Constructs comprising UTR sequences that improve mRNA intracellular stability and biosynthesis and uses thereof

Abstract

Unlike DNA, the advantage of mRNA is that the desired protein can be transiently expressed directly in the cytoplasm without entry into the nucleus, so that the attempt to use the mRNA vaccine is positively proceeding. However, the structural stability of mRNA is lower than that of DNA, so that the mRNA has limitation in industrial application. Therefore, the present invention relates to a UTR sequence that improves intracellular stability and biosynthesis of mRNA, and a composition for stabilizing a nucleic acid comprising the UTR sequence. The UTR sequence disclosed by the invention has excellent effects of stabilizing nucleic acid and promoting expression of target protein, so that the UTR sequence is expected to be widely applied to the field of health / medicine.

Description

Technical Field

[0001] The present invention relates to a sequence for stabilizing nucleic acid, in particular to an untranslated region (UTR) sequence for improving the intracellular stability and biosynthesis of mRNA, and a composition for stabilizing nucleic acid comprising the UTR sequence. Background Art

[0002] Unlike DNA, mRNA has the advantage of being able to transiently express the desired protein directly in the cytoplasm without entering the nucleus, and therefore has attracted attention as a new treatment method in the health / medical field. In particular, attempts to treat and prevent diseases using mRNA vaccines are being actively carried out based on global biotechnology and pharmaceutical companies, but mRNA has lower structural stability than DNA, so it has limitations in industrial applications due to its lower structural stability than DNA. Therefore, recently, in order to solve these problems, attempts have been made to increase the intracellular stability of mRNA and improve the biosynthesis efficiency of proteins by using UTRs. UTRs play an auxiliary role in enabling the efficient synthesis of peptides or proteins as therapeutic targets, and are independent of the gene sequence to be synthesized to enable the expression of various peptides or proteins. In particular, in the field of mRNA vaccines, UTRs improve the stability of mRNA vaccines and enable efficient synthesis of antigens (new antigens in the case of cancer vaccines) in vivo, so even small doses of vaccines can be expected to be highly effective in treating and preventing diseases.

[0003] Therefore, the present invention relates to sequences for stabilizing nucleic acids, in particular to untranslated region (UTR) sequences for improving intracellular stability and biosynthesis of mRNA, and compositions for stabilizing nucleic acids comprising UTR sequences. The UTR sequences of the present invention have excellent effects of stabilizing nucleic acids and promoting target protein expression, and are therefore expected to be widely used in the health / medical field. Summary of the invention

[0004] [Technical issues]

[0005] An object of the present invention is to provide a sequence for stabilizing nucleic acids, in particular, an untranslated region (UTR) sequence for improving the intracellular stability and biosynthesis of mRNA; and a method for selecting the same.

[0006] Another object of the present invention is to provide an artificial nucleic acid and a method for producing the same, wherein the artificial nucleic acid comprises a sequence for stabilizing nucleic acid, particularly an untranslated region (UTR) sequence for improving the intracellular stability and biosynthesis of mRNA.

[0007] Another object of the present invention is to provide a vector comprising an artificial nucleic acid and a method for producing the same.

[0008] Another object of the present invention is to provide a transfectant transfected with a vector containing an artificial nucleic acid and a method for producing the same.

[0009] Still another object of the present invention is to provide a composition comprising an artificial nucleic acid for stabilizing nucleic acid and a method for producing the same.

[0010] Another object of the present invention is to provide an immunopotentiating composition comprising artificial nucleic acid and a method for producing the same.

[0011] Still another object of the present invention is to provide a kit for detecting nucleic acid comprising artificial nucleic acid and a method for producing the same.

[0012] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating a disease comprising an artificial nucleic acid and a method for producing the same.

[0013] However, the technical objectives of the present invention are not limited to the above objectives, and those skilled in the art can clearly understand other objectives not mentioned through the following description.

[0014] [Technical solution]

[0015] Hereinafter, various embodiments described herein are described with reference to the accompanying drawings. In the following description, various specific details, such as specific forms, compositions and processes are set forth to provide a thorough understanding of the present invention. However, specific embodiments may be implemented with one or more of these specific details or with other known methods and formats. In other examples, known processes and manufacturing techniques are not described as specific details, so as not to obscure the present invention unnecessarily. "One embodiment" or "one embodiment" mentioned throughout this specification means that the specific features, shapes, compositions or characteristics described in conjunction with the embodiment are included in one or more embodiments of the present invention. Therefore, the details of "one embodiment" or "one embodiment" represented in various positions of this specification do not necessarily refer to the same embodiment of the present invention. In addition, in one or more embodiments, specific features, shapes, compositions or characteristics may be combined in any suitable manner.

