Constructs Comprising UTR Sequences That Improve the Intracellular Stability and Biosynthesis of mRNA and Their Use

Abstract

Unlike DNA, mRNA has the advantage that the desired protein can be immediately and temporarily expressed in the cytoplasm without the need to enter the nucleus. Therefore, attempts to utilize mRNA vaccines are actively underway. However, mRNA is structurally less stable than DNA and has limitations for industrial use. Accordingly, the present invention relates to a UTR sequence that improves the intracellular stability and biosynthesis of mRNA, and a nucleic acid stabilization composition containing the same. Since the UTR sequence of the present invention is excellent in nucleic acid stabilization and the effect of enhancing the expression of target proteins, it is expected to be widely used in the healthcare / medical field.

Description

Technical Field

[0001] The present invention relates to a nucleic acid stabilizing sequence, specifically, a UTR (Untranslated region) sequence that improves the intracellular stability and biosynthesis of mRNA, and a nucleic acid stabilizing composition containing the same.

Background Art

[0002] Unlike DNA, mRNA has the advantage that it can immediately and temporarily express a desired protein in the cytoplasm without the need to enter the nucleus, so it has attracted attention as a new treatment method in the healthcare / medical field. In particular, attempts to use mRNA vaccines for the treatment and prevention of diseases are actively underway, mainly by global biotech and pharmaceutical companies. However, mRNA has a lower structural stability compared to DNA and has limitations in industrial use. Therefore, in recent years, attempts have been made to use UTR (Untranslational region) to enhance the intracellular stability of mRNA and improve the protein biosynthesis efficiency in order to solve such problems. The UTR plays an auxiliary role so that a peptide or protein serving as a therapeutic target can be effectively synthesized, and is independent of the sequence of the gene to be synthesized, enabling the expression of various peptides or proteins. In particular, in the field of mRNA vaccines, the UTR improves the stability of mRNA vaccines and enables efficient synthesis of antigens (neoantigens in the case of cancer vaccines) in vivo, so high effects in the treatment and prevention of diseases can be expected even with a small amount of vaccine administration.

[0003] Therefore, the present invention relates to a nucleic acid stabilizing sequence, specifically, a UTR (Untranslated region) sequence that improves the intracellular stability and biosynthesis of mRNA, and a nucleic acid stabilizing composition containing the same. Since the UTR sequence of the present invention is excellent in nucleic acid stabilization and the effect of enhancing the expression of target proteins, it is expected to be widely used in the healthcare / medical field.

Summary of the Invention

Problems to be Solved by the Invention

[0004] An object of the present invention is to provide a nucleic acid stabilization sequence, specifically, a UTR (Untranslated region) sequence that improves the intracellular stability and biosynthesis of mRNA, and a method for selecting the same.

[0005] Another object of the present invention is to provide an artificial nucleic acid containing a nucleic acid stabilization sequence, specifically, a UTR (Untranslated region) sequence that improves the intracellular stability and biosynthesis of mRNA, and a method for producing the same.

[0006] Still another object of the present invention is to provide a vector containing the artificial nucleic acid, and a method for producing the same. Still another object of the present invention is to provide a transformant transformed with a vector containing the artificial nucleic acid, and a method for producing the same.

[0007] Still another object of the present invention is to provide a nucleic acid stabilization composition containing the artificial nucleic acid, and a method for producing the same. Still another object of the present invention is to provide an immune-enhancing composition containing the artificial nucleic acid, and a method for producing the same.

[0008] Still another object of the present invention is to provide a nucleic acid detection kit containing the artificial nucleic acid, and a method for producing the same. Still another object of the present invention is to provide a pharmaceutical composition for preventing or treating a disease containing the artificial nucleic acid, and a method for producing the same.

[0009] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those with ordinary knowledge in the art from the following description.

Means for Solving the Problems

[0010] Various embodiments described in the present application will be described with reference to the drawings. In the following description, for a complete understanding of the present invention, various specific details, such as specific forms, compositions, and processes, etc., are described. However, a particular embodiment may be implemented with one or more of these specific details, or with other known methods and forms. In other instances, known processes and manufacturing techniques are not described in specific detail so as not to unnecessarily obscure the present invention. References throughout this specification to "one embodiment" or "an embodiment" mean that a particular feature, form, composition, or characteristic described in connection with the embodiment is included in one or more embodiments of the present invention. Thus, the occurrences of "one embodiment" or "an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment of the present invention. Further, the particular features, forms, compositions, or characteristics may be combined in a suitable manner in one or more embodiments.

