5'-utr containing mRNA constructs with improved translation efficiency and vaccine compositions comprising same

Abstract

The present invention relates to an mRNA construct containing 5'-UTR with improved translation efficiency and a vaccine composition comprising same, and more particularly to an mRNA construct containing 5'-UTR with improved translation efficiency that have been engineered to include specific motifs, a codon-optimized signal sequence and an antigen encoding sequence, and a vaccine composition comprising the same. The mRNA construct according to the present invention contains a 5'-UTR with improved translation efficiency, which can effectively induce the expression of an antigenic polypeptide and is useful for vaccine development as it can be expected to increase immunogenicity as a vaccine.

Description

5'-UTR CONTAINING MRNA CONSTRUCTS WITH IMPROVED TRANSLATION EFFICIENCY AND VACCINE COMPOSITIONS COMPRISING SAME

[0001] The present invention relates to an mRNA construct containing 5'-UTR with improved translation efficiency and a vaccine composition comprising same, and more particularly to an mRNA construct containing 5'-UTR with improved translation efficiency that have been engineered to include specific motifs, a codon-optimized signal sequence and an antigen encoding sequence, and a vaccine composition comprising the same.

[0002]

[0003] Vaccines have been used since the 1500s, with the first specifically reported in the article on a smallpox vaccine in 1798, and since then, many different types of vaccines for treating numerous diseases have been developed and used to prevent diseases (Pollard, A.J et al., Nat Rev Immunol Vol. 21, pp. 83-100, 2021).

[0004] Vaccines can be categorized into live (live, attenuated), inactivated (killed), toxoid, subunit, VLP, other membrane-based, protein-polysaccharide conjugate, viral vector-based, and genetic vaccines.

[0005] Live vaccines were first used for smallpox, toxoid vaccines were first used for diphtheria, subunit vaccines were first used for anthrax, VLP vaccines were first used for hepatitis B, other membrane-based vaccines were first used for group B meningococcal, protein-polysaccharide conjugate vaccines were first used for influenza type B, viral vector-based vaccines were first used for Ebola, and mRNA vaccines were first used for SARS-CoV-2 (Pollard, A.J et al, Nat Rev Immunol Vol. 21, pp. 83-100, 2021).

[0006] Influenza virus (IV) is a single-stranded, negative-sense RNA virus composed of eight RNA segments of a single negative strand that expresses 10 to 13 different proteins and belongs to the Orthomyxoviridae family. Based on the antigenicity of the nucleoprotein (NP) and matrix (M) proteins, which are structural proteins attached to the surface of the virus, it is classified into types A, B, and C (Lamb RA et al., Fields Virology, pp. 1487-1531, 2001), and mainly types A and B are known to be pathogenic in humans. Type A can infect not only humans but also pigs and birds. Type B is the sole host of humans.

[0007] Influenza viruses are composed of two surface glycoproteins (surface antigens), hemagglutinin and neuraminidase. Influenza viruses are divided into subtypes based on the type and combination of hemagglutinin, an antigenic projection that binds to a receptor (sialic acid) of the host cell, and neuraminidase, which cleaves the sialic acid to release the sialic acid-bound virus into the cell. These subtypes are mainly categorized into influenza A. There are currently 18 different hemagglutinins, i.e., H1 to H18, and 11 different neuraminidases, i.e., N1 to N11, that can theoretically be combined to form 198 different subtypes of influenza A. Influenza B viruses are divided into two strains, Victoria and Yamagata, based on antigenic type.

[0008] Influenza is characterized by antigenic variation, which, as is typical of RNA viruses, occurs almost every year, either slightly or dramatically. Antigenic drift occurs almost every year, especially for influenza A and B, causing seasonal epidemics. Therefore, the World Health Organization (WHO) integrates information on the prevalence of the virus and publishes vaccine recommendations for the current season around February each year. Antigenic drift is a point mutation within the same subtype that results in a new hemagglutinin or neuraminidase with slightly altered antigenicity through a minor antigenic variation, and antigenic shift is a subtype change, such as H3N2 → H2N2, that results in a new virus. In addition to these two modifications, culturing in embryonated eggs can cause mutations that can lead to differences in antigenicity, which are not found in cell culture, so that vaccines using viruses cultured in mammalian cells are preferred.

[0009] Influenza vaccine should be given annually because of antigenic drift and a duration of effectiveness of less than one year. In Northern Hemisphere, the recommended time to get vaccinated is October through December of each year due to the length of the influenza season (December through April) and the duration of the vaccine's effectiveness (6 months on average).

[0010] Current influenza vaccines include inactivated killed vaccine and live attenuated influenza vaccine (LAIV). Inactivated killed vaccines are produced by inactivating viruses cultured in fertilized eggs (hatching eggs) with formalin or by inactivating viruses by growing them in cell culture with formalin, harvesting surface antigens, and injecting them intramuscularly (i.m.) as vaccine antigens. Live attenuated vaccines are administered by spraying into the nasal cavity.

[0011] Killed vaccines include whole virus vaccine, which uses the whole virus; split vaccine (subvirion), which is prepared by removing the viral envelope with Triton X-100; and subunit vaccine, which is prepared by purifying the hemagglutinin and neuraminidase components. Hemagglutinin and neuraminidase are antigens that directly trigger a neutralizing antibody response, and hemagglutinin is the primary neutralizing antigen. Whole virus vaccines are not widely used in Korea and globally, and are only available in a few countries, because they cause side effects in children. On the other hand, component vaccines, such as split or subunit vaccines, are very safe and effective, and are the most commonly used.

[0012] In addition, vaccines containing immune adjuvants such as MF-59 or virosome vaccines, which form virus-like vesicles, have been developed to enhance the immune response and are being used in some countries. In order to provide a sufficient degree of immune defense against influenza virus infection, especially for prophylactic or therapeutic purposes, it is necessary to incorporate potent and safe adjuvants into vaccines.

[0013] Antibodies against a particular subtype or influenza virus, acquired through natural infection or vaccination, cannot usually produce antibodies which are protective against other types or subtypes of the influenza virus, and may not be sufficiently immunogenic against new variants within a single antigen.

[0014] There is a challenge of yearly vaccination because influenza viruses undergo major and minor mutations from year to year, resulting in a change in the prevalence of the disease each year, making it difficult to expect protection effects from the previous year's vaccine.

[0015] Current seasonal influenza vaccines only provide protective immunity against the virus strain used in the vaccine, so there is a need to develop an affordable and effective influenza vaccine that can provide a sufficient degree of cross-protective immunity across various subtypes. To develop a universal vaccine with cross-protective immunity, antigens with minimal antigenic variation or methods that stimulate mucosal immunity are needed. One such approach is to use one or more HA2 domains of HA with low antigenic variation as vaccine antigens (Korean Patent No. 10-1637955).

[0016] Gene vaccines, on the other hand, began to be developed since it was reported that, when DNA or RNA encoding a target gene is directly injected into an animal, the target gene would be expressed in the living animal, and immunity would be conferred by this expression (Wolff JA et al. Science, 247:1465-8, 1990).

[0017] DNA and mRNA can be used as Active pharmaceutical ingredients (API) nucleic acid molecules for in gene vaccines, and DNA is known to be relatively stable and easy to handle compared to mRNA. However, in the case of DNA, there is a potential risk that the DNA-fragment administered into the patient's genome may be inserted at an unwanted location, resulting in gene damage. Furthermore, unwanted anti-DNA antibodies may appear. Another problem is that the expression level of the peptide or protein expressed by DNA administration and subsequent transcription / translation is limited. The presence of specific transcription factors that regulate DNA transcription has a major impact on the level of expression of the administered DNA, and in the absence of specific transcription factors, DNA transcription does not produce sufficient amounts of mRNA, and consequently, the level of translated peptides or proteins is limited.

[0018] On the other hand, when mRNA is used as an API for gene delivery, mRNA does not require transcription and can synthesize proteins directly in the cytoplasm without the need to enter the nucleus like DNA does, thus eliminating the risk of getting stuck in the cell's chromosomes and causing unwanted genetic damage. It also has a shorter half-life compared to DNA, so it does not induce long-term genetic modifications (Sayour EJ, et al., J Immunother Cancer Vol. 3, 13, 2015). Typical mRNA vaccines, when delivered into cells, are only activated for a short period of time to express the target protein and are destroyed by enzymatic reactions within a few days, leaving a specific immune response to the expressed target antigen (protein).

[0019] In addition, when using mRNA as a tool for gene delivery, it does not need to cross the nuclear membrane to work, only the cell membrane, so it can be used in smaller amounts than DNA to express the same amount of target protein as DNA. In addition, mRNA has immunostimulative properties of its own, so a smaller amount of mRNA is required to produce the same immune effect as DNA. By using mRNA instead of DNA for gene vaccination, the risk of unwanted genomic integration and the generation of anti-DNA antibodies is minimized or avoided. However, mRNA is considered to be a fairly unstable molecular species that can be readily degraded by ubiquitous RNase.

