KR20190061114A - 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 which uses an expression regulatory sequence derived from viruses having an internal ribosome binding site (IRES) or eukaryotic cells to express interest genes, an expression vector having the nucleic acid molecule inserted thereto, and an application of the same. The nucleic acid molecule of the present in vention is used to express interest genes efficiently without a complicated capping process relevant to expression of interest genes. According to the present invention, the nucleic acid molecule connected with multiple adenosines or thymines to be adjacent to an expression regulatory sequence is used to express efficiently desired interest genes. With the application of an expression system having nucleic acid molecules according to the present invention, the present invention is expected to be used as a gene vaccine or a gene therapy agent expressing protein or peptide relevant to disease treatments.

Description

[0001] The present invention relates to a nucleic acid molecule having an expression control sequence inserted therein and an expression vector containing the nucleic acid molecule. [0002]

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

As biotechnology advances, a variety of expression systems are known that can efficiently express the gene of interest (GOI). As such expression systems, cell-based expression systems utilize microbial or eukaryotic expression systems, and cell-free expression systems typically use purified RNA polymerases, ribosomes, tRNAs and ribonucleotides. In particular, proteins derived from eukaryotic cells undergo post-translational modification such as phosphorylation, methylation, and glycosylation even after expression. Since microorganisms do not have such a post-translational modification mechanism, expression of proteins derived from eukaryotic cells Eukaryotic expression systems have been used.

Can be utilized as a gene therapy agent or a gene vaccine by using an eukaryotic expression system. In the case of a gene therapy agent, an interest gene consisting of an open reading frame (ORF) encoding a peptide or a protein capable of treating various diseases is inserted into an expression system, and in the case of a gene vaccine, a peptide or protein Into an expression system of interest.

In order to efficiently express a gene of interest, it is common to use a sequence that regulates transcription and translation of a gene of interest. A transcription efficiency can be improved by using a promoter having an excellent transcription efficiency and a capping system peculiar to a eukaryotic cell is used in connection with the translation of a gene of interest.

Specifically, the capping system used in connection with efficient translation from a gene of interest (GOI) to a protein or peptide includes 7-methyl-guanosine (m ⁷ G) at the 5 'end of the gene of interest (GOI) Cap (5'cap) structure. A transcription initiation complex consisting of eIF4A, eIF4E, and eIF4G, which function as translation regulators of eukaryotic cells, recognizes the 5 'cap and binds to the 5' cap site to form a capping structure, . When a capping structure is formed at the site where translation starts, protein synthesis is initiated and mRNA breakdown is prevented from nuclease action.

The IVT process for preparing a nucleic acid molecule having a capping structure is performed by linearly treating pDNA (plasmid DNA) with a restriction enzyme, and then m ⁷ G (5 ') - ppp- (5 ') G (regular cap analog) to create capped mRNA. However, these cap analogs often bind in the opposite direction and the m ⁷ G nucleotide does not act on the cap, and about one-third of the mRNA produced does not have methylation in the cap, and such mRNA can not synthesize proteins.

In order to prevent this, there is a method of carrying out cap reaction using commercially available vaccinia virus capping enzyme after performing IVT without cap analog, and there is a method of 'anti-reverse cap analogue (ARCA) (ARCA-capped mRNA) ARCA-capped mRNA is known to produce protein more than twice as efficiently as regular cap analog-capped mRNA, and the half-life of mRNA is also known to be long. However, since such an artificial capping reaction is required, the cost for expressing the gene of interest is greatly increased. Therefore, in order to utilize the expression system as a gene vaccine or a gene therapy agent, a method for improving the gene vaccination is required.

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 a complicated and expensive process and an expression vector into which such a nucleic acid molecule is inserted.

According to an aspect of the present invention, there is provided a method of preparing a ribozyme comprising the steps of: (a) providing at least one expression control sequence comprising a base sequence having an internal ribosomal entry site (IRES) activity; And at least one coding region operably linked to the expression control sequence and encoding a protein or peptide, wherein 20 to 400 adenosines or thymines are linked adjacent to the at least one expression control sequence Lt; / RTI > nucleic acid molecule.

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

Wherein the nucleic acid molecule further comprises a 3'-untranslated region (3'-UTR) located at the 3'-end of the 5'-UTR and the coding region is between the 5'-UTR and the 3'-UTR Lt; / RTI >

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

For example, the viral IRES base sequence may be selected from the group consisting of picornaviridae virus, Togaviridae virus, Dicistroviridae virus, Flaviridae virus, Retroviridae virus, A viral IRES base sequence derived from a virus selected from the group consisting of Herpesviridae viruses and combinations thereof.

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

Herein, the viral IRES base sequence derived from the picornaviridae virus may be selected from the group consisting of Enterovirus virus, Cardiovirus virus, Aptovirus virus, Hepatovirus Hepatovirus virus, Teschovirus virus, and combinations thereof, wherein the viral IRES base sequence derived from the Dicistroviridae virus is derived from a virus selected from the group consisting of Crepia It may be from a Cripavirus virus.

More specifically, the IRES nucleotide sequence derived from the picornaviridae virus comprises an IRES nucleotide sequence derived from a cockskill virus, and the IRES nucleotide sequence derived from the descovirus and the virus is derived from cricket paralytic virus ( Cricket paralysis virus, CPV).

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

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

In another exemplary embodiment, the at least one expression control sequence comprises a first expression control sequence having a base sequence of IRES and a second expression control sequence located 3 ' of the first expression control sequence and having a base sequence of IRES Sequence.

Here, 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 3 'to the second expression control sequence .

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

At this time, 20 to 400 adenosine or thymine may be bound to adjacent to at least one expression control sequence selected from the first expression control sequence and the second expression control.

For example, 30-100 adenosines may be joined adjacent to the first expression control sequence.

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

Preferably, the nucleic acid molecule further comprises a transcription control sequence located 5 'to the expression control sequence and a polyadenosine (poly (A)) sequence located 3' to the coding region .

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

In another aspect of the present invention, the present invention provides a recombinant expression vector into which the aforementioned nucleic acid molecule is inserted. For example, the recombinant expression vector may be derived from a plasmid or may be derived from a virus.

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

In another aspect of the present invention, there is provided a method for producing the protein or peptide by injecting the recombinant gene vector described above into a living body as a gene carrier and expressing the protein or peptide from the gene carrier.

In another aspect of the present invention, there is provided a host cell transformed with a protein or peptide expressed from the above-described nucleic acid molecule and / or recombinant expression vector.

According to the present invention there is provided a nucleic acid molecule having at least one expression control sequence having an internal ribosome binding site (IRES) for expression of a gene of interest, i. E. A coding region and having a plurality of adenosine or thymine linked adjacent to at least one expression control .

In order to efficiently express a gene of interest using the conventional capping structure, there has been a problem that an expensive enzyme has to be used, and only one peptide or protein has to 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 without using an expensive enzyme. In addition, the present invention includes IRES as an expression control sequence, so that, if necessary, the same or different peptides or proteins can be operatively linked with other IRES sequences to simultaneously produce desired peptides and proteins in a single nucleic acid molecule And can increase the expression efficiency.

For example, the present invention can be applied as a gene vaccine by applying a nucleic acid molecule of the present invention and / or a recombinant expression vector inserted with the nucleic acid molecule of the present invention into which the gene of interest is introduced into the coding region to induce various immune responses in vivo .

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

According to an alternative embodiment, the nucleic acid molecule of the present invention may be prepared in the form of RNA and applied as a vaccine. The RNA vaccine according to the present invention can be rapidly produced and produced only with a small amount of template DNA, and it is not necessary to directly deal with the infectious agent, so that stability can be secured.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram schematically illustrating the construction 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 schematically illustrating the construction of a polynucleotide or nucleic acid molecule capable of efficiently expressing a plurality of genes of interest according to an exemplary embodiment of the present invention.
FIGS. 3A and 3B are graphs showing the results of measuring the expression levels of a gene of interest after administering nucleic acid molecules in the form of an RNA platform containing various virus-derived IRES according to an exemplary embodiment of the present invention to a cell line . FIG. 3A shows the expression level of the A204 cell line, and FIG. 3B shows the expression level of the 293T cell line.
4A and 4B are graphs showing the results of measuring the expression level of a gene of interest after administering nucleic acid molecules in the form of an RNA platform containing various eukaryotic cell-derived IRES according to an exemplary embodiment of the present invention, to be. FIG. 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 the result of measuring the expression level of a gene of interest after administering nucleic acid molecules in the form of an RNA platform containing various virus-derived IRES to various eukaryotic cell lines according to an exemplary embodiment of the present invention, The results are shown in FIG.
FIG. 6 is a graph showing the results of measuring the expression level of a gene of interest after administering nucleic acid molecules containing various virus-derived IRES synthesized according to an exemplary embodiment of the present invention to a Nor10 cell line.
FIGS. 7A and 7B are graphs showing the results of measuring the expression levels of a gene of interest after administering nucleic acid molecules containing IRES derived from various viruses to various cell lines according to an exemplary embodiment of the present invention. FIG. FIG. 7A shows the expression level in the A204 cell line, and FIG. 7B shows the expression level in the 293T cell line.
FIG. 8 is a graph showing the results of measuring the expression levels of a gene of interest after administering nucleic acid molecules containing IRES derived from a plurality of viruses to various cell lines according to an exemplary embodiment of the present invention. .
9A and 9B are graphs showing the expression levels of a gene of interest after administering nucleic acid molecules containing IRES derived from a plurality of viruses to various cell lines according to an exemplary embodiment of the present invention. FIG. 9A shows the expression level in the 293T cell line, and FIG. 9B shows the expression level in the Nor10 cell line.

Hereinafter, the present invention will be described with reference to the accompanying drawings where necessary.

Definition of Terms

As used herein, the term " amino acid " is used in its broadest sense and is intended to include naturally occurring L-amino acids or residues. One-letter abbreviations and / or three-letter abbreviations commonly used for naturally-occurring amino acids may be used herein. Amino acids are not only D-amino acids but also chemically-modified amino acids such as amino acid analogs, chemically-modified amino acids that are characteristic of natural-occurring amino acids that are not normally incorporated into proteins, such as norleucine, - Synthesized compounds. For example, analogs or mimetics of phenylalanine or proline that allow restriction of the stereostructure of the same peptide compound as native Phe or Pro are included within the definition of amino acids. Such analogs and mimetics are referred to herein as " functional equivalents " of amino acids. 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 that are encoded by the gene, thus allowing for a wider variety of substitutions for a given amino acid. Amino acids not coded 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; Homo arginine for Arg and Lys (Har); 2,3-diaminopropionic acid (Dap) against Lys, Arg and His; N-ethylglycine (EtGly) for Gly, Pro and Ala; N-ethylglycine (EtGly) for Gly, Pro and Ala; N-ethyl asparagine (AsAsn) for Asn and Gln; Hydroxylysine (Hyl) for Lys; Alohydroxylysine (AHyl) for Lys; 3- (and 4-) hydroxyproline (3Hyp, 4Hyp) for Pro, Ser and Thr; Allo-isoleucine (AIle) for Ile, Leu and Val; 4-amidinophenylalanine to Arg; N-methylglycine for Gly, Pro and Ala (MeGly, sarcosine); 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 proteins, protein fragments, and peptides that have been isolated from naturally occurring or recombinant techniques or chemically synthesized. For example, the peptide of the present invention is composed of at least 5, preferably 10 amino acids.

In certain embodiments, variants of the compound are provided, such as peptide variants having one or more amino acid substitutions. As used herein, the term " peptide variants " means that one or more amino acids are substituted, deleted, added, and / or inserted into the amino acid sequence of the peptide, ≪ / RTI > and the like. The peptide variant should have an identity of 70% or more, preferably 90% or more, more preferably 95% or more, with the original peptide. Such substituents may include amino acid substituents known as " conservative ". Variants may also include nonconservative changes. In a preferred embodiment, the sequence of the variant polypeptide differs from the original sequence by substitution, deletion, addition or insertion of five or fewer amino acids. Variants can also be altered by deletion or addition of amino acids that have minimal effect on the immunogenicity, secondary structure and hydropathic nature of the peptide.

&Quot; Conservative " substitution means that even when one amino acid is substituted with another amino acid, there is no significant change in characteristics such as the secondary structure and hydropathic nature of the polypeptide. The amino acid variation is based on the relative similarity of the amino acid side chain substituents, such as polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphipathic nature .

