KR20000053320A - Recombinant bicistron adenovirus for treating pathological conditions linked with dyslipoproteinemia - Google Patents

Abstract

PURPOSE: A defective recombinant virus and preferably an adenovirus is provided which is used for treating pathological conditions linked with dyslipoproteinemia. CONSTITUTION: A defective recombinant virus and preferably an adenovirus is characterized in that it comprises at least two nucleic acids coding for distinct enzymes, proteins and/or co-factors involved in the reverse transfer of cholesterol, the nucleic acids being operationally bound to a transcriptional promoter and mutually separated by a sequence coding for an internal entry site of the IRES ribosome. Further, provided is plasmid constructs useful for preparing these adenovirus, and cells transformed by these plasmids or adenovirus and pharmaceutical compositions containing the adenovirus.

Description

RECOMBINANT BICISTRON ADENOVIRUS FOR TREATING PATHOLOGICAL CONDITIONS LINKED WITH DYSLIPOPROTEINEMIA}

Dyslipoproteinemia is a metabolic disorder of lipoproteins responsible for the transport of lipids such as cholesterol and triglycerides in the blood and surrounding fluids. Dyslipoproteinemia causes major pathologies associated with hypercholesterolemia or hypertriglyceridemia, for example, atherosclerosis, in particular.

Atherosclerosis is a multigenic complex disease defined from a histological point of view by deposition of lipids and other blood derivatives (lipid or fibrolipid plaques) on the walls of the large arteries (aorta, coronary, carotid). These plaques, which calcify to some extent with the progress of this process, can be associated with lesions and are associated with the accumulation in the arteries of fatty deposits consisting essentially of cholesterol esters. These plaques are accompanied by thickening of the arterial walls with hypertrophy of smooth muscle, the appearance of foamy cells and the accumulation of fibrous tissue. Atheromatous plaques are very distinct in the walls and confer stenosis characteristics responsible for occlusion of blood vessels by atherosclerosis, thrombosis or embolism occurring in most diseased patients.

Therefore, hypercholesterolemia can cause very serious cardiovascular pathologies such as infarction, sudden death, subject insufficiency, cerebrovascular seizures and the like.

Therefore, in certain pathological conditions, useful treatments can be made to reduce plasma cholesterol or stimulate the outflow of cholesterol in surrounding tissues in order to release cells with accumulated cholesterol within the context of atheromatous plaque formation. This is especially important. Cholesterol is transported to the blood by various lipoproteins, including low-density lipoprotein (LDL) and high-density lipoprotein (HDL). LDL is synthesized in the liver and makes it possible to supply cholesterol to surrounding tissues. In contrast, HDL captures cholesterol from surrounding tissue and transports it to the liver for storage or degradation.

The most common dyslipidemia is characterized by high levels of LDL (low-density lipoprotein) cholesterol and low alpha lipoproteinemia. Low alpha lipoproteinemia is characterized by HDL (high-density lipoprotein) cholesterol levels below 35 mg / dl and represents 40% of cases of steatosis (Genest et al., 1992). Hypoalphalipoproteinemia appears to be related to the genetic deficiency of one or more proteins involved in the synthesis, maturation and disruption of HDL particles, the result of which is often the early appearance of cardiovascular disease (Dammermann et al., 1995). In general, there is an inverse correlation between the incidence of cardiovascular disease and the level of HDL particles (Miller et al., 1987).

The protective effect of HDL against cardiovascular disease is demonstrated by in vivo gene transfer experiments in mice to develop atherosclerotic lesions, and an increase in HDL particle counts indicates the development of such lesions (Rubin et al., 1991). Plump et al., 1994). It has been suggested that the protective effect of HDL particles may be due to their role in the back transport of cholesterol (Reiche et al., 1989). Back transport is the process by which excess cholesterol is delivered to the liver for removal from surrounding tissues (FIG. 1). It consists of a series of steps that involve the capture and esterification of cholesterol in HDL particles and the transfer to low-density lipoproteins that are subsequently recaptured by the liver and this process, as well as many proteins such as CETP, cholesterol acyl Involves precipitation of enzymes including transferases and hepatic lipases (and / or cofactors thereof, such as apolipoprotein AI and AIV).

While effective treatment exists to reduce the levels of LDL cholesterol, among triglycerides based on hypolipidemia and antihypertensive drugs, current treatments for hypoalphalipoproteinemia are provided with only limited efficacy.

The present invention specifically relates to the treatment by gene therapy of pathologies associated with hypoalphalipoproteinemia.

The therapeutic approach adopted in the present invention aims to increase the kinetics of the reverse transport of cholesterol in order to induce or otherwise prevent the formation of atherosclerotic lesions. Advantageously, this object is achieved by the simultaneous effective expression of at least two proteins, enzymes or cofactors involved in cholesterol transport in the cells to be treated according to the invention.

For the purposes of the present invention, it is contemplated that they are involved in the reverse transport of cholesterol as long as the breakdown at the cell concentration level of one of the proteins, enzymes and / or its cofactors automatically affects the reverse process of cholesterol.

As examples of accompanying enzymes, lecithin cholesterol acyltransferase and hepatic lipase may be mentioned in more detail.

Lecithin cholesterol acyltransferase (LCAT) is a 67 kDa glycoprotein synthesized in the liver and catalyzes the transfer of acyl groups from lecithin to cholesterol, resulting in lysophosphatidylcholine and cholesterol esters (Glomset, 1968). The cofactor for this enzyme is apolipoprotein AI bound to the surface of HDL particles. By maintaining a cholesterol concentration gradient between the surrounding cells and HDL, this plays an important role in the first stage of cholesterol back transport.

Human liver lipase (HL) is a 66 kDa glycoprotein with triglyceride hydrolase and phospholipase activity and is synthesized and secreted by hepatocytes. Binding to the hepatocyte surface, by proteoglycan heparin sulfate, it participates in the metabolism of the chiromicron of IDL (medium density lipoprotein) and HDL. HL has a special affinity for large HDL particles that degrade phospholipids and triglycerides, and produces discoid particles of HDL that have the property of receiving cellular cholesterol. In humans, HL deficiency is characterized by the initial development of triglyceride-rich large amounts of LDL, large HDL particles, and atherosclerosis (Hegele et al., 1993).

With regard to proteins, for the purposes of the present invention, more preferably one of the cholesterol ester transferase protein (CETP), apolipoprotein AI and AIV or a variant thereof is present.

Cholesterol ester transfer protein (CETP) is a 74 kDa glycoprotein synthesized in adipose tissue and liver. In plasma, this is mainly associated with HDL. This is the case when it catalyzes the transfer of cholesterol esters from HDL to low-density lipoproteins. Following this delivery, triglycerides pass from the low-density lipoprotein into HDL. Overexpression of CETP in transgene hypertriglyceridemic mice inhibits the production of atherosclerotic lesions (Hayek et al., 1995). This experiment is consistent with observations in individuals deficient in CETP of early development of cardiovascular disease (Zhong et al., 1996).

