MXPA00009046A - Nucleic acid transfer vectors, compositions containing same and uses - Google Patents

Abstract

The invention concerns novel vectors and their use for nucleic acid transfer. More particularly it concerns novel vectors capable of directing nucleic acids towards specific cells or cell compartments.

Description

VECTORS OF TRANSFER OF NUCLEIC ACIDS, COMPOSITIONS THAT CONTAIN THEM AND THEIR USE DESCRIPTION OF THE INVENTION The present invention relates to new vectors and their use for the transfer of nucleic acids. More particularly, the present invention relates to new vectors capable of directing nucleic acids towards specific cells or cell compartments. The transfer of nucleic acids is a basic technique in all the major applications of biotechnology and the increase in the efficiency of the transfer of nucleic acids, constitutes a very important advance for the development of these applications. The efficiency of the transfer of nucleic acids depends on numerous factors, among which are the ability of the nucleic acids to reach the target cell, their ability to cross the plasma membrane and their ability to be transported from the heart of the body. cell to its core. One of the main obstacles to the efficient transfer of nucleic acids comes from the fact that genetic information is often little or not directed towards the target organ to which REF, 122034 is intended. On the other hand, once the nucleic acid has penetrated the target cell, it must still be directed towards the nucleus in order to be expressed. Furthermore, in the case of the transfer of nucleic acids in differentiated cells or at rest, the nucleus is limited by a nuclear envelope that constitutes a supplementary barrier to the passage of these nucleic acids. The recombinant viruses used as vectors have evolved and effective mechanisms to guide the nucleic acids to the nucleus. However, these viral vectors have certain drawbacks inherent to their viral nature, which unfortunately can not be excluded in their entirety. Another strategy is either to transfer DNA? 1, or to use non-viral agents capable of promoting the transfer of DNA in eukaryotic cells. However, nonviral vectors do not possess signals of subcellular or nuclear targets. Thus, the passage of naked DNA, or in combination with a non-viral agent, from the cytoplasm to the nucleus, for example, is by a stage that presents very poor efficacy (Zabner et al., 1995). Different attempts have been made to connect signals directed to the target. Primarily target peptide fragments have been covalently linked to oligonucleotides in a strategy to target an antisense oligonucleotide [Eritja et al., Synthesis of defined peptide-oligonucleotide hybrids containing a nuclear transport signal sequence, Tetrahedron, Vol. 47, No. 24, pp. 4113-4120, 1991]. The complexes formed in this way are good candidates as potential inhibitors of the expression of endogenous genes. Transfection vectors comprising a synthetic polypeptide coupled by electrostatic interactions with a DNA sequence have been reported in International Patent Application WO 95/31557, wherein said polypeptide is constituted by a polymeric chain of basic amino acids, an NLS peptide and a hinge region that connects the NLS peptide with the polymer chain and allows to avoid steric interactions.

