 and 2 with human and mouse sequences, respectively. A position having a filtration score associated with the transcription factor binding site identified at 1111 bp of the sequence of SEQ ID NO: 2 according to the present invention, as well as the transcription factor binding site identified at 1220 bp of the sequence of SEQ ID NO: 5 according to the present invention Is shown in Table 3. Also, various binding sites are shown schematically in FIG.
[0056]
The position of the starting nucleotide at each transcription factor binding site is based on the numbering of the nucleotides of the sequences SEQ ID NO: 2 and NO. FIG.
FIG. 2 shows the sequence of SEQ ID NO: 1, which comprises the sequence of SEQ ID NO: 2. In addition, the first nucleotide at the 5 'position in the sequence of FIG. 2 is also the first nucleotide at the 5' position of any one of the nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2. In FIG. 2, the transcription factor binding sites are shown in bold to delimit their respective positions, and their respective representations are shown in the respective corresponding boxes above. The numbering of the nucleotides in the sequence shown in FIG. 2 indicates that the transcription start site is numbered "+1" and the nucleotide 5 'of nucleotide +1 is relative to having itself numbered "-1". It was done.
[0057]
FIG. 3 shows the sequence of SEQ ID NO: 4, which comprises SEQ ID NO: 5. The first nucleotide at the 5 'position in the sequence of Fig. 3 is also the first nucleotide at the 5' position of any one of the nucleic acid sequences of SEQ ID NO: 4 and SEQ ID NO: 5. In FIG. 3, the transcription factor binding sites are shown in bold to delimit their respective locations, and their respective representations are shown in the respective corresponding boxes above. The numbering of the nucleotides in the sequence shown in FIG. 3 indicates that the transcription start site is numbered "+1" and the nucleotide 5 'of nucleotide +1 is relative to its own being numbered "-1". It was done.
[0058]
Genomic analysis of the nucleic acids that regulate the human and mouse sequences of SEQ ID NOs: 2 and 5 revealed two regulatory modules, described as Module 1 and Module 2, which are particularly conserved in human and mouse. These two regulatory modules contain ubiquitous transcription factor binding sites (eg, NF1, NFY and AP4), as well as liver-specific transcription factor binding sites (eg, CEBP and HNF3B). This is consistent with the experimental expression data shown in Example 3 below, and further with the expression data provided by Kaminski et al. (Described above), where the expression of the ABCA7 gene in human fetal liver tissue Is seen.
[0059]
Also, the two regulatory modules conserved in mouse and human may contain transcription factor binding sites (eg, GF11 and NFkappaB (NFkB)), which are essentially present in lymphoid organs.
The description of the characteristics of the binding site of each transcription factor shown in FIGS. 2 and 3 and Table 3 can be easily found by those skilled in the art. A brief description of some of them is provided below.
[0060]
NF1 factor;
The binding properties of the NF1 factor can be found in particular in the following Medline database entries: 88319994, 91219459, 86140112, 87237877, 90174951, 8928287, 90151633, 89261836, 86274639, 870644414, 89263791. The NF1 element recognizes the following repeat sequence "TGGGCANNNTGCCA (NNTTGGCNNNNNNNNNNCCNN)", which is present in viral and cellular promoters at the level of the adenovirus type 2 replication origin. These proteins can activate transcription and replication. The NF1 factor binds to DNA in the form of a homodimer.
[0061]
NFY factor:
The NFY factor is described in particular in the Swissprot database at entry number P25.208. The NFY element is an element that recognizes a “CCAAT” unit in a promoter sequence (for example, a promoter sequence of a gene encoding type 1 collagen, albumin and β-actin). NFY factor is a stimulator of transcription.
[0062]
AP4 factor:
The person skilled in the art can advantageously refer to the articles corresponding to the following entries in the Medline database: 2123466, 2833704, 8530024. The AP4 element contains a domain that binds to "helix-loop-helix" (bHLH) type DNA, and two dimerization domains. The consensus site of the AP4 factor is “CWCAGCTGGN” below, and the latter half usually overlaps the binding site of the AP1 factor.
[0063]
CEBP
Characteristics of binding to CEBP factors can be found, inter alia, in the following entries in the Medline database: 9315489, 91248826, 94193722, 93211931, 92390404, 90258633, 94088523, 90269225 and 96133958. CEBP factors are important transcriptional activators in the regulation of genes involved in immune and inflammatory responses. CEBP factor specifically binds to an IL-1 response element in the gene for IL-6. CEBP factors are probably involved in the regulation of the acute phase of inflammation and hematopoiesis. The consensus recognition site is: “T (T / G) NNGNAA (T / G)”.
[0064]
HNF3B factor:
Those skilled in the art are aware of the papers by OVERDIER et al. (1994, Mol. Cell Biol. Vol. 14: 2755-2766), as well as the following entries in the Medline database: And 94218249 can be advantageously referred to. This transcription factor acts as an activator of a number of genes in the liver (eg, the AFT gene) and genes for albumin and tyrosine aminotransferase, and interacts with the cis-acting regulatory regions of these genes.
[0065]
GFI1
Features relating to the binding of GFI1 factors can be found, inter alia, in the following entries in the Medline database: 10762661, 9931446, 9571157, 9285685, 9070650 and 7789186. The GFI1 gene encodes a zinc finger protein that is involved in the regulation of transcription, more particularly in the interleukin-2 signaling pathway. The consensus recognition site is “NNNNNNNAAATCANNGNNNNNNNN” below.
[0066]
NF-kappa-B factor;
One of ordinary skill in the art will recognize the following entries in the Medline database: Can be advantageously referred to. NF-kappa-B factor is a heterodimer, consisting of a first 50 kDa subunit and a second 65 kDa subunit.
[0067]
Two heterodimers can form an unstable tetramer. Its binding to DNA is zinc (Zn).⁺⁺) Depends on the presence. NF-kappa-B factor is induced by a number of factors such as, for example, TNF, PKA or PKC. NF-kappa-B factor is an important regulator of genes involved in infection, inflammation and stress response.
[0068]
The main characteristic of the regulatory nucleic acid according to the present invention, more particularly the main characteristic of the sequence located upstream of the transcription initiation site contained in both the sequence of SEQ ID NO: 2 and the sequence of SEQ ID NO: 5, is T lymphocyte Is the presence of a motif characteristic for putative binding sites to transcription factors involved in the gene expression of (eg, transcription factors CEBP, NFKB, and GFI1).
[0069]
GFI1 is a proto-oncogene that encodes a zinc finger nucleoprotein involved in the cytokine signaling pathway and clonal expansion of T cells (Zweidler-McKay, et al., Mol. Cell. Biol. (1966), 16). (8), 4024-4034). The transcription factor GFI1 acts as a transcription repressor for genes that suppress the activation of T cells and tumorigenesis. The transcription factor GFI1 is present especially in the thymus, spleen and T lymphocytes.
[0070]
The transcription factors CEBP and NF-kappaB expressed in the thymus, spleen and T lymphocytes are well known to those skilled in the art, and include T lymphocytes (Runch et al., 1994) and HepB3 cells (Shimizu et al., Gene, (1994)). 149, 305-310).
[0071]
The starting nucleotide position is at the transcription start site at -498 and -469 for the CEBP site, -260 for the NFkB site, and -787 and -760 for the CEBP site, and -301 for the NFKB site on the human regulatory module. In contrast, it can be seen that the two regulatory sites are more distant in the mouse promoter. However, the two sites appear to be closer in three-dimensional structure so that they can be co-activated by the two factors CEBP and NFkB.
[0072]
The presence of these potential binding sites for CEBP and NFkb in a manner that is conserved in humans and mice with respect to the regulatory nucleic acids according to the present invention is consistent with the observations, which indicate that the human ABCA7 protein encodes Gene expression is predominant in hematopoietic tissue and T lymphocytes and is most likely to be involved in cellular mediation of immunity, particularly the pathogenesis of atherosclerosis (Kaminski, supra).
[0073]
As already mentioned above, the present invention relates to a nucleic acid comprising a polynucleotide having at least 20 contiguous nucleotides of any one of the nucleotide sequences of SEQ ID NO: 1 or 2 and SEQ ID NO: 4 or 5, as well as the complementary sequence The nucleic acid to have.
[0074]
Nucleic acids comprising one or more "biologically active" fragments of any one of the sequences of SEQ ID NO: 1 or 2 and SEQ ID NO: 4 or 5 are included in the above definition. Those skilled in the art can readily obtain biologically active fragments of these sequences, particularly by referring to Table 3 above, and FIGS. 2 and 3, which contain the ABCA7 gene. There are various characteristic units of the array. Thus, one skilled in the art would be able to synthesize such biologically active polynucleotides by chemically or completely synthesizing the corresponding polynucleotides, or by acting with restriction endonucleases to obtain the desired DNA fragment. Fragments can be obtained, and restriction sites present in the sequences of SEQ ID NO: 1 to SEQ ID NO: 5 are easily found from sequence information with the aid of a general-purpose restriction mapping software package (eg, GCG version 9.1 map module). be able to.
[0075]
The production of nucleic acid fragments defined with the aid of restriction endonucleases is described, for example, in manuals such as SAMROOK (Molecular Cloning; a laboratory manual, 2ed. Cold Spring Harbor Laboratory, Cold spring Harbor, N.K., Canada). ).
[0076]
Therefore, the invention also relates to a nucleic acid as defined above, which is able to modulate the transcription of a polynucleotide placed under its control.
[0077]
According to a first preferred embodiment, the biologically active fragment of the transcription-regulating nucleic acid according to the invention comprises a first conserved module (module 2), The first nucleotide transcribed comprises the nucleotide at position 1112 of the nucleotide sequence of SEQ ID NO: 1 or the nucleotide of SEQ ID NO: 4 It is the nucleotide at position 1221 of the nucleotide sequence.
[0078]
According to a second embodiment, a biologically active fragment of a transcription-regulating nucleic acid according to the invention comprises a conserved module 1 of nucleotides -1 to -860 relative to the transcription start site and 2 (FIG. 1), the first nucleotide transcribed is the nucleotide at position 1112 of the nucleotide sequence of SEQ ID NO: 1, or the nucleotide at position 1221 of the nucleotide sequence set forth in SEQ ID NO: 4.
[0079]
According to a third embodiment, such a biologically active fragment of a transcription-regulating nucleic acid according to the present invention may also contain other regulatory elements (eg, various regulatory elements) in addition to the core promoter and nearby regulatory elements. The first nucleotide to be transcribed extends from the nucleotide at position -1 to the nucleotide at position -1111 with respect to the transcription start site, and the first nucleotide to be transcribed is the nucleotide sequence of SEQ ID NO: 1. And extending from the nucleotide at position -1 to the nucleotide at position -1220 relative to the transcription start site, the first nucleotide transcribed is the nucleotide at position 1221 in the nucleotide sequence of SEQ ID NO: 4. Nucleotides.
[0080]
Exon 1 analysis
Applicants have also identified nucleotide sequences located downstream of the transcription initiation site and corresponding to the 5 'end of exon 1 as human and mouse genes encoding the ABCA7 protein.
More precisely, the 5 'end of exon 1 is 1210 nucleotides in size, starting at nucleotide 1112 of the sequence of SEQ ID NO: 1 and ending at nucleotide 2322 of the sequence of SEQ ID NO: 1. The 5 'end of exon 1 is identified as the sequence of SEQ ID NO: 3, and the complete sequence of exon 1 is identified as the sequence of SEQ ID NO: 6.
[0081]
Exon 1 includes the start of the open reading phase of the human ABCA7 gene, nucleotide A of the ATG codon is located at position 1208 of the sequence of SEQ ID NOs: 3 and 6. Exon 1 encodes a polypeptide having the sequence of SEQ ID NO: 7.
Exon 1 may include elements that regulate the expression of the ABCA7 gene, especially enhancer-type elements and / or silencer or suppressor-type elements that amplify.
As a result, the transcription-regulating nucleic acids according to the present invention may also comprise, in addition to the biologically active fragments of the sequence of SEQ ID NO: 1, nucleotide fragments or of the sequences of SEQ ID NOs: 2-3 and 6 It may include the whole.
[0082]
Similar to the nucleotide sequences of SEQ ID NO: 1 to SEQ ID NO: 3 and 6, fragments thereof specifically regulate nucleotide probes or primers or the ABCA7 gene to detect the presence of at least one copy of the ABCA7 gene in a sample. Can be used as a nucleotide probe or primer to amplify a target sequence defined in the target sequence.
Therefore, the object of the present invention also has at least 80% nucleotide identity with the nucleic acid as defined above, especially obtained from any one of the sequences SEQ ID No. 1 to SEQ ID No. 3 and 6. It is a nucleic acid.
[0083]
The present invention also relates to a nucleic acid according to the present invention, particularly a nucleic acid obtained from a sequence selected from the sequences of SEQ ID NOS: 1 to 3 and 6, and hybridized under high stringency conditions. To nucleic acids.
The invention also relates to a nucleic acid as defined above, which is additionally characterized in that it can modulate the transcription of a given polynucleotide placed under its control.
[0084]
According to a first aspect, such a nucleic acid is capable of activating the transcription of a given polynucleotide placed under its control.
According to a second aspect, the regulatory nucleic acid according to the present invention may be characterized in that it can inhibit transcription of a given polynucleotide placed under its control.
Preferably, the nucleic acid that regulates transcription according to the present invention, when properly positioned with respect to the predetermined polynucleotide for which expression is sought, causes the predetermined polynucleotide to be transcribed, either structurally or inducibly. obtain.
