Delivery system useful for topical administration of RNA

Abstract

The present invention relates to a composition useful for the topical administration of RNA, wherein the composition comprises (i) an aqueous phase containing RNA and (ii) an oil phase, which in one embodiment are in mixture with two specific surfactants. Particularly, the present invention pertains to the formulation of mRNA encoding for an antigenic protein of interest. More particular, a microemulsion to cross the skin barrier is provided, with allows for alternative routes for mRNA vaccine applications. The microemulsion may be applied topically and can go through the skin via the transfollicular route. Thereby a delivery system is enabled which allows to (1) avoid the use of needles for vaccination and (2) target more specifically the mucosal component of immunity.

Description

[0001] Delivery system useful for topical administration of RNA

[0002] TECHNICAL FIELD

[0003] The present invention relates to a composition useful for the topical administration of RNA, wherein the composition comprises (i) an aqueous phase containing RNA and (ii) an oil phase, which in one embodiment are in mixture with two specific surfactants. Particularly, the present invention pertains to the formulation of mRNA encoding for an antigenic protein of interest. More particular, a microemulsion to cross the skin barrier is provided, with allows for alternative routes for mRNA vaccine applications. The microemulsion may be applied topically and can go through the skin via the transfollicular route. Thereby a delivery system is enabled which allows to (1) avoid the use of needles for vaccination and (2) target more specifically the mucosal component of immunity.

[0004] BACKGROUND INFORMATION

[0005] RNA vaccines and therapeutics are a growing area of interest in vaccinology and gene therapy. The use of nucleic acid-encoded antigens as the basis for a vaccine platform has numerous advantages: Purification is relatively streamlined and RNA constructs can be built in days using DNA synthesis technologies followed by RNA transcription and capping. This allows for rapid responses to emerging pathogen threats, pivoting changes in manufacturing to adapt to new circulating strains, or for personalizing therapeutic interventions for a variety of diseases.

[0006] Viral infection remains an important health problem in animals with adverse economic consequences. For example, there are a number of viral pathogens that cause disease in economically important livestock animals such as pigs. Viruses infecting pigs include, for example, Swine Influenza A Virus (SIAV).

[0007] Swine influenza is an acute respiratory viral disease caused by influenza A virus (IAV) of the Orthomyxovirus family that decreases health and welfare of pigs. Clinical signs of influenza in pigs can display a range of severity, but often occur as mild respiratory disease with high morbidity and rapid recovery, with rare fatal cases. However, the disease has substantial economic burden as it results in 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 of swine and its high prevalence in swine herds worldwide directly correlates to the economic impact of the disease. In Europe there are currently four major Influenza A virus subtypes circulating in farmed pigs and causing economic losses, as described by Brown (Vet Microbiol. 74(1-2):29-46 (2000)), wherein this and the following publications referred to herein are incorporated by reference in their entirety. H1 N1 ("avian" subtype) and H3N2 swine influenza viruses have been enzootic in major swine producing countries since the 1980s. H1 N2 viruses have been introduced in European swine about twenty years ago (Brown et al. Vet Rec. 13:328-329 (1995)) and the H1 N1 "pandemic" subtype (HlpdmNI) has been introduced into swine populations by transmission from humans to pigs in the course of the 2009 human H1 N1 pandemic and has continuously spread globally in swine populations with an estimated average European prevalence of 8% among all swine Influenza virus infections (Watson et al. J Virol. 89(19): 9920-9931 (2015)). In addition swine Influenza also has implications for human health since IVA is well known for its zoonosis potential (Thacker & Janke. J Infect Dis. 15:197 Suppl 1 :S19-24 (2008)).

[0008] Conventional vaccine approaches for SIAV (inactivated virus injected intramuscularly) provide durable protection but fail to induce a protective immunity in a context of maternal derived immunity.

[0009] Several features of influenza virus are considered to be highly similar to severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), including (I) its ability to efficiently replicate in the mucosa of upper respiratory tract, (II) the presence of many asymptomatic and paucisymptomatic infected animals producing infectious virus, and (III) its short incubation time. As known, there are mRNA vaccines against SARS-CoV-2 available.

[0010] Messenger RNA (mRNA) is a transient intermediate between DNA and proteins and is composed of RNA which includes the protein coding region flanked by non-coding regions. There are three main functional regions in an mRNA: the 5' untranslated region (5'-UTR), the coding region (also termed coding sequence (CDS) or ORF region) and finally the 3' untranslated region (3'-UTR). The two untranslated regions (UTR regions) often contain signals for expression or RNA processing. In particular, mRNA further usually contains a 5' cap and a 3' poly(A) tail, which allow efficient translation of mRNA into protein. The information carried by the coding region of the mRNA consists of a series of codons, consecutive triplets of nucleotides which each code for an amino acid residue of the corresponding protein. In the 1980’s, mRNA was in vitro transcribed and emerged as a candidate for therapeutic purposes, including immunotherapies, viral vaccines, genome editing and cellular reprogramming. mRNA can in principle meet all genetic information requirements to encode and express all kinds of proteins.

[0011] Vaccine efficiency can be optimized by modifying mRNA sequence, which is a more convenient way compared to other kinds of vaccine modification (Xu et al. Int J Mol Sci. 21 :6582 (2020)). Furthermore, most of the mRNA vaccine production and purification processes are quite similar despite different encoded antigens. A potential standardization of manufacturing processes could be developed thanks to this technology rather than specifically develop a specific manufacturing process for each new vaccine target.

[0012] Moreover, mRNA has self-adjuvating properties which activate (A) innate immunity via RIG- I / MDA5, TLR3, TLR7 and TLR8 receptors (Verbeke et al. Immunity. 55(11): 1993-2005 (2022)) and (B) strong and long-lasting adaptive immune responses through tumor necrosis factor a (TNFa), interferon a (IFNa) and other cytokines secretion by immune cells, whereas polypeptide and protein-based vaccines usually need extra adjuvants to achieve a similar goal (lavarone et al. Expert Rev Vaccines. 16(9):871-881 (2017)).

[0013] The desired immunologic effects using mRNA as antigenic source can be achieved only if the mRNA molecule reaches target cells, in the right compartment and produces enough protein antigen to induce a protective immunity.

[0014] The development of drugs or vaccines based on mRNAs is sought, in order to express a protein of interest in a host cell. Once in the cytoplasm of the host cell, the mRNA is read by the ribosome and translated into a protein that has the desired therapeutic activity.

[0015] The delivery systems used include lipid nanoparticles (LNP) or liposomes, making it possible to protect the mRNA by encapsulating it within a nanoparticle or a lipid vesicle. The review article Guevara et al. (Front Chem. 8:589959 (2020)) reports the use of LNPs in which mRNA is encapsulated as a vehicle for mRNA delivery and its use for cancer treatment.

[0016] The recent SARS-CoV-2 pandemic has accelerated the research for rapid vaccine solutions. mRNA vaccine technology presents many advantages, compared to conventional approaches, such as their potential for rapid development and large-scale deployment in pandemic situations. The development of RNA therapeutics should take in 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 cytoplasmic membrane, and (3) the strong immunogenicity of exogenous RNA potentially causing cell toxicity and impairing translation into therapeutic proteins (Damase et al. Front Bioeng Biotechnol. 9:628137 (2021)).

[0017] A variety of mRNA delivery methods have been developed to protect mRNA from ribonucleases and neutralize or revert its negative charge to increase the cellular uptake and thus increase the production of the protein of interest. So far, lipids and polymer-based materials are the major non-viral mRNA delivery systems. Typically, cationic lipids and polymers are used to form complexes based on electrostatic and hydrophobic interactions with the negatively charged mRNA. These particles act as a shield to prevent mRNA from enzymatic degradation and invert the polarity to enhance the efficiency of transfection (Chaudhary et al. Nat Rev Drug Discov. 20(11):817-838 (2021)).

[0018] The utility of lipid nanoparticles to deliver mRNA has been successfully demonstrated with COVID-19 vaccines, such as mRNA-1273 and BNT162b. Of all the mRNA vaccines in clinical trials, only mRNA-1273 and BNT162b with LNP-mRNA formulation injected via intramuscular route was so far approved for human use (Wilson & Geetha. J Drug Deliv Sci Technol. 74:103553 (2022)).

[0019] The intramuscular route of administration was retained for mRNA vaccine administration even if intravenous, subcutaneous, intradermal and intranodal injections are considered (Nikita et al. 2022). Intramuscular route is used for decades for vaccine administration and is easy to perform compared to subcutaneous injection. This route includes the opportunity to inject a relatively large amount of vaccine and a reduction in pain and local irritation compared with subcutaneous injections. But intramuscular route could also induce adverse events such as percutaneous injuries and is a source of inflammation or contamination after skin barrier disruption.

[0020] Moreover, while several reports have assessed the generation of vaccine-induced IgG neutralizing antibodies in the serum of immunized individuals, few data are available regarding the activation of a specific mucosal immunity. The respiratory tract and the oral mucosa represent the primary route of entry of influenza virus, thus anti-SlAV vaccines inducing specific immune response in these tissues might become a crucial tool to increase the vaccinal immune response and overcome the maternal derived immunity. In pigs, the concept of common mucosal immune system was highlighted only recently compared to other mammals such as mice. According to this concept, immunization or inoculation using one mucosal route, triggers the immune system both locally and at distance of mucosal sites (Wilson & Obradovic. Mol Immunol. 66:22-34 (2015)). This phenomenon could be used to trigger a mucosal immunity from the skin to the lungs, the main target of the influenza virus.

[0021] The skin contains important early-warning components of the immune system, including powerful antigen presenting cells (APCs), such as Langerhans and dendritic cells, and immunologically active keratinocytes (Levin et al. Hum Vacc Immunother. 11(4):991-997 (2015). However, the very low capability of antigenic compounds (usually biomacromolecules with a high molecular weight) to cross the stratum corneum to reach the basal epidermis and the dermis, in which the immune cells are located, represents a major hurdle.

[0022] Transcutaneous immunization with mannosylated-chitosan-modified ethosomes loaded with mRNA has been described for transfecting dendritic cells (DCs) and for inducing their maturation: Transdermal patches loaded with mRNA vaccines or siRNA and electrosprayed as microspheres containing mRNA- or siRNA- onto the surface of silk fibroin matrices (under the conditions of: voltage 18 kV, extrusion rate 1 ml / h, receiving distance 15 cm, temperature 40 °C, and humidity 8%) allowed the delivery of nucleic acid molecules into the deep layers of skin in a syngeneic mouse model for melanoma (Wang et al. Composites Part B: Engineering. 233:109648 (2022)). For this purpose the transdermal patches loaded with mRNA vaccines were attached to the bare belly skin of the mice and fixed with medical tape for 3 days, which was repeated on the 7th and 14th day in order to boost the immunization.

[0023] However, for non-invasive mRNA delivery an easily producible composition would be needed, the administration of which can be simply practiced, in particular in a farming environment where a large number of animals need to be vaccinated in short period of time.

[0024] WO2023144352 A 1 discloses an oil-in-water nanoemulsion having an oil phase containing an Active Pharmaceutical Ingredient (API) which comprises RNA or a hydrophobic drug, and an aqueous phase. This oil-in-water nanoemulsion comprises, in addition to the API, a high oleic edible oil, plant protein, surfactant, and water. The oil in water emulsion is then combined with a suspension of denatured plant protein and a calcium salt chelating agent to form microparticles. Such microparticles may be administered orally to a subject, e.g. in the form of a tablet, which are said to then pass through the stomach to the ileum where the protein matrix breaks down to release the oil droplets containing the API, which is then absorbed.

[0025] However, since usually the delivery of a tablet to an animal is difficult in practice, an easy non- invasive administration system is desired for delivering RNA to an animal, such as by topical administration. Topical administration would also have the advantage that it allows to deliver the RNA to specific locations of the body, as compared to being adsorbed into the bloodstream resulting from oral administration.

[0026] DESCRIPTION OF THE INVENTION

[0027] The solution to the above technical problems is achieved by the description and the embodiments characterized in the claims.

[0028] Thus, the invention in its different aspects is implemented according to the claims.

[0029] The invention is based on the surprising finding that the topical administration of compositions comprising an aqueous phase containing mRNA which encodes an enzyme or an immunogen, respectively, and an oil phase to swine allowed the cellular intake and translation of said mRNA, resulting in specific enzymatic activity or induction of immunity, respectively, in said swine.

[0030] In a first aspect, the invention thus relates to a composition comprising an aqueous phase containing a ribonucleic acid (RNA), and an oil phase, and wherein said composition is also termed “the composition of the present invention” hereinafter.

[0031] Said aqueous phase containing an RNA, which is also termed the “aqueous phase according to the present invention” hereinafter, is preferably free of solid nanoparticles or, respectively, is in particular free of lipid nanoparticles (LNPs) and / or lipid-like nanoparticles (LLNs). Said RNA contained in said aqueous phase is also termed “the RNA according to the present invention” hereinafter.

[0032] Said oil phase, which is also termed the “the oil phase according to the present invention” hereinafter, is preferably liquid or a liquid, respectively, at room temperature.

[0033] The term “aqueous phase” in particular refers to the aqueous portion of a water-in-oil emulsion. The term “oil phase” in particular refers to the oil portion of a water-in-oil emulsion. More particular, the term “aqueous phase”, as used herein, means a liquid phase which comprises water and can additionally comprise hydrophilic co-solvents and water soluble substances, and which is immiscible with the oil phase. It is particularly understood that said liquid phase which comprises water is a phase in which RNA is soluble. The term “oil phase” as used herein, more particular means a liquid phase which is immiscible with water.

[0034] The term “RNA contained in said aqueous phase”, as used herein, in particular means that the RNA is in contact with said liquid phase which comprises water.

[0035] The term "liquid phase", as mentioned herein in particular refers to a phase that is liquid at room temperature (25°C) and atmospheric pressure (760 mmHg).

[0036] The term “aqueous phase”, as used herein, is in particular understood to be equivalent to “aqueous solution”. The term “oil phase”, as used herein, is in particular understood to be equivalent to “oil”.

[0037] The term “ribonucleic acid” or "RNA", respectively, as used herein, particularly means a molecule comprising at least one ribonucleotide residue and includes single stranded RNA, double stranded RNA, isolated RNA, partially purified, pure or synthetic RNA, recombinantly produced RNA, as well as altered RNA or analogs of naturally occurring RNA, e.g. altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. By "ribonucleotide" is meant a nucleotide with a hydroxyl group at the 2' position of a p-D-ribo-furanose moiety. Nucleotides in the RNA molecules of the instant disclosure can also comprise non-standard nucleotides, such as non- naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides.

[0038] It is in particular understood that the term “a ribonucleic acid” or “an RNA”, respectively, as used herein, also includes plural reference, and thus also encompasses a plurality of a ribonucleic acid or of an RNA, respectively, or encompasses “one or more ribonucleic acids” or “one or more RNAs”, respectively. The term “ribonucleic acid” and “RNA”, respectively, is in particular understood to be equivalent to “ribonucleic acid molecule” and “RNA molecule”, respectively.

[0039] Preferably, the RNA contained in the aqueous phase is dissolved in said aqueous phase. According to this aspect, the aqueous phase containing an RNA is thus an aqueous phase in which an RNA is dissolved. “Dissolved” in this context is particularly understood to mean “freely dissolved”, i.e. not bound to colloids or particles. Thus, in particular, the RNA according to the present invention is freely movable within the aqueous phase and / or is not associated to nanoparticles, such as to a lipid nanoparticles (LNPs) and / or lipid-like nanoparticles (LLNs).

[0040] According to a preferred aspect, the composition of the present invention comprises one or more surfactants.

[0041] Preferably, said one or more surfactants is, or are, selected from any of the first surfactant or second surfactant mentioned hereinafter, or is a mixture of these.

[0042] The term “surfactant”, as used herein, in particular means a compound that contains a lipophilic segment and a hydrophilic segment, which when added to water or solvents, reduces the surface tension of the system.

[0043] In particular, the composition of the present invention comprises two surfactants, being also termed “first surfactant” and “second surfactant” herein, wherein said first and said second surfactant are different from each other. Hence, the herein mentioned second surfactant is different from the herein mentioned first surfactant.

[0044] Thus, the composition of the present invention in particular comprises or consists of an aqueous phase containing an RNA, an oil phase, a first surfactant, and a second surfactant.

