Nucleic acid, drug composition, and method of producing nucleic acid

Abstract

Provided are: a nucleic acid including a protein-encoding region that has been modified, wherein the total proportion of uracil residues and adenine residues in the modified protein-encoding region is reduced in comparison to that in the protein-encoding region prior to modification; and a method of producing the nucleic acid.

Description

Nucleic acids, pharmaceutical compositions, and methods for producing nucleic acids

[0001] The present invention relates to nucleic acids, pharmaceutical compositions, and methods for producing nucleic acids. This application claims priority based on Japanese Patent Application No. 2023-196002, filed November 17, 2023, the contents of which are incorporated herein by reference.

[0002] In recent years, the practical application of nucleic acid medicines such as nucleic acids for gene therapy and mRNA vaccines has progressed. In addition to DNA medicines such as DNA plasmids and viral vectors, the use of messenger RNA (mRNA) has been proposed as a nucleic acid for gene therapy. However, the use of exogenously supplied RNA has been limited due to immunogenicity issues. In order to reduce the immunogenicity of RNA, it has been proposed to reduce the uridine content in mRNA by, for example, replacing uridine with modified uridine (Patent Document 1).

[0003] Special table 2018-525410 publication

[0004] In nucleic acid medicines containing a protein coding region, it is necessary to suppress cytotoxic activity due to immunogenicity, etc., as well as to improve the production amount of the protein.

[0005] Therefore, an object of the present invention is to provide a nucleic acid that has low cytotoxic activity and improves protein production, a pharmaceutical composition containing the nucleic acid, and a method for producing the nucleic acid.

[0006] The present invention includes the following aspects: [1] A nucleic acid comprising a modified protein-coding region, wherein the total proportion of uracil residues and adenine residues in the modified protein-coding region is reduced compared to the protein-coding region before modification. [2] The nucleic acid according to [1], wherein at least a portion of the codons contained in the protein-coding region before modification have been substituted with codons encoding the same amino acid but with a lower proportion of uracil and adenine. [3] The nucleic acid according to [1] or [2], wherein, among the codons contained in the protein-coding region before modification, at least a portion of the codons whose first base is uracil have been substituted with codons encoding the same amino acid but with adenine as the first base. [4] The nucleic acid according to any one of [1] to [3], wherein at least a portion of the codons contained in the protein-coding region before modification have been substituted with codons encoding the same amino acid but with a higher frequency of use in a cell into which the nucleic acid is introduced. [5] The nucleic acid according to any one of [1] to [4], wherein at least a portion of the uracil residues contained in the nucleic acid are residues derived from modified uracil nucleotides. [6] The nucleic acid according to [5], wherein the modified uracil nucleotide is at least one selected from the group consisting of 5-methoxyuridine-5'-triphosphate, 5-methyluridine-5'-triphosphate, pseudouridine-5'-triphosphate, N1-methylpseudouridine-5'-triphosphate, N1-methyl-2'-O-methylpseudouridine-5'-triphosphate, N1-methoxymethylpseudouridine-5'-triphosphate, N1-propylpseudouridine-5'-triphosphate, biotin-16-aminoallyluridine-5'-triphosphate, 2'-O-methyluridine-5'-triphosphate, 5-bromouridine-5'-triphosphate, 5-iodouridine-5'-triphosphate, 4-thiouridine-5'-triphosphate, and 1-thiouridine-5'-triphosphate. [7] The nucleic acid according to any one of [1] to [6], wherein the nucleic acid is mRNA. [8] The nucleic acid according to any one of [1] to [7], wherein the protein coding region encodes hepatocyte growth factor.[9] The nucleic acid according to [8], wherein the nucleotide sequence of the protein-coding region is the nucleotide sequence set forth in SEQ ID NO: 1.

[10] A pharmaceutical composition comprising the nucleic acid according to any one of [1] to [9].

[11] A pharmaceutical composition for treating or preventing an ischemic disease, comprising the nucleic acid according to [8] or [9].

[12] The pharmaceutical composition according to

[11] , wherein the ischemic disease is chronic arterial occlusion disease, chronic obstructive pulmonary disease, interstitial pneumonia, acute lung injury, ophthalmologic disease, optic nerve injury disease, or intractable skin ulcer.

[13] A method for producing a nucleic acid comprising a protein-coding region, the method comprising: (a) determining the nucleotide sequence of the protein-coding region in accordance with the following principles (a1) to (a4): (a1) codon-optimizing the protein-coding region for a cell into which the nucleic acid is to be introduced; (a2) selecting codons so as to reduce the total proportion of uracil residues and adenine residues in the protein-coding region; (a3) ​​when the options for the first base of a codon encoding the same amino acid are uracil and adenine, selecting a codon whose first base is adenine; and (a4) when the options for the third base of a codon encoding the same amino acid include guanine and cytosine, selecting a codon whose third base is guanine or cytosine and which is more frequently used in the cell; and (b) producing a nucleic acid comprising the protein-coding region of the nucleotide sequence determined in step (a).

