AU2002257904B2 - Polythiourea lipid derivatives - Google Patents

Description

WO 02/092558 PCT/FR02/01626 LIPID DERIVATIVES OF POLYTHIOUREA The present invention relates to novel compounds which make it possible to transfer nucleic acids into cells. More precisely, these novel compounds are lipid derivatives of polythiourea. They are useful for the in vitro, ex vivo or in vivo transfection of nucleic acids into various cell types.

With the development of biotechnology, the possibility of effectively transferring nucleic acids into cells has become a necessity. It may involve the transfer of nucleic acids into cells in vitro, for example, for the production of recombinant proteins, or in the laboratory for studying the regulation of the expression of genes, the cloning of genes, or any other manipulation involving DNA. It may also involve the transfer of nucleic acids into cells in vivo, for example for the creation of transgenic animals, the production of vaccines, labeling studies or also therapeutic approaches. It may also involve the transfer of nucleic acids into cells ex vivo, in approaches including bone marrow transplants, immunotherapy or other methods involving the transfer of genes into cells collected from an organism for the purpose of their subsequent readministration.

Nowadays, several methods have been proposed for the intracellular delivery of exogenous genetic material. One of them, in particular, is based on the use of nonviral vectors which constitute a highly advantageous alternative to the viral methods which are not completely risk free. These synthetic vectors have two main functions: to complex and to compact the nucleic acid to be transfected, and to promote its passage across the plasma membrane and possibly across the nuclear envelope.

Several families of synthetic vectors have thus been developed, such as for example polymers or alternatively biochemical vectors (consisting of a cationic protein combined with a cellular receptor ligand), but a major advance has in particular been made with the development of lipofectants and more particularly of cationic lipids. It has thus been demonstrated that cationic lipids, because of their overall positive charge, spontaneously interfere with DNA which is globally negative, forming nucleolipid complexes capable both of protecting the DNA against nucleases and of binding to the cellular membranes for intracellular release of the DNA.

Various types of cationic lipids have been synthesized to date: lipids comprising a quaternal ammonium group (for example DOTMA, DOTAP, DMRIE, DLRIE, and the like), lipopolyamines such as for example DOGS, DC-Chol or alternatively the lipopolyamines disclosed in Patent Application WO 97/18185, lipids combining both a quaternary ammonium group and a polyamine such as DOSPA, or alternatively lipids comprising various other cationic entities, in particular amidinium groups (for example ADPDE, ADODE or the lipids of patent application WO 97/31935).

However, the use of these cationic lipids as transfection agent still poses numerous problems, and their efficiency remains to be improved. In particular, it has been observed that to obtain efficient and stable nucleolipid complexes, it is in general necessary for these complexes to be highly cationic.

However, it would be desirable to be able to have available vectors which are not cationic so as to form, with the nucleic acid, particles which are globally neutral or negative. Indeed, it has been observed that the globally cationic complexes formed between the nucleic acid and the cationic lipids tend to be captured by the reticuloendothelial system, which induces their elimination. In addition, the plasma proteins tend to become adsorbed at their surface because of the overall positive charge of the complexes formed, and this results in a loss of the transfection power. Furthermore, in a context of local injection, the presence of a large overall positive charge prevents the diffusion of the nucleic acid complexes away from the site of administration because the complexes become adsorbed onto the extracellular 4 matrices; the complexes can therefore no longer reach Z the target cells, which consequently causes a decrease in the transfer efficiency in relation to the injected quantity of complexes. Finally, it has also been observed, in many instances, that cationic lipids have V an inflammatory effect.

O Advantageously, one or more embodiments of the present q invention may precisely provide novel transfecting compounds which are innovative by virtue of their polythiourea functional group and which are capable of being efficiently used for the in vitro, ex vivo or in vivo transfection of nucleic acids. These novel compounds are particularly advantageous because: the absence of positive charges from their structure makes it possible to solve the many problems raised by the use of cationic vectors discussed above, just like cationic lipids, they are capable of complexing and compacting nucleic acids and of promoting their transfection.

A first subject of the present invention is thus transfecting compounds characterized in that they consist of a polythiourea part linked to a lipid via a spacer.

In particular, the subject of the present invention is transfecting compounds of general formula S 0 X-N N-(CHm N Y L (I) H H I nL 1

RR'

in which: P is an integer chosen from 0 and 1, n is an integer chosen from 1, 2, 3, 4, 5 and 6, m is an integer chosen from 2, 3 and 4, it being possible for m to take different values within the different groups -[NH-CS-NH-(CH)m]-, R' represents a group of general formula (II):

S

N N 2 H H in which q is an integer chosen from 1, 2, 3, 4, 5 and 6, and p is an integer chosen from 2, 3 and 4, it being possible for p to take different values within the different groups -[(CH 2 )p-NH-CS-NH]-, R represents either a hydrogen atom or a group of general formula (II) as defined above, it being understood that when n is 1 and I is 0, then at least one group R is of formula (II), X, in the formulae and represents a saturated or unsaturated, linear or cyclic aliphatic group, comprising 1 to 8 carbon atoms, a mercaptomethyl

(-CH

2 SH) group, or alternatively a hydrophilic chain chosen from the groups: -(CH2)x-(CHOH)u-H with x an integer between 0 and and u an integer chosen from 1, 2, 3, 4, 5 and 6, or,

-(OCH

2

CH

2 0)v-H with v an integer chosen from 1, 2 and 3, it being understood that no more than one substituent X, both in the formulae and represents a hydrophilic chain, Y represents a spacer, and L represents: either a group -N(RI)R 2 with Ri and R 2 which represent, independently of each other, a hydrogen atom or alternatively a fatty aliphatic chain, or alternatively a group of formula -(CH 2 )t-OZ with t representing an integer chosen from 11, 12, 13, 14 or 15 and Z represents a sugar, a polyol or a PEG, it being understood that at least one of Ri and R 2 is different from hydrogen, or a group -OR 3 with R 3 which represents a steroid derivative.

According to the present invention, the term "spacer" is understood to mean any chemical group which makes it possible both to provide the linkage between the polythiourea part and the lipid part of the molecule, and to keep these two parts apart so as to attenuate any undesirable interruption between them.

Preferred spacers may for example consist of one or more chemical functional groups chosen from alkyls having 1 to 6 carbon atoms, ketone, ester, ether, amide, amidine, carbamate or thiocarbamate functional groups, glycerol, urea, thiourea, or else aromatic rings. For example, the spacer Y may be chosen from the groups of formula:

-NH-C(O)-CH

2

-CH

2 or: -(CH2-)i-W-(CH2) j in which i and j are integers chosen between 1 and 6 inclusive and W is a group chosen from ketone, ester, ether, amide, amidine, carbamate or thiocarbamate functional groups, glycerol, urea, thiourea, or alternatively aromatic rings.

For the purposes of the present invention, the expression "fatty aliphatic chains" is understood to mean alkyl groups containing 10 to 22 carbon atoms which are saturated or unsaturated and optionally containing one or more heteroatoms, provided that said fatty aliphatic chains exhibit lipid properties.

Preferably, they are linear or branched alkyl groups containing 10 to 22 carbon atoms and i, 2 or 3 unsaturations. Preferably, said alkyl groups comprise 12, 14, 16, 18, 20 or 22 carbon atoms. There may be mentioned more particularly the aliphatic groups

(CH

2 1CH 3

(CH

2 13

CH

3

(CH

2 )15CH 3 and -(CH 2 )1 7

CH

3 The term "sugar" is understood to mean, for the purposes of the invention, any molecule consisting of one or more saccharides. There may be mentioned, by way of example, sugars such as pyranoses and furanoses, for example glucose, mannose, rhamnose, galactose, fructose or alternatively maltose, lactose, saccharose, sucrose, fucose, cellobiose, allose, laminarabiose, gentiobiose, sophorose, melibiose, and the like.

Preferably, the sugar(s) are chosen from glucose, mannose, rhamnose, galactose, fructose, lactose, saccharose and cellobiose. Furthermore, it may also involve so-called "complex" sugars, that is to say several sugars which are covalently coupled to each other, each sugar being preferably chosen from the list cited above. As suitable polysaccharides, there may be mentioned dextran, a-amylose, amylopectin, fructans, mannans, xylans and arabinans. Some preferred sugars may in addition interact with the cell receptors, such as for example certain types of lectin.

According to the invention, the term "polyol" is also understood to mean any linear, branched or cyclic hydrocarbon molecule comprising at least two hydroxyl functional groups. There may be mentioned by way of example glycerol, ethylene glycol, propylene glycol, tetritols, pentitols, cyclic pentitols (or quercitols), hexitols such as mannitol, sorbitol, dulcitols, cyclic hexitols or inositols, and the like (Stanek et al., The Monosaccharides Academic Press, pp.

621-655 and pp. 778-855). According to a preferred aspect, the polyols are chosen from the alcohols of general formula: HO OH

OH

for which s is chosen from 2, 3, 4, 5 and 6.

When the compounds of general formula (I) according to the invention contain a polyethylene glycol (PEG) group, the latter generally comprises between 2 and 120 -OCH 2

CH

2 0- units, and preferably between 2 and 80 -OCH 2

CH

2 0- units. This may include simple PEGs, that is to say whose chain ending ends with a hydroxyl group, or else PEG whose terminal group is chosen from alkyls, for example methyl.

For the purposes of the present invention, the expression "steroid derivatives" is understood to mean polycyclic compounds of the cholestane type. These compounds may be natural or otherwise and are more preferably chosen from cholesterol, cholestanol, cyclo-5-a-cholestan-6--ol, cholic acid, cholesteryl formate, chotestanyl formate, 3a,5-cyclo-5a-cholestan- 63-yl formate, cholesterylamine, 6-(1,5-dimethylhexyl)- [2,3]cyclopenta[l,2-f]naphthalen-10-ylamine, or cholestanylamine.

According to a preferred variant of the invention, the transfecting compounds have the general formula (III): S O Xj: A

R

N N-(CH, Y N (III) H H n

R,

in which X, m, n and Y are as defined above in general formula with the exception of n which is different from 1, and RI and R 2 represent, independently of each other, a hydrogen atom or else a fatty aliphatic chain, it being understood that at least one of Ri and R 2 is different from hydrogen.

More preferably still, the transfecting compounds of the invention have the general formula (IV) S 0 H3C N-(CH, Y N

(IV)

H H

R,

in which m, n and Y are as defined above in general formula with the exception of n which is different from 1, and RI and R 2 represent, independently of each other, a hydrogen atom or else a fatty aliphatic chain, it being understood that at least one of R 1 and R 2 is different from hydrogen.

It is understood that the present invention also relates to the isomers of the products of general formula when they exist, as well as mixtures thereof.

The preparation of the compounds of general formula according to the present invention is carried out using the following steps, in the order presented or according to any other known and equally suitable variant, using conventional organic synthesis techniques, in solution or on solid supports, which are well known to a person skilled in the art: 1) Production of the lipid part L When the lipid part L of the compounds of general formula is represented by a group -N(RI)R 2 with RI and/or R 2 which represent a fatty aliphatic chain, the amine of formula HN(RI)R 2 is first of all formed. Said amine may be obtained by condensing a carboxylic acid and an amine, one containing the substituent RI and the other the substituent R 2 to form the corresponding amide, followed by reduction of said amide thus obtained.

Amide formation is advantageously carried out by mixing constituents and melting, by heating at a temperature of greater than the melting point of the substances involved, in general between 20°C and 2000C, followed by elimination of the water produced by dehydrating the medium; or more advantageously in the presence of a desiccating agent such as for example phosphorus pentoxide or any other substance which can absorb water. The formation of this intermediate amide may also be carried out using a variant of this method or another method for forming an amide (such as for example peptide-coupling type) involving carboxylic acids or derivatives thereof, and varying conditions and reagents Larock, Comprehensive Organic Transformations, VCH Publishers] well known to a person skilled in the art.

The reduction of the amide previously obtained to an amine of formula HN(RI)R 2 may be carried out for example using a reducing agent such as lithium aluminum hydride, or any other hydride or reducing agent effective in this case. The procedure is then preferably carried out in an aprotic solvent (for example tetrahydrofuran or ethers) at a temperature below the boiling point of the solvent or under a dry and/or inert atmosphere.

According to another variant, the lipid part designated as HN(RI)R 2 may be commercially available.

When RI and/or R 2 represent(s) a group of formula -(CH 2 )t-OZ, the procedure is carried out as described above for forming the alkyl part, followed by simple coupling with a commercial PEG, polyol or sugar according to conventional techniques known to a person skilled in the art.

When the lipid part L of the compounds of the general formula is represented by a group -OR 3 the latter is preferably chosen from commercially available products.

2) Grafting of the spacer Y The spacer Y is then attached to the lipid part L obtained in the preceding stage according to conventional techniques known to a person skilled in the art. According to a preferred variant, an amide bond is made by N-acylation of the lipid part L in an appropriate solvent such as dichloromethane, chloroform, tetrahydrofuran, or any other ether, at a temperature below the boiling point of the solvent, and under a dry and/or inert atmosphere. This reaction is preferably carried out in the presence of an aminecontaining base such as N,N-dimethylaminopyridine, or in the presence of this base mixed with nonnucleophilic amine-containing bases such as triethylamine or else ethyl diisopropylamine. Pyridine may also be used, alone or mixed with another base, diluted with one of the solvents mentioned or used itself as solvent.

3) Formation of the polythiourea chain The third part of the synthesis of the compounds of general formula consists in the successive introduction of the thiourea units. This will be carried out in a series of reactions which may be repeated as many times as necessary in order to obtain the desired polythiourea part. According to a preferred method, the procedure is carried out in the following manner: A) There is first of all grafted onto the Y-C(O)-L obtained in the preceding stage the first part of the unit in the form of a member group.

