KR102007926B1 - Nucleic acid molecules inserted expression regulation sequences and expression vector comprising nucleic acid molecules - Google Patents

Abstract

The present invention relates to a nucleic acid molecule capable of expressing a gene of interest using an expression control sequence derived from a virus or eukaryotic cell having an internal ribosomal binding site (IRES), an expression vector into which the nucleic acid molecule is inserted, and an application thereof. The nucleic acid molecules of the invention can be used to efficiently express a gene of interest without the complex capping process associated with expression of the gene of interest. According to the present invention, a nucleic acid molecule having a plurality of adenosine or thymine linked to an expression control sequence can be used to efficiently express a gene of interest. By applying an expression system having a nucleic acid molecule according to the present invention, it is expected to be used as a gene vaccine or as a gene therapy agent for expressing a protein or peptide related to disease treatment.

Description

Nucleic acid molecule having an expression control sequence inserted therein and an expression vector comprising the same {NUCLEIC ACID MOLECULES INSERTED EXPRESSION REGULATION SEQUENCES AND EXPRESSION VECTOR COMPRISING NUCLEIC ACID MOLECULES}

The present invention relates to a nucleic acid molecule, and more particularly, to a nucleic acid molecule with improved expression efficiency, an expression vector including the same, and an application thereof.

As biotechnology advances, various expression systems capable of efficiently expressing genes of interest (GOI) are known. As such an expression system, a cell-based expression system uses an expression system of microorganisms or eukaryotic cells, and an expression system containing no cells generally uses purified RNA polymerase, ribosomes, tRNAs and ribonucleotides. In particular, proteins derived from eukaryotic cells post-translational modification such as phosphorylation, methylation, and glycosylation occur even after expression, and microorganisms express proteins derived from eukaryotic cells because they do not have such mechanisms. If desired, eukaryotic cell expression systems are used.

The expression system derived from eukaryotic cells can be used as a gene therapy agent or gene vaccine. In gene therapy products, a gene of interest consisting of an open reading frame (ORF) encoding a peptide or protein capable of treating various diseases is inserted into an expression system, and in the case of a gene vaccine, a peptide or protein that can be expressed as an antigen is encoded. The gene of interest consisting of an open reading frame is inserted into the expression system.

In order to efficiently express a gene of interest, it is common to use a sequence that regulates the transcription and translation of the gene of interest. In general, promoters having excellent transcriptional efficiency can be used to improve transcriptional efficiency, and eukaryotic cell-specific capping systems are used in connection with translation of the gene of interest.

Specifically, the capping system used in connection with efficient translation from a gene of interest (GOI) to a protein or peptide is a 7-methyl guanosine (m ⁷ G) at the 5 'end of the gene of interest (GOI). It has a 5 'cap structure. Translation initation complex consisting of eIF4A, eIF4E, eIF4G, etc., which acts as a translational regulator of eukaryotic cells, recognizes the 5 'cap and binds to the 5' cap to form a capping structure while It starts. When the capping structure is formed at the site where translation is initiated, protein synthesis is initiated and the destruction of mRNA is prevented from the action of nuclease.

IVT process for preparing nucleic acid molecules having a capping structure was performed by linearizing by processing restriction enzymes on pDNA (plasmid DNA), followed by m ⁷ G (5 ')-ppp- (5 ') G (called regular cap analog) is attached to make capped mRNA. However, these cap analogs often bind in the opposite direction, preventing m ⁷ G nucleotides from acting on the cap, and about one-third of the mRNA produced has no methylation in the cap, and these mRNAs cannot synthesize protein.

To prevent this, there is a method that performs IVT without a cap analog, and then performs a cap reaction using a commercially available vaccinia virus capping enzyme, and an 'anti-reverse' cap analog (ARCA) that prevents the reverse reaction of the cap. Induces protein synthesis using (ARCA-capped mRNA) ARCA-capped mRNA is known to have twice the efficiency of protein synthesis than regular cap analog-capped mRNA and has a long half-life of mRNA. However, since such an artificial capping reaction is required, the cost for expressing the gene of interest is greatly increased. Therefore, there is a need for a method for improving the expression system to be used as a gene vaccine or gene therapy.

It is an object of the present invention to provide an expression system such as a nucleic acid molecule capable of efficiently expressing a desired peptide or protein in vivo without complicated and expensive processes and an expression vector into which such nucleic acid molecule is inserted.

According to an aspect of the present invention, the present invention provides at least one expression control sequence comprising a base sequence having an internal ribosomal entry site (IRS) activity; And at least one coding region operably linked with the expression control sequence, the coding region encoding an protein or peptide, wherein 20 to 400 adenosine or thymine are linked adjacent to the at least one expression control sequence. Providing nucleic acid molecules.

In one example, the at least one expression control sequence may form a 5'-untranslated region (5'-UTR).

In this case, the nucleic acid molecule further comprises a 3'-untranslated region (3'-UTR) located on the 3 'side of the 5'-UTR, the coding region is between the 5'-UTR and the 3'-UTR It can be located at

In one exemplary embodiment, the at least one expression control sequence may comprise a viral IRES base sequence.

In one example, the viral IRES base sequence is Picornaviridae virus, Togaviridae virus, Dicistroviridae virus, Flaviridae virus, Retroviridae virus, Herpes Viral IRES sequences derived from a virus selected from the group consisting of the herpesviridae virus and combinations thereof.

For example, the viral IRES base sequence may be derived from a virus selected from the group consisting of the Picornaviridae virus, the Dicistroviridae virus, and combinations thereof.

At this time, the viral IRES base sequence derived from the Picornaviridae virus is Enterovirus virus, Cardiovirus virus, Aptovirus virus, Hepattovirus genus ( Hepatovirus virus, Teschovirus virus, and combinations thereof are selected from the group of piconaviruses virus selected from the group consisting of, and the viral IRES base sequence derived from the Dicistroviridae virus is Creepa It may be derived from a Cripavirus virus.

More specifically, the IRES nucleotide sequence derived from the Picornaviridae virus includes an IRES nucleotide sequence derived from coxsackievirus, and the IRES nucleotide sequence derived from the dyscivirus virus includes a cricket palsy virus ( Cricket paralysis virus (CPV) may include the IRES base sequence.

According to one exemplary embodiment, the coding region may encode an antigen or fragment thereof.

In another alternative embodiment, the coding region may encode a protein or fragment thereof for treating a disease.

In another exemplary embodiment, the at least one expression control sequence is a first expression control sequence having a base sequence of IRES and a second expression control located 3 ′ side of the first expression control sequence and having a base sequence of IRES Sequences may be included.

In this case, the coding region may include a first coding region located between the first expression control sequence and the second expression control sequence, and a second coding region located on the 3 'side of the second expression control sequence. .

If necessary, the first coding region and the second coding region may encode different peptides or proteins.

In this case, 20 to 400 adenosine or thymine may be coupled to the first expression control sequence and at least one expression control sequence selected from the second expression control sequence.

For example, 30 to 100 adenosine may be bound adjacent to the first expression control sequence.

In one exemplary embodiment, the first expression control sequence comprises an IRES base sequence derived from Coxsachie virus B3 (CVB3) or Cricket paralysis virus (CPV), and wherein the second The expression control sequence may comprise an IRES base sequence derived from Enncephalomyocarditis virus (EMCV).

Preferably, the nucleic acid molecule may further comprise a transcriptional regulatory sequence located on the 5 'side of the expression control sequence and a poly adenosine (poly (A)) sequence located on the 3' side of the coding region. .

In one example, the nucleic acid molecule may be RNA.

In another aspect of the invention, the invention provides a recombinant expression vector into which the above-described nucleic acid molecule is inserted. In one example, the recombinant expression vector may be from a plasmid or from a virus.

In another aspect of the present invention, the present invention provides a method for preparing the protein or peptide by injecting the above-described nucleic acid molecule into a living body and expressing the protein or peptide from the injected nucleic acid molecule.

In another aspect of the present invention, the present invention provides a method for preparing the protein or peptide by injecting the above-described recombinant gene vector in vivo as a gene carrier and expressing the protein or peptide from the gene carrier.

In another aspect of the invention, the invention provides host cells transformed with proteins or peptides expressed from the above-described nucleic acid molecules and / or recombinant expression vectors.

According to the present invention, a nucleic acid molecule having at least one expression control sequence having an internal ribosomal binding site (IRES) for expression of a gene of interest, i. To present.

In order to efficiently express a gene of interest using a conventional capping structure, a problem of using expensive enzymes has arisen, and only one peptide or protein can be expressed in one expression system.

However, the nucleic acid molecule according to the present invention can efficiently express the desired peptide or protein in vivo even without using an expensive enzyme. In addition, since the present invention includes IRES as an expression control sequence, by operably linking the same or different peptides or proteins with other kinds of IRES sequences, the peptides and proteins desired at the same time in one nucleic acid molecule can be simultaneously linked. Can express and increase expression efficiency.

For example, a nucleic acid molecule of the present invention which introduces a gene sequence of interest that induces various immune responses in vivo to a coding region and / or a recombinant expression vector into which the nucleic acid molecule is inserted can be used as a gene vaccine. .

Alternatively, by introducing a gene sequence of interest that encodes a peptide and / or protein associated with the treatment of various diseases in the coding region, it may be utilized as a gene therapy agent or the like.

According to an alternative embodiment, the nucleic acid molecules of the invention can be made in RNA form and applied in the form of a vaccine. The RNA vaccine according to the present invention can be produced and produced quickly with only a small amount of template DNA, and stability can be secured because it does not need to deal directly with an infectious agent.

1 is a schematic diagram showing the configuration of a polynucleotide or nucleic acid molecule capable of efficiently expressing one gene of interest according to an exemplary embodiment of the present invention.
FIG. 2 is a schematic diagram showing the configuration of a polynucleotide or nucleic acid molecule capable of efficiently expressing a plurality of genes of interest in accordance with exemplary embodiments of the present invention. FIG.
3A and 3B are graphs showing the results of measuring expression levels of genes of interest after administration of a nucleic acid molecule in the form of an RNA platform containing IRES from various viruses, respectively, according to an exemplary embodiment of the present invention. . 3A shows the expression level in the A204 cell line, and FIG. 3B shows the expression level in the 293T cell line.
4A and 4B are graphs showing the results of measuring the expression levels of genes of interest after administration of a nucleic acid molecule in the form of an RNA platform containing IRES derived from various eukaryotic cells, respectively, according to an exemplary embodiment of the present invention. to be. 4A shows the expression level in the A204 cell line, and FIG. 4B shows the expression level in the 293T cell line.
FIG. 5 is a graph showing a result of measuring the expression level of a gene of interest after administering a nucleic acid molecule in the form of an RNA platform including IRES from various viruses, according to an exemplary embodiment of the present invention. FIG. The result of measuring the expression amount in is shown.
6 is a graph showing the results of measuring expression levels of genes of interest after administration of a nucleic acid molecule comprising IRES derived from various viruses synthesized according to an exemplary embodiment of the present invention to a Nor10 cell line.
7A and 7B are graphs showing the results of measuring expression levels of genes of interest after administering nucleic acid molecules containing IRESs of various viruses, respectively, according to exemplary embodiments of the present invention to various cell lines. 7A shows the expression level in the A204 cell line, and FIG. 7B shows the expression level in the 293T cell line.
8 is a graph showing the results of measuring expression levels of genes of interest after administration of nucleic acid molecules comprising a plurality of virus-derived IRESs to various cell lines according to an exemplary embodiment of the present invention. Indicates that was measured.
9A and 9B are graphs showing the expression levels of genes of interest after administration of various cell lines with nucleic acid molecules comprising a plurality of virus-derived IRESs, respectively, according to an exemplary embodiment of the present invention. 9A shows the expression level in 293T cell line, and FIG. 9B shows the expression level in Nor10 cell line.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated, referring drawings attached if needed.

Definition of Terms

The term "amino acid" is used herein in its broadest sense and is intended to include naturally-occurring L-amino acids or residues. One-letter abbreviations and / or three-letter abbreviations commonly used for naturally-occurring amino acids can be used herein. Amino acids are chemically possessing properties known in the art to be characteristic of not only D-amino acids but also chemically-modified amino acids such as amino acid analogs, naturally-occurring amino acids such as norleucine, and amino acids that are not commonly incorporated into proteins. Synthetic compounds. For example, within the definition of amino acids are analogs or mimetics of phenylalanine or proline that allow conformational limitations of the same peptide compounds as native Phe or Pro. Such analogs and mimetics are referred to herein as "functional equivalents" of amino acids. Other examples of amino acids are described in Roberts and Vellaccio, The Peptides: Analysis, Synthesis, Biology, Eds. Gross and Meiehofer, Vol. 5, p. 341 (Academic Press, Inc .: N.Y. 1983).

For example, synthetic peptides synthesized by standard solid-phase synthesis techniques are not limited to amino acids encoded by genes, thus allowing 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" and are described, for example, in WO 90/01940. For example, amino acid analogs include 2-amino adipic 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 (AHyl) for Lys; 3- (and 4-) hydroxyproline (3Hyp, 4Hyp) for Pro, Ser and Thr; Allo-isoleucine (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.

As used herein, the term “peptide” includes all proteins, protein fragments and peptides that have been separated from those present in nature, or synthesized by recombinant techniques or chemically synthesized. For example, the peptide of the present invention consists of at least 5, preferably 10 amino acids.

In certain embodiments, variants of a compound are provided, such as peptide variants having one or more amino acid substitutions. As used herein, “peptide variants” refers to the original amino acid while one or more amino acids are substituted, deleted, added, and / or inserted into the amino acid sequence of the peptide. It is said to exert almost the same biological function as the peptide consisting of. Peptide variants should have at least 70% identity, preferably at least 90% and more preferably at least 95% identity with the original peptide. Such substituents may include amino acid substituents known as “conservatives”. Variants may also include changes in nonconservative. In a preferred embodiment, the sequence of the variant polypeptide differs from the original sequence by replacing, deleting, adding or inserting five or fewer amino acids. Variants may also be altered by deletion or addition of amino acids with minimal impact on the immunogenicity, secondary structure and hydropathic nature of the peptide.

“Conservative” substitutions mean that even when one amino acid is substituted with another amino acid, there is no significant change in the properties such as the secondary structure and hydropathic nature of the polypeptide. Amino acid variations are based on the relative similarities of amino acid side chain substituents, such as polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphipathic nature. Can be obtained.

For example, amino acids may be characterized by their common side chain properties: i) hydrophobicity (norleucine, methionine, alanine, valine, leucine, isoleucine); ii) neutral hydrophilicity (cysteine, serine, threonine, asparagine, glutamine), Acids, glutamic acid), iv) basic (histidine, lysine, arginine), iv) residues (glycine, proline) that affect the chain orientation, iii) aromatics (tryptophan, tyrosine, phenylalanine). Conservative substitutions will entail exchanging a member of one of each of these classes for another member of the same class.

By analysis of the size, shape and type of amino acid side chain substituents, arginine, lysine and histidine are all positively charged residues; Alanine, glycine and serine have similar sizes; It can be seen that phenylalanine, tryptophan and tyrosine have a similar shape. Thus, based on these considerations, arginine, lysine and histidine; Alanine, glycine and serine; Phenylalanine, tryptophan, and tyrosine are biologically equivalent functions.

In introducing variants, hydropathic idex of amino acids can be considered. Each amino acid is assigned a hydrophobicity index depending on its hydrophobicity and charge: isoleucine (+4.5); Valine (+4.2); Leucine (+3.8); Phenylalanine (+2.8); Cysteine / cysteine (+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).

The hydrophobic amino acid index is very important in conferring the interactive biological function of proteins. It is known that substitution with amino acids having similar hydrophobicity indexes can retain similar biological activity. When introducing mutations with reference to the hydrophobicity index, substitutions are made between amino acids which exhibit a hydrophobicity index difference of preferably within ± 2, more preferably within ± 1, even more preferably within ± 0.5.

On the other hand, it is also well known that substitutions between amino acids having similar hydrophilicity values result in proteins with equivalent biological activity. As disclosed in US Pat. No. 4,554,101, the following hydrophilicity values are assigned to each amino acid residue: arginine (+3.0); Lysine (+3.0); Aspartic acid (+ 3.0 ± 1); Glutamic acid (+ 3.0 ± 1); Serine (+0.3); Asparagine (+0.2); Glutamine (+0.2); Glycine (0); Threonine (-0.4); Proline (-0.5 ± 1); Alanine (-0.5); Histidine (-0.5); Cysteine (-1.0); Methionine (-1.3); Valine (-1.5); Lysine (-1.8); Isoleucine (-1.8); Tyrosine (-2.3); Phenylalanine (-2.5); Tryptophan (-3.4). When introducing mutations with reference to hydrophilicity values, substitutions are made between amino acids which exhibit a hydrophilicity value difference of preferably within ± 2, more preferably within ± 1 and even more preferably within ± 0.5.

Amino acid exchange in proteins that do not alter the activity of the molecule as a whole is known in the art (H. Neurode, R. L. Hill, The Proteins, Academic Press, New York, 1979). The most commonly occurring exchanges are amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Exchange between Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, Asp / Gly.

In general, the peptides (including fusion proteins) and polynucleotides mentioned herein are isolated. An “isolated” peptide or polynucleotide is one that is removed from its original environment. For example, proteins that exist in nature are separated by removing some or all of the substances that exist together in that state. Such polypeptides should be at least 90% pure, preferably at least 95%, more preferably at least 99% pure. Polynucleotides are isolated by cloning in a vector.

