FR3121360A1 - Formulation for messenger RNA delivery - Google Patents

Abstract

Formulation for the delivery of messenger RNA The invention relates to a formulation in the form of a nanoemulsion, comprising a continuous aqueous phase and at least one dispersed phase in the form of lipid nanoparticles in which at least one messenger RNA is complexed on the surface of the lipid nanoparticles , its method of preparation and its uses for the prevention and/or treatment of a disease. Figure for abstract: 1

Description

Formulation for messenger RNA delivery

The present invention relates to a nanoemulsion type formulation useful for the delivery of messenger RNA (mRNA).

Messenger ribonucleic acid, messenger RNA or mRNA, is a transient copy of a portion of DNA corresponding to one or more genes and coding for proteins.

mRNA is used as an intermediate by cells for protein synthesis.

mRNA is a linear single-stranded copy of DNA and is composed of RNA, which includes the protein coding region flanked by non-coding regions. There are three main functional regions in an mRNA: the 5′ untranslated region (5′-UTR), the coding cistron(s) (ORF region) and finally the 3′ untranslated region (3′-UTR). The two untranslated regions or UTR regions (from the English “untranslated regions”) often contain signals for expression or RNA processing. mRNA further contains a 5′ cap and a 3′ poly(A) tail, which allow efficient translation of mRNA into protein. The information carried by the ORF region of the mRNA consists of a series of codons, consecutive triplets of nucleotides which each code for an amino acid of the corresponding protein.

The development of drugs or vaccines based on mRNAs is sought, in order to express a protein of interest in a host cell. Once in the cytoplasm of the host cell, the mRNA is read by the ribosome and translated into a protein that has the desired therapeutic activity.

The prerequisite is therefore that the mRNA reaches the cytoplasm.

There are two major obstacles to the development of mRNA-based vaccines or therapies: their instability in vivo, in particular because of their degradation for RNases, and their instability during storage, which hinders the development of mRNA vaccines. . Increasing the mRNA concentration to compensate for the degraded mRNA is not an option, since the vaccine would then become much too expensive.

The delivery systems used include lipid nanoparticles (LNP) or liposomes, which protect mRNA by encapsulating it within a nanoparticle or a lipid vesicle. The encapsulation of the mRNA at the heart of an LNP advantageously makes it possible to protect it from degradation by nucleases. The review Guevara et al. Frontiers in Chemistry 2020, 8, 589959 reports the use of LNPs in which mRNA is encapsulated as a vehicle for mRNA delivery and its use for the treatment of cancer.

The literature describes a few delivery systems based on the use of LNPs on the surface of which nucleotide sequences are complexed. Thus, application WO 2014/032953 describes a formulation in the form of a nanoemulsion, comprising a continuous aqueous phase and at least one dispersed phase, and comprising an amphiphilic lipid, a cationic surfactant, a co-surfactant, a solubilizing lipid, and a sequence nucleotide capable of modulating the interference mechanisms. This nucleotide sequence is short, since it comprises less than 200 bases for a single-stranded nucleotide sequence or less than 200 base pairs for a double-stranded nucleotide sequence. It is preferably microRNA or SiRNA. In the examples, siRNA GFP-22 siRNA rhodamine, siGFP or miRIDIAN Mimic Human has-miR612 microRNA were used. Thus, this application only shows the feasibility of this approach with very short RNAs, and does not suggest that the vehicle can be adapted for the delivery of sequences having more than 200 bases.

There are indeed prejudices for those skilled in the art to use a delivery system in which a large nucleotide sequence is located on the crown of lipid nanoparticles.

Indeed, contrary to the encapsulation of the mRNA in the core of the lipid nanoparticles, the complexation of the mRNA on the surface of these results in exposing it to the surface of the nanoparticle. However, mRNA is a fragile molecule and sensitive to RNA degrading enzymes (RNase) which are present in most biological media. Complexing the mRNA on the surface of the lipid nanoparticles therefore makes it accessible to RNases, all the more so since it is a large molecule which, once complexed on the lipid nanoparticle, exceeds well beyond the lipid nanoparticle. For this reason, the person skilled in the art is discouraged from complexing large nucleotide sequences on the surface of lipid nanoparticles.

Another prejudice against the complexation method is the strong affinity of the electrostatic interaction between a large (negatively charged) nucleic acid, such as mRNA, and a cationic (positively charged) lipid nanoparticle, which can interfere with the release of mRNA from the mRNA-LNP complex once it is in the cytoplasm of the host cell. Because it is not only necessary for the mRNA to reach the cytoplasm, but also for it to be released so that it can perform its role.

There is a need for a delivery system for large nucleotide sequences, in particular mRNA, which is sufficiently stable in storage and in vivo to allow efficient delivery.

To this end, according to a first object, the invention relates to a formulation in the form of a nanoemulsion, comprising a continuous aqueous phase and at least one dispersed phase in the form of lipid nanoparticles, and comprising:

- at least 5% molar of amphiphilic lipid,

- from 15 to 70% molar of cationic surfactant bearing at least one positive charge comprising:

- at least one lipophilic group chosen from:

- an R or R-(C=O)- group, where R represents a linear hydrocarbon chain comprising from 11 to 23 carbon atoms,

- an ester or an amide of fatty acids comprising from 12 to 24 carbon atoms and of phosphatidylethanolamine, and

• a poly(propylene oxide), and

- at least one hydrophilic group comprising at least one cationic group chosen from:

• a linear or branched alkyl group comprising from 1 to 12 carbon atoms and interrupted and/or substituted by at least one cationic group, and
• a hydrophilic polymeric group comprising at least one cationic group, and
• from 10% to 55% molar of co-surfactant comprising at least one poly(ethylene oxide) chain comprising at least 25 ethylene oxide units,
• a solubilizing lipid,
• possibly a fusogenic lipid,

where the molar percentages of amphiphilic lipid, cationic surfactant and co-surfactant are relative to the cumulative molar proportions of amphiphilic lipid, cationic surfactant, co-surfactant and any fusogenic lipid,

in which at least one messenger RNA, the single-stranded nucleotide sequence of which comprises at least 300 bases, is complexed to the surface of the lipid nanoparticles,

the N/P ratio of the quantity of positive charges of the cationic surfactant relative to the quantity of negative charges of the messenger RNA being greater than or equal to 2/1.

Surprisingly, such a formulation is stable and has the ability to protect from the external biological environment and to efficiently deliver mRNA so that it can be translated into protein by a eukaryotic cell. The formulation advantageously has good bioavailability and makes it possible to limit the degradation of the mRNA generally observed with other delivery systems, in particular to limit the degradation by RNases.

In the formulation of the invention, the mRNA is complexed on the surface of a lipid nanoparticle, which is advantageously very stable on storage. The mRNA-lipid nanoparticle complex can be stored for at least two weeks at 4°C without any degradation being observed.

The emulsion is an oil-in-water type emulsion. It can be single or multiple, in particular by comprising in the dispersed phase a second aqueous phase. Preferably, it is simple.

Cationic surfactant

The formulation according to the invention comprises a cationic surfactant comprising:

- at least one lipophilic group chosen from:

- a group R representing a linear hydrocarbon chain comprising from 11 to 23 carbon atoms,

- an ester or an amide of fatty acids comprising from 12 to 24 carbon atoms and of phosphatidylethanolamine, such as distearyl phosphatidylethanolamine (DSPE), and

- a poly(propylene oxide), and

- at least one hydrophilic group comprising at least one cationic group chosen from:

- a linear or branched alkyl group comprising from 1 to 12 carbon atoms and interrupted and/or substituted by at least one cationic group, and

- a hydrophilic polymeric group comprising at least one cationic group, said polymeric group being chosen in particular from:

- a poly(ethylene oxide) typically comprising from 3 to 500 ethylene oxide units, preferably from 20 to 200 ethylene oxide units, and comprising at least one cationic group.

- a polysaccharide, such as dextran, cellulose or chitosan, having in particular molecular masses between 0.5 and 20 kDa, for example between 1 and 12 kDa,

- a polyamine, such as a chitosan or a polylysine, having in particular molecular masses of between 0.5 and 20 kDa, for example between 1 and 12 kDa.

By “ester or amide of fatty acids comprising from 12 to 24 carbon atoms and of phosphatidylethanolamine”, is meant a group of formula:

in which

- R ₃ and R ₄ independently represent a linear hydrocarbon chain comprising from 11 to 23 carbon atoms,

- A ₃ and A ₄ represent O or NH, and

- M represents H or a cation.

The cationic groups of the cationic surfactant are typically:

- oniums chosen from ammonium, imidazolium, pyridinium, pyrrolidinium, piperidinium, phosphonium or sulfonium groups, or

- metal complexes between a radical of a mono- or multi-dentate chelating organic group, for example phenanthroline, pyridine, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), porphyrins, phthalocyanines, clorines, bacteriochlorines complexed with an inorganic cation, such as Ca ²⁺ , Al ³⁺ , Ni ⁺ , Zn ²⁺ _, Fe ²⁺ , Fe ³⁺ or Cu ²⁺ ,

- ammonium groups, in particular - ⁺ NMe ₃ , - ⁺ NHMe ₂ , - ⁺ NH ₂ Me and - ⁺ NH ₃ , in particular - ⁺ NH ₃ , being particularly preferred.

Of course, anions are associated with the cationic group(s) so that the formulation is electrically neutral. The nature of the anions is not limited. By way of illustration, mention may be made of halides, in particular chloride or bromide, or trifluoroacetate.

In the cationic surfactant, the nature of the linking group linking the lipophilic group(s) to the hydrophilic group(s) comprising at least one cationic group is not limited. Examples of linking groups are provided below (group L).

In one embodiment, the cationic surfactant has the following formula (A):

[(Lipo) _l -L-(Hydro) _h ] ⁿ⁺ , (n/m) [A] ^m- (A)

in which:

- l and h represent whole numbers independently between 1 and 4,

- n is an integer greater than or equal to 1, generally between 1 and 50,

- Lipo represents a lipophilic group as defined above,

- Hydro represents a hydrophilic group as defined above comprising at least one cationic group,

- L represents a linking group,

- A represents an anion,

- m is an integer representing the charge of the anion,

- vn is an integer representing the charge of the cation [(Lipo) _l -L-(Hydro) _h ].

