AU758406B2 - Vectors for transferring nucleic acids, compositions containing them and their uses - Google Patents

Description

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION NAME OF APPLICANT(S): Aventis Pharma S.A.

4 ADDRESS FOR SERVICE: DAVIES COLLISON CAVE Patent Attorneys 1 Little Collins Street, Melbourne, 3000.

INVENTION TITLE: Vectors for transferring nucleic acids, compositions containing them and their uses The following statement is a full description of this invention, including the best method of performing it known to me/us:a. a a The present invention relates to new vectors and to their use for transferring nucleic acids. More particularly, the invention relates to new vectors capable of directing nucleic acids towards cells or specific cellular compartments.

The transfer of nucleic acids is a technique which forms the basis of all the major applications of 10 biotechnology, and increasing the efficiency of transferring nucleic acids constitutes a very important challenge for the development of these applications.

The efficiency of transferring nucleic acids depends on numerous factors, among which are the capacity of the nucleic acids to to cross the plasma membrane and their capacity to be transported within the cell up to the e* nucleus.

One of the major obstacles to the efficiency of transferring nucleic acids stems from the fact that the genetic information is often poorly or not directed towards the target organ for which it is intended.

Moreover, once the nucleic acid has penetrated into the target cell, it must still be directed towards the nucleus so as to be expressed there. Furthermore, in the case of the transfer of nucleic acids into differentiated or quiescent cells, the nucleus is delimited by a nuclear envelope which constitutes an additional barrier to the passage of these nucleic acids.

The recombinant viruses used as vectors possess sophisticated and efficient mechanisms for guiding the nucleic acids up to the nucleus. However, viral vectors have certain disadvantages inherent to their viral nature, which unfortunately cannot be completed excluded. Another strategy consists either in transfecting naked DNA, or in using nonviral agents capable of promoting the transfer of DNA into 10 eukaryotic cells. However, the nonviral vectors do not possess subcellular or nuclear targeting signals. Thus the passage of naked DNA, or in combination with its nonviral agent, from the cytoplasm to the nucleus is, for example, 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 in an antisense-oligonucleotidetargeting strategy [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 expression of endogenous genes.

Transfection vectors comprising a synthetic polypeptide coupled by electrostatic interactions to a DNA sequence have also been described in patent application WO 95/31557, the said polypeptide consisting of a polymeric chain of basic amino acids, of an NLS peptide and of a hinge region which connects the NLS peptide to the polymeric chain and makes it possible to avoid steric interactions. However, this type of construct poses a problem of stability because the interactions called into play between the DNA and the targeting signal are of an electrostatic nature.

o. Moreover, nucleic acid-specific targeting peptide chimeras exist which are described in patent application WO 95/34664, the binding between the two S•being of a chemical nature. However, this method involves in particular enzymatic steps which are ••go difficult to control and which do not make it possible to produce large quantities of nucleic acids.

Finally, it has been shown that it is gee• possible to attach an NLS ("Nuclear Localization Signal") sequence to a plasmid DNA via a cyclopropapyrroloindole (Nature Biotechnology, Volume 16, pp. 80-85, January 1998). However, a complete inhibition of the transcription of the gene of interest because of the random attachment of several hundreds of NLS sequences onto the plasmid was observed. One solution proposed by the authors to remedy this consists in linking the NLS sequences onto linear fragments of DNA and then in coupling these modified fragments with other nonmodified fragments.

However, this technique, like the preceding one, has 4 the disadvantage of involving at least one enzymatic step.

Thus, all the methods proposed up until now do not make it possible to solve satisfactorily the difficulties linked to the targeting of double-stranded DNAs.

The present invention provides an advantageous solution to these problems. More particularly, the present invention uses oligonucleotides which are conjugated with targeting signals and which are capable of forming triple helices with one or more specific sequences present on a *.double-stranded DNA molecule.

o Such a vector has the advantage of being able to direct a double-stranded DNA towards cells or specific cellular compartments by means of the oooo targeting signal, without genetic expression being S"inhibited. The applicant has indeed shown that, by virtue of the formation of stable site-specific triple helices, it is now possible to link a targeting signal to a double-stranded DNA in a site-specific manner.

Consequently, it is possible to attach the targeting signal outside the expression cassette for the gene to be transferred. The applicant has thus shown that genetic expression in the cell is not inhibited in spite of the chemical modification qf the DNA.

Furthermore, the presence of a triple helix as a means for binding the targeting signal to the DNA is particularly advantageous because it makes it possible to preserve a DNA size which is appropriate for the transfection.

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

10 Another advantage of the invention is to make it possible to couple the DNA to be transferred to targeting signals whose number and nature are both controlled. Indeed, it is possible to control the number of targeting signals linked to each doublestranded DNA molecule by introducing a suitable number of specific sequences appropriate for the formation of a.o triple helices into the said double-stranded DNA .o molecule. Likewise, it is possible to introduce into the same double-stranded DNA molecule several oligonucleotides linked to different targeting signals (intracellular and/or extracellular), and in this case it is also possible to determine beforehand the respective proportions thereof. In addition, these various targeting signals may be attached to the double-stranded DNA molecule in a manner stable to a greater or lesser degree depending on whether the triple helix is formed with or without a covalent bond (that is to say with or without the use of an alkylating agent).

Finally, the functionalized triple helix obtained results only from chemical conversion steps and may therefore be obtained in a simple manner, reproducibly and in very large, in particular industrial, quantities.

A first subject of the invention therefore relates to a vector useful in transfection, which is S. :capable of targeting a cell and/or a specific cellular compartment. Accordingly, the invention provides a vector for transferring nucleic acids, characterized in that it comprises a double-stranded DNA molecule and at least one oligonucleotide which is coupled to a targeting signal forming a triple helix with a specific sequence present on said double-stranded DNA molecule.

For the purposes of the invention, "doublestranded DNA" is understood to mean a double-stranded deoxyribonucleic acid which may be of human, animal, .i plant, bacterial or viral origin, and the like. It may be obtained by any technique known to persons skilled in the art, and in particular by screening a library, by chemical or enzymatic synthesis of sequences obtained by screening libraries. It may be chemically or enzymatically modified.

This double-stranded DNA may 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 form and in a supercoiled conformation.

The double-stranded DNA may also carry a replication origin which is functional or otherwise in the target cell, one or more marker genes, sequences for regulating transcription or replication, genes of therapeutic interest, anti-sense sequences which are modified or otherwise, regions for binding to other cellular components, and the like. Preferably, the double-stranded DNA comprises an expression cassette eoo 10 consisting of one or more genes of interest under the control of one or more promoters and of a transcriptional terminator which are active in the target cells.

For the purposes of the invention, "expression cassette for a gene of interest" is understood to mean a DNA fragment which may be inserted into a vector at specific restriction sites. The DNA fragment comprises a nucleic acid sequence encoding an RNA or a polypeptide of interest and comprises, in addition, the sequences necessary for the expression (enhancer(s), promoter(s), polyadenylation sequences and the like) of the said sequence. The cassette and the restriction sites are designed to ensure insertion of the expression cassette into an open reading frame appropriate for transcription and translation.

It is generally a plasmid or an episome carrying one or more genes of therapeutic interest. By way of example, there may cited the plasmids described 8 in Patent Applications WO 96/26270 and WO 97/10343 which are incorporated into the present by reference.

For the purposes of the invention, gene of therapeutic interest is understood to mean in particular any gene encoding a protein product having a therapeutic effect. The therapeutic product thus encoded may be in particular a protein or peptide. This protein product may be homologous in relation to the target cell (that is to say a product which is normally expressed in the target cell when the latter has no apathological condition). In this case, the expression of a protein makes it possible, for example, to palliate an insufficient expression in the cell or the expression of a protein which is inactive or weakly 15 active because of a modification, or to overexpress the said protein. The gene of therapeutic interest may also a encode a mutant of a cellular protein, having increased stability, a modified activity, and the like. The protein product may also be heterologous in relation to the target cell. In this case, an expressed protein may, for example, supplement or provide an activity which is deficient in the cell, allowing it to combat a pathological condition, or to stimulate an immune response.

Among the therapeutic products for the purposes of the present invention, tlhere may be mentioned more particularly enzymes, blood derivatives, hormones, lymphokines [interleukins, interferons, TNF,

I

9 and the like (FR 92/03120)], growth factors, neurotransmitters or their precursors or synthesis enzymes, trophic factors [BDNF, CNTF, NGF, IGF, GMF, aFGF, pFGF, NT3, NT5, HARP/pleiotrophin, and the like, dystrophin or a minidystrophin (FR 91/11947)], the CFTR protein associated with cystic fibrosis, tumour suppressor genes [p53, Rb, RaplA, DCC, k-rev, and the like (FR 93/04745)], the genes encoding factors involved in coagulation [factors VII, VIII, IX], the genes involved in DNA repair, suicide genes [thymidine kinase, cytosine deaminase], the genes for haemoglobin or other protein carriers, the genes corresponding to the proteins involved in the metabolism of lipids, of the apolipoprotein type chosen from apolipoproteins 15 A-l, A-II, A-IV, B, C-I, C-II, C-III, D, E, F, G, H, J and apo(a), metabolic enzymes such as, for example, lipoprotein lipase, hepatic lipase, lecithin cholesterol acyl transferase, 7-alpha cholesterol hydroxylase, phosphatidic acid phosphatase, or lipid transfer proteins such as cholesterol ester transfer protein and phospholipid transfer protein, an HDLbinding protein or a receptor chosen, for example, from the LDL receptors, the remnant chylomicron receptors and the scavenger receptors, and the like.