Peptides can be isolated by preparation through recombinant means or chemical synthesis. Recombinant peptides encoded by the nucleic acid sequences mentioned herein can be readily prepared by known methods using any of a number of known expression vectors. Expression can be carried out in an appropriate host cell transformed with an expression vector comprising a DNA sequence encoding the recombinant protein. Suitable host cells include prokaryotes, yeasts and eukaryotes. For example, it is preferable to use a cell line derived from a eukaryotes such as a yeast or mammalian cell line (such as Cos or CHO) as a host cell. For purification of the recombinant protein, the supernatant containing the recombinant protein secreted into the culture, first obtained in a water-soluble host / vector system, is concentrated using a commercially available filter. In the next step, the concentrate obtained above is purified using an appropriate purification matrix such as an affinity matrix or an ion exchange resin. Finally, pure recombinant protein can be obtained by performing one or several reverse phase HPLC.

Peptides or proteins described herein can be secreted into and recovered from the periplasm of the host cell. Typically, recovery of the peptides and / or proteins generally involves grinding the microorganisms by means such as osmotic shock, sonication or lysis. Once the cells are destroyed, cell debris or whole cells can be removed by centrifugation or filtration. Peptides and / or proteins can be further purified, for example, by affinity resin chromatography. Alternatively, peptides and / or proteins can be transferred to and isolated from the culture medium. The cells can be removed from the culture and the culture supernatant filtered and concentrated to further purify the peptides and / or proteins produced. Expressed polypeptides are commonly known methods such as fractional distillation on immunoaffinity or ion-exchange columns; Ethanol precipitation; Reverse phase HPLC; Chromatography on silica or cation exchange resins such as DEAE; Chromatofocusing; SDS-PAGE; Ammonium sulfate precipitation; Gel filtration using, for example, Sephadex G-75; It can be further isolated and identified using hydrophobic affinity resins, ligand affinity using suitable antigens immobilized on the matrix and Western blot assays.

In addition, the peptides produced can be purified to obtain a substantially homogeneous formulation for further assays and uses. Standard protein purification methods known in the art can be used. The following procedures are examples of suitable purification procedures: fractional distillation on immunoaffinity or ion-exchange columns, ethanol precipitation, reverse phase HPLC, silica or cation-exchange resins, such as chromatography on DEAE, chromatographic focusing, SDS-PAGE, ammonium sulfate Precipitation, and gel filtration using, for example, Sephadex G-75.

As used herein, “polynucleotide” or “nucleic acid” is used interchangeably and refers to a polymer of nucleotides of any length and encompasses DNA (such as cDNA) and RNA molecules inclusively. The “nucleotide”, the constituent unit of a nucleic acid molecule, can be incorporated into a polymer by deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or analogs thereof, or by DNA or RNA polymerase, or by synthetic reactions. It can be any substrate. Polynucleotides can include analogs with modified nucleotides, sugars or bases, such as methylated nucleotides and analogs thereof (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews , 90: 543-584 (1990).

Variations in nucleotides do not result in mutations in proteins. Such nucleic acids include functionally equivalent codons or codons that encode the same amino acid (eg, by degeneracy of the codon, six codons for arginine or serine), or codons that encode biologically equivalent amino acids. And nucleic acid molecules.

In addition, variations in nucleotides may result in changes in the protein itself. Even in the case of mutations that bring about changes in amino acids of proteins, those exhibiting almost the same activity as the proteins of the present invention can be obtained.

To the extent that the nucleic acids or polynucleotides of the present invention have a feature, for example, as an RNA vaccine and / or an adjuvant, the peptides and nucleic acid molecules of the present invention are not limited to the amino acid sequences or base sequences described in the sequence listing. It is clear to the person skilled in the art. For example, the biological functional equivalents that may be included in the recombinant protein of the present invention may be a peptide having a variation in the amino acid sequence exerting an equivalent biological activity with the recombinant protein of the present invention.

Considering the above-described variations with biologically equivalent activity, nucleic acid molecules encoding peptides and / or proteins according to the present invention are also construed to include sequences that exhibit substantial identity with the sequences set forth in the Sequence Listing. The above substantial identity is at least 61% when the sequence of the present invention is aligned as closely as possible with any other sequence, and the aligned sequence is analyzed using algorithms commonly used in the art. By a sequence that shows homology, more preferably 70% homology, even more preferably 80% homology, and most preferably 90% homology. Alignment methods for sequence comparison are known in the art.

As used herein, the term “vector” refers to a construct that is capable of delivery to a host cell and preferably enables expression of one or more desired genes or sequences. For example, a vector may include viral vectors, DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA linked to cationic condensing agents, or RNA expression vectors, DNA or RNA expression vectors packaged in liposomes, specific eukaryotic cells such as producer cells, and the like.

As used herein, "expression control / regulation sequence" or "expression control / regulation element" refers to the transcription of nucleic acids and / or translation from nucleic acid in transcript form. It can mean a nucleic acid sequence. Meanwhile, "transcription control / regulation sequence" or "trnacription control / regulation element" refers to a nucleic acid sequence that regulates transcription of a nucleic acid. Transcriptional regulatory sequences include promoters such as constitutie promoters or inducible promoters or enhancers. In addition, the term "translation control / regulation sequence" or "translation control / regulation element" is used for nucleic acid sequences that control translation from transcript-type nucleic acids to proteins or peptides. Can be. These expression control sequences / elements, transcriptional regulatory sequences / elements and / or translational regulatory sequences / elements are operatively linked to the sequence to be expressed, eg, the nucleic acid sequence to be transcribed or translated.

As used herein, the term “operatively linked” refers to a functional binding between a nucleic acid expression control sequence (eg, promoter, signal sequence, ribosomal binding site, transcription termination sequence, etc.) and another nucleic acid sequence, The regulatory sequence thereby controls the transcription and / or translation of the other nucleic acid sequence.

Nucleic acid molecule

According to an aspect of the present invention, the present invention provides an expression control sequence for expressing a gene of interest (GOI) constituting a coding region, specifically an internal ribosomal entry site (IRES) as an expression control sequence. It relates to a polynucleotide or nucleic acid molecule comprising a nucleotide sequence of). 1 is a schematic diagram showing the configuration of a polynucleotide or nucleic acid molecule capable of expressing one gene of interest located in a coding region according to an exemplary embodiment of the present invention.

As shown in FIG. 1, a nucleic acid molecule according to an exemplary embodiment of the present invention includes an expression control sequence (expressed as ECS) including a nucleotide sequence having IRES activity, and the expression control sequence (ECS). It is operably linked to and may comprise a coding region (CR) consisting of an open reading frame (ORF) of a gene of interest (GOI) that encodes a peptide or protein. In one exemplary embodiment, an expression control sequence (ECS) can form a 5'-Untranslated Region (5'-UTR) comprising an IRES sequence.

In one example, the coding region (CR) may be located on the 3 'side of the 5'-UTR comprising an expression control sequence (ECS), eg, an IRES sequence. On the other hand, the nucleic acid molecule may be DNA or RNA. In one exemplary embodiment, the nucleic acid molecule according to the invention may have an RNA form. If the nucleic acid molecule is in RNA form, the coding region (CR) may consist of the transcript sequence of the gene of interest.

The expression control sequence (ECS) includes an IRES base sequence operably linked to a gene of interest (GOI) inserted into an open reading frame, and may have a structure of 5′-UTR including an IRES base sequence. 5'-UTR is the region where the translational initiation complex associated with the translation of the gene of interest (GOI) into proteins and / or peptides is bound. IRES complexes the secondary and tertiary structures. Cis-acting base sequence that induces translation of (GOI).

In one exemplary embodiment, an expression control sequence (ECS) having an IRES base sequence may comprise an IRES base sequence derived from a virus (viral) and / or an IRES base sequence derived from a eukaryotic cell (eukaryotic). Can be. An expression control sequence (ECS) comprising a viral IRES base sequence and / or a eukaryotic IRES base sequence may have a 5'-UTR structure comprising a viral IRES base sequence and / or a eukaryotic IRES base sequence. .

In one exemplary embodiment, the viral IRES base sequence is Picornaviridae, Togaviridae, Dicistroviridae, Flaviridae, Retroviridae and / or It may be derived from the virus of the herpesviridae (Herpesviridae).

For example, the viral IRES nucleotide sequence belonging to the family of piconaviruses is a virus belonging to the genus Enterovirus, a virus belonging to the Cardiovirus, a virus belonging to the Aptovirus, and a hepattovirus genus. It may be derived from a virus belonging to (Hepatovirus) and / or a virus belonging to the genus Teschovirus (Teschovirus). In one example, the viruses belonging to the genus Enterovirus may be derived from viruses classified into enterovirus A to enterovirus J type, and rhinovirus A to rhinovirus C type.

In a non-limiting embodiment, the viral IRES base sequence belonging to the family of piconaviruses is 1) poliovirus (PV), rhinovirus (RV), cossachievirus, eg from the genus enteroviruses, eg For example, Coxsachie virus B3 (CVB3), Enterovirus 71 (Enterovirus 71, EV71), 2) Cardiovirus belonging to the cardiovirus (Encephalomyocarditis virus, EMCV), mouse encephalomyelitis virus (Theiler murine) encephalomyelitis virus (TMEV), 3) Foot-and-mouth disease virus (FMDV), 4) Hepatitis A Virus (HAV), and / or 5) Porcine teschovirus belonging to the genus Tesco virus (Porcine teschovirus, PTV, such as PTV-1) and combinations thereof, but the present invention is not limited thereto.

In another alternative embodiment, the viral IRES base sequence belonging to the Togaviraceae family may be derived from Sindbis virus (SV) belonging to the genus Alphavirus. In addition, the viral IRES nucleotide sequence belonging to the Distrovirus family is, for example, Plautia stali intestine virus (PSIV), Cricket paralysis virus (CPV) belonging to the genus Cripavirus. ), Triatoma virus, and / or Rhopalosiphum padi virus (RPXD).

On the other hand, in an exemplary embodiment, the viral IRES base sequence belonging to the Flavivirus family is hepatitis C virus (HCV) belonging to the Hepacivirus, Japan belonging to the Flavivirus. It can be derived from Japanese Encephalitis Virus (JEV), Classic swine fever virus (CSFV) belonging to Pestivirus and / or Bovine viral diarrhea virus (BVDV). . In another exemplary embodiment, the viral IRES nucleotide sequence belonging to the family of retrovirals is Friend murine leukemia virus (FMLV), Moloney murine leukemia virus, belonging to the genus Gammaretrovirus, MMLV) and / or Rous sarcoma virus (RSV) belonging to the genus Alpharetrovirus. The viral IRES base sequence belonging to the herpesvirus family may be derived from Marek's disease virus (MDV) belonging to the genus Madardivirus, but the present invention is not limited thereto.

In one example, the expression control sequence (ECS) having the viral IRES base sequence may be from a virus belonging to the family Picornaviridae and / or Dicystroiridae. In one example, an expression control sequence (ECS) having a viral IRES nucleotide sequence belonging to the family of piconaviruses may be derived from a virus belonging to the genus Enterovirus, Cardiovirus or Atovirus, and particularly preferably to the genus Enterovirus. May be derived from a belonging virus. For example, an expression control sequence (ECS) having an IRES base sequence belonging to the genus Enteroviruses may be derived from a virus of the genus Enteroviruses, such as a coxsackie virus such as CVB3, or may be derived from a virus of the genus Cardioviruses, such as EMCV.

In addition, an expression control sequence (ECS) having a viral IRES nucleotide sequence belonging to the dyscirovirus family may be derived from PSIV or CPV belonging to the genus Creepavirus.

In one exemplary embodiment, the expression control sequence (ECS) may have an IRES base sequence from a virus selected from the group consisting of SV, CVB3, EMCV, JEV, CPV, and combinations thereof, It is not limited. In one example, the expression control sequence (ECS) is a 5'-UTR (SEQ ID NO: 1) comprising an IRES base sequence derived from SV, and a 5'-UTR (SEQ ID NO: 1) comprising an IRES base sequence derived from CVB3. 2), 5'-UTR comprising an IRES nucleotide sequence derived from EMCV (SEQ ID NO: 3), 5'-UTR comprising an IRES nucleotide sequence derived from JEV (SEQ ID NO: 4) and / or 5'-UTR (SEQ ID NO: 5) comprising the IRES base sequence derived from CPV, but the present invention is not limited thereto.

In other exemplary embodiments, the eukaryotic IRES base sequence may have an IRES base sequence derived from the cellular mRNA of the eukaryotic cell. In an exemplary embodiment, the eukaryotic IRES sequence is a growth factor, transcription factors, translation factors, oncogenes, transporter / receptor, cell plan. IRES sequences related to activators of apoptosis, inhibitors of apoptosis, proteins localized in neuronal dendrites and / or other peptides / proteins Although it may include, the present invention is not limited thereto.

For example, growth factors having an IRES nucleotide sequence may include fibroblast growth factor (FGF, for example, FGF-1, FGF-2), platelet-derived growth factor-B (PDGF). -B), Vascular endothelial growth factor (VEGF), Insulin-like groth factor-2 (IGF-2), and the like.

Transcription factors with IRES sequences include antennapedia (antp), ultrarabirasax (ubx), MYT-2, NF-κB repressing factor (NRF), and acute myeloid leukemia protein. -1 (acute myelod leukemia 1 protein, AML1; Runt-related transcription factor 1, RUNX1) and the like.

Translation factors having an IRES sequence include eukaryotic initiation factor 4G (eIF4G), eukarytoic initiation factor 4GI (eIF4GI), and eukaryotic translation initiation factor 4 gamma-2 (Eukaryotic transcription). initiation factor 4 gamma 2, eIF4G2).

Cancer genes having an IRES sequence include c-myc, L-myc, and p53. Carriers / receptors having an IRES sequence include cationic amino acid transporters (eg, SLC7A1) Potassium channel (Voltage-gated potassium channel) and the like.

Cytoplasmic activators with IRES sequences include Apoptotic Activation Factor 1 (Apopa-1), and cytoplasmic inhibitors with IRES sequences include cytoplasmic X-link inhibitors. (X-linked inhibitor of apoptosis protein, XIAP) and Bcl-2. Proteins / peptides localized to dendrites of neurons include fragile X mental retardation 1 (FMR1), activity-regulated cytoskeleton-associated protein (ARC), and microtubule associations. Protein-2 (Microtubule-associated protein 2, MAP2), neuroranin (neurogranin, RC3), amyloid precursor protein (Amyloid precursor protein) and the like.

In addition, eukaryotic IRES sequences include immunoglobulin heavy chaing binding protein (BiP), connexin32, connexin43, and adenomatous polyposis coils. ARES base sequence derived from APC), Ornithine decarboxylase, Hypoxia-inducible factor 1-alpha (HIF1α), and the like.

In one exemplary embodiment, the expression control sequence (ECS) may have an IRES base sequence derived from a eukaryotic cell selected from the group consisting of eIF4G, FMR1, CN43, and combinations thereof, but the invention is not so limited. . In one example, the expression control sequence (ECS) is a 5'-UTR (SEQ ID NO: 6) comprising an IRES base sequence derived from eIF4G, and a 5'-UTR (SEQ ID NO: 1) comprising an IRES base sequence derived from FMR1. : 7) and / or 5'-UTR (SEQ ID NO: 8) comprising the IRES base sequence derived from CN43, but the present invention is not limited thereto.

IRES has a unique secondary or tertiary structure and can be classified into Group 1 to Group 4 based on the need for special translation factors required for translation and the location of the translation start codon. have. For example, the virus-derived IRES is 1) the simplest structure, and does not require translation factors in particular and does not require methyonyl-tRNA (Met-tRNA _i ), a translation initiation tRNA, from viruses such as CPV and PSIV. Group 1, 2) IIF structure, original translation initiation factor such as eIF2, eIF3 and Met-tRNA _i , HCV belonging to the genus Apthovirus, PTV-1 belonging to the genus Teschovirus, In addition to Group 2, 3) eIFs and Met-tRNA _i , which are IRES structures derived from viruses such as CSFV belonging to the Flavivirus family, IRES trans-activating factors (ITAFs) are required, and at the 3 'end of IRES The translation is initiated in the AUG codon located downstream of Group 3, 4) IRES, which is an IRES structure derived from a virus such as FMDV belonging to the genus Aptovirus or EMCV or TMEV belonging to the cardiovirs, which initiates translation. Hepatov virus irus) can be divided into Group 4, an IRES structure derived from viruses such as HAV, Enterovirus, poliovirus (PV), and rhinovirus (RV). If desired, the 5 'end of the IRES derived from some viruses may be modified to have a Cap-like structure derived from a viral protein, but the invention is not so limited.

On the other hand, the nucleic acid molecule according to the present invention may be further inserted a nucleic acid sequence that can increase the expression efficiency of the gene of interest (GOI). In one example, multiple adenosine or thymine may be inserted adjacent to the expression control sequence (ECS), which illustrates the insertion of multiple adenosine (pMA) on the 5 'side of the expression control sequence (ECS). In this case, since adenosine (pMA) represents a transcript form, the nucleic acid molecule in the DNA state is adjacent to the expression control sequence (ECS), for example, on the 5 'side of the expression control sequence (ECS). pMT) may be interpreted as being inserted. In one exemplary embodiment, 20-400, preferably 30-300, more preferably 30-200, most preferably 30-, adjacent to an expression control sequence (ECS) having an IRES base sequence. 100 consecutive adenosine or thymine can be inserted.

In this case, the amount of expression of the gene of interest (GOI) can be increased or maintained at an equivalent level compared to a nucleic acid molecule in which a plurality of adenosine or thymine is not inserted adjacent to the expression control sequence (ECS). According to one exemplary embodiment, an expression control sequence (ECS) wherein multiple adenosine or thymines can be inserted contiguously to increase or maintain the level of expression of a gene of interest (GOI) is described in the viral IRES described above. For example, it may be derived from the viruses of the Picornaviridae, the Togaviridae, the Dicistroviridae, the Flaviridae, the Retroviridae and / or the Herpesviridae. have.

