MXPA98001920A - Pharmaceutical composition useful for the transfection of nucleic acids and its u - Google Patents

Abstract

The present invention relates to a pharmaceutical composition useful for the transfection of a nucleic acid characterized in that it contains in addition to said nucleic acid and at least one transfection agent, at least one compound that associates the DNA binding properties with a nuclear vectoring capacity of this DNA The use of said composition for the in vitro, ex vivo and / or in vivo transfer of nucleic acids is also reported.

Description

PHARMACEUTICAL COMPOSITION USEFUL FOR THE TRANSFECTION OF NUCLEIC ACIDS AND THEIR USES The present invention relates to the domain of gene therapy and is more particularly interested in in vitro, ex vivo and / or in vivo transfer of genetic material. It proposes in particular a new pharmaceutical composition useful for efficiently transfecting the cells. It is also related to the uses of this composition. Chromosomal deficiencies and / or anomalies (mutation, aberrant expression, etc.) are the origin of numerous diseases, a hereditary character or not. For a long time, conventional medicine remained impossible in its field. Currently, with the development of gene therapy, it is expected that this type of chromosomal aberration can be corrected or prevented in the future. This new medication consists of introducing a genetic information, in the affected cell or organ, in order to correct this deficiency or anomaly or even to express it in a protein of therapeutic interest. The main obstacle to the penetration of a nucleic acid into a cell or into a receptor organ lies in the size and polyanionic nature of this nucleic acid that opposes its passage through cell membranes. To overcome this difficulty, they are currently proposed REF: 26844 various techniques of which in particular the transfection of naked DNA through the plasmid membrane in vivo (WO90 / 11092) and the transfection of DNA via a transfection vector. In regard to the transfection of naked DNA, its efficiency remains very small. The naked nucleic acids have a short plasma half-life due to their degradation by enzymes and their elimination by the urinary tract. For what is the second technique, it proposes two strategies: The first uses the natural transfection vectors that are viruses. Thus, it is proposed to use adenoviruses, herpes viruses, retroviruses and, more recently, adeno-associated viruses. These vectors are tested by acting on the transfection plan, but unfortunately, some risks of pathogenicity, replication and / or immunogenicity, inherent in their viral nature, can not be totally excluded in their field. The second strategy is to advantageously use non-viral agents capable of promoting the transfer and expression of DNA in eukaryotic cells. The object of the present invention is particularly inscribed in this second strategy. The chemical or biochemical vectors represent an advantageous alternative with natural viruses in particular for this absence of immune response and / or viral recombination. They do not possess the pathogenic power, the risk of multiplication of DNA in these vectors is null and they are not subject to any theoretical limit in what refers to the size of the DNA to be transfected. These synthetic vectors have two main functions, to condense the DNA to be transfected and to promote its cellular fixation as well as its passage through the plasmic membrane and, if necessary, the two nuclear membranes. On the part of its polyanionic nature, DNA has no natural affinity for the plasmic membrane of cells of an equally polyanionic nature. To reduce this drawback, non-viral vectors generally have all the polycationic charges. Among the developed synthetic vectors, the cationic polymers of the polylysine and DEAE type dextran or even the cationic or lipofectant lipids are the most advantageous. They have the property of condensing DNA and promoting its association with the cell membrane. More recently, the concept of directed transfection, mediated by a receptor, was developed. This technique offers the benefit of the principle of condensing the DNA thanks to the cationic polymer, all of which is directed to the fixation of the complex in the membrane with the help of a chemical coupling eriirrc or the cationic polymer and the ligand of a membranal receptor., present on the surface of the cell type to be inserted. Screening of the transferrin receptor, insulin or the receptor for the asialoglycoproteins of hepatocytes have been described. However, the synthetic vectors proposed today are still far from acting as well as the viral vectors. In particular this may be the consequence of insufficient condensation of the DNA to be transfected and / or of the difficulties encountered by the transfected DNA to exit the endosome and penetrate the cell nucleus. Indeed, the transport of DNA in the nucleus of a resting eukaryotic cell exposes an obvious problem since the dimensions of the nuclear pores do not allow more than the diffusion of proteins of lower molecular weight to 60000 Da. (I. Davis et al., Ann.Rev. Biochem. 1995; 64; 865-896): A plasmid DNA having a molecular weight greater than 10c can therefore not naturally penetrate the cell nucleus by simple diffusion.

The object of the present invention is precisely to propose an advantageous solution to the aforementioned problem. More precisely, the present invention proposes a pharmaceutical composition useful for the transfection of at least one nucleic acid characterized in that it contains, in addition to said nucleic acid and at least one transfection agent, at least one compound that associates the DNA binding properties with a nuclear vectorization capacity of this DNA.

