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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
  • C12N15/86—Viral vectors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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  • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
  • 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
  • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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  • C12N2810/00—Vectors comprising a targeting moiety
  • C12N2810/10—Vectors comprising a non-peptidic targeting moiety
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  • C12N2810/00—Vectors comprising a targeting moiety
  • C12N2810/40—Vectors comprising a peptide as targeting moiety, e.g. a synthetic peptide, from undefined source
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  • C12N2810/00—Vectors comprising a targeting moiety
  • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N2810/00—Vectors comprising a targeting moiety
  • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
  • C12N2810/80—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates
  • C12N2810/85—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian
  • C12N2810/859—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian from immunoglobulins

Definitions

• the present invention relates to the field of gene therapy and is more particularly concerned with the in vitro, ex vivo and / or in vivo transfer of genetic material.
• it proposes a new composition useful for efficiently transfecting cells.
• Chromosomal deficiencies and / or anomalies are the cause of many diseases, whether inherited or not. For a long time, conventional medicine was powerless against them. Today, with the development of gene therapy, we hope to be able to correct or prevent this type of chromosomal aberration from now on. This new medication consists in introducing genetic information into the affected cell or organ, with a view to correcting this deficiency or anomaly, or even expressing a protein of interest there.
• nucleic acid The major obstacle to the penetration of a nucleic acid into a target cell or organ, rests on the size and polyanionic nature of this nucleic acid which oppose its passage through cell membranes.
• the first uses the natural transfection vectors that are viruses and the second relies on the use of chemical or biochemical vectors.
• vectors of viral origin they are today widely used for cloning, transfer and expression of genes in vitro (for the production of recombinant proteins, the performance of screening tests, the study regulation of genes, etc.), ex vivo or in vivo (for the creation of animal models, or in gene transfer approaches of therapeutic interest).
• viruses there may be mentioned in particular adenoviruses, adeno-associated viruses (AAV), retroviruses, herpes viruses or vaccinia.
• Adenoviridae The family of Adenoviridae is widespread in mammals and birds and includes more than one hundred different serotypes of non-enveloped double-stranded DNA viruses, having a capsid of icosahedral symmetry (Horwitz, In: Fields BN, Knipe DM, Howley PM, ed Virology, Third Edition, Philadelphia Ed: Raven Publishers, 1996: 2149-2171).
• Their genome includes in particular a repeated inverted sequence (ITR) at each end, an encapsidation sequence (Psi), early genes and late genes.
• ITR inverted sequence
• Psi encapsidation sequence
• the main early genes are contained in the E1, E2, E3 and E4 regions. Among these, the genes contained in the E1 region are necessary for viral propagation.
• the main late genes are contained in regions L1 to L5.
• Ad5 adenovirus The genome of the Ad5 adenovirus has been fully sequenced and is accessible on the database (see in particular Genebank M73260). Likewise, parts or even all of other adenoviral genomes (Ad2, Ad7, Ad 12, etc.) have also been sequenced.
• the adenovirus has a very wide cellular tropism. Unlike the retrovirus whose cycle is dependent on cell division, it can advantageously infect cells in active division such as quiescent cells and its genome is maintained in episomal form. In addition it can be produced with high titers (10 ⁇ pfu / ml). These major assets have made it a vector of choice for the cloning and expression of heterologous genes.
• Group C adenoviruses, in particular type 2 and 5, as well as canine adenoviruses of CAV-2 type, the molecular biology of which is best known, are at the origin of the vectors currently used.
• adenoviral vectors have been used for the cloning and expression of genes in vitro (Gluzman et al., Cold Spring Harbor, New York 1 1724, p. 187), for the creation of transgenic animals ( WO95 / 22616) for the transfer of genes into cells ex vivo (WO95 / 14785; WO95 / 06120) or also for the transfer of genes into cells in vivo (see in particular WO93 / 19191, WO94 / 24297, WO94 / 08026) .
• AAV adeno-associated viruses
• vectors derived from AAVs for gene transfer in vitro and in vivo has been described in the literature (see in particular WO 91/18088; WO 93/09239; US 4,797,368, US 5,139,941, EP 488,528).
• These vectors and more particularly the adenoviruses prove to be efficient in terms of transfection.
• the process of infection of cells with a viral transfection vector generally takes place in two stages. First, there is recognition of target receptors on the surface of the cells to allow attachment of the recombinant virus.
• the second step then consists in an interaction of the viral protein penton, at the base of the fiber, via a peptide motif RGD with the cellular integrins avb3 and / or avb5 leading to the internalization of the virion.
• the efficiency with which recombinant viruses can infect a cell is very variable depending on the cell types studied in culture or the tissues whose targeting is important for in vivo treatment. In all cases, it is the first step of attachment that determines the internalization efficiency of the virion.
• the quantity of receptors on the cell surface is therefore the limiting factor in the infection process and it is clear that any approach aimed at circumventing or making easier this attachment step will overcome this obstacle.
