SK156397A3 - Novel variants of apolipoprotein a-i and a pharmaceutical composition containing same - Google Patents

Abstract

The present invention relates to variants of the human apoloprotein A-I comprising a cystein in position 151, the corresponding nucleic acids and the vectors containing them. It also relates to pharmaceutical compositions comprising said elements and their utilization, particularly in genic therapy.

Description

Apolipoprotein A-I variants and a pharmaceutical composition containing them. .

Technical field

The present invention relates to novel variants of apolipoprotein

I

A-I. It also applies to all nucleic acids encoding these new variants. In particular, the invention relates to the use of these proteins or nucleic acids in therapy. It also relates to novel variants of apolipoprotein A-I comprising, in particular, a mutation at position 151.

BACKGROUND OF THE INVENTION

Apolipoprotein A-I (apoA-I) is primarily composed of high density lipoproteins (HDL), which are macromolecular complexes containing cholesterol, phospholipids and triglycerides. Apolipoprotein A-I is a protein consisting of 243 amino acids, synthesized in the ‘form of a 267 residue preprotein having a molecular weight of 28,000 daltons. The prepro apoA-I form is synthesized in humans by the liver and intestine at the same time. This form of protein is then cleaved into a preprotein that is secreted into the plasma. In the vascular system, proapoA-I is transformed into a final protein (243 amino acids) by calcium-dependent protease activity. Apolipoprotein A-I has a structural and active role in lipoprotein metabolism: apoA-I is in particular the cofactor of lecithin cholesterol acyltransferase (LCAT), responsible for esterification of plasmid cholesterol.

The level of cholesterol contained in the HDL fraction and the plasma concentration of apoA-I are dangerous negative factors in the development of atherosclerosis in humans. In fact, epidemiological studies should show a correlation of inversion between HDL cholesterol concentration and apoA-I and the incidence of cardiovascular disease (E.G. Miller et al., Lancet, 1977: 965-968). In contrast, longevity will be associated with high, HDL cholesterol. Recently, the protective role of apoA-I has been demonstrated in a model of transgenic mouse expressing human apoA-I (Rubin et al., Nature). Infusion of HDL in rabbits induces regression of disorders (Badimon et al., J. Clin. Invest. 85, 1990, 1234-1241). Various mechanisms have been proposed to explain the effect of the HDL protector, as well as the role of HDL in inverse cholesterol transport (Frichart et al., Circulation, 87) 1993, 22-27) and the activity of the HDL antioxidant (Forte T., Current Opinion in Lipidology, 5: 354 -

364 (1994). « He was cloned a sequenced gene encoding apoA- I (Sharpe et f? al., Nucleic Acids Res., 12 ( 9), 1984, 3917). this gene with length • R- 1863 bp, contains 4 exons and 3 introns. She was Too described cDNA coding apoA - I (Law et al., A. S. 81, 1984, 66 ). This cDNA It contains 840 bp (see SEQ ID NO . 1). In addition to the standard forms of apoA-I

various natural variants have been described whose differences with respect to wild-type protein are shown in the following table.

variant mutation variant mutation Milan Argl73Cys norway Glul36Lys Marburg Lysl07 Prol65Arg Munster2B Alal58Glu for 3 H Giessen Prol43Arg ArglOLeu Munster3A AsplO3Asn Gly2 6Arg Munster3B Pro4Arg Asp89Glu Munster3C Pro3Arg LyslO7Met Munster3D Asp213Gly Glul39Gly Munster4 Glul98Lys Glul47Val Yame Aspl3Tyr Alal58Glu Asp213Gly Glul69Gln Argl77His

SUMMARY OF THE INVENTION

The present invention results from the demonstration of a new series of variants of apolipoprotein A-I. In particular, this series of variants represents the substitution of an arginine residue at position 151 by a cysteine residue. The apoA-I variant of the present invention offers unique therapeutic properties. It has particularly important anti-atherogenic protection properties. In the situation of extremely low cholesterol in the HDL fraction associated with hypertriglyceridemia, the presence of this variant prevents the development of atherosclerosis, thus demonstrating its very strong protective role, specific for this mutated apoA-I. The presence of cysteine on apoA-I according to the invention causes the formation of dimers and other disulfide bridge complexes. This apoA-I is found in free form in plasma, bound in dimers or bound to apolipoprotein A-II, which is another important HDL-associated protein and which also has cysteine in its sequence. Otherwise, the loss of the arginine charge bound at position 151 causes this mutant to be visible by electrospraying the plasma proteins of the apoA-I immunological discovery.

These. The novel proteins of the invention offer an important therapeutic advantage in the treatment and prevention of cardiovascular diseases.

A first object of the invention relates to a series of variants of the human apolipoprotein A-I containing cysteine at position 151. The amino acid sequence of wild-type apolipoprotein A-I is described in the literature (see Law Préciteé). This sequence, including the prepro region (residues 1 to 24), is represented by SEQ ID NO: 1. Thus, the characteristics of the variants of the invention are based on the presence of cysteine at position 151 of the final apoA-I (corresponding to position 175 in SEQ ID No. 1) in the substitution of arginine present in the standard sequence. A preferred variant comprises the peptide sequence of SEQ ID NO. 2, especially the pentapeptide sequence comprised between residues 68-267 of SEQ ID NO. 1 ,. residue 175 is replaced by cysteine.

In particular, variants of the invention are represented by apoA-I Paris, i.e. apoA-I having a cysteine at position 151 relative to native apoA-I. Variants ^! according to the invention they may carry further structural modification with respect to the standard apolipoprotein A1, in particular other mutations, deletions and / or additions. Variants of the invention also include other mutations resulting in the replacement of individual residues by cysteine. Another variant combines the mutations present in the apoAI Paris and apoA-I milano variants. Other mutations may also be present

However, K * does not significantly modify the properties of apoA-I. In particular, the activity of these variants can be verified by a cholesterol assay.

Variants of the invention can be obtained in various ways. In particular, they can be chemically synthesized thanks to techniques used by those skilled in the art of peptide synthesis. They can also be obtained from standard apoA-I mutations or mutations. Preferably, they are recombinant proteins obtained by expressing the corresponding nucleic acid into a host cell as described below.

As indicated above, the variants of the invention may be in the form of monomers or in the form of dimers. The presence of cysteine in at least the sequences of the variants of the invention actually allows the formation of dimers by disulfide bonding. These may be - * homodimers, i. dimers corresponding to two variants of the invention (e.g., diapoA-I Paris), or heterodimers i.e. dimers comprising a variant of the invention and another molecule having a free cysteine (e.g., apoA-I Paris: apoA-II).

Another object of the invention is to provide a nucleic acid encoding a variant of apolipoprotein A-I as hereinbefore defined. The nucleic acid of the present invention may be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The plurality of deoxyribonucleic acids may be complementary DNA (cDNA), genomic DNA (gDNA), hybrid or synthetic sequences, or semisynthetic sequences. It may further be a chemically modified nucleic acid, e.g. in view of enhancing its nuclease resistance, in terms of increasing its penetration or its cellular targeting, in terms of enhancing its therapeutic effect, etc. These nucleic acids may be of human, animal, plant, bacterial, synthetic, etc. origin. They can be obtained by all techniques known to those skilled in the art, in particular by bank sorting, chemical synthesis or enzymatic synthesis of the sequences obtained by bank sorting.

A preferred nucleic acid is cDNA or gDNA.

The nucleic acid of the invention comprises the sequence of SEQ ID NO.

