ZA200110545B - Nucleic and proteinic acids corresponding to human gene ABC1. - Google Patents

Description

® 2G 54 5 ¢ WO 00/78970 PCT/FR00/01595

NUCLEIC ACIDS AND PROTEINS CORRESPONDING TO THE HUMAN ABC1 GENE

The present invention relates to nucleic acids corresponding to the various exons and introns of the ABC1 gene, for which it is now demonstrated that it is a causal gene for pathologies linked to a cholesterol metabolism dysfunction inducing diseases such as atherosclerosis, more particularly disruption in the reverse transport of cholesterol, and more particularly familial

HDL deficiencies (FHD), such as Tangier disease. The invention also relates to means for the detection of polymorphisms in general, and of mutations in particular, in the ABC1 gene or in the corresponding protein produced by the allelic form of the ABC1 gene. The invention also provides pharmaceutical compositions comprising a nucleic acid containing the coding region of the

ABC1 gene and pharmaceutical compositions containing the ABC1 protein intended for the treatment of diseases linked to a deficiency in the reverse transport of cholesterol, such as Tangier disease. The invention also provides methods for screening small molecules acting on the ABC1 protein which may by itself constitute products acting on the reverse transport of cholesterol and as such may make it possible to effectively combat atherosclerosis from a therapeutic point of view.

High-density lipoproteins (HDL) are one of the four major classes of lipoproteins circulating in blood plasma.

These lipoproteins are involved in various metabolic pathways such as lipid transport, the formation of bile acids, steroidogenesis, cell proliferation and, in addition, interfere with the plasma proteinase systems.

HDLs are perfect free cholesterol acceptors and, in combination with the 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, that is to say the transport of excess

® ® 2 cholesterol in the peripheral cells to the liver for its elimination from the body in the form of bile acid.

It has been demonstrated that the HDLs 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 cause a degradation of the HDLs.

Nevertheless, for Tangier disease, the exact nature of the defect has not yet been precisely defined.

In Tangier disease, this cellular defect leads to a disruption in the lipoprotein metabolism. The HDL particles not incorporating cholesterol from the peripheral cells and not being able to be metabolized correctly, are rapidly eliminated from the body. The plasma HDL concentration in these patients is therefore extremely reduced and the HDLs no longer ensure the return of cholesterol to the liver. This cholesterol accumulates in these peripheral cells and cause characteristic clinical manifestations such as the formation of orange-colored tonsils. Furthermore, other lipoprotein disruptions such as overproduction of triglycerides as well as increased synthesis and intracellular catabolism of phospholipids are observed.

Tangier disease, whose symptoms have been described above, is classified among the familial conditions linked to the metabolism of HDLs which are the ones most commonly detected in patients affected by coronary diseases.

Numerous studies have shown that a reduced level of HDL cholesterol is an excellent risk factor which makes it possible to detect a coronary condition.

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In this context, syndromes linked to HDL deficiencies have been of increasing interest for the past decade because they make it possible to increase understanding of the role of HDLs in atherogenesis.

Several mutations in the apo A-l gene have been characterized. These mutations are rare and may lead to a lack of production of apo A-I.

Mutations in the genes encoding lipoprotein lipase (LPL) or its activator apo C-ll are associated with severe hypertriglyceridemias and substantially reduced HDL-c levels.

Mutations in the gene encoding the enzyme Iecithin:cholesterol acyltransferase (LCAT) are also associated with a severe HDL deficiency.

Furthermore, dysfunctions in the reverse transport of cholesterol may be induced by physiological deficiencies affecting one or more of the steps in the transport of stored cholesterol, from the intracellular vesicles to the membrane surface where it is accepted by the HDLs.

An increasing need therefore exists in the state of the art to identify genes involved in any of the steps in the metabolism of cholesterol and/or lipoproteins, and in particular genes associated with dysfunctions in the reverse transport of cholesterol from the peripheral cells to the liver.

Recently, a study was carried out of the segregation of different 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 has 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 9931 locus of human chromosome 9 which is statistically associated with the condition (Rust S. et al., Nature Genetics, vol. 20, September 1998, pages 96-98).

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However, the study by RUST et al. only defines a wide region of the genome whose impairments are likely to be associated with Tangier disease. It is simply specified that the relevant 9q31-34 region contains ESTs but no known gene.

It has since been shown according to the invention that a region of about 1cM situated in the 9q31 locus in humans was generally associated with familial

HDL deficiencies.

More precisely, it has been shown that a gene encoding a protein of the family of ABC transporters, which is located precisely in the region of 1 cM of the 9q31 locus, was involved in pathologies linked to a deficiency in the reverse transport of cholesterol.

More particularly, it has been shown according to the invention that the gene encoding the ABC-1 transporter was mutated in patients impaired in the reverse transport of cholesterol, and most particularly in patients suffering from

Tangier disease.

The ABC ("ATP-binding cassette”) transport proteins constitute a family of proteins which are extremely well conserved during evolution, from bacterial to humans.

The ABC transport 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 had a common general structure, in particular two nucleotide binding folds (NBF) with

Walker A and B type units 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

® ® 5 the structure of the transmembrane domains, whereas the energy necessary for the transport activity is provided by the degradation of ATP at the level of the

NBF fold.

Several ABC transport proteins which have been identified in humans have been associated with various diseases.

For example, cystic fibrosis is caused by mutations in the CFTR (cystic fibrosis transmembrane conductance regulator) gene.

Moreover, some multiple 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 (patent US No. 5,858,719) or potentially involved in diseases caused by impairment of the homeostasis of metals, such as the ABC-3 protein.

Likewise, another transport ABC, designated PFIC2, appears to involve in a progressive familial intrahepatic cholestasia form, this protein being potentially responsible, in humans, for the export of bile salts.

In 1994, a cDNA encoding a new mouse ABC transporter was identified and designated 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 two NBF units.

In humans, a partial cDNA comprising the entire open reading frame of the human ABC1 transporter has been identified (Langmann 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 as well as the fetal tissues.

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These authors have also shown that the expression of the gene encoding the human ABC1 protein was induced during the differentiation of the monocytes into macrophages in vitro. Furthermore, the expression of the gene encoding the ABC1 protein is increased when the human macrophages are incubated in the presence of acetylated low-density lipoproteins (AcLDLs).

However, the exact role of the human ABC1 protein in the lipid transport system is completely unknown. It is simply assumed that the ABC1 protein has a translocase activity for phospholipids.

It has now been shown, according to the invention, that patients suffering from Tangier disease had a mutated ABC1 gene. Several mutations distributed : in different exons of the ABC1 gene have been identified in the genome of various patients, in particular patients suffering from a severe form of the disease associated with coronary disorders. Moreover, various polymorphisms have been found both in the exons and in the introns of the ABC1 gene in patients suffering from the mildest forms of the disease, indicating that these patients carry particular alleles of the gene, distinct from the “wild-type” allele(s).

Such alleles, partly characterizable by these polymorphisms, are moreover likely to contain substitutions, additions or deletions of nucleotides in the noncoding regions located respectively on the 5 side of the first exon or alternatively on the 3’ side of the last exon of the gene, in particular in the regulatory regions, for example in the promoter sequences or alternatively in the enhancer sequences, of the type which induces defects - increase or decrease - in the synthesis of the ABC1 polypeptide.

A first particular mutation has thus been identified in a patient suffering from Tangier disease, in the ABC-1 gene, which is located in exon 13, and which consists of a substitution of a nucleotide causing the introduction of a codon for premature termination of translation into the open reading frame,

® ® 7 leading to the synthesis of a truncated polypeptide comprising about a quarter of the amino acid sequence of the polypeptide synthesized in patients not affected by Tangier disease.

A second particular mutation in the ABC1 gene has been found, which consists in an insertion of a fragment of 100 nucleotides into exon 12, leading to the synthesis of a polypeptide which is abnormal in that it contains a deletion of 6 residues and an insertion of 38 amino acids, at position 468 of the sequence of the protein.

It has, in addition, been confirmed according to the invention that the

ABC1 gene was positively regulated by the acetylated low-density lipoproteins (AcLDLs).

GENERAL DEFINITIONS

The term “isolated” for the purposes of the present invention designates a biological material (nucleic acid or protein) which has been removed from its original environment (the environment in which it is naturally present).

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 in which it is naturally inserted in the genome of the plant or animal is considered as being “isolated”.

Such a polynucleotide may be included in a vector and/or such a polynucleotide may be included in a composition and remains nevertheless in the isolated state because of the fact that the vector or the composition does not constitute its natural environment.

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The term “purified” does not require the material to be present in a form exhibiting absolute purity, exclusive of 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 of the natural material by at least one order of magnitude, preferably 2 or 3 and preferably 4 or 5 orders of magnitude.

For the purposes of the present description, the expression “nucleotide sequence” may be used to designate either a polynucleotide or a nucleic acid.

The expression “nucleotide sequence” covers the genetic material itself and is therefore not restricted to the information relating to its sequence.

The terms “nucleic acid”, “polynucleotide”, “oligonucleotide” or “nucleotide sequence” cover RNA, DNA or cDNA sequences or alternatively

RNA/DNA hybrid sequences of more than one nucleotide, either in the single- chain form or in the duplex form.

The term “nucleotide” designates both the natural nucleotides (A, T, G,

C) as well as the modified nucleotides which comprise at least one modification such as (1) an analog of a purine, (2) an analog of a pyrimidine, or (3) an analogous sugar, examples of such modified nucleotides being described, for example, in the 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 whose orientation is reversed. The complementary bases are Aand T (or A and U), or C and G. “Variant” of a nucleic acid according to the invention will be understood to mean a nucleic acid which differs by one or more bases relative to the reference polynucleotide. A variant nucleic acid may be of natural origin, such

® ® as an allelic variant which exists naturally, or it may also be a nonnatural variant obtained, for example, by mutagenic 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 similar and, in many regions, identical. The nucleotide modifications present in a variant nucleic acid may be silent, which means that they do not alter the amino acid sequences encoded by said variant nucleic acid.

However, the changes in nucleotides in a variant nucleic acid may also result in substitutions, additions or deletions in the polypeptide encoded by the variant nucleic acid in relation to the peptides encoded by the reference nucleic acid. In addition, nucleotide modifications in the coding regions may produce conservative or nonconservative substitutions in the amino acid sequence.

Preferably, the variant nucleic acids according to the invention encode polypeptides which substantially conserve the same function or biological activity as the polypeptide of the reference nucleic acid or alternatively 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 whose systematic study will make it possible to deduce structure- activity relationships of the proteins in question. Knowledge of these variants in relation to the disease studied is essential since it makes it possible to understand the molecular cause of the pathology. “Fragment” will be understood to mean a reference nucleic acid according to the invention, a nucleotide sequence of reduced length relative to the reference nucleic acid and comprising, over the common portion, a nucleotide sequence identical to the reference nucleic acid.

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Such a nucleic acid “fragment” according to the invention may be, where appropriate, included in a larger polynucleotide of which it is a constituent.

Such fragments comprise, or alternatively consist of, oligonucleotides ranging in length from 8, 10, 12, 15, 18, 20 to 25, 30, 40, 50, 70, 80, 100, 200, 500, 1000 or 1500 consecutive nucleotides of a nucleic acid according to the invention. “Variant” of a polypeptide according to the invention will be understood to mean mainly a polypeptide whose amino acid sequence contains one or more substitutions, additions or deletions of at least one amino acid residue, relative to the amino acid sequence of the reference polypeptide, it being understood that the amino acid substitutions may be either conservative or nonconservative. “Fragment” of a polypeptide according to the invention will be understood to mean a polypeptide whose amino acid sequence is shorter than that of the reference polypeptide and which comprises, over the entire portion with these reference polypeptides, an identical amino acid sequence.