[14] The method according to

[13] , wherein a modified uracil nucleotide is used as the uracil nucleotide in the step (b).

[15] The method of

[14] , wherein the modified uracil nucleotide is at least one selected from the group consisting of 5-methoxyuridine-5'-triphosphate, 5-methyluridine-5'-triphosphate, pseudouridine-5'-triphosphate, N1-methylpseudouridine-5'-triphosphate, N1-methyl-2'-O-methylpseudouridine-5'-triphosphate, N1-methoxymethylpseudouridine-5'-triphosphate, N1-propylpseudouridine-5'-triphosphate, biotin-16-aminoallyluridine-5'-triphosphate, 2'-O-methyluridine-5'-triphosphate, 5-bromouridine-5'-triphosphate, 5-iodouridine-5'-triphosphate, 4-thiouridine-5'-triphosphate, and 1-thiouridine-5'-triphosphate.

[0007] According to the present invention, there are provided a nucleic acid, a pharmaceutical composition containing the nucleic acid, and a method for producing the nucleic acid, which have low cytotoxic activity and improved protein production.

[0008] The Cap1 structure of the 5' cap structure of mRNA is shown. The structure of mRNA for human HGF production (AG-mRNA) synthesized in the examples is shown. The amount of human HGF produced at each amount of nucleic acid introduced is shown. The dose-response regression line of human HGF production with AG-mRNA, created from the results of AG-mRNA shown in Figure 3, is shown. Cell viability at each amount of nucleic acid introduced is shown. An outline of the test schedule for the continuous nucleic acid exposure test is shown. The time course of human HGF concentration in the culture supernatant in the continuous nucleic acid exposure test is shown. The time course of intracellular human HGF concentration in the continuous nucleic acid exposure test is shown. An outline of the test schedule for the 48-hour nucleic acid exposure test is shown. The amount of human HGF released per day from intracellular to extracellular in the 48-hour nucleic acid exposure test is shown. The cumulative amount of human HGF released from intracellular to extracellular in the 48-hour nucleic acid exposure test is shown.

[0009] The terms "polynucleotide" and "nucleic acid" are used interchangeably and refer to a nucleotide polymer in which nucleotides are linked by phosphodiester bonds. A "polynucleotide" and a "nucleic acid" may be deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or a combination of DNA and RNA. A "polynucleotide" and a "nucleic acid" may be a polymer of natural nucleotides, a polymer of natural nucleotides and non-natural nucleotides (an analog of a natural nucleotide, a nucleotide in which at least one of the base moiety, sugar moiety, and phosphate moiety is modified), or a polymer of non-natural nucleotides. The nucleotide sequence of a "polynucleotide" or a "nucleic acid" is written in the commonly accepted single-letter code unless otherwise specified. Unless otherwise specified, the nucleotide sequence is written from the 5' to the 3' end. The nucleotide residues constituting a "polynucleotide" or a "nucleic acid" may be simply written as adenine, thymine, cytosine, guanine, uracil, etc., or their single-letter codes (A, T, C, G, U).

[0010] The terms "polypeptide," "peptide," and "protein" are used interchangeably and refer to a polymer of amino acids linked by amide bonds. A "polypeptide," "peptide," or "protein" may be a polymer of natural amino acids, a polymer of natural and non-natural amino acids (e.g., chemical analogs or modified derivatives of natural amino acids), or a polymer of non-natural amino acids. The amino acid sequence of a "polypeptide," "peptide," or "protein" is written using the commonly accepted one-letter or three-letter code unless otherwise specified. Unless otherwise specified, the amino acid sequence is written from the N-terminus to the C-terminus.

[0011] "Gene" refers to a polynucleotide containing at least one open reading frame (ORF) that encodes a particular protein. A gene can contain both exons and introns.

[0012] The term "protein coding region" refers to a region in a nucleic acid that is translated into a protein. More specifically, it refers to a region in an mRNA that is translated into a protein and is sandwiched between an initiation codon and a termination codon, and a corresponding region in DNA that serves as a template for the mRNA. The protein coding region may be divided into multiple exons.

[0013] The term "residue" refers to a structure (structural unit) derived from each monomer in a polymer formed by bonding monomers.

