Lipid nanoparticles for nucleic acid delivery

Abstract

Provided are lipid nanoparticles containing mRNA useful as an mRNA-based vaccine. 【Solution means】Cationic lipid according to formula (I): R a -A-R b Formula (I) Or a salt, prodrug, or stereoisomer thereof, a composition comprising the cationic lipid useful for delivering nucleic acid to living cells. In formula (I), R a Has the following structure JPEG2026065025000178.jpg43170 -R 1 -N(H)-C(O)-R 3 -R 4 And R b Has the following structure etc., JPEG2026065025000179.jpg22170 A is -S-S-, -NH-C(O)-, etc.

Description

[Technical Field]

[0001] This invention relates to mRNA-containing lipid nanoparticles useful as mRNA-based vaccines. Furthermore, this invention relates to compositions comprising mRNA-containing lipid nanoparticles, and to the use of mRNA-containing lipid nanoparticles or compositions for preparing pharmaceutical compositions, particularly vaccines, for use in the prevention or treatment of infectious diseases, tumors or cancers, allergies or autoimmune diseases, for example. This invention further describes methods for treating or preventing the above diseases. [Background technology]

[0002] Gene therapy and genetic vaccination belong to the most promising and rapidly developing methods of modern medicine. They can offer highly specific and individualized treatment options for a wide variety of diseases. Genetic vaccination allows for the elicitation of a desired immune response against selected antigens, such as characteristic components like bacterial surfaces, viral particles, and tumor antigens. In general, vaccination is one of the important achievements of modern medicine. However, effective vaccines are currently available for only a limited number of diseases. Therefore, millions of people still contract infectious diseases that cannot be prevented by vaccination each year.

[0003] Generally, vaccines can be subdivided into “first,” “second,” and “third” generation vaccines. “First generation” vaccines are typically whole-organism vaccines. These are based on either live attenuated pathogens or dead pathogens, such as viruses or bacteria. The main drawback of live attenuated vaccines is the risk of reversion to life-threatening variants. Thus, such pathogens, although attenuated, can still inherently carry unpredictable risks. Dead pathogens may not be as effective as desired to elicit 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 pathogens.

[0004] Genetic vaccines, or vaccines for genetic vaccination, are generally understood as "third-generation" vaccines. These typically consist of genetically engineered nucleic acid molecules that enable the expression of peptides or protein (antigen) fragments characteristic of a pathogen or tumor antigen in vivo. Once administered to a patient, genetic vaccines are expressed after being taken up by target cells. The expression of the administered nucleic acid molecules leads to the production of encoded proteins. When these proteins are recognized as exogenous by the patient's immune system, an immune response is triggered.

[0005] DNA and 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 leading to mutagenic events such as loss of function of the affected gene. A further risk is the unwanted production of anti-DNA antibodies. Another drawback is that the expression level of the encoded peptide or protein achievable with DNA administration is limited because the DNA must enter the nucleus to be transcribed before the resulting mRNA can be translated. Among several reasons, the level of expression of 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 a satisfactory amount of RNA. As a result, the level of translated peptide or protein obtained is limited.

[0006] Using RNA instead of DNA in genetic vaccination minimizes or avoids the risk of unwanted genomic integration and the production of anti-DNA antibodies. However, RNA is considered a fairly unstable molecular species that can be readily degraded by ubiquitous RNases.

[0007] mRNA vaccines containing antigen-coding mRNA complexed with protamine have already been described in the prior art (e.g., Non-Patent Document 1, Non-Patent Document 2, Patent Document 1, Patent Document 2, Patent Document 3, Patent Document 4, and Patent Document 5). Patent Document 6 also describes lipid nanoparticle compositions containing nucleoside-modified RNA encoding various antigens.

[0008] Despite the significant progress made in recent years, there remains a need in this field to provide efficient methods for mRNA vaccination that enable adaptive immune responses, where administration is not severely impaired by premature antigen degradation or insufficient mRNA translation due to inadequate release of mRNA in cells. Furthermore, there is an urgent need to reduce the dosage of mRNA vaccines to mitigate potential safety concerns and make vaccines available in the developing world.

[0009] Many challenges exist in delivering nucleic acids to produce desired responses in biological systems. Nucleic acid-based therapies, such as vaccines, have enormous potential, but realizing this potential requires more effective delivery of nucleic acids to the appropriate sites within cells or organisms.

[0010] However, the use of nucleic acids in therapeutic settings currently faces two challenges. First, free RNA is susceptible to nuclease digestion in plasma. Second, free RNA has a limited ability to gain access to intracellular compartments where the relevant translation mechanisms reside. Lipid nanoparticles formed from cationic and neutral lipids, cholesterol, PEG, PEGylated lipids, and other lipid components, as well as oligonucleotides, have been used to block RNA degradation in plasma and promote the uptake of oligonucleotides into cells.

[0011] Improved cationic lipids and lipid nanoparticles are needed for the delivery of oligonucleotides. Preferably, these lipid nanoparticles would provide an optimal drug-to-lipid ratio, protect nucleic acids from degradation and clearance in serum, be suitable for systemic or topical delivery, and provide intracellular delivery of nucleic acids. Furthermore, these lipid-nucleic acid particles should be well-tolerated and provide a sufficient therapeutic index so that treatment of a patient with an effective dose of nucleic acid does not involve unacceptable toxicity and / or risk to the patient. The present invention provides these and related advantages. [Prior art documents] [Patent Documents]

[0012] [Patent Document 1] European Patent No. 1083232 [Patent Document 2] International Publication No. 2010 / 037539 [Patent Document 3] International Publication No. 2012 / 116811 [Patent Document 4] International Publication No. 2012 / 116810 [Patent Document 5] International Publication No. 2015 / 024665 [Patent Document 6] International Publication No. 2016 / 176330 [Non-patent literature]

[0013] [Non-Patent Document 1] PMID:27336830 [Non-Patent Document 2] PMID:23159882 [Overview of the Initiative] [Means for solving the problem]

[0014] In one aspect, the present invention relates to novel cationic lipids useful for the delivery of nucleic acids to living cells. The cationic lipid is a compound according to formula (I): R a -A-R b Formula (I) (wherein R a is

[0015]

Chem.

[0016] -R 1 -N(H)-C(O)-R 3 -R 4 selected from; R b is

[0017]

Chem.

[0018] -R 1 -N(H)-C(O)-R 3 -R 4 or -R 1 -N(CH3)2 selected from; A is -S-, -S-S-, -S-C(O)-, -NH-C(O)-, -NH-C(O)O-, -NH-C(O)-NH-, -S-C(O)-N(H)-, -C(O)O-, or -O-P(O)(OH)-O-; R 1 is optionally substituted ethanediyl, propanediyl, butanediyl, or a straight-chain or unbranched alkanediyl having 2 to 8 carbon atoms; R 2 is an alkanediyl having 2 to 8 carbon atoms; R 3 is optional, and when present, -R 5 -C(O)-O-, or -R 5-OC(O)-, -R 5 -C(O)-NH-, -R 5 -OC(O)-NH-, or R 5 -NH-C(O)O-; R 4 It is a lipophilic substituent having 12 to 36 carbon atoms; R 5 It is an alkanediyl having 1 to 6 carbon atoms; X is a carbon or nitrogen atom; All choices are independent of each other. Depending on the case, R 1 , R 2 and R 5 All of them are ethane, A is SS-, and R a and R b If they are the same, R 4 teeth,

[0019] [ka]

[0020] isn't it) That is the case. Regarding this point, Arcanziel is (-C n H 2n -) is a term referring to a group; therefore, for example, "alkanediyl having 2 to 8 carbon atoms" is a group with the formula -C2H4-, -C3H6-, -C4H8-, -C5H 10 -, -C6H 12 -, -C7H 14 -, or -C8H respectively 16 It is equivalent to an alkanediyl group having -. In other words, an alkanediyl is a group derived from aliphatic hydrocarbons with the general formula C n H 2n These are a series of divalent radicals. Unless otherwise specified, such alkanediyls include substituted alkanediyls.

[0021] In another embodiment, R 1 , R 2 and R5 All of them are ethane, A is SS-, and R a and R b If they are the same, R 4 teeth

[0022] [ka]

[0023] In each embodiment, the lipid according to formula (I) is not the lipid C23 disclosed in Table 1 of this specification, nor is it the lipid SS-EC described below herein (to avoid doubt, i.e., in some selected embodiments, the cationic lipid COATSOME® SS-EC is waived from the embodiments relating to the cationic lipid according to formula (I)).

[0024] In another embodiment, the present invention provides a novel composition incorporating a cationic lipid, such as the novel cationic lipid defined above. Cationic lipids and compositions have been found to be particularly effective in introducing nucleic acids into living cells. For example, they enable improved RNA (e.g., mRNA) vaccines, i.e., mRNA-based vaccines against certain infectious diseases or tumors.

[0025] In a further embodiment, the present invention provides the use of compositions incorporating cationic lipids and nucleic acid compounds as pharmaceuticals and, in particular, vaccines, as well as methods for vaccination based on these vaccines.

[0026] In another aspect of the present invention, the present invention also provides a kit, in particular a kit of parts, comprising an mRNA compound comprising an mRNA sequence as defined herein and a lipid according to at least one formula (I) or formula (II) as defined herein.

[0027] definition For clarity and ease of reading, the following scientific background information and definitions are provided. Any technical features referred to or disclosed herein can be part of, or be read in, each and every embodiment of the present invention. Additional definitions and explanations can be provided in the context of this disclosure.

[0028] Unless otherwise defined or the specific context requires, all technical terms used herein have the same meaning as commonly understood by those skilled in the relevant technical fields.

[0029] Unless the context dictates otherwise or requires, the words "comprise", "comprises" and "comprising", and similar expressions, should be construed in this specification and the claims as having an open, inclusive meaning of "including, but not limited to".

[0030] Expressions such as "one embodiment", "an embodiment", "a specific embodiment", etc. mean that a particular feature, property or characteristic, or a particular group or combination of features, properties or characteristics, referred to in combination with each such expression, is present in at least one of the embodiments of the present invention. The appearance of these expressions at various places throughout this specification does not necessarily refer to the same embodiment. Furthermore, a particular feature, property or characteristic can be combined in any suitable way in one or more embodiments.

[0031] The singular forms "a", "an" and "the" should be understood to include plural references unless the context clearly dictates otherwise. Percentages in the context of numbers should be understood as being relative to the total number of each item. In other cases, unless the context indicates otherwise, percentages should be understood as weight percentages (wt%).

[0032] As used herein, "compound" means a chemical substance that is a material composed of molecules having essentially the same chemical structure and properties. For low molecular weight compounds, the molecules are typically identical with respect to their atomic composition and structural arrangement. For high molecular weight or polymeric compounds, the molecules of the compound are very similar, but not all of them are necessarily identical. For example, a segment of a polymer specified as consisting of 50 monomer units may also contain individual molecules having, for example, 48 or 53 monomer units.

[0033] 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 to include the ionized form of each 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 to include the non-ionized form of each compound or moiety. For example, a disclosure of a compound having a carboxyl group should be interpreted as referring to each compound having a non-ionized carboxyl group or an ionized carboxylate group.

[0034] As used herein, "physiological conditions" refers to an aqueous environment having a pH 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 may also refer to near-neutral conditions such as from about pH 5 to about pH 8.5, or from about 5.5 to about pH 8.

[0035] Lipidoid compounds, also simply called lipidoids, are lipid-like compounds, that is, amphiphilic compounds that possess lipid-like physical properties. In the context of this invention, the term lipid is considered to encompass lipids.

[0036] In the context of the present invention, the phrase "selected from the group consisting of..." followed by a specific group of elements (e.g., "A, B, and C") is not intended to be limited to that group within the context of the present invention. In other words, such a phrase does not indicate that the disclosure is closed to elements not listed; that is, alternative meanings are also included in the group following this term. Therefore, in the context of the present invention, the phrase "selected from the group consisting of..." followed by a specific group of elements (e.g., "A, B, and C") should be understood as "selected from A, B, and C" or "A, B, or C," encompassing other structurally and functionally related and unrelated elements that are not mentioned.

[0037] The term "approximately" is used when a parameter or value does not necessarily have to be identical, i.e., 100% the same. Thus, "approximately" means that a parameter or value may deviate by 0.1% to 20%, preferably 0.1% to 10%; in particular, by 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, and 20%. Those skilled in the art will understand, for example, that a particular parameter or value may vary slightly depending on the method used to determine the parameter. For example, if a particular parameter or value is defined herein as having a length of, for example, "about 1000 nucleotides," then its length may deviate by 0.1% to 20%, preferably 0.1% to 10%, in particular by 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, and 20%. Thus, those skilled in the art will see that in specific examples, its length may deviate by 1 to 200 nucleotides, preferably 1 to 100 nucleotides, in particular by 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, and 200 nucleotides.

[0038] The term "cationic" means that each structure has a positive charge, permanently or not permanently, depending on certain conditions such as pH, unless a different meaning is evident from the specific context. Therefore, the term "cationic" encompasses both "permanently cationic" and "cationisable." As used herein, the term "cationisable" means that a compound, 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, cationisable compounds, groups, or atoms are positively charged at high hydrogen ion concentrations and uncharged at low concentrations or active hydrogen ions. This refers to the individual properties of cationisable or polycationisable compounds, particularly the pK of each cationisable group or atom, whether it is charged or uncharged at its pH or hydrogen ion concentration. a It depends on the pK of the compound or part. In a diluted aqueous environment, the fraction of positively charged cationizable compounds, groups, or atoms can be estimated using the so-called Henderson-Hasselbalch formula, which is well known to those skilled in the art. For example, if the compound or part is cationizable, it is preferable that it is positively charged at a pH of about 1 to 9, preferably 4 to 9, 5 to 8 or even 6 to 8, more preferably 9 or less, 8 or less, 7 or less, most preferably a physiological pH, for example about 7.3 to 7.4, i.e., under physiological conditions, particularly under physiological salt conditions of cells in vivo. In some embodiments, it is preferable that the cationizable compound or part is mainly neutral at a physiological pH, for example about 7.0 to 7.4, but positively charged at lower pH values. In some embodiments, the pK of the cationizable compound or part a The preferred range is about 5 to about 7. In some embodiments, the protonatable lipid has a pK of protonatable groups in the range of about 4 to about 11. a For example, approximately 5 to approximately 7 pK a It holds.

[0039] Unless a different meaning is evident from the specific context, the term "cationic" means that each structure has a positive charge, either permanently or not permanently, depending on certain conditions such as pH. Therefore, the term "cationic" encompasses both "permanently cationic" and "cationizable." 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 is therefore cationic, and more specifically, cationizable.

[0040] As used herein, “permanently cationic” means that each compound, or group or atom, is positively charged at any pH value or hydrogen ion activity in its environment. Often, the positive charge arises from the presence of a quaternary nitrogen atom. If a compound has multiple such positive charges, it may be called permanently polycationic, a subcategory of permanently cationic compounds.

[0041] Similarly, the terms “anionic,” “anionizable,” and “persistently anionic” are used to have similar meanings to “cationic,” “cationizable,” and “persistently cationic,” except that the charge of the respective compound, group, or atom is negative rather than positive.

[0042] The term "neutral," when applied to compounds such as lipids or steroids, or to a group or part thereof, means that it is neither cationic nor anionic, for example, a hydrocarbon, that does not have functional groups that can be ionized under physiological conditions; or it means that it is both cationic and anionic under typical physiological conditions, i.e., zwitterionic, such as a typical natural phosphatidylcholine.

[0043] As used herein, “lipids” refer to a group of organic compounds characterized by being derivatives of fatty acids (e.g., esters) and generally insoluble in water but soluble in many organic solvents. Lipids are usually divided into at least three classes: (1) “simple lipids” including fats, oils, and waxes; (2) “compound lipids” including phospholipids and glycolipids; and (3) “derived lipids” such as steroids. With respect to glycolipids, in certain embodiments, the LNP comprises a glycolipid (e.g., monosialoganglioside GM1).

[0044] In this context, the prefix "poly-" refers to multiple atoms or groups in a compound that possess different properties. When placed in parentheses, the presence of multiple atoms or groups is optional. For example, (poly)cationic means cationic and / or polycationic. However, the absence of the prefix should not be interpreted as excluding multiples. For example, a polycationic compound is also a cationic compound and can be called as such.

[0045] The term “nucleic acid” means any compound comprising DNA or RNA, or any compound comprising them. The term may also be used for polynucleotides and / or oligonucleotides. Whenever a nucleic acid or nucleic acid sequence encoding a particular protein and / or peptide is referred to herein, such nucleic acid or nucleic acid sequence preferably also includes a regulatory sequence that enables its expression, i.e., transcription and / or translation of the nucleic acid sequence encoding the particular protein or peptide, in a suitable host, e.g., human.

[0046] In the context of the present invention, the term "nucleoside modification" refers to nucleic acids such as mRNA compounds or molecules that contain nucleosides not normally present in natural mRNA, preferably non-natural nucleosides. In particular, this term preferably refers to mRNA nucleosides other than adenine, guanine, cytosine, uracil, and thymine.

[0047] 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 contains a phosphate group attached to a sugar.

[0048] A "peptide" refers to an oligomer or polymer of at least two amino acid monomers linked by peptide bonds. This term does not limit the length of the amino acid polymer chain. A peptide may contain, for example, fewer than 50 monomer units. Longer peptides, typically having 50 to 600 monomer units, or more specifically 50 to 300 monomer units, are also called polypeptides.

[0049] A "protein" is defined as comprising or consisting of one or more polypeptides folded into a three-dimensional form that facilitate biological functions. An influenza pandemic, or pandemic flu, can occur when a non-human (novel) influenza virus acquires the ability to efficiently and persistently transmit from person to person and subsequently spreads globally. Influenza viruses that have the potential to cause a pandemic are called "pandemic potential influenza viruses" or "pandemic influenza viruses."

[0050] Examples of influenza viruses with pandemic potential include two different "bird flu" viruses, avian influenza A (H5N1) and avian influenza A (H7N9). Since these are non - human viruses (i.e., they are new among humans and are circulating in birds in some parts of the world), people have little to no immunity to these viruses. Human infections with these viruses are rare, but if either of these viruses were to change such that it could easily infect humans and spread easily from person to person, an influenza pandemic could occur.

[0051] Vaccine for pandemic influenza / influenza or pandemic influenza / influenza vaccine: A vaccine directed against a pandemic influenza virus is referred to herein as a vaccine for pandemic influenza / influenza or a pandemic influenza / influenza vaccine.

[0052] Influenza / influenza epidemic period: The influenza epidemic period is a period that recurs annually and is characterized by the occurrence of an influenza (flu) epidemic. The epidemic period occurs during the cold season in each hemisphere. Influenza activity can sometimes be geographically predicted and even tracked. The start of the main influenza activity in each epidemic period varies by location, but in any given location, these minor epidemics usually take about 3 weeks to reach a peak and another 3 weeks to significantly decline. Influenza vaccination is used to reduce the effects of the influenza epidemic period; pneumococcal vaccination further reduces the effects and complications of the influenza epidemic period. Since there is winter at different times of the year in the Northern and Southern Hemispheres, there are actually two influenza epidemic periods each year.

[0053] Seasonal influenza / flu vaccine or seasonal influenza / flu vaccine: A vaccine directed against the seasonal influenza virus during the influenza season is referred to herein as "seasonal influenza / flu vaccine or seasonal influenza / flu vaccine."

[0054] Immune System: The immune system can protect an organism from infection. When a pathogen breaches the organism's physical barriers and invades, the innate immune system provides an immediate but nonspecific response. If the pathogen evades this innate response, the vertebrate develops an adaptive immune system, which is a second layer of defense. Here, the immune system adapts its response to improve its recognition of the pathogen during infection. Thus, even after the pathogen has been 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 it encounters the pathogen. According to this, the immune system consists of the innate immune system and the adaptive immune system. Each of these two parts contains so-called humoral and cellular components.

[0055] Immune Response: An immune response can typically be either a specific response of the adaptive immune system to a particular antigen (a so-called specific or adaptive immune response) or a non-specific response of the innate immune system (a so-called non-specific or innate immune response). This invention relates to the core of the specific response of the adaptive immune system (adaptive immune response). In particular, this invention relates to an adaptive immune response to infection by a virus, such as the influenza virus. However, this specific response may be supported by an additional non-specific response (innate immune response). Therefore, this invention also relates to compounds for the simultaneous stimulation of the innate and adaptive immune systems to elicit an efficient adaptive immune response.

[0056] Adaptive Immune System: The adaptive immune system consists of highly specialized systemic cells and processes that eliminate or prevent pathogenic proliferation. Adaptive immune responses provide the vertebrate immune system with the ability to recognize and remember (generate immunity to) specific pathogens and initiate a stronger attack each time it encounters them. This system is highly adaptable due to somatic hypermutation (processes with increased frequency of somatic mutations) and V(D)J gene rearrangements (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. Since gene rearrangements result in irreversible changes in the DNA of each cell, all of that cell's progeny (offspring) inherit genes encoding the same receptor specificity, including memory B cells and memory T cells, which are key to longevity-specific immunity. Immune network theory is a theory of how the adaptive immune system works, based on the interaction between the variable regions of T cells, B cells, and molecules produced by T cells and B cells that possess these variable regions.

[0057] Adaptive Immune Response: Adaptive immune responses are typically understood to be antigen-specific. Antigen specificity allows for the generation of a response adapted to a specific antigen, pathogen, or pathogen-infected cell. The ability to initiate these adapted responses is maintained in the body by "memory cells." If a pathogen infects the body more than once, these specific memory cells are used to rapidly eliminate this pathogen. In this context, the first step of an adaptive immune response is the activation of different immune cells that can induce an antigen-specific immune response by naive antigen-specific T cells or antigen-presenting cells. This occurs in lymphoid tissues and organs through which naive T cells constantly pass. Cell types that can act as antigen-presenting cells include, among others, dendritic cells, macrophages, and B cells. Each of these cells has a distinct function in inducing an immune response. Dendritic cells take up antigens by phagocytosis and macropinocytosis, and are stimulated, for example, by contact with a foreign antigen, 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 soluble protein antigens via their receptors and internalize them can also be important in inducing T cells. Antigen presentation on MHC molecules leads to T cell activation, 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, 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 do not directly recognize and bind to antigens, but instead recognize antigens by T cell receptors which recognize short peptide fragments of pathogen-inducing protein antigens that are bound to MHC molecules on the surface of other cells, for example.

[0058] Cellular immunity / cellular immune response: Cellular immunity typically relates to the activation of macrophages, natural killer cells (NKs), antigen-specific cytotoxic T lymphocytes, and the release of various cytokines in response to antigens. More generally, cellular immunity relates to the activation of cells in the immune system, without the involvement of antibodies. A cellular immune response is characterized by the activation of antigen-specific cytotoxic T lymphocytes that can induce apoptosis in somatic cells that exhibit antigen epitopes on their surface, such as virus-infected cells, cells with intracellular bacteria, and cancer cells exhibiting tumor antigens; the activation of macrophages and natural killer cells so that they can destroy pathogens; and the stimulation of cells to secrete various cytokines that affect the function of other cells involved in adaptive and innate immune responses.

[0059] Humoral immunity / humoral immune response: Humoral immunity typically refers to antibody production and any associated auxiliary processes. Humoral immune responses typically may be characterized by, for example, Th2 activation and cytokine production, germinal center formation and isotype switching, affinity maturation and memory cell production. Humoral immunity may also typically refer to the effector functions of antibodies, including pathogen and toxin neutralization, classical complement activation, and phagocytic opsonin enhancement and pathogen elimination.

[0060] Innate immune system: Also known as the nonspecific immune system, the innate immune system includes cells and mechanisms that nonspecifically protect the host from infection by other organisms. This means that cells in the innate system recognize and respond to pathogens in a general way, but unlike the adaptive immune system, they do not confer persistent immunity or protective immunity to the host. The innate immune system responds to various factors, including pathogen-associated molecular pattern (PAMP) receptors, ligands for Toll-like receptors (TLRs), lipopolysaccharides, TNF-alpha, CD40 ligands, cytokines, monokines, lymphokines, interleukins, or chemokines, such as 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, and IFN-alpha. It can be activated by other adjuncts such as IFN-beta, IFN-gamma, GM-CSF, G-CSF, M-CSF, LT-beta, TNF-alpha, growth factors, and 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, immunostimulant nucleic acids, immunostimulant RNA (isRNA), CpG-DNA, antibacterial agents, or antiviral agents. Typically, the innate immune response includes the recruitment of immune cells to the site of infection through the production of chemical factors, including specialized chemical mediators called cytokines; activation of the complement cascade; identification and removal of foreign substances present in organs, tissues, blood, and lymph by specialized leukocytes; activation of the adaptive immune system through a process known as antigen presentation; and / or acting as a physical and chemical barrier to infectious agents.

[0061] Adjuvant / Adjuvant Component: An adjuvant or adjuvant component is, in a broad sense, a drug or composition that can modify, for example, enhance the effectiveness of other agents, such as drugs or vaccines (e.g., pharmacologically or immunologically). Conventionally, in the context of the present invention, this term refers to a compound or composition that acts as an immunostimulant carrier or auxiliary and / or other pharmaceutically active compound. This term should be interpreted broadly and refers to a wide range of substances that can increase the immunogenicity of an antigen incorporated with 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 activator of the present invention. Typically, "adjuvant" and "adjuvant component" have the same meaning and can be used interchangeably. Adjuvants can be divided into, for example, immunostimulants, antigenic delivery systems, or even combinations thereof.

[0062] The term "adjuvant" is typically understood to refer to a drug that does not confer immunity itself. Adjuvants nonspecifically support the immune system to enhance antigen-specific immune responses, for example, by promoting antigen presentation to the immune system or induction of nonspecific innate immune responses. Furthermore, adjuvants can modulate antigen-specific immune responses, preferably by shifting a dominant Th2-based antigen-specific response to a more Th1-based antigen-specific response, or vice versa. Thus, adjuvants can favorably modulate cytokine expression / secretion, antigen presentation, type of immune response, etc.

[0063] Immunostimulating RNA: In the context of this invention, immunostimulating RNA (isRNA) is typically RNA capable of inducing the innate immune response itself. It usually does not possess an open reading frame and therefore does not provide peptide antigens or immunogens, but it induces an innate immune response by binding, for example, to a specific type of Toll-like receptor (TLR) or other suitable receptor. However, of course, mRNA that possesses an open reading frame and encodes peptides / proteins (e.g., antigenic function) can also induce an innate immune response.

[0064] As used herein, the term “antibody” includes both intact antibodies and antibody fragments. Typically, an intact “antibody” is an immunoglobulin that specifically binds to a particular antigen. An antibody may 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” chain and a “heavy” chain. An “antibody fragment” includes a portion of an intact antibody, such as the antigen-binding or variable region of the antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; tribes; tetras; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. For example, an antibody fragment includes an isolated fragment, an "Fv" fragment consisting of a heavy chain variable region and a light chain variable region, a recombinant single-chain polypeptide molecule ("ScFv protein") in which the light chain variable region and the heavy chain variable region are linked by a peptide linker, and a minimal recognition unit consisting of amino acid residues that mimic a hypervariable region. Examples of antigen-binding fragments of an antibody include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, scFv fragments, Fv fragments, dsFv diabodies, dAb fragments, Fd' fragments, Fd fragments, and isolated complementarity-determining regions (CDRs). Suitable antibodies that can be encoded by the therapeutic RNA of the present invention include monoclonal antibodies, polyclonal antibodies, antibody mixtures or cocktails, human or humanized antibodies, chimeric antibodies, Fab fragments, or bispecific antibodies. In the context of the present invention, an antibody may be provided by at least one therapeutic RNA of the combination / composition of the present invention.

[0065] In the context of this invention, the term “antigen” typically refers to a substance that can be recognized by the immune system, preferably the adaptive immune system, and can induce an antigen-specific immune response, for example, by the formation of antibodies and / or antigen-specific T cells as part of an adaptive immune response. Typically, an antigen may be, or include, a peptide or protein that can be presented to T cells by MHC. In the sense of this invention, an antigen may be a product of translation of a provided nucleic acid molecule, preferably mRNA as defined herein. In this context, peptide and protein fragments, variants, and derivatives containing 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, and can be recognized, for example, by the immune system, preferably the adaptive immune system, and can induce an antigen-specific immune response, for example, by the formation of antibodies and / or antigen-specific T cells as part of an adaptive immune response. Typically, an antigen may be, or include, a peptide or protein that can be presented to T cells by MHC. Peptide or protein fragments, variants, and derivatives derived from, for example, cancer antigens, containing at least one epitope can also be understood as antigens. In the context of the present invention, the antigen may be the product of translation of a provided therapeutic RNA (e.g., coding RNA, replicon RNA, mRNA). The term “antigenic peptide or protein” is intended to be recognized and understood by those skilled in the art and to refer to a peptide or protein derived from an (antigenic) protein that can stimulate the body’s adaptive immune system to produce an adaptive immune response, for example. Thus, “antigenic peptide or protein” comprises at least one epitope or antigen (e.g., tumor antigen, viral antigen, bacterial antigen, protozoan antigen) of the protein from which it is derived. In the context of the present invention, the antigen may be provided by at least one therapeutic RNA of the combination / composition of the present invention.

[0066] In the context of nucleic acids, that is, with respect to nucleic acids "derived from" another nucleic acid, the term "derived from" as used herein means that a nucleic acid derived from another nucleic acid shares 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. Those skilled in the art will notice that sequence identity is typically calculated for nucleic acids of the same kind, i.e., DNA sequences or RNA sequences. Thus, when DNA is "derived from" RNA, or RNA is "derived from" DNA, it is understood that in the first step, the RNA sequence is converted to the corresponding DNA sequence (in particular by substituting U with T throughout the sequence), or vice versa, a DNA sequence is converted to the corresponding RNA sequence (in particular by substituting T with U throughout the sequence). The sequence identity of the DNA sequence or the RNA sequence is then determined. Preferably, a nucleic acid “derived from” also refers to a nucleic acid that is modified compared to the nucleic acid from which it is derived, for example, to further increase RNA stability 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 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.

[0067] Epitope (also called "antigen determinant"): In the context of the present invention, a T cell epitope or portion of a protein may include a fragment having a length of about 6 to about 20 or more amino acids, for example, preferably about 8 to about 10, for example 8, 9 or 10 (or even 11 or 12 amino acids), which is processed and presented by an MHC class I molecule, or a fragment having a length of about 13 or more amino acids, for example 13, 14, 15, 16, 17, 18, 19, 20 or more, which is processed and presented by an MHC class II molecule, 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 a peptide fragment and an MHC molecule.

