EP0854930A1 - Pharmaceutical composition useful for nucleic acid transfection, and use thereof - Google Patents

Pharmaceutical composition useful for nucleic acid transfection, and use thereof

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Publication number
EP0854930A1
EP0854930A1 EP96932668A EP96932668A EP0854930A1 EP 0854930 A1 EP0854930 A1 EP 0854930A1 EP 96932668 A EP96932668 A EP 96932668A EP 96932668 A EP96932668 A EP 96932668A EP 0854930 A1 EP0854930 A1 EP 0854930A1
Authority
EP
European Patent Office
Prior art keywords
pharmaceutical composition
composition according
nucleic acid
compound
transfection
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP96932668A
Other languages
German (de)
French (fr)
Inventor
Francis Blanche
Béatrice Cameron
Jo[L Crouzet
Vincent Thuillier
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Aventis Pharma SA
Original Assignee
Rhone Poulenc Rorer SA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Rhone Poulenc Rorer SA filed Critical Rhone Poulenc Rorer SA
Publication of EP0854930A1 publication Critical patent/EP0854930A1/en
Withdrawn legal-status Critical Current

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    • 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.

Abstract

A pharmaceutical composition useful for nucleic acid transfection is disclosed. The composition contains, in addition to a nucleic acid and at least one transfection agent, at least one compound that combines DNA binding properties with a nuclear DNA vectorisation capability, and preferably belongs to the HMG ('High mobility group') protein family. The use of said composition for in vitro, ex vivo and/or in vivo nucleic acid transfer is also disclosed.

Description

COMPOSITION PHARMACEUTIQUE UTILE POUR LA TRANSFECTION D'ACIDES NUCLEIQUES ET SES UTILISATIONS PHARMACEUTICAL COMPOSITION USEFUL FOR TRANSFECTING NUCLEIC ACIDS AND USES THEREOF
La présente invention concerne le domaine de la thérapie génique et s'intéresse plus particulièrement au transfert in vitro, ex vivo et/ou in vivo du matériel génétique. Elle propose notamment une nouvelle composition pharmaceutique utile pour transfecter efficacement des cellules. Elle se rapporte également aux utilisations de cette composition.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. In particular, it proposes a new pharmaceutical composition useful for efficiently transfecting cells. It also relates to the uses of this composition.
Des déficiences et/ou anomalies (mutation, expression aberrante, ete) chrornosomiques sont à l'origine de nombreuses maladies, à caractère héréditaire ou non. Pendant longtemps, la médecine conventionnelle est demeurée impuissante à leur égard. Aujourd'hui, avec le développement de la thérapie génique, on espère pouvoir désormais corriger ou prévenir ce type d'aberration chromosomique. Cette nouvelle médication consiste à introduire une information génétique, dans la cellule ou l'organe affecté, en vu de corriger cette déficience ou anomalie ou encore, d'y exprimer une protéine d'intérêt thérapeutique.Chrornosomal deficiencies and / or anomalies (mutation, outlier, etc.) 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.
L'obstacle majeur à la pénétration d'un acide nucléique dans une cellule ou un organe cible, repose sur la taille et la nature polyanionique de cet acide nucléique qui s'opposent à son passage à travers les membranes cellulaires.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.
Pour lever cette difficulté, diverses techniques sont aujourd'hui proposées dont plus particulièrement la transfection d'ADN nu à travers la membrane plasmique in vivo(WO90/l 1092) et la transfection d'ADN via un vecteur de transfection.To overcome this difficulty, various techniques are now proposed, including more particularly the transfection of naked DNA through the plasma membrane in vivo (WO90 / 1092) and the transfection of DNA via a transfection vector.
En ce qui concerne, la transfection d'ADN nu, son efficacité demeure encore très faible. Les acides nucléiques nus possèdent une demi vie plasmatique courte en raison de leur dégradation par les enzymes et de leur élimination par les voies urinaires.Regarding the transfection of naked DNA, its efficiency is still very low. Naked nucleic acids have a short plasma half-life due to their degradation by enzymes and their elimination through the urinary tract.
Pour ce qui est de la seconde technique, elle propose principalement deux stratégies:As for the second technique, it mainly proposes two strategies:
La première met en oeuvre les vecteurs de transfection naturels que sont les virus. Il est ainsi proposé d'utiliser les adenovirus, les herpès virus, les retrovirus et plus récemment les virus adéno associés. Ces vecteurs s'avèrent performants sur le plan de la transfection mais on ne peut malheureusement exclure totalement à leur égard certains risques de pathogénicité, réplication et/ou immunogénicité, inhérents à leur nature virale.The first uses the natural transfection vectors that are viruses. It is thus proposed to use adenoviruses, herpes viruses, retroviruses and more recently associated adeno viruses. These vectors prove to be efficient in terms of transfection but unfortunately we cannot totally exclude from them certain risks of pathogenicity, replication and / or immunogenicity, inherent in their viral nature.
La seconde stratégie consiste avantageusement à utiliser des agents non viraux capables de promouvoir le transfert et l'expression d'ADN dans des cellules eucaryotes.The second strategy advantageously consists in using non-viral agents capable of promoting the transfer and expression of DNA in eukaryotic cells.
L'objet de la présente invention s'inscrit plus particulièrement dans cette seconde stratégie.The object of the present invention is more particularly part of this second strategy.
Les vecteurs chimiques ou biochimiques représentent une alternative avantageuse aux virus naturels en particulier pour cette absence de réponse immunologique et/ou recombinaison virale. Ils ne possèdent pas de pouvoir pathogène, le risque de multiplication de l'ADN au sein de ces vecteurs est nul et il ne leur est attaché aucune limite théorique en ce qui concerne la taille de l'ADN à transf ecter. Ces vecteurs synthétiques ont deux fonctions principales, condenser l'ADN à transfecter et promouvoir sa fixation cellulaire ainsi que son passage à travers la membrane plasmique et, le cas échéant, les deux membranes nucléaires.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.
De part sa nature polyanionique, l'ADN n'a naturellement aucune affinité pour la membrane plasmique des cellules de nature également polyanionique. Pour pallier à cet inconvénient, les vecteurs non viraux possèdent généralement tous des charges polycationiques.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.
Parmi les vecteurs synthétiques développés, les polymères cationiques de type polylysine et DEAE dextran ou encore les lipides cationiques ou lipofectants sont les plus avantageux. Ils possèdent la propriété de condenser l'ADN et de promouvoir son association avec la membrane cellulaire. Plus récemment, il a été développé le concept de la transfection ciblée, médiée par un récepteur. Cette technique met à profit le principe de condenser 1ΑDN, grâce au polymère cationique, tout en dirigeant la fixation du complexe à la membrane à l'aide d'un couplage chimique entre le polymère cationique et le ligand d'un récepteur membranaire, présent à la surface du type cellulaire que l'on veut greffer. Les criblages du récepteur à la transferrine, à l'insuline ou du récepteur des asialoglycoprotéines des hépatocytes ont ainsi été décrits. Toutefois, les vecteurs synthétiques proposés aujourd'hui sont encore loin d'être aussi performants que les vecteurs viraux. Ceci peut notamment être la conséquence d'une condensation insuffisante de l'ADN à tranfecter et/ou des difficultés rencontrées par l'ADN transfecté pour sortir de l'endosome et pénétrer dans le noyau cellulaire. En effet, le transport d'ADN dans le noyau d'une cellule eucaryote au repos pose un problème évident puisque les dimensions des pores nucléaires ne permettent que la diffusion de protéines de poids moléculaire inférieur à 60 OOODa. (I. Davis et al., Ann. Rev. Biochem. 1995; 64; 865-896). Un ADN plasmidique possédant un poids moléculaire supérieur à IO6 ne peut donc naturellement pénétrer dans le noyau cellulaire par simple diffusion.Among the synthetic vectors developed, 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 1ΑDN, 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. However, 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.
La présente invention a précisément pour objectif de proposer une solution avantageuse au problème précité.The present invention specifically aims to provide an advantageous solution to the above problem.
Plus précisément, la présente invention propose une composition pharmaceutique utile pour la transfection d'au moins un acide nucléique caractérisée en ce qu'elle contient outre ledit acide nucléique et au moins un agent de transfection, au moins un composé associant des propriétés de fixation de l'ADN à une capacité de vectorisation nucléaire de cet ADN.More specifically, 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.
