WO2016005099A1 - Hypotonic aqueous composition comprising mrna nanoparticles, kit, and preparation method - Google Patents

Abstract

The invention relates to an aqueous composition comprising nanoparticles of messenger RNA (mRNA) coding for a therapeutic protein, characterized in that - the composition is hypotonic and has an osmolarity of less than 300 mOsm, - the mRNA nanoparticles contain mRNA condensed in an aqueous medium in hypotonic conditions with cationic compounds of type A by forming non-covalent bonds with the mRNA that are reversible in the cytoplasm of mammal cells. The invention also relates to a kit comprising mRNA nanoparticles and a reconstituted hypotonic aqueous solution. The invention finally relates to the therapeutic use of said hypotonic compositions.

Description

 Hypotonic aqueous composition comprising nanoparticles of A Nm, kit and method of preparation

The expression of therapeutic proteins in pathological tissue can currently be obtained by the administration of recombinant gene or protein vectors produced in vitro.

 Gene vectors have three major defects. First, the transgene can integrate randomly into the genome of a cell. This increases the risk of cellular transformation, causing the occurrence of cancer. The maximum level of expression of the protein encoded by the transgene is reached 72 hours after the injection of the vector. Treatment of some conditions would require that the peak of expression be reached within the first 24 hours after administration of the drug product. In addition, the transgene is expressed durably, which is not appropriate for the treatment of acute diseases, which requires transient expression of a therapeutic protein.

 The production of recombinant proteins is in full swing. However, these proteins hardly accumulate in pathological tissue from the blood stream and the residence time of a therapeutic protein in a tissue is short-lived. However, the treatment of acute conditions requires the local expression of a protein for several days, in order to obtain a clinical benefit.

 Thus, there is a demand for a transient expression system of extracellular therapeutic proteins, membrane or intracellular. The alternative strategy to recombinant protein and gene therapy is the transfer of messenger RNA (mRNA) encoding a therapeutic protein.

 The benefits of messenger RNA:

 Transfer of mRNA into a cell results in transient expression of the protein of interest. Indeed, mRNA and the protein encoded by it are fully degraded into nucleotides and amino acids within a few days. The reversibility of protein expression is a guarantee of safety for the patient. This transient expression of therapeutic proteins would avoid the deleterious effects, which can result from a lasting and uncontrolled expression.

An mRNA can not integrate into the genome, unlike a DNA molecule. The total absence of genotoxicity of the mRNA vectors represents a weight advantage over the gene vectors. This also contributes to the high safety of this type of vector for the patient. The mRNA is introduced into the cytosol of the target cell, while the DNA of a gene vector has to be transferred to the nucleus. In addition, the mRNA is readily accessible to ribosomes to be translated into protein. Thus, the expression of the protein of interest is earlier in the case of an mRNA vector. This represents an advantage for the treatment of an acute pathology, which requires that the therapeutic protein appear as early as possible in the target tissue.

 In vitro messenger RNA production:

 In a first step, a plasmid must be constructed using standard molecular biology techniques. The gene coding for the protein of interest is inserted into the plasmid downstream of the phage T7 RNA polymerase promoter. The plasmid is amplified, purified and linearized downstream of the gene of interest by a restriction enzyme.

 The linearized plasmid is then incubated with recombinant T7 RNA polymerase, the four ribonucleotide triphosphates and a capping analog. The enzyme binds to its promoter and transcribes the gene to the end of the DNA.

 The mRNA thus produced is then purified by affinity chromatography and by precipitation with lithium chloride. The concentration of purified mRNA is determined by measuring its absorbance at 260 nm.

 General characteristics of a messenger RNA vector:

 First, the mRNA must be protected and condensed in the form of nanometric particles, constituting a vector, in order to be out of reach of the extracellular and lysosomal nucleases.

 The nanoparticle must also advantageously expose on its surface a molecule capable of interacting with one or more components of the plasma membrane of the target cells. This ligand thus allows the adsorption of the nanoparticle on the cell surface, in order to promote its internalization by endocytosis.

The invention relates to an aqueous composition comprising messenger RNA nanoparticles (mRNA) encoding a therapeutic protein.

 It has been demonstrated that it is possible to obtain mRNA nanoparticles comprising mRNA fused with cationic compounds under hypotonic conditions. These nanoparticles comprising condensed mRNA will be able to penetrate the mammalian cells where they will release the mRNA.

Surprisingly, it has been found a synergistic effect between a hypotonic aqueous solution, advantageously containing 40 mM KCl, 20 mM Hepes pH 5.5 and 100 μl trifluoroacetate, and the formation of nanoparticles mRNA / cationic peptide dissolved at a high concentration in this aqueous solution, for the transfer of A Nm in mammalian cells.

 A hypotonic solution is a solution whose tonicity, i.e. the effective osmolarity, is much lower than that of the intracellular medium of the target cell.

 The subject of the invention is an aqueous composition comprising messenger RNA nanoparticles (mRNAs) encoding a therapeutic protein, characterized in that

 the composition is hypotonic and has an osmolarity of less than 300 mosM,

 the mRNA nanoparticles comprise mRNA condensed in an aqueous medium under hypotonic conditions with cationic compounds of the type

A by formation of noncovalent bonds with mRNA, reversible in the cytoplasm of mammalian cells.

A synergistic effect between the composition and the nanoparticles is observed: significant improvement in mRNA transfer when the nanoparticles are administered in a hypotonic composition.

 The nanoparticles are suspended in the aqueous composition, advantageously without they precipitate. The composition advantageously comprises said mRNA nanoparticles in a concentration such that the concentration of mRNA is greater than 50 μg / ml, more preferably greater than 100 μg / ml, and still more advantageously greater than 200 μg / ml. For example, the volume of the hypotonic solution can be adjusted so that the final concentration of mRNA is 250 μg RNA / ml. The maximum concentration depends on the solubility of the nanoparticles in the hypotonic solution. This relatively high concentration of vector makes it possible not to inject a too high volume of aqueous solution into an animal or human tissue. The volume to be injected can vary from 10 μΙ to 5 ml.

 The invention also relates to the use of a hypo-osmotic solution capable of inducing a hypotonic shock. The hypotonic aqueous composition advantageously has an osmolarity of less than 300 mosM, advantageously less than 200 mosM, for example between 0 mosM and 200 mosM.

 Preferably a composition free of multivalent cations is used. In the composition according to the invention, the cation concentration is advantageously less than 150 mM, more advantageously less than 100 mM.

The composition advantageously comprises potassium chloride and / or sodium chloride in a concentration ranging from 5 mM to 100 mM, more preferably from 20 mM to 60 mM. Potassium chloride is preferred, preferably at a concentration of 40 mM. The composition advantageously comprises Hepes buffer making it possible to adjust the pH of the aqueous composition to between 4 and 7; the terminals being included. An essential feature of the buffer is that it does not contain divalent or multivalent ions. Depending on the target pH and the desired osmolarity, the Hepes buffer will be used in its acid form and / or in its basic form.

 The composition advantageously comprises Hepes buffer in a concentration ranging from 1 mM to 80 mM, more preferably from 5 mM to 50 mM, still more preferably from 8 mM to 40 mM, still more preferably from 10 mM to 30 mM. A concentration of 20 mM is particularly preferred, especially when the Hepes buffer is used in its acid form.

 In particular, for a hypotonic solution containing 40 mM potassium chloride buffered with Hepes, in its acid form, it has been shown that the maximum efficiency of mRNA transfer is achieved at the Hepes concentration of 20 mM. mM. At this Hepes concentration, the pH of the solution is 5.5. The expression "Hepes pH X" will be used to designate a Hepes buffer that makes it possible to adjust the pH of the aqueous composition to X.

 It has been demonstrated that the optimum pH in terms of mRNA transfer efficiency is 6.5 for a composition comprising KCI and Hepes buffer.

 It has surprisingly been found that the addition to the trifluoroacetic acid composition makes it possible to significantly increase the efficiency of the transfer of mRNA with respect to the same vector prepared with a cationic peptide batch that is devoid of acid. trifluoroacetic.