• a first objective of the present invention aims precisely to increase the transfection efficiency of these viral vectors by optimizing their internalization at the level of the target cell to be treated.
• Another objective targeted and achieved by the present invention relates to the reduction or even the suppression of the immune response conventionally manifested by the cells treated against the viral vector.
• the administration of recombinant viruses such as recombinant adenoviruses defective for replication (Yang et al., PNAS (1994) 4407) induces an important immune response.
• One of the major roles of the immune system is to destroy the non-self or self-altered elements.
• the administration of a gene therapy vector of viral origin introduces non-self motifs into the body.
• cells infected with such a vector and thereby expressing an exogenous gene become self-altering elements.
• the present invention makes it possible to achieve these two objectives by associating with the recombinant virus considered a chemical or biochemical compound which assists it effectively in its transduction and protects it from the reactions of the immune system manifested against it.
• viral vectors As mentioned previously, in addition to viral vectors, it is also developed in gene therapy for non-viral transfection vectors. More precisely, these are chemical or biochemical vectors. These synthetic vectors have two main functions, to compact the DNA to be transfected and to promote its cellular fixation as well as its passage through the plasma membrane and, where appropriate, through the two nuclear membranes.
• these chemical vectors are on the other hand more advantageous on the immune level. They do not show pathogenic power, the risk of DNA multiplication within these vectors is zero and it does not there is no theoretical limit attached to them with regard to the size of the DNA to be transfected.
• cationic polymers of polylysine and DEAE dextran type or else lipofectants are the most advantageous. They have the property of condensing DNA and promoting its association with the cell membrane. More recently, the concept of targeted transfection mediated by a receptor has been developed. This technique takes advantage of the principle of condensing DNA, thanks to the cationic polymer, while directing the binding of the complex to the membrane using a chemical coupling between the cationic polymer and the ligand of a membrane receptor, present on the surface of the cell type that we want to graft. The targeting of the transferrin, insulin receptor or the hepatocyte asialoglycoprotein receptor has thus been described.
• the main object of the present invention is precisely based on the association of at least one of these chemical or biochemical vectors with a recombinant virus comprising in its genome at least one exogenous nucleic sequence with a view to more effectively promoting cell transduction of this recombinant virus.
• the present invention advantageously takes advantage of both the property of vehicles of the liposome or lipofectant type chemical vectors capable of fusing with a wide range of cell types and the efficiency of internalization of the recombinant viruses such as more particularly the adenovirus.
• the virion as a transducing agent of a gene of therapeutic interest
• the lipofectant as a transfection assistant. This synergy manifests itself advantageously on two levels.
• the present invention therefore has as its first object a composition useful in gene therapy, characterized in that it combines with one or more viruses which are not recombinant envelopes comprising in their genome at least one exogenous nucleic acid at least one non-viral and non-plasmid transfection agent.
• non-viral transfection agent it is preferably chosen from cationic polymers and lipofectants.
• lipofectants it is intended to cover, within the meaning of the invention, under this name, any compound of lipid nature and already proposed as active agent with regard to cellular transfection of nucleic acids.
• they are amphiphilic molecules comprising at least one lipophilic region associated or not with a hydrophilic region.
• lipids capable of forming liposomes such as POPC, phosphatidylserine, phosphatidylcholine, cholesterol, lipofectamine, maleimidophenylbutyrylphosphatidylethanolamine, lactosylceramide in the presence or not of polyethylene glycol to form liposomes with or without antibodies or ligands, to form targeted immunoliposomes or liposomes.
• the lipid agent used has a cationic region.
• This cationic region preferably polyamine, cationically charged, is probably associated in a reversible manner with the recombinant virus.
• DOTMA lipophilic group associated with an amino group via a so-called "spacer” arm
• DOTMA lipophilic group associated with an amino group via a so-called "spacer” arm
• DOTAP, DOBT or ChOTB can in particular be cited as representative of this category of cationic lipids.
• Other compounds, such as DOSC and ChOSC are characterized by the presence of a choline group in place of the quaternary ammonium group.
• the lipofectants suitable for the invention can also be chosen from lipopolyamines whose polyamine region corresponds to the general formula
• m is an integer greater than or equal to 2 and n is an integer greater than or equal to 1, m being able to vary between the different carbon groups comprised between 2 amines, this polyamine region being covalently associated with a lipophilic region of the hydrocarbon chain type, saturated or not, cholesterol, or a natural or synthetic lipid capable of forming lamellar or hexagonal phases.
• This polyamine region is more preferably represented by spermine, thermine or one of their analogs having retained its binding properties to nucleic acid.
• Patent application EP 394 111 describes lipopolyamines of this family capable of being used in the context of the present invention. Mention may more particularly be made, as representative of these lipopolyamines, of dioctadecylamidoglycyl spermine (DOGS) and 5-carboxyspermylamide of palmitoylphosphatidylethanolamine (DPPES).
