Novel lipid nanoparticles for delivery of nucleic acids containing phosphatidylserine

Abstract

The present invention relates to a vaccine composition comprising a) at least one nucleic acid encoding at least one antigen or a fragment or variant thereof and b) a carrier composition, the carrier composition comprising the phospholipid phosphatidylserine. The present invention further relates to a pharmaceutical composition comprising the vaccine composition and a pharma- ceutically acceptable carrier, diluent or excipient, as well as to a vaccine composition or pharmaceutical composition for use in the treatment or prevention (and corresponding treatment methods) of infectious diseases; cancer or tumor diseases, disorders or conditions; certain liver diseases; allergies; or autoimmune diseases, disorders or conditions in a subject. Still further, the present invention relates to a kit or kit-of-parts comprising the vaccine composition or pharmaceutical composition, as well as a method of inducing an immune response in a subject. Finally, the present invention relates to the use of the vaccine composition or pharmaceutical composition or kit or kit-of-parts for (i) inducing an immune response and (ii) inducing an antigen-specific T cell response in a subject.

Description

[Technical field]

[0001] The present invention relates to a vaccine composition comprising at least one nucleic acid encoding at least one antigen or its fragment or variant and a carrier composition, the carrier composition comprising the phospholipid phosphatidylserine.The at least one antigen or its fragment or variant can be derived from a pathogenic antigen, a tumor antigen, an allergenic antigen or an autoimmune autoantigen, so that the vaccine composition or the pharmaceutical composition comprising the vaccine composition can be used for the treatment and prevention of infectious disease; cancer or tumor disease, disorder or condition; certain liver disease; allergy; or autoimmune disease, disorder or condition in a subject.The present invention also relates to a corresponding kit or kit-of-parts, a corresponding method of inducing immune response in a subject, and its use for inducing immune response and for inducing antigen-specific T cell response in a subject. [Background technology]

[0002] Generally, vaccines can be subdivided into "first", "second" and "third" generation vaccines. "First generation" vaccines are typically whole organism vaccines. They are based on either live attenuated or killed pathogens, e.g., viruses, bacteria, etc. The main drawback of live attenuated vaccines is the risk of reversion to life-threatening mutations. Thus, such pathogens, although attenuated, may still inherently have unpredictable risks. Killed pathogens may not be as effective as desired to generate a specific immune response. To minimize these risks, "second generation" vaccines were developed. These are typically subunit vaccines, consisting of defined antigens or recombinant protein components derived from the pathogen.

[0003] Genetic vaccines, i.e. vaccines for genetic vaccination, are usually understood as "third generation" vaccines. They are typically composed of genetically engineered nucleic acid molecules that allow the expression of peptides or protein (antigen) fragments characteristic of pathogens or tumor antigens in vivo. When administered to a patient, the genetic vaccines are expressed after uptake by target cells. Expression of the administered nucleic acid molecule leads to the production of the encoded proteins. If these proteins are recognized as foreign by the patient's immune system, an immune response is elicited.

[0004] DNA as well as RNA can be used as nucleic acid molecules for administration in the context of genetic vaccination. DNA is known to be relatively stable and easy to handle. However, the use of DNA carries the risk of unwanted insertion of the administered DNA fragment into the patient's genome, potentially resulting in mutagenic events such as loss of function of the impaired gene. An additional risk has emerged in the unwanted production of anti-DNA antibodies. Another drawback is that DNA administration can only achieve limited levels of expression of the encoded peptide or protein, since DNA must enter the nucleus to be transcribed before the resulting mRNA can be translated. Among other reasons, the level of expression of the administered DNA depends on the presence of specific transcription factors that regulate DNA transcription. In the absence of such factors, DNA transcription does not produce satisfactory amounts of RNA. As a result, the level of translated peptide or protein obtained is limited.

[0005] The use of messenger RNA (mRNA) to deliver genetic information to target cells offers an attractive alternative to DNA. Advantages of using mRNA include transient expression and non-transforming properties. mRNA does not need to enter the nucleus to be expressed, and moreover cannot integrate into the host genome, thereby eliminating the risk of carcinogenesis. Thus, by using RNA instead of DNA for genetic vaccination, the risk of unwanted genomic integration and the production of anti-DNA antibodies is minimized or avoided. Two main problems with the use of mRNA in vaccines are related to degradation and intracellular access. Thus, free RNA is susceptible to nuclease digestion in plasma, and free RNA has limited ability to gain access to intracellular compartments where the relevant translation machinery resides.

[0006] Lipid nanoparticles (LNPs) formed from cationic lipids and other lipid components such as neutral lipids, cholesterol, and polymer-conjugated lipids, as well as mRNA, have been used to block RNA degradation in plasma and promote cellular uptake of oligonucleotides.Patent Document 1 and Patent Document 2 describe lipid nanoparticle compositions that contain unmodified and nucleotide-modified RNA that code for different antigens in this respect.

[0007] Although the use of lipid nanoparticles described above has already been a major step forward towards the effective use of mRNA-based vaccines, there is a need to further improve mRNA-based vaccines, for example, by adding at least one further component to the mRNA-based vaccine so that the vaccine produces a greater immune response against the antigen encoded by the mRNA.

[0008] Moreover, standard LNPs usually target the liver. This can be disadvantageous in some cases, for example in immunotherapy, since immune responses against these organs can be elicited. Thus, there is a need for mRNA formulations that can be administered systemically, avoiding liver targeting. Furthermore, it would be advantageous to provide LNPs that target the spleen, macrophages, or DCs, respectively. In this regard, nucleic acids such as mRNAs are of great interest for various therapeutic interventions in patients, for example tumor treatment approaches, based on tumor antigen expression by encoding mRNA in antigen-presenting cells (APCs) to induce T-cell responses against tumors. Target cells for such interventions are, for example, dendritic cells (DCs) present in lymph nodes (LNs) or spleens. Thus, mRNAs encoding polypeptides containing one or more epitopes can be used to deliver epitopes derived from tumor-associated antigens encoded by excessively upregulated RNA transcripts to patients. Dendritic cells (DCs) present in the spleen represent antigen-presenting cells of particular interest for mRNA expression of epitopes. Therefore, one objective of the present invention is to develop an injectable RNA formulation with high spleen selectivity that meets the product criteria for application to patients. The claims of the present invention provide a solution to the above problems or objectives. According to the present invention, LNPs containing phosphatidylserine surprisingly lead to substantial mRNA expression in the spleen or dendritic cells, respectively, after administration of those LNPs. Strong expression of the reporter gene in target cells (spleen) was measured, while expression in other organs was low. This was unexpected, since LNPs usually show major expression in the liver upon administration.

[0009] Thus, despite all prior art, there is still a need for alternative lipid nanoparticle formulations, including alternative lipids, that provide one or more of the following properties: reduced cytotoxicity of molecules, such as nucleic acids, better targeting ability, enhanced short-term and / or long-term immunity, or promotion of endosomal escape.Thus, despite the enormous amount of research that has been carried out to date in the field of lipid nanoparticle formulations, it is desirable to develop further lipid nanoparticle formulations that can improve or eliminate one or more of the above problems, or the in vivo efficacy, toxicity, cost and simplicity of design of transfection process.

[0010] The object of the present invention can therefore also be seen in terms of the provision of novel lipid nanoparticle formulations that improve or eliminate one or more of the above problems or the in vivo efficacy, toxicity, cost and design simplicity of the transfection process. These problems and the further problems described in the "Background of the Invention" have been solved by the subject matter and claims of the present invention. One exemplary solution to the problems of the present invention is: a) at least one nucleic acid encoding at least one antigen or a fragment or variant thereof; b) a carrier composition comprising the phospholipid phosphatidylserine, the amount of phosphatidylserine being 9 mol % or less, preferably 5 mol % or less, of the total molar amount of all lipid excipients in the carrier composition; The present invention provides a vaccine composition comprising:

[0011] Furthermore, the above problems were solved by adding a phospholipid with a shorter alkyl chain, specifically (07:0)PC (DHPC; 1,2-diheptanoyl-sn-glycero-3-phosphocholine), to the LNP formulation, as disclosed herein. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] International Publication No. 2018078053 [Patent Document 2] International Publication No. 2016176330 Summary of the Invention

[0013] The inventors have surprisingly solved the above need in that they have found that the addition of the phospholipid phosphatidylserine to a vaccine composition comprising a carrier composition (preferably lipid nanoparticles) comprising a nucleic acid encoding an antigen, most likely targets the vaccine composition for phagocytosis by phagocytes of the immune system, without wishing to be bound by theory, ultimately resulting in the presence of the encoded antigen or a fragment or variant thereof in the phagocytes, thereby resulting in a more effective vaccine.

[0014] Moreover, it has been surprisingly found that the above-mentioned addition of phosphatidylserine to the vaccine composition results in a substantial increase in the targeting of the vaccine composition to antigen-presenting cells (APCs), such as DCs, macrophages, or the spleen itself, when compared with a vaccine composition that does not contain phosphatidylserine, preferably a vaccine composition that is an mRNA-LNP vaccine composition.It has also been surprisingly found that the above-mentioned addition of phosphatidylserine to the mRNA-LNP vaccine composition results in a substantial increase in the targeting of the mRNA-LNP vaccine composition to lymph nodes, when compared with a mRNA-LNP vaccine composition that does not contain phosphatidylserine.

[0015] In a first aspect, the present invention provides a) at least one nucleic acid encoding at least one antigen or a fragment or variant thereof; b) a carrier composition comprising the phospholipid phosphatidylserine; The present invention relates to a vaccine composition comprising:

[0016] In another aspect, the present invention relates to a method for delivering a vaccine composition comprising at least one nucleic acid encoding at least one antigen or a fragment or variant thereof to the spleen or lymph nodes, wherein the carrier composition comprises the phospholipid phosphatidylserine, as compared to a vaccine composition not comprising phosphatidylserine.

[0017] In an embodiment of the first aspect, the antigen is derived from a pathogenic antigen, a tumor antigen, an allergen antigen, or an autoimmune self-antigen. In another embodiment of the first aspect, the amount of phosphatidylserine is no more than 9 mol %, preferably no more than 5 mol % of the total molar amount of all lipid excipients in the composition.

[0018] In another embodiment of the first aspect, the carrier composition is a lipid nanoparticle composition. In yet another embodiment, the lipid nanoparticle composition comprises: (i) cationic or ionizable lipids; (ii) steroids; (iii) additional phospholipids in addition to phosphatidylserine; and (iv) Polymer-conjugated lipids Further includes:

[0019] In a second aspect, the present invention relates to a pharmaceutical composition comprising the vaccine composition of the first aspect and a pharma- ceutically acceptable carrier, diluent or excipient, preferably wherein the pharmaceutical composition is a sterile solid composition for reconstitution with a sterile liquid carrier, and further comprises one or more inactive ingredients selected from pH correctors, bulking agents, stabilizers, non-ionic surfactants and antioxidants, and wherein the sterile liquid carrier is an aqueous carrier.

[0020] In a third aspect, the present invention relates to the vaccine composition of the first aspect or the pharmaceutical composition of the second aspect for use in treating or preventing an infectious disease, a cancer or tumor disease, disorder or condition, a liver disease selected from the group consisting of liver fibrosis, liver cirrhosis, and liver cancer, an allergy, or an autoimmune disease, disorder or condition in a subject.In a highly preferred embodiment of the third aspect of the present invention, the present invention relates to a vaccine composition for use in treating or preventing a cancer or tumor disease.

[0021] In a fourth aspect, the present invention relates to a kit or kit of parts comprising the vaccine composition of the first aspect or the pharmaceutical composition of the second aspect, optionally with a liquid vehicle for solubilization and optionally with technical instructions providing information on the use and dosage of the components.

[0022] In a fifth aspect, the present invention provides a method of treating or preventing an infectious disease; a cancer or neoplastic disease, disorder or condition; a liver disease selected from the group consisting of liver fibrosis, liver cirrhosis, and liver cancer; an allergy; or an autoimmune disease, disorder or condition in a subject, comprising: a) providing a vaccine composition according to the first aspect or a pharmaceutical composition according to the second aspect or a kit or kit-of-parts according to the fourth aspect; b) applying or administering the vaccine composition or pharmaceutical composition or the kit or kit-of-parts to a tissue or organism of interest; The present invention relates to a method comprising the steps of:

[0023] In a sixth aspect, the present invention relates to a method of inducing an immune response in a subject comprising administering to the subject a vaccine composition of the first aspect or a pharmaceutical composition of the second aspect in an amount effective to generate an antigen-specific immune response in the subject.

[0024] In a seventh aspect, the present invention relates to the use of a vaccine composition of the first aspect or a pharmaceutical composition of the second aspect or a kit or kit-of-parts of the fourth aspect for inducing (i) an immune response, (ii) an antigen-specific T cell response or preferably (iii) a CD8+ T cell response in a subject.

[0025] definition For clarity and readability, the following scientific background information and definitions are provided. Any technical feature mentioned in or disclosed herein can be part of or can be read in each and every embodiment of the present invention. Additional definitions and explanations can be provided in the context of this disclosure.

[0026] Unless otherwise defined or required by specific context, all technical terms used herein have the same meaning as commonly understood by those of ordinary skill in the relevant art.

[0027] Unless the context indicates or requires otherwise, the words "comprise", "comprises" and "comprising" and similar expressions are to be interpreted in the present specification and claims in an open and inclusive sense as "including, but not limited to". For the purposes of the present invention, it should also be understood that the term "consisting of" is considered to be a preferred embodiment of the term "comprising". Hereinafter, when a group is defined as including at least a certain number of embodiments, this is also intended to encompass a group that preferably consists of only these embodiments.

[0028] The phrases "one embodiment," "embodiment," "specific embodiment," and the like mean that the particular feature, characteristic or characteristic, or particular group or combination of features, characteristics or characteristics, that is recited in conjunction with the respective phrase, is present in at least one embodiment of the invention. The appearances of these phrases in various places throughout this specification do not necessarily refer to the same embodiment. Furthermore, the particular features, characteristics or characteristics may be combined in any suitable manner in one or more embodiments.

[0029] The singular forms "a," "an," and "the" are to be understood as including plural references unless the context clearly dictates otherwise. Percentages in numerical contexts should be understood as relative to the total number of the respective item. In other cases, unless the context dictates otherwise, percentages should be understood as percentages by weight (wt.%).

[0030] As used herein, "compound" refers to a chemical substance, which is a material that consists of molecules with essentially the same chemical structure and properties.For small molecule compounds, the molecules are typically identical in terms of their atomic composition and structural arrangement.For macromolecular or polymeric compounds, the molecules of a compound are very similar, but not necessarily all of them are identical.For example, a segment of a polymer that is specified as consisting of 50 monomer units may also contain individual molecules with, for example, 48 or 53 monomer units.

[0031] The term "molecule" may be used as a synonym for "compound" or an individual (i.e., single) molecule. Any reference to a compound or moiety having a functional group that is ionizable under physiological conditions should be understood as including the ionized form of the respective compound or moiety. Conversely, any reference to a compound or moiety having an ionizable functional group that may also exist in a non-ionized form under physiological conditions should be understood as including the non-ionized form of the respective compound or moiety. For example, the disclosure of a compound having a carboxyl group should be interpreted as referring to the respective compound having a non-ionized carboxyl group or having an ionized carboxylate group.

[0032] As used herein, "physiological conditions" refers to an aqueous environment having a pH that is within the pH range known from human physiology, including both extracellular and intracellular conditions. An approximation of this pH range is from about pH 1 to about pH 9. Depending on the context, physiological conditions can also refer to near neutral conditions, such as from about pH 5 to about pH 8.5, or from about pH 5.5 to about pH 8.

[0033] Lipidoid compounds, also simply called lipidoids, are lipid-like compounds, i.e. amphipathic compounds that have lipid-like physical properties. In the context of the present invention, the term lipid is considered to encompass lipidoids.

[0034] In the context of the present invention, the term "selected from the group consisting of" followed by a certain group of elements (e.g., "A, B, and C") is intended to be non-limiting within the context of the present invention. In other words, such terms do not indicate that the disclosure is closed to the elements not listed, i.e., alternative meanings are also included within the group following the term. Thus, in the context of the present invention, the term "selected from the group consisting of" followed by a certain group of elements (i.e., "A, B, and C") should be understood as "selected from A, B, and C" or "is A, B, or C," which encompasses other structurally and functionally related as well as unrelated, unmentioned elements.

[0035] The term "about" is used when the parameter or value does not necessarily have to be identical, i.e., 100% the same. Thus, "about" means that the parameter or value may deviate from 0.1% to 20%, preferably 0.1% to 10%; in particular, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%. Those skilled in the art will appreciate that, for example, a certain parameter or value may vary slightly based on the method by which the parameter is determined. For example, if a particular parameter or value is defined herein as having a length of, for example, "about 1000 nucleotides," that length may deviate by 0.1% to 20%, preferably 0.1% to 10%, in particular 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%. Thus, the skilled artisan will appreciate that in that embodiment, the length may deviate by 1 to 200 nucleotides, preferably 1 to 100 nucleotides, in particular 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 nucleotides.

[0036] The term "cationic" means that the respective structure has a positive charge, either permanently or not, depending on certain conditions, e.g. pH, unless a different meaning is clear from the specific context. Thus, the term "cationic" encompasses both "permanently cationic" and "cationisable". The term "cationisable" as used herein means that a compound, or a group or atom, is positively charged in a low pH environment and uncharged in a high pH environment. Even in non-aqueous environments where the pH value cannot be determined, a cationisable compound, group or atom is positively charged at high hydrogen ion concentration and uncharged at low or active hydrogen ion concentration. This depends on the individual properties of the cationisable or polycationisable compound, in particular the pKa of each cationisable group or atom, which is charged or uncharged at that pH or hydrogen ion concentration. In a dilute aqueous environment, the fraction of cationisable compounds, groups or atoms with a positive charge can be estimated using the so-called Henderson-Hasselbalch equation, which is well known to those skilled in the art. For example, if the compound or moiety is cationizable, it is preferred that it is positively charged at a pH value of about 1-9, preferably 4-9, 5-8 or even 6-8, more preferably a pH value of 9 or less, 8 or less, 7 or less, most preferably a physiological pH value, e.g., about 7.3-7.4, i.e. under physiological conditions, particularly under physiological salt conditions of cells in vivo. In embodiments, it is preferred that the cationizable compound or moiety is primarily neutral at physiological pH values, e.g., about 7.0-7.4, but positively charged at lower pH values. In some embodiments, the preferred range of pKa of the cationizable compound or moiety is about 5 to about 7. In some embodiments, the protonizable lipid has a pKa of the protonizable group in the range of about 4 to about 11, e.g., a pKa of about 5 to about 7.

[0037] Unless a different meaning is clear from the specific context, the term "cationic" means that the respective structure has a positive charge, either permanently or not, depending on certain conditions such as pH. Thus, the term "cationic" encompasses both "permanently cationic" and "cationisable". For example, a compound or moiety having a primary, secondary or tertiary amino group can exist in a predominantly positively charged state under physiological conditions, and therefore is cationic, or more specifically, cationisable.

[0038] As used herein, "permanently cationic" means that each compound, or group or atom, is positively charged at any pH value or hydrogen ion activity of its environment.In most cases, the positive charge is caused by the presence of quaternary nitrogen atoms.When a compound has multiple such positive charges, it can be called permanently polycationic, which is a subcategory of permanently cationic.

[0039] Similarly, the terms "anionic", "anionizable" and "permanently anionic" are used analogously to "cationic", "cationizable" and "permanently cationic", except that the charge on the respective compounds, groups or atoms is negative rather than positive.

[0040] The term "neutral" when applied to a compound, such as a lipid or steroid, or to a group or moiety, means that it is neither cationic nor anionic, e.g., a compound that does not have functional groups that are ionizable under physiological conditions, such as a hydrocarbon; or that it is both cationic and anionic, i.e., zwitterionic, under typical physiological conditions, such as a typical naturally occurring phosphatidylcholine.

[0041] "Lipid" as used herein refers to a group of organic compounds that are derivatives (e.g., esters) of fatty acids and are generally insoluble in water, but soluble in many organic solvents. Lipids are generally divided into at least three classes: (1) "simple lipids," which include fats and oils, as well as waxes; (2) "compound lipids," which include phospholipids and glycolipids; and (3) "derived lipids," such as steroids. With respect to glycolipids, in certain embodiments, the LNPs are prepared from glycolipids (e.g., monosialoganglioside GM 1 ).

[0042] In this context, the prefix "poly-" refers to multiple atoms or groups having respective properties in a compound. When placed within parentheses, the presence of the multiple is optional. For example, (poly)cationic means cationic and / or polycationic. However, the absence of the prefix should not be construed as excluding the multiple, etc. For example, polycationic compounds are also cationic compounds and may be referred to as such.

[0043] The term "nucleic acid" refers to any compound that comprises or consists of DNA or RNA. This term can be used for polynucleotides and / or oligonucleotides. Whenever a nucleic acid or a nucleic acid sequence that codes for a particular protein and / or peptide is mentioned herein, said nucleic acid or nucleic acid sequence, respectively, preferably also comprises a control sequence that allows its expression, i.e., the transcription and / or translation of the nucleic acid sequence that codes for the particular protein or peptide, in a suitable host, e.g., a human. Preferably, the nucleic acid according to the present invention is not a cyclic dinucleotide, such as a Toll-like receptor (TLR9) ligand CpG oligonucleotide (ODN) or cyclic guanosine monophosphate-adenosine monophosphate (cGAMP).

[0044] In a highly preferred embodiment, the "nucleic acid" of the present invention is an "artificial mRNA" or "isolated mRNA". The term "artificial mRNA" (sequence) can be understood to be an mRNA molecule that typically does not occur in nature. In other words, an artificial mRNA molecule can be understood as a non-natural mRNA molecule. Such an mRNA molecule can be non-natural because of its individual sequence (not occurring in nature) and / or because of other modifications, such as structural modifications of nucleotides that do not occur in nature. Typically, an artificial mRNA molecule can be designed and / or created by genetic engineering methods that correspond to a desired artificial sequence of nucleotides (heterologous sequence). In this context, an artificial sequence is usually a sequence that cannot occur in nature, i.e., the artificial sequence differs from the wild-type sequence by at least one nucleotide. The term "wild-type" can be understood as a naturally occurring sequence. Furthermore, the term "artificial nucleic acid molecule" is not limited to mean "one single molecule" but is typically understood to include an ensemble of identical molecules. Thus, the term can relate to multiple identical molecules contained in an aliquot.

[0045] In a highly preferred embodiment, the nucleic acid of the invention is an "isolated" mRNA. "Isolated": As used herein, the term "isolated" in reference to a nucleic acid molecule, preferably an isolated mRNA or polypeptide, means that the nucleic acid molecule, preferably an isolated mRNA or polypeptide, is in a state other than its natural environment, such as away from blood and / or animal tissue. In some embodiments, the isolated nucleic acid molecule, preferably an isolated mRNA or polypeptide, is substantially free of other nucleic acid molecules or other polypeptides, particularly other nucleic acid molecules or polypeptides of animal origin. In some embodiments, the nucleic acid molecule, preferably an isolated mRNA or polypeptide, may be in a highly purified form, i.e., more than 95% pure or more than 99% pure. When used in this context, the term "isolated" does not exclude the presence of the same nucleic acid molecule or polypeptide in alternative physical forms, such as dimers or alternatively phosphorylated or derivatized forms. Isolated materials may also have various levels of purity with respect to the materials with which they were associated. Isolated substances and / or entities may also be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more of other components with which they were initially associated. In some embodiments, the isolated agents are about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more pure. As used herein, a substance is "pure" if it is substantially free of other components. In the context of the present invention, the specification and claims, the term "mRNA" preferably means "isolated mRNA" and vice versa.

[0046] In a particularly preferred embodiment, the artificial nucleic acid, nucleic acid or RNA is mRNA, more preferably isolated mRNA.In the context of the present invention, mRNA technology is particularly preferred, since compared with viral systems, mRNA allows for regulated dosage, transient and controlled expression, complete degradation of mRNA after protein synthesis, and does not bring about the risk of insertion mutation.

[0047] In the context of the present invention, the term "nucleoside modification" refers to a nucleic acid, such as an mRNA compound or molecule, that contains a nucleoside that is not normally present in natural mRNA, preferably a non-natural nucleoside. In particular, the term preferably refers to an mRNA nucleoside other than adenine, guanine, cytosine, uracil and thymine.

[0048] The term "nucleoside" generally refers to a compound consisting of a sugar, usually ribose or deoxyribose, and a purine or pyrimidine base. The term "nucleotide" generally refers to a nucleoside that includes a phosphate group attached to the sugar.

[0049] "Peptide" means an oligomer or polymer of at least two amino acid monomers linked by peptide bonds. The term does not limit the length of the polymer chain of amino acids. A peptide may, for example, contain less than 50 monomer units. Longer peptides, typically having 50-600 monomer units, more specifically 50-300 monomer units, are also called polypeptides.

[0050] A "protein" comprises or consists of one or more polypeptides folded into a three-dimensional form that facilitates a biological function. Immune system: The immune system can protect an organism from infection. When a pathogen breaks the physical barriers of an organism and invades the organism, the innate immune system provides an immediate but non-specific response. If a pathogen evades this innate response, vertebrates are equipped with a second layer of defense: the adaptive immune system. Here, the immune system adapts its response to improve the recognition of the pathogen during infection. Therefore, even after the pathogen is eliminated, this improved response is retained in the form of immunological memory, allowing the adaptive immune system to launch a faster and more powerful attack each time the pathogen is encountered. According to this, the immune system includes the innate immune system and the adaptive immune system. Each of these two parts contains the so-called humoral and cellular components.

[0051] Immune response: An immune response can typically be either a specific reaction of the adaptive immune system against a particular antigen (the so-called specific or adaptive immune response) or a non-specific reaction of the innate immune system (the so-called non-specific or innate immune response). The present invention relates to the core of the specific reaction of the adaptive immune system (adaptive immune response). In particular, the present invention relates to the adaptive immune response against infection by a virus, such as influenza virus. However, this specific response can be supported by an additional non-specific reaction (innate immune response). Thus, the present invention also relates to compounds for the simultaneous stimulation of the innate and adaptive immune systems to trigger an efficient adaptive immune response.

[0052] Adaptive immune system: The adaptive immune system is composed of highly specialized systemic cells and processes that eliminate or prevent pathogenic proliferation. The adaptive immune response provides the vertebrate immune system with the ability to recognize and remember specific pathogens (generating immunity) and to launch a stronger attack each time the pathogen is encountered. The system is highly adaptable due to somatic hypermutation (a process in which somatic mutations occur at an increased frequency), and V(D)J gene rearrangement (irreversible genetic recombination of antigen receptor gene segments). This mechanism allows a small number of genes to generate a vast number of different antigen receptors, which are then uniquely expressed on each individual lymphocyte. Gene rearrangement results in irreversible changes in each cell's DNA, so that all of that cell's progeny (descendants) inherit genes that code for the same receptor specificity, including memory B cells and memory T cells, which are key to long-lived specific immunity. The immune network theory is a theory of how the adaptive immune system works that is based on the interactions between the variable regions of the receptors of T cells, B cells, and molecules made by T cells and B cells that carry the variable regions.

[0053] Adaptive immune response: Adaptive immune responses are typically understood to be antigen-specific. Antigen specificity allows the generation of responses adapted to specific antigens, pathogens or pathogen-infected cells. The ability to mount these adapted responses is maintained in the body by "memory cells". Once a pathogen has infected the body more than once, these specific memory cells are used to rapidly eliminate this pathogen. In this context, the first step of the adaptive immune response is the activation of different immune cells capable of inducing an antigen-specific immune response by naive antigen-specific T cells or antigen-presenting cells (APCs). This occurs in lymphoid tissues and organs through which naive T cells constantly pass. Cell types that can act as antigen-presenting cells are, among others, dendritic cells, macrophages and B cells. Each of these cells has a distinct function in eliciting an immune response. Dendritic cells take up antigens by phagocytosis and macropinocytosis and are stimulated, for example by contact with foreign antigens, to migrate to local lymphoid tissues where they differentiate into mature dendritic cells. Macrophages ingest particulate antigens such as bacteria and are induced to express MHC molecules by infectious agents or other appropriate stimuli. The unique ability of B cells to bind and internalize soluble protein antigens via their receptors may also be important in inducing T cells. Presentation of antigens on MHC molecules leads to activation of T cells, thereby inducing their proliferation and differentiation into armed effector T cells. The most important functions of effector T cells are the killing of infected cells by CD8+ cytotoxic T cells and the activation of macrophages by Th1 cells, which together constitute cell-mediated immunity, and the activation of B cells by both Th2 and Th1 cells to produce different classes of antibodies and thus drive the humoral immune response. T cells recognize antigens by T cell receptors that do not directly recognize and bind to antigens, but instead recognize short peptide fragments of pathogen-derived protein antigens that are bound to, for example, MHC molecules on the surface of other cells.

[0054] Cellular immunity / cell-mediated immune response: Cellular immunity typically involves the activation of macrophages, natural killer cells (NK), antigen-specific cytotoxic T lymphocytes, and the release of various cytokines in response to antigens. In a more general way, cellular immunity is not related to antibodies but to the activation of cells of the immune system. Cellular immune responses are characterized by activating antigen-specific cytotoxic T lymphocytes that can induce apoptosis in somatic cells that display epitopes of the antigen on their surface, such as virus-infected cells, cells with intracellular bacteria, and cancer cells that display tumor antigens; activating macrophages and natural killer cells so that they can destroy pathogens; and stimulating cells to secrete various cytokines that affect the function of other cells involved in adaptive and innate immune responses.

[0055] Humoral immunity / humoral immune response: Humoral immunity typically refers to antibody production and the auxiliary processes that may accompany it. Humoral immune response typically can be characterized by, for example, Th2 activation and cytokine production, germinal center formation and isotype switching, affinity maturation and memory cell production. Humoral immunity also typically refers to the effector functions of antibody, including pathogen and toxin neutralization, classical complement activation, and opsonization promotion of phagocytosis and pathogen elimination.

[0056] Innate immune system: This means that the cells of the innate system recognize and respond to pathogens in a general way, but unlike the adaptive immune system, they do not confer long-lasting or protective immunity to the host. The innate immune system may be, for example, responsive to pathogen-associated molecular pattern (PAMP) receptors, such as ligands for Toll-like receptors (TLRs), or to lipopolysaccharide, TNF-alpha, CD40 ligand, or to cytokines, monokines, lymphokines, interleukins or chemokines, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IFN-alpha, , IFN-beta, IFN-gamma, GM-CSF, G-CSF, M-CSF, LT-beta, TNF-alpha, growth factors, and other auxiliary substances such as hGH, ligands for human Toll-like receptors TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, ligands for mouse Toll-like receptors TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13, ligands for NOD-like receptors, ligands for RIG-I-like receptors, immunostimulatory nucleic acids, immunostimulatory RNA (isRNA), CpG-DNA, antibacterial agents, or antiviral agents. Typically, the response of the innate immune system involves recruitment of immune cells to the site of infection through the production of chemical factors, including specialized chemical messengers called cytokines; activation of the complement cascade; recognition and removal of foreign substances present in organs, tissues, blood and lymph by specialized white blood cells; activation of the adaptive immune system through a process known as antigen presentation; and / or acting as a physical and chemical barrier against infectious agents.

[0057] Adjuvant / Adjuvant Component: Adjuvant or adjuvant component, in a broad sense, is an agent or composition (e.g., pharmacological or immunological) that can modify, e.g., enhance, the effectiveness of other agents, such as drugs or vaccines. Conventionally, the term refers to a compound or composition that acts as a carrier or auxiliary substance for immunogens and / or other pharma- ceutical active compounds in the context of the present invention. The term should be interpreted broadly and refers to a wide range of substances that can increase the immunogenicity of the antigen that is incorporated or co-administered with the adjuvant in question. In the context of the present invention, an adjuvant preferably enhances the specific immunogenic effect of the active agent of the present invention. Typically, "adjuvant" or "adjuvant component" have the same meaning and can be used interchangeably. Adjuvants can be divided, for example, into immunostimulants, antigenic delivery systems, or even combinations thereof.

[0058] The term "adjuvant" is understood to not include agents that typically confer immunity themselves. Adjuvants non-specifically support the immune system, for example to enhance antigen-specific immune responses by promoting the presentation of antigens to the immune system or the induction of non-specific innate immune responses. In addition, adjuvants can preferably modulate antigen-specific immune responses, for example by shifting a predominantly Th2-based antigen-specific response to a more Th1-based antigen-specific response, or vice versa. Thus, adjuvants can advantageously modulate cytokine expression / secretion, antigen presentation, type of immune response, etc.

[0059] The term "antibody" as used herein includes both intact antibodies and antibody fragments. Typically, an intact "antibody" is an immunoglobulin that specifically binds to a particular antigen. An antibody can be a member of any immunoglobulin class, including any of the human classes: IgG, IgM, IgE, IgA and IgD. Typically, an intact antibody is a tetramer. Each tetramer consists of two identical pairs of polypeptide chains, each pair having a "light" and a "heavy" chain. The term "antigen" in the context of the present invention refers to a substance that can be recognized, typically by the immune system, preferably by the adaptive immune system, and that can elicit an antigen-specific immune response, for example by the formation of antibodies and / or antigen-specific T cells as part of the adaptive immune response. Typically, an antigen can be or include a peptide or protein that can be presented to T cells by MHC. In the sense of the present invention, an antigen can be the product of translation of a provided nucleic acid molecule, preferably an mRNA as defined herein. In this context, fragments, variants and derivatives of peptides and proteins that contain at least one epitope are also understood as antigens. Thus, the term "antigen" as used herein is intended to refer to a substance that is recognized and understood by those skilled in the art, e.g., that can be recognized by the immune system, preferably the adaptive immune system, and that can elicit an antigen-specific immune response, e.g., by the formation of antibodies and / or antigen-specific T cells as part of the adaptive immune response. Typically, an antigen can be or include a peptide or protein that can be presented to T cells by MHC. Fragments, variants and derivatives of peptides or proteins derived from, e.g., cancer antigens, that contain at least one epitope, can also be understood as antigens. In the context of the present invention, an antigen can be a product of translation of a provided RNA (e.g., coding RNA, replicon RNA, mRNA). The term "antigenic peptide or protein" is intended to refer to a peptide or protein derived from, e.g., an (antigenic) protein that can stimulate the body's adaptive immune system to result in an adaptive immune response.Thus, an "antigenic peptide or protein" includes at least one epitope or antigen of the protein (eg, a tumor antigen, a viral antigen, a bacterial antigen, a protozoan antigen) from which it is derived.

[0060] In the context of nucleic acids, i.e., for nucleic acids "derived from" (another) nucleic acid, the term "derived from" as used throughout the specification means that the nucleic acid derived from (another) nucleic acid shares, for example, at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or about 99% sequence identity with the nucleic acid from which it is derived. The skilled artisan is aware that sequence identity is typically calculated for the same type of nucleic acid, i.e., for DNA or RNA sequences. Thus, when DNA is "derived from" RNA or RNA is "derived from" DNA, it is understood that in a first step, the RNA sequence is converted to the corresponding DNA sequence (particularly by replacing U by T throughout the sequence) or vice versa, the DNA sequence is converted to the corresponding RNA sequence (particularly by replacing T by U throughout the sequence). The sequence identity of the DNA sequence or the sequence identity of the RNA sequence is then determined. Preferably, a nucleic acid "derived from" a nucleic acid also refers to a nucleic acid that has been modified compared to the nucleic acid from which it is derived, e.g. to increase RNA stability even further and / or to prolong and / or increase protein production. In the context of amino acid sequences, the term "derived from" means that an amino acid sequence derived from (another) amino acid sequence shares, e.g., at least about 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% sequence identity with the amino acid sequence from which it is derived.

[0061] Epitope (also called "antigenic determinant"): T cell epitopes or portions of a protein in the context of the present invention may comprise fragments having a length of preferably about 6 to about 20 or even more amino acids, such as fragments having a length of preferably about 8 to about 10, such as 8, 9 or 10 (or even 11 or 12 amino acids), which are processed and presented by MHC class I molecules, or fragments having a length of preferably about 13 or more amino acids, such as 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids, which are processed and presented by MHC class II molecules, which fragments may be selected from any part of the amino acid sequence. These fragments are typically recognized by T cells in the form of a complex consisting of the peptide fragment and the MHC molecule.