For that, the procedure is advantageously carried out starting with a diamine-containing member of formula

H

2 N-(CHR)m-NH 2 in the presence of a coupling agent, for example 1-benzotriazolyloxytris(pyrrolidino)phosphonium hexafluorophosphate (PyBOP), l-benzotriazolyloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), 0-(lH-benzotriazol-l-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate or tetrafluoroborate (HBTU or TBTU), dicyclohexylcarbodiimide (DCC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), or else 1-(3trimethylammoniopropyl)-3-ethylcarbodiimide iodide, supported or otherwise. This coupling is carried out in a suitable solvent, for example dichloromethane, chloroform, tetrahydrofuran or any other ether, at a temperature below the boiling point of a solvent, and under a dry and/or inert atmosphere. The procedure is also carried out in the presence of a non-nucleophilic amine-containing base, for example ethyldiisopropylamine, triethylamine or else triisopropylamine. If the nature of the lipid part and of the spacer is compatible, a sequence of the SCN-(CHR)m- type, or a precursor, may be grafted, thus making it possible to continue the synthesis through a stage such as that described below in C), B) The product obtained in the preceding stage is then converted, according to a preferred technique, to isothiocyanate by treating with carbon disulfide (CS 2 or with any other reagent known to the person skilled in the art for obtaining such a functionality Ulrich, Chemistry and Technology of Isocyanates, Wiley (1996). The Chemistry of Cyanates and their Thio Derivatives, S. Patai Ed., Wiley (1977).

S. Ozaki, Recent Advances in Isocyanate Chemistry, Chem. Rev. 72, 457 (1972)]. The reaction is advantageously carried out in a solvent such as for example tetrahydrofuran, or any other compatible ether solvent, at a temperature varying between that of the cooling mixtures and about 20 0 C. The procedure is also carried out in the presence of an agent capable of promoting the reaction and/or of trapping the hydrogen sulfide released during the reaction, for example dicyclohexylcarbodiimide (DCC).

C) The thiourea unit is then formed from the isothiocyanate obtained in the preceding stage so as to allow, where appropriate, the introduction of another segment of formula Advantageously, a diamine of formula H 2 N-(CHR)m-NH 2 optionally protected, is reacted, in its neutral form or in the form of an acid salt, with the isothiocyanate obtained in the preceding stage. This reaction is optionally carried out in the presence of a non-nucleophilic amine-containing base, for example triethylamine, ethyldiisopropylamine, triisopropylamine or else 1,8-diazabicyclo[5.4.0]undec- 7-ene (DBU). The procedure is preferably carried out in a suitable solvent such as dichloromethane, chloroform, tetrahydrofuran or any other compatible ether or solvent, at a temperature which may be between that of the cooling mixtures and the reflux temperature of the solvent.

Stages B) and C) described above are then repeated sequentially and in the required order until the desired structure is obtained, so as to introduce the desired unit in n copies. To obtain branched structures, the procedure is carried out in a similar manner by introducing, at the appropriate time, the molecule(s) required to obtain a substitution R as

I

described by formula (II).

4) Ending of the polythiourea part by introducing the substituent X The last stage allowing the ending of the polythiourea-type chain(s) consists in introducing the substituent X. For that, conventional grafting methods known to a person skilled in the art, chosen according to the nature of the substituent X, are used. For example, when X represents an alkyl, the procedure is carried out by reacting an alkyl isothiocyanate, in the presence, when necessary, of a non-nucleophilic aminecontaining base such as for example triethylamine, ethyldiisopropylamine, triisopropylamine or else 1,8diazabicyclo[5.4.0]undec-7-ene (DBU). The reaction is performed in a suitable solvent, for example dichloromethane, chloroform, tetrahydrofuran or any other compatible ether, at a temperature between the temperature of the cooling mixtures and the reflux temperature of the solvent.

Naturally, when the various substituents can interfere with the reaction, it is preferable to protect them beforehand with compatible radicals which can be put in place and removed without affecting the remainder of the molecule. For that, the procedure is carried out according to conventional methods known to a person skilled in the art, and in particular

I

according to the methods described in T.W. Greene, Protective Groups in Organic Synthesis, Wiley- Interscience, in McOmie, Protective Groups in Organic Chemistry, Plenum Press, or in P.J. Kocienski, Protecting Groups, Thieme.

Moreover, each stage of the method of preparation may be followed, where appropriate, by stages for separating and purifying the compound obtained according to any method known to a person skilled in the art.

The preferred compounds according to the present invention are 3-(2-{3-[2-(3-{2-[3-(ditetradecylcarbamoyl)propionylamino]ethyl}thioureido)ethyl]thioureido}ethyl)-l-methylthiourea or DT-3TU, which comprises three thioureas and corresponds to formula in which X is a -CH3, m is equal to 2, n is equal to 3, R is a hydrogen, P is equal to 0, L

N(RI)R

2 where RI R2 C 14

H

29 and Y NH-CO-CH 2

-CH

2 3-(2-{3-[2-(3-{2-[3-(2-{3-[ditetradecylcarbamoyl]propionylamino}ethyl)thioureido]ethyl}thioureido)ethyl]-thioureido}ethyl)-l-methylthiourea or DT-4TU, which contains four thiourea groups and corresponds to general formula in which X is a -CH 3 m is equal to 2, n is equal to 4, R is a hydrogen, P is equal to 0, L -N(RI)R 2 where RI R2 C 14

H

29 and Y NH-CO-CH 2

-CH

2 The compound 2-(3-{2-[3-(ditetradecylcarbamoyl)propionylamino]ethyl}thioureido)ethyl]propane-1,2-diol or DT-2TU diol corresponds to general formula in which X Ho m 2; R H; n 2; I 0; L -N(Ri)R 2 where R1 C 14

H

29 and Y NH-CO-CH 2

-CH

2 The compound tetradecylcarbamoyl)propionylamino)ethyl]thioureido}ethyl)thioureido]ethyl}thioureido)ethyl]propane- 1,2-diol or DT-3TU diol corresponds to formula in which X HO m 2; R H; n 3; 0; L -N(RI)R 2 where RI R2 C 14

H

2 9 and Y NH-CO-CH 2

-CH

2 Another subject of the invention relates to the compositions comprising a transfecting compound according to the invention and a nucleic acid. The respective quantities of each component may be easily adjusted by a person skilled in the art according to the transfecting compound used, the nucleic acid and the desired applications (in particular the type of cells to be transfected).

For the purposes of the invention, the expression "nucleic acid" is understood to mean both a deoxyribonucleic acid and a ribonucleic acid. They may be natural or artificial sequences, and in particular genomic DNA (gDNA), complementary DNA (cDNA), messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), hybrid sequences such as DNA/RNA chimeroplasts or synthetic or semisynthetic sequences, and oligonucleotides which are modified or otherwise. These nucleic acids may be of human, animal, plant, bacterial or viral origin and the like. They may be obtained by any technique known to persons skilled in the art, and in particular by the screening of libraries, by chemical synthesis or by mixed methods including the chemical or enzymatic modification of sequences obtained by the screening of libraries. They may be chemically modified. In general, they contain at least 20, 50 or 100 consecutive nucleotides, and preferably at least 200 consecutive nucleotides. More preferably still, they contain at least 500 consecutive nucleotides.

As regards more particularly deoxyribonucleic acids, they may be single- or double-stranded, as well as short oligonucleotides or longer sequences. In particular, the nucleic acids advantageously consist of plasmids, vectors, episomes, expression cassettes and the like. These deoxyribonucleic acids may carry a prokaryotic or eukaryotic replication origin which is functional or otherwise in the target cell, one or more marker genes, sequences for regulating transcription or replication, genes of therapeutic interest, anti-sense sequences which are modified or otherwise, regions for binding to other cellular components, and the like.

Preferably, the nucleic acid comprises one or more genes of therapeutic interest under the control of regulatory sequences, for example one or more promoters and a transcriptional terminator which are active in the target cells.

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

Among the therapeutic products for the purposes of the present invention, there may be mentioned more particularly enzymes, blood derivatives, hormones, lymphokines and cytokines as well as their inhibitors or their antagonists: interleukins, interferons, TNF, antagonists of interleukin 1, soluble receptors for interleukin 1 or TNFa, and the like (FR 92/03120), growth factors, neuro-transmitters or their precursors or synthesis enzymes, trophic factors (BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, VEGF-B, NT3, HARP/pleiotrophin and the like), apolipoproteins (ApoAI, ApoAIV, ApoE, and the like, FR 93/05125), dystrophin or a minidystrophin (FR 91/11947), the CFTR protein associated with cystic fibrosis, tumor suppressor genes (p53, Rb, RaplA, DCC, k-rev, and the like, FR 93/04745), genes encoding factors involved in coagulation (Factors VII, VIII, IX), the genes involved in DNA repair, suicide genes (thymidine kinase, cytosine deaminase), the genes for hemoglobin or other protein carriers, metabolic enzymes, catabolic enzymes and the like.

The nucleic acid of therapeutic interest may also be a gene or an anti-sense sequence or a DNA encoding an RNA with ribzyome function, whose expression in the target cell makes it possible to control the expression of genes or the transcription of cellular mRNAs. Such sequences can, for example, be transcribed in the target cell into RNAs which are complementary to cellular mRNAs and thus block their translation to protein, according to the technique described in Patent EP 140 308. The therapeutic genes also comprise the sequences encoding ribozymes, which are capable of selectively destroying target RNAs (EP 321 201).

As indicated above, the nucleic acid may also comprise one or more genes encoding an antigenic peptide, which is capable of generating an immune response in humans or in animals. In this specific embodiment, the invention allows the production of vaccines or the carrying out of immunotherapeutic treatments applied to humans or to animals, in particular for treating or preventing infections, for example viral or bacterial infections, or cancerous states.

They may be in particular antigenic peptides specific for the Epstein-Barr virus, the HIV virus, the hepatitis B virus (EP 185 573), the pseudo-rabies virus, the syncitia forming virus, other viruses, or antigenic peptides specific for tumors (EP 259 212).

Preferably, the nucleic acid also comprises sequences allowing the expression of the gene of therapeutic interest and/or the gene encoding the antigenic peptide in the desired cell or organ. They may be sequences which are naturally responsible for the expression of the gene considered when these sequences are capable of functioning in the infected cell. They may also be sequences of different origin (responsible for the expression of other proteins, or even synthetic). In particular, they may be promoter sequences of eukaryotic or viral genes. For example, they may be promoter sequences derived from the genome of the cell which it is desired to infect. Likewise, they may be promoter sequences derived from the genome of a virus. In this regard, there may be mentioned, for example, the promoters of the ElA, MLP, CMV and RSV genes, and the like. In addition, these expression sequences may be modified by the addition of activating or regulatory sequences, and the like. The promoter may also be inducible or repressible.

Moreover, the nucleic acid may also comprise, in particular upstream of the gene of therapeutic interest, a signal sequence directing the therapeutic product synthesized in the secretory pathways of the target cell. This signal sequence may be the natural signal sequence of the therapeutic product, but it may also be any other functional signal sequence, or an artificial signal sequence. The nucleic acid may also comprise a signal sequence directing the synthesized therapeutic product towards a particular compartment of the cell.

The compositions according to the invention may, in addition, comprise one or more adjuvants capable of combining with the transfecting compound/nucleic acid complexes and of improving the transfecting power thereof. In another embodiment, the present invention therefore relates to compositions comprising a nucleic acid, a transfecting compound as defined above and at least one adjuvant capable of combining with the transfecting compound/nucleic acid complexes and of improving the transfecting power thereof. The presence of this type of adjuvant (lipids, peptides, proteins or polymers for example) may make it possible advantageously to increase the transfecting power of the compounds. In this regard, the compositions of the invention may comprise, as adjuvant, one or more neutral lipids, which possess in particular the property of forming lipid aggregates.

The term "lipid aggregate" is a generic term which includes liposomes of all types (both unilamellar and multilamellar) as well as micelles or else more amorphous aggregates.

More preferably, the neutral lipids used within the framework of the present invention are lipids containing two fatty chains. In a particularly advantageous manner, natural or synthetic lipids which are zwitterionic or lacking ionic charge under physiological conditions are used. They may be chosen more particularly from dioleoylphosphatidylethanolamine (DOPE), oleoylpalmitoylphosphatidylethanolamine (POPE), di-stearoyl, -palmitoyl, -myristoylphosphatidylethanolamines as well as their derivatives which are N-methylated 1 to 3 times, phosphatidylglycerols, diacylglycerols, glycosyldiacylglycerols, cerebrosides (such as in particular galactocerebrosides), sphingolipids (such as in particular sphingomyelins) or asialogangliosides (such as in particular asialoGM1 and GM2). Advantageously, the lipid adjuvants used in the context of the present invention are chosen from DOPE, DOPC or cholesterol.

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

Preferably, the compositions of the invention comprise from 0.01 to 20 equivalents of adjuvants for one equivalent of nucleic acid in mol/mol and, more preferably, from 0.5 to 5 molar equivalents.

According to another alternative, the adjuvants mentioned above making it possible to improve the transfecting power of the compositions according to the present invention, in particular the peptides, proteins or certain polymers, such as polyethylene glycol, may be conjugated with the transfecting compounds according to the invention, and not simply mixed. In this case, they are covalently linked either to the substituent X in the general formula or to the end of the alkyl chain(s) RI and/or R 2 when the latter are fatty aliphatic chains. It is also advantageous to use, as adjuvant, polyethylene glycol covalently linked to cholesterol (chol-PEG). Indeed, when the latter is conjugated with the transfecting compounds according to the present invention, it makes it possible to obtain particles of reduced sizes, and thus to avoid their aggregation and to increase the half-life period of the particles in the bloodstream.