[0011] Unless otherwise defined in the specification, all scientific and technical terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0012] In the present invention, the term "untranslated region (UTR)" refers to a part 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 called 5'-UTR, and the untranslated region located in the 3'-direction of the nucleic acid is called 3'-UTR. Specifically, the 5'-UTR is located at the 5' (i.e., "upstream") of the open reading frame, and is typically understood as a specific section of messenger RNA (mRNA) located at the 5' of the open reading frame of the mRNA. Preferably, the 5'-UTR has a length of 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 can contain elements for regulating gene expression, so-called regulatory elements, and without being limited thereto, such regulatory elements may be ribosome binding sites. The 5'-UTR can be post-transcriptionally modified through the addition of a 5'-CAP. The 5'-UTR of the mRNA is not translated into an amino acid sequence, and the 5'-UTR sequence is generally encoded by the gene transcribed into each mRNA during the gene expression process. The genomic sequence can be transcribed including alternative introns and then modified by processes such as 5'-capping or splicing. The 5'-UTR is a nucleotide adjacent to the 5'-CAP of the modified mRNA and typically corresponds to the transcription start site. Here, "corresponds" means that the 5'-UTR sequence can be an RNA sequence such as the mRNA sequence or a DNA sequence corresponding to such an RNA sequence. The 3'-UTR is located at the 3' (i.e., "downstream") of the open reading frame, and is typically understood as a specific section of messenger RNA (mRNA) located at the 3' of the open reading frame of the mRNA. Preferably, the 3'-UTR has a length of 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). It is located between the region encoding the protein of the mRNA and the polyA sequence.The 3'-UTR sequence is generally encoded by the gene transcribed into each mRNA during the gene expression process. After the genetic sequence is transcribed including selective introns, it can be modified by processes such as splicing or 3'-polyadenylation. The 3'-UTR is the nucleotide close to the poly-A sequence of the modified mRNA and typically corresponds to the transcription termination site. Here, "corresponding" means that the 3'-UTR sequence can be an RNA sequence like the mRNA sequence or a DNA sequence corresponding to such an RNA sequence.

[0013] In the present invention, the term "nucleic acid" is a concept including DNA (deoxyribonucleic acid) and RNA (ribonucleic acid). It is used synonymously with the term "polynucleotide". Preferably, it is a polymer comprising nucleotide monomers covalently bonded to each other by phosphodiester bonds of the sugar / phosphate backbone. Or it includes DNA or RNA with modified bases, modified sugars, or modified backbones.

[0014] In the present invention, the term "stabilizing nucleic acid" is a concept that prevents the disintegration or deformation of the nucleic acid structure inside or outside the cell and stabilizes the nucleic acid structure. Generally, RNA consists of a single strand and has lower structural stability compared to DNA because of its higher activity. Therefore, the nucleic acid stabilization in the present invention is for preventing the structural disintegration or deformation of DNA, RNA, more preferably RNA, and even more preferably mRNA. The sequences represented by SEQ ID NO: 1 to SEQ ID NO: 18 and SEQ ID NO: 20 to SEQ ID NO: 37 in the present invention were each evaluated to have excellent nucleic acid stabilization effects.

[0015] In the present invention, the term "sequence homology" refers to the percentage indicating the degree to which two sequences to be compared are identical. The sequences to be compared in order to determine the degree of homology are preferably of the same length. However, when the sequence lengths are different, the calculation is based on the longer sequence among the sequences to be compared. For example, a 10-nt sequence has 80% homology with an 8-nt sequence that is represented as the same sequence as a part thereof.

[0016] In the present invention, the artificial nucleic acid having excellent nucleic acid stabilization effect of the present invention may be an artificial nucleic acid containing 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 containing 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 containing 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 containing 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 containing a sequence having 100% 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.

[0017] In the present invention, the term "artificial nucleic acid" can be understood as a non-natural nucleic acid molecule that does not originally exist in nature. The artificial nucleic acid can be composed entirely of sequences that do not exist in nature, or can be composed of a mixture of a part of a sequence that exists in nature (wild type) and a part of a sequence that does not exist in nature. When composed of a mixture of a part of a sequence that exists in nature and a part of a sequence that does not exist in nature, the sequence that exists in nature can be a mixture of sequences derived from one or more species. The artificial nucleic acid can be non-natural due to non-naturally occurring modifications of its individual sequences, for example, structural modifications of nucleotides that do not occur naturally. Also, the artificial nucleic acid can be a DNA molecule, an RNA molecule, or a hybrid molecule containing DNA and RNA moieties.

[0018] In the present invention, the term "vector" can be understood as a kind of the artificial nucleic acid. In the context of the present invention, a vector is an artificial nucleic acid selected for nucleic acid stabilization, specifically, 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 having a homology of 70% or more with a sequence included therein. Such a vector may be a storage vector, an expression vector, a cloning vector, a transport vector, etc. A storage vector is a vector that allows for convenient storage of nucleic acid molecules, for example, mRNA molecules. An expression vector can be used for the production of expression products such as RNA, for example, mRNA, or peptides, polypeptides or proteins. A cloning vector is a vector that typically contains a cloning site that can be used to incorporate a nucleic acid sequence into the vector. A cloning vector may be, for example, a plasmid vector or a bacteriophage vector. A transport vector is a vector suitable for transporting nucleic acid molecules into cells or organisms and may be, for example, a viral vector. In the context of the present invention, a vector may be, for example, an RNA vector or a DNA vector. Preferably, the vector is a DNA molecule. Preferably, the vector of the present invention is an artificial nucleic acid selected for nucleic acid stabilization, specifically, 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 having a homology of 70% or more with a sequence included therein and includes a sequence of a target gene (a sequence encoding a target protein) for the purpose of expression regulation. This is preferably a plasmid vector, but is not limited thereto.

