Nucleic acid expression platform with increased expression efficiency

Abstract

The present disclosure relates to a nucleic acid molecule having multiple translational regulatory elements inserted downstream of a coding region, and a nucleic acid expression platform or a nucleic acid expression system, such as a recombinant expression vector into which the nucleic acid molecule is inserted. The nucleic acid molecule of the present disclosure can be used to efficiently express a gene of interest inserted into the coding region. The expression platform can be used to improve the expression efficiency of a reporter gene, an antigen, and / or a therapeutic protein or peptide.

Description

[Technical Field]

[0001] This patent application relates to the results of the "Development of a new mRNA vaccine platform" project (supervising agency: The Catholic University of Korea Industry-Academia Collaboration Foundation; project number: 1711158916) as part of the "New and Variant Infectious Disease Response Platform Core Technology Development Project" of the Ministry of Science, ICT and Communications of the Republic of Korea, and the "Research on the Development of Toxicity Assessment Technology for mRNA Vaccines, etc. (1)" project (supervising agency: The Catholic University of Korea Industry-Academia Collaboration Foundation; project number: 1475013322) as part of the "Innovative Technology Support Research Project for Infectious Disease Response" of the Ministry of Food and Drug Safety of the Republic of Korea.

[0002] The present disclosure relates to nucleic acid expression platforms, and more particularly to nucleic acid expression platforms or systems such as nucleic acid molecules with increased expression efficiency and recombinant expression vectors containing the same. [Background technology]

[0003] Since the advent of recombinant DNA technology in the 1970s, expression systems capable of expressing a gene of interest (GOI) have become available. Cell-based expression systems, such as those based on microorganisms or eukaryotes, are common, while cell-free expression systems typically use purified RNA polymerase, ribosomes, tRNA, and ribonucleotides.

[0004] In particular, proteins derived from eukaryotic cells undergo post-translational modifications such as phosphorylation, methylation, and glycosylation after expression, but microorganisms do not possess such post-translational modification mechanisms, so when attempting to express proteins derived from eukaryotic cells, a eukaryotic cell expression system must be used.

[0005] Eukaryotic cell-induced expression systems can be used for gene therapy agents, nucleic acid vaccines, etc. In the case of gene therapy agents, a gene of interest consisting of an open reading frame (ORF) encoding a peptide or protein that can treat various diseases is inserted into the expression system. In the case of nucleic acid vaccines, a gene of interest consisting of an open reading frame encoding a peptide or protein that can be expressed as an antigen is inserted into the expression system. However, there is a constant demand in related technical fields for nucleic acid expression systems that can improve the expression efficiency of a gene of interest. Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present disclosure is to provide a nucleic acid expression platform or a nucleic acid expression system that can improve the expression efficiency of a target gene. [Means for solving the problem]

[0007] According to one aspect, coding region and the above coding region operably linked to Translational regulatory elements wherein the translational regulatory element comprises an upstream translational regulatory element located upstream of the coding region and a downstream translational regulatory element located downstream of the coding region, and the downstream translational regulatory element comprises a plurality of downstream translational regulatory elements.

[0008] In exemplary embodiments, the downstream translational regulatory element may include a first downstream translational regulatory element and a second downstream translational regulatory element located downstream of the coding region.

[0009] As an example, the multiple downstream translational regulatory elements (e.g., the first downstream translational regulatory element and the second downstream translational regulatory element) may each comprise a translational regulatory element or a transcript sequence derived from any one gene selected from the group consisting of human troponin T1, slow skeletal type (human TNNT1), human albumin, human ferritin light chain (human FTL), human CC motif chemokine ligand (human CCL19), human vascular-associated migratory cell protein (human AAMP), human ribosomal protein S27 (human RPS27), and human defensin alpha 5 (human DEFA5).

[0010] The plurality of downstream translational regulatory elements (e.g., the first downstream translational regulatory element and the second downstream translational regulatory element) may each independently comprise a translational regulatory element or a transcript sequence thereof selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14.

[0011] In an exemplary embodiment, the first downstream translational regulatory element may comprise a translational regulatory element derived from the human TNNT1 or a transcript sequence thereof, and the second downstream translational regulatory element may comprise a translational regulatory element derived from any one selected from the group consisting of the human albumin, the human FTL, the human CCL19, the human AAMP, the human RPS27, and the human DEFA5 or a transcript sequence thereof.

[0012] In another exemplary embodiment, the first downstream translational regulatory element may comprise a translational regulatory element derived from human albumin or a transcript sequence thereof, and the second downstream translational regulatory element may comprise a translational regulatory element derived from human DEFA5 or a transcript sequence thereof.

[0013] In yet another exemplary embodiment, the first downstream translational regulatory element may comprise a translational regulatory element derived from the human FTL or a transcript sequence thereof, and the second downstream translational regulatory element may comprise a translational regulatory element derived from any one selected from the group consisting of the human TNNT1, the human FTL, the human RPS27, and the human DEFA5, or a transcript sequence thereof.

[0014] In yet another exemplary embodiment, the first downstream translational regulatory element may comprise a translational regulatory element derived from the human CCL19 or a transcript sequence thereof, and the second downstream translational regulatory element may comprise a translational regulatory element derived from any one selected from the group consisting of the human TNNT1, the human FTL, the human CCL19, the human RPS27, and the human DEFA5, or a transcript sequence thereof.

[0015] In yet another exemplary embodiment, the first downstream translational regulatory element may comprise a translational regulatory element derived from the human AAMP or its transcript sequence, and the second downstream translational regulatory element may comprise a translational regulatory element derived from any one selected from the group consisting of the human albumin, the human FTL, the human RPS27, and the human DEFA5 or its transcript sequence.

[0016] In yet another exemplary embodiment, the first downstream translation regulatory element may comprise a translation regulatory element derived from human RPS27 or a transcript sequence thereof, and the second downstream translation regulatory element may comprise a translation regulatory element derived from any one selected from the group consisting of human TNNT1, human albumin, human FTL, human CCL19, human AAMP, human RPS27, and human DEFA5, or a transcript sequence thereof.

[0017] In yet another exemplary embodiment, the first downstream translational regulatory element may comprise a translational regulatory element derived from human DEFA5 or a transcript sequence thereof, and the second downstream translational regulatory element may comprise a translational regulatory element derived from any one selected from the group consisting of human TNNT1, human albumin, human FTL, human CCL19, human AAMP, and human RPS27, or a transcript sequence thereof.

[0018] In yet another exemplary embodiment, the downstream translational regulatory element may further include a third downstream translational regulatory element located downstream of the second downstream translational regulatory element.

[0019] The third downstream translational regulatory element may comprise a translational regulatory element or a transcript sequence thereof derived from any one selected from the group consisting of human troponin T1, slow skeletal type (TNNT1), human albumin, human ferritin light chain (FTL), human CC motif chemokine ligand (CCL19), human vascular-associated migratory cell protein (AAMP), human ribosomal protein S27 (RPS27) and human defensin alpha 5 (DEFA5).

[0020] For example, the first downstream translational regulatory element may comprise a translational regulatory element derived from human TNNT1 or a transcript sequence thereof, the second downstream translational regulatory element may comprise a translational regulatory element derived from human DEFA5 or a transcript sequence thereof, and the third downstream translational regulatory element may comprise a translational regulatory element derived from human FTL or human RPS27 or a transcript sequence thereof.

[0021] In another exemplary embodiment, the first downstream translational regulatory element may comprise a translational regulatory element derived from human albumin or a transcript sequence thereof, the second downstream translational regulatory element may comprise a translational regulatory element derived from human DEFA5 or a transcript sequence thereof, and the third downstream translational regulatory element may comprise a translational regulatory element derived from human RPS27 or a transcript sequence thereof.

[0022] In yet another exemplary embodiment, the first downstream translation regulatory element may comprise a translation regulatory element derived from human RPS27 or a transcript sequence thereof, the second downstream translation regulatory element may comprise a translation regulatory element derived from human FTL or a transcript sequence thereof, and the third downstream translation regulatory element may comprise a translation regulatory element derived from any one selected from the group consisting of human CCL19, human RPS27, and human DEFA5, or a transcript sequence thereof.

[0023] In yet another exemplary embodiment, the first downstream translational regulatory element may comprise a transcriptional translational regulatory element derived from human DEFA5 or its transcript sequence, the second downstream translational regulatory element may comprise a translational regulatory element derived from human TNNT1 or its transcript sequence, and the third downstream translational regulatory element may comprise a translational regulatory element derived from human FTL or its transcript sequence.

[0024] The upstream translational regulatory element may comprise a translational regulatory element or a transcript sequence thereof derived from any one selected from the group consisting of human troponin T1, slow backbone type (human TNNT1), human albumin, human ferritin light chain (human FTL), human CC motif chemokine ligand (human CCL19), human vascular-associated migratory cell protein (human AAMP), human ribosomal protein S27 (human RPS27), and human defensin alpha 5 (human DEFA5).

[0025] As an example, the upstream translational regulatory element may comprise a translational regulatory element or transcript sequence thereof selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7.

[0026] The nucleic acid molecule may be RNA.

[0027] The nucleic acid molecule may further comprise a transcriptional regulatory element operably linked to the coding region.

[0028] In alternative embodiments, the nucleic acid molecule may further comprise a polyadenylation signal sequence or a polyadenosine sequence located downstream of the downstream translational regulatory sequence.

[0029] In exemplary embodiments, the coding region may encode at least one of a reporter protein or fragment thereof, and a marker or selection protein or fragment thereof.

[0030] In another exemplary embodiment, the coding region may encode an antigen or a fragment thereof.

[0031] For example, the antigen may comprise a pathogen antigen, a tumor antigen, or a peptide that is a variant or derivative thereof.

[0032] In yet another exemplary embodiment, the coding region may encode a peptide or fragment thereof for the treatment of a disease.

[0033] In another aspect, a recombinant expression vector is disclosed into which the above-described nucleic acid molecule has been inserted.

[0034] In yet another aspect, a method for producing a protein or peptide is disclosed, which comprises injecting the nucleic acid molecule described above or a recombinant expression vector into which the nucleic acid molecule described above has been inserted into a living body, and expressing the protein or peptide from the injected nucleic acid molecule. [Effects of the Invention]

[0035] The nucleic acid expression platform of the present disclosure is designed to place multiple translational regulatory sequences downstream of the coding region of the open reading frame of a gene of interest. By introducing the nucleic acid expression platform, the expressed transcript can be stably maintained in vivo. This allows the application of the nucleic acid expression platform of the present disclosure to ultimately improve the efficiency of expression of the gene of interest.

[0036] The expression platform can be used to improve the expression efficiency of various genes of interest, such as reporter genes, infectious antigens, tumor antigens, and / or peptides or proteins used for therapeutic purposes. Therefore, the expression platform of the present disclosure can be used to detect or analyze the presence or absence of specific substances, as well as to produce vaccines, such as gene therapy agents, mRNA vaccines, and / or protein subunit vaccines. [Brief explanation of the drawings]

[0037] FIG. 1 is a schematic diagram illustrating the structure of a nucleic acid molecule or polynucleotide that can efficiently express a gene of interest according to an exemplary embodiment of the present disclosure.

[0038] FIG. 2 is a schematic diagram illustrating the structure of a nucleic acid molecule or polynucleotide that can efficiently express a gene of interest according to another exemplary embodiment of the present disclosure.

[0039] 3 and 4 are graphs showing the results of measuring the expression level of Renilla luciferase 6 hours and 24 hours after transfecting a nucleic acid molecule having a coding region encoding Renilla luciferase into Nor10 cells, a mouse muscle-derived cell line, according to an exemplary embodiment of the present disclosure.

[0040] 5 and 6 are graphs showing the results of measuring the expression level of Renilla luciferase 6 hours and 24 hours after transforming a nucleic acid molecule having a coding region encoding Renilla luciferase according to an exemplary embodiment of the present disclosure into HeLa cells, a human induced cell line.

[0041] 7 and 8 are graphs showing the results of measuring the expression level of Renilla luciferase 6 hours and 24 hours after transforming a nucleic acid molecule having a coding region encoding Renilla luciferase according to an exemplary embodiment of the present disclosure into 293A cells, a human derived cell line.

[0042] 9 and 10 are graphs showing the results of measuring the expression level of Renilla luciferase 6 hours after injecting a nucleic acid molecule having a coding region encoding Renilla luciferase into the ear of a mouse according to an exemplary embodiment of the present disclosure.

[0043] 11 and 12 are graphs showing the results of measuring the expression level of Renilla luciferase 6 hours and 24 hours after transforming a nucleic acid molecule having a coding region encoding Renilla luciferase according to an exemplary embodiment of the present disclosure into 293A cells, a human derived cell line.

[0044] In Figures 3 to 12, A to G each indicate a downstream translation regulatory sequence. The first letter indicates the first downstream translation regulatory element, the second letter indicates the second downstream translation regulatory element, and the third letter indicates the third downstream translation regulatory element. A indicates a downstream translation regulatory element derived from human TNNT1 (SEQ ID NO: 8), B indicates a translation regulatory element derived from human albumin (SEQ ID NO: 9), C indicates a translation regulatory element derived from human FTL (SEQ ID NO: 10), D indicates a translation regulatory element derived from human CCL19 (SEQ ID NO: 11), E indicates a translation regulatory element derived from human AAMP (SEQ ID NO: 12), F indicates a translation regulatory element derived from human RPS27 (SEQ ID NO: 13), and G indicates a translation regulatory element derived from human DEFA5 (SEQ ID NO: 14). The expression levels of peptides or proteins expressed from the pHJ5 nucleic acid molecule used as a comparative example were also compared. DETAILED DESCRIPTION OF THE INVENTION

[0045] Definition of Terms As used herein, the term "amino acid" is used in the 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 that have the characteristics of amino acids and properties known in the art. For example, analogs or mimetics of phenylalanine or proline that allow for the same stereochemical restriction of peptide compounds as natural Phe or Pro are included within the definition of amino acids. Such analogs and mimetics are referred to herein as "functional equivalents" of amino acids.