On the other hand, the coding region (CR) may be composed of a nucleic acid sequence of the gene of interest (GOI) that can be expressed as a protein or peptide. The expression control sequence (ECS) may be operably linked with the coding region (CR). In one exemplary embodiment, the coding region (CR) may be located 3 'side of the expression control sequence (ECS), but the position of the coding region (CR) is not limited thereto.

In one exemplary embodiment, the coding region (CR) may consist of an open reading frame (ORF) encoding a reporter protein / peptide, a marker or a selection protein / peptide. For example, an open reading frame (ORF) of a coding region (CR) capable of encoding a reporter protein or peptide may include Renilla luciferase (eg, SEQ ID NO: 19 or SEQ ID NO: 20) and And / or luciferase, such as Firefly luciferase (eg, SEQ ID NO: 21), Green Fluorescent Protein (GFP), Enhanced Green Fluorescent Protein (EGFP), and / or Beta-galactosidase can be encoded. In addition, the open reading frame encoding the marker or select protein / peptide may comprise nucleic acid sequences encoding alpha-globin, galactokinase and / or xanthine guanine phosphoribosyl transferase and the like. However, in addition, other reporter proteins / peptides and / or open reading frames encoding markers or selection proteins / peptides may be inserted into the coding region (CR).

In another exemplary embodiment, the coding region (CR) may consist of an open reading frame that encodes an antigen, fragment, variant or derivative thereof. As antigens, for example, tumor antigens, animal antigens, vegetative antigens, viral antigens, bacterial antigens, fungal and protozoan antigens, autoimmune antigens or allergens can be considered. Secreted forms of surface antigens and surface antigens of tumor cells, in particular viral pathogens, bacterial pathogens, fungal pathogens or protozoal pathogens, are preferred.

If desired, the antigen may, for example, be present in the nucleic acid molecule according to the invention or in the form of a haptene bound to a suitable carrier. Other antigenic components, such as inactivated or attenuated pathogens, may also be used.

Specific examples of tumor antigens are, among others, tumor-specific surface antigens (TSSA), for example 5T4, 707-AP, 9D7, AFP, AlbZIP HPG1, alpha-5-beta-1-integrin, alpha-5 -Beta-6-integrin, alpha-actinine-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, CA 19-9, CA72-4, CA125, calreticulin, CAMEL, CASP-8 / m, cathepsin B, cathepsin L, CD19, CD20, CD22, CD25, CDE30, CD33, CD4, CD52, CD55, CD56, CD80, CDC27 / m, CDK4 / m, CDKN2A / m, CEA, CLCA2, CML28, CML66, COA-1 / m, coactosin-like protein, collage XXIII, COX-2, CT-9 / BRD6, Cten, cyclin B1, cyclin D1, cyp-B, CYPB1, DAM -10, DAM-6, DEK-CAN, EFTUD2 / m, EGFR, ELF2 / m, EMMPRIN, EpCam, EphA2, EphA3, ErbB3, ETV6-AML1, EZH2, FGF-5, FN, Frau-1, G250, GAGE -1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE7b, GAGE-8, GDEP, GnT-V, gp100, GPC3, GPNMB / m, HA GE, HAST-2, Hepsin, Her2 / neu, HERV-K-MEL, HLA-A * 0201-R17I, HLA-A11 / m, HLA-A2 / m, HNE, Homeobox NKX3.1, HOM-TES -14 / SCP-1, HOM-TES-85, HPV-E6, HPV-E7, HSP70-2M, HST-2, hTERT, iCE, IGF-1R, IL-13Ra2, IL-2R, IL-5, immature Laminin receptor, kallikrein-2, kallikrein-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 , Mamaglobin A, MART-1 / melan-A, MART-2, MART-2 / m, matrix protein 22, MC1R, M-CSF, ME1 / m, mesothelin, MG50 / PXDN, MMP11, MN / CA IX-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 (glucosaminyltransferase-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, PART-1, PATE, PDEF, Pim-1-kinase, Pin-1, Pml / PARalpha, POTE, PRAME, PRDX5 / m, prostein, Proteinase-3, PSA, PSCA, PSGR, PSM, PSMA, PTPRK / m, RAGE-1, RBAF600 / m, RHAMM / 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, Survivin, Survivin-2B, SYT-SSX -1, SYT-SSX-2, TA-90, TAG-72, TARP, TEL-AML1, TGFbeta, TGFbetaRII, TGM-4, TPI / m, TRAG-3, TRG, TRP-1, TRP- 2 / 6b, TRP / INT2, TRP-p8, tyrosinase, UPA, VEGF, VEGFR-2 / FLK-1 and WT1. The class of tumor antigens is suitable for tumor antigens known to be involved in neovascularization, affecting the purposes of the present invention, for example, extracellular matrix structures and the like. Tumor antigens may be provided in pharmaceutical compositions as protein or peptide antigens or as tumor antigens or epitopes thereof, preferably as mRNA or DNA encoding said tumor antigens.

For example, pathogenic antigens may be derived from pathogenic organisms that induce an immune response by an individual, for example a mammalian individual, in particular a human. In one example, the pathogenic antigen may be derived from bacterial, viral or protozoan (multicellular) pathogenic organisms. In one exemplary embodiment, the pathogenic antigens are surface antigens, eg proteins (or fragments of proteins, eg, external portions of the surface house) located on the surface of an organism such as a virus, bacteria or protozoa. Can be.

In one exemplary embodiment, the pathogenic antigens that can be encoded by the coding region (CR) are protein or peptide antigens derived from pathogens associated with infectious disease. Non-limiting examples of pathogens associated with infectious diseases include: Acinetobacter baumannii, Anaplasma genus, Anaplasma phagocytophilum, Ancylostoma braziliense , Ancylostoma duodenale, Hemolytic Acanobacterium haemolyticum, Ascaris lumbricoides, Aspergillus genus, Astroviridae, Babesia genus ), Bacillus anthracis, Bacillus cereus, Bartonella henselae, BK virus, Blastocystis hominis, Blastomyces Blastomyces dermatitidis, Bordetella pertussis, Borrelia burgdorferi, Borreli Borrelia genus, Borrelia spp, Brucella genus, Bruia malayi, Bunyaviridae family, Burkholderia cepacia and other Burkholderia cepacia Lia species, Burkholderia mallei, Burkholderia pseudomallei, Caliciviridae family, Campylobacter genus, Candida albicans, Candida Species spd, Chlamydia trachomatis, Chlamydophila pneumonia, Chlamydophila psittaci, CJD prion, CJD prion, Clolonchis sinensis Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium perfringens dium perfringens, Clostridium spp, Clostridium tetani, Coccidioides spp, Coronaviruses, Corynebacterium diphtheria, Cocciella Conetella 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, Ebolavirus (EBOV), Echinococcus genus, Erlichia Ehrlichia chaffeensis, Ehrlichia ewingii, Ehrlichia genus, Entamoeba histolytica, Enterococcus genus, Enterovirus genus terovirus genus, Enteroviruses, mainly Coxsackie A virus and Enterovirus 71 (EVox), Epidermophyton spp, Epstein-Barr Virus (EBV)), E. coli O157: H7, O111 and O104: H4 (Escherichia coli O157: H7, O111 and O104: H4), Fasciola hepatica and Fasciola gigantica, FFI prion (FFI prion), Filarioidea superfamily, Flaviviruses, Francisella tularensis, Fusobacterium genus, Geotrichum candidum ), Giardia intestinalis, Gnathostoma spp, GSS prion, Guanarito virus, Haemophilus ducreyi, Hemophil Haemophilus influenza, Helicobact Helicobacter pylori, Henipavirus (Hendra virus Nipah virus), Hepatitis A Virus, Hepatitis B Virus (HBV), Hepatitis C 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 (Hmpv), human papillomavirus ( HPV)), human parainfluenza virus (Human p) arainfluenza viruses (HPIV), Japanese encephalitis virus, JC virus, Jin virus, Junin virus, Kingella kingae, Klebsiella granulomatis, Kuru prion, Lassa virus, Legionella pneumophila, Leishmania genus, Leptospira genus, Listeria monocytogenes, Lymphoblastic meningitis Virus (Lymphocytic choriomeningitis virus (LCMV)), Machupo virus (Machupo virus), Malassezia spp, Marburg virus, Measles virus, Yokogawa insects (Metagonimus yokagawai) , Microsporidia phylum, Middle East respiratory syndrome coronavirus (MERS-CoV), Molucocum contagisum virus (Molluscum contagiosum virus (MCV)), mumps virus, Mycobacterium leprae and Mycobacterium lepromatosis, Mycobacterium tuberculosis , Mycobacterium ulcerans, Pneumonia Mycoplasma pneumonia, Naegleria fowleri, Necator americanus, Neisseria gonorrhoeae, Neisseria meningitidis meningitides ), Nocardia asteroids, Nocardia spp, Onchocerca volvulus, Orientia tsutsugamushi, Orthomyxoviridae family (Influenza), Paracoccidioides brasiliensis, Pararagonimus spp, and Pararagonimus westerma ni), Parvovirus B19, Pasteurella genus, Plasmodium genus, Pneumocystis jirovecii, Poliovirus, Rabies virus Respiratory syncytial virus (RSV), rhinovirus, rhinoviruses, Rickettsia akari, Rickettsia genus, Rickettsia prowazekii, Rickettsia rickettsia, Rickettsia typhi, Rift Valley fever virus, Rotavirus, Rubella virus, Sabia virus, Salmonella genus (Salmonella genus), Sarcoptes scabiei, SARS coronavirus, Schistosoma genus, Shigella genus, Shin Nombreva Sin Nombre virus, Hantavirus, Sporotrix rix schenckii, Staphylococcus genus, Staphylococcus genus, Streptococcus agalactia agalactiae, Streptococcus pneumonia, Streptococcus pyogenes, Strongyloides stercoralis, Taenia genus, Taenia solium ), Tick-borne encephalitis virus (TBEV), Toxocara canis or Toxocara cati, Toxoplasma gondii, Treponema pallidum, Trichy Trichinella spiralis, Trichomonas vaginalis, Trichophyton spp, Trichoris trichiura, Tripanoso Trypanosoma brucei, Trypanosoma cruzi, Ureaplasma urealyticum, Varicella zoster virus (VZV), Varicella zoster virus (VZV) ), Variola major or Variola minor, vCJD prion, Venezuelan equphalitis virus, Vibrio cholera, West Nile virus Western equine encephalitis virus, Wuchereria bancrofti, Yellow fever virus, Yersinia enterocolitica, Yersinia pestis, Ysinia pseudodo Tuberculosis (Yersinia pseudotuberculosis) and Zika virus.

Particularly preferred in this regard are influenza virus, respiratory cell fusion virus (RSV), herpes simplex virus (HSV), human papilloma virus (HPV), human immunodeficiency virus (HIV), plasmidum, Staphylococcus aureus, Dengue fever virus, Chlamydia trachomatis, cytomegalovirus (CMV), hepatitis B virus (HBV), mycobacterium tuberculosis, rabies virus, yellow fever virus, Middle East Respiratory Syndrome Coronavirus (MERS-CoV) and / or Zika virus Antigens derived from pathogens selected from.

That is, in one exemplary embodiment, the coding region (CR) is a protein and / or peptide capable of inducing an immune response in vivo, such as an antigen, ie, a gene of interest (GOI) that encodes an immune protein and / or peptide. Or a transcript sequence of a gene of interest (GOI). Accordingly, the nucleic acid molecule according to the present invention can function as a gene vaccine or the like.

According to one exemplary embodiment, the nucleic acid molecule according to the invention may have an RNA form. In this case, when the coding region (CR) has a transcript sequence of a gene of interest (GOI) that encodes a protein and / or peptide capable of inducing an immune response in vivo, such as the aforementioned pathogenic antigen, the nucleic acid molecule according to the present invention May be utilized as an RNA vaccine and injected directly in vivo. Such RNA vaccines may have the following advantages over DNA vaccines.

First, the human body is safer than DNA vaccines. RNA vaccines, by their nature, can synthesize desired peptides or proteins in the cytoplasm without having to enter the nucleus for transcription into mRNA, so RNA vaccines are superior to DNA vaccines. In other words, RNA vaccines are unlikely to penetrate into the host chromosome in the nucleus, and antibiotic resistance genes, which are screening markers used for selective production in host cells, are unnecessary for RNA vaccine production, and RNA has a short half-life compared to DNA. It does not induce long term genetic transformation. That is, a general RNA vaccine is delivered into a cell and activated for a short time to express a target peptide / protein and break down by an enzymatic reaction in a few days, but specific immune responses to the originally expressed target peptide / protein remain.

Second, RNA vaccines can induce a desired in vivo immune response even with relatively small amounts compared to DNA vaccines. Because RNA vaccines do not need to enter the nucleus, unlike DNA vaccines, they only need to pass through the cell membrane, so RNA vaccines can express the same degree of target peptide / protein even with lesser amounts than DNA vaccines. In addition, RNA itself may be utilized as an adjuvant.

Third, because all manufacturing processes can be artificially controlled, vaccines can be produced safely in small good manufacturing practice (GMP) facilities without the risk of biological contamination. In other words, RNA vaccines require only small-scale GMP laboratory-level facilities and do not have to deal directly with infectious agents (viruses or pathogenic microorganisms), enabling rapid production and production of various vaccines.

Fourth, RNA vaccines can induce stronger immune responses than naked DNA vaccines. This indicates that the innate immune response induced by the stem-loop structure and RNA itself of the RNA vaccine and the acquired immune response induced by the target peptide / protein expressed in the immunized host cell are cooperatively represented. I think. In addition, the RNA vaccine itself produces a complex antigen within the cell, which can serve as an ideal vaccine by accessing the major histocompatiblity complex (MHC) class II of antigen presenting cells.

Fifth, RNA vaccines are easier to produce than conventional vaccines. As described above, when producing an RNA vaccine, there is no need to deal directly with an infectious agent, instead artificially synthesizing only the nucleic acid sequence of the neutralizing antibody inducing part of the infectious agent to be expressed (neutralizing epitope), and in vitro transcription, IVT) alone can produce large quantities of RNA vaccines. In recent years, reagents associated with IVT reactions, particularly DNA-dependent RNA polymerases, have been improved to enable rapid production of large amounts of RNA in a week or two using small amounts of DNA templates. In addition, it is possible to immunize after mixing a plurality of antigens to induce an immune response at the same time, there is no particular restriction on the gene length of the antigen to be expressed, the applicability and simplicity of RNA vaccine production increased.

Since the nucleic acid molecule of the present invention contains an IRES base sequence as an expression control sequence (ECS), which does not require such a complicated capping process, it can solve the problems caused by the capping process, and efficiently the gene of interest (GOI). Can induce expression.

In another embodiment, a transcript sequence of a gene of interest (GOI) or a gene of interest (GOI) that encodes a protein and / or peptide, or fragment thereof, associated with disease treatment may be inserted into the coding region (CR). Accordingly, the nucleic acid molecule according to the present invention can be utilized as a gene therapy agent. In an exemplary embodiment, the protein or peptide associated with treating the disease is a therapeutic protein / peptide used to treat metabolic or endocrine diseases; Therapeutic proteins / peptides used to treat blood diseases, circulatory diseases, respiratory diseases, cancer or tumor diseases, infectious diseases or immunodeficiency; Therapeutic proteins / peptides used in hormone replacement therapy; Therapeutic proteins / peptides used to reverse differentiate somatic cells into pluripotent stem cells or pluripotent stem cells; Therapeutic proteins / peptides and / or antibodies selected from adjuvants or immune stimulating proteins. Specific examples of proteins or peptides associated with the treatment of diseases are described in Korean Patent Publication No. 2014-0125434.

In one example, the protein or peptide used in the treatment of metabolic or endocrine disease is acid sphingomyelinase, adipotide, agalsidase-beta, alglucosidase, alpha-galactosidase A, alpha-glucose Cedarase, alpha-L-eduronidase, alpha-N-acetylglucosaminidase, ampyregulin, angiopoietin (Ang1, Ang2, Ang3, Ang4, ANGPTL2, ANGPTL3, ANGPTL4, ANGPTL5, ANGPTL6, ANGPTL7 ), Betacellulin, beta-glucuronidase, bone forming protein BMP (BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP10, BMP15), CLN6 protein, epidermal growth factor (EGF) ), Epigen, epiregulin, 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-17, FGF-18, FGF-19, FGF-20, FGF-21, FGF-22, FGF-23), gallspase, ghrelin, glucocerebrosidase, GM-CSF, heparin-binding EGF-like growth factor (HB-EGF), hepatocytes Enteric Factor HGF, Hepcidin, Human Albumin, Increased Albumin Loss, Idursulfase (Eduronate-2-Sulfatase), Integrins αVβ3, αVβ5 and αVβ1, Irononate Sulfatase, Laronidase, N-acetylgalactto Samine-4-sulfatase (rhASB; galsulfase, arylsulfatase A (ARSA), arylsulfatase B (ARSB)), N-acetylglucosamine-6-sulfatase, nerve growth factor (NGF, brain derived neurotrophic factor ( BDNF), Neurotropin-3 (NT-3), Neurotropin 4/5 (NT-4 / 5), Neuroregulin (NRG1, NRG2, NRG3, NRG4), Neurophylline (NRP-1, NRP-2 ), Ovestatin, platelet-derived growth factor (PDGF (PDFF-A, PDGF-B, PDGF-C, PDGF-D), TGF beta receptors (endoglin, TGF-beta 1 receptor, TGF-beta 2 receptor, TGF- Beta 3 receptor), thrombopoietin (THPO) (megakaryotic growth and development factor (MGDF)), tumor growth factor (TGF (TGF-a, TGF-beta (TGFbeta1, TGFbeta2, and TGFbeta3)) ), VEGF (VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and PIGF), neciridide, Lysine, corticosteroids (ACTH), atrial natriuretic peptide (ANP), cholecystokinin, gastrin, leptin, oxytocin, somatostatin, vasopressin (antidiuretic hormone), calcitonin, exenatide, growth hormone (GH), somatotrot Pin, insulin, insulin-like growth factor 1 (IGF-1), mechasermin linpabate, IGF-1 analogues, mechasamine, IGF-1 analogues, pegbisomants, pramlintide, teriparatide (human parathyroid hormone residues) 1-34), becaplamine, dibotermin-alpha (bone-forming protein 2), histerin acetate (gonadotropin releasing hormone; GnRH), octreotide and palipermin (keratin cell growth factor; KGF).