¹ 2. Even more preferably, it is a sequence of SEQ ID NO. 10th

Preferably, the nucleic acid of the invention comprises a functional transcription promoter region in a cell or target organism, as well as a region located at the 3 'end that specifies a transcription termination signal and a polyadenylation site. Together these elements form an expression cassette. It may be a promoter region naturally responsible for the expression of the apolipoprotein A-I gene or the apoA-I variant when it is capable of functioning in a cell or in an organism. They may also be regions of different origin (responsible for the expression of other proteins, even synthetic ones). These may be promoter sequences of eukaryotic or viral genes. For example, they may be promoter sequences derived from the target cell genome. Among the eukaryotic promoters, all promoters or derived sequences that stimulate or suppress gene transcription, in a specific manner or non-specific, inductive or non-inductive, strong or weak, may be used. These may be promoters (e.g., HPRT, PGK, vimentin, α-actin, tubulin, etc.) promoter, therapeutic gene promoters (e.g., MDR, CFTR, Fasteur VII, etc.), tissue-specific promoters (pyruvate gene promoter) kinases, witch hazel, intestinal fatty acid binding protein, α-smooth muscle actin, etc.) or promoters responsible for stimulation (steroid hormone receptor, retinoic acid receptor, etc.). It may be a sequence of promoters derived from the genome of the virus, e.g. it is a promoter of the E1A and MLP genes of adenoviruses, followed by the CMV promoter, the RSV LTR promoter, etc. These promoter regions may be modified by the addition of active sequences, regulatory sequences, or sequences that permit expression of a tissue specific or a major sequence.

The nucleic acids may also comprise a signal sequence directing the synthesized product into the secretory pathways of the target cell. This signal sequence may be the natural apoipoprotein apoA-I signal sequence, but may also be any other functional signal sequence, or an artificial signal sequence.

The nucleic acid of the invention can be used to generate recombinant variants of ApoA-I by expression into a host recombinant cell or directly as a medicament in the application of gene therapy or cell therapy.

For generating recombinant variants of the invention, the nucleic acid is preferably incorporated into a plasmid vector or a viral vector, which may be autonomous replication or integrative. This vector is then used to transfect or infect a selected cell population. The transfected or infected cells thus obtained are cultured under conditions permitting expression of the nucleic acid and the recombinant apoA-I variant of the invention is then isolated. The host cells used to produce variants of the invention in a recombinant manner are both eukaryotic and prokaryotic. Among the host eukaryotic organisms that are suitable, animal cells, yeasts, fungi may be cited. For yeasts, the genera Saccharomyces, Kluyveromyces, Pichia, Schwanniomyces or Hansenula may be cited. For animal cells, COS, CHO, CI27, NIH-3T3, etc. may be cited. In the case of fungi, Aspergillus ssp. or Trichoderma ssp. Preferably, the following bacteria can be used as host prokaryotic cells: Escherichia coli, Bacillus or Streptomyces. The variant thus isolated may then be modified in view of its therapeutic use.

The nucleic acid of the invention is used directly in the name of the medicament, in gene therapy or cell therapy applications. In this regard, it can be administered by injection at the site of treatment or by incubation with the cells for administration. It has been reported that nus nucleic acids can penetrate cells without a special vector. Nevertheless, it is preferred, within the scope of the present invention, to use the applied vectors to improve (i) cell penetration efficiency, (ii) targeting, (iii) extra- and intracellular stability.

Thus, the present invention relates to vectors comprising a nucleic acid as defined above.

Different types of vectors can be used. They may be viral or non-viral vectors.

According to the invention, a viral vector is preferred.

The use of viral vectors rests on the natural properties of viral transfection. It is also possible to use adenoviruses, herpes viruses, retroviruses, adeno-associated viruses or smallpox viruses. These vectors appear to be transfection efficient.

These are in particular adenoviruses, various serotypes whose structure and properties have been characterized. Among these serotypes, human adenoviruses of type 2 or 5 (Ad 2 or Ad 5) or adenoviruses of animal origin are preferably used in the present invention (see WO 94/26914). Among adenoviruses of animal origin. Adenoviruses of canine, bovine, murine (for example: Mavl, Beard et al., Virology 75, 1990, 81), sheep, porcine, avian, and monkey (for example: SAV) origin may be cited for use in the present invention. In the case of an adenovirus of animal origin, preference is given to a canine adenovirus, in particular a CAV2 adenovirus (e.g. the Manhattan strain or A26 / 61 (ATCC VR800)). Adenoviruses of human or canine origin, or mixtures thereof, are preferably used in the present invention.

The defective adenovirus of the invention comprises an ITR, an encapsidation-enabling sequence and a nucleic acid. In the genome of the adenovirus of the present invention, the E1 region is non-functional. Functionality of the viral gene of interest can be restored by any technique known to those skilled in the art, in particular by total removal, substitution, partial deletion or addition of one or more bases in the gene or genes of interest. Such modifications may be obtained in vitro (on isolated DNA) or in situ, e.g. through techniques of the genetic discipline or by modification through mutagenic agents. Other regions may be modified, in particular the E3 (WO 95/02697), E2 (WO 94/28938), E4 (WO 94/28152, WO 94/12649, WO 95/02697) and L5 (WO 95/02697). Preferably, recombinant adenovirus representing the deletion of all or part of the E1 and E4 regions is preferably used in the field of the present invention. This type of vector offers advantageous security properties.

The defective recombinant adenoviruses of the invention can be prepared by any technique known to those skilled in the art (Levrero et al., Gene 101, 1991, 195, EP 185 573, Graham, EMBO J., 3, 1984,

2917). They may be prepared by recombination of homologues between adenoviruses and plasmids carrying, inter alia, an amino acid or cassette of the invention. A recombinant homologue is generated after >

cotransfecting said adenovirus and plasmid in a suitable cell line. In particular, the cell line used must (i) be transformable with said elements and (ii) contain sequences capable of complementing a portion of the genome of defective adenoviruses, particularly in integrated form, in order to avoid the risk of recombination. By way of example, the human embryonic kidney line 293 (Graham et al., J. Gen. Virol. 36, 1977, 59), which contains, in particular, the left portion of the Ad5 adenovirus genome (12%) incorporated into its genome. Another line has been described in patent application no. WO 94/26914 and WO 95/02697.

Adenoviruses, which are multiplets, are obtained and purified according to classical molecular biology techniques, as illustrated in the examples.

Adeno-associated viruses (AAVs) are DNA viruses of relatively reduced size that integrate into the genome of cells that infect in a stable manner and at a specific site. They are capable of infecting a broad spectrum of cells without inducing an effect on growth, morphology or cell differentiation. Otherwise, they do not appear to be involved in human pathologies. The AAV genome has been cloned, sequenced and characterized. It comprises about 4700 bases and contains, at each end, a repeated altered region (ITR) of 145 bases having a replication origin for the virus. The remainder of the genome is divided into two regions carrying encapsidation functions: the left part of the genome that contains the rep gene implicated in viral replication and expression of the viral genes, and the right part of the genome that contains the cap gene encoding the capsidation proteins of the virus.

The use of AAV-derived vectors for gene transfer in vitro and in vivo has been described in the literature (see in particular WO 91/18088, WO 93/09239, US 4, 797, 368, US 5, 139, 949, EP 488 528). These inventions disclose various AAV-derived constructs in which the rep and / or cap genes are deleted and replaced with the gene of interest and their use for transferring said gene of interest in vitro (on cells in culture) or in vivo (directly in the body). The recombinant defective AAVs of the invention can be prepared by cotransfection in the infected cell line with helper human viruses (e.g., adenoviruses), a plasmid containing the nucleic acid or cassette of the invention flanked by two repeated AAV inverse regions (ITR) and a plasmid carrying the encapsidation genes ) AAV. The recombinant AAV products are then purified by conventional techniques (see in particular WO 95/06743).

With regard to herpes and retrovirus viruses, the construction of recombinant vectors has been widely described in the literature: see especially Breakfield et al., New Biologist 3, 1991, 203, EP 453242, EP 178220, Bernstein et al., Genet. Eng. 7, 1985, 235, McCormick, Biotechnology 3, 1985, 689, and so on. Retroviruses are integrative viruses, selectively infecting cells during division. They thus create the desired vectors for cancer application. In particular, the retrovirus gene comprises two LTRs, one encapsidation sequence and three coding regions (gag, pol and env). In recombinant vectors derived from retroviruses, the gag, pol and env genes are generally deleted in part or in full and replaced with the desired heterologous nucleic acid sequence. These vectors can be realized from various types of retroviruses, such as in particular MoMuLV (Murine Moloney Leukemia Virus, still named MoMLV), MSV (Murine Moloney Sarcoma Virus), HaSV (Harvey Sarcoma Virus), Spleen Necrosis Virus SNV ), RSV (Rous Sarcoma Virus), or Friend virus.

