# Patent

## Title
Regulatory nucleic acid for the ABC1 gene, molecules modifying its activity and therapeutic uses

## Abstract

The present invention concerns a nucleic acid which is capable of regulating the transcription of the ABC1 gene, which is a causal gene for pathologies linked to a dysfunctioning of cholesterol metabolism, inducing diseases such as atherosclerosis. The invention also relates to nucleotide constructs comprising a polynucleotide which encodes a polypeptide or a nucleic acid of interest, placed under the control a regulatory nucleic acid for the ABC1 gene. The invention also relates to recombinant vectors, transformed host cells and nonhuman transgenic mammals comprising a nucleic acid which regulates the transcription of the ABC1 gene or an abovementioned nucleotide construct, as well as methods for screening molecules or substances which are capable of modifying the activity of the regulatory nucleic acid for the ABC1 gene.

## Background
The instant application claims benefit of U.S. provisional application 60/201,280 filed 2 May 2000.


BACKGROUND
The present invention concerns a nucleic acid which is capable of regulating the transcription of the ABC1 gene, which is a causal gene for pathologies linked to a dysfunctioning of cholesterol metabolism, inducing diseases such as atherosclerosis.
The invention also relates to nucleotide constructs comprising a polynucleotide which encodes a polypeptide or a nucleic acid of interest, placed under the control of a regulatory nucleic acid for the ABC1 gene.
The invention also relates to recombinant vectors, transformed host cells and non-human transgenic mammals comprising a nucleic acid which regulates the transcription of the ABC1 gene or an above-mentioned nucleotide construct, as well as to methods for screening molecules or substances which are capable of modifying the activity of the regulatory nucleic acid for the ABC1 gene.
The invention also relates to methods for detecting an impairment in the transcription of the ABC1 gene in an at-risk individual.
A subject of the invention is also substances or molecules which modify the activity of the nucleic acid which regulates the transcription of the ABC1 gene, as well as pharmaceutical compositions containing such substances or such molecules.
High density lipoproteins (HDLs) are one of the four major classes of lipoprotein which circulate in the blood plasma.
These lipoproteins are involved in various metabolic pathways, such as lipid transport, bile acid formation, steroidogenesis or cell proliferation, and also interfere with plasmatic proteinase systems.
HDLs are perfect free cholesterol acceptors, and in combination with cholesterol ester transfer proteins (CETP), lipoprotein lipase (LPL), hepatic lipase (HL) and lecithin-cholesterol acyltransferase (LCAT), play a major role in the reverse transport of cholesterol, i.e. the transport of excess cholesterol in the peripheral cells to the liver, for its removal from the body in the form of bile acid.
It has been demonstrated that the HDLs generally play a central role in the transport of cholesterol from the peripheral tissues to the liver.
Various diseases linked to an HDL deficiency have been described, including Tangier disease, HDL deficiency and LCAT deficiency.
The deficiency involved in Tangier disease is linked to a cellular defect in the translocation of cellular cholesterol, which leads to a degradation of the HDLs.
In Tangier disease, this cellular defect leads to a disruption of lipoprotein metabolism. The HDL particles in Tangier disease, which do not incorporate cholesterol from the peripheral cells, and which are not able to be correctly metabolized, are rapidly eliminated from the body. The plasma HDL concentration in these patients is thus extremely reduced, and the HDLs no longer contribute to the return of cholesterol to the liver. This cholesterol accumulates in these peripheral cells and causes characteristic clinical manifestations such as the formation of orange-colored tonsils. Furthermore, other lipoprotein disruptions such as an overproduction of triglycerides and an increased synthesis and intracellular catabolism of phospholipids are generally observed.
Tangier disease, the symptoms of which have been described above, is classified among the familial conditions linked to metabolism of the HDLs which are commonly detected in patients affected by coronary diseases.
Numerous studies have shown that a reduced level of HDL cholesterol is a risk factor which is useful for detecting a coronary condition.
In this context, syndromes linked to HDL deficiencies have been of increasing interest for the past decade, since they make it possible to increase the understanding of the role of HDLs in atherogenesis.
Several mutations in the apo A-1 gene have been characterized. These mutations are rare and can lead to an absence of production of apo A-1.
Mutation in the genes encoding lipoprotein lipase (LPL) or its activator apoC-II are associated with severe hypertriglyceridemias and substantially reduced levels of HDL-c.
Mutations in the gene encoding the enzyme lecithin-cholesterol acyltransferase (LCAT) are also associated with severe HDL deficiency.
Furthermore, dysfunctions in the reverse transport of cholesterol might be induced by physiological deficiencies affecting at least one of the steps for transporting stored cholesterol from the intracellular vesicles toward the membrane surface, where it is accepted by the HDLs.
Recently, a study was carried out on the segregation of various allelic forms of 343 microsatellite markers distributed over the entire genome and distant from each other by 10.3 cM on average.
The linkage study was carried out on a family which had been well characterized over eleven generations, in which many members are affected by Tangier disease, the family comprising five consanguineous lines.
This study made it possible to identify a region located in the 9q31 locus of human chromosome 9 which is statistically linked to the condition (Rust S. et al., Nature Genetics Vol. 20, September 1998, pages 96-98).
However, the study by Rust et al. only characterizes a wide region of the genome in which impairments are likely to be associated with Tangier disease. The study simply stated that the relevant 9q31-34 region contains ESTs, but no known gene.
It has been shown that a region spanning 1 cM, situated in the 9q31 locus in humans, is generally associated with familial HDL deficiencies (Rust et al., 1999).
Furthermore, it has been shown that a gene encoding a protein of the family of ABC transporters, which is located precisely in the 1 cM region of the 9q31 locus, is involved in pathologies linked to a deficiency in the reverse transport of cholesterol.
For example, it has been shown that the gene encoding the ABC-1 transporter is mutated in patients with affected reverse transport of cholesterol, such as in patients suffering from Tangier disease.
The ABC (“ATP-binding cassette”) transporter proteins constitute a family of proteins which are extremely conserved in evolution, from bacteria to humans.
The ABC transporter proteins are involved in the membrane transport of various substrates, for example, ions, amino acids, peptides, sugars, vitamins or steroid hormones.
The characterization of the complete amino acid sequence of some ABC transporters has made it possible to determine that these proteins have a common general structure, for example, two nucleotide-binding folds (Nucleotide Binding Fold or NBF) with moieties of Walker A type and Walker B type, as well as two transmembrane domains, each of the transmembrane domains consisting of six helices. The specificity of the ABC transporters for the various transported molecules appears to be determined by the structure of the transmembrane domains, whereas the energy required for the transport activity is provided by degrading ATP at the NBF fold.
Several of the ABC transporter proteins which have been identified in humans have been associated with various diseases.
For example, cystic fibrosis is caused by mutations in the CFTLR (cystic fibrosis transmembrane conductance regulator) gene.
Moreover, some multi-drug resistance phenotypes in tumor cells have been associated with mutations in the gene encoding the MDR (multi-drug resistance) protein which also has an ABC transporter structure.
Other ABC transporters have been associated with neuronal and tumor conditions (U.S. Pat. No. 5,858,719), or potentially implicated in diseases caused by an impairment of the homeostasis of metals, for example, the ABC-3 protein.
Similarly, another ABC transporter, referred to as PFIC2, seems to be involved in a form of progressive familial intrahepatic cholestasis, this protein being potentially responsible, in humans, for the export of bile salts.
In 1994, a cDNA encoding a novel mouse ABC transporter was identified and referred to as ABC1 (Luciani et al., 1994). This protein is characteristic of the ABC transporters in that it has a symmetrical structure comprising two transmembrane domains linked to a highly hydrophobic segment and to two NBF moieties.
In humans, a partial cDNA comprising the entire open reading frame of the human ABC1 transporter has been identified (Lanigmann et al., 1999).
It has also been shown that the gene encoding the human ABC1 protein is expressed in various tissues, and more particularly at high levels in the placenta, the liver, the lungs, the adrenal glands and the fetal tissues.
These authors have also shown that the expression of the gene encoding the human ABC1 protein is induced during the differentiation of monocytes into macrophages in vitro. Furthermore, the expression of the gene encoding the ABC1 protein is increased when human macrophages are incubated in the presence of acetylated low-density lipoproteins (AcLDLs).
The work of Rust S. et al., 1999, Brooks-Wilson A. et al., 1999, Bodzioch M. et al., 1999, Remaley A. et al., 1999 and of Marcil M. et al., 1999 has shown that patients suffering from Tangier disease and from HDL deficiencies (FHD; familial HDL deficiency) have a mutated ABC1 gene. Several mutations, which are distributed in various regions of the ABC1 gene, have been identified in the genome of various patients, for example, of patients with a severe form of the disease associated with coronary disorders. Moreover, diverse polymorphisms have been found, both in the exons and in the introns of the ABC1 gene, in patients suffering from milder forms of the disease, indicating that these patients carry specific alleles of the gene, which are distinct from the “wild-type” allele(s).
Although the expression of the human ABC1 gene seems to be regulated according to the type of cell or to the metabolic situation of a given cell type, the sequence(s) which make(s) it possible to regulate this gene were not known.
Thus, there exists a need in the state of the art to identify these regulatory sequences, for the two principal reasons below:
a) These sequences are likely to be mutated in patients suffering from a pathology linked to a deficiency in cholesterol transport, for example, in patients suffering from Tangier disease, or likely to develop such pathologies.
The characterization of the regulatory sequences of the human ABC1 gene would make it possible, firstly, to detect mutations in patients, and, for example, also to diagnose the individuals who belong to at-risk familial groups. In addition, the isolation of these regulatory sequences would make it possible to complement the mutated sequence with a functional sequence capable of overcoming the metabolic dysfunctions induced by the mutation(s) diagnosed, through the construction of targeted therapeutic means, such as means intended for gene therapy.
b) The characterization of the regulatory sequences of the ABC1 gene would place at the disposal of persons skilled in the art means capable of allowing the construction, by genetic engineering, and then the expression, of given genes in the cell types in which the ABC1 gene is expressed.
c) Moreover, some portions of the regulatory sequences of the ABC1 gene might constitute high expression-level constitutive promoter sequences, which are liable to enable the construction of novel means for expressing given sequences in cells, completing an already existing set of means.
To date, despite the efforts undertaken, the regulatory sequences of the ABC1 gene have remained totally unknown.
The inventors have henceforth isolated and then sequenced a genomic DNA comprising the first two exons of the ABC1 gene (respectively exon 1A and exon 1B), as well as a non-transcribed region of approximately 2.9 kb, which is located on the 5′ side of exon 1A, and which comprises regulation signals for the ABC1 gene.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1: illustrates a portion of the sequence SEQ ID No. 3, which starts with the nucleotide at position 1 of the sequence SEQ ID No. 3. The position of each of the characteristic binding moieties for various transcription factors is represented by boxes, the designation of the transcription vector specific for the corresponding sequence being indicated above the nucleotide sequence.
FIG. 2A: is a schematic illustration that shows the location of the point mutations introduced into the −200 bp promoter region of the human ABC1 gene;
FIG. 2B: displays the luciferase activity in RAW cells after transfection with wildtype and mutant constructs. The data shown represents the mean of 3 independent transfection studies. Values are expressed relative to the wildtype construct;
FIG. 3: RAW 264.7 cells were transfected with wild type construct (p200-L) or mutant LXR construct (p200-LXRm) along with a β-galactosidase expression plasmid. Three hours after transfection, cells were refed with fresh media containing 10% FCS. Sixteen hours later, cells were washed with PBS and replaced with DMEM media containing 0.1% BSA and added 50 μg/ml cholesterol, 2 μg/ml 22(R)-HOch, 10 μM 9CRA or, 2 μg/ml 22(R)-HOch plus 10 μM 9CRA for 24 hours. Cell lysates were analyzed for luciferase and β-galactosidase activity. Luciferase values were normalized to β-galactosidase and expressed as mean±SEM.
FIG. 4: RAW 264.7 cells were transfected with wild type construct (p200-L), mutant E-box construct (p200-EBm) or the deleted E-box construct (p200-EBd) along with a β-galactosidase expression plasmid. Addition of 50 μg/ml cholesterol, 2 μg/ml 22(R)-HOch, 10 μM 9CRA or, 2 μg/ml 22(R)-HOch plus 10 μM 9CRA were performed. Cell lysates were analyzed for luciferase and β-galactosidase activity. Luciferase values were normalized to β-galactosidase activity and expressed as mean±SEM.
FIG. 5: A radiolabelled fragment (−171 to −77 bp) of the human ABC1 promoter was digested with different concentrations of DNaseI in the presence (+) or absence (−) of RAW cell nuclear extract (NE). The G and G+A ladders from Maxam and Gilbert sequence of the radiolabelled fragment are shown. The nucleotide position relative to the transcriptional start site and location of the Sp1, AP1 and EB motifs in the hABC1 promoter are indicated on the right;
FIG. 6A: illustrates the probes used for the gel shift analysis. Fragment A (100 bp) includes binding motifs for Sp1 and AP1 and the E-box. Fragment EB (27 bp) contains the E-box and fragment EBm (not shown) contains a mutated E-box. In panels B and C, the labeled fragments (fragments A, EB or EBm) used for the gel shift study are shown on top of each gel. Incubation of the radiolabelled probe with RAW cell nuclear extract (NE) is indicated (+);
FIG. 6B: shows the gel-shift analysis performed by incubating RAW cell nuclear extracts with radiolabelled Fragment A (left), EB (middle) or EBm (right) in the presence or absence of specific competitors (unlabelled fragment A, EB or EBm);
FIG. 6C: shows supershift analysis of Fragments A or EB with antibodies specific to the amino (N) or carboxyl (C) ends of USF1 and USF2. Arrows indicate the position of probe complexed with protein and arrows with an asterisk indicates the position of antibody-supershifted complex.



DETAILED DESCRIPTION OF THE INVENTION
General Definitions
For the purpose of the present invention, the term “isolated” refers to a biological material (nucleic acid or protein) which has been abstracted from its environment of origin (the environment in which it is naturally located).
For example, a polynucleotide present in the natural state in a plant or an animal is not isolated. The same polynucleotide separated from the adjacent nucleic acids among which it is naturally inserted in the genome of the plant or the animal is considered to be “isolated”.
Such a polynucleotide can be included in a vector and/or such a polynucleotide can be included in a composition, and can remain, however, in the isolated state, because the vector or the composition does not constitute its natural environment.
The term “purified” does not require the material to be present in a form of absolute purity, excluding the presence of other compounds. It is, rather, a relative definition.
A polynucleotide is in the “purified” state after purification of the starting material, or after purification of the natural material, wherein impurities are present after the purification in an amount of at least one order of magnitude less than the amount of impurities before the purification. In one embodiment, impurities are present after the purification in an amount of 2 or 3 orders of magnitude less than the amount of impurities before the purification. In another embodiment, impurities are present after the purification in an amount of 4 or 5 orders of magnitude less than the amount of impurities before the purification.
For the purposes of the present description, the expression “nucleotide sequence” can be used to refer indiscriminately to a polynucleotide or a nucleic acid. The expression “nucleotide sequence” encompasses the genetic material itself, and is thus not restricted to the information concerning its sequence.
The terms “nucleic acid”, “polynucleotide”, “oligonucleotide” and “nucleotide sequence” encompass RNA, DNA or cDNA sequences, or DNA/RNA hybrid sequences of more than one nucleotide, indiscriminately in the single chain form or in the duplex form.
The term “nucleotide” refers to both natural nucleotides (A, T, G, C) and modified nucleotides, which comprise at least one modification such as (1) a purine analog, (2) a pyrimidine analog or (3) a similar sugar, examples of such modified nucleotides being described for example, in PCT application No. WO 95/04 064.
For the purposes of the present invention, a first polynucleotide is considered as being “complementary” to a second polynucleotide when each base of the first nucleotide is paired with the complementary base of the second polynucleotide, the orientation of which is inversed. The complementary bases are A and T (or A and U), or C and G.
“Variant” of a nucleic acid according to the invention will be intended to mean a nucleic acid which differs by one or more bases with respect to the reference polynucleotide. A variant nucleic acid can be of natural origin, such as an allelic variant found naturally, or can also be an unnatural variant obtained for example, by mutagenesis techniques.
In general, the differences between the reference nucleic acid and the variant nucleic acid are small, such that the nucleotide sequences of the reference nucleic acid and of the variant nucleic acid are very close and, in many regions, identical. The nucleotide modifications present in a variant nucleic acid can be silent, which means that they do not alter the amino acid sequences encoded by said variant nucleic acid.
However, the nucleotide changes in a variant nucleic acid can also result in substitutions, additions or deletions in the polypeptide encoded by the variant nucleic acid, with respect to the peptides encoded by the reference nucleic acid. In addition, nucleotide modifications in the coding regions can produce substitutions, which may be conservative or non-conservative in the amino acid sequence.
For example, the variant nucleic acids according to the invention encode polypeptides which conserve more or less the same function or biological activity as the polypeptide of the reference nucleic acid, or the capacity to be recognized by antibodies directed against the polypeptides encoded by the initial nucleic acid.
Some variant nucleic acids will thus encode mutated forms of the polypeptides, the systematic study of which will make it possible to deduce structure-activity relationships for the proteins in question. Knowledge of these variants with respect to the disease studied is fundamental, since it makes it possible to understand the molecular cause of the pathology.
A “fragment” of a reference nucleic acid according to the invention, will be intended to mean a nucleotide sequence which is shorter in length than the nucleotide sequence of the reference nucleic acid and which comprises a nucleotide sequence which is identical to a portion of the nucleotide sequence of the reference nucleic acid. Such a “fragment” of nucleic acid according to the invention can be, if needed, included in the nucleotide sequence of a second polynucleotide different from the reference nucleic acid. The resulting nucleotide which comprises the “fragment” and the second polynucleotide may have a nucleotide sequence that is longer than, the same length as, or shorter than the nucleotide sequence of the reference nucleic acid. Such fragments comprise, or alternatively consist of, oligonucleotides of lengths ranging from 20 to 25, 30, 40, 50, 70, 80, 100, 200, 500, 1000 or 1500 consecutive nucleotides of a nucleic acid according to the invention.
“Biologically active fragment” of an acid which regulates transcription according to the invention is intended to mean a nucleic acid which is capable of modifying the transcription of a sequence of DNA placed under its control. Such a biologically active fragment comprises a basic promoter and/or a regulatory element, as defined in the present description.
“Regulatory nucleic acid” according to the invention is intended to mean a nucleic acid which activates and/or regulates the expression of a DNA sequence which is selected and placed under its control.
“Promoter” is intended to mean a DNA sequence recognized by the proteins of the cell which are involved in initiating the transcription of a gene. The basic promoter is the minimum regulatory nucleic acid which is capable of initiating the transcription of a given DNA sequence which is placed under its control. In general, the basic promoter consists a region of genomic DNA upstream of the transcription start site, where a sequence CAAT (to which one or more protein transcription factors bind), as well as, except in rare cases such as in certain housekeeping genes, the sequence TATA, or “TATA box”, or a related box, are very often found. An RNA polymerase, as well as one or more transcription factors, such as “TATA” box binding proteins (or TBPs), bind to this box.
A nucleotide sequence is “placed under the control” of a regulatory nucleic acid when this regulatory nucleic acid is located, with respect to the nucleotide sequence, in such a way as to control the initiation of the transcription of the nucleotide sequence with an RNA polymerase.
For the purpose of the invention, “regulatory element” and “regulatory sequence” are intended to mean a nucleic acid comprising elements capable of modifying the transcription initiated by a basic promoter, such as binding sites for diverse transcription factors, “enhancer” sequences for increasing transcription or “silencer” sequences for inhibiting transcription.
“Enhancer” sequence is intended to mean a DNA sequence included in a regulatory nucleic acid, which is capable of increasing or of stimulating the transcription initiated by a basic promoter.
“Silencer” sequence is intended to mean a DNA sequence included in a regulatory acid, which is capable of decreasing or of inhibiting the transcription initiated by a basic a promoter.
Regulatory elements can be present outside the sequence which is located on the 5′ side of the transcription start site, for example, in the introns and the exons, including in the coding sequences.
The terms “basic promoter” and “regulatory element” can be “specific for one or more tissues” if they allow the transcription of a given DNA sequence, placed under their control, such as in certain cells (for example, the cells specific for a tissue), i.e. either exclusively in the cells of certain tissues, or at different levels of transcription according to the tissues.
“Transcription factor” is intended to mean proteins which preferentially interact with regulatory elements of a regulatory nucleic acid according to the invention, and which stimulate or, on the contrary, suppress transcription. Some transcription factors are active in the form of monomers, others being active in the form of homo- or heterodimers.
The term “modification” is directed toward either a positive regulation (increase, stimulation) of transcription, or a negative regulation (decrease, inhibition, blocking) of transcription.
For the purpose of the present invention, the “percentage of identity” between two sequences of nucleotides or amino acids can be determined by comparing two optimally aligned sequences, through a comparison window.
The portion of the nucleotide sequence or polypeptide which is in the comparison window can thus comprise additions or deletions (for example, “gaps”), with respect to the reference sequence (which does not comprises these additions or these deletions), in such a way as to obtain an optimal alignment of the two sequences.
The percentage is calculated by determining the number of positions at which an identical nucleic base or amino acid residue is observed for the two sequences (nucleic or peptide) compared, then by dividing the number of positions at which there is identity between the two bases or amino acid residues by the total number of positions in the comparison window, and then by multiplying the result by 100 to obtain the percentage of sequence identity.
The optimal sequence alignment for the comparison can be carried out using a computer with the aid of known algorithms in the package from the company Wisconsin Genetics Software Package, Genetics Computer Groups (GCG), 575 Science Doctor, Madison, Wis.
By way of illustration, it will be possible to produce the percentage of sequence identity with the aid of the BLAST software (Versions BLAST 1.4.9 of March 1996, BLAST 2.0.4 of February 1998 and BLAST 2.0.6 of September 1998), using exclusively the default parameters (S. F. Altschul et al., J. Mol. Biol. 1990 215: 403-410, S. F. Altschul et al., Nucleic Acids Res. 1997 25: 3389-3402). Blast searches for sequences which are similar/homologous to a “request” sequence of reference, with the aid of the algorithm of Altschul et al. The request sequence and the databases used can be of peptide or nucleic type, any combination being possible.
For the purposes of the present invention, “high stringency hybridization conditions” are intended to mean the following conditions:
1—Membrane Competition and Prehybridization:


