mRNA for protein expression and templates for it

Abstract

The present application relates to mRNA for protein expression and a template therefor, and provides an mRNA transcription vector including a gene construct according to one embodiment, a method for producing an mRNA molecule using the mRNA transcription vector, the method including a step of in vitro transcription, an mRNA molecule produced by the method, and an immunogenic composition including the mRNA molecule as an active ingredient.

Description

[Technical field]

[0001] The present invention relates to mRNA for protein expression and templates therefor.

[0002] This patent application claims priority to Korean Patent Application No. 10-2021-0098684 filed with the Korean Intellectual Property Office on July 27, 2021, and Korean Patent Application No. 10-2022-0062306 filed with the Korean Intellectual Property Office on May 20, 2022, the disclosures of which are incorporated herein by reference. [Background technology]

[0003] When nucleic acid-based therapeutic and vaccine development technologies are applied to a target individual beyond the cellular level, they have insufficient transcription and translation efficiency, and show insufficient efficacy as therapeutic agents, which has been pointed out as a limitation of nucleic acid-based therapeutic agent development. Therefore, enhancing the intracellular / extracellular expression ability of antigenic proteins for the treatment of infectious diseases is one of the essential requirements for the development of pharmaceuticals using artificial nucleic acid molecules. In addition, in the field of nucleic acid-based therapeutic and vaccine development, securing an mRNA sequence with high stability and translation efficiency has been highlighted as an essential requirement for mRNA-based therapeutic agents.

[0004] In addition, an mRNA vaccine is a drug that uses a protein coded by mRNA as an antigen and is used for the prevention and treatment of cancer, infectious diseases, autoimmune diseases, etc. Compared to DNA vaccines, the mRNA vaccine has the advantage of being stable and easy to mass-produce, and is expected to be widely used in future anti-cancer vaccines and infectious disease vaccine platforms in pandemic situations. The mRNA vaccine contains an artificial RNA molecule made by mimicking the natural mRNA structure as an active ingredient, and the main goal is to strengthen the individual's immune system using the RNA molecule. Currently, Moderna's mRNA-1273, BioNTech / Pfizer's BNT162b2, etc. have been commercialized through emergency use approval procedures, but such mRNA vaccines require technical elements for delivery to the cytoplasm due to the instability of the RNA molecule, and after the RNA molecule is delivered to the cytoplasm, damage to the RNA molecule due to an excessive innate immune response or a decrease in translation efficiency must be minimized, so multifaceted research is required in the field of mRNA vaccine technology. In particular, the immune activation properties of naked mRNA, which induce the stimulation of adjuvants such as Toll-like receptor agonists (TLR agonists), are known to inhibit the expression of sufficient amounts of mRNA antigens to exhibit pharmaceutical activity by interfering with the mRNA transcription signal transduction system. In order to solve this problem, active attempts are being made to produce vaccines using modified mRNA, which has eliminated or weakened immunogenicity. However, in such cases, due to the low immunogenicity of mRNA, there is a limit to the induction of effective humoral and / or cellular immune responses.

[0005] Against this technical background, as part of the development to improve the utility of mRNA molecules for medicinal purposes, multifaceted research is being conducted to improve the expression of mRNA molecules or enhance their immunogenicity (Korean Patent Publication No. 10-2022-0017377), but the reality is that there are still shortcomings. Summary of the Invention [Problem to be solved by the invention]

[0006] One aspect provides an mRNA transcription vector comprising a promoter region recognized by an RNA polymerase and a gene construct capable of enhancing expression of a target mRNA antigen.

[0007] Another aspect provides a method for producing an mRNA molecule that utilizes an mRNA transcription vector as a template, and the mRNA molecule produced by said method.

[0008] Yet another aspect provides an immunogenic composition comprising the mRNA molecule and a pharma- ceutically acceptable excipient as an active ingredient.

[0009] Other objects and advantages of the present application will become more apparent from the following detailed description taken in conjunction with the appended claims and drawings. Contents not described in this specification can be fully understood and inferred by those skilled in the technical field of the present application or a similar technical field, so the description thereof will be omitted. [Means for solving the problem]

[0010] One embodiment is an mRNA transcription vector comprising a promoter region recognized by an RNA polymerase and a gene construct operably linked to the promoter region,

[0011] The gene construct provides an mRNA transcription vector comprising a 5'-untranslated region (5'-UTR: 5'-untranslated region region) consisting of SEQ ID NO: 1 or a nucleotide sequence having 90% or more sequence identity thereto; an open reading frame (ORF: open reading frame) region containing a nucleotide sequence encoding a target antigen, operably linked to the 5'-UTR region; and a 3'-untranslated region (3'-UTR: 3'-untranslated region) region in which a nucleotide sequence consisting of SEQ ID NO: 2 or a nucleotide sequence having 90% or more sequence identity thereto is repeated twice, operably linked to the open reading frame region.

[0012] As used herein, the term "nucleotide sequence" refers to a polymeric substance containing a plurality of nucleotide units, specifically a polymer in which the plurality of nucleotide units are linked to each other by phosphodiester bonds of a sugar / phosphate backbone, and may be used interchangeably with the terms "polynucleotide," "nucleic acid," and "nucleic acid molecule." The polynucleotide is an essential biopolymer for living organisms, and may be RNA or DNA that encodes genetic information through a unique base sequence. The polynucleotide may be isolated, artificially synthesized, or non-naturally occurring or engineered, and the term "non-naturally occurring or engineered" refers to a state in which the polynucleotide is artificially modified rather than in the natural state. Here, the artificial modification is also intended to mimic the structure of natural mRNA, specifically mature mRNA, and improve the expression of a target antigen in cells.

[0013] As used herein, the term "messenger RNA (mRNA)" refers to RNA that is transcribed from a DNA template and transfers the genetic information of the DNA to ribosomes in the cytoplasm. The transcription process in eukaryotic organisms takes place in the nucleus of the cell and involves processing premature RNA. Specifically, such processes are called post-transcriptional modification and include processes such as splicing, 5' capping, polyadenylation, and export from the nucleus or mitochondria. As a result of such processes, mature mRNA is generated that contains a nucleotide sequence that can be translated into the amino acid sequence of a specific peptide or protein. Generally, the mature mRNA may optionally contain a 5' cap, a 5'-UTR, an open reading frame, a 3'-UTR, and a polyA tail. The mRNA can be synthesized by any of a variety of known methods, for example, the mRNA can be synthesized via in vitro transcription (IVT).