Such fragments may, where appropriate, be included in a larger polypeptide of which they are a part.

Such fragments of a polypeptide according to the invention may have a length of 10, 15, 20, 30 to 40, 50, 100, 200 or 300 amino acids.

The “percentage identity” between two nucleotide or amino acid sequences, for the purposes of the present invention, may be determined by comparing two sequences aligned optimally, through a window for comparison.

The portion of the nucleotide or polypeptide sequence in the window for comparison may thus comprise additions or deletions (for example “gaps”) relative to the reference sequence (which does not comprise these additions or these deletions) so as to obtain an optimum alignment of the two sequences.

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The percentage is calculated by determining the number of positions at which an identical nucleic base or an identical amino acid residue is observed for the two sequences (nucleic or peptide) compared, and 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 window for comparison, and then multiplying the result by 100 in order to obtain the percentage sequence identity.

The optimum sequence alignment for the comparison may be achieved using a computer with the aid of known algorithms contained in the package from the company WISCONSIN GENETICS SOFTWARE PACKAGE,

GENETICS COMPUTER GROUP (GCG), 575 Science Doctor , Madison,

WISCONSIN.

By way of illustration, it will be possible to produce the percentage sequence identity with the aid of the BLAST software (versions BLAST 1.4.9 of

March 1996, BLAST 2.0.4 of February1998 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 similar/fhomologous to a reference “request” sequence, with the aid of the Altschul et al. algorithm. The request sequence and the databases used may be of the peptide or nucleic types, any combination being possible. “High stringency hybridization conditions” for the purposes of the present invention will be understood to mean the following conditions:

1- Membrane competition and PREHYBRIDIZATION: - Mix: 40 pl salmon sperm DNA (10 mg/ml) + 40 pl human placental DNA (10 mg/m)

- Denature for 5 min at 96°C, then immerse the mixture in ice. - Remove the 2X SSC and pour 4 ml of formamide mix in the hybridization tube containing the membranes.

- Add the mixture of the two denatured DNAs. - Incubation at 42°C for 5 to 6 hours, with rotation. 2- Labeled probe competition: - Add to the labeled and purified probe 10 to 50 ul Cot | DNA, depending on the quantity of repeats. - Denature for 7 to 10 min at 95°C. - Incubate at 65°C for 2 to 5 hours. 3- HYBRIDIZATION:

- Remove the prehybridization mix.

- Mix 40 pl salmon sperm DNA + 40 ul human placental DNA; denature for min at 96°C, then immerse in ice. - Add to the hybridization tube 4 ml of formamide mix, the mixture of the two 5 DNAs and the denatured labeled probe/Cot | DNA . - Incubate 15 to 20 hours at 42°C, with rotation. 4- Washes: - One wash at room temperature in 2X SSC, to rinse. - Twice 5 minutes at room temperature 2X SSC and 0.1% SDS at 65°C. - Twice 15 minutes at 65°C 1X SSC and 0.1% SDS at 65°C.

Envelope the membranes in Saran and expose.

The hybridization conditions described above are adapted to hybridization, under high stringency conditions, of a molecule of nucleic acid of varying length from 20 nucleotides to several hundreds of nucleotides.

It goes without saying that the hybridization conditions described above may be adjusted as a function of the length of the nucleic acid whose hybridization is sought or of the type of labeling chosen, according to techniques known to persons skilled in the art.

Suitable hybridization conditions may for example be adjusted according to the teaching contained in the manual by HAMES and HIGGINS (1985) or in the manual by F. AUSUBEL et al (1999).

NUCLEIC ACIDS OF THE ABC1 GENE

GENOMIC SEQUENCES

The human ABC1 gene is thought to comprise 48 exons and 47 introns, if reference is made in particular to the structure of the orthologous ABC1 gene in mice.

Several partial genomic nucleotide sequences of the ABC1 gene have been isolated and characterized according to the invention, these genomic sequences comprising both new exonic sequences and intronic sequences which may be used in particular for the production of various means of detection of the ABC1 gene or of its nucleotide expression products in a sample. These partial genomic sequences are represented in Table 1 below.

Table

Partial genomic sequences of the human ABC1 gene 156

SEQ ID NO

1 [intron 10(p), exon 11(s 2 Intron 11(p), exon 12, intron 12, exon 13, intron 13, exon 14, intron 14, exon 15, intron 15, exon 16, intron 16, exon 17(p

Exon 17(p), intron 17(p intron 18(p), exon 19, intron 19(p 5 Intron 19(p), exon 20, intron 20, exon 21, intron 21, exon 22, intron 22, exon 23, intron 23, exon 24, intron 24, exon 25, intron 25, exon 26, intron 26(p

Intron 26(p), exon 27, intron 27, exon 28, intron 28, exon 29, intron 29, exon 30, intron 30(p 7 Intron 30(p), exon 31, intron 31, exon _

32, intron 32, exon 33, intron 33, exon 34, intron 34(p 8 intron 34(p), exon 35, intron 35(p 9 [intron B5(p), exon 36, intron 36(p 10 Intron 36(p), exon 37, intron 37, exon 38, intron 38(p 11 Intron 39(p), exon 40, intron 40, exon 41, intron 41(p intron 41(p), exon 42, intron 42(¢

Intron 46(p), exon 47, intron 47(¢ 14 Last exon(p), sequence in 3’ of the last exon

Thus, a first subject of the invention consists in a nucleic acid comprising at least 245 consecutive nucleotides of a polynucleotide chosen from the group consisting of the nucleotide sequences SEQ ID NO 1-14, or a nucleic acid having a complementary sequence.

The invention also relates to a nucleic acid having at least 80%, advantageously 90%, preferably 95% and most preferably 98% nucleotide identity with a nucleic acid comprising at least 245 consecutive nucleotides of a polynucleotide chosen from the group consisting of the nucleotide sequences

SEQ ID NO 1-14, or a nucleic acid having a complementary sequence.

Thirty two exons of the ABC1 gene have been characterized, at least partially, by their nucleotide sequence, as indicated in Table ll below. /

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Table II

NO SEQ ID NO nucleotide in 5’ nucleotide in 3’ 27 [31 le [set Jroe 28 laa le [oas9 Joass

Iss le [ara [sz

EE CO =

ER EE = I 36 lao lo Jess Ties

EE CE I

® 1d 17 3' end

Thus, the invention also relates to a nucleic acid comprising a polynucleotide chosen from the group consisting of the nucleotide sequences

SEQ ID NO 15-47, or a nucleic acid having a complementary sequence.

Moreover, thirty-five introns of the ABC1 gene have been isolated and characterized, at least partially. The nucleotide sequences of the introns of the

ABC1 gene, as well as their fragments and their variants may also be used as nucleotide probes or primers for detecting the presence of at least one copy of the ABC1 gene in a sample, “or alternatively for amplifying a given target sequence in the ABC1 gene.

The references to the intronic sequences of the ABC1 gene are indicated in Table Hl below.

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Table Il]

NO SEQ ID NO nucleotide in 5’ | nucleotide in 3’ 21 leo [5s [76s Jia 26(3end) 66 6 [1 [seo 27 ler le rio Ja3ss 28 les 6 Joasa Jama e096 seta lesar s05end) [70 |6 lrosr [rar 34@end) 76 [8 Jt Ja3s ss(end) [77 ls laos Jes

35(@end) [78 jo [1 Toss 6(Eend [79 lo [1167 1664 36(end) [80 10 [1 saa 38 (5' end 1279 39 (3' end 41(5' end 1124 41(end) [86 J12 [1 Jess 42 (5 end 46 (3' end 47(5end) 89 [13 fez1 ra

Distal 14 1238 3501 sequence in 3’ of the last exon

The invention also relates to a nucleic acid comprising at least 8 consecutive nucleotides of a polynucleotide chosen from the group consisting of the nucleotide sequences SEQ ID NO 48-89, or a nucleic acid having a complementary sequence.

The subject of the invention is, in addition, a nucleic acid having at least 80% nucleotide identity with a polynucleotide chosen from the group consisting of the nucleotide sequences SEQ ID NO 48-89, or a nucleic acid having a complementary sequence.

The invention also relates to a nucleic acid hybridizing, under high stringency conditions, with a polynucleotide chosen from the group consisting of the nucleotide sequences SEQ ID NO 48-89, or a nucleic acid having a complementary sequence.

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In addition, a potentially regulatory genomic nucleotide sequence located downstream of the 3’ end of the last exon of the ABC1 gene has been isolated.

It is a polynucleotide having the sequence SEQ ID NO 90. The characterization of polymorphisms in this potentially regulatory sequence (possible presence of regulatory sequences of the “enhancer” type), in particular in patients suffering from mild forms of deficiency in the reverse transport of cholesterol, in particular mild forms of Tangier disease, would be of the type allowing the production of appropriate means of detection, probes or primers, specific for some of these polymorphisms capable of inducing defects in the regulation of the expression of the ABC1 gene.

In order to identify the biologically active polynucleotide fragments of the sequence SEQ ID NO 90, persons skilled in the art can advantageously refer to the book by Sambrook et al. (1989) which describes the use of a recombinant vector carrying a marker gene (for example B-galactosidase, chloramphenicol acetyl transferase and the like) whose expression may be detected when this marker gene is placed under the control of a suitable promoter and of a biologically active fragment of the polynucleotide having the sequence

SEQ ID NO 90. Such biologically active fragments of the sequence

SEQ ID NO 90 may be in particular cloned into appropriate selection vectors having regulatory sequences, such as one of the vectors pSEAP-Basic, pSEAP-Enhancer, ppgal-Basic, ppgal-Enhancer, or pEGFP-1, marketed by the company Clontech.

The subject of the invention is, in addition, a nucleic acid having at least 80% nucleotide identity with a polynucleotide chosen from the group consisting of the nucleotide sequences SEQ ID NO 90, or a nucleic acid having a complementary sequence.

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The invention also relates to a nucleic acid hybridizing, under high stringency conditions, with a polynucleotide chosen from the group consisting of the nucleotide sequences SEQID NO 90, or a nucleic acid having a complementary sequence.

COMPLETE cDNA

As already indicated above, a partial cDNA sequence corresponding to the expression of the ABC1 gene has been identified by Langman et al. (1999).

This partial cDNA sequence of ABC1 comprises 6880 nucleotides and contains the entire open reading frame corresponding to the ABC1 protein produced in subjects not affected by disorders linked to the reverse transport of cholesterol.

The cDNA sequence described by Langmann et al. (1999) contains, in addition, a portion of the 5-UTR region (nucleotides 1 to 120) and a portion of the 3’-UTR region (nucleotides 6727 to 6880).

The entire complete cDNA corresponding to the ABC1 gene, which corresponds to a new 3-UTR region, which constitutes a major nucleic region, in particular from the point of view of the stability of the messenger RNAs in the cell, has now been isolated and characterized according to the invention.

The analyses of expression of the transcript having the sequence

SEQ ID NO 91 were carried out by RT-PCR, as described in Example 1. These analyses carried out starting with the polyA+ RNA of various tissues have made it possible to ensure that the ABC1 gene was expressed in the fetal brain, the brain, the heart, the placenta and the uterus.

Consequently, the invention also relates to a nucleic acid comprising a polynucleotide having the sequence SEQ ID NO 91 of the cDNA of the human

ABC1 gene, or a nucleic acid having a complementary sequence.

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The cDNA of the human ABC1 gene having the sequence

SEQ ID NO 91 comprises an open reading frame going from the nucleotide at position 121 (base A of the ATG codon for initiation of translation) to the nucleotide at position 6723 of the sequence SEQ ID NO 91. A polyadenylation signal (having the sequence ATTAAA) is present, starting at the nucleotide at position 9454 of the sequence SEQ ID NO 91.

