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  • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
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  • C12N2830/42—Vector systems having a special element relevant for transcription being an intron or intervening sequence for splicing and/or stability of RNA

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 new recombinant viruses comprising an inserted gene coding for all or part of lecithin cholesterol acyltransferase (LCAT) or a variant thereof.
• LCAT lecithin cholesterol acyltransferase
• the present invention also relates to pharmaceutical compositions comprising said recombinant viruses. More particularly, the present invention relates to defective recombinant viruses 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 hyperttriglyceridérnie, 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). 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.
• 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 cardio- very serious vascular events such as infarction, sudden death, cardiac decompensation, stroke, etc.
• LDL low density lipoproteins
• HDL high density lipoproteins
• dyslipidaemia and in particular hypercholesterolaemia are mainly treated by means of compounds acting either on the biosynthesis of cholesterol (hydroxymethylglutaryl-coenzymeA reductase inhibitors, statins), or on the uptake and elimination of bile cholesterol (sequestrants or resins), or again on lipolysis by a mode of action which remains to be elucidated on the molecular level (fibrates). Consequently, all the major classes of drugs, which have been used in this indication (sequestrants, fibrates or statins), are intended only for the preventive aspect of the formation of atheroma plaque and not in fact for the treatment of the atheroma.
• the current treatments for atheroma, post coronary accident are only palliative since they do not intervene on the homeostasis of cholesterol and they are surgical acts (coronary bypass, angioplasty).
• a first approach for the treatment of these pathologies by gene therapy has been described in application WO94 / 25073.
• This approach is based in particular on the direct transfer of genes coding for apolipoproteins.
• the present invention constitutes a new therapeutic approach for the treatment of pathologies linked to dyslipoproteinemias. It is based more particularly on the transfer of genes coding for enzymes involved in the catabolism of cholesterol.
• the transfer and expression in vivo of the LCAT according to the invention advantageously makes it possible to act not only on the level of circulating HDL, but also on their enzymatic activity linked to the reverse transport of cholesterol. This approach therefore has a double stimulating effect aimed at bringing cholesterol back to the liver.
• the present invention also relies on the use of viruses which make it possible to transfer and express genes coding for enzymes of cholesterol metabolism in the liver, and to secrete said enzymes in the circulatory system where they exercise their activity with a great efficiency.
• viruses which make it possible to transfer and express genes coding for enzymes of cholesterol metabolism in the liver, and to secrete said enzymes in the circulatory system where they exercise their activity with a great efficiency.
• the examples presented below indicate in particular that the adenoviruses are capable, depending on the mode of administration, of transferring and expressing efficiently, for a significant period and without cytopathological effect, the gene coding for lecithin cholesterol acyltransferase (LCAT).
• LCAT lecithin cholesterol acyltransferase
• a first object of the invention therefore resides in a defective recombinant virus containing at least one inserted gene coding for all or part of lecithin cholesterol acyltransferase (LCAT) or a variant thereof.
• LCAT lecithin cholesterol acyltransferase
• the invention also relates to the use of such a defective recombinant virus for the preparation of a pharmaceutical composition intended for the treatment or prevention of pathologies linked to dyslipoproteinemias.
• LCAT Human lecithi-cholesterol acyltransferase
• LCAT is an enzyme that catalyzes the esterification of free cholesterol by transfer of an acyl group of phosphatidyleholine to a hydroxyl residue of cholesterol, with the formation of cholesterol ester and lysophosphatidyleholine.
• LCAT high density lipoproteins
• the inserted gene can be a fragment of complementary DNA (cDNA), of genomic DNA (gDNA), or a hybrid construct 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 the LCAT or a variant thereof. Within the meaning of the present invention, the term variant denotes any mutant, fragment or peptide having at least one biological property of LCAT, as well as all natural variants of LCAT.
• 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, or even by cloning by expression, allowing the selection of variants according to their biological activity. . Genetic modifications include deletions, deletions, mutations, etc.
• the gene inserted within the meaning of the invention is preferably the gene coding for all or part of the human LCAT. It is more preferably a cDNA or a gDNA.
• the inserted gene also includes sequences allowing its expression in the infected cell. These may be sequences which are naturally responsible for the expression of said gene when 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 genes 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, CMV promoter, LTR-RSV, 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 VTII, etc.) tissue-specific promoters (pyruvate kinase, villin, promoter of the intestinal fatty acid binding protein, promoter of smooth muscle cell actin, specific promoters for the liver; Apo AI, Apo AU, human albumin etc) or the promoters responding to a stimulus (steroid hormone receptor, retinoic acid receptor, etc.).
