EP0948638A1 - Method for preparing compositions for transferring nucleic acids - Google Patents

Abstract

The invention concerns a method for preparing a composition for transferring nucleic acids consisting in contacting a nucleic acid with a cationic lipid, whereby, previous to contacting, the cationic lipid is subjected to a heating step. The invention also concerns the resulting compositions and their use.

Description

 PROCESS FOR THE PREPARATION OF COMPOSITIONS FOR TRANSFERRING NUCLEIC ACIDS

The present invention relates to a process for the preparation of compositions for the transfer of nucleic acids. The compositions obtained can be used for the transfer of nucleic acids into cells in vitro, ex vivo or in vivo.

The cellular penetration of a naked nucleic acid (generally of high molecular mass and negatively charged) is a rare phenomenon and which, in general, only leads to the transfection of the cell with very limited effectiveness. For this reason, different types of vectors and techniques have been described in the prior art for carrying out this transfer. Two large families can be distinguished in this respect: viral vectors and physical techniques.

Among the viral vectors, mention may be made, for example, of retroviruses, adenoviruses, AAVs, herpes viruses, baculoviruses, etc. The transfer efficiency obtained with viral vectors is generally very good. However, their construction and production are difficult, the cloning capacity of these vectors is sometimes limited, and their use can in certain cases have certain drawbacks inherent in the use of viruses (dissemination, pathogenicity, etc.).

For these reasons, various physical techniques for transferring nucleic acids have been developed. Examples include electroporation, coprecipitation or the use of particle guns.

Electroporation involves applying an electric field to a suspension of cells containing DNA. However, if this technique gives in some cases good results, it is difficult to optimize because of the risk of irreversible damage to the cell membrane. In the particle gun technique, particles (gold, tungsten) are coated with nucleic acid and then projected onto the cells. This method is however promising mainly for walled cells, such as plant cells. Furthermore, the coprecipitation of DNA with certain polymers (DEAE-dextran) or calcium phosphate has the drawback of being not very reproducible and sometimes cytotoxic.

To overcome these drawbacks, synthetic transfer vectors have been developed. The role of these vectors is essentially to present the nucleic acid in a form conducive to cell penetration, to facilitate this penetration (into the cytoplasm then into the nucleus) as well as to preserve it from cytoplasmic nucleases.

Among these vectors, cationic lipids have interesting properties. These vectors consist of a polar cationic part allowing the condensation of nucleic acids and a hydrophobic lipid part stabilizing the ionic interaction. An excess of lipid, due to the ionization of its polar part, would also promote interaction with the cell membrane. Particular examples of cationic lipids are in particular lipopolylysine, monocationic lipids (DOTMA:

® Lipofectin); certain cationic detergents (DDAB); iipospermins

(DOGS, DPPES, etc.), lipothermins, etc.

The use of this type of vector, possibly in combination with a fusogenic lipid (DOPE, etc.), has shown that they have good nucleic acid transfer properties in vitro, ex vivo and in vivo on many cell types. . These vectors therefore constitute an advantageous alternative to viral vectors and to physical techniques for transferring nucleic acids. However, the exploitation of these vectors at the industrial level is currently limited. In particular, the methods of preparation of these vectors described in the prior art are empirical, not very industrializable, not very reproducible, and lead to ill-defined mixtures and the composition of which does not give optimal properties. Thus, it is known to prepare compositions by mixing the various lipid components in chloroform, drying under nitrogen flow for 20 minutes then drying under vacuum. The lipid film thus obtained is taken up in deionized water [Hui 96], [Felgner 87]. The mixture is then vortexed to resuspend the lipid and sonicated in a sonication bath for 10 minutes until clarification of the suspension which can then be diluted. The compaction is carried out immediately before addition to the cells and left for 4 hours at 37 ° C. in an F10 culture medium. Variants of this technique can be encountered. For example after dissolution with chloroform and drying, the film can be taken up in a 20 mM NaCl solution [Guershon 93] or 20 mM Hepes buffer [Gao 91]. The suspension is generally vortexed (immediately after resumption of the film or after a 24-hour hydration phase at 4 ° C [Gao 91]) and sonicated for variable times. Sometimes the ionic strength and the nature of the ions of the complex can be modified after compaction [Xu 1996]. The sonication operation itself has variations since some authors use titanium sonication probes [Gustafsson 1995]. The method of preparation consisting of the formation of a film by dissolution with chloroform, evaporation, drying, recovery by an aqueous medium and sonication is also frequently encountered for the manufacture of liposomes composed of a neutral lipid associated with a cationic surfactant [ Pinnaduwage 89],

Another method of preparation consists in directly diluting the commercial lipids (in ethanolic solution, aqueous suspension or liposomal formulation) in MEM culture medium. After identical dilution of the DNA, the two components are mixed by inversion and the suspension is left at room temperature for 15 to 30 minutes. Then a further dilution is made before contacting the cells for 6 hours [Fasbender 95]. Commercial lipid and DNA can also be simply diluted separately in distilled water before placing in contact by simple shaking and possible incubation at room temperature for 15 minutes [Bringham 89].

According to other methods, the initial form of the lipid is an ethanolic solution of variable concentration which is diluted extemporaneously in culture medium just before contact with DNA [Behr 89]. The ethanolic solution can also be diluted in other media (water, NaCl at various concentrations) immediately or 10 minutes before compaction. At the end of the compaction and a possible wait of 10 minutes, a new dilution can take place in a medium of variable ionic composition. The transfection is carried out after a time of 0 or 10 minutes after the last dilution [Barthel 93]. The ethanolic solution can also be introduced directly into the aqueous suspension containing the DNA and the mixture can subsequently undergo sonication in a bath [Demeneix 91].

It is also known to dissolve cationic liposomes composed of a DOSPA / DOPE mixture [Hofland 96] with a 1% solution of Octylglucoside (OG) in a 10 mM solution of Tris (pH 7.4). After contact with the DNA, the complex is dialyzed three times (in order to remove the excess micelles of GM) against 2000 volumes of 10 mM Tris / 5% dextrose (pH 7.4) for more than 48 hours at a temperature 4 ° C. The transfection is carried out for 4 to 5 hours at 37 ° C. in DMEM devoid of serum.

