WO2001049869A1

WO2001049869A1 - Compositions comprising nucleic acids incorporated in bilaminar mineral particles

Info

Publication number: WO2001049869A1
Application number: PCT/FR2000/003702
Authority: WO
Inventors: Bruno Pitard
Original assignee: Aventis Pharma S.A.
Priority date: 1999-12-30
Filing date: 2000-12-27
Publication date: 2001-07-12
Also published as: EP1246931A1; JP2003519241A; AU2860401A; IL150476A0; MXPA02006528A; CA2395742A1

Abstract

The invention concerns compositions comprising at least a nucleic acid and a mineral particle having an interchangeable foliate structure, the method for preparing them and their uses for in vivo, in vitro and/or ex vivo nucleic acid transfection. Said mineral particles preferably correspond to general formula (I): [M<II>1-xM<III>x(OH)2]<x+>[X<m->x/m nH2O]<x->.

Description

COMPOSITIONS COMPRISING NUCLEIC ACIDS INCORPORATED IN BILAMELLAR MINERAL PARTICLES

The present invention relates to compositions comprising at least one nucleic acid and a mineral particle having a structure with exchangeable sheets, their preparation process and their applications for the in vivo, in vitro and / or ex vivo transfection of nucleic acids .

With the development of biotechnologies, the possibility of efficiently transferring nucleic acids into cells (that is to say of introducing them into cells so that the gene of interest which they carry is expressed there) is 0 has become a basic technique with many biotechnological applications. It may be the transfer of nucleic acids into cells in vitro, for example for the production of recombinant proteins, or in the laboratory for the study of the regulation of gene expression, the cloning of genes or any other manipulation involving DNA. It may also involve the transfer of nucleic acids into cells in vivo, for example for the production of vaccines, labeling studies or also therapeutic approaches. It may also involve the transfer of genes ex vivo into cells taken from an organism, with a view to their subsequent re-administration, for example for the creation of transgenic animals.

To date, there are many methods for transferring 0 nucleic acids into cells: calcium phosphate precipitation, electroporation, micro injection, viral infection, injection of naked DNA, or even use of non-viral vectors, such as, for example, cationic polymers, biochemical vectors (consisting of a cationic protein associated with a cellular receptor), or else lipofectants, and more particularly cationic lipids.

However, these techniques developed to date have numerous drawbacks which limit the efficiency of the transfection and do not make it possible to satisfactorily resolve the difficulties associated with the transfer of genes into cells and / or the organism. Thus, calcium phosphate precipitation is a a method that is quite ineffective, particularly compared to the injection of naked DNA, and has therefore been abandoned for a long time. If it has been shown that the injection of “naked”, that is to say unformulated, nucleic acids makes it possible to obtain acceptable levels of transfection in certain cell types in vivo (see in particular the request for WO90 / 11092), naked nucleic acids, however, only enjoy a short plasma half-life, due to their degradation by enzymes and their elimination via the urinary tract. The level of efficiency of gene transfer by injection of naked nucleic acid remains too low for most of the applications envisaged. The use of polymers or cationic lipids thus constitutes a possible remedy vis-à-vis the enzymatic degradations which the DNA undergoes to be transfected, but it has often been observed that their use more or less strongly inhibits the efficiency of transfection in muscle and they also induce toxicity towards cells. Although recombinant viruses make it possible to obtain good nucleic acid transfer efficiency, their use nevertheless presents certain risks linked to their viral nature such as pathogenicity, transmission, replication, recombination, transformation, immunogenicity, etc. .. It is therefore preferable to avoid viral transfection techniques. Finally, techniques such as electroporation constitute an interesting but limited alternative to local administration. They often cause irreversible damage to cell membranes. It therefore appears that it would be desirable to have a transfer vector which makes it possible both to protect the nucleic acids from enzymatic degradations and to obtain satisfactory transfer efficiency in the cells, without however damaging them or inducing toxicity.

The present invention provides an advantageous solution to these problems. The Applicant has indeed shown that the compositions comprising a nucleic acid and a mineral particle having a structure with exchangeable sheets allow the transfer in vitro, ex vivo or in vivo of said nucleic acid into a cell and / or an organ with an efficiency greater than those obtained to date with conventional non-viral transfer techniques. The compositions of the invention further allow avoid the drawbacks associated with the use of viral vectors or physical transfection techniques.