The quantity of transfectant, for example DT-3TU, used according to the present invention is such that the particles have sizes of less than 500 nm. Preferably, the quantity of transfectant, such as DT-3TU, used is at least 40 nmol of DT-3TU lipids/pg of DNA (see Examples 11, 13 and 14 below).

According to a particularly advantageous embodiment, the compositions of the present invention comprise, in addition, a targeting element which makes it possible to orient the transfer of the nucleic acid.

This targeting element may be an extracellular targeting element which makes it possible to orient the transfer of the nucleic acid toward certain cell types or certain desired tissues (tumor cells, hepatic cells, hematopoietic cells and the like). It may also be an intracellular targeting element which makes it possible to orient the transfer of the nucleic acid toward certain preferred cellular compartments (mitochondria, nucleus and the like). The targeting element may be mixed with the transfecting compounds according to the invention and with the nucleic acids, and in this case, the targeting element is preferably covalently linked to a fatty alkyl chain (at least 10 carbon atoms) or to a polyethylene glycol. According to another alternative, the targeting element is covalently linked to the transfecting compound according to the invention either at the level of the substituent X or on the spacer Y, or else at the end of RI and/or R 2 when the latter represent fatty aliphatic chains. Finally, the targeting element may also be linked to the nucleic acid as was specified above.

Among the targeting elements which may be used within the framework of the invention, there may be mentioned sugars, peptides, proteins, oligonucleotides, lipids, neuromediators, hormones, vitamins or derivatives thereof. Preferably, they are sugars, 29 peptides, vitamins or proteins such as for example

O

Z antibodies or antibody fragments, ligands of cell c- receptors or fragments thereof, receptors or receptor fragments. For example, they may be ligands of growth O 5 factor receptors, cytokine receptors, cellular lectintype receptors, folate receptors, or RGD sequence- Cq containing ligands with an affinity for the receptors Sfor adhesion proteins such as the integrins. There may also be mentioned the receptors for transferin, HDLs and LDLs, or the folate transporter. The targeting element may also be a sugar which makes it possible to target lectins such as the receptors for asialoglycoproteins or for sialydes, such as the Sialyl Lewis X, or alternatively an Fab fragment of antibodies, or a single-chain antibody (ScFv).

The subject of the invention is also the use of the transfecting compounds as defined above for transferring nucleic acids into cells in vitro, in vivo or ex vivo. More precisely, an aspect of the present invention is the use of the transfecting compounds according to the invention for the preparation of a medicament intended for treating diseases. A further aspect relates to a method of treating a disease in a subject in need thereof comprising the administration to said subject a compound according to the invention. In particular diseases result from a deficiency in a protein or nucleic product. The polynucleotide contained in said medicament encodes said protein or nucleic product, or constitutes said nucleic product, capable of correcting said diseases in vivo or ex vivo.

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

Preferably, the compositions of the invention contain a vehicle which is pharmaceutically acceptable for an injectable formulation, in particular a direct injection into the desired organ, or for administration by the topical route (on the skin and/or the mucous membrane). They may be in particular isotonic sterile solutions, or dry, in particular freeze-dried, compositions which, upon addition, depending on the case, of sterilized water or of physiological saline, allow the constitution of injectable solutions. The nucleic acid doses used for the injection as well as the number of administrations may be adapted according to various parameters, and in particular according to the mode of administration used, the relevant pathological condition, the gene to be expressed, or the desired duration of treatment. As regards more particularly the mode of administration, it may be either a direct injection into the tissues, for example at the level of the tumors, or an injection into the circulatory system, or a treatment of cells in culture followed by their reimplantation in vivo by injection or transplantation. The relevant tissues within the framework of the present invention are, for example, the muscles, skin, brain, lungs, liver, spleen, bone marrow, thymus, heart, lymph, blood, bones, cartilages, pancreas, kidneys, bladder, stomach, intestines, testicles, ovaries, rectum, nervous system, eyes, glands, connective tissues, and the like.

Another subject of the present invention relates to a method of transferring nucleic acids into cells comprising the following steps: bringing the nucleic acid into contact with a transfecting compound according to the present invention, to form a complex, and bringing the cells into contact with the complex formed in The invention relates, in addition, to a method of treating the human or animal body comprising the following steps: bringing the nucleic acid into contact with a transfecting compound according to the present invention, to form a complex, and bringing the cells of the human or animal body into contact with the complex formed in The cells may be brought into contact with the complex by incubating the cells with said complex (for uses in vitro or ex vivo), or by injecting the complex into an organism (for uses in vivo). In general, the quantity of nucleic acid intended to be administered depends on numerous factors such as for example the disease to be treated or to be prevented, the actual nature of the nucleic acid, the strength of the promoter, the biological activity of the product expressed by the nucleic acid, the physical condition of the individual or of the animal (weight, age and the like), the mode of administration and the type of formulation. In general, the incubation is preferably carried out in the presence, for example, of 0.01 to 1000 Ag of nucleic acid per 106 cells. For administration in vivo, nucleic acid doses ranging from 0.01 to 50 mg may for example be used. The administration may be carried out as a single dose or repeated at intervals.

In the case where the compositions of the invention contain, in addition, one or more adjuvants as defined above, the adjuvant(s) may be mixed beforehand with the transfecting compound according to the invention and/or the nucleic acid. Alternatively, the adjuvant(s) may be administered before the administration of the nucleolipid complexes.

According to another advantageous alternative, the tissues may be subjected to a chemical or physical treatment intended to improve the transfection. In the case of the physical treatment, the latter may use electrical pulses as in the case of electrotransfer, or else mechanical forces as in the case of sodoporation.

The present invention thus provides a particularly advantageous method for transferring nucleic acids in vivo, in particular for the treatment of diseases, comprising the in vivo or in vitro administration of a nucleic acid encoding a protein or which can be transcribed into a nucleic acid capable of correcting said disease, said nucleic acid being combined with a transfecting compound according to the invention under the conditions defined above.

The transfecting compounds of the invention are particularly useful for transferring nucleic acids into primary cells or into established lines. They may be fibroblast cells, muscle cells, nerve cells (neurons, astrocytes, glial cells), hepatic cells, hematopoietic cells (lymphocytes, CD34, dendritic cells, and the like), epithelial cells and the like, in differentiated or pluripotent form (precursors).

Another subject of the present invention also relates to the transfection kits which comprise one or more transfecting compounds according to the invention and/or mixtures thereof. Such kits may be provided in the form of a packaging which is compartmented so as to receive various containers such as for example vials or tubes. Each of these containers comprises the various elements necessary to carry out the transfection, individually or mixed: for example one or more transfecting compounds according to the invention, one or more nucleic acids, one or more adjuvants, cells, and the like.

In addition to the preceding arrangements, the present invention also comprises other characteristics and advantages which will emerge from the examples and figures below, which should be considered as illustrating the invention without limiting its scope. In particular, the applicant proposes, without limitation, an operating protocol as well as reaction intermediates which may be used to prepare the transfecting compounds according to the invention. Of course, it is within the capability of persons skilled in the art to draw inspiration from this protocol or intermediate products to develop similar methods so as to arrive at these same compounds.

ABBREVIATIONS USED EtBr: ethidium bromide DCC: dicyclohexylcarbodiimide DPPC: 1,2-dipalmitoyl-sn-glycero-3-phosphocholine DTTU: 3-(2-{3-[2-(3-{2-[3-(ditetradecylcarbamoyl)propionylamino]ethyl}thioureido)ethyl]thioureido}ethyl)-1-methylthiourea In the case where the DTTU molecule comprises 3 thioureas, it will be designated as DT-3TU, 4 thioureas DT-4TU, and so on.

EPC: L-a-phosphatidylcholine 95% (egg) PyBOP: benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate TBE: tris-borate-EDTA TFA: trifluoroacetic acid THF: tetrahydrofuran LEGEND TO THE FIGURES Figure 1: Variation of the level of fluorescence (in as a function of the quantity of EPC/DT-3TU mixture (in nmol) per gg of nucleic acid and as a function of the quantity of EPC alone (in nmol) per ug of nucleic acid (control mixture).

Figure 2: Variation of the level of fluorescence (in as a function of the quantity of DPPC/DT-3TU mixture (in nmol) per mg of nucleic acid and as a function of the quantity of DPPC alone (in nmol) per gg of nucleic acid (control mixture).

Figure 3: Agarose gel (0.8%/TBE) showing the compaction of the plasmid pXL3031 (gg) as a function of the quantity of EPC/DT-3TU liposome (in nmol) used.

Figure 4: Zeta potential (in mV) corresponding to the potential at the surface of the DPPC/DT-3TU-DNA liposomes as a function of the quantity of lipid (nmol) per gg of nucleic acid.

Figure 5: Efficiency of in vitro transfection of HeLa cells with complexes formed between the DNA and the DT-3TU/DPPC liposomes at various lipid/DNA ratios in nmol/gg, with or without serum.

The y-axis represents the expression of luciferase in RLU/gg of protein.

The x-axis indicates the quantity of DT-3TU (in nmol) per jg of DNA.

Figure 6: Level of proteins (as absorbance) of HeLa cells not treated or treated with EPC+DT-3TU/DNA liposomes at various lipid/DNA ratios in nmol/gg.

Figure 7: Schematic representation of the plasmid pXL3031.

Figure 8: Agarose gel (0.8%/TBE) showing the compaction of the plasmid pXL3031 (pg) as a function of the quantity of DT-3TU/DPPC nanoemulsion (in nmol) used.

Figure 9: Agarose gel (0.8%/TBE) showing the compaction of the plasmid pXL3031 (pg) as a function of the quantity of DT-3TU/DPPC/Chol-PEG nanoemulsion (in nmol) used.

Figure 10: Variation of the percentage of compacted DNA as a function of the quantity of DT-4TU/DPPC mixture (in nmol) per pg of nucleic acid compared with different quantities of DT-3TU/DPPC mixture (in nmol) per pg of nucleic acid.

Figure 11: Agarose gel (0.8%/TBE) showing the protection of the plasmid pXL3031 (gg) by the mixture of DT-3TU/DPPC lipids against DNases. The control DNA in the presence of DNases was deposited in well 2, the DNA in the presence of 30, 40 nmol of lipid/g of DNA and 40 nmol/gg 6% chol-PEG treated with DNase were deposited in wells 3, 4 and 5 respectively.

Figure 12: Agarose gel (0.8%/TBE) showing the protection of the plasmid pXL3031 (gg) by the mixture of DT-3TU/DPPC lipids against serum. Well 1 corresponds to the DNA alone, wells 2, and 3 to the DNA alone and in the presence of 40 nmolgg of nanoemulsions of DT-3TU/DPPC in 150 mM NaC1 and then in 20% serum (wells 4 and 5) and in 100% serum (wells 6 and 7).

Figure 13: Efficiency of in vivo transfection into the muscle with complexes formed between the DNA and the DT-3TU/DPPC liposomes at various lipid/DNA ratios in nmol/tg, with and without electrotransfer Figure 14: Biodistribution in vivo in mice of DT-3TU/DPPC/DOPE-Rh/DNA complexes after 30 min, 1 h and 6 h in blood, the lungs and the RES system. This figure shows the furtive character of these particles: 50% of complexes were found after 30 minutes in the bloodstream.

EXAMPLES

Customary reagents and catalysts such as triethylamine, trifluoroacetic acid, p-toluenesulfonic acid, benzotriazol-l-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), dicyclohexylcarbodiimide (DCC), carbon disulfide, tetradecylamine, di-tert-butyl dicarbonate, 4-dimethylaminopyridine, or diisopropylethylamine are commercially available.

The proton nuclear magnetic resonance 1 H NMR) spectra were recorded on Bruker 300, 400 and 600 MHz spectrometers. The chemical shifts are expressed in ppm (parts per million) and the multiplicities by the customary abbreviations.

In the text which follows, the nucleic acid used is the plasmid pXL3031 described in the publication Gene Therapy (1999) 6, pp. 1482-1488, which contains the luc gene encoding luciferase under the control of the cytomegalovirus CMV E/P promoter. This plasmid is represented in Figure 7. Its size is 3671 bp. The plasmid solution used is diluted to 1.262 g/l in water for injection.

EXAMPLE 1: Synthesis of DT-3TU 3-(2-{3-12-(3-{2-[3-Ditetradecylcarbamoyl)propionylamino]ethyl}thioureido)ethyl]thioureido}ethyl)-1-methylthiourea, or DT-3TU, is according to the general formula in which: X -CH3; m 2; R H; n 3; P 0; Y NH-CO-CH 2

-CH

2 and L -N(RI)R 2 in which RI R2 C 1 4

H

29 a) Synthesis of ditetradecylamide (1) 131.6 mmol of tetradecanoic acid (30 g) and 140.8 mmol of tetradecylamine (30 g) are mixed in a round-bottomed flask equipped with a magnetic stirrer connected to a collecting flask containing a drying agent (P 2 0 5 The reaction mixture is then heated for 4 hours to 1700C under reduced pressure (50 mmHg). The crude material is then solubilized in THF (700 ml; heated slightly in order to aid solubilization) and then 4 equivalents of Amberlyst-15 resin (8 g) are added in order to bind the excess amine. After stirring for 20 minutes, the solution is filtered and the filtrate is then concentrated to give 55.11 g of a white solid (yield: 99%).

