CA2323831A1 - 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 FOR TRANSFERRING ACES NUCLEIOUES. COMPOSITIONS
CONTAINING. AND THEIR USES
The present invention relates to new vectors and their use for the transfer of nucleic acids. More particularly, the invention relates to new vectors capable of directing nucleic acids towards cells or specific cell compartments.
Nucleic acid transfer is a technique that underpins all major applications of biotechnology and increasing efficiency of the transfer nucleic acids is a very important issue for development of these Io applications. The efficiency of nucleic acid transfer depends on numerous 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 stems from the fact that genetic information is often little or not directed towards the target organ for which it is intended. By the way, once the acid nucleic a penetrated into the target cell it still has to be directed to the nucleus so to be there Express. In addition, in the case of transfer of nucleic acids into cells differentiated or quiescent, the nucleus is limited by a nuclear envelope who 2o constitutes an additional barrier to the passage of these nucleic acids.
Recombinant viruses used as vectors have mechanisms evolved and effective in guiding nucleic acids to the nucleus.
However, the viral vectors have certain inherent drawbacks viral, that it unfortunately is not possible to exclude completely. Another strategy consists either to transfect naked DNA, or to use non-viral agents able to promote the transfer of DNA into eukaryotic cells. But the vectors no Virals do not have subcellular or nuclear targeting signals.
So the passage of naked DNA, or in combination with a non-viral agent, from the cytoplasm towards

2 the nucleus is for example a step which has a very low efficiency (Zabner et al., 1995).
Different attempts to attach targeting signals have thus been do.
In particular, peptide fragments for targeting have been attached covaIently to oligonucleotides in an oligonucleotide targeting strategy antisense [Eritja et al., Synthesis of deftned peptide-oligonucleotide hybrids containing a nuclear transport signal seguence, Tetrahedron, Vol. 47, No. 24, pp.
4113-4120, 1991]. The complexes thus formed are good candidates as inhibitors endogenous gene expression potentials.
1o Transfection vectors comprising a synthetic polypeptide coupled through electrostatic interactions to a DNA sequence have also been described in patent application WO 95/31557, said polypeptide consisting of chain polymer of basic amino acids, an NLS peptide, and a hinge region who connects the NLS peptide to the polymer chain and makes it possible to avoid interactions 1s sterics. But this kind of construction poses a problem of stability because the interactions between DNA and the targeting signal are of a nature electrostatic.
There are also nucleic acid-targeting peptide chimeras specific described in patent application WO 95/34664, the connection between the two being from 2o chemical nature. But this method notably involves steps enzymatic difficult to control and unable to produce large quantities nucleic acids.
Finally, it has been shown that it is possible to attach an NLS sequence ("
Nuclear Signal Localization ”) to a plasmid DNA via a 25 cyclopropapyrroloindole (Nature Biotechnoiogy, Volume 16, pp. 80-85, January 1998).
But total inhibition of the transcription of the gene of interest due to attachment randomly several hundred NLS sequences on the plasmid has been observed. A
solution proposed by the authors to remedy this consists in linking the NLS sequences on linear DNA fragments and then to couple these modified fragments with others

3 unmodified fragments. However, this technique presents as before the disadvantage of going through at least one enzymatic step.
Thus, all the methods proposed so far do not allow satisfactorily resolve the challenges of targeting DNA
double strand.
The present invention provides an advantageous solution to these problems. More in particular, the present invention uses ogonucleotides combined with targeting signals and capable of forming triple helices with one / more sequences) specific) present) on a double-stranded DNA molecule.
Such a vector has the advantage of being able to direct double-stranded DNA towards 1o specific cells or cell compartments thanks to the signal targeting, without gene expression being inhibited. The plaintiff has indeed shown that through the formation of stable site-specific triple helices it is from now on possible to link targeting signal to double stranded DNA site-specific. In consequence, it is possible to set the targeting signal outside the cassette expression of the gene to be transferred. The plaintiff has thus shown that the expression genetics in the cell is not inhibited despite the chemical modification of DNA.
In addition, the presence of a triple helix as a means to link the signal targeting at DNA is particularly advantageous because it keeps a size DNA
suitable for transfection.
The vector obtained also has the advantage of incorporating targeting that are very stably linked to double stranded DNA, especially when the oligonucleotide capable of forming the triple helix is modified by the presence of a alkylating agent.
Another advantage of the invention is that it makes it possible to couple the DNA to to transfer targeting signals whose number and nature are controlled at the same time. In effect it is possible to control the number of targeting signals linked to each molecule double stranded DNA by introducing an appropriate number of specific sequences conducive to the formation of triple helices on said double DNA molecule strand. Of the

4 same way, it is possible to introduce on the same double DNA molecule strand several oligonucleotides linked to different targeting signals (intracellular and / or extracellular), and in this case it is also possible to determine previously the respective proportions. In addition, these different targeting signals can be more or less stable attached to the double-stranded DNA molecules the triple helix is formed with or without covalent bond (i.e. with or without the use of an alkylating agent).
Finally, the functionalized triple helix obtained is only the result of chemical transformations and can therefore be obtained in a simple way, reproducible, and lo in very large quantities, especially industrial.
A first object of the invention therefore relates to a vector useful in transfection capable of targeting a specific cell and / or cell compartment.
More in particular, the vector according to the invention comprises a DNA molecule double strand and at least one oligonucleotide coupled to a targeting signal and capable to train by hybridization a triple helix with a specific sequence present on said double stranded DNA molecule.
For the purposes of the invention, the expression “double-stranded DNA” means an acid deoxyribonucleic double strand which can be of human, animal, vegetable, bacterial origin, viral, etc ... It can be obtained by any technique known to those skilled in the art, and in particular by bank screening, by chemical or enzymatic synthesis of sequences obtained by screening of libraries. II can be chemically modified or enzymat ~ cally.
This double stranded DNA can be in linear or circular form. In this last case, double stranded DNA may be in a supercoiled or relaxed state.
Preferably, the DNA molecule is circular in shape and in a supercoiled conformation.
Double stranded DNA can also carry an origin of replication functional or not in the target cell, one or more marker genes, of regulatory sequences for transcription or replication, genes of interest WO 99! 49067 PCT / FR99 / 00643 therapeutic, antisense sequences modified or not, binding regions at other cellular components, etc. Preferably double stranded DNA
includes a expression cassette consisting of one or more genes of interest under control of one or several promoters and a transcriptional terminator active in the target cells.

5 For the purposes of the invention, the expression “gene expression cassette”
of interest "a DNA fragment that can be inserted into a vector at sites of specific restrictions. DNA fragment includes an acid sequence nucleic encoding an RNA or a polypeptide of interest and further comprises the sequences necessary for expression (enhancer (s), promoter (s), sequences of polyadenylation ...) of said sequence. The cassette and the restriction are designed to ensure insertion of the expression cassette into a frame reading suitable for transcription and translation.
It is generally a plasmid or an 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 through reference.
Within the meaning of the invention, the term gene of therapeutic interest in particular any gene encoding a protein product having a therapeutic effect. The product therapeutic thus coded can be in particular a protein or a peptide. This product 2o protein can be homologous with respect to the target cell (i.e.
a product that is normally expressed in the target cell when it does any pathology). In this case, the expression of a protein allows for example overcome a insufficient expression in the cell or expression of a protein inactive or weakly active due to modification, or overexpression said protein. The gene of therapeutic interest can also code for a mutant of a cellular protein, having increased stability, modified activity, etc.
The product protein can also be heterologous towards the target cell. In this case, an expressed protein can for example supplement or provide an activity deficient

6 in the cell, allowing it to fight against a pathology, or stimulate answer immune.
Among the therapeutic products within the meaning of the present invention, one can more particularly mention enzymes, blood derivatives, hormones, the iymphokines [interleukins, interferons, TNF, etc. (FR 92/03120)], the factors of neurotransmitters or their precursors or enzymes synthesis, the trophic factors [BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NTS, HARP / pleiotrophin, etc., dystrophin or a minidystrophin (FR 91/11947)], the CFTR protein associated with cystic fibrosis, tumor suppressor genes [p53, 1o 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 DNA repair, suicide genes [thymidine kinase, cytosine deaminase], the genes for hemoglobin or other protein transporters, the genes corresponding proteins involved in lipid metabolism, such as apolipoprotein l3 chosen from apolipoproteins AI, A-II, A-IV, B, CI, C-II, C-III, D, E F G H, J and apo (a), metabolism enzymes such as lipoprotein lipase, the Hepatic lipase, lecithin cholesterol acyltransferase, 7 alpha cholesterol hydroxylase, phosphatidic acid phosphatase, or even proteins transfer lipids such as the cholesterol ester transfer protein and the protein 2o transfer of phospholipids, an HDL binding protein or even a receiver chosen for example from LDL receptors, chylomicron receptors-remnants and scavenger receivers, etc ...
The DNA of therapeutic interest can also be a gene or a sequence antisense, whose expression in the target cell makes it possible to control the expression of 25 genes or transcription of cellular mRNA. Such sequences can, through example, be transcribed in the target cell into RNA complementary to mRNA
cells and thus block their translation into protein, according to the technique described in Patent EP 140,308. Genes of therapeutic interest also include the sequences encoding ribozymes, which are capable of destroying selectively WO 99/4906