The preparation of cationic liposomes can also be carried out by rehydration of the lipid film followed by successive extrusions through polycarbonate membranes of calibrated porosity [Duzgϋnes 89] or microfluidization [Sternberg 94].

All these preparation methods have drawbacks for extrapolation to an industrial scale. Sonication can hardly be used on such a scale and the stability of the vesicles thus formed is often insufficient. In addition, the probes used for sonication, of metallic origin, can release in the medium annoying particles for pharmaceutical applications. Similarly, filtration gives only liposomes of size defined by the porosity of the membrane after filtration and whose stability over time is not guaranteed. The use of lipids in the form of an ethanolic solution, in addition to the problem of using an organic solvent, appears to induce the formation of very polydisperse particles.

The Applicant has now developed a new process for the preparation of transfecting compositions.

This process derives more particularly from the characterization by the applicant of the physicochemical properties of the lipid vectors. The Applicant has thus shown that, depending on the conditions, these vectors exist in different physicochemical states (micellar form, form of mature aggregates, etc.). The Applicant has made it possible more particularly to characterize the different physicochemical states of the lipid vectors, it has also highlighted means making it possible to control the passage between these different states, and to stop maturation in defined states, conducive to complexation with l And cell transfection.

The present invention thus describes a reproducible process allowing the preparation at industrial level of characterized and calibrated transfecting compositions.

The process of the invention is based more particularly on carrying out a preliminary treatment of the cationic lipid, making it possible to obtain a homogeneous micellar suspension. Thus, a first subject of the invention relates to a process for the preparation of a composition for the transfer of nucleic acids comprising bringing a nucleic acid into contact with a cationic lipid, characterized in that, prior to placing contact, the cationic lipid is subjected to a heating step.

The Applicant has studied the physicochemical characteristics of the various cationic lipids. The results obtained show that, according to the ionic strength of the medium, depending on the pH of the medium or even depending on the temperature, cationic lipids exist in different physical states. The examples presented below describe in particular phase diagrams of different cationic lipids (lipospermins, lipothermins) as well as a study of the optical density as a function of pH and temperature, which clearly show different physicochemical states depending on the conditions. . The Applicant has now shown that these different states have different DNA compaction and cell transfection properties, and that it is advantageous to have a method allowing reproducible control of access to these different states.

The method according to the invention, notably involving the heating of the lipid suspension, has the advantage of a simple method which is easily transposable to the constraints of industrial exploitation. It also allows, by controlling the organizational kinetics, the control of the structure adopted by the lipid before and after it is brought into contact with DNA. The process of the invention also allows standardization of the preparation methodology, essential on an industrial scale.

More particularly, the preliminary step consists in heating the cationic lipid until the formation of a micellar solution. Even more preferably, the lipid vector is heated to a temperature higher than its phase transition temperature. The phase transition temperature corresponds to the temperature at which the lipid chains melt. This temperature can be determined, for each lipid vector, by techniques known to those skilled in the art. In particular, it is possible to use the Differential Thermal Analysis method (or DSC: "Differential Scanning Calorimetry") using for example the DSC4 device (Perkin Elmer) following the manufacturer's recommendations, as illustrated in the examples.

The process of the invention is applicable to any type of cationic lipid. It is particularly suitable for the preparation of lipopolyamines. The lipopolyamines are amphiphilic molecules comprising at least one hydrophilic polyamine region and one lipophilic region covalently linked together by a chemical arm. Advantageously, the polyamine region corresponds to the general formula H2N - (- (CH) _m -NH-) | -H in which m is an integer greater than or equal to 2 and I is an integer greater than or equal to 1, m can vary between different carbon groups between two amines. Preferably, m is between 2 and 6 inclusive and I is between 1 and 5 inclusive. Even more preferably, the polyamine region is represented by spermine or by thermine, or by an analog having retained its DNA binding properties.

The lipophilic region can be one or more hydrocarbon chains, saturated or not, cholesterol, a natural lipid or a synthetic lipid capable of forming lamellar or hexagonal phases. Advantageously, the lipophilic region comprises two hydrocarbon chains.

In addition, the cationic lipid can be combined with lipid adjuvants, as indicated below.

Preferred examples of cationic lipids are in particular lipospermins, in particular dioctadecylamidoglycyl spermine (DOGS) or 5-carboxyspermylamide of palmitoylphosphatidylethanolamine (DPPES), the preparation of which has been described, for example, in patent application EP 394 111. Another preferred family consists of lipothermins, among which there may be mentioned more particularly RPR120531 and RPR120535 (FR95 / 13490, WO96 / 17823 incorporated herein by reference). The structure of these compounds is shown in Figure 1.

For the implementation of the process of the invention, the lipid, in the form of crystalline powder or film, is taken up in pure water or a saline medium whose pH is optionally adjusted. The homogenization is carried out by heating above the transition temperature of the lipid, ie the temperature from which the aliphatic chains melt. As indicated above, this phase transition temperature can be determined by differential thermal analysis. In the case of DOGS for example, it is 38 ° C in water at pH 7.5, and it is 44 ° C at a pH of 8.8. No transition is observed in pure water at acid pH: the lipid is in micellar form whatever the temperature. The phase transition temperature is also a function of the ionic strength of the medium. For example, the temperature of DOGS in a NaCl solution is 44.4 ° C at acid pH, 42 ° C at pH 7.5 and 39.8 ° C at pH 9.9. This transition temperature is of course variable from one lipid to another (see the examples). In particular, for RPR120531, the phase transition temperature as determined by DSC and turbidimetry is 51.5 ° C (powder) and 30 ° C (hydrated, pH 7.5). For RPR120535, it is around 43 ° C (hydrated, pH 7.5). Heating can be obtained in a water bath, by direct heating, or by any means which makes it possible to raise the temperature. It allows the formation of a micellar solution characterized by the clarification of the suspension, if the pH conditions allow it. The presence of micelles has been demonstrated by techniques such as turbidimetry, scattering / diffraction of X-rays at small angles and by transmission electron microscopy.