[0045] Preferably, said first surfactant is a non-ionic surfactant or said second surfactant is a nonionic surfactant. In particular, said first surfactant is a non-ionic surfactant and said second surfactant is a non-ionic surfactant. A “non-ionic surfactant”, as mentioned herein, in particular refers to a surfactant that does not carry an electrical charge. It is thus particularly understood that a non-ionic surfactant does not react with ions in solution and does not ionize in water.

[0046] According to a preferred aspect, said first surfactant is a water-soluble surfactant.

[0047] The term “water-soluble surfactant”, as mentioned herein, in particular means a surfactant that has an hydrophile-lipophile balance (HLB) value of more than 10.

[0048] As used herein, the term “HLB value” refers to the hydrophile-lipophile balance (HLB) scale devised by Griffin in 1949 (Griffin, W.C. (1949) Classification of Surface-Active Agents by “HLB”. Journal of Cosmetic Science, 1 , 311-326), which is a scale from 0-20 (20 being Hydrophilic) used to characterize the nature of molecules, such as surfactants.

[0049] The HLB of a molecule is calculated as follows:

[0050] HLB=20* / / W where Mh is the molecular mass of the hydrophilic portion of the molecule, and M is the molecular mass of the whole molecule, giving a result 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, W. C. Calculation of HLB values of Nonionic Surfactants, J. Soc. Cosmet. Chem. 1954, 5, 249- 256. The HLB values for commonly-used surfactants are readily available in the literature (e.g., HLB Index in McCutcheon's Emulsifiers and Detergents, MC Publishing Co., 2004).

[0051] According to a preferred aspect, said first surfactant has an HLB value of more than 10.

[0052] Said second surfactant is preferably a co-surfactant.

[0053] The term “co-surfactant”, as mentioned herein, is in particular understood to mean a surfactant acting in addition to a first surfactant to further reduce the surface tension of a liquid.

[0054] Preferably, said second surfactant is a water-insoluble surfactant.

[0055] The term “water-insoluble surfactant”, as mentioned herein, in particular means a surfactant that has an HLB value of less than 10.

[0056] Said second surfactant preferably has an HLB value of less than 10.

[0057] According to a further preferred aspect, the aqueous phase according to the present invention comprises a solvent selected from the group consisting of water, and mixture of water with any other water-miscible solvent. A “water-miscible solvent”, as mentioned herein, in particular means a solvent (including mixtures of solvent compounds) other than water that is liquid at room temperature and that mixes with water in all proportions to form a homogenous solution. Examples of a water- miscible solvent include ethanol, ethylene glycol and / or glycerin.

[0058] Preferably, the aqueous phase according to the invention comprises water.

[0059] According to another preferred aspect, the oil phase according to the invention is selected from the group consisting of mineral oil and vegetable oil.

[0060] The term "mineral oil", as used herein, in particular refers to a mixture of liquid hydrocarbons obtainable from petrolatum by distillation. The mineral oil can be synthetically prepared or purified from petroleum products. The term “vegetable oil”, as used herein, refers to any oil that can be processed from a vegetable.

[0061] Preferably, the oil phase according to the present invention is a mineral oil.

[0062] According to a particularly preferred aspect, the composition of the present invention comprises or consists of an aqueous phase containing an RNA, wherein said aqueous phase comprises water; an oil phase, wherein said oil phase is a mineral oil; a first surfactant having an HLB value of more than 10; and a second surfactant having an HLB value of less than 10.

[0063] According to another preferred aspect, said first surfactant is selected from the group consisting of polyethylene glycol derivatives, polyethylene glycols, glycerol-based surfactants, and polyethylene glycol (PEG) esters.

[0064] Preferably, said first surfactant has an HLB value of from 11 to 16.

[0065] According to a further preferred aspect, said first surfactant comprises PEG-8 caprylic / capric glycerides or PEG-6 caprylic / capric glycerides.

[0066] In a still further preferred aspect, said first surfactant has an HLB value of from 11 to 13.

[0067] Preferably, said first surfactant comprises PEG-8 caprylic / capric glycerides.

[0068] In another preferred aspect, said first surfactant comprises mono-, di- and / or triglycerides.

[0069] In a further preferred aspect, said first surfactant is mainly composed of PEG-8 caprylic / capric glycerides. Most preferably, said first surfactant comprises or consists of monoesters and diesters of polyethylene glycol with mean relative molecular weight between 200 and 400, and monoesters, diesters, and triesters of glycerol.

[0070] In one preferred aspect, the herein mentioned first surfactant comprises or consists of

[0071] PEG-8 (MW 400) mono- and diesters of caprylic (Cs) and capric (Cw) acids, and mono-, di- and triglycerides.

[0072] In another preferred aspect, the herein mentioned first surfactant consists of a small fraction of mono-, di- and triglycerides and mainly PEG-8 (MW 400) mono- and diesters of caprylic (Cs) and capric (Cw) acids.

[0073] In one example, the commercially available product Labrasol® (Gattefosse SA, Saint-Priest, France) is used as said first surfactant, but other surfactants are suitable as well. Thus, in one preferred example, said first surfactant is Labrasol® or, respectively, said first surfactant is the mixture of components included in Labrasol®.

[0074] Said second surfactant is preferably a polyglycerol fatty acid ester.

[0075] According to a preferred aspect, said second surfactant has a HLB value of from about 3 to about 8.

[0076] In another preferred aspect, said second surfactant is selected from the group consisting of polyglyceryl-3 dioleate and polyglyceryl-6 dioleate.

[0077] According to a still further preferred aspect, said second surfactant has a HLB value of from about 3 to about 6. According to one preferred example, said second surfactant has a HLB value of about 3. According to another preferred example, said second surfactant has a HLB value of about 6.

[0078] Preferably, said second surfactant is Polyglyceryl-3 dioleate.

[0079] In one example, the commercially available Polyglyceryl-3 dioleate Plurol® Oleique CO 497 (Gattefosse SA, Saint-Priest, France)) is used as said second surfactant, but other surfactants are suitable as well. Thus, in one preferred example, said second surfactant is Plurol® Oleique CC 497 or, respectively, said second surfactant is the mixture of components included in Plurol® Oleique CC 497.

[0080] In a preferred aspect, the aqueous phase according to the present invention is selected from the group consisting of aqueous buffer solution containing an RNA,

[0081] - water containing an RNA, and mixture of

[0082] • water or

[0083] • an aqueous buffer solution with any other water-miscible solvent, wherein said mixture contains an RNA.

[0084] Preferably, the aqueous phase comprises or consists of an aqueous buffer solution.

[0085] According to a particularly preferred aspect, the aqueous phase according to the present invention is an aqueous buffer solution containing an RNA.

[0086] The oil phase according to the present invention is preferably a mineral oil selected from the group consisting of liquid paraffin, and oily liquid that is composed of saturated hydrocarbons derived from petroleum like hexadecane or methyl oleate or ethyl oleate.

[0087] Preferably, said oil phase is liquid paraffin.

[0088] In a preferred aspect, the composition of the present invention comprises or consists of an aqueous phase containing an RNA, wherein said aqueous phase containing an RNA is an aqueous buffer solution containing an RNA; an oil phase, wherein said oil phase is liquid paraffin; a first surfactant, wherein said first surfactant comprises or is mainly composed of PEG-8 caprylic / capric glycerides; and a second surfactant, wherein said second surfactant is Polyglyceryl-3 dioleate. In particular, it is preferred if the oil phase according to the present invention is a purified mixture of liquid saturated hydrocarbons. Preferably, said oil phase is obtained from petroleum through purification by catalytic hydrogenation.

[0089] Preferably, the oil phase according to the present invention comprises from about 67% to about 68% paraffinic carbons, and / or from about 32% to about 33% naphtenic carbons.

[0090] According to another preferred aspect, the oil phase according to the present invention has a dynamic viscosity at 20°C of no more than about 100 mPa s, preferably of no more than about 50 mPa s, more preferably of no more than about 30 mPa s, and most preferably of no more than about 20 mPa s. In a particular preferred aspect, the oil phase according to the present invention has a dynamic viscosity at 20°C of about 10 mPa s to about 100 mPa s, preferably of about 10 mPa s to about 50 mPa s, more preferably of about 10 mPa s to about 30 mPa s, and most preferably of about 10 mPa s to about 20 mPa s. According to a preferred example, the oil phase according to the present invention has a dynamic viscosity at 20°C of about 12 mPa s.

[0091] In one example, the commercially available mineral oil Marcol® 52 (Exxon, USA) is used as the oil phase according to the present invention, but other oils are suitable as well.

[0092] Thus, in one preferred example, the oil phase according to the present invention is Marcol® 52 or, respectively, the oil phase according to the present invention is the mixture of components included in Marcol® 52.

[0093] Preferably, the herein mentioned aqueous buffer solution is selected from the group consisting of citrate buffer, PBS buffer and Tris buffer.

[0094] In a particular preferred aspect, the aqueous phase according to the present invention is a citrate buffer containing an RNA.

[0095] As used herein, the term "citrate buffer" in particular refers to a buffer (i.e. a solution that can resist pH change when an acidic or basic components are added to it) containing a citrate ion. Here, specific embodiments of the citrate buffer may be any one selected from the group consisting of a sodium citrate buffer, a potassium citrate buffer, a calcium citrate buffer, and a magnesium citrate buffer, but are not limited thereto. Preferably, said citrate buffer has a pH value of from 5.0 to 6.8, most preferably a pH value of 6.4 ± 0.2.

[0096] Preferably, the composition of the present invention is an emulsion.

[0097] The term "emulsion", as used herein, in particular refers to a mixture of two or more liquids that are normally immiscible (unmixable or unblendable). In an emulsion, one liquid (the dispersed phase) is dispersed in the other (continuous phase). Examples of emulsions include vinaigrettes, homogenized milk, mayonnaise, and the like.

[0098] In one preferred aspect, the composition of the present invention is an emulsion in which the aqueous phase is dispersed into the oil phase. Thus, preferably, the aqueous phase of the present invention is the dispersed phase and the oil phase of the present invention is the continuous phase.

[0099] The composition of the present invention is preferably a water-in-oil emulsion.

[0100] A “water-in-oil emulsion”, as mentioned herein, in particular refers to a water-in-oil mixture in which the oil forms a continuous phase and the water is in discontinuous droplets. A water-in- oil emulsion can be distinguished from an oil-in-water emulsion by using an electrical emulsion tester. An oil-in-water emulsion will conduct electricity with relatively low resistance since water forms its external or continuous phase, whereas a water-in- oil emulsion will not conduct, or very poorly conduct, electricity.

[0101] In a preferred aspect, the composition of the present invention is a microemulsion.

[0102] As used herein, the term “microemulsion” is given its ordinary meaning in the art and refers to dispersions of one immiscible liquid in another, in the form of droplets, with diameters approximately in the range of about between about 1 and about 1000 nm, most preferably below about 350 nm. The microemulsion of the present invention preferably relates to a dispersion of the aqueous phase according to the present invention in the oil phase according to the present invention, in the form of droplets, with diameters approximately in the range of about between about 1 and about 500 nm, or between about 1 and about 350 nm, or between about 5 and about 500 nm, or between about 5 and about 350 nm, or most preferably between about 5 and about 200 nm. Said range is also termed “droplet size distribution” herein. Preferably, said microemulsion is a homogeneous thermodynamically stable system.

[0103] Preferably, the aqueous phase according to the present invention is dispersed in the oil phase according to 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. Said average diameter is also termed “average droplet size” herein.

[0104] The droplet size distribution or the average droplet size can be determined by a person skilled in the art using the dynamic light-scattering (DLS) technique as described in Example 4. In cases of controversial results and in any question of doubt, the herein mentioned droplet size distribution and average droplet size, refer to those which are / can be estimated by the DLS technique as described in Example 4.

[0105] In a preferred aspect, the composition of the present invention is an emulsion, in particular a microemulsion, wherein said emulsion is thermodynamically stable.

[0106] In a further preferred aspect, the composition of the present invention is an emulsion, in particular a microemulsion, wherein said emulsion is isotropic.

[0107] In still a further preferred aspect, the composition of the present invention is an emulsion, in particular a microemulsion, wherein said emulsion is optically transparent.

[0108] In yet a further preferred aspect, the composition of the present invention is an emulsion, in particular a microemulsion, wherein said emulsion is opalescent.

[0109] 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.

[0110] In one preferred aspect, in the composition of the present invention the aqueous phase of the present invention is in a final concentration of about 5% to 30% v / v (volume per volume).

[0111] Most preferably, in the composition of the present invention the aqueous phase of the present invention is in a final concentration of about 14.3% to 24.3% v / v.

[0112] According to another preferred aspect, in the composition of the present invention the oil phase of the present invention is in a final concentration of about 5% to 30% v / v.

[0113] Most preferably, in the composition of the present invention the oil phase of the present invention is in a final concentration of about 8.5% to 18.5% v / v. In a further preferred aspect, in the composition of the present invention said first surfactant is in a final concentration of about 30.7% to 70.7% v / v.

[0114] Most preferably, in the composition of the present invention said first surfactant is in a final concentration of about 31 .9% to 41 .9% v / v.

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

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

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

[0118] According to a further preferred aspect, the final concentration of the RNA according to the present invention in the composition of the present invention is about 0.1 to 500 pg per ml (pg / ml), wherein it is understood that in said concentration (pg / ml) said mass (pg) pertains to the RNA according to the present invention and said volume (ml) pertains to the composition of the present invention.

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

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

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

[0122] According to a further preferred aspect, the final concentration of the RNA according to the present invention in the composition of the present invention is about 1 to 400 pg / ml, and most preferably about 30 to 300 pg / ml. More particular, it is preferred that in the composition of the present invention the RNA according to the present invention is in a final concentration of about 200 pg / ml.

[0123] The RNA according to the present invention is preferably selected from the group consisting of messenger RNA (mRNA), small interfering RNA (siRNA) and microRNA (miRNA).

[0124] As used herein, the term "messenger RNA" (mRNA) in particular refers to any polynucleotide, which may be synthetic, which encodes a polypeptide, e.g., an immunogenic protein, and which is capable of being translated to produce the encoded polypeptide in vitro, in vivo, in situ or ex vivo. The herein used term „small interfering RNA” (siRNA) in particular means a low molecular weight (usually 19-27 base pairs) double-stranded RNA involved in RNAi. As used herein, the term "RNAi" means RNA interference. The term “microRNA” (miRNA), as mentioned herein, particularly refers to a non-coding RNA molecule about 22 nucleotides long that functions in post-transcriptional regulation of gene expression by forming base pairs with the 3' untranslated region (UTR) of mRNAs.

[0125] Most preferably, the RNA according to the present invention is an mRNA, and wherein said mRNA is also termed “mRNA according to the present invention” hereinafter.

[0126] In particular, the mRNA according to the present invention comprises a 5'-UTR, a coding region encoding a polypeptide, and a 3'-UTR. The term “coding region”, as used herein, in particular refers to a portion or region of the mRNA that can be translated into a chain of amino acids, i.e., two or more amino acids linked by peptide bonds. More particular, said coding region is flanked by said 5'-UTR and said 3'-UTR. According to a preferred aspect, the mRNA according to the present invention comprises a poly(A) tail, wherein said poly(A) tail is preferably located following said 3'-UTR in 3'direction. Preferably, said 3'-UTR is flanked by a poly(A) tail. Most preferably, the mRNA according to the present invention comprises a coding region encoding a polypeptide, wherein said coding region is flanked by a 5'-UTR and a 3'-UTR, and wherein said 3'-UTR is flanked by a poly(A) tail. It is in particular understood that the coding region, as mentioned herein, also comprises at least one stop codon, in particular at the 3' end of the coding region. Preferably, said 3'-UTR is located following said stop codon, and in particular flanks said stop codon, in 3' direction.

[0127] Most preferably, the mRNA according to the present invention comprises (from 5' in 3' direction) a 5'-UTR, a coding region, a 3'-UTR, and a poly(A) tail. The term "untranslated region" or "UTR" as used in accordance with the present invention relates to sections of an mRNA upstream of the start codon and downstream of the stop codon that are not translated, and are, therefore, termed the five prime untranslated region (5' UTR) and three prime untranslated region (3' UTR), respectively. These regions are transcribed with the coding region and thus are exonic as they are present in the mature mRNA.

