WO1996037608A1

WO1996037608A1 - Novel variants of apolipoprotein a-i

Info

Publication number: WO1996037608A1
Application number: PCT/FR1996/000747
Authority: WO
Inventors: Gerd Assmann; Patrick Benoit; Eric Bruckert; Patrice Denefle; Nicolas Duverger; Jean-Charles Fruchart; Gérald Luc; Gérard Turpin; Harald Funke
Original assignee: Rhone-Poulenc Rorer S.A.; Institut Pasteur De Lille; Universite Pierre Et Marie Curie
Priority date: 1995-05-22
Filing date: 1996-05-20
Publication date: 1996-11-28
Also published as: TW434260B; MX9708727A; IL118336A0; SK156397A3; AU717202B2; CZ291376B6; CA2218759A1; HUP9802926A2; JPH11505712A; EP0827538A1; AU5904896A; BR9608813A; KR19990021828A; HUP9802926A3; CZ367197A3; NO975367D0; FR2734568A1; FR2734568B1; NO975367L; ZA964097B

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

NEW APOLIPOPROTEIN A-I VARIANTS

The present invention relates to a new variant of apolipoprotein A-I. It also relates to any nucleic acid coding for this new variant. It also relates to the use of these proteins or nucleic acids for therapeutic purposes. More particularly, the invention relates to a new variant of apolipoprotein A-I comprising in particular a mutation in position 151.

Apolipoprotein A-I (apoA-1) is the major constituent of high density lipoproteins (HDL), which are macromolecular complexes composed of cholesterol, phospholipids and triglycerides. ApoA-1 is a protein made up of 243 amino acids, synthesized in the form of a preproprotein with 267 residues, having a molecular mass of 28,000 daltons. The prepro form of apoA-1 is synthesized in humans by both the liver and the intestine. This form of protein is then cleaved into proprotein which is secreted in the plasma. In the vascular compartment, proapoA-1 is then transformed into mature protein (243 amino acids) by the action of a calcium-dependent protease. ApoA-1 has a structural role and an active role in lipoprotein metabolism: apoA-1 is in particular a cofactor of lecithin cholesterol acyltransferase (LCAT), responsible for the esterification of plasma cholesterol.

The level of cholesterol in the HDL fraction and the plasma concentration of apoA-1 are negative risk factors for the development of atherosclerosis in humans. Epidemiological studies have indeed shown an inverse correlation between HDL cholesterol and apoA-1 concentrations and the incidence of cardiovascular disease (EG Miller et al. Lancet, 1977: 965-968). However, longevity is associated with high HDL cholesterol. Recently, the protective role of apoA-1 has been demonstrated in a model of transgenic mice expressing human apolipoprotein AI (Rubin et al. Nature). Similarly, the infusion of HDL in rabbits induces a regression of lesions (Badimon et al. J. Clin. Invest. 85, 1234-41, 1990). Different mechanisms have been proposed to explain the protective effect of HDL, and in particular a role of HDL in the reverse transport of cholesterol (Fruchart et al. Circulation, 87: 22-27, 1993) and an antioxidant action of HDL (Forte T ., Current Opinion in Lipidology, 5: 354-364, 1994)).

The gene encoding apoA-1 has been cloned and sequenced (Sharpe et al., Nucleic Acids Res. 12 (9) (1984) 3917). This 1863 bp gene comprises 4 exons and 3 introns. The cDNA encoding apoA-1 has also been described (Law et al., PNAS 81 (1984) 66). This cDNA comprises 840 bp (see SEQ ID No. 1). In addition to the wild form of apoA-1, various natural variants have been described in the prior art, the differences from which are compared to the wild protein are given in the table below.

Variant: Mutation Variant Mutation

Milano Arg173Cys Norway Glu136Lys

Marburg Lys1 O70 Pro165Arg

Munster2B Ala158Glu Pro3His

Giessen Pro143Arg ArglOLeu

Munster3A Asp103Asn Gly26Arg

Munster3B Pro4Arg Asp89Glu

Munster3C Pro3Arg Lys107Met

Munster3D Asp213Gly Glu139Gly

Munster4 Glu198Lys Glu147Val

Yame Asp13Tyr Ala158Glu

Asp213Gly Glu169Gln Arg177His The present invention stems from the discovery of a new series of variants of the apolipoprotein AI. This series of variants presents in particular a substitution of the arginine residue at position 151 by a cysteine residue. The apoA-1 variant according to the invention has remarkable therapeutic properties. In particular, it has particularly important anti-atherogenic protective properties. Thus, in a situation of extremely low cholesterol levels in the HDL fraction, associated with hypertriglyceridemia, the presence of this variant prevents the development of any atherosclerosis, testifying to a very powerful protective role, specific to this mutated apoA-1. In addition, the presence of a cysteine on the apoA-1 according to the invention leads to the formation of dimers and other complexes linked by a disulfide bridge. This apoA-1 is found in free form in plasma, linked in dimer to itself or associated with apolipoprotein A-ll which is another important protein associated with HDL and which also has a cysteine in its sequence. Furthermore, the loss of charge linked to arginine at position 151 leads to the visualization of this mutant by electroisofocusing of the plasma proteins followed by an immunological revelation of apoA-1.

Given its particularly remarkable anti-atherogenic properties, this new protein according to the invention offers an important therapeutic advantage in the treatment and prevention of cardiovascular pathologies.

A first subject of the invention therefore relates to a series of variants of human apolipoprotein AI comprising a cysteine at position 151. The amino acid sequence of the reference apoA-1 is described in the literature (Cf Law above). . This sequence, including the prepro region (residues 1 to 24), is presented on the sequence SEQ ID No. 1. A characteristic of the variants according to the invention therefore resides in the presence of a cysteine in position 151 of apoA- l mature (corresponding to position 175 on the sequence SEQ ID No. 1), in substitution for arginine present in the reference sequence. A preferred variant according to the invention comprises the peptide sequence SEQ ID No. 2, and, even more preferably, the peptide sequence comprised between residues 68 to 267 of the sequence SEQ ID No. 1, the residue 175 being substituted by a cysteine .

The variants according to the invention are represented in particular by apoA-1 Paris, that is to say an apoA-1 having a cysteine in position 151 relative to the native apoA-1. The variants according to the invention can also carry other structural modifications with respect to the reference apolipoprotein A-I, and in particular other mutations, deletions and / or additions. According to a particular embodiment, the variants of the invention also include other mutations leading to the replacement of residues by cysteines. Thus, another particular variant combines the mutations present in the variant apoA-l Paris and apoA-l milano. Other mutations may also be present affecting residues which do not significantly modify the properties of apoA-1. The activity of these variants can be verified in particular by a cholesterol efflux test.

The variants according to the invention can be obtained in different ways. They can first of all be chemically synthesized, using the techniques of a person skilled in the art using peptide synthesizers. They can also be obtained from the reference apoA-1, by mutation (s). Advantageously, they are recombinant proteins, that is to say obtained by expression in a cellular host of a corresponding nucleic acid as described below.

As indicated above, the variants according to the invention can be in monomeric form or in the form of a dimer. The presence of a cysteine at least in the sequence of the variants of the invention in fact allows the production of dimers by disulfide bond. They may be homodimers, that is to say dimers comprising two variants according to the invention (example: diApoA-l Paris); or heterodimers, that is to say dimers comprising a variant according to the invention and another molecule having a free cysteine (example: ApoA-1 Paris: ApoAII).

Another subject of the invention resides in a nucleic acid coding for a variant of apolipoprotein A-I as defined above. The nucleic acid of the present invention can be a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA). Among the DNAs, it may be a complementary DNA (cDNA), a genomic DNA (gDNA), a hybrid sequence or a synthetic or semi-synthetic sequence. It may also be a chemically modified nucleic acid, for example with a view to increasing its resistance to nucleases, its penetration or cellular targeting, its therapeutic efficacy, etc. These nucleic acids can be of human, animal, plant, bacterial, viral, synthetic, etc. origin. They can be obtained by any technique known to those skilled in the art, and in particular by screening of banks, by chemical synthesis, or also by mixed methods including chemical or enzymatic modification of sequences obtained by screening of banks.