Apolipoprotein AI is a protein of 243 amino acids and is synthesized in the form of 267 residues prepropeptide having a molecular weight of 28,000 Daltons. It is specifically synthesized in the liver and small intestine in humans and it constitutes the essential protein of HDL particles (70% of protein mass). It is abundant in plasma (1.0 to 1.2 g / l). Its most biochemically characterized activity is the activation of lecithin-cholesterol acyltransferase (LCAT), but a number of other activities, for example stimulation of the outflow of cellular cholesterol, are the result. Apolipoprotein AI plays a major role in resistance to atherosclerosis, which is associated with the reverse transport of cholesterol. 1863 bp long genes were cloned and sequenced. Sharpe et al., Nucleic Acids Res. 12 (9) (1984) 3917]. Among the protein products having apolipoprotein AI type activity, mention may be made in particular of the natural variants described in the prior art.

Apolipoprotein AIV (apoAIV) is a protein consisting of 376 amino acids and is synthesized in the small intestine in the form of precursors of 396 residues. With regard to its physiological activity, it can activate lecithin-cholesterol acyltransferase (LCAT) in vitro and see Steinmetz et al., 1985, J. Biol. Chem., 260: 2258-2264], which may interfere with the binding of HDL particles to small arterial endothelial cells, such as apolipoprotein AI (Savion et al., 1987, Eur. J. Biochem., 257: 4171-4178. These two activities indicate that apoAIV can act as a vehicle for the reverse transport of cholesterol. The apoAIV gene has been cloned and described in the prior art (see in particular WO 92/05253). Among protein products having apolipoprotein AIV activity, fragments and derivatives described in patent application FR 92 00806 may be mentioned.

The present invention relates to novel recombinant viruses for their in vivo delivery and expression of the desired genes, their preparation and their use in gene therapy. More specifically, the present invention relates to recombinant viruses wherein the expression product comprises at least two insertion genes used for the back transport of cholesterol. The invention also relates to shuttle plasmids useful for the production of adenoviruses according to the invention. More specifically, the present invention relates to defective recombinant adenoviruses and their use for the prevention or treatment of pathologies associated with dyslipoproteinemia, which is known for serious consequences at the cardiovascular and neurological levels.

1: Schematic diagram of the putative mechanism for the reverse transport of cholesterol.

2: Schematic diagram of a protocol for generating adenovirus by homologous recombination in E. coli.

Figure 3: Construction protocol of bicistron shuttle plasmid pXL2970 comprising RSV-LCAT-IRES-CETP.

Figure 4: Construction protocol of the prokaryotic plasmid pXL2974 comprising RSV-LCAT-IRES-CETP.

5: Construction protocol of plasmid pXL2984 comprising RSV-LCAT-IRES-HL.

6: Representative diagram of the prokaryotic plasmid pXL3058 comprising RSV-ApoAI-IRES-LCAT.

I materials and methods

I-1 materials

1) The plasmids used for the construction of the bicistronic recombinant adenoviruses LCAT-IRES-CETP, LCAT-IRES-HL and LCAT-IRES-ApoAI were:

pSK IRES (denoted NOVAGEN)

pXL2616 LCAT cDNA (Seguret-Mace et al. Circulation 1996, 94 (9): 2177-21841)

pCRII (indicated by INVITROGEN)

-pXL2794 (WO96 / 25506)

-pXL2757 (WO96 / 25506)

2) total RNA obtained from hepatocytes and HepG2 cells

3) Cells containing a gene encoding adenovirus E1 protein 293, human kidney cells (Graham et al., 1977)

4) Cell E. coli: Genotype EndA1, recA1, hsdR17, supE44, I-, thy-1, gyrA, rel A1, lacZD M15, deoR +, F +, dam +, dcm + subtype DH5α (Woodcock et al., 1989)

5) Enzymes and restriction buffers are provided by New England Biolabs.

I-2 method

General Molecular Biology Technology

Methods commonly used in molecular biology, such as preparation sampling of plasmid DNA, centrifugation of plasmid DNA in a cesium chloride gradient, electrophoresis of agarose or acrylamide gels, purification of DNA fragments by electrolysis, phenols of proteins Or phenol-chloroform extraction, ethanol or isopropanol precipitation of DNA in saline medium, transformation in this coli, and the like are well known to those skilled in the art and described in Maniatis T. et al., Molecular Cloning, a Laboratory Manual ″, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982; Ausubel F.M. et al. (eds), ″ Current Protocols in Molecular Biology ″, John-Wiley & Sons, New York, 1987.

pBR322 and pUC plasmids and M13 phages are of commercial origin (Bethesda Research Laboratories).

For ligation, DNA fragments are sized by agarose or acrylamide gel electrophoresis, extracted with a phenol or phenol / chloroform mixture, precipitated with ethanol and then phage T4 DNA ligase (Biolabs) as recommended by the supplier. Is cultured in the presence of.

Filling of the protruding 5 'end can be performed using a Klenow fragment of E. coli DNA polymerase I (Biolabs) according to the supplier's details. Destruction of the overhanging 3 'ends is carried out in the presence of phage T4 DNA polymerase (Biolabs) used according to the manufacturer's recommendations. Destruction of the protruding 5 ′ end is performed by regulatory treatment with S1 nucleases.

Site-directed mutations by synthetic oligodeoxynucleotides in vitro can be performed by a method developed by Taylor et al. (Nuleic Acids Res. 13 (1985) 8749-8764) using a kit provided by Amersham. have.

So-called PCR techniques [polymerase-catalyzed chain Reaction, Saiki R. K. et al., Science 230 (1985) 1350-1354; Mullis K.B. and Faloona F.A., Meth. Enzym. 155 (1987) 335-350, enzymatic amplification of DNA fragments can be performed using a DNA thermal cycler (Perkin Elmer Cetus) according to the manufacturer's details.

Identification of the nucleotide sequence was performed by Sanger et al., Proc. Natl. Acad. Sci. USA, 74 (1977) 5463-5467.