[0046] For example, synthetic peptides synthesized by standard solid-phase synthesis techniques are not limited to the amino acids encoded by genes, thereby allowing for a wider variety of substitutions for a given amino acid. Amino acids not encoded by the genetic code are referred to herein as "amino acid analogs."

[0047] By way of 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; allohydroxylysine (Ab) for Lys. OH; 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 trifluorylphenylalanine for Phe.

[0048] As used herein, the term "peptide" includes proteins, protein fragments, and peptides isolated from nature, synthesized by recombinant techniques, or chemically synthesized. For example, peptides of the present disclosure are composed of at least five, and for example, at least ten, amino acids.

[0049] In certain embodiments, compound variants, such as peptide variants with one or more amino acid substitutions, are provided. As used herein, the term "peptide variant" refers to a peptide 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 exhibits substantially the same biological function as the peptide composed of the original amino acids. Peptide variants must have at least 70% identity with the original peptide, preferably at least 90%, and more preferably at least 95% identity.

[0050] Such substitutions may include what are known as "conservative" amino acid substitutions. Variants can also include nonconservative changes. In exemplary embodiments, the sequence of a variant polypeptide differs from the original sequence by the substitution, deletion, addition, or insertion of five or fewer amino acids. Variants can also vary by the deletion or addition of amino acids that have minimal effect on the immunogenicity, secondary structure, and hydropathic nature of the peptide.

[0051] A "conservative" substitution means that the substitution of one amino acid for another does not significantly change the secondary structure of the polypeptide, etc. Amino acid mutations are obtained based on the relative similarity of the amino acid side chain substitutes, such as similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature.

[0052] For example, amino acids can be classified by common side chain properties as follows: 1) hydrophobic (norleucine, methionine, alanine, valine, leucine, isoleucine), 2) neutral hydrophilic (cysteine, serine, threonine, asparagine, glutamine), 3) acidic (aspartic acid, glutamic acid), 4) basic (histidine, lysine, arginine), 5) residues that influence chain direction (glycine, proline), and 6) aromatic (tryptophan, tyrosine, phenylalanine). Conservative substitutions involve exchanging one member of each of these classes for another member of the same class.

[0053] Analysis of the size, shape, and type of amino acid side chain substitutions 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. Therefore, based on these considerations, arginine, lysine, and histidine; alanine, glycine, and serine; and phenylalanine, tryptophan, and tyrosine can be considered biologically functional equivalents.

[0054] When introducing mutations, the hydropathic index of amino acids can be considered. Each amino acid is assigned a hydrophobic index based on its 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).

[0055] The hydrophobic amino acid index is very important in imparting interactive biological functions to proteins. It is well known that substitution with an amino acid having a similar hydrophobic index can retain similar biological activity. When introducing mutations based on the hydrophobic index, substitutions are made between amino acids showing a difference in hydrophobic index preferably within ±2, more preferably within ±1, and even more preferably within ±0.5.

[0056] It is also well known that substitutions of amino acids with similar hydrophilicity values ​​result in proteins with equivalent biological activity. The following hydrophilicity values ​​have been 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); lysine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). When mutations are introduced with reference to the hydrophilicity value, substitutions are preferably made between amino acids showing a difference in hydrophilicity value within ±2, more preferably within ±1, and more preferably within ±0.5.

[0057] 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 between 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, and Asp / Gly.

[0058] Generally, 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 present in its natural state is isolated by removing all or part of the materials with which it is present in that state. Such polypeptides should be at least 90% pure, preferably 95%, and more preferably 99% pure. Polynucleotides are isolated by cloning into a vector. Recombinant peptides encoded by the nucleotide sequences referred to herein can be readily produced by known methods using any of many known expression vectors. Expression can be carried out in a suitable host cell transformed with an expression vector containing a DNA sequence encoding the recombinant protein. Suitable host cells include prokaryotes, yeast, and eukaryotes. By way of example, yeast, insect cells, or eukaryotic-derived cell lines such as mammalian cell lines (e.g., Cos or CHO) can be used as host cells.

[0059] As used herein, the terms "polynucleotide" and "nucleic acid" are used interchangeably and refer to a polymer of nucleotides of any length, encompassing DNA (e.g., cDNA) and RNA molecules. A "nucleotide," the building block of a nucleic acid molecule, can be a deoxyribonucleotide, a ribonucleotide, a modified nucleotide or base, and / or its analog, or any substrate that can be incorporated into a polymer by a DNA or RNA polymerase or by a synthetic reaction. A polynucleotide may also include modified nucleotides, sugar- or base-modified analogs, such as methylated nucleotides and their analogs.

[0060] Some mutations in nucleotides do not result in mutations in the protein. Such nucleic acids include nucleic acid molecules containing functionally equivalent codons or codons encoding the same amino acid (for example, due to codon degeneracy, there are six codons for arginine or serine), or codons encoding biologically equivalent amino acids. Nucleic acid mutations can also result in changes in the protein itself. Mutations that result in changes in the amino acids of the protein can also result in proteins that exhibit approximately the same activity as the proteins of the present disclosure.

[0061] It will be clear to those skilled in the art that the peptides and nucleic acid molecules of the present disclosure are not limited to the amino acid or nucleotide sequences set forth in the sequence listing, so long as they retain the characteristics of the nucleic acid molecules or polynucleotides of the present disclosure, for example, their effectiveness as vaccines and / or immunostimulants. For example, biologically functional equivalents that may be contained in a coding region operably linked to an expression control sequence and / or a recombinant protein / peptide expressed therefrom may be polynucleotides having a nucleotide sequence mutation and / or proteins / peptides having an amino acid sequence mutation that exhibit biological activity equivalent to that of the aforementioned coding region and / or recombinant protein.

[0062] Considering the aforementioned biologically equivalent variants, nucleic acid molecules encoding peptides and / or proteins are understood to include sequences that exhibit substantial identity to the sequences set forth in the sequence listing. The term "substantial identity" refers to sequences that exhibit at least 61% homology, more preferably 70% homology, even more preferably 80% homology, and most preferably 90% homology when the sequences disclosed herein are aligned with any other sequence for maximum correspondence and analyzed using algorithms commonly used in the art. Alignment methods for sequence comparison are well known in the art. As used herein, the term "vector" refers to a construct that can be transferred to a host cell, preferably to express one or more genes or sequences of interest. Examples of vectors include viral vectors, DNA or RNA expression vectors, plasmids, cosmids, phage vectors, DNA or RNA expression vectors linked with cationic condensing agents (CCA), DNA or RNA expression vectors encapsulated in liposomes, and specific eukaryotic cells such as producer cells.

[0063] As used herein, "expression control / regulation sequence" or "expression control / regulation element" may refer to a nucleic acid sequence that regulates the transcription of a nucleic acid molecule and / or the translation of a nucleic acid in the form of a transcript. Furthermore, "transcription control / regulation sequence" or "transcription control / regulation element" refers to a nucleic acid sequence that regulates the transcription of a nucleic acid. Transcription control elements include promoters, such as constitutive promoters or inducible promoters, and enhancers.

[0064] The terms "translation control / regulation sequence" or "translation control / regulation element" can also be used for nucleic acid sequences that regulate the translation of a nucleic acid in the form of a transcript into a protein or peptide. These expression control sequences / elements, transcriptional and / or translational control sequences / elements are operatively linked to the sequence to be expressed, e.g., the nucleic acid sequence to be transcribed or translated.

[0065] As used herein, the term "operatively linked" refers to a functional connection between a nucleic acid expression control sequence (e.g., a promoter, a signal sequence, a ribosomal binding site, a transcription termination sequence, etc.) and another nucleic acid sequence, whereby the control sequence controls the transcription and / or translation of the other nucleic acid sequence.

[0066] nucleic acid molecule The present disclosure is based on a nucleic acid molecule-based expression platform or expression system, such as a nucleic acid molecule that has improved expression efficiency of a coding region operably linked to an expression regulatory sequence, and a vector containing the same. Figure 1 is a schematic diagram illustrating the structure of a nucleic acid molecule or polynucleotide that can efficiently express a gene of interest according to an exemplary embodiment of the present disclosure.

[0067] As shown in FIG. 1, the nucleic acid molecule may include a translation control element (TLCE) containing a nucleotide sequence having translation initiation activity, and a coding region (CR) operably linked to the translation control element (TLCE) and consisting of an open reading frame (ORF) of a gene of interest (GOI).

[0068] Optionally, the nucleic acid molecule may comprise at least one nucleotide sequence of a polyadenylation signal sequence or a polyadenosine sequence (PA) located downstream of a transcriptional regulatory element (TCCE) and / or a translational regulatory element (TLCE) operably linked to the coding region (CR).

[0069] The translational regulatory element (TLCE) may comprise a nucleotide sequence having translation initiation activity operably linked to a coding region (CR) inserted in the form of an open reading frame (ORF). For example, the translational regulatory element (TLCE) may comprise an upstream translational regulatory element (U-TLCE) located upstream of the coding region (CR) and / or a downstream translational regulatory element (D-TLCE) located downstream of the coding region (CR).

[0070] In other words, the upstream translational regulatory element (U-TLCE) can be located upstream of the coding region (CR), and the downstream translational 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 upstream translational regulatory element (U-TLCE) and the downstream translational regulatory element (D-TLCE), forming a target sequence (TS) consisting of the open reading frame of the gene of interest.

[0071] The upstream translational regulatory element (U-TLCE) can be all or part of a 5'-untranslated region (5'-UTR) that has cap-dependent translation initiation activity.

[0072] Most eukaryotic mRNAs have a 7-methyl-guanosine (cap, m7G) at their 5' ends, and the translation initiation complex recognizes the cap at the 5' end and advances to the AUG initiation codon to begin protein synthesis. In other words, the cap structure at the 5' end of mRNA not only initiates protein synthesis but also protects the mRNA from destruction by nucleases.

[0073] For example, in vitro transcription can be performed by linearizing pDNA (plasmid DNA) with a restriction enzyme, followed by the addition of m7G(5')-ppp(5')G (called a regular cap analog) to the mRNA produced using an appropriate RNA polymerase, creating capped mRNA. Alternatively, in vitro transcription can be performed without a cap analog, followed by the capping reaction using commercially available vaccinia virus-derived capping enzymes. An "anti-reverse" cap analog (ARCA) can be used to prevent the reverse reaction of the cap. With ARCA, only the 3'-O-methylated guanosine nucleotides can be attached to unmethylated guanosine nucleotides.

[0074] Exemplary modified 5'-Cap structures include Cap1 (methylation of the ribose in the nucleotide adjacent to mG), Cap2 (methylation of the ribose of the nucleotide located second downstream of mG), Cap3 (methylation of the ribose of the nucleotide located third downstream of mG), Cap4 (methylation of the ribose of the nucleotide located fourth downstream of mG), ARCA (anti-reverse cap analog, modified ARCA (e.g., phosphorothioate-modified ARCA), inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-diaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0075] For example, the upstream translational regulatory element (U-TLCE) and / or downstream translational regulatory element (D-TLCE) can each consist of a nucleotide sequence having cap-dependent translation initiation activity derived from an animal, for example, a mammal, specifically a primate, more specifically a human, or its transcript sequence. The upstream translational regulatory element (U-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 sequence that induces translation of the coding region (CR).

[0076] When the upstream translational regulatory element (U-TLCE) is all or part of a nucleotide sequence with cap-dependent translation initiation activity, it may also contain a downstream translational regulatory element (D-TLCE) located downstream of the coding region (CR). A nucleic acid molecule containing an upstream translational regulatory element (U-TLCE) and a downstream translational regulatory element (D-TLCE) can further improve the expression efficiency of the open reading frame (ORF) that constitutes the coding region (CR). In other words, the upstream translational regulatory element (U-TLCE) and the downstream translational regulatory element (D-TLCE) improve the translation efficiency of the open reading frame of the gene of interest (GOI) that forms the coding region (CR) or its transcript, and play an important role in ensuring the stable maintenance of the transcript, mRNA, in cells without destruction.

[0077] In exemplary embodiments, the upstream translational regulatory element (U-TLCE) may include, but is not limited to, a translational regulatory element derived from any one selected from the group consisting of human troponin T1, slow backbone type (human TNNT1), human albumin (human ALB), human ferritin light chain (human FTL), human CC motif chemokine ligand 19 (human CCL19), human vascular-associated migratory cell protein (human AAMP), human ribosomal protein S27 (human RPS27), and human defensin alpha 5 (human DEFA5).

[0078] For example, an upstream translational regulatory element (U-TLCE) derived from human TNNT1 may comprise the translational regulatory element of SEQ ID NO: 1 or its transcript sequence. An upstream translational regulatory element (U-TLCE) derived from human ALB may comprise the translational regulatory element of SEQ ID NO: 2 or its transcript sequence. An upstream translational regulatory element (U-TLCE) derived from human FTL may comprise the translational regulatory element of SEQ ID NO: 3 or its transcript sequence. An upstream translational regulatory element (U-TLCE) derived from human CCL19 may comprise the translational regulatory element of SEQ ID NO: 4 or its transcript sequence. An upstream translational regulatory element (U-TLCE) derived from human AAMP may comprise the translational regulatory element of SEQ ID NO: 5 or its transcript sequence. An upstream translational regulatory element (U-TLCE) derived from human RPS27 may comprise the translational regulatory element of SEQ ID NO: 6 or its transcript sequence. An upstream translational regulatory element (U-TLCE) derived from human DEFA5 may comprise the translational regulatory element of SEQ ID NO: 7 or its transcript sequence. However, the upstream translational regulatory element (U-TLCE) is not limited to a particular nucleotide sequence or its transcript sequence.