Au sens de l'invention, un composé possédant des propriétés de fixation de l'ADN, couvre tout composé capable de se fixer au moins partiellement à l'ADN à vectoriser. En ce qui concerne plus particulièrement son aptitude à vectoriser cet ADN, elle se traduit au sens de l'invention par une efficacité à diriger cet ADN efficacement à travers les différentes membranes cellulaires et/ou nucléaires pour le conduire jusqu'au sein du noyau de la cellule à traiter. Le composé selon l'invention peut également se présenter par exemple sous l'aspect d'une molécule chimère associant un domaine de fixation à l'ADN, à un domaine permettant l'import nucléaire. Dans ce cas particulier, on peut sélectionner parmi des domaines de fixation à l'ADN, ceux issus de protéines régulatrices capables de se lier par exemple à des séquences spécifiques ou au contraire de protéines connues pour posséder une affinité non séquence dépendante pour l'ADN. En ce qui concerne plus particulièrement le domaine impliqué dans l'import nucléaire, il peut être par exemple figuré par une séquence dites nls (Nuclear localisation Séquence). De telles séquences pourront être apparentées ou dérivées au consensus bipartite déduit de la nucléoplasmine ou au consensus de l'antigène T de SV40.Within the meaning of the invention, 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. . As regards more particularly the field involved in nuclear import, it can for example be represented by a 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.
Selon un mode de réalisation particulier, l'invention concerne une composition pharmaceutique utile pour la transfection d'au moins un acide nucléique caractérisée en ce qu'elle contient outre ledit acide nucléique et au moins un agent de transfection, au moins un composé appartenant à la famille des HMG ou l'un de ses dérivés.According to a particular embodiment, 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.
Les protéines de type HMG, pour "High Mobility Group" sont des protéines riches en acides aminés chargés et possédant une masse moléculaire inférieure à 30000 Da. Solubles dans l'acide perchlorique 2-5%, elles sont classiquement extraites de la chromatine par 0,35 M NaCl.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.
Classiquement, on distingue 3 familles de protéines HMG: les protéines de type HMG1/2 de masse moléculaire voisine de 25000, HMG14/17 de masse moléculaire voisine de 10-12000, et HMGI/Y de composition voisine des protéines de type HMG14/17 mais dont la répartition tissulaire et au cours de l'ontogenèse est différente. On sait que la séquence primaire des protéines est conservée au cours de l'évolution au sein de chacune de ces trois familles.Conventionally, there are 3 families of 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.
En ce qui concerne plus particulièrement les protéines de la famille HMG 1/2, elles se caractérisent par la présence d'une séquence de 80 acides aminés à prédominance basique (charge nette +20), dite "boîte HMG", qui constitue un domaine de liaison à l'ADN. Dans cette famille on distingue les protéines capables de se lier à des séquences spécifiques de l'ADN double brin et les protéines dont la spécificité de liaison réside dans une stucture tridimensionnelle particulière de l'ADN. Dans la première catégorie on trouve les protéines, UBF, SRY, TCF1, ABF2, qui stimulent la transcription de gènes spécifiques (Greiss E.A. et al. J. Mol. Evol. (1993) 37:204-210). La séquence de chacune de ces protéines contient une ou plusieurs "boîtes HMG". La deuxième catégorie est représentée par les protéines HMG1 et HMG2. Leur séquence primaire est caractérisée par la présence de deux "boîtes HMG" et d'une séquence acide en C-terminal (Bustin M. B.B.A. (1990) 1049:231- 243). Ces protéines se lient de façon spécifique sur les séquences d'ADN palindromiques extrudées en structure cruciforme (appariement mtabrin au niveau du palindrome) ou sur les séquences d'ADN qui présentent une forte courbure. Ces deux types de structure ont en commun d'élargir le petit sillon de l'ADN qui devient alors capable d'accomoder la liaison des protéines de type HMG 1/2. Le rôle physiologique des protéines HMG1 et HMG2 est à ce jour peu élucidé. D a toutefois été montré que la protéine HMG1 de veau est transportée de manière active dans le noyau des cellules de mammifères. (L. Kuehl et al ; J. Biol. Chem. 1985 ; 260, 10361-10368).As regards more particularly the proteins of the HMG 1/2 family, they 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. In this family, a distinction is made between 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. In the first category are the proteins, UBF, SRY, TCF1, ABF2, which stimulate the transcription of specific genes (Greiss EA et al. J. Mol. Evol. (1993) 37: 204-210). The sequence of each of these proteins contains one or more "HMG boxes". The second category is represented by the proteins HMG1 and HMG2. Their primary sequence is characterized by the presence of two "boxes HMG "and a C-terminal acid sequence (Bustin MBBA (1990) 1049: 231-243). These proteins bind specifically to the palindromic DNA sequences extruded into a cruciform structure (mtabrin pairing at the palindrome level ) or on DNA sequences which have a strong curvature. These two types of structure have in common the widening of the small DNA groove which then becomes capable of accommodating the binding of proteins of the HMG 1/2 type. The physiological role of the HMG1 and HMG2 proteins has not been fully elucidated to date, but it has been shown that calf HMG1 protein is actively transported in the nucleus of mammalian cells (L. Kuehl et al; J. Biol. Chem. 1985; 260, 10361-10368).
De manière inattendue, la Demanderesse a mis en évidence qu'il était possible de mettre à profit ces facultés des protéines HMG, à savoir leurs aptitudes à fixer l'ADN et à être transportées de manière active dans le noyau, pour promouvoir efficacement la transfection dans le noyau de cellules à traiter de séquences nucléiques hétérologues, associées à au moins un agent transfectant. Au sens de la présente invention, le terme dérivé désigne tout peptide, pseudopeptide (peptide incorporant des éléments non biochimiques) ou protéine différant du composé tel que défini précédemment, obtenu par une ou plusieurs modifications de nature génétique et/ou chimique. Par modification de nature génétique et/ou chimique, on peut entendre toute mutation, substitution, délétion, addition et/ou modification d'un ou plusieurs résidus par exemple de la protéine considérée. Plus précisément, par modification chimique, on entend toute modification du peptide ou protéine générée par réaction chimique ou par greffage chimique de molécule (s), biologique(s) ou non, sur un nombre quelconque de résidus de la protéine. Par modification génétique, on entend toute séquence peptidique dont l'ADN hybride avec ces séquences ou des fragments de celles-ci et dont le produit possède les activités indiquées. De tels dérivés peuvent être générés dans des buts différents, tels que notamment celui d'augmenter raffinité du polypeptide correspondant pour son ligand d'ADN, celui d'améliorer ses niveaux de production, celui d'augmenter sa résistance à des protéases, celui d'augmenter et/ou de modifier l'une de ses activités, ou celui de lui conférer de nouvelles propriétés pharmacocinétiques et/ou biologiques. Parmi les dérivés résultant d'une addition, on peut citer par exemple les séquences peptidiques chimères comportant une partie hétérologue supplémentaire liée à une extrémité. Le terme dérivé comprend également les séquences proteiques homologues à la séquence considérée, issues d'autres sources cellulaires et notamment de cellules d'origine humaine, ou d'autres organismes, et possédant une activité du même type. De telles séquences homologues peuvent être obtenues par des expériences d'hybridation de l'ADN correspondant. Les hybridations peuvent être réalisées à partir de banques d'acides nucléiques, en utilisant comme sonde la séquence native ou un fragment de celle-ci, dans des conditions de stringence conventionnelles (Maniatis et al.), (Cf techniques générales de biologie moléculaire), ou, de préférence, dans des conditions de stringence élevées.Unexpectedly, 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. Within the meaning of the present invention, 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. More precisely, by chemical modification is meant 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. By 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. Among the 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.
En outre, il est également envisageable dans le cadre de la présente invention de mettre à profit la forte affinité de certaines des protéines de la famille HMG, ou de leurs dérivées pour des structures secondaires présentes sur de l'ADN double brin. E peut notament s'agir de structures à quatre brin pour lesquelles il a notamment été montré que l'HMGl de rat possède une très forte affinité (Bianchi et al. Sciences, 1989, 243, 1056-1059). De telles structures peuvent également être obtenues à partir de séquences naturelles telles que les ITRs du virus associé aux adenovirus ou bien être complètement synthétiques obtenues à partir de palyndromes artificiels.In addition, it is also possible in the context of the present invention to take advantage of the high affinity of some of the proteins of the HMG family, or their derivatives, for secondary structures present on double stranded DNA. 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.
