Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
  • A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
  • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
  • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
  • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • 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
  • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • 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
  • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
  • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
• • 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

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 pharmaceutical composition useful for efficiently transfecting cells. It also relates to the uses of this composition.
• Chrornosomal 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 therapeutic 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.
• naked nucleic acids have a short plasma half-life due to their degradation by enzymes and their elimination through the urinary tract.
• the second strategy advantageously consists in using non-viral agents capable of promoting the transfer and expression of DNA in eukaryotic cells.
• the object of the present invention is more particularly part of this second strategy.
• Chemical or biochemical vectors represent an advantageous alternative to natural viruses, in particular for this lack of immunological response and / or viral recombination. They have no pathogenic power, the risk of multiplication of DNA within these vectors is zero and there is no theoretical limit attached to them as regards the size of the DNA to be transfected. These synthetic vectors have two main functions, to condense the DNA to be transfected and to promote its cellular fixation as well as its passage through the plasma membrane and, where appropriate, the two nuclear membranes.
• non-viral vectors Due to its polyanionic nature, DNA naturally has no affinity for the plasma membrane of cells of a polyanionic nature as well. To overcome this drawback, the non-viral vectors generally all have polycationic charges.
• cationic polymers of polylysine and DEAE dextran type or else cationic or lipofectant lipids 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 1 ⁇ DN, 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 at the surface of the cell type that we want to graft. Screenings of the transferrin, insulin receptor or the hepatocyte asialoglycoprotein receptor have thus been described.
• the synthetic vectors offered today are still far from being as efficient as the viral vectors. This can in particular be the consequence of insufficient condensation of the DNA to be transfected and / or of the difficulties encountered by the transfected DNA to leave the endosome and enter the cell nucleus. Indeed, the transport of DNA in the nucleus of a resting eukaryotic cell poses an obvious problem since the dimensions of the nuclear pores only allow the diffusion of proteins of molecular weight below 60 OOODa. (I. Davis et al., Ann. Rev. Biochem. 1995; 64; 865-896). Plasmid DNA having a molecular weight greater than 10 6 cannot therefore naturally enter the cell nucleus by simple diffusion.
• the present invention specifically aims to provide an advantageous solution to the above problem.
• the present invention provides a pharmaceutical composition useful for the transfection of at least one nucleic acid, characterized in that it contains, in addition to said nucleic acid and at least one transfection agent, at least one compound combining the binding properties of DNA has a nuclear vectorization capacity for this DNA.
• a compound having DNA binding properties covers any compound capable of binding at least partially to the DNA to be vectorized. With regard more particularly to its ability to vectorize this DNA, it is reflected in the sense of the invention by an efficiency in directing this DNA efficiently through the various cell and / or nuclear membranes to conduct it as far as the nucleus of the cell to be treated.
• the compound according to the invention can also be presented, for example, in the form of a chimeric molecule associating a domain of attachment to DNA, with a domain allowing nuclear import. In this particular case, it is possible to select from DNA binding domains, those derived from regulatory proteins capable of binding for example to specific sequences or, on the contrary, from proteins known to have a non-sequence dependent affinity for DNA.
• nls sequence Nuclear localization Sequence
• Such sequences could be related to or derived from the bipartite consensus deduced from nucleoplasmin or from the consensus of the SV40 T antigen.
• the invention relates to a pharmaceutical composition useful for the transfection of at least one nucleic acid, characterized in that it contains, in addition to said nucleic acid and at least one transfection agent, at least one compound belonging to the HMG family or one of its derivatives.
• Proteins of the HMG type for "High Mobility Group” are proteins rich in charged amino acids and having a molecular mass of less than 30,000 Da. Soluble in 2-5% perchloric acid, they are conventionally extracted from chromatin with 0.35 M NaCl.
• HMG proteins proteins of the HMG1 / 2 type with a molecular mass close to 25,000, HMG14 / 17 with a molecular mass close to 10-12,000, and HMGI / Y with a composition close to the proteins of the HMG14 / 17 type. but whose tissue distribution and during ontogenesis is different. We know that the primary sequence of proteins is conserved during evolution within each of these three families.