    
    
        Mixed: 40 μ1 salmon sperm DNA (10 mg/ml)
        
            +40 μl human placental DNA (10 mg/ml)
        
        
        Denatured for 5 min at 96° C., then immersed the mixture in ice.
        Removed the 2×SSC buffer and poured 4 ml of formamide mix into the hybridization tube containing the membranes.
        Added the mixture of the two denatured DNAs.
        Incubation at 42° C. for 5 to 6 hours, with rotation.

2—Labeled Probe Competition:

        Added 10 to 50 μl Cot I DNA to the labeled and purified probe, according to the amount of nonspecific hybridization.
        Denatured for 7 to 10 min at 95° C.
        Incubated at 65° C. for 2 to 5 hours.

3—Hybridization:

        Removed the prehybridization mix.
        Mixed 40 μ1 salmon sperm DNA+40 μl human placental DNA; denatured 5 min at 96° C., then immersed in ice.
        Added 4 ml of formamide mix, the mixture of the two DNAs and the denatured labeled probe/Cot I DNA to the hybridization tube.
        Incubated for 15 to 20 hours at 42° C., with rotation.

4—Washes:

        One wash at room temperature in 2×SSC, to rinse.
        Twice 5 minutes at room temperature, 2×SSC and 0.1% SDS, at 65° C.
        Twice 15 minutes at 65° C., 1×SSC and 0.1% SD)S, at 65° C.

Wrapped the membranes in Saranwrap and exposed

    
    


The hybridization conditions described above are adapted to hybridization, under high stringency conditions, of a molecule of nucleic acid of variable length, from 20 nucleotides to several hundred nucleotides.
The hybridization conditions described above can be adapted as a function of the length of the nucleic acid whose hybridization is desired, or of the type of labeling chosen, according to techniques known to persons skilled in the art.
Suitable hybridization conditions may for example, be adapted according to the teaching contained in the work by Hames and Higgins (1985) or in the work by F. Ausubel et al. (1999).
For the purposes of the present invention, “transformation” is intended to mean the introduction of a nucleic acid (or of a recombinant vector) into a host cell. The term “transformation also encompasses a situation in which the genotype of a cell has been modified by an exogenous nucleic acid, and in which this cell thus transformed expresses said exogenous nucleic acid, for example, in the form of a recombinant polypeptide or in the form of a sense or antisense nucleic acid.
For the purposes of the invention, “transgenic animal” is intended to mean a nonhuman animal, such as a mammal, in which one or more cells contain a heterologous nucleic acid which has been introduced through human intervention, such as by transgenesis techniques well known to persons skilled in the art. The heterologous nucleic acid is introduced directly or indirectly into the cell or the precursor of the cell, by genetic manipulation such as microinjection or infection with a recombinant virus. The heterologous nucleic acid can be integrated into the chromosome, or can be in the form of DNA which replicates extrachromosomally.
Regulatory Nucleic Acid for the ABC1 Gene
The inventors have succeeded in isolating a regulatory nucleic acid for the human ABC1 gene from vector libraries of BAC type prepared from human genomic material.
According to the sequence analysis carried out, the inventors have determined that the nucleic acid which regulates the transcription of the ABC1 gene, when it is defined in the broadest way, consists of a polynucleotide comprising, from the 5′ end toward the 3′ end:

    
    
        a nontranscribed region of approximately 2.9 kb located upstream of the transcription start site of the ABC1 gene;
        the nucleotide sequence of the first exon of the ABC1 gene, also referred to as exon 1A;
        the partial nucleotide sequence of the first intron of the ABC1 gene, also referred to under the name intron 1A; and
        the nucleotide sequence of the second exon of the human ABC1 gene, also referred to as exon 1B.
        the partial nucleotide sequence of the second intron of the ABC1 gene, also referred under the name intron 1B;
    
    


Under a general definition, the nucleic acid which regulates the transcription of the ABC1 gene comprises all the nucleotide regions as defined above, and is identified as the sequence SEQ ID No. 1 according to the invention.
Preferentially, the nucleic acid which regulates the transcription factor of the ABC1 gene comprises all the nucleotide regions comprising nucleotide −2228 to nucleotide +108 with respect of the transcription start site of the ABC1 gene, i.e., a region comprised in nucleotides 654 to 3001 of sequence SEQ ID NO: 1.
Thus, a first subject of the invention consists of a nucleic acid comprising a polynucleotide which has at least 20 consecutive nucleotides of the nucleotide sequence SEQ ID No. 1, or a nucleic acid of complementary sequence.
The region of approximately 2.9 kb, which is located upstream of the transcription start site of the ABC1 gene, and which comprises the basic promoter and multiple regulatory elements for transcription, is also included in the sequence identified as SEQ ID No. 3 according to the invention.
More precisely, the nucleotide in position 1 of the sequence SEQ ID No. 3 is the nucleotide in position −2893, with respect to the transcription start site of the ABC1 gene.
According to a second aspect, the invention relates to a nucleic acid comprising a polynucleotide which has at least 20 consecutive nucleotides of the nucleotide sequence SEQ ID No. 3, or to a nucleic acid of complementary sequence.
As already specified above, in addition to a 5′ non transcribed regulatory region, the nucleic acid which regulates the transcription of the ABC1 gene, of sequence SEQ ID No. 1, also comprises the first exon and the 5′ portion of the first intron of the human ABC1 gene.
The first exon of the ABC1 gene, also referred to as exon 1A, is defined as the sequence SEQ ID No. 4.
The sequence of the intron 1a has been partially characterized. The 5′ end of intron 1a is defined as the nucleotide sequence SEQ ID No. 6. The 3′ end of intron 1a is defined as the sequence SEQ ID No. 7.
The second exon of the human ABC1 gene, also referred to as exon 1B, is defined as the sequence SEQ ID No. 5.
According to a third aspect, the invention relates to a nucleic acid comprising a polynucleotide which has at least 20 consecutive nucleotides of a nucleotide sequence chosen from the sequences SEQ ID No. 3 to 7, or to a nucleic acid of complementary sequence.
In some embodiments of the invention, a nucleic acid according to the invention will be in an isolated and/or purified form.
Any “biologically active” fragment of a nucleic acid as defined above also forms part of the invention.
According to another aspect, the invention concerns a nucleic acid having at least 80% nucleotide identity with a nucleic acid as defined above.
The invention also encompasses a nucleic acid that hybridizes, under high stringency conditions, with any one of the nucleic acids according to the invention.
The invention also concerns a nucleic acid having at least 80%, for example, 90%, 95% or 98%, nucleotide identity with a nucleic acid comprising at least 20 consecutive nucleotides of a polynucleotide chosen from nucleotide sequences SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8.
Detailed Analysis of the Sequence SEQ ID NO. 3
The nucleic acid of sequence SEQ ID No. 3, included in the regulatory nucleic acid for the ABC1 gene of sequence SEQ ID No. 1, comprises the constituent elements of a basic promoter, respectively a “TATA” box and a homeobox, represented in FIG. 1.
The regulatory sequence SEQ ID No. 3 also comprises many binding sites for diverse transcription factors which are capable of positively or negatively regulating the activity of the basic promoter.
Thus, the various sequences which are characteristic of the binding sites for diverse transcription factors in the sequence SEQ ID No. 3 have been identified by the inventors, in the manner detailed below.
The sequence SEQ ID No. 3 was used as a reference sequence and processed according to the algorithms of the BLAST 2, version 2.10, software, and compared to the data listed in several databases, and the presence, as well as the location, of the various characteristic sites of the sequence SEQ ID No. 3, and, for example, the binding sites for transcription factors, were determined according to methods well known to persons skilled in the art.
Furthermore, a detailed analysis was carried out on the 1.3 kb upstream of the start site, in which a total of 1900 sequences corresponding to binding sites for transcription factors was identified during the first step of the search. After compilation and filtering as described above, only 79 binding sites, specific for 27 different transcription factors, were retained. These sites are presented in Table 1 below.
Table 1 represents the binding sites for the transcription factors identified in the 1318 nt in the 3′ portion of the sequence SEQ ID No. 3 according to the invention.
The positions of the start and end nucleotildes of each of the binding sites for transcription factors are referred with reference to the numbering of the nucleotides of the sequence SEQ ID No. 3, as represented in FIG. 1 (+strand) or of the nucleotides on the complementary sequence of the sequence SEQ ID No. 3 (−strand).












TABLE 1







Transcription factor
Start
End
Strand





















RAR
−1299
−1294
+



TFIID
−1251
−1246
+



AP2
−1203
−1192
+



MYB
−1177
−1170
+



GR
−1166
−1153
+



T3R
−1110
−1095
−



AP1 (C-JUN)
−1052
−1046
+



CEBP (CEBPA CEBPB)
−1015
−1008
+



SP1
−992
−986
+



HNF (HNF3, HNF5)
−951
−940
−



MYB
−941
−934
+



T3R
−933
−928
+



CP2
−924
−918
−



TFID
−869
−864
+



HNF (HNF3, HNF5)
−865
−854
−



GATA1 (NF-E1A)
−844
−835
−



GATA1 (NF-E1A)
−829
−820
+−



SP1
−803
−794
+



GR
−788
−775
+



HNF (HNF3, HNF5)
−782
−750
+



AP1 (C-JUN)
−731
−725
+−



PEA3
−731
−726
+−



PU.1 (NF-JB)
−730
−725
−



PU.1 (NF-JB)
−696
−691
−



AP2
−675
−665
−



AP2
−671
−666
+



H-APF-1
−658
−652
+



NE-kappaB
−658
−647
+



GATA1 (NF-E1A)
−631
−626
+−



SP1
−565
−559
−



CAC-bf (htbeta)
−557
−547
+



SP1
−551
−542
−



TFIID
−537
−532
−



NF-E (NF-E1C)
−509
−505
−



CCAT-bf (CBF)
−508
−502
+−



GATA1 (NF-E1A)
−508
−504
+



CTF/CBP (NF1)
−507
−502
+



CP2
−506
−502
−



NF-Y
−506
−502
+



CCAAT-bf (CBF)
−464
−458
+−



CTF/CBP (NF1)
−464
−459
−



NF-Y
−464
−460
−



SP1
−462
−453
−



SP1
−459
−453
+



AP2
−458
−447
+



PU.1 (NF-JB)
−445
−440
+



PEA3
−444
−439
+−



SRY
−439
−428
+



PEA3
−431
−426
+−



CEBP (CEBPA CEBPB)
−420
−412
−



PU.1 (NF-JB)
−400
−395
+



PEA3
−399
−394
+−



AP2
−305
−294
−



SP1
−302
−287
+



AP2
−291
−282
−



CEBP (CEBPA CEBPB)
−272
−261
+



PEBP2
−272
−267
+



MYOD
−257
−250
+



E2A
−255
−249
−



SP1
−244
−235
−



CAC-bf (htbeta)
−240
−211
+−



SP1
−238
−223
+−



GATA1 (NF-E1A)
−225
−219
+



SP1
−225
−215
+



SP1
−221
−209
−



MYB
−211
−204
+



GR
−197
−192
−



SP1
−189
−183
+−



MYB
−174
−167
−



SP1
−166
−152
+−



MYC/MAX
−151
−138
+−



USF 1 and 2
−147
−142
+



(MYOD) (E-box)