Cell culture technology

a) culture of cells

Cells 293 are cultured in Eagle medium (MEM, Gibco BRL) supplemented with 10% fetal calf serum (FCS, Gibco BRL) at 37 ° C. and 5% CO ₂ .

b) Transfection of Cultured Cells

Cells 293 cultured in 100 mm dishes on MEM medium and supplemented with 10% fetal calf serum are transfected with 1 mg of DNA using lipofectamine (Gibco BRL). After 6 hours, the medium is transferred and the cells are incubated in complete medium (MEM + 10% FCS). Supernatants of cultured cells transfected with the recombinant plasmids RSV LCAT-IRES-CETP, RSV LCAT-IRES-ApoAI, and RSV LCAT-IRES-HL and β-galactosidase controls, and the supernatants of untransfected cells 72 hours after transfection. Recover at bay and store at 4 ° C. Activity tests of the enzymes LCAT, CETP, ApoAI and HL are performed in 10-30 ml fractions of cell supernatant using human plasma as a control.

c) generation and purification of recombinant adenoviruses

Bicistronic adenovirus DNA is obtained by homologous recombination in E. coli. This is then linearized with PacI and virus generated after transfection of cellular IP3. Cell 293 trans-complementing the El protein is transfected with 10 mg of linearized adenovirus DNA using lipofectamine. Cells in MEM medium supplemented with 4% FCS were harvested using agar and incubated at 37 ° C. for 8 days, after which plaques containing the recombinant virus were collected and analyzed for their restriction profiles.

d) infection of cultured cells;

Infection with supernatant 0.25, 0.5 and 1 ml containing bicistronic recombinant adenovirus is performed on a 12 well plate containing approximately 4 x 10 ⁵ cells 293. After 72 hours of incubation in 2 ml of MEM supplemented with 2% FCS, supernatants of infected cells were harvested and analyzed for enzyme activity.

Biochemical Confirmation of In Vitro Results

a) Assay of LCAT Activity

And as a substrate by the method described by Chen et al. (1982), using proteosome liposomes by measuring the conversion of ¹⁴ to ¹⁴ C- cholesterol esterification in cholesterol C- assesses the LCAT activity. Proteoliposomes are prepared from phosphatidylcholine, cholesterol, ¹⁴ C-cholesterol and apolipoprotein AI and incubated with 20 ml of the transfection culture supernatant to be tested. The reaction products (free ¹⁴ C-cholesterol and esterified ¹⁴ C-cholesterol) are separated by thin layer chromatography using solubility difference in organic solvent and detected by radiograph on Instant Imager (Packard). LCAT activity is expressed as time and% of ¹⁴ C-cholesterol esterified per 20 ml of supernatant tested.

b) CETP activity assay

CETP activity is determined by measuring the protein's ability to deliver cholesterol esters from high density lipoprotein particles (donors) to low density lipoprotein particles (receptors). Substrates for this reaction are provided by the Wak-Chemie kit, Medical GmbH. The fluorescence of cholesterol linoleate contained in the donor particles appears only in the presence of active CETP which will disappear in the latter natural state and will catalyze the delivery of fluorescent molecules to the receptor particles, and fluorescence can be detected at 535 nm. CETP activity is expressed as the fluorescence intensity value emitted per hour at 535 nm for 30 ml of culture supernatant tested.

HL activity assay

Liver lipase activity is assessed using synthetic triglyceride substrates provided by the Progen Biotechnik GmbH kit. These substrates contain fluorescent pyrene groups that are concealed by trinitrophenol in their natural state and the effect of the latter hydrolysis is fluorescence emission at 400 nm. After incubating the substrate with the sample to be tested, the fluorescence intensity emitted at 400 nm is measured and the standard plasma provided by the kit is used to assess the amount of HL (pmol / min) contained in 20 ml of the supernatant.

d) apolipoprotein AI analysis

Anti-ApoAI monoclonal antibodies are used to assess ApoAI activity. To this end, Immulon II plaques (Dynatech) were coated with anti-ApoAI monoclonal antibody (10 mg / ml in carbonate buffer pH 9.6) by incubating overnight at 4 ° C., followed by 2 in PBS pH 7.4 at 37 ° C. for 1 hour. Saturate with% BSA. The cell supernatant is then optionally diluted in PBS 2% BSA and incubated at 37 ° C. for 1 hour. Subsequently, the cells were incubated for 1 hour at 37 ° C with an anti-ApoAI monoclonal antibody mixture labeled with peroxidase, and then diluted to 1/5000. Binding of the peroxylated antibody is finally revealed by incubating with 250 μl of TMB (KPL) and reading the plaque at 630 nm.

Shuttle plasmid construction technology

Methods of constructing non-cistronic plasmids are depicted in detail in FIGS. The non-cistronic plasmids obtained are simultaneously shuttle vectors because they contain pIX-IVa2, an adenovirus sequence required for recombination with the viral genome. This recombination process results in cotransfection with a truncated genome derived from viral βgalactosidase, or E. coli. It occurs by double recombination in E. coli (coli shuttle vector).

Sequence similarity of various plasmid constructs is examined by analyzing their restriction profiles. These tests enable the selection of recombinant clones to be used for subsequent cloning and the identification of cloning results. Restriction enzymes are chosen to build the most complete information on the fully cloned cDNA and the number of strategic sites for cloning. However, such regulation does not exclude the presence of certain mutations such as nonsense or substitution point mutations that may occur in the cloning step. Such mutations can only be detected by complete sequencing of the cDNA of interest. Final regulation of the effectiveness of the obtained constructs is by in vitro biochemical testing of the enzymatic activity of LCAT, CETP and HL, or by detection of the presence of ApoAI.

More specifically, the present invention is based on the use of recombinant viruses that enable the delivery and expression of at least two nucleic acids encoding enzymes, proteins and / or cofactors involved in the transport of cholesterol.

Unexpectedly, Applicants have demonstrated that incorporating such nucleic acids into viruses in the form of non-cistron units makes it possible to effectively provide for the delivery and expression of at least two nucleic acids from the same recombinant virus. It is clear that the use of a single virus rather than two has various advantages in terms of treatment.

Above all, it is more advantageous to construct a single recombinant virus incorporating two genes than two respective recombinant viruses.

In addition, from a therapeutic point of view, the use of the claimed vector makes it possible to reduce the amount of recombinant virus required for gene expression by half. This is most particularly beneficial in terms of the immune response typically shown towards cells infected by recombinant viruses. This immune response usually causes the destruction of infected cells and / or a major inflammatory response. It is evident that these two signs are very detrimental to the duration of therapeutic gene expression and thus the level of anticipated therapeutic effect.

Finally, efficient delivery in two separate recombinant viruses at equal concentrations is an unclear event and therefore needs to be adjusted by further work. When using the recombinant virus according to the invention, this type of regulation may advantageously not be necessary.

Advantageously, the claimed virus can deliver and efficiently express two nucleic acids encoding proteins, enzymes and / or cofactors involved in the back transport of cholesterol for a long time without cytopathic effects.

Therefore, the first subject matter of the present invention is separated from each other by a sequence operably linked to a transcriptional promoter encoding a separate enzyme, protein and / or cofactor involved in the back transport of cholesterol and encoding an internal ribosome entry site IRES. In a defective recombinant virus comprising at least two nucleic acids.