But this type of constructions has a stability problem since the interactions between the DNA and the target signal are electrostatic in nature. On the other hand, there are nucleic acid-peptide chimeras directed to a specific target described in Patent Application WO 95/34664, in which the union between the two is of a chemical nature. But this method passes through difficult controllable enzymatic steps and does not allow the production of large quantities of nucleic acids. Finally, it has been demonstrated that it is possible to join an NLS ("Nuclear Localisation Signal") sequence to a plasmid DNA by means of a cyclopropanopyrroloindole (Nature Biotechnology, Volume 16, pp. 80-85, January 1998). But a total inhibition of the transcription of the gene of interest has been observed due to the random binding of several hundreds of NLS sequences on the plasmid. A solution proposed by the authors to remedy this situation consists of ligating the NLS sequences on linear fragments of DNA and then coupling these modified fragments with other unmodified ones. However, this technique has, as before, the disadvantage of going through at least one enzymatic step. Thus, all the methods proposed up to the present do not allow satisfactorily solving the difficulties involved in directing double-stranded DNA molecules to the target. The present invention proposes an advantageous solution to these problems. More particularly, the present invention relates to oligonucleotides conjugated with signals directed to the target and capable of forming triple helices with one or several specific sequences present in a double-stranded DNA molecule. - ~ - Such a vector has the advantage of being able to direct a double-stranded DNA towards the cells or the specific cellular compartments, thanks to the signal directed to the target, without the genetic expression being inhibited. The applicant has shown that, thanks to the formation of stable and site-specific triple helices, it will henceforth be possible to bind a target signal to a double-stranded DNA in a site-specific manner. Accordingly, it is possible to fix the targeted signal to the blank apart from the expression cartridge of the gene to be transferred. The applicant, in this way, has shown that genetic expression in the cell is not inhibited by chemical modification of the DNA. In addition, the presence of a triple helix as a means of binding the target signal to the DNA is particularly advantageous since it allows to preserve a size of DNA adapted for transfection. The vector obtained also has the advantage of incorporating signals directed to the target which are very stable in the double-stranded DNA, in particular when the oligonucleotide capable of forming the triple helix is modified by the presence of an alkylating agent. Another advantage of the present invention is that it allows the DNA to be coupled by transferring signals directed to the target while controlling the number and nature. In effect, it is possible to control the number of target-directed signals linked to each double-stranded DNA molecule, by introducing a suitable number of specific sequences appropriate for the formation of triple helices in said double-stranded DNA molecule. In the same way, it is possible to introduce in the same double-stranded DNA molecule, several oligonucleotides linked with different targeting signals (intracellular and / or extracellular) and in this case, it is also possible to previously determine the respective proportions. In addition, these different target-directed signals can be fixed to the double-stranded DNA molecules in a more or less stable manner, depending on whether the triple helix is formed, with or without covalent linkages (ie, with or without the use of a alkylating agent). Finally, the functionalized triple helix obtained is not the result more than stages of chemical transformations and can be obtained in a simple, reproducible and in large quantities, mainly industrial quantities. A first objective of the present invention relates to a vector useful in transfection capable of targeting a cell and / or a specific cell compartment. More particularly, the vector according to the present invention comprises a double-stranded DNA molecule and at least one oligonucleotide coupled to a target signal and capable of forming, by hybridization, a triple helix with a specific sequence present in said molecule of double-stranded DNA. In the sense of the present invention, the term "double-stranded DNA" is to be understood as a double-chain deoxyribonucleic acid which may be of human origin, animal, vegetable, bacterial, viral, et cetera. It can be obtained by any technique known to those skilled in the art and mainly by screening, by chemical or enzymatic synthesis of sequences obtained by screening. It can be modified chemically or enzymatically. This double stranded DNA can be in linear or circular form. In this latter case, the double-stranded DNA may be in a supercoiled or relaxed state. Preferably, the DNA molecule is circular in shape and is in a supercoiled conformation. The double-stranded DNA can also carry an origin of replication, functional or not, of the target cell, one or a plurality of genetic markers, transcriptional or replication regulatory sequences, genes of therapeutic interest, modified antisense sequences or no, binding regions with other cellular components, et cetera. Preferably, the double-stranded DNA comprises an expression cartridge consisting of one or a plurality of genes of interest under the control of one or a plurality of promoters and of an active transcription terminator in the target cells. In accordance with the present invention, the term "expression cartridge of a gene of interest" refers to a DNA fragment that can be inserted into a vector having specific restriction sites. The DNA fragment comprises a nucleic acid sequence coding for an RNA or a polypeptide of interest and further comprises the sequences necessary for the expression (enhancers, promoters, polyadenylation sequences ...) of said sequence. Restriction sites are known to serve to ensure an insertion of the expression cartridge into an appropriate reading frame for transcription and translation In general, it is a plasmid or an episome carrying one or a plurality of genes of interest As an example, mention may be made of the plasmids described in International Patent Applications WO 96/26270 and WO 97/10343, which are incorporated herein by reference In the sense of the present invention, the term "gene of interest" The term "therapeutic" is understood as any gene that codes for a protein product that has a therapeutic effect. way, it can be a protein or a peptide. This protein product can be homologous to the target cell (ie, a product that is normally expressed in the target cell when it does not present any pathology). In this case, the expression of a protein makes it possible, for example, to ally an insufficient expression in the cell or the expression of an inactive or weakly active protein due to some modification, or even to overexpress said protein. The gene of therapeutic interest may also code for some mutant of a cellular protein having a better stability, a modified activity, and so on. The protein product can also be heterologous against the target cell. In this case, an expressed protein, for example, can complete or provide a deficient activity in the cell, which allows it to fight against a pathology, or stimulate an immune response. Among the therapeutic products in the sense of the present invention, mention may be made more particularly of enzymes, blood derivatives, hormones, lymphokines [interleukins, interferons, TNF, etc.]. (French Patent FR 92/03120)], growth factors, neurotransmitters or their precursors or synthetic enzymes, trophic factors [BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NT5, HARP / pleiotrophin, etc. , dystrophin or a minidistrofin (French Patent FR 91/11947)], CFTR protein associated with ucoviscidosis, tumor suppressor genes [p53, Rb, RaplA, DCC, k-rev, etc., (French Patent FR 93 / 04745)], genes that code for factors involved in coagulation [Factors VII, VIII, IX], genes involved in DNA repair, suicide genes [thymidine kinase, cytosine deaminase], hemoglobin genes or other protein transporters, genes that correspond to the proteins involved in lipid metabolism, of the apolipoprotein type, which are selected from the group consisting of apolipoproteins AI, A- II, A-IV, B, CI, C-II, C-III, D, E, F, G, H, J and apo (a), enzymes of metabolism such as for example lipoprotein lipase, hepatic lipase, lecithin cholesterol acyltransferase, 7-alpha-cholesterol hydroxylase, phosphatidic acid phosphatase or even lipid transfer proteins such as the cholesterol ester transfer protein and the phospholipid transfer protein, an HDL binding protein or even a receptor that is selected for example of the group consisting of LDL receptors, remnant chylomicron receptors and waste receptors, and so on. The DNA of therapeutic interest can also be a gene or an antisense sequence whose expression in the target cell allows controlling the expression of genes or the transcription of cellular mRNAs. Such sequences, for example, can be transcribed in the target cell into RNA complementary to the cellular mRNA and thus block its translation into proteins, according to the technique described in European Patent EP 140 308. Genes of therapeutic interest also comprise the sequences coding for ribozymes, which are capable of selectively destroying target RNAs (European Patent EP 321 201) or sequences encoding single chain intracellular antibodies, such as for example ScFv antibodies. As indicated above, the deoxyribonucleic acid may also comprise one or a plurality of genes encoding an antigenic peptide, capable of generating an immune response in humans or animals. In this particular embodiment, the present invention allows the realization either of vaccines, or of immunotherapeutic treatments applied to humans or animals, mainly against microorganisms, viruses or cancers. It can be mainly antigenic peptides specific against the Epstein Barr virus, against the HIV virus, the hepatitis B virus (European Patent EP 185 573), the pseudorabies virus, the "syncytia-forming virus", the influenza, cytomegalovirus (CMV), other viruses or even tumor specific viruses (European Patent EP 259 212). Preferably, the deoxyribonucleic acid also comprises sequences that allow the expression of the gene of therapeutic interest and / or of the gene coding for the antigenic peptide in the desired cell or organ. These may be sequences that are naturally responsible for the expression of the gene considered when these sequences are capable of functioning in the transfected cell. In the same way, it can be sequences of different origin (responsible for the expression of other proteins, or equally synthetic). Primarily, it can be promoter sequences of eukaryotic or viral genes. For example, they can be promoter sequences of the genome of the cell to be infected. Likewise, it can be promoter sequences from the genome of a virus. In this regard, the promoters of the genes E1 ° A, MLP, CMV, RSV, etc. can be cited as an example. On the other hand, these expression sequences can be modified by the addition of activation, regulation and other sequences. They can also be inducible or repressible promoters. A triple helix corresponds to the fixation of a modified oligonucleotide or not on double-stranded DNA by means of hydrogen bonds termed "Hoogsteen" between the bases of the third chain and those of the region in the double helix. These couplings occur in the gran- zurzo of the double helix and are specific to the sequence considered [Frank-Kamenetski, M.D., Triplex DNA Structures, Ann. Rev. Biochem., 1995, 64, pop. 65-95]. The specific sequence in the double helix can be a homopuric-homopyrimidic sequence. Two categories of triple helices are distinguished according to the nature of the bases of the third chain [Sun, J. and C. Héléne, Oligonucleotide-directed triple-helix formation, Curr. Opin. Struct. Biol., 1993, 3, pp. 345-356]: The purine bases allow to obtain matings C-G * G and T-A * A and the pirimídicas bases allow to obtain matings C-G * C and T-A * T (the symbol * corresponds to the mating with the third chain). These structures are characterized from the physicochemical point of view thanks to numerous NMR studies (Nuclear Magnetic Resonance), hybridization temperature or nuclease protection, which allow to define their properties and stability conditions. For triple helices with a third chain, this chain is antiparallel with respect to the DNA strand of the DNA and the formation of the triple helix depends to a large extent on the concentration of divalent ions: Ions such as Mg 2+ stabilize the structure formed with the third chain. For triple helices with a third homopyrimidic chain, it is parallel with respect to the chain and the formation of the triple helix depends on the pH: An acid pH below 6 allows the protonation of cytokines and the formation of a supplementary hydrogen bond It also stabilizes the triplet CG * C "Likewise, there are triple" mixed "helices for which the third chain carries purine and pirimidic bases, in this case, the orientation of the third chain depends on the base sequence of the region. The oligonucleotides used in the present invention are oligonucleotides that hybridize directly with the double-stranded DNA These oligonucleotides can contain the following bases: thymidine (T), which is capable of forming triplets with the DNA doublets AT double chain (Rajagopal et al., Biochem 28 81989) 7859), - adenine (A), which is capable of forming triplets with the AT doublets of the DNA of double chain, - guanine (G), which is capable of forming triplets with the doublets GC of double-stranded DNA, - protonated cytokine (C), which is capable of forming triplets with doublets GC of double-stranded DNA ( Rajagopal et al, supra), - uracil (U) which is capable of forming triplets with base pairs AU or AT To allow the formation of a triple helix by hybridization, it is important that the oligonucleotide and the specific sequence present in the DNA are complementary. In this regard, to obtain the best fixations and the best selectivity, a perfectly complementary oligonucleotide and specific sequence is used for the vector according to the present invention. It can be in particular a poly-CTT oligonucleotide and a poly-GAA specific sequence. For example, one can cite the oTL sequence gonucleotide: 5'-GAGGCTTCTTCTTCTTCTTCTTCTT-3 '_ (GAGG (CTT) 7, SEQ ID No. 1), where the GAGG bases do not form multiple helices, but allow to separate the oligonucleotide of the coupling arm. The sequence can also be cited (CTT) (SEQ ID No. 2). These oligonucleotides are capable of forming a triple helix with a specific sequence comprising the complementary portions (GAA). It can be in particular a region comprising 7, 14 or 17 GAA portions. Another sequence of specific interest is the sequence: 5 '-AAGGGAGGGAGGAGAGGAA-3' (SEQ ID NO 3). This sequence forms a triple helix with the oligonucleotides: 5'-AAGGAGAGGAGGGAGGGAA-3 ') SEQ ID No. 4) or 5'-TTGGTGTGGTGGGTGGGTT-3' (SEQ ID No. 5). In this case, the oligonucleotide is fixed in an antiparallel orientation to the polyuric chain. These triple helices are not stable except in the presence of MG 2+, as previously stated (Vasquez et al., Biochemistry, 1995, 34, 7343,7251; Beal et Dervan, Science, 1991, 251, 1360-1363). The specific sequence may be a sequence of natural origin in the double-stranded DNA, or a synthetic or naturally-occurring sequence introduced artificially. It is particularly interesting to use an oligonucleotide capable of forming a triple helix with a sequence naturally occurring in the double-stranded DNA. Indeed, this advantageously makes it possible to obtain the vectors according to the present invention with unmodified plasmids, mainly commercial plasmids of the pUC type, pBR322, pSV, and so on. Among the homopuric-opirimidic sequences naturally present in double-stranded DNA, a sequence comprising all or a part of the sequence 5 '-CTTCCCGAAGGGAGAAAGG-3' (SEQ ID No. 6) present in the ColEl origin of E. coli replication can be cited. In this case, the oligonucleotide forming the triple helix possesses the sequence: 5 'GAAGGGTTCTTCCCTCTTTCC-3' (SEQ ID No. 7) and is alternatively fixed on the two chains of the double helix, as described by Beal and Dervan (J. Am. Chem. Soc. 1992, 114.4976-4982) and Jayasena and Johnston (Nucleic Acids Res. 1992, 20, 5279-5288). Mention may also be made of the sequence 5 'GAAAAAGGAAGAG-3' (SEQ ID No. 8) of the β-lactamase gene of plasmid pBR322 (Duva-Valentin et al., Proc. Nati. Acad. Sci. USA, 1992, 89 , 504-508). Another sequence is the AAGAAAAAAAAGAA (SEQ ID No. 9) present in the origin of replication? of plasmids with origin of conditional replication, such as the plasmid pCOR. Although perfectly complementary sequences are preferred, it should be understood that a certain mismatch can be tolerated between the sequence of the oligonucleotide and the sequence present in the DNA, which do not lead to any great loss of affinity. Mention may be made of the sequence 5'-AAAAAAGGGAATAAGGG-3 '(SEQ ID No. 10) present in the E. coli β-lactamase gene. In this case, the thymine that interrupts the polyuric sequence can be recognized by a guanine of the third chain, thus forming a triplet ATG that is stable when it is framed between two TAT triplets (Kiessling et al., Biochemistry, 1992, 31 , 2829-2834).