AP1 (C-JUN)
−131
−121
+



SP1
−100
−86
+−



CEBP (CEBPA CEBPB)
−90
−83
+



GR
−80
−67
−



LXR
−69
−55
+



TFIID (TBP TATA BOX)
−31
−26
+



SIF
−3
2
+



SP1
22
31
+










FIG. 1 represents a portion of the sequence SEQ ID No. 3. The first nucleotide in the 5′ position of the sequence in FIG. 1 is also the first nucleotide in the 5′ position of one or the nucleic acid sequences SEQ ID No. 1 and SEQ ID No. 3. In the figure, the binding sites for transcription factors are illustrated with boxes which delimit their respective start and end positions, and their respective designations is indicated above each of the corresponding boxes. The numbering of the nucleotides of the sequence represented in FIG. 1 was carried out with respect to the transcription start site, numbered “+1”, the nucleotide 5′ to the nucleotide +1 being itself numbered “−1”.
The description of the characteristics of the binding sites for each of the transcription factors referred to in FIG. 1 and Table 1 can easily be found by persons skilled in the art. A short description of some of them is produced below.
Factor CAC: The characteristics of a binding site for the factor CAC can be found, for example, in the article by Schuele et al., (1988, Nature, Vol. 332: 87-90), entry No. T00077 of the EMBL database, the article by Mantovani et al., (1988, Nucleic Acids Research Vol. 16: 4299-4313), the article by Catala et al., (1989, Nucleic Acid Research, Vol. 17: 3811-3827) and the article by Wang et al., (1993, Mol. Cell Biol., Vol. 13: 5691-5701). The binding of this factor has been shown on the regulatory regions of several genes, including the promoter for the β-globin gene and the gamma-globin gene. This factor appears to act in cooperation with the glucocorticoid receptor.
Factor C/EBP:
C/EBP-α
The characteristics of a binding site for the factor C/EPB-α can be found, for example, in the articles corresponding to the following entries in the Medline database: 892020040, 94023981, 96194262, 96003748. This factor inhibits cell proliferation by increasing the level of p21 (WAF-1) due to an increased expression of the gene and to a posttranslational stabilization of p21.
C/EBP-β
The binding characteristics for the factor C/EBP β can be found, for example, in the following entries in the Medline database: 93315489, 91248826, 94193722, 93211931, 92390404, 90258863, 94088523, 90269225 and 96133958. It is a transcription activator which is involved in regulating genes involved in immune and inflammatory responses. It binds specifically to an IL-1 response element in the IL-6 gene. It is believed to play a role in regulating the acute phase of inflammation and in hemopoiesis. The consensus recognition site is as follows: “T(T/G) NNGNAA(T/G)”.
Factor c-Myb:
The characteristics of a binding site for the factor c-Myb can be found in the following entries in the Medline database: 91122626, 87092302, 93131991, 86261774, 92049347, 90044066, 90265605, 93101590, 94316485 and 90090611. This factor specifically recognizes the sequence “YAAC(G/T)G”. It plays a role in controlling the proliferation and differentiation of hematopoietic precursor cells.
Factor CP2:
The characteristics of the binding sites for the factor CP2 can be found, for example, in the articles by Kim et al., (1990), Mol Cell Biol, Vol. 10: 5958-5966 and Lim et al., (1992), J. Biol. Chem. Vol. 268: 18008-18017.
Factor CTF:
The binding characteristics of the factor CTF can be found, for example, in the following entries in the Medline database: 88319941, 91219459, 86140112, 87237877, 90174951, 89282387, 90151633, 892618136, 86274639, 87064414, 89263791. The factor CTF/NF-I recognizes the following palindromic sequence: “TGGCANNNTGCCA” (SEQ ID NO: 21), which is present in viral and cellular promoters and at the origin of replication of type 2 adenoviruses. These proteins are capable of activating transcription and replication. They bind to DNA in the form of a homodimer.
Factor E2A:
The characteristics of a binding site for factor E2A can be found, for example, in the articles corresponding to the following entries in the Medline database: 91160969, 91331308, 91115096, 91117219, 90346284, 89168418, 90150281. This factor binds to a KAPPA-E2 site of the enhancer element of the KAPPA immunoglobulin gene. It forms a heterodimer with the protein ASH1. It belongs to the family of transcription factors of helix-loop-helix type.
Factor GRα:
The characteristics of a binding site for the GRα factor can be found, for example, in the following entries in the Medline database: 88264449, 93024441, 89091080, 90319784, 92020837, 90381775, 86298392, 91131612, 86092211, 86092206. It is a glucocorticoid receptor which is involved in regulating the expression of eukaryotic genes and which affects the proliferation and differentiation of target tissues. This factor binds to the target site of type “GRE”. It is composed of three domains and belongs to the NR3 subfamily of hormone nuclear receptors.
Factor LXR:
The characteristics of the binding sites for the factor LXR (Liver X Receptor) have been described by Apfel et al., (Moll. Cell. Biol., 1994), Song et al., (Proc. Natl. Acad. Sci USA, 1994) and Willy et al., (Genes Dev., 1995). Oxysterols are the physiological ligand of LXR elements (Janowski et al., Proc. Natl. Acda. Sci USA, 1999). Like other receptors, LXR heterodimerizes with RXR (Retinoid X Receptor). Our LXR response elements have been identified at positions −1729/−1714 and −69/−55 (FIG. 1).
Factor NF-EIC:
The characteristics of a binding site for the factor NF-EIC can be found, for example, in the following entries in the Medline database: 91266910, 91216113, 91334450, 91203899, 91029498 and 910655813. It is a transcriptional activator which binds to an enhancer element of the α and δ genes of the T cell receptor. It binds to the following consensus sequence: “AGATAG”. It belongs to the family of transcription factors of the type GATA.
Factor HNF5:
Persons skilled in the art may, for example, refer to the article by Grange et al. (1991, Nucleic Acids Res. Vol. 19: 131-139) for this transcription factor.
Factor HNF3B:
Persons skilled in the art may advantageously refer to the article by Overdier et al., (1994, Mol. Cell Biol. Vol. 14: 2755-2766).
Factor Nfkappa-B:
Persons skilled in the art may advantageously refer to the articles corresponding to the following entries in the Medline database: 95369245, 91204058, 94280766, 89345587, 93024383, 888248039, 94173892, 91088538, 91239561, 91218850, 92390404, 90156535, 93377072, 92097536, 93309429, 93267517, 92037544, 914266911, 91105848 and 95073993. The factor Nfkappa-B is a heterodimer consisting of a first 50-kDa subunit and a second 65-kDa subunit. Two heterodimers can form a labile tetramer. Its DNA-binding depends on the presence of zinc (Zn++). It can be induced by many agents, such as TNF, PKA or PKC. It is generally a regulator of genes involved in responses to infection, inflammation and stress.
Factor NFY:
The factor NFY is described, for example, in entry No. P25.208 of the Swissprot database. It is a factor which recognizes a “CCAAT” moiety in promoter sequences such as those of the gene encoding type 1 collagen, of albumin and of β-actin. It is a transcription stimulator.
Factor PEA3:
Persons skilled in the art may advantageously refer to the articles corresponding to the following entries in the Medline database: 90059931, 90309995, 90291989, 93181246 and 90384794. This transcription factor binds to an “AGGAAG” PEA3 moiety, and can play a regulatory role during embryogenesis.
Factor PEBP2:
Persons skilled in the art may advantageously refer to the articles corresponding to the following entries in the Medline database: 95199266, 94217721, 97188387, 97325753 and 95347606. This factor binds to a “PYGTYGGT” site in many enhancer and promoter elements.
Factor TFIID:
Persons skilled in the art may advantageously refer to the following articles: Fikes et al., (1990, Nature, Vol. 346: 291-294), GILL et al., (1991, Cell, Vol. 65: 333-340), Hoffmann et al., (1990, Genes Dev. Vol. 4: 1141-1148). This factor plays a major role in activating the eukaryotic genes which are transcribed by RNA polymerase II. This factor binds specifically to the TATA promoter element located close to the transcription start site.
Factor T3R:
Persons skilled in the art may advantageously refer to the articles corresponding to the following entries in the Medline database: 92017776, 90242396, 870903752, 91212192. This factor has a strong affinity for triiodothyroine. It is composed of three domains and belongs to the family of hormone nuclear receptors.
Factor SIF:
Persons skilled in the art may advantageously refer to the article by Wagner et al., (1990, EMBO J., Vol. 9: 4477-4784). This factor activates the expression of the c-fos gene.
Factor RAR:
Persons skilled in the art may advantageously refer to the articles corresponding to the following entries in the Medline database: 91216109, 92017791, 92127595, 91219411, 92103690, 93321869, 91092269, 91029504, 90242395, 91029504. This factor is a retinic acid receptor. This factor controls cellular functions by directly regulating gene expression. It belongs to the family of hormone nuclear receptors.
Factor PU:
Persons skilled in the art may advantageously refer to the articles corresponding to the following entries in the Medline database: 92107189, 93165739, 95317607, 92318913, 92275360, 93028372, 93206099, 90199884, 87257848 and 93275657. This factor binds to the PU box, which is a purine-rich DNA sequence, such as the sequence “GAGGAA”, which can act as a lymphoid cell-specific enhancer element.
It is a transcription activation factor which can be specifically involved in the activation or differentiation of macrophages or B cells.
SITE AP1:
The characteristics of a binding site for the transcription factor AP1 can be found in various articles corresponding to the following entries in the Medline database: Numbers 89125693, 89252809, 90318391, 91175677, 911458338, 89313776, 88217909, 911662, 91121514, 89017284, 88070595, 90097934, 88189275, 87301729, 88151062, 90291989, 91330875, 89051877 and 91219459. This factor binds to an enhancer element of the type “TGA(C/G)TCA.
Factor AP2:
The characteristics of a binding site for the transcription factor AP2 can be found, for example, in the articles corresponding to the following entries in the Medline database: 90127451, 90174951 and 91009310.
This factor binds to enhancer-type elements, in order to stimulate the transcription of certain genes. For example, the factor AP2 binds to the following consensus sequence: “CCCCAGGC”.
Factor CCAAT:
A characteristic of the binding site for the factor CCAAT can be found in the article by Lum et al., (1990, Mol Cell Biol. Vol. 10: 6709-6717) and in entry No. T 00086 of the EMBL database. This factor has, for example, been shown to be a stimulator of the transcription of the human hsp70 gene promoter.
Factor GATA-1:
The characteristics of the binding site for the factor GATA-1 can be found, for example, in the following entries in the Medline database: 91340773, 91093039, 91266910, 90114418, 89385992, 91268074, 89118131, 91224987, 89218991 and 91081330. It is known to be a “switch” factor in erythroid development. It binds to DNA on the following consensus sequence: “(A/T)GATA(A/G)”, in regulatory regions of globin genes and of other genes expressed in erythroid cells.
Factor MyoD-Myf-3:
The characteristics of a binding site for the factor MyoD-Myf-3 can be found, for example, in the article by Rosenthal et al., (1990, Nucleic Acids Res., Vol. 18: 6239). This transcription factor induces differentiation of fibroblasts into myoblasts, activates muscle specific promoters and interacts with, and is inhibited by, the protein twist.
Factor MYC/MAX:
The characteristics of a binding site for the factor Nyc/MAX can be found, for example, in the following entries in the Medline database: 94040733, 93101610, 92229468, 92112037, 93145325, 93026389, 93157390, 92366516, 93145324 and 91173288. This transcription factor binds to DNA nonspecifically, but also binds to DNA specifically by recognizing the sequence CAC[GA]TG. This factor appears to activate the transcription of genes associated with growth.
Factor HNF3:
Persons skilled in the art may advantageously refer to the following entries in the Medline database: 91352065, 91032994, 92345837, 89160814, 91187609, 91160974, 91029477, 94301798 and 94218249. This transcription factor acts as an activator of many genes of the liver, such as AFT, albumin and tyrosine aminotransferase genes, and interacts with regulatory regions which are cis-acting with respect to these genes.
Factor SRY:
Persons skilled in the art may advantageously refer to the articles corresponding to the following entries in the Medline database: 92132550, 95292338, 95112822, 93049201. This factor is responsible, for example, for initiating male sex determination.
Factor Sp1:
Persons skilled in the art may advantageously refer to the articles corresponding to the following entries in the Medline database: 852707437, 89091123, 89039842, 89384647, 85061571, 88111565, 91224491, 91095025, 91357479, 91139695. This factor activates the synthesis of messenger RNA from genes containing functional recognition sites; it can interact with G/C base-rich moieties of the promoter of the serotonin receptor gene.
Factor USF:
USF belongs to the helix-loop-helix family of transcription factors that bind the E-box motif (CACGTG) and include inter alia Myc, Mad1, Max, MyoD (Littlewood et al., Oxford University Press, New York, 1 pp. 1998). Although it has a well-established role as a transcription activator (Ghosh et al., Oncogene, 1997, 14:589-594), it is clear that in some promoter contexts USF does have the capacity to act as a transcription repressor. USF1 has been shown to be important for developmental repression of the LpS1 gene of Lytechinus pictus in all cell types except aboral ectoderm cells that do not express USF (Seid et al., J. Mol Biol, 1996, 264:7-19). USF1 inhibits autoactivation of the Xenopus MyoD gene whose product binds to the E-box (Lun et al., Cell Growth Differ., 1997, 8:275-282) and also represses transcription of the CYP1A1 gene by competing with the stimulatory AhR.Arnt complex for binding to the xenobiotic-responsive element (XRE) that contains an E-box like motif (Takahashi et al., J Biol Chem, 1997, 272: 30025-30031). Carter et al. demonstrated that the absence of a strong activation domain in USF1 leads to transcriptional repression of the immunoglobulin heavy-chain (IgH) gene (Carter et al., Mol Cell Biol, 1997, 17:18-23). Truncated and splice variants of USF2 which abrogate E-box activity repress the expression of the major histocompatibility complex class I (Howcroft et al., Mol Cell Biol (1999) 19:4788-4797), ATPA (Breen et al., J Biol Chem, 1997, 272:10528-10542) and prostaglandin G/H synthase-2 genes. Harris et al (J Biol Chem, 2000, 275:28539-28548) have provided evidence that the close proximity between the AP-1 and E-box elements on the FGF-BP promoter facilitates transcriptional repression via interactions between USF1, USF2 and the AP-1 binding proteins. In addition, overexpression of human USF decreases AP-1 dependent transcription in murine teratocarcinoma F9 cells (Pognonec et al., Oncogene, 1997, 14:2091-2098) and USF binding to a complex consisting of AP-1 factors, Fra2 and CREB represses the chicken alphaA-crystallin gene (Cvekl et al., 1994, Mol Cell Biol, 147363-7376). USF1 and USF2 homodimers inhibit transcription of the ribosomal RNA gene (Sirito et al, Gen Expr, 1992, 2:231-240). USF binding motifs have also been shown to act as negative regulatory elements in the promoters of the Protease Nexin-1 (Erno et al, Mol Cell Neurosci, 1996, 8:28-37) and the HLA-B (Gobin et al., J Immunol, 1999, 163:1428-1434) genes. Interestingly, apolipoprotein CIII, which like ABC1 is involved in lipid metabolism, is also repressed by USF2 (Navantkasattusas et al., Mol Cell biol, 1994, 14: 7331-7339). Finally, USF can both positively and negatively regulate the MLC-2v gene and ribosomal RNA gene transcription. Several studies indicate that USF may also function as a constitutively bound protein that cooperates with basal factors such as TAFII55 (Chiang et al., Science, 1995, 267:531-536) or inducible factors such as USA (Meisterernst et al., Cell, 1991, 66:981-993), PC5 (Halle et al, J Biol Chem, 1995, 270:21307-21311), c-Myc, Max (Harris et al.), CREB and JunD (Cvekl et al.) to mediate transcriptional induction or repression. Thus, USF can function to either activate or repress gene transcription.
Without wishing to be bound by any theory, the applicant thinks that USF may for example inhibit gene transcription by competing for binding to the E-box with transcriptional activators (Lun et al.; Takahashi et al. above cited). However, the applicant has demonstrated in Example 5 that transfection of EBm (mutated E-box) and EBdel (deleted E-box) constructs in RAW cells led to increased rather than decreased ABC1 promoter activity, consistent with E-box mediated gene repression. Furthermore, gel shift assays using antibodies specific to different E-box binding activators failed to demonstrate binding to the E-box motif. Western blot analysis were also performed to determine truncated and splice variants of USF2 that lack the transactivation domain (Liu et al., Sirito et al., et Howcroft et al.,) and lead to gene repression. Under these conditions, these USF variants were not detected in RAW cell or 293 cell nuclear extracts.
Because USF can also function as a repressor through specific protein-protein interaction with transcriptional activators that bind DNA motifs distinct from the E-box (Harris et al., Pognonec et al., Cvekl et al., above-cited). As previously described, in the human ABC1 promoter, the E-box motif is flanked by two Sp1 sites and an AP1 motif. It is well known in the art that transcription factors which bind to Sp1 and AP1 have been implicated in the transcriptional regulation of other genes involved in lipid metabolism including apoA-II (Ribeiro et al., J Biol Chem, 265:1216-1225), apoC-III (Ogami et al, J. Biol Chem, 1990, 265:9808-9815), chicken vitellogenin II (Seal et al., Mol Cell Biol, 1991, 11:2704-2717), fatty acid synthase (Casado et al., J Biol Chem, 1999, 274:2009-2013) and the LDL receptor (Sanchez et al., J Biol Chem, 1995, 270:1161-1169) and the LDL receptor related protein (LRP) (Gaeta et al., BBA, 1994,1219:307-313.)
An preferred characteristic of the regulatory nucleic acid according to the invention, and characteristic of the sequence located upstream of the transcription start site, included in both the sequence SEQ ID No. 1 and in the sequence SEQ ID No. 3, is the presence of eight moieties which are characteristic of a putative binding site for the proteins PPAR. The PPARs, also referred to as peroxisome proliferator-activated receptors, which can be of type α,δ(β) and γ, form a subfamily belonging to the family of nuclear receptor genes. All PPARs are activated by fatty acids and derivatives thereof. For example, the PPAR of type α binds to hypolipidemic fibrates, whereas antidiabetic glitazones are ligands for the PPAR of type gamma. Activation of the PPAR of type α induces pleiotropic effects such as the stimulation of lipid oxidation, the impairment of lipoprotein metabolism and the inhibition of vascular inflammation. Activators of PPARα increase hepatic absorption and esterification of free fatty acids by stimulating the expression of the fatty acid transport protein and of the acyl-CoA synthetase. In the skeletal muscle and the heart, PPARα increases mitochondrial absorption of free fatty acids, and their oxidation, by stimulating muscle-specific carnitine palmitoyl transferase I. The effect of fibrates on the metabolism of triglyceride-rich lipoproteins is due to the stimulation of lipoprotein lipase, this stimulation being dependent on PPARα, and to the inhibition of apolipoprotein C-III, whereas the increase in plasmatic cholesterol, in the form of HDL, depends on an overexpression of apolipoprotein A-I and apolipoprotein A-II.
PPARαs are also expressed in atherosclerotic adhesions. PPARα inhibits inducible nitric oxide synthase in macrophages and prevents IL-1-induced cyclooxygenase-2 and IL-6 expression, as well as thrombin-induced endothelin-1 expression, which results from a negative transcriptional regulation of the nuclear factor of the signaling pathways of the nuclear factor NF-KAPPA B and of activating protein-1.
The activation of PPARα also induces apoptosis in monocyte-derived macrophages, probably by inhibiting the activity of NFKAPPA B. Thus, the pleiotropic effects of the PPARα activators on the plasmatic lipid profile certainly participate in inhibiting the development of atherosclerosis. PPARα activators, such as fibrates, inhibit the development of atherosclerosis because of their normolipidemic activities.
The presence of eight potential PPAR binding sites (positions of the start and end nucleotides, with respect to the transcription start site, for each of the sites: −1280-1276/−1270-1264, −889-883/−878-872, −584-578/−575-569 and −366-360/−358-352 [also FIG. 1]) on a regulatory nucleic acid according to the invention is compatible with the observation according to which the expression of the gene encoding the human ABC1 protein is induced during the differentiation of monocytes into macrophages in vitro. It is also compatible with the prior observation of the regulation of the ABC1 gene by fibrates. It is also compatible with experimental results demonstrating that the expression of the ABC1 gene is increased when human macrophages are incubated in the presence of acetylated low density lipoproteins (AcLDLs).
Without wishing to be bound by any theory, the applicant thinks that the PPAR binding sites identified according to the invention on the regulatory nucleic acid of sequence SEQ ID No. 1 are highly involved in the tissue specific regulation, and in the specific regulation of the cellular metabolic situation, of the ABC1 gene, and that as a result, a regulatory sequence which comprises at least 4, for example, at least 5, 6, 7 or all of the 8 PPAR binding sites (FIG. 1)(of the sequence SEQ ID No. 1, and which also comprises a basic promoter element, is useful as a regulatory sequence for a polynucleotide whose expression is desired in the liver, the lungs, the adrenal glands, the monocytes/macrophages, the placenta or the fetal tissues, or for a polynucleotide whose expression is desired in response to a specific stimulation of the cell, in relation with cholesterol metabolism, such as the presence, in the cellular environment, of acetylated low density lipoproteins (Ac LDLs).
In addition, it has been shown according to the invention that a regulatory nucleic acid for the human ABC1 gene, as defined above, which comprises all the abovementioned PPAR sites, is capable of regulating the expression of a coding sequence placed under its control, in a manner which is dependent on the presence of cholesterol in the cellular environment. The results are presented in Example 4 below.
As previously mentioned, the invention concerns a nucleic acid comprising a polynucleotide which has at least 20 consecutive nucleotides of either of the nucleotide sequences SEQ ID No. 1 and SEQ ID No. 2, as well as a nucleic acid of complementary sequence.
The nucleic acids comprising one or more “biologically active” fragments of either of the sequences SEQ ID No. 1 and SEQ ID No. 2 are encompassed in the definition above. Persons skilled in the art can easily obtain biologically active fragments of these sequences by referring, for example, to Table 1 below and to FIG. 1, in which are presented the various moieties which are characteristic of the regulatory sequence for the ABC1 gene. Persons skilled in the art may thus obtain such biologically active fragments by totally or partially chemically synthesizing the corresponding polynucleotides or by using restriction endonucleases to obtain desired DNA fragments, the restriction sites present on the sequences SEQ ID No. 1 and SEQ ID No. 1 being able to be easily found from the sequence information, with the aid of current restriction mapping software such as GCG version 9.1 module map.
The production of nucleic acid fragments determined using restriction endonucleases is described, for example, in the work by Sambrook et al. (1989).
The invention thus also relates to a nucleic acid as defined above, which is capable of modulating the transcription of a polynucleotide placed under its control.
According to one embodiment, a biologically active fragment of a transcription-regulating acid according to the invention comprises the basic promoter (TATA box and homeobox) ranging from the nucleotide at position −1 to the nucleotide at position −300, with respect to the transcription start site, the first nucleotide transcribed being the nucleotide in position 2894 of the nucleotide sequence SEQ ID No. 1.
According to a second embodiment, a biologically active fragment of a transcription-regulating nucleic acid according to the invention comprises both the basic promoter and the proximal regulatory elements, ranging from the nucleotide at position −1 to the nucleotide at position −600, with respect to the transcription start site, the first nucleotide transcribed being the nucleotide in position 2894 of the nucleotide sequence SEQ ID No. 1.
According to a third embodiment, a biologically active fragment of a transcription-regulating nucleic acid according to the invention comprises besides the basic promoter (core promoter) and the proximal 200 bp of the ABC1 gene promoter which is rich in binding sites for transcription factors, i.e., Sp1, AP1, LXR, and E-box, that are likely involved in modulating human ABC1 gene expression.
According to a fourth embodiment, such a biologically active fragment of a transcription-regulating acid according to the invention also comprises, besides the basic promoter (core promoter) and the proximal regulatory elements, other regulatory elements such as the various PPARα sites, and stretches from the nucleotide at position −1 to the nucleotide at position −2894, with respect to the transcription start site, the first nucleotide transcribed being the nucleotide in position 2894 of the nucleotide sequence SEQ ID No. 1.
According to a fifth embodiment, such a biologically active fragment of a transcription-regulating acid according to the invention, which also comprises, besides the basic promoter (core promoter) and the proximal regulatory elements, other regulatory elements such as the various PPARα sites, stretches from the nucleotide at position +120 to the nucleotide at position −995, with respect to the transcription start site, the first nucleotide transcribed being the nucleotide in position 2894 of the nucleotide sequence SEQ ID No. 1.