The nucleic acid is preferably selected from one of lecithin cholesterol acyltransferase (LCAT), cholesterol ester transfer protein (CETP), liver lipase (HL), apolipoprotein AI and AIV or variants thereof.

The inserted nucleic acid can be a complementary DNA fragment (cDNA), genomic DNA (gDNA) or a hybrid construct consisting of, for example, one or more introns inserted cDNA. These may be synthetic or semisynthetic sequences. As noted above, the gene may encode all or part of one of the enzymes, proteins and / or cofactors or variants thereof involved in the back transport of cholesterol. For the purposes of the present invention, the term variant refers to a mutant, fragment or peptide having at least one biological property of the protein product under consideration and optionally each natural variant thereof.

These fragments and variants may be obtained by techniques known to those skilled in the art, in particular genetic and / or chemical and / or enzymatic modifications. Genetic modifications include oppression, deletions, mutations, and the like.

Inserted nucleic acids for the purposes of the present invention are preferably genes encoding all or part of human enzymes, proteins and / or cofactors. These are more preferably cDNA or gDNA.

Each inserted nucleic acid may also include an activation or regulatory sequence, and the like. It also generally includes a signal sequence upstream of the coding sequence that directs the synthesized polypeptide in the secretory pathway of the target cell. Such signal sequences may be natural signal sequences, but they may also be other functional signal sequences or artificial signal sequences.

According to certain embodiments of the invention, the claimed recombinant virus comprises at least one nucleic acid encoding LCAT. More preferably, the second inserted nucleic acid encodes HL, CETP or ApoAI.

As explicitly mentioned above, the coexpression of the two nucleic acids under consideration is temporarily ensured by the formation of a single RNA that is translated and then produces two respective enzymes. For this reason, recombinant viruses include at least one transcriptional promoter, polyadenylation site and IRES sequence in addition to two nucleic acid sequences.

This transcriptional promoter is operably linked to a coding nucleic acid to produce bicistronic mRNA and to induce the expression of two respective enzymes from the mRNA. According to a preferred embodiment of the invention, it is disposed directly upstream of the first nucleic acid.

Such transcriptional promoters may be selected from among those sequences that are essentially responsible for the expression of the nucleic acid, particularly with respect to their placement upstream, provided that these sequences may of course act on the infected cell. They may also be sequences of different origin (may be responsible for the expression of other proteins or synthesized). In particular, they may be eukaryotic or viral nucleic acid sequences or derived sequences that specifically or inductively stimulate or inhibit transcription of the gene. By way of example, they can be promoter sequences derived from the genome of the cells to be infected or from the viral genome, in particular promoters of the adenovirus MLP and E1A genes, CMV, RSV-LTR, MT-1 and SV40 promoters and the like. Among eukaryotic promoters, also ubiquitous promoters (HPRT, bimentin, α-actin, tubulin, etc.), promoters for intermediate filaments (desmin, neurofilament, keratin, GFAP, etc.), therapeutic genes (type MDR, CFTR, factor) VIII, etc.), tissue specific promoters (pyruvate kinases, borrowers, promoters for fatty acid-binding visceral proteins, promoters for α-actin of smooth muscle cells, liver specific promoters: ApoAI, ApoAII, human albumin, etc.) or stimulation Promoters that respond to (steroid hormone receptors, retinoic acid receptors, etc.) may be mentioned. In addition, these expression sequences can be modified by addition of activation and regulatory sequences and the like.

With regard to the IRES sequence, this is preferably derived from piconaviruses. More specifically, these piconavirus IRES sequences are derived from cerebral myocarditis virus or poliovirus. It is more preferably a fragment derived from the cardiomyocardial virus present in the vector pCITE-2a + from NOVAGEN.

For clarity, a construct defined by a transcriptional promoter, two nucleic acids, a polyadenylation site and an IRES sequence between two nucleic acids will be defined below by the name bicistron cassette.

The virus according to the invention is defective, ie, cannot spontaneously replicate in target cells. In general, the genome of a defective virus is used within the framework of the present invention and thus lacks the minimum sequence necessary for viral replication in infected cells. Such regions may be removed (completely or partially) nonfunctional or substituted by other sequences and in particular by the bicistron cassette as defined above. Preferably, the defective virus nevertheless preserves the sequence of the genome essential for the encapsidation of the viral particles.

The virus according to the invention can be derived from adenoviruses, adeno-associated viruses (AAV) or retroviruses. In a preferred embodiment, it is an adenovirus.

There are several adenovirus antigen types that vary somewhat in structure and properties. Among these antigenic types, type 2 or 5 human adenoviruses (Ad 2 or Ad 5) or adenoviruses of human origin (application WO94 / 26914) are preferably used within the framework of the invention. Among adenoviruses of animal origin that can be used within the framework of the present invention, dogs, cattle, mice (eg Mav1, Beard et al., Virology 75 (1990) 81), sheep, pigs, birds, or monkeys (eg SAV) origin may be mentioned. Preferably, the adenovirus of animal origin is canine adenovirus, more preferably CAV2 adenovirus [Manhattan or A26 / 61 strain (ATCC VR-800)]. Preferably, adenoviruses of human or dog or mixed origin are used within the framework of the present invention.

Preferably, in the adenovirus genome of the invention, at least the El region is made nonfunctional. Viral genes of interest can be made nonfunctional by techniques known to those of skill in the art, in particular by total repression, substitutions, partial deletions or addition of one or more bases into the gene of interest. Such modifications can be obtained in vitro (on isolated DNA) or in situ, for example by genetic engineering techniques or by treatment with a mutant. Other regions, in particular the E3 (WO95 / 02697), E2 (WO94 / 28938), E4 (WO94 / 28152, WO94 / 12649, WO95 / 02697) and L5 (WO95 / 02697) regions can also be modified. In a preferred embodiment, the adenovirus according to the invention comprises at least one deletion in the El region and a deletion in the E3 region. In the virus of the invention, the deletion of the El region is preferably extended from nucleotides 455 to 3329 on the Ad5 adenovirus sequence. In another preferred embodiment, the bicistronic cassette is inserted at the level of deletion in the El region.

Therefore, certain modalities of the present invention are at least separated from each other by sequences encoding an internal ribosome entry site IRES and operably linked to a transcriptional promoter that encodes separate enzymes, proteins and / or cofactors involved in the back transport of cholesterol. It relates to a defective recombinant adenovirus characterized in that it comprises two nucleic acids.

Preferred recombinant adenoviruses according to the invention may be mentioned, more particularly, defective recombinant adenoviruses comprising at least one nucleic acid encoding LCAT and one nucleic acid encoding HL, CETP or ApoAI, wherein the nucleic acid is transcribed. It is operably linked to a promoter and is separated from each other by a sequence encoding an internal ribosomal entry site IRES.