The DNA of therapeutic interest may also be a gene or an anti-sense sequence, whose expression in the target cell makes it possible to control the expression of genes or the transcription of cellular mRNAs. Such i!" sequences can, for example, be transcribed in the target cell into RNAs which are complementary to cellular mRNAs and thus block their transcription to protein, according to the technique described in Patent EP 140 308. The genes of therapeutic interest also comprise the sequences encoding ribozymes, which are capable of selectively destroying target RNAs (EP 321 201), or the sequences encoding single-chain intracellular antibodies such as, for example, ScFv.

As indicated above, the deoxyribonucleic acid may also comprise one or more genes encoding an antigenic peptide, which is capable of generating an immune response in humans or in animals. In this specific embodiment, the invention therefore allows the 15 production of vaccines or the carrying out of immunotherapeutic treatments applied to humans or to animals, in particular against microorganisms, viruses .oe.ei or cancers. They may be in particular antigenic peptides specific for the Epstein-Barr virus, the HIV virus, the hepatitis B virus (EP 185 573), the pseudorabies virus, the syncitia forming virus, the influenza virus, the cytomegalovirus (CMV), other viruses, or specific for tumours (EP 259 212) 0L- Preferably, the deoxyribonucleic acid also comprises sequences allowing the expression of the gene of therapeutic interest and/or the qene encoding the antigenic peptide in the desired cell or organ. They may be sequences which are naturally responsible for the expression of the gene considered when the sequences are capable of functioning in the infected cell. They may also be sequences of plant 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 which it is desired to infect. Likewise, they may be promoter sequences derived from the genome 10 of the virus. In this regard, there may be mentioned, for example, the promoters of the E1A, MLP, CNV and RSV genes, and the like. In addition, these expression sequences may be modified by the addition of activating or regulatory sequences, and the like. The promoter may 15 also be inducible or repressible.

A triple helix corresponds to the attachment of an oligonucleotide, modified or otherwise, to the double-stranded DNA by so-called "Hoogsteen" hydrogen bonds between the bases of the third strand and those of the region forming the double helix. These pairings occur in the large spiral of the double helix and are specific for the sequence considered [Frank-Kamenetski, Triplex DNA Structures, Ann. Rev. Biochem., 1995, 64, pp 65-95]. The specific sequence in the form of a double helix may be in particular a homopurinehomopyrimidine sequence. Two categories of triple helices can be distinguished according to the nature of the bases of the third strand [Sun, J. and C. H616ne, Oligonucleotide-directed triple-helix formation, Curr.

Opin. Struct. Biol., 1993, 3, pp. 345-356]: the purine bases make it possible to obtain C-G*G and T-A*A pairings, and the pyrimidine bases make it possible to obtain C-G*G and T-A*T pairings (the symbol corresponds to a pairing with the third strand).

These structures have been characterized from S. the physicochemical point of view by means of numerous NMR (Nuclear Magnetic Resonance), hybridization 10 temperature or nuclease protection studies, which makes it possible to define their properties and the conditions for their stability. For the triple helices with the third purine strand, this strand is antiparallel relative to the purine strand of the DNA 15 and the formation of the triple helix highly depends on the concentration of divalent ions: ions such as Mg 2 *o stabilize the structure formed with the third strand.

For the triple helices with a third homopyrimidine strand, the latter is parallel relative to the purine strand, and the formation of the triple helix is dependent on the pH: an acidic pH of less than six allows protonation of the cytosines and the formation of an additional hydrogen bond stabilizing the triplet C-G C "Mixed" triple helices also exist for which the third strand carries purine and pyrimidine bases. In this case, the orientation of this third strand depends on the base sequence of the homopurine region.

The oligonucleotides used in the present invention are oligonucleotides which hybridize directly with the double-stranded DNA. These oligonucleotides may contain the following bases: thymidine which is capable of forming triplets with the doublets A.T of the double-stranded DNA (Rajagopal et al., Biochem 28 (1989) 7859); adenine which is capable of forming triplets with the doublets A.T of the double-stranded

DNA;

10 guanine which is capable of forming triplets with the doublets G.C of the double-stranded

DNA;

protonated cytosine which is capable of forming triplets with the doublets G.C of the S: 15 double-stranded DNA (Rajagopal et al., cited above); uracil which is capable of forming triplets with the base pairs A.U or A.T.

STo 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, in order to obtain the best attachment and the best selectivity, an oligonucleotide and a specific sequence which are perfectly complementary are used for the vector according to the invention. This may be in particular an oligonucleotide poly-CTT and a specific sequence poly-GAA. By way of example, there may be mentioned the oligonucleotide having the sequence: 14 5'-GAGGCTTTTCTTCTTCTTCTTCCTT-3' (GAGG(CTT)7, SEQ ID No.1), in which the bases GAGG do not form triple helices but make it possible to separate the oligonucleotide from the coupling arm. The sequence

(CTT)

7 (SEQ ID No.2) may also be mentioned. These oligonucleotides are capable of forming a triple helix with a specific sequence comprising complementary units (GAA). These may be in particular a region comprising 7, 14 or 17 GAA units. Another specific sequence of 10 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 In this case, the oligonucleotide binds in an orientation which is antiparallel to the polypurine strand. These triple helices are unstable in the o* 2+ presence of Mg as is mentioned above (Vasquez et al., Biochemistry, 1995, 34, 7243-7251; Beal and Dervan, Science, 1991, 251, 1360-1363).

The specific sequence may be a sequence which is naturally present on the double-stranded DNA, or a synthetic sequence or a sequence of natural origin artificially introduced into it. It is particularly advantageous to use an oligonucleotide capable of forming a triple helix with a sequence which is naturally present on the 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 and pSV type, and the like. Among the natural homopurine-homopyrimidine sequences present in the double-stranded DNA, there may be mentioned a sequence comprising all or part of the sequence 5'-CTTCCCGAAGGGAGAAAGG-3' (SEQ ID No.6) present in the replication origin ColEl of E. coli. In this case, the oligonucleotide forming the triple helix has the sequence: 5'-GAAGGGTTCTTCCCTCTTTCC-3' (SEQ ID No.7) and 10 binds alternately to the two strands of the double i* helix, as described by Beal and Dervan Am. Chem.

Soc. 1992, 114, 4976-4982) and Jayasena and Johnston (Nucleic Acids Res. 1992, 20, 5279-5288). There may also be mentioned the sequence 5'-GAAAAAGGAAGAG-3' (SEQ ID No.8) of the p-lactamase gene of the plasmid pBR322 (Duval-Valentin et al., Proc. Natl. Acad. Sci. USA, 1922, 89, 504-508). Another sequence is AAGAAAAAAAAGAA (SEQ ID No. 9) present in the replication origin y of the plasmids with a conditional replication origin such as pCOR.

Although perfectly complementary sequences are preferred, it is understood, however, that certain mismatches may be tolerated between the sequence of the oligonucleotide and the sequence present on the DNA, as long as they do not lead to an excessive loss of affinity. There may be mentioned the sequence 5'-AAAAAAGGGAATAAGGG-3' (SEQ ID No. 10) present in the E. coli P-lactamase gene. In this case, the thymine i n t errupting the polypurine sequence may be recognized by a guanine of the third strand, thus forming a triplet ATG which is stable when it is surrounded by two TAT triplets (Kiessling et al., Biochemistry, 1992, 31, 2829-2834).

The oligonucleotide used may be natural (composed of natural bases which are unmodified or chemically modified). In particular, the oligonucleotide may advantageously exhibit certain chemical modifications which make it possible to S: increase its resistance or its protection in relation to nucleases, or its affinity in relation to the *o.

specific sequence or which also make it possible to provide other additional properties Goodchild, Conjugates of Oligonucleotides and Modified Oligonucleotides: A Review of their Synthesis and Properties, Bioconjugate Chemistry, Vol. 1 No. 3, 1990, pp. 165-187).

According to the present invention, oligonucleotide is also understood to mean any succession of nucleosides having undergone a modification of the backbone. Among the possible modifications, there may be mentioned oligonucjeotide phosphorothioates which are capable of forming triple helices with DNA (Xodo et al., Nucleic Acids Res., 1994, 22, 3322-3330), likewise the oligonucleotides possessing formacetal or methylphosphonate backbones (Matteucci et al., J. Am. Chem. Soc., 1991, 113, 7766- 7768). It is also possible to use the oligonucleotides synthesized with a-anomers of nucleotides, which also form triple helices with DNA (Le Doan et al., Nucleic Acids Res., 1987, 15, 7749-7760). Another modification of the backbone is the phosphoramidate bond. There may be mentioned, for example, the internucleotide bond phosphoramidate described by Gryaznov and Chen, which gives oligonucleotides forming particularly stable triple helices with DNA Am. Chem. Soc., 10 1994, 116, 3143-3144). Among the other modifications of :'the backbone, it is also possible to mention the use of ribonucleotides, of 2'-O-methylribose, of phosphotriester, and the like (Sun and H616ne, Curr.

