mRNA and templates for protein expression

Abstract

The present invention relates to an mRNA for protein expression and a template therefor, and provides an mRNA transcription vector including a gene construct according to one embodiment, a method for producing an mRNA molecule including a step of performing transcription using the mRNA transcription vector, an mRNA molecule produced by the method, and a pharmaceutical composition including the mRNA molecule as an active ingredient.

Description

[Technical field]

[0001] The present invention relates to mRNA and templates therefor for protein expression. [Background technology]

[0002] In recent years, mRNA has been attracting attention as an active pharmaceutical ingredient (API). There are three major advantages to vaccines using mRNA. First, in terms of production, the process is relatively simple and it can be produced in a short period of time, allowing for rapid and flexible responses to emergencies such as pandemics and mutations. Second, in terms of functionality, if intracellular delivery is effective, it can induce both humoral immunity and cellular immunity, and is expected to prevent diseases more effectively than other types of vaccines. Third, in terms of safety, there are major advantages in that it has few side effects and, unlike DNA, RNA does not enter the nucleus and modify the genes of the human body. Therefore, there is a demand for the development of an appropriate expression system for mass production of mRNA. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Stephen F. Altschul, et al (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402 [Non-Patent Document 2] Smith, TF & Waterman, MS (1981) "Identification of common molecular subsequences." J. Mol. Biol. 147:195-197 [Non-Patent Document 3] Needleman, SB & Wunsch, CD (1970) "A general method applicable to the search for similarities in the amino acid sequences of two proteins." J. Mol. Biol. 48:443-453. [Non-Patent Document 4] Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988 [Non-Patent Document 5] Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993 [Non-Patent Document 6] Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987 [Non-Patent Document 7] Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994 [Non-Patent Document 8] Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991 [Non-Patent Document 9] CABIOS, 1989, 4:11-17 [Non-Patent Document 10] Carillo, H., and Lipman, D., SIAM J Applied Math., 48:1073 (1988) [Non-Patent Document 11] Devereux, J., et al., Nucleic Acids Research, 12(1), 387 (1984) [Non-Patent Document 12] SF et al., J. Molec. Biol., 215, 403 (1990) Summary of the Invention [Problem to be solved by the invention]

[0004] The present invention relates to mRNA and templates therefor for protein expression. [Means for solving the problem]

[0005] An object of the present invention is to provide an mRNA transcription vector comprising a promoter region recognized by RNA polymerase and a gene construct operably linked to the promoter region.

[0006] Another object of the present invention is to provide a method for producing an mRNA molecule, which comprises a step of performing transcription using the mRNA transcription vector as a template.

[0007] A further object of the present invention is to provide an mRNA molecule produced as described above.

[0008] A further object of the present invention is to provide a pharmaceutical composition comprising the mRNA molecule produced as described above as an active ingredient.

[0009] Another object of the present invention is to provide a use of the mRNA transcription vector for producing mRNA and for producing a pharmaceutical composition. Effect of the Invention

[0010] The mRNA transcription vector containing a specific UTR combination of the present invention can increase the expression level of a target protein, so that a desired protein can be expressed in large amounts. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 shows the mRNA transcription vector prepared in Example 1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] One aspect of the present invention is an mRNA transcription vector.

[0013] The mRNA transcription vector comprises a promoter region recognized by RNA polymerase and a gene construct operably linked to the promoter region.

[0014] In one specific example, the gene construct is characterized by comprising: (1) a 5'-untranslated region (5'-UTR); (2) an open reading frame (ORF) region operably linked to the 5'UTR region, the open reading frame (ORF) region including a nucleotide sequence encoding a target protein; and (3) a 3'-untranslated region (3'-UTR) operably linked to the open reading frame region.

[0015] In another specific example, the 3'UTR region is characterized in that it consists of SEQ ID NO: 1 or a nucleotide sequence having 90% or more sequence identity thereto, or the 5'UTR region is characterized in that it consists of SEQ ID NO: 47 or a nucleotide sequence having 90% or more sequence identity thereto.

[0016] The 3'UTR region contained in the mRNA expression vector according to any of the above-mentioned specific examples is characterized in that it consists of SEQ ID NO: 1 or a nucleotide sequence having 90% or more sequence identity thereto, and the 5'UTR is selected from SEQ ID NO: 5, 6, 7, 8, 9, 11, 12, 13, 14, 16, 19, 20, 21, 22, 23, 42 or a nucleotide sequence having 90% or more sequence identity thereto.

[0017] The 5'UTR region contained in the mRNA expression vector according to any of the above-mentioned specific examples is characterized in that it consists of SEQ ID NO: 47 or a nucleotide sequence having 90% or more sequence identity thereto, and the 3'UTR region is selected from SEQ ID NO: 50, 51, 53, 54, 55, 60, 64, 65, 66, 67, 75, 91 or a nucleotide sequence having 90% or more sequence identity thereto.