The cDNA having the sequence SEQ ID NO 91 encodes the ABC1 polypeptide of 2201 amino acids in length, and having the amino acid sequence

SEQ ID NO 139.

The invention also relates to a nucleic acid comprising at least eight consecutive nucleotides of a polynucleotide having the sequence

SEQ ID NO 92, a biologically active fragment thereof or a nucleic acid having a complementary sequence.

The subject of the invention is also a nucleic acid having at least 80% nucleotide identity with a polynucleotide having the sequence SEQ ID NO 92, a biologically active fragment thereof or a nucleic acid having a complementary sequence.

Another subject of the invention consists in a nucleic acid hybridizing, under high stringency conditions, with a polynucleotide having the sequence

SEQ ID NO 92, a biologically active fragment thereof or a nucleic acid having a complementary sequence.

POLYMORPHISMS WITHIN THE ABC1 GENE

MUTATIONS

According to the invention, several mutations have been identified in the sequence of the ABC1 gene, these mutations leading to major structural impairments of the ABC1 polypeptide encoded by the mutated sequences.

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These mutations have been found particularly in patients suffering from severe forms of Tangier disease, associated with serious coronary disorders. Two particularly deleterious mutations are described below. 1. Mutation in exon 12

This mutation consists both in a deletion of a localized segment of 14 nucleotides (“TGAGAGGAAGTTCT") from the nucleotide at position 472 to the nucleotide at position 485 of the normal genomic DNA having the sequence

SEQ ID NO 2 and in an insertion of an Alu-type sequence of 110 nucleotides into the sequence of exon 12 of the ABC1 gene, upstream of the nucleotide at position 486 of the normal genomic DNA having the sequence SEQ ID NO 2.

The exon 12 carrying this deletion/insertion mutation has the nucleotide sequence SEQ ID NO 93.

The corresponding mutated cDNA has the nucleotide sequence

SEQ ID NO 94, encodes a mutated ABC1 polypeptide of 2233 amino acids in length, having the sequence SEQ ID NO 140, whose structure is substantially altered compared with the normal ABC1 polypeptide having the sequence

SEQ iD NO 139.

The nucleotide sequences SEQID NO 93 and 94 as well as the polypeptide sequence SEQ ID NO 140 also form part of the invention. 2 Mutation in exon 13

This mutation consists of a deletion of the nucleotide (G) at position 1232 of the genomic sequence SEQ ID NO 2, which is located in exon 13 (nucleotide

G at position 106 of the sequence of exon 13 SEQ ID NO 17). This point deletion of one base introduces a stop codon in the normal reading frame in the mRNA of the ABC1 gene.

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The sequence of exon 13 of the ABC1 gene carrying this mutation is the polynucleotide having the sequence SEQ ID NO 95.

The cDNA corresponding to this mutation in exon 13 of the ABC1 gene is represented in the nucleotide sequence SEQ ID NO 96.

The mutated protein encoded by the mutated ABC1 gene having a length of 574 amino acids, that is to say about a quarter of the length in terms of amino acids of the normal protein. The truncated polypeptide has the amino acid sequence SEQ ID NO 141.

The structural characteristics which make it possible to differentiate the normal sequences from the mutated sequences of ABC1 (genomic sequences, messenger RNAs, cDNA) may be exploited in order to produce means of detection of the mutated sequences of ABC1 in a sample, in particular probes specifically hybridizing with the mutated sequences of ABC1 or pairs of primers making it possible to selectively amplify the regions of the ABC1 gene carrying the mutations described above, it being possible to carry out the detection of the presence of these mutations in particular by distinguishing the length of the amplified nucleic acid fragments, by hybridization of the amplified fragments with the aid of the specific probes described above, or by direct sequencing of these amplified fragments.

Thus, a further subject of the invention is a nucleic acid having at least eight consecutive nucleotides of a polynucleotide chosen from the group consisting of the nucleotide sequences SEQ ID NO 93-96, or a nucleic acid -— - ————having-a complementary sequence:—/———"——-—""-—"—"————————

Preferably, such a nucleic acid comprises: a) either at least two consecutive nucleotides of the Alu sequence located in the sequences SEQ ID NO 93 and 94, preferably 5, 10,

® ® 25 15, 20, 25, 30, 35, 40, 50 or 100 consecutive nucleotides of the

Alu sequence located in the sequences SEQ ID NO 93 and 94; b) or at least the two “CT” nucleotides situated on either side of the deleted G base, in the sequences SEQ ID NO 94 and 95.

The primers hybridizing with a nucleic sequence located in the region of an ABC1 sequence (genomic sequence, messenger RNA) carrying either of the two mutations described above also form part of the invention.

The invention relates, in addition, to a nucleic acid having at least 80% nucleotide identity with a polynucleotide chosen from the group consisting of the nucleotide sequences SEQ ID NO 93-96, or a nucleic acid having a complementary sequence.

The invention also relates to a nucleic acid hybridizing, under high stringency conditions, with a polynucleotide chosen from the group consisting of the nucleotide sequences SEQ ID NO 93-96, or a nucleic acid having a complementary sequence.

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OTHER POLYMORPHISMS

Other polymorphisms have been found within the sequence of the ABC1 gene, in particular nucleotide substitutions located both in the coding regions (exons) and in the noncoding regions.

As regards the polymorphisms found in the coding regions, they are essentially substitutions of a single nucleotide located on the third base of the codons of the open reading frame of ABC1, these substitutions causing no modification as regards the nature of the amino acid encoded, taking into account the rules of genetic degeneration in humans, which are well known to persons skilled in the art.

These polymorphisms are represented in the present description in the form of nucleotide sequences of 41 bases in length, the polymorphic base being located at the center of the polymorphic fragment. For each of the polymorphisms identified, each allele is thus represented as a sequence of 41 bases, the polymorphism itself being defined by the two nucleotide sequences corresponding respectively to each of the forms. The polymorphisms identified in the ABC1 gene are represented in Table IV below.

Table IV

Polymorphisms found in the ABC1 gene

Designation Position in the | Allele 1 Allele 2 Polymorphic

No. sequence SEQ ID NO SEQ ID NO base

SEQIDNO 2 Aliele1/Allele 2 1 ser Jor Je Jem 1324 ER Er 7 3028 3234 3390 6 [essa

The detection of these polymorphisms within a DNA sample obtained from a subject may, for example, be carried out by a specific amplification of the nucleotide region of ABC1 containing the polymorphic base, and then sequencing the amplified fragment in order to determine the nature of the allele or of the alleles carried by said subject.

The detection of these polymorphisms in a DNA sample obtained from a subject may also be carried out with the aid of nucleotide probes or primers specifically hybridizing with a given allele containing one of the polymorphic bases of a polymorphism of the ABC1 gene according to the invention.

By way of illustration, appropriate nucleotide primers are for example primers whose base at the 3’ end hybridizes with the base located immediately on the § side of the polymorphic base of the fragment comprising said polymorphism. After the step of hybridization of the specific primer, a step of extension with a mixture of the two dideoxynucleotides complementary to the polymorphic base of said polymorphism, for example differentially labeled by fluorescence, and then a step detection of the fluorescence signal obtained

® 28 makes it possible to determine which of the two differentially labeled fluorescent dideoxynucleotides has been incorporated and to directly deduce the nature of the polymorphic base present at the level of this polymorphism.

Various approaches may be used for the labeling and detection of the dideoxynucleotides. A method in homogeneous phase based on FRET ("Fluorescence resonance energy transfer") has been described by Chen and

Kwok (1997). According to this method, the amplified fragments of genomic

DNA containing polymorphisms are incubated with a primer labeled with fluorescein at the 5'end in the presence of labeled dideoxynucleotide triphosphate and a modified Taq polymerase. The labeled primer is extended by one base by incorporation of the labeled dideoxynucleotide specific for the allele present on the complementary genomic DNA sequence. At the end of this genotyping reaction, the fluorescence intensities for the two labeling compounds for the labeled dideoxynucleotides are directly analyzed without separation or purification. All these steps may be carried out in the same tube and the modifications of the fluorescence signal monitored in real time.

According to another embodiment, the extended primer may be analyzed by

MALDI-TOF type mass spectrometry. The base located at the level of the polymorphic site is identified by measuring the mass added to the microsequencing primer (Haff and Smirnov, 1997).

Such nucleotide primers may, for example, be demobilized on a support.

Furthermore, it is possible to immobilize on a support, for example in an orderly manner, multiple specific primers as described above, each of the primers being suited to the detection of one of the polymorphisms of the ABC1 gene according to the invention.

® ug 29

The polymorphisms of the ABC1 gene according to the invention are useful in particular as genetic markers in studies of association between the presence of a given allele in a subject and the predisposition of this subject to a given pathology, in particular to one of the pathologies already associated with the chromosomal region 9q31 preferably with a pathology linked to a dysfunction in the reverse transport of cholesterol.

The methods for the genetic analysis of complex characters (phenotypes) are of various types (Lander and Schork, 1994). In general, the bialleleic polymorphisms according to the invention are useful in any of the methods described in the state of the art intended to demonstrate a statistically significant correlation between a genotype and a phenotype. The bialleleic polymorphisms may be used in linkage analyses and in allele sharing methods.

Preferably, the bialleleic polymorphisms according to the invention are used to identify genes associated with detectable characters (phenotypes) in use for studies of association, an approach which does not require the use of families affected by the character, and which allows, in addition, the identification of genes associated with complex and sporadic characters.

Other statistical methods using bialleleic polymorphisms according to the invention are for example those described by Forsell et al. (1997), Xiong et al. (1999), Horvath et al. (1998), Sham et al. (1995) or Nickerson et al. (1992).

According to another aspect, the invention also relates to the nucleotide sequences of the ABC1 gene comprising at least one bialleleic polymorphism as described above.

Thus, the invention also relates to a nucleic acid having at least eight consecutive nucleotides of a polynucleotide chosen from the group consisting of the nucleotide sequences SEQ ID NO 97-108 and comprising the polymorphic base, or a nucleic acid having a complementary sequence.

_

NUCLEOTIDE PROBES AND PRIMERS

The nucleic acid fragments derived from any one of the nucleotide sequences SEQ ID NO 1-14, 15-47, 48-89, 90, 92, 94-96 and 97-108 are useful for the detection of the presence of at least one copy of a nucleotide sequence of the ABC1 gene or of a fragment or of a variant (containing a mutation or a polymorphism) thereof 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 1-14, 15-47, 48-89, 90, 92, 93-96 and 97-108, or of a nucleic acid having a complementary sequence.

Preferably, nucleotide probes or primers according to the invention will have a length of 10, 12, 15, 18 or 20 to 25, 35, 40, 50, 70, 80, 100, 200, 500, 1000, 1500 consecutive nucleotides of a nucleic acid according to the invention, in particular a nucleic acid having a nucleotide sequence chosen from the sequences SEQ ID NO 1-14, 15-47, 48-89, 90, 92, 93-96 and 97-108, or of a nucleic acid having a complementary sequence.

Alternatively, a nucleotide probe or primer according to the invention will consist of and/or comprise the fragments having a length of 12, 15, 18, 20, 25, 35, 40, 50, 100, 200, 500, 1000, 1500 consecutive nucleotides of a nucleic acid according to the invention, more particularly of a nucleic acid chosen from the sequences SEQ ID NO 1-14, 15-47, 48-89, 90, 92, 93-96 and 97-108, or of a nucleic acid having a complementary sequence.

The definition of a nucleotide probe or primer according to the invention therefore covers oligonucleotides which hybridize, under the high stringency hybridization conditions defined above, with a nucleic acid chosen from the

® ® 31 sequences SEQ ID NO 1-14, 15-47, 48-89, 90, 92, 93-96 and 97-108 or with a sequence complementary thereto.

