Expression system and nucleic acid-based pharmaceutical composition containing the same

Abstract

Disclosed is a nucleic acid molecule comprising a translational regulatory element having translation initiation activity and a coding region consisting of nucleotides operably linked to the translational regulatory element and encoding an immunogen or a fragment thereof of influenza virus or severe fever with thrombocytopenia syndrome virus (SFTSV). The nucleic acid molecule or an expression system into which the nucleic acid molecule is inserted can be utilized as a pharmaceutical composition, such as an mRNA vaccine or a gene therapy platform, for treating or preventing influenza or SFTS.

Description

Technical Field

[0001] This patent application claims the benefit of priority of Korean Patent Application No. 10-2021-0100099, filed on July 29, 2021, and Korean Patent Application No. 10-2021-0177977, filed on December 13, 2021. The present disclosure relates to an expression system, and more particularly, to a viral immunogen expression system and a nucleic acid-based pharmaceutical composition using the same.

Background Art

[0002] Various infectious diseases that spread to humans have been well known since before modern times, and sometimes these infectious diseases have led to severe pandemics. Many of the European population died due to the Black Death that prevailed in medieval Europe. Such pandemics of infectious diseases have continued into modern times. It is known that at the beginning of the 20th century, more people died from the Spanish flu than those who died in combat in World War I. In addition, COVID-19, which occurred in Wuhan, China in 2019, has undergone various mutations and is still causing a global pandemic.

[0003] Vaccines are widely used as a strategy for preventing such infectious diseases. Traditionally manufactured vaccines are protein vaccines that attenuate or inactivate the antigens of the virus or bacteria that cause infectious diseases. Vaccines manufactured in the traditional way have effectively protected humans from many infectious diseases, but there are also limitations. In particular, it is difficult to develop effective vaccines against various infectious pathogens that can avoid the acquired immune response. In addition, a major difficulty in the development of most new vaccines is the need for rapid development and large-scale distribution of the vaccines rather than their effectiveness. Therefore, vaccine development using nucleic acids has become a good option. As a result, during the COVID-19 pandemic that occurred in 2019, nucleic acid-based mRNA vaccines were rapidly developed, received emergency use authorization in various countries, and mRNA vaccinations were carried out worldwide. Therefore, in addition to COVID-19, it is necessary to develop a nucleic acid-based vaccine platform that can efficiently prevent infectious diseases.

Summary of the Invention

Problems to be Solved by the Invention

[0004] An object of the present disclosure is to provide a nucleic acid molecule and a recombinant expression vector capable of efficiently expressing an immunogenic protein or peptide related to an infectious disease, and a nucleic acid-based pharmaceutical composition using the same.

Means for Solving the Problems

[0005] In one aspect, the present disclosure provides a nucleic acid molecule comprising a translation regulator derived from troponin T1 (TNNT1) and a coding region operably linked to the translation regulator, wherein the coding region contains nucleotides encoding an immunogen of influenza virus or severe fever with thrombocytopenia syndrome virus (SFTSV) or a fragment thereof.

[0006] The translation regulatory factor may include a first translation regulatory factor located upstream of the coding region and / or a second translation regulatory factor located downstream of the coding region. As an example, the first translation regulatory factor may consist of the nucleotides of SEQ ID NO: 1 or its transcript, and the second translation regulatory factor may consist of the nucleotides of SEQ ID NO: 2 or its transcript.

[0007] In an exemplary embodiment, the immunogen of the influenza virus may be the hemagglutinin of the influenza virus or a fragment thereof. The immunogen of the influenza virus may consist of the amino acids of SEQ ID NO: 3. The nucleotide encoding the immunogen of the influenza virus or a fragment thereof may consist of the nucleotides of SEQ ID NO: 4 or its transcript.

[0008] In an alternative embodiment, the immunogen of the severe fever with thrombocytopenia syndrome virus may be the glycoprotein N of the severe fever with thrombocytopenia syndrome virus or a fragment thereof. The immunogen of the severe fever with thrombocytopenia syndrome virus may consist of amino acids selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9.

[0009] The nucleotide encoding the immunogen of the severe fever with thrombocytopenia syndrome virus or a fragment thereof may consist of nucleotides selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 10 or their transcripts. As an example, the nucleic acid molecule may be a nucleic acid molecule in the form of RNA. The nucleic acid molecule may further include at least one nucleotide of a transcription regulatory factor operably linked to the coding region and a polyadenylation signal sequence or polyadenosine sequence located downstream of the transcription regulatory factor.

[0010] In another aspect, the present disclosure provides a recombinant expression vector comprising the nucleic acid molecule described above. In yet another aspect, the present disclosure provides a pharmaceutical composition for treating or preventing influenza or severe fever with thrombocytopenia syndrome, comprising the nucleic acid molecule described above or an expression construct into which the nucleic acid molecule is inserted as an active ingredient. For example, the pharmaceutical composition can be a vaccine composition.

[0011] Optionally, the pharmaceutical composition may further comprise at least one of an immunopotentiator, a nucleic acid stabilizer, and lipid nanoparticles. In yet another aspect, the present disclosure provides a method for treating or preventing influenza or severe fever with thrombocytopenia syndrome, comprising administering to a subject the nucleic acid molecule described above that is pharmaceutically acceptable or an expression construct into which the nucleic acid molecule is inserted.

Advantages of the Invention

[0012] Through the nucleic acid molecule of the present disclosure and the expression system into which the nucleic acid molecule is inserted, proteins and / or peptides derived from influenza virus or severe fever with thrombocytopenia syndrome virus (SFTSV) can be efficiently expressed. The nucleic acid molecule and / or expression construct of the present disclosure can be utilized as a nucleic acid-based pharmaceutically active material and as a pharmaceutical composition for treating and / or preventing influenza caused by influenza virus or severe fever with thrombocytopenia syndrome (SVTS). By administering the nucleic acid molecule and / or expression construct to a living body, a Th1 immune response and a Th2 immune response can be efficiently induced in vivo.

[0013] Therefore, the nucleic acid molecule and / or the expression construct containing the same according to the present disclosure can be utilized as an active ingredient of a drug such as a nucleic acid-based vaccine that can efficiently prevent or treat influenza caused by influenza virus or SFTS.

Brief Description of the Drawings

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[0015] Definition of Terms As used herein, the term "amino acid" is used in its broadest sense and is intended to include naturally occurring L-amino acids or residues. Amino acids include not only D-amino acids, but also chemically modified amino acids, such as amino acid analogs, naturally occurring amino acids that are not normally incorporated into proteins, such as norleucine, and chemically synthesized compounds having properties known in the art as characteristic of amino acids. For example, analogs or mimics of phenylalanine or proline that allow the same conformational restrictions of peptide compounds as natural Phe or Pro are included within the definition of amino acids. Such analogs and mimics are referred to herein as "functional equivalents" of amino acids.

[0016] For example, synthetic peptides synthesized by standard solid-phase synthesis techniques are not restricted to the amino acids encoded by genes, thereby allowing a wider variety of substitutions for a given amino acid. Amino acids not encoded by the genetic code may be referred to herein as "amino acid analogs". For example, amino acid analogs include 2-aminoadipic acid (Aad) for Glu and Asp; 2-aminopimelic acid (Apm) for Glu and Asp; 2-aminobutyric acid (Abu) for Met, Leu and other aliphatic amino acids; 2-aminoheptanoic acid (Ahe) for Met, Leu and other aliphatic amino acids; 2-aminobutyric acid (Aib) for Gly; cyclohexylalanine (Cha) for Val, Leu and Ile; homoarginine (Har) for Arg and Lys; 2,3-diaminopropionic acid (Dap) for Lys, Arg and His; N-ethylglycine (EtGly) for Gly, Pro and Ala; N-ethylglycine (EtGly) for Gly, Pro and Ala; N-ethylasparagine (EtAsn) for Asn and Gln; hydroxylysine (Hyl) for Lys; allo-hydroxylysine (AHyl) for Lys; 3-(and 4-)hydroxyproline (3Hyp, 4Hyp) for Pro, Ser and Thr; alloisoleucine (AIle) for Ile, Leu and Val; 4-amidinophenylalanine for Arg; N-methylglycine (MeGly, sarcosine) for Gly, Pro and Ala; N-methylisoleucine (MeIle) for Ile; norvaline (Nva) for Met and other aliphatic amino acids; norleucine (Nle) for Met and other aliphatic amino acids; ornithine (Orn) for Lys, Arg and His; citrulline (Cit) and methionine sulfoxide (MSO) for Thr, Asn and Gln; and N-methylphenylalanine (MePhe), trimethylphenylalanine, halo-(F-, Cl-, Br- or I-)phenylalanine or trifluorolylphenylalanine for Phe.

[0017] As used herein, the term "peptide" encompasses all proteins, protein fragments, and peptides that are isolated from naturally occurring sources, produced by recombinant technique, or chemically synthesized. For example, the peptides of the present disclosure are composed of at least 5 amino acids, and by way of example, at least 10 amino acids. In certain embodiments, variants of the compounds are provided, such as peptide variants having one or more amino acid substitutions. As used herein, "peptide variants" are those in which one or more amino acids have been substituted, deleted, added, and / or inserted into the amino acid sequence of the peptide, and which exhibit substantially the same biological function as the peptide composed of the original amino acids. Peptide variants must have at least 70%, preferably at least 90%, more preferably at least 95% identity with the original peptide.

[0018] Such variants may include amino acid substitutions known as "conservative" substitutions. Variants can also include nonconservative changes. By way of example, the sequence of the variant polypeptide differs from the original sequence by the substitution, deletion, addition, or insertion of 5 or fewer amino acids. Variants can also be changed by the deletion or addition of amino acids that have minimal effect on the immunogenicity, secondary structure, and hydropathic nature of the peptide.

[0019] "Conservative" substitution means that even when one amino acid is substituted for another, there are no significant changes in the secondary structure of the polypeptide and properties such as its hydropathic nature. Amino acid mutations are based on the relative similarity of the amino acid side chain substituents, for example, similarities such as polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphipathic nature.

[0020] For example, amino acids can be classified by common side chain properties into: a) hydrophobic (norleucine, methionine, alanine, valine, leucine, isoleucine); b) neutral hydrophilic (cysteine, serine, threonine, asparagine, glutamine); c) acidic (aspartic acid, glutamic acid); d) basic (histidine, lysine, arginine); e) residues that affect the chain direction (glycine, proline); f) aromatic (tryptophan, tyrosine, phenylalanine). Conservative substitutions involve exchanging one member of each of these classes for another member of the same class.

[0021] Analysis of the size, shape and type of amino acid side chain substituents reveals that arginine, lysine and histidine are all positively charged residues; alanine, glycine and serine have similar sizes; and phenylalanine, tryptophan and tyrosine have similar shapes. Thus, based on such considerations, arginine, lysine and histidine; alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine can be said to be biologically functional equivalents.

[0022] In introducing mutations, the hydropathic index of amino acids can be considered. Each amino acid is assigned a hydropathic index based on hydrophobicity and charge: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0023] In conferring the interactive biological function of proteins, the hydrophobic amino acid index is very important. It is a known fact that when substituting with amino acids having similar hydropathic indices, similar biological activities can be retained. When introducing mutations with reference to the hydropathic index, preferably, substitutions are made between amino acids showing a difference in hydropathic index within ±2, more preferably within ±1, and even more preferably within ±0.5.