• DOGS dioctadecylamidoglycyl spermine
• DPES 5-carboxyspermylamide of palmitoylphosphatidylethanolamine
• lipopolyamines described in patent application WO96 / 17823 can also be used advantageously according to the invention. They are represented by the general formula H2N - (- (CH) m-NH-) nH R
• -X and X ' represent, independently of one another, an oxygen atom, a methylene group - (CH2) q- with q equal to 0, 1, 2 or 3, or an amino group -NH - or -NR'- with R representing a C 1 -C 4 alkyl group
• - R3, R4 and R5 represent, independently of one another, a hydrogen atom or an alkyl radical, substituted or unsubstituted, C 1 to C 4, with p being able to vary between 0 and 5
• - R5 represents a cholesterol derivative or a group amino alkyl -NR1R2 with R1 and R2 representing, independently of one another, an aliphatic radical, saturated or unsaturated, linear or branched at C 2 to C 22.
• lipopolyamines As a representative of these lipopolyamines, mention may very particularly be made of 2-5-bis- (3-amino-propylamino) -pentyl (Dioctadecyl-carbamoylmethoxy) acetate and 1,3-bis (Dioctadecyl-carbamoylmethoxy) -acetate - (3-amino-propylamino) -2 propyl.
• - RI, R2 and R3 represent independently of each other a hydrogen atom or a group - (CH2) q-NRR 'with q which can vary between 1, 2, 3, 4, 5 and 6 this independently between the different groups RI, R2 and R3 and R and R 'independently of one another representing a hydrogen atom or a group - (CH 2 ) q'-NH 2 , q' which can vary between 1, 2, 3, 4, 5 and 6 this independently between the different groups R and R ', - m, n and p represent, independently of one another, an integer which can vary between 0 and 6 with when n is greater than 1, m can take different values and R3 different meanings within the general formula and -R4 represents a grouping of general formula
• - R6 and R7 independently of one another represent a hydrogen atom or an aliphatic radical, saturated or not, in CIO to C22 with at least one of the two groups being different from hydrogen
• u is an integer chosen between 0 and 10 with when u is an integer greater than 1 R5
• X, Y and r may have different meanings within the different units [X- (CHR5) rY], -X represents an atom d oxygen, sulfur or an amino group monoalkylated or not, - Y represents a carbonyl group or a methylene group
• - R5 represents a hydrogen atom or a side chain of natural amino acid, optionally substituted and -r represents an integer varying between 1 and 10 with when r is equal to 1, R5 representing a side chain of substituted or unsubstituted natural amino acid and when r is greater than 1, R5 representing a hydrogen atom.
• Mention may more particularly be made, as representative of these lipopolyamines, of the following:
• the non-viral vector is represented by at least one cationic polymer and more preferably by a compound of general formula
• R can be a hydrogen atom or a group of formula
• n is an integer between 2 and 10, p and q whole numbers, it being understood that the sum p + q is such that the average molecular weight of the polymer is between 100 and 10 ⁇ Da.
• n is an integer between 2 and 10
• p and q whole numbers it being understood that the sum p + q is such that the average molecular weight of the polymer is between 100 and 10 ⁇ Da.
• polymers of polyethylene imine (PEI) and polypropylene imine (PPI) have quite advantageous properties.
• the polymers preferred for the implementation of the present invention are those whose molecular weight is between 10 ⁇ and 5.10 ⁇ .
• PEI50K or PEI800K are commercially available.
• the other polymers represented by the general formula above they can be prepared according to the process described in patent application FR 94 08735.
• lipofectamine dioctadecylamidoglycyl spermine (DOGS), 5-carboxyspermylamide of palmitoylphosphatidylethanolamine (DPPES), (2-5-bis-dioctadecyl-carbamoylmethoxy) -acetate
• DOGS dioctadecylamidoglycyl spermine
• DPES 5-carboxyspermylamide of palmitoylphosphatidylethanolamine
• (2-5-bis-dioctadecyl-carbamoylmethoxy) -acetate can be used in the context of the invention.
• the recombinant viruses used 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 nucleic acid.
• the defective virus retains nevertheless the sequences of its genome which are necessary for the packaging of the viral particles.
• the recombinant virus used in the context of the present invention derives from an enveloped virus.
• an enveloped virus As a representative and non-limiting example of this type of virus, mention may more particularly be made of adenoviruses and adeno-associated viruses (AAV). According to a preferred embodiment, it is an adenovirus.
• adenoviruses of type 2 or 5 Ad 2 or Ad 5
• Ad 2 or Ad 5 adenoviruses of animal origin
• adenoviruses of animal origin mention may be made of adenoviruses of canine, bovine, murine origin (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.
• 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 comprises a deletion in the El and E4 regions.
• it comprises a deletion in the E1 region at the level of which the E4 region and the coding sequence are inserted.
• the deletion in the E1 region preferably extends from nucleotides 455 to 3329 on the sequence of the adenovirus Ad5.
• the exogenous nucleic acid sequence is inserted at the level of the deletion in the E1 region.