Thus, a first object of the present invention relates to the compositions comprising at least one nucleic acid and one mineral particle having a structure with exchangeable sheets.

Within the meaning of the invention, the term “mineral particles having a structure with exchangeable sheets” means mineral particles whose structure is based on a stack of sheets of composition M (OH) ₂ similar to those well known in brucite (Mg (OH) ₂ ), M representing a divalent metal, but in which part of the divalent metals is replaced by trivalent metals so as to make the sheets generally cationic. The inter-sheet spaces include anionic entities solvated by water molecules. Such a structure is shown in Figure 1 and is also called "double lamellar hydroxide". These particles have the general formula:

in which M ^u represents a divalent metal cation, M ¹¹¹ represents a trivalent metal cation, X ^{m ~} represents an interfoliar anion and m is an integer greater than or equal to 1, x is between 0 and 1 strictly, and n is strictly greater at 0.

Such mineral particles have already been described for applications in many fields such as catalysis or the environment. Indeed, they form nanostructured materials where molecular or colloidal species are interposed in a lamellar host structure. The properties of these structures can thus be used in heterogeneous or homogeneous catalysis on a support, in exchange and separation techniques, in particular optical isomers, for the design of membranes which may be selective for filtration and permeation, for trapping and controlled restitution of molecules, or for the design of electro-active materials and deposits, electrodes and electronic devices. Thus the article by Choy et al (1999, J. Am. Chem. Soc. 121 (6), 1399-1400) describes the preparation of hybrid nanoparticles composed of sheets of a Double Lamellar Hydroxide (HDL), pristine ( Mg ₂ Al (NO ₃ ) -LDH), between which are inserted monophoshated nucleosides or DNA fragments of herring testes. The nucleic molecules intercalated in the particles described in this article have a size less than 1000 base pairs. No use of these molecules is described.

However, such mineral particles had never been used for the transfer of nucleic acids and there was no indication that it would be possible to obtain a very marked improvement in the transfer efficiency, in particular with respect to the results of transfection obtained by injection of naked DNA or formulated with conventional synthetic vectors such as cationic lipids.

The mineral particles having a structure with exchangeable sheets usable within the framework of the present invention are often named after the nature of the major metal cations of the sheets: for example hydrotalcite (Mg-Al), pyroaurite (Mg-Fe) , stichtite (Mg-Cr) or takovite (Ni-Al). The general formula indicated above underpins the wide variety of mineral particles which it is possible to prepare by varying the nature of the two metal cations M ¹¹ and M ^πι , their respective proportions with possible extension to more than two types of cations (eg Mg, Zn and Al), the nature of the interfoliar anions, or the state of hydration. In addition, the richness of the general formula indicated above is further increased by a structural diversity resulting from the existence of polytypes differing by the sequence of stacking of the sheets as well as from the appearance of over-structures due for example to a classification cationic in the hydroxylated sheet.

The divalent metal cations M ^π can be chosen for example from magnesium (Mg), chromium (Cr), manganese (Mn), iron (Fe), nickel (Ni), cobalt (Co), copper (Cu), calcium (Ca) or even zinc (Zn). Preferably, the divalent metal cation is magnesium. Trivalent metal cations M ^πι may be chosen for example from aluminum (Al), chromium (Cr), manganese (Mn), iron (Fe), nickel (Ni), cobalt (Co) or even gallium (Ga). Preferably, the trivalent metal cation is aluminum. In addition, the two members of the couple M ^π and M can also correspond to the same element (for example Fe ^π / Fe ^ffl or Mn ^π / Mn ^ιπ ).

The interfoliar anions X ^″ may for example be chosen from halides, oxoanions, iso- and heteroanions, complex anions or even organic anions. Examples include fluorides, chlorides, bromides, carbonates , nitrates, sulfates or tetrahedral oxoanions such as CrO ^{2 "} . Preferably, the anion is chosen from carbonates

∞ ₃ ² -.