The oligonucleotide used can be natural (composed of natural, unmodified bases) or chemically modified. In particular, the oligonucleotide can advantageously have certain chemical modifications that increase its resistance, its protection against nucleases, its affinity for the specific sequence or even allow for additional properties (J. Goodchild, Conjugates jof Oligonucleotides and Modified Oligonucleotides : A Review of their Synthesis and Properties, Bioconjugate Chemistry, Vol. 1 No. 3, 1990, pp. 165-187). According to the present invention, oligonucleotide is also understood to be any chain of nucleosides that have undergone skeletal modification. Among the possible modifications, mention may be made of oligonucleotides phosphothionates which are capable of forming triple helices with DNA (Xodo et al., Nucleic Acids Res., 1994, 22, 3322-3330), as well as oligonucleotides possessing skeletons of formacetal or methylphosphonate (Matteucci et al., J. Am. Chem. Soc., 1991, 113, _7767-7768). In the same way, oligonucleotides synthesized with a-nucleotide α-anomers can be used, which also form triple helices with the DNA (Le Doan et al., Nucleic Acids Res., 1987, 1J5, 7749-7760). Another modification of the skeleton is the phosphora-idato bond. Mention may be made, for example, of the internucleotide link N3 '-P5' phosphoramidate described by Gryaznov and Chen, which provides oligonucleotides which form particularly stable triple helices with DNA (J. Am. Chem. Soc., 1994 116, 3143-3144). Among the other modifications of the skeleton, mention may also be made of the use of ribonucleotides, 2'-O-methylribose, phosphotriester, etc., (Sun and Héléne, Curr Opinion Struct. Biol., 116, 3143-3144). Finally, the phosphorated skeleton can be replaced by a polyamide skeleton as in the PNA (Peptide Nucleic Acid), which can in the same way form triple helices (Nielsen et al., Science, 1991, 254, 1497-1500; Kim et al. al., J. Am. Chem. Soc., 1993, 115, 6477-6481) or by a guanidine-based skeleton, as in the DNG (deoxyribonucleic guanidine, Proc. Nati. Acad. Sci. USA, 1995, 92 , 6097-6101), polycationic DNA analogues that also form triple helices. The thymine of the third chain can also be replaced by a 5-bromouracil, which increases the affinity of the oligonucleotide for the DNA (Povsic and Dervan, J. Am. Chem. Soc., 1989, 111, 3059-3061). The third chain may also contain non-natural bases, among which may be mentioned 7-deaza-2'-deoxyxantosine (Milligan et al., Nucleic Acids Res., 1993, 21, 327-333), 1- (2 -deoxy-bD-ribofuranosyl) -3-methyl-5-amino-1H-pyrazolo [4, 3-d] -pyrimidin-7-one (Koh and Dervan, J. Am. Chem. Soc., - 1992, 114 , 1470-1478), 8-oxoadenine _, _ 2-aminopurine, 2'-O-methyl-pseudoisocitidine or any other modification known to those skilled in the art (see reference Sun and Héléne, Curr. Opinion Struct. Biol., 1993, 3, 345-356). Another type of modification of the oligonucleotide is more particularly aimed at improving the interaction and / or the affinity between the oligonucleotide and the specific sequence. In particular, an advantageous modification consists in coupling an alkylating agent with the oligonucleotide. The link can be made either chemically or photochemically using a functional group as an intermediate. photoreactive Advantageous alkylating agents are mainly photoactivatable alkylating agents, for example psoralen. Under the action of light, they form covalent bonds at the level of the pyrimidic bases of DNA. When these molecules are intercalated at the level of the 5'-ApT-3 'or 5'-TpA-3' sequences in a double-stranded DNA fragment, they form bonds with the two chains. This light-induced binding reaction can occur at a specific site in the plasmid. As noted above, an advantage of the present invention is the possibility of forming very stable and specific triple helices between the oligonucleotide and a specific sequence of double-stranded DNA, thanks to a covalent bond formed through an alkylating agent. The length of the oligonucleotide used in the method according to the present invention is at least three bases and preferably between 5 and 30 bases. An oligonucleotide of a length comprised between 10 and 30 bases is advantageously used. It should be understood that the length can be adapted, as the case may be, by those skilled in the art, depending on the desired selectivity and interaction stability. The oligonucleotides according to the present invention can be synthesized by any known technique. In particular, they can be prepared by means of nucleic acid synthesizers. Any other method known to those skilled in the art can also be used. In the sense of the present invention, the term "signal directed to the target" means molecules directed to the target, which are of varied nature. In most cases they are polypeptides known for their targets. They can also be used to interact with an extracellular matrix component, with a plasma membrane receptor, to target some intracellular compartment or to improve intracellular DNA trafficking, at the time of non-viral gene transfer in therapy gene These targeting signals may comprise, for example, growth factors (EGF, PDGF, TGFb, NGF, IGF I, FGF), cytokines (IL-1, IL-2, TNF, interferon, CSF), hormones (insulin, growth hormone, prolactin, glucagon, thyroid hormone, steroid hormones), sugars that recognize lectins, immunoglobulins, ScFv, transferrin, lipoproteins, vitamins such as vitamin B12, peptide hormones or neuropeptides (tachykinins, neurotensin, VIP, endothelin, CGRP, CCK , etc.,) or any portion recognized by the integrins, for example the RGD peptide, or by any other extrinsic protein of the cell membrane. Complete proteins or peptide sequences derived from these proteins can be used, or even peptides that are fixed to their receptor and obtained by the "phage display" technique or by combination synthesis. The intracellular target signals are also included. Many of the sequences of nuclear approach (NLS) of diverse composition of amino acids, have been identified and allow to direct different proteins involved in the nuclear transport of proteins or nucleic acids. These sequences include mainly the short sequences (the NLS of the SV40 virus virus T antigen (PKKKRKV, SEQ ID No. 11) is an example), bipartite sequences (the NLS of the nucleoplasmin containing two domains essential for nuclear transport). : KRPAATKKAGQAKKKKLDK, SEQ ID No. 12), or the sequence M9 (NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY, SEQ ID No. 13) of the hnRNPAl protein. The carrier proteins of these NLS sequences are bound to their specific receptors, such as the receptors of the importin family or carioferins, for example. The function of this sequence is to direct the DNA, into the nucleus, where it is later available to the transcription machinery and can be expressed. The signals directed to the target "mixed"; that is, they can simultaneously serve to direct an intracellular and extracellular target, they are also within the framework of the present invention. For example, sugars that target lectins that are located on the cell membrane, but also at the level of the nuclear pores, can be cited. The targeting caused by these sugars refers to both extracellular targeting and nuclear import. Other signals are involved in the direction of the mitochondrial target (for example the N-terminal portion of the rat ornithine transcarbamylase (OTC), which allows mitochondria to target) or in the direction towards the endoplasmic reticulum. Finally, certain signals allow nuclear retention or at the level of the endoplasmic reticulum (such as the KDEL sequence). Advantageously, signals directed to the target according to the present invention allow specifically directing the double-stranded DNA towards certain cells or certain cellular compartments. For example, targeting signals according to the present invention can target receptors or ligands to the cell surface, mainly insulin receptors, transferrin, folic acid or any other growth factor, cytokines or vitamins or particular polysaccharides, on the surface of the cell or on the extracellular matrix. The synthesis of target-directed oligonucleotide-chimeras is carried out in solid phase or in solution, taking into account the very different chemical stability properties of the oligonucleotides and target-directed signals [Erijita, R. et al., Synthesis of defined peptide-oligonucleotide hybrids containing a nuclear transport signal sequence, Tetrahedron, 1991, 47 (24), pp. 4113-4120]. In solution, one-step couplings can be made: the targeting signal, for example, can be synthesized with a disulfide, maleimide, amine, carboxyl, ester, epoxide, cyanogen bromide or aldehyde carrier group, and be coupled to an oligonucleotide modified by a thiol, amine or carboxyl terminal group at the 3 'or 5' position. These couplings are formed to stabilize the disulfide, thioether, ester, amide or amine bonds between the oligonucleotide and the target signal. Any other method known to those skilled in the art, such as bifunctional coupling reagents, can also be used. Another object of the present invention relates to compositions comprising a vector such as that defined above. Advantageously, the vectors according to the present invention can be associated with one or a plurality of known agents for transfecting DNA. As examples, mention may be made of cationic lipids possessing interesting properties. These vectors, in fact, are constituted by a polar, cationic portion, interacting with the DNA, and another lipid portion, hydrophobic, that favors cellular penetration and facilitates the ionic interaction with the DNA insensitive to the external environment. Particular examples of cationic lipids are monocationic lipids (DOTMA: Lipofectin®, certain cationic detergents (DDAB), lipopoly acids and in particular dioctadecylaminoglycyl-spermine (DOGS) or 5'-carboxypermyl amide of palmitoylphosphatidylethanolamine (DPPES), the preparation of which is described, for example, in European Patent Application EP 394 111. Another interesting family of lipopolyamines is represented by the compounds described in International Patent Application WO 97/18185, which is incorporated herein by reference. A number of other cationic lipids which can be used with the vectors according to the present invention Among the synthetic transfection agents developed, the cationic polymers of the polylysine and DEAE dextran type are equally advantageous. It is also possible to use polyethyleneimine polymers ( PEÍ) and polypropyleneimine (PPI), which are commercially available and can be prepared in accordance with the procedure described in International Patent Application WO 56/02655. Generally, any synthetic agent known to transfect nucleic acid can be associated with the vectors of the present invention. The compositions may further comprise adjuvants capable of associating with the complex vectors according to the present invention / transfection agents and of improving the transfectant power. In another embodiment, the present invention relates to compositions comprising a vector such as that defined above, one or a plurality of transfection agents such as those defined below and one or a plurality of adjuvants capable of associating with complexes vectors according to the present invention / transfection agents and to improve the transfectant power. The presence of this type of adjuvants (lipids, peptides or proteins, for example) can advantageously make it possible to increase the transfectant power of the compounds. In this view, the compositions of the present invention may comprise as adjuvant one or a plurality of neutral lipids, the use of which is particularly advantageous. The Applicant has demonstrated that the addition of a neutral lipid allows to improve the formation of nucleolipid particles and favors the penetration of the particle into the cell, destabilizing its membrane. Preferably, the neutral lipids used in the context of the present invention are lipids with two fatty chains. Particularly advantageously, natural or synthetic lipids, zwitterionic or devoid of ionic charge, are used under physiological conditions. They may be selected more particularly from the group consisting of dioleylphosphatidylethanolamine (DOPE), oleylpalmitodylphosphatidylethanolamine (POPE), di-stearyl-di-palmitoyl, di-myristoylphosphatidylethanolamines, as well as their N-methylated 1 to 3-fold derivatives, phosphatidylglycerols, diacylglycerols, glycosyldiacylglycerols, cerebrosides (such as galactocerebrosides), sphingolipids (such as sphingomyelins) or even asialogangliosides (such as asialoGMl and GM2). These different lipids can be obtained either by synthesis, or by extraction from organs (for example: The brain) or eggs, by classical techniques known to those skilled in the art. In particular, the extraction of the natural lipids can be carried out by means of organic solvents (see also Lehninger, Biochemistry). The Applicant has shown that it is particularly advantageous to use as a adjuvant a compound that directly intervenes or not at the level of DNA condensation (International Patent WO 96/25508).