Peptides or proteins used in the treatment of blood diseases, circulatory disorders, respiratory disorders, cancer or tumor diseases, infectious diseases or immunodeficiency include alteplases (tissue plasminogen activating factor; tPA), anestreplases, antithrombin III (AT-III), vivalidin, darbepoetin-alpha, drotrecogin-alpha (activated protein C), erythropoietin, epoetin-alpha, erythropoietin, erthropoietin, factor IX, factor VIIa , Factor VIII, lepirudine, protein C concentrate, reteplase (deleted mutant protein of tPA), streptokinase, tennectease, urokinase, angiostatin, anti-CD22 immunotoxin, denirukine diphthytox, immunonocyanin , MPS (metallopanstimullin), aflibercept, endostatin, collagenase, human deoxyribonuclease I, dornase, hyaluronidase, papain, L-asparaginase, Peg-asparagi Naze, Lars Buricase, Human Chorionic Gonadotropin (HCG), Human Follicle Stimulating Hormone (FSH), Lutropin-alpha, Prolactin, Alpha-1-Protease Inhibitor, Lactase, Pancreatic Enzymes (Lipase, Amylase, Protease ), Adenosine deaminase (bovine pegasadese, PEG-ADA), Avatarcept, alefacept, anakinra, etanercept, interleukin-1 (IL-1) receptor antagonist, anakinra, timulline, TNF-alpha antagonist , Enfuvirtide and thymosin α1.

In addition, human adjuvant proteins, in particular TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, which are pattern recognition receptors; NOD1, NOD2, NOD3, NOD4, NOD5, NALP1, NALP2, NALP3, NALP4, NALP5, NALP6, NALP6, NALP7, NALP7, NALP8, NALP9, NALP10, NALP11, NALP12, NALP13, NALP14, IIPAF, NAIP, CIITA, RIG- Signal transduction agents for TLR signal transduction, including adapter proteins including I, MDA5 and LGP2, such as Trif and Cardif; Components of small-GTPase signaling (RhoA, Ras, Rac1, Cdc42, Rab, etc.), components of PIP signaling (PI3K, Src-kinase, etc.), components of MyD88-dependent signaling (MyD88, IRAK1, IRAK2) , IRAK4, TIRAP, TRAF6, etc.), components of MyD88-independent signaling (TICAM1, TICAM2, TRAF6, TBK1, IRF3, TAK1, IRAK1, etc.); Activated kinases including, for example, Akt, MEKK1, MKK1, MKK3, MKK4, MKK6, MKK7, ERK1, ERK2, GSK3, PKC kinase, PKD kinase, GSK3 kinase, JNK, p38MAPK, TAK1, IKK and TAK1; For example NF-κB, c-Fos, c-Jun, c-Myc, CREB, AP-1, Elk-1, ATF2, IRF-3, IRF-7, HSP10, HSP60, HSP65, HSP70, HSP75 and HSP90 Activated transcription factors comprising a Typ III repeat extra domain A of a heat shock protein such as, gp96, fibrinogen, fibronectin; Or C1q, MBL, C1r, C1s, C2b, Bb, D, MASP-1, MASP-2, C4b, C3b, C5a, C3a, C4a, C5b, C6, C7, C8, C9, CR1, CR2, CR3, CR4 Inducible target genes comprising components of the complement system, including C1qR, C1INH, C4bp, MCP, DAF, H, I, P and CD59, or for example beta-defensin, cell surface proteins; Or trif, flt-3 ligand, Gp96 or fibronectin, IL-1 alpha, IL-1 beta, IL-2, IL-6, IL-7, IL-8, IL-9, IL-12, IL-13, Including IL-15, IL-16, IL-17, IL-18, IL-21, IL-23, TNFalpha, IFNalpha, IFNbeta, IFNgamma, GM-CSF, G-CSF, M-CSF Human adjuvant proteins, including cytokines, which induce or enhance an auditory immune response; Chemokines including IL-8, IP-10, MCP-1, MIP-1alpha, RANTES, eotaxin, CCL21; Cytokines released from macrophages, including IL-1, IL-6, IL-8, IL-12 and TNF-alpha; And IL-1R1 and IL-1alpha; Bacterial heat shock proteins or chaperones, including bacterial (adjuvant) proteins, in particular Hsp60, Hsp70, Hsp90, Hsp100; Gram-negative bacteria-derived OmpA (external membrane protein); Bacterial porins, including OmpF; Bordetella pertussis-derived pertussis toxin (PT), Bordetella pertussis-derived pertussis adenylate cyclase toxin CyaA and CyaC, pertussis toxin-derived PT-9K / 129G mutant, Bordetella pertussis Derived pertussis adenylate cyclase toxin CyaA and CyaC, tetanus toxin, cholera toxin (CT), cholera toxin B subunit, cholera toxin-derived CTK63 mutant, CT-derived CTE112K mutant, Escherichia coli heat labile toxin (LT), bacterial toxins, including reduced toxicity Escherichia coli heat labile toxin toxin mutants, including heat labile enterotoxin derived B subunit (LTB), LTK63 and LTR72; Phenol soluble modulins; Helicobacter pylori derived neutrophil activating protein (HP-NAP); Surfactant protein D; Outer surface protein A lipoprotein derived from Borrelia bergdorferi, Ag38 (38kDa antigen) derived from Mycobacterium tuberculosis; Bacterial cilia derived proteins; Intestinal toxin CT, Gram-negative Bacterial Piline, and Surfactant Protein A and Bacterial Flagellin, Protozoan (Adjuvant) Proteins in Vibrio Cholerae LeIF from Mania species, profiling-like protein derived from Toxoplasma gondi, virus (adjuvant) protein, in particular respiratory cell fusion virus fusion glycoprotein (F-protein), MMT virus-derived envelope protein, mouse leukemia virus protein, wild type measles virus Magglutinin protein, fungal (adjuvant) protein, in particular fungal immune regulatory protein (FIP; LZ-8); And a therapeutic protein selected from adjuvants or immune stimulating proteins, including keyhole limpet hemocyanin (KLH), OspA; Therapeutic proteins used in hormone replacement therapy, in particular estrogen, progesterone or progestin, and testosterone; Therapeutic proteins used to dedifferentiate somatic cells into pluripotent stem cells or pluripotent stem cells, in particular Oct-3 / 4, the Sox family of genes (Sox1, Sox2, Sox3 and Sox15), the Klf group (Klf1, Klf2, Klf4 and Klf5), Myc family (c-myc, L-myc and N-myc), Nanog and LIN28; And therapeutic antibodies selected from antibodies used for the treatment of cancer or tumor diseases, in particular 131I-tocitumomab, 3F8, 8H9, abagobomap, adecatumumab, aputuzumab, alacizumab pigol, alemtuzumab, flax Tuximab, AME-133v, AMG 102, Anatomomap Mapenatox, Apolizumab, Bobituximab, Vectumomap, Belimumab, Bevacizumab, Vivatuzumab Mertansine, Brinatomomap, Bren Tuximab bedotin, cantuzu map, cantuzu map mertansine, cantuzu map rabtansin, capromap pendetide, kalumap, katumaksomap, cetuximab, sitatuzumab bogatox, drinking water tumamap, kibatuzumap tet Laxetane, CNTO 328, CNTO 95, Konatomu Map, Dasetuzuma Map, Dalo Tzu Map, Deno Su Map, De Tomo Map, Drozito Map, Ecromexi Map, Edre Colo Map, Elotuz Map, El Silimo Map , Enabatuzu Map, Encituximab, Epratuzu Map, Ertumaksomap, Ertumakso Map, Etarasiju Map, Arm Project map, FBTA05, Piclatuz map, Piggy to mu map, Flanvo tu map, Garlic map, Garlic sea map, Gany tu map, GC1008, Gem tuju map, Gem tuju map ozogamicin, Giren tuxima map, Glembatum mu map bedotin , GS6624, HuC242-DM4, HuHMFG1, HuN901-DM1, Ibritumomab, Icrucumab, ID09C3, Indatuximab Rabtansin, Inotuzumab Ozogamicin, Intetumumab, Ipilimumab, Iratumumab, Labetuzu Map, Lexatu Map, Lintuju Map, Robotuju Map Mertansin, Lucatu Map, Rumilixi Map, Mapatumu Map, Matuju Map, MDX-060, MEDI 522, Mitumo Map, Moga Physical Map, MORab-003 , MORab-009, Moxetomomap Fasudotox, MT103, Nacolomap Tapenatox, Naptumomap Estapenatox, Narnatumap, Nesitumumab, Nimotuzumab, Nimotuzumamap, Olatuumap, Onartu Main Map, Opportuz Map Monatox, Oregovo Map, Oregovo Map, PAM4, Panitum Map, Partitum Map, Femtumo Map, Pertuzuma Map, Freetomu Map, Laco-Tumo Map, Ladre Tuma Map, Lamusi Map, Lilo-Tumuma Map, Rituxima Map, Lova-Tumuma Map, Samalizu Map, SGN-30, SGN-40, Cibro-Tzu Map, Siltu Map, Tavalu Map, Taka Tuzumate Map Tetraxetane, Tapli Tumomap Poptox, Tenatumo Map, Teprotumum Map, TGN1412, Tishlimium Map (= Tremelimu Map), Tigatum Map, TNX-650, Tossi Tomo Map, Trastuz Map , TRBS07, Tremelimumab, TRU-016, TRU-016, Tucotuzumab Selmorukin, Eublituximab, Urelumab, Beltuzumab, Beltuzumab (IMMU-106), Bolosik Map, Botumumab, WX-G250, zalutumumab and zanolimumap; Antibodies used in the treatment of immune diseases, in particular epalizumab, epratuzumab, etrolizumab, pontolizumab, ixezizumab, mepolizumab, milatuzumab, pooled immunoglobulins, priximab, rituxima Map, rontali map, luplice map, salilu map, bedoliz map, visil map, lesley map, adali mu map, asselizu map, atinuma map, ateli map, butil mu map, besiliso map, BMS- 945429, Briakinumap, Brodalumap, Canakinumap, Canakinumap, Sutolizu Map Phigol, Alliju Map, Pezakinu Map, Golimu Map, Gomilicimab Map, Infliximab Map, Mabrilimu Map, Natalie Map, Okrelic Map , Odulimo Map, Opatumu Map, Ozo Liju Map, Pexel Ju Map, Lovel Iju Map, SBI-087, SBI-087, Sekyukinu Map, Shiruku Map, Taliju Map, Tosilli Ju Map, Tali Ju Map, TRU-015 , TRU-016, Eustekinumab, Eustekinumab, Bepalimob, Zolimomap Aritox, Cipalimumap, Rumilicsi Called immune glue and the like; Antibodies used in the treatment of infectious diseases, in particular Afelimomap, CR6261, Edobarcomap, Epungumap, Exbibimap, Pelbizumab, Porabirumap, Ivalizumab, Libibimap, Motabizumab, Nevakumap , Tobiru Map, Ultoksaju Map, Bobituxi Map, Pagi Box Map, Palivivi Map, Panovaku Map, PRO 140, Lapivir Map, Laksibak Map, Legaviru Map, Sebiru Map, Defizu Map and Tapeiva Main map; Antibodies used in the treatment of blood diseases, in particular absicimab, atorolimumab, eculizumab, mepolizumab and milatuzumab; Antibodies used in immunomodulation, in particular anti-thymocyte globulin, basiliximab, cedelizumab, daclijumab, gabilimomap, inolimomap, muromonab-CD3, muromonab-CD3, odulimomap and Siplizu map; Antibodies used in the treatment of diabetes, in particular gebokizumab, otelixizumab and teplizumab; Antibodies used in the treatment of Alzheimer's disease, in particular bapinizumab, crenezumab, gantenerumab, ponezumab, R1450 and solanezumab; Antibodies used in the treatment of asthma, in particular benralizumab, enokizoomap, kelicimap, lebrikizumab, omalizumab, oxelumab, paskolizumab and tralokinumab; And therapeutic proteins, fragments, variants or the like selected from antibodies used in the treatment of various diseases, in particular blozojumab, caroRx, presolumabuma, fulanumab, lomosojumab, stamulumab, tanezumab and ranibizumab. Derivatives may be included.

The length of the open reading frame constituting the coding region (CR) is not limited, and the expression efficiency according to the length of the open reading frame (ORF) is determined by the nucleic acid molecule according to the present invention, a recombinant expression vector using the same, and a nucleic acid for treatment or prevention. This is not a big consideration in the development of vaccines. Codon usage can usually affect protein / peptide expression in various species, but human codon usage bias is not known to affect protein / peptide expression normally. Therefore, nucleic acid vaccines or gene therapies for humans can be developed. When not considered. However, the start codon must have a Kozak sequence and also need to optimize the sequences near the end codon. If necessary, the third portion of the codon sequence of the gene of interest (GOI) or transcript thereof to be expressed may be changed to GC to increase the stability of the mRNA by increasing the GC% of the target gene without changing the amino acid.

On the other hand, when the 5'-UTR including the IRES base sequence as an expression control sequence (ECS), the nucleic acid molecule of the present invention may further include a 3'-UTR in order to improve the expression efficiency of the gene of interest (GOI). have. In this case, the coding region CR may be located between 5′-UTR and 3′-UTR. The 3'-UTR, together with the 5'-UTR, improves the translational efficiency of the gene of interest (GOI) or its transcript, and plays an important role in maintaining the transcript mRNA stable without being disrupted in the cell.

In one exemplary embodiment, the 3'-UTR derived from the same virus and / or eukaryotic cell as the 5'-UTR derived from the viral and / or eukaryotic cell having the aforementioned IRES base sequence as the 3'-UTR is used. Can be. In this case, the 3'-UTR may be derived from the same or different source than the 5'-UTR having the above-described IRES.

In one exemplary embodiment, the viral 3′-UTR has a viral IRES base sequence of Picornaviridae, Togaviridae, Dicistroviridae, Flaviridae, retro It may be derived from a virus of the family Virtoviridae and / or Herpesviridae.

Viral 3'-UTR belonging to the family of piconaviruses is 1) PV, RV) derived from the enterovirus genus, Cossachievirus, for example, CVB3, EV71, 2) EMCV to Cardiovirus. , TMEV, 3) FMDV belonging to the genus Aptovirus, 4) HAV belonging to the genus Hepatovirus, and / or 5) PTVs belonging to the genus Tescovirus, for example PTV-1 and combinations thereof. This is not limited to this.

In addition, the viral 3′-UTR belonging to the Togavirus family can be derived from SV, and the viral 3-UTR belonging to the Distroviraceae family PSIV, CPV, Triatoma virus (Triatoma) belonging to the genus Cripavirus. virus), and / or RXPD. In an alternative embodiment, the viral 3′-UTR belonging to the Flavivirus family is HCV belonging to the Hepacivirus, JEV belonging to the Flavivirus, CSFV belonging to the Pestivirus and And / or from BVDV. In another exemplary embodiment, the viral 3′-UTR belonging to the Retroviraceae family may be from an RSV belonging to the FMLV, MMLV, and / or Alpharetrovirus belonging to the Gammmaretrovirus. In addition, viral 3-UTR belonging to the herpesvirus family may be derived from MDV belonging to the genus Madardivirus, but the present invention is not limited thereto.

In one exemplary embodiment, the 3'-UTR is 3'-UTR (SEQ ID NO: 10) derived from SV, 3'-UTR (SEQ ID NO: 11) derived from CVB3, 3 derived from EMCV '-UTR (SEQ ID NO: 12), 3'-UTR (SEQ ID NO: 13) derived from JEV and / or 3'-UTR (SEQ ID NO: 14) derived from CPV, but The invention is not limited to this.

In another exemplary embodiment, the 3′-UTR is a growth factor, transcription factors, translation factors, oncogenes, transporter / receptor, cell plan. May be derived from activators of apoptosis, inhibitors of apoptosis, proteins localized in neuronal dendrites and / or other peptides / proteins However, the present invention is not limited to this.

In one exemplary embodiment the 3′-UTR may be a 3′-UTR from a eukaryotic cell selected from the group consisting of eIF4G, FMR1, CN43 alc combinations thereof. In one example, the 3'-UTR is a 3'-UTR derived from eIF4G (SEQ ID NO: 15), a 3'-UTR derived from FMR1 (SEQ ID NO: 16) and / or a 3'-UTR derived from CN43. (SEQ ID NO: 17), but the present invention is not limited thereto.

Meanwhile, the nucleic acid molecule may have a transcription control control sequence (TCS) that promotes transcription of the polynucleotide adjacent to the expression control sequence. In one example, the transcriptional control sequence (TCS) may be located on the 5 'side of the expression control sequence (ECS). Such transcriptional regulatory sequences (TCS) are not particularly limited and will be described in detail in the section of recombinant expression vectors described below to avoid duplication of description.

