Delivery system for topical application of RNA

Abstract

The present invention relates to a composition useful for the topical application of RNA, wherein the composition comprises (i) an aqueous phase containing RNA and (ii) an oil phase, in one embodiment the aqueous and oil phase are mixed with two specific surfactants. In particular, the present invention relates to the formulation of mRNA encoding a target antigenic protein. More particularly, a microemulsion for crossing the skin barrier is provided which allows an alternative route for mRNA vaccine applications. The microemulsion can be applied topically and can cross the skin via the hair follicle route. Thereby a delivery system is achieved which allows (1) to avoid the use of needles in vaccination and (2) to more specifically target the mucosal component of the immune.

Description

Technical Field

[0001] This invention relates to a composition for topical RNA administration, wherein the composition comprises (i) an aqueous phase containing RNA and (ii) an oil phase, wherein in one embodiment the aqueous and oil phases are mixed with two specific surfactants. In particular, this invention relates to formulations of mRNA encoding target antigenic proteins. More particularly, a microemulsion for crossing the skin barrier is provided, allowing for an alternative route for mRNA vaccine application. The microemulsion can be applied topically and can penetrate the skin via the hair follicle route. This achieves a delivery system that allows (1) the avoidance of needles in vaccination and (2) more specific targeting of mucosal components of the immune system.

[0002] Background Information RNA vaccines and therapeutics are an increasingly important area of ​​research in vaccinology and gene therapy. Using nucleic acid-encoded antigens as the basis for a vaccine platform offers numerous advantages: purification is relatively simplified, and RNA constructs can be built within days using DNA synthesis techniques followed by RNA transcription and capping. This enables rapid responses to emerging pathogen threats, the conversion of production to accommodate new circulating strains, or personalized therapeutic interventions for multiple diseases.

[0003] Viral infections remain a significant health problem in animals, with adverse economic consequences. For example, a variety of viral pathogens cause disease in economically important livestock animals such as pigs. Viruses that infect pigs include, for example, swine influenza A virus (SIAV).

[0004] Swine influenza is an acute respiratory viral disease caused by influenza A virus (IAV) of the Orthomyxoviridae family, which reduces the health and welfare of pigs. Clinical symptoms of swine influenza can vary in severity, but it typically presents as a mild respiratory illness with high morbidity and rapid recovery, and rare mortality. However, the disease has a considerable economic burden as it leads to weight loss, reduced weight gain, and, in some cases, reproductive failure in sows due to high fever. Swine influenza A virus (SIAV) is one of the most important respiratory pathogens in pigs, and its high prevalence in global swine populations is directly related to the economic impact of the disease. In Europe, there are currently four main subtypes of influenza A virus that are circulating in farmed pig populations and causing economic losses, as described by Brown (Vet Microbiol. 74(1-2):29-46 (2000)), which, along with the following publications cited herein, are incorporated herein by reference in their entirety. Since the 1980s, H1N1 (“avian” subtype) and H3N2 swine influenza viruses have been endemic in major pig-producing countries. The H1N2 virus was introduced into European pig populations approximately two decades ago (Brown et al., VetRec. 13:328-329 (1995)), while the H1N1 pandemic subtype (H1pdmN1) was introduced into pig populations during the 2009 human H1N1 pandemic through transmission from humans to pigs and has been continuously spreading in pig populations globally, with an estimated average prevalence of 8% among all swine influenza virus infections in Europe (Watson et al., J Virol. 89(19):9920-9931 (2015)). In addition, swine influenza also has impacts on human health, as IVA is known for its zoonotic potential (Thacker & Janke, J InfectDis. 15:197 Suppl 1:S19-24 (2008)).

[0005] Traditional vaccine methods for SIAV (intramuscular injection of inactivated virus) provide durable protection, but fail to induce protective immunity in the context of maternal immunity.

[0006] Several characteristics of influenza viruses are considered highly similar to those of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), including (I) its ability to replicate effectively in the mucous membranes of the upper respiratory tract, (II) the presence of numerous asymptomatic and mildly asymptomatic infected animals that produce infectious virus, and (III) its short incubation period. To the known extent, mRNA vaccines against SARS-CoV-2 are available.

[0007] Messenger RNA (mRNA) is a transient intermediate between DNA and protein, composed of RNA that includes protein-coding regions flanked by non-coding regions. mRNA has three main functional regions: the 5' untranslated region (5'-UTR), the coding region (also called the coding sequence (CDS) or ORF region), and the final 3' untranslated region (3'-UTR). The two untranslated regions (UTRs) typically contain signals for expression or RNA processing. In particular, mRNA usually also contains a 5' cap and a 3' poly(A) tail, which allows for efficient translation of mRNA into proteins. The information carried by the coding region of mRNA consists of a series of codons, i.e., consecutive triplet nucleotides, each codon encoding one amino acid residue of the corresponding protein. In the 1980s, mRNA was transcribed in vitro and emerged as a candidate for therapeutic uses, including immunotherapy, viral vaccines, genome editing, and cell reprogramming. In principle, mRNA can fulfill all the genetic information requirements for encoding and expressing a wide variety of proteins.

[0008] Vaccine efficiency can be optimized by modifying the mRNA sequence, which is a more convenient approach compared to other types of vaccine modification (Xu et al., Int J Mol Sci. 21:6582 (2020)). Furthermore, despite encoding different antigens, the production and purification processes for most mRNA vaccines are quite similar. This technology allows for the development of potentially standardized production processes, rather than developing specific processes for each new vaccine target.

[0009] In addition, mRNA has self-adjuvant properties, which activate (A) innate immunity through RIG-I / MDA5, TLR3, TLR7 and TLR8 receptors (Verbeke et al., Immunity. 55(11):1993-2005 (2022)) and induce (B) strong and durable adaptive immune responses through the secretion of tumor necrosis factor α (TNFα), interferon α (IFNα) and other cytokines by immune cells, while peptide and protein vaccines usually require additional adjuvants to achieve similar purposes (Iavarone et al., Expert RevVaccines. 16(9):871-881 (2017)).

[0010] The desired immune effect of using mRNA as an antigen source can only be achieved when mRNA molecules reach the target cells, are in the correct compartments, and produce sufficient protein antigens to induce protective immunity.

[0011] The aim is to develop mRNA-based drugs or vaccines that express target proteins in host cells. Once inside the host cell's cytoplasm, the mRNA is read by ribosomes and translated into proteins with the desired therapeutic activity.

[0012] The delivery systems used include lipid nanoparticles (LNPs) or liposomes, which protect mRNA by encapsulating it within nanoparticles or lipid vesicles. A review article by Guevara et al. (Front Chem. 8:589959 (2020)) reported the use of LNPs encapsulated with mRNA as mRNA delivery vectors and their application in cancer therapy.

[0013] The recent SARS-CoV-2 pandemic has accelerated research into rapid vaccine solutions. Compared to traditional methods, mRNA vaccine technology offers many advantages, such as the potential for rapid development and large-scale deployment in pandemic situations.

[0014] The development of RNA therapeutics should take into account several drawbacks, such as (1) the rapid degradation of exogenous RNA by RNases that are ubiquitous in the environment and tissues, (2) the delivery of negatively charged RNA across hydrophobic cell membranes, and (3) the strong immunogenicity of exogenous RNA may lead to cytotoxicity and impair the translation of therapeutic proteins (Damase et al., Front Bioeng Biotechnol. 9:628137 (2021)).

[0015] Various mRNA delivery methods have been developed to protect mRNA from ribonuclease degradation and to neutralize or reverse its negative charge to increase cellular uptake, thereby increasing the production of target proteins. To date, lipid and polymer-based materials have been the main nonviral mRNA delivery systems. Typically, cationic lipids and polymers are used to form complexes through electrostatic and hydrophobic interactions with negatively charged mRNA. These particles act as shields to prevent mRNA from being degraded by enzymes and reverse polarity to improve transfection efficiency (Chaudhary et al., Nat Rev Drug Discov. 20(11):817-838 (2021)).

[0016] The practicality of delivering mRNA using lipid nanoparticles has been successfully demonstrated in COVID-19 vaccines, such as mRNA-1273 and BNT162b. Of all the mRNA vaccines in clinical trials, only mRNA-1273 and BNT162b, which are LNP-mRNA formulations administered intramuscularly, have been approved for human use to date (Wilson & Geetha, J Drug Deliv SciTechnol. 74:103553 (2022)).

[0017] Although intravenous, subcutaneous, intradermal, and intranodal injections have been considered, the intramuscular route of administration remains reserved for mRNA vaccine delivery (Nikita et al., 2022). The intramuscular route has been used for vaccine delivery for decades and is easier to administer compared to subcutaneous injection. This route includes the ability to inject relatively large amounts of vaccine and less pain and local irritation compared to subcutaneous injection. However, the intramuscular route can also cause adverse events such as percutaneous injury and can be a source of inflammation or contamination following skin barrier disruption.

[0018] Furthermore, while several reports have assessed the production of vaccine-induced IgG neutralizing antibodies in the serum of immunized individuals, data on specific mucosal immune activation are scarce.

[0019] The respiratory and oral mucosa are the primary routes of entry for influenza viruses; therefore, anti-SIAV vaccines that induce specific immune responses in these tissues may be a key tool for enhancing vaccine-mediated immune responses and overcoming maternal immunity. In pigs, the concept of a shared mucosal immune system has only recently gained attention compared to other mammals, such as mice. According to this concept, immunization or vaccination via a mucosal route triggers the immune system at mucosal sites both locally and remotely (Wilson & Obradovic, Mol Immunol. 66:22-34 (2015)). This phenomenon can be used to trigger mucosal immunity from the skin to the lungs (a major target of influenza viruses).

[0020] The skin contains important early warning components of the immune system, including powerful antigen-presenting cells (APCs) such as Langerhans cells and dendritic cells, as well as immune-active keratinocytes (Levin et al., Hum VaccImmunother. 11(4):991-997 (2015)). However, the ability of antigenic compounds (usually high molecular weight biomacromolecules) to cross the stratum corneum to reach the basal epidermis and dermis (where immune cells are present) is very low, which is a major obstacle.

[0021] Transdermal immunization with mannosyl-chitosan-modified mRNA-loaded protoplasts to transfect dendritic cells (DCs) and induce their maturation has been described: transdermal patches loaded with mRNA vaccines or siRNA, and microspheres containing mRNA or siRNA sprayed onto a silk fibroin matrix surface via electrospraying (conditions: voltage 18 kV, extrusion rate 1 ml / h, receiving distance 15 cm, temperature 40 °C, humidity 8%), have allowed for the delivery of nucleic acid molecules to the deep skin layers in a melanoma syngeneic mouse model (Wang et al., Composites Part B: Engineering. 233:109648 (2022)). For this purpose, transdermal patches loaded with mRNA vaccines were applied to the bare abdominal skin of mice and secured with medical tape for 3 days, repeated on days 7 and 14 for booster immunization.

[0022] However, for non-invasive mRNA delivery, there is a need for an easily produced composition that can be simply administered, especially in breeding environments where large numbers of animals need to be vaccinated in a short period of time.

[0023] WO2023144352A1 discloses an oil-in-water nanoemulsion having an oil phase containing an active pharmaceutical ingredient (API) (the API comprising RNA or a hydrophobic drug) and an aqueous phase. In addition to the API, the oil-in-water nanoemulsion also contains high-oleic edible oil, plant protein, surfactant, and water. This oil-in-water emulsion is then combined with a suspension of denatured plant protein and a calcium salt chelating agent to form microparticles. These microparticles can be orally administered to a recipient, for example in tablet form, and are claimed to subsequently pass through the stomach to the ileum, where the protein matrix breaks down to release oil droplets containing the API, which are then absorbed.

[0024] However, since delivering tablets to animals is typically difficult in practice, an easy-to-use, non-invasive administration system is desired for delivering RNA to animals, such as through local administration. Local administration also has the advantage of allowing RNA to be delivered to a specific location in the body, compared to oral administration where it is adsorbed into the bloodstream. Summary of the Invention

[0025] The solutions to the above-mentioned technical problems are achieved through the embodiments characterized in the specification and claims.

[0026] Therefore, the present invention is implemented in various aspects according to the claims.

[0027] The present invention is based on the following surprising discovery: Topical application of a composition comprising an aqueous phase and an oil phase containing mRNA encoding an enzyme or an immunogen, respectively, to pigs allows for cellular uptake and translation of the mRNA, thereby generating specific enzyme activity or inducing immunity in the pigs, respectively.

[0028] In a first aspect, the present invention therefore relates to a composition comprising - An aqueous phase containing ribonucleic acid (RNA), and - oil phase, The composition described herein is also referred to hereinafter as "the composition of the present invention".

[0029] The aqueous phase containing RNA, hereinafter also referred to as the "aqueous phase of the present invention", preferably does not contain solid nanoparticles, or specifically, in particular, does not contain lipid nanoparticles (LNPs) and / or lipid-like nanoparticles (LLNs). The RNA contained in the aqueous phase is hereinafter also referred to as the "RNA of the present invention".

[0030] The oil phase, also referred to below as the "oil phase of the present invention", is preferably a liquid or a liquid at room temperature.

[0031] The term "aqueous phase" specifically refers to the aqueous portion of a water-in-oil emulsion. The term "oil phase" specifically refers to the oily portion of a water-in-oil emulsion. More specifically, as used herein, the term "aqueous phase" means a liquid phase containing water and may additionally contain a hydrophilic co-solvent and a water-soluble substance, and which is immiscible with the oil phase. It should be particularly understood that the water-containing liquid phase is an RNA-soluble phase. As used herein, the term "oil phase" more specifically means a liquid phase immiscible with water.

[0032] The term “RNA contained in the aqueous phase” as used herein specifically refers to RNA in contact with the aqueous liquid phase.

[0033] The term "liquid phase" as used in this article specifically refers to a phase that is liquid at room temperature (25°C) and atmospheric pressure (760 mmHg).

[0034] The term "aqueous phase" as used in this article should be specifically understood as equivalent to "aqueous solution". The term "oil phase" as used in this article should be specifically understood as equivalent to "oil".

[0035] As used herein, the terms "ribonucleic acid" or "RNA" specifically refer to molecules containing at least one ribonucleotide residue, and include single-stranded RNA, double-stranded RNA, isolated RNA, partially purified, pure, or synthetic RNA, recombinant RNA, and altered RNA or analogs of naturally occurring RNA, such as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. "Ribononucleotide" means a nucleotide having a hydroxyl group at the 2' position of the β-D-furanose moiety. The nucleotides in the RNA molecules of this disclosure may also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides.

[0036] It should be particularly understood that the terms "a type of ribonucleic acid" or "a type of RNA" used in this article also include plural references, and therefore also cover multiple types of ribonucleic acids or multiple types of RNA, or respectively cover "one or more types of ribonucleic acids" or "one or more types of RNA". The terms "ribonucleic acid" and "RNA" should be specifically understood to be equivalent to "ribonucleic acid molecule" and "RNA molecule", respectively.

[0037] Preferably, the RNA contained in the aqueous phase is dissolved in the aqueous phase. According to this aspect, the aqueous phase containing RNA is therefore an aqueous phase in which RNA is dissolved. In this context, "dissolved" should be specifically understood as "freely dissolved," i.e., not bound to colloids or particles. Therefore, in particular, the RNA of the present invention is free to move within the aqueous phase and / or does not bind to nanoparticles (such as lipid nanoparticles (LNPs) and / or lipid-like nanoparticles (LLNs)).

[0038] According to a preferred aspect, the compositions of the present invention comprise one or more surfactants.

[0039] Preferably, the one or more surfactants are selected from any one of the first surfactants or the second surfactant mentioned below, or a mixture thereof.

[0040] The term "surfactant" as used in this article specifically refers to a compound containing both lipophilic and hydrophilic segments that reduces the surface tension of the system when added to water or a solvent.

[0041] Specifically, the compositions of the present invention comprise two surfactants, referred herein as "first surfactant" and "second surfactant," wherein the first and second surfactants are different from each other. Therefore, the second surfactant mentioned herein is different from the first surfactant mentioned herein.

[0042] Therefore, the compositions of the present invention specifically comprise or consist of the following: - An aqueous phase containing RNA - oil phase, - The first surfactant, and - Second surfactant.

[0043] Preferably, the first surfactant is a nonionic surfactant, or the second surfactant is a nonionic surfactant. Specifically, both the first surfactant and the second surfactant are nonionic surfactants.

[0044] The term "nonionic surfactant" as used in this article specifically refers to surfactants that are uncharged. Therefore, it should be understood that nonionic surfactants do not react with ions in solution and do not ionize in water.

[0045] According to a preferred aspect, the first surfactant is a water-soluble surfactant.

[0046] The term “water-soluble surfactant” as used in this article specifically refers to surfactants with a hydrophilic-lipophilic balance (HLB) value greater than 10.

[0047] The term "HLB value" used in this article refers to the hydrophilic-lipophilic balance (HLB) scale established by Griffin in 1949 (Griffin, WC (1949) Classification of Surface-Active Agents by "HLB". Journal of Cosmetic Science, 1, 311-326), which is a scale of 0-20 (20 being hydrophilic) used to characterize the properties of molecules (such as surfactants).