[0079] The downstream translational regulatory element (D-TLCE) may consist of a first downstream translational regulatory element (D-TLCE1) and a second downstream translational regulatory element (D-TLCE2) inserted sequentially downstream of the coding region (CR). The first downstream translational regulatory element (D-TLCE1) and the second downstream translational regulatory element (D-TLCE2) may be the same or different from each other.

[0080] As an example, the first downstream translational regulatory element (D-TLCE1) and the second downstream translational regulatory element (D-TLCE2) may each independently comprise a downstream translational regulatory element derived from any one selected from the group consisting of human TNNT1, human ALB, human FTL, human CCL19, human AAMP, human RPS27 and human DEFA5.

[0081] For example, the first and second downstream translational regulatory elements (D-TLCE1, D-TLCE2) derived from human TNNT1 may each comprise the translational regulatory element of SEQ ID NO: 7 or a transcript sequence thereof. The first and second downstream translational regulatory elements (D-TLCE1, D-TLCE2) derived from human ALB may each comprise the translational regulatory element of SEQ ID NO: 9 or a transcript sequence thereof. The first and second downstream translational regulatory elements (D-TLCE1, D-TLCE2) derived from human FTL may each comprise the translational regulatory element of SEQ ID NO: 10 or a transcript sequence thereof. The first and second downstream translational regulatory elements (D-TLCE1, D-TLCE2) derived from human CCL19 may each comprise the translational regulatory element of SEQ ID NO: 11 or a transcript sequence thereof. The first and second downstream translational regulatory elements (D-TLCE1, D-TLCE2) derived from human AAMP may each comprise the translational regulatory element of SEQ ID NO: 12 or a transcript sequence thereof. The first and second downstream translational regulatory elements (D-TLCE1, D-TLCE2) derived from human RPS27 may comprise the translational regulatory element of SEQ ID NO: 13 or its transcript sequence. The first and second downstream translational regulatory elements (D-TLCE1, D-TLCE2) derived from human DEFA5 may comprise the translational regulatory element of SEQ ID NO: 14 or its transcript sequence. However, the first and second downstream translational regulatory elements (D-TLCE1, D-TLCE2) are not limited to a particular nucleotide sequence or its transcript sequence.

[0082] In an exemplary embodiment, the first downstream translational regulatory element (D-TLCE1) may comprise a downstream translational regulatory element derived from human TNNT1 or its transcript sequence, and the second downstream translational regulatory element (D-TLCE2) may comprise a downstream translational regulatory element derived from any one selected from the group consisting of human ALB, human FTL, human CCL19, human AAMP, human RPS27, and human DEFA5, or its transcript sequence. For example, when the first downstream translational regulatory element (D-TLCE1) comprises a downstream translational regulatory element derived from human TNNT1 or its transcript sequence, the second downstream translational regulatory element (D-TLCE2) may be derived from any one selected from the group consisting of human FTL, human CCL19, and human DEFA5.

[0083] In alternative embodiments, when the first downstream translational regulatory element (D-TLCE1) comprises a downstream translational regulatory element derived from human TNNT1 or a transcript sequence thereof, the second downstream translational regulatory element (D-TLCE2) may comprise a translational regulatory element derived from human FTL or human CCL19 or a transcript sequence thereof. In other alternative embodiments, when the first downstream translational regulatory element (D-TLCE1) comprises a downstream translational regulatory element derived from human TNNT1 or a transcript sequence thereof, the second downstream translational regulatory element (D-TLCE2) may comprise a lower translational regulatory element derived from human CCL19 or human RPS27 or a transcript sequence thereof.

[0084] In another exemplary embodiment, the first downstream translational regulatory element (D-TLCE1) may comprise a downstream translational regulatory element derived from human ALB or its transcript sequence, and the second downstream translational regulatory element (D-TLCE2) may comprise a downstream translational regulatory element derived from human DEFA5 or its transcript sequence.

[0085] In yet another exemplary embodiment, the first downstream translation regulatory element (D-TLCE1) may comprise a downstream translation regulatory element derived from human FTL or its transcript sequence, and the second downstream translation regulatory element (D-TLCE2) may comprise a downstream translation regulatory element derived from any one selected from the group consisting of human TNNT1, human FTL, human RPS27, and human DEFA5 or its transcript sequence. For example, when the first downstream translation regulatory element (D-TLCE1) comprises a downstream translation regulatory element derived from human FTL or its transcript sequence, the second downstream translation regulatory element (D-TLCE2) may comprise a downstream translation regulatory element derived from any one selected from the group consisting of human TNNT1, human FTL, the human RPS27, and the human DEFA5 or its transcript sequence.

[0086] In alternative embodiments, when the first downstream translational regulatory element (D-TLCE1) comprises a downstream translational regulatory element derived from human FTL or its transcript sequence, the second downstream translational regulatory element (D-TLCE2) may comprise a downstream translational regulatory element derived from any one selected from the group consisting of human TNNT1, human FTL, and human RPS27, or its transcript sequence. In other alternative embodiments, when the first downstream translational regulatory element (D-TLCE1) comprises a downstream translational regulatory element derived from human FTL or its transcript sequence, the second downstream translational regulatory element (D-TLCE2) may comprise a downstream translational regulatory element derived from human FTL or its transcript sequence.

[0087] In yet another exemplary embodiment, the first downstream translational regulatory element (D-TLCE1) may comprise a downstream translational regulatory element derived from human CCL19 or its transcript sequence, and the second downstream translational regulatory element (D-TLCE2) may comprise a downstream translational regulatory element derived from any one selected from the group consisting of human TNNT1, human FTL, human CCL19, human RPS27, and human DEFA5, or its transcript sequence. For example, when the first downstream translational regulatory element (D-TLCE1) comprises a downstream translational regulatory element derived from human CCL19 or its transcript sequence, the second downstream translational regulatory element (D-TLCE2) may comprise a translational regulatory element derived from any one selected from the group consisting of human FTL, human CCL19, human RPS27, and the human DEFA5.

[0088] In alternative embodiments, when the first downstream translational regulatory element (D-TLCE1) comprises a downstream translational regulatory element or a transcript sequence thereof derived from human CCL19, the second downstream translational regulatory element (D-TLCE2) may comprise a translational regulatory element or a transcript sequence thereof derived from human FTL or human RPS27. In other alternative embodiments, when the first downstream translational regulatory element (D-TLCE1) comprises a downstream translational regulatory element or a transcript sequence thereof derived from human CCL19, the second downstream translational regulatory element (D-TLCE2) may comprise a downstream translational regulatory element or a transcript sequence thereof derived from human CCL19. In yet other alternative embodiments, when the first downstream translational regulatory element (D-TLCE1) comprises a downstream translational regulatory element or a transcript sequence thereof derived from human CCL19, the second downstream translational regulatory element (D-TLCE2) may comprise a downstream translational regulatory element or a transcript sequence thereof derived from human CCL19 or human RPS27.

[0089] In yet another alternative embodiment, when the first downstream translational regulatory element (D-TLCE1) comprises a downstream translational regulatory element derived from human CCL19 or its transcript sequence, the second downstream translational regulatory element (D-TLCE2) may comprise a lower translational regulatory element derived from human FTL or human CCL19 or its transcript sequence.

[0090] In yet another exemplary embodiment, the first downstream translational regulatory element (D-TLCE1) may comprise a downstream translational regulatory element derived from human AAMP or its transcript sequence, and the second downstream translational regulatory element (D-TLCE2) may comprise a downstream translational regulatory element derived from any one selected from the group consisting of human ALB, human FTL, human RPS27, and human DEFA5, or its transcript sequence.

[0091] In yet another exemplary embodiment, the first downstream translational regulatory element (D-TLCE1) may comprise a downstream translational regulatory element derived from human RPS27 or a transcript sequence thereof, and the second downstream translational regulatory element (D-TLCE2) may comprise a downstream translational regulatory element derived from any one selected from the group consisting of human TNNT1, human ALB, human FTL, human CCL19, human AAMP, human RPS27, and human DEFA5 or a transcript sequence thereof. For example, when the first downstream translational regulatory element (D-TLCE1) comprises a downstream translational regulatory element derived from human RSP27 or a transcript sequence thereof, the second downstream translational regulatory element (D-TLCE2) may comprise a translational regulatory element derived from any one selected from the group consisting of human TNNT1, human FTL1, human CCL19, human AAMP, human RPS27, and human DEFA5 or a transcript sequence thereof.

[0092] In an alternative embodiment, when the first downstream translational regulatory element (D-TLCE1) comprises a downstream translational regulatory element derived from human RSP27 or a transcript sequence thereof, the second downstream translational regulatory element (D-TLCE2) may comprise a translational regulatory element derived from any one selected from the group consisting of human FTL, human CCL19, human RPS27, and human DEFA5 or a transcript sequence thereof. In another alternative embodiment, when the first downstream translational regulatory element (D-TLCE1) comprises a downstream translational regulatory element derived from human RSP27 or a transcript sequence thereof, the second downstream translational regulatory element (D-TLCE2) may comprise a translational regulatory element derived from any one selected from the group consisting of human FTL, human RPS27, and human DEFA5 or a transcript sequence thereof.

[0093] In another alternative embodiment, when the first downstream translational regulatory element (D-TLCE1) comprises a downstream translational regulatory element derived from human RSP27 or its transcript sequence, the second downstream translational regulatory element (D-TLCE2) may comprise a lower translational regulatory element derived from any one selected from the group consisting of human TNNT1, human FTL, human RPS27, and human DEFA5, or its transcript sequence. In yet another alternative embodiment, when the first downstream translational regulatory element (D-TLCE1) comprises a downstream translational regulatory element derived from human RSP27 or its transcript sequence, the second downstream translational regulatory element (D-TLCE2) may comprise a lower translational regulatory element derived from human RPS27 or human DEFA5, or its transcript sequence. In yet another alternative embodiment, when the first downstream translational regulatory element (D-TLCE1) comprises a downstream translational regulatory element derived from human RSP27 or its transcript sequence, the second downstream translational regulatory element (D-TLCE2) may comprise a lower translational regulatory element derived from human FTL, or its transcript sequence.

[0094] In yet another exemplary embodiment, the first downstream translational regulatory element (D-TLCE1) may comprise a downstream translational regulatory element derived from human DEFA5 or its transcript sequence, and the second downstream translational regulatory element (D-TLCE2) may comprise a downstream translational regulatory element derived from any one selected from the group consisting of human TNNT1, human ALB, human FTL, human CCL19, human AAMP, and human RPS27 or its transcript sequence. For example, when the first downstream translational regulatory element (D-TLCE1) comprises a downstream translational regulatory element derived from human DEFA5 or its transcript sequence, the second downstream translational regulatory element (D-TLCE2) may comprise a downstream translational regulatory element derived from any one selected from the group consisting of human TNNT1, human ALB, the human FTL, human AAMP, and human RPS27 or its transcript sequence.

[0095] In alternative embodiments, when the first downstream translational regulatory element (D-TLCE1) comprises a downstream translational regulatory element derived from human DEFA5 or a transcript sequence thereof, the second downstream translational regulatory element (D-TLCE2) may comprise a translational regulatory element derived from any one selected from the group consisting of human ALB, human FTL, human AAMP, and human RPS27, or a transcript sequence thereof. In other alternative embodiments, when the first downstream translational regulatory element (D-TLCE1) comprises a downstream translational regulatory element derived from human DEFA5 or a transcript sequence thereof, the second downstream translational regulatory element (D-TLCE2) may comprise a translational regulatory element derived from any one selected from the group consisting of human ALB, human FTL, and human RPS27, or a transcript sequence thereof.

[0096] In yet another alternative embodiment, when the first downstream translation regulatory element (D-TLCE1) comprises a downstream translation regulatory element derived from human DEFA5 or its transcript sequence, the second downstream translation regulatory element (D-TLCE2) may comprise a downstream translation regulatory element derived from any one selected from the group consisting of human FTL, human CCL9, human AAMP, and human RPS27, or its transcript sequence. In yet another alternative embodiment, when the first downstream translation regulatory element (D-TLCE1) comprises a downstream translation regulatory element derived from human DEFA5 or its transcript sequence, the second downstream translation regulatory element (D-TLCE2) may comprise a downstream translation regulatory element derived from human FTL or human RPS27, or its transcript sequence.

[0097] The coding region (CR) is not particularly limited as long as it contains the open reading frame (ORF) of the intended gene of interest (GOI). In an exemplary embodiment, the coding region (CR) can consist of an open reading frame (ORF) that is a nucleotide sequence or a transcript sequence encoding at least one of a reporter protein or a fragment thereof, a marker or selection protein or a fragment thereof, an antigen or a fragment thereof, and a protein or a fragment thereof for treating a disease.

[0098] By way of example, the coding region (CR) may consist of an open reading frame (ORF) encoding a reporter protein / reporter peptide or a fragment thereof, such as a luciferase protein (e.g., renilla luciferase), a green fluorescent protein (GFP), an enhanced green fluorescent protein (EGFP), a glucuronidase such as beta-glucuronidase, and / or a galactosidase such as beta-galactosidase.

[0099] In one exemplary embodiment, the coding region (CR) can consist of an open reading frame encoding a marker or selection protein / peptide or fragment thereof, such as a globin protein, such as alpha-globin and / or beta-globin, galactokinase and / or hypoxanthine guanine phosphoribosyltransferase, etc. Alternatively, open reading frames (ORFs) encoding other reporter proteins / peptides and / or marker or selection proteins / peptides can be inserted into the coding region (CR).