7 PCT / FR99 / 00643 Target RNA (EP 321 20I), or the sequences encoding antibodies intracellular to simple chain like for example the ScFv.
As indicated above, deoxyribonucleic acid can also contain one or more genes coding for an antigenic peptide capable of generate an immune response in humans or animals. In this mode particular implementation, the invention therefore allows the production of either vaccines or of immunotherapeutic treatments applied to humans or animals, in particular against microorganisms, viruses or cancers. It can be especially from antigenic peptides specific for Epstein Barr virus, HIV virus, virus 1o hepatitis B (EP 185 573), of the pseudo-rabies virus, of "syncitia forming virus ", from influenza virus, cytomegalovirus (CMV), other viruses or specific of tumors (EP 259,212).
Preferably, deoxyribonucleic acid also comprises sequences allowing the expression of the gene of therapeutic interest and / or of the uncomfortable encoding the antigenic peptide in the desired cell or organ. he can to be sequences that are naturally responsible for gene expression considered when these sequences are likely to work in the cell transfected. he may also be sequences of different origin (responsible for the expression other proteins, or even synthetic). In particular, it may be sequences promoters of eukaryotic or viral genes. For example, it could be sequences promoters from the genome of the cell to be infected. Likewise, he can be promoter sequences from the genome of a virus. In this regard, we can cite for example the promoters of the genes E 1 A, MLP, CMV, RS V, etc. In Besides, these expression sequences can be modified by adding sequences activation, regulation, etc ... It can also be promoter, inducible or repressible.
A triple helix corresponds to the binding of a modified or unmodified oligonucleotide on double stranded DNA by so-called "Hoogsteen" hydrogen bonds between the bases of the third strand and those of the region in double helix. These pairings occur in the large groove of the double helix and are specific to the sequence 3o considered [Frank-Kamenetski, MD, Triplex DNA Structures, Ann. Rev.
Biochem.,

8 1995, 64, pp. 65-95). The specific double helix sequence can in particular to be a homopuric-homopyrimidic sequence. There are two categories of triplets propellers according to the nature of the bases of the third strand [Sun, J. and C. Hélène, Oligorrucleotide, 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- pairings A * T (the symbol * corresponds to the pairing with the third strand).
These structures have been characterized from the physico-chemical point of view thanks to numerous NMR (Nuclear Magnetic Resonance), temperature studies 1o of hybridization or protection to nucleases, which made it possible to define their properties and their stability conditions. For triple propellers with a third strand purine, this strand is antiparallel compared to the purine strand of DNA and the training of the triple helix strongly depends on the concentration of divalent ions:
ions such that Mg2 + stabilize the structure formed with the third strand. For the triple propellers with a third homopyrimide strand, this is parallel to the strand purine, and the formation of the triple helix is dependent on pH: a pH
lower acid at six allows protonation of cytosines and bond formation hydrogen additional stabilizing triplet CG * C +. There are also triples propellers "mixed" for which the third strand carries purine bases and pyrimides.
2o In this case, the orientation of the third strand depends on the base sequence of the region homopuric.
The oligonucleotides used in the present invention are oligonucleotides hybridizing directly with double stranded DNA. These oligonucleotides may contain the following bases - thymidine (T), which is able to form triplets with AT doublets double stranded DNA (Rajagopal et al, Biochem 28 (1989) 7859), - adenine (A), which is capable of forming triplets with the AT doublets of double stranded DNA, - guanine (G), which is capable of forming triplets with the GC doublets of double stranded DNA,

9 - protonated cytosine (C +), which is able to form triplets with doublets G. C of double stranded DNA (Rajagopal et al above), - uracil (U) which is able to form 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 on the DNA are complementary. In this regard, to obtain the best fixings and the better selectivity, an oligonucleotide is used for the vector according to the invention and an specific sequence perfectly complementary. It can be in particular of a poly-CTT oligonucleotide and a specific poly-GAA sequence. As example, mention may be made of the foligonucleotide of sequence 5'-GAGGCTTCTTCTTCTTCTTCTTCTT-3 '(GAGG (CTT) ,, SEQ ID N ° 1), in which the GAGG bases do not form triple helices but allow to space the oligonucleotide of the coupling arm. We can also cite the sequence (CTT), (SEQ ID N ° 2). These oligonucleotides are capable of forming a triple propeller with a specific sequence that includes complementary patterns (GAA). he can act in particular of a region comprising 7, 14, or 17 GA.A. Another sequence of specific interest is the sequence: S'-AAGGGAGGGAGGAGAGGAA-3 '(SEQ ID
N ° 3). This sequence forms a triple helix with the oligonucleotides 5'-AAGGAGAGGAGGGAGGGAA-3 '(SEQ ID N ° 4) or 5'-TTGGTGTGGTGGGTGGGTT-3 '(SEQ ID N ° 5).
In this case, the oligonucleotide binds in an antiparallel orientation to the strand polypuric. These triple helices are only stable in the presence of Mg2 + as this has was previously specified (Vasquez et al., Biochemistry, 1995, 34, 7243-7251;
Beal & Dervan, Science, 1991, 251, 1360-1363).
The specific sequence can be a sequence naturally present on double stranded DNA, or a synthetic or naturally occurring sequence introduced artificially in this one. It is particularly interesting to use a oligonucleotide capable of forming a triple helix with a sequence present naturally on double stranded DNA. Indeed, this advantageously allows to get the vectors according to the invention with unmodified plasmids, in particular commercial plasmids of pUC, pBR322, pSV type, etc. Among the sequences natural homopuric-homopyrimidine present on double stranded DNA, we can 5 cite a sequence comprising all or part of the sequence 5'-CTTCCCGAAGGGAGAAAGG-3 '(SEQ m N ° 6) present in the origin of ColEl replication of E. coli. In this case, the oligonucleotide forming the triple propeller has the sequence: 5'-GAAGGGTTCTTCCCTCTTTCC-3 '(SEQ 1D N ° 7) and is fixed alternately on the two strands of the double helix, as described by Beal and lo Dervan (J. Am. Chem. Soc. 1992, 114, 4976-4982) and Jayasena and Johnston (Nucleic Acids Res. 1992, 20, 5279-5288). We can also cite. the sequence 5'-GAA.AAAGGAAGAG-3 '(SEQ ID No. 8) of the ~ 3-lactamase gene from plasmid pBR322 (Duval-Valentin et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 504-508).
A
other sequence is AAGAAAAAAAAGAA (SEQ 1D N ° 9) present in the origin of y replication of plasmids from conditional replication such as pCOR.
Although perfectly complementary sequences are preferred, it is understood however that some mismatches may be iterated between the sequence of the oligonucleotide and the sequence present on the DNA, since they do not drive not too much loss of affinity. We can quote the sequence 5'-2o AAAAAAGGGAATAAGGG-3 '(SEQ mn ° 10) present in the gene for J3-lactamase from E. coli. In this case, the thymine interrupting the sequence polypuric can be recognized by a third strand guanine, thus forming a triplet ATG who is stable when it is framed by two TAT triplets (Kiessling et al., Biochemistry, 1992, 31, 2829-2834).
The oligonucleotide used can be natural (composed of natural bases, not modified) or chemically modified. In particular, foligonucleotide can present advantageously certain chemical modifications making it possible to increase its resistance, its protection against nucleases, its affinity for the sequence specific or allowing to bring other additional properties (J.
3U Goodchild, Corrjugates of Oligonucleotides a »d Modified Oligoriucleotides:
AT

Review of their Synthesis and Properties, Bioconjugate Chemistry, Vol. 1 N ° 3, 1990, pp. 165-187).
According to the present invention, the term “oligonucleotide” also means any sequence of skeleton-modified nucleosides. Among the possible modifications include, phosphorothioate oligonucleotides mistletoe are capable of forming triple helices with DNA (Xodo et al., Nucleic Acids Res., 1994, 22, 3322-3330), as well as the oligonucleotides having skeletons formacetal or methylphosphonate (Matteucci et al., J. Am. Chem. Soc., 1991, 113.
7767-7768). It is also possible to use the oligonucleotides synthesized with of his lo nucleotide anomers, which also form triple helices with DNA (The Doan et al., Nucleic Acids Res., 1987, 15, 7749-7760). Another modification of backbone is the phosphoramidate bond. One can quote for example the connection internucleotide N3'-P5 'phosphoramidate described by Gryaznov and Chen, who given oligonucleotides which, with DNA, form particularly triple helices stable (J.
Am. Chem. Soc., 1994, 116. 3143-3144). Among the other modifications of the skeleton, we can also cite the use of ribonucleotides, 2'-O-methylribose, of phosphotriester, ... (Sun and Hélène, Curr. Opinion Struct. Biol., 116. 3143-3144). The phosphorus skeleton can finally be replaced by a polyamide skeleton like in PNA (Peptide Nucleic Acid), which can also form triples propellers (Nielsen et al., Science, 1991, 254. 1497-1500; Kim et al., J. Am. Chem.
Soc., 1993, 115. 6477-6481) or by a guanidine-based skeleton, as in the DNG
(deoxyribonucleic guanidine, Proc. Natl. Acad. Sci. USA, 1995, 92 6097-6101), polycationic analogs of DNA, which also form triple helices.
The third strand thymine can also be replaced by a 5-bromouracil, which increases the f ~ oligonucleotide for DNA (Povsic and Dervan, J.
Am.
Chem. Soc., 1989, l I l, 3059-3061). The third strand may also contain of non-natural bases, among which we can cite 7-deaza-2'-deoxyxanthosine (Milligan et al., ~ Nucleic Acids Res., 1993, 21, 327-333), I- (2-deoxy-bD-ribofuranosyl) -3-methyl-5-amino-1H-pyrazolo [4,3-d] pyrimidine-7-one (Koh and Dervan, J. Am. Chem. Soc., 1992, 114, 1470-1478), 8-oxoadenine, 2-WO 99/49067 PCT / FR99 / OOb43 aminopurine, 2'-O-methyl-pseudoisocytidine, or any other modification known to a person skilled in the art (see Sun and Hélène for review, Curr. Opinion Struct. Biol., 1993, 3 345-356).
Another type of modification of the oligonucleotide has more particularly for object of improving the interaction and / or the affinity between the oligonucleotide and the sequence specific. In particular, an entirely advantageous modification consists of to couple an oligonucleotide alkylating agent. The connection can be made either chemically either photochemically via a functional group photoreactive. Of advantageous alkylating agents are in particular alkylating agents photoactivable, by lo example psoralens. Under the action of light, they form covalent bonds at the level of pyrimidine bases of DNA. When these molecules are interposed at 5'-ApT-3 'or 5'-TpA-3' sequence level in a double-stranded DNA fragment, they form links with the two strands. This induced binding reaction by light can occur at a specific site on the plasmid.
1 ~ As noted earlier, an advantage of this invention is therefore the possibility of forming very stable triple helices and site-between the oligonucleotide and a specific sequence of double DNA
strand thanks to a covalent bond formed via an alkylating agent.
The length of the oligonucleotide used in the process of the invention is from at 2o minus 3 bases, and preferably between 5 and 30 bases. We use so advantageous an oligonucleotide of length between 10 and 30 bases. The length can of course be adapted on a case-by-case basis by a person skilled in the art in function of the selectivity and the stability of the interaction sought.
The oligonucleotides according to the invention can be synthesized by any 25 known technique. In particular, they can be prepared by means of synthesizers nucleic acids. Any other method known to a person skilled in the art can also be used.