For example, amino acids can be classified as i) hydrophobic (norleucine, methionine, alanine, valine, leucine, isoleucine), ii) neutral hydrophilic (cysteine, serine, threonine, asparagine, glutamine) Acid, glutamic acid), iv) basic (histidine, lysine, arginine), v) residues affecting the chain direction (glycine, proline) and vi) aromatic (tryptophan, tyrosine and phenylalanine). Conservative substitutions will involve exchanging members of one 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 both positively charged residues; Alanine, glycine and serine have similar sizes; Phenylalanine, tryptophan and tyrosine have similar shapes. Based on these considerations, therefore, arginine, lysine and histidine; Alanine, glycine and serine; And phenylalanine, tryptophan and tyrosine are biologically functional equivalents.

In introducing the mutation, the hydrophobic index of the amino acid can be considered. Each amino acid is given a hydrophobic index according to 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 imparting the interactive biological function of proteins. It is known that substitution with an amino acid having a similar hydrophobicity index can retain similar biological activities. When a mutation is introduced with reference to a hydrophobic index, substitution is made between amino acids showing a hydrophobic index difference preferably within ± 2, more preferably within ± 1, even more preferably within ± 0.5.

On the other hand, it is also well known that the substitution between amino acids having similar hydrophilicity values leads to proteins with homogeneous biological activity. As disclosed in U.S. Patent 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 a mutation is introduced with reference to the hydrophilicity value, the amino acid is substituted preferably within ± 2, more preferably within ± 1, even more preferably within ± 0.5.

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

Generally, the peptides (including fusion proteins) and polynucleotides referred to herein are isolated. A " isolated " peptide or polynucleotide is one that has been removed from its original environment. For example, a protein in its natural state is separated by removing all or some of the substances that are present together. Such polypeptides should have a purity of at least 90%, preferably 95%, more preferably 99% or more. Polynucleotides are separated by cloning in a vector.

Peptides may be separated by recombinant means or by chemical synthesis. Recombinant peptides encoded by the nucleic acid sequences referred to herein may be readily prepared in a known manner using any of a number of known expression vectors. Expression can be carried out in a suitable host cell transformed with an expression vector comprising a DNA sequence encoding a recombinant protein. Suitable host cells include prokaryotes, yeast, and eukaryotes. For example, eukaryotic cell lines such as yeast or mammalian cell lines (such as Cos or CHO) are preferably used as host cells. For the purification of the recombinant protein, the supernatant containing the recombinant protein secreted in the culture medium, first obtained in an aqueous 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 step or several steps of reverse phase HPLC.

The peptides or proteins described herein can be secreted into and recovered from the surrounding cytoplasm of the host cell. Typically, the recovery of the peptide and / or protein generally involves pulverizing the microorganism by means such as osmotic shock, sonication or dissolution. When the cells are destroyed, cell debris or whole cells can be removed by centrifugation or filtration. The peptides and / or proteins may be further purified, for example, by affinity resin chromatography. Alternatively, the peptides and / or proteins can be transferred to and isolated from the culture medium. The cells may be removed from the culture, the culture supernatant may be filtered and concentrated to further purify the resulting peptides and / or proteins. The expressed polypeptides are routinely purified by known methods such as fractional distillation on immunoaffinity or ion-exchange columns; Ethanol precipitation; Reversed phase HPLC; Chromatography on silica or cation exchange resins, such as DEAE; Chromatographic focusing; SDS-PAGE; Ammonium sulfate precipitation; Gel filtration using, for example, Sephadex G-75; Hydrophobic affinity resins, ligand affinities using suitable antigens immobilized on a matrix, and western blot assays.

In addition, the produced peptides can be purified to obtain substantially homogeneous preparations for further assay and use. Standard protein purification methods known in the art can be used. The following procedures are examples of suitable purification procedures: immunoaffinity or fractional distillation on ion-exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica or cation-exchange resins such as DEAE, chromatofocusing, 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 (eg, cDNA) and RNA molecules. A " nucleotide " which is a constituent unit of a nucleic acid molecule can be introduced into the polymer by a deoxyribonucleotide, a ribonucleotide, a modified nucleotide or base, and / or an analogue thereof, or a DNA or RNA polymerase, It can be any substrate. Polynucleotides can include modified nucleotides, analogues of sugars or bases that are modified, such as methylated nucleotides and their analogs (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews , ≪ / RTI > 90: 543-584 (1990)).

Variations in nucleotides do not cause mutations in the protein. Such nucleic acids include functionally equivalent codons or codons that encode the same amino acid (e.g., by codon degeneration, six codons for arginine or serine), or codons that encode biologically equivalent amino acids ≪ / RTI >

Also, mutations in the nucleotides may result in changes in the protein itself. Even when the amino acid of the protein is a mutation that causes a change, it can be obtained that exhibits almost the same activity as the protein of the present invention.

The peptide and the nucleic acid molecule of the present invention are not limited to the amino acid sequence or the base sequence described in the Sequence Listing insofar as the nucleic acid or polynucleotide of the present invention has an effect as an RNA vaccine and / It is clear to a person skilled in the art. For example, the biological functional equivalent that may be included in the recombinant protein of the present invention may be a peptide having a mutation in the amino acid sequence exhibiting equivalent biological activity to the recombinant protein of the present invention.

Considering the mutation having the above-mentioned biological equivalent activity, the nucleic acid molecule encoding the peptide and / or protein according to the present invention is interpreted to include a sequence showing substantial identity with the sequence described in the sequence listing. The above-mentioned substantial identity is determined by aligning the above-described sequence of the present invention with any other sequence as much as possible and analyzing the aligned sequence using an algorithm commonly used in the art. Homology, more preferably 70% homology, even more preferably 80% homology, and most preferably 90% homology. Alignment methods for sequence comparison are well known in the art.

The term " vector " as used herein refers to a construct capable of transferring to a host cell, and preferably capable of expressing at least one gene or sequence of interest. For example, the vector may comprise DNA or viral vectors, DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA linked with cationic condensing agents (CCA) RNA expression vectors, DNA or RNA expression vectors packaged in liposomes, specific eukaryotic cells such as producer cells, and the like.

As used herein, the term "expression control / regulation sequence" or "expression control / regulation element" refers to a nucleic acid that transcription of a nucleic acid and / or regulates translation from a transcript- ≪ / RTI > On the other hand, "transcription control / regulation sequence" or "trnacription control / regulation element" means a nucleic acid sequence which regulates the transcription of a nucleic acid. The transcriptional regulatory sequence may be a promoter such as a constitutie promoter or an inducible promoter, or an enhancer. Also, the term " translation control / regulation sequence " or " translation control / regulation element " will be used for nucleic acid sequences that control translation from a nucleic acid in the transcript form to a protein or peptide . These expression control sequences / elements, transcription control sequences / elements and / or translation control sequences / elements are operatively linked to the sequence to be expressed, for example, the transcription or nucleic acid sequence to be translated.

As used herein, the term " operatively linked " refers to a functional linkage between a nucleic acid expression control sequence (e.g., a promoter, a signal sequence, a ribosome binding site, a transcription termination sequence, Whereby the regulatory sequence regulates the transcription and / or translation of the different nucleic acid sequences.

Nucleic acid molecule

According to one 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 ) Or polynucleotide or nucleic acid molecule comprising the nucleotide sequence. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram schematically illustrating the construction 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. FIG.

1, a nucleic acid molecule according to an exemplary embodiment of the present invention includes an expression control sequence (denoted by ECS) comprising a nucleotide sequence having IRES activity, an expression control sequence (ECS) And may comprise a coding region (CR) consisting of an open reading frame (ORF) of the gene of interest (GOI) encoding the peptide or protein. In one exemplary embodiment, an expression control sequence (ECS) can form a 5'-translated 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), such as 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 present invention may have an RNA form. When the nucleic acid molecule is in the form of an RNA, the coding region (CR) may consist of the transcript sequence of the gene of interest.

The expression control sequence (ECS) comprises an IRES nucleotide sequence operatively linked to the gene of interest (GOI) inserted in open reading frame format and may have the structure of a 5'-UTR comprising an IRES nucleotide sequence. The 5'-UTR is a region to which a translational initiation complex related to the translation of a gene of interest (GOI) into a protein and / or peptide is bound, and the IRES complexes secondary and tertiary structures, Lt; / RTI > is a cis-acting base sequence that induces translation of GOI.

In one exemplary embodiment, an expression control sequence (ECS) having an IRES base sequence comprises a viral (viral) IRES base sequence and / or a eukaryotic (eukaryotic) IRES base sequence . 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 selected from the group consisting of picornaviridae, Togaviridae, Dicistroviridae, Flaviridae, Retroviridae, and / or May be derived from the virus of the Herpesviridae.

For example, the viral IRES base sequence belonging to the picornaviridae belongs to the virus belonging to the Enterovirus, the virus belonging to the Cardiovirus, the virus belonging to the Apthovirus, A virus belonging to the genus Hepatovirus and / or a virus belonging to the genus Teschovirus. For example, the virus belonging to the genus Enterovirus may originate from an Enterovirus A to enterovirus J type and a virus classified as Rhinovirus A to Lynovirus C type.

In a non-limiting embodiment, the viral IRES base sequences belonging to the picornaviridae are selected from the group consisting of 1) Poliovirus (PV), Rhinovirus (RV), Cossachievirus, For example, Coxsachie virus B3, CVB3, Enterovirus71, EV71, Encephalomyocarditis virus (EMCV) belonging to Cardiovirus, Theiler murine encephalomyelitis virus (TMEV), 3) foot-and-mouth disease virus (FMDV), 4) hepatitis A virus (HAV) belonging to the genus Hepatovirus, and / or 5) Porcine teschovirus belonging to the genus Tesco virus (PTV, for example, 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 Saury virus family can be derived from the Sindbis virus (SV) belonging to the alphavirus family. In addition, the viral IRES base sequence belonging to the disturbed virus can be obtained, for example, from the genus Plautia stali intestine virus (PSIV) belonging to the genus Cripavirus, Cricket paralysis virus (CPV) ), Triatoma virus, and / or Rhopalosiphum padi virus (RXPD).

On the other hand, in the exemplary embodiment, the viral IRES base sequence belonging to Flaviviridae belongs to Hepatitis C virus (HCV) belonging to the genus Hepacivirus, HCV belonging to the genus Flavivirus May originate from the Japanese encephalitis virus (JEV), the classical swine fever virus belonging to the pestivirus (CSFV) and / or the bovine viral diarrhea virus (BVDV) . In another exemplary embodiment, the viral IRES base sequence belonging to the retroviral family is selected from the group consisting of Friend murine leukemia virus (FMLV) belonging to the gamma retrovirus genus (Gammaretrovirus), Moloney murine leukemia virus MMLV) and / or Rous sarcoma virus (RSV) belonging to the alpha retrovirus genus (Alpharetrovirus). In addition, the viral IRES base sequence belonging to the herpes virus family can be derived from Marek's disease virus (MDV) belonging to the MardiVirus, but the present invention is not limited thereto.

For example, the expression control sequence (ECS) having a viral IRES base sequence may be derived from a virus belonging to the picornaviridae and / or Dicistroviridae. For example, an expression regulatory sequence (ECS) having a viral IRES base sequence belonging to the picornaviridae may be derived from a virus belonging to the genus Enterobacteriaceae, the cardiovirus genus or the genus Aftoviridae, It can originate from the virus to which it belongs. For example, an expression regulatory sequence (ECS) having an IRES nucleotide sequence belonging to the genus of enterovirus may originate from a virus of an enterovirus such as Coxsacki virus such as CVB3, or may be derived from a virus of a cardiovirus such as EMCV.

In addition, the expression control sequence (ECS) having a viral IRES base sequence belonging to dsisterivirus may be derived from PSIV or CPV belonging to the genus Crepavirus.

In one exemplary embodiment, the expression control sequence (ECS) may have a viral-derived IRES nucleotide sequence 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) comprises a 5'-UTR (SEQ ID NO: 1) comprising an IRES nucleotide sequence derived from SV, a 5'-UTR comprising an IRES nucleotide sequence derived from CVB3 : 5'-UTR (SEQ ID NO: 3) containing the IRES nucleotide sequence derived from EMCV, 5'-UTR (SEQ ID NO: 4) containing the IRES nucleotide sequence derived from JEV and / or 5'-UTR (SEQ ID NO: 5) containing an IRES nucleotide sequence derived from CPV, but the present invention is not limited thereto.

In another exemplary embodiment, the eukaryotic IRES base sequence may have an IRES base sequence derived from eukaryotic cellular mRNA. In an exemplary embodiment, the eukaryotic IRES base sequence is selected from the group consisting of growth factors, transcription factors, translation factors, oncogenes, transporter / receptor, Activators of apoptosis, inhibitors of apoptosis, proteins localized in neuronal dendrites and / or other peptides / proteins in the dendrites of neurons, But the present invention is not limited thereto.