The value of x is strictly between 0 and 1, that is, 0 and 1 are excluded values. Preferably, x is between 0.05 and 0.95, and preferably between 0.1 and 0.9. Even more preferably, x is between 0.2 and 0.85. For example, x can be equal to 0.25. As regards the value of n, this indicates the state of hydration of the particle in question and it is strictly greater than 0. Preferably, n is greater than or equal to 0.1. For example, n can be equal to 0.5. m is an integer greater than or equal to 1 which represents the charge of the interfoliar anion. For example, m can be 1, 2, 3, 4, 5 or 6.

Mineral particles having a structure with exchangeable sheets according to the present invention can be chosen for example from the hydrotalcite of formula Mg ₆ Al ₂ CO ₃ (OH) ι ₆ .4H ₂ O, the manasseite of formula Mg ₆ Al ₂ CO ₃ (OH) ι ₆ .4H ₂ O, the pyroaurite of formula Mg ₆ Fe ₂ CO ₃ (OH) ι ₆ .4H ₂ θ, the stichtite of formula Mg ₆ Cr ₂ CO ₃ (OH) ι ₆ .4H ₂ O, the takovite of formula Ni ₆ Al ₂ CO ₃ (OH) ι ₆ .4H ₂ 0, the reevesite of formula Ni ₆ Fe ₂ CO ₃ (OH) ι ₆ .4H ₂ θ, or the comblainite of formula Ni ₆ Cθ2CO ₃ (OH) ₁₆ .4H ₂ θ. Other mineral particles having a structure with exchangeable sheets may also be suitable. Preferably, the compositions according to the present invention comprise at least one nucleic acid and hydrotalcite of formula Mg ₆ Al ₂ CO ₃ (OH) ι ₆ .4H ₂ O.

The mineral particles having a structure with exchangeable sheets according to the present invention are either commercial, or they can be prepared according to methods which are similar to known methods known as “soft chemistry”. In particular, the preparation of the mineral particles according to the invention can be based on the controlled precipitation of aqueous solutions or suspensions containing both the metal cations intended to take place in the hydroxylated framework and the anions intended to occupy the interlamellar domains. Under these conditions, there is simultaneously construction of the interlamellar domains consisting of anions and water molecules, and of the sheet. The nature and texture of the phases obtained are highly dependent on the operating conditions (temperature, rate of addition and concentration of the reagents, pH control), but also on the geometry of the reactor and the state of prior association of the metal cations. in reagents (existence of oligomers). The preparation process ultimately always boils down to bringing together, at an appropriate concentration and reactivity, the species leading to the construction of the mineral particles under optimal conditions. These optimal conditions are determined on a case-by-case basis according to the conventional method of trial and error.

For the purposes of the invention, the term “nucleic acid” is understood to mean both a deoxyribonucleic acid and a ribonucleic acid. They can 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 semi-synthetic sequences, of modified or unmodified oligonucleotides. These nucleic acids can be, for example, of human, animal, plant, bacterial or viral origin. They can be obtained by any technique known to a person skilled in the art, and in particular by screening of banks, by chemical synthesis, or even by mixed methods including chemical or enzymatic modification of sequences obtained by screening of libraries. They can be chemically modified.

As regards more particularly deoxyribonucleic acids, they can be single or double stranded or else short oligonucleotides or longer sequences. In particular, the nucleic acids advantageously consist for example of plasmids, vectors, episomes or expression cassettes. These deoxyribonucleic acids may in particular carry an origin of functional or non-functional replication in the target cell, one or more marker genes, transcription or replication regulatory sequences, genes of therapeutic interest, antisense sequences which may or may not be modified or even regions of binding to other cellular components.

Preferably, said nucleic acid has a size greater than 1000 base pairs. Even more preferably, it has a size of between approximately 1000 base pairs and approximately 2,000,000 base pairs (2 Mb), approximately 1,000 base pairs and approximately 100,000 base pairs (100 kb), and approximately 1,000 base pairs and approximately 20,000 base pairs (20 kb).

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

For the purposes of the invention, the term “gene of therapeutic interest” in particular means any gene coding for a protein product having a therapeutic effect. The protein product thus coded can in particular be a protein or a peptide. This protein product can be exogenous homologous or endogenous with respect to the target cell, that is to say a product which is normally expressed in the target cell when the latter presents no pathology. In this case, the expression of a protein makes it possible for example to compensate for an insufficient expression in the cell or the expression of an inactive or weakly active protein due to a modification, or else to overexpress said protein. The gene of therapeutic interest can also code for a mutant of a cellular protein having, for example, increased stability or modified activity. The protein product can also be heterologous towards the target cell. In this case, an expressed protein can, for example, supplement or bring about a deficient activity in the cell, allowing it to fight against a pathology, or stimulate an immune response.