[0014] "Uracil residue" refers to a residue derived from a uracil nucleotide or a modified uracil nucleotide. "Adenine residue" refers to a residue derived from an adenine nucleotide or a modified adenine nucleotide. "Cytosine residue" refers to a residue derived from a cytosine nucleotide or a modified cytosine nucleotide. "Guanine residue" refers to a residue derived from a guanine nucleotide or a modified guanine nucleotide. "Thymine residue" refers to a residue derived from a thymine nucleotide or a modified thymine nucleotide.

[0015] "Uracil nucleotide" refers to a nucleotide containing uracil as a base. "Adenine nucleotide" refers to a nucleotide containing adenine as a base. "Cytosine nucleotide" refers to a nucleotide containing cytosine as a base. "Guanine nucleotide" refers to a nucleotide containing guanine as a base. "Thymine nucleotide" refers to a nucleotide containing thymine as a base.

[0016] A "modified uracil nucleotide" refers to a nucleotide containing uracil or modified uracil as a base, in which at least one site selected from the group consisting of the base (uracil), sugar (ribose), and phosphate group (triphosphate) has been modified. A "modified adenine nucleotide" refers to a nucleotide containing adenine or modified adenine as a base, in which at least one site selected from the group consisting of the base (adenine), sugar (ribose or deoxyribose), and phosphate group (triphosphate) has been modified. A "modified cytosine nucleotide" refers to a nucleotide containing cytosine or modified cytosine as a base, in which at least one site selected from the group consisting of the base (cytosine), sugar (ribose or deoxyribose), and phosphate group (triphosphate) has been modified. A "modified guanine nucleotide" refers to a nucleotide containing guanine or modified guanine as a base, in which at least one site selected from the group consisting of the base (guanine), sugar (ribose or deoxyribose), and phosphate group (triphosphate) has been modified. A "modified thymine nucleotide" refers to a nucleotide containing thymine or modified thymine as a base, in which at least one site selected from the group consisting of the base (thymine), sugar (deoxyribose), and phosphate group (triphosphate) has been modified. In nucleic acids, residues derived from modified uracil nucleotides have the same function as residues derived from uridine triphosphate. In nucleic acids, residues derived from modified adenine nucleotides have the same function as residues derived from adenosine triphosphate or deoxyadenosine triphosphate. In nucleic acids, residues derived from modified cytosine nucleotides have the same function as residues derived from cytidine triphosphate or deoxycytidine triphosphate. In nucleic acids, residues derived from modified guanine nucleotides have the same function as residues derived from guanosine triphosphate or deoxyguanosine triphosphate. Residues derived from modified thymine nucleotides have the same function as residues derived from deoxythymidine triphosphate.As the modified uracil nucleotide, modified adenine nucleotide, modified cytosine nucleotide, modified guanine nucleotide, and modified thymine nucleotide, known nucleotides can be used without any particular limitation.

[0017] "Wild-type" refers to something that exists in nature (naturally). For example, a wild-type nucleic acid refers to a nucleic acid that occurs in nature. For example, a wild-type nucleic acid includes a nucleic acid isolated from a cell or virus of a naturally occurring organism. For example, a wild-type gene can be a gene isolated from a wild-type nucleic acid. For example, a wild-type protein includes a protein isolated from a cell or virus of a naturally occurring organism.

[0018] "Codon optimization" refers to replacing at least one codon in an original nucleotide sequence with a codon that is more frequently used in a target biological species while maintaining the original amino acid sequence. Codon usage tables are readily available, for example, at the "Codon Usage Database" provided by the Kazusa DNA Research Institute (www.kazusa.or.jp / codon / ). For example, codons can be optimized using a codon usage table. Computer algorithms for codon-optimizing a specific sequence for expression in a specific animal species are also known. Computer algorithms for codon optimization are available, for example, in Gene Forge (Aptagen; Jacobus, PA).

[0019] (Nucleic Acid) A first aspect of the present invention is a nucleic acid (hereinafter also referred to as "modified nucleic acid") comprising a modified protein-coding region (hereinafter also referred to as "modified protein-coding region"). In one embodiment, the modified nucleic acid has a reduced total proportion of uracil residues and adenine residues in the modified protein-coding region compared to the protein-coding region before modification (hereinafter also referred to as "protein-coding region before modification").

[0020] The modified nucleic acid may be RNA or DNA. In one embodiment, the modified nucleic acid may be mRNA or DNA containing at least one ORF. When the modified nucleic acid is DNA, the modified nucleic acid may be in the form of a vector such as a plasmid vector or a viral vector. The modified nucleic acid is preferably mRNA. In the following description, when the modified nucleic acid is DNA, uracil (U) is replaced with thymine (T). Alternatively, the mRNA transcribed from the DNA may contain a modified protein coding region as described below.