For example, growth factors having an IRES nucleotide sequence include fibroblast growth factor (FGF) such as FGF-1 and FGF-2, platelet-derived growth factor-B -B), vascular endothelial growth factor (VEGF), insulin-like growth factor-2 (IGF-2), and the like.

Transcription factors with the IRES nucleotide sequence include antennapedia (Antp), Ultrabithorax, ubx, MYT-2, NF-κB repressing factor (NRF), acute myelocytic leukemia protein -1 (acute myelod leukemia 1 protein, AML1, Runt-related transcription factor 1, RUNX1).

The translational factor having the IRES nucleotide sequence is Eukaryotic initiation factor 4G, eIF4G, Eukarytoic initiation factor 4GI, eIF4GI, Eukaryotic transcription factor 4 initiation factor 4 gamma 2, eIF4G2).

The cancer gene having the IRES base sequence includes c-myc, L-myc, and p53. The carrier / receptor having the IRES base sequence is a cationic amino acid transporter (for example, SLC7A1) And a voltage-gated potassium channel.

The cell planarization inhibitor with the IRES nucleotide sequence can be exemplified by the apoptotic protease activation factor 1 (Apaf-1), and the cell line inhibitor with the IRES nucleotide sequence is the X- (X-linked inhibitor of apoptosis protein, XIAP), and Bcl-2. The localized proteins / peptides in the dendrites of nerve cells are composed of fragile X mental retardation 1 (FMR1), activity-regulated cytoskeleton-associated protein (ARC) Protein-2 (MAP2), neurogranin (RC3), amyloid precursor protein, and the like.

In addition, the nucleotide sequence of the eukaryotic IRES is an immunoglobulin heavy chaing binding protein (BiP), Connexin32, connexin43, Adenomatous polyposis coil, APC), an ornithine decarboxylase, an IRES base sequence derived from hypoxia-inducible factor 1-alpha (HIF1?), And the like.

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

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

Meanwhile, the nucleic acid molecule according to the present invention may further include a nucleic acid sequence capable of increasing the expression efficiency of a gene of interest (GOI). For example, a number of adenosines or thymines may be inserted adjacent to an expression control sequence (ECS), which illustrates the insertion of multiple adenosines (pMA) 5 'to the expression control sequence (ECS) In this case, since a plurality of adenosine (pMA) shows a transcript form, the nucleic acid molecule in the DNA state is adjacent to the expression control sequence (ECS), for example, pMT) may be interpreted as being inserted. In one exemplary embodiment, 20 to 400, preferably 30 to 300, more preferably 30 to 200, and most preferably 30 to 200 adjacent to the expression control sequence (ECS) having an IRES base sequence 100 consecutive adenosines or thymines may be inserted.

In this case, the expression level of the gene of interest (GOI) can be increased or maintained at an equivalent level, compared with a nucleic acid molecule in which a plurality of adenosines or thymines are not inserted adjacent to the expression control sequence (ECS). According to one exemplary embodiment, an expression control sequence (ECS), in which a plurality of adenosines or thymines are inserted adjacent to each other to increase the expression level of a gene of interest (GOI) or maintain an equivalent level, For example, it may be derived from the viruses of picornaviridae, Togaviridae, Dicistroviridae, Flaviridae, Retroviridae and / or Herpesviridae have.

On the other hand, the coding region CR can be composed of a nucleic acid sequence of a gene of interest (GOI) which can be expressed as a protein or a peptide. An expression control sequence (ECS) can be operably linked to the coding region (CR). In one exemplary embodiment, the coding region (CR) may be located 3 'to 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) can consist of a reporter protein / peptide, a marker or an open reading frame (ORF) encoding a selectable protein / peptide. For example, an open reading frame (ORF) of a coding region (CR) capable of encoding a reporter protein or peptide can be obtained from Renilla luciferase (e.g., SEQ ID NO: 19 or SEQ ID NO: 20) / RTI > such as luciferase, Green Fluorescent Protein (GFP), Enhanced Green Fluorescent Protein (EGFP) and / or the like, such as Firefly luciferase Beta-galactosidase. ≪ / RTI > In addition, the open reading frame encoding the marker or the selected protein / peptide may include a nucleic acid sequence encoding alpha-globin, galactokinase and / or xanthine guanine phosphoribosyl transferase, and the like. However, an open reading frame encoding another reporter protein / peptide and / or marker or a selectable protein / peptide may be inserted into the coding region (CR).

In another exemplary embodiment, the coding region (CR) may consist of an open reading frame encoding an antigen, fragment, variant or derivative thereof. As an antigen, for example, a tumor antigen, an animal antigen, a plant antigen, a viral antigen, a bacterial antigen, a fungal antigen and a protozoan antigen, an autoimmune antigen or an allergen can be considered. Secreted forms of surface antigens and surface antigens, particularly viral pathogens, bacterial pathogens, fungal pathogens or protozoan pathogens, of tumor cells are preferred.

If desired, the antigen may be present, for example, in a nucleic acid molecule according to the present invention and as a haptene attached 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), such as 5T4, 707-AP, 9D7, AFP, AlbZIP HPG1, alpha-5- MRNA, alpha-methylacyl-coenzyme A racemase, ART-4, ARTC1 / m, B7H4, BAGE-1, BCL-2, bcr- CA12, CA12-9, CA72-4, CA125, calreticulin, CAMEL, < RTI ID = 0.0 > M, CDA2, CDA3, CDA4, CD20, CD33, CD4, CD52, CD56, CD80, CDC27 / m, CDK4 / Cyclin B1, Cyclin D1, cyp-B, CYPB1, DAM, and the like, as well as CMLA2, CML28, CML66, COA-1 / m, coactosin-like protein, collagen XXIII, COX-2, CT-9 / BRD6, Cten, FGF-5, FN, Frau-1, G250, GAGE, EGFR, EGFR, ELF2 / m, EMMPRIN, EpCam, EphA2, EphA3, ErbB3, ETV6-AML1, EZH2, DEK-CAN, 5, GAGE-6, GAGE7b, GAGE-8, GDEP, GnT-V, gp100, GPC3, GPNMB / m, HA HEL-A11 / m, HLA-A2 / m, HNE, homeobox NKX3.1, HOM-TES IL-1R, IL-2R, IL-5, IL-8, IL-8, IL-8, A1, MAGE-A1, MAGE-A2, MAGE-A2, KLK-1, K-Ras / B2, MAGE-B3, MAGE-B4, MAGE-B5, MAGE-B6, MAGE-A10, MAGE-A12, MAGE- M2, MAGE-D1, MAGE-D2, MAGE-D4, MAGE-E1, MAGE-E2, MAGE-F1, MAGE-H1, MAGE-C1, MAGE- MART-1 / melan-A, MART-2, MART-2 / m, matrix protein 22, MC1R, M-CSF, ME1 / m, mesothelin, MG50 / MUN-1 / m, MUM-2 / m, MUM-3 / m, myosin class I / m, NA88-A , N-acetylglucosaminyltransferase-V (Glucosaminyl transferase-V), Neo-PAP, Neo-PAP / m, NFYC / m, NGEP, NMP22, NPM / ALK, N-Ras / , Osteocalcin, osteopontin, p15, p190 minor bcr-abl, p53, osteopontin, osteopontin, PARA, POTE, PRAME, PRDX5 / m, prostane, p53 / m, PAGE-4, PAI-1, PAI-2, PART-1, PATE, PDEF, 1, SART-1, SAGE-2, SAGE-1, SAGE-1, SAGE-1, RAGE-1, RBAF600 / m, RHAMM / CD168, RU1, RU2, S-100, PSA, PSCA, , SART-3, SCC, SIRT2 / m, Sp17, SSX-1, SSX-2 / HOM-MEL-40, SSX-4, STAMP-1, STEAP, 1, TRP-1, TRP-1, SYT-SSX-2, TA-90, TAG-72, TARP, TEL-AML1, TGF beta, TGF beta RII, TGM- 2 / 6b, TRP / INT2, TRP-p8, tyrosinase, UPA, VEGF, VEGFR-2 / FLK-1 and WT1. A family of tumor antigens is suitable for tumor antigens known to be involved in neovascularization, which affects the purpose of the present invention, for example, extracellular matrix structure and the like. Tumor antigens may be provided in pharmaceutical compositions as protein or peptide antigens or as mRNA or DNA encoding tumor antigens or epitopes thereof, preferably such tumor antigens.

For example, a pathogenic antigen can be derived from an individual, e. G., A mammalian subject, particularly a pathogenic organism that elicits an immune response by humans. As an example, a pathogenic antigen can be derived from bacterial, viral, or protozoan (multicellular) pathogenic organisms. In one exemplary embodiment, a pathogenic antigen is surface antigens, such as proteins (or fragments of a protein, e.g., an outer portion of the surface) located on the surface of an organism such as a virus, bacteria, or protozoa, Lt; / RTI >

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 diseases. Non-limiting examples of pathogens associated with infectious diseases are: Acinetobacter baumannii, Anaplasma genus, Anaplasma phagocytophilum, Ancylostoma braziliense, , Ancylostoma duodenale, Arcanobacterium haemolyticum, Ascaris lumbricoides, Aspergillus genus, Astroviridae, Babesia genus, Aspergillus spp., Aspergillus spp. Bacillus anthracis, Bacillus cereus, Bartonella henselae, BK virus, Blastocystis hominis, Blastocystis hominis, Bacillus anthracis, Blastomyces dermatitidis, Bordetella pertussis, Borrelia burgdorferi, Borrelia burgdorferi, Borrelia genus, Borrelia spp, Brucella genus, Brugia malayi, Bunyaviridae family, Burkholderia cepacia and other burgundy spp. Burkholderia mallei, Burkholderia pseudomallei, Caliciviridae family, Campylobacter genus, Candida albicans, Candida albicans, Candida albicans, Candida albicans, The present invention relates to a method for producing Candida spp, Chlamydia trachomatis, Chlamydophila pneumonia, Chlamydophila psittaci, CJD prion, Clonorchis sinensis, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium perfringens, Clostridium botulinum, diarrhea, dium perfringens, Clostridium spp., Clostridium tetani, Coccidioides spp., coronaviruses, Corynebacterium diphtheria, Cryptococcus neoformans, Cryptosporidium genus, Cytomegalovirus (CMV), dengue fever (Cryptococcus neoformans), Cryptosporidium genus, Cryptococcus spp. Viruses such as Dengue viruses (DEN-1, DEN-2, DEN-3 and DEN-4), Dientamoeba fragilis, Ebolavirus (EBOV), Echinococcus genus, Ehrlichia chaffeensis, Ehrlichia ewingii, Ehrlichia genus, Entamoeba histolytica, Enterococcus genus, En (En terovirus genus, Enteroviruses, mainly Coxsackie A virus and Enterovirus 71 (EV71), Epidermophyton spp, Epstein-Barr (EBV), Escherichia coli O157: H7, O111 and O104: H4, Fasciola hepatica and Fasciola gigantica, FFI prion For example, FFI prion, Filarioidea superfamily, Flaviviruses, Francisella tularensis, Fusobacterium genus, Geotrichum candidum ), Geardia intestinalis, Gnathostoma spp, GSS prion, Guanarito virus, Haemophilus ducreyi, Hemophilus, Haemophilus influenza (Haemophilus influenza), Helicobacter A virus such as Helicobacter pylori, Henipavirus (Hendra virus nipah virus), Hepatitis A virus, Hepatitis B virus (HBV), hepatitis C virus 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 such as arabinfluenza viruses (HPIV), Japanese encephalitis virus, JC virus, Junin virus, Kingella kingae, Klebsiella granulomatis, The present invention relates to a method for the treatment and / or prophylaxis of a disease selected from the group consisting of Kuru prion, Lassa virus, Legionella pneumophila, Leishmania genus, Leptospira genus, Listeria monocytogenes, Malassezia spp., Marburg virus, measles virus, Metagonimus yokagawai, and the like), viruses (Lymphocytic choriomeningitis virus (LCMV), Machupo virus, Malassezia spp. , Microsporidia phylum, Middle East respiratory syndrome coronavirus (MERS-CoV), Molluscum coronagiisorumberia Molluscum contagiosum virus (MCV), Mumps virus, Mycobacterium leprae and Mycobacterium lepromatosis, Mycobacterium tuberculosis, Mycoplasma pneumonia, Naegleria fowleri, Necator americanus, Neisseria gonorrhoeae, Neisseria meningitides, and the like), Mycobacterium ulcerans, Mycoplasma pneumonia, ), Nocardia asteroids, Nocardia spp, Onchocerca volvulus, Orientia tsutsugamushi, Orthomyxoviridae family (Influenza), and the like. Paracoccidioides brasiliensis, Paragonimus spp, Paragonimus 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 spp. For example, Salmonella genus, Sarcoptes scabiei, SARS coronavirus, Schistosoma genus, Shigella genus, For example, Sin Nombre virus, Hantavirus, Sporothrix schenckii, Staphylococcus genus, Staphylococcus genus, Streptococcus agaractia, 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, Trichinella spiralis, Trichomonas vaginalis, Trichophyton spp, Trichuris trichiura, Trypanosoma brucei, Trypanosoma cruzi, Ureaplasma urealyticum, varicella zoster virus (VZV), varicella zoster virus (VZV) Variola major or Variola minor, vCJD prion, Venezuelan equine encephalitis virus, Vibrio cholera, West Nile virus, , Western equine encephalitis virus, Wuchereria bancrofti, Yellow fever virus, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis and Zika virus.

