SK63499A3 - Recombinant bicistron adenovirus for treating pathological conditions linked with dyslipoproteinemia - Google Patents

Abstract

The invention concerns a defective recombinant virus and preferably an adenovirus characterised 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, said 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. The invention further concerns plasmid constructs useful for preparing these adenovirus, and cells transformed by these plasmids or adenovirus and pharmaceutical compositions containing said adenovirus.

Description

Technical field

The present invention relates to novel recombinant viruses, their preparation and their use in gene therapy, for the in vivo transfer and expression of genes of interest. More specifically, it relates to recombinant viruses comprising at least two inserted genes whose expression products are involved in reverse cholesterol transport. It also relates to shuttle plasmids useful for the production of the adenoviruses of the invention. More particularly, the present invention relates to defective recombinant adenoviruses and their use for the prevention or treatment of pathological conditions associated with dyslipoproteinemia, which are known for their severe consequences at cardiovascular and neurological levels.

BACKGROUND OF THE INVENTION

Dyslipoproteinemia is a lipoprotein metabolism disorder that is responsible for the transport of lipids such as cholesterol and triglycerides in blood and peripheral fluids. They result in serious pathological conditions associated with hypercholesterolaemia or hypertriglyceridemia, such as in particular atherosclerosis.

Atherosclerosis is a polygenic complex disease, defined histologically by deposits (lipid or fibrolipid plaques) of lipids and other blood derivatives in the wall of large arteries (aorta, coronary artery, carotid arteries).

These plaques, which are to a greater or lesser extent calculated according to disease progression, may be associated with lesions and are associated with accumulation of fat deposits in the arteries, which consist essentially of cholesterol esters. These plaques are accompanied by thickening of the arterial wall with hypertrophy of smooth muscle, appearance of foam cells and accumulation of fibrous tissue. The atheromatous plaque is placed clearly in the wall relief, which is responsible for the narrowing of the vascular occlusions caused by atheroma, thrombosis or embolism that occur in the most affected patients.

Thus, hypercholesterolemia can result in very serious cardiovascular conditions such as heart attack, sudden death, heart failure, cerebrovascular diseases, and the like.

It is therefore of the utmost importance to provide an available treatment that allows, in certain pathological situations, to lower plasma cholesterol levels or even stimulate cholesterol efflux (reverse cholesterol transport) in peripheral tissues to empty cells that have accumulated cholesterol, all associated with atheromatous plaque formation. . Cholesterol is transmitted in the blood by various lipoproteins, including low density lipoproteins (LDLs) and high density lipoproteins (HDLs) .LDLs are synthesized in the liver and allow cholesterol to be supplied to peripheral tissues. cholesterol in peripheral

tissues and carry it degrades. to the liver, where imposes and / or Among the most common dyslipemia belong to those that are characterized by high levels LDL Low density

lipoprotein) cholesterol and hypoalphalipoproteinemia. It is characterized by a high density liprotein (HDL) cholesterol level of less than 0.35 mg / l (35 mg / dl) and represents 40% of dyslipemia cases (Genest et al., 1992). Hypoalphalipoproteinaemia is likely to be associated with a genetic deficiency in one or more proteins involved in the synthesis, maturation and breakdown of HDL particles, and often results in an early occurrence of cardiovascular disease (Dammerman et al., 1995). In general, there is an opposite correlation between the incidence of these diseases and HDL particle levels (Miller et al., 1987).

The protective effect of HDL against cardiovascular disease has been demonstrated by gene transfer experiments in vivo in strains of mice likely to develop atherosclerotic lesions in which an increase in the amount of HDL particles inhibits the development of these lesions (Rubin et al., 1991; Plump et al., 1994). It is contemplated that the protective effect of HDL particles may be due to their role in reverse cholesterol transport (Reiche et al., 1989). Reverse transport is the process by which excess cholesterol is transferred from peripheral tissues to the liver and removed there (Figure 1). It consists of a series of steps involving the uptake and esterification of cholesterol into HDL particles as well as its transfer to low-density lipoprotein particles, which are then taken up again by the liver, and this process also involves the participation of a number of proteins such as CETP, enzymes including cholesterolacyltransferase and liver lipase and / or ¹ cofactors thereof, such as apolipoproteins A1 and AIV).

While there is an effective treatment based on hypolipidemic and antihypertensive drugs for lowering LDL cholesterol and triglyceride levels, the treatment of hypoalphalipoproteinemia is currently of limited effectiveness.

In particular, the present invention relates to the treatment of pathological conditions associated with hypoalphalipoproteinemia, in particular gene therapy.

The therapeutic approach proposed in the present invention aims at increasing the kinetics of reverse cholesterol transport to induce regression of atherosclerotic lesions or alternatively prevent their formation. This object is preferably achieved by the simultaneous and effective expression of at least two reverse transport proteins to be treated.

enzymes or cofactors of choelsterol in cells involved in having one of its protein, enzyme and / or behind involved in reverse transport any disorder of its cellular level

For the invention, cofactors are considered to be cholesterol if the concentration automatically affects the reverse cholesterol process.

Representatives of such enzymes include, in particular, lecithincholesterolacyltransferase and liver lipase.

Lecithincholesterolacyltransferase (LCAT) is a 67 kD large glycoprotein, synthesized in the liver, which catalyzes the transfer of the acyl group from lecithin to cholesterol, thereby forming lysophosphatidylcholine and cholesterol esters (Glomset, 1968). The cofactor for this enzyme is apolipoprotein A1, which is bound to the surface of HDL particles. By maintaining a cholesterol concentration gradient between peripheral cells and HDL, it plays an essential role in the first stage of reverse cholesterol transport.

Human liver lipase (HL) is a 66 kD glycoprotein with triglyceride hydrolase and phospholipase activity that is synthesized and secreted by hepatocytes. Bound to the surface of hepatic cells, it is involved in the metabolism of intermediate density lipoproteins (HDL) and HDL by the proteoglycans of heparin sulfate. HL has a pronounced affinity for large HDL particles whose phospholipids and triglycerides cleave and thereby form discoid HDL particles that have the property of taking up cellular cholesterol. HL deficiency in humans is characterized by a large number of triglyceride-rich LDLs, large HDL particles, and early development of atherosclerosis (Hegele et al., 1993).

As for proteins, most preferred are cholesterol ester transfer protein (CETP), apolipoproteins A1 and IV or one of their variants.