[0020] Although many advances have been made over the years, there are still challenges in the field of efficient methods for mRNA vaccination that can trigger adaptive immune responses, such as inefficient translation of mRNA due to premature degradation of antigens or inefficient release of mRNA from cells. Furthermore, there is an urgent need to reduce the doses of mRNA vaccines to reduce potential safety concerns and to make vaccines more affordable in the third world.

[0021] There are many challenges associated with delivering nucleic acids to cause desired responses in biological systems. While nucleic acid-based therapeutics, such as vaccines, hold tremendous promise, there is still a need to deliver nucleic acids more effectively to the appropriate sites within a cell or organism in order to realize this potential.

[0022] However, the use of nucleic acids for therapeutic and prophylactic purposes currently faces two challenges. First, naked mRNA is vulnerable to nuclease digestion in plasma. Second, naked mRNA has limited ability to access intracellular compartments where the associated translator resides. To address these issues, lipid nanoparticles(LNP) composed of varous lipid components such as neutral lipids, cholesterol, PEG, pegylated lipids, or oligonucleotides, and cationic lipids have been attempted to block the degradation of mRNA in plasma and facilitate cellular uptake of nucleic acids.

[0023] In addition, an mRNA vaccine containing several lipid nanoparticles in association with influenza vaccines has been reported (Korean Patent Publication No. 10-2018-0096591).

[0024]

[0025] SUMMARY OF INVENTION

[0026] Accordingly, the inventors of the present invention have made good faith efforts to solve the above problems and to develop mRNA constructs containing 5'-UTRs with improved translation efficiency, and have confirmed that by selecting artificial nucleic acid molecules that do not generate secondary structures, have low uridine, and do not contain sequences that reduce stability in a combination of artificial nucleic acid molecules of 30 bp in length, it is possible to obtain 5'-UTRs with improved translation efficiency, and that the performance of mRNA constructs comprising them as vaccines is superior, and have completed the present invention.

[0027] It is an object of the present invention to provide an mRNA construct encoding antigenic polypeptides or immunogenic fragments thereof.

[0028] Another object of the present invention is to provide a vaccine composition comprising the mRNA construct.

[0029] To accomplish the above objectives, the present invention provides an mRNA construct encoding antigenic polypeptides or immunogenic fragments thereof, wherein the mRNA construct comprises:

[0030] in order from 5' to 3',

[0031] a) a 5'-CAP structure;

[0032] b) a 5'-untranslated region (5'-UTR) polynucleotide represented by a nucleic acid sequence according to formula (I) or (II) below:

[0033] Formula (I): AG[N22]GCCACC,

[0034] Formula (II): AGGA[N19]RGCCACC,

[0035] wherein R indicates A, G or U;

[0036] c) a polynucleotide encoding a signal peptide;

[0037] d) a polynucleotide encoding an antigenic polypeptide or an immunogenic protein thereof;

[0038] e) 3'-untranslated region (3'-UTR); and

[0039] f) a poly (A) tail or poly (A) tail-like sequence consisting of 10 to 1000 adenine (A).

[0040] The present invention also provides an influenza vaccine composition comprising the mRNA construct.

[0041] The present invention also provides use of the vaccine composition for the prevention of influenza.

[0042] The present invention also provides a method of preventing influenza, comprising the step of administering the vaccine composition.

[0043] The present invention also provides use of the vaccine composition for the preparation of an agent for the prevention of influenza.

[0044]

[0045] FIG. 1 is a schematic illustration of the components of an mRNA construct according to one embodiment of the present invention.

[0046] FIG. 2 is a schematic diagram illustrating an animal testing process according to one embodiment of the present invention.

[0047] FIG. 3 shows the results of protein expression in mouse muscle of an influenza B / Yamagata HA mRNA construct made according to one embodiment of the present invention.

[0048] FIG. 4 shows the results of protein expression of an influenza B / Victoria HA mRNA construct in mouse muscle lysate, made in accordance with one embodiment of the present invention.

[0049] FIG. 5 shows the results of protein expression of an influenza A / H3N2 HA mRNA construct in mouse muscle lysate, made in accordance with one embodiment of the present invention.

[0050] FIG. 6 shows the results of protein expression an influenza A / H1N1 HA mRNA construct in mouse muscle lysate, made in accordance with one embodiment of the present invention.

[0051] FIG. 7 is a schematic diagram illustrating a process ofin vivoimmonogenicity test according to one embodiment of the present invention.

[0052] FIG. 8 shows the anti-HA specific IgG titer followed by immunization of influenza A / H1N1 HA mRNA construct made in accordance with one embodiment of the present invention.

[0053]

[0054] DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EXAMPLES

[0055] Unless otherwise defined, all technical and scientific terms used herein shall have the same meanings as commonly understood by those skilled in the art. In general, the nomenclature and the experimental methods described herein are well known and commonly used in the art.

[0056] In the present invention, it was sought to determine that an mRNA construct containing 5'-UTR with improved translation efficiency can be used to express influenza antigenic proteins at a higher efficiency than wild-type mRNA.

[0057] That is, in one embodiment of the present invention, it was found that utilizing an mRNA construct (FIG. 1) comprising a 5'-UTR polynucleotide with enhanced translation efficiency dramatically improves the expression efficiency of influenza antigenic proteins (FIGs. 3 to 6).

[0058] Thus, the present invention relates to, in one point of view,

[0059] an mRNA construct encoding an antigenic polypeptide or immunogenic fragment thereof, comprising:

[0060] in order from 5' to 3'

[0061] a) a 5'-CAP structure;

[0062] b) a 5'-untranslated region (5'-UTR) polynucleotide represented by a nucleic acid sequence according to formula (I) or (II) below:

[0063] Formula (I): AG[N22]GCCACC,

[0064] Formula (II): AGGA[N19]RGCCACC,

[0065] wherein R indicates A, G or U;

[0066] c) a polynucleotide encoding a signal peptide;

[0067] d) a polynucleotide encoding an antigenic polypeptide or an immunogenic protein thereof;

[0068] e) 3'-untranslated region (3'-UTR); and

[0069] f) a poly (A) tail or poly (A) tail-like sequence consisting of 10 to 1000 adenine (A).

[0070]

[0071] The 5'-CAP of a natural mRNA accompanies nuclear export, increasing mRNA stability, and is bound to an mRNA cap-binding protein (CBP), which results in mRNA stability at the cellular and translational levels through the association of poly(A) binding proteins with CBP to form a mature cyclic mRNA species. The cap further aids in the removal of introns proximal to the 5' during mRNA splicing.

[0072] 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 named m7GpppN). Additional examples of 5'-CAP structure include glyceryl, inverted deoxy basic (abasic) residue (moiety), 4',5' methylenenucleotide, 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 non-base moiety, 3'-2'-inverted nucleotide moiety, 3'-2'-inverted non-base moiety, 1,4-butanediol phosphate, 3'-phosphoramidate, hexyl phosphate, aminohexyl phosphate, 3'-phosphate, 3'phosphorothioate, phosphorodithioate, or a bridging or non-bridging methylphosphonate moiety.

[0073] These modified 5'-CAP structures can be used to modify the mRNA sequence of the synthetic nucleic acid molecule of the present invention.

[0074] Additional modified 5'-CAP structures that can be used in the present invention include CAP1 (additional methylation of the ribose of the nucleotide adjacent to m7GpppN), CAP2 (additional methylation of the ribose of the second nucleotide of the downstream of m7GpppN), CAP3 (additional methylation of the ribose of the third nucleotide of the downstream of m7GpppN), CAP4 (additional methylation of ribose on the fourth nucleotide of the 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.

[0075] In the present invention, the 5'-CAP structure may be formed from chemical RNA synthesis or in vitro transcription of RNA using a cCAP analog (co-transcriptional capping), or alternatively, the CAP structure may be formed in vitro using a capping enzyme (e.g., a commercially available capping kit).

[0076] As used herein, a CAP analog refers to a non-polymerizing di-nucleotide having a CAP function that, when introduced to the 5' end of an RNA molecule, promotes translation or localization, and / or prevents degradation of the RNA molecule. Non-polymerization means that CAP analogs do not have a 5' triphosphate and are therefore bound only at the 5' end and cannot be extended in the 3' direction by template-dependent RNA polymerases.

[0077] CAP analogs may include chemical structures selected from the group consisting of, but not limited to, m7GpppA, m7GpppAmpG, an dimethylated CAP analog, a trimethylated CAP analog (e.g., m2,2,7GpppA), a dimethylated symmetric CAP analog (e.g., m7Gpppm7A), or an anti-inverted CAP analog (e.g., ARCA; m7,2'OmeGpppA, m7,2'dGpppA, m7,3'OmeGpppA, m7,3'dGpppA, and tetraphosphate derivatives thereof).

[0078] Additional CAP analogs have been previously described (US 7,074,596, WO 2008 / 016473, WO 2008 / 157688, WO 2009 / 149253, WO 2011 / 015347, and WO 2013 / 059475).