Particularly preferred in this connection are influenza viruses, respiratory syncytial virus (RSV), herpes simplex virus (HSV), HPV, HPV, plasmodium, staphylococcus aureus, (CMV), hepatitis B virus (HBV), Mycobacterium tuberculosis, rabies virus, yellow fever virus, Middle respiratory syndrome coronavirus (MERS-CoV), and / or Zicca virus ≪ / RTI >

That is, in one exemplary embodiment, the coding region CR comprises a protein and / or peptide capable of inducing an immune response in vivo, such as an antigen, i. E., A gene of interest (GOI) encoding the immunogenic protein and / 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, a nucleic acid molecule according to the present invention may have an RNA form. At this time, when the coding region CR has a transcript sequence of a gene of interest (GOI) encoding a protein and / or a peptide capable of causing an immune response in vivo such as the above-mentioned pathogenic antigen, Can be used as an RNA vaccine and injected directly into a living body. These RNA vaccines may have the following advantages over DNA vaccines.

First, the human body is superior to the DNA vaccine. Because RNA vaccines, like DNA vaccines, can synthesize the desired peptide or protein in the cytoplasm without the need to enter the nucleus for transcription into mRNA, the RNA vaccine is superior to the DNA vaccine in terms of biological safety. In other words, the RNA vaccine is not likely to break into the chromosome of the host in the nucleus, and the antibiotic resistance gene, which is a selective marker used for selective production in host cells, is unnecessary for the production of RNA vaccines and RNA has a shorter half- It does not induce long-term genetic transformation. In other words, a general RNA vaccine is delivered in a cell and is only activated for a short period of time, expressing the target peptide / protein and destroying in an enzymatic reaction in a few days, but a specific immune response to the originally expressed target peptide / protein remains.

Second, RNA vaccines can induce the desired in vivo immune response even when using a small amount relative to DNA vaccines. Unlike DNA vaccines, RNA vaccines do not need to enter the nucleus and only pass through the cell membrane, so RNA vaccines can express the same amount of target peptide / protein even when using less than the DNA vaccine. In addition, the RNA itself may be utilized as an adjuvant.

Third, because all production processes can be artificially controlled, vaccines can be safely produced in small-scale good manufacturing practice (GMP) production facilities without the risk of biological contamination. In other words, RNA vaccines require only small-scale GMP lab-level facilities and do not need to deal with direct infections (viruses or pathogenic microorganisms), so that various vaccines can be manufactured and produced quickly.

Fourth, RNA vaccines can induce stronger immune responses than naked DNA vaccines. This is due to the mutual co-ordination of the innate immune response induced by the stem-loop structure of the RNA vaccine and the RNA itself and the acquired immune response induced by the target peptide / protein expressed in the immunized host cell I think. In addition, the RNA vaccine itself produces a complex antigen in the cell, which can act as an ideal vaccine by approaching the major histocompatibility complex (MHC) class of antigen-presenting cells.

Fifth, RNA vaccines are easier to produce than conventional vaccines. As it described above, it is not necessary to deal with the source of infection directly in the production of RNA vaccines, synthetic instead of only the nucleic acid sequence of the neutralizing antibody induced relevant part (neutralization epitope) of the infectious agent to be expressed artificially, and in vitro transcription (in vitro transcription, IVT) alone can produce a large amount of RNA vaccine. Recently, reagents related to IVT reaction, especially DNA-dependent RNA polymerase, have been improved and a large amount of RNA can be rapidly produced in 1 to 2 weeks using a small amount of DNA template. In addition, since a large number of antigens to induce an immune response can be produced and mixed at the same time, they can be immunized, and there is no particular restriction on the gene length of an antigen to be expressed, which increases applicability and simplicity of RNA vaccine production.

Since the nucleic acid molecule of the present invention contains an IRES nucleotide sequence which does not require such a complicated capping process as an expression regulatory sequence (ECS), it is possible to solve the problems related to the capping process, Lt; / RTI >

In another embodiment, the coding region CR can be inserted with a transcript sequence of a gene of interest (GOI) or a gene of interest (GOI) that encodes proteins and / or peptides, or fragments thereof, associated with disease treatment. 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 disease treatment is a therapeutic protein / peptide used in the treatment of metabolic or endocrine disorders; Therapeutic proteins / peptides used in the treatment of 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 somatic cells to pluripotent stem cells or ovarian stem cells; A therapeutic protein / peptide and / or antibody selected from an adjuvant or an immunostimulatory protein. Specific examples of proteins or peptides related to disease treatment are disclosed in Korean Patent Publication No. 2014-0125434.

For example, the protein or peptide used in the treatment of metabolic or endocrine disorders is selected from the group consisting of acid sphingomyelinase, adipotide, agalcidase-beta, alglucosidase, alpha-galactosidase A, alpha- Angiopoietin (Ang1, Ang2, Ang3, Ang4, ANGPTL2, ANGPTL3, ANGPTL4, ANGPTL5, ANGPTL6, ANGPTL7 ), Beta-glucuronidase, bone formation protein BMP (BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP10, BMP15), CLN6 protein, epithelial growth factor FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-1, fibroblast growth factor 9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, FGF-16, FGF-17, FGF-17, FGF- FGF-22, FGF-23), galosalphase, ghrelin, glucocerebrosidase, GM-CSF, heparin-binding EGF-like growth factor (HB- (Iduronate-2-sulfatase), integrin? V? 3,? V? 5 and? V? 1, iduronate sulfatase, loridase, N-acetylgalactose (NGF), brain-derived neurotrophic factor (neuropathy), neurotrophic factor (neuropathy, neurofibrillary tangles, neurotrophic factor, BDNF), neurotrophin-3 (NT-3), neurotrophin 4/5 (NT-4/5), neuregulin (NRG1, NRG2, NRG3, NRG4), neurofilin (TGF-beta1 receptor, TGF-beta2 receptor, TGF-beta receptor), angiostatin, platelet-derived growth factor (PDGF, PDGF-B, PDGF-C, PDGF- TGF-beta (TGF beta 1, TGF beta 2 and TGF beta 3)), tumor growth factor (MGDF), tumor growth factor (TGF-a) ), VEGF (VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F and PIGF) (ACTH), atrial natriuretic peptide (ANP), cholestystinin, gastrin, leptin, oxytocin, somatostatin, vasopressin (antidiuretic hormone), calcitonin, exenatide, growth hormone (GH), somatotroph (IGF-1), insulin-like growth factor 1 (IGF-1), mechacillin rinfabate, IGF-1 analogue, mechacillamine, IGF-1 analog, pegvisomant, pramlintide, teriparatide 1 to 34), bequaplumine, dibotomine-alpha (bone formation protein 2), histrelin acetate (gonadotropin releasing hormone; GnRH), octreotide, and palipermin (keratinocyte growth factor (KGF)).

Peptides or proteins used in the treatment of blood diseases, circulatory diseases, respiratory diseases, cancer or tumor diseases, infectious diseases or immunodeficiency diseases include, but are not limited to, alteplase (tissue plasminogen activator; tPA), anistreplase, antithrombin Alpha (activated protein C), erythropoietin, epoetin-alpha, erythropoietin, erthropoietin, Factor IX, factor VIIa (AT-III), bivalirudin, darbepoetin- , Factor VIII, Repirudin, Protein C concentrate, Retapla1 (deletion mutant protein of tPA), Streptokinase, Tenecteplase, Eukinase, Angiostatin, Anti-CD22 immunotoxin, Denirukin diptitox, , MPS (metallopartilemylin), influenzacepha, endostatin, collagenase, human deoxyribonuclease I, dornase, hyaluronidase, papain, L-asparaginase, Peg-asparagine Naje, Ras Human chorionic gonadotropin (HCG), human follicle stimulating hormone (FSH), rouropin-alpha, prolactin, alpha-1-proteinase inhibitor, lactase, pancreatic enzyme (lipase, amylase, protease ), Adenosine deaminase (SOPEGADEMASE, PEG-ADA), Avatacept, Alephecept, Anakinra, etanercept, interleukin-1 , Enfuviride, and thymosin alpha 1.

