AU737579B2 - New class of cationic transfecting agents for nucleic acids - Google Patents

Abstract

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

Description

WO 98/5S490 PCT/FR98/01112 1 NEW CLASS OF CATIONIC TRANSFECTING AGENTS FOR NUCLEIC ACIDS The present invention relates to a new class of cationic transfection agents, to pharmaceutical compositions containing them, their applications for the in vivo, ex vivo and/or in vitro transfection of nucleic acids and methods for preparing them.

With the development of biotechnology, it is now possible to transfer nucleic acids into cells. The efficiency of these transfers appears to be necessary for the correction of defects in expression and/or of abnormal expression of nucleic acids involved in numerous genetic diseases. However, the importance of the transfers of nucleic acids is not limited to gene therapy. In addition, the transfer of nucleic acids may be useful for studying the regulation of the expression of genes, the cloning of genes, or for any other in vitro manipulation involving nucleic acids, as well as for the production of recombinant proteins. It may also be the transfer of nucleic acids in vivo, for example for the creation of transgenic animals, the production of vaccines or studies on the labelling of molecules.

Moreover, the transfer of nucleic acids can be made 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 being subsequently 4 readministered.

2 Nowadays, several methods are proposed for the intracellular delivery of this type of genetic information. One of them in particular is based on the use of chemical or biochemical vectors. These synthetic vectors have two principal functions: to complex the DNA to be transfected and to promote its cellular attachment as well as its passage across the plasma membrane and, where appropriate, across the two nuclear membranes. Among the synthetic vectors developed, the cationic polymers of the polylysine and DEAE-dextran type or alternatively the lipofectants are more advantageous.

Major progress was achieved in this mode of transfection with the development of a technology based on the use of lipofectant-type cationic transfection agents, and more precisely of cationic lipids. It has thus been demonstrated that a positively charged cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,Ntrimethylammonium chloride (DOTMA), inteferred, in the form of liposomes or of small vesicles, spontaneously with DNA, which is negatively charged, to form lipid- DNA complexes capable of fusing with the cell membranes, and thus allowed the intracellular delivery of the DNA.

Since DOTMA, other cationic lipids have been developed on this same structural model, that is to say a lipophilic group coupled to an amino group via an arm also called a "spacer". Among these, there may be 3 mentioned more particularly those comprising, as lipophilic group, fatty acids or a derivative of cholesterol, and containing, in addition, where appropriate, as amino group, a quaternary ammonium group. DOTAP, DOBT or ChOTB may be mentioned in particular as representatives of this category of cationic lipids. Other compounds, such as DOSC and ChOSC, are characterized by the presence of a choline group in place of the quaternary ammonium group.

Another category of lipofectants, lipopolyamines, has also been described. In general, this is an amphiphilic molecule comprising at least one hydrophilic region consisting of a polyamine coupled via a "spacer" to a lipophilic region. The polyamine region, which is positively charged, is capable of reversibly combining with the nucleic acid, which is negatively charged. This interaction greatly compacts the nucleic acid. As for the lipophilic region, it makes this ionic interaction insensitive to the external medium, by coating the complex formed with a lipid film. In this type of compound, the cationic group may be represented by the radical which contains four ammonium groups, two of which are primary and two of which are secondary. DOGS and DPPES in particular belong to this category. These lipopolyamines are most particularly effective for the transfection of primary endocrine cells. As a representative of this latter family of compounds, 1 4 there may be mentioned more particularly the lipopolyamines described, for example, in patent applications WO 96/17823 and WO 97/18185.

However, the efficiency of these synthetic vectors remains to be improved, in particular in terms of charge density and rigidity of the transfecting molecule.

Indeed, it is generally accepted that the activity of these products depends on the charge density which they bring into play. However, the increase in the charges on the aliphatic chains is responsible for the appearance of toxicity.

Stabilization of the charge density on the aliphatic chains could therefore prevent the appearance of toxic factors. Furthermore, the transfection efficiency could be enhanced by an increase in the charge density in a region other than the aliphatic chains.

Moreover, the flexibility of the molecule used for the transfer of a nucleic acid may be an obstacle to sufficiently close interaction between the said nucleic acid and the transfecting agent, preventing the obtaining of optimum compacting of the nucleic acid molecule, the compacting being a prior condition necessary for any transfection. An increased rigidity of the molecule of transfecting agent is thought to ensure a more effective interaction with the nucleic acid, thus improving the transfection.

Thus, it would be particularly advantageous to have transfecting agents having an increased transfection capacity while possessing a reduced or zero toxicity. These are the objectives which the present invention proposes to achieve.

The object of the present invention is indeed precisely to provide a new class of transfecting agents having a novel cationic hydrophilic region which confers on the said transfer agents the specific properties mentioned above. The cationic hydrophilic region consists of at least one heterocycle containing 5 or 6 atoms, substituted with amino groups.

The positively charged hydrophilic region is capable of reversibly combining with a negatively charged nucleic e acid which is thus highly compacted. In addition, the S.novel structure of the cationic part confers an increased S.rigidity on the molecule.

More precisely, the present invention relates to a 20 transfecting agent comprising at least one cationic hydrophilic region coupled to a lipophilic region, in which the cationic hydrophilic region has the general Sformula *o, e eo -6-

Z

(CH

2 X Z

(I)

z z y z

Z

in which: y is an integer equal to 0 or 1, the different y's being independent from each other, X represents an oxygen, nitrogen, sulphur or selenium atom, the Z groups represent, independently of each other, a hydrogen atom, an OR group in which R represents a hydrogen atom, a methyl group or a group (CH2)n-NRiR 2 in S. which n is an integer chosen from 1 to 6 inclusive and R, 20 and R 2 represent, independently of each other, a hydrogen atom or a group (CH2)q-NH 2 it being possible for q to vary from 1 to 6 inclusive, the different q's being independent of each other, a group (CH 2 )m-NRiR 2 in which m is an integer 25 chosen from 0 to 6 inclusive and R, and R 2 are as defined above, or alternatively a "spacer" group allowing the binding of the cationic hydrophilic region to the lipid region, it being understood that at least one of the Z substituents is a "spacer" group and that at least two of the Z substituents carry an amino group.

7 The transfer agents according to the invention contain at least one cationic hydrophilic region of general formula According to one variant of the invention, the transfer agents comprise 2 or more cationic hydrophilic regions which are linked to each other at the level of one of the Z groups.

According to a preferred embodiment of the invention, when one of the Z substituents represents OR and R represents a group (CH 2 )n-NR 1

R

2 then n is preferably chosen from 2, 3 or 4.