 The composition advantageously comprises trifluoroacetic acid in a concentration ranging from 1 μΜ to 1000 μΜ, more preferably 3 μΜ to 300 μΜ.

 Trifluoroacetic acid is advantageously used in its salt form, advantageously sodium trifluoroacetate.

 Thus, by the addition of sodium trifluoroacetate, the efficiency of mRNA transfer has been increased to pH 6.5 and more if the pH is reduced to 5.5. It has been shown that the effective and nontoxic concentration of sodium trifluoroacetate is 100 μΜ.

A most preferred composition is a hypotonic aqueous composition comprising 40 mM KCl, 20 mM Hepes pH 5.5 and 100 μl trifluoroacetate. Advantageously, the composition does not include other source compounds of sodium or potassium cations; and more preferably no other cation source compounds. Also, advantageously, the composition does not comprise other compounds substantially affecting its osmolarity, that is to say an order of magnitude greater than 10 mOsM. This composition may also comprise one or more unloaded molecule (s), such as glucose, mannitol or sucrose. These molecules may be present as soon as the nanoparticles are assembled in the first variant of the preparation process, which will be described below.

 The efficiency of introducing mRNA into cells depends on the degree of hypotonicity of the nanoparticle solution. In vivo, a slightly hypotonic solution provides moderate benefit, while a very low osmolarity solution greatly improves the entry of the nanoparticle into the cells. In vitro, it has been shown that the optimal concentration of sodium chloride, in terms of the efficiency of mRNA transfer in cells in culture, is 40 mM. Substituting sodium chloride with potassium chloride further enhances the efficiency of mRNA transfer. It was then shown that the optimal concentration of potassium chloride (KCl) is also 40 mM.

 When a mammalian cell is incubated in a hypoosmotic medium, it undergoes hypotonic shock. Indeed, the water enters the cell, which stretches its plasma membrane. This traction opens calcium channels, which allow the fusion of intracellular vesicles with the plasma membrane, by exocytosis. This increases the area of the plasma membrane and prevents the cell from bursting.

 Subsequently, ATP is released into the extracellular medium. ATP binds and activates P2Y purinergic receptors. A signal transduction path leads to the opening of potassium and chloride channels. Decreased intracellular osmolarity and increased extracellular osmolarity drain water out of the cell. The volume of the latter decreases, which allows the internalization of the excess plasma membrane resulting from the fusion of intracellular vesicles. This massive endocytosis can be exploited by the mRNA vector to enter the cell.

Inside the cell, the vector reaches intracellular compartments, such as endosomes. It appears that the hypotonic shock transiently permeabilizes the membranes of these intracellular compartments, allowing the vector to escape and reach the cytosol. In the cytosol, the vector progressively releases messenger RNA through reducing agents, such as glutathione. At the ionic strength prevailing in the cytosol, the positive charges of the compounds A and A '(advantageously cationic peptide) are not sufficient to maintain the integrity of the nanoparticle. The mRNA is thus progressively stripped. It becomes accessible to translation factors and ribosomes. The protein of interest is then produced by the transfected cell. The invention is based on two complementary elements, which act synergistically: the mRNA nanoparticle and the hypotonic buffer in which it is in suspension. When the nanoparticle is composed of peptides, these molecules are degraded by the amino acid cells, which can then be recycled by them for the synthesis of cellular proteins.

 During hypotonic shock, potassium ions must exit the cell through a potassium channel. It has been shown that at low pH, the opening of this potassium channel is inhibited. This could inhibit mRNA transfer. It has also been demonstrated that trifluoroacetate activates the opening of this potassium channel (Trifluoroacetic acid activates ATP-sensitive K (+) channels in rabbit ventricular myocytes, Han J et al., Biochem Biophys Res Commun 2001, 285 (5): 1136-1142). Sodium trifluoroacetate would suppress potassium channel inhibition and prevent hypotonic shock inhibition at low pH.

 Hepes at pH 5.5 is internalized by endocytosis by the cells at the same time as the peptide / mRNA nanoparticles. Hepes would buffer the endosomal pH at 5.5 thereby inhibiting the pH drop necessary for the mRNA vector to be transferred to the lysosomes. However, these contain enzymes capable of degrading RNA. Hepes would thus increase the residence time of the mRNA vector in the endosomes, allowing it to escape to the cytosol, where the translation into protein takes place. The subject of the invention is compositions comprising nanoparticles of messenger RNA

(MRNA) encoding a therapeutic protein by the use of a cationic compound, such as a cationic peptide. The latter has two functions: to condense the mRNA into nanoparticles to promote its entry into mammalian cells by endocytosis and to protect the mRNA against RNases, enzymes capable of digesting RNA. These two functions participate in the transfer of mRNA in cells, so that it is translated into protein by ribosomes.

 The nanoparticles advantageously also comprise compounds of the type A'-E-

L, where

 A 'is a cationic compound, which also allows the condensation of mRNA, by non-covalent bonds with mRNA reversible in the cytoplasm of mammalian cells

 E is an unloaded spacer and soluble in water

L is a neutral or near-neutral ligand peptide Neutral or near neutral means that the ligand is not charged or that it contains positive charges and negative charges that balance or almost balance.

The cationic compounds of type A or type A 'correspond to the same generic definition. They can be identical or different, they are advantageously identical.

 The cationic compounds of type A or type A 'have positive charges, which can interact with the negative charges carried by the RNA, which results in the condensation of the latter in nanoparticles.

 A vector is a nanoparticle containing messenger RNA. Within the nanoparticle, mRNA is fused and protected against nucleases. In addition, the nanoparticle may also exhibit a ligand peptide on its surface. The assembly of the nanoparticles therefore implies that interactions are established between the mRNA molecule and the condensation molecule (s) of the RNA. Peptides are particularly described hereinafter because they are biodegradable and easily synthesized chemically. The most frequently used interactions for binding RNA to the condensation molecule of the latter are electrostatic interactions. Indeed, RNA is a long polyanion. A molecule carrying a plurality of positive charges or an assembly of molecules bearing a single positive charge can interact with RNA and form a particle. Other non-covalent interactions can complement these electrostatic interactions, among which the hydrogen, Van der Walls and hydrophobic interactions.

 The stability of the nanoparticles can also be increased by multimerization of the cationic compounds with each other, the bonds formed being however reversible in the cytoplasm of mammalian cells. Thus, for example, disulfide bridges are broken by reducing compounds, such as glutathione. This multimerization may follow an oxidation, for example the thiol groups present at the ends may be oxidized in the presence of oxygen dissolved in the medium to form disulfide bridges. Thus, advantageously, the cationic compound of type A, and / or, where appropriate, the cationic compound of type A ', comprises groups capable of forming reversible intermolecular bonds in the cytoplasm of mammalian cells. The intermolecular bonds are advantageously reversible covalent bonds, in particular disulfide bridges.

Of course, it is preferred to use a cationic compound of type A, and optionally a cationic compound of type A ', soluble in water. The cationic compound of type A or A 'is advantageously chosen from the group consisting of cationic peptides, cationic lipids in the form of liposomes, cationic polymers, and cationic surfactants in the form of micelles.

In particular, the cationic compound of type A or A 'is a cationic peptide of formula X _m - (Y) _n -X'p in which

 X and X 'are the same or different and each represents a molecule bearing a thiol group

 m is 0 or 1

 p is 0 or 1

 Y is an amino acid or an amino acid derivative whose side chain is positively charged, and

 n is an integer ranging from 4 to 20.

 Advantageously, X and X 'each represent an amino acid or an amino acid derivative or homologous amino acid bearing a thiol group, advantageously cysteine or homocysteine.

Y advantageously represents an amino acid or an amino acid derivative of formula NH ₂ -alk-COOH where

 alk represents a linear alkyl chain comprising from 3 to 8 carbon atoms, one of these carbon atoms possibly being substituted by a nitrogen atom

 alk is substituted with a radical, where R represents a positively charged radical at the pH of the medium, advantageously an NH 2 radical;

 alk may also be substituted, in particular by a function = Het, where Het represents O or NH.