[0085]
The inducible characteristic of transcription initiated by a regulatory nucleic acid according to the invention is conferred by one or more regulatory elements, for example one or more as defined above in the sequence SEQ ID No. 1 or SEQ ID No. 2. Is present.
Moreover, tissue-specific expression of a given polynucleotide can be searched for by placing the given polynucleotide under the control of a regulatory nucleic acid according to the present invention, thereby, for example, specifically identifying cells. , For example, cells of hematopoietic tissue (eg, peripheral leukocytes, thymocytes, spleen cells, and bone marrow) can initiate transcription of the given polynucleotide.
[0086]
Preferably, a regulatory nucleic acid according to the present invention may include one or more "discrete" regulatory elements, such as enhancer and silencer elements. In particular, such regulatory nucleic acids may include one or more sites that may bind to the transcription factors defined in FIG.
Further, the regulatory nucleic acid according to the present invention includes a sequence not containing a core promoter, that is, a sequence ranging from nucleotide -1 to nucleotide -25 with respect to the transcription start site.
[0087]
In turn, such regulatory nucleic acids may preferably comprise a polynucleotide comprising a so-called “heterologous” core promoter, ie, a “TATA” box and a “homeo box” that are not derived from a nucleic acid that regulates the ABCA7 gene.
Also, a component of the present invention is a nucleic acid that regulates transcription, including, for example, all or a part of the sequence of SEQ ID NO: 1 that has been modified by addition, deletion, or substitution of one or more nucleotides. Such modifications can modulate transcriptional activity by increasing or conversely decreasing the activity of a promoter or regulatory element.
[0088]
Also, such modifications can affect the tissue specificity of the promoter or regulatory element. That is, for example, the regulatory nucleic acid according to the present invention can be modified so that transcription is stimulated in only one tissue and naturally expressed.
In addition, the nucleic acid that regulates transcription according to the present invention can be modified and further induced by a specific compound, for example by forming an inducible site in the sequence with a specific therapeutic compound. .
[0089]
The alteration of the sequence may include all or part of the sequence of SEQ ID NO: 1, and the inclusion of a promoter or regulatory element may be performed by methods well known to those skilled in the art (eg, mutagenesis). The activity of the modified promoter or regulatory element can then be determined, for example, by cloning the modified promoter upstream of the reporter gene, transfecting the resulting DNA construct into a host cell, and determining the activity of the transfected host cell. The test can be performed by measuring the expression level of the reporter gene. Also, the activity of the modified promoter can be analyzed in vivo in transgenic animals. Libraries of modified fragments that can be screened using functional tests can also be constructed, in which, for example, only promoters or regulatory elements having the desired activity are selected.
[0090]
Such a test may be based, for example, on the use of a reporter gene which confers resistance to a defined compound, for example an antibiotic. Subsequently, the selection of cells having a regulatory nucleic acid / reporter gene structure and containing a promoter or regulatory element with the desired alterations is accomplished by transforming host cells transformed with the above structures into a defined compound, such as a defined antibiotic. By culturing in the presence of
The reporter gene can also encode any easily detectable protein, such as a visually detectable protein (eg, luciferase).
[0091]
As a result, the object of the invention is also
a) a nucleic acid that regulates transcription as defined above;
b) a predetermined polynucleotide encoding a predetermined polypeptide or nucleic acid;
A nucleic acid comprising:
[0092]
According to a first aspect, the given polynucleotide for which transcription is desired encodes a protein or peptide. The protein may be of any type and includes, for example, therapeutic proteins such as cytokines, structural proteins, receptors or transcription factors. For example, if specific transcription in a particular tissue is desired (eg, in hematopoietic tissues, ie, cells of the spleen, bone marrow, or peripheral leukocytes), the nucleic acid that regulates transcription may be a transcription of the sequence of SEQ ID NO: 1 or 2. A nucleic acid ranging from nucleotide -1 to nucleotide -1111 correlated to the start site, and nucleotide -1 to nucleotide -1220 correlated to the transcription start site of the sequence of SEQ ID NO: 4 or 5 Nucleic acids in the range of nucleotides are advantageously included.
In this case, the given polynucleotide may encode a gene involved in eliminating inflammation, such as a receptor for a cytokine or a receptor for superoxide dismutase. Then, if an antitumor effect is desired, one can search for stimulating the number and activation of cytotoxic T lymphocytes specific for a particular tumor antigen.
[0093]
In another embodiment, the regulatory nucleic acid according to the present invention can be used in combination with a predetermined polynucleotide encoding ABCA7 protein.
Further, the predetermined polynucleotide may be a sense-type oligonucleotide.
As already mentioned above, a given polynucleotide can also produce a nucleic acid, for example an antisense nucleic acid specific for a gene, in which case it will look for inhibition of its translation.
[0094]
According to another aspect, a given polynucleotide whose transcription is regulated by a regulatory nucleic acid is a reporter gene, for example, any of the genes encoding a detectable protein.
Among preferred reporter genes, a gene for luciferase, a gene for β-galactosidase (LacZ), a gene for chloramphenicol acetyltransferase (CAT), or confer resistance to a specific compound, particularly an antibiotic. Examples include any of the genes encoding the protein.
[0095]
Recombinant vector
The term "vector" for the purposes of the present invention is understood to mean a circular or linear DNA or RNA molecule, in either single-stranded or double-stranded form.
According to a first embodiment, the recombinant vector according to the invention is used for amplifying a regulatory nucleic acid according to the invention inserted therein after transformation or transfection into a desired host cell. Can be.
According to a second embodiment, said recombinant vector corresponds to an expression vector comprising a regulatory nucleic acid according to the invention and a sequence whose expression is to be searched for in a host cell or a defined multicellular organism.
[0096]
According to an advantageous embodiment, the recombinant vector according to the invention comprises, inter alia, the following elements:
(1) a regulatory nucleic acid according to the present invention;
(2) a predetermined polynucleotide containing a coding sequence contained in a nucleic acid to be inserted into such a vector (the coding sequence is placed in a phase with a regulatory signal according to (1)); and
(3) a sequence suitable for initiation and termination of transcription;
May be included.
In addition, a recombinant vector according to the invention may comprise one or more origins of replication, markers or selectable markers in the host cell whose amplification or expression is to be sought.
[0097]
By way of example, bacterial promoters include the Lacl or LacZ promoter, the T3 or T7 bacteriophage RNA polymerase promoter, the phage lambda PR or PL promoter.
Examples of promoters for eukaryotic cells include the HSV virus thymidine kinase promoter and the mouse metallothionein-L promoter.
In general, for the selection of an appropriate promoter, those skilled in the art will advantageously refer to the textbook of Sambrook et al. (1989) cited above, or the technique described by Fuller et al. (1996; Immunology in Current Protocol in Molecular Biology). can do.
[0098]
When the expression of the ABCA7 gene genomic sequence is to be determined, a vector that can contain a large insertion sequence can be preferably used. In this particular embodiment, a bacteriophage vector, for example a P1 bacteriophage vector such as the vector pl58 or the vector pl58 / neo8, may preferably be used and is described by Sternberg (Trends Genet., (1992) 8: 1). -16; Maimn.Genome (1994) 5: 397-404).
[0099]
Preferred bacterial vectors according to the invention are, for example, the vector pBR322 (ATCC37017) or, for example, pAA223-3 (Pharmacia, Uppsala, Sweden) and pGEM1 (Promega Biotech, Madison, WI, UNITED STATES). Vector.
Also, other commercially available vectors, for example, vectors pQE70, pQE60, pQE9 (Qiagen), psiX174, pBluescriptSA, pNH8A, pNH16A, pNH18A, pNH46A, pWLNEO, pSV2CAT, pOG44, pXTI, pSGi, pSGi and pSGa (StRa)
[0100]
In addition, the recombinant vector PXP1 described in Nordeen SK et al. (Bio Technologies, (1988), 6: 454-457) may also be used.
In addition, a baculovirus-type vector is also exemplified, for example, the vector pVL1392 / 1393 (Pharmingen), which is used for Sf9-based transfected cells derived from Spodoptera frugiperda (ATCC No. CRL1711).
Also, adenovirus vectors such as human adenoviruses of type 2 or 5 can be mentioned.
The recombinant vector according to the present invention also includes a retrovirus vector or an adeno-associated vector (AAV). Such adeno-associated vectors are described, for example, in Flotte et al. , Am. J. Resplr. , Cell Mol. Biol. (1992) 7: 349-356; Samulski et al. , J. et al. Virol. (1989) 63: 3822-3828; or McLaughlin et al. , Am. J. Hum. Genet. (1996) 59: 561-569).
[0101]
In order to express a given polynucleotide under the control of the regulatory nucleic acid according to the present invention, a polynucleotide construct comprising a regulatory sequence and a coding sequence must be introduced into a host cell. The introduction of such a polynucleotide construct of the present invention into a host cell can be carried out in either a basic culture or in a cell-based form, according to techniques well known to those skilled in the art of transforming or transfecting cells. It can be performed in vitro. In addition, the polynucleotide of the present invention can be introduced in vivo or ex vivo for prevention or treatment of a disease associated with defective ABCA7 protein transport.
[0102]
To introduce a polynucleotide or vector into a host cell, one skilled in the art can advantageously refer to various techniques, for example, precipitation techniques using calcium phosphate (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), electro. Polation (Tur-Kaspa et al., Mol. Cel. Biol, (1986) 6: 716-718 .; Potter et al., Proc. Natl. Acad. Sci. USA (1984), 81 (22), 7161. -5) Recto microinjection (Harland et al., J. Cell Biol (1985) 101: 1094-1095), DNA-filled liposomes (Nicolau et al., Methods Enzymol (1987) 149: 157-76; Fraley et al., Natl. Acad. Sci. USA (1979) 76: 3348-3352).
[0103]
Once the polynucleotide has been introduced into the host cell, it can be stably integrated into the genome of the cell. Integration may be achieved at the exact site in the genome by homologous recombination, or may be integrated randomly. In some embodiments, the polynucleotide can be stably maintained in the host cell in the form of an episomal fragment, wherein the episome is isolated, either independently or in a manner that is synchronized with the cell cycle. Contains sequences that allow the retention and replication of the part.
[0104]
According to a particular embodiment, the method of introducing a polynucleotide according to the invention into a host cell, in particular a host cell obtained from a mammal, in vivo comprises, during the process, a pharmaceutically compatible vector and a suitable A preparation comprising a "naked" polynucleotide of the invention, placed under the control of various regulatory sequences, is introduced by local injection at the level of a selected tissue (e.g., smooth muscle tissue), and the The polynucleotide is absorbed by cells of the tissue.
Compositions comprising "naked" polynucleotides for use in vitro and in vivo are described, for example, in PCT application WO 95/11307, and are also described in Tacson et al. (Nature Med. (1996) 2 (8), 888-892). ) And Huygen et al. (Nat. Med. (1996) 2 (8), 893-898).
[0105]
According to certain embodiments of the present invention, compositions are provided for in vivo production of a given protein. The composition comprises, in solution, in a physiologically acceptable vector, a polynucleotide encoding a given polypeptide placed under the control of a regulatory sequence of the present invention.
The amount of vector injected into the selected host organism will vary depending on the site of injection. According to the guide, about 0.1 to about 100 μg of the regulatory sequence / coding sequence polynucleotide construct is injected into the animal body.
When the regulatory nucleic acid according to the present invention is located in a polynucleotide construct (or vector) in such a way that transcription of a sequence comprising an open reading frame encoding an ABCA7 protein can be controlled, preferably the vector comprises an ABCA7 protein Is injected into the body of a patient who may develop a disease associated with a defect in the patient.
[0106]
As a result, the present invention also relates to a pharmaceutical composition intended for the prevention or treatment of a subject suffering from a dysfunction of the ABCA7 protein, said pharmaceutical composition comprising one or more physiologically compatible excipients A regulatory nucleic acid according to the present invention and a predetermined polynucleotide encoding an ABCA7 protein are contained in combination with an agent.
Advantageously, such a composition comprises a regulatory nucleic acid as defined in any 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. obtain.
In addition, an object of the present invention is a pharmaceutical composition intended for the prevention or treatment of a subject suffering from a dysfunction of lipid metabolism, said pharmaceutical composition comprising one or more physiologically compatible enhancers. It comprises a recombinant vector as defined above in combination with a vehicle.
[0107]