[0024] It is also well known that substitutions between amino acids with similar hydrophilicity values result in proteins with equivalent biological activity. The following hydrophilicity values are assigned to each amino acid residue: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0 ± 1); glutamic acid (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). When introducing mutations with reference to hydrophilicity values, substitutions are preferably made between amino acids showing a difference in hydrophilicity values within ±2, more preferably within ±1, and even more preferably within ±0.5.

[0025] Amino acid exchanges in proteins that do not globally alter the activity of the molecule are well known in the art (H. Neurath, R. L. Hill, The Proteins, Academic Press, New York, 1979). The most commonly occurring exchanges are those between the amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, Asp / Gly.

[0026] Generally, the peptides (including fusion proteins) and polynucleotides referred to herein are isolated. An "isolated" peptide or polynucleotide is one that has been removed from its original environment. For example, a protein that exists in a natural state is isolated by removing all or part of the substances that exist together in that state. Such a polypeptide must have a purity of at least 90% or more, preferably 95%, more preferably 99% or more. Polynucleotides are isolated by cloning them into a vector.

[0027] As used herein, the terms "polynucleotide" or "nucleic acid" are used interchangeably and refer to polymers of nucleotides of any length, including DNA (e.g., cDNA) and RNA molecules comprehensively. The "nucleotide" that is the building block of a nucleic acid molecule can be a deoxyribonucleotide, ribonucleotide, modified nucleotide or base, and / or its analog, or any substrate that can be incorporated into the polymer by DNA or RNA polymerase or by a synthetic reaction. Polynucleotides may include modified nucleotides, analogues with modified sugars or bases, such as methylated nucleotides and their analogues.

[0028] For example, nucleotides may include 5-modified cytidine and / or 5-modified uridine. 5-Modified cytidine includes 5-halocytidine (e.g., 5-iodocytidine or 5-bromocytidine), 5-alkynylcytidine and / or 5-heterocyclyluridine. 5-Modified uridine may include 5-halouridine (e.g., 5-iodouridine or 5-bromouridine), 5-alkynyluridine and / or 5-heterocyclyluridine. Optionally, 5-modified uridine is a nucleoside containing 2-deoxyribose.

[0029] Mutations in nucleotides may not result in mutations in the protein. Such nucleic acids include nucleic acid molecules containing codons that are functionally equivalent or codons that encode the same amino acid (for example, due to the degeneracy of codons, there are six codons for arginine or serine), or codons that encode biologically equivalent amino acids. Also, mutations in nucleotides can also result in changes in the protein itself. Even in the case of mutations that result in changes in the amino acids of the protein, those that exhibit substantially the same activity as the proteins of the present disclosure can be obtained.

[0030] It is clear to those of ordinary skill in the art that the peptides and nucleic acid molecules of the present disclosure are not limited to the amino acid sequences or base sequences described in the sequence listing, to the extent that the nucleic acid molecules or polynucleotides of the present disclosure have characteristics, such as effects as vaccines and / or immunopotentiators. For example, biological functional equivalents that may be included in the coding region operably linked to the expression regulatory sequence and / or the recombinant protein / peptide expressed therefrom are polynucleotides having mutations in the base sequences that exhibit biological activities equivalent to those of the aforementioned coding region and / or recombinant protein and / or proteins / peptides having mutations in the amino acid sequences.

[0031] Considering the mutations having the above-described biological equivalent activities, nucleic acid molecules encoding the peptides and / or proteins according to the present disclosure are understood to also include sequences that exhibit substantial identity with the sequences described in the sequence listing. The aforementioned substantial identity means that when the sequences of the present disclosure and any other sequence are aligned to maximize correspondence and the aligned sequences are analyzed using algorithms commonly used in the art, the sequences show a homology of at least 61%, more preferably 70%, even more preferably 80%, and most preferably 90%. Alignment methods for sequence comparison are well known in the art.

[0032] As used herein, the term "vector" means a construct that can be transferred into a host cell and is preferably engineered to enable the expression of one or more target genes or sequences. Also, a particular vector can direct the expression of a gene in ORF form to which the vector is operably linked. Such a vector is referred to herein as a "recombinant expression vector" (or simply, "recombinant vector").

[0033] As used herein, the term "expression control / regulation sequence" or "expression control / regulation element" can mean a nucleic acid sequence that regulates the transcription of a nucleic acid and / or the translation from a nucleic acid in the form of a transcript. Note that the term "transcription control / regulation sequence" or "transcription control / regulation element" means a nucleic acid sequence that regulates the transcription of a nucleic acid. Transcription control sequences include promoters or enhancers such as constitutive promoters or inducible promoters.

[0034] Also, the terms "translation control / regulation sequence" or "translation control / regulation element" can be used for nucleic acid sequences that regulate the translation from a nucleic acid in the form of a transcript to a protein or peptide. These expression control sequences / elements, transcription control sequences / elements, and / or translation control sequences / elements are operably linked to the sequences to be expressed, for example, nucleic acid sequences to be transcribed or translated.

[0035] As used herein, the term "operatively linked" means a functional linkage between a nucleic acid expression control sequence (e.g., a promoter, signal sequence, ribosome binding site, transcription termination sequence, etc.) and another nucleic acid sequence such that the control sequence regulates the transcription and / or translation of the other nucleic acid sequence.

[0036] Nucleic acid molecule The present disclosure is based on the fact that the expression efficiency of an immunogenic target sequence operably linked to an expression control sequence is improved, whereby such an expression system can be utilized as a nucleic acid-based vaccine. FIG. 1 is a schematic diagram schematically showing the configuration of a nucleic acid molecule or polynucleotide capable of efficiently expressing a protein or peptide causing an infectious disease according to an exemplary embodiment of the present disclosure.

[0037] As shown in FIG. 1, the nucleic acid molecule may include an expression control element (ECE) and a coding region (CR) consisting of an open reading frame (ORF) that is operably linked to the expression control element (ECE) and encodes an immunogen derived from a virus. The expression control element (ECE) may include a translation control element (TLCE) derived from troponin 1 (TNNT1, slow skeletal muscle). The coding region (CR) may consist of an ORF that is operably linked to the translation control element (TLCE) and encodes an immunogen derived from Influenza virus and / or Severe fever with thrombocytopenia syndrome virus (SFTSV).

[0038] In one aspect, the expression control element (ECE) may further include a transcription control element (TCCE) operably linked to the coding region (CR) and located upstream of the translation control element (TLCE). Further, the nucleic acid molecule may further include at least one nucleotide of a polyadenylation signal sequence or polyadenosine sequence (PA) located downstream of the expression control element (ECE), for example, downstream of the transcription control element (TLCE).

[0039] The translation control element (TLCE) may include nucleotides derived from TNNT1 operably linked to the coding region (CR) inserted in ORF form. For example, the translation control element (TLCE) may include a first translation control element (U-TLCE) located upstream of the coding region (CR) and / or a second translation control element (D-TLCE) located downstream of the coding region (CR).

[0040] As an example, the first translation control element (U-TLCE) may be all or part of a 5'-Untranslated Region (5'-UTR) having cap-dependent translation initiation activity, and the second translation control element (D-TLCE) may be all or part of a 3'-UTR corresponding to the first translation control element (U-TLCE).

[0041] Most eukaryotic mRNAs have 7-methyl-guanosine (cap) at the 5' end. However, the protein synthesis initiation complex recognizes the cap located at the 5' end and advances to the start codon AUG to initiate protein synthesis. That is, the cap structure located at the 5' end of mRNA not only initiates protein synthesis but also prevents the destruction of mRNA by the action of nuclease.

[0042] In vitro, in the first step of transcription, after treating pDNA (plasmid DNA) with restriction enzymes to linearize it, m7G(5’)-ppp(5’)G (this is called regular cap analog) can be attached to the mRNA produced using an appropriate RNA polymerase to create capped mRNA for use. Optionally, in vitro transcription can be performed without a cap analog, and there is a method of performing a cap reaction using a commercially available vaccinia virus capping enzyme, and an ’anti-reverse’ cap analog (ARCA) that prevents such a reverse cap reaction can be used. When introducing ARCA, only the 3’-O-methylation of methylated guanosine can bind to the nucleotide of guanosine that does not undergo methylation.

[0043] For example, the first translation regulatory element (U-TLCE) and / or the second translation regulatory element (D-TLCE) may each consist of nucleotides having a cap-dependent translation initiation activity derived from an animal, for example, a mammal, specifically, a primate, more specifically, a human. For example, the first translation regulatory element (U-TLCE) may consist of the nucleotide of SEQ ID NO: 1 or its transcript, which may be a part of the 5’-UTR derived from human TNNT1, and the second translation regulatory element (D-TLCE) may consist of the nucleotide of SEQ ID NO: 2 or its transcript, which may be a part of the 3’-UTR derived from TNNT1, but is not limited thereto.

[0044] As an example, the first translation regulatory element (D-TLCE) is a region to which a translation initiation complex binds during the translation process of a peptide and / or protein expressed from the coding region (CR), and can be a cis-acting nucleotide that induces translation of the coding region (CR). When the first translation regulatory element (U-TLCE) is all or part of the nucleotides having cap-dependent translation initiation activity, the nucleic acid molecule is used to improve the expression efficiency of a target gene encoding all or part of a virus-derived immunogen inserted into the coding region (CR) in ORF form, and may include a second translation regulatory element (D-TLCE) that can be located downstream of the coding region (CR). The first translation regulatory element (U-TLCE) and the second translation regulatory element (D-TLCE) play an important role in improving the translation efficiency of an ORF or its transcript encoding a peptide that can function as an immunogen in influenza virus and / or SFTSV forming the coding region (CR), and in stably maintaining the transcript mRNA without being destroyed intracellularly.

[0045] As an example, the first translation regulatory element (U-TLCE) can be located upstream of the coding region (CR), and the second translation regulatory element (D-TLCE) can be located downstream of the coding region (CR). In other words, the coding region (CR) can be located between the first translation regulatory element (U-TLCE) and the second translation regulatory element (D-TLCE), and forms a target sequence encoding a peptide derived from influenza virus or SFTSV, such as an infectious immunogen.

[0046] The influenza virus is a negative-sense RNA virus and there are four types: A, B, C, and D. Among them, types A and B mainly cause diseases in humans. Type A is classified according to the combination of hemagglutinin (HA) and neuraminidase (NA), which are viral surface antigens. Worldwide, among seasonal influenza patients every year, 3 to 5 million people show severe symptoms, and 290,000 to 650,000 people die. There is still no effective therapeutic agent, and prevention using vaccines is the best option. Inactivated influenza vaccines were first approved in the United States in 1945, and currently, influenza vaccines are being developed in various ways using protein vaccines and virus-like particles (VLPs).

[0047] In an exemplary embodiment, the immunogen of the influenza virus encoded by the coding region (CR) can be the hemagglutinin (HA) of the influenza virus or a fragment thereof. For example, the hemagglutinin, which is the immunogen of the influenza virus, may consist of the amino acids of SEQ ID NO: 3. As an example, the nucleotide encoding the immunogen of the influenza virus or a fragment thereof may consist of the nucleotide of SEQ ID NO: 4 or its transcript, but is not limited thereto.