• the defective recombinant adenoviruses according to the invention can be prepared by any technique known to those skilled in the art (Levrero et al., Gene 101 (1991) 195, EP 185 573; Graham, EMBO J. 3 (1984) 2917). In particular, they can be prepared by homologous recombination between an adenovirus and a plasmid carrying inter alia the exogenous nucleic acid. Homologous recombination occurs after co-transfection of said adenovirus and plasmid in an appropriate cell line.
• the cell line used must preferably (i) be transformable by said elements, and (ii), contain the sequences capable of complementing the part of the genome of the defective adenovirus, preferably in integrated form to avoid the risks of recombination.
• a line mention may be made of the human embryonic kidney line 293 (Graham et al., J. Gen. Virol. 36 (1977) 59) which contains in particular, integrated into its genome, the left part of the genome an Ad5 adenovirus (12%) or lines capable of complementing the E1 and E4 functions as described in particular in applications No. WO 94/26914 and WO95 / 02697.
• adenoviruses which have multiplied are recovered and purified according to conventional techniques of molecular biology, as illustrated in the examples.
• the adenoviruses used according to the invention can also be obtained according to an original method described in patent application WO96 / 25506 which uses, as shuttle plasmid, a prokaryotic plasmid comprising a recombinant adenovirus genome bordered by one or more several restriction sites not present in said genome.
• compositions which combines with a recombinant adenovirus comprising in its genome at least one exogenous nucleic acid, a lipofectamine in an amount sufficient to improve its cellular transduction.
• the nucleic acid incorporated into the genome of the recombinant virus can be either a deoxyribonucleic acid or a ribonucleic acid. They may be sequences of natural or artificial origin, and in particular genomic DNA, cDNA, mRNA, tRNA, rRNA, hybrid sequences or synthetic or semi-synthetic sequences of modified or unmodified oligonucleotides. These nucleic acids can be of human, animal, plant, bacterial, viral, etc. origin. They can be obtained by any technique known to those skilled in the art, and in particular by screening of banks, by chemical synthesis, or also by mixed methods including chemical or enzymatic modification of sequences obtained by screening of banks.
• deoxyribonucleic acids can be single or double stranded as well as short oligonuleotides or longer sequences. These deoxyribonucleic acids can carry therapeutic genes, transcription or replication regulatory sequences, modified or unmodified antisense sequences, regions of binding to other cellular components, etc.
• therapeutic gene in particular means any gene coding for a protein product having a therapeutic effect.
• the protein product thus coded can be a protein, a peptide, etc. This protein product can be homologous with respect to the target cell (that is to say a product which is normally expressed in the target cell when the latter presents no pathology).
• the expression of a protein makes it possible for example to compensate for an insufficient expression in the cell or the expression of an inactive or weakly active protein due to a modification, or else to overexpress said protein.
• the therapeutic gene can also code for a mutant of a cellular protein, having increased stability, modified activity, etc.
• the protein product can also be heterologous towards the target cell.
• an expressed protein can, for example, supplement or bring about a deficient activity in the cell, allowing it to fight against a pathology, or stimulate an immune response.
• therapeutic products within the meaning of the present invention, there may be mentioned more particularly enzymes, blood derivatives, hormones, lymphokines: interleukins, interferons, TNF, etc. (FR 9203120), growth factors, neurotransmitters or their precursors or synthetic enzymes, trophic factors: BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NT5, HARP / pleiotrophin, etc.
• enzymes blood derivatives, hormones, lymphokines: interleukins, interferons, TNF, etc. (FR 9203120), growth factors, neurotransmitters or their precursors or synthetic enzymes, trophic factors: BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NT5, HARP / pleiotrophin, etc.
• dystrophin or a minidystrophin FR 9111947
• the CFTR protein associated with cystic fibrosis the genes associated with the arrest of cell division and in particular the gax gene, the tumor suppressor genes: p53, Rb, RaplA, DCC, k-rev, etc.
• FR 93 04745 the genes coding for factors involved in coagulation: Factors VII, VIII, IX, genes involved in DNA repair, suicide genes (thymidine kinase, cytosine deaminase), genes for hemoglobin or other protein transporters, genes corresponding to proteins involved in lipid metabolism, of the apolipoprotein type chosen from the apolipoproteins AI, A-II, A-IV, B, CI, C-II, C-III, D, E, F, G, H, J and apo (a), metabolism enzymes such as, for example, lipoprotein lipase, hepatic lipase, lecithin cholesterol acyltransferase, 7 alpha cholesterol hydroxylase, phosphatidic acid phosphatase, or also lipid transfer proteins such as ester transfer protein cholesterol and the phospholipid transfer protein, an HDL binding protein or a receptor chosen for example from LDL receptors, chylomicron-remnant receptors and s
• the therapeutic nucleic acid can also be an antisense gene or sequence, the expression of which in the target cell makes it possible to control the expression of genes or the transcription of cellular mRNAs.
• Such sequences can, for example, be transcribed in the target cell into RNAs complementary to cellular mRNAs and thus block their translation into protein, according to the technique described in patent EP 140 308.
• the therapeutic genes also include the sequences coding for ribozymes, which are capable of selectively destroying target RNAs (EP 321,201).