• proteins of the HMG 1/2 family are characterized by the presence of a sequence of 80 amino acids predominantly basic (net charge +20), called "HMG box", which constitutes a domain DNA binding.
• HMG box proteins capable of binding to specific sequences of double-stranded DNA and proteins whose specificity of binding lies in a particular three-dimensional structure of DNA.
• the sequence of each of these proteins contains one or more "HMG boxes”.
• the second category is represented by the proteins HMG1 and HMG2.
• the Applicant has highlighted that it is possible to take advantage of these faculties of the HMG proteins, namely their ability to fix DNA and to be actively transported in the nucleus, to effectively promote transfection in the nucleus of cells to be treated with heterologous nucleic acid sequences associated with at least one transfecting agent.
• the term derivative designates any peptide, pseudopeptide (peptide incorporating non-biochemical elements) or protein differing from the compound as defined above, obtained by one or more modifications of a genetic and / or chemical nature. By modification of a genetic and / or chemical nature, one can hear any mutation, substitution, deletion, addition and / or modification of one or more residues, for example of the protein considered.
• chemical modification any modification of the peptide or protein generated by chemical reaction or by chemical grafting of molecule (s), biological (s) or not, onto any number of protein residues.
• genetic modification is meant any peptide sequence whose DNA hybridizes with these sequences or fragments thereof and whose product has the indicated activities.
• Such derivatives can be generated for different purposes, such as that of increasing the refinement of the corresponding polypeptide for its DNA ligand, that of improving its production levels, that of increasing its resistance to proteases, that of '' increase and / or modify one of its activities, or that of giving it new pharmacokinetic and / or biological properties.
• derivatives resulting from an addition mention may, for example, be made of chimeric peptide sequences comprising an additional heterologous part linked at one end.
• the term derivative also includes protein sequences homologous to the sequence under consideration, originating from other cellular sources and in particular from cells of human origin, or from other organisms, and having an activity of the same type. Such homologous sequences can be obtained by hybridization experiments of the corresponding DNA. Hybridizations can be carried out from nucleic acid libraries, using the native sequence or a fragment thereof as a probe, under conventional stringency conditions (Maniatis et al.), (Cf. general molecular biology techniques). , or, preferably, under conditions of high stringency.
• E can in particular be four-stranded structures for which it has been shown in particular that the rat HMGl has a very strong affinity (Bianchi et al. Sciences, 1989, 243, 1056-1059).
• Such structures can also be obtained from natural sequences such as the ITRs of the virus associated with adenoviruses or else be completely synthetic obtained from artificial palyndromes.
• the compound used is chosen from proteins of the HMG type 1, 2, 1, Y, 14 and 17 and their derivatives. It is more preferably represented by all or part of the human HMG1 protein or one of its derivatives or homologs as defined above.
• compositions of the present invention further comprise a targeting element making it possible to direct the transfer of the nucleic acid.
• This targeting element can be an extracellular targeting element, making it possible to direct the transfer of nucleic acid to certain cell types or certain desired tissues (tumor cells, hepatic cells, hematopoietic cells, etc.).
• D can also be an element of intracellular targeting, making it possible to direct the transfer of nucleic acid to certain privileged cellular compartments (mitochondria, nucleus, ete).
• the targeting element is linked, covalently or non-covalently, to the compound according to rinvenhon.
• the targeting element can also be linked to the nucleic acid.
• said compound is associated, via an additional heterologous part linked to one of its ends, with a cell receptor ligand present on the surface of the cell type such as for example a sugar, totLnsferrin, insulin or the asialo-orosomucoid protein. It can also be an intracellular ligand such as a nuclear rental signal sequence, nls, which favors the accumulation of the transfected DNA within the nucleus.
• sugars which can be used in the context of the invention, there may be mentioned sugars, peptides, oligonucleotides or lipids.