Choelsterol ester transfer protein (CETP) is a 74 kD glycoprotein synthesized in adipose tissue and liver. In plasma, it is mainly associated with HDL. Here, it catalyzes the transfer of cholesterol esters from HDL to low density lipoproteins. This transfer is followed by the transition of triglycerides from low density lipoproteins to HDL. Overexpression of CETP in transgenic hypertriglyceridemic mice inhibits the development of atherosclerotic lesions (Hayek et al., 1995). This experiment is consistent with the observation that CETP-deficient individuals soon develop cardiovascular diseases (Zhong et al., 1996).

Apolipoprotein AI is a 243 amino acid protein that is synthesized in the form of a 267 residue pre-propeptide, having a molecular weight of 28,000 D. In man, it is synthesized specifically in the liver and intestine and forms the parent protein of HDL particles (70% of their protein weight) ). It is rich in plasma (1.0 - 1.2 g / l). Its best biochemical activity characterized is the activation of lecithin cholesterol transferase (LCAT), but is attributed to many other activities, namely stimulation of cell cholesterol efflux. Apolipoprotein A1 has a major role in resistance to atherosclerosis, associated with reverse cholesterol transport. Its 1863 bp gene was cloned and sequenced (Sharpe et al., Nucleic Acids Res., 12 (9), 3917, 1984). Among the protein products having apoliporpotein A1 type activity, particular natural variants described in the prior art can be mentioned.

Apolipoprotein AIV (apoAIV) is a 376 amino acid protein that is specifically synthesized in the intestine as a precursor with 396 residues. Regarding its physiological activity, it is known that it can activate in vitro lecithin cholesterol transyltransferase (LCAT) (Steinmetz et al., J. Biol. Chem., 260: 2258-2264, 1985) and that it can, like apolipoprotein AI, interfere with by binding HDL particles to bovine aortic endothelial cells (Savion et al., Eur. J. Biochem. 257: 4171-4178, 1987). These two activities suggest that apoAIV is very likely to act as a mediator of reverse choelsterol transport. The apoAIV gene has been cloned and described in the prior art (see in particular WO92 / 05253). Among the protein products having apolipoprotein AIV type activity, particular fragments and derivatives described in patent application FR 92 00806 may be mentioned.

SUMMARY OF THE INVENTION

More precisely, the present invention is based on the use of recombinant viruses which allow the transfer and expression of at least two nucleic acids encoding enzymes, proteins and / or cofactors involved in cholesterol reversal transport.

The Applicant has unexpectedly demonstrated that it is possible to efficiently assure the transfer and expression of at least two nucleic acids by the same recombinant virus by integrating these nucleic acids into said virus in the form of a bicistronic unit. It is clear that using one virus instead of two has many benefits in terms of treatment.

In particular, it is preferable to construct one recombinant virus by incorporating two genes than the two respective recombinant viruses.

Similarly, from a therapeutic point of view, the use of a vector as claimed in the claims makes it possible to halve the amount of recombinant virus necessary for the expression of said genes. This is most beneficial in particular in terms of the immune response usually manifested against cells infected with recombinant viruses. This immune response results in the normal destruction of infected cells and / or a severe inflammatory response. It is clear that these two manifestations are very detrimental at the level of expression of the therapeutic genes and hence the expected therapeutic effect.

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Finally, the efficient transfer and balanced concentration of two different recombinant viruses is an uncertain phenomenon and must therefore be controlled by other means. In the case of using the recombinant virus according to the invention, we can advantageously dispense with this type of control.

The viruses of the present invention are preferably capable of transferring and expressing efficiently long-term and without any cytopathological action, two nucleic acids encoding proteins, enzymes and / or cofactors involved in reverse cholesterol transport.

Therefore, a first object of the invention is a defective recombinant virus comprising at least two nucleic acids encoding enzymes, proteins and / or cofactors that are different and involved in reverse cholesterol transport, said nucleic acids being operably linked to a transcriptional promoter and separated from one another by an internal coding sequence ribosome entry site (IRES).

Preferably, the nucleic acids are selected from genes encoding all or part of lecithinchoelsterolacyltransferase (LCTA), cholesterol Esters Transfering protein (CETP), liver lipase (HP), apolipoproteins AI and AIV, or one of their variants.

The inserted nucleic acids may be complementary DNA (cDNA) fragments, genomic DNA (gDNA) or hybrid constructs consisting, for example, of a cDNA into which one or more introns have been inserted. They may be synthetic or semi-synthetic sequences. As mentioned above, these sequences may be a gene encoding all or part of one of the enzymes, proteins and / or cofactors involved in reverse cholesterol transport, or variants thereof. For the invention, the term variant refers to each mutant, fragment, or peptide having at least one biological property of the protein product under consideration and also, where appropriate, their respective natural variants.

These fragments and variants can be obtained by any technique known to the person skilled in the art and in particular by genetic and / or chemical and / or enzyme modifications. Gentle modifications include suppressions, deletions, mutations, and the like.

The inserted nucleic acids for the purposes of the invention are preferably genes encoding all or parts of the corresponding human enzymes, proteins and / or cofactors. More preferably, they are cDNA or gDNA.

Each inserted nucleic acid may also contain activation or regulatory sequences or the like. In addition, it generally comprises upstream of the coding sequence a signal sequence of a control polypeptide synthesized in the secretory pathways of the target cell. This signal sequence may be its natural signal sequence, but it may also be any other functional signal sequence or an artificial signal sequence.

According to a specific embodiment of the invention, the recombinant viruses according to the invention at least one acid encoding LLCAT. More preferably, the second insertion comprises a nucleic nucleic acid encoding HL, CETP or ApoAI.

As clearly stated above, the co-expression of the two nucleic acids of interest is pre-ensured by the generation of one RNA, which is then translated to give the two respective enzymes. For these reasons, the recombinant virus comprises, in addition to the two nucleic acid sequences, at least one transcriptional promoter, a polyadenylation site, and an IRES sequence.

The transcriptional promoter is operably linked to the coding nucleic acids to form a bicistronic mRNA and allows expression of two respective enzymes from said mRNA.

According to a preferred embodiment of the invention, it is located directly upstream of the first nucleic acid.