In the aforementioned formula (A), preferably L is such that:

- when l and h represent 1, L is a divalent linking group chosen from:

- a single bond,

- a group Z chosen from –O-, -NH-, -O(OC)-, -(CO)O-, -(CO)NH-, -NH(CO)-, -O-(CO)-O -, -NH-(CO)-O-, -O-(CO)-NH- and –NH-(CO)-NH, -O-PO(OH)-O- or a divalent cyclic radical of 5 to 6 atoms,

- an Alk group being an alkylene comprising from 1 to 6 carbon atoms, and

- a Z-Alk, Alk-Z, Alk-Z-Alk or Z-Alk-Z group where Alk and Z are as defined above and where the two Z groups of the Z-Alk-Z group are identical or different ,

- when one of the groups l or h represents 1, and the other represents 2, L is a trivalent group chosen from a phosphate group OP-(O-) ₃ , a group derived from glycerol of formula –O-CH ₂ -CH-(O-)CH ₂ -O- and a cyclic trivalent radical of 5 to 6 atoms,

- for the other values of l and h, L is a cyclic multivalent radical of 5 to 6 atoms.

Particularly preferably, L is such that:

- when l and h represent 1, L is a divalent linking group chosen from:

- a single bond,

- a group Z chosen from –O-, -NH-, -O(OC)-, -(CO)O-, -(CO)NH-, -NH(CO)-, -O-(CO)-O -, -NH-(CO)-O-, -O-(CO)-NH- and –NH-(CO)-NH or -O-PO(OH)-O-,

- when one of the groups l or h represents 1, and the other represents 2, L is a trivalent group chosen from a phosphate group OP-(O-) ₃ and a group derived from glycerol of formula –O-CH ₂ -CH-(O-)CH ₂ -O-.

In the above formula (A), l and h are preferably independently 1 or 2.

According to a first alternative, the hydrophilic group of the cationic surfactant is a linear or branched alkyl group comprising from 1 to 12 carbon atoms and interrupted and/or substituted by at least one cationic group. As an example of such cationic surfactants, mention may be made of:

1) (Lipo)-(CH ₂ ) _m1 -NR ₃₀ R ₃₁ R ₃₂ , where Lipo is a lipophilic group as defined above, m1 represents 1 or 2 and R ₃₀ , R ₃₁ and R ₃₂ independently represent H, Me or –CH ₂ -CH ₂ -OH,

2) , where each Lipo is independently a lipophilic group as defined above, and R33 represents H, Me or –CH ₂ -CH ₂ -OH,

3) , where Lipo is a lipophilic group as defined above, and R _{3 4} , R _{3 5} , R _{3 6} , R _{3 7} , R _{3 8} , R _{3 9} , R _{4 0} , R _{4 1} , R ₄₂ and R ₄₃ independently represents H, Me or –CH ₂ -CH ₂ -OH.

In one embodiment, the cationic surfactant is chosen from:

- N[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium (DOTMA),

- 1,2-dioleyl-3-trimethylamonium-propane (DOTAP),

- N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy-1-propananium) (DMRIE),

- 1-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)imidazolinium (DOTIM), and

- dioctadecylamidoglycylspermine (DOGS) (in protonated form),

and is preferably 1,2-dioleyl-3-trimethylamonium-propane (DOTAP).

According to a second alternative, the hydrophilic group of the cationic surfactant is a hydrophilic polymeric group comprising at least one cationic group.

When the hydrophilic group of the cationic surfactant is polymeric, the cationic group(s) can be terminal or pendent group(s). For example :

- when the hydrophilic polymeric group is a poly(ethylene oxide), the cationic group(s) is (are) generally located on a terminal group at the end of the poly(oxide) chain of ethylene).

- when the hydrophilic polymeric group is dextran or cellulose, the cationic group(s) is (are) generally located on a terminal group at the end of the polysaccharide chain.

- when the hydrophilic polymeric group is chitosan, the cationic group(s) is (are) generally a pendant group(s), in particular –NH ₃ ⁺ groups present in an acid medium on chitosan.

In one embodiment, the cationic group(s) is (are) a terminal group(s). Indeed, the pendant groups of anionic surfactants adjacent to the surface of the lipid nanoparticles of the dispersed phase repel each other by electrostatic interactions and, consequently, the formulations comprising cationic surfactants whose Hydro group comprises pendant groups are generally less stable.

In another embodiment, the cationic group(s) is (are) pendant group(s). It is advantageously possible to use a cationic surfactant whose hydrophilic group comprises several pendent cationic groups, and therefore to obtain a more positively charged formulation, and which will all the better allow the complexation of negative species such as mRNAs.

The preferred hydrophilic polymeric group is a radical of a poly(ethylene oxide) typically comprising from 3 to 500 ethylene oxide units, preferably from 20 to 200 ethylene oxide units, and comprising at least one cationic group.

Thus, in one embodiment, the cationic surfactant has one of the following formulas:

in which :

- R ₁ , R ₂ , R ₃ and R ₄ independently represent a linear hydrocarbon chain comprising from 11 to 23 carbon atoms,

- A ₁ , A ₂ , A ₃ and A ₄ represent O or NH,

- m, n, o and p independently represent whole numbers from 3 to 500, preferably 20 to 200, and

- a represents an integer from 20 to 120,

- M represents H or a cation,

- A ₁₀ , A ₁₁ , A ₁₂ and A ₁₃ independently represent a group – ⁺ NR ₂₀ R ₂₁ R ₂₂ , in which R ₂₀ , R ₂₁ and R ₂₂ independently represent H, Me or –CH ₂ -CH ₂ -OH.

In one embodiment, in formula (AII), A ₁₁ represents - ⁺ NH ₃ and the cationic surfactant has the following formula:

in which A ₂ , R ₂ and n are as defined above. Preferably, in formula (AII), R ₂ represents C ₁₇ H ₃₅ .

Without wanting to be bound to a particular theory, the presence of the hydrophilic polymeric group would allow:

- to stabilize the formulation, and

- to protect the mRNAs, located on the surface of the lipid nanoparticles, from the proteins of the medium in which the formulation is administered/used, and therefore the degradation of the mRNAs by these proteins.

According to a third alternative, the formulation according to the invention comprises at least two cationic surfactants, including:

- one is chosen from:

- N[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium (DOTMA),

- 1,2-dioleyl-3-trimethylamonium-propane (DOTAP),

- N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy-1-propananium) (DMRIE),

- 1-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)imidazolinium chloride (DOTIM), and

- dioctadecylamidoglycylspermine (DOGS),

and is preferably 1,2-dioleyl-3-trimethylamonium-propane (DOTAP), and

- the other is a cationic surfactant comprising:

- at least one lipophilic group chosen from:

- an R or R-(C=O)- group, where R represents a linear hydrocarbon chain comprising from 11 to 23 carbon atoms,

- an ester or an amide of fatty acids comprising from 12 to 24 carbon atoms and of phosphatidylethanolamine, such as distearyl phosphatidylethanolamine (DSPE), and

- a poly(propylene oxide), and

- a hydrophilic polymeric group comprising at least one cationic group, said polymeric group being chosen from:

- a poly(ethylene oxide) typically comprising from 3 to 500 ethylene oxide units, preferably from 20 to 200 ethylene oxide units, and comprising at least one cationic group,

- a polysaccharide, such as dextran, cellulose or chitosan,

- a polyamine, such as a chitosan or a polylysine,

and is preferably poly(ethylene oxide) comprising at least one cationic group.

In one embodiment, the formulation according to the invention comprises, as cationic surfactants:

- 1,2-dioleyl-3-trimethylamonium-propane, and

- a cationic surfactant comprising:

- at least one lipophilic group chosen from:

- an R or R-(C=O)- group, where R represents a linear hydrocarbon chain comprising from 11 to 23 carbon atoms,

- an ester or an amide of fatty acids comprising from 12 to 24 carbon atoms and of phosphatidylethanolamine, such as distearyl phosphatidylethanolamine (DSPE), and

- a poly(ethylene oxide) typically comprising from 3 to 500 ethylene oxide units, preferably from 20 to 200 ethylene oxide units, and comprising at least one cationic group.

In one embodiment, the formulation according to the invention comprises, as cationic surfactants:

- 1,2-dioleyl-3-trimethylamonium-propane, and

- a compound of formula (AII) as defined above, in particular of formula (AV).

The cationic surfactant is located in the crown of the lipid nanoparticles of the formulation. Thanks to the positive charge(s) of its cationic group(s), it binds by electrostatic interactions to the mRNAs and makes it possible to maintain the mRNAs on the surface of the lipid nanoparticles.

The formulation comprises 15 to 70 molar % of cationic surfactant relative to the cumulative molar proportions of the amphiphilic lipid, the cationic surfactant, the co-surfactant and any fusogenic lipid. Below 15%, the formulation does not include enough positive charges and the subsequent complexation of the "premix" formulation with the mRNA (negatively charged) is insufficient. Above 70%, the formulations are not stable, and generally cannot even be formulated (the formation of the nanoemulsion is not possible because the lipid nanoparticles coalesce to form two phases), and the lipid nanoparticles generally become toxic to cells. When the formulation comprises several cationic surfactants, it is their cumulative molar quantity which must respect the range of 15 to 70% molar. In general, in the sense of the application, when there are several components of the same type, the proportion, mass or quantity of all of them must be taken into account.

These proportions are particularly suitable for obtaining effective complexation of mRNAs, and therefore good delivery.

I ipid fusogenic ("helper lipid" in English)

In one embodiment, the formulation according to the invention comprises a fusogenic lipid, which is capable of facilitating cytosolic release by destabilization of the endosomal membrane, called “helper” lipid. Preferably, this lipid is dioleylphosphatidylethanolamine (DOPE).

This lipid makes it possible to promote the endosomal escape of the lipid nanoparticles of the formulation according to the invention, and therefore of the mRNA that they contain.

The fusogenic lipid is located in the crown of the lipid nanoparticles of the formulation.

I amphiphilic ipid

The formulation comprises at least one amphiphilic lipid, which is located in the crown of the lipid nanoparticles of the formulation.

