5'-UTR-containing mRNA structure with improved translation efficiency and vaccine composition containing the same

Abstract

The present invention relates to an mRNA structure containing 5'-UTR with improved translation efficiency and a vaccine composition containing the same. More specifically, it relates to an mRNA structure containing a 5'-UTR with improved translation efficiency manufactured with a specific motif, a codon-optimized signal sequence, and an antigen-encrypting sequence, and a vaccine composition containing the same. The mRNA structure according to the present invention contains a 5'-UTR polynucleotide with improved translation efficiency, which can effectively induce the expression of antigenic polypeptides, thereby increasing the immunogenicity of the vaccine and making it useful in vaccine development.

Description

[Technical Field]

[0001] The present invention relates to an mRNA structure containing a 5'-UTR with improved translation efficiency and a vaccine composition containing the same. More specifically, it relates to an mRNA structure containing a 5'-UTR manufactured with a specific motif to improve translation efficiency, a codon-optimized signal sequence, and an antigen-encrypting sequence, and a vaccine composition containing the same. [Background technology]

[0002] Vaccines began to be used in the 1500s, and the first specific report to the academic community was in a 1798 paper related to the smallpox vaccine. Since then, a wide variety of vaccines for numerous diseases have been developed and are used for disease prevention (Pollard, AJ et al., Nat Rev Immunol Vol.21, pp.83-100, 2021).

[0003] Vaccines can be classified into several types, including live vaccines (live bacteria, live toxins), inactivated vaccines (dead bacteria, dead toxins), toxoid vaccines, subunit vaccines, VLP vaccines, other membrane-based vaccines, protein-polysaccharide conjugate vaccines, viral vector-based vaccines, and gene vaccines.

[0004] Live vaccines were first used for smallpox, toxoid vaccines for diphtheria, subunit vaccines for anthrax, VLP vaccines for hepatitis B, other membrane-based vaccines for Glucos birachis vaccine, protein-polysaccharide conjugate vaccines for influenza type B vaccine, viral vector-based vaccines for Ebola vaccine, and gene vaccines for SARS-CoV-2 vaccine (Pollard, AJ et al., Nat Rev Immunol Vol.21, pp.83-100, 2021).

[0005] On the other hand, influenza virus (IV) is a single-stranded, negative-sense RNA virus composed of eight RNA segments expressing 10 to 13 different proteins, and belongs to the Orthomyxoviridae family. It is classified into types A, B, and C based on the antigenicity caused by the nuclear antigen (NP) and matrix (M) proteins, which are structural proteins attached to the surface of the virus (Lamb RA et al., Fields Virology, pp. 1487-1531, 2001). In humans, mainly types A and B are known to be pathogenic. Type A can infect not only humans but also pigs and birds. Humans are the sole host for type B.

[0006] Influenza viruses are composed of two surface glycoproteins (surface antigens): hemagglutinin and neuraminidase. Influenza viruses are classified into subtypes based on the type and combination of hemagglutinin (an antigenic projection that binds to a receptor (sialic acid) on the host cell) and neuraminidase (which cleaves sialic acid to release the virus into the cell). While these subtypes are mainly based on influenza A, currently 18 types of hemagglutinin (H1-H18) and 11 types of neuraminidase (N1-N11) have been discovered, and theoretically, 198 subtypes of influenza A can be created by combining these. Influenza B viruses are divided into two lineages, Victoria and Yamagata, based on their antigenic type.

[0007] Influenza viruses, as is typical of RNA viruses, exhibit antigenic mutations that occur almost every year, sometimes gradually and sometimes drastically. Continuous antigenic drift occurs almost every year, primarily in influenza A and B, and is the cause of seasonal influenza epidemics. Therefore, the World Health Organization (WHO) analyzes virus epidemic information and announces the recommended strains for the seasonal vaccine around February each year. Continuous antigenic drift (antigenic drift) refers to point mutations within the same subtype, where slight antigenic changes result in the replacement of the virus with a new hemagglutinin or neuraminidase. Antigenic shift (antigenic shift) is the creation of a new virus, such as H3N2 → H2N2, where the subtype changes. Besides these two types of mutations, mutations can also occur when culturing in embryonated eggs, leading to differences in antigenicity. However, such mutations have not been discovered in cell cultures, and vaccines using viruses cultured in mammalian cells are preferred.

[0008] Influenza vaccines must be administered annually because the duration of their effectiveness is less than one year, during which antigenic continuous variation occurs. In the Northern Hemisphere, the recommended vaccination period is set for October to December each year, taking into account the influenza season (December to April of the following year) and the duration of vaccination effectiveness (average 6 months).

[0009] Current influenza vaccines include inactivated dead vaccines and live attenuated influenza vaccines (LAIVs). Inactivated dead vaccines are produced by inactivating viruses cultured in fertilized eggs (hatched eggs) with formalin, or by growing the virus in cell cultures, inactivating it with formalin, harvesting the surface antigen, and injecting it intramuscularly (im) as the vaccine antigen. Live vaccines are administered by spraying them into the nasal cavity.

[0010] Infectious disease vaccines include whole virus vaccines, which use the entire virus; split vaccines (subvirons), which are manufactured by desorbing the viral envelope using ether or similar agents; and subunit vaccines, which are purified hemagglutinin and neuraminidase components. Hemagglutinin and neuraminidase are antigens that directly induce neutralizing antibody reactions, with hemagglutinin being the primary neutralizing antigen. Whole virus vaccines cause side effects in children and are therefore not widely used domestically or globally, and are currently only used in a limited number of countries. On the other hand, component vaccines such as split vaccines and subunit vaccines are very safe, have proven effectiveness, and are the most widely used.

[0011] In addition, vaccines containing immune enhancers (adjuvants) such as MF-59, and virosome vaccines that form vesicles similar in form to the virus, have been developed and are being used in some countries. In particular, to provide a sufficient degree of immune protection against influenza virus infection for preventive or therapeutic purposes, it is necessary to include a potent and safe adjuvant in the vaccine.

[0012] Antibodies obtained through natural infection or vaccination against specific subtypes or influenza viruses often fail to form protective antibodies against other types or subtypes of influenza viruses, and they also lack sufficient immunogenicity against new variants within a single antigen.

[0013] Because influenza viruses undergo minor and major mutations every year, the circulating strain changes annually. Consequently, it is difficult to rely on the protective effect of the vaccine administered the previous year, making annual vaccination necessary.

[0014] Currently used seasonal influenza vaccines provide protective immunity only against the virus strain used in the vaccine; therefore, there is a need for the development of an economical and effective influenza vaccine that can provide sufficient cross-protective immunity across a variety of subtypes. To develop a general-purpose vaccine with cross-immunity, it is necessary to use an antigen with minimal antigenic variation or a method that stimulates mucosal immunity. In some cases, one or more HA2 domains of HA with low antigenic variation are used as the vaccine antigen (Korean Registered Patent 10-1637955).

[0015] On the other hand, gene vaccines were initially developed after it was reported that directly injecting DNA and RNA encoding a target gene into an animal would cause the target gene to be expressed in the living animal, and that this expression could enable immunity (Wolff JA et al., Science, 247:1465-8, 1990).

[0016] In genetic vaccination, both DNA and RNA can be used as nucleic acid molecules for gene delivery, with DNA being known to be relatively more stable and easier to handle than RNA. However, with DNA, a potential risk arises if the DNA section delivered into the patient's genetic material is inserted at an undesirable location, damaging the gene. Additionally, undesirable anti-DNA antibodies may be expressed. Another problem is the limited expression levels of peptides or proteins expressed by DNA delivery and subsequent transcription / translation. The presence or absence of specific gene transfer factors that regulate DNA transcription significantly affects the expression level of the delivered DNA. In the absence of these factors, insufficient amounts of RNA are produced by DNA transcription, and consequently, the levels of peptides or proteins produced by translation are also limited.

[0017] On the other hand, when RNA is used as a tool for gene delivery, it does not require transcription or, like DNA, must enter the nucleus. It can immediately synthesize proteins in the cytoplasm, eliminating concerns about it entering cell chromosomes and causing undesirable gene damage. Furthermore, it has a shorter half-life than DNA and does not induce long-term gene deformation (Sayour EH, et al., J Immunother Cancer Vol.3, 13, 2015). Generally, when RNA vaccines are delivered into cells, they are activated for a short period, causing the target protein to be expressed. Within a few days, they are destroyed by enzymatic reactions, leaving a specific immune response to the expressed target antigen (protein).

[0018] Furthermore, when RNA is used as a tool for gene delivery, it acts by passing only through the cell membrane without needing to cross the nuclear membrane, allowing for the expression of the same amount of target protein as DNA, even with a smaller amount. Additionally, RNA itself possesses immunogenicity, meaning that administering smaller amounts compared to DNA can produce the same immune effect. Using RNA instead of DNA for gene vaccination minimizes or prevents the risk of undesirable genome integration and the production of anti-DNA antibodies. However, RNA is considered a highly unstable molecular species that can be easily degraded by ubiquitously distributed RNases.

[0019] While much progress has been made in recent years, inefficient methods for mRNA vaccination that can induce adaptive immune responses still remain in this field. These include premature degradation of antigens or inefficient translation of mRNA due to inefficient release of mRNA in cells. Furthermore, there is a pressing need to reduce the volume of mRNA vaccines to mitigate potential safety concerns and to ensure that vaccines can be tolerated in the developing world.

[0020] There are many problems associated with nucleic acid delivery in order to elicit the desired reaction in biological systems. Nucleic acid-based therapeutics such as vaccines have great potential, but in order to realize this potential, there remains a need to more effectively deliver nucleic acids to appropriate sites within cells or organisms.