[0068] A B cell epitope is typically a fragment located on the outer surface of a (natural) protein or peptide antigen, as defined herein, which is recognizable by an antibody, i.e., in its natural form, and preferably has 5 to 15 amino acids, more preferably 5 to 12 amino acids, and even more preferably 6 to 9 amino acids.

[0069] Such epitopes of proteins or peptides may be further selected from any of the variants of such proteins or peptides referred herein. In this context, the antigenic determinant may be a three-dimensional structure or discontinuous epitope composed of segments of the protein or peptide as defined herein, which are discontinuous in the amino acid sequence of the protein or peptide as defined herein but can be assembled into a three-dimensional structure consisting of a single polypeptide chain or into a continuous or linear epitope.

[0070] The term "vaccine" is typically understood to be a preventive or therapeutic material that provides at least one antigen or antigenic function. An antigen or antigenic function can stimulate the body's adaptive immune system to produce an adaptive immune response.

[0071] In the context of this invention, the term “antigen-donating mRNA” is typically an mRNA having at least one open reading frame that can be translated by the cell or organism to which the mRNA is donated. The product of this translation is a peptide or protein that can act as an antigen, preferably an immunogen. The product may also be a fusion protein composed of two or more immunogens, for example, a fusion protein consisting of two or more epitopes, peptides, or proteins derived from the same or different viral proteins, where the epitopes, peptides, or proteins may be linked by linker sequences.

[0072] The term “artificial mRNA” (sequence) can typically be understood as an mRNA molecule that does not exist in nature. In other words, an artificial mRNA molecule can be understood as a non-natural mRNA molecule. Such mRNA molecules may be non-natural due to their individual sequences (which do not exist in nature) and / or other modifications, such as structural modifications of nucleotides that do not exist in nature. Typically, artificial mRNA molecules can be designed and / or produced by genetic engineering methods that correspond to a desired artificial sequence (heterogeneous sequence) of nucleotides. In this context, the artificial sequence is usually a sequence that cannot exist in nature; that is, the artificial sequence differs from the wild-type sequence by at least one nucleotide. The term “wild-type” can be understood as a sequence that exists in nature. Furthermore, the term “artificial nucleic acid molecule” is not limited to meaning “a single molecule,” but is typically understood to include an ensemble of identical molecules. Thus, the term can refer to multiple identical molecules contained in an aliquot.

[0073] As used herein in the context of nucleic acid sequences or amino acid sequences, the terms “heterogeneous” or “heterogeneous sequence” refer to sequences (e.g., DNA, RNA, amino acids) as recognized and understood by those skilled in the art, and are intended to refer to sequences derived from a different gene, a different allele, or a different species. If two sequences cannot be derived from the same gene or the same allele, these sequences are typically understood to be “heterogeneous.” That is, heterogeneous sequences may be derived from the same organism, but they do not naturally exist in the same nucleic acid molecule, such as the same RNA or protein.

[0074] Bi / Multicistronic mRNA: mRNA that typically has two (biscistronic) or more (multicistronic) open reading frames (ORFs) (coding regions or coding sequences). In this context, an open reading frame is a sequence of several nucleotide triplets (codons) that can be translated into a peptide or protein. Translation of such mRNA yields two (biscistronic) or more (multicistronic) distinct translation products (if the ORFs are not identical). For expression in eukaryotes, such mRNA may contain, for example, an internal ribosome entry site (IRES) sequence.

[0075] Monocistronic mRNA: Monocistronic mRNA can be mRNA that typically contains only one open reading frame (coding sequence or coding region). In this context, the open reading frame is a sequence of several nucleotide triplets (codons) that can be translated into a peptide or protein.

[0076] 3' Untranslated Region (3'-UTR): The 3'-UTR is typically the portion of mRNA located between the protein-coding region (i.e., the open reading frame) and the poly(A) sequence. The 3'-UTR of mRNA is not translated into an amino acid sequence. The 3'-UTR sequence is generally encoded by a gene that is transcribed into its respective mRNA during the gene expression process. The genomic sequence is first transcribed into an mRNA precursor, which contains optional introns. The mRNA precursor is then further processed into mature mRNA in the maturation process. This maturation process includes steps such as 5' capping, splicing of the mRNA precursor to remove optional introns, and 3' end modification, including polyadenylation of the 3' end of the mRNA precursor and optional endo- or exonuclease cleavage. In the context of the present invention, the 3'-UTR corresponds to a sequence of mature mRNA located 3' to the stop codon of the protein-coding region, preferably immediately 3' to the stop codon of the protein-coding region, and extending to the nucleotide immediately 5' to the poly(A) sequence, preferably immediately 5' to the poly(A) sequence. The term "corresponding" means that the 3'-UTR sequence may be an RNA sequence in the mRNA used to define the 3'-UTR sequence, for example, or a DNA sequence corresponding 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 the sequence corresponding to the 3'-UTR of the mature mRNA derived from that gene, i.e., the mRNA obtained by gene transcription and maturation of the mRNA precursor. The term "3'-UTR of a gene" encompasses both DNA and RNA sequences of the 3'-UTR.

[0077] 5' Untranslated Region (5'-UTR): The 5'-UTR is typically understood to be a specific section of messenger RNA (mRNA). It is located at 5' of the open reading frame of the mRNA. Typically, the 5'-UTR begins at the transcription start 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 this invention, the 5'-UTR corresponds to a sequence of 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' relative to the 5' cap, preferably a nucleotide immediately 3' relative to the 5' cap, to a nucleotide located 5' relative to the start codon of the protein-coding region, preferably a nucleotide immediately 5' relative to the start codon of the protein-coding region. The nucleotide located immediately 3' to the 5' cap of mature mRNA typically corresponds to the transcription start site. The term “corresponding” means that the 5'-UTR sequence may be an RNA sequence in the mRNA used to define the 5'-UTR sequence, for example, or a DNA sequence corresponding to such an RNA sequence. In the context of this invention, the term “5'-UTR of a gene,” such as “5'-UTR of a TOP gene,” refers to the sequence corresponding to the 5'-UTR of the mature mRNA derived from this gene, i.e., the mRNA obtained by gene transcription and maturation of the mRNA precursor. The term “5'-UTR of a gene” encompasses both the DNA and RNA sequences of the 5'-UTR.

[0078] 5'-terminal oligopyrimidine tract (TOP): A 5'-terminal oligopyrimidine tract (TOP) is an elongation of a pyrimidine nucleotide located in the 5' terminal region of a nucleic acid molecule, typically the 5' terminal region of a particular mRNA molecule or the 5' terminal region of a functional entity of a particular gene, such as the transcribed region. This sequence usually begins with a cytidine, corresponding to the transcription start site, followed by an elongation of approximately 3 to 30 pyrimidine nucleotides. For example, a TOP may 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. Due to the pyrimidine elongation, the 5'-TOP ends at 5' of the first purine nucleotide located downstream of the TOP. Messenger RNAs containing a 5'-terminal oligopyrimidine tract are often called TOP mRNAs. Therefore, genes providing such messenger RNAs are called TOP genes. TOP sequences are found, for example, in genes and mRNAs encoding peptide elongation factors and ribosomal proteins.

[0079] TOP motif: In the context of the present invention, the TOP motif is a nucleic acid sequence corresponding to the 5'-TOP as defined above. Thus, the TOP motif in the context of the present invention is preferably an extension of a pyrimidine nucleotide having a length of 3 to 30 nucleotides. Preferably, the 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, and most preferably at least 8 pyrimidine nucleotides, wherein the pyrimidine nucleotide extension preferably begins with a cytosine nucleotide at its 5' end. In the TOP gene and TOP mRNA, the TOP motif preferably begins with a transcription start site at its 5' end and ends with 1 nucleotide 5' relative to the first purine residue in the gene or mRNA. The TOP motif in the sense of the present invention is preferably located at the 5' end of a sequence representing the 5'-UTR, or at the 5' end of a sequence encoding the 5'-UTR. Therefore, preferably, an extension of three or more pyrimidine nucleotides is referred to as a "TOP sequence" in the sense of the present invention if it is located at the 5' end of the mRNA of the present invention, the 5'-UTR element of the mRNA of the present invention, or a nucleic acid sequence derived from the 5'-UTR of the TOP gene described herein. In other words, an extension 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 somewhere within the 5'-UTR or 5'-UTR element, is preferably not referred to as a "TOP motif".

[0080] TOP genes: TOP genes are typically characterized by the presence of a 5'-terminal oligopyrimidine tract. Furthermore, 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 mature mRNA induced from the TOP gene, preferably extending from a nucleotide located 3' relative to the 5' cap to a nucleotide located 5' relative to the start codon. The 5'-UTR of a TOP gene typically does not contain any start codon, preferably neither an upstream AUG (uAUG) nor an upstream open reading frame (uORF). In this context, the upstream AUG and upstream open reading frame are understood to be the AUG and open reading frame located 5' of the start codon (AUG) of the open reading frame to be translated. The 5'-UTR of a TOP gene is generally quite short. The length of the 5'-UTR of the TOP gene can vary between 20 and up to 500 nucleotides, typically less than about 200 nucleotides, preferably less than about 150 nucleotides, and more preferably less than about 100 nucleotides. An exemplary 5'-UTR of the TOP gene in the sense of the present invention is a nucleic acid sequence extending from the nucleotide at position 5 to the nucleotide immediately 5' relative to the start codon (e.g., ATG) in the sequence or its homolog or variant as described in SEQ ID NOs. 1-1363, SEQ ID NOs. 1395, SEQ ID NOs. 1421 and SEQ ID NOs. 1422 of International Publication No. 2013 / 143700, the disclosure of which is incorporated herein by reference. In this context, a particularly preferred fragment of the 5'-UTR of the TOP gene is the 5'-UTR of the TOP gene lacking the 5'-TOP motif. The term “5'-UTR of the TOP gene” preferably refers to the naturally occurring 5'-UTR of the TOP gene.

[0081] Nucleic acid sequences, particularly fragments of mRNA: A fragment of a nucleic acid sequence consists of sequential extensions of nucleotides corresponding to sequential extensions of nucleotides in the full-length nucleic acid sequence that forms the basis of the fragment's nucleic acid sequence, 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 nucleic acid sequence. In the sense of the present invention, such a fragment is preferably a functional fragment of a full-length nucleic acid sequence.

[0082] In the context of the present invention, a "fragment" or "variant" of a protein or peptide is defined as having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60% of the elongation of at least 10, 20, 30, 50, 75, or 100 amino acids of such a protein or peptide. %, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity. More preferably, a "fragment" or "variant" of a protein or peptide used herein is at least 40%, preferably at least 50%, more preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% identical to the protein or peptide from which the variant is derived.

[0083] Nucleic acid sequence variants, particularly mRNA variants: A nucleic acid sequence variant refers to a variant of a nucleic acid sequence that forms the basis of a nucleic acid sequence. For example, a variant nucleic acid sequence may show deletions, insertions, additions, and / or substitutions of one or more nucleotides compared to the nucleic acid sequence from which the variant is induced. Preferably, a nucleic acid sequence variant is at least 40%, preferably at least 50%, more preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% identical to the nucleic acid sequence from which the variant is induced. Preferably, the variant is a functional variant. A nucleic acid sequence "variant" may have at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% nucleotide identity over an extension of 10, 20, 30, 50, 75, or 100 nucleotides of such a nucleic acid sequence.

[0084] Stabilized nucleic acids, preferably mRNA: Stabilized nucleic acids, preferably mRNA, typically exhibit modifications that increase resistance to in vivo degradation (e.g., degradation by exo- or endo-nucleases) and / or exovivo degradation (e.g., by manufacturing processes prior to vaccine administration, e.g., during the preparation of the vaccine solution to be administered). RNA stabilization can be achieved, for example, by providing a 5' cap structure, a poly-A 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. Various other methods are known in the art and can be considered in the context of the present invention.

[0085] RNA In Vitro Transcription: The term “RNA in vitro transcription” or “in vitro transcription” refers to the process by which RNA is synthesized in a cell-free system (in vitro). DNA, particularly plasmid DNA, is used as a template for producing RNA transcripts. RNA can be obtained by DNA-dependent in vitro transcription of a suitable DNA template, preferably a linear plasmid DNA template, according to the present invention. The promoter for controlling in vitro transcription can be any promoter for any DNA-dependent RNA polymerase. Specific examples of DNA-dependent RNA polymerases include T7, T3, and SP6 RNA polymerases. The DNA template for in vitro RNA transcription can be obtained by cloning nucleic acids, particularly cDNA corresponding to each RNA to be transcribed in vitro, and introducing this into a suitable vector for in vitro transcription, such as plasmid DNA. In a preferred embodiment of the present invention, the DNA template is linearized with a suitable restriction enzyme before being transcribed in vitro. cDNA can be obtained by reverse transcription or chemosynthesis of mRNA. Furthermore, the DNA template for in vitro RNA synthesis can also be obtained by gene synthesis.

[0086] Methods for in vitro transcription are known in the art (see, for example, Geall et al. (2013) Semin.Immunol.25(2):152-159; Brunelle et al. (2013) Methods Enzymol.530:101-14). Reagents used in the above methods typically include: 1) A linearized DNA template having a promoter sequence that has high binding affinity to each RNA polymerase, such as RNA polymerase encoded by bacteriophages; 2) Ribonucleoside triphosphates (NTPs) for four bases (adenine, cytosine, guanine, and uracil); 3) Depending on the case, the capped analog defined above (e.g., m7G(5')ppp(5')G(m7G)); 4) DNA-dependent RNA polymerases (e.g., T7, T3, or SP6 RNA polymerases) that can bind to promoter sequences within a linearized DNA template; 5) Ribonuclease (RNase) inhibitors to inactivate any contaminated RNase, if necessary; 6) Pyrophosphatases that break down pyrophosphate, which may inhibit transcription in some cases; 7) MgCl2 supplying Mg2+ ions as a cofactor for polymerase; 8) A buffer to maintain an appropriate pH value, which may also contain an optimal concentration of antioxidants (e.g., DTT) and / or polyamines such as spermidine.

[0087] Full-length protein: As used herein, the term “full-length protein” typically refers to a protein that substantially contains the entire amino acid sequence of a naturally occurring protein. Nevertheless, amino acid substitutions in a protein, such as those resulting from mutation, are also included in the term “full-length protein.”

[0088] Protein Fragments: In the context of the present invention, a “fragment” of a protein or peptide may include a sequence of a protein or peptide as defined herein that is typically N-terminal and / or C-terminally cleaved with respect to its amino acid sequence (or its coding nucleic acid molecule) compared to the amino acid sequence (or its coding nucleic acid molecule) of the original (natural) protein. Thus, such cleavage may exist at the amino acid level or, correspondingly, at the nucleic acid level. Therefore, sequence identity with respect to such a fragment as defined herein may preferably refer to the entire protein or peptide as defined herein, or the entire (coding) nucleic acid molecule of such a protein or peptide.

[0089] In the context of a gene's nucleic acid sequence, the term “variant” refers to a nucleic acid sequence variant, i.e., a nucleic acid sequence or gene that contains at least one nucleic acid that differs from the reference (or “parent”) nucleic acid or the reference (or “parent”) nucleic acid sequence of the gene. Thus, a variant nucleic acid or gene may preferably contain at least one mutation, substitution, insertion, or deletion in its nucleic acid sequence compared to its respective reference sequence. Preferably, the term “variant” as used herein includes naturally occurring variants of a nucleic acid sequence or gene, and engineered variants. Thus, “variant” as defined herein may be derived from a reference nucleic acid sequence, isolated from a reference nucleic acid sequence, related to a reference nucleic acid sequence, based on a reference nucleic acid sequence, or homologous to a reference nucleic acid sequence. The "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% sequence identity with the nucleic acid sequence of the respective naturally occurring (wild-type) nucleic acid sequence or gene, or its homolog, fragment, or derivative.

[0090] Furthermore, the term “variant” as used herein in the context of proteins or peptides is intended to be recognized and understood by those skilled in the art and to refer to a protein or peptide variant having an amino acid sequence different 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, e.g., its specific antigenic properties, compared to the full-length natural protein. A “variant” of a protein or peptide as defined herein may include conserved amino acid substitutions compared to its natural, i.e., unmutated physiological sequence. These amino acid sequences and their coding nucleotide sequences fall under the term “variant” as defined herein. Substitutions in which amino acids of the same class are exchanged with each other are called conserved substitutions. In particular, these are amino acids having aliphatic side chains, amino acids having positively or negatively charged side chains, amino acids having aromatic groups in their side chains or amino acids, and amino acids having side chains that can enter hydrogen bridges, e.g., hydroxyl functional groups. This means, for example, that an amino acid with a polar side chain may be substituted by another amino acid with a similar polar side chain, or that an amino acid characterized by a hydrophobic side chain may be substituted by another amino acid with a similar hydrophobic side chain (e.g., serine (threonine) by threonine (serine), or leucine (isoleucine) by isoleucine (leucine)). Insertions and substitutions are possible, in particular, at sequence positions that do not cause modification to the three-dimensional structure and do not affect the binding domain. Modification to the three-dimensional structure by insertion or deletion can be easily determined, for example, using CD spectroscopy (circular dichroism spectroscopy). A "variant" of a protein or peptide may have at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% amino acid identity over an elongation of at least 10, 20, 30, 50, 75, or 100 amino acids of such a protein or peptide.Preferably, the protein variant includes a functional variant of the protein, meaning that the variant exhibits the same effect or functionality as the protein from which it is induced, or at least 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the effect or functionality.

[0091] Furthermore, in the context of nucleic acid sequences or genes, the term “fragment” refers to a continuous 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 the corresponding extension within a full-length nucleic acid sequence or gene. This term includes naturally occurring fragments as well as manipulated fragments. In the context of the present invention, a preferred fragment of a sequence consists of a continuous extension of nucleic acid corresponding to the continuous extension of the entity within the nucleic acid or gene from which the fragment is induced, 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 induced. The sequence identity expressed with respect to such a fragment preferably refers to the entire nucleic acid sequence or gene. Preferably, the “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%, most preferably at least 95%, or even more preferably 97% sequence identity with the reference nucleic acid sequence or gene from which it is derived.

[0092] In this context, protein fragments may typically contain amino acid sequences 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%, most preferably at least 95%, or even more preferably at least 97% sequence identity with the amino acid sequence of the respective naturally occurring full-length protein.

[0093] The term “identity” as used herein in the context of nucleic acid sequences or amino acid sequences is intended to be recognized and understood by those skilled in the art and to refer, for example, to the percentage of two sequences that are identical. To determine the percentage of two sequences that are identical, for example, nucleic acid sequences or amino acid (aa) sequences as defined herein, preferably aa sequences encoded by nucleic acid sequences as defined herein, or aa sequences themselves, the sequences can be aligned and then compared with each other. Thus, for example, a position in a first sequence can be compared to a corresponding position in a second sequence. If the position in the first sequence is occupied by the same residue as the position in the second sequence, the two sequences are identical at this position. Otherwise, the sequences are different 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 for 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 for further alignment. Therefore, the percentage of identical arrays is a function obtained by dividing the number of identical positions by the total number of positions that are occupied only in one array. The percentage of identical arrays can be determined using an algorithm, such as the one built into the BLAST program.

[0094] In the context of the present invention, a protein or peptide fragment may further include a sequence of a protein or peptide as defined herein, having, for example, at least 5 amino acid lengths, preferably at least 6 amino acid lengths, preferably at least 7 amino acid lengths, more preferably at least 8 amino acid lengths, even more preferably at least 9 amino acid lengths; even more preferably at least 10 amino acid lengths; even more preferably at least 11 amino acid lengths; even more preferably at least 12 amino acid lengths; even more preferably at least 13 amino acid lengths; even more preferably at least 14 amino acid lengths; even more preferably at least 15 amino acid lengths; even more preferably at least 16 amino acid lengths; even more preferably at least 17 amino acid lengths; even more preferably at least 18 amino acid lengths; even more preferably at least 19 amino acid lengths; even more preferably at least 20 amino acid lengths; even more preferably at least 25 amino acid lengths; even more preferably at least 30 amino acid lengths; even more preferably at least 35 amino acid lengths; even more preferably at least 50 amino acid lengths; and most preferably at least 100 amino acid lengths. For example, such fragments may have a length of about 6 to about 20 or more amino acids, for example preferably about 8 to about 10, for example 8, 9 or 10 amino acids (or even 6, 7, 11 or 12 amino acids), and may be fragments processed and presented by MHC class I molecules, or preferably about 13 or more amino acids, for example 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid lengths, and may be fragments processed and presented by MHC class II molecules, 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. Protein or peptide fragments may contain at least one epitope of these proteins or peptides. Furthermore, it can also be understood that protein domains, such as the extracellular domain, intracellular domain or transmembrane domain of a protein and shortened or cleaved versions of the protein constitute protein fragments.

[0095] Protein Variants: “Variants” of proteins or peptides, as defined in the context of this invention, can be made having an amino acid sequence different from the original sequence due to one or more mutations, such as substitutions, insertions, and / or deletions of amino acids. Preferably, these fragments and / or variants have the same biological function or specific activity, e.g., its specific antigenic properties, compared to the full-length natural protein. “Variants” of proteins or peptides, as defined herein, may include conserved amino acid substitutions compared to their natural, i.e., unmutated physiological sequence. These amino acid sequences and their coding nucleotide sequences fall under the term “variant” as defined herein. Substitutions in which amino acids of the same class are exchanged with each other are called conserved substitutions. In particular, these are amino acids having aliphatic side chains, amino acids having positively or negatively charged side chains, amino acids having aromatic groups in their side chains or amino acids, and amino acids having side chains whose side chains can enter hydrogen bridges, e.g., hydroxyl functional groups. This means, for example, that an amino acid with a polar side chain may be substituted by another amino acid with a similar polar side chain, or that an amino acid characterized by a hydrophobic side chain may be substituted by another amino acid with a similar hydrophobic side chain (e.g., serine (threonine) by threonine (serine), or leucine (isoleucine) by isoleucine (leucine)). Insertions and substitutions are possible, in particular, at sequence positions that do not cause modification to the three-dimensional structure and do not affect the binding domain. Modification to the three-dimensional structure by insertion or deletion can be easily determined, for example, using CD spectroscopy (circular dichroism spectrum) (Urry, 1985, Absorption, Circular Dichroism and ORD of Polypeptides, Modern Physical Methods in Biochemistry, Neuberger et al. (eds.), Elsevier, Amsterdam).

[0096] A "variant" of a protein or peptide may have at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% amino acid identity over an extension of 10, 20, 30, 50, 75, or 100 amino acids of such a protein or peptide.

[0097] Furthermore, variants of proteins or peptides as defined herein, which can be encoded by nucleic acid molecules, may also include sequences in which the nucleotides of the encoding nucleic acid sequence are exchanged by degeneracy of the genetic code without resulting in a change in the respective amino acid sequence of the protein or peptide; that is, the amino acid sequence or at least a portion of it is not different from the original sequence in one or more mutations within the sense described above.

[0098] Sequence Identity: To determine the percentage of identical sequences between two sequences, for example, nucleic acid sequences or amino acid sequences as defined herein, preferably amino acid sequences encoded by the nucleic acid sequence of a polymer carrier as defined herein, or the amino acid sequences themselves, the sequences can be aligned and then compared to each other. For example, a position in a first sequence can be compared to a corresponding position in a second sequence. If a position in the first sequence is occupied by the same component (residue) as a position in the second sequence, the two sequences are identical at that position. Otherwise, the sequences differ at that position. If an insertion occurs in the second sequence compared to the first, a gap can be inserted into the first sequence to allow for further alignment. If a deletion occurs in the second sequence compared to the first, a gap can be inserted into the second sequence to allow for further alignment. Therefore, the percentage of identical sequences is a function of the number of identical positions divided by the total number of positions that include positions occupied only in one sequence. The percentage of identical sequences can be determined using a mathematical algorithm. Preferred but not limited examples of mathematical algorithms that can be used are those of Karlin et al. (1993), PNAS USA, 90:5873-5877 or Altschul et al. (1997), Nucleic Acids Res., 25:3389-3402. Such algorithms are incorporated into the BLAST program. Sequences that are identical to the sequences of the present invention to a certain extent can be identified by this program.

[0099] Derivatives of proteins or peptides: Derivatives of peptides or proteins are typically understood to be molecules derived from another molecule, such as the aforementioned peptide or protein. “Derivatives” of peptides or proteins also include fusions containing the peptide or protein used in this invention. For example, a fusion may include a label, such as an epitope, e.g., a FLAG epitope or a V5 epitope. For example, the epitope is a FLAG epitope. Such tags are useful, for example, for purifying fusion proteins.

[0100] Pharmacologically effective dose: In the context of this invention, the pharmaceutically effective dose is typically understood to be an amount sufficient to induce an immune response. Carrier: In the context of the present invention, a carrier is typically a compound that facilitates the transport and / or complexation of another compound. The carrier may form a complex with the other compound. A polymer carrier is a carrier formed of a polymer.

[0101] Vehicle: Typically, a drug, such as a carrier, that may be used within a pharmaceutical composition or vaccine to facilitate the administration of the components of the pharmaceutical composition or vaccine to an individual. [Brief explanation of the drawing]