[0128] As used in the present invention, the 3' untranslated region (3'-UTR) relates to the section of messenger RNA (mRNA) that immediately follows the translation termination codon. The 3' UTR may comprise regulatory regions within the 3'-untranslated region which are known to influence polyadenylation and stability of the mRNA. Many 3'-UTRs also contain AU-rich elements (AREs). Furthermore, the 3'-UTR may preferably contain the sequence AAUAAA that directs addition of several hundred adenine residues called the poly(A) tail to the end of the mRNA transcript. The 5' untranslated region (5'-UTR) (also known as a Leader Sequence or Leader RNA) is the region of an mRNA that is directly upstream of the start codon. The 5'-UTR begins at the transcription start site and ends one nucleotide (nt) before the start codon (usually AUG in the mRNA) of the coding region. In eukaryotes the length of the 5'-UTR is generally from 100 to several thousand nucleotides long but sometimes also shorter UTRs occur in eukaryotes.

[0129] As used herein, the term "poly(A) tail" or "poly(A) sequence" refers to an uninterrupted or interrupted sequence of adenylate residues which is typically located at the 3'-end of an mRNA molecule. Poly(A) tails or poly(A) sequences are known to those of skill in the art and may follow the 3'-UTR in the RNAs (in particular, mRNAs) described herein. An uninterrupted poly(A) tail is characterized by consecutive adenylate residues. In nature, an uninterrupted poly(A) tail is typical. RNAs (in particular, mRNAs) disclosed herein can have a poly(A) tail attached to the free 3'-end of the RNA by a template-independent RNA polymerase after transcription or a poly(A) tail encoded by DNA and transcribed by a template-dependent RNA polymerase.

[0130] It has been demonstrated that a poly(A) tail of about 120 A nucleotides has a beneficial influence on the levels of mRNA in transfected eukaryotic cells, as well as on the levels of protein that is translated from an open reading frame that is present upstream (5') of the poly(A) tail (Holtkamp et ai. Blood, 108: 4009-4017( 2006)).

[0131] The poly(A) tail may be of any length. In some embodiments, a poly(A) tail comprises, essentially consists of, or consists of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 A nucleotides, and, in particular, about 120 A nucleotides. In this context, "essentially consists of means that most nucleotides in the poly(A) tail, 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% by number of nucleotides in the poly(A) tail are A nucleotides, but permits that remaining nucleotides are nucleotides other than A nucleotides, such as U nucleotides (uridylate), G nucleotides (guanylate), or C nucleotides (cytidylate). In this context, "consists of" means that all nucleotides in the poly(A) tail, i.e., 100% by number of nucleotides in the poly(A) tail, are A nucleotides. The term "A nucleotide" or "A" refers to adenylate.

[0132] In some embodiments, a poly(A) tail is attached during RNA transcription, e.g., during preparation of in vitro transcribed RNA, based on a DNA template comprising repeated dT nucleotides (deoxythymidylate) in the strand complementary to the coding strand. The DNA sequence encoding a poly(A) tail (coding strand) is referred to as poly(A) cassette.

[0133] According to a preferred aspect, the mRNA according to the invention comprises a Kozak sequence. A “Kozak sequence”, as mentioned herein, in particular refers to a consensus sequence included in mRNA which is recognized by the ribosome to initiate translation of a polypeptide.

[0134] The Kozak sequence, as referred to herein, in particular has the RNA sequence (from 5' in 3' direction) RCCALIG, preferably RCCAUGG, wherein AUG is the start codon of the coding region and R is selected from A and G. In one preferred example, the mRNA according to the present invention comprises the Kozak sequence ACCAUGG.

[0135] In one non-limiting example, an mRNA according to the present invention comprising (from 5' in 3' direction) a 5'-UTR, a coding region, a 3'-UTR, and a poly(A) tail, and comprising a Kozak sequence, is the mRNA of SEQ ID NO:3, wherein

[0136] - the nucleotide positions 1 to 359 of SEQ ID NO:3 correspond to a 5'-UTR,

[0137] - the nucleotide positions 360 to 1070 of SEQ ID NO:3 correspond to a coding region,

[0138] - the nucleotide positions 357 to 363 of SEQ ID NO:3 correspond to a Kozak sequence,

[0139] - the nucleotide positions 1071 to 1332 of SEQ ID NO:3 correspond to a 3'-UTR,

[0140] - the nucleotide positions 1333 to 1452 of SEQ ID NO:3 correspond to a poly(A) tail.

[0141] According to a further aspect, the mRNA according to the present invention preferably contains one or more modifications, e.g., in order to increase its stability and / or increase translation efficiency and / or decrease immunogenicity and / or decrease cytotoxicity. For example, in order to increase expression of the mRNA, it may be modified within the coding region, i.e., the sequence encoding the expressed peptide or polypeptide, preferably without altering the sequence of the expressed peptide or polypeptide.

[0142] Such modifications are described, for example, in W02007 / 036366, WO2019 / 175356 or WO2023 / 147352, and include the following: a 5'-cap structure; an extension or truncation of the naturally occurring poly(A) tail; an alteration of the 5'- and / or 3'-untranslated regions (UTR) such as introduction of a UTR which is not related to the coding region of said RNA; the replacement of one or more naturally occurring nucleotides with synthetic nucleotides; and codon optimization (e.g., to alter, preferably increase, the GC content of the RNA).

[0143] According to a particular preferred aspect, the mRNA according to the present invention comprises a 5'-cap structure. In some embodiments, the mRNA according to the present invention does not have uncapped 5'-triphosphates. In some embodiments, the mRNA according to the present invention may comprise a conventional 5'-cap and / or a 5'-cap analog. As used herein, the term “5'-cap structure” is in particular understood to encompass “conventional 5'-cap” as well as “5'-cap analog”. Most preferably, the mRNA according to the present invention comprises a conventional 5'-cap.

[0144] The term "conventional 5'-cap" refers to a cap structure found on the 5'-end of an mRNA molecule and generally consists of a guanosine 5'-triphosphate (Gppp) which is connected via its triphosphate moiety to the 5'-end of the next nucleotide of the mRNA (i.e., the guanosine is connected via a 5' to 5' triphosphate linkage to the rest of the mRNA). The guanosine may be methylated at position N7(resulting in the cap structure m7Gppp). In the context of the present application, the term "capO" or “capO structure”, respectively, means the structure "m7GpppN", wherein N is any nucleoside bearing an OH moiety at position 2'. According to the present application, the term "cap1" or “cap1 structure”, respectively, means the structure "m7GpppNm", wherein Nm is any nucleoside bearing an OCH3 moiety at position 2'. According to the present application, the term "cap2" or “cap2 structure”, respectively, means the structure "m7GpppNmNm", wherein each Nm is independently any nucleoside bearing an OCH3 moiety at position 2'.

[0145] According to one preferred example, the mRNA according to the present invention comprises a cap1 structure, in particular the structure m7pppNm. Thus, the mRNA according to the present invention preferably comprises a 5'-cap having the cap1 structure m7GpppNm, wherein “m7Gppp” represents guanosine 5'-triphosphate in which the guanosine is methylated at position N7, “Nm” represents any nucleoside bearing an OCH3 moiety at position 2', and wherein m7Gppp is connected via its triphosphate moiety to the 5'-end of Nm.

[0146] According to a particularly preferred aspect, the mRNA according to the invention comprises a 5'-cap having the cap1 structure m7GpppAm, wherein Am is an adenosine bearing an OCH3 moiety at position 2'. Thus, most preferably the mRNA according to the invention comprises a m7GpppAm cap. Producing mRNA with said cap may be done, for example, by using the CleanCap® AG reagent (TriLink Biotechnologies, San Diego (GA), USA, product no. N-7113) according to manufacturer's instructions.

[0147] Hence, the mRNA according to the present invention preferably comprises a 5'-cap having the cap1 structure m7GpppAm, wherein “m7Gppp” represents guanosine 5'-triphosphate in which the guanosine is methylated at position N7, “Am” represents adenosine bearing an OCH3 moiety at position 2', and wherein m7Gppp is connected via its triphosphate moiety to the 5'- end of Am.

[0148] The term "5'-cap analog" means a structural derivative of a conventional 5'-cap. For example, in the structure m7Gppp, the guanosine methylated at position N7may comprise a substitution of the hydrogen on the hydroxyl at position C-3' with a methyl group (resulting in the cap structure m7(3'OMe)Gppp). Thus the structure m7(3'OMe)Gppp, as mentioned herein, in particular means the structure m7Gppp, but wherein the guanosine methylated at position N7bears an OCH3 moiety at position 3'. Examples of 5'-cap analogs are for instance described in WO2023 / 147352 or WO2019 / 175356.

[0149] In the context of the present application, the term "capO analog" or “capO analog structure”, respectively, means a structural derivative of the structure "m7GpppN", and wherein said structural derivative is preferably the structure “m7(3'OMe)Gppp”. According to the present application, the term "cap1 analog" or “cap1 analog structure”, respectively, means a structural derivative of the structure "m7GpppNm", and wherein said structural derivative is preferably the structure “m7G(3'OMe)pppNm”. According to the present application, the term "cap2 analog" or “cap2 analog structure”, respectively, means a structural derivative of the structure "m7GpppNmNm", and wherein said structural derivative is preferably the structure “m7G(3'OMe)pppNmNm”.

[0150] According to one preferred example, the mRNA according to the present invention comprises a cap1 analog structure, in particular the structure m7G(3'OMe)pppNm. For example, the mRNA according to the invention may comprise a 5'-cap analog having the cap1 analog structure m7G(3'OMe)pppAm, wherein Am is an adenosine bearing an OCH3 moiety at position 2'. Production of mRNA with said cap may be done, for example, by using the CleanCap® AG 3'0Me reagent (TriLink Biotechnologies, San Diego (GA), USA, product no. N-7413) according to manufacturer's instructions.

[0151] Providing an mRNA with a 5'-cap structure may be achieved by in vitro transcription of a DNA template in presence of a corresponding 5'-cap compound, wherein said 5'-cap structure is co- transcriptionally incorporated into the generated mRNA strand, or the mRNA may be generated, for example, by in vitro transcription, and the 5'-cap structure may be attached to the mRNA post-transcriptionally using capping enzymes, for example, capping enzymes of vaccinia virus. Methods for providing an mRNA with a 5'-cap structure are known to the person skilled in the art and are e.g. summarized in the review articles Muttach et al. Beilstein J Org Chem. 13:2819-2832 (2017) and Ramanathan et al. Nucleic Acids Res. 44(16):7511-7526 (2016).

[0152] Preferably, the mRNA according to the present invention encodes a peptide or polypeptide selected from the group consisting of antigen, functional protein and reporter protein.

[0153] Pursuant to one preferred aspect, the RNA according to the present invention is an mRNA encoding an antigen or a functional protein.

[0154] In the context of the mRNA according to the present invention, the wording “mRNA encoding” or “mRNA encodes”, respectively, is equivalent to “mRNA comprising a coding region which encodes” or “mRNA comprises a coding region encoding”, respectively.

[0155] The term “antigen”, as mentioned herein, generally refers to a substance such as a polypeptide or peptide which, if administered to a host, can elicit an immunological response in the host. As used herein, the term “antigen” is interchangeable with the term “immunogen”. In one example, an antigen, as referred to herein, can be an immunogenic protein or an immunogenic protein domain. The term “immunogenic”, as used herein, means “capable of eliciting a humoral and / or cellular immune response in a host”.

[0156] The herein used term "functional protein" means a polypeptide that is capable to affect the structure and / or appearance of a product when coming in contact therewith, e.g. to function as an enzyme acting upon a substrate. The term “polypeptide” used herein in particular refers to any chain of amino acid residues linked together by peptide bonds, and does not refer to a specific length of the product. For instance, “polypeptide” may refer to a long chain of amino acid residues, e.g. one that is 150 to 600 amino acid residues long or longer. The term “polypeptide” particularly includes polypeptides which are altered by one or more co-translational and / or post-translational modifications, wherein such modifications include, e.g., glycosylation, phosphorylation, lipidation (e.g., myristoylation, etc.), acetylation, ubiquitylation, sulfation, ADP ribosylation, hydroxylation, Cys / Met oxidation, carboxylation, methylation, etc.. The terms "polypeptide" and "protein" are used interchangeably in the context of the present invention.

[0157] As used herein, "mRNA encoding" in particular means that the mRNA is capable, if present in the appropriate environment, preferably within a cell, to be expressed to produce the protein or peptide it encodes.

[0158] Most preferably, the RNA according to the present invention is an mRNA encoding an antigen.

[0159] According to one preferred aspect, the RNA according to the present invention is an mRNA encoding an antigenic peptide or antigenic polypeptide.

[0160] An “antigenic peptide or antigenic polypeptide”, as mentioned herein, in particular refers to a peptide or polypeptide capable of eliciting, producing, or generating an immune response in a host, in particular in an animal. In the context of the present invention it is understood that the term “antigenic” is equivalent to “immunogenic”.

[0161] According to a another preferred aspect, the RNA according to the present invention is an mRNA encoding a reporter protein. As used herein the term “reporter protein” in particular refers to a protein encoded by a reporter gene. The term “reporter gene” refers to any gene that expresses a detectable gene product, in particular to any gene whose expression can be measured, such as the expression of a detectable fluorescent protein or enzyme. In one embodiment of this aspect, the RNA according to the present invention is an mRNA encoding a fluorescent protein, in particular selected from the group consisting of mCherry, GFP (green fluorescent protein), and derivatives of said proteins. Exemplarily, the RNA according to the present invention is an 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%, 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. Said mRNA encoding an mCherry derivative 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 in particular 100% sequence identity with the sequence of SEQ ID NO:2.

[0162] According to a particular preferred aspect, the RNA according to the present invention is an mRNA encoding an enzyme. The term “enzyme” refers to a protein having catalytic activity. For example, catalytic activity includes dephosphorylating activity. In one example, the RNA according to the present invention is an mRNA encoding a hydrolase, such as an alkaline phosphatase.

[0163] In one embodiment of this aspect, the RNA according to the present invention is an mRNA encoding a human alkaline phosphatase. Exemplarily, the RNA according to the present invention is an mRNA encoding a secreted embryonic human alkaline phosphatase (SEAP), i.e. a C-terminal truncated variant of human placental alkaline phosphatase (PLAP). Preferably, the RNA according to the present invention is an mRNA encoding a SEAP comprising or consisting 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:4. The sequence of Seq ID NO:4 can be found under GenBank accession number AAB64400.1. Said mRNA encoding a SEAP 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 in particular 100% sequence identity with the sequence of SEQ ID NO:5.

[0164] It is understood that the wording “consisting of an amino acid sequence" or "consists of an amino acid sequence", respectively, as used herein, in particular also concerns any cotranslational and / or posttranslational modification or modifications of the amino sequence affected by the cell in which the protein or protein domain is expressed. Thus, the wording “consisting of an amino acid sequence" or "consists of an amino acid sequence", respectively, as described herein, is also directed, unless expressly mentioned otherwise, to the amino acid sequence having one or more modifications effected by the cell in which the protein or protein domain is expressed, in particular modifications of amino acid residues effected in the protein biosynthesis and / or protein processing, preferably selected from the group consisting of glycosylations, phosphorylations, and acetylations.

[0165] Regarding the term “at least 90%”, as mentioned in the context of the present invention, it is understood that said term preferably relates to “at least 91%”, more preferably to “at least 92%”, still more preferably to “at least 93%” or in particular to “at least 94%”. Regarding the term “at least 95%” as mentioned in the context of the present invention, it is understood that said term preferably relates to “at least 96%”, more preferably to “at least 97%”, still more preferably to “at least 98%” or in particular to “at least 99%”.

[0166] Regarding the term “at least 99%” as mentioned in the context of the present invention, it is understood that said term preferably relates to “at least 99.2%”, more preferably to “at least 99.4%”, still more preferably to “at least 99.6%” or in particular to “at least 99.8%”.

[0167] The term “having 100% sequence identity”, as used herein, is understood to be equivalent to the term “being identical”.

[0168] Percent sequence identity has an art recognized meaning and there are a number of methods to measure identity between two polypeptide or polynucleotide sequences. See, e.g., 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, M Stockton Press, New York, (1991). Methods for aligning polynucleotides or polypeptides are codified in computer programs, including the GCG program package (Devereux et al., Nuc. Acids Res. 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul et al., J. Molec. Biol. 215:403 (1990)), and Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711) which uses the local homology algorithm of Smith and Waterman (Adv. App. Math., 2:482-489 (1981)). For example, the computer program ALIGN which employs the FASTA algorithm can be used, with an affine gap search with a gap open penalty of -12 and a gap extension penalty of -2. For purposes of the present invention, nucleotide sequences are aligned using Clustal W method in MegAlign software version 11.1.0 (59), 419 by DNASTAR Inc. using the default multiple alignment parameters set in the program (Gap penalty =15.0, gap length penalty =6.66, delay divergent sequence (%)= 30%, DNA transition weight =0.50 and DNA weight matrix= IUB) and, respectively, protein / amino acid sequences are aligned using Clustal W method in MegAlign software version 11.1.0 (59), 419 by DNASTAR Inc. using the default multiple alignment parameters set in the program (Gonnet series protein weight matrix with Gap penalty =10.0, gap length penalty =0.2, and delay divergent sequence (%)= 30%).