1H NMR (CDC1 3 6 (ppm) 0.88 6H, J=6.5 Hz, -CH 3 1.25 42 H, -CH 2 1.47 2H, CO-CH 2

-CH

2 1.60 2H, N-CH 2

-CH

2 2.15 2H, J=7.5 Hz, CO-CH2), 3.23 (dt, 2H, J=7 Hz, N-CH 2 5.50 1H, NH).

SC NMR (CDC1 3 6 (ppm) 14.09 (C 14

+C'

1 3 22.71

(C

13

+C'

12 25.90 (C' 2 26.99 (C 3 29.69 (-CH 2 31.96

(C

12 36.97 39.56 (C 1 160 (CO).

b) Synthesis of ditetradecylamine (2) 47 mmol of ditetradecylamide (20 g) are dissolved in 700 ml of anhydrous THF, under nitrogen, in a round-bottomed flask equipped with a condenser and a drying tube. The mixture is cooled to 0°C and then 89 mmol of lithium aluminum hydride LiAlH 4 (3.4 g) are added. After addition, the mixture is then heated under reflux for 5 hours, with vigorous stirring. Once the reaction is complete, the mixture is cooled to 0°C in order to carry out the hydrolysis by successive addition of 3.4 ml of water, 6.8 ml of 2N sodium hydroxide and 3.4 ml of water. After stirring for 1 hour at room temperature, the crude reaction material is filtered on a Bchner funnel and the filtrate is concentrated. The product obtained is then purified on equivalents of A-15 resin (15 g) in 300 ml of THF, with stirring for 30 minutes. The resin is filtered and redissolved in 300 ml of THF, with addition of 2 equivalents of triethylamine (19.2 ml). After stirring for 30 minutes, the solution is filtered and the filtrate is concentrated to give 17.72 g of a white solid (yield: 92%).

H NMR (CDC13): 6 (ppm) 0.87 6H, J=6.5 Hz, -CH 3 1.25 44 H, -CH 2 1.46 4H, N-CH2-CH 2 2.58 (t, 4H, J=7 Hz, N-CH 2 13C NMR (CDC1 3 6 (ppm) 14.06 (C 14 22.69 (C 13 27.48

(C

3 28.29 (C 2 29.69 (C 4 -CIn), 31.96 (C 12 50.15 c) Synthesis of N,N-ditetradecylsuccinamic acid (3) 12.65 mmol of succinic anhydride (1.266 g), 12.65 mmol of 4-dimethylaminopyridine (1.546 g) and 10.75 mmol of ditetradecylamine (4.407 g) are successively added to 125 ml of dichloromethane in a round-bottomed flask. The reaction mixture is stirred for 18 hours at room temperature. Once the reaction is complete, the mixture is extracted with dichloromethane and hydrochloric acid The organic phase is then washed with brine and dried over magnesium sulfate, filtered and then concentrated to give 4.21 g of product (yield: 66%).

WH NMR (CDC1 3 6 (ppm) 0.85 6H, J=6.3 Hz, -CH3), 1.23 44 H, -CH 2 1.48 4H, N-CH2-CH 2 2.64 (s, 4H, CO-CH2-CH 2 3.22 4H, N-CH2).

13C NMR (CDC1 3 5 (ppm) 14.08 (C 14 22.69 (C 13 27.74

(C

3 28.10 (CO-CH 2 -CH2-CO), 28.92 (C 2 29.67 (C 4 -C11), 30.07 (CO-CH2-CH 2 31.95 (C1 2 46.21 and 47.98 (C1), 4171.46 (CO-NH(C14H 29 2 173.14 (NH-CO) d) Synthesis of the tert-butyl ester of 2-aminoethylcarbamic acid (4) 18.6 mmol of di-tert-butyl dicarbonate (4 g) are added dropwise to 102.83 mmol of ethylenediamine (6.17 g) in solution in chloroform (20 ml), under nitrogen. The reaction mixture is then stirred for 18 hours at room temperature. Once the reaction is complete, the solution is concentrated. The resulting oil, dissolved in dichloromethane, is washed with a saturated aqueous sodium carbonate solution. The organic phase is then dried over magnesium sulfate, filtered and concentrated. The crude product is purified by flash chromatography (dichloromethane/ methanol 2.38 g of product are thus obtained (yield: H NMR (CDC1 3 6 (ppm) 1.40 9H, (CH 3 3 1.52 (s, 2H, NH2), 2.59 2H, J=5.9 Hz, N-CH2), 3.12 2H, 4J=5.4 Hz, NHBoc-CH 2 5.1 1H, NHBoc) 13C NMR (CDC1 3 6 (ppm) 28.15 (CH3) 3 41.67 (CH 2 -NHBoc), 43.46 (CH 2 -NHz), 78.31 (C-(CH 3 3 156.21 e) Synthesis of the tert-butyl ester of 2-[3-(ditetradecylcarbamoyl)propionylamino]ethylcarbamic acid 8.84 mmol of PyBOP (4.601 9.72 mmol of the amine obtained in the preceding stage (1.558 g) and 24.31 mmol of diisopropylethylamine (4.24 ml) are successively added to a solution of 8.84 mmol of the acid obtained above (4.5 g) in 88 ml of dichloromethane. The solution is then stirred for 4 hours at room temperature. At the end of the reaction, the reaction mixture is filtered and then the product is purified by flash chromatography (heptane/ethyl acetate 5:5 and then heptane/ethyl acetate 3.79 g of the ester are thus obtained (yield: 66%).

1H NMR (CDC1 3 8 (ppm) 0.87 6H, J=6.6 Hz, -CH3), 1.25 44H, -CH 2 1.43 9H, (CH 3 3 1,48 4H,

N-CH

2

-CH

2 2.56 2H, J=6.7 Hz, CH 2 2.69 2H, J=6.2 Hz, CH 2 3.28 4H, N-CH 2 3.3 4H, CH 2 1

CH

2 2).

13C NMR (CDC1 3 8 (ppm) 14.07 (C" 14 22.56 (C" 13 26.99

((CH

3 3 27.73 (C" 3 28.29 (C' 2 28.59 (C" 2 29.27

(C"

4 29.55 (C' 3 31.41 (C"t 2 39.85 (C 2 40.39 46.21 and 47.98 78.77 (C(IV)-Boc), 156.33 (CO-Boc), 171.46 (CO-NH(C 1 4H 29 2 173.14 f) Synthesis of 2-[3-(ditetradecylcarbamoyl)propionylamino]ethylamine (6) 12.2 mmol of distilled TFA (0.94 ml) are added to 2.44 ml of the ester obtained in the preceding stage (1.59 After two hours, the reaction is complete. The product is coevaporated twice with cyclohexane in a rotary evaporator in the cold state.

The yield is quantitative.

IH NMR (CDC1 3 8 (ppm) 0.91 6H, J=6.6 Hz, -CH 3 1.29 44H, 1.51 4H, N-CH 2

-CH

2 2,59 2H, J=6.7 Hz, H' 3 2.71 2H, J=6.2 Hz, H' 3 3.29 4H, N-CH 2 3.31 4H, HI, H2).

13C NMR (CDC13): 6 (ppm) 14.00 (C" 14 22.67 27.35 3 27.95 (C' 2 28.53 (C 2 29.65 (C" 4 -C"11), 30.70 (C' 3 31.94 (C" 12 37.83 (C 2 40.08 (C 2 47.85 and 49.42 171.72 (CO-NH(C4H 29 2 173.26 g) Synthesis of ((1,1-dimethylethoxycarbonyl)amino)ethylisothiocyanate (7) 43.69 mmol of DCC (9.015 297.9 mmol of carbon disulfide (17.9 ml) in 27.5 ml of THF are successively added to a round-bottomed flask. The mixture is cooled to -7 0 °C with a bath of ice and ammonium chloride NH 4 Cl 43.69 mmol of the amine obtained above (7 g) dissolved in 20.5 ml of anhydrous THF are then added dropwise over 30 minutes.

The mixture is allowed to return to room temperature and the mixture is kept stirring for 21 hours. After evaporation, diethyl ether is added to precipitate the dicyclohexylurea formed. The solution is filtered, the filtrate is concentrated and then purified by flash chromatography (heptane/ethyl acetate 80:20) to give 6.357 g of desired product (yield: 72%).

'H NMR (CDC1 3 8 (ppm) 1.38 9H, (CH 3 3 3.31 (q, 2H, 4 J=5.8 Hz, NHBoc-CH 2 3.59 2H, J=5.6 Hz, S=C=N-

CH

2 5.16 1H, NHBoc) "1C NMR (CDC13):: 6 (ppm) 28.54 ((CH 3 3 41.03 (CH2)- NHBoc), 45.53 (CH 2 79.71 (C-(CH3)3, 132.72 156.21 h) Synthesis of the tert-butyl ester of (ditetradecylcarbamoyl)propionylamino]ethyl}thioureido) ethylcarbamic acid (8) 9.76 mmol of triethylamine (1.36 ml) are directly added to 2.44 mmol of the amine obtained above (1.62 g) and the mixture is kept stirring for minutes. 24.4 ml of dichloromethane and 2.92 mmol of the isothiocyanate obtained in the preceding stage (0.59 g) are then added. The reaction mixture is stirred at room temperature for 12 hours. The mixture is then evaporated and then purified by flash chromatography (ethyl acetate/heptane 6:4 and then 100% of ethyl acetate). 1.343 g of the desired ester are thus obtained (yield: 73%).

1H NMR (CDC1 3 5 (ppm) 0.67 6H, J=6.4 Hz, -CH 3 1.05 44 H, -CH 2 1.26 9H, CH 3 3 1.35 4H,

N-CH

2

-CH

2 2.31 2H, H 3 3 2.49 2H, H 3 3.06 4H, N-CH 2 3.11 4H, HI, H" 2 3.47 4H, H2, 7.14 (2H, H thiourea).

C NMR (CDC1 3 6 (ppm) 13.95 (C 4 14 22.57 (C 4 13 26.92 ((CH 3 3 27.07 4

'C

3 27.78 (C 3 2 28.39 (C 4 28.82 (C 3 3 29.55 (C 4 '4-C 4 31.83 (C 4 39.45

(C"

2 43.63 (C 2 and C" 1 46.39 and 48.16 79.24 (C(IV)-Boc), 156.53 (CO-Boc) 171.72 (CO-NH(C 14

H

29 2 173.71 (C 3 1 182.97 i) Synthesis of 2-(3-(2-[3-(ditetradecylcarbamoyl)propionylamino]ethyl}thioureido)ethylamine (9) 9.86 mmol of distilled TFA (0.76 ml) are added to 1.98 mmol of the product obtained in the preceding stage (1.5 After 3 hours, the reaction is complete. The product is coevaporated twice with cyclohexane using a rotary evaporator in the cold state. The yield is quantitative.

1 H NMR (CDC1 3 6 (ppm) 0.67 6H, J=6.4 Hz, -CH 3 1.05 44 H, 1.26 9H, CH3) 3 1.31 4H,

N-CH

2

-CH

2 2.31 2H, H 3 3 2.49 2H, H3'3), 3.06 4H, N-CH2), 3.11 4H, HI, 3.44 4H, H 2 7.10, (2H, H thiourea).

13 C NMR (CDC1 3 6 (ppm) 13.85 (C 4 14 22.49 (C 4 13 27.01 (C 4 3 27.72 (C 3 2 28.29 (C 4 2 28.79 (C 3 3 29.49 (C 4 4

-C

4 11 31.77 (C 4 12 39.42 (C" 2 43.75 (C 2 and 46.29 and 48.09 (C 4 171.72 (CO-NH(C 1 4H 29 2 173.71 (C 3 182.97 j) Synthesis of the tert-butyl ester of [3-(ditetradecylcarbamoyl)propionylamino]ethyl)thioureido)ethyl]thioureido}ethylcarbamic acid 7.92 mmol of triethylamine (1.1 ml) are directly added to 1.98 mmol of the amine obtained in the preceding stage (1.47 g) and the mixture is kept stirring for 15 minutes. 19.8 ml of dichloromethane and 2.38 mmol of the isothiocyanate obtained above (0.461 g) are then added and the reaction is allowed to proceed at room temperature, with stirring, for 12 hours. The mixture is then evaporated and then purified by flash chromatography (ethyl acetate/heptane 6:4 and then ethyl acetate/methanol 98:2). 1.136 g of the desired product (10) are thus obtained (yield: 67%).

1H NMR (CDC13): 6 (ppm) 0.74 6H, J=6.6 Hz, -CH 3 1.12 44 H, -CH 2 1.30 9H, CH3 3 1.45 4H,

N-CH

2

-CH

2 2.41 2H, H5'2), 2.58 2H, H 5 2 3.12 4H, N-CH 2 3.25 4H, HI, H 4 2 3.56 8H, H 2 H"i, H"2, H 4 7.14 (4H, H thiourea).

1C NMR (CDC1 3 6 (ppm) 14.06 (C6'4) 22.57 (C 6 '1 3 27.11 (C 6 3 26.93 ((CH 3 3 27.79 (C 5 2 28.38 (C 6 2 28.81 (C 5 3 29.56 (C 6 4

-C

6 31.83 (C6'12), 39.55

(C

4 2 43.66 (C 2

C"

1

C"

2

C

4 46.49 and 48.23 (C 6 79.28 (C(IV)-Boc), 156.61 (CO-Boc), 171.96 (CO- NH(Ci 4

H

29 2 173.72 182.93 k) Synthesis of 2-(3-[2-(3-{2-[3-(ditetradecylcarbamoyl)propionylamino]ethyl}thioureido)ethyl]thioureido)ethylamine (11) 5.84 mmol of distilled TFA (0.45 ml) are added to 1.15 mmol of the product (10) obtained in the preceding stage (1 After 3 hours, the reaction is complete. The product is coevaporated twice with cyclohexane in a rotary evaporator in the cold state.

