mRNA vaccines or combinations of mRNA-encoded therapeutic proteins and immunomodulatory mRNA to improve or reduce immunogenicity and increase efficacy.

Abstract

The present invention relates to a combination comprising two or more mRNA molecules, or a single mRNA molecule encoding a first molecule which is a therapeutic or immunogenic protein or peptide and a second molecule which is a protein or peptide having the ability to modulate an immune response to the first molecule and / or the translation product of the first molecule, wherein the combination comprises at least one first mRNA molecule encoding a first molecule which is a therapeutic or immunogenic protein or peptide, and at least one second mRNA molecule encoding a second molecule which is a protein or peptide having the ability to modulate an immune response to the first molecule and / or the translation product of the first molecule. The present invention also relates to a host cell comprising the combination or single mRNA molecule described in the present invention, a pharmaceutical composition comprising the combination, the single mRNA molecule, or the host cell of the present invention, and a vaccine comprising the combination, the single mRNA molecule, the host cell, or the pharmaceutical composition described in the present invention. Furthermore, kits comprising the combination, the single mRNA molecule, the host cell, the pharmaceutical composition, or the vaccine described in the present invention are provided. The present invention also relates to the combination, the single mRNA molecule, the host cell, the pharmaceutical composition, the vaccine, or the kit described in the present invention for use in pharmaceuticals. Finally, the present invention also relates to the combination, the single mRNA molecule, the host cell, the pharmaceutical composition, the vaccine, or the kit described in the present invention for use in methods for the prevention and / or treatment of infectious, hereditary, or proliferative diseases.

Description

Technical Field

[0001] Field of the Invention The present invention relates to a combination comprising two or more mRNA molecules, or a first molecule that is a therapeutic or immunogenic protein or peptide and a second molecule that is a protein or peptide having the ability to modulate an immune response against the first molecule and / or the translation product of the first molecule, wherein the combination is encoded by a single mRNA molecule, and wherein the combination comprises at least one first mRNA molecule encoding a first molecule that is a therapeutic or immunogenic protein or peptide, and at least one second mRNA molecule encoding a second molecule that is a protein or peptide having the ability to modulate an immune response against the first molecule and / or the translation product of the first molecule. The present invention also relates to host cells comprising the combination or single mRNA molecule according to the present invention, pharmaceutical compositions comprising the combination, the single mRNA molecule, or the host cells of the present invention, and vaccines comprising the combination, the single mRNA molecule, the host cells, or the pharmaceutical composition according to the present invention. Further, a pharmaceutical combination, and a kit comprising the combination, the single mRNA molecule, the host cells, the pharmaceutical composition, the vaccine, or the pharmaceutical combination according to the present invention are provided. The present invention also relates to the combination, the single mRNA molecule, the host cells, the pharmaceutical composition, the vaccine, the pharmaceutical combination, or the kit according to the present invention for use in medicine. Finally, the present invention also relates to the combination, the single mRNA molecule, the host cells, the pharmaceutical composition, the vaccine, the pharmaceutical combination, or the kit according to the present invention for use in methods for the prevention and / or treatment of infectious, genetic or proliferative diseases.

Summary of the Invention

[0002] Description The in vivo delivery of ribonucleic acids that are biologically active or encode therapeutic peptides is at the forefront of modern medicine. This includes gene therapy approaches and gene vaccination. However, because much of the biological process is precisely regulated to achieve optimal activity, in vivo delivery of biologically active ribonucleic acids alone is unlikely to achieve optimal efficacy.

[0003] For example, adaptive immunity is induced as a response to an antigen, and this response is further modulated by other cellular factors that enhance or reduce its activity. In the case of subunit vaccines (e.g., recombinant proteins), optimal adaptive immunity is obtained by adding adjuvants, such as aluminum salts, oil-in-water emulsions, and more recently approved adjuvants such as AS01, AS03, AS04, CpG ODN, and MF59. However, unlike traditional vaccines, there have been no adjuvants combined with clinically approved messenger RNA (mRNA) vaccines to date, possibly due to the adverse effects of the innate response induced by the adjuvant to antigen expression, which is driven by mRNA. Therefore, adaptive immunity induced by mRNA vaccines may achieve suboptimal results in terms of efficacy and / or quality and / or persistence.

[0004] WO2003 / 051401(A2) describes a pharmaceutical composition comprising at least one mRNA containing at least one region encoding a tumor-derived antigen, which is combined with an aqueous solvent and preferably a cytokine such as GM-CSF. The pharmaceutical composition is proposed to be used for the treatment and / or prevention of cancer.

[0005] WO2006 / 008154(A1) discloses an mRNA mixture for vaccination against tumor diseases, wherein at least one type of mRNA comprises at least one tumor antigen coding region; and at least one other mRNA comprises at least one immunogenic protein coding region.

[0006] WO2016 / 170176(A1) relates to RNA comprising a composition for use in the treatment or prevention of tumors and / or cancerous diseases, and to the use of said RNA comprising a composition for the treatment or prevention of tumors and / or cancerous diseases.

[0007] The problem to be solved by the present invention is to improve the effectiveness of gene vaccine or gene therapy approaches. The inventors' solution to this problem is to administer one (or more) other mRNA molecules (regulatory RNA) having direct or indirect immunomodulatory activity together with the mRNA vaccine or therapeutic mRNA. This strategy is based on a combination including the mRNA vaccine or therapeutic mRNA and the regulatory mRNA as a mixture or co-formulation of LNP formulations (both mRNAs contained in the same LNP particle), or on a single mRNA molecule encoding a first molecule which is a therapeutic or immunogenic protein or peptide and a second molecule which is a protein or peptide capable of modulating the immune response to the first molecule and / or the translation product of the first molecule, thereby enabling the spatially and temporally coordinated in vivo expression of the two proteins (failure to do so results in reduced or lost regulation).

[0008] Brief Description of the Invention In general and for a brief explanation, the main aspects of the present invention can be described below:

[0009] In a first embodiment, the present invention relates to a combination comprising two or more mRNA molecules, or to a single mRNA molecule encoding a first molecule which is a therapeutic or immunogenic protein or peptide and a second molecule which is a protein or peptide having the ability to modulate the immune response to the first molecule and / or the translation product of the first molecule.

[0010] In a second aspect, the present invention relates to a host cell comprising the combination or single mRNA molecule described in the first aspect of the present invention.

[0011] In a third aspect, the present invention relates to a pharmaceutical composition comprising a combination or single mRNA molecule as described in the first aspect of the present invention, or a host cell as described in the second aspect of the present invention, and a pharmaceutically acceptable carrier, stabilizer and / or excipient.

[0012] In a fourth aspect, the present invention relates to a vaccine comprising a combination or single mRNA molecule as described in the first aspect of the present invention, a host cell as described in the second aspect of the present invention, or a pharmaceutical composition as described in the third aspect of the present invention.

[0013] In a fifth embodiment, the present invention relates to pharmaceutical combinations.

[0014] In a sixth aspect, the present invention relates to a kit comprising a combination or single mRNA molecule as described in the first aspect of the present invention, a host cell as described in the second aspect of the present invention, a pharmaceutical composition as described in the third aspect of the present invention, a vaccine as described in the fourth aspect of the present invention, or a pharmaceutical combination as described in the fifth aspect of the present invention.

[0015] In a seventh aspect, the present invention relates to a combination or single mRNA molecule according to the first aspect of the present invention, a host cell according to the second aspect of the present invention, a pharmaceutical composition according to the third aspect of the present invention, a vaccine according to the fourth aspect of the present invention, a pharmaceutical combination according to the fifth aspect of the present invention, or a kit according to the sixth aspect of the present invention, for use in pharmaceuticals.

[0016] In the eighth aspect, the present invention relates to a combination or single mRNA molecule according to the first aspect of the present invention, a host cell according to the second aspect of the present invention, a pharmaceutical composition according to the third aspect of the present invention, a vaccine according to the fourth aspect of the present invention, a combination of pharmaceuticals according to the fifth aspect of the present invention, or a kit according to the sixth aspect of the present invention for use in methods for the prevention and / or treatment of infectious, hereditary, or proliferative diseases.

[0017] In a ninth aspect, the present invention relates to combinations or single mRNA molecules, therapeutic proteins, host cells, pharmaceutical compositions, pharmaceutical combinations or kits described in the present invention for use in methods for preventing and / or treating hereditary disorders.

[0018] In a tenth aspect, the present invention relates to combinations or single mRNA molecules, therapeutic proteins, host cells, pharmaceutical compositions, pharmaceutical combinations or kits described in the present invention for use in gene therapy.

[0019] Detailed description of the present invention The components of the present invention are described below. These components are listed together with specific embodiments, but it should be understood that they can be combined in any manner and in any number to make additional embodiments. The various examples and preferred embodiments described should not be construed as limiting the invention to only the explicitly described embodiments. This specification should be understood as supporting and encompassing embodiments that combine two or more explicitly described embodiments, or that combine one or more explicitly described embodiments with any number of disclosed and / or preferred components. Furthermore, unless the context indicates otherwise, all permutations and combinations of all components described in this application should be considered as disclosed by the specification of this application.

[0020] In a first embodiment, the present invention relates to a combination of two or more mRNA molecules, or a single mRNA molecule encoding a first molecule which is a therapeutic or immunogenic protein or peptide and a second molecule which is a protein or peptide which has the ability to modulate the immune response to the first molecule and / or the translation product of the first molecule, wherein the combination is as follows: (i) at least one first mRNA molecule encoding a first molecule which is a therapeutic or immunogenic protein or peptide, and (ii) at least one second mRNA molecule encoding a second molecule which is a protein or peptide capable of modulating the immune response to the first molecule and / or the translation product of the first molecule. The present invention comprises, wherein the at least one first mRNA molecule and the at least one second mRNA molecule are co-formulated into a single vector, or wherein the at least one first mRNA molecule and the at least one second mRNA molecule are formulated into two separate compositions, optionally, wherein the two separate compositions are mixed prior to administration to form a mixed composition, or wherein the first molecule and the second molecule are translated from the single mRNA molecule, for example by an internal ribosome entry site (IRES) or by a self-cleaving peptide (e.g., 2A peptide), optionally, wherein the single mRNA molecule is a bicistronic or polycistronic mRNA molecule. Here, at least one second mRNA molecule or a portion of the single mRNA molecule encoding the second molecule is selected from the group consisting of mRNA molecules encoding cell-bound (or membrane-immobilized) agonist antibodies or agonist antibody fragments, or agonist peptides for cell-bound (or membrane-immobilized) receptors or ligands, mRNA molecules encoding cell-bound (or membrane-immobilized) receptors or ligands, mRNA molecules encoding chemokines or cytokines, mRNA molecules encoding antagonist antibodies or antagonist antibody fragments for immune checkpoint proteins, mRNA molecules encoding immune checkpoint inhibitors, mRNA molecules encoding immune checkpoints, and mRNA molecules encoding transcription factors.

[0021] The term "immune response" as used in this invention refers to either a specific response of the adaptive immune system to a particular antigen (hereinafter referred to as a specific or adaptive immune response) or a nonspecific response of the innate immune system (hereinafter referred to as a nonspecific or innate immune response). Typically, this invention relates to adaptive immune responses. However, such adaptive immune responses may be supported by additional innate immune responses.

[0022] The term "adaptive immune response" as used in this invention refers to an antigen-specific response of the immune system. Importantly, antigen specificity allows for the generation of a response tailored to a particular pathogen or pathogen-infected cell, and this ability is usually maintained in the body by so-called "memory cells." If a pathogen infects the body more than once, these specific memory cells are used to quickly eliminate the pathogen.

[0023] As used herein, the term “adaptive immune system” refers to a system that is essentially solely directed to eliminate or prevent the proliferation of a pathogen. It typically modulates the adaptive immune response by providing the vertebrate immune system with the ability to recognize and remember a particular pathogen (thus generating immunity) and to launch a stronger attack each subsequent encounter with the pathogen.

[0024] "Cell-mediated immunity / cellular immune response", as used herein, generally includes activation of macrophages, natural killer (NK) cells, antigen-specific cytotoxic T lymphocytes in response to an antigen, and release of various cytokines. In more general terms, cell-mediated immunity is not antibody-based but is based on activation of cells of the immune system. In one example, a cell-mediated immune response can be characterized by activation of antigen-specific cytotoxic T lymphocytes that can induce apoptosis in cells presenting epitopes of foreign antigens on their surface, such as certain immune cells like dendritic cells or other cells. Such cells can be virus-infected or intracellular bacterium-infected cells, or cancer cells presenting tumor antigens. Further features of a cell-mediated immune response can be activation of macrophages and natural killer cells (which enables them to destroy pathogens), and stimulation of cells to secrete various cytokines that affect the functions of other cells involved in the adaptive and innate immune responses.

[0025] The term "bicistronic mRNA molecule" or "polycistronic mRNA molecule", as used herein, generally refers to an mRNA molecule having two (bicistronic) or more (multicistronic) open reading frames (ORFs). In contrast, a "monocistronic mRNA molecule" generally contains only one open reading frame. An open reading frame in this context is a sequence of several nucleotide triplets (codons) that can be translated into a peptide or protein.

[0026] The term “at least one” refers to a specific number that is at least one, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or any other number. For example, the term “at least one second mRNA molecule encoding a second molecule which is a protein or peptide having the ability to modulate the immune response to the first molecule and / or the translation product of the first molecule” refers to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or any other number, encoding a second molecule which is a protein or peptide having the ability to modulate the immune response to the first molecule and / or the translation product of the first molecule. In one embodiment, the at least one second mRNA molecule encoding the second molecule which is a protein or peptide having the ability to modulate the immune response to the first molecule and / or the translation product of the first molecule is a single mRNA molecule encoding the second molecule which is a protein or peptide having the ability to modulate the immune response to the first molecule and / or the translation product of the first molecule. Thus, in this embodiment, a single immunomodulator is used. In other embodiments, more than one immunomodulatory factors are used, such as two, three, four, five, or any other number. In further embodiments, at least one of the second mRNA molecules encoding a second molecule which is a protein or peptide capable of modulating the immune response to the first molecule and / or the translation product of the first molecule is not at least a combination of CD40L and CD70. In yet another embodiment, at least one of the second mRNA molecules encoding a second molecule which is a protein or peptide capable of modulating the immune response to the first molecule and / or the translation product of the first molecule is not at least a combination of CD40L, CD70, and a non-wild-type molecule such as constitutively active TLR4. In yet another embodiment, at least one of the second mRNA molecules encoding a second molecule which is a protein or peptide capable of modulating the immune response to the first molecule and / or the translation product of the first molecule is not TGF-beta.

[0027] Importantly, the present invention enables the spatially and / or temporally coordinated in vivo expression of at least one said first molecule, which is a therapeutic or immunogenic protein or peptide, and at least one said second molecule, which is a protein or peptide having the ability to modulate an immune response against the first molecule and / or the translation product of the first molecule. Thus, at least one said second molecule can function as a genetic adjuvant. The term "adjuvant" as used herein refers to a component that can modify, for example enhance, the effect of other agents such as vaccines. The term "adjuvant" as used herein is to be construed broadly and refers to a wide range of substances. Usually, these substances can increase the immunogenicity of an antigen. For example, an adjuvant can be recognized by the innate immune system and can, for example, induce an innate immune response. Alternatively, at least one said second molecule can function as a genetic immunosuppressant.

[0028] In a preferred embodiment, the present invention relates to a combination comprising two or more mRNA molecules, or a single mRNA molecule encoding a first molecule, which is a therapeutic or immunogenic protein or peptide, and a second molecule, which is a protein or peptide having the ability to modulate an immune response against the first molecule and / or the translation product of the first molecule, wherein the combination is as follows: (i) at least one first mRNA molecule encoding a first molecule, which is a therapeutic or immunogenic protein or peptide, and (ii) at least one second mRNA molecule encoding a second molecule, which is a protein or peptide having the ability to modulate an immune response against the first molecule and / or the translation product of the first molecule The present invention comprises, wherein at least one of the first mRNA molecules and at least one of the second mRNA molecules are co-formulated into a single vector, or wherein at least one of the first mRNA molecules and at least one of the second mRNA molecules are formulated into two separate compositions, optionally the two separate compositions being mixed prior to administration to form a mixed composition, or wherein the first and second molecules are translated from the single mRNA molecule, for example by an internal ribosome entry site (IRES) or by a self-cleaving peptide (e.g., 2A peptide), optionally the single mRNA molecule being a bicistronic or polycistronic mRNA molecule. Here, at least one of the second mRNA molecules, or a portion of the single mRNA molecule encoding the second molecule, is selected from the group consisting of an mRNA molecule encoding a cell-bound (or membrane-immobilized) agonist antibody or agonist antibody fragment, or an mRNA molecule encoding an agonist peptide for a cell-bound (or membrane-immobilized) receptor or ligand, or an mRNA molecule encoding a cell-bound (or membrane-immobilized) receptor or ligand.

[0029] To our surprise, we discovered that combinations of antigens and mRNA molecules encoding cell-bound (or membrane-immobilized) agonist antibodies, cell-bound (or membrane-immobilized) receptors, or cell-bound (or membrane-immobilized) ligands as immune adjuvant RNAs need to be cell-bound for optimal activity. For example, as shown in Figure 5, OX86 agonist antibodies co-administered in protein form did not enhance the immune response to the vaccine. Conversely, a transmembrane version of OX86 encoded by mRNA / LNP ("TM-OX86") improved vaccine immunogenicity to a degree comparable to that of the membrane-immobilized natural ligand OX40L encoded by mRNA / LNP. Soluble OX86 antibodies encoded by mRNA / LNP also improved vaccine immunogenicity, albeit to a lower degree.

[0030] In a preferred embodiment, at least one of the second mRNA molecules, or a portion of the single mRNA molecule encoding the second molecule, encodes a protein having positive immunomodulatory activity, such as a protein having the ability to increase the potency and / or quality and / or persistence of an immune response, or, in this embodiment, at least one of the second mRNA molecules, or a portion of the single mRNA molecule encoding the second molecule, encodes a protein having negative immunomodulatory activity, such as a protein having the ability to decrease the potency and / or quality and / or persistence of an immune response. Notably, Figures 1 to 7 and 9 relate to at least one second mRNA molecule or a portion of the single mRNA molecule encoding the second molecule, encoding a protein having positive immunomodulatory activity, such as a protein having the ability to increase the potency and / or quality and / or persistence of an immune response, whereas Figure 8 relates to at least one second mRNA molecule or a portion of the single mRNA molecule encoding the second molecule, encoding a protein having negative immunomodulatory activity, such as a protein having the ability to decrease the potency and / or quality and / or persistence of an immune response.