• the nucleic acid can also contain one or more genes coding for an antigenic peptide, capable of generating in humans or animals an immune response.
• the invention therefore makes it possible to produce either vaccines or immunotherapeutic treatments applied to humans or animals, in particular against microorganisms, viruses or cancers.
• These may in particular be antigenic peptides specific for the Epstein Barr virus, the HIV virus, the hepatitis B virus (EP 185 573), the pseudo-rabies virus, the "syncitia forming virus, other viruses or even specific for tumors (EP 259 212).
• the nucleic acid also comprises sequences allowing the expression of the therapeutic gene and / or of the gene coding for the antigenic peptide in the desired cell or organ.
• These may be sequences which are naturally responsible for the expression of the gene considered 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).
• they may be promoter sequences of eukaryotic or viral genes.
• they may be promoter sequences originating from the genome of the cell which it is desired to infect.
• they may be promoter sequences originating from the genome of a virus.
• promoters of the El A, MLP, CMV, RSV, etc. genes can be modified by adding activation, regulation sequences, etc. It can also be a promoter, inducible or repressible.
• the nucleic acid can also comprise, in particular upstream of the therapeutic gene, a signal sequence directing the therapeutic product synthesized in the secretory pathways of the target cell.
• This signal sequence may be the natural signal sequence of the therapeutic product, but it may also be any other functional signal sequence, or an artificial signal sequence.
• the nucleic acid may also include a signal sequence directing the synthesized therapeutic product to a particular compartment of the cell.
• the claimed compositions may also comprise an adjuvant of the dioleoylphosphatidylethanolamine (DOPE) type, oleoyl-palmitoylphos-phatidylethanolamine (POPE), di-stearoyl, - palmitoyl, -mirystoyl phosphatidylethanolamines as well as their N-methylated derivatives 1 to 3 times; phosphatidylglycerols, diacylglycerols, glycosyldiacylglycerols, cerebrosides (such as in particular galactocerebrosides), sphingolipids (such as in particular sphingomyelins) or also asialogangliosides (such as in particular asialoGMl and GM2).
• DOPE dioleoylphosphatidylethanolamine
• POPE oleoyl-palmitoylphos-phatidylethanolamine
• a compound intervening, directly or indirectly, in the condensation of nucleic acids consists, in whole or in part, from peptide units (KTPKKAKKP) and / or (ATPAKKAA), the number of units can vary between 2 and 10.
• peptide units KTPKKAKKP
• ATPAKKAA peptide units
• Such an agent can also derive in whole or in part from a histone, a nucleolin, d a protamine and / or one of their derivatives (WO96 / 25508)
• Such adjuvants can be used in order to improve the intracellular trafficking of the recombinant virus in the claimed compositions.
• compositions according to the invention can also use one or more targeting element making it possible to direct the viral and non-viral vectors towards receptors or ligands on the surface of the cells.
• the composition of the present invention may comprise one or more antibodies directed against molecules on the cell surface, or also one or more ligands of membrane receptors such as insulin, transferrin, folic acid or any other growth factor, cytokines or vitamins.
• the composition can use lectins, modified or not, in order to target particular polysaccharides on the surface of the cell or on the neighboring extracellular matrix.
• Proteins with an RGD motif, peptides containing a tandem of RGD motifs, cyclic or not, as well as polylysine peptides can thus be used.
• These targeting agents can be conjugated with either the recombinant virus and / or the non-viral transfection vector.
• the doses of virus used for administration 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 10 * 4 pfu / ml.
• the term pfu (“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 amounts of associated non-viral transfection vector they obviously vary according to its nature, the nature of the viral vector which is associated with it as well as the cell type targeted. Generally, an amount which can vary between 50 ng and 1 ⁇ g is used in vitro. In ex-vivo applications the quantity of associated non-viral transfection vector varies according to the quantity and type of tissue cells which it is desired to transfect. This quantity can vary between 60 ng / ml and 500 ⁇ g / ml.
• the combinations according to the invention can be formulated for topical, cutaneous, oral, rectal, vaginal, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal, etc. administration.
• the pharmaceutical combinations of the invention contain a pharmaceutically acceptable vehicle for an injectable formulation, in particular for a direct injection into the desired organ, or for topical administration (on the skin and / or mucosa).
• a pharmaceutically acceptable vehicle for an injectable formulation in particular for a direct injection into the desired organ, or for topical administration (on the skin and / or mucosa).
• injectable formulation in particular for a direct injection into the desired organ, or for topical administration (on the skin and / or mucosa).
• They may in particular be sterile, isotonic solutions, 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 nucleic acid used for the injection as well as the number of administrations can be adapted according to different parameters, and in particular according to the mode of administration used, the pathology concerned, the gene to be expressed, or even the duration of the treatment sought.
• the mode of administration it may be either a direct injection into the tissues or the circulatory pathways, or a treatment of cells in culture followed by their reimplantation in vivo, by injection or graft.
• the present invention therefore also relates to cells treated with the composition of the invention for ex-vivo use.
