MXPA99010765A - Novel class of nucleic acid cationic transfecting agents - Google Patents

Abstract

The invention concerns a novel class of nucleic acid cationic transfecting agents, characterised in that the transfecting agents comprise at least a lipophilic region associated with a cationic hydrophilic region, said cationic hydrophilic region consisting of a heterocycle with 5 or 6 atoms substituted by amino groups.

Description

NEW CLASS OF TRANSFEC AGENTS BEFORE CATIONIC OF NUCLEIC ACIDS.

The present invention relates to a new class of cationic transfectants, to pharmaceutical compositions containing them, to their applications for in vivo, ex vivo and / or in vitro transfection of nucleic acids and their methods of preparation.

With the development of biotechnologies, it is from this that it is possible to transfer nucleic acids into cells. The efficiency of these transfers seems necessary for the correction of expression defects and / or abnormal expression of nucleic acids involved in numerous genetic diseases. However, the interest of nucleic acid transfers may be useful for the. study of the regulation of gene expression, cloning of genes, or for any other manipulation in vi tro that involves nucleic acids, as well as for the production of recombinant proteins. It can also be nucleic acids in vivo, for example for the preparation of transgenic animals, the production of vaccines, and the labeling of molecules. On the other hand, the acid transfer REF. 31920 nucleic acids can be performed on ex vivo cells, on attempts at bone marrow grafts, on immunotraping or on other methods that involve the transfer of genes into cells sampled from an organism in order to be readministered later.

Currently, several methods are proposed for the intracellular release of this type of genetic information. One of them, in particular, relies on the use of chemical or biochemical vectors. These synthetic vectors have two main functions: to complex the DNA to be transfected and to promote its cellular fixation as well as its passage through the plasmic membrane and, if necessary, through the two nuclear membranes. Among the synthetic vectors developed, the cationic polymers of the polylysine and DEAE dextran type or even the lipofectants are the most advantageous.

An important progress has been achieved in this mode of transfection with the development of a technology based on the use of cationic transfection agents of lipofectant type, and more precisely of cationic lipids. Thus it has been shown that a positively charged cationic lipid, N- [1- (2, 3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride (DOTMA), interfered, in the form of liposomes or of small bladders, spontaneously with DNA, which is negatively charged, to form DNA-lipid complexes, capable of fusing with cell membranes, and thus allowing the intracellular release of DNA.

After DOTMA, other cationic lipids have been developed on this same structure model, ie a lipophilic group coupled to an amino group via an arm still called * spacer. "Among these, we can more particularly cite those comprising lipophilic group fatty acids or a derivative of cholesterol, and which also contains, if necessary, as an amino group, a quaternary ammonium group, DOTAP, DOBT or ChOTB can be mentioned in particular as representative of this category of cationic lipids Other compounds, such as DOSC and ChOSC, which are characterized by the presence of a choline group instead of the quaternary ammonium group.

Another category of lipofectants, lipopolyamines, has also been described. Generally, it is an amphiphilic molecule comprising at least one hydrophilic region constituted by a polyamine coupled via a "spacer" to a lipophilic region. The polyamine region, positively charged, is capable of reversibly associating with the negatively charged nucleic acid. This interaction strongly compacts the nucleic acid. As for the lipophilic region, it converts this insensitive ionic interaction to the external medium, coating the formed complex of a lipid film. In this type of compounds, the cationic group can be represented by the radical L-5-carboxiespermine, which contains four ammonium groups, two primary and two secondary. The DOGS and the DPPES are particularly part of it. These lipopolyamines are very particularly effective for the transfection of primary endocrine cells. Representative of this last family of compounds is the lipopolyamines described, for example, in patent applications WO 96/17823 and WO 97/18185.

However, the efficiency of these synthetic vectors remains to be improved particularly in terms of charge density and stiffness of the transfectant molecule.

In fact, it is generally accepted that the activity of these products depends on the density of load they cause to intervene. However, the increase of the charges of the aliphatic chains is the origin of the appearance of the toxicity. A stabilization of the charge density of the aliphatic chains would therefore prevent the appearance of toxic factors. In addition, the efficiency of the transfection would be increased by Tm increase in charge density in a region different from that of the aliphatic chains.

On the other hand, the flexibility of the molecule used for the transfer of a nucleic acid can be an obstacle to a sufficiently close interaction between said nucleic acid and the transfectant agent, preventing obtaining an optimal compaction of the nucleic acid molecule, being the compaction a necessary condition for all transfection An increased rigidity of the transfectant agent molecule will ensure a more efficient interaction with the nucleic acid, thus improving transfection.

Thus, it would be particularly interesting to have transfectants that have an increased transfection capacity, always having a reduced or no toxicity. These are the objectives that the present invention aims to achieve.

The object of the present invention is precisely to propose a new class of transfectant agents which have an original cationic hydrophilic region which confers said properties with the aforementioned particular properties.

More precisely, the present invention concerns a transfectant agent comprising at least one cationic hydrophilic region coupled to a lyophilic region, said cationic hydrophilic region, which is constituted by at least one heterocycle of 5 or 6 atoms substituted by amino groups.

The positively charged hydrophilic region is capable of reversibly associating with a negatively charged nucleic acid, which is thus strongly compacted. In addition, the original structure of the cationic part gives the molecule an increased rigidity.

The cationic hydrophilic region, intermediate in the synthesis of the nucleic acid transfer agents according to the invention, is more particularly represented by the general formula (I).

(I) in which: . and is an integer equal to 0 or 1, the different ones that are independent of each other, . X represents an atom of oxygen, nitrogen, sulfur or selenium, Z groups represent, independently of each other, a hydrogen atom, an OR group in which R represents a hydrogen atom, a methyl group, or a group (CH2) n-NR? R2 in which n is an integer selected from 1 to 6 included and Ri and R2 represent, independently one on the other, a hydrogen atom or a group (CH2) q-NH2, q that can vary from 1 to 6 included, the different q that are different from each other, - a group (CH2) m-NR? R2, in which m is an integer selected from 0 to 6 included and Ri and R2 are defined as above, or a group * spacer "that allows the union of the hydrophilic region cationic to the lipid region, it being understood that at least one of the substituents Z carries an amino group.

The transfer agents according to the invention contain at least one cationic hydrophilic region of general formula (I). According to a variant of the invention, the transfer agents comprise 2 or several cationic hydrophilic regions which are linked to one another at the level of one. of the Z groups.

According to a preferred embodiment of the invention, when one of the substituents Z represents OR and R represents a group (CH2) n-NR? R2, when n is preferentially selected from 2, 3 or 4.

According to an advantageous variant of the invention, the transfectant agents contain a cationic hydrophilic region of general formula (I) in which X represents an oxygen atom. The hydrophilic cationic part is then constituted by a glycoside of pyranose or furanose form. The pyranose form is particularly interesting and has been described in a non-limiting manner in the examples.

According to a second advantageous variant of the present invention, the transfectant agents comprise a cationic hydrophilic region of general formula (I) composed of a 6-membered aminated glycoside (and present on the cycle is equal to), X is an oxygen atom and at least one of the substituents Z contains an amino group. Even more preferably, at least two of the substituents Z contain an amino group.