[0031] In one embodiment, the single vector comprises at least one of the first mRNA molecules and at least one of the second mRNA molecules, or the single mRNA molecule is contained in a nanoparticle, the nanoparticle is preferably liponoparticle (LNP) or non-lipid nanoparticle (e.g., nanoparticle composed of polymer), and / or the two separate compositions, each comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules, are selected from nanoparticles, synthetic particles for in vivo transduction, extracellular vesicles and viral vectors, preferably the nanoparticle is LNP or non-lipid. The base nanoparticles are (for example, nanoparticles composed of polymers), where the LNPs are preferably composed of ionizable cationic lipids, neutral lipids (helper lipids), cholesterol, and PEGylated lipids, wherein the molar lipid ratio (%) of ionizable cationic lipids:neutral lipids:cholesterol:PEGylated lipids in the composition is 50:10:38.5:1.5 or 46.3:9.4:42.7:1.6, and the N / P ratio is preferably (but not limited to) 3, 6, 4, or 8, where N is an ionizable cationic lipid (nitrogen) and P is a nucleotide (phosphate).

[0032] In the LNP described in the present invention, at least one of the first mRNA molecules and at least one of the second mRNA molecules, or the single mRNA molecule, form a complex with one or more lipids, thereby forming liposomes, lipid nanoparticles and / or lipoplexes.

[0033] One embodiment relates to a combination comprising two or more mRNA molecules, or to a single mRNA molecule encoding a first molecule which is a therapeutic or immunogenic protein or peptide and a second molecule which is a protein or peptide which has the ability to modulate the immune response to the first molecule and / or the translation product of the first molecule, wherein the combination is as follows: (i) at least one first mRNA molecule encoding a first molecule which is a therapeutic or immunogenic protein or peptide, and (ii) at least one second mRNA molecule encoding a second molecule which is a protein or peptide capable of modulating the immune response to the first molecule and / or the translation product of the first molecule. The present invention comprises, wherein at least one of the first mRNA molecules and at least one of the second mRNA molecules are co-formulated into a single vector, or wherein at least one of the first mRNA molecules and at least one of the second mRNA molecules are formulated into two separate compositions, optionally wherein the two separate compositions are mixed prior to administration to form a mixed composition, or wherein the first and second molecules are translated from the single mRNA molecule, for example by an internal ribosome entry site (IRES) or by a self-cleaving peptide (e.g., 2A peptide), optionally wherein the single mRNA molecule is a bicistronic or polycistronic mRNA molecule. Here, at least one of the aforementioned second mRNA molecules, or a portion of the single mRNA molecule encoding the aforementioned second molecule, is selected from the group consisting of an mRNA molecule encoding a cell-bound (or membrane-immobilized) agonist antibody or agonist antibody fragment, or an agonist peptide for a cell-bound (or membrane-immobilized) receptor or ligand, an mRNA molecule encoding a cell-bound (or membrane-immobilized) receptor or ligand, an mRNA molecule encoding a chemokine or cytokine, an mRNA molecule encoding an antagonist antibody or antagonist antibody fragment for an immune checkpoint protein, an mRNA molecule encoding an immune checkpoint inhibitor, an mRNA molecule encoding an immune checkpoint, and an mRNA molecule encoding a transcription factor. Herein, the single vector comprises at least one of the first mRNA molecules and at least one of the second mRNA molecules, or the single mRNA molecule is contained in a nanoparticle, the nanoparticle is preferably lipoponoparticle (LNP) or non-lipid nanoparticle (e.g., nanoparticles composed of polymers), and / or herein, the two separate compositions comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules respectively are lipoponoparticles (LNP) or non-lipid nanoparticles (e.g., nanoparticles composed of polymers), where the LNP is preferably composed of ionizable cationic lipids, neutral lipids (helper lipids), cholesterol and PEGylated lipids, optionally herein, the composition The molar lipid ratio (%) of ionizable cationic lipids:neutral lipids:cholesterol:PEGylated lipids is 50:10:38.5:1.5 or 46.3:9.4:42.7:1.6, and the N / P ratio is preferably (but not limited to) 3, 6, 4 or 8, where N is an ionizable cationic lipid (nitrogen) and P is a nucleotide (phosphate), and / or the single vector comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules, or each of the two separate compositions comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules, or the single mRNA molecule being self-replicating RNA or circular RNA. Furthermore / or the single vector comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules, or each of the two separate compositions comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules, or the single mRNA molecule being a viral vector.

[0034] According to the present invention, the term “mRNA-encoded antigen” refers to at least one such first mRNA molecule encoding a first molecule which is a therapeutic or immunogenic protein or peptide, and the terms “mRNA-encoded antigen” and “mRNA molecule encoding a first molecule” may be used interchangeably throughout this application.

[0035] According to the present invention, the term “mRNA-encoded immunomodulator” refers to at least one second mRNA molecule encoding a second molecule which is a protein or peptide having the ability to modulate an immune response to the first molecule and / or the translation product of the first molecule, and the terms “mRNA-encoded immunomodulator” and “mRNA molecule encoding a second molecule” may be used interchangeably throughout this application.

[0036] In another embodiment, at least one of the second mRNA molecules, or a portion of the single mRNA molecule encoding the second molecule, encodes the following: i) Chemokines or cytokines (soluble, cell-bound, or membrane-immobilized) selected preferentially from the following list, but not limited to these: leptin, IL21, Ccl19, Ada, IL4, Ccl5, Cxcl9, Cxcl10, IL7, Ccl20, Ccl21a, Cxcl12, Ccl2, HMGB1, Ccl3, IL2, Efnb2, Ifng, IL12a, IL12b, IL1a, IL1b, IL36, TL1A, Cxcl13, Cxcl16, Ifnb1, IL13, IL17a, IL1 8, IL23a, IL6, Nlrp3, Ccl12, Ccl6, Ccl7, Ccl8, chemerin, Csf2, Cxcl11, IL5, Ccl11, Ccl24, Ccl26, Ccl4, Csf1, IL11, Ccl17, Ccl22, Ccl25, Ccl9, Cxcl1, Cxcl14, Cxcl15, Cxcl2, Cxcl3, Cxcl5, IL22, IL3, Ccl27a, Ccl28, IL-12 single chain, IFNα, GM-CSF, IL15, IL-15sushi and IL-15 receptor α chain chimeric protein, or ii) Cell-binding (or membrane-immobilized) ligands preferred from, but not limited to, the following list: LIGHT-Tnfsf14, OX40L-Tnfsf4, GITRL-Tnfsf18, 41BBL-Tnfsf9, Cd40lg, Cd70, ICOSL, Cd80, Icam1, Flt3l, CALR, Cd86, APRIL-Tnfsf13, CD166, LFA3, CD30L-Tnfsf8, Cd48, Cd74, Cd320, Cd83, Dpp4, Cd1d1, Cd1d2, Clec4n, Clec5a, ITGAL, Slc11a1, Vcam1, CAV1, Cd14, PLXNB2, and TWEAK-Tnfsf12, or iii) an agonist antibody (preferably cell-bound or membrane-immobilized), an agonist antibody fragment (preferably cell-bound or membrane-immobilized), or an agonist peptide (preferably cell-bound or membrane-immobilized) having the same target as the molecule contained in i) and / or ii).

[0037] To our surprise, we discovered that co-administration of the IL4 cytokine as part of at least one of the second mRNA molecules or as part of the single mRNA molecule encoding the second molecule increases anti-spike adaptive immunity to the co-administered SARS-CoV-2 spike protein used as an antigen encoded by mRNA formulated in LNPs (Figure 9).

[0038] Furthermore, the inventors surprisingly also discovered that the T cell response induced by the COVID mRNA vaccine functions using a combination of mRNAs comprising mRNA vaccines and mRNAs encoding different regulatory peptides. To demonstrate the adjuvant effect of chemokines or cytokines and RNA-modulating cell-binding ligands on the immunological activity of mRNA vaccines, the inventors immunized mouse populations with mRNA vaccines alone or with mRNA vaccines mixed with mRNAs encoding immunomodulatory peptides (see Figure 11). The mouse populations were immunized intramuscularly with mRNA formulated in liponopanoparticles (LNPs) encoding the SARS-CoV-2 spike protein (Gr1-Spike); a mixture (combination) containing mRNA formulated in LNPs encoding the SARS-CoV-2 spike protein and mRNA in LNPs encoding OX40L or CD80; and a mixture (combination) containing mRNA in LNPs encoding the SARS-CoV-2 spike protein and mRNA in LNPs encoding IL7, IL21, or IL15.

[0039] Therefore, our data clearly support the use of RNA-encoded antagonist anti-CTLA4 antibodies to enhance the immune response to at least one first mRNA molecule encoding a first molecule that is a therapeutic or immunogenic protein or peptide in gene vaccine applications. Furthermore, our data also support the use of RNA-encoded CTLA4 to reduce and / or eliminate the immune response to at least one first mRNA molecule encoding a first molecule that is a therapeutic or immunogenic protein or peptide in gene therapy applications.

[0040] In one embodiment, at least one of the second mRNA molecules encoding a second molecule which is a protein or peptide capable of modulating an immune response to the first molecule and / or the translation product of the first molecule is an mRNA-encoded antagonist anti-CTLA4 antibody, and the at least one second mRNA molecule is used in combination with the at least one first mRNA molecule encoding a first molecule which is a therapeutic or immunogenic protein or peptide in a gene vaccine application.

[0041] In another embodiment, the at least one second mRNA molecule encoding a second molecule which is a protein or peptide having the ability to modulate an immune response to the first molecule and / or the translation product of the first molecule is CTLA4, and the at least one second mRNA molecule is used in gene therapy applications in combination with the at least one first mRNA molecule encoding a first molecule which is a therapeutic or immunogenic protein or peptide.

[0042] In one particularly preferred embodiment relating to a first aspect of the present invention, at least one of the second mRNA molecules, or a portion of the single mRNA molecule encoding the second molecule, encodes a cell-bound (membrane-immobilized) OX40 ligand, a cell-bound (membrane-immobilized) ICOS ligand, a cell-bound (membrane-immobilized) GITR ligand, a cell-bound (membrane-immobilized) OX40-conjugated antibody, a cell-bound (membrane-immobilized) ICOS-conjugated antibody, and / or a cell-bound (membrane-immobilized) GITR-conjugated antibody.

[0043] In one embodiment relating to a first aspect of the present invention, at least one of the second mRNA molecules, or a portion of the single mRNA molecule encoding the second molecule, encodes a cell-bound (membrane-immobilized) receptor or ligand having immunomodulatory activity from the TNF superfamily.

[0044] In another embodiment, at least one of the second mRNA molecules or the portion of the single mRNA molecule encoding the second molecule is an mRNA molecule encoding an antagonist antibody or antagonist antibody fragment or antagonist peptide against an immune checkpoint protein, or an mRNA molecule encoding an immune checkpoint inhibitor, or an mRNA molecule encoding an immune checkpoint, wherein the immune checkpoint protein is preferredly selected from, but not limited to, one of PD-1, cd47, cd31, PD-L1, PD-L2, BTLA, HVEM, lag3, tigit, prv, cd155, cd112, galectin-9, tim3, tim4, gitr, gitrl, tigit, Vista, cd276, cd39, cd73, CTLA4, IL10, IL35, ido, vip, IL27, and IL37.

[0045] Further embodiments relate to the combination or single mRNA molecule described in the first aspect of the present invention, wherein at least one of the second mRNA molecules or a portion of the single mRNA molecule encoding the second molecule encodes a transcription factor, wherein the transcription factor is preferredly selected from, but not limited to, one of Tcf1, TFEB, TFE3, NLRC5, CIITA, Lef1, Bcl11b, Gata3, Snai3, Ets1, IRF3, IRF7, Zbtb1, irf4, stat5b, runx3, bcl11a, foxo1, ikzf3, ets1, Tcf4, pax5, Bhlhe41, irf3, irf5, irf8, eomes, Stat1, Stat3, Stat5, Cebpd, and Cebpa.

[0046] To our surprise, we discovered that co-administration of the transcription factor Tcf1 as part of at least one of the second mRNA molecules or as part of the single mRNA molecule encoding the second molecule increased anti-spike adaptive immunity to the co-administered SARS-CoV-2 spike protein used as an antigen encoded by mRNA formulated into LNPs (Figure 9).

[0047] In further embodiments, at least one of the second mRNA molecules, or a portion of the single mRNA molecule encoding the second molecule, encodes an antagonist antibody or antagonist antibody fragment against an immune checkpoint protein, or encodes an immune checkpoint inhibitor, and here: (i) The antagonist antibody or the antagonist antibody fragment targets the PD-1 / PD-L1 pathway, preferably wherein the mRNA molecule encodes an antibody selected from atezolizumab, avelumab, semiprimab, dostallimab, durvalumab, nivolumab, and pembrolizumab; (ii) The antagonist antibody or the antagonist antibody fragment targets the CTLA-4 pathway, preferably where the mRNA molecule encodes the antibody ipilimumab, and / or (iii) The antagonist antibody or the antagonist antibody fragment targets the LAG-3 pathway, preferably the mRNA molecule encoding the antibody relatrimab.

[0048] Atezolizumab (Tecentriq®) is a checkpoint inhibitor that targets the PD-1 / PD-L1 pathway; it is approved for use in some patients with bladder cancer, breast cancer, liver cancer, lung cancer, and melanoma.

[0049] Avelumab (Bavencio®) is a checkpoint inhibitor that targets the PD-1 / PD-L1 pathway; it is approved for use in some patients with bladder cancer, kidney cancer, and Merkel cell carcinoma, a type of skin cancer.

[0050] Semiprimab (ributayo®) is a checkpoint inhibitor that targets the PD-1 / PD-L1 pathway; it is approved for use in some patients with cutaneous squamous cell carcinoma, basal cell carcinoma, and lung cancer.

[0051] Dostallumab (Jemperli) is a checkpoint inhibitor that targets the PD-1 pathway; it is approved for use in some patients with uterine (endometrial) cancer.

[0052] Durvalumab (Imfinzi) TM ) is a checkpoint inhibitor that targets the PD-1 / PD-L1 pathway; it is approved for use in some patients with bladder cancer and lung cancer.

[0053] Nivolumab (Opdivo®) is a checkpoint inhibitor that targets the PD-1 / PD-L1 pathway; it is approved for use in some patients with bladder cancer, colorectal cancer, esophageal cancer, gastric cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, lymphoma, melanoma, and mesothelioma.

[0054] Pembrolizumab (Keytruda®) is a checkpoint inhibitor that targets the PD-1 / PD-L1 pathway; it is approved for some patients with bladder cancer, breast cancer, cervical cancer, colorectal cancer, cutaneous squamous cell carcinoma, esophageal cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, lymphoma, melanoma, Merkel cell carcinoma, and gastric cancer. It is also approved for the treatment of some patients with any type of cancer that exhibits certain hereditary mutations (MSI-H, dMMR, or TMB-H).

[0055] Ipilimumab (Yervoy®) is a checkpoint inhibitor that targets the CTLA-4 pathway; it is approved for use in some patients with melanoma, mesothelioma, liver cancer, and lung cancer.

[0056] Reratrimab is a checkpoint inhibitor that targets the LAG-3 pathway; in combination with nivolumab (Opdualag) in some patients with melanoma. TM It is approved as (known as)

[0057] Another embodiment relates to the combination or single mRNA molecule described in the first aspect of the present invention, wherein at least one of the second mRNA molecules or a portion of the single mRNA molecule encoding the second molecule encodes a chemokine or cytokine, and here: (i) The cytokine targets the IL-2 / IL-2R pathway, preferably where the mRNA molecule encodes the cytokine aldesleukin; (ii) The mRNA molecule encodes an immunomodulatory cytokine, such as granulocyte-macrophage colony-stimulating factor (GM-CSF), and / or (iii) The cytokine targets the IFNAR1 / 2 pathway.

[0058] Aldesleukin (Proleukin®) is a cytokine that targets the IL-2 / IL-2R pathway; it is approved for use in some patients with renal cancer and melanoma.

[0059] In one embodiment, at least one of the second mRNA molecules or a portion of the single mRNA molecule encoding the second molecule is not a miRNA, such as an organ-specific miRNA.

[0060] In a further embodiment, at least one of the first mRNA molecules or a portion of the single mRNA molecule encoding the first molecule encodes a therapeutic or immunogenic protein or peptide, which is preferably an infectious antigen such as a bacterial antigen, such as a Gram-positive or Gram-negative bacterial antigen, a protozoan antigen, a viral antigen, a fungal antigen, or a parasitic antigen selected from the group consisting of a single amino acid mutation peptide, a frameshift peptide, a read-through mutation peptide, a splice site mutation peptide, and / or an aging-related antigen, or a therapeutic or immunogenic protein or peptide against a cancer antigen, tumor antigen, and / or tumor neoantigen selected from the group consisting of a single amino acid mutation peptide, a frameshift peptide, a read-through mutation peptide, a splice site mutation peptide, and / or an aging-related antigen, or, in this embodiment, at least one of the first mRNA molecules or a portion of the single mRNA molecule encoding the first molecule encodes a CAS protein, such as a CAS9 protein, or, in this embodiment, at least one of the first mRNA molecules or a portion of the single mRNA molecule encoding the first molecule encodes a therapeutic protein that has the ability to correct a disease caused by the absence of an endogenous gene encoding a therapeutic protein, a mutation in an endogenous gene encoding a therapeutic protein, or inactivation of an endogenous gene encoding a therapeutic protein.

[0061] In the context of the present invention, the term “antigen,” as used herein, refers to a peptide or protein that can be recognized by the immune system, preferably the adaptive immune system. Antigens typically have the ability to provoke 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. Antigens typically comprise at least one epitope, which can be presented to T cells by MHC. In the sense of the present invention, an antigen may be the product of translation of a provided RNA, preferably mRNA as defined herein. In this context, peptide and protein fragments, variants, and derivatives comprising at least one epitope should also be understood as antigens. In the context of the present invention, particularly preferred are infectious antigens, such as antigens selected from the group consisting of bacterial antigens, protozoan antigens, viral antigens, fungal antigens, and parasitic antigens, or therapeutic or immunogenic proteins or peptides against cancer antigens, tumor antigens, and / or tumor neoantigens as defined herein.