• Figure 1 Effect of lipofectamine on the transduction efficiency of an Ad- ⁇ gal in CMLV cells.
• Figure 2 Comparison of the transduction efficiency of an Ad-luc in the presence and absence of lipofectamine.
• Figure 3 Crystal violet detection of CMV cells transduced by Ad-Tk and treated with GCV, in the presence and absence of 0.125 ⁇ g of lipofectamine.
• Figures 4 Evaluation of the effect of lipofectamine on the neutralization of Ad-luc in S VF or SHA medium for doses varying from 0 to 0.5 ⁇ g of lipofectamine (FIG. 4A) and from 1 to 10 ⁇ g of lipofectamine ( Figure 4B).
• Figure 5 Inhibition of proliferation of smooth muscle cells by overexpression of the Gax protein using an adenovirus alone (I) or using CLAAT (II).
• Figure 6 Ex vivo transfection of an adenovirus on isolated arteries in association with increasing doses of lipofectamine.
• LVMC vascular smooth muscle cells
• the CMLV are obtained by enzymatic digestion of rabbit aorta according to a method adapted from Chamley et al (Cell Tissue Res. 197,177, 503-522) and then placed in primary culture. To do this, the rabbit aorta is removed and incubated for 45 minutes in the presence of collagenase (Collagenase II, Cooper Biomedical) at 37 ° C. A second digestion in the presence of collagenase and elastase (Biosys) is carried out for 2 hours at 37 ° C. These two digestions make it possible to obtain a cell suspension consisting essentially of CMLV.
• collagenase Collagenase II, Cooper Biomedical
• CMLVs are maintained in culture in DMEM medium (Gibco) containing 20% fetal calf serum (SVF, Gibco) and used for all of the experiments before the tenth passage. Every passage phenotyping of CMLV is carried out by immunofluorescence using an antibody directed against alpha-actin specific for smooth muscle cells (Sigma).
• CMLV transduction Different recombinant adenoviruses are used for the different examples of CMLV transduction. All the recombinant Ads used are first generation, deficient in replication by deletion of the El region.
• the Ad-Luc carries an expression cassette containing the luciferase gene under the control of the CMV promoter.
• This expression cassette comes from a commercial plasmid pUT650 (Cayla, Toulouse France) and contains the luciferase gene fused to the zeo resistance gene under the control of the CMV promoter.
• the expression cassette was inserted into the E1 region of the adenovirus In340 (Feldman et al., Improved efficiency of arterial gene transfer by use of poloxamer 407 as a vehicle for adenoviral vectors Gene Ther, 1997. 4: 189-98 ; Robert, JJ et al Gene Neurochemistry., 1997, Vol 68, pp 2152-2160; Hearing et al 1983, Cell 33 p695-703.).
• the Ad-BGal carries a cassette coding for the gene for ⁇ -galctosidase, from Echerichia Coli, fused in its N-terminal part to a nuclear localization sequence (NLS) identical to that of the T antigen of the SV40 virus. , under the control of the promoter LTR-RSV (Stratford-Perricaudet et al. Widespread long-term gene transfer to mouse skeletal muscles and heart. J. Clin.Inves, 1992. 90: 626-630).
• the Ad-tk codes for the gene for thymidine kinase, from the Herpes simplex virus, under the control of LTR-RSV (Maron et al., Gene therapy of rat C6 glioma using adenovirus- mediated transfer of the herpes simplex virus thymidine kinase gene: long-term follow-up by magnetic resonance imaging. Gene Ther, 1996. 3: 315-22).
• the VMCs are seeded in 24-well culture plates (Falcon) at a density of 50,000 cells per well in 1 ml of DMEM containing 10% S VF (DMEM-S VF). After 20 to 24 hours the DMEM-SVF of the CMLV is replaced by 300 ⁇ l of DMEM without serum. The cells are then infected with the recombinant viruses, alone or complexed with liposomes, at multiplicities of infection (MOI) which vary according to the experiments.
• MOI multiplicities of infection
• This adenoviral preparation is added, after an incubation of 30 minutes at room temperature, in a suspension of 100 ⁇ l of water or phosphate buffer (PB S) containing either Ad alone, at the desired MOI, or Ad with variable amounts cationic liposomes (Lipofectamine, Gibco).
• the 100 ⁇ l of Ad or the Ad-lipofectamine complex are directly added to the CMLV in the 300 ⁇ l of DMEM.
• HAS neutralizing human serum
• the transduction mixture is replaced by DMEM 0.5% FCS. 24 h after infection, cell proliferation is induced by adding medium rich in serum (DMEM 10% S VF). Cell viability is estimated 72 h post-infection using the Alamar Blue test (Biosource).
• CMLV transduced by Ad-tk the cells are placed in the presence of 25 ⁇ M of Ganciclovir (GCV) during the 48 hours of culture. At the same time, cells not infected or transduced by Ad-BGal serve as controls, treated or not treated by the GCV. D- Transfer of the adenovirus encoding the luciferase gene to isolated arteries
• the abdominal arteries of New Zealand White rabbits are removed after euthanasia by overdose of sodium pentobarbital.