[0014] The term "vector" as used herein refers to a gene construct that is capable of expressing a target antigen in a suitable host cell and includes regulatory elements operably linked to allow a gene insert to be expressed. The vector according to one embodiment may include expression regulatory elements such as a promoter, an operator, an initiation codon, a termination codon, a polyadenylation signal, and / or an enhancer, and the promoter of the vector may be constitutive or inducible. The vector may also be an expression vector that is capable of stably expressing a target antigen in a host cell. The expression vector may be any of those commonly used in the art for expressing foreign proteins in plants, animals, or microorganisms, and the vector may be constructed through various methods known in the art. In the vector, the gene construct sequence is also operably linked to a promoter. The term "operatively linked" may refer to nucleotide sequences linked together on a single nucleic acid fragment such that the function of one is affected by the other. In one embodiment, the vector is also a vector capable of expressing an mRNA molecule as a target antigen in a host cell, i.e., also referred to as an mRNA transcription vector. The mRNA transcription vector is constructed as a DNA-type gene construct, and also referred to as a template DNA for mRNA production. To this end, the mRNA transcription vector contains a promoter that is recognized by an RNA polymerase, and can generate an mRNA molecule through a transcription process mediated by the promoter.

[0015] In one embodiment, the mRNA transcription vector is in the form of a plasmid, and is also a linearized vector, specifically a linearized plasmid. The linearized plasmid is obtained by contacting the plasmid DNA with a restriction enzyme under suitable conditions, where the restriction enzyme cuts the plasmid DNA at the recognition site and destroys the plasmid structure. The linearized plasmid includes a free 5' end and a free 3' end, which are also for carrying out the subsequent in vitro transcription process. In addition, the elements of the plasmid, such as the type of plasmid and the restriction enzyme recognition site, can be applied by techniques known in the art without limitation.

[0016] As used herein, the term "RNA polymerase" refers to an enzyme that synthesizes a primary transcript RNA from DNA. The RNA polymerase may be, for example, T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, or mitochondrial polymerase (POLRMT). The term "promoter region recognized by RNA polymerase" may refer to a DNA sequence region that acts as a template that can generate a promoter mRNA recognized by RNA polymerase through the transcription process.

[0017] In one embodiment, the promoter region is also one that is recognized by an RNA polymerase acting in the cytoplasm, for example one that is recognized by T7 RNA polymerase, and is comprised of the nucleotide sequence of SEQ ID NO: 4 or SEQ ID NO: 15, or a sequence having at least 90% or more sequence identity with said nucleotide sequence.

[0018] The term "identity" as used herein refers to the overall relatedness between polymer molecules, e.g., between nucleic acids (e.g., DNA molecules and / or RNA molecules) and / or between polypeptides. For example, the polypeptides are considered to be "substantially identical" to each other if their amino acid sequences are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical. Calculation of the percent identity of two nucleic acid or polypeptide sequences can be performed, for example, by aligning the two sequences for optimal comparison purposes (e.g., for optimal alignment, gaps can be introduced in one or both of the first and second sequences, and non-identical sequences can be ignored for comparison purposes). For example, the length of the aligned sequences for comparison purposes is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of the reference sequence. The nucleic acid or polypeptide sequences at corresponding positions are then compared. The determination of percent identity between two sequences and the comparison of sequences can be accomplished using mathematical algorithms. As is well known to those skilled in the art, amino acid or nucleic acid sequences can be compared using any of a variety of algorithms, including those available in commercial computer programs, such as BLASTN for nucleotide sequences, and BLASTP, gapped BLAST, and PSIBLAST for amino acid sequences.

[0019] In this specification, with respect to the position of a nucleotide sequence, unless otherwise specified, it indicates that it is linked or located in the direction from the 5' end to the 3' end.

[0020] The gene construct is also a technical element for improving the expression of an mRNA molecule or an mRNA antigen, and includes a 5' untranslated region, an open reading frame region containing a nucleotide sequence encoding a target antigen, and a 3' untranslated region.

[0021] As used herein, the term "open reading frame" generally refers to a sequence of various nucleotide triplets that can be translated into a peptide or protein, and may be used interchangeably with the term "protein coding region." The open reading frame preferably includes a start codon, i.e., a combination of three nucleotide sequences (ATG or AUG) that code for the amino acid methionine, in the subsequent region beginning at its 5' end, and preferably includes a stop codon (e.g., TAA, TAG, TGA, or UAA, UAG, UGA) that directs the end of translation in the region toward the 3' end. The term "open reading frame region" may refer to a DNA sequence region that acts as a template that can generate an open reading frame mRNA, i.e., a target mRNA antigen, through the aforementioned transcription process.

[0022] As used herein, the term "5'-untranslated region" refers to the region located at the 5' end of the open reading frame, specifically the mRNA region upstream from the initiation codon. The term "5'-untranslated region" may refer to a region of DNA sequence that acts as a template capable of generating 5'-untranslated mRNA through the transcription process described above.

[0023] As used herein, the term "3'-untranslated region (3'-UTR)" refers to the region located at the 3' end of the open reading frame, specifically the mRNA region downstream from the stop codon. The term "3'-untranslated region" may refer to a DNA sequence region that acts as a template capable of generating 3'-untranslated region mRNA through the transcription process described above.

[0024] In one embodiment, the open reading frame region also includes a nucleotide sequence encoding an antigen derived from a pathogen, which may be selected from the group consisting of virus, bacteria, prion, fungus, protozoon, viroid, and parasite, but is not limited thereto. For example, the open reading frame region may be a nucleotide sequence encoding a viral surface protein or a functional domain thereof, but may be extended to any mRNA antigen capable of infecting mammals, including humans, and causing pathological symptoms.

[0025] In one specific example, the 5' untranslated region is a modified sequence derived from human hemoglobin subunit α2 (HBA2) and consists of the nucleotide sequence of SEQ ID NO: 1 or a sequence having at least 90% sequence identity with the nucleotide sequence.

[0026] In one embodiment, the 3' untranslated region is a modified sequence derived from human hemoglobin subunit beta (HBA2), and is a repeat of two nucleotide sequences consisting of the nucleotide sequence of SEQ ID NO: 2 or a sequence having at least 90% sequence identity with the nucleotide sequence. Here, the 3' untranslated region is a nucleotide sequence to which the nucleotide sequences are directly linked or linked via a linker sequence. The 3' untranslated region is also a nucleotide sequence of SEQ ID NO: 3 or a sequence having at least 90% sequence identity with the nucleotide sequence.