• these are sugars and / or peptides such as antibodies or antibody fragments, ligands of cellular receptors or fragments thereof, receptors or fragments of receptors, etc.
• they may be ligands for growth factor receptors, cytokine receptors, cellular lectin receptors or adhesion protein receptors.
• the targeting element can also be a sugar making it possible to target lectins such as asialoglycoprotein receptors, or alternatively an Fab fragment of antibodies making it possible to target the receptor for the Fc fragment of immunoglobulins.
• the compound according to the invention can also be polyglycosylated, sulfonated and / or phosphorylated and / or grafted to complex sugars or to a lipophilic compound such as for example a polycarbon chain or a cholesterol derivative.
• composition according to the invention can of course comprise several compounds according to the invention, of different nature. Similarly, it turns out to be possible to combine with the compound according to the invention, in addition to the nuclear targeting compound. ⁇
• the compound according to the invention is present in an amount sufficient to act with the nucleic acid according to the invention.
• the compound / nucleic acid ratio (expressed by weight) can be between 0.01 and 5 and more preferably between 0.25 and 0.5.
• transfection agent present in the composition according to the invention, it is preferably chosen from cationic polymers and lipofectants.
• the cationic polymer is preferably a compound of general formula I,
• - R can be a hydrogen atom or a group of formula
• - n is an integer between 2 and 10;
• n is between 2 and 5.
• the polymers of polyethylene imine (PEI) and polypropylene irnine (PPI) have entirely 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 general formula I they can be prepared according to the process described in patent application FR 9408735.
• these are amphiphilic molecules comprising at least one cationic hydrophilic region, for example polyamine, and one lipophilic region.
• the cationic region preferably polyamine, cationically charged, is capable of associating reversibly with the negatively charged nucleic acid. This interaction strongly compacts the nucleic acid.
• the lipophilic region makes this ionic interaction inaccessible to the external aqueous medium, by covering the nucleolipid particle formed with a lipid film.
• these lipofectants can also be chosen from lipopolyamines whose polyamine region corresponds to the general formula ⁇
• the polyamine region is represented by spermine, thermine or one of their analogs which have retained DNA binding properties.
• the lipophilic region it is represented by at least one hydrocarbon chain, saturated or not, of cholesterol, a natural lipid or a synthetic lipid capable of forming lamellar or hexagonal phases, covalently linked to the hydrophilic region.
• Patent application EP 394 111 describes other Hpopolyarnines of general formula III capable of being used in the context of the present invention
• R represents in particular a radical of general formula (RjR2) N-CO- .
• lipopolyamines of dioctadecylamidoglycyl spermine (DOGS) and 5-carboxyspermylamide of palmitoylphosphatidylethanolamine (DPPES).
• DOGS dioctadecylamidoglycyl spermine
• DPES 5-carboxyspermylamide of palmitoylphosphatidylethanolamine
• lipopolyamines described in patent application FR 94 14596 can also be used advantageously as a transfection agent according to the invention. They are represented by the general formula HI above in which R represents
• R ' Representing a C 1 -C 4 alkyl group
• - R3, R4 and R5 independently of one another representing a hydrogen atom or an alkyl radical, substituted or unsubstituted, at C j to C4, with p being able to vary between 0 and 5,
• R5 representing a cholesterol derivative or an amino alkyl group -NR1R2 with Rj and R 2 independently of one another representing an aliphatic radical, saturated or unsaturated, linear or branched in Cj 2 to C 2 2.
• Patent applications EP 394 111 and FR 94 also describe a process which can be used for the preparation of the corresponding lipopolyamines.
• dioctadecylamidoglycyl spermine DOGS
• DPES palmitoylphosphatidylemanolamine
• Dioctadecyl-carbamoylmethoxy 2-5-bis- (3- acetate) amino-propylamino) -pentyl or 1,3-bis- (3-amino-propylamino) -2 propyl
• DOGS dioctadecylamidoglycyl spermine
• DPES palmitoylphosphatidylemanolamine
• the respective proportions of the polyamine and of the nucleic acid are preferably determined so that the ratio R positive charges of the transfection agent / negative charges of the nucleic acid is between 0.1 and 10 and more preferably between 0.5 and 2.