[0048] Severe fever with thrombocytopenia syndrome (SFTS) is a disease caused by SFTSV, and the first patients were reported in South Korea and Japan in 2013. In the case of South Korea, among 605 patients from 2013 to 2017, the mortality rate reached an average of 20.9%. The symptoms when infected with the virus are mainly high fever, fatigue, headache, muscle pain, vomiting, diarrhea, etc. In severe cases, platelets decrease, leading to a complex decline in the functions of various organs including the kidneys, liver, and heart, and in severe cases, death ensues.

[0049] Severe fever with thrombocytopenia syndrome (SFTS) is mainly caused by infection with ticks carrying severe fever with thrombocytopenia syndrome virus (SFTSV), and occurs frequently among agricultural and forestry workers who engage in a lot of outdoor activities and often come into contact with grass where ticks mainly inhabit. In the case of South Korea, many infections have been reported between July and October, which is the harvest season. Although it is infected by ticks, it can also be transmitted to other animal hosts such as sheep, pigs, dogs, and cats. However, in the case of infected patients, since the viral load in the blood is very high, it is known that human-to-human transmission through blood is also possible. In particular, there is a risk that ticks will move to North America and spread globally beyond the Far East of Asia. Therefore, in 2018, the World Health Organization (WHO) included SFTSV in the list of pathogens requiring attention.

[0050] SFTSV belongs to the Bunyaviridae family and is a negative-strand RNA virus containing three segments. The genome is divided into the L segment, M segment, and S segment. The L segment consists of 6,368 nucleotides and contains the RNA-dependent RNA polymerase gene. The M segment consists of 3,378 nucleotides and contains the genes for glycoprotein N (Gn) and glycoprotein C (Gc) present in the envelope. The S segment consists of 1,744 nucleotides and encodes a nucleoprotein and a non-structural protein.

[0051] To date, no therapeutic agents or vaccines for SFTSV have been commercially developed. The reason for the delay in development is that SFTS is a newly identified infectious disease that has been corrected relatively recently. Since patients are concentrated in Northeast Asia and the number of patients has not been large, there has been little global interest. In addition, the lack of an appropriate animal model is also one of the reasons why therapeutic agents and vaccines for SFTS have not been developed.

[0052] As an example, the immunogen of severe fever with thrombocytopenia syndrome virus (SFTSV) encoded in the coding region (CR) can be the glycoprotein N (Gn) of SFTSV or a fragment thereof. For example, the glycoprotein (Gn), which is an immunogen of SFTSV, may consist of, but is not limited to, a peptide mutated from the glycoprotein N of SFTSV having an amino acid sequence selected from the group consisting of SEQ ID NO: 5 (referred to as Gn herein), SEQ ID NO: 7 (referred to as GnΔTM herein), and SEQ ID NO: 9 (referred to as GnΔSTEM herein). As an example, the nucleotide encoding the immunogen of SFSTV or a fragment thereof may consist of, but is not limited to, a nucleotide selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 10 or a transcript thereof.

[0053] For example, when the nucleic acid molecule is in the RNA form, the coding region (CR) may consist of a transcript having an open reading frame (ORF) encoding an immunogen of influenza virus (e.g., HA) or an immunogen of SFTSV (e.g., glycoprotein) or a fragment thereof. There is no limitation on the length of the ORF constituting the coding region (CR), and the expression efficiency depending on the length of the ORF is not a major consideration in the development of nucleic acid molecules, recombinant expression vectors using the same, and nucleic acid vaccines for treatment or prevention according to the present disclosure. Codon usage can usually affect protein / peptide expression in various species, but it is known that human codon usage bias usually does not have a significant impact on protein / peptide expression, so it is not a consideration when developing nucleic acid vaccines and gene therapy agents for humans.

[0054] Optionally, the start codon may have a Kozak sequence. It is also necessary to optimize the nucleotides near the stop codon. Optionally, the third part of the codon sequence of the mRNA, which is the gene or its transcript to be expressed in the coding region (CR), can be changed to GC without changing the amino acid, increasing the GC% of the target gene and enhancing the stability of the mRNA.

[0055] In alternative embodiments, the nucleic acid molecule may further be inserted with nucleotides that can increase the expression efficiency of the coding region (CR) ligated in ORF form. As an example, the nucleic acid molecule can have a transcription regulatory factor (TCCE) that promotes transcription of the nucleic acid molecule adjacent to a first translation regulatory factor (U-TLCE), for example. For example, the transcription regulatory factor (TCCE) can be located upstream of the first translation regulatory factor (U-TLCE). Such transcription regulatory factors (TCCE) are not particularly limited.

[0056] In an exemplary embodiment, the transcription regulatory factor (TCCE) can be a promoter that promotes transcription of HA derived from influenza virus or Gn derived from SFTSV encoded in ORF form in the coding region (CR). The transcription regulatory factor (TCCE) operates in animal cells, more specifically, mammalian cells, and can regulate the transcription of a virus-derived immunogen encoded in the coding region (CR).

[0057] As an example, the transcriptional regulatory element (TCCE) includes a promoter derived from a mammalian virus, a promoter derived from the genome of a mammalian cell, or a promoter derived from a bacteriophage. For example, the transcriptional regulatory element (TCCE) includes a CMV (cytomegalo virus) promoter, an adenovirus late promoter, a vaccinia virus 7.5K promoter, an SV40 promoter, an HSV tk promoter, a T7 bacteriophage promoter, a T3 bacteriophage promoter, an SM6 promoter, an RSV promoter, an EF1 alpha promoter, a metallothionein promoter, a β-actin promoter, a promoter of the human IL-2 gene, a promoter of the human IFN gene, a promoter of the human IL-4 gene, a promoter of the human lymphotoxin gene, a promoter of the human GM-CSF gene, a cancer cell-specific promoter (e.g., TERT promoter, PSA promoter, PSMA promoter, CEA promoter, E2F promoter, and AFP promoter), and a tissue-specific promoter (e.g., albumin promoter), but is not limited thereto.

[0058] In an exemplary embodiment, any transcriptional regulatory element (TCCE) capable of transcribing mRNA from a linearized DNA template, such as a T7 bacteriophage promoter, a T3 bacteriophage promoter, an SP6 bacteriophage promoter, etc., can be located adjacent to the first translational regulatory element (U-TLCE), specifically, upstream of the first translational regulatory element (U-TLCE).

[0059] In addition, the nucleic acid molecule may be inserted with a nucleotide capable of inducing the expression of an ORF consisting of a gene encoding an immunogenic peptide derived from a virus or its transcript sequence inserted into the coding region (CR), in addition to the aforementioned translational regulatory element (TLCE), coding region (CR), and transcriptional regulatory element (TCCE). In one exemplary embodiment, the nucleic acid molecule may include a Kozak sequence inserted between the transcriptional regulatory element (TCCE) or the first translational regulatory element (U-TLCE) and the start codon of the coding region (CR).

[0060] Optionally, the nucleic acid molecule can further insert a polyadenylation signal sequence and / or a polyadenosine sequence (PA) that stabilizes the nucleic acid molecule transcribed downstream of the coding region (CR), more specifically, downstream of the second translation regulatory element (D-TLCE), and further improves the translation efficiency of an open reading frame (ORF) consisting of a gene encoding a viral-derived antigen peptide present in the coding region (CR) or its transcript sequence.

[0061] For example, when the nucleic acid molecule of the present disclosure consists of a transcript sequence in RNA form, the polyadenosine sequence (PA) can be nucleotides consisting of approximately 25 to approximately 400, for example, 30 to 400, 50 to 250, or 60 to 250 adenosines. In another exemplary embodiment, when the nucleic acid molecule of the present disclosure consists of DNA form, the polyadenylation signal sequence (PA) can be located downstream of the coding region (CR). As an example, the polyadenylation signal sequence (PA) can be derived from SV40, human growth factor (hGH), bovine growth hormone (BGH), rabbit beta-globin (rbGlob), etc., but the present disclosure is not limited thereto.

[0062] Optionally, the polyadenylation signal sequence or the polyadenosine sequence (PA) may consist of a sequence in which a signal sequence or a linker sequence such as 5'-GATCATCAGT-3' is inserted between two nucleotides consisting of a large number of adenosines, for example, 25 to approximately 400, 30 to 400, 50 to 250, or 60 to 250 adenosines or its transcript.

[0063] To insert a coding region (CR) into a nucleic acid molecule, the nucleic acid molecule may contain one or more cloning sites, preferably a Multiple Cloning Site (MCS). The one or more cloning sites may contain one or more restriction endonuclease recognition sites and / or sequences cleaved by restriction enzymes. The restriction enzymes include, of course, natural restriction enzymes found in bacteria and archaea, etc., and may also include artificially produced restriction enzymes (for example, restriction enzymes based on the DNA binding sites of Zinc finger nuclease and TAL effector or PNA-based PNAznymes, etc.).

[0064] For example, naturally occurring restriction enzymes can be classified into 1) Type I restriction enzymes (cleave at a site distant from the recognition site, require ATP, S-adenosyl-L-methionine and magnesium ions), 2) Type II restriction enzymes (cleave at a specific site within or slightly distant from the recognition site, most require magnesium ions), 3) Type III restriction enzymes (cleave at a site slightly distant from the recognition site, require ATP but do not require ATP hydrolysis), 4) Type IV restriction enzymes (target modified sites such as methylation, hydroxymethylation or glucosyl-hydroxymethylation), 5) Type V restriction enzymes (CRISPRs' cas9-gRNA complex), etc.

[0065] For example, the multiple cloning site may include, but is not limited to, sites recognized by the following restriction enzymes and / or restriction enzyme cleavage sites: AngI, AatI, AbaI, BamHI, BbvI, BcgI, BplI, BsmAI, Alw26I, BsrI, ClaI, EarI, Eco57I, EcoRI, EcoRII, EcoRV, FokI, HaeIII, HindIII, HpaIII, HphI, KpnI, MboI, MluI, NaeI, NdeII, NgoMIV, NlaIII, NotI, PacI, PstI, SacI, SacII, SalI, SapI, SfaNI, SmaI, TaqI, XbaI, XhoI, PvuI, and combinations thereof. In one exemplary embodiment, the cloning site may include, but is not limited to, at least one restriction enzyme recognition site and / or restriction enzyme cleavage site of SEQ ID NO: 16 to SEQ ID NO: 21.

[0066] The nucleic acid molecule can be DNA or RNA. According to an exemplary embodiment, the nucleic acid molecule according to the present disclosure may have an RNA form. When the nucleic acid molecule consists of an RNA form, such as when the coding region (CR) consists of the transcript sequence of the ORF encoding the immunogen of influenza virus or SFTSV, it is advantageous compared to the nucleic acid molecule in DNA form. Different from the nucleic acid molecule in DNA form, the nucleic acid molecule in RNA form does not need to enter the nucleus of the host cell for transcription into mRNA. The nucleic acid molecule in RNA form has no possibility of entering the host chromosome in the nucleus, and the antibiotic resistance gene, which is a selection marker used for selective production in the host cell, is unnecessary for the production of the RNA nucleic acid molecule. Since RNA has a shorter half-life than DNA, it does not induce persistent genetic transformation.