Selon un mode de réalisation préféré de l'invention, le composé mis en oeuvre est choisi parmi les protéines de type HMG 1, 2, 1, Y, 14 et 17 et leurs dérivés. Il est plus préférentiellement représenté par tout ou partie de la protéine HMG1 humaine ou l'un de ses dérivés ou homologues tels que définis précédemmentAccording to a preferred embodiment of the invention, 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.
Dans un mode de réalisation particulièrement avantageux, les compositions de la présente invention comprennent en outre un élément de ciblage permettant d'orienter le transfert de l'acide nucléique. Cet élément de ciblage peut être un élément de ciblage extracellulaire, permettant d'orienter le transfert de l'acide nucléique vers certains types cellulaires ou certains tissus souhaités (cellules tumorales, cellules hépatiques, cellules hématopoiétiques, ete). D peut également s'agir d'un élément de ciblage intracellulaire, permettant d'orienter le transfert de l'acide nucléique vers certains compartiments cellulaires privilégiés (mitochondries, noyau, ete).In a particularly advantageous embodiment, the 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).
Plus préférentiellement, l'élément de ciblage est lié, de manière covalente ou non covalente, au composé selon rinvenhon. L'élément de ciblage peut également être lié à l'acide nucléique. Selon un mode privilégié de l'invention, ledit composé est associé, via une partie hétérologue supplémentaire liée à une de ses extrémités, à un ligand de récepteur cellulaire présent à la surface du type cellulaire comme par exemple un sucre, la totLnsférrine, l'insuline ou la protéine asialo-orosomucoïde. Il peut également s'agir d'un ligand de type intracellulaire comme une séquence signal de location nucléaire, nls, qui privilégie l'accumulation de l'ADN transfecté au sein du noyau.More preferably, 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. According to a preferred embodiment of the invention, 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.
Parmi les éléments de ciblage utilisables dans le cadre de l'invention, on peut citer les sucres, les peptides, les oligonucléotides ou les lipides. Préférentiellement, il s'agit de sucres et/ou peptides tels que des anticorps ou fragments d'anticorps, des ligands de récepteurs cellulaires ou des fragments de ceux-ci, des récepteurs ou fragments de récepteurs, etc. En particulier, il peut s'agir de ligands de récepteurs de facteurs de croissance, de récepteurs de cytokines, de récepteurs de lectines cellulaires ou de récepteurs de protéines d'adhésion. On peut également citer le récepteur de la transferrine, des HDL et des LDL. L'élément de ciblage peut également être un sucre permettant de cibler des lectines tels que les récepteurs asialoglycoprotéiques, ou encore un fragment Fab d'anticorps permettant de cibler le récepteur du fragment Fc des immunoglobulines.Among the targeting elements which can be used in the context of the invention, there may be mentioned sugars, peptides, oligonucleotides or lipids. Preferably, 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. In particular, they may be ligands for growth factor receptors, cytokine receptors, cellular lectin receptors or adhesion protein receptors. We can also cite the transferrin receptor, HDL and LDL. 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.
Avantageusement, le composé selon l'invention peut être en outre polyglycosylé, sulfoné et/ou phosphorylé et/ou greffé à des sucres complexes ou à un composé lipophile comme par exemple une chaîne polycarbonée ou un dérivé du cholestérol.Advantageously, 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.
La composition selon l'invention peut bien entendu comprendre plusieurs composés selon l'invention, de nature différente. De même, il s'avère possible d'associer au composé selon l'invention, outre le composé de ciblage nucléaire δThe 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. δ
précédemment décrit, un second composé caractérisé par sa capacité à compacter l'ADN. De tels composés sont notamment décrits dans la demande FR 95/01865.previously described, a second compound characterized by its ability to compact DNA. Such compounds are in particular described in application FR 95/01865.
Le composé selon l'invention est présent en quantité suffisante pour agir avec l'acide nucléique selon l'invention. C'est ainsi que le rapport composé/acide nucléique (exprimé en poids) peut être compris entre 0,01 et 5 et plus préférentiellement entre 0,25 et 0,5.The compound according to the invention is present in an amount sufficient to act with the nucleic acid according to the invention. Thus, the compound / nucleic acid ratio (expressed by weight) can be between 0.01 and 5 and more preferably between 0.25 and 0.5.
En ce qui concerne l'agent de transfection présent dans la composition selon l'invention, il est préférentiellement choisi parmi les polymères cationiques et les lipofectants.As regards the transfection agent present in the composition according to the invention, it is preferably chosen from cationic polymers and lipofectants.
Selon la présente invention, le polymère cationique est de préférence un composé de formule générale I,According to the present invention, the cationic polymer is preferably a compound of general formula I,
dans laquelle in which
- R peut être un atome d'hydrogène ou un groupe de formule- R can be a hydrogen atom or a group of formula
- n est un nombre entier compris entre 2 et 10;- n is an integer between 2 and 10;
- p et q sont des nombres entiers, étant entendu que la somme p+q est telle que le poids moléculaire moyen du polymère soit compris entre 100 et 10? Da. II est entendu que, dans la formule (I) la valeur de n peut varier entre les différents motifs p. Ainsi, la formule (I) regroupe à la fois les homopolymères et les hétéropolymères.- p and q are 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. It is understood that, in formula (I) the value of n can vary between the different units p. Thus, formula (I) groups together both homopolymers and heteropolymers.
Plus préférentiellement, dans la formule (I), n est compris entre 2 et 5. En particulier, les polymères de polyethylene imine (PEI) et polypropylène irnine (PPI) présentent des propriétés tout à fait avantageuses. Les polymères préférés pour la mise en oeuvre de la présente invention sont ceux dont le poids moléculaire est compris entre 10^ et 5.10^. A titre d'exemple, on peut citer le polyethylene irnine de poids moléculaire moyen 50000 Da (PEI50K) ou le polyethylene irnine de poids moléculaire moyen 800000 Da (PEI800K).More preferably, in formula (I), n is between 2 and 5. In particular, 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 ^. By way of example, mention may be made of polyethylene irnine of average molecular weight 50,000 Da (PEI50K) or polyethylene irnine of average molecular weight 800,000 Da (PEI800K).
Le PEI50K ou Le PEI800K sont accessibles commercialement Quant aux autres polymères représentés par la formule générale I, ils peuvent être préparés selon le procédé décrit dans la demande de brevet FR 9408735.PEI50K or PEI800K are commercially available. As for the other polymers represented by general formula I, they can be prepared according to the process described in patent application FR 9408735.
Pour obtenir un effet optimum des compositions de l'invention, les proportions respectives du polymère et de l'acide nucléique sont de préférence déterminées de sorte que le rapport molaire R = aminés du polymère/phosphates de l'acide nucléique soit compris entre 0,5 et 50, plus préférentiellement entre 5 et 30. Des résultats tout particulièrement avantageux sont obtenus en utilisant de 5 à 15 équivalents d'aminés de polymère par charge d'acide nucléique.To obtain an optimum effect of the compositions of the invention, the respective proportions of the polymer and of the nucleic acid are preferably determined so that the molar ratio R = amines of the polymer / phosphates of the nucleic acid is between 0, 5 and 50, more preferably between 5 and 30. Very particularly advantageous results are obtained using 5 to 15 equivalents of polymer amines per charge of nucleic acid.
En ce qui concerne plus particulièrement les lipofectants, il s'agit de molécules amphiphiles comprenant au moins une région hydrophile cationique, par exemple polyamine, et une région lipophile. La région cationique, préférentiellement polyamine, chargée cationiquement, est capable de s'associer de manière réversible avec l'acide nucléique, chargé négativement. Cette interaction compacte fortement l'acide nucléique. La région lipophile rend cette interaction ionique inaccessible au milieu aqueux externe, en recouvrant la particule nucléolipidique formée d'une pellicule lipidique.As regards more particularly the lipofectants, 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.