According to another variant of the present invention, the cationic hydrophilic region is composed of an aminated glycoside of formula (I), in which the two y are equal to, X is an oxygen atom, two of the Z groups represent hydrogen atoms , two other groups Z are nitrogen groups and preferentially amino groups, and the last group Z represents a group 'OR, R defined as above. Preferably, the OR group is located in position -2 on the heterocycle constituting the hydrophilic cationic region of the transfectants according to the invention.

According to another advantageous embodiment of the invention, the cationic hydrophilic region is composed of an aminated glycoside of general formula (I), in which the y present on the cycle is equal to X, is an oxygen atom, at least two groups Z correspond to groups 0- (CH2) q-NH2, q which are defined as above, and at least one of the groups Z represents a group OR, R which are defined as above.

As representative of cationic hydrophilic regions of general formula (I), intermediates useful in the synthesis of the transfer agents according to the invention, the following compounds can be mentioned more particularly: Generally speaking, the transfectants in the sense of the present invention contain at least one cationic hydrophilic region such as that defined above associated with a lipophilic region.

The molecules constituting the lipophilic region in the sense of the invention are selected from lipophilic molecules known to the person skilled in the art. Advantageously, the lipophilic region is constituted by one or more linear or branched aliphatic chains, saturated or not, optionally halogenated. The lipophilic region can also be advantageously selected from the steroid derivatives.

According to a preferred variant of the invention, the lipophilic part consists of one or more aliphatic chains containing 10 to 22 carbon atoms, and even more preferably 12 to 22 carbon atoms. By way of example, mention may be made of aliphatic chains containing 14, 16, 17, 18 or 19 carbon atoms, and particularly (CH 2) 3 3CH 3, (CH 2) 5 5CH 3, (CH 2) 6 6CH 3, (CH 2) 17 CH 3 , Y The lipophilic part of the transfer agents according to the invention can also be advantageously selected from steroid derivatives, for example cholesterol, cholestanol, 3- a-5-cyclo-5- a-cholestan-6-ß-ol, colic acid, cholesteryl formate, cholestanyl formate, 3a, 5-cyclo-5a-cholestan-6-yl formate, cholesteryl amine, 6- (1, 5-dimethylhexyl) -3a, 5a-dimethylhexadecahydro-cyclopenta [a] cyclopropa [2, 3] cyclopenta [1, 2-f] naphatalen-10-ylamine, or cholestanylamine.

According to a preferred embodiment, the hydrophilic cationic region is coupled to the lipophilic region by an intermediate molecule called "spacer." For the purposes of the invention, the term "spacer" is understood to mean any acidic or amine substituent containing hydrolyzables that allow amide, carbamate, ester, ether linkages or via an aromatic cycle between the cationic hydrophilic part, and the lipophilic region, known to the person skilled in the art The coupling between the hydrophilic part and the lipophilic part is preferentially operated at the level of one of the groups Z present on the cationic hydrophilic inte rdiary of formula (I) and useful for the synthesis of the transfer agents according to the invention Advantageously, the coupling between the two hydrophilic and lipophilic regions is effected in position -2 or in position -5 or -6 (according to whether and present on the cycle is equal to 0 or 1) on the hydrophilic part cycle cationic of general formula (I), via a "spacer" group.

Preferably, the "spacer" region contains an aliphatic or aromatic chain. In addition, the "spacer" advantageously contains one or more groups selected from the group amide, carbamate, ester, ether or aromatic rings. "Preferred spacers are particularly those of the formula -O-CO- (CH2) x-C00H, -O- (CH2) x -COOH, -O-CO- (CH2) / -NH2, -O- (CH) X- NH2, -NH- (CH2) X-NH2, with x representing an integer selected from 1 to 6 included.

As representative of transfectant agents according to the invention, compounds' (16), (17) and (8a) and (8b) of formulas can be more particularly cited.

The present invention also concerns the methods of preparing a new class of nucleic acid transfection agents as defined above. More precisely, it concerns the methods of preparing said transfectant agents from a mono- or polysubstituted hetrocycle to which a lipid region is coupled by the intermediary of a "spacer".

The cationic hydrophilic region of general formula (I) can be prepared in different ways, depending on the relative position of the amine functions on the cycle and their number.

When it is desired to obtain a cationic hydrophilic region of general formula (I) for which at least one of the susbstituents Z is a * spacer "that allows attachment to the lipophilic part and at least one of the Z groups is an OR group with R which represents a group (CH2) n-NR? R2, is operated from the corresponding derivative for which all the susbstituent O- (CH2) n-NR? R2 are hydroxy functions.

In a first step, said derivative containing the hydroxy functions undergoes an O-alkylation according to the classical methods known to the person skilled in the art. Particularly, it is operated with the aid of an alkylating agent in basic medium in the conventional alkylation solvents, in the presence of a crown ether and at a temperature comprised between 10 and 60 ° C.

Alkylamines, halogenated derivatives of esters, such as, for example, halogen alkyl acetate, or halogenated derivatives of alcohols are used in particular as the alkylating agent. Preferably, alkyl bromoacetate is used, the alkyl functions are selected depending on the value desired n. For example, to have n equal to 2, ethyl bromoacetate is preferably used.

The solvents used are the conventional solvents of the O-alkylations, for example dimethylformamide, dimethylethylacetamide, dimethylsulfoxide, acetonitrile, tetrahydrofuran (THF), etc. Advantageously, THF is used.

The reaction is carried out in the presence of a base, for example potassium hydroxide or sodium hydride.

In a second step, the carboxy functions of the derivative are reduced in alcohol according to the classical methods known to the person skilled in the art. The action is particularly carried out by the action of a reducing agent in conventional compatible solvents.

As a reducing agent, mention may be made, for example, of borane dimethyl sulphide (BMS), lithium aluminum hydride or sodium borohydride.

Suitable solvents are, for example, ethers or alcohols. Preferably, tetrahydrofuran (THF) is used.

In a third step, the hydroxy functions obtained are azidated by conventional methods known to the person skilled in the art.

The action is particularly carried out by the action of the hydrazoic acid in the presence of triphenylphosphine and of diethylazodicarboxylate in a solvent compatible with the reaction.

The usable solvents are the classic azidation solvents such as for example tetrahydrofuran, benzene, toluene, chloroform, dichloromethane, etc ..., and preferably tetrahydrofuran THF).

Finally the functions azides. they are transformed into amino functions by the classical methods known to the person skilled in the art.

In particular, reduction is carried out in an acid medium, for example by hydrogenation in an acid medium in the presence of palladium on carbon, or by applying the Staudinger reaction, or even by the action of a reducing agent, for example lithium aluminum hydride, sodium borohydride, stannous chloride, etc ...

A cationic hydrophilic compound of general formula (1) is thus obtained for which at least one of the substituents Z is a * spacer "that allows the union of the lipophilic part and at least one of the groups Z is an OR group with R representing a group - (CH2) n-NR? R2.

The coupling in the lipophilic part is then carried out by the classical methods common to those skilled in the art, and particularly by peptide coupling (Bodans iM., Principles and Practice of Peptides Synthesis, Ed. Springe-Verlag). The coupling intervenes at the level of group Z that represents a * spacer. " When one of the Z is a "spacer" group, this is, if necessary, previously protected. It is the same for amine functions carried by the cationic hydrophilic part that are preferably protected prior to peptide coupling. The protection and removal of the protective radicals are carried out according to the usual methods.