[0079] In the present invention, the 5'-CAP structure may be selected from the group consisting of, but not limited to, m7GpppAmpG, m7,3'OmeApppG, and m7GpppA.

[0080]

[0081] In the present invention, the 5'-UTR may be any one of the sequences represented by SEQ ID Nos: 1 to 34 in Table 1 below, and more preferably by any one selected from the group consisting of SEQ ID Nos: 1, 2, 15, 28 and 30, but is not limited thereto.

[0082]

[0083] As used herein, the term "UTR" refers to an "untranslated region" that is located upstream (5') and / or downstream (3') of the coding region of a nucleic acid molecule described herein, and thus typically located on the side of the coding region. Thus, the term "UTR" generally includes the 3' untranslated region ("3'-UTR") and the 5'-untranslated region ("5'-UTR"). UTRs typically contain or may consist of nucleic acid sequences that are not translated into proteins. Typically, a UTR includes a "regulatory element".

[0084] The term "regulatory element" refers to a nucleic acid sequence having a gene regulatory activity, the expression of transcribable nucleic acid sequences which are operably (cis or trans) linked, and in particular the ability to influence transcription or translation. The term includes promoters, enhancers, internal ribosomal entry sites (IRES), introns, leaders, transcription termination signals, such as polyadenylation signals and poly-U sequences, and other regulatory elements of expression. The regulatory elements can act constitutively or in a time- and / or cell-specific manner. Optionally, the regulatory elements can function through the interaction (e.g., mobilization and binding) of regulatory proteins that can regulate (induce, enhance, reduce, discard, or prevent) expression, particularly transcription of genes.

[0085] The UTR is preferably "operably linked" to a coding region, i.e., arranged in a functional relationship, in such a way as to control (i.e., mediate or regulate, preferably enhance) the expression of the coding sequence.

[0086]

[0087] In the present invention, the term "5'-UTR" refers to a portion of a nucleic acid molecule, which is located 5' (i.e., "upstream") of the open reading frame, which is not translated into a protein. In the context of the present invention, a 5'-UTR begins at the transcription start site and ends one nucleotide before the start codon of the open reading frame.

[0088] The 5'-UTR can contain 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 by post-transcriptional modifications, for example by the addition of a 5'-CAP. Thus, the 5'-UTR may preferably correspond to a nucleic acid located between the 5'-CAP and the start codon, in particular a sequence of mature mRNA, and more specifically a nucleotide located at 3' in the 5'-CAP, preferably a nucleotide located at 5' to the start codon (transcription start site) of the protein coding sequence in the nucleotide located immediately after 3' of 5'-CAP, preferably a nucleotide located immeciately before 5' of the start codon (transcription start site) of the protein coding sequence.

[0089] The nucleotides located immediately at 3' of 5'-CAP of mature mRNA typically correspond to the transcription initiation sites. The 5' UTR typically has a length of less than 500, 400, 300, 250, or less than 200 nucleotides. In some examples, its length can be 10, 20, 30, or 40 or more nucleotides, preferably 10 or 50 or less nucleotides.

[0090]

[0091] In the present invention, the signal peptide may be derived from an antigenic polypeptide, immunoglobulin E (IgE), or tissue plasminogen activator (tPA), but is not limited thereto.

[0092]

[0093] In the present invention, the signal peptide of the antigenic polypeptide can be represented by an amino acid sequence of SEQ ID NO: 72 (MKAIIVLLMVVTSNA), SEQ ID NO:73 (MKXIIALSXILCLVFA, wherein X is T, A, Y or N), or SEQ ID NO:77 (MKAILVVXLYTFTTANA, wherein X is L or M). More preferably, it may be represented by an amino acid sequence of, but not limited to, SEQ ID NO: 72 (MKAIIVLLMVVTSNA), SEQ ID NO: 74 (MKTIIALSYILCLVFA), SEQ ID NO: 75 (MKTIIALSNILCLVFA), SEQ ID NO: 76 (MKAIIALSNILCLVFA), SEQ ID NO: 78 (MKAILVVMLYTFTTANA), or SEQ ID NO: 79 (MKAILVVLLYTFTTANA).

[0094] In the present invention, the signal peptide of IgE may be represented by an amino acid sequence of SEQ ID NO: 80, but is not limited thereto.

[0095] In the present invention, the signal peptide of tPA may be represented by the amino acid sequence of SEQ ID NO: 81, but is not limited thereto.

[0096]

[0097] In the present invention, the polynucleotide encoding the signal peptide may be codon optimized, preferably be any one of the sequences represented by SEQ ID Nos: 39 to 50, but not limited thereto.

[0098]

[0099] In the present invention, the polynucleotide encoding the 5'-UTR and the signaling protein may be any one selected from the group consisting of, but not limited to:

[0100] (i) a polynucleotide encoding a 5'-UTR of SEQ ID NO: 1 and a signal peptide of SEQ ID NO: 39;

[0101] (ii) a polynucleotide encoding a 5'-UTR of SEQ ID NO: 1 and a signal peptide of SEQ ID NO: 42;

[0102] (iii) a polynucleotide encoding a 5'-UTR of SEQ ID NO: 1 and a signal peptide of SEQ ID NO: 45;

[0103] (iv) a polynucleotide encoding a 5'-UTR of SEQ ID NO: 1 and a signal peptide of SEQ ID NO: 48;

[0104] (v) a polynucleotide encoding a 5'-UTR of SEQ ID NO: 28 and a signal peptide of SEQ ID NO: 39;

[0105] (vi) a polynucleotide encoding a 5'-UTR of SEQ ID NO: 28 and a signal peptide of SEQ ID NO: 42;

[0106] (vii) a polynucleotide encoding a 5'-UTR of SEQ ID NO: 28 and a signal peptide of SEQ ID NO: 45;

[0107] (viii) a polynucleotide encoding a 5'-UTR of SEQ ID NO: 28 and a signal peptide of SEQ ID NO: 48;

[0108] (ix) a polynucleotide encoding a 5'-UTR of SEQ ID NO: 1 and a signal peptide of SEQ ID NO: 41;

[0109] (x) a polynucleotide encoding a 5'-UTR of SEQ ID NO: 1 and a signal peptide of SEQ ID NO: 44;

[0110] (xi) a polynucleotide encoding a 5'-UTR of SEQ ID NO: 1 and a signal peptide of SEQ ID NO: 47; and

[0111] (xii) a polynucleotide encoding a 5'-UTR of SEQ ID NO: 1 and a signal peptide of SEQ ID NO: 50.

[0112]

[0113] In the present invention, the term "GC00-WT-HA" is an abbreviation for 5'-UTR-signal peptide-ORF, in accordance with one embodiment of the present invention, meaning that, although the same expression is used, different strains have different compositions of 5'-UTRs, signal peptides and ORF sequences, as described in Table 4 to Table 7 of the present invention.

[0114]

[0115] In the present invention, the antigenic polypeptide may one or more selected from the group consisting of, but not limited to, tumor antigens, pathogenic antigens, autoantigens, alloantigens, and allergenic antigens.

[0116]

[0117] 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. The terms "cancer" and "tumor" as used herein are used interchangeably to refer to neoplasms characterized by uncontrolled and generally rapid proliferation of cells that tend to invade surrounding tissues and metastasize to distant body sites. The terms include benign and malignant neoplasms. Malignant tumors are typically characterized by degeneration (anaplasia), invasiveness, and metastasis; benign tumors typically do not have these characteristics. The terms "cancer" and "tumor" refer specifically to cancers of the blood and lymphatic systems as well as neoplasms characterized by tumor growth. A "tumor antigen" is typically derived from a tumor / cancer cell, preferably a mammalian tumor / cancer cell, and may be located in or on the interior or surface of a tumor cell derived from a mammal, preferably a mammalian, preferably a human, or a tumor, e.g., a systemic or solid tumor. "Tumor antigen" generally includes a tumor-specific antigen (TSA) and a tumor-associated antigen (TAA). TSA is typically due to tumor-specific mutations and are specifically expressed by tumor cells. TAA, which is more common, is typically presented by tumor and "normal" (healthy, non-tumor) cells.

[0118]

[0119] In the present invention, the tumor antigen may be a protein or nucleic acid sequence associated with a tumor, wherein each nucleic acid sequence encodes a different peptide or protein; and wherein the at least one nucleic acid sequence may encode 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, A T-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, TGF-beta, TGF-betaRII, 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 an immunoglobulin genotype of a lymphoid cell or a T cell receptor genotype of a lymphocytic cell, or a homolog, fragment, variant, or derivative of the above tumor antigens.

[0120]

[0121] In the present invention, the tumor antigens are selected from the group consisting of, but not limited to, 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.

[0122]

[0123] In the present invention, the pathogenic antigen may be selected from the group consisting of bacterial, viral, fungal and protozoa antigens.