Opinion Struct. Biol., 116, 3143-3144). The phosphoruscontaining backbone can finally be replaced with a polyamide backbone as in the PNAs (Peptide Nucleic •Acid), which can also form triple helices (Nielson et al., Science, 1991, 254, 1497-1500; Kim et al., J. Am Chem. Soc., 1993, 115, 6477-6481) or with a guanidinebased backbone, as in the DNG (deoxyribonucleic guanidine, Proc. Natl. Acad. Sci. USA, 1995, 92, 6097- 6101), polycationic analogues of DNA, which also form triple helices.

The thymine of the third strand may also be replaced with a 5-bromouracil, which increases the affinity of the oligonucleotide for DNA (Povsic and Dervan, L. Am. Chem. Soc., 1989, 111, 3059-3061). The third strand may also contain non natural bases among 18 which there may be mentioned 7-deaza-2' -deoxiyxanthosine (Milligan et al., Nucleic Acids Res., 1993, 21, 327- 333), 1-(2-deoxy-p-D-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, 2aminopurine, 2'-O-methylpseudoisocytidine, or any other modification known to persons skilled in the art (for a review see Sun and H6lene, Curr. Opinion Struct. Biol.,

S*

1993, 3, 345-356).

10 The object of another type of modification of the oligonucleotide is more particularly to improve the interaction and/or the affinity between the oligonucleotide and the specific sequence. In particular, a completely advantageous modification t. 15 consists in coupling an alkylating agent to the oligonucleotide. The binding may take place either chemically or photochemically by means of a photoreactive functional group. Advantageous alkylating agents are in particular photoactivable alkylating agents, for example psoralens. Under the action of light, they form covalent bonds at the level of the pyrimidine bases of the DNA. When these molecules are intercalated at the level of the 5'-ApT-3' or 5'-TpA-3' sequences in a double-stranded DNA fragment, they form bonds with both strands. This light-induced binding reaction can occur at a specific site of the plasmid.

As has been highlighted previously, one advantage of the present invention is therefore the possibility of forming very stable and site-specific triple helices between the oligonucleotide and a specific sequence of the double-stranded DNA by means of a covalent bond formed via an alkylating agent.

The length of the oligonucleotide used in the method of the invention is at least 3 bases, and preferably between 5 and 30 bases. An oligonucleotide having a length of between 10 and 30 bases is advantageously used. The length may of course be 10 adapted on a case-by-case basis by persons skilled in the art according to the desired selectivity and stability of the interaction.

The oligonucleotides according to the invention may be synthesized by any known technique. In particular, they may be prepared by means of nucleic acid synthesizers. Any other method known to persons •skilled in the art may quite obviously be used.

For the purposes of the invention, "targeting signal" is understood to mean targeting molecules of a varied nature. It represents in most cases peptides known for targeting. They may be used to interact with a component of the extracellular matrix, a plasma membrane receptor, in order to target an intracellular compartment or in order to improve the intracellular flow of DNA, during the nonviral transfer of genes in gene therapy.

These targeting signals may comprise, for example, growth factors (EGF, PDGF, TGFp, NGF, IGF, I, FGF), cytokines (TT-1 IL-2, TNIF, Interferon, CSF) hormones (insulin, growth hormone, prolactin, glucagon, thyroid hormone, steroid hormones), sugars which recognize lectins, immunoglobulins, ScFv's, transferrin, lipoproteins, vitamins such as vitamin B12, peptide or neuropeptide hormones (tachykinins, neurotensin, VIP, endothelin, CGRP, CCK, and the like), or any unit recognized by the integrins, for example the peptide RGD, or by other extrinsic proteins of the 10 cell membrane.

It is also possible to use whole proteins, or peptide sequences derived from these proteins, or alternatively peptides which bind to their receptor and which are obtained by the "phage display" technique or by combinatory synthesis.

Intercellular targeting signals may also be S* envisaged. Many nuclear homing sequences (NLS) of varied amino acid compositions have been identified and make it possible to get different proteins involved in the nuclear transport of proteins or of nucleic acids.

Among these are in particular short sequences (the NLS of the SV40 T antigen (PKKKRKV, SEQ ID No. 11) is one example), bipartite sequences (the NLS of nucleoplasmin which contains two essential domains for nuclear transport: KRPAATKKAGQAKKKKLDK, SEQ ID No. 12), or the M9 sequence (NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY, SEQ ID No. 13) of the hnRNPA1 protein. The proteins carrying these NLS sequences bind to specific receptors, such as the receptors of the importin or karyopherin family, for example. The role of these sequences is to direct the DNA inside the nucleus where it is then immediately available for the transcription machinery, and may be expressed.

The "mixed" targeting signals, that is to say Swhich can serve both for intracellular and extracellular targeting, also come within the scope of the present invention. It is possible to mention, for 10 example, the sugars which target lectins which are present on the cell membrane but also at the level of the nuclear pores. Targeting by these sugars therefore also relates both to extracellular targeting and nuclear import.

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

Advantageously, the targeting signals according to the invention make it possible to direct the double-stranded DNA specifically towards certain cells or certain cellular compartments. By way of example, the targeting signals according to the invention can target receptors or ligands at the cell surface, in particular the receptors for insulin, for transferin, for folic acid, or anyther growth factor, cytokines, or vitamins, or particular polysaccharides at the surface of the cell or on the neighbouring extracellular matrix.

The synthesis of oligonucleotide-targeting signal chimaeras occurs on a solid phase or in solution and takes into account the very different stability properties of the oligonucleotides and targeting signals (Erijita, R. et al., Synthesis of defined 10 peptide-oligonucleotide hybrids containing a nuclear transport signal sequence, Tetrahedron, 1991, 47(24), coo pp. 4113-4120]. In solution, it is possible to envisage couplings in one step: the targeting signal may, for example, be synthesized with a group carrying disulphide, maleimide, amine, carboxyl, ester, epoxide, cyanogen bromide or aldehyde functional groups, and may be coupled to an oligonucleotide modified by a thiol, amine or carboxyl terminal group at the 3' or position. These couplings are made by establishing disulphide, thioether, ester, amide or amine bonds between the oligonucleotide and the targeting signal.

Any other method known to persons skilled in the art may be used, such as bifunctional coupling reagents, for example.

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

Advantageously, the vectors according to the invention may also be combined with one or more agents known for transfecting DNA. There may be mentioned by way of example the cationic lipids which possess advantageous properties. These vectors actually consist of a cationic polar part which interacts with the DNA, and a hydrophobic lipid part which promotes cellular penetration and renders the ionic interaction with DNA insensitive to the external medium. Specific examples of cationic lipids are in particular the monocationic lipids (DOTMA: Lipofectin®), some cationic detergents (DDAB), lipopolyamines and in particular dioctadecylamidoglycyl spermine (DOGS) or 5-carboxyspermylamide of palmitoylphosphatidylethanolamine (DPPES), whose preparation has been described, for example, in patent application EP 394 111. Another advantageous lipopolyamine family is S. represented by the compounds described in patent S• application WO 97/18185 incorporated into the present by way of reference. Numerous other cationic lipids have been developed and may be used with the vectors according to the invention.

Among the synthetic transfection agents developed, the cationic polymers of the polylysine and the DEAE-dextran type are also advantageous. It is also possible to use the polyethylenimine (PEI) and polypropylenimine (PPI) polymers which are commercially available and may be prepared according to the method described in patent application WO 96/02655.

In general, any synthetic agent known to transfect nucleic acid may be combined with the vectors according to the invention.

The compositions may, in addition, comprise adjuvants capable of combining with the vector according to the invention/transfection agent complexes and of improving the transfecting power thereof. 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 combining with the vector according to the invention/transfection agent(s) complexes and of improving the transfecting power thereof. The presence of this type of adjuvant (lipids, peptides or proteins, for example) can advantageously ee o eao 15 make it possible to increase the transfecting power of .i the compounds.

S•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 indeed shown that the addition of a neutral lipid makes it possible to enhance the formation of nucleolipid particles and to promote the penetration of the particle into the cell by destabilizing its membrane.

More preferably, the neutral lipids used within the framework of the present invention are lipids containing 2 fatty chains. In a particularly advantageous manner, natural or synthetic lipids which are zwitterionic or lacking ionic charge under physiological conditions are used. They may be chosen more particularly from dioleoylphosphatidylethanolamine (DOPE), oleoylpalmitoylphosphatidylethanolamine (POPE), di-stearoyl, -palmitoyl, -mirystoyl phosphatidylethanolamines as well as their derivatives which are N-methylated 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 asialoGM1 and GM2).