 Y is advantageously chosen from arginine, lysine and ornithine, preferably arginine, positively charged to the pH of the medium. In particular, the cationic peptide is chosen from the peptides of the following sequences:

 (SEQ ID NO: 1 to 24)

Cys (Arg ₈ ) Cys, Cys (Arg ₉ ) Cys, Cys (Arg ₁₀ ) Cys, Cys (Arg _u ) Cys, Cys (Arg ₁₂ ) Cys, Cys (Arg ₁₃ ) Cys, Cys (Arg ₁₄ ) Cys, Cys (Arg ₁₅ ) Cys; Cys (Lys ₈₎ Cys, Cys (Lys ₉₎ Cys, Cys (Lys ₁₀₎ Cys, Cys (Lys _u) Cys, Cys (Lys ₁₂₎ Cys, Cys (Lys ₁₃₎ Cys, Cys (Lys ₁₄₎ Cys, Cys (Lily ₁₅ ) Cys; Cys (Orn ₈ ) Cys, Cys (Orn ₉ ) Cys, Cys (Orn ₁₀ ) Cys, Cys (Ornn) Cys, Cys (Orni ₂ ) Cys, Cys (Orni ₃ ) Cys, Cys (Orni ₄ ) Cys, and Cys (Orni ₅ ) Cys.

In another variant, X represents an organic acid carrying a thiol function, advantageously thioglycolic acid, and X 'represents an aminothiol, advantageously cysteamine. During the assembly of the nanoparticles, an oxidation process takes place between the thiol groups creating new covalent bonds (disulfide bonds). Most often, the dissolved oxygen present in the medium is sufficient for the oxidation reactions to take place. This multimerization of the cationic peptide in contact with A Nm, brings a great stability to the assembly of mRNA and peptides, which makes it possible to increase the stability of the nanoparticles which do not dissociate in the extracellular medium, after their injection into a tissue.

 Cationic peptides with terminal cysteines have been described by Kevin Rice's team (Low Molecular Weight Disulfide Cross-linking Peptides as Nonviral Gene Delivery Carriers, McKenzie DL et al., Bioconjugate Chem., 2000, 11, 901-909). for the transfer of nucleic acids into mammalian cells.

E is a spacer (or English spacer) not loaded and soluble in water. By unloaded, it is meant that the molecule does not include electric charges. It is preferred to use biodegradable molecules whose degradation products are non-toxic metabolites for mammalian cells.

Advantageously, the spacer is a water-soluble polymer, linear or crosslinked, having a size ranging from 0.5 kDa to 50 kDa, advantageously from 0.5 to 20 kDa, more advantageously from 2 kDa to 5 kDa. In order to easily dissolve the compound A'-EL, in freeze-dried form, in the aqueous medium, a water-soluble polymer is advantageously used. Polymers considered to be non-toxic are preferred. More particularly, the spacer is chosen from polyethylene glycol (PEG), poly (hydroxyalkyl) -L-glutamine and poly (hydroxyalkyl) -L-asparagine; where the alkyl groups are advantageously C ₁ -C ₄ alkyls, more preferably the ethyl radical. The degradation products of these last two compounds are ethanolamine and glutamate or aspartate. It is also particularly possible to use the PEG of 2 to 5 kDa, more particularly 3.5 kDa.

The compounds A'-E-L comprise at their end a ligand peptide, L. The latter binds to molecules present in the plasma membrane, which allows the nanoparticle to adsorb on the cell surface.

The messenger RNA nanoparticle and the hypotonic shock serve to promote the introduction of the mRNA into the target cells. The presence of a ligand peptide allows the nanoparticle to attach to the plasma membrane, in order to be subsequently endocytinated. This improves the efficiency of the entry of the mRNA into the cells. The choice of ligand peptide was focused on a peptide penetrating the cells (CPP). The first characteristic of a CPP is to attach to the plasma membrane and be internalized by a cell.

 The ligand peptide is advantageously chosen from peptides of cellular penetration or CPP, in particular from mammalian cells, RGD ligands, transferrin, folate, antibodies, ligands of a receptor (for example: cytokines, hormones, growth), small molecules (e.g. carbohydrates such as mannose or galactose or synthetic ligands), small molecule agonists, receptor inhibitors or antagonists (e.g. RGD peptidomimetic analogues).

 Advantageously, the ligand peptide is of formula PQRDTVGGRTTPPSWGPAKA (SEQ ID No. 25). For this peptide, it has been shown that it is able to bind to the plasma membrane and be internalized by a cell. This has been demonstrated by conjugating a marker compound to CPP, incubating cells in culture with labeled CPP and following the entry of CPP into these cells (Screening of cell-penetrating peptides using mRNA display, Jae-Hun Lee et al. Biotechnol, J 2012, 7, 387-396). In addition, the demonstration was made with a CPP conjugated to a large molecule or particle, in order to prove that the CPP possesses this characteristic of penetration into the cells in the context of a macromolecular assembly. Indeed, it is not necessary that the CPP is effective in isolation and inefficient when it is present on the surface of a nanoparticle mRNA.

 After injection into a tissue, the mRNA nanoparticle must attach to the cells, not only to penetrate, but also so that the nanoparticle diffuses as little as possible outside of this tissue. This retention of the nanoparticle by the tissue into which it has been injected is important because the mRNA must not be introduced into the cells of other organs. There should be the least possible ectopic expression of the protein encoded by the messenger RNA.

 The CPP must not be significantly positively or negatively charged. Indeed, only the cationic peptides of type A and A 'must interact with the phosphodiester groups of the mRNA. The presence of a large number of electrical charges on the CPP would allow it to interact with either the RNA or the cationic compound. This would profoundly alter the formation of mRNA nanoparticles. The CPP chooses, PQRDTVGGRTTPPSWGPAKA, has three positive charges for a negative charge scattered throughout the sequence. It has all the necessary characteristics.

Cationic CPP would also interact in vivo with the extracellular matrix present in the injected tissue. The mRNA vector would thus be retained on the extracellular matrix instead of entering the cells. The efficiency of introducing mRNA into the target cells would therefore be very low. The lyophilized A'-EL compound should readily dissolve in deionized water. This implies that the CPP portion of this compound should advantageously be soluble in water, too. This is the case of the CPP chooses. It is necessary to avoid any hydrophobic CPP containing too much phenylalanine, leucine, isoleucine, alanine, valine, cysteine and methionine, and too few hydrophilic amino acids, such as arginine, lysine, histidine, glutamic acid , aspartic acid, proline, asparagine and glutamine.

In a preferred variant of the invention, the cationic compound of type A is the Cys (Arg ₈ ) Cys cationic peptide (SEQ ID No. 1).

In a preferred variant of the invention, the A'-EL type compound is the cationic Cys (Arg ₈ ) Cys peptide (SEQ ID No. 1) extended at its N-terminus by polyethylene glycol conjugated to a ligand peptide. . PEG is preferred from 2 to 5 kDa, more preferably 3.5 kDa. The ligand peptide is advantageously of formula PQ DTVGG TTPPSWGPAKA (SEQ ID No. 25).

 The nanoparticle is very stable outside the cells, where the environment is oxidizing.

The nanoparticle does not dissociate and does not aggregate outside the cells.

 In the presence of the compounds A'-E-L, the spacer being not loaded, during the assembly of the nanoparticle, the ligand is found preferentially on the surface of the nanoparticle. The ligand can thus be effectively exposed to interact with the plasma membrane of the target cells. Thus, the groups L are predominantly on the outer surface.

 The nanoparticles advantageously have a size of less than 200 nm, more advantageously less than 100 nm. The size of nanoparticles can be measured by electron microscopy. It can also be measured by diffraction of the light of a laser

(dynamic light scattering) issued by a particle sizer.