• these expression sequences can be modified by adding activation sequences, regulation, etc.
• the inserted gene does not contain expression sequences, it can be inserted into the genome of the defective virus downstream of such a sequence.
• the inserted gene 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 may be the natural LCAT signal sequence, but it may also be any other functional signal sequence, or an artificial signal sequence.
• 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 inserted gene.
• 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.
• serotypes of adenoviruses there are different serotypes of adenoviruses, 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 defective adenoviruses of the invention comprise ITRs, a sequence allowing the encapsidation and the nucleic acid of interest.
• the E1 region at least is non-functional.
• the viral gene considered can be made non-functional by any technique known to 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
• the adenovirus according to the invention comprises a deletion in the regions E1 and E4. According to another preferred embodiment, it comprises a deletion in region E1 at the level of which the region E4 and the sequence coding for LCAT are inserted (Cf FR94 13355).
• 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) 0 195, EP 185 573; Graham, EMBO J. 3 (1984) 2917) .
• they can be prepared by homologous recombination between an adenovirus and a plasmid carrying inter alia the DNA sequence of interest. Homologous recombination occurs after co-transfection of said adenovirus and plasmid in an appropriate cell line.
• the cell line used should preferably (i) be transformable by said elements, and (ii), contain the sequences capable of complementing the part of the genome of the defective adenovirus, preferably in integrated form to avoid the risks of recombination.
• a line mention may be made of the human embryonic kidney line 293 (Graham et al., J. Gen. Virol. 36 (1977) 59) which contains in particular, integrated into its genome, the left part of the genome 0 Ad5 adenovirus (12%) or lines capable of complementing the El and E4 as described in particular in applications No. WO 94/26914 and WO95 / 02697.
• the adenoviruses which have multiplied are recovered and purified according to conventional techniques of molecular biology, as illustrated in the examples.
• AAV adeno-associated viruses
• the defective recombinant AAVs according to the invention can be prepared by co-transfection, in a cell line infected with a human helper virus (for example an adenovirus), of a plasmid containing the nucleic sequence of interest bordered by two inverted repeat regions (ITR) from AAV, and a plasmid carrying the packaging genes (rep and cap genes) from AAV.
• a human helper virus for example an adenovirus
• ITR inverted repeat regions
• the recombinant AAVs produced are then purified by conventional techniques.
• the invention therefore also relates to a recombinant virus derived from AAVs, the genome of which comprises a LCAT coding sequence bordered by AAV ITRs.
• the invention also relates to a plasmid comprising a sequence coding for LCAT bordered by two ITRs of an AAV.
• a plasmid can be used as such to transfer the LCAT sequence, optionally incorporated into a liposomal vector (pseudovirus).
• retroviruses are integrative viruses, infecting dividing cells.
• the genome of retroviruses essentially comprises two LTRs, an encapsidation sequence and three coding regions (gag, pol and env).
• gag, pol and env genes are generally deleted, in whole or in part, and replaced by a heterologous nucleic acid sequence of interest.
• These vectors can be produced from different types of retroviruses such as in particular MoMuLV ("murine moloney leukemia virus”; also designated MoMLV), MSV ("murine moloney sarcoma virus”), HaSV ("harvey sarcoma virus”) ; SNV ("spleen necrosis virus”); RSV ("rous sarcoma virus”) or the Friend virus.
• MoMuLV murine moloney leukemia virus
• MSV murine moloney sarcoma virus
• HaSV human moloney sarcoma virus
• SNV spleen necrosis virus
• RSV rous sarcoma virus
• Friend virus Friend virus
• a plasmid comprising in particular the LTRs
• the packaging sequence and said coding sequence is generally constructed, then used to transfect a cell line called packaging, capable to bring in trans retroviral functions deficient in the plasmid.
• packaging lines are therefore capable of expressing the gag, pol and env genes.
• packaging lines have been described in the prior art, and in particular the line PA317 (US4,861,719); the PsiCRIP line (WO90 / 02806) and the GP + envAm-12 line (WO89 / 07150).
• the recombinant retroviruses may include modifications at the level of the LTRs to suppress transcriptional activity, as well as extended packaging sequences, comprising a part of the gag gene (Bender et al., J. Virol. 61 (1987) 1639).
• the recombinant retroviruses produced are then purified by conventional techniques.
• the present invention also relates to a pharmaceutical composition
• a pharmaceutical composition comprising one or more defective recombinant viruses 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 can be in particular saline solutions (monosodium phosphate, disodium, 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 defective recombinant adenoviruses according to the invention can be administered according to different modes, and in particular by intravenous injection. Preferably, they are injected at the portal vein.
