FR2776669A1 - New nucleic acid transfer vector comprising double-stranded DNA linked to oligonucleotide, used for gene therapy - Google Patents

Abstract

Nucleic acid transfer vector (A) comprises a double-stranded DNA molecule (I) and at least one oligonucleotide (II) that is linked to a targeting system (TS) and can form a triplex with a specific sequence within (I). Independent claims are also included for the following: (1) composition containing at least one (A); (2) method for transfer of nucleic acid into cells by: (a) forming a (II)-TS chimera; (b) treating this with (I) to form a triplex; (c) optionally complexing the product with at least one transfection agent and/or adjuvant, and (d) contacting cells with the product, and (3) recombinant cells containing (A).

Description

NUCLEIC ACID TRANSFER VECTORS COMPOSITIONS
CONTAINER AND USES THEREOF
The present invention relates to novel vectors and their use for nucleic acid transfer. More particularly, the invention relates to novel vectors capable of directing nucleic acids to specific cells or cell compartments.

 The transfer of nucleic acids is a technique underlying all the major applications of biotechnology and the increase in the efficiency of nucleic acid transfer is a very important issue for the development of these applications. The efficiency of nucleic acid transfer depends on many factors including the ability of nucleic acids to reach the target cell, their ability to cross the plasma membrane and their ability to be transported within the cell to the nucleus.

 One of the major obstacles to the efficiency of nucleic acid transfer is that the genetic information is often little or not directed to the target organ for which it is intended. On the other hand, once the nucleic acid has entered the target cell, it must still be directed to the nucleus in order to be expressed there. In addition, in the case of nucleic acid transfer in differentiated or quiescent cells, the nucleus is bounded by a nuclear envelope which constitutes an additional barrier to the passage of these nucleic acids.

 Recombinant viruses used as vectors have advanced and efficient mechanisms for guiding nucleic acids to the nucleus. However, viral vectors have certain drawbacks inherent in their viral nature, which it is unfortunately not possible to totally exclude. Another strategy is therefore to transfect the naked DNA or to use non-viral agents capable of promoting the transfer of DNA into eukaryotic cells. But non-viral vectors do not have sub-cellular or nuclear targeting signals. The passage of naked DNA, or in combination with a non-viral agent, from the cytoplasm to the nucleus is thus a step which has a very low efficiency (Zabner et al., 1995).

 Various attempts to attach targeting signals have thus been made.

In particular, peptide fragments for targeting have been covalently attached to oligonucleotides [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 thus formed are good candidates as potential inhibitors of gene expression.

 Transfection vectors comprising a synthetic polypeptide coupled by electrostatic interactions to a DNA sequence have also been described in the patent application WO 95/31557, said polypeptide consisting of a polymer chain of basic amino acids, an NLS peptide, and a hinge region that connects the NLS peptide to the polymer chain and avoids steric interactions. But this kind of construction poses a problem of stability because the interactions involved between the DNA and the targeting signal are of electrostatic nature.

 There are also specific targeting nucleic acid-peptide constructs described in patent application WO 95/34664, the bond between the two being of a chemical nature. However, this method notably involves enzymatic steps that are difficult to control and that do not make it possible to produce large quantities of nucleic acids.

Finally, it has been shown that it is possible to attach an NLS sequence (Nuclear
Signal Localization) to a plasmid DNA (Nature Biotechnology, Volume 16, pp.

80-85, January 1998). But a complete inhibition of the transcription of the gene of interest due to the random attachment of several hundred NLS sequences on the plasmid was observed. One proposed solution is to bind the NLS sequences on linear fragments of DNA, then to couple these modified fragments with other unmodified fragments. However, this technique has the disadvantage of going through at least one enzymatic step.

 Thus, all the methods proposed so far do not solve satisfactorily the difficulties related to the targeting of double-stranded DNAs.

 The present invention provides an advantageous solution to these problems. More particularly, the present invention employs oligonucleotides conjugated to targeting signals and capable of forming triple helices with specific sequence (s) present on a double-stranded DNA molecule.

 Such a vector has the advantage of being able to direct double-stranded DNA to specific cells or cell compartments, without the gene expression being inhibited. The Applicant has in fact shown that the presence of targeting signals does not affect the expression of the genes of interest when the specific sequence at which the triple helix is formed is located outside the gene expression cassette. to transfer.

 The obtained vector furthermore has the advantage of incorporating targeting signals which are stably bound to the double-stranded DNA, in particular when the oligonucleotide capable of forming the triple helix is modified by the presence of an agent. alkylating.

 Another advantage of the invention is that it makes it possible to couple the DNA to be transferred to targeting signals whose number and nature are controlled.

Different targeting signals can be coupled on the same DNA, and in this case it is also possible to determine the respective proportions beforehand.

 Finally, the functionalized triple helix obtained results only from chemical transformation steps and can therefore be obtained simply, reproducibly, and in very large quantities, especially industrial.

 A first object of the invention therefore 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 invention comprises a double-stranded DNA molecule and at least one oligonucleotide coupled to a targeting signal and capable of forming by hybridization a triple helix with a specific sequence present on said double-stranded DNA molecule. .

Within the meaning of the invention, "double-stranded DNA" is intended to mean a double-stranded deoxyribonucleic acid which may be of human, animal, plant, bacterial or viral origin, etc. It may be obtained by any technique known to the those skilled in the art, and in particular by bank screening, by chemical or enzymatic synthesis of sequences obtained by screening libraries. It can be chemically modified.

 This double-stranded DNA can be in linear or circular form. In the latter case, the double-stranded DNA may be in a supercoiled or relaxed state.

Preferably, the DNA molecule is of circular shape and in a supercoiled conformation.

 The double-stranded DNA may also carry an origin of replication functional or otherwise in the target cell, one or more marker genes, regulatory sequences for transcription or replication, genes of therapeutic interest, modified or unmodified antisense sequences. , Binding regions to other cellular components, etc. Preferably, the double-stranded DNA comprises an expression cassette consisting of one or more genes of interest under the control of one or more promoters and a transcriptional terminator active in the target cells.

 This is usually a plasmid or episome carrying one or more genes of therapeutic interest. By way of example, mention may be made of the plasmids described in patent applications WO 96/26270 and WO 97/10343 incorporated herein by reference.

 For the purposes of the invention, the term "gene of therapeutic interest" is intended to mean any gene coding for a protein product having a therapeutic effect. The therapeutic product thus coded may in particular be a protein or a peptide. This protein product may be homologous to the target cell (i.e., a product that is normally expressed in the target cell when it has no pathology). In this case, the expression of a protein makes it possible for example to overcome insufficient expression in the cell or the expression of an inactive or weakly active protein due to a modification, or to overexpress said protein. The gene of therapeutic interest can also code for a mutant of a cellular protein, having increased stability, a modified activity, etc. The protein product can also be heterologous with respect to the target cell. In this case, an expressed protein may, for example, supplement or provide a deficient activity in the cell, enabling it to fight against a pathology, or to stimulate an immune response.

Among the therapeutic products within the meaning of the present invention, mention may be made more particularly of enzymes, blood derivatives, hormones, lymphokines [interleukins, interferons, TNF, etc. (FR 92/03120)] growth factors, neurotransmitters or their precursors or synthetic enzymes, the trophic factors [BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NT5,
HARP / pleiotrophin, etc., dystrophin or a minidystrophin (FR 91/11947)], CFTR protein associated with cystic fibrosis, lp53 tumor suppressor genes,
Rb, RaplA, DCC, k-rev, etc. (FR 93/04745)], the genes coding for factors involved in coagulation [Factors VII, VIII, IX], the genes involved in the repair of DNA, the suicide genes [thymidine kinase, cytosine deaminase], hemoglobin genes or other protein transporters, genes corresponding to proteins involved in lipid metabolism, apolipoprotein type selected from apolipoproteins AI, A-II, A- IV, B, CI, C-II, C-III, D, E, F, G, H, J and apo (a), metabolic enzymes such as lipoprotein lipase, hepatic lipase, lecithin cholesterol acyltransferase , 7 alpha-cholesterol hydroxylase, phosphatidic acid phosphatase, or lipid transfer proteins such as the cholesterol ester transfer protein and the phospholipid transfer protein, an HDL binding protein or a chosen receptor, for example among the LDL receptors , receptors for chylomicrons-remnants and scavenger receptors, etc.

The DNA of therapeutic interest may also be an antisense gene or sequence whose expression in the target cell makes it possible to control the expression of genes or the transcription of cellular mRNAs. Such sequences may, for example, be transcribed into 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 genes of therapeutic interest also comprise the sequences encoding ribozyrans, which are capable of selectively killing
Target RNAs (EP 321 201), or sequences encoding single-chain intracellular antibodies such as for example ScFv.