 At higher saline concentrations, particles are significantly larger than low osmolarity and tend to aggregate with time. However, it is essential that the mRNA vector be of small size (advantageously <200 nm), in order to effectively penetrate the target cells by endocytosis.

 As explained above, the nanoparticles are obtained by condensation and possibly multimerization. The multimers of peptides comprise tens or even hundreds of radicals Y (advantageously arginines).

The presence of the compound AEL is optional. The assembly of the mRNA vector can be obtained by mixing an RNA solution and a solution containing either the only X- (Y) nX cationic peptide, or the cationic peptide and the A'-EL compound. . The amount of peptide complexed to the mRNA has been precisely fixed. There must be an excess of negative charges on the surface of the nanoparticle, from the mRNA, in order to avoid the immobilization of the vector on the extracellular matrix present in the tissues. Indeed, if the nanoparticle were positively charged, it would attach to the glycosaminoglycans of the extracellular matrix at the injection site. The vector could not penetrate abundantly into the cells.

 In addition, if the amount of cationic peptide is sufficient to form a nanoparticle with the mRNA, but is not too high, the mRNA is released more rapidly into the cytosol of the cells, in order to be translated early into protein.

 The amount of A compound and optionally A'-E-L compound mixed with the mRNA depends on the ratio of positive / negative charges that one wishes to achieve. Advantageously, this ratio varies from 1 to 3, more preferably from 1.5 to 2.5. The +/- charge ratio is the ratio between the total number of positive charges carried by all the peptides and the total number of negative charges carried by all the RNA molecules. It has been found that the +/- charge ratio of the mRNA / cationic peptide vector giving the best results, in terms of mRNA transfer, varies from 1.4 to 2.2, more preferably from 1.6 to 2, 2, still more preferably 1.6 to 2.0.

 The compound molar ratio of type A / compound A'-E-L advantageously varies from 99% / 1% to 96% / 4%.

The assembly of the vector can be obtained in different ways.

 The invention particularly relates to a method for preparing messenger RNA nanoparticles (mRNA) encoding a therapeutic protein, comprising the condensation of mRNA with cationic compounds of type A in an aqueous medium under hypotonic conditions, leading to the formation non-covalent linkages with mRNA, reversible in the cytoplasm of mammalian cells.

 It has been discovered that by condensing the mRNA, with the cationic compounds of type A, under hypotonic conditions, stable nanoparticles are obtained which do not precipitate in the medium. By stable is meant that the mRNA assembled with the cationic compounds of type A, otherwise called here nanoparticle or vector, does not dissociate in the extracellular medium.

The medium in which the mRNA is assembled with the cationic compounds of type A is a hypotonic aqueous medium. Advantageously, the pH of the medium is between 6.5 and 8.5 inclusive, advantageously between 7 and 7.5 inclusive. The medium advantageously comprises a hypotonic buffer preferably consisting of one or monovalent cation (s). In particular, the buffer is sodium hepes. A medium free of multivalent cations is preferably used. Advantageously, the vast majority of the cations in the medium is provided by the buffer. The concentration of cations in the medium is advantageously less than 150 mM, more preferably less than 100 mM.

 The medium advantageously has an osmolarity of less than 300 mosM, advantageously between 0 and 200 mosM.

 The assembly, also called condensation, of the A Nm under these conditions makes it possible to obtain mRNA nanoparticles having a size of less than or equal to 200 nm, advantageously less than or equal to 100 nm.

 The method advantageously comprises the following steps:

 at. Prepare a buffered aqueous medium comprising the mRNA,

 b. Prepare a buffered aqueous medium comprising the cationic compound of type A, and optionally the compound A'-E-L, as defined previously,

 vs. Mix for a short time the aqueous medium prepared in step b) and the aqueous medium of step a).

 The two media of steps a) and b) are hypotonic. In particular, the medium of step a) has a cation concentration of less than 150 mM, advantageously less than 100 mM, and the medium of step b) has a cation concentration of less than 150 mM, advantageously less than 100 mM. mM.

In step a) and step b), the buffers are monovalent buffers, such as sodium hepes. The choice fell on Hepes because it can be used in health products administered to patients. However, other buffers can be used, excluding those containing divalent or multivalent ions. For example, the phosphate buffer can not be used because the phosphate is present in a divalent form (PO ₄ ^2- ). This divalent anion interacts, in competition with the mRNA, with the positive charges of the compounds of type A or A '(in particular arginines of the cationic peptides). This also results in the formation of large mRNA particles. Any buffer adjusted to pH 7-7.5, consisting of monovalent ions, can replace sodium hepes.

 The volume-to-volume ratio of step a): medium of step b) advantageously varies from

1: 100 to 100: 1, more preferably it is 1: 1.

The mRNA nanoparticles are assembled by mixing the media of steps a) and b). Advantageously, the medium of step b) is introduced into that of step a). However, the reverse order is possible. After adding, mix as quickly as possible (from 1 to 10 seconds according to the scale of production), in order to obtain a homogeneous mixture. This homogeneity is important because it allows each RNA molecule to complex with a similar number of copies of the A-type compounds and A'-EL compounds and to form a population of nanoparticles having a similar diameter.

 Advantageously, the assembly of the nanoparticles takes place at ambient temperature (ie 20-25 ° C.) for a sufficient time, most often a few hours.

 The assembly of the nanoparticles takes place in a solution with a very low salt concentration, that is to say hypoosmotic or hypotonic. Indeed, the cations and anions dissolved in an aqueous solution are in competition with the positively charged radicals of the compounds A and A '(for example arginines of the cationic peptide) and the phosphodiester groups of the RNA. At saline concentrations greater than or equal to 150 mM (NaCl for example), the peptide / RNA particles are significantly larger than at low osmolarity and tend to aggregate with time.

 The medium of step b) can easily be prepared by adding the cationic compound of type A, and optionally the compound A'-E-L, in freeze-dried form in the buffered aqueous solution.

In another variant, the assembly of the vector is obtained by mixing a solution of mRNA dissolved in demineralized water and a solution of cationic compound, advantageously peptide, dissolved in demineralized water. The mixture is incubated for 15 minutes at room temperature. The mixture of RNA and peptide solutions must be as fast as possible in order to be homogeneous. This homogeneity is important because it allows each RNA molecule to complex with a similar number of peptide copies and to form a population of nanoparticles having a similar diameter.

 The method advantageously comprises the following steps:

 at. Prepare a demineralized aqueous medium including mRNA,

 b. Prepare an aqueous demineralized medium comprising the cationic compound of type A, and optionally the compound A'-E-L, as defined previously, c. Mix for a short time the aqueous medium prepared in step b) and the aqueous medium of step a);

 d. Incubation of the mixture at room temperature, advantageously for 15 mm.

e. Lyophilization of the mixture obtained following the previous step The volume ratio between the two aqueous solutions is more advantageously 1: 1. To promote the formation of small complexes (<100 nm), the concentration of RNA is advantageously less than or equal to 100 μg RNA / ml, at the end of the mixture.

 Lyophilization is carried out according to the procedures known to those skilled in the art. Advantageously, the RNA / peptide mixture is frozen by the use of liquid nitrogen (or any other means allowing rapid freezing) and freeze-dried.

 Aqueous solutions must be demineralized for two reasons. The higher the ionic strength, the more the cationic peptide / mRNA particles are bulky. However, they must be small, in order to effectively enter mammalian cells by endocytosis. Lyophilization makes it possible to preserve the vector, in the long term, in the form of a lyophilizate, at -20 ° C. Lyophilization does not eliminate ions. If the aqueous solution contained ions, the osmolarity of the vector solution would be higher after lyophilization, when the vector is dissolved in a smaller volume of water than that used during assembly.

 Lyophilization also allows extemporaneous reconstitution of the vector suspension.

Thus, in the composition as described above, the nanoparticles may be nanoparticles obtained by the first variant of the process. The composition may be identical or different from that used for the preparation of the nanoparticles. In another variant, the composition is obtained by reconstitution of the lyophilizate of nanoparticles (second variant of the process).