Representative examples of these adenoviruses are indicated in FIG. 2 as containing genes encoding LCAT and CETP, respectively, and mention may be made of homologous recombination between pXL2974 and pXL2822.

Defective recombinant adenoviruses according to the present invention can be prepared by techniques 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 produced, inter alia, by homologous recombination between the adenovirus carrying the bicistron cassette and the plasmid. Homologous recombination occurs after cotransfection of adenoviruses and plasmids into appropriate cell lines. The cell line used should preferably comprise sequences that are in an integrated form to avoid the risk of recombination, preferably (i) transformable by said element and complementary to the defective adenovirus genome portion. Examples of cell lines contain Ad5 adenovirus (12%) or the left region of the cell line's genome, which may, in particular, when integrated into the genome, may be complementary to E1 and E4 functions as detailed in applications WO 94/26914 and WO95 / 02697. One human embryonic kidney cell line 293 (Graham et al., J. Gen. Virol. 36 (1977) 59) may be mentioned. The propagated adenovirus is then recovered and purified by conventional molecular biology techniques, as described in the Examples.

According to a preferred embodiment of the present invention, however, the claimed adenovirus is a shuttle plasmid, which uses a prokaryotic plasmid comprising a recombinant adenovirus genome which is contacted by one or more restriction sites which are not present in the genome. It is prepared according to the novel process described in 25506. This protocol is described in particular in FIG. 2. This process is particularly advantageous because it allows the use of a second construct to provide another part of the viral genome, and the need for recombination steps in the trans complementary cell line.

The second subject of the invention relates precisely to certain prokaryotic plasmids used in the production of the claimed adenoviruses.

According to a preferred embodiment of the present invention, these prokaryotic plasmids incorporate the bicistron cassette described above.

More precisely, the subject of the present invention is also a prokaryotic plasmid comprising at least two nucleic acids encoding the adenovirus genome and the enzymes, proteins and / or cofactors that are distinct and involved in the reverse transport of cholesterol, both of which are directed to the transcriptional promoter. It is operably linked and separated from each other by a sequence encoding the internal ribosomal entry site, IRES.

Preferably, the prokaryotic plasmid according to the invention comprises a first region which allows for replication in prokaryotic cells and a second region which is not present in the genome but which is contacted by one or more restriction sites and which is contacted by There are at least two nucleic acids that are distinct and that encode enzymes, proteins and / or cofactors involved in the reverse transport of cholesterol, which are operably linked to transcriptional promoters and linked to each other by sequences encoding the internal ribosome entry site, IRES. Are separated.

In relation to the definition of what affects the composition of enzymes, proteins and / or cofactors, transcriptional promoters, polyadenylation sites, IRES sequences and cassettes that are distinct and involved in the reverse transport of cholesterol, reference may be made to those described above.

As used in the claimed plasmids, the region allowing replication in prokaryotic cells may be of origin of replication functioning in the selected cell. This may be the origin of replication derived from plasmids of P incompatible groups (eg = pRK290) that allow replication in E. coli poly A strains. More generally, it may be of origin of replication derived from plasmids replicating in prokaryotic cells. This plasmid may be a RK2 derivative, a pBR322 derivative (Bolivar et al., 1977), a pUC derivative (Viera and Messing, 1982) or a derivative of the same incompatible group, ie other plasmids derived from eg ColE1 or pMB1. These plasmids can also be selected from other incompatibility groups that replicate in this coli. These are for example incompatible groups A, B, FI, FII, FIII, FIV, H1, H11, I1, I2, J, K, L, N, OF, P, Q, T, U, W, X, Y It may be a plasmid derived from a plasmid belonging to, Z or 9. Other plasmids can also be used, such as B. subtilis, Streptomyces, P. putida, P. aeruginosa, Pseudomonas melilotti (Rhizobium meliloti), Agrobacterium tumefaciens, Staphylococcus aureus, Streptomyces pristinaespiralis, Enterococcus fascium facium It does not replicate in E. coli except for other hosts such as Clostridium. Preferably, origins of replication derived from plasmids replicating in E. coli are used.

As noted above, the adenovirus genomes present in the plasmids of the present invention advantageously do not require the provision of other regions by recombination or ligation during the generation of the viral stock in the complete or functional genome, ie the selected capsid.

Preferably, the recombinant adenovirus genome comprises at least an ITR sequence or a sequence that allows for encapsidation. In a preferred embodiment of the invention, such adenovirus genomes used lack all or part of the El region. Advantageously, the adenovirus genome used lacks a portion of the El region between nucleotides 454 to 3328 (PvuII-BglII fragments) or 382 to 3446 (HinfII-Sau3A fragments).

In a particularly advantageous embodiment, the adenovirus genome used also lacks all or part of the E3 and / or E4 region. According to certain embodiments of the invention, it is an adenovirus genome lacking the El and E3 regions.

More precisely, the claimed prokaryotic plasmids are directed to the first site of the adenovirus genome, including the viral ITR and ψ sequences, the transcriptional promoter, cholesterol, in the 5′-3 ′ direction. A first nucleic acid encoding an enzyme, protein and / or cofactor involved in back transport, an IRES sequence, a second nucleic acid, a polyadenylation site and a pIX-IVA2 region that encodes a second enzyme, protein and / or cofactor involved in reverse transport of cholesterol And a second site of the adenovirus genome comprising a.

Advantageously, the prokaryotic plasmids claimed in accordance with the invention also comprise a region which allows for the selection of prokaryotic cells containing the plasmid. This region may in particular be composed of genes that confer resistance to products, in particular antibiotics. Thus, for example, kanamycin resistance (Kan ^r ), ampicillin resistance (Amp ^r ), tetracycline resistance (tet ^r ) or spectinomycin resistance commonly used in molecular biology (Maniatis et al., 1989) Referenceing nucleic acid sequences may be mentioned. The selection of plasmids can be performed using nucleic acid sequences excluding genes encoding antibiotic resistance markers. In general, this involves genes that provide the bacterium with a function that it no longer has (also it may correspond to a gene that is deleted or does not function on the chromosome), and the gene on the plasmid restores this function. For example, it may be a gene for transcriptional RNA that restores defective chromosomal function (Somoes et al., 1991).

According to a preferred embodiment, the claimed prokaryotic plasmid comprises at least one nucleic acid encoding LCAT, wherein the second nucleic acid is selected from those encoding CETP, HL or ApoAI.