This transcriptional promoter may be selected, in particular, from sequences that are naturally responsible for the expression of said nucleic acids, in relation to which it is located upstream, but provided that the sequences are capable of functioning in the infected cell. They may also be sequences of different origins (responsible for the expression of other proteins, or even synthetic). Specifically, they may be sequences from eukaryotic or viral nucleic acids or derived sequences that stimulate or inhibit gene transcription in a specific and inducible manner or otherwise. By way of example, these may be promoter sequences derived from the genome of the cell to be infected or from the genome of the virus, in particular the promoters of the MLP and E1A adenoviral genes, the CMV promoter, the RSV-LTR, MT-1 and SV40 and the like. Among the eukaryotic promoters, there may also be mentioned generally occurring promoters (HPRT, vimentin, α-actin, tubulin, etc.), promoters for intermadial filaments (desmin, neurofilaments, keratin, GFAP, etc.), promoters of therapeutic genes (MDR type). , CFTR, factor VIII and the like), tissue specific promoters (pyruvate kinase, villin, intestinal fatty acid binding protein promoter, α-actin smooth muscle cell promoter, liver specific promoters: ApoAI, ApoAII, human albumin, etc.) or alternatively promoters that respond to the stimulus (steroid hormone receptor, retinoic acid receptor, and the like). In addition, these expression sequences can be modified by adding activation and regulatory sequences, and the like.

As regards the IRES sequence, it preferably originates from a picornavirus. More specifically, this piocorna-viral IRES sequence is derived from encephalomyocarditis virus or poliovirus. More preferably, the encephalomyocarditis virus-derived fragment is present in the NOVAGEN pCITE-2a + vector.

For clarity, the construction will be defined by a transcriptional promoter, two nucleic acids, a polyadenylation site, and an IRES sequence that is between two nucleic acids, hereinafter referred to as a bicistronic cassette.

The viruses of the present invention are defective, in other words, they are not capable of autonomously replicating in the target cell. In general, the genome of the defective viruses used in the present invention and therefore does not contain at least the sequences necessary for the replication of said virus in the infected cell. These regions can either be removed (in whole or in part) or rendered inoperative, or substituted with other sequences and in particular with the bicistron cassette as defined above. Preferably, the defective virus has more or less conserved sequences of its genome which are necessary for encapsidation of the viral particles.

The virus of the present invention may be derived from an adenovirus, an adeno-associated virus (AAV), or a retrovirus. According to a preferred embodiment, it is an adenovirus.

There are various serotypes of adenoviruses whose structure and properties differ somewhat. Of these serotypes, types 2 or 5 of human adenoviruses (Ad2 or Ad5) or adenoviruses of animal origin (see application WO94 / 26914) are preferably used in the present invention. Among the adenoviruses of animal origin that can be used in the present invention, canine, bovine, murine (e.g., Mavl, Beard et al., Virology 75, 81, 1990), sheep, swine, avian or, alternatively, monkey, can be mentioned. (example: SAV). The adenovirus of animal origin is preferably a canine adenovirus, more preferably a CAV2 adenovirus (e.g. Manhattan or A26 / 61 strain (ATCC VR-800). Preferably, adenoviruses of human or canine or mixed origin are used in the invention.

Preferably, at least one E1 region is non-functional in the adenovirus genome of the invention. The viral gene contemplated may be rendered inoperative by any technique known to the person skilled in the art, namely complete suppression, substitution, partial deletion or addition of one or more bases to the genes under consideration. Such modifications may be obtained in vitro (on isolated DNA) or in situ, for example by genetic engineering techniques, or alternatively by treatment with mutagenic agents. Other regions may also be modified, in particular E3 (WO95 / 02697), E2 (WO94 / 28938), E4 (WO94 / 28152, WO94 / 12649, WO95 / 02697) and L5 (WO95 / 02697). According to a preferred embodiment, the adenovirus according to the invention comprises at least one deletion in the E1 region and a deletion in the E3 region. In the viruses of the invention, the deletion in the E1 region preferably ranges from nucleotides 455 to 3329 of the Ad5 adenoviral sequence. According to another preferred embodiment, the bicistrone cassette is inserted at the deletion level in the E1 region.

Therefore, the present invention specifically relates to a defective recombinant adenovirus characterized in that it comprises at least two nucleic acids encoding enzymes, proteins and / or cofactors that are different and involved in reverse cholesterol transport, said nucleic acids being operably linked to a transcriptional promoter and separated one from the second internal ribosome entry site (IRES) sequence.

More preferred recombinant adenoviruses of the invention may be defective recombinant adenoviruses comprising at least one LCAT encoding nucleic acid and one nucleic acid encoding either HL, CATP or ApoAI, wherein said nucleic acids are operably linked to a transcriptional promoter and separated from each other by an internal ribosome sequence. entry point (IRES).

Representative of these adenoviruses may be those shown in Figure 2, comprising genes encoding LCTA and CETP and derived from homologous recombination between pXL2974 and pXL2822.

The defective recombinant adenoviruses of the invention can be prepared by any technique known to the person skilled in the art (Levrero et al., Gene 101, 195, 1991, EP 185 573; Graham, EMBO J., 3, 2917, 1984). In particular, they may be prepared by homologous recombination between an adenovirus and a plasmid carrying inter alia a bicistron cassette. Homologous recombination occurs after co-transfection (co-transfection) of said adenoviruses and plasmids into the respective cell line. The cell line used should preferably (i) be transformable with said elements and (ii) include sequences capable of complementing the genomic portions of the defective adenovirus, preferably in integrated form, to avoid the risk of recombination. An example of a line is human embryonic kidney line 293 (Graham et al., J. Gen. Virol. 36, 59, 1977), which specifically comprises an integrated left portion of the Ad5 adenovirus genome (12%) or complementary lines in its genome. functions E1 and E4 as described separately in applications no. WO94 / 26914 and WO95 / 02697. Furthermore, the adenoviruses that have been propagated are recovered and purified by conventional molecular biology techniques as exemplified.

However, according to a preferred embodiment of the invention, the adenoviruses of the invention are prepared according to the novel process described in patent application WO96 / 25506, where a prokaryotic plasmid containing a recombinant adenovirus genome flanked by one or more restriction sites not present in said genome is used in shuttle plasmid function. This protocol is schematically shown in Figure 2. This procedure is particularly advantageous since it allows the step of recombination in the transcomplementation line to be avoided without using a second construct providing another portion of the genome of the virus.

More specifically, the second object of the present invention relates to the specific prokaryotic plasmids used to produce the adenoviruses of the invention.

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

More specifically, the present invention also provides a prokaryotic plasmid comprising the adenovirus genome and at least two nucleic acids encoding enzymes; proteins and / or cofactors that are different and that are involved in reverse cholesterol transport, wherein the two nucleic acids are operably linked to a transcriptional promoter and separated from each other by a sequence encoding an internal ribosome entry site, IRES.

