Nucleic acid molecule with hairpin structure capable of modulating innate immunity and use thereof

Abstract

The present invention relates to a nucleic acid molecule capable of controlling innate immunity by RNA and, more specifically, to a short duplex hairpin RNA (sdhRNA) that binds to protein kinase R (PKR), which is activated upon recognition of RNA, thereby inhibiting the activity of PKR and suppressing PKR-mediated innate immunity and which, when introduced into the 3'-UTR of mRNA, can increase the expression level of an antigen. The sdhRNA motif according to the present invention inhibits the activity of protein kinase R (PKR) by binding to PKR activated by recognizing RNA. Therefore, when administered into cells in combination with exogenous RNA or as an insert into the 3'-UTR of mRNA, the sdhRNA motif exhibits the effect of reducing RNA-indued PKR activation, mitigating RNA-induced cell death, and downregulating the expression of interferon-stimulated gene (ISG) as well as improving the expression efficiency of a protein encoded by the mRNA. With the effects, the motif is applied to various RNA-based uses, for example, vaccines, in vivo / ex vivo gene therapeutic agents, and the like.

Description

Nucleic acid molecules with hairpin structures capable of regulating innate immunity and their uses The present invention relates to a nucleic acid molecule capable of controlling innate immune activity by RNA, and more specifically, to a short duplex hairpin RNA (sdhRNA) that binds to protein kinase R (PKR) that recognizes and is activated by RNA, thereby inhibiting the activity of PKR and suppressing innate immunity by PKR, and that can increase the expression amount of an antigen when introduced into the 3'-UTR of mRNA. Advances in molecular biology have led to the development of nucleic acid (DNA / mRNA) vaccines, which utilize plasmids containing genes encoding protein / peptide antigens that trigger an immune response. Nucleic acid vaccines were proposed in the early 1990s as a powerful alternative to existing vaccines. However, due to shortcomings such as low mRNA stability and inefficient in vivo delivery, they were not widely used until the COVID-19 pandemic. Over the past decade, technological innovations and R&D investments have led to mRNA gaining recognition as a promising therapeutic tool in vaccine development and protein replacement therapy. mRNA vaccines offer numerous advantages over DNA vaccines, as well as subunit killed and live attenuated viral vaccines. The greatest advantage of mRNA vaccines is that because the body's own cells produce antigens, there's no need for traditional manufacturing methods, which involve culturing pathogens and harvesting the vaccine fragments. Furthermore, while traditional vaccines only activate humoral immunity, mRNA vaccines also have the advantage of inducing cell-mediated immunity during the production of intracellular antigens. Furthermore, mRNA vaccines are non-infectious and non-integral platforms, reducing the potential risk of infection or mutations caused by human DNA insertion and minimizing unnecessary immune responses. Furthermore, they are rapidly uptaken and expressed within the cytoplasm, and their immune responses are lower than those of viral approaches, allowing for repeated administration. mRNA vaccines are produced through in vitro transcription (IVT), and their high yields allow for rapid, economical, and large-scale production. Furthermore, mRNA introduced into cells can produce 100- to 1,000-fold more protein, making even small amounts of mRNA effective. However, RNA used in gene therapy, including mRNA vaccines, is recognized as foreign RNA within cells and triggers an innate immune response. Therefore, there is a need to develop technologies to control innate immune activation using therapeutic or vaccine mRNA. mRNA typically consists of a cap, 5' and 3' untranslated regions (UTRs), an open reading frame (ORF), and a poly(A) tail. Modifying the nucleic acid structure can improve protein (antigen) expression efficiency, increase immunogenicity in the body, and minimize unnecessary immune responses. Although many means and methods have been disclosed to increase the stability of mRNA, to reduce the immunogenic response triggered by mRNA administered to a cell or organism, and to increase the expression efficiency (i.e., transcription and / or translation efficiency) (US 10080809, US 2018-0353618, US 2019-0144883), there still exists a need for improvement, particularly with regard to additional or alternative means to increase the expression efficiency (i.e., transcription and / or translation efficiency), as the expression efficiency is an essential parameter for the envisioned medical applications, since it determines, for example, the dosing and dosing interval of the mRNA drug, and ultimately the bioavailability of the final product, i.e., the encoded peptide or protein. At the same time, there continues to be a need to further reduce the manufacturing cost of mRNA drugs, to increase the yield of the resulting mRNA molecules, and to increase the available space within the resulting mRNA molecule for the actual transgene, i.e., for the coding region encoding the desired polypeptide. The delivery of nucleic acids to elicit desired responses in biological systems presents numerous challenges. Nucleic acid-based therapeutics, such as vaccines, hold tremendous promise, but to realize this potential, more efficient delivery of nucleic acids to the appropriate site within cells or organisms remains a pressing need. However, the use of nucleic acids for therapeutic and prophylactic purposes currently faces two challenges. First, free RNA is vulnerable to nuclease digestion in plasma. Second, free RNA has limited access to intracellular compartments where relevant translational machinery resides. Lipid nanoparticles formed from cationic lipids and other lipid components, such as neutral lipids, cholesterol, PEG, PEGylated lipids, and oligonucleotides, are being developed to block RNA degradation in plasma and promote cellular uptake of nucleic acids. Accordingly, the present inventors have made extensive efforts to develop a nucleic acid structure capable of solving the above problems, suppressing innate immune responses, and improving translation efficiency, and as a result, have selected a short duplex hairpin RNA molecule that suppresses the activity of the PKR protein, and have confirmed that when the hairpin RNA molecule is introduced into a cell, the innate immune response can be suppressed and translation efficiency can be improved, thereby completing the present invention. Summary of the invention The purpose of the present invention is to provide a short duplex hairpin nucleic acid molecule capable of suppressing innate immunity and improving translation efficiency. Another object of the present invention is to provide a nucleic acid structure comprising the short duplex hairpin nucleic acid molecule. Another object of the present invention is to provide a vaccine composition comprising the nucleic acid structure. To achieve the above purpose, the present invention provides a short duplex hairpin nucleic acid molecule (sdhNA) represented by any one of the base sequences of SEQ ID NOs: 1 to 8 and which suppresses an innate immune response. The present invention also provides a nucleic acid construct comprising, in order from 5' to 3': a) one or more coding regions; b) a 3'-untranslated region (3'-UTR); c) a short duplex hairpin nucleic acid (sdhNA); and d) 10 to 1000 poly(A) tails or poly(A) tail-like sequences. The present invention also provides a vaccine composition comprising the nucleic acid structure. The present invention also provides a use of the vaccine composition for disease prevention. The present invention also provides a method for preventing a disease comprising a step of administering the vaccine composition. The present invention also provides the use of the vaccine composition for the manufacture of a medicament for the prevention of a disease. Figure 1 is a conceptual diagram showing suppression of innate immune response and increase in antigen expression by mRNA administration through sdhRNA of the present invention. Figure 2 shows the results of analyzing antigen expression of EGFP mRNA injected into A549 lung cancer cells from which PKR was deleted and cell death induced by external mRNA injection. (A) shows the results of analyzing EGFP expression, PKR activity (pPKR), and peIF2α, a substrate of PKR, in A549 cells (PKR WT) and PKR-deleted A549 cells (PKR KO) using western blotting, where TUBB was used as a loading control. (B) shows the results of analyzing EGFP fluorescence after EGFP mRNA injection into A549 WT and PKR KO A549 cells using a fluorescence microscope, and (C) shows the results of analyzing cell death in the two cell lines after EGFP mRNA injection. Figure 3 shows the results of analyzing antigen expression and interferon response when nc886 RNA was administered to A549 cells together with the 3' UTR of EGFP mRNA or EGFP mRNA. (A) is the result of measuring EGFP fluorescence when EGFP mRNA, RNA containing the nc886 sequence in the 3' UTR of EGFP mRNA, and EGFP mRNA and nc886 were separately administered to cells. (B) is the result of analyzing PKR activity (pPKR) through western blotting, and TUBB was used as a loading control. (C) is the result of measuring the expression of IFNβ1, IL8, and ISG15 mRNA through RT-qPCR. Figure 4 shows the results of inserting three nc886 sequences into EGFP 3' UTR and analyzing the effects. (A) is an experimental schematic and EGFP mRNA synthesis results, (B) is the result of analyzing EGFP expression and PKR activation after administering EGFP mRNA with one nc886 sequence and EGFP mRNA with three nc886 sequences to A549 cells, (C) is the result of analyzing EGFP fluorescence of each construct, and (D) is the result of analyzing IFNβ1, IL8, and ISG15 mRNA, which are representative interferon genes, by RT-qPCR. Figure 5 shows the results of analyzing the PKR binding affinity for 1,840 hairpin sdhRNAs produced by IVT. 1,840 hairpin RNAs were incubated with Flag-PKR obtained from HEK-293T, and then immunoprecipitation was performed. The obtained PKR-sdhRNA complexes were analyzed by RNA-seq together