[0018] The gene construct contained in the mRNA expression vector according to any of the above-mentioned embodiments includes: (1) a 5'-untranslated region (5'-UTR); (2) an open reading frame (ORF) region operably linked to the 5'UTR region, the open reading frame (ORF) region including a nucleotide sequence encoding a target protein; and (3) a 3'-untranslated region (3'-UTR) operably linked to the ORF region. and 3'-UTR), wherein the 3'UTR region consists of SEQ ID NO: 1 or a nucleotide sequence having 90% or more sequence identity thereto, or the 5'UTR region consists of SEQ ID NO: 47 or a nucleotide sequence having 90% or more sequence identity thereto, and when the 3'UTR region consists of SEQ ID NO: 1 or a nucleotide sequence having 90% or more sequence identity thereto, the 5'UTR region is selected from SEQ ID NO: 5, 6, 7, 8, 9, 11, 12, 13, 14, 16, 19, 20, 21, 22, 23, 42 or a nucleotide sequence having 90% or more sequence identity thereto, and when the 5'UTR region consists of SEQ ID NO: 47 or a nucleotide sequence having 90% or more sequence identity thereto, the 3'UTR region is selected from SEQ ID NO: 50, 51, 53, 54, 55, 60, 64, 65, 66, 67, 75, 91 or a nucleotide sequence having 90% or more sequence identity thereto.

[0019] The open reading frame region contained in the mRNA expression vector according to any of the above-mentioned embodiments is characterized in that it contains a nucleotide sequence encoding an antigen.

[0020] The open reading frame region contained in the mRNA expression vector according to any of the above-mentioned embodiments is characterized in that it contains a nucleotide sequence encoding an antigen derived from a pathogen.

[0021] In the mRNA expression vector according to any of the above-mentioned specific examples, the pathogen is characterized in that it is selected from the group consisting of a virus, a bacterium, a prion, a fungus, a protozoon, a viroid and a parasite.

[0022] The gene construct contained in the mRNA expression vector according to any of the above-mentioned specific examples is characterized in that it further comprises a nucleotide sequence operably linked to the 5'UTR that is transcribed into a 5'Cap.

[0023] The gene construct contained in the mRNA expression vector according to any of the above-mentioned specific examples is characterized in that it further comprises a nucleotide sequence operably linked to the 3'UTR region that is transcribed into a polyA tail.

[0024] The poly A tail contained in the mRNA expression vector according to any of the above-mentioned specific examples is characterized by containing 20 to 200 adenines.

[0025] The mRNA expression vector according to any of the above-mentioned embodiments is characterized in that it is a plasmid.

[0026] The mRNA expression vector according to any of the above-mentioned embodiments is characterized in that it is linearized.

[0027] Another aspect of the invention is a method for producing an mRNA molecule.

[0028] In one specific example, the method is characterized by comprising a step of performing transcription using the mRNA transcription vector of the above-mentioned embodiment as a template.

[0029] In another embodiment, the method for producing mRNA comprises a step of in vitro transcription using the mRNA transcription vector of the above-mentioned embodiment as a template.

[0030] Yet another embodiment of the present invention is an mRNA molecule produced by the method for producing an mRNA molecule of the above-mentioned embodiment.

[0031] Yet another embodiment of the present invention is a pharmaceutical composition comprising as an active ingredient the mRNA molecule produced according to the above-mentioned embodiment.

[0032] In one embodiment, the pharmaceutical composition is a pharmaceutical composition for preventing or treating a disease.

[0033] In another embodiment, the pharmaceutical composition comprises a pharma- ceutically acceptable excipient.

[0034] In an embodiment according to any of the above embodiments, the pharmaceutical composition is an immunogenic composition.

[0035] The mRNA molecule contained in the pharmaceutical composition according to any of the above-mentioned embodiments is characterized in that it is complexed with at least one lipid component.

[0036] The mRNA molecules of the pharmaceutical composition according to any of the above-mentioned embodiments are characterized in that they are complexed with at least one lipid component to form lipidoids, liposomes, lipid nanoparticles and / or lipoplexes.

[0037] The pharmaceutical composition according to any of the above embodiments may be administered intramuscularly or subcutaneously.

[0038] Yet another aspect of the present invention is a method for producing a pharmaceutical composition containing mRNA as an active ingredient.

[0039] In one specific example, the method is characterized by comprising a step of obtaining mRNA by performing transcription using the mRNA transcription vector of the above-mentioned embodiment as a template.

[0040] Yet another aspect of the present invention is the use of the mRNA transcription vector for the production of mRNA.

[0041] Yet another aspect of the present invention is a composition for producing a pharmaceutical composition comprising the mRNA transcription vector.

[0042] Yet another aspect of the present invention is the use of the mRNA transcription vector for the preparation of a pharmaceutical composition.

[0043] Yet another aspect of the present invention is a genetic construct comprising: (1) a 5'-untranslated region (5'-UTR); (2) an open reading frame (ORF) region operably linked to the 5'UTR region, the open reading frame (ORF) region comprising a nucleotide sequence encoding a target protein; and (3) a 3'-untranslated region (3'-UTR) operably linked to the open reading frame region.

[0044] In one specific example, the gene construct is characterized by comprising a 3'UTR region consisting of SEQ ID NO: 1 or a nucleotide sequence having 90% or more sequence identity thereto, and a 5'UTR region consisting of a nucleotide sequence selected from SEQ ID NO: 5, 6, 7, 8, 9, 11, 12, 13, 14, 16, 19, 20, 21, 22, 23, 42 or a nucleotide sequence having 90% or more sequence identity thereto.

[0045] In another specific example, the gene construct is characterized in that it comprises a 5'UTR region consisting of SEQ ID NO: 47 or a nucleotide sequence having 90% or more sequence identity thereto, and a 3'UTR region consisting of a nucleotide sequence selected from SEQ ID NO: 50, 51, 53, 54, 55, 60, 64, 65, 66, 67, 75, 91 or a nucleotide sequence having 90% or more sequence identity thereto.