 As indicated above, the deoxyribonucleic acid may also comprise one or more genes coding for an antigenic peptide, capable of generating in human or animal an immune response. In this particular mode of implementation, the invention thus allows the realization of either vaccines or immunotherapeutic treatments applied to humans or animals, especially against microorganisms, viruses or cancers. It may be in particular antigenic peptides specific for Epstein Barr virus, HIV virus, hepatitis B virus (EP 185 573), pseudo-rabies virus, syncitia-forming virus, influenza virus, cytomegalovirus (CMV), other viruses or specific tumors (EP 259 212).

 Preferably, the deoxyribonucleic acid also comprises sequences allowing the expression of the gene of therapeutic interest and / or of the gene encoding the antigenic peptide in the desired cell or organ. They may be sequences that are naturally responsible for the expression of the gene when these sequences are likely to function in the transfected 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 derived from the genome of the cell that it is desired to infect. Similarly, they may be promoter sequences derived from the genome of a virus. In this respect, mention may be made, for example, of the promoters of the E1A, MLP, CMV, RSV genes, etc. In addition, these expression sequences can be modified by addition of activation sequences, regulation, etc. It can also be promoter, inducible or repressible.

A triple helix corresponds to the attachment of an oligonucleotide modified or not on double-stranded DNA by so-called "Hoogsteen" hydrogen bonds between the bases of the third strand and those of the double helix region. These pairings occur in the broad groove of the double helix and are specific to the sequence under consideration [Frank-Kamenetski, MD, Triplex DNA Structures, Ann. Rev. Biochem., 1995, 64, pp. 65-95]. The specific double-helix sequence may in particular be a homopuric-homopyrimidine sequence. Two categories of triple helices are distinguished according to the nature of the bases of the third strand [Sun, J. and C. Hélène,
Oligonucleotide-directed triple-helix formation, Curr. Opin. Struct. Biol., 1993, 3, pp.

345-356]: the purine bases make it possible to obtain CG * G and T pairings
A * A, and the pyrimidine bases make it possible to obtain CG * C + and T
A * T (the symbol * corresponds to the pairing with the third strand).

 These structures have been characterized from a physico-chemical point of view thanks to numerous NMR (Nuclear Magnetic Resonance), hybridization temperature or nuclease protection studies, which allowed to define their properties and stability conditions. . For triple helices with a purine third strand, this strand is antiparallel to the purine strand of DNA and the formation of the triple helix strongly depends on the concentration of divalent ions ions such as Mg2 + stabilize the structure formed with the third strand. For the triple helices with a homopyrimidine third strand, this is parallel to the purine strand, and the formation of the triple helix is pH dependent: an acidic pH of less than six allows the protonation of cytosines and the formation of a additional hydrogen bond stabilizing the CG * C + triplet. There are also "mixed" triple helices for which the third strand bears purine and pyrimidine bases. In this case, the orientation of the third strand depends on the base sequence of the homopuric region.

Oligonucleotides used in the present invention are oligonucleotides that directly hybridize with double-stranded DNA. These oligonucleotides may contain the following bases:
thymidine (T), which is capable of forming triplets with AT doublets of double-stranded DNA (Rajagopal et al., Biochem 28 (1989) 7859);
adenine (A), which is capable of forming triplets with AT doublets of double-stranded DNA;
guanine (G), which is capable of forming triplets with GC doublets of double-stranded DNA;
protonated cytosine (C +), which is capable of forming triplets with GC doublets of double-stranded DNA (Rajagopal et al);
- uracil (U) which is able to form triplets with base pairs
AU or AT

 To allow triple helix formation by hybridization, it is important that the oligonucleotide and the specific sequence present on the DNA be complementary. In this respect, to obtain the best bindings and the best selectivity, a perfectly complementary oligonucleotide and specific sequence is used for the vector according to the invention. It may be in particular a poly-CTT oligonucleotide and a specific poly-GAA sequence. By way of example, mention may be made of the oligonucleotide of sequence 5'-GAGGCTTCTTCTTCTTCTTCTTCTT-3 '(GAGG (CTT) 7, SEQ ID No. 1), in which the GAGG bases do not form triple helices but allow spacing the oligonucleotide from the coupling arm, or the sequence (C1T) 7 (SEQ ID NO: 2). These oligonucleotides are capable of forming a triple helix with a specific sequence comprising complementary motifs (GAA). It may be in particular a region comprising 7, 14, or 17 GAA patterns. 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 binds in an antiparallel orientation to the polypuric strand. These triple helices are stable only in the presence of Mg 2+ as previously stated (Vasquez et al., Biochemistry, 1995, 34, 7243-7251, Beal and Dervan, Science, 1991, 251, 1360-1363).

The specific sequence may be a naturally occurring sequence on double-stranded DNA, or a synthetic or naturally occurring sequence artificially introduced into it. It is particularly interesting to use an oligonucleotide capable of forming a triple helix with a sequence naturally present on double-stranded DNA. Indeed, this advantageously makes it possible to obtain the vectors according to the invention with unmodified plasmids, in particular commercial plasmids of the pUC, pBR322, pSV, etc. type. Among the natural homopuric-homopynmidic sequences present on the DNA. double strand, there may be mentioned a sequence comprising all or part of the 5 'sequence
CTTCCCGAAGGGAGAAAGG-3 '(SEQ ID NO: 6) present in the ColEl origin of replication of E. coli In this case, the triple helix-forming oligonucleotide has the sequence: 5'-GAAGGGTTCTTCCCTCTTTCC-3' (SEQ ID NO. "7) and is fixed alternately on the two strands 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). We can also mention the 5 'sequence
GAAAAAGGAAGAG-3 '(SEQ ID NO: 8) of the p-lactamase gene of plasmid pBR322 (Duval-Valentin et al., Proc Natl Acad Sci USA, 1992, 89, 504-508). sequence is AAGAAAAAAAAGAA (SEQ ID NO: 9) present in the origin of replication y plasmids with conditional replication origin such as pCOR.

Although perfectly complementary sequences are preferred, it is understood, however, that certain mismatches may be tolerated between the oligonucleotide sequence and the sequence present on the DNA, as long as they do not lead to an excessive loss of DNA. affinity. We can cite the 5 'sequence
AAAAAAGGGAATAAGGG-3 '(SEQ ID No. 10) present in the E. coli lactamase gene. In this case, the thymine interrupting the polypuric sequence can be recognized by a guanine of the third strand, thus forming an ATG triplet which is stable when it is flanked by two TAT triplets (Kiessling et al., Biochemistry 1992 31, 289-2834). .

 The oligonucleotide used can be natural (composed of natural bases, unmodified) or chemically modified. In particular, the oligonucleotide may advantageously have certain chemical modifications making it possible to increase its resistance or its protection vis-à-vis the nucleases, or its affinity with respect to the specific sequence.

According to the present invention is also meant by oligonucleotide any sequence of nucleosides having undergone a modification of the backbone. Among the possible modifications are phosphorothiolated oligonucleotides which are capable of forming triple helices with DNA (Xodo et al., Nucleic Acids Res., 1994, 22, 3322-3330), as well as oligonucleotides having formacetal skeletons or methylphosphonate (Matteucci et al., J. Am Chem Soc., 1991, 113, 7767-7768). Oligonucleotides synthesized with nucleotide anomers, which also form triple helices with DNA can also be used.
Doan et al., Nucleic Acids Res. 1987 15, 7749-7760). Another modification of the backbone is the phosphoramidate linkage. For example, mention may be made of the N3'-P5 phosphoramidate internucleotide linkage described by Gryaznov and Chen, which gives oligonucleotides forming particularly stable triple helices with DNA (J. Arn, Chem., Soc., 1994, 116, 3143- 3144). Other modifications of the backbone also include the use of ribonucleotides, 2'-Omethylribose, phosphotriester, etc. (Sun and Helene, Opinion Opinion Biol., 116, 3143-3144). The phosphorus backbone can finally be replaced by a polyamide backbone as in PNA (Peptide Nucleic Acid), which can also form triple helices (Nielsen et al., Science, 1991, 254, 1497-1500, Kim et al.,
J. Am. Chem. Soc., 1993, 115, 6477-6481) or by a guanidine backbone, as in DNG (deoxyribonucleic guanidine, Proc Natl Acad Sci USA, 1995 92 6097-6101), polycationic analogs of DNA , which also form triple helices.