According to an advantageous variant of the invention, the transfecting agents comprise a cationic hydrophilic region of general formula in which X represents an oxygen atom. The cationic hydrophilic part then consists of a glycoside of the pyranose or furanose form. The pyranose form is particularly advantageous and has been described in a non-limiting manner in the examples.

According to a second advantageous variant of the present invention, the transfecting agents comprise a cationic hydrophilic region of general formula (I) composed of a 6-membered amino glycoside (y which is present on the ring is equal to X is an oxygen atom and at least one of the Z substituents comprises an amino group. Still more preferably, at least two of the Z substituents comprise an amino group.

According to another variant of the present invention, the cationic hydrophilic region is composed 8 of an amino glycoside of formula in which the two y's are equal to 1, X is an oxygen atom, two of the Z groups represent hydrogen atoms, two other Z groups are nitrogen-containing groups and preferably amino groups, and the last Z group represents an OR group, R being as defined above. Preferably, the OR group is situated at the 2-position on the heterocycle constituting cationic the hydrophilic region of the transfecting agents according to the invention.

According to another advantageous embodiment of the invention, the cationic hydrophilic region is composed of an amino glycoside of general formula in which the y present on the ring is equal to i, X is an oxygen atom, at least two of the Z groups correspond to groups 0- (CH 2 )q-NH 2 q being defined as above, and at least one of the Z groups represents an OR group, R being as defined above.

As representative of cationic hydrophilic regions of general formula which are intermediates useful in the synthesis of transfer agents according to the invention, there may be mentioned more particularly the following compounds:

OH

0 NH. 2 9

NH,

NH, H N H O

O

0 ^^LOH

HN

In general, the transfecting agents for the purposes of the present invention comprise at least one cationic hydrophilic region as defined above combined with a lipophilic region.

The molecules constituting the lipophilic region for the purposes of the invention are chosen from the lipophilic molecules known to persons skilled in the art. Advantageously, the lipophilic region consists of one or more optionally halogenated saturated or unsaturated, linear or branched aliphatic chains. The lipophilic region may also be advantageously chosen from the steroid derivatives.

According to a preferred variant of the invention, the lipophilic part consists of one or more aliphatic chains containing 10 to 22 carbon atoms, and still more preferably 12 to 22 carbon atoms. By way of example, there may be mentioned the aliphatic chains containing 14, 16, 17, 18 or 19 carbon atoms, and in Sparticular (CH2) 13

CH

3

(CH

2 sCH 3 (CH2) 16

CH

3

(CH

2 1

CH

3 and 10

(CH

2 1

CH

3 The lipophilic part of the transfer agents according to the invention may also be advantageously chosen from steroid derivatives such as, for example, cholesterol, cholestanol, 3-a-5-cyclo-5-a-cholestancholic acid, cholesteryl formate, cholestanyl formate, 3a,5-cyclo-5a-cholestan-6p-yl formate, cholesterylamine, 6-(1,5-dimethylhexyl)-3a,5adimethylhexadecahydrocyclopenta[alcyclopropa[2,3]cyclopenta[1,2-f]naphthalen-10-ylamine, or cholestanylamine.

According to a preferred embodiment, the cationic hydrophilic region is coupled to the lipophilic region by a so-called "spacer" intermediate molecule. "Spacer" group is understood to mean, for.the purposes of the invention, any acidic or amino substituent comprising hydrolysable functional groups which make it possible to obtain bonds of the amide, carbamate, ester or ether type or via an aromatic ring between the cationic hydrophilic part and the lipophilic region, which is known to persons skilled in the art. The coupling between the hydrophilic part and the lipophilic part is preferably performed at the level of one of the Z groups which is present on the cationic hydrophilic intermediate of formula and which is useful for the synthesis of transfer agents according to the invention. Advantageously, the coupling between the hydrophilic and lipophilic regions is performed at the 2-position or at the 5- or 11 6-position (depending on whether y present on the ring is equal to 0 or 1) on the ring of the cationic hydrophilic part of general formula via a "spacer" group.

Preferably, the "spacer" region comprises an aliphatic or aromatic chain. In action, the "spacer" advantageously comprises one or more groups chosen from the amide, carbamate, ester or ether groups or aromatic rings. Preferred "spacers" are in particular those of formula -O-CO-(CH 2 -COOH, -O-(CH 2 )x-COOH, -O-CO-(CH 2 )x-

NH

2

-O-(CH

2 )x-NH 2

-NH-(CH

2 )x-NH 2 with x representing an integer chosen from 1 to 6 inclusive.

As representatives of transfecting agents according to the invention, there may be mentioned more particularly the compounds (8a) and (8b) of formulae: -12- 0 O O NN OH

L

O C,H, 0 0 0 NC H7

CISH,

(8b) H 2N--O' NH0 The present invention also relates to the methods of preparing a new class of nucleic acid transfection agents such as defined above. More precisely, it relates to a method of preparing a transfecting agent as defined above in which a lipophilic region is coupled by means of a "spacer" to a heterocycle of general formula as defined above.

The cationic hydrophilic region of general formula may be prepared in various ways, depending 13 on the relative position of the amino functional groups on the ring and their number.

When it is desired to obtain a cationic hydrophilic region of general formula for which at least one of the Z substituents is a "spacer" allowing the binding to the lipophilic part and at least one of the Z groups is an OR group with R representing a group

(CH

2 )n-NRIR 2 the procedure is carried out starting with the corresponding derivative for which all the substituents O-(CH 2 )n-NRiR 2 are hydroxyl functional groups.

In a first step, the said derivative carrying the hydroxyl functional groups undergoes O-alkylation according to conventional methods known to persons skilled in the art. In particular, the procedure is carried out with the aid of an alkylating agent in a basic medium in conventional alkylation solvents, in the presence of a crown ether and at a temperature of between 10 and 600C.

There are used in particular as alkylating agents alkylamines, halogenated derivatives of esters such as, for example, alkyl haloacetate, or halogenated derivatives of alcohols. Preferably, alkyl bromoacetate is used. The alkyl functional groups are chosen according to the value which it is desired to give n.

For example, to have n equal to 2, ethyl bromoacetate is preferably used.

The solvents used are conventional 14 O-alkylation solvents, for example dimethylformamide, dimethylacetamide, dimethyl sulphoxide, acetonitrile, tetrahydrofuran (THF) and the like. Advantageously,

THF

is used.

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

In a second step, the carboxyl functional groups of the derivative obtained are reduced to an alcohol according to conventional methods known to persons skilled in the art. The procedure is carried out in particular by the action of a reducing agent in compatible conventional solvents.

By way of reducing agent, there may be mentioned for example borane-dimethyl sulphide (BMS), lithium aluminium hydride or sodium borohydride.