For the construction of recombinant retroviruses containing a nucleic acid or cassette according to the invention, a plasmid containing, in particular, an LTR, an encapsidation sequence and a nucleic acid or cassette is constructed, then used to transfect a line of cells called encapsidation capable of delivering inadequate retroviral functions into the plasmid. Thus, the general encapsidation lines are capable of expressing the gag, pol and env genes. Several such encapsidation lines have been described in the prior art, in particular the PA 317 line (US 4,861,719), the PsiCRIP line (WO 90/02806) and the GP + envAm-12 line (WO 89/07150). Recombinant retroviruses may contain modifications at the LTR level to suppress transcriptional activity as well as an extensive encapsidation sequence containing part of the gag gene (Bender et al., J. Virol. 61, 1987, 1639). The recombinant retroviruses are then purified by conventional techniques.

Adenoviruses or recombinant defective petroviruses are preferably used in the present invention. In fact, these vectors have properties of particular interest for the transfer of genes encoding apolipoproteins. The adenoviral vectors of the invention are particularly advantageous for direct administration of an in vivo pure suspension or for the transformation of ex vivo cells, in particular autologous in view of their implantation. The adenoviral vectors of the invention have other important properties, in particular they have a very high infectious ability to allow infection from weak volumes of the viral suspension.

In this regard, the invention relates in particular to recombinant defective adenoviruses containing DNA encoding a variant of apolipoprotein A-I as defined above inserted into its genome.

Particularly preferably, a line that produces rettoviral vectors comprising a sequence encoding the ApoA-I variant for in vivo implantation is used particularly preferably for carrying out the invention. For this purpose, useful lines, in particular PA317 (US 4,861,719), PsiCRIP (WO 90/02806) and GP + envAm-12 (US 5,278,056) cells, modified to allow the production of retroviruses containing the nucleic acid sequence encoding ApoA.1 variants of the invention.

According to another aspect of the invention, the vector used is a chemical vector. Indeed, the vector of the invention may be a non-viral agent capable of promoting the transfer and expression of nucleic acids in eukaryotic cells. Chemical vectors or biochemical represent an interesting alternative to natural viruses, in particular for advantageous reasons, safety and the possible absence of a theoretical limit on the size of the transfected DNA.

These synthetic vectors have two basic functions, to thicken the nucleic acid for transfection and to promote its cellular fixation as well as its passage through the plasmid membrane, if necessary through two nucleic membranes. By suppressing the polyanionic nature of nucleic acids, non-viral vectors have a total polycationic charge.

Among the synthetic vectors developed, most preferred are cationic polymers of the polylysine type (LKLK) n, (LKLK) n, polyethyleneimine and DEAE dextran or cationic lipids or lipofectants. They have the ability to condense DNA and promote their binding to the cell membrane. Among the latter, lipopolyamines (lipofecamine, transfectam, etc.) and various cationic lipids or neutral lipids (DOTMA, DOGS, DOPE, etc.) may be cited. A receptor-driven cell transfection concept has been developed that utilizes the principle of DNA condensation by a cationic polymer controlling the fixation of the complex to the membrane due to the chemical bond between the cationic polymer and the membrane receptor ligand that is present on the cell-type surface that it wants to cleave. The transferrin, insulin, or hepatocyte asialoglycoprotein receptor has also been described.

In particular, the invention relates to all genetically modified cells by insertion of a nucleic acid encoding an apolipoprotein A-I variant as defined above. Preferably, they are mammalian cells capable of being administered or implanted in vivo. These may be, in particular, fibroblasts, myoblasts, hepatocytes, keratinocytes, endothelial cells, epithelial cells, etc. The cells are preferably of human origin. Preferably, they are autologous cells, cells taken from a patient modified with an ex vivo nucleic acid of the invention in view of conferring their therapeutic properties, and then re-administered to the patient.

The cells of the invention may be primary cell tissues. They can be harvested by any technique known to those skilled in the art, then introduced into the culture under conditions that allow their proliferation. They are especially fibroblasts, they can be easily obtained from biopsies e.g. according to the technique described by Ham (Methods Cell. Biol. 21a, 1980, 255). These cells can be used directly to insert a nucleic acid of the invention (via a viral or chemical vector) or conserved e.g. freezing on autologous bank equipment for further use. The cells of the invention may be secondary cells obtained e.g. from predetermined banks (see e.g. EP 228458, EP 289034, EP 400047, EP 456640).

In particular, cells in culture may be infected with the recombinant viruses of the invention to compare their ability to produce a biologically active variant of apoA-I. The infection is carried out in vitro, according to techniques known to those skilled in the art. In particular, depending on the type of cells used and the number of copies of viruses desired by the cell, the skilled person can adapt the multiplicity of infection and, optionally, the number of cycles of infection performed. These stages are expected to be performed under acceptable sterile conditions until the cells are ready for administration in vivo. The doses of the recombinant virus used to infect the cells can be adapted to those skilled in the art according to the objective of the study. The conditions previously described for in vivo administration may be applied to an in vitro infection. In particular, co-culture of the cells to be infected with the cells producing the recombinant retroviruses of the invention can be used for retrovirus infection. This makes it possible to bypass the purification of retroviruses.

Another object of the invention relates to an implant comprising mammalian cells genetically modified by insertion of a nucleic acid as defined above and of an extracellular matrix. The implants of the invention contain 10 ⁵ to 10 ¹⁰ cells. Even more preferred are implants containing 10 ⁶ to 10 ® cells. In particular, the cells may be cells that produce recombinant viruses containing a nucleic acid as defined above inserted in their genome. 'i

I · · *

In the implants of the invention, the extracellular matrix comprises a gelling agent and optionally a cell permitting anchorage.

Various types of gelatinous substances can be used to prepare the implants of the invention. These substances are used to incorporate cells into a matrix having a gel constitution and to favor anchoring the cells to the substrate if necessary. Thus, various cell adhesion agents, such as gelatinous agents, e.g. collagen, gelatin, glycosaminoglycan, fibronectin, lectin, etc. Preferably, collagen is used in the present invention. It may be a collagen of human, bovine or murine origin. Collagen type I is preferably used.

As mentioned above, the composition of the invention preferably comprises a cell-anchoring substrate. The term anchorage describes all forms of biological and / or chemical and / or physical interactions promoting cell adhesion and / or fixation to the substrate. Otherwise, the cells may either be coated with the substrate used, or may penetrate to the internal substrate, or both may occur. Within the scope of the invention, it is preferred to use a solid non-toxic and / or biocompatible substrate. In particular, polytetrafluoroethylene (PTFE) fibers or a substrate of biological origin (coral, bone, collagen, etc.) may be used.

The implants of the invention may be implants from different sites of the body. In particular, the implants may be performed at the level of the peritoneal cavity, in the subcutaneous tissue (suprapubic region, lumbar region or groin region) in the organ, muscle, tumor, nervous center system, or also under the mucosa. The implants of the invention are particularly advantageous in that they allow the release of the ApoA-I variant from the body to be controlled. This is primarily determined by the multiplicity of infection and the number of implanted cells. The release can be controlled either by removing the implant that stops definitive treatment or by using controllable expression systems to induce or suppress the expression of the therapeutic gene.

Thus, the nucleic acids create a new medicament in the treatment and prevention of cardiovascular pathologies (atherosclerosis, restenosis, etc.) because of the anti-atherogenic properties of the variants of the invention.

The invention also relates to all pharmaceutical compositions comprising an apoA-I and / or a nucleic acid variant and / or a vector and / or a genetically modified cell as described above.

Thus, the present invention provides a novel composition for the treatment or prevention of pathologies associated with dislipoproteinaemia, particularly in the field of cardiovascular diseases such as myocardial infarction, sudden death, restenosis, cardiac decompensation and cerebrovascular events. In general, these approaches offer great hope through therapeutic intervention in any case where the deficiency of apolipoprotein A-I genetic order or metabolism can be corrected.