According to a sixth embodiment, a biologically active fragment of a transcription-regulating nucleic acid according to the invention comprises a region ranging from the nucleotide at position +108 to the nucleotide at position −2228, with respect to the transcription start site as set forth in sequence SEQ ID No. 1.
Exons 1A and 1B and Introns 1A and 1B
The applicant has also identified the nucleotide sequences located downstream of the transcription start site, and corresponding respectively, from the 5′ end to the 3′ end, to exon 1A, intron 1A, exon 1B and intron 1B of the human gene encoding the ABC1 protein.
More precisely, exon 1A, which is 221 nucleotides long, starts at the nucleotide at position 2894 of the sequence SEQ ID No. 1 and ends at the nucleotide at position 3114 of the sequence SEQ ID No. 1. Exon 1A is identified as the sequence SEQ ID No. 4.
Exon 1B, which is 109 nucleotides long, starts at the nucleotide at position 100 and ends at the nucleotide at position 258 of the sequence SEQ ID No. 2. Exon 1B is identified as the sequence SEQ ID No. 5.
Intron 1A has been partially sequenced. The 5′ end of intron 1A starts at the nucleotide at position 3115 and ends at the nucleotide at position 3231 of the nucleotide sequence SEQ ID No. 1, and is also defined as the sequence SEQ ID No. 6. The 3′ end of intron 1A starts at the nucleotide at position 1 and ends at the nucleotide at position 99 of the nucleotide sequence SEQ ID No. 2, and is also identified as the sequence SEQ ID No. 7.
Intron 1B has been partially sequenced. The 5′ end of intron 1B starts at the nucleotide at position 259 and ends at the nucleotide at position 357 of the sequence SEQ ID No. 2. This sequence is also identified as the sequence SEQ ID No. 8.
Exon 1B contains the start of the open reading frame of the human ABC1 gene, the nucleotide A of the ATG codon being located in start position at position 94 of the sequence SEQ ID No. 5. Exon 1B encodes the polypeptide of sequence SEQ ID No. 9.
Exons 1A and 1B, as well as introns 1A and 1B, can contain elements for regulating the expression of the ABC1 gene, for example, elements of enhancer type and/or elements of silencer type.
Consequently, a transcription-regulating nucleic acid according to the invention can also contain, besides biologically active fragments of the sequence SEQ ID No. 1, nucleotide fragments, or even all, of the sequences SEQ ID No. 2 to SEQ ID No. 8.
The nucleotide sequences SEQ ID No. 1 to SEQ ID No. 8, as well as fragments thereof, can, for example, be used as nucleotide probes or primers for detecting the presence of at least one copy of the ABC1 gene in a sample, or for amplifying a given target sequence in the regulatory sequence for the ABC1 gene.
An individual of the invention is thus a nucleic acid having at least 80% nucleotide identity with a nucleic acid as defined above, for example, originating from one of the sequences SEQ ID No. 1 to SEQ ID No. 8.
The invention also concerns a nucleic acid which hybridizes, under high stringency conditions, with any one of the nucleic acids according to the invention, for example, a nucleic acid originating from a sequence chosen from the sequences SEQ ID No. 1 to SEQ ID No. 8.
The invention also relates to a nucleic acid as defined above and also further characterized in that it is capable of modifying the transcription of a polynucleotide of interest placed under its control.
According to a first aspect, such a nucleic acid is capable of activating the transcription of the polynucleotide of interest placed under its control.
According to a second aspect, a regulatory nucleic acid according to the invention can be characterized in that it is capable of inhibiting the transcription of the polynucleotide of interest placed under its control.
For example, a transcription-regulating nucleic acid according to the invention, when it is suitably located with respect to a polynucleotide of interest whose expression is desired, will allow the transcription of said polynucleotide of interest, either constitutively or inducibly.
The inducible nature of the transcription initiated by a regulatory nucleic acid according to the invention can be conferred by one or more of the regulatory elements that it contains, for example, the presence of one or more PPARα sites, such as at least 4 PPARα sites, or at least 5, 6, 7 PPARα sites or the 8 PPARα sites of the sequence SEQ ID No. 1 or SEQ ID No. 3.
In addition, a tissue specific expression of the polynucleotide of interest can be sought by placing this polynucleotide of interest under the control of a regulatory nucleic acid according to the invention which is capable, for example, of initiating the transcription of this polynucleotide of interest specifically in certain categories of cells, for example, cells from the liver, placenta cells or macrophages.
In general, a regulatory nucleic acid according to the invention can comprise one or more “discrete” regulatory elements, such as enhancer and silencer elements. For example, such a regulatory nucleic acid can comprise one or more potential transcription factor binding sites as defined in FIG. 1.
A regulatory acid according to the invention also encompasses a sequence which does not comprise the basic promoter, i.e. the sequence ranging from the nucleotide at position −1 to the nucleotide at position −300, with respect to the transcription start site.
Such a regulatory nucleic acid will then generally comprise a so-called “heterologous” basic promoter, i.e. a polynucleotide comprising a “TATA” box and a “homeobox”, which does not originate from the regulatory nucleic acid for the ABC1 gene.
A transcription-regulating nucleic acid comprising all or part of the sequence SEQ ID No. 1 which has been modified, for example, by addition, deletion or substitution of one or more nucleotides, also forms part of the invention. Such modifications can modify the transcriptional activity by causing an increase, or on the contrary a decrease, in the activity of the promoter or of the regulatory element.
Such a modification can also affect the tissue specificity of the promoter or of the regulatory element. Thus, for example, a regulatory nucleic acid according to the invention can be modified in order to stimulate transcription in only one of the tissues in which it is naturally expressed.
A transcription-regulating acid according to the invention can also be modified and be made inducible by a specific compound, for example, by creating, in the sequence, a site which is inducible by a given therapeutic compound.
The modifications in a sequence which comprises all or part of the sequence SEQ ID No. 1 and which comprises the promoter or a regulatory element can be carried out using methods that are well known to persons skilled in the art, such as mutagenesis. The activity of the modified promoter or regulatory element can then be tested, for example, by cloning the modified promoter upstream of a reporter gene, by transfecting the resulting DNA construct into a host cell and by measuring the level of expression of the reporter gene in the transfected host cell. The activity of the modified promoter can also be analyzed in vivo in transgenic animals. It is also possible to construct libraries of modified fragments, which can be screened using functional tests in which, for example, only the promoters or the regulatory elements having the desired activity will be selected.
Such assays can be based, for example, on the use of reporter genes which confer resistance to given compounds, for example, to antibiotics. The selection of cells which have a regulatory nucleic acid/reporter gene construct, and which contain a promoter or a regulatory element with the desired modification, can then be isolated by culturing the transformed host cells having such a construct, in the presence of the given compound, for example, of the given antibiotic.
The reporter gene can also encode any easily detectable protein, for example, an optically detectable protein such as luciferase.
Consequently, an individual of the invention is also a nucleic acid comprising:
a) a transcription-regulating nucleic acid as defined above; and
b) a polynucleotide of interest encoding a polypeptide or a nucleic acid of interest.
According to a first aspect, the polynucleotide of interest whose transcription is desired encodes a protein or a peptide. The protein can be of any nature, for example, a protein of therapeutic interest, including cytokines, structural proteins, receptors or transcription factors. For example, when transcription specifically in certain tissues is desired, such as for example, in liver, macrophage or placenta cells, the transcription-regulating nucleic acid will advantageously comprise a nucleic acid ranging from the nucleotide at position −1 to the nucleotide at position −1318, with respect to the transcription start site of the sequence SEQ ID No. 1 or SEQ ID No. 3.
In this case, the polynucleotide of interest will encode a gene involved in combating inflammation, such as a cytokine receptor, or a superoxide dismutase. If an antitumoral effect is desired, then stimulation of the number and the activation of cytotoxic T lymphocytes specific for a given tumoral antigen will be sought.
Alternatively, the regulatory nucleic acid advantageously comprises a nucleic acid ranging from the nucleotide at position +108 to the nucleotide −2228, with respect to the transcription start site of the sequence SEQ ID NO: 1.
In another embodiment, a regulatory nucleic acid according to the invention will be used in combination with a polynucleotide of interest encoding the ABC1 protein.
As already mentioned, the polynucleotide of interest can also encode a nucleic acid, such as an antisense nucleic acid specific for a gene the inhibition of whose translation is desired.
According to another aspect, the polynucleotide of interest, whose transcription is regulated by the regulatory nucleic acid, is a reporter gene, such as any gene encoding a detectable protein.
Among the exemplified reporter genes, mention may be made for example, of the luciferase, the β-galactosidase (LacZ) or the chloramphenicol acetyl transferase (CAT) gene, or any gene encoding a protein which confers resistance to a specific compound, such as to an antibiotic.
Recombinant Vectors
For the purposes of the present invention, “vector” will be intended to mean a linear or circular RNA or DNA molecule which is indifferently in the single-stranded or double-stranded form.
According to a first embodiment, a recombinant vector according to the invention is used to amplify the regulatory nucleic acid according to the invention, which is inserted therein after transformation or transfection of the desired host cell.
According to a second embodiment, they are expression vectors comprising, besides a regulatory nucleic acid in accordance with the invention, sequences whose expression is sought in a host cell or in a given multicellular organism.
According to an advantageous embodiment, a recombinant vector according to the invention will, for example, comprise the following elements:
(1) a regulatory nucleic acid according to the invention;
(2) a polynucleotide of interest comprising a coding sequence included in the nucleic acid to be inserted into such a vector, said coding sequence being placed in frame with the regulatory signals described in (1); and
(3) suitable transcription start and stop sequences.
In addition, the recombinant vectors according to the invention may include one or more origins of replication in the host cells in which their amplification or their expression is desired, markers or selection markers.
By way of examples, the bacterial promoters may be the promoters LacI or LacZ, the T3 or T7 bacteriophage RNA polymerase promoters, or the lambda phage PR or PL promoters.
The promoters for eukaryotic cells may comprise the thymidine kinase promoter of the virus HSV or the mouse metallothionine-L promoter.
In general, for the choice of a suitable promoter, persons skilled in the art may advantageously refer to the abovementioned work by Sambrook et al. (1989) or to the techniques described by Fuller et al. (1996).
When the expression of the genomic sequence of the ABC1 gene is desired, vectors which are capable of including large insertion sequences will, for example, be used. In this specific embodiment, bacteriophage vectors, such as the P1 bacteriophage vectors like the vector p158 or the vector p158/neo8 described by Sternberg (1992, 1994), will, for example, be used.
Bacterial vectors according to the invention can be, for example, the vectors pBR322(ATCC37017) or vectors such as pAA2233 (Pharmacia, Uppsala, Sweden) and pGEMI (Promega Biotech, Madison, Wis., USA,).
Mention may also be made of other commercialized vectors, such as the vectors pQE70, pQE60, pQE9 (Qiagen), psiX174, pBluescript SA, pNH8A, pNH16A, pNH18A, pNH46A, pWLNEO, pSV2CAT, pOG44, pXTI, pSG(Stratagene).
It can also be the recombinant vector PXP1 described by Nordeen SK et al. (1988, Bio Techniques, 6: 454-457).
They can also be vectors of Baculovirus type, such as the vector pVL1392/1393 (Pharmingen) used for transfecting cells of the Sf9 line (ATCC No. CRL 1711) derived from Spodoptera frugiperda. 
They can also be adenoviral vectors, such as the type 2 or 5 human adenovirus.
A recombinant vector according to the invention can also be a retroviral vector or an adeno-associated vector (AAV). Such adeno-associated vectors are described, for example, by Flotte et al. (1992), Samulski et al. (1989) or McLaughlin B A et al. (1996).
To enable the expression of a polynucleotide of interest under the control of a regulatory nucleic acid according to the invention, the polynucleotide construct comprising the regulatory sequence and the coding sequence may be introduced into a host cell. The introduction of such a polynucleotide construct according to the invention into a host cell can be carried out in vitro, according to the techniques well known to persons skilled in the art for transforming or transfecting cells, either in primary culture or in the form of cell lines. The introduction of the polynucleotides according to the invention can also be carried out in vivo or ex vivo, for preventing or treating diseases linked to a deficiency in the reverse transport of cholesterol.
To introduce the polynucleotides or the vectors into a host cell, persons skilled in the art may advantageously refer to various techniques, such as the calcium phosphate precipitation technique (Graham et al., 1973, Chen et al., 1987), DEAE Dextran (Gopal, 1985), electroporation (Tur-Kaspa, 1896; Potter et al., 1984), direct microinjection (Harland et al., 1985) or DNA-loaded liposomes (Nicolau et al., 1982, Fraley et al., 1979).
Once the polynucleotide has been introduced into the host cell, it can be stably integrated into the genome of the cell. The integration can be carried out at a specific place in the genome, by homologous recombination, or it can be randomly integrated. In some embodiments, the polynucleotide can be stably maintained in the host cell in the form of an episome fragment, the episome comprising sequences which allow the latter to be maintained and replicated, either independently or in synchrony with the cell cycle.
According to one embodiment, one method for introducing a polynucleotide according to the invention into a host cell, such as a host cell originating from a mammal, in vivo, comprises a preparation comprising a pharmaceutically compatible vector and a “naked” polynucleotide according to the invention, placed under the control of suitable regulatory sequences, are introduced by local injection into the chosen tissue for example, a smooth muscle tissue, the “naked” polynucleotide being absorbed by the cells of this tissue.
Compositions for use in vitro and in vivo comprising “naked” polynucleotides are described for example, in PCT Application No. WO 95/11307 (Pasteur Institute, Inserm, University of Ottawa) and in the articles by Tacson et al. (1996) and by Huygen et al. (1996).
According to one specific embodiment of the invention, a composition is provided for producing a protein of interest in vivo. This composition comprises a polynucleotide encoding the polypeptide of interest, placed under the control of a regulatory sequence according to the invention, in solution in a physiologically acceptable vector.
The amount of vector which is injected into the chosen host organism varies according to the site of injection. By way of indication, between approximately 0.1 and approximately 100 μg of the regulatory sequence/coding sequence polynucleotide construct can be injected into the body of an animal.
When the regulatory nucleic acid according to the invention is located, on the polynucleotide construct (or vector), so as to control the transcription of a sequence comprising an open reading frame encoding the ABC1 protein, the vector may be injected into the body of a patient likely to develop a disease linked to a deficiency in the reverse transport of cholesterol, or who has already developed this disease, for example, a patient with a predisposition for Tangier disease, or who has already developed the disease.
Consequently, the invention also concerns a pharmaceutical composition intended for preventing, or for treating individuals affected by, a dysfunction of the reverse transport of cholesterol, comprising a regulatory nucleic acid according to the invention and a polynucleotide of interest encoding the ABC1 protein, in combination with one or more physiologically compatible excipients.
Advantageously, such a composition will comprise the regulatory nucleic acid defined by either of the sequences SEQ ID No. 1 and SEQ ID No. 2, or a biologically active fragment of this regulatory nucleic acid.
A subject of the invention is also a pharmaceutical composition intended for preventing, or for treating individuals affected by, a dysfunction of the reverse transport of cholesterol, comprising a recombinant vector as defined above in association with one or more physiologically compatible excipients.
The invention also relates to the use of a polynucleotide construct in accordance with the invention which comprises a regulatory nucleic acid for the ABC1 gene, as well as a sequence encoding the ABC1 protein, for manufacturing a medicinal product intended for preventing atherosclerosis in various forms or, for example, for treating individuals affected by a dysfunction of the reverse transport of cholesterol.
The invention also relates to the use of a recombinant vector according to the invention, comprising, besides a regulatory nucleic acid of the invention, a nucleic acid encoding the ABC1 protein, for manufacturing a medicinal product intended for preventing atherosclerosis in various forms or, for example, for treating individuals affected by a dysfunction of the reverse transport of cholesterol.
Vectors which are Useful in Somatic Gene Therapy Methods and Compositions Containing such Vectors
The present invention also concerns a novel therapeutic approach for treating pathologies linked to cholesterol transport. It proposes an advantageous solution to the drawbacks of the prior art, by demonstrating the possibility of treating pathologies, for example, pathologies linked to cholesterol transport, by gene therapy, by transferring and expressing, in vivo, a polynucleotide construct comprising, besides a regulatory nucleic acid according to the invention, a sequence encoding an ABC1 protein involved in cholesterol transport and metabolism. The invention also offers a simple means which allows specific and effective treatment of the associated pathologies such as for example, atherosclerosis.
Gene therapy consists in correcting a deficiency or an abnormality (mutation, aberrant expression, etc.) or in effecting the expression of a therapeutic protein of interest by introducing genetic information into the affected cell or organ. This genetic information can be introduced either ex vivo, into a cell extracted from the organ, the modified cell then being reintroduced into the body, or directly in vivo into the appropriate tissue. In this second case, various techniques exist, among which diverse techniques of transfection involving complexes of DNA and of DEAE-dextran (Pagano et al., J. Virol. 1 (1967) 891), of DNA and of nuclear proteins (Kaneda et al., Science 243 (1989) 375), and of DNA and of lipids (Felgner et al., PNAS 84 (1987) 7413), the use of liposomes (Fraley et al., J. Biol. Chem. 255 (1980) 10431), etc. More recently, the use of viruses as vectors for transferring genes has appeared as a promising alternative to these physical techniques of transfection. In this respect, various viruses have been tested for their capacity to infect certain cell populations. For example, retroviruses (RSV, HMS, MMS, etc.), the HSV virus, adeno-associated viruses and adenoviruses.
The present invention thus also relates to a novel therapeutic approach for treating pathologies linked to cholesterol transport, consisting in transferring and in expressing, in vivo, genes encoding ABC1 placed under the control of a regulatory acid according to the invention. The applicant has now advantageously shown that it is possible to construct recombinant viruses which contain a DNA sequence comprising a regulatory nucleic acid according to the invention and a sequence encoding an ABC1 protein involved in cholesterol metabolism, and to administer these recombinant viruses in vivo, and that this administration enables an expression of a biologically active ABC1 protein in vivo, which is stable and effective, and which is without cytopathological effect.
Adenoviruses constitute vectors which are efficient for transferring and expressing the ABC1 gene. For example, the use of recombinant adenoviruses as vectors makes it possible to obtain levels of expression of the gene of interest which are sufficiently high to produce the desired therapeutic effect. Other viral vectors, such as retroviruses or adeno-associated viruses (AAVs), which enable a stable expression of the gene, are also claimed.
The present invention thus offers a novel approach for treating and preventing cardiovascular and neurological pathologies linked to the abnormalities in cholesterol transport.
A subject of the invention is thus a defective recombinant virus comprising a regulatory nucleic acid according to the invention and a nucleic acid sequence encoding an ABC1 protein involved in cholesterol metabolism.
The invention also relates to the use of such a defective recombinant virus for preparing a pharmaceutical composition intended for treating and/or for preventing cardiovascular diseases.
The present invention also concerns the use of cells which are genetically modified ex vivo with a virus as described above, or of producer cells for such viruses, which are implanted in the body, enabling a protracted and effective in vivo expression of a biologically active ABC1 protein.
The present invention shows that it is possible to incorporate a DNA sequence encoding ABC1, under the control of a regulatory nucleic acid as defined above, into a viral vector, and that these vectors make it possible to effectively express a biologically active mature form. For example, the invention shows that the in vivo expression of ABC1 can be obtained by direct administration of an adenovirus or by implantation of a producer cell or a cell which is genetically modified with an adenovirus or with a retrovirus incorporating such a DNA.
The present invention is also advantageous because it makes it possible to induce an expression of ABC1 which is controlled and without harmful effects, in organs which the expression of this protein does not normally concern. For example, a significant release of the ABC1 protein is obtained by implantation of cells which produce vectors of the invention, or which are infected ex vivo with vectors of the invention.
The cholesterol transporter activity produced in the context of the present invention can be of human or animal ABC1 type. The nucleic acid sequence used in the context of the present invention can be a cDNA, a genomic DNA (gDNA) an RNA (in the case of retroviruses) or a hybrid construct consisting for example, of a cDNA into which one or more introns would be inserted. It can also be synthetic or semisynthetic sequences. For example, a cDNA or a gDNA is used. For example, the use of a gDNA allows better expression in human cells. To allow their incorporation in a viral vector according to the invention, these sequences are advantageously modified, for example, by site-directed mutagenesis, for example, for inserting suitable restriction sites. The sequences described in the prior art are not in fact constructed for a use according to the invention, and prior adjustments may prove to be necessary, so as to obtain substantial expressions. In the context of the present invention, use of a nucleic acid sequence encoding a human ABC1 protein is one example. Moreover, it is also possible to use a construct encoding a derivative of these ABC1 proteins. A derivative of these ABC1 proteins comprises, for example, any sequence which is obtained by mutation, deletion and/or addition, with respect to the native sequence, and which encodes a product which conserves cholesterol transporter activity. These modifications can be carried out by the techniques known to persons skilled in the art (see the general molecular biology techniques below). The biological activity of the derivatives thus obtained can then be easily determined, as indicated, for example, in the examples describing the measurement of the afflux of cholesterol from cells. For the purposes of the invention, the derivatives can also be obtained by hybridization using nucleic acid libraries, using the native sequence or a fragment of this sequence as probe.
These derivatives are, for example, molecules with greater affinity for their binding sites, molecules having greater resistance to protease, or molecules with a greater therapeutic efficacy or fewer side effects, or optionally, novel biological properties. The derivatives also include the modified DNA sequences which allow improved expression in vivo.
In a first embodiment, the present invention concerns a defective recombinant virus comprising a regulatory nucleic acid according to the invention and a cDNA sequence encoding an ABC1 protein involved in cholesterol transport and metabolism. In another embodiment of the invention, the DNA sequence is a gDNA sequence. The cDNA sequence which encodes the ABC1 protein, and which can be used in a vector according to the invention, is advantageously the sequence SEQ ID No. 10.