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

In a third stage, the hydroxyl functional groups obtained are azidated by conventional methods known to persons skilled in the art.

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

The solvents which can be used are the conventional azidation solvents such as for example tetrahydrofuran, benzene, toluene, chloroform, 15 dichloromethane, and the like, and preferably tetrahydrofuran (THF).

Finally, the azide functional groups are converted to amino functional groups by conventional methods known to persons skilled in the art.

The procedure is carried out in particular by reduction in an acid medium, for example by hydrogenation in an acid medium in the presence of palladium on carbon, or using the Staudinger reaction, or alternatively by the action of a reducing agent, for example lithium aluminium hydride, sodium borohydride, tin(II) chloride, and the like.

A cationic hydrophilic compound of general formula is thus obtained for which at least one of the Z substituents is a "spacer" allowing binding to the lipophilic part and at least one of the Z groups is an OR group with R representing a group -(CH 2 )n-NR 1

R

2 The coupling to the lipophilic part is then carried out by conventional methods known to persons skilled in the art, and in particular by peptide coupling (Bodanski Principles and Practices of Peptides Synthesis, Ed. Springe-Verlag). The coupling occurs at the level of the Z group representing a "spacer" When one of the Z groups is a "spacer" group, the latter is, where appropriate, protected beforehand.

The same applies to the amino functional groups carried by the cationic hydrophilic parts which are preferably 16 protected prior to the peptide coupling. The protection and the removal of the protective radicals are carried out according to the customary methods.

The protection may be achieved by any compatible group whose use and removal does not adversely affect the remainder of the molecule. In particular, the procedure is carried out according to the methods described by T.W. GREENE, Protective Groups in Organic Syntheses, A. Wiley-Interscience Publication (1981), or by Mc OMIE, Protective Groups in Organic Chemistry, Plenum Press (1973).

By way of example, the protective groups may be chosen from the trimethylsilyl, benzhydryl, tetrahydropyranyl, formyl, acetyl, chloroacetyl, trichloroacetyl, trifluoroacetyl, ethoxycarbonyl, tert-butoxycarbonyl or trichloroethoxycarbonyl radicals, and the like.

Another route of synthesis makes it possible to arrive at a second series of cationic hydrophilic compounds. This second embodiment differs from the first in that the carbohydrate backbone is converted.

Indeed, the starting material used is a glycal of general formula (II): 17

S(II)

Z'

for which the Z' substituents are acetoxy functional groups. The glycals used are commercially available or alternatively are obtained by any method known to persons skilled in the art from commercially available carbohydrates, and in particular by reacting the corresponding acetobromosugars with the zinc/copper pair.

In a first step, the said acetylated glycal is alkylated according to conventional methods known to persons skilled in the art.

In particular, the Ferrier reaction is used by the action, in the presence of a Lewis acid, of an amino alcohol, an alkoxycarbonylalcohol, a carboxyalcohol, or any other alcohol functionalized by a group allowing coupling to a lipophilic region.

Preferably, the reaction is performed in the presence of boron trifluoride in a compatible solvent, for example an ether such as ethyl ether.

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

In particular, a reduction is carried out in 18 an acid medium, for example by hydrogenation in an acid medium in the presence of palladium on carbon.

In a third step, the acetoxy functional groups present on the ring are then converted to hydroxyl functional groups by any method known to persons skilled in the art.

In particular, the procedure is carried out by transesterification with the aid of an alkoxide, for example sodium methoxide.

In a fourth step, the hydroxyl functional groups present on the ring are converted to azide functional groups by any method known to persons skilled in the art.

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

The solvents which can be used are conventional azidation solvents, and preferbly tetrahydrofuran (THF).

There is thus obtained a cationic hydrophilic region of general formula (III): z" 0 x (CHO

-NRR,

n(III) 19 for which Z" is either a hydrogen atom or an azide group, at least one of the Z" groups being different from hydrogen.

The lipophilic part may be coupled to the amino functional group -NRiR 2 according to methods known to persons skilled in the art, in particular by peptide coupling (Bodanski Principles and Practices of Peptides Synthesis, Ed. Springe-Verlag), or alternatively by condensation.

Since the alcohol used during the first step comprises an amino or ester functional group or any other functional group allowing coupling to a lipophilic region, it is preferable to protect it beforehand. The protection and removal of the protective radical before the coupling with the lipophilic region are carried out according to the customary methods.

The protection may be achieved by any compatible group whose use and removal does not adversely affect the remainder of the molecule. In particular, the procedure is carried out according to the methods described by T.W. GREENE, Protective Groups in Organic Syntheses, A. Wiley-Interscience Publication (1981), or by Mc OMIE, Protective Groups in Organic Chemistry, Plenum Press (1973).

By way of example, the protective groups may be chosen from the trimethylsilyl, benzhydryl, tetrahydropyranyl, formyl, acetyl, chloroacetyl, trichloroacetyl, trifluoroacetyl, ethoxycarbonyl, tert-butoxycarbonyl, trichloroethoxycarbonyl or phtalimido radical, and the like.

Finally, in a last step, the azide functional groups carried by the 5- or 6-membered ring are converted to amino functional groups by conventional methods known to persons skilled in the art which do not adversely affect the remainder of the molecule. Preferably, the procedure is carried out by the action of triphenylphosphine in the presence of water.

Any other method known to persons skilled in the art leading to the nucleic acid transfer agents according to the present invention also falls within the scope of the o invention.

By way of non-limiting example, the first embodiment o of the invention leads to the transfer agents of formula (8a) and whereas the second embodiment leads to the 0" 20 transfer agents of formula (16) and as is specified in the "Examples" section of the present description.

The importance of this second way of synthesizing cationic hydrophilic regions lies mainly in the few steps necessary for the synthesis and in the versatility of the 25 amino synthon obtained. The same cationic head can thus be condensed with various hydrophobic substituents.

Another subject of the invention relates to a composition comprising a transfecting agent as 21 defined above and a nucleic acid. Preferably, the compositions comprise a ratio of 0.1 to 50 nanomoles of vector per Vg of nucleic acid. Advantageously, this ratio is between 2 and 20 nanomoles of vector per jig of nucleic acid and still more preferably between 4 and 12 nanomoles of vector per jig of nucleic acid. More particularly, the transfer agent and the nucleic acid are present in a ratio of 8 to 12 nanomoles of vector per jg of nucleic acid.

For the purposes of the invention, "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 or synthetic or semisynthetic sequences, 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.

As regards more particularly deoxyribonucleic acids, they may be single- or double-stranded, as well 22 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 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 an expression cassette consisting of one or more genes of interest under the control of one or more promoters and of a transcriptional terminator which are active in the target cells.