[0046] These will be described in detail below. Note that each description and embodiment disclosed in the present invention is also applicable to other descriptions and embodiments. In other words, all combinations of various elements disclosed in the present invention are included in the present invention. In addition, the present invention is not limited to the following specific description.

[0047] Additionally, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the present invention.

[0048] Furthermore, many papers and patent documents are referred to throughout this specification, and citations are provided thereto, the disclosures of which are incorporated herein by reference in their entirety in order to more clearly describe the state of the art to which the present invention pertains and the contents of the present invention.

[0049] In the present invention, the term "nucleotide sequence" refers to a nucleotide polymer in which nucleotide monomers are linked in a long chain by covalent bonds, and is also called a "polynucleotide" or "base sequence," which is a DNA or RNA chain longer than a certain length.

[0050] The polynucleotide of the present invention includes modified nucleotides. In the present invention, the term "modified nucleotide" refers to any non-classical nucleoside, nucleotide, or its corresponding phosphorylated form. Modified nucleotides may have one or more backbone or base modifications. Examples of modified nucleotides include dI, dU, 8-oxo-dG, dX, and THF, as well as other natural or non-natural nucleotides known in the art.

[0051] In one embodiment of the present invention, uridines present in mRNA may be present as thymidines in the respective DNA encoding the mRNA. Also, the polynucleotide sequences of the present invention use "T" in representative DNA sequences, but it is recognized that when the sequence represents RNA, "T" is replaced by "U".

[0052] "Non-natural" in the present invention refers to a polynucleotide, polypeptide, carbohydrate, lipid, or composition that does not exist in nature. Such a polynucleotide, polypeptide, carbohydrate, lipid, or composition differs from a naturally occurring polynucleotide, polypeptide, carbohydrate, lipid, or composition in one or more respects. For example, a polymer (e.g., a polynucleotide, polypeptide, or carbohydrate) differs in the type and sequence (e.g., nucleotide sequence, amino acid sequence, or sugar molecule) of its constituent building blocks. A polymer differs from a naturally occurring polymer in the molecules that link it.

[0053] In the present invention, "operably linked" refers to a configuration in which the regulatory sequences are appropriately positioned so that they control the transcription of the coding sequence, and for purposes of the present invention, refers to nucleotide sequences that are functionally linked so that mRNA transcription occurs.

[0054] In the present invention, the term "mRNA transcription vector" refers to a vector that contains a template strand for mRNA transcription.

[0055] For example, the vector of the present invention may contain a promoter that initiates transcription, an operator sequence for regulating the promoter, a sequence that codes for the target mRNA, and a sequence that regulates the termination of transcription and translation. As an example, the vector of the present invention may contain a restriction enzyme binding site that facilitates cloning.

[0056] For example, the promoter contained in the vector of the present invention is any promoter for any DNA-dependent RNA polymerase. The promoter is selected and used from a promoter sequence having high binding affinity to the RNA polymerase, but is not necessarily limited thereto.

[0057] Said vectors are obtained by cloning a polynucleotide, for example a cDNA, and introducing it into an expression cassette or vector suitable for transcription. The DNA template is obtained by reverse transcription of mRNA or by gene synthesis.

[0058] The vector of the present invention comprises a promoter region recognized by RNA polymerase and a gene construct operably linked to the promoter region, and comprises (1) a 5'-untranslated region (5'-UTR), (2) an open reading frame (ORF) region operably linked to the 5'UTR region and including a nucleotide sequence encoding a target protein, and (3) a 3'-untranslated region (3'-UTR) operably linked to the open reading frame region.

[0059] In the present invention, the term "5'UTR" refers to an untranslated region located upstream of an initiation codon. The 5'UTR is involved in regulating RNA translation and / or stabilizing mRNA.

[0060] The 5'UTR sequence of the present invention may be modified or optimized to improve the stability and / or translation accuracy of mRNA. For example, by avoiding a gene sequence similar to the start of an ORF in the 5'UTR, it is possible to effectively prevent the frame from starting erroneously during mRNA translation. In addition, in order to improve the stability and translation accuracy of mRNA, some specific sequences may be added to the 5'UTR. For example, a Kozak sequence may be inserted to configure the translation process to start more accurately.

[0061] In the present invention, "3'UTR" refers to a non-translated region located downstream of the coding region of an mRNA.

[0062] The 3'UTR sequence of the present invention may be modified or optimized to improve mRNA stability and / or translation accuracy. For example, appropriate sequences in the 3'UTR can be used to improve mRNA stability and extend half-life.

[0063] The mRNA transcription vector of the present invention is characterized in that it contains a specific combination of 5'UTR and 3'UTR.

[0064] In one embodiment, the mRNA transcription vector of the present invention comprises a 3'UTR sequence comprising SEQ ID NO:1 or a nucleotide sequence having 90% or more sequence identity thereto, and a 5'UTR sequence comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:5, 6, 7, 8, 9, 11, 12, 13, 14, 16, 19, 20, 21, 22, 23, 42, or any sequence selected from these sequences having at least 90% or more sequence identity thereto.

[0065] In one embodiment, the mRNA transcription vector of the present invention comprises a 5'UTR sequence comprising SEQ ID NO: 47 or a nucleotide sequence having 90% or more sequence identity thereto, and a 3'UTR sequence comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 50, 51, 53, 54, 55, 60, 64, 65, 66, 67, 75, 91, or a nucleotide sequence having at least 90% or more sequence identity thereto.