[0062] In one example, at least one of the first mRNA molecules or a portion of the single mRNA molecule encoding the first molecule encodes at least one protein or peptide of one of the viruses or bacteria listed below: influenza virus type A or B or any other orthomyxovirus (influenza type C), picornaviruses such as rhinovirus or hepatitis A virus, togaviruses such as alphavirus or rubivirus, sisdovis virus, semliki forest virus or rubeolavirus, rubella virus, coronavirus, rhabdoviruses such as rabies virus, paramyxoviruses such as mumps virus, and rotaviruses such as group A, B or C. Reoviruses, hepadnaviruses such as hepatitis B virus, papoviruses such as any serotype of human papillomavirus, adenoviruses, herpesviruses such as herpes simplex virus 1, herpes simplex virus 2, or herpes simplex virus 3, cytomegalovirus (preferably CMVpp65), Epstein-Barr virus, vaccinia virus, Chlamydia pneumoniae, flaviviruses such as dengue virus types 1 to 4, yellow fever virus, West Nile virus, Japanese encephalitis virus, hepatitis C virus, caliciviruses, filoviruses such as Ebola virus, borna virus, bunyaviruses, arenaviruses such as lymphocytic choriomeningitis virus or hemorrhagic fever virus, retroviruses such as HIV, or parvoviruses.

[0063] Notably, another example of suboptimal activity achieved by prior art mRNA vectors can be exemplified by excessive immunoreactivity to the translation products of therapeutic mRNA delivered in vivo, such as that induced against peptides with molecular scissor activity (i.e., Cas9 or zinc finger nucleases), thereby suppressing or reducing their activity and therapeutic efficacy. Similarly, in vivo delivery of mRNA encoding therapeutic proteins required for gene replacement therapy can induce excessive adaptive immunity that limits therapeutic efficacy.

[0064] In one embodiment, at least one of the first mRNA molecules or a portion of the single mRNA molecule encoding the first molecule encodes a genome editing molecule / molecular scissors (e.g., a CAS protein such as CAS9, or a Zn finger nuclease).

[0065] A further embodiment relates to the combination or single mRNA molecule described in the first aspect of the present invention, wherein at least one of the first mRNA molecules and at least one of the second mRNA molecules, or the single mRNA molecule, is a synthetic and / or modified mRNA molecule, preferably wherein a chemically modified nucleoside is preferentially incorporated into the uracil nucleotide bases and / or cytidine nucleotide bases in at least one of the first mRNA molecules and at least one of the second mRNA molecules, or the single mRNA molecule, wherein all of the uracil nucleotide bases and / or all of the cytidine nucleotide bases in at least one of the first mRNA molecules and at least one of the second mRNA molecules, or the single mRNA molecule, or the at least one of the first mRNA molecules and at least one of the second mRNA A given proportion of uracil nucleosides and / or cytidine nucleosides in molecule A or in the single mRNA molecule are replaced with chemically modified nucleosides, such as N1-methylpseudridine, pseudouridine(ψ), or 5-methylcytidine(m5C), and / or thereof, at least one of the first mRNA molecules and at least one of the second mRNA molecules, or the single mRNA molecule, in the context of linear or circular RNA, preferably comprises an optimized Kosack sequence, optimized codon usage frequency, optimized 5' and / or 3' untranslated region, poly-A tail, 5' cap (preferably selected from, but not limited to, anti-reverse cap analogs (ARCA), CAP0, CAP1:m7GpppNmpN, CAP2:m7GpppNmpNm), and / or internal ribosome entry site (IRES).

[0066] As used herein, “poly(A) tail” may also be referred to as “3'-poly(A) tail,” which is typically a long sequence of adenine nucleotides, such as up to about 400 adenosine nucleotides, e.g., about 25 to about 400, preferably about 50 to about 400, more preferably about 50 to about 300, even more preferably about 50 to about 250, and most preferably about 60 to about 250 adenosine nucleotides, and is attached to the 3' end of a nucleic acid sequence, preferably mRNA. The poly(A) tail may be located at the 3' of a coding region (e.g., mRNA) composed of nucleic acids.

[0067] As used herein, the 5' cap is typically a modified nucleotide (particularly a guanine nucleotide) that is attached to the 5' end of an RNA molecule. In a preferred embodiment, the 5'-cap is attached using a 5'-5'-triphosphate bond.

[0068] In another embodiment, the single vector comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules, or each of the two separate compositions comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules, or the single mRNA molecule is a self-replicating RNA or a circular RNA.

[0069] In one embodiment, the single vector comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules, or each of the two separate compositions comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules, or the single mRNA molecule is a nonviral RNA molecule.

[0070] In a further embodiment, the single vector comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules, or each of the two separate compositions comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules, or the single mRNA molecule is a viral vector.

[0071] In a second aspect, the present invention relates to a host cell comprising the combination or single mRNA molecule described in the first aspect of the present invention, wherein optionally, the host cell is an antigen-presenting cell, preferably a professional antigen-presenting cell, or a lymphocyte, preferably a T lymphocyte or T lymphocyte progenitor cell, more preferably a CD4 or CD8-positive T cell.

[0072] In a third aspect, the present invention relates to a pharmaceutical composition comprising a combination or single mRNA molecule as described in the first aspect of the present invention, or a host cell as described in the second aspect of the present invention, and a pharmaceutically acceptable carrier, stabilizer and / or excipient.

[0073] In a fourth aspect, the present invention relates to a vaccine comprising a combination or single mRNA molecule as described in the first aspect of the present invention, a host cell as described in the second aspect of the present invention, or a pharmaceutical composition as described in the third aspect of the present invention.

[0074] As used herein, the term “vaccine” typically refers to a prophylactic or therapeutic substance that provides at least one antigen, preferably an immunogen. “Providing at least one antigen” means, for example, that the vaccine contains the antigen, or that the vaccine contains, for example, a molecule encoding the antigen, or a molecule containing the antigen. For example, the vaccine may contain a nucleic acid, such as RNA, that encodes a peptide or protein containing the antigen. The antigen or immunogen may be derived from any substance suitable for vaccination. For example, the antigen or immunogen may be derived from a pathogen, such as a bacterium or viral particle, or from a tumor or cancerous tissue. The antigen or immunogen stimulates the body’s adaptive immune system to provide an adaptive immune response.

[0075] In a preferred embodiment, the vaccine is an RNA vaccine. The term “RNA vaccine,” as used herein, is defined herein as a vaccine comprising at least one RNA molecule containing at least one open reading frame (ORF) encoding at least one antigen. In the context of the present invention, the at least one RNA molecule comprising the vaccine is preferably an isolated RNA molecule. This at least one RNA is preferably viral RNA, a self-replicating RNA (replicon), or most preferably mRNA.

[0076] In one embodiment relating to a fourth aspect of the present invention, the vaccine induces an adaptive immune response, preferably a virus (for example, a virus selected from, but not limited to, the following list: coronavirus, SARS coronavirus, SARS-CoV-2 coronavirus, respiratory syncytial virus (RSV), measles virus, influenza virus, rabies virus, dengue virus, HIV (human immunodeficiency virus), hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus, hepatitis E virus, foot-and-mouth disease virus (FMDV), bovine leukemia virus (BLV), bovine parainfluenza virus, human parainfluenza virus (HPIV), bovine respiratory syncytial virus, porcine reproductive and respiratory disorder syndrome virus, respiratory syncytial virus (RSV) Viruses, vesicular stomatitis virus, bovine viral diarrhea virus, bovine coronavirus, BHV1 virus, equine arteritis virus, Nipah virus, BK virus, porcine respiratory coronavirus infection, Yargsigte sheep retrovirus, infectious bronchitis virus, avian pneumovirus, calicivirus, enterovirus, Epstein-Barr virus (EBV), Newcastle disease virus, astrovirus, influenza virus A (avian influenza) H5N1 subtype, herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), heartland virus, boca Protective adaptive immune responses to viruses (HBoV, human herpesvirus 6 (HHV-6), human herpesvirus 7 (HHV-7), human metapneumovirus (hMPV), human papillomavirus (HPV), Japanese encephalitis virus, JC virus, Junin virus, cytomegalovirus (CMV), Machupovirus, Marburg virus, Molluscum contagiosum virus (MCV), Lassa virus, mumps virus, rhinovirus, rotavirus, varicella-zoster virus, Venezuelan encephalitis virus, West Nile virus, Western equine encephalitis virus, and yellow fever virus, filovirus),and / or bacteria (for example, selected from the list below, but not limited to: Acinetobacter, Acinetobacter baumannii, Actinobacillus, Anaplasma, Ankylostomata duodenale, Hemolytic Alcholderia viralidae, Babesia, Bacillus, Anthrax, Bacillus cereus, Bacteroides, Burkholderia, Burkholderia hensele, Glanders, Bordetella partasis, Bordetella, Burkholderia brizaris, Brucella, Campylobacter, Candida albicans, Chlamydia, Chlamydia trachomatis, Chlamydophila, Chlamydia pneumoniae, Clostridium botulinum, Clostridium difficile, Clostridium candididium, Clostridium tetanus, Clostridium diphtheriae, Clostridium, Clostridium brunetii, Cyanobacteria, Dientamoeba fragilis, Escherichia coli, Ehrlichia, Ehrlichia schaffensis, Ehrlichia euingii, Enterobacter, Enterococcus, Erwinia, Francisella, Fusobacteria, Haemophilus, Haemophilus influenzae, Helicobacter, He Hemophilus, Histoplasma capsulatum, Kingera, Kingella, Kingella granulomatis, Klebsiella, Klebsiella granulomatis, Legionella, Legionella pneumophila, Listeria, Neisseria meningitidis, Moraxella, Mycobacterium lepromatosis, Mycobacterium tuberculosis, Mycobacterium ulcerans mulcerans, Moraxella, Mycoplasma pneumoniae, Neisseria, Pasteurella, Prevotella, Proteus It induces a protective adaptive immune response against Pseudomonas, Rickettsia, Salmonella, Serratia, Sigella, Staphylococcus, Streptococcus, Treponema, Vibrio, Yersinia, and escape pathogens, and / or a protective adaptive immune response against parasites (e.g., selected from the list below, but not limited to: Malaria, Leshmania, Cryptosolidium, Entamoeba, Taenia solium, Acaris lumbricoides, Echinococcus, Trypanosoma cruzi, Trypanosoma brusey, Schistosoma).

[0077] In another embodiment relating to a fourth aspect of the present invention, the vaccine induces an adaptive immune response, preferably for example, 1A01 HLA-A / m;1A02;5T4;ACRBP;AFP;AKAP4;alpha-actinin-4 / m;alpha-methylacyl-CoA racemase;ANDR;ART-4;ARTCl / m;AURKB;B2MG;B3GN5;B4GN1;B7H4;BAGE-1;BASI;BCL-2;bcr / abl;beta-catenin / m;BING-4;BIRC7;BRCAl / m;BY55;calreticulin;CAMEL;CASPA;caspase_8;cathepsin_B;cathepsin_L;CD1A;CD1B; CD1C; CD1D; CD1E; CD20; CD22; CD276; CD33; CD3E; CD3Z; CD4; CD44 isoform 1; CD44 elsoform 6 (CD44_lsoform_6); CD52; CD55; CD56; CD80; CD86; CD8A; CDC27 / m; CDE30; CDK4 / m; CDKN2A / m; CEA; CEAM6; CH3L2; CLCA2; CML28; CML66; COA-1 / m; Coactosin-like protein; Collagen XXIII; COX-2; CP1B1; CSAG2; CT-9 / BRD6;CT45A1;CT55;CTAG2 isoform LAGE-1A;CTAG2 isoform LAGE-1B;CTCFL;Cten;CyclinBl(cyclin_Bl);CyclinD1;cyp-B;DAM-10;DEP1A;E7;EF1A2;EFTUD2 / m;EGFR;EGLN3;ELF2 / m;EMMPRIN;EpCam;EphA2;EphA3;ErbB3;ERBB4;ERG;ETV6;EWS;EZH2;FABP7;FCGR3A_Version_1;F CGR3A_Version_2;FGF5;FGFR2;Fibronectin;FOS;FOXP3;FUT1;G250;GAGE-1;GAGE-2;GAGE-3;GAGE-4;GAGE-5;GAGE-6;GAGE7b;GAGE-8JGAGE-2D);GASR;GnT-V;GPC3;GPNMB / m;GRM3;HAGE;hepsin;Her2 / neu;HLA-A2 / m;homeobox_NKX3.1;HOM-TES-85;HPG1;HS71A;HS71B;HST-2;hTERT;iCE;IF2B3;IL-10;IL-13Ra2;IL2-RA;IL2-RB;IL2-RG;IL-5;IMP3;ITA5;I TB1;ITB6;カリクレイン-2;カイリクレイン-4(kailikrein-4);KI20A;KIAA0205;KIF2C; KK-LC-1;LDLR;LGMN;LIRB2;LY6K;MAGA5;MAGA8;MAGAB;MAGE-_B1;MAGE-_E1;MAGE-A1;MAGE-A10;MAGE-A12;MAGE-A2;MAGE-A3;MAGE-A4;MAGE-A6;MAGE-A6;MAGE-A1; GE-A9;MAGE-BIO;MAGE-B16;MAGE-B17;MAGE-B2;MAGE-B3;MAGE-B4;MAGE-B5;MAGE-B6;MAGE-C1;MAGE-C2;MAGE-C3;MAGE-D1;MAGE-D2;MAGE-D4;MAGE -E1JMAGE1);MAGE-E2;MAGE-F1;MAGE-HI;MAGEL2;マンマグロビンA;MART-l / mela n-A;MART-2;MC1R;M-CSF;メソテリン;MITF;MMP1;MMP7;UC-1;MUM-l / m;MUM-2 / m ;MY01A;MY01B;MY01C;MY01D;MY01E;MY01F;MY01G;MY01H;NA17;NA88-A;Neo-PAP;NFYC / m;NGEP;N-myc;NPM;NRCAM;NSE;NUF2;NY-ESO1;OS1;9; p53;PAGE-4;PAI-1;PAI-2;PAP;PATE;PAX3;PAX5;PD1 L1;PDCD1;PDEF;PECA1;PGCB;PGFRB;Pim-1-Kinase;Pin-1;PLAC1;PMEL;PM L;POTE;POTEF;PRAME;PRDX5 / m;PRM2;prostein;プロテイナーゼ-3;PSA;PSB9;PSCA;PSGR;PSM;PTPRC;RAB8A;RAGE-1;RARA;RASH;RASK;RASN;RHAMSC5;RHAMSC5; D168;RHOC;RSSA;RU1;RU2;RUNX1;S-100;SAGE;SART-1;SART-2;SART-3;SEPR;SERPINB5;SIA7F;SIA8A;SIAT9;SIRT2 / m;SOX10;SP17;SPNXA;SPXIM3;It induces a protective adaptive immune response to cancer antigens or tumor antigens such as cancer antigens or tumor antigens selected from SSX-1;SSX-2;SSX3;SSX-4;ST1A1;STAG 2;STAMP-1;STEAP-1;Survivin;Survivin-2B;SYCP1;SYT-SSX-1;SYT-SSX-2;TARP;TCRg;TF2AA;TGFbeta1;TGFR2;TGM-4;TIE2;TKTL1;TPI / m;TRGV11;TRGV9;TRPC1;TRP-p8;TSG10;TSPY1;TVCJTRGV3;TX101;Tyrosinase;TYRP1;TYRP2;UPA;VEGFR1 and XAGE1; and / or tumor neoantigens selected from the group consisting of single amino acid mutant peptides, frameshift peptides, readthrough mutant peptides, splice site mutant peptides, and / or aging-related antigens.

[0078] In a fifth embodiment, the present invention relates to pharmaceutical combinations including: (i) a combination or single mRNA molecule as described in the first aspect of the present invention, a host cell as described in the second aspect of the present invention, a pharmaceutical composition as described in the third aspect of the present invention, or a vaccine as described in the fourth aspect of the present invention, and (ii) Checkpoint inhibitor, optionally selected from atezolizumab, avelumab, semiprimab, dostallimab, durvalumab, nivolumab, pembrolizumab, ipilimumab, and reratrimab. (iii) an antagonist antibody or antagonist antibody fragment or antagonist peptide against an immune checkpoint protein, wherein the immune checkpoint protein is optionally preferredly selected from, but not limited to, one of PD-1, cd47, cd31, PD-L1, PD-L2, BTLA, HVEM, lag3, tigit, prv, cd155, cd112, galectin-9, tim3, tim4, gitr, gitrl, tigit, Vista, cd276, cd39, cd73, CTLA4, IL10, IL35, ido, vip, IL27 and IL37, and / or (iv) an agonist antibody, agonist antibody fragment, or agonist peptide against an immune checkpoint protein, wherein the immune checkpoint protein is preferably selected from, but is not limited to, one of PD-1, cd47, cd31, PD-L1, PD-L2, BTLA, HVEM, lag3, tigit, prv, cd155, cd112, galectin-9, tim3, tim4, gitr, gitrl, tigit, Vista, cd276, cd39, cd73, CTLA4, IL10, IL35, ido, vip, IL27, and IL37.

[0079] In a preferred embodiment, the pharmaceutical combination includes: (i) A vaccine according to a fourth aspect of the present invention, wherein the vaccine induces a protective adaptive immune response against a cancer antigen or tumor antigen, (ii) A checkpoint inhibitor, wherein the checkpoint inhibitor is optionally selected from atezolizumab, avelumab, semiprimab, dostallimab, durvalumab, nivolumab, pembrolizumab, ipilimumab, and relatrimab. (iii) an antagonist antibody, antagonist antibody fragment, or antagonist peptide against an immune checkpoint protein, wherein the immune checkpoint protein is preferably selected from, but not limited to, one of the following: PD-1, cd47, cd31, PD-L1, PD-L2, BTLA, HVEM, lag3, tigit, prv, cd155, cd112, galectin-9, tim3, tim4, gitr, gitrl, tigit, Vista, cd276, cd39, cd73, CTLA4, IL10, IL35, ido, vip, IL27, and IL37, and / or (iv) an agonist antibody, agonist antibody fragment, or agonist peptide against an immune checkpoint protein, wherein the immune checkpoint protein is preferably selected from, but is not limited to, one of PD-1, cd47, cd31, PD-L1, PD-L2, BTLA, HVEM, lag3, tigit, prv, cd155, cd112, galectin-9, tim3, tim4, gitr, gitrl, tigit, Vista, cd276, cd39, cd73, CTLA4, IL10, IL35, ido, vip, IL27, and IL37.