The following examples illustrate the invention in more detail without limiting its scope in any way.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Restriction map of plasmid pXL2116

Figure 2: Construction of M13 phage carrying exon 4 of the patient

Figure 3: Construction of the vector carrying the mutated PXL2116

Figure 4: Temperature-dependent turbidimetry study

4a: turbidimetric association of various ApoA-I and DPMH in the absence of the GndHCI standard, recombinant, plasma-like,

- Paris - Paris)

4b: turbidimetric association of ApoA-I and DPMC in the presence

GndHCI (- standard, recombinant, - <- plasma,

Paris)

4c: turbidimetric coupling of ApoA-I and DPMC (in the absence of GndHCI, in the presence of GndHCI (0.5 M))

4d: turbidimetric association of recombinant ApoA-I and DPMC (- in the absence of GndHCI, in the presence of GndHCI (0.5 M))

I

4e: turbidimetric association of plasma Apo-I and DPMC (in the absence of GndHCl, in the presence of GndHCl (0.5 M))

4f: effect of GndHCl (0.5 M) on ApoA-I / DPMC junction (-in absence of GndHCI, in the presence of GndHCl (0.5 M))

Figure 5: Relative fluorescence of Superose 6 PG fractions (-POPC / AIParis, POPC / AI recombinant, POPC / AI plasma)

Figure 6: 6a: Tryptophan fluorescence and phospholipid concentration for POPC / AI Paris complex (- relative fluorescence, - □ - phospholipids)

6b: Tryptophan fluorescence and phospholipid concentration for POPC / AI complex recombinant (- relative fluorescence, - □ - phospholipids)

SUBSTITUTE SHEET

DETAILED DESCRIPTION OF THE INVENTION

Example 1: Evidence of the ApoAI variant

The ApoA-I Paris variant was identified and isolated from the patient due to his own lipid balance. The patient represents the following lipid balance (nature of collection: serum).

normal cholesterol 4.59 mmol / l 4.54-6, 97 triglycerides 2,82 mmmol 0.74-1.71 HDL-cholesterol 0.25 mmo1 / l 0.88 to 1.6 LDL-cholesterol 3.06 mmo1 / l 2.79 to 4.85 apoliprotein A-I 0.5 g / l 1.20-2.15 apoliprotein B 1.38 g / l 0.55-1.3 apoE phenotype: 3/3

Unexpectedly, this patient showed no evidence of atherosclerosis despite extremely low HDL concentrations, nor hypertriglyceridemia. This leads the applicant to seek the origin of this protection.

Isolation of HDL containing ApoA-I Paris from plasma

Blood (overnight) was drawn from subjects carrying the apoA-I Paris gene. Plasma was prepared by slow centrifugation of blood at 4 ° C (2000 g, over 30 minutes). High density lipoproteins were prepared by sequential ultracentrifugation at a density of 1.063-1.21 g / ml (Havel, J. Clin. Invest. 34, 1345-54, 1995). The fraction containing HDL was then dialyzed against Tris-HCl buffer (10 mM), with the addition of 0.01% sodium azide, pH 7.4,

Evidence of apoA-I Pariš dimer

Lipids were removed from the HDL fraction in diethyl ether / ethanol (3/1, v / v), and protein concentration was determined by the Lowry method (Lowry et al., J. Biol. Chem., 193, 265-275, 1951). . HDL fraction proteins were subjected to migration on a polyacrylamide gel in the presence of SDS under non-reducing conditions. This migration allows the different HDL protein sizes of the patient to be detected. In addition, the proteins present correspond to the normal size ApoA-I and ApoA-II, this analysis also reveals the presence of the highest molecular weight proteins corresponding to the apoA-I dimer and the apoA-I and apoA-II complexes. The presence of apoA-I and apoA-II in these complexes was verified by a specific immunological method.

Evidence of different charge of mutated apoA-I

Detection of mutated apoA-I directly from plasma was performed according to the following protocol (Menzel, HJ and Utermann, G., Electroforesis, 7, 492-495, 1986): 20 ml of plasma from which lipids were removed overnight with ethanol / ether, was suspended in the buffer. A 5 ml aliquot was electrophoresed on an isoelectrically adjusted gel (pH 4-6.5, Pharmolyte), then the proteins were transferred to a nylon membrane. Bands corresponding to apoA-I were detected by an immunological reaction with a human anti-apoA-I antibody.

Detection of mutated apoA-I may also be performed from the HDL protein. Lipids in the diethyl ether / ethanol (3/1, v / v) mixture were removed from the dialyzed HDL fraction and the protein concentration was determined by the Lowry method (Lowry et al., J. Biol. Chem., 193, 265-275, 1951). ). 100 ml of proteins were subjected to electrophoresis on an isoelectrically adjusted gel (pH 4 - 6.5, Pharmolyte) and the proteins were then visualized by staining with Coomassie Brilliant Blue. This technique demonstrates that the most important apoA-I isoforms of the patient carrying the mutation were placed at the anode, corresponding to a charge difference of -1 relative to the charge of normal apoA-I.

Example 2: Gene Identification and Mutation

The patient's genomic DNA was isolated from blood according to the Madisen technique (Madisen et al., Amer. J. Med. Genet., 27, 379-390, 1987). The apoA-I gene was then amplified by PCR. In this regard, amplification reactions were performed with 1 mg of pure genomic DNA inserted into the following mixture:

- 10 ml of 10X buffer (100 mM Tris-HCl, pH 8,3, 500 mM KCl, 15 mM MgCl, 0,1% (w / v) gelatin)

10 ml dNTP (dATP, dGTP, dCTP, dTTP) 2 mM

- 2.5 U Taq polymerase (Perkin - Elmer)

- qsp 100 ml _H2O

The sequences used for amplification are as follows:

Sq5490: 5 ' -AAGGCACCCCACTCAGCCAGG-3 ' (SEQ ID No.3) Sq5491: 5 ' -TTCAACATCATCCCACAGGCCTCT (SEQ ID No.4) Sq5492: 5 ' -CTGATAGGCTGGGGCGCTGG-3 ' (SEQ ID No.5) Sq5493: 5 ' -CGCCTCACTGGGTGTTGAGC-3 ' (SEQ ID no. 6)

The Sq5490 and Sq5491 sequences amplify a 508 bp fragment corresponding to exons 2 and 3 of the apoA-I gene, and the Sq5492 and Sq5493 tools amplify a 664 bp fragment corresponding to exon 4 of this gene.

The amplification products (two PCR fragments 508 and 664 bp) were sequenced. Therefore, a method directly involving sequencing using a PCR fragment sequencing kit (Amersham) was used. The sequences used for sequencing are PCR sequences, but may also be internal fragment sequences (cf. S4, S6 and S8 sequences below).

The second sequencing technique involves cloning the PCR fragments into the M13 mp28 vector. The double stranded DNA of the M13 vector was digested with EcoRV and then dephosphorylated. The PCR fragment was engineered with the Klonow fragment, phosphorylated and linked to the M13 vector. White areas were then removed and single stranded amplified DNA then purified on Catalyst (Applied Biosystem) was sequenced with a -20 fluorescent sequence (PRISM dye primer kit and protocol # 401386, Applied Biosystem) or internal fragment sequences. The dideoxynucleotide fluorescence technique (DyeDeoxyTerminator Kit and Protocol No. 401388, Applied Biosystem) was used.

S8 5 ' -TGG GAT CGA GTG AAG GAT CTG-3 ' (SEQ ID No.7) S4 5 ' -CGC CAG AAG CTG CAC CAG CTG-3 ' (SEQ ID No. 8) S6 5 ' -GCG CTG GCG CAG CTC GTC GCT-3 ' (SEQ ID No. 9)

The resulting sequences of multiple clones were then compiled and compared to ApoA-I. The final amino acid sequence of 148-154 ApoAI is shown below (corresponds to residues 172-178 of SEQ ID NO: 1).