[0027] The gene construct may also additionally comprise a nucleotide sequence operably linked to the 5'-UTR that is transcribed into a 5' cap, and / or a nucleotide sequence operably linked to the 3'-UTR region that is transcribed into a polyA tail.

[0028] As used herein, the term "5' cap" refers to a structure located at the 5' end region of a polynucleotide that affects stability and expression efficiency, and can generally be formed by a modified nucleotide, particularly a derivative of a guanine nucleotide. The 5' cap can be formed by any of the following: 7 G(5')ppp(5')(2'OMeA)pG,m 7 Gppp, Gppp, m 7 (3'OMeG)(5')ppp(5')(2'OMeA)pG, m 7 (3'OMeG)(5')ppp(5')(2'OMeG)pG, G(5')ppp(5')G, m 7 G(5')ppp(5')G, 3'-O-Me-m 7 G(5')ppp(5')G,m 7 G(5')ppp(5')(2'OMeA)pG or m 7 G(5')ppp(5')(2'OMeA)pU, but any known 5' cap having the same functionality as described above can be used, without limitation.

[0029] As used herein, the term "poly A tail" refers to a structure located in the 3'-terminal region that delays the degradation process of RNA exo-nuclease, extends the stability and in vivo half-life of a polynucleotide, and affects expression efficiency, and is generally formed by a sequence of multiple adenine nucleotides. In one embodiment, the polyA tail is comprised of 20 to 200 adenines, for example, 20 to 190, 20 to 170, 20 to 150, 20 to 130, 20 to 110, 20 to 90, 20 to 70, 20 to 50, 20 to 30, 30 to 190, 30 to 170, 30 to 150, 30 to 130, 30 to 110, 30 to 90, 30 to 70, or 30 to 50 repeating adenine nucleotide sequences. The polyA tail may also be comprised of multiple units linked by a linker.

[0030] In one embodiment, the mRNA transcription vector comprises a promoter region and a gene construct comprising the nucleotide sequence of SEQ ID NO:4 or SEQ ID NO:15, and the gene construct is also a plasmid comprising a 5'-UTR region comprising the nucleotide sequence of SEQ ID NO:1, an ORF region operably linked to the 5'-UTR region, and a 3'-UTR region comprising the nucleotide sequence of SEQ ID NO:3.

[0031] According to one embodiment, an mRNA transcription vector according to one embodiment including a gene construct having a combination of a specific 5'-UTR region and a specific 3'-UTR region can express luciferase mRNA with higher efficiency than existing vectors. In addition, an mRNA molecule according to one embodiment, which is prepared by adopting a coronavirus spike protein as a target mRNA antigen, induces a high level of immune response when administered to an animal model in the form of a vaccine formulation, and also shows effective immunogenicity even in mRNA antigens including various forms of deformation. The mRNA transcription vector can be used in the fields of preparing mRNA molecules and therapeutic agents or vaccines including the mRNA molecules.

[0032] Another aspect provides a method for producing an mRNA molecule comprising the step of performing in vitro transcription using the mRNA transcription vector as a template, and the mRNA molecule produced by the method.

[0033] Among the terms and elements referred to in the method for producing an mRNA molecule and the mRNA molecule produced by the method, those referred to in the description of the mRNA transcription vector are the same as those described above.

[0034] As used herein, the term "in vitro transcription" refers to a process in which a desired mRNA molecule is synthesized in a cell-free system (in a test tube), preferably in which DNA constituting a transcription vector is used as a template for the production of an mRNA transcript, and an RNA polymerase can be used to control the in vitro transcription process. Generally, in in vitro RNA transcription, the DNA template for the RNA is obtained by cloning a specific cDNA corresponding to each RNA to be in vitro transcribed and introducing it into a suitable vector for RNA in vitro transcription.

[0035] In one embodiment, the in vitro transcription step may be performed using an mRNA transcription vector according to one embodiment including a gene construct having a combination of the specific 5'-UTR region and 3'-UTR region described above, and other performance conditions or the configuration of the ORF region may be appropriately changed depending on the purpose.

[0036] Yet other aspects provide an immunogenic composition comprising the mRNA molecule and a pharma- ceutically acceptable excipient as an active ingredient, the mRNA transcription vector for producing an immunogenic composition, or the pharmaceutical use of the mRNA molecule produced by the transcription vector, or a method for stimulating an immune response comprising administering the immunogenic composition to an individual.

[0037] Among the terms or elements referred to in the immunogenic composition below, those mentioned in the description of the mRNA transcription vector, the method for producing an mRNA molecule, and the mRNA molecule produced by the method are as described above.

[0038] As used herein, the term "immunogenic composition" refers to a substance containing an active ingredient, or an effective amount thereof, effective to induce a specific degree of immunity in a subject against a specific pathogen or disease, and may be used interchangeably with the terms "vaccine," "vaccine formulation," and "vaccine composition." The immunogenic composition is also a pharmaceutical composition that induces a reduction in the severity, duration, or other symptoms associated with a disease or infection by a pathogen. Thus, the immunogenic composition may optionally include a pharma- ceutical acceptable carrier, diluent, excipient, buffer, salt, surfactant, cryoprotectant, and the like.

[0039] As used herein, the term "immunogenicity" refers to the ability of a composition to elicit an immune response against a particular pathogen, whether that be a cellular immune response mediated primarily by cytotoxic T-cells and cytokine-producing T-cells, or a humoral immune response mediated primarily by helper T cells, which subsequently activates B cells to produce antibodies.

[0040] As used herein, the term "pharmaceutical acceptable excipient" also includes any substance that, when combined / mixed with an mRNA molecule, maintains the activity of the mRNA molecule and does not react with the subject's immune system. Examples include, but are not limited to, any standard pharmaceutical excipient such as a buffer system such as phosphate buffered saline, surfactants, water, emulsions such as oil / water emulsions, and various forms of wetting agents, starch, milk, sugar, certain forms of clay, gelatin, stearic acid or its salts, magnesium or calcium stearate, talc, vegetable oils, gums, glycols, or other known excipients.

[0041] The immunogenic composition may be in any form known in the art, such as, but not limited to, a liquid or an injection. The liquid or injection may contain 10-40% propylene glycol, etc., as necessary. The liquid or injection may contain any diluent or buffer known in the art. The immunogenic composition may be prepared just before use by storing a formulation containing an active ingredient in a container such as a vial, and adding a necessary carrier or adjuvant, saline, etc. to the injection before use.

