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  • C12N9/10—Transferases (2.)
  • C12N9/1025—Acyltransferases (2.3)
  • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
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  • C12N9/14—Hydrolases (3)
  • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
  • C12N9/18—Carboxylic ester hydrolases (3.1.1)
  • C12N9/20—Triglyceride splitting, e.g. by means of lipase
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  • C12N2710/00011—Details
  • C12N2710/10011—Adenoviridae
  • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
  • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
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  • C12N2830/00—Vector systems having a special element relevant for transcription
  • C12N2830/42—Vector systems having a special element relevant for transcription being an intron or intervening sequence for splicing and/or stability of RNA
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  • C12N2840/00—Vectors comprising a special translation-regulating system
  • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
  • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES

Definitions

• the present invention relates to new recombinant viruses, their preparation and their use in gene therapy, for the transfer and expression in vivo of desired genes. More specifically, it relates to recombinant viruses comprising at least two inserted genes and whose expression products are involved in the reverse transport of cholesterol. It also relates to shuttle plasmids useful for the production of adenoviruses in accordance with the invention. More particularly, the present invention relates to defective recombinant adenoviruses and their use for the prevention or treatment of pathologies linked to dyslipoproteinemias, which are known for their serious consequences at the cardiovascular and neurological level.
• Dyslipoproteinemias are disorders of the metabolism of lipoproteins, responsible for the transport, in the blood and peripheral fluids, of lipids such as cholesterol and triglycerides. They lead to significant pathologies, respectively linked to hypercholesterolemia or hypertriglyceridemia, such as in particular atherosclerosis
• Atherosclerosis is a complex, polygenic disease which is defined histologically by deposits (lipid or fibro-lipid plaques) of lipids and other blood derivatives in the wall of the large arteries (aorta, coronary arteries, carotid artery). ). These plaques, more or less calcified depending on the progress of the process, can be combined with lesions and are linked to the accumulation in the arteries of fatty deposits consisting essentially of cholesterol esters. These plaques are accompanied by a thickening of the arterial wall, with enlargement of the smooth muscle, appearance of foam cells and accumulation of fibrous tissue. The atherosclerotic plaque is very clearly in relief on the wall, which gives it a stenosing character responsible for vascular occlusions by atheroma, thrombosis or embolism which occur in the most affected patients.
• Hypercholesterolaemia can therefore lead to cardiovascular pathologies very serious such as infarction, sudden death, heart decompensation, stroke, etc.
• LDL low density lipoproteins
• HDL high density lipoproteins
• dyslipemias those characterized by high LDL cholesterol (low density lipoprotein) and hypoalphalipoproteinemia.
• the latter is characterized by a HDL (high density lipoprotein) cholesterol level of less than 35 mg / dl and it represents 40% of cases of dyslipemia (Genest et al., 1992).
• Hypoalphalipoproteinemia seems to be linked to a genetic deficiency in one or more proteins involved in the synthesis, maturation and catabolism of HDL particles and often results in the premature appearance of cardiovascular diseases (Dammermann et al., 1995). In general, there is an inverse correlation between the incidence of the latter and the level of HDL particles (Miller et al., 1987).
• hypoalphalipoproteinemia While there are effective treatments for lowering LDL cholesterol and triglycerides based on hypolipidemic and antihypertensive drugs, current care for hypoalphalipoproteinemia is only of limited effectiveness.
• the present invention is specifically concerned with the treatment, by gene therapy, of pathologies linked to hypoalphalipoproteinemia.
• the therapeutic approach adopted by the present invention aims to increase the kinetics of the reverse transport of cholesterol in order to induce the regression of atherosclerotic lesions or else to prevent their formation.
• this objective is achieved according to the invention via a simultaneous and effective expression, in the cells to be treated, of at least two of the proteins, enzymes or co-factors involved in the reverse transport of cholesterol.
• a protein, enzyme and / or one of their co-factor is considered to be involved in the reverse transport of cholesterol insofar as any disturbance in its cell concentration, automatically affects the process reverse cholesterol.
• Lecithin cholesterol acyltransferase is a 67 kDa glycoprotein, synthesized by the liver, which catalyzes the transfer of an acyl group from lecithin to cholesterol by producing lysophosphatidylcholine and esters of cholesterol (Glomset, 1968).
• the cofactor of this enzyme is apolipoprotein AI which is linked to the surface of HDL particles.
• LH Human hepatic lipase
• IDL intermediate density lipoproteins
• HDL high density lipoproteins
• LH deficiency in humans is characterized by a high amount of LDLs rich in triglycerides, large HDL particles and the early development of atherosclerosis (Hegele et al., 1993).
• cholesterol ester transfer protein CETP
• apolipoproteins AI and AIV or one of their variants.