In order to form a stable nanoemulsion, it is necessary to include in the composition at least one amphiphilic lipid as a surfactant. The amphiphilic nature of the surfactant ensures the stabilization of the lipid nanoparticles within the aqueous continuous phase. Below 5 molar % of amphiphilic lipid relative to the cumulative molar proportions of amphiphilic lipid, cationic surfactant, co-surfactant and any fusogenic lipid, the formulations are not stable, and generally cannot even be formulated (the formation of the nanoemulsion is not possible because the lipid nanoparticles coalesce to form two phases).

Generally, the formulation comprises from 5 to 85% molar, preferably from 5 to 75% molar, in particular from 5 to 50% molar and very particularly from 8 to 30% molar of amphiphilic lipid relative to the cumulative molar proportions of the amphiphilic lipid , cationic surfactant, co-surfactant and optional fusogenic lipid.

The amount of amphiphilic lipid advantageously contributes to controlling the size of the dispersed phase of the nanoemulsion.

Amphiphilic lipids have a hydrophilic part and a lipophilic part. They are generally chosen from compounds whose lipophilic part comprises a saturated or unsaturated, linear or branched chain, having from 8 to 30 carbon atoms. They can be chosen from phospholipids, cholesterols, lysolipids, sphingomyelins, tocopherols, glucolipids, stearylamines, cardiolipins of natural or synthetic origin; molecules composed of a fatty acid coupled to a hydrophilic group via an ether or ester function, such as sorbitan esters such as, for example, sorbitan monooleate and monolaurate sold under the names ^Span® by the company Sigma; polymerized lipids; lipids conjugated with short chains of polyethylene oxide (PEG) such as the nonionic surfactants sold under the trade names ^Tween® by the company ICI Americas, Inc. and ^Triton® by the company Union Carbide Corp.; sugar esters such as mono- and di-laurate, mono- and di-palmitate, mono- and distearate of sucrose; said surfactants being able to be used alone or in mixtures.

Phospholipids are the preferred amphiphilic lipids.

Lecithin is the particularly preferred amphiphilic lipid.

I solubilizing ipid

The formulation also comprises a solubilizing lipid comprising at least one fatty acid glyceride, which is located in the dispersed phase of the nanoemulsion, more precisely in the core of the lipid nanoparticles. The main purpose of this compound is to solubilize the poorly soluble amphiphilic lipid in the dispersed phase of the nanoemulsion.

The solubilizing lipid is a lipid exhibiting an affinity with the amphiphilic lipid sufficient to allow its solubilization. Preferably, the solubilizing lipid is solid at room temperature (20°C).

In the case where the amphiphilic lipid is a phospholipid, it may be in particular:

- esters of fatty acids and fatty alcohol, such as cetylpalmitate, or

- glycerol derivatives, and in particular glycerides obtained by esterification of glycerol with fatty acids.

The solubilizing lipid used is advantageously chosen according to the amphiphilic lipid used. It will generally have a similar chemical structure, in order to ensure the desired solubilization. It can be an oil or a wax. Preferably, the solubilizing lipid is solid at room temperature (20°C), but liquid at body temperature (37°C).

Preferred solubilizing lipids, in particular for phospholipids, are esters of fatty acids and of fatty alcohol, such as cetylpalmitate, or glycerides of fatty acids, in particular of saturated fatty acids, and in particular of fatty acids saturated containing 8 to 18 carbon atoms, more preferably 12 to 18 carbon atoms. Advantageously, it is a mixture of different glycerides.

Preferably, they are saturated fatty acid glycerides comprising at least 10% by weight of C12 fatty acids, at least 5% by weight of C14 fatty acids, at least 5% by weight of C16 fatty acids and at least 5% by weight of C18 fatty acids.

Preferably, they are saturated fatty acid glycerides comprising 0% to 20% by weight of C8 fatty acids, 0% to 20% by weight of C10 fatty acids, 10% to 70% by weight of C12 fatty acids, 5% to 30% by weight of C14 fatty acids, 5% to 30% by weight of C16 fatty acids and 5% to 30% by weight of C18 fatty acids.

Particularly preferred are the mixtures of semi-synthetic glycerides which are solid at room temperature sold under the trade name ^Suppocire® NC by the company Gattefossé and approved for injection into humans. Type N Suppocire ^® are obtained by direct esterification of fatty acids and glycerol. These are semi-synthetic glycerides of saturated fatty acids from C8 to C18, the qualitative and quantitative composition of which is indicated in table 1.

Chain length [% in weight] C8 0.1 to 0.9 C10 0.1 to 0.9 C12 25 to 50 C14 10 to 24.9 C16 10 to 24.9 C18 10 to 24.9

Table 1: Fatty acid composition of Suppocire ^® NC from Gattefossé

The aforementioned solubilizing lipids make it possible to obtain a formulation in the form of an advantageously stable nanoemulsion. Without wanting to be bound to a particular theory, it is assumed that the aforementioned solubilizing lipids make it possible to obtain lipid nanoparticles with an amorphous core. The core thus obtained has a high internal viscosity without however having any crystallinity. However, crystallization is harmful for the stability of the nanoemulsion because it generally leads to an aggregation of the lipid nanoparticles and/or to an expulsion of the encapsulated molecules outside the lipid nanoparticles. These physical properties promote the physical stability of the nanoemulsion.

The amount of solubilizing lipid can vary widely depending on the nature and amount of amphiphilic lipid present in the dispersed phase. Generally, the core of the lipid nanoparticles (comprising the solubilizing lipid, the possible oil, the possible imaging agent, the possible therapeutic agent if it is lipophilic) comprises from 1 to 100% by weight, preferably from 5 to 80% by weight and most particularly from 40 to 75% by weight of solubilizing lipid.

H useful

The dispersed phase may also comprise one or more other oils, which are located in the core of the lipid nanoparticles.

The oils used preferably have a hydrophilic-lipophilic balance (HLB) of less than 8 and even more preferably between 3 and 6. Advantageously, the oils are used without chemical or physical modification prior to the formation of the emulsion.

The oils are generally chosen from biocompatible oils, and in particular from oils of natural (vegetable or animal) or synthetic origin. Among such oils, mention may in particular be made of oils of natural vegetable origin, among which are in particular soybean, linseed, palm, peanut, olive, grapeseed and sunflower oils; synthetic oils among which include in particular triglycerides, diglycerides and monoglycerides. These oils can be first expressions, refined or interesterified.

Preferred oils are soybean oil and flaxseed oil.

Generally, if present, the oil will be contained in the core of the lipid nanoparticles (including the solubilizing lipid, the possible oil, the possible imaging agent, the possible therapeutic agent if it is lipophilic) in a proportion ranging from from 1 to 80% by weight, preferably between 5 and 50% by weight and very particularly 10 to 30% by weight.

Preferably, the formulation is free of squalene.

The dispersed phase may additionally contain other additives such as colorants, stabilizers, preservatives or other active principles, in an appropriate amount.

Co-surfactant

The formulation includes a co-surfactant, which stabilizes the nanoemulsion.

The co-surfactants that can be used in the formulations used in the invention are generally water-soluble surfactants. They comprise at least one poly(ethylene oxide) chain comprising at least 25, in particular at least 30, preferably at least 35, ethylene oxide units. The number of ethylene oxide units is usually less than 500.

Formulations comprising a co-surfactant comprising a poly(ethylene oxide) chain comprising less than 25 ethylene oxide units are in fact not stable. Generally, it is not even possible to prepare the nanoemulsion.

These numbers of units are in fact particularly suitable for preventing the escape of mRNAs from the lipid nanoparticles.

Indeed, the inventors have observed that a formulation not comprising a co-surfactant is not sufficiently stable.

Moreover, without wanting to be bound to a particular theory, the presence of the chain composed of ethylene oxide units of the co-surfactant would make it possible to protect the mRNAs, located on the surface of the lipid nanoparticles, from the nucleases of the medium in which the formulation is administered/used, and therefore the degradation of said mRNAs by these nucleases.

By way of example of co-surfactants, mention may in particular be made of the conjugated polyethylene glycol/phosphatidyl-ethanolamine (PEG-PE) compounds, fatty acid and polyethylene glycol ethers such as the products sold under the trade names Brij ^® ( for example Brij ^® 35, 58, 78 or 98) by the company ICI Americas Inc., fatty acid and polyethylene glycol esters such as the products sold under the trade names Myrj ^® by the company ICI Americas Inc. (for example Myrj ^® 45, 52, 53 or 59) and block copolymers of ethylene oxide and propylene oxide such as the products sold under the trade names Pluronic ^® by the company BASF AG (for example Pluronic ^® F68, F127 , L64, L61, 10R4, 17R2, 17R4, 25R2 or 25R4) or the products sold under the trade name ^Synperonic® by the company Unichema Chemie BV (for example ^Synperonic® PE/F68, PE/L61 or PE/L64).

Thus, the co-surfactant is located both in the continuous aqueous phase and in the dispersed phase. Indeed, the hydrophobic part of the co-surfactant is inserted into the lipid nanoparticles of the dispersed phase, while the polyalkoxylated chains are in the continuous aqueous phase. In the present application, the described molar or mass percentages of dispersed phase are calculated considering that the co-surfactant belongs to the dispersed phase.

The formulation comprises from 10% to 55% molar of co-surfactant with respect to the cumulated molar proportions of the amphiphilic lipid, of the cationic surfactant, of the co-surfactant and of any fusogenic lipid. Below 10%, the formulations are not stable, and generally cannot even be formulated (the formation of the nanoemulsion is not possible because the lipid nanoparticles coalesce to form two phases). Beyond 55%, the subsequent complexation of the "premix" formation with the mRNA does not take place, probably because the positive charges of the cationic surfactant are masked by the poly(ethylene oxide) chains of the co-surfactant. , and therefore more accessible to bind by electrostatic binding to mRNA.

The co-surfactant may also have other effects in the intended application of the nanoemulsion.

According to one embodiment, the dispersed phase of the nanoemulsion is surface-grafted with molecules of interest such as biological ligands, in order to increase the specific targeting of an organ. Such grafting allows specific recognition of certain cells or certain organs by promoting the internalization of the lipid nanoparticles of the formulation according to the invention by the target cells which express the receptor on the surface. Preferably, the surface grafting is carried out by coupling the molecules of interest or their precursors with an amphiphilic compound, in particular with the co-surfactant. The nanoemulsion then comprises a grafted co-surfactant. In this case, the co-surfactant plays the role of a spacer making it possible to accommodate the molecules of interest on the surface.