Thymine of the third strand can also be replaced by a bromouracil, which increases the affinity of the oligonucleotide for DNA (Povsic and
Dervan, J. Am. Chem. Soc., 1989, 111, 3059-3061). The third strand may also contain non-natural bases, among which may be mentioned 7-deaza-2'-deoxyxanthosine (Milligan et al., Nucleic Acids Res., 1993, 21, 327-333), 1- (2-deoxy) -bD-nbofuranosyl) -3-methyl-5-amino-1H-pyrazoloC4,3-d] pyrimidin-7-one (Koh and Dervan, J. Am Chem Soc., 1992, 114, 1470-1478) 8-oxoadenine, 2aminopurine, 2'-O-methyl-pseudoisocytidine, or any other modification known to those skilled in the art (see Sun and Helen, Journal of Opinion Structural Biol. 356).

 Another type of modification of the oligonucleotide is more particularly intended to improve the interaction and / or the affinity between the oligonucleotide and the specific sequence. In particular, a quite advantageous modification consists in coupling an alkylating agent to the oligonucleotide. The bond can be either chemically or photochemically via a photoreactive functional group. Advantageous alkylating agents are in particular photoactivatable, for example psoralenes. Under the action of light, they form covalent bonds at the level of pyrimidine bases of the DNA. When these molecules are intercalated at the 5'-ApT-3 'or 5'-TpA-3' sequences in a double-stranded DNA fragment, they form bonds with the two strands. This light-induced binding reaction can occur specifically at one site of the plasmid.

 An advantage of the present invention is therefore the possibility of forming very stable and site-specific triple helices between the oligonucleotide and the double-stranded DNA through a covalent bond formed via an alkylating agent.

 The length of the oligonucleotide used in the process of the invention is at least 3 bases, and preferably between 5 and 30 bases. An oligonucleotide longer than 10 bases is advantageously used. The length can be adapted case by case by the skilled person depending on the desired selectivity and stability of the interaction.

 The oligonucleotides according to the 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 may of course be used.

 The oligonucleotides according to the invention also have another type of modification whose purpose is to direct the double-stranded DNA to specific cells or cell compartments. In particular, this modification consists in coupling a targeting signal to the free end of the oligonucleotide.

 Another advantage of the present invention is that it is now possible, thanks to the formation of very stable site-specific triple helices, to bind a targeting signal to a double-stranded DNA in a site-specific manner. Accordingly, it is possible to set the targeting signal outside the expression cassette of the gene to be transferred. The applicant has thus shown that gene expression in the cell is not inhibited despite the chemical modification of the DNA. In addition, the presence of a triple helix as a means for binding the targeting signal to the DNA is particularly advantageous because it allows to maintain a suitable size of DNA to be transfected.

 According to another variant of the invention, it is possible to control the number of targeting signals bound to each double-stranded DNA molecule by introducing an appropriate number of specific sequences onto said double-stranded DNA molecule. In the same way, it is possible to introduce on the same molecule of double-stranded DNA several oligonucleotides linked to different targeting signals (intracellular and / or extracellular). In addition, these different targeting signals can be attached to the double-stranded DNA molecules with different degrees of stability in their binding depending on whether the triple helix is formed with or without a covalent link via an alkylating agent.

 Targeting signals can be very varied in nature. They can be used to interact with a component of the extracellular matrix, a plasma membrane receptor, to target an intracellular compartment or to enhance intracellular DNA trafficking, during nonviral gene transfer into gene therapy.

These targeting signals may comprise, for example, growth factors (EGF, PDGF, TGFb, NGF, IGF I, FGF), cytokines (IL-I, 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, ...), or any motif recognized by integrins, for example RGD peptide, or by other extrinsic proteins of the membrane cellular.

 Whole proteins, or peptide sequences derived from these proteins, or peptides that bind to their receptor and obtained by the "phage display" technique or by combinatorial synthesis can be used.

Intracellular targeting signals can be envisioned. Many nuclear addressing sequences (NLS) of varied amino acid composition have been identified and allow to target different proteins involved in the nuclear transport of proteins or nucleic acids. These sequences include short sequences (the NLS of the SV40 T antigen (PKKKRKV,
SEQ ID No. 11) is an example), bipartite sequences (the NLS of nucleoplasmine which contains two essential domains for nuclear transport KRPAATKKAGQAKKKKLDK, SEQ ID No. 12), or the sequence M9 (NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY, SEQ ID NO: 13) of hnRNPA1 protein Proteins bearing these NLS sequences bind to specific receptors, such as receptors of the importin or karyopherin family, for example.The role of these sequences is of the nucleus, where it is immediately available for the machinery. transcription, and can be expressed.

 "Mixed" targeting signals, i.e., both for intracellular and extracellular targeting, are also within the scope of the present invention. It is possible, for example, to mention sugars which target lectins which are located on the cell membrane but also at the level of the nuclear pores. Targeting by these sugars therefore concerns both extracellular targeting and nuclear import.

 Other signals are involved in mitochondrial targeting (for example, the N-terminal part of ornithine transcarbamylase (OTC) from rat allows mitochondrial targeting) or targeting to the endoplasmic reticulum. Finally, some signals allow nuclear retention or at the level of the endoplasmic reticulum (such as the KDEL sequence).

 Advantageously, the peptides according to the invention make it possible to specifically target certain cells or certain cell compartments. By way of example, the peptides according to the invention can target receptors or ligands on the surface of the cell, in particular the insulin, transferrin, folic acid or any other growth factor receptors. , cytokines, or vitamins, or particular polysaccharides on the surface of the cell or on the surrounding extracellular matrix.

 The synthesis of oligonucleotide-peptide chimeras is in solid phase or in solution and takes into account the very different stability properties of oligonucleotides and peptides [Erijita, R. et al., Synthesis of hybrid peptide-oligonucleotide containing a nuclear transport signal sequence, Tetrahedron, 1991, 47 (24), pp. 41134120]. In solution, couplings in one step are conceivable: the targeting signal may for example be synthesized with a group bearing disulfide, maleimide, amine, carboxyl, ester, epoxide, cyanogen bromide or aldehyde functions, and be coupled to a oligonucleotide modified with a terminal thiol, amine or carboxyl group at the 3 'or 5' position. These couplings are formed by establishing disulfide, thioether, ester, amide, or amine bonds between the oligonucleotide and the targeting signal. Any other method known to those skilled in the art can be used, such as bifunctional coupling reagents for example.

 Another subject of the invention relates to pharmaceutical compositions comprising a vector as defined above.

Advantageously, the vectors according to the invention may be combined with one or more agents known to transfect the DNA. By way of example, mention may be made of cationic lipids which have advantageous properties. These vectors consist of a polar part, cationic, interacting with the DNA, and a lipid part, hydrophobic, promoting cell penetration. Particular examples of cationic lipids are in particular monocationic lipids (DOTA Lipofectint)), certain cationic detergents (DDAB), lipopolyamines and in particular dioctadecylamidoglycyl spermine (DOGS) or 5-carboxyspermylamide of palmitoylphosphatidylethanolamine (DPPES) whose preparation has has been described for example in the patent application EP 394 111. Another interesting family of lipopolyamines is represented by the compounds described in the patent application.
WO 97/18185 incorporated herein by reference. Many other cationic lipids have been developed and can be used with the vectors according to the invention.

 Among the synthetic transfection agents developed, cationic polymers of polylysine and DEAE dextran type are also advantageous. It is also possible to use polyethylene imine (PEI) and polypropylene imine (PPI) polymers which are commercially accessible and can be prepared according to the process described in patent application WO 96/02655.

 In general, any agent known to transfect the nucleic acid can be associated with the vectors according to the invention.

 The compositions may further comprise adjuvants capable of associating with the vector complexes according to the invention / transfection agent and of improving their transfecting capacity. In another embodiment, the present invention therefore relates to compositions comprising a vector as defined above, one or more transfection agents as defined above and one or more adjuvants capable of associating with the vector / complexes. transfection agent (s) and to improve transfecting capacity. The presence of this type of adjuvant (lipids, peptides or proteins, for example) may advantageously make it possible to increase the transfecting power of the compounds.

 In this regard, the compositions of the invention may comprise as adjuvant, one or more neutral lipids whose use is particularly advantageous. The Applicant has in fact shown that the addition of a neutral lipid makes it possible to improve the formation of the nucleolipid particles and to promote the penetration of the particle into the cell by destabilizing its membrane.

 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 may be chosen more particularly from dioleoylphosphatidylethanolamine (DOPE), oleoyl palmitoylphosphatidylethanolamine (POPE), di-stearoyl, -palmitoyl, mirystoyl phosphatidylethanolamines and their N-methylated derivatives 1 to 3 times, phosphatidylglycerols, diacylglycerols, glycosyldiacylglycerols, cerebrosides (such as in particular galactocerebrosides), sphingolipids (such as in particular sphingomyelins) or asialogangliosides (such as in particular AsialoGM 1 and GM2).