The prokaryotic plasmids of the invention preferably comprise a first region allowing replication in prokaryotic cells and a second region comprising an adenoviral genome flanked by one or two restriction sites not present in said genome, and wherein there are at least two nucleic acids encoding enzymes, proteins and / or cofactors. differently and which are involved in reverse cholesterol transport, the two nucleic acids being operably linked to a transcriptional promoter and separated from each other by an internal ribosome entry site, IRES.

As regards the definition of enzymes, proteins and / or cofactors that are different and which are involved in reverse cholesterol transport, the transcriptional promoter, the polyadenylation site, the IRES sequence, and also what affects their organization in said cassette, reference is made to what has been described above.

The region allowing replication in prokaryotic cells, which is used in patented plasmids, can be any origin of replication that is functional in selected cells. It may be an origin of replication derived from a plasmid from the group of incompatibility P (example = pRK290), which allows replication in E. coli polA strains. More generally, it can be any origin of replication originating from a plasmid that replicates in prokaryotic cells. This plasmid may be a derivative of RK2, pBR322 (Bolivar et al., 1977), a derivative of pUC (Vieira and Messing, 1982), or other plasmids that originate from the same class of incompatibilities, i. for example from ColEl or pMB1. In addition, these plasmids may be selected from other incompatibility classes replicating in Escherichia coli. They may be plasmids derived from plasmids belonging to the incompatibility groups, for example A, B, FI, FII, FIII, FIV, H1, Hli, II, 12, J, K, L, N, OF, P, Q, T, U, W, X, Y, Z or 9. Other plasmids may be used, among other plasmids that do not replicate in E. coli, but in other hosts if they are B. subtilis, Streptomyces, P. putida, P. aeruginosa, Rhizobium meliloti, Agrobacterium tumefaciens, Staphylococcus aureus, Streptomyces pristinaespiralis, Enterococcus faecium or Clostridium. Preferably, origins of replication derived from plasmids replicating in E. coli are used.

As mentioned above, the adenoviral genome present in the plasmids of the invention is preferably a complete or functional genome, i. it does not require the provision of other regions by recombination or ligation to generate viral stocks in selected encapsidation lines.

Preferably, the recombinant adenoviral genome comprises at least the ITR or encapsidation-enabling sequence. In a preferred embodiment, the adenoviral genome used does not contain all or part of the E1 region. The adenoviral genome used preferably does not contain the E1 regions between nucleotides 454 to 3328 (PvuII-BglII fragment) or 382 to 3446 (HinfII-Sau3A fragment).

According to a particularly preferred embodiment, the adenoviral genome used does not contain all or part of the E3 and / or E4 region. According to a specific embodiment of the invention, it is an adenoviral genome lacking the E1 and E3 regions.

More specifically, patent prokaryotic plasmids contain at least one origin of replication functional in prokaryotic cells in a 5'-3 'orientation, a first part of an adenoviral genome comprising a viral ITR and a ψ sequence, a transcriptional promoter, a first nucleic acid encoding an enzyme, a protein and / or cofactor involved in reverse cholesterol, an IRES sequence, a second nucleic acid encoding a second enzyme, a protein and / or a cofactor involved in reverse cholesterol transport, a polyadenylation site, and a second portion of the adenoviral genome comprising the pIX-IVA2 region.

The prokaryotic plasmids of the invention preferably also contain regions allowing selection of prokaryotic cells containing said plasmid. In particular, this region may consist of any gene conferring resistance to kanamycin (Kan ^r ), ampicillin (Amp ^r ), tetracycline (tet ^r ) or spectinomycin, for example, which are commonly used in molecular biology (Maniatis et al., 1989). Selection of plasmids can be performed with nucleic acid sequences other than genes encoding antibiotic resistance markers. Generally, this includes a gene that provides a bacterium with a function it no longer has (it may coincide with a gene that has been removed or inactivated from the chromosome), ie a gene in a plasmid that restores this function. As an example, it may be a transfer RNA gene that restores defective chromosome function (Somoes et al., 1991).

According to a preferred embodiment of the invention, the prokaryotic plasmid comprises at least one LCAT encoding nucleic acid, the second nucleic acid being selected from CETP, HL or ApoAI encoding acids.

Representative of these prokaryotic plasmids may be mentioned in particular plasmids pXL2974 and pXL3058 shown in Figures 4 and 6. Plasmid pXL2974 contains a 5 '-3 * origin of replication, a spectinomycin resistance gene, a SacB gene for sucrose sensitivity, and a viral ITR. ψ sequence, RSV promoter, two LCAT and CETP transgenes separated by IRES, polyadenylation site, and pIX and IVA2 viral sequences. Plasmid pXL3058 contains a 5'3 'origin of replication, kanamycin resistance gene, viral ITR and ψ sequences, the RSV promoter, two LCAT and intron + ApoAI transgenes separated by IRES, a polyadenylation site, the pIX and IVA2 viral sequences, and the SacB gene for SacB sucrose sensitivity.

The present invention also relates to plasmid constructs used to construct these prokaryotic plasmids and which also comprise a bicistron cassette as defined by the invention.

In particular, the prokaryotic plasmids of the invention may be obtained by transforming an initial shuttle plasmid comprising a bicistron cassette as defined by the invention, in particular comprising a sequence encoding a transcriptional promoter operably linked to two nucleic acids encoding two enzymes, proteins and / or cofactors that are differently involved and cholesterol transport, a polyadenylation site, an IRES sequence located between said nucleic acids.

Representative of these shuttle plasmids may be the particular plasmids pXL2970 and pXL2984 described in Figures 3 and 5.

Regarding the definitions of the enzymes involved in reverse cholesterol transport, the transcriptional promoter and the IRES sequence, as well as what affects their organization in said cassette, reference is made to what has already been described above.

Another object of the present invention relates to any prokaryotic cell containing a prokaryotic plasmid as defined above. Specifically, it can be any bacterium for which there is a vector system into which recombinant DNA can be introduced. For example, Escherlchia coli, Salmonella typhimurium, Bacillus subtilis, Pseudomonas putida, Pseudomonas aeruginosa, Agrobacter tumefaciens, Rhizobium meliloti, or bacteria of the genus Streptomyces may be mentioned. These cells are preferably obtained by transformation by techniques known to those skilled in the art.

