Novel nucleic acid construct comprising poly(a) tail having secondary or tertiary structure and containing non-a sequence at terminus, and uses thereof

Abstract

The present invention relates to a novel nucleic acid construct with improved stability and protein expression levels and uses thereof and, more specifically, to a nucleic acid construct comprising: a coding region encoding a polypeptide or a protein; a poly(A) tail having a secondary or tertiary structure; and a non-A sequence at the 3'-terminal region, and to a pharmaceutical composition for vaccine or gene therapy comprising said nucleic acid construct. The nucleic acid construct platform according to the present invention exhibits superior stability and protein expression levels in cells without interference with CDS and UTR sequences, or other genetic elements, and thus may be broadly and advantageously used in fields such as gene therapy and vaccine applications.

Description

Novel nucleic acid construct comprising a poly A tail having a secondary or tertiary structure and containing a non-A sequence at the end, and uses thereof

[0001] The present invention relates to a novel nucleic acid structure with improved stability and protein expression levels and its use, and more specifically, to a nucleic acid structure comprising: a coding region encoding a polypeptide or protein; a poly(A) tail having a secondary or tertiary structure; and a non-A sequence at a 3′ polyA terminus, and a pharmaceutical composition for a vaccine or gene therapy comprising said nucleic acid structure.

[0002]

[0003] mRNA is attracting attention as a potential new drug class due to its advantages over protein therapeutics, such as a simpler purification method and shorter development time, which have recently led to its application in vaccines or gene therapies. Unlike DNA, mRNA therapeutics are not transported to the nucleus upon injection into the human body; therefore, they induce therapeutic effects by temporarily expressing desired proteins without genetic modification, offering significant advantages in terms of safety.

[0004] In vitro transcription (IVT) is a standardized method for producing mRNA that begins with a pDNA vector for IVT as a template. Such pDNA vectors for IVT can have the following structures: a 5′ RNA polymerase promoter for RNA transcription, 3′ and / or 5′ untranslated regions (UTRs), and a 3′ polyadenyl cassette containing A nucleotides (=poly(A) region). pDNA with these structures exists in a circular form and is linearized using type IIS restriction enzymes for IVT.

[0005] However, mRNA is a single-stranded nucleic acid that can be easily degraded by common RNA degrading enzymes from production to injection into the cell, and thus can be severely limited by its short half-life. While there are various reasons for the short half-life of mRNA, the mechanism most commonly known in the academic community is that it begins when the poly(A) region, a homopolymer region of the single strand located at the 3′ end, is attacked by degrading enzymes.

[0006] The poly(A) region is a critical site for mRNA half-life and protein expression, and without it, the mRNA is rapidly degraded. Therefore, for mRNA to be fully expressed as a specific protein, the poly(A) region must remain intact and protected.

[0007] mRNA, which must be produced artificially, is even more susceptible to these unstable factors. To produce mRNA, pDNA containing an accurate sequence must be secured. During the initial stage of mRNA production, which involves creating template pDNA, the poly(A) region at the 3′ end is a homopolymer structure consisting of repeating A sequences. Since shortening can occur within bacterial subclones due to various enzymes before the pDNA vector for IVT is produced on a large scale via E. coli colliferation, a large number of subclones must be tested to obtain a single clone containing a 3′ polyadenyl cassette of the correct length. Even if a single clone containing an intact poly(A) is found, there is a high probability that shortening will occur again during large-scale fermentation. Such results affect the purity of the mRNA subsequently produced by IVT and can ultimately lead to problems with drug efficacy.

[0008] Therefore, developers of therapeutic mRNA formulations are continuously conducting research to overcome the instability of poly(A).

[0009] These shortening effects occurring from the pDNA template stage also affect the QC verification stage for IND profiling. Even during the emergency approval of COVID-19 mRNA vaccines, regulatory agencies required the determination of the 3′ poly(A) tail length, and this must be clearly determined for all mRNA preparations in the same way today.

[0010] Therefore, for successful IND profiling as well as development, a clear analysis of the poly(A) tail length after production is required.

[0011] The inventors have previously developed a novel nucleic acid structure containing a poly(A) tail having a secondary or tertiary structure, and demonstrated that such shortening phenomenon in bacterial subclones is improved, and that the protein expression rate is increased when mRNA is injected into cells without the occurrence of immunogenicity caused by the complementary binding sequence inserted to form the secondary or tertiary structure.

[0012] The inventors have continuously developed poly(A) structures to ensure that clearer poly(A) sequences are secured in both DNA and RNA states, and that protein expression rates can be increased. As a result, a structure has been developed that improves mRNA stability and protein expression rates compared to previously developed poly(A) structures.

[0013]

[0014] The information described above in the background section is intended solely to enhance understanding of the background of the present invention and may not include information that constitutes prior art already known to those skilled in the art to which the present invention belongs.

[0015]

[0016] Summary of the Invention

[0017] The objective of the present invention is to provide a novel nucleic acid structure with enhanced intracellular stability and protein expression levels.

[0018] Another objective of the present invention is to provide a vaccine and a vaccine therapy comprising the nucleic acid structure.

[0019] Another objective of the present invention is to provide a pharmaceutical composition for gene therapy and a gene therapy method comprising the nucleic acid structure.

[0020] Another objective of the present invention is to provide the use of the nucleic acid structure for vaccine therapy and gene therapy, and the use of the nucleic acid structure for the manufacture of vaccines or gene therapy agents.

[0021]

[0022] To achieve the above objective, the present invention provides a nucleic acid structure comprising: a coding region (CDS) encoding a polypeptide or protein; and a poly(A) tail having the structure of formulas (I) to (III).

[0023] The present invention also provides a pharmaceutical composition for a vaccine or gene therapy comprising the nucleic acid structure.

[0024] The present invention also provides a vaccine therapy or gene therapy method comprising the step of administering the nucleic acid structure.

[0025] The present invention also provides the use of the nucleic acid structure for vaccine therapy and gene therapy.

[0026] The present invention also provides the use of the nucleic acid structure for the manufacture of vaccines or gene therapy drugs.

[0027]

[0028] Figure 1 is a schematic diagram of an mRNA expression cassette construct.

[0029] Figure 2 is a graph of experimental results comparing the protein expression rates of chemical formulas (I) to (III) for the selection of structures with improved efficacy.

[0030] Figure 3 is a graph of experimental results comparing the protein expression rates of selected structures produced using pDNA obtained through a scale-up DNA prep process as a template.

[0031] Figure 4 is a graph of experimental results comparing protein expression rates by changing the CDS of selected structures.

[0032] Figure 5 is a graph showing the results of measuring the half-life of selected structures at the mRNA level.

[0033] Figure 6 is a graph showing the results of measuring the pDNA stability of selected structures using a bacterial system.

[0034] Figure 7 is a diagram showing the results of measuring the immunogenicity of selected structures using a cell line.

[0035] Figure 8 is a diagram showing the results of measuring the poly(A) tail length of the selected structure using RP-HPLC-MS.

[0036] Figure 9 shows the results of measuring the protein expression rate in an in vivo mouse model by intravenously injecting mRNA-LNP containing selected structures.

[0037] Figure 10 shows the results of measuring the protein expression rate in an in vivo mouse model by intramuscularly injecting mRNA-LNP containing selected structures.

[0038] Figure 11 shows the results of measuring immunogenicity in an in vivo mouse model by intravenously injecting mRNA-LNP containing selected structures.

[0039]

[0040] Detailed Description of the Invention and Preferred Embodiments

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a skilled expert in the art to which this invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

[0042]

[0043] In one embodiment of the present invention, a secondary or tertiary structure in the form of a stem loop structure, bulged stem structure, pseudoknot structure, or complete complementary binding structure is formed using a complementary binding sequence (CS) inside or outside the poly(A) of the mRNA structure, and a platform is developed that increases the stability of intracellular mRNA and protein expression rate by using a structure with a Non-A combination at the 3′ end, and can always maintain high efficiency consistently in any mRNA sequence without interference with UTR or CDS sequences.

[0044]

[0045] Accordingly, in one aspect, the present invention relates to a nucleic acid structure comprising: a coding region (CDS) encoding a polypeptide or protein; and a poly(A) tail having any one of the following chemical formulas (I) to (III).

[0046]

[0047] In the present invention, G / C means G (guanine) or C (cytosine). A structure comprising G / C includes a sequence combination named Mixed tail or Non-A having a functionally similar action to poly(A).

[0048] In one embodiment of the present invention, A may be adenine or a random base that maintains the structure and function of a poly(A) tail.

[0049] In the above formulas (I) to (III), m may be an integer from 5 to 200, n may be an integer from 3 to 200, o may be an integer from 1 to 10, p may be an integer from 1 to 200, q may be an integer from 1 to 20, and r may be an integer from 3 to 200, but is not limited thereto.

[0050] In the present invention, at least one of m or n may be an integer of 20 or less, but is not limited thereto.

