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 for protein expression and templates therefor. [Background technology]

[0002] In recent years, mRNA has attracted attention as an active pharmaceutical ingredient (API). There are three major advantages to mRNA-based vaccines. First, the production process is relatively simple and can be completed in a short period of time, allowing for rapid and flexible responses to emergencies such as pandemics and mutations. Second, functionally, effective intracellular delivery can induce both humoral and cellular immunity, potentially providing more effective disease prevention than other types of vaccines. Third, safety is a major advantage: it has few side effects, and unlike DNA, RNA does not enter the nucleus and modify the human body's genes. Therefore, there is a need to develop 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 for protein expression and templates therefor. [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] Another object of the present invention is to provide a pharmaceutical composition containing the mRNA molecule prepared as described above as an active ingredient.

[0009] Another object of the present invention is to provide uses of the mRNA transcription vector for producing mRNA and for producing pharmaceutical compositions. [Effects 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, allowing the desired protein to be expressed in large amounts. [Brief explanation of the drawings]

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

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

[0013] The mRNA transcription vector comprises a promoter region recognized by an 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 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.

[0015] In another specific example, 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.

[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 comprises: (1) a 5'-untranslated region (5'-UTR); (2) an open reading frame (ORF) region operably linked to the 5'UTR region and containing 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 NOs: 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 NOs: 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 any of the above-mentioned embodiments of the mRNA expression vector, 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 embodiments is characterized in that it further comprises a nucleotide sequence operably linked to the 5'UTR that is transcribed into a 5' cap (5' Cap).

[0023] The gene construct contained in the mRNA expression vector according to any of the above-mentioned embodiments 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-described embodiment as a template.

[0030] A further aspect of the present invention is an mRNA molecule produced by the method for producing an mRNA molecule according to the above-mentioned aspect.

[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-described 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 pharmaceutically 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 is characterized in that it is 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 producing mRNA.

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

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

[0043] Yet another aspect of the present invention is a gene 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 ORF 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 NOs: 5, 6, 7, 8, 9, 11, 12, 13, 14, 16, 19, 20, 21, 22, 23, and 42 or a nucleotide sequence having 90% or more sequence identity thereto.

[0045] In another specific example, the gene construct 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 NOs: 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 also applies to other descriptions and embodiments. In other words, all combinations of various elements disclosed in the present invention are included in the present invention. Furthermore, 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, throughout this specification, many papers and patent documents are referenced and citations are provided, the disclosures of which are incorporated herein by reference in their entirety to more clearly explain the state of the art to which the present invention pertains and the content of the present invention.

[0049] In the present invention, the term "nucleotide sequence" refers to a polymer of nucleotides 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 polynucleotides of the present invention include modified nucleotides. The term "modified nucleotide" as used herein refers to any non-standard 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, uridine present in mRNA may be present as thymidine in each DNA encoding the mRNA. Also, while the polynucleotide sequences of the present invention use "T" in representative DNA sequences, it is recognized that when the sequence represents RNA, "T" is replaced with "U."

[0052] As used herein, "non-natural" refers to a polynucleotide, polypeptide, carbohydrate, lipid, or composition that does not exist in nature. Such polynucleotides, polypeptides, carbohydrates, lipids, or compositions differ in one or more respects from naturally occurring polynucleotides, polypeptides, carbohydrates, lipids, or compositions. For example, a polymer (e.g., a polynucleotide, polypeptide, or carbohydrate) differs in the type and sequence of its constituent building blocks (e.g., nucleotide sequence, amino acid sequence, or sugar molecules). A polymer differs from naturally occurring polymers in the molecules that link it.

[0053] As used herein, "operably linked" refers to a configuration in which regulatory sequences are appropriately positioned so that they control 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 containing 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 encoding the target mRNA, and a sequence for regulating 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 may be any promoter for any DNA-dependent RNA polymerase, and the promoter may be selected from promoter sequences with high binding affinity to the RNA polymerase, but is not necessarily limited thereto.