Advantageously, the nucleic acid is a cDNA or a gDNA.

Preferably, the nucleic acid according to the invention comprises the sequence SEQ ID No. 2. Even more preferably, it comprises the sequence SEQ ID No. 10.

The nucleic acid according to the invention advantageously comprises a promoter region for functional transcription in the target cell or organism, as well as a region located at 3 ′, which specifies a transcriptional end signal and a polyadenylation site. All of these elements constitute the expression cassette. Regarding the promoter region, it may be the promoter region naturally responsible for the expression of the apoA-1 gene or an ApoA-1 variant when the latter is likely to function in the cell or organism concerned. They can also be regions of different origin (responsible for the expression of other proteins, or even synthetic). In particular, they may be promoter sequences of eukaryotic or viral genes. For example, they may be promoter sequences originating from the genome of the target cell. Among the eukaryotic promoters, one can use any promoter or derived sequence stimulating or repressing the transcription of a gene in a specific way or not, inducible or not, strong or weak. They may in particular be ubiquitous promoters (promoter of the HPRT, PGK genes, vimentin, α-actin, tubulin, etc.), promoters of therapeutic genes (for example the promoter of the MDR, CFTR, Factor VIII genes, etc.) tissue-specific promoters (promoter of the pyruvate kinase gene, villin, intestinal fatty acid binding protein, smooth muscle α-actin, etc.) or promoters responding to a stimulus (steroid hormone receptor, acid receptor retinoic, etc.). Likewise, they may be promoter sequences originating from the genome of a virus, such as for example the promoters of the E1A and MLP genes of adenovirus, the CMV early promoter, the RSV LTR promoter, etc. In addition, these promoter regions can be modified by adding activation or regulatory sequences, or allowing tissue-specific or majority expression.

Furthermore, the nucleic acid may also include a signal sequence directing the product synthesized in the secretory pathways of the target cell. This signal sequence may be the natural signal sequence of apoA-1, but it may also be any other functional signal sequence, or an artificial signal sequence.

The nucleic acid according to the invention can be used to produce the recombinant ApoA-1 variants by expression in a recombinant host cell, or directly as a drug in gene or cell therapy applications. For the production of recombinant variants according to the invention, the nucleic acid is advantageously incorporated into a plasmid or viral vector, which can be autonomous or integrative replication. This vector is then used to transfect or infect a selected cell population. The transfected or infected cells thus obtained are then cultured under conditions allowing expression of the nucleic acid, and the recombinant apoA-1 variant according to the invention is isolated. The cellular hosts which can be used for the production of the variants of the invention by the recombinant route are both eukaryotic and prokaryotic hosts. Among suitable eukaryotic hosts, there may be mentioned animal cells, yeasts, or fungi. In particular, as regards yeasts, mention may be made of yeasts of the genus Saccharomyces, Kluyveromyces, Pichia, Schwanniomyces, or Hansenula. As regards animal cells, mention may be made of COS, CHO, C127, NIH-3T3 cells, etc. Among the mushrooms, there may be mentioned more particularly Aspergillus ssp. or Trichoderma ssp. As prokaryotic hosts, it is preferred to use the following bacteria E.coli, Bacillus, or Streptomyces. The variant thus isolated can then be packaged for its therapeutic use.

According to a particularly advantageous mode, the nucleic acid according to the invention is used directly as a medicament, in gene or cell therapy applications. In this regard, it can be used as it is, by injection at the site to be treated or incubation with cells for their administration. It has in fact been described that naked nucleic acids can penetrate cells without any particular vector. Nevertheless, it is preferred in the context of the present invention to use an administration vector, making it possible to improve (i) the efficiency of cell penetration, (ii) targeting (iii) extra- and intracellular stability.

According to a particular embodiment, the present invention therefore relates to a vector comprising a nucleic acid as defined above. Different types of vectors can be used. They can be viral or non-viral vectors.

In a preferred embodiment, the vector of the invention is a viral vector.

The use of viral vectors is based on the natural properties of transfection of viruses. It is thus possible to use adenoviruses, herpes viruses, retroviruses, associated adeno viruses or even vaccinia virus. These vectors are particularly effective in terms of transfection.

With regard more particularly to adenoviruses, various serotypes, whose structure and properties vary somewhat, have been characterized.

Among these serotypes, it is preferred to use, in the context of the present invention, human adenoviruses of type 2 or 5 (Ad 2 or Ad 5) or adenoviruses of animal origin (see application WO94 / 26914). Among the adenoviruses of animal origin which can be used in the context of the present invention, mention may be made of adenoviruses of canine, bovine, murine origin,

(example: Mav1, Beard et al., Virology 75 (1990) 81), sheep, pigs, avian or even simian (example: SAV). Preferably, the adenovirus of animal origin is a canine adenovirus, more preferably an adenovirus

CAV2 [Manhattan strain or A26 / 61 (ATCC VR-800) for example]. Preferably, in the context of the invention, adenoviruses of human or canine or mixed origin are used.

Preferably, the defective adenoviruses of the invention comprise ITRs, a sequence allowing the encapsidation and the nucleic acid of interest. Even more preferably, in the genome of the adenoviruses of the invention, the E1 region at least is non-functional. The viral gene considered can be made non-functional by any technique known to a person skilled in the art, and in particular by total suppression, substitution, partial deletion, or addition of one or more bases in the gene or genes considered. Such modifications can be obtained in vitro (on isolated DNA) or in situ, for example, by means of genetic engineering techniques, or by treatment with mutagenic agents. Other regions can also be modified, and in particular the region E3 (WO95 / 02697), E2 (W094 / 28938), E4 (W094 / 28152, W094 / 12649, WO95 / 02697) and L5 (WO95 / 02697). According to a particularly preferred embodiment, a recombinant adenovirus having a deletion of all or part of the E1 and E4 regions is used in the context of the present invention. This type of vector indeed offers particularly advantageous security properties.

The defective recombinant adenoviruses according to 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). In particular, they can be prepared by homologous recombination between an adenovirus and a plasmid carrying, inter alia, the nucleic acid or the cassette of the invention. Homologous recombination occurs after co-transfection of said adenovirus and plasmid in 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 genome of the defective adenovirus, preferably in integrated form to avoid the risks of recombination. As an example of a line, mention may be made of 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 an Ad5 adenovirus (12%). Other lines have been described in applications No. WO 94/26914 and WO95 / 02697.

Then, the adenoviruses which have multiplied are recovered and purified according to conventional techniques of molecular biology, as illustrated in the examples. Concerning adeno-associated viruses (AAV), these are relatively small DNA viruses, which integrate into the genome of the cells it infects, in a stable and site-specific manner. They are capable of infecting a broad spectrum of cells, without inducing an effect on cell growth, morphology or differentiation. Furthermore, they do not seem to be involved in pathologies in humans. The AAV genome has been cloned, sequenced and characterized. It comprises approximately 4,700 bases, and contains at each end an inverted repeat region (ITR) of approximately 145 bases, serving as an origin of replication for the virus. The rest of the genome is divided into 2 essential regions carrying the packaging functions: the left part of the genome, which contains the rep gene involved in viral replication and the expression of viral genes; the right part of the genome, which contains the cap gene coding for the capsid proteins of the virus.

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

WO 93/09239; US 4,797,368, US 5,139,941, EP 488,528). 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 (onto cells in culture) or in vivo (directly into an organism ) said gene of interest. The defective recombinant AAVs according to the invention can be prepared by co-transfection, in a cell line infected with a human helper virus (for example an adenovirus), of a plasmid containing the nucleic acid or the cassette of the invention bordered two inverted repeat regions (ITR) of AAV, and a plasmid carrying the packaging genes (rep and cap genes) of AAV. The

Recombinant AAV products are then purified by conventional techniques (see in particular WO95 / 06743).