The yield is quantitative.

IH NMR (CDC1 3 8 (ppm) 0.85 6H, J=6.6 Hz, -CH 3 1.25 40 H, -CH 2 1.48 4H, N-CH 2

-CH

2 1.52 (m, 4H, N-CH 2

-CH

2

-CH

2 2.65 2H, H 5 2 2.77 2H,

H

5 3 3.26 4H, N-CH2), 3.43 4H, HI, H 4 2 3.85 8H, H 2

H"

1

H"

2 H4'I), 7.44 (4H, H thiourea).

13C NMR (CDC13): (ppm) 14.05 (C 6 22.68 (C 6 13 27.05 (C 6 3 27.58 (C5' 2 28.71 (C 6 2 29.36 (C 5 3 29.67 (C 6 4-C 6 1 1 31.94 (C 6 '1 2 40.49 43.49 (C in a of 47.40 and 49.07 (C 6 172.16 (CO-

NH(C

14

H

29 2 174.00 (NH-CO), 182.73 1) Synthesis of DT-3TU (12) 1.12 mmol of triethylamine (0.16 ml) are directly added to 0.28 mmol of the amine (11) obtained in the preceding stage (0.24 g) and the mixture is kept stirred for 15 minutes. 2.8 ml of dichloromethane and 0.34 mmol of methyl isothiocyanate (0.024 g) are then added and the reaction is allowed to proceed at room temperature, with stirring for 12 hours. The mixture is then evaporated and then purified by HPLC (highperformance liquid chromatography) on a C 4 column with the following gradient: initially a water/methanol 95:5 mixture up to 100% of methanol. The product obtained is again purified on a small silica column (ethyl acetate/heptane 80:20 and then 100% of ethyl acetate).

118 mg of DTTU are thus obtained (yield: 51%).

IH NMR (CDC1 3 6 (ppm) 0.86 6H, J=6.7 Hz, -CH 3 1.24 40 H, 1.44 4H, N-CH 2

-CH

2 1.54 (m, 4H, N-CH 2 -CH2-CH 2 2.52 2H, H 6 2 2.67 2H,

H

6 2 3.05 3H, terminal -CH3), 3.21 4H, N-CH2), 3.32 2H, H 5 2 3.75 10H, H'i, H' 2

H

3 1

H

3 2

H

5 1 7.14 (6H, H thiourea).

13C NMR (CDC1 3 6 (ppm) 14.09 (C 7 '1 4 22.69 (C 7 13 27.24 (C 7 3 27.89 (C 6 2 28.87 (C 7 2 29.38 (C6 3), 29.67 (C 7 4

_C

7 11 31.26 (terminal CH 3 31.94 (C 7 12 39.71 (C 5 2 43.63 (C' 1

C'

2

C

3 1

C

3 2

C

5 1 46.67 and 48.40 (C 7 172.16 (CO-NH(C 4

H

29 2 174.00 (C 6 1 182.73 EXAMPLE 2: Synthesis of DT-4TU DT-4TU or tetradecylcarbamoyl]propionylamino}ethyl)thioureido]ethyl}thioureido)ethyl]thioureido}ethyl)-1-methylthiourea. DT-4TU, is according to general formula in which: X -CH 3 m 2; R H; n 4; f 0; Y NH-CO-CH 2

-CH

2 and L -N(RI)R 2 where RI R2 C 14

H

2 9 To synthesize DT-4TU, the procedure will likewise be carried out starting with the amine (11).

a) Synthesis of the tert-butyl ester of {3-[2-(3-(ditetradecylcarbamoyl)propionylamino)ethyl]thioureidoethyl)thoureidoethylthioureidedo)ethylcarbamic acid (13) s Y s o o o Triethylamine (0.643 ml, 4.6 mmol) is added to the amine (11) (0.8 g, 92 mmol) and the mixture is kept stirring for 15 min. CH 2 C12 (9.2 ml) is then added.

Isothiocyanate (0.224 g, 1.104 mmol) is then added and the reaction is allowed to proceed at RT, while stirring for 20 h. The mixture is then evaporated and then purified by chromatography (ethyl acetate/heptane 8:2 and then ethyl acetate/methanol 90:10); 252 mg of the desired product are obtained (yield: 46%).

H NMR (CDC1 3 6 (ppm) 0.86 6H, J=6 Hz, H-14'), 1.25 44 H, 1.42 9H, CH3)3), 1.45 4H, 2.55 2H, 2.69 2H, H-3), 3.22 4H, 3.31 4H, H-5 and H-15), 3.74 12H, H-6, H-8, H-9, H-11, H-12, H-14), 7.31 (6H, H thiourea).

"C NMR (CDC13): 8 (ppm) 14.06 22.66 27.21 27.88 28.50 ((CH 3 3 28.87 29.64 31.27 31.92 39.639 40.44 43.74 C-8, C-9, C-11, C-12, C-14), 46.64 and 48.38 79.10 156.63 172.35 174.04 182.65 C-13).

b) Synthesis of 2-(3-(2-[3-(2-{3-[2-(3-(ditetradecylcarbamoyl)propionylamino)ethyl] thioureido)ethyl) thioureido]ethyl}thioureido)ethylamine (14) s s 0 CFCOo- Hp >2 Distilled TFA (0.142 ml, 1.84 mmol) is added to the amine boc (13) obtained in the preceding stage (0.22 g, 0.23 mmol). After 6 hours, the reaction is complete.

The product is coevaporated twice with cyclohexane in a rotary evaporator in the cold state and placed in a desiccator over sodium hydroxide pellets overnight. The yield is quantitative.

H NMR (CDC1 3 6 (ppm) 0.74 6H, J=6.6 Hz, H-14'), 1.12 44 H, 1.45 4H, 2.41 2H, 2.58 2H, 3.12 4H, 3.25 4H, H-5 and H-15), 3.56 12H, H-6, H-8, H-9, H-11, H-12, H-14), 7.14 (6H, H thiourea).

"C NMR (CDC13): 6 (ppm) 14.06 22.57 27.11 27.79 28.38 28.81 29.56 31.83 39.55 43.66 C-8, C-9, C-11, C-12, C-14), 46.49 and 48.23 171.96 173.72 182.93 C-10, C-13).

c) Synthesis of decylcarbamoyl]propionylamino)ethyl)thioureido]ethyl}thioureido)ethyl]thioureido}ethyl)-l-methylthiourea (DT-4TU) 8 o 1.38 mmol of triethylamine (0.19 ml) are directly added to 0.23 mmol of the amine (14) obtained in the preceding stage (0.224 g) and the reaction is allowed to proceed for 15 minutes. 2.3 ml of dichloromethane and 0.46 mmol of methyl isothiocyanate (0.034 g) are then added and the reaction is allowed to proceed at room temperature, with stirring, for 12 hours. The mixture is then evaporated and then purified by flash chromatography (100% ethyl acetate and then ethyl acetate/methanol 95:5). 109 mg of DT4TU are thus obtained (yield: 51%).

1 H NMR (CDC1 3 6 (ppm) 0.88 6H, J=6.3 Hz, H-14'), 1.26 44 H, 1.45 1.45 4H, 1.58 4H, 2.57 2H, 2.73 2H, 3.03 3H, H-17), 3.23 4H, 3.31 (m, 2H, 3.73 14H, H-6, H-8, H-9, H-ll, H-12, H-14, H-15), 7.14 (8H, H thiourea).

13C NMR (CDC1 3 6 (ppm) 14.09 22.69 27.24 27.89 28.87 29.38 29.67 31.26 31.94 39.71 43.63 C-8, C-9, C-1l, C-12, C-14, 46.67 and 48.40 172.16 174.00 182.73 C-10, C-13, C-16).

Example 3: Synthesis of DT-2TU diol The compound 2-(3-{2-[3-(ditetradecylcarbamoyl)propionylamino]ethyl}thioureido)ethyl]propane-1,2-diol or DT-2TU corresponds to general formula in which 'rC HO OH m 2; R H; n 2; 1 0; Y NH-CO-CH2-CH 2 and L -N(RI)R 2 where RI R2 C 1 4

H

29 a) Synthesis of 4-isothiocyanatomethyl-2,2-dimethyl- [1,3]dioxalane (16) H 4S

SC

X

DCC (3.146 g, 15.25 mmol), carbon disulfide (6.253 ml, 104.005 mmol) in THF (9.6075 ml) are successively added to a round-bottomed flask. The mixture is cooled to -7°C with a bath of ice/NH4C1 2,2-Dimethyl- 1,2-dioxalane-4-methanamine (2 g, 15.25 mmol), dissolved in anhydrous THF (7.1675 ml), is then added dropwise over 30 minutes. The mixture is allowed to return to room temperature and the mixture is kept stirring for 21 h. After evaporation, diethyl ether is added. The mixture is filtered, evaporated and chromatographed.

H NMR (CDC1 3 6 (ppm) 1.33 and 1.44 3H, H-5, H-6), 3.57 (dd, 1H, J 4.8 Hz, J 14.4 Hz, 3.69 (dd, 1H, J 4.9 Hz, J 14.4 Hz, 3.80 (dd, 1H, J 5.4 Hz and J 8.7 Hz, 4.09 (dd, 1H, J 6.3 Hz and J 8.7 Hz, 4.28 1H, H-2).

C NMR (CDC1 3 6 (ppm) 25.17, 26.77 47.49 66.55 73.70 110.29 132.76 b) Synthesis of 2-(3-{2-[3-(ditetradecylcarbamoyl)propionylamino]ethyl}thioureido)ethyl-4-ylmethyl- 2,2-dimethyl[1,3]dioxalane (17) 13

H

8 0 0.95 mmol of diisopropylethylamine(0.165 ml) is directly added to 0.19 mmol of the amine obtained in the preceding stage (0.146 and the reaction is kept stirring for 15 minutes. 1.9 ml of dichloromethane and 0.209 mmol of isothiocyanate (16) (0.027 g) are then added and the reaction is allowed to proceed at room temperature, with stirring, for 12 hours. The mixture is then evaporated and then purified over a C8 reversed phase cartridge with a gradient 100% water up to 100% acetonitrile. The product (17) is obtained with a yield of 49%.

"1H NMR (CDC1 3 8 (ppm) 0.88 6H, J=6.3 Hz, H-14'), 1.25 44 H, 1.34 and 1.43 3H, H-16), 1.48 4H, 1.56 4H, 2.52 (m, 2H, 2.68 2H, 3.23 4H, 3.37

I

2H, 3.73 8H, H-6, H-8, H-9, 4.05 2H, H-13), 4.33 1H, H-12), 7.14 (4H, H thiourea).

13C NMR (CDC13): 6 (ppm) 14.06 22.67 25.32 27.00 and 27.17 (C-15, C-16), 27.80 28.56 28.84 29.66 C-1l'), 31.25 31.92 39.78 43.66 (C-6, C-8, 44.53 46.73 and 47.13 66.87 (C-136), 74.62 109.05 172.21 174.14 183.32 C-10, C-13, C-16).

c) Synthesis of [2-(3-(2-[3-(ditetradecylcarbamoyl)propionylamino]ethyl}thioureido)ethyl]propane-1,2-diol (DT-2TU diol) (18) S O H H HO OH s 0 The protected diol (17) (0.05 g, 0.05 mmol) is dissolved in 1 ml of 1N HCl/THF solution at RT; the mixture is stirred for 18 h. The crude product is then extracted with dichloromethane (2 x 5 ml). The organic phases are combined and then neutralized with a sodium hydrogen carbonate solution. The aqueous phases are extracted with dichloromethane. The organic phases are assembled and then dried over magnesium sulfate and then evaporated. The crude product is chromatographed on a C8 reversed phase column with a gradient 100% water up to 100% acetonitrile. The product (18) is obtained with a yield of 49%.

"H NMR (CDC1 3 6 (ppm) 0.88 6H, J=6.3 Hz, H-14'), 1.26 44 H, 1.43 4H, 1.59 4H, 1.79 2H, OH), 2.50 2H, H-2), 2.70 2H, 3.24 4H, 3.39 2H, 3.72 6H, H-6, H-8, 3.9 2H, H-ll), 4.22 2H, H-13) 4.58 1H, H-12), 7.14 (4H, H thiourea).

13C NMR (CDC1 3 6 (ppm) 14.06 22.67 25.32 27.80 28.56 28.84 29.66 31.25 31.92 39.78 43.66 C-8, 45.86 46.73 and 47.13 63.54 70.97 172.21 174.14 183.32 EXAMPLE 4: Synthesis of DT-3TU diol The compound decylcarbamoyl)propionylamino)ethyl]thioureido}ethyl)thioureido]ethyl}thioureido)ethyl]propane-1,2-diol or DT-3TU corresponds to formula in which HO OH m= 2; R H; n 3; S= 0; Y NH-CO-CH 2

-CH

2 and L -N(RI)R 2 where RI R2 C 14

H

29 To synthesize DT-3TU diol, the procedure will likewise be carried out starting with the amine (11).

a) Synthesis of decylcarbamoyl)propionylamino)ethyl]thioureido}ethyl)thioureido]ethyl)thioureido)ethyl]-4-ylmethyl-2,2-dimethyl[1,3]dioxalane (19) 11 o /H H kY Ytcwcz 0.95 mmol of diisopropylethylamine (0.165 ml) is directly added to 0.19 mmol of the amine (11) obtained in the preceding stage (0.165 g) and the mixture is kept stirring for 15 minutes. 1.9 ml of dichloromethane and 0.209 mmol of isothiocyanate (16) (0.027 g) are then added and the reaction is allowed to proceed at room temperature, with stirring, for 12 hours. The mixture is then evaporated and then purified on a C8 reversed phase cartridge with a gradient 100% water up to 100% acetonitrile. The product (19) is obtained with a yield of 49%.