[0080] In a sixth aspect, the present invention relates to a kit comprising a combination or single mRNA molecule as described in a first aspect of the present invention, or a host cell as described in a second aspect of the present invention, a pharmaceutical composition as described in a third aspect of the present invention, a vaccine as described in a fourth aspect of the present invention, or a pharmaceutical combination as described in a fifth aspect of the present invention, and optionally comprising a liquid solvent for solubilization, and further optionally comprising technical instructions providing information regarding the administration and / or dosage of the components.

[0081] A seventh aspect of the present invention relates to a combination or single mRNA molecule described in the first aspect of the present invention, or a host cell described in the second aspect of the present invention, a pharmaceutical composition described in the third aspect of the present invention, a vaccine described in the fourth aspect of the present invention, a pharmaceutical combination described in the fifth aspect of the present invention, or a kit described in the sixth aspect of the present invention, for use in pharmaceuticals.

[0082] An eighth aspect of the present invention relates to a combination or single mRNA molecule described in the first aspect of the present invention, or a host cell described in the second aspect of the present invention, a pharmaceutical composition described in the third aspect of the present invention, a vaccine described in the fourth aspect of the present invention, a pharmaceutical combination described in the fifth aspect of the present invention, or a kit described in the sixth aspect of the present invention for use in a method of preventing and / or treating infectious, hereditary or proliferative diseases, wherein the infectious disease is preferably a viral infection such as an infection caused by a coronavirus (e.g., COVID-19 disease), a bacterial infection, a fungal infection, a protozoan infection and / or a parasitic infection, and wherein the proliferative disease is preferably cancer, more preferably pancreatic cancer, bladder cancer, breast cancer, liver cancer, lung cancer, cutaneous squamous cell carcinoma, basal cell carcinoma, melanoma, uterine (endometrial) cancer, kidney cancer, skin cancer such as Merkel cell carcinoma, colorectal cancer, esophageal cancer, gastric cancer, head and neck cancer, neuroblastoma, leukemia, sarcoma, kidney cancer These cancers are selected from cancers exhibiting specific hereditary mutations (e.g., MSI-H, dMMR, or TMB-H), including cervical cancer, stomach cancer, and cancers exhibiting certain hereditary mutations.

[0083] In another embodiment, the cancers are preferably esophageal cancer; thyroid cancer; lung cancer; stomach cancer; pancreatic cancer; kidney cancer; cervical cancer; breast cancer; neuroendocrine cancer; endometrial cancer; vaginal cancers; hematological cancers; glioma; bone sarcoma; cervix cancer; synovial cancer; sarcoma; acute lymphoblastic leukemia; acute myeloid leukemia; adrenocortical carcinoma; AIDS-related lymphoma; AIDS-related malignancies; anal cancer; astrocytoma; bile duct cancer, extrahepatic bile duct Cancer, Extrahepatic; Bladder cancer; Bone cancer, Osteosarcoma / Malignant Fibrous Histiocytoma; Brainstem glioma; Cerebellar astrocytoma; Brain tumor, Cerebral astrocytoma / Malignant glioma; Ependymoma; Medulloblastoma; Brain tumor, Supratentorial Primitive Neuroectodermal Tumors; Visual Pathway and Hypothalamic Glioma, Bronchial adenoma / Carcinoid; Carcinoma; Adrenocortical; Central Nervous System Lymphoma, Primary; Chronic Lymphocytic Leukemia; Chronic Myelogenous Leukemia Leukemia; Chronic Myeloproliferative Disorders; Clear cell sarcoma of the tendon sheath; Colon cancer;Cutaneous T-cell lymphoma; Endometrial cancer; Ependymoma; Epithelial cancer; Ovarian cancer; Esophageal cancer, Ewing's Family of Tumors; Extracranial germ cell tumor; Extragonadal germ cell tumor; Extrahepatic bile duct cancer; Eye cancer, Intraocular melanoma; Eye cancer, Retinoblastoma; Gallbladder cancer; Gastric (Stomach) cancer; Gastrointestinal carcinoid tumor; Germ cell tumor, Extracranial; Germ cell tumor, Extragonadal; Germ cell tumor, Ovarian Ovarian; Gestational Trophoblastic Tumor; Hair cell leukemia; Head and neck cancer; Hodgkin lymphoma, hypopharyngeal cancer; Hypothalamic and Visual Pathway Glioma; Intraocular melanoma; Islet cell carcinoma (endocrine pancreas); Kaposi's sarcoma; Kidney cancer; Laryngeal cancer, acute lymphoblastic; Leukemia, acute myeloid; Chronic lymphocytic; Chronic myelogenous; Hair cell; Lip and Oral Cavity Cancer; Lymphoblastic leukemia; Lymphoma, AIDS-related; Lymphoma, Central Nervous System (primary) System (Primary); Lymphoma, Cutaneous T-Cell;Lymphoma, primary central nervous system lymphoma; macroglobulinemia, malignant mesothelioma; malignant thymoma; medulloblastoma; melanoma; intraocular melanoma; Merkel cell carcinoma; malignant mesothelioma; metastatic cervical squamous cell carcinoma of unknown primary origin; multiple endocrine neoplasia syndrome; multiple myeloma / plasma cell neoplasm; mycosis fungoides; myelodysplastic syndrome; chronic myelogenous leukemia; myeloid leukemia; multiple myeloma; myeloproliferative disorder Disorders, Chronic; Nasal Cavity and Paranasal Sinus Cancer; Nasopharyngeal Cancer; Neurofibroma; Non-Hodgkin Lymphoma, Non-Small Cell Lung Cancer; Oral Cavity and Lip Cancer; Oropharyngeal Cancer; Osteosarcoma / Malignant Fibrous Histiocytoma of Bone; Epithelial Ovarian Cancer; Ovarian Germ Cell Tumor; Low-Grade Ovarian Tumor; Pancreatic Cancer; Paranasal Sinus and Nasal Cavity Cancer; Parathyroid Cancer; Penile Cancer; Pheochromocytoma; Pineal and Supratentorial Primitive Neuroectodermal Tumors; Pituitary Tumor; Plasma Cell Neoplasm / Multiple Myeloma Myeloma); pleuropulmonary blastoma;Primary Central Nervous System Lymphoma; Primary Liver Cancer; Prostate Cancer; Rectal Cancer; Renal Cell (Kidney) Cancer; Renal Pelvis and Ureter, Transitional Cell Cancer; Retinoblastoma; Rhabdomyosarcoma; Salivary Gland Cancer; Sarcoma, Ewing's Family of Tumors; Sarcoma, Kaposi's; Sarcoma (Osteosarcoma) / Malignant Fibrous Histiocytoma of Bone Bone; Sarcoma, rhabdomyosarcoma; Sarcoma, soft tissue; Sézary syndrome; Skin cancer (melanoma); Skin carcinoma, Merkel cells; Small cell lung cancer; Small intestine cancer; Soft tissue sarcoma; Cervical squamous cell carcinoma of unknown primary origin, metastatic; Stomach (Gastric) Cancer; Supratentorial Primitive Neuroectodermal Tumors; T-cell lymphoma, cutaneous; Testicular cancer; Thymoma; Thyroid cancer; Transitional Cell Cancer of the Renal Pelvis and Ureter; Trophoblastic Tumor, Gestational; Ureter and Renal Pelvis, Transitional Cell Cancer; Urethral cancer; Uterine sarcoma; Vaginal cancer Cancer; selected from one or more of the following: visual pathway and hypothalamic glioma and vulvar cancer.

[0084] In a ninth aspect, the present invention relates to a combination or single mRNA molecule, therapeutic protein, host cell, pharmaceutical composition, pharmaceutical combination, or kit described in the present invention for use in methods for preventing and / or treating hereditary disorders.

[0085] One embodiment of the present invention relates to a combination or single mRNA molecule described in the first aspect of the present invention, a host cell described in the second aspect of the present invention, a pharmaceutical composition described in the third aspect of the present invention, a vaccine described in the fourth aspect of the present invention, a pharmaceutical combination described in the fifth aspect of the present invention, or a kit described in the sixth aspect of the present invention, for use as described in the seventh aspect, wherein the method involves applying to a subject a combination or single mRNA molecule described in the first aspect of the present invention, or a host cell described in the second aspect of the present invention, a pharmaceutical composition described in the third aspect of the present invention, a vaccine described in the fourth aspect of the present invention, a pharmaceutical combination described in the fifth aspect of the present invention, or a kit described in the sixth aspect of the present invention, at least one of the first mRNA molecules and at least one of the second mRNA molecules The administration comprises administering the single vector containing the NA molecule, the two separate compositions each containing at least one of the first mRNA molecules and at least one of the second mRNA molecules, or the mixed composition, or the single mRNA molecule, optionally administering the single vector each containing at least one of the first mRNA molecules and at least one of the second mRNA molecules, or the two separate compositions each containing at least one of the first mRNA molecules and at least one of the second mRNA molecules, or the mixed composition, or the single mRNA molecule to a subject, and further optionally administering the two separate compositions each containing at least one of the first mRNA molecules and at least one of the second mRNA molecules separately, and here i) The two distinct compositions are administered separately to a first site and a second site, where the first site is within 20 cm, 17.5 cm, 15 cm, 12.5 cm, 10 cm, 7.5 cm, 5 cm, 2.5 cm, 1 cm, 0.5 cm, 0.25 cm or 0.1 cm of the second site, preferably where the outflow lymphatic system of the first site flows to the same lymph nodes as the lymphatic system of the second site, or where the first site and the second site are the same, and / or ii) The two distinct compositions are administered separately within a time interval of 1 hour or less, 30 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, 5 minutes or less, 3 minutes or less, or 1 minute or less. This enables the spatially and / or temporally coordinated in vivo expression of at least one first molecule, which is a therapeutic or immunogenic protein or peptide, and at least one second molecule, which is a protein or peptide capable of modulating the immune response to the first molecule and / or the translation product of the first molecule.

[0086] Further preferred embodiments of the present invention relate to a combination or single mRNA molecule described in the first aspect of the present invention, or a host cell described in the second aspect of the present invention, a pharmaceutical composition described in the third aspect of the present invention, a vaccine described in the fourth aspect of the present invention, a pharmaceutical combination described in the fifth aspect of the present invention, or a kit described in the sixth aspect of the present invention, for use as described in the seventh aspect, wherein the method involves, to a subject, a combination or single mRNA molecule described in the first aspect of the present invention, or a host cell described in the second aspect of the present invention, a pharmaceutical composition described in the third aspect of the present invention, a vaccine described in the fourth aspect of the present invention, a pharmaceutical combination described in the fifth aspect of the present invention, or a kit described in the sixth aspect of the present invention, at least one of the first mRNA molecules and at least one The administration comprises the administration of the single vector containing the second mRNA molecule, the two separate compositions each containing at least one first mRNA molecule and at least one second mRNA molecule, or the mixed composition, or the single mRNA molecule, wherein the single vector containing at least one first mRNA molecule and at least one second mRNA molecule, or the two separate compositions each containing at least one first mRNA molecule and at least one second mRNA molecule, or the mixed composition, or the single mRNA molecule is administered to the subject, wherein the two separate compositions each containing at least one first mRNA molecule and at least one second mRNA molecule are administered separately, and here i) The two distinct compositions are administered separately to a first site and a second site, where the first site is within 20 cm, 17.5 cm, 15 cm, 12.5 cm, 10 cm, 7.5 cm, 5 cm, 2.5 cm, 1 cm, 0.5 cm, 0.25 cm or 0.1 cm of the second site, preferably where the outflow lymphatic system of the first site flows to the same lymph nodes as the lymphatic system of the second site, or where the first site and the second site are the same, and / or ii) The two distinct compositions are administered separately within a time interval of 1 hour or less, 30 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, 5 minutes or less, 3 minutes or less, or 1 minute or less. This enables the spatially and / or temporally coordinated in vivo expression of at least one of the first molecules, which is a therapeutic or immunogenic protein or peptide, and at least one of the second molecules, which is a protein or peptide capable of modulating the immune response to the first molecule and / or the translation product of the first molecule.

[0087] To our surprise, the inventors discovered that the mRNA-encoded antigen and the mRNA-encoded immunomodulator should be administered as close together in time and space as possible to achieve spatially and / or temporally coordinated in vivo expression of the mRNA-encoded antigen and the mRNA-encoded immunomodulator. Specifically, the mRNA-encoded antigen and the mRNA-encoded immunomodulator should not be administered sequentially (i.e., at different times), nor at different sites (and / or by different routes), as this would lead to a loss of the mRNA-encoded immunomodulator's biological effect.

[0088] In a preferred embodiment, the first mRNA molecule encoding a first molecule which is a therapeutic or immunogenic protein or peptide, and the second mRNA molecule encoding a second molecule which is a protein or peptide capable of modulating an immune response to the first molecule and / or the translation product of the first molecule, are not administered sequentially, for example, at different times.

[0089] In another preferred embodiment, at least one of the first mRNA molecules encoding a first molecule which is a therapeutic or immunogenic protein or peptide, and at least one of the second mRNA molecules encoding a second molecule which is a protein or peptide having the ability to modulate an immune response to the first molecule and / or the translation product of the first molecule, are administered simultaneously. The term “simultaneously” as used herein means “at the same time.”

[0090] In a more preferred embodiment, at least one of the first mRNA molecules encoding a first molecule which is a therapeutic or immunogenic protein or peptide, and at least one of the second mRNA molecules encoding a second molecule which is a protein or peptide capable of modulating an immune response to the first molecule and / or the translation product of the first molecule, are not administered at different sites.

[0091] In another preferred embodiment, at least one of the first mRNA molecules encoding a first molecule which is a therapeutic or immunogenic protein or peptide, and at least one of the second mRNA molecules encoding a second molecule which is a protein or peptide capable of modulating an immune response to the first molecule and / or the translation product of the first molecule, are administered to the same site.

[0092] In yet another preferred embodiment, at least one of the first mRNA molecules encoding a first molecule which is a therapeutic or immunogenic protein or peptide, and at least one of the second mRNA molecules encoding a second molecule which is a protein or peptide having the ability to modulate an immune response to the first molecule and / or the translation product of the first molecule, are not administered by different routes.

[0093] In a more preferred embodiment, at least one of the first mRNA molecules encoding a first molecule which is a therapeutic or immunogenic protein or peptide, and at least one of the second mRNA molecules encoding a second molecule which is a protein or peptide capable of modulating an immune response to the first molecule and / or the translation product of the first molecule, are administered via the same route.

[0094] Figures 3 and 4 demonstrate that the activity of immunomodulatory RNA requires co-formulation or co-administration with therapeutic RNA (assessed by B cell response in Figure 3 and T cell response in Figure 4). Importantly, antibody titers were significantly improved when mice were immunized with mRNA encoding spike protein and mRNA encoding Ox40 ligand protein co-formulated with the same LNP, and when the spike-encoding mRNA was co-administered with mRNA encoding OX40L. However, contralateral administration of therapeutic and regulatory mRNAs completely abolished this enhancement (Figure 3). Furthermore, T cell responsiveness was also clearly enhanced when mice were immunized with mRNA encoding spike protein and mRNA encoding Ox40 ligand protein co-formulated with the same LNP, and when the therapeutic mRNA was co-administered with mRNA encoding OX40L. However, contralateral administration of therapeutic and regulatory mRNAs completely abolished this enhancement (Figure 4). Therefore, if mRNA-encoded antigens and mRNA-encoded immunomodulators are delivered simultaneously but to different sites, an improved immune response cannot be observed.

[0095] In conclusion, delivery of the two components at different sites, even when administered simultaneously, does not lead to an improved immune response. This is because the immunomodulator must be expressed in the same microenvironment (and better, in the same cells) in which the antigen is expressed in order to target the immune cells that react to the antigen. Expression at distant sites leads to a decrease / absence of the immunomodulator concentration at sites where antigen-stimulated immune cells can be found.

[0096] In one embodiment, at least one first mRNA molecule encoding a first molecule which is a therapeutic or immunogenic protein or peptide, and at least one second mRNA molecule encoding a second molecule which is a protein or peptide having the ability to modulate an immune response to the first molecule and / or the translation product of the first molecule, are expressed in the same cell.

[0097] In a preferred embodiment, the present invention relates to a combination comprising two or more mRNA molecules for use as described in the seventh aspect, or a single mRNA molecule encoding a first molecule which is a therapeutic or immunogenic protein or peptide and a second molecule which is a protein or peptide which has the ability to modulate an immune response to the first molecule and / or the translation product of the first molecule, a host cell comprising the combination or the single mRNA molecule, a pharmaceutical composition comprising the combination or the single mRNA molecule, the host cell or the pharmaceutical composition, a vaccine comprising the combination or the single mRNA molecule, the host cell or the pharmaceutical composition, the pharmaceutical combination described in the present invention, or a kit comprising the combination or the single mRNA molecule, the host cell, the pharmaceutical composition, the vaccine or the pharmaceutical combination. The aforementioned combinations are as follows: (i) at least one first mRNA molecule encoding a first molecule which is a therapeutic or immunogenic protein or peptide, and (ii) at least one second mRNA molecule encoding a second molecule which is a protein or peptide capable of modulating the immune response to the first molecule and / or the translation product of the first molecule. The vector comprises, wherein at least one of the first mRNA molecules and at least one of the second mRNA molecules are co-formulated into a single vector, or wherein at least one of the first mRNA molecules and at least one of the second mRNA molecules are formulated into two separate compositions, optionally wherein the two separate compositions are mixed prior to administration to form a mixed composition, or wherein the first molecule and the second molecule are translated from the single mRNA molecule, for example by an internal ribosome entry site (IRES) or by a self-cleaving peptide (e.g., 2A peptide), optionally wherein the single mRNA molecule is a bicistronic or polycistronic mRNA molecule. Here, at least one of the second mRNA molecules, or a portion of the single mRNA molecule encoding the second molecule, is selected from the group consisting of an mRNA molecule encoding a cell-bound (or membrane-immobilized) agonist antibody or agonist antibody fragment, or an mRNA molecule encoding an agonist peptide for a cell-bound (or membrane-immobilized) receptor or ligand, and an mRNA molecule encoding a cell-bound (or membrane-immobilized) receptor or ligand. Furthermore, the method hereby includes administering to a subject a combination or single mRNA molecule according to the first aspect of the present invention, or a host cell according to the second aspect of the present invention, a pharmaceutical composition according to the third aspect of the present invention, a vaccine according to the fourth aspect of the present invention, a pharmaceutical combination according to the fifth aspect of the present invention, a kit according to the sixth aspect of the present invention, the single vector comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules, the two separate compositions each comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules, or the mixed composition, or the single mRNA molecule, to the subject, and hereby administering the two separate compositions each comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules separately, and further, i) The two distinct compositions are administered separately to a first site and a second site, where the first site is within 20 cm, 17.5 cm, 15 cm, 12.5 cm, 10 cm, 7.5 cm, 5 cm, 2.5 cm, 1 cm, 0.5 cm, 0.25 cm, or 0.1 cm of the second site, preferably where the outflow lymphatic system of the first site flows to the same lymph nodes as the lymphatic system of the second site, or where the first site and the second site are the same, and / or ii) The two distinct compositions are administered separately within a time interval of 1 hour or less, 30 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, 5 minutes or less, 3 minutes or less, or 1 minute or less. This enables the spatially and / or temporally coordinated in vivo expression of at least one of the first molecules, which is a therapeutic or immunogenic protein or peptide, and at least one of the second molecules, which is a protein or peptide capable of modulating the immune response to the first molecule and / or the translation product of the first molecule.