[0100] In other exemplary embodiments, the antigen gene inserted into the coding region (CR) can consist of an open reading frame (ORF) encoding a protein / peptide, including a pathogenic antigen or fragment thereof, a tumor antigen, or a variant or derivative thereof.

[0101] Pathogenic antigens are derived from pathogenic organisms, particularly bacterial, viral, or protozoan (multicellular) pathogenic organisms, that provoke an immunological response in an individual, particularly a mammalian individual, more particularly a human. More specifically, a pathogenic antigen can be a surface antigen, e.g., a protein (or a fragment of a protein, e.g., an external portion of a surface antigen), located on the surface of a viral, bacterial, or protozoan organism.

[0102] By way of example, a pathogenic antigen can be a peptide or protein antigen derived from a pathogen associated with an infectious disease. More specifically, the pathogenic antigens include Acinetobacter baumannii, Anaplasma genus, Anaplasma phagocytophilum, Ancylostoma braziliense, Ancylostoma duodenale, Arcanobacterium haemolyticum, Ascaris lumbricoides, Aspergillus genus, Astroviridae, Babesia genus, Bacillus anthracis, Bacillus cereus, Bartonella henselae, and henselae, BK virus, Blastocystis hominis, Blastomyces dermatitidis, Bordetella pertussis, Borrelia burgdorferi, Borrelia genus, Borrelia spp, Brucella genus, Brugia malayi, Bunyaviridae family, Burkholderia cepacia and other Burkholderia species, Burkholderia mallei, Burkholderia pseudomallei, Caliciviridae family, Campylobacter genus, Candida albicans, Candida spp, Chlamydia trachomatistrachomatis, Chlamydophila pneumoniae, Chlamydophila psittaci, CJD prion, Clonorchis sinensis, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium difficile, Clostridium spp, Clostridium tetani, Coccidioides spp, coronaviruses, Corynebacterium diphtheria, Coxiella burnetii burnetii, Crimean-Congo hemorrhagic fever virus, Cryptococcus neoformans, Cryptosporidium genus, Cytomegalovirus (CMV), Dengue viruses (DEN-1, DEN-2, DEN-3 and DEN-4), Dientamoeba fragilis, Ebola virus (EBOV), Echinococcus genus, Ehrlichia chaffeensis, Ehrlichia ewingii, Ehrlichia genus, Entamoeba histolytica histolytica, Enterococcus genus, Enterovirus genus, Enteroviruses, mainly Coxsackie A virusvirus and Enterovirus 71 (EV71), Epidermophyton spp., Epstein-Barr Virus (EBV), Escherichia coli O157:H7, O111, and O104:H4, Fasciola hepatica and Fasciola gigantica, FFI prions, Filarioidea superfamily, Flaviviruses, Francisella tularensis, Fusobacterium genus, Geotrichum candidum, Giardia intestinalis, Gnathostoma spp., GSS prions, Guanarito virus virus, Haemophilus ducreyi, Haemophilus influenza, Helicobacter pylori, Henipavirus (Hendra virus Nipah virus), Hepatitis A virus, Hepatitis B virus (HBV), Hepatitis C virus (HCV), Hepatitis D virus, Hepatitis E virus, Herpes simplex virus 1 and 2 (HSV-1 and HSV-2), Histoplasma capsulatum, HIV (Human immunodeficiency virus), Hortaea werneckii, Human bocavirus (HBoV), Human herpesvirus 6 (HHV-6) and Human herpesvirus 7 (HHV-7), Human metapneumovirus (Human metapneumovirus (hMPV), human papillomavirus (HPV), human parainfluenza virus (HPV)HPIV, Japanese encephalitis virus, JC virus, Junin virus, Kingella kingae, Klebsiella granulomatis, Kuru prion, Lassa virus, Legionella pneumophila, Leishmania genus, Leptospira genus, Listeria monocytogenes, Lymphocytic choriomeningitis virus (LCMV), Machupo virus, Malassezia spp., Marburg virus, Measles virus virus, Metagonimus yokogawai, Microsporidia phylum, Molluscum contagiosum virus (MCV), Mumps virus, Mycobacterium leprae and Mycobacterium lepromatosis, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumoniae, Naegleria fowleri, Necator americanus, Neisseria gonorrhoeae, Neisseria meningitidis meningitides, Nocardia asteroids, Nocardia spp, Onchocerca volvulusvolvulus, Orientia tsutsugamushi, Orthomyxoviridae family (influenza), Paracoccidioides brasiliensis, Paragonimus spp., Paragonimus westermani, Parvovirus B19, Pasteurella genus, Plasmodium genus, Pneumocystis jirovecii, Poliovirus, Rabies virus, Respiratory syncytial virus (RSV), Rhinovirus, Rickettsia akari akari, Rickettsia genus, Rickettsia prowazekii, Rickettsia rickettsia, Rickettsia typhi, Rift Valley fever virus, Rotavirus, Rubella virus, Sabia virus, Salmonella genus, Sarcoptes scabiei, SARS coronavirus, Schistosoma genus, Severe fever with thrombocytopenia syndrome virus (SFTSV), Shigella genus, Sin Nombre virus virus, Hantavirus, Sporothrix schenckii, Staphylococcusgenus), Staphylococcus genus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Strongyloides stercoralis, Taenia genus, Taenia solium, Tickborne encephalitis virus (TBE) V), Toxocara canis or Toxocara cati, Toxoplasma gondii, Treponema pallidum, Trichinella spiralis, Trichomonas vaginalis, Trichophyton spp, Trichuris trichiura, Trypanosoma brucei, Trypanosoma cruzi, Ureaplasma urealyticum, Varicella zoster virus (VZV), Variola major or Variola minor The virus may be derived from, but is not limited to, Vibrio minor, vCJD prion, Venezuelan equine encephalitis virus, Vibrio cholera, West Nile virus, Western equine encephalitis virus, Wuchereria bancrofti, Yellow fever virus, Yersinia enterocolitica, Yersinia pestis, and Yersinia pseudotuberculosis.

[0103] In an alternative aspect, the coding region (CR) can comprise an open reading frame (ORF) encoding a protein or peptide that is a tumor antigen, or a fragment, variant, or derivative thereof. The tumor antigen can be a melanocyte-specific antigen, a testicular cancer antigen, or a tumor-specific antigen, such as a CT-X antigen, a non-CT-X antigen, a binding partner for a CT-X antigen, or a binding partner for a non-CT-X antigen or a tumor-specific antigen, or a variant or derivative of a tumor-specific antigen, tumor antigen.

[0104] Open reading frames (ORFs) encoding such tumor antigens include 5T4, 707-AP, 9D7, AFP, AlbZIP HPG1, alpha-5-beta-1-integrin, alpha-5-beta-6-integrin, alpha-actinin-4 / m, alpha-methylacyl-coenzyme A racemase, ART-4, ARTC1 / m, B7H4, BAGE-1, BCL-2, bcr / abl, beta-catenin / m, BING-4, BRCA1 / m, BRCA2 / m, CA 15-3 / CA 27-29, CA19-9, CA72-4, CA125, calreticulin, CAMEL, CASP-8 / m, and cathepsin B. B), cathepsin L, CD19, CD20, CD22, CD25, CDE30, CD33, CD4, CD52, CD55, CD56, CD80, CDC27 / m, CDK4 / m, CDKN2A / m, CEA, CLCA2, CML28, CML66, COA-1 / m, coactosin-like protein, collageXXIII, COX-2, CT-9 / BRD6, Cten, cyclin B1, cyclin D1, cyp-B, CYPB1, DAM-10, DAM-6, DEKCAN, EFTUD2 / m, EGFR, ELF2 / m, EMMPRIN, EpCam, EphA2, EphA3, ErbB3, ETV6-AML1, EZH2, FGF-5, FN, Frau-1, G250, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE7b, GAGE-8, GDEP, GnT-V, gp100, GPC3, GPNMB / m, HAGE, HAST-2, hepsin, Her2 / neu, HERV-K-MEL, HLA-A*0201-R17I, HLA-A1 1 / m, HLA-A2 / m, HNE, Homeobox NKX3.1, HOM-TES-14 / SCP-1, HOM-TES-85, HPV-E6, HPV-E7, HSP70-2M, HST-2, hTERT, iCE, IGF-1 R, IL-13Ra2, IL-2R, IL-5, immature laminin receptor, kallikrein-2, krein-4, Ki67, KIAA0205, KIAA0205 / m, KK-LC-1, K-Ras / m, LAGE-A1, LDLR-FUT, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-A10, MAGE-A12, MAGE-B1, MAGE-B2, MAGE- B3, MAGE-B4, MAGE-B5, MAGE-B6, MAGE-B10, MAGE-B16, MAGE-B17, MAGE-C1, MAGE-C2, MAGE-C3, MAGE-D1, MAGE-D2, MAGE-D4, MAGE-E1, MAGE-E2, MAGE-F1, MAGE-H1, MAGEL2, mammaglobin A, MART-1 / melan-A, MART-2, MART-2 / m, matrix protein 22, MC1R, M-CSF, ME1 / m, mesothelin, MG50 / PXDN, MMP11, MN / CAIX-antigen, MRP-3, MUC-1, MUC-2, MUM-1 / m, MUM-2 / m, MUM-3 / m, myosin class I / m, NA88-A, N-acetylglucosaminyltransferase-V, neo-PAP, neo-PAP / m, NFYC / m, NGEP, NMP22, NPM / ALK, N-Ras / m, NSE, NY-ESO-1, NY-ESO-B, OA1, OFA-iLRP, OGT, OGT / m, OS-9, OS-9 / m, Osteocalcin, osteopontin, p15, p190 minor, bcr-abl, p53, p53 / m, PAGE-4, PAI-1, PAI-2, PAP, PART-1, PATE, PDEF, Pim-1-kinase, Pin-1, Pml / PAR alpha, POTE, PRAME, PRDX5 / m, prostein, proteinase-3, PSA, PSCA, PSGR, PSM, PSMA, PTPRK / m, RAGE-1, RBAF600 / m, RHAMM / CD168, RU1, RU2, S-100, SAGE, SART-1, SART-2, SART-3, SCC, SIRT2 / m, Sp17, SSX-1, SSX-2 / HOM-MEL-40, SSX- 4, STAMP-1, STEAP-1, survivin, survivin-2B, SYT-SSX-1, SYT-SSX-2, TA-90, TAG-72, TARP, TEL-AML1, TGF beta, TGF beta RII, TG It may consist of, but is not limited to, a nucleotide sequence encoding M-4, TPI / m, TRAG-3, TRG, TRP-1, TRP-2 / 6b, TRP / INT2, TRP-p8, tyrosinase, UPA, VEGFR1, VEGFR-2 / FLK-1, WT1, and a lymphocyte blood cell immunoglobulin genotype or a lymphocyte blood cell T-cell receptor genotype, or a fragment, variant or derivative thereof, or a transcript sequence thereof.

[0105] In yet other exemplary embodiments, the coding region (CR) can consist of an open reading frame (ORF) that encodes a protein / peptide that is a therapeutic protein / peptide or a fragment, variant, or derivative thereof.

[0106] Therapeutic proteins can play an important role in the generation of therapeutic agents that can modify and treat genetic defects, destroy cancer cells or pathogen-infected cells, treat immune system disorders, and treat metabolic or endocrine disorders. By way of example, therapeutic proteins can be used for a variety of purposes, including the treatment of various disorders, whether genetic or acquired, such as infectious diseases, neoplasms (e.g., cancer or tumor disorders), disorders of the blood and blood-forming organs, endocrine, nutritional, and metabolic disorders, nervous system disorders, circulatory system disorders, respiratory system disorders, digestive system disorders, skin and subcutaneous tissue disorders, musculoskeletal and connective tissue disorders, and genitourinary system disorders.