• the cholesterol ester transfer protein (CETP) is a 74 kDa glycoprotein synthesized in adipose tissue and the liver. In plasma, it is mainly associated with HDL. This is where it catalyzes the transfer of cholesterol esters from HDL to low density lipoproteins. This transfer is followed by the passage of triglycerides from low density lipoproteins to HDL.
• the overexpression of CETP in hypertriglycemidic transgenic mice inhibits the development of atherosclerotic lesions (Hayek et al., 1995). This experience is in agreement with the observation, in individuals deficient in CETP. early development of cardiovascular disease (Zhong et al, 1996).
• Apolipoprotein AI is a protein made up of 243 amino acids, synthesized in the form of a prepropeptide of 267 residues, having a molecular mass of 28,000 daltons. It is synthesized in humans specifically in the liver and the intestine and it constitutes the essential protein of HDL particles (70% of their mass in proteins). It is abundant in plasma (1.0-1.2 g / 1). Its activity which is best characterized biochemically is the activation of lecithin cholesterol acyl transferase (LCAT), but many other activities are attributed to it, such as in particular the stimulation of the efflux of cellular cholesterol. Apolipoprotein AI plays a major role in resistance to atherosclerosis linked to the reverse transport of cholesterol.
• LCAT lecithin cholesterol acyl transferase
• Apolipoprotein AIV is a protein made up of 376 amino acids, synthesized specifically in the intestine in the form of a precursor of 396 residue. As regards its physiological activity, it is known that it can activate lecithin-cholesterol acyltransferase (LCAT) in vitro (LCAT) (Steinmetz et al., 1985, J. Biol. Chem., 260: 2258-2264) and that like apolipoprotein AI, it can interfere with the fixation of HDL particles on bovine aortic endothelial cells (Savion et al., 1987, Eur. J. Biochem., 257: 4171-4178).
• LCAT lecithin-cholesterol acyltransferase
• LCAT lecithin-cholesterol acyltransferase
• LCAT lecithin-cholesterol acyltransferase
• LCAT lecithin-cholesterol acyltransferase
• apoAIV most likely acts as a mediator of reverse cholesterol transport.
• the apoAIV gene was cloned and described in the previous year (see in particular WO 92/05253).
• apolipoprotein AIN type activity mention may in particular be made of the fragments and derivatives described in patent application FR 92 00806.
• the present invention is based on the use of recombinant viruses which make it possible to transfer and express at least two nucleic acids coding for enzymes, proteins and / or co-factors involved in the reverse transport of cholesterol.
• the applicant has thus demonstrated that it was possible to efficiently ensure the transfer and expression of at least two nucleic acids from the same recombinant virus, by integrating these nucleic acids. in said virus in the form of a bicistronic unit. It is clear that the use of a single virus and not of two presents many therapeutic advantages.
• the therapeutic use of a vector as claimed makes it possible to reduce by half the quantities of recombinant viruses necessary for the expression of said genes. This is particularly beneficial with regard to the immune response conventionally manifested with regard to cells infected with recombinant viruses. This immune response usually results in destruction of infected cells and / or a significant inflammatory response. It is clear that these two manifestations are highly detrimental in terms of the duration of expression of the therapeutic genes and therefore of the expected therapeutic effect.
• the claimed viruses are capable of efficiently transferring and expressing, for a considerable period of time and without cytopathological effect, two nucleic acids coding for proteins, enzymes and / or cofactors involved in the reverse transport of cholesterol.
• a first object of the invention therefore resides in a defective recombinant virus comprising at least two nucleic acids coding for enzymes, proteins and / or co-factors, distinct and involved in the reverse transport of cholesterol, said nucleic acids being operatively linked to a transcriptional promoter and separated from each other by a sequence coding for an internal entry site of the IRES ribosome.
• the nucleic acids are preferably chosen from the genes coding for all or part of lecithin cholesterol acyltransferase (LCAT), the cholesterol ester transfer protein (CETP), hepatic lipase (LH), the apolipoproteins AI and AIV or l 'one of their variants.
• the inserted nucleic acids can be fragments of complementary DNA (cDNA), genomic DNA (gDNA), or hybrid constructs consisting, for example, of a cDNA into which one or more introns would be inserted. They can also be synthetic or semi-synthetic sequences. As indicated above, it may be a gene encoding all or part of one of the enzymes, proteins and / or co-factors involved in the reverse transport of cholesterol or a variant thereof.
• the term variant designates any mutant, fragment or peptide having at least one biological property of the protein product under consideration, as well as, where appropriate, their respective natural variants.
• fragments and variants can be obtained by any technique known to those skilled in the art, and in particular by genetic and / or chemical and / or enzymatic modifications. Genetic modifications include deletions, deletions. mutations, etc.