Examples of primers and pairs of primers which make it possible to amplify various regions of the ABC1 gene are presented in Table V below.

Table V

Primers for the amplification of nucleic fragments of the ABC1 gene

SEQ ID sequence of the hybridization primer 1496

IEEE i 3468

ET

5362 9 | 2 | 6240-6262 | 117 | introns _ 6603 1770 4262 | 6 | 3587-3610 | 123 | intron2s

I a a

17 | 6 | ersae7r5 Intron 29 18 Comp 7112- 126 Intron 30 7134 1060-1082 intron 30 20 Comp 1377- 128 Intron 31 1399 3574-3596 intron 32 22 Comp 3900- 130 Intron 33 3931 5161-5183 intron 33 24 7 Comp 5463- 132 Intron 34 5485 25 | 8 | 100-122 Intron 34 26 | 8 | compars4g7 Intron 35 27 | 9 | satge1 intron 35 1271 455-477 Intron 36

Comp 966-983 Intron 38

According to a first embodiment of preferred probes and primers according to the invention, they comprise all or part of a polynucleotide chosen from the nucleotide sequences SEQ ID NO 109-138, or nucleic acids having a complementary sequence.

A nucleotide primer or probe according to the invention may be prepared by any suitable method well known to persons skilled in the art, including by cloning and action of restriction enzymes or by direct chemical synthesis according to techniques such as the phosphodiester method by Narang et al. (1979) or by Brownetal. (1979), the diethylphosphoramidite method by

Beaucage et al. (1980) or the technique on a solid support described in EU patent No. EP 0,707,592.

® ® 33

Each of the nucleic acids according to the invention, including the oligonucleotide probes and primers described above, may be labeled, if desired, by incorporating a marker which can be detected by spectroscopic, photochemical, biochemical, immunochemical or chemical means.

For example, such markers may consist of radioactive isotopes (32P, 33P, 3H, 35S), fluorescent molecules (5-bromodeoxyuridine, fluorescein, acetylaminofluorene, digoxigenin) or ligands such as biotin.

The labeling of the probes is preferably carried out by incorporating labeled molecules into the polynucleotides by primer extension, or alternatively by addition to the 5’ or 3’ ends.

Examples of nonradioactive labeling of nucleic acid fragments are described in particular in French patent No. 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 may have structural characteristics of the type to allow amplification of the signal, such as the probes described by Urdea et al. (1991) or alternatively in European patent

No. EP-0,225,807 (CHIRON).

The oligonucleotide probes according to the invention may be used in particular in Southern-type hybridizations with the genomic DNA or alternatively in hybridizations with the corresponding messenger RNA when the expression of the corresponding transcript is sought in a sample.

The probes according to the invention may also be used for the detection of products of PCR amplification or alternatively for the detection of mismatches.

Nucleotide probes or primers according to the invention may be immobilized on a solid support. Such solid supports are well known to persons skilled in the art and comprise surfaces of wells of microtiter plates, polystyrene

® og 34 beds, magnetic beds, nitrocellulose bands or microparticles such as latex particles.

Consequently, the present invention also relates to a method of detecting the presence of a nucleic acid as described above in a sample, said method comprising the steps of: 1) bringing one or more nucleotide probes according to the invention into contact with the sample to be tested; 2) detecting the complex which may have formed between the probe(s) and the nucleic acid present in the sample.

According to a specific embodiment of the method of detection according to the invention, the oligonucleotide probes are immobilized on a support.

According to another aspect, the oligonucleotide probes comprise a detectable marker.

The invention relates, in addition, to a box or kit for detecting the presence of a nucleic acid according to the invention in a sample, said box comprising: a) one or more nucleotide probes as described above; b) where appropriate, the reagents necessary for the hybridization reaction.

According to a first aspect, the detection box or kit is characterized in that the probe(s) are immobilized on a support.

According to a second aspect, the detection box or kit is characterized in that the oligonucleotide probes comprise a detectable marker.

According to a specific embodiment of the detection kit described above, such a kit will comprise a plurality of oligonucleotide probes in accordance with

® ® 35 the invention which may be used to detect target sequences of interest or alternatively to detect mutations in the coding regions or the noncoding regions of the nucleic acids according to the invention, more particularly of the nucleic acids having the sequences SEQ ID NO 1-14, 15-47, 48-89, 90, 92, 93-96 and 97-108 or the nucleic acids having a complementary sequence.

Thus, the probes according to the invention, immobilized on a support, may be ordered into matrices such as “DNA chips”. Such ordered matrices have in particular been described in US patent No. 5,143,854, in PCT applications No. WO 90/150 70 and 92/10092.

Support matrices on which oligonucleotide probes have been immobilized at a high density are for example described in US patents

No. 5,412,087 and in PCT application No. WO 95/11995.

The nucleotide primers according to the invention may be used to amplify any one of the nucleic acids according to the invention, and more particularly all or part of a nucleic acid having the sequences SEQ ID NO 1-14, 15-47, 48-89, 90, 92, 93-96 and 97-108, or alternatively a variant thereof.

Another subject of the invention relates to a method of amplifying a nucleic acid according to the invention, and more particularly a nucleic acid having the sequences SEQ ID NO 1-14, 15-47, 48-89, 90, 92, 93-96 and 97-108 or a fragment or a variant thereof contained in a sample, said method comprising the steps of: a) bringing the sample in which the presence of the target nucleic acid is suspected into contact with a pair of nucleotide primers whose 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 necessary for the amplification reaction; and b) detecting the amplified nucleic acids.

® ® 36

To carry out the amplification method as defined above, use will be advantageously made of any of the nucleotide primers described above.

The subject of the invention is, in addition, a box or kit for amplifying a nucleic acid according to the invention, and more particularly all or part of a nucleic acid having the sequences SEQ ID NO 1-14, 15-47, 48-89, 90, 92, 93-96 and 97-108, said box or kit comprising: a) a pair of nucleotide primers in accordance with the invention, whose hybridization position is located respectively on the 5’ side and 3’ side of the target nucleic acid whose amplification is sought; b) where appropriate, the reagents necessary for the amplification reaction.

Such an amplification box or kit will advantageously comprise at least one pair of nucleotide primers as described above.

According to a first preferred embodiment, primers according to the invention comprise all or part of a polynucleotide chosen from the nucleotide sequences SEQ ID NO 109 and 110, making it possible to amplify the region of exon 12 of the ABC1 gene carrying the first mutation (deletion/insertion) described above, or nucleic acids having a complementary sequence.

According to a second preferred embodiment, primers according to the invention comprise all or part of a polynucleotide chosen from the nucleotide sequences SEQ ID NO 111 and 112, making it possible to amplify the region of exon 13 of the ABC1 gene carrying the second mutation (deletion of a G base) described above, or nucleic acids having a complementary sequence.

According to a third preferred embodiment, primers according to the invention comprise, generally, all or part of a polynucleotide chosen from the

® ® 37 nucleotide sequences SEQ ID NO 109-138, or nucleic acids having a complementary sequence.

According to a fourth preferred embodiment, the invention also relates to nucleotide primers comprising at least 15 consecutive nucleotides of a nucleic acid chosen from the group consisting of the sequences SEQ ID NO 97-108 or a nucleic acid having a complementary sequence, the base of the 3’ end of these primers being complementary to the nucleotide located immediately on the 5’ side of the polymorphic base of one of the sequences SEQ ID NO 97-108 or of their complementary sequences.

According to another aspect, the invention also relates to nucleotide primers comprising at least 15 consecutive nucleotides of a nucleic acid chosen from the group consisting of the sequences SEQ ID NO 97-108 or a nucleic acid having a complementary sequence, the base of the 3' end of these primers being complementary to a nucleotide situated at 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 15. 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or more on the 5 side of the polymorphic base of one of the sequences SEQ ID NO 97-108 or of their complementary sequences. To construct primers whose nucleotide at the 3’ end is complementary to a nucleotide located -at more than 20 nucleotides on the 5’ side of the polymorphic base of one of the sequences SEQ ID NO 97- 108, persons skilled in the art will advantageously refer to the corresponding genomic sequence among the sequences SEQ ID NO 1-14 or SEQ ID NO 15- 47 and 48-90, comprising the polymorphism for which the nature of the allele is sought.

Such primers are particularly useful in the context of methods of genotyping subjects and/or of genotyping populations, in particular in the context of studies of association between particular allele forms or particular forms of groups of alleles (haplotypes) in subjects and the existence of a

® . particular phenotype (character) in these subjects, for example the predisposition of these subjects to develop diseases linked to a deficiency in the reverse transport of cholesterol, or alternatively the predisposition of these subjects to develop a pathology whose candidate chromosomal region is situated on chromosome 9, more precisely on the 9q arm and still more precisely in the 9q31 locus.

RECOMBINANT VECTORS

The invention also relates to a recombinant vector comprising a nucleic acid according to the invention.

Advantageously, such a recombinant vector will comprise a nucleic acid chosen from the following nucleic acids: a) a nucleic acid having the sequence SEQ ID NO 92 or a biologically active fragment thereof; b) a nucleic acid comprising a polynucleotide having the sequence SEQ

ID NO 91,94 or 96 ; c) a nucleic acid comprising a polynucleotide chosen from the group consisting of the nucleotide sequences SEQ ID NO 15-47 and 48-90 d) a nucleic acid having at least 80% nucleotide identity with a nucleic acid chosen from the group consisting of the sequences SEQ ID NO15-47 and 48-90 or a fragment or a variant thereof ; d) a nucleic acid hybridizing, under high stringency hybridization conditions, with a nucleic acid having the sequences SEQ ID NO 15-47 and 48- 90, or a fragment or a variant thereof.

® ® 39 “Vector” for the purposes of the present invention will be understood to mean a circular or linear DNA or RNA molecule which is either in single- stranded or double-stranded form.

According to a first embodiment, a recombinat vector according to the invention is used to amplify the nucleic acid which is inserted therein after transformation or transfection of the desired cellular host.

According to a second embodiment, it corresponds to expression vectors comprising, in addition to a nucleic acid in accordance with the invention, regulatory sequences which make it possible to direct the transcription and/or translation thereof.

According to an advantageous embodiment, a recombinant vector according to the invention will comprise in particular the following elements: (1) elements for regulating the expression of the nucleic acid to be inserted, such as promoters and enhancer sequences; (2) the coding sequence contained in the nucleic acid in accordance with the invention to be inserted into such a vector, said coding sequence being placed in phase with the regulatory signals described in (1); and (3) appropriate sequences for initiation and termination of the transcription.

In addition, the recombinant vectors according to the invention may include one or more origins for replication in the cellular hosts in which their amplification or their expression is sought, markers or selectable markers.

By way of example, the bacterial promoters may be the Lacl or LacZ promoters, the T3 or T7 bacteriophage RNA polymerase promoters, the lambda phage PR or PL promoters.

® 40

The promoters for eukaryotic cells will comprise the HSV virus thymidine kinase promoter or alternatively the mouse metallothionein-L promoter.

Generally, for the choice of a suitable promoter, persons skilled in the art can advantageously refer to the book by Sambrook et al. (1989) cited above or to the techniques described by Fuller et al. (1996).

When the expression of the genomic sequence of the ABC1 gene will be sought, use will preferably be made of the vectors capable of containing large insertion sequences. In this particular embodiment, bacteriophage vectors such as the P1 bacteriophage vectors such as the vector p158 or the vector p158/neo8 described by Sternberg (1992, 1994) will be preferably used.

The preferred bacterial vectors according to the invention are for example the vectors pBR322(ATCC37017) or alternatively vectors such as pAA223-3 (Pharmacia, Uppsala, Sweden), and pGEM1 (Promega Biotech,

Madison, WI, UNITED STATES).