Avantageusement, ces lipofectants peuvent également être choisis parmi des lipopolyamines dont la région polyamine répond à la formule générale πAdvantageously, these lipofectants can also be chosen from lipopolyamines whose polyamine region corresponds to the general formula π
H2N-(-(ÇH)m-NH-)n-H IIH 2 N - (- (ÇH) m -NH-) n -H II
dans laquelle m est un nombre entier supérieur ou égal à 2 et n est un nombre entier supérieur ou égal à 1, m pouvant varier entre les différents groupes de carbone compris entre deux aminés. Préférentiellement, m est compris entre 2 et 6 inclusivement et n est compris entre 1 et 5 inclusivement. Encore plus préférentiellement, la région polyamine est représentée par la spermine, la thermine ou un de leurs analogues ayant conservé des propriétés de liaison à l'ADN. Quant à la région lipophile, elle est représentée par au moins une chaîne hydrocarbonée, saturée ou non, du cholestérol, un lipide naturel ou un lipide synthétique capable de former des phases lamellaires ou hexagonales, liée de manière covalente à la région hydrophile.in which 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 between two amines. Preferably, m is between 2 and 6 inclusive and n is between 1 and 5 inclusive. Even more preferably, the polyamine region is represented by spermine, thermine or one of their analogs which have retained DNA binding properties. As for 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.
La demande de brevet EP 394 111 décrit d'autres Hpopolyarnines de formule générale III susceptibles d'être mises en oeuvre dans le cadre de la présente inventionPatent application EP 394 111 describes other Hpopolyarnines of general formula III capable of being used in the context of the present invention
H2N-(-(ÇH)m-NH-)n-H IIH 2 N - (- (ÇH) m -NH-) n -H II
dans laquelle R représente notamment un radical de formule générale (RjR2) N-CO- .in which R represents in particular a radical of general formula (RjR2) N-CO- .
A titre représentatif de ces lipopolyamines on peut plus particulièrement citer la dioctadécylamidoglycyl spermine (DOGS) et la 5-carboxyspermylamide de la palmitoylphosphatidylethanolamine (DPPES).Mention may more particularly be made, as representative of these lipopolyamines, of dioctadecylamidoglycyl spermine (DOGS) and 5-carboxyspermylamide of palmitoylphosphatidylethanolamine (DPPES).
Les lipopolyamines décrites dans la demande de brevet FR 94 14596 peuvent également être utilisées avantageusement à titre d'agent de transfection selon l'invention. Elles sont représentées par la formule générale HI ci-dessus dans laquelle R représenteThe 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
avec with
- X et X' représentant, indépendamment l'un de l'autre, un atome d'oxygène, un groupement méthylène -(CH2)q- avec q égal à 0, 1, 2 ou 3, ou un groupement amino- X and X 'representing, 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- ou -NR'- avec R' représentant un groupement alkyle en C\ à C4, - Y et Y' représentant indépendamment l'un de l'autre un groupement méthylène, un groupement carbonyle ou un groupement C=S, - R3, R4 et R5 représentant indépendamment l'un de l'autre un atome d'hydrogène ou un radical alkyle, substitué ou non, en Cjà C4, avec p pouvant varier entre 0 et 5,-NH- or -NR'- with R 'representing a C 1 -C 4 alkyl group, - Y and Y' representing independently of one another a methylene group, a carbonyl group or a C = S 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 représentant un dérivé du cholestérol ou un groupement alkyle amino -NR1R2 avec Rj et R2 représentant indépendamment l'un de l'autre un radical aliphatique, saturé ou non, linéaire ou ramifié en Cj2 à C22.- 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.
A titre représentatif de ces kpcpolyamines on peut tout particulièrement citer le (Dioctadécyl-carbamoylméthoxy)-acétate de 2-5-bJs-(3-amino-prc>pylarnino)-pentyle et le (Dioctadécyl-carbamoylméthoxy)-acétate de l,3-bis-(3-ammo-propylarnino)-2 propyle.As a representative of these kpcpolyamines, mention may very particularly be made of (Dioctadecyl-carbamoylmethoxy) 2-5-bJs- (3-amino-prc> pylarnino) -pentyl acetate and (Dioctadecyl-carbamoylmethoxy) -acetate -bis- (3-ammo-propylarnino) -2 propyl.
Les demandes de brevet EP 394 111 et FR 94 décrivent également un procédé utilisable pour la préparation des lipopolyamines correspondantes.Patent applications EP 394 111 and FR 94 also describe a process which can be used for the preparation of the corresponding lipopolyamines.
De manière particulièrement avantageuse, on peut utiliser dans le cadre de l'invention la dioctadécylamidoglycyl spermine (DOGS) la 5-carboxyspermylamide de la palmitoylphosphatidylemanolamine (DPPES), le (Dioctadécyl-carbamoylméthoxy)- acétate de 2-5-bis-(3-amino-propylamino)-pentyle ou le (Dioctadécyl-carbamoyl- méthoxy) -acétate de l,3-bis-(3-amino-propylamino)-2 propyle.Particularly advantageously, it is possible to use in the context of the invention dioctadecylamidoglycyl spermine (DOGS) 5-carboxyspermylamide of palmitoylphosphatidylemanolamine (DPPES), (Dioctadecyl-carbamoylmethoxy) - 2-5-bis- (3- acetate) amino-propylamino) -pentyl or 1,3-bis- (3-amino-propylamino) -2 propyl (Dioctadecyl-carbamoyl-methoxy) -acetate.
Pour obtenir un effet optimum des compositions de l'invention, les proportions respectives de la polyamine et de l'acide nucléique sont de préférence déterminées de sorte que le rapport R charges positives de l'agent de transfection/ charges négatives de l'acide nucléique soit compris entre 0,1 et 10 et plus préférentiellement entre 0,5 et 2.To obtain an optimum effect of the compositions of the invention, 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.
La présence d'un composé selon l'invention au sein d'une composition transfectante est avantageuse à plusieurs titres. En particulier, il s'en suit une toxicité nettement amoindrie qui rend désormais possible par exemple la transfection de cellules sensibles à l'origine à l'agent de transfection comme par exemple les cellules hématopoïétiques avec les lipopolyamines. Dans les compositions de la présente invention, l'acide nucléique peut être aussi bien un acide désoxyribonucléique qu'un acide ribonucléique. D peut s'agir de séquences d'origine naturelle ou artificielle, et notamment d'ADN génomique, d'ADNc, d'ARNm, d'ARNt, d'ARNr, de séquences hybrides ou de séquences synthétiques ou semi-synthétiques. Ces acides nucléiques peuvent être d'origine humaine, animale, végétale, bactérienne, virale, etc. Us peuvent être obtenus par toute technique connue de l'homme du métier, et notamment par criblage de banques, par synthèse chimique, ou encore par des méthodes mixtes incluant la modification chimique ou enzymatique de séquences obtenues par criblage de banques. Ds peuvent par ailleurs être incorporés dans des vecteurs, tels que des vecteurs plasmidiques.The presence of a compound according to the invention within a transfecting composition is advantageous for several reasons. In particular, there follows a markedly reduced toxicity which henceforth makes possible, for example, the transfection of cells sensitive at the origin to the transfection agent such as, for example, hematopoietic cells with lipopolyamines. In the compositions of the present invention, 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.
Concernant plus particulièrement les acides désoxyribonucléiques, ils peuvent être simple ou double brin. Ces acides désoxyribonucléiques peuvent coder pour des gènes thérapeutiques, des séquences régulatrices de la transcription ou de la réplication, des séquences antisens, des régions de liaison à d'autres composants cellulaires, etc.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.