In the sense of the invention, a compound that possesses the DNA binding properties covers any compound capable of at least partially binding to the DNA to be vectorized. As regards more particularly its aptitude to vectorize this DNA, it is translated in the sense of the invention by an efficiency of directing this DNA efficiently through the different cellular and / or nuclear membranes to lead up to inside the nucleus of the cell to treat. The compound according to the invention can also be presented, for example, under the aspect of a chimeric molecule that associates a DNA binding domain with a domain that allows the nuclear amount. In this particular case, one can select between the DNA binding domains, those leaving regulatory proteins capable of binding, for example, to specific sequences or, conversely, of proteins known to possess a non-sequence dependent affinity for DNA. As regards more particularly the domain involved in the nuclear amount, it can be for example by a sequence of said nls (Nuclear localization Sequence). Such sequences could be apparent or derived from the bipartite consensus derived from the nucleoplasmin or the consensus of the SV40 T antigen.

According to a particular embodiment, the invention relates to a pharmaceutical composition useful for the transfection of at least one nucleic acid characterized in that it contains, in addition to said nucleic acid and at least one transfection agent, at least one compound belonging to the family of the HMG or one of its derivatives.

The HMG type proteins, by "High Mobility Group" are proteins rich in charged amino acids and have a molecular mass below 30000 Da. Soluble in 2-5% perchloric acid, they are extracted from the chromatin classically with 0.35 M NaCl.

Classically, 3 families of HMG proteins are distinguished: the proteins of the HMG1 / 2 type of neighboring molecular mass of 25000, HMG14 / 17 of neighboring molecular mass of 10-12000, and HMGI / Y of neighboring composition of the HMG14 type proteins / 17 but in which the tissue distribution and the development of ontogenesis is different. It is known that the primary sequence of proteins was conserved during evolution within one of these three families.

As regards more particularly the proteins of the HMG1 / 2 family, they are characterized by the presence of a sequence of 80 amino acids with basic predominance (net charge +20), ie "HMG box", which constitutes a binding domain of DNA In this family we can distinguish proteins capable of binding to specific sequences of double-stranded DNA and proteins of which the binding specificity resides in a particular three-dimensional structure of DNA. In the first category are the proteins UBF, SRY, TCF1, ABF2, which stimulate the transcription of specific genes (greiss E.A. et al., J. Mol, Evol. (1993) 37: 204-210). The sequence of each of these proteins contains one or more "HMG boxes". The tenth category is represented by the HMG1 and HMG2 proteins. Its primary sequence is characterized by the presence of two "HMG boxes" and one acid sequence in the C-term (Bustin M. B.B.A. (1990) 1049: 231-243). These proteins bind in a specific way on palindromic DNA sequences extruded in a cruciform structure (coupled intrahebra at the level of the palindrome) or on DNA sequences with a strong curvature. These two types of structure have in common to widen the small groove of DNA that then becomes able to accommodate the binding of HMG1 / 2 type proteins. The physiological role of the HMG1 and HMG2 proteins is currently poorly elucidated. It has been shown, however, that the calf HMG1 protein is actively transported in the nucleus of mammalian cells. (L. Kuehl et al.; J. Biol. Chem. 1985; 260, 10361-10368).

Unexpectedly, the applicant firm has demonstrated that it would be possible to benefit from these faculties of HMG proteins, to know their ability to fix DNA and to be transported actively in the nucleus, to efficiently promote transfection in the nucleus of the proteins. cells to be treated with heterologous nucleic sequences, associated with at least one transfectant agent. In the sense of the present invention, the term derivative designates any peptide, pseudopeptide (peptide incorporating the non-biochemical elements) or protein that differs from the compound as defined above, obtained by one or more modifications of a genetic and / or chemical nature . By modification of genetic and / or chemical nature can be understood any mutation, substitution, elimination, addition and / or modification of one or more residues for example of the considered protein. More precisely, by chemical modification, is meant any modification of the peptide or protein generated by chemical reaction chemical insertion of molecule (s), biological (s) or not, into any number of residues of the protein. By genetic modification, is meant any peptide sequence from which the hybrid DNA with sequences or fragments and from which the product possesses the indicated activities. Such derivatives can be generated for different purposes, such as particularly increasing the affinity of the corresponding polypeptide for its DNA ligand, improving its production levels, increasing its resistance to proteases, increasing and / or modifying one of its activities, or conferring new properties pharmacokinetics and / or biological. Among the derivatives resulting from an addition, there may be mentioned, for example, chimeric peptide sequences containing a supplementary heterologous part attached to a limb. The term derivative also comprises the homologous protein sequences of the considered sequence, obtained from other cellular sources and particularly cells of human origin, or from other organisms, and which possess an activity of the same type. Such homologous sequences can be obtained by hybridization experiments of the corresponding DNA. Hybridizations can be performed from nucleic acid libraries, using as a probe the native sequence or a fragment thereof, under the conditions of conventional astringency (Maniatis et al.), Or, preferably, molecular biology techniques. , in conditions of high astringency. Furthermore, it is also considerable within the framework of the present invention to benefit from the strong affinity of certain proteins of the HMG family, or their derivatives, by the secondary structures present in the double-stranded DNA. It can be in particular four-stranded structures for which it has been shown in particular that rat HMG1 possesses a strong affinity (Bianchi et al., Sciences, 1989, 243, 1056-1059). Such structures can also be obtained from natural sequences such as the ITRs of the virus associated with the adenoviruses or they can be completely synthetic obtained from artificial palindromas.