[0169] As used herein, it is in particular understood that the term “sequence identity with the sequence of SEQ ID NO:X” is equivalent to the term “sequence identity with the sequence of SEQ ID NO:X over the length of SEQ ID NO:X” or to the term “sequence identity with the sequence of SEQ ID NO:X over the whole length of SEQ ID NO:X”, respectively. In this context, “X” is any integer selected from 1 to 7 so that “SEQ ID NO:X” represents any of the SEQ ID NOs mentioned herein.

[0170] Pursuant to a preferred aspect, the RNA according to the present invention is an mRNA encoding an antigen of a pathogen. An “antigen of a pathogen”, as mentioned herein, in particular refers to an antigen which is encoded by the genome of a pathogen or derived therefrom. As used herein, the term “pathogen” refers to a biological agent that causes a disease state (e.g., infection, sepsis, etc.) in a host. “Pathogens” include, but are not limited to, viruses, bacteria, archaea, fungi, protozoans, mycoplasma, prions, and parasitic organisms.

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

[0172] In a particular preferred aspect, the RNA according to the present invention is an mRNA encoding a viral antigen.

[0173] As used herein, the term “viral antigen” in particular means an antigen derived from a virus or a component thereof or both of them, said viral antigen being capable of inducing an immune response. Thus, said viral antigen, is in particular a virus protein or an immunogenic fragment thereof. The term “virus protein” encompasses any protein that is derived from a virus. Specific examples include a virus glycoprotein, a virus capsid protein, a virus coat protein, a virus envelope protein, and a virus core protein.

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

[0175] In a particular aspect, the RNA according to the present invention is an mRNA encoding a virus glycoprotein or a virus capsid protein.

[0176] Preferably, the RNA according to the present invention is an mRNA encoding a virus glycoprotein.

[0177] The term "glycoprotein" is herein used in its normal scientific meaning and particularly refers to a protein modified to comprise one or more monosaccharide or oligosaccharide chains ("glycans") covalently bonded to the protein. A glycan may be attached to a hydroxyl group on the protein (O-linked-glycan), e.g. to the hydroxyl group of serine, threonine, tyrosine, hydroxylysine or hydroxyproline, or to an amide function on the protein (N- glycoprotein), e.g. asparagine or arginine, or to a carbon on the protein (C-glycoprotein), e.g. tryptophan. A glycoprotein may comprise more than one glycan, may comprise a combination of one or more monosaccharide and one or more oligosaccharide glycans, and may comprise a combination of N-linked, O-linked and C-linked glycans. A “virus glycoprotein”, as referred to herein, in particular means a glycoprotein encoded by the genome of a virus or being derived therefrom.

[0178] In one preferred example the virus referred to herein is an influenza virus. Thus, in the context of the present invention, the viral antigen mentioned herein is exemplarily an influenza virus antigen, and the herein mentioned virus glycoprotein is preferably an influenza virus glycoprotein.

[0179] Specifically, the virus referred to herein is preferably an influenza A virus. The term “influenza A virus” is understood to be in particular equivalent to “influenza virus type A” or “influenza virus A”, as frequently used in the context of influenza virus, wherein “A” denotes the antigenic type of the virus (based on the antigenic specificity of the nucleoprotein). Thus, in the context of the present invention, the viral antigen mentioned herein is exemplarily an influenza A virus antigen, and the herein mentioned virus glycoprotein is preferably an influenza A virus glycoprotein.

[0180] More specifically, the virus referred to herein is preferably a swine influenza virus. Thus, in the context of the present invention, the viral antigen mentioned herein is exemplarily a swine influenza virus antigen, and the herein mentioned virus glycoprotein is preferably a swine influenza virus glycoprotein.

[0181] Still more specifically, the virus referred to herein is preferably a swine influenza A virus. Thus, in the context of the present invention, the viral antigen mentioned herein is exemplarily a swine influenza A virus (SIAV) antigen, and the herein mentioned virus glycoprotein is preferably a SIAV glycoprotein. The term “swine influenza A virus” and “SIAV”, respectively, as used herein, is understood to be interchangeable with “swine influenza virus A” and “swine influenza virus type A”, respectively, wherein “A” denotes the antigenic type of the influenza virus.

[0182] For instance, the term “swine influenza virus A antigen” or “SIAV antigen”, respectively, in particular means a peptide or polypeptide derived from a Swine Influenza A Virus (SIAV) or a component thereof or both of them, said peptide or polypeptide being capable of inducing an immune response. Thus, in one example, the RNA according to the present invention is an mRNA encoding a SIAV antigen. Or, more particular, the RNA according to the present invention is an mRNA encoding, e.g., a swine influenza A virus (SIAV) glycoprotein or an immunogenic fragment thereof. Hence, in one example, said mRNA encodes a SIAV glycoprotein.

[0183] Even more specifically, the virus referred to herein is preferably a swine influenza A virus subtype H3N2. Thus, in the context of the present invention, the viral antigen mentioned herein is exemplarily a swine influenza virus A subtype H3N2 antigen, and the herein mentioned virus glycoprotein is preferably a swine influenza virus A subtype H3N2 glycoprotein.

[0184] The antigenic type (e.g. “A”, as expressed in the terms “influenza A virus” and “influenza virus type A”, respectively), subtype (e.g. “H3N2”), and antigenic character of the hemagglutinin (e.g. “H3”), of an influenza virus, as used herein, in particular relate to the internationally established system of nomenclature for influenza viruses published in the Bulletin of the World Health Organization, 58(4):585-591 (1980).

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

[0186] In particular, the RNA according to the present invention is an mRNA encoding a hemagglutinin 3 (H3). According to another preferred aspect, the RNA according to the present invention is an mRNA encoding an immunogenic fragment thereof, i.e. an immunogenic fragment of a H3. The term “hemagglutinin 3” and “H3”, respectively, as used herein, is understood to be equivalent to “hemagglutinin 3 protein” and “H3 protein”, respectively.

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

[0188] The term “immunogenic fragment”, as mentioned herein, is in particular understood to refer to a fragment of a protein, which at least partially retains the immunogenicity of the protein from which it is derived. Thus, for instance an “immunogenic fragment of a H3 of SIAV” is particularly understood to refer to a fragment of a H3 of SIAV, which at least partially retains the immunogenicity of the full length H3 protein.

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

[0190] Said mRNA encoding a 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 in particular 100% sequence identity with the sequence of SEQ ID NO:7.

[0191] According to one further preferred aspect of the present invention, the herein mentioned mRNA is a synthetic mRNA.

[0192] By “synthetic mRNA” is meant an mRNA that is produced outside of a cell or that is produced inside of a cell using bioengineering, by way of non-limiting example, an mRNA that is produced in an in vitro-transcription reaction, an mRNA that is produced by direct chemical synthesis or an mRNA that is produced in a genetically-engineered E. coli cell.

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

[0194] The term “nucleoside-modified RNA” refers to an mRNA containing one or more nucleosides modified as compared to the equivalent RNA nucleoside by the introduction of one or more modifications of the sugar moiety or the (nucleo)base moiety. In a preferred embodiment the modified nucleoside comprise a modified sugar moiety. In one example, a nucleoside-modified mRNA is a mRNA in which at least one uridine is replaced by pseudouridine.

[0195] Pursuant to a further preferred aspect, the mRNA according to the present invention is at least 300 nucleotides in length, preferably at least 500 nucleotides in length, more preferably at least 1000 nucleotides in length, and most preferably at least 1500 nucleotides in length.

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

[0197] The present invention further provides the composition of the present invention for use as a medicament, preferably as a vaccine, or for use in a diagnostic method practised on the body.

[0198] In still another aspect, the invention also relates to the use of the composition of the present invention in the preparation of a medicament, preferably of a vaccine, or in the preparation of a diagnostic agent. The term "vaccine", as used herein refers, in particular refers to a pharmaceutical composition comprising at least one immunologically active component that, when administered to an animal, induces an immune response in the animal.

[0199] Furthermore, the present invention provides the composition of the present invention for use in a method for delivering the RNA according to the present invention into a subject, wherein said method comprises applying the composition of the present invention topically to said subject.

[0200] In still another aspect, the present invention relates to the composition of the present invention for use in a method for delivering the RNA according to the present invention through the skin into a subject, wherein said method comprises applying the composition of the present invention to the skin of said subject.

[0201] The present invention furthermore provides a method for delivering a ribonucleic acid (RNA) into a subject, comprising the step of topically applying the composition of the present invention to said subject.

[0202] Also, the present invention provides a method for delivering a ribonucleic acid (RNA) through the skin into a subject, comprising the step of applying the composition of the present invention to the skin of said subject.

[0203] In each of said methods for delivering a ribonucleic acid (RNA) preferably the aqueous phase of the composition of the present contains the RNA to be delivered to said subject. Thus, in said methods for delivering a ribonucleic acid (RNA), the RNA to be delivered to said subject is preferably the RNA according to the present invention.

[0204] In any of the medical uses and methods for delivering, as described herein in the context of the present invention, it is particularly preferred that the composition of the present invention is topically applied to the surface of an ear of the subject. Most preferably, the composition of the present invention is topically applied to the inner pinna of the subject. Thereby, a particularly easy administration of RNA to a subject is enabled.

[0205] The present invention further provides the composition of the present invention for use in a method for inducing an immune response in a subject. Said immune response is in particular an immune response against an antigen encoded by the mRNA according to the present invention. The herein-described “immune response” can be a cellular and / or antibody-mediated immune response, in particular against antigen(s) encoded by the mRNA according to the present invention and / or against pathogen(s) from which said antigen(s) is (or are) derived.

[0206] Usually, an "immune response" includes but is not limited to one or more of the following effects: the production or activation of antibodies, B cells, helper T cells, suppressor T cells, and / or cytotoxic T cells and / or gamma-delta T cells, directed specifically to an antigen or antigens encoded by the RNA according to included in the immunogenic composition according to the present invention. Preferably, the subject to which the composition of the present invention, which includes mRNA according to the present invention encoding an antigen, will be administered will display either a protective immune response or a therapeutic response.

[0207] A "protective immune response" will be demonstrated by either a reduction or lack of one or more clinical signs normally displayed by an infected host, a quicker recovery time and / or a lowered duration of infectivity or lowered pathogen titer in the tissues or body fluids or excretions of the infected host.

[0208] The present invention in particular provides the composition of the present invention for use in a method for inducing an immune response in a subject, wherein said immune response is an immune response against an antigen encoded by the mRNA according to the present invention.

[0209] The present invention further provides the composition of the present invention for use in a method for inducing an immune response in a subject, wherein said immune response is preferably an immune response against a pathogen.

[0210] In particular, the composition of the present invention is for use in a method for inducing an immune response against a pathogen selected from the group consisting of a virus, bacteria and parasite.

[0211] In one aspect, the composition of the present invention is for use in a method for inducing an immune response against a virus in a subject.

[0212] In one aspect, the composition of the present invention is for use in a method for inducing an immune response against an influenza virus in a subject. In one aspect, the composition of the present invention is for use in a method for inducing an immune response against a swine influenza virus in a subject.

[0213] In one aspect, the composition of the present invention is for use in a method for inducing an immune response against a swine influenza A virus (SIAV) in a subject.

[0214] In one aspect, the composition of the present invention is for use in a method for inducing an immune response against an influenza virus comprising H3.

[0215] In one aspect, the composition of the present invention is for use in a method for inducing an immune response against an H3N2 influenza virus in a subject.

[0216] In one aspect, the composition of the present invention is for use in a method for inducing an immune response against a SIAV subtype H3N2 in a subject.

[0217] Pursuant to another preferred aspect, the herein mentioned immune response is a mucosal immune response.

[0218] Preferably, any of the above mentioned medical uses and methods comprises administering one or more doses of the composition of the present invention to said subject.

[0219] Preferably, any of the above mentioned medical uses and methods comprises administering two doses of the composition of the present invention to said subject.

[0220] Preferably, one dose of the composition of the present invention has a volume of about 0.01 ml to 1 ml.

[0221] Preferably, one dose of the composition of the present invention has a volume of about 0.05 ml to 0.5 ml.

[0222] Preferably, one dose of the composition of the present invention has a volume about 0.1 ml to 0.25 ml.

[0223] The subject, as mentioned herein in the context of the present invention, is preferably a mammal.

[0224] Preferably, said subject is an animal.

[0225] Preferably, said subject is a swine, in particular a piglet. The invention also provides a method of producing the composition of the present invention, comprising the step(s) of mixing:

[0226] - an oil phase,

[0227] - a first surfactant,

[0228] - a second surfactant, and

[0229] - an aqueous phase containing an RNA in a container.

[0230] The invention further provides a method of producing the composition of the present invention, comprising the steps of

[0231] (a) mixing an oil phase, a first surfactant, and a second surfactant, and

[0232] (b) adding an aqueous phase containing an RNA to the mixture obtained in step (a).

[0233] Preferably, in said methods of producing the composition of the present invention

[0234] - said oil phase is the oil phase according to the present invention,

[0235] - said first surfactant is the first surfactant according to the present invention,

[0236] - said second surfactant is the second surfactant according to the present invention, and

[0237] - said aqueous phase is the aqueous phase according to the present invention.

[0238] More specifically, with reference to the clauses disclosed further below, in said methods of producing the composition of the present invention

[0239] - said oil phase is the oil phase as specified in any one of clauses 8, 9, 17, 18, 35 to 49, 85, 86, and 91 ,

[0240] - said first surfactant is the first surfactant as specified in any one of clauses 10 to 12, 17 to 25, 37, 38, 53 to 57, 87, 88, and 91 ,

[0241] - said second surfactant is the second surfactant as specified in any one of clauses 13 to 18, 26 to 30, 37, 38, 58 to 61 , 89 to 91 , and / or

[0242] - said aqueous phase is the aqueous phase as specified in any one of clauses 6, 7, 17, 18, 31 to 33, 37, 38, 51 , 52, 82 to 84, and 91 , and wherein preferably said RNA is the RNA as specified in any one of clauses 92 to 155. Furthermore, in the context of the present invention, a hemagglutinin 3 (H3) is provided, wherein said H3 comprises or consists of an amino acid sequence having at least 90% sequence identity with the sequence of SEQ ID NO:6, and wherein said H3 is also termed the “H3 related to the present invention” hereinafter.

[0243] In one aspect, the H3 related to the present invention is a H3 comprising an amino acid sequence having at least 90% sequence identity with the sequence of SEQ ID NO:6.

[0244] In another aspect, the H3 related to the present invention is a H3 consisting of an amino acid sequence having at least 90% sequence identity with the sequence of SEQ ID NO:6.

[0245] In a preferred aspect, the H3 related to the present invention has at amino acid position 295 a tyrosine residue.

[0246] In another preferred aspect, the H3 related to the present invention has at amino acid position 176 an alanine residue.

[0247] In a further preferred aspect, the H3 related to the present invention has at amino acid position 188 an asparagine residue.

[0248] More preferably, the H3 related to the present invention has at amino acid position 176 an alanine residue at amino acid position 188 an asparagine residue, and at amino acid position 295 a tyrosine residue.

[0249] As described herein, the numbering of amino acid positions refers to the amino acid sequence of full length wild type H3 protein (SEQ ID NO:6). Hence, the numbering of the amino positions as mentioned herein is with reference to a wild type H3 sequence having 566 amino acid residues, including a methionine residue at the (N-terminal) amino acid position 1.

[0250] Thus, the phrase "wherein the numbering of the amino acid positions refers to the amino acid sequence of wild type H3", as used herein, relates to the sequence of a naturally occurring H3, as exemplarily set forth in SEQ ID NO:6. According to one preferred aspect, the H3 related to the present invention has at amino acid position 180 a glutamine residue.

[0251] According to another preferred aspect, the H3 related to the present invention has at amino acid position 21 an arginine residue.

[0252] According to a further preferred aspect, the H3 related to the present invention has at amino acid position 291 an aspartate residue.

[0253] According to yet another preferred aspect, the H3 related to the present invention has at amino acid position 69 a lysine residue.