[0102] [Figure 1-1] (Chemical Structure of HEXA Lipids) - (A) A diagram showing the structure of the HEXA lipid compound of the present invention as described herein, i.e., lipid compound C1 HEXA-C4DE-PipSS. (B) A diagram showing the structure of the HEXA lipid compound of the present invention as described herein, i.e., lipid compound C2 HEXA-C5DE-PipSS. (Complete details can be found in Example 2.1). [Figure 1-2] (Chemical structure of HEXA lipids) - (C) A diagram showing the structure of the HEXA lipid compound of the present invention as described herein, i.e., lipid compound C3 HEXA-C6DE-PipSS. (D) A diagram showing the structure of the HEXA lipid compound of the present invention as described herein, i.e., lipid compound C4 HEXA-C7DE-PipSS. (Complete details can be found in Example 2.1). [Figure 1-3] (Chemical structure of HEXA lipids)-(E) A diagram showing the structure of the HEXA lipid compound of the present invention as described herein, namely the lipid compound C5 HEXA-C8DE-PipSS. (F) A diagram showing the structure of the HEXA lipid compound of the present invention as described herein, namely the lipid compound C6 HEXACA-C3ME-PipSS. (Complete details can be found in Example 2.1). [Figure 1-4] (Chemical structure of HEXA lipids)-(G) A diagram showing the structure of the HEXA lipid compound of the present invention as described herein, i.e., lipid compound C7 HEXACA-C4ME-PipSS. (H) A diagram showing the structure of the HEXA lipid compound of the present invention as described herein, i.e., lipid compound C8 HEXACA-C6ME-PipSS. (Complete details can be found in Example 2.1). [Figure 1-5] (Chemical structure of HEXA lipid)-(I) A diagram showing the structure of the HEXA lipid compound of the present invention as described herein, i.e., the lipid compound C9 HEXACA-C8ME-PipSS. (Complete details can be found in Example 2.1). [Figure 2-1] (Protonation Profile / pKa of HEXA Lipids) - Figure showing the measurement of the degree of protonation of the HEXA lipid compounds of the present invention via TNS (dye 2-p-toluidinylnaphthalene-6-sulfonate) fluorescence. (A) TNS fluorescence of LNP1, LNP2, LNP3 and LNP4 in Composition 1 (DSPC). (B) TNS fluorescence of LNP5, LNP6, LNP7 and GN01 in Composition 1 (DSPC). (Complete details can be found in Example 2.1.2 / Table Ex 4). [Figure 2-2](Protonation Profile / pKa of HEXA Lipids) - Figure showing the measurement of the degree of protonation of the HEXA lipid compounds of the present invention via TNS (dye 2-p-toluidinylnaphthalene-6-sulfonate) fluorescence. (C) TNS fluorescence of LNP8, LNP9, LNP10 and LNP11 in composition 21 (DPhyPE or 4ME16:0PE). (D) TNS fluorescence of LNP12, LNP13, LNP14 and GN01 in composition 2 (DPhyPE). (Complete details can be found in Example 2.1.2 / Table Example 4). [Figure 2-3] (Protonation Profile / pKa of HEXA Lipids) - Figure showing the measurement of the degree of protonation of the HEXA lipid compounds of the present invention via TNS (dye 2-p-toluidinylnaphthalene-6-sulfonate) fluorescence. (E) TNS fluorescence of LNP15 (in Composition 1-DSPC), LNP16 (in Composition 2-DPhyPE), LNP17 (in Composition 2-DSPC), LNP18 (in Composition 2-DPhyPE), and GN01. (Complete details can be found in Example 2.1.2 / Table Example 4). [Figure 3-1] (Structure of HEAD lipids)-(A) A diagram showing the structure of the HEAD lipid compound of the present invention as described herein, i.e., the lipid compound CISE. (Complete details can be found in Example 2.1 / Table Example 6). [Figure 3-2] (Structure of HEAD lipids)-(B) A diagram showing the structure of the HEAD lipid compound of the present invention as described herein, i.e., the lipid compound CPZE. (Complete details can be found in Example 2.1 / Table Example 6). [Figure 3-3] (Structure of HEAD lipid) - (C) A diagram showing the structure of the HEAD lipid compound of the present invention as described herein, i.e., the lipid compound ESTER. (Complete details can be found in Example 2.1 / Table Example 6). [Figure 4-1](Protonation Profile / pKa of HEAD Lipids) - Figure showing the measurement of the degree of protonation of the HEAD lipid compounds of the present invention via TNS (dye 2-p-toluidinylnaphthalene-6-sulfonate) fluorescence. (A) TNS fluorescence of HEAD lipid CISE in composition A (DSPC) and composition B (DPhyPE) compared with GN01. (B) TNS fluorescence of HEAD lipid CPZE in composition A (DSPC) and composition B (DPhyPE) compared with GN01. (Complete details can be found in Example 2.2.2 / Table Example 9). [Figure 4-2] (Protonation Profile / pKa of HEAD Lipids) - Figure showing the measurement of the degree of protonation of the HEAD lipid compounds of the present invention via TNS (dye 2-p-toluidinylnaphthalene-6-sulfonate) fluorescence. TNS fluorescence of HEAD lipid ESTER in composition A (DSPC) and composition B (DPhyPE) compared with GN01. (Complete details can be found in Example 2.2.2 / Table Example 9). [Figure 5-1] (PpLuc expression in HeLa and HepG2)-HEXA lipids 1-7 were formulated as LNPs using composition 1 or 2 and transfected into HeLa or HepG2 cells together with PpLuc mRNA. Relative luminescence (RLU) was measured 24 hours after transfection. (A) Figure showing transfection of LNP1-LNP7 (composition 1-DSPC) and GN01 into HeLa cells. (B) Figure showing transfection of LNP8-LNP14 (composition 2-DPhyPE) and GN01 into HeLa cells. (Complete details can be found in Example 3.1.1). [Figure 5-2](PpLuc Expression in HeLa and HepG2)-HEXA lipids 1-7 were formulated as LNPs using composition 1 or 2 and transfected with PpLuc mRNA into HeLa or HepG2 cells. Relative luminescence (RLU) was measured 24 hours after transfection. (C) Figure showing transfection of LNP1-LNP7 (composition 1-DSPC) and GN01 into HepG2 cells. (D) Figure showing transfection of LNP8-LNP14 (composition 2-DPhyPE) and GN01 into HepG2 cells. This indicates that mRNA formulated with lipids and compositions showed very good and even better PpLuc expression in HeLa and mice. (Complete details can be found in Example 3.1.1). [Figure 6] (PpLuc expression in HeLa of HEAD lipids)-DPhyPE-containing compositions also demonstrate a clear advantage over DSPC, which has been used in the art to date as a standard neutral lipid in virtually all prior art LNP compositions, as shown in Example 3.1.1 and Figure 5. HEAD lipids CISE, CPZE, and ESTER were formulated as LNPs using compositions A and B and transfected into HeLa cells with PpLuc mRNA. Relative luminescence (RLU) was measured 24 hours after transfection. (Full details can be found in Example 3.2.1). [Figure 7] (hEPO expression of HEXA lipids 1-7 in HeLa cells) - Figure showing superior in vitro hEPO expression in treated HeLa cells. HEXA lipid-DPhyPE, prepared as LNP using GN01 and composition 2, was formulated with mRNA encoding hPEO and transfected into HeLa cells. hEpo ELISA was performed 24 hours after transfection to show transfection efficiency (complete details can be found in Example 4.1.1). [Figure 8](hEPO expression of HEXA lipids 1-7 in mice) - Figure showing that in vivo analysis of the compositions of the present invention resulted in clearly high hEPO expression 6 and 24 hours after injection. Using GN01 and composition 2, HEXA lipid-DPhyPE prepared as LNPs was formulated with the mRNA encoding hEPO and injected into Balb / C mice (5 mice / group) at a dose of 0.5 mg / kg. HsEpo levels were measured in plasma 6 and 24 hours after injection using ELISA. (Full details can be found in Example 4.1.1). [Figure 9] (HEXA Lipid Tolerance - Liver Enzymes) - Figure showing that none of the tested animals showed significantly elevated AST and ALT liver enzyme activity compared to the buffer control. To analyze the tolerance of HEXA lipids, ALT and AST levels were measured 24 hours after intravenous transfection of each LNP into Balb / C mice. (A) ALT and AST levels of LNPs and GN01 containing HEXA lipids 1-9 compared to the buffer. (B) ALT and AST levels of lipid 2 m / m ratio (m / m20, m / m30, m / m40) and GN01 compared to the buffer. (Full details can be found in Example 4.1.2). [Figure 10-1] (Tolerance and Immunostimulation of HEXA Lipids) - Figure showing that none of the tested lipid compounds induced significantly elevated cytokine levels. To analyze the immunostimulatory properties of HEXA lipids, a CBA assay was performed using serum samples collected from Balb / C mice 6 hours after injection of HEXA lipid-containing LNPs and GN01 LNPs. Serum IFN-α levels were determined by ELISA. (A) MCP-1 from HEXA lipids 1-7 and GN01. (B) IL-6 from HEXA lipids 1-7 and GN01. (C) MP1-β from HEXA lipids 1-7 and GN01. (D) INF-α from HEXA lipids 1-7 and GN01. (Complete details can be found in Example 4.1.2). [Figure 10-2](Tolerance of HEXA Lipids - Immunostimulation) - Figure showing that none of the tested lipid compounds induced significantly elevated cytokine levels. To analyze the immunostimulatory properties of HEXA lipids, a CBA assay was performed using serum samples collected from Balb / C mice 6 hours after injection of HEXA lipid-containing LNPs and GN01 LNPs. Serum IFN-α levels were determined by ELISA. (E) MCP-1 in HEXA lipids 8, 9, and lipid 2 m / m ratios (m / m20, m / m30, m / m40) and GN01. (F) IL-6 in HEXA lipids 8, 9, and lipid 2 m / m ratios (m / m20, m / m30, m / m40) and GN01. (G) MP1-β in HEXA lipids 8, 9, and lipid 2 m / m ratios (m / m20, m / m30, m / m40) and GN01. (H)HEXA lipid 8, 9, and lipid 2 in m / m ratios (m / m20, m / m30, m / m40) and INF-α of GN01. (Complete details can be found in Example 4.1.2). [Figure 11] (LNP-tumor antigen Trp2 id injection for prophylactic and therapeutic vaccine approaches) - Vaccination with GN01, GN02, and CISE LNPs containing trp2 mRNA (full details can be seen in Example 4.2.1) showed stable IgG1 and IgG2 titers (Figures 11A and 11B), as well as low T cell response with GN01, GN02, and CISE LNPs (Figures 11C and 11D). Specifically, C57 / BL6 mice were intradermally (id) injected into the back with formulated mRNA encoding the tumor antigen Trp2 and GN01, GN02, or CISE. Immunization was performed on days 0, 7, and 14. Blood samples were collected at 14 hours, and blood and organ samples were collected 21 days after the first vaccination. T cell response and humoral immune response were measured using ELISA. (A) Figure showing IgG1 endpoint titer. (B) Figure showing IgG2a[b] endpoint titer. (C) Diagram showing %TNFα+ / IFNγ+ in CD4+ cells. (D) Diagram showing %TNFα+ / IFNγ+ in CD8+ cells. (Complete details can be found in Example 4.2.1). [Figure 12.1](HEAD Lipid Tolerance - Liver Enzymes) - Figure showing that none of the tested animals showed significantly elevated AST and ALT liver enzyme activity compared to the buffer control. To analyze the tolerance of HEAD lipids, ALT and AST levels were measured 24 hours after intravenous transfection into Balb / C mice. ALT and AST levels for HAED lipid ESTER (m / m40) and CPZE (m / m20) were measured and compared to the levels of CISE / Lipid 2 (m / m30), Lipid 2 (m / m30), GN01 and buffer. Different molar / mass (m / m) ratios (m / m20, m / m30, m / m40) were used (full details can be found in Example 4.2.2). [Figure 12.2] (HEAD Lipid Tolerance - Immunostimulation) - Figure showing that none of the tested lipid compounds induced significantly elevated cytokine levels. To analyze the immunostimulatory properties of HEAD lipids (CPZE, ESTER) compared to HEXA lipids (Lipid 2), GN01, and a mixture of both (CISE / Lipid 2), a CBA assay was performed using serum samples collected from Balb / C mice 6 hours after injection of HEXA lipids into the mice. Serum IFN-α levels were determined by ELISA. (A) Figure showing MCP-1. (B) Figure showing IL-6. (C) Figure showing MP1-β. (D) Figure showing IFN-α. (Complete details can be found in Example 4.2.2). [Figure 13](Stability of the LNP of the present invention after long-term storage) Figure showing analysis of the integrity and biophysical properties of GN01 LNPs stored for long periods at -4°C and -80°C. As is clear, the particles and RNA were stable and no significant changes were observed - i.e., changes in the biophysical properties and mRNA integrity of the LNPs may be shown on the agarose gel after gel electrophoresis. GN01 LNPs were formulated with hEPO mRNA and stored at 4°C and -80°C for 1.5 months or 6 months. The LNPs were destroyed for analysis by gel electrophoresis so that the incorporated mRNA could be shown on the gel. (A) After 1.5 months - Figure showing degradation condition 1 (LNPs were degraded using a combination of heparin and Triton; C1 in the figure) and degradation condition 2 (LNPs were degraded using a combination of heparin and Pluronic® and heating to 45°C for 15 minutes; C2 in the figure). (B) After 6 months - Figure showing degradation conditions 1 (LNPs degraded using a combination of heparin and Triton; C1 in the figure) and degradation condition 2 (LNPs degraded using a combination of heparin and Pluronic® and heating to 45°C for 15 minutes; C2 in the figure). (Complete details can be found in Example 5.1). [Figure 14.1] (Biological activity of GN01 after 10 weeks of storage at -80°C) - Figure showing analysis of formulated GN01 mRNA after 10 weeks of storage compared to 1 week of storage (resulting in even higher expression efficiency 6 and 24 hours after injection). In other words, the biological activity of formulated GN01 was evaluated by ELISA. GN01 LNP was formulated with hEPO mRNA and frozen for 1 week and 10 weeks, respectively. It was intravenously injected into Balb / C mice (5 mice / group). Plasma samples were collected and analyzed 6 and 24 hours after injection (complete details can be found in Example 5.2). [Figure 14.2](Biological activity of GN01 LNP after different F / T cycles) - Plasma samples were analyzed by comparing them after one freeze / thaw cycle (1F / T) followed by two F / T cycles (both after being stored at -80°C for one week). - The results showed the biological activity of HsEpo for all test approaches, as shown in the figure for the second evaluation. Specifically, the biological activity of formulated GN01 was evaluated by ELISA. GN01 LNP was formulated with hEPO mRNA, frozen for one week, and intravenously injected into Balb / C mice (5 mice / group). (A) Figure showing analysis of Elisa plasma levels after one F / T cycle. (B) Figure showing analysis of Elisa plasma levels after two F / T cycles. (Complete details can be found in Example 5.2). [Figure 15.1] (Variations in phospholipid components and their effect on the composition of the present invention) - Figure showing that the incorporation of DPhyPE resulted in higher expression compared to standard neutral lipid DSPC - i.e., LNPs were prepared using different phospholipids (DPhyPE, DSPC, DPhyPE+DSPC(1+1)). (A) Lipid 1 (lipid compound C1) and GN01 LNPs were formulated into PpLuc mRNA and transfected into HeLa cells. Transfection efficiency was analyzed 24 hours after transfection by measuring RLU intensity. (B) Measurement of protonation degree of GN01 and lipid compound C1 containing LNPs with different phospholipids via TNS (dye 2-p-toluidinylnaphthalene-6-sulfonate) fluorescence. Dotted line = GN01; dashed line = DPhyPE; normal line = DSPC, stepped line to the right of the dashed line = DSPC / DPhyPE (complete details can be seen in Example 6). [Figure 15.2](Variations in PEG component and their effect on the composition of the present invention) - Figure showing that a composition containing a polymer conjugate lipid with a shorter alkyl chain (C8 tail, C8-ceramide-PEG, shown in the figure as Cer8) was more efficient than a polymer conjugate lipid with a longer alkyl chain (C14=C14 DMG-PEG=1,2-dimiristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 (DMG-PEG2000)). Expression of PpLuc in HepG2 cells - Filled bars show results after 1 hour, and empty bars show results after 4 hours (full details can be seen in Example 7). [Figure 16] (Anti-rabies mAb expression in GN01 after single IV injection) - Figure showing the expression of formulated anti-rabies monoclonal antibody in GN01 LNPs after a single IV injection. As is clear, very potent anti-rabies mAb expression was detected 6 hours and 24 hours after IV injection (full details can be seen in Figure 8). [Figure 17-1] (Immunogenicity of GN01 and GN02: In vivo T cell response - VNT analysis) - (A) Figure showing that single-dose im-immunization with 5 μg of GN01- and GN02-LNP-formulated RABV-G mRNA had already induced very robust VNT well above a protective titer of 0.5 IU / ml in all animals at 35 days post-prime vaccination. (B) Figure showing that the GN01 and GN02-LNP-formulated RABV-G mRNA vaccines of the present invention induced a specific cellular response after vaccination - an effect not observed in control LNP-vaccinated animals. RABV-G specific CD4+ T cells were observed for both mRNA formulations GN01 and GN2. (Full details can be seen in Example 9). [Figure 17-2](Immunogenicity of GN01 and GN02: In vivo T cell response - VNT analysis) (C) Figure showing that the GN01 and GN02-LNP-formulated RABV-G mRNA vaccines of the present invention induced specific cellular responses after vaccination - effects not observed in control LNP-vaccinated animals. RABV-G specific CD8+T was also observed for both mRNA formulations GN01 and GN2. (Full details can be seen in Example 9). [Figure 18] Figures illustrating the high reactogenicity of GN01-formulated RNA for a monotope vaccine approach. (A) Vaccination with a monotope construct containing PADRE via GN01 LNP increased the number of splenocytes. (B) The monotope construct, combined with GN01 formulation and intradermal application, resulted in a very potent CD8 T cell response. (C) The monotope construct, combined with GN01 formulation and intradermal application, resulted in a very potent CD8 T cell response. (D) The monotope construct, combined with GN01 formulation and intradermal application, resulted in a very potent CD8 T cell response. (Full details can be found in Example 10). [Figure 19] A single im-immunization with 10 μg of GN01 LNP-formulated HA-mRNA (GN01-H3N2) for influenza vaccination induced a protective HI titer well above 40 in all animals 21 days after prime vaccination, and the figure shows that the boost with 10 μg of GN01 LNP-formulated HA-mRNA induced a multiple increase in the humoral immune response (full details can be seen in Example 11). [Figure 20](GN01 for rabies tested in a calf animal model)-Figure showing that intramuscular vaccination of calves with GN01-formulated RABV-G coded mRNA already resulted in very potent induction of neutralizing antibodies 14 days after prime vaccination with a dose of only 30 μg of mRNA (0.5 IU / ml WHO standard shown as a dashed line; empty bars = Rabisin control, filled bars = GN01-formulated mRNA) (full details can be seen in Example 12). [Figure 21.1] Figure showing that a GN01-formulated mRNA malaria vaccine encoding CSP (GN01-endpoint titer for in vivo malaria vaccination) induced a very potent humoral immune response in mice using an ELISA assay. (A) Coated: [NANP]7 peptide, IgG1 and IgG2a endpoint titers at 21 and 35 days post-priming. (B) Coated C-terminal peptide, IgG1 and IgG2a endpoint titers at 21 and 35 days post-priming. Group 1: GN01-LNP containing CSP vaccine; Group 2: GN01-LNP containing unrelated mRNA. (Full details can be seen in Example 13). [Figure 21.2] Figure showing that a GN01-formulated mRNA malaria vaccine encoding (GN01-ICS for in vivo malaria vaccination)-CSP induced a cellular immune response (CD8+ and / or CD4+ T cell response) in mice using an intracellular cytokine staining assay (35 days post-vaccination). Group 1: GN01-LNP containing the CSP vaccine; Group 2: GN01-LNP containing unrelated mRNA (full details can be seen in Example 13). [Figure 22] (GN01 for in vivo expression of FGF21) - Figure showing that FGF21 mRNA formulated in the LNP of the present invention resulted in high FGF21 concentrations after administration of a low dose of 0.25 mg / kg to mice via IV injection (full details can be seen in Example 14). [Figure 23](GN01 for in vivo expression of FGF21) - Figure showing that FGF21 mRNA formulated in the LNP of the present invention resulted in very high FGF21 concentrations after administration of a high dose of 1 mg / kg to mice via IV injection (full details can be seen in Example 14). [Figure 24] (Chemical Structure of HEXA Lipids) - (A) A diagram showing the structure of the HEXA lipid compound of the present invention as described herein, i.e., lipid compound C24 HEXA-C5DE-reverse PipSS. (B) A diagram showing the structure of the HEXA lipid compound of the present invention as described herein, i.e., lipid compound C25 HEXA-C5DE-Pip-C3 thioether. (Complete details can be found in Example 20.1). [Figure 25] (Structure of HEAD lipids) - (A) A diagram showing the structure of the HEAD lipid compound of the present invention as described herein (complete details can be seen in Example 20.2), namely the lipid compound THIOETHER. (B) A diagram showing the structure of the HEAD lipid compound of the present invention as described herein (complete details can be seen in Example 20.2), namely the lipid compound C3SS. [Figure 26] (Immunogenicity of C2-containing LNP in in vivo T cell response - VNT analysis) - Figure showing that single-dose im-immunization with 5 μg of LNP (C2-containing) formulated RABV-G mRNA had already induced very robust VNT well above a 0.5 IU / ml protective titer in all animals 21 days after prime vaccination. Full details can be found in Example 21. [Figure 27A] (Immunogenicity-VNT analysis of LNPs containing different cationic lipids in in vivo T cell response) - Figure showing that single im-immunization with 5 μg of LNP-formulated RABV-G mRNA had already induced very robust VNTs well above a 0.5 IU / ml protective titer in all animals 21 days after prime vaccination. The LNPs were formulated using different cationic lipids according to the present invention as shown in Example 21. [Figure 27B](Immunogenicity of LNPs containing different cationic lipids in in vivo T cell response - VNT analysis) - Figure showing that the LNP-formulated RABV-G mRNA vaccine of the present invention induced a specific cellular response in restimulated splenocytes compared to unstimulated splenocytes after vaccination. RABV-G specific CD4+ T cells are shown in (i) restimulated and (ii) unstimulated settings. [Figure 27C] (Immunogenicity of LNPs containing different cationic lipids in in vivo T cell response - VNT analysis) - Figure showing that the LNP-formulated RABV-G mRNA vaccine of the present invention induced a specific cellular response in restimulated splenocytes compared to unstimulated splenocytes after vaccination. RABV-G specific CD8+ T cells are shown in (i) restimulated and (ii) unstimulated settings. Complete details can be found in Example 21. [Figure 28] (Immunogenicity of LNP containing THIOETHER in in vivo T cell response - VNT analysis) - Figure showing that single-dose im-immunization with 1 μg of LNP (containing THIOETHER) formulated RABV-G mRNA had already induced very robust VNT well above a protective titer of 0.5 IU / ml in all animals 21 days after prime vaccination. Full details can be found in Example 21. [Figure 29] This figure shows that a GN02-like formulated mRNA malaria vaccine encoding (GN02-like LNP-ICS)-CSP for in vivo malaria vaccination induced a cellular immune response (CD4+ T cell response) in mice using an intracellular cytokine staining assay (35 days post-vaccination). RABV-G specific CD4+ T cells are shown in (i) restorative and (ii) unstimulated settings. Full details can be found in Example 22. [Figure 30]This figure shows that a GN02-like formulated mRNA malaria vaccine encoding (GN02-like LNP-ICS)-CSP for in vivo malaria vaccination induced a cellular immune response (CD8+ T cell response) in mice using an intracellular cytokine staining assay (35 days post-vaccination). RABV-G specific CD8+ T cells are shown in (i) restorative and (ii) unstimulated settings. Full details can be found in Example 22. [Figure 31] A figure showing that a GN02-like formulated mRNA malaria vaccine encoding a GN02-like LNP-IgG total titer-CSP for in vivo malaria vaccination induced a very potent humoral immune response in mice using an ELISA assay (coating: [NANP]7 peptide, IgG total endpoint titer at 35 days post-priming) (full details can be seen in Example 22). [Figure 32] Figure showing that an LNP-formulated mRNA malaria vaccine encoding a different cationic lipid of the present invention for in vivo malaria vaccination (LNP-ICS)-CSP induced a cellular immune response (CD4+ T cell response shown for (i) restorative and (ii) non-stimulatory settings) in mice using an intracellular cytokine staining assay (35 days post-vaccination). Full details can be found in Example 22. [Figure 33] Figure showing that an LNP-formulated mRNA malaria vaccine encoding a different cationic lipid-containing LNP-ICS (for in vivo malaria vaccination)-CSP of the present invention induced a cellular immune response (CD8+ T cell response shown for (i) restorative and (ii) non-stimulatory settings) in mice using an intracellular cytokine staining assay (35 days post-vaccination). Full details can be found in Example 22. [Figure 34]A figure showing that the LNP-formulated mRNA malaria vaccine encoding the LNP-IgG total titer (LNP-IgG total titer)-CSP containing different cationic lipids of the present invention for in vivo malaria vaccination induced a very potent humoral immune response in mice using an ELISA assay (coating: [NANP]7 peptide, IgG total endpoint titer at 35 days post-priming) (full details can be seen in Example 22). [Figure 35] A figure showing that the LNP-formulated mRNA malaria vaccine encoding (LNP-IgG1 titer containing the C26 lipid of the present invention for in vivo malaria vaccination)-CSP induced a very potent humoral immune response in mice using an ELISA assay (coating: [NANP]7 peptide, IgG1 endpoint titer at 35 days post-priming) (full details can be seen in Example 22). [Modes for carrying out the invention]

[0103] This invention is based on the inventors' remarkable discovery that the use of novel cationic lipids and / or lipid nanoparticles (LNPs) is highly effective in delivering nucleic acids, such as mRNA, to living organisms, such as human individuals. In particular, intracellular delivery of such nucleic acids is enhanced. This has made it possible for the inventors to produce improved vaccines that deliver, for example, mRNA compounds encoding antigenic peptides or proteins, and induce antigen-specific immune responses very efficiently at very low doses. A further advantage achieved by this invention is, quite remarkably, that the inventors have discovered a class of formulations for delivering mRNA vaccines in vivo that, according to aspects and embodiments of this invention, result in a significantly enhanced, and in many ways synergistic, immune response, including functional antibody production with enhanced antigen-generating and neutralizing capabilities. These results can be achieved even when administering significantly lower doses of mRNA compared to the mRNA doses used in other classes of lipid-based formulations. The formulations of this invention have demonstrated significant and unpredictable in vivo immune responses sufficient to establish the efficacy of functional mRNA vaccines as prophylactic and therapeutic agents. Typically, self-replicating RNA vaccines rely on the viral replication pathway to deliver enough RNA to cells to produce an immunogenic response. The formulation of the present invention does not require viral replication to produce enough protein to produce a potent immune response. Therefore, preferably, the mRNA of the present invention is not self-replicating RNA and does not contain components necessary for viral replication.

[0104] lipid composition In a first embodiment, the present invention is directed toward compositions comprising cationic lipids as described herein below. All options and preferences disclosed for cationic lipids themselves are also applicable to compositions in this aspect of the present invention. In other words, the specifically disclosed embodiments of cationic lipids, and particularly preferred cationic lipids, should be understood to also define specific preferred embodiments of compositions according to the present invention, i.e., compositions characterized by comprising a cationic lipid according to one of the specific selections described herein. The compositions may comprise further active and / or inactive excipients as described below. In one specific embodiment, in addition to cationic lipids, the composition comprises one or more lipids selected from the group consisting of (a) steroids; (b) neutral lipids; and (c) polymer conjugate lipids, preferably pegylated lipids.

[0105] Cationic lipids Cationic lipids are preferably cationizable, i.e., the pH is the pK of the ionizable group of the lipid. a As the pH decreases further, it becomes protonated, but at higher pH values ​​it gradually becomes more neutral. When positively charged, lipids can associate with negatively charged nucleic acids. In certain embodiments, cationic lipids include zwitterionic lipids that become positively charged as the pH decreases.

[0106] In one embodiment, the present invention relates to a compound according to formula (I): R a -AR b Equation (I) (In the formula, R a teeth,

[0107] [ka]

[0108] -R 1 -N(H)-C(O)-R 3 -R 4 Selected from; R b teeth,

[0109] [ka]

[0110] -R 1 -N(H)-C(O)-R 3 -R 4 ,or -R 1 -N(CH3)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 These are optionally substituted ethanediyl, propanediyl, butanediyl, or linear or unbranched alkanediyl having 2 to 8 carbon atoms; R 2 It is an alkanediyl having 2 to 8 carbon atoms; R 3 This is optional, and if present, select -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 It is a lipophilic substituent having 12 to 36 carbon atoms; R 5 It is an alkanediyl having 1 to 6 carbon atoms; X is a carbon or nitrogen atom; All choices are independent of each other. Depending on the case, R 1 , R 2 and R 5 All of them are linear unsubstituted ethanediyls, A is SS-, and R a and R b If they are the same, R 4 teeth,

[0111] [ka]

[0112] isn't it) This invention provides a novel cationic lipid defined as such. In another embodiment, the present invention relates to a novel cationic lipid useful for the delivery of nucleic acids to living cells. The cationic lipid is a compound according to formula (I): R a -AR b Equation (I) (In the formula, R a teeth,

[0113] [ka]

[0114] -R 1 -N(H)-C(O)-R 3 -R 4 Selected from; R b teeth,

[0115] [ka]

[0116] -R 1 -N(H)-C(O)-R 3 -R 4 ,or -R 1 -N(CH3)2 Selected from; A is -S-, -SS-, -SC(O)-, -NH-C(O)-, -NH-C(O)O-, -NH-C(O)-NH-, -SC(O)-N(H)-, -C(O)O-, or -OP(O)(OH)-O-; R 1is optionally substituted ethanediyl, propanediyl, butanediyl, or a straight-chain or unbranched alkanediyl having 2 to 8 carbon atoms; R 2 is an alkanediyl having 2 to 8 carbon atoms; R 3 is optional, and when present, -R 5 -C(O)-O-, or -R 5 -O-C(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 or nitrogen atom; All selections are independent of each other, and optionally, R 1 , R 2 and R 5 are all ethanediyl, A is S-S-, and R a and R b are the same, then R 4 is,

[0117]

Chemical formula

[0118] is not) is. In yet another aspect, in aspect A, the present invention provides a compound according to formula (I): R a -A-R b Formula (I) or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof (wherein, R a is,

[0119]

Chemical formula

[0120] or -R 1 -N(H)-C(O)-R 3 -R 4 Selected from; R b teeth,

[0121] [ka]

[0122] -R 1 -N(H)-C(O)-R 3 -R 4 , or -R 1 -N(CH3)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 These are ethanediyl, propanediyl, butanediyl, or linear or unbranched alkanediyl having 2 to 8 carbon atoms, where each substituted carbon atom is either unsubstituted or substituted with one or more C1-C4 alkyl, C1-C4 alkenylene, C3-C8 cycloalkylene, or C3-C8 cycloalkenylene; R 2 It is an alkanediyl having 2 to 8 carbon atoms; R 3 This is optional, and if present, select -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 4A lipophilic substituent having 12 to 36 carbon atoms is either a linear or branched alkyl or alkenyl having 12 to 25 carbon atoms, or is derived from alpha-tocopherol; R 5 It is an alkanediyl having 1 to 6 carbon atoms; X is a carbon atom (CH) or nitrogen atom bonded to a hydrogen atom; All choices are independent of one another; Depending on the case, (i)R 3 ga-R 5 (ii)R 1 and R 2 (iii)R 5 (iv)A is -SS-, and (v)R a and R b If they are the same, R 4 teeth,

[0123] [ka]

[0124] Instead, Furthermore, (i)R 3 (ii)R is nonexistent. 1 and R 2 (iii) A is a linear unsubstituted ethanediyl, and (iv) R a and R b If they are the same, R 4 teeth,

[0125] [ka]

[0126] but

[0127] [ka]

[0128] But not; Alternatively, as an alternative to the above proviso, cationic lipids may, in some cases,

[0129] [ka]

[0130] (Not a lipid selected from the group consisting of) This invention provides a novel cationic lipid defined as such. In novel cationic lipids, the degradable / biodegradable moiety A may consist of two structures R which may be the same or different. a and R b Connect R a and R b Each of these contains at least one basic, i.e., cationic, tertiary nitrogen atom-containing moiety. a and R b At least one of them has a substantially lipophilic tail structure and at least one ester group.

[0131] The degradable / biodegradable moiety A may be selected from the following functional groups: -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-. In one preferred embodiment, A is a moiety or group containing one or more sulfur atoms, such as -S-, -SS-, or -SC(O)-N(H)-. In another preferred embodiment, A is a disulfide group (-SS-), and the cationic lipid is R a -SSR b (In the formula, R a and R b (which can be selected as defined above) can be represented as. In particular, in yet another preferred embodiment of the above embodiment A, A is -S- and the cationic lipid is R a -SR b(In the formula, R a and R b (which can be selected as defined above) can be represented as

[0132] While we do not wish to be constrained by theory, we currently believe that the degradability of portion A may play a significant role in the apparent biological efficacy of novel lipids. For example, if A is a disulfide moiety, and the lipid is used with other excipients, as described in more detail below, to form liposomes or lipid nanoparticles (LNPs) carrying nucleic acid compounds as cargo, such liposomes or LNPs would be effectively taken up by cells via endocytosis. Within the endocytic vesicles, the disulfide group of the cationic lipid is reduced to two thiol moieties (possibly in the presence of glutathione), simultaneously resulting in the cleavage of the cationic lipid molecule into two smaller cationic species. Potentially, high concentrations of thiols in the cell may also lead to further degradation of the lipid through thioesterification, etc.

[0133] R a and R b These may differ from each other depending on the circumstances, and can therefore be chosen independently. As mentioned, R a teeth,

[0134] [ka]

[0135] (Preferably, X is CH), or -R 1 -N(H)-C(O)-R 3 -R 4 Selectable from; R b teeth,

[0136] [ka]

[0137] (Preferably, X is CH), -R1 -N(H)-C(O)-R 3 -R 4 , or -R 1 -N(CH3)2 may be selected. Furthermore, as mentioned, R a teeth,

[0138] [ka]

[0139] (Preferably, X is CH) R can be selected from b teeth,

[0140] [ka]

[0141] (Preferably, X is CH) It can be selected from the following. In one preferred embodiment, R a and R b At least one of them is R 2 and A or R 1 It includes a piperidine- or piperazine-derived 6-membered ring structure between A and B. This includes at least one tertiary nitrogen atom adjacent to part A and at least one spacer (R 2 ) Lipophilic tail structure R by any ester group 4 It means to exist and be located away from the ester group (or R). 3 The potential advantages of multiple ester groups (if present) relate to the further enhanced degradation of lipids in physiological environments, such as intracellular environments, provided by hydrolyzable ester bonds.