[0042] In one embodiment, the mRNA molecules are also complexed with at least one lipid component to form liposomes, lipid nanoparticles and / or lipoplexes.

[0043] In one embodiment, the lipid nanoparticles contain an ionizable cationic lipid as one component and may also contain other components such as helper lipids and stabilizers that aid in encapsulation of the mRNA and improve delivery efficiency and stabilization.

[0044] In one embodiment, the lipid nanoparticles also include cationic and neutral lipids (eg, phospholipids), sterols or steroids (eg, cholesterol), PEG-conjugated lipids, and the like. In this case, specific examples of the cationic lipid include ALC-0315 ([(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate)), SM-102 (9-Heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate), N,N-dioleyl-N,N-dimethyIammonium chloride(DODAC), N-(l-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride(DOTMA), N-(l-(2,3-dioleoyoxy)propyl)-N,N,N- trimethylammonium chloride (DOTAP), N,N-dimethyl-(2,3-dioleyloxy)propylamine(DODMA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), DLin-KC2-DMA, (6Z,9Z,28Z,31Z)-Heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLinMC3-DMA, CAS 1224606-06-7), ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), and the like can be used, but are not limited thereto.

[0045] In one embodiment, the lipid nanoparticles may comprise a cationic lipid (in the range of 35 to 60 mol % of the total lipid), a phospholipid (in the range of 5 to 15 mol % of the total lipid), cholesterol (in the range of 30 to 50 mol % of the total lipid), DMG-PEG, as described above. 2000 The present invention also includes, but is not limited to, PEG-conjugated lipids such as those listed above (in the range of 0.5 to 2.5 mol % of the total lipids).

[0046] In one embodiment, the lipid nanoparticles may be applied with components known in the art, and may include cationic lipids, helper lipids, and PEG-conjugated lipids, where the cationic lipids are, for example, ALC-0315 and / or SM-102, the helper lipids are, for example, DSPC (distearoylphosphatidylcholine) and / or cholesterol, and the PEG-conjugated lipids are, for example, ALC-0159 and / or PEG-DMG (PEG-dimyristoyl glycerol), but may also be particles composed of, for example, ALC-0315:DSPC:Chol:ALC-0159 (47.5 mol%:10 mol%:40.8 mol%:1.7 mol%), but are not limited thereto.

[0047] In one embodiment, the lipid of the lipid nanoparticles may be any of the lipid compounds disclosed in WO2014 / 172045A1, WO2020 / 252589A1, and WO2021 / 000041A1 without any particular limitation, and may be PNI123 / DSPC / cholesterol / PEG-DMG (50 mol%:10 mol%:38.5 mol% or 37.5 mol%:1.5 mol% or 2.5 mol%), PNI123 / DOPE / cholesterol / PEG-DMG, but is not limited thereto.

[0048] As used herein, the term "individual" refers to a subject in need of treatment or prevention of disease, and more specifically refers to mammals such as human or non-human primates, mice, dogs, cats, horses, and cows. Effect of the Invention

[0049] According to one embodiment of the mRNA transcription vector and the method for producing an mRNA molecule using the transcription vector, a specific gene construct is contained, thereby enabling highly efficient expression of a target mRNA molecule. Furthermore, it has been confirmed that the mRNA molecule produced by the method induces a high level of immune response in an animal model and can be applied to the treatment or prevention of various infectious diseases.

[0050] Therefore, the mRNA molecule-related technology according to one embodiment can be utilized in the production of mRNA molecules, and in the field of therapeutic agents or vaccines that include the mRNA molecules. [Brief description of the drawings]

[0051] [Figure 1] FIG. 1 is a simplified diagram showing the structure of an mRNA transcription vector according to one embodiment. [Diagram 2] FIG. 1 shows a simplified diagram of the structures of an mRNA transcription vector according to one embodiment and an mRNA transcription vector prepared for comparing intracellular antigen expression levels, in which A shows pSKBS-luciferase, B shows pMod-luciferase, and C shows pBNT-luciferase. [Diagram 3] 1 shows the results of confirming luciferase activity after transfecting HEK293 cells with a luciferase mRNA molecule prepared using an mRNA transcription vector according to one embodiment. [Figure 4] The results show that an immunogenic composition including a coronavirus spike mRNA molecule according to one embodiment was administered to mice, and then the IgG antibody titer in the serum of the mice induced by the composition was confirmed. [Diagram 5]This is a result of comparing the IgG antibody titers in the serum of mice induced by an immunogenic composition containing a full-length coronavirus spike mRNA molecule (spike) or RBD mRNA molecule (RBD) according to one embodiment after the composition was administered to the mice. [Figure 6] 1 shows a comparison of the antibody titers of IgG in the serum of mice induced by an immunogenic composition comprising a spike mRNA molecule with a modified polyA tail sequence according to one embodiment, after the composition was administered to the mice. [Figure 7] FIG. 1 shows a comparison of the antibody titers of IgG in the serum of mice induced by an immunogenic composition comprising an mRNA molecule in which the uridine sequence in the spike mRNA molecule has been modified, according to one embodiment. [Figure 8] According to one embodiment, changes in the ratio of CD103+, CD62Lhigh, CD44high, and CD4+ T cells due to external antigen stimulation were confirmed in animal models (G1 to G5) administered with each vaccine formulation. [Figure 9] According to one embodiment, changes in the ratio of KI-67+, CD62Lhigh, CD44high, and CD4+ T cells due to external antigen stimulation were confirmed in animal models (G1 to G5) administered with each vaccine formulation. [Figure 10] According to one embodiment, changes in the ratio of KI-67+, CD62Lhigh, CD44high, CD8+ T cells due to external antigen stimulation were confirmed in animal models (G1 to G5) administered with each vaccine formulation. [Figure 11] According to one embodiment, changes in the ratio of KI-67+, CD62Llow, CD44high, and CD8+ T cells due to external antigen stimulation were confirmed in animal models (G1 to G5) administered with each vaccine formulation. [Figure 12]FIG. 1 shows the results of evaluating the immunogenicity of omicron spike protein antigen over time in animal models (G1 to G5) administered with each vaccine formulation according to one embodiment. (A) shows the results of confirming the antibody titer level of omicron RBD IgG1, and (B) shows the results of confirming the antibody titer level of omicron RBD IgG2a. [Figure 13] According to one embodiment, these are results of evaluating the immunogenicity against the Wuhan spike protein antigen over time in animal models (G1 to G5) administered with each vaccine formulation, where A is the result of confirming the antibody titer level of Wuhan RBD IgG1, B is the result of confirming the antibody titer level of Wuhan RBD IgG2a, C is the result of confirming the antibody titer level of Wuhan S IgG1, and D is the result of confirming the antibody titer level of Wuhan S IgG2a. [Figure 14] According to one embodiment, the neutralizing ability against pseudoviruses was evaluated in animal models (G1 to G5) administered with each vaccine formulation, and serum dilution factor (VNT50) was confirmed for a Vero cell line infected with a VSV pseudotyped virus (REVACC SCIENTIFIC). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0052] In the following, preferred examples are presented to aid in understanding the present invention. However, the following examples are provided only to facilitate understanding of the present invention, and are not intended to limit the scope of the present invention.