In addition, the nucleic acid molecule may induce the expression of genes of interest (GOI) present in the coding region (ECS) in addition to the expression control sequences (ECS), coding region (CR), 3′-UTR and transcriptional control sequences (TCS) described above. Nucleic acid sequences can be inserted. In one exemplary embodiment, a Kozak sequence may be inserted between an expression control sequence having an IRES base sequence (eg, 5'-UTR having an IRES base sequence) and an initiation codon of a coding region (CR). If necessary, the downstream hairpin structur (DLP) may be located 3 'side of the expression control sequence (ECS). For example, when the 5'-UTR derived from SV (SEQ ID NO: 1) is applied as an expression control sequence (ECS), the DLP nucleic acid sequence derived from SV (e.g., SEQ ID NO: 9) is selected from the 5'-UTR. It can be inserted between coding regions.

In another alternative embodiment, a base sequence (eg, CCTGCT) is inserted into the 3 'side of the expression control sequence (ECS) and / or inserted into the 3' side of the expression control sequence (ECS). A recognition sequence (eg, ATGGCAGCTCAA) and the like, which can improve the expression efficiency of GOI), may be inserted.

In addition, the gene of interest (GOI) present in the coding region (CR) is stabilized while stabilizing the nucleic acid molecule transcribed onto the 3 'side of the coding region (CR), if the 3'-UTR is used. ), A polyadenosine (poly (A)) sequence can be further inserted which can further improve the translation efficiency of In one example, the poly (A) sequence is a nucleic acid sequence consisting of approximately 25 to approximately 400, preferably 30 to 400, more preferably 50 to 250, most preferably 60 to 250 adenosine nucleotides Can be.

Meanwhile, FIG. 1 illustrates a nucleic acid molecule including one expression control sequence and one coding region. In other embodiments, the nucleic acid molecules of the invention will comprise a plurality of expression control sequences having an IRES base sequence and a plurality of coding regions encoding proteins and / or peptides that can be expressed by either expression control sequence. Can be. 2 schematically shows a nucleic acid molecule comprising a plurality of expression control sequences and a plurality of coding regions.

As shown in FIG. 2, a nucleic acid molecule according to another embodiment of the present invention includes a first expression control sequence (ECS 1; eg, 5′-UTR 1) having an IRES base sequence, and a first expression control sequence (ECS). And a second expression control sequence (ECS2; eg, 5′-UTR 2) located on the 3 ′ side of 1). In one exemplary embodiment, the first expression control sequence (ECS 1) and the second expression control sequence (ECS 2) are each 5 ′ having the viral IRES base sequence and / or eukaryotic IRES base sequence described above. May be -UTR. In this case, the first expression control sequence (ECS 1) and the second expression control sequence (ECS 2) may be from the same source or may be from different sources.

Meanwhile, a nucleic acid molecule according to an exemplary embodiment of the present invention is adjacent to, for example, a plurality of expression control sequences (ECS 1) of at least one of the plurality of expression control sequences (ECS 1, ECS 2). , Adenosine or thymine sequence is inserted on the 5 'side of the expression control sequence of at least one of ECS 2). Figure 2 shows the insertion of a number of adenosine or thymine sequences on the 5 'side of the first expression control sequence (ECS 1) located proximal to the transcriptional control sequence (TCS). However, a number of adenosine or thymine sequences that can improve the expression efficiency of genes of interest (GOI 1, GOI 2) may be inserted on the 5 'side of the second expression control sequence (ECS 2), or the first expression control sequence ( Both ECS 1) and the second expression control sequence (ECS 2) may be inserted into the 5 'side.

In one exemplary embodiment, at least one of the first expression control sequence (ECS 1) and the second expression control sequence (ECS 2) may comprise an IRES base sequence derived from a virus. More specifically, the first expression control sequence (ECS 1) and / or the second expression control sequence (ECS 2) may be the piconaviridae family, Togaviridae family, the Dicistroviridae family, the Flavivirus family family. (Flaviridae), Retroviridae and / or Herpesviridae virus may include the IRES base sequence from.

In other alternative embodiments, the first expression control sequence (ECS 1) and / or the second expression control sequence (ECS 2) may have an IRES base sequence derived from a eukaryotic cell. Since the viral IRES base sequence, the type of eukaryotic IRES base sequence, and the like overlap with those described with reference to FIG. 1, detailed descriptions thereof will be omitted.

In one exemplary embodiment, the first expression control sequence (ECS 1) and / or the second expression control sequence (ECS 2) having the viral IRES base sequence is selected from the family of Picornaviridae and / or dysciroviruses ( It may be derived from a virus belonging to (Dicistroviridae). In one example, at least one of the first / second expression control sequences (ECS 1 / ECS 2) having a viral IRES nucleotide sequence belonging to the family of piconaviruses is in a virus belonging to the genus Enterovirus, Cardiovirus or Aptovirus And particularly preferably from viruses belonging to the genus Enteroviruses. For example, at least one of the first / second expression control sequences (ECS 1 / ECS 2) having an IRES base sequence belonging to the genus Enterovirus is derived from a virus of the genus Enteroviruses such as coxsackie virus such as CVB3, or It can be derived from viruses of the same cardiovirus.

In addition, at least one of the first / second expression control sequences (ECS 1, ECS 2) having a viral IRES base sequence belonging to the dyscirovirus family may be derived from PSIV or CPV belonging to the genus Creepavirus.

On the other hand, in an exemplary embodiment, the coding region is a first coding region (CR 1) located between the first expression control sequence (ECS 1) and the second expression control sequence (ECS 2) and the second expression control It may comprise a second coding region (CR 2) located on the 3 'side of the sequence (ECS 2). For example, the first coding region CR 1 and the second coding region CR 2 may be formed as an open reading frame of the first gene of interest GOI 1 and the second gene of interest GOI 2, respectively. The first coding region GOI 1 and the second coding region GOI 2 each encode a reporter protein / peptide, a marker or identification protein / peptide, an antigen and / or a protein / peptide associated with disease treatment, or the like, respectively. It can consist of an open reading frame (ORF).

According to one exemplary embodiment, the first coding region (CR 1) is operably linked with a first expression control sequence (ECS 1; eg 5′-UTR 1) and the second coding region (CR 2) May be operably linked with a second expression control sequence (ECS 2; eg, 5′-UTR 2).

The first gene of interest GOI 1 constituting the first coding region CR 1 and the second gene of interest GOI 2 constituting the second coding region CR 2 are each open to encode different proteins or peptides. It may consist of a reading frame. In this case, one protein can express different proteins or peptides. In one exemplary embodiment, when the first coding region CR 1 and the second coding region CR 2 each consist of an open reading frame encoding different antigens or fragments thereof, the nucleic acid molecule according to the invention or Recombinant expression vectors can be used to express different antigens and can be utilized as genetic vaccines to prevent a number of diseases.

In another exemplary embodiment, when the first coding region CR 1 and the second coding region CR 2 each consist of an open reading frame encoding different therapeutic proteins or peptides, the nucleic acid molecule or recombinant according to the invention. Expression vectors can be used to treat different diseases.

Similar to that described in FIG. 1, a nucleic acid molecule comprising a plurality of expression control sequences (ECS 1, ECS 2) and a plurality of coding regions (CR1, CR 2) shown in FIG. 2 may be a gene of interest (GOI 1, GOI 2). It may further comprise a nucleic acid sequence capable of improving the expression. In one example, if the first expression control sequence (ECS 1) and / or the second expression control sequence (ECS 2) are each a viral or eukaryotic 5'-UTR having an IRES base sequence, the nucleic acid sequence is 3'-. It may further comprise a UTR. In this case, the 3'-UTR may be preferably located on the 3 'side of the second coding region CR 2. The 3'-UTR is not particularly limited, but viral and / or eukaryotic 3'-UTR as described with reference to FIG. 1 may be used.

For example, the 3'-UTR may be derived from a 5'-UTR 2 which may be a 5'-UTR 1 or a second expression control sequence (ECS 2) which may be a first expression control sequence (ECS 1). have. In one exemplary embodiment, the 3′-UTR may use one derived from the same source as the 5′-UTR 1, but the invention is not so limited.

In addition, the nucleic acid molecules shown in FIG. 2 are operable to the transcriptional control sequences (TCE) located adjacent to the expression control sequences (ECS 1, ECS 2), and each of the expression control sequences (ECS 1, ECS 2), respectively. Kozak sequences between linked coding regions (CR 1, CR 2). In addition, if necessary, 3LP's of each expression control sequence (ECS 1, ECS 2) may be inserted with a DLP nucleic acid sequence, a sequence which may act as an initiation codon, and / or a recognition sequence which may improve expression efficiency. . Also, in one exemplary embodiment, the poly (A) sequence can be further inserted on the 3 'side of the second coding region (CR 2), eg 3'-UTR if the 3'-UTR is inserted. have.

In FIG. 2, the expression control sequences (ECS 1, ECS 2) and the coding regions (CR 1, CR 2) are respectively divided into two, but the number and / or expression of expression control sequences having an IRES base control sequence The number of coding regions operably linked with regulatory sequences may be greater than two.

Recombinant Expression Vectors and Applications

The nucleic acid molecules shown in FIGS. 1 and 2 can be inserted into a vector. Vectors also include expression control sequences, eg, transcriptional control sequences, linked to nucleic acid molecules of the invention. In embodiments of the invention, the vector may comprise a nucleic acid molecule encoding a protein or peptide of interest. In this case, the nucleic acid molecule may bind to another nucleic acid to encode a fusion protein or a fusion peptide. Accordingly, according to another aspect of the present invention, the present invention relates to a recombinant expression vector into which the nucleic acid molecule of the present invention is inserted.

For example, a vector may include viral vectors, DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA linked to cationic condensing agents, or RNA expression vectors, DNA or RNA expression vectors packaged in liposomes, specific eukaryotic cells such as producer cells, and the like.

Illustratively, the nucleic acid molecules of the present invention are formulated to enter into and express in mammalian cells. Such compositions are particularly useful for use for therapeutic purposes. There are many ways to express nucleic acid molecules in host cells, and any suitable method can be used. For example, the nucleic acid molecule according to the present invention may be adenovirus, adeno-associated virus, retrovirus, vaccinia or other pox virus (eq, avian pox virus). It can be inserted into such viral vectors.

According to one exemplary embodiment, the above-described nucleic acid molecule may be inserted into an appropriate vector and then modified into nucleic acid molecules in RNA form through in vitro transcription (IVT).

Techniques for inserting nucleic acid molecules such as DNA into such vectors are well known. Retroviral vectors include targeting sites such as genes for selectable markers that facilitate the identification or selection of transduced cells and / or genes that encode ligands that act as receptors for specific target cells. can additionally insert a targeting moiety. Targeting can also be accomplished by known methods using antibodies.

Many vectors available and known in the art can be used for the purposes of the present invention. Selection of the appropriate vector will depend primarily on the size of the nucleic acid molecule inserted into the vector and the particular host cell transformed with the vector. Each vector contains various components depending on its function (amplification or expression of heterologous polynucleotides, or both) and compatibility with the particular host cell in which the vector is present. Vector components generally include, but are not limited to, origin of replication (particularly when the vector is inserted into prokaryotic cells), selectable marker genes, promoters, ribosomal binding sites (RBS), signal sequences, heterologous nucleic acid inserts and transcription termination sequences. .

For example, recombinant expression vectors according to the present invention may be used for expression control sequences that may affect the expression of proteins or peptides, such as initiation codons, termination codons, polyadenylation signals, enhancers, membrane targeting or secretion. Signal sequence and the like. Polyadenylation signals increase the stability of transcripts or facilitate cellular transport. Enhancer sequences are nucleic acid sequences that are located at various sites in the promoter and increase transcriptional activity as compared to the transcriptional activity by the promoter in the absence of the enhancer sequence. The signal sequence includes PhoA signal sequence and OmpA signal sequence when the host is Escherichia spp., And α-amylase signal sequence and subtilisin signal sequence when the host is Bacillus sp . In the case of the yeast, MF-α signal sequence, SUC2 signal sequence, etc., if the host is an animal cell, insulin signal sequence, a-interferon signal sequence, antibody molecular signal sequence, etc. may be used, but the present invention is not limited thereto. Do not.

One type of vector is a “plasmid” that refers to a circular double stranded DNA loop into which additional DNA segments can be ligated therein. Another type of vector is a phage vector. Another type of vector is a viral vector in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors and episomal mammalian vectors with bacterial origins of replication). Other vectors (eg, non-episomal mammalian vectors) can be integrated into the genome of the host cell upon introduction into the host cell and thereby replicate with the host genome. In addition, certain vectors may direct expression of the gene to which the vector is operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "recombinant vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.

In a specific embodiment of the invention, the nucleic acid molecule is inserted into a host cell using a virus expression system (vaccinia or other pox virus, retrovirus or adenovirus). Exemplary viral vectors include retroviral vectors derived from HIV, SIV, murine retroviruses, gibbon ape leukemia virus, adeno-associate viruses, and adenoviruses, and the like. But not limited thereto (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). Retroviruses derived from rodent leukemia virus (MulVe), basic leukemia virus (GIBBON ape leukemia virus, GaLV), ecotropic retroviruses, simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), etc. Vectors 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, WE Paul (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).

The vector system according to the present invention can be constructed through various methods known in the art, and specific methods thereof are disclosed in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001). This document is incorporated herein by reference.

For example, a vector of the present invention can typically be constructed as a vector for cloning or a vector for expression. In addition, the vector of the present invention can be constructed using prokaryotic or eukaryotic cells as hosts. The nucleic acid molecule of the present invention is derived from prokaryotic cells, and it is preferable to use prokaryotic cells as a host in consideration of the convenience of culture. Vectors of the present invention can typically be constructed as vectors for cloning or vectors for expression. For example, vectors that can be used in the present invention include plasmids often used in the art (eg pSC101, ColE1, pBR322, pUC8 / 9, pHC79, pUC19, pET, etc.), phage (eg λgt4λB, λ-Charon , λΔz1, λGEM.TM.-11 and M13, etc.) or viruses (eg SV40, etc.).

Constitutive or inducible promoters can be used in the present invention depending on the needs of a particular situation that can be identified by one skilled in the art. Many promoters that are recognized by a variety of possible host cells are well known. The selected promoter is a nucleic acid molecule having a coding region (CR) consisting of an open reading frame (ORF) of the appropriate gene of interest (GOI) by removing the promoter from the source nucleic acid molecule via restriction enzyme digestion and inserting the isolated promoter sequence into the selection vector. Can be operatively connected. Both native promoter sequences and multiple heterologous promoters can be used to direct amplification and / or expression of the gene of interest (GOI). However, heterologous promoters are generally preferred because they allow for greater transcription and higher yield of the expressed gene of interest as compared to the native promoter.

For example, when the vector of the present invention is an expression vector and the prokaryotic cell is a host, a strong promoter capable of promoting transcription (for example, a tac promoter, a lac promoter, a lacUV5 promoter, an lpp promoter, a pLλ promoter, a pRλ promoter) , rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter, etc.), expression control sequences (ECS) having an IRES to initiate translation, and transcription / translation termination sequences. 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 leftward promoter of phage λ (pLλ) Promoter, Herskowitz, I. and Hagen, D., Ann. Rev. Genet., 14: 399-445 (1980)) can be used as regulatory sites.

On the other hand, when the vector of the present invention is an expression vector and the eukaryotic cell is a host, a promoter derived from the mammalian cell genome (for example, metallothionine promoter) or a promoter derived from a mammalian virus (for example, adeno) Late viral promoters, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter and tk promoter of HSV) or promoters derived from bacteriophages (e.g., T7 promoter, T3 promoter, SM6 promoter) can be used and transcription termination It generally has a polyadenylation sequence as a sequence.

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

Various in vitro amplification techniques are known for amplifying a subcloned sequence in an expression vector. Such techniques include polymerase chain reaction (PCR), ligand chain reaction (LCR), Qβ-replicase amplification, and other RNA polymerases (Sambrook et al., 1989, Molecular Cloning-A Laboratory Manual). (2nd Ed) 1-3; US 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 US Patent No. 5,426,039.

The present invention also provides a host cell transformed with the recombinant expression vector of the present invention. Transformed host cells can be used in the methods of producing proteins or peptides in accordance with the present invention. The method includes culturing the host cell and recovering the produced protein or peptide. The recovered protein or peptide can be purified from the culture supernatant.

The present invention also provides a recombinant microorganism genetically modified to express a protein or peptide from an open reading frame constituting the coding region of a nucleic acid molecule described above. The recombinant microorganism may be used as a vaccine or gene therapy agent. In one example, the recombinant microorganism can be prepared according to the method for producing live attenuated vaccines.

Vectors of the invention may be fused with other sequences to facilitate purification of the recombinant protein or peptide expressed therefrom. Sequences to be fused include, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA) and 6x His (hexahistidine; Quiagen, USA), and most preferably Is 6 × His. Because of the additional sequence for this purification, the protein expressed in the host is purified quickly and easily through affinity chromatography. If desired, sequences encoding Fc fragments may be fused to facilitate extracellular secretion of these recombinant proteins.

According to a preferred embodiment of the present invention, 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, which is a substrate of this enzyme, can be used, and when 6x His is used, a desired recombination is performed using a Ni-NTA His-binding resin column (Novagen, USA). Proteins can be obtained quickly and easily.