[0142] In a further embodiment, R a and R b Both contain a piperidine- or piperazine-derived 6-membered cyclic structure. a and R bBoth are selected independently, or R a and R b They are identical,

[0143] [ka]

[0144] (Preferably, X is CH) Cationic lipids are also preferred. As mentioned, R 1 R is optionally substituted ethanediyl, propanediyl, butanediyl, or a linear or unbranched alkanediyl having 2 to 8 carbon atoms. Propanediyl is preferably n-propanediyl, i.e., -CH2-CH2-CH2- (wherein one or more hydrogen atoms are optionally substituted). Butanediyl is preferably n-butanediyl, i.e., -CH2-CH2-CH2-CH2- (wherein one or more hydrogen atoms are optionally substituted). However, preferably, one or fewer hydrogen atoms of ethanediyl, propanediyl, or butanediyl are substituted. In some embodiments, R a R inside 1 substituents and R b R inside 1 The substituents are either the same or different. In some embodiments, R a R inside 1 substituents and R b R inside 1 Both substituents are ethanediyl. In other embodiments, R a R inside 1 substituents and R b R inside 1 Both substituents are propanediyl. In other embodiments, R a R inside 1 substituents and R b R inside 1 Both substituents are butanediyl. In some embodiments, R a R inside 1 The substituent is ethanediyl, R b R inside1 The substituent is propanediyl. In other embodiments, R a R inside 1 The substituent is propanediyl, R b R inside 1 The substituent is ethanediyl. In some embodiments, R a R inside 1 The substituent is butanediyl, R b R inside 1 The substituent is propanediyl. In other embodiments, R a R inside 1 The substituent is butanediyl, R b R inside 1 The substituent is ethanediyl. In certain other embodiments, in particular R 1 In this regard, the term "optionally substituted" indicates that each substituted carbon atom may be independently substituted with one or more C1-C4 alkyl, C1-C4 alkenylene, C3-C8 cycloalkylene, or C3-C8 cycloalkenylene.

[0145] Similarly, in some embodiments, R a R inside 2 substituents and R b R inside 2 The substituents are either the same or different. In some embodiments, R a R inside 2 substituents and R b R inside 2 The substituents are both ethanediyl, propanediyl, butanediyl, pentanediyl, hexanediyl, heptanediyl, or octanediyl. In other embodiments, R a R inside 2 The substituent is propanediyl, R b R inside 2 The substituent is heptanediyl. In other embodiments, R a R inside 2 The substituent is heptanediyl, R b R inside 2 The substituent is propanediyl. In particular, in some embodiments of the above embodiment A, R a R inside2 substituents and R b R inside 2 Both substituents are ethanediyl.

[0146] Furthermore, R 3 R a and R b In some embodiments present in both, R a R inside 5 substituents and R b R inside 5 The substituents are either the same or different. In some embodiments, R a R inside 5 substituents and R b R inside 5 The substituents are all methanediyl, ethanediyl, propanediyl, butanediyl, pentanediyl, or hexanediyl. In other embodiments, R a R inside 5 The substituent is ethanediyl, R b R inside 5 The substituent is hexanediyl. In other embodiments, R a R inside 5 The substituent is hexanediyl, R b R inside 5 The substituent is ethanediyl. 3 R a and R b In both, and in particular in some embodiments of the above embodiment A, R a R inside 5 substituents and R b R inside 5 Both substituents are ethanediyl.

[0147] The substituents may optionally include further functional groups such as ester or amide groups, i.e., any linear or branched alkyl, aryl, heteroalkyl, or heteroaromatic structure.

[0148] In particular, -R 1 -N(H)-C(O)-R 3 -R 4 R a and / or Rb If selected, R 1 It is preferable that the -R is a substituted ethanediyl, propanediyl, butanediyl, or a linear or unbranched alkanediyl having 2 to 8 carbon atoms, such as a substituted propanediyl. 1 -N(H)-C(O)-R 3 -R 4 When R is used, 1 The substituents preferably include such amino groups; optionally, such amino groups may be part of a cyclic structure, such as a six-membered ring structure derived from piperidine or piperazine. Optionally, the cyclic structure, characterized by a cationic nitrogen atom, is linked to an ethanediyl or propanediyl via a decomposable group, such as an ester group.

[0149] R a teeth

[0150] [ka]

[0151] (Preferably, X is CH) is, and / or R b teeth

[0152] [ka]

[0153] (Preferably, X is CH) In one embodiment, R 2 It acts as a linker or spacer between the respective basic piperidine- or piperazine derivative ring structure and the ester group. As mentioned, R 2 It is defined as an alkanediyl having 2 to 8 carbon atoms. 2 R can be linear or branched, and otherwise (i.e., except branched), preferably unsubstituted. In one embodiment, R 2R is a linear unsubstituted alkanediyl having 2, 3, 4, 5, 6, 7, or 8 carbon atoms. In another embodiment, R 2 R is a linear unsubstituted alkanediyl having 2 to 6 carbon atoms. In a further preferred embodiment, R 2 R is a linear unsubstituted ethanediyl or propanediyl. For example, R a and R b Both are

[0154] [ka]

[0155] (Preferably, X is CH and R 2 (This is selected from straight-chain unsubstituted alkanediyls having 2 to 6 carbon atoms, such as ethanediyl or propanediyl.) It is possible.

[0156] R a teeth

[0157] [ka]

[0158] (Preferably, X is CH) is, and / or R b teeth

[0159] [ka]

[0160] (Preferably, X is CH) In a further embodiment, R 2 It acts as a linker or spacer between the respective basic piperidine- or piperazine derivative ring structure and the ester group. As mentioned, R 2 It is defined as an alkanediyl having 2 to 8 carbon atoms. 2R can be linear or branched, and otherwise (i.e., except branched), preferably unsubstituted. In one embodiment, R 2 R is a linear unsubstituted alkanediyl having 2, 3, 4, 5, 6, 7, or 8 carbon atoms. In another embodiment, R 2 R is a linear unsubstituted alkanediyl having 2 to 6 carbon atoms. In a further preferred embodiment, R 2 R is a linear unsubstituted ethanediyl or propanediyl. For example, R a and R b Both are

[0161] [ka]

[0162] (Preferably, X is CH and R 2 (This is selected from straight-chain unsubstituted alkanediyls having 2 to 6 carbon atoms, such as ethanediyl or propanediyl.) It is possible.

[0163] Arbitrary Selection Structure R 3 It contains an ester group that can further enhance the degradation of cationic lipids into lower molecular weight species under physiological, e.g., intracellular conditions. As defined above, R 3 If present, in particular, -R 5 -C(O)-O- or -R 5 -OC(O)-(wherein, R 5 It is defined as a spacer consisting of an alkanediyl having 1 to 6 carbon atoms. In other words, the ester group can have any orientation. Preferably, R 5 R is an unsubstituted linear alkanediyl having 1, 2, 3, 4, 5, or 6 carbon atoms. In another preferred embodiment, R 3 There exists, R 5 It is an unsubstituted linear alkanediyl having two or three carbon atoms, or three to six carbon atoms.

[0164] R 4R is defined as a lipophilic substituent having 12 to 36 carbon atoms. a And in some cases, also R b (R b ga-R 1 This "tail" end (unless it is N(CH3)2) is thought to provide the lipophilicity typically required for molecules to cross biological membranes. Therefore, R 4 In principle, R can be any structure that is substantially lipophilic. For example, hydrocarbon structures are lipophilic. In one embodiment, R 4 In at least one of its appearances, it may consist only of carbon and hydrogen atoms. In one preferred embodiment, R 4 R preferably represents a linear or branched alkyl or alkenyl having 12 to 25 carbon atoms. The branched alkyl or alkenyl may have multiple side chains, such as two, three, four or more methyl side chains. In another embodiment, R 4 This can be an alkyl or alkenyl containing a single alkyl or alkenyl side chain having, for example, 2 to 10 carbon atoms. For example, R 4 This can be 1-n-hexyl-n-nonyl (or 7-n-pentadecyl) or 2-n-hexyl-n-decyl. In other embodiments, the lipophilic substituent may optionally comprise one or more heteroatoms such as O, S, or N. In other embodiments, the lipophilic substituent may optionally comprise one or more saturated, unsaturated, or aromatic ring structures, which may optionally comprise one or more heteroatoms such as O, S, or N.

[0165] R 4 Furthermore, it may contain a small number of heteroatoms, such as oxygen atoms, as long as its primarily lipophilic properties are maintained. In one embodiment, R 4 It contains one or more oxygen atoms and does not contain any other heteroatoms. 4 It may also include cyclic structures, such as aromatic or aliphatic ring structures, which may contain one or more oxygen atoms. If present, heteroatoms and / or cyclic structures are directed towards the ends of the "tail" rather than towards the R of choice.3 It is preferable that it be positioned toward the structure. In one embodiment, R 4 is a lipophilic group derived from tocopherol or tocotrienol. In one embodiment, particularly R 1 , R 2 and R 5 Not all of them are straight-chain unsubstituted ethanediyl molecules, and A is -SS-, R a and R b If they are the same, R 4 This is a lipophilic group derived from alpha-tocopherol, particularly

[0166] [ka]

[0167] That is the case. As used herein, “lipophilic groups derived from tocopherol or tocotrienol” include derivatives of tocopherol and tocotrienol, particularly derivatives having the structures shown in Scheme 1 below, namely derivatives derived from alpha-tocopherol, beta-tocopherol, gamma-tocopherol, delta-tocopherol, alpha-tocotrienol, beta-tocotrienol, gamma-tocotrienol, and delta-tocotrienol.

[0168] [ka]

[0169] [Table 1]

[0170] In particular, in the preferred embodiment of the above-described aspect A, R 4The group is either a linear or branched alkyl or alkenyl having 12 to 25 carbon atoms, or a lipophilic group selected from the group consisting of alpha-tocopherol, beta-tocopherol, gamma-tocopherol, delta-tocopherol, alpha-tocotrienol, beta-tocotrienol, gamma-tocotrienol, and delta-tocotrienol derivatives as shown herein in Scheme 1.

[0171] In particular, in yet another preferred embodiment of the above embodiment A, R 4 is a linear or branched alkyl or alkenyl having 12 to 25 carbon atoms, or

[0172] [ka]

[0173] That is the case. In particular, in yet another preferred embodiment of the above embodiment A, R 4 teeth

[0174] [ka]

[0175] It is selected from the group consisting of the following. As mentioned, X is a carbon or nitrogen atom independently selected in each occurrence. In one embodiment, X is a carbon atom. In another embodiment, R a and R b Both are structures containing X, preferably where X is a carbon atom in each occurrence. Alternatively, X is a nitrogen atom; for example, R a and R bBoth are structures containing X, and in each appearance, the nitrogen atom is selected for X. Whenever X is referred to as a carbon atom in this specification, this is understood to mean that the carbon atom is bonded to a hydrogen atom, i.e., CH. In some examples in this specification, X has already been referred to as CH.

[0176] According to a further specific embodiment, R a teeth

[0177] [ka]

[0178] (Preferably, X is CH) or -R 1 -N(H)-C(O)-R 3 -R 4 Selected from; R b teeth

[0179] [ka]

[0180] (Preferably, X is CH) -R 1 -N(H)-C(O)-R 3 -R 4 , or -R 1 -N(CH3)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 These are optionally substituted ethanediyl, propanediyl, butanediyl, or linear or unbranched alkanediyl having 2 to 8 carbon atoms; R 2 It is an alkanediyl having 2 to 8 carbon atoms; R 3 If it is optional and exists, then -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 It is an alkanediyl having 3 to 6 carbon atoms; X is a carbon or nitrogen atom; All choices are independent of each other. A cationic lipid having formula (I) is provided. In some cases, R 2 and / or R 5 Alkanediyls represented by this symbol are linear and unsubstituted.

[0181] According to a further specific embodiment, R a teeth

[0182] [ka]

[0183] (Preferably, X is CH) or -R 1 -N(H)-C(O)-R 3 -R 4 Selected from; R b teeth

[0184] [ka]

[0185] (Preferably, X is CH) -R 1 -N(H)-C(O)-R 3 -R 4 , or -R1 -N(CH3)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 These are optionally substituted ethanediyl, propanediyl, butanediyl, or linear or unbranched alkanediyl having 2 to 8 carbon atoms; R 2 It is an alkanediyl having 2 to 8 carbon atoms; R 3 If it is optional and exists, then -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 an alkyl or alkenyl having 12 to 25 carbon atoms; R 5 It is an alkanediyl having 1 to 6 carbon atoms; X is a carbon or nitrogen atom; All choices are independent of each other. A cationic lipid of formula (I) is provided. In some cases, R 2 and / or R 5 Alkanediyls represented by this symbol are linear and unsubstituted.

[0186] Furthermore, in another embodiment, R a and R b teeth

[0187] [ka]

[0188] (Preferably, X is CH) or -R1 -N(H)-C(O)-R 3 -R 4 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 These are optionally substituted ethanediyl, propanediyl, butanediyl, or linear or unbranched alkanediyl having 2 to 8 carbon atoms; R 2 It is an alkanediyl having 2 to 8 carbon atoms; R 3 If it is optional and exists, then -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 It is an alkanediyl having 1 to 6 carbon atoms; X is a carbon or nitrogen atom; All choices are independent of each other. Depending on the case, R 1 , R 2 and R 5 All are linear unsubstituted ethanediyl molecules, A is -SS-, and R a and R b If they are the same, R 4 but

[0189] [ka]

[0190] isn't it, A cationic lipid according to formula (I) is provided. In this embodiment, R 2 and / or R 5 The alkanediyl represented by can be linear and unsubstituted; in some cases, R 5 It contains 2 to 6 carbon atoms, R a and R b Each of them

[0191] [ka]

[0192] (Preferably, X is CH) That is the case. In a further specific embodiment, R a and R b Each of them

[0193] [ka]

[0194] (Preferably, X is CH) and; A is -SS-; R 1 These are optionally substituted ethanediyl, propanediyl, butanediyl, or linear or unbranched alkanediyl having 2 to 8 carbon atoms; R 2 It is an alkanediyl having 2 to 8 carbon atoms; R 3 If it is optional and exists, then -R 5 -C(O)-O- or -R 5 -OC(O)-; R 4 is a lipophilic substituent having 12 to 36 carbon atoms; R 5 It is an alkanediyl having 2 to 6 carbon atoms; X is a carbon or nitrogen atom; All choices are independent of each other. Depending on the case, R 1, R 2 and R 5 All are linear unsubstituted ethanediyl molecules, A is -SS-, and R a and R b If they are the same, R 4 but

[0195] [ka]

[0196] isn't it, A cationic lipid according to formula (I) is provided. Also, R 2 and / or R 5 The alkanediyl represented by can be linear and unsubstituted.

[0197] Further embodiments include R a and R b Each of them

[0198] [ka]

[0199] (Preferably, X is CH) and; A is -SS-; R 1 These are optionally substituted ethanediyl, propanediyl, butanediyl, or linear or unbranched alkanediyl having 2 to 8 carbon atoms; R 2 It is an alkanediyl having 2 to 8 carbon atoms; R 3 If it is optional and exists, then -R 5 -C(O)-O- or -R 5 -OC(O)-; R 4 is an alkyl or alkenyl having 12 to 25 carbon atoms; R 5 It is an alkanediyl having 2 to 6 carbon atoms; X is a carbon or nitrogen atom; All choices are independent of each other. R 2 and / or R 5 Alkanediyls represented by can be linear and unsubstituted. A cationic lipid according to formula (I) is provided.

[0200] In another embodiment, R a and R b Each of them

[0201] [ka]

[0202] (Preferably, X is CH) and; A is -SS-; R 1 These are optionally substituted ethanediyl, propanediyl, butanediyl, or linear or unbranched alkanediyl having 2 to 8 carbon atoms; R 2 It is an alkanediyl having 2 to 8 carbon atoms; R 3 If it is optional and exists, then -R 5 -C(O)-O- or -R 5 -OC(O)-; R 4 but

[0203] [ka]

[0204] It is an alkyl selected from; R 5 It is an alkanediyl having 2 to 6 carbon atoms; X is a carbon or nitrogen atom; All choices are independent of each other, R 2 and / or R 5Alkanediyls represented by can be linear and unsubstituted. A cationic lipid according to formula (I) is provided.

[0205] Further embodiments include R a and R b Each of them

[0206] [ka]

[0207] (Preferably, X is CH) and; A is -SS-; R 1 These are optionally substituted ethanediyl, propanediyl, butanediyl, or linear or unbranched alkanediyl having 2 to 8 carbon atoms; R 2 It is an alkanediyl having 2 to 8 carbon atoms; R 3 ga-R 5 -C(O)-O- or -R 5 -OC(O)-; R 4 but

[0208] [ka]

[0209] and; R 5 It is an alkanediyl having 2 to 6 carbon atoms; X is a carbon or nitrogen atom; R a and R b They are identical, and all other choices are independent of each other, R 2 and / or R 5 The alkanediyl represented by can be linear and unsubstituted. This relates to cationic lipids according to formula (I).

[0210] In a further embodiment, the present invention is R a and R b Each of them

[0211] [ka]

[0212] (Preferably, X is CH) and; A is -SS-; R 1 These are optionally substituted ethanediyl, propanediyl, butanediyl, or linear or unbranched alkanediyl having 2 to 8 carbon atoms; R 2 It is an alkanediyl having 2 to 8 carbon atoms; R 3 ga-R 5 -C(O)-O-; R 4 but

[0213] [ka]

[0214] and; R 5 It is an alkanediyl having 2 to 6 carbon atoms; X is a carbon or nitrogen atom; R a and R b They are identical, and all other choices are independent of each other, R 2 and / or R 5 Alkanediyls represented by can be linear and unsubstituted. A cationic lipid according to formula (I) is provided.

[0215] According to further embodiments, the cationic lipids provided by the present invention are R a and R b Each of them

[0216] [ka]

[0217] (Preferably, X is CH) and; A is -SS-; R 1 These are optionally substituted ethanediyl, propanediyl, butanediyl, or linear or unbranched alkanediyl having 2 to 8 carbon atoms; R 2 It is an alkanediyl having 2 to 8 carbon atoms; R 3 ga-R 5 -C(O)-O-; R 4 but

[0218] [ka]

[0219] and; R 5 It is an alkanediyl having 2 to 6 carbon atoms; X is a carbon atom; R a and R b They are identical, and all other choices are independent of each other, R 2 and / or R 5 Alkanediyls represented by can be linear and unsubstituted. It is a compound of formula (I).

[0220] In another specific embodiment, the cationic lipid provided by the present invention is R a and R b Each of them

[0221] [ka]

[0222] (Preferably, X is CH) and; A is -SS-; R 1 is ethane; R 2 It is an alkanediyl having 2 to 8 carbon atoms; R 3 ga-R 5 -C(O)-O-; R 4 but

[0223] [ka]

[0224] and; R 5 It is an alkanediyl having 2 to 6 carbon atoms; X is a carbon atom; R a and R b They are identical, and all other choices are independent of each other, R 2 and / or R 5 The alkanediyl represented by can be linear and unsubstituted; preferably, R 1 The ethanediyl is also linear and unsubstituted. It is a compound according to formula (I).

[0225] In another preferred embodiment, the cationic lipid is independently selected in each appearance and has the following characteristics: (i)R 1 is an unsubstituted ethanediyl, propanediyl, or butanediyl; (ii)R 2 It is a straight-chain, unbranched alkanediyl having 2 to 8 carbon atoms; (iii)R 3 ga-R 5 -C(O)-O- or -R 5 -OC(O)-; (iv)R 4It is an alkyl or alkenyl having 12 to 25 carbon atoms; (v)R 5 is an alkanediyl having 2 to 6 carbon atoms; and / or (vi) X is a carbon atom, It has one or more of these.

[0226] In particular, in another preferred embodiment of the above embodiment A, R 3 It exists, -R 5 -C(O)-O-, -R 5 -OC(O)-, -R 5 -C(O)-NH-, -R 5 -OC(O)-NH-, and R 5 Selected from the group consisting of -NH-C(O)O-; R 4 R is a linear or branched alkyl or alkenyl having 12 to 25 carbon atoms. In this embodiment, R 3 is R 5 -C(O)-O- or -R 5 It is particularly preferred that it be -OC(O)-. In this embodiment, R 4 teeth

[0227] [ka]

[0228] It may be even more preferable to select from the group consisting of the following. In particular, in yet another preferred embodiment of the above embodiment A, A is -S-. In this embodiment, R a and R b They are identical,

[0229] [ka]

[0230] (X is preferably CH) It may be preferred that R 3 It exists, -R5 -C(O)-O- or -R 5 It may be even more preferable to select from -OC(O)-. In this embodiment, R 4 teeth

[0231] [ka]

[0232] It may also be preferred that R a R 4 and R b R 4 It is even more preferable that they be identical. Finally, in this embodiment, R 1 , R 2 and R 3 It is also preferable that one or all of them are alkanediyls having 1 to 6 carbon atoms, especially 2, 3, or 3 carbon atoms.

[0233] In further embodiments, the cationic lipid is preferably selected from the cationic lipids listed in Table 1.

[0234] [Table 2-1]

[0235] [Table 2-2]

[0236] [Table 2-3]

[0237] [Table 2-4]

[0238] [Table 2-5]

[0239] [Table 2-6]

[0240] [Table 2-7]

[0241] [Table 2-8]

[0242] [Table 2-9]

[0243] [Table 2-10]

[0244] [Table 2-11]

[0245] Therefore, the present invention is directed toward compositions comprising the cationic lipids described above. For example, the composition may contain cationic lipids selected from compounds C1 to C27 in Table 1. In a further specific embodiment, the cationic or cationizable lipid may be any of several lipid species that contain a tertiary or quaternary nitrogen / amino group, or have a net positive charge at a selective pH such as physiological pH. Therefore, in one embodiment, cationic lipids containing a tertiary or quaternary nitrogen / amino group, or cationic lipids having a net positive charge at physiological pH, include, but are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); N,N-distearyl-N,N-dimethylammonium bromide (DDAB); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); 3-(N-(N',N'-dimethylaminoethane) Selected from the group consisting of rubamoyl)cholesterol (DC-Chol); N-(1-(2,3-dioleoyloxy)propyl)N-2-(sperminecarboxamide)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA); dioctadecylamideglycylcarboxyspermine (DOGS); 1,2-dioleoyl-3-dimethylammoniumpropane (DODAP); N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA); and N-(1,2-dimyristyloxypropane-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE).

[0246] In further embodiments, cationic lipids containing tertiary or quaternary nitrogen / amino groups, or cationic lipids having a net positive charge at physiological pH, are selected from the group consisting of amino lipids, but are not limited to these. In another embodiment, suitable amino acids include those described in International Publication 2012 / 016184, which is incorporated in whole into this specification.

[0320] Other suitable additional cationic lipids for use in the composition include cholesterol-based cationic lipids.

[0247] Further representative aminolipids include, but are not limited to, (i) Formula:

[0248] [ka]

[0249] (In the formula, R1 and R2 are the same or different, and are independently substituted as applicable by C) 10 ~C 24 Alkyl, and optionally substituted C 10 ~C 24 Alkenyl, optionally substituted C 10 ~C 24 Alkynyl, or optionally substituted C 10 ~C 24 It is an acyl; R3 and R4 are the same or different, independently of optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, or optionally substituted C2-C6 alkynyl, or R3 and R4 may bond to form an optionally substituted heterocyclic ring of 4-6 carbon atoms and one or two heteroatoms selected from nitrogen and oxygen; R5 is absent or present, and if present, is hydrogen or a C1-C6 alkyl; m, n, and p are the same or different, independently of 0 or 1, provided that m, n, and p are not simultaneously 0; q is 0, 1, 2, 3, or 4; Y and Z are the same or different, independently of O, S, or NH. Having the following characteristics: In one embodiment, R1 and R2 are linoleyl, and the aminolipid is a dilinoleyl aminolipid; or (ii) Dilinoleyl aminolipids; 1,2-dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC); 1,2-dilinoleyoxy-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); 1,2-dilinoleoyl- Selected from the group consisting of 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); 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA); and DLin-KC2-DMA (where n is 2, the same as the above DLin-K-DMA), It includes.

[0250] The cationic lipids described herein may exist in the form of salts, such as acid addition salts, or, in certain cases, salts of organic and inorganic bases, such as carboxylates, sulfonates, and phosphates. All such salts are within the scope of the present invention, and references to the cationic lipids and compounds of formula (I) and their subgroups include the salt forms of these compounds.

[0251] In further embodiments, commercially prepared cationic lipids may be used in the present invention. These include, for example, LIPOFECTIN® (commercially available cationic liposomes containing DOTMA and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), manufactured by GIBCO / BRL [Grand Island, New York]); LIPOFECTAMINE® (commercially available cationic liposomes containing N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamide)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA) and DOPE, manufactured by GIBCO / BRL); and TRANSFECTAM® (commercially available cationic lipids containing dioctadecylamideglycylcarboxyspermine in ethanol (DOGS), manufactured by Promega Corp. [Madison, Wisconsin]).

[0252] In further embodiments, the composition comprises imidazole cholesterol esters or "ICE" as disclosed in paragraphs

[0320] and

[0339] -

[0340] of International Publication No. 2019226925, which is incorporated entirely by the present specification. Other suitable (cationic) lipids are all incorporated entirely by the present specification, as disclosed in International Publication Nos. 2009 / 086558, 2009 / 127060, 2010 / 048536, 2010 / 054406, 2010 / 088537, 2010 / 129709, 2011 / 153493, and U.S. Patent Application Publication No. 2011 / 0256175. The patent is disclosed in U.S. Patent Publication No. 2012 / 0128760, U.S. Patent Publication No. 2012 / 0027803, 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, and International Publication No. 2017112865. In some embodiments, the cationic lipid is selected from the group consisting of 98N12-5, C12-200, and ckk-E12, all of which are disclosed in International Publication No. 2017049074, U.S. Patent No. 9512073, International Publication No. 2015200465, and U.S. Patent Application Publication No. 20150376144, all of which are incorporated in their entirety in this specification.

[0253] Cationic lipids can constitute 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 70 mol% of the total lipids present in the composition or lipid nanoparticles of the present invention. In another embodiment, the lipid nanoparticles contain about 25% to about 75% on a molar basis, for example, 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% on a molar basis (based on 100% of the total moles of lipids in the lipid nanoparticles) cationic lipids.

[0254] Generally, the compositions according to the present invention may contain other excipients such as one or more further lipids. In one embodiment, the composition may contain further cationic lipids, i.e., second, third, and so on cationic lipids. Such further cationic lipids may optionally be cationic lipids disclosed herein. Alternatively, it may be any other cationic lipid suitable for pharmaceutical compositions, particularly compositions containing an active ingredient selected from nucleic acid compounds such as mRNA.

[0255] In one specific embodiment, the further cationic lipid is a permanently cationic lipid containing at least one quaternary nitrogen atom. In this case, the first cationic lipid is preferably a lipid that is cationizable rather than permanently cationic.

[0256] Pharmacovigilant salts of basic cationic lipids can be derived from inorganic or organic acids. For example, salts from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, and nitric acid, as well as salts from organic acids such as acetic acid, propanoic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid (fumatic), toluenesulfonic acid, methanesulfonic acid, ethanesulfonic acid, naphthalenesulfonic acid, benzenesulfonic acid, and trifluoroacetic acid. Further examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and so on. Typical acid addition salts include acetate, acetic acid, adipine, alginate, ascorbate, aspartate, benzenesulfonate, benzenesulfonic acid, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, and 2-hydroxy-ethanol. This includes sulfonates, lactobionates, lactates, laurates, lauryl sulfates, malates, maleates, malons, methanesulfons, 2-naphthalenesulfons, nicotinates, nitrates, oleates, oxalates, palmitates, pamoates, pectins, persulfates, 3-phenylpropionates, phosphates, picrates, pivalates, propions, stearates, succinates, sulfates, tartrates, thiocyans, toluenesulfons, undecanoates, valersates, and others.Typical alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and, but are not limited to, non-toxic ammonium, quaternary ammonium, and amine cations, including ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, and ethylamine. The pharmaceutically acceptable salts of this disclosure include, for example, conventional non-toxic salts of parent compounds formed from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of this disclosure can be synthesized from parent compounds containing basic or acidic moieties by conventional chemical methods. Generally, such salts can be prepared by reacting the free acidic or base form of these compounds with a stoichiometric amount of a suitable base or acid in water, an organic solvent, or a mixture thereof; generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. A list of suitable salts can be found in Remington's Pharmaceutical Sciences, 17th edition, Mack Publishing Company [Easton, Pennsylvania], 1985, pp. 1418, each incorporated in its entirety in this specification; Pharmaceutical Salts: Properties, Selection and Use, PHStahl and CGWermuth (eds.), Wiley-VCH, 2008; and Berge et al., Journal of Pharmaceutical Science, 66, 1-19 (1977).

[0257] Polymer-conjugated lipids, PEGylated lipids In some embodiments, the LNP includes a lipid conjugate, preferably a polymer-conjugated lipid. The term "polymer-conjugated lipid" refers to a molecule containing both a lipid and a polymer portion. Preferably, the polymer-conjugated lipid is a PEG-lipid. The term "PEG-lipid" refers to a molecule containing both a lipid and a polyethylene glycol portion. PEG-lipids are well known in the art and include PEG-DMG, among others.

[0258] In a specific embodiment, the polymer conjugate lipid is a compound according to formula (II): PAL formula (II) (In the formula, P is the hydrophilic polymer moiety, A is an optional linker or spacer, and L is the lipid moiety.) It is defined as follows.

[0259] Hydrophilic polymer part P The hydrophilic polymer moiety P in the polymer-conjugated lipid according to formula (II) may be a polyethylene glycol ("PEG") moiety. In specific embodiments, the PEG moiety has an average molecular mass between 1 kDa and 3 kDa, for example, between 1.5 and 2.5 kDa, between 1.7 and 2.3 kDa, between 1.8 and 2.2 kDa, between 1.9 and 2.1 kDa, or 2 kDa. Thus, the PEG may be the PEG commonly known as "PEG2000" or "PEG2k", but shorter "PEG1000" and longer "PEG3000" can also be used. The PEG moiety usually contains a linear polymer chain, but in some embodiments, the PEG moiety may contain a branched polymer chain. Alternatively, the intended PEG-modified lipid may include, but is not limited to, polyethylene glycol chains covalently bonded to the lipid, with lengths up to 2 kDa, up to 3 kDa, up to 4 kDa, or up to 5 kDa.

[0260] In another embodiment, the hydrophilic polymer moiety P in the polymer conjugate lipid may also be a substantially hydrophilic polymer different from the hydrophilic polymer moiety described above, i.e., the hydrophilic polymer moiety P in the polymer conjugate lipid may be based on poly(propylene oxide), poly(vinylpyrrolidone), poly(vinyl alcohol), poly-N-(2-hydroxypropyl)methacrylamide, a hesylation process (according to PMID24681396), a PASylation approach (i.e., proline-alanine-serine), an XTEN approach known in the art (i.e., peptide-based PEG), polysarcosine, or poly(vinyl acetate).