For the purposes of the invention, therapeutic gene of interest is understood to mean in particular any gene encoding a protein product having a therapeutic effect. The protein product thus encoded may 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 the 23 said protein. The therapeutic gene of interest may also encode a mutant of a cellular protein, having increased stability, a modified activity and the like. The protein product may also be heterologous in relation to the target cell. In this case, an expressed protein may, for example, supplement or provide an activity which is deficient in the cell, allowing it to combat a pathological condition, or to stimulate an immune response.

Among the therapeutic products for the purposes of the present invention, there may be mentioned more particularly enzymes, blood derivatives, hormones, lymphokines: interleukins, interferons, TNF, and the like (FR 92/03120), growth factors, neurotransmitters or their precursors or synthesis enzymes, trophic factors (BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NT5, 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, tumour 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 haemoglobin or other protein carriers, metabolic enzymes, catabolic enzymes and the like.

Nucleic acid of therapeutic interest may also 24 be a gene or an anti-sense sequence, whose expression in the target cell makes it possible to control the expression of genes or the transcription of cellular mRNAs. Such 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 therefore allows the production of vaccines or the carrying out of immunotherapeutic treatments applied to humans or to animals, in particular against microorganisms, viruses or cancers. They may be in particular antigenic peptides specific for the Epstein-Barr virus, the HIV virus, the hepatitis B virus (EP 185 573), the pseudorabies virus, the syncytia forming virus, other viruses, or antigenic peptides specific for tumours (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 25 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 therapeutic gene, 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 may, in addition, comprise adjuvants capable of combining with the transfer 26 agent/nucleic acid complexes and of improving its transfecting power. In another embodiment, the present invention therefore relates to compositions comprising a nucleic acid, a transfer agent as defined above and one or more adjuvants capable of combining with the nucleolipid complexes and of improving the transfecting power thereof.

In this regard, the compositions according to the invention may comprise, as adjuvant, one or more neutral lipids. Such compositions are particularly advantageous, in particular when the transfer agent/nucleic acid charge ratio is low. The Applicant has indeed shown that the addition of a neutral lipid makes it possible to improve the formation of nucleolipid particles and to promote the penetration of the particle into the cell by destabilizing its membrane.

More preferably, the neutral lipids used within the framework of the present invention are lipids containing 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, 1-t N-methylated 1 to 3 times, phosphatidylglycerols, m 27 diacylglycerols, glycosyldiacylglycerols, cerebrosides (such as in particular galactocerebrosides), sphingolipids (such as in particular sphingomyelins), or asialogangliosides (such as in particular asialoGM1 and GM2).

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

More recently, the Applicant has demonstrated that it was also particularly advantageous to use, as adjuvant, a compound directly involved or otherwise in the condensation of the said nucleic acid (WO 96/25508). The presence of such a compound in a composition according to the invention makes it possible to reduce the quantity of transfecting agent, with the beneficial consequences resulting therefrom from the toxicological point of view, without any damaging effect on the transfecting activity. Compound involved in the condensation of the nucleic acid is intended to define a compound which compacts, directly or otherwise, the nucleic acid. More precisely, this compound may either act directly at the level of the Snucleic acid to be transfected, or may be involved at the level of an additional compound which is directly the level of an additional compound which is directly 28 involved in the condensation of this nucleic acid.

Preferably, it acts directly at the level of the nucleic acid. For example, the precompacting agent may be any polycation, for example polylysine. According to a preferred embodiment, this agent is involved in the condensation of the nucleic acid derived partly or completely from a protamine, a histone, or a nucleoline and/or from one of their derivatives. Such an agent may also consist, as a whole or in part, of peptide units (KTPKKAKKP) and/or (ATPAKKAA), it being possible for the number of units to vary between 2 and 10. In the structure of the compound according to the invention, these units may be repeated continuously or otherwise.

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

In 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 extracelluar targeting element which makes it possible to orient the transfer of DNA towards certain cell types or certain cell tissues (tumour cells, hepatic cells, haematopoietic cells and the like). It may also be an intracellular targeting element which makes it possible to orient the transfer of the nucleic acid towards certain preferred 1 cellular compartments (mitochondria, nucleus and the 29 like). The targeting element may be linked to the nucleic acid transfer agent according to the invention or also to the nucleic acid as 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, peptides or proteins such as antibodies or antibody fragments, ligands of cell receptors or fragments thereof, receptors or receptor fragments, and the like. In particular, they may be ligands of growth factor receptors, cytokine receptors, cellular lectinetype receptors, or RGD sequence-containing ligands with an affinity for the receptors for adhesion proteins such as the integrins. There may also be mentioned the receptors for transferrin, 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 sialyde Lewis X, or alternatively an Fab fragment of antibodies, or a single-chain antibody (ScFv). More recently, natural or synthetic ligand peptides have also been described which are advantageous in particular for their selectivity towards specific cells and which are capable of efficiently promoting internalization in these cells (Bary et al. Nature Medicine, 2, 1996, 299-305).

30 The combination of the targeting elements with the nucleolipid complexes may be made by any technique known to the person skilled in the art, for example by coupling to a hydrophobic part or to a part which interacts with the nucleic acid of the transfer agent according to the invention, or alternatively to a group which interacts with the transfer agent according to the invention or with the nucleic acid. The interactions in question may be, according to a preferred mode, of an ionic or covalent nature.

According to another variant, the compositions of the invention may also optionally incorporate at least one nonionic surfactant in a sufficient quantity to stabilize the size of the particles of nucleolipid complexes. The introduction of nonionic surfactants prevents the formation of aggregates, which makes the composition more suitable for an in vivo administration. The compositions according to the invention incorporating such surfactants have an advantage from the point of view of safety. They also have an additional advantage in the sense that they reduce the risk of interference with other proteins, given the reduction in the overall charge of the compositions of nucleolipid complexes.

The surfactants advantageously consist of at least one hydrophobic segment, and at least one hydrophilic segment. Preferably, the hydrophobic segment is chosen from aliphatic chains, hydrophobic 31 polyoxyalkylenes, alkylidene polyesters, polyethylene glycols with a benzyl polyether head and cholesterol, and the hydrophilic segment is advantageously chosen from hydrophilic polyoxyalkylenes, polyvinyl alcohols, polyvinylpyrrolidones, or saccharides. Such nonionic surfactants have been described in application PCT/FR 98/00222.

The subject of the present invention is also the use of the transfer agents described above for transferring nucleic acids (and more generally of polyanions) into cells. Such a use is particularly advantageous because these transfecting agents increase the transfection efficiency while having a zero or highly reduced cell toxicity.