The subject of the invention is also a kit comprising:

 i. A hypotonic aqueous solution, of osmolarity lower than 300 mosM, of concentration in cations lower than 150 mM,

 ii. Messenger RNA nanoparticles (mRNA) encoding a therapeutic protein, as defined above.

This hypotonic aqueous solution may be called a reconstitution solution. In particular, the hypotonic solution of step i) is as previously described. Advantageously, it comprises:

Potassium chloride and / or sodium chloride in a concentration ranging from 5 mM to 100 mM - Hepes buffer, to adjust the pH of the aqueous composition between 4 and 7, the terminals being included

 - Trifluoroacetate in a concentration ranging from 1 μΜ to 1000 μΜ Advantageously, the hypotonic solution of step i) does not include other compounds source of sodium or potassium cations; and more preferably no other cation source compounds. Likewise, advantageously, the hypotonic solution of step i) does not comprise other compounds substantially affecting its osmolarity, that is to say of an order of magnitude greater than 10 mOsM.

 This hypotonic solution of step i) may also comprise one or more unloaded molecule (s), such as glucose, mannitol or sucrose.

 The nanoparticles are advantageously in a lyophilized form. The lyophilizate is prepared as described above. In particular, freeze-dried aqueous solutions do not include ions. The invention finally relates to a composition according to the invention for its use as a medicament. The therapeutic indication will of course depend on the protein that will be encoded by the mRNA.

 The invention has the great advantage that, unlike gene therapy, mRNA transfer forces a tissue to produce a therapeutic protein only for a few days.

 In particular, the composition may be used for treating or preventing diseases, disorders or pathological conditions chosen from cardiac pathologies such as myocardial infarction, angina pectoris, respiratory pathologies such as asthma, disorders caused by a viral infection (especially by the respiratory route or by nebulization), disorders due to an inflammatory reaction (local injection instead of inflammation), disorders due to a bacterial infection, for the healing of diabetic wounds, bedsores.

 The composition according to the invention can also be used for the manufacture of mRNA vaccines, for the treatment of genetic diseases, autoimmune diseases and cancer.

 The composition according to the invention may very particularly be used for the treatment of myocardial infarction.

Myocardial infarction (MI) is a destruction of the heart muscle due to occlusive thrombosis of a coronary artery. This acute coronary pathology most often occurs on an atheromatous plaque that has become unstable following erosion or fissuring. The rupture of an atheromatous plaque or the erosion of the vascular endothelium platelet adhesion and initiation of the coagulation cascade, resulting in the formation of platelet aggregates and fibrin, capable of reducing the lumen of the blood vessel or obstructing it completely. This embolism causes a sharp drop in the supply of oxygen and nutrients to myocardial cells, mainly cardiomyocytes and endothelial cells. The part of the myocardium affected by this ischemic episode is called the infarcted area. The destruction of the blood clot by thrombolysis or crushing by primary angioplasty causes reperfusion of the infarcted zone of the myocardium. The cardiomyocytes can thus return to a normal metabolism and function, after a period of stunning during which they have only a limited mechanical activity.

 Management of IDM begins with emergency hospitalization of patients with symptoms of the disease in a cardiac intensive care unit. Reperfusion of the obstructed coronary is the priority. Indeed, it is a question of restoring as soon as possible the blood circulation in the artery responsible for the IDM. For this, the preferred technique is primary coronary angioplasty.

 Limiting the size of the infarcted area can be achieved by inhibiting apoptotic cell death and stimulating the formation of new blood vessels, neoangiogenesis. In fact, the increase in vascularization leads to an improvement in the oxygen and nutrients supply. The viability of the cells, having survived ischemia and reperfusion, is thus maintained. At present, no anti-apoptotic and angiogenic treatment is used in clinical routine.

 The biotechnology of A Nm transfer in the myocardium is particularly well adapted. Inhibition of apoptosis and formation of new blood vessels can be achieved by transient expression of a protein of the family of growth factors. Unlike gene therapy, mRNA transfer technology indeed forces a tissue to produce a therapeutic protein for only a few days.

 The composition according to the invention can also be used to improve the well-being of a patient, and in particular to fight against pain (especially postoperatively), for the healing of the skin. The invention therefore also relates to the use of the composition according to the invention for combating pain or for promoting skin healing, especially in non-pathological conditions.

The invention also relates to a method of therapeutic treatment of one of these diseases or conditions comprising administering an effective amount of the composition to a patient in need thereof. The invention finally relates to the use of a hypotonic composition comprising mRNA nanoparticles as defined above for improving the transfer efficiency of said mRNA in mammalian cells.

 In particular, the hypotonic composition is of osmolarity lower than 300 mosM, with a cation concentration of less than 150 mM. Advantageously, it comprises:

 potassium chloride and / or sodium chloride in a concentration ranging from 5 mM to 100 mM

 - Hepes buffer for adjusting the pH of the aqueous composition between 4 and 7 - trifluoroacetate in a concentration ranging from 1 μΜ to 1000 μΜ

The invention will now be illustrated by the nonlimiting examples below. Description of the figures:

 Figure 1: Transfection of H9c2 cells with mRNA nanoparticles in the presence or absence of hypotonic shock. Measurement of luciferase activity (RLU / mg protein). From left to right: mRNA / pepMB1, isotonicity; MRNA / pepMB1 / pepMB2, isotonicity; Naked mRNA, hypotonic shock; MRNA / pepMB1, hypotonic shock; MRNA / pepMB1 / pepMB2, hypotonic shock

Figure 2: Transfection of H9c2 cells with naked mRNA or mRNA nanoparticles by hypotonic shock. Measurement of luciferase activity (RLU / mg protein). Left: naked mRNA; Then, mRNA / pepMB1 / pepMB2 nanoparticles with a molar percentage of pepMB2 of 0 or 1.25 or 2.5 or 3.75

 Figure 3: Transfection of H9c2 cells with naked mRNA or mRNA nanoparticles into a solution containing 0 mM Hepes, pH5.5 and sodium chloride at a concentration of up to 145 mM. From left to right: [NaCl] = 0 mM - mRNA nanoparticles; [NaCl] = 20 mM - mRNA nanoparticles; [NaCl] = 40 mM - mRNA nanoparticles; [NaCl] = 55 mM - mRNA nanoparticles; [NaCl] = 100 mM - mRNA nanoparticles; [NaCl] = 145 mM - mRNA nanoparticles; [NaCl] = 145 mM - nude mRNA

 Figure 4: Transfection of H9c2 cells with mRNA nanoparticles into a solution containing 40 mM NaCl and Hepes at a concentration of up to 100 mM. From left to right: [Hepes] = 0 mM; [Hepes] = 10 mM; [Hepes] = 20 mM; [Hepes] = 30 mM; [Hepes] = 100 mM

Figure 5: Transfection of H9c2 cells with mRNA nanoparticles in a solution containing 30, 40 and 50 mM KCl and 20 mM Hepes, pH 5.5. From left to right: NaCl salt at 40 mM; KCI salt at 30 mM; KCl salt at 40 mM; 50mM KCl salt Figure 6: Transfection of H9c2 cells with mRNA nanoparticles into a solution containing 20 mM Hepes and 40 mM KCI, at a pH ranging from 5.5 to 8.5 without or with TFA. From left to right: No TFA, pH 5.5; ΙΟμΜ TFA, pH 5.5; ΙΟΟμΜ TFA, pH 5.5; 1 mM TFA, pH 5.5; No TFA, pH 6.5; ΙΟμΜ TFA, pH 6.5; ΙΟΟμΜ TFA, pH 6.5; 1 mM TFA, pH 6.5; No TFA, pH 7.5; No TFA, pH 8.5

 Figure 7: Transfection of H9c2 cells with mRNA nanoparticles in a solution containing 20 mM Hepes, 40 mM KCl and 100 μl sodium trifluoroacetate at a final RNA concentration of 6.6 μg / ml or 250 μg / ml. From left to right: load ratio 2, [mRNA] = 6.6 μg / ml; loading ratio 2.2, [mRNA] = 250 μg / ml; load ratio 2, [mRNA] = 250 μg / ml; charge ratio 1.8, [mRNA] = 250 μg / ml.