The presence of such a compound, within a composition according to the present invention, makes it possible to decrease the amount of the transfectant compound, with the beneficial consequences that derive in the toxicological level, without having any prejudice about the transfectant activity. As an intervening compound at the level of the nucleic acid condensation, a compacting compound must be understood, directly or not, of the DNA. More precisely, this compound can act directly at the level of the DNA to be transfected, or it can intervene at the level of an annex compound that is directly involved in the condensation of the nucleic acid. Preferably, it acts directly at the DNA level. Mainly, this agent intervening in the condensation of DNA, can be any polycation, for example. the polylysine. According to a preferred embodiment, this agent can also be any compound that is wholly or partially derived from a histone, a nucleolin, a protamine and / or any of its derivatives. In the same way, such agent may be constituted, in whole or in part, of peptide portions (KTPKKAKKP) and / or (ATPAKKAA), wherein the number of portions can vary between 2 and 10. In the structure of the compound according to the present invention, these portions can be repeated continuously or not. Thus, these may be separated by ligands of a biochemical nature, for example by one or a plurality of amino acids, or of a chemical nature. Likewise, the present invention aims at the use of vectors such as those defined herein, to manufacture a medicament intended to treat diseases by transfection of DNA in primary cells or in stable cell lines. They can be fibroblastic, muscular, nervous (neurons, astrocytes, gual cells), hepatic cells, of the hopoietic line (lymphocytes, CD34, dendritic cells, etc.), epithelial cells, etc., in differentiated or pluripotential forms (precursors). The vectors of the present invention, by way of example, can be used for the transfection in vi tro, ex vivo or in vivo, of DNA coding for proteins or polypeptides. For use in vivo, either in therapy or for the study of gene regulation or the creation of animal models of pathologies, the compositions according to the present invention can be formulated in forms for topical, cutaneous, oral administration, rectal, vaginal, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal, intratracheal, intraperitoneal, and so on. Preferably, the compositions of the present invention contain a pharmaceutically acceptable carrier for the injectable formulation, primarily for direct injection at the level of the desired organ, or for topical administration (on the skin and / or mucosa). It is possible in particular to treat sterile, isotonic solutions or dry compositions, in particular lyophilized, which by the addition, as the case may be, of sterilized water or physiological saline, allow the constitution of injectable solutes. The doses of nucleic acid used for injection, as well as the number of administrations, can be adapted according to different parameters, mainly depending on the route of administration used, the pathology being treated, the gene to be expressed, or even the duration of the desired treatment. As regards more particularly the mode of administration, it can be a direct injection in the tissue or in the circulatory route, or it can be a treatment of cells in culture followed by its reimplantation in vivo, by means of an injection or a graft. The present invention further relates to a method of transfecting DNA into cells, which comprises the following steps: (1) The synthesis of the target-directed oligonucleotide-chimera, in accordance with the procedure described above, (2) in contact the chimera synthesized in step (1), with a double-stranded DNA to form triple helices, (3) eventually, complex the vector obtained in step (2) with one or a plurality of transfection agents and / or one or a plurality of adjuvants; and (4) contacting the cells with the complex formed in step (2) or step (3). Contacting the cells with the complex can be done by incubating the cells with said complex (for in vivo or ex vivo uses), or by injecting the complex into an organism (for in vivo use). The present invention also includes a particularly advantageous method for the treatment of diseases, by administering a vector according to the present invention which contains a nucleic acid capable of correcting said disease. More particularly, this method is applicable to diseases that are the result of a deficiency of a protein or peptide product, wherein the DNA administered encodes said protein or peptide product. The present invention extends to any use of a vector according to the present invention, for transfection in vivo, ex vivo or in vitro cell. The present invention also relates to any recombinant cell that contains a vector such as that defined above. Preferably, they are eukaryotic cells, for example yeasts, animal cells, and the like. These cells are obtained by any technique that allows the introduction of a DNA into a given cell and that is known to those skilled in the art. The following examples are intended to illustrate the present invention without limiting it, allowing to highlight other features and advantages of the present invention. "FIGURES Figure 1: Coupling of an oligonucleotide (ODN) and the maleimide-NLS peptide Figure 2: 15% polyacrylamide gel analysis of the oligonucleotide-peptide chimera (Pso-GA1? -NLS), by proteolytic action of trypsin 1 = oligo Pso-GA19-SH 2 = chimera oligonucleotide-peptide Pso-GAxa-NLS 3 = chimera oligonucleotide-peptide Pso-GA? 9-NLS after trypsin digestion Figure 3: Schematic representation of pXL2813 plasmid Figure 4 Schematic representation of plasmid pXL2652 Figure 5: 15% p-acrylamide gel analysis of triple helix formation between the plasmid pXL2813 and the oligonucleotide-peptide Pso-GA? 3-NLS. Pso-GAio-NLS and the plasmid are mixed in a buffer solution containing 100 mM MgCli The excess molarity of the oligonucleotide with respect to the plasmid varies from 0 to 200. The mixture is photo-active overnight at 37 ° C, then digested c on two restriction enzymes that cut the plasmid in one part of the triple helices formation region. 1 = without oligonucleotide 2 = excess of molarity of the oligonucleotide with respect to the plasmid of 15 3 = excess of molarity of the oligonucleotide with respect to the plasmid of 4 = excess of molarity of the oligonucleotide with respect to the plasmid of 100 5 = excess of molarity of the oligonucleotide with respect to the plasmid of 200 6 = oligonucleotide only photoactivated 7 = oligonucleotide alone without photoactivating 8 = excess of molarity of the oligonucleotide with respect to the plasmid of 30, where the mixture has not been photoactivated. Figure 6: In vitro expression of the transgene (β-galactosidase) for the tests carried out with the plasmid pXL2813, vector pXL2813-Pso-GA19-NLS and without plasmid. Figure 7: Characterization of the peptide part of the oligonucleotide-peptide Pso-GA? 9-NLS, by interaction with the i-port 60-GST (analysis in 15% polyacrylamide gel). 1 = oligo Pso-GAi9 (1 μg) 2 = oligonucleotide-peptide Pso-GA? 5-NLS (1 μg) 3 = supernatant recovered after incubation of glutathione particles coated with importin 60 and the oligonucleotide Pso-GAt. a, and separation of the particle residue (containing the elements that interact with the importins) of the supernatant 4 = residue recovered after the incubation of glutathione particles coated with importin 60 and the oligonucleotide Pso-GAls, and separation of the particle residue (containing the elements that interact with the importins) of the supernatant 5 = supernatant recovered after incubation of glutathione particles coated with importin 60 and the oligonucleotide-peptide Pso-GA? 9-NLS and separation of the particle residue (containing the elements that interact with the importins) of the supernatant 6 = residue recovered after incubation of the glutathione particles coated with impo rtina 60 and the oligonucleotide-peptide Pso-GA? 9-NLS and separation of the particle residue (containing the elements that interact with the importins) of the supernatant Figure 8: 15% polyacrylamide gel analysis of the oligonucleotide-chimera peptide (GAi-NLS) by the proteolytic action of trypsin. 1 = oligo GA19-SH (200 ng) 2 = oligonucleotide-peptide GA19-NLS peptide (1 μg) before purification by high pressure liquid chromatography 3 = oligonucleotide-peptide GA19-NLS (1 μg) after purification 4 = oligonucleotide-peptide GA19-NLS chimera after digestion with trypsin (1 μg). Figure 9: Graphic representation of the formation kinetics of the triple helices (% of triple-helical sites occupied as a function of time) between the plasmid pXL2813 and the GA? - NLS chimera. Figure 10: Characterization of the peptide portion of the oligonucleotide-peptide chain GA19-NIS, by interaction with importin 60-GST. 1 = oligonucleotide-peptide GA? 9-NLS, 2 = oligo GAig, 3 = supernatant recovered after incubation of glutathione particles coated with importin 60 and the oligonucleotide-peptide GA19-NLS, and separation of the particle residue ( containing the elements that interact with the importins) of the supernatant, 4 = residue recovered after incubation of glutathione particles coated with importin 60 and the oligonucleotide-GAIA-NLS peptide chimera, and separation of the particle residue (containing the elements that interact with the importins) of the supernatant, 5 = supernatant recovered after incubation of glutathione particles coated with importin 60 and oligonucleotide GA? 9, and separation of the particle residue (containing the elements that interact with the importins) of the supernatant, 6 = recovered residue after incubation of glutathione particles coated with importin 60 and the oligonucleotide GAi9, and separation of the particle residue (containing the elements that interact with the importins) of the supernatant. Figure 11: Schematic representation of plasmid pXL2997.

Figure 12: Graphic representation of the kinetics of triple bond formation (% of triple helical sites occupied as a function of time) between the plasmid pXL29S7 and the pim-NLS chimera. Figure 13: Histogram representing the activity of β-galactosidase in vivo in human lung tumors H1299, plasmids pXL2813 (indicated as Bgal in the figure) and pXL2813-Pso-GAβ9-NLS (indicated as NLS-Bgal in the figure ) in ULR ("Relative Light Units") per tumor. The transfection was performed using electrotransfer techniques such as those described in International Patent Applications WO99 / 01157 and WO 99/01158. MATERIALS AND METHODS 1. Coupling of oligonucleotides and peptides Oligonucleotides The oligonucleotides used are either the sequence 5 '-AAGGAGAGGAGGGAGGGAA-3' (SEA ID No. 4) of a length of 19 bases and referred to as "GA "9" in the text, or the sequence 5 '-GGGGAGGGGGAGG-3' (SEQ ID No. 15) of a length of 13 bases and referred to by the name of "pi" in the text (since it is the proto-oncogene sequence pim-1). The oligonucleotides referred to as co? GA? 9-SH or pim-SH are the same sequences as GAag and pim, respectively, and a thiol group at the 5 'end, with a 6-carbon separator between the thiol and the phosphate of the extremity 5' . Oligonucleotides referred to as Pso-GA? 9-SH are a thiol group at the 3 'end (SH) and in addition a psoralen at the 5' end (Pso), with a six carbon spacer between the psoralen and the phosphate end 5' . Oligonucleotides referred to as Pso-GA? 9 do not have a thiol group. The nomenclature used is summarized in Table I, which is presented below. TABLE I These lyophilized oligonucleotides are resuspended in a buffer solution of 100 mM triethylammonium acetate, pH 0 7.