[0016] Unless explicitly defined otherwise in the specification, all scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0017] As used herein, the term "untranslated region (UTR)" means a portion of a nucleic acid molecule that is not translated into a protein. The untranslated region located in the 5' direction of the nucleic acid is referred to as 5'-UTR, and the untranslated region located in the 3' direction of the nucleic acid is referred to as 3'-UTR. In detail, the 5'-UTR is located at the 5' end (i.e., "upstream") of the open reading frame, and is generally understood to be a specific fragment of the messenger RNA (mRNA) located at the 5' end of the mRNA open reading frame. Preferably, the length of the 5'-URT is 10 to 100, 20 to 100, 20 to 80, 20 to 60, 20 to 50, 20 to 40, or 20 to 30 nucleotides (nt). The 5'-UTR may include elements for regulating gene expression, so-called regulatory elements, but is not limited thereto, and the regulatory element may be a ribosome binding site. The 5'-UTR may be modified by adding a 5'-CAP after transcription. The 5'-UTR of mRNA is not translated into an amino acid sequence, and the 5'-UTR sequence is generally encoded by a gene that is transcribed into each mRNA during gene expression. The genomic sequence can be modified after transcription by including alternative introns through processes such as 5'-capping or splicing. 5'-UTR is the nucleotide at the proximal end of the 5'-CAP of the modified mRNA, usually corresponding to the transcription start site. Here, "corresponding" means that the 5'-UTR sequence can be an RNA sequence, such as an mRNA sequence, or a DNA sequence corresponding to such an RNA sequence. 3'-UTR is located at the 3' end (i.e., "downstream") of the open reading frame and is generally understood to be a specific fragment of the messenger RNA (mRNA) located at the 3' end of the mRNA open reading frame. Preferably, the length of 3'-UTR is 10 to 1000, 10 to 500, 20 to 500, 20 to 300, 20 to 200, 20 to 100, 20 to 80, 20 to 60, 20 to 50, 20 to 40 or 20 to 30 nucleotides (nt). 3'-UTR is located between the protein coding region and the poly A sequence of the mRNA. The 3'-UTR sequence is usually encoded by the gene that is transcribed into each mRNA during gene expression, and the genomic sequence can be modified after transcription by including alternative introns through processes such as splicing or 3'-polyadenylation. The 3'-UTR is the nucleotides proximal to the poly A sequence of the modified mRNA, usually corresponding to the transcription termination site. Here, "corresponding" means that the 3'-UTR sequence can be an RNA sequence, such as an mRNA sequence, or a DNA sequence corresponding to such an RNA sequence.

[0018] As used herein, the term "nucleic acid" is a concept that includes deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). "Nucleic acid" is synonymous with "polynucleotide". Preferably, nucleic acid is a polymer formed by nucleotide monomers covalently bound to each other by phosphodiester bonds of a sugar / phosphate backbone. Alternatively, nucleic acid includes base-modified, sugar-modified or backbone-modified DNA or RNA.

[0019] As used herein, the term "stable nucleic acid" is a concept of stabilizing the structure of nucleic acids by preventing the collapse or modification of nucleic acid structures inside and outside cells. Generally speaking, RNA is composed of a single strand, has higher activity than DNA and has lower structural stability. Therefore, the stabilization of nucleic acids in the present invention is to prevent the collapse or modification of the structure of DNA or RNA, preferably RNA, and more preferably mRNA, and the sequences represented by SEQ ID NO: 1 to SEQ ID NO: 18 and SEQ ID NO: 20 to SEQ ID NO: 37 of the present invention are all evaluated to have excellent nucleic acid stabilization effects.

[0020] As used herein, the term sequence homology refers to a percentage representing the degree of identity of two sequences to be compared. To determine the degree of homology, it is preferred that the sequences to be compared have the same length, but if the sequence lengths are different, the degree of homology is calculated based on the longer sequence in the sequence to be compared. For example, a 10nt sequence has 80% homology with an 8nt sequence, and the 8nt sequence is represented by a sequence that is partially identical to it.

[0021] In the present invention, the artificial nucleic acid having an excellent nucleic acid stabilizing effect of the present invention may be an artificial nucleic acid including a sequence having 70% or more homology with any one selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 18 and SEQ ID NO: 20 to SEQ ID NO: 37. Alternatively, the artificial nucleic acid of the present invention may be an artificial nucleic acid including a sequence having 80% or more homology with any one selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 18 and SEQ ID NO: 20 to SEQ ID NO: 37. Alternatively, the artificial nucleic acid of the present invention may be an artificial nucleic acid including a sequence having 90% or more homology with any one selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 18 and SEQ ID NO: 20 to SEQ ID NO: 37. Alternatively, the artificial nucleic acid of the present invention may be an artificial nucleic acid including a sequence having 95% or more homology with any one selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 18 and SEQ ID NO: 20 to SEQ ID NO: 37. Alternatively, the artificial nucleic acid of the present invention may be an artificial nucleic acid including a sequence having 100% homology to any one selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 18 and SEQ ID NO: 20 to SEQ ID NO: 37.

[0022] As used herein, the term "artificial nucleic acid" can be understood as a non-natural nucleic acid molecule that does not exist in nature. An artificial nucleic acid can be 100% composed of a sequence that does not exist in nature, or can be composed of a portion of a sequence that exists in nature (wild type) and a portion of a sequence that does not exist in nature. In the case where a sequence is mixed with a portion of a sequence that exists in nature and a portion of a sequence that does not exist in nature, the sequence that exists in nature can be a mixture of sequences from one or more species. Artificial nucleic acids can be non-natural due to non-natural modifications of their individual sequences (e.g., structural modifications of non-naturally occurring nucleotides). In addition, an artificial nucleic acid can be a DNA molecule, an RNA molecule, or a hybrid molecule comprising DNA and RNA portions.