[0021] However, the use of nucleic acids for therapeutic and prophylactic purposes currently faces two problems. One, free RNA is vulnerable to nuclease digestion in plasma. Two, free RNA has limited ability to access intracellular compartments where the associated translation machinery resides. Lipid nanoparticles formed from another lipid component such as neutral lipids, cholesterol, PEG, pegylated lipids and oligonucleotides and cationic lipids have been tried to block the degradation of RNA in plasma and promote the cellular uptake of nucleic acids.

[0022] Both, mRNA vaccines containing several lipid nanoparticles have been reported in relation to influenza vaccines (Korean Patent 10-2018-0096591).

[0023] Therefore, the inventors have made intensive efforts to solve the above problems and develop an mRNA construct containing a 5'-UTR with improved translation efficiency. As a result, when selecting an artificial nucleic acid molecule that does not generate a secondary structure, has few uridines, and does not contain a sequence that reduces stability in a 30bp-length artificial nucleic acid molecule combination, not only is a 5'-UTR with improved translation efficiency obtained, but it was confirmed that the performance of the mRNA construct containing this as a vaccine is very excellent, and the present invention was completed.

Summary of the Invention

Problems to be Solved by the Invention

[0024] An object of the present invention is to provide an mRNA construct encoding an antigenic polypeptide or an immunogenic fragment thereof.

[0025] Another object of the present invention is to provide a vaccine composition containing the above mRNA construct.

Means for Solving the Problem

[0026] To achieve the above object, the present invention provides an mRNA construct encoding an antigenic polypeptide or an immunogenic fragment thereof, which comprises, in order from 5' to 3': a) a 5'-CAP structure; b) a 5'-untranslated region (5'-UTR) polypeptide represented by a nucleic acid sequence according to the following chemical formula (I) or (II): Chemical formula (I): AG[N

[0029] , , [Figure 1] ,

[0031] , , , , , , , ,

[0030] , GCCACC, Chemical formula (II): AGGA[N 19 RGCCACC, where R represents A, G or U; c) a polypeptide encoding a signal peptide; d) a polynucleotide encoding an antigenic polypeptide or an immunogenic protein thereof; e) a 3'-untranslated region (3'-UTR); and f) a poly(A) tail or a poly(A) tail-like sequence consisting of 10 to 1000 adenines (A). The present invention also provides an influenza vaccine composition comprising the above mRNA construct.

[0027] The present invention also provides the use of the above vaccine composition for preventing influenza.

[0028] The present invention also provides a method for preventing influenza, which comprises administering the above vaccine composition.

[0029] The present invention also provides the use of the above vaccine composition for the manufacture of a medicament for preventing influenza.

[0030]

Brief Description of the Drawings

[0031] [Figure 1]This diagram illustrates the components of an mRNA structure according to one embodiment of the present invention. [Figure 2] This is a schematic diagram showing the process of an animal experiment according to one embodiment of the present invention. [Figure 3] This is the result of confirming the protein expression level of influenza B / Yamagata HA mRNA structure produced according to one embodiment of the present invention within the muscles of mice. [Figure 4] This is the result of confirming the protein expression level of influenza B / Victoria HA mRNA structures produced according to one embodiment of the present invention within mouse muscles. [Figure 5] This is the result of confirming the protein expression level of influenza A / H3N2 HA mRNA structure produced according to one embodiment of the present invention within mouse muscle. [Figure 6] This is the result of confirming the protein expression level of influenza A / H1N1 HA mRNA structure produced according to one embodiment of the present invention within mouse muscle. [Figure 7] This is a schematic diagram showing the in vivo immunogenicity experiment process according to one embodiment of the present invention. [Figure 8] This is the result of confirming the anti-HA specific IgG titer of the influenza A / H1N1 HA mRNA structure produced according to one embodiment of the present invention. [Modes for carrying out the invention]

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by experts skilled in the art to which this invention pertains. The nomenclature used herein and the experimental methods described below are generally well known and commonly used in the art.

[0033] In this invention, we aimed to confirm that when using an mRNA structure containing a 5'-UTR with improved translation efficiency, influenza antigenic protein can be expressed with higher efficiency than wild-type mRNA.

[0034] In other words, in one embodiment of the present invention, it was confirmed that the expression efficiency of influenza antigenic protein is dramatically improved when an mRNA structure containing a 5'-UTR polynucleotide with improved translation efficiency (Figure 1) is used (Figures 3 to 6).

[0035] Therefore, in one view, the present invention is As an antigenic polypeptide or mRNA structure encoding this immunogenic fragment, From 5' to 3' in order a) 5'-CAP structure; b) 5'-untranslated region (5'-UTR) polynucleotide represented by the nucleic acid sequence of the following chemical formula (I) or (II): Chemical formula (I):AG[N 22 ]GCCACC, Chemical formula (II):AGGA[N 19 RGCCACC, Here, R means A, G, or U; c) Polynucleotides encoding signal peptides; d) Influenza antigenic polypeptides or polynucleotides encoding this immunogenic protein; e) 3'-untranslated region (3'-UTR); and f) A poly(A) tail or poly(A) tail-like sequence consisting of 10 to 1000 adenine (A) units; This concerns mRNA structures that include [specific element].

[0036] The 5'-CAP of natural mRNA increases mRNA stability upon nuclear efflux and binds to mRNA cap-binding protein (CBP), which leads to mRNA stability at the cellular and translational stages through association of CBP with poly(A)-binding protein to form a mature cyclic mRNA species. The cap further assists in the removal of 5' proximal introns during mRNA splicing.

[0037] In this invention, 5'-CAP is typically a modified nucleotide (CAP analogue), particularly a guanine nucleotide added to the 5' end of an mRNA molecule. Preferably, 5'-CAP is added using a 5'-5'-triphosphate linkage (named m7GpppN). Additional examples of 5'-CAP structures include glyceryl, inverted deoxy-abasic residue (moyati), 4',5' methylene nucleotide, 1-(beta-D-erythropuranosyl) nucleotide, 4'-thionucleotide, 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 The material comprises a rheotide, an acyclic 3,5-dihydroxypentyl nucleotide, a 3'-3'-reversed nucleotide moisture, a 3'-3'-reversed non-basic moisture, a 3'-2'-reversed nucleotide moisture, a 3'-2'-reversed non-basic moisture, a 1,4-butanediol phosphate, a 3'-phosphoramidite, a hexyl phosphate, an aminohexyl phosphate, a 3'-phosphate, a 3'-phosphorothioate, a phosphorodithioate, or a bridging or non-bridging methylphosphonate moisture.

[0038] These modified 5'-CAP structures can be used to deform the mRNA sequence of the synthetic nucleic acid molecule of the present invention.

[0039] Additional modified 5'-CAP structures that can be used in the present invention are CAP1 (additional methylation of ribose at the adjacent nucleotide of m7GpppN), CAP2 (additional methylation of ribose at the second downstream nucleotide of m7GpppN), CAP3 (additional methylation of ribose at the third downstream nucleotide of m7GpppN), CAP4 (additional methylation of ribose at the fourth downstream nucleotide of m7GpppN), ARCA (anti-reverse CAP analogue), 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.

[0040] In this invention, the 5'-CAP structure can be formed by chemical RNA synthesis or RNA in vitro transmission (co-transmission capping) using a cCAP analog, or the CAP structure can be formed in vitro using a capping enzyme (e.g., a commercially available capping kit).

[0041] In this invention, a CAP analog refers to a non-polymerizable dinucleotide that, when introduced to the 5' end of an RNA molecule, has a CAP function that promotes translation or localization and / or prevents the degradation of the RNA molecule. Non-polymerizable means that because the CAP analog cannot have a 5' triphosphate, it is bound only at the 5' end and therefore cannot be extended in the 3' direction by template-dependent RNA polymerases.

[0042] CAP analogues are m7GpppA, m 7 GpppA mThe chemical structures include, but are not limited to, those selected from the group consisting of pG, unmethylated CAP analogs; dimethylated CAP analogs, trimethylated CAP analogs (e.g., m2, 2, 7GpppA), dimethylated symmetric CAP analogs (e.g., m7Gpppm7A), or anti-reverse CAP analogs (e.g., ARCA; m7, 2'PmeGpppA, m7, 2'dGpppA, m7, 3'OmeGpppA, m7, 3'dGpppA, and their tetraphosphate derivatives).

[0043] Additional CAP analogues have been described previously (US7, 074, 596, WO2008 / 016473, WO2008 / 157688, WO2009 / 149253, WO2011 / 015347, and WO2013 / 059475).

[0044] In the present invention, the above 5'-CAP structure is m 7 GpppA m The selection can be characterized by being chosen from the group consisting of pG, m7, 3'OmeApppG, and m7GpppA, but is not limited to this.

[0045] In the present invention, the 5'-UTR may be one of the base sequences represented by SEQ ID NOs: 1 to 34 shown in Table 1 below, and more preferably one selected from the group consisting of SEQ ID NOs: 1, 2, 15, 29, and 31, but is not limited thereto.

[0046] [Table 1]

[0047] In this invention, the term "UTR" means an "untranslated region" located upstream (5') and / or downstream (3') of the coding region of a nucleic acid molecule described herein, thereby typically on the side of the coding region. Accordingly, the term "UTR" generally includes the 3' untranslated region ("3'-UTR") and the 5'-untranslated region ("5'-UTR"). A UTR can typically contain or consist of nucleic acid sequences that are not translated into proteins. Typically, a UTR contains "regulatory elements".

[0048] The term “regulatory element” refers to a nucleic acid sequence that has the ability to influence gene regulatory activity, specifically the expression of a transmissible nucleic acid sequence that is operablely linked (cis or trans), particularly its transduction or translation. The above term includes promoters, enhancers, internal ribosome entry sites (IRESs), introns, leaders, transduction termination signals, such as polyadenylation signals and poly-U sequences, and other expression regulatory elements. Regulatory elements can act constitutively or in a time- and / or cell-specific manner. Selectively, regulatory elements can exert their function through interactions (e.g., recruitment and binding) with regulatory proteins that can regulate (induce, enhance, reduce, discard, or prevent) gene expression, particularly gene transduction.