Au sens de l'invention, on entend par gène thérapeutique notamment tout gène codant pour un produit protéique ayant un effet thérapeutique. Le produit protéique ainsi codé peut être une protéine, un peptide, etc. Ce produit protéique peut être homologue vis-à-vis de la cellule cible (c'est-à-dire un produit qui est normalement exprimé dans la cellule cible lorsque celle-ci ne présente aucune pathologie). Dans ce cas, l'expression d'une protéine permet par exemple de pallier une expression insuffisante dans la cellule ou l'expression d'une protéine inactive ou faiblement active en raison d'une modification, ou encore de surexprimer ladite protéine. Le gène thérapeutique peut aussi coder pour un mutant d'une protéine cellulaire, ayant une stabilité accrue, une activité modifiée, etc. Le produit protéique peut également être hétérologue vis-à-vis de la cellule cible. Dans ce cas, une protéine exprimée peut par exemple compléter ou apporter une activité déficiente dans la cellule, lui permettant de lutter contre une pathologie, ou stimuler une réponse immunitaire. Parmi les produits thérapeutiques au sens de la présente invention, on peut citer plus particulièrement les enzymes, les dérivés sanguins, les hormones, les lymphokines : interleukines, interférons, TNF, ete (FR 9203120), les facteurs de croissance, les neurotransmetteurs ou leurs précurseurs ou enzymes de synthèse, les facteurs trophiques : BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NT5, HARP/pléiotrcφhine, ete; les apolipoprotéines : ApoAI, ApoAIV, ApoE, ete (FR 93 05125), la dystrophine ou une minidystrophine (FR 9111947), la protéine CFTR associée à la mucoviscidose, les gènes suppresseurs de tumeurs : p53, Rb, RaplA, DCC, k-rev, ete (FR 93 04745), les gènes codant pour des facteurs impliqués dans la coagulation : Facteurs VII, VTII, IX, les gènes intervenant dans la réparation de l'ADN, les gènes suicides (thymidine kinase, cytosine déaminase), etc.For the purposes of the invention, 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). In this case, 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. In this case, 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. Among the 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 / pleiotrcφhine, 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, DCC, k- rev, ete (FR 93 04745), genes coding for factors involved in coagulation: Factors VII, VTII, IX, genes involved in DNA repair, suicide genes (thymidine kinase, cytosine deaminase), etc. .
Le gène thérapeutique peut également être un gène ou une séquence antisens, dont l'expression dans la cellule cible permet de contrôler l'expression de gènes ou la transcription d'ARNm cellulaires. De telles séquences peuvent, par exemple, être transcrites dans la cellule cible en ARN complémentaires d'ARNm cellulaires et bloquer ainsi leur traduction en protéine, selon la technique décrite dans le brevet EP 140 308. Les antisens comprennent également les séquences codant pour des ribozymes, qui sont capables de détruire sélectivement des ARN cibles (EP 321 201).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).
Comme indiqué plus haut, l'acide nucléique peut également comporter un ou plusieurs gènes codant pour un peptide antigénique, capable de générer chez l'homme ou l'animal une réponse immunitaire. Dans ce mode particulier de mise en oeuvre, l'invention permet donc la réalisation soit de vaccins soit de traitements immunothérapeutiques appliqués à l'homme ou à l'animal, notamment contre des microorganismes, des virus ou des cancers. Il peut s'agir notamment de peptides antigéniques spécifiques du virus d'Epstein Barr, du virus HIV, du virus de l'hépatite B (EP 185 573), du virus de la pseudo-rage, ou encore spécifiques de tumeurs (EP 259 212).As indicated above, 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. In this particular mode of implementation, 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).
Préférentiellement, l'acide nucléique comprend également des séquences permettant l'expression du gène thérapeutique et/ou du gène codant pour le peptide antigénique dans la cellule ou l'organe désiré. 11 peut s'agir des séquences qui sont naturellement responsables de l'expression du gène considéré lorsque ces séquences sont susceptibles de fonctionner dans la cellule infectée. Il peut également s'agir de séquences d'origine différente (responsables de l'expression d'autres protéines, ou même synthétiques). Notamment, il peut s'agir de séquences promotrices de gènes eucaryotes ou viraux. Par exemple, il peut s'agir de séquences promotrices issues du génome de la cellule que l'on désire infecter. De même, il peut s'agir de séquences promotrices issues du génome d'un virus. A cet égard, on peut citer par exemple les promoteurs des gènes E1A, MLP, CMV, RSV, etc. En outre, ces séquences d'expression peuvent être modifiées par addition de séquences d'activation, de régulation, etc.Preferably, 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). In particular, they may be promoter sequences of eukaryotic or viral genes. For example, they may be promoter sequences originating from the genome of the cell which it is desired to infect. Likewise, they may be promoter sequences originating from the genome of a virus. In this regard, mention may be made, for example, of the promoters of the E1A, MLP, CMV, RSV, etc. genes. In addition, these expression sequences can be modified by adding activation, regulation sequences, etc.
Par ailleurs, l'acide nucléique peut également comporter, en particulier en amont du gène thérapeutique, une séquence signal dirigeant le produit thérapeutique synthétisé dans les voies de sécrétion de la cellule cible. Cette séquence signal peut être la séquence signal naturelle du produit thérapeutique, mais il peut également s'agir de toute autre séquence signal fonctionnelle, ou d'une séquence signal artificielle.Furthermore, 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.
Plus préférentiellement, les compositions de l'invention comprennent en outre, un ou plusieurs lipides neutres. De telles compositions sont particulièrement avantageuses, notamment lorsque le rapport R est faible. La demanderesse a en effet montré que l'addition d'un lipide neutre permet d'améliorer la formation des particules nucléolipidiques et, de manière surprenante, de favoriser la pénétration de la particule dans la cellule en déstabilisant sa membrane.More preferably, the 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.
Plus préférentiellement, les lipides neutres utilisés dans le cadre de la présente invention sont des lipides à 2 chaînes grasses. De manière particulièrement avantageuse, on utilise des lipides naturels ou synthétiques, zwitterioniques ou dépourvus de charge ionique dans les conditions physiologique. Il peuvent être choisis plus particulièrement parmi la dioleoylphospha- tidylémanolamine (DOPE), le oléoyl-palrrûtoylphos-phatidyléttianolamine (POPE), le di-stéaroyl, -palmitoyl, -mirystoyl phosphatidyléthanolamine ainsi que leurs dérivé N- méthyles 1 à 3 fois; les phosphatidylglycérols, les diacylglycérols, les glycosyldiacyl- glycérols, les cérébrosides (tels que notamment les galactocérébrosides), les sphingo- lipides (tels que notamment les sphingomyélines)ou encore les asialogangliosides (tels que notamment les asialoGMl et GM2).More preferably, the neutral lipids used in the context of the present invention are lipids with 2 fatty chains. Particularly advantageously, 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 GM2).
Ces différents lipides peuvent être obtenus soit par synthèse, soit par extraction à partir d'organes (exemple : le cerveau) ou d'oeufs, par des techniques classiques bien connues de l'homme du métier. En particulier, l'extraction des lipides naturels peut être réalisée au moyen de solvants organiques (voir également Lehninger,These different 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. In particular, the extraction of natural lipids can be carried out using organic solvents (see also Lehninger,
Biochemistry).Biochemistry).
Préférentiellement, les compositions de l'invention, mettant en oeuvre à titre d'agent de transfection un lipofectant, comprennent de 0,1 à 20 équivalents de lipide neutre pour 1 équivalent de Upopolyarnine, et, plus préférentiellement, de 1 à 5. Dans le cas où l'agent de transfection est un polymère cationique, les compositions de l'invention comprennent, en plus du polymère cationique dans les rapports cités ci- avant, de 0,1 à 20 équivalents molaires de lipide neutre pour 1 équivalent molaire de phosphate de l'acide nucléique, et, plus préférentiellement, de 1 à 5.Preferably, 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. In in the case where 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.
Les compositions selon l'invention peuvent être formulées en vue d'administrations par voie topique, cutanée, orale, rectale, vaginale, parentérale, intranasale, intraveineuse, intramusculaire, sous-cutanée, intraoculaire, transdermique, etc.. De préférence, les compositions pharmaceutiques de l'invention contiennent un véhicule pharmaceutiquement acceptable pour une formulation injectable, notamment pour une injection directe au niveau de l'organe désiré, ou pour une administration par voie topique (sur peau et/ou muqueuse). Il peut s'agir en particulier de solutions stériles, isotoniques, ou de compositions sèches, notamment lyophilisées, qui, par addition selon le cas d'eau stérilisée ou de sérum physiologique, permettent la constitution de solutés injectables. Les doses d'acide nucléique utilisées pour l'injection ainsi que le nombre d'administrations peuvent être adaptées en fonction de différents paramètres, et notamment en fonction du mode d'administration utilisé, de la pathologie concernée, du gène à exprimer, ou encore de la durée du traitement recherchée. Elles peuvent être avantageusement utilisées pour transfecter une grande variété de type cellulaire comme par exemple les ceUules hématopoïétiques, les lymphocytes, les hépatocytes, les cellules endothéliales, les cellules de mélanomes, de carcinomes et de sarcomes, les cellules musculaires lisses, les neurones et les astrocytes.The 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. 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. 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.