Among the therapeutic products within the meaning of the present invention, there may be mentioned more particularly enzymes, blood derivatives, hormones, lymphokines (for example interleukins, interferons or TNF: see FR 92/03120), growth, neurotransmitters or their synthetic precursors or enzymes, trophic factors (for example BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, VEGF, NT3, NT5 or HARP / pleiotrophin), apolipoproteins (for example ApoAI, ApoAIV or ApoE: see FR 93/05125), dystrophin or a minidystrophin (FR 91/11947), the CFTR protein associated with cystic fibrosis, tumor suppressor genes (for example p53, Rb, RaplA, DCC or k-rev : see FR 93/04745), the genes coding for factors involved in coagulation (Factors VII, VIII, IX), the genes involved in DNA repair, the suicide genes (thymidine kinase, cytosine deaminase), hemoglobin or other transp genes protein speakers, enzymes of metabolism or catabolism.

The nucleic acid of therapeutic interest can also be an antisense gene or sequence, the expression of which 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 complementary to cellular mRNAs and thus block their translation into protein, according to the technique described in patent EP 140 308. The therapeutic genes also include the sequences coding for ribozymes, which are capable of selectively destroying target RNAs (EP 321,201). As indicated above, the nucleic acid can also contain one or more genes coding for an antigenic peptide, capable of generating in humans or animals an immune response. In this particular mode of implementation, the invention allows the production of either vaccines or immunotherapeutic treatments applied to humans or animals, in particular against microorganisms, viruses or cancers. These may in particular be antigenic peptides specific for the Epstein Barr virus, the HIV virus, the hepatitis B virus (EP 185 573), the pseudo-rabies virus, the "syncitia forming virus", d other viruses or tumor-specific antigenic peptides (EP 259,212).

As indicated above, the nucleic acid preferably also comprises sequences allowing the expression of the gene of therapeutic interest and / or of the gene coding for the antigenic peptide in the desired cell or organ. These 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. It can 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 originating from the genome of the cell which it is desired to infect. Likewise, they may be promoter sequences originating from the genome of a virus. In this regard, mention may be made, for example, of promoters of the El A, MLP, CMV, RSV or HSV genes. In addition, these expression sequences can be modified, for example, by adding activation or regulation sequences. They can also be inducible or repressible promoters.

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

The compositions according to the present invention can be prepared by mixing an aqueous solution containing a mineral particle having a structure with exchangeable sheets and a solution containing the nucleic acids. More specifically, the compositions according to the present invention can be prepared by bringing the mineral particles having a structure with exchangeable sheets in aqueous solution at a pH close to neutral (for example between 6 and 8, and more preferably between 6.5 and 7 , 5), then by adding the aqueous solution thus obtained to a solution containing the nucleic acids or else by adding said solution containing the nucleic acids to the aqueous solution of mineral particles having a structure with exchangeable sheets. The solution containing the nucleic acids is preferably an isotonic solution in sodium chloride or in glucose. Preferably, the mineral particles having a structure with exchangeable sheets and the nucleic acids are introduced into the composition in quantities such that the mass ratio between the mineral particles having a structure with exchangeable sheets and the nucleic acids is between 0.01 and 1000 , preferably between 0.01 and 500, more preferably between 0.01 and 100, and even more preferably between 0.5 and 100. In any event, the respective amounts of each component can be easily adjusted and optimized by l he skilled in the art by the usual method of trial and error, as a function of the mineral particle having a structure with exchangeable sheets used, of the nucleic acid, and of the desired applications (in particular of the cell type to be transfected).

A subject of the invention is also the compositions as defined above for use as a medicament.

The invention also relates to the use of the compositions as defined above for the transfer of nucleic acids into cells in vitro, in vivo or ex vivo. More specifically, the subject of the present invention is the use of the compositions as defined above for the preparation of a medicament intended to treat diseases, in particular diseases which result from a deficiency in a protein or nucleic product. The nucleic acid contained in said medicament encodes said protein or nucleic product, or constitutes said nucleic product, capable of correcting said diseases in vivo or ex vivo. Said medicament can also be a vaccine and in this case, the transfected nucleic acid induces an immune response.