[0049] The UTR is preferably "operably linked" to the coding region, i.e., arranged in a functional relationship, to control (i.e., arbitrate or modulate, preferably enhance) the expression of the coding sequence.

[0050] In this invention, the term "5'-UTR" refers to a portion of a nucleic acid molecule located at 5' (i.e., "upstream") of the open reading frame, which is not translated into protein. In the context of this invention, the 5'-UTR begins at the propagation start site and terminates one nucleotide before the start codon of the open reading frame.

[0051] The 5'-UTR may contain elements that regulate gene expression, which are called "regulatory elements." Such regulatory elements may be, for example, ribosome binding sites. The 5'-UTR can be deformed after propagation, for example by the addition of a 5'-CAP. Therefore, the 5'-UTR may correspond to a nucleic acid located between the 5'-CAP and the start codon, particularly the sequence of mature mRNA, and more specifically, a nucleotide located 3' to the 5'-CAP, preferably a nucleotide located 3' immediately after the 5'-CAP, to a nucleotide located 5' to the start codon (propagation initiation site) of the proteincoding sequence, preferably a nucleotide located 5' immediately before the start codon (propagation initiation site) of the proteincoding sequence.

[0052] The nucleotide located immediately 3' from the 5'-CAP of mature mRNA typically corresponds to the propagation start site. The length of the 5'UTR generally has 500, 400, 300, fewer than 250, or fewer than 200 nucleotides. In some examples, this length can be in the range of 10, 20, 30, or 40 or more nucleotides, preferably 10 or 50 or fewer.

[0053] In the present invention, the signal peptide may be derived from an antigenic polypeptide, immunoglobuline E (IgE), or tissue plasminogen activator (tPA), but is not limited thereto.

[0054] In the present invention, the signal peptide of the antigenic polypeptide can be characterized by being represented by the amino acid sequence of SEQ ID NO: 72 (MKAIIVLLMVVTSNA), SEQ ID NO: 73 (MKXIIALSXILCLVFA, where X is T, A, Y, or N), SEQ ID NO: 77 (MKAILVVXLYTFTTANA, where X is L or M), and more preferably by being represented by the amino acid sequence of SEQ ID NO: 72 (MKAIIVLLMVVTSNA), SEQ ID NO: 74 (MKTIIALSYILCLVFA), SEQ ID NO: 75 (MKTIIALSNILCLVFA), SEQ ID NO: 76 (MKAIIALSNIKCLVFA), SEQ ID NO: 78 (MKAILVVMLYTFTTANA), or SEQ ID NO: 79 (MKAILVVLLYTFTTANA), but is not limited thereto.

[0055] In the present invention, the IgE signal peptide can be characterized by being represented by the amino acid sequence of SEQ ID NO: 80, but is not limited thereto.

[0056] In the present invention, the signal peptide of tPA is characterized by being represented by the amino acid sequence of SEQ ID NO: 81, but is not limited thereto.

[0057] [Table 2]

[0058] In the present invention, the polynucleotide encoding the signal peptide is characterized by being codon-optimized, and preferably by being one of the base sequences represented by SEQ ID NOs. 39 to 50, but is not limited thereto.

[0059] In the present invention, the 5'-UTR and the polynucleotide encoding the signal peptide are (i) Polynucleotides encoding the signal peptide represented by the 5'-UTR of SEQ ID NO: 1 and SEQ ID NO: 39; (ii) Polynucleotides encoding the signal peptide represented by the 5'-UTR of SEQ ID NO: 1 and SEQ ID NO: 42; (iii) Polynucleotides encoding the signal peptide represented by the 5'-UTR of SEQ ID NO: 1 and SEQ ID NO: 45; (iv) Polynucleotides encoding the signal peptide represented by the 5'-UTR of SEQ ID NO: 1 and SEQ ID NO: 48; (v) Polynucleotides encoding the signal peptide represented by the 5'-UTR of SEQ ID NO. 28 and SEQ ID NO. 39; (vi) Polynucleotides encoding the signal peptide represented by the 5'-UTR of SEQ ID NO. 28 and SEQ ID NO. 42; (vii) Polynucleotides encoding the signal peptide represented by the 5'-UTR of SEQ ID NO. 28 and SEQ ID NO. 45; (viii) Polynucleotides encoding the signal peptide represented by the 5'-UTR of SEQ ID NO. 28 and SEQ ID NO. 48; (ix) A polynucleotide encoding the signal peptide represented by the 5'-UTR of SEQ ID NO: 1 and SEQ ID NO: 41; (x) Polynucleotides encoding the signal peptide represented by the 5'-UTR of SEQ ID NO: 1 and SEQ ID NO: 44; (xi) a polynucleotide encoding the 5'-UTR of SEQ ID NO: 1 and the signal peptide represented by SEQ ID NO: 47; and (xii) A polynucleotide encoding the signal peptide represented by the 5'-UTR of SEQ ID NO: 1 and SEQ ID NO: 50; It can be characterized as being one of the selected groups, but is not limited to this.

[0060] In this invention, terms such as "GCOO-WT-HA" are abbreviations representing 5'-UTR-signal sequence-ORF according to one embodiment of this invention. Even when the same expression is used, it means that different strains have different configurations of 5'-UTR, signal peptide, and ORF sequences, as described in Tables 4 to 7 of this invention.

[0061] In the present invention, the antigenic polypeptide may be characterized by being one or more selected from the group consisting of tumor antigens, pathogenic antigens, autoantigens, alloantigens, and allergic antigens, but is not limited thereto.

[0062] In this invention, the term “tumor antigen” means an antigenic (poly)peptide or protein derived from or associated with (preferably malignant) tumors or cancers. As used herein, the terms “cancer” and “tumor” are used interchangeably to refer to neoplasms, which are generally characterized by uncontrolled and generally rapid growth of cells that tend to invade surrounding tissues and metastasize to distant parts of the body. This term includes positive and malignant neoplasms. Malignant tumors are typically characterized by anaplasia, invasiveness, and metastasis; positive tumors typically do not possess these characteristics. The terms “cancer” and “tumor” refer not only to neoplasms characterized in particular by tumor growth, but also to cancers of the blood and lymphatic systems. “Tumor antigens” are typically derived from tumor / cancer cells, preferably mammalian tumors / cancer cells, and can be located inside or on the surface of tumor cells, tumors, etc., systemically or on solid tumors, derived from mammals, preferably mammals, preferably humans. “Tumor antigens” generally include tumor-specific antigens (TSAs) and tumor-associated antigens (TAAs). TSAs are typically expressed specifically by tumor cells, resulting from tumor-specific mutations. More commonly, TAAs are presented by tumor and "normal" (healthy, non-tumor) cells.

[0063] In the present invention, the tumor antigen may be a tumor-associated protein or nucleic acid sequence, each nucleic acid sequence coding for another peptide or protein; and at least one of the above nucleic acid sequences may be 5T4, 707-AP, 9D7, AFP, A1BZIP HPG1, alpha-5-beta-1-integrin, alpha-5-beta-6-integrin, alpha-actinin-4 / m, alpha-methylacyl-coenzyme A racemase, A T-4, ARTC1 / m, B7H4, BAGE-1, BCL-2, bcr / abl, beta-catenin / m, BING-4, BRCA1 / m, BRCA2 / m, CA1 5-3 / CA27-29, CA19-9, 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 D 1, cyp-B, CYPB1, DAM-10, DAM-6, DEK-CAN, EFTUD2 / m, DGFR, 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-R17I, HLA-A11 / 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-1R, IL-13Ra2, IL-2R, IL-5, Immature Laminin Containment, 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-B16, MAGE-B17, MAGE-C1, MAGE-C2, MAGE-C3, MAGE-D1, MAGE-D2, MAGE-D4, MAGE-E1, MAGE-E2, MAGE-F1, MAGE-HI, MAGE-L2, Mammaglobin A, MART-1 / Melan-A, MART-2, MART-2 / m, Substrate Protein 22, MC1R, M-CSF, ME1 / m, mesothelin, MG50 / PCDN, MMP11, MN / CAIX-antigen, MRP-3, MUC-1, MUC-2, MUM-1 / m, MUM-2 / m, MUM-3 / m, myosin class 1 / 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, pi5, 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, TGF-beta, TGF-beta-RII, 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 genotypes of lymphoid blood cells or T cell receptor genotypes of lymphoid blood cells, or homologs, fragments, variants or derivatives of the above tumor antigens.

[0064] In the present invention, the tumor antigen may be selected from the group consisting of NY-ESO-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.

[0065] In the present invention, the pathogenic antigen can be selected from the group consisting of bacteria, viruses, fungi, and protist antigens.

[0066] 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, Chlamydia trachomatis, cytomegalovirus (CMV), hepatitis B virus (HBV), Mycobacterium tuberculosis, rabies virus, and yellow fever virus, or any isomorph, homolog, fragment, variant, or derivative of these proteins.

[0067] In the present invention, the antigenic polypeptide or immunogenic protein may be characterized as an influenza virus antigenic polypeptide by being at least one selected from the group consisting of hemagglutinin (HA), also known as HA1, HA2, or a defined antigenic subdomain of a combination of HA1 and HA2, and neuraminidase (NA), nucleoprotein (NP), substrate protein 1 (M1), substrate protein 2 (M2), non-structural protein 1 (NS1), and non-structural protein 2 (NS2).

[0068] In the present invention, the influenza virus antigenic polypeptide may be characterized as influenza hemagglutinin 1 (HA1), hemagglutinin 2 (HA2), or an immunogenic fragment of HA1 or HA2, but is not limited thereto.