[0254] According to yet a further preferred aspect, the H3 related to the present invention has at amino acid position 214 a valine residue.

[0255] In one preferred aspect, the H3 related to the present invention has at amino acid position 176 an alanine residue.

[0256] In another preferred aspect, the H3 related to the present invention has at amino acid position 188 an asparagine residue.

[0257] In a further preferred aspect, the H3 related to the present invention has at amino acid position 140 an aspartate residue.

[0258] In yet another preferred aspect, the H3 related to the present invention has at amino acid position 208 a threonine residue.

[0259] In yet a further preferred aspect, the H3 related to the present invention has at amino acid position 10 a valine residue.

[0260] Preferably, the H3 related to the present invention has at amino acid position 19 a phenylalanine residue.

[0261] In another aspect, the H3 related to the present invention has at amino acid position 323 a histidine residue.

[0262] In a further aspect, the H3 related to the present invention has at amino acid position 546 an isoleucine residue. In yet another aspect, the H3 related to the present invention has at amino acid position 552 an isoleucine residue.

[0263] Preferably, the H3 related to the present invention is a H3 of a swine influenza A virus (SIAV).

[0264] Preferably, the H3 related to the present invention is a H3 of a swine influenza A virus (SIAV) subtype H3N2.

[0265] According to a preferred aspect, the H3 related to 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.

[0266] According to another preferred aspect, the H3 related to the present invention comprises or consists of an amino acid sequence having 99% sequence identity with the sequence of SEQ ID NO:6.

[0267] According to a further preferred aspect, the H3 related to 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 in particular 100% sequence identity with the sequence of SEQ ID NO:6.

[0268] Further, an immunogenic composition is provided which comprises the H3 related to the present invention

[0269] Moreover, a polynucleotide is provided which comprises a sequence encoding the H3 related to the present invention, and furthermore a vector comprising said polynucleotide, or a plasmid comprising the polynucleotide.

[0270] Also, an mRNA is provided which comprises a sequence encoding the H3 related to the present invention.

[0271] Further, in the context of the H3 related to the present invention, preferably said H3 said immunogenic composition, said polynucleotide, said mRNA, said vector, and / or said plasmid is used for the preparation of a medicament, in particular of a vaccine.

[0272] Furthermore, in the context of the H3 related to the present invention, a method for inducing an immune response against a virus in a subject is provided, wherein the method comprises administering said H3 said immunogenic composition, said polynucleotide, said mRNA, said vector, and / or said plasmid to the subject, and wherein said immune response against a virus is preferably an immune response against an influenza virus, a swine influenza virus, a swine influenza A virus (SIAV) virus, an influenza virus comprising H3, an H3N2 influenza virus, and / or a SIAV subtype H3N2. Also, an isolated influenza virus is provided which comprises the H3 related to the present invention and / or whose genome encodes the H3 related to the present invention, and wherein said influenza virus is preferably a swine influenza A virus (SIAV) virus, and / or

[0273] - a SIAV subtype H3N2.

[0274] In this context, "isolated" means altered "by the hand of man" from its natural state, i.e., if it occurs in nature, it has been changed or removed from its original environment, or both. For example, a virus naturally present in a living organism is not "isolated", but the same virus separated from the coexisting materials of its natural state is "isolated", as the term is employed herein.

[0275] EXAMPLES

[0276] The following examples are only intended to illustrate the present disclosure. They shall not limit the scope of the claims in any way.

[0277] Example 1

[0278] Production of a test emulsion with mCherry derivative encoding mRNA at a final concentration of 1 pg / ml.

[0279] This test composition was produced, as described in the following protocol:

[0280] (a) under laminar flow in a 100 ml beaker, under agitation with a magnetic stirrer, add 21.81 g of the surfactant Labrasol® (Gattefosse SA, Saint-Priest, France), then 17.88 g of Polyglyceryl-3 dioleate (Plurol® Oleique CC 497 (Gattefosse SA, Saint-Priest, France)), then 7.97 g of mineral oil (Marcol® 52 (Exxon, USA));

[0281] (b) prepare separately the aqueous phase containing mRNA: this aqueous phase is prepared by the addition of 30pl of mRNA (SEQ ID NO:3 (i.e. an mRNA comprising a 5'-UTR, a coding region encoding the mCherry derivative of SEQ ID NO:1, a Kozak sequence (around the start codon of said coding region), a 3'-UTR, and a poly(A) tail) without a 5'-cap) at a concentration of 155.4 pg / ml in 870 pl of RNAse free water, resulting in the aqueous phase containing the mRNA in a concentration of 5.18 pg / ml; (c) in a 5 ml glass vial, to 3.347 ml of the mixture obtained in step (a) above, add 0.8 ml of the aqueous phase (containing mRNA at 5.18 pg / ml) obtained in step (b) above. The magnetic stirring is continued 5 minutes at room temperature. The resulting emulsion is opalescent.

[0282] This emulsion was further tested as described in Example 4.

[0283] Example 2

[0284] Formulation of an emulsion which comprises mRNA encoding a reporter protein (human secreted embryonic alkaline phosphatase (SEAP)), topical administration of this formulation to, and subsequent expression of the reporter protein in, piglets.

[0285] The composition to be tested was produced, as described in the following protocol:

[0286] Under laminar flow in a 10 ml glass vial, under agitation with a magnetic stirrer, add 1.372 ml of the surfactant Labrasol® (Gattefosse SA, Saint-Priest, France) which consists of a small fraction of mono-, di- and triglycerides and mainly PEG-8 (MW 400) mono- and diesters of caprylic (Cs) and capric (C ) acids, then 1.126 ml of Polyglyceryl-3 dioleate (Plurol® Oleique CO 497 (Gattefosse SA, Saint-Priest, France)), then 0.502 ml of mineral oil (Marcol® 52 (Exxon, USA)), then 0.717 ml of the aqueous phase (1 mM citrate buffer pH 6.4) containing mRNA encoding SEAP (SEQ ID NO:4) at a concentration of 1 mg / ml, said mRNA having a structure comparable to the mRNA of Example 1 , but having a coding region (SEQ ID NO:5) encoding SEAP instead of the mCherry derivative, and having a 5 '-cap with the cap1 structure m7GpppAm (producable by using the CleanCap® AG reagent (TriLink Biotechnologies, San Diego (CA), USA, product no. N-7113) according to manufacturer's instructions). The magnetic stirring is continued for 5 minutes at room temperature. The resulting emulsion is opalescent.

[0287] Subsequently a study was conducted in order to test if topically administering the composition to be tested to animals would allow the effective delivery of the RNA contained in it through the skin into the animals. In particular, the aim of this animal study was to evaluate (i) the safety of such topical administration to, and (ii) the efficiency of the production of a protein of interest resulting from such topical administration in, piglets. In this study, specific pathogen free (SPF) piglets being approximately 10 weeks of age on study day 0 (DO) received, on DO, either 0.25 ml of a reference product (corresponding to, and produced as, the composition to be tested but not containing RNA) (Group 1 (G1)), or 0.10 ml (Group 2 (G2)) or 0.25 ml (Group 3 (G3)) of the composition to be tested (also termed “product” or ’’products”, respectively, in the following) on the inner face of the ear flap (i.e. the inner pinna) of their right ear. For this, the reference product and the composition to be tested, respectively, were applied by means of a 1 ml syringe without needle, thereby spreading it evenly over the respective skin surface of the ear by a circular massage. Each of the tested groups (G1 , G2, and G3) included five piglets.

[0288] Blood was collected on D1 and on D2 (i.e. one day and two days after said auricular administration), and ear tissues on D2, and the collected samples were evaluated by SEAP enzymatic activity quantification as described below. An overview of the experimental design of the study is provided in Table 1.

[0289] Table 1 Experimental design

[0290] SEAP enzymatic activity, resulting from the cellular intake and translation of the mRNA, delivered by topical application, in piglet cells, was monitored in serum and after extraction from the ears tissues, respectively.

[0291] Briefly, whole blood was collected in Heparine-Sodium collection tubes and centrifuged 10 minutes at 1 ,100 g at 20°C. Sera were collected and immediately tested / quantified for SEAP enzymatic activity as described below.. Weighted piece of ears, sampled in the aera of product application, were suspended in PBS (PBS w / o Ca2+ and Mg2+) and dissociated using gentleMACS™ M Tubes in combination with the gentleMACS™ Dissociator (Miltenyi Biotec) using the preloaded program m_heart_01. Homogenized samples were centrifuged 10 minutes at 1 ,100 g at 20°C. Supernatants were collected and immediately tested / quantified for SEAP enzymatic activity as described below. Measured enzymatic activity from each ear sample was standardized according to its weight.

[0292] SEAP enzymatic activity was quantified by using a commercial kit (NovaBright™ PhosphaLight™ EXP Assay Kit for SEAP (Secreted Placental Alkaline Phosphatase) Reporter Gene Detection, Invitrogen) according to the manufacturer's instructions. This kit / assay applies the end-point method using the chloro- 5-substituted adamantyl-1 ,2-dioxetane phosphate (CSPD) as substrate. The chemiluminescent substrate CSPD is dephosphorylated by secreted embryonic alkaline phosphatase (SEAP), resulting in an unstable dioxetane anion that decomposes and emits light. The light emission has maximal activity at a wavelength of 477 nm.

[0293] To measure the light emission (from a 96- well microplate (96 Well White Plate, Thermo Scientific)) , an EnVision plate reader (Perkin-Elmer), set for luminescence measurement, was used.

[0294] Results of this study are presented in Fig. 1 (“SEAP concentration per g of ears”, showing the SEAP activity on study day 2 in supernatant (per 1 gram of processed ear tissue) as measured by chemiluminescence), and Fig. 2 (“SEAP concentration in serum”, showing the SEAP activity on study day 1 and study day 2 as measured by chemiluminescence).

[0295] In conclusion, two days after application, mRNA encoding SEAP, formulated in a microemulsion as described herein, was efficiently uptaken by the porcine cells and translated into active SEAP. Secreted enzyme was detected not only in the local tissues but also in the systemic circulation two days after application. Considering the size and the weight of a 10 week old piglet (approx. 20 kg), this demonstrates a surprisingly high production rate of the protein of interest.

[0296] Furthermore, no visible adverse events (i.e. Fever, redness, swelling) were noticed after applying the products to the piglets. Example 3

[0297] Formulation of an emulsion which comprises mRNA encoding an antigen (hemagglutinin 3 (H3) of Swine Influenza A Virus (SIAV) H3N2), topical administration of this formulation to, and subsequent induction of immunity in, piglets.

[0298] The composition to be tested was produced, as described in the following protocol:

[0299] Under laminar flow in a 5 ml glass vial, under agitation with a magnetic stirrer, add 686 l of the surfactant Labrasol® (Gattefosse SA, Saint-Priest, France), then 563 pl ml of Polyglyceryl-3 dioleate (Plurol® Oleique CC 497 (Gattefosse SA, Saint-Priest, France)), then 251 pl of mineral oil (Marcol® 52 (Exxon, USA)), then 359 pl of the aqueous phase (1 mM citrate buffer pH 6.4) containing mRNA encoding H3 of SIAV (SEQ ID NO:6) at a concentration of 1 mg / ml, said mRNA having a structure comparable to the mRNA of Example 1 , but having a coding region (SEQ ID NO:7) encoding H3 of SIAV instead of the mCherry derivative, and having a 5'-cap with the cap1 structure m7GpppAm (producible by using the CleanCap® AG reagent (TriLink Biotechnologies, San Diego (CA), USA, product no. N-7113) according to manufacturer's instructions). The magnetic stirring is continued 5 minutes at room temperature. The resulting emulsion is opalescent.

[0300] An animal study was conducted in order to test if topically administering the composition to be tested to animals would allow to induce an immune response in the animals against the antigen encoded by the RNA contained in said composition.

[0301] In this study, conventional pigs being approximately 7 weeks of age on study day 0 (DO) received, on DO and on study day 21 (D21), either 0.25 ml of a reference product (corresponding to, and produced as, the composition to be tested but not containing RNA) (Group 1 (G1)), or 0.2 ml of the composition to be tested (Group 2 (G2)), on the inner face of the ear flap of their both ears (100 pl each). For this, the reference product and the composition to be tested, respectively, were applied by means of a 1ml syringe without needle, thereby spreading it evenly over the respective skin surface of the ear by a circular massage. Each of the tested groups (G1 and G2) included five piglets.

[0302] Blood was collected twenty-one days after the second application (on study day 42 (D42)).

[0303] In addition, bronco alveolar liquid lavages (BALF) were collected to monitor the mucosal immunity potentially induces. An overview of the experimental design of the study is provided in Table 2. Specific IgG in serum, after blood centrifugation 10 minutes at 1 ,000g, and specific IgA in BALF after centrifugation 10 minutes at 1 ,000g were quantified by ELISA using recombinant H3 ectodomain as target antigen and anti pig-IgG or anti pig-lgA secondary antibodies for the revelation, as described in the following:

[0304] Briefly 120 pl of a solution of a H3 ectodomain (corresponding to amino acid residues 1-529 of SEQ ID NO:6) recombinant protein at 167 pg / ml in carbonate buffer was coated overnight at 4°C and washed. After saturation 1h in 5% in PBS, 1 / 4 dilutions and subsequent 1 / 5 dilutions of sera in T ris Elisa Buffer (TEB) + 1 % milk were applied over 1.5 hours at 37°C. After washing 5 times with Tris buffer + 0.1% Tween 20, 100 pl of HRP labelled secondary antibody (Conjugate goat anti Swine IgG-HRP - BIORAD AAI41 P or Goat anti-swine IgA-HRP - Fortis Life Science A100-102P), diluted according manufacturer instructions, were applied over 1.5 hour at 37°C and plates were revealed using TMB (Symbiotics, France) substrate for 30 minutes at 20°C. The reaction was stopped by the addition of concentrated sulphuric acid and plates were read at 630 / 450 nm with the Bio-Teck Instruments Power Wave 340 plate reader. Titres were expressed as the OD max obtained for the minimal dilution obtained before reaching a plateau of OD. Each groups are compared at the same dilution ratio.

[0305] Table 2 Experimental design

[0306] As shown in Fig. 3, topical application of the composition to be tested, as described, resulted in an increased level of specific anti SIAV H3 IgG antibodies in the serum of tested animals on As shown in Fig. 4, topical application of the composition to be tested, as described, resulted in an increased level of specific anti SIAV H3 IgA antibodies in the BALF of tested animals on D42.

[0307] As result of this study it was found that the topical mRNA-H3 application induced the production of a specific humoral response in piglets.

[0308] Example 4

[0309] Determination of the average droplet size of the aqueous phase dispersed in the oil phase in various microemulsions containing different amounts of mRNA of different natures.

[0310] The average droplet size and polydispersity index were determined using the dynamic lightscattering (DLS) technique on a Nano ZS Zetasizer (ZEN3600, Malvern Instruments Ltd., Enigma Business Park, Grovewood Road, UK) at 20 °C . Average droplet sizes were calculated by using Zetasizer software (Malvern Instruments Ltd.).

[0311] Emulsions, as described below (A)-(D)), were diluted 5-fold in volume in Marcol® 52. Three measurements (studies (A)-(C)) or four measurements (study (D)), respectively, for each sample were performed and the results are expressed as the mean of the three or four measurements, respectively.

[0312] The Rl index of the dispersant was 1.460 and the material Rl 1.33. The viscosity 16.0000 cP. The duration of acquisition was fixed at 60 seconds.

[0313] (A) Results with no mRNA in the aqueous phase

[0314] This test emulsion with no mRNA in the aqueous phase was produced, as described in the following protocol:

[0315] (a) under laminar flow in a 100 ml beaker, under agitation with a magnetic stirrer, add 21.81 g of the surfactant Labrasol® (Gattefosse SA, Saint-Priest, France), then 17.88 g of Polyglyceryl-3 dioleate (Plurol® Oleique CO 497 (Gattefosse SA, Saint-Priest, France)), then 7.97 g of mineral oil (Marcol® 52 (Exxon, USA)); (b) in a 10 ml glass vial, to 3 ml of the mixture obtained in step (a) above, add 0.717 ml of Phosphate Buffer Saline w / o Ca2+ and Mg2+. The magnetic stirring is continued 5 minutes at room temperature. The resulting emulsion is opalescent.

[0316] In this emulsion the average droplet size of the aqueous phase dispersed in the oil phase was 57.33 nm with a Standard deviation (St dev) of 14.56 nm, as measured by DLS (Fig. 5 A). The polydispersity index (PDI) value was 0.173.