In addition, human adjuvant proteins, particularly pattern recognition receptors TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11; NALP6, NALP6, NALP7, NALP7, NALP8, NALP9, NALP10, NALP11, NALP12, NALP13, NALP14, IIPAF, NAIP, CIITA, RIG- I, MDA5 and LGP2, for example, Trif and Cardif; The components of small-GTPase signaling (RhoA, Ras, Rac1, Cdc42, Rab etc.), constituents of PIP signaling (PI3K, Src-kinase etc.), constituents of MyD88-dependent signaling (MyD88, IRAK1, IRAK2 , IRAK4, TIRAP, TRAF6, etc.), MyD88-independent signaling components (TICAM1, TICAM2, TRAF6, TBK1, IRF3, TAK1, IRAK1, etc.); An activated kinase 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 , An activated transcription factor including the Typ III repeat extra domain A of gp96, fibrinogen, fibronectin; Or MASP-1, MASP-2, C4b, C3b, C5a, C3a, C4a, C5b, C6, C7, C8, C9, CR1, CR2, CR3, CR4 , C1qR, C1INH, C4bp, MCP, DAF, H, I, P and CD59, or an inducible target gene comprising, for example, beta -depentin, a cell surface protein; IL-1 beta, IL-2, IL-6, IL-7, IL-8, IL-9, IL-12, IL-13, Including but not limited to IL-15, IL-16, IL-17, IL-18, IL-21, IL-23, TNF alpha, IFN alpha, IFN beta, IFN gamma, GM-CSF, G- , Human adjuvant proteins including cytokines that induce or augment innate immune responses; Chemokines including IL-8, IP-10, MCP-I, MIP-I alpha, 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, particularly Hsp60, Hsp70, Hsp90, Hsp100; Gram-negative bacterial origin OmpA (outer membrane protein); Bacterial forin containing OmpF; (PT) derived from Bordetella pertussis, pertussis adenylate cyclase toxins CyaA and CyaC derived from Bordetella pertussis, PT-9K / 129G mutant derived from pertussis toxin, Bordetella pertussis (CT), cholera toxin B subunit, cholera toxin-derived CTK63 mutant, CT-derived CTE112K mutant, Escherichia coli heat-labile intestinal toxin A bacterial toxin comprising a toxin-reduced Escherichia coli heat labile intestinal toxin mutant, including LT (LT), heat labile intestinal toxin derived B subunit (LTB), LTK63 and LTR72; Phenol solubility modulus; Helicobacter pylori-derived neutrophil activation protein (HP-NAP); Surfactant protein D; Outer surface protein A lipoprotein derived from Borrelia burgdorferi, Ag38 (38 kDa antigen) derived from Mycobacterium tuberculosis; Bacterial ciliated proteins; Intestinal toxin CT on V. cholera, gram-negative bacterial keratin-derived fillin, and surface active protein A and bacterial plasmin, protozoal proteins, especially Tc52 derived from Trypanosoma krugi, PFTG derived from Trypanosoma gondii, protozoan heat shock protein, (F-protein), MMT virus-derived coat protein, mouse leukemia virus protein, wild type measles virus-derived Lei, Toxoplasma gondii-derived profiling-like protein, virus (adjuvant) A muglutinin protein, a fungal (adjuvant) protein, especially a fungal immunomodulatory protein (FIP; LZ-8); And a therapeutic protein selected from an adjuvant or immunostimulatory protein, including keyhole limpet hemocyanin (KLH), OspA; Therapeutic proteins used in hormone replacement therapy, particularly estrogen, progesterone or progestin, and testosterone; (Sox1, Sox2, Sox3 and Sox15), the Klf group (Klf1, Klf2, Klf4 and Klf5), which are used for the reprogramming of somatic cells into pluripotent stem cells or ovarian stem cells, Myc family (c-myc, L-myc and N-myc), Nanog and LIN28; And therapeutic antibodies selected from the antibodies used in the treatment of cancer or tumor diseases, in particular the therapeutic antibodies selected from the group consisting of 131I-Tositumomap, 3F8, 8H9, Avagobomap, Adekatumumap, Aputujumap, Alashizapagol, Tuxi map, AME-133v, AMG 102, Anaatumo map Maepanatox, Apolyumu map, Bobby Tuxi map, Beck tomomo map, Belimu map, Bebashir map, Bivatu zap martensin, Blena tomomap, Bren Kantoumapombetide, Kaloomm, Kattoumarsomaps, Cetuximaps, Sittuzumab Borgotox, Drinking Water Tumumap, Klimtujumapatte, Kwangwoonjungmatan, Lecce Tan, CNTO 328, CNTO 95, Konatumu Map, Dasetou Zummap, Dalotou Zummap, Denosu Map, Des Tomomaps, Drawing Map, Ecrom Mex Map, Ed Recol Map, , Enabatu Zoom map, Enshitux map, Efratu Zoom map, Er Tu Tu Sole map, Er Tu Tu Sole map, Gigantu Mapum, Gilgen Tuxi Map, Glemt Baumum Mapidotin, Gigantu Mapum, Maple Map, Gagliot Map, GC1008, , GS6624, HuC242-DM4, HuHMFG1, HuN901-DM1, ibritumomap, icculum map, ID09C3, indatux mapabtansine, inotoumaps ozogamicin, intetumu map, MORAB-003, MEDI 522, MITUMO MAP, MOGA PHYSICAL MAP, MORAB-003, MELAB-003, MELAB-003 , MORab-009, Mokhapomo Map Pohang Tox, MT103, Naho Colomap Tapanatox, Lead Tumo Map Estapenatox, Nernatum Map, Nesitoumu Map, Nemotou Map Map, Nemotou Map Map, Oregon map, Oregon map, Oregon map, PAM4, Panny toum map, Patriot map, Femtomo map, Peru map, Preitum map, SGN-30, SGN-40, Sivurouzumapu, Syltuxi Map, Tavaluma Map, Takaru Map, Ratatoumo Map, Radturupmap, Ramushiru Map, Lilo Tumemap, Rituximap, Rovatumumapu, TNT-TTM-650, TNT-TTM-MAP, TTTToMuMap, TGN1412, Ticilmum Map (Trammellimu Map) TRU-016, TRU-016, Tuco to Zuem Cell Morookin, Yubulituxi Map, Ulrelu Map, Beltu Map, IMU-106, Boloshi Map, Bautomum Map, WX-G250, Zalutumumap and Zanorium mapp; Antibodies used in the treatment of immune disorders, particularly antibodies used in the treatment of immune diseases, especially antibodies used in the treatment of immune disorders, such as antibodies, such as antibodies, immunoglobulins, immunoglobulins, Bismillah Map, Bismillah Map, Leslie's Map, Adalmum Map, Asceli Mapum, Artinu Map, Artemis Map, Virtirem Map, Bismillah Map, Bismillah Map, 945429, briquinuke map, broodal map, kanakinu map, kanakinu map, cortolite zip mapol, early zip map, pesah kinu map, golimum map, omilik city map, infix map, SBI-087, SBI-087, Sekyukinu Map, Sirukumapu, Tariu Zipmap, Tosalu Zipmap, Torii Zipmap, TRU-015, Oulurimu Map, Ozulurimu Map, , TRU-016, Yuseki Kin Map, Yuseki Kin Map, Bep Palymo Map, Jolimom Map Aritox, Shifa Lim Map, Called immune glue and the like; Antibodies used in the treatment of infectious diseases, in particular Aphelmimag, CR6261, Edobakomaps, Efun Guramm, Xbibiroomm, Pelvisimap, Poraviramap, Ivar Lijimap, Libbyvirap, Motavivapap, Nevekap , PRO TO 140, Rafibiru Map, Lakshivak Map, Levabiru Map, Cebiru Map, Defibu Map and Tefiba are all available on the map. Main map; Antibodies used in the treatment of blood disorders, in particular, emmisimaps, atorolimu maps, ecuriujimaps, mepolipmaps and milafujumaps; Antibodies used for immunomodulation, in particular antitumour cell globulin, basiliximag, sedelijumap, daclizumap, gabilimomap, inolimomaps, mylomolab-cd3, mylomolab-cd3, Shipyour Map; Antibodies used in the treatment of diabetes, in particular Geboky's map, Othelic's map and Tepley's map; Antibodies used in the treatment of Alzheimer ' s disease, in particular Bapphynuemaps, Creteumplum, Gentenemaps, Ponzemaps, R1450 and Solanumplum; Antibodies used in the treatment of asthma, in particular Benrali Jumap, Enoki Jumap, Kellysium Map, Rebricki Jumap, Oomari Jumap, Oxeluma Map, Pascoal Jumap and Trilokinumap; And a therapeutic protein selected from the antibodies used in the treatment of various diseases, in particular a blooming map, a carro Rx, a presoleum map, a furan map, a Lomo shochu map, a star mulap map, a tanemu map and a Ranibizum map, Derivatives thereof.

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, the recombinant expression vector using the same, It is not a big consideration in the development of vaccines. Codon usage is usually known to affect protein / peptide expression in a variety of species, but human codon usage bias is usually not known to have a significant effect on protein / peptide expression, so developing humanized nucleic acid vaccines or gene therapy products This is not the case. However, the start codon should have the Kozak sequence, and the nucleotide sequence near the stop codon should also be optimized. If necessary, the third portion of the codon sequence of the gene of interest (GOI) or its transcript, which is to be expressed, may be replaced with GC to increase the GC% of the target gene without changing the amino acid, thereby increasing the stability of the mRNA.

On the other hand, in the case of having a 5'-UTR containing an IRES nucleotide sequence as an expression control sequence (ECS), the nucleic acid molecule of the present invention may further include 3'-UTR to improve the expression efficiency of a gene of interest (GOI) have. At this time, the coding region CR may be located between the 5'-UTR and the 3'-UTR. The 3'-UTR enhances the translation efficiency of the gene of interest (GOI) or its transcript with the 5'-UTR, and plays an important role in keeping the transcriptional mRNA stable in the cell without destruction.

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

In one exemplary embodiment, the viral 3'-UTR comprises a viral IRES base sequence selected from the group consisting of picornaviridae, Togaviridae, Dicistroviridae, Flaviridae, May be derived from the viruses of the virus family (Retroviridae) and / or the herpesviruses (Herpesviridae).

The viral 3'-UTRs belonging to the picornaviridae are classified as follows: 1) PV, RV derived from the genus Enterovirus, Cossachievirus such as CVB3, EV71, 2) Cardiovirus (EMCV , TMEV, 3) FMDV belonging to the genus Afthovirus, 4) HAV belonging to the genus of hepatovirus, and / or 5) PTV belonging to the genus Tesvikovirus, for example PTV-1 and combinations thereof. But is not limited thereto.

In addition, the viral 3'-UTR belonging to toovirus can originate from SV, while the viral 3-UTR belonging to disturbed virus belongs to Cripavirus PSIV, CPV, triatomavirus (Triatoma virus, and / or RXPD. In an alternative embodiment, the viral 3'-UTRs belonging to the flavivirus family are HCV belonging to the Hepacivirus, JEV belonging to the Flavivirus, CSFV belonging to the Pestivirus and / / RTI > and / or BVDV. In another exemplary embodiment, the viral 3'-UTRs belonging to the retroviral family can be derived from FMLV, MMLV, and / or RSV belonging to the alpha retroviral genus (Alpharetrovirus) belonging to the gamma retroviral genus (Gammaretrovirus). In addition, the viral 3-UTR belonging to the herpes virus family can be derived from MDV belonging to the MardiVirus family, but the present invention is not limited thereto.

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

In another exemplary embodiment, the 3'-UTR is selected from the group consisting of growth factors, transcription factors, translation factors, oncogenes, transporter / receptor, May be derived from activators of apoptosis, inhibitors of apoptosis, proteins localized in neuronal dendrites and / or other peptides / proteins in the dendrites of neurons , But the present invention is not limited thereto.

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

On the other hand, the nucleic acid molecule may have a transcription control sequence (TCS) that promotes the transcription of the polynucleotide adjacent to the expression control sequence. For example, the transcriptional control sequence (TCS) may be located 5 'to the expression control sequence (ECS). Such transcriptional regulatory sequence (TCS) is not particularly limited, and will be described in detail in the following recombinant expression vector part in order to avoid duplication of explanation.

In addition, the nucleic acid molecule induces the expression of the gene 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 Can be inserted. In one exemplary embodiment, a Kozak sequence can be inserted between the initiation codon of the coding region (CR) and the expression control sequence (5'-UTR with an IRS nucleotide sequence) that has an IRES nucleotide sequence. If necessary, a downstream hairpin structure (DLP) may be located 3 'to the expression control sequence (ECS). For example, when a 5'-UTR derived from SV (SEQ ID NO: 1) is applied as an expression control sequence (ECS), the SV-derived DLP nucleic acid sequence (e.g., SEQ ID NO: 9) Coding region.

In another alternative embodiment, a nucleotide sequence (e.g., CCTGCT) that can act as an initiation codon is inserted and / or inserted into the 3 'side of the expression control sequence (ECS) (E.g., ATGGCAGCTCAA) capable of enhancing the expression efficiency of the GOI.

In addition, when the 3'-UTR is used, the 3'-side of the 3'-UTR of the coding region CR can be stabilized while the target gene (GOI (Poly (A)) sequence capable of further improving the translation efficiency of the polyadenosine (poly (A)) can be further inserted. In one example, the poly (A) sequence is a nucleic acid sequence comprising about 25 to about 400, preferably 30 to 400, more preferably 50 to 250, most preferably 60 to 250 adenosine nucleotides .

On the other hand, FIG. 1 shows a nucleic acid molecule containing one coding region and one expression control sequence. In another embodiment, the nucleic acid molecule of the invention comprises a plurality of coding regions that encode a plurality of expression control sequences having an IRES base sequence and a protein and / or peptide that can be expressed by either expression control sequence . Figure 2 schematically depicts nucleic acid molecules comprising a plurality of expression control sequences and a plurality of coding regions.

2, a nucleic acid molecule according to another embodiment of the present invention comprises a first expression control sequence (ECS 1; for example, 5'-UTR 1) having an IRES base sequence and a first expression control sequence (ECS 1) and a second expression control sequence (ECS2; e.g., 5'-UTR2) located on the 3 'side of SEQ ID NO: 1. In one exemplary embodiment, the first expression control sequence (ECS 1) and the second expression control sequence (ECS 2) are each a 5 'amino acid sequence having the viral IRES nucleotide sequence and / or the eukaryotic IRES nucleotide sequence described above, -UTR. At this time, the first expression control regulatory sequence (ECS 1) and the second expression control sequence (ECS 2) may originate from the same source or may originate from different sources.

Meanwhile, a nucleic acid molecule according to an exemplary embodiment of the present invention may include, for example, a plurality of expression control sequences (ECS 1, ECS 2) adjacent to an expression control sequence of at least one of a plurality of expression control sequences , ECS 2), a plurality of adenosine or thymine sequences are inserted at the 5 'side of at least one of the expression control sequences. In Figure 2, a number of adenosine or thymine sequences are inserted at the 5 'side of the first expression control sequence (ECS 1) located close to the transcription control sequence (TCS). However, a plurality of adenosine or thymine sequences capable of enhancing the expression efficiency of the gene of interest (GOI 1, GOI 2) may be inserted 5 'to the second expression control sequence (ECS 2), or the first expression control sequence ECS 1) and the second expression control sequence (ECS 2).

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) are selected from the group consisting of picornaviridae, Togaviridae, Dicistroviridae, Flaviridae, Retroviridae and / or Herpesviridae viruses.

In another alternative embodiment, the first expression control sequence (ECS 1) and / or the second expression control sequence (ECS 2) may have an IRES nucleotide sequence derived from a eukaryotic cell. The viral IRES nucleotide sequence, the type of eukaryotic IRES nucleotide sequence, and the like are the same as those described with reference to FIG. 1, and therefore, detailed description thereof will be omitted.

In one exemplary embodiment, a first expression control sequence (ECS 1) and / or a second expression control sequence (ECS 2) having a viral IRES base sequence is selected from the group consisting of picornaviridae and / Dicistroviridae. ≪ / RTI > For example, at least one of the first / second expression control sequences (ECS 1 / ECS 2) having a viral IRES base sequence belonging to the picornaviridae is selected from the group consisting of enterovirus, cardiovirus or virus belonging to the genus Afthoviridae , And particularly preferably from a virus belonging to the genus Enterovirus. For example, at least one of the first / second expression control sequences (ECS 1 / ECS 2) having an IRES nucleotide sequence belonging to the genus of enterovirus is derived from an enterovirus virus such as Coxsack virus such as CVB3, Or from viruses in the same cardiovirus.