Also an object of the present invention is a pharmaceutical composition for the prevention or treatment of a subject suffering from a dysfunction of processes including the immune system and inflammation, said pharmaceutical composition comprising one or more physiologically It comprises a recombinant vector as defined above, in combination with a compatible excipient.
The present invention also relates to the regulation of the ABCA7 gene for the manufacture of a medicament for the prevention or treatment of a subject suffering from dysfunction of lipid metabolism or a subject suffering from immunological or inflammatory causes. And use of a polynucleotide construct according to the invention comprising a sequence encoding the ABCA7 protein.
The present invention also relates to the use of the recombinant vector according to the invention, which is intended for the prevention or treatment of a subject suffering from a dysfunction of a regulatory nucleic acid according to the invention and of processes including the immune system and inflammation. And a nucleic acid encoding an ABCA7 protein for producing a pharmaceutical.
[0108]
Vectors useful in body gene therapy methods, and compositions containing such vectors
The present invention also provides novel therapies for the treatment and / or prevention of conditions related to lipid metabolism, as well as for the treatment and / or prevention of dysfunction in lymphocyte-mediated mechanisms of inflammation. About the method. The present invention relates to the prior art by demonstrating the potential for treating conditions associated with dysfunction of lipid metabolism in myeloid lymphoid tissue, particularly by gene therapy, in vivo delivery and expression of polynucleotide constructs. It provides an advantageous solution to the disadvantages, wherein the polynucleotide construct encodes a regulatory nucleic acid according to the invention and an ABCA7 protein likely to be involved in lipid transport and / or lipid metabolism Contains the sequence Thus, the present invention provides a simple means to enable specific and effective treatment of subjects suffering from dysfunction of processes including the immune system and inflammation.
Gene therapy is the correction of defects or abnormalities (mutations, abnormal expression, etc.) by introducing genetic information into diseased cells or organs, or generating the expression of a protein of therapeutic interest. This genetic information is either introduced ex vivo into cells extracted from the organ and then the modified cells are reintroduced into the body, or are introduced directly into the appropriate tissue in vivo.
[0109]
In this second case, various techniques exist, among which are complexes of DNA and DEAE-dextran (Pagano et al., J. Virol. 1 (1967) 891), complexes of DNA and nuclear proteins ( Various transfection techniques using Kaneda et al., Science 243 (1989) 375), a complex of DNA and lipids (Felgner et al., PNAS 84 (1987) 7413), use of liposomes (Fraley et al., J. Biol. Chem, 255 (1980) 10431). More recently, the use of viruses as vectors for gene transfer has emerged as a promising alternative to these physical transfection techniques. In that regard, various viruses were tested for their ability to infect specific cell populations. In particular, retroviruses (RSV, HMS, MMS, etc.), HSV viruses, adeno-associated viruses and adenoviruses.
[0110]
Therefore, the present invention also relates to a novel therapeutic method for the treatment of conditions associated with lipid transport, wherein the gene encoding ABCA7 placed under the control of the regulatory nucleic acids according to the present invention is transported and expressed in vivo. Including. It is particularly advantageous to construct recombinant viruses comprising a DNA sequence comprising the regulatory nucleic acid according to the invention and a sequence encoding the ABCA7 protein involved in lipid metabolism, and to administer these recombinant viruses in vivo. Administration allows stable and effective expression of the biologically active ABCA7 protein in vivo without cytopathological effects.
[0111]
Adenoviruses constitute, in particular, an efficient vector for the transport and expression of the ABCA7 gene. In particular, by using a recombinant adenovirus as a vector, it is possible to obtain a sufficiently high level of a given gene expression to obtain the desired therapeutic effect. Other viral vectors, such as retroviruses or adeno-associated virus (AAV), that allow for stable expression of the gene are also claimed.
Accordingly, the present invention provides novel methods of treating and preventing pathologies associated with dysfunction of lipid metabolism and dysfunction of signaling pathways by lymphocytes for inflammation.
Therefore, an object of the present invention is also a defective recombinant virus, which comprises a regulatory nucleic acid according to the invention and an ABCA7 involved in lipid metabolism or involved in processes including the immune system and inflammation. A nucleic acid sequence encoding a protein.
[0112]
The invention also relates to the use of such a defective recombinant virus for preparing a pharmaceutical composition for the treatment and / or prevention of a dysfunction of inflammatory signal transduction by lymphocytes.
The invention also relates to the use of cells that have been genetically modified with the above-mentioned viruses ex vivo, or cells that produce such viruses, by transplanting them into the body to obtain in vivo biologically Allows for sustained and efficient expression of highly active ABCA7 protein.
[0113]
The present invention allows the DNA sequences encoding ABCA7 to be incorporated into viral vectors under the control of regulatory nucleic acids as defined above, and these vectors are effective in effecting the biologically active mature form. It can be expressed in a specific manner. More specifically, the present invention has shown that the expression of ABCA7 in vivo can be achieved by direct administration of adenovirus or by production cells or by adenovirus or retrovirus integrated into such DNA. It can be obtained by transplanting chemically modified cells.
[0114]
The present invention is particularly advantageous because it allows controlled ABCA7 expression without adverse effects to be induced in organs not normally involved in the expression of this protein. In particular, significant release of the ABCA7 protein is obtained by implanting cells producing the vector of the invention or cells infected ex vivo with the vector of the invention.
[0115]
The mediator activity of lipid metabolism produced in connection with the present invention may be of human or animal ABCA7 type. Nucleic acid sequences used in the context of the present invention can be cDNA, genomic DNA (gDNA), RNA (in the case of retroviruses), or hybrid structures containing, for example, cDNA with one or more introns inserted. The invention may also include synthetic or semi-synthetic sequences. In a particularly advantageous form, cDNA or gDNA is used. In particular, the use of gDNA allows for better expression in human cells. By incorporating them into a viral vector according to the invention, these sequences are advantageously modified, in particular by inserting appropriate restriction sites, for example by site-directed mutagenesis. In practice, the sequences described in the prior art are not configured for use in accordance with the present invention, and may need to be verified for substantial expression before adaptation. In the context of the present invention, the use of a nucleic acid sequence encoding the human ABCA7 protein is preferred. Moreover, it is also possible to use constructs encoding these ABCA7 protein derivatives, which are, for example, all sequences obtained by mutation, deletion and / or addition to the wild-type sequence, and And a product encoding a product that maintains the activity of a mediator of lipid metabolism. These modifications can be made by techniques known to those skilled in the art (see general molecular biology techniques below). The biological activity of the derivative thus obtained can then be easily measured, as shown in the examples which specifically describe the measurement of lipid efflux from cells. In addition, derivatives for the purpose of the present invention can be obtained by hybridization from a nucleic acid library using the wild-type sequence or a fragment thereof as a probe.
[0116]
These derivatives are particularly useful for molecules that have a higher affinity for their binding site, molecules that exhibit greater protease resistance, molecules that have a higher therapeutic efficiency, or fewer side effects, or where necessary, novel biological It is a molecule with specific properties. The derivatives also include modified DNA sequences that can improve expression in vivo.
[0117]
In a first embodiment, the present invention relates to a defective recombinant virus comprising a regulatory nucleic acid according to the present invention and a cDNA sequence encoding an ABCA7 protein involved in cholesterol transport and metabolism. In another preferred embodiment of the invention, the DNA sequence is a gDNA sequence. The cDNA sequence encoding the ABCA7 protein which can be used in the vector according to the invention is advantageously the sequence of SEQ ID NO: 8.
[0118]
The vectors of the present invention can be prepared from various types of viruses. Preferably, vectors derived from adenovirus, adeno-associated virus (AAV) / herpes virus (HSV) or retrovirus are used. It is most particularly advantageous to use adenoviruses for direct administration or for ex vivo modification of cells to be transplanted, or retroviruses for transplantation of producer cells.
[0119]
The virus according to the present invention is defective, ie it cannot replicate autonomously in target cells. Usually, the genome of the defective virus used in the present invention therefore lacks at least the sequences necessary to replicate the virus in infected cells. These regions are either removed (completely or partially) or rendered non-functional or replaced with other sequences, in particular with nucleic acid sequences encoding the ABCA7 protein. obtain. Preferably, the defective virus still retains the sequences necessary for encapsidation of the viral particles of its genome.
[0120]
More specifically, with respect to adenoviruses, they are characterized by different serotypes, and their structures and properties are somewhat diverse. Among these serotypes, human adenoviruses of type 2 or 5 (Ad2 or Ad5) or adenoviruses of animal origin (see application WO 94/26914) are preferably used in the present invention. Among adenoviruses of animal origin in the context of the present invention, dog, bovine, mouse (eg, Mav1: Beard et al., Virology 75 (1990) 81), sheep, pig, bird or monkey (eg SAV) origin Adenovirus. An adenovirus of animal origin is preferably a dog adenovirus, more preferably a CAV2 adenovirus [eg, Manhattan or A26 / 61 strain (ATCC @ VR-800)]. Preferably, human or dog, or adenoviruses of complex origin, are used in connection with the present invention. Preferably, a defective adenovirus of the invention comprises an ITR, a sequence to be encapsidated, and a sequence encoding an ABCA7 protein placed under the control of a nucleic acid according to the invention. In the genome of the adenovirus according to the invention, advantageously at least the E1 region is non-functional. In the genome of the adenovirus of the present invention, even more preferably, the E1 gene and at least one of the E2, E4 and L1-L5 genes are non-functional. The viral gene considered can be rendered non-functional by any technique known to the person skilled in the art, in particular, by repressing, replacing, partially deleting or adding one or more bases in the gene considered. , Can be non-functional. Such modifications can be achieved in vitro (to isolated DNA) or in situ, for example, by genetic engineering techniques, or by treatment with a mutagen. Other regions can also be modified, particularly the E3 (W095 / 02697), E2 (W094 / 28938), E4 (W094 / 28152, W094 / 12649, W095 / 02697) and L5 (W095 / 02697) regions. It is. According to a preferred embodiment, the adenovirus according to the invention comprises a deletion in the E1 and E4 regions, and the sequence encoding ABCA7 is inserted at the level of the inactivated E1 region. According to another preferred embodiment, the adenovirus according to the invention comprises a deletion in the E1 region at the level where the E4 region and the sequence coding for ABCA7 (FR 9413355) are inserted.
[0121]
The defective recombinant adenovirus according to the present invention can be prepared by any technique known to those skilled in the art (Levrero et al., Gene (1991) 101: 195, EP185573; Graham, EMBO J. (1984) 3: 2917). In particular, the adenovirus can be prepared by homologous recombination between the adenovirus and, inter alia, a plasmid containing a DNA sequence encoding the ABCA7 protein. Homologous recombination is achieved by co-transfecting the adenovirus and plasmid into a suitable cell line. The cell line used preferably has (i) a sequence which can complement a part of the defective adenovirus genome, in order to avoid the risk of recombination, and (ii) avoid the risk of recombination, It should preferably be included in an integrated form. An example of a system is the human fetal kidney system 293 (Graham et al., J. Gen. Virol. (1977) 36:59), which is particularly useful for integrating the Ad5 adenovirus into its genome. Or a system that can complement the functions of E1 and E4 as described in application numbers WO94 / 26914 and WO95 / 02697, among others.
[0122]
The grown adenovirus is then recovered and purified according to conventional molecular biology techniques, as described in the examples.
With respect to adeno-associated virus (AAV), they are relatively small-sized DNA viruses that integrate into the genome of the cells they infect in a stable, site-specific manner. Adeno-associated virus can infect a wide range of cells without inducing any effects on cellular growth, morphology or differentiation. Moreover, adeno-associated virus does not appear to be involved in human pathologies. The genome of AAV has been cloned, sequenced and characterized. AAV consists of about 4700 bases and at each end contains an inverted repeat region (ITR) of about 145 bases that acts as a viral origin of replication. The rest of the genome is divided into two major regions with encapsidation functions, the left part of the genome containing the rep genes involved in viral replication and viral gene expression, and the right part of the genome. Contains the cap gene encoding the viral capsid protein.
[0123]