• retroviruses it may be advantageous to use infected cells ex vivo for their reimplantation in vivo, possibly in the form of neo-organs (WO94 / 24298).
• the doses of virus used for the injection can be adapted according to different parameters, and in particular according to the mode of administration used, the pathology concerned or the duration of the treatment sought.
• the recombinant viruses according to the invention are formulated and administered in the form of doses of between 10 4 and 10 14 pfu / ml.
• doses of 10 6 to 10 10 pfu / ml can also be used.
• pfu plaque forming unit
• compositions of the invention may also contain one or more defective recombinant adenoviruses containing an inserted gene coding for an apolipoprotein.
• the association of these two types of genes exerts a synergistic effect on the activity of HDL and thus on the reverse transport of cholesterol.
• the construction of adenovirus containing an inserted gene coding for an apolipoprotein was described in application WO94 / 25073.
• a preferred combination comprises an adenovirus according to the invention and an adenovirus containing a gene coding for apolipoprotein AI or apolipoprotein AIV.
• the present invention provides a new, very effective means for the treatment or prevention of pathologies linked to dyslipoproteinemias, in particular in the field of cardiovascular affections such as myocardial infarction, angina, sudden death, cardiac decompensation, accidents. cerebrovascular, atherosclerosis or restenosis. More generally, this approach offers a very promising therapeutic intervention for each case where a genetic or metabolic deficit of the LCAT can be corrected.
• this treatment can concern both humans and any animal such as sheep, cattle, domestic animals (dogs, cats, etc.), horses, fish, etc.
• Figure 1 Representation of the plasmid pXL2639.
• FIG. 2 Representation of the plasmid pXL2640.
• Flgure3 Transfection of Hep3B cells with an adeno AdCMV hLCAT.
• the Hep3B cells were infected with an adeno AdCMV hLCAT (empty squares) or adeno AdCMV ⁇ gal (filled squares) at multiplicities of infection of 10, 25, 50, 100, 250 and 500.
• the LCAT activity was measured in the supernatant at 72 h. The determinations were made in duplicate and each value represents the mean ⁇ standard deviation.
• FIG. 4 Analysis of TARN by Northern blot, isolated from the liver of infected or non-infected mice.
• the total RNA comes from the livers of control mice (1), infected with the adeno AdCMV ⁇ gal (2) and the adeno AdCMV hLCAT (3). 10 ⁇ g of RNA were separated by formaldehyde-1.2% agarose electrophoresis, transferred to a nylon membrane and hybridized with various human LCAT and mouse apoE probes
• FIGS 5A and 5B Effect of the transfer of the human LCAT gene on plasma concentrations of total cholesterol and HDL cholesterol.
• Plasma concentrations of total cholesterol and HDL-cholesterol mean ⁇ standard deviation
• control mice empty squares
• 1 x 10 9 pfu of adeno AdCMV hLCAT empty circles
• 1 x 10 9 pfu d adeno AdCMV ⁇ gal filled squares
• transgenic mice expressing human apolipoprotein AI.
• FIG. 6 Effect of human LCAT gene transfer on plasma concentrations of human apoA-I.
• Plasma concentrations of human apoA-I mean ⁇ standard deviation
• control mice empty squares
• 1 ⁇ 10 9 pfu of adeno AdCMV hLCAT empty circles
• 1 ⁇ 10 9 pfu of adeno AdCMV ⁇ gal filled squares
• transgenic mice expressing human apolipoprotein AI.
• Figure 8 Effect of the transfer of the human LCAT gene on the sizes of HDL particles.
• the plasma comes from the mice, 5 days after the injection of 1 ⁇ 10 9 pfu of adeno AdCMV hLCAT (solid line) and controls (broken line).
• the plasma was separated on a polyacrylamide gel (gradient 4-20%) and transferred by Western blot and human apoA-I is then revealed by specific antibodies against human apoA-I. The blot is then scanned by densitometry.
• Figure 9 Effect of the transfer of the human LCAT gene on the mobility of particles containing apoA-I.
• the plasma comes from the mice, 5 days after the injection of 1 ⁇ 10 9 pfu of adeno AdCMV ⁇ gal (1), 5 ⁇ 10 8 pfu of adeno AdCMV hLCAT (2) or 1 ⁇ 10 9 pfu of adeno AdCMV hLCAT (3).
• 2 ⁇ l of plasma are used to separate the HDLs by electrophoresis on agarose gel followed by staining of the lipids with black Sudan.
• Figure 10 Effect of the transfer of the human LCAT gene on the ability of the serum to promote efflux of cholesterol.