These different lipids may be obtained either by synthesis or by extraction from organs (for example the brain) or from eggs, by conventional techniques well known to persons skilled in the art. In oeo particular, the extraction of the neutral lipids may be carried out by means of organic solvents (see also Lehninger, Biochemistry).

The applicant has demonstrated that it was also particularly advantageous to use, as adjuvant, a compound directly involved or otherwise in the condensation of the DNA (WO 96/25508). The presence of such a compound in the composition according to the invention makes it possible to reduce the quantity of transfecting compound, with the beneficial consequences resulting therefrom from the toxicological point of view, without any damaging effect on the transfecting activity. Comnnond involvedm in the condensation of the nucleic acid is intended to define a compound which compacts, directly or otherwise, the DNA. More precisely, this compound may either act directly at the level of the DNA to be transfected, or may be involved at the level of an additional compound which is directly involved in the condensation of this nucleic acid. Preferably, it acts directly at the level of the DNA. In particular, this agent which is involved in the 0 condensation of the DNA may be any polycation, for example polylysine. According to a preferred embodiment, this agent may also be any compound which is derived, as a whole or in part, from a histone, a nucleolin, a protamine and/or from one of their derivatives, as a whole or in part, of peptide units (KTPKKAKKP) and/or (ATPAKKAA), it being possible for the number of units to vary between 2 and 10. In the structure of the compound according to the invention, these units may be repeated continuously or otherwise.

They may thus be separated by linkages of a biochemical nature, for example one or more amino acids, or of a chemical nature.

The subject of the invention is also the use of the vectors as defined above for manufacturing a medicament intended to treat diseases by transfection of DNA into primary cells or into established cell lines. They may be fibroblast cells, muscle cells, nerve cells (neurons, astrocytes, glial cells), hepatic cells, haematopoietic cell line (lymphocytes, CD34, dendritic cells, and the like), epithelial cells and the like, in differentiated or pluripotent form (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 polypeptides.

For usage in vivo, either in therapy or for 10 studying the regulation of the genes or the creation of animal models of pathological conditions, the compositions according to the invention can be formulated for administration by the topical, cutaneous, oral, rectal, vaginal, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal, intratracheal or intraperitoneal route, and the like.

Preferably, the compositions of the invention contain a vehicle which is pharmaceutically acceptable for an injectable formulation, in particular a direct injection into the desired organ, or for administration by the topical route (on the skin and/or the mucous membrane). They may be in particular isotonic sterile solutions, or dry, in particular freeze-dried, compositions which, upon addition, depending on the case, of sterilized water or of physiological saline, 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, in particular according to the mode of administration used, the relevant pathological condition, the gene to be expressed, or the desired duration of treatment. As regards more particularly the mode of administration, it may be either a direct injection into the tissues or the circulatory system, or a treatment of cells in culture followed by their reimplantation in vivo by injection or transplantation.

*o The invention relates, in addition, to a 10 method of transfection of DNA into cells comprising the following steps: synthesis of the oligonucleotide-targeting signal chimera according to the method described above, bringing the chimera synthesized in into contact with a double-stranded DNA so as to form triple helices, optionally, complexing the vector obtained in with one or more transfection agents and/or one more adjuvants, and bringing the cells into contact with the complex formed in or, if applicable, in (3) The cells may be brought into contact with the complex by incubating the cells with the said complex (for uses in vitro or ex vivo), or by injecting the complex into an organism (for uses in vivo) The present invention thus, provides a particularly advantageous method for the treatment of diseases by administration of a vector according to the invention containing a nucleic acid capable of correcting the said disease. More particularly, this method is applicable to diseases resulting from a deficiency in a protein or peptide product, the administered DNA encoding the said protein or peptide product. The present invention extends to any use of a vector according to the invention for the in vivo, ex vivo or in vitro transfection of cells.

The invention also relates to any recombinant 10 cell containing a vector as defined above. It involves preferably eukaryotic cells, for example yeast, animal cells, and the like. These cells are obtained by any technique allowing the introduction of a DNA into a S. given cell known to persons skilled in the art.

The following examples which are 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 the peptide maleimide-NLS.

Figure 2: Analysis, on a 15% polyacrylamide gel, of the oligonucleotide-peptide (Pso-GA 19 -NLS) chimera by proteolytic action of trypsin.

1 oligo Pso-GA 19

-SH

2 oligonucleotide-peptide Pso-GA 19 -NLS chimera 3 oligonucleotide-peptide Pso-GA 19 -NLS chimera after digestion with trypsin Figure 3: schematic representation of the plasmid pXL2813.

Figure 4: schematic representation of the plasmid pXL2652.

Figure 5: analysis on a 15% polyacrylamide gel of the formation of triple helices between the plasmid pXL2813 and the oligonucleotide-peptide Pso-GA 1 9 -NLS chimera.

The oligonucleotide pso-GAi 9 -NLS and the plasmid are 10 mixed in a buffer containing 100 mM MgCl 2 The molar excess of oligonucleotide relative to the plasmid eo varies from 0 to 200. The mixture is photoactivated, after leaving overnight at 370C, and then digested with two restriction enzymes which cut the plasmid on either side of the region of formation of the triple helices.

1 no oligonucleotide 2 molar excess of oligonucleotide relative to the plasmid of 3 molar excess of oligonucleotide relative to the plasmid of 4 molar excess of oligonucleotide relative to the plasmid of 100 molar excess of oligonucleotide relative to the plasmid of 200 6 photoactivated oligonucleotide alone 7 nonphotoactivated oligonucleotide alone 8 molar excess of oligonucleotide relative to the plasmid of 30, the mixture not having been photoactivated.

Figure 6: Expression in vitro of the transgene (Pgalactosidase) for tests carried out with the plasmid pXL2813 alone, the vector pXL2813-Pso-GA 9 -NLS, and without plasmid.

Figure 7: Characterization of the peptide part of the oligonucleotide-peptide Pso-GA 19 -NLS chimera by .r interaction with importin 60-GST (analysis on a polyacrylamide gel).

o. 10 1 oligo Pso-GA 19 (l[g) 2 oligonucleotide-peptide Pso-GA 19 -NLS chimera (1lg) 3 supernatant recovered after incubation of glutathione-beads coated with importins 60 and the oligonucleotide Pso-GA 19 and separation of the pellet of beads (containing the components which interact with the importins) from the supernatant 4 pellet recovered after incubation of glutathionebeads coated with importins 60 and the oligonucleotide Pso-GA 19 and separation of the pellet of beads (containing the components which interact with the importins) from the supernatant supernatant recovered after incubation of the glutathione-beads coated with importins 60 and the oligonucleotide-peptide Pso-GA 19 -NLS chimera, and separation of the pellet of beads (containing the components which react with the importins) from the supernatant 6 pellet recovered after incubation of the glutathione-beads coated with importins 60 and the oligonucleotide-peptide Pso-GA19-NLS chimera, and separation of the pellet of beads (containing the components which interact with the importins) from the supernatant.

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

1 oligo GA 19 -SH (200 ng) 10 2 oligonucleotide-peptide GA 19 -NLS chimera (1ng) before purification by high-performance liquid chromatography 3 oligonucleotide-peptide GA 1 9 -NLS chimera 1 lg) after purification 15 4 oligonucleotide-peptide GA19-NLS chimera after digestion with trypsin (lg).

Figure 9: Graphical representation of the kinetics of formation of the triple helices of triple helix sites occupied as a function of time) between the plasmid pXL2813 and the chimera GA 19

-NLS.

Figure 10: Characterization of the peptide part of the oligonucleotide-peptide GA 19 -NLS chimera by interaction with the importin 1 oligonucleotide-peptide GA 19 -NLS chimera, 2 oligo GA1 9 3 supernatant recovered after incubation of the glutathione-beads coated with importins 60 and the oligonucleotide-peptide GA 19 -NLS chimera, and separation of the pellet of beads (containing the components which interact with the importins) from the supernatant, 4 pellet recovered after incubation of the glutathione-beads coated with importins 60 and the oligonucleotide-peptide GA 1 9 -NLS chimera, and separation of the pellet of beads (containing the components which interact with the importins) from the supernatant, .o 5 supernatant recovered after incubation of the glutathione-beads coated with importins 60 and the 10 oligonucleotide-peptide GA19 and separation of the e o pellet of beads (containing the components which interact with the importins) from the supernatant, 6 pellet recovered after incubation of the glutathione-beads coated with importins 60 and the oligonucleotide GA 19 and separation of the pellet of beads (containing the components which interact with the importins) from the supernatant.

Figure 11: Schematic representation of the plasmid pXL2997.

Figure 12: Graphical representation of the kinetics of formation of the triple helices of triple helix sites occupied as a function of time) between the plasmid pXL2997 and the chimera pim-NLS.

Figure 13: Histogram representing the p-galactosidase activity in vivo in human pulmonary tumours H1299 of the plasmids pXL2813 (indicated Bgal in the figure) and pXL2813-Pso-GAi 9 -NLS (indicated NLS-Bgal in the figure) in RLU ("Relative Light Unit") per tumour.

The transfection was performed using the electrotransfer techniques as described in applications WO 99/01157 and WO 99/01158.