The use of AAV-derived vectors for gene transfer in vitro and in vivo has been described in the literature (see, inter alia, WO 91/18088; WO 93/09239; US 4,797,368; US 5,139,941, EP 488528). reference). These documents describe various constructs from AAV, in which the rep and / or cap genes have been removed and replaced with a given gene, and their use has been made to replace said given gene. For transport in vitro (to cells in culture) or in vivo (directly to organisms). However, none of these documents describe or suggest the use of recombinant AAV for transport and expression of ABCA7 protein in vivo or ex vivo, or the benefits of such transport. A defective recombinant AAV according to the present invention comprises a plasmid comprising a sequence encoding the ABCA7 protein flanked by two AAV inverted repeat regions (ITRs), into a cell line infected with a human helper virus (eg, an adenovirus); It can be prepared by co-transfection with a plasmid containing the AAV encapsidation genes (rep and cap genes). Next, the produced recombinant AAV is purified by a conventional technique.
[0124]
With respect to herpesviruses and retroviruses, the construction of recombinant vectors has been extensively described in the literature, in particular Breakfield et al. (New Biologist 3 (1991) 203); EP453242, EP178220, Bernstein et al. (Genet. Eng. 7 ( 1985) 235); McCormick, (BioTechnology 3 (1985) 689).
[0125]
In particular, retroviruses are integrating viruses that infect dividing cells. The retroviral genome essentially contains two LTRs, an encapsidation sequence, and three coding regions (gag, pol and env). In a retrovirus-derived recombinant vector, the gag, pol and env genes are generally completely or partially deleted and replaced with a predetermined heterologous nucleic acid sequence. These vectors may contain various types of retroviruses, for example, MoMuLV (also referred to as “Mouse Moloney Leukemia Virus” or MoMLV), MSV (“Mouse Moloney Sarcoma Virus”), HaSV (“Harvey Sarcoma Virus”); SNV, among others. ("Spleen necrosis virus"); can be produced from RSV ("Rous sarcoma virus") or a friend virus.
[0126]
To construct a recombinant retrovirus comprising a sequence encoding the ABCA7 protein placed under the control of a regulatory nucleic acid according to the present invention, one generally constructs, inter alia, a plasmid comprising the LTR, the encapsidation sequence and said coding sequence. A retroviral function deficiency in the plasmid can then be provided at the trans position and used to transfect so-called encapsidated cell lines. Usually, the encapsidation system is therefore capable of expressing the gag, pol and env genes. Such encapsidation systems have been described in the prior art, in particular the PA317 system (US Pat. No. 4,861,719), the PsiCRIP system (WO90 / 02806), and the GP + envAm-12 system (WO89 / 07150). Moreover, recombinant retroviruses can include modifications at the level of the LTR to suppress transcriptional activity, as well as extended capsidation sequences that include portions of the gag gene. (Bender et al., J. Virol. 61 (1987) 1639). The produced recombinant retrovirus is then purified by conventional techniques.
[0127]
It is most particularly advantageous to use a defective recombinant adenovirus to carry out the invention. The results obtained below show indeed particularly advantageous properties of adenoviruses that express proteins having lipid metabolic mediator activity in vivo. The adenovirus vectors according to the invention are particularly advantageous for direct administration of the purified suspension in vivo or for ex vivo transformation of cells, especially autologous cells, in view of their transplantation. Moreover, the adenoviral vectors according to the invention show, in addition to a number of advantages, considerable benefits, for example, in particular, their very high efficiency of infection, whereby infection with small amounts of viral suspension is possible. It is possible to do.
[0128]
According to another particularly advantageous embodiment of the invention, a system for producing a retroviral vector comprising a regulatory nucleic acid according to the invention and a sequence encoding the ABCA7 protein is used for in vivo transplantation. Systems that can be used for this purpose are, in particular, PA317 (US Pat. No. 4,861,719), PsiCrip (WO90 / 02806) and GP + envAm-12 (US Pat. No. 5,278,056), which are used to express the ABCA7 protein according to the invention. A cell that has been modified to produce a retrovirus containing the encoding nucleic acid sequence. For example, totipotent stem cells, precursors of blood cell lines, can be recovered from a subject and isolated. When culturing these cells, the cells can then be infected with a retroviral vector containing a sequence encoding the ABCA7 protein under the control of its own promoter. The cells are then reintroduced into the subject. The differentiation of these cells may involve cells of hematopoietic tissues that express the ABCA7 protein, especially T lymphocytes involved in inflammatory signaling.
[0129]
Advantageously, in the vector according to the invention, the sequence coding for the ABCA7 protein is a regulatory nucleic acid according to the invention comprising regulatory elements that allow it to be expressed in infected cells, most particularly NF kappa B, CEBP and GFI1 type regulatory elements. Under the control of
As mentioned above, the invention also relates to the preparation of a pharmaceutical composition for the treatment and / or prevention of pathologies related to lipid metabolism or dysfunctions related to processes involving the immune system and inflammation. , Regarding all of the uses of the above mentioned viruses.
[0130]
The invention also relates to a pharmaceutical composition comprising one or more of the above-described defective recombinant viruses. These pharmaceutical compositions can be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, ocular or transdermal administration and the like. Preferably, the pharmaceutical composition of the invention comprises a pharmaceutically acceptable excipient for injectable preparations, in particular for example for intravenous injection into the patient's portal vein. They especially relate to isotonic sterile solutions, or to dry compositions, especially lyophilized compositions, which can be prepared in sterile water or saline to prepare solutions for injection. Direct injection into the patient's portal vein is advantageous because it allows to target infection at the level of the liver, thereby increasing the efficacy of treatment at the level of this organ.
[0131]
The dose of the defective recombinant virus used for injection will be adjusted depending on the function of the various parameters and, in particular, the function of the viral vector used, the mode of administration, the relevant condition or the desired duration of treatment. can do. Generally, the recombinant adenovirus according to the present invention comprises 10⁴-10¹⁴pfu / ml, preferably 10⁶-10¹⁰It is formulated and administered in the form of a dose of pfu / ml. The term pfu ("plaque forming unit") corresponds to the infectivity of the virus solution and is measured by infecting the appropriate cell culture and measuring the plaque count of the infected cells, typically after 48 hours. Techniques for measuring pfu titer of virus solutions are well described in the literature.
[0132]
With respect to retroviruses, the compositions according to the invention may, for the purpose of their transplantation, directly contain producer cells.
In this context, another object of the invention relates to all mammalian cells infected with one or more of the defective recombinant viruses mentioned above. More particularly, the invention relates to all human cell populations infected with these viruses. Mammalian cells include, in particular, cells of blood origin (totipotent stem cells or precursors), fibroblasts, myoblasts, hepatocytes, keratinocytes, smooth muscle and endothelial cells, glial cells, and the like.
[0133]
The cells according to the invention can be obtained from primary cultures. These can be recovered by any technique known in the art and then cultured under conditions that allow them to grow. More particularly with respect to fibroblasts, they can be easily obtained from biopsies, for example according to the technique described by Ham (Methods Cell Biol (1980) 21a: 255). These cells may be used directly for viral infection or may be stored for autologous library construction, eg, by freezing, for subsequent use. The cells according to the present invention may be, for example, secondary cultures obtained from a library that has been previously constructed (see, for example, EP 228458, EP 289034, EP 400047, EP 456640).
[0134]
The cells in culture are then infected with the recombinant virus, giving them the ability to produce a biologically active ABCA7 protein. Infection is performed in vitro according to techniques known to those skilled in the art. In particular, depending on the type of cells used and the number of virus copies per cell sought, the person skilled in the art can adjust the multiplicity of infection and, if necessary, the number of infection cycles produced. . It goes without saying that these steps must be performed under suitable sterile conditions if the cells are intended to be administered in vivo. The dose of the recombinant virus used to infect the cells can be adjusted by those skilled in the art according to the desired purpose. The conditions for in vivo administration described above can be adapted for infection in vitro. With respect to infection with a retrovirus, cells that are desirably infected with cells that produce the recombinant retrovirus of the present invention can also be co-cultured. Thereby, it is possible to omit retrovirus purification.
[0135]
Another object of the invention relates to a mammalian cell infected with one or more of the above-mentioned defective recombinant viruses, or a cell producing the recombinant virus, and an implant comprising an extracellular matrix. Preferably, the implant according to the invention comprises 10⁵-10¹⁰Cells. More preferably, they are⁶-10⁸Cells.
More specifically, in the implant of the present invention, the extracellular matrix includes a gelling compound and, if necessary, a support for fixing cells.
[0136]
Various types of gelling agents can be used for the preparation of the implant according to the invention. Gelling agents are used to encapsulate the cells in a matrix having the properties of a gel and, if necessary, to enhance the fixation of the cells to the support. Therefore, various cell adhesives can be used as gelling agents, for example, especially collagen, gelatin, glycosaminoglycan, fibronectin, lectin and the like. Preferably, collagen is used in the present invention. This may be collagen from humans, cows or mice. More preferably, type I collagen is used.
[0137]
As mentioned above, the composition according to the invention advantageously comprises a support on which the cells are fixed. The term fixation refers to any form of biological and / or chemical and / or physical interaction that causes the attachment and / or attachment of cells to a support. Moreover, the cells can either coat the support used, or penetrate the interior of the support, or both. For the present invention, it is preferred to use a solid non-toxic and / or biocompatible support. In particular, it is possible to use polytetrafluoroethylene (PTFE) fibers or supports of biological origin.
[0138]
Accordingly, the present invention relates to the treatment or prevention of conditions associated with the transport of cholesterol (especially obesity, hypertriglyceridemia) or cardiovascular conditions, myocardial infarction, stomatitis, sudden death, heart failure and cerebral vascular attacks. Provide very effective means for treatment or prevention in the field of
In addition, this treatment can be applied to both humans and all animals (eg, sheep, cattle, livestock (dogs, cats, etc.), horses, fish, etc.).
[0139]
Recombinant host cells
The present invention also relates to a recombinant host cell comprising any one of the nucleic acids of the present invention having the sequence of SEQ ID NO: 1 to SEQ ID NO: 6, and more specifically, to the recombinant host cell having the sequence of SEQ ID NO: 1 to SEQ ID NO: 3. Related to nucleic acids.
According to another aspect, the present invention also relates to a recombinant host cell comprising a recombinant vector as described above.
Preferred host cells according to the present invention include, for example, the following.
a) Prokaryotic host cells: Escherichia coil strain (DH5-CX strain), Bacillus subtlls strain, Salmonella typhimurium strain, or strains of, for example, Pseudomonas, Streptomyces and Staphylococcus species;
b) Eukaryotic host cells: HeLa cells (ATCC No. CCL2), Cv1 cells (ATCC No. CCL70), COS cells (ATCC No. CRL1650), Sf-9 cells (ATCC No. CRL1711), CHO cells (ATCC No. CCL-61). Or 3T3 cells (ATCC No. CRL-6361), or Hepa1-6 type cells, which are referred to by American Type Culture Collection (ATCC, Rockville, MD, United States of America);
c) Primary culture cells were obtained from individuals whose expression of a given nucleic acid was placed under the control of a regulatory nucleic acid according to the present invention.
d) Indefinitely grown cells (cell lines) are obtained from the above-mentioned c) primary culture cells according to techniques well known in the art.
[0140]
Screening method
In vitro screening method
The present invention provides a method of treating a subject suffering from a condition associated with ABCA7 protein expression levels. In particular, such a method of treatment comprises administering to the subject a compound that modulates the expression of the ABCA7 gene, the method comprising identifying by a variety of in vitro screening methods, as defined below. Can be.
The first method consists in identifying compounds that modulate the expression of the ABCA7 gene.
According to such a method, cells expressing the ABCA7 gene are incubated with the candidate substance or molecule to be tested, and subsequently the expression level of messenger RNA for ABCA7 or the production level of ABCA7 protein is measured.
[0141]
Messenger RNA levels for ABCA7 can be measured by Northern-type gel hybridization well known to those skilled in the art. The messenger RNA level for ABCA7 can also be measured by a method using PCR or a technique described by WEBB (Journal of Biomolecular Screening (1996), vol. 1: 119).
The production level of ABCA7 protein can be measured by immunoprecipitation or immunochemistry using an antibody that specifically recognizes ABCA7 protein.
[0142]