The peptides The peptides used for the couplings are synthesized in an automatic solid-phase synthesizer. - Contain: - either the nuclear localization sequence of the SV40 T antigen (PKKKRKV, SEQ ID No. 11), - or, the same mutated sequence (PKMKRKV, SEQ ID No. 14) that does not allow targeting or to nuclear transport (due to mutation). These peptides are also carriers of a separator of amino acid at the N-terminal: KGAG. The N-terminal lysine is chemically modified: It contains a maleimide group on carbon e and a protective group 9-fluorenylmethyloxycarbonyl (Fmoc) on the amine of carbon a. This Fmoc group absorbs at 260 nm, which allows the peptide to be followed by reverse phase high pressure liquid chromatography. The C-terminal group is also protected (CONHc group), the protection being added at the end of the peptide synthesis. The representation of the peptides is indicated in Table II, which is presented below.

TABLE II The lyophilized peptides are resuspended in a buffer solution of 100 mM triethylammonium acetate, pH = 7. The concentration is 0.4 mg / ml. Couplings _ For coupling with oligonucleotides, the strategy used is to react the thiol group carried by the oligonucleotide with the maleimide group carried by the peptide [Eritja, R., et al., Synthesis of defined peptide-oligonucleotide hybrids containing a nuclear transport signal sequence, Tetrahedron, 1991, 47 (24), pp. 4113-4120]. The oligonucleotide is added to the peptide solution in an equimolar way and the reaction medium is allowed to stand for 2 hours at room temperature. The oligonucleotide-peptide chimera is purified by reversed-phase high-pressure liquid chromatography on a Vydac C8 column containing steroidal silica of 5 μm in diameter and a porosity of 300 μA. A 0.1 M triethylammonium acetate (TEAA) buffer solution and an acetonitrile gradient ranging from 5 to 50% in 35 minutes are used. The products are detected at 260 nm. Analysis of chimeras by digestion with trypsin Oligonucleotide-peptide conjugates are subjected to the proteolytic action of trypsin, which allows to highlight the peptide portion of the chimera [Reed, M.W. et al, Synthesis and evaluation of nuclear targeting peptide-natisens oligodeoxinucleotide conjugates, Bioconjugate Chemistry, 1995, 6, pp. 101-108]. The solutions, which contain 1 μg of the oligonucleotide-peptide in 7 μl of 0.1 M triethylammonium purification buffer (TEAA), are mixed with 1 μl of a trypsin solution (5 mg / ml). Add 1 μl of buffer solution Tris 100 mM, HCl pH = 9 and 1 μl of 500 mM EDTA. After a one-hour digestion, the samples are placed in wells of a 15% polyacrylamide gel with 7 M urea. The electrophoresis is carried out in a buffer solution of 100 mM Tris, 90 mM boric acid, 1 mM EDTA, pH = 8.3 . The nucleic acids are revealed by a staining with silver, by means of a commercial package of Biorad. 2. Formation of triple helices with the chimeras The plasmid The plasmid used to study the formation of the triple helices with the GA? 9-peptide chimeras is called pXL2813 (from 7257 bp, see figure 3). This plasmid expresses the ß-galactosidase gene under the control of the strong promoter of early cytomegalovirus (CMV) genes, as well as the ampicillin resistance gene. Upstream of the promoter, between positions 7238 and 7256, the sequence GA19 (5'-AAGGAGAGGAGGGAGGGAA-3 ', SEQ ID No. 4) is cloned according to the classical methods of molecular biology. This is cloned into the plasmid pXL2652 (of 7391 bp whose representation is schematized in Figure 4) which expresses the β-galactosidase gene under the control of the strong promoter of the early cytomegalovirus genes (CMV), as well as the ampicillin resistance gene.

This promoter comes from the pCDNA3 plasmid, the LacZ gene and its polyA come from the pCHUO plasmid and the rest comes from the plasmid pGL2. The sequences are cloned upstream of the promoter, between the unique break sites of the Muñí and Xmal enzymes. For this, the two complementary oligonucleotides containing the sequences to be cloned 6651 (5'-AATTGATTCCTCTCCTCCCTCCCTTAC-3 ') and 6652 (3'-CTAAGGAGAGGAGGGAGGGAATGGG-5'), are heated for 5 minutes at 95 ° C and then hybridized allowing the temperature drops slowly. The plasmid pXL2652, subsequently, is digested with the enzymes Muñí and Xmal for 2 hours at 37 ° C and the products of this double digestion are separated by electrophoresis in 1% agarose gel and stained with ethidium bromide. The fragment of interest for cloning is eluted according to the Jetsorb protocol (Genomed) and 200 ng of this fragment is bound with 10 ng of the mixture of oligonucleotides hybridized by the T4 ligase, for 16 hours at 16 ° C. Competent E. coli bacteria of strain DH5a are transformed by electroporation with the reaction product and inoculated into petri dishes containing LB medium and ampicillin. Clones resistant to ampicillin are selected and the DNA is extracted by alkaline lysis and analyzed on a 1% agarose gel. A clone corresponding in size to the expected product is subjected to sequencing. In the case where the oligonucleotide is pim, the plasmid used to study the formation of the triple helix is pXL2997, represented in Figure 11. This plasmid expresses the β-galactosidase gene under the control of the strong promoter of the genes early cytomegalovirus (CMV), as well as the ampicillin resistance gene. Upstream of the promoter, the pim sequence is cloned (SEQ ID No. 15) according to the classical methods of molecular biology. Formation of triple helices _ The formation of the triple helices is carried out by mixing the plasmid pXL2813 (3 pmol of the plasmid, or 15 μg) or the plasmid pXL2997, as the case may be, with variable amounts of oligonucleotides or of chimaeras, in a solution regulator Tris 100 mM, HCl pH = 7.5 and 100 mM MgCl2. Photoactivation of the triple helices. After a night of incubation, the mixture is irradiated on ice for 15 minutes, using a monochromatic lamp with a wavelength of 365_ nm (Biorad). The product of the photoactivation is digested with the restriction enzymes Mfel and Spel and analyzed on a 15% polyacrylamide gel with 7M urea, with a migration buffer of 100 mM Tris, 90 mM boric acid, 1 mM ADTE, pH = 8.3. The nucleic acids are revealed by silver staining with a commercial package from Biorad 'Musso, M., J.C. Wang, and M.W.V. Dyke, in vivo persistence of DNA triple helices containing psoralen-conjugated oligodeoxiribonucleotides, Nucleic Acid Research, 1996, 24 (24), pp. 4924-4932]. Method of studying triple helices _ This method is based on the principle of exclusion chromatography of solutions containing the plasmid and the oligonucleotide, capable of forming a triple helix. The exclusion columns used (Linkers 6 columns from Boehringer Mannheim) are composed of sepharose pellets and have an exclusion limit of 194 base pairs, which allows the oligonucleotides unpaired to the plasmids to be retained in the column. At first, the oligonucleotides are radioactively labeled at their 3 'end by the terminal transferase with the help of dATPa5S. The protocol used comes from Amersham: 10 pmol of oligonucleotides are incubated for 2 hours at 37 ° C with 50 μCi of dATPa35S in the presence of 10 units of terminal transferase, in a volume of 50 μl of buffer containing sodium cacodylate. The percentage of labeled oligonucleotides is evaluated according to the following method: 1 μl of a diluted sample 1/100 of the solution after labeling, is deposited on Whatman paper DE81, in duplicate. One of the two papers is washed twice for 5 minutes with 2xSSC, for 30 seconds with water and for 2 minutes with ethanol. The radioactivities of the two papers are compared. The radioactivity of the washed paper corresponds to the 35S that was incorporated effectively. The formation of the triple helices is carried out in a volume of 35 μl. The concentrations of the plasmids (40 nM) and of the oligonucleotides (20 nM), the buffer solution used (100 mM Tris-HCl, pH = 7.5, MgCl_ 50. mM) and temperature (37 ° C) are fixed, while that the incubation time varies from 1 to 24 hours. The sepharose columns are equilibrated before use with the reaction buffer and centrifuged at 2200 rpm for 4 minutes to agglomerate. 25 μl of the reaction medium is deposited in the columns and these are centrifuged under the same conditions as mentioned above. Then 25 μl of the reaction buffer solution is deposited in the columns, which are centrifuged again. The eluate is recovered. The radioactivity contained in 5 μl of the reaction medium, recorded in cpm (of the deposit) and that contained in the eluate, recorded in cpm (of the eluate), are evaluated, which allows estimating the percentage of eluted oligonucleotides:% of oligonucleotides eluted = cpm (eluate) / [5 x cpm (deposit)] xlOO. The percentage of plasmids that elute effectively in the course of the experiments is evaluated by estimating the optical density at 260 nm of the eluate and that of the deposit, which allows calculating the percentage of oligonucleotides that were fixed in all the plasmids: % of fixed oligonucleotides = - [% of eluted oligonucleotides /% eluted plasmids] xl00. This parameter allows to evaluate the percentage of triple helices formation sites (there is one per plasmid pXL2813 or pX12997) that are effectively occupied, taking into account the concentrations of the plasmids (annotated as [plasmid]) and of the oligonucleotides (annotated as [ oligo]) used during the formation reaction of the triple helices:% of occupied triple helix sites =% of oligonucleotides fixed by [ligo] / [plasmid]. 3. Interaction with the importins The 'recombinant proteins The importin 60 subunit used to study the interaction with the oligonucleotide-peptide conjugates (NLS or mutated NLS) is of murine origin and is fused with glutathione S-Transferase (GST). The sequence of importin 60 was cloned into a vector pGEX-2T to merge it into the GST. The recombinant protein was produced in Escherichia coll _ [Imamoto, N. et al., In vivo evidence for involvement of a 58kDa component of nuclearpore-targeting complex in nuclear protein import, The EMBO Journal, 1995, 14 (15), p. 3617-3626]. Interactions with recombinant proteins All interaction experiments were carried out in the fixation buffer solution (20 mM HEPES, pH 0 6.8, 150 mM potassium acetate, 2 mM magnesium acetate, 2 mM DTT and bovine serum albumin (ASB) 100 μg / ml). In a first stage, the recombinant proteins were incubated in the presence of sepharose pellets coated with glutathione groups (Pharmacia Biotech), 1 μg of recombinant protein per 10 μl of pellets was used. After incubating for. 30 ^ at room temperature, in 500 μl of fixing buffer, the mixture was centrifuged at 2000 G for 30 seconds and the supernatant was removed. The pellets were washed 5 times for resuspension in 500 μl of binding buffer and centrifuged in the manner previously described. The pellets were resuspended in a fixing buffer solution to obtain a suspension containing 50% pellets coated with recombinant proteins.