The protection can be carried out by any compatible group and whose application and elimination does not alter the rest of the molecule. Particularly, one proceeds according to the methods described by T. W. GREENE, Protective Groups in Organic Syntheses, A. Wiley-Interscience Publication (1981), or by Me OMIE, Protective Groups in Organic Chemistry, Plenum Press (1973).

By way of example, the protecting groups may be selected from the group consisting of trimethylsilyl, benzyl, tetrahydropyranyl, formyl, acetyl, chloracetyl, trichloroacetyl, trifluoroacetyl, ethoxycarbonyl, tert-butoxycarbonyl, trichloroethoxycarbonyl, etc.

Another synthesis route allows to conclude in a second series of cationic hydrophilic compounds. This second embodiment-differs from the first in which the carbohydrate skeleton is transformed.

In effect, the starting product used is a glycal of general formula (II): for which _ the substituents Z 'are acetoxy functions. The glycols used are commercially available or are obtained by any method known to those skilled in the art from commercial glucides, and in particular by reactions of the corresponding acetobromoazugars with the zinc / copper couple.

In a first step, said acetylated glycal is rented according to the classical methods known to the person skilled in the art.

Particularly, the Ferrier reaction is applied per action, in the presence of a Lewis acid, an aminoalcohol, an alkylcarbonylalcohol, a carboxyalcohol, or any other alcohol functionalized by a group that allows coupling in a lipophilic region.

In a second step, the unsaturated glycoside obtained is reduced according to the classical methods known to the person skilled in the art.

In particular, a reduction is carried out in an acid medium, for example by hydrogenation in an acid medium in the presence of palladium on carbon.

Then, in a third stage, the acetoxy functions present on the cycle are transformed into hydroxy functions by any method known to the person skilled in the art.

In particular, the process is carried out by transesterification with the aid of an alcoholate, for example sodium methanoate.

In a fourth step, the hydroxy functions present on the cycle are transformed into azide functions by any method known to the person skilled in the art.

In particular, it is carried out, for example, by the action of hydrazoic acid in the presence of triphenylphosphine and diethylazodicarboxylate in a solvent compatible with the reaction.

The usable solvents are the classical azidation solvents, and preferably tetrahydrofuran (THF).

* A cationic hydrophilic region of general formula (III) is thus obtained: (III) for which Z "is either a hydrogen atom, or an azide group, at least one of the Z" that is different from hydrogen.

The lipophilic part can be coupled to the amino function -NR1R2 according to methods known to those skilled in the art, particularly by peptide coupling (Bodanski M., Principies and Practices of Peptides Synthesis, Ed. Springe-Verlag), or even by condensation .

The alcohol applied in the course of the first stage containing an amino function, ester or any other function that allows coupling in a lipophilic region, it is preferable to previously protect it.

The protection and removal of the protective radical before coupling with the lipophilic region are carried out according to the usual methods.

The protection can be carried out by any compatible group and whose application and elimination does not alter the rest of the molecule. Particularly, one proceeds according to the methods described by T. W. GREENE, Protective Groups in Organic Syntheses, A. Wiley-Interscience Publication (1981), or by Me OMIE, Protective Groups in Organic Chemistry, Plenum Press (1973).

By way of example, the protecting groups may be selected from the group consisting of trimethylsilyl, benzyl, tetrahydropyranyl, formyl, acetyl, chloracetyl, trichloroacetyl, trifluoroacetyl, ethoxycarbonyl, tert-butoxycarbonyl, trichloroethoxycarbonyl, fatlimid, etc ...

Finally, in a first stage, the azide functions contained in the 5 or 6 link cycle are transformed into amino functions by the classical methods known to those skilled in the art that do not alter the rest of the molecule. Preferentially, it proceeds by the action of triphenylphosphine in the presence of water.

Any method known to the person skilled in the art that leads to the nucleic acid transfer agents according to the present invention also falls within the scope of the invention.

By way of non-limiting example, the first embodiment of the invention leads to the transfer agents of formula (8a) and (8b), while the second embodiment leads to agents of formula (16) and ( 17), as what is specified in the "Examples" part of the present description.

The interest of this second route of synthesis of hydrophilic cationic regions resides mainly in the few necessary steps and in the versatility of the aminated concordance obtained. The same cationic head can thus be condensed with different hydrophobic substituents.

Another object of the invention concerns a composition comprising a nucleic acid transfer agent as defined above and a nucleic acid. Preferably, the compositions comprise a ratio of 0.1 to 50 nahomoles of vector per μg of nucleic acid. Advantageously, this ratio is between 2 and 20 nanomoles of vector per μg of acid and even more preferably between 4 and 12 nanomoles of vector per μg of nucleic acid, more particularly the transfer agent and the nucleic acid are present in a ratio from 8 to 12 nanomoles of vector per μg of nucleic acid.

In the sense of the invention, "dexyribonucleic acid" and "ribonucleic acid" are understood as meaning "nucleic acid". These may be natural or artificial sequences, and particularly genomic DNA (gDNA), complementary DNA (cDNA), messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), hybrid sequences or of synthetic or semi-synthetic sequences, of oligonucleotides modified or not. These nucleic acids can 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 particularly by screening of banks, by chemical synthesis, or even by methods mixed that include the chemical or enzymatic modification of sequences obtained by bank screening. They can be chemically modified. _ As regards, more particularly, the deoxyribonucleic acids, they can be single or double filaments as well as short oligonucleotides or longer sequences. In particular, the nucleic acids are advantageously constituted by plasmids, vectors, episomes, expression cartridges, etc. These deoxyribonucleic acids can contain a replication origin functional or not in the target cell, one or more indicator genes, regulatory sequences of the transcription or of the replication, genes of therapeutic interest, antisense sequences modified or not, regions of binding to other cellular components, etc ...

Preferably, the nucleic acid comprises an expression cartridge constituted of one or several genes of interest under the control of one or more promoters and of a transcriptional terminator active in the target cells.

In the sense of the invention, the term "gene of therapeutic interest" is understood particularly as any gene coding for a protein product having a therapeutic effect. The therapeutic product thus encoded may be in particular a protein or a peptide. This protein product thus encoded can be, in particular, a protein or a peptide. This protein product can be exogenous homologous or endogenous in relation 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 deactivate an insufficient expression in the cell or the expression of an inactive or weakly active protein due to a modification, or even to over-express said protein. The gene of therapeutic interest can thus encode a mutant of a cellular protein, which has an increased stability, a modified activity, etc ... The protein product can also be heterologous in relation to the target cell. In this case, an expressed protein can, for example, complete or provide a deficient activity in the cell, allowing it to fight against a pathology, or stimulate an immune response.

Among the therapeutic products in the sense of the present invention, mention may be made more particularly of enzymes, blood products, hormones, lymphokines: interleukins, interferons, TNF, etc. (FR 92/03120), growth factors , neurotransmitters or precursors or synthesis enzymes, trophic factors (BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NT5, HARP / pleiotrophin, etc.) apolipoproteins (ApoAI, ApoAIV, ApoE, etc ..., FR 93/05125), dystrophin or a minidistrofin (FR 91/11947), CFTR protein associated with mucoviscidosis, tumor suppressor genes (p53, Rb, RaplA, DCC, k-rev, etc. , FR 93/04745), the genes that code for factors involved in coagulation (Factors VII, VIII, IX), the genes involved in DNA repair, the suicide genes (thymidine kinase, cytosine deaminase), the genes of hemoglobin or other protein transporters, enzymes of metabolism, catabolism, etc ...