1H NMR (CDC1 3 6 (ppm) 0.88 6H, J=6.3 Hz, H-14'), 1.25 44 H, 1.34 and 1.43 3H, H-18, H-19), 1.48 4H, 1.56 4H, 2.52 (m, 2H, 2.68 2H, 3.23 4H, 3.37 2H, 3.73 12H, H-6, H-8, H-9, H-ll, H-12, H-14), 4.05 2H, H-16), 4.33 1H, H-15), 7.14 (6H, H thiourea).

3C NMR (CDC1 3 8 (ppm) 14.06 22.67 25.32 27.00 and 27.17 (C-18, C-19), 27.80 28.56 28.84 29.66 C-ll'), 31.25 31.92 39.78 43.66 (C-6, C-8, C-9, C-ll), 44.53 46.73 and 47.13 66.87 74.62 109.05 172.21 174.14 183.32 C-10, C-13, C-16).

b) Synthesis of 2-(3-(2-[3-(2-(3-[2-(3-(ditetradecylcarbamoyl)propionylamino)ethyl]thioureido}ethyl)thioureido]ethyl)thioureido)ethyl]propane-1,2-diol (DT-3TU diol) i 12 H H The protected diol (19) (0.05 g, 0.05 mmol) is dissolved in 1 ml of 1N HC1/THF solution at RT; the mixture is stirred for 18 h. The crude product is then extracted with dichloromethane (2 x 5 ml). The organic phases are combined and then neutralized with a sodium hydrogen carbonate solution. The aqueous phases are extracted with dichloromethane. The organic phases are combined and then dried over magnesium sulfate and then evaporated. The crude product is chromatographed on a C8 reversed phase column with a gradient 100% water up to 100% acetonitrile. The product (20) is obtained with a yield of H NMR (CDC13): 6 (ppm) 0.88 6H, J=6.3 Hz, H-14'), 1.26 44 H, 1.43 4H, 1.59 4H, 1.79 2H, OH), 2.50 2H, H-2), 2.70 2H, 3.24 4H, 3.39 2H, 3.72 10H, H-6, H-8, H-9, H-1, H-12), 3.9 (m, 2H, H-14), 4.22 2H, H-16), 4.58 1H, H-15), 7.14 (6H, H thiourea).

1C NMR (CDC1 3 6 (ppm) 14.06 22.67 25.32 27.80 28.56 28.84 29.66 31.25 31.92 39.78 43.66 C-8, C-9, C-ll), 44.53 46.73 and 47.13 63.54 70.97 109.05 172.21 174.14 183.32 C-13, C-16).

EXAMPLE 5: Compaction of the nucleic acid in the presence of DT-3TU (12) The aim of this example is to illustrate the capacity of the transfecting compounds according to the invention to combine with the nucleic acids.

This can be easily demonstrated by a fluorescence test with ethidium bromide: the absence of fluorescence indicates the absence of free nucleic acid, which means that the nucleic acid is compacted by the transfecting compound.

The nucleic acid is brought into contact with increasing quantities of DT-3TU by equivolumetric mixing of lipid solutions of various titers in the solutions of nucleic acid. Samples of 800 g1 of nucleic acid complexes with a concentration of 0.01 pg/ml are thus prepared in a 150 mM sodium chloride solution with increasing quantities of DT-3TU (12).

In the same manner, a control was prepared by bringing the nucleic acid into contact with increasing quantities of EPC (see Figure 1) or of DPPC (see Figure by equivolumetric mixing of lipid solutions of various titers in the solutions of nucleic acid.

Samples of 800 1 of nucleic acid complexes with a concentration of 0.01 pg/mm are thus prepared in a 150 mM sodium chloride solution with increasing quantities of EPC or of DPPC (Figures 1 and 2 respectively) The ethidium bromide fluorescence is measured over time (measured at 20 0 C) using a FluoroMax-2 (Jobin Yvon-Spex) with excitation and emission wavelengths of 260 nm and 590 nm respectively. The slit widths for excitation and emission are set at 5 nm. The fluorescence value is recorded after addition of 3 gl of ethidium bromide to 1 g/l per ml of DNA/lipid solution (at 0.01 mg of DNA/ml).

The results are summarized in Figures 1 and 2.

In Figure i, the curve with squares shows that the addition of an increasing quantity of DT-3TU/EPC lipid mixture (0.75 to 20 nmol of DT-3TU) relative to a fixed quantity of nucleic acid (8 gg) induces a reduction in fluorescence linked to the reduction in the insertion of ethidium bromide between the base pairs of the DNA. This indicates that the combination between the DT-3TU/EPC liposomes and the DNA is sufficiently strong to exclude the ethidium bromide from the complexes. We were thus able to obtain up to 90% exclusion of fluorescence, that is DNA-DT-3TU/EPC lipid combination. To show the active role of the DT-3TU lipid in this lipid/DNA combination, a control was prepared. It consists in observing the interaction between the EPC lipid and the DNA, this is represented by the curve with the diamonds. When the EPC is brought into contact with the DNA under conditions identical to those used for the study of the DT-3TU/EPC-DNA complexes, only a weak decrease in fluorescence is observed (about which may be attributed to the increase in the turbidity of the mixture. This control therefore reflects the absence of combination of EPC alone with the DNA under the abovementioned experimental conditions.

This example thus illustrates the capacity of the DT-3TU lipid to combine with the nucleic acid.

In the same manner, in Figure 2, the curve with squares shows that the addition of an increasing quantity of DT-3TU/DPPC lipid mixture (0.75 to 20 nmol of DT-3TU) relative to a fixed quantity of nucleic acid (8 gg) induces a reduction in fluorescence when an identical quantity of ethidium bromide is added to the various samples. This indicates that the combination between the DT-3TU/DPPC liposomes and the DNA is sufficiently strong to exclude the ethidium bromide from the complexes. We were thus able to obtain up to exclusion of fluorescence, that is DNA-DT-3TU/DPPC combination. To show the active role of the-DT-3TU lipid in this lipid/DNA combination, a control was prepared. It consists in observing the interaction between the DPPC lipid and the DNA, this is represented by the curve with the diamonds. When the DPPC is brought into contact with the DNA under conditions identical to those used for the study of the DT-3TU/DPPC-DNA complexes, only a weak decrease in fluorescence is observed (about This control therefore reflects the absence of the combination of DPPC alone with DNA under the abovementioned experimental conditions.

This example thus illustrates the capacity of the DT-3TU lipid to combine with the nucleic acid.

EXAMPLE 6: Compaction of the DNA by DT-3TU/EPC complexes The aim of this example is to illustrate the capacity of the transfecting compounds according to the invention to compact the nucleic acids.

This may be easily demonstrated by a test of electrophoretic retardation on agarose gel of the DNA visualized by the use of ethidium bromide (EtBr): the absence of migration of the nucleic acid on the gel indicates the compaction of the nucleic acid. The free nucleic acid, for its part, is not subject to gel retardation.

Various DNA/DT-3TU samples comprising increasing quantities of DT-3TU lipid relative to the DNA were deposited on an agarose gel agarose in IN TBE). The gel was subjected to an electric current for one and a half hours at 70 V and 70 mA in order to cause the DNA to migrate by electrophoresis. The bands were revealed with EtBr and by absorption under a UV lamp. The results are represented in Figure 3.

The gel shows the electrophoretic migration of the DNA when it is not combined with the lipids (well and then its difference in retention when it is combined with the lipids. Wells 2 to 6 represent the DNA (0.01 g/l) combined with increasing quantities of DT-3TU/EPC liposomes: 0.75 then 5 then 10 then 15 and finally 20 nmol of DT-3TU lipid. Comparison between well 1 and the other wells indicates that the higher the increase in the quantity of DT-3TU lipid, the more DNA is retained on the gel which is completely retarded from 3 nmol of DT-3TU/g of DNA, zone of aggregation of the complexes. Wells 8 to 13 correspond respectively to the DNA alone (0.1 g/l, 1 pg for the gel), the lipoplexes formed at the concentration of 0.1 g/l of DNA at the lipid/DNA ratios: 0.75 or 5 or 10 or 15 and finally 20 nmol/lg of DNA. In the same manner, it can be observed that at this concentration of DNA compatible with in vivo experiments, the DNA is compacted from ratios of 5 nmol lipid/pg of DNA.

This example thus illustrates the capacity of the DT-3TU lipid to compact the nucleic acid.

EXAMPLE 7: Measurement of the Zeta potential of the DT-3TU/DNA compositions The aim of this example is to illustrate the capacity of the transfecting compounds according to the invention to compact the nucleic acids while preserving a globally anionic, neutral or very weakly cationic structure.

This may be demonstrated by a measurement of the Zeta potential; the measurement given in mV indicates the surface charge of the particle relative to the electrophoretic mobility of the sample.

The nucleic acid is brought into contact with increasing quantities of the DT-3TU/EPC lipid mixture by equivolumetric mixing of lipid solutions of various titers in the solutions of nucleic acid. Samples of 2 ml of nucleic acid complexes with a concentration of 0.01 g/l are thus prepared in a 20 mM sodium chloride solution with increasing quantities of DT-3TU.

The measurement of the Zeta potential (mV) is carried out using a zetasizer 3000 Hsa (Malvern). The value of the potential is determined 3 times in succession on 2 ml of DT-3TU/EPC-DNA sample. The results are summarized in Figure 4.

The DT-3TU/EPC liposomes are added to the DNA in a zone ranging from 0.75 nmol to 20 nmol of lipids per Ag of DNA. In this zone of variation of the quantity of lipid, the Zeta potential varies from mV to +15 mV. The negative part corresponds to what is shown in Figures 1, 2 and 3, namely that the Zeta potential is negative when the DNA is not completely compacted. The more lipid added, the more the DNA is compacted and the more the Zeta potential approaches zero, the lipoplexes then exhibit a practically zero surface potential. The Zeta potential then becomes slightly positive toward 8 nmol of lipid/gg of DNA. The relativity of this measurement should take into account the comparison of the various samples during the same experiment. It is thus important to note the evolution of the Zeta potential as a function of the increase in the quantity of lipid up to a weakly positive value.

This example thus confirms the compaction of the DNA by the transfecting compounds according to the invention, in particular DT-3TU, and show that the lipoplexes formed exhibit a surface potential close to neutrality.

EXAMPLE 8: In vitro transfection of the DT-3TU/DNA compositions The aim of this example is to illustrate the capacity of the transfecting compounds according to the invention to transfect cells in vitro.

This study was carried out for lipoplexes comprising various quantities of DT-3TU: 1.5 or 5 or or 15 or 20 nmol of DT-3TU/gg of DNA. Each of these conditions was tested with and without fetal calf serum Serum" or Serum").

The cell culture: HeLa cells (American type Culture Collection (ATCC) Rockville, MD, USA) derived from a carcinoma of human cervical epithelium, are cultured in the presence of an MEM ("minimum essential medium") type medium with addition of 2 mM L-glutamine, units/ml of penicillin and 50 units/ml of streptomycin. The medium and the additives are from Gibco-BRL Life Technologies (Gaithersburg, MD, USA).

The cells are cultured in flasks at 370C and at carbon dioxide in an incubator.

Transfection: one day before the transfection, the HeLa cells are transferred into 24-well plates with a cell number of 30,000 to 50,000 per well. These dilutions represent approximately confluence after 24 hours. For the transfection, the cells are washed twice and incubated at 370C with 500 p1 of medium with serum (10% FCS v/v) or without serum.

p1 of complexes containing 0.5 pg of plasmid DNA are added to each well (the complexes are prepared at least minutes before addition to the wells). After 2 hours at 370C, the plates without serum are supplemented with FCS ("Fetal Calf Serum").

All the plates are placed for 24 hours at 370C and at 5% carbon dioxide.

Determination of luciferase activity: Briefly, the transfected cells are washed twice with 500 pl of PBS (phosphate buffer) and then lysed with 250 p1 of reagent (Promega cell culture lysis reagent, of the Luciferase Assay System kit).

An aliquot of 10 pl of supernatant of the lysate centrifuged (12,000 x g) for 5 minutes at 40C is measured with a Wallace Victor 2 luminometer (1420 Multilabel couter).

The luciferase activity is assayed by the light emission in the presence of luciferin, coenzyme A and ATP for 10 seconds and expressed relative to 2000 treated cells. The luciferase activity is then expressed in relative light units (RLU) and normalized with the concentration of proteins in the sample obtained using a Pierce BCA kit (Rockford, IL, USA).

The results summarized in Figure 5 show an optimum transfection efficiency for the lipoplexes comprising 5 or 10 nmol of DT-3TU per pg of DNA. The presence of serum induces a weak inhibition of transfection in all cases.

EXAMPLE 9: Determination of the toxicity of the DT-3TU/DNA lipoplexes toward the cells The aim of this example is to illustrate the absence of toxicity of the transfecting compounds according to the invention.

The protein level is measured after transfection. The transfection protocol is identical to that described in Example 8.

Determination of the protein level: Briefly, the transfected cells are washed twice with 500 pl of PBS (phosphate buffer) and then lysed with 250 pl of reagent (Promega cell culture lysis reagent, of the Luciferase Assay System kit).