According to a preferred embodiment of the invention, the compound used is selected from the HMG 1, 2, I, Y, 14 and 17 type proteins and their derivatives. It is most preferably represented by all or part of the human HMG1 protein or one of its derivatives or homologs as defined above.

In a particularly advantageous embodiment, the compositions of the present invention additionally contain a targeting element which enables the transfer of the nucleic acid to be oriented. This targeting element can be an extracellular targeting element, which allows guiding the transfer of the nucleic acid towards certain cell types or certain desired tissues (tumor cells, liver cells, hematopoietic cells, etc.). It can also be an intracellular targeting element, which allows directing the transfer of nucleic acid to certain privileged cell compartments (mitochondria, nucleus, etc). More preferably, the targeting element is bound, covalently or non-covalently, to the compound according to the invention. The targeting element can also be linked to the nucleic acid. According to a preferred embodiment of the invention, said compound is associated, via a supplementary heterologous part joined to one of its extremities, to a ligand of the cellular receptor present on the surface of the cell type such as, for example, a sugar, transferrin , insulin or asialo-orosomucoid protein. It can also be an intracellular ligand type as a nuclear localization signal sequence, which privileges the accumulation of transfected DNA inside the nucleus.

Among the targeting elements useful within the framework of the invention, mention may be made of sugars, peptides, oligonucleotides or lipids. Preferably, these are sugars and / or peptides such as antibodies or fragments of antibodies, ligands of cellular receptors or fragments thereof, receptors or fragments of receptors, etc. In particular, they can be ligands of growth factor receptors, cytokine receptors, cell lectin receptors or adhesion protein receptors. The receptor for transferrin, HDL and LDL can also be mentioned. The targeting element can also be a sugar that allows targeting lectins such as asialoglycoproteic receptors, or even a Fab fragment of antibodies that allow targeting of the immunoglobulin Fe fragment receptor.

Advantageously, the compound according to the invention can also be polyglycosylated, sulphonated and / or phosphorylated and / or inserted into complex sugars or a lipophilic compound such as a polycarbonate chain or a cholesterol derivative.

The composition according to the invention can of course contain compounds according to the invention, of a different nature. Likewise, it is proved to be able to associate to the compound according to the invention, another nuclear targeting compound described above, a second compound characterized by its ability to compact the DNA. Such compounds are described in particular in the application FR 95/01865.

The compound according to the invention is present in an amount sufficient to act with the nucleic acid according to the invention. Thus, the compound / nucleic acid ratio (expressed by weight) can be between 0.01 and 5 and more preferably between 0.25 and 0.5.

As far as the transfection agent present in the composition according to the invention is concerned, it is preferably chosen from cationic polymers and lipofectants.

According to the present invention, the cationic polymer is preferably a compound of general formula I, wherein - R can be a hydrogen atom or a group of formula - n is an integer between 2 and 10; - p and q are integers, it being understood that the sum p + q is such that the average molecular weight of the polymer is between 100 and 107 Da. It is understood that, in the formula (I) the value of n can vary between the different elements p. Thus, formula (I) regroups both homopolymers and heteropolymers at the same time.

More preferably, in the formula (I), n is between 2 and 5. In particular, the polymers of polyethylene imine (PEI) and polypropylene imine (PPl) have all the advantageous properties. Preferred polymers for employing the present invention are those in which the molecular weight is between 103 and 5,106. By way of example, mention may be made of the polyethylene imine of average molecular weight 50 000 Da (PEI50K) or the polyethylene imine of average molecular weight 800 000 Da (PEI800K).

The PEI50K or PEI800K are commercially available.

As for the other polymers represented by the general formula I, they can be prepared according to the procedure described in the patent application FR 94 08735.

To obtain an optimum effect of the compositions of the invention, the respective proportions of the polymer and the nucleic acid are preferably determined in such a way that the molar ratio R = amines of the polymer / nucleic acid phosphates is between 0.5 and 50, more preferably between 5 and 30. All particularly advantageous results are obtained using 5 to 15 equivalents of polymer amines per nucleic acid charge.

As regards more particularly the lipofectants, they are amphiphilic molecules containing at least one cationic hydrophilic region, for example polyamine, and a lipophilic region. The cationic region, preferably polyamine, cationically charged, is capable of reversibly associating with the negatively charged nucleic acid. This interaction strongly compacts nucleic acid. The lipophilic region makes this ionic interaction inaccessible to the external aqueous medium, coating the nucleolipid particle forming a lipid film.