[0098] A more preferred embodiment relates to a kit comprising a combination of two or more mRNA molecules for use as described in the seventh aspect, or a single mRNA molecule encoding a first molecule which is a therapeutic or immunogenic protein or peptide and a second molecule which is a protein or peptide which has the ability to modulate an immune response to the first molecule and / or the translation product of the first molecule, a host cell comprising the combination or the single mRNA molecule, a pharmaceutical composition comprising the combination or the single mRNA molecule, the host cell or the pharmaceutical composition, a pharmaceutical combination as described in the present invention, the combination or the single mRNA molecule, the host cell, the pharmaceutical composition, the vaccine or the pharmaceutical combination, The aforementioned combinations are as follows: (i) at least one first mRNA molecule encoding a first molecule which is a therapeutic or immunogenic protein or peptide, and (ii) at least one second mRNA molecule encoding a second molecule which is a protein or peptide capable of modulating the immune response to the first molecule and / or the translation product of the first molecule. The present invention comprises, wherein at least one of the first mRNA molecules and at least one of the second mRNA molecules are co-formulated into a single vector, or wherein at least one of the first mRNA molecules and at least one of the second mRNA molecules are formulated into two separate compositions, optionally wherein the two separate compositions are mixed prior to administration to form a mixed composition, or wherein the first and second molecules are translated from the single mRNA molecule, for example by an internal ribosome entry site (IRES) or by a self-cleaving peptide (e.g., 2A peptide), optionally wherein the single mRNA molecule is a bicistronic or polycistronic mRNA molecule. Here, at least one of the second mRNA molecules or a portion of the single mRNA molecule encoding the second molecule is selected from the group consisting of an mRNA molecule encoding a cell-bound (or membrane-immobilized) agonist antibody or agonist antibody fragment, or an mRNA molecule encoding an agonist peptide for a cell-bound (or membrane-immobilized) receptor or ligand, or an mRNA molecule encoding a cell-bound (or membrane-immobilized) receptor or ligand. Herein, the single vector or single mRNA molecule comprising at least one first mRNA molecule and at least one second mRNA molecule is contained in a nanoparticle, the nanoparticle is preferably liponoparticle (LNP) or non-lipid nanoparticle (e.g., nanoparticles composed of polymers), and / or herein, the two separate compositions comprising at least one first mRNA molecule and at least one second mRNA molecule respectively are liponoparticle (LNP) or non-lipid nanoparticle (e.g., nanoparticles composed of polymers), where the LNP is preferably composed of ionizable cationic lipids, neutral lipids (helper lipids), cholesterol and PEGylated lipids, optionally herein, the ionized lipids in the composition The molar lipid ratio (%) of ionizable cationic lipids:neutral lipids:cholesterol:PEGylated lipids is 50:10:38.5:1.5 or 46.3:9.4:42.7:1.6, and the N / P ratio is preferably (but not limited to) 3, 6, 4 or 8, where N is an ionizable cationic lipid (nitrogen) and P is a nucleotide (phosphate), and / or the single vector comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules, or each of the two separate compositions comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules, or the single mRNA molecule being self-replicating RNA or circular RNA. Furthermore / or, herein, the single vector comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules, or each of the two separate compositions comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules, or the single mRNA molecule is a viral vector. Furthermore, the method hereby includes administering to a subject a combination or single mRNA molecule according to the first aspect of the present invention, or a host cell according to the second aspect of the present invention, a pharmaceutical composition according to the third aspect of the present invention, a vaccine according to the fourth aspect of the present invention, a pharmaceutical combination according to the fifth aspect of the present invention, a kit according to the sixth aspect of the present invention, the single vector comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules, the two separate compositions each comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules, or the mixed composition, or the single mRNA molecule, where the single vector comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules, or the two separate compositions each comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules, or the mixed composition, or the single mRNA molecule, where the two separate compositions each comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules are administered separately, and here i) The two distinct compositions are administered separately to a first site and a second site, where the first site is within 20 cm, 17.5 cm, 15 cm, 12.5 cm, 10 cm, 7.5 cm, 5 cm, 2.5 cm, 1 cm, 0.5 cm, 0.25 cm, or 0.1 cm of the second site, preferably where the outflow lymphatic system of the first site flows to the same lymph nodes as the lymphatic system of the second site, or where the first site and the second site are the same, and / or ii) The two distinct compositions are administered separately within a time interval of 1 hour or less, 30 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, 5 minutes or less, 3 minutes or less, or 1 minute or less. This enables the spatially and / or temporally coordinated in vivo expression of at least one of the first molecules, which is a therapeutic or immunogenic protein or peptide, and at least one of the second molecules, which is a protein or peptide capable of modulating the immune response to the first molecule and / or the translation product of the first molecule.

[0099] The subjects described in the present invention are preferably mammals such as humans, mice, dogs, cats, rabbits, and monkeys. In a preferred embodiment, the subject is a human. In a more preferred embodiment, the subject is a human patient suffering from a disease, or a human suspected of being at risk of developing a disease in the future.

[0100] Another embodiment of the present invention relates to a combination or single mRNA molecule described in the first aspect of the present invention, or a host cell described in the second aspect of the present invention, a pharmaceutical composition described in the third aspect of the present invention, a vaccine described in the fourth aspect of the present invention, a pharmaceutical combination described in the fifth aspect of the present invention, or a kit described in the sixth aspect of the present invention, wherein the combination or single mRNA molecule described in the first aspect of the present invention, wherein the treatment further includes radiotherapy and / or surgery.

[0101] Another embodiment of the present invention relates to a combination or single mRNA molecule described in the first aspect of the present invention, or a host cell described in the second aspect of the present invention, a pharmaceutical composition described in the third aspect of the present invention, a vaccine described in the fourth aspect of the present invention, a pharmaceutical combination described in the fifth aspect of the present invention, or a kit described in the sixth aspect of the present invention, for use as described in the seventh aspect, wherein the combination or single mRNA molecule described in the first aspect of the present invention, or a host cell described in the second aspect of the present invention, a pharmaceutical composition described in the third aspect of the present invention, a vaccine described in the fourth aspect of the present invention, a pharmaceutical combination described in the fifth aspect of the present invention, Alternatively, the kit according to the sixth aspect of the present invention, the single vector comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules, the two separate compositions each comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules, or the mixed composition, or the single mRNA molecule, is administered to the subject by intramuscular, subcutaneous, intradermal, intraperitoneal, intravenous and / or intrathoracic injection, where preferably the two separate compositions each comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules are administered via the same route.

[0102] Preferred embodiments of the present invention relate to a combination or single mRNA molecule described in the first aspect of the present invention, a host cell described in the second aspect of the present invention, a pharmaceutical composition described in the third aspect of the present invention, a vaccine described in the fourth aspect of the present invention, a pharmaceutical combination described in the fifth aspect of the present invention, or a kit described in the sixth aspect of the present invention, wherein the combination or single mRNA molecule described in the first aspect of the present invention, or a host cell described in the second aspect of the present invention, a pharmaceutical composition described in the third aspect of the present invention, a vaccine described in the fourth aspect of the present invention, a pharmaceutical combination described in the fifth aspect of the present invention, or a kit described in the sixth aspect of the present invention, the single vector comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules, the two separate compositions each comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules, or the mixed composition, or the single mRNA molecule, are administered to the subject by intramuscular injection, wherein preferably the two separate compositions each comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules are administered via the same route.

[0103] A more preferred embodiment of the present invention relates to a combination or single mRNA molecule described in the first aspect of the present invention, or a host cell described in the second aspect of the present invention, a pharmaceutical composition described in the third aspect of the present invention, a vaccine described in the fourth aspect of the present invention, a pharmaceutical combination described in the fifth aspect of the present invention, or a kit described in the sixth aspect of the present invention, for use as described in the seventh aspect, wherein the combination or single mRNA molecule described in the first aspect of the present invention, or a host cell described in the second aspect of the present invention, a pharmaceutical composition described in the third aspect of the present invention, a vaccine described in the fourth aspect of the present invention, a pharmaceutical combination described in the fifth aspect of the present invention, and The kit according to the sixth aspect of the present invention, the single vector comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules, the two separate compositions each comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules, or the mixed composition, or the single mRNA molecule, is not administered to the subject by intratumoral injection, nor is it delivered locally near the tumor and / or into the tumor nutrient supply vessels, where preferably the two separate compositions each comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules are administered via the same route.

[0104] Therefore, according to a preferred embodiment of the present invention, the antigen encoded in the mRNA and / or the immunomodulatory factor encoded in the mRNA are not delivered by intratumor injection.

[0105] According to a more preferred embodiment of the present invention, the antigen encoded in the mRNA and / or the immunomodulatory factor encoded in the mRNA are not delivered locally near the tumor.

[0106] Further embodiments relate to the delivery of antigens and / or immunomodulatory factors encoded in the mRNA, which are not delivered to tumor nutrient supply vessels.

[0107] A further preferred embodiment of the present invention relates to a combination or single mRNA molecule described in the first aspect of the present invention, or a host cell described in the second aspect of the present invention, a pharmaceutical composition described in the third aspect of the present invention, a vaccine described in the fourth aspect of the present invention, a pharmaceutical combination described in the fifth aspect of the present invention, or a kit described in the sixth aspect of the present invention, for use as described in the seventh aspect, wherein the combination or single mRNA molecule described in the first aspect of the present invention, or a host cell described in the second aspect of the present invention, a pharmaceutical composition described in the third aspect of the present invention, a vaccine described in the fourth aspect of the present invention, a pharmaceutical combination described in the fifth aspect of the present invention, or a kit described in the sixth aspect of the present invention, the single vector comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules, the two separate compositions each comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules, or the mixed composition, or the single mRNA molecule is not delivered and / or administered to the subject by electroporation, where preferably two separate compositions each comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules are administered by the same route.

[0108] A more preferred embodiment of the present invention relates to a combination or single mRNA molecule described in the first aspect of the present invention, or a host cell described in the second aspect of the present invention, a pharmaceutical composition described in the third aspect of the present invention, a vaccine described in the fourth aspect of the present invention, a pharmaceutical combination described in the fifth aspect of the present invention, or a kit described in the sixth aspect of the present invention, wherein the combination or single mRNA molecule described in the first aspect of the present invention, or a host cell described in the second aspect of the present invention, a pharmaceutical composition described in the third aspect of the present invention, a vaccine described in the fourth aspect of the present invention, a pharmaceutical combination described in the fifth aspect of the present invention, or a kit described in the sixth aspect of the present invention, the single vector comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules, the two separate compositions comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules respectively, or the mixed composition, or the single mRNA molecule is an amino-lipidated peptoid. The two separate compositions, each preferably comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules, are administered via the same route, rather than being delivered by (delivery).

[0109] Another preferred embodiment of the present invention relates to a combination or single mRNA molecule described in the first aspect of the present invention, or a host cell described in the second aspect of the present invention, a pharmaceutical composition described in the third aspect of the present invention, a vaccine described in the fourth aspect of the present invention, a pharmaceutical combination described in the fifth aspect of the present invention, or a kit described in the sixth aspect of the present invention for use as described in the seventh aspect, wherein the combination or single mRNA molecule described in the first aspect of the present invention, or a host cell described in the second aspect of the present invention, a pharmaceutical composition described in the third aspect of the present invention, a vaccine described in the fourth aspect of the present invention, or a pharmaceutical combination described in the fifth aspect of the present invention. A combination, or the kit according to the sixth aspect of the present invention, the single vector comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules, the two separate compositions each comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules, or the mixed composition, or the single mRNA molecule, is administered to the subject by intramuscular, subcutaneous, intradermal, intraperitoneal, intravenous and / or intrathoracic injection, wherein the two separate compositions each comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules are administered via the same route.

[0110] A more preferred embodiment of the present invention relates to a combination or single mRNA molecule described in the first aspect of the present invention, or a host cell described in the second aspect of the present invention, a pharmaceutical composition described in the third aspect of the present invention, a vaccine described in the fourth aspect of the present invention, a pharmaceutical combination described in the fifth aspect of the present invention, or a kit described in the sixth aspect of the present invention, wherein the combination or single mRNA molecule described in the first aspect of the present invention, or a host cell described in the second aspect of the present invention, a pharmaceutical composition described in the third aspect of the present invention, a vaccine described in the fourth aspect of the present invention, a pharmaceutical combination described in the fifth aspect of the present invention, or a kit described in the sixth aspect of the present invention, the single vector comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules, the two separate compositions each comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules, or the mixed composition, or the single mRNA molecule, is administered to the subject by intramuscular injection, wherein the two separate compositions each comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules are administered via the same route.

[0111] Another aspect of the present invention, which may be combined with any other particularly preferred embodiments and / or aspects of the present invention, relates to a method for preventing and / or treating infectious, hereditary or proliferative diseases, comprising administering to a subject a combination or single mRNA molecule described in the first aspect of the present invention, or a host cell described in the second aspect of the present invention, a pharmaceutical composition described in the third aspect of the present invention, a vaccine described in the fourth aspect of the present invention, a pharmaceutical combination described in the fifth aspect of the present invention, or a kit described in the sixth aspect of the present invention, wherein the infectious disease is preferably a viral infection such as an infection caused by a coronavirus (e.g., COVID-19 disease), a bacterial infection, a fungal infection, a protozoan infection and / or a parasitic infection, and wherein the proliferative disease is preferably cancer, more preferably pancreatic cancer, bladder cancer, breast cancer, liver cancer, lung cancer, cutaneous squamous cell carcinoma, basal cell carcinoma, melanoma, uterine (endometrial) cancer, kidney cancer, skin cancer such as Merkel cell carcinoma, colorectal cancer, esophageal cancer, gastric cancer Cancers selected from head and neck cancer, neuroblastoma, leukemia, sarcoma, kidney cancer, lymphoma, mesothelioma, cervical cancer, stomach cancer, and cancers exhibiting specific hereditary mutations (e.g., MSI-H, dMMR, or TMB-H).

[0112] The subject is preferably a mammal such as a human, mouse, dog, cat, rabbit, or monkey. In a preferred embodiment, the subject is a human. In a more preferred embodiment, the subject is a human patient suffering from a disease, or a human suspected of being at risk of developing the disease in the future.

[0113] In all aspects of the present invention and in a tenth aspect which may be combined with all embodiments, the present invention relates to combinations or single mRNA molecules, therapeutic proteins, host cells, pharmaceutical compositions, pharmaceutical combinations or kits described in the present invention for use in gene therapy.

[0114] Further aspects of the present invention may be combined with any other particularly preferred embodiments and / or aspects of the present invention in the manufacture of a medicament for the treatment of infectious, hereditary, or proliferative diseases, relating to a combination or single mRNA molecule described in the first aspect of the present invention, or a host cell described in the second aspect of the present invention, a pharmaceutical composition described in the third aspect of the present invention, a vaccine described in the fourth aspect of the present invention, a pharmaceutical combination described in the fifth aspect of the present invention, or a kit described in the sixth aspect of the present invention, wherein the infectious disease is preferably a viral infection such as an infection caused by a coronavirus (e.g., COVID-19 disease), a bacterial infection, a fungal infection, a protozoan infection and / or a parasitic infection, and wherein the proliferative disease is preferably cancer, more preferably pancreatic cancer, bladder cancer, breast cancer, liver cancer, lung cancer, cutaneous squamous cell carcinoma, basal cell carcinoma, melanoma, uterine (endometrial) cancer, kidney cancer, skin cancer such as Merkel cell carcinoma, colorectal cancer, esophageal cancer, gastric cancer, head and neck cancer, neuroblastoma, leukemia, sarcoma, kidney cancer These cancers are selected from cancers exhibiting specific hereditary mutations (e.g., MSI-H, dMMR, or TMB-H), including cervical cancer, stomach cancer, and cancers exhibiting certain hereditary mutations.

[0115] Another aspect of the present invention relates to a combination or single mRNA molecule described in the first aspect of the present invention, or a host cell described in the second aspect of the present invention, a pharmaceutical composition described in the third aspect of the present invention, a vaccine described in the fourth aspect of the present invention, a pharmaceutical combination described in the fifth aspect of the present invention, or a kit described in the sixth aspect of the present invention for use in the prevention and / or treatment of infectious, hereditary or proliferative diseases, wherein the combination or single mRNA molecule, the host cell, the pharmaceutical composition, the vaccine, the pharmaceutical combination, or the kit stimulates neutralizing antibodies and / or other protective antibodies and / or T cells against infectious antigens and / or cancer antigens in the subject.

[0116] Another aspect of the present invention relates to a method for producing a combination or single mRNA molecule as described in the first aspect of the present invention, or a host cell as described in the second aspect of the present invention, a pharmaceutical composition as described in the third aspect of the present invention, a vaccine as described in the fourth aspect of the present invention, a pharmaceutical combination as described in the fifth aspect of the present invention, or a kit as described in the sixth aspect of the present invention.

[0117] When used herein, terms such as “of the present invention,” “in accordance with the invention,” and “according to the invention” are intended to refer to all aspects and embodiments of the invention described herein and / or in the claims.