[0057] The vector is obtained by cloning a polynucleotide, such as 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 further comprises: (1) a 5'-untranslated region (5'-UTR); (2) an open reading frame (ORF) region operably linked to the 5'UTR region and comprising 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, "5'UTR" refers to an untranslated region located upstream of the start 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 mRNA stability and / or translation accuracy. For example, avoiding gene sequences similar to the start of an ORF in the 5' UTR can effectively prevent incorrect initiation of mRNA translation. Furthermore, to improve mRNA stability and translation accuracy, certain specific sequences may be added to the 5' UTR. For example, a Kozak sequence may be inserted to more accurately initiate the translation process.

[0061] In the present invention, "3'UTR" refers to the untranslated region located downstream of the coding region of mRNA.

[0062] The 3' UTR sequences 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 by comprising 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 a nucleotide sequence 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 NOs: 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 NOs: 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 with the nucleotide sequence of a specific sequence number and in which a portion of the sequence has been deleted, modified, substituted or added, as long as the nucleotide sequence exhibits the same activity as the nucleotide sequence consisting of the 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] Sequence identity calculations can be performed, for example, by aligning two sequences for optimal comparison purposes (e.g., gaps are introduced into 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 alignment tools 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 common global alignment method based on dynamic programming is the Needleman-Wunsch algorithm (Non-Patent Document 3). In addition to optimal global alignment methods such as the Needleman-Wunsch algorithm, more recent reports have shown that 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 percent identity of two sequences can be accomplished using mathematical algorithms. For example, the percent identity of two nucleic acid sequences can be determined similarly to that described in the literature [Non-Patent Documents 4, 5, 6, 7, and 8]. As an example, the percent identity of two nucleic acid sequences can be determined using the Meyers and Miller algorithm (Non-Patent Document 9) incorporated into 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 percent 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 percent sequence identity 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, "open reading frame" (ORF), which may be abbreviated as "ORF," refers to a segment or region of an mRNA molecule that encodes a protein or polypeptide of interest. An ORF is a continuous stretch of DNA that begins with an initiation codon and ends with a termination codon, and is translated by the ribosome.

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

[0075] The term "antigen" as used herein 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 contains at least one epitope (antigenic determinant).

[0076] For the purposes of the present invention, the antigen may be the translation product of the aforementioned 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] By way of 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 cancer-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), and tumor-specific mutant antigens (e.g., Mum-1, β-Catenin, or CDK4). Such antigens are personalized cancer antigens or tissue-specific. However, they are not limited to the above examples.

[0081] In another example, the target protein may be a protein involved in the prevention or treatment of a disease. For 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. For another example, the protein may be involved in the prevention or treatment of a metabolic disease, such as obesity. Examples of such target proteins include, but are not limited to, fibroblast growth factor 21 (FGF21) 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. In one example, the 5' Cap may be methylated.

[0084] For example, a cap analog may be added during in vitro transcription, allowing capping to 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. In another example, capping may be performed by an enzymatic reaction after in vitro transcription. Such enzymatic reactions involve enzymes such as triphosphatase and guanyltransferase. However, without being limited thereto, mRNA can be provided with a 5' Cap using methods known in the art.

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

[0086] As used herein, a "polyA tail" refers to a continuous or discontinuous sequence of adenylate residues typically located at the 3' end of an RNA molecule. The polyA tail may follow the 3'UTR of an mRNA of the present invention. Such a polyA tail may be composed of or contain 20 or more, 25 or more, 40 or more, 60 or more, 80 or more, 100 or more, or about 200 or more adenylate (A) nucleotides. 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% of the nucleotides in the polyA tail are A nucleotides, with the remaining nucleotides being nucleotides other than A nucleotides, such as U nucleotides (uridylic acid), G nucleotides (guanylic acid), or C nucleotides (cytidylic acid). For example, the polyA tail may contain modifications that retard mRNA degradation. As an example, the poly A 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 will survive or exhibit 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 before 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] In the present invention, the term "pharmaceutical composition" 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. Administration of the immunogenic composition of the present invention to an individual can prevent disease. Therefore, the "pharmaceutical composition for preventing disease" in the present invention may be an "immunogenic composition" or a "vaccine composition." The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, excipient, binder, carrier, preservative, buffer, isotonicity agent, emulsifier, or wetting agent.