ATG CGC GAC Met Arg 151 C-> T mutation in first base codon encoding amino acid 151, which encodes cysteine (cf. SEQ ID No. 2)

below) was found again in part of the sequenced clone. This mutation is present in a heterozygous state in the selected patient.

A cDNA file encoding the apoA-I Paris variant of the invention was sequenced. Part of this sequence is present in SEQ ID NO. 10th

Example 3: Construction of Plasmid Expression Vector (pXL2116mute)

ApoA-I Paris represents a mutation precisely situated on the exon 4 sequence of the patient's apoA-I gene. The strategy for constructing the expression vector is to substitute the expression vector apoA-I (vector pXL2116, Figure 1) for regions corresponding to exon 4 derived from the patient's gene.

3.1 Use of exon 4 of a patient cloned in M13

Exon 4 was produced by PCR from the patient's DNA cells and inserted at the EcoRV site level of the M13mp28 tag polylinker (Figure 2).

Mutagenesis is observed by relocating the apoA-I fragment with the M13-derived fragment carrying the mutation. For selection, the enzymes are suitable for two vectors such that the vector derived from pXL2116 produces cohesive ends, and for insertion from double stranded M13.

Selection of restriction enzymes

Bsu361 has a unique restriction site against mutation in M13 and pXL2116

- BamHI digestion having a unique restriction site at the 3 'end of apoA-I, performed by the cloning technique, having a restriction site in the M13 polylinker allows us to:

1) binding of a vector derived from pXL2116 and insertions derived from M13

2) to incorporate a polylinker fragment that will be useful to verify insertion of the M13-derived mutated fragment by enzyme digestion

It may be noted that the presence of this polylinker fragment does not alter any apoA-I coding sequence when it is located after the termination codon. .

Thus, a histidine rich polypeptide whose nucleotide sequence has been cloned at the 5 'end of apoA-I will also be synthesized.

3.3 Vector Construction (Figure 3)

The double stranded M13 is digested with Bsu361 / BamHI and the digestion product applied to the gel. The advertisement thus generated is recovered in sufficient quantity and present in a size of 1 Db.

-pXL2116 is digested by the same enzymes but unclean. Digestion is performed with XhoI, which recognizes two restriction sites to the internal Bsu361 / BamHI fragment to avoid religation of the digestion products.

. The dephosphorylation, which was first tested, gave very poor results on the dish and gave no positive clone of the 24 tested.

The amount of vector and optimal insertion information (engineer) as well as binding was estimated to be 50 ng of the vector per 15 ng of insertion information (insert).

DH5α strains were transformed with the products of the following three ligands:

Bsu361 and BamHI digested vector without ligase (negative ligation control)

- cleavage product + ligase

- cleavage product + insert + ligase

Competent cells were transformed with pUC19 to test transformation efficiency.

The results of the transformed cells on a dish on LB medium with chloramphenicol (selection from base BL21), ampicillin (selection from base including plasmid) are reproduced below.

Results obtained on Petri dish

DH5α + transformation the results interpretation PUC19 200 clones evidence of transformation vector (+ fragment) 0 klenov complete digestion, no plasmid vector (+ fragment) · 160 clones background noise very important; 1 post-cleavage strategy not very effective vector (+ fragment) + ligase + insert 120 clones the ligase effect often remained bajktérie are not mandatory transformed with the right mutated sequence

clones are again isolated from dish 4.

To repeat the generation of positive clones (having inserted the mutated M13 fragment), plasmid DNA obtained by purification on all 48 clones is digested with NdeI.

If the M13-derived insert was well inserted, there would be two fragments on the gel: one 4.5 kb and the other 1 kb.

If another ligation result is obtained, i. other product, either more bands or a simple linear plasmid (general case) are obtained.

According to the gel results, clones 7, 8, 13, 16, 26 appear to be correct. Choose a clone that gives a clear result.

The presence of the polylinker, i.e. the insert, is thus verified, but the result of the ligation must also be verified. Obviously, the ligation is performed correctly since the size of the plasmid is correct, i.e. the same as the size of pXL2116.

In addition to the result of a very rare mutation, the newly cloned apoA-I sequence must be accurate.

Thus, the clone 8 carries the correctly mutated plasmid px12116.

Example 4: Expression of recombinant variants of apoA-I

This example describes the process of producing recombinant variants of apoA-I. This process was carried out in bacteria. Other expression systems (yeast, animal cells, etc.) were used for this purpose.

4.1 Principle

Plasmid expression in the middle of the bacterium was placed under the control of the T7 promoter and terminator. In this system, isopropyl-β-thiogalactopyranoside (IPTG), an indicator of the lactose operon, induces the synthesis of RNA polymerase T7, which is then specifically fixed to the T7 promoter and initiates transcription of the recombinant protein gene. RNA polymerase is stopped by the T7 terminator, thus avoiding the transcriptional flow and not exceeding the desired sequence.

Rifampicin is an antibiotic that inhibits endogenous E. Coli RNA polymerase activity. Thus, it inhibits the synthesis of bacterial proteins, i.e. proteases, those that limit degradation of the desired protein and bacterial contaminating proteins, such as to amplify expression of the protein.

4.2 Protocol

The base used for expression is Escherichia coli BL21 DE3 pLys

S. Plasmid DNA is introduced by dd E. coli by transformation according to classical techniques. The culture is preserved in. as a frozen suspension at -20 ° C in the presence of 25% glycerol in 500 ml aliquots.

. The inoculum is initiated by adding a few drops of the frozen suspension to 10 ml of M9 medium with ampicillin, then incubating overnight at 37 ° C. The inoculum was inoculated into 1 liter Erlenmeyer flasks which were placed on a shaker at 37 ° C until a DO was obtained between 0.5 and 1 at 610 nm. IPTG (Bachem ref Q-1280) is then added in an amount to give a final concentration of 1 mM. After incubation for 15 minutes at 37 ° C, rifampicin (Sigma) is added at a final concentration of 100 mg / ml. After one hour of culture, the cells are recovered by centrifugation (15 minutes, 800 rpm) and expression is verified by electrophoresis under denaturing conditions on a 15% acrylamide gel and immunopaked.

The results obtained show that the mutated protein is expressed according to a good expression ratio and that it is efficiently proven by polycloned anti-apoA-I antibodies.

Example 5: Purification of recombinant apoA-I variants

This example describes an effective method for purifying the recombinant apoA-I variants of the invention. It is contemplated that other methods may be used.

Proteins that are expressed in the cytoplasm of Escherichia coli are first extracted and then cell lysis is performed followed by nucleic acid elimination.

5.1 Lysis of bacteria

After centrifuging the cells, the bacterial sediment is resuspended under gentle agitation in the buffer in the presence of protease inhibitors and β-mercaptomethanol to effect lysis.

β-mercaptomethanol is a reducing agent that cleaves disulfide bridges formed between two cysteines. No disulfide bridge is formed in the cytoplasm of Escherichia coli, and the presence of a reducing agent now appears necessary for the once extracted proteins. Indeed, the addition of β-mercaptomethanol to the lysis buffer prevents the formation of bridges between the cysteine of the bacterial protein and the cysteine of the recombinant proteins.

The cell lysate is harvested 3 times for 5 minutes by freeze-drying (Vibracells sinics material, pulsed mode, output control 5), then centrifuged (10000 rpm, 1 hour, 4 ° C on Beckman J2-21 M / E, JA10 rotor) and then cell debris can be eliminated. The amount of protein is determined from the supernatant by the Bradford colorimetric method.

5.2 Nucleic acid precipitation

Precipitation is effected from the lysis supernatant with a 10% streptomycin sulfate solution at a rate of 10 ml per 10 g of proteins under gentle magnetic stirring for one hour at 4 ° C. Nucleic acids are eliminated by centrifugation at 10,000 rpm (1 hr, 4 ° C on Beckman J2-21 M / E, JA10 rotor) and protein concentration in the supernatant is determined by a colorimetric method.

From these two steps, a protein solution of known concentration is obtained, in which all intracellular proteins, including the desired protein, are included. These proteins were purified by chromatographic methods.