with the immunoprecipitation input RNA. The number of sequencing reads obtained by the PKR immunoprecipitation method for each RNA was divided by the input RNA sequencing reads to analyze the PKR binding affinity. Figure 6 shows the results of confirming PKR activation in cells after administering sdhRNA having a binding affinity to seven different PKRs to A549 cells and then administering poly(I:C) that induces PKR activity. Figure 7 shows the results of analyzing PKR activity after administering nc886, sdhRNA_mid_1, and sdhRNA_mid_2 together with poly (I:C) to A549 cells. Figure 8 shows a schematic diagram of EGFP mRNA synthesis with nc886, sdhRNA_mid_1, sdhRNA_mid_2, and sdhRNA_ctrl loaded in the 3' UTR, and the results of mRNA synthesis confirmed using an electrophoresis device. Figure 9 shows the results of analyzing PKR activity after administering EGFP mRNA synthesized in Figure 8 to A549 cells. Figure 10 shows the results of RT-qPCR analysis of IFNB1 and CXCL10 mRNA in cells administered with four types of EGFP mRNA in Figure 9. Figure 11 shows the results of analyzing EGFP expression and apoptosis in A549 cells administered with the four EGFP mRNAs used in Figure 9. (A) is the result of analyzing EGFP protein fluorescence, (B) is the result of analyzing EGFP expression by western blotting, and (C) is the result of analyzing apoptosis for EGFP mRNA. Figure 12 shows the results of analyzing the mRNA expression level after administering EGFP mRNA loaded with sdhRNA_mid_1, sdhRNA_mid_2, and sdhRNA_ctrl to A549. The left panel shows the change in the relative mRNA expression level over time after cell administration, and the right panel shows the mRNA expression level remaining in the cell 24 hours later. Figure 13 shows the results of analyzing EGFP expression, PKR activity, and cell death after administering EGFP mRNA loaded with nc886, sdhRNA_mid_1, sdhRNA_mid_2, and sdhRNA_ctrl to A549 cells using lipid nanoparticles. (A) is a schematic diagram of the experiment, (B) is the result of analyzing EGFP expression and PKR activity by EGFP mRNA loaded with sdhRNA through western blotting, and (C) is the result of analyzing cell death by EGFP mRNA loaded with sdhRNA. Figure 14 shows the results of analyzing the binding affinity with PKR after producing a total of 7 sdhRNAs and nc886 using existing uridine or pseudouridine to analyze the influence of pseudouridine. Figure 15 shows the results of analyzing the EGFP expression level and PKR activity after synthesizing EGFP mRNA loaded with nc886, sdhRNA_mid_2, and sdhRNA_ctrl using uridine or pseudouridine and administering it to A549 cells. (A) is the result of analyzing the EGFP expression level and PKR activity through western blotting, and (B) is the result of analyzing EGFP fluorescence using a fluorescence microscope. Figure 16 shows the results of analyzing the apoptotic effect of EGFP mRNA synthesis using uridine or pseudouridine synthesized in Figure 15. The upper panel is the result of analyzing cleaved PARP, an apoptosis marker, through Western blotting, and the lower panel is the result of analyzing apoptosis through an MTT experiment. Figure 17 shows the results of analyzing spike protein expression and PKR activity after inserting nc886, sdhRNA_ctrl, sdhRNA_mid_1, or sdhRNA_mid_2 into the 3' UTR of mRNA capable of producing the spike protein of the SARS-CoV-2 virus and administering it to A549 cells. Figure 18 shows the results of analyzing the number of antigen-specific T cells after two inoculations into mice after inserting nc886, sdhRNA_mid_1, and sdhRNA_mid_2 into the 3' UTR of SARS-CoV-2 virus spike mRNA and loading it into lipid nanoparticles. Figure 19 shows the results of analyzing antigen-specific antibodies after inserting nc886, sdhRNA_mid_1, and sdhRNA_mid_2 into the 3' UTR of SARS-CoV-2 virus spike mRNA, loading it into lipid nanoparticles, and inoculating mice twice. Detailed description of the invention and preferred embodiments Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used herein and the experimental methods described below are well known and commonly used in the art. In the present invention, when sdhRNA is injected into cells together with external RNA, the RNA is recognized by innate immune response proteins such as protein kinase R (PKR), melanoma-differentiation associated gene 5 (MDA5), and retinoic acid-inducible gene I (RIG-I), inducing an immune response, inducing cell death, inhibiting protein translation, and promoting ISG expression. It was intended to confirm whether this could inhibit the process and increase the expression amount. That is, in one embodiment of the present invention, the PKR binding affinity for 1,840 hairpin sdhRNAs was analyzed to select sdhRNAs that inhibit PKR activity and increase the expression of a target protein (Fig. 1). Therefore, the present invention, from one point of view, It relates to a short duplex hairpin nucleic acid molecule (sdhNA) represented by any one of the base sequences of SEQ ID NOs: 1 to 8 and which suppresses an innate immune response. The present invention also provides: From 5' to 3' in order a) one or more coding areas; b) 3'-untranslated region (3'-UTR); c) the short duplex hairpin nucleic acid molecule; and d) 10 to 1000 poly(A) tails or poly(A) tail-like sequences; It relates to a nucleic acid structure including . In the present invention, the short duplex hairpin nucleic acid molecule (sdhNA) may be characterized as being a short duplex hairpin RNA (sdhRNA). In the present invention, the sdhRNA may be introduced into a cell alone or by binding to the 3'-UTR of mRNA. In the present invention, the nucleic acid structure may be characterized by further comprising a 5'-CAP structure and a 5'-UTR polynucleotide 5' upstream of at least one coding region. As used herein, the term "UTR" refers to an "untranslated region" located upstream (5') and / or downstream (3') of the coding region of a nucleic acid molecule described herein, and thus typically flanking the coding region. Thus, the term "UTR" generally encompasses a 3' untranslated region ("3'-UTR") and a 5' untranslated region ("5'-UTR"). A UTR typically comprises or may consist of a nucleic acid sequence that is not translated into a protein. Typically, a UTR comprises a "regulatory element." The term "regulatory element" refers to a nucleic acid sequence that has the ability to influence gene regulatory activity, expression, particularly transcription or translation, of a transcribable nucleic acid sequence to which it is operably (cis- or trans-) linked. The term includes promoters, enhancers, internal ribosome entry sites (IRES), introns, leaders, transcription termination signals such as polyadenylation signals and poly-U sequences, and other expression regulatory elements. Regulatory elements may act constitutively or in a time- and / or cell-specific manner. Alternatively, regulatory elements may exert their function through interaction (e.g., recruitment and binding) with regulatory proteins that can regulate (induce, enhance, reduce, abrogate, or prevent) expression, particularly transcription, of a gene. A UTR is preferably "operably linked" to a coding region, i.e., positioned in a functional relationship, such that it controls (i.e., mediates or regulates, preferably enhances) the expression of said coding sequence. The term "5'-UTR" as used herein refers to a portion of a nucleic acid molecule that is located 5' (i.e., "upstream") of the open reading frame and is not translated into protein. In the context of the present invention, the 5'-UTR begins at the transcription start site and ends one nucleotide before the start codon of the open reading frame. The 5'-UTR may comprise elements that regulate gene expression, so-called "regulatory elements". Such regulatory elements may be, for example, ribosome binding sites. The 5'-UTR may be modified post-transcriptionally, for example by the addition of a 5'-CAP. Thus, the 5'-UTR may preferably correspond to a sequence of nucleic acids located between the 5'-CAP and the start codon, in particular a sequence of a mature mRNA, and more particularly a sequence extending from a nucleotide located 3' to the 5'-CAP, preferably a nucleotide located immediately 3' to the 5'-CAP, to a nucleotide located 5' to the start codon (transcription start site) of a protein coding sequence, preferably a nucleotide located immediately 5' to the start codon (transcription start site) of the protein coding sequence. The nucleotide immediately 3' to the 5'-CAP of a mature mRNA typically corresponds to the transcription initiation site. The 5' UTR typically has a length of less than 500, 400, 300, 250, or 200 nucleotides. In some embodiments, its length can range from 10, 20, 30, or 40 nucleotides, and preferably from 10 or 50 nucleotides. In the present invention, the 5'-UTR polynucleotide may be characterized by being selected from the group consisting of α-globin 5'UTR; Hsp70 5'UTR; axon dynein heavy chain 2 (DNAH2) 5'UTR; hydroxysteroid dehydrogenase (3β-HSD) 5'UTR; 5'-UTR represented by the base sequence of SEQ ID NO: 11; and 5'-UTR represented by the base sequence of SEQ ID NO: 12, but is not limited thereto. The 5'-CAP of native mRNA is involved in nuclear export, increasing mRNA stability and binding to mRNA cap binding protein (CBP), which leads to mRNA stability during cellular and translational stages through the association of poly(A) binding protein and CBP to form the mature cyclic mRNA species. The cap further assists in the removal of 5'-proximal introns during mRNA splicing. In the present invention, 5'-CAP is typically a modified nucleotide (CAP analog), particularly a guanine nucleotide added to the 5' end of an mRNA molecule. Preferably, the 5'-CAP is added using a 5'-5'-triphosphate linkage (also referred to as m7GpppN). Additional examples of 5'-CAP structures include glyceryl, inverted deoxy abasic moiety, 4',5' methylene nucleotide, 1-(beta-D-erythrofuranosyl) nucleotide, 4'-thio nucleotide, carbocyclic nucleotide, 1,5-anhydrohexitol nucleotide, L-nucleotide, alpha-nucleotide, modified base nucleotide, threo-pentofuranosyl nucleotide, acyclic 3',4'-seco nucleotide, acyclic 3,4-dihydroxybutyl nucleotide, acyclic 3,5 dihydroxypentyl nucleotide, 3'-3'-inverted nucleotide moiety, 3'-3'-inverted