[0019] In the present invention, the term "transfection" means introducing a nucleic acid molecule such as a DNA or RNA (e.g., mRNA) molecule into a cell, preferably into a eukaryotic cell, to change the original traits of the object to be introduced. The transfection can be carried out by a method known to an ordinary technician for introducing a nucleic acid molecule into a cell, preferably into a eukaryotic cell such as a mammalian cell. Such methods include, for example, electroporation, lipofection 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, the transfection can be carried out using a virus, for example, a lentivirus. An object to be introduced that has been transfected by such a method and whose original traits have changed is named a transformant.

[0020] In the present invention, the term "immunity-boosting" means that a desired immune response is amplified against selected antigens such as characteristic components of bacterial surfaces, virus particles, tumor antigens, etc. The immune response can be amplified naturally or induced to be amplified artificially, and in the case of artificial amplification of the immune response, vaccination is the most common method. A vaccine is typically understood to be a prophylactic or therapeutic substance that provides at least one antigen, preferably an immunogen. The antigen or immunogen can be derived from a substance suitable for vaccination. For example, the antigen or immunogen can be derived from bacteria or virus particles, etc., or from tumors or cancer tissues. The antigen or immunogen stimulates the adaptive immune system in vivo. Conventionally, vaccines that utilize proteins or peptides derived from antigens or immunogens as stimulants of the adaptive immune system in vivo have been dominant, but in recent years, attempts to utilize nucleic acids as stimulants have been ongoing. In particular, mRNA has the advantage that, unlike DNA, it can immediately and temporarily express a desired protein in the cytoplasm without the need to enter the nucleus, and thus has attracted attention as a new treatment method in the healthcare / medical field. However, mRNA is structurally less stable than DNA and has limitations for industrial use. Therefore, in recent years, attempts have been made to improve the intracellular stability of mRNA and enhance the biosynthesis efficiency of proteins by utilizing the UTR (Untranslational region) to solve such problems. In such a context, any one 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, which has excellent nucleic acid stabilization effects of the present invention, or a sequence having 70% or more homology thereto can be administered as a vaccine together with a nucleic acid derived from an antigen or immunogen suitable for the intended immunity-boosting.In such a case, any one 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 having 70% or more homology therewith, is preferably located before / after the antigen genetic information in the nucleic acid vaccine, and it is expected to have an effect of boosting so that the nucleic acid vaccine is protected from degrading enzymes in vivo and the nucleic acid sequence is effectively translated as an antigen. In addition, since the stability of the nucleic acid vaccine is improved and antigens can be efficiently synthesized in vivo, high effects can be expected for the treatment and prevention of diseases even with a small amount of vaccine administration. Also, since the volume of the vaccine administered to satisfy the expected therapeutic effect is reduced, an effect of reducing the production cost of the vaccine can be expected. The vaccine can further contain an adjuvant component for boosting the expected effect. An adjuvant is typically a pharmaceutical and / or immunological formulation that can modify or enhance the effect of other formulations, such as drugs or vaccines. This is interpreted in a broad sense and indicates a wide range of substances. Typically, these substances can increase the immunogenicity of antigens. For example, an adjuvant can be recognized by the innate immune system and can induce an innate immune response. The vaccine of the present invention containing 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, and nucleic acids derived from antigens or immunogens suitable for the intended immune enhancement can have its uses infinitely extended depending on the types of antigens or immunogens contained. For example, when nucleic acids derived from cancer tissues are included as antigens or immunogens, the vaccine is a cancer vaccine. Here, the cancer vaccine can mean an active immunotherapy method that activates the in vivo immune function by administering cancer-specific antigens possessed by cancer cells to cancer patients to activate the immune system and attacks cancer cells to remove cancer. When the vaccine (or vaccine composition) of the present invention is used as a cancer vaccine, the nucleic acids derived from the cancer tissues are preferably, but not limited to, neoantigen nucleic acids (specific antigens that appear only in cancer cells).The cancer may mean a tumor composed of undifferentiated cells that grow unrestrictedly regardless of order within a tissue, or a disease that forms a tumor. It may mean a condition in which abnormal cells that should die proliferate excessively, and in some cases invade surrounding tissues and organs to form a tumor, destroying or deforming the existing structure. Ultimately, it may be a general term for a group of diseases that infiltrate and destroy surrounding normal tissues and organs, can metastasize to any organ of an individual from the primary lesion to create a new growth site, and can deprive the individual of life. Also, the cancer may be any one selected from the group consisting of oral cancer, liver cancer, gastric cancer, colon cancer, breast cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head cancer, neck cancer, skin cancer, melanoma, cervical cancer, ovarian cancer, colorectal cancer, small intestine cancer, rectal cancer, fallopian tube carcinoma, cancer near the anus, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, adenocarcinoma of the lymph nodes, bladder cancer, gallbladder cancer, endocrine adenocarcinoma, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic leukemia, acute leukemia, lymphocyte lymphoma, kidney cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma, but is not limited thereto.