[0062] A B-cell epitope is typically a fragment located on the outer surface of a (natural) protein or peptide antigen as defined herein that can be recognised by an antibody, i.e. in its native form, preferably having 5 to 15 amino acids, more preferably having 5 to 12 amino acids, even more preferably having 6 to 9 amino acids.

[0063] Such epitopes of proteins or peptides may further be selected from any of the variants mentioned herein of such proteins or peptides. In this context, an antigenic determinant may be a conformational or discontinuous epitope composed of segments of a protein or peptide as defined herein that are discontinuous in the amino acid sequence of the protein or peptide as defined herein, but are grouped together in a three-dimensional structure composed of a single polypeptide chain, or a continuous or linear epitope.

[0064] A "tolerogenic composition" is a composition which promotes immune tolerance in a cell or cell line against an antigen, which may be a self or non-self antigen. In other words, there is no or a reduced immune response against the antigen. In contrast, a vaccine composition according to the invention induces an immune response against a specific antigen or a fragment or variant thereof, i.e. an antigen or a fragment or variant thereof encoded by at least one nucleic acid. The antigen may also be a self or non-self antigen, the overall aim of the vaccine composition of the invention being to generate a (strong) immune response against this antigen or a fragment or variant thereof, and the overall aim of a tolerogenic composition being to at least partially, and at best completely, suppress the immune response against this antigen.

[0065] A "tolerogenic nucleic acid" is a nucleic acid that promotes immune tolerance in a cell or cell line to an antigen, the nucleic acid may be a chemically modified mRNA and / or may encode a tolerogenic polypeptide. Conversely, at least one nucleic acid used in the present invention encodes at least one antigen or a fragment or variant thereof against which a (strong) immune response is desired and / or induced upon administration.

[0066] A "tolerogenic polypeptide" is a polypeptide that promotes immune tolerance in a cell or cell line, typically by reducing the immune response through acting on an underlying pathway, in particular by inhibiting an underlying mediator in such pathway. Thus, a tolerogenic polypeptide can be an inhibitor of mTOR, IL-2, IL-10 or an antibody reactive with CD3 or CD40. Conversely, at least one antigen or a fragment or variant thereof according to the invention does not induce immune tolerance in a cell or cell line, but induces a (strong) immune response against itself.

[0067] The tolerogenic composition may in particular comprise a tolerogenic nucleic acid that promotes the above-mentioned tolerance. The tolerogenic composition may further comprise a specific antigen, so that due to the presence of the tolerogenic nucleic acid, there is no immune response to this specific antigen or the immune response to this specific antigen is reduced. On the contrary, the vaccine composition according to the invention does not comprise an antigen or a fragment or variant thereof, in a preferred embodiment, since it is the overall aim of the vaccine composition of the invention to reveal a (strong) immune response directed to the encoded at least one antigen or a fragment or variant thereof (and not, as is the aim of the tolerogenic composition, to block or reduce the immune response directed to the co-administered antigen), but of course still comprises at least one nucleic acid encoding at least one antigen or a fragment or variant thereof. In yet another preferred embodiment, the vaccine composition according to the invention comprises at least one nucleic acid encoding at least one antigen or a fragment or variant thereof as the only payload and therefore cannot comprise an antigen (as is the case for the tolerogenic composition discussed in this paragraph in addition to the tolerogenic nucleic acid).

[0068] The term "vaccine" or "vaccine composition" is understood to be a prophylactic or therapeutic material that typically provides at least one antigen or antigenic function, or a nucleic acid encoding an antigen or a fragment or variant thereof, that can stimulate the body's adaptive immune system to produce an adaptive immune response.

[0069] The term "antigen-providing mRNA" in the context of the present invention may typically be an mRNA having at least one open reading frame that can be translated by the cell or organism that is provided with the mRNA. The product of this translation is an antigen, preferably a peptide or protein that can act as an immunogen. The product may also be a fusion protein composed of two or more immunogens, such as a fusion protein composed of two or more epitopes, peptides or proteins derived from the same or different viral proteins, and the epitopes, peptides or proteins may be linked by a linker sequence.

[0070] The term "heterologous" or "heterologous sequence" as used throughout this specification in the context of a nucleic acid or amino acid sequence refers to a sequence (e.g., DNA, RNA, amino acid) that is recognized and understood by those of skill in the art and is intended to refer to a sequence derived from another gene, another allele, another species. If the two sequences cannot be derived from the same gene or the same allele, then these sequences are typically understood to be "heterologous". That is, a heterologous sequence may be derived from the same organism, but does not naturally (naturally) occur in the same nucleic acid molecule, e.g., the same RNA or protein.

[0071] Bi / multicistronic mRNA: An mRNA that typically has two (bicistronic) or more (multicistronic) open reading frames (ORFs) (coding regions or coding sequences). An open reading frame in this context is a sequence of several nucleotide triplets (codons) that can be translated into a peptide or protein. Translation of such an mRNA results in two (bicistronic) or more (multicistronic) separate translation products (if the ORFs are not identical). For expression in eukaryotes, such an mRNA may, for example, contain an internal ribosome entry site (IRES) sequence.

[0072] Monocistronic mRNA: A monocistronic mRNA can be an mRNA that typically contains only one open reading frame (coding sequence or coding region). An open reading frame in this context is a sequence of several nucleotide triplets (codons) that can be translated into a peptide or protein.

[0073] 3' untranslated region (3'-UTR): 3'-UTR is a portion of an mRNA that is typically located between the protein coding region (i.e., open reading frame) and the poly(A) sequence of the mRNA. The 3'-UTR of an mRNA is not translated into an amino acid sequence. The 3'-UTR sequence is generally encoded by a gene that is transcribed into the respective mRNA during the gene expression process. The genomic sequence is first transcribed into a pre-mRNA, which contains an optional intron. The pre-mRNA is then further processed into a mature mRNA in a maturation process. This maturation process includes the steps of 5' capping, splicing of the pre-mRNA to excise the optional intron, and modification of the 3' end, such as polyadenylation of the 3' end of the pre-mRNA and optional endo- or exonuclease cleavage. In the context of the present invention, the 3'-UTR corresponds to the sequence of a mature mRNA that is located 3' to the stop codon of the protein coding region, preferably immediately 3' to the stop codon of the protein coding region, and extends to the nucleotide 5' to the poly(A) sequence, preferably immediately 5' to the poly(A) sequence. The term "corresponding" means that the 3'-UTR sequence can be an RNA sequence, for example in the mRNA used to define the 3'-UTR sequence, or a DNA sequence that corresponds to such an RNA sequence. In the context of the present invention, the term "3'-UTR of a gene", such as "3'-UTR of the albumin gene", is a sequence that corresponds to the 3'-UTR of the mature mRNA derived from this gene, i.e., the mRNA obtained by transcription of the gene and maturation of the mRNA precursor. The term "3'-UTR of a gene" encompasses the DNA and RNA sequences of the 3'-UTR.

[0074] 5' untranslated region (5'-UTR): 5'-UTR is typically understood to be a specific section of a messenger RNA (mRNA). It is located 5' of the open reading frame of the mRNA. Typically, the 5'-UTR starts at the transcription initiation site and ends one nucleotide before the start codon of the open reading frame. The 5'-UTR may contain elements for controlling gene expression, also called regulatory elements. Such regulatory elements may be, for example, a ribosome binding site or a 5' terminal oligopyrimidine tract. The 5'-UTR may be post-transcriptionally modified, for example by the addition of a 5' cap. In the context of the present invention, the 5'-UTR corresponds to a sequence of the mature mRNA located between the 5' cap and the start codon. Preferably, the 5'-UTR corresponds to a sequence extending from a nucleotide located 3' to the 5' cap, preferably immediately 3' to the 5' cap, to a nucleotide located 5' to the start codon of the protein coding region, preferably immediately 5' to the start codon of the protein coding region. The nucleotides located immediately 3' to the 5' cap of a mature mRNA typically correspond to the transcription start site. The term "corresponding" means that the 5'-UTR sequence may be an RNA sequence, for example in the mRNA used to define the 5'-UTR sequence, or a DNA sequence corresponding to such an RNA sequence. In the context of the present invention, the term "5'-UTR of a gene", such as "5'-UTR of a TOP gene", is a sequence corresponding to the 5'-UTR of the mature mRNA derived from this gene, i.e., the mRNA obtained by transcription of the gene and maturation of the mRNA precursor. The term "5'-UTR of a gene" encompasses the DNA and RNA sequences of the 5'-UTR.

[0075] 5'-Terminal Oligopyrimidine Tract (TOP): A 5'-Terminal Oligopyrimidine Tract (TOP) is a stretch of pyrimidine nucleotides typically located in the 5'-terminal region of a nucleic acid molecule, such as the 5'-terminal region of a particular mRNA molecule or the functional entity of a particular gene, e.g., the 5'-terminal region of a transcribed region. This sequence begins with a cytidine, usually corresponding to the transcription start site, followed by a stretch of pyrimidine nucleotides, usually about 3 to 30. For example, a TOP can contain 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or even more nucleotides. The pyrimidine stretch, thus the 5'-TOP, ends one nucleotide 5' to the first purine nucleotide located downstream of the TOP. Messenger RNAs containing 5'-terminal oligopyrimidine tracts are often called TOP mRNAs. Thus, genes that provide such messenger RNAs are called TOP genes. TOP sequences are found, for example, in genes and mRNAs that code for peptide elongation factors and ribosomal proteins.

[0076] TOP motif: In the context of the present invention, a TOP motif is a nucleic acid sequence corresponding to a 5'-TOP as defined above. Thus, a TOP motif in the context of the present invention is a stretch of pyrimidine nucleotides, preferably having a length of 3 to 30 nucleotides. Preferably, a TOP motif consists of at least 3 pyrimidine nucleotides, preferably at least 4 pyrimidine nucleotides, preferably at least 5 pyrimidine nucleotides, more preferably at least 6 nucleotides, more preferably at least 7 nucleotides, most preferably at least 8 pyrimidine nucleotides, the stretch of pyrimidine nucleotides preferably starting at its 5' end with a cytosine nucleotide. In TOP genes and TOP mRNAs, the TOP motif preferably starts at its 5' end with the transcription start site and ends one nucleotide 5' to the first purine residue in said gene or mRNA. A TOP motif in the sense of the present invention is preferably located at the 5' end of a sequence representing a 5'-UTR or at the 5' end of a sequence encoding a 5'-UTR. Thus, preferably, when a stretch of three or more pyrimidine nucleotides is located at the 5'-end of the respective sequence, such as the mRNA of the invention, the 5'-UTR element of the mRNA of the invention, or the nucleic acid sequence derived from the 5'-UTR of the TOP gene described herein, it is called a "TOP motif" in the sense of the present invention. In other words, a stretch of three or more pyrimidine nucleotides that is not located at the 5'-end of the 5'-UTR or 5'-UTR element, but is located anywhere within the 5'-UTR or 5'-UTR element, is preferably not called a "TOP motif".

[0077] TOP genes: TOP genes are typically characterized by the presence of a 5'-terminal oligopyrimidine tract. Moreover, most TOP genes are characterized by growth-related translational regulation. However, TOP genes with tissue-specific translational regulation are also known. As defined above, the 5'-UTR of a TOP gene corresponds to the sequence of the 5'-UTR of the mature mRNA derived from the TOP gene, preferably extending from the nucleotide located 3' to the 5' cap to the nucleotide located 5' to the start codon. The 5'-UTR of a TOP gene typically does not include any start codon, preferably an upstream AUG (uAUG) or an upstream open reading frame (uORF). Therein, the upstream AUG and the upstream open reading frame are typically understood to be the AUG and the open reading frame that are present 5' to the start codon (AUG) of the open reading frame to be translated. The 5'-UTR of a TOP gene is generally rather short. The length of the 5'-UTR of a TOP gene can vary between 20 nucleotides and up to 500 nucleotides, typically less than about 200 nucleotides, preferably less than about 150 nucleotides, more preferably less than about 100 nucleotides. Exemplary 5'-UTRs of TOP genes in the sense of the present invention are nucleic acid sequences extending from the nucleotide at position 5 in the sequences according to SEQ ID NOs: 1-1363, 1395, 1421 and 1422 of WO2013143700, or homologs or variants thereof, the disclosures of which are incorporated herein by reference, to the nucleotide located immediately 5' to the start codon (e.g., ATG). In this context, a particularly preferred fragment of the 5'-UTR of a TOP gene is a 5'-UTR of a TOP gene lacking the 5'-TOP motif. The term "5'-UTR of a TOP gene" preferably refers to a 5'-UTR of a naturally occurring TOP gene.

[0078] Stabilized nucleic acid, preferably mRNA: Stabilized nucleic acid, preferably mRNA, typically exhibits modifications that increase resistance to in vivo degradation (e.g., exo- or endo-nuclease degradation) and / or ex vivo degradation (e.g., by manufacturing steps prior to vaccine administration, e.g., during the preparation of the vaccine solution to be administered). Stabilization of RNA can be achieved, for example, by providing a 5' cap structure, a polyA tail, or any other UTR modification. This can also be achieved by chemical modification or modification of the G / C content of the nucleic acid. A variety of other methods are known in the art and are considered in the context of the present invention.

[0079] RNA in vitro transcription: The term "RNA in vitro transcription" or "in vitro transcription" refers to a process in which RNA is synthesized in a cell-free system (in vitro). DNA, in particular plasmid DNA, is used as a template to generate the RNA transcript. RNA can be obtained by DNA-dependent in vitro transcription of a suitable DNA template, which according to the invention is preferably a linear plasmid DNA template. The promoter for controlling the in vitro transcription can be any promoter for any DNA-dependent RNA polymerase. Specific examples of DNA-dependent RNA polymerases are T7, T3 and SP6 RNA polymerases. The DNA template for in vitro RNA transcription can be obtained by cloning a nucleic acid, in particular a cDNA corresponding to the respective RNA to be in vitro transcribed, and introducing it into a suitable vector for in vitro transcription, for example a plasmid DNA. In a preferred embodiment of the invention, the DNA template is linearized by a suitable restriction enzyme before being transcribed in vitro. The cDNA can be obtained by reverse transcription of mRNA or by chemical synthesis. Furthermore, the DNA template for in vitro RNA synthesis can also be obtained by gene synthesis.

[0080] Methods for in vitro transcription are known in the art (see, e.g., Geall et al. (2013) Semin. Immunol. 25(2):152-159; Brunelle et al. (2013) Methods Enzymol. 530:101-14). Reagents used in the methods typically include: 1) a linearized DNA template carrying a promoter sequence with high binding affinity for a respective RNA polymerase, such as a bacteriophage-encoded RNA polymerase; 2) ribonucleoside triphosphates (NTPs) for the four bases (adenine, cytosine, guanine, and uracil); 3) optionally a cap analog as defined above (e.g., m7G(5')ppp(5')G(m7G)); 4) a DNA-dependent RNA polymerase (e.g., T7, T3, or SP6 RNA polymerase) capable of binding to a promoter sequence within a linearized DNA template; 5) optionally, a ribonuclease (RNase) inhibitor to inactivate any contaminating RNases; 6) pyrophosphatase, which breaks down pyrophosphate, which can potentially inhibit transcription; 7) Mg as a cofactor for polymerases 2+ MgCl provides ions 2 ; 8) A buffer to maintain a suitable pH value, which may also contain optimal concentrations of antioxidants (e.g., DTT) and / or polyamines such as spermidine.

[0081] Full-length protein: As used herein, the term "full-length protein" refers to a protein that contains substantially the entire amino acid sequence of a typically naturally occurring protein. Nevertheless, amino acid substitutions in a protein, for example by mutation, are also encompassed by the term full-length protein.

[0082] Fragment of a protein: A "fragment" of a protein or peptide in the context of the present invention may include a sequence of a protein or peptide as defined herein, which is typically N-terminally and / or C-terminally truncated with respect to its amino acid sequence (or its encoding nucleic acid molecule) compared to the amino acid sequence of the original (natural) protein (or its encoding nucleic acid molecule). Such truncation may thus be present at the amino acid level or, correspondingly, at the nucleic acid level. Thus, sequence identity with respect to such fragments as defined herein may preferably refer to the entire protein or peptide as defined herein, or to the entire (encoding) nucleic acid molecule of such protein or peptide.

[0083] The term "variant" in the context of a nucleic acid sequence of a gene refers to a nucleic acid sequence variant, i.e., a nucleic acid sequence or gene that comprises a nucleic acid sequence that differs from the reference (or "parent") nucleic acid sequence of the reference (or "parent") nucleic acid or gene by at least one nucleic acid. Thus, a variant nucleic acid or gene may preferably include at least one mutation, substitution, insertion or deletion in its nucleic acid sequence compared to the respective reference sequence. Preferably, the term "variant" as used herein includes naturally occurring variants of a nucleic acid sequence or gene, as well as engineered variants. Thus, a "variant" as defined herein may be derived from, isolated from, related to, based on, or homologous to a reference nucleic acid sequence. "Variant" may preferably have at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably at least 70%, more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95% or even 97% sequence identity with the respective naturally occurring (wild type) nucleic acid sequence or gene, or the nucleic acid sequence of a homologue, fragment or derivative thereof.

[0084] The term "variant" as used throughout the present specification in the context of a protein or peptide is also intended to refer to a protein or peptide variant that has an amino acid sequence that differs from the original sequence in one or more mutations, such as, for example, one or more substitutions, insertions and / or deletions of amino acids, as recognized and understood by those skilled in the art. Preferably, these fragments and / or variants have the same biological function or specific activity, such as its specific antigenic properties, as compared to the full-length native protein. A "variant" of a protein or peptide as defined herein may contain conservative amino acid substitutions as compared to its native, i.e., non-mutated physiological sequence. These amino acid sequences as well as their coding nucleotide sequences particularly fall under the term variant as defined herein. Substitutions in which amino acids from the same class are exchanged for one another are called conservative substitutions. In particular, these are amino acids with aliphatic side chains, amino acids with positively or negatively charged side chains, amino acids with aromatic groups in the side chain or amino acid, amino acids with side chains whose side chains can enter into hydrogen bridges, for example with hydroxyl functions. This means that, for example, an amino acid with a polar side chain is replaced by another amino acid with a similar polar side chain, or, for example, an amino acid characterized by a hydrophobic side chain is replaced by another amino acid with a similar hydrophobic side chain (e.g., serine (threonine) by threonine (serine) or leucine (isoleucine) by isoleucine (leucine). In particular, insertions and substitutions are possible at sequence positions that do not cause modifications to the three-dimensional structure or affect the binding region. Modifications to the three-dimensional structure by insertions or deletions can be easily determined, for example, using CD spectra (circular dichroism spectra). A "variant" of a protein or peptide may have at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid identity over a stretch of at least 10, 20, 30, 50, 75 or 100 amino acids of such a protein or peptide.Preferably, a variant of a protein includes a functional variant of a protein, meaning that the variant exerts the same effect or functionality or at least 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the effect or functionality of the protein from which it is derived.

[0085] Also, the term "fragment" in the context of a nucleic acid sequence or gene refers to a contiguous subsequence of a full-length reference (or "parent") nucleic acid sequence or gene. In other words, a "fragment" can typically be a shorter portion of a full-length nucleic acid sequence or gene. Thus, a fragment typically consists of a sequence that is identical to a corresponding stretch within a full-length nucleic acid sequence or gene. The term includes naturally occurring fragments as well as engineered fragments. A preferred fragment of a sequence in the context of the present invention consists of a contiguous stretch of nucleic acid that corresponds to a contiguous stretch of an entity in the nucleic acid or gene from which the fragment is derived, representing at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, and most preferably at least 80% of the entire (i.e., full-length) nucleic acid sequence or gene from which the fragment is derived. The sequence identity indicated for such fragments preferably refers to the entire nucleic acid sequence or gene. Preferably, a "fragment" may comprise a nucleic acid sequence having at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably at least 70%, more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95% or even 97% sequence identity to a reference nucleic acid sequence or gene from which it is derived.

[0086] Also, in this context, a fragment of a protein may typically comprise an amino acid sequence having at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably at least 70%, more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95% or even 97% sequence identity with the amino acid sequence of the respective naturally occurring full-length protein.

[0087] A fragment of a protein or peptide in the context of the present invention may further comprise a protein or peptide sequence as defined herein, e.g. having a length of at least 5 amino acids, preferably at least 6 amino acids, preferably at least 7 amino acids, more preferably at least 8 amino acids, even more preferably at least 9 amino acids; even more preferably at least 10 amino acids; even more preferably at least 11 amino acids; even more preferably at least 12 amino acids; even more preferably at least 13 amino acids; even more preferably at least 14 amino acids; even more preferably at least 15 amino acids; even more preferably at least 16 amino acids; even more preferably at least 17 amino acids; even more preferably at least 18 amino acids; even more preferably at least 19 amino acids; even more preferably at least 20 amino acids; even more preferably at least 25 amino acids; even more preferably at least 30 amino acids; even more preferably at least 35 amino acids; even more preferably at least 50 amino acids; and most preferably at least 100 amino acids. For example, such fragments may have a length of about 6 to about 20 or more amino acids, e.g., fragments processed and presented by MHC class I molecules, preferably having a length of about 8 to about 10, e.g., 8, 9 or 10 amino acids (or even 6, 7, 11, or 12 amino acids), or fragments processed and presented by MHC class II molecules, preferably having a length of about 13 or more amino acids, e.g., 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids, and these fragments may be selected from any part of the amino acid sequence. These fragments are typically recognized by T cells in the form of a complex consisting of the peptide fragment and the MHC molecule, i.e., the fragments are typically not recognized in their native form. Fragments of proteins or peptides may comprise at least one epitope of these proteins or peptides. Furthermore, domains of proteins, e.g., extracellular, intracellular or transmembrane domains of proteins and truncated or truncated versions may be understood to constitute fragments of proteins.

[0088] Protein variant: "Variant" of a protein or peptide as defined in the context of the present invention may be produced, which has an amino acid sequence that differs from the original sequence in one or more mutations, such as one or more substitutions, insertions and / or deletions of amino acids. Preferably, these fragments and / or variants have the same biological function or specific activity, such as its specific antigenic properties, as compared to the full-length natural protein. "Variant" of a protein or peptide as defined in the context of the present specification may contain conservative amino acid substitutions as compared to its natural, i.e. non-mutated physiological sequence. These amino acid sequences as well as their coding nucleotide sequences are particularly included in the term variant as defined herein. Substitutions in which amino acids from the same class are exchanged for one another are called conservative substitutions. In particular, these are amino acids with aliphatic side chains, amino acids with positively or negatively charged side chains, amino acids with aromatic groups in the side chain or amino acid, amino acids with side chains whose side chains can enter into hydrogen bridges, for example with hydroxyl functions. This means, for example, that an amino acid with a polar side chain is replaced by another amino acid with a similar polar side chain, or that an amino acid featuring, for example, a hydrophobic side chain is replaced by another amino acid with a similar hydrophobic side chain (for example, serine (threonine) by threonine (serine) or leucine (isoleucine) by isoleucine (leucine). In particular, insertions and substitutions are possible at sequence positions that do not cause modifications to the three-dimensional structure or affect the binding regions. Modifications to the three-dimensional structure due to insertions or deletions can be easily determined, for example, using CD spectroscopy (circular dichroism spectroscopy) (Urry, 1985, Absorption, Circular Dichroism and ORD of Polypeptides, Modern Physical Methods in Biochemistry, Neuberger et al. (eds.), Elsevier, Amsterdam).

[0089] A "variant" of a protein or peptide may have at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid identity over a stretch of 10, 20, 30, 50, 75 or 100 amino acids of such protein or peptide.

[0090] Furthermore, variants of proteins or peptides as defined herein that may be encoded by a nucleic acid molecule may also include sequences in which the nucleotides of the encoding nucleic acid sequence have been exchanged due to the degeneracy of the genetic code without resulting in a change in the respective amino acid sequence of the protein or peptide, i.e. the amino acid sequence or at least a part thereof does not differ from the original sequence in one or more mutations within the above meaning.

[0091] Sequence identity: To determine the percentage of identity between two sequences, such as the nucleic acid sequences or amino acid sequences defined herein, preferably the amino acid sequences encoded by the nucleic acid sequences of the polymeric carriers defined herein, or the amino acid sequences themselves, the sequences can be aligned and then compared to each other. Thus, for example, a position of a first sequence can be compared to the corresponding position of a second sequence. If a position in the first sequence is occupied by the same component (residue) as in the position in the second sequence, the two sequences are identical at this position. Otherwise, the sequences differ at this position. If an insertion occurs in the second sequence compared to the first sequence, a gap can be inserted into the first sequence to allow further alignment. If a deletion occurs in the second sequence compared to the first sequence, a gap can be inserted into the second sequence to allow further alignment. Thus, the percentage of identity between two sequences is a function of the number of identical positions divided by the total number of positions, including positions that are only occupied in one sequence. The percentage of identity between two sequences can be determined using a mathematical algorithm. A preferred, but non-limiting example of a mathematical algorithm that can be used is the algorithm of Karlin et al. (1993), PNAS USA, 90:5873-5877 or Altschul et al. (1997), Nucleic Acids Res., 25:3389-3402. Such an algorithm is incorporated into the BLAST program. Sequences that are identical to the sequences of the present invention to a certain degree can be identified by this program.

[0092] Derivative of protein or peptide: Derivative of peptide or protein is understood to be a molecule that is typically derived from another molecule, such as said peptide or protein. "Derivative" of peptide or protein also includes fusion that contains peptide or protein used in the present invention. For example, fusion contains tag, such as epitope, such as FLAG epitope or V5 epitope. For example, epitope is FLAG epitope. Such tag is useful, for example, to purify fusion protein.

[0093] Pharmaceutically effective amount: A pharma- ceutical effective amount in the context of the present invention is typically understood to be an amount sufficient to induce an immune response. Carrier: A "carrier" or a "carrier composition" in the context of the present invention is a compound or compounds that facilitates the transport and / or complexation of another compound, e.g., a nucleic acid. The carrier may form a complex with the other compound, e.g., a nucleic acid. Preferably, the carrier or carrier composition is a lipid nanoparticle or a lipid nanoparticle composition, as described herein below, e.g., in the paragraph "Lipid Nanoparticle Composition". A polymeric carrier is a carrier formed of a polymer, e.g., a cationic polymer comprising an amino acid with a positive charge (e.g., a peptide comprising the amino acids G, H or R, and preferably further comprising cysteine). Protamine as a carrier is disclosed, for example, in PMID 27336830 or 23159882, EP 1083232, WO 2010037539, WO 2012116811, WO 2012116810, and WO 2015024665.

[0094] Vehicle: Typically an agent, such as a carrier, used within a pharmaceutical composition or vaccine to facilitate administration of the components of the pharmaceutical composition or vaccine to an individual. [Brief description of the drawings]

[0095] [Figure 1](Chemical Structures of Cationic Lipids) - The structures of HEXA1 (HEXA-C5DE-PipSS, see FIG. 1A), HEXA2 (HEXA-C5DE-PipC3SS, see FIG. 1B), and THIOETHER (ViTE-C4DE-Pip-thioether, see FIG. 1C) are shown - full details can be found in Example 1). [Diagram 2] (LNP-IgG total titers for in vivo malaria vaccination) - shows that the tested mRNA malaria vaccine encoding CSP induced a very strong humoral immune response in mice using an ELISA assay (Coating: [NANP]7 peptide, IgG total detection signal at fixed titers 35 days post-prime (full details can be found in Example 3)). [Diagram 3] (LNP-Intracellular Staining (ICS) for In Vivo Malaria Vaccination) - shows that LNP-formulated mRNA malaria vaccine encoding CSP induced cellular immune responses in mice: CD8+ T cell responses are shown for the restimulation setting in FIG. 3A and CD4+ T cell responses are shown for the restimulation setting in FIG. 3B. An intracellular cytokine staining assay (day 35 post-vaccination) was used. Full details can be found in Example 3. [Figure 4] 1 shows exemplary PMOZ-lipids from Example 4. [Diagram 5] (Organ Targeting of LNP)-This figure shows the results of PpLuc expression in different tissue lysates after organ harvest at two different time points, namely 4 hours and 24 hours, of LNP8-formulated mRNA encoding PpLuc, where LNP8 contains DPhyPS. At both time points, the highest expression is observed in spleen (expression levels are shown as pg of total luciferase protein / organ). Full details can be found in Example 5. [Figure 6](Organ Targeting of LNPs) - Results of PpLuc expression in pancreas (see FIG. 6A) and liver (see FIG. 6B) of LNP8-formulated mRNA encoding PpLuc at two different time points, namely 4 hours and 24 hours (expression levels are normalized to total protein and given as pg / mg of total protein; note that the y-axes of FIG. 6A and FIG. 6B are different to account for the fact that expression in the spleen is much higher than that in the liver). Full details can be found in Example 5. [Figure 7A] (LNP immunogenicity / VNT analysis) - The figure shows that intramuscular (im) immunization with 1 μg of LNP-formulated RABV-G-mRNA induced very strong VNT, well above the protective titer of 0.5 IU / ml (indicated by dashed line) in all animals on day 28, 7 days after the boost vaccination (Figure 7A). Full details can be found in Example 6. [Figure 7B]The number of spots showing the induction of RABV-G protein-specific T cells, including cytotoxic T cells (CTLs), in an experiment with mice after vaccination with mRNA encoding the RABV-G protein is shown. One week after the last boost vaccination, 28 days after prime, antigen-specific cells in the spleens of vaccinated mice were analyzed by ELISpot (enzyme-linked immunospot assay) analysis. Splenocytes were restimulated with a RABV-G peptide mix (PepMix) of 129 peptides (final concentration of 5 μg / peptide). The y-axis shows the number of detected IFN-gamma spots per mio. cell: the bars represent the mean. IFN-gamma (interferon-gamma) is considered to be the prototypic proinflammatory cytokine produced by various immune cells under inflammatory conditions, in particular by T cells and natural killer (NK) cells. IFN-gamma plays a key role in host defense by promoting the development and activation of type 1 helper T cells (Th1 cells - resulting in an increase in cell-mediated responses), chemoattraction and activation of monocytes and macrophages, and upregulation of antigen-presenting molecules. IFN-gamma also exhibits antiviral, antiproliferative, and apoptotic effects. Results: As can be seen, the addition of PS to certain LNP formulations greatly increased the number of detected IFN-gamma spots, indicating the induction of RABV-G protein-specific T cells, including cytotoxic T cells (CTLs), compared to the control LNP formulation. Full details of this experiment can be found in Example 6. [Figure 8A] (LNP for in vivo malaria vaccination - long term experiment) - shows that the tested mRNA malaria vaccine encoding CSP induced a humoral immune response in mice using an ELISA assay (Coating: [NANP]7 peptide, IgG total titer 35 days post prime (full details can be found in Example 7)). [Figure 8B](LNP for in vivo malaria vaccination - long term experiments) - shows that the tested mRNA malaria vaccine encoding CSP induced a very strong humoral immune response in mice using an ELISA assay (Coating: [NANP]7 peptide, IgG total titers 105 days post prime (full details can be found in Example 7)). [Figure 9] Organ distribution of PpLuc after intramuscular LNP injection - shows PpLuc expression in different tissue lysates after organ harvest of LNP-formulated mRNA encoding PpLuc at two different time points (4 hours and 24 hours). Expression levels are shown as pg of total luciferase protein / organ. Full details can be found in Example 12; the table below is the legend corresponding to FIG. 9.

[0096] [Table 1] [Figure 10] Organ distribution of PpLuc after intradermal LNP injection - shows PpLuc expression in different tissue lysates after organ harvest of LNP-formulated mRNA encoding PpLuc at two different time points (4 hours and 24 hours). Expression levels are shown as pg of total luciferase protein / organ; full details can be found in Example 13; the table below is the legend corresponding to FIG. 10.

[0097] [Table 2] [Figure 11] (Mouse immunization with tyrosine-related protein 2 (Trp2) formulated in LNPs containing PS) - shows that after peptide restimulation with the Trp2 immunodominant epitope, LNPs containing DPhyPS (closed squares) and LNPs containing DHPC (closed circles) showed significantly higher CD8+ T cell responses compared to LNPs without DPhyPS or DHPC (open circles); full details can be found in Example 14. [Figure 12](Mouse immunization with tyrosine-related protein 2 (Trp2) formulated in LNPs with PS or DHPC) - shows that LNPs containing DPhyPS (closed squares) and LNPs containing DHPC (closed circles) showed significantly higher IgG2a endpoint titers compared to LNPs without DPhyPS or DHPC (open circles); full details can be found in Example 14. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0098] The present disclosure will be described in detail below, but it should be understood that the present disclosure is not limited to the specific methodology, protocol and reagent described herein, which may vary.It should also be understood that the terms used herein are for the purpose of describing specific embodiments only, and are not intended to limit the scope of the present disclosure, which is limited only by the scope of the appended claims.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as technical and scientific terms commonly understood by those skilled in the art.

[0099] The present invention is based on the inventors' surprising discovery that phosphatidylserine-containing nucleic acid-based vaccines (particularly vaccines that contain nucleic acids at least partially encapsulated by lipid nanoparticles, where the lipid nanoparticles contain phosphatidylserine) have improved properties compared to vaccines that do not contain the phospholipid phosphatidylserine. Without wishing to be bound by theory, the inventors currently believe that this may be due to the role of the phospholipid phosphatidylserine during apoptosis. Thus, while in healthy cells phosphatidylserine is exclusively located in the inner lipid layer of the lipid bilayer membrane of the cell, phosphatidylserine is transferred to the outer lipid layer of the lipid bilayer membrane during apoptosis, so that the head group of phosphatidylserine is exposed on the cell surface of apoptotic cells. This exposed head group of phosphatidylserine serves as a marker for the rapid uptake of apoptotic cells by cells of the immune system, i.e. phagocytes, in particular macrophages and dendritic cells. Thus, the marker phosphatidylserine may increase the process of phagocytosis of the vaccine composition and thus the process of introducing at least partially encapsulated nucleic acid into phagocytes, which are translated and ultimately result in the presence of the encoded antigen or fragments or variants thereof in the phagocytes. Again, without wishing to be bound by theory, the inventors currently believe that this may explain, inter alia, the observed targeting of phosphatidylserine-containing vaccines to the spleen, which is the center of activity of the mononuclear phagocyte system and serves as the main reservoir of lymphocytes (see Example 5 of the present application).

[0100] Phospholipid Phosphatidylserine The term "phosphatidylserine" as used herein relates to a compound consisting of a head group which is serine linked via a phosphodiester to a carbon atom of glycerine and one or more tail groups. Preferably, said tail group is a fatty acid linked via an ester to another carbon atom of glycerine. Preferably, the term "phosphatidylserine" as used herein relates to a compound consisting of a head group which is serine linked via a phosphodiester to a carbon atom of glycerine and one or more tail groups, wherein the tail group is a fatty acid linked via an ester to another carbon atom of glycerine. The fatty acid may be a saturated fatty acid preferably selected from the group consisting of caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, and cerotic acid. The fatty acid may also be an unsaturated fatty acid, preferably selected from the group consisting of myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, alpha-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, and docosahexanoic acid. The fatty acid may also be a branched chain fatty acid, in particular phytanic acid.

[0101] Examples are given below, for example, DPhyPS, WT-PS (i.e., 1-stearoyl-2-oleoyl-sn-glycero-3-phospho-L-serine or 18:0-18:1PS, with two different fatty acid / alkyl chains of WT-PS, widely distributed among animals, plants, and microorganisms), 16:0-PS, 14:0-PS, 10:0-PS, 6:0-PS, and 18:1-PS DOPS, where serine is linked to the first carbon atom of glycerol via a phosphodiester, and the second and third carbon atoms of glycerol are each linked to a fatty acid via an ester. In this arrangement, the two fatty acids may be the same (see, for example, DPhyPS, 16:0PS, 14:0-PS, 10:0-PS, 6:0-PS, and 18:1-PS DOPS) or different (see, for example, WT-PS or 18:0-18:1PS). In other examples, for example, 18:1-Lyso PS and 18:0-Lyso PS, serine is also linked to the first carbon atom of glycerol via phosphodiester, and only one additional carbon atom of glycerol is linked to fatty acid via ester, leaving a single OH group on the remaining carbon atom of glycerol.This arrangement is typically called "lysophosphatidylserine", which, in the light of the above definition, is included in the term "phosphatidylserine" used herein.