The vectors of the invention can be prepared from various types of virus. For example, vectors derived from adenoviruses, adeno-associated viruses (AAV), herpesviruses (HSV) or retroviruses are used. It is also advantageous to use an adenovirus for a direct administration or for modifying, ex vivo, cells intended for implanting, or a retrovirus for implanting producer cells.
The viruses according to the invention are defective, i.e. they are incapable of replicating autonomously in the target cell. Generally, the genome of the defective viruses used in the context of the present invention is thus lacking at least the sequences required for replication of said virus in the infected cell. These regions can be either removed (totally or partially), or rendered nonfunctional or substituted with other sequences, and, for example, with the nucleic acid sequence encoding the ABC1 protein. For example, however, the defective virus conserves the sequences in its genome which are required for encapsidation of viral particles.
In regards to adenoviruses, various serotypes, whose structure and properties vary somewhat, have been characterized. For example, among these serotypes, in the context of the present invention, type 2 or 5 human adenoviruses (Ad 2 or Ad 5) or adenoviruses of animal origin (see application WO 94/26914) may be used. Among the adenoviruses of animal origin which can be used in the context of the present invention, mention may be made of the adenoviruses of canine, bovine, murine (example: Mav1, Beard et al., Virology 75 (1990) 81), ovine, porcine, avian or simian (example: SAV) origin. For example, the adenovirus of animal origin may be a canine adenovirus, such as a CAV2 adenovirus [Manhattan or A26/61 strain (ATCC VR-800) for example]. For example, in the context of the invention, adenoviruses of human or canine or mixed origin may be used. In addition, the defective adenoviruses of the invention may also comprise ITRs, a sequence enabling the encapsidation and the sequence encoding the ABC1 protein placed under the control of a nucleic acid according to the invention. In one embodiment of the present invention, in the genome of the adenoviruses of the invention, the E1 region at least is rendered nonfunctional. In another embodiment, in the genome of the adenoviruses of the invention, the E1 gene and at least one of the genes E2, E4 and L1-L5 are nonfunctional. The viral gene under consideration can be rendered nonfunctional by any technique known to persons skilled in the art, and, for example, by total elimination, substitution, partial deletion, or addition of one or more bases in the gene(s) under consideration. Such modifications can be obtained in vitro (on isolated DNA) or in situ, for example, by means of the techniques of genetic engineering or by treatment with mutagenic agents. Other regions can also be modified, and, for example, the E3 region (WO 95/02697), E2 (WO 94/28938), E4 (WO 94/28152, WO 94/12649, WO 95/02697) and L5 (WO 95/02697). According to one embodiment, the adenovirus according to the invention comprises a deletion in the E1 and E4 regions, and the sequence encoding ABC1 is inserted into the inactivated E1 region. According to another embodiment, it comprises a deletion in the E1 region, into which are inserted the E4 region and the sequence encoding ABC1 (French Patent Application FR 94 13355).
The defective recombinant adenoviruses according to the invention can be prepared by any technique known to persons skilled in the art (Levrero et al., Gene 101 (1991) 195, EP 185 573; Graham, EMBO J. 3 (1984) 2917). For example, they can be prepared by homologous recombination between an adenovirus and a plasmid carrying, inter alia, the DNA sequence encoding the ABC1 protein. The homologous recombination takes place, for example, after cotransfection of said adenovirus and plasmid into a suitable cell line. The cell line used should, for example, (i) be transformable by said elements, and (ii) have the sequences which are capable of complementing the portion of the genome of the defective adenovirus, such as in an integrated form to avoid the risks of recombination. By way of example of a line, mention may be made of the 293 human embryonic kidney line (Graham et al., J. Gen. Virol. 36 (1977) 59), which comprises, for example, integrated into its genome, the left-hand portion of the genome of an adenovirus Ad5 (12%) or lines which are capable of complementing the E1 and E4 functions, as described, for example, in applications No. WO 94/26914 and WO 95/02697.
Next, the adenoviruses which have multiplied may be recovered and purified according to the conventional techniques of molecular biology, as illustrated in the examples.
As regards the adeno-associated viruses (AAVs), they are relatively small DNA viruses, which integrate into the genome of the cells that they infect, in a stable and site-specific way. They are capable of infecting a broad spectrum of cells, without inducing effects on cell growth, morphology or differentiation. Moreover, they do not seem to be involved in pathologies in humans. The AAV genome has been cloned, sequenced and characterized. It comprises approximately 4700 bases and contains, at each end, an inverted repeat region (ITR) of 145 bases approximately which serves as an origin of replication for the virus. The rest of the genome is divided into two essential regions carrying the encapsidation functions: the left-hand portion of the genome, which contains the rep gene which is involved in viral replication and expression of viral genes; the right-hand portion of the genome, which contains the cap gene which encodes the capsid proteins of the virus.
The use of AAV-derived vectors for transferring genes in vitro and in vivo has been described in the literature (see, for example, WO 91/18088; WO 93/09239; U.S. Pat. Nos. 4,797,368, 5,139,941, EP 488 528). These documents describe various AAV-derived constructs, in which the rep and/or cap genes are deleted and replaced with a gene of interest, and their use for transferring said gene of interest in vitro (on culture cells) or in vivo (directly in an organism). However, none of these documents describes or suggests using a recombinant AAV for transferring and expressing an ABC1 protein in vivo or ex vivo, or the advantages of such a transfer. The defective recombinant AAVs according to the invention can be prepared by cotransfecting, into a cell line which has been infected with a human helper virus (for example, an adenovirus), a plasmid containing the sequence encoding the ABC1 protein, bordered by two AAV inverted repeat regions (ITR), and a plasmid carrying the AAV encapsidation genes (rep and cap genes). The recombinant AAVs produced may then be purified by conventional techniques.
As regards herpes viruses and retroviruses, the construction of recombinant vectors has been widely described in the literature: see, for example, Breakfield et al., New Biologist 3 (1991) 203; EP 453242, EP 178220, Bernstein et al. Genet. Eng. 7 (1985) 235; McCormick, Bio Technology 3 (1985) 689, etc.
For example, retroviruses are integrating viruses which infect dividing cells. The retrovirus genome comprises two LTRs, an encapsidation sequence and three coding regions (gag, pol and env). In retrovirus-derived recombinant vectors, the gag, pol and env genes are generally totally or partially deleted and replaced with a heterologous nucleic acid sequence of interest. These vectors can be prepared from various types of retrovirus, such as, for example, MoMuLV (“murine moloney leukemia virus”; also referred to as MoMLV), MSV (“murine moloney sarcoma virus”), HaSV (“harvey sarcoma virus”), SNV (“spleen necrosis virus”), RSV (“rous sarcoma virus”) or the Friend virus.
To construct recombinant retroviruses comprising a sequence encoding the ABC1 protein, placed under the control of a regulatory nucleic acid according to the invention, a plasmid comprising, for example, the LTRs, the encapsidation sequence and said coding sequence is generally constructed, and then used for transfecting a so-called encapsidation cell line which is capable of providing, in trans, the retroviral functions deficient in the plasmid. Generally, encapsidation lines are thus capable of expressing gag, pol and env genes. Such encapsidation lines have been described in the prior art, for example, the line PA317 (U.S. Pat. No. 4,861,719); the line PsiCRIP (WO 90/02806) and the line GP+envAm-12 (WO 89/07150). Moreover, the recombinant retroviruses can comprise modifications in the LTRs so as to eliminate transcriptional activity, as well as extended encapsidation sequences comprising a portion of the gag gene (Bender et al., J. Virol. 61 (1987) 1639). The recombinant retroviruses produced may then be purified by conventional techniques.
To implement the present invention, it is advantageous to use a defective recombinant adenovirus. The results given below indeed demonstrate the properties of adenoviruses which are advantageous for expressing, in vivo, a protein having cholesterol transport activity. The adenoviral vectors according to the invention are advantageous for direct administration in vivo of a purified substance or for transforming cells, for example, autologous cells, ex vivo, with a view to implanting them. In addition, the adenoviral vectors according to the invention also have advantages, such as, for example, their very high infection efficiency, which makes it possible to carry out infections using small volumes of viral suspension.
According to another embodiment of the invention, a producer line for retroviral vectors containing a regulatory nucleic acid according to the invention and the sequence encoding the ABC1 protein is used for an in vivo implantation. The lines which can be used to this end are, for example, the cells PA317 (U.S. Pat. No. 4,861,719), PsiCrip (WO 90/02806) and GP+envAm-12 (U.S. Pat. No. 5,278,056), which are modified to allow production of a retrovirus containing a nucleic acid sequence encoding an ABC1 protein according to the invention. For example, totipotent stem cells, which are precursors of the blood cell lines, can be sampled and isolated from the individual. These cells, which are put into culture, can then be transfected with the retroviral vector containing the sequence encoding the ABC1 protein, under the control of its own promoter. These cells are then reintroduced into the individual. The differentiation of these cells will be the origin of blood cells which express the ABC1 protein, for example, the origin of monocytes which, when transformed into macrophages, participate in removing cholesterol from the arterial wall. These macrophages expressing the ABC1 protein will have an increased capacity for metabolizing excess cholesterol, and will make it available at the cell surface for its removal by the primary acceptors of membrane cholesterol.
In one embodiment of the present invention, in the vectors of the invention, the sequence encoding the ABC1 protein is placed under the control of a regulatory nucleic acid according to the invention, comprising the regulatory elements which allow its expression in the infected cells and, for example, the regulatory elements of type PPAR.
In another embodiment, the vectors of the invention comprise the sequence encoding the ABC1 protein which is placed under the control of a regulatory nucleic acid comprising a region ranging from the nucleotide at position +108 to the nucleotide at position −2228, with respect to the transcription start site as set forth in sequence SEQ ID No. 1.
Again, in another embodiment, the vectors of the invention comprise the sequence encoding the ABC1 protein which is placed under the control of a regulatory nucleic acid comprising the core promoter sequence and the proximal 200 bp of the ABC1 gene promoter.
As indicated above, the present invention also concerns any use of a virus as described above for preparing a pharmaceutical composition intended for treating and/or for preventing pathologies linked to cholesterol transport.
The present invention also concerns a pharmaceutical composition comprising one or more defective recombinant viruses as described above. These pharmaceutical compositions can be formulated with a view to topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal, etc. administration. In one embodiment of the present invention, the pharmaceutical compositions of the invention contain a pharmaceutically acceptable vehicle for an injectable formulation, for example, for an intravenous injection, such as for example, into the portal vein of the patient. They can be, for example, isotonic, sterile solutions or dry, for example, freeze-dried, compositions which, upon addition, depending on the case, of sterilized water or physiological saline, allow the constitution of injectable solutions. Direct injection into the portal vein of the patient is advantageous since it makes it possible to target the infection in the liver, and thus to concentrate the therapeutic effect on this organ.
The doses of defective recombinant viruses used for the injection can be adjusted as a function of various parameters, and, for example, as a function of the viral vector, the method of administration used, the pathology concerned or the desired duration of the treatment. In general, the recombinant adenoviruses according to the invention are formulated and administered in the form of doses between 104 and 1014 pfu/ml, and for example, 106 to 1010 pfu/ml. The term pfu (“plaque forming unit”) corresponds to the infectious power of a viral solution, and is determined by infecting a suitable cell culture and measuring, generally after 48 hours, the number of plaques of infected cells. The techniques for determining the pfu titer of a viral solution are well documented in the literature.
As regards retroviruses, the compositions according to the invention can directly comprise the producer cells, with a view to implanting them.
In this respect, another embodiment of the invention concerns any mammalian cell infected with one or more defective recombinant viruses as described above. For example, the invention concerns any population of human cells infected by these viruses. They can be, for example, cells of blood origin (totipotent stem cells or precursors), fibroblasts, myoblasts, hepatocytes, keratinocytes, smooth muscle cells, endothelial cells, glial cells, etc.
The cells according to the invention can be derived from primary cultures. These primary cultures can be sampled by any technique known to persons skilled in the art, and then put into culture under conditions which allow their proliferation. As regards to fibroblasts, they can easily be obtained from biopsies, for example, according to the technique described by Ham [Methods Cell. Biol. 21a (1980) 255]. These cells can be used directly for the infection with the viruses or stored, for example, by freezing, to establish autologous banks, with a view to later use. The cells according to the invention can also be secondary cultures, obtained for example, from pre-established banks (see for example, EP 228458, EP 289034, EP 400047, EP 456640).
The cultured cells are then infected with the recombinant viruses, to confer upon them the capacity of producing a biologically active ABC1 protein. The infection is carried out in vitro according to techniques known to persons skilled in the art. For example, according to the type of cell used and the virus copy number desired per cell, persons skilled in the art can adjust the multiplicity of infection, and optionally, the number of infection cycles carried out. It is clearly understood that the method should be performed under suitable sterile conditions when the cells are intended for an administration in vivo. The doses of recombinant virus used for the infection of the cells can be adjusted by persons skilled in the art according to the desired aim. The conditions described above for the administration in vivo can be applied to the infection in vitro. For the infection with retroviruses, it is also possible to coculture the cells whose infection is desired with producer cells for the recombinant retroviruses according to the invention. This makes it possible to dispense with purifying the retroviruses.
Another subject of the invention concerns an implant comprising mammalian cells infected with one or more defective recombinant viruses as described above or recombinant virus producer cells, and an extracellular matrix. In one embodiment of the present invention, the implants according to the invention comprise 105 to 1010 cells. For example, they may comprise 106 to 108 cells.
In addition, in the implants of the invention, the extracellular matrix may comprise a gelling compound and optionally a support for anchoring the cells.
Various types of gelling agent can be used to prepare the implants according to the invention. The gelling agents may be used for embedding the cells in a matrix having the constitution of a gel, and to promote the anchoring of the cells onto the support, when needed. Various cell adhesion agents can thus be used as gelling agents, such as, for example, collagen, gelatin, glycosaminoglycans, fibronectin, lectins, etc. In one embodiment of the present invention, collagen is chosen as a gelling agent. It can be collagen of human, bovine or murine origin. For example, type I collagen is used.
As indicated above, the compositions according to the invention may advantageously comprise a support for anchoring the cells. The term “anchoring” refers to any form of biological and/or chemical and/or physical interaction which leads to the adhesion and/or binding of the cells onto the support. Moreover, the cells can either cover the support used, or penetrate inside this support, or both. In the context of the invention, a nontoxic and/or biocompatible solid support may be used. For example, polytetrafluoroethylene (PTFE) fibers or a support of biological origin can be used.
The present invention thus offers an effective means for treating or preventing the pathologies linked to cholesterol transport, such as obesity, hypertriglyceridemia or, in the field of cardiovascular disorders, myocardial infarction, angina, sudden death, heart failure and cerebrovascular accidents.
In addition, this treatment can concern both humans and any animal such as sheep, cattle, domestic animals (dogs, cats, etc.), horses, fish, etc.
Recombinant Host Cells
The invention also concerns a recombinant host cell comprising at least one of the nucleic acids of the invention chosen from sequence SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8, for example, a nucleic acid of sequence chosen from SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4 and SEQ ID No. 5.
According to another aspect, the invention also relates to a recombinant host cell comprising a recombinant vector as described above.
Host cells according to the invention are, for example, as follows:
a) prokaryotic host cells: strains of Escherichia coli (strain DH5-α), of Bacillus subtilis, of Salmonella typhimurium, or strains from species such as Pseudomonas, Streptomyces and Staphylococus; 
b) eukaryotic host cells: HeLa cells (ATCC No. CCL2), Cv 1 cells (ATCC No. CCL70), COS cells (ATCC No. CRL 1650), Sf-9 cells (ATCC No. CRL 1711), CHO cells (ATCC No. CCL-61) or 3T3 cells (ATCC No. CRL-6361), or cells of the line Hepa1-6 referenced at the American Type Culture Collection (ATCC, Rockville, Md., United States of America).
c) cells in primary culture, originating from an individual in whom the expression of a nucleic acid of interest, placed under the control of a regulatory nucleic acid according to the invention, is desired.
d) indefinitely-multiplying cells (cell lines), obtained from the cells in primary culture in c) above, according to the techniques well known to persons skilled in the art.
Screening Methods
In vitro Screening Method
The invention provides methods for treating an individual affected by a pathology linked to the level of expression of the ABC1 protein. For example, such a treatment method consists in administering to the individual a compound which modifies the expression of the ABC1 gene, and which can be identified according to diverse in vitro screening methods as defined below.
A first method consists in identifying compounds which modify the expression of the ABC1 gene. According to such a method, cells expressing the ABC1 gene are incubated with a candidate substance or molecule to be tested, and the level of expression of the ABC1 messenger RNA, or the level of production of the ABC1 protein, is then determined.
The levels of ABC1 messenger RNA can be determined with Northern-type gel hybridization, which is well known to persons skilled in the art. The levels of ABC1 messenger RNA can also be determined by methods using PCR or the technique described by WEBB and HURSKAINEN (1996, Journal of Biomolecular Screening, vol. 1: 119).
The levels of production of the ABC1 protein can be determined by immunoprecipitation or immunochemistry, using an antibody which specifically recognizes the ABC1 protein.
According to another method for screening a candidate molecule or substance which modifies the activity of a regulatory nucleic acid according to the invention, a nucleotide construct as defined above, comprising a regulatory nucleic acid according to the invention and a reporter polynucleotide placed under the control of the regulatory nucleic acid, is used, said regulatory nucleic acid comprising at least one basic promoter and at least one regulatory element from one of the sequences SEQ ID No. 1 and SEQ ID No. 2, a regulatory nucleic acid comprising a region ranging from nucleotides −2228 to +108 of sequence SEQ ID NO: 1, or a regulatory nucleic acid comprising the core promoter and the 200 pb proximal of the ABC1 gene promoter The reporter polynucleotide can be a gene encoding a detectable protein, such as a gene encoding a luciferase.
According to such a screening method, the cells are stably or transiently transfected with the polynucleotide construct containing the regulatory nucleic acid according to the invention and the reporter polynucleotide.
The transformed cells are then incubated in the presence or absence of the candidate molecule or substance to be tested, for a sufficient time, and then the level of expression of the reporter gene is determined. The compounds which induce a statistically significant change in the expression of the reporter gene (either an increase or, on the contrary, a decrease in the expression of the reporter gene) are then identified and, where appropriate, selected.
Thus, a subject of the invention is also a method for screening, in vitro, a molecule or a substance which modifies the activity of a regulatory nucleic acid according to the invention, for example, which modifies the transcription of the reporter polynucleotide which is a constituent of a polynucleotide construct according to the invention, characterized in that it comprises:
a) culturing a recombinant host cell comprising a polynucleotide of interest placed under the control of a regulatory nucleic acid according to the invention;
b) incubating the recombinant host cell with the substance or molecule to be tested;
c) detecting the expression of the polynucleotide of interest;
d) comparing the results obtained in c) with the results obtained when the recombinant host cell is cultured in the absence of the candidate molecule or substance to be tested.
The invention also concerns a kit or pack for screening, in vitro, a candidate molecule or substance which is capable of modifying the activity of a regulatory nucleic acid according to the invention, comprising:
a) a host cell transformed with a polynucleotide construct as defined above, comprising a reporter polynucleotide of interest placed under the control of a regulatory nucleic acid according to the invention; and
b) optionally, means for detecting the expression of the reporter polynucleotide of interest.
In one embodiment of the present invention, the reporter polynucleotide of interest is the sequence encoding a luciferase. In this embodiment, the regulatory nucleic acid according to the invention is inserted into a vector, upstream of the sequence encoding the luciferase. It can be for example, the vector pGL3-basic (pGL3-b) sold by the company PROMEGA (Madison, Wis., USA).
In this embodiment, the recombinant vector which comprises the sequence encoding the luciferase, placed under the control of a regulatory nucleic acid according to the invention, is transfected into hepatocellular carcinoma cells, such as the cells of the line HepG2, whose luciferase activity is then determined after culturing in the presence or absence of the candidate substance or molecule to be tested.
In this embodiment, pGL3-b vectors containing either the cytomegalovirus (CMV) promoter, the ApoAI promoter or no promoter can be used as controls. For the luciferase activity assay, the transfected cells are washed with a PBS buffer and lysed with 500 μl of lysis buffer (50 mM tris, 150 mM NaCl, 0.02% of sodium azide, 1% of NP-40, 100 μg/ml of AEBSF and 5 μg/ml of leupeptin).
50 μl of the cell lysate obtained are then added to 100 μl of the luciferase substrate (Promega) and the activity measurements are carried out on a spectrophotometric microplate reader, 5 minutes after adding the cell lysate.
The data are expressed in relative units of luciferase activity. The polynucleotide constructs which produce high levels of luciferase activity in the transfected cells are those which contain a regulatory nucleic acid according to the invention, included in the sequence SEQ ID No. 1 which is capable of stimulating transcription.
For the measurements of the levels of expression of messenger RNA in a screening method according to the invention, probes specific for the messenger RNA of the reporter polynucleotide of interest are first prepared, for example, using the kit multiprime labeling kit (sold by the company Amersham Life Sciences, Cleveland, Ohio, USA).
In vivo Screening Method
According to another aspect of the invention, the compositions which modify the activity of a regulatory nucleic acid according to the invention can be identified in vivo in non-human transgenic animals.
According to such a method, a non-human transgenic animal, for example, a mouse, is treated with a candidate molecule or substance to be tested, for example, a candidate substance or molecule which has been selected beforehand by an in vitro screening method as defined above.