As representative examples of these prokaryotic plasmids, mention may be made in particular of the plasmids pXL2974 and pXL3058 indicated in FIGS. 4 and 6, respectively. Plasmid pXL2974, in the 5'-3 'direction, has two transgenes LCAT and CETP isolated by origin of replication, spectinomycin resistance gene, Sac B gene for sucrose sensitivity, viral ITR and ψ sequence, RSV promoter, IRES , Polyadenylation sites and viral sequences pIX and IVA2. Plasmid pXL3058, in the 5'-3 'direction, originated from replication, kanamycin resistance gene, viral ITR and ψ sequence, RSV promoter, two transgene LCATs isolated by IRES and intron + ApoAI, polyadenylation site, viral sequence pXI And the Sac B gene for IVA2 and sucrose sensitivity.

The present invention also encompasses bicistron cassettes defined in accordance with the present invention and extended to plasmid constructs used in the construction of these prokaryotic plasmids.

The prokaryotic plasmids claimed according to the invention in particular comprise a bicistronic cassette defined according to the invention, ie operably on two nucleic acids which distinguish two enzymes, proteins and / or cofactors which are involved in the transport of cholesterol. It can be obtained by the transformation of the initial shuttle plasmid containing the sequence encoding the transcriptional promoter linked to, the polyadenylation site, the IRES sequence located between the nucleic acid.

As representative examples of these shuttle plasmids, mention may be made in particular of the plasmids pXL2970 and pXL2984 described in FIGS. 3 and 5, respectively.

In defining enzymes, transcriptional promoters and IRES sequences and those involved in the cassettes involved in the reverse transport of cholesterol, reference may be made to what is described above.

Another subject of the present application relates to prokaryotic cells containing a prokaryotic plasmid as defined above. This may in particular be a bacterium which is a vector system into which recombinant DNA can be introduced. For example, cells of the genus E. coli, Salmonella typhimurium, Bacillus subtilis, Pseudomonas putida, Pseudomonas aeruginosa, Agrobacterium tumefaciens, Raizobium melelloti or Streptomyces May be mentioned. These cells are advantageously obtained by transformation by techniques known to those skilled in the art.

It is also defined above as a recombinant virus, defective recombinant virus or shuttle plasmid construct for the preparation of a pharmaceutical composition for the treatment or prevention of pathologies associated with hypoalphalipoproteinemia, more particularly including atherosclerosis and / or restenosis. It relates to the use of.

The invention also relates to a pharmaceutical composition comprising one or more deleted recombinant viruses comprising the adenovirus described above. Such compositions can be formulated for administration by topical, oral, parenteral, intranasal, endovascular, intramuscular, subcutaneous or intraocular routes.

Preferably, the composition according to the invention contains a pharmaceutically acceptable vehicle for injection formulation. They are particularly isotonic in sterile saline (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride, etc., or mixtures of these salts), or dry to allow the reconstitution of the injection solution upon addition of sterile water or, optionally, physiological saline. , In particular freeze-drying compositions.

The viral dosage used for injection can be adjusted according to various parameters, in particular the dosage form used, the pathology involved or the desired duration of treatment. In general, the recombinant virus according to the invention is formulated and infused in a dosage form of 10 ⁴ to 10 ¹⁴ pfu / ml. The term pfu (″ plaque forming unit ″) corresponds to the infectivity of a virion suspension and is determined by measuring the number of infected cell plaques, usually 48 hours after infection with an appropriate cell culture. Techniques for measuring the pfu titer of viral solutions are well provided in the literature.

The present invention provides a new, highly effective method for the treatment or prevention of pathologies associated with hypoalphalipoproteinemia, especially in the field of cardiovascular disease such as myocardial infarction, laryngitis, sudden death, cardiac breathing, cerebrovascular accident, atherosclerosis or restenosis. to provide.

In addition, this treatment can be applied to both humans and animals such as sheep, cattle, livestock (dogs, cats, etc.), horses, fish, and the like.

The invention is described in more detail by the following examples which are considered to be illustrative and non-limiting.

Example 1

Construction of shuttle plasmid LCAT-IRES-CETP

1) construction of the usual shuttle vector pXL2970

The expression vector pXL2968 RSV LCAT polyA bGH was obtained by cleaving the plasmid pXL2616 containing LCAT cNDA, whereas the plasmid pXL LPL under the control of the RSV promoter and upstream of the bovine growth hormone polyadenylation signal was restricted to Sal and Obtained by cleavage with ClaI and the resulting fragments linked by T4 DNA ligase.

for teeth; Buffer 4 (20 mM tris acetate, 10 mM Mg-acetate, 50 mM K-acetate and 1 mM DTT) supplemented with 100 μg / ml of acetylated BSA, each of plasmids pXL RSV LPL (2 μg) and pXL2616 (2 μg) It decompose | disassembles in ClaI10 unit (u) for 90 minutes at 37 degreeC. 100 mM NaCl and 10 u of SalI are added and the reaction mixture is incubated for another 90 minutes at 37 ° C. After electrophoretic migration on 0.7% agarose gel of the degradation product, 6.5 and 1.7 Kb bands corresponding to pXLRSV and LCAT cDNA are cut from the gel and extracted with Qiaquick kit. Both bands are incubated overnight at 14 ° C. and then linked with 400 u of T4 DNA ligase.

Recombinant plasmid pXL2969 IRES-CETP was generated from a plasmid bluescript containing modified CETP cDNA at the 5 ′ end by introducing 5 ′ GCCTGATAAC CATGGTGGCT GCCACAG 3 ′ (SEQ ID NO: 1) into the NcoI site using PCR. The modified plasmid is cleaved with NcoI and Sal and cloned downstream of the adenovirus IRES sequence contained in the plasmid pSKIRES cleaved by the same restriction enzyme in the following manner.

2.5 μg of plasmid CETP and 2.5 μg of plasmid pSKIRES were digested with 10 u of NcoI for 90 min at 37 ° C. in Buffer 3 (50 mM Tris-HCl, 10 mM MgCl ₂ , 100 mM NaCl and 1 mM DTT), followed by 90 at 37 ° C. Decompose with SalI 10 u for min. The digested product was electrophoresed and the bands of 1,5 Kbp (CETP cNDA) and 3.5 Kbp were extracted and linked under the same conditions as above.

To obtain plasmid pXL2970, the procedure is carried out as follows:

4 μg of plasmid pXL2968 is linearized with SalI 10 u (buffer 3 + BSA, 90 min at 37 ° C.). DNA is then extracted using Qiaquick kit and recovered in 50 μl of water preheated to 50 ° C. The sticky ends resulting from SalI cleavage are smoothed by incubation with 5 u of Klenow polymerase at 25 ° C. for 15 minutes. Re-extract DNA with Qiaquick kit and dephosphorylate with 2 u of calf intestinal phosphatase (CIP) for 60 minutes at 37 ° C. Plasmid pXL2969 (4 μg) was first digested with SmaI (buffer 4, 90 min at 25 ° C.) followed by HincII (buffer 3 + BSA, 100 mM NaCl, 90 min at 37 ° C.). The degradation products of both plasmids were electrophoresed on a 0.7% agarose gel and DNA bands of 8.5 Kbp (pXL2968) and 2.2 Kbp (IRES CETP cDNA) were extracted using the Qiaquick kit and then linked to 400 u of T4 DNA ligase ( 3).