MATERIALS AND METHODS 1. Coupling of the oligonucleotides and the peptides Oligonucleotides The oligonucleotides used are the sequence 5'-AAGGAGAGGAGGGAGGGAA-3' (SEQ ID No. 4) 19 bases long and referenced under the name "GA 19 in the text which follows, or the sequence 5'-GGGGAGGGGGAGG-3' (SEQ ID No. 15) 13 bases long and referenced under the name "pim" in the text which follows (because it is the 15 sequence of the protooncogene pim-1).

The oligonucleotides noted GA 19 -SH or pim-SH have the same sequences as GA 19 and pim, respectively, and a thiol group at the 5' end, with a spacer of six carbon between the thiol and the phosphate of the end. The oligonucleotides noted Pso-GA 19 -SH have a thiol group at the 3' (SH) end, and in addition a psoralen at the 5' (Pso) end, with a spacer of six carbons between the psoralen and the phosphate of the 5' end. The oligonucleotides noted Pso-GA 19 do not have a thiol group.

The nomenclature used is summarized in Table I below:

S

S

S

name of the modification of Imodification of oligonucleotide the 3' end the 5' end

GA

19 none none pim none none

GA

19 -SH none thiol group pim-SH none thiol group Psi-GA 1 9 none psoralen Pso-GA 19 -SH thiol group psoralen Table 1 These freeze-dried oligonucleotides are taken up in a 100 mM trimethylammonium acetate buffer, pH 7.

Peptides The peptides used for the couplings are synthesized by an automatic machine in the solid phase.

They contain: either the nuclear localization sequence of the T antigen (PKKKRKV, SEQ ID No. 11), or the same sequence, mutated (PKNKRKV, SEQ ID No. 14) which allows neither targeting nor 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 and the e carbon and a protecting group 9-fluorenylmethyloxycarbonyl (Fmoc) on the amine of the a carbon. This Fmoc group absorbs at 260 nm, which a makes it possible to monitor the peptide by reversedphase high-performance liquid chromatography. The C-terminal group is also protected (CONH 2 group), the protection being added at the end of peptide synthesis.

The representation of the peptides is indicated in Table II below: name of the peptide sequence and modifications maleimide-NLS maleimide-KGAGPKKKRKV-CONH 2 _moc maleimide-NLSmutated maleimide-KGAGPKNKRKV-CONH 2 Fmoc Table II The freeze-dried peptides are picked up in a 100 mM triethylammonium acetate buffer, pH 7. The concentration is 0.4 mg/ml.

Couplings For the coupling with the oligonucleotides, the strategy used consists in reacting the thiol group carried by the oligonucleotide and the maleimide group carried by the peptide [Eritja, et al., Synthesis of defined peptide-oligonucleotide hybrids containing a nuclear transport signal sequence, Tetrahedron, 1991, 47(24), pp. 4113-4120].

The oligonucleotide is added to the peptide solution in an equimolar quantity and the reaction medium is left for 2 hours at room temperature. The oligonucleotide-peptide chimera is purified by reversed-phase high-pressure liquid chromatography on a Vydac C8 column containing spheroidal silica having a diameter of 5 nm and a porosity of 300 A. Use is made of a 0.1 M triethylammonium acetate (TEAA) buffer and an acetonitrile gradient passing from 5% to 50% over minutes. The products are detected at 260 nm.

Analysis of the chimeras by digestion with trypsin 10 The oligonucleotide-peptide conjugates are subjected to the proteolytic action of trypsin which .makes it possible to reveal the peptide part of the chimera [Reed, M.W. et al., Synthesis and evaluation of nuclear targeting peptide-anti-sense 15 oligodeoxynucleotide conjugates, Bioconjugate Chemistry, 1995, 6, pp.101-108]. Solutions containing 1 1 g of oligonucleotide-peptide in 7 l1 of 0.1 M triethylammonium (TEAA) purification buffer are mixed with 1 nl of a trypsin solution (5 mg/ml). 1 pl of 100 mM Tris-HC1 buffer, pH 9, and 1 1 of 500 mM EDTA are added thereto. After digesting for one hour, the samples are deposited into the wells of a polyacrylamide, 7 M urea, gel. The electrophoresis is performed in 100 mM Tris buffer, pH 8.3 containing 90 mM boric acid and 1 mM EDTA. The nucleic acids are visualized by silver staining using a Biorad kit.

2. Formation of triple helices with the chimeras Plasmid The plasmid used to study the formation of triple helices with the GA 19 -peptide chimeras is called pXL2813 (7257 pb, cf Figure This plasmid expresses the p-galactosidase gene under the control of the strong promoter of the cytomegalovirus (CMV) early S-genes, as well as the ampicillin resistance gene.

10 Upstream of the promoter, between positions 7238 and 7256, the GA 19 sequence AAGGAGAGGAGGGAGGGAA-3', SEQ ID No. 4) was cloned according to conventional molecular biological techniques.

It was cloned into the plasmid pXL2652 (of S. 7391 bp and which is schematically represented in Figure 4) which expresses the p-galactosidase gene under the control of the strong promoter of the cytomegalovirus (CMV) early genes, as well as the ampicillin resistance gene. This promoter is derived from the pCDNA3, the LacZ and its polyA are derived from the pCH110, and the remainder is derived from pGL2.

The sequences were cloned upstream of the promoter between the unique cleavage sites for the enzymes MunI and XmaI. For that, the two complementary oligonucleotides containing the sequence to be cloned 6651 (5'-AATTGATTCCTCTCCTCCCTCCCTTAC-3') and 6652 were heated for minutes at 95 0 C and then hybridized by allowing the temperature to fall 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 a 1% agarose gel followed by staining with ethidium bromide.

S' The fragment of interest for the remainder of the cloning was eluted according to the Jetsorb 10 protocol (Genomed) and 200 ng of this fragment were linked to 10 ng of the mixture of oligonucleotides hybridized by T4 ligase, for 16 hours at 160C.

Competent E. coli bacteria of the DH5a strain were transformed by eletroporation with the reaction *15 product, plated on Petri dishes containing LB medium and ampicillin. The ampicillin-resistant clones were *4 selected and the DNA was extracted by alkaline lysis q and analysed on a 1% agarose gel. A clone corresponding in size to the expected product was sequenced.

In the case where the oligonucleotide is pim, the plasmid used to study priple helix formation is pXL2997 represented in Figure 11. This plasmid expresses the gene for p-galactosidase under the control of the strong promoter of the Cytomegalovirus (CMV) early genes, as well as the gene for resistance to Ampicillin.

Upstream of the promoter, the sequence pim (SEQ ID No. 15) was cloned according to conventional molecular biology methods.

Formation of the triple helices The formation of the triple helices is performed by mixing the plasmid pXL2813 (3 pmol of plasmid, that is to say 15 ng) or pXL2997, depending on the case, and variable quantities of oligonucleotides or of chimeras in a 100 mM Tris-HCl buffer, pH -containing 100 mM MgCl2.

Photoactivation of the triple helices After incubating overnight, the mixture is irradiated, in ice, for 15 minutes, using a S. monochromatic lamp having a wavelength of 365 nm (Biorad). The product of photoactivation is digested with the restriction enzymes MfeI and Spel and analysed on a 15% polyacrylamide, 7 M urea, gel with 100 mM Tris migration buffer, pH 8.3, containing 90 mM boric acid and 1 mM EDTA. The nucleic acids are visualized by silver staining using a Biorad kit [Musso, M., J.C. 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].

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

In a first instance, the oligonucleotides are radioactively labelled at their 3' end with Terminal 10 Transferase with the aid of S]dATP. The protocol used is from Amersham: 10 pmol of oligonucleotides are incubated for 2 hours at 370C, with 50 jCi of 3[a-5S]dATP in the presence of 10 units of Terminal Transferase, in a volume of 50 [l of buffer containing 15 sodium cacodylate. The percentage of labelled oligonucleotides is evaluated according to the following method: 1 tl of a sample, diluted 1/100, of the solution after labelling is deposited on Whatman DE81 paper, in duplicate. One of the two papers is washed twice for 5 minutes with 2xSSC, for 30 seconds with water and for 2 minutes with ethanol. The radioactivities of the two papers are compared. The radioactivity of the washed paper corresponds to the effectively incorporated.

The formation of the triple helices occurs in a volume of 35 P1. The concentrations of plasmids nM) and of oligonucleotides (20 nM), the buffer used (100 mM Tris-HCl pH 7.5, 50 mM of MgCl 2 and the temperature (370C) are fixed whereas the incubation time varies from 1 to 24 hours. The Sepharose columns are equilibrated, before use, with the reaction buffer and centrifuged at 2200 rpm for 4 minutes, in order to cause them to settle. 25 1 of the reaction medium are deposited on the columns and the latter are centrifuged under the same conditions as above. 25 n1 of the reaction buffer are then deposited on the columns which are again centrifuged. The eluate is recovered.

10 The radioactivity contained in 5 ul 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 oligonucleotides eluted: of oligonucleotides eluted cpm(eluate)/ [5 x cpm(deposit)] x 100.