[0118] Where used herein, the term “comprising” should be interpreted as encompassing both “including” and “consisting of,” both meanings being particularly intended and therefore the individually disclosed embodiments relating to the present invention. Where used herein, “and / or” should be taken as each specific disclosure of two specified features or components, whether having the other or not. For example, “A and / or B” should be taken as each specific disclosure of (i) A, (ii) B, and (iii) A and B, as if each were individually described herein. In the context of the present invention, the terms “about” and “approximately” indicate intervals of precision that a person skilled in the art would understand to still guarantee the technical effect of the feature in question. The terms typically indicate deviations of ±20%, ±15%, ±10%, and, for example, ±5% from the given numerical value. As a person skilled in the art would understand, such specific deviations of numerical values ​​for a given technical effect depend on the nature of that technical effect. For example, natural or biological technical effects may generally have greater deviations than artificial or engineered technical effects. As will be understood by those skilled in the art, such specific deviations in numerical values ​​for a given technical effect depend on the nature of that technical effect. For example, natural or biological technical effects may generally have greater deviations than artificial or engineered technical effects. When an indefinite or definite article is used when referring to a singular noun, such as "a," "an," or "the," this includes the plural form of that noun unless otherwise specified.

[0119] It should be understood that the application of the teachings of the present invention to specific problems or environments, and the inclusion of variations of the invention or additional features therein (e.g., further aspects and embodiments) are within the scope of the ability of a person with ordinary skill in the art, in light of the teachings contained herein.

[0120] Unless otherwise specified by the context, the above descriptions and definitions of features are not limited to any particular aspect or embodiment of the present invention and apply equally to all aspects and embodiments described.

[0121] All references, patents, and publications cited herein are incorporated herein by reference in their entirety. [Brief explanation of the drawing]

[0122] The drawing is shown below:

[0123] [Figure 1] Figure 1 shows the improvement in humoral response induced by COVID mRNA vaccines using combinations of mRNA vaccines and mRNA encoding regulatory peptides. The mouse population was immunized intramuscularly with: 7 μg of mRNA formulated in liponopanoparticles (LNPs) encoding the SARS-CoV2 spike protein (Gr1-Spike); a mixture (combination) containing 7 μg of mRNA formulated in LNPs encoding the SARS-CoV2 spike protein and 21 μg of mRNA in LNPs encoding the Ox40 ligand protein (Gr2-Spike+Ox40L); a mixture (combination) containing 7 μg of mRNA in LNPs encoding the SARS-CoV2 spike protein and 7 μg of mRNA in LNPs encoding an antibody against CTLA4 (Gr3-Spike+CTLA4); and a mixture (combination) containing 7 μg of mRNA in LNPs encoding the SARS-CoV2 spike protein and 7 μg of mRNA in LNPs encoding the ICOS ligand protein (Gr4-Spike+ICOSL). Figure 1A: Serum from mice immunized with LNP mRNA was tested for antibody titers against the SARS-CoV-2 receptor-binding domain (RBD) by ELISA at 3 weeks post-immunization. Figure 1B: Serum from mice immunized with LNP mRNA was tested for antibody titers against the SARS-CoV-2 receptor-binding domain (RBD) by ELISA at 5 weeks post-immunization. [Figure 2] Figure 2 shows the improvement in cellular response induced by COVID mRNA vaccines using combinations of mRNA vaccines and mRNA encoding regulatory peptides. The mouse population was immunized intramuscularly with: 7 μg of mRNA formulated in liponopanoparticles (LNPs) encoding the SARS-CoV2 spike protein (Gr1-Spike); a mixture (combination) containing 7 μg of mRNA formulated in LNPs encoding the SARS-CoV2 spike protein and 21 μg of mRNA in LNPs encoding the Ox40 ligand protein (Gr2-Spike+Ox40L); a mixture (combination) containing 7 μg of mRNA in LNPs encoding the SARS-CoV2 spike protein and 21 μg of mRNA in LNPs encoding an antibody against CTLA4 (Gr3-Spike+CTLA4); and a mixture (combination) containing 7 μg of mRNA in LNPs encoding the SARS-CoV2 spike protein and 21 μg of mRNA in LNPs encoding the ICOS ligand protein (Gr4-Spike+ICOSL). The T cell response in immunized mice was tested using ex vivo IFNgamma ELIspot, which employs a synthetic peptide that replicates the amino acid sequence of the spike protein. [Figure 3]Figure 3 shows that the activity of immunomodulatory RNA requires co-formulation or co-administration with therapeutic RNA (evaluation by B cell response). Control mice were immunized with LNP-Spike alone (Gr1-Spike). In the second group of mice, LNPs containing mRNA encoding the SARS-CoV2 spike protein (LNP-Spike) and LNPs containing mRNA encoding the Ox40 ligand protein (LNP-Ox40L) were prepared separately and then mixed prior to administration to the quadriceps femoris muscle of the same mice (Gr2-Spike+Ox40L co-administration). In the third group of mice, the same amounts of LNP-Spike and LNP-Ox40L were injected contralaterally into the left and right quadriceps femoris muscles, respectively (Gr3 Spike&OX40L contralateral). The fourth mouse group was immunized with the same amount of mRNA encoding the spike protein and mRNA encoding the Ox40 ligand protein co-formulated in the same LNP (Gr3 Spike&OX40L co-formulation). B cell responses were evaluated in serum from immunized mice 5 weeks post-immunization, and antibody titers against the SARS-CoV-2 receptor-binding domain (RBD) were tested by ELISA. [Figure 4]Figure 4 shows that the activity of immunomodulatory RNA requires co-formulation or co-administration with therapeutic RNA (assessment by T cell response). Control mice were immunized with LNP-Spike alone (Gr1-Spike). In the second group of mice, LNPs containing mRNA encoding the SARS-CoV2 spike protein (LNP-Spike) and LNPs containing mRNA encoding the Ox40 ligand protein (LNP-Ox40L) were prepared separately and then mixed prior to administration to the quadriceps femoris muscle of the same mice (Gr2-Spike+Ox40L co-administration). In the third group of mice, the same amounts of LNP-Spike and LNP-Ox40L were injected contralaterally into the left and right quadriceps femoris muscles, respectively (Gr3 Spike&OX40L contralateral). The fourth mouse group was immunized with the same amount of mRNA encoding the spike protein and mRNA encoding the Ox40 ligand protein co-formulated in the same LNP (Gr3 Spike&OX40L co-formulation). T cell responses were measured 5 weeks after immunization using ex vivo IFNgamma ELIspot with spike peptide. [Figure 5]Figure 5 shows that mRNA encoding positive immunomodulators in the cell-bound receptor or ligand category (natural protein or agonist antibody or agonist peptide) requires a cell-bound state for optimal activity (assessed by T cell response). Control mice were immunized with spike RNA vaccine only (mRNA vaccine). In the second group of mice, the spike RNA vaccine was administered with the protein form of the OX40 agonist antibody (OX86) (mRNA vaccine & OX86 mAb). The third group was inoculated with the same mRNA / LNP vaccine along with mRNA / LNP encoding the OX86 antibody (mRNA vaccine & OX86 mRNA). The fourth group was inoculated with the same mRNA / LNP vaccine along with mRNA / LNP encoding the membrane-immobilized version of the OX86 antibody (TM-OX86) (mRNA vaccine & OX86-TM mRNA). The final group of mice was inoculated with the same mRNA / LNP vaccine along with mRNA / LNP encoding the natural OX40 ligand (mRNA vaccine & OX40L mRNA). Anti-spike cell immunity (T cell response) was measured by ex vivo IFNgamma ELIspot 5 weeks after immunization. [Figure 6] Figure 6 shows that the activity of nucleic acid-encoded immunomodulators is dependent on and unpredictable by vector / nucleic acid type (DNA-encoded OX40L does not improve the immunogenicity of co-delivered DNA vaccines). Mice in the first control group were immunized intramuscularly by in vivo electroporation with a DNA plasmid encoding the SARS-CoV-2 spike protein, mixed with a pseudonon-coding DNA plasmid. Mice in the second group were similarly immunized by in vivo electroporation with a DNA plasmid encoding the SARS-CoV-2 spike protein, mixed with a plasmid encoding OX40L. T cell responses were assessed by ex vivo IFNgamma ELIspot (A); endpoint antibody titers of anti-spike IgG were measured by ELISA (B). [Figure 7]Figure 7 shows the kinetics of RNA-encoded, LNP-formulated aCTLA4 antibody concentrations from serum of immunized mice. Mice were inoculated with a spike RNA vaccine along with two different doses of mRNA in LNPs encoding antibodies against CTLA4, as reported in Figures 1 and 2. Serum was collected before intramuscular administration of RNA and at 3 and 5 weeks post-injection. Anti-CTLA4 antibodies were measured by an ELISA assay using plates coated with CTLA4 as the capture antigen. [Figure 8] Figure 8 shows the effect of mRNA-encoded negative immunomodulators. In this experiment, the first control group was inoculated with physiological saline ("PBS"). The second control group was inoculated with mRNA encoding the model antigen alone ("antigen alone"). Furthermore, the third control group was co-administered with mRNA encoding highly sensitive green fluorescent protein ("antigen + eGFP"). Finally, the fourth group of mice were intramuscularly administered mRNA encoding the model antigen (SARS-CoV-2 spike) and mRNA encoding the immune checkpoint PDL1 (programmed death ligand 1) in a ratio of 3:1 (antigen mRNA to immunomodulator mRNA). The anti-spike T cell response was measured by IFNgamma ELIspot in mouse splenocytes. [Figure 9]Figure 9 shows the improvement in T cell response induced by a COVID mRNA vaccine using a combination of mRNA vaccine and mRNA encoding a regulatory peptide. To demonstrate the adjuvant effects of cytokines and transcription factor regulatory RNAs on the immunological activity of mRNA vaccines, the inventors immunized a group of mice with either the mRNA vaccine alone or the mRNA vaccine mixed with mRNA encoding an immunomodulatory peptide. The mouse population was immunized intramuscularly with: a) 1.25 μg of mRNA (Gr1-Spike) formulated in liponopanoparticles (LNPs) encoding the SARS-CoV-2 spike protein; b) a mixture (combination) containing 1.25 μg of mRNA formulated in LNPs encoding the SARS-CoV-2 spike protein and 3.75 μg of mRNA in LNPs encoding the IL4 cytokine (Gr2-Spike+IL4); c) a mixture (combination) containing 1.25 μg of mRNA in LNPs encoding the SARS-CoV-2 spike protein and 3.75 μg of mRNA in LNPs encoding the TCF7 transcription factor (Gr3-Spike+TCF7). All mRNAs were transcribed in vitro using N1mPseudoU and synthetic CAP1. The LNPs were composed of ionizable cationic lipids, neutral lipids, cholesterol, and PEGylated lipids in appropriate proportions. [Figure 10]Figure 10 shows the improvement in T cell response induced by a COVID mRNA vaccine using a combination of two immunomodulatory peptides. To demonstrate the enhanced adjuvant effect of the two regulatory RNAs on the immunological activity of the mRNA vaccine, we immunized a group of mice with the mRNA vaccine alone or with mRNA vaccines mixed with mRNA encoding a single immunomodulatory peptide (i.e., IL21 and OX40L) or a combination thereof (i.e., IL21 + OX40L). The mouse population was immunized intramuscularly with the following: a) 1.25 mg of mRNA (Gr1-Spike) formulated in liponopanoparticles (LNPs) encoding the SARS-CoV-2 spike protein; b) a mixture (combination) containing 1.25 mg of mRNA formulated in LNPs encoding the SARS-CoV-2 spike protein and 3.75 mg of mRNA in LNPs encoding the IL21 cytokine (Gr2-Spike+IL21); c) a mixture (combination) containing 1.25 mg of mRNA in LNPs encoding the SARS-CoV-2 spike protein and 3.75 mg of mRNA in LNPs encoding OX40L (Gr3-Spike+TCF7); d) a mixture (combination) containing 1.25 mg of mRNA formulated in LNPs encoding the SARS-CoV-2 spike protein and 3.75 mg of mRNA in LNPs encoding the IL21 cytokine and OX40L ligand. All mRNAs were transcribed in vitro using N1mPseudoU and synthetic CAP1. LNPs were composed of ionizable cationic lipids, neutral lipids, cholesterol, and PEGylated lipids in appropriate proportions. [Figure 11]Figure 11 shows the improvement in T cell response induced by a COVID mRNA vaccine using a combination of mRNA vaccine and mRNA encoding different regulatory peptides. To demonstrate the adjuvant effect of chemokines or cytokines and cell-binding ligand regulatory RNAs on the immunological activity of mRNA vaccines, we immunized a group of mice with mRNA vaccine alone or with mRNA vaccine mixed with mRNA encoding immunomodulatory peptides. The mouse population was immunized intramuscularly with: a) 1.25 μg of mRNA (Gr1-Spike) formulated in liponopanoparticles (LNPs) encoding the SARS-CoV-2 spike protein; b) a mixture containing 1.25 μg of mRNA formulated in LNPs encoding the SARS-CoV-2 spike protein and 3.75 μg of mRNA in LNPs encoding OX40L or CD80; c) a mixture containing 1.25 μg of mRNA in LNPs encoding the SARS-CoV-2 spike protein and 3.75 μg of mRNA in LNPs encoding IL7, IL21, or IL15. All mRNAs were transcribed in vitro using N1mPseudoU and synthetic CAP1. The LNPs were composed of ionizable cationic lipids, neutral lipids, cholesterol, and PEGylated lipids in appropriate proportions. [Examples]

[0124] Specific aspects and embodiments of the present invention are described below by example and with reference to the description, drawings, and tables set forth herein. Such examples of the methods, uses, and other aspects of the present invention are representative examples only and should not be taken as limiting the scope of the present invention to such representative examples.

[0125] Examples are shown below:

[0126] Example 1: Improvement of humoral response induced by COVID mRNA vaccine using a combination of mRNA vaccine and mRNA encoding a regulatory peptide. To demonstrate the adjuvant effect of regulatory RNA on the immunological activity of mRNA vaccines, the inventors immunized a group of mice with mRNA vaccine alone or with mRNA vaccine mixed with mRNA encoding an immunomodulatory peptide.

[0127] The mouse population was immunized intramuscularly with the following: a) 7 μg of mRNA formulated in liponopanoparticles (LNPs) encoding the SARS-CoV-2 spike protein (Gr1-Spike); b) a mixture (combination) containing 7 μg of mRNA formulated in LNPs encoding the SARS-CoV-2 spike protein and 21 μg of mRNA in LNPs encoding the Ox40 ligand protein (Gr2-Spike+Ox40L); c) a mixture (combination) containing 7 μg of mRNA in LNPs encoding the SARS-CoV-2 spike protein and 7 μg of mRNA in LNPs encoding an antibody against CTLA4 (Gr3-Spike+CTLA4); d) a mixture (combination) containing 7 μg of mRNA in LNPs encoding the SARS-CoV-2 spike protein and 7 μg of mRNA in LNPs encoding the ICOS ligand protein (Gr4-Spike+ICOSL).

[0128] All mRNAs were transcribed in vitro using N1mPseudoU and synthetic CAP1. LNPs consisted of ionizable cationic lipids, neutral lipids, cholesterol, and PEGylated lipids in appropriate proportions. Serum from immunized mice was tested for antibody titers against the SARS-CoV-2 receptor-binding domain (RBD) by ELISA at 3 and 5 weeks post-immunization (Figure 1).

[0129] The results shown in Figure 1 demonstrate that co-administration of different immunomodulatory mRNAs increases anti-RBD antibody titers at both time points, indicating that our approach improves both the efficacy and persistence of vaccine-induced B-cell adaptive immunity.

[0130] Example 2: Improvement of cellular response induced by COVID mRNA vaccine using a combination of mRNA vaccine and mRNA encoding a regulatory peptide. To demonstrate that the adjuvant effect of regulatory RNA on the immunological activity of mRNA vaccines also acts on vaccine-induced T cell immunity, the inventors immunized a group of mice with either spike mRNA vaccine alone or spike mRNA vaccine mixed with mRNA encoding an immunomodulatory peptide, and measured anti-spike cell immunity 5 weeks after immunization using ex vivo IFNgamma ELIspot.

[0131] The mouse population was immunized intramuscularly with the following: a) 7 μg of mRNA formulated in liponopanoparticles (LNPs) encoding the SARS-CoV-2 spike protein (Gr1-Spike); b) a mixture (combination) containing 7 μg of mRNA formulated in LNPs encoding the SARS-CoV-2 spike protein and 21 μg of mRNA in LNPs encoding the Ox40 ligand protein (Gr2-Spike+Ox40L); c) a mixture (combination) containing 7 μg of mRNA in LNPs encoding the SARS-CoV-2 spike protein and 21 μg of mRNA in LNPs encoding an antibody against CTLA4 (Gr3-Spike+CTLA4); d) a mixture (combination) containing 7 μg of mRNA in LNPs encoding the SARS-CoV-2 spike protein and 21 μg of mRNA in LNPs encoding the ICOS ligand protein (Gr4-Spike+ICOSL).

[0132] All mRNAs were transcribed in vitro using N1mPseudoU and synthetic CAP1. LNPs were composed of ionizable cationic lipids, neutral lipids, cholesterol, and PEGylated lipids in appropriate proportions.

[0133] Splenocytes from immunized mice were tested using ex vivo IFNgamma ELIspot, which employs a synthetic peptide that replicates the amino acid sequence of the spike protein (described in SEQ ID NO: 1).

[0134] The results shown in Figure 2 indicate that co-administration of different immunomodulatory mRNAs increases the anti-spike T cell immune response.

[0135] Example 3: The activity of immunomodulatory RNA requires co-formulation or co-administration with therapeutic RNA. To demonstrate that enhanced adaptive immunity requires the coordinated expression of therapeutic and regulatory RNAs, the inventors immunized mice using different administration methods. In the first group of mice, the control group was immunized with LNP-Spike alone (Gr1-Spike). In the second group, LNPs containing mRNA encoding the SARS-CoV2 spike protein (LNP-Spike) and LNPs containing mRNA encoding the Ox40 ligand protein (LNP-Ox40L) were prepared separately and then mixed prior to administration to the quadriceps femoris muscle of the same mice (Gr2-Spike+Ox40L co-administration). In the third group of mice, the same amounts of LNP-Spike and LNP-Ox40L were injected contralaterally into the left and right quadriceps femoris muscles, respectively (Gr3 Spike&OX40L contralateral). Group 4 mice were immunized with the same amount of mRNA encoding the spike protein and mRNA encoding the Ox40 ligand protein co-formulated on the same LNP (Gr3 Spike&OX40L co-formulation).