According to another method for screening a candidate molecule or substance that modulates the activity of a regulatory nucleic acid according to the present invention, a nucleotide construct as defined above is used, wherein the construct comprises a regulatory nucleic acid according to the present invention and a regulatory nucleic acid Wherein the regulatory nucleic acid comprises at least one core promoter and at least one element that regulates any one of the sequences of SEQ ID NOS: 1-3. The reporter polynucleotide can be a gene encoding a detectable protein, for example, a gene encoding luciferase.
According to such a screening method, cells are infected with a polynucleotide construct comprising a regulatory nucleic acid according to the present invention and a reporter polynucleotide in a stable and transient manner.
The transformed cells are then incubated for a sufficient time in the presence or absence of the candidate molecule or substance to be tested, and then the level of reporter gene expression is measured. Next, compounds that induce a statistically significant change in reporter gene expression (either increasing or decreasing reporter gene expression) are identified and, where appropriate, selected.
[0143]
Accordingly, an object of the invention is also a molecule or substance that modulates the activity of a regulatory nucleic acid according to the invention, in particular a molecule or substance that modulates the transcription of a constitutive reporter polynucleotide of a polynucleotide construct according to the invention. Is a method of screening in vitro, the method comprising:
a) culturing a recombinant host cell containing a given polynucleotide placed under the control of a regulatory nucleic acid according to the present invention;
b) incubating the recombinant host cells with the substance or molecule to be tested;
c) detecting the expression of a given polynucleotide;
d) comparing the result obtained in step c) with the result obtained when the recombinant host cell is cultured in the absence of the candidate molecule or substance to be tested;
Characterized by including a step consisting of:
[0144]
The present invention also relates to a kit or box for in vitro screening for a candidate molecule or substance capable of modulating the activity of the regulatory nucleic acid according to the present invention, wherein the kit or box comprises:
a) a host cell transformed with a polynucleotide construct as defined above, comprising a predetermined reporter polynucleotide placed under the control of a regulatory nucleic acid according to the present invention; and
b) if necessary, means for detecting the expression of the given reporter polynucleotide,
including.
Preferably, a given reporter polynucleotide is a luciferase coding sequence. In this case, the regulatory nucleic acid according to the present invention is inserted into the vector upstream of the luciferase-encoding sequence. This can be, for example, the vector pGL3-basic (pGL3-b) marketed by Promega (Madison, Wisconsin, United States).
[0145]
In this case, the recombinant vector containing the luciferase coding sequence placed under the control of the regulatory nucleic acid according to the invention is transfected into a recombinant host cell and then in the presence or absence of the candidate substance or molecule to be tested. After culturing in the presence, its luciferase activity is measured.
In this case, either a cytomegalovirus (CMV) promoter, a pGL3-b vector containing an ApoAI promoter, or a pGL3-b vector containing no promoter can be used as a control. To test luciferase activity, transfected cells were washed with PBS buffer and 500 μl lysis buffer (50 mM Tris, 150 mM NaCl, 0.02% sodium azide, 1% NP-40, 100 μg / Ml AEBSF and 5 μg / ml leupeptin).
[0146]
50 μl of the obtained cell lysate is then added to 100 μl of luciferase substrate (Promega), and the activity is measured with a spectrophotometric microplate reader for 5 minutes after the addition of the cell lysate.
The data is expressed in relative units of luciferase activity. Polynucleotide constructs that produce high levels of luciferase activity in the transfected cells include a regulatory nucleic acid according to the present invention comprised in the sequence of SEQ ID NO: 1, wherein the regulatory nucleic acid stimulates transcription. be able to.
In the screening method according to the present invention, in order to measure the expression level of messenger RNA, a probe specific to a messenger RNA for a predetermined reporter polynucleotide is first prepared, and for example, a multiprime labeling kit (Amersham Life Sciences) (Commercially available from Cleveland, Ohio, United States).
[0147]
In vivo screening method
In accordance with another aspect of the present invention, compositions that modulate the activity of a regulatory nucleic acid of the present invention can be identified in vivo in non-human transgenic animals.
According to such a method, a non-human transgenic animal, such as a mouse, is treated with a candidate molecule or substance to be tested, for example, with a candidate substance or molecule previously selected by an in vitro screening method as defined above. Is done.
After a predetermined period of time, the activity level of the regulatory nucleic acid according to the present invention is measured and compared with the activity of the same non-human transgenic animal that has not taken up the candidate molecule or substance, for example the same transgenic mouse.
The activity of a regulatory nucleic acid according to the present invention that is functional in a transgenic animal can be measured in various ways, for example, Northern hybridization, or in situ hybridization, or non-invasive biophotoimaging. The level of messenger RNA corresponding to a given reporter polynucleotide placed under the control of the regulatory nucleic acid is determined by (noninvasive biophotonic imaging) (Xenogen Corporation).
[0148]
Alternatively, the activity of a regulatory nucleic acid according to the present invention can be determined by measuring the expression level of a protein encoded by a given reporter polynucleotide, for example, by immunohistochemistry, In that case, a given reporter polynucleotide will include an open reading frame that encodes a protein detectable by such techniques.
In order to carry out the method of in vivo screening for a candidate substance or molecule that modulates the activity of a regulatory nucleic acid according to the present invention, non-human mammals, such as mice, rats, guinea pigs or rabbits, are preferred. Has been modified by inserting a polynucleotide construct comprising a given reporter polynucleotide placed under the control of a regulatory nucleic acid according to the present invention.
[0149]
The transgenic animals according to the invention contain a transgene, ie a polynucleotide construct as described above, in a large number of their somatic and / or germ cells.
The construction of the transgenic animal according to the present invention can be performed according to conventional techniques well known to those skilled in the art. Those skilled in the art will be aware of, inter alia, U.S. Pat. No. 4,873,191 (patented Oct. 10, 1989), U.S. Pat. No. 5,464,764 (patented Nov. 7, 1995) and U.S. Pat. Reference may be made to the production of transgenic animals, in particular the production of transgenic mice, as described in US Pat. No. 5,789,215 (patented Aug. 4, 1998), the contents of which are incorporated herein by reference. Incorporated by reference in the book.
Briefly, a polynucleotide construct comprising a regulatory nucleic acid according to the present invention and a given reporter polynucleotide placed under control is inserted into an ES type stem cell line. Insertion of the polynucleotide construct is preferably performed by electroporation as described by Thomas et al. (1987, Cell, Vol. 51: 503-512).
[0150]
The cells that have undergone the electroporation step are then screened for the presence of a polynucleotide construct (eg, by marker-assisted selection, or by PCR, or Southern-type analysis of the DNA on an electrophoretic gel). Positive cells can be selected in which the foreign polynucleotide constructs have been integrated into their genome, optionally followed by a homologous recombination step. Such a technique is described, for example, by MANSOUR et al. (Nature (1988) 336: 348-352).
The cells selected as positive are then isolated, cloned, and injected into 3.5 day old mouse blastocysts, which are described in BRADLEY (1987, Production and Analysis of Chloramic acid. In: EJ. ROBERTSON (Ed., Teratocarcinomas and embryonic stem cells: A practical approach. IRL press, Oxford, page 113). Continued to develop.
Alternatively, positively selected ES-type cells are brought into contact with 8-16 cell stages (morula embryos) of 2.5-day-old embryos, as described in WOOD et al. (1993, Proc. Natal. Acad. Sci. USA, vol. 90: 4582-4585) or NAGY et al. (1993, Proc. Natl. Acad. Sci. USA, vol. 90: 8424-8428). The blastocysts containing the cells to be produced are taken up so that they can colonize a wide area.
[0151]
Next, progeny are tested to determine progeny that have integrated the polynucleotide construct (transgene).
Therefore, an object of the present invention is also a non-human transgenic animal, the somatic and / or germ cells of which have been transformed with a nucleic acid or polynucleotide construct according to the present invention.
The present invention also relates to a recombinant host cell obtained from the above-mentioned transgenic animal. Recombinant cell lines obtained from the transgenic animals according to the invention can be used in long-term cultures from all tissues of such transgenic animals, for example by transfecting a primary cell culture with a vector expressing an oncogene. It can be established, for example, the SV40 large T antigen, such as CHOU (1989, Mol. Endocrinol. Vol. 3: 1511-1514) and SCHAY et al. (1991, Biochem. Biophys. Acta, vol. 1072: 1-1-1). 7).
The present invention also relates to a method of in vivo screening for a candidate molecule or substance that modulates the activity of a regulatory nucleic acid according to the present invention, the method comprising the following steps: a) identifying the candidate substance or molecule as defined above. B) detecting the expression level of a given reporter polynucleotide placed under the control of a regulatory nucleic acid; c) incorporating the results obtained in b) and the candidate substance or molecule. Comparing the results obtained with non-transgenic animals.
[0152]
In addition, the present invention relates to a method for in vivo screening for a substance or molecule that modulates the transcription of a predetermined polynucleotide containing a nucleic acid according to the present invention, wherein the method comprises the steps of: Administering to a defined non-human transgenic mammal; (b) detecting expression of a given polynucleotide in the transgenic mammal treated in step a); and (c) step (b). And comparing the results of the detection with the results observed in a non-human transgenic mammal to which the above-defined candidate substance or molecule has not been administered.
The invention also relates to a kit or box for in vivo screening for candidate molecules or substances that modulate the activity of a regulatory nucleic acid according to the invention, the kit or box comprising: a) a transgenic animal as defined above. B) if necessary, means for detecting the expression level of a given reporter polynucleotide.
[0153]
Pharmaceutical compounds and pharmaceutical compositions
The present invention also relates to a pharmaceutical composition for the purpose of preventing or treating defects in lipid metabolism or dysfunction of processes including the immune system and inflammation.
First, an object of the present invention is also a candidate substance or molecule that modulates the activity of a regulatory nucleic acid according to the present invention.
Most preferably, the invention also relates to a candidate substance or a substance characterized by increasing the activity of a regulatory nucleic acid according to the invention, most particularly the activity of a regulatory nucleic acid comprising the sequence of SEQ ID NO: 1, 2, 4 or 5. About molecules.
Preferably, such substances or molecules capable of modulating the activity of a regulatory nucleic acid according to the invention have been selected according to any one of the in vitro or in vivo screening methods defined above.
Accordingly, a subject having a disorder in lipid metabolism or immune signaling is treated by administering to the subject an effective amount of a compound that modulates the activity of a regulatory nucleic acid according to the present invention.
Accordingly, patients with weak ABCA7 promoter activity can be treated with the molecules or agents described above to increase ABCA7 promoter activity.
[0154]
Alternatively, patients with abnormally high ABCA7 promoter activity can be treated with compounds that can reduce or block ABCA7 promoter activity.
Accordingly, the present invention also relates to substances or molecules used as active ingredients in pharmaceuticals.
Such a compound may be a compound that modulates the interaction of at least one transcription factor with an ABCA7 promoter or a regulatory element of a regulatory nucleic acid according to the present invention.
For example, the compound can inhibit the interaction of any one of the transcription factors listed in Table 1 with the regulatory nucleic acid of the present invention.
[0155]
In addition, the compound may be a compound that modulates the activity of a transcription factor that binds to the ABCA7 promoter or a regulatory element located behind it.
The therapeutic compound of the present invention may be a compound that modulates the interaction between the first transcription factor and the second transcription factor.
As described in detail in the analysis of various transcription factors that can bind to the sequence of SEQ ID NO: 1, 2, 4, or 5, some transcription factors are only active when combined with other transcription factors.
The compound for therapeutic purposes according to the present invention is preferably selected from nucleic acids, peptides, and low molecular substances.
For example, such a compound can be an antisense nucleic acid that specifically binds to one region of the ABCA7 promoter or a regulatory element of a nucleic acid that regulates ABCA7 and inhibits or reduces promoter activity.
[0156]