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).

 The Applicant has demonstrated that it is also particularly advantageous to use as an adjuvant, a compound that may or may not be directly involved in the condensation of DNA (WO 96/25508). The presence of such a compound, within a composition according to the invention makes it possible to reduce the amount of transfecting compound, with the beneficial consequences that derive therefrom from a toxicological point of view, without causing any harm to the transfecting activity. By compound involved in the condensation of the nucleic acid is meant to define a compacting compound, directly or indirectly, DNA. More specifically, this compound can either act directly at the level of the DNA to be transfected or intervene at the level of an additional compound directly involved in the condensation of this nucleic acid. Preferably, it acts directly on the DNA. According to a preferred embodiment, this agent involved in the condensation of DNA consists, in whole or in part, of peptide units (KTPKKAKKP) and / or (ATPAKKAA), the number of patterns may vary between 2 and 10. In the structure of the compound according to the invention, these units can be repeated continuously or not. Thus they can be separated by links of a biochemical nature, for example one or more amino acids or of a chemical nature. Such an agent may also derive in whole or in part from a histone, a nucleolin, a protamine and / or one of their derivatives.

 The subject of the invention is also the use of the vectors as defined above for the transfection of DNA into primary cells or into established cell lines. It can be fibroblastic, muscular, nerve cells (neurons, astrocytes, glial cells), liver cells, haematopoietic line (lymphocytes, CD34, dendritic, etc.), epithelial cells, etc., in differentiated form. or pluripotent (precursors).

 The vectors of the invention may, for illustrative purposes, be used for the in vitro or in vivo transfection of DNA encoding proteins or polypeptides.

For uses in vivo, either in therapy or for the study of the regulation of genes or the creation of animal models of pathologies, the compositions according to
The invention may be formulated for administration by topical, cutaneous, oral, rectal, vaginal, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal, intratracheal, intraperitoneal, etc. Preferably, the compositions of the invention contain a pharmaceutically acceptable vehicle for an injectable formulation, in particular for direct injection into the desired organ, or for topical administration (to skin and / or mucosa). It may be in particular sterile solutions, isotonic, or dry compositions, including freeze-dried, which, by the addition of sterilized water or saline solution, allow the constitution of injectable solutions. The nucleic acid doses used for the injection as well as the number of administrations may be adapted according to various parameters, and in particular according to the mode of administration used, the pathology concerned, the gene to be expressed, or the duration of the desired treatment. As regards more particularly the mode of administration, it may be either a direct injection into the tissues or the circulatory pathways, or a treatment of cells in culture followed by their reimplantation in vivo, by injection or graft.

The invention further relates to a method of transfection of DNA into cells comprising the following steps
(1) synthesis of the oligonucleotide chimera - targeting signal according to the method described above,
(2) contacting the chimera synthesized in (1) with a double-stranded DNA to form triple helices,
(3) complexation of the vector obtained in (2) with one or more transfection agents and / or one or more adjuvant (s), and
(4) contacting the cells with the complex formed in (3).

 The cells can be brought into contact with the complex by incubating the cells with said complex (for in vitro or ex vivo uses), or by injecting the complex into an organism (for in vivo uses).

 The present invention thus provides a particularly advantageous method for transfection of DNA in vivo, or in vitro, in particular for the treatment of diseases, comprising the in vivo or in vitro administration of a vector according to the invention containing a nucleic acid. adapted to correct said disease. More particularly, this method is applicable to diseases resulting from a deficiency in a protein or peptide product, the administered DNA coding for said protein or peptide product. The present invention extends to any use of a vector according to the invention for the in vivo or in vitro transfection of cells.

 The invention also relates to any recombinant cell containing a vector as defined above. These are preferably eukaryotic cells, for example yeasts, animal cells, etc. These cells are obtained by any technique allowing the introduction of a DNA into a given cell and known to those skilled in the art.

 The following examples intended to illustrate the invention without limiting its scope, make it possible to demonstrate other characteristics and advantages of the present invention.

FIGURES
Figure 1: Coupling of an oligonucleotide (ODN) and maleimide-NLS peptide.

Figure 2: 15% polyacrylamide gel analysis of oligonucleotide peptide chimera (Pso-GA19-NLS) by proteolytic action of trypsin.

1 = oligo Pso-GA19-SH 2 = oligonucleotide-peptide chimera Pso-GA19-NLS 3 = oligonucleotide-peptide chimera P so-GA 1 s-NL S after digestion with trypsin
Figure 3: Schematic representation of plasmid pXL2813.

Figure 4: Schematic representation of the plasmid pXL2652.

The pso-GAls-NLS oligonucleotide and the plasmid are mixed in a buffer containing 100 mM MgCl 2. The molar excess of the oligonucleotide relative to the plasmid varies from 0 to 200. The mixture is photoactivated, after one night at 370C, and then digested with two restriction enzymes which cut the plasmid on both sides of the region. training of triple helices.

1 = no oligonucleotide 2 = oligonucleotide molar excess relative to the plasmid of 3 = molar oligonucleotide excess relative to the plasmid of 50 4 = oligonucleotide molar excess relative to the plasmid of 100 5 = oligonucleotide molar excess over plasmid of 200 6 = oligonucleotide alone photoactivated 7 = oligonucleotide alone non-photoactivated 8 = molar excess of oligonucleotide relative to plasmid of 30, the mixture not being photoactivated.

Figure 6: Transgene expression (s-galactosidase) for assays performed with plasmid pXL2813 alone, vector pXL2813-Pso-GA19-NLS, and without plasmid.

Figure 7: Characterization of the peptide part of the Pso-GA19-NLS oligonucleotide peptide chimera by interaction with importin 60-GST (15% polyacrylamide gel analysis).

1 = oligo Pso-GA19 (1cl) 2 = oligonucleotide-peptide Pso-GA19-NLS (1zg) 3 = supernatant recovered after incubation of the glutathione-coated glutathione beads 60 and the oligonucleotide Pso-GAls, and separation of the bead pellet (containing the elements interacting with the importers) of the supernatant 4 = pellet recovered after incubation of the glutathione-coated glutathione beads 60 and the oligonucleotide Pso-GAls, and separation of the pellet of beads (containing the elements interacting with the supernatants 5 = supernatant recovered after incubation of the importin 60-coated glutathione beads and the Pso-GA19-NLS oligonucleotide-peptide chimera, and separation of the pellets pellet (containing the importin interacting elements) from the supernatant 6 = pellet recovered after incubation of the glutathione-coated glutathione beads 60 and the oligonucleotide-peptide Pso-GA19-NLS chimera, and separation of the pellet from beads ( containing the elements interacting with the imports) of the supernatant.

Figure 8: 15% polyacrylamide gel analysis of the oligonucleotide peptide (GA19-NLS) chimera by proteolytic action of trypsin.

1 = GA19-SH oligo (200 ng) 2 = GA19-NLS oligonucleotide-peptide chimera (1cl) before purification by high-pressure liquid chromatography 3 = GA19-NLS oligonucleotide-peptide chimera (1zg) after purification 4 = oligonucleotide-peptide chimera GA19-NLS after digestion with trypsin (1Clg).

FIG. 9: Graphical representation of the kinetics of formation of the triple helices (% of sites of triple helices occupied as a function of time) between the plasmid pXL2813 and the chimera GA19-NLS.

Figure 10: Characterization of the peptide part of the GA19-NLS oligonucleotide peptide chimera by interaction with the importin 60-GST.

 1 = oligonucleotide-peptide chimeric GA19-NLS, 2 = oligo GA19, 3 = supernatant recovered after incubation of the glutathione-coated glutathione beads 60 and the oligonucleotide-peptide chimeric GA19-NLS, and separation of the pellet from the beads (containing the elements interacting with the importines) of the supernatant, 4 = pellet recovered after incubation of the glutathione-glutathione beads coated with importines 60 and the oligonucleotide-peptide chimera GA19-NLS, and separation of the pellet of beads (containing the elements interacting with the iminins) supernatant, 5 = supernatant recovered after incubation of the importin 60-coated glutathione beads and GA19 oligonucleotide and separation of the pellet pellet (containing the importin interacting elements) from the supernatant, 6 = pellet recovered after incubation of the beads -glutathion coated with importines 60 and oligonucleotide GA19, and separation of the pellet from beads (containing the elements interagi with the imports) of the supernatant.

MATERIAL AND METHODS 1. Coupling of oligonucleotides and peptides
Oligonucleotides
The oligonucleotides used have a length of 19 bases and their common sequence is 5 '-AAGGAGAGGAGGGAGGGAA-3' (SEQ ID NO: 4) These oligonucleotides are referred to under the name GA19 in the rest of the text.