[0051] In the present invention, m may be an integer from 10 to 200, preferably from 10 to 150, more preferably from 20 to 100, and most preferably from 30 to 80, but is not limited thereto.

[0052] In the present invention, n may be an integer from 3 to 200, preferably from 5 to 100, more preferably from 8 to 80, and most preferably from 10 to 50, but is not limited thereto.

[0053] In the present invention, the above o may be characterized as being an integer from 1 to 10, but is not limited thereto.

[0054] In the present invention, p may be an integer from 1 to 200, preferably from 1 to 100, and more preferably from 1 to 30, but is not limited thereto.

[0055] In the present invention, the above q may be an integer from 1 to 20, preferably an integer from 1 to 12, but is not limited thereto.

[0056] In the present invention, r may be an integer from 3 to 100, preferably from 5 to 50, more preferably from 8 to 30, and most preferably from 10 to 25, but is not limited thereto.

[0057] In the above formulas (I) to (III), n, r, and p are preferably integers of 20 or less, and more preferably integers of 3 to 20, 4 to 19, and 10 to 18, but are not limited thereto.

[0058] Preferably, the poly A tail has the structure of the formula (I), wherein m is an integer from 10 to 100, n is an integer from 10 to 50, o is an integer from 1 to 5, p is an integer from 1 to 30, and q is an integer from 1 to 12, and more preferably, p is an integer from 3 to 19 and q is an integer from 3 to 9, but is not limited thereto.

[0059] In the above chemical formulas (I) to (III), CS1 and CS2 may be characterized by being complementarily bonded to each other, and may be characterized by forming a stem-loop structure, a bulged stem structure, a pseudoknot structure, or a complete complementary bond structure, and may be characterized by forming a secondary or tertiary structure through such a structure, but are not limited thereto.

[0060] The above CS1 and CS2 may each be characterized by being composed of 5 to 100 nucleotides, preferably 10 to 80, more preferably 20 to 60, and most preferably 30 to 50 nucleotides, but are not limited thereto.

[0061] In the present invention, the CS1 and CS2 may be characterized by being coupled complementarily to each other to form a stem-loop structure, a bulged stem structure, or a pseudoknot structure, or by being coupled 30% to 100% complementarily.

[0062] The above CS1 and CS2 may be characterized by being combined complementarily by 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, 95% to 100%, 96% to 100%, 97% to 100%, 98% to 100%, 99% to 100%, or 100%, but are not limited thereto.

[0063] In the present invention, the CS1 may be characterized as being represented by the nucleotide sequence of SEQ ID NO. 1 or SEQ ID NO. 2, and the CS2 may be characterized as being represented by the nucleotide sequence of SEQ ID NO. 3 or SEQ ID NO. 4, but is not limited thereto.

[0064] Preferably, the above CS1 and CS2 may be sequences that do not cause pathogenic effects derived from RNA viruses, and more preferably, the above CS1 and CS2 may be characterized as being conserved sequences derived from dengue virus (DENV), but are not limited thereto.

[0065] In the present invention, the poly A tail is divided into two regions, one in front of the CS1 sequence and the other in front of the CS2 sequence, and at the end there exists a Non-A sequence mixed with a Non-A structure, and may be characterized as being represented by any one of the nucleotide sequences, but is not limited thereto.

[0066] In the present invention, the poly A tail may be characterized by being represented by any one of the nucleotide sequences of SEQ ID NOs 5 to 83, but is not limited thereto.

[0067] In this specification, the term "nucleic acid" preferably means DNA or RNA. In the present invention, the term "nucleic acid structure" may be used to include DNA structures, RNA structures, mRNA structures, etc., and preferably may be an mRNA structure, but is not limited thereto.

[0068] In relation to the above nucleic acid, "polynucleotide," "nucleotide," "nucleotide sequence," and "oligonucleotide" are used interchangeably. It may include polymer forms of nucleotides of any length, deoxyribonucleotides or ribonucleotides, or analogs thereof. The polynucleotide may have any three-dimensional structure and may perform any known or unknown function. The polynucleotide may include one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. Modification of the nucleotide structure may be possible before or after the assembly of the polymer.

[0069] Preferably, the nucleic acid is a polymer comprising or composed of nucleotide monomers covalently bonded to each other by phosphodiester bonds of sugar / phosphate backbones. The “nucleic acid” may include modified nucleic acids, such as base-modified, sugar-modified, or backbone-modified DNA or RNA molecules.

[0070] In the present invention, the nucleic acid may be characterized by comprising an unmodified or modified nucleic acid. The modified nucleic acid may be characterized by being selected from the group consisting of pseudouridine (Ψ), N1-methyl-pseudouridine (m1Ψ), 5-methyluridine (m5U), 2-thiouridine (s2U), 2'-O-methyluridine (2′-O-methyl-U, Um), 5-methylcytidine (m5C), and 5-methoxyuridine (5moU), but is not limited thereto.

[0071] In this specification, the term 'DNA' is an abbreviation for deoxyribonucleic acid. It is a nucleic acid molecule, that is, a polymer composed of nucleotides. These nucleotides are typically deoxy-adenosine-monophosphate, deoxy-thymidine-monophosphate, deoxy-guanosine-monophosphate, and deoxy-cytidine-monophosphate monomers, composed of a sugar (deoxyribose), a base, and a phosphate, and polymerized by a characteristic backbone structure. The backbone structure is typically formed by phosphodiester bonds between adjacent monomers and the sugar portion of the first nucleotide, namely deoxyribose and the phosphate portion of the second. A specific sequence of monomers, that is, the sequence of bases connected to the sugar / phosphate backbone, is called the DNA sequence. DNA can be single-stranded or double-stranded. In the double-stranded form, nucleotides of the first strand typically hybridize with nucleotides of the second strand, for example, through A / T base pairs and G / C base pairs.

[0072] In this specification, the term 'RNA' is an abbreviation for ribonucleic acid, a nucleic acid molecule, that is, a polymer composed of nucleotides. Examples include mRNA (messenger RNA). The nucleotides are typically adenosine-monophosphate, uridine-monophosphate, guanosine-monophosphate, and cytidine monophosphate monomers linked together through a so-called backbone. The backbone is formed first by a sugar, e.g., ribose, and second by phosphodiester bonds between adjacent monomers, a portion of the phosphate. A specific sequence of monomers is called an RNA sequence. Messenger RNA typically provides a nucleotide sequence that can be translated into the amino acid sequence of a specific peptide or protein. Typically, mRNA includes a 5′-cap, 5′-UTR, ORF (or CDS), 3′-UTR, and poly(A) sequence. Aside from messenger RNA, there exist several non-coding forms of RNA that may be involved in the regulation of transcription and / or translation.

[0073]

[0074] In the present invention, the nucleic acid structure may be characterized by further comprising a 5′-untranslated region (5′-UTR) and a 3′-untranslated region (3′-UTR) connected to both ends of the coding region; and a 5′-cap or IRES connected to the 5′-untranslated region, but is not limited thereto. Preferably, it may be characterized by having a structure of the following formula (IV) or formula (V), but is not limited thereto.

[0075] Chemical formula (IV): 5′-cap - 5′-UTR - CDS - 3′-UTR - [Chemical formula (I) to Chemical formula (III)] -3'

[0076] Chemical formula (V): 5′ - IRES - 5′-UTR - CDS - 3′-UTR - [Chemical formula (I) to Chemical formula (III)] -3'

[0077] In the present invention, the term “5′-untranslated region (5′-UTR)” refers to a nucleic acid molecule located at 5′ (i.e. “upstream”) of the coding region that is not translated into a protein. Generally, the 5′-UTR starts at the transcription start site and terminates one nucleotide before the start codon of the coding region. Preferably, the 5′-UTR has a length of 20, 30, 40, or 50 or more nucleotides. The 5′-UTR may contain elements for regulating gene expression, so-called regulatory elements. Such regulatory elements may be, for example, ribosome binding sites. The 5′-UTR may be modified post-transcriptionally, for example, by the addition of a 5′-cap. The 5′-UTR of the mRNA is not translated into an amino acid sequence. The 5′-UTR sequence is generally encoded by the gene that is transcribed into the respective mRNA during the process of gene expression. The genomic sequence is first transcribed into premature mRNA containing selective introns. Premature mRNA is subsequently further processed into mature mRNA during the maturation process. This maturation process includes 3′-terminal modification steps, such as 5′-capping, splicing of premature mRNA to cut into selective introns, and polyadenylation of the 3′-terminus of premature mRNA. In the present invention, the 5′-UTR corresponds to the sequence of the mature mRNA located between the start codon and, for example, the 5′-cap. Preferably, the 5′-UTR corresponds to a sequence extending from a nucleotide located 3′ to the 5′-cap, more preferably a nucleotide located immediately next to 3′ to the 5′-cap, to a nucleotide located 5′ to the start codon of the protein-coding region, preferably a nucleotide located immediately next to the start codon of the protein-coding region. The nucleotide located immediately next to 3′ to the 5′-cap of the mature mRNA typically corresponds to the transcription start site.The term "correspondence" means that the 5′-UTR sequence may be an RNA sequence such as the mRNA sequence used to define the 5′-UTR sequence, or a DNA sequence corresponding to such RNA sequence.