For in vivo uses, for example in therapy or for the study of gene regulation or the creation of animal models of pathologies, the compositions according to the invention can be formulated for administration in particular by topical or cutaneous route, oral, rectal, vaginal, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal, intratracheal or intraperitoneal. Preferably, the compositions of the invention 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 on the mucous membrane). 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. In addition, the compositions according to the invention may contain one or more adjuvants conventionally used in pharmacy. The doses of nucleic acids used for the injection as well as the number of administrations can be adapted according to various parameters, and in particular according to the mode of administration used, the pathology concerned, the gene to be expressed, or the duration of the treatment sought. As regards more particularly the mode of administration, it may be either a direct injection into the tissues, for example at the level of tumors, or into the circulatory tract, or a treatment of cultured cells followed of their reimplantation in vivo, by injection or graft. The tissues concerned in the context of the present invention are for example the muscles, the skin, the brain, the lungs, the liver, the spleen, the bone marrow, the thymus, the heart, the lymph, the blood, the bones, cartilage, pancreas, kidneys, bladder, stomach, intestines, testes, ovaries, rectum, nervous system, eyes, glands or connective tissue. Another object of the present invention relates to a method for transferring nucleic acids into cells, comprising the following steps:

(1) the formation of a composition comprising at least one nucleic acid and one mineral particle having a structure with exchangeable sheets as defined above, and

(2) bringing the cells into contact with the composition formed in (1).

More particularly, the present invention relates to a method of therapeutic treatment of the human or animal body comprising the following steps:

(2) bringing the cells of the human or animal body into contact with the composition formed in (1).

The contacting of the cells with the composition according to the present invention can be carried out by incubation of the cells with said composition or by injection of the composition into the organism or part of the organism. The incubation is preferably carried out in the presence of, for example, from 0.01 to 1000 μg of nucleic acid per 10 cells. For administration in vivo, doses of nucleic acid ranging from 0.01 to 10 mg can for example be used.

The compositions of the invention may further contain one or more pharmaceutically acceptable adjuvants. In this case, the adjuvant (s) are mixed beforehand with the aqueous solution containing the mineral particle having a structure with exchangeable sheets according to the invention and / or with the solution of nucleic acid (s).

The present invention thus provides a particularly advantageous method for the transfer of nucleic acids, in particular for the treatment of diseases, comprising the administration in vivo or ex vivo of a nucleic acid coding for a protein or capable of being transcribed into a nucleic acid. able to correct said disease, said nucleic acid being associated with a mineral particle having a structure with exchangeable sheets as defined above under the conditions defined above.

The compositions according to the present invention are particularly useful for the transfer of nucleic acids into primary cells or into established lines. They can be, for example, fibroblastic, muscular, nervous cells (neurons, astrocytes, glial cells), hepatic, hematopoietic (for example lymphocytes, CD34, or dendritics) or epithelial, in differentiated or pluripotent forms (precursors).

In addition to the foregoing provisions, the present invention also includes other characteristics and advantages which will emerge from the examples and figures which follow, and which should be considered as illustrating the invention without limiting its scope.

FIGURES Figure 1: idealized structure of the mineral particles having a structure with exchangeable sheets (double lamellar hydroxides) according to the present invention.

Figure 2: schematic representation of the hydrotalcite Mg ₆ Al ₂ CO ₃ (OH) ι ₆ .4H ₂ O.

Figure 3: measurement of the zeta potential of hydrotalcite particles alone in solution.

Figure 4: measurement of the zeta potential of compositions containing hydrotalcite particles and DNA in a mass ratio equal to 0.5.

Figure 5: Measurement of the percentage of DNA remaining in the supernatant relative to the initial amount of DNA introduced, as a function of the hydrotalcite / DNA mass ratio. The measurements were made for three different concentrations of DNA introduced: 20 μg of DNA / ml, 50 μg of DNA / ml, and 100 μg of DNA / ml. Figure 6: Electrophoresis on agarose gel of different DNA formulations. The column "a" constitutes the control (DNA not formulated and not subjected to nucleases), the column "b" represents the unformulated DNA subjected to nucleases, and the columns "C" to "f" represent hydrotalcite / DNA compositions at mass ratios of 0.125 or 0.25 or 0.5 or 1 which are subjected to nucleases.