[0021] In the present invention, the term "markers" refers to indicators that can detect changes in the body using proteins, DNA, RNA, metabolites, etc. Specifically, tumor markers (TMs), or cancer cell markers, are substances that indicate the presence of cancer cells, including substances produced by cancer cells or substances produced by normal cells in the body reacting with cancer cells, and substances that can be detected in blood, tissues, excreta, etc. and are useful as indicators for cancer diagnosis and treatment. As an example, CEA (Carcinoembryonic Antigen), a type of glycoprotein that significantly increases in colorectal cancer, pancreatic cancer, gastric cancer, breast cancer, etc. and is a pan-tumor marker, AFP (Alfa-Fetoprotein), a marker for primary liver cancer, CA 19-9, a marker for pancreatic cancer and biliary tract cancer, CA 125, a marker for ovarian cancer, PSA (Prostate Specific Antigen), a marker for prostate cancer, etc. When a sequence having 70% or more homology with any one selected from the group consisting of the artificial nucleic acids selected for nucleic acid stabilization of the present invention, specifically, SEQ ID NO: 1 to SEQ ID NO: 18 and SEQ ID NO: 20 to SEQ ID NO: 37, is used as a labeling composition for the diagnosis or treatment of a specific disease, although not limited thereto, the disease may be a cancer disease, and an artificial nucleic acid containing 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 of the present invention can be used as a cancer cell labeling composition together with an antibody suitable for cancer cell labeling or a nucleic acid sequence complementary to a nucleic acid derived from cancer cells. In such a case, the cancer cell labeling composition can be used as a vaccine composition and can further contain an adjuvant for boosting the expected effect. Specific limitations regarding the above-mentioned cancer, vaccine, or adjuvant overlap with those described in the above "immune enhancement" and are omitted below to avoid excessive complexity of this specification.

[0022] In the present invention, the term "kit" is a concept of a set including all or part of a preparation for detecting a target substance. The kit may be applied to a biological sample separated from an individual. The biological sample separated from the individual may be one or more selected from the group consisting of blood, saliva, tissue, cells, sputum, bronchial cell washing fluid, and urine. For the biological sample, a liquid or tissue sample in a dried form, or a sample contained in a liquid may be used. In the present invention, the purpose of the kit may be to confirm the presence or absence of a specific gene or nucleic acid in a biological sample, and the kit preferably contains 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 sequence having a homology of 70% or more. For example, in the case of a kit for detecting gene A or a nucleic acid in a biological sample separated from an individual, which kit contains, as all or part of a preparation, nucleic acid B that binds complementarily to gene A or the nucleic acid, the kit may contain, together with nucleic acid B, a sequence having a homology of 70% or more 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 for stabilizing nucleic acid B.