[0078] In the present invention, the term “3′-untranslated region (3′-UTR)” refers to a portion of an artificial nucleic acid molecule located at 3′ (i.e., “downstream”) of a coding region that is not translated into a protein. Typically, the 3′-UTR is a region of mRNA located between the protein-coding region (ORF or CDS) and the poly(A) sequence of the mRNA. In the present invention, the term 3′-UTR may include elements that are not encoded by the template from which the RNA was transcribed. The 3′-UTR sequence is usually encoded by a gene that is transcribed into mRNA during the process of gene expression. In the present invention, the 3′-UTR corresponds to a sequence of mature mRNA located between stop codons of the protein-coding region, preferably 3′ immediately adjacent to the stop codon of the protein-coding region. The term “corresponds” means that the 3′-UTR sequence may be an RNA sequence, as in the mRNA sequence used to define the 3′-UTR sequence, or a DNA sequence corresponding to such an RNA sequence. Preferably, the 3′-UTR has a length of 20, 30, 40, or 50 nucleotides or more.

[0079] In the present invention, the term “5′-cap” is an independent component that “caps” the 5′-terminus located at the 5′ starting site of mRNA. Generally, the cap structure serves to initiate protein synthesis and protect the mRNA from the action of nucleases. The 5′-cap can typically be formed by a modified nucleotide, in particular by a derivative of a guanine nucleotide. Preferably, the 5′-cap is connected to the 5′-terminus via a 5′-5′-triphosphate linkage. The 5′-cap is in a methylated form, for example, m7GpppN, where N is the terminal 5′ nucleotide of the nucleic acid connected to the 5′-cap, typically the 5′-terminus of RNA.

[0080] Additional examples of 5′-cap structures include glyceryl, inverted deoxy abasic residue (some), 4',5′ methylene nucleotide, 1-(beta-D-erythrofuranosyl) nucleotide, 4'-thio nucleotide, carbocyclic nucleotide, 1,5-anhydrohexitol nucleotide, L-nucleotide, alpha-nucleotide, modified base nucleotide, threo-pentofuranosyl nucleotide, acyclic 3′,4'-seconucleotide, acyclic 3,4-dihydroxybutyl nucleotide, acyclic 3,5-dihydroxypentyl nucleotide, some 3′-3′-inverted nucleotides, some 3′-3′-inverted abasic residues, some 3′-2'-inverted nucleotides, and 3′-2'-inverted abasic residues. It includes some, 1,4-butanediol phosphate, 3′-phosphoramidate, hexyl phosphate, aminohexyl phosphate, 3′-phosphate, 3′phosphorothioate, phosphorodithioate, or bridging or non-bridging methylphosphonate.

[0081] In the present invention, the term "IRES (Internal Ribosome Entry Site)" is known to be an internal ribosome entry site or ribosome binding site that forms a loop structure on mRNA and induces the initiation of translation through a mechanism independent of the Cap. By utilizing this, when an IRES is positioned between two or more genes to produce a single vector, two genes can be expressed simultaneously on a single mRNA. Accordingly, inserting an IRES region to induce the expression of a target gene is widely used in gene recombination technology.

[0082]

[0083] In the present invention, “CDS (coding sequence)” refers to a coding region encoding a target polypeptide or protein, and is used interchangeably with ORF (open reading frame) in this specification.

[0084] The polypeptide or protein may be (a) a therapeutically active protein or peptide; or (b) an antigen selected from the group consisting of tumor antigens, pathogenic antigens, viral antigens, protozoal antigens, bacterial antigens, allergic antigens and autoimmune antigens, but is not limited thereto. Depending on the gene to be inserted into the coding region, the nucleic acid structure according to the present invention may be used for gene vaccine therapy or gene therapy for autoimmune diseases, infectious diseases, cancer or tumor-related diseases, inflammatory diseases, etc.

[0085] Specifically, when the nucleic acid structure according to the present invention is used as a vaccine for vaccine therapy, the CDS may be a region encoding all or part of a viral antigen capable of causing infection in humans and animals, such as influenza virus, coronavirus, varicella zoster virus, human papillomavirus, Zika virus, herpes virus, AIDS virus, SFTS virus, measles virus, Ebola virus, MERS virus, hepatitis virus, avian influenza, rabies virus, foot-and-mouth disease virus, respiratory syncytial virus, etc., but is not limited thereto.

[0086] In addition, when the nucleic acid structure of the present invention is used as a concept of a gene therapy agent by expressing a protein or antigen, the CDS may be a region encoding all or part of the sequence of protein drugs, antibodies, disease-related antigens, etc., but is not limited thereto.

[0087] The above disease-related antigens are 4-1BB, integrin, amyloid beta, angiopoetin (angiopoetin 1 or 2), angiopoetin analog 3, B-cell activating factor (BAFF), BAFF-R, BCMA, B7-H3, complement 5, CCR4, CD3, CD4, CD6, CD11a, CD19, CD20, CD22, CD30, CD33, CD38, CD52, CD62, CD79b, CD80, CGRP, Claudin-18, complement factor D, CTLA4, DLL3, EGF receptor, hemophilia factor, FGF23, folate receptor, GD2, GM-CSF, HER2, HER3, interferon receptor, interferon gamma, It may be selected from the group consisting of IgE, IGF-1 receptor, interleukin 1, interleukin 2, interleukin 2 receptor, interleukin 4, interleukin 4 receptor, interleukin 5, interleukin 5 receptor, interleukin 6, interleukin 6 receptor, interleukin 7, interleukin 12 / 23, interleukin 13, interleukin 17A, interleukin 17 receptor A, interleukin 31 receptor, interleukin 36 receptor, TGF-beta, TGF-beta receptor, LAG3, LFA3, NGF, PVSK9, PD-1, PD-L1, TIGIT, TIM3, GITR, KLRG1, OX40, OX40L, RANK-L, SLAMF7, tissue factor, TNF, VEGF, and VEGF receptor, but is not limited thereto.

[0088] The above antibody may be used without restriction as long as it is an antibody used for treating a disease-related condition, preferably a therapeutic antibody against the disease-related antigen, and more preferably an antibody drug currently on the market or in the clinical trial phase, but is not limited thereto.

[0089] In addition, the protein drug mentioned above is a drug composed of amino acids that exhibits a therapeutic or preventive effect against disease through the activity of the protein, and refers to a drug composed of proteins including antibody drugs, and may be selected from the group consisting of cytokines, therapeutic enzymes, hormones, soluble receptors and their fusion proteins, insulin or its analogues, BMP (Bone Morphogenetic Protein), EPO (erythropoietin), and serum-derived proteins, but is not limited thereto.

[0090] As specific examples, the above cytokine may be selected from the group consisting of interferon, interleukin, CSF (colony stimulating factor), TNF (tumor necrosis factor) and TGF (tissue growth factor), but is not limited thereto, and the above therapeutic enzyme may be exemplified by beta-glucocerebrosidase and agalsidase β, but is not limited thereto.

[0091] In addition, the above-mentioned soluble receptor refers to the extracellular domain of the receptor, and the fusion protein refers to a protein in which the Fc region of an antibody, etc., is fused to the said soluble receptor; examples include a form in which the Fc region is fused to a TNF-α soluble receptor (e.g., a product with the active ingredient name Etanercept, etc.), a form in which the Fc region is fused to a VEGF soluble receptor (a product with the active ingredient name Aflibercept and similar forms), a form in which the Fc region is fused to CTLA-4 (e.g., a product with the active ingredient name Abatacept or Beladacept and similar forms), a form in which the Fc region is fused to an interleukin 1 soluble receptor (e.g., a product with the active ingredient name Rilonacept and similar forms), and a form in which the Fc region is fused to an LFA3 soluble receptor (e.g., a product with the active ingredient name Alefacept and similar forms). However, it is not limited to this.

[0092] The above-mentioned hormone refers to a hormone or an analog thereof injected from outside the body for the treatment or prevention of diseases caused by hormone deficiency, and examples may include, but are not limited to, human growth hormone, estrogen, and progesterone; the above-mentioned plasma-derived protein refers to a protein present in plasma, including both those extracted from plasma and those produced by recombinantation, and examples may include, but are not limited to, fibrinogen, von Willebrand Factor, albumin, thrombin, FII (Factor II), FV (Factor V), FVII (Factor VII), FVIII (Factor VIII), FIX (Factor IX), FX (Factor X), and FXI (Factor XI).

[0093]

[0094] In the present invention, a template DNA (e.g., plasmid DNA (pDNA)) can be prepared to produce a nucleic acid structure containing mRNA of a gene encoding a target polypeptide or protein by an in-vitrotranscription (IVT) method.