Another unexpected advantage of the process of the invention lies in the efficiency of transfection of the compositions. Thus, particularly advantageously, the compositions obtained allow the transfection of cells independently of the presence of serum. It is in fact known that serum (fetal calf serum for example) exerts an inhibitory effect on transfection with cationic lipids (in vitro, ex vivo or in vivo). The results presented in the examples show that the process of the invention can make it possible, by prior heating of the lipid, to improve the transfection efficiency in the presence of serum. Thus, the compositions according to the invention have also a good transfection efficiency in the presence and in the absence of serum. The nucleic acid can be brought into contact with the lipid solution directly after obtaining the micellar solution, and the obtained nucleolipid complex used directly for cell transfection. The advantage of such compositions lies in particular in their homogeneous, defined and reproducible nature. However, the applicant has also shown that to further improve the properties of the compositions, in particular the transfection properties, it is advantageous to proceed with a maturation of the cationic lipid. before contacting with the nucleic acid (pre-compaction maturation) and / or of the nucleolipid complex after contacting (post-compaction maturation). The maturation of the lipid / complex makes it possible to reorganize the cationic lipid in terms of its structure and in terms of its size. The Applicant has indeed shown that a lipid in micellar solution as obtained during the first step of the process of the invention can evolve to form organized bodies of different size and structure. The Applicant has also shown that the state of organization of the cationic lipid has an influence on its capacity to complex (or compact) nucleic acids, as well as on its transfection properties.

A method of the invention further comprises, between the heating step and the bringing into contact, a pre-compaction maturing step of the lipid vector.

A method of the invention further comprises, after contacting, a step of post-compaction maturation of the nucleolipid complex.

Starting from the micellar solution, it is possible, by promoting the interactions between the different lipid molecules and the supramolecular reorganization, to form different organized bodies. The examples presented below show in particular that the cationic lipids pass through unexpected organized states of the lamellar, tubular or vermicular type. When the concentration is increased, states of size more important appear, such as columnar or hexagonal states. These different structures have different properties.

For the implementation of the invention, a pre-compaction maturation step is advantageously carried out. This step is advantageously carried out until the appearance of organized aggregates of the lamellar, tubular, vermicular, columnar and / or hexagonal type. More preferably, this step is carried out until the appearance of organized aggregates of the lamellar, tubular and / or vermicular type. This type of body has an original structure, which is particularly conducive to complexation with nucleic acids.

Maturation can be carried out by any means making it possible to increase the interactions between the cationic lipid molecules, or to promote intramolecular reorganization. According to the invention, the maturation is advantageously carried out by decreasing the temperature, increasing the pH of the medium and / or increasing the ionic strength of the medium.

By lowering the temperature, the fatty aliphatic chains reorganize and make it possible to restructure the cationic lipid. The results presented in FIG. 2 show the clear evolution of the optical density of a lipid solution, reflecting the internal architecture of said lipid, as a function of the temperature of the medium. By increasing the pH or the ionic strength of the medium, it is possible to modify the state of the positive ionic charges present on the cationic part of the vector. Thus, by neutralizing (pH) or by gradually protecting (shielding, ionic strength) the charges, the Applicant has shown that the forces of electrostatic repulsion between the lipid molecules are progressively reduced, and that favored interactions and reorganization into structured aggregates. The results presented in Figures 4 and 5 show the effect of pH and ionic strength on the state of organization of the lipid. The photos presented in Figure 3 illustrate the different bodies highlighted. Advantageously, the pre-compaction maturation is carried out by reducing the temperature. Even more preferably, it is carried out by incubation of the solution at room temperature. This allows a gradual and slow maturation (aggregation and reorganization) kinetics, conducive to compaction with nucleic acids. Maturation can be continued for a variable period depending on the lipid and according to the pH and ionic strength conditions. The examples below show that this maturation can be carried out over a period ranging from a few hours to 1 month. The duration of the pre-compaction maturation is determined in particular by the appearance of structured bodies of the lamellar, tubular or vermicular type. However, if the pH and / or the ionic strength during the condensation stage are sufficiently high, and / or if a post-compaction maturation is carried out, it is advantageous not to carry out a pre-compaction maturation. . The micellar form of the lipid makes it possible in this case to obtain nucleolipid complexes which are smaller and more homogeneous in size, more effective for transfection in the presence or in the absence of serum.

Maturation can also be carried out by increasing the ionic strength of the medium. In this regard, it is advantageous to place, either during the solubilization, or during the maturation, or during the bringing into contact (compaction), in a solution of ionic strength between 0 and 0.5M. Preferably, a solution close to isotonia is used (0.15 M), that is to say an ionic strength of between 0.05 and 0.2 M. Different salts can be used (NaCl; KN03, Kl , etc). In addition, it is also possible to play on the pH during the maturation phase. It is advantageous, during maturation or during compaction, to work in a pH zone of between 3 and 9, preferably between 6 and 9. The pH is of course adjusted by the skilled person as a function of the pK lipid vectors used and the lipid phase diagram. An advantageous pH zone is that at which at least 30% of the amines of the cationic vector are deprotonated, preferably at least 40%. Generally speaking, independently of the pH and of the ionic strength, the pre-compaction maturation is carried out by decreasing the temperature below the phase transition temperature of the lipid. This decrease may be accompanied by changes in pH and ionic strength, either during maturation or during the compaction itself.

By way of illustration, under the conditions of intermediate pH and ionic strength (for example for DOGS in 150 mM NaCl at a pH <8), the following states of organization are observed:

A suspension of DOGS in 150 mM NaCl at pH 7.5 is composed: - essentially of spherical micelles, immediately after heating to approximately 50 ° C. (FIG. 3A),

- spherical micelles of small rods and vermicles of variable length, three hours after heating and cooling to room temperature (Figure 3B), - platelets of lamellar structure whose size is variable, one week after heating and cooling to room temperature ( Figure 3C),

- only large plates of lamellar structure, one month after heating and cooling to room temperature (Figure 3D).