[0048] The HLB of the molecule is calculated as follows: HLB=20* Mh / M Where Mh is the molecular mass of the hydrophilic portion of the molecule, and M is the molecular mass of the entire molecule, the results are on a scale of 0 to 20. An HLB value of 0 corresponds to a completely lipophilic / hydrophobic molecule, and a value of 20 corresponds to a completely hydrophilic / lipophobic molecule. See Griffin, WC Calculation of HLB values ​​of Nonionic Surfactants, J. Soc.Cosmet. Chem. 1954, 5, 249-256. HLB values ​​of commonly used surfactants can be readily obtained from the literature (e.g., the HLB index in McCutcheon's Emulsifiers and Detergents, MC Publishing Co., 2004).

[0049] According to a preferred aspect, the HLB value of the first surfactant is greater than 10.

[0050] The second surfactant is preferably a co-surfactant.

[0051] The term “co-surfactant” used in this article should be specifically understood as a surfactant that, in addition to the primary surfactant, also functions to further reduce the surface tension of the liquid.

[0052] Preferably, the second surfactant is a water-insoluble surfactant.

[0053] The term "water-insoluble surfactant" as used in this article specifically refers to surfactants with an HLB value of less than 10.

[0054] The second surfactant preferably has an HLB value of less than 10.

[0055] According to another preferred aspect, the aqueous phase of the present invention comprises a solvent selected from a mixture of water and other water-miscible solvents that can be mixed with water in any way.

[0056] The term "water-miscible solvent" as used herein specifically refers to solvents other than water (including mixtures of solvent compounds) that are liquid at room temperature and can be mixed with water in any proportion to form a homogeneous solution. Examples of water-miscible solvents include ethanol, ethylene glycol, and / or glycerol.

[0057] Preferably, the aqueous phase of the present invention contains water.

[0058] According to another preferred aspect, the oil phase of the present invention is selected from mineral oil and vegetable oil.

[0059] As used herein, the term "mineral oil" specifically refers to a mixture of liquid hydrocarbons obtainable from petroleum lipids through distillation. Mineral oils can be synthesized or purified from petroleum products. The term "vegetable oil" as used herein refers to any oil obtainable from vegetable processing.

[0060] Preferably, the oil phase of the present invention is mineral oil.

[0061] According to a particularly preferred aspect, the compositions of the present invention comprise or consist of the following: - An aqueous phase containing RNA, wherein the aqueous phase contains water; - Oil phase, wherein the oil phase is mineral oil; - The first surfactant with an HLB value greater than 10; and - A second surfactant with an HLB value less than 10.

[0062] According to another preferred aspect, the first surfactant is selected from polyethylene glycol derivatives, polyethylene glycol, glycerol-based surfactants, and polyethylene glycol (PEG) esters.

[0063] Preferably, the HLB value of the first surfactant is 11 to 16.

[0064] According to another preferred aspect, the first surfactant comprises PEG-8 caprylic / capric glyceride or PEG-6 caprylic / capric glyceride.

[0065] In yet another preferred aspect, the HLB value of the first surfactant is 11 to 13.

[0066] Preferably, the first surfactant comprises PEG-8 caprylic / capric glyceride.

[0067] In another preferred aspect, the first surfactant comprises monoglycerides, diglycerides, and / or triglycerides.

[0068] In another preferred aspect, the first surfactant is primarily composed of PEG-8 caprylic / capric glyceride.

[0069] Most preferably, the first surfactant comprises or is composed of the following: - Monoesters and diesters of polyethylene glycol with an average relative molecular weight between 200 and 400, and - Glyceryl monoesters, diesters, and triesters.

[0070] In a preferred aspect, the first surfactant mentioned herein comprises or consists of the following: - Caprylic acid (C 8) and decanoic acid (C 10 PEG-8 (MW 400) monoesters and diesters, and - Monoglycerides, diglycerides, and triglycerides.

[0071] In another preferred aspect, the first surfactant mentioned herein comprises small amounts of monoglycerides, diglycerides, and triglycerides, and mainly caprylic acid (C8) and capric acid (C8). 10 It consists of PEG-8 (MW 400) monoester and diester.

[0072] In one instance, the commercially available product Labrasol® (Gattefosse SA, Saint-Priest, France) is used as the first surfactant, but other surfactants are also suitable. Therefore, in a preferred embodiment, the first surfactant is Labrasol®, or, separately, the first surfactant is a mixture of the components contained in Labrasol®.

[0073] The second surfactant is preferably a polyglycerol fatty acid ester.

[0074] According to a preferred aspect, the HLB value of the second surfactant is from about 3 to about 8.

[0075] In another preferred aspect, the second surfactant is selected from polyglycerol-3 dioleate and polyglycerol-6 dioleate.

[0076] According to yet another preferred aspect, the HLB value of the second surfactant is from about 3 to about 6. According to one preferred embodiment, the HLB value of the second surfactant is about 3. According to another preferred embodiment, the HLB value of the second surfactant is about 6.

[0077] Preferably, the second surfactant is polyglycerol 3-dioleate.

[0078] In one instance, commercially available polyglycerol 3-dioleate Plurol® Oleique CC 497 (Gattefosse SA, Saint-Priest, France) was used as the second surfactant, but other surfactants may also be used.

[0079] Therefore, in a preferred embodiment, the second surfactant is Plurol® Oleique CC 497, or, respectively, the second surfactant is a mixture of the components contained in Plurol® Oleique CC 497.

[0080] In a preferred aspect, the aqueous phase of the present invention is selected from: - An aqueous buffer solution containing RNA. - Water containing RNA, and - A mixture of the following substances: • water or • Aqueous buffer solution With any other water-miscible solvent, The mixture contained RNA.

[0081] Preferably, the aqueous phase comprises or consists of an aqueous buffer solution.

[0082] According to a particularly preferred aspect, the aqueous phase of the present invention is an aqueous buffer solution containing RNA.

[0083] The oil phase of this invention is preferably mineral oil, selected from liquid paraffin, and an oily liquid composed of petroleum-derived saturated hydrocarbons (such as hexadecane, methyl oleate, or ethyl oleate).

[0084] Preferably, the oil phase is liquid paraffin.

[0085] In a preferred aspect, the composition of the present invention comprises or consists of the following: - An aqueous phase containing RNA, wherein the aqueous phase containing RNA is an aqueous buffer solution containing RNA; - An oil phase, wherein the oil phase is liquid paraffin; - A first surfactant, wherein the first surfactant comprises or is primarily composed of PEG-8 caprylic / capric glyceride; and - A second surfactant, wherein the second surfactant is polyglycerol-3 dioleate.

[0086] In particular, the oil phase of the present invention is preferably a purified mixture of liquid saturated hydrocarbons. Preferably, the oil phase is obtained from petroleum through catalytic hydrogenation purification.

[0087] Preferably, the oil phase of the present invention comprises Approximately 67% to approximately 68% of alkane carbon, and / or Approximately 32% to approximately 33% of the carbon in cycloalkanes.

[0088] According to another preferred aspect, the dynamic viscosity of the oil phase of the present invention at 20°C is not more than about 100 mPa·s, preferably not more than about 50 mPa·s, more preferably not more than about 30 mPa·s, and most preferably not more than about 20 mPa·s. In a particularly preferred aspect, the dynamic viscosity of the oil phase of the present invention at 20°C is about 10 mPa·s to about 100 mPa·s, preferably about 10 mPa·s to about 50 mPa·s, more preferably about 10 mPa·s to about 30 mPa·s, and most preferably about 10 mPa·s to about 20 mPa·s. According to a preferred example, the dynamic viscosity of the oil phase of the present invention at 20°C is about 12 mPa·s.

[0089] In one instance, commercially available mineral oil Marcol® 52 (Exxon, USA) was used as the oil phase in this invention, but other oils are also applicable.

[0090] Therefore, in a preferred embodiment, the oil phase of the present invention is Marcol® 52, or, respectively, the oil phase of the present invention is a mixture of components contained in Marcol® 52.

[0091] Preferably, the aqueous buffer solutions mentioned herein are selected from citrate buffer, PBS buffer, and Tris buffer.

[0092] In a particularly preferred aspect, the aqueous phase of the present invention is a citrate buffer containing RNA.

[0093] As used herein, the term "citrate buffer" specifically refers to a buffer solution containing citrate ions (i.e., a solution that resists pH changes when acidic or basic components are added). Specific implementations of the citrate buffer solution may be selected from, but are not limited to, sodium citrate buffer, potassium citrate buffer, calcium citrate buffer, and magnesium citrate buffer.

[0094] Preferably, the citrate buffer solution has a pH of 5.0 to 6.8, and most preferably a pH of 6.4 ± 0.2.

[0095] Preferably, the composition of the present invention is an emulsion.

[0096] As used herein, the term "emulsion" specifically refers to a mixture of two or more liquids that are normally immiscible (immiscible or non-mixable). In an emulsion, one liquid (dispersed phase) is dispersed in another liquid (continuous phase). Examples of emulsions include vinaigrette, homogenized milk, mayonnaise, etc.

[0097] In a preferred aspect, the composition of the present invention is an emulsion in which an aqueous phase is dispersed in an oil phase. Therefore, preferably, the aqueous phase of the present invention is a dispersed phase, and the oil phase of the present invention is a continuous phase.

[0098] The composition of the present invention is preferably a water-in-oil emulsion.

[0099] The term "water-in-oil emulsion" as used in this article specifically refers to a water-in-oil mixture, in which the oil forms a continuous phase and the water exists as discontinuous droplets. Water-in-oil emulsions can be distinguished from oil-in-water emulsions using an electroemulsion tester. Oil-in-water emulsions have relatively low electrical resistance and conductivity because water forms its external or continuous phase, while water-in-oil emulsions are non-conductive or have very poor conductivity.

[0100] In a preferred aspect, the composition of the present invention is a microemulsion.

[0101] As used herein, the term "microemulsion" has its common meaning in the art, referring to a dispersion of an immiscible liquid in another liquid, in the form of droplets, with a diameter in the range of about 1 to about 1000 nm, most preferably less than about 350 nm. The microemulsions of the present invention preferably relate to a dispersion of the aqueous phase of the present invention in the oil phase of the present invention, in the form of droplets, with a diameter in the range of about 1 to about 500 nm, or about 1 to about 350 nm, or about 5 to about 500 nm, or about 5 to about 350 nm, or most preferably about 5 to about 200 nm. This range is also referred to herein as "droplet size distribution". Preferably, the microemulsion is a homogeneous thermodynamically stable system.

[0102] Preferably, the aqueous phase of the present invention is dispersed in the oil phase of the present invention in the form of droplets with an average diameter in the range of 5 to 95 nm, more preferably 10 to 90 nm, even more preferably 15 to 85 nm, still more preferably 20 to 80 nm, and most preferably 25 to 75 nm. The average diameter is also referred to herein as the "average droplet size".

[0103] The droplet size distribution or average droplet size can be determined by those skilled in the art using the dynamic light scattering (DLS) technique described in Example 4. In the event of disputed results or any doubt, the droplet size distribution and average droplet size mentioned herein refer to those that can be estimated using the DLS technique described in Example 4.

[0104] In a preferred aspect, the compositions of the present invention are emulsions, particularly microemulsions, wherein the emulsions are thermodynamically stable.

[0105] In another preferred aspect, the compositions of the invention are emulsions, particularly microemulsions, wherein the emulsions are isotropic.

[0106] In yet another preferred aspect, the composition of the invention is an emulsion, particularly a microemulsion, wherein the emulsion is optically transparent.

[0107] In another preferred aspect, the composition of the invention is an emulsion, particularly a microemulsion, wherein the emulsion is opalescent.

[0108] Most preferably, the composition of the present invention is a thermodynamically stable, optically transparent, and isotropic liquid mixture. In particular, the composition of the present invention is a water-in-oil microemulsion.

[0109] In a preferred aspect, the final concentration of the aqueous phase in the composition of the present invention is about 5% to 30% v / v (volume / volume).

[0110] Most preferably, in the composition of the present invention, the final concentration of the aqueous phase is about 14.3% to 24.3% v / v.

[0111] According to another preferred aspect, in the composition of the present invention, the final concentration of the oil phase is about 5% to 30% v / v.

[0112] Most preferably, in the composition of the present invention, the final concentration of the oil phase is about 8.5% to 18.5% v / v.

[0113] In another preferred aspect, in the composition of the present invention, the final concentration of the first surfactant is about 30.7% to 70.7% v / v.

[0114] Most preferably, in the composition of the present invention, the final concentration of the first surfactant is about 31.9% to 41.9% v / v.

[0115] According to another preferred aspect, in the composition of the present invention, the final concentration of the second surfactant is about 17.5% to 57.5% v / v.

[0116] Most preferably, in the composition of the present invention, the final concentration of the second surfactant is about 25.2% to 35.2% v / v.

[0117] According to a particularly preferred aspect, in the composition of the present invention: - The final concentration of the aqueous phase in this invention is approximately 19% v / v. - The final concentration of the oil phase in this invention is approximately 14% v / v. - The final concentration of the first surfactant is approximately 37% v / v, and - The final concentration of the second surfactant is approximately 30% v / v.

[0118] According to another preferred aspect, the final concentration of the RNA of the present invention in the composition of the present invention is about 0.1 to 500 µg / ml (µg / ml), wherein it should be understood that at said concentration (µg / ml), the mass (µg) refers to the RNA of the present invention, and the volume (ml) refers to the composition of the present invention.

[0119] Preferably, the final concentration of the RNA of the present invention in the composition of the present invention is at least about 1 µg / ml.

[0120] According to another preferred aspect, the final concentration of the RNA in the composition of the present invention is at least about 30 µg / ml.

[0121] In particular, the final concentration of the RNA of the present invention in the composition of the present invention is preferably about 1 to 500 µg / ml, especially about 30 to 500 µg / ml.

[0122] According to another preferred aspect, the final concentration of the RNA of the present invention in the composition of the present invention is about 1 to 400 µg / ml, most preferably about 30 to 300 µg / ml.

[0123] More particularly, it is preferred that the final concentration of the RNA of the present invention in the composition of the present invention is about 200 µg / ml.

[0124] The RNA used in this invention is preferably selected from spontaneous RNA (mRNA), small interfering RNA (siRNA), and microRNA (miRNA).

[0125] As used in this article, “messenger RNA” (mRNA) specifically refers to any polynucleotide, which may be synthetic, encodes a polypeptide (e.g., an immunogenic protein), and is capable of being translated in vitro, in vivo, in situ, or ex vivo to produce the encoded polypeptide. “Small interfering RNA” (siRNA) specifically refers to low-molecular-weight (typically 19–27 base pairs) double-stranded RNA involved in RNAi. “RNAi” as used in this article means RNA interference. “MicroRNA” (miRNA) specifically refers to a non-coding RNA molecule approximately 22 nucleotides long that plays a role in the post-transcriptional regulation of gene expression by forming a base pair with the 3' untranslated region (UTR) of mRNA.

[0126] Most preferably, the RNA of the present invention is mRNA, and the mRNA is also referred to below as "the mRNA of the present invention".

[0127] Specifically, the mRNA of the present invention comprises a 5'-UTR, a coding region encoding a polypeptide, and a 3'-UTR. The term "coding region" as used herein specifically refers to a portion or region of the mRNA that can be translated into an amino acid chain (i.e., two or more amino acids linked by peptide bonds). More specifically, the coding region is flanked by the 5'-UTR and the 3'-UTR. According to a preferred aspect, the mRNA of the present invention comprises a poly(A) tail, wherein the poly(A) tail is preferably located after the 3'-UTR along the 3' direction. Preferably, the 3'-UTR is flanked by a poly(A) tail. Most preferably, the mRNA of the present invention comprises a coding region encoding a polypeptide, wherein the coding region is flanked by the 5'-UTR and the 3'-UTR, and wherein the 3'-UTR is flanked by a poly(A) tail. It should be particularly understood that the coding region mentioned herein also comprises at least one stop codon, particularly at the 3' end of the coding region. Preferably, the 3'-UTR is located after the stop codon and is particularly flanked by the stop codon in the 3' direction.

[0128] Most preferably, the mRNA of the present invention comprises (from 5' to 3' direction) a 5'-UTR, a coding region, a 3'-UTR, and a poly(A) tail.

[0129] The term "untranslated region" or "UTR" as used in this article refers to the untranslated regions in mRNA upstream of the start codon and downstream of the stop codon, hence referred to as the 5' UTR and 3' UTR, respectively. These regions are transcribed along with the coding regions and are therefore exons because they are present in mature mRNA.

[0130] In this invention, the 3' untranslated region (3'-UTR) refers to the segment of messenger RNA (mRNA) immediately following the translation stop codon. The 3' UTR may contain regulatory regions within the 3'-UTR that are known to affect polyadenylation and stability of mRNA. Many 3'-UTRs also contain AU-rich elements (AREs). Furthermore, the 3'-UTR preferably contains the sequence AAUAAA, which directs the addition of hundreds of adenine residues, known as poly(A) tails, to the end of the mRNA transcript. The 5' untranslated region (5'-UTR) (also called the leader sequence or leader RNA) is the region of mRNA immediately upstream of the start codon. The 5' UTR begins at the transcription start site and ends one nucleotide (nt) before the coding start codon (typically AUG in mRNA). In eukaryotes, the length of the 5'-UTR is typically 100 to several thousand nucleotides, but shorter UTRs are sometimes observed in eukaryotes.