[0124] 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 chlamydiae, cytomegalovirus (CMV), hepatitis B virus (HBV), mycobacterium tuberculosis, rabies virus, and yellow fever virus, or any homolog, homologue, fragment, variant, or derivative of the above proteins.

[0125]

[0126] In the present invention, the antigenic polypeptide or immunogenic protein thereof is an influenza virus antigenic polypeptide, which is at least one selected from the group consisting of, but not limited to, a defined antigenic subdomain of hemagglutinin (HA), also known as HA1, HA2, or a combination of HA1 and HA2, and neuraminidase (NA), nucleoprotein (NP), matrix protein 1 (M1), matrix protein 2 (M2), non-structural protein 1 (NS1), and non-structural protein 2 (NS2).

[0127] In the present invention, the influenza virus antigenic polypeptide may be, but is not limited to, influenza hemagglutinin 1 (HA1), hemagglutinin 2 (HA2), or an immunogenic fragment of HA1 or HA2.

[0128] In the present invention, the influenza antigenic polypeptide or immunogenic protein thereof 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, more preferably may be an HA protein derived from each strain, but is not limited thereto.

[0129]

[0130] In the present invention, the polynucleotide encoding the influenza antigenic polypeptide or immunogenic protein thereof may be codon optimized, preferably any one of the sequences represented by SEQ ID Nos. 55 to 66, but not limited thereto.

[0131]

[0132] In the present invention, a polynucleotide encoding the 5'-UTR and a signal peptide; and a polynucleotide encoding an antigenic polypeptide or an immunogenic protein thereof are any one selected from the group consisting of, but not limited to:

[0133] (i) a polynucleotide encoding a 5'-UTR of SEQ ID NO: 1 and a signal peptide of SEQ ID NO: 39; and a polynucleotide encoding an antigenic polypeptide or an immunogenic protein thereof of SEQ ID NO: 55;

[0134] (ii) a polynucleotide encoding a 5'-UTR of SEQ ID NO: 1 and a signal peptide of SEQ ID NO: 42; and a polynucleotide encoding an antigenic polypeptide or an immunogenic protein thereof of SEQ ID NO: 58;

[0135] (iii) a polynucleotide encoding a 5'-UTR of SEQ ID NO: 1 and a signal peptide of SEQ ID NO: 45; and a polynucleotide encoding an antigenic polypeptide or an immunogenic protein thereof of SEQ ID NO: 61;

[0136] (iv) a polynucleotide encoding a 5'-UTR of SEQ ID NO: 1 and a signal peptide of SEQ ID NO: 48; and a polynucleotide encoding an antigenic polypeptide or an immunogenic protein thereof of SEQ ID NO: 64;

[0137] (v) a polynucleotide encoding a 5'-UTR of SEQ ID NO: 28 and a signal peptide of SEQ ID NO: 39; and a polynucleotide encoding an antigenic polypeptide or an immunogenic protein thereof of SEQ ID NO: 55;

[0138] (vi) a polynucleotide encoding a 5'-UTR of SEQ ID NO: 28 and a signal peptide of SEQ ID NO: 42; and a polynucleotide encoding an antigenic polypeptide or an immunogenic protein thereof of SEQ ID NO: 58;

[0139] (vii) a polynucleotide encoding a 5'-UTR of SEQ ID NO: 28 and a signal peptide of SEQ ID NO: 45; and a polynucleotide encoding an antigenic polypeptide or an immunogenic protein thereof of SEQ ID NO: 61;

[0140] (viii) a polynucleotide encoding a 5'-UTR of SEQ ID NO: 28 and a signal peptide of SEQ ID NO: 48; and a polynucleotide encoding an antigenic polypeptide or an immunogenic protein thereof of SEQ ID NO: 64;

[0141] (ix) a polynucleotide encoding a 5'-UTR of SEQ ID NO: 1 and a signal peptide of SEQ ID NO: 41; and a polynucleotide encoding an antigenic polypeptide or an immunogenic protein thereof of SEQ ID NO: 57;

[0142] (x) a polynucleotide encoding a 5'-UTR of SEQ ID NO: 1 and a signal peptide of SEQ ID NO: 44; and a polynucleotide encoding an antigenic polypeptide or an immunogenic protein thereof of SEQ ID NO: 60;

[0143] (xi) a polynucleotide encoding a 5'-UTR of SEQ ID NO: 1 and a signal peptide of SEQ ID NO: 47; and a polynucleotide encoding an antigenic polypeptide or an immunogenic protein thereof of SEQ ID NO: 63; and

[0144] (xii) a polynucleotide encoding a 5'-UTR of SEQ ID NO: 1 and a signal peptide of SEQ ID NO: 50; and a polynucleotide encoding an antigenic polypeptide or an immunogenic protein thereof of SEQ ID NO: 66.

[0145]

[0146] In the present invention, the 3'-UTR is selected from the group consisting of, but not limited to, 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 barley yellow dwarf virus (BYDV-PAV) 3'-UTR; elongation factor 1 α1 (EEF1A1) 3'-UTR; manganese superoxide dismutase (MnSOD) 3'-UTR; β subunit of mitochondrial H(+)-ATP synthase (β-mRNA) 3'-UTR; GLUT1 3'-UTR; MEF2A 3'-UTR; β-F1-ATPase 3'-UTR; functional fragments thereof and combinations thereof.

[0147] In the present invention, the 3'-UTR may be represented by a sequence of SEQ ID NO: 71.

[0148]

[0149] As used herein, the term "3'-UTR" typically refers to a portion of mRNA located between the protein-coding region of the mRNA (i.e., open reading frame, coding region) 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 genes that are transcribed into their respective mRNAs during the process of gene expression. This genomic sequence is first transcribed into immature mRNA, which contains optional introns. The immature mRNA is then further processed into mature mRNA in a maturation process. This maturation process includes steps such as 5'-capping, splicing of the immature mRNA to excise selective introns, and modification of the 3' end, such as polyadenylation of the 3' end of the immature mRNA, and selective endo- or exonuclease cleavage.

[0150] In the present invention, a 3'-UTR corresponds to a sequence in mature mRNA that is located at 3' to a stop codon of a protein coding region, preferably at 3' immediately next to the stop codon of the protein coding region, and extends to the 5' of the poly(A) sequence, preferably nucleotide immediately next to 5' to the poly(A) sequence. The term "corresponding" means that the 3'-UTR sequence may be an RNA sequence, as in the mRNA sequence used to define the 3'-UTR sequence, or a DNA sequence corresponding to such an RNA sequence.

[0151]

[0152] In the present invention, the poly(A) tail or poly(A) tail-like sequence further comprises a poly(A) tail. In further embodiments, the terminal groups on the poly(A) tail may be incorporated for stabilization. In another embodiment, the poly(A) tail comprises a des-3' hydroxyl tail.

[0153] During RNA processing, long chains 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 transcript can be cleaved to free the 3' hydroxyl. Subsequently, poly-A polymerase adds an adenine nucleotide chain to the RNA. A process called polyadenylation adds a poly(A) tail, which can be, for example, about 80 to about 250 residues in length (including about 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 residues in length). The poly(A) tail can also be added after the product has been effluxed from the nucleus.

[0154] According to the present invention, the terminal groups on the poly(A) tail may be incorporated for stabilization. The polynucleotides of the present invention may comprise a des-3' hydroxyl tail. They may also include structural moieties or 2'-O-methyl modifications as taught by Junjie Li et al. (Current Biology, Vol. 15, 1501-1507, August 23, 2005, the entirety of which is incorporated herein by reference)

[0155] The unique poly(A) tail length provides certain advantages for the polynucleotides of the present invention. Typically, the length of the poly(A) tail, 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).

[0156] In some embodiments, the polynucleotide or region thereof comprises about 30 to about 3,000 nucleotides (e.g., 30 to 50, 30 to 100, 30 to 250, 30 to 500, 30 to 750, 30 to 1,000, 30 to 1,500, 30 to 2,000, 30 to 2,500, 50 to 100, 50 to 250, 50 to 500, 50 to 750, 50 to 1,000, 50 to 1,500, 50 to 2,000, 50 to 2,500, 50 to 3,000, 100 to 500, 100 to 750, 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 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).

[0157] 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 coding region, the length of a specific feature or region, or the length of the ultimate product expressed from the polynucleotide.

[0158] In this connection, the poly(A) tail may be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% larger in length than the polynucleotide or feature thereof. The poly(A) tail can also be designed as a fraction of the polynucleotide to which it belongs. In this regard, the poly(A) tail can be at least 10, 20, 30, 40, 50, 60, 70, 80, or 90% of a total length of the fragment, a region of the fragment, or a total length of the fragment - the length of the poly(A) tail. Further, engineered binding sites and conjugation of polynucleotides to the poly-A binding protein can enhance expression.

[0159] Additionally, multiple distinct polynucleotides can be linked together via a poly-A binding protein (PABP) via 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 tested by ELISA at 12 hours, 24 hours, 48 hours, 72 hours, and 7 days after the transfection.