• nucleic acids inserted within the meaning of the invention are preferably the genes coding for all or part of the corresponding enzymes, proteins and / or cofactors. It is more preferably cDNA or gDNA.
• Each inserted nucleic acid can also include activation, regulation sequences, etc. Furthermore, it generally comprises, upstream of the coding sequence, a signal sequence directing the polypeptide synthesized in the secretory pathways of the target cell.
• This signal sequence can be its natural signal sequence, but it can also be any other functional signal sequence, or an artificial signal sequence.
• the claimed recombinant viruses comprise at least one nucleic acid coding for LCAT. More preferably, the second inserted nucleic acid codes for LH, CETP or ApoAI.
• the co-expression of the two nucleic acids considered is ensured via the formation of a single RNA which is then translated to lead to the two respective enzymes.
• the recombinant virus incorporates, in addition to the two nucleic acid sequences, at least one transcriptional promoter, a polyadenylation site and an IRES sequence.
• This transcriptional promoter is operationally linked to the nucleic acids coding so as to produce the bicistronic mRNA and to drive the expression of the two respective enzymes from said mRNA. According to a preferred embodiment of the invention, it is placed directly upstream of the first nucleic acid.
• This transcriptional promoter can in particular be chosen from the sequences which are naturally responsible for the expression of said nucleic acid with respect to which it is placed upstream provided of course that these sequences are capable of functioning in the infected cell. It can also be sequences of different origin (responsible for the expression of other proteins, or even synthetic). In particular, they may be sequences of eukaryotic or viral nucleic acid sequences or derived sequences, stimulating or repressing the transcription of a gene in a specific way or not and in an inducible way or not.
• they may be promoter sequences originating from the genome of the cell which it is desired to infect, or from the genome of a virus, and in particular, the promoters of the El A, MLP genes of adenovirus, promoter CMV, LTR-RSV, MT-1, SV40 etc.
• eukaryotic promoters include ubiquitous promoters (HPRT, vimentin, ⁇ -actin, tubulin, etc.), promoters of intermediate filaments (desmin, neurofilaments, keratin, GFAP, etc.) promoters of therapeutic genes (MDR type , CFTR, factor VIII, etc.) specific promoters tissues (pyruvate kinase, villin, promoter of the intestinal fatty acid binding protein, promoter of actin ⁇ of smooth muscle cells, specific promoters for the liver; Apo AI, Apo AU, Human albumin, etc.) or promoters responding to a stimulus (steroid hormone receptor, retinoic acid receptor, etc.).
• these expression sequences can be modified by adding activation, regulation, etc. sequences.
• this picornavirus IRES sequence preferably derives from a picornavirus. More specifically, this picornavirus IRES sequence is derived either from the encephalomyocarditis virus or from the poliovirus. It is more preferably the fragment from encephalomyocarditis present in the vector pCITE-2a + of
• the construction defined by the transcriptional promoter, the two nucleic acids, the polyadenylation site and the IRES sequence present between the two nucleic acids will be identified below under the name bicistronic cassette.
• the viruses according to the present invention are defective, that is to say incapable of replicating autonomously in the target cell.
• the genome of the defective viruses used in the context of the present invention is therefore devoid of at least the sequences necessary for the replication of said virus in the infected cell. These regions can be either eliminated (in whole or in part), or made non-functional, or substituted by other sequences and in particular by the bicistronic cassette defined above.
• the defective virus nevertheless retains the sequences of its genome which are necessary for the packaging of the viral particles.
• the virus according to the invention can be derived from an adenovirus, an adeno-associated virus (AAV) or a retrovirus. According to a preferred embodiment, it is an adenovirus.
• AAV adeno-associated virus
• serotypes of adenovirus the structure and properties of which vary somewhat. Among these serotypes, it is preferred to use, within the framework of the present invention, human adenoviruses of type 2 or 5 (Ad 2 or Ad 5) or adenoviruses of animal origin (see application WO94 / 26914).
• adenoviruses of animal origin which can be used in the context of the present invention, mention may be made of adenoviruses of canine, bovine, murine origin (example: Mavl, Beard et al., Virology 75 (1990) 81), ovine, porcine , avian or even simian (example: after-sales service).
• the adenovirus of animal origin is a canine adenovirus, more preferably a CAV2 adenovirus [Manhattan strain or A26 / 61 (ATCC VR-800) for example].
• adenoviruses of human or canine or mixed origin are used.
• the El region at least is non-functional.
• the viral gene considered can be made non-functional by any technique known to those skilled in the art, and in particular by total suppression, substitution, partial deletion, or addition of one or more bases in the gene or genes considered. Such modifications can be obtained in vitro (on isolated DNA) or in situ, for example, by means of genetic engineering techniques, or by treatment with mutagenic agents.