[0053] Example 1. Preparation of mRNA antigen expression system and comparison of expression levels of target antigens In this example, we attempted to confirm whether the expression level of a target antigen in cells could be improved using the mRNA antigen expression system according to one embodiment. To this end, we used luciferase mRNA as a target antigen, prepared an mRNA transcription vector according to one embodiment, transfected the mRNA molecules prepared therethrough into HEK293 cells, and quantitatively compared their expression levels.

[0054] 1-1. Preparation of mRNA transcription vector As shown in Figure 1, an mRNA transcription vector was prepared based on a structure in which a promoter region sequence, a 5'-UTR region sequence, an ORF region sequence, a 3'-UTR region sequence, and a polyA-tail region sequence are operably linked in sequence from the 5' end to the 3' end. In this example, a pUC19 plasmid was used, and a T7 promoter and a 5'-UTR region sequence (SEQ ID NO: 6) were inserted between the KpnI and BamHI recognition sites (GGTACC to GGATCC), and then a 3'-UTR and A30LA70 (SEQ ID NO: 7) were inserted between the XbaI and HindIII recognition sites (TCTAGA to AAGCTT) of the plasmid to prepare a pSKBS plasmid (SEQ ID NO: 8). Then, in order to express the target antigen luciferase mRNA, the luciferase gene (SEQ ID NO: 9) was inserted into the BamHI and XbaI recognition sites (GGATCC~TCTAGA) of the plasmid to prepare the pSKBS·luciferase plasmid (SEQ ID NO: 10). The genetic information of the main functional structures in the pSKBS·luciferase plasmid is shown in Table 1 below.

[0055] [Table 1]

[0056] In addition, to compare the expression efficiency of the pSKBS·luciferase plasmid according to one embodiment with that of the mRNA antigen, a pMod·luciferase plasmid containing 5'-UTR and 3'-UTR from Modna (Comparative Example 1) and a pBNT·luciferase plasmid containing 5'-UTR and 3'-UTR from BioNTech (Comparative Example 2) were prepared (2004, NAR, A simple, rapid, high-fidelity and cost effective PCR based two step DNA synthesis method for long gene sequence). Specifically, the pMod·luciferase plasmid was prepared by inserting the T7 promoter and the 5'-UTR sequence (SEQ ID NO: 11) of Modona into the KpnI and BamHI recognition site (GGTACC-GGATCC) of the pUC19 plasmid, inserting the 3'-UTR sequence (SEQ ID NO: 12) of Modona into the XbaI and SacII recognition site (TCTAGA-CCGCGG) of the plasmid, and inserting the luciferase gene into the BamHI and XbaI recognition site (GGATCC-TCTAGA) of the plasmid. The pBNT·luciferase plasmid also adopted the recognition site used in the preparation of the pMod·luciferase plasmid, and inserted the 5'-UTR sequence (SEQ ID NO: 13) and 3'-UTR sequence (SEQ ID NO: 14) of BioNTech in the same manner. The structure of the mRNA transcription vector prepared in this example is shown in FIG. 2.

[0057] 1-2. Evaluation of intracellular target antigen expression levels E. coli transformed with the plasmid prepared in Example 1-1 was cultured, the plasmid was isolated therefrom, and the plasmid was linearized using EcoRV restriction enzyme. The linearized plasmid was recovered using PCI ethanol precipitation and then used in an in vitro transcription (IVT) reaction for mRNA production. To this end, the linearized plasmid DNA template was mixed under the conditions described in Table 2 and reacted at 37°C for 2 hours. After that, after the reaction was terminated, DNase1 at a concentration of 2KU / mL was added thereto and reacted at 37°C for 15 minutes to remove the linearized plasmid. Then, in order to prepare an mRNA molecule suitable for transfection, the synthesized mRNA was purified using MEGAclear™ Transcription Clean-Up Kit (Thermo Fisher). Then, the purified mRNA was transfected into HEK293 cell line using Lipofectamine™ MessengerMAX (Thermo Fisher). 24 hours after the transfection, the activity of luciferase expressed in the HEK293 cell line was measured using the ONE-Glo™ Luciferase Assay System (Promega), and the relative expression level of luciferase was evaluated based on the measured activity. A group not transfected (mock) was used as a control.

[0058] [Table 2]

[0059] 3 shows the results of transfecting HEK293 cells with luciferase mRNA molecules prepared using an mRNA transcription vector according to one embodiment and then confirming the luciferase activity. As shown in the above Fig. 3, the luciferase mRNA molecules prepared using an mRNA transcription vector according to one embodiment showed high levels of luciferase activity when transfected into host cells, thereby confirming excellent intracellular expression efficiency of the target mRNA antigen.

[0060] Example 2. Efficacy evaluation of immunogenic compositions using mRNA antigen expression systems In this example, the mRNA antigen expression system according to one embodiment was used to confirm whether an effective immune response could be induced in an animal model. To this end, corona spike mRNA was used as a target antigen, an mRNA transcription vector according to one embodiment was prepared, and the mRNA molecule prepared therethrough was formulated into a lipid mRNA-LNP (mRNA-lipid nanoparticle) vaccine formulation, which was then administered to an animal model to evaluate the immunogenicity thereof, specifically, the antibody titer formation level.