[0023] In the present invention, the term "pharmaceutical composition" is a concept of a composition administered for the prevention or treatment of a target disease. The above-mentioned disease can be selected without limitation as long as prevention or treatment is required, but for the purpose of the present invention, it may be one that is prevented or treated by regulating gene expression. The gene here may be, for example, a gene that directly affects the prevention or treatment of a disease such as a genetic disease, or, for example, one that can be expected to have an indirect preventive or therapeutic effect on a disease through enhancing in vivo immunity such as immune cell stimulation. In the present invention, the pharmaceutical composition may contain 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 having a homology of 70% or more for the regulation of the target gene expression. For example, in the case of a pharmaceutical composition for cancer prevention or treatment, it may be a pharmaceutical composition containing a sequence having a homology of 70% or more 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 nucleic acid for suppressing the expression of an oncogene. Or, it may be a pharmaceutical composition containing a sequence having a homology of 70% or more 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 nucleic acid for enhancing the expression of an antioncogene. Or, it may be a pharmaceutical composition containing a sequence having a homology of 70% or more 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 nucleic acid for immune cell stimulation. Or, it may be a pharmaceutical composition containing a sequence having a homology of 70% or more 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 nucleic acid for cancer cell labeling. Or, it may be a pharmaceutical composition containing a sequence having a homology of 70% or more 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 patient-specific neoantigen.Alternatively, it may be a pharmaceutical composition of a synthetic long peptide (SLP) containing a sequence having at least 70% 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 plurality of patient-specific neoantigens. The pharmaceutical composition may be characterized in that it is in the form of a capsule, tablet, granule, injection, ointment, powder or beverage, and may be characterized in that it is intended for human use. The pharmaceutical composition of the present invention can be formulated into oral dosage forms such as powders, granules, capsules, tablets, aqueous suspensions, etc., external preparations, suppositories and injections according to conventional methods, but is preferably a suppository or an injection. In addition, the pharmaceutical composition of the present invention can contain a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers can include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, dyes, flavors, etc. for oral administration, and buffers, preservatives, soothing agents, solubilizers, isotonic agents, stabilizers, etc. can be mixed and used for injections, and bases, excipients, lubricants, preservatives, etc. can be used for topical administration. The dosage form of the pharmaceutical composition of the present invention can be manufactured in various ways by mixing with pharmaceutically acceptable carriers as described above. For example, for oral administration, it can be manufactured in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and for injections, it can be manufactured in unit-dose ampoules or multiple-dose forms. In addition, it can be formulated into solutions, suspensions, tablets, capsules, sustained-release formulations, etc. On the other hand, examples of carriers, excipients and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil, etc. It can also further contain fillers, anticoagulants, lubricants, wetting agents, flavors, emulsifiers, preservatives, etc.The pharmaceutical composition according to the present invention can be administered through oral, intravenous, intramuscular, intra-arterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal routes, but parenteral administration is preferred as described above. The parenteral administration includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intradural, 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 vary diversely depending on various factors including the activity of the specific compound used, age, weight, general health, sex, formulation, administration time, administration route, excretion rate, drug combination and severity of the specific disease to be prevented or treated, and the dosage of the pharmaceutical composition varies depending on the patient's condition, weight, degree of disease, drug form, administration route and period, but can be appropriately selected by those skilled in the art. Administration may be once a day or divided into several times. The dosage does not limit the scope of the present invention in any interpretation.

[0024] In one specific example of the present invention, there is provided an artificial nucleic acid comprising a sequence having at least 70% 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, wherein the sequence is of an untranslated region sequence, there is provided an artificial nucleic acid wherein the sequence has at least 90% 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, there is provided an artificial nucleic acid wherein the sequence has at least 95% 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, the artificial nucleic acid comprises a sequence having at least 70% homology with any one selected from SEQ ID NO: 1 to SEQ ID NO: 18; and a sequence having at least 70% homology with any one selected from SEQ ID NO: 20 to SEQ ID NO: 37; there is provided an artificial nucleic acid wherein a sequence having at least 70% homology with any one selected from SEQ ID NO: 1 to SEQ ID NO: 18 is located at the 5'-end and a sequence having at least 70% homology with any one selected from SEQ ID NO: 20 to SEQ ID NO: 37 is located at the 3'-end, and further, there is provided an artificial nucleic acid which further comprises any one or more of an open reading frame sequence, a promoter sequence, a Kozak sequence, and a poly A tail sequence.

[0025] In another specific example of the present invention, there is provided a vector comprising the artificial nucleic acid, and there is provided a vector comprising the artificial nucleic acid, which is a plasmid vector or a viral vector.

[0026] In still another specific example of the present invention, there is provided a transformant transformed with the vector, and there is provided a transformant transformed with the vector, which is a virus, a bacterium, a plant cell, or an animal cell.

[0027] In yet another specific example of the present invention, there is provided a composition for nucleic acid stabilization containing the artificial nucleic acid. In yet another specific example of the present invention, there is provided a composition for immune enhancement containing the artificial nucleic acid, and the composition for immune enhancement provides a composition for immune enhancement containing the artificial nucleic acid which is for a vaccine composition.

[0028] In yet another specific example of the present invention, there is provided a composition for cancer cell labeling containing the artificial nucleic acid, and the composition for cancer cell labeling provides a composition for cancer cell labeling containing the artificial nucleic acid which is for a vaccine composition.

[0029] In yet another specific example of the present invention, there is provided a kit for nucleic acid detection containing the artificial nucleic acid. In yet another specific example of the present invention, there is provided a pharmaceutical composition for preventing or treating a disease containing the artificial nucleic acid.

[0030] In yet another specific example of the present invention, there is provided a method for selecting an untranslated region sequence for nucleic acid stabilization, including: (a) a step of selecting genes with an expression level in the top 20% from a database; (b) a step of primarily selecting the untranslated region (UTR) sequences of the selected genes; and (c) a step of secondarily selecting untranslated region sequences without a secondary structure among the selected untranslated region sequences. The method for selecting untranslated region sequences without a secondary structure in the step (c) may be to calculate the Minimum Free Energy (MFE) and select a 5’UTR sequence with a value greater than -10.