[0107] For example, proteins that can be used to treat metabolic or endocrine disorders (specific diseases for which protein therapeutics are used) include acid sphingomyelinase (Niemann-Pick disease), adipotide (obesity), agalsidase-beta (human galactosidase A) (Fabry disease; prevents lipid accumulation that leads to renal and cardiovascular complications), alglucosidase (Pompe disease; glycogen storage disease type II), alpha-galactosidase A (alpha-GAL A, agalsidase alpha) (Fabry disease), alpha-glucosidase (glycogen storage disease (GSD), Pompe disease (Morbus Pompe)), alpha-L-iduronidase (mucopolysaccharidosis (MPS), Hurler syndrome), and alpha-L-iduronidase (mucopolysaccharidosis (MPS), Hurler syndrome). syndrome, Scheie syndrome), alpha-N-acetylglucosaminidase (Sanfilippo syndrome), amphiregulin (cancer, metabolic disorders), angiopoietin (Ang1, Ang2, Ang3, Ang4, ANGPTL2, ANGPTL3, ANGPTL4, ANGPTL5, ANGPTL6, ANGPTL7) (angiogenesis, vascular stability), betacellulin (metabolic disorders), beta-glucuronidase (Sly syndrome), bone morphogenetic proteins BMPs (BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP10, BMP15) (regenerative effects, bone-related conditions, chronic kidney disease (CKD)), CLN6 protein (CLN6 disease - atypical late infantile, late onset variants)Variant), Early Juvenile, Neuronal Ceroid Lipofuscinosis (NCL), Epidermal Growth Factor (EGF) (Wound healing, regulation of cell growth, proliferation and differentiation), Epigen (Metabolic Disorders), Epiregulin (Metabolic Disorders), Fibroblast Growth Factor FGF, FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, FGF-16, FGF-17, FGF-18, FGF-19, FGF-20, FGF-21, FGF-22, FGF-23 (wound healing, angiogenesis, endocrine disorders, tissue regeneration), Galsulphase (Mucopolysaccharidosis type VI), Ghrelin (irritable bowel syndrome (IBS), obesity, Prader-Willi syndrome, type 2 diabetes mellitus) mellitus, Glucocerebrosidase (Gaucher's disease), GM-CSF (regenerative effects, white blood cell production, cancer), Heparin-binding EGF-like growth factor (HB-EGF) (wound healing, cardiac hypertrophy and cardiac development and function), Hepatocyte growth factor factor, HGF) (regenerative effect, wound healing), hepcidin (iron metabolism disorder, beta-thalassemia), human albumin (decreased production of albumin (hypoproteinemia), decreased production of increased albumin (renal syndrome, hypovolemia, hyperbilirubinemia), idursulfase (iduronate-2-sulfatase) (mucopolysaccharidosis type II (Hunter syndrome, Hunter syndrome)syndrome), integrins αVβ3, αVβ5, and α5β1 (Bind matrix macromolecules and proteinases, angiogenesis), iduronate sulfatase (Hunter syndrome), laronidase (Hurler and Hurler-Scheie forms of mucopolysaccharidosis type I), N-acetylgalactosamine-4-sulfatase (rhASB; galsulfase, arylsulfatase A (ARSA), arylsulfatase B (ARSB)) (arylsulfatase B deficiency, Maroteaux-Lamy syndrome, mucopolysaccharidosis type VI), N-acetylglucosamine-6-sulfatase (Sanfilippo syndrome), syndrome), nerve growth factor (NGF, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 / 5 (NT-4 / 5) (regenerative effects, cardiovascular disease, coronary angioplasty, coronary atherosclerosis, obesity, type 2 diabetes mellitus, metabolic syndrome, acute coronary syndrome, dementia, depression, schizophrenia, autism, Rett syndrome, anorexia nervosa, bulimia nervosa) nervosa), wound healing, skin ulcers, corneal ulcers, Alzheimer's disease), Neuregulin (NRG1, NRG2, NRG3, NRG4) (metabolic diseases, schizophrenia), Neuropilin (NRP-1, NRP-2) (angiogenesis, axon guidance) guidance), cell survival, migration), obestatin (irritable bowel syndrome), obestatin (irritable bowel syndrome),syndrome, IBS, obesity, Prader-Willi syndrome, type 2 diabetes mellitus), Platelet Derived Growth Factor (PDGF (PDFF-A, PDGF-B, PDGF-C, PDGF-D) (regenerative effects, wound healing, angiogenic diseases, arteriosclerosis, fibrosis, cancer), TGF beta receptors (endoglin, TGF-beta 1 receptor, TGF-beta 2 receptor, TGF-beta 3 receptor) (renal fibrosis, kidney disease, diabetes, end-stage renal disease (ESRD), angiogenesis), thrombopoietin (THPO) (Megakaryocyte growth and development factor (MGDF)) (platelet disorders, platelet donation, platelet count recovery after myelosuppressive chemotherapy, transforming growth factor TGF (TGF-alpha, TGF-beta (TGF-beta1, TGF-beta2, and TGF-beta3)) (regenerative effects, wound healing, immunity, cancer, heart disease, diabetes, Marfan syndrome, Loeys-Dietz syndrome), VEGF (VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and PIGF) (regenerative effects, angiogenesis, wound healing, cancer, permeability), nesiritide (acute decompensated congestive heart failure), trypsin (decubitus ulcer, varicose ulcer, eschar debridement, dehiscence wound) wound, sunburn, meconium ileus), adrenocorticotrophic hormone (ACTH) (Addison's disease, small cell carcinoma, adrenoleukodystrophy, congenital adrenal hyperplasia)hyperplasia, Cushing's syndrome, Nelson's syndrome, infantile spasms), atrial-natriuretic peptide (ANP) (endocrine disorders), cholecystokinin (various), gastrin (hypogastrinemia), leptin (diabetes, hypertriglyceridemia, diabetes), oxytocin (stimulation of breastfeeding, non-progression of labor), somatostatin (symptomatic treatment of carcinoid syndrome, acute variceal bleeding) bleeding, and acromegaly, polycystic disease of the liver and kidney, acromegaly and symptoms due to neuroendocrine tumors), vasopressin (antidiuretic hormone) (diabetes insipidus), calcitonin (postmenopausal osteoporosis, hypercalcemia, Paget's disease, bone metastases, phantom limb pain, spinal stenosis), exenatide (type 2 diabetes resistant to treatment with metformin and sulfonylureas), growth hormone (GH), somatotropin (growth failure due to GH deficiency or chronic renal insufficiency), Prader-Willi syndrome, Turner syndrome, AIDS wasting wasting or antiviral therapy with cachexia), insulin (diabetes mellitus, diabetic ketoacidosis)ketoacidosis, hyperkalaemia), insulin-like growth factor 1, IGF-1 (childhood growth failure due to GH gene deficiency or severe primary IGF1 deficiency, neurodegenerative disease, cardiovascular disease, heart failure), mecasermin rinfabate, IGF-1 analog (GH gene deficiency or Childhood growth failure due to severe congenital IGF-1 deficiency, neurodegenerative diseases, cardiovascular disease, and heart failure), Mecasermin, IGF-1 analogs (childhood growth failure due to GH gene deficiency or severe congenital IGF-1 deficiency, neurodegenerative diseases, cardiovascular disease, and heart failure), Pegvisomant (acromegaly), Pramlintide (diabetes mellitus, in combination with insulin), Teriparatide (human parathyroid hormone residues 1-34) (severe osteoporosis), Becaplermin (debridement adjunct for diabetic ulcers), Dibotermin-alpha (bone morphogenetic protein 2) (spinal fusion) These may include, but are not limited to, steroids (for surgery, bone wound healing), histrelin acetate (gonadotropin releasing hormone (GnRH)) (precocious puberty), octreotide (for symptomatic relief of acromegaly, VIP-secreting adenoma and metastatic carcinoid tumors), and palifermin (keratinocyte growth factor (KGF)) (for severe oral mucositis, wound healing in patients undergoing chemotherapy).

[0108] In addition, proteins related to blood disorders, circulatory system disorders, respiratory system disorders, cancer or tumor disorders, infectious diseases, or immunodeficiency disorders (specific diseases for which protein therapeutics are used) include Alteplase (tissue plasminogen activator; tPA) (pulmonary embolism, myocardial infarction, acute ischemic stroke, occlusion of central venous access devices), Anistreplase (thrombolysis), Antithrombin III (AT-III) (genetic AT-III deficiency, thromboembolism), Bivalirudin (reduction of blood clotting risk in coronary angioplasty and heparin-induced thrombocytopenia), Darbepoetin-alpha (chronic renal insufficiency and chronic renal failure), and Treatment of anemia in patients with dialysis failure (+ / - dialysis), Drotrecogin-alpha (activated protein C) (severe sepsis with high mortality), Erythropoietin, Epoetin-alpha, erythropoietin (anemia of chronic disease, myelodysplasia, anemia due to renal failure or chemotherapy, preoperative preparation), Factor IX (hemophilia B), Factor VIIa (bleeding in patients with hemophilia A or B and inhibitors of factor VIII or factor IX), Factor VIII (hemophilia A), Lepirudin (heparin-induced hypothrombocytosis), Protein C concentrate (venous thrombosis, purpura fulminans)fulminans), Reteplase (deletion mutation of tPA) (regulation of acute myocardial infarction, enhancement of left ventricular function), Streptokinase (acute evolving transmural myocardial infarction, pulmonary embolism, deep vein thrombosis, arterial thrombosis or embolism, occlusion of arteriovenous cannula), Tenecteplase (acute myocardial infarction), Urokinase (pulmonary embolism), Angiostatin (cancer), Anti-CD22 immunotoxin (relapsed CD33+ acute myeloid leukemia), Denileukin diftitox (cutaneous T-cell lymphoma) lymphoma, CTCL), Immunocyanin (bladder and prostate cancer), MPS (Metallopanstimulin) (cancer), Aflibercept (non-small cell lung cancer (NSCLC), metastatic colorectal cancer (mCRC), hormone-refractory metastatic prostate cancer, wet macular degeneration), Endostatin (cancer, rheumatoid arthritis, as well as inflammatory diseases such as Crohn's disease, diabetic retinopathy, psoriasis, and endometriosis), Collagenase (debridement of chronic skin ulcers and severe burns, Dupuytren's contracture, Peyronie's disease), Human deoxyribonuclease I, Dornase (Cystic fibrosis) fibrosis; reduction of respiratory infections in selected patients with an FVC greater than 40% predicted), hyaluronidase (used as an adjuvant to improve absorption and dispersion of injected drugs, especially in ophthalmic surgery and certain contrast agents), papain (pressure ulcers, varicose and diabetic ulcers, burns, postoperative wounds, pilonidal cyst wounds)Debridement of necrotic tissue or slough in acute and chronic lesions such as carbuncles, subcutaneous tissue inflammation, and other wounds), L-asparaginase (acute lymphoblastic leukemia requiring exogenous asparagine for growth), Peg-asparaginase (acute lymphoblastic leukemia requiring exogenous asparagine for growth), Rasburicase (pediatric patients with leukemia, lymphoma, and solid tumors undergoing anticancer treatment can develop tumor lysis syndrome), Human chorionic gonadotropin (HCG) (assisted reproduction), Human follicle-stimulating hormone (HFS) FSH (assisted reproduction), Lutropin-alpha (infertility with luteinizing hormone deficiency), Prolactin (hypoprolactinemia, serum prolactin deficiency, ovarian dysfunction in women, anxiety, arteriogenic erectile dysfunction in men, premature ejaculation, oligozoospermia, asthenospermia, seminal vesicles dysfunction, hypoandrogenism), Alpha-1-proteinase inhibitor (congenital antitrypsin deficiency), Lactase (gas, bloating, cramps, and diarrhea due to lactose indigestion), Pancreatic enzymes enzymes) (lipase, amylase, protease) (cystic fibrosis, chronic pancreatitis, pancreatic insufficiency)insufficiency, post-Billroth II gastric bypass surgery, pancreatic duct obstruction, steatorrhoea, poor digestion, gas, bloating, adenosine deaminase (pegademase bovine, PEG-ADA) (severe combined immunodeficiency due to adenosine deaminase deficiency), abatacept (rheumatoid arthritis, especially when refractory to TNF-alpha inhibitors), alefacept (plaque psoriasis), anakinra (rheumatoid arthritis), etanercept (rheumatoid arthritis, polyarticular-course juvenile rheumatoid arthritis) These agents may include, but are not limited to, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, plaque psoriasis, ankylosing spondylitis), interleukin-1 (IL-1) receptor antagonists, Anakinra (inflammation and cartilage degeneration associated with rheumatoid arthritis), Thymulin (neurodegenerative diseases, rheumatoid arthritis, anorexia nervosa), TNF-alpha antagonists (autoimmune diseases such as rheumatoid arthritis, ankylosing spondylitis, Crohn's disease, psoriasis, hidradenitis suppurativa, and refractory asthma), Enfuvirtide (HIV-1 infection), and Thymosin alpha 1 (hepatitis B and C).

[0109] The length of the open reading frame (ORF) constituting the coding region (CR) is not limited, and expression efficiency due to the length of the ORF is not a major consideration in the development of the nucleic acid molecules disclosed herein, recombinant expression vectors using the same, and therapeutic or prophylactic nucleic acid vaccines. While codon usage can generally affect protein / peptide expression in various species, codon usage bias in humans is known to generally not significantly affect protein / peptide expression, and is therefore not a consideration when developing nucleic acid vaccines or gene therapy agents for humans. However, the initial codon should have a Kozak sequence (e.g., GCCACC), and the nucleotide sequence near the stop codon should also be optimized. If necessary, the third codon in the codon sequence of the gene to be expressed in the coding region (CR) or its transcript, mRNA, can be changed to GC to increase the GC% of the target gene without changing the amino acids, thereby improving mRNA stability.

[0110] In an alternative embodiment, the nucleic acid molecule may further include a nucleotide sequence that can increase the expression efficiency of the coding region (CR) linked in the form of an open reading frame (ORF). For example, the nucleic acid molecule may have a transcription control element (TCCE) adjacent to a translation control element, e.g., an upstream translation control element (U-TLCE), that promotes transcription of the nucleic acid molecule. For example, the transcription control element (TCCE) may be located upstream of the upstream translation control element (U-TLCE). Such transcription control elements (TCCEs) are not particularly limited, and to avoid repetition, they will be described in detail in the section on recombinant expression vectors below.

[0111] In addition, in addition to the above-mentioned translational regulatory element (TLCE), coding region (CR), and transcriptional regulatory element (TCCE), the nucleic acid molecule can also contain a nucleotide sequence capable of inducing expression of an open reading frame (ORF) consisting of a gene constituting the coding region (CR) or its transcript sequence. In one exemplary embodiment, a Kozak sequence can be inserted between the upstream translational regulatory element (U-TLCE) and the start codon of the coding region (CR).

[0112] In addition, downstream of the coding region (CR), more specifically downstream of the second downstream translational regulatory element (D-TLCE2), a polyadenylation signal sequence and / or polyadenosine sequence (PA) can be further inserted, which can stabilize the nucleic acid molecule while further improving the translation efficiency of the open reading frame (ORF) consisting of the gene present in the coding region (CR) or its transcript sequence.

[0113] For example, when the nucleic acid molecule of the present disclosure consists of an RNA transcript sequence, the polyadenosine sequence (PA) can be a nucleotide sequence consisting of approximately 25 to approximately 400 adenosines, for example, 30 to 400, 50 to 250, or 60 to 250 adenosines.

[0114] In another exemplary embodiment, when the nucleic acid molecule of the present disclosure is composed of a nucleotide sequence in the form of DNA, a polyadenylation signal sequence (PA) can be located downstream of the second downstream translation control sequence (D-TLCE2). As an example, the polyadenylation signal sequence (PA) can be derived from SV40, human growth factor (hGH), bovine growth hormone (BGH), or rabbit beta-globin (rbGlob), but the present disclosure is not limited thereto.