abasic moiety, A 3'-2'-inverted nucleotide moiety, a 3'-2'-inverted abasic moiety, 1,4-butanediol phosphate, 3'-phosphoramidate, hexylphosphate, aminohexyl phosphate, 3'-phosphate, 3'phosphorothioate, phosphorodithioate, or a bridging or non-bridging methylphosphonate moiety. These modified 5'-CAP structures can be used to modify the mRNA sequence of the nucleic acid construct of the present invention. Additional modified 5'-CAP structures that can be used in the present invention are CAP1 (additional methylation of the ribose of the adjacent nucleotide of m7GpppN), CAP2 (additional methylation of the ribose of the second nucleotide downstream of m7GpppN), CAP3 (additional methylation of the ribose of the third nucleotide downstream of m7GpppN), CAP4 (additional methylation of the ribose of the fourth nucleotide downstream of m7GpppN), ARCA (anti-reverse CAP analog), modified ARCA (e.g., phosphothioate modified ARCA), inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine and 2-azido-guanosine. In the present invention, the 5'-CAP structure may be formed by chemical RNA synthesis or in vitro transcription of RNA using a cCAP analogue (co-transcriptional capping), or the CAP structure may be formed in vitro using a capping enzyme (e.g., a commercially available capping kit). In the present invention, a CAP analogue refers to a non-polymerizable dinucleotide having a CAP function that promotes translation or localization and / or prevents degradation of an RNA molecule when introduced to the 5' end. Non-polymerizable means that the CAP analogue cannot be extended in the 3' direction by a template-dependent RNA polymerase because it is incorporated only at the 5' end, as it does not have a 5' triphosphate. CAP analogues are m7GpppA, m 7 GpppA mpG, unmethylated CAP analogues; dimethylated CAP analogues, trimethylated CAP analogues (e.g., m2,2,7GpppA), dimethylated symmetrical CAP analogues (e.g., m7Gpppm7A), or anti-inverted CAP analogues (e.g., ARCA; m7,2'OmeGpppA, m7,2'dGpppA, m7,3'OmeGpppA, m7,3'dGpppA and tetraphosphate derivatives thereof). Additional CAP analogues have been previously described (US7,074,596, WO2008 / 016473, WO2008 / 157688, WO2009 / 149253, WO2011 / 015347 and WO2013 / 059475). In the present invention, the 5'-CAP structure is an anti-inverted Cap analogue (m2 7,3'-O It may be characterized by being selected from the group consisting of G(5')ppp(5')G), but is not limited thereto. In the present invention, the coding region may be characterized by encoding at least one protein selected from the group consisting of an antigenic protein, an allergenic protein, a therapeutic protein, and a fragment, variant, or derivative of the protein, but is not limited thereto. In the present invention, the antigenic protein may be characterized by being at least one selected from the group consisting of tumor antigens, pathogenic antigens, autoantigens, alloantigens, and allergic antigens, but is not limited thereto. As used herein, the term "tumor antigen" refers to an antigenic (poly-)peptide or protein derived from or associated with a (preferably malignant) tumor or cancer disease. The terms "cancer" and "tumor" are used interchangeably herein to refer to a neoplasm characterized by uncontrolled, usually rapid proliferation of cells that tends to invade surrounding tissues and metastasize to distant sites in the body. The term encompasses benign and malignant neoplasms. Malignant tumors are typically characterized by anaplasia, invasiveness, and metastasis; benign malignant tumors typically lack these characteristics. The terms "cancer" and "tumor" specifically refer to neoplasms characterized by tumor growth, as well as cancers of the blood and lymphatic systems. A "tumor antigen" is typically derived from a tumor / cancer cell, preferably a mammalian tumor / cancer cell, and may be located within or on a tumor cell, e.g., a systemic or solid tumor, derived from a mammal, preferably a mammalian, preferably a human. "Tumor antigens" generally include tumor-specific antigens (TSAs) and tumor-associated antigens (TAAs). TSAs are typically due to tumor-specific mutations and are specifically expressed by tumor cells. TAAs, which are more common, are generally expressed by tumors and "normal" (healthy, non-tumor) cells. In the present invention, the antigen may be a protein or nucleic acid sequence associated with a tumor, each nucleic acid sequence encoding a different peptide or protein; And said at least one nucleic acid sequence is 5T4, 707-AP, 9D7, AFP, AlbZIP HPG1, alpha-5-beta-1-integrin, alpha-5-beta-6-integrin, alpha-actinin-4 / m, alpha-methylacyl-coenzyme A racemase, AT-4, ARTC1 / m, B7H4, BAGE-1, BCL-2, bcr / abl, beta-catenin / m, BING-4, BRCA1 / m, BRCA2 / m, CA 1 5-3 / CA 27-29, CA 19-9, CA72-4, CA125, calreticulin, CAMEL, CASP-8 / m, cathepsin B, cathepsin L, CD19, CD20, CD22, CD25, CDE30, CD33, CD4, CD52, CD55, CD56, CD80, CDC27 / m, CDK4 / m, CDKN2A / m, CEA, CLCA2, CML28, CML66, COA-1 / m, coactosin-like protein, collage XXIII, COX-2, CT-9 / BRD6, Cten, cyclin B1, cyclin D1, cyp-B, CYPB1, DAM-10, DAM-6, DEK-CAN, EFTUD2 / m, EGFR, ELF2 / m, EMMPRIN, EpCam, EphA2, EphA3, ErbB3, ETV6-AML1, EZH2, FGF-5, FN, Frau-1, G250, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE7b, GAGE-8, GDEP, GnT-V, gp100, GPC3, GPNMB / m, HAGE, HAST-2, hepsin, Her2 / neu, HERVK-MEL, HLA-A*0201 - R1 7I, HLA-A1 1 / m, HLA-A2 / m, HNE, homeobox NKX3.1, HOM-TES-14 / SCP-1, HOM-TES-85, HPV-E6, HPV-E7,HSP70-2M, HST-2, hTERT, iCE, IGF-1 R, IL-13Ra2, IL-2R, IL-5, immature laminin receptor, kallikrein-2, kallikrein-4, i67, KIAA0205, KIAA0205 / m, KK-LC- 1, K-Ras / m, LAGE-A1, LDLR-FUT, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-A10, MAGE-A12, MAGE-B1, MAGE-B2, MAGE-B3, MAGE-B4, MAGE-B5, MAGE-B6, MAGE-B10, MAGE-B1 6, MAGE-B1 7, MAGE-C1, MAGE-C2, MAGE-C3, MAGE- D1, MAGED2, MAGE-D4, MAGE-E1, MAGE-E2, MAGE-F1, MAGE-H I, MAGEL2, mammaglobin A, MART-1 / melan-A, MART-2, MART-2 / m, matrix protein 22, MC1 R, M-CSF, ME 1 / m, mesothelin, MG50 / PXDN, MMP1 1, MN / CA IX-antigen, MRP-3, MUC-1, MUC-2, MUM-1 / m, MUM-2 / m, MUM-3 / m, myosin class l / m, NA88-A, N-acetylglucosaminyltransferase- V, neo-PAP, neo-PAP / m, NFYC / m, NGEP, NMP22, NPM / ALK, N-Ras / m, NSE, NY-ESO-1, NY-ESOB, OA1, OFA-iLRP, OGT, OGT / m, OS-9, OS- 9 / m, osteocalcin, osteopontin, pi 5, p190 minor bcr-abl, p53, p53 / m, PAGE-4, PAI-1, PAI-2, PAP, PART-1, PATE, PDEF, Pim-1 -Kinase, Pin-1, Pml / PARalpha, POTE, PRAME, PRDX5 / m, prostein, proteinase-3, PSA, PSCA, PSGR, PSM,PSMA, PTPRK / m, RAGE-1, RBAF600 / m, RHAMM / CD1 68, RU1, RU2, S-100, SAGE, SART-1, SART-2, SART-3, SCC, SIRT2 / m, Sp1 7, SSX-1, SSX-2 / HOM-MEL-40, SSX-4, STAMP-1, STEAP-1, survivin, survivin-2B, SYT-SSX-1, SYT-SSX-2, TA-90, TAG-72, TARP, TEL-AML1, TGFbeta, TGFbetaRII, TGM-4, TPI / m, TRAG- 3, TRG, TRP-1, TRP-2 / 6b, TRP / INT2, TRP-p8, tyrosinase, UPA, VEGFR1, VEGFR-2 / FLK-1, WT1 and immunoglobulin genotype of lymphoid blood cells or T cell receptor genotype of lymphoid blood cells, or a homolog, fragment, variant or derivative of the above tumor antigen. In the present invention, the tumor antigen may be selected from the group consisting of NYESO-1, HER-2 / neu, MAGE-1, Tyrosinase, MUC1, CEA, Mam-A, hTERT, Syalyl-Tn, WT1, alpha-fetopotein, CA-125, gp-100, p53, Ras, Src, EGFRvIII, PSMA, GD2, Bcr-abl, Survivin, PSA, EphA2, PAP, AFP, EpCAM, ALK, Mesothelin, PSCA, MART-1, Melan-A, SCP-1, SPAG9, AKAP4, and OY-TES-1, but is not limited thereto. In the present invention, the pathogenic antigen may be characterized by being selected from the group consisting of bacterial, viral, fungal and protozoan antigens. In the present invention, the pathogenic antigen may be derived from influenza virus, respiratory syncytial virus (RSV), coronavirus, herpes simplex virus (HSV), human papillomavirus (HPV), human immunodeficiency virus (HIV), Plasmodium, Staphylococcus aureus, dengue virus, trachoma chlamydia, cytomegalovirus (CMV), hepatitis B virus (HBV), Mycobacterium tuberculosis, rabies virus, and yellow fever virus, or any isoform, homolog, fragment, variant, or derivative of these proteins. In the present invention, the viral antigen may be characterized as being a coronavirus, but is not limited thereto. In the present invention, the coronavirus may be human coronavirus 229E (HCoV-229E), human coronavirus OC43 (HCoV-OC43), severe acute respiratory syndrome coronavirus (SARS-CoV), human coronavirus NL63 (HCoV-NL63, New Haven coronavirus, human coronavirus HKU1, Middle East respiratory syndrome coronavirus (MERS-CoV), or severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2). In the present invention, the 3'-UTR is β-globin 3'UTR; CYBA 3'UTR; albumin 3'UTR; growth hormone (GH) 3'UTR; VEEV 3'UTR; hepatitis B virus (HBV) 3'UTR; α-globin 3'UTR; DEN 3'UTR; PAV barley yellow dwarf virus (BYDV-PAV) 3'UTR; elongation factor 1 α1 (EEF1A1) 3'UTR; manganese superoxide dismutase (MnSOD) 3'UTR; mitochondrial H(+)-ATP synthase β subunit (β-mRNA) 3'UTR; GLUT1 3'UTR; MEF2A 3'UTR; β-F1-ATPase 3'UTR; It may be characterized by being selected from the group consisting of a 3'UTR represented by the base sequence of SEQ ID NO: 13; a 3'UTR represented by the base sequence of SEQ ID NO: 14; a functional fragment thereof, and a combination thereof, but is not limited thereto. The term “3'-UTR” in the present invention typically refers to a portion of an mRNA located between the protein-coding region (i.e., open reading frame, coding region) of the mRNA and the poly(A) sequence. The 3'-UTR of an mRNA is not translated into an amino acid sequence. The 3'-UTR sequence is typically encoded by a gene, which is transcribed into each mRNA during the gene expression process. This genomic sequence is first transcribed into an immature mRNA containing an optional intron. The immature mRNA is then further processed into a mature mRNA during a maturation process. This maturation process includes steps such as 5'-capping, splicing of the immature mRNA to excise the optional intron, and modifications of the 3' terminus, such as polyadenylation of the 3' terminus of the immature mRNA, and optional endo- or exonuclease cleavage. In the present invention, the 3'-UTR corresponds to a sequence of a mature mRNA that is located 3' to the stop codon of