[0067] Nucleic acid molecules in RNA form can induce a desired in vivo immune response even when using a relatively small amount compared to nucleic acid molecules in DNA form. Also, when producing nucleic acid molecules in RNA form, all production processes can be artificially controlled, so they can be safely produced in a small-scale GMP (good manufacturing practice) production facility without the risk of biological contamination. When producing RNA nucleic acid molecules, there is no need to directly handle the infection source. Instead, only the nucleic acid sequence of the neutralizing antibody-inducing related part (neutralizing epitope) of the infection source to be expressed is artificially synthesized, and a large amount of RNA nucleic acid molecules can be produced only by in vitro transcription (IVT). Recently, reagents related to the IVT reaction, especially DNA-dependent RNA polymerase, have been improved, and a large amount of RNA can be rapidly produced within 1 to 2 weeks using a small amount of DNA template.

[0068] Nucleic acid molecules in RNA form can induce a stronger immune response compared to naked DNA nucleic acid molecules. The RNA nucleic acid molecules themselves produce complex antigen complexes inside cells, which can approach MHC (major histocompatibility complex) class II of antigen-presenting cells and function as an ideal immune enhancer. Also, multiple antigens whose immune responses are to be induced can be simultaneously produced and mixed before immunization, and there are no special restrictions on the gene length of the antigens to be expressed, so the applicability and simplicity of RNA nucleic acid molecule production can be increased.

[0069] When attempting to utilize a nucleic acid molecule that may be in the form of RNA, an appropriate transcriptional control element (TCCE) that enables in vitro transcription (IVT) can be positioned upstream of the first translational control element (U-TLCE). Since nucleic acid molecules in RNA form can be synthesized through the IVT process, there is no need to directly handle live viruses or pathogenic microorganisms used in the production of general live vaccines and killed bacteria vaccines, nor is it necessary to culture host cells such as yeast, Escherichia coli, and insect cells that must be used to produce recombinant proteins / peptides.

[0070] When transcribing a nucleic acid molecule in DNA form inserted into a vector into mRNA form through the IVT process, a nucleic acid molecule in RNA form can be synthesized in vitro by RNA polymerase using linearized DNA with its ends transferred by restriction enzymes or the like as a template. For transcription from linearized DNA to RNA, a transcriptional control element (TCCE), such as a promoter sequence derived from bacteriophage, for example, can be positioned upstream of the translational control element (TLCE).

[0071] Recombinant expression vectors, expression constructs, and nucleic acid molecule injection The nucleic acid molecule shown in FIG. 1 can be inserted into a recombinant expression vector. The recombinant expression vector may include a translational control element (TLCE) having translational initiation activity, a coding region (CR), and may also include a transcriptional control element (TCCE) and / or a polyadenylation signal sequence (PA). That is, the recombinant expression vector may include the nucleic acid molecule described with reference to FIG. 1, but this nucleic acid molecule can also be combined with other nucleic acids to encode a fusion protein or a fusion peptide. In this case, the nucleic acid molecule can be injected into a living body in the form of an expression construct that is a gene delivery vector.

[0072] Vectors that can be used as gene delivery vehicles can be produced in various forms, including viral vectors, DNA or RNA expression vectors, plasmids, cosmids, or phage vectors, DNA or RNA expression vectors linked to cationic condensing agents (CCAs), such as DNA or RNA expression vectors encapsulated in liposomes or niosomes that encapsulate plasmids, and certain eukaryotic cells such as producer cells.

[0073] Exemplarily, the nucleic acid molecules of the present disclosure may be configured to enter and express in mammalian cells. Such a configuration is particularly useful for the treatment and / or prevention of infectious diseases. There are many ways to express nucleic acid molecules in host cells, and any suitable method can be used. For example, the nucleic acid molecules according to the present disclosure can be inserted into any vector that constitutes an expression construct or a gene delivery system.

[0074] One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a phage vector. Still another type of vector is a viral vector into which additional DNA segments can be ligated within the viral genome. One particular vector is capable of autonomous replication within the host cell into which it is introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of the host cell upon introduction into the host cell and are thereby replicated with the host genome. Also, generally, expression vectors useful in recombinant DNA technology often exist in the form of plasmids.

[0075] As an example, a nucleic acid molecule can be inserted into a host cell using a viral gene delivery system. Viral vectors into which the nucleic acid molecule can be inserted may include vectors derived from adenovirus, adeno-associated virus (AAV), retrovirus, vaccinia, or other poxviruses (e.g., avian pox virus), lentivirus, herpes simplex virus. For example, viral vectors include vectors derived from lentiviruses such as human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV); retroviral vectors derived from murine retroviruses, gibbon ape leukemia virus, adeno-associated viruses (AAVs), and adenoviruses, etc., but are not limited thereto. In addition, retroviral vectors derived from murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), ecotropic retroviruses, etc. are widely used. The vector system according to the present disclosure can be constructed using various methods well known in the art.

[0076] Optionally, after inserting the aforementioned nucleic acid molecule into an appropriate vector, it can be modified into a nucleic acid molecule in the form of RNA through in vitro transcription (IVT). Techniques for inserting nucleic acid molecules, such as DNA, into such vectors are already well known. Retroviral vectors can additionally have targeting moieties inserted, such as genes for selectable markers that facilitate identification or selection of transduced cells and / or genes encoding ligands that serve as receptors for specific target cells. Targeting can also be performed by known methods using antibodies.

[0077] A number of vectors that are available and well known in the art to which this disclosure pertains can be used for the purposes of this disclosure. The selection of an appropriate vector will mainly vary depending on the size of the nucleic acid molecule to be inserted into the vector and the particular host cell to be transformed with the vector. Each vector contains various components depending on its function (amplification and / or expression of a heterologous polynucleotide, or both) and its compatibility with the particular host cell in which the vector is present. Vector components generally include, but are not limited to, an origin of replication (especially when the vector is to be inserted into a prokaryotic cell), a selectable marker gene, a promoter, a ribosome binding site (RBS), a signal sequence, a heterologous nucleic acid insert, and / or a transcription termination sequence.

[0078] For example, an expression vector according to the present disclosure may include expression regulators that can affect the expression of influenza virus or SFTSV antigens, encoded within a coding region (CR), such as start codons, stop codons, polyadenylation signal sequences, enhancers, signal sequences for membrane targeting or secretion, and the like. An enhancer sequence is a nucleotide sequence that is located at various sites in a promoter and increases the transcriptional activity as compared to the transcriptional activity by the promoter in the absence of the enhancer sequence.

[0079] When the host is a bacterium of the genus Escherichia, signal sequences such as the PhoA signal sequence and the OmpA signal sequence can be used. When the host is a bacterium of the genus Bacillus, the α-amylase signal sequence, the subtilisin signal sequence, etc. can be used. When the host is yeast, signal sequences such as the MF-α signal sequence and the SUC2 signal sequence can be used. When the host is an animal cell, the insulin signal sequence, the α-interferon signal sequence, the antibody molecule signal sequence, etc. can be used, but the present disclosure is not limited thereto.

[0080] Any vector (e.g., a bacterial vector having a bacterial replication origin and an episomal mammalian vector) can autonomously replicate within the host cell into which it is introduced. Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of the host cell upon introduction into the host cell, thereby replicating with the genome of the host.

[0081] The vector system according to the present disclosure can be constructed using various methods well known in the technical field to which the present disclosure pertains. Also, the vectors of the present disclosure can typically be constructed as vectors for cloning or vectors for expression. Further, the vectors of the present disclosure can be constructed using prokaryotic cells or eukaryotic cells as hosts. For example, vectors that can be used in the present disclosure can be produced by manipulating plasmids (e.g., pSC101, ColE1, pBR322, pUC8 / 9, pHC79, pUC19, pET, etc.), phages (e.g., λgt4λB, λ-Charon, λΔz1, λGEM.TM.-11, and M13, etc.) or viruses (e.g., SV40, etc.) that are frequently used in the technical field to which the present disclosure pertains.

[0082] Constitutive or inducible promoters can be used in the present disclosure as needed for specific situations that can be recognized by an ordinary technician. A number of promoters recognized by various possible host cells are widely known. The selected promoter can be operably linked to a nucleic acid molecule having a coding region (CR) consisting of an open reading frame (ORF) of a gene or transcript encoding an immunogenic peptide or protein by removing the promoter from the source nucleic acid molecule through restriction enzyme digestion and inserting the isolated promoter sequence into a selection vector. Both natural promoter sequences and a number of heterologous promoters can be used to direct the amplification and / or expression of the gene or transcript constituting the coding region (CR). Heterologous promoters are generally preferred because they can obtain a greater transcription and a higher yield of the expressed gene of interest compared to natural promoters.

[0083] For example, when the vector of the present disclosure is an expression vector and a prokaryotic cell is used as the host, it generally includes a transcriptional regulatory factor (TCCE) such as a strong promoter capable of promoting transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, and T7 promoter, etc.), translation regulatory sequences (TLCE1, TLCE2) for initiating translation, and transcription / translation termination sequences. When E. coli is used as the host cell, the promoter and operator sites of the E. coli tryptophan biosynthesis pathway (Yanofsky, C., J. Bacteriol., 158: 1018-1024 (1984)) and the leftward promoter of phage λ can be used as expression regulatory sites.

[0084] In addition, when the vector of the present disclosure is an expression vector and uses eukaryotic cells as a host, a promoter derived from the genome of mammalian cells (e.g., metallothionein promoter) or a promoter derived from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, and HSV tk promoter) or a promoter derived from bacteriophage (e.g., T7 promoter, T3 promoter, SM6 promoter) can be used, and it generally has a polyadenylation signal sequence as a transcription termination sequence.

[0085] In addition, when the recombinant vector of the present disclosure is a replicable expression vector, it may contain a replication origin, which is a specific nucleic acid sequence at which replication is initiated. Further, the recombinant vector may contain a selection marker. The selection marker is for selecting cells transformed with the vector, and markers that confer selectable phenotypes such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of surface proteins can be used. The vector of the present disclosure contains an antibiotic resistance gene commonly used in the art as a selection label. For example, there are resistance genes for ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline. Since only cells expressing the selection marker survive in an environment treated with a selective agent, transformed cells can be selected. Representative examples of selection markers include auxotrophic markers such as ura4, leu1, and his3, but the types of selection markers that can be used in the present disclosure are not limited by the above examples.

[0086] There are various in vitro amplification techniques for amplifying an array subcloned into an expression vector. Such techniques include PCR (polymerase chain reaction), LCR (ligase chain reaction), Qβ-replicase amplification, and techniques using other RNA polymerases.

[0087] The vectors of the present disclosure can also be fused with other sequences to facilitate the purification of the recombinant proteins or peptides expressed therefrom. Examples of the sequences for fusion include glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA), and 6x His (hexahistidine; Quiagen, USA), and most preferably, 6x His. Due to the additional sequences for the purification, the proteins expressed in the host can be rapidly and easily purified through affinity chromatography. Optionally, a sequence encoding an Fc fragment can also be fused so as to promote the extracellular secretion of these recombinant proteins.

[0088] According to an exemplary embodiment of the present disclosure, a fusion protein expressed by a vector containing the fusion sequence is purified by affinity chromatography. For example, when glutathione-S-transferase is fused, glutathione, which is a substrate of this enzyme, can be used, and when 6x His is used, the desired recombinant protein can be rapidly and easily obtained using a Ni-NTA His-binding resin column (Novagen, USA).

[0089] Host cells capable of stably and continuously cloning and expressing the aforementioned vectors can be any host cells well known in the technical field to which the present disclosure pertains. For example, E. coli JM109, E. coli BL21(DE3), E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus strains such as Bacillus thuringiensis, and enterobacteria and strains such as Salmonella typhimurium, Serratia marcescens, and various Pseudomonas species can be used. Also, when transforming the vectors of the present disclosure into eukaryotic cells, host cells such as yeast (Saccharomyces cerevisiae), insect cells (e.g., SF9 cells), and human cells (e.g., CHO cell line (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN, and MDCK cell lines) can be used.