The molecules of interest can be for example:

- biological targeting ligands such as antibodies, peptides, saccharides, aptamers, oligonucleotides or compounds such as folic acid;

- a stealth agent: an entity added in order to give the nanoemulsion stealth vis-à-vis the immune system, to increase its circulation time in the body, and to slow down its elimination.

For example, when the biological ligand is a peptide comprising one or more cysteine (for example to use the formulation for the treatment of a disease), the grafting to the alkylene oxide chain of the surfactant can be ensured by thiol maleimide coupling.

AT imaging gent

In one embodiment, the formulation comprises an imaging agent, which advantageously makes it possible to visualize the distribution of the lipid nanoparticles in the cells or the body of the patient, and therefore the distribution of the mRNAs.

The imaging agent can in particular be used in imaging of the type:

- positron emission tomography (Positron Emission Tomography (PET) in English) (the imaging agent may be a compound comprising a radionuclide, such as ¹⁸ F, ¹¹ C, a chelate of metal cations ⁶⁸ Ga, ⁶⁴ Cu) ,

- Single photon emission computed tomography (SPECT) (TEMP) (the imaging agent may be a compound comprising a radionuclide, for example ¹²³ I, or a chelate of ^99m Tc or ¹¹¹ In) ,

- magnetic resonance imaging (MRI) (the imaging agent may be a gadolinium chelate or a magnetic nanocrystal such as an iron oxide, manganese oxide or iron-platinum FePt nanocrystal),

- optical imaging or X-ray imaging (the imaging agent may be a lipophilic fluorophore or a contrast agent, for example an iodine molecule such as iopamidol, amidotrizoate, or gold nanoparticles).

Preferably, the imaging agent is a lipophilic fluorophore making it possible to carry out optical imaging.

The nature of the lipophilic fluorophore(s) that can be used is not critical as long as they are compatible with in vivo imaging (i.e. they are biocompatible and non-toxic). Preferably, the fluorophores used as imaging agent absorb and emit in the visible or near infrared. For non-invasive imaging in a living tissue or organism (animal, human) the preferred fluorophores absorb and emit in the near infrared. Indeed, so that the excitation light and the light emitted by the fluorophore can better cross the tissues, it is advisable to use fluorophores absorbing and emitting in the near infrared, that is to say at a length of wave between 640 and 900 nm.

By way of lipophilic fluorophore, mention may for example be made of the compounds described in chapter 13 (“Probes for Lipids and Membranes”) of the InVitrogen catalog. More specifically, mention may in particular be made, by way of fluorophore, of indocyanine green (ICG), fatty acid analogues and phospholipids functionalized with a fluorescent group, such as the fluorescent products sold under the trade names Bodipy ( R) such as Bodipy (R) 665/676 (Ex/Em.); lipophilic derivatives of carbocyanins such as 1,1′--dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine (DiD) perchlorate, for example sold under the reference D-307, 3,3′ perchlorate -dihexadecyloxacarbocyanine (DiO), for example sold under the reference D1125, 1,1′-dihexadecyl-3,3,3′,3′-tetramethylindocarbocyanine (DiI) perchlorate, for example sold under the reference D384; fluorescent probes derived from sphingolipids, steroids or lipopolysaccharides such as the products sold under the trade names BODIPY® TR ceramides, BODIPY® FL C5-lactosylceramide, BODIPY® FL C5-ganglioside, BODIPY® FL cerebrosides; amphiphilic derivatives of cyanines, rhodamines, fluoresceins or coumarins such as octadecyl rhodamine B, octadecyl fluorescein ester and 4-heptadecyl-7-hydroxycoumarin; and diphenylhexatriene (DPH) and its derivatives; all of these products being sold by the company Invitrogen.

According to a preferred embodiment of the invention, the fluorophore is indocyanine green, 1,1'--dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate, 3,3'-perchlorate -dihexadecyloxacarbocyanine, or 1,1′-dihexadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate.

AT therapeutic agent

The formulation according to the invention may comprise a therapeutic agent.

The therapeutic agents capable of being encapsulated in the nanoemulsion according to the invention comprise in particular the active principles acting by chemical, biological or physical means. Thus, they may be pharmaceutical active ingredients or biological agents such as DNA, proteins, peptides or antibodies or even agents useful for physical therapies such as compounds useful for thermotherapy, compounds releasing singlet oxygen when excited by light useful for phototherapy and radioactive agents. Preferably, they are active principles administered parenterally.

Depending on its lipophilic or amphiphilic affinity, the therapeutic agent will be encapsulated by the dispersed phase or will be located at the interface of the two phases.

The nature of the therapeutic agents encapsulated in the nanoemulsion is not particularly limited. However, the nanoemulsion is particularly interesting for poorly soluble compounds, which are difficult to formulate in conventional administration systems and for active principles useful for phototherapy, whose quantum yield can be preserved.

Due to the mild conditions of the preparation process, the formulation described is particularly advantageous for the encapsulation of therapeutic agents which degrade at high temperature .

Among the pharmaceutical active principles of interest as therapeutic agents, mention may be made in particular of the agents used in the treatment of AIDS, the agents used in the treatment of heart diseases, analgesics, anesthetics, anorectics, anthelmintics, antiallergics, antianginals, antiarrhythmics, anticholinergics, anticoagulants, antidepressants, antidiabetics, antidiuretics, antiemetics, anticonvulsants, antifungals, antihistamines, antihypertensives, anti-inflammatories, antimigraine drugs, antimuscarinics, antimycobacterials , anticancer drugs including antiparkinsonians, antithyroid drugs, antivirals, astringents, blocking agents, blood products, blood substitutes, cardiac inotropic agents, cardiovascular agents, central nervous system agents, chelating agents, chemotherapy, colleges hematopoietic growth promoters, corticosteroids, antitussives, dermatological agents, diuretics, dopaminergics, elastase inhibitors, endocrine agents, ergot alkaloids, expectorants, gastrointestinal agents, genitourinary agents, growth hormone inducing factor, growth hormones, hematologic agents, hematopoietic agents, hemostats, hormones, immunosuppressants, interleukins, interleukin analogues, regulating agents lipids, gonadotropins, muscle relaxants, narcotic antagonists, nutrients, nutritive agents, oncological therapies, organic nitrates, vagomimetics, prostaglandins, antibiotics, renal agents, respiratory agents, sedatives, hormones stimulants, including immunostimulants, adjuvants, Toll-like receptor agonists (TLRs), sym pathomimetics, systemic anti-infectives, tacrolimus, thrombolytic agents, thyroid agents, treatments for attention disorders, vasodilators, xanthines, cholesterol lowering agents. Particularly targeted are anti-cancer drugs such as taxol (paclitaxel), doxorubicin and cisplatin in particular for therapeutic applications, or immunostimulating agents, adjuvants, TLR agonists, in particular for prophylactic applications (vaccines)

Among the physical or chemical agents, mention may in particular be made of radioactive isotopes and photosensitizers.

Among the photosensitizers, there may be mentioned in particular those belonging to the class of tetrapyrroles such as porphyrins, bacteriochlorins, phthalocyanines, chlorins, purpurines, porphycenes, pheophorbides, or those belonging to the class of texaphyrins or hypericins. Among the first-generation photosensitizers, mention may be made of hemato-porphyrin and a mixture of hemato-porphyrin (HpD) derivatives (sold under the trade name ^Photofrin® by Axcan Pharma). Among the second-generation photosensitizers, mention may be made of meta-tetra-hydroxyphenyl chlorine (mTHPC; trade name Foscan ^® , Biolitec AG) and the monoacid derivative of cycle A of benzoporphyrin (BPD-MA sold under the trade name Visudyne ^® by QLT and Novartis Opthalmics). The formulations of second-generation photosensitizers which combine with these photosensitizers a molecule (lipid, peptide, sugar, etc.) qualified as a carrier which allows their selective delivery to the level of the tumor tissue are called third-generation photosensitizers.

Among the biological agents, mention may be made of oligonucleotides, DNA, RNA, siRNAs, peptides and proteins and saccharides, preferably peptides and proteins and saccharides. Preferably, the formulation is free of SiRNA and/or microRNA, and more generally free of nucleotide sequence capable of modulating endogenous RNA interference mechanisms, or even free of RNA other than mRNA, or even free of nucleotide sequence other than mRNA.

Of course, the therapeutic agent can be formulated directly in its active form or in the form of a prodrug. Further, it is contemplated that several therapeutic agents may be formulated in combination in the nanoemulsion.

The amount of therapeutic agent depends on the intended application as well as the nature of the agent. However, it will generally be sought to formulate the nanoemulsion with a maximum concentration of therapeutic agent, in particular when it comes to poorly soluble therapeutic agents, in order to limit the volume and/or the duration of administration to the patient.

However, it has been observed that the presence of the solubilizing lipid in the dispersed phase makes it possible to incorporate a large quantity of even hydrophobic or amphiphilic compounds.

P proportion of heart in the lipid nanoparticles

Generally, the molar proportion of the core components of the lipid nanoparticles relative to the components of the lipid nanoparticles, without taking into account the molar amount of the mRNA(s), is 10 to 80%, especially 25 to 75%, of preferably from 33.35 to 73.99%. In other words, the molar proportion (mol/mol) of the cumulated molar quantities of solubilizing lipid, of the possible oil, of the possible imaging agent, of the possible lipophilic therapeutic agent) compared to the molar quantity of the dispersed phase (i.e. the cumulated molar quantity of all the components of the dispersed phase, except that of the mRNA(s), i.e. the cumulated molar quantity of solubilizing lipid, any oil , possible imaging agent, possible lipophilic therapeutic agent, amphiphilic lipid, cationic surfactant, co-surfactant, possible fusogenic lipid, possible amphiphilic therapeutic agent, is generally from 10 to 80%, in particular from 25 to 75%, preferably from 33 .35 to 73.99%.