[0095] mRNA produced by the IVT method can be purified by purification methods known in the art, and purification methods may include, but are not limited to, Multimodal chromatography, RP-HPLC (Reversed-phase high-performance liquid chromatography), SEC (Size-exclusion HPLC), IEC (Ion exchange HPLC), Cellulose chromatography, Oligo-dT (oligo-deoxythymidine acid), or TFF (Tangential flow filtration).

[0096] In the present invention, the plasmid DNA for expressing the nucleic acid structure by the IVT method may further include a promoter sequence. The promoter may be located upstream of the 5′-UTR. The promoter may include elements necessary for transcription, such as, for example, an RNA polymerase promoter. It may include a phage RNA polymerase promoter such as T7, SP6, or T3, preferably a T7 or SP6 promoter encoding an mRNA sequence.

[0097]

[0098] In another aspect, the present invention relates to a pharmaceutical composition for a vaccine or gene therapy comprising the nucleic acid structure.

[0099] In another aspect, the present invention relates to a vaccine therapy or gene therapy method comprising the step of administering the nucleic acid structure to an individual requiring treatment or prevention.

[0100] In another aspect, the present invention relates to the use of the nucleic acid structure for vaccine therapy and gene therapy.

[0101] In another aspect, the present invention relates to the use of the nucleic acid structure for the manufacture of vaccines or gene therapy drugs.

[0102]

[0103] In the present invention, the term “vaccine” is understood as a preventive or therapeutic substance that provides at least one antigen, preferably an immunogen. The antigen or immunogen may be derived from any substance suitable for vaccination. For example, the antigen or immunogen may be derived from pathogens, such as bacterial or viral particles, or from tumor or cancer tissue. The antigen or immunogen stimulates the body’s adaptive immune system to provide an adaptive immune response.

[0104] The term “prevention” in the present invention refers to any act of preventing the onset of a disease or delaying its progression through the administration of the above-mentioned composition. Furthermore, the term “treatment” used in the present invention refers to any act of improving, alleviating, or completely curing the symptoms of a disease through the administration of the composition.

[0105] The term “gene therapy agent” of the present invention may be an anticancer agent, an immunotherapy agent, a CAR-T therapy agent, or a therapy agent using CRISPR and its derivatives, but is not limited thereto, and the description of cases where the nucleic acid structure of the present invention is used as a concept of a gene therapy agent may be applied.

[0106]

[0107] In the present invention, the pharmaceutical composition may include a delivery means for delivering mRNA.

[0108] The nucleic acid structure according to the present invention can be delivered via liposomes, lipid nanoparticles (LNPs), or various nanoparticles.

[0109] Liposomes or LNPs include cationic lipids, non-cationic lipids, or neutral lipids, and may additionally include other lipids such as PEG (polyethylene glycol) or cholesterol. Such mRNA carriers are specifically described in US 2018 / 0311176 A, US 2019 / 0032051 A, US 2021 / 0046192 A, WO 2018 / 081480 A, WO 2020 / 097540 A, WO 2020 / 097548 A, WO 2021 / 007278 A, etc., and are incorporated herein by reference.

[0110] 상기 양이온성 지질은 US 2018 / 0311176 A, US 2019 / 0032051 A 등을 통해 구체적으로 예시되어 있으며, 예를 들어 N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-1,2-propanedio (DOAP), 1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) 또는 이들의 유사체, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9, 12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), 1, 1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (Tech G1), 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane, β-L-arginyl-2, 3-L-diaminopropionic acid-N-palmityl-N-oleylamide trihydrochloride, N′,N′-dioctadecyl-N-4, 8-diaza-10-aminodecanoylglycine amide

[0071] , 1,2-dilinoleyloxy-3-dimethylaminopropane, DLin-KC2-DMA, amino lipid 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA, 1), 1,2-distearloxy- / V,N-dimethylaminopropane (DSDMA), dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA), DLin-D-DMA, C12-200, 98N12-5, (20Z,23Z)-N,N-dimethylnonacosa-20,23-dien-10-amine, (17Z,20Z)-N,N-dimemylhexacosa-17,20-dien-9-amine, (1Z,19Z)-N5N-dimethylpentacosa-16,19-dien-8-amine, (13Z,16Z)-N,N-dimethyldocosa-13,16-dien-5-amine, (12Z,15Z)-N,N-dimethylhenicosa-12,15-dien-4-amine, (14Z,17Z)-N,N-dimethyltricosa-14,17-dien-6-amine, (15Z,18Z)-N,N-dimethyltetracosa-15,18-dien-7-amine, (18Z,21Z)-N,N-dimethylheptacosa-18,21-dien-10-amine, (15Z,18Z)-N,N-dimethyltetracosa-15,18-dien-5-amine, (14Z,17Z)-N,N-dimethyltricosa-14,17-dien-4-amine, (19Z,22Z)-N,N-dimethyloctacosa-19,22-dien-9-amine, (18Z,21Z)-N,N-dimethylheptacosa-18,21-dien-8-amine, (17Z,20Z)-N,N-dimethylhexacosa-17,20-dien-7-amine, (16Z,19Z)-N,N-dimethylpentacosa-16,19-dien-6-amine, (22Z,25Z)-N,N-dimethylhentriaconta-22,25-dien-10-amine, (21Z,24Z)-N,N-dimethyltriaconta-21,24-dien-9-amine, (18Z)-N,N-dimethylheptacos-18-en-10-amine, (17Z)-N,N-dimethylhexacos-17-en-9-amine, (19Z,22Z)-N,N-dimethyloctacosa-19,22-dien-7-amine, N,N-dimethylheptacosan-10-amine, (20Z,23Z)-N-ethyl-N-methylnonacosa-20,23-dien-10-amine, 1-[(11Z,14Z)-1-nonylicosa-11,14-dien-1-yl]pyrrolidine, (20Z)-N,N-dimethylheptacos-20-en-10-amine, (15Z)-N,N-dimethyleptacos-15-en-10-amine, (14Z)-N,N-dimethylnonacos-14-en-10-amine, (17Z)-N,N-dimethylnonacos-17-en-10-amine, (24Z)-N,N-dimethyltritriacont-24-en-10-amine, (20Z)-N,N-dimethylnonacos-20-en-10-amine, (22Z)-N,N-dimethylhentriacont-22-en-10-amine, (16Z)-N,N-dimethylpentacos-16-en-8-amine, (12Z,15Z)-N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine, (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]eptadecan-8-amine, 1-[(1S,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadecan-10-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]nonadecan-10-amine, N,N-dimethyl-21-[(1S,2R)-2-octylcyclopropyl]henicosan-10-amine,N,N-dimeth-yl-1-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]nonadecan-10-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexadecan-8-amine, N,N-dimethyl-[(1R,2S)-2-undecylcyclopropyl]tetradecan-5-amine, N,N-dimethyl-3-{7-[(1 S,2R)-2-octylcyclopropyl]heptyl}dodecan-1-amine, 1-[(1R,2S)-2-heptylcyclopropyl]-N,N-dimethyloctadecan-9-amine, 1-[(1S,2R)-2-decylcyclopropyl]-N,N-dimethylpentadecan-6-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]pentadecan-8-amine, R-N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propa-n-2-amine, S-N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octy-loxy)propan-2-amine, 1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-Roctyloxy)methyl]ethyl}pyrro-lidine, (2S)-N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-[(5Z)-oct-5-en-1-yloxy]propan-2-amine, 1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}azet-idine, (2S)-1-(hexyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-ylo-xy]propan-2-amine, (2S)-1-(heptyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]pr-opan-2-amine, N,N-dimethyl-1-(nonyloxy)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, N,N-dimethyl-1-[(9Z)-octadec-9-en-1-yloxy]-3-(octyloxy)propan-2-am-ine; (2S)-N,N-dimethyl-1-[(6Z,9Z,12Z)-octadeca-6,9,12-trien-1-yloxy]-3-(o-ctyloxy)propan-2-amine, (2 S)-1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(pentyloxy)propa-n-2-amine, (2S)-1-(hexyloxy)-3-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-di-methylpropan-2-amine, 1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, 1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)pr-opan-2-amine, (2S)-1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-3-(hexyloxy)-N,N-dimethylpro-pan-2-amine, (2S)-1-[(13Z)-docos-13-en-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amin-e, 1-[(13Z)-docos-13-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, 1-[(9Z)-hexadec-9-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, (2R)-N,N-dimethyl-H(1-metoyloctyl)oxy]-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, (2R)-1-[(3,7-dimethyloctyl)oxy]-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-di-en-1-yloxy]propan-2-amine, N,N-dimethyl-1-(octyloxy)-3-({8-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]-methyl}cyclopropyl]octyl}oxy)propan-2-amine, N,N-dimethyl-1-{[8-(2-oclylcyclopropyl)octyl]oxy}-3-(octyloxy)propan-2-am-ine and (11E,20Z,23Z)-N,N-dimethylnonacosa-11,20,2-trien-10-amine, 5-carboxyspermylglycine dioctaoleoylamide ("DOGS"), dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide ("DPPES"), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide (DMRIE), DMRIE-HP, Lipofectamine (DOSPA), 3b-(N-(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol ("DC-Choi"), N-(1,2-dimyhstyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide ("DMRIE"), 1,2-Dioleoyl-3-dimethylammonium-propane ("DODAP"), DMDMA, cationic lipid-based transfection reagents TransIT-TKO,LIPOFECTIN, Lipofectamine, OLIGOFECTAMINE or DHARMAFECT, DSDMA, DODMA, DLinDMA, DLenDMA, gamma-DLenDMA, DLin-K-DMA, DLin-K-C2-DMA (also known as DLin-C2K-DMA, XTC2, and C2K), DLin-K-C3-DM A, DLin-K-C4-DMA, DLen-C2K-DMA, y-DLen-C2K-DMA, DLin-M-C2-DMA (also known as MC2), DLin-M-C3-DMA (also known as MC3) or (DLin-MP-DMA)(also known as 1-B11), 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester (also known as SM-102), It may be 2-hexyl-decanoic acid, 1,1'-[[(4-hydroxybutyl)imino]di-6,1-hexanediyl] ester (also known as ALC-0315) or a mixture thereof, but is not limited thereto.