[0160] In some embodiments, the polynucleotides of the present invention are designed to include a polyA-G quartet region. A G-quartet is a cyclic hydrogen-bonded secondary structure of four guanine nucleotides that can be formed by G-rich sequences in both DNA and RNA. In this experiment, the 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 poly-A-G quartet results in protein production from mRNA equivalent to at least 75% of what could be found using a poly(A) tail of 120 nucleotides alone.

[0161]

[0162] In the present invention, the poly(A) tail-like sequence may be any nucleic acid sequence capable of performing the function of a poly(A) tail, and may be preferably characterized that one or more nucleotides other than adenines 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.

[0163]

[0164] In the present invention, the mRNA constructs may comprise one or more backbone-modified, sugar-modified, or base-modified nucleic acids, but are not limited thereto.

[0165] Sugar modification:

[0166] Modified nucleosides and nucleotides that may be incorporated into a modified mRNA compound comprising an mRNA sequence as described herein may be modified at a 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 may include, but not limited to, alkoxy or allyloxy (-OR, e.g., R = H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar); polyethylene glycol (PEG), -O(CH2CH2O)nCH2CH2OR; "locked" nucleic acid (LNA) in which the 2' hydroxyl is linked to the 4' carbon of the same ribose sugar, e.g., by methylene cross-linking; and an amino group (-O-amino, wherein the amino group, e.g., NRR, may be alkylamino, dialkylamino, heterocyclic, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, polyamino) or aminoalkoxy.

[0167] The "deoxy" modification may comprise hydrogen, amino (e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); or an amino group may be attached to the sugar via a linker, wherein the linker comprises at least one of the atoms C, N and O.

[0168] The sugar group can also contain one or more carbons having an opposite stereochemical arrangement compared to the corresponding carbons in ribose. Thus, the modified mRNA can comprise a nucleotide containing, for example, arabinose such as a sugar.

[0169] Backbone modification:

[0170] The phosphate backbone can be further modified in modified nucleosides and nucleotides, which can be incorporated into a modified mRNA compound comprising mRNA sequences as described herein. The phosphate group of the backbone can be modified by replacing one or more of the oxygen atoms with other substituents. In addition, modified nucleosides and nucleotides can include the complete replacement of an 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-bridging oxygens replaced by sulfur.

[0171] Phosphate linkers can also be modified by substitution of the linking oxygen with nitrogen (crosslinked phosphoroamidates), sulfur (crosslinked phosphorothioates), and carbon (crosslinked methylene-phosphonates).

[0172] Base modification:

[0173] Modified nucleosides and nucleotides that may be incorporated into a modified mRNA compound comprising an mRNA sequence as described herein may be further modified at a 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 can be chemically modified at the major groove face. In some embodiments, the major groove chemical modification can include an amino group, a thiol group, an alkyl group, or a halo group.

[0174] In a particularly preferred embodiment of the present invention, the nucleotide analogs / modifications are preferably selected from 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-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-chloropurine riboside-5'-triphosphate, 7-deazaadenosine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 8-azadenosine-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.

[0175] Particularly preferred are nucleotides for base modifications selected from the group consisting of nucleotides whose base is modified, comprising 5-methylcytidine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, and pseudoridine-5'-triphosphate.

[0176] In some embodiments, the modified nucleoside includes 5-pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-methoxyuridine, 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.

[0177] In some embodiments, the modified nucleoside includes 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, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-l-methyl-pseudoisocytidine.

[0178] In other embodiments, the modified nucleoside includes 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-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine.

[0179] In other embodiments, the modified nucleoside includes inosine, 1-methyl-inosine, wyosine, wybutosine, 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.

[0180] In some embodiments, the nucleotide may be modified at the major groove face and may include replacement of the hydrogen at C-5 of the uracil with a methyl or 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.

[0181] In an additional specific embodiment, the modified mRNA may include a nucleoside modification selected from 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, α-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytidine, 8-oxo-guanosine, 7-deaza-guanosine, N1-methyl-adenosine, 2-amino-6-chloro-purine, N6-methyl-2-amino-purine, pseudo-iso-cytidine, 6-chloro-purine, N6-methyl-adenosine, α-thio-adenosine, 8-azido-adenosine, and 7-deaza-adenosine.

[0182]

[0183] In another aspect, the present invention relates to an influenza vaccine composition comprising the mRNA construct.

[0184] As used in the present invention, a "vaccine" is typically understood to be a prophylactic or therapeutic substance that provides at least one antigen, preferably an antigenic peptide or protein. "Providing to at least an antigen" means, for example, that the vaccine comprises an antigen or that the vaccine comprises, for example, a molecule encoding an antigen. Thus, it is particularly envisaged that the vaccines of the present invention comprise 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 alloantigens, and which, when expressed and presented to the immune system, preferably induces an immune response to the respective antigen. However, synthetic nucleic acid (RNA) molecules encoding non-antigenic (poly-)peptides or proteins of interest may also be used in the vaccines of the present invention.

[0185]

[0186] In the present invention, the mRNA construct of the vaccine composition may be complexed with one or more lipids to form lipid nanoparticles or liposomes.

[0187] The lipid nanoparticles may include, but not limited to, cationic lipids, PEG-modified lipids, sterols, and non-cationic lipids.

[0188] In the present invention, the mRNA construct may be provided in a complexed, i.e. complexed or associated with one or more (poly-)cationic compounds, preferably (poly-)cationic polymers, (poly-)cationic peptides or proteins, for example protamines, (poly-)cationic polysaccharides and / or (poly-)cationic lipids. In this connection, the term "complexed" or "associated" refers to an intrinsically stable combination of at least one synthetic nucleic acid (RNA) molecule with one or more of the above compounds in a larger complex or assembly without covalent linkage.

[0189] Lipids

[0190] According to preferred embodiments, the mRNA construct of the present invention is complexed or conjugated with lipids (in particular cationic and / or neutral lipids) to form one or more lipid nanoparticles or liposomes. Thus, in some embodiments, the synthetic nucleic acid (RNA) molecules of the present invention can be provided in the form of lipid-based formulations, in particular liposomes comprising the synthetic nucleic acid (RNA) molecules, and / or lipid nanoparticles.

[0191] Lipid nanoparticles

[0192] According to some preferred embodiments, the mRNA construct of the present invention is complexed or conjugated with lipids (particularly cationic and / or neutral lipids) to form one or more lipid nanoparticles.

[0193] Preferably, the lipid nanoparticles (LNPs) may comprise: (a) at least one mRNA complex of the present 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.

[0194] In some embodiments, the LNP comprises, in addition to the at least one mRNA construct of the present invention, (i) at least one cationic lipid; (ii) a neutral lipid; (iii) a sterol, e.g., cholesterol; and a PEG-lipid, which may be included in a molar ratio of about 20-60% cationic lipids: 5-25% neutral lipids: 25-55% sterols; 0.5-15% PEG-lipids.

[0195] In some embodiments, the mRNA construct of the present invention can be formulated as aminoalcohol lipidoids. Aminoalcohol lipidoids that can be used in the present invention can be prepared by the method described in U.S. Patent No. 8,450,298, the entirety of which is incorporated herein by reference.

[0196] Liposomes

[0197] In some embodiments, the mRNA construct of the present invention is formulated into liposomes. Cationic lipid-based liposomes can form complexes with negatively charged nucleic acids (e.g., RNA) via electrostatic interactions, resulting in complexes that provide biocompatibility, low toxicity, and the potential for large-scale production required for in vivo clinical applications. Liposomes can be fused with plasma membranes for uptake; in the cell, the liposomes are processed through the phagocytosis pathway and nucleic acids are subsequently released from the endosome / carrier into the cytoplasm. Given that liposomes are essentially analogs of biological membranes and can be prepared from both natural and synthetic phospholipids, liposomes have long been recognized as drug delivery vehicles due to their excellent biocompatibility.

[0198] Liposomes typically consist of a lipid bilayer, which can be composed of cationic, anionic, or neutral (phosphorus) lipids and cholesterol, surrounding an 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, referred to as unilamellar, or multi-layered, referred to as multilamellar.

[0199] In vivo, liposome properties and behavior can be modified by adding hydrophilic polymeric coatings, such as polyethylene glycol (PEG), to the liposome surface to provide steric stability. In addition, liposomes can be used for specific targeting by attaching ligands (e.g., antibodies, peptides, and carbohydrates) to their surface or to the ends of attached PEG chains.

[0200] Liposomes typically exist as spherical vesicles and can have a size of 20 nm to several microns. Liposomes can be of different sizes, including, but not limited to, multilamellar vesicles (MLVs), which can be hundreds of nanometers in diameter and contain a series of concentric bilayers separated by narrow aqueous compartments; small unicellular vesicles (SUVs), which can be smaller than 50 nm in diameter; and large unilamellar vesicles (LUVs), which can be between 50 and 500 nm in diameter. Liposome designs may include, but are not limited to, opsonins or ligands to improve liposome attachment to unhealthy tissue or to activate events such as, but not limited to, endocytosis. The liposomes can include a low or high pH to enhance delivery of the pharmaceutical formulation.