[0066] The UTR sequence may comprise, consist essentially of, or consist of a nucleotide sequence represented by a specific SEQ ID NO. In one embodiment of the present invention, the 5'UTR and 3'UTR sequences consist of a nucleotide sequence represented by a specific SEQ ID NO or a nucleotide sequence having 90% or more sequence identity thereto.

[0067] In one embodiment, the mRNA transcription vector of the present invention comprises a 3'UTR sequence consisting of SEQ ID NO: 1 or a nucleotide sequence having 90% or more sequence identity thereto, and a 5'UTR sequence consisting of a nucleotide sequence selected from the group consisting of SEQ ID NO: 5, 6, 7, 8, 9, 11, 12, 13, 14, 16, 19, 20, 21, 22, 23, 42, or any sequence selected from these sequences having at least 90% or more sequence identity thereto.

[0068] In one embodiment, the mRNA transcription vector of the present invention comprises a 5'UTR sequence consisting of SEQ ID NO: 47 or a nucleotide sequence having 90% or more sequence identity thereto, and a 3'UTR sequence consisting of a nucleotide sequence selected from the group consisting of SEQ ID NO: 50, 51, 53, 54, 55, 60, 64, 65, 66, 67, 75, 91, or any sequence selected from these sequences having at least 90% or more sequence identity thereto.

[0069] The present invention also includes nucleotide sequences that have the above-mentioned homology or identity to a nucleotide sequence of a specific sequence number and in which a portion of the sequence has been deleted, modified, substituted or added, so long as the nucleotide sequence exhibits the same activity as a nucleotide sequence consisting of the above-mentioned sequence number.

[0070] "Homology" or "identity" refers to the degree of similarity between two given amino acid or nucleotide sequences, expressed as a percentage. Homology and identity are often used interchangeably.

[0071] Calculation of sequence identity can be performed, for example, by aligning the two sequences for optimal comparison purposes (e.g., gaps are introduced in either or both of the first and second nucleic acid sequences for optimal alignment, and non-identical sequences are ignored for comparison purposes). In certain embodiments, the length of the sequences aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence. Nucleotides are then compared at corresponding nucleotide positions. Such tools for alignment include the BLAST suite of tools (Non-Patent Document 1). Another popular local alignment method is based on the Smith-Waterman algorithm (Non-Patent Document 2). A popular global alignment method based on dynamic programming is the Needleman-Wunsch algorithm (Non-Patent Document 3). More recently, in addition to optimal global alignment methods such as the Needleman-Wunsch algorithm, a fast and optimal global sequence alignment algorithm (FOGSAA) has been developed to generate global alignments of nucleotide and protein sequences.

[0072] Comparison of sequences and determination of the percentage of identity of two sequences can be accomplished using mathematical algorithms. For example, the percentage of identity of two nucleic acid sequences can be determined similarly to those described in the literature [Non-Patent Documents 4, 5, 6, 7 and 8]. As an example, the percentage of identity of two nucleic acid sequences can be determined using the Meyers and Miller algorithm (Non-Patent Document 9) implemented in the ALIGN program (version 2.0) using a PAM120 weighted residue table, a gap length penalty of 12 and a gap penalty of 4. Alternatively, the percentage of identity of two nucleic acid sequences can be determined using the GAP program in the GCG software package using the NWSgapdna.CMP matrix. Commonly used methods for determining the percentage of identity of sequences include, but are not limited to, those disclosed in Non-Patent Document 10. Examples of computer software for determining the homology of two sequences include, but are not limited to, the GCG program package, Non-Patent Document 11, BLASTP, BLASTN and FASTA Altschul, Non-Patent Document 12.

[0073] In the present invention, the term "open reading frame" is sometimes abbreviated as "ORF" and refers to a segment or region of an mRNA molecule that encodes a protein or polypeptide of interest. The ORF is a continuous stretch of DNA that begins with a start codon and ends with a stop codon, and is translated by the ribosome.

[0074] In one example, the protein or polypeptide of interest may be an antigen.

[0075] An "antigen" in the present invention refers to a substance that is recognized by the immune system and triggers an antigen-specific immune response. For example, the antigen is recognized by the adaptive immune system and triggers an immune response by forming antibodies and / or antigen-specific T cells as part of the adaptive immune response. As an example, the antigen may comprise a peptide or protein that is presented to T cells by MHC. For example, the antigen may be a peptide / protein fragment, variant or derivative thereof that comprises at least one epitope (antigenic determinant).

[0076] For the purposes of the present invention, said antigen may be the translation product of the above mentioned mRNA.

[0077] For example, the antigen may be derived from a pathogen.

[0078] For example, the pathogen may be selected from the group consisting of a virus, a bacterium, a prion, a fungus, a protozoon, a viroid, and a parasite, but is not limited thereto.

[0079] As an example, the antigen may be a peptide or protein antigen derived from a pathogen associated with an infectious disease.

[0080] In another example, the antigen of the present invention may be a tumor-associated antigen. Examples of cancer-associated antigens include tissue differentiation antigens, cancer-testis antigens (e.g., NY-ESO-1 or MAGE-3), normal proteins overexpressed by tumor cells (e.g., EGFR, Muc-1, Her2 / neu), oncoviral proteins (e.g., EBV, HPV), tumor-specific mutant antigens (e.g., Mum-1, β-Catenin, or CDK4). Such antigens are personalized cancer antigens or tissue-specific. However, the above examples are not limited to these.