In a second step, 60 μl of the suspension containing 50% pellets coated with recombinant protein was incubated with 2 μg of oligonucleotide or oligonucleotide-peptide in a volume of 500 μl of binding buffer. After incubating for 30 minutes at room temperature, the mixture was centrifuged at 2000 G for 30 seconds and the supernatant was removed. 30 μl of the supernatant was collected to analyze the fraction not fixed on the pellets. The pellets were washed 5 times to resuspend them in 500 μl of binding buffer and centrifuged in the manner described above. The pellets were resuspended in 15 μl of charge buffer solution (0.05% bromophenol blue, 40% sucrose, 0.1M EDTA, pH = 8, 0.5% sodium lauryl sulphate) and heated for 10 minutes at 90 ° C. . The content of the supernatant and the residue was analyzed on 15% polyacrylamide gel, 7M urea, with a migration buffer of 100 mM Tris, 90 mM boric acid, 1 mM ADTE, pH = 8.3. Nucleic acids were revealed by silver staining with a commercial package of Biorad [Rexach, M and G Blobel, Protein import into nuclei: associati- on and dissociation reactions invol ving transport substrate, transport factors, and nucleoporins,, Cell, 1995, 83, pp. 683-692].

. Transfection of cells Cell culture The cell type used was NIH3T3 (ATCC CRL-1658). These are mouse fibroblasts. These cells were cultured in Dulbecco's medium modified with glucose 4.5 g / 1 (DMEM-Gibco), 2 mM glutamine, penicillin (100 U / ml) and streptomycin (100 μg / ml), and 10% fetal bovine serum (Gibco) . These were incubated at 37 ° C in an oven with 5% CO_. Transfection __ One day before transfection, the wells of a 24-well plate were filled with 50,000 cells per well. The vectors were diluted in 150 mM NaCl and mixed with a cationic lipid (the condensed formula compound HN (CH_) 3 NH (CH2) 4 NH (CH2) 3 NHCHCOGlyN [(CHz) i7CH3] 2 described in International Patent Application WO 97 / 18185 with the number (6)), is diluted with 150 mM NaCl. The mixture is made with 6 nmol of lipid per microgram of plasmid. This mixture is diluted 1/10 with culture medium without serum and is deposited on the cells. After incubation at 37 ° C in an oven with 5% CO; for 2 hours, 10% fetal bovine serum is added. Quantification of β-galactosidase _ After incubation for 48 hours, the cells are washed twice with PBS and lysed with 250 μl of lysis buffer (Promega). The β-galactosidase was quantified according to the protocol "Lu igal β-Galactosidase genetic repórter system" (Clontech). The activity was measured in a luminometer Lumat LB9501 (Berthold). The amount of proteins was measured with the commercial BCA package (from Pierce). EXAMPLES Example 1 This example illustrates the possibility of coupling the oligonucleotide Pso-GA? 9-SH with the maleimide-NLS peptide. The oligonucleotide PSO-GAIQ-SH, which has the sequence 5 '-AAGGAGAGGAGGGAGGGAA-3', with a thiol group at the 3 'end, was coupled with the NLS peptide carrying a maleimide group at its N-terminal end, in accordance with the method described above in part corresponding to "Materials and Methods" in the section "coupling of oligonucleotides and peptides". The coupling was followed by reverse phase high pressure liquid chromatography (CLAP). It is carried out with an equimolar stoichiometry: In two hours, the coupling is complete and the chimera is purified by CLAP. The coupling reaction scheme is shown in Figure 1. The oligonucleotide-peptide chimera (Pso-GAig-NLS) was analyzed by polyacrylamide gel electrophoresis after the proteolytic action of trypsin, which allowed to demonstrate the presence of the peptide portion of the chimera after having migrated in the polyacrylamide gel, denaturation and nucleic acid staining by silver stain (as indicated in the corresponding part to "Materials and Methods" in the "Analysis of chimeras by digestion with trypsin "). The Pso-GA19-NLS chimera shows a delayed electrophoretic migration with respect to the Pso-GA oligonucleotide: a-SH and the product of the proteolytic digestion is visualized at an intermediate level between the Pso-GAig-NLS and Pso-migration levels. GAig-SH, as illustrated in Figure 2. The Pso-GA? 9-NLS chimera contains a peptide portion accessible to trypsin. These results clearly demonstrate that the coupling between the oligonucleotide and the peptide operated and that coupling performance is important. Example 2 This example illustrates the formation of triple helices between the plasmid pXL2813 and the Pso-_GA? 9-NLS chimera which is modified by a photoactivatable alkylating agent. This example also indicates the proportion of plasmids modified according to the excess molarity of the oligonucleotides, with respect to the plasmid. The plasmid pXL2813, represented in Figure 3, comprises the homologous complementary sequence of GA? O which is capable of forming a triple helix with the oligonucleotides GA19, Pso-GA19, Pso-GA? 9-SH or Pso-GAig-NLS. Divalent cations such as Mg ~ +, stabilize the triple helices. The oligonucleotide Pso-GAβ9-NLS and the plasmid are mixed in a buffer solution containing MgCl. 100 mM. The excess molarity of the oligonucleotide with respect to the plasmid varies from 0 to 200. After incubation for 12 hours at 37 ° C, the mixture is photoactivated for 15 minutes (as indicated in the part corresponding to "Materials and Methods" in the section "Photoactivation of the triple helices") and then digested with the two restriction enzymes Mfel and Spel that cut the plasmid in one part of the triple helices formation region. In this manner, a fragment of nucleic acids containing 70 base pairs is released after digestion of the unmodified plasmid pXL2813. The fragment obtained with the plasmid and the oligonucleotide form a triple helix covalently linked to the double helix consisting of 70 base pairs plus 19 bases of the oligonucleotide. By migration in a denaturing polyacrylamide gel, these fragments of different size can also be separated: The fragments that come from plasmids modified by a triple helix have a shorter migration distance than the fragments from unmodified plasmids. Thus, by denaturing polyacrylamide gel electrophoresis, the proportion of plasmids modified according to the excess molarity of the oligonucleotide with respect to the plasmid can be quantified. The results are indicated in Figure 5. For an excess molarity of the oligonucleotide with respect to the plasmid greater than 50, all plasmids are modified and are associated with an oligonucleotide Pso-GAt.9-NLS. Without photoactivation, the delay of the denaturing gel digestion fragment is lost. In this way, it seems that the triple helices formed with the oligonucleotides Pso-GA ??? SH or Pso-GA? 9-NLS are well linked covalently with the double helix after photoactivation. In addition, by digesting the rest of the plasmid skeleton and analyzing in the manner described above, it can be verified that the photoactivation does not produce non-specific covalent bonds of the Pso-GAig-NLS oligonucleotide outside the region containing the sequence capable of forming a triple propeller.