[0023] As used herein, the term "vector" can be understood as an artificial nucleic acid. In the context of the present invention, a vector is a concept comprising a sequence having at least 70% homology with an artificial nucleic acid selected for stabilizing nucleic acid (specifically any one of the group consisting of SEQ ID NO: 1 to SEQ ID NO: 18 and SEQ ID NO: 20 to SEQ ID NO: 37). Such a vector can be a storage vector, an expression vector, a cloning vector, a transfer vector, etc. A storage vector is a vector that allows convenient storage of nucleic acid molecules (e.g., mRNA molecules). An expression vector can be used to produce RNA, such as mRNA, or an expression product, such as a peptide, polypeptide, or protein. A cloning vector is a vector that generally includes a cloning site that can be used to incorporate a nucleic acid sequence into a vector. A cloning vector can be, for example, a plasmid vector or a phage vector. A transfer vector can be a vector suitable for transferring a nucleic acid molecule into a cell or an organism, such as a viral vector. In the context of the present invention, a vector can be, for example, an RNA vector or a DNA vector. Preferably, the vector is a DNA molecule. Preferably, the vector of the present invention comprises an artificial nucleic acid selected for stabilizing the nucleic acid, specifically a sequence having 70% or higher homology with any one selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 18 and SEQ ID NO: 20 to SEQ ID NO: 37, and a target gene sequence (sequence encoding a target protein) for expression regulation. The vector is preferably a plasmid vector, but is not limited thereto.

[0024] As used herein, the term "transfection" means introducing nucleic acid molecules such as DNA or RNA (such as mRNA) molecules into cells, preferably eukaryotic cells, to change the original phenotype of the subject to be introduced. Transfection can be carried out by any method known to those skilled in the art, for introducing nucleic acid molecules into cells, preferably eukaryotic cells, such as mammalian cells. This method includes, for example, electroporation, lipid transfection based on cationic lipids and / or liposomes, calcium phosphate precipitation, nanoparticle-based transfection, virus-based transfection or cationic polymer-based transfection, such as DEAE-dextran or polyethyleneimine. Alternatively, a virus (such as a slow virus) can be used for transfection. The subject to be introduced, whose original phenotype has been changed by this method of transfection, is called a transfectant.

[0025] As used herein, the term "enhancing immunity" means to amplify the desired immune response for selected antigens (such as characteristic components of bacterial surfaces, virus particles, tumor antigens, etc.).Immunoenhancement can be natural or artificially induced to enhance immune response. In the case of artificial immune response amplification, vaccination is the most common method. Vaccines are generally understood to provide at least one antigen, preferably a preventive or therapeutic substance of an immunogen. Antigens or immunogens can be derived from any substance suitable for vaccination. For example, antigens or immunogens can be derived from bacteria or virus particles, etc., or from tumors or cancer tissues. Antigens or immunogens stimulate the adaptive immune system in vivo. In the related art, vaccines using proteins or peptides derived from antigens or immunogens as stimulants of the adaptive immune system in vivo are dominant, but recently attempts have continued to use nucleic acids as stimulants. In particular, unlike DNA, mRNA has the advantage of being able to transiently express desired proteins directly in the cytoplasm without entering the nucleus, and therefore has attracted attention as a new treatment method in the field of health / medicine. However, the structural stability of mRNA is lower than that of DNA, so it has limitations in industrial applications. Therefore, recently, in order to solve these problems, people have tried to improve the intracellular stability of mRNA and improve the biosynthesis efficiency of proteins by using UTR. In this context, any sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 18 and SEQ ID NO: 20 to SEQ ID NO: 37, or a sequence with 70% or more homology thereto, with excellent nucleic acid stabilization effect of the present invention, can be administered as a vaccine together with a nucleic acid derived from an antigen or immunogen suitable for the desired immune enhancement. In this case, any sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 18 and SEQ ID NO: 20 to SEQ ID NO: 37 of the present invention, or a sequence with 70% or more homology thereto, is preferably located before / after the antigen genetic information in the nucleic acid vaccine, and the nucleic acid vaccine is expected to have the effect of protecting the nucleic acid vaccine from in vivo degradative enzymes and enhancing the nucleic acid sequence as an effectively translated antigen. In addition, since the stability of the nucleic acid vaccine is improved and the antigen can be effectively synthesized in vivo, even if a small amount of vaccine is administered, a highly effective effect in disease treatment and prevention can be expected. In addition, owing to reducing the dosage of the vaccine used to reach the expected therapeutic effect, it is expected that the effect of reducing the cost of vaccine production can be achieved. Vaccines can additionally include adjuvants (ingredients) to enhance the expected effect. Adjuvants are generally medicines and / or immune reagents that can modify or enhance the effect of another agent (such as medicine or vaccine). This adjuvant is interpreted in a broad sense and represents a wide range of substances. Usually, these substances can increase the immunogenicity of antigens. For example, adjuvants can be recognized by the innate immune system and can induce innate immune response.The vaccine of the present invention includes a sequence having at least 70% homology with any one of the groups selected from SEQ ID NO: 1 to SEQ ID NO: 18 and SEQ ID NO: 20 to SEQ ID NO: 37, and a nucleic acid derived from an antigen or immunogen suitable for the desired immune enhancement, which can be infinitely amplified in use according to the type of antigen or immunogen included. For example, when a nucleic acid derived from cancer tissue is included as an antigen or immunogen, the vaccine is a cancer vaccine. The cancer vaccine herein may refer to an active immunotherapy that activates the immune system by administering cancer-specific antigens of cancer cells to cancer patients to activate the immune function in the body to attack cancer cells and remove cancer. When the vaccine (or vaccine composition) of the present invention is used as a cancer vaccine, the nucleic acid derived from cancer tissue is preferably a new antigen (specific antigen produced only by cancer cells) nucleic acid, but is not limited thereto. Cancer may refer to a disease that forms a mass or tumor composed of undifferentiated cells, which proliferate indefinitely in the tissue without distinction of order. Cancer may refer to an abnormal cell to be killed that proliferates excessively, and in some cases, invades surrounding tissues and organs to form a mass, destroying or changing existing structures. Ultimately, cancer can refer to a group of diseases that can infiltrate and destroy surrounding normal tissues or organs and metastasize from the primary site to any organ of the subject to produce new growth sites, thereby taking the life of the subject. In addition, the cancer can be any one selected from the group consisting of oral cancer, liver cancer, stomach cancer, colon cancer, breast cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head cancer, cervical cancer, skin cancer, melanoma, cervical cancer, ovarian cancer, colorectal cancer, small intestine cancer, rectal cancer, fallopian tube cancer, anal cancer, endometrial cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, lymphoma, bladder cancer, gallbladder cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic leukemia, acute leukemia, lymphocytic lymphoma, kidney cancer, ureteral cancer, renal cell carcinoma, renal pelvis cancer, central nervous system tumors, primary central nervous system lymphoma, spinal cord tumor, brain stem glioma and pituitary tumor, but is not limited to these.