• Other regions can also be modified, and in particular the region E3 (WO95 / 02697), E2 (WO94 / 28938), E4 (WO94 / 28152, WO94 / 12649, WO95 / 02697) and L5 (WO95 / 02697).
• the adenovirus according to the invention comprises at least one deletion in the region E 1 and a deletion in the region E3.
• the deletion in the E1 region preferably extends from nucleotides 455 to 3329 on the sequence of the adenovirus Ad5.
• the bicistronic cassette is inserted at the level of the deletion in the region E 1.
• a particular mode of the present invention therefore relates to a defective recombinant adenovirus characterized in that it comprises at least two nucleic acids coding for enzymes, proteins and / or co-factors distinct and involved in the reverse transport of cholesterol, said nucleic acids being linked operationally to a transcriptional promoter and separated from each other by a sequence coding for an internal entry site of the IRES ribosome.
• adenoviruses comprising at least one nucleic acid coding for LCAT and one nucleic acid coding for either LH, CETP or ApoAI, said nucleic acids being operably linked to a transcriptional promoter and separated from each other by a sequence coding for an internal entry site of the IRES ribosome.
• the defective recombinant adenoviruses according to the invention can be prepared by any technique known to a person skilled in the art (Levrero et al, Gene 101 (1991) 195, EP 185 573; Graham, EMBO J. 3 (1984) 2917). In particular, they can be prepared by homologous recombination between an adenovirus and a plasmid carrying, inter alia, the bicistronic cassette. Homologous recombination occurs after co-transfection of said adenovirus and plasmid in an appropriate cell line.
• the cell line used should preferably (î) be transformable by said elements, and (it), include the sequences capable of complementing the part of the genome of the defective adenovirus, preferably in integrated form to avoid the risks of recombination.
• a line mention may be made of the human embryonic kidney line 293 (Graham et al., J Gen. Virol 36 (1977) 59) which contains in particular, integrated into its genome, the left part of the genome of an Ad5 adenovirus (12%) or lines capable of complementing the E1 and E4 functions as described in particular in applications No. WO 94/26914 and WO95 / 02697.
• the adenoviruses which have multiplied are recovered and purified according to the conventional techniques of molecular biology, as illustrated in the examples.
• the claimed adenoviruses are prepared according to an original method described in patent application WO96 / 25506 which uses, as shuttle plasmid, a prokaryotic plasmid comprising a genome of bordered recombinant adenovirus by one or more restriction sites not present in said genome.
• This protocol is illustrated in particular in FIG. 2. This process is particularly interesting since it makes it possible to dispense with the use of a second construct providing another part of the viral genome, and the recombination step in the line of transcomplementation.
• a second object of the present invention relates specifically to particular prokaryotic plasmids used for the production of the claimed adenoviruses.
• these prokaryotic plasmids integrate the bicistronic cassette previously described.
• the present invention also relates to a prokaryotic plasmid comprising an adenovirus genome and at least two nucleic acids coding for enzymes, proteins and / or co-factors distinct and involved in the reverse transport of cholesterol, the two acids nucleic acids being operatively linked to a transcriptional promoter and separated from each other by a sequence coding for an internal ribosome entry site, IRES.
• the prokaryotic plasmids according to the invention comprise a first region allowing replication in prokaryotic cells and a second region comprising the adenoviral genome bordered by one or more restriction sites not present in said genome and in which at least two nucleic acids coding for enzymes, proteins and / or co-factors distinct and involved in the reverse transport of cholesterol are present, these two nucleic acids being operatively linked to a transcriptional promoter and separated from each other by a sequence encoding an internal ribosome entry site, IRES.
• IRES internal ribosome entry site
• This plasmid can be a derivative of RK2, of pBR322 (Bolivar et al., 1977), a derivative of pUC (Viera and Messing, 1982), or other plasmids derived from the same incompatibility group, that is to say ColEl or pMBl for example.
• These plasmids can also be chosen from other incompatibility groups which replicate in Escherichia coli. They may be plasmids derived from plasmids belonging to the incompatibility groups A, B, FI, Fil, FUI, FIV, Hl, Hl 1, II, 12, J, K, L, N, OF. P, Q, T, U, W, X, Y, Z or 9 for example.
• Plasmids can also be used, among which plasmids which do not replicate in E. coli but in other hosts such as B. subtilis, Streptomyces, P. putida, P. aeruginosa. Rhizobium meliloti, Agrobacterium tumefaciens, Staphylococcus aureus, Streptomyces pristinaespiralis, Enterococcus faecium or Clostridium.
• the origins of replication from plasmids replicating in E. coli are used.