These results clearly show the conditions that allow to associate a peptide sequence to a plasmid in a specific and covalent manner, this outside the promoter that regulates the expression of the transgene, which prevents the expression of the transgene to be affected. Example 3 This example illustrates the ability of the transgene to be expressed in vi tro although the plasmid is modified. The expression of ß-galactosidase by plasmids pXL2813 unmodified or associated with an oligonucleotide Pso-GA? 9-NLS was compared by transfection of NIH3T3 cells. The results are presented in Figure 6 and the averages of the values obtained (± standard deviation) are presented in Table III below: TABLE III It is found that the expression of the transgene increases the modification provided by the covalent attachment of a triple helix upstream of the promoter. - _-- These results clearly show that the formation of the triple helices does not affect the expression of the transgene in vi tro. On the contrary, thanks to the nuclear targeting of the plasmid due to its coupling with the targeted NLS target, more plasmids reach the nucleus, which results in an increase in the efficiency of the transfection. Example 4 This example aims to verify in vivo expression not inhibited by the presence of a target signal associated with the plasmid through a triple helix. The experiment consists of transfecting the plasmids pXL2813 and pXL2813-Pso-GA19-NLS into human lung tumors H1299, using the electrotransfer techniques described in the International Patent Applications WO 99/01157 and WO 99/01158. The experiment was performed on nude female mice of 18 to 20 g of body weight. The mice were implanted monolaterally with 20 mm H1299 tumor grafts. "The tumors developed to a volume of 200-300 mm3 The mice were selected based on the size of their tumors and divided into homogeneous batches. with a mixture of Cetamine / Xylazine (available in the trade). The plasmid solution (8 μg of DNA / 40 μl of 150 mM sodium chloride) was injected longitudinally into the periphery of the tumor, with the aid of a Hamilton syringe. The lateral faces of the tumors were coated with conductive gel and the tumors were placed between two plates of stainless steel electrodes with a separation of 0.45 to 0.7 cm. The electrical impulses were applied with the help of a commercial square pulse generator (Electro-pulsateur PS 15, Jouan, France) 20 to 30 seconds after the injection. An oscilloscope allowed to control the intensity in volts, the duration in milliseconds and the frequency in hertz of the impulses that were applied at 500 V / cm for 20 milliseconds at 1 hertz. For the evaluation of the tumor transfection, 10 and 7 mice, respectively for the plasmid pXL2813 and the pXL2813-Pso-GA? 9-NLS were sacrificed two hours after the injection of the plasmid. The tumors were removed, weighed and placed in a lysis buffer (Promega) added with antiproteases (Complete, Boehringer). The obtained suspension was centrifuged in order to obtain a clear supernatant. After incubation of 10 μl of this supernatant with 250 μl of reaction buffer (Clontech) for 1 hour in the dark, the activity of β-galactosidase was measured with the aid of a commercial luminometer. The results are expressed in total ULR ("Relative Light Units") per tumor. It was found that the plasmid pXL2813-Pso-GA? 9-NLS expresses the transgene in vivo at a level greater than or equal to that obtained with the unmodified plasmid pXL2813 (see Figure 13). Example 5 This example illustrates the characterization of the peptide portion of the Pso-GAig-NLS conjugates; that is, the verification of the targeted properties of the NLS peptide associated with the constructions of nonconformity with the present invention. The peptide sequence used, the NLS signal of the SV40 T antigen, is recognized by the receptors of the a-carioferin family. The murine equivalent, called importin 60, fused with a glutathione S-transferase group, was used to characterize the oligonucleotide-peptide conjugates. We proceeded in accordance with the method described in the part corresponding to "Materials and Methods" in the section "Interactions with importins". The interactions between the pellets coated with glutathione of importin 60 and the oligonucleotides GA? 9-NLS or Pso-GAig-NLS were studied. After incubation for 30 minutes at room temperature, the pellet residue (containing the elements that interact with the importins) was separated from the supernatant. The result of this characterization is presented in Figure 7. This figure indicates that the oligonucleotide Pso-GAi9-NLS is associated with the glutathione pellets, while the oligonucleotide Pso-GAig is left in the supernatant. This clearly demonstrates the ability of the oligonucleotide Pso-GA? 9-NLS to interact with the importin. The peptide portion of the oligonucleotide-peptide chimeras, then, is recognized by its receptor, which means that the peptide effectively performs its targeting function. . EXAMPLE 6 This example illustrates the possibility of coupling oligonucleotide GA? 9-SH to the maleimide-NLS peptide. Contrary to Example 1, the chimera was not modified by a photoactivatable activating agent. The oligonucleotide GAig-SH, whose sequence 5'-AAGGAGAGGAGGGAGGGAA-3 '(SEQ ID No. 4), with a thiol group at the 5' end, was coupled with the maleimide-NLS peptide having a maleimide group at its N-terminus -terminal, under the same conditions used for the oligonucleotide Pso-GA? 9-SH (see example 1). The coupling was followed by reverse phase high pressure liquid chromatography (CLAP). It was carried out with an equimolar stereochemistry: In two hours, the coupling was total. The chimera was subsequently purified by CLAP. The oligonucleotide-peptide chimera was analyzed by the proteolytic action of trypsin, in the manner described in the part corresponding to "Materials and Methods". The result is presented in Figure 8. This is comparable to that obtained with the oligonucleotide Pso-GAj ..- SH. Example 7 This example illustrates the possibility of forming triple helices between the plasmid pXL2813 and the GAIG-NLS chimera, in the absence of an alkylating agent. A kinetics of triple helices formation was digested and studied between the plasmid pXL2813 and the GA19-NLS chimera obtained in the manner described in example 5, in accordance with the technique described e - the part corresponding to "Materials and Methods" in the section "Formation of triple helices with the chimaera". Figure 9 represents the formation kinetics of the triple helices.

It is evident that under the conditions of plasmid concentrations of 40 nM and of the oligonucleotide of 20 nM, a stable triple helix is formed. Example 8 This example illustrates the characterization of the peptide portion of the GA19-NLS chimera. __The peptide sequence used, the NLS signal of the SV40 T antigen, is recognized by the receptors of the a-carioferin family, as mentioned in example 4. The murine equivalent, called importin 60, fused to a glutathione S group -transferase was used to characterize the oligonucleotide-peptide conjugates. We proceeded in the manner described in the part corresponding to "Materials and Methods" in the section "Interactions with the importins". The interactions between pellets coated with glutathione of importin 60 and oligonucleotides GAi9 or GA? 9-NLS were studied. After incubation for 30 minutes at room temperature, the pellet residue (containing the elements that interact with the importins) was removed from the supernatant. The results are presented in Figure 10. It is evident that the oligonucleotide GAi_-NLS associates with the glutathione pellets, while the oligonucleotide GA9 remains in the supernatant. This clearly demonstrates the ability of the oligonucleotide GAig-NLS to interact with the importin a. The peptide portion of the oligonucleotide-peptide chimeras, then, is recognized by its receptor and performs its targeting function. Example 9 This example illustrates the possibility of coupling the oligonucleotide pi -SH with the maleimide-NLS peptide. Oligonucleotide pim-SH, whose sequence is 5'-GGGGAGGGGGAGG-3 '(SEQ ID No. 15), with a thiol group at the 5' end, was coupled with the maleimide-NLS peptide which has a maleimide group at its end N-terminal, under the same conditions as those used for the oligonucleotide GA19-SH (see example 5). The coupling was followed by reverse phase high pressure liquid chromatography (CLAP). This was done with an equimolar stoichiometry: In two hours, the coupling was total. The chimera was subsequently purified by CLAP. Example 10 This example illustrates the possibility of forming triple helices between the plasmid pXL2997 and the pim-NLS chimera, in the absence of an alkylating agent. A kinetics of triple helical formation between the plasmid pXL2997 and the pim-NLS chimera was studied and studied (the formation was obtained in the manner described in example 8), in accordance with the technique described in the part corresponding to "Materials and Methods "in the section" Formation of the triple helices with the chimera ". Figure 12 represents the formation kinetics of the triple helices. It is evident that under the conditions of plasmid concentrations of 40 nM and of oligonucleotide of 20 nM, a stable triple helix is formed. Although the present invention has been fully described, it is clear that it is possible to make numerous modifications to the present invention without departing from the spirit and scope of it and the following claims.

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: RHONE POULENC RORER (B) STREET: 20 AVENUE RAYMOND ARON (C) CITY: ANTONY CEDEX (E) COUNTRY: FRANCE (F) POSTAL CODE: 92165 (G) TELEPHONE: 01.55.71.73.26 (H) TELEX: 01.55.71.72.91 (ii) TITLE OF THE INVENTION: NUCLEIC CIDOS TRANSFER VECTORS, COMPOSITIONS THAT CONTAIN THEM AND THEIR USE (iii) NUMBER OF SEQUENCES: 15 (iv) COMPUTER LEGIBLE FORM _ (A) TYPE OF MEDIA: Tape (B) COMPUTER: IBM compatible PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Relay # 1.0, Version # 1.30 (EPO) (2) INFORMATION FOR SEQ ID NO. 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 25 base pairs (B) TYPE: nucleotide (C) TYPE OF CHAIN: simple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 1: GAGGCTTCTT CTTCTTCTTC TTCTT (2) INFORMATION FOR SEQ ID NO. 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 base pairs (B) TYPE: nucleotide (C) TYPE OF CHAIN: simple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 2: CTTCTTCTTC TTCTTCTTCTT ._. (2) INFORMATION FOR SEQ ID NO. 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleotide (C) CHAIN TYPE: simple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: cDNA (xi) ) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 3: AAGGGAGGGA GGAGAGGA (2) INFORMATION FOR SEQ ID NO. 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleotide (C) CHAIN TYPE: simple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 4: AAGGAGAGGA GGGAGGGAA (2) INFORMATION FOR SEQ ID NO. 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 19 base pairs (B) TYPE: nucleotide (C) TYPE OF CHAIN: simple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 5: TTGGTGTGGT GGGTGGGTT (2) INFORMATION FOR SEQ ID NO. 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleotide (C) CHAIN TYPE: simple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 6: CTTCCCGAAG GGAGAAAGG (2) INFORMATION FOR SEQ ID NO. 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 base pairs (B) TYPE: nucleotide (C) TYPE OF CHAIN: simple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 7: GAAGGGTTCT TCCCTCTTTC C (2) INFORMATION FOR SEQ ID NO. 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 13 base pairs (B) TYPE: nucleotide (C) TYPE OF CHAIN: simple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 8: GAAAAAGGAA GAG (2) INFORMATION FOR SEQ ID NO. 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 14 base pairs (B) TYPE: nucleotide (C) TYPE OF CHAIN: simple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 9: AAGAAAAAAA AGAA (2) INFORMATION FOR SEQ ID NO. 10: • (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleotide (C) CHAIN TYPE: simple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: cDNA (xi) ) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. TU: AAAAAAGGGA ATAAGGG (2) INFORMATION FOR SEQ ID NO. 11: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 7 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. eleven: Pro Lys Lys Lys Arg Lys Val 1 5 (2) INFORMATION FOR SEQ ID NO. 12: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 19 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 12: Lys Arg Pro Wing Wing Thr Lys Lys Wing Gly Gln Wing Lys Lys Lys Lys 10 15 Leu Asp Lys (2) INFORMATION FOR SEQ ID NO. 13: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 38 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 13: Asn Gln Ser Being Asn Phe Gly Pro Met Lys Gly Gly Asn Phe Gly Gly 1 5 10 15 Arg Being Ser Gly Pro Tyr Gly Gly Gly Gly Gln Tyr Phe Wing Lys Pro 20 25 30 Arg Asn Gln Gly Gly Tyr 35 (2) INFORMATION FOR SEQ ID NO. 14: (i). SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 14: Pro Lys Asn Lys Arg Lys Val 1 5 (2) INFORMATION FOR SEQ ID NO. 15: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 13 base pairs (B) TYPE: nucleotide (C) TYPE OF CHAIN: simple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. fifteen: GGGGAGGGGG AGG It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (42)