Advantageously, these lipofectants can also be chosen from lipopolyamines in which the polyamine region corresponds to general formula II where m is an integer greater than or equal to 2 and n is an integer greater than or equal to 1, m may vary between the different carbon groups comprised between two amines. Preferably, m is comprised between 2 and 6 inclusive and n is comprised between 1 and 5 inclusive. Even more preferably, the polyamine region is represented by the spermine, the terminus or one of its analogues which have retained the DNA binding properties. As regards the lipophilic region, it is represented by at least one hydrocarbon chain, saturated or otherwise, of cholesterol, a natural lipid or a synthetic lipid capable of forming the lamellar or hexagonal phases, covalently bound to the hydrophilic region.

Patent application EP 394 111 describes other lipopolyamines of general formula III which can be used in the context of the present invention H, N - (- (CH) rn-NH-) n-H II wherein R represents in particular a radical of the general formula (R: R2) N-CO-CH2-NH-CO-.

Representative of these lipopolyamines are, more particularly, dioctadecylamidoglycyl spermine (DOGS) and 5-carboxyespermilamide of palmitoylphosphatidylethanolamine (DPPES).

The lipopolyamines described in the patent application FR 94 14596 can also be used advantageously as the transfection agent according to the invention. They are represented by the general formula III above in which R represents with -X and X1 representing, independently of one another, an oxygen atom, a methylene group - (CH2) q- with q equal to 0, 1, 2 or 3, or an amino group -NH- or -NR ' - with R 'representing an alkyl group of Cx to C4, - Y and Y' independently representing one of the other a methylene group, a carbonyl group or a group C = S, - R3, R4 and R5 independently representing one another hydrogen atom or an alkyl radical, substituted or not, of Cx to C4, with p which may vary between 0 and 5, - R6 represents a derivative of the cholesterol or an amino-NR ^ alkyl group with Rx and R2 independently representing one of the another an aliphatic radical, saturated or not, linear or branched from C12 to C22.

Representative of these lipopolyamines are, in particular, all 1,3-bis- (3-amino-propylamino) -2-propyl (Dioctadecyl-carbamoylmethoxy) -acetate.

Patent applications EP 394 111 and FR 94 also describe a process useful for the preparation of the corresponding lipopolyamines.

In a particularly advantageous manner, the dioctadecyl idoglycyl spermine (DOGS), the 5-carboxyespermilamide of palmitoylphosphatidylethanolamine (DPPES), the 2,5-bis- (3-) dioctadecyl-carbamoylmethoxy) -acetate can be used within the framework of the invention. amino-propylamino) -pentyl or the 1,3-bis- (3-amino-propylamino) -2-propyl (Dioctadecyl-carbamoylmethoxy) -acetate.

To obtain an optimum effect of the compositions of the invention, the respective proportions of the polyamine and the nucleic acid are preferably determined in such a way that the ratio R positive charges of the transfection agent / negative charges of the nucleic acid is between 0.1 and 10 and more preferably between 0.5 and 2.

The presence of a compound according to the invention within a transfectant composition is advantageous in several ways. In particular, it follows a clearly diminished toxicity which makes possible for example the transfection of cells sensitive to the origin of the transfection agent, such as, for example, the hematopoietic cells with the lipopolyamines.

In the compositions of the present invention, the nucleic acid can be either a deoxyribonucleic acid or a ribonucleic acid. These may be sequences of natural or artificial origin, and in particular of genomic DNA, cDNA, MRNA, tRNA, rRNA, hybrid sequences or synthetic or semi-synthetic sequences. These nucleic acids may be of human, animal, plant, bacterial, viral, etc. origin. They can be obtained by any technique known to the person skilled in the art, and in particular by screening of banks, by chemical synthesis or even by mixed methods including chemical or enzymatic modification of sequences obtained by screening of banks. On the other hand they can be incorporated into vectors, such as plasmid vectors.

With respect more particularly to the deoxyribonucleic acids, they can be single or double stranded. These deoxyribonucleic acids can code for therapeutic genes, transcriptional or replication regulatory sequences, antisense sequences, regions of attachment to other cellular components, etc. In the sense of the invention, the therapeutic gene is understood in particular as any gene coding for a protein product having a therapeutic effect. The protein product thus encoded can be a protein, a peptide, etc. This protein product can be homologous to the target cell (ie a product that is normally expressed in the target cell when it does not present any pathology). In this case, the expression of a protein makes it possible, for example, to alleviate insufficient expression within the cell or the expression of an inactive or small active protein due to a modification, or even to overexpress said protein. The therapeutic gene can also encode a mutant of a cellular protein, which has an increased stability, a modified activity, etc. The protein product can also be heterologous to the target cell. In this case, an expressed protein can, for example, complete or provide a deficient activity inside the cell, which makes it possible to combat a pathology, or to stimulate an immune response.