At a native pH (about 3.5), the same suspension is composed of spherical micelles and vermicles one day after heating and cooling to room temperature.

In a DMEM medium adjusted to pH 5 a suspension of DOGS is composed: - only of spherical micelles, immediately after heating,

- spherical micelles and vermicles, 5 hours or a week after heating and cooling to room temperature.

These observations show how the maturation time can be adapted as a function of the lipid and the environmental conditions to form aggregates. organized like lamellar or vermicular. Insofar as it is now possible to know the structure adopted by the lipid as a function of the conditions of medium and time after heating, it is possible to control the lipid structure which is subsequently brought into contact with the DNA. Furthermore, the reversibility of the heating / cooling / "pre-compaction maturation" process is total. Whatever the time of maturation having been carried out, it is therefore possible to reheat the suspension to return to a micellar state common to all conditions.

After the heating step and the possible pre-compaction maturation, the lipid is brought into contact with the nucleic acid.

The nucleic acid can be DNA or RNA. It may be a linear or circular DNA, supercoiled or relaxed, of the plasmid type or not, carrying different genetic elements (coding phase, promoters, terminators, link sites, origins of replication, etc.). The nucleic acid can be of various origins (human, animal, lower eukaryote, proceryote, plant, viral, phage, etc.). It can also be a synthetic or semi-synthetic nucleic acid. The size of the nucleic acid can be very variable (from the oligonucleotide to the complete genome). The advantage of the compositions of the invention also lies in their application to the transfer of nucleic acids of any size. According to a particular embodiment, the nucleic acid is DNA (for example a plasmid or a DNA fragment) carrying an expression cassette for a particular protein or RNA. It can be a therapeutic or food-processing protein (enzyme, amino acid, etc.), with a view to producing said protein or said RNA in vitro, ex vivo or in vivo. In addition, the nucleic acid used can be a mixture of different nucleic acids, having different properties. The nucleic acid can be prepared by all the techniques known to those skilled in the art (bank screening, artificial synthesis, mixed methods, etc.). For contacting (compaction), the nucleic acid (the nucleic acid composition) is dissolved in an aqueous medium. The composition, ionic strength and pH of this medium can be adjusted to improve the efficiency of the compaction and the transfer properties of the final compositions. In particular, it has been found in studies carried out with DOGS that a compaction carried out in a medium where the lipid could be in the micellar state during its contact with DNA (that is to say in a medium of very low ionic strength and / or low pH), regardless of its state prior to the contacting step, did not allow good transfection efficiency in the presence of fetal calf serum. It has been shown that the structure of the complexes formed under these conditions of low pH and ionic strength is not of the lamellar type. Thus, advantageously, the compaction is carried out in a saline medium, in a pH zone of between 4 and 10. The optimal conditions depend on the lipid and in particular on its ability to adopt, in the presence of nucleic acid, phases lamellar type. Advantageously, the compaction is carried out in a medium with a pH of between 6 and 9. Concerning the salinity of the medium, it is advantageously between 0 and 2M, preferably between 0.01 and 0.5M, even more preferably between 0.05 and 0 , 2M. An advantageous medium for compaction is a medium close to isotonia (0.15 M), the pH of which is between 6 and 9. These conditions can be those of the lipid heating medium or those of the precompaction maturation. Under these conditions, compaction can take place without modifying the medium. If the conditions of the pre-compaction maturation are different, it is advantageous to adjust the composition of the medium as indicated above. The results presented in the examples indeed show that, under these conditions, it is possible to obtain compositions having particularly unexpected and advantageous transfection properties. In particular, it is possible to obtain compositions which are insensitive to the inhibitory effect by serum, which is particularly advantageous for vitro (SVF) or vivo uses. For contacting, the respective amounts and concentrations of nucleic acid and lipid may vary. These quantities and concentrations are those described in the prior art for the use of these vectors. In particular, the respective amounts of nucleic acid and lipid used are determined by the ratio of positive charges of the lipid and negative charges of the nucleic acid. This charge ratio can vary within a range of 0.01 to 100, and can be adapted by those skilled in the art, depending on the lipid selected, the nucleic acid selected, and depending on whether the use is of the type in vitro or in vivo. Advantageously, it is between 0.01 and 20.

The nucleolipid complex thus obtained can be used as it is for transfection. It can also be subjected to a so-called "post-compaction" maturation, making it possible to optimize the structural organization of the complex with a view to improving its transfer properties. Indeed, the states of organization of the lipids allowing a good compaction with the nucleic acid are not necessarily those giving the best levels of transfection. Thus, it is advantageous to carry out a post-compaction maturation until the appearance of organized aggregates of the lamellar, columnar and / or hexagonal type. When such aggregates are initiated during compaction, it may be advantageous to carry out post-compaction maturation to homogenize the composition. The conditions and the means of post-compaction maturation are similar to those of pre-compaction maturation. The duration of this maturation is adapted by a person skilled in the art as a function of the pre-compaction maturation, and of the lipid vector.

The compositions obtained can be used extemporaneously for the transfer of nucleic acids. They can also be stored and preserved, for example in lyophilized or frozen form, for later use. As indicated above, the compositions of the invention have numerous advantages for industrial use. They are calibrated, homogeneous, reproducible, stable and offer high transfer powers. An object of the invention resides in particular in a composition for the transfer of nucleic acids comprising a nucleic acid complexed with a cationic lipid, characterized in that the transfer efficiency is practically not altered by the presence of serum and in that it can be obtained by the process described above.

The composition preferably comprises, as cationic lipid, a lipopolyamine. Even more preferably, it is a lipothermin or a lipospermin, preferably having 2 fatty chains. Furthermore, certain adjuvants can be added to the compositions, and in particular lipid adjuvants. More preferably, the lipid adjuvants are neutral lipids having 2 fatty chains.