[0131] As used herein, the terms “poly(A) tail” or “poly(A) sequence” refer to a continuous or discontinuous sequence of adenosine residues, typically located at the 3' end of an mRNA molecule. Poly(A) tails or poly(A) sequences are known to those skilled in the art and can be located after the 3'-UTR in the RNA (particularly mRNA) described herein. A continuous poly(A) tail is characterized by a continuous sequence of adenosine residues. Continuous poly(A) tails are typical in nature. The RNA (particularly mRNA) disclosed herein may have a poly(A) tail attached to the free 3' end of the RNA post-transcriptionally by a template-independent RNA polymerase, or the poly(A) tail may be encoded by DNA and transcribed by a template-dependent RNA polymerase.

[0132] The poly(A) tail of approximately 120 A nucleotides has been shown to have a beneficial effect on the level of mRNA transfected in eukaryotic cells and on the level of protein translated from an open reading frame located upstream (5') of the poly(A) tail (Holtkamp et al., Blood, 108:4009-4017 (2006)).

[0133] The poly(A) tail can be of any length. In some embodiments, the poly(A) tail comprises, is substantially composed of, or consists of at least 20, at least 30, at least 40, at least 80, or at least 100 and at most 500, at most 400, at most 300, at most 200, or at most 150 A nucleotides, particularly about 120 A nucleotides. In this context, "substantially composed of" means that the majority of the nucleotides in the poly(A) tail are typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the nucleotides in the poly(A) tail are A nucleotides, but the remaining nucleotides are allowed to be nucleotides other than A nucleotides, such as U nucleotides (uridine monophosphate), G nucleotides (guanosine monophosphate), or C nucleotides (cytidine monophosphate). In this context, "composed of" means that all the nucleotides in the poly(A) tail are 100% A nucleotides. The term "A nucleotide" or "A" refers to adenosine monophosphate.

[0134] In some implementations, the poly(A) tail is ligated to a DNA template containing repeating dT nucleotides (deoxythymidines) in the complementary strand of the coding strand during RNA transcription (e.g., during the preparation of RNA transcribed in vitro). The DNA sequence encoding the poly(A) tail (coding strand) is called a poly(A) cassette.

[0135] According to a preferred aspect, the mRNA of the present invention contains a Kozak sequence. The term "Kozak sequence" as used herein specifically refers to a common sequence contained in the mRNA that is recognized by ribosomes to initiate polypeptide translation.

[0136] The Kozak sequence mentioned herein specifically has an RNA sequence (from 5' to 3' direction) RCCAUG, preferably RCCAUGG, where AUG is the start codon of the coding region and R is selected from A and G. In a preferred embodiment, the mRNA of the present invention contains the Kozak sequence ACCAUGG.

[0137] In one non-limiting example, the mRNA of the present invention comprises a 5'-UTR (from 5' to 3' direction), a coding region, a 3'-UTR, and a poly(A) tail, and includes a Kozak sequence, which is the mRNA of SEQ ID NO:3, wherein: - Nucleotides 1 to 359 of SEQ ID NO:3 correspond to the 5´-UTR. - Nucleotides 360 to 1070 of SEQ ID NO:3 correspond to the coding region. - Nucleotides 357 to 363 of SEQ ID NO:3 correspond to the Kozak sequence. - Nucleotides 1071 to 1332 of SEQ ID NO:3 correspond to the 3´-UTR. - Nucleotides 1333 to 1452 of SEQ ID NO:3 correspond to poly(A) tails.

[0138] According to another aspect, the mRNA of the present invention preferably contains one or more modifications, for example, to increase its stability and / or increase translation efficiency and / or reduce immunogenicity and / or reduce cytotoxicity. For example, to increase mRNA expression, it may be modified within the coding region (i.e., within the sequence encoding the expressed peptide or polypeptide), preferably without altering the sequence of the expressed peptide or polypeptide.

[0139] Such modifications are described, for example, in WO2007 / 036366, WO2019 / 175356 or WO2023 / 147352, and include the following: 5'-cap structure; extension or truncation of naturally occurring poly(A) tails; alteration of the 5'- and / or 3'-untranslated regions (UTRs), such as the introduction of a UTR unrelated to the coding region of the RNA; replacement of one or more naturally occurring nucleotides with synthetic nucleotides; and codon optimization (e.g., alteration, preferably an increase in the GC content of the RNA).

[0140] According to a particularly preferred aspect, the mRNA of the present invention comprises a 5'-cap structure. In some embodiments, the mRNA of the present invention does not have an uncapped 5'-triphosphate. In some embodiments, the mRNA of the present invention may comprise a conventional 5'-cap and / or a 5'-cap analogue. The term "5'-cap structure" as used herein should be specifically understood to encompass both "conventional 5'-cap" and "5'-cap analogue". Most preferably, the mRNA of the present invention comprises a conventional 5'-cap.

[0141] The term "conventional 5'-cap" refers to the cap structure found at the 5' end of an mRNA molecule, typically composed of guanosine 5'-triphosphate (Gppp), which is linked to the 5' end of the next nucleotide in the mRNA via its triphosphate portion (i.e., guanosine is linked to the rest of the mRNA via a 5'-to-5' triphosphate bond). Guanosine can be found in N... 7 Methylation at position (resulting in a cap structure m7Gppp). In the context of this application, the terms "cap0" or "cap0 structure" respectively refer to the structure "m 7 "GpppN", where N is any nucleoside with an OH moiety at the 2' position. According to this application, the terms "cap1" or "cap1 structure" respectively refer to the structure "m 7 "GpppNm", where Nm is any nucleotide with an OCH3 moiety at the 2' position. According to this application, the terms "cap2" or "cap2 structure" respectively refer to the structure "m 7 "GpppNmNm", where each Nm is independently any nucleoside with an OCH3 motif at the 2' position.

[0142] According to a preferred embodiment, the mRNA of the present invention comprises a cap1 structure, particularly the structure m7GpppNm. Therefore, the mRNA of the present invention preferably comprises a 5' cap having the cap1 structure m7GpppNm, wherein "m7Gppp" represents that the guanosine is present in the N... 7 5'-triphosphate of guanosine methylated at position 2', where "Nm" represents any nucleoside with an OCH3 moiety at position 2', and where m7Gppp is attached to the 5' end of Nm via its triphosphate moiety.

[0143] According to a particularly preferred aspect, the mRNA of the present invention comprises a 5' cap having a cap1 structure m7GpppAm, wherein Am is an adenosine with an OCH3 moiety at the 2' position. Therefore, it is most preferred that the mRNA of the present invention comprises an m7GpppAm cap. The production of mRNA having said cap can be performed, for example, using CleanCap® AG reagent (TriLink Biotechnologies, San Diego (CA), USA, Product No. N-7113) according to the manufacturer's instructions.

[0144] Therefore, the mRNA of the present invention preferably contains a 5´-cap having a cap1 structure m7GpppAm, wherein "m7Gppp" represents that the guanosine is present in the N... 7 5'-triphosphate of guanosine with methylation at position 2', where "Am" represents adenosine with an OCH3 moiety at position 2', and where m7Gppp is attached to the 5' end of Am via its triphosphate moiety.

[0145] The term "5'-cap analogue" refers to a structural derivative with a conventional 5'-cap. For example, in the structure m7Gppp, in N... 7 Methylated guanosines can contain hydrogen atoms at the C-3' hydroxyl group that are replaced by methyl groups (producing the cap structure m7(3'OMe)Gppp). Therefore, the structure m7(3'OMe)Gppp mentioned herein specifically refers to the structure m7Gppp, but where the N-methyl group is methylated... 7 The methylated guanosine has an OCH3 moiety at the 3' position. Examples of 5'-cap analogues are described, for example, in WO2023 / 147352 or WO2019 / 175356.

[0146] In the context of this application, the terms "cap0 analogue" or "cap0 analogue structure" respectively refer to the structure "m 7 The structural derivative of “GpppN”, and wherein the structural derivative is preferably the structure “m7(3´OMe)Gppp”. According to this application, the terms “cap1 analog” or “cap1 analog structure” respectively refer to the structure “m…” 7 The structural derivative of “GpppNm”, and wherein the structural derivative is preferably the structure “m7G(3´OMe)pppNm”. According to this application, the terms “cap2 analog” or “cap2 analog structure” respectively refer to the structure “m”. 7 The structural derivative of “GpppNmNm” is preferably “m7G(3´OMe)pppNmNm”.

[0147] According to a preferred embodiment, the mRNA of the present invention comprises a cap1 analog structure, particularly the structure m7G(3´OMe)pppNm.

[0148] For example, the mRNA of the present invention may comprise a 5'-cap analog having a cap1 analog structure m7G(3'OMe)pppAm, wherein Am is an adenosine with an OCH3 moiety at the 2' position. The production of mRNA having said cap can be performed, for example, using CleanCap® AG 3'OMe reagent (TriLink Biotechnologies, San Diego (CA), USA, product number N-7413) according to the manufacturer's instructions.

[0149] Providing mRNA with a 5'-cap structure can be achieved by in vitro transcription of a DNA template in the presence of a suitable 5'-cap compound, wherein the 5'-cap structure is co-transcribed into the resulting mRNA strand, or the mRNA can be generated, for example, by in vitro transcription, and the 5'-cap structure can be ligated to the mRNA post-transcriptionally using a capping enzyme (e.g., a capping enzyme of vaccinia virus). Methods for providing 5'-cap structures to mRNA are known to those skilled in the art and are summarized, for example, in review articles such as Muttach et al., Beilstein J Org Chem. 13:2819-2832 (2017) and Ramanathan et al., Nucleic Acids Res. 44(16):7511-7526 (2016).

[0150] Preferably, the mRNA of the present invention encodes a peptide or polypeptide selected from antigens, functional proteins, and reporter proteins.

[0151] According to a preferred aspect, the RNA of the present invention is mRNA encoding an antigen or a functional protein.

[0152] In the context of the mRNA of this invention, the terms "encoded mRNA" or "mRNA encoding" are equivalent to "mRNA containing a coding region encoding..." or "mRNA containing a coding region encoding...", respectively.

[0153] The term "antigen" as used herein generally refers to a substance, such as a polypeptide or peptide, that, when applied to a host, can elicit an immune response in the host. The term "antigen" as used herein is interchangeable with the term "immunogen." In one instance, the antigen referred to herein may be an immunogenic protein or an immunogenic protein domain. The term "immunogenicity" as used herein means "the ability to elicit a humoral and / or cellular immune response in a host."

[0154] As used in this article, the term "functional protein" refers to a polypeptide that, when in contact with a substrate, can affect the structure and / or appearance of the product, such as an enzyme that functions as a substrate.

[0155] As used herein, the term "peptide" specifically refers to any chain of amino acid residues linked together by peptide bonds and does not refer to a product of a specific length. For example, "peptide" can refer to a long chain of amino acid residues, such as 150 to 600 amino acid residues or longer. The term "peptide" specifically includes peptides that have been altered by one or more co-translational and / or post-translational modifications, wherein such modifications include, for example, glycosylation, phosphorylation, esterification (e.g., myristylation), acetylation, ubiquitination, sulfation, ADP ribosylation, hydroxylation, Cys / Met oxidation, carboxylation, methylation, etc. The terms "peptide" and "protein" are used interchangeably in the context of this invention.

[0156] As used herein, “encoded mRNA” specifically means mRNA that, if present in a suitable environment (preferably within a cell), can be expressed to produce the protein or peptide it encodes.

[0157] Most preferably, the RNA of the present invention is mRNA encoding an antigen.

[0158] According to a preferred aspect, the RNA of the present invention is mRNA encoding an antigenic peptide or antigenic polypeptide.

[0159] The term "antigenic peptide or antigenic polypeptide" as used herein specifically refers to peptides or polypeptides capable of inducing, generating, or producing an immune response in a host (particularly an animal). In the context of this invention, the term "antigenic" should be understood to be equivalent to "immunogenicity."

[0160] According to another preferred aspect, the RNA of the present invention is mRNA encoding a reporter protein. The term "reporter protein" as used herein specifically refers to a protein encoded by a reporter gene. The term "reporter gene" refers to any gene that expresses a detectable gene product, particularly any gene whose expression can be measured, such as the expression of a fluorescent protein or enzyme. In one embodiment of this aspect, the RNA of the present invention is mRNA encoding a fluorescent protein, particularly selected from mCherry, GFP (green fluorescent protein), and derivatives of said proteins. Exemplarily, the RNA of the present invention is mRNA encoding an mCherry derivative, wherein said mCherry derivative comprises or consists of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, and still more preferably at least 95% sequence identity with the sequence of SEQ ID NO:1. The sequence of SEQ ID NO:1 can be found under GenBank accession number AY678264.1. The mRNA encoding the mCherry derivative preferably comprises a nucleotide sequence having at least 70%, preferably at least 80%, more preferably at least 90%, and still more preferably at least 95%, or particularly 100% sequence identity with the sequence of SEQ ID NO:2.

[0161] According to a particularly preferred aspect, the RNA of the present invention is mRNA encoding an enzyme. The term "enzyme" refers to a protein having catalytic activity. For example, catalytic activity includes dephosphorylation activity. In one example, the RNA of the present invention is mRNA encoding a hydrolase (such as alkaline phosphatase).

[0162] In one embodiment of this aspect, the RNA of the present invention is an mRNA encoding human alkaline phosphatase. Exemplarily, the RNA of the present invention is an mRNA encoding secretory embryonic human alkaline phosphatase (SEAP), specifically a C-terminal truncated variant of human placental alkaline phosphatase (PLAP). Preferably, the RNA of the present invention is an mRNA encoding SEAP, which comprises or consists of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, and still more preferably at least 95% sequence identity with the sequence of SEQ ID NO:4. The sequence of SEQ ID NO:4 can be found under GenBank accession number AAB64400.1. The mRNA encoding SEAP preferably comprises a nucleotide sequence having at least 70%, preferably at least 80%, more preferably at least 90%, and still more preferably at least 95%, or particularly 100% sequence identity with the sequence of SEQ ID NO:5.

[0163] It should be understood that the terms "composed of an amino acid sequence" or "composed of an amino acid sequence" as used herein also specifically refer to any co-translational and / or post-translational modifications of the amino acid sequence affected by the cells expressing the protein or protein domain. Therefore, the terms "composed of an amino acid sequence" or "composed of an amino acid sequence" as used herein also refer to (unless explicitly stated otherwise) amino acid sequences having one or more modifications affected by the cells expressing the protein or protein domain, particularly amino acid residue modifications affecting protein biosynthesis and / or protein processing, preferably selected from glycosylation, phosphorylation, and acetylation.

[0164] Regarding the term "at least 90%" as used in the context of this invention, it should be understood that the term preferably refers to "at least 91%", more preferably to "at least 92%", still more preferably to "at least 93%" or particularly "at least 94%".

[0165] Regarding the term "at least 95%" as used in the context of this invention, it should be understood that the term preferably refers to "at least 96%", more preferably to "at least 97%", still more preferably to "at least 98%" or particularly "at least 99%".

[0166] Regarding the term "at least 99%" as used in the context of this invention, it should be understood that the term preferably refers to "at least 99.2%", more preferably to "at least 99.4%", still more preferably to "at least 99.6%" or particularly "at least 99.8%".

[0167] The term “100% sequence identical” used in this article should be understood as equivalent to the term “identical”.

[0168] Percentage sequence identity has a generally accepted meaning in the field, and there are various methods to measure the identity between two polypeptide or polynucleotide sequences. See, for example, Lesk, Ed., Computational Molecular Biology, Oxford University Press, New York, (1988); Smith, Ed., Biocomputing: Informatics And Genome Projects, Academic Press, New York, (1993); Griffin & Griffin, Eds., Computer Analysis Of Sequence Data, Part I, Humana Press, New Jersey, (1994); von Heinje, Sequence Analysis In Molecular Biology, Academic Press, (1987); and Gribskov & Devereux, Eds., Sequence Analysis Primer, Stockton Press, New York, (1991). Methods for aligning polynucleotides or peptides are encoded in computer programs, including the GCG package (Devereux et al., Nuc. Acids Res. 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul et al., J. Molec. Biol. 215:403 (1990)), and the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711), which uses Smith and Waterman's local homology algorithm (Adv. App. Math., 2:482-489 (1981)). For example, the computer program ALIGN, which uses the FASTA algorithm, employs affine space search with a space open penalty of -12 and a space extension penalty of -2.For the purposes of this invention, nucleotide sequences were aligned using the Clustal W method in MegAlign software version 11.1.0 (59), 419 of DNASTAR Inc., using the default multiple alignment parameters set in the program (vacancy penalty = 15.0, vacancy length penalty = 6.66, delayed divergent sequence (%) = 30%, DNA conversion weight = 0.50, DNA weight matrix = IUB), and protein / amino acid sequences were aligned using the Clustal W method in MegAlign software version 11.1.0 (59), 419 of DNASTAR Inc., using the default multiple alignment parameters set in the program (Gonnet series protein weight matrix, vacancy penalty = 10.0, vacancy length penalty = 0.2, delayed divergent sequence (%) = 30%).