• the nucleic acid can be either a deoxyribonucleic acid or a ribonucleic acid.
• D can be sequences of natural or artificial origin, and in particular genomic DNA, cDNA, mRNA, tRNA, rRNA, hybrid sequences or synthetic or semi-synthetic sequences. 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 libraries, by chemical synthesis, or also by mixed methods including chemical or enzymatic modification of sequences obtained by screening of libraries. They can also be incorporated into vectors, such as plasmid vectors.
• deoxyribonucleic acids With regard more particularly to deoxyribonucleic acids, they can be single or double stranded. These deoxyribonucleic acids can code for therapeutic genes, transcription or replication regulatory sequences, antisense sequences, regions of binding to other cellular components, etc.
• the term “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, ete (FR 9203120), growth factors, neurotransmitters or their precursors or synthetic enzymes, trophic factors: BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NT5, HARP / pleiotrc ⁇ hine, ete; apolipoproteins: ApoAI, ApoAIV, ApoE, ete (FR 93 05125), dystrophin or a minidystrophin (FR 9111947), the CFTR protein associated with cystic fibrosis, tumor suppressor genes: p53, Rb, RaplA, D
• the therapeutic gene 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 antisenses 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, 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 peptide antigen in the desired cell or organ.
• They may be the 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.
• these expression sequences can be modified by adding activation, regulation sequences, etc.
• 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.
• compositions of the invention further comprise one or more neutral lipids.
• Such compositions are particularly advantageous, especially when the ratio R is low.
• the Applicant has indeed shown that the addition of a neutral lipid makes it possible to improve the formation of nucleolipid particles and, surprisingly, to promote the penetration of the particle into the cell by destabilizing its membrane.
• the neutral lipids used in the context of the present invention are lipids with 2 fatty chains.
• natural or synthetic lipids, zwitterionic or lacking ionic charge under physiological conditions are used. They can be chosen more particularly from dioleoylphosphatidylemanolamine (DOPE), oleoyl-palrrutoylphos-phatidyléttianolamine (POPE), di-stearoyl, -palmitoyl, -mirystoyl phosphatidylethanolamine as well as their N-methyl derivatives 1; phosphatidylglycerols, diacylglycerols, glycosyldiacyl- glycerols, cerebrosides (such as in particular galactocerebrosides), sphingolipids (such as in particular sphingomyelins) or alternatively asialogangliosides (such as in particular asialoGMl and
• DOPE dio
• lipids can be obtained either by synthesis or by extraction from organs (example: the brain) or eggs, by conventional techniques well known to those skilled in the art.
• extraction of natural lipids can be carried out using organic solvents (see also Lehninger,
• the compositions of the invention using a lipofectant as transfection agent, comprise from 0.1 to 20 equivalents of neutral lipid per 1 equivalent of Upopolyarnine, and, more preferably, from 1 to 5.
• the transfection agent is a cationic polymer
• the compositions of the invention comprise, in addition to the cationic polymer in the ratios cited above, from 0.1 to 20 molar equivalents of neutral lipid per 1 molar equivalent of phosphate of the nucleic acid, and, more preferably, from 1 to 5.
• compositions according to the invention can be formulated for topical, cutaneous, oral, rectal, vaginal, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal, etc. administration.
• Pharmaceuticals 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). 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.
• 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. They can advantageously be used to transfect a wide variety of cell types such as, for example, hematopoietic cells, lymphocytes, hepatocytes, endothelial cells, melanoma, carcinoma and sarcoma cells, smooth muscle cells, neurons and astrocytes.
• the present invention thus provides a particularly advantageous method for the treatment of diseases using the transfection in vitro, ex vivo or in vivo of a nucleic acid capable of correcting said disease in association with a transfection agent of cationic polymer or lipofectant type, and a compound as defined above More particularly, this method is applicable to diseases resulting from a deficiency in a protein or nucleic product and the administered nucleic acid codes for said protein product or contains the sequence corresponding to said nucleic product.