[0261] Optional linker or spacer A The optional linker or spacer A in the polymer conjugate lipid according to formula (II) can be any useful spacer structure, such as those generally known to be useful in pegylated lipids, for example, spacers selected from, but not limited to, succinimide, amine, ether, ester, anhydride, aldehyde, ketone, amide, carbamate linker or combination thereof.

[0262] Lipid part L The lipid moiety L in polymer conjugate lipids according to formula (II) can be derived from phospholipids, sphingolipids, or ceramides. As used herein, the expression “derived from phospholipids or ceramides” includes both phospholipid and ceramide groups. Examples include polymer conjugate lipids containing phosphatidylethanolamine or phosphatidylglycerol moieties.

[0263] In specific embodiments, the polymer conjugate lipid is a PEGylated lipid. In more specific embodiments, the polymer conjugate lipid included in the composition of the present invention is 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]} (also called PEG-Ceramide 8, C8-Ceramide-PEG, PEG-Cer8, C8PEG2000 Ceramide or Ceramide 8PEG, containing N-Octanoyl-D-erythro-sphingosine (d18:1 / 8:0)); 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)butanediate (PEG-S-DMG); 2-mPEG2000-n,n-ditetradecylacetamide; N-[(methoxypoly(ethylene glycol)2000) The polymer conjugate lipid is selected from the group consisting of rubamil]-1,2-dimyristyloxypropyl-3-amine (PEG-c-DMA); ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecaneoxy)propyl)carbamate; PEG lipids disclosed in International Publication No. 2018126084, International Publication No. 2020093061, or International Publication No. 2020219941 (all three references are incorporated herein by reference); PEGylated cholesterol or PEGylated cholesterol derivatives disclosed herein; and 2,3-di(tetradecaneoxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate.

[0264] In a more preferred embodiment, the lipid portion L comprises one, two, three, four, or more hydrophobic fatty acids ("tails," corresponding to an aliphatic chain containing an even number of carbon atoms). In a more preferred embodiment, the lipid portion L comprises two hydrophobic fatty acids ("tails") having the same or different numbers of carbon atoms.

[0265] Preferably, the lipid portion L contains fatty acids ("tails") having 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 combinations thereof. More preferably, the lipid portion L contains fatty acids ("tails") having 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 carbon atoms or combinations thereof. In a more specific embodiment, the lipid portion L includes a fatty acid ("tail") selected from the group consisting of caprylic acid 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 serotic acid (26:0).

[0266] In a more preferred embodiment, the lipid portion L includes at least one fatty acid ("tail") having 8, 10, or 12 carbon atoms, preferably 8 or 10 carbon atoms.

[0267] In a more preferred embodiment, the composition is a polymer-conjugate lipid. - 1,2-Dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 (DMG-PEG2000)

[0268] [ka]

[0269] Includes. 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 approximately 97:3.

[0270] In a more specific embodiment, the composition is - 1,2-Dicapryl-rac-Glycero-3-methylpolyoxyethylene glycol 2000(C 10 -PEG2000)

[0271] [ka]

[0272] and - N-octanoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)2000]}(Cer8-PEG2000)

[0273] [ka]

[0274] It contains polymer conjugate lipids selected from the group consisting of the following. In a further embodiment, the composition has the following chemical structure:

[0275] [ka]

[0276] It contains polymer conjugate lipids selected from the group consisting of the following structures similar to "C8-PEG2000" having the following characteristics. In a specific embodiment of the present invention, "C 10 Each composition disclosed herein, including "-PEG2000", is referred to as "C 10 It is also possible to formulate the product using "C8-PEG2000" instead of "-PEG2000".

[0277] Therefore, as an example, a polymer conjugate lipid, or each lipid portion L, may have two fatty acid tails, including saturated fatty acids, unsaturated fatty acids, or combinations thereof ("tails"), for example, Cer8-PEG2000, which contains one saturated fatty acid chain (8:0; caprylic acid or octanoic acid, respectively) and one unsaturated fatty acid of different lengths having nine or more carbon atoms.

[0278] Specifically, a more remarkable discovery made by the inventors is the advantageous use of polymer conjugate lipids having shorter alkyl chains (e.g., Cer8) as disclosed herein, preferably in combination with DPhyPE as a neutral lipid instead of the lipids of the present invention disclosed herein, e.g., Table 1, and / or DSPC, for delivering mRNA vaccines in vivo, resulting in a significantly enhanced immune response, similar to specific aspects and embodiments of the present invention.

[0279] steroid A "steroid" is an organic compound that has four rings arranged in a specific molecular configuration. Steroids contain the following carbon skeletons:

[0280] [ka]

[0281] Steroids and neutral steroids both include naturally occurring steroids and their analogues (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” as provided herein, a neutral steroid may be a steroid that has neither ionizable atoms nor groups under physiological conditions, or it may be a zwitterionic steroid. In one preferred embodiment, the neutral steroid contains neither ionizable atoms nor groups under physiological conditions. In some preferred embodiments, the steroid or steroid analogue is cholesterol. The terms “steroid” and “neutral steroid” are used interchangeably herein.

[0282] In further embodiments, the steroid is an imidazole cholesterol ester or "ICE" as disclosed in paragraphs

[0320] and

[0339] -

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

[0283] neutral lipids, neutral phospholipids The “neutral lipids,” also called “helper lipids,” according to the present invention are preferably phospholipids or neutral phospholipids. As used herein, “neutral phospholipids” are typically amphiphilic compounds comprising molecules having two hydrophobic fatty acid “tails” and a hydrophilic “head” containing 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 make up a significant proportion of additives in biological membranes. As used herein, the terms “phospholipids” or “neutral phospholipids” encompass both natural and synthetic phospholipids.

[0284] The terms “neutral lipid,” “neutral phospholipid,” or “zwitterionic compound,” as used interchangeably herein, refer to any one of several lipid species that exist at physiological pH uncharged or in a neutral zwitterionic form. Representative neutral lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramides, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides, which are further described below herein.

[0285] According to one preferred embodiment, the composition comprises a zwitterionic neutral lipid such as phosphatidylcholine or phosphatidylethanolamine. Suitable examples of phosphatidylcholine include natural or purified mixtures, sometimes called "lecithin" or "phosphatidylcholine," typically derived from egg yolk or soy; or highly purified or semi-synthetic compounds such as phosphatidylcholine having two fatty acyl moieties selected from myristoyl, palmitoyl, stearoyl, oleoyl, etc.

[0286] In another preferred embodiment, the neutral lipid or neutral phospholipid is, but is not limited to, 1,2-difitanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE; also known as 1,2-di-(3,7,11,15-tetramethylhexadecanoyl)-sn-glycero-3-phosphoethanolamine), 1,2-difitanoyl-sn-glycero-3-phosphocholine (DPhyPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC; also known as dioleoylphosphatidylcholine), 1,2-dipalmi Toyl-sn-glycero-3-phosphocholine (also called DPPC, 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), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DLoPE), distearoylphosphatidylethanolamine (DSPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine Phosphoethanolamine (POPE), 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-linoleyl-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) (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-difytanol-sn-glycero-3-phospho-L-serine (sodium salt), 1-O-hexadecanyl-2-O-(9Z-oc Tadenyl-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-cholesteryl-hemisuccinoyl-sn-glycero-3-phosphocholine (PChemsPC), 1,2-dicholesterylhemisuccinoyl It is a zwitterionic compound selected from the group consisting of 2-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).

[0287] In preferred embodiments, the neutral lipid according to the present 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 more preferred embodiments, the neutral lipid according to the present invention is 1,2-difitanoyl-sn-glycero-3-phosphocholine (DPhyPC). In even more preferred, particularly preferred embodiments, the neutral lipid according to the present invention is 1,2-difitanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE). An advantage of the present invention related to the use of DPhyPE is its high membrane fusion ability due to its bulky tail, which enables high-level fusion with endosomal lipids.

[0288] Specifically, the advantageous use of 1,2-difitanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE), preferably in combination with the lipids of the present invention disclosed herein, for example in Table 1, for delivering mRNA vaccines in vivo, resulting in a significantly enhanced immune response, is a remarkable discovery made by the inventors and is similar to specific aspects and embodiments of the present invention. In other words, the inventors have surprisingly found that the use of DPhyPE offers a clear advantage over DSPC, which has been used in the art to date as a standard neutral lipid in virtually all prior art LNP compositions for vaccine settings, specifically, but not limited to mRNA and also siRNA. In other words, the compositions of the present invention have highly advantageous and unpredictable behavior in vivo, resulting in a highly enhanced immune response.

[0289] Interestingly, the inventors found that compositions containing DPhyPE instead of DSPC exhibited superior expression profiles both in vitro and in vivo, even when compared to the GN01 composition. Therefore, surprisingly, the use of DPhyPE was found to offer a clear advantage over DSPC, which has been used in the art to date as a standard neutral lipid in almost all prior art LNP compositions.

[0290] Importantly, the inventors have found that one of the advantageous features of the compositions and lipid nanoparticles of the present invention, for example, the GN01 formulation, is that it can induce a potent CD8+ T cell response. This is because, for example in the case of malaria, an effective malaria vaccine should induce a potent CD8+ T cell response, since CD8+ T cells are the primary protective immune mechanism against intracellular infections caused by the malaria parasite.

[0291] Furthermore, the data presented in the examples demonstrate that the significantly enhanced immune response using the compositions of the present invention, i.e., all RNA vaccines of the present invention, is more useful than the present invention. Surprisingly, in contrast to prior art knowledge indicating that DSPC is the most common and undoubtedly neutral lipid for lipid nanoparticles, the inventors found that it is preferable to use DPhyPE in the mRNA formulation of the composition for producing the vaccine.

[0292] The inventors have also found, to their surprise, that the immune response can be enhanced by adding at least one further neutral lipid, particularly a second neutral lipid, to the above neutral lipid (see Figures 28-31 and corresponding examples). As described above, it is preferable for the (first) neutral lipid of the present invention to have two fatty acyl moieties selected from myristoyl, palmitoyl, stearoyl, oleoyl, etc. (this means in particular that the fatty acyl moieties are fairly long, starting with a moiety having 14 carbon atoms). The inventors have found that, in particular, when the additional neutral lipid has two fatty acid moieties selected from pentanoyl, hexanoyl, heptanol, octanoyl, nonanoyl, and decanoyl, i.e., moieties having up to 10 carbon atoms, the addition of a neutral lipid having a shorter fatty acyl moiety provides a beneficial effect. A particularly preferred additional neutral lipid is 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC), but 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-dinonanyl-sn-glycero-3-phosphocholine), and 10:0PC (1,2-dihexanoyl-sn-glycero-3-phosphocholine) can also be used.

[0293] Therefore, in one embodiment of the present invention, the lipid nanoparticles of the present invention are C5, C6, C7, C8, C9, C 10 , C 11 , C 12 , C 13 or C 14 Having a length, preferably C6, C7, C8, C9, or C 10 The present invention comprises a neutral lipid or phospholipid having at least one alkyl chain having a length of C5, C6, C7, C8, most preferably C7. In another embodiment of the present invention, the lipid nanoparticles of the present invention have each alkyl chain independently of C5, C6, C7, C8, C9, C10 , C 11 , C 12 , C 13 or C 14 Having a length, preferably C6, C7, C8, C9, or C 10 The lipid nanoparticles include a neutral lipid or phospholipid having at least two alkyl chains having a length of C6, C7, or C8, more preferably C7. In a preferred embodiment, the lipid nanoparticles of the present invention further include DHPC. In a further embodiment, one or more alkyl chains may include a carbon double bond. In other embodiments, the lipid nanoparticles include additional phospholipids 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-dinonanyl-sn-glycero-3-phosphocholine).

[0294] Lipid nanoparticle composition The terms “lipid nanoparticle composition” and “composition” are used interchangeably herein. In the context of the present invention, lipid nanoparticles are not limited to any particular form and should be interpreted as including any form produced by combining cationic lipids and optionally one or more further lipids, for example, in an aqueous environment and / or in the presence of nucleic acid compounds. For example, liposomes, lipid complexes, lipoplexes, etc., fall within the range of lipid nanoparticles.

[0295] In the context of the present invention, “composition” means any type of composition in which the specified components may be incorporated together with any further excipients, and usually with at least one pharmaceutically acceptable carrier or excipient. Thus, the composition may be a dry composition such as a powder or granules, or a solid unit such as a lyophilized form or a tablet. Alternatively, the composition may be in liquid form, and each excipient may be incorporated independently in dissolved or dispersed (e.g., suspension 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 versions of the composition containing nucleic acid cargo, which are described in more detail below.

[0296] In the composition of the present invention, cationic lipids may be present within or as part of lipid nanoparticles (LNPs). In other words, such a composition contains lipid nanoparticles, and the cationic lipids are present within the lipid nanoparticles.

[0297] As used herein, “nanoparticles” are submicron particles having any structure or form. Submicron particles may also be called colloids or colloidal. With respect to the material on which nanoparticles are based and their structure or form, nanoparticles can be classified, to name only a few of the possible names for certain types of nanoparticles, for example, as nanocapsules, vesicles, liposomes, lipid nanoparticles, micelles, cross-linked micelles, lipoplexes, polyplexes, mixtures or hybrid complexes. “Lipid nanoparticles” (LNPs) are nanoparticles formed by lipids, typically comprising at least one amphiphilic membrane-forming lipid and possibly other lipids, and possibly cargo material such as nucleic acid compounds. As used herein, the expressions “lipid nanoparticles” or “LNPs” include any subtypes and forms of nanoparticles formed or co-formed by lipids, such as liposomes and lipoplexes.

[0298] As defined above, lipid nanoparticles include any type of nanoparticles formed or co-formed by lipids. 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.

[0299] In some embodiments, such lipid nanoparticles comprise a cationic lipid (e.g., a lipid of formula (I)) and one or more excipients selected from neutral lipids, charged lipids, steroids, and polymer-conjugated lipids (e.g., pegylated lipids such as pegylated lipids of formula (II)). Compositions comprising the cationic lipids, steroids, neutral lipids, and polymer-conjugated lipids of formula (II) as defined herein are currently considered by the inventors to exist, at least in an aqueous environment, as compositions comprising lipid nanoparticles typically formed by these excipients.

[0300] LNPs may contain any lipids capable of forming particles to which one or more nucleic acid molecules are bound or to which one or more nucleic acid molecules are encapsulated. In some embodiments, mRNA, or a portion thereof, is encapsulated in an aqueous space surrounded by the lipid portion of the lipid nanoparticle, or some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by mechanisms of the host organism or cell, such as adverse immune responses. In some embodiments, mRNA, or a portion thereof, is associated with the lipid nanoparticle.

[0301] As noted herein, compositions comprising lipid excipients typically form lipid nanoparticles, at least in an aqueous environment. As defined herein, the nanoparticles are primarily submicron in size. In certain embodiments, 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-mean 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 from about 30 nm to about 800 nm. In various embodiments, lipid nanoparticles are available in the following ranges: approximately 30nm to 150nm, approximately 50nm to 200nm, approximately 60nm to 200nm, approximately 70nm to 200nm, approximately 80nm to 200nm, approximately 90nm to 200nm, approximately 90nm to 200nm, approximately 90nm to 190nm, approximately 90nm to 180nm, approximately 90nm to 170nm, approximately 90nm to 160nm, approximately 90nm to 150nm, approximately 90nm to 140nm, approximately 90nm to 130nm, approximately 90nm to 120nm, approximately 90nm to 100nm, and approximately 70nm to 9nm. They have an average diameter of 0 nm, approximately 80 nm to 90 nm, approximately 70 nm to 80 nm, or approximately 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.

[0302] Compositions comprising the lipid excipients described herein that yield the lipid nanoparticles of the present invention may be relatively homogeneous. The polydispersity index (PDI) can be used to indicate the homogeneity of the nanoparticle composition, for example, the particle size distribution of the nanoparticle composition. A small polydispersity index (e.g., less than 0.3) generally indicates a narrow particle size distribution. The nanoparticle compositions of the present invention may have polydispersity indices ranging from 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 may be about 0.1 to about 0.2.

[0303] Various optional features, choices, and preferences relating to the compositions of the present invention are generally described herein; all of these also apply to lipid nanoparticles, as will be clearly understood by those skilled in the art. Similarly, the choices and preferences also apply to compositions comprising such lipid nanoparticles.

[0304] For example, lipid nanoparticles according to one preferred embodiment include a neutral lipid which may be DphyPE, a steroid which may be cholesterol, and a polymer conjugate lipid which may be 1,2-dimiristoyl-rac-glycero-3-methoxypolyethylene glycol (PEG-DMG), combined with a cationic lipid as defined above, a second neutral lipid which may optionally be DHPC; the cationic lipid may optionally be selected from the compounds listed in Table 1.

[0305] Therefore, in the context of the present invention, mRNA is preferably contained in a liquid or semiliquid composition, and the mRNA is complexed or associated with lipid nanoparticles according to one of the preferred embodiments. That is, in a preferred embodiment, the liquid or semiliquid composition contains a complex comprising mRNA, the complex preferably existing as lipid nanoparticles as defined herein.

[0306] Regarding the amounts of each excipient, it is preferable that the cationic lipid is incorporated into the lipid nanoparticles or composition according to the present invention in a relatively high molar amount compared to the molar amount of polymer conjugate lipid present. Furthermore, the molar amount of cationic lipid is also preferably higher than the molar amount of neutral lipid in the composition or nanoparticles. In addition, the molar amount of steroid may be higher than the molar amount of polymer conjugate lipid.

[0307] In one particular embodiment, polymer conjugate lipids are present in the LNP in an amount of about 1 mol% to about 10 mol% relative to the total lipid content of the nanoparticles. In one embodiment, polymer conjugate lipids are present in the LNP in an amount of about 1 mol% to about 5 mol%. In one embodiment, polymer conjugate lipids are present in the LNP in an amount of about 1 mol% or about 1.5 mol%.

[0308] In various embodiments, the molar ratio of cationic lipids (e.g., lipids of formula (I)) to polymer conjugate lipids is in the range of about 100:1 to about 25:1, about 50:1 to about 25:1, or about 40:1 to about 25:1.

[0309] In certain embodiments, LNPs include one or more additional lipids that stabilize their formation during particle formation. Suitable stabilizing lipids include neutral and anionic lipids. In various embodiments, the molar ratio of cationic lipids (e.g., lipids of formula (I)) to neutral lipids is in the range of about 2:1 to about 8:1, about 3:1 to about 7:1, or about 4:1 to about 6:1.

[0310] As used herein, lipid nanoparticles are typically formed by their respective excipients and reflect the same quantitative ratio of excipients as the entire composition containing the nanoparticles; therefore, references to the molar amount of lipid excipients in the composition of the present invention should also be understood as describing the molar amount of each excipient in the lipid nanoparticles contained in the composition.

[0311] Generally, the amount of cationic lipids in a composition (and thus in lipid nanoparticles) is typically at least about 20 mol% of the total molar amount of all lipid excipients in the composition (or nanoparticles). In another embodiment, the amount of cationic lipids is at least about 25 mol%, or at least 30 mol%, respectively. In another preferred embodiment, the amount of cationic lipids in a 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%; for example, about 30, 35, 40, 45, 50, 55, 60, 65, or 70 mol%, respectively, or about 40 mol% to about 60 mol%; 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.

[0312] The amount of steroids in the composition may be at least about 10 mol%, or in the range of about 10 mol% to about 60 mol%, or about 20 mol% to about 50 mol%, or about 25 mol% to about 45 mol%, respectively; for example, it may be about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 mol%, respectively. Also, to avoid any doubt, the molar percentages are relative to the total molar amount of all lipid excipients in the composition.

[0313] Neutral lipids may be present in amounts of at least about 5 mol%. In some embodiments, the amount of neutral lipids in the composition ranges from about 5 mol% to about 25 mol%, or about 5 mol% to about 15 mol%, or about 8 mol% to about 12 mol%, using the same basis of molar percentages; for example, about 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, 10 mol%, 11 mol%, 12 mol%, 13 mol%, 14 mol%, 15 mol%, 16 mol%, 17 mol%, 18 mol%, 19 mol%, 20 mol%, 21 mol%, 22 mol%, 23 mol%, 24 mol%, or 25 mol%, respectively. This amount is the total amount of neutral lipids, i.e., this amount may be the sum obtained from the amounts of two neutral lipids, such as DPhyPE and DHPC.

[0314] The amount of polymer-conjugated lipids in the composition or lipid nanoparticles may be selected to be, for example, about 0.1 mol% or more. In certain embodiments, the amount of polymer-conjugated lipids is in the range of about 0.5 mol% to about 5 mol%, or about 1 mol% to about 3 mol%, for example, about 0.1, 0.3, 0.5, 1, 2, 3, 4, or 5 mol%, respectively, using the total molar amount of all lipid excipients as the basis for molar percentage. In other specific embodiments, the composition or lipid nanoparticles are 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 mol% or more than 7 mol% of polymer conjugate lipids. In a preferred embodiment, the content of polymer conjugate lipids or pegylated lipids is about 1 to 5 mol% of the total lipid content of the formulation. As an unlimiting example, lipid nanoparticles contain 1.5% polymer conjugate lipids. As another unlimiting example, lipid nanoparticles contain 1.7% polymer conjugate lipids. As yet another unlimiting example, lipid nanoparticles contain 3% polymer conjugate lipids. As yet another example, lipid nanoparticles contain 5% polymer-conjugated lipids.

[0315] In one embodiment, the composition is (a) Cationic lipids according to formula (I) or as described herein in an amount of 30 to 70 mol%; (b) Steroids in amounts of 20-50 mol%; (c) 5-25 mol% of neutral lipids; and (d) 0.5-5 mol% of polymer-conjugate lipids Contains lipid nanoparticles; Each amount represents the total molar amount of all lipid excipients in the lipid nanoparticles.

[0316] In another embodiment, the composition is (a) Cationic lipids according to formula (I) or as described herein in an amount of 40-70 mol%; (b) Steroids in amounts of 20-50 mol%; (c) 5-15 mol% of neutral lipids; and (d) 0.5-5 mol% of polymer-conjugate lipids Contains lipid nanoparticles; Each amount represents the total molar amount of all lipid excipients in the lipid nanoparticles.

[0317] In one embodiment, the composition is (a) Cationic lipids according to formula (I) or as described herein in an amount of 20 to 60 mol%; (b) Steroids in amounts of 25-55 mol%; (c) 5-25 mol% of neutral lipids; and (d) 0.5 to 15 mol% of polymer-conjugate lipids Contains lipid nanoparticles; Each amount represents the total molar amount of all lipid excipients in the lipid nanoparticles.

[0318] In a further embodiment, the composition, (a) Cationic lipids according to formula (I) or as described herein in an amount of 45-65 mol%; (b) Steroids in amounts of 25-45 mol%; (c) 8-12 mol% of neutral lipids; and (d) 1-3 mol% of polymer-conjugate lipids Contains lipid nanoparticles; Each amount represents the total molar amount of all lipid excipients in the lipid nanoparticles.

[0319] In a more preferred embodiment, the composition is (a) Cationic lipids according to formula (I) or as described herein in an amount of 45-65 mol%; (b) Cholesterol in an amount of 25-45 mol%; (c) 8-12 mol% of neutral lipids; and (d) 1-3 mol% of polymer-conjugate lipids Contains lipid nanoparticles; Each amount represents the total molar amount of all lipid excipients in the lipid nanoparticles.

[0320] In a more preferred embodiment, the composition is (a) Cationic lipids according to formula (I) or as described herein in an amount of 45-65 mol%; (b) Cholesterol in an amount of 25-45 mol%; (c) 8-12 mol% of DPhyPE and optionally 1-10 mol% of DHPC; and (d) 1-3 mol% of polymer-conjugate lipids Contains lipid nanoparticles containing; Each amount represents the total molar amount of all lipid excipients in the lipid nanoparticles.

[0321] In a more preferred embodiment, the composition is (a) Cationic lipids according to formula (I) or as described herein in an amount of 45-65 mol%; (b) Cholesterol in an amount of 25-45 mol%; (c) 8-12 mol% of DPhyPE and optionally 1-10 mol% of DHPC; and (d) PEG-DMG2000 in amounts of 1-3 mol% Contains lipid nanoparticles containing; Each amount represents the total molar amount of all lipid excipients in the lipid nanoparticles.

[0322] 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 may be selected from the compounds listed in Table 1. Furthermore, these embodiments may also include steroids, neutral lipids, and / or polymer conjugate lipids selected according to any one of the preferences disclosed herein. In all embodiments in which compositions or lipid nanoparticles described herein are listed and mol% values ​​are given for each excipient, each amount should be considered to be relative to the total molar amount of all lipid excipients in the lipid nanoparticles.

[0323] In a more preferred embodiment, the composition or lipid nanoparticles described herein comprise 59 mol% of a cationic lipid according to formula (I) of the present invention, 10 mol% of a neutral lipid, 29.3 mol% of a steroid, and 1.7 mol% of a polymer conjugate lipid.

[0324] In one embodiment, the composition or lipid nanoparticles described herein contain 59 mol% of a cationic lipid according to formula (I) of the present invention, 10 mol% of DPhyPE, 29.3 mol% of cholesterol, and 1.7 mol% of DMG-PEG2000.

[0325] In one embodiment, the composition or lipid nanoparticles described herein contain 59 mol% of a cationic lipid according to formula (I) of the present invention, 10 mol% of DPhyPE, 29.3 mol% of cholesterol, and 1.7 mol% of C10-PEG2000. In one embodiment, the composition or lipid nanoparticles described herein contain 59 mol% of a cationic lipid according to formula (I) of the present invention, 10 mol% of DPhyPE, 29.3 mol% of cholesterol, and 1.7 mol% of Cer8-PEG2000.

[0326] In another embodiment, the composition or lipid nanoparticles described herein comprise 47.4 mol% of a cationic lipid according to formula (I) of the present invention, 10 mol% of a neutral lipid, 40.9 mol% of a steroid, and 1.7 mol% of a polymer conjugate lipid.

[0327] In further embodiments, the composition or lipid nanoparticles described herein contain 47.4 mol% of a cationic lipid according to formula (I) of the present invention, 10 mol% of DPhyPE, 40.9 mol% of cholesterol, and 1.7 mol% of DMG-PEG2000. In one embodiment, the composition or lipid nanoparticles described herein contain 47.4 mol% of a cationic lipid according to formula (I) of the present invention, 10 mol% of DPhyPE, 40.9 mol% of cholesterol, and 1.7 mol% of C 10 - Contains PEG2000. In one embodiment, the composition or lipid nanoparticles described herein contain 47.4 mol% of a cationic lipid according to formula (I) of the present invention, 10 mol% of DPhyPE, 40.9 mol% of cholesterol, and 1.7 mol% of Cer8-PEG2000.

[0328] In another embodiment, the composition or lipid nanoparticles described herein comprise 59 mol% of a cationic lipid according to formula (I) of the present invention, 11 mol% of a neutral lipid, 28.3 mol% of a steroid, and 1.7 mol% of a polymer conjugate lipid.

[0329] In one embodiment, the composition or lipid nanoparticles described herein contain 59 mol% of a cationic lipid according to formula (I) of the present invention, 10 mol% of DphyPE and 1 mol% of DHPC, 28.3 mol% of cholesterol and 1.7 mol% of DMG-PEG2000. In one embodiment, the composition or lipid nanoparticles described herein contain 59 mol% of a cationic lipid according to formula (I) of the present invention, 10 mol% of DphyPE and 1 mol% of DHPC, 28.3 mol% of cholesterol and 1.7 mol% of C 10- Contains PEG2000. In one embodiment, the composition or lipid nanoparticles described herein contain 59 mol% of a cationic lipid according to formula (I) of the present invention, 10 mol% of DphyPE and 1 mol% of DHPC, 29.3 mol% of cholesterol and 1.7 mol% of Cer8-PEG2000.

[0330] In another embodiment, the composition or lipid nanoparticles described herein comprise 49 mol% of a cationic lipid according to formula (I) of the present invention, 20 mol% of a neutral lipid, 29.3 mol% of a steroid, and 1.7 mol% of a polymer conjugate lipid.

[0331] In one embodiment, the composition or lipid nanoparticles described herein contain 49 mol% of a cationic lipid according to formula (I) of the present invention, 10 mol% of DphyPE and 10 mol% of DHPC, 29.3 mol% of cholesterol and 1.7 mol% of DMG-PEG2000. In one embodiment, the composition or lipid nanoparticles described herein contain 49 mol% of a cationic lipid according to formula (I) of the present invention, 10 mol% of DphyPE and 10 mol% of DHPC, 29.3 mol% of cholesterol and 1.7 mol% of C 10 -Includes PEG2000. In one embodiment, the composition or lipid nanoparticles described herein contain 49 mol% of a cationic lipid according to formula (I) of the present invention, 10 mol% of DphyPE and 10 mol% of DHPC, 29.3 mol% of cholesterol and 1.7 mol% of Cer8-PEG2000.

[0332] In any of the embodiments described above in this section that disclose specific compositions or lipid nanoparticles having distinct percentage values ​​for excipients, if 1,2-difitanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE) is referred to as a neutral lipid, in further embodiments, DPhyPE may be replaced with another neutral lipid, preferably 1,2-difitanoyl-sn-glycero-3-phosphocholine (DPhyPC). Furthermore, in any of the embodiments described above in this section that disclose specific compositions or lipid nanoparticles having separate percentage values ​​for excipients, if 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE) is referred to as a neutral lipid, in further embodiments, DPhyPE may be replaced with another neutral lipid, preferably 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC; also known as dioleoylphosphatidylcholine) or instead 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

[0333] A more preferred lipid composition according to a further specific embodiment of the present invention comprises at least four lipid excipients disclosed herein in Table E. For example, a preferred lipid composition comprises the excipients disclosed in row "E1," which are "C1" (disclosed herein in Table 1) as a cationic lipid, DPhyPE as a neutral lipid, cholesterol as a sterol, and DMG-PEG2000 as a polymer conjugate lipid excipient. Another example of a preferred lipid composition is "C12" (disclosed herein in Table 1) as a cationic lipid, DPhyPE as a neutral lipid, cholesterol as a sterol, and C as a polymer conjugate lipid excipient. 10 -Includes the excipient disclosed in line "E35", which is PEG2000.

[0334] [Table 3-1]

[0335] [Table 3-2]

[0336] Furthermore, Table F shows preferred lipid formulations of the present invention, indicating distinct molar percentages of at least four lipid excipients in the compositions of the present invention. For example, a preferred lipid composition comprises the molar percentages of lipids disclosed in row "F1," i.e., 59 mol% cationic lipids, 29.3 mol% sterols, 10 mol% neutral lipids, and 1.7 mol% polymer conjugate lipids. Another example of a preferred lipid composition comprises the molar percentages of lipids disclosed in row "F31," i.e., 45 mol% cationic lipids, 43.5 mol% sterols, 10 mol% neutral lipids, and 1.5 mol% polymer conjugate lipids.