• the plasma comes from mice, 5 days after the injection of 1 ⁇ 10 9 pfu of adeno AdCMV hLCAT (open circles), 1 ⁇ 10 9 pfu of adeno AdCMV ⁇ gal (solid squares) or control mice (open squares).
• the cholesterol efflux is calculated by measuring the radioactivity in the medium and in the cells after the incubation of the serum diluted to 2.5% with Fu5Ah cells preloaded with radioactive cholesterol.
• the plasmids of the pBR322, pUC type and the phages of the Ml 3 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.
• phage T4 ligase Biolabs
• the filling of the prominent 5 ′ ends can be carried out by the lenow fragment of DNA Polymerase I of E. coli (Biolabs) according to the supplier's specifications.
• the destruction of the protruding 3 ′ ends is carried out in the presence of the DNA polymerase of phage T4 (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. 13 (1985) 8749-8764] using the kit distributed by Amersham.
• PCR Polymerase-catalyzed Chain Reaction, Saiki RK et al., Science 230 (1985) 1350- 1354; Mullis KB and Faloona FA, Meth. Enzym. 1 __ ⁇ (1987) 335-350]
• PCR Polymerase-catalyzed Chain Reaction, Saiki RK et al., Science 230 (1985) 1350- 1354; Mullis KB and Faloona FA, Meth. Enzym. 1 __ ⁇ (1987) 335-350
• DNA thermal cycler Perkin El er Cetus
• Verification of the nucleotide sequences can be carried out by the method developed by Sanger et al. [Proc. Natl. Acad. Sci. USA, 74 (1977) 5463-5477] using the kit distributed by Amersham.
• Example 1 Construction of a defective recombinant adenovirus containing the human lecithin cholesterol acyltransferase (hLCAT) gene:
• the defective recombinant adenoviruses were prepared by homologous recombination between an adenovirus and a plasmid carrying, inter alia, the gene which it is desired to insert, after co-transfection into an appropriate cell line.
• Plasmid pXL2616 contains the cDNA encoding human lecithin cholesterol acyltransferase.
• the DNA fragment corresponding to the LCAT cDNA was isolated by the RT-PCR technique from the total RNA of HepG2 cells (First-Strand cDNA synthesis Kit, Pharmacia).
• the cDNAs were produced by reverse transcription of the polyadenylated RNAs using hexanucleotide primers.
• a PCR reaction was then carried out on these cDNAs with the oligonucleotides Sq5209: CCC TCG AGG CCA TCG ATG AGG CCT GAC TTT TTC AAT AAA (SEQ ID n ° 1) and Sq5287: GCG TCG ACA GCT CAG TCC CAG GCC TCA GAC GAG (SEQ ID No.
• Plasmids pXL2639 and pXL2640 contain the human LCAT cDNA, under the control of the CMV early promoter and the RSV virus LTR promoter, respectively.
• the DNA was introduced via a calcium phosphate-DNA complex according to the method of Wilger et al., Cell, 11 (1977) 223.
• LCAT activity was measured on cell supernatants 60 hours after transfection, according to the method of Chen and Albers, JLR, 23 (1982) 680.
• the measurement is based on the use of proteoliposomes as exogenous substrate, prepared by incubation of 30 minutes of apoA-I, 14C cholesterol, phosphatidylcholine at a molar ratio of 0.8: 12.5: 250 at 37 ° C.
• the activity is determined by measuring the conversion of 14C-cholesterol into 14C-cholesterol ester after incubation of the substrate with 4 ⁇ l of plasma or culture supernatant for 2 hours at 37 ° C.
• the esters formed are separated by thin layer chromatography on silica plates using a petroleum ether-diethyl ether-acetic acid 76: 20: 1 mixture and the radioactivity is determined by liquid scintillation spectrometry.
• the plasmids prepared in A were then linearized and cotransfected for recombination with the deficient adenoviral vector, in helper cells (line 293) providing in trans the functions encoded by the El (El A and E1B) o adenovirus regions.
• the Ad.CMVLCAT adenovirus was obtained by homologous in vivo recombination between the Ad.RSV ⁇ gal adenovirus (Stratford-Perricaudet et al., J. Clin. Invest 90 (1992) 626) and the plasmid pXL2639 according to the following protocol: plasmid pXL2639 linearized by the enzyme Xmnl and the adenovirus Ad.RSV ⁇ gal linearized by Clal are co-transfected in line 293 in the presence of calcium phosphate to allow homologous recombination. The recombinant adenoviruses thus generated are selected by plaque purification.
• the recombinant adenovirus is amplified in cell line 293, which leads to a culture supernatant containing the unpurified recombinant defective adenovirus having a titer of approximately 10 10 0 pfu / ml.