• the abrasion of the arteries is carried out using the fogarty 3F probe inflated with 0.15 ml of air.
• the arteries are cut into 5 mm fragments and then opened longitudinally. Each fragment is deposited in the wells of a 12-well plate in 1 ml of serum-free medium.
• Infection takes place on the day of collection.
• the volume of the adenovirus mixture (AVi . OCMVluc or AVi.oCMV ⁇ Gal, 10 8 pfu) and lipofectamine is adjusted to 80 ⁇ l / well with PB S.
• the solution is then incubated for 30 min at room temperature and then deposited on the artery fragments contained in 1 ml of serum-free medium in a 12-well plate well.
• the fragments are then incubated for 1 hour at 37 ° C., then they are transferred to a new 12-well plate and incubated in 2 ml of DMEM 20% S VF medium.
• the evaluation of the transfer is made either by measuring the luciferase activity (AVi.oCMVluc) or by histochemistry (AVi.oCMV ⁇ Gal) 3 days post infection. Several doses of lipofectamine were tested for a constant MOI of AVi . oCMVluc.
• CMLV transduced by Ad-Luc are collected in 200 ⁇ l of lysis buffer for luciferase test (Promega). After a period of 15 minutes of lysis, 10 ⁇ l of each sample is used for the determination of the luciferase activity using a kit (Luciferase assay Kit, Promega) and a luminometer (Berthold). The lucierase activity is expressed in RLU (Relative Right Unit s). The measurement of the luciferase activity on the isolated arteries requires that the artery fragments be ground in 500 ⁇ l of lysis buffer containing anti-proteases and then incubated for 20 min at room temperature with shaking. They are then centrifuged for 5 min at 10,000 revolutions. The reading is carried out on 10 ⁇ l of extract in the presence of 50 ⁇ l of substrate for 10 seconds.
• CMLVs transduced by Ad-BGal The ⁇ -galctosidase activity in CMLVs transduced by Ad-BGal is assayed by chemiluminescence according to the Galacto-Light plus Kit from Tropix, Inc. Briefly, the cells are washed in PBS and then harvested in 250 ⁇ l of a lysis solution comprising 100mM potassium phosphate pH 7.8 and 0.2% Triton X-100. The lysates are centrifuged for 2 minutes at 10,000 g, then 5 ⁇ l of each sample are mixed with 50 ⁇ l of the Galcton-Plus reaction buffer and incubated for 1 hour at room temperature. For luminescence measurement, the mixtures are placed in a luminometer (Berthold) then receive 50 ⁇ l of accelerator (Light Emission Accelerator) and the luminescence is measured and expressed in RLU.
• CMLV on the culture support or take off by treatment with trypsin, are washed with PBS and then fixed in PBS containing 4% formaldehyde. The fixing solution is removed after 10 min, then the cells are stained for 1 to 4 hours at 37 ° C. in PBS containing 4 mM potassium ferricyanide, 4 mM potassium ferrocyanide, 200 mM magesium chloride and 0.4 mg / ml of X-Gal substrate. The percentage of blue cells is calculated from the preparations of CMLV in suspension while the CMLV staining on the culture support are photographed.
• the LVMCs are subjected to treatment with GCV as described above. Viable cells, at the end of this treatment, are washed with PBS and then fixed in PBS containing 4% formaldehyde. The fixing solution is removed after 15 min, then the cells are stained for 20 minutes in a solution of 0.1% "crystal violet" in water. After staining, the cells are washed three times in water and then photographed.
• This example shows the increase in CMLV transduction when Ad- ⁇ gal is associated with a cationic liposome such as lipofectamine.
• CMLVs are transduced according to the protocol described in MATERIALS AND METHODS, by Ad- ⁇ gal at an MOI of 10 in the presence of increasing doses of lipofectamine ranging from 0 to 0.5 ⁇ g.
• the histochemical revelation of ⁇ galactosidase activity clearly shows that from the dose of 0.125 ⁇ g lipofectamine significantly increases the efficiency of transduction of CMLV by Ad- ⁇ gal.
• the percentage of cells positive for ⁇ galactosidase activity is determined after histochemical staining of the cells in suspension according to the protocol described in MATERIAL AND METHOD. Figure 1 illustrates these results.
• Example 2 made it possible to determine a dose of lipofectamine making it possible to increase the transduction of CMLV by Ad- ⁇ gal to an MOI of 10.
• Ad-Luc is used at variable MOIs for the transduction of CMLV and a luciferase activity is then assayed as described in MATERIAL AND METHOD.
• the luciferase activity increases almost linearly as a function of the MOIs of Ad-Luc used to transduce the CMLVs (FIG. 2, empty circles).
• the addition of 0.125 ⁇ g of lipofectamine to Ad-Luc (FIG. 2, solid diamonds) induces an increase of a factor of 100 in the luciferase activity recorded. This shows that, up to an MOI 100 of Ad-Luc, a small amount of lipofectamine, ie 0.125 ⁇ g is sufficient to very significantly potentiate the transduction of CMLV.