[0090] The vectors of the present disclosure can be used to genetically modify cells in vivo, ex vivo, or in vitro. As methods for genetically modifying cells, there are various known methods including infecting or transducing cells with viral vectors, calcium phosphate precipitation, fusing recipient cells with bacterial protoplasts containing DNA, treating recipient cells with liposomes or microspheres containing DNA, DEAE dextran, receptor-mediated endocytosis, electroporation, micro-injection, and the like.

[0091] For example, when the host cell is a prokaryotic cell, CaCl 2It can be carried out by methods such as the Hanahan method and / or electroporation. When the host cell is a eukaryotic cell, the vector can be injected into the host cell by methods such as microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, and / or gene bombardment. The vector injected into the host cell can be expressed in the host cell, and in such a case, a large amount of recombinant protein or recombinant peptide can be obtained. For example, when the expression vector contains the lac promoter, the host cell can be treated with IPTG to induce gene expression.

[0092] Pharmaceutical composition According to another aspect, the present disclosure relates to a pharmaceutical composition for preventing or treating influenza or SFTS, for example, a vaccine composition, which contains, as an active ingredient, the aforementioned nucleic acid molecule and / or an expression construct in which the aforementioned nucleic acid molecule is inserted as a gene delivery agent. That is, the aforementioned nucleic acid molecule or an expression construct containing the same can function as an immunogen and can be an active ingredient of a pharmaceutical composition for preventing or treating influenza or SFTS.

[0093] Therefore, according to another aspect of the present disclosure, there is disclosed a pharmaceutical composition for preventing or treating influenza or SFTS, which contains a pharmaceutically effective amount of the aforementioned nucleic acid molecule and optionally contains a pharmaceutically acceptable carrier. In this case, the aforementioned nucleic acid molecule is directly administered to the subject. As used herein, "pharmaceutically effective amount" means an amount sufficient to achieve the efficacy or activity of the nucleic acid molecule according to the present disclosure.

[0094] According to another aspect of the present disclosure, the present disclosure discloses a pharmaceutical composition for preventing or treating influenza or SFTS, which comprises an expression construct into which the nucleic acid molecule of the present disclosure described above is inserted, and optionally a pharmaceutically acceptable carrier. In still another aspect, the present disclosure relates to a method for preventing or treating influenza or SFTS, which comprises administering to a subject a pharmaceutically effective amount of the nucleic acid molecule or expression construct described above.

[0095] The pharmaceutical composition containing a pharmaceutically effective amount of the nucleic acid molecule or expression construct according to the present disclosure comprises a carrier, a diluent and / or an excipient. In one exemplary aspect, the nucleic acid molecule or expression construct as the active ingredient is formulated by being mixed with a physiologically acceptable carrier, that is, a carrier that is non-toxic to the recipient at the dosage and concentration used in the herbal medicine dosage form, at ambient temperature, at an appropriate pH, and with the desired degree of purity. The pH of the formulation mainly varies depending on the specific use and concentration of the compound, but is preferably in the range of about 3 to about 8. In still other embodiments, the compound is sterilized. The compound can be stored, for example, as a solid or amorphous composition, a lyophilized formulation or an aqueous solution.

[0096] In one exemplary embodiment, the nucleic acid molecule or expression construct described above can be stabilized in the pharmaceutical composition using a nucleic acid stabilizer such as a cationic polymer, a cationic peptide or a cationic polypeptide. The cationic (poly)peptide that can be used as a nucleic acid stabilizer can be a multi-cationic polymer such as polylysine or polyarginine, a cationic lipid or a lipofectant. More specifically, the stabilizer can be histone, nucleolin, protamine, oligofectamine, spermine or spermidine and cationic polysaccharides, especially chitosan, TDM, MDP, muramyl dipeptide, pluronic® and / or derivatives thereof. Histone and protamine are cationic proteins that naturally compact DNA.

[0097] As an example, histones that can form a complex with a nucleic acid molecule or an expression construct include histones H1, H2a, H3, and H4, and protamines, preferably protamine P1 or P2, particularly the cationic partial sequence of protamine. Optionally, other compounds that can form a complex with the nucleic acid molecule according to the present disclosure can further be an adjuvant used. Further adjuvants can improve the immunogenicity of the pharmaceutically active substance and the nucleic acid molecule.

[0098] In one exemplary embodiment, the immunopotentiator is a combination of protamine, nucleolin, spermine, spermidine, and cationic polysaccharides, and a stabilized cationic peptide or polypeptide, particularly chitosan, TDM, MDP, muramyl dipeptide, pluronic, alum solution, aluminum hydroxide, ADJUMER (polyphosphazene); aluminum phosphate gel; glucan from algae; algammulin; aluminum hydroxide gel (alum); high protein-adsorbing aluminum hydroxide gel; low-viscosity aluminum hydroxide gel; AF or SPT (emulsion of squalane (5%), Tween-80 (0.2%), PLURONIC®-L121 (1.25%), phosphate-buffered saline, pH 7.4); AVRIDINE (propylenediamine); BAY R1005 ((N-(2-deoxy-2-L-leucylamino-β-D-glucopyranosyl)-N-octadecyl dodecanoyl-amide hydroacetate); CALCITRIOL (1α,25-dihydroxy-vitamin D3); calcium phosphate gel; CAPTM (calcium phosphate nanoparticles); cholera holotoxin, cholera-toxin-A1-protein-A-D-fragment fusion protein, subunit B of cholera toxin; CRL 1005 (block copolymer P1205); cytokine-containing liposome; DDA (dimethyl octadecyl ammonium bromide); DHEA (dehydroepiandrosterone); DMPC (dimyristoyl phosphatidylcholine); DMPG (dimyristoyl phosphatidylglycerol); DOC / alum complex (sodium deoxycholate salt); Freund's complete adjuvant; Freund's incomplete adjuvant; gamma-inulin; Gerbu adjuvant (mixture of N-acetylglucosaminyl-(β1-4)-N-acetylmuramyl-L-alanyl-D-glutamine (GMDP), dimethyloctadecyl ammonium chloride (DDA), zinc-L-proline salt complex (ZnPro-8); GM-CSF); GMDP (N-acetylglucosaminyl-(β1-4)-N-acetylmuramyl-L-alanyl-D-isoglutamine); macimorelin (1-(2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-amine); (N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Ala-glycerol dipalmitate); DRV (immunoliposome produced from dehydrated-rehydration vesicles); interferon-gamma; interleukin-1 beta; interleukin-2; interleukin-7; interleukin-12; ISCOMS ("Immunostimulating Complexes"); ISCOPREP 7.0.3.; liposome; LOXORIBINE (7-allyl-8-oxoguanosine (guanine)); LT oral adjuvant (E.coli labile enterotoxin - prototoxin); microspheres and microparticles of the composition; MF59; (squalene - water emulsion); MONTANIDE ISA 51 (purified incomplete Freund adjuvant); MONTANIDE ISA 720 (metabolizable oil adjuvant); MPL (3 - Q - desacyl - 4’ - monophosphoryl lipid A); MTP - PE and MTP - PE liposomes ((N - acetyl - L - alanyl - D - isoglutaminyl - L - alanine - 2 - (1,2 - dipalmitoyl - sn - glycero - 3 - (hydroxyphosphoryloxy)) - ethylamide, monosodium salt); MURAMETIDE (Nac - Mur - L - Ala - D - Gln - OCH3); MURA PALMITINE and D - MURAPALMITINE (Nac - Mur - L - Thr - D - isoGIn - sn - glycerol dipalmitoyl); NAGO (neuraminidase - galactose oxidase); nanospheres or nanoparticles of the composition; NISV (non - ionic surfactant vesicle); PLEURAN (β - glucan); PLGA, PGA and PLA (homo - and copolymers of lactic and glycolic acids; microspheres / nanospheres); PLURONIC L121; PMMA (polymethyl methacrylate); PODDS (proteoid microspheres); polyethylene carbamate derivatives; poly - rA: poly - rU (polyadenylic acid - polyuridylic acid complex); polysorbate 80 (Tween 80); protein cochleate (Avanti Polar Lipids, Inc., Alabaster, AL); STIMULON (QS-21); Quil-A (Quil-A saponin); S-28463 (4-amino-otec-dimethyl-2-ethoxymethyl-1H-imidazo[4,5-c]-quinolin-1-ethanol); SAF-1 (“Syntex adjuvant formulation”); Sendai proteoliposome and Sendai-containing lipid matrix; Span-85 (sorbitan trioleate); Specol (emulsion of Marcol 52, Span 85 and Tween 85); squalene or Robane (2,6,10,15,19,23-hexamethyltetracosane and 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexane); stearyl tyrosine (octadecyl tyrosine hydrochloride); Thermamid (N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Ala-dipalmitoxypropylamide); threonyl-MDP (Termurtide or [thr-1]-MDP; N-acetylmuramyl-L-threonyl-D-isoglutamine); Ty particles (Ty-VLP or virus-like particles); Walter-Reed liposome (liposome containing lipid A adsorbed to aluminum hydroxide), and the like similar thereto, etc. are included.

[0099] As an example, the immunopotentiator contained in the pharmaceutical composition is alum (Th 2Inducing an immune response to enhance the humoral immune response); oil-in-water emulsion type immune enhancers such as MF59, AS03, AS04 (a mixture of MPL, which is a TLR-4 agonist, and alum, GSK), AddaVax (squalene-based; InvivoGen) (enhancing the antigenic immune response and inducing a well-balanced Th1 immune response); agonists such as Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), and NOD-like receptors (NLRs) of pattern recognition receptors (PRRs) such as LPS (lipopolysaccharide), Poly:C, imidazoquinolines (imiquinod and R848), CpG oligonucleotides, etc. may be included, but are not limited thereto.

[0100] When mixing the nucleic acid molecule or expression construct according to the present disclosure with an immune enhancer, their mixing ratios are not particularly limited. As an example, the nucleic acid molecule or expression construct according to the present disclosure and the immune enhancer can be formulated at a weight ratio of 100:1 to 1:100, preferably 10:1 to 1:10, more preferably 5:1 to 1:5, and most preferably 3:1 to 1:3.

[0101] Optionally, the pharmaceutical composition can be manufactured into a sustained release formulation. Suitable examples of the sustained release formulation include a semi-permeable matrix of a solid hydrophobic polymer containing a nucleic acid molecule or a gene delivery vector, and this matrix is in the form of a molded article, for example, a film or a microcapsule. Examples of the sustained release matrix include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate), or poly(vinyl alcohol)), polylactide, copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, and poly-D-(-)-3-hydroxybutyric acid.

[0102] In alternative embodiments, the pharmaceutical composition may include lipid nano particles (LNP) that can protect the nucleic acid molecule used as the active ingredient and improve the in vivo injection activity. Lipid nano particles include a number of lipid molecules physically associated with each other, but include microspheres (including monolayer and multilayer vesicles, such as liposomes), phases dispersed in emulsions, micelles, or the internal phase of a suspension. Lipid nano particles can be used to encapsulate the nucleic acid molecules of the present disclosure or peptides (proteins) expressed from the nucleic acid molecules for delivery.