[0337] [Table 4]

[0338] Therefore, in a more preferred embodiment of the present invention, the composition of the present invention is a formulation name F1, F2, F3, F4, F5, F6, F7, F8, F9, F10, F11, F12, F13, F14, F15, F16, F17, F 18, F19, F20, F21, F22, F23, F24, F25, F26, F27, F28, F29, F30, F31, F32, F 33, F34, F35, F36, F37, F38, F39, F40, F41, F42, F43, F44, F45, F46, F47, F 48, F49, F50, F51, F52, F53, F54, F55, F56, F57, F58, F59, F60, F61 and F62 The excipient combination designations of distinct mol percentages disclosed in Table F, selected from the group consisting of the following: 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, E 30, 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, E 57, 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 It comprises an excipient selected from the group consisting of the following, as disclosed in Table E.

[0339] A particularly preferred embodiment of the lipid nanoparticles of the present invention is given when the combination F1 × E23 according to Tables E and F, i.e., 59 mol% of the cationic lipid C23 disclosed in Table 1, i.e., COATSOME® SS-EC (formerly known as SS-33 / 4PE-15, as is evident from the examples section; NOF Corporation, Tokyo, Japan), 29.3 mol% of cholesterol as a steroid, 10 mol% of DPhyPE as a neutral lipid / phospholipid, and 1.7 mol% of DMG-PEG2000 as a polymer conjugate lipid, is used to formulate the lipid nanoparticles. The LNP composition is referred to as "GN01" herein and in the examples. SS-EC has a positive charge at pH 4 and a neutral charge at pH 7, which is advantageous for the LNP and formulation / composition of the present invention. For "GN01", the N / P (mol ratio of lipids to mRNA) is preferably 14, and the total lipid / mRNA mass ratio is preferably between about 20 and about 60, more preferably between about 30 and about 50, and most preferably 40 (m / m).

[0340] A more particularly preferred embodiment of the lipid nanoparticles of the present invention is given when the combination F1×E2 according to Tables E and F, i.e., C2 lipids disclosed in Table 1 as 59 mol% cationic lipids (i.e., HEXA-C5DE-PipSS as is evident from the Examples section and Figure 1B), cholesterol as 29.3 mol% steroids, DPhyPE as 10 mol% neutral lipid / phospholipid and DMG-PEG2000 as 1.7 mol% polymer conjugate lipid is used to formulate the lipid nanoparticles. The LNP composition is referred to herein and in the Examples as "GN02". For "GN02", the N / P (mol ratio of lipid to mRNA) is preferably 17.5, and the total lipid / mRNA mass ratio is preferably between about 20 and about 60, more preferably between about 30 and about 50, and most preferably 40 (m / m).

[0341] Another particularly preferred embodiment of the lipid nanoparticles of the present invention is given when the combination F1×E23 according to Tables E and F, i.e., 59 mol% of the cationic lipid C23 disclosed in Table 1, i.e., COATSOME® SS-EC (formerly known as SS-33 / 4PE-15, as is evident from the Examples section; NOF Corporation, Tokyo, Japan), 26 mol% cholesterol as a steroid, 10 mol% DPhyPE as a neutral lipid / phospholipid, and 5 mol% Cer8 as a polymer conjugate lipid containing a shorter alkyl chain, is used to formulate the lipid nanoparticles. The LNP composition is referred to herein and in the Examples as "GN01-C8". For "GN01-C8", the N / P (mol ratio of lipid to mRNA) is preferably 14, and the total lipid / mRNA mass ratio is preferably between about 20 and about 60, more preferably between about 30 and about 50, and most preferably 40 (m / m).

[0342] A more particularly preferred embodiment of the lipid nanoparticles of the present invention is given when the combination F1 × E72 according to Tables E and F, i.e., C26 lipid disclosed in Table 1 as 59 mol% cationic lipid (i.e., THIOETHER as is evident from the Examples section, Figure 25A), cholesterol as 29.3 mol% steroid, DPhyPE as 10 mol% neutral lipid / phospholipid and DMG-PEG2000 as 1.7 mol% polymer conjugate lipid, is used to formulate the lipid nanoparticles. The LNP composition is referred to herein and in the Examples as "LNP28". For "LNP28", the N / P (mol ratio of lipid to mRNA) is preferably 14, and the total lipid / mRNA mass ratio is preferably between about 20 and about 60, more preferably between about 30 and about 50, and most preferably 40 (m / m).

[0343] Furthermore, regarding preferred compositions, (i) Cationic lipids may be selected from the compounds in Table 1; and / or (ii) Neutral lipids or neutral phospholipids, sometimes combined with 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC), such as 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE; also called 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 Phosphorus (DOPC; also called dioleoylphosphatidylcholine), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, also called 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), dipalmitoylphosphatidylethanolamine (D PPE), dimystoylphosphoethanolamine (DMPE), 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine (DLoPE), distearoylphosphatidylethanolamine (DSPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 16-O-monomethylphosphoethanolamine, 16-O-dimethylphosphatidylethanolamine, 1,2-Diellcoyl-sn-glycero-3-phosphoethanolamine (DEPE), 18-1-transphosphatidylethanolamine, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), 1,2-disqualeoyl-sn-glycero-3-phosphoethanolamine (DSQPE), 1,2-diellidoyl-sn-glycero-3-phosphoethanolamine (transDOPE), 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) (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) (sodium salt), 1,2-diphytanyl-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-cholesteryl-hemisuccinoyl-sn-glycero-3-phosphocholine It is a zwitterionic compound selected from the group consisting of (PChemsPC), 1,2-dicholesterylhemisuccinoyl-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); and / or, (iii) Polymer conjugate lipids include: pegylated diacylglycerol lipids (PEG-DAG); pegylated ceramide lipids (PEG-Cer); pegylated phosphatidylethanolamine lipids (PEG-PE); pegylated succinate diacylglycerol lipids (PEG-S-DAG); pegylated dialkoxypropyl carbamate lipids; 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol ("PEG-DMG" or "DMG-PEG"); 1,2-dicapryl-rac-glycero-3-methylpolyoxyethylene glycol (C 10 The following can be selected from the group consisting of diacylglycerol PEG; N-octanoyl-sphingosine-1-succinyl[methoxy(polyethylene glycol)](PEG-ceramide 8 or PEG-Cer8); 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)butanediate (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(tetradecaneoxy)propyl)carbamate; and 2,3-di(tetradecaneoxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate.

[0344] Alternatively, the composition may be provided in solid form. In particular, the composition may be provided as a sterile solid composition for reconstitution with a sterile liquid carrier; in this case, the solid composition may further contain one or more inert components selected from pH modifiers, fillers, stabilizers, nonionic surfactants, and antioxidants. In this embodiment, the sterile liquid carrier is preferably an aqueous carrier.

[0345] The zeta potential of a nanoparticle composition can be used to indicate the interfacial dynamic potential of the composition. For example, the zeta potential may describe the surface charge of the nanoparticle composition. Lipid nanoparticles according to the present invention may 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 using, for example, the Malvern Zetasizer Nano (Malvern Instruments Ltd.; located in Malvern, UK). Depending on the amount and nature of the charged compounds in the lipid nanoparticles, the nanoparticles may be characterized by their zeta potential. In a preferred embodiment, the zeta potential is in the range of about -50 mV to about +50 mV. In another preferred embodiment, 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 approximately -10mV to approximately +20mV, approximately -10mV to approximately +15mV, approximately -10mV to approximately +10mV, approximately -10mV to approximately +5mV, approximately -10mV to approximately 0mV, approximately -10mV to approximately -5mV, approximately -5mV to approximately +20mV, approximately -5mV to approximately +15mV, approximately -5mV to approximately +10mV, approximately -5mV to approximately +5mV, approximately -5mV to approximately 0mV, approximately 0mV to approximately +20mV, approximately 0mV to approximately +15mV, approximately 0mV to approximately +10mV, approximately 0mV to approximately +5mV, approximately +5mV to approximately +20mV, approximately +5mV to approximately +15mV, or approximately +5mV to approximately +10mV.

[0346] In certain embodiments, the LNP comprises one or more targeting moieties that can target the LNP to a cell or a population of cells. For example, in one embodiment, the targeting moiety is a ligand that directs the LNP to a receptor found on the cell surface.

[0347] In certain embodiments, the LNP comprises one or more internalization domains. For example, in one embodiment, the LNP comprises one or more domains that bind to cells and induce the internalization of the LNP. For example, in one embodiment, one or more internalization domains bind to receptors found on the cell surface to induce receptor-mediated uptake of the LNP. In certain embodiments, the LNP can bind to a biomolecule in vivo, thereby allowing the LNP-bound biomolecule to be recognized by a cell surface receptor and induce internalization. For example, in one embodiment, the LNP binds to systemic ApoE, which leads to the uptake of the LNP and associated cargo. In certain embodiments of the present invention, ApoE can be supplemented in the medium or pharmaceutical composition used.

[0348] Preferably, in one embodiment, the composition of the present invention further comprises a bioactive ingredient. bioactive ingredients As used herein, bioactive ingredient means any compound or material having bioactivity, and for that purpose, this compound or material is potentially useful for preventing, managing, improving, treating or curing diseases or conditions in subjects such as animals, and especially in human subjects.

[0349] In one preferred embodiment, the active compound is a nucleic acid compound. Examples of nucleic acid compounds that are potentially useful for carrying out the present invention include nucleic acid compounds selected from the group consisting of chemically modified or unmodified messenger RNA (mRNA), chemically modified or unmodified RNA, single-stranded or double-stranded RNA, coding or non-coding RNA, viral RNA, replicon RNA, and self-replicating RNA, or any combination thereof; preferably, the biologically active component is mRNA.

[0350] In preferred embodiments, nucleic acid compounds complex or associate with one or more lipids (e.g., cationic lipids and / or neutral lipids) to form liposomes, lipid nanoparticles (LNPs), lipoplexes, and / or nanoliposomes. In this context, the terms “complexed” or “associated” refer to an intrinsically stable combination of the nucleic acid compound of the first embodiment and one or more lipids to form a larger complex or aggregate, without the use of covalent bonds.

[0351] In specific embodiments, the active ingredient may include CRISPR RNA (crRNA) + tracer RNA (tracrRNA), guide RNA (gRNA), or single guide RNA (sgRNA) and / or donor DNA combined with a CRISPR endonuclease. Preferably, the CRISPR endonuclease may be provided as a protein or polypeptide, or as mRNA encoding the CRISPR endonuclease. Compositions or formulations containing this combination are suitable for delivering CRISPR gene-editing activity to target cells. In one embodiment, the composition according to the present invention may provide gRNA and mRNA encoding a CRISPR endonuclease for separate, sequential, or simultaneous administration. That is, the gRNA and mRNA may be provided in the same formulation or lipid nanoparticles according to the present invention, or in separate lipid nanoparticles for separate, simultaneous, or sequential administration. Preferably, the ratio of gRNA to mRNA for administration is, for example, 1:1, 1:3, 1:9, 1:19 (i.e., 50%, 25%, 10%, and 5% guide RNA). In one embodiment, gRNA and mRNA encoding a CRISPR endonuclease such as Cas9 are co-packed into the formulation according to the present invention. Advantageously, co-packing makes it possible to obtain better encapsulation efficiency (EE). Preferably, the formulation or pharmaceutical composition according to the present invention, co-packed with gRNA and mRNA, contains LNPs having an average diameter between 80 and 160 nm. In one embodiment, the gRNA may be a modified gRNA sequence. Suitable modifications are described, for example, in International Publication No. 2016 / 089433 and PCT / GB2016 / 053312. Other suitable modifications are well known to those skilled in the art.

[0352] "CRISPR endonuclease" refers to an endonuclease that can be used in a CRISPR gene editing composition. Suitable "CRISPR endonucleases" include cas9 and its variants and modified forms. Therefore, the mRNA for use in combination with the gRNA is one that encodes a CRISPR endonuclease, preferably cas9. Other "CRISPR endonucleases" include, for example, cpf1. Those skilled in the art will notice that a gRNA forms a pair with a specific "CRISPR endonuclease". Therefore, the present invention aims to provide a composition using a suitable gRNA / endonuclease pair. Preferably, the gRNA is specific to a target gene, and preferably, the target gene is a gene associated with liver disease.

[0353] In another embodiment, the peptide or protein expressed by a 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 for improving the condition of an individual. In particular, among its many functions, therapeutic proteins play a crucial role in the design of novel therapeutic agents that can modify and repair gene defects, destroy cancer cells or pathogen-infected cells, treat or prevent immune system disorders, or treat or prevent metabolic or endocrine disorders.

[0354] In another embodiment, the antigen is a peptide or protein expressed by a nucleic acid compound. As defined more specifically above herein, the antigen is a compound or material that can be recognized by the immune system, preferably by the adaptive immune system, and can induce, for example, an antigen-specific immune response.

[0355] In some embodiments, the active ingredient is siRNA. siRNA is a small interfering RNA, as described, for example, in international patent application PCT / EP03 / 08666. These molecules consist of a double-stranded RNA structure containing 15 to 25, preferably 18 to 23, nucleotide pairs that are essentially complementary to each other, typically mediated by Watson-Crick base pairing. One strand of this double-stranded RNA molecule is essentially complementary to the target nucleic acid, preferably mRNA, and the second strand of the double-stranded RNA molecule is essentially identical to the elongation of the target nucleic acid. The siRNA molecule may have several additional nucleotides adjacent to each other, each on each side and in each elongation, which do not necessarily have to be base-paired to each other.

[0356] In some embodiments, the active ingredient is RNAi. RNAi has essentially the same design as siRNA, but the molecule is significantly longer than siRNA. RNAi molecules typically contain 50 or more nucleotides and base pairs.

[0357] In some embodiments, the active ingredient is an antisense nucleic acid. Preferably, the antisense nucleic acid used herein is an oligonucleotide that hybridizes with target RNA, preferably mRNA, based on base complementarity, thereby activating RNase H. RNase H is activated by both phosphodiester-bound DNA and phosphothioate-bound DNA. However, phosphodiester-bound DNA is rapidly degraded by cellular nucleases, while phosphothioate-bound DNA is not. Therefore, antisense polynucleotides are effective only as DNA-RNA hybrid complexes. The preferred length of the antisense nucleic acid is in the range of 16 to 23 nucleotides. Examples of this type of antisense oligonucleotide are described, among others, in U.S. Patent No. 5,849,902 and U.S. Patent No. 5,989,912.

[0358] In some embodiments, the active ingredient is a ribozyme. A ribozyme is preferably a catalytically active nucleic acid consisting of RNA that essentially comprises two parts. The first part exhibits catalytic activity, and the second part is responsible for specific interaction with the target nucleic acid. Typically, interaction between the target nucleic acid and the aforementioned part of the ribozyme occurs through hybridization of essentially complementary extensions of bases on two hybridizing strands and Watson-Crick base pairing, which can activate the catalytically active part, meaning that this part cleaves the target nucleic acid intramolecularly or intermolecularly, if the catalytic activity of the ribozyme is phosphodiesterase activity. The principles of ribozymes, their use, and design are known to those skilled in the art and are described, for example, in Doherty and Doudna (Annu. Ref. Biophys. Biomolstruc. 2000:30:457-75).

[0359] In some embodiments, the active ingredient is an aptamer. An aptamer is a single-stranded or double-stranded D-nucleotide that specifically interacts with a target molecule. The preparation or selection of aptamers is described, for example, in European Patent No. 0533838. In contrast to RNAi, siRNA, antisense nucleotides, and ribozymes, aptamers do not degrade any target mRNA but specifically interact with the secondary and tertiary structures of target compounds, such as proteins. Upon interaction with a target, the target typically exhibits a change in its biological activity. The length of aptamers typically ranges from a minimum of 15 to a maximum of about 80 nucleotides, preferably in the range of about 20 to about 50 nucleotides.

[0360] In some embodiments, the active ingredient is a spiegelmer, which is described, for example, in International Publication No. 98 / 08856. Spiegelmers are molecules similar to aptamers; however, in contrast to aptamers, spiegelmers consist entirely or almost entirely of L-nucleotides rather than D-nucleotides. In other respects, particularly with regard to the possible length of a spiegelmer, the same things outlined for aptamers apply to spiegelmers.

[0361] mRNA In one preferred embodiment, the nucleic acid compound is mRNA or an mRNA compound. As discovered by the inventors, the lipids and compositions according to the present invention are particularly suitable for in vivo delivery of mRNA compounds expressing antigens, thus enabling highly effective, potent, versatile, and safe vaccines that can be developed rapidly at an affordable cost. Specific antigens of interest for carrying out the present invention are described in further detail below. The mRNA compounds according to the present invention are encapsulated in lipid nanoparticles or associated with lipid nanoparticles.

[0362] The advantages of including at least one antigenic peptide or protein-coding mRNA in lipid nanoparticles (LNPs) are: - Induction of a strong humoral immune response; - Induction of B-cell memory; - Faster onset of immune defense; - Longer lifespan of induced immune responses; - Induction of widespread cellular T cell responses; - Induction of a (localized and transient) pro-inflammatory environment; - No induction of a systemic cytokine or chemokine response; - Excellent tolerability, no side effects, non-toxic; - Favorable stability features; - Many different antigens and compatible formulations: A larger antigen cocktail is feasible based on the same (manufacturing) technology; - No vector immunization is required; that is, the technology can be used to vaccinate the same target multiple times against multiple (different) antigens; - Speed, adaptability, simplicity, and scalability of manufacturing; That is the case.

[0363] In a particular embodiment, lipid nanoparticles are at least (i) cationic lipids and / or polymer conjugate lipids as defined herein; and (ii) mRNA compounds containing mRNA sequences encoding antigenic peptides or proteins Includes.

[0364] In other specific embodiments, the lipid nanoparticle composition is (a) Cationic lipids according to formula (I) as described herein; (b) Steroids; (c) neutral lipids; (d) Polymer conjugate lipids which are compounds of formula (II) as described herein; and (e) mRNA compounds encoding peptides or proteins Includes.

[0365] With respect to cationic lipids, steroids, neutral lipids, polymer-conjugated lipids, and mRNA compounds encoding peptides or proteins, the same choices, preferences, and alternatives described above for these features apply. For example, in one preferred embodiment, the peptide or protein expressed by the mRNA compound is an antigen.

[0366] The amount of cationic lipid to the amount of mRNA compound in lipid nanoparticles can also be expressed as a weight ratio (e.g., abbreviated as "m / m"). For example, lipid nanoparticles contain the mRNA compound in an amount such that they achieve a lipid-to-mRNA weight ratio in the range of about 20 to about 60, or about 10 to about 50. In other embodiments, the mass ratio is in the range of about 30 to about 50. In other embodiments, the ratio of cationic 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 very preferred embodiment of the present invention, the total lipid / mRNA mass ratio is about 40 or 40, i.e., about 40 or 40-fold excess mass 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.

[0367] Furthermore, the amount of cationic lipids may be selected considering the amount of nucleic acid cargo, such as mRNA compounds. In one embodiment, the N / P ratio can be in the range of about 1 to about 50. In another embodiment, the ranges are about 1 to about 20, about 1 to about 10, and about 1 to about 5. In one preferred embodiment, these amounts are selected to result in an N / P ratio of lipid nanoparticles or composition in the range of about 10 to about 20. In a further very preferred embodiment, the N / P is 14 (i.e., 14 mol excess positive charge to ensure mRNA encapsulation). In another very preferred embodiment, the N / P is 17.5 (i.e., 17.5 mol excess positive charge to ensure mRNA encapsulation).

[0368] In this context, the N / P ratio is defined as the molar ratio of nitrogen atoms ("N") of the basic nitrogen-containing group of a cationic lipid to the phosphate groups ("P") of nucleic acids incorporated into or associated with the lipid nanoparticle as bioactive cargo. The N / P ratio can be calculated, for example, based on the fact that 1 μg of RNA typically contains about 3 nmols of phosphate residues, given that 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 cationic groups. If two or more cationic lipids are present, the N value should be calculated based on all cationic lipids contained in the lipid nanoparticle.

[0369] The total amount of mRNA in lipid nanoparticles varies and can be defined according to the mRNA-to-total lipid w / w ratio. In one embodiment of the present invention, the mRNA-to-total lipid ratio is less than 0.06 w / w, preferably between 0.03 and 0.04 w / w.

[0370] 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, and even more preferably about 500 to about 5,000.

[0371] As mentioned, peptides or proteins expressed by mRNA compounds can be antigens. In other words, a composition comprises an mRNA compound containing mRNA encoding an antigenic peptide or protein, or a fragment, variant, or derivative thereof. Such antigens, or antigenic peptides or proteins, may preferably be derived from pathogenic antigens, tumor antigens, allergen antigens, or autoimmune autoantigens, or fragments or variants thereof, as defined herein.

[0372] pathogenic antigen Pathogenic antigens are derived from pathogenic organisms, particularly bacteria, viruses, and protozoan (multicellular) pathogenic organisms, and evoke immunological responses in targets, especially mammalian targets, and more particularly in humans. More specifically, pathogenic antigens are preferably surface antigens located on the surface of viruses, bacteria, or protozoan organisms, such as proteins (or fragments of proteins, such as the outer part of a surface antigen).

[0373] Therefore, in some preferred embodiments, an artificial nucleic acid (RNA) molecule may encode, in at least one coding region, at least one pathogenic antigen selected from bacterial, viral, fungal, or protozoan antigens. The encoded (poly)peptide or protein may consist of, or contain, a fragment, variant, or derivative of the pathogenic antigen or the same.

[0374] The pathogenic antigen is preferably, but not limited to, a peptide or protein antigen derived from a pathogen associated with an infectious disease, selected from the group of pathogen-derived antigens disclosed on pages 21-35 of International Publication No. 2018 / 078053; International Publication No. 2018 / 078053 in its entirety is incorporated herein by reference. Furthermore, the pathogenic antigen is preferably, but not limited to, a peptide or protein antigen derived from a pathogen associated with an infectious disease, selected from the group of pathogen-derived antigens disclosed on pages 57, paragraph 3 to 63, paragraph 2 of International Publication No. 2019 / 077001; International Publication No. 2019 / 077001 in its entirety is incorporated herein by reference.

[0375] Further pathogenic antigens are preferably, but not limited to, peptides or proteins derived from pathogens associated with infectious diseases, selected from the group of antigens derived from pathogens disclosed on pages 32, line 26 to 34, line 27 of International Publication No. 2013120628. In this regard, pathogenic antigens (antigens derived from pathogens associated with infectious diseases) may be preferably, but not limited to, antigens derived from antigens selected from the group of antigens disclosed on pages 34, line 29 to 59, line 5 of International Publication No. 2013120628 (wherein parentheses are the family of pathogens from which a particular pathogen or antigen is derived and the infectious disease associated with the pathogen); International Publication No. 2013120628 as a whole is incorporated herein by reference.

[0376] Preferred antigens expressed by mRNA compounds incorporated into the composition of the present invention include, but are not limited to, SARS coronavirus 2 (SARS-CoV-2), nCoV-2019 coronavirus, SARS coronavirus (SARS-CoV), Bunyavirales viruses, cytomegalovirus (CMV), dengue virus (DEN-1, DEN-2, DEN-3 and DEN-4), Ebola virus, 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, extraenteropathogenic Escherichia coli (E. coli), Lassa mammarenavirus (LASV), MERS coronavirus, and Mycobacterium tuberculosis. The pathogen is selected from the group consisting of tuberculosis), nipah virus, norovirus, rabies virus, synovial respiratory virus (RSV), rhinovirus, rotavirus, vaccinia virus, yellow fever virus, Zika virus, Chlamydia trachomatis (i.e., Chlamydia, the bacterium that causes chlamydia), and malaria parasites (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 parasites, influenza virus, or rabies virus.

[0377] Furthermore, pathogenic antigens are more preferably, but not limited to, Acinetobacter baumannii, Anaplasma, Anaplasma phagocytophilum, Ancylostoma braziliense, Ancylostoma duodenale, Arcanobacterium haemolyticum, Ascaris lumbricoides, Aspergillus, Astroviridae, Babesia, Bacillus anthracis, Bacillus cereus, and Bartonella hensele. henselae), BK virus, Blastocystis hominis, Blastomyces dermatitidis, Bordetella pertussis, Borrelia burgdorferi, Borrelia genus, Borrelia species, Brucella genus, Brugia malayi, Bunyaviridae family, Burkholderia cepacia and other Burkholderia species, Glanders bacillus, Burkholderia meridian (pseudomallei), Caliciviridae, Campylobacter genus, Candida albicans, Candida species, Chlamydia trachomatis, Chlamydophila pneumoniae, Chlamydophila psittacosispsittaci), CJD prion, liver fluke (Clonorchis sinensis), Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium species, Clostridium tetani, Coccidioides species, coronavirus, Corynebacterium diphtheriae, Coxiella burnetii, Crimean-Congo hemorrhagic fever virus, Cryptococcus neoformans, Cryptosporidium, cytomegalovirus (CMV), dikaryocytovirus (Dientamoeba) fragilis), Ebola virus (EBOV - e.g., envelope glycoprotein), Echinococcus, Ehrlichia chaffeensis, Ehrlichia ewingii, Ehrlichia, Entamoeba histolytica, Enterococcus, Enterovirus, Enterovirus, mainly Coxsackie A virus and Enterovirus 71 (EV71), Epidermophyton species, Epstein-Barr virus (EBV), Escherichia coli O1 57:H7, O1 1 1 and O1 04:H4, hepatitis flea (Fasciola hepatica) and giant hepatitis flea (Fasciola FFI prion, feline immunodeficiency virus (FIV), filariasis, flavivirus, Francisella tularensis, Fusobacterium, Geotrichum candidumGiardia candidum, Giardia intestinalis, Gnathostoma species, GSS prion, Guanarito virus, Haemophilus ducreyi, Haemophilus influenzae, Helicobacter pylori, Henipavirus (Henclra virus, Nipah virus), Hepatitis A virus, Hepatitis B virus (HBV), Hepatitis C virus (HCV), Hepatitis D virus, Hepatitis E virus, Histoplasma capsulatum, Hortaea werneckii werneckii), human bocavirus (HBoV), human metapneumovirus (hMPV), human parainfluenza virus (HPIV), Japanese encephalitis virus, JC virus, Junin virus, Kingella kingae, Klebsiella granulomatis, Couloprin, Lassa virus, Legionella pneumophila, Leishmania, Leptospira, Listeria monocytogenes, lymphocytic choriomeningitis virus (LCMV), Machupo virus, Malassezia species, Marburg virus, measles virus, Metagonimus yokagawai, Microsporidia Mycobacterium phylum), molluscum contagiosum virus (MCV), mumps virus, Mycobacterium leprae and Mycobacterium lepromatosis, Mycobacterium tuberculosistuberculosis), Mycobacterium ulcerans, Mycoplasma pneumoniae, Naegleria fowleri, Necator americanus, Neisseria gonorrhoeae, Neisseria meningitidis, Nocardia asteroides, Nocardia species, Onchocerca volvulus, Orientia tsutsugamushi, Orthomyxoviridae (influenza), Paracoccidioides brasiliensis, Paragonimus species, Paragonimus westermani Westernan), parvovirus B19, Pasteurella, Plasmodium, Pneumocystis jirovecii, poliovirus, rhinovirus, rhinovirus, Rickettsia akari, Rickettsia, Rickettsia prowazekii, Rickettsia rickettsii, Rickettsia typhi, Rift Valley fever virus, rotavirus (preferably, e.g., VP8 antigen), rubella virus, Sabia virus, Salmonella, Sarcoptes Schistosoma scabiei, SARS coronavirus, Schistosoma, Shigella, Sin Nombre virus, Hantavirus, Sporothrix schenkyiStaphylococcus schenckii, Staphylococcus, 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, Trichomonas vaginalis Vaginalis), Trichophyton species, whipworm (Trichuris trichiura), Trypanosoma brucei, Trypanosoma cruzi, Ureaplasma urealyticum, Vaccinia virus (preferably, for example, 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 enterocolitis Enterocolitica), Yersinia pestis, and Yersinia pseudotuberculosis, Zika virus, Zika SPH2015 - Brazil, Z1106033 - Suriname, MR766 - Uganda or NatalAntigens derived from pathogens selected from the group consisting of RGN, or isoforms, homologs, fragments, variants, or derivatives of any of these proteins, may be selected. Particularly preferred pathogenic antigens are those derived from the pathogen SARS coronavirus, in particular the SARS coronavirus spike protein (S).