1. CLAIMS Having described the invention as an antecedent, the content of the following claims is claimed as property: 1. A nucleic acid transfer vector, characterized in that it comprises a double-stranded DNA molecule and at least one oligonucleotide coupled with a directed signal to the target and able to form a triple helix with a specific sequence present in the double-stranded DNA molecule.
2. 2. The nucleic acid transfer vector according to claim 1, characterized in that the double-stranded DNA is a plasmid or an episome.
3. 3. The nucleic acid transfer vector according to claim 2, characterized in that the double-stranded DNA is a plasmid in a circular form and in a supercoiled state.
4. 4. The nucleic acid transfer vector according to any of claims 1 to 3, characterized in that the double-stranded DNA molecule comprises an expression cartridge constituted by one or a plurality of genes of interest, under the control of one or a plurality of promoters and an active transcription terminator in mammalian cells.
5. 5. The nucleic acid transfer vector according to claim 4, characterized in that the gene of interest is a nucleic acid encoding a therapeutic product.
6. 6. The nucleic acid transfer vector according to any of claims 1 to 5, characterized in that the specific sequence present in the double-stranded DNA molecule is a homopuric-homopyrimidic sequence.
7. 7. The nucleic acid transfer vector according to any of claims 1 to 6, characterized in that the specific sequence present in the double-stranded DNA molecule is a sequence naturally present in the double-stranded DNA or a synthetic sequence or of natural origin artificially introduced into double-stranded DNA.
8. 8. The nucleic acid transfer vector according to any of claims 1 to 7, characterized in that the oligonucleotide comprises a poly-CTT sequence; and the specific sequence present in the double-stranded DNA molecule is a poly-GAA sequence.
9. 9. The nucleic acid transfer vector according to any of claims 1 to 7, characterized in that the oligonucleotide comprises the sequence GAGGCTTCTTCTTCTTCTTCTTCTT (SEQ ID No. 1), or the sequence (CTT) 7 (SEQ ID No. 2) ).
10. 10. The nucleic acid transfer vector according to any one of claims 1 to 7, characterized in that the specific sequence present in the double-stranded DNA molecule comprises the sequence 5 '-AAGGGAGGGAGGAGAGGAA-3' (SEQ ID No. 3) and the oligonucleotide comprises the sequence 5'-AAGGAGAGGAGGGAGAGGAGA-3 '(SEQ ID No. 4) or 5'TTGGTGTGGTGGGTGGGTT-3' (SEQ ID No. 5).
11. 11. The nucleic acid transfer vector according to any of claims 1 to 7, characterized in that the specific sequence present in the double-stranded DNA molecule comprises all or a part of the sequence 5'-CTTCCCGAAGGGAGAAAGG-3 '(SEQ ID No. 6) comprised in the ColEl origin of replication of E. coli, and the oligonucleotide possesses the sequence 5'-GAAGGGTTCTTCCCTCTTTCC-3' (SEQ ID No. 7).
12. 12. The nucleic acid transfer vector according to any of claims 1 to 7, characterized in that the specific sequence present in the double-stranded DNA molecule comprises the sequence 5'-GAAAAAGGAAGAG-3 '(SEQ ID No. 8) ) or the sequence 5 '-AAAAAAGGGAATAAGGG-3' (SEQ ID No. 10) of the β-lactamase gene of plasmid pBR322 and E. coli, respectively.
13. 13. The nucleic acid transfer vector according to any of claims 1 to 7, characterized in that the specific sequence present in the double-stranded DNA molecule comprises the sequence 5 '-AAGAAAAAAAAGAA-3' (SEQ ID No. 9) ) present in the origin of replication? of the plasmids, having a conditional origin of replication such as pCOR.
14. 14. The nucleic acid transfer vector according to any of claims 1 to 13, characterized in that the oligonucleotide comps at least 3 bases.
15. 15. The nucleic acid transfer vector according to claim 14, characterized in that the oligonucleotide comps from 5 to 30 bases.
16. 16. The nucleic acid transfer vector according to any of claims 1 to 15, characterized in that the oligonucleotide has at least one chemical modification that makes it resistant or that protects against nucleases, or that increases its affinity with respect to the specific sequence present in the double-stranded DNA molecule.
17. 17. The nucleic acid transfer vector according to any of claims 1 to 16, characterized in that the oligonucleotide is a chain of nucleotides that was subjected to a modification of the backbone.
18. 18. The nucleic acid transfer vector according to any of claims 1 to 16, characterized in that the oligonucleotide is coupled with an alkylating agent that forms a covalent bond at the base level of the double stranded DNA.
19. 19. The nucleic acid transfer vector according to claim 18, characterized in that the alkylating agent is photoactivatable.
20. 20. The nucleic acid transfer vector according to claims 18 and 19, characterized in that the alkylating agent is a psoralen.
21. 21. The nucleic acid transfer vector according to any of claims 1 to 20, characterized in that the target signal interacts with a component of the extracellular matrix, a plasma membrane receptor, targets an intracellular compartment and / or improves the intracellular traffic of double-stranded DNA.
22. 22. The nucleic acid transfer vector according to claim 21, characterized in that the target signal comps growth factors (EGF, PDGF, TGFb, NGF, IGF I, FGF), cytokines (IL-1, IL-2, TNF, interferon, CSF), hormones (insulin, growth hormone, prolactin, glucagon, thyroid hormone, steroid hormones), sugars that recognize lectins, immunoglobulins, transferrin, lipoproteins, vitamins such as vitamin B12, peptide hormones or neuropeptides (tachykinins, neurotensins, VIP, endothelin, CGRP, CCK, etc.) or any other portion recognized by the integrins, for example the RGD peptide, or by other cellular membrane extrinsic proteins.
23. 23. The nucleic acid transfer vector according to claim 21, characterized in that the targeting signal is an intracellular targeting signal, such as a nuclear targeting sequence (NLS).
24. 24. The nucleic acid transfer vector according to claim 23, characterized in that the nuclear targeting signal is the NLS sequence of the SV40 T antigen.
25. 25. The nucleic acid transfer vector according to claim 21, characterized in that the signal directed to the target allows both targeting to extracellular targets and targeting to intracellular targets.
26. 26. The nucleic acid transfer vector according to any of claims 1 to 25, characterized in that the coupling of the signal directed to the target with the oligonucleotide is obtained by synthesis in solid phase or in solution, mainly by establishing disulfide bonds. , thioether, ester, amide or amine.
27. 27. A composition characterized in that it contains at least one vector as defined in any of claims 1 to 26.
28. 28. The composition according to claim 27, characterized in that it also contains one or a plurality of transfectant agents.
29. 29. The composition according to claim 28, characterized in that the transfectant is a cationic lipid, a lipopolyamine or a cationic polymer.
30. 30. The composition according to any of claims 27 to 29, characterized in that it also contains one or a plurality of adjuvants capable of associating with the vector complexes as defined in any of claims 1 to 25 / transfectant agent.
31. 31. The composition according to claim 30, characterized in that the adjuvant is one or a plurality of neutral lipids that are selected from the group consisting of synthetic or natural, zwitterionic or ion-depleted lipids under physiological conditions.
32. 32. The composition according to any of claims 30 or 31, characterized in that the neutral lipid (s) are selected from the group consisting of lipids with two fatty chains.
33. 33. The composition according to any of Claims 30 to 32, characterized in that the neutral lipid (s) are selected from the group consisting of dioleylphosphatidylethanolamine (DOPE), oleylpaltoyl-phosphatidylethanolamine (POPE), distearyl, -palmitoyl, -myristoyl phosphatidylethanolamine. , as well as its N-methylated derivatives of 1 to 3 times, phosphatidylglycerols, diacylglycerols, glucosildiacilgliceroles, cerebrosides (such as galactocerebrosides), sphingolipids (such as sphingomyelins) and asialogangliosides (such as asialoGMl and GM2).
34. 34. The composition according to claim 30, characterized in that the adjuvant is or comprises a compound that intervenes at the level of DNA condensation.
35. 35. The composition according to claim 34, characterized in that the compound is derived in whole or in part from a histone, a nucleolin and / or a protamine, or is formed in whole or in part by peptide portions (KTPKKAKKP) and / or (ATPAKKAA) repeated continuously or not, wherein the number of portions can vary between 2 and 10. The composition according to any of claims 27 to 35, characterized in that it comprises a pharmaceutically acceptable carrier for a injectable formulation. 37. The composition according to any of claims 27 to 35, characterized in that it comprises a pharmaceutically acceptable carrier for application on the skin and / or on the mucous membranes. 38. The use of a nucleic acid transfer vector as defined in any of claims 1 to 26, for the manufacture of a medicament intended to cure diseases. 39. A method of transfection of nucleic acids in cells, characterized in that it comprises the following steps: (1) synthesizing the target-directed oligonucleotide-chimera, (2) contacting the chimera synthesized in the step of part (1) with a double stranded DNA to form triple helices, (3) eventually, complex the vector obtained in the step of part (2) with one a plurality of transfection agents and / or one or a plurality of adjuvants, and (4) put in contact the cells with the complex formed in the stage of subsection (2) or in the stage of subsection (3), as the case may be. 40. A method of treating diseases by administering a nucleic acid transfer vector as defined in any of claims 1 to 26, which contains a double-stranded DNA capable of correcting the disease. 41. A recombinant cell characterized in that it contains a nucleic acid transfer vector as defined in any of claims 1 to 26. 42. The recombinant cell according to claim 41, characterized in that it is a eukaryotic cell.