[0061] 2-1. Preparation of mRNA transcription vector and target mRNA molecule The mRNA transcription vector was prepared in a manner similar to that of Example 1-1. Specifically, in this example, pUC19 plasmid and a T7 promoter (SEQ ID NO: 15) in which the transcription start sequence +1 is A and the transcription start sequence +2 is G were used, and the promoter and 5'-UTR region sequence (SEQ ID NO: 16) were inserted between the KpnI and BamHI recognition sites (GGTACC to GGATCC), and then 3'-UTR and A20 (SEQ ID NO: 17) were inserted between the XbaI and HindIII recognition sites (TCTAGA to AAGCTT) of the plasmid to prepare pSKBS-AG plasmid (SEQ ID NO: 18). Then, the spike gene (SEQ ID NO: 19) of the SARS-CoV2β mutant virus was inserted between the BamHI and XbaI recognition sites (GGATCC to TCTAGA) of the plasmid to prepare pSKBS-spike(SA)-AG plasmid (SEQ ID NO: 20).

[0062] The above-prepared pSKBS-spike(SA)-AG plasmid was used to prepare mRNA. More specifically, E. coli transformed with the above-prepared plasmid was cultured, the plasmid was isolated therefrom, and the plasmid was linearized using EcoRV restriction enzyme. The linearized plasmid was recovered using PCI ethanol precipitation and used in an in vitro transcription reaction for mRNA preparation. To this end, the linearized plasmid DNA template was mixed under the conditions listed in Table 3 and reacted at 37°C for 2 hours. After that, after the reaction was terminated, DNase1 at a concentration of 2KU / mL was added thereto and reacted at 37°C for 15 minutes to remove the linearized plasmid. Then, in order to add a polyA tail to the 3' end of the mRNA, the Poly(A) Polymerase Tailing Kit (Lucigen) was used to mix the substances under the conditions listed in Table 4 and reacted at 37°C for 1 hour. The synthesized mRNA was then purified on an AKTA FPLC (GE Healthcare) using a CIMmultus® Oligo dT (Sartorius) column.

[0063] [Table 3]

[0064] [Table 4]

[0065] 2-2. Manufacturing of mRNA-LNP vaccine formulations The purified mRNA molecule / stock solution of Example 2-1 was formulated into a lipid mixture to produce mRNA-LNP (lipid nanoparticle) in which mRNA was encapsulated in lipid components. Specifically, the mRNA stock solution was diluted to 0.17 mg / mL with a formulation buffer, and GenVoy-ILM (PRECISION NANOSYSTEMS) was diluted 1 / 2 with ethanol, and each of them was filled into a syringe. Then, a fine tube cartridge was attached to an Ignite (trademark) device, and the syringe containing the diluted mRNA solution and the syringe containing the diluted GenVoy-ILM solution were attached, reacted at a volume ratio of 3:1, and the formulation process was carried out under the condition of N / P ratio = 4. After that, the reaction solution after the formulation process was collected and concentrated by buffer exchange with 20 times the volume of PBS using a Centrifugal Ultrafiltration unit (MWCO 50 kDa). The mRNA-LNP stock solution was then stored at 4°C, and the mRNA content and encapsulation ratio were measured, after which it was used in animal experiments.

[0066] 2-3. Immunogenicity assessment 0.1 mL of injection containing 100 μl of stock solution containing 5 μg or 25 μg of mRNA-LNP prepared in Example 2-2 was intramuscularly administered (IM) twice to an animal model (mouse, BALB / c, female) at intervals of 0, 2 or 3 weeks. In addition, serum was separated from blood samples taken from the animal model before and after administration, and the serum was used as a sample to measure the antibody titer of IgG produced by the vaccine formulation by ELISA (enzyme-linked immunosorbent assay). Specifically, the surface of a 96-well plate was coated with 1 μg / mL of antigen and then incubated at 2 to 8° C. for 18±2 hours. The incubated plate was washed and then blocked with BSA solution at room temperature for 1 hour. Then, the plate was washed and the serum dilutions prepared through serial dilutions were dispensed into the plate. The secondary antibody, Goat Anti-Mouse IgG-Alkaline phosphatase conjugates, was diluted and added to the plate, and then reacted at room temperature for 2 hours. The plate was then washed, and 1 mg / mL p-nitrophenylamine buffer was added thereto, and then reacted at room temperature for another 2 hours. Then, 3 M NaOH was added to each well to stop the reaction, and the absorbance at 405 nm and 690 nm was measured, and the OD (optical density) value of the IgG antibody titer was calculated.

[0067] 4 shows the results of administering a vaccine formulation containing a coronavirus spike mRNA molecule according to one embodiment to mice, and then confirming the IgG antibody titer in the serum of the mice induced by the formulation. As shown in the figure, it was confirmed that an effective level of IgG antibody titer was formed by administering the mRNA-LNP vaccine formulation according to one embodiment, and it was found that its efficacy was enhanced by repeated administration.

[0068] 2-4. Immunogenicity evaluation of vaccine formulations including various morphological modifications In the same manner as in Examples 2-1 to 2-3, the immunogenicity of 1) a vaccine preparation containing a full-length spike mRNA molecule (spike) or an RBD mRNA molecule (RBD) in coronavirus, 2) a vaccine preparation containing a spike mRNA molecule with a modified polyA tail sequence, and 3) a vaccine preparation containing an mRNA molecule with a modified uridine sequence in the spike mRNA molecule was evaluated. Specifically, in this example, a 124nt polyA tail (A124, SA) and a polyA tail in which a 30nt polyA and a 70nt polyA are linked by a GCAUAUGACU linker (A30LA70, SAL) were used as the polyA tail, and pseudouridine (Ψ) was used instead of uridine as a modification of the uridine sequence.

[0069] Figures 5 to 7 show the results of comparing the serum IgG titers of mice induced by a vaccine formulation containing a full-length spike mRNA molecule (spike) or RBD mRNA molecule (RBD) in coronavirus according to one embodiment, a vaccine formulation containing a spike mRNA molecule with a modified polyA tail sequence, and a vaccine formulation containing an mRNA molecule with a modified uridine sequence in the spike mRNA molecule, after the vaccine formulation was administered to mice. As shown in Figures 5 to 7, it was confirmed that effective levels of IgG antibody titers were formed in all vaccine formulations containing various modifications.