[0102] In a preferred embodiment, the phosphatidylserine is selected from the group consisting of DPhyPS, WT-PS, 16:0-PS, 14:0-PS, 10:0-PS, 6:0-PS, 18:1-PS DOPS, 18:1-Lyso PS, and 18:0-Lyso PS. Most preferably, the phosphatidylserine is either DPhyPS or WT-PS (18:0-18:1PS).

[0103] The structures of the above phosphatidylserines are as follows (note that all of these lipids are commercially available, e.g., from Avanti Polar Lipids):

[0104] [ka]

[0105] DPhyPS

[0106] [ka]

[0107] WT-PS (18:0-18:1 PS)

[0108] [ka]

[0109] 16:0 PS

[0110] [ka]

[0111] 14:0 PS

[0112] [ka]

[0113] 10:0 PS

[0114] [ka]

[0115] 6:0 PS

[0116] [ka]

[0117] 18:1 PS DOPS

[0118]

change

[0119] 18:1 Lyso PS

[0120]

change

[0121] 18:0 Lyso PS Further examples of saturated phosphatidylserines include 1,2-dilauroyl-sn-glycero-3-phosphoserine (DLPS), 1,2-dimyristoyl-sn-glycero-3-phosphoserine (dimyristoylphosphatidylserine; DMPS), 1,2-distearoyl-sn-glycero-3-phosphoserine (distearoylphosphatidylserine; DSPS), 1,2-dipalmitoyl-sn-glycero-3-phosphoserine (dipalmitoylphosphatidylserine; DPPS), 1-myristoyl-2-palmitoyl-s Examples of phosphatidylserine include n-glycero-3-phosphoserine (MPPC), 1-palmitoyl-2-myristoyl-sn-glycero-3-phosphoserine (PMPS), 1-myristoyl-2-stearoyl-sn-glycero-3-phosphoserine (MSPS), 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphoserine (PSPS), 1-stearoyl-2-palmitoyl-sn-glycero-3-phosphoserine (SPPS), and 1-stearoyl-2-myristoyl-sn-glycero-3-phosphoserine (SMPS). In some embodiments, the phosphatidylserine comprises a stearoyl (18:0) moiety, an oleoyl (18:1) moiety, an eicosatetraenoyl (20:4) moiety, a docosahexaenoyl (22:06) moiety, or a combination thereof. In another embodiment, the PS is L-α-phosphatidylserine (brain, porcine; CAS. Registry No. 383907-32-2).

[0122] Cationic or Ionizable Lipids The cationic or ionizable lipid of LNP is cationizable, i.e., it protonates when the pH is decreased below the pK of the ionizable group of the lipid, but becomes progressively more neutral at higher pH values.At pH values ​​below the pK, the lipid can associate with negatively charged nucleic acid.In certain embodiments, the cationic lipid comprises a zwitterionic lipid, which becomes positively charged at a decreased pH.

[0123] Preferred cationic lipids are defined as compounds according to formula (Cat-I): R a -AR bFormula (Cat-I) (In the formula, R a teeth

[0124] [ka]

[0125] or -R 1 -N(H)-C(O)-R 3 -R 4 Selected from; R b teeth

[0126] [ka]

[0127] -R 1 -N(H)-C(O)-R 3 -R 4 ,or -R 1 -N(CH 3 ) 2 ; Selected from; A is -S-, -SS-, -NH-C(O)-, -NH-C(O)O-, -NH-C(O)-NH-, -SC(O)-N(H)-, -C(O)O-, or -OP(O)(OH)-O-; R 1 is optionally substituted ethanediyl, propanediyl, butanediyl, or a straight-chain or branched alkanediyl having 2 to 8 carbon atoms; R 2 is an alkanediyl having 2 to 8 carbon atoms; R 3 is optional and, if present, -R 5 -C(O)-O-, -R 5 -OC(O)-, -R 5 -C(O)-NH-, -R 5 -OC(O)-NH-, or R 5 -NH-C(O)O-; R 4 is a lipophilic substituent having 12 to 36 carbon atoms; R 5 is an alkanediyl having 1 to 6 carbon atoms; X is a carbon atom or a nitrogen atom; All choices are independent of each other. However, in some cases, R 1 , R 2 , and R 5 are all straight-chain unsubstituted ethanediyl, A is -SS-, and R a and R b If are identical, R 4 teeth

[0128] [ka]

[0129] isn't it). In other embodiments, R 4 teeth

[0130] [ka]

[0131] It is. In one preferred embodiment, A is -S- and R a and R b are identical, and R 4 teeth

[0132] [ka]

[0133] It is. In another preferred embodiment, A is -S- and R 4 teeth

[0134] [ka]

[0135] It is. R in formula (Cat-I) 4 R is defined as a lipophilic substituent having 12 to 36 carbon atoms. a and possibly also R b (R b -R 1 -N(CH 3 ) 2 This "tail" end of the R (unless otherwise specified) is believed to provide the degree of lipophilicity typically required for the molecule to be able to cross biological membranes. 4 R can in principle be any structure that is substantially lipophilic. For example, hydrocarbon structures are lipophilic. In one embodiment, R 4 may consist exclusively of carbon and hydrogen atoms in at least one of its occurrences. In one preferred embodiment, R 4 represents a linear or branched alkyl or alkenyl, preferably having 12 to 25 carbon atoms. The branched alkyl or alkenyl may optionally have multiple side chains, such as two, three, four or more methyl side chains. In another embodiment, R 4 can be, for example, an alkyl or alkenyl containing a single alkyl or alkenyl side chain having from 2 to 10 carbon atoms. For example, R 4 can be 1-n-hexyl-n-nonyl (or 7-n-pentadecyl), or 2-n-hexyl-n-decyl. In other embodiments, the lipophilic substituent can optionally contain one or more heteroatoms such as O, S, or N. In other embodiments, the lipophilic substituent can optionally contain one or more saturated, unsaturated, or aromatic ring structures, which can optionally contain one or more heteroatoms such as O, S, or N.

[0136] R 4 may also contain a small number of heteroatoms, such as oxygen atoms, so long as the predominantly lipophilic nature is maintained. 4 R contains one or more oxygen atoms and no other heteroatoms.4 may also include cyclic structures, such as aromatic or aliphatic ring structures, optionally containing one or more oxygen atoms. When present, the heteroatoms and / or cyclic structures are located at the optional R 3 In one embodiment, R 4 is a lipophilic group derived from tocopherol or tocotrienol. In one embodiment, R 1 , R 2 , and R 5 are not all linear unsubstituted ethanediyl, A is -SS-, and R a and R b If are identical, R 4 is a lipophilic group derived from alpha-tocopherol, especially

[0137] [ka]

[0138] It is. The "lipophilic group derived from tocopherol or tocotrienol" referred to herein includes derivatives of tocopherol and tocotrienol, particularly those having the structure shown in Scheme 1 below, i.e., derivatives derived from alpha-tocopherol, beta-tocopherol, gamma-tocopherol, delta-tocopherol, alpha-tocotrienol, beta-tocotrienol, gamma-tocotrienol and delta-tocotrienol.

[0139] [ka]

[0140] [Table 3]

[0141] Scheme 1: Derivatives of tocopherol have saturated phytyl chains and derivatives of tocotrienol have polyunsaturated phytyl chains. For both derivatives of tocopherol and derivatives of tocotrienol, the isoforms are CH 3 and H. 1 and R 2 Thus, as shown, for example, R 1 CH 3 And R 2 CH 3 The resulting derivatives are the alpha isoforms of tocopherol and tocotrienol, respectively (called alpha-tocopherol and alpha-tocotrienol derivatives, respectively). The OH group is naturally absent in the derivatives, since it is the point of attachment, as shown on the left in the two structures.

[0142] In other preferred embodiments, at least one nucleic acid (e.g., DNA or RNA), preferably at least one mRNA, of the composition is complexed with one or more lipids, thereby forming an LNP, wherein the cationic lipid of the LNP is selected from lipids derived from structures C1 to C23, or C1 to C27, respectively, or formula (I) of Table 1 of PCT patent application PCT / EP2019 / 086825 or any subsequent patent application claiming priority to PCT / EP2019 / 086825, i.e. WO2021123332. In other embodiments, at least one nucleic acid (e.g., DNA or RNA), preferably at least one mRNA, of the composition is complexed with one or more lipids, thereby forming a LNP, wherein the cationic lipid of the LNP is derived from structures C1-C23, or C1-C27, respectively, of Table 1 of PCT patent application PCT / EP2019 / 086825 or any subsequent patent application claiming priority to PCT / EP2019 / 086825, i.e., WO2021123332, and element "A" of formula (I) of PCT / EP2019 / 086825 is -S-. Accordingly, formulas C1 to C23, or C1 to C27, respectively, of PCT / EP2019 / 086825 or any subsequent patent application claiming priority to PCT / EP2019 / 086825, i.e., WO 2021123332, and the specific disclosures relating thereto are incorporated herein by reference.

[0143] In still further embodiments, the cationic lipid is preferably selected from the cationic lipids listed herein in Table 1.

[0144] [Table 4-1]

[0145] [Table 4-2]

[0146] [Table 4-3]

[0147] [Table 4-4]

[0148] [Table 4-5]

[0149] [Table 4-6]

[0150] [Table 4-7]

[0151] [Table 4-8]

[0152] [Table 4-9]

[0153] [Table 4-10]

[0154] [Table 4-11]

[0155] Therefore, the present invention includes a vaccine composition comprising the above-mentioned cationic lipid. For example, the composition may comprise a cationic lipid selected from compounds C1 to C27 in Table 1. In another preferred embodiment, at least one nucleic acid (e.g., DNA or RNA), preferably at least one mRNA, of the composition is complexed with one or more lipids, thereby forming a LNP, and the cationic lipid of the LNP has the structure "C24," which is the most preferred structure for the cationic lipid.

[0156] [ka]

[0157] (C24 or THIOETHER). Cationic, ionizable or cationizable lipids include, but are not limited to, DSDMA, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), 1,2-dioleoyltrimethylammonium propane chloride (DOTAP) (also known as N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride and 1,2-dioleyloxy-3-trimethylaminopropane chloride salt). (1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), ckk-E12, ckk, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-di-y-linolenyloxy-N,N-dimethylaminopropane (γ-DLenDMA), 98N12-5, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (γ-DLenDMA), DLin-C-DAP, 1,2-Dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3 -Dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), ICE (imidazole based), HGT5000, HGT5001, DMDMA, CLinDMA, CpLinDMA, DMOBA, DOcarbDAP, DLincarbDAP, DLinCDAP, KLin-K-DMA, DLin-K-XTC2-DMA, XTC (2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane), HGT4003, 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-1,2-propanediol(propanedio) (DOAP), 1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DM A), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or its analogues, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine, En-19-yl-4-(dimethylamino)butanoate (MC3), ALNY-100 ((3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine)), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amine) 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), NC98-5 (4,7,13-tris(3-oxo-3-(undecylamino) propyl)-N1,N16-diundecyl-4,7,10,13-tetraazahexadecane-l,16-diamide), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-M-C3-DMA), 3-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylpropan-1-amine (MC3 ether), 4-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylbutan-1-amine (MC4 ether), LIPOFECTIN® (commercially available cationic liposomes containing DOTMA and 1,2-dioleoyl-sn-3 phospho-ethanolamine (DOPE), GIBCO / BRL, Grand Island, NY, USA)

[0036] LIPOFECTAMINE® (a commercially available cationic liposome comprising N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE), available from GIBCO / BRL; and TRANSFECTAM® (a commercially available cationic lipid comprising dioctadecylamidoglycylcarboxyspermine (DOGS) in ethanol, available from Promega Corp., Madison, Wisconsin, USA), or any combination of any of the foregoing. Further suitable cationic lipids for use in the compositions and methods of the invention include the cationic lipids described in International Patent Publications WO2010053572 (and in particular CI2-200 described in paragraph

[0225] ) and WO2012170930 (both of which are incorporated herein by reference), HGT4003, HGT5000, HGTS001, HGT5001, HGT5002 (see U.S. Patent Publication No. 20150140070).

[0158] In embodiments, the cationic lipid may be an amino lipid. Representative amino lipids include, but are not limited to, 1,2-dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1, 2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), and 2,2 -Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA); Dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA); MC3 (U.S. Patent Application Publication No. 20100324120).

[0159] In embodiments, the cationic lipid may be an amino alcohol lipidoid. Aminoalcohol lipidoids that may be used in the present invention may be prepared by the methods described in U.S. Patent No. 8,450,298, which is incorporated herein by reference in its entirety. Suitable (ionizable) lipids may also be the compounds disclosed in Tables 1, 2 and 3 of WO2017075531, which is incorporated herein by reference, and as defined in claims 1 to 24 thereof.

[0160] In another embodiment, suitable lipids may also be compounds disclosed in WO2015074085 (i.e., ATX-001 to ATX-032 or compounds specified in claims 1 to 26), U.S. Patent Application Publication Nos. 61 / 905,724 and 15 / 614,499, or U.S. Patent Nos. 9,593,077 and 9,567,296, the entireties of which are incorporated herein by reference.

[0161] In other embodiments, suitable cationic lipids may also be compounds disclosed in WO2017117530, which is incorporated herein by reference in its entirety (i.e., lipids 13, 14, 15, 16, 17, 18, 19, 20, or compounds specified in the claims).

[0162] In a preferred embodiment, the ionizable or cationic lipid may also be selected from the lipids disclosed in WO 2018078053 (i.e., lipids derived from formulas I, II, and III of WO 2018078053, or lipids specified in claims 1-12 of WO 2018078053) (the entire disclosure of WO 2018078053 is incorporated herein by reference). In that context, the lipids disclosed in Table 7 of WO 2018078053 (e.g., lipids derived from formulas I-1 to I-41) and the lipids disclosed in Table 8 of WO 2018078053 (e.g., lipids derived from formulas II-1 to II-36) may be suitably used in the context of the present invention. Therefore, Formulas I-1 to I-41 and II-1 to II-36 of WO 2018078053, and specific disclosures related thereto, are incorporated herein by reference.

[0163] In a preferred embodiment, the cationic lipid can be derived from formula III of published PCT patent application WO2018078053. Accordingly, formula III of WO2018078053 and the specific disclosures related thereto are incorporated herein by reference.

[0164] In a particularly preferred embodiment, at least one nucleic acid (e.g., DNA or RNA), preferably at least one mRNA, of the composition is complexed with one or more lipids, thereby forming an LNP, wherein the cationic lipid of the LNP is selected from structures III-1 to III-36 in Table 9 of published PCT patent application WO 2018078053. Accordingly, formulas III-1 to III-36 of WO 2018078053, and the specific disclosures relating thereto, are incorporated herein by reference.

[0165] In a particularly preferred embodiment of the second aspect, at least one nucleic acid (e.g., DNA or RNA), preferably at least one mRNA, is complexed with one or more lipids, thereby forming an LNP, the LNP comprising:

[0166] [ka]

[0167] Or most preferably of formula III-3 of WO 2018078053, i.e. (4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate):

[0168] [ka]

[0169] The cationic lipids include those based on In certain embodiments, a cationic lipid as defined herein, more preferably cationic lipid compound III-3 ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)), is present in the LNP in an amount of about 30 to about 80 mole percent, preferably about 30 to about 60 mole percent, more preferably about 40 to about 55 mole percent, more preferably about 47.4 mole percent, relative to the total lipid content of the LNP. When more than one cationic lipid is incorporated within the LNP, such percentages apply to the combined cationic lipids.

[0170] In an embodiment, the cationic lipid is present in the LNP in an amount of about 30 to about 70 mole percent. In one embodiment, the cationic lipid is present in the LNP in an amount of about 40 to about 60 mole percent, e.g., about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 mole percent, respectively. In an embodiment, the cationic lipid is present in the LNP in an amount of about 47 to about 48 mole percent, e.g., about 47.0, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9, 50.0 mole percent, respectively, with 47.4 mole percent being particularly preferred.

[0171] In some embodiments, the cationic lipid is present in a ratio of about 20 mol% to about 70 or 75 mol%, or about 45 to about 65 mol%, or about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or about 70 mol% of the total lipid present in the LNP. In further embodiments, the LNP comprises about 25 mol% to about 75 mol% on a molar basis, e.g., about 20 to about 70%, about 35 to about 65%, about 45 to about 65%, about 60%, about 57.5%, about 57.1%, about 50%, or about 40% cationic lipid on a molar basis (based on 100% total moles of lipid in the lipid nanoparticle). In some embodiments, the ratio of cationic lipid to nucleic acid (e.g., coding RNA or DNA) is about 3 to about 15, e.g., about 5 to about 13 or about 7 to about 11.

[0172] Other suitable (cationic or ionizable) lipids are described in WO 2009086558, WO 2009127060, WO 2010048536, WO 2010054406, WO 2010088537, WO 2010129709, WO 2011153493, WO 2013063468, U.S. Patent Application Publication No. 20110256175, U.S. Patent Application Publication No. 20120 No. 128760, U.S. Patent Application Publication No. 20120027803, U.S. Patent No. 8158601, International Publication No. 2016118724, International Publication No. 2016118725, International Publication No. 2017070613, International Publication No. 2017070620, International Publication No. 2017099823, International Publication No. 2012040184, International Publication No. 2011153120, International Publication No. 2011149733, International Publication No. 2011090965, International Publication No. 2011043913, International Publication No. 2011022460, International Publication No. 2012061259, International Publication No. 2012054365, International Publication No. 2012044638, International Publication No. 2010080724, International Publication No. 201021865, International Publication No. 2008103276, International Publication No. 2013086373, International Publication No. 2013086354, U.S. Patent Nos. 7,893,302, 7,404,969, and 7,404,969. Nos. 8,283,333, 8,466,122 and 8,569,256, as well as U.S. Patent Application Publication Nos. 20100036115, 20120202871, 20130064894, 20130129785, 20130150625, 20130178541, 20130225836, 20140039032, and WO 2017112865.In that context, the following publications are specifically related to (cationic) lipids suitable for LNPs: WO 2009086558, WO 2009127060, WO 2010048536, WO 2010054406, WO 2010088537, WO 2010129709, WO 2011153493, WO 2013063468, U.S. Patent Application Publication No. 20110256175, U.S. Patent Application Publication No. 2012010306211, U.S. Patent Application Publication No. 20110256212, U.S. Patent Application Publication No. 20110256213, U.S. Patent Application Publication No. 20110256214, U.S. Patent Application Publication No. 20110256215, U.S. Patent Application Publication No. 20110256216, U.S. Patent Application Publication No. 20110256217, U.S. Patent Application Publication No. 20110256218, U.S. Patent Application Publication No. 20110256219 ... No. 0120128760, U.S. Patent Application Publication No. 20120027803, U.S. Patent No. 8158601, WO 2016118724, WO 2016118725, WO 2017070613, WO 2017070620, WO 2017099823, WO 2012040184, WO 2011153120, WO 2011149733, WO 2011090965, International Publication No. 2011043913, International Publication No. 2011022460, International Publication No. 2012061259, International Publication No. 2012054365, International Publication No. 2012044638, International Publication No. 2010080724, International Publication No. 201021865, International Publication No. 2008103276, International Publication No. 2013086373, International Publication No. 2013086354, U.S. Patent Nos. 7,893,302, 7,404,969, 8,2 The disclosures of U.S. Patent Application Publication Nos. 83,333, 8,466,122 and 8,569,256, as well as U.S. Patent Application Publication Nos. 20100036115, 20120202871, 20130064894, 20130129785, 20130150625, 20130178541, 20130225836 and 20140039032, and International Publication No. WO 2017112865 are incorporated by reference herein.

[0173] In other embodiments, the cationic or ionizable lipid is

[0174] [ka]

[0175] [ka]

[0176] It is. In embodiments, the amino or cationic lipids defined herein have at least one protonatable or deprotonatable group such that the lipid is positively charged at a pH below physiological pH (e.g., pH 7.4) and neutral at a second pH, preferably above physiological pH. Of course, it will be understood that the addition or removal of protons as a function of pH is an equilibrium process, and reference to charged or neutral lipids refers to the nature of the predominant species and does not require that all of the lipids must be present in a charged or neutral form. Lipids having two or more protonatable or deprotonatable groups or being zwitterionic are not excluded and may be suitable in the context of the present invention as well. In some embodiments, the protonatable lipid has a pKa of the protonatable group in the range of about 4 to about 11, e.g., a pKa of about 5 to about 7.

[0177] LNPs can contain two or more (different) cationic lipids as defined herein. Cationic lipids can be selected to contribute various advantageous properties. For example, cationic lipids with different properties such as amine pKa, chemical stability, circulation half-life, tissue half-life, net accumulation in tissue, or toxicity can be used in LNPs. In particular, cationic lipids can be selected so that the properties of mixed LNPs are more desirable than the properties of single LNPs of individual lipids.

[0178] The amount of persistent cationic lipid, lipidoid or preferably ionizable cationic lipid can be selected taking into account the amount of nucleic acid cargo. In one embodiment, these amounts are selected to provide an N / P ratio of the nanoparticle or composition in the range of about 0.1 to about 20, or (i) an amount to achieve an N / P ratio in the range of from about 1 to about 20, preferably from about 2 to about 15, more preferably from about 3 to about 10, even more preferably from about 4 to about 9, and most preferably about 6; (ii) an amount to achieve an N / P ratio in the range of from about 5 to about 20, more preferably from about 10 to about 18, even more preferably from about 12 to about 16, and most preferably about 14; (iii) an amount to achieve a lipid:mRNA weight ratio in the range of 20 to 60, preferably about 3 to about 15, 5 to about 13, about 4 to about 8, or about 7 to about 11; or (iv) an amount to achieve an N / P ratio in the range of about 6 for lipid nanoparticles according to the invention, particularly lipid nanoparticles comprising cationic lipid III-3; or (v) for lipid nanoparticles according to the invention, preferably in an amount to achieve an N / P ratio in the range of about 17 or about 17.5 is selected.

[0179] In this context, the N / P ratio is defined as the molar ratio of the nitrogen atom ("N") of the basic nitrogen-containing group of the lipid or lipidoid to the phosphate group ("P") of the nucleic acid used as cargo. The N / P ratio can be calculated, for example, on the basis that 1 μg of RNA typically contains about 3 nmol of phosphate residues, provided that the RNA exhibits a statistical distribution of bases. The "N" value of a cationic lipid or lipidoid can be calculated based on its molecular weight and the relative content of persistent cationic groups and, if present, cationizable groups. If more than one cationic lipid is present, the N value should be calculated based on all cationic lipids contained in the lipid nanoparticle.

[0180] In other embodiments, the ionizable lipid of the present disclosure is a compound of formula (Cat-II):

[0181] [ka]

[0182] or their N-oxides, or their salts or isomers (In the formula, R 1 is C5-30 alkyl, C5-20 alkenyl, -R *selected from the group consisting of -YR'', -YR'', and -R''M'R''; R 2 and R 3 are independent, H, C 1~14 Alkyl, C 2~14 Alkenyl, -R * YR'', -YR'', and -R * OR″, or R 2 and R 3 form a heterocyclic or carbocyclic ring together with the atoms to which they are attached; R 4 is hydrogen, C 3~6 Carbocycle, -(CH 2 ) n Q, -(CH 2 ) n CHQR, -CHQR, -CQ(R) 2 , and unsubstituted C 1~6 alkyl, and Q is carbocycle, heterocycle, -OR, -O(CH 2 ) n N(R) 2 , -C(O)OR, -OC(O)R, -CX 3 , -CX 2 H, -CXH 2 , -CN, -N(R) 2 , -C(O)N(R) 2 , -N(R)C(O)R, -N(R)S(O) 2 R, -N(R)C(O)N(R) 2 , -N(R)C(S)N(R) 2 , -N(R)R 8 , -N(R)S(O) 2 RS, -O(CH 2 ) n OR, -N(R)C(=NR 9 )N(R) 2 , -N(R)C(=CHR 9 )N(R) 2 , -OC(O)N(R) 2 , -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O) 2 R, -N(OR)C(O)OR, -N(OR)C(O)N(R) 2 , -N(OR)C(S)N(R)2 , -N(OR)C(=NR 9 )N(R) 2 , -N(OR)C(=CHR 9 )N(R) 2 , -C(=NR 9 )N(R) 2 , -C(=NR 9 )R, -C(O)N(R)OR, and -C(R)N(R) 2 C(O)OR, where each n is independently selected from 1, 2, 3, 4, and 5; Each R 5 are independent, C 1~3 Alkyl, C 2~3 alkenyl, and H; Each R 6 are independent, C 1~3 Alkyl, C 2~3 alkenyl, and H; M and M' are independently -C(O)O-, -OC(O)-, -OC(O)-M''-C(O)O-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O). 2 -, -SS-, an aryl group, and a heteroaryl group; M'' is a bond, C 1~13 Alkyl, or C 2~13 alkenyl; R 7 is C 1~3 Alkyl, C 2~3 alkenyl, and H; R 8 is C 3~6 selected from the group consisting of carbocycle and heterocycle; R 9 H, CN, NO 2 , C 1~6 Alkyl, -OR, -S(O) 2 R, -S(O) 2 N(R) 2 , C 2~6 Alkenyl, C 3~6 selected from the group consisting of carbocycle and heterocycle; Each R is independently C1~3 Alkyl, C 2~3 alkenyl, and H; Each R' is independently C 1~18 Alkyl, C 2~18 Alkenyl, -R * selected from the group consisting of YR'', -YR'', and H; Each R'' is independently C 3~15 Alkyl and C 3~15 alkenyl; Each R * is independently C 1~12 Alkyl and C 2~12 alkenyl; Each Y is independently C 3~6 It is a carbocyclic ring; each X is independently selected from the group consisting of F, Cl, Br, and I; m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; R 4 Ga-(CH 2 ) n Q, -(CH 2 ) n CHQR, -CHQR, or -CQ(R) 2 (i) when n is 1, 2, 3, 4 or 5, Q is -N(R) 2 or (ii) when n is 1 or 2, Q is not a 5-, 6-, or 7-membered heterocycloalkyl. may be one or more of:

[0183] As used herein, the term "ionizable lipid" has its ordinary meaning in the art and may refer to a lipid that includes one or more charged moieties. In some embodiments, the ionizable lipid may be positively or negatively charged. The ionizable lipid may be positively charged, in which case it may be referred to as a "cationic lipid". In certain embodiments, the ionizable lipid molecule may include an amine group, which may be referred to as an ionizable amino lipid. As used herein, a "charged moiety" is a chemical moiety that has a formal charge, e.g., monovalent (+1 or -1), divalent (+2 or -2), trivalent (+3 or -3), etc. A charged moiety may be anionic (i.e., negatively charged) or cationic (i.e., positively charged). Examples of positively charged moieties include amine groups (primary, secondary and / or tertiary amines), ammonium groups, pyridinium groups, guanidine groups, and imidazolium groups. In certain embodiments, the charged moiety comprises an amine group. Examples of negatively charged groups or precursors thereof include carboxylate, sulfonate, sulfate, phosphonate, phosphate, hydroxyl, and the like. The charge of a charged moiety may in some cases change with environmental conditions, e.g., a change in pH may change the charge of the moiety and / or cause the moiety to become charged or uncharged. In general, the charge density of a molecule may be selected as desired. It should be understood that the term "charged" or "charged moiety" does not refer to a "partial negative charge" or a "partial positive charge" on a molecule. The terms "partial negative charge" and "partial positive charge" are given their ordinary meaning in the art.A "partial negative charge" can occur when a functional group contains a bond that is polarized such that electron density is pulled toward one atom of the bond, creating a partial negative charge on that atom. Those skilled in the art will generally recognize bonds that can be polarized in this way. In some embodiments, the ionizable lipid is an ionizable amino lipid, which may be referred to in the art as an "ionizable cationic lipid." In one embodiment, the ionizable amino lipid can have a positively charged hydrophilic head and a hydrophobic tail that are connected via a linker structure.

[0184] Further particularly preferred embodiments of the lipid nanoparticles of the present invention are given when the following combinations of excipients are used to formulate the lipid nanoparticles: 59 mol% of the C2 or C15 or C24 lipids disclosed in Table 1 as cationic lipids (i.e., HEXA-C5DE-PipSS, which is cationic lipid compound C2 of Table 1, HEXA-C5DE-PipC3SS, which is cationic lipid compound C15 of Table 1, or VitE-C4DE-piperidine-thioether, which is cationic lipid compound C24 of Table 1, respectively), 29.3 mol% of cholesterol as steroid, a total of 10 mol% of phosphatidylserine in combination with DPhyPE as additional neutral lipid / phospholipid, and 1.7 mol% of DMG-PEG2000 as polymer-conjugated lipid.

[0185] steroid A "steroid" is an organic compound that has four rings arranged in a specific molecular configuration. Steroids contain the following carbon skeleton:

[0186] [ka]

[0187] Steroids and neutral steroids include both naturally occurring steroids and their analogs (e.g., cholesteryl hemisuccinate (CHEMS), an amphiphilic lipid consisting of succinic acid esterified to the beta-hydroxyl group of cholesterol as a cholesterol derivative). Using the definition of "neutral" provided herein, a neutral steroid can be a steroid that has no atoms or groups that are ionizable under physiological conditions, or can be a zwitterionic steroid. In one preferred embodiment, a neutral steroid does not contain any atoms or groups that are ionizable under physiological conditions. In some preferred embodiments, the steroid or steroid analog is cholesterol. The terms "steroid" and "neutral steroid" are used interchangeably herein.In other embodiments, the sterol is a phytosterol, e.g., β-sitosterol, campesterol, stigmasterol, fucosterol, stigmastanol, dihydrocholesterol, ent-cholesterol, epicholesterol, desmosterol, cholestanol, cholestanone, cholestenone, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, 3β-[N-(N'N'-dimethylaminoethyl)carbamoylcholesterol (DC-Chol), 24(S)-hydroxycholine, 1,2-dimethyl- ... esterol, 25-hydroxycholesterol, 25(R)-27-hydroxycholesterol, 22-oxacholesterol, 23-oxacholesterol, 24-oxacholesterol, cycloartenol, 22-ketosterol, 20-hydroxysterol, 7-hydroxycholesterol, 19-hydroxycholesterol, 22-hydroxycholesterol, 25-hydroxycholesterol, 7-dehydrocholesterol, 5a-cholest-7-en-3β-ol, 3,6,9-trioxaoctan-1-ol-cholesteryl- 3e-ol, dehydroergosterol, dehydroepiandrosterone, lanosterol, dihydrolanosterol, lanostenol, lumisterol, cytocalciferol, calcipotriol, coprostanol, cholecalciferol, lupeol, ergocalciferol, 22-dihydroergocalciferol, ergosterol, brassicasterol, tomatidine, tomatine, ursolic acid, cholic acid, chenodeoxycholic acid, zymosterol, diosgenin, fucosterol The cholesterol ester may be selected from the group consisting of cholesteryl, fecosterol or fecosterol or salts or esters thereof, cholesterol, cholesterol succinate, cholesterol sulfate, cholesterol hemisuccinate, cholesterol phthalate, cholesterol phosphate, cholesterol valerate, cholesterol acetate, cholesteryl oleate, cholesteryl linoleate, cholesteryl myristate, cholesteryl palmitate, cholesteryl arachidate, cholesteryl phosphorylcholine, and sodium cholate.

[0188] In a further embodiment, the steroid is an imidazole cholesterol ester or "ICE" as disclosed in paragraphs

[0320] and

[0339] to

[0340] of International Publication No. 2019226925, the entirety of which is incorporated herein by reference.

[0189] More Phospholipids Further phospholipids, which may also be called "neutral lipids" or "helper lipids", are amphipathic compounds consisting of a molecule that typically has two hydrophobic fatty acid "tails" and a hydrophilic "head" that contains a phosphate group. The phosphate group may be modified with simple organic molecules such as choline, ethanolamine or serine. Phospholipids are abundant in nature. For example, phospholipids constitute a significant proportion of the additives of biological membranes. As used herein, the term "phospholipid" or "neutral phospholipid" encompasses both natural and synthetic phospholipids.

[0190] The terms "neutral lipid," "neutral phospholipid," or "zwitterionic compound," as used interchangeably herein, refer to any one of several lipid species that exist in either uncharged or neutral zwitterionic form at physiological pH. Representative neutral lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides, which are further described herein below.

[0191] According to one of the preferred embodiments, the composition comprises zwitterionic neutral lipid, such as phosphatidylcholine or phosphatidylethanolamine.Examples of suitable phosphatidylcholine include natural or purified mixtures, which are usually derived from egg yolk or soybean, and are sometimes called "lecithin" or "phosphatidylcholine"; or highly purified or semi-synthetic compounds, such as phosphatidylcholine with two fatty acyl moieties selected from myristoyl, palmitoyl, stearoyl, oleoyl, etc.

[0192] In another preferred embodiment, the neutral lipid or neutral phospholipid is, but is not limited to, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE; also known as 1,2-di-(3,7,11,15-tetramethylhexadecanoyl)-sn-glycero-3-phosphoethanolamine), 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhyPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC; also known as dioleoylphosphatidylcholine), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPhyP), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC; also known as dioleoylphosphatidylcholine), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPhyP), 1,2-dioleo ... Dioleoyl-sn-glycero-3-phosphocholine (DPPC, also known as dipalmitoylphosphatidylcholine), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), phosphatidylethanolamine, distearoylphosphatidylcholine, dioleoyl-phosphatidylethanolamine (DOPEA), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine ( POPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine (DLoPE), distearoylphosphatidylethanolamine (DSPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (DPPE), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), Phosphatidylethanolamine (POPE), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 16-O-monomethylphosphoethanolamine, 16-O-dimethylphosphatidylethanolamine, 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE), 18-1-transphosphatidylethanolamine, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), 1,2-disqualeoyl-sn-glycero-3-phosphoethanolamine (DSQPE), 1,2-Dielaidoyl-sn-glycero-3-phosphoethanolamine (trans-DOPE), 1-Stearoyl-2-linoleoyl-sn-glycero-3-phosphoethanolamine (SLPE), 1-Tridecanoyl-sn-glycero-3-phospho-L-serine (sodium salt), 1-Oleoyl-2-hydroxy-sn-glycero-3-phospho-L-serine (sodium salt), 1-Palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (sodium salt) Sodium salt) (POPS), 1-1-stearoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (sodium salt), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DOPS), 1,2-distearoyl-sn-glycero-3-phospho-L-serine (sodium salt), 1,2-diphytanoyl-sn-glycero-3-phospho-L-serine (sodium salt), 1-O-hexadecanyl-2-O-(9Z-oleoyl) 1,2-Di-O-phytanyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphatidylcholine or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-di-O-phytanyl-sn-glycero-3-phosphoethanolamine, 1-palmitoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (PChemsPC), 1,2-dicholesterylhemisuccinoyl The zwitterionic compound is selected from the group consisting of 1-O-octadecyl-sn-glycero-3-phosphocholine (DChemsPC), 2-((2,3-bis(oleoyloxy)propyl)dimethylammonio)ethyl hydrogen phosphate (DOCP), 2-((2,3-bis(oleoyloxy)propyl)dimethylammonio)ethyl ethyl phosphate (DOCPe), and 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (edelfosine).