[0136] B-cell responses were evaluated in serum from immunized mice 5 weeks post-immunization, and antibody titers against the SARS-CoV-2 receptor-binding domain (RBD) were tested by ELISA (Figure 3). As expected, antibody titers were significantly improved when the spike-encoding mRNA was co-administered with the OX40L-encoding mRNA. Contralateral administration of therapeutic and regulatory mRNA completely abolished this enhancement (Figure 3).

[0137] The inventors conducted further experiments to evaluate the immunomodulatory activity of different modes of administration on vaccine-induced T cell responses. In the first group of mice, the control group was immunized with LNP-Spike alone (Gr1-Spike). In the second group of mice, LNPs containing mRNA encoding the SARS-CoV-2 spike protein (LNP-Spike) and LNPs containing mRNA encoding the Ox40 ligand protein (LNP-Ox40L) were prepared separately and then mixed prior to administration to the quadriceps femoris muscle of the same mice (Gr2-Spike+Ox40L co-administration). In the third group of mice, the same amounts of LNP-Spike and LNP-Ox40L were injected contralaterally into the left and right quadriceps femoris muscles, respectively (Gr3 Spike&OX40L contralateral). The fourth mouse group was immunized with the same amount of mRNA encoding the spike protein and mRNA encoding the Ox40 ligand protein co-formulated on the same LNP (Gr3 Spike&OX40L co-formulation). Subsequently, T cell responses were measured 5 weeks after immunization using ex vivo IFNgamma ELIspot with spike peptide. Similar to the B cell response, T cell responsiveness was enhanced when therapeutic mRNA was co-administered with regulatory mRNA. Contralateral administration of therapeutic mRNA and regulatory mRNA completely abolished this enhancement (Figure 4).

[0138] These results demonstrate the need to combine therapeutic RNA and regulatory RNA to achieve the desired effect.

[0139] Example 4: mRNA encoding a positive immunomodulator in the cell-bound receptor or ligand category (natural protein or agonist antibody or agonist peptide) requires being in a cell-bound state for optimal activity. To further investigate the mechanisms of action of immunomodulatory factors belonging to the category of mRNA-encoded cell-bound receptors or ligands (i.e., OX40 agonists), the inventors immunized groups of mice with spike mRNA / LNP vaccine alone or with spike mRNA / LNP vaccine mixed with mRNA / LNP encoding different forms of OX40 agonists.

[0140] In the first group of mice, the control group was immunized with spike RNA vaccine alone. In the second group of mice, the spike RNA vaccine was administered with the protein form of the OX40 agonist antibody (OX86). The third group was inoculated with the same mRNA / LNP vaccine along with mRNA / LNP encoding the OX86 antibody. The fourth group was inoculated with the same mRNA / LNP vaccine along with mRNA / LNP encoding a membrane-immobilized version of the OX86 antibody (TM-OX86) obtained by adding the transmembrane region of precursor IgG to the C-terminus of the heavy chain. The last group of mice was inoculated with the same mRNA / LNP vaccine along with mRNA / LNP encoding the natural OX40 ligand. Anti-spike cell immunity was measured by ex vivo IFNgamma ELIspot 5 weeks after immunization.

[0141] As shown in Figure 5, co-administered protein-form OX86 agonist antibodies did not enhance the vaccine immune response. Conversely, the transmembrane version of OX86 encoded in mRNA / LNP (TM-OX86) improved vaccine immunogenicity to a degree comparable to that of the membrane-immobilized natural ligand OX40L encoded in mRNA / LNP. Soluble OX86 antibodies encoded in mRNA / LNP improved vaccine immunogenicity to a lower degree.

[0142] Example 5: The activity of nucleic acid-encoded immunomodulators is dependent on the vector / nucleic acid type and cannot be predicted (DNA-encoded OX40L does not improve the immunogenicity of co-delivered DNA vaccines). To evaluate whether co-delivery of nucleic acid-based vaccines in combination with nucleic acid-based immunomodulators leads to improved immunogenicity independently of the vector or nucleic acid type, the inventors tested the ability of DNA-encoded OX40L to improve the immunogenicity of DNA-encoded spike vaccines.

[0143] As shown in Figure 6, co-delivery of DNA-encoded OX40L did not improve the vaccine-induced immune response in either B cells or T cells.

[0144] Example 6: The activity of the encoded immunomodulator is independent of systemic delivery of the payload. To evaluate whether the activity of immunomodulators is local (at the co-injection site with therapeutic mRNA) or systemic diffusion of the molecule, the inventors tested the concentrations of secreted anti-CTLA4 antibodies used as immunomodulators as reported in Examples 1 and 2 (Figures 1 and 2). As shown in Figure 7, mRNA-encoded, LNP-formulated anti-CTLA4 antibodies were administered intramuscularly at two different doses, and very low systemic concentrations were measured. These concentrations (<30 ng / ml) are far from the concentrations (>1000 ng / ml) required to induce a typical systemic immune response when administered as recombinant protein.

[0145] Example 7: Effects of mRNA-encoded negative immunomodulators. To evaluate whether anti-drug immune responses can be avoided in gene therapy applications using mRNA encoding therapeutic proteins (e.g., Cas9 for genome editing or replacement of deficient genes), the inventors tested the potential benefits of co-administration of therapeutic mRNA with a second mRNA encoding a protein having negative immunomodulatory activity. To address this question, the inventors co-administered mRNA encoding a model antigen (SARS-CoV-2 spike) with mRNA encoding the immune checkpoint PDL1 (programmed death ligand 1). The two mRNAs were administered intramuscularly in a ratio of 3:1 (antigen mRNA versus immunomodulator mRNA; here, in Figure 8, the antigen mRNA is referred to as "antigen" and the immunomodulator mRNA as "PDL1"). Control animals were either inoculated with the same amount of mRNA encoding the model antigen alone (Figure 8, referred to as "antigen alone") or co-administered with three times the amount of mRNA encoding high-sensitivity green fluorescent protein (eGFP) (eGFP; Figure 8, referred to as "antigen + eGFP"). Another control group of animals was inoculated with physiological saline (Figure 8, referred to as "PBS"). Anti-spike T cell responses were measured in mouse splenocytes using IFNg ELIspot. The results showed that co-administration of mRNA encoding the negative immunomodulator PDL1 led to a statistically significant reduction in the antigen-specific immune response compared to that induced by delivery of mRNA encoding the spike protein alone ("antigen alone") or in combination with mRNA encoding eGFP ("antigen + eGFP"), demonstrating the concept of negative immunomodulation by co-delivery of mRNA encoding a protein factor that has the ability to reduce the immune response induced by mRNA encoding an immunogenic protein.

[0146] Example 8: Improvement of T cell response induced by a COVID mRNA vaccine using a combination of mRNA vaccine and mRNA encoding a regulatory peptide. To demonstrate the adjuvant effects of cytokine and transcription factor regulatory RNAs on the immunological activity of mRNA vaccines, the inventors immunized a group of mice with mRNA vaccines alone or mRNA vaccines mixed with mRNA encoding peptides having immunomodulatory activity. The mice were immunized intramuscularly with: a) 1.25 mg of mRNA (Gr1-Spike) formulated in liponopanoparticles (LNPs) encoding the SARS-CoV-2 spike protein; b) a mixture (combination) containing 1.25 mg of mRNA formulated in LNPs encoding the SARS-CoV-2 spike protein and 3.75 mg of mRNA in LNPs encoding the IL4 cytokine (Gr2-Spike+IL4); c) a mixture (combination) containing 1.25 mg of mRNA in LNPs encoding the SARS-CoV-2 spike protein and 3.75 mg of mRNA in LNPs encoding the TCF7 transcription factor (Gr3-Spike+TCF7). All mRNAs were transcribed in vitro using N1mPseudoU and synthetic CAP1. LNPs were composed of ionizable cationic lipids, neutral lipids, cholesterol, and PEGylated lipids in appropriate ratios. The results shown in Figure 9 demonstrate that co-administration of cytokines and transcription factor mRNAs increases anti-spike adaptive immunity.

[0147] Example 9: Improvement of T cell response induced by a COVID mRNA vaccine using a combination of two mRNAs encoding an mRNA vaccine and a regulatory peptide. To demonstrate the additional adjuvant effect of two RNA-encoded immunomodulatory factors on the immunological activity of mRNA vaccines, the inventors immunized mouse groups with mRNA vaccines alone or with mRNA vaccines mixed with single or combined mRNAs encoding peptides with immunomodulatory activity. The mouse population was immunized intramuscularly with the following: a) 1.25 mg of mRNA (Gr1-Spike) formulated in liponopanoparticles (LNPs) encoding the SARS-CoV-2 spike protein; b) a mixture (combination) containing 1.25 mg of mRNA formulated in LNPs encoding the SARS-CoV-2 spike protein and 3.75 mg of mRNA in LNPs encoding the IL21 cytokine (Gr2-Spike + IL21); c) a mixture (combination) containing 1.25 mg of mRNA in LNPs encoding the SARS-CoV-2 spike protein and 3.75 mg of mRNA in LNPs encoding OX40L (Gr3-Spike + TCF7); d) a combination of 1.25 mg of mRNA formulated in LNPs encoding the SARS-CoV-2 spike protein and 3.75 mg of mRNA in LNPs encoding IL21 and OX40L. All mRNAs were transcribed in vitro using N1mPseudoU and synthetic CAP1. LNP was composed of ionizable cationic lipids, neutral lipids, cholesterol, and PEGylated lipids in appropriate proportions. The results shown in Figure 10 indicate that co-administration of cytokines and transcription factor mRNA increases anti-spike adaptive immunity.

[0148] Example 10: The improvement of the T cell response induced by the COVID mRNA vaccine functions using a combination consisting of the mRNA vaccine and mRNA encoding different regulatory peptides. To demonstrate the adjuvant effect of chemokines or cytokines and cell-binding ligand regulatory RNAs on the immunological activity of mRNA vaccines, the inventors immunized a group of mice with mRNA vaccines alone or mRNA vaccines mixed with mRNA encoding peptides having immunomodulatory activity (see Figure 11). The mice were immunized intramuscularly with: a) 1.25 μg of mRNA (Gr1-Spike) formulated in liponopanoparticles (LNPs) encoding the SARS-CoV-2 spike protein; b) a mixture (combination) containing 1.25 μg of mRNA formulated in LNPs encoding the SARS-CoV-2 spike protein and 3.75 μg of mRNA in LNPs encoding OX40L or CD80; c) a mixture (combination) containing 1.25 μg of mRNA in LNPs encoding the SARS-CoV-2 spike protein and 3.75 μg of mRNA in LNPs encoding IL7, IL21, or IL15. All mRNAs were transcribed in vitro using N1mPseudoU and synthetic CAP1. LNPs were composed of ionizable cationic lipids, neutral lipids, cholesterol, and PEGylated lipids in appropriate proportions.

Claims

1. A combination comprising two or more mRNA molecules, or a single mRNA molecule encoding a first molecule which is a therapeutic or immunogenic protein or peptide and a second molecule which is a protein or peptide capable of modulating the immune response to the first molecule and / or the translation product of the first molecule, wherein the combination is as follows: (i) at least one first mRNA molecule encoding a first molecule which is a therapeutic or immunogenic protein or peptide, and (ii) At least one second mRNA molecule encoding a second molecule which is a protein or peptide capable of modulating the immune response to the first molecule and / or the translation product of the first molecule. The vector comprises, wherein at least one of the first mRNA molecules and at least one of the second mRNA molecules are co-formulated into a single vector, or wherein at least one of the first mRNA molecules and at least one of the second mRNA molecules are formulated into two separate compositions, optionally, wherein the two separate compositions are mixed prior to administration to form a mixed composition, or wherein the first and second molecules are translated from the single mRNA molecule, for example by an internal ribosome entry site (IRES) or by a self-cleaving peptide (e.g., a 2A peptide), optionally, wherein the single mRNA molecule is a bicistronic or polycistronic mRNA molecule. Herein, the combination or the single mRNA molecule is selected from the group consisting of at least one of the second mRNA molecules or a portion of the single mRNA molecule encoding the second molecule, which is an mRNA molecule encoding a cell-bound (or membrane-immobilized) agonist antibody or agonist antibody fragment, or an agonist peptide for a cell-bound (or membrane-immobilized) receptor or ligand, an mRNA molecule encoding a cell-bound (or membrane-immobilized) receptor or ligand, an mRNA molecule encoding a chemokine or cytokine, an mRNA molecule encoding an antagonist antibody or antagonist antibody fragment for an immune checkpoint protein, an mRNA molecule encoding an immune checkpoint inhibitor, an mRNA molecule encoding an immune checkpoint, and an mRNA molecule encoding a transcription factor.

2. The combination or single mRNA molecule according to claim 1, wherein at least one of the second mRNA molecules or a portion of the single mRNA molecule encoding the second molecule encodes a protein having positive immunomodulatory activity, such as a protein having the ability to increase the potency and / or quality and / or persistence of an immune response, or wherein at least one of the second mRNA molecules or a portion of the single mRNA molecule encoding the second molecule encodes a protein having negative immunomodulatory activity, such as a protein having the ability to decrease the potency and / or quality and / or persistence of an immune response.

3. The single vector comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules, or the single mRNA molecule comprising nanoparticles, wherein the nanoparticles are preferably liponoparticles (LNPs) or non-lipid nanoparticles (e.g., nanoparticles composed of polymers), and / or the two distinct compositions comprising, respectively, at least one of the first mRNA molecules and at least one of the second mRNA molecules, are selected from nanoparticles, synthetic particles for in vivo transduction, extracellular vesicles and viral vectors, wherein the nanoparticles are preferably LNPs or non-lipid nanoparticles (e.g., nanoparticles composed of polymers), wherein the LNPs are preferably ionizable cationic lipids, neutral lipids (helper lipids), cholesterol and PEGylated lipids The composition comprises, optionally, the molar lipid ratio (%) of ionizable cationic lipids:neutral lipids:cholesterol:PEGylated lipids in the composition being 50:10:38.5:1.5 or 46.3:9.4:42.7:1.6, and the N / P ratio being preferably (but not limited to) 3, 6, 4 or 8, where N is an ionizable cationic lipid (nitrogen) and P is a nucleotide (phosphate), and / or the single vector comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules, or each of the two separate compositions comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules, or the single mRNA molecule being self-replicating RNA or circular RNA. The combination or single mRNA molecule according to claim 1 or 2, wherein the single vector comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules, or each of the two distinct compositions comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules, or the single mRNA molecule is a viral vector.

4. At least one of the second mRNA molecules or a portion of the single mRNA molecule encoding the second molecule encodes the following: i) Chemokines or cytokines (soluble, cell-bound, or membrane-immobilized) preferred from the following list, but not limited to these: leptin, IL21, Ccl19, Ada, IL4, Ccl5, Cxcl9, Cxcl10, IL7, Ccl20, Ccl21a, Cxcl12, Ccl2, HMGB1, Ccl3, IL2, Efnb2, Ifng, IL12a, IL12b, IL1a, IL1b, IL36, TL1A, Cxcl13, Cxcl16, Ifnb1, IL13, IL17a, IL1 8, IL23a, IL6, Nlrp3, Ccl12, Ccl6, Ccl7, Ccl8, chemerin, Csf2, Cxcl11, IL5, Ccl11, Ccl24, Ccl26, Ccl4, Csf1, IL11, Ccl17, Ccl22, Ccl25, Ccl9, Cxcl1, Cxcl14, Cxcl15, Cxcl2, Cxcl3, Cxcl5, IL22, IL3, Ccl27a, Ccl28, IL-12 single chain, IFNα, GM-CSF, IL15, IL-15sushi and IL-15 receptor α chain chimeric protein, or ii) Cell-bound (or membrane-immobilized) ligands, preferred but not limited to the following list: LIGHT-Tnfsf14, OX40L-Tnfsf4, GITRL-Tnfsf18, 41BBL-Tnfsf9, Cd40lg, Cd70, ICOSL, Cd80, Icam1, Flt3l, CALR, Cd8 6. APRIL-Tnfsf13, CD166, LFA3, CD30L-Tnfsf8, Cd48, Cd74, Cd320, Cd83, Dpp4, Cd1d1, Cd1d2, Clec4n, Clec5a, ITGAL, Slc11a1, Vcam1, CAV1, Cd14, PLXNB2 and TWEAK-Tnfsf12, or iii) an agonist antibody (preferably cell-bound or membrane-immobilized) or agonist antibody fragment (preferably cell-bound or membrane-immobilized) or agonist peptide (preferably cell-bound or membrane-immobilized) having the same target as the molecules included in iii) i) and / or ii), A combination or a single mRNA molecule according to any one of claims 1 to 3.

5. At least one of the second mRNA molecules or a portion of the single mRNA molecule encoding the second molecule encodes an antagonist antibody or antagonist antibody fragment or antagonist peptide against an immune checkpoint protein, or an mRNA molecule encoding an immune checkpoint inhibitor, or an mRNA molecule encoding an immune checkpoint, where the immune checkpoint protein is PD-1, cd47, c A combination or single mRNA molecule according to any one of claims 1 to 3, preferably selected from, but not limited to, one of d31, PD-L1, PD-L2, BTLA, HVEM, lag3, tigit, prv, cd155, cd112, galectin-9, tim3, tim4, gitr, gitrl, tigit, Vista, cd276, cd39, cd73, CTLA4, IL10, IL35, ido, vip, IL27, and IL37.

6. A combination or single mRNA molecule according to any one of claims 1 to 3, wherein at least one of the second mRNA molecules or a portion of the single mRNA molecule encoding the second molecule encodes a transcription factor, wherein the transcription factor is preferentially selected from, but not limited to, one of Tcf1, TFEB, TFE3, NLRC5, CIITA, Lef1, Bcl11b, Gata3, Snai3, Ets1, IRF3, IRF7, Zbtb1, irf4, stat5b, runx3, bcl11a, foxo1, ikzf3, ets1, Tcf4, pax5, Bhlhe41, irf3, irf5, irf8, eomes, Stat1, Stat3, Stat5, Cebpd, and Cebpa.

7. At least one of the second mRNA molecules or a portion of the single mRNA molecule encoding the second molecule encodes an antagonist antibody or antagonist antibody fragment against an immune checkpoint protein, or encodes an immune checkpoint inhibitor, and here: (i) The antagonist antibody or the antagonist antibody fragment targets the PD-1 / PD-L1 pathway, preferably the mRNA molecule encoding an antibody selected from atezolizumab, avelumab, semiprimab, dostallimab, durvalumab, nivolumab, and pembrolizumab; (ii) The antagonist antibody or the antagonist antibody fragment targets the CTLA-4 pathway, preferably the mRNA molecule encoding the antibody ipilimumab, and / or (iii) The antagonist antibody or the antagonist antibody fragment targets the LAG-3 pathway, preferably the mRNA molecule encoding the antibody relatrimab. A combination or single mRNA molecule according to any one of claims 1 to 3 or 5.