 Figure 8: Transfection of healthy rat myocardium with luciferase mRNA nanoparticles dissolved in solutions of variable tonicity. Measurement of luciferase activity (RLU / mg protein). The last histogram on the right corresponds to an isotonic solution. The others correspond to hypotonic solutions.

 Figure 9: Rat model of myocardial infarction. In A, representative section of a rat heart with a heart attack and placebo. In B, a representative section of a rat heart having undergone infarction and received the mRNA vector encoding a growth factor. The asterisk indicates the position of the infarct.

Efficiency of messenger RNA transfer:

 The amount of mRNA introduced into cultured cells or cells of a tissue can be accurately estimated using the mRNA encoding a protein-label. Luciferase is the most used protein-marker. It is detected by a specialized spectrophotometer, the luminometer.

 There is a direct relationship between the amount of luciferase mRNA transferred into a cell and the amount of luciferase protein produced by that cell. The cationic peptides A and A'-EL, where A and A 'each represents the peptide of SEQ ID No. 1, E is the PEG of 3.5 kDa and L is the peptide of SEQ ID No. 25, were mixed. to luciferase mRNA, to form nanoparticles. These have been shown to be very effective in transferring mRNA into cells of the H9c2 line in vitro.

The transfer of luciferase mRNA into the healthy myocardium of Sprague-Dawley rats necessitated the development of the various parameters of vector administration. Indeed, the latter is injected into the wall of the left ventricle through a thoracotomy. The injection volume, the RNA dose, the saline concentration and the injection rate were optimized, which made it possible to introduce a large quantity of luciferase mRNA into myocardial cells. EXAMPLE 1 PREPARATION OF THE MESSENGER RNA VECTOR

 a) Linearization of the plasmid

 Fifty micrograms of a plasmid, containing the luciferase gene (Firefly) under the control of the phage T7 promoter, were digested per hundred units of the Sspl-HF restriction enzyme (New England Biolabs) in NEBuffer 4 buffer. (50mM Potassium Acetate, 20mM Tris Acetate, 10mM Magnesium Acetate, 1mM DTT, pH 7.9 at 25 ° C), for four hours, at 37 ° C. An Sspl restriction site is downstream of the luciferase gene. The linearized DNA was then precipitated with 5 μl of 0.5 M EDTA, 10 μl of 3 M sodium acetate and 235 μl of 100% ethanol. The mixture was cooled to -20 ° C for one hour, before being centrifuged at maximum speed, for thirty minutes. The DNA pellet was then resuspended in 50 μΙ TE pH 8.0. The concentration of the DNA solution was determined by measuring the absorbance at 260 nm using a spectrophotometer.

 (b) In vitro transcription

 The in vitro transcription of the linearized plasmid was carried out using the mMessage mMachine kit (Ambion). 4.65 μl of pure water were mixed with 10 μl of NTP / CAP 2 ×, 2 μl of 10 × reaction buffer, 1.35 μl of linearized plasmid (1 μg of DNA) and 2 μl of enzyme mix. RNA synthesis was performed at 37 ° C for two hours in a dry bath. T7 RNA polymerase transcribed the luciferase gene through the T7 promoter located upstream of the gene. After transcription of the luciferase gene, a μΙ of TURBO DNase was added, in order to degrade the plasmid and thus facilitate the subsequent purification of the mRNA.

 c) Purification of the messenger RNA

 Purification of the luciferase messenger RNA was performed using the MegaClear kit (Ambion). Seventy-nine μΙ of Elution Solution, 350 μΙ of Binding Solution Concentrate and 250 μΙ of 100% ethanol were added to the 21 μΙ of the previous mixture. These 700 μΙ were deposited on a Filter Cartridge and centrifuged at 10000 g for one minute. The filter retained the messenger RNA. Two washings were carried out with 500 μl of Wash Solution, centrifuging at 10,000 g for one minute. The RNA was then eluted from the filter by adding, twice, 50 μl of Elution Solution and heating at 70 ° C for ten minutes in a dry bath. Elution was obtained by centrifugation at 10,000 g for one minute.

 A second purification step was carried out by lithium chloride precipitation.

Sixty μΙ of LiCI Precipitation Solution was added to the 100 μΙ eluate. The mixture was cooled to -20 ° C for 45 minutes, before being centrifuged at maximum speed at 4 ° C for 15 minutes. The pellet was washed with 500 μl of 70% ethanol and a last centrifugation was carried out at maximum speed at 4 ° C. for 5 minutes. The messenger RNA pellet, dried during a few minutes in the air, was resuspended in Hepes 20 mM pH 7.5. The concentration of the mRNA solution was determined by measuring the absorbance at 260 nm, using a spectrophotometer.

 d) Peptides A and A'-E-L: pepMB1 and pepMB2

 The two peptides capable of interacting with the mRNA were synthesized by Proteogenix. The amino acid sequence of A is: CRRRRRRRRC (pepMB1). The lyophilizate was resuspended in 20 mM Hepes pH 7.5 at 1 mg / ml.

 The sequence of L-E-A 'is as follows: PQRDTVGGRTTPPSWGPAKA-PEG (3.5 kDa) - CRRRRRRRRC (pepMB2). PEG (3.5 kDa) is 3.5 kilodaltons polyethylene glycol. The lyophilizate of L-E-A 'was resuspended in 20 mM Hepes pH 7.5 at 100 μg / ml.

 e) Assembly of the messenger RNA nanoparticles

 The concentrated solution of messenger RNA luciferase was diluted in 20 mM Hepes pH 7.5 to reach 100 μg / ml. The solutions of pepMB1 and pepMB2 were successively mixed with 20 mM Hepes pH 7.5, in order to reach the necessary amount of peptides to condense the luciferase messenger RNA. A volume of the peptide solution was then rapidly mixed by pipetting to one volume of the diluted RNA solution. The assembly of the nanoparticles took place at room temperature for four hours. The binding of the peptides to the RNA is very fast, whereas the oxidation of the cysteines at the ends of the peptides by the oxygen dissolved in the buffer is much slower. This oxidation causes the formation of disulfide bridges between the peptides, which results in their multimerization in contact with the RNA. The nanoparticle consisting of mRNA, trapped in a gangue of multimerized peptides, is very stable in the extracellular medium.

 The amount of peptides mixed with the luciferase messenger RNA depends on the +/- charge ratio that one wishes to achieve. At a + / - charge ratio of 2, enough peptides must be added so that there are two arginines on the peptides, for a phosphodiester bond at the level of the mRNA.

Example 2: IN VITRO TRANSFECTION (Protocol 1)

 a) Culture and plating of the H9c2 line

All cell manipulations were performed under a laminar flow hood. The H9c2 cell line derived from rat cardiomyoblasts (ECACC) was cultured in DMEM Glutamax (Gibco) supplemented with a mixture of penicillin and streptomycin and fetal calf serum (10% final). The culture was carried out at 37 ° C in flasks of 75 cm ² (Corning). When the number of cells required for inoculation of a 48-well plate (Corning) was reached, the cells were detached from the bottom of the flask using 2.5 ml of TryPLE Select (Gibco) to 37 ° C, for 5 to 10 minutes. 7.5 ml of DMEM was added to neutralize the TryPLE Select. The cells were centrifuged at 100 g for 10 minutes at room temperature. The cell pellet was then resuspended in DMEM. 250 μl of this cell suspension was added to each well of a 48-well plate and the latter was placed in an incubator at 37 ° C., containing 5% CO 2. About 16 hours later, the culture medium was replaced with 250 μl of DMEM, preheated to 37 ° C.

 b) Transfection of H9c2 cells

 Each transfection was performed in three different wells. 160 μl of luciferase mRNA (16 μg) were mixed with 160 μl of pepMB1 alone or with 160 μl of a pepMB1 / pepMB2 mixture (pepMB1 and pepMB2 are as defined in example 1). Assembly of the complexes took place at room temperature for four hours. Alternatively, the mRNA solution was diluted with 160 μl of 20 mM Hepes pH7.5 in order to compare the naked RNA with the nanoparticles. 160 μl of DMEM IX containing neither serum nor antibiotics were added to the solutions of naked RNA or nanoparticles, in order to constitute the hypotonic solutions of mRNA (the concentration in cations is approximately 55 mM). The isotonic solutions (the cation concentration is approximately 160 mM) were obtained by mixing 320 μΙ of nanoparticles in 20 mM Hepes pH 7.5 to 160 μl of DMEM 3X. The DMEM (IX) culture medium is isotonic with respect to the interior of the cells. The DMEM 3X medium is three times more concentrated: approximately 480 mM of cations.