[0111] The above non-cationic lipids are specifically exemplified in US 2018 / 0311176 A, US 2019 / 0032051 A, etc., and may be, for example, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoyl phosphatidylethanoloamine, N-succinyl phosphatidylethanolamine, N-glutaryl phosphatidylethanolamine, or lysylphosphatidylglycerol.경우에 따라서, 상기 비양이온성 지질은 예를 들어, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, phosphatidylglycerols, cardiolipins, diacylphosphatidylserines, diacylphosphatidic acids, N-dodecanoyl phosphatidylethanolamines, N-succinyl phosphatidylethanolamines, N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoyloleyolphosphatidylglycerol (POPG)일 수 있으나, 이에 제한되는 것은 아니다.

[0112] The above neutral lipids are specifically exemplified in US 2018 / 0311176 A, US 2019 / 0032051 A, etc., and may include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, or cerebrosides, but are not limited thereto.

[0113] PEG lipids may be included to prevent the aggregation of particles generated during the delivery of the mRNA. PEG lipids are specifically exemplified in US 2018 / 0311176 A, US 2019 / 0032051 A, etc., and include, for example, PEG-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), specifically α-[3-[[[2,3-bis[(1-oxotetradecyl)oxy]propoxy]carbonyl]amino]propyl]-ω-methoxy-poly(oxy-1,2-ethanediyl) (also known as PEG2000-c-DMG), α-[(2R)-2,3-bis[(1-oxotetradecyl)oxy]propyl]-ω-methoxy-poly(oxy-1,2-ethanediyl) (also known as DMG-PEG2000), It may be α-[2-(ditetradecylamino)-2-oxoethyl]-ω-methoxy-poly(oxy-1,2-ethanediyl) (also known as ALC-0159), or a mixture thereof, but is not limited thereto.

[0114] In another aspect, the nucleic acid structure according to the present invention may be delivered via nanoparticles, for example, via gold nanoparticles. The surface of the gold nanoparticles may be modified. Specific examples of modification are specifically exemplified in Acc Chem Res. 2019 June 18; 52(6): 1496-1506 and Pharmaceutics 2021, 13, 900, etc., which may be incorporated herein by reference.

[0115] Gold nanoparticles can be attached to the nucleic acid structure and formed into a complex with a cationic endosomal disruptive polymer to be delivered to cells (Nature Biomedical Engineering volume 1, pages 889-901 (2017)). The cationic endosomal disruptive polymer may be, for example, polyethylene imine, poly(arginine), poly(lysine), poly(histidine), poly-[2-{(2-aminoethyl)amino}-ethyl-aspartamide] (pAsp(DET)), a block co-polymer of poly(ethylene glycol) (PEG) and poly(arginine), a block co-polymer of PEG and poly(lysine), or a block co-polymer of PEG and poly{N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} (PEG-pAsp(DET)).

[0116] In some cases, gold particles with a surface modified with arginine can be used. The arginine-modified gold particles can be assembled with a nuclease or a polynucleotide encoding it and / or a cleavage factor or a polynucleotide encoding it. This allows them to fuse with the membrane of a target cell and move into the cytoplasm (ACS Nano. 2017, 11:2452-2458).

[0117] In another aspect, a peptide may be used for the delivery of a nucleic acid structure according to the present invention. The peptide must have a cation to electrostatically interact with the anionic phosphate group of the nucleic acid and may include a positively charged amino acid to electrostatically interact with the phosphate group. Specific details regarding peptides usable for mRNA delivery are described in AIMS Biophysics, 7(4): 323-338 and may be incorporated by reference into the present invention.

[0118] The peptides usable for the delivery of the above nucleic acid structure may include protamine. Protamine is a small nuclear protein rich in cationic arginine that contributes to the stability of DNA during sperm formation in the testis, and protamine can efficiently deliver mRNA molecules by stabilizing them. A protamine-mRNA complex is specifically described in US 9352028 B and may be incorporated herein by reference.

[0119] Cell-permeable peptides (CPPs) may also be promising cationic molecules for the delivery of nucleic acid structures. Non-limiting examples of the above cationic molecules may include, but are not limited to, amphiphilic CPPs such as the arginine-rich RALA peptide (WEARLARALARALARHLARALARALRACEA: SEQ ID NO. 92), LAH4 (KKALLALALHHLAHLALHLALALKKA: SEQ ID NO. 93), and LAH4-L1 (KKALLAHALHLLALLALHLAHALKKA: SEQ ID NO. 94).

[0120] If necessary, in addition to the liposomes or LNPs mentioned above, peptides may be additionally included for mRNA delivery. The peptides may provide nucleic acid packaging functions and prevent DNA or RNA from being degraded inside or outside the cell. Examples of such peptides are specifically described in US 2021 / 0170046 A and may be incorporated herein by reference, but are not limited thereto.

[0121]

[0122] The pharmaceutical composition according to the present invention may comprise the nucleic acid structure alone in a pharmaceutically effective amount, or may comprise one or more pharmaceutically acceptable carriers, excipients, or diluents. In the above, a pharmaceutically effective amount refers to an amount sufficient to prevent, improve, and treat a target disease.

[0123] The term “pharmaceuticalally acceptable” means physiologically acceptable and, when administered to humans, does not typically cause allergic reactions such as gastrointestinal disorders or dizziness, or similar reactions. Examples of the carrier, excipient, and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. Additionally, the pharmaceutical composition may further include fillers, anticoagulants, lubricants, wetting agents, flavorings, emulsifiers, and preservatives.

[0124] The term “carrier” is defined as a compound that facilitates the addition of nucleic acid structures into cells or tissues. For example, dimethyl sulfoxide (DMSO) is a commonly used carrier that facilitates the introduction of many organic compounds into the cells or tissues of an organism.

[0125] The term “diluent” is defined as a compound that is diluted in water, which not only stabilizes the biologically active form of a target compound but also dissolves the compound. Salts dissolved in buffer solutions are used as diluents in this field. Commonly used buffer solutions are phosphate-buffered saline solutions, as they mimic the salt state of human fluids. Since buffer salts can control the pH of a solution at low concentrations, it is rare for buffer diluents to alter the biological activity of a compound.

[0126] A pharmaceutical composition containing a nucleic acid structure according to the present invention may be administered to a patient as itself, or as a pharmaceutical composition mixed with other active ingredients, such as in combination therapy, or with a suitable carrier or excipient.

[0127]

[0128] The pharmaceutical composition of the present invention may be administered via various routes including oral, transdermal, subcutaneous, intravenous, or intramuscular, and the dosage of the active ingredient may be appropriately selected according to various factors such as the route of administration, the patient's age, gender, weight, and severity of the patient.

[0129] Pharmaceutical compositions suitable for use in the present invention include compositions containing a nucleic acid structure encoding an active ingredient in an amount effective for achieving the intended purpose. More specifically, a therapeutically effective dose refers to an amount of compound effective for prolonging the survival of the subject to be treated, or for preventing, alleviating, or mitigating the symptoms of a disease. The determination of the therapeutically effective dose is within the scope of the ability of a person skilled in the art, particularly in terms of the detailed disclosure provided herein.

[0130] In the present invention, “individual” means a mammal suffering from or at risk of having a condition or disease that can be alleviated, suppressed, or treated by administering a nucleic acid structure according to the present invention, and preferably means a human.

[0131] The dosage of the nucleic acid structure of the present invention to the human body may vary depending on the patient's age, body weight, gender, form of administration, health condition, and degree of disease.