[0069] In the present invention, the influenza antigenic polypeptide or immunogenic protein may be characterized by being derived from an influenza virus strain selected from the group consisting of influenza B Yamagata, influenza B Victoria, influenza A H3N2, and influenza A H3N1, and more preferably from an HA protein derived from each strain, but is not limited thereto.

[0070] In the present invention, the influenza antigenic polypeptide or the polynucleotide encoding this immunogenic protein may be characterized by being codon-optimized, and preferably by being one of the base sequences represented by SEQ ID NOs. 55 to 66, but is not limited thereto.

[0071] In the present invention, the 5'-UTR; polynucleotide encoding the signal peptide; and the antigenic polypeptide or polynucleotide encoding the immunogenic protein are (i) the 5'-UTR of SEQ ID NO: 1; a polynucleotide encoding the signal peptide represented by SEQ ID NO: 39; and a polynucleotide encoding the antigenic polypeptide represented by SEQ ID NO: 55 or the immunogenic protein thereof; (ii) the 5'-UTR of SEQ ID NO: 1; a polynucleotide encoding the signal peptide represented by SEQ ID NO: 42; and a polynucleotide encoding the antigenic polypeptide represented by SEQ ID NO: 58 or the immunogenic protein thereof; (iii) the 5'-UTR of SEQ ID NO: 1; a polynucleotide encoding the signal peptide represented by SEQ ID NO: 45; and a polynucleotide encoding the antigenic polypeptide represented by SEQ ID NO: 61 or the immunogenic protein thereof; (iv) the 5'-UTR of SEQ ID NO: 1; a polynucleotide encoding the signal peptide represented by SEQ ID NO: 48; and a polynucleotide encoding the antigenic polypeptide represented by SEQ ID NO: 64 or the immunogenic protein thereof; (v) the 5'-UTR of SEQ ID NO: 28; a polynucleotide encoding the signal peptide represented by SEQ ID NO: 39; and a polynucleotide encoding the antigenic polypeptide represented by SEQ ID NO: 55 or the immunogenic protein thereof; (vi) the 5'-UTR of SEQ ID NO: 28; a polynucleotide encoding the signal peptide represented by SEQ ID NO: 42; and a polynucleotide encoding the antigenic polypeptide represented by SEQ ID NO: 58 or the immunogenic protein thereof; (vii) the 5'-UTR of SEQ ID NO: 28; a polynucleotide encoding the signal peptide represented by SEQ ID NO: 45; and a polynucleotide encoding the antigenic polypeptide represented by SEQ ID NO: 61 or the immunogenic protein thereof; (viii) the 5'-UTR of SEQ ID NO: 28; a polynucleotide encoding the signal peptide represented by SEQ ID NO: 48; and a polynucleotide encoding the antigenic polypeptide represented by SEQ ID NO: 64 or the immunogenic protein thereof; (ix) the 5'-UTR of SEQ ID NO: 1; a polynucleotide encoding the signal peptide represented by SEQ ID NO: 41; and a polynucleotide encoding the antigenic polypeptide represented by SEQ ID NO: 57 or the immunogenic protein thereof; (x) 5'-UTR of SEQ ID NO: 1; polynucleotide encoding the signal peptide represented by SEQ ID NO: 44; and the antigenic polypeptide represented by SEQ ID NO: 60 or polynucleotide encoding this immunogenic protein; (xi) the 5'-UTR of SEQ ID NO: 1; a polynucleotide encoding the signal peptide represented by SEQ ID NO: 47; and a polynucleotide encoding the antigenic polypeptide represented by SEQ ID NO: 63 or the immunogenic protein thereof; (xii) 5'-UTR of SEQ ID NO: 1; polynucleotide encoding the signal peptide represented by SEQ ID NO: 50; and polynucleotide encoding the antigenic polypeptide represented by SEQ ID NO: 66 or the immunogenic protein thereof; It can be characterized by being selected from a group consisting of, but is not limited to, this.

[0072] In the present invention, the above 3'-UTR may be selected from, but is not limited to, the group consisting of human α-globin 3'-UTR; 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 (Bathynomide-Amur virulence virus) 3'-UTR; extension factor 1α1 (EEF1A1) 3'-UTR; manganese peroxide dismutase (MnSOD) 3'-UTR; β-subunit of mitochondrial H(+)-ATPase (β-mRNA) 3'-UTR; GLUT1 3'-UTR; MEF2A 3'-UTR; β-F1-ATPase 3'-UTR; and combinations thereof.

[0073] In the present invention, the 3'-UTR can be characterized by being represented by the base sequence of SEQ ID NO: 62.

[0074] In this invention, the term "3'-UTR" typically refers to a portion of mRNA located between the protein-encrypting region (i.e., the open reading frame, coding region) and the poly(A) sequence. The 3'-UTR of mRNA is not translated into an amino acid sequence. The 3'-UTR sequence is generally encoded by the gene that is transcribed into each mRNA during gene expression. This genomic sequence is first transcribed into immature mRNA containing alternative introns. The immature mRNA is then further modified into mature mRNA during the maturation process. Such a maturation process includes steps such as 5'-capping, splicing of immature mRNA to suppress alternative introns, and 3'-end modification such as polyadenylation of the 3' end of immature mRNA, as well as alternative endo- or exonuclease cleavage.

[0075] In this invention, the 3'-UTR corresponds to a sequence in mature mRNA that is located 3' to the stop codon of the protein coding region, preferably 3' immediately adjacent to the stop codon of the protein coding region, and traces back to the 5' side of the poly(A) sequence, preferably to the 5' immediately adjacent nucleotide relative to the poly(A) sequence. The term "corresponds" means that the 3'-UTR sequence can be an RNA sequence, such as the mRNA sequence used to define the 3'-UTR sequence, or a DNA sequence that corresponds to such an RNA sequence.

[0076] The present invention further includes the above-mentioned poly(A) tail or poly(A) tail-like sequence. In additional embodiments, terminal groups on the poly(A) tail may be introduced for stabilization. In other embodiments, the poly(A) tail includes a des-3' hydroxyl tail.

[0077] During RNA processing, a long chain of adenine nucleotides (poly(A) tails) can be added to polynucleotides, such as mRNA molecules, to increase stability. Immediately after transcription, the 3' end of the transduction body can be cleaved to release the 3' hydroxyl. Subsequently, poly(A) polymerase adds the adenine nucleotide chain to the RNA. The process called polyadenylation involves adding a poly(A) tail, which can be, for example, approximately 80 to approximately 250 residue lengths (including approximately 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 residue lengths). Poly(A) tails can also be added after the product has been leached out of the nucleus.

[0078] According to the present invention, terminal groups on the poly(A) tail can be introduced for stabilization. The polynucleotides of the present invention may contain a des-3' hydroxyl tail. They may also contain structural moieties or 2'-O-methyl variants, such as those taught by Junjie Li et al. (Current Biology, Vol. 15, 1501-1507, August 23, 2005, the full text of which is incorporated herein by reference).

[0079] The unique poly(A) tail length provides certain advantages to the polynucleotides of the present invention. Generally, the poly(A) tail length, if present, is a length of more than 30 nucleotides. In other embodiments, the poly(A) tail is a length of more than 35 nucleotides (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, 2,0000, 2,500 and more than 3,000 nucleotides).

[0080] In some embodiments, the polynucleotide or region contains approximately 30 to approximately 3,000 nucleotides (for example, 30-50, 30-100, 30-250, 30-500, 30-750, 30-1,000, 30-1,500, 30-2,000, 30-2,500, 50-100, 50-250, 50-500, 50-750, 50-1,000, 50-1,500, 50-2,000, 50-2,500, 50-3,000, 100-500, 100-750, 100-1,000, 100- This includes 1,500, 100-2,000, 100-2,500, 100-3,000, 500-750, 500-1,000, 500-1,500, 500-2,000, 500-2,500, 500-3,000, 1,000-1,500, 1,000-2,000, 1,000-2,500, 1,000-3,000, 1,500-2,000, 1,500-2,500, 1,500-3,000, 2,000-3,000, 2,000-2,500, and 2,500-3,000).

[0081] In some embodiments, the poly(A)tail is designed for the length of the entire polynucleotide or for the length of a specific region of the polynucleotide. This design can be based on the length of the cryptographic region, the length of a specific feature or region, or the length of the ultimate product expressed from the polynucleotide.

[0082] In connection with this, the poly(A) tail can be 10, 20, 30, 40, 50, 60, 70, 80, 90, or even 100% longer than the polynucleotide or this feature. The poly(A) tail can also be designed as a fraction of the polynucleotide to which it belongs. In connection with this, the poly(A) tail can be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the total length of the artifact, the artifact region, or the total length of the artifact - poly(A) tail. In addition, manipulated binding sites and conjugations of polynucleotides to poly(A) binding proteins can improve expression.

[0083] Additionally, multiple distinct polynucleotides can be linked together via PABP (poly(A)-binding protein) through the 3' end using nucleotides modified at the 3' end of the poly(A) tail. Transfection experiments can be performed in appropriate cell lines, and protein production can be assessed by ELISA at 12, 24, 48, 72, and 7 hours after transfection.

[0084] 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 assay of four guanine nucleotides that can be formed by a G-rich sequence in both DNA and RNA. In this experiment, the G-quartet is introduced into the end of a poly(A) tail. The resulting polynucleotides are assayed for stability, protein production, and other parameters including half-life at various time points. We found that the polyAG quartet induces protein production from at least 75% equivalent mRNA, which can be determined using a single poly(A) tail of 120 nucleotides.

[0085] In the present invention, the poly(A) tail-like sequence can be used without limitation as long as it is a nucleic acid sequence capable of performing the function of a poly(A) tail. Preferably, it is characterized by having one or more nucleotides other than adenines, selected from the group consisting of uracil (U), cytosine (C), and guanine (G), inserted between multiple adenines or at the poly(A) tail end, but is not limited thereto.