LCAT and CETP activity of plasmid pXL2970 is analyzed in 293 cell cultures three days after transfection.

The LCAT activity of this plasmid corresponds to 3.5% of the cholesterol esters formed per hour and the CETP activity corresponds to 120% (Table 1). This activity shows that plasmid pXL2970 synthesizes LCAT and CETP, which are actually catalytically active.

2) E. coli shuttle plasmid LCAT-IRES-CETP

In this technique, the shuttle vector is

The sequence ITR-reverse terminal repeat and ψcapsidation sequence required for homologous recombination in E. coli, surrounding the -LCAT-IRES-CETP sequence,

Col E1 replication origin, which makes the plasmid non-replicating in E. coli C2110 strain and allows cloning ability of the recombinant plasmid,

Suicide gene sucrose B (fatal to bacteria in sucrose in medium) and spectinomycin resistance gene that allows selection of recombinant clones.

To this end, the noncistronic shuttle plasmid pXL2970 RSV LCAT-IRES-CETP is cleaved with BstEII and SpeI to introduce the ITR and ψ sequences of the adenovirus genome to obtain a coli shuttle plasmid. ITR and ψ sequences are isolated by digesting plasmid pXL2794 with BstEII and XbaI. Smooth-terminal fragments containing the spectinomycin sucrose B cassette obtained by digesting pXL2757 with EcoRV and SmaI were introduced into shuttle plasmid pXL2970 + 2794 linearized with bFspI (FIG. 4).

Experimentally, the procedure is performed according to the following protocol;

3 μg of plasmid pXL2970 was digested with 20 u of BstEII (buffer 2: 10 mM Tris HCl, 10 mM MgCl ₂ , 50 mM NaCl, 1 mM DTT; 90 min at 60 ° C.) followed by 10 u of SpeI (90 min at 37 ° C.) Let's do it.

The resulting DNA fragments are extracted with Qiaquick kit and dephosphorylated with 2 u of CIP (60 min at 37 ° C.). Plasmid pXL2794 is digested with BstEII 20 u (buffer 2, 90 min at 60 ° C.) followed by 20 u of XbaI (90 min at 37 ° C.). The bands of 6.5 Kbp (pXL2970) and 2.9 Kbp (ITR ψ and Kan ^r of pXL2794) extracted after electrophoretic transfer were connected using T4 DNA ligase (40 Ou).

The resulting plasmid pXL2970 + 2794 (1.5 μg) was digested with FspI (buffer 4, 60 min at 37 ° C.) and extracted using a Qiaquick kit, then plasmid pXL2757 10 u (buffer 4, 90 min at 25 ° C.), This was followed by ligation into EcoRV 10 u (90 min at 37 ° C.) and linked to 3.8 Kbp DNA fragment extracted from the gel containing the sacB-spect ^r cassette (T4 DNA ligase, 400 u).

Shuttle plasmid pXL2974 LCAT-IRES-CETP is first screened on spectinomycin medium and spectinomycin + sucrose medium. NcoI (6.2 + 3 + 2.2 + 2), NotI (13.5 Kbp) and EcoRV (1 + 3 + 9.5 Kbp) digestion results are consistent with the construction map of the plasmid.

The LCAT activity of plasmid pXL2974 corresponds to 2% of the cholesterol esters formed per hour and that of CETP corresponds to 114% (FIG. 1). LCAT and CETP activity was found at the level of E. coli shuttle plasmid LCAT-IRES-CETP.

LCAT active CETP activity Shuttle Plasmid LCAT-IRES-CETP 3.5 +/- 0.2% 120 +/- 2% E. coli shuttle plasmid LCAT-IRES-CETP 2 +/- 0.2% 114 +/- 2%

Example 2

Construction of shuttle plasmid RSV LCAT-IRES-HL

The HL cDNA is cloned after IRES in Vector BlueScript and the IRES-HL fragments are included in a similar manner to the vector LCAT-IRES-CETP according to the following protocol.

Plasmid pXL2971 (4 μg) was digested with BglII 40 I.U. (buffer 3, 90 min at 37 ° C.) followed by SalI 40 u at 37 ° C. for 90 min to remove the NcoI site. 2.5 Kbp DNA fragments (approximately 0.5 μg mass) derived from this digestion are digested with NcoI (0.1-1 u NcoI / DNA μg) for 60 min at 37 ° C. in Buffer 4 after migration and extraction on the gel. The degradation product was analyzed on a 0.7% agarose gel and the 1.5 Kbp band containing HL cDNA ABC, obtained with 0.5 u of NcoI, was obtained by pSK IRES resulting from NcoI and SalI digestion (1 u of each enzyme, buffer 3, 37 ° C.). Connect to fragment (1.3 μg) (T4 DNA ligase, 400 u).

The final vector is shown in FIG. The corresponding noncistronic shuttle plasmid LCAT-IRES-HL is named pXL2984.

LCAT active HL active Shuttle Plasmid LCAT-IRES-HL 1.2 +/- 0.2% 46 +/- 2%

Example 3

Construction of shuttle plasmid ApoAI-IRES-LCAT

General principles for this construction are the same as described above. LCAT is mutated by PCR, including an additional NcoI site that allows ligation to the right after IRES. Fully review its sequence. The IRES-LCAT fragment is then linked behind ApoAI. The resulting vector is derived directly from the coli technique and named pXL3058 (FIG. 6). After conventional double recombination, the resulting viral vector is tested for activity. ApoAI was detected by western blotting and LCAT activity was estimated at 1.3% (background noise at 0.2%).