For uses in vivo, for example for studying the regulation of genes, the creation of animal models of pathological conditions, or in therapy, 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 pharmaceutical 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 Sthe mucous membrane). They may be in particular -32isotonic sterile solutions, or dry, in particular freezedried, 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 tumours, or the circulatory system, or a treatment of ot 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, 20 bone marrow, thymus, heart, lymph, blood, bones, cartliages, pancreas, kidneys, bladder, stomach, intestines, testicles, ovaries, rectum, nervous system, eyes, glands, connective tissues, and the like.

The invention relates, in addition, to a method of 25 transferring nucleic acids into cells comprising the following steps: bringing the nucleic acid into contact with a transfecting agent as defined above and, where appropriate, with one or more adjuvants, to form a nucleolipid complex, and 33bringing the cells into contact with the complex formed in (1) The cells may be brought into contact with the complex by incubating the cells with the said nucleolipid complex (for uses in vitro or in vivo), or by injecting the complex into an organism (for uses in vivo). The incubation is carried out preferably in the presence, for example, of 0.01 to 1000 pg of nucleic acid per 106 cells. For administration in vivo, nucleic acid doses of between 0.01 and 10 mg may for example be used.

In the case where the compositions of the invention contain, in addition, one or more adjuvanits as defined above, the adjuvant(s) is (are) previously mixed with the lipid according to the invention or with the nucleic acid.

The present invention thus provides a particularly advantageous method for transferring nucleic acids, in particular for the treatment of diseases, comprising the in vivo, ex vivo or in vitro administration of a nucleic acid encoding the protein or which can be transcribed into a nucleic acid capable of correcting the said disease, the said nucleic acid being combined with a compound of the general formula under the conditions defined above. More particularly, this method is applicable to diseases resulting from a deficiency in the protein or nucleic product, the administered nucleic acid encoding the -34said protein product or being transcribed into a nucleic product or alternatively constituting the said nucleic product.

Accordingly, the invention also provides the use of a transfecting agent as defined above for the manufacture of a medicament containing at least one nucleic acid to be transferred.

The nucleic acid transfer agents of the invention can be used in particular 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, haematopoietic cell line (lymphocytes, CD34, dendritic cells, and the go: like), epithelial cells and the like, in differentiated or pluripotent form (precursors) S. :In addition to the preceding arrangements, the o present invention also comprises other characteristics 20 and advantages which will emerge from the examples and figures below, which should be considered as illustrating the invention without limiting its scope.

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

35 Figure 3/5: Scheme for the synthesis of compounds to (16).

Figure 4/5: Measurement of the transfection efficiency in vitro of compounds (8a) and (8b) by means of measurement of the luciferase activity in Hela cells, in the absence of serum. The measurements were made at different nanomoles of vector/pig DNA ratios, and are represented by the bars. The protein assay, for each of the compositions, was also carried out and is represented on the same graph by a curve in the form of a solid line.

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

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

The fluorescence measurements were carried out on Perkin Elmer LS50B, using excitation and emission wavelengths of 260 nm and 590 nm respectively. The slit widths for excitation and emission are set at 5 nm. The 36 fluorescence value is recorded after adding 5 pg of ethidium bromide/ml as final concentration.

The nuclear magnetic resonance (NMR) spectra were recorded at 300.13 MHz for the proton and at 75.47 MHz for carbon, on a Bruker MSL 300 apparatus in deuterated chloroform.

EXAMPLES

EXAMPLE 1: Synthesis of the transfer agent 8(a) from 6methylbenzyloxygalactopyranose The different reaction steps are shown diagrammatically in figures 1/5 and a) Synthesis of 6-methylbenzyloxy-2,3,4-tri-O-(2'carboxyethyl)-P-D-galactopyranoside (2) 28.4 g (0.1 mol) of 6-methylbenzyloxy--Dgalactopyranside are dissolved in 400 ml of THF and placed at 0°C, with stirring. 32 g (5.7 equivalents) of finely browned potassium hydroxide, 0.5 g of 18- Cr-6 crown ether and 32.5 ml (1.5 equivalents) of ethylbromoacetate are added. After two hours, the mixture is evaporated to dryness, redissolved in water and the solution obtained is neutralized with 2N hydrochloric acid. 32 g of product are thus obtained (yield: which product is extracted with dichloromethane.

37 13C NMR: 6, 172.94; 136.87; 127.93; 127.41; 98.89; 97.22; 78.74; 76.17; 72.97; 69.52; 67.93; 63.59; 54.75.

b) Synthesis of 6-methylbenzyloxy-2,3,4-tri-0-(2'hydroxyethyl) -,D-galactopyranoside (3) g (0.065 mol) of the preceding triacid (2) are dissolved in 400 ml of THF and placed at 0°C under nitrogen. 40 ml of borane-dimethyl sulphide are added dropwise while stirring. The mixture is stirred for a further two hours at room temperature and then methanol is carefully added in excess. The solution is then evaporated to dryness and the solid obtained is redissolved several times in methanol. 24.5 g of final product are thus obtained (yield: which product is purified on a silica column eluted with ethyl acetate.

c) Synthesis of 6-methylbenzyloxy-2,3,4-tri-O-(2'azidoethyl) D-galactopyranoside (4) 20 g (0.048 mol) of triol obtained in the preceding step are dissolved in 500 ml of THF. 40 g (3.2 equivalents) of triphenylphosphine, 160 ml (3.7 equivalents) of a 1.1 M hydrazoic acid solution in toluene, and then 24.2 ml (3.2 equivalents) of diethylazodicarboxylate are added. After one hour, the solution is evaporated and purified on a silica gel Scolumn eluted with a heptane/ethyl acetate (6:4) 38 mixture. 17.6 g of product are thus obtained (yield 1C NMR: 5, 128.47; 127.80; 98.23; 79.05; 76.27; 73.55; 71.92; 70.15; 69.95; 68.77; 68.14; 55.37; 51.41; 51.11; 50.87.

d) Synthesis of methyl-2,3,4-tri-0- (2'-aminoethyl) -P-Dgalactopyranoside, trishydrochloride g (0.03 mol) of triazide are dissolved in 200 ml of ethanol. 7.5 ml of 12 N hydrochloric acid are added and then the mixture is treated with hydrogen in the presence of palladium on carbon for three hours.