La présente invention fournit ainsi une méthode particulièrement avantageuse pour le traitement de maladies utilisant la transfection in vitro, ex vivo ou in vivo d'un acide nucléique apte à corriger ladite maladie en association avec un agent de transfection de type polymère cationique ou lipofectant, et un composé tel que défini ci-avant Plus particulièrement, cette méthode est applicable aux maladies résultant d'une déficience en un produit protéique ou nucléique et l'acide nucléique administré code pour ledit produit protéique ou contient la séquence correspondant audit produit nucléique. Les compositions selon l'invention sont particulièrement intéressantes pour leur biodisponibilité et leur niveau de transfection.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.
La présente invention concerne également toute utilisation d'un composé selon l'invention couplé à un ligand de récepteur cellulaire, un anticorps ou dérivé d'anticorps, cibler un acide nucléique vers des cellules exprimant les récepteurs ou anti-gènes correspondants. Dans cette perspective, un ligand, anticorps ou dérivé d'anticorps potentiel est couplé audit composé et l'on apprécie le pouvoir de transfection de cette molécule chimère comparativement au composé seul.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. In this perspective, 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.
La présente invention sera plus complètement décrite à l'aide des exemples qui suivent, qui doivent être considérés comme illustratifs et non limitatifs.The present invention will be more fully described with the aid of the following examples, which should be considered as illustrative and not limiting.
FIGURES:FIGURES:
Figure 1: Représentation en intensités liunineuses de l'efficacité de transf ections réalisées selon l'invention dans diiférents types cellulaires.Figure 1: Representation in line intensities of the efficiency of transf ections carried out according to the invention in different cell types.
Figure 2: Appréciation de l'efficacité de transf ections réalisées en présence et en absence de HMG 1. MATERIEL ET METHODES :Figure 2: Assessment of the efficiency of transf ections performed in the presence and absence of HMG 1. MATERIAL AND METHODS :
La construction utilisée pour mettre en évidence l'activité des compositions de l'invention est un plasmide comportant le gène codant pour la luciférase (Luc), pCMV- Luc.The construction used to demonstrate the activity of the compositions of the invention is a plasmid comprising the gene coding for luciferase (Luc), pCMV-Luc.
Le plasmide pCMV-luc comporte le promoteur du Cytomégalovirus (CMV), extrait du plasmide vecteur pcDNA3 (Invitrogen) par coupure avec les enzymes de restriction Mlu I et Hindiπ, situé en amont du gène codant pour la luciférase, inséré aux sites Mlul et Hindlϋ dans le vecteur pGL basic Vector (Promega).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 Hindiπ, located upstream of the gene coding for luciferase, inserted at the Mlul and Hindlϋ sites in the vector pGL basic Vector (Promega).
EXEMPLE 1; PREPARATION DE LA PROTEINE HMG1 DE RAT 1.1 Expression de la protéine HMG1 chez E. coliEXAMPLE 1; PREPARATION OF THE RAT HMG1 PROTEIN 1.1 Expression of the HMG1 protein in E. coli
Cette protéine recombinante provenant d'un mammifère a été préparée par surexpression chez E. coli.This recombinant protein from a mammal was prepared by overexpression in E. coli.
Le plasmide T7-RNHMG1 codant pour la protéine HMG1 de rat (M. E. Bianchi, Gène, 104 (1991) 271-275) est introduit dans la souche d'E. coli BL21(DE3). La souche est par la suite cultivée à 37°C en milieu LB+ Ampicilline (25 mg/L). Une préculture est obtenue à partir d'une colonie isolée. Elle permet d'ensemencer une culture de 500 ml. Lorsque l'absorbance à 600nm de la culture atteint la valeur de 0,7, la synthèse de HMG1 est induite par addition d'IPTG à 0,5 mM final. La souche productrice de HMG1 est encore cultivée 2h30 en présence d'IPTG. Les cellules sont ensuite récoltées par centrifugation (5000 x g, 20 minutes), rincées par 200 ml d'eau distillée centrifugées de nouveau. Le culot de cellules est conservé à -80°C jusq'à purification.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.
1.2; purification de la protéine HMG1 recombinante1.2; purification of the recombinant HMG1 protein
La purification de la protéine HMG1 peut être effectuée par chromatographie à partir d'une culture de la souche de E. coli décrite dans l'exemple 1.1, par exemple en utilisant le protocole suivant : Sauf indication contraire, l'ensemble de la purification décrite ci-dessous est effectuée à 4°C. Les cellules obtenues à partir de 500 ml de culture sont resuspendues dans 15tml de tampon 50 mM Tris/HCl pH 7,7 contenant 500 μM EDTA, 5 mM DTT, 200tμM Pefabloc SC [4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride], et 10 % (poids/volume) glycerol. Après cassage des cellules par sonication durant 15tmin, les débris cellulaires sont séparés par centrifugation (50 000 x g; 1 h). L'extrait acellulaire est ensuite chromatographie à travers une colonne de Sephadex G- 25 (Pharmacia) équilibrée et éluée avec du tampon A [20 mM Hepes pH 7,9 contenant 400 mM chlorure de sodium, 200 mM EDTA, 1 mM DTT, 200 μM Pefabloc SC, 0,2 % Nonidet P40, et 10 % glycerol]. La fraction contenant les protéines est récoltée et chromatographiee à travers une colonne de gel DEAE Sephadex A-25 (Pharmacia) équilibrée dans le tampon A. La fraction protéique non retenue sur cette colonne est mélangée progressivement avec du sulfate d'ammonium solide jusqu'à une concentration finale de 2,8 M. Après 2 h, cette suspension est centrifugée (30 000 x g; 15 min). Le surnageant est chromatographie à 20°C à travers une colonne Phényl- Superose HR 5/5 (Pharmacia) équilibrée dans du tampon 20 mM Hepes pH 7,9 contenant 200 mM EDTA, 500 μM DTT, et 2,8 M sulfate d'ammonium. Les protéines sont éluées de la colonne avec un gradient linéaire décroissant de sulfate d'ammonium (2,8 M à 0 M) dans le même tampon. Les fractions contenant la protéine HMGl sont regroupées et dialysées extensivement contre du tampon 50 mM Tris/HCl pH 7,7 contenant 1 mM EDTA et 500 μM DTT. Cet échantillon est ensuite injecté sur une colonne MonoQ HR 5/5 (Pharmacia) qui est ensuite éluée avec un gradient linéaire de 0 à 0,5 M de chlorure de sodium dans du tampon 50 mM Tris/HCl pH 7,7-500 μM DTT. La protéine HMGl, qui forme un pic d'absorbance symétrique à 280 nm, est collectée dans ce tampon. La protéine HMGl est ensuite reprise dans du tampon lOmM Mes pH6,2-140 mM NaCl-500 μM DTT après concentration par centrifugation dans Centrikon 10. La protéine HMGl est stockée à -80°C jusqu'à utilisation. Cette préparation présente une seule bande protéique migrant à un poids moléculaire apparent de 31 000 lorsqu'elle est analysée par électrophorese en conditions dénaturantes (SDS) et révélation au Coomassie. Le rendement global de la purification est de 850 μg de protéine HMGl pure pour 500 ml de culture de départ. EXEMPLE 2: TRANSFERT D'ACIDE NUCLEIQUE IN VITRO DANS DES CELLULES DE MAMMIFEREThe 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 μM EDTA, 5 mM DTT, 200 t μM Pefabloc SC [4- (2-aminoethyl) -benzenesulfonyl fluoride hydrochloride] , and 10% (weight / volume) glycerol. After breaking the cells by sonication for 15 minutes, the cell debris are separated by centrifugation (50,000 xg; 1 h). 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 μM 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 μM 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 μM 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 μM 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 μM 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 μg of pure HMGl protein per 500 ml of starting culture. EXAMPLE 2 TRANSFER OF NUCLEIC ACID IN VITRO IN MAMMALIAN CELLS
Cet exemple montre comment une protéine, de type HMGl, se liant à l'ADN et étant importée dans le noyau de manière active, peut être utilisée pour stimuler la transfection d'ADN plasmidique.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.
La construction utilisée pour mettre en évidence l'activité des compositions de l'invention est le plasmide comportant le gène codant pour la luciférase (Luc) décrit précédemment.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.