• the compositions according to the invention are particularly advantageous for their bioavailability and their level of transfection.
• the present invention also relates to any use of a compound according to the invention coupled to a cell receptor ligand, an antibody or antibody derivative, to target a nucleic acid towards cells expressing the corresponding receptors or anti-genes.
• a ligand, antibody or derivative of potential antibody is coupled to said compound and we appreciate the transfection power of this chimeric molecule compared to the compound alone.
• FIGURES are a diagrammatic representation of FIGURES.
• Figure 1 Representation in line intensities of the efficiency of transf ections carried out according to the invention in different cell types.
• compositions of the invention is a plasmid comprising the gene coding for luciferase (Luc), pCMV-Luc.
• the plasmid pCMV-luc comprises the promoter of Cytomegalovirus (CMV), extracted from the vector plasmid pcDNA3 (Invitrogen) by cleavage with the restriction enzymes Mlu I and Hindi ⁇ , located upstream of the gene coding for luciferase, inserted at the Mlul and Hindl ⁇ sites in the vector pGL basic Vector (Promega).
• CMV Cytomegalovirus
• This recombinant protein from a mammal was prepared by overexpression in E. coli.
• Plasmid T7-RNHMG1 coding for the rat HMG1 protein (M. E. Bianchi, Gene, 104 (1991) 271-275) is introduced into the strain of E. coli BL21 (DE3). The strain is subsequently cultivated at 37 ° C. in LB + Ampicillin medium (25 mg / L). A preculture is obtained from an isolated colony. It allows a 500 ml culture to be sown. When the absorbance at 600 nm of the culture reaches the value of 0.7, the synthesis of HMG1 is induced by the addition of IPTG at 0.5 mM final. The HMG1 producing strain is still cultivated for 2 h 30 min in the presence of IPTG. The cells are then harvested by centrifugation (5000 x g, 20 minutes), rinsed with 200 ml of distilled water, centrifuged again. The cell pellet is stored at -80 ° C until purification.
• the purification of the HMG1 protein can be carried out by chromatography from a culture of the E. coli strain described in Example 1.1, for example using the following protocol: Unless otherwise indicated, all of the purification described below is carried out at 4 ° C.
• the cells obtained from 500 ml of culture are resuspended in 15 ml of 50 mM Tris / HCl buffer pH 7.7 containing 500 ⁇ M EDTA, 5 mM DTT, 200 t ⁇ M Pefabloc SC [4- (2-aminoethyl) -benzenesulfonyl fluoride hydrochloride] , and 10% (weight / volume) glycerol.
• the acellular extract is then chromatographed through a column of Sephadex G-25 (Pharmacia) balanced and eluted with buffer A [20 mM Hepes pH 7.9 containing 400 mM sodium chloride, 200 mM EDTA, 1 mM DTT, 200 ⁇ M Pefabloc SC, 0.2% Nonidet P40, and 10% glycerol].
• buffer A 20 mM Hepes pH 7.9 containing 400 mM sodium chloride, 200 mM EDTA, 1 mM DTT, 200 ⁇ M Pefabloc SC, 0.2% Nonidet P40, and 10% glycerol.
• the protein-containing fraction is harvested and chromatographed through a column of DEAE Sephadex A-25 gel (Pharmacia) equilibrated in buffer A.
• the protein fraction not retained on this column is gradually mixed with solid ammonium sulfate until a final concentration of 2.8 M. After 2 h, this suspension is centrifuged (30,000 xg; 15 min). The supernatant is chromatographed at 20 ° C. through a Phenyl-Superose HR 5/5 column (Pharmacia) equilibrated in 20 mM Hepes buffer pH 7.9 containing 200 mM EDTA, 500 ⁇ M DTT, and 2.8 M sulfate of ammonium. The proteins are eluted from the column with a decreasing linear gradient of ammonium sulfate (2.8 M to 0 M) in the same buffer.