[0081] In another embodiment, the target protein may be a protein involved in the prevention or treatment of a disease. As an example, the protein may be selected from a chimeric antigen receptor (CAR) capable of recognizing a target antigen, an immunostimulatory cytokine polypeptide, and a telomerase reverse transcriptase. As another example, the protein may be related to the prevention or treatment of a metabolic disease, including obesity. Examples of such target proteins include, but are not limited to, FGF21 (Fibroblast growth factor 21) and leptin.

[0082] In one embodiment, the mRNA transcription vector of the present invention may further comprise a nucleotide sequence that is transcribed into the 5' Cap.

[0083] In the present invention, "5' Cap" refers to a modified structure that caps the end of an mRNA. The 5' cap is generally formed by a modification, such as a guanine derivative. For example, the 5' cap may be linked to the 5' end by a 5'→5' triphosphate bond. As an example, the 5' cap may be methylated.

[0084] As an example, a cap analogue may be added during in vitro transcription, and capping may occur simultaneously with transcription. This is called co-transcriptional capping. In the co-transcriptional method, a cap is incorporated into an RNA molecule simultaneously with transcription during the in vitro transcription process. As another example, capping may occur by an enzymatic reaction after in vitro transcription. Such enzymatic reactions involve enzymes such as triphosphatase and guanyltransferase. However, the present invention is not limited to these, and any method known in the art may be used to cause an mRNA to have a 5' Cap.

[0085] In one embodiment, an mRNA transcription vector of the invention may include a polyA tail.

[0086] In the present invention, the term "poly A tail" refers to a continuous or discontinuous sequence of adenylic acid residues typically located at the 3' end of an RNA molecule. The poly A tail may follow the 3'UTR of an mRNA of the present invention. Such a poly A tail may be composed of or may include 20 or more, 25 or more, 40 or more, 60 or more, 80 or more, 100 or more, about 200 or more adenyl (A) nucleotides. Based on the number of nucleotides in the poly A tail, typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% are A nucleotides, but the remaining nucleotides may be nucleotides other than A nucleotides, such as U nucleotides (uridylic acid), G nucleotides (guanylic acid), or C nucleotides (cytidylic acid). For example, the poly A tail may include modifications that retard degradation of the mRNA. As an example, the polyA tail may contain 20 to 200 adenines.

[0087] For example, the vector of the present invention may contain a selection marker to confirm whether or not it has been transformed. Markers that confer a selectable phenotype, such as drug resistance, auxotrophy, or resistance to cytotoxic agents, are used. In an environment treated with a selective agent, only cells expressing the selection marker survive or show a different phenotype, allowing transformed cells to be selected. However, the above examples are not limiting.

[0088] In the present invention, "in vitro transcription" refers to the process of synthesizing RNA in a cell-free system (in a test tube). In the method of the present invention, the vector of the present invention is used as a template to produce mRNA.

[0089] For example, a vector may be linearized prior to in vitro transcription by treating the vector with an appropriate restriction enzyme.

[0090] For example, the vector of the present invention is a plasmid, but is not particularly limited thereto, and any vector known in the art can be used.

[0091] The term "pharmaceutical composition" as used herein means a composition used for the prevention or treatment of a disease.

[0092] In the present invention, "immunogenicity" refers to the property of inducing an immune response when introduced into the body. When the immunogenic composition of the present invention is administered to an individual, it can prevent a disease. Therefore, the "pharmaceutical composition for preventing a disease" in the present invention may be an "immunogenic composition" or a "vaccine composition". The pharmaceutical composition may further include a pharmaceutical acceptable carrier, excipient, binder, carrier, preservative, buffer, isotonicity agent, emulsifier, or wetting agent.

[0093] The RNA contained in the pharmaceutical composition of the invention may be formulated with one or more excipients to improve stability, increase cell transfection, allow sustained or delayed release (e.g., from a depot dosage form), alter biodistribution (e.g., targeting to specific tissues or cell types), increase translation of the in vivo encoded protein, and / or modify the release profile of the in vivo encoded protein (antigen). Thus, the pharmaceutical composition of the invention may include a pharma- ceutically acceptable excipient.

[0094] The pharmaceutical composition may be formulated. Such formulations may be in solid, liquid or a combination thereof.

[0095] For example, the mRNA contained in the pharmaceutical composition of the present invention may be in a naked form, in a form contained in a vector, or in a form complexed with a delivery system.

[0096] For example, the pharmaceutical composition of the present invention may contain a lipidoid, liposome, lipoplex, lipid nanoparticle, polymer of compound, peptide / protein, cell, nanoparticle mimic or nanotube as a delivery agent for delivering the mRNA. The mRNA and the delivery agent may form a complex.

[0097] Examples of polymers that may be used as carriers for mRNA include polyamidoamine (PAA), poly(beta-amino-ester) (PBAE), poly(ethylenimine) (PEI), poly(l-lysine) (PLL), spermine, chitosan, and / or polyurethane. Examples of nanoparticles include ferritin, platelet membrane-coated nanoparticles (PNP), etc. An example of a peptide / protein is protamine.

[0098] For example, lipid nanoparticles for delivering mRNA can include phospholipids, structured lipids, PEG lipids, ionizable lipids, and / or quaternary amine compounds.