[0193] In a preferred embodiment, the neutral lipid according to the invention is 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). In a more preferred embodiment, the neutral lipid according to the invention is 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhyPC). In an even more preferred, particularly preferred embodiment, the neutral lipid according to the invention is 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE). An advantage of the invention related to the use of DPhyPE is the high membrane fusion ability due to the bulky tail, which allows for a high level of fusion with endosomal lipids. In other words, another advantage of the invention related to the use of DPhyPE is the high membrane fusion ability due to the bulky tail, which allows for a high level of fusion with endosomal lipids. Thus, in another embodiment, the present invention provides lipid-based carriers or nucleic acid-lipid particles, preferably DPhyPE as shown herein:

[0194] [ka]

[0195] The present invention relates to the use of lipids having high membrane fusion properties in The terms "fusogenic" or "fusogenicity" are intended to refer to lipids that aid in the fusion of a lipid-based carrier or nucleic acid-lipid particle with a cell membrane, thereby aiding in the entry of the nucleic acid contained in the lipid-based carrier or nucleic acid-lipid particle into the cell.

[0196] DSPC, DOPC or DOPE, which are routinely used in the art as phospholipids in LNPs, each have the structure shown herein below:

[0197] [ka]

[0198] As is clear from the two C 18 It has a chain side arm. Surprisingly, in a further aspect of the present invention, the present inventors have found that the addition of phospholipids with shorter alkyl chains than, for example, the DSPC or DOPE of the prior art is highly beneficial to the efficacy of the lipid nanoparticles of the present invention comprising polymer-conjugated lipids according to formula (I).In particular, the advantageous use of (07:0)PC (DHPC; 1,2-diheptanoyl-sn-glycero-3-phosphocholine) with shorter alkyl chains than, for example, the DSPC of the prior art disclosed herein, preferably combined with the polymer-conjugated lipid of the present invention disclosed herein, to deliver mRNA vaccines in vivo and result in significantly enhanced immune responses, is a further very surprising discovery made by the present inventors and resembles a specific aspect and embodiment of the present invention.

[0199] The structure of (07:0)PC (DHPC; 1,2-diheptanoyl-sn-glycero-3-phosphocholine) is shown herein below:

[0200] [ka]

[0201] The inventors have further surprisingly found that the immune response can also be enhanced by adding at least one further neutral lipid, in particular a third neutral lipid, to the neutral lipid (see corresponding examples).As mentioned above, it is preferred for the further (second) neutral lipid of the present invention to have two fatty acyl moieties selected from myristoyl, palmitoyl, stearoyl, oleoyl, etc., which means that the fatty acyl moiety is a rather long moiety starting from a moiety with 14 carbon atoms.The inventors have found that the addition of a neutral lipid with a shorter fatty acyl moiety provides beneficial effects, especially when the further neutral lipid has two fatty acid moieties selected from pentanoyl, hexanoyl, heptanoyl, octanoyl, nonaoyl and decanoyl, i.e. a moiety with up to 10 carbon atoms. A particularly preferred additional neutral lipid is 1,2-diheptanoyl-sn-glycero-3-phosphocholine, although related neutral lipids such as 05:0PC (1,2-dipentanoyl-sn-glycero-3-phosphocholine), 06:0PC (1,2-dihexanoyl-sn-glycero-3-phosphocholine), 08:0PC (1,2-dioctanoyl-sn-glycero-3-phosphocholine), 09:0PC (1,2-dinonanoyl-sn-glycero-3-phosphocholine), and 10:0PC (1,2-dihexanoyl-sn-glycero-3-phosphocholine) can be used as well.

[0202] Thus, in one aspect of the present invention, the lipid nanoparticles of the present invention are 5 , C 6 , C 7 , C 8 , C 9 , C 10 , C 11 , C 12 , C 13 or C 14 Preferably, the length is C 6 , C 7 , C 8 , C 9 , or C 10 More preferably, the length of 6 , C 7, C 8 Most preferably C 7 In another embodiment of the present invention, the lipid nanoparticles of the present invention comprise a neutral lipid or phospholipid having at least one alkyl chain having a length of 5 , C 6 , C 7 , C 8 , C 9 , C 10 , C 11 , C 12 , C 13 or C 14 Preferably, the length is C 6 , C 7 , C 8 , C 9 , or C 10 More preferably, the length of 6 , C 7 , C 8 Most preferably C 7 In a preferred embodiment, the lipid nanoparticles of the present invention further comprise DHPC. In a further embodiment, one or more of the alkyl chains may include a carbon double bond.

[0203] In other embodiments, the lipid nanoparticles comprise an additional phospholipid selected from the group consisting of 05:0PC (1,2-dipentanoyl-sn-glycero-3-phosphocholine), 04:0PC (1,2-dibutyryl-sn-glycero-3-phosphocholine), 06:0PC (DHPC, 1,2-dihexanoyl-sn-glycero-3-phosphocholine), 08:0PC (1,2-dioctanoyl-sn-glycero-3-phosphocholine), and 09:0PC (1,2-dinonanoyl-sn-glycero-3-phosphocholine).

[0204] Polymer-conjugated lipids In some embodiments, the LNP comprises a lipid conjugate, preferably a polymer-conjugated lipid. The term "polymer conjugated lipid" refers to a molecule that comprises both a lipid portion and a polymer portion.

[0205] PEG-lipids The polymer-conjugated lipid can be a PEGylated lipid or a PEG-lipid. The term "PEGylated lipid" or "PEG-lipid" refers to a molecule that contains both a lipid portion and a polyethylene glycol portion. PEGylated lipids are known in the art and include PEG-DMG and the like.

[0206] In certain embodiments, the polymer-conjugated lipid is defined as a compound according to formula (I): PAL formula (I) where P is a hydrophilic polymer moiety, A is an optional linker or spacer, and L is a lipid moiety.

[0207] Hydrophilic polymer part P The hydrophilic polymer moiety P in the polymer-conjugated lipid according to formula (I) can be a polyethylene glycol ("PEG") moiety. In certain embodiments, the PEG moiety has an average molecular weight of 1 kDa to 3 kDa, e.g., 1.5 to 2.5 kDa, 1.7 to 2.3 kDa, 1.8 to 2.2 kDa, 1.9 to 2.1 kDa, or 2 kDa. Thus, the PEG can be PEG commonly known as "PEG2000" or "PEG2k", although the shorter "PEG1000" and longer "PEG3000" can also be used. The PEG moiety typically comprises a linear polymer chain, although in some embodiments, the PEG moiety can comprise a branched polymer chain. Alternatively, contemplated PEG-modified lipids include, but are not limited to, polyethylene glycol chains of up to 2 kDa, up to 3 kDa, up to 4 kDa, or up to 5 kDa in length covalently attached to the lipid.

[0208] In another embodiment, the hydrophilic polymer moiety P of the polymer-conjugated lipid may be a substantially hydrophilic polymer different from the hydrophilic polymer moieties described above, i.e., the hydrophilic polymer moiety P in the polymer-conjugated lipid may be based on poly(propylene oxide), poly(vinylpyrrolidone), poly(vinyl alcohol), poly-N-(2-hydroxypropyl)methacrylamide, the hesylation-process (according to PMID 24681396), the PASylation approach (i.e., proline-alanine-serine), the XTEN approach known in the art (i.e., peptide-based PEG), polysarcosine or poly(vinyl acetate).

[0209] Optional Linker or Spacer A The optional linker or spacer A in the polymer-conjugated lipid according to formula (I) can be any useful spacer structure, such as a spacer selected from spacers commonly found to be useful in PEGylated lipids, including, but not limited to, succinimide, amine, ether, ester, anhydride, aldehyde, ketone, amide, carbamate linkers, or combinations thereof.

[0210] Lipid part L The lipid moiety L in the polymer-conjugated lipid according to formula (I) can be derived from phospholipid, sphingolipid, or ceramide.As used herein, the expression "derived from phospholipid or ceramide" includes phospholipid and ceramide groups.Examples are polymer-conjugated lipids that contain phosphatidylethanolamine or phosphatidylglycerol moieties.

[0211] In certain embodiments, the polymer-conjugated lipid is a PEGylated lipid. In more specific embodiments, the polymer-conjugated lipid included in the composition of the present invention is a PEGylated diacylglycerol lipid (PEG-DAG); PEGylated ceramide lipid (PEG-Cer); PEGylated phosphatidylethanolamine lipid (PEG-PE); PEGylated succinate diacylglycerol lipid (PEG-S-DAG); PEGylated dialkoxypropyl carbamate lipid; 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol ("PEG-DMG" or "DMG-PEG"); 1,2-dicapryl-rac-glycero-3-methylpolyoxyethylene glycol (C 10 Diacylglycerol PEG; N-octanoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol) 2000]} (PEG-ceramide 8, C 8 -Ceramide-PEG, PEG-Cer8, C 8 PEG2000 ceramide, also known as ceramide 8PEG, contains N-octanoyl-D-erythro-sphingosine (d18:1 / 8:0); 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG); 2-mPEG2000-n,n ditetradecylacetamide; N-[(methoxypoly(ethylene glycol)2000)carbamyl]-1,2-dimyristyloxypropyl-3-amine (PEG-c-DMA); ω-methoxy(polyethoxy )ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate; a PEG-lipid disclosed in WO2018126084, WO2020093061, or WO2020219941 (all three references are incorporated herein by reference), a PEGylated cholesterol or PEGylated cholesterol derivative disclosed herein, and 2,3-di(tetradecanoxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate.

[0212] In further preferred embodiments, the lipid moiety L comprises one, two, three, four or more hydrophobic fatty acids ("tails" corresponding to aliphatic chains containing an even number of carbon atoms). In a more preferred embodiment, the lipid moiety L comprises two hydrophobic fatty acids ("tails") having the same or different number of carbon atoms.

[0213] Preferably, the lipid moiety L comprises a fatty acid ("tail") that comprises 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 carbon atoms, or a combination thereof. More preferably, the lipid moiety L comprises a fatty acid ("tail") that comprises 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 carbon atoms, or a combination thereof. In more specific embodiments, the lipid moiety L comprises a fatty acid ("tail") selected from the group consisting of caprylic or octanoic acid (8:0); capric acid (10:0); lauric acid (12:0); myristic acid (14:0); palmitic acid (16:0); stearic acid (18:0); arachidic acid (20:0); behenic acid (22:0); lignoceric acid (24:0); and cerotic acid (26:0).

[0214] In an even more preferred embodiment, the lipid moiety L comprises at least one fatty acid (the "tail") containing 8, 10 or 12 carbon atoms, preferably 8 or 10 carbon atoms.

[0215] In a further preferred embodiment, the composition comprises a polymer-conjugated lipid. -1,2-Dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 (DMG-PEG2000)

[0216] [ka]

[0217] Preferably, as used in the art, "DMG-PEG2000" is considered to be a mixture of 1,2-DMG PEG2000 and 1,3-DMG PEG2000 in a ratio of about 97:3.

[0218] In further particular embodiments, the composition comprises: -1,2-Dicapryl-rac-glycero-3-methylpolyoxyethylene glycol 2000 (C 10 -PEG2000

[0219] [ka]

[0220] and -N-Octanoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)2000]} (Cer8-PEG2000)

[0221] [ka]

[0222] The polymer conjugated lipid comprises a polymer conjugated lipid selected from the group consisting of: In a further embodiment, the composition has the following chemical structure:

[0223] [ka]

[0224] "C 8 The polymer conjugated lipid includes a polymer conjugated lipid selected from the group consisting of the following structures similar to "-PEG2000": In certain embodiments of the present invention, "C 10 Each composition disclosed herein, including "C-PEG2000," 10 -PEG2000" instead of "C 8 -PEG2000" can also be used to formulate the drug.

[0225] Thus, by way of example, the polymer-conjugated lipid, or respectively the lipid moiety L, may have two fatty acid tails ("tails") comprising saturated fatty acids, unsaturated fatty acids, or a combination thereof, such as Cer8-PEG2000, which comprises one saturated fatty acid chain (8:0; caprylic acid or octanoic acid, respectively) and one unsaturated fatty acid chain of different length having more than 8 carbon atoms.

[0226] POZ-Lipid The polymer-conjugated lipid may be a POZ-lipid defined as a compound according to formula (II): [H]-[Linker]-[M] Formula (II) (In the formula, [H] is at least one polyoxazoline (POZ) monomer unit.

[0227] [ka]

[0228] (R is C 1~9 Alkyl or C 2~9 alkenyl, where n has an average value in the range of 2 to 200, preferably 20 to 100, more preferably 24 to 26 or 45 to 50. is a homopolymer moiety comprising [linker] is an optional linker group, [M] is the lipid moiety).

[0229] In an embodiment, [H] is Poly(2-methyl-2-oxazoline) (PMOZ)

[0230] [ka]

[0231] Poly(2-ethyl-2-oxazoline) (PEOZ)

[0232] [ka]

[0233] Poly(2-propyl-2-oxazoline) (PPOZ)

[0234] [ka]

[0235] Poly(2-butyl-2-oxazoline) (PBOZ)

[0236] [ka]

[0237] Poly(2-isopropyl-2-oxazoline) (PIPOZ)

[0238] [ka]

[0239] Poly(2-methoxymethyl-2-oxazoline) (PMeOMeOx), and Poly(2-dimethylamino-2-oxazoline) (PDMAOx) a heteropolymer or homopolymer moiety comprising a plurality of monomer units selected from the group consisting of Preferably, [H] is a homopolymer moiety comprising a plurality of PMOZ or PEOZ monomer units, more preferably, [H] comprises a plurality of PMOZ monomer units, or preferably consists of a plurality of PMOZ monomer units; (i) n has an average value in the range of 2 to 200, preferably 20 to 100, more preferably 24 to 26 or 45 to 50; or (ii) n is selected so that the [H] moiety has an average molecular weight of 1.5 to 22 kDa, more preferably 2 to 19 kDa, even more preferably about 7.5 kDa or about 15 kDa, preferably 1 to 15 kDa, more preferably 2 to 12.5 kDa, more preferably about 5 kDa or about 10 kDa.

[0240] In another embodiment, [H] is

[0241] [ka]

[0242] The monomer unit is a heteropolymer or homopolymer moiety comprising a plurality of monomer units selected from the group consisting of: In yet another embodiment, the [H] of the polymer-conjugated lipid according to formula (II) is selected from the group consisting of poly(2-methoxymethyl-2-oxazoline) (PMeOMeOx) and poly(2-dimethylamino-2-oxazoline) (PDMAOx).

[0243] In still further embodiments, the polymer conjugated lipid according to formula (II) is selected from the group consisting of POZ-monoacylglycerol conjugates, POZ-diacylglycerol conjugates, POZ-dialkyloxypropyl conjugates, POZ-steroid or POZ-sterol conjugates, POZ-phospholipid conjugates, POZ-ceramide conjugates, PMOZ-lipids as shown in FIG. 4, and mixtures thereof.

[0244] In one embodiment, the lipid moiety [M] shown in formula (II) comprises at least one linear or branched, saturated or unsaturated alkyl chain containing 6 to 30 carbon atoms, preferably the lipid moiety [M] comprises at least one linear or branched saturated alkyl chain, the alkyl chain being optionally interrupted by one or more biodegradable groups and / or optionally comprising one terminal biodegradable group, the biodegradable group being selected from the group consisting of, but not limited to, pH-sensitive moieties, zwitterionic linkers, non-ester-containing Linker moieties and ester-containing linker moieties (-C(O)O- or -OC(O)-), amide (-C(O)NH-), disulfide (-SS-), carbonyl (-C(O)-), ether (-O-), thioether (-S-), oxime (e.g., -C(H)=NO- or -ON=C(H)-), carbamate (-NHC(O)O-), urea (-NHC(O)NH-), succinyl (-(O)CCH2CH2C(O)-), succinamidyl (-NHC(O)CH2CH2C(O)NH-), -C(R 5 )=N-, -N=C(R 5 )-, -C(R 5 )=NO-, -ON=C(R 5 )-, -OC(O)O-, -C(O)N(R 5 ), -N(R 5 )C(O)-, -C(S)(NR 5 )-, (NR 5 )C(S)-, -N(R 5 )C(O)N(R 5 )-, -C(O)S-, -SC(O)-, -C(S)O-, -OC(S)-, -OSi(R 5 ) 2 O-, -C(O)(CR 3 R 4 )C(O)O-, or -OC(O)(CR 3 R 4 )C(O)-, carbonate (-OC(O)O-), succinoyl, phosphate ester (-O-(O)POH-O-), cyclic compounds, heterocyclic compounds, piperidine, pyrazine, pyridine, piperazine, and sulfonate ester, and combinations thereof; R 3 , R 4, and R 5 are independently H or alkyl (e.g., C 1 ~C 4 alkyl).

[0245] In another embodiment, the lipid moiety [M] comprises at least one linear or branched, saturated or unsaturated alkyl chain comprising 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 carbon atoms, preferably in the range of 10-20 carbon atoms, more preferably in the range of 12-18 carbon atoms, even more preferably 14, 16 or 18 carbon atoms, even more preferably 16 or 18 carbon atoms, most preferably 14 carbon atoms, all selections being independent of each other.

[0246] In one embodiment, the linker group [linker] shown in formula (II) includes, but is not limited to, pH-sensitive moieties, zwitterionic linkers, non-ester and ester-containing linker moieties (-C(O)O- or -OC(O)-), amide (-C(O)NH-), disulfide (-SS-), carbonyl (-C(O)-), ether (-O-), thioether (-S-), oxime (e.g., -C(H)=NO- or -ON=C(H)-), carbamate (-NHC(O)O-), urea (-NHC(O)NH-), succinyl (-(O)CCHCHC(O)-), succinamidyl (-NHC(O)CHCHC(O)NH-), -C(R 5 )=N-, -N=C(R 5 )-, -C(R 5 )=NO-, -ON=C(R 5 )-, -OC(O)O-, -C(O)N(R 5 ), -N(R 5 )C(O)-, -C(S)(NR 5 )-, (NR 5 )C(S)-, -N(R 5 )C(O)N(R 5 )-, -C(O)S-, -SC(O)-, -C(S)O-, -OC(S)-, -OSi(R 5 )2 O-, -C(O)(CR 3 R 4 )C(O)O-, or -OC(O)(CR 3 R 4 )C(O)-, carbonate (-OC(O)O-), succinoyl, phosphate ester (-O-(O)POH-O-), and sulfonate ester, and combinations thereof; R 3 , R 4 , and R 5 are independently H or alkyl (e.g., C 1 ~C 4 alkyl).

[0247] In a highly preferred embodiment, the polymer conjugated lipid is

[0248] [ka]

[0249] It has the structure: In another highly preferred embodiment, the polymer-conjugated lipid is

[0250] [ka]

[0251] It has the structure: In another preferred embodiment, the polymer-conjugated lipid has the structure "PMOZ2" with n=50, ie, 50 monomer repeats.

[0252] In an even more preferred embodiment, the polymer-conjugated lipid is

[0253] [ka]

[0254] It has the structure: In another preferred embodiment, the polymer-conjugated lipid is

[0255] [ka]

[0256] It has the structure: In an even more highly preferred embodiment, the polymer-conjugated lipid is

[0257] [ka]

[0258] Preferably n=50, i.e. having 50 monomer repeats, i.e.

[0259] [ka]

[0260] It has the structure: For "PMOZ1" to "PMOZ5", preferably, n has an average value in the range of 2 to 200, preferably 20 to 100, more preferably 24 to 26, even more preferably about 100, or even more preferably 45 to 50, and most preferably 50, or n is selected such that the [P] portion has an average molecular weight of about 4.2 kDa to about 4.4 kDa, or most preferably about 4.3 kDa.

[0261] In another highly preferred embodiment, the linker group [linker] preferably comprises an amide linker moiety. In a further highly preferred embodiment the linker group [linker] preferably comprises an ester linker moiety.

[0262] In a further highly preferred embodiment, the linker group [linker] preferably comprises a succinate linker moiety. In another highly preferred embodiment, the linker group [linker] includes both ester and amide linker moieties. In another preferred embodiment, the linker group [linker] includes all of an ester linker moiety, an amine linker moiety, and an amide linker moiety.

[0263] In this specification, all chemical compounds referred to throughout the specification may be prepared through methods known to those skilled in the art; it is understood that the starting materials and / or reagents used in these methods can be obtained through the routine knowledge of those skilled in the art based on common general knowledge (e.g., from textbooks or patent applications WO2022173667, WO2009043027, WO2013067199, WO2010006282, WO2009089542, WO2016019340, WO2008106186, WO2020264505, and WO2020023947, the complete disclosures of which are incorporated herein by reference).

[0264] In yet a further embodiment, the lipid nanoparticles do not comprise polyethylene glycol-(PEG)-lipid conjugates or conjugates of PEG and lipid-like materials, preferably do not comprise PEG, and / or (ii) the polymer-conjugated lipids of the present invention do not comprise a sulfur group (-S-), a terminal nucleophile, and / or are covalently coupled to a biologically active component which is a nucleic acid compound selected from the group consisting of RNA, artificial mRNA, chemically modified or unmodified messenger RNA (mRNA) comprising at least one coding sequence, self-replicating RNA, circular RNA, viral RNA, and replicon RNA; or any combination thereof, preferably the biologically active component is a chemically modified or unmodified mRNA, more preferably the biologically active component is a chemically unmodified mRNA.

[0265] In another highly preferred embodiment, the polymer-conjugated lipids of the invention contain no sulfur (S) or sulfur groups (-S-). Lipid Nanoparticle Compositions The terms " lipid nanoparticle composition " and " composition " are used interchangeably in this section.In the context of the present invention, lipid nanoparticle is not limited to any particular form, but should be interpreted as including any form that is produced when cationic lipid and optionally one or more additional lipids are combined, for example, in an aqueous environment and / or in the presence of nucleic acid compound.For example, liposome, lipid complex, lipoplex, etc. are within the scope of lipid nanoparticle.

[0266] In the context of the present invention, "composition" refers to any type of composition in which the specified components can be incorporated, optionally with any further excipients, usually with at least one pharma- ceutically acceptable carrier or excipient. Thus, the composition can be a dry composition, such as a powder or granules, or a solid unit, such as a lyophilized form or tablet. Alternatively, the composition can be in liquid form, and each excipient can be incorporated independently in dissolved or dispersed (e.g., suspended or emulsified) form. In one preferred embodiment, the composition is formulated as a sterile solid composition, such as a powder or lyophilized form for reconstitution with an aqueous liquid carrier. Such formulations are also preferred for the versions of the composition that contain nucleic acid cargo, which are described in more detail below.

[0267] A "nanoparticle" as used herein is a submicron particle having any structure or morphology. Submicron particles may also be referred to as colloids or colloidal. With respect to the material on which the nanoparticles are based and the structure or morphology, they may be classified as, for example, nanocapsules, vesicles, liposomes, lipid nanoparticles, micelles, crosslinked micelles, lipoplexes, polyplexes, mixed or hybrid complexes, to mention just a few of the possible designations of certain types of nanoparticles. A "lipid nanoparticle" (LNP) is a nanoparticle formed by lipids, typically comprising at least one amphiphilic membrane-forming lipid, and optionally other lipids, and optionally further comprising a cargo material, such as a nucleic acid compound. As used herein, the term "lipid nanoparticle" or "LNP" includes any subtype and form of nanoparticle formed or co-formed by lipids, such as liposomes and lipoplexes.

[0268] As defined above, lipid nanoparticles include any kind of nanoparticles formed or co-formed by lipid.In particular, lipid nanoparticles can be co-formed by a combination of lipids, including at least one amphiphilic, vesicle-forming lipid.Liposomes and lipoplexes are examples of lipid nanoparticles.

[0269] LNPs according to the invention comprise the phospholipid phosphatidylserine, preferably in combination with further lipids as outlined herein. In principle, LNP can comprise any lipid that can form particles with one or more nucleic acid molecules bound or encapsulated.In some embodiments, mRNA, or a part thereof, is encapsulated in the aqueous space that is enveloped by the lipid portion of lipid nanoparticles, or by a part or all of the lipid portion of lipid nanoparticles, thereby protecting it from enzymatic degradation or other undesirable effects induced by the host organism or cellular mechanisms, such as adverse immune response.In some embodiments, mRNA, or a part thereof, is associated with lipid nanoparticles.

[0270] As mentioned, compositions comprising the lipid excipients described herein typically form lipid nanoparticles, at least in an aqueous environment. As defined herein, nanoparticles have a predominantly submicron size. In certain embodiments, the mRNA, when present in lipid nanoparticles, is resistant to degradation by nucleases in aqueous solution. As used herein, the average diameter may be represented by the z-average determined by dynamic light scattering. In one embodiment, the composition is a sterile liquid composition comprising lipid nanoparticles having an average hydrodynamic diameter (or average particle size) determined by dynamic laser scattering of about 30 nm to about 800 nm. In various embodiments, the lipid nanoparticles may be from about 30 nm to about 150 nm, from about 50 nm to about 200 nm, from about 60 nm to about 200 nm, from about 70 nm to about 200 nm, from about 80 nm to about 200 nm, from about 90 nm to about 200 nm, from about 90 nm to about 190 nm, from about 90 nm to about 180 nm, from about 90 nm to about 170 nm, from about 90 nm to about 160 nm, from about 90 nm to about 150 nm, from about 90 nm to about 140 nm, from about 90 nm to about 130 nm, from about 90 nm to about 120 nm, from about 90 nm to about 100 nm, about 70 nm to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, or 200 nm, and are substantially non-toxic. In another preferred embodiment of the present invention, the lipid nanoparticles have a hydrodynamic diameter in the range of about 50 nm to about 300 nm, or about 60 nm to about 250 nm, about 60 nm to about 150 nm, or about 60 nm to about 120 nm, or about 80 nm to about 160 nm, or about 90 nm to about 140 nm, 50 nm to about 300 nm, or about 60 nm to about 250 nm, or about 60 nm to about 200 nm, or about 70 nm to 200 nm, or about 75 nm to about 160 nm, or about 100 nm to about 140 nm, or about 90 nm to about 140 nm. The range of about 50 nm to about 60 nm or the range of about 60 nm to about 80 nm is also preferred.

[0271] The composition comprising the lipid excipients described herein that produces the lipid nanoparticles of the present invention can be relatively homogeneous.Polydispersity index (PDI) can be used to indicate the homogeneity of nanoparticle composition, for example, the particle size distribution of nanoparticle composition.A small polydispersity index (e.g., less than 0.3) generally indicates a narrow particle size distribution. The nanoparticle compositions of the invention can have a polydispersity index of about 0 to about 0.35, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, or 0.35. In some embodiments, the polydispersity index (PDI) of the nanoparticle composition can be about 0.1 to about 0.2.

[0272] Various optional features, choices and preferences for the compositions of the present invention are generally described herein; as will be clearly understood by those skilled in the art, all of these also apply to lipid nanoparticles. Similarly, the choices and preferences also apply to compositions comprising such lipid nanoparticles.

[0273] With regard to the amount of each excipient, it is preferred that cationic lipid is incorporated in the lipid nanoparticle or composition according to the present invention in a relatively high molar amount compared to the molar amount of polymer-conjugated lipid according to formula (I) present.Moreover, the molar amount of cationic lipid is also preferably higher than the molar amount of neutral lipid in the composition or nanoparticle, respectively.Moreover, the molar amount of steroid is optionally higher than the molar amount of polymer-conjugated lipid according to formula (I).

[0274] In certain embodiments, the polymer-conjugated lipid is present in the LNP in an amount of about 1 mol% to about 10 mol% based on the total lipid content of the nanoparticle. In one embodiment, the polymer-conjugated lipid is present in the LNP in an amount of about 1 mol% to about 5 mol%. In one embodiment, the polymer-conjugated lipid is present in the LNP in an amount of about 1 mol% or about 1.5 mol%. In a preferred embodiment, the polymer-conjugated lipid is present in the LNP in an amount of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mol%; preferably in an amount of 5 mol%, more preferably in an amount of 2.5 mol% or also preferably in an amount of 1.7 mol%, based on the composition of all lipid components or excipients in mol percentages relative to 100%.

[0275] In various embodiments, the molar ratio of cationic lipid to polymer-conjugated lipid ranges from about 100:1 to about 25:1, from about 50:1 to about 25:1, or from about 40:1 to about 25:1.

[0276] In certain embodiments, the LNP comprises one or more additional lipids that stabilize the particle during its formation. Suitable stabilizing lipids include neutral lipids and anionic lipids. In various embodiments, the molar ratio of cationic lipid to neutral lipid ranges from about 2:1 to about 8:1, about 3:1 to about 7:1, or about 4:1 to about 6:1.

[0277] As used herein, since lipid nanoparticles are typically formed with the respective excipients and reflect the same quantitative ratio of excipients as in the overall composition containing the nanoparticles, references to the molar amount of lipid excipients in the compositions of the invention should also be understood as describing the molar amount of each excipient in the lipid nanoparticles contained in the composition.

[0278] Phospholipid phosphatidylserine may be present in the composition in an amount ranging from about 1 mol% to about 15 mol%, or from about 2 mol% to about 10 mol%, respectively, using the same criteria of molar percentage; for example, about 1 mol%, 2 mol%, 3 mol%, 4 mol%, about 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, 10 mol%, 11 mol%, 12 mol%, 13 mol%, 14 mol%, or 15 mol%. In some embodiments, the amount of phosphatidylserine is less than or about 10 mol% of the total molar amount of all lipid excipients in the composition. In some embodiments, the amount of phosphatidylserine is less than or about 9 mol% of the total molar amount of all lipid excipients in the composition. In some embodiments, the amount of phosphatidylserine is less than or about 8 mol% of the total molar amount of all lipid excipients in the composition. In some embodiments, the amount of phosphatidylserine is less than or about 7mol% of the total molar amount of all lipid excipients in the composition.In some embodiments, the amount of phosphatidylserine is less than or about 6mol% of the total molar amount of all lipid excipients in the composition.In other embodiments, the amount of phosphatidylserine is less than or about 5mol% of the total molar amount of all lipid excipients in the composition.

[0279] In general, the amount of cationic lipid in the composition (and thus in the lipid nanoparticle) is typically at least about 20 mol% relative to the total molar amount of all lipid excipients in the composition (or nanoparticle). In another embodiment, the amount of cationic lipid is at least about 25 mol%, or at least 30 mol%, respectively. In another preferred embodiment, the amount of cationic lipid in the composition is about 30 mol% to about 70 mol%, or about 40 mol% to about 70 mol%, or about 45 mol% to about 65 mol%, respectively; for example, about 30, 35, 40, 45, 50, 55, 60, 65, or 70 mol%, or about 40 mol% to about 60 mol%, respectively; for example, about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 mol%, respectively.

[0280] The amount of steroid in the composition may optionally be at least about 10 mol%, or may range from about 10 mol% to about 60 mol%, or from about 20 mol% to about 50 mol%, or from about 25 mol% to about 45 mol%, respectively; for example about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 mol%, respectively. For the avoidance of doubt, the molar percentages are relative to the total molar amount of all lipid excipients in the composition.

[0281] Neutral lipid may be present in an amount of at least about 5mol% in some cases.In some embodiments, the amount of neutral lipid in the composition is about 5mol% to about 25mol%, or about 5mol% to about 15mol%, or about 8mol% to about 12mol%, respectively, using the same standard of molar percentage; for example, about 5mol%, 6mol%, 7mol%, 8mol%, 9mol%, 10mol%, 11mol%, 12mol%, 13mol%, 14mol%, 15mol%, 16mol%, 17mol%, 18mol%, 19mol%, 20mol%, 21mol%, 22mol%, 23mol%, 24mol% or 25mol% respectively.

[0282] The amount of polymer-conjugated lipid in the composition or lipid nanoparticles can be selected to be, for example, about 0.1 mol% or more. In certain embodiments, the amount of polymer-conjugated lipid is about 1 mol% to about 15 mol%, or about 2 mol% to about 12 mol%, also using the total molar amount of all lipid excipients as a molar percentage basis. In other certain embodiments, the composition or lipid nanoparticles contain 0.1; 0.2; 0.3; 0.4; 0.5; 0.6; 0.7; 0.8; 0.9; 1.0; 1.1; 1.2; 1.3; 1.4; 1.5; 1.6; 1.7; 1.8; 1.9; 2.0; 2.1; 2.2; 2.3; 2.4; 2.5; 2.6; 2.7; 2.8;2.9;3.0;3.1;3.2;3.3;3.4;3.5;3.6;3.7;3.8;3.9;4.0;4.1;4.2;4.3;4.4;4.5;4.6;4.7;4.8;4.9;5.0;5.1;5.2;5.3;5.4;5.5;5.6;5.7;5.8;5.9;6;6.1;6.2;6 .3;6.4;6.5;6.6;6.7;6.8;6.9;7;7.1;7.2;7.3;7.4;7.5;7.6;7.7;7.8;7.9;8;8.1;8.2;8.3;8.4;8.5;8.6;8.7;8.8;8.9;9;9.1;9.2;9.3;9.4;9.5;9.6;9.7;9.8;9 .9;10;10.1;10.2;10.3;10.4;10.5;10.6;10.7;10.8;10.9;11;11.1;11.2;11.3;11.4;11.5;11.6;11.7;11.8;11.9; or 12 mol% or more of polymer-conjugated lipid. In a preferred embodiment, the content of polymer-conjugated lipid is about 1 mol% to 5 mol%, preferably 1.7 mol% or 2.5 mol% of the total lipid content of the formulation. As a non-limiting preferred example, the lipid nanoparticle comprises 5 mol% of polymer-conjugated lipid. As another preferred non-limiting example, the lipid nanoparticle comprises 10 mol% of polymer-conjugated lipid. As another non-limiting example, the lipid nanoparticle comprises 7.5 mol% of polymer-conjugated lipid.

[0283] In one embodiment, the composition comprises: (a) cationic lipid in an amount of 30-70 mol %; (b) steroids in amounts of 20–50 mol %; (c) phospholipids in an amount of 5 to 25 mol %; and (d) a polymer-conjugated lipid according to formula (I) in an amount of 0.5 to 5 mol % (Each amount is relative to the total molar amount of all lipid excipients in the lipid nanoparticles) The lipid nanoparticles include:

[0284] In another embodiment, the composition comprises: (a) cationic lipid in an amount of 40-60 mol %; (b) steroids in amounts of 20–40 mol %; (c) phospholipids in an amount of 10 to 20 mol %; and (d) a polymer-conjugated lipid according to formula (I) in an amount of 1-2 mol % (Each amount is relative to the total molar amount of all lipid excipients in the lipid nanoparticles) The lipid nanoparticles include:

[0285] In one embodiment, the composition comprises: (a) cationic lipid in an amount of 30-70 mol %; (b) cholesterol in an amount of 20–50 mol %; (c) the phospholipid phosphatidylserine and a further phospholipid (preferably DPhyPE) in an amount of 5 to 25 mol %; and (d) a polymer-conjugated lipid (preferably DMG-PEG2000 or DSG-PEG2000) in an amount of 0.5-5 mol %; (Each amount is relative to the total molar amount of all lipid excipients in the lipid nanoparticles) The lipid nanoparticles include:

[0286] In a further embodiment, the composition comprises: (a) cationic lipid in an amount of 40-60 mol %; (b) cholesterol in an amount of 20–40 mol%; (c) the phospholipid phosphatidylserine in an amount of 10 to 20 mol % and a further phospholipid (preferably DPhyPE); and (d) a polymer-conjugated lipid (preferably DMG-PEG2000 or DSG-PEG2000) in an amount of 1-2 mol%, preferably 1.7 mol%; (Each amount is relative to the total molar amount of all lipid excipients in the lipid nanoparticles) The lipid nanoparticles include:

[0287] In one embodiment, the composition comprises: (a) cationic lipid in an amount of 30-70 mol %; (b) cholesterol in an amount of 20–50 mol %; (c) the phospholipids phosphatidylserine, DPhyPE and DHPC in amounts of 5-25 mol %; and (d) a polymer-conjugated lipid (preferably DMG-PEG2000 or DSG-PEG2000) in an amount of 0.5-5 mol %; (Each amount is relative to the total molar amount of all lipid excipients in the lipid nanoparticles) The lipid nanoparticles include:

[0288] In a further embodiment, the composition comprises: (a) cationic lipid in an amount of 40-60 mol %; (b) cholesterol in an amount of 20–40 mol%; (c) the phospholipids phosphatidylserine, DPhyPE and DHPC in amounts of 10-20 mol %; and (d) a polymer-conjugated lipid (preferably DMG-PEG2000 or DSG-PEG2000) in an amount of 1-2 mol%, preferably 1.7 mol%; (Each amount is relative to the total molar amount of all lipid excipients in the lipid nanoparticles) The lipid nanoparticles include:

[0289] In these embodiments, the cationic lipid is preferably a compound selected according to any one of the preferences disclosed herein. For example, the cationic lipid can be selected from the compounds listed in Table 1. In addition, these embodiments can also include a steroid, a phospholipid, and / or a polymer-conjugated lipid selected according to any one of the preferences disclosed herein. In all embodiments in which the compositions or lipid nanoparticles described herein are listed and mol% values ​​are given for each excipient, each amount should be considered as being relative to the total molar amount of all lipid excipients in the lipid nanoparticle.