[0317] (B) Results with mCherry derivative encoding mRNA at a final concentration of 1 ug / ml or 30 ug / ml in the emulsion

[0318] The 1 pg mRNA / ml target emulsion was produced as described in Example 1.

[0319] The 30 pg mRNA / ml target emulsion was produced, as described in the following protocol:

[0320] (a) under laminar flow in a 100 ml beaker, under agitation with a magnetic stirrer, add 21.81 g of the surfactant Labrasol® (Gattefosse SA, Saint-Priest, France), then 17.88 g of Polyglyceryl-3 dioleate (Plurol® Oleique CC 497 (Gattefosse SA, Saint-Priest, France)), then 7.97 g of mineral oil (Marcol® 52 (Exxon, USA));

[0321] (b) prepare separately an aqueous phase containing mRNA: this aqueous phase is prepared by the addition of 17pl of mRNA (SEQ ID NO:3 (i.e. an mRNA comprising a 5'-UTR, a coding region encoding the mCherry derivative of SEQ ID NO:1, a Kozak sequence (around the start codon of said coding region), a 3'-UTR, and a poly(A) tail) without a 5'-cap) at a concentration of 2 mg / ml in 202 pl of RNAse free water, resulting in the aqueous phase containing the mRNA in a concentration of 155.4 pg / ml;

[0322] (c) in a 5 ml glass vial, to 0.791 ml of the mixture obtained in step (a) above, add 0.189 ml of the aqueous phase (containing mRNA at 155.4 pg / ml) obtained in step (b) above. The magnetic stirring is continued 5 minutes at room temperature. The resulting emulsion is opalescent.

[0323] In the emulsion with the mRNA at a final concentration of 1 pg / ml the average droplet size of the aqueous phase dispersed in the oil phase was 35.44 nm with a St dev of 8.506 nm, as measured by DLS (Fig. 5 B). The PDI value was 0.068. In the emulsion with the mRNA at a final concentration at 30 pg / ml the average droplet size of the aqueous phase dispersed in the oil phase was 32.45 nm with a St dev of 10.23 nm, as measured by DLS (Fig. 5 C). The PDI value was 0.130.

[0324] (C) Results with SEAP encoding mRNA at a final concentration of 200 uq / ml in the emulsion

[0325] The composition to be tested was produced, as described in the following protocol:

[0326] (a) under laminar flow in a 100 ml beaker, under agitation with a magnetic stirrer, add 21.81 g of the surfactant Labrasol® (Gattefosse SA, Saint-Priest, France), then 17.88 g of Polyglyceryl-3 dioleate (Plurol® Oleique CC 497 (Gattefosse SA, Saint-Priest, France)), then 7.97 g of mineral oil (Marcol® 52 (Exxon, USA));

[0327] (b) in a 10 ml glass vial, to 3 ml of the mixture obtained in step (a) above, add 0.717 ml of the aqueous phase described in Example 2 (1 mM citrate buffer pH 6.4 containing mRNA encoding SEAP (SEQ ID NO:4) at a concentration of 1 mg / ml). The magnetic stirring is continued 5 minutes at room temperature. The resulting emulsion is opalescent.

[0328] In this emulsion the average droplet size of the aqueous phase dispersed in the oil phase was 35.74 nm with a St dev of 19.44 nm, as measured by DLS (Fig. 5 D). The PDI value was 0.220.

[0329] (D) Results with SIAV H3 encoding mRNA at a final concentration of 200 uq / ml in the emulsion

[0330] The composition to be tested was produced, as described in the following protocol:

[0331] (a) under laminar flow in a 100 ml beaker, under agitation with a magnetic stirrer, add 21.81 g of the surfactant Labrasol® (Gattefosse SA, Saint-Priest, France), then 17.88 g of Polyglyceryl-3 dioleate (Plurol® Oleique CC 497 (Gattefosse SA, Saint-Priest, France)), then 7.97 g of mineral oil (Marcol® 52 (Exxon, USA));

[0332] (b) in a 10 ml glass vial, to 3 ml of the mixture obtained in step (a) above, add 0.717 ml of the aqueous phase described in Example 3 (1 mM citrate buffer pH 6.4 containing mRNA encoding H3 of SIAV (SEQ ID NO:6) at a concentration of 1 mg / ml). The magnetic stirring is continued 5 minutes at room temperature. The resulting emulsion is opalescent. In this emulsion the average droplet size of the aqueous phase dispersed in the oil phase was 44.28 nm with a St dev. of 11.40 nm, as measured by DLS (Fig. 5 E). The PDI value was 0.106.

[0333] LIST OF FIGURES:

[0334] Fig. 1 : “SEAP concentration per g of ears”, showing the SEAP activity on study day 2 in supernatant (per 1 gram of processed ear tissue) as measured by chemiluminescence.

[0335] Fig. 2 : “SEAP concentration in serum”, showing the SEAP activity on study day 1 and study day 2 as measured by chemiluminescence.

[0336] Fig. 3: Anti-SIAV H3 IgG ELISA response on D42 (21 days after the second application).

[0337] Fig. 4: Anti-SIAV H3 IgA ELISA response in Bronco Alveolar Lavage Fluids (BALF) at D42 (21 days after the second application).

[0338] Fig. 5: (A)-(E) Dynamic light scattering (DLS) spectra showing the droplet size distribution of the aqueous phase dispersed in the oil phase in an emulsion without mRNA in the aqueous phase (Fig. 5 A), emulsion with mCherry derivative encoding mRNA at a final concentration of 1 pg / ml (Fig. 5 B), emulsion with mCherry derivative encoding mRNA at a final concentration of 30 pg / ml (Fig. 5 C), emulsion with SEAP encoding mRNA at a final concentration of 200 pg / ml (Fig. 5 D), emulsion with SIAV H3 encoding mRNA at a final concentration of 200 pg / ml (Fig. 5 E).

[0339] IN THE SEQUENCE LISTING / source and geographical origin (where applicable):

[0340] SEQ ID NO:1 corresponds to an amino acid sequence of an mCherry derivative,

[0341] SEQ ID NO:2 (RNA) corresponds to an mRNA coding region encoding the mCherry derivative of SEQ ID NO: 1 ,

[0342] SEQ ID NO:3 (RNA) corresponds to an exemplary mRNA sequence comprising a 5'-UTR, the mRNA coding region of SEQ ID NO:2, a Kozak sequence (around the start codon of said coding region), a 3'-UTR, and a poly(A) tail, SEQ ID N0:4 corresponds to an amino acid sequence of a secreted embryonic human alkaline phosphatase (SEAP),

[0343] SEQ ID NO:5 (RNA) corresponds to an mRNA coding region encoding the SEAP of SEQ ID NO:4,

[0344] SEQ ID NO:6 corresponds to an amino acid sequence of a hemagglutinin 3 (H3) of Swine Influenza A Virus (SI AV) H3N2 isolate A / swine / Germany / AR452 / 2014 (isolated 2014 from pigs in Germany),

[0345] SEQ ID NO:7 (RNA) corresponds to an mRNA coding region encoding the H3 of SEQ ID NO:6.

[0346] In the RNA sequences set forth in the accompanying st.26 standard sequence listing an uracil nucleotide is represented by the symbol “t”.

[0347] The following clauses are also disclosed herein. Thus, the present disclosure further includes aspects as featured by the following clauses:

[0348] 1. A composition comprising an aqueous phase containing a ribonucleic acid (RNA), and an oil phase.

[0349] 2. The composition of clause 1 , wherein the composition comprises one or more surfactants.

[0350] 3. The composition of any clauses 1 or 2, wherein said composition comprises an aqueous phase containing an RNA, an oil phase, a first surfactant, and a second surfactant.

[0351] 4. The composition of any one of clauses 1 to 3, wherein said composition consists of an aqueous phase containing an RNA, an oil phase, a first surfactant, and a second surfactant.

[0352] 5. The composition of any one of clauses 1 to 4, wherein said composition is a mixture.

[0353] 6. The composition of any one of clauses 1 to 5, wherein the aqueous phase comprises a solvent selected from the group consisting of water, and mixture of water with any other water-miscible solvent.

[0354] 7. The composition of any one of clauses 1 to 6, wherein the aqueous phase comprises water, or wherein the aqueous phase is free of solid nanoparticles or, respectively, is in particular free of lipid nanoparticles (LNPs) and / or lipid-like nanoparticles (LLNs).

[0355] 8. The composition of any one of clauses 1 to 7, wherein said oil phase is selected from the group consisting of mineral oil and vegetable oil.

[0356] 9. The composition of any one of clauses 1 to 8, wherein said oil phase is a mineral oil.

[0357] 10. The composition of any one of clauses 3 to 9, wherein said first surfactant is a nonionic surfactant.

[0358] 11. The composition of any one of clauses 3 to 10, wherein said first surfactant is a water-soluble surfactant.

[0359] 12. The composition of any one of clauses 3 to 11, wherein said first surfactant has a hydrophilic-lipophilic balance (HLB) value of more than 10.

[0360] 13. The composition of any one of clauses 3 to 12, wherein said second surfactant is a co-surfactant.

[0361] 14. The composition of any one of clauses 3 to 13, wherein said second surfactant is a non-ionic surfactant. 15. The composition of any one of clauses 3 to 14, wherein said second surfactant is a water-insoluble surfactant.

[0362] 16. The composition of any one of clauses 3 to 15, wherein said second surfactant has an HLB value of less than 10.

[0363] 17. The composition of any one of clauses 3 to 16, wherein said composition comprises an aqueous phase containing an RNA, wherein said aqueous phase comprises water; an oil phase, wherein said oil phase is a mineral oil; a first surfactant having an HLB value of more than 10; and a second surfactant having an HLB value of less than 10.

[0364] 18. The composition of any one of clauses 3 to 17, wherein said composition consists of an aqueous phase containing an RNA, wherein said aqueous phase comprises water; an oil phase, wherein said oil phase is a mineral oil; a first surfactant having an HLB value of more than 10; and a second surfactant having an HLB value of less than 10.

[0365] 19. The composition of any one of clauses 3 to 18, wherein said first surfactant is selected from the group consisting of polyethylene glycol derivatives, polyethylene glycols, glycerol-based surfactants, polyethylene glycol (PEG) esters.

[0366] 20. The composition of any one of clauses 3 to 19, wherein said first surfactant has an HLB value of 11 to 16.

[0367] 21. The composition of any one of clauses 3 to 20, wherein said first surfactant comprises PEG-8 caprylic / capric glycerides or PEG-6 caprylic / capric glycerides.

[0368] 22. The composition of any one of clauses 3 to 21, wherein said first surfactant has an HLB value of 11 to 13.

[0369] 23. The composition of any one of clauses 3 to 22, wherein said first surfactant comprises PEG-8 caprylic / capric glycerides. 24. The composition of any one of clauses 3 to 23, wherein said first surfactant comprises mono-, di- and / or triglycerides.

[0370] 25. The composition of any one of clauses 3 to 24, wherein said first surfactant is mainly composed of PEG-8 caprylic / capric glycerides.

[0371] 26. The composition of any one of clauses 3 to 25, wherein said second surfactant is a polyglycerol fatty acid ester.

[0372] 27. The composition of any one of clauses 3 to 26, wherein said second surfactant has a HLB value of about 3 to about 8.

[0373] 28. The composition of any one of clauses 3 to 27, wherein said second surfactant is selected from the group consisting of polyglyceryl-3 dioleate and polyglyceryl-6 dioleate.

[0374] 29. The composition of any one of clauses 3 to 28, wherein said second surfactant has a HLB value of about 3 to about 6.

[0375] 30. The composition of any one of clauses 3 to 29, wherein said second surfactant is polyglyceryl-3 dioleate.

[0376] 31. The composition of any one of clauses 1 to 30, wherein said aqueous phase containing an RNA is selected from the group consisting of aqueous buffer solution containing an RNA,

[0377] - water containing an RNA, and mixture of

[0378] • water or

[0379] • an aqueous buffer solution with any other water-miscible solvent, wherein said mixture contains an RNA. The composition of any one of clauses 1 to 31, wherein the aqueous phase comprises an aqueous buffer solution. The composition of any one of clauses 1 to 32, wherein the aqueous phase containing an RNA is an aqueous buffer solution containing an RNA. The composition of any one of clauses 6 to 33, wherein said any other water- miscible solvent is one or more solvents selected from the group consisting of ethanol, ethylene glycol, and glycerin. The composition of any one of clauses 1 to 34, wherein said oil phase is a mineral oil selected from the group consisting of liquid paraffin, and oily liquid that is composed of saturated hydrocarbons derived from petroleum like hexadecane or methyl oleate or ethyl oleate. The composition of any one of clauses 1 to 35, wherein said oil phase is liquid paraffin. The composition of any one of clauses 1 to 36, wherein said composition comprises an aqueous phase containing an RNA, wherein said aqueous phase containing an RNA is an aqueous buffer solution containing an RNA; an oil phase, wherein said oil phase is liquid paraffin; a first surfactant, wherein said first surfactant comprises or is mainly composed of PEG-8 caprylic / capric glycerides; and a second surfactant, wherein said second surfactant is Polyglyceryl-3 dioleate. The composition of any one of clauses 1 to 37, wherein said composition consists of an aqueous phase containing an RNA, wherein said aqueous phase containing an RNA is an aqueous buffer solution containing an RNA; an oil phase, wherein said oil phase is liquid paraffin; a first surfactant, wherein said first surfactant comprises or is mainly composed of PEG-8 caprylic / capric glycerides; and a second surfactant, wherein said second surfactant is Polyglyceryl-3 dioleate. 39. The composition of any one of clauses 1 to 38, wherein said oil phase is a purified mixture of liquid saturated hydrocarbons.

[0380] 40. The composition of any one of clauses 1 to 39, wherein said oil phase is obtained from petroleum through purification by catalytic hydrogenation.

[0381] 41. The composition of any one of clauses 1 to 40, wherein said oil phase comprises from about 67% to about 68% paraffinic carbons.

[0382] 42. The composition of any one of clauses 1 to 41 , wherein said oil phase comprises from about 32% to about 33% naphtenic carbons.

[0383] 43. The composition of any one of clauses 1 to 42, wherein said oil phase has a dynamic viscosity at 20°C of no more than about 100 mPa s, preferably of no more than about 50 mPa s, more preferably of no more than about 30 mPa s, and most preferably of no more than about 20 mPa s.

[0384] 44. The composition of any one of clauses 1 to 43, wherein said oil phase has a dynamic viscosity at 20°C of about 10 mPa s to about 100 mPa s.

[0385] 45. The composition of any one of clauses 1 to 44, wherein said oil phase has a dynamic viscosity at 20°C of about 10 mPa s to about 50 mPa s.

[0386] 46. The composition of any one of clauses 1 to 45, wherein said oil phase has a dynamic viscosity at 20°C of about 10 mPa s to about 30 mPa s.

[0387] 47. The composition of any one of clauses 1 to 46, wherein said oil phase has a dynamic viscosity at 20°C of about 10 mPa s to about 20 mPa s.

[0388] 48. The composition of any one of clauses 1 to 47, wherein said oil phase is the mixture of components included in Marcol 52.

[0389] 49. The composition of any one of clauses 1 to 48, wherein said oil phase is Marcol 52.

[0390] 50. The composition of any one of clauses 31 to 49, wherein said aqueous buffer solution is selected from the group consisting of citrate buffer, PBS buffer and Tris Buffer. 51. The composition of any one of clauses 1 to 50, wherein the aqueous phase containing an RNA is a citrate buffer containing an RNA.

[0391] 52. The composition of any one of clauses 50 or 51 , wherein the aqueous phase containing an RNA is a citrate buffer containing an RNA, and wherein said citrate buffer has a pH value of 6.4 ± 0.2.

[0392] 53. The composition of any one of clauses 3 to 52, wherein said first surfactant comprises or consists of monoesters and diesters of polyethylene glycol with mean relative molecular weight between 200 and 400, and monoesters, diesters, and triesters of glycerol.

[0393] 54. The composition of any one of clauses 3 to 53, wherein said first surfactant comprises or consists of

[0394] PEG-8 (MW 400) mono- and diesters of caprylic (Cs) and capric (Cw) acids, and mono-, di- and triglycerides.

[0395] 55. The composition of any one of clauses 3 to 54, wherein said first surfactant consists of a small fraction of mono-, di- and triglycerides and mainly PEG-8 (MW 400) mono- and diesters of caprylic (Cs) and capric (Cw) acids.

[0396] 56. The composition of any one of clauses 3 to 55, wherein said first surfactant is the mixture of components included in Labrasol.