• the adenoviral genome present in the plasmids of the invention is advantageously a complete or functional genome, that is to say not requiring the contribution of other regions, by recombination or ligation, for the production of viral stocks in the selected packaging lines.
• the recombinant adenoviral genome comprises at least ITR sequences and a sequence allowing the packaging.
• this genome of the adenovirus used is devoid of all or part of the E1 region.
• the genome of the adenovirus used is devoid of a part of the E1 region comprised between nucleotides 454 to 3328 (fragment PvuII-BglII) or 382 to 3446 (fragment HinfII-Sau3A).
• the genome of the adenovirus used is also devoid of all or part of the E3 and / or E4 region. According to a particular embodiment of the invention, it is an adenovirus genome lacking in regions E1 and E3.
• the prokaryotic plasmids claimed comprise in 5'-3 'orientation at least one origin of functional replication in prokaryotic cells, a first part of an adenoviral genome comprising the ITR ⁇ viral sequences, a transcriptional promoter, a first nucleic acid. coding for an enzyme, protein and / or a co-factor involved in the reverse transport of cholesterol, an IRES sequence, a second nucleic acid coding for a second enzyme, protein and / or a co-factor involved in the reverse transport of cholesterol , a polyadenylation site and a second part of an adenoviral genome comprising the pIX-IVA2 region.
• the prokaryotic plasmids claimed according to the invention also comprise a region allowing the selection of the prokaryotic cells containing said plasmid.
• This region can be constituted in particular by any gene conferring resistance to a product, and in particular to an antibiotic.
• the claimed prokaryotic plasmid comprises at least one nucleic acid coding for LCAT, the second nucleic acid being chosen from those coding for CETP, LH or ApoAI.
• the plasmid pXL2974 comprises in 5'-3 'orientation, an origin of replication, a gene for resistance to spectinomycin, the Sac B gene for sensitivity to sucrose.
• the plasmid pXL3058 comprises in 5'-3 'orientation, an origin of replication, a kanamycin resistance gene, the ITR ⁇ viral sequences, the RSV promoter, the two LCAT and intron + ApoAI transgenes separated by TIRES, the site of polyadenylation. the viral sequences pIX and IV A2 and the Sac B gene for sensitivity to sucrose.
• the present invention also extends to the plasmid constructs used for the construction of these prokaryotic plasmids and which also comprise the bicistronic cassette defined according to the invention.
• the prokaryotic plasmids claimed according to the invention can in particular be obtained by transformation of an initial shuttle plasmid comprising the bicistronic cassette defined according to the invention, namely comprising the sequence coding for a transcriptional promoter operatively linked to two nucleic acids coding for two enzymes, distinct proteins and / or co-factors involved in the reverse transport of cholesterol, a polyadenylation site and an IRES sequence located between said nucleic acids.
• prokaryotic cell containing a prokaryotic plasmid as defined above. It can in particular be any bacterium for which there is a vector system into which recombinant DNA can be introduced. Examples include Escherichia coli, Salmonella typhimurium, Bacillus subtilis, Pseudomonas putida, Pseudomonas aeruginosa, Agrobacterium tumefaciens, Rhizobium meliloti or bacteria of the genus Streptomyces. These cells are advantageously obtained by transformation according to techniques known to those skilled in the art.
• It also relates to the use of a recombinant virus, of a defective recombinant adenovirus or of a shuttle plasmid construction as defined above for the preparation of a pharmaceutical composition intended for the treatment or prevention of pathologies linked to hypoalphalipoproteinemia including more particularly atherosclerosis and / or restenosis.
• the present invention also relates to a pharmaceutical composition
• a pharmaceutical composition comprising one or more defective recombinant viruses including adenovirus as described above.
• Such compositions can be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, etc. administration.
• the composition according to the invention contains pharmaceutically acceptable vehicles for an injectable formulation.
• pharmaceutically acceptable vehicles for an injectable formulation may in particular be saline solutions (monosodium phosphate, disodium phosphate, sodium chloride, potassium, calcium or magnesium, etc., or mixtures of such salts), sterile, isotonic, or dry compositions, in particular lyophilized, which, by addition, as appropriate, of sterilized water or physiological saline, allow the constitution of injectable solutes.
• the doses of virus used for the injection can be adapted according to different parameters, and in particular according to the mode of administration used, the pathology concerned or the duration of the treatment sought.
• the recombinant viruses according to the invention are formulated and administered in the form of doses of between 10 ⁇ and 1014 pfu / ml.
• pfu plaque forming unit
• plaque forming unit corresponds to the infectious power of a suspension of virions, and is determined by infection of an appropriate cell culture, and measures, generally after 48 hours, the number of plaques of infected cells. The techniques for determining the pfu titer of a viral solution are well documented in the literature.