The nucleic acid of therapeutic interest can also be a gene or an antisense sequence, whose expression in the target cell allows controlling the expression of genes or the transcription of cellular RNAs. Such sequences can, for example, be transcribed in the target cell in RNA complementary to cellular RNA and thus block its translation into protein, according to the technique described in EP 140 308. The therapeutic genes also include the sequences coding for ribozymes, which are capable of selectively destroying target RNAs (EP 321 201).

As indicated above, the nucleic acid may also contain one or more genes encoding an antigenic peptide, capable of generating an immune response in man or animal. In this particular embodiment, the invention allows the realization either of vaccines or of immunotherapeutic treatments applied to man or animal, particularly against microorganisms, viruses or cancers. It can be particularly antigenic peptides specific to Epstein Barr virus, HIV virus, hepatitis B virus (EP 185 573), pseudo rabies virus, syncitia forming virus ", other viruses or even specific anigenic peptides. of tumors (EP 259 212).

Preferably, the nucleic acid also comprises sequences that allow the expression of the gene of therapeutic interest and / or of the gene coding for the antigenic peptide in the cell or the desired organ. These may be sequences that are naturally responsible for the expression of the gene considered when these sequences are capable of functioning in the infected cell. It can also be sequences of different origin (responsible for the expression of other proteinsor also synthetic). In particular, they may be promoter sequences of eukaryotic or viral genes. For example, they may be promoter sequences originating from the genome of the cell to be infected. Likewise, they may be promoter sequences originating from the genome of a virus. In this regard, we can mention, for example, the gene promoters E1A, MLP, CMV, RSV, etc ... In addition, these expression sequences can be modified by the addition of activation sequences, regulation, etc ... be promoter, inducible or repressible.

On the other hand, the nucleic acid can also contain, in particular towards the start of the gene of therapeutic interest, an indicator sequence that directs the therapeutic product synthesized in the secretion pathways of the target cell. This indicator sequence may be the natural indicator sequence of the therapeutic product, but may also be another functional indicator sequence, or an artificial indicator sequence. The nucleic acid may also contain an indicator sequence which directs the synthesized therapeutic product into a particular compartment of the cell.

The compositions of the invention may further contain adjuvants capable of associating with the transfer / nucleic acid complexes and improving the transfectant power. In another embodiment, the present invention thus concerns compositions comprising a nucleic acid, a transfer agent as defined above and one or more adjuvants capable of associating with the nucleolipid complexes and improving the transfectant power.

In this view, the compositions of the invention can comprise, as an adjuvant, one or more neutral lipids. Such compositions are particularly advantageous, particularly when the charge ratio transfer agent / nucleic acid is weak. The Applicant has in fact shown that the addition of a neutral lipid allows to improve the formation of the nucleolipid particles and to favor the penetration of the particle in the cell by destabilizing its membrane.

More preferably, the neutral lipids used in the context of the present invention are lipids of two fatty chains. Particularly advantageously, natural or synthetic lipids, amphoteric or devoid of ionic charge are used under physiological conditions. They can be selected more particularly between. dioleoylphosphatidylethanolamine (DOPE), oleoyl-palmitoylphosphatidylethanolamine (POPE), di-stearoyl, -palmitoyl, -miristoyl, phosphatidylglycerols, diacylglycerols, glycosyldiacylglycerols, cerebrosides (such as particularly galactocerebrosides), sphingolipids (such as particularly sphingomyelins) or even asialogangliosides (such as, in particular, asialoGM1 and GM2).

These different lipids can be obtained by synthesis, either by extraction from organs (example: brain) or eggs, by 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 (see also Lhenninger, Biochemistry).

More recently, the Applicant has shown that it is equally advantageous to employ as an adjuvant, a compound that directly or not intervenes at the level of the condensation of said nucleic acid (WO 96/25508). The presence of such a compound, within a composition "according to the invention, makes it possible to decrease the amount of transfectant agent, with the beneficial consequences that arise on the toxicological level, without prejudice to the transfectant activity. intervenes at the level of the condensation of the nucellic acid it is understood to define a compound that compacts, directly or not, the nucleic acid More precisely, this compound can either act directly at the level of the nucleic acid to be transfected or intervene at the level of a an adjunct compound that is directly involved in the condensation of this nucleic acid Preferably, it acts directly at the level of the nucleic acid For example, the precompacting agent can be all polycation, for example polylysine, According to a preferred embodiment, this agent that intervenes at the level of the condensation of the total or partial derivative nucleic acid of a protamine, a histone, or a nucleolin and / or one of its derivatives. Such an agent can also consist, in whole or in part, of peptide motifs (KTPKKAKKP) and / or (ATPAKKAA), the number of motifs which can vary between 2 and 10. In the structure of the compound according to the invention, these motifs can be Thus, they can be separated by ligatures of a biochemical nature, for example by one or several amino acids, or by chemical nature.

In a particularly advantageous embodiment, the compositions of the present invention also comprise a screening element that allows the transfer of the nucleic acid to be oriented. This screening element can be an extracellular screening element that allows guiding the transfer of DNA to certain, cell types or certain desired tissues (tumor cells, liver cells, hematopoietic cells ...). It can also be an intracellular screening element that allows guiding the transfer of nucleic acid to certain privileged cell compartments (mitochondria, nuclei, etc.). the screening element can be linked to the nucleic acid transfer agent according to the invention or also to the nucleic acid as previously defined.

Among the screening elements that can be used in the context of the invention, mention may be made of sugars, peptides, proteins, oligonucleotides, lipids, neuromediators, hormones, vitamins or their derivatives.

Preferentially, these are sugars of peptides or proteins such as antibodies or fragments of antibodies, ligands of cellular receptors or fragments thereof, receptors or fragments of receptors, etc ... In particular, it can they are ligands of growth factor receptors, cytokine receptors, cell lectin receptors, or RGD sequence ligands with an affinity for adhesion protein receptors such as integrins. It is also possible to cite transferrin receptors, HDL and LDL, or the folate transporter. The element • Screening can also be a sugar that allows to screen lectins such as asialoglycoprotein or sialylated receptors such as Lewis X sialide, or even a Fab fragment of antibody, or a single chain antibody (ScFv). More recently, it has also been described peptides of ligands, natural or synthetic, which are of particular interest for their selectivity in relation to specific cells and capable of effectively promoting internalization at the level of cells. (Bary et al. Nature Medicine, 2, 1996, 299-305).

The association of the screening elements to the nucleolipid complexes can be carried out by any technique known to those skilled in the art, for example by coupling to a hydrophobic part or to a part which interacts with the nucleic acid of the transfer agent according to the invention , or even to a group that interacts with the transfer agent according to the invention or with the nucleic acid. The interactions in question can be, according to a preferred embodiment, ionic or covalent in nature.

According to another variant, the compositions of the invention can also optionally incorporate at least one nonionic surface agent in an amount sufficient to stabilize the size of the nucleolipid complex particles. The introduction of non-ionic surface agents prevents the formation of aggregates, which makes the composition more particularly adapted to in vivo administration. The compositions according to the invention incorporating such surface agents have an advantage over the safety plane. They also present an additional advantage in this sense that they reduce the risk of interference with other nucleolipidic proteins.