The present invention also relates to the use of a recombinant virus, a defective recombinant virus or a shuttle plasmid construct as defined above for the preparation of a pharmaceutical composition intended for the treatment or prevention of pathological conditions associated with hypoalphalipoproteinemia, including in particular atherosclerosis and / or restenosis.

The present invention also relates to a pharmaceutical composition comprising one or more defective recombinant viruses, including adeoviruses, as described above. Such compositions may be formulated for administration topically, orally, parenterally, intranasally, intravenously, intramuscularly, subcutaneously or intraocularly and the like.

The formulation of the invention preferably comprises pharmaceutically acceptable carriers for the injectable formulation. They may be, in particular, isotonic sterile saline solutions (disodium hydrogen phosphate or dihydrogen phosphate, sodium, potassium, calcium or magnesium chloride and the like, or mixtures thereof) or dry, in particular lyophilized preparations, which allow reconstitution of injectable solutions after addition of sterile water or physiological solution, depending on the particular case being treated.

The doses of virus for injection may be adjusted according to various parameters, in particular according to the route of administration used, the respective pathological condition or the desired duration of treatment. In general, the recombinant viruses of the invention are formulated and administered in the form of doses between 10 ⁴ and 10 ¹⁴ pfu / ml. The term pfu (plaque forming unit) refers to the infectivity of the virion suspension and is determined by infection of the respective cell culture and by measuring, generally after 48 hours, the number of plaques of the infected cells. Techniques for determining the pfu titre of a viral solution are well described in the literature.

The present invention provides a novel, very effective preparation for the treatment or prevention of pathological conditions associated with hypoalphalipoproteinemia, in particular in the field of cardiovascular conditions such as pectoris, sudden death, heart failure, myocardium, angina cerebrovascular accidents, atherosclerosis or restenosis.

In addition, the treatment can be applied to both human and any animal, such as e.g. sheep, cattle, pets (dogs, cats, etc.), horses, fish and the like.

The present invention is described in more detail by the following examples, which are to be considered illustrative and not limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Schematic representation of a putative mechanism of cholesterol transport.

Figure 2: Representation of the adenovirus production protocol by homologous recombination in E. coli.

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

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

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

Figure 6: Representation of the prokaryotic plasmid pXL3058 containing the RSV-ApoAI-IRES-LCAT.

DETAILED DESCRIPTION OF THE INVENTION

I. MATERIAL AND METHODS

1-1. MATERIAL

1) The plasmids used to construct the bicistrone recombinant adenoviruses LCAT-IRES-CETP, LCAT-IRES-HL and LCAT-IRES-ApoAI are:

- pSK IRES (supplied by NOVAGEN)

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

- pCRII (supplied by INVITROGEN)

2) Total RNA obtained from hepatocytes and HepG2 cells

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

4) Escherichia coli: DH5α subtype with endAl, recAl, hsdR17, supE44, I-, thy-1, gyrA, relAl, lacZAM15, deoR ⁺ , F ⁺ , dam ⁺ , dcm ⁺ genotype (Woodcock et al., 1989)

5) Enzymes and restriction buffers were from New England Biolabs.

METHODS

General techniques of molecular biology

Methods commonly used in molecular biology, such as preparative plasmid DNA extraction, plasmid DNA centrifugation in cesium chloride gradient, agarose or acrylamide gel electrophoresis, purification of DNA fragments by electroelution, phenol or phenol-chloroform protein extraction with ethanol or isopropanol, ethanol or isopropanol E. coli transformation and the like are well known to those skilled in the art and are described in detail in the literature (Maniatis T. et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982; Ausubel FM and (eds., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1987).

Plasmids of the pBR322 and pUC type and phages of the M13 series are of commercial origin (Bethesda Research Laboratories).

For ligation, the DNA fragments can be separated according to their size by agarose or acrylamide gel electrophoresis, extracted with phenol or phenol / chloroform, precipitated with ethanol, and then incubated in the presence of phage T4 DNA ligase (Biolabs) as recommended by the supplier.

Addition of the overhanging 5 'ends can be performed with a Klenow fragment of E. coli DNA polymerase I (Biolabs) according to the supplier's instructions. Removal of the protruding 3 'ends can be performed in the presence of phage T4 DNA polymerase (Biolabs) used as recommended by the supplier. Removal of the protruding 5 'ends is accomplished by controlled removal with SI nuclease.

Site-directed mutagenesis in vitro using synthetic oligodeoxynucleotides can be performed according to the method developed by Taylor et al. (Nucleic Acids Res. 13, 8749-8764, 1985) using a kit supplied by Amersham.

Enzyme amplification of DNA fragments by the so-called Polymerase-Catalyzed Chain Reaction (PCR) technique, Saiki R. K. et al., Science 230, 1350-1354,

1985 Mullis K.B ', and Faloon F.A., Meth. Enzyme. 155, 335-350

1987) can be performed using a DNA thermal cycler i (Perkin Elmer Cetus) as recommended by the manufacturer.

Verification of nucleotide sequences can be performed by the method developed by Sanger et al. (Proc. Natl. Acad. Sci. USA 74 (1977) 5463-5467) using a kit supplied by

Amersham.

Cell culture techniques

a) Cell culture

293 cells were grown in Eagle nutrient 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

293 cells grown on 100 mm dishes on MEM medium supplemented with 10% fetal calf serum are transfected with 1 mg DNA using lipofectamine (Gibco BRL). Six hours later, the medium is removed and the cells are incubated in complete medium (MEM + 10% FCS). Cultured cell supernatants transfected with recombinant plasmids RSV LCAT-IRES-CETP, RSV LCAT-IRESApoAI and RSV LCAT-IRES-HK as well as the β-galactosidase control and the untransfected cell supernatant were obtained 72 hours after transfection and stored at 4 ° C. The assays for LCAT, CETP, ApoAI and HL enzymes were performed on 10-30 ml cell supernatant fractions using human plasma as a control.

c) Production and purification of recombinant adenoviruses

Bicistronic adenoviral DNA is obtained by homologous recombination in E. coli. It is then linearized with PacI and the virus is produced after transfection of 293 cells. j. cells transcomplenting the E1 protein are transfected using lipofectamine with 10 mg of linearized adenovirus DNA. Subsequently, plaques containing recombinant virus are evaluated on restriction profile and analyzed on 293-cell agar in MEM supplemented with 4% FCS and incubated for 8 days at 37 ° C.

d) Infection of cultured cells

Infection with 0.25, 0.5 and 1 ml of the supernatant containing the bicistrone recombinant adenovirus is performed in a 12-well plate containing approximately 4 x 10 ⁵ cells 293. After 72 h incubation in 2 ml MEM supplemented with 2% FCS, the supernatant was harvested from the infected cells and tested. enzyme activity.