[0031] In yet another specific example of the present invention, a method for selecting an untranslated region sequence for nucleic acid stabilization includes: (a) a step of selecting genes with an expression level in the top 20% from a database; (b) a step of primarily selecting the untranslated region (UTR) sequences of the selected genes; and (c) a step of secondarily selecting untranslated region sequences that do not contain an ARE (AU rich element) among the selected untranslated region sequences. After the step (b), in the method for selecting the untranslated region sequence for nucleic acid stabilization, when various isoforms of the same gene exist among the selected untranslated region sequences, a step of secondarily selecting the shortest untranslated region sequence; and / or a step of clustering miRNAs using the binding energy between the selected UTR sequence and miRNAs, and then additionally selecting the untranslated region sequence based on the relevant information can be further included.

[0032] In yet another specific example of the present invention, a method for manufacturing an artificial nucleic acid includes a step of manufacturing an artificial nucleic acid such that the sequence has at least 70% homology with the untranslated region sequence selected by any one or more of the methods for selecting the untranslated region sequence for nucleic acid stabilization.

[0033] Hereinafter, the present invention will be described in detail with reference to examples.

Advantages of the Invention

[0034] Since the UTR sequence of the present invention is excellent in nucleic acid stabilization and the effect of enhancing the expression of target proteins, it is expected to be widely used in the health / medical field.

Brief Description of the Drawings

[0035]

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Mode for Carrying Out the Invention

[0036] Hereinafter, the present invention will be described in more detail through examples. It will be apparent to those having ordinary knowledge in the art that these examples are solely for the purpose of more specifically explaining the present invention, and the scope of the present invention is not limited by these examples according to the gist of the present invention.

Examples

[0037] Example 1. UTR Candidate Sequence Design and Selection To select UTR sequences that enhance the intracellular stability and translation efficiency of mRNA vaccines, 1,689 highly expressed genes were selected by an in silico method based on the Genotype-Tissue Expression (GTEx) database, which is a gene expression information database in normal tissues, under the assumption that highly expressed genes in vivo would contain UTRs that enhance the stability of mRNA in vivo and have high translation efficiency. The 1,689 genes correspond to the top 10% of the 16,903 genes in the database. Subsequently, candidate UTR sequences for nucleic acid stabilization were selected for the 5'UTR and 3'UTR sequences of the selected genes.

[0038] Specifically, in the case of the 5’UTR, relatively short sequences of 20 nt to 60 nt were selected. When the length of the 5’UTR is long, difficulties in vaccine synthesis may occur, and undesired structural biological reactions may occur. If an upstream AUG exists in the selected sequence, an antigen different from the design may be synthesized, so those with an upstream AUG were excluded. In addition, to further remove 5’UTRs having structural characteristics (formation of secondary structure within the 5’UTR) that inhibit translation, the Minimum Free Energy (MFE) was calculated, and 5’UTR sequences with a value greater than -10 were selected. As a result, a total of 360 5’UTR candidate sequences were selected. In the case of the 3’UTR, 3’UTRs with a length satisfying 20 nt to 300 nt were first selected for efficient in vitro transcription (IVT). If there are various isoforms of the same gene, the shortest 3’UTR among them was selected, and initially, 1,077 3’UTR candidate sequences were selected. To understand the binding mode with miRNAs that affect the stability of mRNA for these, the binding energies between 1,077 candidate 3’UTR sequences and 2,657 miRNAs were calculated (IntaRNA 2.0), and clustering was performed using the cola R package using the types of bound miRNAs and their energies. Here, UTR sequences classified into the same cluster as UTR sequences known through various prior literatures in the art were secondarily selected, and thereafter, genes containing an AU rich element (ARE) involved in mRNA degradation in the 3’UTR were excluded with reference to the AU-Rich Element Database (ARED-Plus). As a result, a total of 217 3’UTR candidate sequences were selected.

[0039] Example 2. Optimization of the Selected UTR Candidates Out of the 360 types of 5’UTRs and 217 types of 3’UTR sequences selected in Example 1 above, 40 types of 5’UTRs and 3’UTRs derived from the same gene were selected. Among them, considering the gene expression levels, 9 types of 5’UTRs and 3’UTRs were reselected each, and sequence optimization was carried out to have a strong Kozak sequence. As a result, the selected genes were CLPS (Colipase), APOC3 (Apolipoprotein C-III), REG3A (Regenerating Family Member 3 Alpha), TNNC1 (Troponin C type 1), HPX (Hemopexin), RGS5 (Regulator Of G Protein Signaling 5), IFI30 (IFI30 Lysosomal Thiol Reductase), CYP11A1 (Cytochrome P450 Family 11 Subfamily A Member 1), and CYC1 (Cytochrome C1). The UTR sequences with optimized Kozak sequences among the UTRs of the said genes are shown in Table 1 (5’UTR) and Table 2 (3’UTR) below. As the control group UTRs for these, UTR sequences conventionally known in the art were used.