[0169] As used herein, the term “sequence identity with the sequence of SEQ ID NO:X” should be understood in particular to be equivalent to the term “sequence identity with the sequence of SEQ ID NO:X over the entire length of SEQ ID NO:X” or equivalent to the term “sequence identity with the sequence of SEQ ID NO:X over the entire length of SEQ ID NO:X”. In this context, “X” is any integer selected from 1 to 7, and therefore “SEQ ID NO:X” refers to any SEQ ID NO mentioned herein.

[0170] According to a preferred aspect, the RNA of the present invention is mRNA encoding pathogen antigens. The term "pathogen antigen" as used herein specifically refers to antigens encoded by or derived from the genome of a pathogen. The term "pathogen" as used herein refers to a biological agent that causes a disease state (e.g., infection, sepsis, etc.) in a host. "Pathogen" includes, but is not limited to, viruses, bacteria, archaea, fungi, protozoa, mycoplasma, prions, and parasites.

[0171] Preferably, the RNA of the present invention is an mRNA encoding a viral antigen, a bacterial antigen, and an antigen derived from a parasite.

[0172] In a particularly preferred aspect, the RNA of the present invention is mRNA encoding viral antigens.

[0173] As used herein, the term "viral antigen" specifically refers to an antigen derived from a virus or its components or both, which is capable of inducing an immune response. Therefore, the viral antigen particularly refers to a viral protein or an immunogenic fragment thereof. The term "viral protein" encompasses any protein derived from a virus. Specific examples include viral glycoproteins, viral capsid proteins, viral outer coat proteins, viral envelope proteins, and viral core proteins.

[0174] Preferably, the viral antigen is an RNA virus antigen. The term "RNA virus" refers to a virus whose genome contains RNA. In particular, the virus is a single-stranded (ss) RNA virus, therefore the viral antigen is preferably an ss RNA virus antigen.

[0175] In one particular aspect, the RNA of the present invention is mRNA encoding viral glycoproteins or viral capsid proteins.

[0176] Preferably, the RNA of the present invention is mRNA encoding a viral glycoprotein.

[0177] The term "glycoprotein" is used herein in its normal scientific sense, specifically referring to proteins modified to include one or more monosaccharide or oligosaccharide chains ("glycans") covalently bound to a protein. Glycans may be attached to a hydroxyl group on a protein (O-linked glycans), such as those attached to serine, threonine, tyrosine, hydroxylysine, or hydroxyproline; or to an amide functional group on a protein (N-glycoproteins), such as asparagine or arginine; or to a carbon atom on a protein (C-glycoproteins), such as tryptophan. Glycans may contain more than one glycan, may contain combinations of one or more monosaccharides and one or more oligosaccharides, and may contain combinations of N-linked, O-linked, and C-linked glycans. The term "viral glycoprotein" as used herein specifically refers to glycoproteins encoded by or derived from a viral genome.

[0178] In a preferred embodiment, the virus referred to herein is an influenza virus. Therefore, in the context of this invention, the viral antigen referred to herein is exemplarily an influenza virus antigen, and the viral glycoprotein referred to herein is preferably an influenza virus glycoprotein.

[0179] Specifically, the virus mentioned herein is preferably an influenza A virus. The term "influenza A virus" should be understood to be particularly equivalent to "influenza virus type A" or "influenza virus A," as is commonly used in the context of influenza viruses, where "A" indicates the viral antigen type (based on the antigenic specificity of the nucleoprotein). Therefore, in the context of this invention, the viral antigen mentioned herein is exemplary an influenza A virus antigen, and the viral glycoprotein mentioned herein is preferably an influenza A virus glycoprotein.

[0180] More specifically, the virus mentioned herein is preferably a swine influenza virus. Therefore, in the context of this invention, the viral antigen mentioned herein is exemplary a swine influenza virus antigen, and the viral glycoprotein mentioned herein is preferably a swine influenza virus glycoprotein.

[0181] More specifically, the virus mentioned herein is preferably a swine influenza A virus. Therefore, in the context of this invention, the viral antigen mentioned herein is exemplarily a swine influenza A virus (SIAV) antigen, and the viral glycoprotein mentioned herein is preferably a SIAV glycoprotein. The terms "swine influenza A virus" and "SIAV" as used herein should be understood to be interchangeable with "swine influenza virus A" and "swine influenza A virus," respectively, where "A" indicates the antigenic type of the influenza virus.

[0182] For example, the terms "swine influenza A virus antigen" or "SIAV antigen" specifically refer to peptides or polypeptides derived from swine influenza A virus (SIAV) or its components or both, which are capable of inducing an immune response. Thus, in one example, the RNA of the present invention is mRNA encoding a SIAV antigen. Or more specifically, the RNA of the present invention is mRNA encoding, for example, a swine influenza A virus (SIAV) glycoprotein or an immunogenic fragment thereof. Thus, in one example, the mRNA encodes a SIAV glycoprotein.

[0183] More specifically, the virus mentioned herein is preferably the H3N2 subtype of swine influenza A virus. Therefore, in the context of this invention, the viral antigen mentioned herein is exemplary to be the H3N2 antigen of swine influenza A virus subtype A, and the viral glycoprotein mentioned herein is preferably the H3N2 glycoprotein of swine influenza A virus subtype A.

[0184] The antigenic types (e.g., “A”, as expressed in the terms “influenza A virus” and “influenza A virus”), subtypes (e.g., “H3N2”) and hemagglutinin antigenic characteristics (e.g., “H3”) of influenza viruses used in this article refer in particular to the internationally recognized influenza virus nomenclature system published in the World Health Organization Bulletin, 58(4):585-591 (1980).

[0185] According to a preferred embodiment, the RNA of the present invention is an mRNA encoding hemagglutinin or an immunogenic fragment thereof. In one embodiment, the mRNA encodes hemagglutinin.

[0186] Specifically, the RNA of the present invention is an mRNA encoding hemagglutinin 3 (H3). According to another preferred aspect, the RNA of the present invention is an mRNA encoding its immunogenic fragment (i.e., the immunogenic fragment of H3). The terms "hemagglutinin 3" and "H3" as used herein should be understood as equivalent to "hemagglutinin 3 protein" and "H3 protein," respectively.

[0187] Most preferably, the RNA of the present invention is an mRNA encoding SIAV H3 or an immunogenic fragment thereof. In one example, the RNA encodes SIAV H3.

[0188] The term "immunogenic fragment" as used herein should be specifically understood to refer to a fragment of a protein that at least partially retains the immunogenicity of the protein from which it is derived. Thus, for example, "immunogenic fragment of SIAV H3" should be specifically understood to refer to a fragment of SIAV H3 that at least partially retains the immunogenicity of the full-length H3 protein.

[0189] According to a preferred aspect, the RNA of the present invention is an mRNA encoding a hemagglutinin, said hemagglutinin comprising or consisting of an amino acid sequence having at least 95%, preferably at least 98%, more preferably at least 99%, or particularly 100% sequence identity with the sequence of SEQ ID NO:6.

[0190] The mRNA encoding hemagglutinin preferably comprises a nucleotide sequence having at least 70%, preferably at least 80%, more preferably at least 90%, still more preferably at least 95%, or particularly 100% sequence identity with the sequence of SEQ ID NO:7.

[0191] According to another preferred aspect of the invention, the mRNA mentioned herein is synthetic mRNA.

[0192] "Synthetic mRNA" refers to mRNA produced outside the cell, or mRNA produced inside the cell using bioengineering, including mRNA produced in an in vitro transcription reaction by non-restrictive examples, mRNA produced by direct chemical synthesis, or mRNA produced in genetically engineered E. coli cells.

[0193] According to another particularly preferred aspect of the invention, the mRNA mentioned herein is nucleoside-modified mRNA.

[0194] The term "nucleoside-modified RNA" refers to mRNA containing one or more nucleosides modified relative to an equivalent RNA nucleoside by introducing one or more modifications to a sugar moiety or a (nucleo)base moiety. In a preferred embodiment, the modified nucleoside comprises a modified sugar moiety. In one example, the nucleoside-modified mRNA is an mRNA in which at least one uridine is replaced by a pseudouridine.

[0195] According to another preferred aspect, the length of the mRNA of the present invention is at least 300 nucleotides, preferably at least 500 nucleotides, more preferably at least 1000 nucleotides, and most preferably at least 1500 nucleotides.

[0196] In another preferred aspect, the mRNA of the present invention is 300 to 5000 nucleotides in length, preferably 500 to 4000 nucleotides, and most preferably 1000 to 3000 nucleotides.

[0197] The present invention also provides compositions thereof for use as medicines, preferably as vaccines, or for use as diagnostic methods implemented in the body.

[0198] In another aspect, the present invention also relates to the use of the compositions of the invention in the preparation of pharmaceuticals, preferably vaccines, or in the preparation of diagnostic agents.

[0199] As used in this article, the term "vaccine" specifically refers to a pharmaceutical composition containing at least one immunologically active component that, when administered to animals, induces an immune response in the animals.

[0200] Furthermore, the present invention provides a composition of the present invention in a method for delivering the RNA of the present invention to a object, wherein the method includes locally applying the composition of the present invention to the object.

[0201] In another aspect, the present invention relates to the composition of the present invention in a method for delivering the RNA of the present invention to a subject through the skin, wherein the method includes applying the composition of the present invention to the skin of the subject.

[0202] The present invention also provides a method for delivering ribonucleic acid (RNA) to a subject, comprising the step of locally applying the composition of the present invention to the subject.

[0203] Meanwhile, the present invention provides a method for delivering ribonucleic acid (RNA) to a subject through the skin, including the step of applying the composition of the present invention to the skin of the subject.

[0204] In each of the methods for delivering ribonucleic acid (RNA), the aqueous phase of the composition of the present invention preferably contains the RNA to be delivered to the target. Therefore, in the methods for delivering ribonucleic acid (RNA), the RNA to be delivered to the target is preferably the RNA of the present invention.

[0205] In any medical use and delivery method within the context of this invention, it is particularly preferred to apply the composition of the invention topically to the surface of the ear of the subject. Most preferably, the composition of the invention is applied topically to the inner ear of the subject. This allows for a particularly simple application of RNA to the subject.

[0206] The present invention also provides the compositions thereof for use in methods of inducing an immune response in a subject. The immune response is particularly an immune response against antigens encoded by the mRNA of the present invention.

[0207] The “immune response” referred to herein can be a cell- and / or antibody-mediated immune response, particularly an immune response against antigens encoded by the mRNA of this invention and / or against pathogens from which said antigens originate.

[0208] Typically, an "immune response" includes, but is not limited to, one or more of the following effects: the generation or activation of antibodies, B cells, helper T cells, suppressor T cells and / or cytotoxic T cells and / or γδ T cells specifically targeting one or more antigens encoded by the RNA contained in the immunogenic composition of the present invention. Preferably, subjects administered the composition of the present invention containing the mRNA of the present invention encoding the antigen will exhibit a protective immune response or a therapeutic response.

[0209] A “protective immune response” will be demonstrated by a reduction or absence of one or more clinical signs typically exhibited by the infected host, faster recovery time and / or shorter duration of infectivity, or a decrease in pathogen titers in the infected host’s tissues, body fluids or excretions.

[0210] The present invention particularly provides a composition of the invention for a method of inducing an immune response in a subject, wherein the immune response is an immune response against an antigen encoded by the mRNA of the present invention.

[0211] The present invention also provides the composition of the invention in a method for inducing an immune response in a subject, wherein the immune response is preferably an immune response against a pathogen.

[0212] In particular, the compositions of the present invention are used in methods for inducing an immune response in a subject against pathogens selected from viruses, bacteria, and parasites.

[0213] In one aspect, the composition of the present invention is used in a method for inducing an immune response against a virus in a subject.

[0214] In one aspect, the composition of the present invention is used in a method for inducing an immune response against an influenza virus in a subject.

[0215] In one aspect, the composition of the present invention is used in a method for inducing an immune response against a swine influenza virus in a subject.

[0216] In one aspect, the composition of the present invention is used in a method for inducing an immune response against swine influenza A virus (SIAV) in a subject.

[0217] In one aspect, the composition of the present invention is used in a method for inducing an immune response against an influenza virus containing H3 in a subject.

[0218] In one aspect, the composition of the present invention is used in a method for inducing an immune response against H3N2 influenza virus in a subject.

[0219] In one aspect, the composition of the present invention is used in a method for inducing an immune response against the SIAV H3N2 subtype in a subject.

[0220] According to another preferred aspect, the immune response mentioned herein is a mucosal immune response.

[0221] Preferably, any of the above medical uses and methods include administering one or more doses of the composition of the present invention to the subject.

[0222] Preferably, any of the above medical uses and methods include administering two doses of the composition of the present invention to the subject.

[0223] Preferably, the volume of one dose of the composition of the present invention is about 0.01 ml to 1 ml.

[0224] Preferably, the volume of one dose of the composition of the present invention is about 0.05 ml to 0.5 ml.

[0225] Preferably, the volume of one dose of the composition of the present invention is about 0.1 ml to 0.25 ml.

[0226] In the context of this invention, the preferred object is a mammal.

[0227] Preferably, the object is an animal.

[0228] Preferably, the object is a pig, especially a piglet.

[0229] The present invention also provides a method for producing the composition of the present invention, comprising the step of mixing the following substances in a container: - oil phase, - First surfactant, - The second surfactant, and - An aqueous phase containing RNA.

[0230] The present invention also provides a method for producing the composition of the present invention, comprising the following steps: (a) The mixed oil phase, the first surfactant, and the second surfactant, and (b) Add an aqueous phase containing RNA to the mixture obtained in step (a).

[0231] Preferably, in the method for producing the composition of the present invention: - The oil phase is the oil phase of this invention. - The first surfactant is the first surfactant of this invention. - The second surfactant is the second surfactant of the present invention, and - The aqueous phase is the aqueous phase of this invention.

[0232] More specifically, referring to the further disclosed terms below, in the method of producing the composition of the invention: - The oil phase is the oil phase specified in any of clauses 8, 9, 17, 18, 35 to 49, 85, 86 and 91. - The first surfactant is the first surfactant specified in any one of clauses 10 to 12, 17 to 25, 37, 38, 53 to 57, 87, 88 and 91. - The second surfactant is the second surfactant specified in any one of clauses 13 to 18, 26 to 30, 37, 38, 58 to 61, 89 to 91, and / or - The aqueous phase is the aqueous phase specified in any of clauses 6, 7, 17, 18, 31 to 33, 37, 38, 51, 52, 82 to 84 and 91. And preferably, the RNA is the RNA specified in any one of clauses 92 to 155.

[0233] Furthermore, in the context of this invention, a hemagglutinin 3 (H3) is provided, wherein the H3 comprises or consists of an amino acid sequence having at least 90% sequence identity with the sequence of SEQ ID NO:6, and the H3 is also referred to below as "H3 related to this invention".

[0234] In one aspect, the relevant H3 of the present invention is an H3 containing an amino acid sequence having at least 90% sequence identity with the sequence of SEQ ID NO:6.

[0235] On the other hand, the present invention relates to H3, which is composed of an amino acid sequence having at least 90% sequence identity with the sequence of SEQ ID NO:6.

[0236] In a preferred aspect, the relevant H3 of the present invention has a tyrosine residue at position 295 of the amino acid.

[0237] In another preferred aspect, the present invention's related H3 has an alanine residue at position 176 of the amino acid.

[0238] In another preferred aspect, the relevant H3 of the present invention has an asparagine residue at position 188 of the amino acid.

[0239] More preferably, the related H3 of the present invention has: - At the alanine residue at amino acid position 176, - The asparagine residue at amino acid position 188, and - The tyrosine residue at position 295 of the amino acid.

[0240] As described herein, the amino acid positions are numbered with reference to the amino acid sequence of the full-length wild-type H3 protein (SEQ ID NO:6). Therefore, the amino acid positions mentioned herein refer to the wild-type H3 sequence with 566 amino acid residues, including the methionine residue at the first (N-terminal) amino acid position.

[0241] Therefore, the phrase “wherein the amino acid positions are numbered with reference to the amino acid sequence of wild-type H3” as used herein refers to the sequence of naturally occurring H3, as exemplarily shown in SEQ ID NO:6.

[0242] According to a preferred aspect, the relevant H3 of the present invention has a glutamine residue at position 180 of the amino acid.

[0243] According to another preferred aspect, the relevant H3 of the present invention has an arginine residue at the 21st position of the amino acid.

[0244] According to yet another preferred aspect, the relevant H3 of the present invention has an aspartic acid residue at position 291 of the amino acid.

[0245] According to another preferred aspect, the relevant H3 of the present invention has a lysine residue at position 69 of the amino acid.

[0246] According to yet another preferred aspect, the relevant H3 of the present invention has a valine residue at position 214 of the amino acid.

[0247] In a preferred aspect, the present invention's related H3 has an alanine residue at position 176 of the amino acid.