Biochemical verification of results in vitro

a) LCAT activity assay

LCAT activity is determined by measuring the conversion of free 14 ^C cholesterol to esterified 14 ^C -cholesterol using proteoliposomes as substrate as described by Chen et al., (1982). Proteoliposomes are prepared from phosphatidylcholine, cholesterol, 14 ^C- cholesterol, and apolipoprotein A1 and incubated with 20 ml of the transfection culture supernatant to be tested. The products (free 14 ^c -cholesterol) and esterified 14 ^c- cholesterol) reactions were separated by thin layer chromatography and detected by autoradiography on an Instant Imager (Packard). LCAT activity is expressed as percentage of esterified 14 ^C -cholesterol per hour and per 20 ml of test supernatant.

b) CETP activity assay

CETP activity is determined by measuring the capacity of this enzyme to transfer cholesterol esters from the HDL particle (donor) to the LDL particle (acceptor). Substrates for this reaction were provided by kits from Wak-Chemie, Medical GmbH. The fluorescence of the cholesterol linoleate found in the donor particles is quenched in the native state of linoleate and only in the presence of active CETP, which catalyzes in the presence of active CETP, which catalyzes the transfer of the fluorescent molecule to the acceptor particle. CETP activity is expressed as the value of fluorescence intensity emitted at 535 nm per 30 ml of tissue supernatant tested and per hour.

c) HL activity assay

Liver lipase activity is determined using a synthetic triglyceride substrate from the Progen Biotechnik GmbH kit. This substrate contains a fluorescent pyrene moiety which is masked in the native state of the molecule by trinitrophenol and the result of hydrolysis of the trinitrophenol is the emission of fluorescence at 400 nm. The amount of HL (in pmol / L) contained in 20 ml of the supernatant is determined by measuring the fluorescence intensity emitted at 400 nm after incubating the substrate with the test sample and using standard plasma from the kit.

d) Apolipoprotein A1 Assay

ApoAI activity is determined using a monoclonal anti-ApoAI antibody. Immulon II plates (Dynatech) were coated with anti-ApoAI monoclonal antibody (10 mg / ml in carbonate buffer pH 9.6), incubated overnight at 4 ° C and then saturated with 2% BSA in PBS pH 7.4 hour at 37 ° C. The cell supernatants are then incubated for one hour at 37 ° C, optionally incubating for one hour at 37 ° C with a mixture of peroxidase-labeled anti-ApoAI monoclonal antibodies and diluted 1/5000. Peroxidized antibody binding is finally detected by incubation with 250 μΐ TMB (KPL) and plate counting.

Shuttle plasmid construction technique

I

Constructs of the bicistron plasmids are detailed in Figures 3 to 5. The obtained bicistron plasmids are also shuttle plasmids because they contain the adenoviral pIX-IVa2 sequences necessary for recombination with the viral genome. This recombination occurs either by co-transfection (co-transfection) with the cleaved genome derived from viral β-galactosidase (conventional shuttle vector) or by double recombination in E. coli (coli shuttle vector).

The sequence identity of the different plasmid structures is analyzed by their restriction profile. This analysis makes it possible to select recombinant clones to serve for subsequent cloning as well as to verify cloning results. Restriction enzymes are selected to deliver as complete as possible the entire cloned cDNA and number of cloning strategic sites. However, this control does not exclude point mutations that may occur at each stage of cloning. Such mutations can only be detected by complete sequencing of the respective cDNA. Final validation of the obtained constructs is done by in vitro biochemical assays of LACT, CETP and HL enzyme activities, or by detecting the presence of ApoAI.

Example 1. '

LCAT-IRES-CETP shuttle plasmid construction

1) Construction of the conventional shuttle vector pXL2970

The pXL2968 RSV PCAT polyA bGH expression vector was obtained on the one hand by digesting the plasmid pXL2616 containing the LACT cDNA and, on the other hand, the pXL LPL plasmid, under the control of the RSV promoter and upstream of the bovine growth hormone polyadenylation signal. T4 DNA ligase.

To obtain the vector, the following were performed:

Each of the plasmids pXL RSV LPL (2 μg) and pXL2616 (2 μς) was digested with 10 units (U) of Clal in buffer 4 (20 mM Tris acetate, 10 mM Mg-acetate, 50 mM K-acetate and 1 mM DTT). supplemented with 100 μς / πιΐ of acetylated BSA, for 90 minutes at 37 ° C. 100 mM NaCl and 10 U Sal were added and the reaction mixture was incubated again at 37 ° C for another 90 minutes. After electrophoretic separation of the cleavage products on a 0.7% agarose gel, the bands were sized

6.5 and 1.7 kb, corresponding to pXLRSV and LCAT cDNA, were excised from the gel and extracted with a Qiaquick kit. The two bands were ligated with 400 U T4 DNA ligase after incubation overnight at 14 ° C.

The recombinant plasmid pXL2969 IRES-CETP originated from a Bluescript plasmid having a CETP cDNA modified at its 5 'end by introducing an NcoI site using the PCR primer 5' GCCTGATAAC CATGGTGGCT GCCACAG 3 '(SEQ ID NO .: 1). The plasmid thus modified was digested with NcoI and SalI and cloned downstream of the adenovirus IRES sequence found in the plasmid pSKIRES, which had been digested with the same restriction enzymes as follows:

2.5 gg of plasmid CETP and 2.5 gg of plasmid pSKIRES were digested with 10 U Ncol for 90 minutes at 37 ° C in buffer 3 (50 mM Tris-HCl, 10 MgCl 2, 100 mM NaCl and 1 mM DTT) followed by digestion with 10 U Sali 9 minutes at 37 ° C. The digestion products were resolved by electrophoresis and 1.5 kb (CETP cDNA) and 3.5 kb bands were extracted and ligated under the same conditions as described above.