Host cells capable of stably and continuously cloning and expressing the aforementioned vectors are known in the art and can be used with any host cell, for example E. coli JM109, E. coli BL21 (DE3), E. coli RR1, Bacillus sp. Strains such as E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus thuringiensis, and Salmonella typhimurium, Serratia marsonsons, and various Pseudomonas species Enterobacteria and strains, such as. In addition, when transforming a vector of the present invention into eukaryotic cells, as host cells, yeast ( Saccharomyce cerevisiae ), insect cells (such as SF9 cells) and human cells (such as CHO cell lines (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cell lines) and the like.

The vectors of the present invention can be used to genetically modify cells in vitro, ex vivo or in vitro. Methods for genetically modifying cells include infection or transduction of cells with viral vectors, calcium phosphate precipitation, bacterial protoplasts containing DNA of receptor cells. Fusion methods, treatment of liposomes or microspheres containing DNA in receptor cells, DEAE dextran, receptor-mediated endocytosis, electroporation, microinjection Various methods are known, including micro-injection.

For example, if the host cell is a prokaryotic cell, the CaCl ₂ method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9: 2110-2114, 1973), one method (Hanahan, D., J. Mol. Biol., 166: 557-580, 1983) and / or electroporation methods (Dower, WJ et al., Nucleic. Acids Res., 16: 6127-6145, 1988) and the like. . In addition, when the host cell is a eukaryotic cell, micro-injection (Capecchi, MR, Cell, 22: 479, 1980), calcium phosphate precipitation method (Graham, FL et al., Virology, 52: 456, 11973), electricity Perforation (Neumann, E. et al., EMBO J., 1: 841, 1982), liposome-mediated transfection (Wong, TK et al., Gene, 10:87, 1980), DEAE-dextran treatment (Gopal, Mol. Cell Biol., 5: 1188-1190, 1985), and gene balm (Yang et al., Proc. Natl. Acad. Sci., 87: 9568-9572, 1990) and the like. May be injected into a host cell.

Vectors injected into a host cell can be expressed in the host cell, in which case a large amount of recombinant protein or recombinant peptide is obtained. For example, when the expression vector contains a lac promoter, the host cell may be treated with IPTG to induce gene expression.

According to another aspect of the present invention, the present invention provides a pharmaceutical composition as a nucleic acid vaccine comprising a pharmaceutically effective amount of the above-described nucleic acid molecule and a pharmaceutically acceptable carrier, if appropriate, appropriate adjuvant. In this case, the nucleic acid molecule described above is administered directly to the subject.

According to another aspect of the present invention, the present invention provides a gene carrier comprising a nucleic acid molecule of the present invention described above; And it provides a pharmaceutical composition as a therapeutic agent for diseases comprising a pharmaceutically acceptable carrier. This is for the purpose of gene therapy.

Pharmaceutical compositions (nucleic acid vaccines and / or gene therapeutics) comprising a nucleic acid molecule or a gene carrier in which a nucleic acid molecule is inserted are preferably administered systemically. Routes of administration of such pharmaceutical compositions / vaccines are typically oral, subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal , Intrahepatic, intralesional, intracranial, transdermal, intradermal, intrapulmonal, intraperitoneal, intracardial, intraarterial ), Sublingual topical and / or intranasal routes. Optionally, the vaccine or pharmaceutical composition of the present invention may be administered by intradermal, subcutaneous or intramuscular routes. Thus, pharmaceutical compositions / vaccines are generally formulated preferably in liquid or solid form as defined above for pharmaceutical compositions.

If necessary, an adjuvant may be included in the pharmaceutical composition to enhance the immunogenicity of the vaccine. Adjuvants can be incorporated into the vaccine according to the invention. Auxiliary components such as adjuvant are selected according to immunogenicity and other properties of the pharmaceutically active component of the vaccine according to the present invention.

Gene carriers, such as nucleic acid molecules or recombinant expression vectors of the invention, may be any suitable means, for example oral, topical (including buccal and sublingual), rectal, vaginal, transdermal, parenteral, subcutaneous, intraperitoneal, intrapulmonary, intradermal, Intradural and epidural and intranasal, and if desired, by topical treatment, by means for intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration.

The pharmaceutical compositions according to the invention can be administered in any convenient dosage form, for example tablets, powders, capsules, solutions, dispersions, suspensions, syrups, sprays, suppositories, gels, emulsions, patches and the like. Such compositions may contain components conventional to pharmaceutical formulations, such as diluents, carriers, pH adjusters, sweeteners, bulking agents and additional active agents.

Typical formulations are prepared by mixing the nucleic acid molecules or gene carriers of the invention with a carrier or excipient. Suitable carriers and excipients are well known to those skilled in the art and are described, for example, in Ansel, Howard C., et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems. Philadelphia: Lippincott, Williams & Wilkins, 2004; Gennaro, Alfonso R., et al. Remington: The Science and Practice of Pharmacy. Philadelphia: Lippincott, Williams & Wilkins, 2000; And Rowe, Raymond C. Handbook of Pharmaceutical Excipients, Chicago, Pharmaceutical Press, 2005.

For example, pharmaceutically acceptable carriers are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline Sex cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like.

The formulations also contain one or more buffers, stabilizers, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, opacifiers, glidants, processing aids, colorants, sweeteners, flavorings, flavoring agents, diluents, and drugs (Ie, nucleic acid molecules, gene carriers or pharmaceutical compositions thereof, which are the active ingredients of the present invention) or may contain other known additives to aid in the manufacture of pharmaceutical products (ie, medicaments).

The pharmaceutical compositions of the present invention may be prepared in unit dosage form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporating into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or an aqueous medium, or may be in the form of extracts, powders, granules, tablets or capsules, and may further include a dispersant or stabilizer.

On the other hand, the formulations herein may also contain more than one active compound, preferably compounds with complementary activities which do not adversely affect each other when necessary for the particular indication to be treated. Alternatively or additionally, the composition may comprise agents that enhance its function, such as, for example, cytotoxic agents, cytokines, chemotherapeutic agents, or growth-inhibiting agents, or growth-enhancing agents. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.

In addition, gene carriers comprising a nucleic acid molecule of the invention comprising a coding region encoding a gene of interest may be included in a pharmaceutical composition. The gene carrier is designed to transport and express the nucleic acid molecule of interest, and details of the nucleic acid molecule of interest as a carrier object are omitted in order to avoid overlapping with the contents described above.

To prepare a gene carrier, the transcript of the gene of interest is preferably present in a suitable expression construct. In the expression construct, the transcript of the gene of interest is preferably operably linked to a promoter. In the present invention, a promoter bound to a transcript of a gene of interest is preferably capable of controlling transcription of a nucleic acid molecule of interest by operating in an animal cell, more preferably a mammalian cell, which is derived from a mammalian virus. Promoters, promoters derived from the genome of mammalian cells, or promoters derived from bacteriophages. For example, CMV (cytomegalo virus) promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, tk promoter of HSV, T7 promoter, T3 promoter, SM6 promoter, RSV promoter, EF1 alpha promoter, metallothio Nin promoter, beta-actin promoter, promoter of human IL-2 gene, promoter of human IFN gene, promoter of human IL-4 gene, promoter of human lymphotoxin gene, promoter of human GM-CSF gene, cancer cell specific promoter ( Such as, but not limited to, TERT promoter, PSA promoter, PSMA promoter, CEA promoter, E2F promoter and AFP promoter) and tissue specific promoters (eg, albumin promoter). Preferably, the expression construct used in the present invention comprises a polyaninylation sequence (eg, a glial growth hormone terminator and a SV40 derived poly adenylation sequence).

For example, where a nucleic acid molecule according to the present invention is intended to be utilized as an RNA vaccine, an appropriate transcriptional regulatory sequence that enables IVT can optionally be located on the 5 'side of the expression regulatory sequence (ECE). As described above, the nucleic acid molecule in the form of RNA that can be utilized as an RNA vaccine, etc. can be synthesized through the IVT process, there is no need to deal directly with live viruses or pathogenic microorganisms used for the production of common live or dead vaccines, There is no need for culturing host cells such as yeast, E. coli, insect cells, etc., which must be used to produce recombinant proteins / peptides.

For example, in order to prepare an RNA vaccine using a nucleic acid molecule according to the present invention, when a polynucleotide inserted into a plasmid in the form of DNA is transcribed into an mRNA form through an IVT process, the terminal is transferred to a restriction enzyme or the like and linearized. RNA is synthesized in vitro by RNA polymerase using DNA as a template. For transcription from linearized DNA to RNA, transcriptional regulatory sequences, such as, for example, promoter sequences derived from bacteriophages and the like, may be located on the 5 'side of the 5'-UTR. In addition to the promoter derived from T7 bacteriophage as such a transcriptional regulatory sequence, any transcriptional regulatory sequence (TES) capable of transcription of a mRNA from a linearized DNA template, such as a promoter derived from T3 bacteriophage or a promoter derived from SP6 bacteriophage, is expressed as an expression control sequence (ECS). It can be located nearby.

Gene carriers can be produced in a variety of forms, such as plasmids, viral vectors, or in the form of liposomes or niosomes containing plasmids. In one example, the transcript of the gene of interest can be applied to all gene delivery systems used in conventional gene therapy, preferably plasmids, adenoviruses (Lockett LJ, et al., Clin. Cancer Res. 3: 2075-2080). , 1997)), Adeno-associated viruses (AAV, Lashford LS., Et al., Gene Therapy Technologies, Applications and Regulations Ed.A. Meager, 1999), retroviruses (Gunzburg WH, et al. , Retroviral vectors.Gene Therapy Technologies, Applications and Regulations Ed.A. Meager, 1999), lentiviruses (Wang G. et al., J. Clin. Invest. 104 (11): R55-62 (1999)), herpes Simplex virus (Chamber R., et al., Proc. Natl. Acad. Sci USA 92: 1411-1415, 1995), vaccinia virus (Puhlmann M. et al., Human Gene Therapy 10: 649-657, 1999 )), Liposomes (Methods in Molecular Biology, Vol 199, SC Basu and M. Basu (Eds.), Human Press 2002) or niosomes.

On the other hand, the method of introducing the above-described gene carrier into the cell can be carried out through various methods known in the art. In the present invention, when the gene carrier is produced based on a viral vector, it is carried out according to a virus infection method known in the art. Infection of host cells with viral vectors is described in the references cited above.

In addition, in the present invention, when the gene delivery system is a naked recombinant DNA molecule or plasmid, micro-injection method (Capecchi, M.R., Cell, 22: 479, 1980); And Harland and Weintraub, J. Cell Biol. 101: 1094-1099, 1985)), calcium phosphate precipitation (Graham, F.L. et al., Virology, 52: 456, 1973); And Chen and Okayama, Mol. Cell. Biol. 7: 2745-2752, 1987), electroporation (Neumann, E. et al., EMBO J., 1: 841, 1982; and Tur-Kaspa et al., Mol. Cell Biol., 6: 716-718 (1986)), liposome-mediated transfection (Wong, TK et al., Gene, 10:87, 1980); Nicolau and Sene, Biochim. Biophys. Acta, 721: 185-190, 1982; And Nicolau et al., Methods Enzymol., 149: 157-176, 1987)), DEAE-dextran treatment (Gopal, Mol. Cell Biol., 5: 1188-1190, 1985)), and gene bombardment ( Yang et al., Proc. Natl. Acad. Sci., 87: 9568-9572, 1990)) can be used to introduce the gene into the cell.

EMBODIMENT OF THE INVENTION Hereinafter, although this invention is demonstrated through exemplary embodiment, this invention is not limited to the technical idea described in the following Example.

Example 1: Nucleic Acid Molecular Construction of RNA Platforms

An RNA polynucleotide having a 5'-UTR having an IRES derived from Sindbis virus was prepared. The template DNA sequence was designed as follows.

5'- restriction enzyme recognition sequence ( GGATCC ) -T7 promoter (SEQ ID NO: 18)-5'-UTR (SEQ ID NO: 1) having an IRES from SV-SV DLP structure sequence (SEQ ID NO: 9) - restriction enzyme recognition sequence (GGATCC) Kozak sequence (GACC) Renilla Luciferase (R / L) encrypted ORF sequences as GOI gene (SEQ ID NO: 19) restriction enzyme recognition sequence (GAATTC) of the SV-derived 3 ' UTR (SEQ ID NO: 10)-Polyadenosine Sequence (50 Adenosines) -Restriction Enzyme Recognition Sequence ( GCGGCCGC ).

The template DNA was cloned into a pGH vector and linearized with restriction enzymes to obtain nucleic acid molecules in the form of RNA platforms through in vitro trasncrption (IVT). A nucleic acid molecule in the form of an RNA platform prepared according to the present example is named 'pnon-SV-R / L' below.

Example 2: Nucleic Acid Molecular Construction of RNA Platforms

As the 5'-UTR of the template DNA, a 5'-UTR (SEQ ID NO: 2) having an IRES derived from CVB3 having no DLP structure sequence was used, and a sequence capable of improving expression efficiency on the 3 'side of the 5'-UTR ( ATGGCAGCTCAA) was inserted, the restriction enzyme recognition sequence ( CCGCGG CGATCG) was inserted at 3 'of ORF, and 3'-UTR derived from CVB3 (SEQ ID NO: 11) was used as 3'-UTR, and 5'-UTR was used. The nucleic acid molecules of the RNA platform were prepared by repeating the procedure of Example 1 except that capping was carried out using anti-reverse cap analog (ARCA) at 5 '. A nucleic acid molecule in the form of an RNA platform prepared according to this example is named 'pCAP-CVB3-R / L'.

Example 3: Nucleic Acid Molecular Construction of RNA Platforms

A 5'-UTR (SEQ ID NO: 3) having an IRES from EMCV without a DLP structure sequence was used as the 5'-UTR of the template DNA, and a restriction enzyme recognition sequence ( GTCGAC CGATCG) was inserted into 3 'of the ORF. Nucleic acid molecules of the RNA platform were prepared by repeating the procedure of Example 1, except that 3'-UTR (SEQ ID NO: 12) derived from EMCV was used as the 3'-UTR. The nucleic acid molecule in the form of an RNA platform prepared according to the present embodiment is named 'pEMCV-R / L' below.

Example 4: Nucleic Acid Molecular Construction of RNA Platforms

A 5'-UTR (SEQ ID NO: 4) having an IRES from JEV without a DLP structure sequence was used as the 5'-UTR of the template DNA, and a restriction enzyme recognition sequence ( GTCGAC CGATCG) was inserted into 3 'of the ORF. 3'-UTR derived from JEV (SEQ ID NO: 13) as 3'-UTR, except that 5'-UTR's capping using anti-reverse cap analog (ARCA) The procedure of Example 1 was repeated to prepare nucleic acid molecules of the RNA platform. A nucleic acid molecule in the form of an RNA platform prepared according to this Example is named 'pCAP-JEV-R / L' below.

Example 5: Nucleic Acid Molecular Construction of RNA Platforms

A 5'-UTR (SEQ ID NO: 5) having an IRES derived from CPV without a DLP structure sequence was used as the 5'-UTR of the template DNA, and the sequence acted as a start codon of CPV on the 3 'side of the 5'-UTR. (CCTGCT) was inserted, 3'-UTR derived from CPV as 3'-UTR without using the R / L coding ORF sequence of SEQ ID NO: 20 as the GOI gene, and the restriction enzyme recognition sequence inserted into the 3 'side of the ORF. The nucleic acid molecules of the RNA platform were prepared by repeating the procedure of Example 1 except using -UTR (SEQ ID NO: 14). A nucleic acid molecule in the form of an RNA platform prepared according to the present example is named 'pCPV-R / L' below.

Comparative Example 1 Construction of Nucleic Acid Molecules on an RNA Platform

Nucleic acid molecules of the cap-dependent RNA platform disclosed in WO 2015/101414 to CureVac AG were constructed. The rpl32 sequence (SEQ ID NO: 22) having a stem-loop structure was used as the 5'-UTR of the template DNA, and the sequence (SEQ ID NO: 23) having the histone stem-loop structure was used as the 3'-UTR. Except for the procedure of Example 1, the nucleic acid molecules of the RNA platform were constructed. The nucleic acid molecule in the form of an RNA platform prepared according to Comparative Example 1 is referred to below as 'pCAP-curevac-R / L'.

Experimental Example 1 Measurement of Expression Efficiency of Nucleic Acid Molecules

Nucleic acid molecules in the form of RNA platforms prepared in Examples 1 to 5 and Comparative Example 1, respectively, were transfected into human muscle cell line (A204) and human embryonic kidney cell line (293T), respectively. Expression level was measured. The measurement results are shown in FIGS. 3A and 3B, respectively. As shown in FIGS. 3A and 3B, the 5'-UTR-inserted nucleic acid molecule (pEMCV-R / L) having an IRES derived from EMCV has a higher expression efficiency than pCAP-curevac-R / L used as a positive control. have. In addition, the 5'-UTR-inserted nucleic acid molecule (pCPV-R / L) having an IRES derived from CPV had an expression efficiency close to that of the positive control, and the 5'-UTR-inserted nucleic acid having an IRES derived from the remaining viruses was found. Molecules are lower than positive controls but have good expression efficiency.

Example 6: Nucleic Acid Molecular Construction of RNA Platforms

Nucleic acid molecules of an RNA platform having IRES derived from eukaryotic cells were prepared. As a 5'-UTR of template DNA, a 5'-UTR (SEQ ID NO: 6) having an IRES derived from eIF4G without a DLP structure sequence was used, and a sequence capable of improving expression efficiency on the 3 'side of the 5'-UTR ( Example 1 except inserting 3'-UTR (SEQ ID NO: 15) derived from eIF4G as a 3'-UTR, using a 3'-restriction enzyme recognition sequence ( GGCGCC ) of the ORF. The procedure was repeated to prepare nucleic acid molecules of the RNA platform. A nucleic acid molecule in the form of an RNA platform prepared according to this example is named 'peIF4G-R / L' below.