• the adenovirus used in this example is the Ad-TK adenovirus, used for the cytotoxic property of the herpes simplex thymidine kinase gene in the presence of ganciclovir (GCV).
• the CMLVs were transduced with MOIs of 1.10 and 100 of Ad-TK and treated with GCV as described in MATERIAL AND METHOD.
• Ad- ⁇ gal at MOI 100 is used as a control.
• Ad-TK and AD- ⁇ gal are used either alone or complexed with 0.125 ⁇ g of lipofectamine.
• FIG. 3 shows a staining of crystal violet CMLVau after transduction and treatment with GCV.
• GCV has no toxicity with respect to cells transduced by Ad- ⁇ gal in the presence or absence of lipofectamine.
• Ad-TK is complexed with 0.125 ⁇ g of lipofectamine treatment with GCV at an efficiency at MOI 1 comparable to that obtained with Ad-TK alone at MOI 100.
• the Ad-TK associated with lipofectamine is highly toxic in the presence of GCV. This example shows the interest of the present formulation. It makes it possible both to significantly reduce the amount of recombinant Ad-TK virus to be administered and this for a therapeutic efficacy equal or greater compared to that observed with Ad-TK alone.
• one of the major obstacles to optimal efficiency of a recombinant adenovirus is its neutralization by the immune system. Indeed, following a cell lysis after a first transfer to animals or following a first contact with an adenovirus in humans, the immune system can effectively neutralize the virus in the circulation thus reducing the possibilities of reinjection of virus.
• This example reproduces in vitro the neutralization of the recombinant adenovirus by a pool of human sera, and advantageously shows that the association of a recombinant adenovirus with liposomes makes it possible to repeal this neutralization and, depending on the dose of liposomes, the transduction may be greater than that observed with the virus alone.
• FIGS. 4A and B use CMLVs transduced with Ad-Luc at MOI 100 in the presence of fetal calf serum (SVF) or adult human serum (SHA) in the DMEM culture medium.
• SVF fetal calf serum
• SHA adult human serum
• This example therefore shows that the claimed composition makes it possible, on the one hand, to completely repeal the neutralization of a recombinant adenovirus by neutralizing antibodies or serum and, on the other hand, to increase the transduction efficiency, in the presence of neutralizing serum. , as already shown in the previous examples.
• This example shows the increase in transduction of a therapeutic gene (gax) in rabbit smooth muscle cells by association of the adenovirus.
• AVi.oCMVrGax and a cationic lipid such as lipofectin (CLAAT).
• CLAAT lipofectin
• Smooth rabbit muscle cells are seeded at the rate of 5 ⁇ 10 4 cells per well of a MW 48 in DMEM 10% FCS medium.
• the infection is carried out 24 hours later in DMEM medium without serum.
• Different dilutions of adenovirus are mixed with 60 ng of lipofectamine in a final volume of 25 ⁇ l adjusted with PBS. This solution is incubated for 30 min at room temperature.
• the culture medium is replaced with 175 ⁇ l of serum-free medium per well and the mixture of adenovirus and lipofectamine is added to the cells.
• the infection lasts 1 hour at 37 ° C.
• the medium containing the virus is replaced by DMEM 0.5% SVF.
• 24 h after infection cell proliferation is induced by adding medium rich in serum (DMEM 10% FCS).
• Cell viability is estimated 72 h post-infection using the Alamar Blue test (Biosource).
• FIG. 5 compares the inhibition of proliferation of smooth muscle cells by overexpression of the Gax protein using an adenovirus alone (I) or using CLAAT (II).
• the cells were infected at MOIs varying between 0 and 10 5 PV / cell with PAV1.0CMV (a) or with AVi.oCMVrGax alone (b).
• the MOIs vary from 0 to 3 10 4 PV / cell with the same viruses (Il-a and b). After infection, the cells are incubated in DMEM 0.5% FCS medium for 24 hours. The medium is then changed to medium rich in serum. Cell viability is estimated 72 hours post infection.
• This example describes the transfer of genes to fragments of arteries isolated using the association of an adenovirus encoding luciferase or ⁇ Galactosidase and lipofectamine (CLAAT).
• CLAAT adenovirus encoding luciferase or ⁇ Galactosidase and lipofectamine
• the artery fragments are ground in 500 ⁇ l of lysis buffer containing anti-proteases and then incubated for 20 min at room temperature with shaking. They are then centrifuged for 5 min at 10,000 revolutions. The reading is carried out on 10 ⁇ l of extract in the presence of 50 ⁇ l of substrate for 10 seconds using a kit (Luciférase assay Kit, Promega) and a luminometer (Berthold).
• This example demonstrates that gene transfer is possible on isolated arteries using CLAAT.
• this technique makes it possible on the one hand to improve gene transfer and on the other hand to reach the cells of the adventitia. He can be consider using this technique for the treatment of venous grafts using genes of interest, such as for example the FGF factor, carried by an adenoviral vector.

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