Concerning herpes viruses and 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, Bemstein et al. Broom. Eng. 7 (1985) 235; McCormic BioTechnology 3 (1985) 689, etc. In particular, retroviruses are integrative viruses, selectively infecting dividing cells. They therefore constitute vectors of interest for cancer applications. The retrovirus genome essentially comprises two LTRs, an 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 whole or in part, and replaced by a heterologous nucleic acid sequence of interest. These vectors can be produced from different types of retroviruses such as in particular MoMuL ("murine moloney leukemia virus"; also designated MoMLV), MSV ("murine moloney sarcoma virus"), HaSV ("harvey sarcoma virus"); SNV ("splee necrosis virus"); RSV ("rous sarcoma virus") or the Friend virus.

To construct recombinant retroviruses comprising the nucleic acid or the cassette of the invention, a plasmid comprising in particular the LTRs, the packaging sequence and the nucleic acid or the cassette is constructed, then used to transfect a cell line called d packaging, capable of providing trans retroviral functions deficient in the plasmid. Generally, the packaging lines are therefore capable of expressing the gag, pol and env genes. Such packaging lines have been described in the prior art, and in particular the line

PA317 (US4,861,719); the PsiCRIP line (WO90 / 02806) and the line

GP + envAm-12 (WO89 / 07150). Furthermore, the recombinant retroviruses may include modifications at the level of the LTRs to suppress transcriptional activity, as well as extended encapsidatio sequences, comprising a part of the gag gene (Bender et al., J. Virol. 61

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

For the implementation of the present invention, it is always particularly advantageous to use an adenovirus or a retrovirus defective recombinant. These vectors indeed have particularly advantageous properties for the transfer of genes coding for apolipoproteins. The adenoviral vectors according to the invention are particularly advantageous for direct administration in vivo of a purified suspension, or for the ex vivo transformation of cells, in particular autologous, with a view to their implantation. In addition, the adenoviral vectors according to the invention also have significant advantages, such as in particular their very high infection efficiency, making it possible to carry out infections from small volumes of viral suspension.

In this regard, the invention preferably relates to a defective recombinant adenovirus comprising, inserted into its genome, a DNA coding for a variant of apolipoprotein A-I as defined above.

According to another particularly advantageous embodiment of the invention, a line producing retroviral vectors containing the sequence coding for the ApoA-1 variant is used for implantation in vivo. The lines which can be used for this purpose are in particular the cells PA317 (US4,861,719), PsiCrip (WO90 / 02806) and GP + envAm-12 (US5,278,056), modified to allow the production of a retrovirus containing a nucleic sequence coding for a variant of ApoA-1 according to the invention.

According to another aspect of the invention, the vector used is a chemical vector. The vector according to the invention can indeed be a non-viral agent capable of promoting the transfer and expression of nucleic acids in eukaryotic cells. Chemical or biochemical vectors represent an interesting alternative to natural viruses, in particular for reasons of convenience, safety and also by the absence of theoretical limit as regards the size of the DNA to be transfected. These synthetic vectors have two main functions, to compact the nucleic acid to be transfected and to promote its cellular fixation as well as its passage through the plasma membrane and, where appropriate, the two nuclear membranes. To compensate for the polyanionic nature of nucleic acids, the non-viral vectors all have polycationic charges.

Among the synthetic vectors developed, cationic polymers of polylysine type, (LKLK) n, (LKKL) n, polyethylene immine and DEAE dextran or else cationic lipids or lipofectants are the most advantageous. They have the property of condensing DNA and promoting its association with the cell membrane. Among the latter, mention may be made of lipopolyamines (lipofectamine, transfectam, etc.) and various cationic or neutral lipids (DOTMA, DOGS, DOPE, etc.). More recently, the concept of targeted transfection, mediated by a receptor, has been developed which takes advantage of the principle of condensing DNA thanks to the cationic polymer while directing the binding of the complex to the membrane thanks to a chemical coupling between the cationic polymer and the ligand of a membrane receptor, present on the surface of the cell type that we want to graft. The targeting of the transferrin, insulin receptor or the hepatocyte asialogiycoprotein receptor has thus been described.

The invention also relates to any cell genetically modified by insertion of a nucleic acid coding for a variant of apolipoprotein AI as defined above. Advantageously, these are mammalian cells capable of being administered or implanted in vivo. They may in particular be fibroblasts, myoblasts, hepatocytes, keratinocytes, endothelial, epithelial, glial cells, etc. The cells are preferably of human origin. In a particularly advantageous manner, these are autologous cells, that is to say cells taken from a patient, modified ex vivo by a nucleic acid according to the invention in to give them therapeutic properties, then re-administered to the patient.

The cells according to the invention can come from primary cultures. These can be removed by any technique known to those skilled in the art, then cultured under conditions allowing their proliferation. As regards more particularly fibroblasts, these can be easily obtained from biopsies, for example according to the technique described by Ham [Methods Cell.Biol. 21a (1980) 255]. These cells can be used directly for the insertion of the nucleic acid of the invention (by means of a viral or chemical vector), or preserved, for example by freezing, for the establishment of autologous libraries, with a view to 'further use. The cells according to the invention can also be secondary cultures, obtained for example from pre-established banks (see for example EP 228458, EP 289034, EP 400047, EP 456640).

The cells in culture can in particular be infected with the recombinant viruses of the invention to give them the capacity to produce a variant of the biologically active apoA-1. The infection is carried out in vitro according to techniques known to those skilled in the art. In particular, according to the type of cells used and the number of copies of virus per cell desired, a person skilled in the art can adapt the multiplicity of infection and possibly the number of infection cycles carried out. It is understood that these steps must be carried out under conditions of appropriate sterility when the cells are intended for administration in vivo. The doses of recombinant virus used for infection of the cells can be adapted by a person skilled in the art according to the aim sought. The conditions described above for administration in vivo can be applied to infection in vitro. For infection by retroviruses, it is also possible to co-cultivate the cells which it is desired to infect with cells producing retroviruses. recombinants according to the invention. This makes it possible to dispense with the purification of retroviruses.

Another subject of the invention relates to an implant comprising mammalian cells genetically modified by insertion of a nucleic acid as defined above, and an extracellular matrix. Preferably, the implants according to the invention comprise 10 ^ to 10 ^" O cells. More preferably, they comprise 10 ^ to 10 ^ _ The cells can also be cells producing recombinant viruses containing inserted in their genome a nucleic acid such as defined above.

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

For the preparation of the implants according to the invention, different types of gelling agents can be used. The gelling agents are used for the inclusion of cells in a matrix having the constitution of a gel, and to promote the anchoring of the cells on the support, if necessary. Different cell adhesion agents can therefore be used as gelling agents, such as in particular collagen, gelatin, glycosaminoglycans, fibronectin, lectins, etc. Preferably, in the context of the present invention, collagen is used. It can be collagen of human, bovine or murine origin. More preferably, type I collagen is used.

As indicated above, the compositions according to the invention advantageously comprise a support allowing the anchoring of the cells. The term anchoring designates any form of biological and / or chemical and / or physical interaction resulting in the adhesion and / or fixing of the cells on the support. Furthermore, the cells can either cover the support used, or penetrate inside this support, or both. We prefer to use in the framework of the invention a solid, non-toxic and / or biocompatible support. In particular, polytetrafluoroethylene (PTFE) fibers or a support of biological origin (coral, bone, collagen, etc.) can be used.

The implants according to the invention can be implanted at different sites in the body. In particular, the implantation can be carried out in the peritoneal cavity, in the subcutaneous tissue (suprapubic region, iliac or inguinal fossa, etc.), in an organ, a muscle, a tumor, the central nervous system , or under a mucous membrane. The implants according to the invention are particularly advantageous in that they make it possible to control the release of the apoA-1 variant in the body: This is first of all determined by the multiplicity of infection and by the number of cells implanted. Then, the release can be controlled either by the withdrawal of the implant, which definitively stops the treatment, or by the use of regulable expression systems, making it possible to induce or repress the expression of the therapeutic genes.

Due to the particularly remarkable anti-atherogenic properties of the variants of the invention, nucleic acids thus constitute a new drug in the treatment and prevention of cardiovascular pathologies (atherosclerosis, restenosis, etc.).

To this end, the invention relates to any pharmaceutical composition comprising a variant of apolipoprotein A-I and / or a nucleic acid and / or a vector and / or a genetically modified cell as described above.