[0070] Example 3. Evaluation of immune response induction efficacy using animal models In this example, the mRNA antigen expression system according to one embodiment was used to confirm whether an effective immune response could be induced in an animal model as an action against a specific external antigen. To this end, changes at the cellular level involved in the immune response were evaluated, and cross-reactivity due to the characteristics of coronavirus-derived antigens was confirmed. The mRNA transcription vector was prepared by introducing the SARS-CoV2 omicron spike·2P prefusion gene (SEQ ID NO: 21), the SARS-CoV2 omicron spike·6P prefusion gene (SEQ ID NO: 22), or the SARS-CoV2 Wuhan spike·2P prefusion gene (SEQ ID NO: 23) into the pSKBS-AG plasmid (SEQ ID NO: 18) of Example 2-1 between the BamHI and XbaI recognition sites (GGATCC~TCTAGA) of the plasmid, and in this Example 3, N1-methyl pseudouridine (m1Ψ) was applied instead of uridine as a modification of the uridine sequence. The mRNA produced through this was formulated, and 0.1 mL of injection containing 100 μl of the original solution containing 10 μg of mRNA-LNP was intramuscularly administered to an animal model (mouse, BALB / c, female).

[0071] In this example, the subjects were classified into a total of 5 groups based on the type of active ingredient contained in the administered preparation, as shown in Table 5 below, and the experiment was performed.

[0072] [Table 5]

[0073] The animal experimental groups were then further classified according to the type of stimulation with external antigens into a group that received no additional stimulation (non), a group that received stimulation with a SARS-CoV2 antigen mixture (SARS-CoV-2 (S1), scanning (3629-1), mebtech.com) (peptide pool), a group that received stimulation with SARS-CoV2 Wuhan spike antigen (RBD Wuhan), and a group that received stimulation with SARS-CoV2 omicron spike antigen (RBD omicron), and the efficacy of each antigen in inducing an immune response was evaluated.

[0074] 3-1. Cell-mediated immune analysis (T cell analysis) The prepared mRNA-LNP stock solution was administered intramuscularly (IM) to an animal model (mouse, BALB / c, female). Specifically, it was administered twice at 2-week intervals on weeks 0 and 2, and 1 week after the secondary immunization, the spleen was removed via autopsy, red blood cells were removed from the removed spleen, and spleen cells were obtained. In order to analyze the cell lines that react to the antigen in the obtained spleen cells, they were treated with Wuhan, Omicron-mutated SARS-CoV2 RBD protein, or SARS-CoV2 CD8 epitope prediction peptide pool (Sinobiological), stimulated, and then labeled with KI-67 dye and CD62L, CD44, and CD8 antibodies. In the spleen cells prepared as described above, CD4 T cells and CD8 T cells that react to the antigen were analyzed via FACS (fluorescence-activated cell sorting), and the results are shown in Figures 8 to 11.

[0075] FIG. 8 and FIG. 9 show the CD103 expression level induced by external antigen stimulation in animal models (G1 to G5) administered with each vaccine formulation according to one embodiment. + ,CD62L high ,CD44 high ,CD4 + T cells and KI-67 + ,CD62L high ,CD44 high ,CD4 +FIG. 10 and FIG. 11 show the results of changes in the ratio of T cells. The results are shown in FIG. 10 and FIG. 11. The changes in the ratio of T cells were observed in the animal models (G1 to G5) administered with each vaccine preparation according to one embodiment. + ,CD62L high ,CD44 high ,CD8 + T cells and KI-67 + ,CD62L low ,CD44 high ,CD8 + These are the results of confirming changes in the ratio of T cells.

[0076] As shown in Figures 8 and 9, the immune response to external antigens, i.e., spike antigens of SARS-CoV2 antigen mixture, SARS-CoV2 Wuhan, or SARS-CoV2 Omicron, was confirmed to increase in the ratio of resident memory CD4 T cells and CD4 memory proliferation T cells. Also, as shown in Figures 10 and 11, the immune response to external antigens was confirmed to increase in the ratio of proliferation memory CD8 T cells and effector proliferation CD8 T cells.

[0077] 3-2.Humoral immune analysis (ELISA analysis) The prepared mRNA-LNP stock solution was administered intramuscularly (IM) to an animal model (mouse, BALB / c, female). It was administered twice at two-strain intervals on weeks 0 and 2, and serum was isolated from blood samples taken before administration and on weeks 2 and 4 after administration. Then, using the isolated serum as a sample, the IgG antibody titer generated by the vaccine formulation was measured by enzyme-linked immunosorbent assay (ELISA) in the same manner as in Example 2-3. Then, the RBD protein and spike protein of each strain were coated on antigens, and the associated Ig antibody titer was measured, and the results are shown in Figures 12 and 13.

[0078] Figure 12 shows the results of an evaluation over time of the immunogenicity against the omicron spike protein antigen in animal models (G1 to G5) administered with each vaccine formulation according to one embodiment, and Figure 13 shows the results of an evaluation over time of the immunogenicity against the Wuhan spike protein antigen in animal models (G1 to G5) administered with each vaccine formulation according to one embodiment.

[0079] As shown in Figures 12 and 13, when viewed in light of the above-mentioned antibody titer formation levels, the vaccine formulation prepared according to one embodiment confirmed that groups G1 and G2 administered the SARS-CoV2 omicron spike mRNA vaccine formulation showed cross-reactivity to the Wuhan spike protein antigen across all time points, and group G5 administered the SARS-CoV2 Wuhan spike mRNA vaccine formulation also showed cross-reactivity to the omicron spike protein antigen.

[0080] 3-3.Humoral immunoassay (PBNA:pseudovirion-based neutralization assay) The above-mentioned mRNA-LNP stock solution was administered intramuscularly (IM) to an animal model (mouse, BALB / c, female). It was administered twice at 2-week intervals at week 0 and week 2, and serum was isolated from the blood sample two weeks after administration. The above-mentioned isolated serum was then used as a sample to proceed with neutralizing antibody analysis (PBNA). SARS-CoV2 Wuhan (D614G), a VSV pseudotyped virus (REVACC SCIENTIFIC) with an omicron-mutated spike protein and containing a luciferase gene, was appropriately diluted. They were then mixed with the above-mentioned serially diluted serum sample, treated with a Vero cell line, and infected for 24 hours, after which luciferase activity was measured, and the results are shown in Figure 14.

[0081] Figure 14 shows the serum dilution factor (VNT) of cultured cells at 50% infection. 50 , 50% neutralization titers), confirming that the antibodies produced in the groups vaccinated with the vaccine formulation (G1, G2, and G5) had neutralizing ability against pseudoviruses expressing different mutant spike proteins.