There may also be cited other commercially available vectors such as the vectors pQE70, pQE60, pQE9 (Qiagen), psiX174, pBluescript SA, pNHS8A, pNH16A, pNH18A, pNH46A, pWLNEO, pSV2CAT, pOG44, pXTl, pSG(Stratagene).

They may also be vectors of the baculovirus type such as the vector pVL1392/1393 (Pharmingen) used to transfect cells of the Sf9 line (ATCC

No. CRL 1711) derived from Spodoptera frugiperda.

They may also be adenoviral vectors such as the human adenovirus of type 2 or 5.

A recombinant vector according to the invention may also be a retroviral vector or an adeno-associated vector (AAV). Such adeno-associated vectors are for example described by Flotte et al. (1992), Samulski et al. (1989), or

McLaughlin BA et al. (1996).

® ® 41

To allow the expression of the polynucleotides according to the invention, the latter must be introduced into a host cell. The introduction of the polynucleotides according to the invention into a host cell may be carried out in vitro, according to the techniques well known to persons skilled in the art for transforming or transfecting cells, either in primer culture, or in the form of cell lines. It is also possible to carry out the introduction of the polynucleotides according to the invention in vivo or ex vivo, for the prevention or treatment of 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 can 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), liposomes charged with

DNA (Nicolau et al., 1982, Fraley et al., 1979).

Once the polynucleotide has been introduced into the host cell, it may be stably integrated into the genome of the cell. The intregration may be achieved at a precise site of the genome, by homologous recombination, or it may be randomly integrated. In some embodiments, the polynucleotide may be stably maintained in the host cell in the form of an episome fragment, the episome comprising sequences allowing the retention and the replication of the latter, either independently, or in a synchronized manner with the cell cycle.

According to a specific embodiment, a method of introducing a polynucleotide according to the invention into a host cell, in particular a host cell obtained from a mammal, in vivo, comprises a step during which a preparation comprising a pharmaceutically compatible vector and a “naked” polynucleotide according to the invention, placed under the control of appropriate regulatory sequences, is introduced by local injection at the level of the chosen tissue, for

® ® 42 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 for example described in PCT Application

No. WO 95/11307 (Institut Pasteur, Inserm, University of Ottawa) as well as in the articles by Tacson et al. (1996) and Huygen et al. (1996) .

According to a specific embodiment of the invention, a composition is provided for the in vivo production of the ABC1 protein. This composition comprises a polynucleotide encoding the ABC1 polypeptide placed under the control of appropriate regulatory sequences, in solution in a physiologically acceptable vector.

The quantity of vector which is injected into the host organism chosen varies according to the site of the injection. As a guide, there may be injected between about 0.1 and about 100 pg of polynucleotide encoding the ABC protein years the body of an animal, preferably 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, in particular a patient having a predisposition to

Tangier disease or who has already developed the disease.

Consequently, the invention also relates to a pharmaceutical composition intended for the prevention of or treatment of subjects affected by, a dysfunction in the reverse transport of cholesterol, comprising a nucleic acid encoding the ABC1 protein, in combination with one or more physiologically compatible excipients.

Advantageously, such a composition will comprise the polynucleotide having the sequence SEQ ID NO 91, placed under the control of appropriate regulatory elements.

® ® 43

The subject of the invention is, in addition, a pharmaceutical composition intended for the prevention of or treatment of subjects affected by, a dysfunction in the reverse transport of cholesterol, comprising a recombinant vector according to the invention, in combination with one or more physiologically compatible excipients.

The invention also relates to the use of a nucelic acid according to the invention, encoding the ABC1 protein, for the manufacture of a medicament intended for the prevention of Atherosclerosis in various forms or more particularly for the treatment of subjects affected by a dysfunction in the reverse transport of cholesterol.

The invention also relates to the use of a recombinant vector according to the invention, comprising a nucleic acid encoding the ABC1 protein, for the manufacture of a medicament intended for the prevention of Atherosclerosis in various forms or more particularly for the treatment of subjects affected by a dysfunction in the reverse transport of cholesterol.

Vectors useful in methods of somatic gene therapy and compositions containing such vectors

The present invention also relates to a new therapeutic approach for the treatment of pathologies linked to the transport of cholesterol. It provides an advantageous solution to the disadvantages of the prior art, by demonstrating the possibility of treating the pathologies linked to the transport of cholesterol by gene therapy, by the transfer and expression in vivo of a gene encoding an

ABC1 protein involved in the transport and the metabolism of cholesterol. The invention thus offers a simple means allowing a specific and effective treatment of related pathologies such as, for example, atherosclerosis.

® ® 44

Gene therapy consists in correcting a deficiency or an abnormality (mutation, aberrant expression and the like) and in bringing about the expression of a protein of therapeutic interest by introducing genetic information into the affected cell or organ. This genetic information may 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 various transfection techniques involving complexes of DNA and DEAE-dextran (Pagano et al,

J.Virol. 1 (1967) 891), of DNA and nuclear proteins (Kaneda et al., Science 243 (1989) 375), of DNA and lipids (Felgner et al., PNAS 84 (1987) 7413), the use of liposomes (Fraley et al., J.Biol.Chem. 255 (1980) 10431), and the like. More recently, the use of viruses as vectors for the transfer of genes has appeared as a promising alternative to these physical transfection techniques. In this regard, various viruses have been tested for their capacity to infect certain cell populations. In particular, the retroviruses (RSV, HMS, MMS, and the like), the

HSV virus, the adeno-associated viruses and the adenoviruses.

The present invention therefore also relates to a new therapeutic approach for the treatment of pathologies linked to the transport of cholesterol, consisting in transferring and in expressing in vivo genes encoding ABC1. In a particularly advantageous manner, the applicant has now found that it is possible to construct recombinant viruses containing a DNA sequence encoding an ABC1 protein involved in the metabolism of cholesterol, to administer these recombinant viruses in vivo, and that this administration allows ‘a stable and effective expression of a biologically active ABC1 protein in vivo, with no cytopathological effect.

The present invention also results from the demonstration that adenoviruses constitute particularly efficient vectors for the transfer and the

® ® 45 expression of the ABC1 gene. In particular, the present invention shows that the use of recombinant adenoviruses as vectors makes it possible to obtain sufficiently high levels of expression of this gene to produce the desired therapeutic effect. Other viral vectors such as retroviruses or adeno-associated viruses (AAV) allowing a stable expression of the gene are also claimed.

The present invention thus offers a new approach for the treatment and prevention of cardiovascular and neurological pathologies linked to the abnormalities of the transport of cholesterol.

The subject of the invention is therefore also a defective recombinant virus comprising a nucleic sequence encoding an ABC1 protein involved in the metabolism of cholesterol.

The invention also relates to the use of such a defective recombinant virus for the preparation of a pharmaceutical composition intended for the treatment and/or for the prevention of cardiovascular diseases.

The present invention also relates to the use of cells genetically modified ex vivo with a virus as described above, or of producing cells such as viruses, implanted in the body, allowing a prolonged and effective expression in vivo of a biologically active ABC1 protein.

The present invention shows that it is possible to incorporate a DNA sequence encoding ABC1 into a viral vector, and that these vectors make it possible to effectively express a biologically active, mature form. More particularly, the invention shows that the in vivo expression of ABC1 may be obtained by direct administration of an adenovirus or by implantation of a producing cell or of a cell genetically modified by an adenovirus or by a retrovirus incorporating such a DNA.

® 46

The present invention is particularly advantageous because it makes it possible to induce a controlled expression, and with no harmful effect, of ABC1 in organs which are not normally involved in the expression of this protein. In particular, a significant release of the ABC1 protein is obtained by implantation of cells producing vectors of the invention, or infected ex vivo with vectors of the invention.

The activity of transport of cholesterol produced in the context of the present invention may be of the human or animal ABC1 type. The nucleic sequence used in the context of the present invention may 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 may also involve synthetic or semisynthetic sequences. In a particularly advantageous manner, a cDNA or a gDNA is used. In particular, the use of a gDNA allows a better expression in human cells. To allow their incorporation into a viral vector according to the invention, these sequences are advantageously modified, for example by site-directed mutagenesis, in particular for the insertion of appropriate restriction sites. The sequences described in the prior art are indeed not constructed for use according to the invention, and prior adaptations may prove necessary, in order to obtain substantial expressions. In the context of the present invention, the use of a nucleic sequence encoding a human ABC1 protein is preferred. 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 obtained by mutation, deletion and/or addition relative to the native sequence, and encoding a product retaining the cholesterol transport activity. These modifications may be made by techniques known to a person skilled in the art (see general molecular biological techniques below). The biological activity of

@ ® 47 the derivatives thus obtained can then be easily determined, as indicated in particular in the examples of the measurement of the efflux of cholesterol from cells. The derivatives for the purposes of the invention may also be obtained by hybridization from nucleic acid libraries, using as probe the native sequence or a fragment thereof.

These derivatives are in particular molecules having a higher affinity for their binding sites, molecules exhibiting greater resistance to proteases, molecules having a higher therapeutic efficacy or fewer side effects, or optionally new biological properties. The derivatives also include the modified

DNA sequences allowing improved expression in vivo.

In a first embodiment, the present invention relates to a defective recombinant virus comprising a cDNA sequence encoding an ABC1 protein involved in the transport and metabolism of cholesterol. In another preferred embodiment of the invention, the DNA sequence is a gDNA sequence.

The vectors of the invention may be prepared from various types of viruses. Preferably, vectors derived from adenoviruses, adeno-associated viruses (AAV), herpesviruses (HSV) or retroviruses are used. It is most particularly advantageous to use an adenovirus, for direct administration or for the ex vivo modification of cells intended to be implanted, or a retrovirus, for the implantation of producing cells.

The viruses according to the invention are defective, that is to say that they are incapable of autonomously replicating in the target cell. Generally, the genome of the defective viruses used in the context of the present invention therefore lacks at least the sequences necessary for the replication of said virus in the infected cell. These regions may be either eliminated (completely or partially), or made nonfunctional, or substituted with other sequences and in particular with the nucleic sequence encoding the ABC1 protein. Preferably, the

® ® 48 defective virus retains, nevertheless, the sequences of its genome which are necessary for the encapsidation of the viral particles.

As regards more particularly adenoviruses, various serotypes, whose structure and properties vary somewhat, have been characterized. Among these serotypes, human adenoviruses of type 2 or 5 (Ad 2 or Ad 5) or adenoviruses of animal origin (see Application WO 94/26914) are preferably used in the context of the present invention. Among the adenoviruses of animal origin which can be used in the context of the present invention, there may be mentioned adenoviruses of canine, bovine, murine (example: Mav1, Beard et al, Virology 75 (1990) 81), ovine, porcine, avian or simian (example: SAV) origin. Preferably, the adenovirus of animal origin is a canine adenovirus, more preferably a CAV2 adenovirus [Manhattan or A26/61 strain (ATCC VR-800) for example]. Preferably, adenoviruses of human or canine or mixed origin are used in the context of the invention. Preferably, the defective adenoviruses of the invention comprise the ITRs, a sequence allowing the encapsidation and the sequence encoding the ABC1 protein. Advantageously, in the genome of the adenoviruses of the invention, the E1 region at least is made nonfunctional.

Still more preferably, in the genome of the adenoviruses of the invention, the E1 gene and at least one of the E2, E4 and L1-L5 genes are nonfunctional. The viral gene considered may be made nonfunctional by any technique known to a person skilled in the art, and in particular by total suppression, by substitution, by partial deletion or by addition of one or more bases in the gene(s) considered. Such modifications may be obtained in vitro (on the isolated DNA) or in situ, for example, by means of genetic engineering techniques, or by treatment by means of mutagenic agents. Other regions may also be modified, and in particular the E3 (W085/02697), E2 (W094/28938), E4 (W094/28152,

W094/12649, WO95/02697) and LS (WO95/02697) region. According to a

® 49 preferred embodiment, the adenovirus according to the invention comprises a deletion in the E1 and E4 regions and the sequence encoding ABC1 is inserted at the level of the inactivated E1 region. According to another preferred embodiment, it comprises a deletion in the E1 region at the level of which the

E4 region and the sequence encoding ABC1 (French Patent Application

FR94 13355) are inserted.