[0248] In another preferred aspect, the present invention has an asparagine residue at position 188 of the amino acid H3.

[0249] In another preferred aspect, the relevant H3 of the present invention has an aspartic acid residue at position 140 of the amino acid.

[0250] In another preferred aspect, the relevant H3 of the present invention has a threonine residue at position 208 of the amino acid.

[0251] In yet another preferred aspect, the present invention has a valine residue at the 10th position of the amino acid H3.

[0252] Preferably, the H3 of the present invention has a phenylalanine residue at the 19th position of the amino acid.

[0253] On the other hand, the H3 of the present invention has a histidine residue at position 323 of the amino acid.

[0254] In another aspect, the present invention relates to H3 having an isoleucine residue at position 546 of the amino acid.

[0255] In another aspect, the relevant H3 of the present invention has an isoleucine residue at position 552 of the amino acid.

[0256] Preferably, the relevant H3 of the present invention is the H3 of swine influenza A virus (SIAV).

[0257] Preferably, the H3 associated with this invention is H3 of the swine influenza A virus (SIAV) H3N2 subtype.

[0258] According to a preferred aspect, the relevant H3 of the present invention comprises or consists of an amino acid sequence having at least 95% sequence identity with the sequence of SEQ ID NO:6.

[0259] According to another preferred aspect, the relevant H3 of the present invention comprises or consists of an amino acid sequence having 99% sequence identity with the sequence of SEQ ID NO:6.

[0260] According to yet another preferred aspect, the relevant H3 of the present invention comprises or consists of an amino acid sequence having at least 99.3%, preferably at least 99.5%, more preferably at least 99.7%, or particularly 100% sequence identity with the sequence of SEQ ID NO:6.

[0261] In addition, an immunogenic composition is provided that contains the H3 related to this invention.

[0262] In addition, a polynucleotide is provided comprising a sequence encoding the H3 related to this invention, and also provides: - A carrier containing the polynucleotide, or - A plasmid containing the polynucleotide.

[0263] An mRNA is also provided that contains a sequence encoding the H3 related to this invention.

[0264] Furthermore, in the context of H3 related to this invention, it is preferred that: - The H3, - The immunogenic composition, - The polynucleotide, - The mRNA - The carrier, and / or - The plasmid Used in the preparation of medicines, especially vaccines.

[0265] Furthermore, within the context of H3 of this invention, a method for inducing an immune response against a virus in an object is provided, wherein the method includes administering to the object: - The H3, - The immunogenic composition, - The polynucleotide, - The mRNA - The carrier, and / or - The plasmid Furthermore, the immune response against the virus is preferably an immune response against the following: - Influenza virus, - Swine flu virus, - Swine influenza A virus (SIAV) - Influenza viruses containing H3, - H3N2 influenza virus, and / or - SIAV H3N2 subtype.

[0266] An isolated influenza virus is also provided, comprising the invention-related H3 and / or its genome encoding the invention-related H3, wherein the influenza virus is preferably: - Swine influenza A virus (SIAV), and / or - SIAV H3N2 subtype.

[0267] In this context, "separated" means altered from its natural state "artificially," that is, if it exists in nature, it has been altered or removed from its original environment, or both. For example, a virus naturally present in a living organism is not "separated," but the separation of the same virus from its natural coexisting material is "separated," as the term is used here.

[0268] Example The following examples are intended to illustrate this disclosure only. They do not limit the scope of the claims in any way.

[0269] Example 1 A test emulsion containing mRNA encoding an mCherry derivative was produced at a final concentration of 1 µg / ml.

[0270] The test composition was produced according to the following protocol: (a) Under laminar flow, in a 100 ml beaker, with stirring on a magnetic stirrer, add 21.81 g of surfactant Labrasol® (Gattefosse SA, Saint-Priest, France), then add 17.88 g of polyglycerol 3-dioleate (Plurol® Oleique CC 497 (Gattefosse SA, Saint-Priest, France)), then add 7.97 g of mineral oil (Marcol® 52 (Exxon, USA)); (b) Preparation of aqueous phases containing mRNA: The aqueous phases were prepared by adding 30 µl of mRNA (SEQ ID NO:3, i.e., containing 5´-UTR, coding region encoding the mCherry derivative of SEQ ID NO:1, Kozak sequence (located around the start codon of the coding region), 3´-UTR and poly(A) tail, without 5´-cap)) to 870 µl of RNase-free water, resulting in an aqueous phase containing 5.18 µg / ml of mRNA. (c) In a 5 ml glass vial, add 0.8 ml of the aqueous phase (containing 5.18 µg / ml mRNA) obtained in step (b) above to the 3.347 ml mixture obtained in step (a) above. Continue magnetic stirring at room temperature for 5 minutes. The resulting emulsion is opalescent in color.

[0271] The emulsion was further tested as described in Example 4.

[0272] Example 2 An emulsion containing mRNA encoding a reporter protein (human secretory embryonic alkaline phosphatase (SEAP)) was formulated, the formulation was applied topically to piglets, and the expression of the reporter protein in the piglets was subsequently studied.

[0273] The composition to be tested was produced according to the following protocol: Under laminar flow conditions, in a 10 ml glass vial with magnetic stirring, 1.372 ml of surfactant Labrasol® (Gattefosse SA, Saint-Priest, France) (composed of small amounts of monoglycerides, diglycerides, and triglycerides, and PEG-8 (MW 400) monoesters and diesters, primarily caprylic acid (C8) and capric acid (C10)) was added, followed by 1.126 ml of polyglycerol 3-dioleate (Plurol® Oleique CC 497 (Gattefosse SA, Saint-Priest, France)), then 0.502 ml of mineral oil (Marcol® 52 (Exxon, USA)), and finally 0.717 ml of an aqueous phase (1 mM citrate buffer, pH 6.4) containing 1 mg / ml of mRNA encoding SEAP (SEQ ID NO:4), the mRNA having a structure equivalent to that of the mRNA in Example 1, but with a coding region encoding SEAP (rather than an mCherry derivative) (SEQ ID NO:4). NO:5), and has a 5'-cap with a cap1 structure m7GpppAm (can be produced using CleanCap® AG reagent (TriLink Biotechnologies, San Diego (CA), USA, product number N-7113) according to the manufacturer's instructions). Continue magnetic stirring at room temperature for 5 minutes. The resulting emulsion is opalescent in color.

[0274] A subsequent study was conducted to test whether topical application of the test composition to animals could effectively deliver the contained RNA through the skin into the animal body. Specifically, the animal study aimed to evaluate (i) the safety of this topical application to piglets and (ii) the production efficiency of the target protein resulting from this topical application.

[0275] In this study, specific pathogen-free (SPF) piglets, approximately 10 weeks old on study day 0 (D0), received 0.25 ml of the reference product (corresponding to the test composition but without RNA and manufactured in the same manner) (Group 1 (G1)), or 0.10 ml (Group 2 (G2)) or 0.25 ml (Group 3 (G3)) of the test composition (hereinafter also referred to as the “product”) on the inner surface of the right ear flap (i.e., the inner auricle). For this purpose, the reference product and the test composition were administered using a needle-free 1 ml syringe and evenly applied to the corresponding skin surface of the ear by circumferential massage. Each test group (G1, G2, and G3) consisted of five piglets.

[0276] Blood was collected on D1 and D2 (i.e., one day and two days after ear application), and ear tissue was collected on D2. The collected samples were quantitatively assessed by SEAP enzyme activity as described below. An overview of the experimental design is shown in Table 1.

[0277] Table 1 Experimental Design

[0278] SEAP enzyme activity was monitored in serum and after extraction from ear tissue. This activity is generated by cellular uptake and translation of locally administered mRNA in piglet cells.

[0279] In short, whole blood was collected in heparin sodium collection tubes and centrifuged at 1,100 g for 10 minutes at 20°C. Serum was collected and SEAP enzyme activity was immediately tested / quantified as described below.

[0280] Ear tissue samples from the application area were weighed and suspended in PBS (Ca2+ and Mg2+-free) and dissociated using a gentleMACS™ M-tube with a gentleMACS™ dissociator (Miltenyi Biotec) using the preloaded program m_heart_01. The homogenized samples were centrifuged at 1,100 g for 10 minutes at 20°C. The supernatant was collected and SEAP enzyme activity was immediately tested / quantified as described below. Enzyme activity measured for each ear sample was normalized to weight.

[0281] SEAP enzyme activity was quantified using a commercial kit (NovaBright™ Phospha-Light™ EXP Assay Kit for SEAP (Secreted Placental Alkaline Phosphatase) Reporter GeneDetection, Invitrogen) according to the manufacturer's instructions. This kit / assay employed an endpoint method using chloro-5-substituted adamantyl-1,2-dioxane phosphate (CSPD) as the substrate. The chemiluminescent substrate CSPD was dephosphorylated by secreted embryonic alkaline phosphatase (SEAP), generating an unstable dioxane anion, which decomposed and emitted light. Maximum activity was observed at a wavelength of 477 nm.

[0282] To measure light emission (from a 96-well microplate (96 Well White Plate, Thermo Scientific)), an EnVision reader (Perkin-Elmer) set to measure light emission was used.

[0283] The results of this study can be found in Figure 1("SEAP concentration per gram of ear" shows the SEAP activity in the supernatant (per gram of treated ear tissue) on day 2 of the study, measured by chemiluminescence) and Figure 2 (“SEAP concentration in serum”, showing SEAP activity on day 1 and day 2 of the study, measured by chemiluminescence).

[0284] Conclusion: Two days after administration, the mRNA encoding SEAP, formulated in the microemulsion described herein, was efficiently taken up by porcine cells and translated into active SEAP. The secreted enzyme was detected not only in local tissues but also in systemic circulation two days after administration. Considering the size and weight of 10-week-old piglets (approximately 20 kg), this indicates a remarkably high rate of production of the target protein.

[0285] Furthermore, no significant adverse events (i.e., fever, redness, swelling) were observed after the product was applied to piglets.

[0286] Example 3 An emulsion containing mRNA encoding hemagglutinin 3 (H3) of swine influenza A virus (SIAV) H3N2 was formulated, and the formulation was applied topically to piglets, followed by induction of immunization in the piglets.

[0287] The composition to be tested was produced according to the following protocol: Under laminar flow conditions, in a 5 ml glass vial with magnetic stirring, 686 µl of surfactant Labrasol® (Gattefosse SA, Saint-Priest, France) was added, followed by 563 µl of polyglycerol 3-dioleate (Plurol® Oleique CC 497 (Gattefosse SA, Saint-Priest, France)), then 251 µl of mineral oil (Marcol® 52 (Exxon, USA)), and finally 359 µl of aqueous phase (1 mM citrate buffer, pH 6.4) containing 1 mg / ml of mRNA encoding SIAV H3 (SEQ ID NO: 6). This mRNA has a structure comparable to that of the mRNA in Example 1, but contains the coding region for SIAV H3 (not the mCherry derivative) (SEQ ID NO: 7) and a 5'-cap of the cap1 structure m7GpppAm (which can be achieved using CleanCap® AG reagent (TriLink Biotechnologies, San Diego (CA)). (Made in the USA, product number N-7113, according to manufacturer's instructions). Continue magnetic stirring for 5 minutes at room temperature. The resulting emulsion is milky in color.

[0288] An animal study was conducted to test whether topical application of the test composition to animals could induce an immune response against an RNA-encoded antigen contained in the composition.

[0289] In this study, conventionally grown pigs, approximately 7 weeks old on study day 0 (D0), received either 0.25 ml of the reference product (corresponding to the test composition but without RNA and manufactured in the same manner) (Group 1 (G1)) or 0.2 ml of the test composition (Group 2 (G2)) on the inner surface of both ear flaps (100 µl per ear) on both days 0 and 21 (D21). For this purpose, the reference product and the test composition were administered using a needle-free 1 ml syringe and evenly applied to the corresponding skin surface of the ear by circumferential massage. Each test group (G1 and G2) consisted of five piglets.

[0290] Blood was collected 21 days after the second administration (day 42 of the study (D42)).

[0291] In addition, bronchoalveolar lavage fluid (BALF) was collected to monitor potential induced mucosal immunity. An overview of the study design is shown in Table 2.

[0292] Specific IgG in serum (blood centrifuged at 1,000g for 10 minutes) and specific IgA in BALF (blood centrifuged at 1,000g for 10 minutes) were quantified by ELISA, using the recombinant H3 extracellular domain as the target antigen, and colorimetric analysis was performed using anti-pig IgG or anti-pig IgA secondary antibody, as described below: In short, 120 µl of a solution of recombinant H3 extracellular domain (corresponding to amino acid residues 1-529 of SEQ ID NO:6) at a concentration of 167 µg / ml in carbonate buffer was coated overnight at 4°C and washed. After saturation in 5% PBS for 1 hour, serum was diluted 1 / 4 and then 1 / 5 in Tris ELISA buffer (TEB) + 1% milk and incubated at 37°C for 1.5 hours. After washing 5 times with Tris buffer + 0.1% Tween 20, 100 µl of HRP-labeled secondary antibody (Conjugate goat anti-Swine IgG-HRP - BIORAD AAI41P or Goat anti-swine IgA-HRP - Fortis Life Science A100-102P), diluted according to the manufacturer's instructions, was incubated at 37°C for 1.5 hours, and then developed with TMB (Symbiotics, France) substrate at 20°C for 30 minutes. The reaction was terminated by adding concentrated sulfuric acid, and reads were taken at 630 / 450 nm on a Bio-Teck Instruments Power Wave 340 plate reader. Titers are expressed as the maximum OD obtained at the minimum dilution before reaching the OD plateau. Each group was compared at the same dilution ratio.

[0293] Table 2 Experimental Design

[0294] like Figure 3 As shown, topical application of the test composition resulted in an increase in the level of specific anti-SIAVH3 IgG antibodies in the serum of D42 test animals.

[0295] like Figure 4 As shown, topical application of the test composition resulted in an increase in the level of specific anti-SIAVH3 IgA antibody in the BALF of D42 test animals.

[0296] As a result of the study, it was found that local application of mRNA-H3 induced specific humoral responses in piglets.

[0297] Example 4 The average droplet size of the aqueous phase dispersed in the oil phase in various microemulsions containing different properties and amounts of mRNA was determined.

[0298] The average droplet size and polydispersity index were determined at 20 °C using dynamic light scattering (DLS) on a Nano ZS Zetasizer (ZEN3600, Malvern Instruments Ltd., Enigma Business Park, Grovewood Road, UK). The average droplet size was calculated using Zetasizer software (Malvern Instruments Ltd.).

[0299] The emulsions described below (A)-(D) were diluted 5-fold by volume with Marcol® 52. Each sample was measured three times (Studies (A)-(C)) or four times (Study (D)), and the results are expressed as the average of the three or four measurements, respectively.

[0300] The dispersant's RI index is 1.460, and the material's RI is 1.33. The viscosity is 16.0000 cP. The sampling time is fixed at 60 seconds.

[0301] (A) Results showing no mRNA in the aqueous phase The following protocol was used to produce the test emulsion in the aqueous phase that was free of mRNA: (a) Under laminar flow, in a 100 ml beaker, with stirring on a magnetic stirrer, add 21.81 g of surfactant Labrasol® (Gattefosse SA, Saint-Priest, France), then add 17.88 g of polyglycerol 3-dioleate (Plurol® Oleique CC 497 (Gattefosse SA, Saint-Priest, France)), then add 7.97 g of mineral oil (Marcol® 52 (Exxon, USA)); (b) In a 10 ml glass vial, add 0.717 ml of Ca2+ and Mg2+-free phosphate-buffered saline solution to the 3 ml mixture obtained in step (a) above. Continue magnetic stirring at room temperature for 5 minutes. The resulting emulsion is opalescent in color.

[0302] In this emulsion, the average droplet size of the aqueous phase dispersed in the oil phase, as measured by DLS, was 57.33 nm, with a standard deviation (St dev) of 14.56 nm. Figure 5A The polydispersity index (PDI) value is 0.173.

[0303] (B) Results of emulsions containing mRNA encoding mCherry derivatives at final concentrations of 1 µg / ml or 30 µg / ml The 1 µg mRNA / ml target emulsion was produced as described in Example 1.

[0304] The 30 µg mRNA / ml target emulsion was produced according to the following protocol: (a) Under laminar flow, in a 100 ml beaker, with stirring on a magnetic stirrer, add 21.81 g of surfactant Labrasol® (Gattefosse SA, Saint-Priest, France), then add 17.88 g of polyglycerol 3-dioleate (Plurol® Oleique CC 497 (Gattefosse SA, Saint-Priest, France)), then add 7.97 g of mineral oil (Marcol® 52 (Exxon, USA)); (b) Preparation of aqueous phases containing mRNA: The aqueous phases were prepared by adding 17 µl of mRNA (SEQ ID NO:3, i.e., containing 5´-UTR, coding region encoding the mCherry derivative of SEQ ID NO:1, Kozak sequence (located around the start codon of the coding region), 3´-UTR and poly(A) tail, without 5´-cap)) to 202 µl of RNase-free water, resulting in an aqueous phase containing 155.4 µg / ml of mRNA. (c) In a 5 ml glass vial, add 0.189 ml of the aqueous phase (containing 155.4 µg / ml mRNA) obtained in step (b) above to the 0.791 ml mixture obtained in step (a) above. Continue magnetic stirring at room temperature for 5 minutes. The resulting emulsion is opalescent in color.