[0132] The toxicity and therapeutic efficacy of compositions comprising the nucleic acid structures described herein may be determined by standard pharmaceutical procedures in cell cultures or experimental animals to determine, for example, the LD50 (lethal dose for 50% of the population), ED50 (dose with therapeutic effect for 50% of the population), and IC50 (dose with therapeutic inhibitory effect for 50% of the population). The dose ratio between toxicity and therapeutic effect is the therapeutic index, which can be expressed as the ratio between the LD50 and the ED50 (or IC50). Compounds exhibiting a high therapeutic index are preferred. Data obtained from these cell culture analyses may be used to determine the range of doses for use in humans. The dosage or application amount of such compounds is preferably within a range of circulating concentrations containing an ED50 (or IC50) in a state of no or minimal toxicity.

[0133] The term “administration” in this invention refers to the act of introducing the pharmaceutical composition of this invention into an individual by any appropriate method, and the route of administration may be administered through various oral or parenteral routes as long as it can reach the target tissue.

[0134] The pharmaceutical composition of the present invention may be administered via any general route as long as it can reach the target tissue. The pharmaceutical composition of the present invention may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, intranasally, intrapulmonaryly, or rectally, although it is not particularly limited thereto, as intended. Additionally, the composition may be administered by any device capable of delivering the active substance to target cells.

[0135] The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. It may also be administered as a single or multiple doses. It is important to consider all of the above factors and administer an amount that obtains maximum effect with a minimum amount without side effects.

[0136]

[0137] The pharmaceutical composition according to the present invention may be formulated into an injectable formulation such as an aqueous solution, suspension, or emulsion, but is not limited thereto; preferably, it is provided in a lyophilized formulation. Methods commonly known in the art to which the present invention belongs may be used to prepare the lyophilized formulation, and a stabilizer for lyophilization may be added. Furthermore, it may be preferably formulated according to each disease or component using appropriate methods in the art or methods disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton, PA).

[0138]

[0139] The present invention will be described in more detail below through examples. These examples are intended solely to illustrate the present invention, and it will be obvious to those skilled in the art that the scope of the present invention is not to be interpreted as being limited by these examples.

[0140]

[0141] Example 1: mRNA expression cassette construct

[0142] We confirmed that a structure using a CS (Complementary sequence) previously developed by the inventors to protect poly(A) from exonuclease (deadenylase) increased not only pDNA stability but also intracellular mRNA stability, thereby enhancing protein expression rates. Based on this technology, we aimed to develop an additional poly(A) structure that can further delay deadenylase attack at the CS terminus and increase protein expression rates, and can pass the Quality Control process—the final stage of development as a therapeutic agent—without issues.

[0143] When an appropriate combination of Non-A (C, G, T) sequences is present in the middle of a continuous Adenine sequence, they not only possess a poly(A) function similar to inducing protein expression but also cause structural modifications compared to sequences where only Adenine is repeated, which can delay the time of deadenylase attack (Nat Struct Mol Biol. 2019 Jun;26(6):433-442. doi: 10.1038 / s41594-019-0227-9. Epub 2019 May 20).

[0144] Accordingly, the inventors obtained a poly(A) structure that exhibits significantly increased efficacy compared to the previously developed structure by inserting an additional Non-A structure into the previously developed CS end (Fig. 1).

[0145]

[0146] Example 2: Design of construct sequences for screening poly(A) structures with improved efficacy

[0147] Structures with a Non-A combination at the end were designed and selected by applying a previously developed CS sequence to the Poly(A) region (Tables 1 and 2).

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160]

[0161]

[0162]

[0163]

[0164] Example 3: Comparison of protein expression rates for the selection of structures with improved efficacy

[0165] To verify the effect of various non-A poly(A) tail constructs on increasing protein expression, pDNA of the constructs (Table 2) was synthesized, mRNA was obtained via IVT, and transfection was performed on HeLa cells using MessengerMAX™. After transfection, the fluorescence value of d2EGFP was measured every 2 hours for up to 48 hours using IncuCyte®, and the total fluorescence value of d2EGFP over 48 hours was normalized to the fluorescence value of co-transfected d2mCherry. Additionally, to confirm whether efficacy was improved compared to the existing poly(A) construct, the values ​​were normalized to the control group (PLA310) (Normalized EGFP AUC (au)) (Fig. 2).

[0166] Compared to the previously developed poly(A) structure PLA310, it was confirmed that the poly(A) tail containing the non-A at the end of chemical formulas (I) to (III) exhibited equivalent or higher protein expression efficiency over a total of 48 hours (Figs. 2A to 2C). In particular, according to the time-based result graph, the protein expression rate per hour was found to improve significantly starting from 6 hours (Fig. 2D). It is believed that the in vivo stability of the corresponding mRNA was increased by the non-A sequence, and in particular by the poly(A) tail structure containing poly(C) (chemical formula (I)), resulting in increased protein production (Cheuk Yin Li, et al., Mol Ther Nucleic Acids., 2022).

[0167]

[0168] Example 4: Confirmation of protein expression rate through scale-up of pDNA of selected constructs

[0169] When culturing E. coli, sequences consisting of repeated identical sequences exceeding a certain length, such as poly(A) tails, are frequently not fully maintained and partially lost due to recombination or deletion phenomena. Furthermore, this phenomenon varies depending on the culture environment and conditions.

[0170] Therefore, unlike the experimental conditions of Example 3, where pDNA preparation was performed at the mini-prep level to confirm that the protein expression enhancement efficacy of the structures of the present invention, developed for poly(A) repeat sequences, appears independent of the protein culture environment, the pDNA of the selected structures (PC162, PC171, PC180, PC187, PC223, PC236) was secured at the maxi-prep level. Subsequently, under the same conditions as the previous experiment, mRNA synthesized via IVT was transfected into HeLa, and the fluorescence values ​​of d2EGFP observed for 48 hours were normalized to d2mCherry values ​​and then normalized once more to the control group, PLA310. Similar to the results in Example 3, all of the selected structures of the present invention showed higher protein expression rates compared to PLA310, even when mRNA was produced using the pDNA secured at the maxi-prep level as a template (Fig. 3).

[0171] Meanwhile, regarding mRNA stability, previous inventions (US2021 / 0128716A and EP2831240B) proposed a method of inserting a linker to maintain the length of the poly(A) to 100, but in the present invention, the poly(A) tail of A30L70 was used as another control to verify whether equivalent or greater protein expression efficiency is achieved even if the length of the poly(A) does not reach 100. The selected constructs showed equivalent protein expression rates compared to A30L70. In the graph of results over 48 hours, improved protein expression efficiency was observed from 6 hours onwards compared to PLA310, and higher protein expression efficiency was observed from 24 hours onwards compared to A30L70 (Fig. 3). Through this, it was confirmed that even if the total number of As in the poly(A) tails of the selected constructs is small, protein expression levels equivalent to or greater than those of previously reported long poly(A) tails are maintained.

[0172]

[0173] Example 5: Modification of the coding site of a selected structure and confirmation of the resulting protein expression rate

[0174] To demonstrate that the application of the Poly(A) structure of the present invention increases protein expression rates regardless of the sequence of the coding region, the CDS (Coding sequence) region of the structures selected in Example 3 (PC162, PC171, PC180, PC187, PC223, PC236) was replaced to encode Firefly luciferase (FLuc).

[0175] mRNA obtained through in vitro transcription (IVT) was formulated into mRNA-LNP (mRNA-lipid nanoparticle) preparations for cellular introduction. The lipids used for the mRNA-LNP formulation were ALC-0315, DSPC, cholesterol, and ALC-0159, with a composition ratio of 46.3:9.4:42.7:1.6. The mRNA and lipids to be used for formulation were dissolved in citrate buffer (pH 4) and ethanol, respectively, and then mixed using NanoAssemblr™ Ignite™ to prepare the mRNA-LNP. The formulation conditions were N / P=6, total flow rate (TFR) = 12 ml / min, and flow rate ratio (FFR) = 3. The formulated mRNA-LNP was filtered to remove impurities and used in the experiment.

[0176] The mRNA-LNP complex obtained through the above process was introduced into THP-1 cell-derived macrophages differentiated into PMA (forbol-12-myristate-13-acetate) at a concentration of 500 ng RNA / ml. Cells were obtained at 6, 24, 36, and 48 hours after introduction, and Firefly luciferase expression levels were evaluated using the Dual-Luciferase® Reporter Assay System (Promega). To compare protein expression rates with a construct (A120) having a Poly(A) tail consisting of 120 consecutive A's, representing the morphology of a normal poly(A) tail found in nature, the results were normalized by the FLuc expression levels of the A120 construct at each time point (Relative luciferase activity (A120)). The selected constructs showed equivalent or superior expression compared to the A120 tail at all measured time points (Fig. 4).