- gel filtration chromatography

affinity chromatography

5.3 Gel filtration chromatography The aim of this ¹ stage is to remove EDTA, a molecule interfering with the required conditions for affinity chromatography. Therefore, tris-acrylic GF 05 (Sepracor) is used as carrier. This gel allows the separation of molecules whose molecular weight (MM) is comprised between 300 and 2500 daltons and the exclusion of molecules, including proteins, with a molecular weight at the upper limit, ie 2500 daltons. This gel, representing another part of interest, has good pressure resistance, allowing to operate at elevated pressure from the beginning of the method, without modifying the method. Bacterial lysate chromatography is performed in pH 8 phosphate buffer. The protein concentration in the excluded volume is determined and adjusted to 4 mg / ml by dissolution in pH 8 phosphate buffer.

This stage can be replaced by dialysis against 2 x 10 liters PBS. The protein solution is then left in the presence of Hecameg 25 mM, this detergent assisting the subsequent purification step by reducing protein-protein interactions.

5.4 Affinity chromatography

principle

The presence of six consecutive histidine residues associated with the recombinant protein gives the protein an affinity which is partially enhanced by nickel ions (Ni ²⁺ ) (15). These Ni ²⁺ ions are fixed to the agarose matrix by nitriloacetic acid (NTA). Between the imidazole of histidine and nickel ions, a metal chelate bond is formed, which is stronger than the ionic bond and weaker than the covalent bond.

protocol

The ability to fix the NiNTA agarose gel (Qiagen) is 2 mg protein per ml gel. This gel is equilibrated in pH 8 buffer by the addition of 25 mM Hecameg. Bacterial contaminating proteins are not captured at pH 8, are eliminated at pH 6, and the desired protein is recovered at pH 5. These stages are performed in the presence of 25 mM Hecameg.

The eluted fractions (2 mL) at pH 5 are added to:

- 60 μΐ 1 M NaOH (neutralization)

- 10 μΐ 0.2 M PMSF

- 40 μΐ 0.1 M EDTA

Fractions are pooled relative to. the concentration and purity of the eluted protein. These characteristics were analyzed by electrophoresis (15% PAGE-SDS).

Elution at pH 5 yields proteins with nickel which is effective in binding Ni ²⁺ -NTA. Thus, it is necessary to dissociate the cation of the recombinant protein. This step is accomplished by competition by incubating with gentle stirring on a magnetic stirrer at 4 ° C for one hour in the presence of 50 mM histidine.

5.5 Dialysis

Dialysis allows the elimination of histidine and nickel. The sample is dialyzed (Spectra / por MWCO 12-14000 dalton membrane) at 4 ° C for 5 hours overnight against 2 x 10 L of 2 mM PBS EDTA buffer. Finally, the amount of protein is determined.

This method makes it possible to obtain the apoA-I Paris protein in a pure form, particularly free of contaminated proteins.

Example 6: Physico-chemical properties of apoA-I Paris and normal recombinant apoA-I.

6.1. Turbidimetric measurements

6.1.1 Turbidimetry as a function of temperature

Measurement of DMPC absorbance at 325 nm in the presence of apoA-I is a measurement of the formation of small size proteolipid complexes. Analysis of the temperature change between 19-28 ° C shows us a decrease in absorbance around the phospholipid conversion temperature (23 ° C), suggesting complex formation. Figure 4 (in the presence or absence of Gdnhd1 to avoid dimer formation) shows a comparison of the formation of these complexes with normal apoA-I and recombinant apoA-I Paris, as well as native apoA-I. These three proteins have quite similar behavior, but in apoA-I Paris one can notice its tendency to associate with itself to form dimers.

6.1.2 Turbidimetry as a function of time

The decrease in DMPC turbidimetry after apoA-I incubation was temperature related as a function of time in the presence or absence of GdnHDL. The temperature constant (l / tl / 2, which corresponds to a 50% decrease in initial turbidimetry) is estimated as a function of 1 / T (temperature in degrees Kelvin). Coupling rate is fast for native apoAI, weaker for recombinant apoA-I, especially for apoA-I Paris. Otherwise, the addition of GdnHDL increases protein-lipid binding. This method is very important for recombinant apoA-I, especially for apoA-I Paris.

6.2 Spectrum emission spectra of tryptophan

The emission spectra of tryptophan fluorescence in various apoA-I were measured at 300 and 400 nm (excitation at 295 nm). The maximum emission is given in Table 1 for apoA-I and apoA-I / cholesterol / POPC complexes. The maximum emission of tryptophan fluorescence in the various apoA-I and complexes are identical, suggesting that tryptophanes are in the same environment in the three proteins.

Table 1

Product Maximum emission Al Paris 335 POPC / C / AI Paris 332 recombinant A1 334 POPC / C / AI rec 332 Al plasma 336 .POP.C / C / AI plasma. 333.

6.3 Isolation and characterization of apoA-I / lipid complexes

The complexes with apoA-I and POPC were prepared by a chelate technique. The apoA-I complexes were separated by chromatography on a Superose 6PG column and analyzed for composition. Gel filtration profiles are depicted in Figure 5. The homogeneous peak itself. obtained for complexes formed with native apoA-I, while heterogeneous populations were observed with recombinant apoA-I. Free apoA-I elutes in fractions 20-24. The phospholipid concentration and fluorescence of tryptophan fractions for the various complexes are shown in Figure 6.

Example 7: Construction of an adenoviral vector for expression of a mutated ApoA-I cDNA encoding a variant of the invention containing an Arg Cys mutation at position 151 of ApoA-I is obtained by PCR.

Sequences used:

almlov: AT C GAT ACC GCC ATG AAA GCT GCG GTG CTG (SEQ ID No.11) ALM 2: ATG GGC GCG CGC GCA GTC GCG CAT CTC CTC (SEQ ID No.12) ALM3: GAG GAG ATG CGC GAC TGC GCG CGC GCC CAT (SEQ ID No.13) Alm4: GTC GAC GGC GCC TCA CTG GGT GTT GAG CTT (SEQ ID No. 14)

The Alml and Alm4 sequences insert the Call at 5 'and SalI at the 3' cDNA, while the Alm2 and Alm3 sequences that are complementary insert the mutation. The PCR reaction with Alml-Alm2 and Alm3-Alm4 pairs is first performed on non-mutated ApoA-I cDNA. The PCR-generated fragments are then reinserted into a third PCR in the presence of Alml and Alm4, which form an 822 bp fragment which is then cloned in pCRII (Invitrogen) for sequence verification. The Cla1 / SalI fragment containing the mutated cDNA is then inserted into the same restriction site into the pXL-RSV-LPL vector containing the LPL cDNA under the control of the LTR-RSV promoter and the bovine growth hormone polyadenyl position to replace the LPL cDNA (FR9406759) . Thus, another vector may also be used. The resulting vector is then released and cotransfected into 293 to obtain a recombinant adenovirus. The adenoviruses thus obtained can be amplified, purified (especially cesium chloride), then preserved by freezing, e.g. in glycerol. For therapeutic use, they may be mixed with a pharmaceutically acceptable vehicle. These may be, in particular, salt solutions (sodium phosphate, disodium phosphate, sodium chloride, potassium chloride, calcium or magnesium chloride, etc. Or mixtures of these salts), sterile, isotonic or dry compositions, in particular lyophilized, which by addition, under sterile water or physiological conditions, solution to give an injectable consistency. For use in the treatment of pathologies associated with dyslipoproteinemia, the recombinant defective adenoviruses of the invention can be administered according to a variety of methods, particularly by intravenous injection. They are injected at the level of the portal vein. The doses of virus used for injection may be adapted to various parameters, in particular the mode of administration used, the parameters related to the pathologies, or else, and the duration of the treatment being investigated. The recombinant viruses of the invention are formulated and administered in the form of doses containing between 10 ⁴ and 10 ¹⁴ pfu / ml. Doses of 10 ⁶ and 10 ¹⁰ pfu / ml can be used for AAV and adenoviruses.