the protein coding region, preferably immediately 3' to the stop codon of the protein coding region, and extends to the 5' side of the poly(A) sequence, preferably to the nucleotide immediately 5' to the poly(A) sequence. The term "corresponding" means that the 3'-UTR sequence can be an RNA sequence, such as in the mRNA sequence used to define the 3'-UTR sequence, or a DNA sequence corresponding to such an RNA sequence. In the present invention, the nucleic acid structure further comprises a poly(A) tail or a poly(A) tail-like sequence. In a further embodiment, a terminal group on the poly-A tail may be incorporated for stabilization. In another embodiment, the poly-A tail comprises a dec-3' hydroxyl tail. During RNA processing, a long chain of adenine nucleotides (a poly-A tail) may be added to a polynucleotide, such as an mRNA molecule, to increase its stability. Immediately after transcription, the 3' end of the transcript may be cleaved to release a 3' hydroxyl group. Poly-A polymerase then adds a chain of adenine nucleotides to the RNA. This process, called polyadenylation, adds a poly-A tail that may be, for example, about 80 to about 250 residues long (including about 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 residues long). The polyA tail can also be added after the construct has been exported from the nucleus. According to the present invention, a terminal group on the poly A tail may be incorporated for stabilization. The polynucleotides of the present invention may include a des-3' hydroxyl tail. They may also include structural moieties or 2'-Omethyl modifications, as taught by Junjie Li et al. (Current Biology, Vol. 15, 1501-1507, August 23, 2005, the contents of which are incorporated herein by reference in their entirety). The unique poly-A tail length provides certain advantages to the polynucleotides of the present invention. Typically, the poly-A tail length, if present, is greater than 30 nucleotides in length. In other embodiments, the poly-A tail is greater than 35 nucleotides in length (e.g., at least about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and greater than 3,000 nucleotides). In some embodiments, the polynucleotide or region thereof comprises from about 30 to about 3,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 750, from 30 to 1,000, from 30 to 1,500, from 30 to 2,000, from 30 to 2,500, from 50 to 100, from 50 to 250, from 50 to 500, from 50 to 750, from 50 to 1,000, from 50 to 1,500, from 50 to 2,000, from 50 to 2,500, from 50 to 3,000, from 100 to 500, from 100 to 750, from 100 to 1,000, 100 to 1,500, 100 to 2,000, 100 to 2,500, 100 to 3,000, 500 to 750, 500 to 1,000, 500 to 1,500, 500 to 2,000, 500 to 2,500, 500 to 3,000, 1,000 to 1,500, 1,000 to 2,000, 1,000 to 2,500, 1,000 to 3,000, 1,500 to 2,000, 1,500 to 2,500, 1,500 to 3,000, 2,000 to 3,000, 2,000 to 2,500, and 2,500 to 3,000). In some embodiments, the poly-A tail is designed for the entire length of the polynucleotide or for a specific region of the polynucleotide. This design may be based on the length of the coding region, the length of a specific feature or region, or the length of the ultimate product expressed from the polynucleotide. In this regard, the poly-A tail may be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% longer than the polynucleotide or its features. The poly-A tail may also be designed as a fraction of the polynucleotide to which it belongs. In this regard, the poly-A tail may be greater than or equal to 10, 20, 30, 40, 50, 60, 70, 80, or 90% of the total length of the construct, a region of the construct, or the total length of the construct minus the poly-A tail. Additionally, the engineered binding site and conjugation of the polynucleotide to a poly-A binding protein may enhance expression. Additionally, multiple distinct polynucleotides can be linked together via PABP (poly-A binding protein) at their 3'-ends using modified nucleotides at the 3'-end of the poly-A tail. Transfection experiments can be performed in appropriate cell lines, and protein production can be assayed by ELISA at 12, 24, 48, 72 hours, and 7 days after transfection. In some embodiments, the polynucleotides of the present invention are designed to include a polyAG quartet region. A G-quartet is a cyclic hydrogen-bonded sequence of four guanine nucleotides that can be formed by G-rich sequences in both DNA and RNA. In this experiment, a G-quartet is incorporated into the end of a poly-A tail. The resulting polynucleotide is tested for stability, protein production, and other parameters, including half-life at various time points. It was found that the polyAG quartet results in protein production from mRNA that is at least 75% equivalent to that obtained using a poly-A tail of 120 nucleotides alone. In the present invention, the poly (A) tail-like sequence may be used without limitation as long as it is a nucleic acid sequence capable of performing the function of a poly (A) tail, and is preferably characterized in that one or more nucleotides other than adenine selected from the group consisting of uracil (U), cytosine (C) and guanine (G) are inserted between a plurality of adenines or at the end of the poly (A) tail, but is not limited thereto. In the present invention, the nucleic acid structure may be characterized as being RNA. In the present invention, the RNA may be characterized by being selected from the group consisting of mRNA, viral RNA, self-replicating RNA, and replicon RNA, but is not limited thereto. In the present invention, the nucleic acid structure may be characterized by including, but is not limited to, one or more backbone-modified, sugar-modified, or base-modified nucleic acids. Party Variation: Modified nucleosides and nucleotides that may be incorporated into modified mRNA compounds comprising mRNA sequences as described herein may be modified at the sugar moiety. For example, the 2' hydroxyl group (OH) may be modified or replaced with a number of different "oxy" or "deoxy" substituents. Examples of "oxy"-2' hydroxyl group modifications include alkoxy or allyloxy (-OR, e.g., where R = H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar); polyethylene glycol (PEG), -O(CH2CH2O)nCH2CH2OR; "locked" nucleic acids (LNA), in which the 2' hydroxyl is linked to the 4' carbon of the same ribose sugar, e.g., by a methylene bridge; and amino groups (-O-amino, wherein the amino group, for example, NRR, may be alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, polyamino) or aminoalkoxy, but are not limited thereto. A "deoxy" modification includes hydrogen, amino (e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); or the amino group may be attached to the sugar via a linker, wherein the linker includes one or more of the atoms C, N, and O. The sugar group may also contain one or more carbons with the opposite stereochemical configuration compared to the corresponding carbons in ribose. Thus, the modified mRNA may include nucleotides containing, for example, arabinose as a sugar. Backbone Variants: The phosphate backbone can be further modified in modified nucleosides and nucleotides that can be incorporated into modified mRNA compounds comprising an mRNA sequence as described herein. The phosphate group of the backbone can be modified by replacing one or more of the oxygen atoms with another substituent. Furthermore, the modified nucleosides and nucleotides can include complete replacement of the unmodified phosphate moiety with a modified phosphate as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioates, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates, and phosphotriesters. Phosphorodithioates have both non-linked oxygens replaced by sulfur. Phosphate linkers can also be modified by substitution of the linking oxygen with nitrogen (bridged phosphoroamidate), sulfur (bridged phosphorothioate), and carbon (bridged methylene-phosphonate). Base modification: Modified nucleosides and nucleotides that can be incorporated into modified mRNA compounds comprising an mRNA sequence as described herein may be further modified at the nucleobase moiety. Examples of nucleobases found in mRNA include, but are not limited to, adenine, guanine, cytosine, and uracil. For example, the nucleosides and nucleotides described herein may be chemically modified at the major groove face. In some embodiments, the major groove chemical modification may include an amino group, a thiol group, an alkyl group, or a halo group. In a particularly preferred embodiment of the present invention, the nucleotide analogue / modification is preferably 2-amino-6-chloropurineriboside-5'-triphosphate, 2-aminopurine-riboside-5'-triphosphate; 2-Aminoadenosine-5'-triphosphate, 2'-amino-2'-deoxycytidine-triphosphate, 2-thiocytidine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 2'-fluorothymidine-5'-triphosphate, 2'-O-methyl-inosine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 5-aminoallylcytidine-5'-triphosphate, 5-aminoallyluridine-5'-triphosphate, 5-bromosytidine-5'-triphosphate, 5-bromouridine-5'-triphosphate, 5-bromo-2'-deoxycytidine-5'-triphosphate, 5-bromo-2'-deoxyuridine-5'-triphosphate, 5-Iodocytidine-5'-triphosphate, 5-iodo-2'-deoxycytidine-5'-triphosphate, 5-iodouridine-5'-triphosphate, 5-iodo-2'-deoxyuridine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, 5-methyluridine-5'-triphosphate, 5-propynyl-2'-deoxycytidine-5'-triphosphate, 5-propynyl-2'-deoxyuridine-5'-triphosphate, 6-azacytidine-5'-triphosphate, 6-azauridine-5'-triphosphate, 6-chloropurineriboside-5'-triphosphate, 7-deazaadenosine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 8-azaadenosine-5'-triphosphate, 8-azidoadenosine-5'-triphosphate, benzimidazole-riboside-5'-triphosphate, N1-methyladenosine-5'-triphosphate, N1-methylguanosine-5'-triphosphate, N6-methyladenosine-5'-triphosphate, O6-methylguanosine-5'-triphosphate, pseudouridine-5'-triphosphate, or puromycin-5'-triphosphate, xanthosine-5'-triphosphate. Particularly preferred are nucleotides for base modification selected from the group of base modified nucleotides consisting of 5-methylcytidine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 5-bromosytidine-5'-triphosphate, and pseudouridine-5'-triphosphate. In some embodiments, the modified nucleoside is pyridin-4-one.