FIG. 7: visualization in the form of a histogram of the efficiency of gene transfer in vivo (by injection into the cranial tibial muscle of mice) of DNA / hydrotalcite compositions in different mass ratios, compared with the injection of naked DNA.

Figure 8: Schematic representation of the plasmid pXL3031 used in DNA transfer experiments in cells

EXAMPLES

Al MATERIALS AND METHODS

Mice used;

The mice used in the in vivo gene transfer experiments are 8 week old female C57B16 mice, divided into 9 groups of 12 mice.

Mineral particle according to the invention used the mineral particle having a structure with exchangeable sheets used in the various tests is hydrotalcite in aqueous solution at a concentration of 20 g / 1 and at pH 7. This hydrotalcite is sold by the company Sud- Chemie AG (Germany).

DNA

The plasmid used is pXL3031 which contains the read gene coding for luciferase under the control of the P / E CMV promoter of the cytomegalovirus, represented in FIG. 8. Its size is 3671 bp. The plasmid solution used contains 320 μg of DNA / ml in a 150 mM solution of sodium chloride (NaCl).

Hydrotalcite / DNA complexes

The complexes are prepared by equivolumetric mixing of two solutions: one containing hydrotalcite and the other plasmid DNA.

EXAMPLE 1 Measurement of the Zeta Potentials of Hydrotalcite Alone and of DNA / Hydrotalcite Complexes

The purpose of this example is to show that DNA is capable of associating with a mineral particle having a structure with exchangeable sheets such as hydrotalcite. The measurement of the zeta potential provides information on the nature of the surface charge of a particle placed in a liquid. The zeta potential is not a direct measure of the surface charge, but is the potential that exists around the particle when it moves in a solution when it is subjected to an electric field. Therefore if the particles have a positive surface charge, the zeta potential, which is the only measurable quantity, will also be positive. Zeta potential plays a role determining in the colloidal stability of the particles in solution. Indeed, when the zeta potential is strongly positive or negative, the particles do not aggregate because forces of electrostatic repulsions keep them isolated. On the other hand, the particles which have a zeta potential close to zero are not stable because the Van Der Waals forces attract the particles together, and cause them to precipitate.

The zeta potential of hydrotalcite and of hydrotalcite / DNA complexes was measured by a Coulter brand zetameter (Delsa 440 SX). The hydrotalcite solution contained 0.15 mg hydrotalcite / ml in a 20 mM solution of sodium chloride (NaCl), giving a conductivity of 2.3 mS / cm. The hydrotalcite / DNA complexes were prepared at concentrations of 250 μg of DNA / ml in a 20 mM solution of sodium chloride (NaCl), at a mass ratio of 0.5. The measurement was made by applying an electric field of 1.5 mA for 60 seconds.

Figure 3 shows the zeta potential of hydrotalcite alone before being mixed with DNA. This zeta potential is + 32 mV and therefore indicates an overall cationic surface charge, which is consistent with the structure and composition of the hydrotalcite (see Figures 1 and 2). FIG. 4 represents the zeta potential of hydrotalcite / DNA complexes with a mass ratio of 0.5. In this case, the zeta potential is - 41 mV. The surface charge has therefore been modified and is now generally anionic, which indicates that the anionic DNA molecules have associated on the surface of the hydrotalcite thus modifying its surface charge.

EXAMPLE 2 Demonstration of the Association Between the DNA and the Mineral Particles Having a Structure with Exchangeable Sheets such as Hydrotalcite

The purpose of this example is to show that mineral particles having a structure with exchangeable sheets such as hydrotalcite associate with DNA. Adsorption isotherms are achieved by mixing increasing amounts of hydrotalcite with a constant amount of DNA. Hydrotalcite / DNA mixtures are prepared by equivolumetric mixing of a solution containing DNA and a solution containing hydrotalcite. All of these mixtures are then ultracentrifuged at 50,000 rpm for 10 minutes (Ultracentrifuge: Beckman TL100). The graph in FIG. 5 indicates that the quantity of DNA present in the supernatant gradually decreases with the increase in the hydrotalcite / DNA mass ratio. At a hydrotalcite / DNA ratio of 50 w / w, there is no longer any DNA present in the supernatant. This indicates that all DNA molecules are associated with hydrotalcite and are in the pellet after ultracentrifugation. The same phenomenon was observed at three different DNA concentrations: 20, 50 and 100 μg of DNA / ml. Consequently, we can deduce that there is indeed an association between hydrotalcite and DNA.