[0040]

Table 1-1

[0041]

Table 1-2

[0042]

Table 2-1

[0043]

Table 2-2

[0044]

Table 2-3

[0045]

Table 2-4

[0046]

Table 2-5

[0047]

Table 2-6

[0048]

Table 2-7

[0049] Example 3. Performance Evaluation of Selected UTR Candidates Example 3-1. Preparation of plasmid DNA for IVT mRNA synthesis with an endogenous UTR For the performance evaluation of the UTR optimization candidate sequences selected in Example 2 above, after producing IVT mRNA through an RNA polymerase (T7 polymerase) reaction, this was transfected into the K562 cell line to compare and analyze the intracellular mRNA stability and protein expression ability, and thus an IVT template DNA was prepared. Specifically, plasmid DNA for IVT mRNA production in the form in which each UTR candidate sequence was inserted was prepared, and based on the pUC57-kan IVT plasmid vector backbone DNA, a vector for IVT mRNA production was designed and prepared considering the Linearization method and the Cap binding site. The preparation of the DNA construct was carried out using the In-fusion cloning technique. When amplifying the insert fragment, it was carried out with a primer in a form in which the corresponding sequence was extended so as to share the 3' and 5' terminal sequences (15 bp) of the ligation site. After PCR amplification, the terminal sites were ligated and cloned using the linearized plasmid vector and the In fusion cloning kit. Since the reaction-induced plasmid DNA contained a kanamycin resistance gene, it was carried out through single colony selection in an antibiotic medium supplemented with kanamycin, and d2EGFP having a short half-life in the CDS (Coding sequence) region was inserted so as to facilitate the comparison of protein expression between candidates. Thereafter, based on the completed IVT vector, DNA in the form in which each selected gene form was inserted was prepared to prepare the final version of the IVT template. The prepared UTR candidate construct has the same sequence except for the UTR sequence. The plasmid DNA construct structure for IVT mRNA synthesis incorporating the prepared UTR is shown in FIGS. 1 and 2. FIG. 2 shows a schematic diagram of a plasmid vector using the RGS5 gene as a representative, but plasmid vectors of other genes also show the same structure except that only the gene is changed.

[0050] Example 3-2. Plasmid DNA linearization and confirmation Eight plasmid DNA circular forms containing the Control plasmid DNA and the selected gene UTR candidate group were linearized. Plasmid DNA linearization was carried out using a restriction enzyme, and for this purpose, the SmaI restriction enzyme (Enzynomics, cat# R015S) was used. The linearized plasmid DNA cut by the restriction enzyme SmaI was confirmed for the change to the linearized form through agarose gel electrophoresis. As a result of the experiment, it was confirmed that all plasmid DNAs containing the selected gene UTR sequence were changed from the circular form to the linear form by the SmaI restriction enzyme. The above results are shown in Figure 3.

[0051] Example 3-3. mRNA production and performance evaluation using the linearized plasmid DNA (1)In vitro transcription IVT mRNA synthesis was carried out using the mMESSAGE mMACHINE® T7 Transcription Kit (Invitrogen, cat# AM1344) with 8 kinds of linear plasmid DNA containing the selected gene UTR sequences. IVT proceeded with RNA polymerase T7 polymerase to synthesize mRNA, and the 8 kinds of linear plasmid DNA contained the T7 promoter sequence. Nucleoside triphosphate (NTPs) and Cap analog were added during the IVT process to attach a 5’ cap to the 5’ end of the IVT mRNA by the co-transcriptional capping method. The finally synthesized IVT mRNA was measured for concentration using a Nanodrop instrument and purified using the MEGAclear™ Transcription Clean-Up Kit (Invitrogen, cat# AM1908).

[0052] (2) IVT mRNA transfection The translation efficiency was evaluated by transfecting the UTR candidate group IVT mRNA encoding the d2EGFP sequence, a marker reporter gene, into the human myeloid leukemia cell line (K-562; human lymphoblast). The UTR candidate group IVT mRNA transfection was carried out using Lipofectamine MessengerMax (Invitrogen, cat# LMRNA015).