[0026] As used herein, the term "marker" means an indicator that can detect changes in the body using proteins, DNA, RNA, metabolites, etc. Specifically, a tumor marker (TM) or cancer cell marker is a substance that indicates the presence of cancer cells, and means that the examination of a substance in blood, tissue, or excrement is helpful as a diagnostic or therapeutic indicator of cancer in a substance produced by cancer cells or when normal cells in the body react with cancer cells. For example, markers include: carcinoembryonic antigen (CEA), which is a glycoprotein that is a pan-tumor marker that is significantly increased in colon cancer, pancreatic cancer, gastric cancer, breast cancer, etc.; alpha-fetoprotein (AFP), as a marker for primary liver cancer; CA 19-9, as a marker for pancreatic cancer and bile duct cancer; CA 125, as a marker for ovarian cancer; and prostate-specific antigen (PSA), as a marker for prostate cancer. In the case where an artificial nucleic acid (specifically a sequence having 70% or more homology with any one of the groups consisting of SEQ ID NO: 1 to SEQ ID NO: 18 and SEQ ID NO: 20 to SEQ ID NO: 37 of the present invention) is selected for stabilizing nucleic acid as a marker composition for diagnosis or treatment of a specific disease, the disease may be cancer, but is not limited thereto. An artificial nucleic acid comprising a sequence having 70% or more homology with any one of the groups consisting of SEQ ID NO: 1 to SEQ ID NO: 18 and SEQ ID NO: 20 to SEQ ID NO: 37 of the present invention can be used as a cancer cell marker composition together with an antibody suitable for a cancer cell marker or a nucleic acid sequence complementary to a nucleic acid derived from a cancer cell. In this case, the cancer cell marker composition can be used as a vaccine composition and may further include an adjuvant (component) to enhance the desired effect. Specific restrictions on cancer, vaccines or adjuvants overlap with the restrictions described in "immune enhancement" and will be omitted below to avoid overly complicated instructions.

[0027] As used herein, the term "kit" is a concept of a set of preparations including all or part of the preparations for detecting a target substance. The kit can be applied to a biological sample separated from a subject. The biological sample separated from the subject can be one or more selected from the group consisting of blood, saliva, tissue, cells, sputum, bronchoalveolar lavage fluid and urine. The biological sample can be a dry type of liquid or tissue sample, or a sample contained in a liquid. In the present invention, the purpose of the kit can be to confirm whether a specific gene or nucleic acid is present in a biological sample, and the kit can preferably include a sequence having 70% or higher homology with any one of the group consisting of SEQ ID NO: 1 to SEQ ID NO: 18 and SEQ ID NO: 20 to SEQ ID NO: 37 selected from the present invention. For example, as a kit for detecting the A gene or nucleic acid in a biological sample isolated from a subject, a kit including a B nucleic acid that complementarily binds to the A gene or nucleotide as a whole or part of the preparation may include a sequence having 70% or higher homology with any one selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 18 and SEQ ID NO: 20 to SEQ ID NO: 37 of the present invention, and a B nucleic acid for stabilizing the B nucleic acid.