After a given duration, the level of activity of the regulatory nucleic acid according to the invention is determined and compared to the activity of an identical nonhuman transgenic animal, for example, an identical transgenic mouse, which has not received the candidate molecule or substance.
The activity of the regulatory nucleic acid according to the invention, which is functional in the transgenic animal, can be determined by diverse methods, for example, measuring the levels of messenger RNA corresponding to the reporter polynucleotides of interest placed under the control of said regulatory nucleic acid, by Northern-type hybridization or by in situ hybridization.
According to one alternative, the activity of the regulatory nucleic acid according to the invention can be determined by measuring the levels of expression of protein encoded by the reporter polynucleotides of interest, for example, by immunohistochemistry, when the polynucleotide reporter of interest comprises an open reading frame encoding a protein which is detectable by such a technique.
For implementing a method for screening, in vivo, a candidate substance or molecule which modifies the activity of a regulatory nucleic acid according to the invention, nonhuman mammals may be used, such as mice, rats or guinea pigs or rabbits, whose genome is modified by inserting a polynucleotide construct comprising a reporter polynucleotide of interest placed under the control of a regulatory nucleic acid according to the invention.
The transgenic animals according to the invention comprise the transgene, i.e. the above-mentioned polynucleotide construct, in a plurality of their somatic and/or germ cells.
The construction of transgenic animals according to the invention can be carried out according to conventional techniques well known to persons skilled in the art. Persons skilled in the art may refer, for example, to the production of transgenic animals, such as to the production of transgenic mice, as described in U.S. Pat. No. 4,873,191 (granted on 10 Oct. 1989), U.S. Pat. No. 5,464,764 (granted on 7 Nov. 1995) and U.S. Pat. No. 5,789,215 (granted on 4 Aug. 1998), the contents of these documents being incorporated herein by way of reference.
Briefly, a polynucleotide construct comprising a regulatory nucleic acid according to the invention and a reporter polynucleotide of interest, placed under the control of the latter, is inserted into a line of stem cells of ES type. The insertion of the polynucleotide construct is carried out for example, by electroporation, as described by Thomas et al. (1987, Cell, Vol. 51:503-512).
The cells which have undergone the electroporation are then screened for the presence of the polynucleotide construct (for example, by selection with the aid of markers, or by PCR, or by Southern-type analysis of DNA on electrophoresis gels), in order to select the positive cells which have integrated the exogenous polynucleotide construct into their genome, when appropriate, following a homologous recombination event. Such a technique is described for example, by Mansour et al. (1988, Nature, Vol. 336:348-352).
Next, the positively selected cells are isolated, cloned and injected into 3.5-day old mouse blastocysts, as is described by Bradley (1987, Production and Analysis of Chimaeric mice. In: E. J. Robertson (Ed., Teratocarcinomas and embryonic stem cells: A practical approach. IRL Press, Oxford, page 113). Blastocysts are then introduced into a female host animal, and the development of the embryo is monitored until full term.
According to one alternative, positively selected cells of ES type are brought into contact with 2.5-day old embryos at an 8- to 16-cell stage (morulae), as described by Wood et al. (1993, Proc. Natl. Acad. Sci. USA, Vol. 90:4582-4585) or by Nagy et al. (1993, Proc. Natl. Acad. Sci. USA, Vol. 90:8424-8428), the ES cells being internalized so as to extensively colonize the blastocyst, including the cells which give rise to the germ line.
The descendants are then tested to determine those which have integrated the polynucleotide construct (the transgene).
A subject of the invention is thus also a non-human transgenic animal whose somatic and/or germ cells have been transformed with a nucleic acid or a polynucleotide construct according to the invention.
The invention also relates to recombinant host cells obtained from a transgenic animal as described above.
Recombinant cell lines originating from a transgenic animal according to the invention can be established in long term culture starting from any tissue from such a transgenic animal, for example, by transfecting primary cell cultures with vectors expressing oncogenes such as the large T antigen of SV40, as described for example, by Chou (1989, Mol. Endocrinol. Vol. 3:1511-1514) and Schay et al. (1991, Biochem. Biophys. Acta, Vol. 1072:1-7).
The invention also relates to a method for screening, in vivo, a candidate molecule or substance which modifies the activity of a regulatory nucleic acid according to the invention, comprising:
a) administering the candidate substance or molecule to a transgenic animal as described above;
b) detecting the level of expression of a reporter polynucleotide of interest placed under the control of the regulatory nucleic acid;
c) comparing the results obtained in b) with the results obtained in a transgenic animal which has not received the candidate substance or molecule.
The invention also relates to a kit or pack for screening, in vivo, a candidate molecule or substance which modifies the activity of a regulatory nucleic acid according to the invention, comprising:
a) a transgenic animal as defined above;
b) optionally, the means for detecting the level of expression of the reporter polynucleotide of interest.
Pharmaceutical Compositions and Compounds
The invention also concerns pharmaceutical compositions intended for preventing or for treating a deficiency in cholesterol metabolism, such as atherosclerosis, for example, in cholesterol transport, and in the reverse transport of cholesterol.
Firstly, a subject of the invention is also a candidate substance or molecule which modifies the activity of a regulatory nucleic acid according to the invention.
The invention also concerns a candidate substance or molecule characterized in that it increases the activity of a regulatory nucleic acid according to the invention, and for example, of a regulatory nucleic acid comprising the sequence SEQ ID No. 1 or SEQ ID No. 3, a region comprising the sequence ranging from nucleotides −2228 to +108 of sequence SEQ ID NO: 1, or a region comprising the core promoter and the 200 pb proximal of the ABC1 gene promoter.
For example, such a substance or molecule which is capable of modifying the activity of a regulatory nucleic acid according to the invention has been selected according to one of the in vitro or in vivo screening methods defined above.
Thus, an individual whose cholesterol metabolism is affected, for example, an individual affected by Tangier disease, is treated by administering to this individual an effective amount of a compound which modifies the activity of a regulatory nucleic acid according to the invention.
Thus, a patient with a weak activity of the ABC1 promoter can be treated with an abovementioned molecule or substance to increase the activity of the ABC1 promoter.
Alternatively, a patient with an abnormally high activity of the ABC1 promoter can be treated with a compound which is capable of decreasing or blocking the activity of the ABC1 promoter.
Such a compound can be a compound which modifies the interaction of at least one transcription factor with the ABC1 promoter or a regulatory element of a regulatory nucleic acid according to the invention.
For example, the compound can inhibit the interaction of one of the transcription factors listed in Table 1 with a regulatory nucleic acid according to the invention.
The compound can also be a compound which modifies the activity of a transcription factor which binds to the ABC1 promoter, or of a regulatory element present on this latter.
A compound of therapeutic interest according to the invention can also be a compound which modifies the interaction of a first transcription factor with a second transcription factor.
As detailed in the analysis of the various transcription factors which are capable of binding to the sequence SEQ ID No. 3, some transcription factors are active only if they are associated with another transcription factor.
A compound of therapeutic interest according to the invention is for example, chosen from nucleic acids, peptides and small molecules. For example, such a compound can be an antisense nucleic acid which binds specifically to a region of the ABC1 promoter or to a regulatory element of a regulatory nucleic acid of ABC1, and which inhibits or decreases the activity of the promoter.
This compound of therapeutic interest can also be an antisense nucleic acid which interacts specifically with a gene encoding a transcription factor which modifies the activity of the ABC1 promoter, such that the interaction of the antisense nucleic acid with the gene encoding the transcription factor which binds to the ABC1 promoter decreases the production of this transcription factor, resulting in an increase or a decrease in the activity of the ABC1 promoter, according to whether the transcription factor increases or, on the contrary, reduces the activity of the ABC1 promoter.
The toxicity and the therapeutic efficacy of the therapeutic compounds according to the invention can be determined according to the standard pharmaceutical protocols, in cells in culture or in experimental animals, for example, to determine the lethal dose LD50 (i.e. the dose which is lethal for 50% of the population tested) and the effective dose ED50 (i.e. the dose which is therapeutically effective in 50% of the population tested).
For all the compounds of therapeutic interest according to the invention, the effective therapeutic dose can be estimated initially from tests carried out in cell cultures in vitro.
A subject of the invention is also pharmaceutical compositions comprising a therapeutically effective amount of a substance or molecule of therapeutic interest according to the invention.
Such pharmaceutical compositions can be formulated conventionally, using one or more physiologically acceptable vectors or excipients.
Thus, the compounds of therapeutic interest according to the invention, as well as physiologically acceptable salts and solvates thereof, can be formulated for administration by injection or inhalation, or by oral, buccal, parenteral or rectal administration.
Techniques for preparing pharmaceutical compositions according to the invention can be easily found by persons skilled in the art, for example, in the work Remmington's Pharmaceutical Sciences, Mead Publishing Co., Easton, Pa., USA.
For a systemic administration, an injection, including intramuscular, intravenous, intraperitoneal and subcutaneous injections, may be used. In this case, the pharmaceutical compositions according to the invention can be formulated in the form of liquid solutions, for example, in physiologically compatible solutions or buffers.
Method for Detecting an Impairment of the Transcription of the Human ABC1 Gene
A subject of the invention is also methods for determining whether an individual presents a risk of developing a pathology linked to a deficiency in cholesterol metabolism, such as atherosclerosis, for example, in cholesterol transport, and in the reverse transport of cholesterol, such as a risk of developing Tangier disease.
Such methods comprise detecting, in cells from a biological sample originating from an individual to be tested, the presence or absence of a genetic alteration characterized by an impairment of the expression of a gene whose expression is regulated by the ABC1 promoter.
By way of illustration, such genetic alterations can be detected, in order to determine the existence of a deletion of one or more nucleotides in the sequence of a regulatory nucleic acid for ABC1 of sequence SEQ ID No. 1 or SEQ ID No. 2, of the addition of one or more nucleotides or of the substitution of one or more nucleotides in said sequence SEQ ID No. 1 or SEQ ID No. 2.
According to one embodiment of a method for detecting an impairment of the transcription of the ABC1 gene in an individual, the genetic alteration is identified according to a method comprising sequencing all or part of the sequence SEQ ID No. 1, or alternatively all or part of at least the sequence SEQ ID No. 2.
Sequencing primers can be constructed in order to hybridize with a given region of the sequence SEQ ID No. 1. Such sequencing primers are for example, constructed so as to amplify fragments of approximately 250 to approximately 300 nucleotides of the sequence SEQ ID No. 1 or of a complementary sequence.
The fragments amplified, for example, by the PCR method, are then sequenced, and the sequence obtained is compared with the reference sequence SEQ ID No. 1 in order to determine whether one or more deletions, additions or substitutions of nucleotides are found in the sequence amplified from the DNA contained in the biological sample originating from the individual tested.
The invention thus also concerns a method for detecting an impairment of the transcription of the ABC1 gene in an individual, comprising:
a) sequencing a nucleic acid fragment which can be amplified with the aid of at least one nucleotide primer which hybridizes with the sequence SEQ ID No. 1 or SEQ ID No. 2 according to the invention;
b) aligning the sequence obtained in a) with the sequence SEQ ID No. 1 or the SEQ ID No. 2;
c) determining the presence of one or more deletions, additions or substitutions of at least one nucleotide in the sequence of the nucleic acid fragment, with respect to the reference sequence SEQ ID No. 1 or SEQ ID NO. 2.
Oligonucleotide probes which hybridize with a region of the sequence SEQ ID No. 1 or the sequence SEQ ID No. 2 in which an alteration in the sequence has been determined during the implementation of the detection method described above also form part of the invention.
Alternatively, oligonucleotide probes which hybridize specifically with a corresponding region of the sequence SEQ ID No. 1 or of the sequence SEQ ID No. 2, for which one or more deletions, additions or substitutions of at least one nucleotide have been determined in an individual., also form part of the invention.
Such oligonucleotide probes constitute means for detecting alterations in the regulatory sequence for the ABC1 gene and thus also means for detecting a predisposition to developing a pathology linked to a deficiency in cholesterol metabolism, such as atherosclerosis or Tangier disease.
A subject of the invention is thus also a kit or pack for detecting an impairment of the transcription of the ABC1 gene in an individual, comprising:
a) one or more primers which hybridize with a region of the sequence SEQ ID No. 1 or of the sequence SEQ ID No. 2;
b) optionally, the means required for carrying out an amplification reaction.
A subject of the invention is also a kit or pack for detecting an impairment of the transcription of the ABC1 gene in an individual, comprising:
a) one or more oligonucleotide probes as defined above;
b) optionally, the reagents required for carrying out a hybridization reaction.
The nucleic acid fragments derived from any one of the nucleotide sequences SEQ ID No. 1 to 8 are thus useful for detecting the presence of at least one copy of a regulatory nucleotide sequence for the ABC1 gene or of a fragment or of a variant (containing a mutation or a polymorphism) of the latter, in a sample.
The nucleotide probes or primers according to the invention comprise at least 8 consecutive nucleotides of a nucleic acid chosen from the group consisting of the sequences SEQ ID No. 1 to 8, or of a nucleic acid of complementary sequence.
For example, nucleotide probes or primers according to the invention will have a length chosen from 10, 12, 15, 18, 20 to 25, 35, 40, 50, 70, 80, 100, 200, 500, 1000, or 1500 consecutive nucleotides of a nucleic acid according to the invention, such as a nucleic acid of nucleotide sequence chosen from the sequences SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8.
Alternatively, a nucleotide probe or primer according to the invention will consist of and/or will comprise the fragments with a length chosen from 12, 15, 18, 20, 25, 35, 40, 50, 100, 200, 500, 1000, and 1500 consecutive nucleotides of a nucleic acid according to the invention, or a nucleic acid chosen from the sequences SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8, or of a nucleic acid of complementary sequence.
The definition of a nucleotide probe and primer according to the invention thus encompasses oligonucleotides which hybridize, under the high stringency hybridization conditions defined above, with a nucleic acid chosen from the sequences SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8 or with a complementary sequence of these sequences.
Examples of primers and primer pairs for amplifying various regions of the ABC1 gene are represented below.
It is for example, the primer pair represented by the primer of sequence SEQ ID No. 12: 5′-TTG CCG TCG ACT GTT TTG GGT AGT TT-3′ and the primer of sequence SEQ ID No. 13: 5′-GCC CTG TCG ACC GGC TCT GTT GGT G-3′.
A nucleotide primer or probe according to the invention can be prepared by any suitable method well known to persons skilled in the art, including by cloning and restriction enzyme action, or by direct chemical synthesis according to techniques such as the phosphodiester method of Narang et al. (1979) or of Brown et al. (1979), the diethylphosphoramidite method of Beaucage et al. (1980) or the technique on a solid support described in EU Patent No. EP 0 707 592.
Each of the nucleic acids according to the invention, including the oligonucleotide probes and primers described above, can be labeled, if desired, by incorporating a detectable label, by spectroscopic, photochemical, biochemical, immunochemical or chemical means.
For example, such labels can comprise radioactive isotopes (32P, 33P, 3H, 35S), fluorescent molecules (5-bromodeoxyuridine, fluorescein, acetyl-aminofluorene, digoxigenin) or ligands such as biotin.
The labeling of the probes is for example, carried out by incorporation of labeled molecules into the polynucleotides by primer extension, or by addition onto the 5′ or 3′ ends.
Examples of nonradioactive labeling of nucleic acid fragments are described, for example, in French Patent No. FR 78 109 75 or in the articles by Urdea et al. (1988) or Sanchez-pescador et al. (1988).
Advantageously, the probes according to the invention can have structural properties of a type which enables an amplification of the signal, such as the probes described by Urdea et al. (1991), or in European Patent No. EP-0 225 807 (Chiron).
The oligonucleotide probes according to the invention can be used, for example, in Southern-type hybridizations to genomic DNA.
The probes according to the invention can also be used for detecting PCR amplification products or for detecting mismatches.
Nucleotide probes or primers according to the invention can be immobilized on a solid support. Such solid supports are well known to persons skilled in the art, and comprise surfaces of the wells of microtitration plates, polystyrene beds, magnetic beds, nitrocellulose bands or microparticles such as latex particles.
Consequently, the present invention also concerns a method for detecting the presence of a nucleic acid as described above in a sample, said method comprising:
1) bringing one or more nucleotide probes according to the invention into contact with the sample to be tested;
2) detecting the complex possibly formed between the probe(s) and the nucleic acid present in the sample.
According to one embodiment of the detection method according to the invention, the oligonucleotide probe(s) are immobilized on a support.
According to another aspect, the oligonucleotide probes comprise a detectable label.
The invention also concerns a pack or kit for detecting the presence of a nucleic acid according to the invention in a sample, said pack comprising:
a) one or more nucleotide probes as described above;
b) optionally, the reagents required for the hybridization reaction.
According to a first aspect, the detection pack or kit is characterized in that the probe(s) is (are) immobilized on a support.
According to a second aspect, the detection pack or kit is characterized in that the oligonucleotide probes comprise a detectable label.
According to one embodiment of the detection kit described above, such a kit will comprise a plurality of oligonucleotide probes in accordance with the invention, which may be used for detecting target sequences of interest, or alternatively detecting mutations in the coding regions or the noncoding regions of the nucleic acids according to the invention, for example, of the nucleic acids of sequences chosen from SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, and SEQ ID No. 5 or the nucleic acids of complementary sequence.
Thus, the probes according to the invention which are immobilized on a support can be ordered in matrices such as “DNA chips”. Such ordered matrices have been described, for example, in U.S. Pat. No. 5,143,854 and in PCT Applications No. WO 90/150 70 and 92/10092.
Support matrices onto which oligonucleotide probes have been immobilized at a high density are, for example, described in U.S. Pat. No. 5,412,087 and in PCT Application No. WO 95/11995.
The nucleotide primers according to the invention can be used to amplify any one of the nucleic acids according to the invention, and for example, all or part of a nucleic acid of sequences chosen from SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, and SEQ ID No. 5, or a variant of this nucleic acid.
Another subject of the invention concerns a method for amplifying a nucleic acid according to the invention, and for example, a nucleic acid sequence chosen from SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, and SEQ ID No. 5, or a fragment or a variant of this nucleic acid, contained in a sample, said method comprising:
a) bringing the sample, in which the presence of the target nucleic acid is suspected, into contact with a pair of nucleotide primers, of which the hybridization position is located respectively on the 5′ side and on the 3′ side of the region of the target nucleic acid whose amplification is sought, in the presence of the reagents required for the amplification reaction; and
b) detecting the amplified nucleic acids.
To implement the amplification method as described above, any one of the nucleotide primers described above will advantageously be used.
A subject of the invention is also a pack or kit for amplifying a nucleic acid according to the invention, and for example, all or part of a nucleic acid sequence chosen from SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, and SEQ ID No. 5, said pack or kit comprising:
a) a pair of nucleotide primers in accordance with the invention, the hybridization position of which is located respectively on the 5′ side and on the 3′ side of the target nucleic acid whose amplification is sought;
b) optionally, the reagents required for the amplification reaction.
Such an amplification pack or kit will advantageously comprise at least one pair of nucleotide primers as described above.
The following examples are intended to illustrate the invention without limiting the scope thereof.
EXAMPLES
Example 1 
Tissue Distribution of the ABC1 Gene Transcripts According to the Invention
The expression profile of the polynucleotides according to the present invention was determined according to the reverse transcription-coupled PCR and Northern blot analysis protocols described, for example, by Sambrook et al. (ref. CSH Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). “Molecular Cloning: A Laboratory Manual”, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
For example, in the case of an analysis by reverse transcription, a pair of primers which have been synthesized from the human ABC1 gene full-length cDNA of sequence SEQ ID No. 10 was used for detecting the corresponding cDNA.
The polymerase chain reaction (PCR) was carried out on matrices of cDNAs corresponding to reverse-transcribed polyA+ mRNAs (Clontech). The reverse transcription into cDNA was carried out with the enzyme Superscript II (GibcoBRL, Life Technologies), according to the conditions described by the manufacturer.
The polymerase chain reaction was carried out according to standard conditions, in 20 μl of reaction mixture, with 25 ng of the cDNA preparation. The reaction mixture was composed of 400 μM of each of the dNTPs, of 2 units of Thermus aquaticus (Taq) DNA polymerase (Ampli Taq Gold; Perkin Elmer), of 0.5 μM of each primer, of 2.5 mM MgCl2 and of PCR buffer. Thirtyfour cycles of PCR (denaturation for 30 s at 94° C., hybridization of 30 s, broken down as follows during 34 cycles: 64° C. 2 cycles, 61° C. 2 cycles, 58° C. 2 cycles and 55° C. 28 cycles, and an elongation of one minute per kilobase at 72° C.) were carried out after a first denaturation at 94° C. for 10 min, in a Perkin Elmer 9700 thermocycler machine. The PCR reactions were visualized on agarose gels by electrophoresis. The cDNA fragments obtained could be used as probes for a Northern blot analysis and could also be used for exactly determining the polynucleotide sequence.
In the case of an analysis by Northern blot, a cDNA probe produced as described above was labeled with 32P, using the High Prime DNA labeling system (Boehringer), according to the instructions indicated by the manufacturer. After labeling, the probe was purified on a Sephadex G50 microcolumn (Pharmacia), according to the instructions indicated by the manufacturer. The labeled and purified probe was then used for detecting the expression of the mRNAs in various tissues.
The Northern blot which contained samples of RNA from various human tissues ((Multiple Tissue Northern, MTN, Clontech) Blot 2, reference 77759-1) was hybridized with the labeled probe.
The protocol which was followed for the hybridizations and washes were either directly that described by the manufacturer (Instruction manual PT1200-1), or an adaptation of this protocol using the methods which are known to persons skilled in the art, and which are described for example, in F. Ausubel et al. (1999). For example, the temperatures of prehybridization and of hybridization in the presence of formamide may thus be varied.
For example, the following protocol was used:
1—Membrane Competition and Prehybridization:


    
    
        Mixed: 40 μl salmon sperm DNA (10 mg/ml)
        
            +40 μl human placental DNA (10 mg/ml)
        
        
        Denatured for 5 min at 96° C., then immersed the mixture in ice.
        Removed the 2×SSC buffer and poured 4 ml of formamide mix into the hybridization tube containing the membranes.
        Added the mixture of the two denatured DNAs.
        Incubation at 42° C. for 5 to 6 hours, with rotation.

2—Labeled Probe Competition:

        Added 10 to 50 μl Cot I DNA to the labeled and purified probe, according to the amount of repeated sequences.
        Denatured for 7 to 10 min at 95° C.
        Incubated at 65° C. for 2 to 5 hours.

3—Hybridization:

        Removed the prehybridization mix.
        Mixed 40 μl salmon sperm DNA +40 μl human placental DNA; denatured 5 min at 96° C., then immersed in ice.
        Added 4 ml of formamide mix, the mixture of the two DNAs and the denatured labeled probe/Cot I DNA to the hybridization tube.
        Incubated for 15 to 20 hours at 42° C., with rotation.

4—Washes:

        One wash at room temperature in 2×SSC to rinse.
        Twice 5 minutes at room temperature, 2×SSC and 0.1% SDS, at 65° C.
        Twice 15 minutes at 65° C., 1×SSC and 0.1% SDS, at 65° C.
    
    


After hybridization and washing, the blot was analyzed after overnight exposure in contact with a phosphor screen, which was revealed with the aid of Storm (Molecular Dynamics, Sunnyvale, Calif.).
Example 2
Analysis of the Profile of Gene Expression for Tangier Disease
Verification of the impairment in the expression level of the ABC1 gene, which leads to the Tangier cellular phenotype determined by hybridization of these sequences with probes corresponding to the mRNAs which originate from fibroblasts from individuals possibly suffering from the disease, according to the methods described below:
1. Preparation of Total RNAs, of Poly(A)+ mRNAs and of cDNA Probes
The total RNAs were obtained from cell cultures of the fibroblasts from individuals who were normal or suffering from Tangier disease, by the guanidine isothiocyanate method (Chomczynski & Sacchi, 1987). The poly(A)+ mRNAs were obtained by affinity chromatography on cellulose-oligo(dT) columns (Sambrook et al., 1989), and the cDNAs used as probes were obtained by RT-PCR (DeRisi et al., 1997) with oligonucleotides which were labeled with a fluorescent product (Amersham Pharmacia Biotech; CyDyeTM).
2. Hybridization and Detection of Expression Levels
The glass fiber membranes containing the sequences according to the present invention which correspond to the Tangier gene were hybridized with the cDNA probes, which were obtained from fibroblasts (Lyer et al., 1999). Using the Amersham/Molecular Dynamics system (Avalanche MicroscannerTM) allowed the quantification of the expressions of the sequence products in the healthy or affected cell type.
Example 3 
Use of IL-1Beta-Expressing THP-1 Macrophages for Screening Molecules which Activate or Inhibit the Expression of the ABC-1 Gene
The principle of this assay is that any substance which modifies the synthesis activity of the ABC1 protein has repercussions on the synthesis of IL-1beta.
a) The macrophage cells of the THP-1 lines, which are human monocytic leukemia cells, are a model of differentiated macrophages. These cells were cultured in an RPMI 1640 medium supplemented with 10% of fetal calf serum, in multiwell plates, at a density of 2 105 cells per wells.
b) For the assay per se, the cells were then washed and placed in an RPMI 1640 medium containing 1 mg/ml of purified human albumin fraction IV.
c) The products were added into the extracellular medium. Simultaneously, the cells were then activated by addition of lipopolysaccharides (LPS) for 3 hours, at 1 μg/ml, followed by an incubation of 30 minutes in the presence of ATP at 5 mmol/L.
d) The concentrations of IL-1beta and of control IL-1alpha, tumor necrosis factor alpha (TNFalpha) and IL-6 were determined with ELISA kits, according to the manufacturers' instructions (R&D System; human IL-1beta Chemiluminescent ELISA reference QLB00). The variations in IL-1beta mRNA, which was not supposed to be affected, were evaluated by the Northern blot technique, using the corresponding probe.
Example 4
Expression of a Gene of Interest Under the Control of a Regulatory Nucleic Acid for the Human ABC1 Gene According to the Invention
4.1 Materials and Methods
4.1.1 Construction of Expression Plasmids Containing a Regulatory Nucleic Acid for the Human ABC1 Gene.
The region of the regulatory acid for the human ABC1 gene which ranges from the nucleotide at position −-995 up to the nucleotide at position +120, with respect to the transcription start site, was amplified by the PCR technique, with the aid of the following pair of primers:

    forward primer S995 (SEQ ID No. 12), of sequence 5′-TTG CCG TCG ACT GTT TTG GGT AGT TT-3′; and
    reverse primer +220R (SEQ ID No. 13), of sequence 5′-GCC CTG TCG ACC GGC TCT GTT GGT-3′,
    From human genomic DNA present in a BAC vector from a collection of human BAC vectors.


The DNA fragment amplified was digested with the restriction endonuclease Sal 1, and then inserted into the vector PXP1 described by Nordeen et al. (1988, BioTechniques, 6: 454-457), at the Sal 1 restriction site of this vector.
The insert was then sequenced.
4.1.2. Cell Culture and Transfection
Cells of the Hepa1-6 line (ATCC, Rockville, Md., USA) were cultured in the medium E-MEM (Minimum Essential Medium with Earle's Salts), to which 10% (v/v) of fetal calf serum (BioWhittaker, Walkersville, Md.) was added.
Approximately 1.5×105 cells were distributed into each of the wells of a 12-well (2.5 cm) culture plate, and were cultured until approximately 50 to 70% confluence, and were then cotransformed with 1 μg of the plasmid Sal-Lucif and 0.5 μg of the control vector pBetagel (CloneTech Laboratories Inc., Palo Alto, Calif., USA) using the Superfectin Reagent Kit pack (QIAGEN Inc., Valencia, Calif., USA). Two hours after adding the DNA, the culture medium was removed, and replaced with complete AMEM (Minimum Essential Medium Eagle's Alpha Modification) medium.
After a duration of twenty hours, the cells were placed in fresh medium such as DMEM (Dulbecco's Minimum Essential Medium), to which 2 μg/ml of glutamine, 100 units/ml of streptomycin and 0.1% of bovine serum albumin (BSA, Fraction V) were added, in the presence or absence of 50 μg/ml of cholesterol (Sigma Chemical Co., St Louis, Missouri, Mo., USA).
The cells were recovered 16 hours after the final change of medium using a lysis solution from the Tropix Luciferase Assay Kit pack (Tropix Inc., Bedford, Mass., USA).
The cell lysate was divided into aliquot fractions which were stored at −70° C.
Freshly thawed aliquot fractions were used to quantify the proteins, using the MicroBCA Kit pack (Pierce, Rockford, Ill., USA), as well as to quantify the luciferase and beta-galactosidase production, using, respectively, the Tropix Luciferase Assay Kit and Galacto-Light Plus Kit packs. The assays were carried out according to the manufacturer's recommendations.
4.2 Results
The results are represented in Table 2 below:







TABLE 2







Luciferase production by the cells transfected with a vector containing the


luciferase gene placed under the control of a regulatory nucleic acid for the


human ABC1 gene according to the invention, in the presence or absence


of cholesterol.