Within the meaning of the invention, the term “targeting signal” means molecules of targeting of various kinds. In most cases, these are known peptides for the targeting. They can be used to interact with a component of the matrix extracellular, a plasma membrane receptor, to target a compartment intracellular or to improve intracellular DNA trafficking, when transfer no viral gene therapy in gene therapy.
These targeting signals may include, for example, growth (EGF, PDGF, TGFb, NGF, IGF I, FGF), cytokines (IL-1, IL-2, TNF, Interferon, CSF), hormones (insulin, growth hormone, prolactin, glucagon, lo thyroid hormone, steroid hormones), sugars that recognize of lectins, immunoglobulins, ScFv, transferrin, lipoproteins, vitamins such as vitamin B 12, peptide hormones or neuropeptides (tachykinins, neurotensin, VIP, endothelin, CGRP, CCK, ...), or any reason recognized by integrins, for example the peptide RGD, or by others protein extrinsic of the cell membrane.
We can use whole proteins, or peptide sequences derived of these proteins, or peptides that bind to their receptor and obtained by the "phage display" technique or by combinatorial synthesis.
Intracellular targeting signals can be considered. A lot of 2o nuclear addressing sequences (NLS) of amino acid composition varied have been identified and allow to target different proteins involved in the transport nuclear of proteins or nucleic acids. Among these sequences are in particular short sequences (the NLS of the SV40 T antigen (PKKICRICV, SEQ 117 N ° I 1) is an example), bipartite sequences (the NLS of nucleoplasmin which contains two essential areas for nuclear transport KRPAATKKAGQAKKKKLDK, SEQ ID No. 12), or ia sequence M9 (NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY, SEQ ID N ° 13) from hnRNPAI protein. The proteins carrying these sequences are NT.C ~ P ~ YP., R ~ ", ~
, ~ p ~
specific receptors, such as the importin family receptors or karyopherins for example. The role of these sequences is to direct DNA to the interior of the nucleus, where it is then immediately available for the machinery of transcription, and can be expressed.
Targeting signals "mixed", that is to say that can both serve for the intracellular and extracellular targeting, are also part of the current invention. We can for example quote sugars which target lectins which situate on the cell membrane but also at the nuclear pore level. The targeting by these sugars therefore concerns both extracellular targeting and the import nuclear.
Other signals are involved in mitochondrial targeting (for example, N-terminal part of rat ornithine transcarbamylase (OTC) allows the targeting mitochondria) or in addressing to the endoplasmic reticulum. Finally, some signals allow nuclear retention or at the reticulum level endoplasmic (such as the KDEL sequence).
Advantageously, the targeting signals according to the invention make it possible to lead specifically double stranded DNA to certain cells or certain compartments cellular. By way of example, the targeting signals according to the invention can target receptors or ligands on the cell surface, especially receptors of insulin, transferrin, folic acid, or any other factor growth, 2o cytokines, or vitamins, or specific polysaccharides on the surface of the cell or on the surrounding extracellular matrix.
The synthesis of oligonucleotide-targeting signal chimeras takes place in phase soda or in solution and takes into account the properties of chemical stability very different from oligonucleotides and targeting signals [Erijita, R. and al., Synthesis 2s of defrned peptide-oligonucleotide hybrids containing a nuclear transport signal sequence, Tetrahedron, 1991, 47 (24), pp. 4113-4120]. In solution, couplings in a step can be envisaged: the targeting signal can for example be synthesized with a group carrying disulfirre, maleimide, amine functions, carboxyl, ester, epoxide, cyanogen bromide, or aldehyde, and be coupled to a oligonucleotide modified by a terminal thiol, amine or carboxyl group in position 3 'or 5'. These couplings are formed by establishing links disulfide, thioether, ester, amide, or amine between the oligonucleotide and the signal targeting. Any other method known to those skilled in the art can be used, such as reagents 5 bifunctional coupling for example.
Another subject of the invention relates to the compositions comprising a vector as defined above.
Advantageously, the vectors according to the invention can also be associated with one or more agents known to transfect DNA. We can cite as

10 example cationic lipids which have properties interesting. These vectors are in fact made up of a polar, cationic part, interacting with DNA, and a lipid, hydrophobic part, promoting penetration cell and making the ionic interaction with DNA insensitive to the external environment. Of examples particular of cationic lipids are in particular monocationic lipids 15 (DOTMA: Lipofectin ~), certain cationic detergents (DDAB), lipopolyamines and in particular your dioctadecylamidoglycyl spermine (DOGS) or 5-palmitoylphosphatidylethanolamine carboxyspermylamide (DPPES), the preparation has been described for example in patent application EP 394 111.
A
another interesting family of lipopolyamines is represented by the compounds described in patent application WO 97/18185 incorporated herein by reference.
Of many other cationic lipids have been developed and can be used with vectors according to the invention.
Among the synthetic transfection agents developed, polymers cationic polylysine and DEAE dextran are also advantageous. II
East also possible to use polymers of polyethylene imine (PEI) and polypropylene imine (PPI) which are commercially available and can be prepared according to process described in patent application WO 96/02655.
Generally, any synthetic agent known to transfect acid nucleic acid can be associated with the vectors according to the invention.

WO 99! 49067 PCT / FR99 / 00643 The compositions can also comprise adjuvants capable of associate with the vector complexes according to the invention / transfection agent and from improve the transfecting power. In another embodiment, the present the invention therefore relates to compositions comprising a vector as defined previously, one or more transfection agents as defined above and one or several adjuvants capable of associating with the vector complexes according to the invention / transfection agent (s) and improve its potency transfectant. The presence of this type of adjuvant (lipids, peptides or proteins for example) can advantageously allow to increase the transfecting power of the compounds.
1o With this in mind, the compositions of the invention can comprise as an adjuvant, one or more neutral lipids the use of which is particularly advantageous. The Applicant has indeed shown that the addition of a lipid neutral improves the formation of nucleolipid particles and promote the penetration of the particle into the cell by destabilizing its membrane.
More preferably, the neutral lipids used in the context of present invention are lipids with 2 fatty chains. Particularly advantageous, natural or synthetic, zwitterionic lipids are used or free of ionic charge under physiological conditions. They can be choose more particularly among dioleoylphosphatidylethanolamine (DOPE), oleoyl-2U palmitoylphosphatidylethanolamine (POPE), di-stearoyl, -palmitoyl, -mirystoyl phosphatidylethanolamines as well as their N-methylated derivative 1 to 3 times, the phosphatidylglycerols, diacylglycerols, glycosyldiacylglycerols, cerebrosides (such as in particular galactocerebrosides), sphingolipids (such as especially 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 techniques well known to those skilled in the art. In particular, the extraction of lipids natural can be carried out using organic solvents (see also Lehninger, 3o Biochemistry).

The Applicant has demonstrated that it is also particularly advantageous to use as an adjuvant, a compound intervening or not directly at the level of DNA condensation (WO 96/25508). The presence of such compound, within a composition according to the invention makes it possible to reduce the amount of compound s transfectant, with the resulting beneficial consequences toxicological, without causing any harm to the transfecting activity.
Through compound involved in the condensation of nucleic acid, we hears define a compacting compound, directly or indirectly, DNA. Specifically, this compound can either act directly on the DNA to be transfected or intervene at 1o level of an annexed compound which is directly involved in the condensation of this nucleic acid. Preferably, it acts directly on. DNA level.
In particular, this agent involved in the condensation of DNA can be any polycation, for example polylysine. According to a preferred embodiment, this agent can also be any compound derived in whole or in part from a histone, a nucleolin, 15 of a protamine and / or one of their derivatives. Such an agent may also to be consisting, in whole or in part, of peptide motifs (KTPKKAICKP) and / or (ATPAKKAA), the number of patterns can vary between 2 and 10. In the structure of the compound according to the invention, these units can be repeated so continue or no. This is how they can be separated by nature links biochemical, by 2o for example with one or more amino acids, or of a chemical nature.
The subject of the invention is also the use of the vectors as defined this-before to make a medicine to treat illnesses by transfection of DNA in primary cells or in established cell lines. he can be fibroblast, muscle, nerve cells (neurons, astrocytes, cells 25 glial), hepatic, of the hematopoietic line (lymphocytes, CD34, dendritics, etc ...), epithelial, etc ..., in differentiated or pluripotent forms (precursors).
The vectors of the invention may, by way of illustration, be used for the in vitro, ex vivo or in vivo transfection of DNA encoding proteins or of polypeptides.

