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  • C07K14/775—Apolipopeptides
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  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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  • C12N2799/00—Uses of viruses
  • C12N2799/02—Uses of viruses as vector
  • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
  • C12N2799/022—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from an adenovirus

Definitions

• 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 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.
• 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.
• LCAT lecithin cholesterol acyltransfera
• 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).
• longevity is associated with high HDL cholesterol.
• the protective role of apoA-1 has been demonstrated in a model of transgenic mice expressing human apolipoprotein AI (Rubin et al. Nature).
• the infusion of HDL in rabbits induces a regression of lesions (Badimon et al. J. Clin. Invest. 85, 1234-41, 1990).
• 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).
• 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.
• 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.
• the presence of this variant prevents the development of any atherosclerosis, testifying to a very powerful protective role, specific to this mutated apoA-1.
• a cysteine on the apoA-1 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.
• 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.
• 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.
• the variants of the invention also include other mutations leading to the replacement of residues by cysteines.
• 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.
• 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).
• the nucleic acid of the present invention can be a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA).
• DNA deoxyribonucleic acid
• RNA ribonucleic acid
• 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.
• the nucleic acid is a cDNA or a gDNA.
• 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.
• 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).
• they may be promoter sequences of eukaryotic or viral genes.
• they may be promoter sequences originating from the genome of the target cell.
• promoters 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, ⁇ -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 ⁇ -actin, etc.) or promoters responding to a stimulus (steroid hormone receptor, acid receptor retinoic, etc.).
• ubiquitous promoters promoter of the HPRT, PGK genes, vimentin, ⁇ -actin, tubulin, etc.
• promoters of therapeutic genes for example the promoter of the MDR, CFTR, Factor VIII genes, etc.
• tissue-specific promoters promoter of
• 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.
• these promoter regions can be modified by adding activation or regulatory sequences, or allowing tissue-specific or majority expression.
• 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.
• 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.
• suitable eukaryotic hosts there may be mentioned animal cells, yeasts, or fungi.
• yeasts mention may be made of yeasts of the genus Saccharomyces, Kluyveromyces, Pichia, Schwanniomyces, or Hansenula.
• animal cells mention may be made of COS, CHO, C127, NIH-3T3 cells, etc.
• the mushrooms there may be mentioned more particularly Aspergillus ssp. or Trichoderma ssp.
• 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.
• the nucleic acid according to the invention is used directly as a medicament, in gene or cell therapy applications.
• it can be used as it is, by injection at the site to be treated or incubation with cells for their administration.
• 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.
• the present invention therefore relates to a vector comprising a nucleic acid as defined above.
• vectors can be used. They can be viral or non-viral vectors.
• the vector of the invention is a viral vector.
• viral vectors are 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.
• adenoviruses of type 2 or 5 Ad 2 or Ad 5
• Ad 2 or Ad 5 adenoviruses of animal origin
• adenoviruses of animal origin mention may be made of adenoviruses of canine, bovine, murine origin,
• the adenovirus of animal origin is a canine adenovirus, more preferably an adenovirus
• CAV2 Manhattan strain or A26 / 61 (ATCC VR-800) for example.
• adenoviruses of human or canine or mixed origin are used.
• the defective adenoviruses of the invention comprise ITRs, a sequence allowing the encapsidation and the nucleic acid of interest.
• 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.
• 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).
• 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.
• 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.
• AAV adeno-associated viruses
• 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.
• a human helper virus for example an adenovirus
• ITR inverted repeat regions
• Recombinant AAV products are then purified by conventional techniques (see in particular WO95 / 06743).
• 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).
• 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.
• 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.
• d packaging a cell line called d packaging
• the packaging lines are therefore capable of expressing the gag, pol and env genes.
• PA317 (US4,861,719); the PsiCRIP line (WO90 / 02806) and the line
• 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
• adenovirus or a retrovirus defective recombinant 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.
• 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.
• 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.
• 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.
• 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.
• the non-viral vectors all have polycationic charges.
• 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.).
• lipopolyamines lipofectamine, transfectam, etc.
• DOTMA cationic or neutral lipids
• 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.
• 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.
• 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 retrovirus
• an implant comprising mammalian cells genetically modified by insertion of a nucleic acid as defined above, and an extracellular matrix.
• 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.