Generally, the mass proportion (wt/wt) of the core components of the lipid nanoparticles relative to the components of the lipid nanoparticles, without taking into account the weight of the mRNA(s), is 10 to 60%, in particular 20 to 60 %, preferably from 23.53 to 59.51%. In other words, the mass proportion of the cumulated masses of the solubilizing lipid, of the possible oil, of the possible imaging agent, of the possible lipophilic therapeutic agent with respect to the dispersed phase (i.e. to the mass of all components of the dispersed phase except mRNA, i.e. the cumulative masses of solubilizing lipid / possible oil / possible imaging agent / possible lipophilic therapeutic agent / amphiphilic lipid / cationic surfactant / co surfactant/possible fusogenic lipid, possible amphiphilic therapeutic agent, is generally from 10 to 60%, in particular from 20 to 60%, preferably from 23.53 to 59.51%.

These molar and/or mass proportions are particularly suitable for the "premix" formulation (that used to prepare the formulation according to the invention, before complexation of the mRNA) to be stable on storage, in particular for it to be stable while being stored more than 2 years at 4°C. The stability can in particular be measured by following the size of the lipid nanoparticles, their polydispersity index and/or their zeta potential (for example by quasi-elastic light scattering, in particular by a device of the ZetaSizer type, Malvern). This stability of the “premix” formulation is important for the applications envisaged. It is particularly interesting to be able to store the “premix” formulation and to carry out the complexation with the mRNA just before using the formulation according to the invention.

Aqueous phase

The aqueous phase of the nanoemulsion used in the invention preferably consists of water and/or a buffer such as a phosphate buffer such as for example PBS ("Phosphate Buffer Saline") or a saline solution, especially sodium chloride.

According to one embodiment, the continuous aqueous phase also comprises a thickening agent such as glycerol, a saccharide, oligosaccharide or polysaccharide, a gum or even a protein, preferably glycerol. Indeed, the use of a continuous phase of higher viscosity facilitates the emulsification and thus makes it possible to reduce the sonication time.

The aqueous phase advantageously comprises from 0 to 50% by weight, preferably from 1 to 30% by weight and very particularly from 5 to 20% by weight of thickening agent.

Of course, the aqueous phase may also contain other additives such as colorants, stabilizers and preservatives in appropriate quantities.

The proportion of dispersed phase and aqueous phase is highly variable. However, most often, the nanoemulsions will be prepared with 1 to 50%, preferably 5 to 40% and very particularly 10 to 30% by weight of dispersed phase (without taking into account the mRNA) and 50 to 99%, of preferably 60 to 95% and very particularly 70 to 90% by weight of aqueous phase.

AR NOT messenger (mRNA)

In the formulation, at least one messenger RNA (mRNA), the single-stranded nucleotide sequence of which comprises at least 300 bases, is complexed to the surface of the lipid nanoparticles.

This mRNA can be translated into protein by a eukaryotic cell.

Its nucleotide sequence is single-stranded, and it comprises at least 300 bases, typically at least 400 bases, in particular at least 500 bases, preferably at least 1000 bases, or even more than 10,000 bases. Generally, it comprises less than 50,000 bases.

mRNA typically includes (and usually consists of) a 5' cap, a 5'-UTR region, an ORF region, a 3'-UTR region, and a 3' poly(A) tail.

mRNA can be natural or synthetic.

The mRNA may in particular be a synthetic mRNA transcribed in vitro (IVT mRNA), for example as described in the article Sahin et al. Nature Reviews 13, 2014, 759.

The synthetic mRNA may comprise one or more non-natural bases, in particular chosen from uridine, pseudoridine, N1-methyl-pseudouridine, 5-methoxy-uridine and 5-methyl-cytidine, provided that the mRNA remains capable of being translated into protein by a eukaryotic cell.

Its 3'-UTR region can be stabilized, in particular by a stabilization sequence rich in pyrimidines. Typically the 3'UTR region is that of α- or β-globin. Usual modifications of synthetic mRNA are described in the paragraph “Improving the translation and stability of mRNA” of the article Sahin et al. Nature Reviews 13, 2014, 759.

Synthetic mRNA usually includes a 3' poly(A) tail.

In the formulation, the mRNA is not covalently linked to the other components of the lipid nanoparticles. In particular, the mRNA is not covalently linked either to the co-surfactant, or to the amphiphilic lipid, or to the possible imaging agent. This is very advantageous because the mRNAs, once released in their site of action, are not denatured and can play the expected role.

An mRNA includes phosphodiester bonds, which include negatively charged phosphate groups. mRNA therefore naturally carries negative charges.

Thanks to their negative charges, the mRNAs are maintained on the surface of the lipid nanoparticles of the dispersed phase of the formulation thanks to electrostatic interactions with the positive charges of the cationic surfactants.

They are therefore located on the surface of the lipid nanoparticles, at the level of the crown of the lipid nanoparticles, on the hydrophilic side of the crown.

In the formulation according to the invention, the N/P ratio of the quantity of positive charges of the cationic surfactant relative to the quantity of negative charges of the messenger RNA is greater than or equal to 2/1, in particular greater than or equal to 4 /1, in particular greater than or equal to 5/1, preferably greater than or equal to 6/1, particularly preferably greater than or equal to 8/1. Lower N/P ratios lead to insufficient mRNA delivery. The higher the N/P ratio, the more the lipid nanoparticles are loaded with mRNA. The N/P ratio is generally less than 2000.

Generally, mRNA is not likely to modulate endogenous RNA interference mechanisms.

Size

The lipid nanoparticles of the formulation generally have a diameter of between 20 and 250 nm, typically between 40 and 200 nm. This diameter can in particular be measured by quasi-elastic light scattering on a ZetaSizer device, Malvern.

Location of the components of the lipid nanoparticles

As illustrated on the , the lipid nanoparticles of the formulation according to the invention are organized in the form of a heart – crown, where:

- the heart includes:

• the solubilizing lipid,
• any oil,
• the possible imaging agent,
• the possible therapeutic agent if it is lipophilic,
• the crown includes:
• the amphiphilic lipid,
• the cationic surfactant,
• the co-surfactant (possibly grafted with a molecule of interest),
• mRNA,
• the possible fusogenic lipid,
• the possible therapeutic agent if it is amphiphilic,
• the optional surfactant of formula (I).

Preferably, the core of the lipid nanoparticles of the formulation is free of mRNA, or even of RNA, or even of nucleotide sequence. In other words, there is generally no mRNA, or even RNA, or even nucleotide sequence, encapsulated inside the lipid nanoparticles. The localization of the mRNAs can for example be verified by transmission electron microscopy (TEM) or by cryo-microscopy (cryo-TEM).

[Preparation process]

According to a second object, the invention relates to the process for preparing the formulation defined above.

Typically, the various oily constituents are first mixed to prepare an oily premix for the dispersed phase of the nanoemulsion, then it is dispersed in an aqueous phase under the effect of shearing, and finally the mRNA is complexed .

The preparation process typically includes the following steps:

1. prepare an oily phase comprising the solubilizing lipid, the amphiphilic lipid, the cationic surfactant, the possible fusogenic lipid;
2. preparing an aqueous phase comprising the co-surfactant;
3. disperse the oily phase in the aqueous phase under the action of sufficient shear to form a formulation in the form of a nanoemulsion; Then
4. add the messenger RNA whose single-stranded nucleotide sequence comprises at least 300 bases to the formulation obtained in step (iii), then

(v) recovering the formulation thus formed.

Step (i)

Generally, the preparation of the oily phase comprises mixing the oily components of the formulation (solubilizing lipid/amphiphilic lipid/cationic surfactant). When the formulation comprises a lipid capable of facilitating cytosolic release by destabilization of the endosomal membrane and/or an oil, and/or an imaging agent and/or a therapeutic agent, these are generally introduced into the oily phase during of step (i).

Mixing may optionally be facilitated by dissolving one of the constituents or the complete mixture in an appropriate organic solvent. The organic solvent is then evaporated, to obtain a homogeneous oily premix for the dispersed phase.

Furthermore, it is preferred to carry out the premix (step (i)) at a temperature at which all the ingredients are liquid.

Step (i ii )

Advantageously, the oily phase is dispersed in the aqueous phase in the liquid state. If one of the phases solidifies at room temperature, it is preferable to carry out the mixture with one or preferably both phases heated to a temperature greater than or equal to the melting temperature.

The emulsification under the shear effect is preferably carried out using a sonicator or a microfluidizer. Preferably, the aqueous phase and then the oily phase are introduced in the desired proportions into a suitable cylindrical container, then the sonicator is immersed in the medium and turned on for a sufficient time to obtain a nanoemulsion, most often a few minutes.

At the end of step (iii), a homogeneous nanoemulsion is generally obtained in which:

- the average diameter of the lipid nanoparticles, measured by quasi-elastic light scattering, for example on a ZetaSizer device, Malvern, is generally greater than 10 nm and less than 200 nm, in particular from 20 to 200 nm, preferably from 30 at 190 nm, and

- the zeta potential is greater than 20 mV, generally between 25 mV and 60 mV, preferably between 40 and 55 mV, measured by electrophoretic light scattering (ELS), for example on a ZetaSizer device, Malvern, preferably when the aqueous phase of the formulation is an aqueous solution of 0.15 mM NaCl.

The nanoemulsion obtained at the end of step (iii) (therefore before the complexation with the mRNA) corresponds to the so-called “premix” formulation.

For a premix formulation comprising lipid nanoparticles of size between 20 and 40 nm, it will be preferred to use a formulation comprising at least 5% molar of amphiphilic lipid and:

• from 25 to 45% molar of co-surfactant (below 25% molar, the formulation may present stability problems), and/or
• from 15 to 50% molar of cationic surfactant.

For a premix formulation comprising lipid nanoparticles of size between 40 and 100 nm, it will be preferred to use a formulation comprising at least 5% molar of amphiphilic lipid and:

• from 45 to 50% molar of co-surfactant (below 45% molar, the formulation may present stability problems. Beyond 50%, the release of mRNA from the LNP-mRNA complex is less efficient, and /Or
• from 30 to 40% molar of cationic surfactant. (below 30% molar, the release of mRNA from the LNP-mRNA complex is less efficient. Above 40%, the formulation may present stability problems).

For a premix formulation comprising lipid nanoparticles of size between 130 and 175 nm, it will be preferred to use a formulation comprising

- at least 5% molar of amphiphilic lipid and 15 to 70% molar of at least one cationic surfactant and:

- from 10 to 25% molar, in particular from 10 to 15% of co-surfactant.