Among the therapeutic products in the sense of the present invention, mention may be made more particularly of enzymes, blood derivatives, hormones, lymphokines: interleukins, interferons, TNF, etc. (FR 9203120), growth factors, neurotransmitters or their precursors or synthetic enzymes, trophic factors: BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NT5, HARP / pleiotrophin, etc; the apolipoproteins: ApoAI, ApoAIV, ApoE, etc (FR 93 05125), dystrophin or a minidistrofine (FR 9111947), the CFTR protein associated with mucoviscidosis, the tumor suppressor genes: p53, Rb, RaplA, DCC, -rev, etc. (FR 93 04745), the genes that code for factors involved in coagulation: Factors VII , VIII, IX, genes involved in DNA repair, suicide genes (thymidine kinase, cytosine deaminase), etc.

The therapeutic genes may also be a gene or an antisense sequence, of which expression within the target cell allows to control the expression of genes or the transcription of cellular mRNAs. Such sequences can, for example, be transcribed within the target cell into RNAs complementary to cellular mRNAs and thus block their translation into protein, according to the technique described in EP 140 308. The antisense also contains the sequences coding for ribosomes, which are capable of selectively destroying white RNAs (EP 321 201).

As indicated below, the nucleic acid may also contain one or more genes encoding an antigenic peptide, capable of generating an immune response in man or animal. Within this particular mode of use, the invention allows, as a consequence, the realization either of vaccines or of immunotherapeutic treatments applied to man or animal, particularly against microorganisms, viruses or cancers. It can be in particular antigenic peptides specific to Epstein Barr virus, HIV virus, hepatitis B virus (EP 185 573), pseudo-rabies virus, or even tumor-specific (EP 259 212).

Preferably the nucleic acid also contains the sequences that allow the expression of the therapeutic gene and / or the gene encoding the antigenic peptide within the desired cell or organ. These may be sequences that are naturally responsible for the expression of the gene considered when these sequences are capable of functioning within the infected cell. They may also be sequences of different origin (responsible for the expression of other proteins, or also synthetic). In particular, they may be promoter sequences of eukaryotic or viral genes. For example, they may be promoter sequences obtained from the genome of the cell to be infected. Also, they may be promoter sequences obtained from the genome of a virus. In this regard, mention may be made, for example, of the promoters of the genes E1A, MLP, CMV, RSV, etc. In addition, these expression sequences can be modified by addition of activation sequences, regulation sequences, etc. On the other hand, the nucleic acid may also contain, in particular higher than the therapeutic gene, a signal sequence that directs the therapeutic product synthesized in the secretion pathways of the target cell. This signal sequence may be the natural signal sequence of the therapeutic product, but it may also be another functional signal sequence, or an artificial signal sequence. More preferably, the compositions of the invention also contain one or more neutral lipids. Such compositions are particularly advantageous, in particular when the ratio R is small. The applicant has indeed demonstrated that the addition of a neutral lipid allows to improve the formation of nucleolipid particles and, surprisingly, to favor the penetration of the particle in the cell by destabilizing its membrane. More preferably, the neutral lipids used within the framework of the present invention are lipids of two fatty chains. Particularly advantageously, natural or synthetic lipids, zwitterionic or devoid of ionic charge under physiological conditions are used. They can be chosen more particularly between dioleoylphosphatidylethanolamine (DOPE), oleoyl-palmitoylphos-fatidylethanolamine (POPE), di-stearoyl, -palmitoyl, -myristoyl phosphatidylethanolamine as well as their N-methylated derivatives 1 to 3 times; phosphatidylglycerols, diacylglycerols, glycosyldiacylglycerols, cerebrosides (particularly such as galactocerebrosides), sphingolipids (particularly such as sphingomyelins) or even asialogangliosides (particularly such as asialoGMl and GM2). These different lipids can be obtained either by synthesis, either by extraction from organs (eg, the brain) or from eggs, by the classical techniques well known to the person skilled in the art. In particular, the extraction of natural lipids can be carried out by means of organic solvents (also see Lehninger, Biochemistry). Preferably, the compositions of the invention employ a lipofectant as the transfection agent, contain from 0.1 to 20 equivalents of neutral lipid per 1 equivalent of lipopolyamine, and, more preferably, from 1 to 5. In the case where the transfection agent is a cationic polymer, the compositions of the invention contain, more than the cationic polymer in the ratios cited above, from 0.1 to 20 molar equivalents of neutral lipid per 1 molar equivalent of nucleic acid phosphate, and, more preferably, from 1 to 5 .

The compositions according to the invention can be formulated for the purpose of topical, cutaneous, oral, rectal, vaginal, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal, etc. administration. Preferably, the pharmaceutical compositions of the invention contain an acceptable pharmaceutical carrier for an injectable formulation, in particular for a direct injection at the level of the desired organ, or for topical administration (in skin and / or mucosa). It can be, in particular, sterile, isotonic solutions or dry compositions, in particular freeze-dried, which, by addition according to the case of sterilized water or physiological saline, allow the constitution of injectable solutes. The doses of nucleic acid used for the injection as well as the number of administrations can be adapted according to the manner of administration used, the relevant pathology, the gene to be expressed, or even the duration of the investigated treatment. They can be used advantageously to transfect a wide variety of cell type such as, for example, hematopoietic cells, lymphocytes, hepatocytes, endothelial cells, melanoma, carcinoma and sarcoma cells, smooth muscle cells, neurons and astrocytes.