[0086] In the present invention, the mRNA structure may be characterized by containing one or more nucleic acids that have undergone backbone modification, sugar modification, or base modification.

[0087] Sugar deformation: Modified nucleosides and nucleotides that can be incorporated into a modified mRNA compound containing an mRNA sequence as described herein can be modified in the sugar moiety. For example, the 2'-hydroxyl group (OH) can be modified or replaced with a number of different "oxy" or "deoxy" substituents. Examples of "oxy"-2'-hydroxyl group modifications include alkoxy or aryloxy (-OR, where R = H, alkyl, cycloalkyl, allyl, aralkyl, heteroaryl or sugar); polyethylene glycol (PEG), -O(CH2CH2O) n CH2CH2OR; "locked" nucleic acids (LNA) in which the 2'-hydroxyl is linked to the 4'-carbon of the same ribose sugar, for example by a methylene bridge; and amino groups (-O-amino, where the amino group, for example NRR can be alkylamino, dialkylamino, heterocycle, allylamino, diallylamino, heteroallylamino, or diheteroallylamino, ethylenediamine, polyamino) or aminoalkoxy, but are not limited thereto.

[0088] "Deoxy" modifications can include hydrogen, amino (e.g., NH2; alkylamino, dialkylamino, heterocycle, allylamino, diallylamino, heteroallylamino, diheteroallylamino, or amino acid); or an amino group can be attached to the sugar through a linker, where the linker contains one or more of the atoms C, N, and O.

[0089] The sugar group can also contain one or more carbons with opposite stereochemical configuration values compared to the corresponding carbons in ribose. Thus, the modified mRNA can include nucleotides containing arabinose, for example, as the sugar.

[0090] Backbone deformation: The phosphate backbone can be further modified with modified nucleosides and nucleotides, which can be incorporated into modified mRNA compounds containing mRNA sequences as described herein. The phosphate group of the backbone can be modified by replacing one or more oxygen atoms with other substituents. Modified nucleosides and nucleotides may also include the complete replacement of unmodified phosphate moieties with the modified phosphates described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, alkyl or allyl phosphonates, and phosphotryesters. Phosphonates have both uncoupled oxygen atoms replaced by sulfur.

[0091] Phosphate linkers can also be modified by substituting linked oxygen with nitrogen (bridged phosphoramide), sulfur (bridged phosphorothioate), and carbon (bridged methylene phosphonate).

[0092] Base transformation: Modified nucleosides and nucleotides that can be incorporated into modified mRNA compounds containing mRNA sequences as described herein can be further modified at the nuclear base moiate. Examples of nuclear bases found in mRNA include, but are not limited to, adenine, guanine, cytosine, and uracil. For example, the nucleosides and nucleotides described herein can be chemically modified 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.

[0093] In a particularly preferred embodiment of the present invention, the creotide analogs / modifications are preferably 2-amino-6-chloropurine riboside-5'-triphosphate, 2-aminopurine-riboside-5'-triphosphate; 2-aminoadenosine-5'-triphosphate, 2'-amino-2'-deoxycytidine-triphosphate, 2-thiocytidine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 2'fluorothymidine-5'- Triphosphate, 2'-O-methyl-inosine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 5-aminoallylcytidine-5'-triphosphate, 5-aminoallyluridine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, 5-bromouridine-5'-triphosphate, 5-bromo-2'-deoxycytidine-5'-triphosphate, 5-bromo-2'-deoxyuridine n-5'-triphosphate, 5-iodocytidine-5'-triphosphate, 5-iodo-2'-deoxycytidine-5'-triphosphate, 5-iodouridine-5'-triphosphate, 5-iodo-2'-deoxyuridine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, 5-methyluridine-5'-triphosphate, 5-propynyl-2'-deoxycytidine-5'-triphosphate, 5- Propynyl-2'-deoxyuridine-5'-triphosphate, 6-azacitidine-5'-triphosphate, 6-azacitidine-5'-triphosphate, 6-chloropurine riboside-5'-triphosphate, 7-deazaadenosine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 8-azaadenosine-5'-triphosphate, 8-azidoadenosine-5'-triphosphate, benzimidazole Selected from base variants selected from -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.

[0094] 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-bromocytidine-5'-triphosphate, and pseudouridine-5'-triphosphate.

[0095] In some concrete examples, the modified nucleosides are 5-pyridine-4-onyribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudridine, 2-thio-pseudridine, 5-hydroxyuridine, 3-methyluridine, 5-methoxyuridine, 5-carboxymethyluridine, 1-carboxymethyl-pseudridine, 5-propynyluridine, 1-propynyl-pseudridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudridine, 5-taurinomethyl-2-thiouridine, 1- This includes taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseuduridine, 4-thio-1-methyl-pseuduridine, 2-thio-1-methyl-pseuduridine, 1-methyl-1-deaza-pseuduridine, 2-thio-1-methyl-1-deaza-pseuduridine, dihydrouridine, dihydropseuduridine, 2-thio-dihydrouridine, 2-thio-dihydropseuduridine, 2-methoxyuridine, 2-methoxy-4-thiouridine, 4-methoxy-pseuduridine, and 4-methoxy-2-thio-pseuduridine.

[0096] In some concrete examples, the modified nucleosides are 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrole-cytidine, pyrrole-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methylcytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudo It contains socytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularinr, 5-aza-zebralin, 5-methyl-zebralin, 5-aza-2-thio-zebralin, 2-thio-zebralin, 2-methoxycytidine, 2-methoxy-5-methylcytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine.

[0097] In other manifestations, the modified nucleosides are 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, N This product contains 6-(cis-hydroxyisopentyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6, N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxyadenine.

[0098] In other embodiments, the modified nucleosides include inosine, 1-methylinosine, waiosine, waibutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methylguanosine, 6-thio-7-methylguanosine, 7-methylinosine, 6-methoxyguanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thioguanosine, N2-methyl-6-thioguanosine, and N2,N2-dimethyl-6-thioguanosine.

[0099] In some embodiments, nucleotides can be deformed at the major groove face, including substitution of a hydrogen atom at C-5 of uracil with a methyl or halo group. In certain embodiments, the deformed nucleosides are 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.

[0100] In additional specific examples, the modified mRNAs include 6-aza-cytidine, 2-thio-cytidine, α-thio-cytidine, pseudo-iso-cytidine, 5-aminoallyl-uridine, 5-iodo-uridine, N1-methyl-pseudouridine, 5,6-dihydrouridine, α-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxy-thymidine, 5-methyl-uridine, pyrrolo-cytidine, inosine, and α It may contain nucleoside variations selected from -thio-guanosine, 6-methyl-guanosine, 5-methyl-cytidine, 8-oxo-guanosine, 7-deaza-guanosine, N1-methyl-adenosine, 2-amino-6-chloropurine, N6-methyl-2-aminopurine, pseudo-isocytidine, 6-chloropurine, N6-methyl-adenosine, α-thio-adenosine, 8-azido-adenosine, and 7-deaza-adenosine.

[0101] In another respect, the present invention also relates to an influenza vaccine composition comprising the above mRNA structure.

[0102] In the present invention, “vaccine” is typically understood as a preventive or therapeutic substance that provides at least one antigen, preferably an antigenic peptide or protein. “Providing at least an antigen” means, for example, that the vaccine contains an antigen or that the vaccine contains, for example, a molecule encoding an antigen. Therefore, the vaccines of the present invention can be derived from, for example, tumor antigens, bacteria, viruses, fungal or protozoan antigens, autoantigens, allergens, or allogeneic antigens, and are particularly expected to contain at least one synthetic nucleic acid (RNA) molecule encoding at least one antigenic (poly-)peptide or protein as defined herein, which, when expressed and presented to the immune system, induces an immune response to each antigen. However, synthetic nucleic acid (RNA) molecules encoding non-antigenic (poly-)peptides or proteins of interest can also be used in the vaccines of the present invention.

[0103] In the present invention, the mRNA structure of the vaccine composition is characterized by being complexed with one or more lipids to form lipid nanoparticles or liposomes.

[0104] The above-mentioned lipid nanoparticles may be characterized by including, but are not limited to, cationic lipids, PEG-modified lipids, sterols, and non-cateionic lipids.

[0105] In the present invention, the mRNA structure can be provided in a complexed form, i.e., in a form complexed or associated 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 terms “complexed” or “associated” mean, in this sense, that at least one synthetic nucleic acid (RNA) molecule is in an essentially stable combination with the one or more compounds in a larger complex or assembly without covalent bonding.

[0106] Lipids In preferred embodiments, the mRNA structure of the present invention is complexed or bound to lipids (particularly cationic and / or neutral lipids) to form one or more lipid nanoparticles or liposomes. Accordingly, in some embodiments, the synthetic nucleic acid (RNA) molecule of the present invention can be provided in the form of a lipid-based formulation, particularly in the form of liposomes and / or lipid nanoparticles containing the above-mentioned synthetic nucleic acid (RNA) molecule.

[0107] Lipid nanoparticles According to some preferred embodiments, the mRNA structure of the present invention is complexed or bound to lipids (particularly cationic and / or neutral lipids) to form one or more lipid nanoparticles.

[0108] Preferably, the lipid nanoparticle (LNP) proposal may include: (a) at least one mRNA complex of the present invention, (b) a cationic lipid, (c) an aggregation-reducing agent (e.g., polyethylene glycol (PEG) lipid or PEG-modified lipid), (d) selectively a non-cationic lipid (e.g., a neutral lipid), and (e) selectively a sterol.

[0109] In some embodiments, the LNP may contain, in addition to at least one mRNA structure of the present invention, (i) at least one cationic lipid; (ii) a neutral lipid; (iii) a sterol, such as cholesterol; and PEG-lipids, in a molar ratio of about 20-60% cationic lipids: 5-25% neutral lipids: 25-55% sterols; and 0.5-15% PEG-lipids.