[0115] Alternatively, the polyadenylation signal sequence or polyadenosine sequence (PA) can consist of a sequence with a signal sequence such as 5'-GATCATCAGT-3' inserted between two nucleotides of a large number of adenosines, for example, 25 to approximately 400, 30 to 400, 50 to 250, or 60 to 250 adenosines, or their transcriptional sequence.

[0116] To insert a coding region (CR) into the nucleic acid molecule, the nucleic acid molecule may contain one or more cloning sites, preferably multiple cloning sites (MCS). The one or more cloning sites may contain one or more restriction endonuclease recognition sequences and / or sequences cleaved by restriction enzymes. Restriction enzymes may include natural restriction enzymes found in bacteria and archaea, as well as artificially engineered restriction enzymes (e.g., zinc finger nucleases, restriction enzymes based on the DNA binding sites of TAL effectors, or DNA-based DNA enzymes).

[0117] For example, naturally occurring restriction enzymes can be classified into 1) Type I restriction enzymes (which cleave at sites distant from the recognition site and require ATP, S-adenosyl-L-methionine, and magnesium ions), 2) Type II restriction enzymes (which cleave at specific sites within or slightly distant from the recognition site and mostly require magnesium ions), 3) Type III restriction enzymes (which cleave at sites slightly distant from the recognition site and require ATP but not ATP hydrolysis), 4) Type IV restriction enzymes (which target modification sites such as methylation, hydroxymethylation, or glucosyl-hydroxymethylation), and 5) Type V restriction enzymes (the cas9-gRNA complex of CRISPRs).

[0118] For example, the following restriction enzyme recognition sites and / or restriction enzyme cleavage sites (restriction enzymes) can be used: 5'-ATCGAT-3' (AngI), 5'-AGGCCT-3' (AatI), 5'-TGATCA-3' (AbaI), 5'-GGATCC-3' (BamHI), 5'-GCAGC(N)8-3' (BbvI), 5'-(N) 10 CGA(N)6TGC(N) 12 -3'(BcgI), 5'-(N)8GAG(N)5CTC(N) 13 -3'(BplI), 5'-GTCTC(N)-3'(BsmAI;Alw26I), 5'-ACTGGN-3'(BsrI), 5'-ATCGAT-3'(ClaI), 5'-CTCT TCN-3'(EarI), 5'-CTGAAG(N)16-3'(Eco57I), 5'-GAATTC-3'(EcoRI), 5'-CCWGG-3'(EcoRII; W is A or T ), 5'-GATATC-3' (EcoRV), 5'-GGATG(N)9-3' (FokI), 5'-GGCC-3' (HaeIII), 5'-AAGCTT-3' (HindIII) , 5'-CCGG-3'(HpaIII), 5'-GGTGA(N)8-3'(HphI), 5'-GGTACC-3'(KpnI), 5'-GATC-3'(MboI), 5'-ACGC GT-3'(MluI), 5'-GCCGGC-3'(NaeI), 5'-GATATG-3'(NdeII), 5'-GCCGGC-3'(NgoMIV), 5'-CATG-3'(N laIII), 5'-GCGGCCGC-3' (NotI), 5'-TTAATTAA-3' (PacI), 5'-CTGCAG-3' (PstI), 5'-GAGCTC-3' (SacI ), 5'-CCGCGG-3'(SacII), 5'-GTCGAC-3'(SalI), 5'-GCATC(N)5-3'(SfaNI), 5'-CCCGGG-3'(SmaI), 5'-TCGA-3'(TaqI), 5'-TCTAGA-3'(XbaI), 5'-CTCGAG-3'(XhoI), 5'-CGATCG-3'(PvuI), and combinations thereof.

[0119] In another exemplary embodiment, the cloning site may consist of SEQ ID NO:19.

[0120] The nucleic acid molecule may be DNA or RNA. According to an exemplary embodiment, the nucleic acid molecule of the present disclosure may be in the form of RNA. The RNA form of the nucleic acid molecule, such as the coding region (CR) consisting of the transcript sequence of the open reading frame (ORF) encoding the aforementioned protein or peptide, is advantageous over the DNA form of the nucleic acid molecule.

[0121] First, unlike DNA nucleic acid molecules, RNA nucleic acid molecules do not need to enter the nucleus of the host cell for transcription into mRNA. Therefore, they are safe because they have less potential risk of mutagenesis and are quickly degraded in vivo. RNA nucleic acid molecules cannot be incorporated into the host's chromosomes within the nucleus. Antibiotic resistance genes, which are selection markers used for selective production in host cells, are not required for the production of RNA nucleic acid molecules. Furthermore, RNA has a shorter half-life than DNA, so it does not induce persistent genetic transformation. In other words, conventional nucleic acid molecules are delivered into cells, become activated for a short period of time, express target peptides / proteins, and are destroyed by enzymatic reactions within a few days, but the specific immune response to the initially expressed target peptides / proteins remains.

[0122] Second, RNA-type nucleic acid molecules can induce a desired in vivo immune response even when used in relatively smaller amounts than DNA-type nucleic acid molecules. As mentioned above, RNA-type nucleic acid molecules, unlike DNA-type nucleic acid molecules, do not need to enter the nucleus but only need to pass through the cell membrane. Therefore, when RNA-type nucleic acid molecules are used, the same level of target peptide expression can be achieved even when used in smaller amounts than DNA-type nucleic acid molecules.

[0123] Third, because all manufacturing processes can be artificially controlled, vaccines can be safely produced in small-scale GMP (good manufacturing practice) production facilities without the risk of biological contamination. RNA-based nucleic acid molecules only require small-scale GMP laboratory-level facilities and do not require direct handling of infectious agents (viruses or pathogenic microorganisms), allowing for the rapid manufacture and production of a variety of vaccines.

[0124] Fourth, RNA nucleic acid molecules can induce a stronger immune response than naked DNA nucleic acid molecules. This is thought to be due to the cooperation of the innate immune response induced by the stem-loop structure of RNA nucleic acid molecules and the properties of RNA itself, and the adaptive immune response induced by target peptides / proteins expressed in immunized host cells. Furthermore, RNA nucleic acid molecules themselves produce complex antigens within cells, which can approach the MHC (major histocompatibility complex) class II of antigen-presenting cells and act as an ideal vaccine.

[0125] Fifth, RNA nucleic acid molecules are easier to produce than existing DNA nucleic acid molecules. As mentioned above, producing RNA nucleic acid molecules does not require direct manipulation of the infectious agent. Instead, only the nucleic acid sequence of the neutralizing antibody-inducing portion (neutralizing epitope) of the infectious agent to be expressed is artificially synthesized, and large amounts of RNA nucleic acid molecules can be produced through in vitro transcription (IVT). Recent improvements in IVT-related reagents, particularly DNA-dependent RNA polymerase, have made it possible to rapidly produce large amounts of RNA within one to two weeks using a small amount of DNA template. Furthermore, multiple antigens to induce an immune response can be simultaneously produced and mixed prior to immunization. Since there are no specific restrictions on the gene length of the antigen to be expressed, the applicability and ease of producing RNA nucleic acid molecules has increased.

[0126] In another exemplary embodiment, the nucleic acid molecule may comprise three or more downstream translational regulatory elements. Figure 2 is a schematic diagram illustrating the configuration of a nucleic acid molecule or polynucleotide that can efficiently express a gene of interest according to another exemplary embodiment of the present disclosure.

[0127] As shown in Figure 2, the nucleic acid molecule includes a translational regulatory element (TLCE) containing a nucleotide sequence having translation initiation activity and a coding region (CR) operably linked to the TLCE and comprising an open reading frame (ORF) of a gene of interest (GOI). The nucleic acid molecule may optionally include a transcriptional regulatory element (TCCE) and / or a polyadenylation signal sequence (PA) located upstream of the TLCE. The TLCE includes an upstream translational regulatory element (U-TLCE) located upstream of the coding region (CR) and a downstream translational regulatory element (D-TLCE) located downstream of the coding region (CR). In this embodiment, the downstream translational regulatory element (D-TLCE) includes a first downstream translational regulatory element (D-TLCE1), a second downstream translational regulatory element (D-TLCE2), and a third downstream translational regulatory element (D-TLCE3) located sequentially between the coding region (CR) and the polyadenylation signal sequence (PA). The downstream translational regulatory element (D-TLCE) may be the same as the nucleic acid molecule of the first embodiment described with reference to FIG. 1, except that it includes a third downstream translational regulatory element (D-TLCE3).

[0128] As an example, the first downstream translational regulatory element (D-TLCE1), the second downstream translational regulatory element (D-TLCE2), and the third downstream translational regulatory element (D-TLCE3) may each independently comprise a translational regulatory element or a transcript sequence derived from any one selected from the group consisting of human troponin T1, slow backbone type (TNNT1), human albumin, human ferritin light chain (FTL), human CC motif chemokine ligand (CCL19), human vascular-associated migratory cell protein (AAMP), human ribosomal protein S27 (RPS27), and human defensin alpha 5 (DEFA5).

[0129] For example, the first downstream translational regulatory element (D-TLCE1), the second downstream translational regulatory element (D-TLCE2), and the third downstream translational regulatory element (D-TLCE3) may each independently comprise a translational regulatory element or transcript sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14.

[0130] In exemplary embodiments, the first downstream translational regulatory element (D-TLCE1) may comprise a translational regulatory element derived from human TNNT1 or its transcript sequence, the second downstream translational regulatory element (D-TLCE2) may comprise a translational regulatory element derived from human DEFA5 or its transcript sequence, and the third downstream translational regulatory element (D-TLCE3) may comprise a translational regulatory element derived from human FTL or human RPS27 or its transcript sequence.

[0131] In other exemplary embodiments, the first downstream translational regulatory element (D-TLCE1) may comprise a translational regulatory element derived from human albumin or its transcript sequence, the second downstream translational regulatory element (D-TLCE2) may comprise a translational regulatory element derived from human DEFA5 or its transcript sequence, and the third downstream translational regulatory element (D-TLCE3) may comprise a translational regulatory element derived from human RPS27 or its transcript sequence.

[0132] In yet another exemplary embodiment, the first downstream translational regulatory element (D-TLCE1) may comprise a translational regulatory element derived from human RPS27 or its transcript sequence, the second downstream translational regulatory element (D-TLCE2) may comprise a translational regulatory element derived from human FTL or its transcript sequence, and the third downstream translational regulatory element (D-TLCE3) may comprise a translational regulatory element derived from any one selected from the group consisting of human CCL19, human RPS27, and human DEFA5, or its transcript sequence.

[0133] In yet another exemplary embodiment, the first downstream translational regulatory element (D-TLCE1) may comprise a transcriptional translational regulatory element or its transcript sequence derived from human DEFA5, the second downstream translational regulatory element (D-TLCE2) may comprise a translational regulatory element or its transcript sequence derived from human TNNT1, and the third downstream translational regulatory element (D-TLCE3) may comprise a translational regulatory element or its transcript sequence derived from said human FTL.

[0134] Recombinant expression vectors, expression constructs The nucleic acid molecules shown in Figures 1 and 2, respectively, can be inserted into a recombinant expression vector. The recombinant expression vector contains a translational regulatory element (TLCE; e.g., U-TLCE and D-TLCE) having translation initiation activity and a coding region (CR), and may contain a transcriptional regulatory element (TCCE) and / or a polyadenylation signal sequence (PA). That is, the recombinant expression vector may contain the nucleic acid molecule described with reference to Figures 1 and 2, but this nucleic acid molecule may also be linked to other nucleic acids to encode a fusion protein or fusion peptide.

[0135] For example, vectors include viral vectors, DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors linked with CCAs (cationic condensing agents), DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells such as producer cells.

[0136] Illustratively, the nucleic acid molecules of the present disclosure may be configured to be introduced into mammalian cells and expressed. Such configurations are particularly useful for treating and / or preventing infectious diseases. Numerous methods are available for expressing nucleic acid molecules in host cells, and any suitable method can be used. For example, nucleic acid molecules of the present disclosure can be inserted into viral vectors such as adenovirus, adeno-associated virus, retrovirus, vaccinia, or other poxviruses. According to one exemplary embodiment, the nucleic acid molecules described above can be inserted into an appropriate vector and then modified into RNA-based nucleic acid molecules through in vitro transcription (IVT).

[0137] Techniques for inserting nucleic acid molecules, e.g., DNA, into such vectors are well known. Retroviral vectors can additionally contain targeting moieties, such as genes for selectable markers that facilitate identification or selection of transduced cells and / or genes encoding ligands that act as receptors for specific target cells. Targeting can also be achieved by known methods using antibodies.

[0138] Numerous vectors are available and generally known in the art to which this disclosure pertains and can be used for the purposes of this disclosure. Selection of an appropriate vector will depend primarily 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 or expression of a heterologous polynucleotide, or both) and compatibility with the particular host cell in which the vector will reside. Vector components generally include, but are not limited to, an origin of replication (particularly if the vector is to be inserted into a prokaryotic cell), a selectable marker gene, a promoter, a ribosome binding site (RBS), a signal sequence, the heterologous nucleic acid insert, and / or a transcription termination sequence.

[0139] For example, an expression vector according to the present disclosure may include expression regulatory elements, such as a start codon, a stop codon, a polyadenylation signal sequence, an enhancer, a signal sequence for membrane targeting or secretion, that can affect the expression of a protein and / or peptide (e.g., a reporter protein / peptide, an antigen, or a therapeutic protein / peptide) encoded within the coding region (CR). A polyadenylation signal sequence (PA) increases the stability of the transcript or facilitates cytoplasmic transport. An enhancer sequence is a nucleic acid sequence located at various sites in a promoter that increases transcriptional activity compared to the transcriptional activity of the promoter in the absence of the enhancer sequence.