[0305] In an emulsion with a final mRNA concentration of 1 µg / ml, the average droplet size of the aqueous phase dispersed in the oil phase was 35.44 nm, and the St dev was 8.506 nm, as measured by DLS. Figure 5B The PDI value is 0.068.

[0306] In an emulsion with a final mRNA concentration of 30 µg / ml, the average droplet size of the aqueous phase dispersed in the oil phase was 32.45 nm, and the St dev was 10.23 nm, as measured by DLS. Figure 5C The PDI value is 0.130.

[0307] (C) Results of an emulsion containing a final concentration of 200 µg / ml of mRNA encoding SEAP. The composition to be tested was produced according to the following protocol: (a) Under laminar flow, in a 100 ml beaker, with stirring on a magnetic stirrer, add 21.81 g of surfactant Labrasol® (Gattefosse SA, Saint-Priest, France), then add 17.88 g of polyglycerol 3-dioleate (Plurol® Oleique CC 497 (Gattefosse SA, Saint-Priest, France)), then add 7.97 g of mineral oil (Marcol® 52 (Exxon, USA)); (b) In a 10 ml glass vial, add 0.717 ml of the aqueous phase described in Example 2 (1 mM citrate buffer, pH 6.4, containing 1 mg / ml mRNA encoding SEAP (SEQ ID NO:4)) to the 3 ml mixture obtained in step (a) above. Continue magnetic stirring at room temperature for 5 minutes. The resulting emulsion is opalescent in color.

[0308] In this emulsion, the average droplet size of the aqueous phase dispersed in the oil phase, as measured by DLS, was 35.74 nm, and the Stdev was 19.44 nm. Figure 5D The PDI value is 0.220.

[0309] (D) Results of an emulsion containing a final concentration of 200 µg / ml of mRNA encoding SIAV H3. The composition to be tested was produced according to the following protocol: (a) Under laminar flow, in a 100 ml beaker, with stirring on a magnetic stirrer, add 21.81 g of surfactant Labrasol® (Gattefosse SA, Saint-Priest, France), then add 17.88 g of polyglycerol 3-dioleate (Plurol® Oleique CC 497 (Gattefosse SA, Saint-Priest, France)), then add 7.97 g of mineral oil (Marcol® 52 (Exxon, USA)); (b) In a 10 ml glass vial, add 0.717 ml of the aqueous phase described in Example 3 (1 mM citrate buffer, pH 6.4, containing 1 mg / ml of mRNA encoding SIAV H3 (SEQ ID NO: 6)) to the 3 ml mixture obtained in step (a) above. Continue magnetic stirring for 5 minutes at room temperature. The resulting emulsion is opalescent in color.

[0310] In this emulsion, the average droplet size of the aqueous phase dispersed in the oil phase, as measured by DLS, was 44.28 nm, and the Stdev was 11.40 nm. Figure 5EThe PDI value is 0.106.

[0311] List of Attachments Figure 1 "SEAP concentration per gram of ear" indicates the SEAP activity in the supernatant (per gram of treated ear tissue) on day 2 of the study, measured by chemiluminescence.

[0312] Figure 2 "SEAP concentration in serum", showing SEAP activity on day 1 and day 2 of the study, measured by chemiluminescence.

[0313] Figure 3 Anti-SIAV H3 IgG ELISA response at D42 (21 days after the second administration).

[0314] Figure 4 Anti-SIAV H3 IgA ELISA response in bronchoalveolar lavage fluid (BALF) of D42 (21 days after the second administration).

[0315] Figure 5: (A)-(E) Dynamic light scattering (DLS) spectra, showing the droplet size distribution of the aqueous phase dispersed in the oil phase in the following emulsions: - Emulsions without mRNA in the aqueous phase ( Figure 5A ), - An emulsion containing mRNA encoding an mCherry derivative at a final concentration of 1 µg / ml. Figure 5B ), - An emulsion containing mRNA encoding an mCherry derivative at a final concentration of 30 µg / ml. Figure 5C ), - An emulsion containing mRNA encoding SEAP at a final concentration of 200 µg / ml. Figure 5D ), - An emulsion containing mRNA encoding SIAV H3 at a final concentration of 200 µg / ml. Figure 5E ).

[0316] Sequence listing / origin and geographic origin (if applicable): SEQ ID NO:1 corresponds to the amino acid sequence of the mCherry derivative. SEQ ID NO:2 (RNA) corresponds to the mRNA coding region encoding the mCherry derivative of SEQ ID NO:1. SEQ ID NO:3 (RNA) corresponds to the exemplary mRNA sequence, comprising a 5'-UTR, the mRNA coding region of SEQ ID NO:2, a Kozak sequence (located around the start codon of the coding region), a 3'-UTR, and a poly(A) tail. SEQ ID NO:4 corresponds to the amino acid sequence of secretory embryonic human alkaline phosphatase (SEAP). SEQ ID NO:5 (RNA) corresponds to the mRNA coding region of SEAP encoded by SEQ ID NO:4. SEQ ID NO:6 corresponds to the amino acid sequence of hemagglutinin 3 (H3) of the swine influenza A virus (SIAV) H3N2 isolate A / swine / Germany / AR452 / 2014 (isolated from German pigs in 2014). SEQ ID NO:7(RNA) corresponds to the mRNA coding region encoding H3 of SEQ ID NO:6.

[0317] In the RNA sequences listed in the attached st.26 standard sequence listing, uracil nucleotides are represented by the symbol "t".

[0318] The following terms are also disclosed herein. Therefore, this disclosure also includes aspects reflected in the following terms: 1. A composition comprising • An aqueous phase containing ribonucleic acid (RNA), and ·Oil phase.

[0319] 2. The composition of Clause 1, wherein the composition comprises • One or more surfactants.

[0320] 3. The composition of clause 1 or 2, wherein the composition comprises • Aqueous phase containing RNA ·Oil phase, • The first surfactant, and • Second surfactant.

[0321] 4. The composition of any one of clauses 1 to 3, wherein said composition comprises: • Aqueous phase containing RNA ·Oil phase, • The first surfactant, and • Second surfactant.

[0322] 5. A composition of any one of clauses 1 to 4, wherein the composition is a mixture.

[0323] 6. A composition of any one of clauses 1 to 5, wherein the aqueous phase comprises a solvent selected from water and mixtures of water with any other water-miscible solvent.

[0324] 7. The composition of any one of clauses 1 to 6, The aqueous phase contains water. Or the aqueous phase may be free of solid nanoparticles, or, in particular, free of lipid nanoparticles (LNPs) and / or lipid-like nanoparticles (LLNs).

[0325] 8. A composition of any one of clauses 1 to 7, wherein the oil phase is selected from mineral oil and vegetable oil.

[0326] 9. The composition of any one of clauses 1 to 8, wherein the oil phase is mineral oil.

[0327] 10. The composition of any one of clauses 3 to 9, wherein the first surfactant is a nonionic surfactant.

[0328] 11. The composition of any one of clauses 3 to 10, wherein the first surfactant is a water-soluble surfactant.

[0329] 12. The composition of any one of clauses 3 to 11, wherein the first surfactant has a hydrophilic-lipophilic balance (HLB) value greater than 10.

[0330] 13. The composition of any one of clauses 3 to 12, wherein the second surfactant is a co-surfactant.

[0331] 14. The composition of any one of clauses 3 to 13, wherein the second surfactant is a nonionic surfactant.

[0332] 15. The composition of any one of clauses 3 to 14, wherein the second surfactant is a water-insoluble surfactant.

[0333] 16. The composition of any one of clauses 3 to 15, wherein the HLB value of the second surfactant is less than 10.

[0334] 17. The composition of any one of clauses 3 to 16, wherein the composition comprises: • An aqueous phase containing RNA, wherein the aqueous phase contains water; • Oil phase, wherein the oil phase is mineral oil; • The first surfactant with an HLB value greater than 10; and • A second surfactant with an HLB value less than 10.

[0335] 18. A composition of any one of clauses 3 to 17, wherein said composition comprises: • An aqueous phase containing RNA, wherein the aqueous phase contains water; • Oil phase, wherein the oil phase is mineral oil; • The first surfactant with an HLB value greater than 10; and • A second surfactant with an HLB value less than 10.

[0336] 19. A composition of any one of clauses 3 to 18, wherein the first surfactant is selected from polyethylene glycol derivatives, polyethylene glycol, glycerol-based surfactants, and polyethylene glycol (PEG) esters.

[0337] 20. The composition of any one of clauses 3 to 19, wherein the first surfactant has an HLB value of 11 to 16.

[0338] 21. The composition of any one of clauses 3 to 20, wherein the first surfactant comprises PEG-8 caprylic / capric glyceride or PEG-6 caprylic / capric glyceride.

[0339] 22. The composition of any one of clauses 3 to 21, wherein the first surfactant has an HLB value of 11 to 13.

[0340] 23. The composition of any one of clauses 3 to 22, wherein the first surfactant comprises PEG-8 caprylic / capric glyceride.

[0341] 24. The composition of any one of clauses 3 to 23, wherein the first surfactant comprises a monoglyceride, a diglyceride, and / or a triglyceride.

[0342] 25. The composition of any one of clauses 3 to 24, wherein the first surfactant is composed primarily of PEG-8 caprylic / capric glyceride.

[0343] 26. The composition of any one of clauses 3 to 25, wherein the second surfactant is a polyglycerol fatty acid ester.

[0344] 27. The composition of any one of clauses 3 to 26, wherein the HLB value of the second surfactant is from about 3 to about 8.

[0345] 28. The composition of any one of clauses 3 to 27, wherein the second surfactant is selected from polyglycerol-3 dioleate and polyglycerol-6 dioleate.

[0346] 29. The composition of any one of clauses 3 to 28, wherein the HLB value of the second surfactant is from about 3 to about 6.

[0347] 30. The composition of any one of clauses 3 to 29, wherein the second surfactant is polyglycerol 3-dioleate.

[0348] 31. The composition of any one of clauses 1 to 30, wherein the aqueous phase containing RNA is selected from: - An aqueous buffer solution containing RNA. - Water containing RNA, and - A mixture of the following substances: • water or • Aqueous buffer solution It is miscible with any other water-soluble solvent, wherein the mixture contains RNA.

[0349] 32. The composition of any one of clauses 1 to 31, wherein the aqueous phase comprises an aqueous buffer solution.

[0350] 33. The composition of any one of clauses 1 to 32, wherein the aqueous phase containing RNA is an aqueous buffer solution containing RNA.

[0351] 34. The composition of any one of clauses 6 to 33, wherein the other water-miscible solvent is one or more solvents selected from ethanol, ethylene glycol and glycerol.

[0352] 35. A composition of any one of clauses 1 to 34, wherein the oil phase is a mineral oil selected from liquid paraffin and an oily liquid composed of petroleum-derived saturated hydrocarbons (such as hexadecane, methyl oleate, or ethyl oleate).

[0353] 36. The composition of any one of clauses 1 to 35, wherein the oil phase is liquid paraffin.

[0354] 37. The composition of any one of clauses 1 to 36, wherein the composition comprises: • An aqueous phase containing RNA, wherein the aqueous phase containing RNA is an aqueous buffer solution containing RNA; • Oil phase, wherein the oil phase is liquid paraffin; • A first surfactant, wherein the first surfactant comprises or is primarily composed of PEG-8 octanoic acid / capric acid glyceride; and • A second surfactant, wherein the second surfactant is polyglycerol-3 dioleate.

[0355] 38. A composition of any one of clauses 1 to 37, wherein said composition comprises: • An aqueous phase containing RNA, wherein the aqueous phase containing RNA is an aqueous buffer solution containing RNA; • Oil phase, wherein the oil phase is liquid paraffin; • A first surfactant, wherein the first surfactant comprises or is primarily composed of PEG-8 octanoic acid / capric acid glyceride; and • A second surfactant, wherein the second surfactant is polyglycerol-3 dioleate.

[0356] 39. The composition of any one of clauses 1 to 38, wherein the oil phase is a purified mixture of liquid saturated hydrocarbons.

[0357] 40. A composition of any one of clauses 1 to 39, wherein the oil phase is obtained from petroleum by catalytic hydrogenation purification.

[0358] 41. The composition of any one of clauses 1 to 40, wherein the oil phase comprises about 67% to about 68% alkane carbon.

[0359] 42. The composition of any one of clauses 1 to 41, wherein the oil phase comprises about 32% to about 33% cycloalkane carbon.

[0360] 43. The composition of any one of clauses 1 to 42, wherein the dynamic viscosity of the oil phase at 20°C is not more than about 100 mPa·s, preferably not more than about 50 mPa·s, more preferably not more than about 30 mPa·s, and most preferably not more than about 20 mPa·s.

[0361] 44. The composition of any one of clauses 1 to 43, wherein the dynamic viscosity of the oil phase at 20°C is about 10 mPa·s to about 100 mPa·s.

[0362] 45. The composition of any one of clauses 1 to 44, wherein the dynamic viscosity of the oil phase at 20°C is about 10 mPa·s to about 50 mPa·s.

[0363] 46. ​​The composition of any one of clauses 1 to 45, wherein the dynamic viscosity of the oil phase at 20°C is about 10 mPa·s to about 30 mPa·s.

[0364] 47. The composition of any one of clauses 1 to 46, wherein the dynamic viscosity of the oil phase at 20°C is about 10 mPa·s to about 20 mPa·s.

[0365] 48. A composition of any one of clauses 1 to 47, wherein the oil phase is a mixture of the components contained in Marcol 52.

[0366] 49. The composition of any one of clauses 1 to 48, wherein the oil phase is Marcol 52.

[0367] 50. The composition of any one of clauses 31 to 49, wherein the aqueous buffer solution is selected from citrate buffer, PBS buffer and Tris buffer.

[0368] 51. The composition of any one of clauses 1 to 50, wherein the aqueous phase containing RNA is a citrate buffer containing RNA.

[0369] 52. The composition of clause 50 or 51, wherein the aqueous phase containing RNA is a citrate buffer containing RNA, and wherein the pH of the citrate buffer is 6.4 ± 0.2.

[0370] 53. The composition of any one of clauses 3 to 52, wherein the first surfactant comprises or is composed of: • Monoesters and diesters of polyethylene glycol with an average relative molecular weight between 200 and 400, and • Glyceryl monoesters, diesters and triesters.

[0371] 54. The composition of any one of clauses 3 to 53, wherein the first surfactant comprises or is composed of: • PEG-8 (MW 400) monoesters and diesters of caprylic acid (C8) and decanoic acid (C10), and • Monoglycerides, diglycerides, and triglycerides.

[0372] 55. The composition of any one of clauses 3 to 54, wherein the first surfactant comprises small amounts of monoglycerides, diglycerides, and triglycerides, and is primarily composed of caprylic acid (C8) and capric acid (C9). 10 It consists of PEG-8 (MW 400) monoester and diester.

[0373] 56. A composition of any one of clauses 3 to 55, wherein the first surfactant is a mixture of the components contained in Labrasol.

[0374] 57. A composition of any one of clauses 3 to 56, wherein the first surfactant is Labrasol.

[0375] 58. The composition of any one of clauses 3 to 57, wherein the second surfactant has an HLB value of about 3.

[0376] 59. The composition of any one of clauses 3 to 58, wherein the second surfactant has an HLB value of about 6.

[0377] 60. A composition of any one of clauses 3 to 59, wherein the second surfactant is a mixture of the components contained in Plurol Oleique CC 497.

[0378] 61. The composition of any one of clauses 3 to 60, wherein the second surfactant is Plurol Oleique CC 497.

[0379] 62. A composition of any one of clauses 1 to 61, wherein said composition is an emulsion.

[0380] 63. A composition of any one of clauses 1 to 62, wherein the composition is an emulsion in which an aqueous phase is dispersed in an oil phase.

[0381] 64. A composition of any one of clauses 1 to 63, wherein the composition is a water-in-oil emulsion.

[0382] 65. A composition of any one of clauses 62 to 64, wherein the emulsion is an emulsion selected from microemulsions and nanoemulsions.

[0383] 66. A composition of any one of clauses 1 to 65, wherein the composition is an emulsion in which the aqueous phase is dispersed in the oil phase in the form of droplets with a diameter in the range of about 1 to about 500 nm.

[0384] 67. A composition of any one of clauses 1 to 66, wherein the composition is an emulsion in which the aqueous phase is dispersed in the oil phase in the form of droplets with a diameter in the range of about 1 to about 350 nm.

[0385] 68. A composition of any one of clauses 1 to 66, wherein the composition is an emulsion in which the aqueous phase is dispersed in the oil phase in the form of droplets with diameters in the range of about 5 to about 500 nm.

[0386] 69. A composition of any one of clauses 1 to 68, wherein the composition is an emulsion in which the aqueous phase is dispersed in the oil phase in the form of droplets with diameters ranging from about 5 to about 350 nm.