• the present invention offers a new, very effective means for the treatment or prevention of pathologies linked to hypoalphalipoproteinemia, in particular in the field of cardiovascular diseases such as myocardial infarction, Tangor, sudden death, cardiac decompensation, accidents. cerebrovascular, atherosclerosis or restenosis.
• cardiovascular diseases such as myocardial infarction, Tangor, sudden death, cardiac decompensation, accidents. cerebrovascular, atherosclerosis or restenosis.
• this treatment can concern both humans and any animal such as sheep, cattle, domestic animals (dogs, cats, etc.). horses, fish, etc.
• Figure 1 Schematic representation of the presumed mechanism of reverse cholesterol transport.
• Figure 2 Representation of the adenovirus production protocol by homologous recombination in E. Coli.
• Figure 3 Protocol for the construction of the bicistronic shuttle plasmid pXL 2970 comprising RSV-LCAT-IRES-CETP.
• Figure 4 Protocol for the construction of the prokaryotic plasmid pXL2974 comprising RSV-LCAT-IRES-CETP.
• Figure 5 Protocol for the construction of the plasmid pXL2984 comprising RSV-LCAT-IRES-LH.
• Figure 6 Representation of the prokaryotic plasmid pXL3058 comprising RSV-
• the plasmids used for the construction of the bicistronic recombinant adenoviruses LCAT-IRES-CETP, LCAT-IRES-LH and LCAT-IRES-ApoAI are: -pSK IRES (marketed by NOVAGEN) -pXL 2616 cDNA LCAT Séguret-Macé et al. Circulation 1996, 94 (9): 2177-2184
• Escherichia coli bacteria DH5 ⁇ subtype of genome EndAl, recAl, hsd
• the plasmids of type pBR322, pUC and the phages of the M 13 series are of commercial origin (Bethesda Research Laboratories).
• the DNA fragments can be separated according to their size by electrophoresis in agarose or acrylamide gels, extracted with phenol or with a phenol / chloroform mixture, precipitated with ethanol and then incubated in the presence of DNA ligase.
• phage T4 Biolabs
• the filling of the protruding 5 ′ ends can be carried out by the Klenow fragment of DNA DNA Polymerase I. coli (Biolabs) according to the supplier's specifications.
• the destruction of the prominent 3 'ends is carried out in the presence of T4 phage T4 DNA Polymerase (Biolabs) used according to the manufacturer's recommendations.
• the destruction of the protruding 5 ′ ends is carried out by gentle treatment with nuclease SI.
• Mutagenesis directed in vitro by synthetic oligodeoxynucleotides can be carried out according to the method developed by Taylor et al. [Nucleic Acids Res. J_3 (1985) 8749-8764] using the kit distributed by Amersham.
• the 293 cells are cultured on Eagle medium (MEM, Gibco BRL) supplemented with 10% fetal calf serum (SVF, Gibco BRL) at 37 ° C. and 5% CO 2.
• MEM Eagle medium
• SVF fetal calf serum
• the 293 cells cultured in 100 mm dishes on MEM medium and supplemented with 10% fetal calf serum are transfected with 1 ⁇ g of DNA with Hpofectamine (Gibco BRL). Six hours later, the medium is removed and the cells are incubated in complete medium (MEM + 10% FCS).
• the culture supernatants of cells transfected with the recombinant plasmids RSV LCAT-IRES-CETP, RSV LCAT-IRES-ApoAI and RSV LCAT-IRES-LH as well as the ⁇ -galactosidase controls and the supernatant of the non-transfected cells were collected 72 h after transfection and stored at 4 ° C.
• the activity tests of the enzymes LCAT, CETP, ApoAI and LH are carried out on fractions of 10 to 30 ⁇ l of cell supernatant using human plasma as a control.
• Bicistronic adenoviral DNA is obtained by homologous recombination in e. coli. Thereafter, it is linearized by Pacl and the virus produced after transfection of the 293 cells.
• the 293 cells which are complementary to the protein E1, are transfected, by lipofectamine, with 10 ⁇ g of linearized adenoviral DNA.
• the plaques containing the recombinant virus are removed and their restriction profile analyzed.
• LCAT activity is estimated by measuring the conversion of free C-cholesterol to
• proteoliposomes as a substrate as described by Chen et al. (1982).
• the proteoliposomes are prepared from phosphatidyl-choline, cholesterol, C-cholesterol and rapolipoprotein Al and incubated with 20 ⁇ l of transfection culture supernatant to be tested.
• the reaction products (free C-cholesterol and esterified C-cholesterol) are separated, by difference of their solubility in organic solvent, by thin layer chromatography and detected by autoradiography on Instant Imager (Packard).
• the LCAT activity is expressed as a percentage of the C-cholesterol esterified per hour and for 20 ⁇ l of the supernatant tested.