The molar percentages of amphiphilic lipid, cationic surfactant and co-surfactant are relative to the cumulative molar proportions of amphiphilic lipid, cationic surfactant, co-surfactant and any fusogenic lipid.

Step (i v )

By complexing the “premix” formulation defined above with mRNA, the formulation according to the invention is obtained.

Step (iv) then makes it possible to prepare the formulation by complexation of said mRNAs on the “premix” formulation obtained at the end of step (iii).

Generally, the mRNAs are added to the nanoemulsion formed and the mixture obtained is mixed at ambient temperature (from 20 to 25° C.), for example from 5 to 30 minutes.

During step (iv), the mRNAs, carrying negatively charged phosphate groups, bind by electrostatic bonds to the lipid nanoparticles whose surface is positively charged thanks to the cationic groups of the cationic surfactants. Complexes are thus formed between the mRNAs and the lipid nanoparticles of the nanoemulsion. mRNAs are usually added in aqueous solution, e.g. nuclease-free water, cell culture media, culture media. Step (iv) is typically carried out at room temperature (25° C.), after simple homogenization or with stirring (for example between 100 and 1000 revolutions per minute), for a period of between 5 minutes and 2 hours, for example the order of 30 minutes.

During step (iv), the amount of mRNA introduced is such that the ratio between the amount of positive charges due to the cationic surfactant in the “premix” formulation to the amount of negative charges provided by the nucleotide sequences introduced into the middle is greater than or equal to 2/1. A person skilled in the art is able to calculate the quantity of negative charges provided by the nucleotide sequences introduced into the medium, and the quantity of positive charges due to the cationic surfactant in the “premix” formulation.

Step (iv) can be followed by various methods, for example by:

• agarose gel electrophoresis, which makes it possible to observe the migration of mRNAs. If there is good complexation, then the mRNAs complexed with the lipid nanoparticles are heavier and are visualized in the wells. If the complexation is less important, free mRNAs will migrate to another position.
• dynamic light scattering (DLS), by observing the impact of complexation on the hydrodynamic diameter. The more efficient the complexation, the more the profile is oriented towards a monomodal distribution.

At the end of step (iv), a homogeneous nanoemulsion is generally obtained in which the average diameter of the lipid nanoparticles is generally greater than 10 nm and less than 200 nm, preferably between 60 and 200 nm.

Advantageously, the process for preparing the formulation does not require chemically modifying the mRNAs, and in particular does not require them to be grafted covalently to another component of the formulation. It is therefore very easy to prepare in parallel numerous formulations according to the invention comprising mRNAs.

The addition of the mRNA after the manufacture of the lipid nanoparticles makes it possible to rationalize the large-scale manufacture of a premix formulation, which will be later complexed with distinct mRNAs. This brings a strong improvement compared to the encapsulation of mRNA at the heart of LNP, which requires to prepare the LNP again as soon as one wishes to change the nature of the mRNA.

Possible next steps

The formulation can be purified, for example by column purification or by dialysis.

Before conditioning, the emulsion can be diluted and/or sterilized, for example by filtration or dialysis. This step makes it possible to eliminate any aggregates which may have formed during the preparation of the emulsion.

The emulsion thus obtained is ready for use, if necessary after dilution.

According to a third object, the invention relates to the formulation capable of being obtained by the method defined above.

[kit]

According to a fourth object, the invention relates to a kit comprising the “premix” formulation as defined above, and separately, at least one mRNA.

In the kit, the “premix” formulation is physically separated from the mRNA. It is for example possible to use a kit comprising at least two containers, one for the “premix” formulation, and at least one for an mRNA, preferably several each containing mRNAs of a different nature.

As explained above, the "premix" formulation, in particular thanks to:

- the molar proportion of cationic surfactant in the crown of the lipid nanoparticles,

- the molar proportion of amphiphilic lipid in the crown of the lipid nanoparticles,

- the molar proportion of co-surfactant in the crown of the lipid nanoparticles,

- the fact that the co-surfactant comprises a poly(ethylene oxide) chain comprising at least 25 ethylene oxide units,

- the presence of solubilizing lipid in the core of the lipid nanoparticles, and/or

- the molar and/or mass proportion of core in the lipid nanoparticles,

is advantageously stable. This stability of the “premix” formulation makes it possible to store the “premix” formulation for the long term, in particular more than two years at 4° C., which is an enormous advantage for being able to store and market the kit defined above.

It is thus advantageously possible to prepare the formulation according to the invention from the “premix” formulation and from the mRNA just before using the formulation according to the invention. The “premix” formulation can be stored. Thus, it is not necessary to prepare the “premix” emulsion each time one wishes to use the formulation according to the invention, which is an advantage in terms of saving time and cost.

[ Usage ]

The formulation is advantageously stable and low in toxicity. It can be used as an in vitro or in vivo mRNA delivery system.

Thus, according to a fifth object, the invention relates to the formulation defined above for its use for the prevention and/or treatment of a disease.

A preferred use of the formulation according to the invention is the delivery of an mRNA sequence coding for an antigenic protein with the aim of inducing a prophylactic immune response against pathogens. These pathologies can be, for example, inflammatory pathologies, cancers (cancer, solid tumors, metastases but also chronic myeloid leukemia), or even infectious diseases in the case of prophylactic therapy. The infectious disease can be that induced by the hepatitis C virus (HCV), by the human immunodeficiency virus (HIV), by the respiratory syncytial virus (RSV), by a coronavirus, in particular by the strain of coronavirus SARS -CoV-2, for example COVID19, or the flu.

When the formulation comprises an imaging agent, it is advantageously possible to monitor the delivery of the mRNAs, for example by monitoring the fluorescence when the imaging agent is a lipophilic fluorophore.

Advantageously, as explained above, the formulation according to the invention protects the mRNAs of the body of the patient (and of these nucleases) to which the formulation is administered.

The formulation may comprise a therapeutic agent making it possible to treat the disease, which makes it possible to benefit from a double therapeutic or prophylactic effect: that induced by the mRNAs and that induced by the therapeutic agent.

Because it can be prepared exclusively from constituents approved for humans, it is attractive for parenteral administration. However, it is also possible to envisage administration by other routes, in particular by the oral route, by the topical route, or by the ophthalmological route.

A method of preventing and/or treating a disease comprising the administration to a mammal, preferably a human, in need thereof of an effective amount of the formulation as defined above is also one of the objects of the present invention.

[Definitions]

In the context of the present application, the term “nanoemulsion” is understood to mean a composition having at least two phases, generally an oily phase and an aqueous phase, in which the average size of the dispersed phase is less than 1 micron, preferably from 10 to 500 nm and in particular from 20 to 200 nm, and very preferably from 50 to 200 nm (see article C. Solans, P. Izquierdo, J. Nolla, N. Azemar and MJ Garcia-Celma, Curr Opi n Colloid In , 2005, 10, 102-110).

Within the meaning of the present application, the expression "dispersed phase" means the lipid nanoparticles comprising the possible oil / the cationic surfactant / the solubilizing lipid / the amphiphilic lipid / the co-surfactant / the possible surfactant of formula (I) / the possible fusogenic lipid / the possible imaging agent / the possible therapeutic agent / the mRNA. The dispersed phase is generally free of aqueous phase.

The term "lipid nanoparticles" encompasses both liquid oil droplets themselves as well as solid particles from oil-in-water type emulsions in which the dispersed phase is solid. In the latter case, it is often also referred to as a solid emulsion.

The term “lipid” refers in the context of this presentation to all fatty substances or substances containing fatty acids present in fats of animal origin and in vegetable oils. They are hydrophobic or amphiphilic molecules mainly made up of carbon, hydrogen and oxygen and having a lower density than water. Lipids can be in the solid state at room temperature (25°C), as in waxes, or liquid, as in oils.

The term "phospholipid" refers to lipids having a phosphate group, in particular phosphoglycerides. Most often, phospholipids have a hydrophilic end formed by the optionally substituted phosphate group and two hydrophobic ends formed by chains of fatty acids. Among the phospholipids, mention will be made in particular of phosphatidylcholine, phosphatidyl ethanolamine, phophatidyl inositol, phosphatidyl serine and sphingomyelin.

The term "lecithin" refers to phosphatidylcholine, which is a lipid formed from a choline, a phosphate, a glycerol and two fatty acids. It more broadly covers phospholipids extracted from living organisms, of plant or animal origin, insofar as they are mainly made up of phosphatidylcholine. These lecithins generally constitute mixtures of lecithins bearing different fatty acids.

The term “fatty acid” is understood to denote aliphatic carboxylic acids having a carbon chain of at least 4 carbon atoms. Natural fatty acids have a carbon chain of 4 to 28 carbon atoms (usually an even number). We speak of long chain fatty acid for a length of 14 to 22 carbons and very long chain if there are more than 22 carbons.

The term “surfactant” is understood to mean compounds with an amphiphilic structure which gives them a particular affinity for interfaces of the oil/water and water/oil type, which gives them the ability to lower the free energy of these interfaces and to stabilize scattered systems.

By the term "co-surfactant" is meant a surfactant acting in addition to a surfactant to further lower the interface energy.

The term "hydrocarbon chain" means a chain composed of carbon and hydrogen atoms, saturated or unsaturated (double or triple bond). Preferred hydrocarbon chains are alkyl or alkenyl.

The term “alkylene” is understood to denote a divalent hydrocarbon-based, saturated, linear or branched, preferably linear, aliphatic group.

By “cyclic radical” is meant a radical resulting from a cycle. For example, a phenylene radical is a divalent radical derived from a benzene group. By "cycle" is meant a carbocycle or a heterocycle, saturated, unsaturated or aromatic (aryl or heteroaryl),

- a carbocycle: a cycle composed of carbon atoms, saturated (the preferred saturated carbocycles being in particular a cycloalkyl, such as a cyclopentyl or a cyclohexyl), unsaturated (for example a cyclohexene) or aromatic (that is to say a phenyl),

- a heterocycle: a cyclic group comprising, unless otherwise stated, from 5 to 6 atoms, and comprising one or more heteroatoms chosen from O, N and/or S. Said heterocycle may be saturated or partially unsaturated and comprise one or more double bonds . This is called a heterocycloalkyl group. It can also be aromatic, comprising, unless otherwise stated, from 5 to 6 atoms and then represent a heteroaryl group.