[0387] 70. A composition of any one of clauses 1 to 69, wherein the composition is an emulsion in which the aqueous phase is dispersed in the oil phase in the form of droplets with a diameter in the range of about 5 to about 200 nm.

[0388] 71. A composition of any one of clauses 1 to 70, wherein the composition is an emulsion in which the aqueous phase is dispersed in the oil phase in the form of droplets with an average diameter in the range of 5 to 95 nm.

[0389] 72. A composition of any one of clauses 1 to 71, wherein the composition is an emulsion in which the aqueous phase is dispersed in the oil phase in the form of droplets with an average diameter in the range of 10 to 90 nm.

[0390] 73. A composition of any one of clauses 1 to 72, wherein the composition is an emulsion in which the aqueous phase is dispersed in the oil phase in the form of droplets with an average diameter in the range of 15 to 85 nm.

[0391] 74. A composition of any one of clauses 1 to 73, wherein the composition is an emulsion in which the aqueous phase is dispersed in the oil phase in the form of droplets with an average diameter in the range of 20 to 80 nm.

[0392] 75. A composition of any one of clauses 1 to 74, wherein the composition is an emulsion in which the aqueous phase is dispersed in the oil phase in the form of droplets with an average diameter in the range of 25 to 75 nm.

[0393] 76. The composition of any one of clauses 62 to 75, wherein the emulsion is a microemulsion.

[0394] 77. The composition of any one of clauses 62 to 76, wherein the emulsion is thermodynamically stable.

[0395] 78. A composition of any one of clauses 62 to 77, wherein the emulsion is isotropic.

[0396] 79. The composition of any one of clauses 62 to 78, wherein the emulsion is transparent.

[0397] 80. The composition of any one of clauses 62 to 79, wherein the emulsion is opalescent.

[0398] 81. A composition of any one of clauses 1 to 80, wherein the emulsion is a water-in-oil microemulsion.

[0399] 82. A composition of any one of clauses 1 to 81, wherein the final concentration of the aqueous phase containing RNA in the composition is about 5% to 30% v / v.

[0400] 83. A composition of any one of clauses 1 to 82, wherein the final concentration of the aqueous phase in the composition is about 14% to 24% v / v.

[0401] 84. A composition of any one of clauses 1 to 83, wherein the final concentration of the aqueous phase in the composition is about 14.3% to 24.3% v / v.

[0402] 85. A composition of any one of clauses 1 to 84, wherein the final concentration of the oil phase in the composition is about 5% to 30% v / v.

[0403] 86. A composition of any one of clauses 1 to 85, wherein the final concentration of the oil phase in the composition is about 8.5% to 18.5% v / v.

[0404] 87. A composition of any one of clauses 3 to 86, wherein the final concentration of the first surfactant in the composition is about 30.7% to 70.7% v / v.

[0405] 88. A composition of any one of clauses 3 to 87, wherein the final concentration of the first surfactant in the composition is about 31.9% to 41.9% v / v.

[0406] 89. A composition of any one of clauses 3 to 88, wherein the final concentration of the second surfactant in the composition is about 17.5% to 57.5% v / v.

[0407] 90. A composition of any one of clauses 3 to 89, wherein the final concentration of the second surfactant in the composition is about 25.2% to 35.2% v / v.

[0408] 91. A composition of any one of clauses 3 to 90, wherein in said composition: The final concentration of the aqueous phase containing RNA was approximately 19% v / v. The final concentration of the oil phase is approximately 14% v / v. The final concentration of the first surfactant is approximately 37% v / v, and The final concentration of the second surfactant is approximately 30% v / v.

[0409] 92. A composition of any one of clauses 1 to 91, wherein the final concentration of said RNA in the composition is about 0.1 to 500 µg / ml.

[0410] 93. A composition of any one of clauses 1 to 92, wherein the final concentration of said RNA in the composition is at least about 1 µg / ml.

[0411] 94. A composition of any one of clauses 1 to 93, wherein the final concentration of said RNA in the composition is at least about 30 µg / ml.

[0412] 95. A composition of any one of clauses 1 to 94, wherein the final concentration of the RNA in the composition is about 200 µg / ml.

[0413] 96. A composition of any one of clauses 1 to 95, wherein the RNA is selected from spontaneous RNA (mRNA), small interfering RNA (siRNA), and microRNA (miRNA).

[0414] 97. A composition of any one of clauses 1 to 96, wherein the RNA is mRNA.

[0415] 98. A composition of any one of clauses 1 to 97, wherein the RNA is mRNA encoding a peptide or polypeptide selected from antigens, functional proteins and reporter proteins.

[0416] 99. A composition of any one of clauses 1 to 98, wherein the RNA is mRNA encoding a reporter protein.

[0417] 100. A composition of any one of clauses 1 to 99, wherein the RNA is mRNA encoding a fluorescent protein.

[0418] 101. The composition of Clause 100, wherein the fluorescent protein is selected from mCherry, GFP (green fluorescent protein), and derivatives of mCherry or GFP.

[0419] 102. The composition of clause 100 or 101, wherein the fluorescent protein is an mCherry derivative, wherein the mCherry derivative comprises or consists of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, still more preferably at least 95% sequence identity with the sequence of SEQ ID NO:1.

[0420] 103. The composition of any one of clauses 100 to 102, wherein the mRNA encoding the mCherry derivative comprises a nucleotide sequence having at least 70%, preferably at least 80%, more preferably at least 90%, still more preferably at least 95%, or particularly 100% sequence identity with the sequence of SEQ ID NO:2.

[0421] 104. The composition of any one of clauses 100 to 103, wherein the mRNA encoding the mCherry derivative comprises or consists of a nucleotide sequence having at least 70%, preferably at least 80%, more preferably at least 90%, still more preferably at least 95%, or particularly 100% sequence identity with the sequence of SEQ ID NO:3.

[0422] 105. A composition of any one of clauses 1 to 98, wherein the RNA is mRNA encoding an antigen, or wherein the RNA is mRNA encoding a functional protein.

[0423] 106. A composition of any one of clauses 1 to 98 and 105, wherein said RNA is mRNA encoding a functional protein.

[0424] 107. A composition of any one of clauses 1 to 98, 105 and 106, wherein said RNA is mRNA encoding an enzyme.

[0425] 108. A composition of any one of clauses 1 to 98 and 105 to 107, wherein said RNA is mRNA encoding secretory embryonic human alkaline phosphatase (SEAP).

[0426] 109. The composition of Clause 108, wherein the SEAP comprises or consists of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, and still more preferably at least 95% sequence identity with the sequence of SEQ ID NO:4.

[0427] 110. The composition of clause 108 or 109, wherein the mRNA encoding SEAP comprises a nucleotide sequence having at least 70%, preferably at least 80%, more preferably at least 90%, still more preferably at least 95%, or particularly 100% sequence identity with the sequence of SEQ ID NO:5.

[0428] 111. A composition of any one of clauses 1 to 98 and 105, wherein said RNA is mRNA encoding an antigen.

[0429] 112. A composition of any one of clauses 1 to 98, 105 and 111, wherein said RNA is mRNA encoding a pathogen antigen.

[0430] 113. The composition of any one of clauses 1 to 98, 105, 111 and 112, wherein the RNA is mRNA encoding a viral antigen, a bacterial antigen and an antigen derived from a parasite.

[0431] 114. The composition of any one of clauses 1 to 98, 105 and 111 to 113, wherein said RNA is mRNA encoding a viral antigen.

[0432] 115. The composition of clause 113 or 114, wherein the viral antigen is a viral glycoprotein, or wherein the viral antigen is a viral capsid protein.

[0433] 116. A composition of any one of clauses 113 to 115, wherein the viral antigen is a viral glycoprotein.

[0434] 117. The composition of clauses 115 or 116, wherein the virus is an RNA virus.

[0435] 118. A composition of any one of clauses 113 to 117, wherein the viral antigen is an RNA viral antigen.

[0436] 119. A composition of any one of clauses 113 to 118, wherein the viral antigen is an RNA viral glycoprotein.

[0437] 120. A composition of any one of clauses 115 to 119, wherein the virus is an influenza virus.

[0438] 121. A composition of any one of clauses 113 to 120, wherein the viral antigen is an influenza virus antigen.

[0439] 122. The composition of any one of clauses 113 to 121, wherein the viral antigen is an influenza virus glycoprotein.

[0440] 123. A composition of any one of clauses 115 to 122, wherein the virus is a swine influenza virus.

[0441] 124. The composition of any one of clauses 113 to 123, wherein the viral antigen is a swine influenza virus antigen.

[0442] 125. A composition of any one of clauses 113 to 124, wherein the viral antigen is a swine influenza virus glycoprotein.

[0443] 126. The composition of any one of clauses 115 to 125, wherein the virus is a swine influenza A virus (SIAV).

[0444] 127. The composition of any one of clauses 113 to 126, wherein the viral antigen is a swine influenza A virus (SIAV) antigen.

[0445] 128. A composition of any one of clauses 113 to 127, wherein the viral antigen is a swine influenza A virus (SIAV) glycoprotein.

[0446] 129. A composition of any one of clauses 115 to 128, wherein the virus is a subtype of swine influenza A virus (SIAV) H3N2.

[0447] 130. A composition of any one of clauses 113 to 129, wherein the viral antigen is a SIAV H3N2 subtype antigen.

[0448] 131. A composition of any one of clauses 113 to 130, wherein the viral antigen is a SIAV H3N2 subtype glycoprotein.

[0449] 132. The composition of any one of clauses 1 to 131, wherein the RNA is mRNA encoding hemagglutinin or an immunogenic fragment thereof.

[0450] 133. A composition of any one of clauses 1 to 132, wherein the RNA is mRNA encoding hemagglutinin.

[0451] 134. The composition of clause 132 or 133, wherein the hemagglutinin is hemagglutinin 3 (H3).

[0452] 135. The composition of any one of clauses 132 to 134, wherein the hemagglutinin comprises or consists of an amino acid sequence having at least 95%, preferably at least 98%, more preferably at least 99%, or particularly 100% sequence identity with the sequence of SEQ ID NO:6.

[0453] 136. A composition of any one of clauses 1 to 98, 105 and 111 to 135, wherein the RNA is an mRNA comprising a nucleotide sequence having at least 70%, preferably at least 80%, more preferably at least 90%, still more preferably at least 95% or particularly 100% sequence identity with the sequence of SEQ ID NO:7.

[0454] 137. A composition of any one of clauses 96 to 136, wherein the mRNA is a synthetic mRNA.

[0455] 138. The composition of any one of clauses 96 to 137, wherein the mRNA is a nucleoside-modified mRNA.

[0456] 139. A composition of any one of clauses 96 to 138, wherein the mRNA is at least 300 nucleotides in length.

[0457] 140. A composition of any one of clauses 96 to 139, wherein the mRNA is at least 500 nucleotides in length.

[0458] 141. The composition of any one of clauses 96 to 140, wherein the mRNA is at least 1,000 nucleotides in length.

[0459] 142. The composition of any one of clauses 96 to 141, wherein the mRNA is at least 1,500 nucleotides in length.

[0460] 143. The composition of any one of clauses 96 to 142, wherein the mRNA is 300 to 5000 nucleotides in length.

[0461] 144. The composition of any one of clauses 96 to 143, wherein the mRNA is 500 to 4000 nucleotides in length.

[0462] 145. The composition of any one of clauses 96 to 144, wherein the mRNA is 1,000 to 3,000 nucleotides in length.

[0463] 146. A composition of any one of clauses 96 to 145, wherein the mRNA comprises a 5'-UTR, a coding region encoding a polypeptide, and a 3'-UTR.

[0464] 147. The composition of Clause 146, wherein the coding region flanks are the 5´-UTR and the 3´-UTR.

[0465] 148. A composition of any one of clauses 96 to 147, wherein the mRNA comprises a poly(A) tail, wherein the poly(A) tail is preferably located after the 3'-UTR along the 3' direction.

[0466] 149. The composition of any one of clauses 146 to 148, wherein the 3´-UTR flank is a poly(A) tail.

[0467] 150. The composition of clauses 148 or 149, wherein the poly(A) tail comprises or consists of about 120 A nucleotides.

[0468] 151. A composition of any one of clauses 96 to 150, wherein the mRNA comprises a Kozak sequence.

[0469] 152. A composition of any one of clauses 96 to 151, wherein the mRNA comprises a 5'-cap structure.

[0470] 153. A composition of any one of clauses 96 to 152, wherein the mRNA comprises a cap1 structure.

[0471] 154. A composition of any one of clauses 96 to 153, wherein the mRNA comprises a 5'-cap having a cap1 structure m7GpppNm.

[0472] 155. A composition of any one of clauses 96 to 154, wherein the mRNA comprises a 5'-cap having a cap1 structure m7GpppAm.

[0473] 156. A composition of any one of clauses 1 to 155, used as a medicine or for use in a diagnostic method performed on the body.

[0474] 157. A composition of any one of clauses 1 to 98, 105 to 107 and 111 to 155, used as a medicine.

[0475] 158. A composition of any one of clauses 1 to 98, 105 and 111 to 155, for use as a vaccine.

[0476] 159. A composition of any one of clauses 1 to 110, used for a diagnostic method performed on the body.

[0477] 160. A composition of any one of clauses 1 to 155, used in a method of delivering said RNA to a object, wherein said method includes applying said composition locally to said object.

[0478] 161. A composition of any one of clauses 1 to 155 for use in a method of delivering said RNA to a subject through the skin, wherein said method includes applying said composition to the skin of said subject.

[0479] 162. The composition of any one of clauses 111 to 155, used in a method of inducing an immune response in a subject.

[0480] 163. The composition of any one of clauses 111 to 155, used in a method of inducing an immune response against a pathogen in a subject.

[0481] 164. The composition used in the method according to Clause 163, wherein the pathogen is selected from viruses, bacteria and parasites.

[0482] 165. The composition of any one of clauses 114 to 155, used in a method of inducing an immune response against a virus in a subject.

[0483] 166. The composition of any one of clauses 120 to 155, used in a method of inducing an immune response against an influenza virus in a subject.

[0484] 167. The composition of any one of clauses 123 to 155, used in a method of inducing an immune response against swine influenza virus in a subject.

[0485] 168. The composition of any one of clauses 126 to 155, used in a method of inducing an immune response against swine influenza A virus (SIAV) in a subject.

[0486] 169. The composition of any one of clauses 129 to 155, used in a method of inducing an immune response against an influenza virus containing H3 in a subject.

[0487] 170. The composition of any one of clauses 129 to 155, used in a method of inducing an immune response against H3N2 influenza virus in a subject.

[0488] 171. The composition of any one of clauses 129 to 155, used in a method for inducing an immune response against the SIAV H3N2 subtype in a subject.

[0489] 172. The composition used in the method of any one of clauses 162 to 171, wherein the immune response is a mucosal immune response.

[0490] 173. A method of delivering ribonucleic acid (RNA) to a subject, comprising the step of topically applying a composition of any one of clauses 1 to 155 to said subject.

[0491] 174. A method of delivering ribonucleic acid (RNA) to a subject through the skin, comprising the step of applying a composition of any one of clauses 1 to 155 to the skin of the subject.

[0492] 175. The method of clause 173 or 174, wherein the aqueous phase of the composition of any one of clauses 1 to 155 contains RNA to be delivered to the subject.

[0493] 176. The method of any one of Clauses 173 to 175, wherein the method is specified in any one of Clauses 160 to 172.

[0494] 177. The method of any one of clauses 173 to 176, wherein the composition is specified as in any one of clauses 160 to 171.

[0495] 178. Use of any of the compositions of clauses 1 to 155 in the preparation of a pharmaceutical or diagnostic agent.

[0496] 179. Use of the composition of any one of clauses 1 to 98, 105 to 107 and 111 to 155 in the preparation of a medicament.

[0497] 180. Use of any of the compositions of clauses 1 to 98, 105 and 111 to 155 in the preparation of vaccines.

[0498] 181. Use of any composition of any one of clauses 1 to 110 in the preparation of diagnostic agents.

[0499] 182. Use of any of the provisions 178 to 180, wherein the drug or vaccine is used in the manner specified in any of the provisions 160 to 177.

[0500] 183. Use of any one of Clauses 178 to 182, wherein the composition is specified as in any one of Clauses 160 to 171.

[0501] 184. The composition used in the method of any one of Clauses 156 to 172, the method of any one of Clauses 173 to 177, or the use of the composition in any one of Clauses 178 to 183, wherein the method includes applying the composition topically to the object.

[0502] 185. The composition used in any of the methods of Clauses 156 to 172 and 184, the method of any of Clauses 173 to 177 and 184, or the use of any of Clauses 178 to 184, wherein said composition is applied topically to the ear surface of said object.

[0503] 186. The composition used in any of the methods of clauses 156 to 172 and 184 to 185, the method of any of clauses 173 to 177 and 184 to 185, or the use of any of clauses 178 to 185, wherein said composition is applied topically to the inner ear of said object.

[0504] 187. The composition used in any of the methods of clauses 156 to 172 and 184 to 186, the method of any of clauses 173 to 177 and 184 to 186, or the use of the composition in any of clauses 178 to 186, wherein the method comprises applying one or more doses of the composition to the subject.