After filtration; the solvent is evaporated off and the residue obtained (12.6 g, yield: 95%) is used directly in the next step.

e) Synthesis of methyl-2,3,4-tri-- (2'-tertbutylcarbamidoethyl) D-galactopyranoside (6) 12 g (0.028 mol) of hydrochloride are dissolved in a mixture of 100 ml of dioxane and 85 ml of 1 N sodium hydroxide. 18.7 g (3.3 equivalents) of di-tert-butyl dicarbonate are added and the mixture is stirred at room temperature for two hours. The reaction mixture is evaporated off and then extracted with dichloromethane (3 times 50 ml). The organic phases are combined and dried over sodium sulphate and evaporated off. 15.56 g (yield: 90%) of residue are thus 39 obtained, which residue is purified on a silica gel column eluted with ethyl acetate.

f) Synthesis of 6-methyldioctadecylamidosuccinyl-2,3,4tri-O-(2'-tert-butylcarbamidoethyl) D-galactopyranoside (7a) g (0.016 mol) of the preceding product (6) are dissolved in 300 ml of dichloromethane. 14.6 g equivalents) of dioctadecylamidosuccinic acid, 1.95 g (1 equivalent) of dimethylaminopyridine and 6.5 g (2 equivalents) of dicyclohexylcarbodiimide are added successively. The dioctadecylamidosuccinic acid is obtained by reacting dioctadecylamine with succinic anhydride. After one night, 10.ml (1.7 g) of a solution of oxalic acid in methanol are carefully added and the mixture is kept stirred for one hour (evolution of carbon monoxide). The solution is filtered and evaporated to dryness, and the product obtained is purified on a silica gel column eluted with a heptane/ethyl acetate solution. 12.8 g of product (7a) are thus obtained (yield: g) Synthesis of 6 -methyl-dioctadecylamidosuccinyl- 2,3,4-tri-O-(2'-aminoethyl) -,D-galactopyranoside, tristrifluoroacetate (8a) 10 g (0.008 mol) of product (7a) are dissolved in 20 ml of 90% trifluoroacetate acid. After half an hour, the solution is evaporated off and the residue is taken up in three times 20 ml of toluene.

40 The residue (8a) obtained is purified by HPLC (RP4; water/acetonitrile; 0->100%; 20 minutes). 9.7 g of product (8a) are thus obtained (yield MH =1304.

EXAMPLE 2: Synthesis of compound 8(b) from 6methylbenzyloxygalactopyranose The different reaction steps are shown diagrammatically in Figures 1/5 and The first phases of the synthesis are identical to synthesis steps a) to e) described in Example 1. Next, the procedure is continued in the following manner: f) Synthesis of 6-methyldioctadecylamidophthalyl-2,3,4tri-O-(2'-tert-butylcarbamidoethyl) D-galactopyranoside (7b) This product is obtained in the same manner as product (7a) but using dioctyldecylamidophthalic acid in place of dioctadecylamidosuccinic acid.

g) Synthesis of 6-methyldioctadecylamidophthalyl-2,3,4tri-O-(2'-aminoethyl) D-galactopyranoside, tristrifluoroacetate (8b) Compound (8b) is obtained from product (7b) obtained in the preceding step, in the same manner as product (8a).

SMH=1317 41 EXAMPLE 3: Synthesis of the transfer agent (16) from triacetyl glucal a) Synthesis of phthalimidopropyl-4,6-di-O-acetyl-2,3bisdeoxy-p-D-erythro-2-hexenopyranoside The different reaction steps are represented in figure 13.17 g (0.048 mol) of triacetyl-D-glucal (9) are dissolved in 250 ml of dichloromethane. 11 g (1.1 equivalents) of phthalimidopropanol and 1.56 ml (0.26 equivalent) of boron trifluoride etherate are added.

After one hour at room temperature, the mixture is neutralized with a saturated solution of sodium bicarbonate, the solution is decanted off, dried and evaporated. 16 g of product (10) are thus obtained (yield: which product is purified by chromatography on a silica gel column eluted with a heptane/ethyl acetate solution.

b) Synthesis of phthalimidopropyl-4,6-di-O-acetyl-2,3bisdeoxy-p-D-glucopyranoside (11) 16 g (0.038 mol) of glycoside (10) are dissolved in 200 ml of ethanol. 0.16 g of 10% palladium on carbon is added, and the mixture is treated with hydrogen for two hours. After filtration, the solution is evaporated off. 15.78 g of product (11) are thus obtained (yield: 99%).

42 c) Synthesis of phthalimidopropyl-2,3-bisdeoxy-p-Dglucopyranoside (12) g (0.036 mol) of the preceding product (11) are dissolved in 250 ml of methanol, and then 3.5 ml (0.1 equivalent) of 1 N sodium methoxide are added. After two hours, the mixture is neutralized with IR120 amberlite resin. 11.5 g of product (12) are obtained after filtration and evaporation (yield: 96%).

d) Synthesis of phthalimidopropyl-4,6-diazido-2,3,4,6tetradeoxy-P-D-glucopyranoside (13) 11 g (0.033 mol) of deacetylated product (12) are dissolved in 250 ml of THF. 21.5 g equivalents) of triphenylphosphine and 110.5 ml (3.7 equivalents) of 1.1 M hydrazoic acid in toluene are successively added, while stirring. 12.88 ml equivalents) of diethylazodicarboxylate are added at a rate such that the temperature of the mixture does not exceed 30 0 C. After one hour, the solution is evaporated to dryness and the product obtained is purified on a silica gel column eluted with a heptane/ethyl acetate mixture. 7.2 g of product (13) are thus obtained (yield: 57%).

e) Synthesis of aminopropyl-4,6-diazido-2,3,4,6tetradeoxy-p-D-glucopyranoside (14) 7g (0.018 mol) of product (13) are dissolved in 500 ml of ethanol and then 2.6 ml of hydrazine 43 hydrate are added, and the mixture is stirred at 40 0

C

for two hours. After evaporation, the residue is redissolved in dichloromethane and the phthalylhydrazide which is formed is allowed to crystallize. The solution is filtered and evaporated off. 4.17 g of product (14) are thus obtained (yield: f) Synthesis of cholesteryl-O-formamidopropyl)-4,6diazido-2,3,4, 6 -tetradeoxy-p-D-glucopyranoside 6.34 g (0.9 equivalents) of cholesteryl chloroformate and 4 g (0.016 mol) of product (14) are dissolved in 200 ml of ethyl ether. A precipitate is immediately formed, and 50 ml of 1 N sodium hydroxide are then added. After a few minutes, the solution becomes clear again. It is kept stirring for a further minutes and then decanted off, washed twice with ml of water, and the solution is evaporated to dryness. 9.4 g of product (15) are thus obtained, which product is purified on a column eluted with heptane/ethyl acetate solution (yield: g) Synthesis of (cholesteryl-O-formamidopropyl)-4,6diamino-2,3,4,6-tetradeoxy-P-D-glucopyranoside (16) 9 g (0.013 mol) of diazide (15) are dissolved in 150 ml of 80% THF, and then 14.2 g (4 equivalents) of triphenylphosphine are added and the mixture is heated at 500C for one hour. After cooling, the mixture 44 is loaded onto a column of IRC-50 COOH anionic resin and washed with 80% THF. The product is then eluted in acetate form with the THF/water/acetic acid (64:16:10) mixture. 8.9 g of product (16) are thus obtained (yield: 1C NMR: 6, 176.55; 156.65; 139.84; 132.02; 128.46; 125.46; 122.44; 97.11; 74.28; 56.66; 56.12; 50.02; 43.70; 42.30; 39.51; 38.57; 36.98; 36.55; 36.16; 35.76; 31.87; 30.30; 29.89; 28.18; 23.82; 22.78; 22.53; 21.03; 19.31; 18.70; 11.84.