The oligonucleotides labeled GA19-SH have the same sequence and a thiol group at the 5 'end, with a six-carbon spacer between the thiol and the 5' end phosphate. The oligonucleotides labeled Pso-GAI9-SH have a thiol group at the 3 'end, and in addition, a psoralen at the 5' end, with a six-carbon spacer between psoralen and 5 'end phosphate. . The oligonucleotides labeled Pso-GAls do not have a thiol group.

The nomenclature used is summarized in Table I below

<tb> name <SEP> of <SEP> oligonucleotide <SEP> modification <SEP> of <SEP> end <SEP> 3 '<SEP> modification <SEP> of <SEP> end <SEP> 5
<tb><SEP> GA19 <SEP> none <SEP> none
<tb><SEP> GA19-SH <SEP> no <SEP> group <SEP> thiol
<tb><SEP> Pso-GA19 <SEP> no <SEP> psoralen
<tb><SEP> Pso-GA19-SH <SEP> group <SEP> thiol <SEP> i <SEP> psoralen
<Tb>
Table I
These lyophilized oligonucleotides are taken up in a 100 mM triethylammonium acetate buffer, pH = 7.

Peptides
The peptides used for the couplings are synthesized by a solid phase automaton. They contain: - either the nuclear localization sequence of the SV40 T antigen (PKKKRKV,
SEQ ID NO: 1), or the same mutated sequence (PKNKRKV, SEQ ID No. 14) which does not allow targeting or nuclear transport (because of the mutation).

These peptides also carry a spacer of four amino acids at the N-terminal end: KGAG. The N-terminal lysine is chemically modified: it contains a maleimide group on carbon E and a fluorenylmethyloxycarbonyl (Fmoc) protecting group on the amine of carbon a. This Fmoc group absorbs at 260 nm which makes it possible to follow the peptide by reverse phase high pressure liquid chromatography. The C-terminal group is also protected (CONH2 group), the protection being added at the end of peptide synthesis.

The representation of the peptides is shown in Table II below

<tb><SEP> name <SEP> of <SEP> peptide <SEP> sequence <SEP> and <SEP> changes
<tb><SEP> Maleimide <SEP> KGAGPKKKRKV-CONH,
<tb><SEP> maleimide-NLS <SEP> cl
<tb><SEP> F <SEP> moz
<tb><SEP> F [oc
<tb> malnmide <SEP> KGAGPKNKRKV-CONH,
<tb><SEP>) moc
<Tb>
Table II
The lyophilized peptides are taken up in a 100 mM triethylammonium acetate buffer, pH = 7. The concentration is 0.4 mg / ml.

The counlages
For oligonucleotide coupling, the strategy employed is to react the thiol moiety carried by the oligonucleotide and the maleimide moiety carried by containing a nuclear transport signal sequence, Tetrahedron, 1991, 47 (24), pp. 4113 4120].

The oligonucleotide is added to the peptide solution equimolarly and the reaction medium is left for two hours at room temperature. The oligonucleotide-peptide chimera is purified by reversed-phase high-pressure liquid chromatography on a Vydac C8 column containing spheroidal silica 5 m in diameter and 300 A porosity. 0.1 M Triethylammonium acetate (TEAA) buffer is used. and a gradient of acetonitrile from 5% to 50% in 35 minutes. The products are detected at 260 nm.

Analysis of chimeras by diestion with trypsin
The oligonucleotide-peptide conjugates are subjected to the proteolytic action of trypsin which makes it possible to highlight the peptide part of the chimera [Reed
MW et al, Synthesis and Evaluation of Nuclear Targeting Peptide-Antisense Oligodeoxynucleotide Conjugates, Bioconjugate Chemistry, 1995, 6, pp.101-108]. Solutions containing 1 μg of oligonucleotide-peptide in 7 μl of 0.1M trethylammonium purification buffer (TEAA) are mixed with 1 μl of a trypsin solution (5 mg / ml). 1 μl of 100 mM Tris buffer, pH = 9 HCl, and 1 μl of 500 mM EDTA are added. After digestion for one hour, the samples are placed in the wells of a 15% polyacrylamide gel, 7M urea. The electrophoresis is made in 100 mM Tris buffer, 90 mM boric acid, 1 mM EDTA, pH = 8.3. The nucleic acids are revealed by silver staining with a Biorad kit.

2. Formation of triple helices with the chimeras
The plasmid
The plasmid used to study the formation of triple helices with the oligonucleotide-peptide chimeras is called pXL2813 (7257 bp). This plasmid expresses the β-galactosidase gene under the control of the strong promoter of the early genes of the
Cytomegalovirus (CMV), as well as the resistance gene to Ampicillin.

Upstream of the promoter, between positions 7238 and 7256, the sequence GA19 (5 '
AAGGAGAGGAGGGAGGGAA-3 ', SEQ ID NO2) was cloned according to standard methods of molecular biology.

It was cloned into the plasmid pXL2652 (of 7391 bp and whose representation is shown schematically in FIG. 4) which expresses the ss-Galactosidase gene under the control of the strong promoter of the early genes of Cytomegalovirus (CMV), as well as the gene resistance to Ampicillin. This promoter comes from pCDNA3, the LacZ gene and its polyA come from pCH1 10, and the rest comes from pGL2.

The sequences were cloned upstream of the promoter between the unique cleavage sites of MunI and XmaI enzymes. For this purpose, the two complementary oligonucleotides containing the sequence to be cloned 6651 (5'-AATTGATTCCTCTCCTCCCTCCCTTAC-3 ') and 6652 (3'
CTAAGGAGAGGAGGGAGGGAATGGG-5 ') were heated for 5 minutes at 95 ° C. and then hybridized, allowing the temperature to drop slowly. The plasmid pXL2652 was then digested with the MunI and XmaI enzymes, for 2 hours at 37 ° C., and the products of this double digestion were separated by electrophoresis on 1% agarose gel and staining with ethidium bromide.

The fragment of interest for the continuation of the cloning was eluted according to the Jetsorb protocol (Genomed) and 200 ng of this fragment were connected to 10 ng of the mixture of oligonucleotides hybridized with T4 ligase, for 16 hours at 16 C.

Competent E. coli bacteria of DHSa strain were electroporated with the reaction product, plated on Petri dishes containing LB medium and Ampicillin. Ampicillin resistant clones were selected and the DNA was extracted by alkaline lysis and analyzed on 1% agarose gel.

A clone corresponding in size to the expected product was sequenced.

Formation of triple helices
The formation of the triple helices is carried out by mixing the plasmid pXL2813 (3 pmol of plasmid is still 15 Hg) and variable amounts of oligonucleotides or chimeras in a 100 mM Tris buffer, HCl pH = 7.5, and MgCl 2 100 mM.

Photoactivation of triple helices
After overnight incubation, the mixture is irradiated in ice for 15 minutes, using a 365 nm wavelength monochromatic lamp (Biorad). The photoactivation product is digested with the restriction enzymes MfeI and SpeI and analyzed on a 15% polyacrylamide gel, 7 M urea with a migration buffer.
100 mM Tris, 90 mM boric acid, 1 mM EDTA, and pH = 8.3. Nucleic acids are revealed by silver staining using a Biorad kit [Musso, M., JC

Wang, and M.W.V.Dyke, In vivo Persistence of DNA triple helices containing psoralen-conjugated oligodeoxyribonucleotides, Nucleic Acid Research, 1996, 24 (24), pp.4924-4932].

The method of study of triple helices
This method is based on the principle of exclusion chromatography of solutions containing the plasmid and oligonucleotide capable of forming a triple helix. The exclusion columns used (Columns, Linkers 6 and Boehringer Mannheim) are composed of sepharose beads and have the exclusion limit of 194 base pairs, which makes it possible to retain in the column the oligonucleotides which are not paired with the plasmids.

In a first step, the oligonucleotides are radioactively labeled at their 3 'end by the Terminal Transferase using dATPa35S. The protocol used comes from Amersham: 10 pmol of oligonucleotides are incubated for 2 hours at 37 ° C., with 50 μl 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: I 1 of a sample diluted to 1/100 of the solution after marking is deposited on paper
Whatman DE81, 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 actually incorporated.

 HCl pH = 7.5, 50 mM MgCl 2) and the temperature (37 ° C) are fixed while the incubation time varies from 1 to 24 hours The sepharose columns are equilibrated, before use, with the buffer of reaction and centrifuged at 2200 rpm for 4 minutes, to settle them, 25 μl of the reaction medium are deposited on the columns and these are centrifuged under the same conditions as above.