Also, at least one of the first / second expression control sequences (ECS 1, ECS 2) having the viral IRES base sequence belonging to the dysthymic virus family may be derived from PSIV or CPV belonging to the genus Crepavirus.

Meanwhile, in an exemplary embodiment, the coding region comprises a first coding region (CR 1) located between the first expression control sequence (ECS 1) and the second expression control sequence (ECS 2) And 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 by an open decoding of the first gene of interest (GOI 1) and the second gene of interest (GOI 2), respectively. The first coding region GOIl and the second coding region GOI2 are regions of interest for coding the reporter proteins / peptides, markers or identifying proteins / peptides, antigens and / or proteins / The open reading frame (ORF).

According to one exemplary embodiment, a first coding region CR 1 is operably linked to a first expression control sequence (ECS 1; e.g., 5'-UTR 1), a second coding region (CR 2) May be operably linked to a second expression control sequence (ECS 2; e.g., 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 respectively open to encode different proteins or peptides It can be done as a decryption framework. In this case, one nucleic acid molecule can express a different protein or peptide. In one exemplary embodiment, when the first coding region (CR 1) and the second coding region (CR 2) are each made up of an open reading frame encoding different antigens or fragments thereof, the nucleic acid molecule Recombinant expression vectors can be used to express different antigens and can be used as gene vaccines to prevent many diseases.

In another exemplary embodiment, when the first coding region (CR 1) and the second coding region (CR 2) are each made of an open reading frame encoding a different therapeutic protein or peptide, the nucleic acid molecule or recombinant Expression vectors can be used to treat different diseases.

1, a nucleic acid molecule comprising a plurality of expression regulatory sequences (ECS 1, ECS 2) and a plurality of coding regions (CR1, CR2) shown in FIG. 2 is inserted into the gene of interest (GOI 1, GOI 2 Lt; RTI ID = 0.0 > expression). ≪ / RTI > For 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 nucleotide sequence, UTR < / RTI > At this time, the 3'-UTR may preferably be located on the 3 'side of the second coding region CR 2. The 3'-UTR is not particularly limited, but it is possible to use the viral and / or eukaryotic 3'-UTR described with reference to FIG.

For example, the 3'-UTR can be derived from 5'-UTR2, which can be the 5'-UTR1 or the second expression control sequence (ECS2), which can be the first expression control sequence (ECS1) have. In one exemplary embodiment, the 3'-UTR can be derived from the same source as 5'-UTR 1, but the invention is not so limited.

In addition, the nucleic acid molecule shown in FIG. 2 is operably linked to a transcription control sequence (TCE) located adjacent to the expression control sequences (ECS 1, ECS 2), to respective expression control sequences (ECS 1, ECS 2) And a Kozak sequence between linked coding regions (CR1, CR2). If necessary, the 3 'side of each of the expression control sequences (ECS 1, ECS 2) may be inserted with a DLP nucleic acid sequence, a sequence capable of acting as an initiation codon and / or a recognition sequence capable of improving expression efficiency, etc. . Also, in one exemplary embodiment, if the 3'-side of the second coding region (CR 2), for example the 3'-UTR, is inserted, a poly (A) sequence may be further inserted on the 3'- have.

2, the expression control sequences (ECS 1, ECS 2) and the coding regions (CR 1, CR 2) are shown to be divided into two, respectively. However, the number of expression control sequences having the IRES base control sequence and / The number of coding regions operatively linked to the regulatory sequence may exceed two.

Recombinant expression vectors and applications

The nucleic acid molecules shown in Figures 1 and 2 can be inserted into a vector. The vector also includes an expression control sequence, such as a transcriptional control sequence, linked to the nucleic acid molecule of the invention. In an embodiment of the invention, the vector may comprise a nucleic acid molecule encoding the desired protein or peptide. At this time, the nucleic acid molecule may bind to another nucleic acid to encode the fusion protein or the fusion peptide. Therefore, according to another aspect of the present invention, the present invention relates to a recombinant expression vector into which the aforementioned nucleic acid molecule of the present invention is inserted.

For example, the vector may comprise DNA or viral vectors, DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA linked with cationic condensing agents (CCA) 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 molecule of the invention is engineered to enter and express in a mammalian cell. Such a composition is particularly useful for therapeutic use. There are many methods for expressing a nucleic acid molecule in a host cell, and any suitable method can be used. For example, the nucleic acid molecule according to the present invention may be used in conjunction with an adenovirus, an adeno-associated virus, a retrovirus, vaccinia or other avian pox virus (eq) And the like.

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

Techniques for inserting nucleic acid molecules, such as DNA, into such vectors are well known. The retroviral vector may contain a targeting region (e.g., a gene for a selectable marker that facilitates identification or selection of the transfected cells and / or a gene that encodes a ligand that acts as a receptor for a particular target cell targeting moiety < / RTI > Targeting can also be accomplished by known methods using antibodies.

A number of vectors available and known in the art can be used for the purposes of the present invention. The choice of the appropriate vector will depend largely on the size and vector of the nucleic acid molecule inserted into the vector and the particular host cell being transformed. Each vector contains various components depending on its function (amplification or expression of the heterologous polynucleotide, or both) and compatibility with the particular host cell in which the vector is present. Vector components generally include, but are not limited to, the origin of replication (particularly when the vector is inserted into a prokaryotic cell), a selectable marker gene, a promoter, a ribosome binding site (RBS), a signal sequence, a heterologous nucleic acid insert and a transcription termination sequence .

For example, a recombinant expression vector according to the present invention may be used for expression control sequences that may affect the expression of a protein or peptide, such as an initiation codon, a stop codon, a polyadenylation signal, an enhancer, a membrane targeting or secretion Signal sequences, and the like. The polyadenylation signal increases the stability of the transcript or facilitates cellular transport. The enhancer sequence is a nucleotide sequence that is located at various sites in the promoter and increases the transcriptional activity compared with the promoter-mediated transcriptional activity in the absence of the enhancer sequence. When the host is a Escherichia genus, the signal sequence includes a PhoA signal sequence and an OmpA signal sequence. When the host is a genus of Bacillus , an α-amylase signal sequence, a subtilisin signal sequence, The IFN-? Signal sequence, the SUC2 signal sequence, and the like. When the host is an animal cell, the insulin signal sequence, the a-interferon signal sequence, the antibody molecule signal sequence, and the like can be used. Do not.

One type of vector is a " plasmid " in which one type of vector refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a phage vector. Another type of vector is a viral vector in which additional DNA fragments can be ligated into the viral genome. Certain vectors are capable of autonomous replication within host cells into which they are introduced (e. G. Bacterial vectors having bacterial replication origin and episomal mammalian vectors). Other vectors (e. G., Non-episomal mammalian vectors) can be integrated into the genome of the host cell upon introduction into the host cell, thereby replicating with the host genome. In addition, a particular vector may direct expression of a gene in which the vector is operably linked. Such vectors are referred to herein as " recombinant expression vectors " (or simply, " recombinant vectors "). Generally, expression vectors useful in recombinant DNA technology are often present in the form of plasmids.

In a specific embodiment of the invention, the nucleic acid molecule is inserted into the host cell using a virus expression system (vaccinia or other poxvirus, retrovirus or adenovirus). Illustratively, viral vectors include retroviral vectors derived from HIV, SIV, murine retroviruses, gibbon ape leukemia virus, adeno-associate viruses (AAVs), and adenoviruses (Miller et al., 1990, Mol. Cell Biol. 10: 4239; J. Kolberg 1992, NIH Res. 4:43; Cornetta et al., 1991, Hum. Gene Ther. 215). Retroviruses derived from murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), ecotropic retroviruses, simian immunodeficiency virus (SIV), human immunodeficiency virus 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. , Miller et al., 1991, J. Virol. 65: 2220-2224, Rosenberg and Fauci 1993, < RTI ID = 0.0 > 10: 4239, R. Kolberg 1992, J. NIH (Ed.), Fundamentals of Immunology, Third Edition, WE Paul (ed.) Raven Press, 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 for this are disclosed in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001) , Which is incorporated herein by reference.

For example, the vector of the present invention can typically be constructed as a vector for cloning or as a vector for expression. In addition, the vector of the present invention can be constructed by using prokaryotic cells or eukaryotic cells as hosts. It is preferable that the nucleic acid molecule of the present invention is derived from a prokaryotic cell and that the prokaryotic cell is used as a host in consideration of the convenience of cultivation. The vector of the present invention can typically be constructed as a vector for cloning or as a vector for expression. For example, vectors that may be used in the present invention include plasmids such as pSClOl, ColEl, pBR322, pUC8 / 9, pHC79, pUC19, pET etc., phage such as λgt4λB, λ-Charon ,?? z1,? GEM.TM.-11 and M13, etc.) or a virus (such as SV40).

Constitutive or inducible promoters may be used in the present invention depending on the needs of the particular situation that can be ascertained by those skilled in the art. A number of promoters 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 the open reading frame (ORF) of the appropriate gene of interest (GOI) by removing the promoter from the source nucleic acid molecule through restriction enzyme digestion and inserting the isolated promoter sequence into the selection vector As shown in FIG. Both natural promoter sequences and a number of heterologous promoters can be used to direct amplification and / or expression of a gene of interest (GOI). However, heterologous promoters are generally preferred because they permit greater transcription and higher yields of genes of interest expressed compared to native promoters.

For example, when the vector of the present invention is an expression vector and a prokaryotic cell is used as a host, a strong promoter capable of promoting transcription (such as a tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pL 貫 promoter, , rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter), an expression control sequence (ECS) with an IRES to initiate translation, and a transcription / translation termination sequence. When E. coli is used as a host cell, the promoter and operator site of the E. coli tryptophan biosynthetic pathway (Yanofsky, C., J. Bacteriol., 158: 1018-1024 (1984)) and the left promoter of phage l Promoter, Herskowitz, I. and Hagen, D., Ann. Rev. Genet., 14: 399-445 (1980)).

On the other hand, when the vector of the present invention is an expression vector and the eukaryotic cell is used as a host, a promoter derived from the genome of a mammalian cell (e.g., a metallothionein promoter) or a mammalian virus A promoter derived from a bacteriophage (e.g., T7 promoter, T3 promoter, SM6 promoter) may be used as the transcription termination promoter, viral late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter and HSV tk promoter. Lt; RTI ID = 0.0 > polyadenylation < / RTI >

In addition, when the recombinant vector of the present invention is a replicable expression vector, it may contain a replication origin, which is a specific nucleic acid sequence at which replication is initiated. In addition, the recombinant vector may comprise a selection marker. Selection markers are for selecting cells transfected with a vector, and markers conferring selectable phenotypes such as drug resistance, nutritional requirements, tolerance to cytotoxic agents, or expression of surface proteins may be used. The vector of the present invention is a selection marker and includes an antibiotic resistance gene commonly used in the technical field. Examples thereof include ampicillin, gentamycin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, There is a resistance gene for tetracycline. In the environment treated with the selective agent, only the cells expressing the selection marker survive, so that the transformed cells can be selected. Representative examples of selectable markers include uro4, leu1, and his3, which are auxotrophic markers. However, the types of selectable markers that can be used in the present invention are not limited by the above examples.

Various in vitro amplification techniques for amplifying subcloned sequences in expression vectors are known. These techniques include polymerase chain reaction (PCR), ligase chain reaction (LCR), Qβ-replicase amplification and other RNA polymerase-based techniques (Sambrook et al., 1989, Molecular Cloning-A Laboratory Manual (2nd Ed) 1-3; 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 described in U.S. Patent No. 5,426,039).

The present invention also provides a host cell transformed with the recombinant expression vector of the present invention. The transformed host cells can be used in a method of producing a protein or peptide according to 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 the nucleic acid molecule described above. The recombinant microorganism may be used as a vaccine or a gene therapy agent. For example, the recombinant microorganism may be prepared according to the method for producing live attenuated vaccines.

The vector of the present invention may be fused with other sequences to facilitate purification of the recombinant protein or peptide expressed therefrom. Fusion sequences include, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA), and 6x His (hexahistidine; Quiagen, USA) Is 6x His. Because of the additional sequence for such purification, proteins expressed in the host are rapidly and easily purified through affinity chromatography. Sequences that encode Fc fragments may be fused to facilitate the extracellular secretion of these recombinant proteins where necessary.

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 the enzyme, can be used. When 6x His is used, Ni-NTA His-conjugated resin column (Novagen, USA) Proteins can be obtained quickly and easily.