The therapeutic compound may reduce the production of this transcription factor by interaction of the antisense nucleic acid with a gene encoding a transcription factor that binds to the ABCA7 promoter, and the transcription factor may increase ABCA7 promoter activity, Alternatively, it may be an antisense nucleic acid that specifically interacts with a gene encoding a transcription factor that modulates ABCA7 promoter activity in such a way as to increase or decrease ABCA7 promoter activity, depending on whether it is reduced.
The toxicity and therapeutic efficacy of the therapeutic compounds according to the invention can be measured in cells in culture or in experimental animals according to standard pharmaceutical protocols, for example, a lethal dose LD.₅₀(Ie the dose at which 50% of the tested population dies) and the effective dose ED₅₀(Ie, the dose that is therapeutically effective in 50% of the population tested) can be measured.
[0157]
For all therapeutic compounds according to the present invention, the therapeutically effective amount can be estimated initially from in vitro tests performed in cell culture.
Also an object of the present invention is a pharmaceutical composition comprising a therapeutically effective amount of a therapeutic substance or molecule according to the present invention.
The pharmaceutical composition according to the invention is more particularly intended for the treatment and / or prevention of a deficiency in lipid metabolism or a deficiency in mechanisms including the immune system and inflammation.
Such pharmaceutical compositions can be formulated in a conventional manner using one or more physiologically acceptable vectors or excipients.
Thus, the therapeutic compounds according to the invention, as well as their physiologically acceptable salts and solvates, are administered by injection or inhalation or by oral, buccal, parenteral or rectal administration. Can be blended for Techniques for preparing a pharmaceutical composition according to the present invention can be readily found by those skilled in the art, and include, for example, Remmington's Pharmaceutical Sciences, Mead Publishing Co. , Easton, PA, United States.
For systemic administration, injection is preferred, for example, intramuscular, intravenous, intraperitoneal and subcutaneous injection. In this case, the pharmaceutical composition according to the invention can be formulated in the form of a solution, preferably in the form of a physiologically compatible solution or buffer.
[0158]
Method of detecting transcriptional defect of human ABCA7 gene
Further, an object of the present invention is a method of determining whether a subject is at risk for developing a condition associated with a defect in lipid metabolism or a process associated with defects in the immune system and inflammation, including inflammation.
Such a method is characterized by the presence or absence of a genetic defect in cells of a biological sample obtained from the subject to be tested, wherein the expression of the gene whose expression is regulated by the ABCA7 promoter is defective. As detecting.
By way of example, in the sequence SEQ ID No. 1, 2, 3 or 6, the presence of the deletion of one or more nucleotides in the sequence of the nucleic acid regulating ABCA7, the presence of the addition of one or more nucleotides, or Such genetic defects can be detected for the purpose of determining the presence of one or more nucleotide substitutions.
[0159]
In accordance with certain embodiments of the method of detecting a defect in ABCA7 gene transcription in a subject, the genetic defect is sequence analysis of all or a portion of the sequence of SEQ ID NO: 1, or at least all or a portion of the sequence of SEQ ID NO: 2. Are identified according to a method comprising:
The primer for sequence analysis can be constructed so as to hybridize with the defined region of the sequence of SEQ ID NO: 1. Such a primer for sequence analysis can be preferably constructed to amplify a fragment of about 300 to about 500 nucleotides or a complementary sequence fragment of the sequence of SEQ ID NO: 1.
The DNA contained in the biological sample obtained from the tested subject is then analyzed, for example by fragment analysis of the fragment amplified by the PCR method and comparing the obtained sequence with the sequence of the reference SEQ ID NO: 1. Can be determined if one or more deletions, additions or substitutions of nucleotides are found in the amplified sequence from.
[0160]
Therefore, the present invention also relates to a method for detecting a defect in ABCA7 gene transcription in a subject, the method comprising the following steps:
a) sequence analysis of a nucleic acid fragment that can be amplified with the aid of at least one nucleotide primer that hybridizes with the sequence of SEQ ID NO: 1 or SEQ ID NO: 2 according to the present invention;
b) aligning the sequence obtained in a) with the sequence of 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 said nucleic acid fragment relative to the reference SEQ ID NO: 1 or SEQ ID NO: 2,
including.
[0161]
The present invention also relates to a kit or box for detecting a defect in ABCA7 gene transcription in a subject, wherein the kit or box includes one or more primers for sequence analysis, and the primer has a sequence of SEQ ID NO: 1. Can hybridize to the region, thereby allowing sequence analysis of a polynucleotide located upstream of the start site of ABCA7 gene transcription in a subject being tested.
In addition, a component of the invention is also an oligonucleotide probe that hybridizes to a region of the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, wherein the defects in the sequence are determined during the execution of the detection method described above. Have been.
[0162]
Alternatively, a component of the invention may also be specific in a subject to a corresponding region of the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, wherein one or more deletions, additions or substitutions of at least one nucleotide have been determined. Oligonucleotide probes that hybridize specifically.
Such oligonucleotide probes constitute a means of detecting a defect in the sequence that regulates the ABCA7 gene and, therefore, may be used to identify conditions associated with defects in lipid metabolism or dysfunction in processes including the immune system and inflammation. It also constitutes means for detecting the tendency to develop.
[0163]
Therefore, an object of the present invention is also a kit or box for detecting a defect in ABCA7 gene transcription in a subject, said kit or box comprising:
a) one or more primers that hybridize to a region of the sequence of SEQ ID NO: 1 or SEQ ID NO: 2;
b) if necessary, the means necessary to carry out the amplification reaction;
including.
[0164]
An object of the present invention is also a kit or box for detecting a transcriptional defect of the ABCA7 gene in a subject, wherein the kit or box comprises:
a) one or more oligonucleotide probes as defined above;
b) if necessary, reagents required to carry out the hybridization reaction,
including.
[0165]
An object of the present invention is also a kit or box for detecting a defect of ABCA7 gene transcription in a subject, wherein the kit or box hybridizes with a region of the sequence represented by SEQ ID NO: 1 or the region of the sequence of SEQ ID NO: 2. Or more probes, thereby allowing quantification of messenger RNA for ABCA7 in biological material obtained from said subject to be tested.
Therefore, a nucleic acid fragment derived from any one of the nucleotide sequences of SEQ ID NOs: 1 to 6 may contain, in a sample, a nucleotide sequence that regulates the ABCA7 gene, or a fragment or variant thereof (including a mutation or polymorphism). Useful for detecting the presence of at least one copy of
[0166]
The nucleotide probe or primer according to the present invention contains at least eight consecutive nucleotides of a nucleic acid selected from the group consisting of the sequences of SEQ ID NOS: 1 to 5, or a nucleic acid having a complementary sequence.
Preferably, the nucleotide probe or primer according to the invention has 10, 12, 15, 18 or 20 to 25, 35, 40, 50, 70, 80, 100, 200, 500, 1000, 1500 according to the invention. It has a length of contiguous nucleotides of the nucleic acid, especially of a nucleic acid having a nucleotide sequence selected from the sequences of SEQ ID NOs: 1-5.
[0167]
Alternatively, a nucleotide probe or primer according to the present invention comprises a sequence of 12, 15, 18, 20, 25, 35, 40, 50, 100, 200, 500, 1000, 1500 nucleic acids according to the present invention, more particularly the sequence Consisting of and / or including a fragment of a nucleic acid selected from the sequences of Nos. 1 to 5 or of a nucleic acid having a complementary sequence, having a continuous nucleotide length.
Therefore, the definition of the nucleotide probe or primer according to the present invention refers to a nucleic acid selected from the sequences of SEQ ID NOs: 1 to 5, 6 or 8 under high stringent hybridization conditions as defined above, or Oligonucleotides that hybridize with the complementary sequence of
[0168]
Examples of primers and primer pairs capable of amplifying various regions of the ABCA7 gene are shown below.
For example, a primer having the sequence of SEQ ID NO: 9: AGCCAGCAACGCAATCCCTC, and a primer having the sequence of SEQ ID NO: 10: a primer pair represented by CGCACCCATGTCAATGAGCCC.
[0169]
The nucleotide primers or probes according to the invention can be prepared by all suitable methods known to the person skilled in the art, for example by cloning or by the action of restriction enzymes, or, for example, by Narang et al. (Methods Enzymol (1979) 68: 90-98) or the phosphodiester method according to Brown et al. (Methods Enzymol (1979) 68: 109-151) and the diethylphosphoramidate method according to Beaucage et al. (Tetrahedron Lett (1980) 22: 1859-1862). Can be prepared by conventional chemical synthesis or by the technique for solid supports described in Patent EP 0,707,592.
[0170]
Any of the nucleic acids according to the present invention, including the above-described oligonucleotide probes and primers, optionally incorporates a detectable marker by spectroscopic, photochemical, biochemical, immunochemical or chemical means. By doing so, it can be labeled.
For example, such markers include radioactive isotopes (³²P,³³P,³H,³⁵S), a fluorescent molecule (5-bromodeoxyuridine, fluorescein, acetylaminofluorene, digoxigenin), or a ligand such as biotin.
[0171]
Labeling of the probe is preferably carried out by incorporating the labeled molecule into the polynucleotide by extension of the primer, or by addition to the 5 'or 3' end, or "nick translation".
Examples of non-radioactive labeling of nucleic acid fragments are, inter alia, French Patent 7810975 or Urdea et al. (Nucleic Acid Res (1988) 11: 4937-4957) or Sanchez-pescador et al. (J. Clin Microbiol (1988)). ) 26 (10) 1934-1938).
Advantageously, the probes according to the invention may have structural features of the type that amplify the signal, such as, for example, Urdea et al. (Mol. Cell. Blol., (1991) 6: 716-718), or And EP 0,225,807 (CHIRON).
[0172]
The oligonucleotide probe according to the present invention can be used particularly in Southern hybridization using genomic DNA or Northern hybridization using RNA.
The probe according to the present invention can be used for detection of a PCR amplification product or mismatch detection.
The nucleotide probe or primer according to the present invention can be immobilized on a solid support. Such solid supports are well known to those skilled in the art, and include the well surface of a microtiter plate, a polystyrene bed, a magnetic bed, a nitrocellulose band, or ultrafine particles (eg, latex particles).
[0173]
Consequently, the present invention also relates to a method for detecting the presence of a nucleic acid as described above in a sample, comprising the steps:
1) contacting one or more nucleotide probes according to the invention with the sample to be tested;
2) detecting a complex that can be formed between the probe and the nucleic acid present in the sample;
including.
[0174]
According to a particular embodiment of the detection method according to the invention, said oligonucleotide probe is immobilized on a support.
According to another aspect, the oligonucleotide probe comprises a detectable marker.
[0175]
In addition, the present invention relates to a box or kit for detecting the presence of a nucleic acid according to the present invention in a sample, wherein the box comprises:
a) one or more of the above nucleotide probes;
b) if necessary, reagents required for the hybridization reaction,
including.
[0176]
According to a first aspect, the detection box or kit is characterized in that the probe is fixed to a support.
According to a second aspect, the detection box or kit is characterized in that the oligonucleotide probe contains a detectable marker.
According to a particular embodiment of the detection kit described above, such a kit comprises a number of oligonucleotide probes according to the invention, said oligonucleotide probes detecting a given target sequence or according to the invention. It can be used to detect mutations in the coding or non-coding regions of nucleic acids, more specifically nucleic acids having the sequences of SEQ ID NOS: 1-5, 6 and 8, or nucleic acids having complementary sequences.
[0177]
Therefore, the probes according to the present invention immobilized on the support can be arranged in a matrix such as a “DNA chip”. Such ordered matrices are described, inter alia, in US Pat. No. 5,143,854, PCT Applications WO 90/15070 and 92/10092.
Support matrices to which oligonucleotide probes are immobilized at high density are described, for example, in US Pat. No. 5,412,087 and PCT application WO 95/11995.
[0178]
The nucleotide primer according to the present invention is used for amplifying any one of the nucleic acids according to the present invention, more specifically, all or a part of the nucleic acid having the sequence of SEQ ID NOS: 1 to 5, or a mutant thereof. Can be used.
Another object of the present invention relates to a method for amplifying a nucleic acid according to the present invention, more particularly a nucleic acid having the sequence of SEQ ID NOS: 1 to 5, or a fragment or variant thereof, contained in a sample, The method comprises:
a) contacting a sample suspected of containing a target nucleic acid with a nucleotide primer pair in the presence of a reagent necessary for the amplification reaction, wherein the hybridization position of the nucleotide primer pair is searched for amplification, Located 5 'and 3' to the region of the target nucleic acid: and
b) detecting the amplified nucleic acid;
Including the step of:
[0179]