[0356] Ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine. In some embodiments, the modified nucleoside is 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, Includes 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine and 4-methoxy-l-methyl-pseudoisocytidine. In another embodiment, the modified nucleoside is 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentyladenosine, N6-(cis-hydroxyisopentyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl Includes carbamoyl adenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine. In another embodiment, the modified nucleoside is inosine, 1-methyl-inosine, wiosine, wibutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine. In some embodiments, the nucleotide may be modified at the major groove face and may include replacement of the hydrogen at C-5 of uracil with a methyl group or a halo group. In certain embodiments, the modified nucleoside is 5'-O-(1-thiophosphate)-adenosine, 5'-O-(1-thiophosphate)-cytidine, 5'-O-(1-thiophosphate)-guanosine, 5'-O-(1-thiophosphate)-uridine, or 5'-O-(1-thiophosphate)-pseudouridine. In a further specific embodiment, the modified mRNA is selected from the group consisting of 6-aza-cytidine, 2-thio-cytidine, α-thio-cytidine, pseudo-iso-cytidine, 5-aminoallyl-uridine, 5-iodo-uridine, N1-methyl-pseudorudine, 5,6-dihydrouridine, α-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxy-thymidine, 5-methyl-uridine, pyrrolo-cytidine, inosine, α-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytidine, 8-oxo-guanosine, 7-deaza-guanosine, N1-methyl-adenosine, 2-amino-6-chloro-purine, N6-methyl-2-amino-purine, It may include a nucleoside modification selected from pseudo-iso-cytidine, 6-chloro-purine, N6-methyl-adenosine, α-thiothio-adenosine, 8-azido-adenosine, 7-deaza-adenosine. In the present invention, the base-modified nucleic acid may be characterized by being selected from the group consisting of pseudouridine (Ψ), N1-methyl-pseudouridine (N1-methylpseudouridine, m1Ψ), 5-methyl-uridine (5-methyluridine, m5U), 2-thio-uridine (2-thiouridine, s2U), 2'-O-methyl-uridine (2'-O-methyl-U, Um), 5-methyl-cytidine (5-methylcytidine, m5C), and 5-methoxy-uridine (5-methoxyuridine, 5moU), but is not limited thereto. In another aspect, the present invention relates to a vaccine composition comprising a nucleic acid structure. In the present invention, a "vaccine" is typically understood to be a prophylactic or therapeutic agent that provides at least one antigen, preferably an antigenic peptide or protein. "Providing at least an antigen" means, for example, that the vaccine comprises the antigen, or that the vaccine comprises, for example, a molecule encoding the antigen. Thus, it is particularly contemplated that the vaccine of the present invention comprises at least one synthetic nucleic acid (RNA) molecule encoding at least one antigenic (poly-)peptide or protein, as defined herein, which may be derived from, for example, tumor antigens, bacterial, viral, fungal, or protozoal antigens, autoantigens, allergens, or allogeneic antigens, and which preferably induces an immune response to the respective antigen when expressed and presented to the immune system. However, synthetic nucleic acid (RNA) molecules encoding non-antigenic (poly-)peptides or proteins of interest may also be used in the vaccine of the present invention. In the present invention, the nucleic acid structure may be characterized in that it is complexed with one or more lipids to form a lipid nanoparticle or liposome. In the present invention, the synthetic nucleic acid (RNA) molecule may be provided in a complexed, i.e., complexed or associated, form with one or more (poly-)cationic compounds, preferably (poly-)cationic polymers, (poly-)cationic peptides or proteins, such as protamine, (poly-)cationic polysaccharides and / or (poly-)cationic lipids. In this context, the term "complexed" or "associated" means an essentially stable combination of at least one synthetic nucleic acid (RNA) molecule into a larger complex or assembly without covalent bonding with said one or more compounds. Geology In a preferred embodiment, the synthetic nucleic acid (RNA) molecules of the present invention are complexed or associated with a lipid (particularly a cationic and / or neutral lipid) to form one or more lipid nanoparticles or liposomes. Thus, in some embodiments, the synthetic nucleic acid (RNA) molecules of the present invention may be provided in the form of a lipid-based formulation, particularly in the form of liposomes and / or lipid nanoparticles comprising the synthetic nucleic acid (RNA) molecules. lipid nanoparticles In some preferred embodiments, the synthetic nucleic acid (RNA) molecules of the present invention are complexed or associated with a lipid (particularly a cationic and / or neutral lipid) to form one or more lipid nanoparticles. Preferably, the lipid nanoparticle (LNP) may comprise: (a) at least one nucleic acid construct (RNA) of the invention, (b) a cationic lipid, (c) an aggregation reducing agent (e.g., a polyethylene glycol (PEG) lipid or a PEG-modified lipid), (d) optionally a non-cationic lipid (e.g., a neutral lipid), and (e) optionally, a sterol. In some embodiments, the LNP may comprise, in addition to at least one nucleic acid construct (RNA) of the invention, (i) at least one cationic lipid; (ii) a neutral lipid; (iii) a sterol, e.g., cholesterol; and a PEG-lipid, in a molar ratio of about 20-60% cationic lipid: 5-25% neutral lipid: 25-55% sterol; 0.5-15% PEG-lipid. In some embodiments, the nucleic acid construct (RNA) of the present invention may be formulated as an aminoalcohol lipidoid. Aminoalcohol lipidoids usable in the present invention may be prepared by the methods described in U.S. Patent No. 8,450,298, the entire disclosure of which is incorporated herein by reference. liposomes In some embodiments, the synthetic nucleic acid (RNA) molecules of the present invention are formulated as liposomes. Cationic lipid-based liposomes can form complexes with negatively charged nucleic acids (e.g., RNA) through electrostatic interactions, resulting in complexes that offer biocompatibility, low toxicity, and the potential for large-scale production for in vivo clinical applications. Liposomes can fuse with the plasma membrane for uptake; once inside the cell, the liposomes are processed via the phagocytosis pathway, and the nucleic acids are then released from the endosome / carrier into the cytoplasm. Liposomes have long been recognized as drug delivery vehicles due to their excellent biocompatibility, as they are essentially analogs of biological membranes and can be prepared from both natural and synthetic phospholipids. Liposomes typically consist of a lipid bilayer, which may be composed of cationic, anionic, or neutral (phospho)lipids and cholesterol, surrounding an aqueous core. Both the lipid bilayer and the aqueous space may contain hydrophobic or hydrophilic compounds, respectively. Liposomes may have one or more lipid membranes. Liposomes may be unilamellar, referred to as unilamellar, or multilamellar, referred to as multilamellar. In vivo, liposome properties and behavior can be modified by adding a hydrophilic polymer coating, such as polyethylene glycol (PEG), to the liposome surface to provide steric stability. Furthermore, liposomes can be used for specific targeting by attaching ligands (e.g., antibodies, peptides, and carbohydrates) to their surface or to the ends of the attached PEG chains. Liposomes typically exist as spherical vesicles and can range in size from 20 nm to several microns. Liposomes can be of different sizes, including, but not limited to, multilamellar vesicles (MLV), which can be hundreds of nanometers in diameter and contain a series of concentric bilayers separated by narrow aqueous compartments; small unicellular vesicles (SUV), which have a diameter less than 50 nm; and large unilamellar vesicles (LUV), which can have a diameter between 50 and 500 nm. Liposome designs can include, but are not limited to, opsonins or ligands to enhance liposome attachment to unhealthy tissue or to activate events such as, but not limited to, endocytosis. Liposomes can also contain a low or high pH to enhance the delivery of pharmaceutical formulations. In the present invention, the vaccine composition may be characterized by additionally comprising one or more adjuvants or activators. In the broadest sense, an "adjuvant" or "adjuvant component" is typically a pharmacological and / or immunological agent capable of modifying, for example, enhancing, the effects of another active agent, such as a therapeutic agent or vaccine. In this context, an "adjuvant" can be understood as any compound suitable for supporting the administration and delivery of the vaccine composition of the present invention. Specifically, an adjuvant can preferably enhance the immunostimulatory properties of the vaccine to which it is added. Furthermore, such adjuvants, without being bound to the vaccine, can initiate or enhance an immune response of the innate immune system, i.e., a non-specific immune response. "Adjuvants" typically do not induce an adaptive immune response. To date, "adjuvants" have not been qualified as antigens. That is, when administered, the vaccines of the present invention typically initiate an adaptive immune response due to antigenic peptides or proteins encoded by at least one coding sequence of a synthetic nucleic acid (RNA) molecule contained in the vaccine. Suitable adjuvants are known to the skilled person and may be selected from any adjuvant that is suitable in the present case, i.e. helps in inducing an immune response in a mammal, and may include, but are not limited to, TDM, MDP, muramyl dipeptide, pluronic, alum solution, aluminum hydroxide, ADJUMER™ (polyphosphazene); aluminum phosphate gel; glucan from algae; algamulin; aluminum hydroxide gel (alum); high protein-adsorbing aluminum hydroxide gel; low viscosity aluminum hydroxide gel; AF or SPT (emulsion of squalane (5%), Tween 80 (0.2%), Pluronic L121 (1.25%), phosphate-buffered saline, pH 7.4); or AVRIDINE™ (propanediamine). In another aspect, the present invention relates to the use of the vaccine composition for disease prevention. In the present invention, 'disease' means an abnormal phenomenon caused by a tumor antigen, bacteria, virus, fungal or protozoan antigen, autoantigen, allergen, or allogeneic antigen, and may include, but is not limited to, cancer, tumor, autoimmune disease, inflammatory disease, viral infection, bacterial infection, fungal infection, and protozoan infection. In the present invention, ‘prevention’ means any act of suppressing a disease or delaying its progression by administering the vaccine composition of the present invention. In another aspect, the present invention relates to a method for preventing a disease comprising a step of administering the vaccine composition. In another aspect, the present invention relates to the use of the vaccine composition for the manufacture of a medicament for the prevention of a disease. Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples. The experimental methods commonly used in the following examples are specifically as follows. Experiment-1: PCR for IVT template DNA preparation Template DNA for in vitro transcription (IVT) is obtained by PCR using a plasmid containing the EGFP or Spike ORF sequence and a primer containing the T7 promoter sequence using KAPA HIFI hotstart ready mix (Roche KK2601). After PCR, confirm the PCR template on a 1–1.5% agarose gel and purify the T7 template DNA for IVT using the Qiaquick PCR purification kit (Qiagen 28104). The IVT PCR template sequence for EGFP or Spike mRNA synthesis is as follows. Experiment 2: In vitro transcription In vitro transcription is the process of synthesizing mRNA or sdhRNA. For mRNA, react with prepared template DNA, T7 RNA polymerase, buffer, NTPs (natural, chemically modified, and other necessary components for IVT) at 37°C for 30 minutes (min) according to the manufacturer's suggested composition (HiScribe™ T7 ARCA mRNA Kit (with tailing), NEB E2060S). Once the reaction is complete, remove template DNA by treating with 1 U of Dnase I per 1 μg of DNA and incubating at 37°C for 15 minutes (min). After removing template DNA, poly A tailing reaction is additionally performed on the synthesized mRNA using E. coli ploy A polymerase at 37°C for 30 minutes (min). For sdhRNA, react with prepared template DNA, T7 RNA polymerase, buffer, NTPs (natural and chemically modified), and other necessary components for IVT according to the manufacturer's instructions at 37°C for 6 hours (hr) (MEGAscript™ T7 Transcription Kit, Invitrogen™ AM1334). After the reaction is complete, remove template DNA by treating with 1 U of Dnase I per 1 μg of DNA and incubating at 37°C for 15 minutes (min). After the reaction is complete, the IVT product is purified using the Monarch RNA Cleanup Kit (NEB 2040L) according to the manufacturer's instructions. The purified mRNA or sdhRNA is then quantified using Nanodrop or UV / Vis absorbance, and the band pattern is confirmed on an agarose gel or urea-PAGE gel. Experiment 3: sdhRNA 3' UTR cloning To insert the sdhRNA sequence into the 3' UTR of EGFP or Spike mRNA, a restriction enzyme is used to digest the plasmid containing the EGFP or Spike sequence, and then ligation with the sdhRNA containing the cloning site is used. For EGFP mRNA, BspEI and BglII are used, and for Spike mRNA, AflII and XbaI are used to digest the plasmid, and then the digested plasmid is purified using the Qiaquick gel extraction kit (Qiagen 28704). The purified digested plasmid and the sdhRNA containing the cloning site are ligated using T4 DNA ligase (NEB M0202S). After transformation and colony picking in the DH5α strain, the plasmid is extracted and Sanger sequenced to confirm that the sdhRNA cloning was successful. Afterwards, using the plasmid loaded with sdhRNA, template PCR (Experiment-1) and IVT (Experiment-2) were performed in the same manner to synthesize sdhRNA-loaded mRNA. Experiment 4: Western blotting Cells transfected with mRNA and sdhRNA are collected, lysed through sonication, and protein is quantified using Pierce™ BCA Protein Assay Reagent (Thermo fisher 23223) for BCA assay. After gel electrophoresis on a 10% SDS-PAGE gel, Amersham transfer is performed. Primary and secondary antibody reactions are then performed against the corresponding proteins, and finally, protein expression intensity is measured using Pierce™ ECL Western Blotting Substrate. The primary antibody information of the proteins measured in this development is as follows: PKR (Cell signaling technology 12297), pPKR (Abcam 81303), eIF2α (Cell signaling technology 5324), peIF2α (Cell signaling technology 9721), TUBB (Cell signaling technology 86298), PARP & Cleaved PARP (Cell signaling technology 9542), GFP (sigma Aldrich G1544), Spike protein (Genetex GTX632604). Experiment 5: RNA extraction and RT-qPCR Cells transfected with mRNA and sdhRNA are treated with Trizol (Ambion 15596018) and IPA precipitation, followed by DNase I (Takara 2270) treatment to remove any remaining DNA within the cells. Following EtOH precipitation, washing, and drying, the RNA is dissolved in TDW. cDNA is synthesized using the extracted RNA, Reverse Transcriptase (Thermo Fisher EP0442), buffer, and NTP. Real-time quantitative PCR is then performed using the SensiFAST SYBR Lo-Rox Kit (Bioline BIO-94020) to compare gene expression. Example 1: Confirming the importance of PRK regulation in protein expression induced by exogenous mRNA. In the present invention, we propose the introduction of sdhRNA to modulate the activity of the innate immune protein PKR using mRNA therapeutic / vaccine RNA construct element technology. To demonstrate the importance of PKR in the expression of antigens encoded by exogenous mRNA and in innate immune activation induced by exogenous RNA, we generated and utilized a PKR knockout (KO) cell line in which PKR was deleted using the CRISPR-Cas9 system. After EGFP mRNA (see Experiment 2) was administered to A549 lung cancer cell line (ATCC) and PKR KO A549 cells using lipofectamine 3000 transfection reagent (Invitrogen™ L3000015), the expression pattern of EGFP antigen and cell death were analyzed. As a result, we confirmed that PKR expression was completely eliminated in PKR KO cells through western blotting (see Experiment-4), and when EGFP mRNA was administered, PKR activation occurred only in A549 wildtype (WT) cells, and phosphorylation of eIF2α (peIF2α), a downstream factor of PKR activation, did not occur in PKR KO cells (Fig. 2A). In addition, we confirmed that the expression of EGFP protein expressed from EGFP mRNA increased in PKR KO cells through fluorescence microscopy observation and western blotting (see Experiment 4) in PKR KO cells (Fig. 2B), and when the cytotoxicity of mRNA administration was evaluated using the CCK8 assay kit (Dojindo CK04) according to the method suggested by the manufacturer, a much higher cell viability was confirmed in PKR KO cells, confirming the importance of PKR inhibition in mRNA therapy technology (Fig. 2C). Example 2: Confirmation of improved antigen expression and innate immune modulation effect of nc886-loaded EGFP mRNA. The nc886 sequence (SEQ ID NO: 9), known to inhibit the activity of the innate immune protein PKR, was incorporated into the 3' UTR of the EGFP nucleic acid construct, and a T7 promoter-attached PCR product was constructed using KAPA HIFI hotstart ready mix (Roche KK2601) to enable IVT (see Experiments 1 and 3). To verify whether the regulation of innate immune activity by nc886 is effective in the immune response to external mRNA, EGFP mRNA with the nc866 sequence incorporated into the 3' UTR was synthesized using the HiScribe™ T7 ARCA mRNA Kit (with tailing), (NEB E2060S) (see Experiment 2). EGFP mRNA was administered to A549 cells at a concentration of 400 ng / ml using lipofectamine 3000 transfection reagent (Invitrogen™ L3000015), and after 24 hours (hr), the expression of EGFP protein and innate immune response were analyzed through fluorescence microscopy, western blotting (see Experiment-4), and RT-qPCR (see Experiment-5). As a result, we compared experiments in which mRNA and nc886 RNA loaded with nc886 in the 3' UTR were synthesized separately with EGFP mRNA and then administered to cells separately. When nc886 RNA and EGFP mRNA were administered together, it was confirmed that antigen (EGFP) expression was significantly increased (Fig. 3A, B) and pPKR was suppressed (Fig. 3C). Example 3: Confirmation of the improved antigen expression and innate immune modulation effect of nc886 multiple-loaded EGFP mRNA. We did not observe any significant improvement in antigen expression or innate immune suppression in EGFP mRNA loaded with a single nc886 sequence. Therefore, to determine whether there is an improvement effect when multiple nc886s are loaded, we ligated three nc886 template DNAs and cloned them (see Experiment 3) to design an EGFP nucleic acid construct (EGFP-nc886X3) loaded with three repeating nc886 sequences in the 3' UTR. We confirmed that three nc886 sequences were loaded through Sanger sequencing and 1% agarose gel (Fig. 4A). Afterwards, EGFP-nc886 mRNA and EGFP-nc886X3 mRNA were synthesized through IVT (see Experiment 2), respectively, and then administered to A549 cells using lipofectamine 3000 transfection reagent (Invitrogen™ L3000015). Then, EGFP protein expression and innate immune response were compared through fluorescence microscopy, western blotting (see Experiment 4), and RT-qPCR (see Experiment 5) experiments. As a result, it was confirmed that when EGFP-nc886X3 mRNA was administered, PKR activity and downstream eIF2α phosphorylation were most reduced (Fig. 4B to D). Example 4: Development of immunomodulatory sdhRNA We synthesized sdhRNAs using a DNA library of 1,840 sdhRNA sequences with various hairpin structures that can bind to PKR (Kim K et al. A quantitative map of human primary microRNA processing sites. Mol Cell. 2021 Aug 34320405) as template DNA by IVT (see Experiment 2), and analyzed the binding affinity between them and PKR. To this end, Flag-PKR plasmid was transfected into HEK-293T cells (ATCC) using Fugene HD transfection reagent (Promega E2311) to express FLAG-PKR, and after cell lysis, immunoprecipitation was performed using Anti-FLAG M2 Affinity Gel (Sigma Aldrich A2220), and PKR protein was eluted and purified using Pierce™ 3x DYKDDDDK Peptide (Thermo Fisher A36805). PKR protein and hairpin RNA synthesized by IVT were incubated at 4°C for 2 hours (hr), and immunoprecipitation was performed again with PKR antibody (Cell signaling technology 12297) to secure PKR-RNA complex. In addition, after creating a sequencing library for the input RNA and PKR-RNA complex, RNA sequencing was performed using a NovaSeq6000 instrument. Afterwards, the sequencing reads were aligned using Hisat2, and the number of sequencing reads and fold change of input RNA and PKR-bound RNA were calculated using FeatureCounts and DESeq2 programs to analyze the binding affinity of each sdhRNA to PKR. As a result, sdhRNAs of sequence numbers 1 to 8 in Table 1 were obtained (Fig. 5). Among the sdhRNAs with various PKR binding affinities derived from the above screening, a total of 7 sdhRNAs were analyzed for their ability to inhibit PKR activity. To this end, sdhRNAs were pre-transfected into A549 cells at a concentration of 50 nM using lipofectamine 3000 transfection reagent (Invitrogen™ L3000015), and then 4 hours later, poly (I:C) (Sigma aldrich P9582), which can induce PKR activity, was administered at a concentration of 1000 ng / ml using lipofectamine 3000 transfection reagent (Invitrogen™ L3000015). 