- as non-aromatic or heterocycloalkyl heterocycle, mention may be made of pyrazolidinyl, imidazolidinyl, tetrahydrothiophenyl, dithiolanyl, thiazolidinyl, tetrahydropyranyl, dioxanyl, morpholinyl, piperidyl, piperazinyl, tetrahydrothiopyranyl, thiomorpholinyl, dihydrofuranyl, 2-imidazolinyl, 2,-3-pyrrolinyl, pyrazolinyl, dihydrothiophenyl, dihydropyranyl, pyranyl, tetrahydropyridyl, dihydropyridyl, tetrahydropyrinidinyl, dihydrothiopyranyl, isoxazolidinyl,

- as heteroaryl, mention may in particular be made of the following representative groups: furyl, imidazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, pyrazinyl, pyridazinyl, pyrazolyl, pyridyl, pyrimidinyl, pyrrolyl, 1,3,4-thiadiazolyl, 1,2,3-thiadiazolyl, thiazolyl, thienyl, triazolyl, 1,2,3-triazolyl and 1,2,4-triazolyl.

By "activated ester" is meant a group of formula -CO-LG, and by "activated carbonate" is meant a group of formula -O-CO-LG where LG is a good leaving group chosen in particular from a chlorine, a imidazolyl, a pentafluorophenolate, a pentachlorophenolate, a 2,4,5-trichlorophenolate, 2,4,6-trichlorophenolate, an -O-succinimidyl group, -O-benzotriazolyl, -O-(7-aza-benzotriazolyl) and -O -(4-nitrophenyl).

The invention will be described in more detail by means of the appended figures and the examples which follow.

FIGURES

There depicts a schematic of the complexation of mRNA with a lipid nanoparticle ("LNP") having a core/crown structure to form an mRNA-LNP complex. In the crown of the lipid nanoparticle are illustrated the phospholipids (lecithin as an amphiphilic lipid and DOTAP as a cationic surfactant) and the co-surfactant whose folding poly(ethylene oxide) chain is represented in the aqueous phase. In the mRNA-LNP complex, the mRNA localizes to the crown of the lipid nanoparticles.

There represents the percentage of GFP-positive cells determined from confocal microscopy images for each experiment, the experiments differing from each other by the N/P ratio of mRNA-LNP complexes used and/or by the dose of mRNA complexes -LNP per well (Example 2).

*P<0.05, **P<0.01, ***P<0.001. NT, Untreated.

There represents the percentage of cells positive for GFP as a function of the number of lipid nanoparticles (“LNPs”) per cell (mean, ± SEM, N=3 independent experiments) (Example 2).

There represents the percentage of GFP-positive cells determined from confocal microscopy images for each experiment (Example 3).

n.s., non-significant, * P < 0.05, ** P < 0.01, *** P < 0.001.

NT, Untreated.

Lipo: Lipofectamine (positive control),

LNP2018: mRNA-LNP complex prepared from LNP having been stored for 2 years at +4°C before complexation with mRNA,

LNP 2020: mRNA-LNP complex prepared from LNPs that were prepared just before complexation with mRNA

There represents the percentage of GFP-positive cells determined from confocal microscopy images for each experiment (Example 4).

* P < 0.05, NT, Untreated.

There represents the percentage of GFP-positive cells determined from confocal microscopy images for each experiment (Example 5).

*** P < 0.001. NT, Untreated.

EXAMPLES

Example 1: Preparation of mRNA-LNP complexes

In the following examples, cationic nanoparticles LNP are those of emulsion A3 of table 1 of patent application WO 2014/032953 following the preparation process described on p. 65, l. 30 to p. 66, l. 16.

An mRNA coding for a fluorescent protein (GFP, Green Fluorescent Protein, Tebu-Bio) was used.

mRNA complexation was achieved by mixing the prepared formulation with a solution of mRNA in OptiMEM medium.

The mixture was stirred for 10 minutes at 600 rpm at room temperature (about 25°C).

There schematizes the complexation step between the lipid nanoparticles and the mRNA in order to form the LNP-mRNA complex.

Example 2: Determination of mRNA-LNP complexation conditions for optimal mRNA delivery

An mRNA coding for a fluorescent protein (GFP, Green Fluorescent Protein) was used to demonstrate the ability of mRNA-LNP complexes to deliver mRNA.

Cells from epithelial cells (PC3, prostate adenocarcinoma, ATCC) were seeded on a 96-well plate at a density of 8400 cells per well 24 h before incubation with the different complexes.

mRNA-LNP complexes were prepared extemporaneously with N/P ratios of 2/1, 4/1, 8/1, 16/1, 24/1 or 36/1. The N/P ratio is the ratio of the amount of positive charges due to the cationic surfactant in the formulation before complexation (due to the charged nitrogen, hence the N of N/P) compared to the amount of negative charges provided by mRNA (due to mRNA phosphate, hence the P of N/P).

In each case, a dose of mRNA-LNP complexes per well was added to the PC3 cells in RPMI medium supplemented with HEPES pH7.3 at 20 mM so that the dose of mRNA per well was 7.8 ng, 15.8ng, 31.25ng, 62.5ng, 125ng or 250ng.

Lipofectamine (Lipo) reagent (Sigma) was used as a positive control (reference) method for mRNA delivery. mRNA alone (mRNA) was used as a negative control.

After 6 h of incubation, the medium was aspirated and replaced with RPMI medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. The cell nuclei were then labeled 24 hours after incubation with the complexes with Hoechst 33342 (2 μM) for 20 min then the cells were fixed with a 4% paraformaldehyde solution for 20 min. Finally, the cells were observed under a confocal microscope (Zeiss LSM880) by automated imaging (25 images per well, illumination time of 4 µsec per pixel). Images were segmented using Cell Profiler software and the positivity of each cell for GFP was assessed against normalized fluorescence intensity to correct for background noise using a Python script ( >2000 cells per condition). Finally, the percentage of GFP-positive cells was quantified (mean, ± S.E.M., N = 3 independent experiments). P-values were determined by an ANOVA test. *P<0.05, **P<0.01, ***P<0.001.

The results are provided to the , where LNP NPX corresponds to the results for the mRNA-LNP complex prepared with an N/P ratio of X/1, where X is the number indicated. For example, LNP NP2 means mRNA-LNP complex prepared with an N/P ratio of 2:1.

It has been observed that the fluorescence intensity is variable depending on the N/P ratio of the mRNA-LNP complexes, corresponding to the ratio between the positive charges carried by the amine groups of the LNPs and the phosphate groups of each phosphodiester bond of the mRNA.

A systematic quantification of the number of GFP-positive cells was carried out to determine an optimal range of N/P ratio allowing good delivery efficiency of GFP mRNA. This optimal range of the N/P ratio is dependent on the amount of mRNA per well.

By expressing the transfection efficiency as a function of the number of LNPs per cell, which is the product of the N/P ratio and the amount of mRNA, we can identify an optimal amount of nanoparticles per cell to use for mRNA delivery. . This optimal number can be determined from the and amounts to 8.32 million LNP per cell in the case of PC3 epithelial cells.

This optimal number is of course variable depending on the type of cells. This method for determining the parameters of the complexes formed for optimal delivery of mRNA thus allows rapid development and is applicable to different cell types of interest.

Example 3: Stability study at LNP storage (before complexation)

To demonstrate the storage stability of LNPs at +4°C, LNP-mRNA complexes were prepared from:

- either LNPs prepared in 2018, or two years before the complexation, the LNPs having been stored at +4°C

- either of LNPs prepared in 2020, or just before the complexation.

Experiments were carried out under the optimal conditions determined on the PC3 epithelial cells in Example 2, that is to say with a number of LNPs per cell constant at 8 million.

PC3 cells were seeded on a 96-well plate at a density of 8400 cells per well 24 h before incubation with the different LNP-mRNA complexes. The mRNA-LNP complexes were made extemporaneously at N/P ratios of 8/1, 16/1, 32/1, 64/1, 128/1, 256/1, 512/1 or 1024/1 and these were added to the PC3 cells in RPMI medium supplemented with HEPES pH7.3 at 20 mM at a dose of mRNA-LNP complexes per well such that the dose of mRNA is respectively 100 ng, 50 ng, 25 ng, 12, 5 ng, 6.25 ng, 3.13 ng, 1.56 ng and 0.78 ng per well, which allowed a constant number of 8 million LNPs per cell.

Lipofectamine (Lipo) reagent was used as the gold standard positive control method for mRNA delivery, while mRNA alone (mRNA) was used as the negative control.

After 6 h of incubation, the medium was aspirated and replaced with RPMI medium supplemented with 10% fetal calf serum and 1% penicillin/streptomycin. 24 hours after incubation with the complexes, the cell nuclei were labeled with Hoechst 33342 (2 μM) for 20 min then the cells were fixed with a 4% paraformaldehyde solution for 20 min. The fixed cells were observed under a confocal microscope (Zeiss LSM880) by automated imaging (25 images per well, illumination time of 4 μsec per pixel). Images were segmented using Cell Profiler software and the positivity of each cell for GFP was assessed against normalized fluorescence intensity to correct for background noise using a Python script ( >2000 cells per condition). Finally, the percentage of GFP-positive cells was quantified (mean, ± SEM, N = 3 independent experiments). P-values were determined by an ANOVA test.

The results are provided to the .

The results show a high and statistically significant percentage of GFP-positive cells compared to the untreated control at up to 3 ng of mRNA per well, when the mRNA is delivered by LNPs. This clearly confirms the effectiveness of the parameters determined by the method described in Example 2.

In addition, LNPs manufactured in March 2018 or September 2020 were used, without noting statistically significant differences (n.s., P-Values displayed) between these two groups. Thus, LNP-mRNA complexes are efficient for mRNA delivery, with an efficiency similar to the commercial positive control (Lipofectamine, Lipo). This example demonstrates the very high stability over time of the LNPs, and the possibility of storing the non-complexed LNPs at +4° C. for at least 2 years.

Example 4 : Study of storage stability at +4°C of complex s mRNA-LNP

In example 3, it is the LNPs before complexation which had been stored at 4°C for two years.