The surface agents advantageously consist of at least one hydrophobic segment, and of at least one hydrophilic segment. Preferably, the hydrophobic segment is selected from the aliphatic chains, the hydrophobic polyoxyalkylenes, the alkylidene polyester, the benzylic polyester-headed polyethylene glycol and the cholesterol, and the hydrophilic segment is advantageously selected from hydrophilic polyoxyalkylenes, polyvinyl alcohols, polyvinylpyrrolidones. or the saccharides. Such non-ionic surface agents have been described in the application PCT / FR 98/00222.

The subject of the present invention is also the use of the transfer agents described above for the transfer of nucleic acids (and more generally polyanions) into cells. Such a use is particularly advantageous since these transfectants increase the efficiency of the transfection always presenting a zero or very low cellular toxicity.

For in vivo uses, for example for the study of gene regulation, the creation of animal models of pathologies or in therapy, the compositions according to the invention can be formulated contemplating administrations by topical, cutaneous, oral, rectal, vaginal, parenteral , intranasal, intravenous, intramuscular, subcutaneous, infraocular, transdermal, intratracheal, intraperitoneal, etc ... Preferably, the pharmaceutical compositions of the invention contain a pharmaceutically acceptable carrier for an injectable formulation, particularly for a direct injection at the level of the desired organ , or for topical administration (on skin and / or mucosa). It can be, in particular, sterile, isotonic, or dry compositions, particularly lyophilized, which, by addition according to the case of 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 different parameters, and particularly depending on the mode of administration used, the pathology concerned, the gene to be expressed, or even the duration of the investigated treatment. As regards more particularly the mode of administration, it can be either a direct injection into the tissues, for example at the level of the tumors or the circulatory pathways, or else a treatment of cells in culture followed by their reimplantation in vivo. , by injection or graft. The tissues concerned in the context of the present invention are, for example, the muscles, the skin, the brain, the lungs, the liver, the spleen, the bone marrow, the thymus, the heart, the lymph, the blood, the bones, cartilage, pancreas, kidneys, gallbladder, stomach, intestines, testicles, ovaries, rectum, nervous system, eyes, glands, connective tissues, etc ...

The invention also concerns a method of transferring nucleic acids in cells comprising the following steps: (1) contacting the nucleic acid with a transfer agent such as defined above, to form a nucleolipid complex, and (2) the contact of the cells with the complex formed in (1).

The contacting of the cells with the complex can be carried out by incubating the cells with said nucleolipid complex (for in vitro or ex vivo use), or by injection of the complex in an organism (for in vivo use). Incubation is preferably carried out in the presence of, for example, 0.01 to 1000 μg of nucleic acid per 10d cells. For an administration in vivo, doses of nucleic acid comprised between 0.01 and 10 mg can for example be used.

In the case in which the compositions of the invention also contain one or more adjuvants as defined above, the adjuvant or adjuvants are previously mixed with the lipid according to the invention or the nucleic acid.

The present invention thus provides an advantageous method for the transfer of nucleic acids, particularly for the treatment of diseases, comprising the administration in vivo, ex vivo or in vi tro of a nucleic acid that encodes a protein or that can be transcribed in a nucleic acid apt to correct said disease, said nucleic acid which is associated with a compound of general formula (I) under the conditions defined above. More particularly, this method is applicable to diseases "that result from a deficiency in a protein or nucleic product, the nucleic acid administered that encodes said protein product or that is transcribed in a nucleic product or even that constitutes said nucleic product. .

The invention is extended to any use of a nucleic acid transfer agent according to the invention for the transfection of cells.

The nucleic acid transfer agents of the invention are particularly usable for the transfer of nucleic acids in primary cells or in established lines. It can be fibroblastic, muscular, nervous (neurons, astrocytes, gual cells), hepatic cells, of the hematopoietic line, (lymphocytes, CD34, dendritic, etc ...), epithelial etc ..., under differentiated or pluripotent forms ( precursors).

In addition to the foregoing provisions, the present invention also comprises other features and advantages which will be emphasized from the examples and figures which follow, and which should be considered as illustrative of the invention without limiting the scope.

LIST OF FIGURES Figure 1/5: Synthesis scheme of the compounds (1) a (5) Figure 2/5: Synthesis scheme of the compounds (5) to (8a) and (8b).

Figure 3/5: Synthesis scheme of the compounds (9) a (16) Figure 4/5: Measurement of the efficiency of the in vitro transfection of the compounds (8a) and (8b) by the intermediary of the measurement of luciferase activity in HeLa cells, in the absence of serum. The measurements have been made at different ratios in vector nanomoles / μg of DNA, and are represented by the bars. The protein dosage, for each of the compositions, has also been made and is represented by a curve the full line on the same graph.

Figure 5/5: Measurement of the efficiency of transfection in vi tro of compounds (8a) and (8b) by the intermediary of the measurement of luciferase activity in NIH 3T3 cells, in the absence of serum. The measurements have been made at different ratios in vector nanomoles / μg of DNA, and are represented by the bars.

MATERIALS AND METHODS . Purifications on HPLC (High Performance Liquid Chromatography, "High Performance Liquid Chromatography") were carried out with a Waters LC 4000 device, on a Vydac C4 column eluted by a gradient of acetonitrile in water.

. Fluorescence measurements were performed on Perkin-Elmer LS50B, using excitation and emission wavelengths respectively of 260 nm and 590 nm. Slit amplitudes for excitation and emission are regulated at 5 nm. The fluorescence value is recorded after the addition of 5 μg of etidium bromide / ml in final concentration.

. The nuclear magnetic resonance (NMR) spectra have been recorded, at 300.13 MHz for the proton or at 75.47 MHz for the carbon, on a Bruker MSL 300 apparatus in deuterium chloroform.

EXAMPLES EXAMPLE 1: Synthesis of the transfer agent (8a) from methylbenzyloxy-6-galactopyranosyl.