In order to obtain plasmid pXL2970, the following procedure was performed:

gg of plasmid pXL2968 was linearized with 10 U SalI (buffer 3 + BSA 90 minutes at 37 ^e C). The DNA was then extracted using a Qiaquick kit and recovered in 50 g of water previously heated to 50 ° C. The cohesive ends resulting from cleavage with SalI were blunt incubated with 5 U Klenow polymerase for 15 minutes at 25 ° C. DNA was re-extracted with the kit

Qiaquick and dephosphorylated with 2 U calf intestinal phosphatase (CIP) for 60 minutes at 37 ° C. Plasmid pXL2969 (4 gg) was first digested with 5 U SmaI (buffer 4, 90 minutes at 37 ° C).

The two plasmid digestion products were electrophoretically resolved in a 0.7% agarose gel and 8.5 kb (pXL2968) and 2.2 kb (IRES CETP cDNA) bands were extracted with Qiaquick and ligated with 400 U T4 DNA ligase ( Figure 3).

LCAT and CETP activities of plasmid pXL 2970 were tested in 293 cell culture supernatant three days after transfection.

The LCAT activity of this plasmid corresponds to 3.5% of the cholesterol esters produced per hour and the CETP activity to 120% (Table 1 below). These activity values indicate that plasmid pXL2970 indeed synthesizes LACT and CETP, which are catalytically active.

2) LCAT-IRES-CETP coli shuttle plasmid

With this technology, the shuttle vector should contain:

- inverted terminal repeat (ITR) sequences and encapsidation sequences necessary for homologous recombination in E. coli flanking the LCAT-IRES-CETP sequence,

- an origin of replication of ColE1 that does not allow the plasmid to replicate in the E. coli strain C2110 and thus allows the clonality of the recombinant plasmid,

- a suicide gene for sucrose B (lethal for bacteria grown on sucrose) and a gene for spectinomycin resistance that allows for the selection of a recombinant clone.

To obtain the vector, the bicistron shuttle plasmid pXL2970 RSV LCAT-IRES-CETP was digested with BstEII and SpeI to introduce the ITR and adenoviral genome sequences to obtain the coli shuttle plasmid. The ITR and sequences were isolated by digesting plasmid pXL2794 with BstEII and XbaI. The blunt end fragment containing the spectinomycin-sucrose B cassette obtained by digestion of pXL2757 with EcoRV and SmaI was introduced into the shuttle plasmid pXL2970 + 2794 linearized with FspI (Figure 4).

The procedure was carried out experimentally according to the following protocol:

gg of plasmid pXL2970 was digested with 20 U BstEII (buffer 2: 10 mM Tris HCl, 10 mM MgCl ₂ , 50 mM NaCl, 1 mM DTT; 90 minutes at 60 ° C) and then with 10 U Spel (90 minutes at 37 ° C). C).

The resulting DNA fragment was extracted with Qiaquick and dephosphorylated with 2 U CIP (60 minutes at 37 ° C). Plasmid pXL2794 was digested with 20 U BstEII (buffer 2, 90 minutes at 60 ° C), then with 20 U XbaI (9 minutes at 37 ° C). The 6.5 kb bands (pXL2970) and 2.9 kb (ITR Ψ and Kan ^r from pXL2794), extracted after electrophoretic separation, were ligated from T4 DNA ligase (40 U).

The resulting plasmid pXL2970 + 2794 (1.5 gg) was digested with 5U

FspI (buffer 4, 60 min at 37 ° C), extracted with Qiaquick kit and ligated (T4 DNA ligase, 400 U) with a 3.8 kb DNA fragment extracted from the gel containing the sacB-spect ^r cassette resulting from the digestion plasmid pXL2757 with 10 U Smal (buffer 4, 90 minutes at 25 ° C), followed by 10 U EcoRV (90 minutes at 37 ° C).

The shuttle plasmid pXL2974 LCAT-IRES-CETP was selected on spectinomycin medium and spectinomycin + sucrose medium. The results of digestion with NcoI (6.2 + 3.3 + 2.2 kb), NotI (13.5 kb) and EcoRV (1 + 3 + 9.5 kb) are consistent with the restriction map of the plasmid.

LCAT activity of plasmid pXL2974 corresponds to 2% of cholesterol esters produced per hour and CETP activity of 114% (Table 2).

1). LCTA and CETP activities were detected at the coli level of the LCAT-IRES-CETP shuttle plasmid.

Table 1

LCAT activity CETP activity shuttle plasmid LCTA-IRES-CETP 3.5 ± 0.2% 120 ± 2% coli shuttle plasmid LCAT-IRES.-CETP 2 ± 0.2% 114 ± 2%

Example 2

Construction of the RSV LCAT-IRES-HL shuttle plasmid

The HL cDNA was cloned behind the IRES in the Bluescript vector, and the IRES-HL fragment was then inserted in a manner similar to the LCAT-IRES-CETP vector according to the following protocol:

Plasmid pXL2971 (4 gg) was digested to remove the NcoI site with 40 U of BglII (buffer 3, 90 minutes at 37 ° C) and then with 40 U of SalI for 90 minutes at 37 ° C. The 2.5 kb DNA fragment (approximate weight 0.5 gg) resulting from these digestions was digested in a controlled manner with NcoI (0.1-1 U NcoI / gg DNA) in a 466 min buffer at migration and gel extraction. Deň: 32 ° C. The cleavage product is analyzed by splicing in a 0.7% agarose gel and a 1.5 kb band containing the HL cDNA ABC, obtained with 0.5 U NcoI, is ligated (T4 DNA ligase, 400 U) with the pSK IRES fragment (1, 3 gg) derived from Ncol and SalI cleavage (1 U of each enzyme, buffer 2, 37 ° C).

The final vector is shown in Figure 5. The corresponding LCIST-IRES-HL bicistron shuttle plasmid was named pXL2984.

Table 2

LCAT activity HL activity Shuttle plasmid LCAT-IRES-HL 1.2 ± 0.2% 46 ± 2%

Example 3

Construction of ApoAI-IRES-LCAT shuttle plasmid

The general principle for this construction is the same as the previous one. LCAT was mutated by PCR, including an additional NcoI site, allowing its ligation to the correct site after the IRES. Its sequence was completely checked. The IRESLCAT fragment was then ligated to ApoAI. The resulting vector is derived directly from coli technology and was called pXL3058 (Figure 6). After the usual double recombination, the resulting viral vector was checked for activity. ApoAI was detected by Western blotting and LCAT activity was evaluated to 1.3% (background noise 0.2%).