EXAMPLE 4: Synthesis of compound (17) a) Synthesis of benzyl(5-O-acetyl-6-acetoxymethyl- 5,6-dihydro-2H-pyran-2-yloxy)acetate 2.8 g of triacetyl D-glucal (0.01 mol) and 1.6 ml of benzyl glycolate (1.1 equivalents) are dissolved in 80 ml of chloroform. The mixture is cooled on an ice bath and 0.651 ml of boron trifluoride etherate (0.5 equivalents) is added. After 45 minutes, the mixture is neutralized with a saturated aqueous sodium bicarbonate solution, dried over sodium sulphate, filtered and concentrated. The mixture is purified by chromatography on a silica gel column, eluted with a heptane/ethyl acetate mixture. The yield is 96%.

45 b) Synthesis of 5 -O-acetyl-6-acetoxymethyltetrahydropyran-2-yloxy)acetic acid: g of the preceding product (0.0093 mol) are hydrogenated at atomospheric pressure and at room temperature, in the presence of 0.35 g of 10% palladium on carbon for 10 hours. The mixture is filtered on celite, concentrated and purified by chromatography on a silica gel column, eluted with ethyl acetate. The yield is c) Synthesis of N,N-dioctadecyl(5-O-acetyl-6acetoxymethyltetrahydropyran-2-yloxy)acetamide: 0.675 g of the preceding product (0.0017 mol) are dissolved in 8.6 ml of chloroform. 0.9 ml of diisopropylethylamine, 0.899 g of dioctadecylamine (1 equivalent) and 0.426 g of dicyclohexylcarbodiimide (1.2 equivalents) are added. After one night at room temperature, the mixture is concentrated and the product is extracted with heptane. The mixture is purified by chromatography on a silica gel column, eluted with a heptane/ethyl acetate mixture. The yield is d) Synthesis of N,N-dioctadecyl(5-hydroxy-6-hydroxymethyltetrahydropyran-2-yloxy)acetamide: 1.57 g of the preceding product (0.00198 mol) are dissolved in 10 ml of methanol. 0.197 ml of sodium methoxide (0.2 equivalent; methanol 2M) is added. After 46 two hours at room temperature, the mixture is neutralized and concentrated. The mixture is purified by chromatography on a silica gel column, eluted with a heptane/ethyl acetate mixture. The yield is 92%.

e) Synthesis of N,N-dioctadecyl(5-azido-6-azidomethyltetrahydropyran-2-yloxy)acetamide (17): 0.6 g of the preceding product (0.00087 mol) are dissolved in 15 ml of THF. 0.48 g of triphenylphosphine (2.1 equivalents) is added followed by 0.288 ml of DEAD (2.1 equivalents). When the exothermic reaction is complete, 1.588 ml of a 1.38 M hydrazoic acid solution in toluene (2.5 equivalents) are added.

At the end of the reaction, the medium is concentrated, taken up in heptane, filtered and concentrated. The mixture is then purified by chromatography on a silica gel column, eluted with a heptane/ethyl acetate (8:2) mixture. The yield is 72%.

1C NMR: 6, 173.21; 172.07; 162.22; 161.77; 118.34; 114.49; 110.65; 108.34; 96.88; 65.18; 64.50; 48.27; 46.54; 40.69; 35.86; 31.78; 30.94; 28.63; 27.56; 26.92; 22.54; 21.84; 13.94.

EXAMPLE 5: Combination of transfecting agents of the invention (8a) and (8b) with DNA This example illustrates the nature of the combination between the transfecting agents of the invention and DNA, as well as the formation of 47 complexes.

The transfecting agents according to the invention were dissolved in water at 10 mM. The solutions thus obtained are transparent and correspond to an organization in the form of cationic lipid micells.

The transfecting agent/DNA complexes were prepared in a ratio of 6 nmoles of transfecting agent/ng of DNA.

The combination of the transfecting agent (8b) with DNA gives a complex with a size equal to 92 nm 27 nm, and a fluorescence of 7% in water and of in sodium chloride NaCI at 300 mM.

The transfecting agent of the invention (8a), combined with DNA, allows the formation of a complex with a size close to 98 nm 20 nm, and a fluorescence of 6% in water and 55% in sodium chloride NaCl at 300 mM.

These results show that the transfecting agents according to the invention are capable of combining with DNA and of forming particles whose mean diameter is about 100 nm, which constitutes a size which is particularly appropriate for the crossing of cell membranes. This combination remains relatively stable when the ionic strength of the medium is increased. Indeed, at 300 mM, more than 50% of the DNA 7 remains combined with the transfecting agents of the invention.

48 EXAMPLE 6: Transfection of DNA in vitro with the transfecting agents of the invention (8a) and (8b) The transfection efficiency was evaluated in vitro at various doses of vectors/pg of DNA.

The genetic material used for these experiments is a plasmid construct pCMV-Luc comprising the "luciferase" reporter gene, which construct is derived from plasmid pGL2-basic Vector [Promega] by inserting an MluI-HindIII fragment containing the human cytomegalovirus (CMV) promoter extracted from the plasmid pcDNA3 [Invitrogen].

Dilute nucleic acid solutions at 40 pg/ml in physiological saline (0.15 M sodium chloride NaCI) were also used.

The products described in the invention are dissolved in water at a concentration varying from gM to 800 pM, and mixed volume for volume with the DNA solution. The final saline concentration is 75 mM so as to freshly prepare cytofectant solutions.

For the transfection, the cells are cultured under the appropriate conditions in 24-well microplates (2 cm 2 /well) and are transfected while in the exponential growth phase and at 50-70% confluence.

The cells are washed with twice 500 il of medium free of serum proteins and again grown in serumfree medium. 50 pl of cytofectant mixture [1 pg of S DNA/well] are added to these cells [3 wells/condition 49 vector-DNA]. The growth medium is supplemented with the appropriate quantity of serum two hours after transfection.