The present invention thus provides a particularly advantageous method for the treatment of diseases utilizing the in vitro, ex vivo or in vivo transfection of a nucleic acid capable of correcting said disease in association with a transfection agent of the cationic polymer or lipofectant type, and a compound as defined above. More particularly, this method is applicable to diseases resulting from a deficiency in a protein or nucleic product and the nucleic acid administered encodes said protein product or contains the sequence corresponding to said nucleic product. The compositions according to the invention are particularly interesting because of their bioavailability and transfection level.

The present invention also relates to any use of a compound according to the invention coupled to a ligand of the cellular receptor, an antibody or antibody derivative, directing a nucleic acid towards the cells expressing the corresponding receptors or anti-genes. Within this perspective, a ligand, antibody or potential antibody derivative is coupled to said compound and the transfection power of this chimeric molecule is appreciated comparatively to the compound alone.

The present invention will be described more fully with the help of the following examples, which should be considered as illustrative and not limiting.

FIGURES: Figure 1: Representation in luminous intensities of the efficiency of transfections carried out according to the invention in different cell types.

Figure 2: Appreciation of the efficiency of transfections performed in the presence and absence of HMGl.

MATERIALS AND METHODS: The construct used to demonstrate the activity of the compositions of the invention is a plasmid containing the gene coding for luciferase (Luc), pCMV-Luc.

The plasmid pCMV-luc contains the Cytomegalovirus (CMV) promoter, extracted from the vector plasmid pcDNA3 (Invitrogen) to cut with the restriction enzymes Mlu I and HindIII, located above the gene coding for luciferase, inserted in the Mlul sites and HindIII in the vector pGL basic Vector (Promega).

EXAMPLE 1: PREPARATION OF THE RAT HMGl PROTEIN 1. 1 Expression of the HMG1 protein in E. coli This recombinant protein that comes from a mammal has been prepared for overexpression in E. coli. Plasmid T7-RNHMG1 coding for the rat HMG1 protein (M. E. Bianchi, Gene, 104 (1991) 271-275) is introduced into E. coli strain BL21 (DE3). The strain is then grown at 37 ° C in LB + ampicillin medium (25 mg / L). A preculture is obtained from an isolated colony. It allows to plant a 500 ml culture. When the absorbance at 600 nm of the culture has the value of 0.7, the synthesis of HMGl is induced by the addition of IPTG. The cells are harvested immediately by centrifugation (5000 x g, 20 minutes), washed with 200 ml of distilled water, centrifuged again. The cell pack is stored at -80 ° C until purification. 1. 2: purification of the recombinant HMGl protein The purification of the HMGl protein can be carried out by chromatography from a culture of the E. coli strain described in example 1.1, for example using the following protocol: Unless otherwise indicated, the set of The purification described below is carried out at 4 ° C. Cells obtained from 500 ml of culture are resuspended in 15 + ml of 50 mM Tris / HCl buffer pH 7.7 containing 500 μM EDTA, 5 mM DTT, Pefabolc SC [4- (2-aminoethyl) -benzenesulfonyl fluroride hydrochloride] 200 + μM, and 10% glycerol (weight / volume). After cell disruption by sonication for 15 + min, the cell debris is separated by centrifugation (50,000 x g; lh). The cell extract is then chromatographed through a Sephadex G-25 column (Pharmacia) equilibrated and eluted with buffer A [20 mM Hepes pH 7.9 containing 400 mM sodium chloride, 200 mM EDTA, 1 mM DTT, 200 μM SC Pefabloc , 0.2% Nonidet P40, and 10% glycerol]. The fraction containing the proteins is collected and chromatographed through a DEAE Sephadex A-25 gel column (Pharmacia) equilibrated with buffer A. The protein fraction not retained in this column is progressively mixed with solid ammonium sulfate to a concentration end of 2.8 M. After 2 h, this suspension is centrifuged (30,000 xg, 15 min). The supernatant is chromatographed at 20 ° C through a Phényl-Superose HR 5/5 column (Pharmacia) equilibrated with the 20 mM Hepes buffer 7.9 containing 200 mM EDTA, 500 μM DTT, and 2.8 M ammonium sulfate. Proteins are eluted from the column with a linear gradient that decreases from ammonium sulfate (2.8 M to 0 M) in the same buffer. The fractions containing the HMGl protein are regrouped and extensively dialyzed against the buffer 50 mM Tris / HCl pH 7.7 containing EDTA lmM and DTT 500 μM. This sample is then injected into a MonoQ HR 5/5 column (Pharmacia) which is then eluted with a linear gradient of 0 to 0.5 M sodium chloride in the buffer 50 mM Tris / HCl pH 7.7-DTT 500 μM. The HMG1 protein, which forms a symmetrical absorbance peak at 280 nm, is collected in this buffer. The HMG1 protein is then placed in a 10 mM Mes buffer pH 6.2-140 mM NaCl-500 μM DTT after concentration by centrifugation in Centrikon 10. The HMG1 protein is stored at -80 ° C until use. This preparation presents a single protein band that migrates with an apparent molecular weight of 31,000 when analyzed by denaturing electrophoresis (SDS) and Coomassie revelation. The overall yield of the purification is 850 μg of pure HMGl protein per 500 ml of starting culture.