[0099] As an example, the pharmaceutical composition of the present invention may comprise an mRNA molecule complexed with at least one lipid component, for example, the mRNA molecule may be complexed with at least one lipid component to form liposomes, lipid nanoparticles and / or lipoplexes.

[0100] For example, when the pharmaceutical composition of the present invention is administered to a subject, the mRNA may be translated in vivo to produce an antigenic polypeptide (antigen).

[0101] As an example, the pharmaceutical compositions of the present invention may be administered by infusion or injection, similar to the administration of vaccines known in the art, including transdermal, oral, parenteral routes (e.g., intradermal, subcutaneous, intravenous, intramuscular, intranodal and / or intraperitoneal injection), or nasal routes of administration (e.g., tracheal inhalation). The compositions may also be administered intranasally, vaginally, rectally, orally, or transdermally. As another example, the vaccine compositions may be administered by a "needle-free" delivery system.

[0102] For example, the pharmaceutical compositions of the present invention may be used to raise antibodies in a mammal.

[0103] The pharmaceutical compositions of the present invention may be administered in a single dose or in a multidose dose. By "multidose" or "multidose" is meant a formulation unit containing more than one (e.g., two or more) vaccine doses to be administered to one or more subjects.

[0104] The immunogenic compositions of the invention may comprise an immunologically effective amount of a polynucleotide. An "immunologically effective amount" is an amount effective to elicit an antibody response to an antigen when administered to an individual. The immunologically effective amount will vary depending on the health and physical condition of the individuals being treated, their age, the capacity of the individuals' immune systems to synthesize antibodies, the level of protection required, the formulation of the composition, the treating physician's evaluation of the medical situation, and other relevant factors.

[0105] The immunogenic composition provided in the present invention may contain an adjuvant. In the present invention, the term "adjuvant" refers to a substance used to enhance the immunogenicity of the immunogenic composition of the present invention. The adjuvant is often used to enhance immune responses.

[0106] The composition for producing the pharmaceutical composition of the present invention may contain components necessary for synthesizing RNA, specifically, components for in vitro transcription (IVT).

[0107] For example, the composition may include an RNA polymerase. For example, the composition may include ATP, GTP, UTP, CTP, or analogues thereof. For example, the composition may include a buffer.

[0108] In the method for producing the pharmaceutical composition of the present invention, the mRNA transcription may be performed in vitro. The in vitro transcription is as described above. EXAMPLES

[0109] The present invention will be described in more detail below with reference to examples and experimental examples. However, these examples and experimental examples are merely illustrative of the present invention, and the present invention is not limited to these examples and experimental examples. EXAMPLES

[0110] Preparation of mRNA transcription vector and mRNA transcripts transcribed by it In this example, we prepared mRNA transcription vectors composed of various combinations of 5' / 3' untranslated region (UTR) sequences and mRNA transcripts transcribed from them. In this example, d2EGFP was used as an example of a target protein to facilitate confirmation of the expression level of the target protein.

[0111] 1-1. Preparation of mRNA transcription vector As shown in Figure 1, an mRNA transcription vector was constructed having a basic structure in which a promoter region sequence, a 5'UTR region sequence, an ORF region sequence, a 3'UTR region sequence, and a poly(A) tail region sequence are operably linked in sequence from the 5' end to the 3' end.

[0112] More specifically, using the pcDNA3.1 plasmid, the T7 promoter sequence was followed by insertion of (TAATACGACTCACTATA), the 5'UTR region sequence, the Kozak sequence (GCCACC), the d2EGFP gene, the 3'UTR region sequence, the poly A sequence, and the Esp3I recognition site to prepare a plasmid.

[0113] Here, various mRNA transcription vectors were prepared by modifying only the UTR sequence. Tables 1 and 2 to 4 show sequence information in which only the 5'UTR was modified while the 3'UTR (α-globin UTR used in the Moderna vaccine) was fixed, Tables 5 and 6 to 12 show sequence information in which only the 3'UTR was modified while the 5'UTR (α-globin UTR used in the Pfizer / BioNTech vaccine) was fixed, and Table 13 shows sequence information in which the vectors were left unchanged in all examples.

[0114] Shown below is sequence information for an example in which only the 5'UTR was changed while the 3'UTR (the α-globin UTR used in the Moderna vaccine) was fixed.

[0115] [Table 1]

[0116] [Table 2]

[0117] [Table 3]

[0118] [Table 4]

[0119] The sequence information of an example in which only the 3'UTR was changed while the 5'UTR (the α-globin UTR used in the Pfizer / BioNTech vaccine) was fixed is shown below.

[0120] [Table 5]

[0121] [Table 6]

[0122] [Table 7]

[0123] [Table 8]

[0124] [Table 9]

[0125] [Table 10]

[0126] [Table 11]

[0127] [Table 12]

[0128] In all the examples below, sequence information is presented which was kept unchanged.

[0129] [Table 13]

[0130] In summary, a total of 91 mRNA transcription vectors were created (specifically, Moderna Ref, A-1 to A-11, B-1 to B-10, C-1 to C-9, D-1 to D-7, E-1 to E-7, Pfizer / BioNTech Ref, F-1 to F-10, G-1 to G-10, H-1 to H-8, I-1 to I-8, J-1 to J-9).