[0110] In some embodiments, the mRNA structure of the present invention can be formulated as an aminoalcohol lipidoid. The aminoalcohol lipidoid that can be used in the present invention can be produced by the method described in U.S. Patent No. 8,450,298, the full text of which is included in this application by reference.

[0111] Liposomes In some embodiments, the mRNA structure of the present invention is drug-formed in liposomes. Cationic lipid-based liposomes can form complexes with negatively charged nucleic acids (e.g., RNA) through electrostatic interactions, producing complexes that offer biocompatibility, low toxicity, and the large-scale production potential necessary for in vivo clinical application. The liposomes can fuse with the plasma membrane for absorption; once inside the cell, the liposomes are processed through a phagocytic pathway, and the nucleic acids are subsequently released from endosomes / carriers 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 produced from both natural and synthetic phospholipids.

[0112] Liposomes consist of a lipid bilayer that can be composed of cationic, anionic, or neutral (phospho)lipids and cholesterol surrounding a typical aqueous core. Both the lipid bilayer and the aqueous space can contain hydrophobic or hydrophilic compounds, respectively. Liposomes can have one or more lipid membranes. Liposomes can be single-layered, called unilamellae, or multi-layered, called multilamellae.

[0113] In vivo, liposome properties and behavior can be altered 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 attached PEG chains.

[0114] Liposomes typically exist as spherical vesicles and can range in size from 20 nm to several microns. Liposomes can vary in size, but are not limited to, multilamellar vesicles (MLVs), which can have diameters of several hundred nanometers and contain a series of concentric bilayers separated by narrow aqueous compartments; small unicellular vesicles (SUVs), which have diameters less than 50 nm; and large unilamellar vesicles (LUVs), which can have diameters between 50 and 500 nm. Liposome design may contain opsonins or ligands to improve liposome adhesion to unhealthy tissues or to activate events such as, but are not limited to, endocytosis. Liposomes may contain low or high pH to enhance the delivery of pharmaceutical dosage forms.

[0115] In the present invention, the vaccine composition may be characterized by further comprising one or more adjuvants or activators.

[0116] In its broadest sense, "adjuvant" or "adjuvant component" is typically a pharmacological and / or immunological preparation that can alter, for example, enhance the effects of other activators, such as therapeutic agents or vaccines. In this context, "adjuvant" can be understood as any compound suitable for assisting the administration and delivery of the vaccine composition of the present invention. Specifically, adjuvants can preferably enhance the immunostimulatory properties of the vaccine to which they are added. Furthermore, such adjuvants can initiate or increase the immune response of the innate immune system, i.e., nonspecific immune responses, without being bound to it.

[0117] Adjuvants typically do not induce an adaptive immune response. To date, adjuvants have not qualified as antigens. That is, when administered, the vaccine of the present invention typically initiates an adaptive immune response through antigenic peptides or proteins encoded by at least one coding sequence of synthetic nucleic acid (RNA) molecules contained in the vaccine.

[0118] Suitable adjuvants are publicly disclosed to those skilled in the art and can be selected from any adjuvants suitable in this case, i.e., any adjuvants that help induce an immune response in mammals, such as TDM, MDP, muramyl dipeptide, pluronic acid, vitiligo solution, aluminum hydroxyl, ADJUMER TM (Polyphosphazene); Aluminum phosphate gel; Glucan from algae; Algamulin; Aluminum hydroxylate gel (Alum); High protein-adsorbent aluminum hydroxylate gel; Low viscosity aluminum hydroxylate gel; AF or SPT (Emulsion of squalene (5%), Tween 80 (0.2%), Pluronic L121 (1.25%), phosphate-buffered saline, pH 7.4); AVRIDINE TM(Propanediamines); substances that correspond to pathogen-associated molecular patterns (PAMPs) and react with pattern recognition receptors (PRRs); CpGDNA; lipoproteins; flagella; poly I:C; saponins; squalene; tricaprin; 3D-MPL; or non-toxic lipooligosaccharides (detoxied lipooligoxassharide, dLOS) may be included, but are not limited to, these.

[0119] In another respect, the present invention relates to the use of the above-mentioned vaccine composition for the prevention of influenza.

[0120] In this invention, "influenza" refers to an infectious disease induced by the influenza virus and can be used interchangeably with "epidemic cold" and "influenza (Flu)."

[0121] In this invention, "prevention" means any action that suppresses influenza or delays its progression by administering the vaccine composition of this invention.

[0122] In another aspect, the present invention relates to a method for preventing influenza, which includes the step of administering the above-mentioned vaccine composition.

[0123] In another respect, the present invention relates to the use of the above vaccine composition for the manufacture of a drug for the prevention of influenza. [Examples]

[0124] The present invention will be described in more detail below through examples. These examples are merely illustrative and it will be obvious to those with ordinary skill in the art that the scope of the present invention is not limited by these examples.

[0125] Example 1. mRNA structure preparation 1-1. mRNA sequencing Thirty-three 5'-UTR sequences selected using the method described in PCT / KR2022 / 019491, along with the additional 5'-UTR sequence of Sequence ID No. 1, were selected for HA protein expression (Table 3).

[0126] Furthermore, signal sequences and ORF sequences were created for each strain using codon optimization based on the wild-type Yamagata, Victoria, HH3N2, and H1N1 signal sequences (sequence numbers 51, 52, 53, and 54 in order) and ORF sequences (sequence numbers 67, 68, 69, and 70 in order) listed in Table 3. In addition, sequences were created by transforming the wild-type strain signal sequences into IgE or tPA through codon optimization.

[0127] [Table 3] TIFF2026513562000005.tif251170TIFF2026513562000006.tif245170TIFF2026513562000007.tif251170 TIFF2026513562000008.tif251170TIFF2026513562000009.tif246170TIFF2026513562000010.tif251170 TIFF2026513562000011.tif251170TIFF2026513562000012.tif251170TIFF2026513562000013.tif251170 TIFF2026513562000014.tif251170TIFF2026513562000015.tif251170TIFF2026513562000016.tif142170

[0128] The mRNA structures for each strain, prepared for in vivo protein expression testing, are shown in Tables 4-7 below.

[0129] [Table 4]

[0130] [Table 5]

[0131] [Table 6]

[0132] [Table 7]

[0133] 1-2. mRNA synthesis and purification The mRNA structures shown in Tables 4 to 7, prepared in Example 1.1 using the method described in PCT / KR2022 / 019491, were purified by in vitro transcription.

[0134] Specifically, after proceeding with IVT under the conditions in Table 8, once the reaction is complete, the IVT product is treated with 1 U of DNase I per 1 μg of DNA and reacted at 37°C for 15-30 minutes (min) to remove the template DNA. TM The sample was purified using the method provided by the manufacturer in the kit (Austin, Tex.).

[0135] [Table 8]

[0136] Example 2. Synthesis of mRNA-LNP complex Ionizable lipids, phospholipids, cholesterol, and PEG-lipid conjugates were dissolved in ethanol in a molar ratio of 50:10:38.5:1.5, and then mixed with citarate buffer (pH 4, 50 mM) containing lysed mRNA in a volume ratio of 1:3. MC3 (MedChemExpress, USA) was used as the ionizable lipid; DSPC (Avanti Polar Lipids, USA) and cholesterol (Sigma Aldrich, USA) were used as the phospholipids; and 1-2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000) (Avanti Polar Lipids, USA) was used as the PEG-lipid conjugate.

[0137] NanoAssemblr Ignite for Lipid Nanoparticle (LNP) Generation TM Mixing was performed using Precision Nanosystems (Inc., Canada) under a total flow rate (TFR) of 12 mL / min to achieve a nitrogen-to-phosphoate ratio (N / P ratio) of 4 between ionizable lipids and mRNA. The prepared lipid nanoparticles were processed using an Amicon Ultra Centrifugal Filter, MWCO 10kDa (Millipore, USA) for ethanol removal, buffer exchange, and concentration, and 1x DPBA (Thermo Scientific, USA) for dilution, concentration, and exchange. A final 300 mM sucrose solution was added as a cryopreservative for frozen storage, and the mixture was stored frozen (-80°C).

[0138] Example 3. Confirmation of mRNA structure performance through animal experiments. 3-1. Method for injecting mRNA constructors into animals and collecting tissue samples for confirmation of HA expression Six-to-seven-week-old BalB / c female mice were purchased (Orient Bio, South Korea) and maintained under conditions free of specific pathogens. One day before injection, the right leg of the mice was shaved, and the mRNA-LNP complex prepared in Example 2 was intramuscularly injected into the right papilla muscle using an insulin syringe. Six hours after injection, the injected right papilla muscle was collected and placed in a 1.7 mL tube and stored in a -70°C ultra-low temperature freezer until the HA protein expression level was evaluated using muscle lysate (Figure 2).

[0139] 3-2. Method for confirming muscle lysates To confirm HA protein expression in the injected muscle, muscle lysate was prepared. To obtain the muscle lysate, the collected tissue was placed in a tube containing tissue disruption buffer (tissue disruption buffer composition: 1x cell lysis buffer (Cell signaling technology, Cat No. #9803), protease and phosphatase inhibitor mini talets, EDTA-Free 1 tablet / 10mL, ThermoFisher Scientific, Cat No. A32961)) and stainless steel beads, and then the tissue was disrupted using a tissue disruptor.

[0140] The tubes containing the disrupted tissue were centrifuged at 13,000 rpm for 10 minutes in a centrifuge set to 4 degrees. The supernatant was then transferred to a new 1.7 mL tube and used for evaluating HA protein expression.