The different reaction stages are schematized in figures 1/5 and 2/5. a) Synthesis of methyl-bensyloxy-6-tri- O- (carboxy-2'-ethyl) -2,3-, 4-ß- D-galactopy- toside (2) 28. 4 g (0.1 mol) of methylbenzyloxy-6- b-D-galactopyranoside (1) are dissolved in 400 ml of THF and placed at 0 ° C under stirring. 32 g (5.7 equivalents) of finely ground potash, 0.5 g (2%) of crown ether 18-Cr-6 and 32.5 ml (1.5 equivalents) of ethyl bromoacetate are added. After two hours, evaporate to dryness, redissolve in water and neutralize the solution obtained with 2N hydrochloric acid. This gives 32 g of product (2) (yield: 70%) which is extracted with dichloromethane. 13 C NMR: ß, 172.94, 136.87; 127.93; 127.41; 98.89; 97.22; 78.74; 76.17; 72.97; 69.52; 67.93; 65.39; 54.75. b) Synthesis of methyl-benzyloxy-6-tri- O- (hydroxy-2'-ethyl) -2,3,4, β-D-galactopyranoside (3) g (0.065 moles) of the preceding triacid (2) are dissolved in 400 ml of THF and placed at 0 ° C under nitrogen. Dripping 40 ml of borane dimethyl sulfide is added dropwise always with stirring. After stirring for 2 hours at room temperature, methanol in excess is added cautiously. The solution is then evaporated to dryness and the solid obtained is redissolved several times in methanol. This gives 24.5 g of final product (3) (yield: 90%) which is purified on a column of silica eluted with ethyl acetate. c) Synthesis of methyl-benzyloxy-6-tri- O- (azido-2'-ethyl) -2,3,4, β-D-galactopyranoside (4) g (0.048 moles) of triol (3) obtained in the preceding step are dissolved in 500 ml of THF. Add 40 g (3.2 equivalents) of triphenylphosphine, 160 ml (3.7 equivalents) of a 1.1 M hydrazoic acid solution in the toluene, then 24.2 ml (3.2 equivalents) of diethylazodicarboxylate. After one hour, the solution is evaporated and purified on a column of silica gel eluted by a heptane / ethyl acetate mixture (6: 4). This gives 17.6 g of product (4) (yield: 90%). 13 C NMR: d ,. 128.47; 127.80; 98.23; 79.05; 76.27; 73.55; 71. 92; 70.15; 69.95; 68.77; 68.14; 55.37; 51.41; 51.11; 50.87. d) Synthesis of methyltri- O- (amino-2'-ethyl) -2,3-, 4--, β-D-galastopyranoside, tris-hydrochloride (5) g (0.03 mol) of triazide (4) are placed in solution in 200 ml of ethanol. 7.5 ml of 12 N hydrochloric acid is added, then treated by hydrogen in the presence of palladium on carbon for 3 hours. After filtration the solvent is evaporated and the residue (5) obtained (12.6 g, yield: 95%) is used directly in the next step. e) Synthesis of methyltri- O- (tert-butylcarbamido-2'-ethyl) -2,3,4, β-D-galactopyranoside (6) 12 g (0.028 mole) of hydrochloride (5) are placed in solution in a mixture of 100 ml of dioxane and 85 ml of 1 N sodium hydroxide. 18.7 g (3.3 equivalents) of di-tert-butyl dicarbonate are added and Stir at room temperature for two hours.

The reaction mixture is evaporated, then extracted by dichloromethane (3 times 50 ml). The organic phases are combined, then dried over sodium sulphate and evaporated. 15.56 g is thus obtained (yield: 90% of residue (6) purified on a column of silica gel eluted by the lime acetate. f) Synthesis of methyl-dioctadecylamido-succinyl-6-tri- O- (tert-butylcarbamido-2'-ethyl) .- 2, 3, 4- ß- D-galactopyranoside (7a) g (0.016 moles) of the preceding product (6) are dissolved in 300 ml of dichloromethane. 14.6 g (1.5 equivalents) of dioctadecylamidosuccinic acid, 1.95 g (1 equivalent) of dimethylaminopyridine and 6.5 g (2 equivalents) of dicyclohexylcarbodiimide are successively added. The dioctadecylamidosuccinic acid is obtained by reaction of the dioctadecylamine and the succinic anhydride. After one night, cautiously add 10 ml (1.7 g) of an oxalic acid solution in the methanol and leave it for one hour (carbon monoxide evolution), filter the solution, evaporate to dryness and purify the product obtained on a column of silica gel eluted with a heptanp / ethyl acetate solution (7: 3), obtaining 12.8 g of product (7a) (yield: 65%). g) Synthesis of methyl-dioctadecylamido-succinyl-6-tri- O- (amino-2'-ethyl) -2,3,4-β-D-galactopyranoside, tris trifluoroacetate (8a) g (0.008 mol) of product (7a) are dissolved in 20 ml of 90% trifluoroacetic acid. After a half hour, the solution is evaporated and 20 ml of toluene are taken again 3 times. The residue (8a) obtained is purified by HPLC (RP4, water / acetonitrile, 0- <100%, 20 minutes). Thus, 9.7 g of product (8a) is obtained (yield: 95%).

MHT = 1304.

E EMPLO 2: Synthesis of compound 8 (b) from methylbenzyloxy-6-galactopyranose.

The different rectional stages are schematized in figures 1/5 and 2/5.

The first phases of synthesis are identical to the synthesis stages a) to e) described in example 1. Then, it is continued as follows: f) Synthesis of methyl. Dioctadecylamido-fatalyl-6-tri- O- (tert-butylcarbamido-2'-ethyl) -2,3,4-b-D-galactopyranoside (7b) This product is obtained in the same way as the product (7a) but using the dioctadecylamidophthalic acid instead of the dioctadecylamidosuccinic acid. g) Synthesis of methyl-dioctadecylamido-fatlyl-6-tri-O- (amino-2'-ethyl) -2,3-, 4-β-D-galactopyranoside, tris trifluoroacetate (8b) Compound (8b) is obtained starting from the product (7b) obtained in the preceding stage, in the same way as the product (8).

MH + = 1317.

EXAMPLE 3: Synthesis of the transfer agent (16) from the triasethyl glycol a) Synthesis of phthalimidopropyl-di-O-acetyl-4,6-bisdeoxy-2,3, b- D-erythro-hexene-2-pyranoside (10) The different reaction stages are represented in Figure 3/5. 13. 17 g (0.048 mol) of triacetyl-D-glycal (9) are dissolved in 250 ml of dichloromethane. 11 g (1.1 equivalents) of phthalimidopropanol and 1.56 ml (0.26 equivalents) of boron trifluoroetherate are added. After one hour at room temperature, the mixture is neutralized by a saturated solution of sodium bicarbonate, decanted, dried and the solution evaporated. This gives 16 g of product (10) (yield: 80% purified by chromatography on a column of silica gel eluted by a heptane / ethyl acetate solution (5: 5). b) Synthesis of phthalimidopropyl-di-O-acetyl-4,6-bisdeoxy-2, 3, β-D-glucopyranoside (11) 16 g (0.038_moles of glycoside (10) are dissolved in 200 ml of ethanol. 0.16 g of 10% palladium on carbon is added and treated by hydrogen for two hours.