An aliquot of 50 i of supernatant of the lysate centrifuged (12,000 x g) for 5 minutes at 4 0 C is transferred into a tube in the presence of 50 g1 of 0.1 M iodoacetamide, 0.1 M hydrochloric acid tris at pH 8.2 and left for 1 hour at 370C. 20 g1 of the preceding solutions are deposited in a 96-well plate and 200 M1 of "BCA protein assay" reagent (Pierce, Montluson, France) are added. The plate is then centrifuged at 2500 revolutions/min and then incubated at 37 0 C for minutes. In parallel, a bovine serum albumin (BSA) range is prepared in order to correlate the absorbance value obtained for the samples with a quantity of protein present in the sample.

The results summarized in Figure 6 show a similar protein level regardless of the condition used, the lipoplexes comprising 0.75 or 5 or 10 or 15 or nmol of DT-3TU per pg of DNA. The presence of DT-3TU lipid does not therefore adversely affect the cell and no toxicity was observed under the conditions used.

This example therefore illustrates one of the major advantages of the transfecting compounds according to the invention, namely their very low toxicity probably linked to the absence of positive charges in their structure.

EXAMPLE 10: Compaction of the nucleic acid with DT-3TU/DPPC nanoemulsions The aim of this example is to illustrate the capacity of the transfecting compounds according to the invention to combine with nucleic acids.

This may be easily demonstrated by a test of electrophoretic retardation on agarose gel of the DNA visualized by the use of ethidium bromide (EtBr): the absence of migration of the nucleic acid on the gel indicates the compaction of the nucleic acid. The free nucleic acid, for its part, is not subject to gel retardation.

Various DNA/DT-3TU samples comprising various formulations of DT-3TU lipid relative to the DNA are deposited on an agarose gel agarose in lN TBE).

The gel is subjected to an electric current for one and a half hours at 70 V and 40 mA in order to cause the DNA to migrate by electrophoresis. The bands were revealed with EtBr and by absorption under a UV lamp.

The results are represented in Figure 8.

Likewise, the capacity of the DT-3TU diol compound to compact the DNA is shown by an agarose gel agarose in 1N TBE) on which various DNA/DT-3TU diol samples comprising various formulations of DT-3TU diol lipid relative to the DNA are deposited. The gel is subjected to an electric current for one and a half hours at 70 V and 40 mA in order to cause the DNA to migrate by electrophoresis. The bands are revealed with EtBr and by absorption under a UV lamp.

The gel shows the electrophoretic migration of the DNA when it is not combined with the lipids (well and then its difference in retention when it is combined with the lipids. Wells 2 to 5 represent the DNA (0.01 g/1) combined with nanoemulsions of DT-3TU/DPPC (60 nmol DT-3TU/gg of DNA) containing or otherwise calcium and ethanol. Well 2 represents nmol/gg of DNA without Ca 2 without EtOH, in well 3 was added 2% EtOH, in well 4, 60 eq Ca2+/PO DNA, in well 5, 2% EtOH and 60 eq of Ca 2 Comparison between well 1 and the other wells indicates that the various DT-3TU formulations studied retard the migration of DNA on the gel, which was also obtained after dialysis of the Ca 2 and EtOH constituents.

This example thus illustrates the capacity of the DT-3TU lipid incorporated into various formulations to compact the nucleic acid.

EXAMPLE 11: Compaction of the nucleic acid by stabilized DT-3TU/DPPC complexes The aim of this example is to illustrate the capacity of the transfecting compounds according to the invention to combine with nucleic acids.

This may be easily demonstrated by a test of electrophoretic retardation on agarose gel of the DNA visualized by the use of ethidium bromide (EtBr): the absence of migration of the nucleic acid on the gel indicates the compaction of the nucleic acid. The free nucleic acid, for its part, is not subject to gel retardation.

Various DNA/DT-3TU/DPPC and DNA/DT-3TUdiol/DPPC samples comprising increasing quantities of DT-3TU lipid relative to the DNA combined or not with cholesterol-PEG were deposited on an agarose gel agarose in 1N TEE). The gel was subjected to an electric current for one hour and a half at 70 V and 40 mA in order to cause the DNA to migrate by electrophoresis. The bands were revealed with EtBr and by absorption under a UV lamp. The results are represented in Figure 9.

The advantage of the insertion of cholesterol-PEG in the DT-3TU/DPPC formulations results from the possibility of reducing the size of the particles to a quantity of lipid which would lead to aggregations without insertion of lipid-PEG. The advantage is to optimize the quantities of transfectants injected in vivo. Indeed, the sizes of the particles required in order to have furtive objects relative to the serum proteins and thus an increased half-life period in the bloodstream ought to be predominantly less than 500 nm. Now, in order to obtain particle sizes of this order, it is necessary to use at least 40 nmol of DT-3TU lipid/pg of DNA. The insertion of lipid-PEG into the DT-3TU lipid formulations makes it possible to reduce the quantity of DT-3TU necessary for compaction of the DNA and for the formation of particles predominantly less than 500 nm.

The gel shows the electrophoretic migration of the DNA when it is not combined with the lipids (well and then its difference in retention when it is combined with the lipids. Wells 2 to 5 represent the DNA (0.01 g/l) combined with increasing quantities of nanoemulsions of DT-3TU/DPPC containing or otherwise cholesterol-PEG (20 ethylene glycol units) as agents for stabilizing the particles. Well 2 represents nmol/gg of DNA 15% chol-PEG, well 3 contains nmol/gg of DNA 20% chol-PEG, well 4 represents 30 nmol/gg of DNA 15% chol-PEG and well 5 represents nmol/g of DNA 20% chol-PEG. Comparison between well 1 and the other wells indicates that the various DT-3TU formulations studied retard the migration of the DNA on the gel, indicating the possibility of incorporating polyethylene glycol polymers into these formulations without releasing the DNA from the complexes, therefore without destabilizing them.

EXAMPLE 12: Compaction of the nucleic acid in the presence of DT-4TU The aim of this example is to illustrate the capacity of the transfecting compounds according to the invention to combine with the nucleic acids.

This can be easily demonstrated by a fluorescence test with ethidium bromide: the absence of fluorescence indicates the absence of free nucleic acid, which means that the nucleic acid is compacted by the transfecting compound.

The nucleic acid is brought into contact with increasing quantities of DT-4TU, by equivolumetric mixing of lipid solutions of various titers in the solutions of nucleic acid. Samples of 800 Al of nucleic acid complexes with a concentration of 0.01 pg/ml are thus prepared in a 150 mM sodium chloride solution with increasing quantities of DT-4TU In the same manner, a control was prepared by bringing the nucleic acid into contact with increasing quantities of DT-3TU (12) in order to compare the efficiencies of complexing of a lipid containing 3 thioureas (see Figure 2) relative to a lipid carrying 4 thioureas, by equivolumetric mixing of lipid solutions of various titers in solutions of nucleic acid. Samples of 800 il of nucleic acid complexes with a concentration of 0.01 gg/ml are thus prepared in a glucose solution with increasing quantities of DPPC.

The ethidium bromide fluorescence is measured using a FluoroMax-2 (Jobin Yvon-Spex), with excitation and emission wavelengths of 260 nm and 590 nm respectively. The slit widths for excitation and emission are set at 5 nm. The fluorescence value is recorded after addition of 3 gl of ethidium bromide (1 g/l) per ml of DNA/lipid solution (0.01 g/l of DNA).

The results are summarized in Figure The cuive with squares shows that the addition of an increasing quantity of DT-3TU/DPPC lipid mixture (0.75 to 30 nmol of DT-3TU) relative to a fixed quantity of nucleic acid (8 gg) induces a reduction in fluorescence linked to the reduction in the insertion of ethidium bromide between the base pairs of the DNA.

This indicates that the combination between the DT-3TU/DPPC liposomes and the DNA is sufficiently strong to exclude the ethidium bromide from the complexes. We were thus able to obtain 70% compaction of the DNA using 30 nmol of DT-3TU/DPPC lipids per gg of DNA. The active role of the DT-3TU lipid in this lipid/DNA combination has been shown in Figures 1 and 2. In the same manner, increasing quantities of DT-4TU/DPPC lipid mixture (0.75 to 30 nmol of DT-3TU) were added to a fixed quantity of nucleic acid (8 gg).

This combination induces a reduction in the fluorescence linked to the reduction in the insertion of ethidium bromide between the base pairs of the DNA.

This indicates that the combination between the DT-4TU/DPPC liposomes and the DNA is sufficiently strong to exclude the ethidium bromide from the complexes. We were thus able to obtain 60% compaction of the DNA for 30 nmol of lipid/ig of DNA (circles, Figure 10), which is comparable with the efficiency of complexing of DT-3TU under the same conditions.

This example thus illustrates the capacity of the DT-4TU lipid to combine with the nucleic acid.

EXAMPLE 13: Protection of the DNA against DNAses by DT-3TU/DPPC complexes The aim of this example is to illustrate the capacity of the transfecting compounds according to the invention to protect the nucleic acids from enzymatic hydrolysis including DNases.

This can be easily demonstrated by a test of electrophoretic retardation on agarose gel of the DNA visualized by the use of ethidium bromide (EtBr): the free DNA or the DNA complexed with the lipid is treated with an appropriate quantity of DNase. The DNA, extracted from the enzymatic digestion medium, is deposited on a gel and its integrity is checked by comparing its migration with that of the untreated nucleic acid.

Various samples of extracted DNA which has been treated beforehand with 2 x 10 4 M DNase (Sigma) were deposited on an agarose gel agarose in 1N TBE). The DNase treatment was carried out on the free DNA and on the DNA complexed with increasing quantities of DT-3TU lipid relative to the DNA. The gel was subjected to an electric current for one hour and a half at 70 V and 70 mA in order to cause the DNA to migrate by electrophoresis. The bands were revealed with EtBr and by absorption under a UV lamp. The results are represented in Figure 11.

The gel shows the electrophoretic migration of the DNA not treated with the DNase (well and then its difference in retention when it has been treated with DNase. Well 2 represents the same quantity of DNA (3 gg) treated with 2 x 10-4 M DNase. Following this treatment (2 x 10 4 M DNase, 30 min, 37 0 the band corresponding to the DNA is not revealed, which indicates a degradation of the DNA. The nucleic acid, complexed with 30 and 40 nmol of DT-3TU lipid per gg of DNA and with 40 nmol of DT-3TU lipid 6% Chol-PEG was treated with 2 x 10 4 M DNase. After extraction of the DNA with a phenol/chloroform mixture and precipitation of the latter, the nucleic acid was deposited on this gel into wells 3, 4 and 5 respectively. The DNA migrates in a manner comparable to the DNA not treated with DNase, which indicates that the DNA is intact, it has not been degraded by the DNase treatment, it has therefore been protected by the DT-3TU lipid. The DNA in the DT-3TU lipid complexes is not therefore accessible to the enzymatic hydrolysis, it is protected from hydrolysis by the DNases.

This example thus illustrates the capacity of

I

the DT-3TU lipid to protect the nucleic acid from enzymatic hydrolysis.

EXAMPLE 14: Protection of the DNA against the serum by DT-3TU/DPPC complexes The aim of this example is to illustrate the capacity of the transfecting compounds according to the invention to protect the nucleic acids from degradation in a serum medium.

This may be easily demonstrated by a test of electrophoretic retardation on agarose gel of DNA visualized by the use of ethidium bromide (EtBr): the free DNA or the DNA complexed with the lipid is incubated in various proportions of serum at 370C. The DNA, extracted from the serum medium, is deposited on a gel and its integrity is checked by comparing its migration with that of the untreated nucleic acid.

Various samples of extracted DNA which has been incubated beforehand in 150 mM NaCl, 20% serum and 100% serum were deposited on an agarose gel (0.8% agarose in 1N TBE). The salt and serum treatments were carried out on the free DNA and on the DNA complexed with increasing quantities of DT-3TU lipid relative to the DNA. The gel was subjected to an electric current for one hour and a half at 70 V and 40 mA in order to cause the DNA to migrate by electrophoresis. The bands were revealed with EtBr and by absorption under a UV lamp. The results are represented in Figure 12.

The gel shows the electrophoretic migration of the control DNA (well and then its difference in retention when it was treated, non-complexed, in salt medium (150 mM NaCI) (well when it is complexed with 40 nmol of DT-3TU lipid/gg of DNA (well The migration of the DNA extracted from the salt medium is comparable for the 2 wells, indicating that the DNA remains intact under these conditions. The following wells show in the same order the DNA (wells 4 and 6) and the DNA 40 nmol of DT-3TU lipid (wells 5 and 7) under two different serum conditions: 20% serum for wells 4 and 5 and 100% serum for wells 6 and 7. When the DNA is free, it is completely degraded under the two serum conditions studied after 30 minutes at 37 0

C

(wells 4 and On the other hand, the complexing of the DNA with the nanoemulsions consisting of DT-3TU/DPPC leads to the protection of the nucleic acid since the migration band corresponding to the DNA is revealed (wells 5 and The DNA in the DT-3TU lipid complexes is therefore protected from degradation in serum medium compared with naked DNA.

This example thus illustrates the capacity of the DT-3TU lipid to protect the nucleic acid from degradation in the serum.

EXAMPLE 15: Transfection in vivo of the DT-3TU/DNA compositions The aim of this example is to illustrate the capacity of the transfecting compounds according to the invention to transfect biological tissues in vivo.

This may be demonstrated by intramuscular injection of DNA complexes encoding luciferase. The muscle samples are then collected 96 h after the injection and the level of expression of luciferase is measured using a luminometer (wallac).