For in vivo uses, either in therapy or for the study of regulation of genes or the creation of animal models of pathologies, the compositions according to the invention can be formulated for topical administration, skin, oral, rectal, vaginal, parenteral, intranasal, intravenous, intramuscular, sub-cutaneous, intraocular, transdermal, intratracheal, intraperitoneal, etc ... From preferably, the compositions of the invention contain a vehicle pharmaceutically acceptable for an injectable formulation, especially for a direct injection at the level of the desired organ, or for administration by track topical (on skin and / or mucosa). These may in particular be solutions sterile, isotonic, or dry compositions, in particular lyophilized, which, by addition depending on the case of sterilized water or physiological saline, allow the constitution of injectable solutions. The doses of nucleic acid used for injection as well as the number of administrations can be adapted according to different settings, and in particular depending on the mode of administration used, the pathology concerned, the gene to be expressed, or the duration of the treatment sought. In what relates more particularly to the mode of administration, it can be either of a direct injection into the tissues or circulatory tract, either of Treatment of cells in culture followed by their reimplantation in vivo, by injection or graft.
The invention further relates to a method for transfection of DNA into 2o cells comprising the following steps (1) synthesis of the oligonucleotide chimera - targeting signal according to the process previously described, (2) bringing the chimera synthesized in (1) into contact with double-stranded DNA
to form triple helices, (3) optionally, complexation of the vector obtained in (2) with one or several transfection agents and / or one or more adjuvant (s), and (4) bringing the cells into contact with the complex formed in (2) or the case appropriate in (3).

The bringing of the cells into contact with the complex can be carried out by incubation of the cells with said complex (for in vitro uses or ex vivo), or by injection of the complex into an organism (for uses in vivo).
The present invention thus provides a particularly advantageous method s for the treatment of diseases by administration of a vector according to the invention containing a nucleic acid capable of correcting said disease. More particularly, this method is applicable to diseases resulting from a deficiency in a product protein or peptide, the administered DNA coding for said protein product or peptide. The present invention extends to any use of a vector according to lo the invention for the in vivo, ex vivo or in vitro transfection of cells.
The invention also relates to any recombinant cell containing a vector as defined above. They are preferably cells eukaryotes, by example of yeasts, animal cells, etc. These cells are obtained by any technique allowing the introduction of DNA into a given and known cell of 15 skilled in the art.
The following examples are intended to illustrate the invention without limiting its scope"
allow to highlight other characteristics and advantages of the present invention.
FIG- USSR
2o Fi ~~ ure 1: Coupling of an oligonucleotide (ODN) and the maleimide peptide-NLS.
Fi ure 2: Analysis on 15% polyacrylamide gel of the chimera oligonucleotide -peptide (Pso-GA19-NLS) by proteolytic action of trypsin.
1 = oligo Pso-GA ~ 9-SH
2 = oligonucleotide-peptide chimera Pso-GA, 9-NLS
25 3 = oligonucleotide-peptide chimera Pso-GA, 9-NLS after digestion with trypsin Figure 3: schematic representation of the plasmid pXL2813.

Figure 4: schematic representation of the plasmid pXL2652.
Figure 5: Analysis on 15% polyacrylamide gel of triple formation propellers between the plasmid pXI, 2813 and the oligonucleotide-peptide chimera Pso-GAl9-NLS.
The oligonucleotide pso-GA, 9-NLS and the plasmid are mixed in a buffer s containing 100 mM MgCl2. The excess in molarity of the oligonucleotide by compared to plasmid ranges from 0 to 200. The mixture is photoactivated, after overnight 37 ° C, then digested by two restriction enzymes which cut the plasmid apart and else from the triple helix formation region.
1 = no oligonucleotide 2 = excess in molarity of oligonucleotide relative to the plasmid of IS
3 = excess in molarity of oligonucleotide relative to the plasmid by 50 4 = excess in molarity of oligonucleotide relative to the plasmid of 100 5 = excess molarity of oligonucleotide compared to the plasmid of 200 6 = single photoactivated oligonucleotide 15 7 = oligonucleotide alone not photoactivated 8 = excess in molarity of oligonucleotide relative to the plasmid of 30, the mixed have not been photoactivated.
Figure 6: In vitro expression of the transgene (J3-galactosidase) for tests made with the plasmid pXL2813 alone, the vector pXL, 2813-Pso-GA, 9-NLS, and without plasmid.
2o Fi ure 7: Characterization of the peptide part of the chimera oligonucleotide-peptide Pso-GA, 9-NLS by interaction with importine 60-GST (gel analysis of polyacrylamide IS%).
1 = oligo Pso-GA19 (I pg) 2 = oligonucleotide-peptide chimera Pso-GAl9-NLS (l ~ cg) 3 = supernatant recovered after incubation of the coated glutathione beads important tines 60 and the oligonucleotide Pso-GA, 9, and separation of the pellet from beads (containing elements interacting with importines) of the supernatant 4 = pellet recovered after incubation of the coated glutathione beads important 60 and the oligonucleotide Pso-GA19, and separation of the pellet from beads (containing the elements interacting with the importines) of the supernatant = supernatant recovered after incubation of the coated glutathione beads important 5 tines 60 and of the oligonucleotide-peptide chimera Pso-GA ~ 9-NLS, and separation of pellet of balls (containing the elements interacting with importines) of supernatant 6 = pellet recovered after incubation of the coated glutathione beads important 60 and the oligonucleotide-peptide chimera Pso-GA19-NLS, and separation of the pellet of beads (containing elements interacting with importines) from supernatant.
Io Fi ure 8: Analysis on 15% polyacrylamide gel of the chimera oIigonucIéotide-peptide (GA, 9-NLS) by proteolytic action of trypsin.
1 = oligo GA, 9-SH (200 ng) 2 = oligonucleotide-peptide GA, 9-NLS chimera (1 pg) before purification by chroma-high pressure liquid tography 3 = oligonucleotide-peptide GA ~ 9-NLS chimera (1 pg) after purification 4 = oligonucleotide-peptide GA19-NLS chimera after digestion with trypsin ( 1 ~ tg).
Fitxure 9: Graphical representation of the kinetics of triple formation propellers (%
of triple helix sites occupied as a function of time) between the plasmid pXL2813 and the chimera GA ~ 9-NLS.
2o F_iRure 10: Characterization of the peptide part of the chimera oligonucleotide-GA19-NLS peptide by interaction with importin 60-GST.
1 = oligonucleotide-peptide GA ~ 9-NLS chimera, 2 = oligo GA, 9, 3 - supernatant recovered after incubation of the coated glutathione beads of importines 60 and of the chimeric oligonucleotide-peptide GAIS-NLS, and separation of pellet of balls (containing the elements interacting with importines) of supernatant, 4 = pellet recovered after incubation of the coated glutathione beads important 60 and of the oligonucleotide-peptide chimera GA19-NLS, and separation of the pellet from marbles (containing the elements interacting with the importins) of the supernatant, - supernatant recovered after incubation of the coated glutathione beads of imports 60 and of the oligonucleotide GA, 9 and separation of the pellet from beads (containing your elements interacting with importines) of the supernatant, 6 = pellet recovered after incubation of the coated glutathione beads important 60 5 and oligonucleotide GA, 9, and separation of the pellet from beads (containing the elements interacting with the importins) of the supernatant.
Figure 1 I: schematic representation of the plasmid pXL, 2997.
Figure 12: Graphical representation of the kinetics of triple formation propellers (% of triple helix sites occupied as a function of time) between the plasmid pXL2997 and the chimera pim-NLS.
Figure 13: histogram representing the ~ 3-galactosidase activity in vivo in of human lung tumors H1299 from plasmids pXI, 2813 (indicated Bgal on the figure) and pXL, 2813-Pso-GA, 9-NLS (indicated NLS-Bgal in the figure) in RLU
("Relative Light Unit") per tumor.
The transfection was done using electrotransfer techniques as described in applications W099 / 01157 and WO 99/01158.
MATERIALS AND METAODS
1. Coupling of oligonucleotides and peptides Olitxonucleotides 2o The oligonucleotides used are either the 5 'sequence AAGGAGAGGAGGGAGGGAA-3 '(SEQ m N ° 4) with a length of 19 bases and referred to as “GA, 9” in the remainder of the text, ie the sequence 5 ′
GGGGAGGGGGAGG-3 '(SEQ) DN ° 15) with a length of 13 bases and referenced under the name "pim" in the rest of the text (because it is the sequence of protooncogene pim-1).
The oligonucleotides noted GA, 9-SH or pim-SH have the same sequences as GA, 9 and pim respectively and a thiol group at the 5 'end, with a spacer of six carbons between tlliol and phosphate at the 5 'end. The oligonucleotides noted Pso-GA19-SH have a thiol group at the 3 'end (SH), and in addition, a psoraiene at the 5 'end (Pso), with a six-carbon spacer between the psoralen and the phosphate from the 5 'end. The oligonucleotides noted Pso-GA, 9 have no thiol group.
The nomenclature used is summarized in Table I below name of oligonucleotide change of end change of end 3 '5' GA, 9 none none Pim none none GA, 9-SH none ~ thiol group pim-SH no thiol group Pso-GA, 9 no psoralne Pso-GA ~ 9-SH psoral thiol group Table I
These lyophilized oligonucleotides are taken up in an acetate buffer of 100 mM triethylammonium, pH = 7.
Peptides lo The peptides used for the couplings are synthesized by an automaton phase solid. They contain - or the nuclear localization sequence of the SV40 T antigen (PKICKRKV, SEQ
ID No. 11), - or the same mutated sequence (PK1VICRKV, SEQ l'D N ° 14) which does not allow neither ie targeting or nuclear transport (due to the mutation).
These peptides also carry a spacer of four amino acids to the N- end Terminal: KGAG. N-terminal lysine is chemically modified: it contains a maleimide group on carbon E and a protective group 9-fluorenylmethyloxycarbonyl (Fmoc) on the carbon amine a. This Fmoc group absorbs at 260 nm which makes it possible to follow the peptide by chromatography liquid to high pressure in reverse phase. The C-terminal group is also protected (CONHZ group), the protection being added at the end of peptide synthesis.
The representation of the peptides is indicated in Table II below peptide sequence name and modifications malimide- ~ GAGPKKKRK ~ -CONHz malimide-NL
S