The present invention thus provides a new means for the treatment or prevention of pathologies linked to dyslipoproteinemias, in particular in the field of cardiovascular affections such as myocardial infarction, angina, sudden death, restenosis, cardiac decompensation and cerebrovascular accidents. More generally, this approach offers a very promising therapeutic intervention for each case where a genetic or metabolic deficit of apolipoprotein AI can be corrected.

The present invention will be described in more detail with the aid of the following examples, which should be considered as illustrative and not limiting.

List of Figures

Fig 1: Restriction map of plasmid PXL2116

Fig 2: Construction of phage M 13 carrying the patient's exon 4.

Fig 3: Construction of the vector carrying mutated PXL2116. Fig 4: Studies of turbidimetry as a function of temperature.

Fig 5: Gel filtration profiles.

Fig 6: Fluorescence of tryptophans and concentration of phospholipids by fraction for the different complexes.

Examples

Example 1 - Demonstration of an ApoAl variant

The ApoA-l Paris variant was identified and isolated from a patient selected because of his particular lipid profile. More specifically, the patient presented the following lipid balance (nature of the sample: serum).

Normal

Total cholesterol 4.59 mmol / l 4.54-6.97

Triglycerides 2.82 mmol / l 0.74-1.71

HDL-cholesterol 0.25 mmol / l 0.88-1.60

LDL-cholesterol 3.06 mmol / l 2.79-4.85 1/17

: List of Figures

Fig 1: Restriction map of the plasmid PXL2116 Fig 2: Construction of the phage M 13 carrying the patient's exon 4. Fig 3: Construction of the vector carrying mutated PXL2116. Fig 4: Studies of turbidimetry as a function of temperature. 4a: turbidimetry of the association of different ApoAl and DPMC in the absence of GndHCI (- “-: control; - ^* -: recombinant; - * -: plasma; - D-: Paris.).

4b: turbidimetry of the association of different ApoAl and DPMC in the presence of GndHCI (-β-: control; - -: recombinant; --Φ--: plasma; -D-: Paris.).

4c: turbidimetry of the association of ApoAl Paris and DPMC (-B-: in the absence of GndHCI; -D-: in the presence of GndHCI (0.5M)).

4d: turbidimetry of the association of the recombinant ApoAl and of DPMC (-B-: in the absence of GnHCI; -D-: in the presence of GnHCI (0.5M)).

4th: turbidimetry of the association of plasma ApoAl and DPMC (-m-: in the absence of GndHCI; -D-: in the presence of GndHCI (0.5M)).

4f: effect of GndHCI (0.5M) on the ApoAl / DPMC association (* •: in the absence of GndHCI; -D-: in the presence of GndHCI)

Fig 5: relative fluorescence of the Superose 6 PG fractions. (- β-: POPC / Al Paris; -D-: POPC / Al recombinant; --Φ- POPC / Al plasma.)

Fig 6: 6a: Fluorescence of tryptophans and concentration of phospholipids for the POPC / Al Paris complex (-B-: relative fluorescence; -D-: phospholipids).

6b: Fluorescence of tryptophans and concentration of phospholipids for the recombinant POPC / Al complex (-B-: relative fluorescence; -D-: phospholipids).

RULE 26 18

Apolipoprotein A-I 0.50 g / 1 1.20-2.15

Apolipoprotein B 1.38 g / l 0.55-1.30

ApoE Phenotype: 3/3

Unexpectedly, this patient showed no signs of atherosclerosis despite an extremely low HDL concentration and hypertriglyceridemia. This led the applicants to investigate the origin of this protection.

Isolation of HDLs containing apoA-l Paris from plasma.

After an overnight fasting, blood is taken from subjects carrying the apoA-1 Paris gene. The plasma is prepared by slow centrifugation of the blood at 4 ° C (2000 g, 30 minutes). The high density lipoproteins are prepared by sequential ultracentrifugation at the density of 1.063-1.21 g / ml (Havel, J. Clin. Invest. 34: 1345-54, 1955). The fraction containing the HDLs is then dialyzed against 10 mM Tris-HCl buffer, 0.01% sodium azide, at pH 7.4.

Highlighting apoA-l Paris dimers.

The dialyzed HDL fraction is defatted in a diethyl ether / ethanol mixture (3/1, v / v) and the protein concentration is estimated by the Lowry method (Lowry et al., J. Biol. Chem., 193: 265- 75, 1951). The proteins of the HDL fration migrate on a polyacrylamide gel in the presence of SDS in a non-reducing condition. This migration makes it possible to highlight the size of the different proteins of the patient's HDLs. In addition to the presence of proteins corresponding to apoA-1 and apoA-ll of normal sizes, this analysis reveals the presence of proteins of higher molecular weights corresponding to dimers of apoA-1 and the complexes apoA-1 and apoA-11. The presence of apoA-1 and apoA-11 in these complexes was verified by a specific immunological revelation. 19

Demonstration of the difference in charge of the mutated apoA-1

The detection of mutated apoA-1 directly from the plasma was carried out according to the following protocol (Menzel, HJ, and Utermann, G., Electroforesis, 7: 492-495, 1986): twenty microliters of plasma are delipidated overnight with the ethanol / ether mixture and resuspended in a deposition buffer. A 5 μl aliquot undergoes electrophoresis on an isoelectric focusing gel (pH 4-6.5, Pharmolyte), and the proteins are then transferred to a nylon membrane. The bands corresponding to apoA-1 are detected by an immunological reaction using anti-human apoA-1 antibodies.

Detection of mutated apoA-1 can also be done from HDL proteins. The dialyzed HDL fraction is defatted in a diethyl ether / ethanol mixture (3/1, v / v) and the protein concentration is estimated by the Lowry method (Lowry et al., J. Biol. Chem., 193: 265- 75, 1951). About 100 μg of proteins are electrophoresed on an isoelectric focusing gel (pH 4-6.5, Pharmolyte), and the proteins are then revealed by staining with Coomassie blue. This technique makes it possible to demonstrate that the most important isoforms of the apoA-1 of the patient with the mutation are displaced towards the anode, which corresponds to a difference in charge of -1 compared to the charge of apoA-l normal.

Example 2 Identification of the gene and the mutation

The patient's genomic DNA was isolated from whole blood according to the technique of Madisen et al. (Amer. J. Med. Genêt, 27: 379-390, 1987). The apoA-1 gene was then amplified by the PCR technique. To this end, the amplification reactions were carried out on 1 μg of purified genomic DNA introduced into the following mixture:

- 10 μl of 10X buffer (100 mM Tris-HCI pH 8.3; 500 mM KCI; 15 mM MgCI2; 0.1% (w / v) gelatin) 20

- 10 μl of dNTP (dATP, dGTP, dCTP, dTTP) 2mM

- 20 pmol of each primer

- 2.5 U of Taq polymerase (Perkin-Elmer)

- qs 100 μl H2O.

The primers used for the amplification are the following:

Sq5490: 5'-AAGGCACCCCACTCAGCCAGG-3 '(SEQ ID n ° 3)

Sq5491: 5'-TTCAACATCATCCCACAGGCCTCT-3 '(SEQ ID n ° 4)

Sq5492: 5'-CTGATAGGCTGGGGCGCTGG-3 '(SEQ ID n ° 5)

Sq5493: 5'-CGCCTCACTGGGTGTTGAGC-3 '(SEQ ID n ° 6)

The primers Sq5490 and Sq5491 amplify a fragment of 508 bp corresponding to exons 2 and 3 of the apoA-1 gene and the primers Sq5492 and Sq5493 amplify a fragment of 664 bp corresponding to exon 4 of this gene.

The amplification products (two PCR fragments of 508 and 664 bp) were then sequenced. For this, a first method was used, consisting in direct sequencing using the PCR fragment sequencing kit (Amersham). The primers used for sequencing are the PCR primers, but they can also be primers internal to the fragments (see primers S4, S6 and S8 below).