• the extracellular matrix comprises a gelling compound and optionally a support allowing the anchoring of the cells.
• 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.
• collagen is used. It can be collagen of human, bovine or murine origin. More preferably, type I collagen is used.
• compositions according to the invention advantageously comprise a support allowing the anchoring of the cells.
• 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.
• the cells can either cover the support used, or penetrate inside this support, or both.
• 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.
• PTFE polytetrafluoroethylene
• the implants according to the invention can be implanted at different sites in the body.
• 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.
• nucleic acids thus constitute a new drug in the treatment and prevention of cardiovascular pathologies (atherosclerosis, restenosis, etc.).
• 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.
• 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.
• 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).
• Triglycerides 2.82 mmol / l 0.74-1.71
• 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.).
• Fig 5 relative fluorescence of the Superose 6 PG fractions. (- ⁇ -: POPC / Al Paris; -D-: POPC / Al recombinant; -- ⁇ - POPC / Al plasma.)
• Fig 6 Fluorescence of tryptophans and concentration of phospholipids for the POPC / Al Paris complex (-B-: relative fluorescence; -D-: phospholipids).
• Apolipoprotein A-I 0.50 g / 1 1.20-2.15
• Apolipoprotein B 1.38 g / l 0.55-1.30
• 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.
• 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.
• 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
• 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 ⁇ l 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 ⁇ g 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.
• 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 ⁇ g of purified genomic DNA introduced into the following mixture:
• 10X buffer 100 mM Tris-HCI pH 8.3; 500 mM KCI; 15 mM MgCI2; 0.1% (w / v) gelatin
• dNTP dATP, dGTP, dCTP, dTTP
• the primers used for the amplification are the following:
• 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.
• 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
• RNA strand amplified and then purified on the Catalyst 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)
• a mutation C-> T at the first base of the codon coding for aa 151 which then codes for a cysteine was found in part of the clone sequences. This mutation is therefore present in the heterozygous state in the selected patient.
• 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.
• 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.
• - Bsu361 has in M13 and in pXL2116 a unique restriction site upstream of the mutation.
• histidine-rich polypeptide whose nucleotide sequence has been cloned 5 'to apo A1 will therefore also be synthesized.
• 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.
• digestion is carried out by Xhol which recognizes two restriction sites inside the Bsu361 / BamHI fragment.
• 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:
• the competent cells are also transformed with pUC19 in order to test the efficiency of the transformation.
• Chloramphenicol selection of BL21 strains
• Ampicillin selection of strains that have integrated the plasmid
• the plasmid DNA obtained by purification on each of the 48 clones is digested with the enzyme NdeI.
• the ligation is that expected since the size of the plasmid is correct, that is to say equal to that of pXL2116.
• Clone 8 therefore carries the correctly mutated px12116 plasmid.
• 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.).
• 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.
• IPTG Isopropyl-b-thiogalactopyranoside
• 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.
• 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.
• 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 ⁇ l 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 Bochem ref Q-1280
• rifampicin Sigma
• 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.
• 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 bacterial pellet After centrifugation of the culture, the bacterial pellet is resuspended with gentle stirring in lysis buffer in the presence of protease inhibitors and ⁇ -mercaptoethanol.
• ⁇ -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 ⁇ 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.
• 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.
• the aim of this step is the elimination of EDTA, a molecule which interferes with the conditions required for affinity chromatography.
• 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.
• Ni2 + nickel ions
• NTA Nitrilo Acetic Acid
• the binding capacity of the agarose NiNTA gel 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.
• fractions are combined according to the concentration and purity of the eluted protein. These characteristics are analyzed by electrophoresis (PAGE-SDS 15%).
• 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.
• Example 6 Physico-chemical properties of the recombinant apoA-l Paris and normal apoA-l.
• 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 shows the comparison of the formation of these complexes with the recombinant apoA-Inormalale and apoA-1 Paris, as well as native apoA-1.
• 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.
• the addition of GdnHDL increases the protein-lipid association. This is very important for the recombinant apoA-l, in particular apoA-l Paris.
• 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.
• 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.
• 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.
• Alm1 ATC GAT ACC GCC ATG AAA GCT GCG GTG CTG (SEQ ID n ° 11),
• the primers Alm1 and Alm4 respectively introduce Clal sites in 5 'and SalI in 3' of the cDNA while the primers Alm2 and 33
• 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.
• 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.
• 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.
• AAVs and 34 AAVs and 34
• adenovirus doses of 10 * 3 to 10 * 10 pfu / ml can also be used.
• pfu plaque forming unit
• the term pfu 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.
• NAME RHONE-POULENC RORER S.A.
• 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
• 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

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