[0290] In a further preferred embodiment, the composition comprises (a) the cationic lipid "THIOETHER" preferably in an amount of 30-70 mol %, more preferably in an amount of 40-60 mol %; (a) cholesterol, preferably in an amount of 20 to 50 mol %, more preferably in an amount of 20 to 40 mol %; (c) the phospholipid phosphatidylserine and further phospholipids (preferably in combination with DPhyPE, optionally DHPC), preferably in an amount of 5 to 25 mol %; more preferably in an amount of 10 to 20 mol %; and (d) a polymer-conjugated lipid "PMOZ2" preferably in an amount of 0.5-5 mol %, more preferably in an amount of 2.5 mol %; (Each amount is relative to the total molar amount of all lipid excipients in the lipid nanoparticles) The lipid nanoparticles include:

[0291] In a particularly preferred embodiment, the composition comprises (a) the cationic lipid "THIOETHER" preferably in an amount of 30-70 mol %, more preferably in an amount of 40-60 mol %; (a) cholesterol, preferably in an amount of 20 to 50 mol %, more preferably in an amount of 20 to 40 mol %; (c) the phospholipid phosphatidylserine and further phospholipids (preferably in combination with DPhyPE, optionally DHPC), preferably in an amount of 5 to 25 mol %; more preferably in an amount of 10 to 20 mol %; and (d) the polymer-conjugated lipid "PMOZ4" preferably in an amount of 0.5-5 mol %, more preferably in an amount of 2.5 mol %; (Each amount is relative to the total molar amount of all lipid excipients in the lipid nanoparticles) The lipid nanoparticles include:

[0292] In a further preferred embodiment, the composition or lipid nanoparticles described herein comprise 59 mol% cationic lipid, 10 mol% phospholipid, 29.3 mol% steroid, and 1.7 mol% polymer-conjugated lipid.In a further preferred embodiment, the composition or lipid nanoparticles described herein comprise 58 mol% cationic lipid, 11 mol% phospholipid, 29.3 mol% steroid, and 1.7 mol% polymer-conjugated lipid.In a further preferred embodiment, the composition or lipid nanoparticles described herein comprise 49 mol% cationic lipid, 20 mol% phospholipid, 29.3 mol% steroid, and 1.7 mol% polymer-conjugated lipid.

[0293] In a further preferred embodiment, the composition or lipid nanoparticles described herein comprise 59 mol% cationic lipid, 10 mol% phosphatidylserine and DPhyPE, 29.3 mol% cholesterol, and 1.7 mol% polymer-conjugated lipid (preferably DMG-PEG2000 or DSG-PEG2000). In a further preferred embodiment, the composition or lipid nanoparticles described herein comprise 58 mol% cationic lipid, 11 mol% phosphatidylserine, DPhyPE, and DHPC, 29.3 mol% cholesterol, and 1.7 mol% polymer-conjugated lipid (preferably DMG-PEG2000 or DSG-PEG2000). In a further preferred embodiment, the compositions or lipid nanoparticles described herein comprise 49 mol% cationic lipid, 20 mol% phosphatidylserine, DPhyPE, and DHPC, 29.3 mol% cholesterol, and 1.7 mol% polymer-conjugated lipid (preferably DMG-PEG2000 or DSG-PEG2000).

[0294] In these embodiments, the cationic lipid is preferably a compound selected according to any one of the preferences disclosed herein. For example, the cationic lipid can be selected from the compounds listed in Table 1.

[0295] In any of the above embodiments in this section disclosing specific compositions or lipid nanoparticles with separate % values ​​for excipients, when 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE) is referred to as the phospholipid, in further embodiments, DPhyPE may be replaced with another phospholipid, preferably 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhyPC). Furthermore, in any of the above embodiments in this section disclosing specific compositions or lipid nanoparticles with separate % values ​​for excipients, when 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE) is referred to as the phospholipid, in still further embodiments, DPhyPE may be replaced with another phospholipid, preferably 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC; also called dioleoylphosphatidylcholine) or alternatively 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

[0296] Further preferred lipid compositions include at least five lipid excipients as disclosed herein in Table E. For example, a preferred lipid composition includes the excipients disclosed in row "E1", which are "C1" (disclosed herein in Table 1) as the cationic lipid, DPhyPE+DPhyPS as the phospholipid combination, cholesterol as the sterol, and DMG-PEG2000 as the polymer-conjugated lipid (and thus a total of five lipid excipients). As another example, a preferred lipid composition includes the excipients disclosed in row "E56", which are "C2" (disclosed herein in Table 1) as the cationic lipid, DPhyPE+DPhyPS+DHPC as the phospholipid combination, cholesterol as the sterol, and DMG-PEG2000 as the polymer-conjugated lipid (and thus a total of six lipid excipients).

[0297] [Table 5-1]

[0298] [Table 5-2]

[0299] [Table 5-3]

[0300] [Table 5-4]

[0301] In a preferred embodiment, the polymer-conjugated lipid DMG-PEG2000 shown in Table E is replaced with a PMOZ-lipid shown in FIG. 4, preferably the polymer-conjugated lipid "PMOZ2", more preferably the polymer-conjugated lipid "PMOZ4", even more preferably the polymer-conjugated lipid "PMOZ4" with n=50, i.e., 50 monomer repeats.

[0302] Further, preferred lipid formulations are shown in Table F, which show the individual mol percentages of at least five lipid excipients. For example, a preferred lipid composition comprises the mol percentages of lipids disclosed in row "F1", i.e., 59 mol% cationic lipid, 29.3 mol% sterol, 10 mol% phospholipid combination, and 1.7 mol% polymer-conjugated lipid. As another example, a preferred lipid composition comprises the mol percentages of lipids disclosed in row "F9", i.e., 49 mol% cationic lipid, 31 mol% sterol, 20 mol% phospholipid combination, and 0 mol% polymer-conjugated lipid. The phospholipid combination may consist of a combination of two or three phospholipids, in each case at least one of the phospholipids is phosphatidylserine.

[0303] [Table 6]

[0304] Therefore, in a further preferred embodiment of the invention, the composition of the invention is F1, F2, F3, F4, F5, F6, F7, F8, F9, F10, F11, F12, and F13 The excipient combination designations, in discrete mol percentages, disclosed in Table F, are selected from the group consisting of: E1, E2, E3, E4, E5, E6, E7, E8, E9, E10, E11, E12, E13, E14, E15, E16, E17, E18, E19, E20, E21, E22, E23, E24, E25, E26, E27, E28, E29, E30, E31, E32, E33, E34, E35, E36, E37, E38, E39, E40, E41, E42, E43, E44, E45, E46, E47, E48, E49, E50, E51, E52, E53, E54, E55, E56, E57, E58, E59, E60, E61, E62, E63, E64, E65, E66, E67, E68, E69, E70, E71, E72, E73, E74, E75, E76, E77, E78, E79, E80, E81, E82, E83, E84, E85, E86, E87, E88, E89, E90, E91, E92, E93, E94, E95, E96, E97, E98, E99, E100, E101, E102, E103, E104, E105, E106, E107, and E108.

[0305] The zeta potential of a nanoparticle composition can be used to indicate the electrokinetic potential of the composition. For example, the zeta potential can describe the surface charge of the nanoparticle composition. Lipid nanoparticles according to the invention can exhibit a relatively neutral zeta potential due to the presence of both negatively and positively charged compounds. The zeta potential (sometimes abbreviated as "charge") can be determined along with the particle size by dynamic light scattering and laser Doppler microelectrophoresis, for example using a Malvern Zetasizer Nano (Malvern Instruments Ltd.; Malvern, UK). Depending on the amount and nature of the charged compounds in the lipid nanoparticle, the nanoparticle can be characterized by a zeta potential. In a preferred embodiment, the zeta potential is in the range of about -50 mV to about +50 mV. In other preferred embodiments, the zeta potential is in the range of about -25 mV to about +25 mV. In some embodiments, the zeta potential of the lipid nanoparticles of the present invention may be about -10 mV to about +20 mV, about -10 mV to about +15 mV, about -10 mV to about +10 mV, about -10 mV to about +5 mV, about -10 mV to about 0 mV, about -10 mV to about -5 mV, about -5 mV to about +20 mV, about -5 mV to about +15 mV, about -5 mV to about +10 mV, about -5 mV to about +5 mV, about -5 mV to about 0 mV, about 0 mV to about +20 mV, about 0 mV to about +15 mV, about 0 mV to about +10 mV, about 0 mV to about +5 mV, about +5 mV to about +20 mV, about +5 mV to about +15 mV, or about +5 mV to about +10 mV.

[0306] In certain embodiments, the LNPs comprise one or more targeting moieties that can target the LNPs to a cell or cell population. For example, in one embodiment, the targeting moiety is a ligand that directs the LNP to a receptor found on the cell surface.

[0307] In certain embodiments, LNPs comprise one or more internalization domains. For example, in one embodiment, LNPs comprise one or more domains that bind to cells and induce internalization of LNPs. For example, in one embodiment, one or more internalization domains bind to receptors found on cell surfaces to induce receptor-mediated uptake of LNPs. In certain embodiments, LNPs can bind to biomolecules in vivo, whereby LNP-bound biomolecules can be recognized by cell surface receptors to induce internalization. For example, in one embodiment, LNPs bind to systemic ApoE, which leads to uptake of LNPs and associated cargo. In certain embodiments of the present invention, ApoE can be supplemented to the vehicle or pharmaceutical composition used.

[0308] Preferably, the LNPs of the invention are (i) at least one cationic lipid; (ii) at least one phospholipid; (iii) at least one steroid or steroid analog, preferably cholesterol; and (iv) at least one polymer-conjugated lipid, preferably a PEG-lipid; Including, (i)-(iv) are molar ratios of approximately 20-60% cationic lipid, 5-25% neutral lipid, 25-55% sterol, and 0.5-15% polymer-conjugated lipid.

[0309] Also preferably, the LNPs of the invention (i) at least one cationic lipid; (ii) at least two phospholipids, preferably DPhyPE and phosphatidylserine; (iii) at least one steroid or steroid analog, preferably cholesterol; and (iv) at least one polymer-conjugated lipid, preferably a PEG-lipid; Including, (i)-(iv) are molar ratios of approximately 20-60% cationic lipid, 5-25% neutral lipid, 25-55% sterol, and 0.5-15% polymer-conjugated lipid.

[0310] Also preferably, the LNPs of the invention (i) at least one cationic lipid; (ii) at least three phospholipids, preferably DPhyPE, DHPC, and phosphatidylserine; (iii) at least one steroid or steroid analog, preferably cholesterol; and (iv) at least one polymer-conjugated lipid, preferably a PEG-lipid; Including, (i)-(iv) are molar ratios of approximately 20-60% cationic lipid, 5-25% neutral lipid, 25-55% sterol, and 0.5-15% polymer-conjugated lipid.

[0311] Also preferably, the LNPs of the invention (i) at least one cationic lipid; (ii) at least two phospholipids, preferably DSPC and phosphatidylserine; (iii) at least one steroid or steroid analog, preferably cholesterol; and (iv) at least one polymer-conjugated lipid, preferably a PEG-lipid; Including, (i)-(iv) are molar ratios of approximately 20-60% cationic lipid, 5-25% neutral lipid, 25-55% sterol, and 0.5-15% polymer-conjugated lipid.

[0312] Encapsulation / complexation into LNPs In a preferred embodiment, at least one nucleic acid (e.g., DNA or RNA), preferably at least one mRNA, is complexed with the phospholipid phosphatidylserine and one or more lipids (e.g., cationic lipids and / or neutral lipids), or encapsulated with the phospholipid phosphatidylserine and one or more lipids, or partially encapsulated with the phospholipid phosphatidylserine and one or more lipids, or associated with the phospholipid phosphatidylserine and one or more lipids, thereby forming a liposome, lipid nanoparticle (LNP), lipoplex, and / or nanoliposome.

[0313] The nucleic acid (e.g., DNA or RNA) incorporated into liposomes, lipid nanoparticles (LNPs), lipoplexes, and / or nanoliposomes may be located completely or partially within the inner space, lipid layer / membrane of liposomes, lipid nanoparticles (LNPs), lipoplexes, and / or nanoliposomes, or may be associated with the outer surface of lipid layer / membrane. The incorporation of nucleic acid into liposomes / LNPs is referred to herein as "encapsulation", where the nucleic acid, e.g., RNA, is completely contained within the inner space of liposomes, lipid nanoparticles (LNPs), lipoplexes, and / or nanoliposomes. The purpose of incorporating nucleic acid into liposomes, lipid nanoparticles (LNPs), lipoplexes, and / or nanoliposomes is to protect the nucleic acid, preferably RNA, from the environment, which may contain enzymes or chemicals or conditions that degrade the nucleic acid and / or systems or receptors that cause the nucleic acid to be rapidly excreted. Furthermore, incorporation of nucleic acids, preferably RNA, into liposomes, lipid nanoparticles (LNPs), lipoplexes, and / or nanoliposomes may facilitate uptake of the nucleic acid and thus enhance the therapeutic effect of the nucleic acid, e.g., RNA encoding an antigenic SARS-CoV-2 (nCoV-2019) protein. Thus, incorporation of nucleic acids, e.g., RNA or DNA, into liposomes, lipid nanoparticles (LNPs), lipoplexes, and / or nanoliposomes may be particularly suitable for coronavirus vaccines (e.g., SARS-CoV-2 vaccines), e.g., for intramuscular and / or intradermal administration.

[0314] In this context, the terms "complexed" or "associated" refer to an essentially stable combination of a nucleic acid and one or more lipids into a larger complex or aggregate without the use of covalent bonds.

[0315] The term "lipid nanoparticle", also referred to as "LNP", is not limited to any particular form and includes any form that is produced when cationic lipids and optionally one or more additional lipids are combined, for example, in an aqueous environment and / or in the presence of nucleic acid, for example, RNA. For example, liposomes, lipid complexes, lipoplexes, etc. are within the scope of lipid nanoparticles (LNPs).

[0316] Liposomes, lipid nanoparticles (LNPs), lipoplexes, and / or nanoliposomes can be of various sizes including, but not limited to, multilamellar vesicles (MLVs), which can be hundreds of nanometers in diameter and contain a series of concentric bilayers separated by narrow aqueous compartments, small unilamellar vesicles (SUVs), which can be less than 50 nm in diameter, and large unilamellar vesicles (LUVs), which can be between 50 nm and 500 nm in diameter.

[0317] The LNPs of the present invention are preferably characterized as microvesicles having an internal aqueous space separated from the external medium by one or more bilayer membranes. The bilayer membrane of LNPs is typically formed by amphiphilic molecules such as lipids of synthetic or natural origin, which contain spatially separated hydrophilic and hydrophobic domains. The bilayer membrane of liposomes can also be formed by amphiphilic polymers and surfactants (e.g., polymerosomes, niosomes, etc.). In the context of the present invention, LNPs typically serve to transport at least one nucleic acid, preferably at least one RNA, to a target tissue. Thus, in a preferred embodiment of the aspect of the present invention, at least one RNA is complexed with one or more lipids, thereby forming a lipid nanoparticle (LNP). LNPs typically include cationic lipids and one or more excipients selected from neutral lipids, charged lipids, steroids, and polymer-conjugated lipids.

[0318] mRNA In one preferred embodiment, the nucleic acid compound is mRNA or mRNA compound.As found by the present inventors, the vaccine composition comprising the carrier composition comprising the phospholipid phosphatidylserine according to the present invention is particularly suitable for in vivo delivery of mRNA compound expressing antigen, thus enabling highly effective, potent, versatile and safe vaccines that can be developed quickly and at reasonable cost.The specific antigens of interest for carrying out the present invention are described in more detail below.The mRNA compound according to the present invention is preferably encapsulated in or associated with lipid nanoparticles.

[0319] The advantages of the phosphatidylserine-containing vaccine comprising mRNA encoding at least one antigen or a fragment or variant thereof are: Spleen targeting Induction of a strong humoral immune response; Induction of B cell memory; Faster onset of immune defense; Longevity of the induced immune response; Induction of broad cellular T cell responses; Induction of a (local and transient) pro-inflammatory environment; Well tolerated, no side effects, non-toxic; Advantageous stability characteristics; Formulations compatible with many different antigens: larger antigen cocktails feasible based on the same (production) technology; There is no vector immunity, i.e. the technique can be used to vaccinate the same subject multiple times against multiple (different) antigens; speed, adaptability, simplicity and scalability of production; Preferential patterns of induction of systemic cytokine or chemokine responses depending on the respective application It is.

[0320] In certain embodiments, the lipid nanoparticle composition comprises, in addition to the phospholipid phosphatidylserine: (a) cationic or ionizable lipids; (b) steroids; (c) further phospholipids in addition to phosphatidylserine; (d) a polymer-conjugated lipid; and / or (e) an mRNA compound encoding an antigen or a fragment or variant thereof Includes.

[0321] In another embodiment, the lipid nanoparticle composition comprises, in addition to the phospholipid phosphatidylserine: (a) cationic or ionizable lipids; (b) steroids; (c) a polymer-conjugated lipid; and (d) an mRNA compound encoding an antigen or a fragment or variant thereof Includes.

[0322] In another embodiment, the lipid nanoparticle composition comprises, in addition to the phospholipid phosphatidylserine: (a) cationic or ionizable lipids; (b) further phospholipids in addition to phosphatidylserine; (c) a polymer-conjugated lipid; and (d) an mRNA compound encoding an antigen or a fragment or variant thereof Includes.

[0323] In certain preferred embodiments, the lipid nanoparticle composition comprises, in addition to the phospholipid phosphatidylserine: (a) cationic or ionizable lipids; (b) steroids; (c) further phospholipids in addition to phosphatidylserine; (d) a polymer-conjugated lipid; and (e) an mRNA compound encoding an antigen or a fragment or variant thereof Includes.

[0324] With respect to the phospholipid phosphatidylserine, cationic or ionizable lipids, steroids, phospholipids, polymer-conjugated lipids, and antigen-encoding mRNA compounds, the same options, preferences and alternatives apply as described with respect to these features above.

[0325] The amount of cationic or ionizable lipid relative to the amount of mRNA compound in the lipid nanoparticle may also be expressed as a weight ratio (e.g., abbreviated as "m / m"). For example, the lipid nanoparticle contains the mRNA compound in an amount to achieve a lipid to mRNA weight ratio ranging from about 20 to about 60, or from about 10 to about 50. In other embodiments, the ratio of cationic or ionizable lipid to nucleic acid or mRNA is about 3 to about 15, such as about 5 to about 13, about 4 to about 8, or about 7 to about 11. In a highly preferred embodiment of the invention, the total lipid / mRNA mass ratio is about 40 or 40, i.e., about 40 or 40-fold mass excess to ensure mRNA encapsulation. Another preferred RNA / lipid ratio is between about 1 to about 10, about 2 to about 5, about 2 to about 4, or preferably about 3.

[0326] Additionally, the amount of cationic or ionizable lipid may be selected taking into consideration the amount of nucleic acid cargo, such as an mRNA compound. In one embodiment, the N / P ratio may range from about 1 to about 50. In another embodiment, the range is from about 1 to about 20, from about 1 to about 10, from about 1 to about 5. In one preferred embodiment, these amounts are selected to result in an N / P ratio of the lipid nanoparticle or composition in the range of about 10 to about 20. In a further highly preferred embodiment, the N / P is 14 (i.e., 14-fold molar excess of positive charges to ensure mRNA encapsulation). In another highly preferred embodiment, the N / P is 17.5 (i.e., 17.5-fold molar excess of positive charges to ensure mRNA encapsulation), or (i) an amount to achieve an N / P ratio in the range of from about 1 to about 20, preferably from about 2 to about 15, more preferably from about 3 to about 10, even more preferably from about 4 to about 9, and most preferably about 6; (ii) an amount to achieve an N / P ratio in the range of from about 5 to about 20, more preferably from about 10 to about 18, even more preferably from about 12 to about 16, and most preferably about 14; or (iii) an amount to achieve a lipid:mRNA weight ratio in the range of 20 to 60, preferably about 3 to about 15, 5 to about 13, about 4 to about 8, or about 7 to about 11. It is.

[0327] In other preferred embodiments, the N / P ratio may range from about 1 to about 50. In other embodiments, the range is from about 1 to about 20, preferably from about 1 to about 15. For the lipid nanoparticles of the present invention, the preferred N / P (molar ratio of lipid to RNA) is about 14 or about 17. A further preferred N / P, i.e., molar ratio of lipid to RNA, is about 6. Another preferred N / P ratio is about 4.85 or 5 (molar ratio of lipid to RNA).

[0328] The total amount of mRNA in the lipid nanoparticles can vary and be defined depending on the mRNA to total lipid w / w ratio. In one embodiment of the invention, the mRNA to total lipid ratio is less than 0.06 w / w, preferably between 0.03 and 0.04 w / w.

[0329] Preferably, the mRNA compound or its coding sequence has a length of about 50 to about 20,000, or 100 to about 20,000 nucleotides, preferably about 250 to about 20,000 nucleotides, more preferably about 500 to about 10,000, even more preferably about 500 to about 5,000.

[0330] Pathogenic or pathogen-derived antigens Pathogenic antigens or pathogen-derived antigens are derived from pathogenic organisms, particularly bacterial, viral, protozoological (multicellular) pathogenic organisms, and provoke an immunological response by a subject, particularly a mammalian subject, more particularly a human. More specifically, pathogenic antigens are preferably surface antigens, such as proteins (or fragments of proteins, e.g., external portions of surface antigens), located on the surface of a virus or a bacterial or protozoological organism.

[0331] Thus, in some preferred embodiments, the mRNA may encode, in at least one coding region thereof, at least one pathogenic antigen selected from a bacterial, viral, fungal, or protozoan antigen, and the encoded (poly)peptide or protein may consist of or comprise the pathogenic antigen or a fragment or variant thereof.

[0332] The pathogenic antigen is preferably a peptide or protein antigen derived from a pathogen associated with an infectious disease, selected from, but not limited to, the group of pathogen-derived antigens disclosed on pages 21-35 of WO2018078053; WO2018078053 is incorporated herein in its entirety by reference. Furthermore, the pathogenic antigen is preferably a peptide or protein antigen derived from a pathogen associated with an infectious disease, selected from, but not limited to, the group of pathogen-derived antigens disclosed on pages 57, paragraph 3 to 63, paragraph 2 of WO2019077001; WO2019077001 is incorporated herein in its entirety by reference.

[0333] Still further pathogenic antigens are peptides or proteins derived from pathogens associated with infectious diseases, preferably selected from antigens derived from pathogens selected from the group of antigens derived from pathogens disclosed in WO2013120628, p. 32, line 26 to p. 34, line 27. Furthermore, in this respect, pathogenic antigens (antigens derived from pathogens associated with infectious diseases) may be preferably selected from antigens derived from antigens selected from the group of antigens disclosed in WO2013120628, p. 34, line 29 to p. 59, line 5 (in parentheses is the particular pathogen or family of pathogens from which the antigen is derived and the infectious disease associated with the pathogen); WO2013120628 is incorporated herein in its entirety by reference.

[0334] Among the preferred antigens expressed by the mRNA compounds incorporated in the compositions of the invention are those including, but not limited to, SARS coronavirus 2 (SARS-CoV-2), nCoV-2019 coronavirus, SARS coronavirus (SARS-CoV), Bunyavirales viruses, cytomegalovirus (CMV), Dengue virus (DENV-1, DENV-2, DENV-3, and DENV-4), Ebola virus (EBOV), Flavivirus, Hepatitis B virus (HBV), Herpes simplex virus (HSV), Human immunodeficiency virus (HIV), Human metapneumovirus (HMPV), Human papillomavirus (HPV), Human parainfluenza virus (HPIV), Influenza virus, Extraintestinal pathogenic E. coli (ExPEC), Lassa mammarenavirus (LASV), MERS coronavirus, Mycobacterium tuberculosis (Mycobacterium tuberculosis), Mycobacterium tuberculosis, ... tuberculosis, Nipah virus, Norovirus, Rabies virus (RABV), Respiratory syncytial virus (RSV), Rhinovirus, Rotavirus, Vaccinia virus, Yellow fever virus (YFV), Zika virus (ZIKV), Chlamydia trachomatis (i.e., the bacterium Chlamydia that causes Chlamydia), and Plasmodium (e.g., Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, or Plasmodium ovale). In another preferred embodiment, the pathogenic antigen is derived from SARS coronavirus 2 (SARS-CoV-2), nCoV-2019 coronavirus, SARS coronavirus (SARS-CoV), malaria parasite, influenza virus or rabies virus (RABV).

[0335] Furthermore, the pathogenic antigen is more preferably selected from the group consisting of, but not limited to, Acinetobacter baumannii, Anaplasma, Anaplasma phagocytophilum, Ancylostoma braziliense, Ancylostoma duodenale, Arcanobacterium haemolyticum, Ascaris lumbricoides, Aspergillus, Astroviridae, Babesia, Bacillus anthracis, Bacillus cereus, Bartonella henselae, and the like. henselae, BK virus, Blastocystis hominis, Blastomyces dermatitidis, Bordetella pertussis, Borrelia burgdorferi, Borrelia, Borrelia spp, Brucella, Brugia malayi, Bunyaviridae, Burkholderia cepacia or other Burkholderia spp, Burkholderia mallei, Burkholderia pseudomallei, Caliciviridae, Campylobacter, Candida albicans, Candida species, Chlamydia trachomatis, Chlamydophila pneumoniae, Chlamydophilapsittaci, CJD prion, Clonorchis sinensis, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium perfringens, Clostridium spp., Clostridium tetani, Coccidioides spp., Coronavirus, Coronaviridae, Corynebacterium diphtheriae, Coxiella burnetii, Crimean-Congo hemorrhagic fever virus, Cryptococcus neoformans neoformans, Cryptosporidium, Cytomegalovirus (CMV), Dientamoeba fragilis, Ebola virus (EBOV - e.g. envelope glycoprotein), Echinococcus, Ehrlichia chaffeensis, Ehrlichia ewingii, Ehrlichia, Entamoeba histolytica, Enterococcus, Enterovirus, Enteroviruses, mainly Coxsackie A virus or Enterovirus 71 (EV71), Epidermophyton species, Epstein-Barr virus (EBV), Escherichia coli coli, E. coli strains O157:H7, O111 or O104:H4, Fasciola hepatica or Fasciola gigantica, FFI prion, Feline immunodeficiency virus (FIV), Filarioidea, Flavivirus, Francisellatularensis, Fusobacterium, Geotrichum candidum, Giardia intestinalis, Gnathostoma spp., GSS prion, Guanarito virus, Haemophilus ducreyi, Haemophilus influenzae, Helicobacter pylori, Henipavirus (Hendra virus, Nipah virus), Hepatitis A virus (HAV), Hepatitis B virus (HBV), Hepatitis C virus (HCV), Hepatitis D virus, Hepatitis E virus, Histoplasma capsulatum, Hortaea wernecki werneckii, Human bocavirus (HBoV), Human metapneumovirus (HMPV), Human parainfluenza virus (HPIV), Japanese encephalitis virus, JC virus, Junin virus, Kingella kingae, Klebsiella granulomatis, Klebsiella pneumoniae, Coulour purine, Lassa virus, Legionella pneumophila, Leishmania spp., Leptospira spp., Listeria monocytogenes, Lymphocytic choriomeningitis virus (LCMV), Machupo virus, Malassezia spp., Marburg virus, Measles virus, Metagonimus yokagawai), Microsporidia phylum, Molluscum contagiosum virus (MCV), Mumps virus, Mycobacterium lepraeleprae or Mycobacterium lepromatosis, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumoniae, Naegleria fowleri, Necator americanus, Neisseria gonorrhoeae, Neisseria meningitidis, Nocardia asteroides, Nocardia species, Onchocerca volvulus, Orientia tsutsugamushi tsutsugamushi, Orthomyxoviridae (Influenza), Paracoccidioides brasiliensis, Paragonimus spp., Paragonimus westermani, Parvovirus B19, Pasteurella spp., Plasmodium spp., Pneumocystis jirovecii, Poliovirus, Rhinovirus, Rickettsia akari, Rickettsia spp., Rickettsia prowazekii, Rickettsia rickettsii, Rickettsia typhus typhi, Rift Valley fever virus, Rotavirus (preferably, e.g., VP8 antigen), Rubella virus, Sabia virus, Salmonella, Sarcoptes scabiei, SARS coronavirus, Schistosoma, Shigella, SinNombre virus, Hantavirus, Sporothrix schenckii, Staphylococcus, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Strongyloides stercoralis, Taenia, Taenia solium, Tick-borne encephalitis virus (TBEV), Toxocara canis or Toxocara cati, Toxoplasma gondii, Treponema pallidum, Trichinella spiralis spiralis, Trichomonas vaginalis, Trichophyton spp., Trichuris trichiura, Trypanosoma brucei, Trypanosoma cruzi, Ureaplasma urealyticum, Vaccinia virus (preferably, e.g., immune evasion proteins E3, K3, or B18), Varicella zoster virus (VZV), Variola major or Variola minor, vCJD prion, Venezuelan equine encephalitis virus, Vibrio cholerae, West Nile virus, Western equine encephalitis virus, Wuchereria bancrofti, Yersinia enterocolitica enterocolitica, Yersinia pestis, or Yersinia pseudotuberculosisThe antigen may be selected from an antigen derived from a pathogen selected from the group consisting of: Coronaviridae, Coronaviridae (CoV), ...

[0336] In a further embodiment, antigens useful for treating infectious diseases (i.e. by administering a nucleic acid, preferably mRNA, encoding the antigen) may be selected from the following antigens (the relevant infectious disease and the relevant pathogen are indicated in brackets after the respective antigen - of course other antigens that may be derived from the following pathogens in brackets may also be derived and used according to the invention): the spike protein (S), envelope protein (E), membrane protein (M) or nucleocapsid protein (N), or an immunogenic fragment or variant of any of these (the infectious disease is "COVID-19 disease"; pathogen: SARS coronavirus 2 (SARS-CoV-2), nCoV-2019 coronavirus, SARS coronavirus (SARS-CoV)); · spike protein (S), spike S1 fragment (S1), envelope protein (E), membrane protein (M) or nucleocapsid protein (N) (the infectious disease is MERS infection; pathogen: Middle East respiratory syndrome coronavirus (MERS coronavirus / MERS-CoV)); · Replication protein E1, regulatory protein E2, protein E3, protein E4, protein E5, protein E6, protein E7, protein E8, major capsid protein L1, minor capsid protein L2 (the infectious disease is a human papillomavirus (HPV) infection; pathogen: human papillomavirus (HPV) or HPV16); fusion protein F, hemagglutinin-neuramidase HN, glycoprotein G, matrix protein M, phosphoprotein P, nucleoprotein N, polymerase L, hemagglutinin-neuraminidase, fusion (F) glycoprotein F0, F1 or F2, recombinant HPIV3 / HPIV1 fusion glycoprotein (F) and hemagglutinin (HN), C protein, phosphoprotein, D protein, matrix protein (M), nucleocapsid protein (N), viral replicase (L), non-structural V protein (the infectious disease is a human parainfluenza virus infection; pathogen: human parainfluenza virus (HPIV / PIV) HPIV-1, HPIV-2, HPIV-3, or HPIV-4 serotype, preferably HPIV-3 serotype, preferably PIV-3); Fusion (F) glycoprotein, glycoprotein G, phosphoprotein P, nucleoprotein N, nucleocapsin protein (Infectious disease: HMPV infection; Pathogen: Human metapneumovirus (HMPV)); Hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), M1 protein, M2 protein, NS1 protein, NS2 protein (NEP protein: nuclear export protein), PA protein, PB1 protein (polymerase basic 1 protein), PB1-F2 protein and PB2 protein, H10N8, H7N9, H10, H1N1, H3N2(X31), H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H1 7, H18, antigenic subdomains of HA, HA1, HA2, neuraminidase (NA), nucleoprotein (NP), matrix protein 1 (M1), matrix protein 2 (M2), nonstructural protein 1 (NS1), nonstructural protein 2 (NS2), HA7 antigen, H7 or H10 and B, pathogen: Orthomyxoviridae, influenza virus (FLU) or influenza A virus (FLUAV) and / or influenza B virus (FLUBV); · Nucleoprotein N, large structural protein L, phosphoprotein P, matrix protein M, glycoprotein G, G protein (the infectious disease is rabies; pathogen: rabies virus (RABV)); · p24 antigen, envelope proteins (Gp120, Gp41, Gp160), polyprotein GAG, negative factor protein Nef, transactivator of transcription Tat, Brec1 (infectious disease HIV; pathogen: human immunodeficiency virus (HIV)); Major outer membrane protein MOMP, likely outer membrane protein PMPC, outer membrane complex protein B OmcB, heat shock protein Hsp60 HSP10, protein IncA, protein of type III secretion system, ribonucleotide reductase small chain protein NrdB, plasmid protein Pgp3, chlamydial outer protein N CopN, antigen CT521, antigen CT425, antigen CT043, antigen TC0052, antigen TC0189, antigen TC0582, antigen TC0660, antigen TC0726, antigen TC0816, antigen TC0828 (Infectious diseases: infections caused by Chlamydia trachomatis; Pathogens: Chlamydia trachomatis; HCMV glycoproteins selected from pp65 antigen, membrane protein pp15, capsid proximal tegument protein pp150, protein M45, DNA polymerase UL54, helicase UL105, glycoprotein gM, glycoprotein gN, glycoprotein H, glycoprotein B gB, protein UL83, protein UL94, protein UL99, gH gL, gB, gO, gN, and gM, HCMV proteins selected from UL83, UL123, UL128, UL130, and UL131A, tegument protein pp150 (pp150), tegument protein pp65 / lower matrix phosphoprotein (pp65), envelope glycoprotein M (UL100), regulatory protein IE1 (UL123), envelope protein (UL128), envelope glycoprotein (130), envelope protein (UL131A), envelope glycoprotein B (UL55), structural glycoprotein N gpUL73 (UL73), structural glycoprotein O gpUL74 (UL74) (infectious disease is cytomegalovirus infection; pathogen: cytomegalovirus (CMV / HCMV)); · Capsid protein C, membrane precursor protein prM, membrane protein M, envelope protein E (domain I, domain II, domain II), protein NS1, protein NS2A, protein NS2B, protein NS3, protein NS4A, protein 2K, protein NS4B, protein NS5 (infectious disease dengue fever; pathogen: dengue virus (DENV-1, DENV-2, DENV-3 and DENV-4)); glycoprotein (GP), surface GP, wild-type proGP, mature GP, secreted wild-type proGP, secreted mature GP, nucleoprotein (NP), RNA polymerase L, and matrix protein selected from VP35, VP40, VP24, and VP30 (infectious disease: Ebola; pathogen: Ebola virus (EBOV)); Hepatitis B surface antigen HBsAg, Hepatitis B core antigen HbcAg, polymerase, protein Hbx, pre-S2, central surface protein, surface protein L, large S protein, viral protein VP1, viral protein VP2, viral protein VP3, viral protein VP4 (the infectious disease is hepatitis B; pathogen: hepatitis B virus (HBV)); · Fusion protein F, F protein, nucleoprotein N, matrix protein M, matrix protein M2-1, matrix protein M2-2, phosphoprotein P, small hydrophobic protein SH, major surface glycoprotein G, polymerase L, nonstructural protein 1 NS1, nonstructural protein 2 NS2, RSV attachment protein (G) (glycoprotein G), fusion (F) glycoprotein (glycoprotein F), nucleoprotein (N), phosphoprotein (P), large polymerase protein (L), matrix protein (M, M2), small hydrophobic protein (SH), nonstructural protein 1 (NS1), nonstructural protein 2 (NS2), membrane-bound RSV F protein, membrane-bound DS-Cavl (stabilized prefusion RSV F protein) (Infectious disease is an infection caused by respiratory syncytial virus (RSV): Pathogen: Respiratory syncytial virus (RSV)); Secretory antigen SssA (Staphylococcus, staphylococcal food poisoning); Secretory antigen SssA (Staphylococcus, e.g. Staphylococcus aureus, staphylococcal infections); Molecular chaperone DnaK, cell surface lipoprotein Mpt83, lipoprotein P23, phosphate transport system permease protein pstA, 14 kDa antigen, fibronectin binding protein C FbpC1, alanine dehydrogenase TB43, glutamine synthetase 1, ESX-1 protein, protein CFP10, TB10.4 protein, protein MPT83, protein MTB12, protein MTB8, Rpf-like protein, protein MTB32, protein MTB39, crystallins, heat shock protein HSP65, protein PST-S (infectious disease is tuberculosis; pathogen: Mycobacterium tuberculosis): Genome polyprotein, protein E, protein M, capsid protein C, protease NS3, protein NS1, protein NS2A, protein AS2B, protein NS4A, protein NS4B, protein NS5 (the infectious disease is yellow fever; pathogen: yellow fever virus (YFV)); Circumsporozoite proteins (CSPs) (infectious disease is malaria; pathogens: P. falciparum and P. vivax); and a Zika virus protein according to WO2017140905, i.e., capsid protein (C), membrane precursor protein (prM), pr protein (pr), membrane protein (M), envelope protein (E), nonstructural protein, prME antigen, capsid protein, membrane precursor / membrane protein, ZIKV nonstructural protein 1, nonstructural protein 2A, nonstructural protein 2B, nonstructural protein 3, nonstructural protein 4A, nonstructural protein 4B, nonstructural protein 5, or Zika virus envelope protein (E) (wherein the fusion loop of domain II is mutated according to WO2017140905); WO2017140905 is incorporated herein by reference in its entirety (the infectious disease is Zika virus infection; pathogen: Zika virus (ZIKV)).