Any host cell known in the art can be used as the host cell capable of continuously cloning and expressing the above-described vector in a stable manner. 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, and Bacillus thuringiensis, and Salmonella typhimurium, Serratia marcesensus and various Pseudomonas species And enterobacteria and strains. When the vector of the present invention is transformed into a eukaryotic cell, Saccharomyce cerevisiae , insect cells (e.g., SF9 cells) and human cells (e.g., CHO cell line (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cell lines).

The vectors of the present invention can be used to genetically transform cells in vitro, in vivo, ex vivo or in vitro. Methods for genetically transforming cells include methods of infecting or transfecting cells with viral vectors, calcium phosphate precipitation, recipient cells in bacterial protoplasts containing DNA, A method of treating liposomes or microspheres containing DNA in receptor cells, a method of fusing with DNA microarrays, such as DEAE, receptor-mediated endocytosis, electroporation, microinjection, (micro-injection) are known.

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) (Dower, WJ et al., Nucleic Acids Res., 16: 6127-6145, 1988)), and the like . In addition, when the host cell is a eukaryotic cell, microinfection (Capecchi, MR, Cell, 22: 479, 1980), calcium phosphate precipitation (Graham, FL et al., Virology, 52: 456, 11973) (Wong, TK et al., Gene, 10: 87, 1980), DEAE-dextran treatment (Neumann, E. et al., EMBO J. 1: 841, 1982), liposome- (Gopal, MoI. Cell Biol., 5: 1188-1190, 1985), and gene bend buddhism (Yang et al., Proc. Natl. Acad. Sci., 87: 9568-9572, 1990) Can be injected into host cells.

The vector injected into the host cell may be expressed in host cells, in which case a large amount of recombinant protein or recombinant peptide is obtained. For example, when the expression vector comprises 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-mentioned nucleic acid molecule and a pharmaceutically acceptable carrier, if necessary, an appropriate adjuvant. In this case, the above-mentioned nucleic acid molecule is directly and directly administered to the subject.

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

The pharmaceutical composition (nucleic acid vaccine and / or gene therapy agent) comprising the gene carrier in which the nucleic acid molecule or the nucleic acid molecule according to the present invention is inserted is preferably administered systemically. The route of administration of such pharmaceutical compositions / vaccines is typically administered orally, subcutaneously, intravenously, intramuscularly, intra-articular, intra-synovial, intrasternal, intrathecal, Intrahepatic, intralesional, intracranial, transdermal, intradermal, intrapulmonal, intraperitoneal, intracardial, intraarterial, intracranial, ), Sublingual topical and / or intranasal routes. Alternatively, the vaccine or pharmaceutical composition of the present invention may be administered by intradermal, subcutaneous or intramuscular routes. Thus, the pharmaceutical composition / vaccine is preferably formulated in liquid or solid form, as defined above, for the pharmaceutical composition in general.

If desired, an adjuvant may be included in the pharmaceutical composition to enhance the immunogenicity of the vaccine. The adjuvant may be incorporated into the vaccine according to the invention. These adjuvant components, such as adjuvants, are selected according to immunogenicity and other properties of the pharmaceutically active ingredient of the vaccine according to the invention.

Gene delivery vehicles, such as nucleic acid molecules or recombinant expression vectors of the invention, can be delivered by any suitable means including, but not limited to, oral, topical (including buccal and sublingual), rectal, vaginal, transdermal, parenteral, subcutaneous, intraperitoneal, Intraperitoneal and intranasal, and, if desired, topical, intra-lesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration.

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

A typical preparation is prepared by mixing a nucleic acid molecule or gene carrier of the present 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 conventionally used in the formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, But are not limited to, cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The formulations may also contain one or more buffers, stabilizers, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, opacifiers, lubricants, processing aids, colorants, sweeteners, flavoring agents, (I. E., Nucleic acid molecules, gene carriers, or pharmaceutical compositions thereof, which are the active ingredients of the present invention) or other known additives that aid in the manufacture of pharmaceutical products (i.e., medicaments).

The pharmaceutical composition of the present invention may be formulated into a unit dosage form by using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by those having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

In the meantime, the formulations herein may also contain more than one active compound, preferably compounds having complementary activity that do not have deleterious effects on each other, if necessary for the particular indication to be treated. Alternatively or additionally, the composition may include an agent that enhances its function, such as a cytotoxic agent, a cytokine, a chemotherapeutic agent, or a growth-inhibitory agent, or a growth-enhancing agent. Such molecules are suitably present in combination in amounts effective for their intended purpose.

In addition, a gene carrier containing a nucleic acid molecule of the present invention including a coding region encoding a gene of interest may be included in the pharmaceutical composition. The gene carrier is prepared for carrying and expressing a nucleic acid molecule of interest. The details of the nucleic acid molecule of interest, which is a transport object, are omitted in order to avoid redundant description with the contents described above.

To produce a gene delivery vehicle, it is preferred that the transcript of interest is present in a suitable expression construct. In such an expression construct, it is preferred that the transcript of the gene of interest is operably linked to a promoter. In the present invention, a promoter bound to a transcript of a gene of interest is preferably one capable of regulating the transcription of a nucleic acid molecule of interest by operating in an animal cell, more preferably a mammalian cell, Promoter, a promoter derived from the genome of a mammalian cell, or a bacteriophage-derived promoter. For example, a promoter such as CMV (cytomegalo virus) promoter, adenovirus late promoter, Vaccinia virus 7.5K promoter, SV40 promoter, HSV tk promoter, T7 promoter, T3 promoter, SM6 promoter, RSV promoter, EF1 alpha promoter, Promoter of the human IL-2 gene, a promoter of the human IFN gene, a promoter of the human IL-4 gene, a promoter of the human lymphotoxin gene, a promoter of the human GM-CSF gene, a cancer cell specific promoter But are not limited to, TERT promoter, PSA promoter, PSMA promoter, CEA promoter, E2F promoter and AFP promoter) and tissue specific promoters (e.g., albumin promoters). Preferably, the expression constructs used in the present invention include polyanenylation sequences (e.g., bovine growth hormone terminator and SV40-derived polyadenylation sequences).

For example, when a nucleic acid molecule according to the present invention is to be used as an RNA vaccine, an appropriate transcription control sequence may be located on the 5 'side of the expression control sequence (ECE) to enable IVT. As described above, nucleic acid molecules in the form of RNA, which can be used as an RNA vaccine or the like, can be synthesized through the IVT process. Therefore, it is not necessary to directly deal with living viruses or pathogenic microorganisms used in general live vaccine or vaccine production, Cultivation of host cells such as yeast, Escherichia coli, and insect cells, which must be necessarily used to produce recombinant proteins / peptides, is unnecessary.

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

Gene delivery can be made in a variety of forms, including plasmids, viral vectors, or liposomes containing plasmids or niobs. For example, a transcript of a gene of interest can be applied to all gene delivery systems used in conventional gene therapy and is preferably a plasmid, an adenovirus (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), lentivirus (Wang G. et al., J. Clin. Invest. 104 (11): R55-62 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.

Meanwhile, the method of introducing the 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 above cited documents.

In addition, microinjection (Capecchi, M.R., Cell, 22: 479, 1980) when the gene delivery system is an naked recombinant DNA molecule or plasmid in the present invention; 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) Yang et al., Proc. Natl. Acad. Sci., 87: 9568-9572, 1990)).

Hereinafter, the present invention will be described with reference to exemplary embodiments, but the present invention is not limited to the technical ideas described in the following embodiments.

Example 1: Generation of nucleic acid molecules of RNA platform

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 having an IRES derived from the SV (SEQ ID NO: 1) - 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 the pGH vector and linearized with restriction enzymes to obtain nucleic acid molecules in the form of RNA platforms via in vitro trasncrption (IVT). The nucleic acid molecule in the form of an RNA platform prepared according to the present embodiment is hereinafter referred to as 'pnon-SV-R / L'.

Example 2: Generation of nucleic acid molecules of RNA platform

UTR (SEQ ID NO: 2) having an IRES derived from CVB3 having no DLP structure sequence was used as the 5'-UTR of the template DNA and a sequence capable of increasing the expression efficiency at the 3'-side of 5'-UTR ( UTR (SEQ ID NO: 11) derived from CVB3 was used as the 3'-UTR, and 5'-UTR (SEQ ID NO: 11) derived from the CVB3 was used as the 3'- UTR and the restriction enzyme recognition sequence ( CCGCGG CGATCG) The procedure of Example 1 was repeated except that capping was performed using an anti-reverse cap analog (ARCA) on the 5 'side of the RNA platform. The nucleic acid molecule in the form of an RNA platform prepared according to the present embodiment is named 'pCAP-CVB3-R / L'.

Example 3: Construction of nucleic acid molecule of RNA platform

(Restriction enzyme recognition sequence ( GTCGAC CGATCG) was inserted in 3 'of the ORF using 5'-UTR (SEQ ID NO: 3) having IRES derived from EMCV having no DLP structure sequence as the 5'-UTR of the template DNA , The procedure of Example 1 was repeated except that the 3'-UTR from EMCV (SEQ ID NO: 12) was used as the 3'-UTR. The nucleic acid molecule in the form of an RNA platform prepared according to this embodiment is hereinafter referred to as 'pEMCV-R / L'.

Example 4: Generation of nucleic acid molecules of RNA platform

A 5'-UTR (SEQ ID NO: 4) having an IRES derived from JEV having no DLP structure sequence was used as the 5'-UTR of the template DNA, a restriction enzyme recognition sequence ( GTCGAC CGATCG) was inserted into 3 ' , Except that 3'-UTR derived from JEV (SEQ ID NO: 13) was used as the 3'-UTR and capping was performed using ARCA (anti-reverse cap analog) at the 5'-side of 5'-UTR The procedure of Example 1 was repeated to produce the nucleic acid molecule of the RNA platform. The nucleic acid molecule in the form of an RNA platform prepared according to this embodiment is hereinafter referred to as 'pCAP-JEV-R / L'.

Example 5: Generation of nucleic acid molecules of RNA platform

A 5'-UTR (SEQ ID NO: 5) having an IRES derived from CPV having no DLP structure sequence was used as the 5'-UTR of the template DNA and a sequence acting as an initiation codon of CPV on the 3 ' (CCTGCT) was inserted, the R / L-encoding ORF sequence of SEQ ID NO: 20 was used as the GOI gene, the restriction enzyme recognition sequence was not inserted at the 3 'end of the ORF and the 3' -UTR (SEQ ID NO: 14) was used to prepare the nucleic acid molecule of the RNA platform. The nucleic acid molecule in the form of an RNA platform prepared according to this embodiment is hereinafter referred to as 'pCPV-R / L'.

Comparative Example 1: Generation of nucleic acid molecule of RNA platform

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

Experimental Example 1: Measurement of expression efficiency of nucleic acid molecule

The RNA platform type nucleic acid molecules prepared in Examples 1 to 5 and Comparative Example 1 were respectively transfected into the human muscle cell line (A204) and the human embryonic kidney cell line (293T), and the transformed Renilla luciferase And the amount of expression 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 the IRES derived from CPV had an expression efficiency close to positive control, and the 5'-UTR inserted nucleic acid The molecule has a lower expression efficiency than the positive control but has a good expression efficiency.

Example 6: Generation of nucleic acid molecules of RNA platform

Nucleic acid molecules of an RNA platform having IRES derived from eukaryotic cells were prepared. A 5'-UTR (SEQ ID NO: 6) having an IRES derived from eIF4G without a DLP structure sequence was used as the 5'-UTR of the template DNA and a sequence capable of increasing the expression efficiency at the 3'-side of 5'-UTR ATGTCTGGG) was inserted and the restriction enzyme recognition sequence ( GGCGCC ) at the 3 'side of the ORF was used and the 3'-UTR derived from eIF4G (SEQ ID NO: 15) was used as the 3'-UTR. Was repeated to prepare the nucleic acid molecule of the RNA platform. The nucleic acid molecule in the form of an RNA platform prepared according to this embodiment is hereinafter referred to as 'peIF4G-R / L'.

Example 7: Generation of nucleic acid molecules of RNA platform

Nucleic acid molecules of an RNA platform having IRES derived from eukaryotic cells were prepared. As the 5'-UTR of the template DNA, a 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) And the 3'-UTR derived from FMR1 (SEQ ID NO: 16) was used as the 3'-UTR (SEQ ID NO: 16). The nucleic acid molecule in the form of an RNA platform prepared according to this embodiment is hereinafter referred to as 'pFMR1-R / L'.