[0201]

[0202] In the present invention, the vaccine composition may further comprise one or more adjuvants or activators.

[0203] In the broadest sense, an "adjuvant" or "adjuvant ingredient" is typically a pharmacologic and / or immunologic agent that can modify, e.g., enhance, the effectiveness of another active agent, e.g., a therapeutic agent or vaccine. In this regard, an "adjuvant" can be understood as any compound suitable to support the administration and delivery of a vaccine composition of the present invention. Specifically, an adjuvant may preferably enhance the immunostimulatory properties of the vaccine to which it is added. Furthermore, such an adjuvant may initiate or increase immune responses of the innate immune system, i.e., non-specific immune responses, without being bound thereto.

[0204] "Adjuvants" typically do not elicit an adaptive immune response. Thus far, the "adjuvant" is not qualified as an antigen. That is, when administered, the vaccine of the present invention typically initiates an adaptive immune response due to an antigenic peptide or protein, encoded by at least one coding sequence of a synthetic nucleic acid (RNA) molecule contained in the vaccine.

[0205] Suitable adjuvants may be selected from any adjuvants known to those skilled in the art and suitable in the present case, i.e., one that aids in the induction of an immune response in a mammal, such as TDM, MDP, muramyl dipeptide, pluronic, vitiligo solution, aluminum hydroxide, ADJUMERTM (polyphosphazene); aluminum phosphate gels; glucan from algae; algamulin; aluminum hydroxide gels (vitiligo); high protein-adsorbing aluminum hydroxide gels; low viscosity aluminum hydroxide gels; AF or SPT (emulsion of squalane (5%), Tween 80 (0.2%), Fluoronic L121 (1.25%), phosphate-buffered saline (pH 7.4); AVRIDINETM(propandiamine); a group of substances corresponding a pathogen-associated molecular pattern (PAMP), which reacts with pattern recognition receptor (PRR); CpG DNA; lipoprotein; flagella; poly I:C; saponin; squalene; tricaprin; 3D-MPL; or detoxified lipooligosaccharide (dLOS).

[0206]

[0207] In another aspect, the present invention relates to the use of the vaccine composition for the prevention of influenza.

[0208] As used herein, "influenza" refers to an infectious disease caused by an influenza virus, and may be used interchangeably with pandemic cold, circulating cold, or flu (FLU).

[0209] As used herein, "preventing" means any act of inhibiting influenza or delaying its progression by administration of a vaccine composition of the present invention.

[0210] In another aspect, the present invention relates to a method of preventing influenza comprising the step of administering the vaccine composition.

[0211] In another aspect, the present invention relates to the use of the vaccine composition for the preparation of an agent for the prevention of influenza.

[0212]

[0213] Examples

[0214] The present invention is described in more detail with reference to the following examples. These examples are intended solely to illustrate the present invention, and it would be apparent to one of ordinary skill in the art that the scope of the present invention is not to be construed as limited by these examples.

[0215]

[0216] Example 1. Preparation of an mRNA construct

[0217] 1-1 Preparation of an mRNA sequence

[0218] The 33 5'-UTRs were screened by the method of PCT / KR2022 / 019491 and an additional 5'-UTR of SEQ ID NO: 1 were selected as 5'-UTR sequences for HA protein expression (Table 3).

[0219] In addition, based on the wild-type Yamagata, Victoria, H3N2, and H1N1 signal sequences (SEQ ID Nos: 51, 52, 53, and 54, respectively) and ORF sequences (SEQ ID Nos: 67, 68, 69, and 70, respectively) listed in Table 3, signal sequences and ORF sequences for each strain were prepared through codon optimization. Furthermore, the signal sequences of the wild-type strains were replaced with codon-optimized IgE or tPA were also prepared.

[0220]

[0221]

[0222]

[0223]

[0224]

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246] The information of the mRNAs produced for in vivo protein expression test is shown in Tables 4 to 7 below.

[0247]

[0248]

[0249]

[0250]

[0251] 1-2. mRNA Synthesis and Purification

[0252] The mRNA constructs of Table 4 to Table 7 prepared in Example 1.1 according to the method described in PCT / KR2022 / 019491 were purified by in vitro transcription.

[0253] Specifically, IVT was performed under the conditions in Table 8, and then, upon completion of the reaction, the template DNA was removed by treating 1 U of Dnase I per 1 ug of DNA and reacting at 37 °C for 15-30 min, and the IVT product was purified by the manufacturer's method using Invitrogen's MEGAclear™ Kit (Austin, Tex.).

[0254]

[0255] Example 2. Preparation of mRNA-LNP complex

[0256] Ionic lipids, phospholipids, cholesterol, and PEG-lipid conjugates were dissolved in ethanol in a molar ratio of 50:10:38.5:1.5 and mixed in a 1:3 volume ratio with citrate buffer (pH 4, 50 mM) in which mRNA was dissolved. MC3 (MedChemExpress, USA) was used as the ionic lipid, DSPC (Avanti Polar Lipids, USA) as the phospholipid, cholesterol (Sigma Aldrich, USA) as the cholesterol, and 1-2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000) (Avanti Polar Lipids, USA) as the PEG-lipid conjugate.

[0257] For the generation of lipid nanoparticles (LNPs), NanoAssemblr IgniteTM (Precision Nanosystems, Inc., Canada) was used to mix ionic lipids and mRNA at a total flow rate (TFR) of 12 mL / min to achieve a nitrogen-to-phosphate ratio (N / P ratio) of 4 between the ionic lipids and mRNA. The prepared lipid nanoparticles were prepared using Amicon Ultra Centrifugal Filter, MWCO 10 kDa (Millipore, USA) for ethanol removal, buffer exchange and concentration, and 1X DPBS (Thermo Scientific, USA) for dilution, concentration and exchange. For cryopreservation, a final 300 mM sucrose solution was added as a cryoprotectant and stored frozen (-80°C).

[0258]

[0259] Example 3. Confirmation of mRNA construct performance in animal experiments

[0260] 3-1. Method of injecting mRNA constructs into animals and harvesting tissue for HA expression

[0261] 6-7 week old Balb / c female mice were purchased (OrientBio, Korea) and maintained under specific pathogen-free conditions. The right leg of the mice was shaved one day before the injection, and the 50 μL of mRNA-LNP complexes prepared in Example 2 were injected intramuscularly in the right tibialis muscle using an insulin syringe. 6 hours later the injection, the injected right tibialis muscle was harvested, collected in 1.7 mL tubes, and stored in an ultra-low temperature freezer at -70°C until the evaluation of HA protein expression level in the muscle lysates (FIG. 2).

[0262] 3-2. Method of obtaining muscle tissue lysate

[0263] The muscle lysates were prepared to detect HA protein in injected muscle. To obtain muscle lysates, the harvested mussle tissues were placed in a tube containing tissue lysis buffer (Tissue lysis buffer composition: 1x cell lysis buffer (Cell signaling technology, Cat No. #9803), protease and phosphatase inhibitor mini talets, EDTA-Free 1 tablet / 10 mL, ThermoFisher scientific, Cat No. A32961) and stainless beads, and then each tissue was lysed using a tissue lysator.

[0264] The tubes containing the lysated tissue were centrifuged at 13,000 rpm for 10 minutes in a 4°C centrifuge at 13,000 rpm, and the supernatant was transferred to a new 1.7 mL tube and used for HA protein expression.

[0265] 3-3. Measurement of HA protein in muscle lysates

[0266] Enzyme-linked immunosorent assay (ELISA) was performed to evaluate HA protein expression in muscle. The ELISA was performed using ELISA kit and capture antibodies from Sinobiological, Inc.(Yamagata: Cat No. 11053-MM09, Victoria: Cat No. 11053-MM06, H3N2: 11056-RP01, H1N1: Cat No. SEK001) were diluted in PBS (phosphate-buffered saline, Lonza, Cat No. 17-516Q) and added to a ELISA plate for protein coating at a final conecntraion of 1 to 2 μg / mL After16 to 20 hours incubation at a 4°C refrigerator, the ELISA plate was washed three times (wash buffer composition: 0.05% Tween-20 in PBS, Tween-20 (Sigma-Aldrich, Cat No. P1379-500 mL)), and satureated with a phosphate-buffered saline (PBS) solution containing 2% bovine serum albumin (bovine serum albumin: Sigma-Aldrich, Cat No. A3803-100G) for 1 hour at room temperature.

[0267] The amount of protein in previously obtained musclelysate was quantified by bicinchoninic acid (BCA) quantification (ThermoFisher Scientific, Cat No. 23225) and an equal amount of protein was used to measure HA protein in muscle.