Le protocole est établi pour des plaques de 24 puits (0 16mm) à récolter 2 jours après transfection (cellules à confluence). Tous les paramètres peuvent être modifiés proportionnellementThe 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
Le jour J les ceUules NIH 3T3 (ATCC: CRL1658), 3LL (Isakov N.et al, JNCID-day NIH 3T3 ceUules (ATCC: CRL1658), 3LL (Isakov N. et al, JNCI
71(1983) 139-145) H460 (Maxwell et al , Oncogène 8 (1993), 3421-3429) sont ensemencées à 10^ cellules par puits. Le jour J+2 les cellules sont rincées avec du PBS (pour éliminer les traces de sérum) et reprises dans 250 ml de RPMI (3LL et H460) ou DMEM (NIH3T3), supplémenté ou non par 10 % Sérum Fetal de Veau (SFV)71 (1983) 139-145) 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 mise en oeuvre pour la transfection:Par équivalent puits, dans un tube on ajoute : - H2O qsp 20 mlComposition used for the transfection: By equivalent well, in a tube is added: - H2O qs 20 ml
- NaCl qsp 140 mM final- NaCl qs 140 mM final
- 0,5 mg d'ADN plasmidique - 125 ng HMGl puis on vortexe modérément et on incube à température ambiante 15 minutes avant d'ajouter 1,5 nmole de lipofectant DOGS. On vortexe à nouveau modérément et incube à température ambiante 15 minutes- 0.5 mg of plasmid DNA - 125 ng HMGl then we vortex moderately and incubate at room temperature 15 minutes before adding 1.5 nmol of lipofectant DOGS. We vortex again moderately and incubate at room temperature for 15 minutes
Les cellules sont transfectées par ajout de 20 ml du mélange ADN/HMGl/lipofectant au milieu de culture, incubées pendant 2 à 4 heures à 37°C. Ce milieu est ensuite remplacé par du milieu complet. Le jour J+4 les cellules sont rincées à température ambiante par 250 ml de PBS, lysées dans 100 ml de tampon ad hoc (Reporter (Promega)+TCK et Arjrotinin). 10 ml de lysat et 50 ml de substrat (Promega) sont utilisés pour mesurer l'activité de la luciférase synthétisée. Les résultats présentés en figures 1 et 2 sont la moyenne de quatre expériences, répétées indépendament 2 fois. Pour ce faire on apprécie les Unitées Lumineuses (UL) obtenues par expression du gène Luc dans les cellules transfectées.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.
La figure 1 rassemble les valeurs obtenues pour les trois types cellulaires précédemment cités, en présence de quantité variable de protéine HMGl. La figure 2 rassemble de manière synoptique les facteurs de stimulation de la transfection obtenus par ajout de différentes quantités de protéines HMGl. L'augmentation de l'efficacité de transfection par la protéine HMGl est variable selon les types cellulaires. On observe ainsi qu'elle est maximale lorsque le milieu contenant la composition nécessaire à la transfection est supplémenté par 10% SFV. Ceci est intéressant car la présence de SFV représente les conditions rencontrées in vivo. D'autre part il est notable que la présence de SFV diminue l'efficacité de transfection, particulièrement en l'absence de protéine HMGl. On peut penser que la présence de SFV dans le milieu de culture diminue la quantité d'ADN susceptible d'être intemalisé par les cellules. Dans ce cadre on peut conclure que HMGl est particulièrement avantageuse pour la transfection dans des conditions où la quantité d'ADN est limitante. Le rapport HMG1/ADN (en masse) optimal pour la transfection est 0,25 à 0,5. De telles conditions ne sont pas décrites comme étant capable de compacter l'ADN (Bôttger M. ét al. , B.B.A. 950 (1988), 221-228); Stros M. et ai , N.A.R. 22 (1994), 1044-1051). En effet le plasmide n'est pas saturé par HMGl (Kohlstaedt LA. et ai , Biochemistry 33 (1994), 12702-12707). L'effet de la protéine HMGl est donc expliqué par sa capacité à lier l'ADN et à être transportée vers le noyau de la cellule. 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. This is interesting because the presence of SFV represents the conditions encountered in vivo. On the other hand, it is notable that 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. In this context it can be concluded that 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). Indeed, 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.

Claims

REVENDICATIONS
1. Composition pharmaceutique utile pour la transfection d'au moins un acide nucléique caractérisée en ce qu'elle contient outre ledit acide nucléique et au moins un agent de transfection au moins un composé associant des propriétés de fixation de l'ADN à une capacité de vectorisation nucléaire de cet ADN .1. 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 DNA binding properties with a capacity for nuclear vectorization of this DNA.
2. Composition pharmaceutique utile pour la transfection d'au moins un acide nucléique caractérisée en ce qu'elle contient outre ledit acide nucléique et au moins un agent de transfection au moins un composé appartenant à la famille des HMG ou l'un de ses dérivés.2. 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 .
3. Composition pharmaceutique selon la revendication 1 ou 2 caractérisée en ce que le composé est choisi parmi les protéines de type HMG 1, 2, 1, Y, 14 et 17 et leurs dérivés.3. Pharmaceutical composition according to claim 1 or 2 characterized in that the compound is chosen from proteins of HMG type 1, 2, 1, Y, 14 and 17 and their derivatives.
4. Composition pharmaceutique selon l'une des revendications précédentes carctérisée en ce que le composé est représenté par tout ou partie de la protéine HMGl humaine, l'un de ses dérivés ou homologues.4. Pharmaceutical composition according to one of the preceding claims characterized in that the compound is represented by all or part of the human HMGl protein, one of its derivatives or homologs.
5. Composition pharmaceutique selon l'une des revendications 1 à 4 caractérisée en ce que ledit composé est en outre polyglycosylé, sulfoné, phosphorylé et/ou greffé à des sucres complexes ou à un agent lipophile.5. Pharmaceutical composition according to one of claims 1 to 4 characterized in that said compound is also polyglycosylated, sulfonated, phosphorylated and / or grafted to complex sugars or to a lipophilic agent.
6. Composition pharmaceutique selon l'une des revendications 1 à 5 caractérisée en ce que ledit composé est en outre associé à un ligand de récepteur cellulaire ou nucléaire.6. Pharmaceutical composition according to one of claims 1 to 5 characterized in that said compound is also associated with a cell or nuclear receptor ligand.
7. Composition pharmaceutique selon l'une des revendications précédentes caractérisée en ce que l'agent de transfection est un polymère cationique ou un lipofectant.7. Pharmaceutical composition according to one of the preceding claims, characterized in that the transfection agent is a cationic polymer or a lipofectant.
8. Composition phaπnaceutique selon la revendication 7 caractérisée en ce que le polymère cationique est de préférence un composé de formule générale (I) : Γ N-(CH?) l ^ n η 8. Pharmaceutical composition according to claim 7 characterized in that the cationic polymer is preferably a compound of general formula (I): Γ N- (CH ? ) L ^ n η
(D dans laquelle(D in which
- R peut être un atome d'hydrogène ou un groupe de formule- R can be a hydrogen atom or a group of formula
- n est un nombre entier compris entre 2 et 10; - n is an integer between 2 and 10;
- p et q sont des nombres entiers, avec la somme p+q étant telle que le poids moléculaire moyen du polymère soit compris entre 100 et 10?.- p and q are whole numbers, with the sum p + q being such that the average molecular weight of the polymer is between 100 and 10 ?.
9. Composition pharmaceutique selon l'une des revendications 7 ou 8 caractérisée en ce que le polymère cationique est choisi parmi le polyethylene imine9. Pharmaceutical composition according to one of claims 7 or 8 characterized in that the cationic polymer is chosen from polyethylene imine
(PEI) et le polypropylène imine (PPI).(PEI) and polypropylene imine (PPI).
10. Composition pharmaceutique selon la revendication 9 caractérisée en ce que le polymère est choisi parmi le polyethylene imine de poids moléculaire moyen 50 000 (PEI50K) et le polyethylene imine de poids moléculaire moyen 800 000 (PEI800K).10. Pharmaceutical composition according to claim 9 characterized in that the polymer is chosen from polyethylene imine of average molecular weight 50,000 (PEI50K) and polyethylene imine of average molecular weight 800,000 (PEI800K).