Example 3 Protection of DNA against Degradation Caused by Nucleases

The purpose of this example is to show that DNA is protected against enzymatic degradations when it is associated with a mineral particle having a structure with exchangeable sheets such as hydrotalcite.

Bare DNA and DNA formulated with hydrotalcite were tested for resistance to nuclease damage. For this, the samples were incubated with a sample of muscle fluid. Figure 6 indicates that when DNA is associated with hydrotalcite at different mass ratios (columns "c", "d", "e" and "f"), it is not degraded by nucleases since it is entirely possible to observe a DNA band on the gel. On the other hand, the column “b” which represents the naked DNA not associated with the hydrotalcite is totally degraded by nucleases because instead of having a well identified DNA band on the gel, we observe a multitude of small fragments which migrate in the gel.

Thus, when DNA is associated with mineral particles of hydrotalcite, it is protected from the action of nuclease degradation, as opposed to naked DNA which is rapidly degraded. This property conferred by mineral particles having a structure with exchangeable sheets such as hydrotalcite is very interesting because a larger quantity of DNA can thus reach the nucleus of cells to be transcribed there, with the beneficial consequences that this implies in terms transfection efficiency. Example 4 Transfection of Hydrotalcite / DNA Complexes in Vivo a) Injections

The injections were carried out bilaterally in the cranial tibial muscle of mice at the rate of 25 μl / muscle, ie 4 μg of DNA / muscle. The animals are anesthetized with 250 μl of Ketamine / Xylazine intraperitoneally (3.9 ml of imalgene 1000, 0.6 ml Rompun at 2%, and 40.5 ml of a solution of sodium chloride NaCl at 150 mM ). Then the animals are shaved and injected into the two cranial tibial muscles. b Sampling of the muscles The cranial tibial muscles are taken up 6 days post-injection in 1 ml of lysis / muscle buffer (Lysis Buffer from Promega E) supplemented with protease inhibitors (coktail tablets - Boehringer). The muscles are taken from the special tubes from BIO101 and stored at -20 ° C, before grinding and reading. c) Extraction of the luciferase expressed by the muscle cells The extraction of the luciferase is carried out with a “Fastprep machine” apparatus under extraction conditions using 1 ml of lysis buffer per tube at a speed of 6.5 m / s, for 30 seconds. The tubes are then placed in ice and centrifuged at 12,000 g for 10 minutes at 4 ° C. d Determination of luciferase activity Luciferase activity is measured using a luciferase test kit (Promega) and a Dynex MLX luminometer. The reading of this activity is measured in RLU ("Relative Light Unit"). in vivo transfection into the muscle

The results of the in vivo gene transfer into the muscle are represented in FIG. 7. These results indicate that at the hydrotalcite / DNA mass ratio of 0.5 an increase in the luciferase activity by a factor of 14 is obtained compared to naked DNA. More generally, the DNA / hydrotalcite compositions have an improved transfection efficiency compared to that obtained by injection of naked DNA.

Claims

1. Composition comprising at least one nucleic acid having a size greater than 1000 base pairs and a mineral particle having a structure with exchangeable sheets.

2. Composition according to claim 1 characterized in that the nucleic acid is a plasmid, a vector, an episome or an expression cassette.

3. Composition according to one of claims 1 and 2 characterized in that said mineral particle having a structure with exchangeable sheets has the general formula:

in which M ^π represents a divalent metal cation, M ^ιπ represents a trivalent metal cation, .

4. Composition according to claim 3 characterized in that the divalent metal cation M ^π can be chosen from magnesium (Mg), chromium (Cr), manganese (Mn), iron (Fe), nickel (Ni) , cobalt (Co), copper (Cu) or zinc (Zn).

5. Composition according to claim 3 characterized in that the divalent metal cation M ^π is magnesium.

6. Composition according to claim 3 characterized in that the trivalent metal cation M ^m can be chosen from aluminum (Al), chromium (Cr), manganese (Mn), iron (Fe), nickel (Ni ), cobalt (Co), or gallium (Ga).