[0141] 3-3. Measurement of HA protein in muscle lysates To evaluate intramuscular HA protein expression, we performed an ELISA (enzyme-linked immunosorent assay). Using Sinobiological's ELISA kit and antibodies, capture antibodies (Yamagata: Cat No. 11053-MM09, Victoria: Cat No. 11053-MM06, H3N2: 11056-RP01, H1N1: Cat No. SEK001) that can confirm HA protein were diluted to a concentration of 1-2 μg / mL using PBS (Phosphate-buffered saline, Lonza, Cat No. 17-516Q), coated onto the ELISA substrate, reacted at 4°C for 16-20 hours, then washed the ELISA substrate three times (washing buffer composition: PBS containing 0.05% Tween-20, Tween-20 (Sigma-Aldrich, Cat No. P1379-500mL)), PBS (Phosphate-buffered saline) containing 2% bovine serum albumin The cells were blocked for one hour at room temperature with saline solution (bovine serum albumin: Sigma-Aldrich, Cat No. A3803-100G).

[0142] The protein quantification of muscle lysates, as previously confirmed, was performed using the bicinchoninic acid (BCA) quantification method (ThermoFisher Scientific, Cat No. 23225), and the same amount of protein was used to evaluate intramuscular HA protein expression.

[0143] After blocking, the ELISA substrate was washed three times with the same washing buffer. The muscle lysate to be evaluated was placed on the ELISA substrate and reacted at room temperature for 2 hours. After washing three more times, detection antibodies capable of detecting HA proteins containing HRP (horseradish peroxidase) or biotin were used. These antibodies were diluted at 0.5-1 μg / mL in PBS solution containing 0.5% bovine serum albumin, and 100 μL of the diluted antibody solution was added to each substrate and reacted at room temperature for 1 hour.

[0144] For H3N2, the ELISA substrate was washed three times with the same washing buffer. Strepravidin-HRP (Sigma-Aldrich, Cat No. S2438-250UG) was diluted 1:5,000 using PBS containing 0.5% bovine serum albumin, and 100 μL of this diluted solution was added to each substrate and allowed to react at room temperature for 20 minutes. After this, the ELISA substrate was washed three more times, and color development was performed using TNB (3,3',5,5'-tetramethylbensidine) solution (ThermoFisher Scientific, Cat No. 34028), followed by reaction at room temperature for 10 minutes. After this, a reaction stop solution (SeraCare, Cat No. 5150-0021) was added to interrupt the color development, and the absorbance was measured at a wavelength of 450 nm using a Molecular Devices microplate reader (VersaMax) to compare the amount of HA protein expression in muscle.

[0145] 3-4. Method for injecting mRNA constructs into animals and collecting serum for evaluation and confirmation of HA-specific immune responses Animal experiments were conducted to evaluate the immune response to mRNA constructs created by the inventors. Female 6-7BalB / c mice (Orient Bio, South Korea) were purchased and maintained under conditions free of specific pathogens. The mice were immunized intramuscularly with 0.1 μg of mRNA-LNP complexes into the right papillosa muscle using an insulin syringe (Figure 7). Blood samples were collected four weeks after immunization to obtain serum.

[0146] Blood samples were left at room temperature for 30 minutes to allow the blood to coagulate, and then centrifuged at 10,000 rpm for 10 minutes in a 4°C refrigerated centrifuge. After centrifugation, the supernatant was transferred to a new 1.7 mL tube and stored in a -20°C freezer before the humoral immune response confirmation test.

[0147] 3-5.HA antigen-specific IgG antibody titer measurement An ELISA (enzyme-linked immunosorbent assay) was performed on serum to measure HA antigen-specific IgG titer. Influenza A / H1N1 virus fractionated vaccine was diluted in PBS and added to an ELISA substrate. The substrate was reacted at 4°C for at least 12 hours to coat the protein. The ELISA substrate was washed three times with PBS containing 0.5% TWEEN-20, and then blocked for 1 hour at room temperature with a PBS (phosphate-buffered saline) solution containing 2% bovine serum albumin (Sigma, Cat No. A3803). After blocking, the ELISA substrate was washed three times, and serum samples diluted in PBS containing 2% bovine serum albumin were added to the ELISA substrate and reacted at room temperature for 2 hours. The ELISA was then washed three times, and HRP (Horseradish peroxidase)-conjugated goat anti-mouse IgG antibody) (Southern Biotech, Cat No. 1031-05) was added and the mixture was allowed to react at room temperature for 1 hour. After that, the ELISA substrate was washed three times, and color development was performed using TMB (3,3',5,5'-tetramethylbensidine, ThermoFisher Scientific, Cat No. 34028) solution, followed by 10 minutes of reaction at room temperature. After that, a stop solution (SeraCare, Cat No. 5150-0021) was added to interrupt the color development, and the optical density was measured using a 450 nm wavelength spectrometer with a Molecular Devices microplate reader (VersaMax).

[0148] Example 4. Comparison of HA expression levels in influenza B / Yamagata strain based on changes in 5'UTR and signal sequence. To increase the immunogenicity of the mRNA structure, ELISA analysis was performed using the method of Example 3 to compare the mRNA sequence prepared in Example 1 with the influenza virus self sequence.

[0149] As a result, as shown in Figure 3, we were able to confirm that the mRNA sequence produced in Example 1 showed a higher in vivo expression level compared to the self-HA sequence of the influenza B / Yamagata strain (Figure 3, control vs each lane).

[0150] Example 5. Comparison of HA expression levels of influenza B / Victoria strain due to changes in 5'UTR and signal sequence. To increase the immunogenicity of the mRNA structure, ELISA analysis was performed using the method of Example 3 to compare the mRNA sequence prepared in Example 1 with the self-sequence of the influenza virus.

[0151] As a result, as shown in Figure 4, we were able to confirm that the mRNA sequence produced in Example 1 showed a higher in vivo expression level compared to the self-HA sequence of the influenza B / Victoria strain (Figure 4, control vs each lane).

[0152] Example 6. Comparison of HA expression levels in influenza A / H3N2 strains based on 5'UTR and signal sequence changes. To increase the immunogenicity of the mRNA structure, ELISA analysis was performed using the method of Example 3 to compare the mRNA sequence prepared in Example 1 with the self-sequence of the influenza virus.

[0153] As a result, as shown in Figure 5, we were able to confirm that the mRNA sequence produced in Example 1 showed a higher in vivo expression level compared to the self-HA sequence of the influenza A / H3N2 strain (Figure 5, control vs each lane).

[0154] Example 7. Comparison of HA expression levels of influenza A / H1N1 strain based on 5'UTR and signal sequence changes. To increase the immunogenicity of the mRNA structure, ELISA analysis was performed using the method of Example 3 to compare the mRNA sequence prepared in Example 1 with the self-sequence of the influenza virus.

[0155] As a result, as shown in Figure 6, we were able to confirm that the mRNA sequence produced in Example 1 showed a higher in vivo expression level compared to the self-HA sequence of the influenza A / H1N1 strain (Figure 6, control vs each lane).

[0156] Example 8. Comparison of immunological responses to influenza A / H1N1 strains based on changes in 5'UTR and signal sequence. To increase the immunogenicity of the mRNA structure, anti-influenza-specific IgG ELISA analysis was performed using the method of Example 3 to compare the mRNA sequence prepared in Example 1 with the influenza virus self sequence.

[0157] As a result, as shown in Figure 8, we were able to confirm that the mRNA sequence produced in Example 1 showed higher immunogenicity compared to the autoantigen of the influenza A / H1N1 strain (Figure 8, control vs each lane).

[0158] Example 9. Selection of 5'UTR and signal sequence combinations with high expression levels and immunogenicity. Based on the results of Examples 4-8, 5'UTR and signal sequence combinations with high in vivo HA expression and high immunogenicity were selected. The criteria for selection were combinations in which the in vivo HA expression was 1.5 times or more than that of the control group, while the immunogenicity was 3 times or more than that of the control group.

[0159] First, the in vivo HA expression levels of Examples 4-7 compared to the control group, organized by fold change, are shown in Table 9 below. In Table 9 below, GCOO-HA has the same meaning as GCOO_WT_HA, GCOO-IgE has the same meaning as GCOO_IgE_HA, and GCOO-tpa has the same meaning as GCOO_tpa_HA. The remaining terms are used with the same meaning.

[0160] [Table 9]

[0161] As shown in Table 9 above, we confirmed that the combinations that showed a 1.5-fold or greater increase in expression levels with any strain were GCOO-HA, GC27-HA, GC29-HA, GCOO-tpa, and GC14-tpa.

[0162] Subsequently, the results of comparing the immunogenicity of the H1N1 strain from Example 8 with the control group, organized by fold change, are shown in Table 10 below.

[0163] [Table 10]

[0164] As shown in Table 10 above, it was confirmed that the combinations with immunogenicity three times or more were GCOO-HA, GCOO1-HA, GC14-HA, GC27-HA, GCOO-tpa, GC01-tpa, GC27-tpa, and GC29-tpa. Among these, it was confirmed that the combinations with HA expression levels of 1.5 times or more were GCOO-HA (GCOO_WT_HA), GC27-HA (GC27_WT_HA), and GCOO-tpa (GCOO-tpa_HA), and these combinations were selected as the final combinations. [Industrial applicability]

[0165] The mRNA structure according to the present invention contains a 5'-UTR polynucleotide with improved translation efficiency, which can effectively induce the expression of antigenic polypeptides, thereby increasing the immunogenicity of the vaccine and making it useful in vaccine development.

[0166] Having described in detail certain aspects of the present invention, it will be clear to any person with ordinary skill in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An antigenic polypeptide or an mRNA structure encoding this immunogenic fragment, From 5' to 3' in order a) 5'-CAP structure; b) 5'-untranslated region (5'-UTR) polynucleotide represented by the nucleic acid sequence of the following chemical formula (I) or (II): Chemical formula (I): AG[N] ２２ ]GCACC, Chemical formula (II): AGGA [N] １９ ]RGCCACC, Here, R means A, G, or U; c) Polynucleotides encoding signal peptides; d) Antigenic polypeptides or polynucleotides encoding these immunogenic proteins; e) 3'-untranslated region (3'-UTR); and f) A poly(A) tail or poly(A) tail-like sequence consisting of 10 to 1000 adenine(A) units; mRNA structure containing this.