To carry out the amplification method as defined above, any one of the nucleotide primers mentioned above can advantageously be used.
In addition, an object of the present invention is a box or kit for amplifying all or a part of the nucleic acid according to the present invention, more particularly, the nucleic acid having the sequence of SEQ ID NOS: 1 to 5, wherein said box or kit is ,
a) the nucleotide primer pair according to the present invention, wherein the hybridization position of the nucleotide primer pair is located on the 5 ′ side and 3 ′ side of the target nucleic acid whose amplification is searched, respectively;
b) If necessary, reagents required for the amplification reaction
including.
Such an amplification box or kit advantageously comprises at least one pair of the above-mentioned nucleotide primer pairs.
The present invention is further illustrated, but not limited, by the following figures and examples.
[0180]
Example:
Example 1: Determination of the 5 'end of cDNA for ABCA7
Amplification of mRNA ends by RT-PCR (RACE) was performed using a SMART® RACE® cDNA amplification kit (Clontech, Palo Alto, Calif.). Using the (polyA) mRNA extracted from human spleen tissue as a template, a SMART5 'cDNA library was prepared according to the manufacturer's instructions. The first amplification primer and the internal primer were selected from the cDNA sequence. Amplifications made using internal primers for PCR amplification were cloned. Next, specific clones were amplified using primers, where the sequences of the primers were (CAGGAAACAGCTATGAC) and (GCCAGTGTGATGGGATAT), respectively, followed by sequence analysis of the two strands. Finally, the end of human ABCA7 cDNA was identified using the following primers: ABCA7L1: GCGGAAAGCAGGTGTTGTTCTCAC (SEQ ID NO: 11) and ABCA7L2: CGATGGCAGTGGCTTGTTTGG (SEQ ID NO: 12).
[0181]
Example 2: Analysis of human and mouse ABCA7 gene promoter
Using three software packages: TSSG and TSSW (Solovyev et al., Ismb (1997) 5, 294-302) and NNPP (Reese MG, et al., 1999), the transcription start site is changed for human and ABCA7. It was placed on the mouse gene promoter. Binding sites for human and mouse transcription factors were predicted using the MatInspector program for motif search (Quandt et al., Nucl. Acid Research (1995) 23 (23) 4878-84). The score calculation for each binding site of the transcription factor is calculated by the formula: (Of-Tf) / (Tf)^1/2Where Of is the frequency at which the motif was observed and Tf is the calculated frequency of the consensus motif. To isolate motifs that seem to be unrelated, the first filtration step was performed with the above Matlnspector program “template similarity” score of 0.85 and the above “core similarity” score of 0.99. This was done by adjusting. Finally, in order to define the transcription module containing sites with similar motifs and the presence of sequences upstream of the ABCA7 gene in both human and mouse sequences, Werner T (Models for prediction and recognition of eukaryotic promoters). , Mammarian Genome (1999) 10: 168-175), and comparative analysis of promoters between species was performed.
[0182]
Example 3: Preferred human and mouse ABCA7 gene expression in hematopoietic tissues
The characteristics of the expression of the polynucleotide according to the present invention were determined according to the protocols of reverse transcription PCR and Northern blot analysis, which are described in particular by Sambrook et al. (Ref. CSH Sambrook, J., Fritsch, EF, and Maniatis) , T. (1989) "Molecular Cloning: A Laboratory Manual," 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
For example, in the case of analysis by reverse transcription, primer pairs were synthesized from each full-length cDNA of the human and mouse ABCA7 genes, and the corresponding cDNA was detected. The sequences of these primers are shown in Table 4.
A polymerase chain reaction (PCR) is performed on the cDNA template corresponding to the retrotranscribed poly A + mRNA. Reverse transcription to cDNA is performed using an enzyme called SUPERSCRIPT II (GibcoBRL, Life Technologies) according to the conditions described by the manufacturer.
[0183]
The polymerase chain reaction is performed under standard conditions in a 20 μl reaction mixture containing 25 ng of the cDNA preparation. The reaction mixture was composed of 400 μM of each dNTP, 2 units of Thermus aquaticus (Taq) DNA polymerase (AmpliTaq Gold; manufactured by PerkinElmer), 0.5 μM of each primer, 2.5 mM of MgCl 2₂, And PCR buffer. After a first denaturation step at 94 ° C. for 10 minutes on a Perkin Elmer 9700 thermocycler, 30 cycles of PCR (denaturation at 94 ° C. for 30 seconds, annealing for 30 seconds (during 30 cycles, (2 cycles at 64 ° C, 2 cycles at 61 ° C, 2 cycles at 58 ° C, and 28 cycles at 55 ° C) and 1 minute extension per kilobase at 72 ° C). The PCR reaction is visualized on an agarose gel by electrophoresis. The obtained cDNA fragment can be used as a probe for Northern blot analysis and used for accurate determination of a polynucleotide sequence.
[0184]
In the case of Northern blot analysis, the cDNA probe prepared as described above was linked to the DNA labeling system High Prime (Boehringer) according to the manufacturer's instructions.³²Labeled with P. After labeling, the probe is purified using a Sephadex G50 microcolumn (Pharmacia) according to the manufacturer's instructions. The labeled purified probe is then used to detect mRNA expression in various tissues.
[0185]
Designate Northern blots (Northern or MTN of multiple tissues) including RNA samples of various human tissues, reference samples (human II, 7759-1, human 7760-1, and human fetal II 7756-1, Clontech) Hybridized with the labeled probe for specific ABCA7 (2637-4881 bp).
For the protocol after hybridization and washing, the protocol described by the manufacturer (manual instruction manual PT1200-1) may be directly used, or this protocol using a method known to those skilled in the art may be applied. F. Ausubel et al. (Currents Protocol in Molecular Biology, Green Publishing Associates and Wiley Interscience NY (1989)). Thus, for example, the prehybridization and hybridization temperatures can be varied in the presence of formamide.
[0186]
For example, the following protocol can be used:
1-membrane competition and pre-hybridization;
Mix 40 μl salmon sperm DNA (10 mg / ml) +40 μl human placental DNA (10 mg / ml),
Denature at 96 ° C. for 5 minutes, then immerse the mixture in ice,
Remove 2 × SSC and pour 4 ml of formamide mix into hybridization tube containing membrane,
Adding a mixture of the two denatured DNAs,
Incubate at 42 ° C. with rotation for 5-6 hours.
2-Competition of labeled probes:
Add 10 to 50 μl of CotI DNA to the labeled purified probe, depending on the amount of repetitive sequence.
Denature at 95 ° C for 7-10 minutes,
Incubate at 65 ° C for 2-5 hours.
[0187]
3-Hybridization:
Remove the prehybridization mix,
Mix 40 μl salmon sperm DNA + 40 μl human placenta DNA, denature at 96 ° C. for 5 minutes, then immerse on ice
To the hybridization tube, add 4 ml of the formamide mix, a mixture of the two DNAs and the denatured labeled probe / CotI DNA,
Incubate at 42 ° C with rotation for 15-20 hours.
4-wash:
Rinse by washing once with 2 × SSC at room temperature,
Wash twice with 2 × SSC and 0.1% SDS at room temperature for 5 minutes,
Wash twice with 0.1 × SSC and 0.1% SDS at 65 ° C. for 15 minutes.
After hybridization and washing, after overnight exposure, blots are analyzed by contact with phosphor screen, which was exposed with the aid of Storm (Molecular Dynamics, Sunnyvale, CA).
[0188]
The results shown in FIG. 5 indicate that the mouse ABCA7 gene was expressed in adult tissues.
Larger amounts of mouse ABCA7 mRNA were detected in hematopoietic tissues (eg, spleen and thymus), and the results are consistent with ABCA7 expression observed in myelomonocytic and lymphocytic systems. No ABCA7 gene expression was detected in the fibroblast cell line.
FIG. 4 shows the expression pattern of the human ABCA7 gene, which is similar but has a strong hybridization signal in fetal liver.
[0189]
Example 4: Analysis of gene expression characteristics related to dysfunction of lipid metabolism or dysfunction of inflammatory signaling
Verification of a defect in the expression level of the ABCA7 gene is performed by hybridizing these sequences with a probe corresponding to mRNA obtained from hematopoietic tissue of a diseased or unaffected subject, according to the method described below. Can be measured.
1. Total RNA, poly (A) ⁺ Preparation of mRNA and cDNA probes
Total RNA is obtained from hematopoietic tissue of normal or highly affected subjects by the guanidine isothiocyanate method (Chomczynski et al., Anal Biochem (1987) 162: 156-159).
Poly (A)⁺mRNA is obtained by affinity chromatography on an oligo (dT) -cellulose column (Sambrook et al., (1989) Molecular Cloning: a laboratory-manual. 2ed. Cold. Spring-Harbor Laboratory, Colorado, NY). The cDNA to be used as a probe is obtained by RT-PCR using an oligonucleotide labeled with a fluorescent product (CyDye: registered trademark of Amersham Pharmacia Biotech) (DeRisi et al., Science (1997)). 278: 680-686).
2. Hybridization and expression detection levels
A slide glass containing a sequence corresponding to the ABCA7 gene according to the present invention is hybridized with a nucleotide probe prepared from messenger RNA of a cell to be analyzed. The expression of the product of the sequence in healthy or diseased cell types can be differentially quantified by the use of Amersham's molecular mechanical system (Avalanche Microscanner®).
[0190]
Example 5: Test for Screening for a Molecule That Activates or Inhibits ABCA7 Gene Expression
Screening tests can identify sequences that can modulate the activity of synthesizing ABCA7 protein.
5.1 Construction of an expression plasmid containing a nucleic acid that regulates the human ABCA7 gene
The region of the nucleic acid that regulates the human ABCA7 gene, ranging from the nucleotide at position -1111 to the nucleotide at position -1 with respect to the transcription start site, was ligated to the human BAC by PCR using a primer pair specific to the above-mentioned region. It can be amplified from human genomic DNA present in the BAC vectors of the vector collection.
The amplified DNA fragment is digested with the restriction endonuclease Sal1, and then added to the vector PXP1 (BioTechniques, (1988) 6: 454-457) described by Nordeen et al. At the level of the Sal1 restriction site of this vector. insert. Next, the inserted portion was subjected to sequence analysis.
[0191]
5.2 Cell culture and transfection
CHO or HELA-based cells (ATCC, Rockville, MD, USA) were added to an E-MEM medium (Earle's salt-containing minimum medium containing 10% (v / v) fetal calf serum (BioWhittaker, Walkersville, MD)). ). About 1.5 × 10⁵Cells are distributed into each well of a 12-well culture plate (2.5 cm), cultured to a population of about 50-70%, 1 μg of plasmid Sal-Lucif and 0.5 μg of the control vector pBetagal (Clontech Laboratories) Co., Palo Alto, Calif., USA) using a Superfectin reagent kit (Qiagen, Valencia, Calif., USA). Two hours after addition of the DNA, the culture is removed and replaced with complete AMEM medium (Eagle's alpha modification of minimal essential medium). Twenty hours later, cells were supplemented with 2 μg / ml glutamine, 100 units / ml streptomycin and 0.1% bovine serum albumin (BSA, fraction V) in the presence or absence of various concentrations of the molecule. Replace with fresh medium of DMEM type (Dulbecco's minimum essential medium).
[0192]
Sixteen hours after the last medium change, cells are harvested using a lysis solution obtained from a Tropix luciferase analysis kit (Tropix, Bedford, Mass., USA). The cell lysate was partitioned into aliquot fractions, which were used to quantitate proteins using the MicroBCA kit (Pierce, Rockford, Ill., USA), which also produced luciferase and β-galactosidase. They are used for quantification using a Tropix luciferase analysis kit and a Galacto-Light Plus kit, respectively. Test according to manufacturer's recommendations. Next, a molecule that is active with respect to the ABCA7 promoter is selected according to the ratio of “luciferase activity / β-galactosidase activity”.
[0193]
Example 6: In situ hybridization experiment
Relevant tissue samples in paraffin were hybridized with a radiolabeled complementary RNA probe. More precisely, a fragment of the ABCA7 gene corresponding to the nucleotide sequence ranging from nucleotide 594 to nucleotide 1055 of the GenBank sequence designated NM-019112 was subcloned into plasmid pCRII (Invitrogen).
next,³⁵Antisense RNA probes labeled with S-uridine triphosphate were generated using RNA polymerases SP6 and T7 and then hybridized to various tissue sections.
[0194]
Various tissue sections were digested with proteinase K and approximately 3.5 × 10⁷Hybridization was carried out at 65 ° C. for 18 hours with the above-mentioned probe at a concentration equal to dpm / ml. The slides were then treated with RNAase A, washed in 0.1 × SSC at 70 ° C. for 2 hours, coated with Kodak NTB-2 photographic emulsion, exposed at 4 ° C. for 7 days, Visualization was performed using Kodak D-19 solution.
Finally, they were stained with hematoxylin and eosin (H & E) and the images were made using a DVC digital photo camera coupled to a Nikon microscope.
[0195]
FIG. 6 is a cross-sectional view of the artery of a 92-year-old man with a cut below the knee and suffering from arteriosclerosis and acute inflammation. Weak specific labeling of macrophages is seen at the site of inflammatory infiltration of thrombus and adventitia.
FIG. 7 is a cross-sectional view of the bronchi obtained at the autopsy of a 63-year-old asthmatic woman, showing weak labeling of lymphocytes and macrophages in submucosal inflammatory infiltrates.
FIG. 8 is a cross-sectional view of the colon obtained during surgery on an 81-year-old woman showing a clinical diagnosis of Crohn's disease. Labeling of the macrophage and lymphocyte subfamily in the lamina propria is observed.
FIG. 9 corresponds to a cross-sectional view of a lymph node obtained during surgery on a 48-year-old man. In reactive germinal centers, ganglion cells are weakly labeled, and isolated macrophages are also labeled in lymph nodes.
FIG. 10 shows a cross-sectional view of the synovium of a 25-year-old woman showing a clinical diagnosis of rheumatoid arthritis, showing strong labeling of subsynovial histiocytes and macrophages.
FIG. 11 shows a cross-sectional view of the skin obtained from a biopsy of a 55-year-old woman with psoriasis, showing moderate labeling of macrophages in inflammatory infiltration around blood vessels. Lymphocytes isolated around blood vessels are also labeled.
[0196]
[Table 1]