In a particularly advantageous manner, natural or synthetic lipids are used, zwitterionic or devoid of ionic charge under physiological conditions. They can be chosen more particularly from dioleoylphosphatidylethanolamine (DOPE), oleoyl-palmitoylphosphatidylethanolamine (POPE), di-stearoyl, -palmitoyl, -mirystoyl phosphatidylethanolamine as well as their N-methyl derivative 1 to 3 times; phosphatidylglycerols, diacylglycerols, glycosyldiacylglycerols, cerebrosides (such as in particular galactocerebrosides), sphingolipids (such as in particular sphingomyelins), cholesterol or also asialogangliosides (such as in particular asialoGMI and GM2).

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

Preferably, the compositions of the invention comprise, in addition to the cationic lipid, from 0.1 to 20 molar equivalents of neutral lipid per 1 molar equivalent of phosphate of the nucleic acid, and, more preferably, from 1 to 5. In a particularly advantageous embodiment, the compositions of the present invention comprise a targeting element making it possible to direct the transfer of the nucleic acid. This targeting element can be an extracellular targeting element, making it possible to direct the transfer of nucleic acid to certain cell types or certain desired tissues (tumor cells, hepatic cells, hematopoietic cells, etc.). It can also be an intracellular targeting element, making it possible to direct the transfer of the nucleic acid towards certain privileged cellular compartments (mitochondria, nucleus, etc.). More preferably, the targeting element is linked, covalently or non-covalently, to the lipid. The bond can in particular be obtained by ionic interaction with ammoniums, or by nucleophilic attack of the amines of the vector on targeting elements comprising a nucleofuge group (halogen, tosylate, etc.), an activated ester (hydroxysuccinimide, etc.) or an isothiocyanate . The targeting element can also be linked to the nucleic acid.

Among the targeting elements which can be used in the context of the invention, there may be mentioned sugars, peptides, oligonucleotides or lipids. Preferably, these are sugars and / or peptides such as antibodies or antibody fragments, ligands of cellular receptors or fragments thereof, receptors or fragments of receptors, etc. In particular, they may be ligands for growth factor receptors, cytokine receptors, cellular lectin receptors or adhesion protein receptors. Mention may also be made of the receptor for transferin, HDL and LDL. The targeting element can also be a sugar making it possible to target the asialoglycoprotein receptors, or alternatively a Fab fragment of antibodies making it possible to target the receptor for the Fc fragment of the immunoglobulins.

The transfecting compositions described can be used for the transfer of nucleic acids in vitro, ex vivo or in vivo. For in vivo applications, they can be formulated for administration topically, skin, oral, rectal, vaginal, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal, etc. Preferably, they contain a pharmaceutically acceptable vehicle for an injectable formulation, in particular for a direct injection into the desired organ, or for topical administration (on the skin and / or mucosa). They may in particular be sterile, isotonic solutions, or dry compositions, in particular lyophilized, which, by addition as appropriate of sterilized water or physiological saline, allow the constitution of injectable solutes. The doses of nucleic acid used for the injection as well as the number of administrations can be adapted according to different parameters, and in particular according to the mode of administration used, the pathology concerned, the gene to be expressed, or even the duration of the treatment sought.

In this regard, another subject of the invention relates to a process for the transfer of nucleic acid into cells in vitro, in vivo or ex vivo comprising bringing said nucleic acid into contact with a suspension of cationic lipid previously heated, and incubating the cells with the resulting nucleolipid complex.

Advantageously, the cationic lipid suspension is previously heated and matured. Still according to a preferred mode, the nucleolipid complex is mature prior to incubation with the cells. Incubation with cells can be done in vitro, on an appropriate support (culture dish, pouch, flask, etc.) possibly under sterile conditions. The quantities of DNA incubated are known to a person skilled in the art (of the order of micrograms for 10 ⁶ cells). For vivo use, the incubation can be carried out by administration of the composition in vivo (local or systemic for example).

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

Figures 1: Chemical structures of cationic lipids. 1A: DOGS, 1 B: RPR 120531, 1C: RPR 120535.

Figure 2: Influence of temperature on the optical density at 400 nm of a suspension of DOGS at 1 mM in NaCl.

Figures 3: Cryo-microscopy images in transmission of a suspension of DOGS at 2 mM in NaCl at pH 7.5. The sample preparation was carried out immediately after heating (3A), 3 hours (3B), 1 week (3C) and 1 month (3D) after heating the suspension and cooling to room temperature.

Figure 4: Influence of pH on the optical density at 400 nm of a suspension of DOGS at 0.6 mM in water or in NaCl at 150 mM. Study temperature: 50 ° C. Figures 5: Phase diagrams of DOGS (5A: water; 5B: 150 mM NaCI), RPR120531 (5C: water; 5D: 150 mM NaCI) and RPR120535 (5E: water; 5F: 150 mM NaCI).

Figure 6: Transfecting power of the compositions of Example 5 Figure 7: Transfecting power of the compositions of Example 6 Figure 8: Transfecting power of the compositions of Example 7 Figure 9: Transfecting power of the compositions of Example 8 Figure 10 : Transfecting power of the compositions of Example 9 Figure 11: Transfecting power of the compositions of Example 10 Figure 12: Transfecting power of the compositions of Example 11

MATERIAL AND METHODS

1. The plasmid used in the following examples (pXL2784) is a ColE1 derivative carrying the Kanamycin resistance gene, and the cer fragment of CoIEL The eukaryotic expression cassette contains the CMV promoter of the plasmid pCDNA3 controlling the gene coding for luciferase (Photinu Pyralis). It is understood that any other nucleic acid can be used. 2. The cationic lipids used in the following examples are DOGS, RPR120531 and RPR120535, the structure of which is shown in FIG. 1. It is understood that the same experiments can be carried out with other cationic lipids.