Sequence list

(1) General Information:

(i) Applicant:

(A) Name: Long franc Laura Societe Anonym

(B) Street: Avenue Lamont Along 20

(C) City: Anthony

(D) Country: France

(E) Postal Code: 92165

(F) Phone Number: 01.55.71.69.22

(G) Fax: 01.55.71.72.96

(ii) Name of the Invention: Non-cistronic recombinant virus useful for the treatment of pathologies associated with dyslipoproteinemia

(iii) Sequence number: 1

(iv) computer readable form:

(A) Medium type: Tape

(B) Computer: IBM PC compatible model

(C) operating system: PC-DOS / MS-DOS

(D) Software: PatentIn Release # 1.0, Version # 1.30 (EPO)

(2) Information about sequence number 1:

(i) Sequence Features:

(A) Length: 27 base pairs

(B) Type: nucleotide

(C) Topology: linear

(ii) Molecular Type: cDNA

(ix) SEQ ID NO: SEQ ID NO 1:

GCCTGATAAC CATGGTGGCT GCCACAG

Claims (35)

At least two nucleic acids that are unique and that encode enzymes, proteins and / or cofactors involved in the reverse transport of cholesterol, are operably linked to transcriptional promoters, and are separated from each other by sequences encoding internal ribosome entry site IRES. Deletion recombinant virus. The method of claim 1, wherein the nucleic acid inserted is preferably all of one of the following: lecithin cholesterol acyltransferase (LCAT), cholesterol ester transfer protein (CETP), liver lipase (HL), apolipoprotein AI and AIV or variants thereof. Or a defective recombinant virus, characterized in that it is selected from among the genes encoding a portion. 3. The defective recombinant virus of claim 1 or 2, wherein the nucleic acid is a gene encoding all or part of the corresponding human enzyme, protein and / or cofactor. 4. The defective recombinant virus of any one of claims 1 to 3, wherein the nucleic acid is preferably cDNA or gDNA. 5. The defective recombinant virus of any one of claims 1 to 4, wherein one of the nucleic acids encodes LCAT. The defective recombinant virus of claim 5, wherein the second nucleic acid encodes CETP, HL or ApoAI. The defective recombinant virus according to any one of claims 1 to 6, wherein the transcriptional promoter is preferably selected from among promoters of adenovirus MLP and E1A nucleic acid sequences and CMV, RSV-LTR, MT-1 and SV40 promoters. . 8. A defective recombinant virus according to any one of claims 1 to 7, wherein the IRES sequence is derived from piconavirus. 9. The defective recombinant virus of claim 8, wherein the piconavirus IRES sequence is derived from a brain myocarditis virus or poliovirus. 10. The defective recombinant virus according to any one of claims 1 to 9, characterized by a lack of at least genomic regions essential for replication in infected cells. The defective recombinant virus according to any one of claims 1 to 10, characterized in that it is preferably an adenovirus of human Ad5 or Ad2 type or animal origin. At least two nucleic acids that are unique and that encode enzymes, proteins and / or cofactors involved in the reverse transport of cholesterol, are operably linked to transcriptional promoters, and are separated from each other by sequences encoding internal ribosome entry site IRES. Defective recombinant adenovirus. 13. The method of claim 12, comprising at least one gene encoding LCAT and one gene encoding HL, wherein said genes are operably linked to a transcriptional promoter and separated from each other by a sequence encoding an internal ribosome entry site IRES. Defective recombinant adenovirus, characterized in that. 13. The method of claim 12, comprising at least one gene encoding LCAT and one gene encoding apoA-I, which genes are operably linked to a transcriptional promoter and by a sequence encoding an internal ribosomal influx site IRES. A defective recombinant adenovirus, characterized in that they are separated from each other. 13. The method of claim 12, comprising at least one gene encoding LCAT and one gene encoding CETP, which genes are operably linked to a transcriptional promoter and separated from each other by a sequence encoding an internal ribosomal influx site IRES. Defective recombinant adenovirus, characterized in that. 16. The defective recombinant adenovirus of claim 15, which is induced by homologous recombination between pXL2974 and pXL2822. 17. The defective recombinant adenovirus of any one of claims 12-16, comprising at least one deletion in the E1 region and one deletion in the E3 region. At least two nucleic acids encoded by the adenovirus genome and sequences that are unique and involved in the reverse transport of cholesterol, are operably linked to a transcriptional promoter, and are separated from each other by an encoding internal ribosomal entry site IRES. Prokaryotic plasmid, characterized in that it comprises a. Bound by one or more restriction sites not present in the genome and the first region that allows replication in prokaryotic cells, encoding enzymes, proteins and / or cofactors that are unique and involved in the reverse transport of cholesterol, and are operative for transcriptional promoters A prokaryotic plasmid consisting of a second region comprising an adenovirus genome, wherein the adenovirus genome is joined to and separated from each other by a sequence encoding an internal ribosome entry site IRES. 20. The prokaryotic plasmid according to claim 18 or 19, wherein the adenovirus genome is deleted in the El and E3 regions. 21. The method of any of claims 18-20. Prokaryotic plasmids comprising viral ITR and ψ sequences. The prokaryotic plasmid according to any one of claims 18 to 21, wherein the origin of replication is derived from a bacterial plasmid selected from RK2, pBR322 and pUC. 5′-3 ′ orientation, the first part of the adenovirus genome comprising viral ITR and ψ sequences functional in prokaryotic cells, enzymes, proteins and / or involved in the back transport of cholesterol A second portion of the adenovirus genome comprising a first nucleic acid encoding a cofactor, an IRES sequence, an enzyme involved in the transport of cholesterol, a second nucleic acid encoding a protein and / or a cofactor, a polyadenylation sequence and a pIX-IVa2 region Prokaryotic plasmid comprising a. The prokaryotic plasmid according to any one of claims 18 to 23, further comprising a prokaryotic selection region containing the plasmid. 25. The prokaryotic plasmid according to any one of claims 18 to 24, wherein at least one of the nucleic acids encodes LCAT and the second nucleic acid is selected from those encoding HL, ApoAI or CETP. 26. The prokaryotic plasmid of claim 25, which is pXL2974 containing nucleic acids encoding LCAT and CETP, respectively. 26. The prokaryotic plasmid of claim 25, which is pXL3058 containing nucleic acids encoding LCAT and ApoAI, respectively. Includes a sequence encoding two nucleic acids, a polyadenylation site, and an internal ribosomal entry site IRES located between the two nucleic acids that are unique and code for enzymes, proteins and / or cofactors involved in the reverse transport of cholesterol, and which are operably linked to transcriptional promoters Shuttle plasmid characterized in that. 29. The shuttle plasmid of claim 28 which is plasmid pXL2984 comprising two nucleic acids encoding HL and LCAT, respectively. 29. The shuttle plasmid of claim 28 which is plasmid pXL2970 comprising two nucleic acids encoding LCAT and CETP, respectively. A prokaryotic cell transformed with the prokaryotic plasmid according to any one of claims 18 to 27. 19. A defective recombinant virus according to any one of claims 1 to 11, a recombinant adenovirus according to any one of claims 12 to 17 for the preparation of a pharmaceutical composition for the treatment or prevention of pathologies associated with hypoalphalipoproteinemia or Use of a plasmid according to any of claims 28 to 30. 33. The use of claim 32 for the manufacture of a pharmaceutical composition for treating atherosclerosis and / or restenosis. A pharmaceutical comprising at least one defective recombinant virus according to any one of claims 1 to 11, an adenovirus according to any one of claims 12 to 17 or a plasmid according to any one of claims 28 to 30. Composition. 35. The pharmaceutical composition of claim 34, which is provided in the form of an injection and comprises adenovirus 10 ⁴ to 10 ¹⁴ pfu / ml.