In example 4, it is the LNP-mRNA complexes (therefore once the complexation has been carried out) which were stored for 2 weeks at +4° C. in order to determine their capacity to deliver a GFP mRNA into target cells after 2 weeks of storage.

PC3 cells were seeded on a 96-well plate at a density of 8400 cells per well 24 h before incubation with the different complexes.

The complexes were produced extemporaneously at an N/P ratio of 8/1 (CL40-NP8 CTRL) or 2 weeks before the experiment at an N/P ratio of 32/1 (CL40-NP32 2 wk-4C), 8 /1 (CL40-NP8 2 weeks-4C) or 4/1 (CL40-NP4 2 weeks-4C). For each of the four types of complexes, and a dose of mRNA per well of 6.25, 12.5, 25, 50 or 100 ng was added to the PC3 cells in RPMI medium supplemented with HEPES pH7.3 at 20mM .

Uncomplexed (mRNA-free) nanoparticles (LNP CTRL) were used as a negative control.

After 6 h of incubation, the medium was aspirated and replaced with RPMI medium supplemented with 10% fetal calf serum and 1% penicillin/streptomycin. 24 hours after incubation with the complexes, the cell nuclei were then labeled with Hoechst 33342 (2 μM) for 20 min and then the cells were fixed with a 4% paraformaldehyde solution for 20 min. The fixed cells were observed under an epifluorescence microscope (TECAN SparkCyto) by automated imaging (5 images per well, X10 objective). Images were segmented using Cell Profiler software and the positivity of each cell for GFP was assessed against normalized fluorescence intensity to correct for background noise using a Python script ( >4000 cells per condition). Finally, the percentage of GFP-positive cells was quantified (mean, ± SEM, N = 3 independent experiments). P-values were determined by an ANOVA test. * P < 0.05. NT, Untreated.

The results are provided to the .

Good delivery efficiency of GFP mRNA for the 100 ng dose is observed, whether with complexes formed extemporaneously at N/P of 8/1 or 2 weeks beforehand at N/P 8/1 and N /P of 32/1. On the other hand, the complexes produced at N/P of 4/1 do not have sufficient delivery efficiency. The amount of LNP-mRNA complexes introduced should be increased.

The LNP-mRNA complexes are therefore stable for at least two weeks and allow good mRNA delivery efficiency. The N/P ratio also seems to play a role in the conservation of mRNA and LNP complexes with a lipid core at +4°C.

Example 5 : Demonstration of resistance to RN To to mRNA complexed on the surface of LNP-mRNA complexes

In this example of use of the invention, the RNase resistance of complexes formed by the mRNA complexed on the surface of the LNPs was tested.

PC3 cells were seeded on a 96-well plate at a density of 8400 cells per well 24 h before incubation with the different complexes.

The complexes were made at a dose of 100 ng ng of mRNA per well, extemporaneously at an N/P ratio of 8/1 or 32/1.

In the case of the "Pre-RNAse" group, RNase A is added to the GFP mRNA alone at a final concentration of 1 ng/mL then incubated for 10 min at 25°C, prior to the addition of the LNPs (comparative) . The mRNA was therefore brought into contact with RNase before complexation with the LNPs.

In the case of the “Post-RNase” group, RNase A is added to the mRNA-LNP complexes at a final concentration of 1 ng/mL then the complexes were incubated for 10 min at 25°C. The mRNA brought into contact with RNase is therefore complexed with the LNPs.

The various complexes or controls were then added to the PC3 cells in RPMI medium supplemented with HEPES pH 7.3 at 20 mM.

The LNPs alone free of mRNA (N/P of 8/1 and N/P of 32/1 alone, free of mRNA, the quantities of LNP (not complexed) introduced corresponding to those introduced respectively for the NP8-mRNA and NP32-mRNA) as well as mRNA alone were used as a negative control.

After 6 h of incubation, the medium was aspirated and replaced with RPMI medium supplemented with 10% fetal calf serum and 1% penicillin/streptomycin. The cell nuclei were then labeled 24 h after incubation with the complexes with Hoechst 33342 (2 μM) for 20 min then the cells were fixed with a 4% paraformaldehyde solution for 20 min. The fixed cells were observed under an epifluorescence microscope (TECAN SparkCyto) by automated imaging (5 images per well, X10 objective). Images were segmented using Cell Profiler software and the positivity of each cell for GFP was assessed against normalized fluorescence intensity to correct for background noise using a Python script ( >4000 cells per condition). Finally, the percentage of GFP-positive cells was quantified (mean, ± SEM, N = 3 independent experiments). P-values were determined by an ANOVA test. *** P < 0.001. NT, Untreated.

The results are provided to the .

Pretreatment of GFP mRNA with RNase A, at a concentration of 1 ng/mL for 10 min at 25°C (Pre-RNAse group), leads to its degradation. Indeed, we do not observe PC3 positive cells for GFP by adding LNP at N/P of 8/1 or N/P of 32/1 after this RNase A pretreatment.

On the other hand, if a quantity of GFP mRNA equivalent to the surface of an LNP (Post-RNAse group) is first complexed, the effect of RNase A at 1 ng/mL for 10 min at 25°C is then strongly reduced and we always observe a significant number of positive cells for GFP at the N/P ratios of 8 or N/P of 32. We also note that the N/P conditions of 8/1 are also more favorable with a higher rate of GFP-positive cells.

This experiment demonstrates that the mRNA is well protected from enzymatic degradation although it is not encapsulated in the core of the LNPs and is exposed on the surface of the LNPs.

LNPs protect the mRNA complexed on their surfaces from degradation by enzymes present in the external environment, such as RNAses (RNA-degrading enzymes), thanks to the protective crown of co-surfactant present on the surface of the LNPs. LNP.

Claims (10)

Formulation in the form of a nanoemulsion, comprising a continuous aqueous phase and at least one phase dispersed in the form of lipid nanoparticles, and comprising:
- at least 5% molar of amphiphilic lipid,
- from 15 to 70% molar of cationic surfactant bearing at least one positive charge comprising:
- at least one lipophilic group chosen from:
- an R or R-(C=O)- group, where R represents a linear hydrocarbon chain comprising from 11 to 23 carbon atoms,
- an ester or an amide of fatty acids comprising from 12 to 24 carbon atoms and of phosphatidylethanolamine, and
- a poly(propylene oxide), and
- at least one hydrophilic group comprising at least one cationic group chosen from:
- a linear or branched alkyl group comprising from 1 to 12 carbon atoms and interrupted and/or substituted by at least one cationic group, and
- a hydrophilic polymeric group comprising at least one cationic group, and
- from 10% to 55% molar of co-surfactant comprising at least one poly(ethylene oxide) chain comprising at least 25 ethylene oxide units,
- a solubilizing lipid,
- possibly a fusogenic lipid,
where the molar percentages of amphiphilic lipid, cationic surfactant and co-surfactant are relative to the cumulative molar proportions of amphiphilic lipid, cationic surfactant, co-surfactant and any fusogenic lipid,
in which at least one messenger RNA, the single-stranded nucleotide sequence of which comprises at least 300 bases, is complexed to the surface of the lipid nanoparticles,
the N/P ratio of the quantity of positive charges of the cationic surfactant relative to the quantity of negative charges of the messenger RNA being greater than or equal to 2/1. Formulation according to Claim 1, in which the cationic surfactant is chosen from:
- N[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium,
- 1,2-dioleyl-3-trimethylamonium-propane,
- N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy-1-propananium),
- 1-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)imidazolinium, and
- dioctadecylamidoglycylspermine. A formulation according to any of claims 1 to 2 comprising a fusogenic lipid, such as 1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine. A formulation according to any of claims 1 to 3, wherein the amphiphilic lipid is a phospholipid. A formulation according to any of claims 1 to 4 comprising an imaging agent and/or a therapeutic agent. Formulation according to any one of Claims 1 to 5, comprising a co-surfactant grafted with a molecule of interest, preferably a biological targeting ligand. Process for the preparation of a formulation according to any one of claims 1 to 6, comprising the following steps:
(i) preparing an oily phase comprising the solubilizing lipid, the amphiphilic lipid, the cationic surfactant, the optional fusogenic lipid;
(ii) preparing an aqueous phase comprising the co-surfactant;
(iii) dispersing the oily phase in the aqueous phase under the action of sufficient shear to form a formulation in the form of a nanoemulsion; Then
(iv) adding the messenger RNA whose single-stranded nucleotide sequence comprises at least 300 bases to the formulation obtained in step (iii), then
(v) recovering the formulation thus formed. Method for introducing in vitro a messenger RNA, the single-stranded nucleotide sequence of which comprises at least 300 bases, into a eukaryotic cell, comprising bringing the eukaryotic cell into contact with a formulation according to any one of Claims 1 to 6 . Formulation according to any one of claims 1 to 6 for its use for the prevention and/or treatment of a disease. Kit including:
- a formulation in the form of a nanoemulsion comprising a continuous aqueous phase and at least one phase dispersed in the form of lipid nanoparticles, and comprising:
- at least 5% molar of amphiphilic lipid,
- from 15 to 70% molar of at least one cationic surfactant bearing at least one positive charge comprising:
- at least one lipophilic group chosen from:
- an R or R-(C=O)- group, where R represents a linear hydrocarbon chain comprising from 11 to 23 carbon atoms,
- an ester or an amide of fatty acids comprising from 12 to 24 carbon atoms and of phosphatidylethanolamine, and
- a poly(propylene oxide), and
- at least one hydrophilic group comprising at least one cationic group chosen from:
- a linear or branched alkyl group comprising from 1 to 12 carbon atoms and interrupted and/or substituted by at least one cationic group, and
- a hydrophilic polymeric group comprising at least one cationic group, and
- from 10% to 55% molar of a co-surfactant comprising at least one poly(ethylene oxide) chain comprising at least 25 ethylene oxide units,
- a solubilizing lipid,
- possibly a fusogenic lipid,
where the molar percentages of amphiphilic lipid, cationic surfactant and co-surfactant are relative to the cumulative molar proportions of amphiphilic lipid, cationic surfactant, co-surfactant and any fusogenic lipid, and
- separately, at least one messenger RNA whose single-strand nucleotide sequence comprises at least 300 bases.