 The wells were emptied of the culture medium that they contained, in order to introduce 150 μΙ of hypotonic or isotonic solution of nanoparticles or bare RNA (5 μg of mRNA per well). Cells were incubated for one hour at 37 ° C in an incubator. The solution of nanoparticles or naked mRNA was then aspirated and replaced with 250 μΙ of DMEM. The cells were then incubated for 16 hours at 37 ° C in an incubator.

 c) Lysis of H9c2 cells and measurement of luciferase activity

The culture medium was aspirated and replaced with 500 μl of Dulbecco's PBS IX. The H9c2 cells were then lysed by the addition of 250 μl of lysis buffer (Luciferase Assay System, Promega). The lysate of each well was centrifuged at maximum speed at 20 ° C for five minutes to clarify. 20 μl of each cell lysate were introduced into a tube adapted to the luminometer (Berthold Technologies). 100 μl of luciferase substrate (Promega) were added to the cell lysate by the luminometer. The latter then measured the amount of light emitted by the enzymatic reaction catalyzed by luciferase. The results are expressed in relative light units (RLU). The amount of luciferase protein produced by H9c2 cells, thanks to to luciferase mRNA, was normalized by assaying total cellular proteins with the 660 nm Protein Assay kit (Pierce). For this, 100 μl of cell lysate were mixed with 1.5 ml of reagent and the absorbance was measured at 660 nm. A calibration range was performed using solutions of bovine serum albumin. Luciferase activity is therefore expressed in RLU per milligram of protein. Each experiment was performed in triplicate

 d) Results

In an isotonic solution, the mRNA vector consisting of the single pepMB1 peptide transferred an extremely small amount of RNA into H9c2 cells (Fig.l). The addition of peptide pepMB2 to the vector significantly improved luciferase mRNA transfer. However, the efficiency of transfection remained very low (<10 ⁵ RLU / mg protein). The same vector consisting of pepMB1 and pepMB2 very efficiently transfected H9c2 cells (> 10 ⁸ RLU / mg protein), when the vector solution was hypotonic (Fig.l).

 In a hypotonic solution, naked mRNA transfected H9c2 cells more than 100-fold less efficiently than the same luciferase mRNA complexed with peptide pepMB1 (Fig.2). The addition of peptide pepMB2 to the vector improved the efficiency of the transfection. The highest increase in luciferase activity was obtained with 2.5 mol% pepMB2 (Fig.2).

 Based on these results, the formation of an mRNA vector, consisting of peptides pepMB1 and pepMB2, and the use of a hypotonic solution had a synergistic effect on the efficiency of transfection of H9c2 cells by the Luciferase mRNA.

Example 3: IN VITRO TRANSFECTION (PROTOCOL 2)

 a) Assembly of the luciferase mRNA nanoparticles

 The luciferase messenger RNA pellets and pepMB1 peptide were dissolved in demineralized water. One volume of the peptide solution was then rapidly mixed by pipetting to one volume of the RNA solution. The charge ratio ranged from 1.8 to 2.2. The assembly took place at room temperature for 15 minutes. Then, the RNA / pepMB1 complex solution was frozen with liquid nitrogen, in order to be lyophilized. The lyophilizate was stored at -20 ° C until use.

 This lyophilizate was resuspended in various aqueous solutions optionally containing Hepes, NaCl, KCl and sodium trifluoroacetate.

 b) Transfection of H9c2 cells

Each transfection was performed in three different wells. The culture medium was aspirated and 150 μl of a solution of nanoparticles mRNA / pepMB1 were introduced into each well. Cells were incubated for 30 minutes at 37 ° C in an incubator. The solution Nanoparticles were then aspirated and replaced with 250 μΙ DMEM. The cells were then incubated for 17 hours, at 37 ° C., in an incubator,

 c) Results

 The luciferase / pepMB1 mRNA complex lyophilizate and a purified mRNA pellet were dissolved in a solution containing 20 mM Hepes, pH 5.5 and sodium chloride at a concentration of up to 145 mM. The concentration of mRNA is 1 μg for 150 μΙ of aqueous solution, that is to say that the concentration of nanoparticles is 13.3 μg / ml.

 The addition of peptide pepMB1 increased the efficiency of transfection more than 100-fold (Fig. 3, last two histograms).

 At the optimal concentration of sodium chloride, 40 mM, luciferase activity was 210 times higher than 145 mM (Fig.3).

 The lyophilizate of RNA / pepMB1 nanoparticles was dissolved in a solution containing 40 mM NaCl and Hepes at a concentration of up to 100 mM. The concentration of mRNA is 1 μg for 150 μΙ of aqueous solution, that is to say that the concentration of nanoparticles is 13.3 μg / ml.

 The transfection efficiency was found to be maximal at 20 mM Hepes (Fig.4).

 The luciferase mRNA vector lyophilizate was then dissolved in a solution containing 30, 40 and 50 mM KCl and 20 mM Hepes, pH 5.5. The concentration of mRNA is 1 μg for 150 μl of aqueous solution, that is to say that the concentration of nanoparticles is 13.3 ng / ml.

 Compared with 40 mM NaCl, the optimal concentration of KCl, 40 mM, moderately improved luciferase activity (Fig. 5).

 The lyophilizate of nanoparticles was dissolved in a solution containing 20 mM Hepes and 40 mM KCl, at pH 5.5 to 8.5. The concentration of mRNA is 1 μg for 150 μΙ of aqueous solution, that is to say that the concentration of nanoparticles is 13.3 μg / ml. The optimum pH was found to be 6.5 (Fig.6).

 At this pH, the addition of sodium trifluoroacetate moderately improved the efficiency of transfection. On the other hand, 100 μΜ sodium trifluoroacetate significantly increased the luciferase activity at pH 5.5 (Fig.6).

The mRNA vector was prepared at a charge ratio of 1.8 to 2.2. The RNA / peptide complex lyophilizates were dissolved in a solution containing 20 mM Hepes, 40 mM KCl and 100 μl sodium trifluoroacetate at a final RNA concentration of 6.6 μg / ml or 250 μg / ml. At a load ratio of 2, the 37.5-fold increase in A Nm vector concentration outside H9c2 cells resulted in an increase in transfection efficiency of 436 fold ( Fig.7).

 The charge ratio for obtaining the best mRNA transfer efficiency was found to be 1.8 (Fig.7).

Example 4: TRANSFECTION IN VIVO

 a) Preparation of luciferase mRNA nanoparticles

 Solutions of mRNA, pepMB1 and pepMB2, dissolved in 20 mM Hepes, were diluted with pure water, before mixing, in order to assemble the nanoparticles (pepMB1 and pepMB2 are such that defined in Example 1). The resulting solution contained 8 mM sodium. The +/- load ratio was 1.75 and the pepMB1 / pepMB2 molar ratio was 39.

 MRNA, pepMB1 and pepMB2 solutions were alternatively diluted in aqueous solutions containing sodium chloride, to obtain 24 mM, 68 mM, 112 mM and 160 mM sodium mRNA nanoparticle solutions. .

 Each experiment was performed on three different rats.

 b) Anesthesia, thoracotomy, transepicardial injection and awakening

 Sprague-Dawley rats were anesthetized by intraperitoneal injection of ketamine (100 mg / kg body weight) and xylazine (10 mg / kg body weight). The animals then underwent intratracheal intubation. The tube was then connected to an artificial respirator.