[0028] As used herein, the term "pharmaceutical composition" refers to the concept of a composition for preventing or treating a desired disease. As long as prevention or treatment is required, the disease can be selected without restriction, but for the purpose of the present invention, it can be prevented or treated by regulating gene expression. Here, the gene can be a gene that directly affects the prevention or treatment of a disease (such as a genetic disease), or it can be a gene that is expected to have an effect of indirectly preventing or treating a disease by enhancing immunity in the body (such as immune cell stimulation). In the present invention, the pharmaceutical composition may include a sequence for regulating the expression of a desired gene with 70% or more homology with any one of the groups consisting of SEQ ID NO: 1 to SEQ ID NO: 18 and SEQ ID NO: 20 to SEQ ID NO: 37 of the present invention. For example, a pharmaceutical composition for preventing or treating cancer may be a pharmaceutical composition comprising the following: a sequence with at least 70% homology with any one of the groups consisting of SEQ ID NO: 1-SEQ ID NO: 18 and SEQ ID NO: 20-SEQ ID NO: 37; and a nucleic acid for inhibiting the expression of an oncogene. Alternatively, the pharmaceutical composition may be a pharmaceutical composition comprising: a sequence having at least 70% homology to any one selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 18 and SEQ ID NO: 20 to SEQ ID NO: 37; and a nucleic acid for enhancing the expression of a tumor suppressor gene. Alternatively, the pharmaceutical composition may be a pharmaceutical composition comprising: a sequence having at least 70% homology to any one selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 18 and SEQ ID NO: 20 to SEQ ID NO: 37; and a nucleic acid for stimulating immune cells. Alternatively, the pharmaceutical composition may be a pharmaceutical composition comprising: a sequence having at least 70% homology to any one selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 18 and SEQ ID NO: 20 to SEQ ID NO: 37; and a nucleic acid used as a cancer cell marker. Alternatively, the pharmaceutical composition may be a pharmaceutical composition comprising a sequence having at least 70% homology to any one selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 18 and SEQ ID NO: 20 to SEQ ID NO: 37; and patient-specific neoantigens. Alternatively, the pharmaceutical composition may be a pharmaceutical composition of a synthetic long peptide (SLP), comprising: a sequence having at least 70% homology to any one selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 18 and SEQ ID NO: 20 to SEQ ID NO: 37; and a plurality of patient-specific neoantigens.The pharmaceutical composition can be in the form of capsules, tablets, granules, injections, ointments, powders or beverages, and can target humans. The pharmaceutical composition of the present invention can be formulated and used in the form of oral preparations according to conventional methods, such as powders, granules, capsules, tablets and aqueous suspensions, external preparations, suppositories and injections, but suppositories or injections are preferred. In addition, the pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. During oral administration, a pharmaceutically acceptable carrier can be used with a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, a pigment, a flavoring, etc.; in the case of injection, it can be mixed and used with a buffer, a preservative, an analgesic, a solubilizer, an isotonic agent, a stabilizer, etc.; in the case of topical administration, it can be used with an alkali, an excipient, a lubricant, a preservative, etc. The preparation of the pharmaceutical composition of the present invention can be prepared differently in combination with the above-mentioned pharmaceutically acceptable carriers. For example, for oral administration, the pharmaceutical composition can be formulated into the forms such as tablets, tablets, capsules, elixirs, suspensions, syrups, thin slices (wafer), and for injection, the pharmaceutical composition can also be formulated into single-dose ampoule forms or multiple dose forms. In addition, the pharmaceutical composition can also be formulated into solutions, suspensions, tablets, capsules, sustained-release agents, etc. Simultaneously, the examples of carriers, excipients and diluents suitable for preparations can be used together with lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talcum, magnesium stearate, mineral oil, etc. In addition, the pharmaceutical composition can also include fillers, anticoagulants, lubricants, wetting agents, flavoring agents, emulsifiers, preservatives, etc. The pharmaceutical composition according to the present invention can be administered by oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal routes, but preferably parenteral administration as described above. Parenteral administration includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention can also be administered in the form of suppositories for rectal administration. The pharmaceutical composition of the present invention can be variously changed according to various factors, including the activity, age, body weight, general health, sex, diet, application time, route of administration, excretion rate, pharmaceutical preparation and the severity of the specific disease to be prevented or treated of the specific compound used. The dosage of the pharmaceutical composition varies according to the patient's situation and body weight, disease extent, drug form and route of administration and cycle, but those skilled in the art can appropriately select. The dosage can be administered once a day or several times a day. The dosage does not limit the scope of the present invention in any aspect.

[0029] In an embodiment of the present invention, an artificial nucleic acid is provided, comprising a sequence having at least 70% homology to any one selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 18 and SEQ ID NO: 20 to SEQ ID NO: 37. The sequence may be an untranslated region sequence. The sequence may be a sequence having at least 90% homology to any one selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 18 and SEQ ID NO: 20 to SEQ ID NO: 37. The sequence may be a sequence having at least 95% homology to any one selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 18 and SEQ ID NO: 20 to SEQ ID NO: 37. The artificial nucleic acid may include a sequence having at least 70% homology to any one selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 18; and a sequence having at least 70% homology to any one selected from the group consisting of SEQ ID NO: 20 to SEQ ID NO: 37. The artificial nucleic acid may include a sequence having at least 70% homology to any one selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 18 and located at the 5' end; and a sequence having at least 70% homology to any one selected from the group consisting of SEQ ID NO: 20 to SEQ ID NO: 37 and located at the 3' end. In addition, the artificial nucleic acid may further include at least one of an open reading frame sequence, a promoter sequence, a Kozak sequence, and a poly A tail sequence.

[0030] In another embodiment of the present invention, a vector comprising an artificial nucleic acid is provided, wherein the vector is a plasmid vector or a viral vector.

[0031] In yet another embodiment of the present invention, a transfectant transfected with a vector is provided, wherein the transfectant is a virus, a bacterium, a plant cell or an animal cell.

[0032] In another embodiment of the present invention, a composition comprising an artificial nucleic acid for stabilizing nucleic acid is provided.

[0033] In yet another embodiment of the present invention, an immune enhancing composition comprising an artificial nucleic acid is provided, wherein the immune enhancing composition is a vaccine composition.

[0034] In another embodiment of the present invention, a cancer cell marker composition comprising an artificial nucleic acid is provided, wherein the cancer cell marker is a vaccine composition.

[0035] In yet another embodiment of the present invention, a kit for detecting nucleic acid comprising an artificial nucleic acid is provided.

[0036] In another embodiment of the present invention, a pharmaceutical composition comprising an artificial nucleic acid for preventing or treating a disease is provided.