The percentage of plasmids which 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 attached to the total number of plasmids.

of oligonucleotides attached oligonucleotides eluted/% plasmids eluted] x 100.

This parameter makes it possible to evaluate the percentage of sites of formatioR of triple helices (there is one per plasmid pXL2813 or pXL2997) which are effectively occupied, taking into account the 43 concentrations of plasmids (noted [plasmid]) and of oligonucleotides (noted [oligo]) used during the reaction of formation of the triple helices: triple helix sites occupied of oligonucleotides attached x [oligo]/[plasmid].

3. Interaction with the importins e.

Recombinant proteins 10 The importin 60 subunit used to study the e interaction with the oligonucleotide-peptide (NLS or NLSmutated) conjugates is of murine origin and is fused with Glutathione S-Transferase (GST). The sequence of importin 60 was cloned into a vector pGEX-2T in order 15 to fuse it with GST. The recombinant protein was produced 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 the recombinant proteins All the interaction experiments are carried out in the binding buffer (20 mM HEPES, pH 6.8, 150 mM potassium acetate, 2 mM magenesium acetate, 2 mM DTT and 100 Vg/ml BSA) In a first instance, the recombinant proteins are incubated in the presence of Sepharose beads coated with glutathion (Pharmacia Biotech), 1 ng of recombinant protein is used for 10 1l of beads. After incubating for 30 minutes at room temperature in 500 nl 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 resuspending in 500 nl of binding buffer followed by centrifugation, as described above. The beads are resuspended in binding buffer in order to obtain a suspension containing eec of beads coated with recombinant proteins.

10 In a second instance, 60 1l of the suspension containing 50% of beads coated with recombinant proteins are incubated with 2 ng of oligonucleotide or of oligonucleotide-peptide, in a volume of 500 nl of i. binding buffer. After incubating for 30 minutes at room 15 temperature, the mixture is centrifuged at 2000 g for 30 seconds, and the supernatant is removed. 30 l of the supernatant are collected in order to analyse the fraction not attached to the beads. The beads are washed five times by resuspending in 500 1l of binding buffer followed by centrifugation, as described above.

The beads are resuspended in 15 nl of loading buffer (0.05% bromophenol blue, 40% sucrose, 0.1 M EDTA, pH 8, 0.5% sodium lauryl sulphate) and heated for minutes at 90°C. The content of the supernatant and of the pellet is analysed on a 15% polyacrylamide, 7 M urea, gel, with a 100 mM Tris migration buffer, pH 8.3, containing 90 mM boric acid and 1 mM EDTA. The nucleic acids are visualized by silver staining using a Biorad kit [Rexach, M. and G. Blobel, Protein import into nuclei: association and dissociation reactions involving transport substrate, transport factors, and nucleoporins, Cell, 1995, 83, pp. 683-692].

4. Transfection of cells Cell culture The cell type used is NIH 3T3 (ATCC CRL- 1658). It consists of mouse fibroblasts. These cells 0 are cultured in a modified Dulbecco's medium, with g/1 glucose (DMEM Gibco), 2 mM glutamine, penicillin (100 U/ml) and streptomycin (100 ug/ml), and 4 10% foetal calf serum (Gibco). They are incubated at 37°C in an incubator containing 5% CO 2 Transfection One day before the transfection, the wells of a 24-well plate are inoculated with 50,000 cells per well. The vectors are diluted in 150 mM NaCl and mixed with a cationic lipid (the compound having the condensed formula

H

2 N (CH2) 3 NH (CH2) 4 NH (CH2) 3

NHCH

2 COGlyN[ (CH2) 17

CH

3 2 described in patent application WO 97/18185 under the number diluted in 150 mM NaC1. 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 incubating at 37°C in an incubator containing 5% CO2 for two hours, 10% foetal calf serum 46 is added.

Quantification of the p-galactosidase After incubating for 48 hours, the cells are washed twice with PBS and lysed with 250 1 of lysis buffer (Promega). The p-galactosidase is quantified according to the protocol "Lumigal p-Galactosidase genetic reporter system" (Clontech). The activity is measured on a Lumat LB9501 luminometer (Berthold). The 10 quantity of proteins is measured with the BCA kit (Pierce).

*555

EXAMPLES

Examples 15 This example illustrates the possibility of coupling the oligonucleotide Pso-GA 19 -SH to the peptide maleimide-NLS.

The oligonucleotide Pso-GA19-SH, of sequence 5'-AAGGAGAGGAGGGAGGGAA-3', with a thiol group at the 3' end, was coupled to the peptide NLS which carries a maleimide group at its N-terminal end according to the method described above in the "Materials and Methods" part under the section "coupling of the oligonucleotides and the peptides".

The coupling is monitored by reversed-phase high-performance liquid chromatography (HPLC). It occurs with an equimolar stoechiometry: in two hours, the coupling is complete and the chimera is purified by

HPLC.

The reaction sheme for the coupling is represented in Figure 1.

The oligonucleotide-peptide (Pso-GA 19

-NLS)

chimera was analysed by polyacrylamide gel electrophoresis after proteolytic action of trypsin which makes it possible to reveal the presence of the peptide part of the chimera, after migration on a denaturing polyacrylamide gel and silver staining of 10 the nucleic acids (as indicated in the "Materials and 0 5 Methods" part under the section "Analysis of the chimeras by digestion with trypsin").

The chimera Pso-GA19-NLS exhibits a retarded electrophoretic migration compared with the 15 oligonucleotide Pso-GA 19 -SH, and the product of proteolytic digestion is visualized at an intermediate level between the levels of migration of Pso-GA19-NLS and of Pso-GA 19 -SH, as shown in Figure 2.

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

Example 2 This example illustrates the formation of triple helices between the plasmid pXL2813 and the chimera Pso-GA 19 -NLS which is modified by a 48 photoactivable alkylating agent. This example also indicates the proportion of plasmids modified according to the molar excess of oligonucleotides relative to the plasmid.

The plasmid pXL2813, represented in Figure 3, comprises the homopurine sequence complementary to GA19 which is capable of forming a triple helix with the *o oligonucleotides GA 19 Pso-GA 19 Pso-GA19-SH or Pso-GA 19 NLS. Divalent cations such as Mg2+ stabilize these 0eO* O. 10 triple helices. The oligonucleotide Pso-GA 19 -NLS and the plasmid are mixed in a buffer containing 100 mM of MgCl 2 The molar excess of oligonucleotide relative to the plasmid varies from 0 to 200.

After incubating for 12 hours at 37 0 C, the 15 mixture is photoactivated for 15 minutes (as indicated :in the "Materials and Methods" part under the section e• "Photoactivation of the triple helices"), and then digested with the two restriction enzymes MfeI and Spel which cut the plasmid on either side of the region of formation of the triple helices. A nucleic acid fragment is thus released which contains 70 base pairs after digestion of the nonmodified plasmid pXL2813. 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 derived from the plasmids modified by a triple helix have a shorter migration distance than the fragments derived from nonmodified plasmids. It is therefore possible, by electrophoresis on a denaturing polyacrylamide gel, to quantify the proportion of modified plasmids according to the molar excess of oligonucleotide relative to the plasmid.

The results are indicated in Figure 5. For a Smolar excess of oligonucleotide relative to the plasmid 10 greater than 50, all the plasmids are modified and are combined with an oligonucleotide Pso-GAi 9 -NLS. Without photoactivation, the retardation of the digestion fragment is lost in a denaturing gel.

It thus appears that the triple helices 15 formed with the oligonucleotides Pso-GA 19 -SH or Pso-GA 19 -NLS are therefore indeed covalently bound to the double helix after photoactivation.

Moreover, by digesting the remainder of the plasmid backbone and by analysing as above, it is possible to check that the photoactivation does not result in a nonspecific covalent binding of the oligonucleotide Pso-GA 19 -NLS outside the region containing the sequence capable of forming a triple helix.

These results clearly demonstrate the conditions which make it possible to specifically and covalently combine a peptide sequence with a plasmid, and is outside the promoter regulating the expression a. of the transgene, which prevents the expression of the transgene from being affected.

Example 3 This example illustrates the capacity of the transgene to be expressed in vitro, although the plasmid is modified.

The expression of p-galactosidase by the plasmids pXL2813 which are nonmodified or which are combined with an oligonucleotide Pso-GA 19 -NLS was thus compared by transfection of NIH3T3 cells.

The results are reported in Figure 6, and the mean values obtained standard deviation) are assembled in the following Table III: expression of the plasmid transgene (in RLU/pg of protein) pXL2813 253759 48545 pXL2813-Pso-GAis-NLS 473984 111476 without plasmid 129 Table III It can be observed that the expression of the transgene increases following the modification provided by the covalent attachment of the triple helix upstream of the promoter.

These results clearly demonstrate that the formation of the triple helices does not affect the expression of the transgene in vitro. In contrast, by virtue of the nuclear homing of the plasmid because of its coupling with the targeting sequence NLS, more plasmids reached the nucleus, resulting in an increase in the transfection efficiency.