[0053] (3) Confirmation of translational efficiency using d2EGFP-encoding IVT mRNA The IVT mRNA synthesized from 8 types of linear plasmid DNA containing the Control UTR and the selected gene UTR sequences contains the marker reporter gene d2EGFP (see Figure 1). After transfection into the human myeloid leukemia cell line, it was confirmed that d2EGFP was expressed by IVT mRNA translation. 2.5 μg of 8 types of d2EGFP-encoding IVT mRNA containing the selected gene UTR sequence was used for the human myeloid leukemia cell line 5×10 5Transfection was carried out using Lipofectamine MessengerMax in cells. Since the translation efficiency of d2EGFP endogenous IVT mRNA varies depending on the selected gene UTR sequence, it was expected that there would also be differences in the d2EGFP expression level. After transfection, fluorescence imaging analysis and flow cytometry analysis were performed at 4, 6, 8, 12, and 24 hours to analyze the changes in d2EGFP expression over time. The results are shown in Figures 4 and 5. In the case of fluorescence imaging analysis, in the negative control group treated with only Lipofectamine MM 12 hours after transfection, no d2EGFP expression was found in previous studies. Therefore, the fluorescence microscope conditions (Exposure time, Gain) were determined through such background confirmation. Also, in the d2EGFP expression confirmation results using the Positive (V2 UTR (pTB0110)) control confirmed in previous studies, almost no d2EGFP expression was shown in the no UTR-control (pTB0112) where the UTR sequence was removed, indicating that the experiment proceeded well. As a result of the experiment, the UTRs of the APOC3, REG3A, TNNC1, HPX, and RGS5 genes showed higher d2EGFP expression than the positive control V2 UTR (pTB0110). In particular, the UTR expressions of the REG3A, HPX, and RGS5 genes were very prominent, and in the case of the UTRs of the REG3A, HPX, and RGS5 genes, it was confirmed that the d2EGFP expression was stably maintained until 24 hours. In the case of flow cytometry analysis, only Live cells (%) among all the cells were selected, and only the K-562 cells expressing d2EGFP were classified and the differences in d2EGFP expression by the selected gene UTR sequences were compared and analyzed. As a result, it was confirmed that d2EGFP-encoding IVT mRNA transfection through lipofection maintained a high cell viability until 24 hours for all UTR sequences.d2EGFP expression was highest in the RGS5 UTR group, but no significant difference was shown among all d2EGFP positive cells over time. The above results are shown in Figure 6.

[0054] As described above in detail for specific parts of the present invention, it is obvious to those with ordinary knowledge in the art that such specific descriptions are merely preferred embodiments, and the scope of the present invention is not limited thereto. Therefore, it can be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An artificial nucleic acid comprising a sequence having at least 70% 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. Artificial nucleic acid.

2. The artificial nucleic acid according to claim 1, wherein the sequence is a non-translated region (untranslated region) sequence.

3. The sequence is The artificial nucleic acid according to claim 1, wherein the sequence has at least 90% 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.

4. The sequence is The artificial nucleic acid according to claim 1, wherein the sequence has at least 95% 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.

5. The artificial nucleic acid is A sequence having at least 70% homology with any one selected from SEQ ID NO: 1 to SEQ ID NO: 18; and A sequence having at least 70% homology with any one selected from SEQ ID NO: 20 to SEQ ID NO: 37; The artificial nucleic acid according to claim 1, which comprises.

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

7. The artificial nucleic acid according to claim 1, further comprising any one or more of an open reading frame (open reading frame) sequence, a promoter (promoter) sequence, a Kozak sequence, and a poly A tail sequence.

8. 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 transformant transformed with the vector according to claim 8.

11. The transformant according to claim 10, wherein the transformant is a virus, a bacterium, a plant cell, or an animal cell.

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

13. A composition for enhancing immunity comprising the artificial nucleic acid according to claim 1.

14. The immunopotentiating composition according to claim 13, which is a composition for a vaccine.

15. A composition for labeling cancer cells, comprising the artificial nucleic acid according to claim 1.

16. The composition for labeling cancer cells according to claim 15, which is a composition for a vaccine.

17. A kit for nucleic acid detection, 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) A step of selecting genes whose expression level is in the top 20% from a database; (b) A step of primarily selecting the untranslated region (UTR) sequences of the selected genes; and (c) A step of secondarily selecting untranslated region sequences that do not have a secondary structure among the selected untranslated region sequences; A method for selecting an untranslated region sequence for nucleic acid stabilization, comprising:

20. (a) A step of selecting genes whose expression level is in the top 20% from a database; (b) A step of primarily selecting the untranslated region (UTR) sequences of the selected genes; and (c) A step of secondarily selecting untranslated region sequences that do not have an ARE (AU rich element) among the selected untranslated region sequences; A method for selecting an untranslated region sequence for nucleic acid stabilization, comprising:

21. (a) A step of selecting an untranslated region sequence for nucleic acid stabilization by the method according to claim 19; (b) A step of selecting an untranslated region sequence for nucleic acid stabilization by the method according to claim 20; and (c) A step of producing an artificial nucleic acid so that the sequence has at least 70% homology with the untranslated region sequence selected in (a) or (b); A method for producing an artificial nucleic acid, comprising:

22. (a) A step of selecting an untranslated region sequence for nucleic acid stabilization by the method according to claim 19; (b) A step of selecting an untranslated region sequence for nucleic acid stabilization by the method according to claim 20; and (c) A step of producing an artificial nucleic acid so that the sequence has at least 70% homology with the untranslated region sequences selected in (a) and (b); A method for producing an artificial nucleic acid, comprising:

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