The defective recombinant adenoviruses according to the invention may 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). In particular, they may be prepared by homologous recombination between an adenovirus and a plasmid carrying, inter alia, the DNA sequence encoding the

ABC1 protein. The homologous recombination occurs after cotransfection of said adenoviruses and plasmid into an appropriate cell line. The cell line used must preferably (i) be transformable by said elements, and (ii), contain the sequences capable of complementing the part of the defective adenovirus genome, preferably in integrated form in order to avoid the risks of recombination. By way of example of a line, there may be mentioned the human embryonic kidney line 293 (Graham et al., J. Gen. Virol. 36 (1977) 59) which contains in particular, integrated into its genome, the left part of the genome of an AdS adenovirus (12%) or lines capable of complementing the E1 and E4 functions as described in particular in Applications No. WO 94/26914 and WO95/02697.

Next, the adenoviruses which have multiplied are recovered and purified according to conventional molecular biological techniques, as illustrated in the examples.

As regards the adeno-associated viruses (AAV), they are DNA viruses of a relatively small size, which integrate into the genome of the cells which they

_ ¢ 50 infect, in a stable and site-specific manner. They are capable of infecting a broad spectrum of cells, without inducing any effect on cellular growth, morphology or differentiation. Moreover, they do not appear to be involved in pathologies in humans. The genome of AAVs has been cloned, sequenced and characterized. It comprises about 4700 bases, and contains at each end an inverted repeat region (ITR) of about 145 bases, serving as replication origin for the virus. The remainder of the genome is divided into 2 essential regions carrying the encapsidation functions: the left hand part of the genome, which contains the rep gene, involved in the viral replication and the expression of the viral genes; the right hand part of the genome, which contains the cap gene encoding the virus capsid proteins.

The use of vectors derived from AAVs for the transfer of genes in vitro and in vivo has been described in the literature (see in particular WO 91/18088;

WO 93/09239; US 4,797,368, US5,139,941, EP 488 528). These applications describe various constructs derived from AAVs, in which the rep and/or cap genes are deleted and replaced by a gene of interest, and their use for : transferring in vitro (on cells in culture) or in vivo (directly into an organism) said gene of interest. However, none of these documents either describes or suggests the use of a recombinant AAV for the transfer and expression in vivo or ex vivo of an ABC1 protein, or the advantages of such a transfer. The defective recombinant AAVs according to the invention may be prepared by cotransfection, into a cell line infected with a human helper virus (for example an adenovirus), of a plasmid containing the sequence encoding the ABC1 protein bordered by two AAV inverted repeat regions (ITR), and of a plasmid carrying the AAV encapsidation genes (rep and cap genes). The recombinant

AAVs produced are then purified by conventional techniques.

@ : ®

As regards the herpesviruses and the retroviruses, the construction of recombinant vectors has been widely described in the literature: see in particular Breakfield et al., New Biologist 3 (1991) 203; EP 453242, EP178220,

Bernstein et al. Genet. Eng. 7 (1985) 235; McCormick, BioTechnology 3 (1985) 689, and the like.

In particular, the retroviruses are integrating viruses, infecting dividing cells. The genome of the retroviruses essentially comprises two LTRs, an encapsidation sequence and three coding regions (gag, pol and env). In the recombinant vectors derived from retroviruses, the gag, pol and env genes are generally deleted, completely or partially, and replaced with a heterologous nucleic acid sequence of interest. These vectors may be produced from various types of retroviruses such as in particular MoMuLV. ("murine moloney leukemia virus"; also called MoMLV), MSV ("murine moloney sarcoma virus"), HaSV ("harvey sarcoma virus"); SNV ("spleen necrosis virus"); RSV ("rous sarcoma virus") or Friend's virus.

To construct recombinant retroviruses containing a sequence encoding the ABC1 protein according to the invention, a plasmid containing in particular the LTRs, the encapsidation sequence and said coding sequence is generally constructed, and then used to transfect a so-called encapsidation cell line, capable of providing in trans the retroviral functions deficient in the plasmid.

Generally, the encapsidation lines are therefore capable of expressing the gag, pol and env genes. Such encapsidation lines have been described in the prior art, and in particular the PA317 line (US 4,861,719), the PsiCRIP line (WO 90 /02806) and the GP+envAm-12 line (WO 89/07150). Moreover, the recombinant retroviruses may contain modifications at the level of the LTRs in order to suppress the transcriptional activity, as well as extended encapsidation sequences, containing a portion of the gag gene (Bender et al., J. Virol. 61

® ® 52 (1987) 1639). The recombinant retroviruses produced are then purified by conventional techniques.

To carry out the present invention, it is most particularly advantageous to use a defective recombinant adenovirus. The results given below indeed demonstrate the particularly advantageous properties of adenoviruses for the in vivo expression of a protein having a cholesterol transport activity. The adenoviral vectors according to the invention are particularly advantageous for a direct administration in vivo of a purified suspension, or for the ex vivo transformation of cells, in particular autologous cells, in view of their implantation. Furthermore, the adenoviral vectors according to the invention exhibit, in addition, considerable advantages, such as in particular their very high infection efficiency, which makes it possible to carry out infections using small volumes of viral suspension.

According to another particularly advantageous embodiment of the invention, a line producing retroviral vectors containing the sequence encoding the ABC1 protein is used for implantation in vivo. The lines which can be used to this end are in particular the PA317 (US 4,861,719), PsiCrip (WO 90/02806) and GP+envAm-12 (US 5,278,056) cells modified so as to allow the production of a retrovirus containing a nucleic sequence encoding an ABC1 protein according to the invention. For example, totipotent stem cells, precursors of - blood cell lines, may be collected and isolated from a subject. These cells, when cultured, may then be transfected with the retroviral vector containing the sequence encoding the ABC1 protein under the control of viral, nonviral or nonviral promoters specific for macrophages or under the control of its own promoter. These cells are then reintroduced into the subject. The differentiation of these cells will be responsible for blood cells expressing the ABC1 protein, in particular for monocytes which, when transformed to macrophages, participate

® ® 53 in the removal of cholesterol from the arterial wall. These macrophages expressing the ABC1 protein will have an increased capacity to metabolize cholesterol in excess and will make it available to the cell surface for its removal by the primary acceptors of membrane cholesterol.

Advantageously, in the vectors of the invention, the sequence encoding the ABC1 protein is placed under the control of signals allowing its expression in the infected cells. These may be expression signals which are homologous or heterologous, that is to say signals different from those which are naturally responsible for the expression of the ABC1 protein. They may also be in particular sequences responsible for the expression of other proteins, or synthetic sequences. In particular, they may be sequences of eukaryotic or viral genes or derived sequences, stimulating or repressing the transcription of a gene in a specific manner or otherwise and in an inducible manner or otherwise. By way of example, they may be promoter sequences derived from the genome of the cell which it is desired to infect, or from the genome of a virus, and in particular the promoters of the E1A or MLP genes of adenoviruses, the CMV promoter, the RSV-LTR and the like. Among the eukaryotic promoters, there may also be mentioned the ubiquitous promoters (HPRT, vimentin, a-actin, tubulin and the like), the promoters of the intermediate filaments (desmin, neurofilaments, keratin, GFAP, and the like), the promoters of therapeutic genes (of the MDR, CFTR or factor VIII type, and the like), tissue-specific promoters (pyruvate kinase, villin, promoter of the fatty acid binding intestinal protein, promoter of the smooth muscle cell a-actin, promoters specific for the liver; Apo Al, Apo All, human albumin and the like) or promoters corresponding to a stimulus (steroid hormone receptor, retinoic acid receptor and the like). In addition, these expression sequences may be modified by addition of enhancer or regulatory sequences and the like. Moreover, when the

® ® 54 inserted gene does not contain expression sequences, it may be inserted into the genome of the defective virus downstream of such a sequence.

In a specific embodiment, the invention relates to a defective recombinant virus comprising a nucleic sequence encoding an ABC1 protein involved in the metabolism of cholesterol under the control of a promoter chosen from RSV-LTR or the CMV early promoter.

As indicated above, the present invention also relates to any use of a virus as described above for the preparation of a pharmaceutical composition for the treatment and/or prevention of pathologies linked to the transport of cholesterol.

The present invention also relates to a pharmaceutical composition comprising one or more defective recombinant viruses as described above.

These pharmaceutical compositions may be formulated for administration by the topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular or transdermal route and the like. Preferably, the pharmaceutical compositions of the invention contain a pharmaceutically acceptable vehicle for an injectable formulation, in particular for an intravenous injection, such as for example into the patient's portal vein. They may relate in particular to isotonic sterile solutions or dry, in particular, freeze-dried, compositions which, upon addition depending on the case of sterilized water or physiological saline, allow the preparation of injectable solutions. Direct injection into the patient's portal vein is advantageous because it makes it possible to target the infection at the level of the liver and thus to concentrate the therapeutic effect at the level of this organ.

The doses of defective recombinant virus used for the injection may be adjusted as a function of various parameters, and in particular as a function of the viral vector, of the mode of administration used, of the relevant pathology or

® ® 55 of the desired duration of treatment. In general, the recombinant adenoviruses according to the invention are formulated and administered in the form of doses of between 104 and 1014 pfu/ml, and preferably 106 to 1010 pfu/ml. The term pfu (“plaque forming unit”) corresponds to the infectivity of a virus solution, and is determined by infecting an appropriate 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 may directly contain the producing cells, with a view to their implantation.

In this regard, another subject of the invention relates to any mammalian cell infected with one or more defective recombinant viruses as described above. More particularly, the invention relates to any population of human cells infected with these viruses. These may be in particular cells of blood origin (totipotent stem cells or precursors), fibroblasts, myoblasts, hepatocytes, keratinocytes, smooth muscle and endothelial cells, glial cells and the like.

The cells according to the invention may be derived from primary cultures. These may be collected by any technique known to persons skilled in the art and then cultured under conditions allowing their proliferation. As regards more particularly fibroblasts, these may be easily obtained from biopsies, for example according to the technique described by Ham [Methods

Cell.Biol. 21a (1980) 255]. These cells may be used directly for infection with the viruses, or stored, for example by freezing, for the establishment of autologous libraries, in view of a subsequent use. The cells according to the invention may be secondary cultures, obtained for example from preestablished libraries (see for example EP 228458, EP 289034, EP 400047, EP 456640).

The cells in culture are then infected with the recombinant viruses, in order to confer on them the capacity to produce a biologically active ABC1

® ® protein. The infection is carried out in vitro according to techniques known to persons skilled in the art. In particular, depending on the type of cells used and the desired number of copies of virus per cell, persons skilled in the art can adjust the multiplicity of infection and optionally the number of infectious cycles produced. It is clearly understood that these steps must be carried out under appropriate conditions of sterility when the cells are intended for administration in vivo. The doses of recombinant virus used for the infection of the cells may be adjusted by persons skilled in the art according to the desired aim. The conditions described above for the administration in vivo may be applied to the infection in vitro. For the infection with retroviruses, it is also possible to coculture the cells which it is desired to infect with cells producing the recombinant retroviruses according to the invention. This makes it possible to dispense with the purification of the retroviruses.