4 hours after poly (I:C) administration, cells were lysed by sonication, and PKR activation was confirmed through western blotting (see Experiment 4) (left panel of Fig. 6). nc886 RNA, which was reported to inhibit PKR activity, was used as a control. As a result, compared to nc886, a marked decrease in PKR activity was confirmed in sdhRNA_high 3, sdhRNA_mid_1, sdhRNA_mid_2, and sdhRNA_low 1 (Fig. 6, right panel). Additional repeat experiments were performed on mid_1 and mid_2 among the four secured sdhRNAs, and a marked decrease in PKR activity was confirmed in both sdhRNAs (Fig. 7). Example 5: Confirmation of improved antigen expression and innate immune modulation effect of EGFP mRNA equipped with sdhRNA structure. In Example 4, the mid_1 and mid_2 sdhRNA sequences secured were obtained and synthesized through cloning of the EGFP mRNA template (see Experiment 3). As a control, low 1 (sdhRNA_ctrl), which is expected not to bind to nc886 and PKR, was used (Fig. 8). Afterwards, EGFP-nc886 mRNA, EGFP-sdhRNA_mid_1 mRNA, EGFP-sdhRNA_mid_2 mRNA, and EGFP-sdhRNA_Ctrl mRNA were synthesized (see Experiment 2), respectively, and transfected into A549 cells at a concentration of 400 ng / ml using lipofectamine 3000 transfection reagent (Invitrogen™ L3000015) (Fig. 9, left panel). After 24 hours, the expression of EGFP protein and the innate immune response were analyzed through western blotting (see Experiment 4) and RT-qPCR (see Experiment 5). Western blotting results showed that PKR and eIF2a activities were most improved in EGFP mRNA loaded with PKR activity-inhibiting sdhRNA compared to EGFP-nc886 mRNA and EGFP mRNA loaded with ctrl sdhRNA (Fig. 9, right panel). In addition, RT-qPCR results showed that the expression of interferon beta and CXCL10, a representative ISG, was lowest in EGFP mRNA loaded with PKR activity-inhibiting sdhRNA (Fig. 10). When the expression of EGFP protein was confirmed through fluorescence microscopy and western blotting (see Experiment 4), it was confirmed that it was most strongly expressed in EGFP mRNA loaded with PKR activity-inhibiting sdhRNA than in EGFP-nc886 mRNA and EGFP mRNA loaded with ctrl sdhRNA (Figs. 11A to B). When cytotoxicity by mRNA administration was evaluated using the CCK8 assay kit (Dojindo CK04) according to the method suggested by the manufacturer, it was confirmed that EGFP mRNA loaded with PKR activity-inhibiting sdhRNA showed the least cytotoxicity (Fig. 11C). To determine whether these effects were due to changes in mRNA uptake efficiency and stability depending on the presence or absence of sdhRNA loading, lipofectamine 3000 transfection reagent (Invitrogen™ L3000015) was used to transfect A549 cells. The expression level of intracellular EGFP mRNA over time was then examined by RT-qPCR (see Experiment 5). As a result, it was confirmed that sdhRNA loading did not significantly affect the efficiency and stability of EGFP mRNA uptake into cells (Fig. 12). Example 6: Confirmation of the delivery effect of sdhRNA-loaded EGFP mRNA using lipid nanoparticles (LNPs). Since lipid nanoparticles, not lipofectamine, are used for actual animal / human administration, it was confirmed whether the innate immune control function similar to the results obtained in the above experimental example was shown when sdhRNA-loaded mRNA was administered into cells through LNP. The LNP used in this example is composed of (6Z,9Z,28Z,31Z)-Heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, 1,2-Distearoyl-sn-glycero-3-phosphocholine, Cholesterol, and 1,2-Dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 in a molar ratio of 50:10:38.5:1.5. EGFP mRNA and LNP loaded with sdhRNA structure were mixed at a mass ratio of 1:40 using ethanol mixing method, and then the buffer was changed to PBS to prepare a stable mRNA-LNP complex. After that, A549 was administered with mRNA concentration of 1000 ng / ml, and when the protein expression level of EGFP was compared through western blotting (see Experiment 4) after 24 hours, LNP-based delivery was more efficient than lipofectamine, and when cytotoxicity was analyzed using CCK8 assay kit (Dojindo CK04), it was confirmed that cytotoxicity was low. That is, as a result of analyzing PKR activity, it was confirmed that PKR activity was greatly reduced when LNP was used (Fig. 13B), and it was confirmed that PKR activity was almost non-existent and cell death was almost non-existent in EGFP mRNA loaded with PKR activity-inhibiting sdhRNA (Fig. 13C). Through this, it was confirmed that delivering sdhRNA-loaded mRNA to cells using LNP can enhance antigen expression while suppressing the innate immune response. Example 7: Analysis of the immune control effect by sdhRNA in mRNA introduced with pseudouridine. In the current mRNA vaccine technology, we analyzed the additional innate immune control effect of sdhRNA loaded in the 3' UTR of mRNA with pseudouridine-modified nucleic acids introduced. First, to analyze the effect of pseudouridine introduction on the binding affinity between sdhRNA and PKR, RNA in which uridine was substituted with pseudouridine (N1-Me-pUTP, GLPBIO GB20016) was synthesized for the eight types of sdhRNA used in the previous experimental examples (see Experiment 2). The synthesized sdhRNA was incubated with PKR protein at 4℃ for 2 hours (hr) and immunoprecipitation was performed with a PKR antibody (Cell signaling technology 12297) to obtain sdhRNA bound to PKR. After that, the binding affinity to PKR was analyzed by comparing the input RNA and PKR-bound RNA through RT-qPCR (see Experiment 5). For most sdhRNAs, it was confirmed that pseudouridine-introduced sdhRNAs also bind to PKR to a similar degree (Fig. 14). EGFP mRNA with PKR activity-inhibiting sdhRNA (sdhRNA_mid_2) introduced into the 3' UTR was synthesized by introducing pseudouridine and sdhRNA together (see Experiment 2). When the mRNA was administered at a concentration of 400 ng / ml to the A549 cell line using lipofectamine 3000 transfection reagent (Invitrogen™ L3000015), the effects on antigen expression and immune activity were analyzed. First, it was confirmed through fluorescence microscopy analysis and western blotting experiments (see Experiment 4) that pseudouridine introduction enhanced antigen expression of EGFP mRNA and inhibited PKR activity (Fig. 15 A, B). In addition, when sdhRNA was loaded into the 3' UTR of the mRNA using such pseudouridine-modified nucleic acid, further increased antigen expression and decreased PKR activity were confirmed. Furthermore, in the previous experimental example, it was confirmed that loading of sdhRNA leads to a decrease in cell death caused by mRNA. Through western blotting (see Experiment 4), it was confirmed that cleaved PARP, a major cell death marker, was the lowest when pseudouridine and sdhRNA-loaded mRNA were administered. When cytotoxicity analysis was performed using a CCK8 assay kit (Dojindo CK04), it was confirmed that this led to a high cell viability rate (Fig. 16). Example 8: Analysis of the effect of increasing antigen production by introducing sdhRNA into SARS-CoV-2 spike mRNA. In order to analyze the possibility of utilizing the PKR inhibitory effect of the developed sdhRNA in mRNA vaccines, the inventors used a plasmid capable of producing the SARS-CoV-2 virus spike protein, loaded nc886 as a control group and sdhRNA_mid_2, which showed the best effect as an experimental group, into the 3' UTR, and then synthesized Spike mRNA (see Experiments 1, 2, and 3). The synthesized Spike mRNA was administered to A549 cells at 1000 ng / ml using the TransIT-mRNA transfection kit (Mirus MIR2250), and Spike antigen expression and PKR activity were confirmed 24 hours later through western blotting (see Experiment 4). As a result, it was confirmed that when sdhRNA was introduced into spike mRNA, spike protein expression increased and PKR activity was inhibited (Fig. 17). Example 9: In vivo efficacy analysis of SARS-CoV-2 spike mRNA containing sdhRNA motifs. The inventors of the present invention encapsulated SARS-CoV-2 virus spike mRNA with the sdhRNA element installed in the 3' UTR into LNPs prepared using a NanoAssemblr Benchtop Instrument (Precision Nanosystems Inc) at a final LNP:RNA weight ratio of 10:1, and administered the resulting mRNA to a mouse model (Samtaco Korea) to evaluate immunogenicity. Specifically, Balb / c mice (4 weeks old, female) were administered two IM injections of 5 μg of mRNA vaccine at three-week intervals, and 2 weeks after the final vaccination, serum and spleen cells were obtained, and cellular immunogenicity and binding antibody titers were measured. In the mouse experiment, mRNA introduced with pseudouridine-modified nucleic acids was used, and nc886, mid_1, and mid_2 were utilized as sdhRNA. To evaluate whether the manufactured SARS-CoV-2 mRNA vaccine induces a cellular immune response, ELISpot was performed to measure T cells secreting virus-specific IFN-γ in the spleen cells of immunized mice. Specifically, the spleen of mice immunized with the SARS-CoV-2 universal mRNA vaccine was isolated in RPMI medium, and the spleen was crushed into single cells using a gentleMACS device. The spleen cells were counted using a LUNA cell counter device. The spleen cells were seeded at 2.5 x 10^5 cells / well in a 96-well plate in the Mouse IFN-γ ELISpot plus kit (XEL 485, R&D SYSTEMS), and the SARS-CoV-2 peptide was diluted to a final concentration of 1 μg / well and dispensed into the wells. After culturing the cell-stimulator mixture at 5% CO2 and 37℃ for 12 to 48 hours, the cell-stimulator mixture is removed and washed with PBS - incubated with primary antibody for 2 hours - washed with PBS - incubated with ALP-labeled secondary antibody for 1 hour - administered substrate solution (BCIP / NBT-plus) and reacted until spots appear - counting the generated spots using CTL automation equipment. As a result of analyzing the number of T cells secreting virus-specific IFN-γ, it was confirmed that sdhRNA_mid_2 showed a superior cellular immune response compared to the two controls (Fig. 18). To investigate whether the manufactured SARS-CoV-2 mRNA vaccine binds to the Spike antigen and generates antibodies, an ELISA was performed using the serum of immunized mice. The initial serum was serially diluted 3-fold from 1:100 for each individual using 3% skim milk in wells coated with 1 μg / 1 ml of SARS-CoV-2 Spike Protein (S1+S2 ECD, 40589-V08B1, Sino Biological), and the standard ELISA process (primary antibody reaction - washing - secondary antibody reaction - washing - TMB solution incubation and reaction) was performed. Finally, the reaction was terminated by adding stop solution (2 M H2SO4), and the OD value was measured at a wavelength of 450 nm using a microplate reader. As a result of analyzing the antibody binding to the Spike antigen, it was confirmed that sdhRNA_mid_2 showed a high binding antibody titer compared to the two controls (Fig. 19). The sdhRNA motif according to the present invention binds to protein kinase R (PKR), which recognizes RNA and is activated, and inhibits the activity of PKR. Therefore, when sdhRNA is injected into a cell together with an external RNA or inserted into the 3' UTR of mRNA, it has the effect of reducing PKR activation by RNA, alleviating apoptosis by RNA, and reducing the expression of interferon-stimulated genes (ISGs). In addition, it has the effect of improving the expression efficiency of proteins encoded by mRNA, and is therefore useful for various RNA-based applications, such as vaccines, in vivo / ex vivo gene therapy, etc. While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents. Electronic file attached.