[0177] This demonstrates that the application of the structure of the present invention can increase the efficiency of protein expression from mRNA by acting independently of the encoding sequence.

[0178]

[0179] Example 6: Verification of mRNA half-life in selected constructs

[0180] When mRNA synthesized in vitro is delivered into the body, the poly(A) tail at the 3′ end of the mRNA undergoes deadenylation by the deadenylase complex present in the cell; as a result, the exposed 3′ end of the mRNA is degraded by nucleases, leading to relatively low persistence in the body. Recent studies indicate that the activity of this deadenylase complex varies depending on the type of nucleic acid, and notably, it exhibits relatively lower activity toward cysteine ​​(C) residues compared to its activity toward adenine (A) (Cheuk Yin Li, et al., Mol Ther Nucleic Acids., 2022). That is, when cysteine ​​(C) is included in the poly(A) tail region of mRNA consisting of a continuous adenine (A) sequence, the rate of deadenylation by the deadenylation complex is slowed down, and as a result, the rate of degradation of the 3′ end by nucleases is reduced, thereby increasing the stability of mRNA in the body.

[0181] It is well known that increased intracellular stability of mRNA is directly correlated with protein production, and that mRNA constructs with improved stability and persistence produce more therapeutic proteins, thereby enhancing efficacy as a therapeutic agent. Therefore, to confirm whether the increased efficacy of the poly(A) tail construct containing the terminal non-A sequence of the present invention—specifically poly(C)—increased total protein production over 48 hours (Fig. 2A) and increased protein production per unit time observed after 6 hours following transfection (Fig. 2D)—is correlated with increased in vivo stability of mRNA, the half-life of the said construct was measured.

[0182] PC180, PC223 (corresponding to chemical formula (I) which exhibited excellent protein expression among poly(A) structures with improved efficacy), and the previously invented structure PLA310 were each produced in-vitro and transfected into HeLa cells. Cells were collected and RNA was extracted at 0, 2, 4, 8, 24, and 48 hours, respectively, based on a time point 1 hour after transfection, and mRNA-specific cDNA was synthesized using primers composed of Oligo dT. Finally, the relative RNA values ​​were analyzed by measuring the PCR products of the synthesized cDNA using Real-Time Quantitative PCR (qPCR). To measure the PCR products of the structures, qPCR was performed using primers that detect d2EGFP, the GOI of the reporter; the measured value for EGFP among the obtained PCR products was corrected to the measured value of human β-actin, a housekeeping gene.

[0183] Half-life of PLA310 (T 1 / 2) is 5.9, the half-life (T of PC180 and PC223, which are poly(A) tail constructs containing non-A terminal sequences and especially poly(C) 1 / 2 ) were 9.9 and 10.9, respectively, and when a non-A sequence, especially a poly(C) sequence, is inserted into a poly(A) structure, the degradation rate of the corresponding mRNA decreases, so the half-life (T 1 / 2 ) increased approximately 1.8 times, from about 5.9 to about 10.9. In addition, the half-life of A30L70 (T 1 / 2 The half-life (T) of the selected structures, even when compared to 4.9. 1 / 2 It was observed that ) increased by 2 times and 2.3 times, respectively (Fig. 5).

[0184] This demonstrates that the protein expression-enhancing efficacy of the structure of the present invention is correlated with increased in vivo safety of the mRNA of the said structure, and at the same time suggests that the efficacy of increasing protein expression levels due to increased intracellular stability and persistence can enhance efficacy as an RNA therapeutic.

[0185]

[0186] Example 7: Verification of the stability of the poly(A) region in pDNA in selected constructs

[0187] Synthesizing mRNA for most therapeutic purposes requires pDNA containing mRNA sequence information, and such pDNA contains a continuous AT base pair region encoding the poly(A) tail of the mRNA. To mass-produce pDNA containing mRNA sequence information, it is cultured after transformation into E. coli lacking recombinant enzymes. However, regions where the same sequence is repeated for more than a certain length, such as the poly(A) tail, are very unstable, and even when cultured using E. coli lacking recombinant enzymes, the poly(A) tail portion is often partially lost. In the case of the present invention, it was expected that the fractionation of the poly(A) tail region within the template pDNA due to the introduction of CS and Non-A bases would make the length of the continuous A sequence relatively shorter, thereby improving the stability of the poly(A) tail region during pDNA production using E. coli culture. To verify this, the finally selected constructs were transformed into the E. coli strain (NEB® Stable Competent E. coli) commonly used for pDNA replication containing mRNA sequences, and pDNA was extracted from 30 clones per construct. The integrity of the poly(A) tail region was confirmed through DNA sequencing analysis, and the degree of instability of the poly(A) tail region was expressed as the ratio of the number of clones with shortened poly(A) tail regions to the total number of clones of each construct (Fig. 6). As a result, it was confirmed that the poly(A) region had high instability, as a portion of the adenine sequence in the poly(A) tail region of pDNA with continuous poly(A) tails (A79 or A120) or the A30L70 construct of the BNT162b2 vaccine was lost. On the other hand, the proportion of poly(A) tail regions that remained intact with the insertion of the construct of the present invention increased compared to the control group, meaning that the degree of instability of the poly(A) region was reduced.This indicates that the stability of the poly(A) region in pDNA is improved when the structure of the present invention is introduced (Fig. 6).

[0188]

[0189] Example 8: Verification of the immunogenicity of the finally selected construct

[0190] RNA induces inflammation through multiple pathways, particularly the innate immune system, which includes numerous sensors that detect RNA. As a result of the innate immune response mediated by RNA, intracellular RNA translation may be reduced, or systemic / local toxicity and inflammatory responses may be induced, which can affect the expected efficacy and safety of pharmaceuticals.

[0191] To evaluate the immunogenicity of the structures of the present invention at the cellular level, the cytokine expression pattern following RNA introduction into macrophages was analyzed. mRNA-LNP complexes containing selected Poly(A) tail structures (PC162, PC171, PC180, PC187, PC223, PC236) were introduced into macrophages derived from THP-1 cells differentiated into PMA (forbol-12-myristate-13-acetate). LNP was used as the delivery means, and the manufacturing process was the same as in Example 5.

[0192] After introduction, culture medium was collected at 6 and 24 hours, and the concentrations of Type I interferon (interferon alpha) and inflammatory cytokines (interleukin 1 beta, interleukin-6, tumor necrosis factor alpha) secreted as a result of the innate immune response were measured. When mRNA coated with the structure of the present invention was introduced into macrophages, similar levels of secretion of Type I interferon and inflammatory cytokines were observed compared to mRNA with a Poly(A) tail consisting of 120 consecutive As (Fig. 7).

[0193] This means that the application of the structure of the present invention does not affect the recognition of mRNA by intracellular innate immune sensors and does not induce an inflammatory response. These characteristics mean that the structure of the present invention is applicable to pharmaceutical compositions for vaccines or gene therapy.

[0194]

[0195] Example 9: Verification of the homogeneity of the Poly(A) tail length of the selected structures

[0196] Biological agents such as mRNA possess molecular heterogeneity. For the safe use of biological agents, it is necessary to accurately determine the degree of homogeneity. The homogeneity of the poly(A) tail length of mRNA structures can be accurately verified using reverse phase high-performance liquid chromatography-mass spectrometry (RP-HPLC-MS) of RNase T1 degradation products (Gau et al., Scientific Reports, 2023). Accordingly, in this example, an experiment was performed to accurately confirm the tail length distribution of Poly(A) structures containing CS1 and CS2 through RP-HPLC-MS experiments.

[0197] For the experimental group of this example, four constructs—PC180, PC171, PC223, and PC236—were used, and for the control group, the A30L70 construct of the A79 and BNT162b2 vaccines was used (US10717982B2, Biontech SE). The experimental method involved first synthesizing pDNA and obtaining mRNA through IVT using N1-methyl-pseudouridine. Next, RNase T1 was used to completely degrade the 3′-terminus of the G in the mRNA sequence. Subsequently, the degraded RNA products were purified using a silica column to obtain the final samples. Table 3 shows the RNase T1 degradation products of each construct. The sites of action of RNase T1 were indicated by a separator ( / ), and fragments containing poly(A) among the separated sequences were highlighted with an underline.

[0198]

[0199]

[0200] RP-HPLC-MS was performed under the following experimental conditions. First, divinylbenzene (DVB) was used as the RP-HPLC stationary phase, and 50 mM hexafluoroisopropanol (HFIP) and 0.2% trimethylamine (TEA) were used as the ion pairing reagents for the mobile phase. The mobile phase gradient was set to start with 0.4% acetonitrile (ACN), followed by 5% ACN, and then 20% ACN. MS was performed using negative ion mode electrospray ionization (ESI) at a spray voltage of -2.5 kV and a resolution of 120 K.