[0337] In another embodiment, an antigen useful for treating an infectious disease (i.e., by administering a nucleic acid, preferably an mRNA, encoding the antigen) may be selected from a pathogenic antigen, preferably selected from the group consisting of a tumor antigen, a viral antigen, a bacterial antigen, and a protozoan antigen.

[0338] In some embodiments of the present invention, disclosures are provided of methods of inducing an antigen-specific immune response in a subject, comprising administering to the subject an effective amount of any of the RNA (e.g., mRNA) vaccines provided herein to produce an antigen-specific immune response.

[0339] In some embodiments, the RNA (e.g., mRNA) vaccine is for use in treating SARS coronavirus 2 (SARS-CoV-2), nCoV-2019 coronavirus, SARS coronavirus (SARS-CoV), Bunyavirales viruses, cytomegalovirus (CMV), Dengue virus (DENV-1, DENV-2, DENV-3, and DENV-4), Ebola virus (EBOV), Flavivirus, Hepatitis B virus (HBV), Herpes simplex virus (HSV), Human immunodeficiency virus (HIV), Human metapneumovirus (HMPV), Human papillomavirus (HPV), Human parainfluenza virus (HPIV), Influenza virus, Extraintestinal pathogenic Escherichia coli (E. coli) (ExPEC), Lassa-Mam arenavirus (LASV), MERS coronavirus, Mycobacterium tuberculosis (Mycobacterium tuberculosis), ... tuberculosis, Nipah virus, Norovirus, Rabies virus (RABV), Respiratory syncytial virus (RSV), Rhinovirus, Rotavirus, Vaccinia virus, Yellow fever virus (YFV), Zika virus (ZIKV), Chlamydia trachomatis (i.e., the bacterium Chlamydia that causes Chlamydia), or Plasmodium (e.g., Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, or Plasmodium ovale) vaccines. In other embodiments, the RNA (e.g., mRNA) vaccine is a rabies, influenza, or malaria vaccine.

[0340] In some embodiments, the RNA (eg, mRNA) vaccine is a combination vaccine that includes a combination of influenza vaccines (broad-spectrum influenza vaccine). In some embodiments, the antigen-specific immune response comprises a T cell response or a B cell response.

[0341] In some embodiments, the method of producing an antigen-specific immune response comprises administering a single dose (i.e., without booster dose) of an antibody or antibody of the present disclosure, such as SARS coronavirus 2 (SARS-CoV-2), nCoV-2019 coronavirus, SARS coronavirus (SARS-CoV), Bunyavirales viruses, cytomegalovirus (CMV), Dengue virus (DENV-1, DENV-2, DENV-3, and DENV-4), Ebola virus (EBOV), Flavivirus, Hepatitis B virus (HBV), Herpes simplex virus (HSV), Human immunodeficiency virus (HIV), Human metapneumovirus (HMPV), Human papillomavirus (HPV), Human parainfluenza virus (HPIV), Influenza virus, Extraintestinal pathogenic Escherichia coli (E. coli) (ExPEC), Lassa-Mam arenavirus (LASV), MERS coronavirus, Mycobacterium tuberculosis (Mycobacterium The method includes administering to the subject an RNA (e.g., mRNA) vaccine against Chlamydia tuberculosis, Nipah virus, Norovirus, Rabies virus (RABV), Respiratory syncytial virus (RSV), Rhinovirus, Rotavirus, Vaccinia virus, Yellow fever virus (YFV), Zika virus (ZIKV), Chlamydia trachomatis (i.e., the bacterium Chlamydia that causes Chlamydia), or Plasmodium (e.g., Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, or Plasmodium ovale).

[0342] In some embodiments, the method further comprises administering a second (booster) dose of SARS coronavirus 2 (SARS-CoV-2), nCoV-2019 coronavirus, SARS coronavirus (SARS-CoV), Bunyavirales viruses, cytomegalovirus (CMV), Dengue virus (DENV-1, DENV-2, DENV-3, and DENV-4), Ebola virus (EBOV), Flavivirus, Hepatitis B virus (HBV), Herpes simplex virus (HSV), Human immunodeficiency virus (HIV), Human metapneumovirus (HMPV), Human papillomavirus (HPV), Human parainfluenza virus (HPIV), Influenza virus, Extraintestinal pathogenic Escherichia coli (E. coli) (ExPEC), Lassa-Mam arenavirus (LASV), MERS coronavirus, Mycobacterium tuberculosis (Mycobacterium tuberculosis), or a combination of these viruses. tuberculosis, Nipah virus, Norovirus, Rabies virus (RABV), Respiratory syncytial virus (RSV), Rhinovirus, Rotavirus, Vaccinia virus, Yellow fever virus (YFV), Zika virus (ZIKV), Chlamydia trachomatis (i.e., the bacterium Chlamydia that causes Chlamydia), or Plasmodium (e.g., Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, or Plasmodium ovale) RNA (e.g., mRNA) vaccine to the subject.

[0343] In some embodiments, subjects exhibit a seroconversion rate of at least 80% (e.g., at least 85%, at least 90%, or at least 95%) after the first or second (booster) dose of the vaccine. Seroconversion is the period during which specific antibodies develop and become detectable in the blood. After seroconversion occurs, the virus can be detected with a blood test for antibodies. During infection or immunization, antigens enter the blood and the immune system responds by producing antibodies. Before seroconversion, the antigen itself may or may not be detectable, but no antibodies are believed to be present. During seroconversion, antibodies are present but not yet detectable. Any time after seroconversion, antibodies can be detected in the blood, indicating a previous or current infection. In some embodiments, the RNA (e.g., mRNA) vaccine is administered to the subject by intradermal, intramuscular, or intranasal injection. In some embodiments, the RNA (e.g., mRNA) vaccine is administered to the subject by intramuscular injection.

[0344] Some embodiments of the present disclosure provide a method for the preparation of a vaccine comprising administering to a subject an effective amount of a vaccine effective to produce an antigen-specific immune response in a subject, the vaccine comprising administering to a subject an effective amount of a vaccine effective to produce an antigen-specific immune response in a subject, the vaccine comprising administering to a subject an effective amount of a vaccine effective to produce an antigen-specific immune response in a subject, the vaccine comprising administering to a subject an effective amount of a vaccine effective to produce an antigen-specific immune response in a subject, the vaccine comprising administering to a subject an effective amount of a vaccine effective to produce an antigen-specific immune response in a subject In one embodiment, the method comprises administering to the subject an RNA (e.g., mRNA) vaccine against Chlamydia tuberculosis, Nipah virus, Norovirus, Rabies virus (RABV), Respiratory syncytial virus (RSV), Rhinovirus, Rotavirus, Vaccinia virus, Yellow fever virus (YFV), Zika virus (ZIKV), Chlamydia trachomatis (i.e., the bacterium Chlamydia that causes Chlamydia), or Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, or Plasmodium ovale.

[0345] The antigen-specific immune response in the subject can, in some embodiments, be determined to be a response to an antigen specific immune response in a subject to a virus ... tuberculosis, Nipah virus, Norovirus, Rabies virus (RABV), Respiratory syncytial virus (RSV), Rhinovirus, Rotavirus, Vaccinia virus, Yellow fever virus (YFV), Zika virus (ZIKV), Chlamydia trachomatis (i.e., the bacterium Chlamydia that causes chlamydia), or malaria parasites (e.g., Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, or Plasmodium ovale). Following administration to a subject of either an ovale) RNA (e.g., mRNA) vaccine, antibody titers (including antibodies against SARS coronavirus 2 (SARS-CoV-2), nCoV-2019 coronavirus, SARS coronavirus (SARS-CoV), Bunyavirales viruses, cytomegalovirus (CMV), dengue virus (DENV-1, DENV-2, DENV-3, and DENV-4), Ebola virus (EBOV), flavivirus, hepatitis B virus (HBV), herpes simplex virus (HSV), human immunodeficiency virus (HIV), human metapneumovirus (HMPV), human papillomavirus (HPV), human parainfluenza virus (HPIV), influenza virus, extraintestinal pathogenic Escherichia coli (E.coli (ExPEC), Lassa-Mam Arenavirus (LASV), MERS coronavirus, Mycobacterium tuberculosis, Nipah virus, norovirus, rabies virus (RABV), respiratory syncytial virus (RSV), rhinovirus, rotavirus, vaccinia virus, yellow fever virus (YFV), Zika virus (ZIKV), Chlamydia trachomatis (i.e., the bacterium Chlamydia that causes chlamydia), or malaria parasites (e.g., Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, or Plasmodium ovale)) antigenic polypeptides). In some embodiments, the anti-antigenic polypeptide antibody titer produced in the subject is increased by at least 1 log relative to a control. In some embodiments, the anti-antigenic polypeptide antibody titer produced in the subject is increased by 1-3 logs relative to a control.

[0346] Tumor antigens In a further preferred embodiment, the mRNA encodes a tumor antigen, preferably as defined herein, or a fragment or variant thereof, and the tumor antigen is preferably selected from the group consisting of, but not limited to, the tumor antigens disclosed on pages 47-51 of WO2018078053; WO2018078053 is incorporated herein by reference in its entirety.

[0347] Further, in the present invention, cytokines, chemokines, suicide enzymes and gene products, apoptosis inducers, endogenous angiogenesis inhibitors, heat shock proteins, tumor antigens, innate immune activators, antibodies directed against proteins associated with tumors or cancer development, useful for example in cancer treatment, are selected from the group of cytokines, chemokines, suicide enzymes and gene products, apoptosis inducers, endogenous angiogenesis inhibitors, heat shock proteins, tumor antigens, innate immune activators, antibodies directed against proteins associated with tumors or cancer development, as disclosed in Tables 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 of WO2016170176; WO2016170176 and in particular Tables 1-12 are incorporated herein by reference in their entirety.

[0348] Other antigens Further antigens useful in the present invention are listed on pages 48-51 of WO2018078053; WO2018078053 is incorporated herein by reference in its entirety.

[0349] Allergen antigens and autoimmune autoantigens As mentioned, the mRNA may code for an antigen that represents an allergen, or an allergen antigen, or an autoantigen, also called an autoantigen or autoimmune antigen. Such antigens and autoantigens (allergens or allergen antigens) associated with allergy or allergic disease are derived or preferably selected from the group of antigens disclosed on pages 59-73 of WO2018078053, including, but not limited to, the entire WO2018078053, which is incorporated herein by reference.

[0350] Molecular therapy - Enzyme replacement therapy - mRNA replacement therapy In other preferred embodiments, the nucleic acid compound is an mRNA comprising at least one coding region encoding a therapeutic protein to replace an absent, missing, or mutated protein; a therapeutic protein beneficial for treating a genetic or acquired disease; an infectious disease, or a neoplasm (e.g., cancer or tumor disease); an adjuvant or immune stimulating therapeutic protein; a therapeutic antibody or antibody fragment, variant, or derivative; a peptide hormone; a gene editing agent; an immune checkpoint inhibitor; a T-cell receptor, or a fragment, variant, or derivative T-cell receptor; and / or an enzyme. In another embodiment, the peptide or protein expressed by the nucleic acid compound is a therapeutic protein, or a fragment or variant thereof, which is useful for treating or preventing a genetic or acquired disease or improving the condition of an individual. In particular, therapeutic proteins play an important role in the design of new therapeutic agents that can, among other functions, modify and repair genetic defects, destroy cancer cells or pathogen-infected cells, treat or prevent immune system disorders, or treat or prevent metabolic or endocrine disorders. Indeed, in one embodiment, an mRNA comprising at least one coding sequence is (a) a peptide or protein, or a fragment or variant thereof, wherein the peptide or protein is an antigen, the antigen being preferably derived from a pathogenic antigen, a tumor antigen, an allergen antigen or an autoimmune autoantigen, or a fragment or variant thereof; or (b) a therapeutic protein, or a fragment or variant thereof The therapeutic protein may encode, for example, (i) Therapeutic proteins for use in enzyme replacement therapy to treat metabolic, endocrine or amino acid disorders, or for use in replacing absent, defective or mutated proteins; (ii) a therapeutic protein for use in the treatment of a blood disorder, a disease of the circulatory system, a disease of the respiratory system, an infectious disease or an immune deficiency; (iii) therapeutic proteins for use in the treatment of cancer or tumor diseases; (iv) therapeutic proteins for use in hormone replacement therapy; (v) therapeutic proteins for use in reprogramming somatic cells into pluripotent or totipotent stem cells; (vi) therapeutic proteins for use as adjuvants or immunostimulants; (vii) a therapeutic protein that is a therapeutic antibody; (viii) a therapeutic protein that is a gene editing agent; and (ix) A therapeutic protein for use in the treatment or prevention of a liver disease selected from the group consisting of liver fibrosis, liver cirrhosis and liver cancer. may be selected from the group consisting of:

[0351] mRNA elements According to certain embodiments of the present invention, the mRNA sequence is preferably mono-, bi- or multicistronic, as defined herein. The coding sequence of the bi- or multicistronic mRNA preferably codes for separate antigens or fragments or variants thereof as defined herein. Preferably, the coding sequence coding for two or more antigens can be separated in the bi- or multicistronic mRNA by at least one IRES (internal ribosome entry site) sequence, as defined below. Thus, the term "coding for two or more antigens" can mean, but is not limited to, that the bi- or even multicistronic mRNA can code for at least two, three, four, five, six or more (preferably different) antigens or fragments or variants thereof, within the definition provided herein. More preferably, but is not limited to, that the bi- or even multicistronic mRNA can code for at least two, three, four, five, six or more (preferably different) antigens, as defined herein, or fragments or variants thereof, as defined herein. In this context, the so-called IRES (internal ribosome entry site) sequence can function as the only ribosome binding site, but can also serve to provide a bi- or even multicistronic mRNA as defined above, which encodes several antigens that are translated by the ribosome independently of each other. Examples of IRES sequences that can be used according to the invention are those derived from picornaviruses (e.g. FMDV), pestiviruses (CFFV), polioviruses (PV), encephalomyocarditis viruses (ECMV), foot and mouth disease viruses (FMDV), hepatitis C viruses (HCV), swine fever viruses (CSFV), murine leukemia viruses (MLV), simian immunodeficiency viruses (SIV) or cricket paralysis viruses (CrPV).

[0352] According to further embodiments, at least one coding region or coding sequence of the mRNA sequence according to the present invention may code for at least two, three, four, five, six, seven, eight and more antigens or fragments or variants thereof as defined herein linked with or without an amino acid linker sequence, said linker sequence may comprise a rigid linker, a flexible linker, a cleavable linker (e.g., a self-cleaving peptide) or a combination thereof. Therein, the antigens may be the same, different or a combination thereof. A particular antigen combination may be encoded by said mRNA encoding at least two antigens as described herein (also referred to herein as a "multi-antigen construct / mRNA").

[0353] In another preferred embodiment, the mRNA encodes a pathogenic antigen whose amino acid sequence is not modified with respect to its respective wild-type amino acid sequence.In this case, the mRNA compound can also include coding regions with nucleic acid sequences that are not modified with respect to their respective wild-type mRNA sequences.For example, the mRNA compound can be natural and unmodified mRNA.As used herein, natural and unmodified mRNA encompasses the mRNA that is produced in vitro, which has neither chemical modification nor sequence change.

[0354] self-replicating RNA In one embodiment, the nucleic acids of the present invention, particularly RNA sequences, are capable of self-replication. Thus, polynucleotides may be capable of self-replication when introduced into a host cell. For example, examples of polynucleotides include self-replicating RNA and DNA selected from, for example, replicons, plasmids, cosmids, phagemids, transposons, viral vectors, artificial chromosomes (e.g., bacteria, yeast, etc.) and other self-replicating species. Polynucleotides include self-replicating polynucleotides inserted with natural or synthetic sequences derived from eukaryotes or prokaryotes (e.g., genomic DNA sequences, genomic RNA sequences, cDNA sequences, etc.). Specific examples of self-replicating polynucleotides include, among others, RNA vector constructs and DNA vector constructs. Sequences that can be expressed include, among others, natural sequences and modifications such as deletions, additions and substitutions (generally conservative in nature) to natural sequences. These modifications may be deliberate, such as through site-directed mutagenesis, or accidental, such as through mutations of the host that produces the antigen. In one aspect, the self-replicating RNA molecule is derived from or based on an alphavirus. In other embodiments, the self-replicating RNA molecule is derived from or based on a virus other than an alphavirus, preferably a positive-stranded RNA virus, more preferably a picornavirus, flavivirus, rubivirus, pestivirus, hepacivirus, calicivirus, or coronavirus. Suitable wild-type alphavirus sequences are well known and available from sequence repositories such as the American Type Culture Collection (Rockville, MD).Representative examples of suitable alphaviruses include Aura (ATCC VR-368), Bebaru virus (ATCC VR-600, ATCC VR-1240), Cabassou (ATCC VR-922), Chikungunya virus (ATCC VR-64, ATCC VR-1241), Eastern equine encephalitis virus (ATCC VR-65, ATCC VR-1242), Fort Morgan (ATCC VR-924), Getah virus (ATCC VR-369, ATCC VR-1243), Kyzylagach (ATCC VR-927), Mayaro (ATCC VR-66), Mayaro virus (ATCC VR-928), and others. VR-1277), Middleburg (ATCC VR-370), Mucambo virus (ATCC VR-580, ATCC VR-1244), Ndumu (ATCC VR-371), Pixuna virus (ATCC VR-372, ATCC VR-1245), Ross River virus (ATCC VR-373, ATCC VR-1246), Semliki Forest (ATCC VR-67, ATCC VR-1247), Sindbis virus (ATCC VR-68, ATCC VR-1248), Tonate (ATCC VR-925), Triniti (ATCC VR-469), Una (ATCC VR-374), Venezuelan equine encephalitis (ATCC VR-69, ATCC VR-923, ATCC VR-1250, ATCC VR-1249, ATCC VR-532), Western equine encephalitis (ATCC VR-70, ATCC VR-1251, ATCC VR-622, ATCC VR-1252), Whataroa (ATCC VR-926), and Y-62-33 (ATCC VR-375).

[0355] mRNA modifications and sequences In another embodiment of the invention, the mRNA compound comprises an artificial mRNA. In this context, an artificial mRNA encompasses an mRNA having a chemical modification, a sequence modification or a non-natural sequence.

[0356] chemical modification According to another embodiment of the present invention, the mRNA compound contained in the composition comprises at least one chemical modification. In one embodiment, the chemical modification can be selected from the group consisting of base modification, sugar modification, backbone modification, and lipid modification. The backbone modification in the present invention is a modification in which the backbone phosphate of the nucleotide contained in the mRNA compound comprising the mRNA sequence defined herein is chemically modified. The sugar modification in the present invention is a chemical modification of the sugar of the nucleotide of the mRNA compound comprising the mRNA sequence defined herein. Furthermore, the base modification in the present invention is a chemical modification of the base portion of the nucleotide of the mRNA compound comprising the mRNA. In this context, the nucleotide analog or modification is preferably selected from nucleotide analogs that are applicable to transcription and / or translation.

[0357] sugar modification Modified nucleosides and nucleotides that can be incorporated into modified mRNA compounds containing the mRNA sequences described herein can have sugar moieties modified. For example, the 2' hydroxyl group (OH) can be modified or replaced with several different "oxy" or "deoxy" substituents. Examples of "oxy"-2' hydroxyl group modifications include, but are not limited to, alkoxy or aryloxy (-OR, e.g., R=H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar); polyethylene glycol (PEG), -O(CH2CHO)nCH2CH2OR; "locked" nucleic acids (LNA), in which the 2' hydroxyl is linked to the 4' carbon of the same ribose sugar, e.g., by a methylene bridge; and amino groups (amino groups, e.g., -O-amino, where NRR can be alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, polyamino) or aminoalkoxy.

[0358] A "deoxy" modification includes hydrogen, amino (e.g., NH; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); or the amino group can be attached to the sugar through a linker, the linker including one or more of the atoms C, N, and O.

[0359] The sugar group can also contain one or more carbons that have the opposite stereochemical configuration to the corresponding carbon in ribose. Thus, modified mRNA can include nucleotides that contain, for example, arabinose as the sugar.

[0360] Backbone Modification The backbone phosphate group can be modified by replacing one or more of the oxygen atoms with different substituents. In addition, modified nucleosides and nucleotides can include complete replacement of unmodified phosphate moieties with modified phosphates as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, alkyl or aryl phosphonates, and phosphotriesters. Both phosphorodithioates have non-linked oxygens replaced by sulfur. Phosphate linkers can also be modified by replacing the linking oxygen with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylene-phosphonates).

[0361] lipid modification A lipid-modified mRNA typically comprises at least one linker covalently linked to the mRNA, and at least one lipid covalently linked to each linker. Alternatively, a lipid-modified mRNA comprises at least one mRNA as defined herein, and at least one (bifunctional) lipid covalently linked to the mRNA (without linker). According to a third alternative, a lipid-modified mRNA comprises an mRNA molecule as defined herein, at least one linker covalently linked to the mRNA, and at least one lipid covalently linked to each linker, and also at least one (bifunctional) lipid covalently linked to the mRNA (without linker). In this context, it is particularly preferred that lipid modification is present at the end of the linear mRNA sequence.

[0362] In another preferred embodiment, the mRNA compound does not contain nucleoside modifications, in particular does not contain base modifications. In a further embodiment, the mRNA compound does not contain 1-methylpseudouridine modifications, pseudouridine modifications, or 5-methyluridine modifications. In one preferred embodiment, the mRNA compound contains only naturally occurring nucleosides. In a further preferred embodiment, the mRNA compound does not contain any chemical modifications, and optionally contains sequence modifications. In a further preferred embodiment of the present invention, the mRNA compound contains only naturally occurring nucleosides adenine, uracil, guanine, and cytosine.

[0363] Base Modification In an alternative embodiment, the mRNA compound comprises at least one base modification. Modified nucleosides and nucleotides that can be incorporated into modified mRNA compounds that contain the mRNA sequences described herein can further be modified at the nucleobase portion.Examples of nucleobases found in mRNA include, but are not limited to, adenine, guanine, cytosine and uracil.For example, the nucleosides and nucleotides described herein can be chemically modified on the major groove surface.In some embodiments, the major groove chemical modification can include an amino group, a thiol group, an alkyl group, or a halo group.

[0364] In a particularly preferred embodiment of the invention, the nucleotide analogue / modification is preferably 2-amino-6-chloropurine riboside-5'-triphosphate, 2-aminopurine-riboside-5'-triphosphate, 2-aminoadenosine-5'-triphosphate, 2'-amino-2'-deoxycytidine-triphosphate, 2-thiocytidine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 2'-fluorothymidine-5'-triphosphate, 2'-O-methyl-inosine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 4-amino-6-chloropurine riboside ... 5'-triphosphate, 5-aminoallylcytidine-5'-triphosphate, 5-aminoallyluridine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, 5-bromouridine-5'-triphosphate, 5-bromo-2'-deoxycytidine-5'-triphosphate, 5-bromo-2'-deoxyuridine-5'-triphosphate, 5-iodocytidine-5'-triphosphate, 5-iodo-2'-deoxycytidine-5'-triphosphate, 5-iodouridine-5'-triphosphate, 5-iodo-2'-deoxycytidine-5'-triphosphate, '-Deoxyuridine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, 5-methyluridine-5'-triphosphate, 5-propynyl-2'-deoxycytidine-5'-triphosphate, 5-propynyl-2'-deoxyuridine-5'-triphosphate, 6-azacytidine-5'-triphosphate, 6-azauridine-5'-triphosphate, 6-chloropurine riboside-5'-triphosphate, 7-deazaadenosine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 8-aza- The base modifications are selected from zaadenosine-5'-triphosphate, 8-azidoadenosine-5'-triphosphate, benzimidazole-riboside-5'-triphosphate, N1-methyladenosine-5'-triphosphate, N1-methylguanosine-5'-triphosphate, N6-methyladenosine-5'-triphosphate, O6-methylguanosine-5'-triphosphate, pseudouridine-5'-triphosphate, or puromycin-5'-triphosphate, xanthosine-5'-triphosphate.Particularly preferred are nucleotides with base modifications selected from the group of base-modified nucleotides consisting of 5-methylcytidine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, and pseudouridine-5'-triphosphate. In some embodiments, the modified nucleoside is pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1 ... pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thiouridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine.In some embodiments, the modified nucleoside is 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl -pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine. In other embodiments, the modified nucleoside is 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyl adenosine, These include N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine.In other embodiments, modified nucleosides include inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine. In some embodiments, the nucleotide may be modified on the major groove face and may include replacement of the hydrogen on C-5 of uracil with a methyl or halo group. In specific embodiments, the modified nucleoside is 5'-O-(1-thiophosphate)-adenosine, 5'-O-(1-thiophosphate)-cytidine, 5'-O-(1-thiophosphate)-guanosine, 5'-O-(1-thiophosphate)-uridine, or 5'-O-(1-thiophosphate)-pseudouridine.

[0365] In more specific embodiments, the modified mRNA is selected from the group consisting of 6-aza-cytidine, 2-thio-cytidine, α-thio-cytidine, pseudo-iso-cytidine, 5-aminoallyl-uridine, 5-iodo-uridine, N1-methyl-pseudouridine, 5,6-dihydrouridine, α-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxy-thymidine, 5-methyl-uridine, pyrrolo-cytidine, inotropic uridine, cytosine ... The nucleoside modifications may include nucleoside modifications selected from: alpha-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytidine, 8-oxo-guanosine, 7-deaza-guanosine, N1-methyl-adenosine, 2-amino-6-chloro-purine, N6-methyl-2-amino-purine, pseudo-iso-cytidine, 6-chloro-purine, N6-methyl-adenosine, alpha-thio-adenosine, 8-azido-adenosine, 7-deaza-adenosine.

[0366] In further embodiments, the chemical modification is selected from pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2'-O-methyluridine.

[0367] In specific embodiments, the chemical modification is selected from the group consisting of pseudouracil (psi or ψ), N1-methylpseudouracil (N1MPU, N1Mpsi or N1Mψ), 1-ethylpseudouracil, 2-thiouracil (s2U), 4-thiouracil, 5-methylcytosine, 5-methyluracil, 5-methoxyuracil, and any combination thereof, and most preferably, the chemical modification is N1-methylpseudouracil (N1MPU, N1Mpsi or N1Mψ).

[0368] Array Modification According to further embodiments, the mRNA compound comprises modified mRNA sequence.For example, the modification of the mRNA sequence can lead to the stabilization of the mRNA sequence.In one embodiment, the mRNA compound comprises a stabilized mRNA sequence comprising at least one coding region as defined herein.In particular, the composition of the invention as described herein can comprise an mRNA compound comprising a coding region that codes for an antigen as defined in any of the embodiments as described herein, said coding region exhibiting sequence modification.

[0369] According to one embodiment, the mRNA compound comprises a "stabilized mRNA sequence", i.e. an mRNA that is essentially resistant to in vivo degradation (e.g. by exo- or endo-nucleases). Such stabilization may be brought about, for example, by a modified phosphate backbone of the mRNA of the present invention. Backbone modifications in the context of the present invention are modifications in which the backbone phosphates of the nucleotides contained in the mRNA are chemically modified. The nucleotides that may be used in this context preferably contain, for example, phosphorothioate modified phosphate backbones, preferably in which at least one of the phosphate oxygens contained in the phosphate backbone is replaced by a sulfur atom. The stabilized mRNA may further comprise, for example: alkyl and aryl phosphonates, in which the charged phosphate oxygen is replaced by an alkyl or aryl group, or non-ionic phosphate analogs such as phosphodiesters and alkyl phosphotriesters, in which the charged oxygen residues are present in alkylated form. Such backbone modifications typically include, without implying any limitation, modifications from the group consisting of methylphosphonates, phosphoramidates and phosphorothioates (e.g. cytidine-5'-O-(1-thiophosphate)).

[0370] Below, specific modifications are described which are preferably capable of "stabilizing" mRNA as defined herein. G / C content modification According to one embodiment, the mRNA compound comprises an mRNA sequence that has been modified, and thus stabilized, by modification of its guanosine / cytosine (G / C) content, such modification, or at least one of these modifications, being located in the coding region of the mRNA compound.

[0371] In one preferred embodiment, the G / C content of the coding region of the mRNA compound is increased compared to the G / C content of the coding region of the respective wild-type mRNA, i.e., unmodified mRNA.At the same time, the amino acid sequence encoded by the mRNA is preferably unmodified compared to the amino acid sequence encoded by the respective wild-type mRNA.For example, the above composition may comprise an mRNA compound that encodes a pathogenic antigen whose amino acid sequence is unmodified with respect to the coding amino acid sequence of the respective wild-type nucleic acid.

[0372] This modification of the mRNA sequence of the present invention is based on the fact that the sequence of any mRNA region to be translated is important for the efficient translation of that mRNA. Thus, the composition of the mRNA and the sequence of the various nucleotides are important. In particular, sequences with an increased G (guanosine) / C (cytosine) content are more stable than those with an increased A (adenosine) / U (uracil) content. Thus, according to the present invention, the codons of the mRNA are varied compared to the respective wild-type mRNA, while retaining the translated amino acid sequence, to contain an increased amount of G / C nucleotides. With regard to the fact that several codons code for one and the same amino acid (the so-called degeneracy of the genetic code), it is possible to determine the codons that are most favorable for stability (the so-called alternative codon usage). Depending on the amino acids coded by the mRNA, there are various possibilities for the modification of the mRNA sequence compared to its wild-type sequence. In the case of amino acids coded by codons containing exclusively G or C nucleotides, no modification of the codons is necessary. Thus, the codons for Pro (CCC or CCG), Arg (CGC or CGG), Ala (GCC or GCG) and Gly (GGC or GGG) do not require modification since neither A nor U is present. In contrast, codons containing A and / or U nucleotides can be modified by substitution of other codons that code for the same amino acid but do not contain A and / or U. Examples of these are: the codon for Pro can be modified from CCU or CCA to CCC or CCG; the codon for Arg can be modified from CGU or CGA or AGA or AGG to CGC or CGG; the codon for Ala can be modified from GCU or GCA to GCC or GCG; the codon for Gly can be modified from GGU or GGA to GGC or GGG. In other cases, neither A nor U nucleotides can be eliminated from the codon, but it is possible to reduce the A and U content by using codons that contain a lower content of A and / or U nucleotides.These include: the codon for Phe may be modified from UUU to UUC; the codon for Leu may be modified from UUA, UUG, CUU or CUA to CUC or CUG; the codon for Ser may be modified from UCU or UCA or AGU to UCC, UCG or AGC; the codon for Tyr may be modified from UAU to UAC; the codon for Cys may be modified from UGU to UGC; the codon for His may be modified from CAU to CAC; the codon for Gln may be modified from CAA to CAG. ; the codon for Ile may be modified from AUU or AUA to AUC; the codon for Thr may be modified from ACU or ACA to ACC or ACG; the codon for Asn may be modified from AAU to AAC; the codon for Lys may be modified from AAA to AAG; the codon for Val may be modified from GUU or GUA to GUC or GUG; the codon for Asp may be modified from GAU to GAC; the codon for Glu may be modified from GAA to GAG; the stop codon UAA may be modified to UAG or UGA. On the other hand, in the case of the codons for Met (AUG) and Trp (UGG), there is no possibility of sequence modification. The substitutions listed above can be used individually or in all possible combinations to increase the G / C content of the mRNA sequence of the invention compared to that particular wild-type mRNA (i.e. the original sequence). Thus, for example, all codons for Thr present in the wild-type sequence may be modified to ACC (or ACG). However, preferably, a combination of the above substitution possibilities is used, for example: - replacement of all codons encoding Thr in the original sequence (wild-type mRNA) with ACC (or ACG), and -Replacement of all codons originally encoding Ser with UCC (or UCG or AGC); - replacement of all codons encoding Ile in the original sequence with AUC, and - the replacement of all codons originally encoding Lys with AAG, and -Replacement of all codons originally encoding Tyr with UAC; - replacement of all codons encoding Val in the original sequence with GUC (or GUG), and -Replacement of all codons originally encoding Glu with GAG; and -Replace all codons originally encoding Ala with GCC (or GCG), and -Replacement of all codons originally encoding Arg with CGC (or CGG); - replacement of all codons encoding Val in the original sequence with GUC (or GUG), and -Replacement of all codons originally encoding Glu with GAG; and -Replace all codons originally encoding Ala with GCC (or GCG), and -Replace all codons originally encoding Gly with GGC (or GGG), and -Replacement of all codons originally encoding Asn with AAC; - replacement of all codons encoding Val in the original sequence with GUC (or GUG), and -Phe, and replacing all codons originally encoding it with UUC. -Replacement of all codons originally encoding Cys with UGC, and -Replace all codons originally encoding Leu with CUG (or CUC), and -replacement of all codons originally encoding Gln with CAG; and -Replacement of all codons originally encoding Pro with CCC (or CCG); etc.