Example 4 This example is intended to verify that the 10 in vivo expression is not inhibited by the presence of a targeting signal associated with the plasmid via a triple helix.

The experiment consists in transfecting pXL2813 and pXL2813-Pso-GAi 9 -NLS into human pulmonary tumours H1299 using the electrotransfer techniques described in applications WO 99/01157 and WO 99/01158.

The experiment was carried out in 18 to 20 g female nude mice.

The mice are monolaterally implanted with 20 mm 3 H1299 tumour grafts. The tumours develop, reaching a volume of 200 to 300 mm 3 The mice are separated according to the size of their tumours and divided into homogeneous groups. The mice are then anaesthetized with a Ketamine/Xylazine mixture (commercially available). The plasmid solution (8 Mg of 1l of 150 mM sodium chloride) is longitudinally injected at the periphery of the tumour with the aid of a Hamilton syringe.

The lateral faces of the tumour are covered with conducting gel and the tumour is placed between two stainless steel flat electrodes 0.45 to 0.7 cm apart. The electrical pulses are applied with the aid of a commercial square pulse generator (Electropulsateur PS 15, Jouan, France) 20 to 30 seconds after the injection. An oscilloscope makes it possible to control the intensity in volts, the duration in milliseconds and the frequency in hertz of the pulses 10 which should be delivered at 500 V/cm for milliseconds at 1 hertz.

For the evaluation of the tumour transfection, 10 and 7 mice respectively for the plasmid pXL2813 and pXL2818-Pso-GAi 9 -NLS are humanely killed 2 days after the injection of the plasmid. The tumours are collected, weighed and ground in a lysis buffer (Promega) supplemented with antiproteases (Complete, Boehringer). The suspension obtained is centrifuged in order to obtain a clear supernatant.

After having incubated 10 u1 of this supernatant with 250 ,l of reaction buffer (Clontech) for 1 hour in the dark, the p-galactosidase is measured with the aid of a commercial luminometer. The results are expressed in total RLU ("Relative Light Unit") per tumour.

It is observed that the plasmid pXL2813-Pso-

GA

19 -NLS expresses the transgene in vivo at a level greater than or equal to that obtained with the unmodified plasmid pXL2813 (see Figure 13).

Example This example illustrates the characterization of the peptide part of the Pso-GA 19 -NLS conjugates, that is to say the verification of the targeting properties of the NLS peptide combined with the constructs according to the invention.

The peptide sequence used, the NLS signal of the SV40 T antigen, is recognized by receptors of the a-karyopherin family. The murine equivalent, called 10 importin 60, fused with a glutothione S-transferase group, was used to characterize the oligonucleotidepeptide conjugates. It was performed according to the method described in the "Materials and Methods" part under the section "Interaction with the importins".

The interactions between the glutathion-beads coated with importins 60 and the oligonucleotides GA1 9 NLS or Pso-GA 19 -NLS were studied. After incubating for 30 minutes at room temperature, the pellet of beads S(containing the components which interact with the importins) is separated from the supernatant.

The result of this characterization is reported in Figure 7. This figure indicates that the oligonucleotide Pso-GAi 9 -NLS is combined with the glutathion beads, whereas the oligonucleotide Pso-GA19 remains in the supernatant.

This clearly shows the capacity of the oligonucleotide Pso-GAi 9 -NLS to interact with the a-importin. The peptide part of the oligonucleotidepeptide chimeras is therefore indeed recognized by its receptor, which means that the peptide effectively fulfils its role of targeting signal.

Example 6 This example illustrates the possibility of coupling the oligonucleotide GA 19 -SH to the peptide maleimide-NLS. Unlike Example 1, the chimera is not modified by a photoactivable alkylating agent.

10 The oligonucleotide GA 19 -SH, of sequence 5'-AAGGAGAGGAGGGAGGGAA-3' (SEQ ID No. with a thiol group at the 5' end, was coupled to the peptide maleimide-NLS which possesses a maleimide group at its N-terminal end under the same conditions as for the oligonucleotide Pso-GA 19 -SH (see Example 1) The coupling is monitored by reversed-phase high-performance liquid chromatography (HPLC). It .occurs with an equimolar stoichiometry: in two hours, the coupling is complete. The chimera is then purified by HPLC.

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

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

Example 7 This example illustrates the possibility of forming triple helices between the plasmid pXL2813 and the GA- 19 -NLS chimera in the absence of alkylating agent.

A kinetics of formation of triple helices between the plasmid pXK2813 and the GA- 19 -NLS chimera, obtained as described in Example 5, was carried out and studied according to the technique described in the *e 10 "Materials and Methods" part, under the section "Formation of triple helices with the chimeras".

Figure 9 represents the kinetics of formation of the triple helices.

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

Example 8 This example illustrates the characterization of the peptide part of the GA 19 -NLS chimera.

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

The interactions between the glutathionebeads coated with importins 60 and the oligonucleotides

GA

19 or GA- 19 -NLS were studied. After incubating for minutes at room temperature, the pellet of beads (containing the components which interact with the importins) is separated from the supernatant.

The results are indicated in Figure 10. It appears that the oligonucleotide GA 19 -NLS is combined 10 with the glutathione beads, whereas the oligonucleotide GA19 remains in the supernatant.

This clearly shows the capacity of the oligonucleotide GA- 19 -NLS to interact with the a-importin. The peptide part of the oligonucleotide- 15 peptide chimeras is therefore indeed recognized by its receptor, and fulfils, here again, its role as targeting signal.

Example 9 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 No. 15), with a thiol group at the 5' end, was coupled to the peptide maleimide-NLS which possesses a maleimide group at its N-terminal end under the same conditions as for the oligonucleotide

GA

19 -SH (see Example The coupling is monitored by reversed-phase high-performance liquid chromatography (HPLC). It occurs with an equimolar stoichiometry: in two hours, the coupling is complete. The chimera is then purified by HPLC.

Example This example illustrates the possibility of forming triple helices between the plasmid pXL2997 and 10 the pim-NLS chimera in the absence of alkylating agent.

A kinetics of formation of triple helices between the plasmid pXL2997 and the pim-NLS chimera (formation obtained as described in Example was carried out and studied according to the technique described in the "Materials and Methods" part, under the section "Formation of triple helices with the chimera".

Figure 12 represents the kinetics of

SSSS*

formation of the triple helices.

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

The invention having now been completely described, it is evident that numerous modifications can be made to the present invention without as a result parting from the spirit of the invention and from the scope of the claims which follow.

SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT

NAME: RHONE POULENC RORER STREET: 20 AVENUE RAYMOND ARON CITY: ANTONY CEDEX COUNTRY: FRANCE POSTAL CODE: 92165 TELEPHONE: 01.55.71.73.26 TELEFAX: 01.55.71.72.91 (ii) TITLE OF INVENTION: VECTORS FOR TRANSFERRING NUCLEIC ACIDS, COMPOSITIONS CONTAINING THEM AND THEIR USES.

(iii) NUMBER OF SEQUENCES: (iv) COMPUTER READABLE FORM: MEDIUM TYPE: Tape COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30 (EPO) INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: GAGGCTTCTT CTTCTTCTTC TTCTT INFORMATION FOR SEQ ID NO: 2: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

S

CTTCTTCTTC TTCTTCTTCTT 21 INFORMATION FOR SEQ ID NO: 3: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs i TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: AAGGGAGGGA GGAGAGGAA 19 INFORMATION FOR SEQ ID NO: 4: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: AAGGAGAGGA GGGAGGGAA 19 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA SEQUENCE DESCRIPTION: SEQ ID NO: TTGGTGTGGT GGGTGGGTT 19 INFORMATION FOR SEQ ID NO: 6: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA 61 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: CTTCCCGAAG GGAGAAAGG 19 INFORMATION FOR SEQ ID NO: 7: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleotide g.

STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA SEQUENCE DESCRIPTION: SEQ ID NO: 7: GAAGGGTTCT TCCCTCTTTC C 21 INFORMATION FOR SEQ ID NO: 8: SEQUENCE CHARACTERISTICS: LENGTH: 13 base pairs TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: GAAAAAGGAA GAG 13 INFORMATION FOR SEQ ID NO: 9: 62 SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: AAGAAAAAAA AGAA 14 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 17 base pairs TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear a (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: AAAAAAGGGA ATAAGGG 17 INFORMATION FOR SEQ ID NO: 11: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear 63 (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: Pro Lys Lys Lys Arg Lys Val 1 INFORMATION FOR SEQ ID NO: 12: SEQUENCE CHARACTERISTICS: LENGTH: 19 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide ;e (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys S1 5 Lys Lys Leu Asp Lys INFORMATION FOR SEQ ID NO: 13: SEQUENCE CHARACTERISTICS: LENGTH: 38 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 64 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: Asn Gin Ser Ser Asn Phe Gly Pro Met Lys Gly Gly Asn Phe 1 5 Gly Gly Arg Ser Ser Gly Pro Tyr Gly Gly Gly Gly Gin Tyr 20 Phe Ala Lys Pro Arg Asn Gin Gly Gly Tyr 30 LENGTH: 7 amino acids TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: Pro Lys Asn Lys Arg Lys Val 1 INFORMATION FOR SEQ ID NO: 14: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: Pro Lys Asn Lys Arg Lys Val 1 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 13 base pairs TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: GGGGAGGGGG AGG Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The reference to any prior art in this e specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that 20 prior art forms part of the common general knowledge in Australia.