[0140] When the host is a bacterium of the genus Escherichia, the signal sequence may be a PhoA signal sequence, an OmpA signal sequence, or the like; when the host is a bacterium of the genus Bacillus, the signal sequence may be an α-amylase signal sequence, a subtilisin signal sequence, or the like; when the host is a yeast, the signal sequence may be an MF-α signal sequence, an SUC2 signal sequence, or the like; when the host is an animal cell, the signal sequence may be an insulin signal sequence, an α-interferon signal sequence, an antibody molecule signal sequence, or the like; however, the present disclosure is not limited thereto.

[0141] 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. Yet another type of vector is a viral vector, into which additional DNA segments can be ligated. Some vectors (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors) are capable of autonomous replication in a host cell into which they are introduced. Other vectors (e.g., non-episomal mammalian vectors) may be integrated into the genome of a host cell upon introduction into the host cell, thereby being replicated along with the host genome. Additionally, some vectors are capable of directing the expression of genes in the form of open reading frames to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "recombinant vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids.

[0142] In a specific embodiment of the present disclosure, the nucleic acid molecule can be inserted into a host cell using a viral expression system (vaccinia or other poxvirus, retrovirus, or adenovirus). Exemplary viral vectors may include, but are not limited to, retroviral vectors derived from HIV, SIV, murine retroviruses, gibbon ape leukemia virus, adeno-associated viruses (AAVs), and adenoviruses (Miller et al., 1990, Mol. Cell Biol. 10:4239; J. Kolberg 1992, NIH Res. 4:43; Cornetta et al., 1991, Hum. Gene Ther. 2:215).Retroviral vectors derived from murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), ecotropic retroviruses, simian immunodeficiency virus (SIV), and human immunodeficiency virus (HIV) are widely used (Buchscher et al., 1992, J. Virol. 66(5):2731-2739; Johann et al., 1992, J. Virol. 66(5):1635-1640; Sommerfelt et al., 1990, Virol. 176:58-59; Wilson et al., 1989, J. Virol. 63:2374-2378; Miller et al., 1991, J. Virol. 65:2220-2224; Rosenberg and Fauci 1993 in Fundermental Immunology,Third Edition,WEPaul(ed.)Raven Press,Ltd.,New York and the references therein;Miller et al.,1990,Mol.Cell.Biol.10:4239;R.Kolberg 1992,J.NIH Res.4:43;Cornetta et al.,1991,Hum.Gene Ther.2:215).

[0143] The vector system according to the present disclosure can be constructed by various methods generally known in the art to which the present disclosure pertains, and specific methods therefor are disclosed in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001), which is incorporated herein by reference.

[0144] For example, the vectors of the present disclosure can typically be constructed as vectors for cloning or expression. Furthermore, the vectors of the present disclosure can be constructed using prokaryotic 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, M13, etc.), or viruses (e.g., SV40, etc.) frequently used in the technical field to which the present disclosure pertains.

[0145] Constitutive or inducible promoters (e.g., transcriptional regulatory elements) can be used in the present disclosure, depending on the needs of a particular situation, ascertainable by one of ordinary skill in the art. Numerous promoters recognized by a variety of potential host cells are widely known. A selected promoter can be operably linked to a nucleic acid molecule having a coding region (CR) comprising the open reading frame (ORF) of a gene or transcript encoding a peptide and / or protein by removing the promoter from the source nucleic acid molecule through restriction enzyme digestion and inserting the isolated promoter sequence into a selected vector. Both the native promoter sequence and many heterologous promoters can be used to direct the amplification and / or expression of the gene or transcript comprising the coding region (CR). However, heterologous promoters are preferred, as they generally result in greater transcription and higher yields of the expressed gene of interest compared to the native promoter.

[0146] For example, when the vector of the present disclosure is an expression vector and a prokaryotic cell is used as the host, it generally contains a transcriptional regulatory element (TCCE) such as a strong promoter capable of driving 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, T7 promoter, etc.), a translational regulatory element (TLCE) for initiating translation, and a transcription / translation termination sequence. When E. coli is used as the host cell, the promoter and operator site of the E. coli tryptophan biosynthetic pathway (Yanofsky, C., J. Bacteriol., 158:1018-1024 (1984)) and the left-handed promoter of phage λ (pLλ promoter, Herskowitz, I. and Hagen, D., Ann. Rev. Genet., 14:399-445 (1980)) can be used as regulatory sites.

[0147] When the vector of the present disclosure is an expression vector and a eukaryotic cell is used as the host, a promoter derived from the genome of a mammalian cell (e.g., a metallothionine promoter), a promoter derived from a mammalian virus (e.g., an adenovirus late promoter, a vaccinia virus 7.5K promoter, an SV40 promoter, a cytomegalovirus promoter, and an HSV tk promoter), or a promoter derived from a bacteriophage (e.g., a T7 promoter, a T3 promoter, an SM6 promoter) can be used, and these generally have a polyadenylation signal sequence (PA) as a transcription termination sequence.

[0148] Furthermore, if 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 from which replication is initiated. The recombinant vector may also contain a selection marker. The selection marker is used to select cells transformed with the vector, and can confer a selectable phenotype, such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of a surface protein. The vector of the present disclosure contains an antibiotic resistance gene commonly used in the art as a selection marker, such as resistance genes to ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline. Transformed cells can be selected because only cells expressing the selection marker survive in an environment treated with a selective agent. Representative examples of selection markers include auxotrophic markers such as ura4, leu1, and his3, but these examples do not limit the types of selection markers that can be used in the present disclosure.

[0149] A variety of in vitro amplification techniques are known for amplifying sequences subcloned into expression vectors, including polymerase chain reaction (PCR), ligase chain reaction (LCR), Qβ-replicase amplification, and other RNA polymerase-based techniques (Sambrook et al., 1989, Molecular Cloning—A Laboratory Manual (2nd Ed) 1–3; U.S. Patent No. 4,683,202; PCR protocols: A Guide to Methods and Applications, Innis et al., eds., Academic Press Inc., San Diego, CA 1990. Improved methods of cloning in vitro amplified nucleic acids are described in U.S. Patent No. 5,426,039).

[0150] The vectors of the present disclosure can also be fused with other sequences to facilitate the purification of recombinant proteins or peptides expressed therefrom. Examples of fused sequences include glutathione S-transferase (Pharmacia, USA), maltose-binding protein (NEB, USA), FLAG (IBI, USA), and 6x His (hexahistidine; Quiagen, USA), with 6x His being the most preferred. Due to the additional purification sequence, proteins expressed in the host can be rapidly and easily purified through affinity chromatography. If necessary, a sequence encoding an Fc segment can also be fused to promote extracellular secretion of these recombinant proteins.

[0151] According to an exemplary embodiment of the present disclosure, the fusion protein expressed by the vector containing the fusion sequence is purified by affinity chromatography. For example, when glutathione-S-transferase is fused, glutathione, 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).

[0152] Host cells capable of stably and continuously cloning and expressing the above-described vectors can be any host cells well known in the art to which the present disclosure pertains, including, for example, Bacillus strains such as E. coli JM109, E. coli BL21(DE3), E. coli RR1, E. coli LE392, E. coli B, E. coli X1776, E. coli W3110, Bacillus subtilis, and Bacillus thuringiensis, as well as Enterobacteriaceae strains such as Salmonella typhimurium, Serratia marcescens, and various Pseudomonas species. Furthermore, when the vectors of the present disclosure are transformed into eukaryotic cells, host cells that can be used include yeast (Saccharomyce cerevisiae), insect cells (e.g., SF9 cells), and human cells (e.g., CHO cell lines (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN, and MDCK cell lines).

[0153] The vectors of the present disclosure can be used to genetically modify cells either in vivo, ex vivo, or in vitro. Various methods for genetically modifying cells are known, including infection or transduction of cells with viral vectors, calcium phosphate precipitation, fusing recipient cells with bacterial protoplasts containing DNA, treating recipient cells with liposomes or microspheres containing DNA, endocytosis (DEAE dextran, receptor-mediated endocytosis), electroporation, microinjection, etc.

[0154] For example, when the host cell is a prokaryotic cell, the conversion can be carried out by the CaCl2 method (Cohen, S. et al., Proc. Natl. Acac. Sci. USA, 9:2110-2114, 1973), the Hanahan method (Hanahan, D., J. Mol. Biol., 166:557-580, 1983), and / or the electroporation method (Dower, W. et al., Nucleic. Acids Res., 16:6127-6145, 1988), etc. Alternatively, when the host cell is a eukaryotic cell, the vector can be injected into the host cell by microinjection (Capecchi, MR, Cell, 22:479, 1980)), calcium phosphate precipitation (Graham, FL et al., Virology, 52:456, 11973), electroporation (Neumann, E. et al., EMBO J., 1:841, 1982), liposome-mediated transformation (Wong, TK et al., Gene, 10:87, 1980), DEAE-dextran treatment (Gopal, Mol. Cell Biol., 5:1188-1190, 1985), and / or gene bombardment (Yang et al., Proc. Natl. Acad. Sci., 87:9568-9572, 1990), or the like. The vector injected into the host cell can be expressed in the host cell, resulting in the production of a large amount of recombinant protein or recombinant peptide. For example, if the expression vector contains a lac promoter, the host cell can be treated with IPTG to induce gene expression.

[0155] The above-mentioned nucleic acid molecules and / or expression constructs, which are expression systems into which the above-mentioned nucleic acid molecules are inserted as gene carriers, can be used as detection systems for detecting the presence or absence of a substance, vaccines for inducing immune responses, and / or therapeutic agents for treating diseases.

[0156] For example, a pharmaceutical composition such as a vaccine or therapeutic agent containing a pharmaceutically effective amount of a nucleic acid molecule or an expression construct containing a nucleic acid molecule may contain a pharmaceutically acceptable carrier, diluent, and / or excipient. As used herein, the term "pharmaceutically effective amount" refers to an amount sufficient to achieve the efficacy or activity of the nucleic acid molecule according to the present disclosure.

[0157] Such pharmaceutical compositions may also contain a stabilizer, such as a cationic polymer, cationic peptide, or cationic polypeptide, capable of stabilizing the nucleic acid molecule or expression construct; at least one immune enhancer capable of enhancing the immune response; a sustained-release formulation; and / or a lipid nanoparticle (LNP) capable of protecting the nucleic acid molecule as an active ingredient and improving its bioactivity.

[0158] The nucleic acid molecules or expression constructs of the disclosure can be administered by any suitable means, for example, oral, topical (including buccal and sublingual), rectal, vaginal, transdermal, parenteral, subcutaneous, intraperitoneal, intrapulmonary, intradermal, intrathecal and epidural, and intranasal, and, if desired, means for local therapeutic, intralesional administration. Parenteral administration includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.

[0159] The nucleic acid molecules or expression constructs of the disclosure can be administered in any convenient dosage form, e.g., tablet, powder, capsule, solution, dispersion, suspension, syrup, spray, suppository, gel, emulsion, patch, etc. Such compositions can contain conventional ingredients of pharmaceutical formulations, e.g., diluents, carriers, pH adjusters, sweeteners, bulking agents, and additional active agents.

[0160] The present disclosure will be described below based on illustrative examples, but the present disclosure is not limited to the technical ideas described in the following examples.

[0161] Comparative Example 1: Preparation of a nucleic acid molecule containing an inserted Renilla luciferase (R / L) coding sequence Renilla luciferase A nucleic acid molecule containing an inserted coding sequence was constructed. The template DNA sequence was designed as follows:

[0162] 5'-KpnI recognition sequence (GGTACC)-T7 promoter (SEQ ID NO: 16)-upstream translational regulatory element derived from human troponin T1 (TNNT1) (SEQ ID NO: 1)-PacI recognition sequence (TTAATTAA)-Kozak sequence (GCCACC)-Renilla luciferase coding sequence (R / L, SEQ ID NO: 15)-ClaI recognition sequence (ATCGAT)-downstream translational regulatory element derived from human troponin T1 (human TNNT1) (SEQ ID NO: 8)-EcoRI recognition sequence (GAATCC)-polyadenylation signal (SEQ ID NO: 17)-restriction enzyme recognition sequence (SEQ ID NO: 19).

[0163] The template DNA was inserted downstream of the pGH vector (SEQ ID NO: 18), cloned, linearized with a restriction enzyme, and subjected to in vitro transcription (IVT) to produce an RNA platform nucleic acid molecule (hereinafter referred to as "pHJ5L").

[0164] Example 1: Construction of a nucleic acid molecule containing a Renilla luciferase (R / L) coding sequence The procedure of the comparative example was repeated, except that instead of the single TNNT1-derived translational regulatory element, two downstream translational regulatory elements were inserted downstream of the coding region encoding Renilla luciferase, each of which was independently selected from the group consisting of SEQ ID NO: 8 (derived from human TNNT1), SEQ ID NO: 9 (derived from human albumin), SEQ ID NO: 10 (derived from human FTL), SEQ ID NO: 11 (derived from human CCL19), SEQ ID NO: 12 (derived from human AAMP), SEQ ID NO: 13 (derived from human RPS27), and SEQ ID NO: 14 (derived from human DEFA5). Hereinafter, the downstream translation regulatory element of SEQ ID NO: 8 will be abbreviated as "A", the downstream translation regulatory element of SEQ ID NO: 9 as "B", the downstream translation regulatory element of SEQ ID NO: 10 as "C", the downstream translation regulatory element of SEQ ID NO: 11 as "D", the downstream translation regulatory element of SEQ ID NO: 12 as "E", the downstream translation regulatory element of SEQ ID NO: 13 as "F", and the downstream translation regulatory element of SEQ ID NO: 14 as "G".