Complexes comprising increasing quantities of DT-3TU lipid per gg of DNA were injected into the two cranial tibial muscles of mice, to which electrical pulses have been applied or otherwise (Bureau, M et al, BBA 2000, 1474(3):535-9).

Complexes comprising increasing quantities and 60 nmol of DT-3TU lipid per gg of DNA are injected in a volume of 30 gl at the rate of 3 gg of DNA per animal into the two cranial tibial muscles of C57bl/6 mice anesthetized with a ketamin/xylazine mixture. This injection was followed or otherwise by the application of transcutaneous electrical pulses applied by means of electrodes placed on either side of the muscle (Bureau, M et al, BBA 2000, 1474(3):535-9).

After humanely killing the mice 96 h post-injection, the muscles are removed and ground in 1 ml of lysis buffer. After centrifugation (10 min, 12 000 rpm, 40C), 10 l1 of supernatant are collected i and deposited in 96-well plates for reading the luciferase after adding 50 pl of luciferase substrate.

The arbitrary luminescence level is read in the supernatant using a luminometer (Wallac, Victor).

The values obtained are indicated in Figure 13. They represent the relative level of expression at the lipid dose associated with the nucleic acid, 20 and 40 nmol of DT-3TU lipid/gg of DNA.

The expression levels obtained are significant, and greater than the background noise represented by the muscle taken as a control which corresponds to 5 x 104.

The DNA complexed with various quantities of DT-3TU lipid is therefore capable of transfecting the muscle tissues with a significant transfection level.

This example illustrates the capacity of the transfecting compounds according to the invention to transfect tissues in vivo.

EXAMPLE 16: Biodistribution in vivo of the DT-3TU/DPPC/DNA complexes The aim of this example is to illustrate the capacity of the transfecting compounds according to the invention to circulate for a long time in the bloodstream in vivo because of their neutral character.

This may be demonstrated by injecting DNA complexes containing a fluorescent lipid into the bloodstream of mice. Blood samples are then collected at various times after the injection and the fluorescence level in the bloodstream is measured using a FluoroMax-2 (Jobin Yvon-Spex).

Complexes containing 40 nmol of DT-3TU lipid per pg of DNA, 1 mol equivalent of DPPC/DT-3TU lipid and 0.7% of lipid-rhodamine relative to the total lipids were injected in a volume of 200 p1 at the rate of 11 pg of DNA per animal into the caudal vein of C57bl/6 mice anesthetized with a ketamin/xylazine mixture.

At 30 minutes, 1 h and 6 h post-injection, the blood is collected by intracardiac puncture on anesthetized mice. After humanely killing the mice, the liver, the spleen and the lungs are immediately removed, weighed and homogenized in PBS (5 pl/mg of tissue). The lipids are extracted from 100 1l of blood and organ homogenates with 3 ml of a chloroform and methanol 1/1 mixture by vigorously stirring for 30 min and then by centrifugation. The fluorescence is read on the supernatant using a FluoroMax-2 (Jobin Yvon-Spex), with excitation and emission wavelengths of 550 nm and 590 nm respectively. The slit widths for excitation and emission are set at 5 nm.

The values obtained are indicated in Figure 14. They represent the percentage of the injected dose obtained in the blood, the lungs and the reticuloendothelial system (liver and spleen cumulatively) 30 minutes, 1 h and 6 h after injection.

The measurement of fluorescence in the blood after minutes represents 50% of the injected dose, which is considerably greater than what can be obtained with surfactants of DNA of the cationic type. After 1 h, 17% of the injected dose could be found, which here again represents a remarkable improvement compared with cationic complexes. The neutral character of these lipid/DNA complexes (very weakly positive zeta: Figure 4) therefore exhibits a real advantage for obtaining furtive particles in relation to the serum proteins. It should also limit their interactions with the macrophages and the kupffer cells of the spleen and of the liver, which can explain the quantity of liposome found at 30 min and 1 h in the blood.

The quantity of lipoplexe found in the lung is low compared with that found in the case of cationic lipoplexes. The neutrality of these liposomes should also reduce the nonspecific interactions with the negative endothelium of the lungs.

This example illustrates the capacity of the transfecting compounds according to the invention to be furtive in relation to the serum proteins.

P \OPER\PDB\Spec. 00225794 spa doc-.2/ I 27 0 -84A-

O

z Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a C 5 stated integer or step or group of integers or steps but not 1 the exclusion of any other integer or step or group of integers or steps.

The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Claims (37)

1. A transfecting compound of general formula: in which: is an integer chosen from 0 and 1, n is an integer chosen from 1, 2, 3, 4, 5 and 6, m is an integer chosen from 2, 3 and 4, it being possible for m to take different values within the different groups -[NH-CS-NH-(CH)m]-, R' represents a group of general formula (II): in which q is an integer chosen from 1, 2, 3, 4, 5 and 6, and.p is an integer chosen from 2, 3 and 4, it being possible for p to take different values within the different groups -[(CH 2 )p-NH-CS-NH]-, R represents either a hydrogen atom or a group of general formula (II) as defined above, it being under- stood that when n is 1 and P is 0, then at least one group R is of formula (II), X, in the formulae and represents a satur- ated or unsaturated, linear or cyclic aliphatic group, comprising 1 to 8 carbon atoms, a mercaptomethyl (-CH 2 SH) group, or alternatively a hydrophilic chain chosen from the groups: -(CH 2 )x-(CHOH)u-H with x is an integer chosen between 0 and 10, and u an integer chosen from 1, 2, 3, 4, 5 and 6, or alternatively, -(OCH 2 CH20)v-H with v an integer chosen from 1, 2 and 3, it being understood that no more than one substituent X, both in the formulae and represents a hydrophilic chain, Y represents a spacer, and L represents: either a group -N(RI)R 2 with RI and R 2 which represent, independently of each other, a hydrogen atom or alternatively a fatty aliphatic chain, or alternatively a group of formula -(CH 2 )t-OZ with t representing an integer chosen from 11, 12, 13, 14 or 15 and Z represents a sugar, a polyol or a PEG, it being understood that at least one of RI and R 2 is different from hydrogen, or a group -OR 3 with R 3 which represents a steroid derivative, where appropriate, in their isomeric forms or their mixtures.

2. The transfecting compound as claimed in claim 1 of general formula: S 0 X- "k R 1 N N-(CH 4I N (III) H H n R2 in which X, m, n and Y are as defined in claim 1, with the exception of n which is different from 1, and RL and R 2 represent, independently of each other, a hydrogen atom or else a fatty aliphatic chain, it being understood that at least one of Ri and R 2 is different from hydrogen, where appropriate, in their isomeric forms or their mixtures.

3. The transfecting compound as claimed in claim 1 or 2 of general formula: S 0 H N N-(CH-Y %(IV) H H R, in which m, n and Y are as defined in claim 1, with the exception of n which is different from 1, and Ri and R 2 represent, independently of each other, a hydrogen atom or else a fatty aliphatic chain, it being understood that at least one of Ri and R 2 is different from hydrogen, where appropriate, in their isomeric forms or their mixtures.

4. The transfecting compound as claimed in one of claims 1 to 3, characterized in that said spacer comprises one or more chemical functional groups chosen from alkyls having 1 to 6 carbon atoms, ketone, ester, ether, amide, amidine, carbamate or thiocarbamate functional groups, glycerol, urea, thiourea, or aromatic rings.

The transfecting compound as claimed in claim 4, characterized in that said spacer is chosen from the groups of formula: -NH-C(0)-CH 2 -CH 2 or: -(CH2-)i-W-(CH2) j in which i and j are integers chosen between 1 and 6 inclusive and W is a group chosen from ketone, ester, ether, amide, amidine, carbamate or thiocarbamate functional groups, glycerol, urea, thiourea, or alter- natively aromatic rings.

6. The transfecting compound as claimed in one of claims 1 to 3, characterized in that the fatty aliphatic chain(s) is/are chosen from alkyl groups containing 10 to 22 carbon atoms and optionally one or more unsaturations.

7. The transfecting compound as claimed in claim 6, characterized in that the fatty aliphatic chain(s) is/are chosen from the aliphatic groups (CH 2 11 CH 3 (CH 2 13 CH 3 -(CH 2 15 CH 3 and 17 CH 3

8. The transfecting compound as claimed in one of the preceding claims, characterized in that said steroid derivative is a polycyclic compound of the cholestane type.

9. The transfecting compound as claimed in claim 8, characterized in that said steroid derivative is chosen from cholesterol, cholestanol, 3-a-5-cyclo-5- a-cholestan-6-3-ol, cholic acid, cholesteryl formate, chotestanyl formate, 3a,5-cyclo-5a-cholestan-6-yl formate, cholesterylamine, 6-(1,5-dimethylhexyl)-3a,5a- dimethylhexadecahydrocyclopenta[a]cyclopropa[2,3]- cyclopenta[l,2-f]naphthalen-10-ylamine, or cholestanylamine.

The transfecting compound as claimed in one of the preceding claims, characterized in that said sugar is chosen from the molecules consisting of one or more saccharides.

11. The transfecting compound as claimed in one of the preceding claims, characterized in that said polyol is chosen from the linear, branched or cyclic hydrocarbon molecules comprising at least two hydroxyl functional groups.

12. A composition, characterized in that it comprises a transfecting compound as claimed in one of claims 1 to 11, and a nucleic acid.

13. The composition as claimed in claim 12, characterized in that the nucleic acid is a deoxyribonucleic acid or a ribonucleic acid.

14. The composition as claimed in claim 12 or 13, characterized in that said nucleic acid comprises one or more genes of therapeutic interest under the control of regulatory sequences.

15. The composition as claimed in claims 12 to 14, characterized in that said nucleic acid is an antisense gene or sequence or a DNA encoding an RNA with ribozyme functions.

16. The composition as claimed in one of claims 12 to 15, characterized in that it comprises, in addition, one or more adjuvants.

17. The composition as claimed in claim 16, characterized in that said adjuvant is chosen from lipids, peptides, proteins or polymers.

18. The composition as claimed in claim 17, characterized in that said polymer is polyethylene glycol.

19. The composition as claimed in claim 17, characterized in that said polymer is polyethylene glycol which is covalently linked to cholesterol.

The composition as claimed in claim 17, characterized in that said adjuvant is chosen from neutral lipids.

21. The composition as claimed in claim characterized in that said neutral lipid is chosen from natural or synthetic lipids which are zwitterionic or lacking ionic charge under physiological conditions.

22. The composition as claimed in claim 21, characterized in that said neutral lipid is chosen from dioleoylphosphatidylethanolamine (DOPE), oleoyl- palmitoylphosphatidylethanolamine (POPE), di-stearoyl, -palmitoyl, -myristoylphosphatidylethanolamines as well as their derivatives which are N-methylated 1 to 3 times, phosphatidylglycerols, diacylglycerols, glycosyldiacylglycerols, cerebrosides (such as in particular galactocerebrosides), sphingolipids (such as in particular sphingomyelins) or asialogangliosides (such as in particular asialoGMl and GM2).

23. The composition as claimed in one of claims 12 to 22, characterized in that it comprises, in addition, an extracellular or intracellular targeting element.

24. The composition as claimed in claim 23, characterized in that said targeting element is chosen from sugars, peptides, proteins, oligonucleotides, lipids, neuromediators, hormones, vitamins or derivatives thereof.

The composition as claimed in claim 23, characterized in that said targeting element is covalently linked to a fatty alkyl chain containing at least 10 carbon atoms or to a polyethylene glycol.

26. The composition as claimed in claim 23, characterized in that said targeting element is covalently linked either to the transfecting compound as claimed in one of claims 1 to 11, or to the nucleic acid.

27. The composition as claimed in one of claims 12 to 26, characterized in that it comprises, in addition, a pharmaceutically acceptable vehicle for an injectable formulation.

28. The composition as claimed in one of claims 12 to 26, characterized in that it comprises, in addition, a pharmaceutically acceptable vehicle for administration to the skin and/or the mucous membranes.

29. Use of the transfecting compound as claimed in one of claims 1 to 11, for the transfer of nucleic acids.

Use of the transfecting compound as claimed in one of claims 1 to 11 to manufacture a medicament intended for treating diseases.

31. A method for transferring nucleic acids into cells, characterized in that it comprises the following steps: bringing the nucleic acid into contact with a transfecting compound as defined in claims 1 to 11, to form a complex, and P\OPER\PDB\Spwl2U002257904 1spa doc-2/1W007 S-93- 0 z bringing the cells into contact with the complex formed in

32. The method for transferring nucleic acids into cells as claimed in claim 31, characterized int hat said transfer agent and/or said nucleic acid are mixed beforehand with one or more adjuvants and/or with a targeting element as defined in claims 16 to 26.

33. The method for transferring nucleic acids into cells as claimed in claim 31, characterized in that one or more adjuvants as defined in claim 16 to 22 are administered to the cells beforehand.

34. The method for transferring nucleic acids into cells as claimed in one of claims 31 to 33, characterized in that the cells are, in addition, subjected to a chemical or physical treatment.

A transfection kit, characterized in that it comprises one or more transfecting compounds as claimed in claims 1 to 11 and/or mixtures thereof.

36. A method of treating a disease in a subject in need thereof comprising the administration to said subject of a transfecting compound according to any one of claims 1 to 11.

37. A transfecting compound according to claim 1; or a composition according to claim 12; or a use according to claim 29 or 30; or a method according to claim 31 or 36; or a transfection kit according to claim substantially as hereinbefore described and/or exemplified and/or illustrated in the Figures.