EXAMPLE 2: TRANSFER OF IN VITRO NUCLEIC ACID IN MAMMALIAN CELLS This example shows how a protein, of HMG1 type, binds to the DNA being actively imported into the nucleus, can be used to stimulate the transfer of plasmid DNA. The construct used to demonstrate the activity of the compositions of the invention is the plasmid containing the gene coding for luciferase (Luc) described above. The protocol is established for 24 well plates (D 16 mm) to collect 2 days after transfection (cells in confluence). All parameters can be modified proportionally. On day J NIH 3T3 cells (ATCC: CRL1658), 3LL (Isakov N et al., JNCI 71 (1983) 139-145) H460 (Maxwell et al., Oncogene 8 (1993), 3421-3429) are seeded with 105 cells per well. On day J + 2 the cells are rinsed with PBS (to remove traces of serum) and put in 250 ml of RPMI (3LL and H460) or DMEM (NIH3T3), supplemented or not with 10% Bovine Fetal Serum ( SFV) Composition used for transfection: By well equivalent, in a tube is added: - H20 qsp 20 ml - NaCl qsp 140 mM final - 0.5 mg of plasmid DNA - 125 ng of HMGl and then vortex moderately and incubated at room temperature 15 minutes before adding 1.5 nmol of lipofectant DOGS. Vortex again moderately and incubate at room temperature for 15 minutes Cells are transfected by adding 20 ml of DNA / HMGl / lipofectant mixture to the culture medium, incubated for 2 to 4 hours at 37 ° C. This medium is replaced immediately by complete means. On day J + 4 the cells are rinsed at room temperature with 250 ml of PBS, lysed in 100 ml of ad hoc buffer (Repórter (Promega) + TCK and Aprotinin). 10 ml of lysate and 50 ml of substrate (Promega) are used to measure the activity of the synthesized luciferase. The results presented in figures 1 and 2 are the average of four experiments, repeated independently 2 times. To do this we can see the Luminous Units (UL) obtained by expressing the Luc gene in the transfected cells.

Figure 1 groups the values obtained by the three cell types mentioned above, in the presence of the variable amount of HMGl protein. Figure 2 groups in a synoptic manner the transfection stimulation factors obtained by adding different amounts of HMGl proteins. The increase in transfection efficiency by the HMG1 protein is variable according to cell types. It is thus observed that it is maximized when the medium containing the composition necessary for the transfection is supplemented with 10% SFV. This is interesting because the presence of SFV represents the conditions found in vi. On the other hand it is notable that the presence of SFV decreases transfection efficiency, particularly in the absence of HMGl protein. It can be thought that the presence of SFV in the culture medium decreases the amount of DNA that can be internalized by the cells. In this framework it can be concluded that HMG1 is particularly advantageous for transfection in conditions where the amount of DNA is limiting. The optimal HMG1 / DNA ratio (mass) for transfection is 0.25 to 0.5. Such conditions are not described as capable of compacting the DNA (Bottger M. et al., B.B.A. 950 (1988), 221-228); Stros M. et al., N.A.R. 22 (1994), 1044-1051). In effect, the plasmid is not saturated by HMG1 (Kohlstaedt L.A. et al., Biochemistry 33 (1994), 12702-12707). Consequently, the effect of the HMG1 protein is explained by its ability to bind to DNA and to be transported to the nucleus of the cell.

Claims (25)

1. A pharmaceutical composition useful for the transfection of at least one nucleic acid, characterized in that it contains, in addition to said nucleic acid and at least one transfection agent, at least one compound that associates the DNA binding properties with a nuclear vectorization capacity of this DNA.

2. A pharmaceutical composition useful for the transfection of at least one nucleic acid, characterized in that it contains, in addition to said nucleic acid and at least one transfection agent, at least one compound belonging to the HMG family or one of its derivatives.

3. Pharmaceutical composition according to claim 1 or 2, characterized in that the compound is chosen from proteins of type HMG 1, 2, I, Y, 14 and 17 and their derivatives.

4. Pharmaceutical composition according to one of the preceding claims, characterized in that the compound is present in all or part of the human HMG1 protein, one of its derivatives or homologs.

5. Pharmaceutical composition according to one of claims 1 to 4, characterized in that said compound is further polyglycosylated, sulfonated, phosphorylated and / or inserted into the complex sugars or a lipophilic agent.