[0093] The RNA contained in the pharmaceutical compositions of the present invention may be formulated with one or more excipients to improve stability, increase cell transfection, allow for 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 compositions of the present invention may include a pharmaceutically acceptable excipient.

[0094] The pharmaceutical composition may be formulated, and such formulation 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 mRNA. The mRNA and the delivery agent may form a complex.

[0097] Examples of polymers that may be used as mRNA carriers 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 and platelet membrane-coated nanoparticles (PNP). 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] For example, the pharmaceutical composition of the present invention may include an mRNA molecule complexed with at least one lipid component, e.g., 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) administrable doses for administration 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 that, when administered to an individual, is effective to elicit an antibody response to an antigen. The immunologically effective amount will vary depending on the health and physical condition of the individual being treated, their age, the capacity of the individual's immune system to synthesize antibodies, the level of protection desired, the formulation of the composition, the treating physician's assessment of the medical situation, and other relevant factors.

[0105] The immunogenic composition provided by the present invention may contain an adjuvant. As used herein, 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 analogs thereof. For example, the composition may include a buffer.

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

[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 are not intended to limit the scope of the present invention. [Example]

[0110] mRNA transcription vectors and the production of mRNA transcripts In this example, we prepared mRNA transcription vectors composed of various combinations of 5' / 3' untranslated region (UTR) sequences, and the 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 the 5'- to 3'-end direction.

[0112] More specifically, using the pcDNA3.1 plasmid, the T7 promoter sequence (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 were inserted after the T7 promoter sequence to prepare a plasmid.

[0113] Here, various mRNA transcription vectors were prepared by varying only the UTR sequence. Tables 1 and 2 to 4 show sequence information for vectors in which only the 5'UTR was varied while the 3'UTR (α-globin UTR used in the Moderna vaccine) was fixed. Tables 5 and 6 to 12 show sequence information for vectors in which only the 3'UTR was varied while the 5'UTR (α-globin UTR used in the Pfizer / BioNTech vaccine) was fixed. Table 13 shows sequence information for vectors in which no changes were made in all examples.

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

[0115] [Table 1]

[0116] [Table 2]

[0117] [Table 3]

[0118] [Table 4]

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

[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 following examples, the sequence information is kept unchanged.

[0129] [Table 13]

[0130] In summary, a total of 91 mRNA transcription vectors were constructed (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 cells were transformed with the mRNA transcription vector plasmid prepared as described above and cultured. The plasmid was isolated using the CompactPrep Plasmid Midi Kit (Qiagen) and then linearized using the Esp3I restriction enzyme. The linearized plasmid was recovered using the MinElute Reaction Cleanup Kit (Qiagen) as described above and then used in an in vitro transcription (IVT) reaction for mRNA production. To do this, the linearized plasmid DNA template was mixed under the conditions shown in Table 14 and then incubated 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] Next, 50 μl of RNA was mixed with 25 μl of LiCl, and the mixture was incubated at -20°C for 30 minutes. The RNA was then precipitated by centrifugation. The RNA precipitate was washed with 70% ethanol, and the remaining ethanol was removed. The mixture was then eluted with nuclease-free HO. To prevent any remaining precipitate, the mixture was incubated at 65°C for 5 minutes to produce mRNA transcripts suitable for transfection. [Example]