[0103] Formulations containing cationic lipids are useful for delivering polyanions such as nucleic acid molecules. Other lipids that may be included are neutral lipids (i.e., uncharged or zwitterionic lipids), anionic lipids, helper lipids that enhance transfection, and stealth lipids that increase the length of time the nanoparticles can exist in vivo. Examples of suitable cationic lipids, neutral lipids, anionic lipids, helper lipids, and stealth lipids are disclosed in WO2016 / 010840A1, which is incorporated herein by reference.

[0104] For example, the lipid for encapsulation can be a cationic lipid, a biodegradable lipid. As an example, such lipids include (9Z,12Z)-3((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, ((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(octane-8,1-diyl)bis(decanoate), 2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3-diyl(9Z,9’Z,12Z,12’Z)-bis(octadeca-9,12-dienoate), 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octanoyloxy)tridecyl 3-octylundecanoate, heptatriaconta-6,9,28,31-tetraene-19-yl 4-(dimethylamino)butanoate (also known as Dlin-MC3-DMA (MC3)).

[0105] Suitable neutral lipids may include neutral, zwitterionic or amphoteric lipids. Neutral phospholipids include 5-heptadecylbenzene-1,3-diol (resorcinol), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), egg yolk phosphatidylcholine (EPC), dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1-myristoyl-2-palmitoylphosphatidylcholine (MPPC), 1-palmitoyl-2-myristoylphosphatidylcholine (PMPC), 1-palmitoyl-2-stearoylphosphatidylcholine (PSPC), 1,2-diarachidonoyl-sn-glycero-3-phosphocholine (DBPC), 1-stearoyl-2-palmitoylphosphatidylcholine (SPPC), 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEPC), palmitoyloleoylphosphatidylcholine (POPC), lysophosphatidylcholine, dioleoylphosphatidylethanolamine (DOPE), etc., but are not limited thereto.

[0106] Helper lipids can enhance transfection / enhance or enhance fusogenicity. Helper lipids include steroids, sterols, and alkylresorcinols. Specifically, helper lipids may include cholesterol, 5-heptadecylresorcinol, and cholesterol hemisuccinate.

[0107] Stealth lipids can assist the formulation process or modulate the pharmacokinetic properties of LNPs by reducing particle aggregation and controlling particle size. As an example, stealth lipids may include polymers with hydrophilic heads such as PEG (polyethylene glycol or polyethylene oxide), poly(oxazoline), poly(vinyl alcohol), poly(glycerol), poly(N-vinylpyrrolidone), polyamino acids, and poly N-(2-hydroxypropyl)methacrylamide. Specifically, stealth lipids may include, but are not limited to, PEG-dilauroylglycerol, PEG-dimyristoyl glycerol (PEG-DMG), PEG-dipalmitoyl glycerol, PEG-distearoyl glycerol (PEG-DSPE), PEG-dilauryl glycamide, PEG-dimyristyl glycamide, PEG-dipalmitoyl glycamide, PEG-distearoyl glycamide, PEG-cholesterol (1-[8’-(cholest-5-en-3[beta]-oxy)carboxamido-3’,6’-dioxaoctanyl]carbamoyl-[omega]-methyl-poly(ethylene glycol)), PEG-DMB (3,4-ditetradecyloxybenzyl-[omega]-methyl-poly(ethylene glycol) ether), etc.

[0108] The nucleic acid molecules of the present disclosure can induce non-antigen-specific immune responses. In relation to the immune response, T-lymphocytes differentiate into T-helper 1 (Th1) cells and T-helper 2 (Th2) cells, and the immune system can destroy intracellular (Th1) and extracellular (Th2) pathogens (e.g., antigens). Th1 cells assist the cellular immune response by the activity of macrophages and cytotoxic T-cells. Note that Th2 cells promote the humoral immune response by enhancing B-cells for conversion into plasma cells and the formation of antibodies (e.g., against antigens). Th 1 / Th 2The ratio is very important in the immune response, but the nucleic acid molecules of the present disclosure enhance and induce a Th1 immune response, i.e., a cell-mediated immune response. Thus, when the nucleic acid molecules according to the present disclosure are administered to a living body together with a pharmaceutically active substance, e.g., an immune enhancing component, the pharmaceutical composition can further enhance a specific immune response induced by the pharmaceutically active substance.

[0109] In one exemplary embodiment, the pharmaceutical composition may further contain a pharmaceutically active substance in addition to the aforementioned nucleic acid molecules. In one embodiment, the pharmaceutically active substance is an immune enhancing component such as an immunogen. As an example, the pharmaceutically active substance can be a compound having a therapeutic and / or prophylactic effect against cancer, infectious diseases, autoimmune diseases or allergies. For example, the pharmaceutically active substance can be a peptide, protein, nucleic acid, therapeutically active low molecular weight organic or inorganic compound, sugar, antigen or antibody, a therapeutic agent well known in the art, antigen-presenting cells, sections of antigen-presenting cells, cell fragments, a pathogen (such as a virus or bacterium) modified (e.g., attenuated or inactivated) by chemical or irradiation.

[0110] As an example, one antigen of the pharmaceutically active substance can be a peptide, polypeptide, protein, cell, cell extract, polysaccharide, complex polysaccharide, lipid, glycolipid and carbohydrate. For example, surface antigens of tumor cells and surface antigens, particularly in the secreted form of viral pathogens, bacterial pathogens, fungal pathogens or protozoan pathogens are preferred. Of course, the antigen can be present, for example, inside the nucleic acid molecules according to the present disclosure, and can also be present as a hapten bound to a suitable carrier. Other antigen components, inactivated or attenuated pathogens can also be used.

[0111] The pharmaceutical composition according to the present disclosure may contain a pharmaceutically acceptable carrier in addition to the nucleic acid molecule and the pharmaceutically active substance. In one exemplary embodiment, when the composition is in liquid form, the pharmaceutically acceptable carrier may be, for example, pyrogen-free water; isotonic saline or a buffered (aqueous) solution such as a buffered solution, for example, phosphate, citrate, etc., vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cacao butter; polyols such as polypropylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; and polyols such as alginic acid. For injecting a liquid vaccine composition into a living body, an aqueous buffer containing sodium salts, calcium salts and optionally potassium salts may be used. The sodium salts, calcium salts and potassium salts may be present in the form of halogens such as chlorine, iodine or bromine, or in the form of hydroxides, carbonates, bicarbonates, or sulfates, etc.

[0112] Also, when the pharmaceutical composition is in solid form, the pharmaceutically acceptable carrier may include a solid carrier such as a solid filler, a liquid filler or a diluent, and an encapsulating compound suitable for administration to a living body may also be used. For example, pharmaceutically acceptable solid carriers include sugars such as lactose, glucose and sucrose; starches such as corn starch or potato starch; celluloses and their derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, cellulose acetate; powdered tragacanth; malt; gelatin; tallow; solid lubricants such as stearic acid, magnesium stearate; calcium sulfate, etc.

[0113] A pharmaceutically acceptable carrier can be selected according to the manner in which the pharmaceutical composition according to the present disclosure is administered. The pharmaceutical composition according to the present disclosure can be administered, for example, systemically. Routes of administration include, for example, oral, subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, intracranial, transdermal, intradermal, intrapulmonal, intraperitoneal, intracardial, intraarterial, and sublingual topical and / or intranasal routes.

[0114] An appropriate amount of the pharmaceutical composition to be used can be determined by routine experimentation using animal models. Such models include, without any limitation, rabbit, sheep, mouse, rat, dog, and non-human primate models. Preferred injectable unit dosage forms include sterile aqueous solutions, physiological saline, or mixtures thereof. The pH of such solutions should be adjusted to about 7.4.

[0115] Carriers suitable for injection include hydrogels, devices for controlled release, devices for delayed release, polylactic acid, and collagen matrices. Pharmaceutically acceptable carriers suitable for topical use include those suitable for use in lotions, creams, gels, and the like. If the compound is administered perorally, tablets, capsules, and the like are preferred unit dosage forms. Pharmaceutically acceptable carriers for the manufacture of unit dosage forms that can be used for oral administration are well known in the art.

[0116] If necessary, in order to further increase the immunogenicity induced by a pharmaceutically active substance and / or a nucleic acid molecule, the pharmaceutical composition according to the present disclosure may further comprise one or more adjuvants. Non-limiting examples of such adjuvants are compounds that permit the maturation of dendritic cells (DCs), such as lipopolysaccharides, TNF-alpha or CD40 ligand, GM-CFS and / or cytokines. Specifically, cytokines such as various interleukins, interferons, GM-CSF, G-CSF, M-CSF, LT-beta, TNF-alpha, growth factors such as hGH, which promote the immune response, monokines, lymphokines, interleukins or chemokines.

[0117] The pharmaceutical composition according to the present disclosure may further contain one or more buffering agents, stabilizers, surfactants, wetting agents, lubricants, emulsifying agents, suspending agents, preservatives, antioxidants, opacifying agents, lubricants, processing aids, colorants, sweeteners, flavors, flavoring agents, diluents, and other known additives that provide an attractive appearance to the drug (i.e., the nucleic acid molecule, gene delivery agent or its vaccine composition which is the active ingredient of the present disclosure) or are useful in the manufacture of pharmaceutical products (i.e., pharmaceuticals). As an example, emulsifying agents such as Tween; wetting agents such as sodium lauryl sulfate, for example; colorants; taste-imparting agents, agents for forming tablets; stabilizers; antioxidants; preservatives.

[0118] The content and concentration of the nucleic acid molecule in the pharmaceutical composition according to the present disclosure are not particularly limited. In particular, when using nucleic acid molecules in the form of RNA platforms, they are rapidly degraded in vivo, so safety and stability can be ensured. In one exemplary embodiment, the nucleic acid molecule according to the present disclosure can be used at a concentration of 1 to 1000 μg / mL, preferably 10 to 1000 μg / mL in the pharmaceutical composition, but is not limited thereto.

[0119] An important factor for an appropriate immune response is the enhancement of other T-cell sub-populations. T-lymphocytes typically consist of two sub-populations, namely T-helper 1 (Th1 ) cells and T-helper 2 (Th 2 ) cells, and said subpopulation has an immune system capable of destroying intracellular (Th 1 ) pathogens and extracellular (Th 2 ) pathogens (e.g., antigens). The two Th cell populations differ in the pattern of effector proteins (cytokines) they produce. Generally, Th 1 cells mainly assist in humoral immunity and cell-mediated immune responses by the activity of macrophages and cytotoxic T-cells. Note that Th 2 cells mainly promote humoral immune responses through the enhancement of B-cells for conversion to plasma cells and the formation of antibodies (e.g., antibodies against antigens) in relation to cell-mediated immunity. Therefore, the Th 1 / Th 2 ratio is very important in the immune response. The nucleic acid molecules according to the present disclosure enhance both Th 1 immune responses and Th 2 immune responses.

[0120] As an example, the pharmaceutical composition according to the present disclosure can be used to induce tumor-specific or pathogen-specific immune responses and prevent tumors and infectious diseases. Optionally, the pharmaceutical composition can be used for the prevention of allergic disorders or diseases, autoimmune diseases, but is not limited thereto. The pharmaceutical composition according to the present disclosure can be administered in any convenient dosage form, such as tablets, powders, capsules, solutions, dispersions, suspensions, syrups, aerosols, suppositories, gels, emulsions, patches, etc. Such compositions can contain the usual components of pharmaceutical formulations, such as diluents, carriers, pH adjusters, sweeteners, bulking agents, and additional active agents.