The term pfu (plaque forming unit) corresponds to the infectious capacity of the viral suspension and determines the infection of the adapted cell culture by measuring the number of infected cells after 48 hours. Techniques for determining the pfu titer of a viral solution are well described in the literature.

List of sequences (1) General information:

(i) the applicant:

(A) Name: RHONE-POULENC RORER S.A.

(B) Street: 20, Raymond ARON (C) City: ANTONY (D) Country: France (E) Postal Code: 92165 (i) Applicant:

(A) Name: UNIVERSITE PIERRE ET MARIE CURIE (B) Street:

(C) City:

(D) Country: France (E) Postal Code:

(i) the applicant:

(A) Name: PASTEUR DE LILLE INSTITUTE (B) Street:

(C) City:

(D) Country: France (E) Postal Code:

(ii) name of the invention: Apolipoprotein A-I variants and a pharmaceutical composition containing them (iii) the number of sequences: 14 (iv) computer reading method:

(A) Media Type: Tape (B) Computer: IBM PC Compatible (C) Utilization System: PC-DOS / MS-DOS (D) Logic: Patentin Relase # 1.0, Version # 1.30 (OEB) (2) Sequence Information SEQ ID NO: 1:

(i) sequence characteristics:

(A) length: 842 base pairs (B) type: nucleotide (C) number of strands: two (D) configuration: linear

(Ii) molecule type: cDNA (Iii) even hypothetical: No. (Iv) antisense: No. (Vi) origin: (A) organism: Homo sapiens (Ix) Features: (A) Name / CLE: CDS (B) location: 1 .. . 842

(C) additional information: / product = human apoA-I cDNA (xi) sequence description: SEQ ID NO: 1:

ATG AAA GCT GCG GTG CTG ACC TTG GCC GTG CTC TTC CTG ACG GGG Met Lys Ala Ala wall Leu Thr Leu Ala wall Leu Phe Leu Thr Gly 1 ^: 5 10 15 AGC CAG GCT CGG CAT TTC TGC CAG CAA GAT GAA CCC CCC CAG 87 Ser gin Ala Arg His Phe Trp gin gin Asp Glu for for gin 20 25 AGC CCC TGG GAT CGA GTG AAG GAC CTG GCC ACT GTG TAC GTG 129 Ser for Trp Asp Arg wall Lys Asp Leu Ala Thr wall Tyr wall 30 35 40 GAT GTG CTC AAA GAC AGC GGC AGA GAC TA.T GTG TCC CAG TTT 171 Asp wall Leu Lys Asp Ser Gly Arg Asp Tyr wall Ser gin Phe 45 50 55 GAA GGC TCC GCC TTG C-GA AAA CAG CTA AAC CTA AAG CTC CTT 213 Glu Gly Ser Ala Leu Gly Lys gin Leu same time Leu Lys Leu Leu 60 65 70 GAC AAC TGG GAC AGC GTC- ACC TCC ACC TTC AGC AAG CTG CGC 255 Asp same time Trp Asp Ser wall Thr Ser Thr Phe Ser Lys Leu Arg

80 85

GAA CAC- CTC GGC CCT GTG ACC CAG GAG TTC TGG C-AT AAC CTG 297 Glu gin Leu Gly for wall Thr A boat Glu Phe Trp Asp same time Leu 90 95 GAA AAG GAG ACA GAG R / τ ' CTG AGG CAG GAG ATC AGC AAG GAT 339 Glu Lys Glu Thr Glu Gly Leu Arg A boat Glu Met Ser Lys Asp 100 105 110 CTG GAG GAG GTG AAG GCC AAC GTG CAG CCC TAC CTG GAC GAC 381 Leu Glu Glu wall Lys Ala Lys wall gin for Tyr only Asp Asp 115 120 125 TTC CAG AAG AAG TGG CAG GAG GAG ATG GAG CTC TAC CGC CAG 423 Phe gin Lys Lys Trp gin Glu Glu Met Glu Leu Tyr Arg gin 130 135 140

AAG GTG GAG CCG. CTG CGC GCA GAG CTC CAA GAG GGC GCG CGC 465 Lys wall Glu for Leu Arg Ala Glu Leu gin Glu Gly Ala Arg 145 150 155 CAG AAG CTG CAC GAG CTG CA » GAG AAG CTG AGC CCA CTG GGC 507 gin Lys Leu His Glu Leu gin Glu Lys Leu Ser for Leu Gly 160 165 GAG GAG ATG CGC GAC CGC GCG CGC GCC CAT GTG GAC GCC CTG 549 Glu Glu Met Arg Asp Arg Ala Arg Ala His wall Asp Ala Leu 170 175 180 CGC ACG CAT CTG GCC CCC TAC AGC GAC GAG CTG CGC CAG CGC 591 Arg Thr His Leu Ala for Tyr Ser Asp Glu Leu Arg gin Arg 185 190 195 TTG GCC GCG CGC CTT GAG GCT CTC AAG GAG AAC GGC GGC C-CC 633 Leu Ala Ala Arg Leu Glu Ala Leu Lys Glu same time Gly Gly Ala 200 205 210 AGA CTG GCC GAG TAC CAC GCC AAG GCC ACC GAG CAT CTG AGC 675 Arg Leu Ala Glu Tyr His Ala Lys Ala Thr Glu His Leu Ser 215 220 225

ACG CTC AGC GAG AAG GCC AAG CCC GCG CTC GAG GAC CTC CGC 717 Thr Leu Ser Glu Lys Ala Lys for Ala Leu Glu Asp Leu Arg 230 235 CAA GGC CTG CTG CCC GTG CTG GAG AGC TTC AAG GTC AGC TTC 759 gin Gly Leu Leu for wall • Leu Glu Ser Phe Lys wall Ser PNE 240 245 250 CTG AGC GCT CTC GAG GAG TAC ACT AAG AAC CTC AAC ACC CAG 801 Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu same time Thr A boat 255 260 265

cDNA

TGA GGCGCCCGCC GCCGCCCCCC TTCCCGGTGC TCAGAATA 842

(2) Information for SEQ ID NO: 2:

(i) sequence characteristics:

(A) length: 21 base pairs (B) type: nucleotide (G) number of strands: two (D) configuration: linear (ii) molecule type: cDNA (iii) hypothetical: No (iv) antisense: No (vi) origin :

(A) organism: Homo sapiens (ix) characteristic:

(A) name / CLE: CDS (B) location: 1 ... 21 (C) additional information: / product = human apoA-I (xi) sequence description: SEQ ID No 2:

Met Arg Asp Cys Ala Arg Ala 15 (2) Information for sequence SEQ ID NO: 3:

(i) sequence characteristics:

(A) length: 21 base pairs (B) type: nucleotide (C) number of strands: one (D) configuration: linear (ii) molecule type: cDNA (ix) characteristic:

(C) additional information: / product = Sq5490 (xi) sequence description: SEQ ID NO. 3:

AAGGCACCCC ACTCAGCCAG G (2) Information for SEQ ID NO: 4:

(i) sequence characteristics:

(A) length: 24 base pairs (B) type: nucleotide (C) number of strands: one (D) configuration: linear (ii) molecule type: cDNA (ix) characteristic:

(D) additional information: / product = Sq5491 (xi) sequence description: SEQ ID NO. 4:

TTCAACATCA TCCCACAGGC CTCT (2) Information for SEQ ID NO: 5:

(i) sequence characteristics:

(A) length: 20 base pairs (B) type: nucleotide (C) number of fibers: one (D) configuration: linear (ii) Type molecules: cDNA

(ix) Characteristics:

(D) additional information: / product = Sq5492 (xi) sequence description: SEQ ID NO: 5:

CTGATAGGCT GGGGCGCTGG (2) Information for SEQ ID NO: 6:

(i) sequence characteristics:

(A) length: 20 base pairs (B) type: nucleotide (C) number of strands: one (D) configuration: linear (ii) molecule type: cDNA (ix) characteristic:

(D) additional information: / product = Sq5493 (xi) sequence description: SEQ ID NO: 6:

CGCCTCACTG GGTGTTGAGC (2) Information for SEQ ID NO: 7:

(i) sequence characteristics:

(A) length: 21 base pairs (B) type: nucleotide (C) number of strands: one (D) configuration: linear (ii) molecule type: cDNA (ix) characteristic:

(D) additional information: / product = S8 (xi) sequence description: SEQ ID NO: 7:

TGGGATCGAG TGAAGGACCT G (2) Information for SEQ ID NO: 8 (i) sequence characteristics:

(A) length: 21 base pairs (B) type: nucleotide (C) number of strands: one (D) configuration: linear (ii) molecule type: cDNA (ix) characteristic:

(D) additional information: / product = S4 (xi) sequence description: SEQ ID NO: 8:

CGCCAGAAGC TGCACCAGCT G (2) Information for SEQ ID NO: 9:

(i) sequence characteristics: (A) length: 21 base pairs (B) type: nucleotide (C) number of strands: one (D) configuration: linear (ii) molecule type: cDNA (ix) characteristic: i (D) Further information: / product = S6 (xi) sequence description: SEQ ID NO: 9:

GCGCTGGCGC AGCTCGTCGC T (2) Information for SEQ ID NO: 10:

(i) sequence characteristics:

(A) length: 603 base pairs (B) type: nucleotide (C) number of strands: two (D) configuration: linear (ii) molecule type: cDNA (vi) origin:

(A) organism: Homo sapiens (ix) characteristic:

(A) name / CLE: CDS (B) location: 1 ... 603 (xi) sequence description: SEQ ID NO: 10:

CTA AAG CTC CTT GAC AAC TGG GAC AGC GTG ACC TCC ACC TTC 42 Leu Lys Leu Leu Asp same time Trp Asp Ser wall Thr Ser Thr The 1 5 10 AGC AAG CTG CGC GAA CAG CTC GGC CCT GTG ACC CAG GAG TTC 84 Ser Lys Leu Arg Glu gin Leu Gly for wall Thr A boat Glu Phe 15 20 25 TGG C-AT AAC CTG GAA AAG GAG ACA GAG GGC CTG AGG CAG GAG 12 6 Trp Asp same time Leu Glu Lys Glu Thr Glu Gly Leu Arg gin C-lu 30 35 40 ATG AGC AAG GAT CTG GAG GAG GTG AAG GCC AAG GTG CAG CCC 163 Met Ser Lys Asp Leu Glu Glu wall Lys Ala Lys wall gin for 45 50 55 TAC CTG GAC GAC TTC CAG AAG AAG TGG CAG GAG GAC- ATG GAG 210 Tyr Leu Asp Asp Phe gin Lys Lys Trp gin Glu C-lu Met Glu 60 65 70 CTC TAC CGC CAG AAG GTG GAG CCG CTG CGC GCA C-AG CTC CAA 252 Leu Tyr Arg gin Lys wall Glu for Leu A_rg Ala Glu Leu gin 75 80 GAG GGC GCG CGC CAG AAG CTG CAC GAG CTG CAA GAG AAC CTC- 294 Glu Gly Ala Arg gin Lys Leu His Glu Leu gin Glu Lys Leu 85 90 95 AGC CCA CTG GGC GAG GAG ATG CGC GAC TGC GCG CGC GCC CAT 336 Ser for Leu Gly Glu Glu Met Arg Asp Cys Ala Arg Ala His 100 105 110 GTG GAC GCG CTG CGC ACG CAT CTG GCC CCC TAC AGC GAC GAG 373 wall Asp Ala Leu Arg Thr His Leu Ala for Tyr Ser Asp Glu 115 120 ¹²⁵ CTG CGC CAG CGC TTG GCC GCG CGC CTT GAG GCT CTC AAG GAG 420 Leu Arg A boat Arg Leu Ala Ala Arg Leu Glu Ara Leu Lys Glu 130 135 140 AAC GGC GGC GCC AGA CTG GCC GAG TAC CAC GCC AAG GCC ACC 462 same time Gly Gly Ala Arg Leu Ala Glu Tyr His Ala Lys Ala Thr 145 150

GAG CAT CTG AGC ACG CTC AGC GAG AAG GCC AAG CCC GCG CTC 504 Glu KÍ 5 Leu Ser Thr Leu Ser Glu Lys Ala Lys for Ala Leu 155 160 165 GAG GAC CTC CGC CAA C-GC CTG CTG CCC GTG CTG GAG AGC TTC 546 Glu Asp LSC. Arg A boat Gly Leu Leu for wall Leu Glu Ser Phe 170 175 180 and AAG GTC AGC TTC CTG AGC GCT CTC GAG GAG TAC AC.T AAG AAG 588 Lys wall Ser Phe Leu Ser ALA Leu Glu Glu Tyr Thr Lys Lys 185 190 195 CTC AAC ACC CAG TGA 603 Leu same time Thr gin X

(2) Information for SEQ ID NO: 11:

(i) sequence characteristics:

¹ (A) length: 30 base pairs (B) type: nucleotide (C) number of strands: one (D) configuration: linear (ii) molecule type: cDNA (ix) characteristic:

(D) additional information: / product = alml (xi) sequence description: SEQ ID NO: 11:

ATCGATACCG CCATGAAAGC TGCGGTGCTG (2) Information for SEQ ID NO: 12 (i) sequence characteristics:

(A) length: 30 base pairs (B) type: nucleotide (C) number of strands: one (D) configuration: linear (ii) type of molecule: cDNA (ix) characteristic:

(D) additional information: / product = alm2 (xi) sequence description: SEQ ID NO: 12:

ATGGGCGCGC GCGCAGTCGC GCATCTCCTC (2) Information for SEQ ID NO: 13:

(i) sequence characteristics:

(A) length: 30 base pairs (B) type: nucleotide (C) number of strands: one (D) configuration: linear (ii) molecule type: cDNA (ix) characteristic:

(D) additional information: / product = alm3 (xi) sequence description: SEQ ID No.13:

GAGGAGATGC GCGACTGCGC GCGCGCCCAT (2) Information for SEQ ID NO: 14:

(i) sequence characteristics:

(A) length: 30 base pairs (B) type: nucleotide (C) number of strands: one (D) configuration: linear (ii) molecule type: cDNA (ix) characteristic:

(D) additional information: / product = alm4 (xi) sequence description: SEQ ID NO: 14:

GTCGACGGCG CCTCACTGGG TGTTGAGCTT

Claims (18)

PATENT CLAIMS 1. A cysteine-containing human apolipoprotein A-I variant at position 151. A variant according to claim 1, characterized in that it comprises the peptide sequence of SEQ ID No. 2. Variant according to claim 1 or 2, characterized in that the variant is apoA-I Paris. A variant according to claims 1 to 3, characterized in that the variant is a recombinant protein. A variant according to claims 1 to 4 in the form of a dimer. A variant according to claim 5, characterized in that the variant is a homodimer. Nucleic acid encoding an apolipoprotein A-I variant according to one of the preceding claims. 8. The nucleic acid of claim 7, wherein the nucleic acid is cDNA. 9. The nucleic acid of claim 7, wherein the nucleic acid is gDNA. 10. The nucleic acid of claim 7, wherein the nucleic acid is RNA. Nucleic acid according to claims 7 to 9, characterized in that it comprises the nucleotide sequence of SEQ ID No. 2. A vector comprising the nucleic acid of claims 7 to 12 13. The vector of claim 11, wherein the vector is a viral vector. 14. The vector of claim 11, wherein the vector is a chemical vector. A defective recombinant adenovirus comprising DNA encoding the apolipoprotein A-I variant of claim 1 inserted into its genome. A cell genetically modified according to claim 7. by inserting a nucleic acid An implant comprising mammalian cells genetically modified by introducing the nucleic acid of claim 7 and an extracellular matrix. A pharmaceutical composition comprising an apolipoprotein A-I variant according to claims 1 to 6 and / or a nucleic acid according to claim 7 and / or a vector according to claim 12 and / or a genetically modified cell according to claim 16.