[0378] In further embodiments, pathogenic antigens useful for treating infectious diseases may be selected from the following antigens (the relevant infectious diseases and associated pathogens are indicated in parentheses after each antigen – naturally, other antigens that can be derived from the pathogens in parentheses below may also be derived and used according to the present invention): • 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 human papillomavirus (HPV) infection; pathogen: human papillomavirus (HPV) or HPV16); • Fusion protein F, hemagglutinin-neuraminidase (HN), glycoprotein G, matrix protein M, phosphoprotein P, nucleoprotein N, polymerase L, hemagglutinin-neuraminidase, fusion (F) glycoprotein F0, F1 or F2, recombinant PIV3 / PIV1 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 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, H1 1, H12, H13, H14, H15, H16, H17, H18, HA antigenic subdomain, HA1, HA2, neuraminidase (NA), nucleoprotein (NP), matrix protein 1 (M1), matrix protein 2 (M2), non-structural protein 1 (NS1), non-structural protein 2 (NS2), HA7 antigen, H7 or H10 and B, pathogen: Orthomyxoviridae, influenza virus (flu); • Nucleoprotein N, macrostructural protein L, phosphoprotein P, matrix protein M, glycoprotein G, G protein (the infectious disease is rabies; pathogen: rabies virus); • HIV p24 antigen, HIV envelope proteins (Gp120, Gp41, Gp160), polyprotein GAG, negative factor protein Nef, transcriptional transactivator Tat, Brec1 (infectious disease HIV; pathogen: human immunodeficiency virus); • Major outer membrane protein MOMP, highly probable outer membrane protein PMPC, outer membrane complex protein B OmcB, heat shock protein Hsp60 HSP10, protein IncA, type III secretory protein, ribonucleotide reductase small chain protein NrdB, plasmid protein Pgp3, Chlamydia 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 disease: Infection caused by Chlamydia trachomatis; Pathogen: Chlamydia trachomatis); · pp65 antigen, membrane protein pp15, capsid proximal envelope protein pp150, protein M45, DNA polymerase UL54, helicase UL105, glycoprotein gM, glycoprotein gN, glycoprotein H, glycoprotein B gB, protein UL83, protein UL94, protein UL99, HCMV glycoprotein selected from gH gL, gB, gO, gN, and gM, HCMV protein selected from UL83, UL123, UL128, UL130, and UL131A, envelope protein pp150 (pp150), envelope 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) (The 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 (DEN-1, DEN-2, DEN-3 and DEN-4)); ·EBOV glycoprotein (GP), surface EBOV GP, wild-type EBOV proGP, mature EBOV GP, secreted wild-type EBOV proGP, secreted mature EBOV GP, EBOV nucleoprotein (NP), RNA polymerase L, and EBOV matrix proteins selected from VP35, VP40, VP24, and VP30 (infectious disease: Ebola; pathogen: Ebola virus); • 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, non-structural protein 1 NS1, non-structural protein 2 NS2, RSV attachment protein (G) (glycoprotein G), fusion (F) glycoprotein (glycoprotein F), nucleoprotein (N), phosphoprotein (P), large polymerase protein (L), matrix proteins (M, M2), small hydrophobic protein (SH), non-structural protein 1 (NS1), non-structural protein 2 (NS2), membrane-bound RSV F protein, membrane-bound DS-Cavl (pre-stabilized fusion RSV F protein) (Infectious diseases are infections caused by respiratory syncytial virus (RSV): pathogen: respiratory syncytial virus (RSV)); • Secretory antigen SssA (Staphylococcus genus, staphylococcal food poisoning); secretory antigen SssA (Staphylococcus genus, e.g., Staphylococcus aureus, staphylococcal infection); molecular chaperone DnaK, cell surface lipoprotein Mpt83, lipoprotein P23, phosphate transporter permease protein pstA, 14kDa antigen, fibronectin-binding protein C FbpC1, alanine dehydrogenase TB43, glutamine synthase 1, ESX-1 protein, protein CFP10, TB10.4 protein, protein MPT83, protein MTB12, protein MTB8, Rpf-like protein, protein MTB32, protein MTB39, crystallin, heat shock protein HSP65, protein PST-S (infectious disease is tuberculosis; pathogen: Mycobacterium tuberculosis): • Genome polyproteins, 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) • Sporozoite surface proteins (CSPs) (the infectious disease is malaria; pathogens: Plasmodium falciparum and Plasmodium vivax); and Zika virus proteins according to International Publication No. 2017 / 140905, namely, Zika virus capsid protein (C), Zika virus membrane precursor protein (prM), Zika virus pr protein (pr), Zika virus membrane protein (M), Zika virus envelope protein (E), Zika virus non-structural protein, ZIKV prME antigen, ZIKV capsid protein, membrane precursor / membrane protein, ZIKV envelope protein, ZIKV non-structural protein 1, ZIKV non-structural protein 2A, ZIKV non-structural protein 2B, ZIKV non-structural protein 3, ZIKV non-structural protein 4A, ZIKV non-structural protein 4B, ZIKV non-structural protein 5, or Zika virus envelope protein (E) (with the fusion loop of domain II mutated according to International Publication No. 2017 / 140905); International Publication No. 2017 / 140905 in its entirety is incorporated herein by reference (the infectious disease is Zika virus infection; pathogen: Zika virus).

[0379] In some embodiments of the present invention, a disclosure is provided of a method for inducing an antigen-specific immune response in a subject, comprising the step of administering to the subject one of the RNA (e.g., mRNA) vaccines provided herein in an amount effective to produce an antigen-specific immune response.

[0380] In some embodiments, RNA (e.g., mRNA) vaccines may target SARS coronavirus 2 (SARS-CoV-2), nCoV-2019 coronavirus, SARS coronavirus (SARS-CoV), Bunyavirales viruses, cytomegalovirus (CMV), dengue virus (DEN-1, DEN-2, DEN-3 and DEN-4), Ebola virus, 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, extraenteropathogenic Escherichia coli (E. coli), Lassa mum arenavirus (LASV), MERS coronavirus, and Mycobacterium tuberculosis. These include tuberculosis), nipah virus, norovirus, rabies virus, synovial respiratory virus (RSV), rhinovirus, rotavirus, vaccinia virus, yellow fever virus, Zika virus, Chlamydia trachomatis (i.e., the bacterium Chlamydia that causes chlamydia), and malaria parasites (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 parasites, influenza virus, or rabies virus vaccine. In other embodiments, RNA (e.g., mRNA) vaccines are COVID-19, rabies, influenza, or malaria vaccines.

[0381] In some embodiments, the RNA (e.g., mRNA) vaccine is a combination vaccine (broad-spectrum influenza vaccine) that includes a combination of influenza vaccines. In some embodiments, the antigen-specific immune response includes a T-cell response or a B-cell response.

[0382] In some embodiments, the method for eliciting an antigen-specific immune response involves a single dose (i.e., without a booster dose) of SARS coronavirus 2 (SARS-CoV-2), nCoV-2019 coronavirus, SARS coronavirus (SARS-CoV), Bunyavirales viruses, cytomegalovirus (CMV), dengue virus (DEN-1, DEN-2, DEN-3 and DEN-4), Ebola virus, 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, extraenteropathogenic Escherichia coli (E. coli), Lassa mum arenavirus (LASV), MERS coronavirus, and Mycobacterium tuberculosis. The process includes administering a target to a parasite (e.g., tuberculosis), nipah virus, norovirus, rabies virus, synovial respiratory virus (RSV), rhinovirus, rotavirus, vaccinia virus, yellow fever virus, Zika virus, Chlamydia trachomatis (i.e., Chlamydia, the bacterium that causes chlamydia), and malaria parasites (e.g., Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, or Plasmodium ovale). In another preferred embodiment, the pathogenic antigen is derived from a SARS coronavirus 2 (SARS-CoV-2), nCoV-2019 coronavirus, SARS coronavirus (SARS-CoV), malaria parasite, influenza virus, or rabies virus (e.g., mRNA) vaccine of the present disclosure.

[0383] In some embodiments, the method involves a second (booster) dose of SARS coronavirus 2 (SARS-CoV-2), nCoV-2019 coronavirus, SARS coronavirus (SARS-CoV), Bunyavirales viruses, cytomegalovirus (CMV), dengue virus (DEN-1, DEN-2, DEN-3 and DEN-4), Ebola virus, 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, extraenteropathogenic Escherichia coli (E. coli), Lassa mum arenavirus (LASV), MERS coronavirus, Mycobacterium tuberculosis. The process further includes administering a target of tuberculosis), nipah virus, norovirus, rabies virus, synovial respiratory virus (RSV), rhinovirus, rotavirus, vaccinia virus, yellow fever virus, Zika virus, Chlamydia trachomatis (i.e., Chlamydia, the bacterium that causes chlamydia), and malaria parasites (e.g., Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, or Plasmodium ovale). In another preferred embodiment, the pathogenic antigen is derived from a SARS coronavirus 2 (SARS-CoV-2), nCoV-2019 coronavirus, SARS coronavirus (SARS-CoV), malaria parasite, influenza virus, or rabies virus RNA (e.g., mRNA) vaccine.

[0384] In some embodiments, subjects exhibit an antibody seroconversion rate of at least 80% (e.g., at least 85%, at least 90%, or at least 95%) after a first or second (booster) dose of the vaccine. Antibody seroconversion is the period during which specific antibodies develop and become detectable in the blood. After antibody seroconversion occurs, the virus can be detected by a blood test for antibodies. During infection or immunization, the antigen enters the bloodstream, and the immune system responds by beginning to produce antibodies. Before antibody seroconversion, the antigen itself may or may not be detectable, but antibodies are not considered to be present. During antibody seroconversion, antibodies are present but not yet detectable. At any time after antibody seroconversion, antibodies can be detected in the blood, indicating a previous or current infection. In some embodiments, RNA (e.g., mRNA) vaccines are administered to subjects by intradermal, intramuscular, or intranasal injection.

[0385] Some embodiments of this disclosure include SARS coronavirus 2 (SARS-CoV-2), nCoV-2019 coronavirus, SARS coronavirus (SARS-CoV), Bunyavirales viruses, cytomegalovirus (CMV), dengue virus (DEN-1, DEN-2, DEN-3 and DEN-4), Ebola virus, 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, extraenteropathogenic Escherichia coli (E. coli), Lassa mum arenavirus (LASV), MERS coronavirus, and Mycobacterium tuberculosis. The present invention provides a method for inducing an antigen-specific immune response in a subject, comprising the step of administering to the subject one of the following: tuberculosis, nipah virus, norovirus, rabies virus, synovial respiratory virus (RSV), rhinovirus, rotavirus, vaccinia virus, yellow fever virus, Zika virus, Chlamydia trachomatis (i.e., Chlamydia, the bacterium that causes chlamydia), and malaria parasites (e.g., Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, or Plasmodium ovale). In another preferred embodiment, the pathogenic antigen is derived from a SARS coronavirus 2 (SARS-CoV-2), nCoV-2019 coronavirus, SARS coronavirus (SARS-CoV), malaria parasite, influenza virus, or rabies virus RNA (e.g., mRNA) vaccine in an amount effective to elicit an antigen-specific immune response in the target.

[0386] Antigen-specific immune responses in the target include, in some embodiments, SARS coronavirus 2 (SARS-CoV-2), nCoV-2019 coronavirus, SARS coronavirus (SARS-CoV), Bunyavirales viruses, cytomegalovirus (CMV), dengue virus (DEN-1, DEN-2, DEN-3 and DEN-4), Ebola virus, 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, extraenteropathogenic Escherichia coli (E. coli), Lassa mum arena virus (LASV), MERS coronavirus, and Mycobacterium tuberculosis. (tuberculosis), Nipah virus, norovirus, rabies virus, synovial respiratory virus (RSV), rhinovirus, rotavirus, vaccinia virus, yellow fever virus, Zika virus, Chlamydia trachomatis (i.e., Chlamydia, the bacterium that causes chlamydia), or malaria parasites (e.g., Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, or Plasmodium ovale) After administration of an ovale))RNA (e.g., mRNA) vaccine to any of the target organisms, antibody titers (SARS coronavirus 2 (SARS-CoV-2), nCoV-2019 coronavirus, SARS coronavirus (SARS-CoV), Bunyavirales viruses, cytomegalovirus (CMV), dengue virus (DEN-1, DEN-2, DEN-3 and DEN-4), Ebola virus, 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, extraenteropathogenic Escherichia coli (E. coli)This can be determined by assaying the titer of antibodies that bind to antigenic polypeptides of Chlamydia trachomatis (i.e., Chlamydia, the bacterium that causes chlamydia), or Plasmodium falciparum (e.g., Plasmodium vivax, Plasmodium malariae, or Plasmodium ovale). In some embodiments, the titer of the anti-antigenic polypeptide antibody produced in the subject is increased by at least 1 log compared to the control. In some embodiments, the titer of anti-antigenic polypeptide antibodies produced in the subject increases by 1 to 3 log compared to the control.

[0387] Tumor antigen In a more preferred embodiment, the mRNA compound comprising mRNA preferably encodes a tumor antigen, or a fragment or variant thereof, as defined herein, the tumor antigen preferably selected from the group of tumor antigens disclosed on pages 45-51 of International Publication 2018 / 078053, but not limited thereto; International Publication 2018 / 078053 in its entirety is incorporated herein by reference.

[0388] Furthermore, the present invention includes, but is not limited to, cytokines, chemokines, suicide enzymes and gene products, apoptosis inducers, endogenous angiogenesis inhibitors, heat shock proteins, tumor antigens, innate immune activators, and antibodies directed against proteins associated with tumors or cancer development, which are useful, for example, in cancer treatment, and are selected from the group disclosed in Tables 1, 2, 3, 4, 5, 6, 7, 8, 10, 11, and 12 of International Publication No. 2016 / 170176; International Publication No. 2016 / 170176 and Tables 1-12 in particular are incorporated herein by reference.

[0389] Therapeutic proteins and uses for treating or preventing genetic or acquired diseases In a further embodiment, the active ingredient is a nucleic acid compound comprising at least one coding sequence, wherein at least one coding sequence codes for a peptide or protein, and the protein is a therapeutic protein, or a fragment or variant of a therapeutic protein. In this context, the therapeutic peptide, protein or fragment thereof may be any peptidolytic compound useful for preventing, managing, improving, treating or mitigating a disease or condition in a subject such as an animal, particularly a human subject.

[0390] In fact, in one embodiment, mRNA containing at least one coding sequence, (a) A peptide or protein is an antigen, and the antigen is preferably a peptide or protein, or a fragment or variant thereof, derived from a pathogenic antigen, a tumor antigen, an allergen antigen or an autoimmune autoantigen, or a fragment or variant thereof; or (b) Therapeutic proteins, or fragments or variants thereof It can encode. Therapeutic proteins are, for example, (i) Therapeutic proteins for use in enzyme replacement therapy to treat metabolic, endocrine, or amino acid disorders, or for use in replacement of absent, deficient, or mutated proteins; (ii) Therapeutic proteins for use in the treatment of blood disorders, circulatory disorders, respiratory disorders, infectious diseases, or immunodeficiency; (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) Therapeutic proteins that are therapeutic antibodies; (viii) Therapeutic proteins that are gene editing agents; and (ix) Therapeutic proteins for use in the treatment or prevention of liver diseases selected from the group consisting of hepatic fibrosis, cirrhosis, and liver cancer. A selection can be made from the group consisting of the following:

[0391] In a specific embodiment, the therapeutic protein, or a fragment or variant thereof, Acid sphingomyelinase, adipotide, agalsidase-beta, alglucosidase, alpha-galactosidase A, alpha-glucosidase, alpha-L-iduronidase, alpha-N-acetylglucosaminidase, amphiregulin, angiopoietin (Ang1, Ang2, Ang3, Ang4, ANGPTL2, ANGPTL3, ANGPTL4, ANGPTL5, ANGPT L6, ANGPTL7), ATPase, Cu(2+) transporting beta polypeptide (ATP7B), argininosuccinate synthase (ASS1), beta-cellulose, beta-glucuronidase, bone morphogenetic proteins (BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP10, BMP15), CLN6 protein, epidermal growth factor (EGF), epigen, epiregulin, fiber Blast growth factors (FGF, FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, FGF-16, FGF-17, FGF-17, FGF-18, FGF-19, FGF-20, FGF-21, FGF-22, FGF-23), fumarylacetoacetate hydrolase (FAH), galsulfase ghrelin, glucocerebrosidase, GM-CSF, heparin-bound EGF-like growth factor (HB-EGF), hepatocyte growth factor (HGF), hepcidin, human albumin, increased albumin loss, idursulfase (iduronate-2-sulfatase), integrin αVβ3, αVβ5 and α5β1, iduronate sulfatase, laronidase, N-acetylgalactosamine-4-sulfatase (rhASB;Galsulfase, arylsulfatase A (ARSA), arylsulfatase B (ARSB), N-acetylglucosamine-6-sulfatase, nerve growth factor (NGF, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 / 5 (NT-4 / 5), neuregulin (NRG1, NRG2, NRG3, NRG4), neuropilin (NRP-1, NRP-2), obestatin, phenylalanine hydroxylase (PAH), phenylalanine ammonia lyase (PAL), platelet-derived growth factor (PDGF (PDGF-A, PDGF-B, PDGF-C, PDGF-D)), TGF-beta receptor (endoglin, TGF-beta-1 receptor, TGF-beta- TGF-2 receptor, TGF-beta3 receptor), thrombopoietin (THPO) (megakaryocyte growth and differentiation factor (MGDF)), transforming growth factors (TGF (TGF-α, TGF-beta (TGF-beta1, TGF-beta2, and TGF-beta3))), VEGF (VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and PIGF)), nesiritide, trypsin, adrenocorticotropic hormone (ACTH), atrial natriuretic peptide (ANP), cholecystokinin, gastrin, leptin, oxytocin, somatostatin, vasopressin (antidiuretic hormone), calcitonin, exenatide, growth hormone (GH), somatotropin, insulin, insulin-like growth factor 1 IGF-1, Mecasermin rinfabate, IGF-1 analog, Mecasermin, IGF-1 analog, Pegvisomant, Pramulintide, Teriparatide (human parathyroid hormone residues 1-34), Becaprelmine, Dibotermin-alpha (bone morphogenetic protein 2), Histrelin acetate (gonadotropin-releasing hormone;Therapeutic proteins for use in enzyme replacement therapy to treat metabolic, endocrine, or amino acid disorders, or for use in replacement of absent, deficient, or mutant proteins, including GnRH, octreotide, hepatocyte nuclear factor 4 alpha (HNF4A), CCAAT / enhancer-binding protein alpha (CEBPA), fibroblast growth factor 21 (EGF21), extracellular matrix protease or human collagenase MMP1, hepatocyte growth factor (HGF), TNF-related apoptosis-inducing ligand (TRAIL), opioid growth factor receptor-like 1 (OGFRL1), Clostridium type II collagenase, relaxin 1 (RLN1), relaxin 2 (RLN2), relaxin 3 (RLN3), and palifermin (keratinocyte growth factor, KGF); Alteplase (tissue plasminogen activator; tPA), anistreplase, antithrombin III (AT-III), bivalirudin, darbepoetin-alpha, drolecogin-alpha (activated protein C), erythropoietin, epoetin-alpha, erythropoietin, erythropoietin, factor IX, factor VIIa, factor VIII, repirudin, protein C concentrate, leteplase (tPA deletion mutant protein), streptokinase, tenecteplase, urokinase, angiostatin, anti-CD22 immunotoxin, denileukin difuticotoxin, immunosia Nin, MPS (Metallopanstimulin), Aflibercept, Endostatin, Collagenase, Star deoxyribonuclease I, Dolnase, Hyaluronidase, Papain, L-Asparaginase, Peg-Asparaginase, Rasburicase, Human chorionic gonadotropin (HCG), Human follicle-stimulating hormone (FSH), Lutropin-Alpha, Prolactin, Alpha-1-proteinase inhibitor, Lactase, Pancreatic enzymes (Lipase, Amylase, Protease), Adenosine deaminase (Pegademase / Bovine drug (Pegademase) Therapeutic proteins for use in the treatment of hematological disorders, circulatory disorders, respiratory disorders, cancer or tumor diseases, infectious diseases or immunodeficiency, including bovine, PEG-ADA, abatacept, alefacept, anakinra, etanercept, interleukin-1 (IL-1) receptor antagonists, anakinra, thymrin, TNF-alpha antagonists, enfuvirtide, and thymosin α1; Therapeutic proteins for use in the treatment of cancer or tumor diseases, including proteins or peptides that bind to cytokines, chemokines, suicide gene products, immunogenic proteins or peptides, apoptosis inducers, angiogenesis inhibitors, heat shock proteins, tumor antigens, beta-catenin inhibitors, STING pathway activators, checkpoint modulators, innate immune activators, antibodies, dominant-negative receptors and decoy receptors, myeloid-derived suppressor cell (MDSC) inhibitors, IDO pathway inhibitors, and apoptosis inhibitors; Human adjuvant proteins, especially pattern recognition receptors TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11; NOD1, NOD2, NOD3, NOD4, NOD5, NALP1, NALP2, NALP3, NALP4, NALP5, NALP6, NALP6, NALP7, NALP7, NALP8, NALP9, NALP10, NALP11, NALP12, NALP13, NALP14, Signaling factors of TLR signaling, including adapter proteins such as IPAF, NAIP, CIITA, RIG-I, MDA5, and LGP2, e.g., Trif and Cardif; components of low molecular weight GTPase signaling (RhoA, Ras, Rac1, Cdc42, Rab, etc.), components of PIP signaling (PI3K, Src kinase, etc.), components of MyD88-dependent signaling (MyD88, IRAK1, IRAK2, IRAK4, TIRAP, TRAF6, etc.), components of MyD88-independent signaling (TICAM1, TICAM2, TRAF6, TBK1, IRF3, TAK1, IRAK1, etc.); e.g., Akt, MEKK1, MKK1, MKK3, MKK4, MKK6, MKK7, ERK1, ERK2, GSK3, PKC kinase, PKD kinase, GSK3 kinase, JNK, p38MAPK, T Activation kinases including AK1, IKK, and TAK1; activation transcription factors including NF-κB, c-Fos, c-Jun, c-Myc, CREB, AP-1, Elk-1, ATF2, IRF-3, IRF-7, etc.; heat shock proteins such as HSP10, HSP60, HSP65, HSP70, HSP75, and HSP90; gp96; fibrinogen; and TypIII repeat extracts of fibrinogen. Rad domain A; or complement system components including C1q, MBL, C1r, C1s, C2b, Bb, D, MASP-1, MASP-2, C4b, C3b, C5a, C3a, C4a, C5b, C6, C7, C8, C9, CR1, CR2, CR3, CR4, C1qR, C1INH, C4bp, MCP, DAF, H, I, P, and CD59, or inducible target genes including, for example, beta-defensins, cell surface proteins;Therapeutic proteins selected from adjuvant or immunostimulatory proteins, including human adjuvant proteins containing trif, flt-3 ligand, Gp96, or fibronectin; cytokines that induce or enhance the innate immune response, including IL-1 alpha, IL-1 beta, IL-2, IL-6, IL-7, IL-8, IL-9, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18, IL-21, IL-23, TNF alpha, IFN alpha, IFN beta, IFN gamma, GM-CSF, G-CSF, and M-CSF; chemokines including IL-8, IP-10, MCP-1, MIP-1 alpha, RANTES, eotaxin, and CCL21; cytokines released from macrophages, including IL-1, IL-6, IL-8, IL-12, and TNF-alpha; and IL-1R1 and IL-1 alpha. Heat shock proteins or chaperones containing Hsp60, Hsp70, Hsp90, and Hsp100; OmpA (outer membrane protein) from Gram-negative bacteria; OspA; bacterial porins containing OmpF; pertussis toxin (PT) from Bordetella pertussis; pertussis adenylyl cyclase toxins CyaA and CyaC from Bordetella pertussis; PT-9K / 129G variant of pertussis toxin; pertussis adenylyl cyclase toxins CyaA and CyaC from Bordetella pertussis; tetanus toxin; cholera toxin (CT); cholera toxin B subunit; CTK63 variant of cholera toxin; CTE112K variant of CT; Escherichia coli heat-labile enterotoxin (LT); heat-labile enterotoxin B subunit (LTB) Reduced toxicity Escherichia coli (Escherichia coli) heat-labile enterotoxin variants including LTK63 and LTR72; phenol-soluble modulin; bacterial toxins including neutrophil-activating protein (HP-NAP) from Helicobacter pylori; surfactant protein D; outer surface protein A lipoprotein from Borrelia burgdorferi; Ag38 (38kDa antigen) from Mycobacterium tuberculosis; bacterial pili-derived proteins; enterotoxin CT from Vibrio cholerae; pyrin derived from Gram-negative bacterial pili; and bacterial (adjuvant) proteins including surfactant protein A and bacterial flagellin; Protozoan (adjuvant) proteins, including Tc52 from Trypanosoma cruzi, PFTG from Trypanosoma gondii, protozoan heat shock proteins, LeIF from Leishmania species, and profiling-like proteins from Toxoplasma gondii; Viral (adjuvant) proteins including respiratory syncytial virus fusion glycoprotein (F protein), MMT virus-derived envelope protein, mouse leukemia virus protein, and wild-type measles virus hemagglutinin protein; Fungal (adjuvant) proteins, including fungal immunomodulatory protein (FIP; LZ-8); Animal-derived protein containing keyhole limpet hemocyanin (KLH); Therapeutic proteins for use in hormone replacement therapy, the hormones of which include estrogen, progesterone or progestin, and testosterone; and Therapeutic proteins used to reprogram somatic cells into pluripotent or totipotent stem cells include Oct-3 / 4, the Sox gene family (Sox1, Sox2, Sox3, and Sox15), the Klf family (Klf1, Klf2, Klf4, and Klf5), the Myc family (c-myc, L-myc, and N-myc), Nanog, and LIN28. Selected from.

[0392] The present invention includes a method for preventing, improving, or treating a disease or condition in a subject, comprising the step of administering a composition described herein to a subject requiring such composition. The compositions of the present invention may be used for the treatment of the body of a human or animal.

[0393] In this context, particularly preferred therapeutic proteins that can be used, in particular, for the treatment of metabolic or endocrine disorders are selected from those disclosed in Table A (combined with Table C) of International Publication 2017 / 191274. Furthermore, diseases that can be treated with the compositions of the present invention, preferably selected from infectious diseases, neoplasms (e.g., cancer or tumor diseases), diseases of the blood and hematopoietic organs, endocrine, nutrition and metabolic diseases, diseases of the nervous system, diseases of the circulatory system, diseases of the respiratory system, diseases of the digestive system, diseases of the skin and subcutaneous tissue, diseases of the musculoskeletal system and connective tissue, and diseases of the genitourinary system, are disclosed on pages 95, line 4 to 103, line 24 of International Publication 2017 / 191274. Even more particularly preferred therapeutic proteins that can be used, in particular, for the treatment of metabolic or endocrine disorders are disclosed in Table 1 of International Publication 2017 / 191274, which also refers to specific target / disease combinations and sequences incorporated herein. International Publication No. 2017 / 191274, including Tables A / C and Table 1, is incorporated herein by reference.

[0394] In preferred embodiments, artificial nucleic acid (RNA) molecules, (pharmaceutical) compositions, or vaccines or kits are used to treat or prevent infectious diseases. The terms “infection” or “infectious disease” relate to the invasion and proliferation of microorganisms such as bacteria, viruses, and parasites that are not normally present in the body. Infections may be asymptomatic and subclinical, or they may be asymptomatic and clinically apparent. Infections may remain locally or they may spread through the blood or lymphatic system and become systemic. In this context, infectious diseases preferably include viral, bacterial, fungal, or protozoological infectious diseases. In particular, infectious diseases are selected from the group disclosed in the section “Infectious Diseases” (ending at page 160), beginning at page 157 of International Publication 2019 / 077001; International Publication 2019 / 077001 in its entirety is incorporated herein by reference.

[0395] In this context, more particularly preferred examples of diseases and / or conditions in which the compositions of the present invention or each of the translatable molecules of the present invention can be used in treatment are disclosed in Table 2 of U.S. Patent Application Publication No. 2019 / 0002906; U.S. Patent Application Publication No. 2019 / 0002906, including Table 2, is incorporated herein by reference.

[0396] Liver diseases or liver-related diseases in animals, and especially in humans, may include, but are not limited to, congenital or acquired diseases, such as viral and parasitic infectious diseases, oncological conditions such as primary tumors and metastases, metabolic, amino acid and / or endocrine disorders, and inflammatory, immune, and autoimmune conditions. Liver diseases that can preferably be treated with the compositions of the present invention are selected from the group consisting of, but are not limited to, hepatitis C, hepatitis B, hepatitis, hepatitis A, cirrhosis, liver cancer, hepatocellular carcinoma, hepatic encephalopathy, autoimmune hepatitis, Wilson's disease, alpha-1 antitrypsin deficiency (AAT deficiency), hepatitis D, phenylketonuria (PKU), Wilson's disease (hepatolenticular degeneration), tyrosinemia type I (FAH deficiency), Alagille syndrome, portal hypertension, fatty liver disease, chronic hepatitis, and hepatitis E.

[0397] In a more preferred embodiment, the compositions of the present invention may be used in a method to treat or prevent a disorder which is a liver disease, preferably selected from the group consisting of hepatic fibrosis, cirrhosis, and liver cancer. Thus, mRNA containing at least one coding sequence may encode a therapeutic protein or a fragment or variant thereof for use in treating or preventing a liver disease selected from the group consisting of hepatic fibrosis, cirrhosis, and liver cancer. Furthermore, preferably, mRNA for treating or preventing liver diseases selected from the group consisting of hepatic fibrosis, cirrhosis, and liver cancer encodes a peptide or protein selected from the group consisting of nuclear factor 4 alpha (HNF4A), CCAAT / enhancer-binding protein alpha (CEBPA), fibroblast growth factor 21 (FGF21), extracellular matrix protease or human collagenase MMP1, hepatocyte growth factor (HGF), TNF-related apoptosis-inducing ligand (TRAIL), opioid growth factor receptor-like 1 (OGFRL1), Clostridium type II collagenase, relaxin 1 (RLN1), relaxin 2 (RLN2), and relaxin 3 (RLN3). In this regard, the specific disclosure of liver diseases in International Publication No. 2018 / 104538 and the sequences disclosed in International Publication No. 2018 / 104538 are incorporated herein by reference.

[0398] Other antigens Further antigens useful for the present invention are listed on pages 48-51 of International Publication No. 2018 / 078053; International Publication No. 2018 / 078053 in its entirety is incorporated herein by reference.

[0399] Allergen antigens and autoimmune autoantigens As mentioned, the mRNA compounds contained in the compositions of the present invention may, according to some embodiments, encode antigens representing allergens, or allergen antigens, or autoantigens also called autoantigens or autoimmune antigens.

[0400] Such antigens and autoantigens (allergens or allergen antigens) associated with allergies or allergic diseases are derived from, or preferably selected from, the group of antigens disclosed on pages 59–73 of International Publication 2018 / 078053, but are not limited to those described herein; International Publication 2018 / 078053 in its entirety is incorporated herein by reference.

[0401] Checkpoint modulator / checkpoint inhibitor In the context of the present invention, immune checkpoint proteins, checkpoint modulators, or checkpoint inhibitors are typically molecules such as proteins (e.g., antibodies), dominant-negative receptors, decoy receptors, or their ligands, fragments, or variants that modulate the function of immune checkpoint proteins, for example, by inhibiting or reducing the activity of a checkpoint inhibitor (or inhibitory checkpoint molecule) or by stimulating or enhancing the activity of a checkpoint stimulant (or stimulating checkpoint molecule). Therefore, checkpoint modulators as defined herein affect the activity of checkpoint molecules. In this context, inhibitory checkpoint molecules are defined and may be used synonymously with checkpoint inhibitors. Furthermore, stimulating checkpoint molecules are defined and may be used synonymously with checkpoint stimulants.

[0402] In a more preferred embodiment, the mRNA compound comprising mRNA preferably encodes an immune checkpoint protein, checkpoint modulator, or checkpoint inhibitor, or a fragment or variant thereof, as defined herein, the immune checkpoint protein, checkpoint modulator, or checkpoint inhibitor preferably, but not limited to, selected from the group consisting of immune checkpoint proteins, checkpoint modulators, or checkpoint inhibitors disclosed on pages 51-56 of International Publication 2018 / 078053; International Publication 2018 / 078053 in its entirety is incorporated herein by reference.