Example 7: Nucleic Acid Molecular Construction of RNA Platforms

Nucleic acid molecules of an RNA platform having IRES derived from eukaryotic cells were prepared. As 5'-UTR of template DNA, 5'-UTR (SEQ ID NO: 7) having an IRES derived from FMR1 without a DLP structure sequence was used, and a restriction enzyme recognition sequence ( CCGCGG GGCGCC) on the 3 'side of the ORF was used. The nucleic acid molecules of the RNA platform were prepared by repeating the procedure of Example 1, except that 3'-UTR (SEQ ID NO: 16) derived from FMR1 was used as the 3'-UTR. A nucleic acid molecule in the form of an RNA platform prepared according to the present example is named 'pFMR1--R / L' below.

Example 8: Nucleic Acid Molecular Construction of RNA Platforms

Nucleic acid molecules of an RNA platform having IRES derived from eukaryotic cells were prepared. 5'-UTR (SEQ ID NO: 8) having an IRES from CN43 without a DLP structure sequence was used as the 5'-UTR of the template DNA, and the restriction enzyme recognition sequence ( GGCGCC ) on the 3 'side of the ORF was used. Nucleic acid molecules of the RNA platform were prepared by repeating the procedure of Example 1, except that 3'-UTR (SEQ ID NO: 17) derived from CN43 was used as the 3'-UTR. A nucleic acid molecule in the form of an RNA platform prepared according to this example is named 'pCN43-R / L' below.

Experimental Example 2 Measurement of Expression Efficiency of Nucleic Acid Molecules

Nucleic acid molecules in the form of RNA platforms prepared in Examples 6 to 8 and Comparative Example 1, respectively, were transfected into human muscle cell line (A204) and human embryonic kidney cell line (293T), respectively. Expression level was measured. The measurement results are shown in FIGS. 4A and 4B, respectively. As shown in FIGS. 4A and 4B, the 5'-UTR-inserted nucleic acid molecule (pCN43-R / L) having an IRES derived from CN43 has a lower expression efficiency than the pCAP-CureVac-R / L used as a positive control. However, the expression rate was higher than that of 5'-UTR inserted nucleic acid molecules having IRES structures (FMR1, eIF4G) derived from two other eukaryotic cells. However, all of the nucleic acid molecules produced in Examples 6 to 8 showed good expression efficiency.

Example 9: Nucleic Acid Molecular Construction of RNA Platforms

The nucleic acid molecules of the RNA platform were prepared by repeating the procedure of Example 2 except that the capping process at the 5 ′ end was not performed using ARCA. A nucleic acid molecule in the form of an RNA platform prepared according to this example is named `` pCVB3-R / L '' below.

Example 10 Nucleic Acid Molecular Construction of RNA Platforms

Nucleic acid molecules of an RNA platform modified to insert multiple adenosine (pMA) at the 5 'side of the 5'-UTR were constructed. The RNA platform was repeated by repeating the procedure of Example 9 except that 50 multiple adenosnine (pMA) sequences were inserted between the T7 promoter and the 5'-UTR derived from CVB3, ie, 5'-UTR 5 'side. Nucleic acid molecules were prepared. A nucleic acid molecule in the form of an RNA platform prepared according to this example is named `` pMA-CVB3-R / L '' below.

Example 11: Nucleic Acid Molecular Construction of RNA Platforms

The nucleic acid molecule of the RNA platform was repeated by repeating the procedure of Example 5 except that 50 multiple adenosnine (pMA) sequences were inserted between the T7 promoter and the 5'-UTR derived from CPV, on the 5 'side of the 5'-UTR. Produced. A nucleic acid molecule in the form of an RNA platform prepared according to this example is referred to below as 'pMA-CPV-R / L'.

Example 12 Nucleic Acid Molecular Construction of RNA Platforms

The nucleic acid of the RNA platform was repeated by repeating the procedure of Example 8 except that 50 multiple adenosnine (pMA) sequences were inserted between the T7 promoter and the 5'-UTR derived from CN43, ie 5'-UTR's 5 'side. Molecules were made. The nucleic acid molecule in the form of an RNA platform prepared according to the present embodiment is named 'pMA-CN43-R / L' below.

Experimental Example 3: Measurement of Expression Efficiency of Nucleic Acid Molecules

Example 2 (pCAP-CVB3-R / L), Example 9 (pCVB3-R / L), Example 10 (pMA-CVB3-R / L), Example 11 (pMA-CPV-R / L), Nucleic acid molecules in the form of RNA platform prepared in Example 12 (pMA-CN43-R / L) and Comparative Example 1, respectively, were transfected into human muscle cell line (A204) or mouse muscle cell line (Nor10), respectively, and used as a gene of interest. The expression level of Renilla luciferase was measured. The measurement result of the expression level of the gene of interest (GOI) using the modified nucleic acid molecule having an IRES derived from CVB3 is shown in FIG. 5 and expressed in the nucleic acid molecule having pMA inserted on the 5 'side of the 5'-UTR. The measurement results for the expression level of the gene of interest are shown in FIG. 6.

As shown in FIG. 5, when the pMA-CVB3-R / L nucleic acid molecule was injected into the cell line, the Renilla luciferase was used as a gene of interest as compared to when the CVB3-R / L nucleic acid molecule was not modified. It can be seen that the expression level is improved. In addition, as shown in Figure 6, the nucleic acid molecule in which a large number of adenosine is inserted into the 5 'side of the 5'-UTR having the IRES was slightly lower than the positive control, but showed good expression efficiency.

Experimental Example 4 Measurement of Expression Efficiency of Nucleic Acid Molecules

Example 2 (pCAP-CVB3-R / L), Example 5 (pCPV-R / L), and Example 8 (pCN43-R / L) which were determined to have relatively high expression efficiency in Experimental Examples 1 to 3 described above. ) And the nucleic acid molecules of the RNA platform prepared in Comparative Example 1 were transfected into human muscle cell line (A204) and human embryonic kidney cell line (293T), respectively, and the expression levels of Renilla luciferase used as the gene of interest were measured. The measurement results are shown in FIGS. 7A and 7B. pCPV-R / L nucleic acid molecules showed similar or slightly lower expression efficiencies than pCureVac-R / L used as a positive control, but with two different types of IRES-inserted nucleic acid molecules (pCAP-CVB3-R / L, pCN43-R / L) was higher than the expression pattern. However, nucleic acid molecules with two other types of IRES structures also showed good expression efficiency.

Example 13: Nucleic Acid Molecular Construction of RNA Platforms

RNA nucleic acid molecules with 5-UTR applied with two different IRES were constructed. The template DNA sequence was designed as follows.

5'- restriction enzyme recognition sequence (GGATCC)-T7 promoter (SEQ ID NO: 19)-restriction enzyme recognition sequence (GTTAAC)-sequence to be transcribed into 50 adenosine (pMA)-restriction enzyme recognition sequence (GTTAAC)-first 5'-UTR derived from CVB3 as 5'-UTR (SEQ ID NO: 2)-Expression efficiency enhancing sequence (ATGGCAGCTCAA)-Restriction enzyme recognition sequence ( GTCGAC ) -Kozak sequence (GACC)-First GOI gene, R / L coding ORF sequence (SEQ ID NO: 20)-5'-UTR from EMCV as the second 5'-UTR (SEQ ID NO: 3)-R / L coding ORF sequence as the second GOI gene (SEQ ID NO: 19) -3'-UTR from CVB3 (SEQ ID NO: 12)-polyadenosine sequence (50 Adenosines) -restriction enzyme recognition sequence ( GCGGCCGC ).

The template DNA was cloned into a pGH vector and linearized with restriction enzymes to obtain nucleic acid molecules in the form of RNA platforms through in vitro trasncrption (IVT). The nucleic acid molecule in the form of an RNA platform prepared according to the present embodiment is named 'pMA-CVB3-R / L-EMCV-R / L' below.

Example 14 Nucleic Acid Molecular Construction of RNA Platforms

Nucleic acid molecules in the form of RNA platforms to which 5-UTRs with two different IRESs were applied were constructed. The template DNA sequence was designed as follows.

5'- restriction enzyme recognition sequence (GGATCC)-T7 promoter (SEQ ID NO: 19)-5'-UTR derived from CPV as the first 5'-UTR (SEQ ID NO: 5)-start codon of CPV (CCTGCT) Restriction enzyme recognition sequence ( GAATTC ) -first GOI gene, R / L encoding ORF sequence (SEQ ID NO: 20)-restriction enzyme recognition sequence ( GAGCTC )-second 5'-UTR, 5'-UTR derived from EMCV (SEQ ID NO: 3)-restriction enzyme recognition sequence (GATATCGTCGAC TTAATTAA ) -Kozak sequence (GACC)-R / L encoding ORF sequence as the second GOI gene (SEQ ID NO: 19) -restriction enzyme recognition sequence ( CCGCGG ) -CPV 3′-UTR (SEQ ID NO: 14)-polyadenosine sequence (50 Adenosines) -restriction enzyme recognition sequence ( GCGGCCGC ).

The template DNA was cloned into a pGH vector and linearized with restriction enzymes to obtain nucleic acid molecules in the form of RNA platforms through in vitro trasncrption (IVT). The nucleic acid molecule in the form of an RNA platform prepared according to the present embodiment is named 'pCPV-R / L-EMCV-R / L' below.

Example 15 Nucleic Acid Molecular Construction of RNA Platforms

The nucleic acid molecule of the RNA platform was prepared by repeating the procedure of Example 13 except for using the ORF sequence (SEQ ID NO: 21) encoding firefly luciferase (F / L) as the second GOI gene. The nucleic acid molecule in the form of an RNA platform prepared according to the present embodiment is named 'pMA-CVB3-R / L-EMCV-F / L' below.

Example 16: Nucleic Acid Molecular Construction of RNA Platforms

The nucleic acid molecule of the RNA platform was prepared by repeating the procedure of Example 14 except for using the ORF sequence (SEQ ID NO: 21) encoding firefly luciferase (F / L) as the second GOI gene. The nucleic acid molecule in the form of an RNA platform prepared according to the present embodiment is named 'pCPV-R / L-EMCV-F / L' below.

Experimental Example 5: Measurement of Expression Efficiency of Nucleic Acid Molecules

Nucleic acid molecules in the form of RNA platforms prepared in Examples 13 and 14 and Comparative Example 1, respectively, were transfected into human embryonic kidney cell lines (293T), and expression levels of Renilla luciferase used as a gene of interest were measured. The result of measuring the expression level of R / L when two identical GOI (R / L) was used is shown in FIG. The highest expression efficiency was shown in the pMA-CVB3-R / L-EMCV-R / L nucleic acid molecule with multiple adenosine inserted on the 5 'side of the 5'-UTR. In addition, the pCPV-R / L-EMCV-R / L nucleic acid molecules also showed slightly higher or similar expression efficiencies when compared to positive controls.

Subsequently, Example 5 (pCVP-R / L) and Example 16 (pCVP-R / L-EMCV-F / L), Example 10 (pMA-CVB3-R / L) and Example 15 (pMA- CVB3-R / L-EMCV-F / L) and nucleic acid molecules of the RNA platform form prepared in Comparative Example 1 were transfected into human embryonic kidney cell line (293T) and mouse muscle cell line (Nor10), respectively. The expression levels of Renilla luciferase and that of Firefly luciferase were compared. The measurement results are shown in Figs. 9A and 9B.

Expression of R / L when different genes of interest are expressed from different IRES base sequences, such as pCPV-R / L-EMCV-F / L and pMA-CVB3-R / L-EMCV-F / L nucleic acid molecules, and pCPV When the expression of one gene of interest, such as -R / L and pMA-CVB3-R / L, the expression level of R / L was compared, the expression level of R / L was not significantly different. However, in expression of F / L whose expression is regulated by 5'-UTR with IRES from EMCV, pCPV-R / L-EMCV-F / L at pMA-CVB3-R / L-EMCV-F / L It was expressed higher than that. This result is related to the high R / L expression efficiency of pMA-CVB3-R / L-EMCV-R / L nucleic acid molecule in FIG. 8 compared to pCVP-R / L-EMCV-R / L nucleic acid molecule. Judging.

The present invention has been described above based on exemplary embodiments and examples of the present invention, but the present invention is not limited to the technical idea described in the above embodiments and examples. Rather, those skilled in the art can easily make various modifications and changes based on the above-described embodiments and examples. However, it is apparent in the appended claims that all such modifications and variations fall within the scope of the present invention.