Sequence Listing (2) Sequence Information SEQ ID NO: 1:

(i) sequence characteristics:

(A) length: 27 base pairs (B) type: nucleotide (C) fiber type: simple (D) topology: linear (ii) molecule type: cDNA (xi) sequence description SEQ ID NO .: 1:

GCCTGATAAC CATGGTGGCT GCCACAG

Claims (35)

PATENT CLAIMS A defective recombinant virus comprising at least two nucleic acids encoding enzymes, proteins and / or cofactors that are different and involved in reverse cholesterol transport, said nucleic acids being operably linked to a transcriptional promoter and separated from each other by a sequence encoding an internal ribosome entry. place (IRES). The defective recombinant virus of claim 1, wherein the nucleic acids are selected from genes encoding all or part of a protein, namely lecithin cholesterol acyltransferase (LCAT), cholesterol ester transfer protein (CETP), liver lipase (HL), apolipoproteins A1 and AIV, or one of their variants. The defective recombinant virus according to claim 1 or 2, wherein the nucleic acids are preferably genes encoding all or part of the corresponding human enzymes, proteins and / or cofactors. The defective recombinant virus according to any one of the preceding claims, wherein the nucleic acids are preferably cDNA or gDNA. The defective recombinant virus according to any one of the preceding claims, 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 the preceding claims, wherein the transcriptional promoter is preferably selected from the promoters of the MLP and Ε1Ά adenoviral nucleic acid sequences and the CMV, RSV-LTR, MT-1 and SV40 promoters. The defective recombinant virus according to any one of the preceding claims, wherein the IRES sequence is derived from picornaviruses. The defective recombinant virus of claim 8, wherein the picornavirus IRES sequence is derived from encephalomyocarditis virus or poliovirus. Defective recombinant virus according to any one of the preceding claims, which lacks at least those regions of its genome which are necessary for its replication in the infected cell. The defective recombinant virus according to any one of the preceding claims, which is preferably an adenovirus, preferably a human adenovirus of the Ad5 or Ad2 type or an adenovirus of animal origin. A defective recombinant adenovirus comprising at least two nucleic acids encoding enzymes, proteins and / or cofactors that are different and involved in reverse cholesterol transport, wherein said nucleic acids are operably linked to a transcriptional promoter and separated from each other by a sequence encoding an internal ribosome entry. place (IRES). The defective recombinant adenovirus according to 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 an internal ribosome entry site (IRES) sequence. The defective recombinant adenovirus according to claim 12, comprising at least one gene encoding LCAT and one gene encoding apoA-I, wherein said genes are operably linked to a transcriptional promoter and separated from each other by an internal ribosome entry site (IRES) sequence. The defective recombinant adenovirus of claim 12, comprising at least one LCAT encoding gene and one CETP encoding gene, wherein said genes are operably linked to a transcriptional promoter and separated from each other by an internal ribosome entry site (IRES) sequence. The defective recombinant adenovirus according to claim 15, which is derived from homologous recombination between pXL2974 and pXL2822. A defective recombinant adenovirus according to any one of claims 12 to 16, comprising at least one deletion in the E1 region and one deletion in the E3 region. comprising an adenovirus genome, nucleic acids encoding enzymes, proteins and / or that are different and involved in reverse cholesterol, said nucleic acids being linked to a transcriptional promoter and separated by a sequence encoding an internal ribosome entry 18. A prokaryotic plasmid, and at least two cofactors, a transport operatively to each other (IRES). 19. A prokaryotic plasmid comprising a first region allowing replication in prokaryotic cells and a second region comprising an adenoviral genome flanked by one or more restriction sites not present in said genome and comprising at least two nucleic acids encoding enzymes, proteins and / or cofactors; which are different and are involved in reverse cholesterol transport, wherein said nucleic acids are operably linked to a transcriptional promoter and separated from each other by an internal ribosome entry site (IRES) coding sequence. The prokaryotic plasmid according to claim 18 or 19, wherein the E1 and E3 regions thereof are removed from the adevirus genome. A prokaryotic plasmid according to any one of claims 18 to 21 20, which contains the ITR viral sequences and Ψ. A prokaryotic plasmid according to any one of claims 18 to 22 21, wherein the origin of replication originates from a bacterial plasmid selected from the group consisting of RK2, pBR322, and pUC. 23. A prokaryotic plasmid comprising at least one origin of replication functional in prokaryotic cells in a 5 '-3 * orientation, a first part of an adenoviral genome comprising the viral ITR and a sequences, a transcriptional promoter, a first nucleic acid encoding an enzyme, a protein and / or a cofactor which are involved in reverse cholesterol transport, an IRES sequence, a second nucleic acid encoding an enzyme, a protein, and / or a cofactor that are involved in reverse cholesterol transport, a polyadenylation site, and a second portion of the adenoviral genome comprising the pIX-IVa2 region. A prokaryotic plasmid according to any one of claims 18 to 24 23, further comprising a region allowing selection of prokaryotic cells containing said plasmid. A prokaryotic plasmid according to any one of claims 18 to 25 24, wherein at least one nucleic acid encodes LCAT and the second nucleic acid is selected from nucleic acids encoding HL, ApoAI, or CETP. The prokaryotic plasmid of claim 25, which is a plasmid pXL2794 comprising two nucleic acids encoding LCAT and CETP. The prokaryotic plasmid of claim 25, which is a plasmid pXL3058 comprising two nucleic acids encoding LCAT and ApoAI. A shuttle plasmid comprising at least two nucleic acids encoding enzymes, proteins and / or cofactors that are different and involved in reverse cholesterol transport, said nucleic acids being operably linked to a transcriptional promoter, a polyadenylation site, and a sequence encoding an internal ribosome entry. site (IRES) located between the two nucleic acids. The shuttle plasmid of claim 28, which is a plasmid pXL2984 comprising two nucleic acids encoding HL and LCAT. The shuttle plasmid of claim 28, which is a plasmid pXL2970 comprising two nucleic acids encoding LCAT and CETP. A prokaryotic cell transformed with a prokaryotic plasmid according to any one of claims 18 to 27. Use of 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 or a plasmid according to any one of claims 28 to 30 for the preparation of a pharmaceutical composition for treating or preventing pathological conditions associated with hypoalphalipoproteinemia. Use according to claim 32 for the preparation of a pharmaceutical composition intended for the treatment or prevention of atherosclerosis and / or restenosis. A pharmaceutical composition comprising one or more defective recombinant viruses 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. 35. The pharmaceutical composition of claim 34, wherein the injectable form comprises 10 ⁴ to 10 ¹⁴ pfu / ml adenovirus.