[0505] 188. The composition used in any of the methods of clauses 156 to 172 and 184 to 187, the method of any of clauses 173 to 177 and 184 to 187, or the use of the composition in any of clauses 178 to 187, wherein the method comprises applying two doses of the composition to the subject.

[0506] 189. The composition used in the method of clause 187 or 188, or the use of the composition according to clause 187 or 188, wherein the volume of one dose of the composition is about 0.01 ml to 1 ml.

[0507] 190. The composition used in the method of any one of Clauses 187 to 189, the method of any one of Clauses 187 to 189, or the use of any one of Clauses 187 to 189, wherein the volume of one dose of said composition is about 0.05 ml to 0.5 ml.

[0508] 191. The composition used in the method of any one of Clauses 187 to 190, the method of any one of Clauses 187 to 190, or the use of any one of Clauses 187 to 190, wherein the volume of one dose of said composition is about 0.1 ml to 0.25 ml.

[0509] 192. The composition used in any of the methods of clauses 160 to 172 and 184 to 191, the method of any of clauses 173 to 177 and 184 to 191, or the use of any of clauses 178 to 191, wherein said object is a mammal.

[0510] 193. The composition used in any of the methods of clauses 160 to 172 and 184 to 192, the methods of any of clauses 173 to 177 and 184 to 192, or the use of any of clauses 178 to 192, wherein said object is an animal.

[0511] 194. The composition used in any of the methods of clauses 160 to 172 and 184 to 193, the methods of any of clauses 173 to 177 and 184 to 193, or the use of any of clauses 178 to 193, wherein the object is a pig.

[0512] 195. A method for producing a composition of any one of clauses 1 to 155, comprising the following steps: (a) The mixed oil phase, the first surfactant, and the second surfactant, and (b) Add an aqueous phase containing RNA to the mixture obtained in step (a).

[0513] 196. The method of Clause 195, wherein the oil phase is the oil phase specified in any one of Clauses 8, 9, 17, 18, 35 to 49, 85, 86 and 91.

[0514] 197. The method of Clause 195 or 196, wherein the first surfactant is the first surfactant specified in any one of Clauses 10 to 12, 17 to 25, 37, 38, 53 to 57, 87, 88 and 91.

[0515] 198. The method of any one of clauses 195 to 197, wherein the second surfactant is the second surfactant specified in any one of clauses 13 to 18, 26 to 30, 37, 38, 58 to 61, 89 to 91.

[0516] 199. The method of any one of Clauses 195 to 198, wherein the aqueous phase is the aqueous phase specified in any one of Clauses 6, 7, 17, 18, 31 to 33, 37, 38, 51, 52, 82 to 84 and 91.

[0517] 200. The method of any one of clauses 195 to 199, wherein the RNA is the RNA specified in any one of clauses 92 to 155.

[0518] 201. A hemagglutinin 3 (H3) comprising an amino acid sequence having at least 90% sequence identity with the sequence of SEQ ID NO:6.

[0519] 202. A hemagglutinin 3 (H3) comprising an amino acid sequence having at least 90% sequence identity with the sequence of SEQ ID NO:6.

[0520] 203. H3 of clause 201 or 202, wherein the H3 has a tyrosine residue at position 295 of the amino acid.

[0521] 204. H3 of any one of clauses 201 to 203, wherein said H3 has an alanine residue at position 176 of an amino acid.

[0522] 205. H3 of any one of clauses 201 to 204, wherein said H3 has an asparagine residue at position 188 of an amino acid.

[0523] 206. H3 of any one of clauses 201 to 205, wherein said H3 has: • At the alanine residue at amino acid position 176, • The asparagine residue at amino acid position 188, and • The tyrosine residue at position 295 of the amino acid.

[0524] 207. H3 of any one of clauses 201 to 206, wherein said H3 has an asparagine residue at position 18 of an amino acid.

[0525] 208. H3 of any one of clauses 201 to 207, wherein said H3 has a glutamine residue at position 180 of an amino acid.

[0526] 209. H3 of any one of clauses 201 to 208, wherein said H3 has an arginine residue at position 21 of an amino acid.

[0527] 210. H3 of any one of clauses 201 to 209, wherein said H3 has an aspartic acid residue at position 291 of an amino acid.

[0528] 211. H3 of any one of clauses 201 to 210, wherein said H3 has a lysine residue at position 69 of an amino acid.

[0529] 212. H3 of any one of clauses 201 to 211, wherein the H3 has a valine residue at position 214 of an amino acid.

[0530] 213. H3 of any one of clauses 201 to 212, wherein said H3 has an aspartic acid residue at position 140 of an amino acid.

[0531] 214. H3 of any one of clauses 201 to 213, wherein the H3 has a threonine residue at position 208 of an amino acid.

[0532] 215. H3 of any one of clauses 201 to 214, wherein said H3 has a valine residue at the 10th position of an amino acid.

[0533] 216. H3 of any one of clauses 201 to 215, wherein said H3 has a phenylalanine residue at position 19 of an amino acid.

[0534] 217. H3 of any one of clauses 201 to 216, wherein said H3 has a histidine residue at position 323 of an amino acid.

[0535] 218. H3 of any one of clauses 201 to 217, wherein said H3 has an isoleucine residue at position 546 of an amino acid.

[0536] 219. H3 of any one of clauses 201 to 218, wherein said H3 has an isoleucine residue at position 552 of an amino acid.

[0537] 220. H3 of any one of clauses 201 to 219, wherein the amino acid position number refers to the amino acid sequence of wild-type H3.

[0538] 221. H3 of any one of clauses 201 to 220, wherein the amino acid position number refers to the amino acid sequence of SEQ ID NO:6.

[0539] 222. The H3 of any one of Articles 201 to 221, wherein the H3 is the H3 of swine influenza A virus (SIAV).

[0540] 223. The H3 of any one of Articles 201 to 222, wherein the H3 is the H3 of the swine influenza A virus (SIAV) H3N2 subtype.

[0541] 224. H3 of any one of clauses 201 to 223, wherein the amino acid sequence has at least 95% sequence identity with the sequence of SEQ ID NO:6.

[0542] 225. H3 of any one of clauses 201 to 224, wherein the amino acid sequence has at least 99% sequence identity with the sequence of SEQ ID NO:6.

[0543] 226. H3 of any one of clauses 201 to 225, wherein the amino acid sequence has at least 99.2% sequence identity with the sequence of SEQ ID NO:6.

[0544] 227. H3 of any one of clauses 201 to 226, wherein the amino acid sequence has at least 99.3%, preferably at least 99.5%, more preferably at least 99.7%, or particularly 100% sequence identity with the sequence of SEQ ID NO:6.

[0545] 228. H3 of any one of clauses 201 to 227, wherein the amino acid sequence has at least 99.4% sequence identity with the sequence of SEQ ID NO:6.

[0546] 229. H3 of any one of clauses 201 to 228, wherein the amino acid sequence has at least 99.6% sequence identity with the sequence of SEQ ID NO:6.

[0547] 230. H3 of any one of clauses 201 to 229, wherein the amino acid sequence has at least 99.8% sequence identity with the sequence of SEQ ID NO:6.

[0548] 231. An immunogenic composition comprising H3 of any one of clauses 201 to 230.

[0549] 232. A polynucleotide comprising a sequence encoding the H3 of any one of clauses 201 to 230.

[0550] 233. An mRNA comprising a sequence encoding H3 of any one of clauses 201 to 230.

[0551] 234. A vector comprising the polynucleotide of clause 232.

[0552] 235. A plasmid comprising the polynucleotides of clause 232.

[0553] 236. The uses of the following substances: • H3 of any one of clauses 201 to 230 • Immunogenic compositions of Clause 231, • The polynucleotides in Clause 232 • Article 233 mRNA, • The carrier of Clause 234, and / or • Plasmid of Clause 235, Used for the preparation of drugs, preferably vaccines.

[0554] 237. A method for inducing an immune response against a virus in a subject, comprising administering to the subject: • H3 of any one of clauses 201 to 230 • Immunogenic compositions of Clause 231, • The polynucleotides in Clause 232 • Article 233 mRNA, • The carrier of Clause 234, and / or • Plasmid of Clause 235.

[0555] 238. The method of Clause 237, wherein the immune response against the virus is an immune response against an influenza virus.

[0556] 239. The method of Articles 237 or 238, wherein the immune response against the virus is an immune response against swine influenza virus.

[0557] 240. The method of any one of clauses 237 to 239, wherein the immune response against the virus is an immune response against swine influenza A virus (SIAV).

[0558] 241. The method of any one of clauses 237 to 240, wherein the immune response against the virus is an immune response against an influenza virus containing H3.

[0559] 242. The method of any one of clauses 237 to 241, wherein the immune response against the virus is an immune response against the H3N2 influenza virus.

[0560] 243. The method of any one of clauses 237 to 242, wherein the immune response against the virus is an immune response against the SIAVH3N2 subtype.

[0561] 244. An isolated influenza virus whose genome encodes H3 of any one of clauses 201 to 230.

[0562] 245. An isolated influenza virus, particularly the isolated influenza virus of clause 244, wherein said isolated influenza virus comprises H3 of any one of clauses 201 to 230.

[0563] 246. An isolated influenza virus as described in Articles 244 or 245, wherein the influenza virus is a swine influenza A virus (SIAV).

[0564] 247. An isolated influenza virus of any one of Articles 244 to 246, wherein the influenza virus is the SIAV H3N2 subtype.

Claims

1. A composition comprising - An aqueous phase containing ribonucleic acid (RNA), and - oil phase, The composition is an emulsion in which an aqueous phase is dispersed in an oil phase.

2. The composition of claim 1, wherein the composition comprises or consists of: - An aqueous phase containing RNA - oil phase, - The first surfactant, and - Second surfactant, And preferably, the oil phase is mineral oil.

3. The composition of claim 1 or 2, wherein the first surfactant It is a water-soluble surfactant, and / or Having a hydrophilic-lipophilic balance (HLB) value greater than 10, and / or It is a nonionic surfactant.

4. The composition of any one of claims 1 to 3, wherein the second surfactant It is a water-insoluble surfactant, and / or Having an HLB value less than 10, and / or It is a co-surfactant, and / or It is a nonionic surfactant.

5. The composition of any one of claims 1 to 4, wherein the composition comprises or consists of: - An aqueous phase containing RNA, wherein the aqueous phase contains water; - Oil phase, wherein the oil phase is mineral oil; - The first surfactant with an HLB value greater than 10; and - A second surfactant with an HLB value less than 10.

6. The composition of any one of claims 2 to 5, wherein the first surfactant: Selected from polyethylene glycol derivatives, polyethylene glycol, glycerol-based surfactants, polyethylene glycol (PEG) esters, and / or With an HLB value of 11 to 16, and / or Contains PEG-8 caprylic / capric triglyceride or PEG-6 caprylic / capric triglyceride, and / or It contains monoglycerides, diglycerides, and / or triglycerides.

7. The composition of any one of claims 2 to 6, wherein the second surfactant: It is a polyglycerol fatty acid ester, and / or Having an HLB value of about 3 to about 8, particularly about 3 to about 6, and / or It is selected from polyglycerol-3 dioleate and polyglycerol-6 dioleate.

8. The composition of any one of claims 1 to 7, wherein the aqueous phase containing RNA is selected from: - An aqueous buffer solution containing RNA. - Water containing RNA, and - A mixture of the following substances: • water or • Aqueous buffer solution It is miscible with any other water-soluble solvent, wherein the mixture contains RNA.

9. The composition of any one of claims 1 to 8, wherein the oil phase is a mineral oil selected from liquid paraffin, and an oily liquid composed of petroleum-derived saturated hydrocarbons such as hexadecane or methyl oleate or ethyl oleate.

10. The composition of any one of claims 1 to 9, wherein the composition comprises or consists of: - An aqueous phase containing RNA, wherein the aqueous phase containing RNA is an aqueous buffer solution containing RNA; - An oil phase, wherein the oil phase is liquid paraffin; - A first surfactant, wherein the first surfactant comprises or is primarily composed of PEG-8 octanoic acid / capric acid glyceride; and - A second surfactant, wherein the second surfactant is polyglycerol-3 dioleate.

11. The composition of any one of claims 1 to 10, wherein the oil phase: It is a purified mixture of liquid saturated hydrocarbons, and / or Obtained from petroleum through catalytic hydrogenation purification, and / or Contains approximately 67% to approximately 68% alkane carbons, and / or Contains approximately 32% to approximately 33% cycloalkane carbons, and / or The dynamic viscosity at 20°C does not exceed approximately 100 mPa·s, and / or It is a mixture of the components contained in Marcol 52.

12. The composition of any one of claims 1 to 11, wherein the aqueous phase containing RNA is an RNA-containing citrate buffer, and wherein preferably the pH of the citrate buffer is 6.4 ± 0.

2.

13. The composition of any one of claims 2 to 12, Wherein the first surfactant It includes or consists of the following: - Monoesters and diesters of polyethylene glycol with an average relative molecular weight between 200 and 400, and - glycerol monoesters, diesters and triesters, and / or It includes or consists of the following: - Caprylic (C8) acid and Decanoic acid 10 ) PEG-8 (MW 400) monoesters and diesters of acid, and - Monoglycerides, diglycerides, and triglycerides, and / or It consists of small amounts of monoglycerides, diglycerides, and triglycerides, as well as mainly caprylic (C8) acid and decanoic acid. 10 The acid is composed of PEG-8 (MW400) monoesters and diesters, and / or It is a mixture of the components contained in Labrasol. And / or the second surfactant thereon Having an HLB value of approximately 3 or approximately 6, and / or It is a mixture of the components contained in Plurol Oleique CC 497.

14. The composition of any one of claims 1 to 13, wherein the composition is: - Water-in-oil emulsions, and / or - Microemulsion, wherein the aqueous phase is preferably dispersed in the oil phase in the form of droplets with diameters ranging from about 1 to about 350 nm.

15. The composition of any one of claims 2 to 14, wherein in said composition: The final concentration of the aqueous phase is approximately 14.3% to 24.3% v / v, and / or The final concentration of the oil phase is approximately 8.5% to 18.5% v / v, and / or The final concentration of the first surfactant is approximately 31.9% to 41.9% v / v, and / or The final concentration of the second surfactant is approximately 25.2% to 35.2% v / v, and / or The final concentration of the RNA is approximately 0.1 to 500 µg / ml, particularly at least approximately 1 µg / ml.

16. The composition of any one of claims 1 to 15, wherein the RNA is: Select self-regulating RNA (mRNA), small interfering RNA (siRNA), and microRNA (miRNA), and / or mRNA encoding a peptide or polypeptide, said peptide or polypeptide being selected from antigens, functional proteins, and reporter proteins, and / or mRNA encoding an antigen, wherein the antigen is selected from viral antigens, bacterial antigens, and antigens derived from parasites.

17. The composition of any one of claims 1 to 16, wherein the RNA is mRNA encoding hemagglutinin, particularly hemagglutinin 3 (H3), and wherein the hemagglutinin preferably comprises an amino acid sequence having at least 95%, preferably at least 98%, more preferably at least 99%, or particularly 100% sequence identity with the sequence of SEQ ID NO:6, or is composed of an amino acid sequence having at least 95%, preferably at least 98%, more preferably at least 99%, or particularly 100% sequence identity with the sequence of SEQ ID NO:

6. and / or The RNA therein is an mRNA containing a nucleotide sequence having at least 70%, preferably at least 80%, more preferably at least 90%, still more preferably at least 95%, or particularly 100% sequence identity with the sequence of SEQ ID NO:

7.

18. The composition of any one of claims 1 to 17, used as a medicine or for use in a diagnostic method performed in the body.

19. The composition of any one of claims 1 to 17, used for: - A method for delivering the RNA to a object, wherein the method includes locally applying the composition to the object, and / or - A method of delivering the RNA to a subject through the skin, wherein the method includes applying the composition to the skin of the subject.

20. The composition of claim 16 or 17, in a method for inducing an immune response, particularly a mucosal immune response, in a subject, wherein the immune response is preferably an immune response against an antigen encoded by the mRNA.

21. The composition of claim 17, used for: - In a method for inducing an immune response in an object against an influenza antigen, particularly against a swine influenza A virus (SIAV) antigen, wherein the influenza antigen is preferably H3, and / or - In a method for inducing an immune response against an influenza virus, particularly against a swine influenza A virus (SIAV), wherein the influenza virus is preferably an H3N2 virus.

22. A method for producing the composition of any one of claims 1 to 17, comprising the following steps: (a) The mixed oil phase, the first surfactant, and the second surfactant, and (b) Add an aqueous phase containing RNA to the mixture obtained in step (a). And among them, the preferred ones are: - The oil phase is the oil phase as defined in any one of claims 2, 5, 9 to 11 and 15; and / or - The first surfactant is a first surfactant as defined in any one of claims 3, 5, 6, 10, 13, and 15; and / or - The second surfactant is a second surfactant as defined in any one of claims 4, 5, 7, 10, 13, and 15; and / or - The aqueous phase is the aqueous phase as defined in any one of claims 5, 8, 10, 12, and 15; and / or - The RNA is the RNA as defined in any one of claims 15 to 17.

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