[0037] In another embodiment of the present invention, a method for selecting a non-translated region sequence for stabilizing nucleic acid is provided, comprising (a) selecting a gene with a top 20% expression level from a database; (b) preliminarily selecting a non-translated region sequence of the selected gene; and (c) secondary selecting a non-translated region sequence without a secondary structure from the selected non-translated region sequence. In step (c), the method for selecting a non-translated region sequence without a secondary structure can be performed by calculating the minimum free energy (MFE) and selecting a 5'UTR sequence with a value greater than -10.

[0038] In another embodiment of the present invention, a method for selecting a non-translated region sequence for stabilizing nucleic acid is provided, comprising (a) selecting a gene with a top 20% expression level from a database; (b) preliminarily selecting a non-translated region sequence of the selected gene; and (c) secondary selection of a non-translated region sequence that does not contain an AU-rich element (ARE) from the selected non-translated region sequence. The method for selecting a non-translated region sequence for stabilizing nucleic acid may also include, after step (b), if there are multiple isoforms of the same gene in the selected non-translated region sequence, secondary selection of the non-translated region sequence of the shortest sequence; and / or clustering miRNA using the binding energy between the selected UTR sequence and miRNA, and then further selecting a non-translated region sequence based on this information.

[0039] In yet another embodiment of the present invention, there is provided a method for producing an artificial nucleic acid, comprising: producing the artificial nucleic acid to comprise a sequence having at least 70% homology to a non-translated region sequence selected by at least one of the methods for selecting a non-translated region sequence for stabilizing a nucleic acid.

[0040] Hereinafter, the present invention will be described in detail by way of examples.

[0041] [Effects of the invention]

[0042] According to the present invention, the UTR sequence has excellent effects of stabilizing nucleic acids and promoting the expression of target proteins, and is therefore expected to be widely used in the health / medical field. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 is a schematic diagram of the structure of a plasmid DNA construct for IVT mRNA synthesis with a selection gene UTR according to an embodiment of the present invention.

[0044] Figure 2Schematic diagram of a plasmid vector using a selection gene according to an embodiment of the present invention. Representatively, an example using the RGS5 gene is shown, but plasmid vectors of other genes also have the same structure, only the gene is changed.

[0045] Figure 3 Shown are the results of linearization of a plasmid DNA vector to confirm a set of UTR candidates prior to in vitro transcription (IVT) according to an embodiment of the present invention.

[0046] Figure 4 The results of analyzing d2eGFP fluorescence expression based on the UTR sequence of selected genes using fluorescence imaging according to an embodiment of the present invention are shown.

[0047] Figure 5 The results of analyzing d2eGFP fluorescence expression based on the UTR sequence of selected genes using flow cytometry according to an embodiment of the present invention are shown.

[0048] Figure 6 Shown are the results of confirming transfection and translation efficiency based on the UTR sequence of selected genes using flow cytometry according to an embodiment of the present invention. DETAILED DESCRIPTION

[0049] Hereinafter, the present invention will be described in more detail with reference to examples. These examples will explain the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention.

[0050] Example 1. Design and selection of UTR candidate sequences

[0051] In order to select UTR sequences that improve the intracellular stability and translation efficiency of mRNA vaccines, 1,689 genes with high expression levels were selected using an in silico method based on genotype tissue expression (GTEx), a database of gene expression information in normal tissues, assuming that genes with high expression levels in vivo will include UTRs that improve mRNA stability in vivo and have high translation efficiency. The 1,689 genes correspond to the top 10% of the 16,903 genes in the database. Thereafter, candidate UTR sequences for stabilizing nucleic acids were selected from the 5'UTR and 3'UTR sequences of the selected genes.

[0052] Specifically, for 5'UTR, relatively short sequences ranging from 20nt to 60nt were selected. If the length of the 5'UTR is longer, vaccine synthesis may be difficult and undesirable structural biological reactions may occur. Since antigens different from the design may be synthesized when the upstream AUG is present in the selected sequence, the presence of the upstream AUG was excluded. In addition, in order to additionally remove 5'UTRs with structural features that inhibit translation (formation of secondary structures in the 5'UTR), the minimum free energy (MFE) was calculated, and 5'UTR sequences with MFE values ​​greater than -10 were selected. As a result, a total of 360 5'UTR candidate sequences were selected. In the case of 3'UTR, 3'UTRs with a length of 20nt to 300nt were first selected for efficient in vitro transcription (IVT). If there are many isoforms of the same gene, 1077 3'UTR candidate sequences were first selected by selecting the shortest 3'UTR among the isoforms. In order to determine the miRNA binding mode that affects the stability of candidate sequence mRNA, the binding energy between 1077 candidate 3'UTR sequences and 2657 miRNAs was calculated (IntaRNA 2.0), and clustering was performed using the cola R package using the binding miRNA type and its energy. Here, the UTR sequences that were classified into the same cluster as the UTR sequences known in various existing literature in the field were secondary selected, and then genes containing AU-rich elements (ARE) involved in 3'UTR mRNA degradation were excluded by referring to the AU-rich element database (ARED-Plus). As a result, a total of 217 3'UTR candidate sequences were selected.