• the viral particles are purified by centrifugation on a cesium chloride gradient according to known techniques (see in particular Graham et al., Virology 52 (1973) 456).
• the Ad.CMVLCAT adenovirus is stored at -80 ° C in 20% glycerol.
• the expression and the functionality of the enzyme were tested after infection of Hep3B cells (cell line of human hepatocytes) by the recombinant adenovirus AdCMV-hLCAT at MOIs of 10, 25, 50, 100, 250 and 500.
• the recombinant adenovirus AdCMV ⁇ gal was used as a control.
• LCAT activity total amount of cholesterol esters produced in 1 hour in 100 ⁇ l of culture medium was measured on cell supernatants 72 hours after infection, according to the method of Chen and Albers, JLR, 23 ( 1982) 680.
• the results (FIG. 3) show that the human LCAT secreted into the culture medium is functional and that the level of expression of the enzyme is dependent on the viral concentration in the cells.
• mice transgenic for human apoA-I were infected by injection into the tail vein of recombinant adenovirus AdCMV-hLCAT (5 x 10 8 or 1 x 10 9 pfu), AdCMV- ⁇ Gal (1 x 10 9 pfu) or non-viral solution.
• Very high levels of LCAT activity were detected in the plasma of mice infected with AdCMV-hLCAT (from 3266 ⁇ 292 to 9068 ⁇ 812 nmol / ml / h) 5 days after injection, while the levels observed in mice not infected or infected with AdCMV- ⁇ Gal correspond to the basal LCAT activity of mouse plasma.
• mice infected with 1 x 10 9 pfu of AdCMV-hLCAT have plasma HDL-cholesterol and total cholesterol (CT) levels 7 and 6 times higher, respectively, than levels obtained in control mice ( Figure 5a and 5b) . These variations are associated with an increase in both esterified cholesterol (EC) and free cholesterol (CL), 8 and 2.5 times respectively compared to the levels obtained in the control mice.
• the increase in plasma CE leads to an increase in the CE / CT ratio in the HDL fraction.
• Mice infected with 1 ⁇ 10 9 pfu of AdCMV-hLCAT show a 2.5-fold increase in the concentration of human apoA-I compared with the control mice (FIG. 6).
• mice Infected mice Infected mice Control infected mice with AdCMV ⁇ gal by AdCMV- by AdCMV-
• esters of 68 ⁇ 8 71 ⁇ 10 319 ⁇ 22 ° 587 ⁇ 41 a cholesterol (CE)
• VLDL + LDL 15 ⁇ 3 20 ⁇ 6 33 ⁇ 12 d 30 ⁇ 3 c - e -TC
• the distribution of cholesterol in the lipoproteic fractions was carried out from pools of mouse plasmas by analytical chromatography by gel filtration (fig. 7). The concentrations of CT and human apoA-I were determined in the eluted fractions. These analyzes reveal a significant accumulation of cholesterol in the HDL fraction as well as an increase in size of HDL for the mice infected with 1 ⁇ 10 9 pfu of AdCMV-hLCAT compared to the control mice. Human apoA-I is found associated with particles the size of HDL.
• apoA-I containing lipoproteins in transgenic mice for human apoA-I has been shown to be bimodal, with peak sizes of 9.4 nm and llnm. While in control mice, this same distribution is conserved, it is altered in mice infected with AdCMV-hLCAT. For the mice having received 1 ⁇ 10 9 pfu of AdCMV-hLCAT, the smaller peak disappears in favor of two larger peaks of 13.3 and 14.2 nm (FIG. 8).
• Plasma lipoproteins were separated by electrophoresis in a non-denaturing agarose gel, followed by lipid detection. As shown in Figure 9, HDLs with pre-alpha mobility appear in the plasmas of mice infected with AdCMV-hLCAT, revealing that not only the size of HDL is affected but also the charges on the surface of HDL.
• Example 6 Effects of Expression of Human LCAT on the Efflux of Cellular Cholesterol
• the efflux of cell cholesterol was determined after incubation of the Fu5AH rat hepatoma cells with pools of plasma from infected or uninfected mice.
• FIG. 10 shows that a 65% increase in efflux is obtained with the plasma of mice infected with AdCMV-hLCAT compared to the plasma of mice infected with AdCMV ⁇ gal. It appears that this increase is related to the higher concentrations of human apoA-I and HDL-cholesterol in mice infected with AdCMV-hLCAT. These results are in favor of greater efficiency of reverse transport of cholesterol resulting from the high expression of human LCAT.
• NAME RHONE POULENC RORER S.A.

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