[0201] Quantitative mass signal values ​​corresponding to the fragments containing poly(A) in Table 3 were extracted from the MS data obtained as described above. The number of A was observed within an error range of 30 to 50 nt, including the number in the original pDNA template. For fragments containing poly(C), the addition of C or C2 was considered. Subsequently, the arithmetic mean of the poly(A) length and the coefficient of variation (CV) were calculated for each poly(A) fragment. Finally, for structures containing multiple poly(A) fragments, the theoretical distribution of the total poly(A) tail length sum for each structure was calculated based on the distributions of each poly(A) fragment.

[0202] The results of the RP-HPLC-MS experiment are shown in Figure 8. First, 1 / 2 poly(A) fragments were normally observed in constructs A79 and A30L70, respectively. The error between the average poly(A) length of the IVT-treated mRNA and the poly(A) length on the pDNA template was approximately 1-5 nt for all fragments, with a coefficient of variation of approximately 10-12%. For construct A30L70, the error in the total calculated poly(A) length was 5.4 nt, with a coefficient of variation of 3.1%. Next, 3 / 3 / 3 / 4 poly(A) fragments were normally observed in constructs PC180, PC171, PC223, and PC236, respectively. The error between the average poly(A) length of the IVT-treated mRNA and the poly(A) length on the pDNA template was approximately 1 nt for all fragments, with a coefficient of variation of approximately 3-11%. The error of the total calculated poly(A) length was approximately 1-3 nt, and the coefficient of variation was approximately 1-3%.

[0203] Through this experiment, it was confirmed that the average tail length distribution of the Poly(A) structure containing CS1 and CS2 is similar to that of the pDNA template, and that its homogeneity is also appropriate.

[0204]

[0205] Example 10: Evaluation of protein expression efficiency in vivo of the finally selected candidates

[0206] To evaluate protein expression efficiency in vivo, mRNA constructs (Luc-mRNA) encoding luciferase containing the A30L70 construct, the previously developed Poly(A) construct (PLA310), and the Poly(A) constructs finally selected in the present invention (PC162, PC171, PC180, PC223, PC236) were delivered into mice via intravenous and intramuscular injection. As a control, Luc-mRNA having 120 consecutive A's as a Poly(A) tail was used.

[0207] LNP was used as the delivery means, and the manufacturing process is the same as in Example 5.

[0208] When injected into the mouse, 5 μg of Luc-mRNA was administered for intravenous injection and 1 μg for intramuscular injection.

[0209] The intracellular delivery and translation efficiency of Luc-mRNA were evaluated using in vivo fluorescence imaging techniques with the IVIS Lumina imaging system (PerkinElmer).

[0210] When administered intravenously, mRNA-LNP is primarily absorbed by hepatocytes through an ApoE-mediated uptake process, and it can be confirmed that luciferase expression occurs in the liver as the translation of mRNA proceeds (Fig. 9A). When mRNA containing the technical structure of the present invention, as well as the previously developed technical structure, is administered into the body, it is found that luciferase expression is similar to that of mRNA having a Poly(A) tail of 120 consecutive As (Figs. 9B and 9C). This suggests that the application of the technical structure of the present invention does not affect normal gene expression from mRNA.

[0211] When injected intramuscularly, mRNA-LNP is primarily absorbed by cells near the site of administration. As the translation of mRNA progresses, luciferase expression increases at the local site of administration, and it can be confirmed that some mRNA-LNP circulates systemically, and luciferase expression is also observed in the liver (Fig. 10A). As with intravenous administration, when mRNA containing the technical structure of the present invention is administered, it can be seen that luciferase expression in the muscle is similar to or higher than that of the control PLA310 (Figs. 10B and 10C). This also suggests that the application of the technical structure of the present invention does not affect normal gene expression from mRNA, implying that it is suitable for application in pharmaceutical compositions for vaccines or gene therapy.

[0212]

[0213] Example 11: Evaluation of in vivo innate immune response of the final selected candidates

[0214] To evaluate the innate immune response in vivo, mRNA (Luc-mRNA) encoding luciferase containing the A30L70 construct, the previously developed Poly(A) construct (PLA310), and the poly(A) constructs finally selected in the present invention (PC162, PC171, PC180) was delivered into mice via intravenous injection. As a control, Luc-mRNA having 120 consecutive A's as a Poly(A) tail was used.

[0215] LNP was used as the delivery means, and the manufacturing process is the same as in Example 5.

[0216] To evaluate the immunogenicity of Luc-mRNA, the concentrations of cytokines in the serum were measured. Serum was collected via whole blood sampling 6 and 24 hours after the administration of Luc-mRNA into the body, and the concentrations of chemokines, inflammatory cytokines, and anti-inflammatory cytokines (MCP-1, MIP-1 alpha, MIP-1 beta, RANTES, IP-10, CCL11, CXCL1, tumor necrosis factor-alpha, interleukin-1 beta, interleukin-6, interleukin-9, interleukin-27, interferon-gamma, interleukin-10) reported to be induced by mRNA introduction were measured (Siri Tahtinen, et al., Nature Immunol., 2022; Cristina Bergamaschi, et al., Cell Reports, 2021). When mRNA containing the technical structure of the present invention, as well as the previously developed technical structure, was administered into the body, a cytokine concentration similar to that of mRNA having 120 consecutive A's as a Poly(A) tail was measured (Fig. 11).

[0217] This means that the application of the structure of the present invention within mRNA does not induce an excessive inflammatory response in experimental animals, and this characteristic can be considered suitable for application of the structure of the present invention in pharmaceutical compositions for vaccines or gene therapy.

[0218]

[0219] The nucleic acid structure platform according to the present invention exhibits enhanced stability and protein expression rates within cells without interference with CDS, UTR sequences, etc., and thus can be universally useful in fields such as gene therapy and vaccines. Furthermore, it can be used to protect against Poly A cleavage phenomena, which are a concern during mRNA production, from the pDNA state, thereby enabling the production of an intact mRNA preparation.

[0220]

[0221] Foregoing, specific parts of the present invention have been described in detail. It will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

[0222]

[0223] I have attached the electronic file.

Claims

1. A nucleic acid structure comprising: a coding region (CDS) encoding a polypeptide or protein; and a poly(A) tail having a structure of any one of the following chemical formulas (I) to (III): In the above chemical formulas (I) to (III), The above G / C means G (guanine) or C (cytosine), and The structure containing the above G / C comprises a sequence combination named Mixed tail or Non-A having a functionally similar action to poly(A), and The above m is an integer from 5 to 200, the above n is an integer from 3 to 200, the above o is an integer from 1 to 10, the above p is an integer from 1 to 200, the above q is an integer from 1 to 20, and the above r is an integer from 3 to 200, and The above CS1 and CS2 are characterized by being combined complementarily with each other.

2. A nucleic acid structure according to claim 1, wherein the poly A tail has the structure of the chemical formula (I), and m is an integer from 10 to 100, n is an integer from 10 to 50, o is an integer from 1 to 5, p is an integer from 11 to 30, and q is an integer from 1 to 12.

3. A nucleic acid structure according to claim 2, characterized in that p is an integer from 3 to 19 and q is an integer from 3 to 9.

4. A nucleic acid structure according to claim 1, wherein the CS1 and CS2 bind complementarily to each other to form a stem-loop structure, a bulged stem structure, or a pseudoknot structure, or bind 30% to 100% complementarily.

5. A nucleic acid structure according to claim 4, wherein the CS1 is represented by the nucleotide sequence of SEQ ID NO. 1 or SEQ ID NO.

2.

6. A nucleic acid structure according to claim 4, wherein the CS2 is represented by the nucleotide sequence of SEQ ID NO. 3 or SEQ ID NO.

4.

7. A nucleic acid structure according to claim 1, characterized in that the poly A tail is represented by any one of the base sequences from SEQ ID NO. 5 to SEQ ID NO.

83.

8. A nucleic acid structure characterized by including a modified nucleic acid in claim 1.

9. A nucleic acid structure according to claim 8, wherein the modified nucleic acid is selected from the group consisting of pseudouridine (Ψ), N1-methyl-pseudouridine (m1Ψ), 5-methyluridine (m5U), 2-thiouridine (s2U), 2'-O-methyluridine (2′-O-methyl-U, Um), 5-methylcytidine (m5C), and 5-methoxyuridine (5moU).

10. A nucleic acid structure according to claim 1, further comprising a 5′-untranslated region (5′-UTR) and a 3′-untranslated region (3′-UTR) connected to both ends of the coding region; and a 5′-cap or IRES connected to the 5′-untranslated region.

11. In paragraph 1, the polypeptide or protein is (a) a therapeutically active protein or peptide; or (b) an antigen selected from the group consisting of tumor antigens, pathogenic antigens, viral antigens, protozoal antigens, bacterial antigens, allergic antigens, and autoimmune antigens; A nucleic acid structure characterized by being.

12. A vaccine comprising a nucleic acid structure according to any one of paragraphs 1 to 11.

13. A pharmaceutical composition for gene therapy comprising a nucleic acid structure according to any one of claims 1 to 11.

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