Example 8: Generation of nucleic acid molecules of RNA platform

Nucleic acid molecules of an RNA platform having IRES derived from eukaryotic cells were prepared. As the 5'-UTR of the template DNA, a 5'-UTR (SEQ ID NO: 8) having an IRES derived from CN43 having no DLP structure sequence was used and a restriction enzyme recognition sequence ( GGCGCC ) at the 3 ' , The procedure of Example 1 was repeated except that 3'-UTR from CN43 (SEQ ID NO: 17) was used as the 3'-UTR to prepare the nucleic acid molecule of the RNA platform. The nucleic acid molecule in the form of an RNA platform prepared according to this embodiment is hereinafter referred to as 'pCN43-R / L'.

Experimental Example 2: Measurement of Expression Efficiency of Nucleic Acid Molecules

The RNA platform type nucleic acid molecules prepared in Examples 6 to 8 and Comparative Example 1 were respectively transfected into a human muscle cell line (A204) and a human embryonic kidney cell line (293T), and the transformed Renilla luciferase And the amount of expression 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 showed lower expression efficiency than pCAP-CureVac-R / L used as a positive control , But showed higher expression patterns than 5'-UTR inserted nucleic acid molecules having IRES structures (FMR1, eIF4G) derived from two other eukaryotic cells. However, all of the nucleic acid molecules prepared in Examples 6 to 8 showed good expression efficiency.

Example 9: Generation of nucleic acid molecules of RNA platform

The procedure of Example 2 was repeated except that the capping at the 5 'end was not carried out using ARCA to prepare nucleic acid molecules of the RNA platform. The nucleic acid molecule in the form of an RNA platform prepared according to this embodiment is hereinafter referred to as 'pCVB3-R / L'.

Example 10: Generation of nucleic acid molecules of RNA platform

A nucleic acid molecule of the modified RNA platform was constructed so that a large number of adenosines (pMA) were inserted into the 5'-side of the 5'-UTR. The procedure of Example 9 was repeated except that 50 multiple adenosine (pMA) sequences were transcribed between the T7 promoter and the 5'-UTR from CVB3, i. E., 5 ' Were prepared. The nucleic acid molecule in the form of an RNA platform prepared according to this embodiment is hereinafter referred to as 'pMA-CVB3-R / L'.

Example 11: Generation of nucleic acid molecule of RNA platform

The procedure of Example 5 was repeated except that 50 multiple adenosine (pMA) sequences were inserted between the 5'-UTR of the 5'-UTR and the 5'-UTR from the T7 promoter and CPV, Respectively. The nucleic acid molecule in the form of an RNA platform prepared according to this embodiment is hereinafter referred to as 'pMA-CPV-R / L'.

Example 12: Generation of nucleic acid molecule of RNA platform

The procedure of Example 8 was repeated except that 50 multiple adenosine (pMA) sequences were inserted between the T7 promoter and the 5'-UTR from CN43, that is, 5 'to the 5'-UTR, Molecule. The nucleic acid molecule in the form of an RNA platform prepared according to this embodiment is hereinafter referred to as 'pMA-CN43-R / L'.

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

(PCAP-CVB3-R / L), Example 9 (pCVB3-R / L), Example 10 (pMA- Nucleic acid molecules in the form of RNA platform prepared in Example 12 (pMA-CN43-R / L) and Comparative Example 1 were respectively transfected into human muscle cell line (A204) or mouse muscle cell line (Nor10) The expression level of Renilla luciferase was measured. FIG. 5 shows the results of measurement of the expression level of a gene of interest (GOI) using the modified nucleic acid molecule with IRES derived from CVB3. In FIG. 5, 5'-UTR is expressed in a nucleic acid molecule into which pMA is inserted The results of measurement of the amount of expression of the gene of interest are shown in Fig.

As shown in FIG. 5, when the pMA-CVB3-R / L nucleic acid molecule was injected into the cell line, the expression of Renilla luciferase used as a gene of interest, as compared with the case of injecting no modified CVB3- Indicating that the expression level is improved. In addition, as shown in FIG. 6, the nucleic acid molecule in which a large number of adenosines were inserted at the 5 'side of the 5'-UTR having IRES showed a slightly lower expression amount of the gene of interest than the positive control, but showed a good expression efficiency.

Experimental Example 4: Measurement of Expression Efficiency of Nucleic Acid Molecules

(PCAP-CVB3-R / L), Example 5 (pCPV-R / L), and Example 8 (pCN43-R / L), which were relatively excellent in the expression efficiency in Experimental Examples 1 to 3, ) And the RNA platform-type nucleic acid molecule prepared in Comparative Example 1 were transfected into human muscle cell line (A204) and human embryonic kidney cell line (293T), respectively, and the expression amount of Renilla luciferase used as a gene of interest was measured. The measurement results are shown in Figs. 7A and 7B. (pCAP-CVB3-R / L, pCN43-R / L), which were similar to or slightly lower in expression efficiency than pCVV-R / L nucleic acid molecules / L), respectively. However, nucleic acid molecules with two other IRES structures also showed good expression efficiency.

Example 13: Generation of nucleic acid molecules of RNA platform

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

Restriction enzyme recognition sequence (GTTAAC) - T7 promoter (SEQ ID NO: 19) - Restriction enzyme recognition sequence (GTTAAC) - Sequence-restriction enzyme recognition sequence (GTTAAC) to be transcribed into 50 adenosines (ATGGCAGCTCAA) -restriction enzyme recognition sequence ( GTCGAC ) -Kozak sequence (GACC) - As the first GOI gene, the 5'-UTR from CVB3 as a 5'- UTR (SEQ ID NO: L encoding ORF sequence (SEQ ID NO: 20) as the second GOI gene; 5'-UTR (SEQ ID NO: 3) derived from EMCV as the second 5'- UTR; - 3'-UTR from CVB3 (SEQ ID NO: 12) - Polyadenosine sequence (50 Adenosines) - Restriction enzyme recognition sequence ( GCGGCCGC ).

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

Example 14: Generation of nucleic acid molecules of RNA platform

Nucleic acid molecules in the form of an RNA platform with 5-UTR with two different IRESs were prepared. The template DNA sequence was designed as follows.

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

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

Example 15: Generation of nucleic acid molecules of RNA platform

The procedure of Example 13 was repeated except that the ORF sequence (SEQ ID NO: 21) encoding firefly luciferase (F / L) was used as the second GOI gene to prepare the nucleic acid molecule of the RNA platform. The nucleic acid molecule in the form of an RNA platform prepared according to this embodiment is hereinafter referred to as 'pMA-CVB3-R / L-EMCV-F / L'.

Example 16: Construction of nucleic acid molecule of RNA platform

The procedure of Example 14 was repeated except that the ORF sequence (SEQ ID NO: 21) encoding firefly luciferase (F / L) was used as the second GOI gene to prepare the nucleic acid molecule of the RNA platform. The nucleic acid molecule in the form of an RNA platform prepared according to the present embodiment is hereinafter referred to as 'pCPV-R / L-EMCV-F / L'.

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

Nucleic acid molecules in the form of RNA platforms prepared in Example 13 and Example 14 and Comparative Example 1 were respectively transfected into a human embryonic kidney cell line (293T), and the amount of expression of Renilla luciferase used as a gene of interest was measured. Fig. 8 shows the results of measurement of the expression level of R / L when two identical GOIs (R / L) were used. The highest expression efficiency was observed in the pMA-CVB3-R / L-EMCV-R / L nucleic acid molecule in which a large number of adenosines were inserted at the 5 'side of 5'-UTR. In addition, the pCPV-R / L-EMCV-R / L nucleic acid molecule also showed slightly higher or similar expression efficiency when compared to the positive control.

Example 10 (pCVP-R / L) and Example 16 (pCVP-R / L-EMCV-F / L) And RNA platform-type nucleic acid molecules 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 Firefly luciferase were compared. The measurement results are shown in Figs. 9A and 9B.

The expression level of R / L when different genes of interest were expressed from different IRES nucleotide sequences such as pCPV-R / L-EMCV-F / L and pMA-CVB3-R / L-EMCV- The expression level of R / L when one gene of interest was expressed, such as -R / L and pMA-CVB3-R / L, was compared, and the expression amount of R / L was not significantly different. However, in the expression of F / L whose expression is regulated by the 5'-UTR with IRES derived from EMCV, pCPV-R / L-EMCV-F / L in pMA-CVB3-R / L-EMCV- . These results indicate that the pMA-CVB3-R / L-EMCV-R / L nucleic acid molecule exhibits a higher R / L expression efficiency than the pCVP-R / L-EMCV-R / .

Although the present invention has been described based on the exemplary embodiments and examples of the present invention, the present invention is not limited to the technical ideas described in the above embodiments and examples. Those skilled in the art will appreciate that various modifications and changes may be made without departing from the spirit and scope of the present invention as defined by the appended claims. It is apparent, however, that the appended claims are intended to cover all such modifications and changes as fall within the true scope of the 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 agggacaaca 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 <213> Luciola 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 operatively linked to said at least one expression control sequence and encoding a protein or peptide,
Wherein 20 to 400 adenosines or thymines are linked adjacent to said at least one expression control sequence.
The method according to claim 1,
Wherein the at least one expression control sequence forms a 5'untranslated region (5'-UTR).
3. The method of claim 2,
Wherein the nucleic acid molecule further comprises a 3'-untranslated region (3'-UTR) located 3 'to the 5'-UTR, the coding region being located between the 5'-UTR and the 3'-UTR A nucleic acid molecule.
The method according to claim 1,
Wherein the at least one expression control sequence comprises a viral IRES base sequence.
5. The method of claim 4,
The viral IRES base sequence may be selected from the group consisting of picornaviridae virus, Togaviridae virus, Dicistroviridae virus, Flaviridae virus, Retroviridae virus, Herpesviridae virus, ) Viruses, and combinations thereof. &Lt; RTI ID = 0.0 &gt; A &lt; / RTI &gt; nucleic acid molecule comprising a viral IRES base sequence derived from a virus selected from the group consisting of:
5. The method of claim 4,
Wherein the viral IRES nucleotide sequence is derived from a virus selected from the group consisting of picornaviridae virus, Dicistroviridae virus, and combinations thereof.
The method according to claim 6,
The viral IRES base sequence derived from the picornaviridae virus may be selected from the group consisting of Enterovirus virus, Cardiovirus virus, Aptovirus virus, Hepatovirus, Viruses, viruses of the genus Teschovirus, viruses of the genus Teschovirus, and combinations thereof,
The viral IRES base sequence derived from the Dicistroviridae virus is a nucleic acid molecule derived from the Cripavirus virus.
8. The method of claim 7,
The IRES nucleotide sequence derived from the picornaviridae virus includes an IRES nucleotide sequence derived from a cockskirus virus,
The IRES nucleotide sequence derived from the Dustrovirus and the virus is a nucleic acid molecule comprising an IRES nucleotide sequence derived from Cricket paralysis virus (CPV).
The method according to claim 1,
Wherein the coding region encodes an antigen or fragment thereof.
The method according to claim 1,
Wherein the coding region is a nucleic acid molecule encoding a protein or fragment thereof for treating disease.
The method according to claim 1,
Wherein the at least one expression control sequence comprises a first expression control sequence having a base sequence of IRES and a second expression control sequence having a base sequence of IRES located 3 'to the first expression control sequence.
12. The method of claim 11,
Wherein the coding region comprises a first coding region located between the first expression control sequence and the second expression control sequence and a second coding region located 3 'to the second expression control sequence.
13. The method of claim 12,
Wherein the first coding region and the second coding region encode different peptides or proteins.
12. The method of claim 11,
Wherein 20 to 400 adenosines or thymines are bound adjacent to at least one expression control sequence selected from the first expression control sequence and the second expression control.
12. The method of claim 11,
Wherein 30 to 100 adenosine or thymine are bound to the first expression control sequence.
12. The method of claim 11,
Wherein the first expression control sequence comprises an IRES nucleotide sequence derived from Coxsachie virus B3, CVB3 or Cricket paralysis virus (CPV), and the second expression control sequence comprises a brain myocarditis virus Encephalomyocarditis virus (EMCV) derived nucleic acid molecule containing the IRES nucleotide sequence.
The method according to claim 1,
Wherein the nucleic acid molecule further comprises a transcription regulatory sequence located 5 'to the expression control sequence and a polyadenosine (poly (A)) sequence located 3' to the coding region.
The method according to claim 1,
Wherein the nucleic acid molecule is RNA.
A recombinant expression vector into which the nucleic acid molecule according to any one of claims 1 to 18 is inserted.
A method for producing a protein or a peptide by injecting a nucleic acid molecule according to any one of claims 1 to 18 into a living body and expressing the protein or peptide from the injected nucleic acid molecule.
20. A method for producing the protein or peptide by injecting a recombinant gene vector according to claim 19 into a living body as a gene carrier and expressing the protein or peptide from the gene carrier.

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