[0053] Example 2. Optimization of selected UTR candidates

[0054] Among the 360 ​​5'UTR and 217 3'UTR sequences selected in Example 1, 40 5'UTR and 3'UTR derived from the same gene were selected. In the UTR, 9 5'UTR and 3'UTR were reselected considering the expression level of the gene, and the sequence was optimized to obtain a strong Kozak sequence. Therefore, the selected genes are CLPS (Escherichia coli protease), APOC3 (apolipoprotein C-III), REG3A (regeneration family member 3α), TNNC1 (troponin C1 type), HPX (heme protein), RGS5 (G protein signaling regulator 5), IFI30 (IFI30 lysosomal thiol reductase), CYP11A1 (cytochrome P450 family 11 subfamily A member 1) and CYC1 (cytochrome C1). In the UTR of the gene, the Kozak sequence optimized UTR sequence is shown in Table 1 (5'UTR) and Table 2 (3'UTR) below. As control UTRs for these sequences, conventional UTR sequences known in the art were used.

[0055] [Table 1]

[0056] [Table 2]

[0057] In the above Tables 1 and 2, if the sequence is RNA, U (uracil) in the sequence can be represented by T in the sequence listing.

[0058] Example 3. Performance evaluation of selected UTR candidates

[0059] Example 3-1. Preparation of plasmid DNA for IVT mRNA synthesis using UTR

[0060] In order to evaluate the performance of the UTR optimization candidate sequences selected in Example 2 above, IVT template DNA was prepared, IVT mRNA was produced by RNA polymerase (T7 polymerase) reaction, and then IVT mRNA was transfected into K562 cell line to compare and analyze the stability of intracellular mRNA and protein expression ability. Specifically, plasmid DNA for producing IVT mRNA in the form of each UTR candidate sequence inserted was prepared, and a vector for producing IVT mRNA was designed and prepared based on the pUC57-kanIVT plasmid vector backbone DNA in consideration of the linearization method and Cap binding site. DNA constructs were prepared using the In-fusion cloning technique and were prepared with primers in the form of extended 3' and 5' terminal sequences (15 bp) of the ligation site so that they could be shared during the insert amplification process. After PCR amplification, the terminal regions were connected and cloned using a linearized plasmid vector and an In-fusion cloning kit. Since the reaction-inducing plasmid DNA contains a kanamycin resistance gene, a vector was designed by performing single colony selection on an antibiotic medium containing kanamycin and inserting the short-lived d2EGFP into the coding sequence (CDS) region to facilitate comparison of protein expression between candidates. Thereafter, based on the prepared IVT vector, DNA was prepared in the form of each selected gene type inserted to produce a final version, namely the IVT template. The prepared UTR candidate constructs have the same sequence except for the UTR sequence. The structure of the plasmid DNA construct for IVT mRNA synthesis containing the prepared UTR is as shown in Figure 1 and Figure 2 shown. Figure 2 A schematic diagram of a plasmid vector using the RGS5 gene is representatively shown, but plasmid vectors of other genes also have the same structure, with only the gene being changed.

[0061] Example 3-2. Plasmid DNA linearization and confirmation

[0062] The control plasmid DNA and 8 plasmid DNA containing the selected gene UTR candidates were linearized in circular form. The plasmid DNA was linearized using a restriction enzyme, and for this purpose, the Sma I restriction enzyme (Enzynomics, cat#R015S) was used. It was confirmed by agarose gel electrophoresis that the linearized plasmid DNA cut by the restriction enzyme Sma I was converted into a linearized form. The experimental results confirmed that all plasmid DNA containing the selected gene UTR sequence were converted from a circular form to a linear form by the Sma I restriction enzyme. The results are shown in Figure 3 shown.

[0063] Example 3-3. mRNA production and performance evaluation using linearized plasmid DNA

[0064] (1) In vitro transcription

[0065] mMESSAGE using 8 linearized plasmid DNAs containing the UTR sequences of the selected genes IVT mRNA synthesis was performed using the T7 transcription kit (Invitrogen, cat#AM1344). IVT was performed using RNA polymerase T7 polymerase to synthesize mRNA, and 8 linear plasmid DNAs contained T7 promoter sequences. Nucleoside triphosphates (NTPs) and Cap analogs were added during the IVT process, and a 5' cap was attached to the 5' end of the IVT mRNA using the co-transcriptional capping method. The concentration of the final synthesized IVT mRNA was measured using a nanodrop device and the concentration was measured using MEGAclear TM The protein was purified using a transcript cleanup kit (Invitrogen, cat# AM1908).

[0066] (2) IVT mRNA transfection

[0067] Translation efficiency was assessed by transfecting UTR candidate IVT mRNA encoding the tagged reporter gene d2EGFP sequence into a human myeloid leukemia cell line (K-562; human lymphoblastoid cells). UTR candidate IVT mRNA transfection was performed using Lipofectamine MessengerMax (Invitrogen, cat#LMRNA015).