• CETP activity is determined by measuring the capacity of this enzyme to transfer cholesterol esters from a high density lipoprotein particle (donor) to a low density lipoprotein particle (acceptor).
• the substrates for this reaction were provided by a Wak-Chemie kit, Medical GmbH.
• the fluorescence of the cholesterol linoleate contained in the donor particle is quenched in the native state of the latter and it is only in the presence of the active CETP, which will catalyze the transfer of the fluorescent molecule to the acceptor particle, that fluorescence can be detected at 535 nm.
• CETP activity is expressed as the fluorescence intensity value emitted at 535 nm for 30 ⁇ l of culture supernatant tested and per hour.
• the hepatic lipase activity is estimated using a synthetic triglyceride substrate supplied by a Progen Biotechnik GmbH kit.
• This substrate contains a pyrene fluorescent group which is masked by trinitrophenol in the native state of the molecule and the hydrolysis of the latter has the effect of emitting fluorescence at 400 nm.
• Apo AI activity is estimated using an anti ApoAI monoclonal antibody.
• Immulon II plates (Dynatech) are coated with an anti ApoAI monoclonal antibody (10 mg / ml in carbonate buffer pH 9.6), by overnight incubation at 4 ° C, then saturated with 2% BSA in PBS pH 7.4 one hour at 37 ° C. The cell supernatants are then incubated for one hour at 37 ° C., optionally after dilution in PBS 2% BSA. The revelation is then carried out by incubation for one hour at 37 ° C. with a mixture of anti ApoAI monoclonal antibodies labeled with peroxidase, and diluted to 1/5000. The fixation of peroxide antibodies is finally revealed by incubation with 250 ⁇ l of TMB (KPL) and reading of the plates at 630 nm.
• KPL TMB
• the constructions of the bicistronic plasmids are detailed in FIGS. 3 to 5.
• the bicistronic plasmids obtained are, at the same time, the shuttle plasmids because they contain the adenoviral sequences pLX-IVa2 necessary for recombination with the viral genome. This recombination occurs either by cotransfection with a cleaved genome coming from a ⁇ -galactosidase virus (conventional shuttle vector), or by double recombination in E-Coli (shuttle vector Coli).
• the sequence conformity of different plasmid structures is examined by analysis of their restriction profile. This examination makes it possible to select recombinant clones which will be used for subsequent cloning as well as to validate the results of the cloning.
• the restriction enzymes are chosen so as to have the most complete information possible on the completeness of the cloned cDNA and the number of strategic sites for cloning. However, this check does not exclude the existence of certain mutations such as point substitution or nonsense mutations which can occur at any stage of cloning. Such mutations can only be demonstrated by complete sequencing of the cDNA in question.
• the ultimate control of the validity of the constructions obtained is made by biochemical tests of enzymatic activities of LCAT, CETP and LH in vitro, or the detection of the presence of TapoA-1.
• the expression vector pXL 2968 RSV LCAT polyA bGH is obtained by cleavage of the plasmids pXL2616, containing LCAT cDNA, on the one hand and of the plasmid pXL LPL, under the control of the RSV promoter and upstream of the polyadenylation signal of the hormone bovine growth, on the other hand, by the restriction enzymes Sal I and ClaI and ligation of the resulting fragments by T4 DNA ligase.
• the plasmids pXL RSV LPL (2 ⁇ g) and pXL 2616 (2 ⁇ g) are each digested with 10 units (u) of Clal, in buffer 4 (20mM Tris acetate, 10mM Mg-acetate, 50mM K-acetate and ImM DTT) supplemented with 100 ⁇ g / ml of acetylated BSA, for 90 'at 37 ° C. 100 mM NaCl and 10 ml of SalI are added and the reaction mixture is incubated again at 37 ° C. for a further 90 minutes.
• the recombinant plasmid pXL 2969 IRES-CETP is generated from the bluescript plasmid having cDNA for CETP modified at its 5 'end by introduction of an Ncol site by PCR primer 5' GCCTGATAAC CATGGTGGCT GCCACAG 3 '(SEQ ID NO: 1) .
• the plasmid thus modified is cleaved by Ncol and Sal and cloned downstream of the adenoviral IRES sequence included in the plasmid pSKIRES cleaved by the same restriction enzymes as follows:
• 2.5 ⁇ g of the plasmid CETP and 2.5 ⁇ g of the plasmid pSK IRES are digested with l or of Ncol for 90 'at 37 ° C in buffer 3 (50mM Tris-HCL, 10 mM MgC 12, 100 mM NaCl and ImM DTT) followed by digestion with Ou de Sali for 90 'at 37 ° C.