[0268] After blocking, the ELISA plate was washed three times with the same washing buffer, and the prepared muscle lysates were added to the ELISA plate, and incubated for 2 hours at room temperature, and then washed three times. the HA detectionantibodies from Sinobiological, which are conjugated with either horseradish peroxidase (HRP) or biotin (Yamagata & Victoria: Cat No. SEK-11053, H3N2: Cat No. 11056-R014B, H1N1: Cat No. SEK001) were diluted at 0.5 to 1 μg / mL in PBS containing 0.5% bovine serum albumin, and 100 μL of the antibody solution was added and incubated for 1 hour at room temperature.

[0269] For H3N2, the ELISA plate was washed three times with the same washing buffer, and Strepravidin-HRP (Sigma-Aldrich, Cat No. S2438-250UG) was diluted 1:5,000 in PBS containing 0.5% bovine serum albumin, and 100 μL of the dilution solution was added and further incubated for 20 minutes at room temperature. The ELISA plate was then washed three times, visualized with TMB (3,3',5,5'-tetramethylbenzidine) solution (ThermoFisher Scientific, Cat No. 34028), at room temperature for 10 minutes. Afterwards, a stop solution (SeraCare, Cat No. 5150-0021) was added to stop the chromogenic reaction, and the absorbance was measured at a wavelength of 450 nm using a microplate reader (VersaMax) from Moleculardeveices to compare HA protein expression in the muscle.

[0270] 3-4. Method of injecting mRNA constructs to animals and collecting a serum for evaluating HA-specific immune responses

[0271] Animal experiments were performed to evaluate immune responses of the mRNA constructs. 6-7 week old Balb / c female mice were purchased (OrientBio, Korea) and maintained under specific pathogen-free conditions. The mice were immunized intramuscularly with 0.1 μg of each mRNA-LNP complexes in the right tibialis muscle at 50 μL using an insulin syringe (FIG. 7). Blood samples were collected 4 weeks after the immunization to obtain serum.

[0272] Blood samples were left at room temperature for 30 minutes to allow blood to clot, and then centrifuged at 10,000 rpm for 10 minutes in a 4°C centrifuge. After centrifugation, the supernatant was transferred to a new 1.7 mL tube and stored in a -20°C freezer until testing for humoral immune response.

[0273] 3-5. Measurement of values of HA antigen-specific IgG antibodies

[0274] An enzyme-linked immunosorbent assay (ELISA) was performed to measure HA antigen-specific IgG titers in serum. Influenza A / H1N1 virus split vaccine Was diluted in PBS and added to a ELISA plate for protein coating. After at least 12hours incubation at 4°C refrigerator, plates were washed three times with phosphate-buffered saline (PBS) containing 0.05% TWEEN-20 and saturated with PBS containing 2% bovine serum albumin (bovine serum albumin. Sigma, Cat No. A3803) for 1 hour at room temperature. After blocking, the ELISA plates were washed three times, and serum samples diluted 2,000-fold in PBS containing 2% bovine serum albumin added to the plate and incubated for 2 hours at room temperature. The plates were then washed three times, and the plates were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody (Southern Biotech, Cat No. 1031-05) for 1 hour at room temperature. The ELISA plates were then washed three times, visualized using TMB (3 3' 5 5'-tetramethylbenzidine. ThermoFisher Scientific, Cat No. 34028), 0 minutes. After visualization, a stop solution (SeraCare, Cat No. 5150-0021) was added to stop the chromogenic reaction, and the optical density was measured by spectroscopy at a wavelength of 450 nm using a microplate reader (VersaMax) from Moleculardeveices.

[0275]

[0276] Example 4. Comparison of HA expression in influenza B / Yamagata strains based on 5'UTR and signal sequence variation

[0277] To enhance the immunogenicity of the mRNA construct, an ELISA assay was performed using the method of Example 3 to compare the mRNA sequence produced in Example 1 with the sequence of influenza virus itself.

[0278] As a result, it was found that the mRNA sequences produced in Example 1 exhibited higher in vivo protein expression compared to the influenza B / Yamagata strain's own HA sequence, as shown in FIG. 3 (FIG. 3, control vs. each lane).

[0279]

[0280] Example 5. Comparison of HA expression in influenza B / Victoria strains based on 5'UTR and signal sequence variation

[0281] To enhance the immunogenicity of the mRNA construct, an ELISA assay was performed using the method of Example 3 to compare the mRNA sequence produced in Example 1 with the sequence of influenza virus itself.

[0282] As a result, it was confirmed that the mRNA sequences produced in Example 1 exhibited higherin vivoprotein expression compared to the influenza B / Victoria strain's own HA sequence, as shown in FIG. 4 (FIG. 4, control vs. each lane).

[0283]

[0284] Example 6. Comparison of HA expression in influenza A / H3N2 strains based on 5'UTR and signal sequence variation

[0285] To enhance the immunogenicity of the mRNA construct, an ELISA assay was performed using the method of Example 3 to compare the mRNA sequence produced in Example 1 with the sequence of influenza virus itself.

[0286] As a result, it was found that the mRNA sequences produced in Example 1 exhibited higher in vivo protein expression compared to the influenza A / H3N2 strain's own HA sequence, as shown in FIG. 5 (FIG. 5, control vs. each lane).

[0287]

[0288] Example 7. Comparison of HA expression in influenza A / H1N1 strains based on 5'UTR and signal sequence variation

[0289] To enhance the immunogenicity of the mRNA construct, an ELISA assay was performed using the method of Example 3 to compare the mRNA sequence produced in Example 1 with the sequence of influenza virus itself.

[0290] As a result, it was found that the mRNA sequences produced in Example 1 exhibited higher in vivo protein expression compared to the influenza A / H1N1 strain's own HA sequence, as shown in FIG. 6 (FIG. 6, control vs. each lane).

[0291]

[0292] Example 8. Comparison of immune responses in influenza A / H1N1 strains based on 5'UTR and signal sequence changes

[0293] To enhance the immunogenicity of the mRNA construct, an anti-influenza specific IgG ELISA assay was performed using the method of Example 3 to compare the mRNA sequence produced in Example 1 with the sequence of influenza virus itself.

[0294] As a result, it was confirmed that the mRNA sequences produced in Example 1 exhibited higher immunogenicity compared to the influenza A / H1N1 strain's own antigen, as shown in FIG. 8 (FIG. 8, control vs. each lane).

[0295]

[0296] Example 9. Screening for 5'UTR and signal sequence combinations with high expression and immunogenicity

[0297] Based on the results of Examples 4 to 8, 5'UTR and signal sequence combinations with high in vivo HA expression and high immunogenicity were screened. The criterion was determined as a combination with at least 1.5-fold higher in vivo HA expression than the control group, and at least 3-fold higher immunogenicity than the control group.

[0298] First, the in vivo HA expression of Examples 4 to 7 summarized as fold change relative to the control group is shown in Table 9 below. In Table 9, GC00-HA is equivalent to GC00_WT_HA, GC00-IgE is equivalent to GC00_IgE_HA, and GC00-tPA is equivalent to GC00_tPA_HA. The rest of the terms are used interchangeably.

[0299]

[0300] As shown in Table 9, it was found that the combinations that increased expression at least 1.5-fold in all strains were GC00-HA, GC27-HA, GC29-HA, GC00-tPA, and GC14-tPA.

[0301] The immunogenicity of the H1N1 strain of Example 8 was then summarized as fold change compared to the control group in Table 10 below.

[0302]

[0303] As shown in Table 10, it was found that the combinations with at least 3-fold immunogenicity were GC00-HA, GC01-HA, GC14-HA, GC27-HA, GC00-tPA, GC01-tPA, GC27-tPA, and GC29-tPA, and among them, the combinations with at least 1.5-fold HA expression were GC00-HA (GC00_WT_HA), GC27-HA (GC27_WT_HA), and GC00-tPA (GC00_tPA_HA), which were selected as the final combinations.

[0304]

[0305] The mRNA construct according to the present invention contains a 5'-UTR polynucleotide with improved translation efficiency, which can effectively induce the expression of an antigenic polypeptide, which is useful for vaccine development as it can be expected to increase the immunogenicity of the vaccine.

[0306]

[0307] While the foregoing has described in detail certain aspects of the present invention, it would be apparent to one of ordinary skill in the art that these specific descriptions are merely preferred examples and are not intended to limit the scope of the present invention. Accordingly, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0308]

[0309] Attached as an electronic file.