• the fractions containing the HMG1 protein are pooled and dialyzed extensively against 50 mM Tris / HCl pH 7.7 buffer containing 1 mM EDTA and 500 ⁇ M DTT. This sample is then injected onto a MonoQ HR 5/5 column (Pharmacia) which is then eluted with a linear gradient from 0 to 0.5 M sodium chloride in 50 mM Tris / HCl buffer pH 7.7-500 ⁇ M DTT.
• the HMG1 protein which forms a symmetrical absorbance peak at 280 nm, is collected in this buffer.
• the HMGl protein is then taken up in 10 mM Mes buffer pH 6.2-240 mM NaCl-500 ⁇ M DTT after concentration by centrifugation in Centrikon 10.
• the HMGl protein is stored at -80 ° C. until use.
• This preparation has a single protein band migrating to an apparent molecular weight of 31,000 when analyzed by electrophoresis under denaturing conditions (SDS) and revelation with Coomassie.
• the overall purification yield is 850 ⁇ g of pure HMGl protein per 500 ml of starting culture.
• EXAMPLE 2 TRANSFER OF NUCLEIC ACID IN VITRO IN MAMMALIAN CELLS
• This example shows how a protein, of the HMG1 type, which binds to DNA and is actively imported into the nucleus, can be used to stimulate the transfection of plasmid DNA.
• compositions of the invention The construction used to demonstrate the activity of the compositions of the invention is the plasmid comprising the gene coding for luciferase (Luc) described above.
• the protocol is established for 24-well plates (0 16mm) to be harvested 2 days after transfection (cells at confluence). All parameters can be changed proportionally
• H460 Maxwell et al, Oncogene 8 (1993), 3421-3429) are seeded at 10 ⁇ cells per well. On day D + 2 the cells are rinsed with PBS (to remove traces of serum) and taken up in 250 ml of RPMI (3LL and H460) or DMEM (NIH3T3), whether or not supplemented with 10% Fetal Calf Serum (SFV )
• composition used for the transfection By equivalent well, in a tube is added: - H2O qs 20 ml
• the cells are transfected by adding 20 ml of the DNA / HMG1 / lipofectant mixture to the culture medium, incubated for 2 to 4 hours at 37 ° C. This medium is then replaced by complete medium. On day D + 4 the cells are rinsed at room temperature with 250 ml of PBS, lysed in 100 ml of ad hoc buffer (Reporter (Promega) + TCK and Arjrotinin). 10 ml of lysate and 50 ml of substrate (Promega) are used to measure the activity of the luciferase synthesized.
• the results presented in Figures 1 and 2 are the average of four experiments, independently repeated 2 times. To do this, we appreciate the Light Units (UL) obtained by expression of the Luc gene in the transfected cells.
• UL Light Units
• FIG. 1 brings together the values obtained for the three cell types mentioned above, in the presence of a variable quantity of HMG1 protein.
• Figure 2 summarizes the transfection stimulation factors obtained by adding different amounts of HMGl proteins.
• the increase in the efficiency of transfection with the HMG1 protein is variable according to the cell types. It is thus observed that it is maximum when the medium containing the composition necessary for the transfection is supplemented with 10% SFV.
• SFV represents the conditions encountered in vivo.
• the presence of SFV decreases the transfection efficiency, particularly in the absence of the HMG1 protein. It may be thought that the presence of SFV in the culture medium decreases the amount of DNA capable of being internalized by the cells.
• HMGl is particularly advantageous for transfection under conditions where the quantity of DNA is limiting.
• the optimal HMG1 / DNA ratio (by mass) for transfection is 0.25 to 0.5.
• Such conditions are not described as being capable of compacting DNA (Bôttger M. et al., B.B.A. 950 (1988), 221-228); Stros M. et ai, N.A.R. 22 (1994), 1044-1051).
• the plasmid is not saturated with HMGl (Kohlstaedt LA. Et ai, Biochemistry 33 (1994), 12702-12707).
• the effect of the HMGl protein is therefore explained by its ability to bind DNA and to be transported to the nucleus of the cell.

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