[0197]
[Table 2]

[0198]
[Table 3]

[0199]
[Table 4]

[0200]
[Table 5]

[0201]
[Table 6]

[0202]
[Table 7]

[0203]
[Table 8]

[0204]
[Table 9]

[0205]
[Table 10]

[0206]
[Table 11]

[0207]
[Table 12]

[0208]
[Table 13]

[0209]
[Table 14]

[0210]
[Table 15]

[0211]
[Table 16]

[0212]
[Table 17]

[0213]
[Table 18]

[0214]
[Table 19]

[0215]
[Table 20]

[0216]
[Table 21]

[0217]
[Table 22]

[0218]
[Table 23]

[0219]
[Table 24]

[0220]
[Table 25]

[0221]
[Table 26]

[0222]
[Table 27]

[0223]
[Table 28]

[0224]
[Table 29]

[0225]
[Table 30]

[0226]
[Table 31]

[0227]
[Table 32]

[Brief description of the drawings]
FIG.
1 is a schematic description showing transcription factor sites in the promoter region of the ABCA7 gene found in humans and mice.
FIG. 2A
The sequence of SEQ ID NO: 1 is shown, the position of each characteristic unit that binds to various transcription factors is shown in bold, and the symbolic designation of the corresponding sequence-specific transcription factor is shown above its nucleotide sequence.
FIG. 2B
It is a continuation of FIG. 2A.
FIG. 3A
The sequence of SEQ ID NO: 4 is shown, the position of each characteristic unit that binds to various transcription factors is shown in bold, and the symbolic designation of the corresponding sequence-specific transcription factor is shown above its nucleotide sequence.
FIG. 3B
It is a continuation of FIG. 3A.
FIG. 4
FIG. 3 shows the expression pattern of the human ABCA7 gene on Northern blots (Clontech) of various adult and fetal tissues hybridized with the amplimers generated using the primers of SEQ ID NOs: 9 and 10 (Table 4).
FIG. 5
FIG. 4 shows the expression pattern of the mouse ABCA7 gene on Northern blots of various adult tissues hybridized with amplimers generated using primers specific for mouse transcripts.
FIG. 6
The profile of the expression of the gene encoding the ABCA7 protein in a cross section of the inflamed artery is shown by in situ hybridization with an ABCA7 antisense probe.
FIG. 7
The profile of the expression of the gene encoding the ABCA7 protein in a cross section of a bronchial tube of an asthmatic patient is shown by in situ hybridization with an ABCA7 antisense probe.
FIG. 8
The profile of the expression of the gene encoding the ABCA7 protein in a cross section of the colon of a patient with Crohn's disease is shown by in situ hybridization with an ABCA7 antisense probe.
FIG. 9
The profile of the expression of the gene encoding the ABCA7 protein in a cross section of the lymph node is shown by in situ hybridization with an ABCA7 antisense probe.
FIG. 10
The profile of the expression of the gene encoding the ABCA7 protein in a cross-sectional view of the synovium of a patient with rheumatoid arthritis is shown by in situ hybridization with an ABCA7 antisense probe.
FIG. 11
A profile of the expression of the gene encoding the ABCA7 protein in a cross section of the skin of a patient with psoriasis is shown by in situ hybridization with an ABCA7 antisense probe.

Claims (27)

A nucleic acid comprising a polynucleotide having at least 20 consecutive nucleotides having a nucleotide sequence selected from SEQ ID NOs: 1 to 5, or a nucleic acid having a complementary sequence. A nucleic acid having at least 80% nucleotide identity with the nucleic acid of claim 1. A nucleic acid that hybridizes with the nucleic acid according to claim 1 or 2 under highly stringent hybridization conditions. The nucleic acid according to any one of claims 1 to 3, wherein the nucleic acid is capable of modulating the transcription of a polynucleotide placed under control. The nucleic acid of claim 4, comprising a polynucleotide ranging from nucleotide -1 to nucleotide -1111 relative to the transcribed first nucleotide, located at position 1112 of the nucleotide set forth in SEQ ID NO: 1. . 5. The nucleic acid of claim 4, wherein the nucleic acid is capable of activating transcription of a given polynucleotide under control. 5. The nucleic acid of claim 4, wherein the nucleic acid is capable of inhibiting transcription of a given polynucleotide under control. a) a nucleic acid according to any one of claims 1 to 7; and
b) a polynucleotide encoding a given polypeptide or nucleic acid,
A nucleic acid comprising: The nucleic acid according to claim 8, wherein the predetermined nucleic acid is a sense or antisense oligonucleotide. A recombinant cloning and / or expression vector comprising the nucleic acid according to any one of claims 1 to 9. A host cell transformed with the nucleic acid according to any one of claims 1 to 9 or the recombinant vector according to claim 10. A non-human transgenic mammal, wherein a somatic cell and / or a germ cell have been transformed with the nucleic acid according to any one of claims 1 to 9 or the recombinant vector according to claim 10. A method for screening a substance or molecule that modulates transcription of a constitutive polynucleotide of a nucleic acid according to claim 8, comprising the steps of:
a) culturing the transformed host cell of claim 11;
b) incubating the transformed host cells in the presence of the candidate substance or molecule;
c) detecting the expression of a given polynucleotide;
d) comparing the detection result obtained in step c) with the detection result obtained by culturing the transformed host cell in the absence of the candidate molecule or substance. The above method. A kit or box for in vitro screening for a candidate molecule or substance that modulates transcription of a predetermined polypeptide encoded by the constitutive polynucleotide of the nucleic acid according to claim 8,
a) a transformed host cell according to claim 11;
b) optionally, means necessary for detecting the transcription of a given constitutive polynucleotide of the nucleic acid according to claim 8,
The kit or box containing the above. A method for in vivo screening for a substance or molecule that modulates transcription of a predetermined constitutive polynucleotide of a nucleic acid according to claim 8, comprising the steps of:
a) administering a candidate substance or molecule to the non-human transgenic mammal according to claim 12;
b) detecting the expression of a given polynucleotide in the transgenic mammal treated in step a);
c) comparing the detection result of step b) with the result obtained in the non-human transgenic mammal according to claim 12, wherein no candidate substance or molecule has been administered;
The above method, comprising: A kit or box for in vivo screening for candidate molecules or substances that modulate the transcription of a given constitutive polynucleotide of the nucleic acid according to claim 8, wherein the kit or box comprises:
a) the non-human transgenic mammal of claim 12;
b) if necessary, means necessary for detecting the transcription of the predetermined polynucleotide,
The kit or box containing the above. A substance or molecule that modulates the transcription of a given constitutive polynucleotide of the nucleic acid of claim 8. 18. A substance or molecule according to claim 17, characterized in that it is selected according to the method of claim 13 or claim 15. A pharmaceutical composition comprising the substance or molecule according to claim 17 or 18 as an active ingredient. 20. Pharmaceutical composition according to claim 19, characterized in that it is intended for the treatment and / or prevention of deficiencies in lipid metabolism or deficiencies in mechanisms including the immune system and inflammation. 19. A substance or molecule according to claim 17 or 18 as an active ingredient of a medicament. Process:
a) extracting total messenger RNA from biological material obtained from the subject to be tested;
b) quantifying messenger RNA for ABCA7 present in the biological material;
c) comparing the amount of messenger RNA for ABCA7 obtained in step b) with the amount of messenger RNA for ABCA7 extracted from a normal subject;
A method for detecting ABCA7 gene transcription defect in a subject, comprising: Process:
a) sequence analysis of a polynucleotide located upstream of the transcription start site of the ABCA7 gene from biological material obtained from a test subject;
b) aligning the nucleotide sequence obtained in a) with SEQ ID NO: 1;
c) determining the various nucleotides between the sequenced polynucleotide obtained from the biological material of the subject to be tested and the reference SEQ ID NO: 1;
A method for detecting ABCA7 gene transcription defect in a subject, comprising: A kit or box for detecting a defect in ABCA7 gene transcription in a subject, comprising a means necessary for quantifying messenger RNA for ABCA7 in a biological material obtained from the subject to be tested. A kit or box for detecting a defect in ABCA7 gene transcription in a subject, comprising the means necessary to sequence a polynucleotide located upstream of the transcription start site of the ABCA7 gene of the subject to be tested. A method for screening a molecule or substance that modulates transcription of a predetermined constitutive polynucleotide of a nucleic acid according to claim 8, comprising the steps of:
a) incubating a nucleic acid according to any one of claims 1 to 9 or a recombinant vector according to claim 10 with a candidate molecule or substance to be tested;
b) detecting a complex formed between the candidate molecule or substance and the candidate molecule or substance;
The above method, comprising: A kit or box for screening a candidate molecule or substance that modulates transcription of a predetermined constitutive polynucleotide of the nucleic acid according to claim 8,
a) a nucleic acid according to any one of claims 1 to 9 or a recombinant vector according to claim 10;
b) if necessary, means necessary for detecting a complex formed between the candidate molecule or substance and the nucleic acid;
The kit or box containing the above.

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