[0131] 1-2. Preparation of mRNA transcripts from mRNA transcription vectors E. coli transformed with the mRNA transcription vector plasmid prepared as described above was cultured, the plasmid was isolated using CompactPrep Plasmid Midi Kit (Qiagen), and then the plasmid was linearized using Esp3I restriction enzyme. The linearized plasmid was recovered using MinElute Reaction Cleanup Kit (Qiagen) as described above, and then used in an in vitro transcription (IVT) reaction for mRNA production. To this end, the linearized plasmid DNA template and other components were mixed under the conditions shown in Table 14, and then reacted at 37°C for 2 hours.

[0132] [Table 14]

[0133] Thereafter, the reaction was terminated, and then 2 units of DNase 1 was added to each sample, followed by reaction at 37° C. for 15 minutes to remove the remaining linearized plasmid DNA template.

[0134] Then, 50 ul of RNA was mixed with 25 ul of LiCl, and reacted at -20°C for 30 minutes, after which the RNA was precipitated by centrifugation. The RNA precipitate was washed with 70% ethanol, after which the remaining ethanol was removed, and then nuclease-free H 2 The mixture was eluted with 0.05% water and incubated at 65° C. for 5 min to remove any residual precipitate, producing mRNA transcripts suitable for transfection. EXAMPLES

[0135] Evaluating protein expression levels by combining UTR sequences In this example, differences in protein expression levels due to various combinations of 5' / 3' untranslated region (UTR) sequences were evaluated.

[0136] Lipofectamine TM A total of 91 mRNA transcripts prepared in Example 1 were independently transfected into Primary Skeletal Muscle cells (ATCC, PCS-950-010) using MessengerMAX (Thermo Fisher). Specifically, Primary Skeletal Muscle cells were placed in a 24-well plate at 2 × 10 4 Seed cells / well, then incubate at 37℃, 5% CO 2 For transfection, 0.75 ul of Lipofectamine was added to 25 ul of OptiMEM (Gibco) per mRNA sample. TMMessengerMax was mixed and incubated at room temperature for 10 minutes to prepare a MessengerMax mixture, and 50ng of RNA transcript was added to 25ul of OptiMEM to prepare an mRNA mixture. 25ul of Messenger Max mixture and 25ul of mRNA mixture were mixed and incubated at room temperature for 5 minutes, after which 50ul of the mixed solution was used to treat Primary Skeletal Muscle Cells. 24 and 48 hours after this, d2EGFP protein expression levels were evaluated using Cytation 7 (BioTek). Specifically, expression levels were measured in the GFP channel (469,525), and then fluorescence was analyzed in an area within a circle with a radius of 3200um from the center of the well. The minimum luminescence value was set to 5000 or more, and the size of the cells to be analyzed was set to 30um to 200um. The total luminescence was then measured by multiplying the analyzed mean fluorescent value by the cell count. Evaluations were performed 4 to 6 times in total for each mRNA transcript, and the results were expressed as average values.

[0137] 2-1. Evaluation of GFP protein expression levels when only the 5'UTR is changed while the 3'UTR (α-globin UTR used in the Moderna vaccine) is fixed Tables 15 and 16 show the GFP protein expression levels when only the 5'UTR was changed while the 3'UTR was fixed.

[0138] [Table 15]

[0139] [Table 16]

[0140] As shown in Tables 15 and 16, among the groups in which only the 5'UTR was changed while the 3'UTR (α-globin UTR used in the Moderna vaccine) was fixed, A-3 to A-7 (SEQ ID NO: 5 to 9), A-9 to A-11 (SEQ ID NO: 11 to 13), B-1 (SEQ ID NO: 14), B-3 (SEQ ID NO: 16), B-6 to B-10 (SEQ ID NO: 19 to 23), and E-3 (SEQ ID NO: 42) showed relatively excellent protein expression efficiency compared to Moderna Ref. and other combinations of UTRs derived from the same tissue / different tissue. In particular, the Fold value in parentheses specifically confirms how much better the protein expression efficiency is compared to Moderna Ref.

[0141] 2-2. Evaluation of GFP protein expression levels when only the 3'UTR was changed while the 5'UTR (α-globin UTR used in the Pfizer / BioNTech vaccine) was fixed Tables 17 and 18 show the GFP protein expression levels when only the 3'UTR was changed while the 5'UTR was fixed.

[0142] [Table 17]

[0143] [Table 18]

[0144] As shown in Tables 17 and 18, among the groups in which only the 3'UTR was changed while the 5'UTR (α-globin UTR used in the Pfizer / BioNTech vaccine) was fixed, F-2 to F-3 (SEQ ID NO: 50 to 51), F-5 to F-7 (SEQ ID NO: 53 to 55), G-2 (SEQ ID NO: 60), G-6 to G-9 (SEQ ID NO: 64 to 67), H-7 (SEQ ID NO: 75), and J-7 (SEQ ID NO: 91) show relatively excellent protein expression efficiency compared to Pfizer / BioNTech Ref. and other combinations of UTRs derived from the same tissue / different tissue. In particular, the Fold value in parentheses specifically confirms how excellent the protein expression efficiency is compared to Pfizer / BioNTech Ref.