• the digestion products are put into electrophoretic migration and the 1.5 Kbp (CETP cDNA) and 3.5 Kbp bands are extracted and ligated under the same conditions as described in the previous paragraph.
• the plasmid pXL 2969 (4 ⁇ g) is first digested with 5U of SmaI (buffer 4, 90 'at 25 ° C) then with 5U of Hincll (buffer 3 + BSA, 100 mM NaCl, 90 'at 37 ° C). The digestion products of the 2 plasmids are subjected to electrophoretic migration on 0.7% agarose gel and the DNA bands of 8.5 Kbp (pXL 2968) and 2.2 Kbp (cDNA IRES CETP) are extracted with the Qiaquick kit and ligated with 400 u of T4 DNA ligase. (Fig. 3).
• the LCAT and CETP activities of the plasmid pXL2970 are assayed on the culture supernatant of 293 cells three days after transfection.
• the LCAT activity of this plasmid corresponds to 3.5% of cholesterol esters formed per hour and the CETP activity to 120% (Table 1 below). These activity values show that the plasmid pXL2970 synthesizes well LCAT and CETP which are catalytically active.
• the shuttle vector must include: the ITR sequences — reverse terminal repeats and ⁇ packaging sequence, necessary for homologous recombination in E. coli, surrounding the sequence
• sucrose B suicide gene lethal for the bacteria in culture on sucrose
• spectinomycin resistance gene which will allow the selection of the recombinant clone.
• the bicistronic shuttle plasmid pXL2970 RSV LCAT-IRES-CETP is cleaved by BstEII and Spel in order to introduce therein ITR and ⁇ sequences of the adenoviral genome and thus obtain a shuttle-coli plasmid.
• the ITR and ⁇ sequences are isolated by digestion of the plasmid pXL 2794 with BstEII and Xbal.
• the blunt-ended fragment containing the spectinomycin-sucrose B cassette, obtained by digestion of pXL2757 with EcoRV and SmaI was introduced into the shuttle plasmid pXL 2970 + 2794 linearized by bFspI (FIG. 4).
• the resulting DNA fragment is extracted with the Qiaquick kit and dephosphorylated with 2 u of the CIP (60 'at 37 ° C).
• the plasmid pXL 2794 is digested with 20 u of BstEII (buffer 2,
• the resulting plasmid pXL 2970 + 2794 (1.5 ⁇ g) is cleaved with 5 u of Fspl (buffer 4,
• the shuttle plasmid pXL2974 LCAT-IRES-CETP undergoes a first selection on spectinomycin medium and spectinomycin medium + sucrose.
• the results of the Ncol (6.2 + 3 + 2.2 + 2), NotI (13.5 Kpb) and EcoRV (1 + 3 + 9.5 Kpb) digestions are in accordance with the restriction map of said plasmid.
• the LCAT activity of the plasmid pXL2974 corresponds to 2% of cholesterol esters formed per hour and that of the CETP to 1 14% (Table 1). LCAT and CETP activities are found in the shuttle-coli plasmid LCAT-IRES-CETP.
• the LH cDNA is cloned behind TIRES in the bluescript vector and the IRES-LH fragment then included in a manner analogous to the LCAT-IRES-CETP vector according to the following protocol:
• the plasmid pXL 2971 (4 ⁇ g) is digested, in order to eliminate an Ncol site, with 40 IU of BglII (buffer 3, 90 'at 37 ° C) and then with 40u of Sali for 90' at 37 ° C.
• the DNA fragment of 2.5 Kbp (of approximate mass of 0.5 ⁇ g) resulting from these digests is subjected, after migration and extraction on gel, to digestion managed by Ncol (0.1-1 u Ncol / ⁇ g DNA) in buffer 4 for 60 'at 37 ° C.
• the digestion products are analyzed by migration on 0.7% agarose gel and the 1.5 Kbp band containing the LH ABC cDNA, obtained with 0.5 u of Ncol, is ligated (T4 DNA ligase, 400 u ) with the pSK IRES fragment (1.3 ⁇ g) resulting from the Ncol and Sali digestions (read from each enzyme, buffer 3, 37 ° C.).
• the final vector is presented in FIG. 5.
• the corresponding bicistronic shuttle plasmid LCAT-IRES-LH bears the name of pXL2984.
• LCAT is mutated by PCR by including an additional Ncol site allowing its ligation in good place behind TIRES. His sequence has been fully verified.
• the IRES-LCAT fragment is then ligated behind TapoA-1.
• the resulting vector is derived directly from Coli technology and bears the name of pXL 3058 (FIG. 6). After the usual double recombinations, the resulting viral vector was checked for activity. ApoA-I was detected in a western blot and the LCAT activity evaluated at 1.3% (background noise at 0.2%).
• NAME RHONE POULENC RORER S.A.

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