Australia.

Claims (41)

1. Vector for transferring nucleic acids, characterized in that it comprises a double-stranded DNA molecule and at least one oligonucleotide which is coupled to a targeting signal forming a triple helix with a specific sequence present on said double- stranded DNA molecule.

2. Vector for transferring nucleic acids according to claim 1, characterized in that the double- stranded DNA molecule is a plasmid or an episome.

3. Vector for transferring nucleic acids according to claim 2, characterized in that the double- stranded DNA molecule is a plasmid in circular form or in a supercoiled state.

4. Vector for transferring nucleic acids according to any one of 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 or of a transcriptional terminator which is active in mammalian cells. Vector for transferring nucleic acids according to claim 4, characterized in that the gene of interest is a nucleic acid encoding a therapeutic product.

6. Vector for transferring nucleic acids according to any one of claims 1 to 5, characterized in that the specific sequence present on the double- stranded DNA molecule is a homopurine-homopyrimidine sequence.

7. Vector for transferring nucleic acids according to any one of claims 1 to 6, characterized in that the specific sequence present on the double- stranded DNA molecule is a sequence which is naturally present on the double-stranded DNA or a synthetic or a natural sequence which is introduced artificially into the double-stranded DNA.

8. Vector for transferring nucleic acids according to any one of claims 1 to 7, characterized in that the oligonucleotide comprises a sequence poly-CTT and the specific sequence present on the double- stranded DNA molecule is a sequence poly-GAA.

9. Vector for transferring nucleic acids according to any 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). Vector for transferring nucleic acids according to any one of claims 1 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. 68

11. Vector for transferring nucleic acids according to any one of claims 1 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) present in the replication origin ColE1 of E. coli, and the oligonucleotide possesses the sequence 5'-GAAGGGTTCTTCCCTCTTTCC-3' (SEQ ID No. 7).

12. Vector for transferring nucleic acids according to any one of claims 1 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 sequence 5'-AAAAAAGGGAATAAGGG-3' (SEQ ID No. 10) present in the p-lactamase gene of the plasmid pBR322 and of E. Coli, respectively.

13. Vector for transferring nucleic acids according to any 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 replication origin y of the plasmids containing a conditional replication origin.

14. Vector for transferring nucleic acids according to any one of claims 1 to 13, characterized in that the oligonucleotide comprises at least 3 base pairs. Vector for transferring nucleic acids according to claim 14, characterized in that the oligonucleotide comprises 5 to 30 base pairs.

16. Vector for transferring nucleic acids according to any one of claims 1 to 15, characterized in that the oligonucleotide exhibits at least one chemical modification making it resistant to, or protecting it from, nucleases, or increasing its affinity towards the specific sequence present on the .i double-stranded DNA molecule.

17. Vector for transferring nucleic acids according to any one of claims 1 to 16, characterized in that the oligonucleotide is a succession of nucleosides which have undergone modification of the backbone.

18. Vector for transferring nucleic acids S""according to any one of claims 1 to 16, characterized in that the oligonucleotide is coupled to an alkylating *agent forming a covalent bond at the level of the bases of the double-stranded DNA.

19. Vector for transferring nucleic acids according to claim 18, characterized in that the said alkylating agent is photoactivable. Vector for transferring nucleic acids according to claim 18 or 19, characterized in that the said alkylating agent is a psoralen.

21. Vector for transferring nucleic acids according to any one of claims 1 to 20, characterized in that the targeting signal interacts with a component of the extracellular matrix or a plasma membrane receptor, (ii) targets an intracellular compartment, and/or (iii) improves the intracellular flow of the double-stranded DNA.

22. Vector for transferring nucleic acids according to claim 21, characterized in that the said targeting signal comprises growth factors, cytokines, hormones, sugars which recognize lectins, immunoglobulins, transferrin, lipoproteins, vitamins, peptide or neuropeptide hormones or any unit recognized by the integrins, or by other extrinsic proteins of the cell membrane.

23. Vector according to claim 22 wherein said targeting signal comprises EGF, PDGF, TGFp, NGF, SIGF, I, FGF,IL-1, IL-2, TNF, Interferon, CSF, insulin, 0* 9 growth hormone, prolactin, glucagon, thyroid hormone, steroid hormones,vitamin B12, tachykinins, neurotensin, VIP, endothelin, CGRP, CCK, or the peptide RGD

24. Vector for transferring nucleic acids according to claim 21, characterized in that the targeting signal is an intracellular targeting signal. Vector according to claim 24 wherein the targeting signal is a nuclear homing sequence (NLS).

26. Vector for transferring nucleic acids according to claim 25, characterized in that the nuclear homing signal is the NLS sequence of the SV40 T antigen.

27. Vector for transferring nucleic acids according to claim 21, characterized in that the targeting signal allows both extracellular targeting and intracellular targeting.

28. Vector for transferring nucleic acids according to any one of claims 1 to 27, characterized :in that the coupling of the targeting signal to the oligonucleotide is obtained by synthesis on a solid ooo oeoo phase or in solution. oooo

29. Vector according to claim 28 wherein said coupling is by establishing disulphide, thioether, ester, amide or amine bonds. Composition characterized in that it ""contains at least one vector as defined in any one of S. claims 1 to 29.

31. Composition according to claim characterized in that it contains, in addition, one or more transfecting agents.

32. Composition according to claim 31, characterized in that the transfecting agent is a cationic lipid, a lipopolyamine or a cationic polymer.

33. Composition according to any one of claims 30 to 32, characterized in that it contains, in addition, one or more adjuvants capable of combining with the vector as defined in any one of claims 1 to 29 to form vector/transfecting agent complexes.

34. Composition according to claim 33, characterized in that the adjuvant is one or more neutral lipids chosen from natural or synthetic lipids which are zwitterionic or which lack ionic charge under physiological conditions. Composition according to claim 34, characterized in that the neutral lipid(s) is(are) chosen from lipids containing two fatty chains.

36. Composition according to claims 34 or characterized in that the neutral lipid(s) is (are) chosen from dioleoylphosphatidylethanolamine (DOPE), oleoylpalmitoylphosphatidylethanolamine (POPE), di-stearoyl, -palmitoyl, -mirystoylphosphatidyl- ethanolamines as well as their derivatives which are N-methylated 1 to 3 times, phosphatidylglycerols, diacylglycerols, glycosyldiacylglycerols, cerebrosides, sphingolipids or asialogangliosides

37. Compostion according to claim 36 wherein the neutral lipid(s) is (are) chosen from galactocerebrosides, sphingomyelins, asialoGM1 and GM2.

38. Composition according to claim 33, characterized in that the adjuvant is or comprises a compound which is involved in the condensation of the DNA.

39. Composition according to claim 38, characterized in that the said compound is derived, as a whole or in part, from a histon, a nucleolin and/or a ^RA protamine, or consists, as a whole or in part, of peptide units (KTPKKAKKP) and/or (ATPAKKAA) repeated continuously or otherwise. Composition according to claim 36 wherein said compound consists, as a whole or in part, of from 2 to 10 of said peptide units.

41. Composition according to any one of claims 30 to 40, characterized in that it comprises a pharmaceutically acceptable vehicle for an injectable formulation.

42. Composition according to any one of claims 30 to 40, characterized in that it comprises a I* pharmaceutically acceptable vehicle for application to the skin and/or to the mucous membranes.

43. Use of a vector for transferring nucleic acids as defined in one of claims 1 to 29 for the "manufacture of a medicament intended to treat diseases. :44. Method of transfecting nucleic acids into cells, characterized in that it comprises the following steps: *p p p synthesis of the oligonucleotide- targeting signal chimera, bringing the chimera synthesized in (1) into contact with a double-stranded DNA so as to form triple helices, optionally, complexing the vector obtained in with one or more transfection agents and/or one or more adjuvants, and bringing the cells into contact with the 74 complex formed in or, if applicable, in Method of treating diseases by administration of a vector for transferring nucleic acids as defined in any one of Claims 1 to 29 containing a double-stranded DNA capable of correcting the said disease.

46. Recombinant cell containing a nucleic acid transfer vector as defined in any one of claims 1 to 29. S* 47. Recombinant cell according to claim 46, characterized in that it is a eukaryotic cell.

48. Vector according to claim 1 substantially as described hereinbefore in any one of the Examples

49. Composition according to claim substantially as described hereinbefore in any one of the Examples

50. Use or method according to claim 43 or substantially as described hereinbefore in any one of the Examples.

51. Method according to claim 44 substantially as described hereinbefore in any one of the Examples

52. Cell according to claim 46 substantially as described hereinbefore in any one of the Examples Dates this 1 1 th day of December 2002. Aventis Pharma S.A. By its Patent Attorneys Davies Collison Cave to 0:1 0* S too* oS