[0397] 57. The composition of any one of clauses 3 to 56, wherein said first surfactant is Labrasol.

[0398] 58. The composition of any one of clauses 3 to 57, wherein said second surfactant has a HLB value of about 3.

[0399] 59. The composition of any one of clauses 3 to 58, wherein said second surfactant has a HLB value of about 6. 60. The composition of any one of clauses 3 to 59, wherein said second surfactant is the mixture of components included in Plurol Oleique CC 497.

[0400] 61. The composition of any one of clauses 3 to 60, wherein said second surfactant is Plurol Oleique CC 497.

[0401] 62. The composition of any one of clauses 1 to 61, wherein the composition is an emulsion.

[0402] 63. The composition of any one of clauses 1 to 62, wherein the composition is an emulsion in which the aqueous phase is dispersed into the oil phase.

[0403] 64. The composition of any one of clauses 1 to 63, wherein the composition is a water-in- oil emulsion.

[0404] 65. The composition of any one of clauses 62 to 64, wherein said emulsion is an emulsion selected from the group consisting of microemulsion and nanoemulsion.

[0405] 66. The composition of any one of clauses 1 to 65, wherein said 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 between about 1 and about 500 nm.

[0406] 67. The 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 between about 1 and about 350 nm.

[0407] 68. The 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 between about 5 and about 500 nm.

[0408] 69. The 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 in the range of about between about 5 and about 350 nm.

[0409] 70. The 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 diameters in the range of about between about 5 and about 200 nm. 71. The 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.

[0410] 72. The 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.

[0411] 73. The 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.

[0412] 74. The 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.

[0413] 75. The 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.

[0414] 76. The composition of any one of clauses 62 to 75, wherein said emulsion is a microemulsion.

[0415] 77. The composition of any one of clauses 62 to 76, wherein said emulsion is thermodynamically stable.

[0416] 78. The composition of any one of clauses 62 to 77, wherein said emulsion is isotropic.

[0417] 79. The composition of any one of clauses 62 to 78, wherein said emulsion is transparent.

[0418] 80. The composition of any one of clauses 62 to 79, wherein said emulsion is opalescent.

[0419] 81. The composition of any one of clauses 1 to 80, wherein said emulsion is a water-in-oil microemulsion. 82. The composition of any one of clauses 1 to 81, wherein in said composition said aqueous phase containing an RNA is in a final concentration of about 5% to 30% v / v.

[0420] 83. The composition of any one of clauses 1 to 82, wherein in the composition said aqueous phase is in a final concentration of about 14% to 24% v / v.

[0421] 84. The composition of any one of clauses 1 to 83, wherein in the composition the aqueous phase is in a final concentration of about 14.3% to 24.3% v / v.

[0422] 85. The composition of any one of clauses 1 to 84, wherein in the composition said oil phase is in a final concentration of about 5% to 30% v / v.

[0423] 86. The composition of any one of clauses 1 to 85, wherein in the composition said oil phase is in a final concentration of about 8.5% to 18.5% v / v.

[0424] 87. The composition of any one of clauses 3 to 86, wherein in the composition said first surfactant is in a final concentration of about 30.7% to 70.7% v / v.

[0425] 88. The composition of any one of clauses 3 to 87, wherein in the composition said first surfactant is in a final concentration of about 31.9% to 41.9% v / v.

[0426] 89. The composition of any one of clauses 3 to 88, wherein in the composition said second surfactant is in a final concentration of about 17.5% to 57.5% v / v.

[0427] 90. The composition of any one of clauses 3 to 89, wherein in the composition said second surfactant is in a final concentration of about 25.2% to 35.2% v / v.

[0428] 91. The composition of any one of clauses 3 to 90, wherein in the composition said aqueous phase containing an RNA is in a final concentration of about

[0429] 19% v / v, said oil phase is in a final concentration of about 14% v / v, said first surfactant is in a final concentration of about 37% v / v, and said second surfactant is in a final concentration of about 30% v / v.

[0430] 92. The composition of any one of clauses 1 to 91 , wherein the final concentration of the RNA in said composition is about 0.1 to 500 pg per ml (pg / ml). 93. The composition of any one of clauses 1 to 92, wherein the final concentration of the RNA in said composition is at least about 1 pg / ml.

[0431] 94. The composition of any one of clauses 1 to 93, wherein the final concentration of the RNA in said composition is at least about 30 pg / ml.

[0432] 95. The composition of any one of clauses 1 to 94, wherein in the composition the RNA is in a final concentration of about 200 pg / ml.

[0433] 96. The composition of any one of any one of clauses 1 to 95, wherein said RNA is selected from the group consisting of messenger RNA (mRNA), small interfering RNA (siRNA) and microRNA (miRNA).

[0434] 97. The composition of any one of clauses 1 to 96, wherein said RNA is an mRNA.

[0435] 98. The composition of any one of clauses 1 to 97, wherein said RNA is an mRNA encoding a peptide or polypeptide selected from the group consisting of antigen, functional protein and reporter protein.

[0436] 99. The composition of any one of clauses 1 to 98, wherein said RNA is an mRNA encoding a reporter protein.

[0437] 100. The composition of any one of clauses 1 to 99, wherein said RNA is an mRNA encoding a fluorescent protein.

[0438] 101. The composition of clause 100, wherein the fluorescent protein is selected from the group consisting of mCherry, GFP (green fluorescent protein), and derivatives of mCherry or GFP.

[0439] 102. The composition of clause 100 or 101 , wherein the fluorescent protein is 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%, still more preferably at least 95% sequence identity with the sequence of SEQ ID NO:1. 103. The composition of any one of clauses 100 to 102, wherein said mRNA encoding an 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 in particular 100% sequence identity with the sequence of SEQ ID NO:2.

[0440] 104. The composition of any one of clauses 100 to 103, wherein said mRNA encoding an 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 in particular 100% sequence identity with the sequence of SEQ ID NO:3.

[0441] 105. The composition of any one of clauses 1 to 98, wherein said RNA is an mRNA encoding an antigen, or wherein said RNA is an mRNA encoding a functional protein.

[0442] 106. The composition of any one of clauses 1 to 98, and 105, wherein said RNA is an mRNA encoding a functional protein.

[0443] 107. The composition of any one of clauses 1 to 98, 105, and 106, wherein said RNA is an mRNA encoding an enzyme.

[0444] 108. The composition of any one of clauses 1 to 98, and 105 to 107, wherein said RNA is an mRNA encoding a secreted embryonic human alkaline phosphatase (SEAP).

[0445] 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%, still more preferably at least 95% sequence identity with the sequence of SEQ ID NO:4.

[0446] 110. The composition of clause 108 or 109, wherein said mRNA encoding a 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 in particular 100% sequence identity with the sequence of SEQ ID NO:5.

[0447] 111. The composition of any one of clauses 1 to 98, and 105, wherein said RNA is an mRNA encoding an antigen. 112. The composition of any one of clauses 1 to 98, 105, and 111, wherein said RNA is an mRNA encoding an antigen of a pathogen.

[0448] 113. The composition of any one of clauses 1 to 98, 105, 111, and 112, wherein said RNA is an mRNA encoding an antigen selected from the group consisting of viral antigen, bacterial antigen, and antigen derived from a parasite.

[0449] 114. The composition of any one of clauses 1 to 98, 105, and 111 to 113, wherein said RNA is an mRNA encoding a viral antigen.

[0450] 115. The composition of clause 113 or 114, wherein said viral antigen is a virus glycoprotein, or wherein said viral antigen is a virus capsid protein.

[0451] 116. The composition of any one of clauses 113 to 115, wherein said viral antigen is a virus glycoprotein.

[0452] 117. The composition of clauses 115 or 116, wherein said virus is an RNA virus.

[0453] 118. The composition of any one of clauses 113 to 117, wherein said viral antigen is an RNA virus antigen.

[0454] 119. The composition of any one of clauses 113 to 118, wherein said viral antigen is an RNA virus glycoprotein.

[0455] 120. The composition of any one of clauses 115 to 119, wherein said virus is an influenza virus.

[0456] 121. The composition of any one of clauses 113 to 120, wherein said viral antigen is an influenza virus antigen.

[0457] 122. The composition of any one of clauses 113 to 121, wherein said viral antigen is an influenza virus glycoprotein.

[0458] 123. The composition of any one of clauses 115 to 122, wherein said virus is a swine influenza virus. 124. The composition of any one of clauses 113 to 123, wherein said viral antigen is a swine influenza virus antigen.

[0459] 125. The composition of any one of clauses 113 to 124, wherein said viral antigen is a swine influenza virus glycoprotein.

[0460] 126. The composition of any one of clauses 115 to 125, wherein said virus is a swine influenza A virus (SIAV).

[0461] 127. The composition of any one of clauses 113 to 126, wherein said viral antigen is a swine influenza A virus (SIAV) antigen.

[0462] 128. The composition of any one of clauses 113 to 127, wherein said viral antigen is a swine influenza A virus (SIAV) glycoprotein.

[0463] 129. The composition of any one of clauses 115 to 128, wherein said virus is a swine influenza A virus (SIAV) subtype H3N2.

[0464] 130. The composition of any one of clauses 113 to 129, wherein said viral antigen is a SIAV subtype H3N2 antigen.

[0465] 131. The composition of any one of clauses 113 to 130, wherein said viral antigen is a SIAV subtype H3N2 glycoprotein.

[0466] 132. The composition of any one of clauses 1 to 131 , wherein said RNA is an mRNA encoding a hemagglutinin or an immunogenic fragment thereof.

[0467] 133. The composition of any one of clauses 1 to 132, wherein said RNA is an mRNA encoding a hemagglutinin.

[0468] 134. The composition of clause 132 or 133, wherein said hemagglutinin is a hemagglutinin 3 (H3). 135. The composition of any one of clauses 132 to 134, wherein said hemagglutinin comprises or consists of an amino acid sequence having at least at least 95%, preferably at least 98%, more preferably at least 99% or in particular 100% sequence identity with the sequence of SEQ ID NO:6.

[0469] 136. The composition of any one of clauses 1 to 98, 105, and 111 to 135, wherein said 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 in particular 100% sequence identity with the sequence of SEQ ID NO:7.

[0470] 137. The composition of any one of clauses 96 to 136, wherein said mRNA is a synthetic mRNA.

[0471] 138. The composition of any one of clauses 96 to 137, wherein said mRNA is a nucleoside-modified mRNA.

[0472] 139. The composition of any one of clauses 96 to 138, wherein said mRNA is at least 300 nucleotides in length.

[0473] 140. The composition of any one of clauses 96 to 139, wherein said mRNA is at least 500 nucleotides in length.

[0474] 141. The composition of any one of clauses 96 to 140, wherein said mRNA is at least 1000 nucleotides in length.

[0475] 142. The composition of any one of clauses 96 to 141, wherein said mRNA is at least 1500 nucleotides in length.

[0476] 143. The composition of any one of clauses 96 to 142, wherein said mRNA is 300 to 5000 nucleotides in length.

[0477] 144. The composition of any one of clauses 96 to 143, wherein said mRNA is 500 to 4000 nucleotides in length. 145. The composition of any one of clauses 96 to 144, wherein said mRNA is 1000 to 3000 nucleotides in length.

[0478] 146. The composition of any one of clauses 96 to 145, wherein said mRNA comprises a 5'-UTR, a coding region encoding a polypeptide, and a 3'-UTR.

[0479] 147. The composition of clause 146, wherein said coding region is flanked by said 5'-UTR and said 3'-UTR.

[0480] 148. The composition of any one of clauses 96 to 147, wherein said mRNA comprises a poly(A) tail, wherein said poly(A) tail is preferably located following said 3'-UTR in 3' direction.

[0481] 149. The composition of any one of clauses 146 to 148, wherein said 3'-UTR is flanked by a poly(A) tail.

[0482] 150. The composition of clause 148 or 149, wherein said poly(A) tail comprises or consists of about 120 A nucleotides.

[0483] 151. The composition of any one of clauses 96 to 150, wherein said mRNA comprises a Kozak sequence.

[0484] 152. The composition of any one of clauses 96 to 151, wherein said mRNA comprises a 5'-cap structure.

[0485] 153. The composition of any one of clauses 96 to 152, wherein said mRNA comprises a cap1 structure.

[0486] 154. The composition of any one of clauses 96 to 153, wherein said mRNA comprises a 5'-cap having the cap1 structure m7GpppNm.

[0487] 155. The composition of any one of clauses 96 to 154, wherein said mRNA comprises a 5'-cap having the cap1 structure m7GpppAm.

[0488] 156. The composition of any one of clauses 1 to 155 for use as a medicament or for use in a diagnostic method practised on the body.

[0489] 157. The composition of any one of clauses 1 to 98, 105 to 107, and 111 to 155 for use as a medicament. 158. The composition of any one of clauses 1 to 98, 105, and 111 to 155 for use as a vaccine.

[0490] 159. The composition of any one of clauses 1 to 110 for use in a diagnostic method practised on the body.

[0491] 160. The composition of any one of clauses 1 to 155 for use in a method for delivering said RNA into a subject, wherein said method comprises applying said composition topically to said subject.

[0492] 161 . The composition of any one of clauses 1 to 155 for use in a method for delivering said RNA through the skin into a subject, wherein said method comprises applying said composition to the skin of said subject.

[0493] 162. The composition of any one of clauses 111 to 155 for use in a method for inducing an immune response in a subject.

[0494] 163. The composition of any one of clauses 111 to 155 for use in a method for inducing an immune response against a pathogen in a subject.

[0495] 164. The composition for use in a method according to clause 163, wherein said pathogen is selected from the group consisting of a virus, bacteria and parasite.

[0496] 165. The composition of any one of clauses 114 to 155 for use in a method for inducing an immune response against a virus in a subject.

[0497] 166. The composition of any one of clauses 120 to 155 for use in a method for inducing an immune response against an influenza virus in a subject.

[0498] 167. The composition of any one of clauses 123 to 155 for use in a method for inducing an immune response against a swine influenza in a subject.

[0499] 168. The composition of any one of clauses 126 to 155 for use in a method for inducing an immune response against a swine influenza A virus (SIAV) virus in a subject.

[0500] 169. The composition of any one of clauses 129 to 155 for use in a method for inducing in a subject an immune response against an influenza virus comprising H3.

[0501] 170. The composition of any one of clauses 129 to 155 for use in a method for inducing an immune response against an H3N2 influenza virus in a subject. 171. The composition of any one of clauses 129 to 155 for use in a method for inducing an immune response against a SIAV subtype H3N2 in a subject.

[0502] 172. The composition for use according to any one of clauses 162 to 171, wherein said immune response is a mucosal immune response.

[0503] 173. Method for delivering a ribonucleic acid (RNA) into a subject, comprising the step of topically applying the composition of any one of clauses 1 to 155 to said subject.

[0504] 174. Method for delivering a ribonucleic acid (RNA) through the skin into a subject, comprising the step of applying the composition of any one of clauses 1 to 155 to the skin of said subject.

[0505] 175. The method of clause 173 or 174, wherein the aqueous phase of the composition of any one of clauses 1 to 155 contains the RNA to be delivered to the subject.

[0506] 176. The method of any one of clauses 173 to 175, wherein said method is the method as specified in any one of clauses 160 to 172.

[0507] 177. The method of any one of clauses 173 to 176, wherein said composition is the composition as specified in any one of clauses 160 to 171.

[0508] 178. Use of the composition of any one of clauses 1 to 155 in the preparation of a medicament or in the preparation of diagnostic agent.

[0509] 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.

[0510] 180. Use of the composition of any one of clauses 1 to 98, 105, and 111 to 155 in the preparation of a vaccine.

[0511] 181. Use of the composition of any one of clauses 1 to 110 in the preparation of a diagnostic agent.

[0512] 182. The use of any one of clauses 178 to 180, wherein said medicament or vaccine is for a method as specified in any one clauses 160 to 177.

[0513] 183. The use of any one of clauses 178 to 182, wherein said composition is the composition as specified in any one of clauses 160 to 171. 184. The composition for use in a method according to any one of clauses 156 to 172, the method according any one of clauses 173 to 177, or the use according to any one of clauses 178 to 183, wherein said method comprises topically administering said composition to said subject.

[0514] 185. The composition for use in a method according to any one of clauses 156 to 172, and 184, the method according any one of clauses 173 to 177, and 184, or the use according to any one of clauses 178 to 184, wherein said composition is topically applied to the surface of an ear of said subject.