[0145] 2-3.Summary From the results of 2-1 above, it was found that among the groups in which only the 5'UTR was changed while the 3'UTR (α-globin UTR used in the Moderna vaccine) was fixed, A-3 to A-7 (SEQ ID NO: 5 to 9), A-9 to A-11 (SEQ ID NO: 11 to 13), B-1 (SEQ ID NO: 14), B-3 (SEQ ID NO: 16), B-6 to B-10 (SEQ ID NO: 19 to 23), and E-3 (SEQ ID NO: 42) had significantly superior protein expression levels. From the results of 2-2 above, it was found that, from the groups in which only the 5'UTR was changed while the 3'UTR (α-globin UTR used in the Moderna vaccine) was fixed, significantly superior protein expression levels were obtained. Among the groups in which only the 3'UTR was changed while the 3'UTR was fixed, it can be seen that significantly superior protein expression levels were obtained in F-2 to F-3 (SEQ ID NO: 50 to 51), F-5 to F-7 (SEQ ID NO: 53 to 55), G-2 (SEQ ID NO: 60), G-6 to G-9 (SEQ ID NO: 64 to 67), H-7 (SEQ ID NO: 75), and J-7 (SEQ ID NO: 91).

[0146] From the above description, a person skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing its technical ideas or essential features. It should be understood that the above examples are merely illustrative and not limiting. The present invention should be interpreted as including all modifications or alterations derived from the meaning and scope of the claims and their equivalent concepts, rather than the specification.

Claims

1. An mRNA transcription vector comprising a promoter region recognized by an RNA polymerase and a gene construct operably linked to the promoter region, The genetic construct comprises: (1) a 5'-untranslated region (5'-UTR); (2) an open reading frame (ORF) region operably linked to the 5'UTR region, the ORF region comprising a nucleotide sequence encoding a target protein; (3) a 3'-untranslated region (3'-UTR) operably linked to the open reading frame region; The 3'UTR region consists of SEQ ID NO: 1 or a nucleotide sequence having 90% or more sequence identity thereto, or the 5'UTR region consists of SEQ ID NO: 47 or a nucleotide sequence having 90% or more sequence identity thereto; When the 3'UTR region consists of SEQ ID NO: 1 or a nucleotide sequence having 90% or more sequence identity thereto, the 5'UTR region is selected from SEQ ID NO: 5, 6, 7, 8, 9, 11, 12, 13, 14, 16, 19, 20, 21, 22, 23, 42 or a nucleotide sequence having 90% or more sequence identity thereto; When the 5'UTR region consists of SEQ ID NO: 47 or a nucleotide sequence having 90% or more sequence identity thereto, the 3'UTR region is selected from SEQ ID NO: 50, 51, 53, 54, 55, 60, 64, 65, 66, 67, 75, 91 or a nucleotide sequence having 90% or more sequence identity thereto; mRNA transcription vector.

2. The mRNA transcription vector of claim 1 , wherein the open reading frame region comprises a nucleotide sequence encoding an antigen derived from a pathogen.

3. The mRNA transcription vector of claim 2, wherein the pathogen is selected from the group consisting of a virus, a bacterium, a prion, a fungus, a protozoon, a viroid, and a parasite.

4. The mRNA transcription vector of claim 1 , wherein the gene construct further comprises a nucleotide sequence to be transcribed into a 5′ Cap operably linked to the 5′ UTR.

5. The mRNA transcription vector of claim 1 , wherein the genetic construct further comprises a nucleotide sequence operably linked to the 3′UTR region that is transcribed into a polyA tail.

6. The mRNA transcription vector of claim 5, wherein the polyA tail comprises 20 to 200 adenines.

7. The mRNA transcription vector of claim 1 , wherein the mRNA transcription vector is a plasmid.

8. The mRNA transcription vector of claim 1 , wherein the mRNA transcription vector is linearized.

9. A method for producing an mRNA molecule, comprising a step of performing transcription using the mRNA transcription vector according to any one of claims 1 to 8 as a template.

10. 10. An mRNA molecule produced by the method of claim 9.

11. A pharmaceutical composition comprising the mRNA molecule of claim 10 as an active ingredient.

12. The pharmaceutical composition of claim 11, wherein the mRNA molecule is complexed with at least one lipid component to form a complex selected from the group consisting of liposomes, lipid nanoparticles, and lipoplexes.

13. The pharmaceutical composition of claim 11, wherein the pharmaceutical composition is administered intramuscularly or subcutaneously.

14. (1) a 5'-untranslated region (5'-UTR); (2) an open reading frame (ORF) region operably linked to the 5'UTR region, the ORF region comprising a nucleotide sequence encoding a target protein; (3) A gene construct comprising a 3'-untranslated region (3'-UTR) operably linked to the open reading frame region, The 3'UTR region consists of SEQ ID NO: 1 or a nucleotide sequence having 90% or more sequence identity thereto, or the 5'UTR region consists of SEQ ID NO: 47 or a nucleotide sequence having 90% or more sequence identity thereto; When the 3'UTR region consists of SEQ ID NO: 1 or a nucleotide sequence having 90% or more sequence identity thereto, the 5'UTR region is selected from SEQ ID NO: 5, 6, 7, 8, 9, 11, 12, 13, 14, 16, 19, 20, 21, 22, 23, 42 or a nucleotide sequence having 90% or more sequence identity thereto; When the 5'UTR region consists of SEQ ID NO: 47 or a nucleotide sequence having 90% or more sequence identity thereto, the 3'UTR region is selected from SEQ ID NO: 50, 51, 53, 54, 55, 60, 64, 65, 66, 67, 75, 91 or a nucleotide sequence having 90% or more sequence identity thereto; Gene constructs.

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