6. Pharmaceutical composition according to one of claims 1 to 5, characterized in that said compound is further associated with a ligand of the cellular or nuclear receptor.

7. Pharmaceutical composition according to one of the preceding claims, characterized in that the transfection agent is a cationic polymer or a lipofectant.

8. Pharmaceutical composition according to claim 7, characterized in that the cationic polymer is preferably a compound of general formula (I): wherein - R can be a hydrogen atom or a group of formula - n is an integer between 2 and 10; - p and q are integers, it being understood that the sum p + q is such that the average molecular weight of the polymer is between 100 and 107.

9. Pharmaceutical composition according to one of claims 7 or 8, characterized in that the cationic polymer is selected from polyethylene imine (PEI) and polypropylene imine (PPl).

10. Pharmaceutical composition according to claim 9, characterized in that the polymer is chosen from polyethylene imine of average molecular weight 50 000 Da (PEI50K) or polyethylene imine of average molecular weight 800 000 Da (PEI800K).

11. Pharmaceutical composition according to claim 7, characterized in that the lipofectant comprises at least one hydrophilic polyamine region of general formula II H2N - (- (CH) m-NH-) n-H II wherein m is an integer greater than or equal to 2 and n is an integer greater than or equal to 1, m may vary between different carbon groups comprised between two amines covalently associated with a lipophilic region of the hydrocarbon chain type, saturated or not, cholesterol, a natural or synthetic lipid capable of forming the lamellar or hexagonal phases.

12. Pharmaceutical composition according to claim 11, characterized in that the polyamine region is represented by the spermine, the end, or one of its analogues that have retained the nucleic acid binding properties.

13. Pharmaceutical composition according to claim 11, characterized in that the lipophilic region and is represented by the general formula IV wherein - X and X 'representing, independently of one another, an oxygen atom, a methylene group - (CH2) q- with q equal to 0, 1, 2 or 3, or an amino group -NH- or -NR'- with R1 representing an alkyl group of Cx to C4, - Y and Y 'independently represent one of the other a methylene group, a carbonyl group or a group C = S, - R3, R4 and R5 independently represent one of the another a hydrogen atom or an alkyl radical, substituted or not, of Cj. to C4, with p which may vary between 0 and 5, - R6 represents a derivative of cholesterol or an amino-NR ^ alkyl group with Rt and R2 independently representing one of an aliphatic, saturated or non-saturated, linear or branched radical of C12 to C22.

14. Pharmaceutical composition according to one of claims 11 to 12 or 13, characterized in that it is preferably a lipopolyamine chosen from dioctadecylamidoglycyl spermine (DOGS), 5-carboxyespermilamide of palmitoylphosphatidylethanolamine (DPPES), (Dioctadecyl-carbamoylmethoxy) - 2, 5-bis- (3-amino-propylamino) -pentyl acetate or the 1,3-bis- (3-amino-propylamino) -2-propyl (Dioctadecyl-carbamoylmethoxy) -acetate.

15. Pharmaceutical composition according to any of claims 1 to 7, 11 to 12 and 14, characterized in that the transfection agent is the dioctadecylamidoglycyl spermine (DOGS).

16. Pharmaceutical composition according to one of the preceding claims, characterized in that the nucleic acid is a deoxyribonucleic acid.

17. Pharmaceutical composition according to one of the preceding claims, characterized in that the nucleic acid is a ribonucleic acid.

18. Pharmaceutical composition according to claim 16 or 17, characterized in that the nucleic acid is chemically modified.

19. Pharmaceutical composition according to claim 16, 17 or 18, characterized in that the nucleic acid is an antisense.

20. Pharmaceutical composition according to one of claims 16 to 19, characterized in that the nucleic acid contains a therapeutic gene.

21. Pharmaceutical composition according to one of the preceding claims which also contains one or more neutral lipids.

22. Pharmaceutical composition according to claim 21, characterized in that the neutral lipid (s) are chosen from synthetic or natural lipids, zwitterionic or devoid of ionic charge under physiological conditions.

23. Pharmaceutical composition according to claim 21 or 22, characterized in that the neutral lipid (s) are chosen from dioleoylphosphatidylethanolamine (DOPE), oleoyl-palmitoylphos-fatidyl-ethanolamine (POPE), di-stearoyl, -palmitoyl, -myristoyl phosphatidylethanolamine as well as their N-methylated derivatives 1 to 3 times; phosphatidylglycerols, diacylglycerols, glycosyldiacylglycerols, cerebrosides (particularly such as galactocerebrosides), sphingolipids (particularly such as sphingomyelins) and asialogangliosides (particularly such as asialoGMl and GM2).

24. Use of a pharmaceutical composition according to one of claims 1 to 23 for the in vitro, ex vivo and / or in vivo transfer of nucleic acids.

25. Use of a compound as defined in claim 1 or 2, coupled to a ligand of the cellular receptor, an antibody or antibody derivative, directing a nucleic acid towards the cells expressing the corresponding receptors or anti-genes.