25 μl of the reaction buffer are then deposited on the columns which are again centrifuged. The eluate is recovered.

The radioactivity contained in 5 Cll of the reaction medium, noted cpm (deposit), and that contained in the eluate, noted cpm (eluate), are evaluated, which makes it possible to estimate the percentage of eluted oligonucleotides:% of eluted oligonucleotides = cpm (eluate) / [5 x cpm (depot)] x10O.

The percentage of plasmids that are effectively eluted during the experiments is evaluated by estimating the optical density at 260 nm of the eluate and that of the deposit, which makes it possible to calculate the percentage of oligonucleotides fixed on all the plasmids:% d fixed oligonucleotides = [% eluted oligonucleotides /% eluted plasmids] xl 00.

This parameter makes it possible to evaluate the percentage of sites of formation of triple helices (there exists one by plasmid pXL2813) which are actually occupied taking into account the concentrations of plasmids (denoted [plasmid]) and oligonucleotides (denoted [oligo] ) used during the triple helix formation reaction% occupied triple helix sites =% fixed oligonucleotides x [oligo] / [plasmi in Escherichia coli [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), pp. 3617-3626].

Interactions with recombinant proteins
All interaction experiments were conducted in the binding buffer (20 mM HEPES, pH = 6.8, 150 mM potassium acetate, 2 mM magnesium acetate, 2 mM DTT, and 100 μg / ml BSA).

In a first step, the recombinant proteins are incubated in the presence of glutathione-coated Sepharose beads (Pharmacia Biotech). 1 g of recombinant protein is used for 10 μl of beads. After incubation for 30 minutes at room temperature in 500 μl of binding buffer, the mixture is centrifuged at 2000 G for 30 seconds, and the supernatant is removed. The beads are washed five times by resuspension in 500 μl of binding buffer and centrifugation as described above. The beads are resuspended in binding buffer to obtain a suspension containing 50% of beads coated with recombinant proteins.

In a second step, 60 μl of the suspension containing 50% of beads coated with recombinant proteins are incubated with 2 g of oligonucleotide or oligonucleotide-peptide, in a volume of 500 μl of binding buffer. After incubation for 30 minutes at room temperature, the mixture is centrifuged at 2000G for 30 seconds, and the supernatant is removed. 30 l of the supernatant are collected to analyze the unbound fraction on the beads. The beads are washed five times by resuspension in 500 μl of binding buffer and centrifugation as described above. The beads are resuspended in 15 l of loading buffer (0.05% bromophenol blue, 40% sucrose, 0.1 M EDTA, pH = 8, 0.5% sodium lauryl sulfate) and heated for 10 minutes at 900 ° C. The contents of the supernatant and the pellet are analyzed on a 15% polyacrylamide gel, 7M urea, with a migration buffer.
100 mM Tris, 90 mM boric acid, 1 mM EDTA, pH = 8.3. The nucleic acids are revealed by silver staining using a Biorad kit [Rexach, M. and G.

Blobel, Protein import into nuclei: association and dissociation reactions involving transport substrate, transportfactors, and nucleoporins, Cell, 1995, 83, pp. 683-692].

4. Transfection of cells
Cellular culture
The cell type used is NIH 3T3 (ATCC CRL-1658). These are mouse fibroblasts. These cells are cultured in modified Dulbecco medium, with glucose 4.5 g / l (DMEM-Gibco), 2 mM glutamine, penicillin (100 U / ml) and streptomycin (100 μg / ml) and 10% fetal calf serum (Gibco). They are incubated at 37 C in an oven at 5% CO2.

Transfection
One day before transfection, the wells of a 24-well plate are inoculated with 50000 cells per well. The vectors are diluted in 150 mM NaCl and mixed with a cationic lipid (RPR 120535 described in patent application WO 97/18185), diluted in 150 mM NaCl. The mixture is made with 6 nmol of lipid per microgram of plasmid. This mixture is diluted 1/10 in serum-free culture medium and deposited on the cells. After incubation at 37 ° C in an oven at 5% CO2 for two hours, 10% fetal calf serum is added.

Quantification of ss-galactosidase
After incubation for 48 hours, the cells are washed twice with PBS and lysed with 250 μl of lysis buffer (Promega). The s-galactosidase is quantified according to the Lumigal ss-Galactosidase genetic reporter system @ (Clontech) protocol.

Activity is measured on a Lumat LB9501 luminometer (Berthold). The amount of protein is measured with the BCA kit (Pierce).

EXAMPLES
Example 1
This example illustrates the possibility of coupling the Pso-GA19-SH oligonucleotide to the maleimide-NLS peptide.

The oligonucleotide Pso-GA1 9-SH, of sequence 5 '-AAGGAGAGGAGGGAGGGAA-3', with a thiol group at the 3 'end, was coupled to the NLS peptide which carries a maleimide group at its N-terminus according to the method previously described in the "Materials and Methods" section under the "Oligonucleotide and Peptide Coupling" section.

Coupling is followed by reverse phase liquid chromatography (HPLC). It is carried out with an equimolar stoichiometry: in two hours, the coupling is total, and the chimera is purified by HPLC.

The reaction scheme of the coupling is shown in Figure 1.

The oligonucleotide-peptide chimera (Pso-GA19-NLS) was analyzed by polyacrylamide gel electrophoresis after proteolytic action of trypsin which makes it possible to demonstrate the presence of the peptide part of the chimera, after migration on denaturing polyacrylamide gel. and staining of the nucleic acids with silver (as indicated in the "Materials and Methods" section under "Trypsin digestion of chimeras").

The Pso-GA1 9-NLS chimera exhibits delayed electrophoretic migration over the Pso-GA19-SH oligonucleotide, and the product of proteolytic digestion is visualized at an intermediate level between the Pso-GAI9-
NLS and Pso-GA19-SH, as shown in Figure 2.

The Pso-GA19-NLS chimera therefore contains a peptide part accessible to trypsin. These results clearly show that the coupling between the oligonucleotide and the peptide has occurred, and the coupling efficiency is important.

EXAMPLE 2
This example illustrates the formation of triple helices between the plasmid pXL2813 and the Pso-GA19-NLS chimera which is modified with a photoactivatable alkylating agent. This example also indicates the proportion of plasmids modified according to the molar excess of oligonucleotides relative to the plasmid. oligonucleotides GA19, Pso-GA19, Pso-GA19-SH or Pso-GA19-NLS. Divalent cations, such as Mg +, stabilize these triple helices. The oligonucleotide Pso-GA19-
NLS and the plasmid are mixed in a buffer containing 100 mM MgCl 2.

The molar excess of oligonucleotide relative to the plasmid ranges from 0 to 200.

After incubation for 12 hours at 370 ° C., the mixture is photoactivated for 15 minutes (as indicated in the "material and methods" section under "Photoactivation of triple helices"), and then digested with both restriction enzymes.
MfeI and Spel which cut the plasmid on both sides of the formation region of the triple helices. A nucleic acid fragment which contains 70 base pairs after digestion of the unmodified plasmid pXL2813 is thus released. The fragment obtained with the plasmid and the oligonucleotide forming a triple helix covalently bound to the double helix has 70 base pairs plus 19 bases of the oligonucleotide. By migration on a denaturing polyacrylamide gel, it is thus possible to separate these fragments of different size: the fragments originating from plasmids modified by a triple helix have a shorter migration distance than the fragments coming from unmodified plasmids. It is therefore possible, by denaturing polyacrylamide gel electrophoresis, to quantify the proportion of plasmids modified according to the excess in molarity of oligonucleotide relative to the plasmid.

The results are shown in FIG. 5. For a molar excess of oligonucleotide relative to the plasmid greater than 50, all the plasmids are modified and are associated with a Pso-GA19-NLS oligonucleotide. Without photoactivation, the delay of the denaturing gel digestion fragment is lost.

It thus appears that the triple helices formed with the oligonucleotides Pso-GAls-
SH or Pso-GA19-NLS are thus covalently bound to the double helix after photoactivation.

Furthermore, by digesting the rest of the plasmid backbone and analyzing it as above, it can be verified that the photoactivation does not lead to non-specific covalent binding of the Pso-GA19-NLS oligonucleotide outside the region containing the sequence likely to form a triple helix.

These results clearly demonstrate the conditions for specifically and covalently associating a peptide sequence with a plasmid, outside of the promoter regulating the expression of the transgene, which prevents the expression of the transgene from being affected.

EXAMPLE 3
This example illustrates the ability of the transgene to be expressed although the plasmid is modified.

The expression of β-galactosidase by the plasmids pXL2813 unmodified or associated with a Pso-GA19-NLS oligonucleotide was thus compared by transfection of NIH3T3 cells.