[0403] RNA element, mRNA element 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 encodes a separate peptide or protein or a fragment or variant thereof, as defined herein. Preferably, a coding sequence encoding two or more peptides or proteins can be separated into bi or multicistronic mRNAs by at least one IRES (Internal Ribosome Entry Site) sequence, as defined below. Thus, the term “encoding two or more peptides or proteins” may mean, but is not limited to, that the bi or even multicistronic mRNA may encode, for example, at least two, three, four, five, six or more (preferably different) peptides or proteins or fragments or variants thereof, as defined herein. More preferably, but is not limited to, the bi or even multicistronic mRNA may encode, for example, at least two, three, four, five, six or more (preferably different) peptides or proteins, or fragments or variants thereof, as defined herein. In this context, the so-called IRES (internal ribosome entry site) sequence defined above can function as a single ribosome binding site, but it can also function to provide the bi or even multicistronic mRNAs defined above, which independently encode several peptides or proteins translated by ribosomes. Examples of IRES sequences that can be used in this invention are those derived from picornaviruses (e.g., FMDV), pestiviruses (CFFV), polioviruses (PV), encephalomyocarditis viruses (ECMV), foot-and-mouth disease viruses (FMDV), hepatitis C virus (HCV), classical swine fever viruses (CSFV), mouse leukemia virus (MLV), simian immunodeficiency virus (SIV), or cricket paralysis virus (CrPV).

[0404] According to further embodiments, at least one coding region of the mRNA sequence according to the present invention may encode at least two, three, four, five, six, seven, eight, and more peptides or proteins (or fragments and derivatives thereof) as defined herein, linked with or without an amino acid linker sequence, the linker sequence may include a rigid linker, a flexible linker, a cleavable linker (e.g., a self-cleaving peptide) or a combination thereof. The peptides or proteins may be identical, different, or a combination thereof. A particular peptide or protein combination may be encoded by the mRNA encoding at least two peptides or proteins as described herein (also referred to herein as a “multi-antigen construct / mRNA”).

[0405] In another preferred embodiment, the mRNA compound contained in the composition encodes a pathogenic antigen whose amino acid sequence is unmodified with respect to its respective wild-type amino acid sequence. In this case, the mRNA compound may include a coding region along with the nucleic acid sequence that is unmodified with respect to its respective wild-type mRNA sequence. For example, the mRNA compound may be natural and unmodified mRNA. As used herein, natural and unmodified mRNA encompasses in vitro-generated mRNA that has neither chemical modifications nor sequence changes.

[0406] mRNA modification and sequence In another embodiment of the present invention, the mRNA compound includes artificial mRNA. In this context, artificial mRNA includes mRNA having chemical modifications, sequence modifications, or non-natural sequences.

[0407] 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 may be selected from the group consisting of base modification, sugar modification, skeletal modification, and lipid modification. Skeletal modification according to the present invention is a modification in which the phosphate of the nucleotide backbone contained in the mRNA compound comprising the mRNA sequence as defined herein is chemically modified. Sugar modification according to the present invention is a chemical modification of the sugar of the nucleotide in the mRNA compound comprising the mRNA sequence as defined herein. Furthermore, base modification according to the present invention is a chemical modification of the base portion of the nucleotide in the mRNA compound comprising mRNA. In this context, the nucleotide analog or modification is preferably selected from nucleotide analogs that are applicable to transcription and / or translation.

[0408] sugar modification Modified nucleosides and nucleotides that can be incorporated into modified mRNA compounds containing mRNA sequences described herein may have their sugar moieties modified. For example, the 2'-hydroxyl group (OH) may be modified or substituted 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(CH2CH2O)nCH2CH2OR; "locked" nucleic acids (LNA) in which the 2'-hydroxyl is bonded to the 4' carbon of the same ribose sugar, for example by a methylene crosslink; and amino groups (-O-amino, amino groups, e.g., NRR may be alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, polyamino) or aminoalkoxy.

[0409] The “deoxy” modification includes hydrogen, an amino (e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); or the amino group may be bonded to the sugar through a linker, the linker containing one or more atoms C, N, and O.

[0410] The sugar group may also contain one or more carbon atoms having the opposite stereochemical configuration to the corresponding carbon of ribose. Therefore, modified mRNA may contain nucleotides containing arabinose as the sugar, for example.

[0411] Skeletal modification The phosphate group of the skeleton may be modified by substituting one or more oxygen atoms with different substituents. Furthermore, modified nucleosides and nucleotides may include complete substitution of the unmodified phosphate moiety with the modified phosphate described herein. Examples of modified phosphate groups, but not limited to, include phosphorothioates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, alkyl or aryl phosphonates, and phosphotryesters. Phosphorothioates all have unbound oxygen atoms substituted with sulfur. The phosphate linker may also be modified by substitution of the bound oxygen with nitrogen (bridged phosphoramidate), sulfur (bridged phosphorothioate), and carbon (bridged methylene phosphonate).

[0412] lipid modification Lipid-modified mRNA typically comprises mRNA as defined herein. Such lipid-modified mRNA as defined herein typically further comprises at least one linker covalently linked to the mRNA, and at least one lipid covalently linked to each linker. Alternatively, lipid-modified mRNA comprises at least one mRNA as defined herein, and at least one (bifunctional) lipid covalently linked to that mRNA (without linkers). According to a third alternative, lipid-modified mRNA comprises an mRNA molecule as defined herein, at least one linker covalently linked to that mRNA, at least one lipid covalently linked to each linker, and also at least one (bifunctional) lipid covalently linked to that mRNA (without linkers). In this context, it is particularly preferred that the lipid modification is located at the ends of a linear mRNA molecule.

[0413] In another preferred embodiment, the mRNA compound is free from nucleoside modifications, particularly base modifications. In a further embodiment, the mRNA compound is free from 1-methylpseudridine modifications, pseudouridine modifications, and 5-methyluridine modifications. In one preferred embodiment, the mRNA comprises only naturally occurring nucleosides. In a more preferred embodiment, the mRNA compound is free from any chemical modifications and optionally includes sequence modifications. In a further preferred embodiment of the present invention, the mRNA compound comprises only naturally occurring nucleosides adenine, uracil, guanine, and cytosine.

[0414] Base modification In an alternative embodiment, the mRNA compound includes at least one base modification. Modified nucleosides and nucleotides that can be incorporated into modified mRNA compounds containing mRNA sequences described herein may have their nucleic acid base portions further modified. Examples of nucleic acid bases found in mRNA include, but are not limited to, adenine, guanine, cytosine, and uracil. For example, the nucleosides and nucleotides described herein may be chemically modified on the main groove surface. In some embodiments, the main groove chemical modification may include an amino group, a thiol group, an alkyl group, or a halo group.

[0415] In a particularly preferred embodiment of the present invention, the nucleotide analogs / modifications are 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-methylinosine-5'-triphosphate, 4-thiouridine- 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 '-deoxyuridine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, 5-methyluridine-5'-triphosphate, 5-propynyl-2'-deoxycytidine-5'-triphosphate, 5-propynyl-2'-deoxyuridine-5'-triphosphate, 6-azacitidine-5'-triphosphate, 6-azacitidine-5'-triphosphate, 6-azacitidine-5'-triphosphate, 6-chloropurine riboside-5'-triphosphate, 7-deazaadenosine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 8-a Selected from base modifications, which include 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.Nucleotides for 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 are particularly preferred. In some embodiments, the modified nucleoside is pyridine-4-onyribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudridine, 2-thio-pseudridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyluridine, 1-carboxymethyl-pseudridine, 5-propynyluridine, 1-propynyl-pseudridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudridine, 5-taurinomethyl-2-thiouridine, 1-taurinomethyl This includes thio-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseuduridine, 4-thio-1-methyl-pseuduridine, 2-thio-1-methyl-pseuduridine, 1-methyl-1-deaza-pseuduridine, 2-thio-1-methyl-1-deaza-pseuduridine, dihydrouridine, dihydropseuduridine, 2-thio-dihydrouridine, 2-thio-dihydropseuduridine, 2-methoxyuridine, 2-methoxy-4-thiouridine, 4-methoxy-pseuduridine, and 4-methoxy-2-thio-pseuduridine.In some embodiments, the modified nucleoside is 5-aza-cytidine, pseudoisocytidine, 3-methylcytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thiocytidine, 2-thio-5-methylcytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl -Includes pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebralin, 5-aza-zebralin, 5-methyl-zebralin, 5-aza-2-thio-zebralin, 2-thio-zebralin, 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-isopentenyladenosine, N This includes 6-(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-methoxyadenine.In other embodiments, the modified nucleosides include inosine, 1-methylinosine, waiosine, waibutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methylguanosine, 6-thio-7-methylguanosine, 7-methylinosine, 6-methoxyguanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thioguanosine, N2-methyl-6-thioguanosine, and N2,N2-dimethyl-6-thioguanosine. In some embodiments, the nucleotide may be modified on the main groove surface, including substitution of the hydrogen atom 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.

[0416] In a more specific embodiment, the modified mRNA is 6-aza-cytidine, 2-thio-cytidine, α-thio-cytidine, pseudo-iso-cytidine, 5-aminoallyl-uridine, 5-iod-uridine, N1-methyl-pseuduridine, 5,6-dihydrouridine, α-thiouridine, 4-thiouridine, 6-aza-uridine, 5-hydroxyuridine, deoxythymidine, 5-methyluridine, pyrrolo-cytidine, inos It may include nucleoside modifications selected from α-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, α-thio-adenosine, 8-azido-adenosine, and 7-deaza-adenosine.

[0417] In further embodiments, the chemical modification is selected from pseudouridine, N1-methylpseudridine, N1-ethylpseudridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deazapsoiduridine, 2-thio-1-methylpsoiduridine, 2-thio-5-azauridine, 2-thio-dihydropsoiduridine, 2-thio-dihydrouridine, 2-thiopsoiduridine, 4-methoxy-2-thiopsoiduridine, 4-methoxypsoiduridine, 4-thio-1-methylpsoiduridine, 4-thiopsoiduridine, 5-azauridine, dihydropsoiduridine, 5-methoxyuridine, and 2'-0-methyluridine.

[0418] In specific embodiments, the chemical modification is selected from the group consisting of pseudouracil (ψ), N1-methylpseudracil (N1Mψ), 1-ethylpseudracil, 2-thiouracil (s2U), 4-thiouracil, 5-methylcytosine, 5-methyluracil, 5-methoxyuracil, and any combination thereof.

[0419] Array modification According to further embodiments, the mRNA compound comprises a modified mRNA sequence. For example, modification of the mRNA sequence may result in 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 compositions of the present invention described herein may comprise an mRNA compound comprising a coding region encoding a peptide or protein, as defined in any of the embodiments described herein, wherein the coding region exhibits sequence modification.

[0420] According to one embodiment, the mRNA compound contains a “stabilized mRNA sequence,” i.e., mRNA that is essentially resistant to in vivo degradation (e.g., by exo- or endo-nucleases). Such stabilization can be brought about, for example, by a modified phosphate backbone of the mRNA according to the present invention. The skeletal modification according to the present invention is a modification in which the phosphate of the nucleotide backbone contained in the mRNA is chemically modified. Preferably, the nucleotide that can be used in this regard contains, for example, a phosphorothioate-modified phosphate backbone, preferably in which at least one of the phosphate oxygens contained in the phosphate backbone is substituted with a sulfur atom. The stabilized mRNA may further include, for example, alkyl and aryl phosphonates in which the charged phosphate oxygen is substituted with an alkyl or aryl group, or nonionic phosphate analogs such as phosphodiesters and alkylphosphotryesters in which the charged oxygen residue is present in an alkylated form. Such skeletal modifications typically include, but are not limited to, modifications from the group consisting of methylphosphonates, phosphoramidates and phosphorothioates (e.g., cytidine-5'-O-(1-thiophosphate)).

[0421] The following describes specific modifications that can preferably "stabilize" mRNA as defined herein. G / C content modification According to one embodiment, the mRNA compound comprises an mRNA sequence that is modified and stabilized by modification of its guanosine / cytosine (G / C) content. Such modification, or at least one of these modifications, is located in the coding region of the mRNA compound.

[0422] 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 include an mRNA compound encoding a pathogenic antigen whose amino acid sequence is unmodified with respect to the coding amino acid sequence of the respective wild-type nucleic acid.

[0423] This modification of the mRNA sequence in the present invention is based on the fact that the sequence of any translated mRNA region is important for the efficient translation of that mRNA. Therefore, the composition of the mRNA and the sequence of various nucleotides are important. In particular, sequences with increased G (guanosine) / C (cytosine) content are more stable than sequences with increased A (adenosine) / U (uracil) content. Thus, according to the present invention, the codons of the mRNA vary compared to their respective wild-type mRNAs, while maintaining the translated amino acid sequence, to contain increased amounts of G / C nucleotides. With respect to the fact that some codons code for one, the same amino acid (so-called degeneracy of the genetic code), it is possible to determine the codon that is most advantageous for stability (so-called alternative codon usage frequency). Depending on the amino acids encoded by the mRNA, there are various possibilities for the modification of the mRNA sequence compared to its wild-type sequence. For amino acids encoded by codons containing only G or C nucleotides, codon modification is not necessary. Therefore, the codons Pro (CCC or CCG), Arg (CGC or CGG), Ala (GCC or GCG), and Gly (GGC or GGG) do not require modification because neither A nor U is present. In contrast, codons containing A and / or U nucleotides can be modified by substitution with other codons that encode the same amino acid but do not contain A and / or U. Examples of these include: the Pro codon can be modified from CCU or CCA to CCC or CCG; the Arg codon can be modified from CGU or CGA or AGA or AGG to CGC or CGG; the Ala codon can be modified from GCU or GCA to GCC or GCG; and the Gly codon can be modified from GGU or GGA to GGC or GGG. In other cases, neither A nor U nucleotides can be excluded from the codon, but it is possible to reduce the A and U content by using codons that contain lower levels of A and / or U nucleotides.Examples of these modifications include: the codon for Phe can be modified from UUU to UUC; the codon for Leu can be modified from UUA, UUG, CUU, or CUA to CUC or CUG; the codon for Ser can be modified from UCU, UCA, or AGU to UCC, UCG, or AGC; the codon for Tyr can be modified from UAU to UAC; the codon for Cys can be modified from UGU to UGC; the codon for His can be modified from CAU to CAC; and the codon for Gln can be modified from CAA to CAG. The codon of Ile can be modified from AUU or AUA to AUC; the codon of Thr can be modified from ACU or ACA to ACC or ACG; the codon of Asn can be modified from AAU to AAC; the codon of Lys can be modified from AAA to AAG; the codon of Val can be modified from GUU or GUA to GUC or GUG; the codon of Asp can be modified from GAU to GAC; the codon of Glu can be modified from GAA to GAG; the stop codon UAA can be modified to UAG or UGA. On the other hand, there is no possibility of sequence modification for the codons of Met (AUG) and Trp (UGG). 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 present invention compared to its particular wild-type mRNA (i.e., the original sequence). Thus, for example, all Thr codons present in the wild-type sequence can be modified to ACC (or ACG). However, preferably, for example, combinations of the above possible substitutions are used: - Substitution of all codons encoding Thr in the original sequence (wild-type mRNA) with ACC (or ACG), and - Substitution of all codons that originally encode Ser with UCC (or UCG or AGC); - Substitution of all codons encoding Ile in the original sequence to their AUC, and - Replacement of all codons that originally code Lys with AAG, and - Replace all codons that originally code Tyr with UAC; - Substitution of all codons encoding Val in the original sequence with GUC (or GUG), and - Replacement of all codons that originally code for Glu with GAG, and - Replace all codons that originally code for Ala with GCC (or GCG), and - Replace all codons that originally code for Arg with CGC (or CGG); - Substitution of all codons encoding Val in the original sequence with GUC (or GUG), and - Replacement of all codons that originally code for Glu with GAG, and - Replace all codons that originally code for Ala with GCC (or GCG), and - Replace all codons that originally code for Gly with GGC (or GGG), and - Replace all codons that originally code for Asn with AAC; - Substitution of all codons encoding Val in the original sequence with GUC (or GUG), and - Replacement of all codons that originally code Phe with UUC, and - Replacement of all codons that originally code Cys with UGC, and - Replace all codons that originally code Leu with CUG (or CUC), and - Replacement of all codons that originally code Gln with CAG, and - Replacing all codons that originally code Pro with CCC (or CCG); etc.

[0424] Preferably, the G / C content of the coding region of an mRNA compound containing the mRNA sequence of the present invention is increased by at least 7%, more preferably at least 15%, and particularly preferably at least 20%, compared to the G / C content of the coding region of a wild-type RNA encoding an antigen or fragment or variant as defined herein. According to specific embodiments, 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 substitutable codons of the region encoding a peptide or protein or fragment or variant as defined herein, or the entire sequence of a wild-type mRNA sequence, are substituted, thereby increasing the G / C content of the sequence. In this context, it is particularly preferable to increase the G / C content of at least one coding region of the mRNA sequence of the present invention, preferably the mRNA sequence according to the present invention, to the maximum extent possible (i.e., 100% of the substitutable codons) compared to the wild-type sequence. According to the present invention, a more preferred modification of the mRNA sequence of the present invention is based on the finding that translation efficiency is also determined by the different frequencies of tRNA appearance in the cell. Therefore, when so-called “rare codons” are present to an increased degree in the mRNA sequence of the present invention, the corresponding modified mRNA sequence is translated to a significantly lower degree than when codons encoding relatively “high-frequency” tRNA are present. According to the present invention, in the modified mRNA sequence of the present invention, the region encoding a peptide or protein or a fragment or variant thereof as defined herein is modified compared to the corresponding region of the wild-type mRNA sequence such that at least one codon encoding a relatively rare tRNA in the cell is replaced with a codon encoding a tRNA that carries the same amino acid as a relatively high-frequency, relatively rare tRNA in the cell. This modification modifies the mRNA sequence of the present invention so that codons available for frequently present tRNA are inserted.In other words, according to the present invention, this modification allows all codons in a wild-type sequence encoding a relatively rare tRNA in the cell to be replaced, in each case, with codons encoding a tRNA that carries the same amino acid as a relatively frequent and, in each case, relatively rare tRNA in the cell. Which tRNAs are relatively frequent and, conversely, relatively rare in the cell is 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, a codon using the most frequently present tRNA, for example, a Gly codon using the most frequently present tRNA in (human) cells, is particularly preferred. According to the present invention, it is particularly preferred that the increased, and especially maximized, continuous G / C content in the modified mRNA sequence of the present invention be linked to a “frequent” codon without modifying the amino acid sequence of the protein encoded by the coding region of the mRNA sequence. This preferred embodiment makes it possible to provide a particularly efficiently translated, stabilized (modified) mRNA sequence of the present invention. The determination of the modified mRNA sequence of the present invention (increased G / C content; tRNA exchange) can be performed using a computer program described in International Publication No. 02 / 098443, the disclosure of which is included in the entire scope of the present invention. Using this computer program, the nucleotide sequence of a desired mRNA sequence can be modified using the genetic code or its degenerate properties, preferably resulting in an amino acid sequence encoded by the unmodified mRNA sequence compared to the unmodified sequence, in combination with the use of codons encoding tRNA that are present at the highest possible frequency in the cell, with the maximum G / C content. Alternatively, it is possible to modify only the G / C content or only the codon usage frequency compared to the original sequence. The Visual Basic 6.0 source code (development environment used: Microsoft Visual Studio Enterprise 6.0 with Service Pack 3) is also described in International Publication No. 02 / 0298443.In a more preferred embodiment of the present invention, the A / U content in the environment of the ribosome binding site of the mRNA sequence of the present 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 ribosome binding to mRNA. Effective binding of ribosomes to the ribosome binding site (Kozak sequence: SEQ ID NO: 1 or SEQ ID NO: 2, or minimal Kozak sequence ACC (AUG forms the start codon)) then has the effect of efficient translation of mRNA. According to a further embodiment of the present invention, the mRNA sequence of the present invention may be modified with respect to potential destabilizing sequence elements. In particular, the coding region and / or 5' and / or 3' untranslated regions of this mRNA sequence may be modified compared to its respective wild-type mRNA such that the coding amino acid sequence of the modified mRNA sequence is preferably not modified compared to its respective wild-type mRNA and does not contain destabilizing sequence elements. For example, it is known that eukaryotic mRNA sequences contain destabilizing sequence elements (DSEs) to which signal proteins bind and which regulate enzymatic degradation of mRNA in vivo. Therefore, one or more such modifications can be made to a modified mRNA sequence, compared to the corresponding region of wild-type mRNA, so that the destabilizing sequence elements are not present, either entirely or substantially, in the region encoding at least one peptide or protein or a fragment or variant as defined herein, for further stabilization of the modified mRNA sequence. According to the present invention, DSEs present in the untranslated region (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 (AURESs) present in the 3'-UTR section of many unstable mRNAs (Caput et al., Proc. Natl. Acad. Sci. USA 1986, 83:1670-1674). Therefore, the mRNA sequences of the present invention are preferably modified compared to their respective wild-type mRNAs so that the mRNA sequences of the present invention do not contain such destabilizing sequences.This also applies to sequence motifs that can be recognized by possible endonucleases, such as the sequence GAACAAG, which is contained in the 3'-UTR segment of genes encoding the transferrin receptor (Binder et al., EMBO J.1994, 13:1969-1980). These sequence motifs are also preferably removed in the mRNA sequence of the present invention.

[0425] According to further embodiments, the mRNA compound comprises an mRNA sequence comprising a coding region comprising one of the RNA sequences disclosed in Tables 1 to 5, Figures 20 to 24 or the sequence listing in International Publication No. 2018 / 078053; Tables 1 to 5 or Figures 20 to 24 of International Publication No. 2018 / 078053; International Publication No. 2018 / 078053 in its entirety is incorporated herein by reference.

[0426] Sequences adapted to hitocodon usage frequency A more preferred modification of mRNA compounds is based on the finding that codons encoding the same amino acid typically exist at different frequencies. In this embodiment, the frequency of codons encoding the same amino acid in the coding region of the mRNA compound differs from the naturally occurring frequency of those codons, for example, according to the human codon usage frequencies shown in Table 2 (Human Codon Usage Frequency Table). For example, in the case of the amino acid alanine (Ala), the wild-type coding region is preferably adapted such that codon "GCC" is used at a frequency of 0.40, codon "GCT" at a frequency of 0.28, codon "GCA" at a frequency of 0.22, and codon "GCG" at a frequency of 0.10 (see Table 2).

[0427] [Table 5]

[0428] Codon-optimized sequences In one embodiment, all codons of a relatively rare, wild-type sequence encoding tRNA in the cell are replaced with codons encoding tRNA that are relatively frequent in the cell and, in each case, carry the same amino acids as the relatively rare tRNA. Therefore, it is particularly preferable that the most frequent codons be used for each coding 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 having an 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, an 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.

[0429] For example, in the case of the amino acid alanine (Ala) present in an amino acid sequence encoded by at least one coding sequence of RNA according to the present invention, the wild-type coding sequence is adapted so that the most frequent human codon "GCC" is always used for the amino acid, or in the case of the amino acid cysteine ​​(Cys), the wild-type sequence is adapted so that the most frequent human codon "TGC" is always used for the amino acid, and so on.

[0430] C-optimized array According to another embodiment, an mRNA compound comprising an mRNA sequence having a modified—particularly increased—cytosine (C) content in the coding region of the mRNA sequence, preferably compared to the C content of the coding region of each wild-type mRNA, i.e., an unmodified mRNA. At the same time, the amino acid sequence encoded by at least one coding region of the mRNA sequence of the present invention is preferably unmodified compared to the amino acid sequence encoded by each wild-type mRNA.

[0431] In a preferred embodiment of the present invention, the modified mRNA sequence is modified to achieve at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the theoretically possible maximum cytosine content, or at least 90%, or even the maximum cytosine content.

[0432] In a more preferred embodiment, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100% of the codons in the "cytosine content-optimizable" target mRNA wild-type sequence are replaced by codons having a higher cytosine content than that present in the wild-type sequence.

[0433] In a more preferred embodiment, some of the codons in the wild-type coding sequence may be further modified such that codons of relatively rare tRNAs in the cell are replaced by codons of relatively rare tRNAs in the cell, provided that the substitution codons of relatively rare tRNAs carry the same amino acid as the relatively rare tRNAs of the original wild-type codon. Preferably, all codons of relatively rare tRNAs are replaced by codons of relatively rare tRNAs in the cell, except for codons encoding amino acids that are exclusively encoded by cytosine-free codons, or glutamine (Gln) encoded by two codon...

Claims

1. (a) Cationic lipid according to formula (I): R ａ -A-R ｂ Formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof (In the formula, R ａ teeth, 【Chemistry 1】 or -R １ -N(H)-C(O)-R ３ -R ４ Selected from; R ｂ teeth, 【Chemistry 2】 -R １ -N(H)-C(O)-R ３ -R ４ or -R １ -N(CH ３ ) ２ Selected from; A is -S-; R １ is an ethanediyl, propanediyl, butanediyl, or an alkanediyl having 2 to 8 carbon atoms, where each substituted carbon atom is either unsubstituted or C １ ~C ４ Alkyl, C １ ~C ４ Alkenylene, C ３ ~C ８ Cycloalkylene and C ３ ~C ８ It is substituted with one or more substituents selected from cycloalkenylenes; R ２ It is an alkanediyl having 2 to 8 carbon atoms; R ３ If present, -R ５ -C(O)-O-, -R ５ -O-C(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, and the lipophilic substituent is (i) A linear or branched alkyl or alkenyl having 12 to 25 carbon atoms, (ii) Selected from tocopherols having saturated phytyl chains or tocotrienols having polyunsaturated phytyl chains; R ５ It is an alkanediyl having 1 to 6 carbon atoms; X is a carbon atom (CH) or nitrogen atom bonded to a hydrogen atom; All choices are independent of each other.) (b) 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE) and (c) mRNA having at least one coding sequence encoding an antigen, or a fragment or variant thereof, A composition containing the following:

2. The composition according to claim 1, further comprising 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC).

3. The composition according to claim 1 or 2, wherein the antigen is derived from a pathogenic antigen, a tumor antigen, an allergen antigen, or an autoimmune autoantigen, or a fragment or variant thereof.

4. The composition according to claim 3, wherein the pathogenic antigen is selected from bacterial antigens, viral antigens, fungal antigens, and protozoan antigens.

5. The pathogenic antigen is SARS coronavirus 2 (SARS-CoV-2), nCoV-2019 coronavirus, SARS coronavirus (SARS-CoV), Bunyavirum viruses, cytomegalovirus (CMV), dengue virus, Ebola virus, flavivirus, hepatitis B virus (HBV), herpes simplex virus (HSV), human immunodeficiency virus (HIV), human metapneumovirus (hMPV), human papillomavirus (HPV) The composition according to claim 4, derived from human parainfluenza virus (HPIV), influenza virus, extraenteropathogenic Escherichia coli (E. coli), Lassa mum arena virus (LASV), MERS coronavirus, Mycobacterium tuberculosis, Nipah virus, norovirus, rabies virus, synovial respiratory virus (RSV), rhinovirus, rotavirus, vaccinia virus, yellow fever virus, Zika virus, Chlamydia trachoma, or malaria parasite.

6. The composition according to any one of claims 1 to 5, wherein the mRNA encodes a viral antigen.

7. A composition according to any one of claims 1 to 6 for treating or preventing a disease selected from infectious diseases, cancer or tumor diseases, liver diseases, autoimmune diseases, allergies, monogenic diseases including genetic diseases, genetic diseases in general, diseases having a genetic background, typically caused by a defined gene defect and inherited according to Mendel's laws, cardiovascular diseases, neurological diseases, respiratory diseases, digestive diseases, skin diseases, musculoskeletal disorders, connective tissue disorders, neoplasms, immunodeficiency, endocrine, nutritional and metabolic diseases, eye diseases, ear diseases, and diseases associated with peptide or protein deficiencies.

8. The composition according to any one of claims 1 to 6 for use as a pharmaceutical.

9. The composition according to any one of claims 1 to 6, comprising an effective amount of mRNA encoding an antigen.

10. A pharmaceutical composition for treating or preventing a disease selected from infectious diseases, cancer or tumor diseases, liver diseases, autoimmune diseases, allergies, monogenic diseases including genetic diseases, genetic diseases in general, diseases having a genetic background, typically caused by a defined gene defect and inherited according to Mendel's laws, cardiovascular diseases, neurological diseases, respiratory diseases, digestive diseases, skin diseases, musculoskeletal disorders, connective tissue disorders, neoplasms, immunodeficiency, endocrine, nutritional and metabolic diseases, eye diseases, ear diseases, and diseases associated with peptide or protein deficiencies, comprising the composition according to any one of claims 1 to 6.

11. The pharmaceutical composition according to claim 10 or the composition according to claim 7, wherein the infectious disease includes viral, bacterial, fungal, or protozoan infectious diseases.

12. The pharmaceutical composition or composition according to claim 10 or 11, or the composition according to claim 7, wherein the pharmaceutical composition or composition is formulated for intravenous, intramuscular, subcutaneous, or intradermal administration.

13. A pharmaceutical composition for use as a pharmaceutical, comprising the composition according to any one of claims 1 to 6.

14. A pharmaceutical composition comprising the composition according to any one of claims 1 to 6, comprising an effective amount of mRNA encoding an antigen.

15. A vaccine comprising the composition according to any one of claims 1 to 6.

16. The vaccine according to claim 15 for preventing or treating infectious diseases, cancer or tumor diseases.

17. The vaccine according to claim 16, wherein the infectious disease includes viral, bacterial, fungal, or protozoan infectious diseases.

18. A vaccine according to any one of claims 15 to 17 for inducing an antigen-specific immune response in a subject.

19. The vaccine according to any one of claims 15 to 18 for inducing an antigen-specific T cell response.

20. A vaccine according to any one of claims 15 to 19 for inducing a CD8+ T cell response.

21. The vaccine according to any one of claims 15 to 20, wherein the vaccine is formulated for intravenous, intramuscular, subcutaneous, or intradermal administration.

22. The vaccine according to any one of claims 15 to 21, comprising an effective amount of mRNA encoding an antigen.

23. A kit comprising the composition according to any one of claims 1 to 6.

24. The kit according to claim 23, further comprising a liquid vehicle for solubilizing the composition.

25. The kit according to claim 23 or claim 24, further comprising instructions providing information about the administration or dosage of the composition.

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