2. The aforementioned 5'-CAP structure is m ７ GPPPA ｍ pG, m ７ The mRNA structure according to claim 1, characterized in that it is selected from the group consisting of GpppApG and m7,3'OmeApppG.

3. The mRNA structure according to claim 1, characterized in that the 5'-UTR is one of the base sequences represented by SEQ ID NOs: 1 to 34.

4. The mRNA structure according to claim 3, characterized in that the 5'-UTR is one selected from the group consisting of SEQ ID NOs: 1, 2, 15, 28, and 30.

5. The mRNA structure according to claim 1, characterized in that the signal peptide is derived from an antigenic polypeptide, immunoglobulin E (IgE), or tissue plasminogen activator (tPA).

6. The mRNA structure according to claim 5, characterized in that the signal peptide is an amino acid represented by SEQ ID NO: 72 (MKAIIVLLMVVTSNA), SEQ ID NO: 73 (MKXIIALSXILCLVFA, where X is T, A, Y, or N), SEQ ID NO: 77 (MKAILVVXLYTFTTANA, where X is L or M), SEQ ID NO: 80 (MDWTWILFVAAAATRVHS), or 81 (MDAMKRGLCCVLLLLCGAVFVSA).

7. The mRNA structure according to claim 1, characterized in that the polynucleotide encoding the signal peptide is codon-optimized.

8. The mRNA structure according to claim 7, characterized in that the polynucleotide encoding the signal peptide is one of the base sequences represented by SEQ ID NOs: 39 to 50.

9. The 5'-UTR and the polynucleotide encoding the signal peptide are (i) Polynucleotides encoding the signal peptide represented by the 5'-UTR of SEQ ID NO: 1 and SEQ ID NO: 39; (ii) Polynucleotides encoding the signal peptide represented by the 5'-UTR of SEQ ID NO: 1 and SEQ ID NO: 42; (iii) Polynucleotides encoding the signal peptide represented by the 5'-UTR of SEQ ID NO: 1 and SEQ ID NO: 45; (iv) Polynucleotides encoding the signal peptide represented by the 5'-UTR of SEQ ID NO: 1 and SEQ ID NO: 48; (v) Polynucleotides encoding the signal peptide represented by the 5'-UTR of SEQ ID NO: 28 and SEQ ID NO: 39; (vi) Polynucleotides encoding the signal peptide represented by the 5'-UTR of SEQ ID NO: 28 and SEQ ID NO: 42; (vii) Polynucleotides encoding the signal peptide represented by the 5'-UTR of SEQ ID NO: 28 and SEQ ID NO: 45; (viii) Polynucleotides encoding the signal peptide represented by the 5'-UTR of SEQ ID NO: 28 and SEQ ID NO: 48; (ix) Polynucleotides encoding the signal peptide represented by the 5'-UTR of SEQ ID NO: 1 and SEQ ID NO: 41; (x) Polynucleotides encoding the signal peptide represented by the 5'-UTR of SEQ ID NO: 1 and SEQ ID NO: 44; (xi) polynucleotides encoding the signal peptide represented by the 5'-UTR of SEQ ID NO: 1 and SEQ ID NO: 47; and (xii) Polynucleotides encoding the signal peptide represented by the 5'-UTR of SEQ ID NO: 1 and SEQ ID NO: 50; The mRNA structure according to claim 1, characterized in that it is one selected from the group consisting of the following.

10. The mRNA structure according to claim 1, characterized in that the antigenic polypeptide is one or more selected from the group consisting of tumor antigens, pathogenic antigens, autoantigens, alloantigens, and allergic antigens.

11. The mRNA structure according to claim 10, characterized in that the tumor antigen is selected from the group consisting of NY-ESO-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.

12. The mRNA structure according to claim 10, characterized in that the pathogenic antigen is selected from the group consisting of bacterial, viral, fungal, and protist antigens.

13. The mRNA structure according to claim 12, characterized in that the virus is an influenza virus.

14. The mRNA structure according to claim 10, characterized in that the antigenic polypeptide is influenza hemagglutinin 1 (HA1), hemagglutinin 2 (HA2), or an immunogenic fragment of HA1 or HA2.

15. The mRNA structure according to claim 14, characterized in that the antigenic polypeptide is derived from an influenza virus strain selected from the group consisting of influenza B Yamagata, influenza B Victoria, influenza A H3N2, and influenza A H1N1.

16. The mRNA structure according to claim 1, characterized in that the antigenic polypeptide or the polynucleotide encoding the immunogenic protein is codon-optimized.

17. The mRNA structure according to claim 16, characterized in that the antigenic polypeptide or the polynucleotide encoding the immunogenic protein is one of the base sequences represented by SEQ ID NOs. 55 to 66.

18. The 5'-UTR; polynucleotide encoding the signal peptide; and the antigenic polypeptide or polynucleotide encoding this immunogenic protein are (i) 5'-UTR of SEQ ID NO: 1; a polynucleotide encoding the signal peptide represented by SEQ ID NO: 39; and a polynucleotide encoding the antigenic polypeptide represented by SEQ ID NO: 55 or the immunogenic protein thereof; (ii) 5'-UTR of SEQ ID NO: 1; polynucleotide encoding the signal peptide represented by SEQ ID NO: 42; and polynucleotide encoding the antigenic polypeptide represented by SEQ ID NO: 58 or the immunogenic protein thereof; (iii) 5'-UTR of SEQ ID NO: 1; polynucleotide encoding the signal peptide represented by SEQ ID NO: 45; and polynucleotide encoding the antigenic polypeptide represented by SEQ ID NO: 61 or the immunogenic protein thereof; (iv) 5'-UTR of SEQ ID NO: 1; polynucleotide encoding the signal peptide represented by SEQ ID NO: 48; and polynucleotide encoding the antigenic polypeptide represented by SEQ ID NO: 64 or the immunogenic protein thereof; (v) 5'-UTR of SEQ ID NO: 28; polynucleotide encoding the signal peptide represented by SEQ ID NO: 39; and polynucleotide encoding the antigenic polypeptide represented by SEQ ID NO: 55 or the immunogenic protein thereof; (vi) 5'-UTR of SEQ ID NO: 28; a polynucleotide encoding the signal peptide represented by SEQ ID NO: 42; and a polynucleotide encoding the antigenic polypeptide represented by SEQ ID NO: 58 or the immunogenic protein thereof; (vii) 5'-UTR of SEQ ID NO: 28; a polynucleotide encoding the signal peptide represented by SEQ ID NO: 45; and a polynucleotide encoding the antigenic polypeptide represented by SEQ ID NO: 61 or the immunogenic protein thereof; (viii) 5'-UTR of SEQ ID NO: 28; a polynucleotide encoding the signal peptide represented by SEQ ID NO: 48; and a polynucleotide encoding the antigenic polypeptide represented by SEQ ID NO: 64 or the immunogenic protein thereof; (ix) 5'-UTR of SEQ ID NO: 1; a polynucleotide encoding the signal peptide represented by SEQ ID NO: 41; and a polynucleotide encoding the antigenic polypeptide represented by SEQ ID NO: 57 or the immunogenic protein thereof; (x) 5'-UTR of SEQ ID NO: 1; a polynucleotide encoding the signal peptide represented by SEQ ID NO: 44; and a polynucleotide encoding the antigenic polypeptide represented by SEQ ID NO: 60 or the immunogenic protein thereof; (xi) 5'-UTR of SEQ ID NO: 1; a polynucleotide encoding the signal peptide represented by SEQ ID NO: 47; and a polynucleotide encoding the antigenic polypeptide represented by SEQ ID NO: 63 or the immunogenic protein thereof; (xii) 5'-UTR of SEQ ID NO: 1; a polynucleotide encoding the signal peptide represented by SEQ ID NO: 50; and a polynucleotide encoding the antigenic polypeptide represented by SEQ ID NO: 66 or the immunogenic protein thereof; The mRNA structure according to claim 1, characterized in that it is one selected from the group consisting of the following.

19. The aforementioned 3'-UTRs are human α-globin 3'-UTR; 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 (bydV-PAV) 3'-UTR; and extension factor 1. The mRNA structure according to claim 1, characterized by being selected from the group consisting of α1(EEF1A1)3'-UTR; manganese peroxide dismutase (MnSOD)3'-UTR; β-subunit of mitochondrial H(+)-ATPase (β-mRNA)3'-UTR; GLUT13'-UTR; MEF2A3'-UTR and β-F1-ATPase3'-UTR.

20. The mRNA structure according to claim 18, characterized in that the 3'-UTR is the base sequence represented by Sequence ID No.

71.

21. The mRNA structure according to claim 1, characterized in that the poly(A) tail-like sequence has one or more nucleotides other than adenine, selected from the group consisting of uracil (U), cytosine (C), and guanine (G), inserted between multiple adenines or at the poly(A) tail end.

22. The mRNA structure according to claim 1, characterized in that it comprises one or more nucleic acids that have undergone backbone modification, sugar modification, or base modification.

23. A vaccine composition comprising the mRNA structure described in any one of claims 1 to 22.

24. The vaccine composition according to claim 23, characterized in that the mRNA structure is complexed with one or more lipids to form lipid nanoparticles or liposomes.

25. The vaccine composition according to claim 24, characterized in that the lipid nanoparticles include cationic lipids, PEG-modified lipids, sterols, and non-cationic lipids.

26. The vaccine composition according to claim 23, characterized in that the vaccine composition further comprises one or more adjuvants or activators.

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