FF '' moc malimide- KGAGPKNKRKV ~ --CONHz malimide-NLSmut ' ~
Fmoc Table II
The lyophilized peptides are taken up in an acetate buffer of triethylammonium 100 mM, pH = 7. The concentration is 0.4 mglml.
The colors For the coupling with the oligonucleotides, the strategy used consists in react the thiol group carried by the oligonucleotide and the maleimide group carried speak peptide [Eritja, R., et al., Synthesis of defrned peptide-oligonucleotide hybrids containing a nuclear transport signal sequence, Tetrahedron, 1991, 47 (24), pp.

4120].
The oligonucleotide is added to the peptide solution equimolarly and the middle reaction is left for two hours at room temperature. The chimera oligonucleotide-peptide is purified by high pressure liquid chromatography in phase reverse, on Vydac C8 column containing spheroidal silica with a diameter of 5 μm and porosity 300 A. 0.1 M triethylammonium acetate buffer (TEAA) and a 2o gradient of acetonitrile passing from 5% to 50% in 35 minutes. Products are detected at 260 nm.

Analysis of chimeras by digestion with trv, _psine The oligonucleotide-peptide conjugates are subjected to the proteolytic action of the trypsin which allows to highlight the peptide part of the chimera [Reed, MW et al, Synthesis and eraluation of nuclear targeting peptide-antisense 5 conjugal oligodeoxynucleotide, Bioconjugate Chemistry, 1995, 6, pp.101-108J. Of solutions containing 1 µg of oligonucleotide-peptide in 7,111 of buffer 0.1 M trethylammonium purification (TEAA) are mixed with 1 μl of a solution of trypsin (5 mg / ml). Add 1 μl of 100 mM Tris buffer, HCl pH = 9, and 1 pl of 500 mM EDTA. After an hour's digestion, the samples are deposited in 10 wells of 15% polyacrylamide gel, 7 M urea. Electrophoresis is made in 100 mM Tris buffer, 90 mM boric acid, 1 mM EDTA, pH = 8.3. Acids nucleic acids are revealed by silver staining using a Biorad kit.
2. Formation of triple helices with chimeras The plasmid The plasmid used to study the formation of triple helices with the chimeras GA, 9-peptide is called pXi, 2813 (7257 bp, see Figure 3). This plasmid expresses the gene Ia (3-galactosidase under the control of the strong promoter of the early genes of Cytomegalovirus (CMV), as well as the Ampicillin resistance gene.
Upstream of the promoter, between positions 7238 and 7256, the sequence GA19 (5'-2o AAGGAGAGGAGGGAGGGAA-3 ', SEQ ID N ° 4) was cloned according to the methods molecular biology classics.
It was cloned into the plasmid pXL2652 (7391 bp and whose representation is shown diagrammatically in FIG. 4) which expresses the gene for [3-Galactosidase under control of the strong promoter of the early genes of Cytomegalovirus (CMV), as well as the uncomfortable 25 resistance to Ampicillin. This promoter comes from pCDNA3, the gene LacZ and his polyA are from pCH110, and the rest are from pGL2.
The sequences were cloned upstream of the promoter between the unique sites of cutting of the Muni and XmaI enzymes. For this, the two oligonucleotides complementary containing the sequence to be cloned 6651 (5'-3o AATTGATTCCTCTCCTCCCTCCCTTAC-3 ') and 6652 (3'-CTAAGGAGAGGAGGGAGGGAATGGG-5 ') were heated for 5 minutes to 95 ° C, then hybridized, allowing the temperature to drop slowly.
The plasmid pXL2652 was then digested with the enzymes Muni and XmaI, 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 further cloning was eluted according to the protocol Jetsorb (Genomed) and 200 ng of this fragment have been linked to 10 ng of the mixture of oligonucleotides hybridized by T4 ligase, for 16 hours at 16 ° C.
Competent E. Coli bacteria of DHSoc strain have been transformed by electroporation with the reaction product, spread on cans Petri dish LB medium and Ampicillin. Ampicillin resistant clones have summer selected and DNA was extracted by alkaline lysis and analyzed on gel 1% agarose.
A clone corresponding in size to the expected product was sequenced.
In cases where the oligonucleotide is pim, the plasmid used to study the training of helical priple is pXL2997 represented in FIG. 11. This plasmid expresses the gene (3-galactosidase under the control of the strong promoter of the early genes of Cytomegalovin. ~ S (CM ~, as well as the Ampicillin resistance gene.
Upstream of the promoter, the pim sequence (SEQ ID No. 15) has been cloned according to classical methods of molecular biology.
zo 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 pg) or pXL2997 depending on the case and quantities variables of egg or chimeras in 100 mM Tris buffer, HCl pH = 7.5, and MgCl2 I 00 mM.
2s Nhotoactivation of triple propellers After an overnight incubation, the mixture is irradiated, in ice, for 15 minutes, using a 365 nm wavelength monochromatic lamp (Biorad). The photoactivation product is digested by restriction enzymes MfeI
and SpeI and analyzed on a 15% polyacrylamide gel, 7 M urea with a buffer of migration 3o Tris 100 mM, boric acid 90 mM, EDTA 1 mM, and pH = 8.3. Acids nucleic are revealed by a silver coloration thanks to a Biorad kit [Musso, M., JC
Wang, and MWV Dyke, Ilt VIVO persistence of DNA triple helices containi »g psoraleu-col jugated oligodeoxyribonucleotides, Nucleic Acid Research, 1996, 24 (24), pp. 4924-4932].
The triple helix study method This method is based on the principle of exclusion chromatography.
solutions containing the plasmid and the oligonucleotide capable of forming a triple propeller. The exclusion columns used (Columns, Linkers 6, Boehringer Mannheim) are composed of sepharose beads and have as exclusion limit 194 pairs of basics this which allows to retain in the column the oligonucleotides not paired with plasmids.
First, the oligonucleotides are radioactively labeled in their 3 'end by Terminal Transferase using dATPa35S. The protocol used comes from Amersham: 10 pmol of oligonucleotides are incubated for 2 hours at 37 ° C, with 50 pCi of dATPa35S in the presence of 10 Terminal units Transferase, in a volume of 50 IU of buffer containing sodium cacodylate. The percentage of labeled oligonucleotides is evaluated according to the following method:

of a sample diluted to 1/100 of the solution after marking is deposited on paper Whatman 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 2o of ethanol. The radioactivities of the two papers are compared. The radioactivity washed paper corresponds to the 35S actually incorporated.
The formation of the triple helices takes place in a volume of 35 μl. The concentrations of plasmids (40 nM) and oligonucleotides (20 nNn, ie buffer used (100 mM
Tris HCI pH = 7.5, 50 mM MgCl2) and the temperature (37 ° C) are fixed while the incubation time varies from 1 to 24 hours. The sepharose columns are balanced, before use, with the reaction buffer and centrifuged at 2200 rpm for 4 minutes, to pack them. 25 μl of the reaction medium are deposited on ies columns and these are centrifuged under the same conditions as above. 25 pl of WO 99149067 PC'T / FR99 / 00643 reaction buffer are then deposited on the columns which are again centrifuged. The eluate is recovered.
The radioactivity contained in 5 μl of the reaction medium, denoted cpm (deposit), and that contained in the eluate, noted cpm (eluate), are evaluated which makes it possible to estimate the percentage of oligonucleotides eluted eluted oligonucleotides = cpm (eluate) / [5 x cpm (deposit}) x100.
The percentage of plasmids that are effectively eluted during experiences is evaluated by estimating the optical density at 260 nm of the eluate and that of the deposit, what calculates the percentage of oligonucleotides attached to all of the plasmids fixed oligonucleotides = [% eluted oligonucleotides /% eluted plasmids] x100.
This parameter is used to evaluate the percentage of triple training sites propellers (there is one by plasnûde pXL2813 or pXL2997) which are actually busy taking into account the concentrations of plasmids (denoted [plasmid]) and oligonucleotides (denoted [oligo]) used during the formation reaction triple propellers occupied triple helix sites =% of fixed oligonucleotides x [oligo] /
[plasmid].
3. Interaction with imnortins Recombinant proteins 2o The importine subunit 60 used to study the interaction with conjugates oligonucleotide-peptide (NLS or NLSmuted) is of murine origin and fused to the Glutathione S-Transferase (GST). Importin sequence 60 has been cloned in one vector pGEX-2T to merge it with the GST. The recombinant protein has been produced in Escherichia coli [Imamoto, N. et al., In vivo evidence for involvement of at 58kDa compose of nuclearpore-targeting complex in rruclear protein import, The EMBO
Journal, 1995, 14 (15), pp. 3617-3626].
Interactions with recombinant proteins All interaction experiments are carried out in the fixation buffer (HEPES
20 mM, pH = 6.8, 150 mM potassium acetate, 2 mM magnesium acetate, DTT
2 mM, and BSA 100 pg / ml).
Initially, the recombinant proteins are incubated in the presence of sepharose beads covered with glutathione groups (Biotech Pharmacy) 1 ug of recombinant protein is used for 10 μl of beads. After one 30 incubation minutes, at room temperature, in 500 µl of fixing buffer, the mixture East centrifuged at 2000 G for 30 seconds, and the supernatant is removed. The balls are washed five times by resuspension in 500 μl of fixing buffer and centrifi. ~ gation, to as described above. The beads are resuspended in buffer of fixing for obtain a suspension containing SO% of protein-coated beads recombinant.
Secondly, 60 μl of the suspension containing 50% of beads covered with recombinant proteins are incubated with 2 µg of oligonucleotide or oligonucleotide-peptide, in a volume of 500 μl of fixing buffer.
After one incubation for 30 minutes, at room temperature, the mixture is centrifuged at for 30 seconds, and the supernatant is removed. 30 µl of the supernatant are collected to analyze the fraction not fixed on the beads. The beads are washed five times a resuspension in 500 μl of fixation buffer and centrifugation, as described this-2o before. The beads are resuspended in 15 μl of loading buffer (blue bromophenol 0.05%, sucrose 40%, EDTA 0.1 M, pH = 8, sodium lauryl sulfate 0.5%) and heated for 10 minutes at 90 ° C. The contents of the supernatant and base is analyzed on a 15% polyacrylamide gel, 7 M urea, with a buffer of migration Tris 100 mM, boric acid 90 mM, EDTA I mM, pH = 8.3. Nucleic acids are revealed by a silver coloration thanks to a Biorad kit [Rexach, M.
and G.
Blobel, Protein import into nuclei: association a ~: d dissociation reactions involving transport substrate, transport factors, and nucleoporins, Cell, 1995, 83, pp.
683-692).
4. Cell transfection Cellular culture The cell type used is NIH3T3 (ATCC CRL-1658). It is fibroblasts mouse. These cells are cultured in a modified Dulbecco medium, with glucose 4.5 g / 1 (DMEM - Gibco), 2 mM glutamine, penicillin (10,000 U / ml) and of the streptomycin (100 pg / ml), and 10% fetal calf serum (Gibco). They are 5 incubated at 37 ° C in an oven with 5% COz.
Transfection One day before transfection, the wells of a 24-well plate are seeded with 50,000 cells per well. The vectors are diluted in NaCI 1 SO mM and mixed with a cationic lipid (the compound of condensed formula 1o H2N (CH2) 3NH (CH2) 4NH (CH2) 3NHCH2COGlyN [(CH2) ~~ CH3] 2 described in Patent application WO 97/18185 under number (6)), diluted in dù 150 mM NaCl. The mixing takes place with 6 nmol of lipid per microgram of plasmid. This mixture East diluted 1/10 in culture medium without serum and deposited on the cells.
After incubation at 37 ° C in an oven at 5% COz for two hours, we add 15 IO% fetal calf serum.
Quantification of (3-galactosidase After a 48 hour incubation, the cells are washed twice with PBS and lysed with 250 111 lysis buffer (Promega). (3-galactosidase is quantified according to the "Lumigal (i-Galactosidase genetic reporter system") protocol (Clontech).
2o The activity is measured on a Lumat LB9501 luminometer (Berthold). The number of protein is measured with the BCA kit (Pierre).
EXAMPLES
Example 1 This example illustrates the possibility of coupling the oligonucleotide Pso-GA19-SH
at 25 maleimide-NLS peptide.
The oligonucleotide Pso-GA, 9-SH, of sequence 5'-AAGGAGAGGAGGGAGGGAA-3 ', with a thiol group at the 3 ′ end, was coupled to the NLS peptide which wear one maleimide group at its N-terminal end according to the method described previously in the "Materials and Methods" section under the "coupling" section of oligonucleotides and peptides ".
The coupling is monitored by high pressure liquid chromatography (HPLC) in phase reverse. It is carried out with an equimolar stoichiometry: in two hours, the coupling is complete, and the chimera is purified by HPLC.
The coupling reaction scheme is shown in Figure 1.
The oligonucleotide-peptide chimera (Pso-GA, 9-NLS) was analyzed by electrophoresis polyacrylamide gel after proteolytic action of trypsin which allows to put highlight the presence of the peptide part of the chimera, after migration on gel 1o of denaturing polyacrylamide and staining of nucleic acids by money (like indicated in the section "Materials and methods" under the section "Analysis of chimeras by trypsin digestion ").
The chimera Pso-GA ~ 9-IVI, S exhibits a delayed electrophoretic migration through compared to the oligonucleotide Pso-GA19-SH, and the product of digestion proteolytic is displayed at an intermediate level between the migration levels of Pso-NLS and Pso-GA, 9-SH, as shown in Figure 2.
The chimera Pso-GA19-NLS therefore contains a peptide part accessible to the trypsin. These results clearly show that the coupling between the oligonucleotide and the peptide has operated, and the coupling yield is important.
2o Example 2 This example illustrates the formation of triple helices between the plasmid pXL2813 and ia Pso-GA19-NLS chimera which is modified by a photoactivatable alkylating agent.
This example also indicates what is the proportion of plasmids modified according to excess in molarity of oligonucleotides relative to the plasmid:
Plasmid pXL2813, shown in Figure 3, has the sequence homopuric complementary to GA19 which is likely to form a triple helix with the oligonucleotides GA, 9, Pso-GA19, Pso-GA ~ 9-SH or Pso-GA, 9-NLS. Cations divalents, such as Mg2 ', stabilize these triple helices. The oligonucleotide Pso-GA, 9-NLS

and the plasmid are mixed in a buffer containing 100 mM MgCl2. Excess in oligonucleotide molarity with respect to the plasmid varies from 0 to 200.
After incubation for 12 hours at 37 ° C, the mixture is photoactivated for 1 S
minutes (as indicated in the "materials and methods" part under the section "Photoactivation of triple helices"), then digested by the two enzymes of restriction MfeI and SpeI which cut the plasmid on either side of the region of training of triple propellers. A nucleic acid fragment which contains 70 pairs of bases after digestion of the plasmid pXL2813 unmodified. The fragment obtained with the plasmid and the oligonucleotide forming a triple helix covalently linked to the double the helix has 70 base pairs plus 19 bases of the oligonucleotide. Through migration on a denaturing polyacrylamide gel, we can thus separate these fragments from cut different: fragments from plasmids modified by a triple propeller have a shorter migration distance than fragments from non-plasmids amended. It is therefore possible, by electrophoresis on polyacrylamide gel denaturing, quantify the proportion of modified plasmids according to excess molarity oligonucleotide relative to the plasmid.
The results are shown in Figure 5. For excess molarity oligonucleotide compared to the plasmid greater than 50, all the plasmids are modified and are associated with an oligonucleotide Pso-GA, 9-NLS. Without photoactivation, we lose the 2o delaying the digestion fragment in denaturing gel.
It thus appears that the triple helices formed with the oligonucleotides Pso-GA, 9-SH
or Pso-GA, 9-NLS are therefore well covalently linked to the double helix after photoactivation.
Furthermore, by digesting the rest of the plasmid skeleton and analyzing it as previously, we can verify that photoactivation does not cause bond non-specific covalent of the oligonucleotide Pso-GA, 9-NLS outside the region containing the sequence capable of forming a triple helix.
These results clearly highlight the conditions allowing to associate specifically and covalently a peptide sequence to a plasmid, and this outside of the promoter regulating the expression of the transgene, which prevents the expression transgene is affected.
Example 3 This example illustrates the ability of the transgene to be expressed in vitro, although the plasmid be modified.
Expression of / 3-galactosidase by unmodified pXL2813 or associated with an oligonucleotide Pso-GA, 9-NLS was thus compared by transfection of lVIFi3T3 cells.
The results are shown in Figure 6, and the means of the values obtained (~ standard deviation) are collated in the following table III
expression of the transgender plasmid (in RLU / pg of protein) pXL2813 253759 48545 pXL2813-Pso-GA19-NLS473984 111476 without plasmid 129 85 Table III
It is noted that the expression of the transgene increases following the modification brought by covalent attachment of a triple helix upstream of the promoter.
These results clearly show that the training of triples propellers does not affect expression of the transgene in vitro. On the contrary, thanks to addressing nuclear of the plasmid due to its coupling to the NLS targeting sequence, more of plasmids have reached the nucleus, and this results in an increase in the effectiveness of transfection.
Example 4 2o Get example aims to verify that the expression in vivo is not inhibited by presence of a targeting signal associated with the plasmid via a triple helix.