<110> THE CATHOLIC UNIVERSITY OF KOREA INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> NUCLEIC ACID MOLECULES INSERTED EXPRESSION REGULATION SEQUENCES          AND EXPRESSION VECTOR COMPRISING NUCLEIC ACID MOLECULES <130> 2434 <160> 23 <170> KoPatentIn 3.0 <210> 1 <211> 59 <212> DNA <213> Sindbis virus <400> 1 attgacggcg tagtacacac tattgaatca aacagccgac caattgcact accatcaca 59 <210> 2 <211> 742 <212> DNA <213> Coxsackievirus B <400> 2 ttaaaacagc ctgtgggttg atcccaccca cagggcctat tgggcgctag cactctggta 60 tcacggtacc tttgtgcgcc tgttttatat cccctccccc aactgtaact tagaagtaac 120 acactccgat caacagtcag cgtggcacac cagccatgtt ttgatcaagc acttctgtta 180 ccccggactg agtatcaata gactgctcac gcggttgaag gagaaagcgt tcgttatccg 240 gccaactact tcgaaaaacc cagtaacacc atagaggttg cagagtgttt cgctcagcac 300 taccccagtg tagaccaggc cgatgagtca ccgcattccc cacgggcgac cgtggcggtg 360 gctgcgttgg cggcctgcct atggggaaac ccataggacg ctctaataca gacatggtgc 420 gaagagtcta ttgagctagt tggtaatcct ccggcccctg aatgcggcta atcctaactg 480 cggacagcac accctcaaac cagagggcag tgtgtcgtaa cgggcaactc tgcagcggaa 540 ccgactactt tgggtgtccg tgtttcattt tattcctata ctggctgctt atggtgacaa 600 ttgagagatt gttaccatat agctattgga ttggccatcc ggtgtctaat agagctatta 660 tatatctctt tgttggattt ataccactta gcttgagaga ggttaaaaca ttacaattca 720 ttgttaaatt gaatacaaca aa 742 <210> 3 <211> 574 <212> DNA <213> Encephalomyocarditis virus <400> 3 cccctctccc tccccccccc ctaacgttac tggccgaagc cgcttggaat aaggccggtg 60 tgcgtttgtc tatatgttat tttccaccat attgccgtct tttggcaatg tgagggcccg 120 gaaacctggc cctgtcttct tgacgagcat tcctaggggt ctttcccctc tcgccaaagg 180 aatgcaaggt ctgttgaatg tcgtgaagga agcagttcct ctggaagctt cttgaagaca 240 aacaacgtct gtagcgaccc tttgcaggca gcggaacccc ccacctggcg acaggtgcct 300 ctgcggccaa aagccacgtg tataagatac acctgcaaag gcggcacaac cccagtgcca 360 cgttgtgagt tggatagttg tggaaagagt caaatggctc tcctcaagcg tattcaacaa 420 ggggctgaag gatgcccaga aggtacccca ttgtatggga tctgatctgg ggcctcggtg 480 cacatgcttt acatgtgttt agtcgaggtt aaaaaacgtc taggcccccc gaaccacggg 540 gacgtggttt tcctttgaaa aacacgatga taat 574 <210> 4 <211> 197 <212> DNA <213> Japanese encephalitis virus <400> 4 agaagtttat ctgtgtgaac ttcttggctt agtatcgttg agaagaatcg agagattagt 60 gcagtttaaa cagtttttta gaacggaaga taaccatgac taaaaaacca ggagggcccg 120 gtaaaaaccg ggctatcaat atgctgaaac gcggcctacc ccgcgtattc ccactagtgg 180 gagtgaagag ggtagta 197 <210> 5 <211> 189 <212> DNA <213> Cricket paralysis virus <400> 5 aaagcaaaaa tgtgatcttg cttgtaaata caattttgag aggttaataa attacaagta 60 gtgctatttt tgtatttagg ttagctattt agctttacgt tccaggatgc ctagtggcag 120 ccccacaata tccaggaagc cctctctgcg gtttttcaga ttaggtagtc gaaaaaccta 180 agaaattta 189 <210> 6 <211> 311 <212> DNA <213> Homo sapiens <400> 6 ggggtaggga tgagggaggg aggggcattg tgatgtacag ggctgctctg tgagatcaag 60 ggtctcttaa gggtgggagc tggggcaggg actacgagag cagccagatg ggctgaaagt 120 ggaactcaag gggtttctgg cacctaccta cctgcttccc gctggggggt ggggagttgg 180 cccagagtct taagattggg gcagggtgga gaggtgggct cttcctgctt cccactcatc 240 ttatagcttt ctttccccag atccgaattc gagatccaaa ccaaggagga aaggatatca 300 cagaggagat c 311 <210> 7 <211> 321 <212> DNA <213> Homo sapiens <400> 7 cagcgttgat cacgtgacgt ggtttcagtg tttacacccg cagcgggccg ggggttcggc 60 ctcagtcagg cgctcagctc cgtttcggtt tcacttccgg tggagggccg cctctgagcg 120 ggcggcgggc cgacggcgag cgcgggcggc ggcggtgacg gaggcgccgc tgccaggggg 180 cgtgcggcag cgcggcggcg gcggcggcgg cggcggcggc ggaggcggcg gcggcggcgg 240 cggcggcggc ggctgggcct cgagcgcccg cagcccacct ctcgggggcg ggctcccggc 300 gctagcaggg ctgaagagaa g 321 <210> 8 <211> 157 <212> DNA <213> Homo sapiens <400> 8 gcgtgaggaa agtaccaaac agcagcggag ttttaaactt taaatagaca ggtctgagtg 60 cctgaacttg ccttttcatt ttacttcatc ctccaaggag ttcaatcact tggcgtgact 120 tcactacttt taagcaaaag agtggtgccc aggcaac 157 <210> 9 <211> 143 <212> DNA <213> Sindbis virus <400> 9 atggagaagc cagtagtaaa cgtagacgta gacccccaga gtccgtttgt cgtgcaactg 60 caaaaaagct tcccgcaatt tgaggtagta gcacagcagg tcactccaaa tgaccatgct 120 aatgccagag cattttcgca tct 143 <210> 10 <211> 319 <212> DNA <213> Sindbis virus <400> 10 ccgctacgcc ccaatgatcc gaccagcaaa actcgatgta cttccgagga actgatgtgc 60 ataatgcatc aggctggtac attagatccc cgcttaccgc gggcaatata gcaacactaa 120 aaactcgatg tacttccgag gaagcgcagt gcataatgct gcgcagtgtt gccacataac 180 cactatatta accatttatc tagcggacgc caaaaactca atgtatttct gaggaagcgt 240 ggtgcataat gccacgcagc gtctgcataa cttttattat ttcttttatt aatcaacaaa 300 attttgtttt taacatttc 319 <210> 11 <211> 103 <212> DNA <213> Coxsackievirus B <400> 11 taaattagag acaatttgat ctgatttgaa ttggcttaac cctactgtac taaccgaact 60 agacaacggt gcagtagggg taaattctcc gcattcggtg cgg 103 <210> 12 <211> 123 <212> DNA <213> Encephalomyocarditis virus <400> 12 tagtgtagtc actggcacaa cgcgttaccc ggtaagccaa tcgggtatac acggtcgtca 60 tactgcagac agggttcttc tactttgcaa gatagtctag agtagtaaaa taaatagata 120 gag 123 <210> 13 <211> 585 <212> DNA <213> Japanese encephalitis virus <400> 13 tagtgtgatt taaagtagaa aagtagacta tgtaaataat gtaaatgaga aaatgcatgc 60 atatggagtc aggccagcaa aagctgccac cggatactgg gtagacggtg ctgtctgcgt 120 ctcagtccca ggaggactgg gttaacaaat ctgacaacag aaagtgagaa agccctcaga 180 accgtctcgg aagtaggtcc ctgctcactg gaagttgaaa gaccaacgtc aggccacaaa 240 tttgtgccac cccgctaggg ggtgcggcct gcgcagcccc aggaggactg ggttaccaaa 300 gccgttgagc ccccacggcc caagcctcgt ctaggatgca atagacgagg tgtaaggact 360 agaggttaga ggagaccccg tggaaacaac aatatgcggc ccaagccccc tcgaagctgt 420 agaggaggtg gaaggactag aggttagagg agaccccgca tttgcatcaa acagcatatt 480 gacacctggg aatagactgg gagatcttct gctctatctc aacatcagct actaggcaca 540 gagcgccgaa gtatgtagct ggtggtgagg aagaacacag gatct 585 <210> 14 <211> 221 <212> DNA <213> Cricket paralysis virus <400> 14 agacatgata agatacattg atgagtttgg acaaaccaca acaagaatgc agtgaaaaaa 60 atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa 120 taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg gggagatgtg 180 ggaggttttt taaagcaagt aaaacctcta caaatgtggt a 221 <210> 15 <211> 456 <212> DNA <213> Homo sapiens <400> 15 tgaccacaac tgagggctgg tggggccggg gacctggagc cccatggaca cacagatggc 60 ccggctagcc gcctggactg caggggggcg gcagcagcgg cggtggcagt gggtgcctgt 120 agtgtgatgt gtctgaacta ataaagtggc tgaagaggca ggatggcttg gggctgcctg 180 ggcccccctc caggatgccg ccaggtgtcc ctctcctccc cctggggcac agagatatat 240 tatatataaa gtcttgaaat ttggtgtgtc ttggggtggg gaggggcacc aacgcctgcc 300 cctggggtcc ttttttttat tttctgaaaa tcactctcgg gactgccgtc ctcgctgctg 360 ggggcatatg ccccagcccc tgtaccaccc ctgctgttgc ctgggcaggg ggaagggggg 420 gcacggtgcc tgtaattatt aaacatgaat tcaatt 456 <210> 16 <211> 517 <212> DNA <213> Homo sapiens <400> 16 tgtgaaggac cttcactcta agatgttata tttttcttaa aaagtaactc ctagtagggg 60 taccactgaa tctgtacaga gccgtaaaaa ctgaagttct gcctctgatg tattttgtga 120 gtttgtttct ttgaattttc attttacagt tacttttcct tgcatacaaa caagcatata 180 aaatggcaac aaactgcaca tgatttcaca aatattaaaa agtcttttaa aaagtattgc 240 caaacattaa tgttgatttc tagttattta ttctgggaat gtatagtatt tgaaaacaga 300 aattggtacc ttgcacacat catctgtaag ctgtttggtt ttaaaatact gtagataatt 360 aaccaaggta gaatgacctt gtaatgtaac tgctcttggg caatattctc tgtacatatt 420 agcgacaaca gattggattt tatgttgaca tttgtttggt tatagtgcaa tatattttgt 480 atgcaagcag tttcaataaa gtttgatctt cctctgc 517 <210> 17 <211> 337 <212> DNA <213> Homo sapiens <400> 17 aattcagaca aggcccacag aataagattt tccatgcatt tgcaaatacg tatattcttt 60 ttccatccac ttgcacaata tcattaccat cactttttca tcattcctca gctactactc 120 acattcattt aatggtttct gtaaacattt ttaagacagt tgggatgtca cttaacattt 180 tttttttttg agctaaagtc agggaatcaa gccatgctta atatttaaca atcacttata 240 tgtgtgtcga agagtttgtt ttgtttgtca tgtattggta caagcagata cagtataaac 300 tcacaaacac agatttgaaa ataatgcaca tatggtg 337 <210> 18 <211> 18 <212> DNA <213> Bacteriophage T7 <400> 18 taatacgact cactatag 18 <210> 19 <211> 936 <212> DNA <213> Renilla reniformis <400> 19 atgacttcga aagtttatga tccagaacaa aggaaacgga tgataactgg tccgcagtgg 60 tgggccagat gtaaacaaat gaatgttctt gattcattta ttaattatta tgattcagaa 120 aaacatgcag aaaatgctgt tattttttta catggtaacg cggcctcttc ttatttatgg 180 cgacatgttg tgccacatat tgagccagta gcgcggtgta ttataccaga ccttattggt 240 atgggcaaat caggcaaatc tggtaatggt tcttataggt tacttgatca ttacaaatat 300 cttactgcat ggtttgaact tcttaattta ccaaagaaga tcatttttgt cggccatgat 360 tggggtgctt gtttggcatt tcattatagc tatgagcatc aagataagat caaagcaata 420 gttcacgctg aaagtgtagt agatgtgatt gaatcatggg atgaatggcc tgatattgaa 480 gaagatattg cgttgatcaa atctgaagaa ggagaaaaaa tggttttgga gaataacttc 540 ttcgtggaaa ccatgttgcc atcaaaaatc atgagaaagt tagaaccaga agaatttgca 600 gcatatcttg aaccattcaa agagaaaggt gaagttcgtc gtccaacatt atcatggcct 660 cgtgaaatcc cgttagtaaa aggtggtaaa cctgacgttg tacaaattgt taggaattat 720 aatgcttatc tacgtgcaag tgatgattta ccaaaaatgt ttattgaatc ggacccagga 780 ttcttttcca atgctattgt tgaaggtgcc aagaagtttc ctaatactga atttgtcaaa 840 gtaaaaggtc ttcatttttc gcaagaagat gcacctgatg aaatgggaaa atatatcaaa 900 tcgttcgttg agcgagttct caaaaatgaa caataa 936 <210> 20 <211> 933 <212> DNA <213> Renilla reniformis <400> 20 acttcgaaag tttatgatcc agaacaaagg aaacggatga taactggtcc gcagtggtgg 60 gccagatgta aacaaatgaa tgttcttgat tcatttatta attattatga ttcagaaaaa 120 catgcagaaa atgctgttat ttttttacat ggtaacgcgg cctcttctta tttatggcga 180 catgttgtgc cacatattga gccagtagcg cggtgtatta taccagacct tattggtatg 240 ggcaaatcag gcaaatctgg taatggttct tataggttac ttgatcatta caaatatctt 300 actgcatggt ttgaacttct taatttacca aagaagatca tttttgtcgg ccatgattgg 360 ggtgcttgtt tggcatttca ttatagctat gagcatcaag ataagatcaa agcaatagtt 420 cacgctgaaa gtgtagtaga tgtgattgaa tcatgggatg aatggcctga tattgaagaa 480 gatattgcgt tgatcaaatc tgaagaagga gaaaaaatgg ttttggagaa taacttcttc 540 gtggaaacca tgttgccatc aaaaatcatg agaaagttag aaccagaaga atttgcagca 600 tatcttgaac cattcaaaga gaaaggtgaa gttcgtcgtc caacattatc atggcctcgt 660 gaaatcccgt tagtaaaagg tggtaaacct gacgttgtac aaattgttag gaattataat 720 gcttatctac gtgcaagtga tgatttacca aaaatgttta ttgaatcgga cccaggattc 780 ttttccaatg ctattgttga aggtgccaag aagtttccta atactgaatt tgtcaaagta 840 aaaggtcttc atttttcgca agaagatgca cctgatgaaa tgggaaaata tatcaaatcg 900 ttcgttgagc gagttctcaa aaatgaacaa taa 933 <210> 21 <211> 1653 <212> DNA Lucia cruciata <400> 21 atggaagatg ccaaaaacat taagaagggc ccagcgccat tctacccact cgaagacggg 60 accgccggcg agcagctgca caaagccatg aagcgctacg ccctggtgcc cggcaccatc 120 gcctttaccg acgcacatat cgaggtggac attacctacg ccgagtactt cgagatgagc 180 gttcggctgg cagaagctat gaagcgctat gggctgaata caaaccatcg gatcgtggtg 240 tgcagcgaga atagcttgca gttcttcatg cccgtgttgg gtgccctgtt catcggtgtg 300 gctgtggccc cagctaacga catctacaac gagcgcgagc tgctgaacag catgggcatc 360 agccagccca ccgtcgtatt cgtgagcaag aaagggctgc aaaagatcct caacgtgcaa 420 aagaagctac cgatcataca aaagatcatc atcatggata gcaagaccga ctaccagggc 480 ttccaaagca tgtacacctt cgtgacttcc catttgccac ccggcttcaa cgagtacgac 540 ttcgtgcccg agagcttcga ccgggacaaa accatcgccc tgatcatgaa cagtagtggc 600 agtaccggat tgcccaaggg cgtagcccta ccgcaccgca ccgcttgtgt ccgattcagt 660 catgcccgcg accccatctt cggcaaccag atcatccccg acaccgctat cctcagcgtg 720 gtgccatttc accacggctt cggcatgttc accacgctgg gctacttgat ctgcggcttt 780 cgggtcgtgc tcatgtaccg cttcgaggag gagctattct tgcgcagctt gcaagactat 840 aagattcaat ctgccctgct ggtgcccaca ctatttagct tcttcgctaa gagcactctc 900 atcgacaagt acgacctaag caacttgcac gagatcgcca gcggcggggc gccgctcagc 960 aaggaggtag gtgaggccgt ggccaaacgc ttccacctac caggcatccg ccagggctac 1020 ggcctgacag aaacaaccag cgccattctg atcacccccg aaggggacga caagcctggc 1080 gcagtaggca aggtggtgcc cttcttcgag gctaaggtgg tggacttgga caccggtaag 1140 acactgggtg tgaaccagcg cggcgagctg tgcgtccgtg gccccatgat catgagcggc 1200 tacgttaaca accccgaggc tacaaacgct ctcatcgaca aggacggctg gctgcacagc 1260 ggcgacatcg cctactggga cgaggacgag cacttcttca tcgtggaccg gctgaagagc 1320 ctgatcaaat acaagggcta ccaggtagcc ccagccgaac tggagagcat cctgctgcaa 1380 caccccaaca tcttcgacgc cggggtcgcc ggcctgcccg acgacgatgc cggcgagctg 1440 cccgccgcag tcgtcgtgct ggaacacggt aaaaccatga ccgagaagga gatcgtggac 1500 tatgtggcca gccaggttac aaccgccaag aagctgcgcg gtggtgttgt gttcgtggac 1560 gaggtgccta aaggactgac cggcaagttg gacgcccgca agatccgcga gattctcatt 1620 aaggccaaga agggcggcaa gatcgccgtg taa 1653 <210> 22 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> RPL32 <400> 22 ggggcgctgc ctacggaggt ggcagccatc tccttctcgg catcaagctt gagg 54 <210> 23 <211> 211 <212> DNA <213> Artificial Sequence <220> <223> Histone stem-loop <400> 23 gactagtgtc cacctgtccc tcctgggctg ctggattgtc tcgttttcct gccaaataaa 60 caggatcagc gctttacaga tctaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 120 aaaaaaaaaa aaaaaaaaaa aaaaaaatgc atcccccccc cccccccccc cccccccccc 180 cccaaaggct cttttcagag ccaccagaat t 211

Claims (21)

At least one expression control sequence comprising a nucleotide sequence having an Internal Ribosomal Entry Site (IRES) activity; And
At least one coding region operably linked with said at least one expression control sequence, encoding a protein or peptide,
20 to 400 adenosine or thymine linked adjacent to the 5 'upstream side of the at least one expression control sequence.
The method of claim 1,
Wherein said at least one expression control sequence forms a 5'-untranslated region (5'-UTR).
The method of claim 2,
The nucleic acid molecule further comprises a 3'-untranslated region (3'-UTR) located 3 'downstream of the 5'-UTR, wherein the coding region comprises the 5'-UTR and the 3'-. Nucleic acid molecules located between UTRs.
The method of claim 1,
Wherein said at least one expression control sequence comprises a viral IRES base sequence.
The method of claim 4, wherein
The viral IRES base sequence is Piconaviridae virus, Togaviridae virus, Dicistroviridae virus, Flaviridae virus, Retroviridae virus, Herpesviridae A nucleic acid molecule comprising a viral IRES base sequence derived from a virus selected from the group consisting of a virus and combinations thereof.
The method of claim 4, wherein
The viral IRES nucleotide sequence is a nucleic acid molecule derived from a virus selected from the group consisting of Picornaviridae virus, Dicistroiridae virus and combinations thereof.
The method of claim 6,
The viral IRES nucleotide sequence derived from the Picornaviridae virus is Enterovirus virus, Cardiovirus virus, Aptovirus virus, Hepatovirus. Is derived from the family of piconaviruses selected from the group consisting of viruses, Teschovirus viruses and combinations thereof,
The viral IRES base sequence derived from the Dicistroviridae virus is a nucleic acid molecule derived from a Creepavirus virus.
The method of claim 7, wherein
The IRES base sequence derived from the Picornaviridae virus includes an IRES base sequence derived from coxsackievirus,
The IRES nucleotide sequence derived from the dyscivirus virus includes a IRES nucleotide sequence derived from Cricket paralysis virus (CPV).
The method of claim 1,
Said coding region is a nucleic acid molecule encoding an antigen or fragment thereof.
The method of claim 1,
The coding region is a nucleic acid molecule encoding a protein or fragment thereof for treating a disease.
The method of claim 1,
Wherein said at least one expression control sequence is a nucleic acid molecule comprising a first expression control sequence having a base sequence of IRES and a second expression control sequence located 3 ′ downstream of the first expression control sequence and having a base sequence of IRES .
The method of claim 11,
Wherein said coding region comprises a first coding region located between said first expression control sequence and said second expression control sequence and a second coding region located 3 'downstream of said second expression control sequence.
The method of claim 12,
Wherein said first coding region and said second coding region encode different peptides or proteins.
The method of claim 11,
20 to 400 adenosine or thymine bound adjacent to the 5 'upstream side of the first expression control sequence and at least one expression control sequence selected from the second expression control sequence.
The method of claim 11,
A nucleic acid molecule having 30 to 100 adenosine or thymine bound adjacent to the 5 'upstream side of the first expression control sequence.
The method of claim 11,
The first expression control sequence comprises an IRES base sequence derived from Coxsachie virus B3 (CVB3) or Cricket paralysis virus (CPV), and the second expression control sequence is brain myocarditis virus ( A nucleic acid molecule comprising an IRES nucleotide sequence derived from Encephalomyocarditis virus (EMCV).
The method of claim 1,
The nucleic acid molecule further comprises a transcriptional regulatory sequence located 5 'upstream of the expression control sequence and a poly adenosine (poly (A)) sequence located 3' downstream of the coding region.
The method of claim 1,
Wherein said nucleic acid molecule is RNA.
The recombinant expression vector which inserted the nucleic acid molecule of any one of Claims 1-18.
A method for producing the protein or peptide by injecting the nucleic acid molecule according to any one of claims 1 to 18 into a living body other than the human body, and expressing the protein or peptide from the injected nucleic acid molecule.
A method for producing the protein or peptide by injecting the recombinant gene vector according to claim 19 into a living body other than a human body as a gene carrier and expressing the protein or peptide from the gene carrier.

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