[0373] Preferably, the G / C content of the coding region of the mRNA compound comprising the mRNA sequence of the invention is increased by at least 7%, more preferably at least 15%, particularly preferably at least 20% compared to the G / C content of the coding region of the wild-type RNA encoding the antigen or fragment or variant thereof as defined herein. According to a specific embodiment, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, more preferably at least 70%, even more preferably at least 80%, most preferably at least 90%, 95% or even 100% of the replaceable codons of the region encoding the peptide or protein or fragment or variant thereof as defined herein or of the entire sequence of the wild-type mRNA sequence are replaced, thereby increasing the G / C content of said sequence. In this context, it is particularly preferred to increase the G / C content of at least one coding region of the mRNA sequence of the invention, preferably the mRNA sequence according to the invention, to the maximum extent (i.e. 100% of the replaceable codons) compared to the wild-type sequence. According to the present invention, a further preferred modification of the mRNA sequence of the present invention is based on the observation that the translation efficiency is also determined by the different frequency of occurrence of tRNAs in a cell. Thus, when so-called "rare codons" are present to an increased extent in the mRNA sequence of the present invention, the corresponding modified mRNA sequence is translated to a significantly lower extent than when codons encoding relatively "frequent" tRNAs are present. According to the present invention, in the modified mRNA sequence of the present invention, the region encoding a peptide or protein as defined herein or a fragment or variant thereof is modified compared to the corresponding region of the wild-type mRNA sequence, such that at least one codon of the wild-type sequence encoding a relatively rare tRNA in a cell is replaced with a codon that is relatively frequent in a cell and that encodes a tRNA carrying the same amino acid as the relatively rare tRNA. This modification modifies the sequence of the mRNA of the present invention to insert a codon that is available for a frequently occurring tRNA.In other words, according to the present invention, by this modification, all codons of the wild-type sequence that code for a tRNA that is relatively rare in the cell can be exchanged for codons that code for a tRNA that is relatively frequent in the cell and that carries the same amino acid as the relatively rare tRNA in each case. Which tRNAs are relatively frequent in the cell and which, in contrast, are relatively rare, are known to those skilled in the art; see, for example, Akashi, Curr. Opin. Genet. Dev. 2001, 11(6):660-666. For a particular amino acid, the codon that uses the most frequently occurring tRNA, for example the Gly codon, that uses the most frequently occurring tRNA in (human) cells, is particularly preferred. According to the present invention, it is particularly preferred to combine an increased, in particular maximized, consecutive G / C content in the modified mRNA sequence of the present invention with a "frequent" codon without modifying the amino acid sequence of the protein encoded by the coding region of the mRNA sequence. This preferred embodiment allows the provision of a particularly efficiently translated, stabilized (modified) mRNA sequence of the invention. The determination of the above-mentioned modified mRNA sequences of the invention (increased G / C content; exchange of tRNA) can be carried out using the computer program described in WO 2002098443, the disclosure content of which is included in the full scope of the present invention. Using this computer program, the nucleotide sequence of the desired mRNA sequence can be modified using the genetic code or its degenerate nature, such that the maximum G / C content, in combination with the use of codons that code for tRNAs present at the highest possible frequency in the cell, results in an amino acid sequence encoded by the modified mRNA sequence that is preferably unmodified compared to the unmodified sequence. Alternatively, it is also possible to modify only the G / C content or only the codon usage compared to the original sequence. The source code in Visual Basic 6.0 (development environment used: Microsoft Visual Studio Enterprise 6.0 with Service Pack 3) is also described in WO 02 / 098443.In a further preferred embodiment of the invention, the A / U content in the environment of the ribosome binding site of an mRNA sequence of the invention is increased compared to the A / U content in the environment of the ribosome binding site of its respective wild-type mRNA. This modification (increased A / U content around the ribosome binding site) increases the efficiency of binding of the ribosome to the mRNA. Effective binding of the ribosome to the ribosome binding site (Kozak sequence: SEQ ID NO: 1 or SEQ ID NO: 2, AUG forms the start codon, or a minimal Kozak binding site ACC) in turn has the effect of an efficient translation of the mRNA. According to a further embodiment of the invention, the mRNA sequence of the invention may be modified with respect to potential destabilizing sequence elements. In particular, the coding region and / or the 5' and / or 3' untranslated regions of this mRNA sequence may be modified compared to the respective wild-type mRNA, such that the coding amino acid sequence of the modified mRNA sequence is preferably unmodified compared to its respective wild-type mRNA and does not contain destabilizing sequence elements. For example, in eukaryotic mRNA sequences, it is known that there are destabilizing sequence elements (DSEs) to which signal proteins bind and regulate the enzymatic degradation of mRNA in vivo. Therefore, for further stabilization of the modified mRNA sequence, possibly in the region coding for at least one peptide or protein as defined herein or a fragment or variant thereof, one or more such modifications can be made in comparison with the corresponding region of the wild-type mRNA so that no or substantially no destabilizing sequence elements are contained therein. According to the present invention, DSEs present in the untranslated regions (3'- and / or 5'-UTR) can also be eliminated from the mRNA sequence of the present invention by such modifications. Such destabilizing sequences are, for example, AU-rich sequences (AURES) present in the 3'-UTR section of many unstable mRNAs (Caput et al., Proc. Natl. Acad. Sci. USA 1986, 83:1670-1674). Thus, the mRNA sequences of the present invention are preferably modified in comparison with the respective wild-type mRNA so that the mRNA sequences of the present invention do not contain such destabilizing sequences.This also applies to sequence motifs recognized by possible endonucleases, such as the sequence GAACAAG, contained in the 3'-UTR segment of the gene encoding the transferrin receptor (Binder et al., EMBO J. 1994, 13:1969-1980). These sequence motifs are also preferably removed in the mRNA sequences of the invention.

[0374] According to further embodiments, the mRNA compound comprises an mRNA sequence comprising a coding region comprising or consisting of any one of the RNA sequences disclosed in Tables 1-5, Figures 20-24 or the Sequence Listing of WO2018078053; Tables 1-5 or Figures 20-24 of WO2018078053; WO2018078053 is incorporated herein by reference in its entirety.

[0375] Human codon usage-matched sequences A further preferred modification of an mRNA compound is based on the observation that codons encoding the same amino acid typically occur at different frequencies. According to this embodiment, the frequency of codons encoding the same amino acid in the coding region of an mRNA compound differs from the naturally occurring frequency of that codon, for example according to the human codon usage frequency shown in Table 2 (human codon usage frequency table). For example, for the amino acid alanine (Ala), the wild-type coding region is preferably adapted such that the codon "GCC" is used at a frequency of 0.40, the codon "GCT" is used at a frequency of 0.28, the codon "GCA" is used at a frequency of 0.22, the codon "GCG" is used at a frequency of 0.10, etc. (see Table 2).

[0376] [Table 7]

[0377] Codon-optimized sequences In one embodiment, all codons of the wild-type sequence encoding a tRNA that is relatively rare in the cell are exchanged for codons encoding a tRNA that is relatively frequent in the cell and that in each case carries the same amino acid as the relatively rare tRNA. It is therefore particularly preferred that the most frequent codon is used for each encoded amino acid (see Table 2). Such an optimization procedure increases the codon adaptation index (CAI) and ultimately maximizes the CAI. In the context of the present invention, sequences with increased or maximized CAI are typically referred to as "codon-optimized" sequences and / or CAI-increased and / or maximized sequences. According to a preferred embodiment, the mRNA compound comprising the mRNA sequence of the present invention comprises at least one coding region, the coding region / sequence being codon-optimized as described herein. More preferably, the codon adaptation index (CAI) of at least one coding sequence is at least 0.5, at least 0.8, at least 0.9 or at least 0.95. Most preferably, the codon adaptation index (CAI) of at least one coding sequence is 1.

[0378] For example, for the amino acid alanine (Ala) present in the amino acid sequence encoded by at least one coding sequence of an RNA according to the invention, the wild-type coding sequence is adapted such that the most frequent human codon "GCC" is always used for said amino acid, or for the amino acid cysteine ​​(Cys), the wild-type sequence is adapted such that the most frequent human codon "TGC" is always used for said amino acid, etc.

[0379] C-optimized arrays According to another embodiment, the mRNA compound comprises an mRNA sequence having a modified, in particular increased, cytosine (C) content, preferably of the coding region of the mRNA sequence, compared to the C content of the coding region of the respective wild-type mRNA, i.e. unmodified mRNA, and at the same time, the amino acid sequence encoded by at least one coding region of the mRNA sequence of the invention is preferably unmodified compared to the amino acid sequence encoded by the respective wild-type mRNA.

[0380] In a preferred embodiment of the invention, the modified mRNA sequence is modified to achieve at least 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80% of the maximum theoretically possible cytosine content, or at least 90% or even the maximum cytosine content.

[0381] In a further preferred embodiment, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or even 100% of the codons of the "cytosine content optimizable" target mRNA wild-type sequence are replaced by codons having a cytosine content higher than the cytosine content present in the wild-type sequence.

[0382] In a further preferred embodiment, some of the codons of the wild-type coding sequence may be further modified so that the codons of the relatively rare tRNA in the cell are replaced by the codons of the relatively frequent tRNA in the cell, provided that the replacement codons of the relatively frequent tRNA carry the same amino acid as the relatively rare tRNA of the original wild-type codon.Preferably, all of the codons of the relatively rare tRNA are replaced by the codons of the relatively frequent tRNA in the cell, except for the codons that code for amino acids that are exclusively coded by codons that do not contain cytosines, or except for glutamine (Gln), which is coded by two codons that each contain the same number of cytosines.

[0383] In further preferred embodiments of the invention, the modified target mRNA is modified such that at least 80%, or at least 90%, or even the maximum theoretically possible cytosine content is achieved by codons encoding tRNAs that are relatively frequent in the cell, and the amino acid sequence remains unchanged.

[0384] Due to the naturally occurring degeneracy of genetic code, more than one codon can code for a particular amino acid. Thus, 18 naturally occurring amino acids out of 20 are coded by more than one codon (Trp and Met are the exceptions), for example, by two codons (e.g., Cys, Asp, Glu), by three codons (e.g., Ile), by four codons (e.g., Al, Gly, Pro), or by six codons (e.g., Leu, Arg, Ser). However, not all codons that code for the same amino acid are used at the same frequency under in vivo conditions. A typical codon usage profile has been established for each single organism.

[0385] The term "cytosine content optimizable codon" as used within the context of the present invention refers to a codon that exhibits a lower content of cytosine than other codons that code for the same amino acid. Thus, any wild-type codon that can be replaced by another codon that codes for the same amino acid and exhibits a higher number of cytosines in that codon is considered to be cytosine-optimizable (C-optimizable). Any such replacement of a C-optimizable wild-type codon in a wild-type coding region with a specific C-optimized codon increases its overall C content, reflecting a C-enriched modified mRNA sequence. According to a preferred embodiment, the mRNA sequence of the present invention, preferably at least one coding region of the mRNA sequence of the present invention, comprises or consists of a C-maximized mRNA sequence that contains a C-optimized codon for every potential C-optimizable codon. Thus, 100% or all of the theoretically replaceable C-optimizable codons are replaced by C-optimized codons, preferably over the entire length of the coding region.

[0386] In this context, a cytosine content optimizable codon is one that contains fewer cytosines than other codons that encode the same amino acid. and / or the codons GCG, GCA, GCU all code for the amino acid Ala and may be replaced by the codon GCC which codes for the same amino acid; The codon UGU encoding Cys may be replaced by the codon UGC encoding the same amino acid, and / or The codon GAU encoding Asp may be replaced by the codon GAC encoding the same amino acid, and / or The codon UUU encoding Phe may be replaced by the codon UUC encoding the same amino acid, and / or Any of the codons GGG, GGA, GGU encoding Gly may be replaced by the codon GGC encoding the same amino acid, and / or The codon CAU encoding His may be replaced by the codon CAC encoding the same amino acid, and / or Any of the codons AUA, AUU encoding Ile may be replaced by the codon AUC, and / or Any of the codons UUG, UUA, CUG, CUA, CUU encoding Leu may be replaced by the codon CUC encoding the same amino acid; and / or The codon AAU encoding Asn may be replaced by the codon AAC encoding the same amino acid, and / or Any of the codons CCG, CCA, CCU encoding Pro may be replaced by the codon CCC encoding the same amino acid, and / or Any of the codons AGG, AGA, CGG, CGA, CGU encoding Arg may be replaced by the codon CGC encoding the same amino acid, and / or Any of the codons AGU, AGC, UCG, UCA, UCU encoding Ser may be replaced by the codon UCC encoding the same amino acid, and / or any of the codons ACG, ACA, and ACU encoding Thr may be replaced by the codon ACC encoding the same amino acid; and / or Any of the codons GUG, GUA, GUU encoding Val may be replaced by the codon GUC encoding the same amino acid, and / or The codon UAU encoding Tyr can be replaced by the codon UAC encoding the same amino acid.

[0387] In any of the above examples, the number of cytosines is increased by 1 per replaced codon. Replacement of all non-C-optimized codons in a coding region (corresponding to C-optimizable codons) results in a C-maximized coding sequence. In the context of the present invention, at least 70%, preferably at least 80%, more preferably at least 90% of the non-C-optimized codons in at least one coding region of an mRNA sequence according to the present invention are replaced by C-optimized codons.

[0388] It may be preferred that for some amino acids the percentage of C-optimizable codons replaced by C-optimized codons is less than 70%, and for other amino acids the percentage of replaced codons is higher than 70%, meeting an overall percentage of C-optimization of at least 70% of all C-optimizable wild-type codons in the coding region.

[0389] Preferably, in the C-optimized mRNA sequence, at least 50% of the C-optimizable wild-type codons for any given amino acid are replaced by C-optimized codons, for example, any modified C-enriched mRNA sequence contains at least 50%, preferably at least 60%, C-optimizable codons at the C-optimizable wild-type codon positions, preferably encoding any one of the amino acids Ala, Cys, Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Arg, Ser, Thr, Val and Tyr mentioned above.

[0390] In this context, a codon that is not cytosine content optimizable but codes for an amino acid that is coded for by at least two codons can be used without any further selection step. However, a codon of the wild-type sequence that codes for a tRNA that is relatively rare in a cell, for example a human cell, can be replaced by a codon that codes for a tRNA that is relatively frequent in a cell (both coding for the same amino acid). Thus, the relatively rare codon GAA that codes for Glu can be replaced by the relatively frequent codon GAG that codes for the same amino acid, and / or The relatively rare codon AAA encoding Lys may be replaced by the relatively frequent codon AAG encoding the same amino acid, and / or The relatively rare codon CAA encoding Gln can be exchanged for the relatively common codon CAG encoding the same amino acid.

[0391] In this context, the amino acids Met (AUG) and Trp (UGG), each of which is encoded by a single codon, remain unchanged. The stop codons are not optimized for cytosine content, but the relatively rare stop codons amber, ochre (UAA, UAG) can be replaced by the relatively frequent stop codon opal (UGA).

[0392] The single substitutions listed above can be used individually as well as in all possible combinations to optimize the cytosine content of the modified mRNA sequence compared to the wild-type mRNA sequence. Thus, at least one coding sequence defined herein may be altered compared to the coding region of the respective wild-type mRNA such that an amino acid is encoded by at least two or more codons, one of which contains an additional cytosine, and such codons may be replaced by C-optimized codons containing an additional cytosine, such that the amino acid is preferably unchanged compared to the wild-type sequence.

[0393] According to a further preferred embodiment, the compositions of the present invention comprise an mRNA compound whose coding region has an increased G / C content compared to the G / C content of the corresponding coding region of the corresponding wild-type mRNA and / or an increased C content compared to the C content of the corresponding coding region of the corresponding wild-type mRNA, and / or the codons in the coding region have been adapted to human codon usage, preferably with an increased or maximized Codon Adaptation Index (CAI), and the amino acid sequence encoded by the mRNA sequence is preferably unmodified compared to the amino acid sequence encoded by the corresponding wild-type mRNA.

[0394] In one preferred embodiment of the invention, the composition comprises an mRNA compound comprising a coding region encoding a peptide or protein, the coding region exhibiting sequence modifications selected from G / C content modifications of the sequence, codon modifications, codon optimization or C optimization.

[0395] In another preferred embodiment, the composition or lipid nanoparticle defined herein comprises an mRNA comprising a coding region encoding a peptide or protein defined herein, which, compared to the coding region of the corresponding wild-type mRNA, - Increased G / C content in coding regions; -The C content of the coding region is increased; - the codon usage of the coding region has been adapted to human codon usage; and / or the Codon Adaptation Index (CAI) has been increased or maximized in the coding region.

[0396] 5' Cap Structure Suitably, the coding RNA may be modified by the addition of a 5'-cap structure, thereby preferably stabilizing the coding RNA and / or enhancing expression of the encoded antigen and / or reducing stimulation of the innate immune system (after administration to a subject). The 5'-cap structure is particularly important in embodiments where the nucleic acid is an RNA, in particular a linear coding RNA, such as a linear mRNA or a linear coding replicon RNA.

[0397] Thus, the RNA, in particular the coding RNA, comprises a 5' cap structure, preferably a cap0, cap1, cap2, modified cap0, or modified cap1 structure. The term "5'-cap structure" as used herein is intended to refer to a 5' modified nucleotide, particularly a guanine nucleotide, that is recognized and understood by those of skill in the art and is located at the 5' end of, for example, an RNA, such as an mRNA. Preferably, the 5' cap structure is attached to the RNA via a 5'-5'-triphosphate bond.

[0398] 5' cap structures that may be suitable in the context of the present invention are cap 0 (methylation of the first nucleobase, e.g., m7GpppN), cap 1 (additional methylation of the ribose of the nucleotide adjacent to m7GpppN), cap 2 (additional methylation of the ribose of the second nucleotide downstream of m7GpppN), cap 3 (additional methylation of the ribose of the third nucleotide downstream of m7GpppN), cap 4 (additional methylation of the ribose of the fourth nucleotide downstream of m7GpppN), ARCA (anti-reverse cap analog), modified ARCA (e.g., phosphothioate modified ARCA), inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0399] The 5' cap (cap0 or cap1) structure can also be formed by chemical RNA synthesis or RNA in vitro transcription using a cap analog (co-transcriptional capping).

[0400] The term "cap analog" as used herein is recognized and understood by those of skill in the art and is intended to refer to a non-polymerizable di- or trinucleotide that has a cap functionality in that, for example, when incorporated at the 5' end of a nucleic acid molecule, it facilitates translation or localization and / or prevents degradation of the nucleic acid molecule, particularly an RNA molecule. Non-polymerizable means that the cap analog is incorporated only at the 5' end because it does not have a 5' triphosphate and therefore cannot be extended in the 3' direction by a template-dependent polymerase, preferably a template-dependent RNA polymerase. Examples of cap analogs include, but are not limited to, chemical structures selected from the group consisting of m7GpppG, m7GpppA, m7GpppC; unmethylated cap analogs (e.g., GpppG); dimethylated cap analogs (e.g., m2,7GpppG), trimethylated cap analogs (e.g., m2,2,7GpppG), dimethylated symmetric cap analogs (e.g., m7Gpppm7G), or anti-reverse cap analogs (e.g., ARCA; m7,2'OmeGpppG, m7,2'dGpppG, m7,3'OmeGpppG, m7,3'dGpppG and their tetraphosphate derivatives). Additionally, cap analogs have been previously described (WO2008016473, WO2008157688, WO2009149253, WO2011015347, and WO2013059475). Further suitable cap analogs in this context are described in WO2017066793, WO2017066781, WO2017066791, WO2017066789, WO2017053297, WO2017066782, WO2018075827, and WO2017066797, the disclosures of which refer to cap analogs are incorporated herein by reference.

[0401] In some embodiments, the modified cap 1 structure is generated using a trinucleotide cap analog as disclosed in WO2017053297, WO2017066793, WO2017066781, WO2017066791, WO2017066789, WO2017066782, WO2018075827, and WO2017066797. In particular, any cap structure derivable from the structures disclosed in claims 1-5 of WO2017053297 may be suitably used to co-transcriptionally generate the modified cap 1 structure. Furthermore, any cap structure derivable from the structures defined in claim 1 or claim 21 of WO2018075827 may be suitably used to co-transcriptionally generate the modified cap 1 structure.

[0402] In a preferred embodiment, the (coding) RNA, in particular the mRNA, comprises a Cap1 structure. In a preferred embodiment, a 5' cap structure may be suitably added co-transcriptionally in an RNA in vitro transcription reaction as defined herein using a trinucleotide cap analog as defined herein.

[0403] In a preferred embodiment, the cap 1 structure of the coding RNA of the present invention is formed using co-transcriptional capping using the trinucleotide cap analog m7G(5')ppp(5')(2'OMeA)pG or m7G(5')ppp(5')(2'OMeG)pG. A preferred cap analog in that context is m7G(5')ppp(5')(2'OMeA)pG.

[0404] In another preferred embodiment, the cap 1 structure of the RNA of the present invention is formed using co-transcriptional capping using the trinucleotide cap analog 3'OMe-m7G(5')ppp(5')(2'OMeA)pG.

[0405] In other embodiments, the cap 0 structure of an RNA of the invention is formed using co-transcriptional capping using the cap analog 3'OMe-m7G(5')ppp(5')G.

[0406] In other embodiments, the 5' cap structure is formed via enzymatic capping using a capping enzyme (e.g., vaccinia virus capping enzyme and / or a cap-dependent 2'-O methyltransferase) to generate a cap0 or cap1 or cap2 structure. The 5' cap structure (cap0 or cap1) can be added using an immobilized capping enzyme and / or a cap-dependent 2'-O methyltransferase using the methods and means disclosed in WO2016193226.

[0407] In preferred embodiments, about 70%, 75%, 80%, 85%, 90%, 95% of the RNA (species) comprise a cap 1 structure as determined using a capping assay. In preferred embodiments, less than about 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% of the RNA (species) do not comprise a cap 1 structure as determined using a capping assay. In other preferred embodiments, about 70%, 75%, 80%, 85%, 90%, 95% of the RNA (species) comprise a cap 0 structure as determined using a capping assay. In preferred embodiments, less than about 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% of the RNA (species) do not comprise a cap 0 structure as determined using a capping assay.

[0408] The term "RNA species" is not limited to mean "one single molecule" but is understood to include an ensemble of essentially identical RNA molecules. Thus, the term can refer to a plurality of substantially identical (coding) RNA molecules.

[0409] To determine the presence / absence of a cap 0 or cap 1 structure, a capping assay may be used as described in published PCT application WO 2015101416, in particular as described in claims 27-46 of published PCT application WO 2015101416. Other capping assays that may be used to determine the presence / absence of a cap 0 or cap 1 structure of an RNA are described in WO 2020127959, or published PCT applications WO 2014152673 and WO 2014152659.

[0410] In a preferred embodiment, the RNA comprises a m7G(5')ppp(5')(2'OMeA)CAP structure. In such an embodiment, the coding RNA comprises a 5'-terminal m7G cap and an additional methylation of the ribose of the nucleotide adjacent to the m7GpppN, in that case a 2'O-methylated adenosine. Preferably, about 70%, 75%, 80%, 85%, 90%, 95% of the RNA (species) comprises such a Cap 1 structure as determined using a capping assay.

[0411] In other preferred embodiments, the RNA comprises a m7G(5')ppp(5')(2'OMeG) cap structure. In such embodiments, the coding RNA comprises a 5'-terminal m7G cap and an additional methylation of the ribose of the adjacent nucleotide, in that case a 2'O-methylated guanosine. Preferably, about 70%, 75%, 80%, 85%, 90%, 95% of the coding RNA (species) comprises such a Cap 1 structure as determined using a capping assay.

[0412] Thus, the first nucleotide of the RNA or mRNA sequence, ie the nucleotide downstream of the m7G(5')ppp structure, may be a 2'O-methylated guanosine or a 2'O-methylated adenosine.

[0413] In one embodiment, the 5' end of the mRNA is "GGGAGA", preferably for mRNAs where an mCap analog is used. In another embodiment, the 5' end of the mRNA is "AGGAGA", preferably for mRNAs where a CleanCap® AG cap analog is used. In a further embodiment, the 5' end of the mRNA is "GGGAGA", preferably for mRNAs where a CleanCap® GG cap analog is used.

[0414] In the context of the present invention, the 5' cap structure can also be formed by chemical RNA synthesis or RNA in vitro transcription using a cap analog (co-transcriptional capping), or the cap structure can be formed in vitro using a capping enzyme. Kits containing capping enzymes are commercially available (e.g., ScriptCap™ Capping Enzyme and ScriptCap™ 2'-O-Methyltransferase, both from CellScript). Thus, the RNA transcript is preferably treated according to the manufacturer's instructions.

[0415] Indeed, a cap analog refers to a non-polymerizable dinucleotide that has a cap functionality in that it facilitates translation or localization and / or prevents degradation of an RNA molecule when incorporated at the 5' end of the RNA molecule. Non-polymerizable means that the cap analog is incorporated only at the 5' end because it does not have a 5' triphosphate and therefore cannot be extended in the 3' direction by a template-dependent RNA polymerase.

[0416] Further examples of 5' cap structures include glyceryl, inverted deoxy abasic residues, residue), 4',5' methylene nucleotide, 1-(beta-D-erythrofuranosyl) nucleotide, 4'-thio nucleotide, carbocyclic nucleotide, 1,5-anhydrohexitol nucleotide, L-nucleotide, alpha-nucleotide, modified base nucleotide, threo-pentofuranosyl nucleotide, acyclic 3',4'-seco nucleotide, acyclic 3,4-dihydroxybutyl nucleotide, acyclic 3,5 dihydroxypentyl nucleotide, 3'-3' inverted nucleotide moiety, 3'-3' inverted abasic moiety, 3'-2'-inverted nucleotide moiety, 3'-2'-inverted abasic moiety, 1,4-butanediol phosphate, 3'-phosphoramidate, hexyl phosphate, aminohexyl phosphate, 3'-phosphate, 3' phosphorothioate, phosphorodithioate, or bridged or non-bridged methylphosphonate moiety. These modified 5' cap structures are considered in this context as at least one modification and, in the context of the present invention, may be used to modify the mRNA sequences of the compositions of the invention.

[0417] Particularly preferred modified 5' cap structures are Cap1 (methylation of the ribose of the nucleotide adjacent to m7G), Cap2 (additional methylation of the ribose of the second nucleotide downstream of m7G), Cap3 (additional methylation of the ribose of the third nucleotide downstream of m7G), Cap4 (methylation of the ribose of the fourth nucleotide downstream of m7G), ARCA (anti-reverse cap analog), modified ARCA (e.g., phosphothioate modified ARCA), CleanCap or m7G(5')ppp(5')(2'OMeA)pG or m7G(5')ppp(5')(2'OMeG)pG (TriLink) and / or the cap structures disclosed in WO2017053297 (hereby incorporated by reference), inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine. In particular, any cap structure derivable from the structures disclosed in claims 1-5 of WO2017053297 may be suitably used to co-transcriptionally generate the modified cap 1 structure. Furthermore, any cap structure derivable from the structure defined in claim 1 or claim 21 of WO2018075827 may be suitably used to co-transcriptionally generate a modified cap 1 structure.

[0418] Additionally, cap analogs have been previously described (U.S. Pat. No. 7,074,596, WO 2008016473, WO 2008157688, WO 2009149253, WO 2011015347, and WO 2013059475). The synthesis of N7-(4-chlorophenoxyethyl) substituted dinucleotide cap analogs has recently been described (Kore et al. (2013) Bioorg. Med. Chem. 21(15):4570-4). Further suitable cap analogs in this context are described in WO2017066793, WO2017066781, WO2017066791, WO2017066789, WO2017066782, WO2018075827 and WO2017066797, the specific disclosures which refer to cap analogs are incorporated herein by reference.

[0419] Poly(A) sequence / polyA tail A polyA tail, also called a "3' poly(A) tail", "polyA sequence" or "poly(A) sequence", is a long adenosine nucleotide sequence of up to about 400 adenosine nucleotides, such as 10 to 200, 10 to 100, 40 to 80, 50 to 70, about 25 to about 400, preferably about 50 to about 400, more preferably about 50 to about 300, even more preferably about 50 to about 250, most preferably about 60 to about 250 adenosine nucleotides, or about 40 to about 150 adenosine nucleotides, typically added to the 3' end of an RNA. In a particularly preferred embodiment, the poly(A) sequence comprises about 64 adenosine nucleotides. In another particularly preferred embodiment, the poly(A) sequence comprises about 100 adenosine nucleotides. Furthermore, poly(A) sequences, or poly(A) tails, can be generated in vitro by enzymatic polyadenylation of RNA, for example using poly(A) polymerase derived from E. coli or yeast. Suitably, the poly(A) sequence of the coding RNA can be long enough to bind at least two, three, four, five or more monomers of polyA binding protein.

[0420] Polyadenylation is typically understood to be the addition of a poly(A) sequence to a nucleic acid molecule, such as an RNA molecule, for example a pre-mRNA. Polyadenylation can be induced by a so-called polyadenylation signal. This signal is preferably located within a stretch of nucleotides at the 3' end of the nucleic acid molecule, such as an RNA molecule, to be polyadenylated. Polyadenylation signals typically comprise a hexamer, preferably the hexamer sequence AAUAAA, consisting of adenine and uracil / thymine nucleotides. Other sequences, preferably hexamer sequences, are also conceivable. Polyadenylation typically occurs during the processing of pre-mRNA (also called pre-mRNA). Typically, RNA maturation (from pre-mRNA to mature mRNA) includes a step of polyadenylation.

[0421] Indeed, according to a further preferred embodiment, the composition comprises an mRNA compound comprising an mRNA sequence that typically contains a poly-A tail at the 3' end of about 10-200 adenosine nucleotides, preferably about 10-100 adenosine nucleotides, more preferably about 40-80 adenosine nucleotides, or even more preferably about 50-70 adenosine nucleotides. Preferably, the poly(A) sequence is derived from a DNA template by RNA in vitro transcription. Alternatively, the poly(A) sequence can also be obtained in vitro by common chemical synthesis methods, without necessarily being transcribed from a DNA precursor. Furthermore, the poly(A) sequence, or the poly(A) tail, can be generated by enzymatic polyadenylation of the RNA according to the invention, using commercially available polyadenylation kits and corresponding protocols known in the art.

[0422] Alternatively, the mRNAs described herein optionally comprise a polyadenylation signal, defined herein as a signal that conveys polyadenylation to the (transcribed) RNA by specific protein factors (e.g., cleavage and polyadenylation specificity factor (CPSF), cleavage stimulation factor (CstF), cleavage factors I and II (CFI and CFII), poly(A) polymerase (PAP)). In this context, consensus polyadenylation signals comprising the NN(U / T)ANA consensus sequence are preferred. In particularly preferred embodiments, the polyadenylation signal comprises one of the following sequences: AA(U / T)AAA or A(U / T)(U / T)AAA, where uridine is typically present in RNA and thymidine is typically present in DNA.

[0423] Poly(C) sequence A poly(C) sequence is typically a long cytosine nucleotide sequence of about 10 to about 200 cytosine nucleotides, preferably about 10 to about 100 cytosine nucleotides, more preferably about 10 to about 70 cytosine nucleotides, or even more preferably about 20 to about 50 or even about 20 to about 30 cytosine nucleotides. The poly(C) sequence may be preferably located 3' of a coding region contained in a nucleic acid.

[0424] Indeed, according to further preferred embodiments, the compositions of the present invention comprise mRNA compounds that typically comprise a 3'-terminal poly(C) tail of about 10-200 cytosine nucleotides, preferably about 10-100 cytosine nucleotides, more preferably about 20-70 cytosine nucleotides or even more preferably about 20-60 or even 10-40 cytosine nucleotides.

[0425] In one preferred embodiment, the mRNA compound preferably has in the 5'→3' direction: a) a 5' cap structure, preferably m7GpppN, more preferably cap1 or m7G(5')ppp(5')(2'OMeA)pG; b) optionally, a 5'-UTR element; c) at least one coding region encoding at least one antigenic peptide or protein; d) optionally a poly(A) sequence, preferably containing 64 adenosines or 100 adenosines; e) optionally a poly(C) sequence, preferably containing 30 cytosines; f) optionally a histone stem loop selected from SEQ ID NO: 3 or 4; and / or g) optionally, a 3'-terminal sequence element selected from SEQ ID NOs: 41 to 70 Includes.

[0426] UTR In a preferred embodiment, the composition comprises an mRNA compound that comprises at least one 5'- or 3'-UTR element. In this context, the UTR element comprises or consists of a nucleic acid sequence derived from the 5'- or 3'-UTR of any naturally occurring gene, or derived from a fragment, homologue or variant of the 5'- or 3'-UTR of a gene. Preferably, the 5'- or 3'-UTR element used according to the present invention is heterologous to at least one coding region of the mRNA sequence of the present invention. Although the 5'- or 3'-UTR element derived from a naturally occurring gene is preferred, synthetically designed UTR elements can also be used in the context of the present invention.

[0427] The term "3'-UTR element" refers to a nucleic acid sequence that typically comprises or consists of a nucleic acid sequence derived from a 3'-UTR or a variant of a 3'-UTR. A 3'-UTR element in the sense of the present invention may represent the 3'-UTR of an RNA, preferably an mRNA. Thus, in the sense of the present invention, a 3'-UTR element may preferably be the 3'-UTR of an RNA, preferably an mRNA, or may be a transcription template of the 3'-UTR of an RNA. Thus, a 3'-UTR element is preferably a nucleic acid sequence that corresponds to the 3'-UTR of an RNA, preferably an mRNA, such as an mRNA obtained by transcription of a genetically engineered vector construct. Preferably, a 3'-UTR element performs the function of a 3'-UTR or codes for a sequence that performs the function of a 3'-UTR.

[0428] Preferably, at least one 3'-UTR element comprises or consists of a nucleic acid sequence derived from the 3'-UTR of a chordate gene, preferably a vertebrate gene, more preferably a mammalian gene, and most preferably a human gene, or a variant of the 3'-UTR of a chordate gene, preferably a vertebrate gene, more preferably a mammalian gene, and most preferably a human gene.

[0429] Preferably, the composition comprises an mRNA compound comprising a 3'-UTR element, such as a 3'-UTR element as defined and described below, which may be derivable from a gene for an mRNA with enhanced half-life (providing a stable mRNA). Preferably, the 3'-UTR element comprises or consists of a nucleic acid sequence derived from the 3'-UTR of a gene, or a homologue, fragment or variant of said gene, preferably encoding a stable mRNA.

[0430] In one preferred embodiment, the UTR combinations disclosed in Table 1, claim 1 and claim 4, claims 6 to 8 and claim 9 of WO 2019077001 are preferred UTR combinations for the mRNA compounds of the present invention. Furthermore, preferably, the UTR combinations disclosed in page 24, the entire second paragraph after Table 1 and in page 24, the last paragraph to page 29, second paragraph of WO 2019077001 are preferred UTR combinations for the mRNA compounds of the present invention. WO 2019077001 is incorporated herein in its entirety by reference.

[0431] In further preferred embodiments, the 3'-UTR element is selected from the group consisting of PSMB3 (SEQ ID NO: 19, SEQ ID NO: 20), ALB / albumin (SEQ ID NO: 13 to SEQ ID NO: 18), α-globin (referred to as "muag", i.e., mutant α-globin 3'-UTR; SEQ ID NO: 11, SEQ ID NO: 12), CASP1 (preferably SEQ ID NO: 81 (DNA) or SEQ ID NO: 82 (RNA)), COX6B1 (preferably SEQ ID NO: 83 (DNA) or SEQ ID NO: 84 (RNA)), GNAS (preferably SEQ ID NO: 85 (DNA) or SEQ ID NO: 86 (RNA)), NDUFA1 (preferably SEQ ID NO: 87 (DNA) or SEQ ID NO: 88 (RNA)) and RPS9 (preferably SEQ ID NO: 79 (DNA) or SEQ ID NO: 80 (RNA)), or the 3'-UTR of a homolog, fragment or variant of any one of these genes (e.g. human albumin / alb 3'-UTR disclosed in SEQ ID NO: 1369 of WO2013143700, which is incorporated herein by reference). In a further preferred embodiment, the 3'-UTR element comprises a nucleic acid sequence derived from a fragment of the human albumin gene according to SEQ ID NO: 1376 of WO2013143700 (albumin / alb 3'-UTR). In a further preferred embodiment, the 3'-UTR element comprises or consists of a nucleic acid sequence derived from the 3'-UTR of an albumin gene, preferably a vertebrate albumin gene, more preferably a mammalian albumin gene, most preferably the 3'-UTR of the human albumin gene, such as the 3'-UTR of the human albumin gene according to GenBank Accession No. NM_000477.5, or a fragment or variant thereof.In another preferred embodiment, the 3'-UTR element comprises or consists of the central, alpha-complex binding portion of the 3'-UTR of an alpha-globin gene, such as preferably a human alpha-globin gene (according to SEQ ID NO:5 or SEQ ID NO:6 (both HBA1) or SEQ ID NO:7 or SEQ ID NO:8 (both HBA2)), or a homologue, fragment or variant of the alpha-globin gene, or the alpha-complex binding portion of the 3'-UTR of an alpha-globin gene (also referred to herein as "muag", SEQ ID NO:11 or SEQ ID NO:12 herein; corresponding to SEQ ID NO:1393 of WO2013143700).