A second sequencing technique was also implemented which consisted in cloning the PCR fragments into an M13 mp28 vector. The double-stranded DNA of M13 was cleaved by EcoRV and then dephosphorylated. the PCR fragments were treated with Klenow, phosphorylated and ligated to the vector M13. The white areas were then removed and the simple DNA 21

strand amplified and then purified on the Catalyst (Applied Biosystem) was sequenced by a fluorescent primer -20 (PRISM dye primer kit and protocol No. 401386, Applied Biosystem) or by primers internal to the fragments after orientations thereof. The fluorescent dideoxynucleotide technique is then used (DyeDeoxyTerminator kit and protocol No. 401388, Applied Biosystem)

S8 5'-TGGGATCGAGTGAAG GACCTG-3 '(SEQ IDn ° 7)

S4 5'-CGCCAGAAGCTGCACCAGCTG-3 '(SEQIDn ° 8)

S6 5'-GCGCTGGCGCAGCTCGTCGCT-3 '(SEQIDn ° 9)

The sequences from several clones were then compiled and compared to the ApoAl sequence. The amino acid sequence 148 to 154 of mature ApoAl is given below (corresponding to residues 172-178 on the sequence SEQ ID No. 1).

ATG CGC GAC CGC GCG CGC GCC Met Arg Asp Arg Ala Arg Ala 151

A mutation C-> T at the first base of the codon coding for aa 151 which then codes for a cysteine (cf. sequence SEQ ID No. 2 below) was found in part of the clone sequences. This mutation is therefore present in the heterozygous state in the selected patient.

ATG CGC GAC TGC GCG CGC GCC

Met Arg Asp Cys Ala Arg Ala

151

All of the cDNA coding for the apoA-1 Paris variant according to the invention has been sequenced. Part of this sequence is presented in SEQ ID No. 10. 22

Example 3 Construction of a Plasmid Expression Vector (pXL2116mute)

ApoAl Paris has a point mutation located in the sequence of exon 4 of the patient's apoAl gene. The strategy for constructing the expression vector consists in substituting, in an apoAl expression vector (the vector pXL2116, FIG. 1), the region corresponding to exon 4 originating from the patient's gene.

3.1. Use of exon 4 of the cloned patient in M13

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

Mutagenesis by replacement of a fragment of apo A1 by a fragment from M13 carrying the mutation is envisaged. We therefore have to choose the enzymes appropriate to the two vectors so that they generate cohesive ends for the vector from digested pXL2116 and for the insert from digested double-stranded M13.

3.2. Choice of restriction enzymes

- Bsu361 has in M13 and in pXL2116 a unique restriction site upstream of the mutation.

- Digestion with BamHI, having a unique restriction site at the 3 'end of apo A1 due to the cloning technique, and having a restriction site in the polylinker of M13, allows us:

1) a ligation of the vector from pXL2116 and the insert from M13 23

2) and this by incorporating a polylinker fragment which will be useful for us to verify by enzymatic digestion the insertion of the mutated fragment originating from M 13.

It should be noted that the presence of this polylinker fragment does not in any way alter the coding sequence of apo A1 since it is located after the stop codon.

The histidine-rich polypeptide whose nucleotide sequence has been cloned 5 'to apo A1 will therefore also be synthesized.

3.3. Construction of the vector (Figure 3).

- The M13 made double strand is digested with Bsu361 / BamHI and the product of digestion is deposited on gel. The insert thus generated is recovered in sufficient quantity and does indeed have a size of 1Db.

- pXL2116 is digested by the same enzymes but not purified.

In order to avoid the religation of the digestion products, digestion is carried out by Xhol which recognizes two restriction sites inside the Bsu361 / BamHI fragment.

Indeed, dephosphosylation was first attempted but gave very poor results on a dish and no positive clone out of 24 tested.

The optimal amounts of vector and insert for ligation have been evaluated at 50 ng of vector for 15 ng of insert.

The DH5a strains are transformed with the products of the following three ligations:

- vector digested with Bsu361 and BamHI without ligase (negative ligation control) 24

- product of digestion + ligase

- digestion product + insert + ligase

The competent cells are also transformed with pUC19 in order to test the efficiency of the transformation.

Results of cells transformed on LB medium dish

Chloramphenicol (selection of BL21 strains) Ampicillin (selection of strains that have integrated the plasmid) are reproduced below.

RESULTS OBTAINED ON PETRI BOX

DH5a TRANSFORMATION + INTERPRETATION RESULTS

PUC19 200 clones Vector transformation control (- (- fragment) 0 clone Perfect digestion, no recircularized vector plasmid (+ fragment) + ligase 160 clones Very important background noise: ineffective additional digestion strategy vector (+ fragment) + ligase 120 clones "Decrease effect" of + insert the ligase often found. Bacteria are not necessarily transformed with a correct mutated sequence

48 clones are re-isolated from box 4.

In order to identify the positive clones (that is to say having inserted the mutated fragment of M13), the plasmid DNA obtained by purification on each of the 48 clones is digested with the enzyme NdeI.

If the insert from M13 has been inserted correctly, we will see on gel two fragments: one of 4.5kb and one of 1kb.

If another ligation event has occurred, we will obtain either several bands, or simply a linearized plasmid (general case). 25

From the gel result, clones 7,8,13,16,26 appear to be correct. We select the clone which gives a net result.

In order to completely verify the sequence of the selected clone, six enzymatic digestions are carried out.

The presence of the polylinker and therefore of the insert is thus verified, but the ligation event must also be verified.

Obviously, the ligation is that expected since the size of the plasmid is correct, that is to say equal to that of pXL2116.

Unless there is a very rare mutation event, the sequence of the newly cloned apoAl should be exact.

Clone 8 therefore carries the correctly mutated px12116 plasmid.

Example 4 Expression of Recombinant ApoAl Variants

This example describes a process for producing recombinant apoAl variants. This process was carried out in a bacterium. Other expression systems can be used for this purpose (yeasts, animal cells, etc.).

4.1. Principle

The expression of the plasmid within the bacterium was placed under the control of the T7 promoter and terminator. Isopropyl-b-thiogalactopyranoside (IPTG), inducer of the lactose operon, induces in this system the synthesis of T7 TARN polymerase which then specifically binds to the T7 promoter and starts the transcription of the gene for the recombinant protein. The RNA polymerase is stopped by the T7 terminator, which prevents the transcriptional flow from overflowing downstream of the sequence of interest. 26

Rifampicin is an antibiotic that inhibits the endogenous RNA polymerase activity of E. coli. It therefore inhibits the synthesis of bacterial proteins, that is to say both proteases, which limits the degradation of the protein of interest; and contaminating bacterial proteins, which enhances the expression of the protein of interest.

4.2. Protocol

The strain used for the expression is Escherichia coli BL21 DE3 pLys S. The plasmid DNA is introduced into E. coli by transformation according to conventional techniques. The culture is stored in the form of a frozen suspension at −20 ° C., in the presence of 25% glycerol, and aliquoted in 500 μl fractions.

A preculture is initiated by the addition of a few drops of frozen suspension in 10 ml of M9 ampicillin medium, then incubated overnight at 37 ° C. The 1 liter erlens are seeded from the preculture and placed in a shaker at 37 ° C until an OD of between 0.5 and 1 at 610 nm is obtained. IPTG (Bachem ref Q-1280) is then added to a final concentration of 1 mM. After 15 minutes of incubation at 37 ° C, rifampicin (Sigma) is added to a final concentration of 100 μg / ml. Then, after 1 hour of culture, the cells are recovered by centrifugation (15 minutes, 8000 rpm) and the expression is checked by electrophoresis under denaturing conditions on a 15% acrylamide gel and by immunoblotting.

The results obtained show that the mutated protein expresses itself at a good level of expression and that this is effectively revealed by anti-apoAI polyclonal antibodies.

EXAMPLE 5 Purification of the Recombinant ApoAl Variants 27

This example describes an efficient method for purifying the recombinant apoAl variants according to the invention. It is understood that other methods can be used.

The proteins being expressed in the cytoplasm of E. coli, their extraction first requires cell lysis followed by elimination of nucleic acids.

5.1. Bacterial lysis

After centrifugation of the culture, the bacterial pellet is resuspended with gentle stirring in lysis buffer in the presence of protease inhibitors and β-mercaptoethanol.