The experiment consists in transfecting pXL2813 and pXI, 2813-Pso-GA19-NLS in H1299 human lung tumors using techniques electrotransfer described in applications WO 99/01157 and WO 99/01158. The experience was performed in nude female mice from 18 to 20 g.
Mice are implanted monolaterally with H1299 tumor grafts of 20 mm3. Tumors grow to reach a volume of 200 to 300 mm3.
The mice are sorted according to their tumor sizes and divided into lots homogeneous.
Then the mice are anesthetized with a Ketamine / Xylazine mixture (available commercially). The plasmid solution (8 μg of DNA / 40 μl of chloride sodium lo I50 mM) is injected longitudinally at the periphery of the tumor using of a Hamilton syringe.
The lateral sides of the tumor are coated with conductive gel and the tumor is placed between two flat stainless steel electrodes 0.45 to 0.7 em apart.
The electrical pulses are applied using a pulse generator squares of trade (PS 15 electro-drainer, Jouan, France) 20 to 30 seconds after injection.
An oscilloscope allows you to control the intensity in volts, the duration in milliseconds and the frequency in hertz of the pulses which must be delivered at 500 V / cm for 20 milliseconds and 1 hertz.
For the evaluation of tumor transfection, 10 and 7 mice respectively for the plasmid pXL, 2813 and pXL2813-Pso-GAl9-NLS are euthanized 2 days after injection of the plasmid. Tumors are removed, weighed and boyaged in a buffer of lysis (Promega) supplemented with antiproteases (Complete, Boehringer). The suspension obtained is centrifuged to obtain a clear supernatant. After incubating 10 ltl of this supernatant with 250 μl of reaction buffer (Clontech) for 1 hour at darkness, ~ i-galactosidase activity is measured using a luminometer of trade. The results are expressed in total Relative Light Unit (RLU) through tumor.
It is 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 plasmid pXL2813 not amended (see figure 13).

Example 5 This example illustrates the characterization of the peptide part of the conjugates Pso-GA ~ 9-NLS, i.e. verification of the targeting properties of the NLS peptide associated with constructions according to the invention.
5 The peptide sequence used, ie the NLS signal of the SV40 T antigen, is recognized by receptors of the karyopherin family a. The murine equivalent, called importine 60, fused to a glutathione S-transferase group, was used for characterize the oligonucleotide-peptide conjugates. He was operated according to the method described in the "Materials and Methods" section under the "Interactions" section with the 10 importines ".
Interactions between glutathione beads coated with 60 and the GA, 9-NLS or Pso-GA19-NLS oligonucleotides were studied. After one incubation 30 minutes at room temperature, the pellet is separated from the beads (containing the elements interacting with the importines) of the supernatant The result of this characterization is given in FIG. 7. This figure indicates that the oligonucleotide Pso-GA, 9-NLS is associated with the glutathione beads, while than the oligonucleotide Pso-GA, 9 remains in the supernatant.
This clearly shows the capacity of the oligonucleotide Pso-GA, 9-NLS to interact with the import a. The peptide part of the oligonucleotide-peptide chimeras is so good 2o recognized by its receptor, which means that the peptide fills actually its targeting signal role.
Example 6 This example illustrates the possibility of coupling the oligonucleotide GA19-SH to the peptide maleimide-NLS. Unlike Example 1, the chimera is not modified by a 25 photoactivatable alkylating agent.
The oligonucleotide GA, 9-SH, of sequence 5'-AAGGAGAGGAGGGAGGGAA-3 ' (SEQ m No. 4), with a thiol group at the 5 'end, was coupled with peptide maleimide-NLS which has a maleimide group at its N-terminal end under the same conditions as for the oligonucleotide Pso-GA, 9-SH (see example 1), WO 99/49067 PCT / E'R99 / 00643 The coupling is monitored by high pressure liquid chromatography (HPLC) in phase reverse. It is performed 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 the 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-GAt9-SH.
Example 7 This example illustrates the possibility of forming triple helices between the plasmid 10 pXL2813 and the chimera GA, 9-NLS in the absence of an alkylating agent.
Kinetics of formation of triple helices between the plasmid pXL, 2813 and the chimera GA, 9-NLS obtained as described in Example 5, was carried out and studied according to the technique described in the "Materials and Methods" part, under the section "Training of triple helices with chimera ".
FIG. 9 represents the kinetics of formation of the triple helices.
It appears that under the conditions of plasmid concentrations of 40 nM and in 20 nM oligonucleotide, a stable triple helix is formed.
Example 8 This example illustrates the characterization of the peptide part of the chimera GAl9-NLS.
2o The peptide sequence used, the IVLS signal of the SV40 T antigen, is recognized by receptors of the karyopherin family a, as has already been mentionned in example 4. The murine equivalent, called importine 60, fused to a group glutathione S-transferase, has been used to characterize conjugates oligonucleotide peptide. It was operated as described in "Materials and Methods" under the section "Interactions with importines".
Interactions between glutathione beads coated with 60 and the GA, 9 or GA, 9-NLS oligonucleotides were studied. After an incubation of 30 minutes at room temperature, the pellet of beads is separated (containing ies elements interacting with the importins) of the supernatant.
The results are shown in Figure 10. It appears that the oligonucleotide GA ~ 9-NLS
is associated with the glutathione beads, while the oligonucleotide GA19 remains in the supernatant.
This clearly shows the ability of the oligonucleotide GA ~ 9-NLS to interact with the import a. The peptide part of the oligonucleotide-peptide chimeras is so good recognized by its receiver, and again fulfills its role of signal of targeting.
Example 9 lo This example illustrates the possibility of coupling the oligonucleotide 'pim-SH to the peptide maleimide-NLS.
The oligonucleotide pim-SH, of sequence 5'-GGGGAGGGGGAGG-3 '(SEQ ID
N ° 15), with a thiol group at the 5 ′ end, was coupled to the maleimide peptide-NLS who has a maleimide group at its N-terminal end in the same conditions as for the GAIS-SH oligonucleotide (see example 5).
The coupling is monitored by high pressure liquid chromatography (HPLC) in phase reverse. It is performed with an equimolar stoichiometry: in two hours, the coupling is total. The chimera is then purified by HPLC.
Example, - ple 10 2o This example illustrates the possibility of forming triple helices between the plasmid pXL2997 and the pim-NLS chimera in the absence of an allryling agent.
Kinetics of formation of triple helices between the plasmid pXL2997 and the chimera pim-NLS (training obtained as described in Example 8), was carried out and studied using the technique described in the "Materials and Methods" section, under the section 2s "Formation of triple helices with chimera".
FIG. 12 represents the kinetics of formation of the triple helices.
It appears that under the conditions of plasmid concentrations of 40 nM and in oligonucleotide of 20 rtM, a stable triple helix is formed.

The invention now being fully described, it is obvious that many modifications may be made to the present invention without, however, step aside the spirit of the invention and the scope of the claims which follow.

WO 99/49067, PCT / FR99 / 00643 SEQUENCE LIST
(1) GENERAL INFORMATION:
(i.) DEPOSITOR:
in) NAME: RHONE POULENC ROBER
(D) STREET: 20 AVENUE RAYMOND ARON
(C) CITY: ANTONY CEDEX
(E) COUNTRY: FRANCE
(F) POSTAL CODE: 92165 (G) PHONE: O.55.71.73.26 (H) FAX: 01.55.71.72.91 (ü) TITLE OF THE INVENTION: NUCLEIC ACID TRANSFER VECTORS, COMPOSITIONS CONTAINING THEM, AND USES THEREOF.
(iii) NUMBER OF SEQUENCES: 15 (iv) COMPUTER-DETACHABLE FORM:
(A) TYPE OF SUPPORT: Tape (B) COMPUTER: IBM PC compatible ' (C) OPERATING SYSTEM: PC-DOS / MS-DOS
(D) SOFTWARE: Pater.tIn Release I (1.0, Version N1.30 (EPO) (2) INFORMATION FOR IA SEQ ID NO: 1:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 25 base pairs (B) TYPE: nucleotide (C) NOHIBER OF STRANDS: simple (D) CONFIGURATION ~ TION: linear (ii) TYPE OF MOLECULE: cDNA
(ai) DESCRIPTIOl4 OF THE SEQUENCE: SEQ ID NO: 1:

(2) INFORMATION FOR SEQ ID NO: 2:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 21 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ü) TYPE OF MOLECULE: cDNA
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2:

SUBSTITUTE SHEET (SET 26) (2) INFORMATION FOR SEQ ID NO: 3:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 21 base pairs (B) TYPE: nucleotide (CI NUMBER OF STRANDS: simple (D) CONFIGURATION: linear (ü) TYPE OF MOLECULE: cDNA
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3:

(2) INFORMATION FOR SEQ ID NO: A:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 29 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ü) 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 STRANDS: single (D) CONFIGURATION: linear (ü) TYPE OF MOLECULE: cDNA
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID N0: 5:
TTGGTGTGGT GGGTGGGTT lg (2) INFORMATION FOR SEQ ID NO: 6:
(i) FEATURES OF THE SEQUENCE:
(A) LENGTH: 19 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear SUBSTITUTE SHEET (GOVERNMENT 26) WO 99! 49067 PCT / FR99 / 00643 (ii) TYPE OF MOLECULE: cDNA
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6:

(2) INFORMATION FOR SEQ ID NO: 7:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 21 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: cDNA
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7:

(2) INFORMATION FOR SEQ ID NO: 8:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 13 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: cDNA
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8:

(2) INFORMATION FOR SEQ ID NO: 9:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 14 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear ü) TYPE OF MOLECULE: cDNA
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 9:

SUBSTITUTE SHEET (SET 26) (2) INFORMATION FOR SEQ ID N0: 10:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 17 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ü) TYPE OF MOLECULE: cDNA
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 10:

(2) INFORMATION FOR SEQ ID NO: 11:
(1) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 7 amino acids (B) TYPE: amino acid (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 11:
Pro Lys Lys Lys Arg Lys Val (2) INFORMATION FOR SEQ ID NO: 12:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 19 amino acids (B) TYPE: amino acid (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ü) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 12:
Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys Leu Asp Lys (2) INFORMATION FOR SEQ ID N0: 13:
SUBSTITUTE SHEET (SET 26) (i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 38 amino acids (B1 TYPE: amino acid (C) NUMBER OF STRANDS: single (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 Azg Ser Ser Gly Pro Tyr Gly Gly Gly Gly Gln Tyr Phe Ala Lys Pro Arg Asn Gln Gly Gly Tyr (2) INFORMATION FOR SEQ ID N0: 14:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 7 amino acids fB) TYPE: amino acid (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 14:
Pro Lys Asn Lys Arg Lys Val (2) INFORMATION FOR SEQ ID N0: 15:
fi) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 13 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: cDNA
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID N0: 15:

SUBSTITUTE SHEET (GOVERNMENT 26)

Claims (42)

39 1. Nucleic acid transfer vector characterized in that it comprises a double-stranded DNA molecule and at least one signal-coupled oligonucleotide of targeting and capable of forming a triple helix with a specific sequence present on said double-stranded DNA molecule. 2. Nucleic acid transfer vector according to claim 1 characterized in that whether 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 what the double stranded DNA molecule includes an expression cassette incorporated of one or more genes of interest under the control of one or more promoters and a active transcriptional terminator in mammalian cells. 5. Nucleic acid transfer vector according to claim 4 characterized in that that the gene of interest is a nucleic acid encoding a product therapeutic. 6. Nucleic acid transfer vector according to one of claims 1 to characterized in that the specific sequence present on the DNA molecule double strand is a homopurine-homopyrimidine sequence. 7. Nucleic acid transfer vector according to one of claims 1 to characterized in that the specific sequence present on the DNA molecule double strand is a sequence naturally present on double-stranded DNA or a sequence synthetic or natural artificially introduced into the double-stranded DNA. 8. Nucleic acid transfer vector according to one of claims 1 to characterized in that the oligonucleotide comprises a poly-CTT sequence and the specific sequence present on the double-stranded DNA molecule is a sequence poly-GAA. 9. Nucleic acid transfer vector according to one of claims 1 to 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 1 at 7 characterized in that the specific sequence present on the DNA molecule double strand comprises the sequence 5'-AAGGGAGGGAGGAGGAGAA-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 1 at 7 characterized in that the specific sequence present on the DNA molecule double strand comprises all or part of the sequence 5'-CTTCCCGAAGGGAGAAAGG-3' (SEQ ID NO 6) included in the CoIE 1 origin of replication of E. coli, and the oligonucleotide has the sequence 5'-GAAGGGTTCTTCCCTCTTTCC-3' (SEQ
ID NO 7). 12. Nucleic acid transfer vector according to one of claims 1 at 7 characterized in that the specific sequence present on the DNA molecule double strand comprises the sequence 5'-GAAAAAGGAAGAG-3' (SEQ ID NO 8) or the sequence 5'-AAAAAAGGGAATAAGGG-3' (SEQ ID NO 10) included in the gene .beta.-lactamase from plasmid pBR322 and E. Coli respectively. 13. Nucleic acid transfer vector according to one of claims 1 at 7 characterized in that the specific sequence present on the DNA molecule double strand comprises the sequence 5'-AAGAAAAAAAAGAA-3' (SEQ ID no 9) present in the .gamma origin of replication. origin of replication plasmids conditional such that pCOR. 14. Nucleic acid transfer vector according to one of claims 1 at 13 characterized in that the oligonucleotide comprises at least 3 bases. 15. Nucleic acid transfer vector according to claim 14 characterized in that that the oligonucleotide comprises 5 to 30 bases. 16. Nucleic acid transfer vector according to one of claims 1 at 15 characterized in that the oligonucleotide has at least one modification chemical the making it resistant to, or protected against nucleases, or increasing its affinity against the specific sequence present on the double DNA molecule strand. 17. Nucleic acid transfer vector according to one of claims 1 at 16 characterized in that the oligonucleotide is a chain of nucleosides having suffered skeletal modification. 18. Nucleic acid transfer vector according to one of claims 1 at 16 characterized in that the oligonucleotide is coupled to an alkylating agent forming a covalent bond at the bases of double-stranded DNA. 19. Nucleic acid transfer vector according to claim 18 characterized in that that said alkylating agent is photoactivatable. 20. Nucleic acid transfer vector according to claims 18 and 19 characterized in that said alkylating agent is a psoralen. 21. Nucleic acid transfer vector according to claims 1 to 20 characterized in that the targeting signal interacts with a component of the matrix extracellular, a plasma membrane receptor, targets an intracellular compartment, and or improves intracellular traffic of double-stranded DNA. 22. Nucleic acid transfer vector according to claim 21 characterized in that that said targeting signal comprises growth factors (EGF, PDGF, TGFb, NGF, IGF I, FGF), cytokines (IL-1, IL-2, TNF, Interferon, CSF), hormone (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 motif recognized by integrins, by example the RGD peptide, or by other extrinsic cell membrane proteins. 23. Nucleic acid transfer vector according to claim 21 characterized in that that the targeting signal is an intracellular targeting signal, such as a sequence Nuclear Addressing System (NLS). 24. Nucleic acid transfer vector according to claim 23 characterized in that said nuclear targeting signal is the NLS sequence of the T antigen of SV40. 25. Nucleic acid transfer vector according to claim 21 characterized in that the targeting signal allows both extracellular targeting and the targeting intracellular. 26. Nucleic acid transfer vector according to claims 1 to 25 characterized in that the coupling of the targeting signal to the oligonucleotide is obtained by synthesis in solid phase or in solution, in particular by the establishment of bonds disulfide, thioether, ester, amide, or amine. 27. Composition characterized in that it contains at least one vector such than defined in one of claims 1 to 26. 28. Composition according to claim 27, characterized in that it contains in addition one or more transfecting agents. 29. Composition according to claim 28, characterized in that the agent transfecting is a cationic lipid, a lipopolyamine, or a cationic polymer. 30. Composition according to claims 27 to 29, characterized in that it contains in in addition to one or more adjuvant(s) capable of associating with the vector complexes such as defined in one of claims 1 to 25/transfecting agent. 31. Composition according to claim 30, characterized in that the adjuvant is a or several neutral lipids chosen from synthetic or natural lipids, zwitterionic or devoid of ionic charge under the conditions physiological. 32. Composition according to one of Claims 30 or 31, characterized in that the OR
the neutral lipids are chosen from lipids with two fatty chains. 33. Composition according to claims 30 to 32, characterized in that the or them neutral lipids are chosen from dioleoylphosphatidylethanolamine (DOPE), oleylpalmitoylphosphatidylethanolamine (POPE), di-stearoyl, -palmitoyl, -mirystoyl phosphatidylethanolamine and their N-methylated derivatives 1 to 3 times, phosphatidylglycerols, diacylglycerols, glycosyldiacylglycerols, cerebrosides (such as in particular galactocerebrosides), sphingolipids (such as notably sphingomyelins), and asialogangliosides (such as in particular asialoGM1 and GM2). 34. Composition according to claim 30, characterized in that the adjuvant is where includes a compound involved in DNA condensation. 35. Composition according to Claim 34, characterized in that the said compound derivative in whole or in part of a histone, a nucleolin, and/or a protamine, west consisting in whole or in part of peptide units (KTPKKAKKKP) and/or (ATPAKKAA) repeated continuously or not, the number of patterns can vary between 2 and 10. 36. Composition according to any one of claims 27 to 35 characterized in this that it comprises a pharmaceutically acceptable carrier for a formulation injectable. 37. Composition according to any one of claims 27 to 35 characterized in this that it comprises a pharmaceutically acceptable carrier for a application on skin and/or mucous membranes. 38. Use of nucleic acid transfer vector as defined in one of claims 1 to 26 for the manufacture of a medicament intended to treat them diseases. 39. Method for Transfection of Nucleic Acids into Cells Characterized in this that it includes the following steps:
(1) synthesis of the oligonucleotide-targeting signal chimera, (2) bringing the chimera synthesized in (1) into contact with a double-stranded DNA
to form triple helices, (3) optionally, complexation of the vector obtained in (2) with one or several transfection agents and/or one or more adjuvant(s), and (4) bringing the cells into contact with the complex formed in (2) or the case applicable in (3). 40. Method of treating diseases by administering a vector of transfer of nucleic acids as defined in one of claims 1 to 26 containing a Double-stranded DNA capable of correcting said disease. 41. Recombinant Cell Containing Nucleic Acid Transfer Vector such as defined in one of claims 1 to 26. 42. Recombinant cell according to claim 41 characterized in that it it is a eukaryotic cell.