3. Transfection protocol. The process described applies to in vitro, ex vivo and in vivo transfections.

The cells used are murine NIH 3T3 fibroblasts, seeded the previous day in 24-well plates, at a density of 50,000 cells per well. The culture medium used is DMEM medium (Dulbecco's Modified Eagle Medium), containing 4.5 g / 1 of glucose, supplemented with 10% fetal calf serum, 1% L-glutamine (200 mM mother solution) , 1% sodium pyruvate, streptomycin (5000 IU / ml) and penicillin (5000 μg / ml) (Gibco). Prior to transfection, the cells are rinsed twice with DMEM not containing fetal calf serum.

The cells are transfected with 50 μl of a transfection suspension containing 0.5 μg of DNA and 3 nmoles of DOGS per well, in

200 μl of culture medium, with or without the addition of 10% fetal calf serum. After a 4 hour incubation at 37 ° C (in a CO2 incubator), the culture medium containing the nucleolipid complex is removed and replaced with 500 μl of DMEM supplemented with 10% fetal calf serum. The cells are then incubated for 48 hours.

4. Measurement of the luciferase activity of eukaryotic cells.

It is performed 48 hours after transfection. Luciferase catalyzes the oxidation of luciferin, in the presence of ATP, Mg2 + and O2, with concomitant production of a photon. The total light emission, measured by a luminometer, is proportional to the luciferase activity of the sample.

The reagents used are supplied by Promega (Luciferase assay System) and used according to a recommended protocol. After double rinsing the cells with PBS, the cells are lysed with 250 μl of lysis buffer and the insoluble fraction of each extract is removed by centrifugation. The assay is carried out on 5 μl of supernatant, diluted or not in the cell lysis buffer.

EXAMPLES

Example 1: Influence of temperature on the molecular organization of cationic lipids.

A colloidal solution of DOGS at 1 mM was prepared by dissolving the DOGS crystallized in chloroform, evaporation of the solvent and drying in a lyophilizer. The film was then taken up in a 0.9% NaCl solution (buffer 10 mM Hepes, pH 7.4). After heating to 50 ° C, the colloidal solution was placed at 4 ° C for one week. The optical density is continuously monitored (Lambda 2 spectrophotometer (Perkin Elmer) with thermostat-controlled cell holder), while the bath temperature (LAUDA RM6), itself connected to the spectrophotometer cells, varies at a determined and controlled speed. 1.25 ° C / min. The tank containing the sample is equipped with a stirring system.

The results obtained are shown in Figure 2. They show a sudden drop in optical density around 40 ° C approximately. This drop in DO reflects a deep rearrangement of the lipid structure.

At the same time, the physical state of DOGS during these temperature variations was studied by transmission cryomicroscopy. For this, a suspension of DOGS at 2 mM in NaCl at pH 7.5 was heated to 50 °. The physical nature of the objects was determined after heating over time, during maturation by incubation at room temperature. The results obtained are presented in FIG. 3: The preparation of the sample was carried out immediately after heating (3A), 3 hours (3B), 1 week (3C) and 1 month (3D) after heating the suspension and cooling at room temperature. The results obtained show an evolution of the structure of DOGS, from the micellar stage to the formation of lamellar bodies, passing through rod and vermicle structures. This example makes it possible to define pre-compaction maturation conditions for the lipid.

Example 2 Influence of pH on the Molecular Organization of Cationic Lipids

A colloidal solution of DOGS at 1 mM was prepared by dissolving the DOGS crystallized in chloroform, evaporation of the solvent and drying in a lyophilizer. The film was then taken up in a 150 mM NaCl solution or in water, in a pH range from 3.0 to 11.0. After heating to 50 ° C, the suspensions are placed at room temperature (25 ° C) at least 3 hours before the start of the measurement. The optical density is determined under the conditions described in Example 1.

The results obtained are presented in FIG. 4. They very clearly show an increase in the optical density as a function of the pH. This increase reflects an increase in interactions between lipid molecules, by a decrease in electrostatic repulsions.

Example 3 Phase Diagrams of Cationic Lipids

To validate the observations made in Examples 1 and 2, phase diagrams were drawn for different lipid vectors. These diagrams describe the physical state of the lipids according to the conditions of pH, ionic strength and temperature. They also highlight the phase transition temperatures and the aggregative behavior of the lipid as a function of its ionization state.

These diagrams are presented in FIGS. 5A and 5B for DOGS, 5C and 5D for RPR 120531 and 5E and 5F for RPR 120535. They show that, in the case where the lipid is solubilized in pure water (pH <6) the micellar solution obtained by heating according to the invention is stable. They allow also to define the ionic strength and pH conditions for which the micellar solution is stable over time.

Example 4: Study of different counterions in the background electrolyte.

Different counterions were tested in the maturation step. For this, different suspensions of DOGS in a medium whose background electrolyte is present at a concentration of 0.1 M and whose pH is not adjusted (native pH: 3.5) were observed one week after heating and cooling at room temperature:

- In KN03, the nanometric structures are of the micellar type and we observe some rare marked edges. Submicron structures are fine filaments with a strong aggregative tendency.

- In Kl, the nanometric structures appear under the aspect of superposition of lamellae whose edges appear very marked. The submicron structures also appear in the form of filaments but whose thickness is greater and with less strong aggregative tendency.

Example 5

A. Complex preparation and transfection protocol:

The lipid (DOGS) is presented to the DNA in the form of a 2 mM suspension in 150 mM NaCl at native pH (3.5) heated and left at room temperature 1 day before contact with the DNA (" 1 day pre-maturation "). The lipid is introduced into a suspension containing the plasmid pXL2784 and composed of 150 mM NaCl adjusted to a pH of 5 - 6.6 or 8.2 (these pHs are measured values). The "post-compaction maturation" is 0 or 3 hours at room temperature and the transfection was carried out in the presence (SVF) and in the absence of fetal calf serum.

B. Results:

The transfection results expressed in RLU (Relative Light Unit) for 5 μl of lysate, ie 1 / 50th of a well, are presented in Figure 6. Each value is a measurement average over 4 wells. Standard deviations are represented by horizontal bars.