11. Composition pharmaceutique selon la revendication 7 caractérisée en ce que le lipofectant comprend au moins une région hydrophile polyamine de formule générale u11. Pharmaceutical composition according to claim 7 characterized in that the lipofectant comprises at least one hydrophilic polyamine region of general formula u
H2N-(-(ÇH)m-NH-)n-H IIH 2 N - (- (ÇH) m -NH-) n -H II
dans laquelle m est un nombre entier supérieur ou égal à 2 et n est un nombre entier supérieur ou égal à 1, m pouvant varier entre les différents groupes de carbone compris entre 2 aminés associée de manière covalente à une région lipophile de type chaîne hydrocarbonée, saturée ou non, du cholestérol, ou un lipide naturel ou synthétique capable de former des phases lamellaires ou hexagonales. in which 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 covalently associated with a lipophilic region of the hydrocarbon chain type, saturated or unsaturated, cholesterol, or a natural or synthetic lipid capable of forming lamellar or hexagonal phases.
12. Composition pharmaceutique selon la revendication 11 caractérisée en ce que la région polyamine est représentée par la spermine, la thermine ou un de leurs analogues ayant conservé ses propriétés de liaison à l'acide nucléique.12. Pharmaceutical composition according to claim 11 characterized in that the polyamine region is represented by spermine, thermine or one of their analogs having retained its nucleic acid binding properties.
13. Composition pharmaceutique selon la revendication 11 caractérisée en ce que la région lipophile y est représentée par la formule générale IV13. Pharmaceutical composition according to claim 11 characterized in that the lipophilic region there is represented by the general formula IV
dans laquelle in which
- X et X' représentent, indépendamment l'un de l'autre, un atome d'oxygène, un groupement méthylène -(CH2)q- avec q égal à 0, 1, 2 ou 3, ou un groupement amino -NH- ou -NR'- avec R' représentant un groupement alkyle en C\ à C4,- X and X 'represent, independently of one another, an oxygen atom, a methylene group - (CH 2 ) 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,
- Y et Y' représentent indépendamment l'un de l'autre un groupement méthylène, un groupement carbonyle ou un groupement C≈S,- Y and Y 'independently of one another represent a methylene group, a carbonyl group or a C≈S group,
- R3, R4 et R5 représentent indépendamment l'un de l'autre un atome d'hydrogène ou un radical alkyle, substitué ou non, en C\ à C4, avec p pouvant varier entre 0 et 5, - RÔ représente un dérivé du cholestérol ou un groupement alkyle amino -NRjR2 avec Rj et R2 représentant indépendamment l'un de l'autre un radical aliphatique, saturé ou non, linéaire ou ramifié en Cι2 à C22 - R3, R4 and R5 independently of one another represent a hydrogen atom or an alkyl radical, substituted or unsubstituted, C \ to C4, with p being able to vary between 0 and 5, - RÔ represents a derivative of cholesterol or an amino alkyl group -NRjR 2 with Rj and R 2 representing, independently of one another, an aliphatic radical, saturated or not, linear or branched in Cι 2 to C 22
14. Composition pharmaceutique selon l'une des revendications 11 à 12 ou 13 caractérisée en ce qu'il s'agit de préférence d'une lipopolyamine choisie parmi la dioctadécylamidoglycyl spermine (DOGS), la 5-carboxyspermylamide de la palmitoylphosphatidylethanolamine (DPPES), le (Dioctadécyl-carbamoylméthoxy)- acétate de 2-5-bis-(3-amino-propylamino)-pentyle ou le (Dioctadécyl-carbamoyl- méthoxy) -acétate de l,3-bis-(3-amino-propylamino)-2 propyle.14. Pharmaceutical composition according to one of claims 11 to 12 or 13 characterized in that it is preferably a lipopolyamine chosen from dioctadecylamidoglycyl spermine (DOGS), 5-carboxyspermylamide from palmitoylphosphatidylethanolamine (DPPES), 2-5-bis- (3-amino-propylamino) -pentyl (Dioctadecyl-carbamoylmethoxy) acetate or 1,3-bis- (3-amino-propylamino) acetate (Dioctadecyl-carbamoyl-methoxy) - 2 propyl.
15. Composition pharmaceutique selon l'une quelconque des revendications 1 à 7, 11 à 12 et 14 caractérisée en ce que l'agent de transfection est la dioctadécylamidoglycyl spermine (DOGS). 15. Pharmaceutical composition according to any one of claims 1 to 7, 11 to 12 and 14 characterized in that the transfection agent is dioctadecylamidoglycyl spermine (DOGS).
16. Composition pharmaceutique selon l'une des revendications précédentes caractérisée en ce que l'acide nucléique est un acide désoxyribonucléique.16. Pharmaceutical composition according to one of the preceding claims, characterized in that the nucleic acid is a deoxyribonucleic acid.
17. Composition pharmaceutique selon l'une des revendications précédentes caractérisée en ce que l'acide nucléique est un acide ribonucléique.17. Pharmaceutical composition according to one of the preceding claims, characterized in that the nucleic acid is a ribonucleic acid.
18. Composition pharmaceutique selon la revendication 16 ou 17 caractérisée en ce que l'acide nucléique est modifié chimiquement18. Pharmaceutical composition according to claim 16 or 17 characterized in that the nucleic acid is chemically modified
19. Composition pharmaceutique selon la revendication 16, 17 ou 18 caractérisée en ce que l'acide nucléique est un antisens.19. Pharmaceutical composition according to claim 16, 17 or 18 characterized in that the nucleic acid is an antisense.
20. Composition pharmaceutique selon l'une des revendications 16 à 19 caractérisée en ce que l'acide nucléique comporte un gène thérapeutique.20. Pharmaceutical composition according to one of claims 16 to 19 characterized in that the nucleic acid comprises a therapeutic gene.
21. Composition pharmaceutique selon l'une des revendications précédentes comprenant en outre un ou plusieurs lipides neutres.21. Pharmaceutical composition according to one of the preceding claims, further comprising one or more neutral lipids.
22. Composition pharmaceutique selon la revendication 21 caractérisée en ce que le ou les lipides neutres sont choisis parmi les lipides synthétiques ou naturels, zwitterioniques ou dépourvus de charge ionique dans les conditions physiologiques.22. Pharmaceutical composition according to claim 21 characterized in that the neutral lipid (s) are chosen from synthetic or natural lipids, zwitterionic or devoid of ionic charge under physiological conditions.
23. Composition pharmaceutique selon la revendication 21 ou 22 caractérisée en ce que le ou les lipides neutres sont choisis parmi la dioléoylphospha- tidyléthanolamine (DOPE), l'oléoyl-palmitoylphos-phatidyl-éthanolamine (POPE), le di-stéaroyl, -palmitoyl, -mirystoyl phosphatidyléthanolamine ainsi que leurs dérivé N-méthylés 1 à 3 fois; les phosphatidylglycérols, les diacylglycérols, les glycosyldiacylglycérols, les cérébrosides (tels que notamment les galactocérébrosides), les sphingolipides (tels que notamment les sphingomyélines) et les asialogangliosides (tels que notamment les asialoGMl et GM2).23. Pharmaceutical composition according to claim 21 or 22 characterized in that the neutral lipid (s) are chosen from dioleoylphosphatididethanolamine (DOPE), oleoyl-palmitoylphos-phatidyl-ethanolamine (POPE), di-stearoyl, -palmitoyl , -mirystoyl phosphatidylethanolamine 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) and asialogangliosides (such as in particular asialoGMl and GM2).
24. Utilisation d'une composition pharmaceutique selon l'une des revendications 1 à 23 pour le transfert in vitro, ex vivo et/ou in vivo d'acides nucléiques. 24. Use of a pharmaceutical composition according to one of claims 1 to 23 for the in vitro, ex vivo and / or in vivo transfer of nucleic acids.
25. Utilisation d'un composé tel que défini en revendication 1 ou 2, couplé à un ligand de récepteur cellulaire, un anticorps ou dérivé d'anticorps, cibler un acide nucléique vers des cellules exprimant les récepteurs ou anti-gènes correspondants. 25. Use of a compound as defined in claim 1 or 2, 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.
EP96932668A 1995-09-28 1996-09-27 Pharmaceutical composition useful for nucleic acid transfection, and use thereof Withdrawn EP0854930A1 (en)

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PCT/FR1996/001516 WO1997012051A1 (en) 1995-09-28 1996-09-27 Pharmaceutical composition useful for nucleic acid transfection, and use thereof

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