[0515] 186. The composition for use in a method according to any one of clauses 156 to 172, and 184 to 185, the method according any one of clauses 173 to 177, and 184 to 185, or the use according to any one of clauses 178 to 185, wherein said composition is topically applied to an inner pinna of the subject.

[0516] 187. The composition for use in a method according to any one of clauses 156 to 172, and 184 to 186, the method according any one of clauses 173 to 177, and 184 to 186, or the use according to any one of clauses 178 to 186, wherein said method comprises administering one or more doses of said composition to said subject.

[0517] 188. The composition for use in a method according to any one of clauses 156 to 172, and 184 to 187, the method according any one of clauses 173 to 177, and 184 to 187, or the use according to any one of clauses 178 to 187, wherein said method comprises administering two doses of said composition to said subject.

[0518] 189. The composition for use in a method according to clause 187 or 188, the method according to clause 187 or 188, or the use according to clause 187 or 188, wherein one dose of said composition has a volume of about 0.01 ml to 1 ml.

[0519] 190. The composition for use in a method according to any one of clauses 187 to 189, the method according to any one of clauses 187 to 189, or the use according to any one of clauses 187 to 189, wherein one dose of said composition has a volume of about 0.05 ml to 0.5 ml.

[0520] 191. The composition for use in a method according to any one of clauses 187 to 190, the method according to any one of clauses 187 to 190, or the use according to any one of clauses 187 to 190, wherein one dose of said composition has a volume of about 0.1 ml to 0.25 ml. 192. The composition for use in a method according to any one of clauses 160 to 172, and 184 to 191 , the method according any one of clauses 173 to 177, and 184 to 191 , or the use according to any one of clauses 178 to 191 , wherein said subject is a mammal.

[0521] 193. The composition for use in a method according to any one of clauses 160 to 172, and 184 to 192, the method according any one of clauses 173 to 177, and 184 to 192, or the use according to any one of clauses 178 to 192, wherein said subject is an animal.

[0522] 194. The composition for use in a method according to any one of clauses 160 to 172, and 184 to 193, the method according any one of clauses 173 to 177, and 184 to 193, or the use according to any one of clauses 178 to 193, wherein said subject is a swine.

[0523] 195. Method of producing the composition of any one of clauses 1 to 155, comprising the steps of

[0524] (a) mixing an oil phase, a first surfactant, and a second surfactant, and

[0525] (b) adding an aqueous phase containing an RNA to the mixture obtained in step (a).

[0526] 196. The method of clause 195, wherein said oil phase is the oil phase as specified in any one of clauses 8, 9, 17, 18, 35 to 49, 85, 86, and 91.

[0527] 197. The method of clause 195 or 196, wherein said first surfactant is the first surfactant as specified in any one of clauses 10 to 12, 17 to 25, 37, 38, 53 to 57, 87, 88, and 91.

[0528] 198. The method of any one of clauses 195 to 197, wherein said second surfactant is the second surfactant as specified in any one of clauses 13 to 18, 26 to 30, 37, 38, 58 to 61 , 89 to 91.

[0529] 199. The method of any one of clauses 195 to 198, wherein said aqueous phase is the aqueous phase as specified in any one of clauses 6, 7, 17, 18, 31 to 33, 37, 38, 51 , 52, 82 to 84, and 91.

[0530] 200. The method of any one of clauses 195 to 199, wherein said RNA is the RNA as specified in any one of clauses 92 to 155.

[0531] 201. A hemagglutinin 3 (H3) comprising an amino acid sequence having at least 90% sequence identity with the sequence of SEQ ID NO:6. 202. A hemagglutinin 3 (H3) consisting of an amino acid sequence having at least 90% sequence identity with the sequence of SEQ ID NO:6.

[0532] 203. The H3 of clause 201 or 202, wherein said H3 has at amino acid position 295 a tyrosine residue.

[0533] 204. The H3 of any one of clauses 201 to 203, wherein said H3 has at amino acid position 176 an alanine residue.

[0534] 205. The H3 of any one of clauses 201 to 204, wherein said H3 has at amino acid position 188 an asparagine residue.

[0535] 206. The H3 of any one of clauses 201 to 205, wherein said H3 has at amino acid position 176 an alanine residue at amino acid position 188 an asparagine residue, and at amino acid position 295 a tyrosine residue.

[0536] 207. The H3 of any one of clauses 201 to 206, wherein said H3 has at amino acid position 18 an asparagine residue.

[0537] 208. The H3 of any one of clauses 201 to 207, wherein said H3 has at amino acid position 180 a glutamine residue.

[0538] 209. The H3 of any one of clauses 201 to 208, wherein said H3 has at amino acid position 21 an arginine residue.

[0539] 210. The H3 of any one of clauses 201 to 209, wherein said H3 has at amino acid position 291 an aspartate residue.

[0540] 211. The H3 of any one of clauses 201 to 210, wherein said H3 has at amino acid position 69 a lysine residue.

[0541] 212. The H3 of any one of clauses 201 to 211 , wherein said H3 has at amino acid position 214 a valine residue.

[0542] 213. The H3 of any one of clauses 201 to 212, wherein said H3 has at amino acid position 140 an aspartate residue. 214. The H3 of any one of clauses 201 to 213, wherein said H3 has at amino acid position 208 a threonine residue.

[0543] 215. The H3 of any one of clauses 201 to 214, wherein said H3 has at amino acid position 10 a valine residue.

[0544] 216. The H3 of any one of clauses 201 to 215, wherein said H3 has at amino acid position 19 a phenylalanine residue.

[0545] 217. The H3 of any one of clauses 201 to 216, wherein said H3 has at amino acid position 323 a histidine residue.

[0546] 218. The H3 of any one of clauses 201 to 217, wherein said H3 has at amino acid position 546 an isoleucine residue.

[0547] 219. The H3 of any one of clauses 201 to 218, wherein said H3 has at amino acid position 552 an isoleucine residue.

[0548] 220. The H3 of any one of clauses 201 to 219, wherein the amino acid position numbering refers to the amino acid sequence of wild type H3.

[0549] 221 . The H3 of any one of clauses 201 to 220, wherein the amino acid position numbering refers to the amino acid sequence of SEQ ID NO:6.

[0550] 222. The H3 of any one of clauses 201 to 221 , wherein said H3 is a H3 of a swine influenza A virus (SIAV).

[0551] 223. The H3 of any one of clauses 201 to 222, wherein said H3 is a H3 of a swine influenza A virus (SIAV) subtype H3N2.

[0552] 224. The H3 of any one of clauses 201 to 223, wherein said amino acid sequence has at least 95% sequence identity with the sequence of SEQ ID NO:6.

[0553] 225. The H3 of any one of clauses 201 to 224, wherein said amino acid sequence has at least 99% sequence identity with the sequence of SEQ ID NO:6.

[0554] 226. The H3 of any one of clauses 201 to 225, wherein said amino acid sequence has at least 99.2% sequence identity with the sequence of SEQ ID NO:6. 227. The H3 of any one of clauses 201 to 226, wherein said amino acid sequence has at least 99.3%, preferably at least 99.5,%, more preferably at least 99.7%, or in particular 100% sequence identity with the sequence of SEQ ID NO:6.

[0555] 228. The H3 of any one of clauses 201 to 227, wherein said amino acid sequence has at least 99.4% sequence identity with the sequence of SEQ ID NO:6.

[0556] 229. The H3 of any one of clauses 201 to 228, wherein said amino acid sequence has at least 99.6% sequence identity with the sequence of SEQ ID NO:6.

[0557] 230. The H3 of any one of clauses 201 to 229, wherein said amino acid sequence has at least 99.8% sequence identity with the sequence of SEQ ID NO:6.

[0558] 231 . An immunogenic composition comprising the H3 of any one of clauses 201 to 230.

[0559] 232. A polynucleotide comprising a sequence which encodes the H3 of any one of clauses 201 to 230.

[0560] 233. An mRNA comprising a sequence which encodes the H3 of any one of clauses 201 to 230.

[0561] 234. A vector comprising the polynucleotide of clause 232.

[0562] 235. A plasmid comprising the polynucleotide of clause 232.

[0563] 236. Use of the H3 of any one of clauses 201 to 230, the immunogenic composition of clause 231 , the polynucleotide of clause 232, the mRNA of clause 233, the vector of clause 234, and / or the plasmid of clause 235 for the preparation of a medicament, preferably of a vaccine. 237. Method for inducing an immune response against a virus in a subject, comprising administering the H3 of any one of clauses 201 to 230, the immunogenic composition of clause 231 , the polynucleotide of clause 232, the mRNA of clause 233, the vector of clause 234, and / or the plasmid of clause 235 to the subject.

[0564] 238. The method of clause 237, wherein said immune response against a virus is an immune response against an influenza virus.

[0565] 239. The method of clause 237 or 238, wherein said immune response against a virus is an immune response against a swine influenza virus.

[0566] 240. The method of any one of clauses 237 to 239, wherein said immune response against a virus is an immune response against a swine influenza A virus (SIAV) virus.

[0567] 241 . The method of any one of clauses 237 to 240, wherein said immune response against a virus is an immune response against an influenza virus comprising H3.

[0568] 242. The method of any one of clauses 237 to 241 , wherein said immune response against a virus is an immune response against an H3N2 influenza virus.

[0569] 243. The method of any one of clauses 237 to 242, wherein said immune response against a virus is an immune response against a SIAV subtype H3N2.

[0570] 244. An isolated influenza virus, whose genome encodes the H3 of any one of clauses 201 to 230.

[0571] 245. An isolated influenza virus, in particular the isolated influenza virus of clause 244, wherein the isolated influenza virus comprises the H3 of any one of clauses 201 to 230. The isolated influenza virus of clause 244 or 245, wherein said influenza virus is a swine influenza A virus (SIAV) virus. The isolated influenza virus of any one of clauses 244 to 246, wherein said influenza virus is a SIAV subtype H3N2.

Claims

CLAIMS:

1. A composition comprising an aqueous phase containing a ribonucleic acid (RNA), and an oil phase, wherein the composition is an emulsion, in which the aqueous phase is dispersed into the oil phase.

2. The composition of claim 1 , wherein said composition comprises or consists of an aqueous phase containing an RNA, an oil phase, a first surfactant, and a second surfactant, and wherein preferably said oil phase is a mineral oil.

3. The composition of claim 1 or 2, wherein said first surfactant is a water-soluble surfactant, and / or has a hydrophilic-lipophilic balance (HLB) value of more than 10, and / or is a non-ionic surfactant.

4. The composition of any one of claims 1 to 3, wherein said second surfactant is a water-insoluble surfactant, and / or has an HLB value of less than 10, and / or is a co-surfactant, and / or is a non-ionic surfactant.

5. The composition of any one of claims 1 to 4, wherein said composition comprises or consists of an aqueous phase containing an RNA, wherein said aqueous phase comprises water; an oil phase, wherein said oil phase is a mineral oil; a first surfactant having an HLB value of more than 10; and a second surfactant having an HLB value of less than 10.

6. The composition of any one of claims 2 to 5, wherein said first surfactant is selected from the group consisting of polyethylene glycol derivatives, polyethylene glycols, glycerol-based surfactants, polyethylene glycol (PEG) esters, and / or has an HLB value of 11 to 16, and / or comprises PEG-8 caprylic / capric glycerides or PEG-6 caprylic / capric glycerides, and / or comprises mono-, di- and / or triglycerides.

7. The composition of any one of claims 2 to 6, wherein said second surfactant is a polyglycerol fatty acid ester, and / or has an HLB value of about 3 to about 8, in particular of about 3 to about 6, and / or is selected from the group consisting of polyglyceryl-3 dioleate and polyglyceryl-6 dioleate.

8. The composition of any one of claims 1 to 7, wherein said aqueous phase containing an RNA is selected from the group consisting of aqueous buffer solution containing an RNA,- water containing an RNA, and mixture of• water or• an aqueous buffer solution with any other water-miscible solvent, wherein said mixture contains an RNA.

9. The composition of any one of claims 1 to 8, wherein said oil phase is a mineral oil selected from the group consisting of liquid paraffin, and oily liquid that is composed of saturated hydrocarbons derived from petroleum like hexadecane or methyl oleate or ethyl oleate.

10. The composition of any one of claims 1 to 9, wherein said composition comprises or consists ofan aqueous phase containing an RNA, wherein said aqueous phase containing an RNA is an aqueous buffer solution containing an RNA; an oil phase, wherein said oil phase is liquid paraffin; a first surfactant, wherein said first surfactant comprises or is mainly composed of PEG-8 caprylic / capric glycerides; and a second surfactant, wherein said second surfactant is Polyglyceryl-3 dioleate.

11. The composition of any one of claims 1 to 10, wherein said oil phase is a purified mixture of liquid saturated hydrocarbons, and / or is obtained from petroleum through purification by catalytic hydrogenation, and / or comprises from about 67% to about 68% paraffinic carbons, and / or comprises from about 32% to about 33% naphtenic carbons, and / or has a dynamic viscosity at 20°C of no more than about 100 mPa s, and / or is the mixture of components included in Marcol 52.

12. The composition of any one of claims 1 to 11, wherein the aqueous phase containing an RNA is a citrate buffer containing an RNA, and wherein preferably said citrate buffer has a pH value of 6.4 ± 0.2.

13. The composition of any one of claims 2 to 12, wherein said first surfactant comprises or consists of- monoesters and diesters of polyethylene glycol with mean relative molecular weight between 200 and 400, and- monoesters, diesters, and triesters of glycerol, and / or comprises or consists of- PEG-8 (MW 400) mono- and diesters of caprylic (Cs) and capric (Cw) acids, and- mono-, di- and triglycerides, and / or consists of a small fraction of mono-, di- and triglycerides and mainly PEG-8 (MW 400) mono- and diesters of caprylic (Cs) and capric (Cw) acids, and / or is the mixture of components included in Labrasol,and / or wherein said second surfactant has a HLB value of about 3 or of about 6, and / or is the mixture of components included in Plurol Oleique CC 497.

14. The composition of any one of claims 1 to 13, wherein the composition is a water-in-oil emulsion, and / or a microemulsion, in which preferably the aqueous phase is dispersed in the oil phase in the form of droplets with diameters in the range of about between about 1 and about 350 nm.

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

16. The composition of any one of claims 1 to 15, wherein said RNA is selected from the group consisting of messenger RNA (mRNA), small interfering RNA (siRNA) and microRNA (miRNA), and / or an mRNA encoding a peptide or polypeptide selected from the group consisting of antigen, functional protein and reporter protein, and / or an mRNA encoding an antigen selected from the group consisting of viral antigen, bacterial antigen, and antigen derived from a parasite.

17. The composition of any one of claims 1 to 16, wherein said RNA is an mRNA encoding a hemagglutinin, in particular a hemagglutinin 3 (H3), and wherein said hemagglutinin preferably comprises or consists of an amino acid sequence having at least at least 95%, preferably at least 98%, more preferably at least 99% or in particular 100% sequence identity with the sequence of SEQ ID NO:6, and / orwherein said 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 in particular 100% sequence identity with the sequence of SEQ ID NO:7.

18. The composition of any one of claims 1 to 17 for use as a medicament, or for use in a diagnostic method practised on the body.

19. The composition of any one of claims 1 to 17 for use in a method for delivering said RNA into a subject, wherein said method comprises applying said composition topically to said subject, and / or a method for delivering said RNA through the skin into a subject, wherein said method comprises applying said composition to the skin of said subject.

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

21. The composition of claim 17 for use in- a method for inducing an immune response against an influenza antigen, in particular against a swine influenza A virus (SIAV) antigen, in a subject, and wherein said influenza antigen is preferably a H3, and / or- a method for inducing an immune response against an influenza virus, in particular against a swine influenza A virus (SIAV), in a subject, and wherein said influenza virus is preferably a H3N2 virus.

22. Method of producing the composition of any one of claims 1 to 17, comprising the steps of(a) mixing an oil phase, a first surfactant, and a second surfactant, and(b) adding an aqueous phase containing an RNA to the mixture obtained in step (a), and wherein preferably said oil phase is the oil phase as specified in any one of claims 2, 5, 9 to 11 , and 15; and / orsaid first surfactant is the first surfactant as specified in any one of claims 3, 5, 6, 10, 13, and 15; and / or said second surfactant is the second surfactant as specified in any one of claims 4, 5, 7, 10, 13 and 15; and / or said aqueous phase is the aqueous phase as specified in any one of claims 5, 8, 10, 12, and 15; and / or said RNA is the RNA as specified in any one of claims 15 to 17.

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