The results are reported in FIG. 6, and the averages of the values obtained (+ standard deviation) are collated in the following table III:

<tb><SEP><SEP> expression of the <SEP> transgene
<tb><SEP> plasmid <SEP> (in <SEP> RLU / pg <SEP> of <SEP> protein)
<tb><SEP> pXL2813 <SEP> 253759 <SEP> t <SEP> 48545
<tb> pXL2813-Pso-TH-NLS <SEP> 473984 <SEP> 111476
<tb><SEP> without <SEP> plasmid <SEP> 129 <SEP> t <SEP> 85
<Tb>
Table m
It is found that the expression of the transgene increases following the modification provided by the covalent attachment of a triple helix upstream of the promoter.

These results clearly show that the formation of triple helices does not affect the expression of the transgene. In contrast, by nuclear addressing of the plasmid due to its coupling to the NLS targeting sequence, more plasmids have reached the nucleus, and an increase in transfection efficiency results.

Example 4
This example illustrates the characterization of the peptide part of the Pso GA1 9-NLS conjugates.

The peptide sequence used, the NLS signal of the SV40 T antigen, is recognized by receptors of the karyopherin a family. The murine equivalent, called importin 60, fused to a glutathione S-transferase moiety, was used to characterize the oligonucleotide-peptide conjugates. It was operated according to the method described in the "Materials and Methods" section under the section "Interactions with Imports
The interactions between glutathione-glutathione coated with importines 60 and oligonucleotides GA19-NLS or Pso-GA19-NLS were studied. After incubation for 30 minutes at room temperature, the pellet (containing the elements interacting with the importins) is separated from the supernatant.
The result of this characterization is reported in FIG. 7. This figure indicates that the Pso-GAls-NLS oligonucleotide is associated with the glutathione beads, while the Pso-GAs9 oligonucleotide remains in the supernatant.

This clearly shows the ability of the Pso-GA19-NLS oligonucleotide to interact with the important a. The peptide part of the oligonucleotide-peptide chimeras is therefore well recognized by its receptor, which means that the peptide effectively fulfills its role as a screening signal.

Example 5
This example illustrates the possibility of coupling the oligonucleotide GA19-SH to the maleimide-NLS peptide. In contrast to Example 1, the chimera is not modified by a photoactivatable alkylating agent.

The oligonucleotide GA1 9-SH, of sequence 5 '-AAGGAGAGGAGGGAGGGAA-3' (SEQ ID NO: 4), with a thiol group at the 5 'end, was coupled to the maleimide-NLS peptide which has a maleimide group at its N-terminal end under the same conditions as for the oligonucleotide Pso-GA19-SH (see Example 1).

Coupling is followed by reverse phase liquid chromatography (HPLC). It is carried out with an equimolar stoichiometry: in two hours, the coupling is total. The chimera is then purified by HPLC.

The oligonucleotide-peptide chimera was analyzed by proteolytic action of trypsin as described in the "Materials and Methods" section.

The result is shown in Figure 8. It is comparable to that obtained with the oligonucleotide Pso-GA19-SH.

EXAMPLE 6
This example illustrates the possibility of forming triple helices between the plasmid pXL2813 and the GA19-NLS chimera in the absence of alkylating agent.

Triple helix formation kinetics between plasmid pXL2813 and the chimera
GA19-NLS obtained as described in Example 5, was conducted and studied according to the technique described in the "Materials and Methods" section, under the section "Formation of triple helices with the chimera".

Figure 9 shows the formation kinetics of the triple helices.

It appears that under the conditions of plasmid concentrations of 40 nM and oligonucleotide of 20 nM, a stable triple helix is formed.

EXAMPLE 7
This example illustrates the characterization of the peptide part of the GA19 chimera
NLS.

The peptide sequence used, the NLS signal of the SV40 T antigen, is recognized by receptors of the karyopherin a family, as already mentioned in Example 4. The murine equivalent, called importin 60, fused to a glutathione S-transferase group, was used to characterize the oligonucleotide-peptide conjugates. n was operated as described in "Materials and Methods" under the section "Interactions with Imports".

The interactions between glutathione-glutathione coated with importines 60 and oligonucleotides GA19 or GA19-NLS were studied. After incubation for 30 minutes at room temperature, the pellet (containing the import interacting elements) is separated from the supernatant.

The results are shown in Figure 10. It appears that the oligonucleotide GA19-NLS is associated with the glutathione beads, whereas the oligonucleotide GA19 remains in the supernatant.

This clearly shows the ability of the oligonucleotide GA19-NLS to interact with the importin a. The peptide portion of the oligonucleotide-peptide chimeras is therefore well recognized by its receptor, and again fulfills its role of targeting signal.

Now that the invention is fully described, it is obvious that many modifications can be made to the present invention without departing from the spirit of the invention and the scope of the following claims.

SEQUENCE LIST
(1) GENERAL INFORMATION:
(i) DEPOSITOR:
(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) FAX: 01.55.71.72.91
(ii) TITLE OF THE INVENTION: NUCLEIC ACID TRANSFER VECTORS,
COMPOSITIONS CONTAINING SAME, AND USES THEREOF.

(iii) NUMBER OF SEQUENCES: 13
(iv) COMPUTER-DEPENDABLE FORM:
(A) TYPE OF SUPPORT: Tape
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS / MS-DOS
(D) SOFTWARE: PatentIn Release &num; 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) NUMBER OF BRINS: simple
(D) CONFIGURATION: linear
(ii) TYPE OF MOLECULE: cDNA
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1:
GAGGCTTCTT CTTCTTCTTC TTCTT 25 (2) INFORMATION FOR SEQ ID NO: 2:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleotide
(C) NUMBER OF BRINS: simple
(D) CONFIGURATION: linear
(ii) TYPE OF MOLECULE: cDNA
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2:
CTTCTTCTTC TTCTTCTTCTT 21
(2) INFORMATION FOR SEQ ID NO: 3:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleotide
(C) NUMBER OF BRINS: simple
(D) CONFIGURATION: linear
(ii) TYPE OF MOLECULE: cDNA
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3:
AAGGGAGGGA GGAGAGGAA 19
(2) INFORMATION FOR SEQ ID NO: 4:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleotide
(C) NUMBER OF BRINS: simple
(D) CONFIGURATION: linear
(ii) TYPE OF MOLECULE: cDNA
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4:
AAGGAGAGGA GGGAGGGAA 19 (2) INFORMATION FOR SEQ ID NO: 5:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleotide
(C) NUMBER OF BRINS: simple
(D) CONFIGURATION: linear
(ii) TYPE OF MOLECULE: cDNA
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5:
TTGGTGTGGT GGGTGGGTT 19
(2) INFORMATION FOR SEQ ID NO: 6:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleotide
(C) NUMBER OF BRINS: simple
(D) CONFIGURATION: linear
(ii) TYPE OF MOLECULE: cDNA
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6:
CTTCCCGAAG GGAGAAAGG 19
(2) INFORMATION FOR SEQ ID NO: 7:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleotide
(C) NUMBER OF BRINS: simple
(D) CONFIGURATION: linear
(ii) TYPE OF MOLECULE: cDNA
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7:
GAAGGGTTCT TCCCTCTTTC C 21
(2) INFORMATION FOR SEQ ID NO: 8:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 13 base pairs
(B) TYPE: nucleotide
(C) NUMBER OF BRINS: simple
(D) CONFIGURATION: linear
(ii) TYPE OF MOLECULE: cDNA
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8:
GAAAAAGGAA GAG 13
(2) INFORMATION FOR SEQ ID NO: 9:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 14 base pairs
(B) TYPE: nucleotide
(C) NUMBER OF BRINS: simple
(D) CONFIGURATION: linear
(ii) TYPE OF MOLECULE: cDNA
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 9: AAGAAAAAAA AGAA 14 (2) INFORMATION FOR SEQ ID NO: 10:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleotide
(C) NUMBER OF BRINS: simple
(D) CONFIGURATION: linear
(ii) TYPE OF MOLECULE: cDNA
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 10: AAAAAAGGGA ATAAGGG 17 (2) INFORMATION FOR SEQ ID NO: 11:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) NUMBER OF BRINS: simple
(D) CONFIGURATION: linear
(ii) TYPE OF MOLECULE: peptide
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 11:
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) NUMBER OF BRINS: simple
(D) CONFIGURATION: linear
(ii) TYPE OF MOLECULE: peptide
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 12:
Lys Arg Pro Ala Ala Lys Lily Lys Ala Gly Ala Lys Lily Lys Lily Lys 1 5 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) NUMBER OF BRINS: simple
(D) CONFIGURATION: linear
(ii) TYPE OF MOLECULE: peptide
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 13:
Asn Gln Ser Ser Asn Phe Gly Pro Met Lys Gly Gly Asn Phe Gly Gly 1 5 10 15
Arg Ser Ser Gly Pro Tyr Gly Gly Gly Gly Gln Tyr Phe Ala Lys Pro
20 25 30
Asn Gln Gly Gly Tyr
35
(2) INFORMATION FOR SEQ ID NO: 14:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) NUMBER OF BRINS: 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