Another subject of the invention relates to an implant comprising mammalian cells infected with one or more defective recombinant viruses as described above or cells producing recombinant viruses, ahd an extracellular matrix. Preferably, the implants according to the invention comprise 10% to 1010 cells. More preferably, they comprise 106 to 108 cells.

More particularly, in the implants of the invention, the extracellular matrix comprises a gelling compound and optionally a support allowing the anchorage of the cells.

For the preparation of the implants ‘according to the invention, various types of gelling agents may be used. The gelling agents are used for the inclusion of the cells in a matrix having the constitution of a gel, and for promoting the anchorage of the cells on the support, where appropriate.

Various cell adhesion agents can therefore be used as gelling agents, such as in particular collagen, gelatin, glycosaminoglycans, fibronectin, lectins and the

® ® like. Preferably, collagen is used in the context of the present invention. This may be collagen of human, bovine or murine origin. More preferably, type collagen is used.

As indicated above, the compositions according to the invention advantageously comprise a support allowing the anchorage of the cells. The term anchorage designates any form of biological and/or chemical and/or physical interaction causing the adhesion and/or the attachment of the cells to the support. Moreover, the cells may either cover the support used, or penetrate inside this support, or both. It is preferable to use in the context of the invention a solid, nontoxic and/or biocompatible support. In particular, it is possible to use polytetrafluoroethylene (PTFE) fibers or a support of biological origin.

The present invention thus offers a very effective means for the treatment or prevention of pathologies linked to the transport of cholesterol, in particular obesity, hypertriglyceridemia, or, in the field of cardiovascular conditions, myocardial infarction, angina, sudden death, cardiac decompensation and cerebrovascular accidents.

In addition, this treatment may be applied to both humans and any animals such as ovines, bovines, domestic animals (dogs, cats and the like), horses, fish and the like.

RECOMBINANT HOST CELLS

The invention also relates to a recombinant host cell comprising any of the nucleic acids of the invention, and more particularly a nucleic acid having the sequence SEQ ID NO 91, 94 or 96

® ug 58

According to another aspect, the invention also relates to a recombinant host cell comprising a recombinant vector as described above.

The preferred host cells according to the invention are for example the following: a) prokaryotic host cells: strains of Escherichia coli (strain DH5-a), of

Bacillus subtilis, of Salmonella typhimurium, or strains of species such as

Pseudomonas, Streptomyces and Staphylococus ; b) eukaryotic host cells: Hela cells (ATCC No. CCL2), Cv 1 cells (ATCC

No. CCL70), COS cells (ATCC No. CRL 1650), Sf-9 cells (ATCC No. CRL 1711), CHO cells (ATCC No. CCL-61) or 3T3 cells (ATCC No. CRL-6361).

MUTATED ABC1 POLYPEPTIDES

According to another aspect, the invention relates to a polypeptide encoded by a mutated ABC1 gene, and more particularly a mutated ABC1 gene in patients suffering from a deficiency in the reverse transport of cholesterol, most particularly in patients suffering from Tangier disease.

As indicated above, two deleterious mutations have been identified in patients suffering from Tangier disease. : The first mutation corresponds to the insertion of a fragment of about one hundred base pairs into the coding sequence, at the level of exon 12 of the

ABC1 gene, leading to the production of a biologically inactive polypeptide of 2233 amino acids having the sequence SEQ ID NO 140. The mutated ABC1 polypeptide having the sequence SEQ ID NO 140 possesses, compared with the normal polypeptide having the sequence SEQ iD NO 139, the following differences:

® ® 59 a) a deletion of a peptide fragment having the sequence “DERKFW” and the replacement of this peptide fragment with the sequence “EYSGVTSAHCNLCLLSSSDSRASASQVAGITAPATTPG” encoded by the inserted Alu-type nucleotide fragment.

The second mutation relates to the introduction of an early stop codon into the first quarter of the coding sequence, at the level of exon 13 of the ABC1 gene, leading to the production of a truncated polypeptide having 574 amino acids having the sequence SEQ ID NO 141. In addition, the deletion of the G base induces a change in the reading frame leading to a protein whose COQH- terminal end is not found in the amino acid sequence of the normal ABC1 polypeptide. This is the COOH-terminal sequence “RAPRRKLVSICNRCPIPVTLMTSFCG” of the mutated ABC1 polypeptide having the sequence SEQ ID NO 141.

These two polypeptides are useful in particular for the preparation of antibodies specifically recognizing them. Such antibodies constitute means of detection of the production of these mutated ABC1 polypeptides in a sample obtained from a subject to be tested, preferably a patient having symptoms characteristic of a deficiency in the reverse transport of cholesterol, and most preferably in a patient having the symptoms characteristic of Tangier disease.

According to another aspect, the invention therefore relates to a polypeptide comprising an amino acid sequence SEQ ID NO 140.

According to another aspect, the invention relates to a polypeptide comprising an amino acid sequence SEQ ID NO 141.

The invention also relates to a polypeptide comprising an amino acid sequence having at least 80% amino acid identity with an amino acid sequence eo ® 60 chosen from the group consisting of the peptides having the sequences SEQ ID

NO 140 and 141, or a peptide fragment thereof.

A first preferred peptide fragment will comprise at least 5 consecutive amino acids of the = peptide fragment having the sequence “EYSGVTSAHCNLCLLSSSDSRASASQVAGITAPATTPG” contained in the mutated ABC1 polypeptide having the sequence SEQ ID NO 140.

A second preferred peptide fragment will comprise at least 5 consecutive amino acids of the peptide fragment having the sequence ‘RAPRRKLVSICNRCPIPVTLMTSFCG” contained in the mutated ABC1 polypeptide having the sequence SEQ ID NO 141. : Avantageously, a polypeptide having at least 85%, 90%, 95% or 99% amino acid identity with an amino acid sequence chosen from the group consisting of the peptides having the sequences SEQ ID NO 140 and 141, or a peptide fragment thereof, forms part of the invention.

Preferably, polypeptides according to the invention will have a length of 15, 18 or 20 to 25, 35, 40, 50, 70, 80, 100 or 200 consecutiuve amino acids of a nucleic acid according to the invention, in particular a polypeptide having an amino acid sequence chosen from the sequences SEQ ID No140 and 141.

Alternatively, a polypeptide according to the invention will consist of and/or will comprise the fragments having a length of 15, 18, 20, 25, 35, 40, 50, 100 or 200 consecutive amino acids of a polypeptide according to the invention, more particularly of a polypeptide chosen from the sequences SEQ ID NO 140 and 141.

@

Generally, the polypeptides according to the invention are provided in an isolated or purified form.

The invention also relates to a method for the production of one of the polypetides having the sequences SEQ ID NO 140 and 141, or of a peptide fragment or of a variant thereof, said method comprising the steps of: a) inserting a nucleic acid encoding said polypeptide into an appropriate vector, b) culturing, in an appropriate culture medium, a previously transformed host cell or transfecting with the recombinant vector of step a) ; c) recovering the conditioned culture medium or lysing the host cell, for example by sonication or by osmotic shock; d) separating and purifying said polypeptide from said culture medium or alternatively from the cell lysates obtained in step c); e) where appropriate, characterizing the recombinant polypeptide produced.

The peptides according to the invention may be characterized by binding to an immunoaffinity chromatography column on which the antibodies directed against this polypeptide or against a fragment or a variant thereof have been previously immobilized.

According to another aspect, a recombinant polypeptide according to the invention may be purified by passing over an appropriate series of chromatography columns, according to methods known to persons skilled in the art and described for example in F.Ausubel et al (1989).

@ ® 62

A polypeptide according to the invention may also be prepared by conventional chemical synthesis techniques either in homogeneous solution or in solid phase.

By way of illustration, a polypeptide according to the invention may be prepared by the technique either in homogeneous solution described by

Houben Weyl (1974) or the solid phase synthesis technique described by

Merrifield (1965a; 1965b).

Polypeptides termed “homologous” to any one of the polypeptides having the amino acid sequences SEQ ID NO 140 and 141, or their fragments or variants, also form part of the invention. - Such homologous polypeptides have amino acid sequences possessing one or more substitutions of an amino acid by an equivalent amino acid, relative to the reference polypeptides.

Equivalent amino acid according to the present invention will be understood to mean for example replacement of a residue in the L form by a residue in the D form or the replacement of a glutamic acid (E) by a pyro- glutamic acid according to techniques well known to persons skilled in the art.

By way of illustration, the synthesis of peptide containing at least one residue in . the D form is described by Koch (1977).

According to another aspect, two amino acids belonging to the same class, that is to say two uncharged polar, nonpolar, basic or acidic amino acids, are also considered as equivalent amino acids.

Polypeptides comprising at least one nonpeptide bond such as a retro- inverse bond (NHCO), a carba bond (CH,CH;) or a ketomethylene bond (CO-CHy) also form part of the invention.

Preferably, the polypeptides according to the invention comprising one or more additions, deletions, substitutions of at least one amino acid will retain

® ® 63 their capacity to be recognized by antibodies directed against the nonmodified polypeptides.

ANTIBODIES

The mutated ABC1 polypeptides according to the invention, in particular the polypeptides having the amino acid sequences SEQ ID NO 140-141] or the fragments thereof as well as the homologous peptides may be used for the preparation of antibodies, in particular for detecting the production of altered forms of the ABC1 polypeptide in a patient.

A first preferred antibody according to the invention is directed against a peptide fragment comprising at least 5 consecutive amino acids of the peptide fragment having the sequence “‘EYSGVTSAHCNLCLLSSSDSRASASQVAGITAPATTPG” contained in the mutated ABC1 polypeptide having the sequence SEQ ID NO 140.

A second preferred antibody according to the invention is directed against a peptide fragment comprising at least 5 consecutive amino acids of the peptide fragment having the sequence “RAPRRKLVSICNRCPIPVTLMTSFCG” contained in the mutated ABC1 polypeptide having the sequence

SEQ ID NO 141. “Antibody” for the purposes of the present invention will be understood to mean in particular polyclonal or monoclonal antibodies or fragments (for example the F (ab), and Fab fragments) or any polypeptide comprising a domain of the initial antibody recognizing the target polypeptide or polypeptide fragment according to the invention.

Monoclonal antibodies may be prepared from hybridomas according to the technique described by Kohler and Milstein (1975).

®

The present invention also relates to antibodies directed against a polypeptide as described above or a fragment or a variant thereof, as produced in the trioma technique or the hybridoma technique described by Kozbor et al. (1983).

The invention also relates to single-chain Fv antibody fragments (ScFv) as described in US patent No. 4,946,778 or by Martineau et al. (1998).

The antibodies according to the invention also comprise antibody fragments obtained with the aid of phage libraries Ridder et al., (1995) or humanized antibodies Reinmann et al. (1997); Leger et al., (1997).

The antibody preparations according to the invention are useful in : immunological detection tests intended for the identification of the presence and/or of the quantity of antigens present in a sample.

An antibody according to the invention may comprise, in addition, a detectable marker which is isotopic or nonisotopic, for example fluorescent, or : may be coupled to a molecule such as biotin, according to techniques well known to persons skilled in the art.

Thus, the subject of the mention is, in addition, a method of detecting the presence of a polypeptide in accordance with the invention in a sample, said method comprising the steps of: a) bringing the sample to be tested into contact with an antibody as described above; b) detecting the antigen/antibody complex formed.

The invention also relates to a box or kit for diagnosis or for detecting the presence of a polypeptide in accordance with the invention in a sample, said box comprising:

Claims (34)

1. Nucleic acid comprising at least 245 consecutive nucleotides of a polynucleotide chosen from the group consisting of the nucleotide sequences SEQID NO 1-14, or a nucleic acid having a complementary sequence.

2. Nucleic acid comprising a polynucleotide chosen from the group consisting of the nucleotide sequences SEQ ID NO 15-47, or a nucleic acid having a complementary sequence.