After filtration, the solution is evaporated. This gives 15.78 g of product (11) (yield: 99%). s) Synthesis of phthalimidopropyl bis-dioxy-2, 3, β-D-glucopyranoside (12) g (0.036 moles) of the preceding product (11) are dissolved in 250 ml of methanol, then 3.5 ml (0.1 equivalent) of 1 N sodium methylate is added. After two hours, it is neutralized by the amberlite resin IR120. 11.5 g of product (12) is obtained after filtration and evaporation (yield: 96%). d) Synthesis of phthalimidopropyl-diazido-4,6-tetra-deoxy-2, 3, 4, 6-β-D-glucopyranoside (13) 11 g (0.033 mol) of deacetylated product (12) are dissolved in 250 ml of THF. While stirring, 21.5 g (2.5 equivalents) of triphenylphosphine and 110.5 ml (3.7 equivalents) of 1.1 M hydrazoic acid are successively added in the toluene. 12.88 ml (2.5 equivalents) of diethylazodicarboxylate are added at a rate such that the temperature of the mixture does not exceed 30 ° C. After one hour, the solution is evaporated to dryness, and the obtained product is purified on a column of silica gel eluted by a hepyne / ethyl acetate mixture (8: 2). In this way, 7.2 g of product (13) is obtained (yield: 57%). e) Synthesis of aminopropyl-diazido-4,6-tetra-deoxy-2, 3, 4, 6-β-D-glucopyranoside (14) 7 g (0.018 mol) of product (13) are dissolved in 500 ml of ethanol, then 2.6 ml of hydrazine hydrate is added, and it is stirred at 40 ° C for two hours. After evaporation, it is redissolved in dichloromethane and the phthalhydrazide which has formed is allowed to crystallize. Filter and evaporate the solution. This gives 4.17 g of product (14) (yield: 90%). f) Synthesis of (cholesteryl-O-formamidopropyl). Diazido- 4, 6-tetra-deoxy-2, 3, 4, 6-β-D-glucopyranoside (15) 6.34 g (0.9 equivalents) of cholesteryl chloroformate and 4 g (0.016 moles) of product (14) are placed in solution in 200 ml of ethyl ether. A precipitate is formed immediately, and then 50 ml of N soda is added. After a few minutes, the solution recovers its limpidity. The mixture is left stirring for 30 minutes, then it is decanted, washed twice with 50 ml of water and the solution evaporated to dryness. 9.4 g of purified product (15) are thus obtained on a column eluted by a heptane / ethyl acetate solution (6: 4). (yield: 90%). g) Synthesis of (cholesteryl-O-formamidopropyl) -diamino-4,6-tetra-deoxy-2, 3, 4, 6-β-D-glucopyranoside (16) 9 g (0.013 moles) of diazide (15) are dissolved in 150 ml of 80% THF, then 14.2 g (4 equivalents) of triphenyl phosphine are added and heated at 50 ° C for one hour. After cooling, it is carried on an anionic resin column IRC-50 COOH, and washed with 80% THF. The product is then eluted in the form of acetate by the THF / water / acetic acid mixture (64: 16: 10). This gives 8.9 g of the product (16) _ (yield: 90%).

NMR 13 d, 176.55; 156.65; 139.84; 132.02; 128.46; 125. 46; 122.44; 97.11; 74.28; 56.66; 56.12; 50.02; 43.70; 42.30; 39.51; 38.57; 36.98; 36.55; 36.16; 35.76; 31.87; 30.30; 29.89; 28.18; 23.82; 22.78; 22.53; 21.03; 19.31; 18.70; 11.84.

EXAMPLE 4: Synthesis of the compound (17) a) Synthesis of benzyl- (5- O -acetyl-6-acetoxymethyl-5,6-dihydro-2H-pyran-2-yloxy) -acetate 2. 8 g of triacetyl D-glucan (0.01 mole) and 1.6 ml of benzylglycolate (1.1 equivalents) are placed in solution in 80 ml of chloroform. It is cooled in an ice bath and 0.651 ml is added. Boron trifluoride etherate (0.5 equivalents). After 45 minutes, neutralize with a saturated aqueous solution of sodium bicarbonate, dry over sodium sulfate, filter and concentrate. Purify by chromatography on a silica gel column, eluted with a heptane / ethyl acetate (6: 4) mixture. The yield is 96%. b) Synthesis of (5- O -acetyl-6-acetoxymethyl-tetrahydro-pyran-2-yloxy) -acetic acid: 35 g of the preceding product (0.0093 mol) are hydrogenated at atmospheric pressure and at room temperature, in the presence of _ 0.035 g of 10% palladium on carbon for 10 hours. It is filtered through celite, concentrated and purified by chromatography on a column of silica gel, eluted by ethyl acetate. The yield is 90%. c) Synthesis of N, N-dioctadecyl- (5- O -acetyl-6-acetoxymethyl-tetrahydro-pyran-2-yloxy) -acetamide: 0. 675 g of the preceding product (0.0017 mol) are dissolved in 8.6 ml of chloroform. 0.9 ml of diisopropylethylamine, 0.899 g of dioctadecylamine (1 equivalent) and 0.426 g of dicyclohexyl carbodiimide (1.2 equivalents) are added. After one night at room temperature, concentrate and extract the product by heptane. Purify by chromatography on a silica gel column, eluted with a heptane / ethyl acetate (8: 2) mixture. The yield is 70%). d) Synthesis of N, N-dioctadecyl- (5-hydroxy-6-hydroxymethyl-tetrahydro-pyran-2-yloxy) -acetamide: 1. 57 g of the preceding product (0.00198 moles) are placed in solution in 10 ml of methanol. 0.197 ml of sodium ethylate (0.2 equivalents, 2M methanol) is added. After two hours at room temperature, neutralize and concentrate. Purify by chromatography on a silica gel column, eluted with a heptane / ethyl acetate (8: 2) mixture. The yield is 92%. e) Synthesis of N, N-diostadesyl- (5-azido-6-azidomethyl-tetrahydro-pyran-2-yloxy) -acetamide (17): 0. 6 g of the preceding product (0.00087 mol) are dissolved in 15 ml of THF. 0.48 g of triphenylphosphine is added (2.1 equivalents) then 0.288 mi of DEAD (2.1 equivalents). When the exothermic reaction has been completed, 1,588 ml of a 1.38 M hydrazoic acid solution in toluene is added. (2.5 equivalents). At the end of the reaction, it is concentrated, taken up again by the heptane, filtered and concentrated. Then, it is purified by chromatography on a column of silica gel, eluted by u: .-. heptane / ethyl acetate (8: 2). The yield is 72%. 13 C NMR: d, 173.21; 172.07; 162.22; 161.77; 118.34, 114. 49; 110.65; 108.34; 96.88; 65.18; 64.50; 48.27; 46.54; 40.69; 35.86; 31.78; 30.94; 28.63; 27.56; 26.92; 22.54; 21.84; 13.'94.

EXAMPLE 5: Association of the transfectant agents of the invention 8 (a) and 8 (b) with the DNA This example illustrates the nature of the association between the transfectant agents of the invention and DNA, as well as the formation of complexes.

The transfectant agents according to the invention have been placed in solution in water at 10 mM.

The solutions thus obtained are transparent and correspond to an organization in the form of micelles of cationic lipid.

The transfectant / DNA complexes have been made following a ratio of 6 nmoles of transfectant agent / μg of DNA.

The association of the transfectant agent 8 (b) with the DNA yields a complex of size equal to 92 nm ± 27 nm, and a fluorescence of 7% in water and 60% in sodium chloride NaCl at 300 nM.

The transfectant agent of the invention 8 (a), associated with DNA, allows the formation of a complex of neighboring size of 98 nm ± 2 O nm, and a fluorescence of 6% in water and 55% in sodium chloride NaCl at 300 nM.

These results show that the transfectants according to the invention are capable of associating with DNA and of forming particles whose average diameter is approximately 100 nm, which constitutes a size particularly adapted to the passage of cell membranes. This association remains relatively stable when the ionic strength of the medium is increased. Indeed, at 300 mM, more than 50% of the DNA remains associated with the transfectant agents of the invention.

EXAMPLE 6: Transfection of DNA in vitro with the transfectant agents of the invention 8 (a) and 8 (b) The efficiency of transfection has been evaluated in vitro at different doses of vectors / μg of DNA.

The genetic material used for these experiments is a pCMV-Luc plasmid construction containing the "luciferase" transporter gene, derived from the plasmid pGL2-basic Vector (Promega) by insertion of a Mlu I-Hind III fragment containing the cytomegalovirus promoter. human (CMV) extracted from plasmid pcDNA3 (Invitrogen).