Claims (40)

 A nucleic acid transfer vector characterized in that it comprises a  double-stranded DNA molecule and at least one oligonucleotide coupled to a  targeting and able to form a triple helix with a specific sequence present  on said double-stranded DNA molecule.  Nucleic acid transfer vector according to claim 1, characterized in that  that the double-stranded DNA molecule is a plasmid or an episome.  3. Nucleic acid transfer vector according to claim 2 characterized in that  that the double-stranded DNA molecule is a plasmid in circular form and in a supercoiled state. 4. Nucleic acid transfer vector according to claims 1 to 3 characterized in that the double-stranded DNA molecule comprises an expression cassette consisting of one or more genes of interest under the control of one or more promoters and an active transcriptional terminator in mammalian cells. 5. Nucleic acid transfer vector according to claim 4 characterized in that the gene of interest is a nucleic acid encoding a therapeutic product. 6. nucleic acid transfer vector according to one of claims 1 to 5 characterized in that the specific sequence present on the double-stranded DNA molecule is a homopunque-homopynmidic sequence. 7. Nucleic acid transfer vector according to one of claims 1 to 6, characterized in that the specific sequence present on the double-stranded DNA molecule is a sequence naturally present on double-stranded DNA or a synthetic sequence or introduced artificially into double-stranded DNA. 8. Nucleic acid transfer vector according to one of claims 1 to 7 characterized in that the oligonucleotide comprises a poly-CTT sequence and the specific sequence present on the double-stranded DNA molecule is a poly-GAA sequence . 9. Nucleic acid transfer vector according to one 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. Nucleic acid transfer vector according to one of claims I to 7, characterized in that the specific sequence present on the double-stranded DNA molecule comprises the sequence 5'-AAGGGAGGGAGGAGAGGAA-3 '(SEQ ID No. 3) and the oligonucleotide comprises the sequence 5'-AAGGAGAGGAGGGAGGGAA-3 '(SEQ ID No. 4) or 5'-TTGGTGTGGTGGGTGGGTT-3 '(SEQ ID NO: 5). 11. Nucleic acid transfer vector according to one of claims I to 7, characterized in that the specific sequence present on the double-stranded DNA molecule comprises all or part of the sequence 5'-CTTCCCGAAGGGAGAAAGG-3 '(SEQ ID No. 6) included in the ColEI origin of E. coli replication, and the oligonucleotide has the sequence 5'-GAAGGGTTCTTCCCTCTTTCC-3 '(SEQ ID n07). 12. Nucleic acid transfer vector according to one of claims I to 7, characterized in that the specific sequence present on the double-stranded DNA molecule comprises the sequence 5'-GAAAAAGGAAGAG-3 '(SEQ ID No. 8) or the 5'-AAAAAAGGGAATAAGGG-3 'sequence (SEQ ID NO: 10) included in the Plactamase gene of plasmid pBR322 and E. coli, respectively. 13. Nucleic acid transfer vector according to one of claims 1 to 7, characterized in that the specific sequence present on the double-stranded DNA molecule comprises the sequence 5'-AAGAAAAAAAAGAA-3 '(SEQ ID No. 9) present in the origin of replication y plasmids with conditional replication origin such as pCOR. 14. Nucleic acid transfer vector according to one of claims 1 to 13 characterized in that the oligonucleotide comprises 5 to 30 bases. 15. Nucleic acid transfer vector according to one of claims 1 to 14 characterized in that the oligonucleotide has at least one chemical modification making it resistant to or protected against nucleases, or increasing its affinity to the specific sequence present on the double-stranded DNA molecule. 16. Nucleic acid transfer vector according to one of claims I to 15 characterized in that the oligonucleotide is a sequence of nucleosides having undergone a modification of the backbone. 17. Nucleic acid transfer vector according to one of claims 1 to 15 characterized in that the oligonucleotide is coupled to an alkylating agent forming a covalent bond at the bases of the double-stranded DNA. 18. Nucleic acid transfer vector according to claim 17 characterized in that said alkylating agent is photoactivatable. 19. Nucleic acid transfer vector according to claims 17 and 18 characterized in that said alkylating agent is psoralen. 20. Nucleic acid transfer vector according to claims 1 to 19 characterized in that the targeting signal interacts with a component of the extracellular matrix, a receptor of the plasma membrane, targets an intracellular compartment, and / or improves the traffic intracellular double-stranded DNA. 21. Nucleic acid transfer vector according to claim 20, characterized in that said targeting signal comprises the growth factors (EGF, PDGF, TGFb, NGF, IGF I, FGF), cytokines (IL-I, 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, neurotensin, VIP, endothelin, CGRP, CCK, ...), or any pattern recognized by the integrins, for example the RGD peptide, or by other extrinsic proteins of the cell membrane. 22. Nucleic acid transfer vector according to claim 20, characterized in that the targeting signal is an intracellular targeting signal, such as a nuclear addressing sequence (NLS). 23. Nucleic acid transfer vector according to claim 22, characterized in that the nuclear addressing signal is the NLS sequence of the SV40 T antigen. 24. Nucleic acid transfer vector according to claim 20, characterized in that the targeting signal allows both extracellular targeting and intracellular targeting. 25. Nucleic acid transfer vector according to claims 1 to 24 characterized in that the coupling of the targeting signal to the oligonucleotide is obtained by solid phase synthesis or in solution, in particular by the establishment of disulfide bonds, thioether , ester, amide, or amine. 26. Pharmaceutical composition characterized in that it contains at least one vector as defined in one of claims 1 to 25. 27. Pharmaceutical composition according to claim 26, characterized in that it further contains one or more transfecting agents. 28. Pharmaceutical composition according to claim 27, characterized in that the transfecting agent is a cationic lipid, a lipopolyamine, or a cationic polymer. 29. Pharmaceutical composition according to claims 27 and 28 characterized in that it further contains one or more adjuvant (s). 30. Pharmaceutical composition according to claim 29 characterized in that the adjuvant is one or more neutral lipids selected from synthetic or natural lipids, zwitterionic or devoid of ionic charge under physiological conditions. 31. Pharmaceutical composition according to claims 29 and 30, characterized in that the neutral lipid or lipids are chosen from lipids with two fatty chains. 32. Pharmaceutical composition according to claims 29 to 31, characterized in that the neutral lipid or lipids are chosen from dioleoylphosphatidylethanolamine (DOPE), oleylpalmitoylphosphatidylethanolamine (POPE), di-stearoyl, -palmitoyl, -mirystoyl phosphatidylethanolamine and their derivatives. N-methylated I to 3 times, phosphatidylglycerols, diacylglycerols, glycosyldiacylglycerols, cerebrosides (such as in particular galactocerebrosides), sphingolipids (such as in particular sphingomyelins), and asialogangliosides (such as in particular AsialoGM1 and GM2). 33. Pharmaceutical composition according to claim 29 characterized in that the adjuvant is or comprises a compound involved in the condensation of DNA. 34. The pharmaceutical composition as claimed in claim 33, characterized in that said compound is derived in whole or in part from a histone, a nucleolin, and / or a protamine, or consists in whole or in part of peptide motifs ( KTPKKAKKP) and / or (ATPAKKAA) repeated continuously or not, the number of patterns may vary between 2 and 10. 35. Composition according to any one of claims 26 to 34, characterized in that it comprises a pharmaceutically acceptable vehicle for an injectable formulation. 36. Composition according to any one of claims 26 to 34 characterized in that it comprises a pharmaceutically acceptable vehicle for application to the skin and / or the mucous membranes. 37. The use of a nucleic acid transfer vector as defined in any one of claims 1 to 25 for the targeted transfection of DNA into cells. 38. Method for in vitro or ex vivo transfection of nucleic acids in cells, characterized in that it comprises the following steps  (1) oligonucleotide chimera synthesis - targeting signal,  (2) contacting the chimera synthesized in (1) with a double-stranded DNA to form triple helices,  (3) complexation of the vector obtained in (2) with one or more transfection agent and / or one or more adjuvant (s), and  (4) contacting the cells with the complex formed in (3). 39. Recombinant cell containing a vector as defined in one of the claims there. 40. Recombinant cell according to claim 39 characterized in that it is a eukaryotic cell.