7. Composition according to claim 3 characterized in that the trivalent metal cation M ^m is aluminum.

8. Composition according to claim 3 characterized in that the interfoliar anion X can be chosen from halides, oxoanions, iso- and heteroanions, complex anions, or organic anions.

9. Composition according to claim 3 or 8 characterized in that the anion m-? interfoliar X is the carbonate CO ₃ .

10. Composition according to claim 3 characterized in that m is equal to 1, 2, 3, 4, 5 or 6.

1 1. Composition according to claim 3 characterized in that x is between 0.05 and 0.95.

12. Composition according to claim 3 characterized in that n is greater than or equal to 0.1.

13. Composition according to one of claims 1 to 3 characterized in that said mineral particle having a structure with exchangeable sheets is chosen from hydrotalcite of formula Mg ₆ Al ₂ CO ₃ (OH)ι _6.4H ₂ θ, the manasseite of formula Mg ₆ Al ₂ CO ₃ (OH)ι ₆ .4H ₂ O, pyroaurite of formula Mg ₆ Fe ₂ CO ₃ (OH)ι ₆ .4H ₂ O, stichtite of formula Mg ₆ Cr ₂ CO ₃ ( OH)j ₆ .4H ₂ O, takovite of formula Ni ₆ Al ₂ CO ₃ (OH)ι ₆ .4H ₂ O, reevesite of formula Ni ₆ Fe ₂ CO ₃ (OH)ι ₆ .4H ₂ O or comblainite of formula Ni ₆ Co ₂ CO ₃ (OH)ι _6.4H ₂ O.

14. Composition according to claim 13 characterized in that said mineral particle having a structure with exchangeable sheets is hydrotalcite of formula

Mg ₆ Al ₂ CO ₃ (OH) _16.4H ₂ O.

15. Composition comprising at least one nucleic acid and a mineral particle having a structure with exchangeable sheets of formula

in which M ¹¹ represents magnesium ^, M ^ra represents aluminum, .

16. Composition according to claim 15 comprising at least one nucleic acid and a mineral particle having a structure with exchangeable sheets of formula

Mg ₆ Al ₂ CO ₃ (OH) _16.4H ₂ O.

17. Composition according to one of claims 1 to 16 characterized in that it further comprises one or more pharmaceutically acceptable adjuvants.

18. Composition according to one of claims 1 to 17 characterized in that it further comprises a pharmaceutically acceptable vehicle for an injectable formulation.

19. Composition according to one of claims 1 to 17 characterized in that it further comprises a pharmaceutically acceptable vehicle for application to the skin and/or mucous membranes.

20. Composition according to one of claims 1 to 19 characterized in that the mass ratio of mineral particles having a structure with exchangeable sheets/nucleic acid(s) is between 0.01 and 1000.

21. Composition according to one of claims 1 to 20 characterized in that the nucleic acid has a size between approximately 1000 base pairs and approximately 2,000,000 base pairs.

22. Composition according to one of claims 1 to 20 characterized in that the nucleic acid has a size between approximately 1000 base pairs and approximately 100,000 base pairs.

23. Composition according to one of claims 1 to 20 characterized in that the nucleic acid has a size between approximately 1000 base pairs and approximately

20,000 base pairs.

24. Composition according to one of claims 1 to 23 for use as a medicine.

25. Use of a composition as defined in one of claims 1 to 24 for the preparation of a medicament intended for the transfection of cells.

26. Process for preparing a composition as defined in one of claims 1 to 24, characterized in that an aqueous solution containing a mineral particle having a structure with exchangeable sheets and a solution containing the nucleic acid(s) is mixed. .

27. Preparation process according to claim 26 characterized in that, in the case where the compositions as defined in the preceding claims also contain one or more pharmaceutically acceptable adjuvants, the adjuvant(s) are mixed beforehand with the aqueous solution containing a mineral particle having a structure with exchangeable sheets and/or to the solution of nucleic acid(s).

28. Method for transferring nucleic acids into cells in vitro or ex vivo comprising the following steps:

(1) the formation of a composition comprising at least one nucleic acid, a mineral particle having an exchangeable sheet structure as defined in one of claims 1 to 24, and (2) bringing the cells into contact with the composition formed in (1).