[0068] (3) Verification of translation efficiency using IVT mRNA encoding d2EGFP

[0069] IVT mRNA synthesized from 8 linear plasmid DNAs containing control UTR and selected gene UTR sequences contains the marker reporter gene d2EGFP (see Figure 1). After transfection into human myeloid leukemia cell lines, d2EGFP was confirmed to be expressed through IVT mRNA translation. Therefore, 2.5 μg of 8 IVT mRNAs encoding d2EGFP containing the UTR sequences of the selected genes were transfected into 5×10 cells of human myeloid leukemia cell lines using Lipofectamine MessengerMax. 5 Since the translation efficiency of IVT mRNA containing d2EGFP varies depending on the selected gene UTR sequence, it is expected that there will be differences even in the d2EGFP expression level. Therefore, the changes in d2EGFP expression over time were analyzed by fluorescence imaging and flow cytometry at time points of 4, 6, 8, 12, and 24 hours after transfection. The results are shown in Figure 4 and Figure 5 As shown. In the case of fluorescence imaging analysis, since no d2EGFP expression was found in the negative control group treated with Lipofectamine MM alone 12 hours after transfection in the previous study, the fluorescence microscope conditions (exposure time, gain) were determined by this background confirmation. In addition, as a result of confirming d2EGFP expression using the positive (V2 UTR (pTB0110)) control confirmed in the previous study, almost no d2EGFP expression was observed in the no-UTR control (pTB0112) in which the UTR sequence was removed, indicating that the experiment was well performed. As a result of the experiment, the UTRs of APOC3, REG3A, TNNC1, HPX, and RGS5 genes showed higher d2EGFP expression than the positive control V2 UTR (pTB0110). In particular, it was confirmed that the UTR expression of the REG3A, HPX, and RGS5 genes was very significant, and in the case of the UTRs of the REG3A, HPX, and RGS5 genes, the d2EGFP expression was stably maintained for up to 24 hours. In the case of flow cytometry analysis, only live cells (%) were selected from total cells, only K-562 expressing d2EGFP were sorted among the cells, and the differences in d2EGFP expression according to the UTR sequences of the selected genes were compared and analyzed. The results confirmed that IVT mRNA transfection encoding d2EGFP by lipofection maintained high cell viability in all UTR sequences for up to 24 hours. d2EGFP expression was highest in the RGS5 UTR group, but there was no significant difference in all d2EGFP-positive cells over time. The results are shown in Figure 2. Figure 6 shown.

[0070] As mentioned above, the specific parts of the present invention have been described in detail, and those skilled in the art will appreciate that these specific technologies are only preferred embodiments, and the scope of the present invention is not limited thereto. Therefore, the essential scope of the present invention will be defined by the appended claims and their equivalents.

Claims

1. An artificial nucleic acid comprising a sequence having at least 70% homology to any one selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 18 and SEQ ID NO: 20 to SEQ ID NO:

37.

2. The artificial nucleic acid according to claim 1, wherein the sequence is an untranslated region sequence.

3. The artificial nucleic acid according to claim 1, wherein the sequence is a sequence having at least 90% homology to any one selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 18 and SEQ ID NO: 20 to SEQ ID NO:

37.

4. The artificial nucleic acid according to claim 1, wherein the sequence is a sequence having at least 95% homology to any one selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 18 and SEQ ID NO: 20 to SEQ ID NO:

37.

5. The artificial nucleic acid according to claim 1, wherein the artificial nucleic acid comprises a sequence having at least 70% homology to any one selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 18; and A sequence having at least 70% homology to any one selected from the group consisting of SEQ ID NO:20 to SEQ ID NO:

37.

6. The artificial nucleic acid according to claim 5, wherein the artificial nucleic acid comprises a sequence having at least 70% homology to any one selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 18 and located at the 5' end; and A sequence having at least 70% homology to any one selected from the group consisting of SEQ ID NO: 20 to SEQ ID NO: 37 and located at the 3' end.

7. The artificial nucleic acid according to claim 1, further comprising: At least one of an open reading frame sequence, a promoter sequence, a Kozak sequence and a poly A tail sequence. A vector comprising the artificial nucleic acid according to claim 1.

9. The vector according to claim 8, wherein the vector is a plasmid vector or a viral vector.

10. A transfectant transfected with the vector according to claim 8. The transfectant according to claim 10 , wherein the transfectant is a virus, a bacterium, a plant cell or an animal cell.

12. A composition for stabilizing nucleic acid, comprising the artificial nucleic acid according to claim 1.

13. An immunopotentiating composition comprising the artificial nucleic acid according to claim 1.

14. The immune enhancing composition according to claim 13, wherein the immune enhancing composition is a vaccine composition. 15 . A cancer cell marker composition comprising the artificial nucleic acid according to claim 1 . The cancer cell marker composition according to claim 15 , wherein the cancer cell marker composition is a vaccine composition.

17. A kit for detecting nucleic acid, comprising the artificial nucleic acid according to claim 1.

18. A pharmaceutical composition for preventing or treating a disease, comprising the artificial nucleic acid according to claim 1.

19. A method for selecting a sequence for stabilizing an untranslated region of a nucleic acid, comprising: (a) Select genes with the top 20% expression levels from the database; (b) preliminarily selecting the untranslated region sequence of the selected gene; as well as (c) secondarily selecting a non-translated region sequence having no secondary structure from the selected non-translated region sequences.

20. A method for selecting a sequence for stabilizing an untranslated region of a nucleic acid, comprising: (a) Select genes with the top 20% expression levels from the database; (b) preliminarily selecting the untranslated region sequence of the selected gene; as well as (c) secondarily selecting untranslated region sequences that do not have an AU-rich element (ARE) from the selected untranslated region sequences.

21. A method for producing an artificial nucleic acid, comprising: (a) selecting the untranslated region sequence for stabilizing the nucleic acid by the method of claim 19; (b) selecting the untranslated region sequence for stabilizing the nucleic acid by the method of claim 20; as well as (c) manufacturing the artificial nucleic acid to include a sequence having at least 70% homology to the untranslated region sequence selected in step (a) or (b).

22. A method for producing an artificial nucleic acid, comprising: (a) selecting the untranslated region sequence for stabilizing the nucleic acid by the method of claim 19; (b) selecting the untranslated region sequence for stabilizing the nucleic acid by the method of claim 20; as well as (c) manufacturing the artificial nucleic acid to include a sequence having at least 70% homology to the untranslated region sequence selected in steps (a) and (b).

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