β-Galacto-

Increase


Culture
Luciferase
sidase
Normalized
in the


conditions
activity
activity
activity
activity














Without
215 523 +/−
29 548 +/−
7.29
—


cholesterol
20 018
1342


In the
500 126 +/−
37 741 +/−
13.25
1.82


presence of
100 069
2813


cholesterol









The results above show the capacity of a regulatory acid for the human ABC1 gene according to the invention to direct the expression of a coding sequence placed under its control.
In addition, the regulatory nucleic acid used, which stretches from the nucleotide at position −995 to the nucleotide at position +120, with respect to the transcription start site, and which contains all the PPAR sites identified, was regulated by cholesterol.
Example 5
Characterization of the Transcription Factor Binding Motifs in the Proximal Human ABC1 Gene Promoter
5.1 Materials and Methods
5.1.1 Construction of Reporter Plasmids for Luciferase Assay
Plasmids containing mutant SP1, AP1, E-box, LXR and deleted E-box fragments were constructed by site-directed mutagenesis using the overlap PCR method and the PXP1 −995 to +120 bp construct (Previato et al., JBC, 1991, 266:18958-63) as template. The primers listed below were used to amplify −200 to +44 bp of the human ABC1 promoter. Upper-case letters represent wild-type sequence whereas lower-case letters represent mutant sequence.










MDistal SP1F
5′TCGCCCGTTTAgGcttgGGcgCCCGGCTC3′
(SEQ ID NO: 14)



MDistal SP1R:
5′GAGCCGGGcgCCcaagCcTAAACGGGCGA3′
(SEQ ID NO: 22)


 


MProximal SP1F:
5′CAGAGGCCGGGAgGcttgGGcgGGAGGGA3′
(SEQ ID NO: 15)


MProximal SP1R:
5′TCCCTCCcgCCcaagCcTCCCGGCCTCTG3′
(SEQ ID NO: 23)


 


MAP1F:
5′CGTGCTTTCTGCTGAGgatgcGAACTAC3′
(SEQ ID NO: 16)


MAP1R:
5′GTAGTTCgcatcCTCAGCAGAAAGCACG3′
(SEQ ID NO: 24)


 


MEBoxF:
5′CGGCTCCtcacggCTTTCTGCTGAGT3′
(SEQ ID NO: 17)


MEBoxR:
5′ACTCAGCAGAAAGccgtgaGGAGCCG3′
(SEQ ID NO: 25)


 


DEboxF:
5′GCCTCCTTTCTGCTGAGTGACTGA3′
(SEQ ID NO: 18)


DEboxR:
5′GAAAGGAGCCGGGGCCCGCCCCA3′
(SEQ ID NO: 26)


 


MLXRF:
5′CTTTGtgtGATAGTAAActaCTGCGCTCGGTGCA
(SEQ ID NO: 19)


MLXRR
5′TGCACCGAGCGCAGtagTTACTATCacaCAAAG
(SEQ ID NO: 27)


 


S224-HindIII:
5′ACTCCCAAGCTTTGTCGTGG3′
(SEQ ID NO: 20)


44-HindIII:
5′GAGAAGCTTCGGCTCGGCTCTG3′
(SEQ ID NO: 28)






S224-HindIII and 44-HindIII were the upstream and downstream primers used for overlap PCR. The Hind III sites are underlined. The resulting fragments which spanned −200 to +44 of the human ABC1 gene were ligated into the HindIII site of the PXP1 luciferase reporter plasmid (Nordeen et al., Biotechniques, 1988, 6:454-457). All constructs were confirmed by sequencing.
5.1.2. Cell Culture and Transfection
Murine macrophage RAW 264.7 cells and human embryonic kidney 293 cells (American Type Culture Collection, Rockville, Md.) were grown in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum (FCS). Approximately 1.5×105 cells were plated in 12-well plates (Costar, Corning, N.Y.), grown to 50-70% confluency and cotransfected with 1.5 μg of the ABC1 promoter-luciferase plasmid and 0.5 μg of the β-galactosidase vector (pCMVβ;Clontech, Palo Alto, Calif.) by using the Superfectin Reagent Kit (Qiagen, Valencia, Calif.). Three hours after addition of DNA, the cells were refed with fresh media containing 10% FCS. Sixteen hours later, cells were washed with phosphate-buffered saline (PBS) and refed with DMEM containing 0.1% bovine serum albumin (BSA) and either 50 μg/ml cholesterol, 2 μg/ml 22-(R)-hydroxycholesterol (22(R)-Hch), 10 μM 9-cis-retinoic acid (9CRA), 10-100 nM estradiol (Sigma, St. Louis, Mo.), 10-100 nM regular insulin (Sigma) or 0.1% ethanol for 24 hours. After harvesting, 10 μl of cell extracts were used for luciferase and β-galactosidase assays by using the Promega dual luciferase assay system (Promega, Madison, Wis.) or the Tropix Galacto-Light Plus Kits, respectively (Tropix, Bedford Mass.). The ratio of luciferase activity in relative light units was divided by the β-galactosidase activity to give a normalized luciferase value.
5.1.3. Gel Mobility Shift Assay
Three double-stranded human ABC1 promoter fragments (Fragment A spanning −171 to −71 bp; Fragment EB spanning −156 to −130 bp and Fragment EB spanning −156 to −130 bp) were end-labeled with α32P-dATP using T4 polynucleotide kinase (Lofstrand, Gaithersburg Md.). Nuclear extracts were isolated from unstimulated RAW 264.7 cell and HepG2 cell as well as RAW 264.7 cells after stimulation with the same concentrations of cholesterol and 22(R)-Hch shown above (Paragon Bioservices Inc. Baltimore, Md.). One ng (10,000 cpm) of radiolabelled probe was added to 2.5 μg nuclear extract in 20 ul of a 20 mM TRIS gel shift buffer (pH 7.9) containing 60 mM KCl, 0.2 mM EDTA, 0.5 mM DTT, 0.25 mM PMS, 1.3 mM MgCl, 10% glycerol, 3% Ficoll and 3 μg of double-stranded poly (dldC) as described (Previato et al., J Biol Chem, 1991, 266:18958-18963) and incubated for 10 minutes on ice followed by 10 minutes at room temperature. The incubated mixture was loaded on a 6% polyacrylamide gel in 0.25×TBE buffer and electrophoresed at 100 V for 90 min followed by autoradiography. For competition assays, nuclear extracts were preincubated for 10 minutes on ice in a 20 μl reaction mixture in the presence or absence of a 100-200-fold excess of double-stranded DNA competitors for Sp1 (−173 to −155 bp), AP1 (−135 to −155 bp), LXR (−54 to −69 bp) and E-box (−158 to −136 bp) before addition of probe. For supershift assays, nuclear extracts were preincubated with antibodies against different E-box binding proteins including Mad1, Mad2, Mad3, Max, c-Myc, MyoD, USF1 and USF2 as well as Sp1, c-Fos, c-Jun, JunB and JunD (Santa Cruz Biotechnology, Santa Cruz, Calif.) on ice for 30 minutes before addition of probe.
5.1.4. DNAse I Protection Assay
End-labelled fragment A was digested with AspHI. The 94 bp fragment was gel-purified from a 10% acrylamide TBE gel (Novex) and added to 14 μl of RAW cell nuclear extract (5.2 μg/μl protein) in gel shift buffer (Example 5.1.3) and incubated on ice for 10 min. One μl of probe (10,000 cpm was added and the mixture was incubated on ice for another 10 min. After 10 min at RT, 20 μl DNaseI digestion buffer (10 mM Tris-HCl pH 8.0, 5 mM CaCl2, 5.0 mM MgCl2) was added, then 15 seconds later DNase I was added and incubated for 1 min 45 sec. DNaseI stop buffer (10 mM Tris-HCl pH 8.0, 0.6M sodium acetate pH 7.0, 0.5% SDS, 100 mM EDTA) was then added. 2.0 μl of protease K at 20 mg/ml were added and the samples were incubated at 37° C. for 30 min. 10 μl of 3M NaOAc and 4.0 μl tRNA (10 mg/ml) were added. Samples were phenol/chloroform extracted and the aqueous phase was precipitated with 2.5 volumes of 100% ethanol. After a 70% ethanol wash, pellets were dissolved in sequencing gel loading buffer, heated and run on an 8% sequencing gel. Naked DNA was digested with DNaseI as described above except that nuclear extract addition and Protease K treatment were omitted. Maxam-Gilbert sequencing was performed as described in Current Protocols in Molecular Biology (Aubusel et al., Current Protocols in Molecular Biology, 1994, 2: 12.1-12.11).
5.1.5 Western Analysis
RAW cell nuclear extracts (35 μg protein per lane) from cells stimulated with cholesterol or 22-R-hydroxycholesterol were loaded onto NuPage Bis-Tris 4-12% gradient gels (Invitrogen, Carlsbad, Calif.) and run according to manufacturer's specifications. Proteins were transferred to Immobilon-P PVDF membranes (Millipore Corp, Bedford, Mass.). Antibodies (2 mg/ml stock) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif.) and were used according to manufacturer's specifications. Anti-USF1 C20X (catalog number sc-229X) antibody was used at a dilution of 1/600 and anti-USF2 antibodies C20X (catalog number sc862X) and N18X (catalog number sc861X) were used at a dilution of 1/1000.
5.2.1 Analysis of Binding Motifs in the Proximal Human ABC1 Gene Promoter
To investigate the role of some of the above described transcription factor binding motifs in this region of the human ABC1 gene promoter, i.e., Sp1 (−100 and −166 bp), AP1 (−131 bp), LXR (−69 bp) and E-box (−147 bp), luciferase reporter constructs under the control of the −200 bp human ABC1 promoter, either wild-type (p200-L) or mutated have been generated according to Example 5.1.1. FIG. 2A shows the locations of point mutations introduced into the −200 bp promoter region of the human ABC1 gene.
The effect of these point mutations on the transcriptional activity of the human ABC1 gene in unstimulated RAW cells is illustrated in FIG. 2B. Mutating the distal Sp1 site, the AP1 site and the proximal Sp1 sites had only a minor effect on promoter activity. In contrast, mutation of the E-box caused a strong and significant increase in promoter activity and mutation of the LXR element caused a strong and significant decrease in promoter activity. These results are consistent with binding of a transcriptional repressor to the human ABC1 E-box and binding of a transcriptional activator to the LXR element.
5.2.2 Mutation of the LXR Element Reduces Transcription of the hABC1 Gene
FIG. 3 shows that the LXR element is implicated as the human ABC1 promoter motif responsive to oxysterols. Mutation of the LXR element at −69 bp caused a significant decrease in transcription for unstimulated cells as well as for cells stimulated by cis-retinoic acid (CRA) and 22(R)-hydroxycholesterol (22OH). Mutation of the LXR element also caused a significant decrease in transcription for cholesterol-stimulated cells, presumably due to the intracellular conversion of cholesterol to oxysterols. Thus, the LXR element mediates responsiveness to cholesterol as well as to cis-retinoic acid and to hydroxysterols.
5.2.3 Mutation of the E-box Increases Transcription of the Human ABC1 Gene
FIG. 4 demonstrates that mutation or deletion of the E-box motif increases transcription of the human ABC1 gene in unstimulated RAW cells by approximately 3-fold and also in RAW cells stimulated with cholesterol (c), cis-retinoic acid (CRA) and oxysterol (22OH) by up to 40-fold. Furthermore, mutation or deletion of the E-box in the proximal human ABC1 promoter had no effect on the stimulatory effect of either CRA or oxysterols. Similar findings were demonstrated for unstimulated and stimulated human embryonal kidney 293 cells. These results indicate that the LXR-mediated activation of ABC1 gene transcription by CRA and oxysterols does not require an intact E-box motif and are consistent with binding of a transcriptional repressor to the E-box in the human ABC1 gene.
5.2.4 Nuclear Transcription Factors Bind the E-Box in the Human ABC1 Gene Promoter
DNaseI footprint analysis of the ABC1 proximal promoter revealed protection of the E-box in the presence of RAW nuclear extracts (FIG. 5) indicative of a protein binding to this region. Consistent with the lack of transcriptional effects observed by independently mutating the AP1 and Sp1 motifs (FIG. 2B), no protection of these potential binding sites in the human ABC1 promoter was evident by footprint analysis (FIG. 5).
To further demonstrate binding of nuclear transcription factors to the E-box motif we performed gel-shift analysis of the human ABC1 promoter (FIG. 6). The probe utilized in FIG. 6B (left) was a 100 bp double-stranded fragment spanning −171 through −71 of the ABC1 promoter (designated Fragment A). Incubation of the radiolabelled probe with nuclear extract isolated from unstimulated RAW cells resulted in a gel shift (FIG. 6B). The shift was abolished when either unlabelled Fragment A (A) or a double-stranded oligonucleotide spanning the E-box (EB) were used as competitors. Competition with the 27 bp fragment encoding a scrambled E-box (EBm) did not abolish the gel shift band. Competition by double-stranded DNA fragments spanning either the proximal or distal Sp1 motifs or the AP1 binding sites did not abolish the gel shift band. Similar results were observed when nuclear extracts from unstimulated 293 cells were utilized.
Gel-shift analysis of the human ABC1 promoter was also performed using either a 27 bp double-stranded probe spanning the E-box (FIG. 6A; Fragment EB; right panel) or an alternative probe containing a scrambled mutant E-box sequence as described above (Fragment EBm). Incubation of the wild-type E-box probe with unstimulated RAW cell nuclear extract resulted in a gel shift, indicating binding of a protein to this probe (FIG. 6B; middle panel). Addition of unlabelled wild-type competitor (EB) eliminated binding. In contrast, competition with the mutant E-box fragment (EBm) did not significantly affect binding to the wild-type E-box. Moreover, using the mutant E-box as a target probe for the binding of cellular nuclear extracts did not result the formation of a gel shift band (FIG. 6B; right panel). This clearly demonstrates specific binding of a protein to the wild-type E-box motif of the human ABC1 gene.
5.2.5 USF Binds the E-Box in the hABC1 Gene Promoter
In the human ABC1 promoter, the E-box is flanked by two C's, leading to a sequence of CCACGTGC. This is a perfect match to the consensus motif for the transcription factor USF. To establish that USF is, in fact the transcription factor that that binds to the E-box in the hABC1 gene promoter, a gel shift analysis utilizing USF-specific antibodies was performed (FIG. 6C). Using the 100-bp fragment as a probe (FIG. 6C, left panel), it was demonstrated that the E-box gel-shifts some protein in the RAW cell nuclear extract. Addition of anti-USF antibodies against either the amino (N) or carboxy (C) terminus of USF1 or USF2 caused a supershift of the gel-shifted probe, confirming the identity of the E-box binding proteins as USF1 and USF2. Antibodies against other E-box binding proteins including Mad1, Mad2, Mad3, Max, c-Myc, and MyoD as well as Sp1, c-Jun, JunB and JunD did not compete or supershift the DNA-protein gel shift band.
Similar results were obtained by preincubating the 27 bp double-stranded fragment spanning the E-box (EB) with anti-USF antibodies (FIG. 6C; right panel). As with the 100 bp gel shift fragment, antibodies specific to other members of the helix-loop-helix family of transcription factors known to also bind the E-box motif did not alter the gel shift band obtained with the 27 bp EB probe. No differences have been shown in the gel shift banding patterns obtained when nuclear extracts isolated from unstimulated RAW cells and RAW cells stimulated with cholesterol or oxysterols were incubated with the EB probe.
This combined data identify USF1 and USF2 as the transcriptional factors that bind to the E-box in the proximal ABC1 promoter and indicate that their binding is not modulated by known activators of ABC1 gene expression.
5.2.6 USF1 and USF2 are Expressed in RAW Cells
The presence of USF in RAW cell nuclear extracts was establish by performing Western blot hybridization analyses utilizing antibodies specific to USF1 (N- and C-terminus) and USF2 (N- and C-terminus). Two major immunoreactive bands of approximately 43 and 44 KDa in size were identified whose expression in RAW cells were not altered by stimulation with cholesterol or oxysterols. Importantly, expression of the 18 kDa mini-USF isoform which lacks the carboxy-terminus transcriptional activating domain (Liu et al., JBC, 1999, 274: 35037-35045; Sirito et al., Nucl Acids Res, 1994, 22:427-433) was not detected in either unstimulated RAW cells or RAW cells incubated for 24 hours with cholesterol or oxysterols.
All articles, patents, and patent applications mentioned in this specification are herein incorporated by reference.
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## Claims

1. An isolated nucleic acid comprising a polynucleotide having the nucleotide sequence from nucleotide 666 to nucleotide 3001 of the nucleotide sequence SEQ ID NO: 1, or the complement of the nucleotide sequence from nucleotide 666 to nucleotide 3001 of SEQ ID NO: 1.

2. An isolated nucleic acid, comprising a polynucleotide comprising nucleotides 1576 to 2893 of SEQ ID No. 3.

3. An isolated nucleic acid, wherein said isolated nucleic acid is a polynucleotide comprising nucleotide 2694 to nucleotide 2893 of the nucleotide sequence SEQ ID NO: 1.

4. The isolated nucleic acid according to claim 3, wherein said isolated nucleic acid is a polynucleotide comprising the nucleotide sequence from nucleotide 2594 to nucleotide 2893 of the nucleotide sequence SEQ ID NO: 1.

5. The isolated nucleic acid according to claim 3, wherein said isolated nucleic acid is a polynucleotide comprising the nucleotide sequence from nucleotide 2294 to nucleotide 2893 of the nucleotide sequence SEQ ID NO: 1.

6. The isolated nucleic acid according to claim 3, wherein said isolated nucleic acid is a polynucleotide comprising the nucleotide sequence from nucleotide 1 to the nucleotide 2893 of the nucleotide sequence SEQ ID NO: 1.

7. An isolated nucleic acid, wherein said isolated nucleic acid is a polynucleotide comprising the nucleotide sequence from nucleotide 1899 to nucleotide 3013 of the nucleotide sequence SEQ ID NO: 1.

8. The isolated nucleic acid according to claim 1, wherein said isolated nucleic acid is a polynucleotide comprising the nucleotide seciuence from nucleotide 654 to nucleotide 3001 of the nucleotide sequence SEQ ID NO: 1 or the complete complement thereof.

9. A recombinant vector comprising at least one isolated nucleic acid according to claim 1.

10. The recombinant vector according to claim 9, wherein said vector is chosen from a recombinant cloning vector and a recombinant expression vector.

11. An isolated host cell transformed with a recombinant vector according to claim 9.