3. Nucleic acid comprising at least 8 consecutive nucleotides of a polynucleotide chosen from the group consisting of the nucleotide sequences SEQ ID NO 48-90, or a nucleic acid having a complementary sequence.

4. Nucleic acid having at least 80% nucleotide identity with a polynucleotide chosen from the group consisting of the nucleotide sequences SEQ ID NO 48-90, or a nucleic acid having a complementary sequence.

5. Nucleic acid hybridizing, under high stringency conditions, with a polynucleotide chosen from the group consisting of the nucleotide sequences SEQ ID NO 48-90, or a nucleic acid having a complementary sequence.

6. Nucleic acid comprising a polynucleotide having the sequence SEQ ID NO 91, or a nucleic acid having a complementary sequence.

7. Nucleic acid comprising at least eight consecutive nucleotides of a polynucleotide chosen from the group consisting of the nucleotide sequences

SEQ ID NO 92, a biologically active fragment thereof or a nucleic acid having a complementary sequence.

8. Nucleic acid having at least 80% nucleotide identity with a polynucleotide chosen from the group consisting of the nucleotide sequences SEQ ID NO 92, a biologically active fragment thereof or a nucleic acid having a complementary sequence.

9. Nucleic acid hybridizing, under high stringency conditions, with a polynucleotide chosen from the group consisting of the nucleotide sequences SEQ ID NO 92, a biologically active fragment thereof or a nucleic acid having a complementary sequence.

10. Nucleic acid having at least eight consecutive nucleotides of a polynucleotide chosen from the group consisting of the nucleotide sequences SEQ ID NO 93-96, or a nucleic acid having a complementary sequence.

11. Nucleic acid having at least 80% nucleotide identity with a polynucleotide chosen from the group consisting of the nucleotide sequences SEQ ID NO 93-96, or a nucleic acid having a complementary sequence.

12. Nucleic acid hybridizing, under high stringency conditions, with a polynucleotide chosen from the group consisting of the nucleotide sequences SEQ ID NO 93-96, or a nucleic acid having a complementary sequence.

13. Nucleic acid having at least eight consecutive nucleotides of a polynucleotide chosen from the group consisting of the nucleotide sequences

SEQ ID NO 97-108 and comprising the polymorphic base, or a nucleic acid having a complementary sequence.

14. Nucleotide probe or primer specific for the ABC1 gene, having a length of at least 15 nucleotides, chosen from the nucleic acids according to any one of claims 1 to 9.

15. Probe or primer according to claim 14, comprising a polynucleotide chosen from the group consisting of the nucleotide sequences SEQ ID NO 109-138, or a nucleic acid having a complementary sequence.

16. Nucleotide probe or primer useful for the detection of a mutation in the ABC1 gene, having a length of at least 15 nucleotides, chosen from the nucleic acids according to either of claims 11 and 12.

17. Nucleotide probe or primer according to claim 16, comprising a polynucleotide chosen from the nucleotide sequences SEQ ID NO 109 — 112, or a nucleic acid having a complementary sequence.

18. Nucleotide probe or primer useful for the detection of a polymorphism in the ABC1 gene, having a length of at least 15 nucleotides, chosen from the nucleic acids according to claim 13.

19. Probe or primer according to claim 18, comprising a polynucleotide chosen from the nucleotide sequences SEQ ID NO 142-149, or a nucleic acid having a complementary sequence.

_ ® 115

20. Nucleotide primer comprising at least 15 consecutive nucleotides of a polynucleotide chosen from the group consisting of the nucleotide sequences SEQ ID NO 97-108 or of their complementary sequences, the base of the 3' end of these primers being complementary to the nucleotide located immediately on the 5' side of the polymorphic base of one of the sequences SEQ ID NO 97-108 or of their complementary sequences.

21. Nucleotide primer comprising at least 15 consecutive nucleotides of a polynucleotide chosen from the group consisting of the nucleotide sequences SEQ ID NO 97-108 or of their complementary sequences, the base of the 3' end of these primers being complementary to a nucleotide situated at 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or more on the 5’ side of the polymorphic base of one of the sequences SEQ ID NO 97-108 or of their complementary sequences.

22. Method of amplifying a nucleic acid according to any one of claims 1 to 13 contained in a sample, said method comprising the steps of: a) bringing the sample in which the presence of the target nucleic acid is suspected into contact with a pair of nucleotide primers whose 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 necessary for the amplification reaction; and b) detecting the amplified nucleic acids.

® ® 116

23. Method of amplification according to claim 22, characterized in that the nucleotide primers are chosen from the primers according to any one of claims 14 to 19.

24. Box for amplifying a nucleic acid according to any one of claims 1 to 13 comprising: a) a pair of nucleotide primers whose hybridization position is located respectively on the 5 side and 3’side of the target nucleic acid whose amplification is sought; b) where appropriate, the reagents necessary for the amplification reaction.

25. Box for amplifying a nucleic acid according to claim 22, characterized in that the nucleotide primers are chosen from the group consisting of the primers according to any one of claims 14 to 19.

26. Nucleotide probe according to any one of claims 14 to 19, characterized in that it comprises a marker compound whose presence is detectable.

27. Method of detecting the presence of a nucleic acid according to any one of claims 1 to 13 in a sample, said method comprising the steps of: a) bringing one or more nucleic probes according to one of claims 14 to 19 into contact with the sample to be tested; b) detecting the complex which may have formed between the probe(s) and the nucleic acid present in the sample.

28. Method of detection according to claim 27, characterized in that the probe(s) are immobilized on a support.

29. Box for detecting the presence of a nucleic acid according to any one of claims 1 to 13 in a sample, said box comprising: a) one or more nucleotide probes according to any one of claims 14 to 19; b) where appropriate, the reagents necessary for the hybridization reaction.

30. Box for detection according to claim 29, characterized in that the probe(s) are immobilized on a suppport.

31. Recombinant vector comprising a nucleic acid according to one of claims 1 to 13.

32. Vector according to claim 31, characterized in that it is an adenovirus.

33. Vector according to either of claims 32 and 33, characterized in that it is ABC1-rldV :

34. Recombinant host cell comprising a nucleic acid according to one of claims 1 to 13 or a recombinant vector according to one of claims 31 to 33.

35. Mutated ABC1 polypeptide, characterized in that it comprises a polypeptide having the amino acid sequence SEQ ID NO 140.

® eo 118 PCT/FR00/01595

36. Mutated ABC1 polypeptide, characterized in that it comprises a polypeptide having the amino acid sequence SEQ ID NO 141.

37. Antibody directed against a mutated ABC1 polypeptide according to either of claims 35 and 36, or a peptide fragment thereof.

38. Antibody according to claim 37, characterized in that it comprises a detectable compound.

39. Method of detecting the presence of a polypeptide according to either of claims 35 and 36 in a sample, comprising the steps of: a) bringing the sample into contact with an antibody according to either of claims 37 and 38; b) detecting the antigen/antibody complex formed.

40. Diagnostic box for detecting the presence of a polypeptide according to either of claims 35 and 36 in a sample, said box comprising: : a) an antibody according to either of claims 37 and 38; b) a reagent allowing the detection of the antigen/antibody complexes formed.

41. A substance or composition for use in a method for the prevention of or treatment of subjects affected by, a dysfunction in the reverse transport of cholesterol, said substance or composition comprising a nucleic acid according to either claims 1 and 6, and said method comprising administering said substance or composition in combination with one or more physiologically compatible excipients. AMENDED SHEET

119 PCT/FRO0/01595

42. A substance or composition for use in a method for the prevention of or treatment of subjects affected by, a dysfunction in the reverse transport of cholesterol, said substance or composition comprising a recombinant vector according to claim 31, and said method comprising administering said substance or composition in combination with one or more physiologically compatible excipients.

43. A use of nucleic acid according to one of claims 1 and 6 for the manufacture of a medicament intended for the prevention of Atherosclerosis in various forms or more particularly for the treatment of subjects affected by a dysfunction in the reverse transport of cholesterol.

44. Use of a recombinant vector according to claim 31 for the manufacture of a medicament intended for the prevention of Atherosclerosis in various forms or more particularly for the treatment of subjects affected by a dysfunction in the reverse transport of cholesterol.

45. Use according to claim 44, characterized in that the vector is ABC1- ridV.

46. Use of ABC1 polypeptide having the sequence SEQ ID NO 139 for the manufacture of a medicament intended for the prevention of Atherosclerosis in various forms or more particularly for the treatment of subjects affected by a dysfunction in the reverse transport of cholesterol.

47. A substance or composition for use in a method for the prevention of or treatment of subjects affected by, a dysfunction in the reverse transport of cholesterol, said substance or composition comprising the polypeptide having the sequence SEQ ID NO 139, and said method comprising administering said substance or composition. AMENDED SHEET y oo 121 PCT/FRO0/01595 d) incubating the cells obtained in step c) with an agonist or antagonist candidate compound for the ABC1 polypeptide; e) measuring the efflux of the labeled anion; f) comparing the value of the efflux of the labeled anion determined in step e) with the value of the efflux of the labeled anion measured with cells which have not been previously incubated in the presence of the agonist or antagonist candidate compound for the ABC1 polypeptide.

51. Method of screening a compound active on the metabolism of cholesterol, an agonist or antagonist of the ABC1 polypeptide, said method comprising the following steps: a) culturing cells of a human monocytic line in an appropriate culture medium, in the presence of purified human albumin; b) incubating the cells of step a) simultaneously in the presence of a compound stimulating the production of IL-1 beta and of the agonist or antagonist candidate compound; c) incubating the cells obtained in step b) in the presence of an appropriate concentration of ATP; d) measuring IL-1 beta released into the cell culture supernatant. e) comparing the value of the release of the IL-1 beta obtained is step d) with the value of the IL-1 beta released into the culture supernatant of cells which have not been previously incubated in the presence of the agonist or antagonist candidate compound.

52. Nucleic acid as claimed in any one of claims 1 to 13, substantially as herein described and illustrated.

53. A probe or primer as claimed in any one of claims 14 to 16, 18, 20, 21 or 26, substantially as herein described and illustrated. AMENDED SHEET

122 PCT/FR00/01595

54. A method as claimed in claim 22, substantially as herein described and illustrated.

55. A box as claimed in claim 24 or claim 25 or claim 29 or claim 40, substantially as herein described and illustrated.

56. A method as claimed in claim 27, substantially as herein described and illustrated.

57. A vector as claimed in claim 31, substantially as herein described and illustrated.

58. A host cell as claimed in claim 34, substantially as herein described and illustrated.

59. A polypeptide as claimed in claim 35 or claim 36, substantially as herein described and illustrated.

60. Antibody as claimed in claim 37, substantially as herein described and illustrated.

61. A method as claimed in claim 39, substantially as herein described and illustrated.

62. A method as claimed in claim 49 or claim 50 or claim 51, substantially as herein described and illustrated.

63. Use as claimed in claim 43 or claim 44 or claim 46, substantially as herein described and illustrated. AMENDED SHEET

123 PCT/FR00/01595

64. A substance or composition for use in a method of prevention or treatment as claimed in claim 41 or claim 42 or claim 47, substantially as herein described and illustrated.

65. Use as claimed in claim 48, substantially as herein described and illustrated.

66. A new nucleic acid, a new probe or primer, a new method of amplifying a nucleic acid, a new box, a new method of detecting the presence of a nucleic acid, a new vector, a new host cell, a new polypeptide, a new antibody, a new method of detecting the presence of a polypeptide, a new method of screening a compound, a new use of a nucleic acid as claimed in any one of claims 1 to 6, a new use of a vector as claimed in claim 31, a new use of ABC1 polypeptide, or a substance or composition for a new use in a method of treatment, substantially as herein described. AMENDED SHEET y /