[0121] The pharmaceutical compositions of the present disclosure can be manufactured in unit dosage form or can be manufactured by being enclosed in a multi-dose container by formulating them by a method that can be easily implemented by those having ordinary knowledge in the technical field to which the present disclosure pertains, using pharmaceutically acceptable carriers and / or excipients. At this time, the dosage form may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may further contain a dispersant or stabilizer.

[0122] In addition, in the present specification, the formulation can also contain more than one active compound, preferably compounds having complementary activities that do not have a harmful effect on each other, if necessary for the specific indication to be treated. Optionally or additionally, the composition may contain an agent that enhances its function, such as a cytotoxic agent, cytokine, chemotherapeutic agent, or growth inhibitor, or growth promoter. Such molecules are appropriately present in combination in an amount effective for the intended purpose.

[0123] In a selective embodiment, a gene delivery vehicle containing the nucleic acid molecule of the present disclosure can be included in the pharmaceutical composition. The gene delivery vehicle is manufactured to carry and express a nucleotide encoding the target immunogen. For manufacturing the gene delivery vehicle, the transcript of the gene of interest is preferably present within a suitable expression construct. In the expression construct, the transcript of the gene of interest encoding a virus-derived immunogen is preferably operably linked to a transcriptional control element (TCCE).

[0124] For example, the expression construct can be an expression vector into which the nucleic acid molecule described above is inserted. At this time, the vector may contain a nucleic acid molecule, and the nucleic acid molecule can also be combined with other nucleotides to encode a fusion protein or fusion peptide. Incidentally, the method of introducing the above-described gene delivery agent into cells can be carried out using various methods well known in the art. In the present disclosure, when the gene delivery agent is produced based on a viral vector, it is carried out by a viral infection method well known in the art. Further, in the present disclosure, when the gene delivery system is a naked recombinant DNA molecule or plasmid, the gene can be introduced into cells by microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, and gene bombardment.

[0125] Example 1: Production of a nucleic acid molecule into which an influenza virus hemagglutinin (HA) coding sequence has been inserted A nucleic acid molecule into which a nucleotide encoding Hemagglutinin (HA) of influenza virus was inserted was produced. The template DNA sequence having a coding region encoding HA of influenza virus from the nucleic acid molecule having MCS was designed as follows.

[0126] 5’-KpnI recognition sequence (GGTACC)-T7 promoter (SEQ ID NO: 11)-translation regulator derived from human troponin T1 (TNNT1) (SEQ ID NO: 1)-PacI recognition sequence (TTAATTAA)-Kozak sequence (GCCACC)-coding sequence encoding influenza virus HA (SEQ ID NO: 3) (HA, SEQ ID NO: 4)-ClaI recognition sequence (ATCGAT)-translation regulator derived from human troponin T1 (TNNT1) (SEQ ID NO: 2)-EcoRI recognition sequence (GAATTC)-polyadenylation signal (SEQ ID NO: 12)-SapI recognition sequence (GAAGAGC)-NotI recognition sequence (GCGGCCGC)-3’.

[0127] The template DNA was inserted into the pGH vector (downstream of SEQ ID NO: 13 and upstream of SEQ ID NO: 14), cloned, linearized with restriction enzymes, and a nucleic acid molecule in the form of an RNA platform (hereinafter referred to as "pHJ5L-HA") was produced through in vitro transcription (IVT).

[0128] Experimental Example 1: In vivo mouse immunization and measurement of antigen-specific immunoglobulins and cytokine production Wild type Balb / c mice were administered saline (Group 1) and the pHJ5L-HA nucleic acid molecule produced in Example 1 (Group 2). The nucleic acid molecule in RNA form was purified with N-methylpseudouridine (m1Ψ) and cellulose. The RNA was modified using a commercially available cap1-type capping reagent and formulated using lipid-based nanoparticles (LNP) used in Moderna's COVID-19 mRNA vaccine.

[0129] After the formulated pHJ5L-HA was intramuscularly injected into the mice twice at two-week intervals, blood was collected from the mice and organs were removed two weeks after the last immunization to confirm the degree of immune induction by the nucleic acid molecule. In this experimental example, the mouse immune groups were as shown in Table 1 below, and five mice per group were immunized.

[0130]

Table 1

[0131] Two weeks after the last immunization, the mice were euthanized with carbon dioxide and then dissected to collect blood from the abdominal aorta. The collected blood was left at room temperature for over 2 hours to agglutinate the blood cells, and then centrifuged at 4,000 g for 15 minutes to separately isolate the serum. After coating 100 ng of HA protein on a 96-well plate, the blood was diluted 1:200 in 1% BSA in PBS as the primary antibody and 50 μl aliquots were added. After reacting at room temperature for 2 hours, it was washed 3 times with 200 μl of 0.5% PBST, and then goat anti-mouse IgG H+I HRP conjugated was diluted 1:3000 in 1% BSA in PBS as the secondary antibody and 100 μl aliquots were added. After reacting at room temperature for 1 hour and then washing, 100 μl aliquots of TMB solution were added to confirm the color development. When the color development had sufficiently progressed, 50 μl aliquots of 2N sulfuric acid were added to stop the reaction, and the absorbance was measured at a wavelength of 450 nm using a spectrophotometer capable of measuring absorbance.

[0132] The results of measuring the amount of antigen-specific antibodies secreted in mouse serum by ELISA according to this example are shown in FIGS. 2, 3, and 4. Different from the negative control group (G1) injected with physiological saline, it was confirmed that a large amount of antibodies specific to HA protein were produced in the group (G2) immunized with the mRNA nucleic acid molecule prepared in Example 1. In particular, both IgG1 involved in the Th2 immune response and IgG2a involved in the Th 1 immune response were produced in large amounts. Even when the serum was diluted 12,800-fold and measured, it showed a high absorbance, confirming that a large amount of antibodies were formed.

[0133] Experimental Example 2: Evaluation of neutralizing ability through Hemagglutin Inhibition (HI) assay Mouse serum was obtained using the same method as in Experimental Example 1. 25 μl of PBS was placed in a 96-well plate. 25 μl of mouse serum was placed in the first column and serially diluted two-fold in the side columns. 25 μl each of influenza virus of the type such as HA encoded by pHJ5L-HA mRNA (PR8 HA) was placed in all wells and then reacted for 30 minutes. Thereafter, 50 μl each of 1% chicken blood was added and the mixture was waited for the red blood cells to agglutinate. The agglutination of red blood cells was due to the HA antigen of the virus, and the non-agglutination of red blood cells was due to the presence of HA antibody in the serum. The analysis results are shown in Fig. 5. As shown in Fig. 5, in the group into which pHJ5L-HA mRNA was introduced, it can be seen that, despite the high dilution ratio, the agglutination of red blood cells was prevented and it had a high antibody titer.

[0134] Experimental Example 3: Measurement of neutralization ability through Microneutralization assay Mouse serum was obtained using the same method as in Experimental Example 1. MDCK cells were seeded in a 96-well plate at 3x10 4 cells / well. Serum was prepared at 12 μl each and inactivated at 56 °C for 30 minutes. The inactivated serum was diluted 10-fold with the medium and 80 μl each was placed in the first row of the empty 96-well plate. 40 μl each of the medium was placed in the other wells, and 40 μl each of the serum in the first row was transferred and diluted two-fold. 100 TCID 50Forty microliters of the virus at a concentration of 40 μl were added, and the reaction was carried out in a 37°C incubator for 1 hour. The reacted virus and serum were each added to the MDCK cells in a 96-well plate from which 50 μl of the medium had been removed, and infection was allowed to occur in a 37°C incubator for 2 hours. After 2 hours, 50 μl of complete medium was added to each well, and it was placed in a 37°C incubator. The cells were observed by time period, and when cytopathic effects occurred, all of the supernatant was removed, 4% formaldehyde solution was added, and fixation was carried out for 3 hours or more. Thereafter, 2% crystal violet solution was added to each well, staining was carried out for 30 minutes or more, and washing was performed to measure the virus titer. The measurement results are shown in Figure 6. It was confirmed that the group immunized with pHJ5L-HA mRNA effectively neutralized the virus.

[0135] Experimental Example 4: Measurement of Antigen-Specific IFN-Gamma Production (1) ELISPOT Analysis When sacrificing the mice immunized according to the immunization schedule of Experimental Example 1, the excised spleen was sieved using a 40 μm strainer to obtain a single cell suspension as much as possible. The sieved spleen cells were added to a pre-coated ELISpot plate at a density of 5x10 5 cells / well, 50 μl each. In the case of non-stimulation (Group 1), 50 μl of RPMI1640 complete medium (10% FBS, 1% antibiotics) was added. In the case of peptide stimulation (Group 2) expressed by pHJ5L-HA mRNA, seven types of HA peptides were added to the complete medium at a concentration of 4 μg / well, 50 μl each. After culturing in a 37°C incubator for 48 hours, the experiment was carried out according to the protocol provided by the manufacturer. The measurement results are shown in Figure 7. It was confirmed that the number of spots increased and the number of cells secreting IFN-gamma increased significantly in the group immunized with pHJ5L-HA mRNA (Group 2) compared with the negative control group (G1).

[0136] (2) Flow Cytometry Using FACS The spleen cells sieved above were added to a round-bottom 96-well plate at a density of 1x106 After placing the cells / well in the appropriate number, similarly, 7 types of HA peptides were added at a concentration of 4 μg / well. After stimulation, the cells were cultured in an incubator at 37 °C for 24 hours. Subsequently, after staining with T cell-specific antibodies and IFN-γ-specific antibodies, T cells that produce IFN-γ were sorted using a flow cytometry device. The analysis results are shown in FIGS. 8 and 9. Compared with the negative control group (G1), the number of T cells that secrete IFN-γ increased significantly in the group immunized with mRNA (G2).

[0137] Example 2: Preparation of a nucleic acid molecule into which the N glycoprotein (Gn) sequence of SFTSV is inserted The procedure of Example 1 was repeated to prepare a nucleic acid molecule in the form of an RNA platform (hereinafter referred to as "pHJ5L-Gn"), except that a modified nucleotide (SEQ ID NO: 6) encoding the glycoprotein N of SFTSV (SEQ ID NO: 5) was inserted instead of the nucleotide encoding influenza HA in the coding region.

[0138] Example 3: Preparation of a nucleic acid molecule into which the N glycoprotein (Gn) deletion sequence of SFTSV is inserted The procedure of Example 1 was repeated to prepare a nucleic acid molecule in the form of an RNA platform (hereinafter referred to as "pHJ5L-GNΔTM"), except that a modified nucleotide (SEQ ID NO: 8) encoding the glycoprotein N deletion protein of SFTSV (SEQ ID NO: 7) was inserted instead of the nucleotide encoding influenza HA in the coding region.

[0139] Example 4: Preparation of a nucleic acid molecule into which the N glycoprotein Gn) deletion sequence of SFTSV is inserted The procedure of Example 1 was repeated, except that a modified nucleotide (SEQ ID NO: 10) encoding the SFTSV glycoprotein N-deficient protein (SEQ ID NO: 9) was inserted instead of the nucleotide encoding influenza HA in the coding region, to produce a nucleic acid molecule in the form of an RNA platform (hereinafter referred to as "pHJ5L-GNΔSTEM").