Claims

1.An mRNA construct encoding an antigenic polypeptide or immunogenic fragment thereof, comprisingin order from 5' to 3',a) a 5'-CAP structure;b) a 5'-untranslated region (5'-UTR) polynucleotide represented by a nucleic acid sequence according to formula (I) or (II) below:Formula (I): AG[N22]GCCACC,Formula (II): AGGA[N19]RGCCACC,wherein R indicates A, G or U;c) a polynucleotide encoding a signal peptide;d) a polynucleotide encoding an antigenic polypeptide or an immunogenic protein thereof;e) 3'-untranslated region (3'-UTR); andf) a poly (A) tail or poly (A) tail-like sequence consisting of 10 to 1000 adenine (A).2.The mRNA construct according to claim 1, wherein the 5'-CAP structure is selected from the group consisting of m7GpppAmpG, m7GpppApG, and m7,3'OmeApppG.3.The mRNA construct according to claim 1, wherein the 5'-UTR is any one of the sequences represented by SEQ ID Nos: 1 to 34.4.The mRNA construct according to claim 3, wherein the 5'-UTR is any one selected from the group consisting of SEQ ID Nos: 1, 2, 15, 28, and 30.5.The mRNA construct according to claim 1, wherein the signal peptide is derived from an antigenic polypeptide, immunoglobulin E (IgE), or tissue plasminogen activator (tPA).6.The mRNA construct according to claim 5, wherein the signal peptide is an amino group represented by SEQ ID NO: 72 (MKAIIVLLMVVTSNA), SEQ ID NO: 73 (MKXIIALSXILCLVFA, wherein X is T, A, Y or N), SEQ ID NO: 77 (MKAILVVXLYTFTTANA, wherein X is L or M), SEQ ID NO: 80 (MDWTWILFLVAAATRVHS), or SEQ ID NO: 81 (MDAMKRGLCCVLLLCGAVFVSA).7.The mRNA construct according to claim 1, wherein the polynucleotide encoding the signal peptide is codon optimized.8.The mRNA construct according to claim 7, wherein the polynucleotide encoding the signal peptide is any one of the sequences represented by SEQ ID NOs: 39 to 50.9.The mRNA construct according to claim 1, wherein the polynucleotide encoding the 5'-UTR and the signal peptide is any one selected from the group consisting of:(i) a polynucleotide encoding a 5'-UTR of SEQ ID NO: 1 and a signal peptide of SEQ ID NO: 39;(ii) a polynucleotide encoding a 5'-UTR of SEQ ID NO: 1 and a signal peptide of SEQ ID NO: 42;(iii) a polynucleotide encoding a 5'-UTR of SEQ ID NO: 1 and a signal peptide of SEQ ID NO: 45;(iv) a polynucleotide encoding a 5'-UTR of SEQ ID NO: 1 and a signal peptide of SEQ ID NO: 48;(v) a polynucleotide encoding a 5'-UTR of SEQ ID NO: 28 and a signal peptide of SEQ ID NO: 39;(vi) a polynucleotide encoding a 5'-UTR of SEQ ID NO: 28 and a signal peptide of SEQ ID NO: 42;(vii) a polynucleotide encoding a 5'-UTR of SEQ ID NO: 28 and a signal peptide of SEQ ID NO: 45;(viii) a polynucleotide encoding a 5'-UTR of SEQ ID NO: 28 and a signal peptide of SEQ ID NO: 48;(ix) a polynucleotide encoding a 5'-UTR of SEQ ID NO: 1 and a signal peptide of SEQ ID NO: 41;(x) a polynucleotide encoding a 5'-UTR of SEQ ID NO: 1 and a signal peptide of SEQ ID NO: 44;(xi) a polynucleotide encoding a 5'-UTR of SEQ ID NO: 1 and a signal peptide of SEQ ID NO: 47; and(xii) a polynucleotide encoding a 5'-UTR of SEQ ID NO: 1 and a signal peptide of SEQ ID NO: 50.10.The mRNA construct according to claim 1, wherein the antigenic polypeptide is at least one selected from the group consisting of tumor antigens, pathogenic antigens, autoantigens, alloantigens, and allergenic antigens.11.The mRNA construct according to claim 10, wherein 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 construct according to claim 10, wherein the pathogenic antigen is selected from the group consisting of bacterial, viral, fungal, and protozoal antigens.13.The mRNA construct according to claim 12, wherein the virus is an influenza virus.14.The mRNA construct according to claim 10, wherein the antigenic polypeptide is an influenza hemagglutinin 1 (HA1), hemagglutinin 2 (HA2), or an immunogenic fragment of HA1 or HA2.15.The mRNA construct according to claim 14, wherein 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 construct according to claim 1, wherein the polynucleotide encoding the antigenic polypeptide or immunogenic protein thereof is codon optimized.17.The mRNA construct according to claim 16, wherein the polynucleotide encoding the antigenic polypeptide or the immunogenic protein thereof is any one of the sequences represented by SEQ ID NOs: 55 to 66.18.The mRNA construct according to claim 1, wherein a polynucleotide encoding the 5'-UTR and a signal peptide; and a polynucleotide encoding an antigenic polypeptide or an immunogenic protein thereof are any one selected from the group consisting of:(i) a polynucleotide encoding a 5'-UTR of SEQ ID NO: 1 and a signal peptide of SEQ ID NO: 39; and a polynucleotide encoding an antigenic polypeptide or an immunogenic protein thereof of SEQ ID NO: 55;(ii) a polynucleotide encoding a 5'-UTR of SEQ ID NO: 1 and a signal peptide of SEQ ID NO: 42; and a polynucleotide encoding an antigenic polypeptide or an immunogenic protein thereof of SEQ ID NO: 58;(iii) a polynucleotide encoding a 5'-UTR of SEQ ID NO: 1 and a signal peptide of SEQ ID NO: 45; and a polynucleotide encoding an antigenic polypeptide or an immunogenic protein thereof of SEQ ID NO: 61;(iv) a polynucleotide encoding a 5'-UTR of SEQ ID NO: 1 and a signal peptide of SEQ ID NO: 48; and a polynucleotide encoding an antigenic polypeptide or an immunogenic protein thereof of SEQ ID NO: 64;(v) a polynucleotide encoding a 5'-UTR of SEQ ID NO: 28 and a signal peptide of SEQ ID NO: 39; and a polynucleotide encoding an antigenic polypeptide or an immunogenic protein thereof of SEQ ID NO: 55;(vi) a polynucleotide encoding a 5'-UTR of SEQ ID NO: 28 and a signal peptide of SEQ ID NO: 42; and a polynucleotide encoding an antigenic polypeptide or an immunogenic protein thereof of SEQ ID NO: 58;(vii) a polynucleotide encoding a 5'-UTR of SEQ ID NO: 28 and a signal peptide of SEQ ID NO: 45; and a polynucleotide encoding an antigenic polypeptide or an immunogenic protein thereof of SEQ ID NO: 61;(viii) a polynucleotide encoding a 5'-UTR of SEQ ID NO: 28 and a signal peptide of SEQ ID NO: 48; and a polynucleotide encoding an antigenic polypeptide or an immunogenic protein thereof of SEQ ID NO: 64;(ix) a polynucleotide encoding a 5'-UTR of SEQ ID NO: 1 and a signal peptide of SEQ ID NO: 41; and a polynucleotide encoding an antigenic polypeptide or an immunogenic protein thereof of SEQ ID NO: 57;(x) a polynucleotide encoding a 5'-UTR of SEQ ID NO: 1 and a signal peptide of SEQ ID NO: 44; and a polynucleotide encoding an antigenic polypeptide or an immunogenic protein thereof of SEQ ID NO: 60;(xi) a polynucleotide encoding a 5'-UTR of SEQ ID NO: 1 and a signal peptide of SEQ ID NO: 47; and a polynucleotide encoding an antigenic polypeptide or an immunogenic protein thereof of SEQ ID NO: 63; and(xii) a polynucleotide encoding a 5'-UTR of SEQ ID NO: 1 and a signal peptide of SEQ ID NO: 50; and a polynucleotide encoding an antigenic polypeptide or an immunogenic protein thereof of SEQ ID NO: 66.19.The mRNA construct according to claim 1, wherein the 3'-UTR is selected from 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 barley yellow dwarf virus (BYDV-PAV) 3'-UTR; elongation factor 1 α1 (EEF1A1) 3'-UTR; manganese superoxide dismutase (MnSOD) 3'-UTR; β subunit of mitochondrial H(+)-ATP synthase (β-mRNA) 3'-UTR; GLUT1 3'-UTR; MEF2A 3'-UTR; and β-F1-ATPase 3'-UTR.20.The mRNA construct according to claim 18, wherein the 3'-UTR is a sequence represented by SEQ ID NO: 71.21.The mRNA construct according to claim 1, wherein in the poly(A) tail-like sequence, one or more nucleotides other than adenines 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.22.The mRNA construct according to claim 1, wherein the mRNA construct comprises one or more backbone-modified, sugar-modified, or base-modified nucleic acids.23.A vaccine composition comprising the mRNA construct of any one of claims 1 to 22.24.The vaccine composition according to claim 23, wherein the mRNA construct is complexed with one or more lipids to form lipid nanoparticles or liposomes.25.The vaccine composition according to claim 24, wherein the lipid nanoparticles comprise cationic lipids, PEG-modified lipids, sterols, and non-cationic lipids.26.The vaccine composition according to claim 23, wherein the vaccine composition further comprises one or more adjuvants or active agents.

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