[0165] Experimental Example 1: In vitro expression Mouse muscle cell line Nor10, human proliferative cell line HeLa, and human embryonic kidney cell line 293A were each plated in a 48-well plate at 8x10 4 The cells were seeded at a concentration of 1000 cells / well. The plate was then placed in a 37°C incubator to allow the cells to attach and grow for 24 hours. Each mRNA prepared in the comparative example and the example was transfected at a concentration of 500 ng / well. Lipofection TM For each 2000 mRNA sample, 1 μl of each mRNA was added to OPTI-MEM medium and mixed thoroughly for at least 30 minutes. TM After mixing the 2000, the 48-well plate with the attached cells was removed, the supernatant was removed, washed with PBS, and then the supernatant was removed. 100 μl of medium was added on top, and the mRNA and lipofection were performed. TM 100 μl of the 2000 mixture was added.

[0166] The negative control group received only 200 μl of medium. The plate was then placed in a 37°C incubator and removed at 6 and 24 hours to confirm expression. After 6 and 24 hours, the plate was removed, the supernatant was completely removed, and the plate was washed once with PBS. 80 μl of Renilla lysis buffer from the Renilla Luciferase Assay System Kit (Promega) was added to each plate to thoroughly disrupt the cells. Renilla luciferase expression was measured using a spectrophotometer as instructed. The level of Renilla luciferase expression after 6 hours in the Nor10 cell line treated with the pHJ5L nucleic acid molecule prepared in the comparative example was 2×10 7 After 24 hours, the expression level of Renilla luciferase was 2X10 6 It was.

[0167] In the HeLa cell line to which the pHJ5L nucleic acid molecule prepared in the comparative example was applied, the expression level of Renilla luciferase after 6 hours was 7x10 7 After 24 hours, the expression level of Renilla luciferase was 5.5X10 6 The expression level of Renilla luciferase in the 293A cell line to which the pHJ5L nucleic acid molecule prepared in the comparative example was applied after 6 hours was 4.3 × 10 7 After 24 hours, the expression level of Renilla luciferase was 2.2X10 6 It was.

[0168] The nucleic acid expression platform used as the base template was pHJ5L prepared in the Comparative Example. The expression level of Renilla luciferase in this platform was set to 1, and the expression level of Renilla luciferase in each RNA expression platform prepared in Example 1 was compared with the expression level in pHJ5L. The measurement results are shown in Figures 3 to 8. As shown in Figures 3 to 8, the nucleic acid molecules prepared in this Example, in which two downstream translation regulatory elements were inserted between the coding region and the 3' end, efficiently enhanced the expression of Renilla luciferase inserted in the coding region in all Nor10, HeLa, and 293A cell lines.

[0169] Experimental Example 2: In vivo expression The mRNA prepared in the Comparative Example and Example 1 was formulated with LNP (the same as that used in Moderna's vaccine) to prepare a composition for bioinjection. Mice were anesthetized using respiratory anesthesia and their ears were immobilized. Each mouse ear was injected with 5 μg / 20 μl of the mRNA-formulated composition prepared in the Comparative Example and Example. Six and 24 hours after injection, mice were sacrificed, and their ears were cut and placed in 300 μl of Renilla lysis buffer and then finely chopped with scissors. The cut mouse ears were further crushed using a homogenizer, and 20 μl of the supernatant was placed in a white 96-well plate. Luminescence was then measured using a spectrophotometer using the Renilla substrate provided in the Renilla luciferase assay kit. The nucleic acid expression platform used as the base template was pHJ5L prepared in the Comparative Example. The expression level of Renilla luciferase in this platform was set to 1, and the expression level of Renilla luciferase in each RNA expression platform prepared in the Examples was compared with the expression level in pHJ5L. The measurement results are shown in Figures 9 and 10. As shown in Figures 9 and 10, the expression level of Renilla luciferase increased from the nucleic acid molecule in which two downstream translation regulatory elements were inserted between the coding region and the 3' end.

[0170] Example 2: Construction of a nucleic acid molecule containing an inserted Renilla luciferase (R / L) coding sequence The procedure of Example 1 was repeated, except that instead of inserting two downstream translational regulatory elements downstream of the coding region encoding Renilla luciferase, three downstream translational regulatory elements were inserted. Each of the three downstream translational regulatory elements was independently the same as that used in Example 1.

[0171] Experimental Example 3: In vitro expression The nucleic acid molecule prepared in Example 2 was transformed into a human embryonic kidney cell line, 293A, using the same method as in Example 1, and the expression level of Renilla luciferase was measured by luminescence using a spectrophotometer. The nucleic acid expression platform used as the base template was pHJ5L prepared in the Comparative Example. The expression level of Renilla luciferase in this platform was set to 1, and the expression level of Renilla luciferase in each RNA expression platform prepared in Example 2 was compared with that in pHJ5L. The measurement results are shown in Figures 11 and 12. As shown in Figures 11 and 12, the nucleic acid molecule prepared in this example, in which three downstream translational regulatory elements were inserted between the coding region and the 3' end, efficiently enhanced the expression of Renilla luciferase inserted in the entire coding region in the 293A cell line.

[0172] Although the present disclosure has been described above based on exemplary embodiments and examples of the present disclosure, the present disclosure is not limited to the technical ideas described in the above embodiments and examples. On the contrary, a person skilled in the art to which the present disclosure pertains can easily consider various modifications and changes based on the above embodiments and examples. However, it is clear from the appended claims that all such modifications and changes are within the scope of the present disclosure.

Claims

1. A coding region; a translational regulatory element operably linked to the coding region, the translational regulatory elements include an upstream translational regulatory element located upstream of the coding region and a downstream translational regulatory element located downstream of the coding region; the downstream translational regulatory elements include a first downstream translational regulatory element and a second downstream translational regulatory element located downstream of the coding region; the upstream translational regulatory element comprises an upstream translational regulatory element derived from human troponin T1, slow skeleton (human TNNT1) or a transcript sequence thereof; the first downstream translational regulatory element comprises a downstream translational regulatory element derived from any one gene selected from the group consisting of human troponin T1, slow backbone type (human TNNT1), human albumin, human ferritin light chain (human FTL), human C-C motif chemokine ligand (human CCL19), human vascular-associated migratory cell protein (human AAMP), human ribosomal protein S27 (human RPS27), and human defensin alpha 5 (human DEFA5), or a transcript sequence thereof; When the first downstream translation regulatory element comprises a downstream translation regulatory element derived from the human TNNT1 or a transcript sequence thereof, the second downstream translation regulatory element comprises a downstream translation regulatory element derived from any one selected from the group consisting of the human albumin, the human FTL, the human CCL19, the human AAMP, the human RPS27, and the human DEFA5 or a transcript sequence thereof; when the first downstream translation regulatory element comprises a downstream translation regulatory element derived from human albumin or a transcript sequence thereof, the second downstream translation regulatory element comprises a downstream translation regulatory element derived from human DEFA5 or a transcript sequence thereof; When the first downstream translation regulatory element comprises a downstream translation regulatory element derived from the human FTL or a transcript sequence thereof, the second downstream translation regulatory element comprises a downstream translation regulatory element derived from any one selected from the group consisting of the human TNNT1, the human FTL, the human RPS27, and the human DEFA5, or a transcript sequence thereof; when the first downstream translation regulatory element comprises a downstream translation regulatory element derived from the human CCL19 or a transcript sequence thereof, the second downstream translation regulatory element comprises a downstream translation regulatory element derived from any one selected from the group consisting of the human TNNT1, the human FTL, the human CCL19, the human RPS27, and the human DEFA5 or a transcript sequence thereof; When the first downstream translation regulatory element comprises a downstream translation regulatory element derived from the human AAMP or a transcript sequence thereof, the second downstream translation regulatory element comprises a downstream translation regulatory element derived from any one selected from the group consisting of the human albumin, the human FTL, the human RPS27, and the human DEFA5, or a transcript sequence thereof; when the first downstream translation regulatory element comprises a downstream translation regulatory element derived from the human RPS27 or a transcript sequence thereof, the second downstream translation regulatory element comprises a downstream translation regulatory element derived from any one selected from the group consisting of the human TNNT1, the human albumin, the human FTL, the human CCL19, the human AAMP, the human RPS27, and the human DEFA5 or a transcript sequence thereof; A nucleic acid molecule, wherein when the first downstream translation regulatory element comprises a downstream translation regulatory element derived from human DEFA5 or a transcript sequence thereof, the second downstream translation regulatory element comprises a downstream translation regulatory element derived from any one selected from the group consisting of human TNNT1, human albumin, human FTL, human CCL19, human AAMP and human RPS27 or a transcript sequence thereof.

2. The nucleic acid molecule of claim 1, wherein the first downstream translational regulatory element comprises a downstream translational regulatory element derived from human TNNT1 or a transcript sequence thereof, and the second downstream translational regulatory element comprises a downstream translational regulatory element derived from any one selected from the group consisting of human FTL, human CCL19, human RPS27 and human DEFA5 or a transcript sequence thereof.

3. The nucleic acid molecule of claim 1, wherein the first downstream translational regulatory element comprises a downstream translational regulatory element derived from the human FTL or a transcript sequence thereof, and the second downstream translational regulatory element comprises a downstream translational regulatory element derived from any one selected from the group consisting of the human FTL, the human RPS27, and the human DEFA5 or a transcript sequence thereof.

4. the upstream translation regulatory element comprises SEQ ID NO:8 or a transcript sequence thereof; 2. The nucleic acid molecule of claim 1, wherein the first downstream translational regulatory element comprises a downstream translational regulatory element selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, or a transcript sequence thereof.

5. The nucleic acid molecule of claim 1, wherein the first downstream translational regulatory element comprises a downstream translational regulatory element derived from the human CCL19 or a transcript sequence thereof, and the second downstream translational regulatory element comprises a downstream translational regulatory element derived from any one selected from the group consisting of the human FTL, the human CCL19, the human RPS27, and the human DEFA5 or a transcript sequence thereof.

6. The nucleic acid molecule of claim 1, wherein the first downstream translational regulatory element comprises a downstream translational regulatory element derived from human RPS27 or a transcript sequence thereof, and the second downstream translational regulatory element comprises a downstream translational regulatory element derived from any one selected from the group consisting of human FTL, human CCL19, human RPS27 and human DEFA5 or a transcript sequence thereof.

7. The nucleic acid molecule of claim 1, wherein the first downstream translational regulatory element comprises a downstream translational regulatory element derived from human DEFA5 or a transcript sequence thereof, and the second downstream translational regulatory element comprises a downstream translational regulatory element derived from any one selected from the group consisting of human albumin, human FTL, and human RPS27 or a transcript sequence thereof.

8. The downstream translation regulatory element further includes a third downstream translation regulatory element located downstream of the second downstream translation regulatory element; the third downstream translation regulatory element comprises a downstream translation regulatory element derived from any one selected from the group consisting of human troponin T1, slow backbone type (TNNT1), human albumin, human ribosomal protein S27 (RPS27), and human defensin alpha 5 (DEFA5), or a transcript sequence thereof; when the first downstream translation regulatory element comprises a downstream translation regulatory element derived from human TNNT1 or a transcript sequence thereof, the second downstream translation regulatory element comprises a downstream translation regulatory element derived from human DEFA5 or a transcript sequence thereof, and the third downstream translation regulatory element comprises a downstream translation regulatory element derived from human FTL or human RPS27 or a transcript sequence thereof; when the first downstream translation regulatory element comprises a downstream translation regulatory element derived from human albumin or a transcript sequence thereof, the second downstream translation regulatory element comprises a downstream translation regulatory element derived from human DEFA5 or a transcript sequence thereof, and the third downstream translation regulatory element comprises a downstream translation regulatory element derived from human RPS27 or a transcript sequence thereof; When the first downstream translation regulatory element comprises a downstream translation regulatory element derived from human RPS27 or a transcript sequence thereof, the second downstream translation regulatory element comprises a downstream translation regulatory element derived from human FTL or a transcript sequence thereof, and the third downstream translation regulatory element comprises a downstream translation regulatory element derived from any one selected from the group consisting of human CCL19, human RPS27, and human DEFA5 or a transcript sequence thereof, or The nucleic acid molecule of claim 1, wherein the first downstream translational regulatory element comprises a downstream translational regulatory element derived from human DEFA5 or a transcript sequence thereof, the second downstream translational regulatory element comprises a downstream translational regulatory element derived from human TNNT1 or a transcript sequence thereof, and the third downstream translational regulatory element comprises a downstream translational regulatory element derived from human FTL or a transcript sequence thereof.

9. The nucleic acid molecule of claim 1 , wherein the nucleic acid molecule is RNA.

10. the nucleic acid molecule comprising a transcriptional regulatory element operably linked to the coding region; and The nucleic acid molecule of claim 1, further comprising at least one of a polyadenylation signal sequence or a polyadenosine sequence located downstream of the downstream translational regulatory element.

11. The nucleic acid molecule of claim 1, wherein the coding region encodes at least one of a reporter protein, or a fragment thereof, or a marker, or a selection protein, or a fragment thereof, or an antigen, or a fragment thereof, or a peptide, or a fragment thereof, for treating a disease.

12. A recombinant expression vector into which the nucleic acid molecule of claim 1 has been inserted.

13. A method for producing a protein or peptide by injecting the nucleic acid molecule of claim 1 or a recombinant expression vector into a living body (excluding humans) and expressing a protein or peptide from the injected nucleic acid molecule.

14. A pharmaceutical composition comprising a pharmaceutically effective amount of the nucleic acid molecule of claim 1 or an expression construct into which the nucleic acid molecule of claim 1 has been inserted.

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