[0140] Experimental Example 5: Confirmation of Expression of Nucleic Acid Molecule The presence or absence of the expression of the target peptide was confirmed from the mRNAs produced in Examples 2 to 4. VERO cells were seeded in a 6-well cell culture plate with 10% FBS DMEM complete medium at 6×10 5 and incubated overnight at 37°C. 10 μg of the nucleic acid molecule was transfected with Lipofectamine2000 (registered trademark) and incubated at 37°C for 30 hours. After incubation, the intracellular protein was quantified and 30 μg each was subjected to SDS PAGE. The presence or absence of the expression of Gn, GnΔTM, and GnΔSTEM proteins was confirmed by Western blot as follows. The SFTS Virus HB29 Antibody (NBP2-41153) was diluted 1:1000 in 5% skim milk in PBST as the primary antibody and incubated overnight at 4°C, and the anti-rabbit IgG HRP antibody was diluted 1:5000 in 5% skim milk in PBST as the secondary antibody and incubated at room temperature for 1 hour. As shown in Figure 10, a protein of approximately 60 kDa was expressed in the pHJ5-Gn nucleic acid molecule, a protein of approximately 50 kDa was expressed in the pHJ5L-GnΔTM nucleic acid molecule, and a protein of approximately 40 kDa was expressed in the pHJ5L-GnΔSTEM nucleic acid molecule.

[0141] Experimental Example 6: In Vivo Mouse Immunization and Measurement of Antigen-Specific Immunoglobulin Production Wild type C57BL / 6 mice were administered saline (Group 1) and the pHJ5L-Gn nucleic acid molecule prepared in Example 2 (Group 2). Otherwise, after intramuscular injection into mice at the same immunization schedule and dosage as in Experimental Example 1, two weeks after the last immunization, the presence or absence of mRNA-induced immunity was confirmed by collecting blood from the mice and removing organs. The mouse groups used in this experimental example were the same as those in Table 1 of Experimental Example 1, and 5 mice per group were immunized.

[0142] Two weeks after the last immunization, the mice were euthanized with carbon dioxide and then dissected to collect blood from the abdominal aorta. The collected blood was left at room temperature for more than 2 hours to aggregate blood cells, and then centrifuged at 4,000 g for 15 minutes to separately isolate serum. After coating 100 ng of Gn protein on a 96-well plate, the amount of antigen-specific antibody secreted in mouse serum was measured by ELISA in the same manner as in Experimental Example 1. The measurement results are shown in FIGS. 11 and 12. Different from the negative control group (G1) injected with physiological saline, it was confirmed that a large amount of antibody specific to Gn protein was produced in the group (G2) immunized with pHJ5L-Gn mRNA. In particular, it was confirmed that a large amount of IgG1 involved in the Th2 immune response and IgG2c involved in the Th 1 immune response were all produced in large amounts.

[0143] Experimental Example 7: Measurement of antigen-specific IFN-gamma production When sacrificing the mice immunized according to the immunization schedule of Experimental Example 6, the excised spleen was sieved using a 40-μm strainer to obtain single cells as much as possible. Subsequently, in the case of peptide stimulation (Group 2) expressed with pHJ5L-Gn mRNA, Gn protein was added to the complete medium at a concentration of 200 ng / well and 50 μl was added to each well. After culturing in a 37°C incubator for 48 hours, the experiment was carried out according to the protocol provided by the manufacturer. The measurement results are shown in Figure 13. It was confirmed that the number of spots increased and the number of cells secreting IFN-gamma increased significantly in the group immunized with pHJ5L-Gn mRNA (Group 2) compared with the negative control group (G1).

[0144] Experimental Example 7: Measurement of neutralizing antibody titer Vero cells were seeded in a 24-well plate at 1.5 x 10 5Inoculate at a concentration of cells / well and grow in a 37°C incubator. 60 μl of serum obtained from rats was added to 540 μl of medium, diluted 1 / 10, and inactivated at 56°C for 30 minutes. 300 μl of the inactivated serum was added to a location containing 300 μl of medium in advance and diluted 1 / 2. In this way, the initially 10-fold diluted serum was diluted up to 320-fold. 200 μl of the diluted serum was mixed with 200 μl of an SFTS virus solution at a concentration of 80 ffu / well. The complete medium of the pre-inoculated cells was removed, washed with PBS, and then 100 μl of the solution in which the serum and the virus were mixed was added to each well and reacted in a 37°C incubator for 1 hour. At this time, shake at 20-minute intervals. After 1 hour, the virus solution was removed, and then 1 mL of a 1.5% carboxymethyl cellulose solution was added to each well and left in a 37°C incubator for 2 days. After 2 days, the supernatant was removed, a 4% formaldehyde solution was added, and left at room temperature for 10 minutes to inactivate the virus. After 10 minutes, the supernatant was removed and washed with PBS. Diluted 1:500 in PBS, 500 μl of an anti-SFTSV NP monoclonal antibody with 0.5% Triton X-100 was added to each well, shaken at room temperature, and reacted for 90 minutes. After removing the supernatant, it was washed with PBS. As a secondary antibody, an HRP-conjugated antibody containing 0.5% Triton X-100 was diluted 1:2000 in PBS, 500 μl was added to each well, and then reacted for 90 minutes while shaking at room temperature. After further removing the supernatant, it was washed with PBS. Then, 500 μl of DAB substrate was added to each well, and after the reaction until the foci turned brown, it was washed with tap water to stop the reaction. At this time, the dilution multiple at which the number of stained spots becomes half of the number of wells containing only the virus without serum is called FRNT50. A higher dilution multiple means more neutralizing antibodies. The measurement results are shown in Figure 14. It can be confirmed that high neutralizing antibodies are present in the serum of the group immunized with mRNA.

[0145] The foregoing has described the present disclosure based on exemplary embodiments and examples of the present disclosure. However, the present disclosure is not limited to the technical ideas described in the foregoing embodiments and examples. Instead, those having ordinary knowledge in the technical field to which the present disclosure pertains can easily conceive of various modifications and changes based on the foregoing embodiments and examples. However, it is clear from the appended claims that all such modifications and changes fall within the scope of the rights of the present disclosure.

Claims

1. A translation regulator derived from troponin T1 (TNNT1), and a coding region operably linked to the translation regulator, wherein the translation regulator includes a first translation regulator located upstream of the coding region and a second translation regulator located downstream of the coding region, the first translation regulator consists of the nucleotide of SEQ ID NO: 1 or its transcript, the second translation regulator consists of the nucleotide of SEQ ID NO: 2 or its transcript, the coding region includes a nucleotide encoding an immunogen of influenza virus or a fragment thereof, the immunogen of the influenza virus is the hemagglutinin of the influenza virus or a fragment thereof, the immunogen of the influenza virus consists of the amino acids of SEQ ID NO: 3, a nucleic acid molecule, wherein the nucleotide encoding the immunogen of the influenza virus or a fragment thereof consists of the nucleotide of SEQ ID NO: 4 or its transcript.

2. A translation regulator derived from troponin T1 (TNNT1), and a coding region operably linked to the translation regulator, wherein the translation regulator includes a first translation regulator located upstream of the coding region and a second translation regulator located downstream of the coding region, the first translation regulator consists of the nucleotide of SEQ ID NO: 1 or its transcript, the second translation regulator consists of the nucleotide of SEQ ID NO: 2 or its transcript, the coding region includes a nucleotide encoding an immunogen of severe fever with thrombocytopenia syndrome virus (SFTSV) or a fragment thereof, the immunogen of the severe fever with thrombocytopenia syndrome virus or a fragment thereof is the glycoprotein N of the severe fever with thrombocytopenia syndrome virus or a fragment thereof, the immunogen of the severe fever with thrombocytopenia syndrome virus consists of amino acids selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9 The nucleotide encoding the immunogen of severe fever with thrombocytopenia syndrome virus or a fragment thereof consists of a nucleotide selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 10, or a transcript thereof, and is a nucleic acid molecule.

3. The nucleic acid molecule according to claim 1 or 2, wherein the nucleic acid molecule is in the form of RNA.

4. The nucleic acid molecule according to claim 1 or 2, further comprising at least one nucleotide of a transcription regulatory factor located upstream of the first translation regulatory factor and a polyadenylation signal sequence or polyadenosine sequence located downstream of the transcription regulatory factor.

5. A recombinant expression vector comprising a nucleic acid molecule, a translation regulatory factor derived from troponin T1 (troponin T1, TNNT1), comprising a coding region operably linked to the translation regulatory factor, wherein the translation regulatory factor comprises a first translation regulatory factor located upstream of the coding region and a second translation regulatory factor located downstream of the coding region, wherein the first translation regulatory factor consists of the nucleotide of SEQ ID NO: 1, wherein the second translation regulatory factor consists of the nucleotide of SEQ ID NO: 2, wherein the coding region comprises a nucleotide encoding an immunogen of influenza virus or a fragment thereof, wherein the immunogen of influenza virus or a fragment thereof is hemagglutinin of the influenza virus or a fragment thereof, wherein the immunogen of influenza virus consists of the amino acid of SEQ ID NO: 3, and the nucleotide encoding the immunogen of influenza virus or a fragment thereof consists of the nucleotide of SEQ ID NO: 4, and is a recombinant expression vector.

6. A recombinant expression vector comprising a nucleic acid molecule, a translation regulatory factor derived from troponin T1 (troponin T1, TNNT1), comprising a coding region operably linked to the translation regulatory factor, wherein the translation regulatory factor comprises a first translation regulatory factor located upstream of the coding region and a second translation regulatory factor located downstream of the coding region, wherein the first translation regulatory factor consists of the nucleotide of SEQ ID NO: 1, wherein the second translation regulatory factor consists of the nucleotide of SEQ ID NO: 2, The coding region contains nucleotides encoding an immunogen of severe fever with thrombocytopenia syndrome virus (SFTSV) or a fragment thereof, The immunogen of the severe fever with thrombocytopenia syndrome virus or a fragment thereof is the glycoprotein (glycoprotein N) of the severe fever with thrombocytopenia syndrome virus or a fragment thereof, The immunogen of the severe fever with thrombocytopenia syndrome virus consists of amino acids selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9, The nucleotide encoding the immunogen of the severe fever with thrombocytopenia syndrome virus or a fragment thereof consists of nucleotides selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 10, and is a recombinant expression vector.

7. The expression vector according to claim 5 or 6, wherein the nucleic acid molecule is a nucleic acid molecule in the form of RNA.

8. The expression vector according to claim 5 or 6, wherein the nucleic acid molecule further comprises at least one nucleotide of a transcription regulatory factor located upstream of the first translation regulatory factor and a polyadenylation signal sequence or a polyadenosine sequence located downstream of the transcription regulatory factor.

9. A pharmaceutical composition for treating or preventing influenza, comprising, as an active ingredient, the nucleic acid molecule according to claim 1 or an expression construct into which the nucleic acid molecule according to claim 1 is inserted.

10. A pharmaceutical composition for treating or preventing severe fever with thrombocytopenia syndrome, comprising, as an active ingredient, the nucleic acid molecule according to claim 2 or an expression construct into which the nucleic acid molecule according to claim 2 is inserted.

11. The pharmaceutical composition according to claim 9 or 10, further comprising at least one of an immunopotentiator, a nucleic acid stabilizer, and lipid nanoparticles.

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