Claims

1. A short duplex hairpin nucleic acid molecule (sdhNA) represented by any one of the base sequences of sequence numbers 1 to 8 and which suppresses an innate immune response. 2.5' to 3' in order a) one or more coding areas; b) 3'-untranslated region (3'-UTR); c) a short duplex hairpin nucleic acid molecule of claim 1; and d) 10 to 1000 poly(A) tail or poly(A) tail-like sequences; A nucleic acid structure comprising:

3. A nucleic acid construct according to claim 2, characterized in that it further comprises a 5'-CAP structure and a 5'-UTR polynucleotide 5' upstream of at least one coding region.

4. In the third paragraph, the 5'-CAP structure is m 7 GpppA m pG, m7,3'OmeApppG, m7GpppA and m2 7,3'-O A nucleic acid structure characterized by being selected from the group consisting of G(5')ppp(5')G (ARCA).

5. A nucleic acid structure according to claim 3, wherein the 5'-UTR polynucleotide is selected from the group consisting of α-globin 5'UTR; Hsp70 5'UTR; axon dynein heavy chain 2 (DNAH2) 5'UTR; hydroxysteroid dehydrogenase (3β-HSD) 5'UTR; a 5'-UTR represented by the base sequence of SEQ ID NO: 11; and a 5'-UTR represented by the base sequence of SEQ ID NO:

12.

6. A nucleic acid structure according to claim 2, wherein the coding region encodes at least one protein selected from the group consisting of an antigenic protein, an allergenic protein, a therapeutic protein, and a fragment, variant or derivative of the protein.

7. A nucleic acid structure according to claim 6, characterized in that the antigenic protein is at least one selected from the group consisting of a tumor antigen, a pathogenic antigen, an autoantigen, an alloantigen, and an allergic antigen.

8. A nucleic acid structure according to claim 7, characterized in that the antigen is selected from the group consisting of EGFP and Spike proteins.

9. A nucleic acid structure according to claim 7, characterized in that the pathogenic antigen is selected from the group consisting of bacterial, viral, fungal and protozoan antigens.

10. A nucleic acid structure according to claim 9, characterized in that the virus is a coronavirus.

11. A nucleic acid structure in claim 1, wherein the poly (A) tail-like sequence is characterized in that at least one nucleotide other than adenine, selected from the group consisting of uracil (U), cytosine (C), and guanine (G), is inserted between a plurality of adenines or at the end of the poly (A) tail.

12. A short duplex hairpin nucleic acid molecule according to claim 1, characterized in that the short duplex hairpin nucleic acid molecule is RNA.

13. A nucleic acid structure according to claim 2, characterized in that the nucleic acid structure is selected from the group consisting of mRNA, viral RNA, self-replicating RNA, and replicon RNA.

14. A nucleic acid structure according to claim 2, characterized in that the nucleic acid structure comprises one or more backbone-modified, sugar-modified or base-modified nucleic acids.

15. A nucleic acid structure according to claim 14, characterized in that the base-modified nucleic acid is selected from the group consisting of pseudouridine (Ψ), N1-methylpseudouridine (m1Ψ), 5-methyluridine (m5U), 2-thiouridine (s2U), 2'-O-methyl-uridine (2'-O-methyl-U, Um), 5-methylcytidine (m5C), and 5-methoxy-uridine (5moU).

16. In the second paragraph, the 3'-UTRβ-globin 3'UTR; CYBA 3'UTR; albumin 3'UTR; growth hormone (GH) 3'UTR; VEEV 3'UTR; hepatitis B virus (HBV) 3'UTR; α-globin 3'UTR; DEN 3'UTR; PAV barley yellow dwarf virus (BYDV-PAV) 3'UTR; elongation factor 1 α1 (EEF1A1) 3'UTR; manganese superoxide dismutase (MnSOD) 3'UTR; mitochondrial H(+)-ATP synthase β subunit (β-mRNA) 3'UTR; GLUT1 3'UTR; MEF2A 3'UTR; β-F1-ATPase 3'UTR; A nucleic acid structure characterized by being selected from the group consisting of a 3'UTR represented by the base sequence of SEQ ID NO: 13; and a 3'UTR represented by the base sequence of SEQ ID NO:

14.

17. A vaccine composition comprising a nucleic acid structure according to any one of claims 2 to 16.

18. A vaccine composition according to claim 17, characterized in that the nucleic acid structure is complexed with one or more lipids to form lipid nanoparticles or liposomes.

19. A vaccine composition according to claim 18, characterized in that the vaccine composition additionally comprises one or more adjuvants or activators.

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