 An incision of the skin and intercostal muscles was performed on the left side of the thorax to access the heart. Spreaders were used to expand the thoracotomy. The pericardium was dissected in order to be able to perform the transepicardial injection.

 An insulin syringe was filled with 60 μl of hypotonic solution of luciferase mRNA nanoparticles, corresponding to 3 μg of RNA per rat. The solution was injected at a rate of 20 μΙ per second into the wall of the left ventricle.

 The thoracotomy was closed with staples. The rats were placed in a cage filled with pure oxygen and watched until they woke up.

 c) Stabulation, euthanasia, myocardial biopsy and measurement of luciferase activity

 After waking up, the rats were returned to their cage. The stabling lasted about 17 hours.

The animals were then given a massive dose of ketamine (500 mg / kg body weight) intraperitoneally. Their heart was explanted and a fragment of the wall of the ventricle left, corresponding to the region injected the day before, was removed and freed from the blood of the animal, using saline. This biopsy was cut into small pieces using a pair of scissors.

 These biopsy pieces were introduced into a tube containing 250 μl of lysis buffer IX (Luciferase Assay System, Promega). This tube was immediately immersed in liquid nitrogen, in order to preserve the biopsy.

 The various transfected myocardial biopsies were thawed at room temperature and refrozen at -80 ° C for 10 minutes. Three freeze / thaw cycles were performed to lyse the myocardial cells and release the luciferase protein encoded by the mRNA. The tissue lysates were centrifuged at 13000 g at 20 ° C for five minutes. The luciferase being present in the supernatant, 20 μΙ of the latter were introduced into a tube for the determination of luciferase activity, using a luminometer. 10 μl of the supernatant were diluted in 90 μl of water for the determination of the total proteins, using the 660 nm Protein Assay kit (Pierce).

 d) Results

 The luciferase mRNA vector dissolved in an isotonic solution, containing 160 mM sodium, gave a very low RNA transfer efficiency, in the myocardial cells of Sprague-Dawley rats (FIG. The lower the sodium concentration, the more hypotonic the mRNA vector solution, the greater the efficiency of transfection (Fig. 8).

 The results obtained in vivo thus confirm the results obtained on the H9c2 line, according to which a hypo-osmotic solution induces a hypotonic shock, which acts in concert with the vectorization of the mRNA, by the peptides pepMB1 and pepMB2, to transfer a large amount of amount of RNA in the mammalian cells. Example 5: Messenger RNA Therapy of Myocardial Infarction in Animals

Infarction was caused on the ventral side of the left ventricle of Sprague-Dawley rats by permanent ligation of the left anterior descending coronary artery via a thoracotomy. 60 μl of a placebo solution (hypotonic buffer) or 60 μl of a hypotonic solution of mRNA nanoparticles, as described in Example 4, coding for a growth factor, was then injected into the lateral face of the left ventricle. . The thoracotomy was closed and the animals sacrificed one week after the operation. The hearts were explanted and histological sections were taken in the area at risk of infarction. Six rats were included in each group. Control animals receiving placebo did experience significant myocardial destruction of their left ventricle. This is evidenced by the thinning of the ventricle wall at the level of the infarcted zone, as well as fibrosis characterized by collagen deposition (Fig. 9A).

 On the other hand, the zone at risk of infarction of the rats having benefited from the vector of A Nm has been preserved from the destruction. Indeed, the wall of the ventricle at this region is not thinned and no sign of fibrosis is visible (Fig.9B).

 The treatment with messenger RNA coding for a growth factor thus protected the myocardial zone at risk of infarction, in this animal model.

Claims

claims

An aqueous composition comprising messenger RNA nanoparticles (mRNA) encoding a therapeutic protein, characterized in that

 the composition is hypotonic and has an osmolarity of less than 300 mosM,

the mRNA nanoparticles comprise mRNA condensed in an aqueous medium under hypotonic conditions with cationic compounds of type A by formation of non-covalent bonds with the mRNA, reversible in the cytoplasm of mammalian cells.

2. A hypotonic aqueous composition according to claim 1, characterized in that its concentration in cations is less than 150 mM, preferably less than 100 mM.

3. Hypotonic aqueous composition according to any one of the preceding claims, characterized in that it comprises potassium chloride and / or sodium chloride in a concentration ranging from 5 mM to 100 mM.

4. Hypotonic aqueous composition according to any one of the preceding claims, characterized in that it comprises Hepes buffer for adjusting the pH of the aqueous composition between 4 and 7.

5. A hypotonic aqueous composition according to any one of the preceding claims, characterized in that it comprises trifluoroacetate in a concentration ranging from 1 μΜ to 1000 μΜ.

6. Hypotonic aqueous composition according to any one of the preceding claims, characterized in that the nanoparticles also comprise compounds of the type A'-E-L, where

 A 'is a cationic compound, also allowing the condensation of mRNA, by non-covalent bonds with mRNA, reversible in the cytoplasm of mammalian cells

 E is an unloaded spacer and soluble in water

L is a neutral or quasi-neutral ligand peptide.

7. A hypotonic aqueous composition according to any one of the preceding claims, characterized in that the cationic compound of type A or A 'is a cationic peptide of formula X _m - (Y) n -X' _P in which

 X and X 'are the same or different and each represents an amino acid or an amino acid derivative or amino acid homolog carrying a thiol group

 m is 0 or 1

 p is 0 or 1

 Y is an amino acid or an amino acid derivative whose side chain is positively charged, and

 n is an integer ranging from 4 to 20.

8. A hypotonic aqueous composition according to claim 7, characterized in that Y is chosen from arginine, lysine and ornithine, preferably arginine.

9. A hypotonic aqueous composition according to any one of claims 6 to 8, characterized in that the spacer is a water-soluble polymer, linear or crosslinked, having a size ranging from 0.5 kDa to 50 kDa, and the peptide ligand is of formula PQRDTVGGRTTPPSWGPAKA (SEQ ID NO: 25).

10. Hypotonic aqueous composition according to any one of the preceding claims, characterized in that the mRNA nanoparticles have a size less than or equal to 200 nm, more preferably less than or equal to 100 nm.

11. A hypotonic aqueous composition according to any one of the preceding claims, characterized in that it comprises said mRNA nanoparticles in an RNA concentration greater than 50 μg / ml.

12. Kit comprising:

at. A hypotonic aqueous solution, of osmolarity lower than 300 mosM, with a cation concentration of less than 150 mM and advantageously comprising - potassium chloride and / or sodium chloride in a concentration ranging from 5 mM to 100 mM - Hepes buffer for adjusting the pH of the aqueous composition between 4 and 7

 Trifluoroacetate in a concentration ranging from 1 μΜ to 1000 μΜ

Messenger RNA (mRNA) nanoparticles encoding a therapeutic protein, as defined in any one of the preceding claims.

13. A composition or kit according to any one of claims 1 to 12 for use as a medicament.

14. A composition according to claim 13 for use in treating or preventing diseases, disorders or pathological conditions selected from cardiac pathologies such as myocardial infarction, angina pectoris, respiratory pathologies such as asthma, disorders due to a viral, respiratory or other infection, disorders due to an inflammatory reaction, disorders due to a bacterial infection, for the healing of diabetic wounds, pressure ulcers; for the manufacture of mRNA vaccines for the treatment of genetic diseases, autoimmune diseases and cancer.

15. Use of a hypotonic osmolarity composition of less than 300 mosM, with a cation concentration of less than 150 mM and advantageously comprising

 Potassium chloride and / or sodium chloride in a concentration ranging from 5 mM to 100 mM

 - Hepes buffer, to adjust the pH of the aqueous composition between 4 and 7

 Trifluoroacetate in a concentration ranging from 1 μΜ to 1000 μΜ and nanoparticles of mRNA as defined in any one of the preceding claims to improve the transfer efficiency of said mRNA in mammalian cells.