[0135] Evaluation of 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, 2 × 10 Primary Skeletal Muscle cells were placed in a 24-well plate. 4 Cells were seeded at 1000 cells / well and then cultured at 37°C for 5 hours or more in a 5% CO environment to allow attachment. For transfection, 0.75 μl of Lipofectamine was added to 25 μl of OptiMEM (Gibco) per mRNA sample. TMMessengerMax was mixed with the cells and incubated at room temperature for 10 minutes to create the MessengerMax mixture. Then, 50 ng of RNA transcripts was added to 25 μl of OptiMEM to create the mRNA mixture. 25 μl of Messenger Max mixture and 25 μl of mRNA mixture were mixed and incubated at room temperature for 5 minutes. Primary skeletal muscle cells were then treated with 50 μl of the mixed solution. d2EGFP protein expression levels were assessed 24 and 48 hours later using Cytation 7 (BioTek). Specifically, expression levels were measured in the GFP channel (469, 525), followed by analysis of fluorescence within a 3200 μm radius circle from the center of the well. The minimum luminescence intensity was set to 5000 or greater, and the cell size for analysis was set to 30 μm to 200 μm. The mean fluorescent value and cell count were then multiplied to calculate the total luminescence. Each mRNA transcript was evaluated four to six times, and the results are shown as the average.

[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 (the α-globin UTR used in the Moderna vaccine) was fixed, A-3 to A-7 (SEQ ID NOs: 5 to 9), A-9 to A-11 (SEQ ID NOs: 11 to 13), B-1 (SEQ ID NO: 14), B-3 (SEQ ID NO: 16), B-6 to B-10 (SEQ ID NOs: 19 to 23), and E-3 (SEQ ID NO: 42) exhibited significantly superior protein expression efficiency compared to Moderna Ref. and other combinations of UTRs derived from the same or different tissues. In particular, the Fold values ​​in parentheses specifically confirm how superior their protein expression efficiency is compared to Moderna Ref.

[0141] 2-2. Evaluation of GFP protein expression levels when only the 3'UTR is changed while the 5'UTR (α-globin UTR used in the Pfizer / BioNTech vaccine) is 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 (the α-globin UTR used in the Pfizer / BioNTech vaccine) was fixed, F-2 to F-3 (SEQ ID NOs: 50-51), F-5 to F-7 (SEQ ID NOs: 53-55), G-2 (SEQ ID NO: 60), G-6 to G-9 (SEQ ID NOs: 64-67), H-7 (SEQ ID NO: 75), and J-7 (SEQ ID NO: 91) exhibited significantly superior protein expression efficiency compared to Pfizer / BioNTech Ref. and other combinations of UTRs derived from the same or different tissues. In particular, the Fold values ​​in parentheses specifically demonstrate how superior 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, significantly superior protein expression levels were obtained in A-3 to A-7 (SEQ ID NOs: 5 to 9), A-9 to A-11 (SEQ ID NOs: 11 to 13), B-1 (SEQ ID NO: 14), B-3 (SEQ ID NO: 16), B-6 to B-10 (SEQ ID NOs: 19 to 23), and E-3 (SEQ ID NO: 42). From the results of 2-2 above, it was found that the 5'UTR (α-globin UTR used in the Pfizer / BioNTech vaccine) was fixed while the 5'UTR was changed. 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 NOs: 50 to 51), F-5 to F-7 (SEQ ID NOs: 53 to 55), G-2 (SEQ ID NO: 60), G-6 to G-9 (SEQ ID NOs: 64 to 67), H-7 (SEQ ID NO: 75), and J-7 (SEQ ID NO: 91).

[0146] From the above description, those skilled in the art to which the present invention pertains will understand that the present invention can be embodied in other specific forms without changing the technical spirit or essential features thereof. It should be understood that the above examples are merely illustrative and not limiting. The present invention should be construed as including all modifications and variations derived from the meaning and scope of the claims, rather than the specification, and their equivalents.

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 gene construct comprises: (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; (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 NOs: 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 (5' Cap) operably linked to the 5' UTR.

5. The mRNA transcription vector of claim 1 , wherein the gene 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 poly A tail contains 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. (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; (3) An mRNA molecule 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; and the corresponding UTR region is selected as defined in claim 1. mRNA molecule.

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

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

13. The pharmaceutical composition according to 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 open reading frame (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 NOs: 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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