The transfection efficiency is evaluated at 48 hours post-transfection by measuring the expression of lucerifase according to the recommendations given for the use of the Promega kit [Luciferase Assay System]. The toxicity of the cytofectant mixtures is estimated by a measurement of the protein concentrations in the cell lysates.

The result obtained for the transfection of HeLa cells are assembled in Figure 4/5, and those obtained on NIH3T3 cells are indicated in Figure From these two figures as well as from the results obtained for vector/DNA ratios greater than nmol/pg of DNA (not shown), it is possible to deduce that the transfection is particularly efficient when the vector/DNA ratio is between 2 and 20 nanomoles of vector per .ig of DNA.

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

Moreover, it is observed that the histograms presenting the dose effect of the vectors can be superposed for products (8a) and (8b) regardless of the Q:'OPERUMS\2238028-169 doc- 18/0601 cell type considered.

The two products of the invention are not toxic at the vector doses studied for the NIH 3T3 cells. Only a maximum loss of 20% of cellular proteins is indeed observed for doses greater than 8 nanomoles of vector per sample of transfected cells.

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

The reference to any prior art in this S 15 specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art Sforms part of the common general knowledge in Australia.

Claims (8)

1. Transfecting agent comprising at least one cationic hydrophilic region coupled to a lipophilic region, in which the cationic hydrophilic region has the general formula z Z X Z (I) y I y ZZZ in which: Y is an integer equal to 0 or 1, the different y's being independent from each other, X represents an oxygen, nitrogen, sulphur or selenium atom, 20 the Z groups represent, independently of each other, S a hydrogen atom, an OR group in which R represents a hydrogen atom, a methyl group or a group (CH2)n-NRR 2 in which n is an integer chosen from 1 to 6 inclusive and Ri and R 2 1 25 represent, independently of each other, a hydrogen atom or a group (CH,)q-NH 2 it being possible for q to vary from 1 to 6 inclusive, the different q's being independent of each other, a group (CH 2 )m-NRIR 2 in which m is an -52- integer chosen from 0 to 6 inclusive and R, and R 2 are as defined above, or alternatively a "spacer" group allowing the binding of the cationic hydrophilic region to the lipid region, it being understood that at least one of the Z substituents is a "spacer" group and that at least two of the z substituents carry an amino group.

2. Transfecting agent according to'Claim 1, in which n is 2, 3 or 4 in the group (CH2)n-NRjR 2 in the general formula defining the cationic hydrophilic region.

3. Transfecting agent according to Claim 1 or 2, in which the cationic hydrophilic region is a glycoside of the pyranose or furanose form.

4. Transfecting agent according to any of the preceding Claims, in which y is equal to 1 and x is an 'oxygen atom, in the general formula defining the cationic hydrophilic region. 20 5. Transfecting agent according to any of the preceding claims, in which the lipophilic region consists of one or more optionally halogenated, saturated, or unsaturated, linear or branched aliphatic chains and/or a steroid derivative. 25 6. Transfecting agent according to Claim 5, in which the aliphatic chains contain 10 to 22 carbon atoms.

7. Transfecting agent according to Claim 6, in which the aliphatic chains contain 14, 16, 17, 18 or 19 carbon atoms.

8. Transfecting agent according to any of Claims to 7, in which the aliphatic chains are(CH2) 13 CH 3 (CH 2 1 5 CH 3 (CH2) 16 CH 3 (CH 2 17 H 3 or (CH 2 18 CH 3 -53-

9. Transfecting agent according to Claim 5, in which the steroid derivative is cholesterol, cholestanol, 3a,5-cyclo-5a-cholestan-6p-yl formate, cholesterylamine, 6-(1,5-dimethyl-hexyl)-3a,5a-dimethylhexadeca-hydrocyclo-

53-penta[a]cyclopropa[2,3]cyclo-penta[1,2-f]-naphthalen- or cholestanylamine. Transfecting agent according to any of the preceding Claims in which the "spacer" group enabling the cationic hydrophilic region to be linked to the lipophilic region consists of an acid or amino group comprising hydrolysable functional groups. 11. Transfecting agent according to Claim 10, in which the "spacer" group consists of an aliphatic or aromatic chain and comprises one or more groups chosen from amides, esters, ethers, carbamates and aromatic rings. 12. Transfecting agent according to Claim 11, in which the "spacer" is -O-CO-(CH 2 )x-COOH, -O-(CH 2 -COOH, -O-CO- (CH 2 -NH 2 -0-(CH 2 -NH 2 or -NH- (CH 2 -NH 2 with x representing an integer from 1 to 6 inclusive. *o• *o oo 13. Transfecting agent according to any of the preceding Claims which is one of the compounds of formula: NH-I 2 H 0 0 0NI-C..SN H,N y 0 CIH7 (17) KNH 2 NH 2 (8a) (8b) 14. Method of preparing a transfecting agent according to any of Claims 1 to 13, in which the lipophilic region is coupled by means of a "spacer" to a heterocycle of general formula as defined in Claim 1. Composition which comprises a transfecting agent as defined in any of Claims 1 to 13 and at least one nucleic acid. 16. Composition according to Claim 15, in which the transfecting agent/nucleic acid ratio is between 0.1 and nanomoles of agent per pg of DNA. 17. Composition according to Claim 15 or 16 which incorporates, in addition, one or more adjuvants. 18. Composition according to Claim 17, in which the one or more adjuvants are neutral lipids. 19. Composition according to Claim 17 or 18 in which the one or more adjuvants are compounds which are involved directly or indirectly in the condensation of 20 the nucleic acid. 20. Composition according to any of Claims 15 to 00. 19, which comprises a pharmaceutically acceptable vehicle for an injectable formulation. •21. Composition according to any of claims 15 to 19, which comprises a pharmaceutically acceptable vehicle for application to the skin and/or the mucous membranes. 22. Method of transferring nucleic acids into cells, the method comprising: bringing the nucleic acid into contact with a transfecting agent as defined in any of Claims 1 to 13 and, where appropriate, with one or more adjuvants, to form a nucleolipid complex; and bringing the cells into contact with the T complex formed in -56- 23. Transfecting agent according to any of Claims 1 to 13 for use in transferring nucleic acids into cells. 24. Use of a transfecting agent as defined in any of claims 1 to 13 for the manufacture of a medicament containing at least one nucleic acid to be transfected. Method of preparing a transfecting agent according to claim 1, substantially as hereinbefore described in any of Examples 1 to 4. 26. Composition according to Claim substantially as.hereinbefore described in Example 27. Method of transferring nucleic acids into cells according to Claim 22, substantially as hereinbefore described in Example 6. Dated this 1 9 th day of June 2001. Rhone-Poulenc Rorer S.A. •By its Patent Attorneys Davies Collison Cave o o o o *oo *oo *go *o *ooo