Β-mercaptoethanol is a reducing agent cleaving the disulfide bridges formed between two cysteine residues. No disulfide bridge is formed in the cytoplasm of E.coli, the presence of a reducing agent is however necessary once the proteins have been extracted. Indeed, the addition of β mercaptoethanol in the lysis buffer makes it possible to avoid the formation of bridges between the cysteine residues of the bacterial proteins and those of the recombinant proteins.

Cell lysis is obtained by 3 times 5 minutes of sonication in ice (Vibracells sonics material, pulsed mode, output control 5), it is followed by centrifugation at 10,000 rpm (1 hour, 4 ° C on Beckman J2-21 M / E, JA10 rotor) to remove cellular debris. A protein assay is carried out on the supernatant by the Bradford colorimetric method.

5.2. Nucleic acid precipitation

It is carried out on the lysis supernatant with a 10% solution of streptomycin sulfate, at a rate of 10 ml per 10 g of protein, under 28

gentle magnetic stirring for 1 hour at 4 ° C. The nucleic acids are removed by centrifugation at 10,000 rpm (1 hour, 4 ° C. on Beckman J2-21 M / E, rotor JA10), and the protein concentration of the supernatant is evaluated by colorimetric assay.

At the end of these two stages, a protein solution of known concentration is obtained, grouping together all of the intracellular proteins, including the protein of interest. This is purified by chromatographic techniques.

-> Chromatography by gel filtration

-> Affinity chromatography

5.3. Gel filtration chromatography

The aim of this step is the elimination of EDTA, a molecule which interferes with the conditions required for affinity chromatography. For this, the Tris-acryl GF 05 support (Sepracor) was chosen. This gel allows the separation of molecules whose Molecular Mass (MM) is between 300 and 2500 daltons, and the exclusion of molecules of MM greater than 2500 daltons, including proteins. This gel also has the advantage of having good resistance to pressure, which makes it possible to work at high flow rate without modifying the resolution. The chromatography of the bacterial lysate is carried out in pH8 phosphate buffer. The protein concentration in the exclusion volume is determined, and optionally adjusted to 4 mg / ml by dilution in the pH8 phosphate buffer.

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

5.4. Affinity Chromatography 29

Principle

The presence of 6 consecutive histidine residues associated with the recombinant protein gives it a particularly high affinity for nickel ions (Ni2 +) (15). These Ni2 + ions are attached to an agarose matrix via Nitrilo Acetic Acid (NTA). Between the imidazole of histidine and the nickel ions occurs a metal chelation bond; this bond is stronger than an ionic bond and weaker than a covalent bond.

Protocol

The binding capacity of the agarose NiNTA gel (Qiagen) is 2 mg of protein per 1 ml of gel. This gel is balanced in pH8 buffer supplemented with 25 mM Hecameg. The contaminating bacterial proteins are not retained at pH8, or eliminated at pH6, and the protein of interest is recovered at pH5. These steps are carried out in the presence of 25 mM hecameg.

The fractions (2 ml) eluted at pH5 are added with:

60 μl of 1 M NaOH (neutralization)

10 μl of 0.2M PMSF

40 μl of 0.1 M EDTA

The fractions are combined according to the concentration and purity of the eluted protein. These characteristics are analyzed by electrophoresis (PAGE-SDS 15%).

Elution at pH5 detaches the protein associated with nickel by acting on the Ni2 + - NTA bond. It is therefore necessary to dissociate this cation from the recombinant protein. This stage is carried out by competition thanks to 30

incubation with gentle magnetic shaking at 4 ° C for 1 hour in the presence of 50 mM histidine.

5.5. Dialysis

They eliminate histidine and nickel. The sample is dialyzed (Spectra / Por membrane MWCO 12-14000 daltons) at 4 ° C for 5 hours then overnight, against 2 times 10 liters of 2 mM PBS EDTA buffer. A protein assay is finally performed.

This process makes it possible to obtain the apoAl Paris protein in purified form, essentially devoid of contaminating proteins.

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

6.1. Turbidimetry measurements

6.1.1. Turbidimetry as a function of temperature

The measurement of the absorbance of the DMPC vesicles at 325 nm in the presence of apoA-1 is a measurement of the formation of small discoid proteolipid complexes. The analysis of temperature variation between 19-28 ° C shows us a decrease in absorbance around the transition temperature of phospholipids (23 ° C), witnessing the formation of complexes. FIG. 4 (whether or not GdnHDL is present to avoid the formation of dimers) shows the comparison of the formation of these complexes with the recombinant apoA-Inormalale and apoA-1 Paris, as well as native apoA-1. These three proteins have fairly similar behaviors, but with apoA-l Paris, there is a tendency to associate with itself to form dimers.

6.1.2. Turbidimetry as a function of time 31

The decrease in turbidimetry of the DMPC vesicles after incubation of the apoA-1 was monitored at given temperature as a function of time in the presence or absence of GdnHDL. The time constant (1 / t1 / 2 which corresponds to 50% decrease in the initial turbidimetry) is evaluated as a function of 1 / T (temperature in Kelvin). The association speed is rapid for the native apoA-l, lower for the recombinant apoA-l, in particular apoA-l Paris. Furthermore, the addition of GdnHDL increases the protein-lipid association. This is very important for the recombinant apoA-l, in particular apoA-l Paris.

6.2. Tryptophan fluorescence emission spectrum

The fluorescence emission spectrum of tryptophans in the different apoA-1 was measured at wavelengths between 300 and 400 nm (excitation at 295 nm). The emission maximums are indicated in Table 1 below for the apoA-1 and for the apoA-1 / cholesterol / POPC complexes. The maximum fluorescence emission of tryptophans in the different apoA-1 and complexes are identical, indicating that the tryptophans are in the same environment in the three proteins.

Table 1

Maximum emission product

Al Paris 335

POPC / C / AI Paris 332

Recombinant Al 334

POPC / C / AI rec 332

Al plasma 336

POPC / C / AI plasma 333 32

6.3. Isolation and characterization of apoA-1 / lipid complexes

Complexes with apoA-1 and POPC were prepared by the cholate technique. The complexes were separated from the free apoA-1 by gel filtration chromatography on a Superose 6PG column and their compositions analyzed. The gel filtration profiles are indicated in FIG. 5. A single homogeneous peak is obtained for the complexes made with the native apoA-1 while with the recombinant apoA-1, heterogeneous populations are observed. The free apoA-1 are eluted in fractions 20 to 24. The phospholipid concentrations and the fluorescence of the tryptophans by fractions for the different complexes are indicated in FIG. 6.

Example 7 Construction of an Adenoviral Vector for Expression of the Mutated ApoAl

A cDNA coding for a variant according to the invention containing the mutation Arg -> Cys in position 151 of the mature ApoAl is obtained by PCR. The primers

Alm1: ATC GAT ACC GCC ATG AAA GCT GCG GTG CTG (SEQ ID n ° 11),

Alm2: ATG GGC GCG CGC GCA GTC GCG CAT CTC CTC (SEQ ID n ° 12),

Alm3: GAG GAG ATG CGC GAC TGC GCG CGC GCC CAT (SEQ ID n ° 13)

and Alm4: GTC GAC GGC GCC TCA CTG GGT GTT GAG CTT (SEQ ID n ° 14)

are used, the primers Alm1 and Alm4 respectively introduce Clal sites in 5 'and SalI in 3' of the cDNA while the primers Alm2 and 33