This study shows that starting from a solution of vermicular micelles, a compaction in a saline medium of high pH (here 8.2) makes it possible to get rid of the inhibitory effect of the serum, whatever the "maturation of post-compaction ". Furthermore, when an intermediate compaction pH (still in a saline medium) is used, "post-compaction maturation" also makes it possible to overcome the inhibitory effect on the transfection of serum.

A similar study was carried out on the lipids RPR 120531 and RPR 120535 (see FIG. 1 for the structures of DOGS, RPR 120531 and RPR 120535). It shows a behavior similar to that of DOGS relative to the influence of the compaction pH and "post-compaction maturation" on the release of the serum effect.

Example 6

A. Protocol for the preparation of the complex and transfection: The lipid (DOGS) is presented to the DNA either in the form of a suspension at 2 mM in 150 mM NaCl at native pH (3.5) heated and left at temperature. ambient 1 day before contact with DNA ("1 day pre-maturation") or in the form of a 2mM solution in ethanol (EtOH). The lipid is introduced into a suspension containing the plasmid pXL2784 and composed of 150 mM NaCl adjusted to a pH of 4.2 - 6.2 - 7.2 or 8.2 (these pHs are measured values) or also in DMEM culture medium. No "post-compaction maturation" is carried out except in the case where the compaction is carried out at a pH of 6.2 (pH 6.2 + mat.). In this case, the "post compaction maturation" is 3 hours at room temperature. The transfection was carried out in the presence (SVF) and in the absence of fetal calf serum (SS SVF). B. Results:

The transfection results expressed in RLU (Relative Light Unit) for 5 μl of lysate, ie 1 / 50th of a well, are shown in Figure 7. Each value is a measurement average over 4 wells. Standard deviations are represented by horizontal bars.

This study shows that the behavior of the compacted DNA particles with respect to the inhibition of fetal calf serum seen in Example 5 (as a function of the compaction pH and of the "post-compaction maturation") is also valid. that DOGS is in the form of a suspension in a saline medium or in the form of an ethanolic solution. It also shows that the "post-compaction maturation" stage makes it possible to overcome inhibition by serum.

Example 7

A. Protocol for the preparation of the complex and transfection: The lipid (DOGS) is presented to the DNA in the form of a suspension at 2 mM in 150 mM NaCl at native pH (3.5) immediately heated (NM: for

Not pre-Matured) before contact with DNA or "pre-matured" 1 day before compaction (U). The lipid is introduced into a suspension containing the plasmid PXL2784 and composed of 150 mM NaCl adjusted to a pH of 6.2 (this pH is a measured value). The "post-compaction maturation" is 0 -

0.5 hour - 3 hours - 1 day when the lipid has not been "pre-matured", it is 3 hours when the lipid has been "pre-matured 1 day" at room temperature (control). The transfection was carried out in the presence and in the absence of fetal calf serum. The results obtained in the absence of fetal calf serum not being significantly different according to the different protocols studied, only the results of the transfections carried out in the presence of fetal calf serum are shown. B. Results:

The transfection results expressed in RLU (Relative Light Unit) for 5 μl of lysate, ie 1 / 50th of a well, are shown in Figure 8. Each value is a measurement average over 4 wells. Standard deviations are represented by horizontal bars.

This study shows that an absence of "pre-maturation" of DOGS (heating immediately before contacting with DNA) obliges to increase the time of "post-compaction maturation" necessary to overcome the inhibitory effect of serum. The structure adopted by the lipid before it is brought into contact with DNA therefore seems to accelerate the organization of the lipid-DNA complex.

It also seems that a compaction carried out with a non-premature lipid but followed by a sufficient "post-compaction maturation" time (IM - 1 D), allows the formation of more effective particles on transfection than if the lipid was premature (1J - 3H), perhaps because the particles formed are smaller and more regular in size.

Example 8

A. Complex preparation and transfection protocol:

The lipid (DOGS) is presented to the DNA in the form of a suspension at 2 mM either in water at pH 7.5 and "pre-matured" 1 day before compaction (water 7.5), or under the form of a suspension in 150 mM NaCl at pH 7.5 also "pre-matured" for 1 day (NaCl 7.5). The lipid is introduced into a suspension containing the plasmid pXL2784 and composed of 150 mM NaCl adjusted to a pH of 7.2 (this pH is a measured value). The "post-compaction maturation" is 0 (0) or 3 hours (3H). The transfection was carried out in the presence of fetal calf serum.

B. Results:

The transfection results expressed in RLU (Relative Light Unit) for 5 μl of lysate, ie 1 / 50th of a well, are presented in Figure 9. Each value is a measurement average over 4 wells. Standard deviations are represented by horizontal bars.

These results show that it is possible to overcome the inhibitory effect of the serum as well as the "post-compaction maturation" by allowing the lipid before compaction to be more structured: the higher pH and ionic strength conditions in the case of NaCI 7.5 are in fact favorable to a certain type of organization. This study also shows that, when the "post-compaction maturation" takes place, it seems preferable for good transfection efficiency to compact the DNA with a lipid whose particle size is not too large (water 7.5) . For good transfection efficiency in the presence of serum, it therefore seems necessary to make a compromise between lipid structure and particle size.

Example 9

A. Protocol for preparation of the complex and transfection: The lipid (DOGS) is presented to the DNA in the form of a suspension at 2 mM in 150 mM NaCl at native pH (3.5) and "pre-matured" 1 day before compaction (1 day) or 4 days before compaction (4 days). The lipid is introduced into a suspension containing the plasmid pXL2784 and composed of 150 mM NaCl adjusted to a pH of 6.6 (this pH is a measured value). The "post-compaction maturation" is 0 (0) or 3 hours (3H). The transfection was carried out in the presence (SVF) and in the absence of fetal calf serum.

B. Results:

The transfection results expressed in RLU (Relative Light Unit) for 5 μl of lysate, ie 1 / 50th of a well, are presented in Figure 10. Each value is a mean of measurement over 4 wells. Standard deviations are represented by horizontal bars.