[0432] In another preferred embodiment, the 3'-UTR element comprises or consists of a nucleic acid sequence derived from the 3'-UTR of an α- or β-globin gene, preferably a vertebrate α- or β-globin gene, preferably a mammalian α- or β-globin gene, preferably a human α- or β-globin gene, according to SEQ ID NO: 5, 7, 9, 11, or the corresponding RNA sequences SEQ ID NO: 6, 8, 10, 12. In another preferred embodiment, the 3'-UTR element comprises or consists of a nucleic acid sequence derived from the 3'-UTR of ALB / albumin (SEQ ID NO: 13 to SEQ ID NO: 18) or PSMB3 (SEQ ID NO: 19 / 20).

[0433] In this context, it is also preferred that the 3'-UTR element of the mRNA sequence according to the invention comprises or consists of the corresponding RNA sequence of the nucleic acid sequence according to SEQ ID NO: 11, or a homologue, fragment or variant thereof, as shown in SEQ ID NO: 12.

[0434] UTR combination SLC7A3 (5'-UTR of mouse solute carrier family 7 (cationic amino acid transporter, y+ system), member 3) / PSMB3: In another preferred embodiment, the mRNA compound comprises a 5'-UTR element comprising or consisting of a nucleic acid sequence derived from the cationic amino acid transporter 3 (solute carrier family 7 member 3, SLC7A3; preferably SEQ ID NO: 77 (DNA) or SEQ ID NO: 78 (RNA)) gene, said 5'-UTR element comprising or consisting of a DNA sequence according to SEQ ID NO: 15 as disclosed in WO2019077001 or an RNA sequence according to SEQ ID NO: 16 as disclosed in WO2019077001, respectively. In another preferred embodiment, the mRNA compound comprises a 3'-UTR element comprising or consisting of a nucleic acid sequence derived from the proteasome subunit type β3 (PSMB3) gene, said 3'-UTR element comprising or consisting of a DNA sequence according to SEQ ID NO: 23 as disclosed in WO2019077001 or an RNA sequence according to SEQ ID NO: 24 as disclosed in WO2019077001, respectively. In a further preferred embodiment, the mRNA compound comprises a UTR combination as disclosed in WO2019077001, i.e. both a 5'-UTR element comprising or consisting of a nucleic acid sequence derived from the SLC7A3 gene and a 3'-UTR element comprising or consisting of a nucleic acid sequence derived from the PSMB3 gene.

[0435] UTR combination RPL31 (5'-UTR of mouse ribosomal protein L31; preferably SEQ ID NO: 75 (DNA) or SEQ ID NO: 76 (RNA)) / RPS9 (3'-UTR of human ribosomal protein S9 (RPS9; preferably SEQ ID NO: 79 (DNA) or SEQ ID NO: 80 (RNA)): In another preferred embodiment, the mRNA compound comprises a 5'-UTR element comprising or consisting of a nucleic acid sequence derived from the 60S ribosomal protein L31 (RPL31) gene, said 5'-UTR element comprising or consisting of a DNA sequence according to SEQ ID NO: 13 as disclosed in WO2019077001 or an RNA sequence according to SEQ ID NO: 14 or preferably SEQ ID NO: 75 / 76 as disclosed in WO2019077001, respectively. Another preferred embodiment In an embodiment, the mRNA compound comprises a 3'-UTR element comprising or consisting of a nucleic acid sequence derived from the 40S ribosomal protein S9 (RPS9) gene, said 3'-UTR element comprising or consisting of a DNA sequence according to SEQ ID NO: 33 as disclosed in WO2019077001 or an RNA sequence according to SEQ ID NO: 34 as disclosed in WO2019077001, respectively. In a further preferred embodiment, the mRNA compound comprises a UTR combination as disclosed in WO2019077001, i.e. both a 5'-UTR element comprising or consisting of a nucleic acid sequence derived from the RPL31 gene and a 3'-UTR element comprising or consisting of a nucleic acid sequence derived from the RPS9 gene (preferably SEQ ID NO: 79 / 80).

[0436] In a highly preferred embodiment, the 5'-UTR element of the mRNA sequence according to the invention comprises or consists of the corresponding RNA sequence of the nucleic acid sequence according to SEQ ID NO: 21 or SEQ ID NO: 22, i.e. HSD17B4. Similarly, in a highly preferred embodiment, the 3'-UTR element of the mRNA sequence according to the invention comprises or consists of the corresponding RNA sequence of the nucleic acid sequence according to SEQ ID NO: 19 or SEQ ID NO: 20, i.e. PSMB3. Similarly, in a highly preferred embodiment, the 5'-UTR and 3'-UTR elements of the mRNA sequence according to the invention comprise or consist of the combination of HSD17B4 and PSMB3-UTR as described above.

[0437] The term "nucleic acid sequence derived from the 3'-UTR of a [...] gene" refers to a nucleic acid sequence based on the 3'-UTR sequence of a [...] gene or a part thereof, such as preferably the albumin gene, the α-globin gene, the β-globin gene, the tyrosine hydroxylase gene, the lipoxygenase gene, or the collagen alpha gene, such as the collagen alpha 1(I) gene, preferably the 3'-UTR of the albumin gene, or a part thereof. The term includes the entire 3'-UTR sequence of a gene, such as the albumin gene, the α-globin gene, the β-globin gene, the tyrosine hydroxylase gene, the lipoxygenase gene, or the collagen alpha gene, such as the collagen alpha 1(I) gene, preferably the albumin gene, i.e. a sequence corresponding to the full-length 3'-UTR of the gene, and a sequence corresponding to a fragment of the 3'-UTR sequence of the gene.

[0438] The term "nucleic acid sequence derived from a variant of the 3'-UTR of a gene [...]" refers to a nucleic acid sequence based on a variant of the 3'-UTR sequence of a gene, such as a variant of the 3'-UTR of the albumin gene, the alpha-globin gene, the beta-globin gene, the tyrosine hydroxylase gene, the lipoxygenase gene or the collagen alpha gene, such as the collagen alpha 1 (I) gene, as described above, or a part thereof. The term includes the entire sequence of the variant of the 3'-UTR of a gene, i.e. a sequence corresponding to the full-length variant 3'-UTR sequence of the gene, as well as a sequence corresponding to a fragment of the variant 3'-UTR sequence of the gene. A fragment in this context consists of a continuous stretch of nucleotides corresponding to a continuous stretch of nucleotides in the full-length variant 3'-UTR, preferably representing at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80% and most preferably at least 90% of the full-length variant 3'-UTR. Fragments of such variants in the sense of the present invention are preferably functional fragments of the variants described herein.

[0439] According to a preferred embodiment, the mRNA compound comprising the mRNA sequence according to the invention comprises a 5' cap structure and / or at least one 3' untranslated region element (3'-UTR element), preferably as defined herein. More preferably, the RNA further comprises a 5'-UTR element as defined herein.

[0440] In one preferred embodiment, the mRNA compound preferably has in the 5'→3' direction: a) a 5' cap structure, preferably m7GpppN, more preferably cap1 or m7G(5')ppp(5')(2'OMeA)pG; b) optionally, a 5'-UTR element; c) at least one coding region encoding at least one antigenic peptide or protein; d) optionally a 3'-UTR element comprising or consisting of a nucleic acid sequence derived from an alpha globin gene, comprising the corresponding RNA sequence of the nucleic acid sequence according to SEQ ID NO: 11, preferably as shown in SEQ ID NO: 12, a homologue, fragment or variant thereof; e) optionally a poly(A) sequence, preferably containing 64 adenosines or 100 adenosines; f) optionally a poly(C) sequence, preferably containing 30 cytosines; g) optionally a histone stem loop selected from SEQ ID NO: 3 or 4; and / or h) optionally, a 3'-terminal sequence element selected from SEQ ID NOs: 41 to 70 Includes.

[0441] In a further preferred embodiment, the mRNA compound preferably has in the 5'→3' direction: a) a 5' cap structure, preferably m7GpppN, more preferably cap1 or m7G(5')ppp(5')(2'OMeA)pG; b) optionally, a 5'-UTR element; c) preferably SARS coronavirus 2 (SARS-CoV-2), nCov-2019 coronavirus, SARS coronavirus (SARS-CoV), Bunyavirales viruses, cytomegalovirus (CMV), Dengue viruses (DENV-1, DENV-2, DENV-3 and DENV-4), Ebola virus (EBOV), Flaviviruses, Hepatitis B virus (HBV), Herpes simplex virus (HSV), Human immunodeficiency virus (HIV), Human metapneumovirus (HMPV), Human papillomavirus (HPV), Human parainfluenza virus (HPIV), Influenza virus, Extraintestinal pathogenic Escherichia coli (E. coli) (ExPEC), Lassa-Mam arenavirus (LASV), MERS coronavirus, Mycobacterium tuberculosis (Mycobacterium at least one coding region encoding at least one antigenic peptide or protein, or a fragment or variant thereof, derived from a protein of Chlamydia tuberculosis, Nipah virus, Norovirus, Rabies virus (RABV), Respiratory syncytial virus (RSV), Rhinovirus, Rotavirus, Vaccinia virus, Yellow fever virus (YFV), Zika virus (ZIKV), Chlamydia trachomatis (i.e., the bacterium Chlamydia that causes Chlamydia), or a malaria parasite (e.g., Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, or Plasmodium ovale); d) optionally a 3'-UTR element comprising or consisting of a nucleic acid sequence derived from an alpha globin gene, comprising the corresponding RNA sequence of the nucleic acid sequence according to SEQ ID NO: 11, preferably as shown in SEQ ID NO: 12, a homologue, fragment or variant thereof; e) optionally a poly(A) sequence, preferably containing 64 adenosines or 100 adenosines; f) optionally a poly(C) sequence, preferably containing 30 cytosines; g) optionally a histone stem loop selected from SEQ ID NO: 3 or 4; and / or h) optionally, a 3'-terminal sequence element selected from SEQ ID NOs: 41 to 70 Includes.

[0442] In a further preferred embodiment, the composition comprises an mRNA compound comprising at least one 5' untranslated region element (5'-UTR element). Preferably, at least one 5'-UTR element comprises or consists of a nucleic acid sequence derived from the 5'-UTR of a TOP gene, or derived from a fragment, homologue or variant of the 5'-UTR of a TOP gene. Preferably, the 5'-UTR element does not comprise a TOP...

Claims

1. a) at least one nucleic acid encoding at least one antigen or a fragment or variant thereof; b) A carrier composition comprising the phospholipid phosphatidylserine, wherein the amount of phosphatidylserine is 5 mol% or less of the total molar amount of all lipid excipients in the carrier composition. A vaccine composition containing the following:

2. The vaccine composition according to claim 1, wherein the at least one nucleic acid is not an immune tolerance-inducing nucleic acid; and / or the at least one nucleic acid does not encode an immune tolerance-inducing polypeptide; and / or the vaccine composition does not contain an antigen or a fragment or variant thereof; and / or the vaccine composition contains the at least one nucleic acid as the sole payload; and / or the vaccine composition is not an immune tolerance-inducing composition.

3. The vaccine composition according to claim 1, wherein the carrier composition at least partially encapsulates the at least one nucleic acid.

4. The vaccine composition according to claim 1, wherein the carrier composition encapsulates the at least one nucleic acid.

5. The vaccine composition according to claim 1, wherein the carrier composition comprises an inner surface and an outer surface facing the outside, and phosphatidylserine is located on the outer surface of the carrier composition.

6. The vaccine composition according to claim 5, wherein the hydrophilic head group of phosphatidylserine is located on the outer surface of the carrier composition.

7. The vaccine composition according to claim 1, wherein the hydrophilic head group of phosphatidylserine contained in the carrier composition is accessible from the outside of the carrier composition.

8. The vaccine composition according to claim 1, wherein phosphatidylserine is selected from the group consisting of DPhyPS, WT-PS, 16:0-PS, 14:0-PS, 10:0-PS, 6:0-PS, 18:1-PS DOPS, 18:1-Lyso PS, and 18:0-Lyso PS.

9. The vaccine composition according to claim 1, wherein the carrier composition is a lipid nanoparticle composition.

10. The lipid nanoparticle composition is (i) Cationic or ionizable lipids; and / or (ii) Steroids; and / or (iii) In addition to phosphatidylserine, further phospholipids, preferably DPhyPE; and / or (iv) Polymer-conjugated lipids The vaccine composition according to claim 9, further comprising:

11. The lipid nanoparticle composition is (i) Cationic or ionizable lipids; (ii) Steroids; (iii) In addition to phosphatidylserine, further phospholipids, preferably DPhyPE; and (iv) Polymer-conjugated lipids The vaccine composition according to claim 9, further comprising:

12. The vaccine composition according to claim 10, wherein the cationic or ionizable lipid has a net positive charge at physiological pH, preferably the cationic or ionizable lipid contains a tertiary nitrogen group or a quaternary nitrogen group, and more preferably the cationic or ionizable lipid is selected from the group consisting of HEXA1, HEXA2, and THIOETHER, each having the structures shown in Figures 1A, 1B, and 1C, respectively.

13. The vaccine composition according to claim 10, wherein the steroid is selected from the group consisting of cholesterol, cholesteryl hemisuccinate (CHEMS), and derivatives thereof, and preferably the steroid is cholesterol.

14. The further phospholipid is 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE; 1,2-di-(3,7,11,15-tetramethylhexadecanoyl)-sn-glycero-3-phosphoethanolamine), 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhyPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC; dioleoylphosphatidylcholine), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC; dipalmito Ilphosphatidylcholine), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), phosphatidylethanolamine, distearoylphosphatidylcholine, dioleoyl-phosphatidylethanolamine (DOPEA), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), 1,2-dipalmitoyl-sn-glycero -3-Phosphoethanolamine (DPPE), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine (DLope), distearoylphosphatidylethanolamine (DSPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (P OPE), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 16-O-monomethylphosphoethanolamine, 16-O-dimethylphosphatidylethanolamine, 1,2-dielucoyl-sn-glycero-3-phosphoethanolamine (DEPE), 18-1-transphosphatidylethanolamine, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), 1,2-disquareoyl-sn-glycero-3-phosphoethanolamine (DSQPE), 1,2-Dierydoyl-sn-glycero-3-phosphoethanolamine (trans-DOPE), 1-Stearoyl-2-linoleoyl-sn-glycero-3-phosphoethanolamine (SLPE), 1-Tridecanoyl-sn-glycero-3-phospho-L-serine (sodium salt), 1-Oleoyl-2-hydroxy-sn-glycero-3-phospho-L-serine (sodium salt), 1-Palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine Phosphorus (sodium salt) (POPS), 1-1-stearoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (sodium salt), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DOPS), 1,2-distearoyl-sn-glycero-3-phospho-L-serine (sodium salt), 1,2-diphytanoyl-sn-glycero-3-phospho-L-serine (sodium salt), 1-O-hexadecanyl-2-O -(9Z-octadecenyl)-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphatidylcholine or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-di-O-phytanyl-sn-glycero-3-phosphoethanolamine, 1-palmitoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (PChemsPC), 1,2-dicholesterol Selected from the group consisting of lylhemisuxinoyl-sn-glycero-3-phosphocholine (DChemsPC), 2-((2,3-bis(oleoyloxy)propyl)dimethylammonio)ethyl hydrogen phosphate (DOCP), 2-((2,3-bis(oleoyloxy)propyl)dimethylammonio)ethyl ethyl phosphate (DOCPe), and 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (edelhosine), Preferably, the further phospholipid is DPhyPE; the phospholipid, preferably DPhyPE, is a phospholipid having at least two alkyl chains, where each alkyl chain independently has a length preferably of C6, C7, C8, C9, or C10, more preferably of C6, C7, or C8, and most preferably of C7; more preferably, DHPC (1,2-difeptanoyl-sn-glycero-3-phosphocholine), 05:0PC (1,2-dipentanoyl-sn-glycero). It exists in combination with a phospholipid selected from the group consisting of 0-3-phosphocholine, 04:0PC (1,2-dibutyryl-sn-glycero-3-phosphocholine), 06:0PC (1,2-dihexanoyl-sn-glycero-3-phosphocholine), 08:0PC (1,2-dioctanoyl-sn-glycero-3-phosphocholine), and 09:0PC (1,2-dinonanyl-sn-glycero-3-phosphocholine), and is most preferred as a phospholipid when DHPC has at least two alkyl chains. The vaccine composition according to claim 10.

15. The vaccine composition according to claim 10, wherein the polymer conjugate lipid is a pegylated lipid or a PMOZ-lipid.

16. (a-i) 30 to 70 mol% of cationic lipids; 20 to 50 mol% of steroids; 5 to 25 mol% of phospholipids; and 0.5 to 5 mol% of polymer conjugate lipids; (a-ii) 40-60 mol% of cationic lipids; 20-40 mol% of steroids; 10-20 mol% of phospholipids; and 1-2 mol% of polymer conjugate lipids; (a-iii) a cationic lipid according to claim 12 in an amount of 30 to 70 mol%; a steroid according to claim 13 in an amount of 20 to 50 mol%; phospholipid phosphatidylserine and phospholipid according to claim 14 in an amount of 5 to 25 mol%; and polymer conjugate lipid according to claim 15 in an amount of 0.5 to 5 mol%; and (a-iv) A cationic lipid according to claim 12 in an amount of 40-60 mol%; a steroid according to claim 13 in an amount of 20-40 mol%; a phospholipid phosphatidylserine and a phospholipid according to claim 14 in an amount of 10-20 mol%; and a polymer conjugate lipid according to claim 15 in an amount of 1-2 mol%; It includes an excipient in a ratio selected from the group consisting of; Preferably, (b-i) 59 mol% of cationic lipids; 29.3 mol% of steroids; 10 mol% of phospholipids; and 1.7 mol% of polymer conjugate lipids; (b-ii) 58 mol% of cationic lipids; 29.3 mol% of steroids; 11 mol% of phospholipids; and 1.7 mol% of polymer conjugate lipids; (b-iii) 49 mol% of cationic lipids; 29.3 mol% of steroids; 20 mol% of phospholipids; and 1.7 mol% of polymer conjugate lipids; (b-iv) 59 mol% of the cationic lipid according to claim 12; 29.3 mol% of the steroid according to claim 13; 10 mol% of the phospholipid phosphatidylserine and the phospholipid according to claim 14; and 1.7 mol% of the polymer conjugate lipid according to claim 15; (b-v) 58 mol% of the cationic lipid according to claim 12; 29.3 mol% of the steroid according to claim 13; 11 mol% of the phospholipid phosphatidylserine and the phospholipid according to claim 14; and 1.7 mol% of the polymer conjugate lipid according to claim 15; and (b-vi) 49 mol% of the cationic lipid according to claim 12; 29.3 mol% of the steroid according to claim 13; 20 mol% of the phospholipid phosphatidylserine and the phospholipid according to claim 14; and 1.7 mol% of the polymer conjugate lipid according to claim 15; It includes an excipient in a ratio selected from the group consisting of; more, (c-i) 59 mol% of the cationic lipid according to claim 12; 29.3 mol% of the steroid according to claim 13; 5 mol% of the phospholipid phosphatidylserine and 5 mol% of DPhyPE; and 1.7 mol% of the polymer conjugate lipid according to claim 15; (c-ii) 59 mol% of the cationic lipid according to claim 12; 29.3 mol% of the steroid according to claim 13; 2 mol% of the phospholipid phosphatidylserine and 8 mol% of DPhyPE; and 1.7 mol% of the polymer conjugate lipid according to claim 15; (c-iii) 58 mol% of the cationic lipid according to claim 12; 29.3 mol% of the steroid according to claim 13; 5 mol% of the phospholipid phosphatidylserine, 5 mol% of DPhyPE, and 1 mol% of DHPC; and 1.7 mol% of the polymer conjugate lipid according to claim 15; (c-iv) 49 mol% of the cationic lipid according to claim 12; 29.3 mol% of the steroid according to claim 13; 5 mol% of the phospholipid phosphatidylserine, 5 mol% of DPhyPE, and 10 mol% of DHPC; and 1.7 mol% of the polymer conjugate lipid according to claim 15; The vaccine composition according to claim 11, comprising an excipient in a ratio selected from the group consisting of the following.

17. The vaccine composition according to claim 1, wherein the at least one nucleic acid is DNA or RNA.

18. The vaccine composition according to claim 17, wherein the at least one nucleic acid is mRNA, preferably comprising a coding sequence encoding at least one antigen or a fragment or variant thereof, and optionally a coding sequence encoding at least one self-renewing enzyme.

19. The lipid nanoparticles (i) an amount that achieves an N / P ratio in the range of 10 to 20, preferably about 2 to about 15, more preferably about 3 to about 10, even more preferably about 4 to about 9, most preferably about 6; or (ii) an amount that achieves an N / P ratio in the range of about 5 to about 20, more preferably about 10 to about 18, even more preferably about 12 to about 16, and most preferably about 14; and / or (iii) an amount that achieves a lipid:mRNA weight ratio in the range of approximately 20 to approximately 60, preferably approximately 3 to approximately 15, approximately 5 to approximately 13, approximately 4 to approximately 8, or approximately 7 to approximately 11. containing mRNA, and / or The lipid nanoparticles have an average hydrodynamic diameter determined by dynamic light scattering in the range of approximately 50 nm to approximately 300 nm, or approximately 60 nm to approximately 250 nm, or approximately 60 nm to approximately 200 nm, or approximately 70 nm to approximately 200 nm, or approximately 75 nm to approximately 160 nm, or approximately 90 nm to approximately 140 nm, or approximately 100 nm to approximately 140 nm; and / or The vaccine composition according to claim 18, wherein the lipid nanoparticles exhibit a zeta potential in the range of -50 mV to +50 mV, preferably in the range of -25 mV to +25 mV, more preferably in the range of -10 mV to +10 mV, and most preferably in the range of -5 mV to +5 mV.

20. The vaccine composition according to claim 18, wherein the mRNA is mono, bi, or multicistronic mRNA.

21. The vaccine composition according to claim 18, wherein the mRNA comprises at least one chemical modification.

22. The vaccine composition according to claim 21, wherein the chemical modification is selected from the group consisting of base modification, sugar modification, skeleton modification, and lipid modification, preferably the chemical modification is base modification, and more preferably the base modification is selected from the group consisting of pseudouridine (psy or ψ), N1-methylpseudracil (N1MPU, N1Mpsy or N1Mψ), 1-ethylpseudracil, 2-thiouracil (s2U), 4-thiouracil, 5-methylcytosine, 5-methyluracil, 5-methoxyuracil, and any combination thereof.

23. The vaccine composition according to claim 18, wherein the code sequence exhibits sequence modification.

24. The sequence modification is selected from G / C content modification, codon modification, codon optimization, or C optimization of the sequence; preferably compared to the coding sequence of the corresponding wild-type mRNA. a) The G / C content of the code sequence is increasing; b) The C content of the code sequence is increasing; c) The codon usage frequency of the code sequence matches the hito-codon usage frequency; and / or d) The Codon Adaptation Index (CAI) is increased or maximized in the aforementioned code sequence. The vaccine composition according to claim 23.

25. The mRNA is a) 5' cap structure, preferably m7GpppN, more preferably cap 1 or m7G(5')ppp(5')(2'OMeA)pG; b) Preferably, at least one miRNA binding site sequence for a microRNA selected from the group consisting of miR-126, miR-142, miR-144, miR-146, miR-150, miR-155, miR-16, miR-21, miR-223, miR-24, miR-27, miR-26a binding sites, preferably miR-122 or miR-142 binding sites, or any combination thereof of the above miRNA binding sites; c) at least one 5'-UTR element; d) at least one 3'-UTR element; e) At least one poly(A) array; f) At least one poly(C) array; g) A histone stem loop selected from sequence number 3 or 4, depending on the circumstances; h) A 3' terminal element selected from sequence numbers 41 to 70, depending on the case; or any combination of these The vaccine composition according to claim 18, further comprising:

26. The vaccine composition according to claim 18, wherein the mRNA comprises a 5' cap structure, preferably an m7G, cap 0, cap 1, cap 2, modified cap 0, or modified cap 1 structure.

27. ​​The at least one coding RNA comprises at least one heterologous 5'-UTR and / or at least one heterologous 3'-UTR, preferably the at least one heterologous 5'-UTR comprising a 5'-UTR of a gene selected from HSD17B4, RPL32, ASAH1, ATP5A1, MP68, NDUFA4, NOSIP, RPL31, SLC7A3, TUBB4B, and UBQLN2, or a nucleic acid sequence derived from a homolog, fragment, or variant of any one of these genes; and / or preferably the at least one heterologous 3'-UTR comprising PSMB3, ALB / albumin, alpha - The vaccine composition according to claim 25, comprising a globin, the 3'-UTR of a gene selected from CASP1 (preferably SEQ ID NO: 81 (DNA) or SEQ ID NO: 82 (RNA)), COX6B1 (preferably SEQ ID NO: 83 (DNA) or SEQ ID NO: 84 (RNA)), GNAS (preferably SEQ ID NO: 85 (DNA) or SEQ ID NO: 86 (RNA)), NDUFA1 (preferably SEQ ID NO: 87 (DNA) or SEQ ID NO: 88 (RNA)), and RPS9 (preferably SEQ ID NO: 79 (DNA) or SEQ ID NO: 80 (RNA)), or a nucleic acid sequence derived from any one of these genes, a homolog, fragment, or variant.

28. The vaccine composition according to claim 27, wherein the at least one coding RNA comprises (i) HSD17B4 5'-UTR and PSMB3 3'-UTR, or (ii) RPL32 5'-UTR and ALB / albumin 3'-UTR, preferably mutant alpha-globin 3'-UTR (SEQ ID NOs. 11, 12), more preferably HSD17B4 5'-UTR (SEQ ID NOs. 21, 22) and PSMB3 3'-UTR (SEQ ID NOs. 19, 20).

29. The mRNA has the following elements in the 5'→3' direction: a) A 5' cap structure preferably selected from the group consisting of m7G(5'), m7G(5')ppp(5')(2'OMeA) and m7G(5')ppp(5')(2'OMeG); b) A 5'-UTR element comprising a nucleic acid sequence derived from the 5'-UTR of the TOP gene, wherein the nucleic acid sequence is preferably the nucleic acid sequence according to SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, or preferably SEQ ID NO: 77 / 78 (SLC7A3) or SEQ ID NO: 75 / 76 (RPL31), or its homolog, fragment, or variant; most preferably a 5'-UTR element comprising an RNA sequence corresponding to the nucleic acid sequence according to SEQ ID NO: 22 (HSD17B4); c) The at least one code sequence; d) A 3'-UTR element comprising a nucleic acid sequence derived from the α-globin gene, wherein the nucleic acid sequence preferably comprises the nucleic acid sequence according to SEQ ID NOs. 6, 8 or SEQ ID NOs. 10, 12, 14, 16, 18 or preferably SEQ ID NOs. 20, or an RNA sequence corresponding to its homolog, fragment, or variant; and / or a 3'-UTR element comprising a nucleic acid sequence derived from the albumin gene, wherein the nucleic acid sequence preferably comprises the nucleic acid sequence according to SEQ ID NOs. 18 (ALB / albumin) or preferably SEQ ID NOs. 79 / 80 (RPS9), or its homolog, fragment, or variant; most preferably comprises an RNA sequence corresponding to the nucleic acid sequence according to SEQ ID NOs. 20 (PSMB3); e) at least one poly(A) sequence, preferably consisting of 10 to 200, 10 to 100, 40 to 80, or 50 to 70 adenosine nucleotides, more preferably at least 70 adenosine nucleotides, and even more preferably about 100 adenosine nucleotides; f) In some cases, preferably at least one poly(C) sequence consisting of 10 to 200, 10 to 100, 20 to 70, 20 to 60 or 10 to 40 cytosine nucleotides; and g) In some cases, preferably including the RNA sequence specified by Sequence ID No. 4, at least one histone stem loop The vaccine composition according to claim 18, comprising:

30. The vaccine composition according to claim 1, wherein the antigen is derived from a pathogenic antigen, a tumor antigen, an allergen antigen, or an autoimmune autoantigen.

31. The vaccine composition according to claim 30, wherein the pathogenic antigen is selected from the group consisting of bacterial antigens, viral antigens, fungal antigens, and protozoan antigens.

32. The pathogenic antigen is (i) SARS coronavirus 2 (SARS-CoV-2), nCoV-2019 coronavirus, SARS coronavirus (SARS-CoV), Bunyavirum viruses, Cytomegalovirus (CMV), Dengue virus (DENV-1, DENV-2, DENV-3 and DENV-4), Ebola virus (EBOV), Flavivirus, Hepatitis B virus (HBV), Herpes simplex virus (HSV), Human immunodeficiency virus (HIV), Human metapneumovirus (HMPV), Human papillomavirus (HPV), Human parainfluenza virus (HPIV), Influenza Induced from lupenza virus, extraenteropathogenic Escherichia coli (ExPEC), Lassa mum arenavirus (LASV), MERS coronavirus, Mycobacterium tuberculosis, Nipah virus, norovirus, rabies virus (RABV), synovial respiratory virus (RSV), rhinovirus, rotavirus, vaccinia virus, yellow fever virus (YFV), Zika virus (ZIKV), Chlamydia trachoma (i.e., Chlamydia bacteria that cause chlamydia), or malaria parasites (e.g., Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, or Plasmodium ovale); and / or (ii) derived from SARS coronavirus 2 (SARS-CoV-2), nCoV-2019 coronavirus, SARS coronavirus (SARS-CoV) structural proteins, accessory proteins, or replicase proteins, or immunogenic fragments or immunogenic variants thereof; and / or (iii) derived from SARS coronavirus 2 (SARS-CoV-2), nCoV-2019 coronavirus, SARS coronavirus (SARS-CoV) spike protein (S), envelope protein (E), membrane protein (M), or nucleocapsid protein (N), or any immunogenic fragment or immunogenic variant thereof (preferably, spike protein (S) includes or consists of spike protein fragment S1 or spike protein fragment S2, more preferably spike protein fragment S1, or immunogenic fragments or immunogenic variants thereof); and / or (iv) Derived from SARS coronavirus 2 (SARS-CoV-2), nCoV-2019 coronavirus, or SARS coronavirus (SARS-CoV) prefusion stabilization spike protein (S) (S_stab) containing at least one prefusion stabilization mutation, The vaccine composition according to claim 30.

33. A pharmaceutical composition comprising the vaccine composition according to claim 30, which is preferably a sterile solid composition for reconstitution with a sterile liquid carrier, further comprising one or more inactive components selected from pH modifiers, bulking agents, stabilizers, nonionic surfactants, and antioxidants, wherein the sterile liquid carrier is an aqueous carrier, and a pharmaceutically acceptable carrier, diluent, or excipient.

34. A vaccine composition according to claim 30 or a pharmaceutical composition comprising the vaccine composition according to claim 30 for use in the treatment or prevention of infectious diseases in a subject; cancer or tumor disease, disorder or condition; liver disease selected from the group consisting of hepatic fibrosis, cirrhosis and liver cancer; allergies; or autoimmune diseases, disorders or conditions.

35. A vaccine composition according to claim 32 or a pharmaceutical composition comprising the vaccine composition according to claim 32, for use in the treatment or prevention of infectious diseases, including viral, bacterial, or protozoan infectious diseases, in a subject.

36. The vaccine composition or pharmaceutical composition is administered by local or local area injection, injection or implantation, particularly intradermal, subcutaneous, intramuscular, anterior chamber, subconjunctival, suprachoroidal injection, subretinal, subtenon's capsule, retroocular, local, near posterior pleura administration, or intrapulmonary inhalation, interstitium, local area, intravitreous, intratumor, intralymphatic, intranodal, intraarticular, intraarticular synovial bursa, periarticular, intraperiperiarticular, intraperitoneal, intracardiac, intracardiac, intrapleural, perineurial, intrathoracic, epidural, intradural, peridural, intramedullary, intramedullary, intracerebral, intracavitary, intracavernosal, intraprostatic, intratesticular, intracartilage, intraosseous, disc The vaccine composition or pharmaceutical composition according to claim 34 or 35, which is administered via intraspinal, intrasacral, intrasacral, intragingival, intragingival, intragingival, intragingival, intragingival, periophthalmos, periodontal, retroocular, subarachnoid, subconjunctival, or choroidal injection, infusion, implantation, transnasal, buccal, sublingual, ear or auricle, eye, conjunctiva, vagina, rectum, intracervix, paranasal sinus, larynx, oropharynx, ureter, or urethral administration, and more preferably the lipid nanoparticles are administered to the target by intramuscular, intravenous, intradermal, subcutaneous, intratumoral, intranasal cavity, or inhalation, preferably by local or local area injection or infusion.

37. A kit or kit of parts comprising the vaccine composition according to claim 30 or the pharmaceutical composition according to claim 33, optionally comprising a liquid vehicle for solubilization, and optionally a technical instruction manual providing information on the use and dosage of the components.

38. a) a step of preparing a vaccine composition or pharmaceutical composition or a kit or kit of parts; b) A step of applying or administering the vaccine composition or the pharmaceutical composition or the kit or kit of parts to a target tissue or organism. A vaccine composition according to claim 30, or a pharmaceutical composition comprising the vaccine composition according to claim 30, or a kit of parts comprising the vaccine composition according to claim 30, for use in a method of treating or preventing an infectious disease in a subject; cancer or tumor disease, disorder or condition; liver disease selected from the group consisting of hepatic fibrosis, cirrhosis and liver cancer; allergy; or autoimmune disease, disorder or condition.

39. A vaccine composition according to claim 30, or a pharmaceutical composition comprising the vaccine composition according to claim 30, for use in a method for inducing an immune response in a subject, comprising the step of administering to a subject an amount of vaccine composition or pharmaceutical composition effective in producing an antigen-specific immune response in the subject.

40. A vaccine composition according to claim 30, or a pharmaceutical composition comprising the vaccine composition according to claim 30, for use in a method of targeting antigen-presenting cells, including dendritic cells and macrophages, and / or the spleen, comprising the step of administering the vaccine composition or pharmaceutical composition to a target, the vaccine composition comprising a step of administering the vaccine composition or pharmaceutical composition to a target, the vaccine composition comprising a step of administering the vaccine composition or pharmaceutical composition to a target, the vaccine composition comprising a step of a step of administering the vaccine composition or pharmaceutical composition to a target, the vaccine composition comprising a vaccine composition according to claim 30, the vaccine composition comprising a

41. A vaccine composition according to claim 30, or a pharmaceutical composition comprising the vaccine composition according to claim 30, or a kit or kit of parts comprising the vaccine composition according to claim 30, wherein the vaccine composition or pharmaceutical composition is used in a subject to (i) induce an immune response, (ii) induce an antigen-specific T cell response, preferably (iii) induce a CD8+ T cell response, and / or to target antigen-presenting cells including dendritic cells and macrophages, and / or the spleen.

42. Use of phosphatidylserine in the preparation of the vaccine composition according to claim 1 or the carrier composition as defined in claim 1, comprising a) at least one nucleic acid, preferably mRNA, encoding at least one antigen or fragment or variant thereof; and b) a carrier composition, preferably a lipid nanoparticle composition, for targeting the vaccine composition to antigen-presenting cells, including dendritic cells and macrophages, and / or the spleen.

43. The vaccine composition or pharmaceutical composition according to claim 34, 35, 38, 39, 40, or 41, wherein the target is a mammal, preferably a human.

44. a) at least one nucleic acid encoding at least one antigen or a fragment or variant thereof; b) A carrier composition containing the phospholipid 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC) A vaccine composition or carrier composition containing the following:

45. Use of DHPC in the preparation of a carrier composition for a vaccine composition, comprising a) at least one nucleic acid, preferably mRNA, encoding at least one antigen or fragment or variant thereof, and b) a vaccine or carrier composition, preferably a lipid nanoparticle composition, for targeting the vaccine composition to antigen-presenting cells, including dendritic cells and macrophages, and / or the spleen.

46. The vaccine composition or pharmaceutical composition according to claim 34, 35, 38, 39, 40, or 41, further comprising DHPC, wherein the target is a mammal, preferably a human.

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