Alm3 which are complementary introduce the mutation. PCR reactions with the primer pairs AIM1-Alm2 and Alm3-Alm4 are first performed on a non-mutated ApoAl cDNA. The fragments from these PCRs are then reintroduced in a third PCR in the presence of the primers Alm1 and Alm4, which generates a fragment of 822pb which is then cloned in pCRI1 (Invitrogen) for verification of its sequence. The ClaI / SalI fragment which contains the mutated cDNA is then introduced by the same restriction sites into the shuttle vector pXL-RSV-LPL which contains the LPL cDNA under the control of an LTR-RSV promoter and with a site bovine growth hormone polyadenylation, replenishment of the LPL cDNA (FR9406759). Any other shuttle vector can obviously be used. The resulting vector is then linearized and cotransfected in 293 to obtain recombinant adenoviruses. The adenoviruses thus obtained can be amplified on plaques, purified (in particular by cesium chloride) and then stored frozen, for example in glycerol. For their therapeutic use, they can be combined with any pharmaceutically acceptable vehicle. They may in particular be saline solutions (monosodium phosphate, disodium phosphate, sodium chloride, potassium, calcium or magnesium, etc., or mixtures of such salts), sterile, isotonic, or dry compositions, in particular lyophilized, which, by addition, as appropriate, of sterilized water or physiological saline, allow the constitution of injectable solutes. In their use for the treatment of pathologies linked to dyslipoproteinemias, the defective recombinant adenoviruses according to the invention can be administered according to different modes, and in particular by intravenous injection. Preferably, they are injected at the portal vein. The doses of virus used for the injection can be adapted according to different parameters, and in particular according to the mode of administration used, the pathology concerned or the duration of the treatment sought. In general, the recombinant viruses according to the invention are formulated and administered in the form of doses of between 10 ⁴ and 10 ¹⁴ pfu / ml. For AAVs and 34

adenovirus, doses of 10 ^* 3 to 10 ^* 10 pfu / ml can also be used. The term pfu ("plaque forming unit") corresponds to the infectious power of a suspension of virions, and is determined by infection of an appropriate cell culture, and measures, generally after 48 hours, the number of plaques of infected cells. The techniques for determining the pfu titer of a viral solution are well documented in the literature.

35

LIST OF SEQUENCES

(1) GENERAL INFORMATION:

(i) DEPOSITOR:

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

(B) STREET: 20, avenue Raymond ARON

(C) CITY: ANTONY

(E) COUNTRY FRANCE

(F) POSTAL CODE: 92165

(i) DEPOSITOR:

(A) NAME: PIERRE ET MARIE CURIE UNIVERSITY

(B) STREET:

(C) CITY:

(E) COUNTRY- FRANCE

(F) POSTAL CODE:

(i) DEPOSITOR:

(A) NAME: INSTITUT PASTEUR DE LILLE

(B) STREET:

(C) CITY:

(E) COUNTRY FRANCE

(F) POSTAL CODE:

(ii) TITLE OF THE INVENTION: New variants of apolipoprotein A- (iii) NUMBER OF SEQUENCES: 14

(iv) COMPUTER-DETACHABLE FORM:

(A) TYPE OF SUPPORT: Tape

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Release # 1.0, Version # 1.30 (EPO)

(2) INFORMATION FOR SEQ ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 842 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: double

(D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: cDNA •

(iii) HYPOTHETIC: NO

(iv) ANTI-SENSE: NO

(vi) ORIGIN: (A) ORGANISM: Homo sapiens

(ix) CHARACTERISTIC:

(A) NAME / KEY: CDS

(B) LOCATION: 1..842 (D) OTHER INFORMATION: / product = "human apo AI cDNA"

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1: ATG AAA GCT GCG GTG CTG ACC TTG GCC GTG CTC TTC CTG ACG GGG AGC 48 Met Lys Ala Ala Val Leu Thr Leu Ala Val Leu Phe Leu Thr Gly Ser 1 5 10 15 36

CAG GCT CGG CAT TTC TGG CAG CAA GAT GAA CCC CCC CAG AGC CCC TGG 96 Gin Ala Arg His Phe Trp Gin Gin Asp Glu Pro Pro Gin Ser Pro Trp 20 25 30 GAT CGA GTG AAG GAC CTG GCC ACT GTG TAC GTG GAT GTG CTC AAA GAC 144 Asp Arg Val Lys Asp Leu Ala Thr Val Tyr Val Asp Val Leu Lys Asp 35 40 45

AGC GGC AGA GAC TAT GTG TCC CAG TTT GAA GGC TCC GCC TTG GGA AAA 192 Ser Gly Arg Asp Tyr Val Ser Gin Phe Glu Gly Ser Ala Leu Gly Lys 50 55 60

CAG CTA AAC CTA AAG CTC CTT GAC AAC TGG GAC AGC GTG ACC TCC ACC 240 Gin Leu Asn Leu Lys Leu Leu Asp Asn Trp Asp Ser Val Thr Ser Thr 65 70 75 80

TTC AGC AAG CTG CGC GAA CAG CTC GGC CCT GTG ACC CAG GAG TTC TGG 288 Phe Ser Lys Leu Arg Glu Gin Leu Gly Pro Val Thr Gin Glu Phe Trp 85 90 95

GAT AAC CTG GAA AAG GAG ACA GAG GGC CTG AGG CAG GAG ATG AGC AAG 336 Asp Asn Leu Glu Lys Glu Thr Glu Gly Leu Arg Gin Glu Met Ser Lys 100 105 110 GAT CTG GAG GAG GTG AAG GCC AAG GTG CAG CCC TAC CTG GAC GAC TTC 384 Asp Leu Glu Glu Val Lys Ala Lys Val Gin Pro Tyr Leu Asp Asp Phe 115 120 125

CAG AAG AAG TGG CAG GAG GAG ATG GAG CTC TAC CGC CAG AAG GTG GAG 432 Gin Lys Lys Trp Gin Glu Glu Met Glu Leu Tyr Arg Gin Lys Val Glu 130 135 140

CCG CTG CGC GCA GAG CTC CAA GAG GGC GCG CGC CAG AAG CTG CAC GAG 480 Pro Leu Arg Ala Glu Leu Gin Glu Gly Ala Arg Gin Lys Leu His Glu 145 150 155 160

CTG CAA GAG AAG CTG AGC CCA CTG GGC GAG GAG ATG CGC GAC CGC GCG 528 Leu Gin Glu ^' Lys Leu Ser Pro Leu Gly Glu Glu Met Arg Asp Arg Ala- 165 170 175

CGC GCC CAT GTG GAC GCG CTG CGC ACG CAT CTG GCC CCC TAC AGC GAC 576 Arg Ala His Val Asp Ala Leu Arg Thr His Leu Ala Pro Tyr Ser Asp 180 185 190

GAG CTG CGC CAG CGC TTG GCC GCG CGC CTT GAG GCT CTC AAG GAG AAC 624 Glu Leu Arg Gin Arg Leu Ala Ala Arg Leu Glu Ala Leu Lys Glu Asn 195 200 205

GGC GGC GCC AGA CTG GCC GAG TAC CAC GCC AAG GCC ACC GAG CAT CTG 672 Gly Gly Ala Arg Leu Ala Glu Tyr His Ala Lys Ala Thr Glu His Leu 210 215 220

AGC ACG CTC AGC GAG AAG GCC AAG CCC GCG CTC GAG GAC ^' CTC CGC CAA 720 Ser Thr Leu Ser Glu Lys Ala Lys Pro Ala Leu Glu Asp Leu Arg Gin 225 230 235 240

GGC CTG CTG CCC GTG CTG GAG AGC TTC AAG GTC AGC TTC CTG AGC GCT 768 Gly Leu Leu Pro Val Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala 245 250 255

CTC GAG GAG TAC ACT AAG AAG CTC AAC ACC CAG TGA GGCGCCCGCC 814

Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gin * 260 265 37

GCCGCCCCCC TTCCCGGTGC TCAGAATA 842

(2) INFORMATION FOR SEQ ID NO: 2:

(i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: double

(D) CONFIGURATION: linear (il) TYPE OF MOLECULE: cDNA (m) HYPOTHETIC: NO (iv) ANTI-SENSE: NO (vi) ORIGIN:

(A) ORGANIZATION: Homo sapiens

(i) CHARACTERISTIC:

(A) NAME / KEY: CDS

(B) LOCATION: 1..21

(D) OTHER INFORMATION: / product = "variant apo AI cDNA '

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2:

ATG CGC GAC TGC GCG CGC GCC 21 Met Arg Asp Cys Ala Arg Ala 1 5

(2) INFORMATION FOR SEQ ID NO: 3:

(l) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: Simple (D) CONFIGURATION: linear

(il) TYPE OF MOLECULE: cDNA (lx) CHARACTERISTIC:

(D) OTHER INFORMATION: / product = Sq5490

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3: AAGGCACCCC ACTCAGCCAG G 21

(2) INFORMATION FOR SEQ ID NO: 4:

(l) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleotide (C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(il) TYPE OF MOLECULE: cDNA (ix) CHARACTERISTIC: (D) OTHER INFORMATION: / product = Sq5491

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4: TTCAACATCA TCCCACAGGC CTCT 24 38

(2) INFORMATION FOR SEQ ID NO: 5: (1) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(il) TYPE OF MOLECULE: cDNA (ix) CHARACTERISTIC:

(D) OTHER INFORMATION: / product = Sq5492 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5:

CTGATAGGCT GGGGCGCTGG 20

(2) INFORMATION FOR SEQ ID NO: 6:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: simple (D) CONFIGURATION: linear

(il) TYPE OF MOLECULE: cDNA (ix) CHARACTERISTIC:

(D) OTHER INFORMATION: / product = Sq5493

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6: CGCCTCACTG GGTGTTGAGC 20

(2) INFORMATION FOR SEQ ID NO: 7:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleotide (C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: cDNA (IX) CHARACTERISTIC: (D) OTHER INFORMATION: / product = S8

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7: TGGGATCGAG TGAAGGACCT G 21

(2) INFORMATION FOR SEQ ID NO: 8:

(i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 base pairs (B) TYPE: nucleotide

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(il) TYPE OF MOLECULE: cDNA (ix) CHARACTERISTIC:

(D) OTHER INFORMATION: / product = S4

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 39

CGCCAGAAGC TGCACCAGCT G 21

(2) INFORMATION FOR SEQ ID NO: 9:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: simple (D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: cDNA (ix) CHARACTERISTIC:

(D) OTHER INFORMATION: / product = S6

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 9: GCGCTGGCGC AGCTCGTCGC T 21

(2) INFORMATION FOR SEQ ID NO: 10:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 603 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: double (D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: cDNA (vi) ORIGIN:

(A) ORGANIZATION: Homo sapiens

(ix) CHARACTERISTIC:

(A) NAME / KEY: CDS

(B) LOCATION: 1..603 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 10:

CTA AAG CTC CTT GAC AAC TGG GAC AGC GTG ACC TCC ACC TTC AGC AAG 48 Leu Lys Leu Leu AΞD Asn Trp Asp Ser Val Thr Ser Thr Phe Ser Lys 1 5 10 15

CTG CGC GAA CAG CTC GGC CCT GTG ACC CAG GAG TTC TGG GAT AAC CTG 96 Leu Arg Glu Gin Leu Gly Pro Val Thr Gin Glu Phe Trp Asp Asn Leu 20 25 30 GAA AAG GAG ACA GAG GGC CTG AGG CAG GAG ATG AGC AAG GAT CTG GAG 144 Glu Lys Glu Thr Glu Gly Leu Arg Gin Glu Met Ser Lys Asp Leu Glu 35 40 45

GAG GTG AAG GCC AAG GTG CAG CCC TAC CTG GAC GAC TTC CAG AAG AAG 192 Glu Val Lys Ala Lys Val Gin Pro Tyr Leu Asp Asp Phe Gin Lys Lys 50 55 60

TGG CAG GAG GAG ATG GAG CTC TAC CGC CAG AAG GTG GAG CCG CTG CGC 240

Trp Gin Glu Glu Met Glu Leu Tyr Arg Gin Lys Val Glu Pro Leu Arg

65 70 75 80

GCA GAG CTC CAA GAG GGC GCG CGC CAG AAG CTG CAC GAG CTG CAA GAG 288 Ala Glu Leu Gin Glu Gly Ala Arg Gin Lys Leu His Glu Leu Gin Glu 85 90 95

AAG CTG AGC CCA CTG GGC GAG GAG ATG CGC GAC TGC GCG CGC GCC CAT 336 Lys Leu Ser Pro Leu Gly Glu Glu Met Arg Asp Cys Ala Arg Ala His 100 105 110 40

GTG GAC GCG CTG CGC ACG CAT CTG GCC CCC TAC AGC GAC GAG CTG CGC 384 Val Asp Ala Leu Arg Thr His Leu Ala Pro Tyr Ser Asp Glu Leu Arg 115 120 125

CAG CGC TTG GCC GCG CGC CTT GAG GCT CTC AAG GAG AAC GGC GGC GCC 432 Gin Arg Leu Ala Ala Arg Leu Glu Ala Leu Lys Glu Asn Gly Gly Ala 130 135 140 AGA CTG GCC GAG TAC CAC GCC AAG GCC ACC GAG CAT CTG AGC ACG CTC 480 Arg Leu Ala Glu Tyr His Ala Lys Ala Thr Glu His Leu Ser Thr Leu 145 150 155 160

AGC GAG AAG GCC AAG CCC GCG CTC GAG GAC CTC CGC CAA GGC CTG CTG 528 Ser Glu Lys Ala Lys Pro Ala Leu Glu Asp Leu Arg Gin Gly Leu Leu

165 170 175

CCC GTG CTG GAG AGC TTC AAG GTC AGC TTC CTG AGC GCT CTC GAG GAG 571 Pro Val Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu 180 185 190

TAC ACT AAG AAG CTC AAC ACC CAG TGA 603

Tyr Thr Lys Lys Leu Asn Thr Gin * 195 200

(2) INFORMATION FOR SEQ ID NO: 11

(i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 30 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear (il) TYPE OF MOLECULE: cDNA (ix) CHARACTERISTIC:

(D) OTHER INFORMATION: / product = alml

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 11: ATCGATACCG CCATGAAAGC TGCGGTGCTG 30

(2) INFORMATION FOR SEQ ID NO: 12:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: simple (D) CONFIGURATION: linear

(il) TYPE OF MOLECULE: cDNA (ix) CHARACTERISTIC:

(D) OTHER INFORMATION: / product = alm2

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 12:

ATGGGCGCGC GCGCAGTCGC GCATCTCCTC 30

(2) INFORMATION FOR SEQ ID NO: 13:

(1) CHARACTERISTICS OF THE SEQUENCE: 41

(A) LENGTH: 30 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: cDNA (ix) CHARACTERISTIC:

(D) OTHER INFORMATION: / product = alm3 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 13:

GAGGAGATGC GCGACTGCGC GCGCGCCCAT 30

(2) INFORMATION FOR SEQ ID NO: 14:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleotide (C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: cDNA (ix) CHARACTERISTIC: (D) OTHER INFORMATION: / product = alm4

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 14:

GTCGACGGCG CCTCACTGGG TGTTGAGCTT 30

Claims

42 CLAIMS

1. Variant of human apolipoprotein A-I comprising a cysteine in position 151.

2. Variant according to claim 1 characterized in that it comprises the peptide sequence SEQ ID No. 2.

3. Variant according to claim 1 or 2 characterized in that it is apoA-l Paris.

4. Variant according to claims 1 to 3 characterized in that it is a recombinant protein.

5. Variant according to one of claims 1 to 4 in the form of a dimer.

6. Variant according to claim 5 characterized in that it is a homodimer.

7. Nucleic acid encoding a variant of apolipoprotein A-I according to one of the preceding claims.

8. Nucleic acid according to claim 7 characterized in that it is a cDNA.

9. Nucleic acid according to claim 7 characterized in that it is a gDNA.

10. Nucleic acid according to claim 7 characterized in that it is an RNA.

11. Nucleic acid according to one of claims 7 to 9 characterized in that it comprises the nucleic sequence SEQ ID No. 2.

12. Vector comprising a nucleic acid according to one of claims 7 to 11. 43

13. Vector according to claim 11 characterized in that it is a viral vector.

14. Vector according to claim 11 characterized in that it is a chemical vector.

15. Defective recombinant adenovirus comprising, inserted into its genome, a DNA coding for a variant of apolipoprotein A-I according to claim 1.

16. A cell genetically modified by insertion of a nucleic acid according to claim 7.

17. Implant comprising mammalian cells genetically modified by insertion of a nucleic acid according to claim 7 and an extracellular matrix.

18. Pharmaceutical composition comprising a variant of apolipoprotein AI according to one of 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.