Nucleic acid solutions diluted at 40 μg / ml in physiological saline (sodium chloride 0.15 M NaCl) have also been used.

The products described in the invention are placed in solution in water at a concentration ranging from 80 μM to 800 μM, and mixed volume by volume with the DNA solution.

The final salt concentration is 75 mM in order to prepare extemporaneously citofectant solutions.

For transfection, the cells are cultured under the appropriate conditions in 14-well microplates (2 cm2 / well) and are transfected when they are in the exponential growth phase and at 50-70% of the confluence.

The cells are washed twice with 500 μl of medium devoid of serum proteins and put back into growth in medium without serum. 50 μl of citofectant mixture (1 μg of DNA / well) are added to the cells (3 wells / vector-DNA condition). The growth medium is supplemented by the appropriate amount of serum 2 hours after transfection.

The transfection efficiency is evaluated at 48 hours post-transfection by a measure of luciferase expression according to the recommendations given for the use of the Promega (Luciferase Assay System). The toxicity of the cytofectant mixtures is estimated by a measurement of the protein concentrations of the cell lysates.

The results obtained by the transfection of the HeLa cells are combined in Figure 4/5, and those obtained on the NIH3T3 cells are indicated in Figure 5/5.

Of these two figures as well as of the results obtained for vector / DNA ratios greater than 20 mmoles / μg of DNA (not shown), it is possible to deduce that transfection is particularly effective when the vector / DNA ratio is between 2 and 20 Vector nanomoles per μg of DNA.

The results presented show that for the two cell types used, the efficiency of the transfection is optimal for a ratio comprised between 4 and 12 nanomoles per μg of DNA, and more precisely between 8 and 12 nanomoles per μg of DNA.

On the other hand, it is verified that the histograms taking into account the dose effect of the vectors are superposable for the products 8 (a) and 8 (b) whatever the cell type considered.

The two products of the invention are non-toxic at the doses of vectors studied for NIH 3T3 cells. In effect, we did not observe more than a maximum loss of 20% of the cellular proteins for doses higher than 8 nanomoles of vector per sample of transfected cells.

Claims (23)

1. Transfectant agent comprising at least one cationic hydrophilic region coupled to a lipophilic region which is characterized in that said hydrophilic cationic region has the following general formula: in which: . and is an integer equal to 0 or 1, the different and independent of each other, . X represents an atom of oxygen, nitrogen, sulfur or selenium, the Z groups represent, independently of each other, - a hydrogen atom, - an OR group in which R represents a hydrogen atom, a methyl group, or a group (CH2) n-NR? R2 in which n is an integer selected from 1 to 6 included and Ri and R2 represent, independently one of the other a hydrogen atom or a group (CH2) q-NH2, q that can vary from 1 to 6 included, the different ones that are independent of each other, . a group (CH2) m-NR? R2, in which m is an integer selected from 0 to 6 included and Ri and 2 are defined as above, or a "spacer" group that allows the union of the cationic hydrophilic region to the lipophilic region, it being understood that at least one of the susbstituents Z is a "spacer" group and that at least two of the Z substituents carry an amino group.

2. Transfectant agent according to claim 1 which is characterized in that, in the general formula defining the hydrophilic cationic region, n is an integer selected from 2, 3 and 4 in the group (CH2) n-NR? R23.

Transfectant agent according to claim 1, characterized in that the general formula defining the hydrophilic cationic region is a pyranose or furanose glycoside.

4. Transfectant agent according to claim 1 which is characterized in that, in the general formula, it defines the hydrophilic cationic region, and is equal to 1 and X is an oxygen atom.

5. Transfectant agent according to claim 1, characterized in that said lipophilic region is constituted by one or several linear or branched aliphatic chains, saturated or not and optionally halogenated, and / or by a steroid derivative.

6. Transfectant agent according to claim 5 which is characterized in that said aliphatic chains contain 10 to 22 carbon atoms, and particularly 14, 16, 17, 18 and / or 19 carbon atoms.

7. Transfectant agent according to claims 5 and 6 which is characterized in that said aliphatic chains are selected from (CH2) i3CH3, (CH2) i5CH3, (CH2) 16CH3, (CH2) i7CH3, (CH2) 18CH3.

8. Transfectant agent according to claim 5 which is characterized in that the steroid derivative is selected from cholesterol, cholestanol, 3- a, -5-cyclo-5- a-cholestan-6-β-ol, cholic acid, cholesteryl formate, cholestanyl formate, 3a, 5-cyclo-5-cholestan-6-yl formate, cholesteryl amine, 6- (1, 5'-dimethylhexyl) -3a, 5a-dimethylhexadecahydro-cyclopenta [a] cyclopropa [2] , 3] cyclopenta [1, 2-f] naphthalene-10-ylamine, or cholestanylamine.

9. Transfectant agent according to claim 1, characterized in that the "spacer" group that allows the union of the cationic hydrophilic region to the lipophilic region is constituted by an acidic or amino group containing hydrolysable functions.

10. transfectant agent according to claim 9 which is characterized in that the "spacer" group is constituted by an aliphatic or aromatic chain and contains one or more groups selected from amides, esters, carbonates and aromatic rings.

11. Transfectant agent according to claim 9 which is characterized in that the "spacer" is selected from -0- CO- (CH2) x-_COOH, -O- (CH2) X- COOH, -O- CO- (CH) S- NH2, -O- (CH2) X-NH2, -NH- (CH2) X-NH2, -with x representing an integer between 1 and 6 included.

12. transfectant agent according to claim 1, characterized in that it is represented by one of the compounds of the formula:

13. Preparation method _ of a transfectant agent according to claims 1 to 12 which is characterized in that it is coupled to the lipophilic region by the intermediary of a "spacer" to the heterocycle of general formula as defined in claim 1.

14. Composition which is characterized in that it comprises a transfectant agent as defined in claims 1 to 12 and at least one nucleic acid.

15. Composition according to claim 21, characterized in that the transfectant agent / nucleic acid ratio is comprised between 0.1 and 50 nanomoles of agent per μg of DNA.

16. composition according to claim 14, characterized in that it also incorporates one or more adjuvants.

17. Composition according to claim 16, characterized in that the adjuvant (s) are neutral lipids.

18. Composition according to claim 16, characterized in that the adjuvant or adjuvants are compounds that directly or not intervene at the level of the condensation of the nucleic acid.

19. Composition according to claims 14 to 18 which is characterized in that it comprises a pharmaceutically acceptable carrier for an injectable formulation.

20. composition according to claims 14 to 18 which is characterized in that it comprises a pharmaceutically acceptable carrier for an application on the skin and / or mucous membranes.

21. Method for transferring nucleic acids in cells which is characterized in that it comprises the following steps: (1) contacting the nucleic acid with a transfer agent as defined in claims 1 to 19 and, if necessary, with or the adjuvants, to form a nucleolipid complex, and (2) the contacting of the cells with the complex formed in (l. -

22. Transfectant agent according to claims 1 to 13 for use in the transfer of nucleic acids in cells.

23. Use of a transfectant agent as defined in the preceding claims for the manufacture of a medicament containing at least one nucleic acid to be transfected.