This study does not show any significant difference between the different protocols when the transfection is carried out in the absence of fetal calf serum. On the other hand, in the presence of serum, it is found that a "pre- "long maturation (4 days) can partially overcome the serum inhibitory effect when a" post-compaction maturation "has not been carried out. This study also shows that when "post-compaction maturation", additional "pre-maturation" proves useless but not harmful, at least in the case of pre-maturation in 150 mM NaCl at native pH, and for the "pre-maturation" times -maturation "described above.

Example 10

A. Protocol for the preparation of the complex and transfection: The lipid (DOGS) is presented to the DNA in the form of a suspension at 2 mM in 150 mM NaCl at pH 7.5 and either heated immediately (IM) or "pre-matured" 1 day (U), or "pre-matured" 1 month (1 M) before compaction. The lipid is introduced into a suspension containing the plasmid pXL2784 and composed of 150 mM NaCl adjusted to a pH of 7.2 (this pH is a measured value). The "post-compaction maturation" is 0 (0) or 3 hours (3H). The transfection was carried out in the presence of fetal calf serum.

B. Results:

The transfection results expressed in RLU (Relative Light Unit) for 5 μl of lysate, ie 1 / 50th of a well, are shown in Figure 11. Each value is a measurement average over 4 wells. Standard deviations are represented by horizontal bars.

This study shows that a "pre-maturation" overcomes in part the inhibitory effect of the serum on the transfection when a "post-compaction maturation" is not carried out. On the other hand, when maturation of the nucleolipid complex is undertaken, it is found that the transfection efficiency obtained is better when the lipid has been heated immediately before it is brought into contact with the DNA and therefore it is in the form of small lipid particles. Example 11

A. Protocol for the preparation of the complex and transfection: The lipid (DOGS) is presented to DNA in the form of a micellar solution at 2 mM in pure water (native pH 3.5). The lipid is introduced into a suspension containing the plasmid pXL2784 and composed of 150 mM NaCl adjusted to a pH of 7.3 (this pH is a measured value). The "post-compaction maturation" is 0 (0) - 0.5 hour (0.5H) - 3 hours (3H) - 1 day (U) or 1 week (1S). The transfection was carried out in the presence of fetal calf serum.

B. Results:

The transfection results expressed in RLU (Relative Light Unit) for 5 μl of lysate, ie 1 / 50th of a well, are presented in Figure 12. Each value is a measurement average over 4 wells. Standard deviations are represented by horizontal bars.

This study confirms that when DOGS is presented to DNA in the form of a micellar solution, "post-compaction maturation" becomes necessary to overcome the inhibitory effect of serum. It is noted in the present case that a "post-compaction" maturation of 3 hours is sufficient.

All of these results show that the process of the invention, involving a prior heating step and advantageous maturation steps, makes it possible to obtain defined, homogeneous, reproducible compositions, the transfecting properties of which are improved. BIBLIOGRAPHY

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Claims

1. A process for preparing a composition for the transfer of nucleic acids comprising bringing a nucleic acid into contact with a cationic lipid, characterized in that, before contacting, the cationic lipid is subjected to a heating stage.

2. Method according to claim 1 characterized in that the cationic lipid is heated until the formation of a micellar solution.

3. Method according to claim 2 characterized in that the cationic lipid is heated to a temperature above its phase transition temperature.

4. Method according to claim 1 characterized in that it further comprises, between the heating step and the contacting step, a pre-compaction maturation step of the cationic lipid.

5. Method according to claim 1 or 4 characterized in that it further comprises, after the contacting step, a step of post-compaction maturation of the nucleolipid complex.

6. Method according to claim 1 or 4 characterized in that the contacting is carried out at pH between 4 and 10, preferably between 6 and 9.

7. Method according to claim 1 or 4 characterized in that the contacting is carried out at an ionic strength of between 0 and 2M, preferably 0.01 and 0.5M, preferably between 0.05 and 0.2M.

8. Method according to claim 4 characterized in that the pre-compaction maturation is carried out by cooling, modification of the ionic strength of the medium and / or modification of the pH of the medium.

9. Method according to claim 8 characterized in that the pre-compaction maturation is carried out by incubation at room temperature.

10. The method of claim 9 characterized in that the pre-compaction maturation is carried out by incubation at room temperature for a period between 0.5 hour and 1 month.

11. The method of claim 8 characterized in that the pre-compaction maturation is carried out until the formation of organized aggregates of vermicular, tubular, lamellar, hexagonal and / or columnar type.

12. Method according to claim 11 characterized in that the pre-compaction maturation is carried out until the appearance of organized aggregates of vermicular and / or tubular type.

13. Method according to claim 5 characterized in that the post-compaction maturation is carried out by cooling.

14. The method of claim 13 characterized in that the cooling is carried out by incubation at room temperature.

15. The method of claim 13 characterized in that the post-compaction maturation is carried out until the formation of organized aggregates of hexagonal type, lamellar and / or columnar, or larger.

16. Method according to any one of the preceding claims, characterized in that the cationic lipid is chosen from lipopolyamines.

17. The method of claim 16 characterized in that the cationic lipid is a lipospermin or a lipothermin.

18. The method of claim 1 characterized in that the nucleic acid is DNA.

19 The method of claim 18 characterized in that the DNA is a plasmid, vector, or linear fragment.

20. The method of claim 1 characterized in that the nucleic acid is an RNA.

21. A method for the transfer of nucleic acid into cells in vitro or ex vivo comprising bringing said nucleic acid into contact with a suspension of cationic lipid previously heated, and incubating the cells with the resulting nucleolipid complex.

22. The method as claimed in claim 21, characterized in that it comprises bringing the nucleic acid into contact with a suspension of cationic lipid previously heated and matured.

23. The method according to claim 21 or 22, characterized in that the nucleolipid complex is mature before incubation with the cells.

24. Composition for the transfer of nucleic acids, characterized in that it is essentially insensitive to the inhibitory effect by serum and in that it can be obtained by the method according to claim 1.

25. Composition according to claim 24 characterized in that it further comprises one or more lipid adjuvants.

26. The method of claim 1 characterized in that the cationic lipid further comprises lipid adjuvants.