8. At least one of the second mRNA molecules or a portion of the single mRNA molecule encoding the second molecule encodes a chemokine or cytokine, and here: (i) The cytokine targets the IL-2 / IL-2R pathway, preferably where the mRNA molecule encodes the cytokine aldesleukin; (ii) The mRNA molecule encodes an immunomodulatory cytokine such as granulocyte-macrophage colony-stimulating factor (GM-CSF), and / or (iii) The cytokine targets the IFNAR1 / 2 pathway, A combination or a single mRNA molecule according to any one of claims 1 to 4.

9. At least one of the first mRNA molecules or a portion of the single mRNA molecule encoding the first molecule encodes a therapeutic or immunogenic protein or peptide, which is preferably an infectious antigen such as a bacterial antigen such as a Gram-positive or Gram-negative bacterial antigen, a protozoan antigen, a viral antigen, a fungal antigen, or a parasitic antigen, or preferably a therapeutic or immunogenic protein against a cancer antigen, tumor antigen, or tumor neoantigen, which is preferably selected from the group consisting of a single amino acid mutation peptide, a frameshift peptide, a readthrough mutation peptide, a splice site mutation peptide, and / or an aging-related antigen. The combination or single mRNA molecule according to any one of claims 1 to 8, wherein is a peptide, or wherein at least one of the first mRNA molecules or a portion of the single mRNA molecule encoding the first molecule encodes a CAS protein, such as the CAS9 protein, or wherein at least one of the first mRNA molecules or a portion of the single mRNA molecule encoding the first molecule encodes a therapeutic protein that has the ability to correct a disease caused by the absence of an endogenous gene encoding a therapeutic protein, a mutation in an endogenous gene encoding a therapeutic protein, or inactivation of an endogenous gene encoding a therapeutic protein.

10. At least one of the first mRNA molecules and at least one of the second mRNA molecules, or the single mRNA molecule, is a synthetic and / or modified mRNA molecule, preferably where chemically modified nucleosides are preferentially incorporated into uracil nucleic acid bases and / or cytidine nucleic acid bases in at least one of the first mRNA molecules and at least one of the second mRNA molecules, or the single mRNA molecule, where all uracil nucleic acid bases and / or all cytidine nucleic acid bases are incorporated in at least one of the first mRNA molecules and at least one of the second mRNA molecules, or the single mRNA molecule, in a given proportion of uracil nucleic acid bases and A combination or single mRNA molecule according to any one of claims 1 to 9, wherein the cytidine nucleic acid base is substituted with a chemically modified nucleoside, such as N1-methylpseudridine, pseudouridine (ψ), or 5-methylcytidine (m5C), and / or thereof, at least one of the first mRNA molecules and at least one of the second mRNA molecules, or the single mRNA molecule, in the context of a linear or circular RNA, preferably comprises an optimized Kosack sequence, an optimized codon usage frequency, an optimized 5' and / or 3' untranslated region, a poly-A tail, a 5' cap (preferably selected from, but not limited to, anti-reverse cap analogs (ARCA), CAP0, CAP1:m7GpppNmpN, CAP2:m7GpppNmpNm), and / or an internal ribosome entry site (IRES).

11. A host cell comprising a combination or single mRNA molecule according to any one of claims 1 to 10, wherein the host cell is optionally an antigen-presenting cell, preferably a professional antigen-presenting cell, or a lymphocyte, preferably a T lymphocyte or T lymphocyte progenitor cell, more preferably a CD4 or CD8-positive T cell.

12. A pharmaceutical composition comprising a combination or single mRNA molecule according to any one of claims 1 to 10, or a host cell according to claim 11, and a pharmaceutically acceptable carrier, stabilizer and / or excipient.

13. A vaccine comprising a combination or single mRNA molecule according to any one of claims 1 to 10, a host cell according to claim 11, or a pharmaceutical composition according to claim 12, wherein the vaccine optionally induces an adaptive immune response, preferably a protective adaptive immune response to the following: (i) For example, viruses selected from the following list but not limited to: coronavirus, SARS coronavirus, SARS-CoV-2 coronavirus, respiratory syncytial virus (RSV), measles virus, influenza virus, rabies virus, dengue virus, HIV (human immunodeficiency virus), hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus, hepatitis E virus, foot-and-mouth disease virus (FMDV), bovine leukemia virus (BLV), bovine parainfluenza virus, human parainfluenza virus (HPIV), bovine respiratory syncytial virus, porcine reproductive and respiratory syndrome virus, respiratory syncytial virus Viruses, vesicular stomatitis virus, bovine viral diarrhea virus, bovine coronavirus, BHV1 virus, equine arteritis virus, Nipah virus, BK virus, porcine respiratory coronavirus infection, Yargsiegte sheep retrovirus, infectious bronchitis virus, avian pneumovirus, calicivirus, enterovirus, Epstein-Barr virus (EBV), Newcastle disease virus, astrovirus, influenza virus A (avian influenza) H5N1 subtype, herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), heartland virus, bocavirus (HBoV), human herpesvirus type 6 (HHV-6), human herpesvirus HHV-7, human metapneumovirus (hMPV), human papillomavirus (HPV), Japanese encephalitis virus, JC virus, Junin virus, cytomegalovirus (CMV), Machupovirus, Marburg virus, Molluscum contagiosum virus (MCV), Lassa virus, mumps virus, rhinovirus, rotavirus, varicella-zoster virus, Venezuelan encephalitis virus, West Nile virus, Western equine encephalitis virus, and yellow fever virus, filovirus and / or bacteria (for example, bacteria selected from but not limited to the following list: Acinetobacter, Acinetobacter baumannii, Actinobacillus, Anaplasma, Ankylostomata duodenale,Hemolytic Alcholderia viridae, Babesia, Bacillus, Anthrax, Bacillus cereus, Bacteroides, Burkholderia, Burkholderia hensele, Glanders, Bordetella partasis, Bordetella, Burkholderia brizaris, Brucella, Campylobacter, Candida albicans, Chlamydia, Chlamydia trachomatis, Chlamydophila, Chlamydia pneumoniae, Clostridium botulinum, Clostridium difficile, Clostridium candididium, Clostridium tetanus, Diphtheria Lactobacillus, Clostridium, Clostridium brunetii, Cyanobacteria, Dientamoeba fragilis, Escherichia, E. coli, Ehrlichia, Ehrlichia schaffensis, Ehrlichia euingii, Enterobacter, Enterococcus, Erwinia, Francisella, Fusobacteria, Haemophilus, Haemophilus influenzae, Helicobacter, Haemophilus, Histoplasma capsulatum, Kingella ( Kingera, Kingella, Kingella granulomatis, Legionella, Legionella pneumophila, Listeria, Neisseria meningitidis, Moraxella, Mycobacterium lepromatosis, Mycobacterium tuberculosis, Mycobacterium ulcerans, Moraxella, Mycoplasma pneumoniae, Neisseria, Pasteurella, Prevotella, Proteus, Pseudomonas , Rickettsia, Salmonella, Serratia, Sigella, Staphylococcus, Streptococcus, Treponema, Vibrio, Yersinia and escape pathogens), and / or parasites (for example, parasites selected from but not limited to the following list: malaria, reshmania, cryptosolidia, Entomoeba, Taenia solium, Acarilumbricoides, Echinococcus, Trypanosoma cruzi, Trypanosoma brusei, Schistosoma, and / or (ii) For example, 1A01 HLA-A / m; 1A02; 5T4; ACRBP; AFP; AKAP4; alpha-actinin-4 / m; alpha-methylacyl-CoA racemase; ANDR; ART-4; ARTCl / m; AURKB; B2MG; B3GN5; B4GN1; B7H4; BAGE-1; BASI; BCL-2; bcr / abl; beta-catenin / m; BING-4; BIRC7; BRCAl / m; BY55; calreticulin; CAMEL; CASPA; caspase_8; cathepsin_B; cathepsin_L; CD1A; CD1B; CD1C; CD1D; CD 1E; CD20; CD22; CD276; CD33; CD3E; CD3Z; CD4; CD44 isoform 1; CD44 elsoform 6 (CD44_lsoform_6); CD52; CD55; CD56; CD80; CD86; CD8A; CDC27 / m; CDE30; CDK4 / m; CDKN2A / m; CEA; CEAM6; CH3L2; CLCA2; CML28; CML66; COA-1 / m; coactosin-like protein; collagen XXIII; COX-2; CP1B1; CSAG2; CT-9 / BRD6; CT45A1; CT55; C TAG2 isoform LAGE-1A; CTAG2 isoform LAGE-1B; CTCFL; Cten; Cyclin Bl (cyclin_Bl); Cyclin D1; cyp-B; DAM-10; DEP1A; E7; EF1A2; EFTUD2 / m; EGFR; EGLN3; ELF2 / m; EMMPRIN; EpCam; EphA2; EphA3; ErbB3; ERBB4; ERG; ETV6; EWS; EZH2; FABP7; FCGR3A_Version_1; FCGR3A_Version_2; FGF5; FGFR2; Fibronectin ;FOS;FOXP3;FUT1;G250;GAGE-1;GAGE-2;GAGE-3;GAGE-4;GAGE-5;GAGE- 6; GAGE7b; GAGE-8JGAGE-2D); GASR; GnT-V; GPC3; GPNMB / m; GRM3; HAGE; hep sin; Her2 / neu; HLA-A2 / m; homeobox_NKX3.1; HOM-TES-85; HPG1; HS71A; HS 71B; HST-2; hTERT; iCE; IF2B3; IL-10; IL-13Ra2; IL2-RA; IL2-RB; IL2-RG;LOVE YOU LOVE YOU LOVE YOU LOVE YOUン20カイリクレインD4(Environmental)14 ROMASHIR20500000000000000000000000000000000000000000000000000000 LOVE LIKE2 LOVE LOVE LOVE LOVE LOVE THIS IS THE FULL VERSION OF THE FULL MOVIE THIS IS THE FULL VERSION OF THE FULL EPISODE THIS IS THE FULL VERSION OF THE FULL EPISODE THIS IS THE ONLY THING YOU CAN DO THIS IS THE FULL VERSION OF THE FULL EPISODE PHYSICS HIGHLIGHTS HIGHLIGHTS HIGHLIGHTS 1ST THIS IS THE FULL VERSION OF THE FULL MOVIE SUBJECT SUBJECT SUBJECT SUBJECT 20マンマグロビンASHASHYASHASHASH 2KSHASHSHASメソテリンSHASHSHAS1 SUBSYBHARBHARHHARH2YSHYS 1000000000000000000000000000000000000000000000000000000000 1000000000000000000000000000000000000000000000000000000000000 THIS IS YOUR LOVE YOU ARE YOUR LOVE ROY2000000000000000000000000000000,000,000,000,000,000,000,000,000,0ンCオステオポンチンD536611 20000000000000000000000000000000000000000000000000 10000000000000000000000000000000000000000000000000000000 THIS IS YOUR LIFE YOU ARE YOUR LIFE ROCK ROCK ROCK ROCK ROCK ROCK ROCK TWO THIS プロテイナーゼTHIS THIS IS YOUR LIFE SHASH SHASH SHASH SHASH SHASH SHASH 66. THIS IS THE ONLY THING YOU CAN KNOW SHYSYS SYS SYS SYS SYS SYS100 SHAS SYS1SYS SYSYS SYSYS STAR STAR STAR STAR STAR STAR S2SYS1000000000000000000000000000000000000000000000000000000000000 C1SYSYSYSYSYSYSYSYSYSYSYS 2SPIRITS11Cancer antigens or tumor antigens selected from STEAP-1; Survivin; Survivin-2B; SYCP1; SYT-SSX-1; SYT-SSX-2; TARP; TCRg; TF2AA; TGF-beta-1; TGFR2; TGM-4; TIE2; TKTL1; TPI / m; TRGV11; TRGV9; TRPC1; TRP-p8; TSG10; TSPY1; TVCJTRGV3; TX101; tyrosinase; TYRP1; TYRP2; UPA; VEGFR1 and XAGE1; and / or tumor neoantigens selected from the group consisting of single amino acid mutation peptides, frameshift peptides, readthrough mutation peptides, splice site mutation peptides, and / or aging-related antigens.

14. The following are examples of medicinal combinations: (i) a combination or single mRNA molecule according to any one of claims 1 to 10, a host cell according to claim 11, a pharmaceutical composition according to claim 12, or a vaccine according to claim 13, and (ii) Checkpoint inhibitor, optionally selected from atezolizumab, avelumab, semiprimab, dostallimab, durvalumab, nivolumab, pembrolizumab, ipilimumab, and reratrimab. (iii) an antagonist antibody, antagonist antibody fragment, or antagonist peptide against an immune checkpoint protein, wherein the immune checkpoint protein is preferably selected from, but not limited to, one of PD-1, cd47, cd31, PD-L1, PD-L2, BTLA, HVEM, lag3, tigit, prv, cd155, cd112, galectin-9, tim3, tim4, gitr, gitrl, tigit, Vista, cd276, cd39, cd73, CTLA4, IL10, IL35, ido, vip, IL27, and IL37, and / or (iv) an agonist antibody, agonist antibody fragment, or agonist peptide against an immune checkpoint protein, wherein the immune checkpoint protein is preferably selected from, but is not limited to, one of PD-1, cd47, cd31, PD-L1, PD-L2, BTLA, HVEM, lag3, tigit, prv, cd155, cd112, galectin-9, tim3, tim4, gitr, gitrl, tigit, Vista, cd276, cd39, cd73, CTLA4, IL10, IL35, ido, vip, IL27, and IL37.

15. A kit comprising a combination or single mRNA molecule according to any one of claims 1 to 10, a host cell according to claim 11, a pharmaceutical composition according to claim 12, a vaccine according to claim 13, or a pharmaceutical combination according to claim 14, and optionally comprising a liquid solvent for solubilization, and further optionally comprising technical instructions providing information regarding the administration and / or dosage of the components.

16. A combination or single mRNA molecule according to any one of claims 1 to 10, a host cell according to claim 11, a pharmaceutical composition according to claim 12, a vaccine according to claim 13, a pharmaceutical combination according to claim 14, or a kit according to claim 15, for use in pharmaceuticals.

17. The infectious disease is preferably a viral infection such as an infection caused by a coronavirus (e.g., COVID-19), a bacterial infection, a fungal infection, a protozoan infection, and / or a parasitic infection, where the proliferative disease is preferably cancer, more preferably pancreatic cancer, bladder cancer, breast cancer, liver cancer, lung cancer, cutaneous squamous cell carcinoma, basal cell carcinoma, melanoma, uterine (endometrial) cancer, kidney cancer, skin cancer such as Merkel cell carcinoma, colorectal cancer, esophageal cancer, gastric cancer, head and neck cancer, neuroblastoma, leukemia, sarcoma, kidney cancer, lymphoma, mesothelioma, cervical cancer, stomach cancer. For use in methods for the prevention and / or treatment of infectious, hereditary or proliferative diseases, which are cancers selected from cancers exhibiting specific hereditary mutations (e.g., MSI-H, dMMR, or TMB-H), a combination or single mRNA molecule according to any one of claims 1 to 10, a host cell according to claim 11, a pharmaceutical composition according to claim 12, a vaccine according to claim 13, a pharmaceutical combination according to claim 14, or a kit according to claim 15.

18. A combination or single mRNA molecule according to any one of claims 1 to 3, 5, 9 or 10, a therapeutic protein according to claim 9, a host cell according to claim 11, a pharmaceutical composition according to claim 12, a pharmaceutical combination according to claim 14, or a kit according to claim 15, for use in a method for preventing and / or treating a hereditary disorder.

19. The method comprises administering to a subject the combination or single mRNA molecule according to any one of claims 1 to 10, the host cell according to claim 11, the pharmaceutical composition according to claim 12, the vaccine according to claim 13, the pharmaceutical combination according to claim 14, the kit according to claim 15, the single vector comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules, the two separate compositions each comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules, or the mixed composition, or the single mRNA molecule, optionally administering to the subject the single vector comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules, or the two separate compositions each comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules, or the mixed composition, or the single mRNA molecule, and optionally administering separately the two separate compositions each comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules, and further optionally administering i) The two distinct compositions are administered separately to a first site and a second site, where the first site is within 20 cm, 17.5 cm, 15 cm, 12.5 cm, 10 cm, 7.5 cm, 5 cm, 2.5 cm, 1 cm, 0.5 cm, 0.25 cm or 0.1 cm of the second site, preferably where the outflow lymphatic system of the first site flows to the same lymph nodes as the lymphatic system of the second site, or where the first site and the second site are the same, and / or ii) The two distinct compositions are administered separately within a time interval of 1 hour or less, 30 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, 5 minutes or less, 3 minutes or less, or 1 minute or less. This enables the spatially and / or temporally coordinated in vivo expression of at least one of the first molecules, which is a therapeutic or immunogenic protein or peptide, and at least one of the second molecules, which is a protein or peptide having the ability to modulate an immune response to the first molecule and / or the translation product of the first molecule, for use according to claims 17 and 18, a combination or single mRNA molecule according to any one of claims 1 to 10, a host cell according to claim 11, a pharmaceutical composition according to claim 12, a vaccine according to claim 13, a pharmaceutical combination according to claim 14, or a kit according to claim 15.

20. A combination or single mRNA molecule according to any one of claims 1 to 10, a host cell according to claim 11, a pharmaceutical composition according to claim 12, a vaccine according to claim 13, a pharmaceutical combination according to claim 14, a kit according to claim 15, the single vector comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules, the two separate compositions each comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules, or the mixed composition, or the single mRNA molecule, administered to a subject by intramuscular, subcutaneous, intradermal, intraperitoneal, intravenous and / or intrathoracic injection, where preferably the two separate compositions each comprising at least one of the first mRNA molecules and at least one of the second mRNA molecules are administered by the same route, for use according to any one of claims 17 to 19.

21. For use in gene therapy, for use according to any one of claims 18 to 20, a combination or single mRNA molecule according to any one of claims 1 to 3, 5, 9 or 10, a therapeutic protein according to claim 9, a host cell according to claim 11, a pharmaceutical composition according to claim 12, a pharmaceutical combination according to claim 14, or a kit according to claim 15.

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