[0082] The above description of the present invention is for illustrative purposes only, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical concept or essential features of the present invention. Therefore, the above-described embodiments should be understood to be illustrative in all respects and not restrictive. (Additional Note) The present disclosure includes the following aspects. Item 1: An mRNA transcription vector comprising a promoter region recognized by an RNA polymerase and a gene construct operably linked to the promoter region, The genetic construct comprises: A 5'-untranslated region (5'-UTR: 5'-untranslated region) consisting of SEQ ID NO: 1 or a nucleotide sequence having 90% or more sequence identity thereto; an open reading frame (ORF) region comprising a nucleotide sequence encoding a target antigen, operably linked to the 5'-UTR region; An mRNA transcription vector comprising: a 3'-untranslated region (3'-UTR) region in which a unit sequence consisting of SEQ ID NO:2 or a nucleotide sequence having 90% or more sequence identity thereto is repeated twice, operably linked to the open reading frame region. Item 2: The mRNA transcription vector according to Item 1, wherein the promoter region is recognized by any one of RNA polymerases including T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, and mitochondrial polymerase (POLRMT). Item 3: The mRNA transcription vector according to Item 1, wherein the open reading frame region comprises a nucleotide sequence encoding an antigen derived from a pathogen. Item 4: The mRNA transcription vector according to item 3, wherein the pathogen is selected from the group consisting of viruses, bacteria, prions, fungi, protozoans, viroids and parasites. Item 5: The mRNA transcription vector according to Item 1, wherein the 3'-UTR region is composed of a nucleotide sequence to which the monomer sequence is directly linked or linked via a linker sequence. Item 6: The mRNA transcription vector according to item 1, wherein the gene construct additionally comprises a nucleotide sequence to be transcribed into a 5' cap, operably linked to the 5'-UTR. Item 7: The mRNA transcription vector according to item 1, wherein the gene construct additionally comprises a nucleotide sequence to be transcribed into a polyA tail, operably linked to the 3'-UTR region. Item 8: The mRNA transcription vector according to Item 7, wherein the poly A tail consists of 20 to 200 adenines. Item 9: The mRNA transcription vector of Item 1, wherein the mRNA transcription vector is a plasmid. Item 10: The mRNA transcription vector of Item 1, wherein the mRNA transcription vector is linearized. Item 11: The mRNA transcription vector comprises a promoter region and a gene construct consisting of the nucleotide sequence of SEQ ID NO: 4 or SEQ ID NO: 15; The genetic construct comprises: A 5'-UTR region consisting of the nucleotide sequence of SEQ ID NO:1; an ORF region operably linked to the 5'-UTR region; 2. The mRNA transcription vector according to item 1, comprising a 3'-UTR region consisting of the nucleotide sequence of SEQ ID NO:3. Item 12: A method for producing an mRNA molecule, comprising a step of in vitro transcription using the mRNA transcription vector according to item 1 as a template. Item 13: An mRNA molecule produced by the method according to Item 12. Item 14: An immunogenic composition comprising the mRNA molecule of Item 13 and a pharma- ceutically acceptable excipient as an active ingredient. Item 15: The immunogenic composition according to item 14, wherein the mRNA molecule is complexed with at least one lipid component to form a liposome, lipid nanoparticle and / or lipoplex. Item 16: The immunogenic composition of item 14, wherein the immunogenic composition is administered intramuscularly or subcutaneously.

Claims

1. An mRNA transcription vector comprising a promoter region recognized by an RNA polymerase and a gene construct operably linked to the promoter region, The genetic construct comprises: A 5'-untranslated region (5'-UTR) region consisting of SEQ ID NO: 1; an open reading frame (ORF) region comprising a nucleotide sequence encoding a target antigen, operably linked to the 5'-UTR region; and a 3'-untranslated region (3'-UTR) region consisting of SEQ ID NO:3, operably linked to the open reading frame region.

2. The mRNA transcription vector of claim 1 , wherein the promoter region is recognized by any one of RNA polymerases including T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, and mitochondrial polymerase (POLRMT).

3. The mRNA transcription vector of claim 1 , wherein the open reading frame region comprises a nucleotide sequence encoding an antigen derived from a pathogen.

4. The mRNA transcription vector of claim 3 , wherein the pathogen is selected from the group consisting of viruses, bacteria, prions, fungi, protozoans, viroids and parasites.

5. The mRNA transcription vector of claim 1, wherein the gene construct further comprises a nucleotide sequence to be transcribed into a 5' cap, operably linked to the 5'-UTR.

6. The mRNA transcription vector of claim 1, wherein the genetic construct further comprises a nucleotide sequence operably linked to the 3'-UTR region that is to be transcribed into a polyA tail.

7. The mRNA transcription vector of claim 6, wherein the poly A tail consists of 20 to 200 adenines.

8. The mRNA transcription vector of claim 1 , wherein the mRNA transcription vector is a plasmid.

9. The mRNA transcription vector of claim 1 , wherein the mRNA transcription vector is linearized.

10. The mRNA transcription vector comprises a promoter region and a gene construct consisting of the nucleotide sequence of SEQ ID NO: 4 or SEQ ID NO: 15; The genetic construct comprises: A 5'-UTR region consisting of the nucleotide sequence of SEQ ID NO:1; an ORF region operably linked to the 5'-UTR region; The mRNA transcription vector of claim 1, comprising a 3'-UTR region consisting of the nucleotide sequence of SEQ ID NO:

3.

11. A method for producing an mRNA molecule, comprising the step of performing in vitro transcription using the mRNA transcription vector of claim 1 as a template.

12. An mRNA transcription vector comprising a promoter region recognized by an RNA polymerase and a gene construct operably linked to the promoter region, the gene construct comprising: a 5'-untranslated region (5'-UTR) region consisting of SEQ ID NO:1; an open reading frame (ORF) region operably linked to the 5'-UTR region, the ORF region comprising a nucleotide sequence encoding a target antigen; and a 3'-untranslated region (3'-UTR) region operably linked to the open reading frame region, the 3'-untranslated region (3'-UTR) region consisting of SEQ ID NO:

3. An mRNA molecule that is a transcript of a

13. The mRNA molecule described in claim 12, in which uridine in the mRNA molecule is replaced with pseudouridine or N1-methylpseudouridine.

14. An immunogenic composition comprising as an active ingredient the mRNA molecule of claim 12 and a pharma- ceutically acceptable excipient.

15. The immunogenic composition of claim 14, wherein the mRNA molecule is complexed with at least one lipid component to form a liposome, lipid nanoparticle and / or lipoplex.

16. The immunogenic composition of claim 14, wherein the immunogenic composition is administered intramuscularly or subcutaneously.

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