[0021] <Modified Protein Coding Region> The modified nucleic acid includes at least one modified protein coding region. The modified protein coding region is obtained by modifying the nucleotide sequence of the pre-modification protein coding region. The pre-modification protein coding region may be the protein coding region of a wild-type nucleic acid, or may be a protein coding region of a wild-type nucleic acid with modifications added. The protein encoded by the pre-modification protein coding region is not particularly limited and may be a desired protein. The protein may be a wild-type protein or a modified protein obtained by modifying a wild-type protein. The protein may be derived from any organism (including animals, plants, protozoa, bacteria, viruses, etc.) and may be any type of protein. An example of a protein encoded by a pre-modification protein coding region is hepatocyte growth factor (HGF). The amino acid sequence of human HGF is shown in SEQ ID NO: 2.

[0022] The modified protein coding region encodes the same protein as the pre-modification protein coding region, and preferably encodes the same amino acid sequence as the pre-modification protein coding region.

[0023] The modified nucleic acid may comprise two or more modified protein coding regions, or may comprise two or more types of modified protein coding regions. If the modified nucleic acid is DNA, the modified protein coding region may be separated into two or more exons by one or more introns.

[0024] The modified protein coding region satisfies the following condition (i). Furthermore, the modified protein coding region preferably satisfies one or more of the following conditions (ii) to (iii): (i) the total proportion of uracil residues and adenine residues is reduced compared to the protein coding region before modification. (ii) among the codons contained in the protein coding region before modification, at least some of the codons whose first base is uracil have been substituted with codons whose first base is adenine that encode the same amino acid. (iii) at least some of the codons contained in the protein coding region before modification have been substituted with codons whose first base is adenine that encode the same amino acid and that are more frequently used in cells into which the modified nucleic acid is introduced.

[0025] Condition (i): The modified protein coding region has been modified so that the total proportion of uracil residues and adenine residues is reduced compared to the protein coding region before modification. For example, the modified protein coding sequence has a reduced total proportion of uracil residues and adenine residues, for example, by 5% or more, 10% or more, 15% or more, or 20% or more, compared to the protein coding region before modification.

[0026] Such modification can be achieved, for example, by substituting at least some of the codons contained in the wild-type protein-coding region with codons having a lower ratio of uracil and adenine. In one embodiment, the modified protein-coding region has 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 100% of the codons containing either or both uracil and adenine contained in the protein-coding region before modification substituted with codons having a lower ratio of uracil and adenine.

[0027] Table 1 shows the codon table.

[0028]

[0029] Examples of codons with a low proportion of uracil and adenine include the codons shown in Table 2. In one embodiment, the codons contained in the modified protein coding region are those in which at least a portion of the codons contained in the protein coding region before modification have been converted to the codons shown in Table 2. In one embodiment, the codons shown in Table 2 are used for 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 100% of the codons contained in the modified protein coding region. Preferably, the codons shown in Table 2 are used for all of the codons contained in the modified protein coding region.

[0030]

[0031] Condition (ii): In the modified protein coding region, at least a portion of the codons in the pre-modification protein coding region that have uracil as the first base may be substituted with codons encoding the same amino acid and having adenine as the first base. In one embodiment, in the modified protein coding region, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 100% of the codons in the wild-type protein coding region that have uracil as the first base are substituted with codons encoding the same amino acid and having adenine as the first base.

[0032] Codons that can be used to satisfy conditions (i) and (ii) are shown in Table 3. In one embodiment, the codons contained in the modified protein coding region are such that at least a portion of the codons contained in the protein coding region before modification are converted to the codons shown in Table 3. In one embodiment, the codons shown in Table 3 are used for 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 100% of the codons contained in the modified protein coding region. Preferably, the codons shown in Table 3 are used for all of the codons contained in the modified protein coding region.

[0033]

[0034] Condition (iii): In the modified protein coding region, at least some of the codons contained in the pre-modification protein coding region may be replaced with codons encoding the same amino acid that are more frequently used in the cell into which the modified nucleic acid is to be introduced (hereinafter also referred to as the "target cell"). For example, when the target cell into which the modified nucleic acid is to be introduced is a human cell, at least some of the codons contained in the pre-modification protein coding region in the modified protein coding region may be replaced with codons more frequently used in humans. A table of codon usage in the target cell is readily available, for example, from the "Codon Usage Database" provided by the Kazusa DNA Research Institute, Public Interest Foundation (www.kazusa.or.jp / codon / ).

[0035] For example, if codons encoding the same amino acid whose third base is guanine are more frequently used in the target cells than codons whose third base is cytosine, codons whose third base is guanine may be selected. In this case, in the modified protein coding region, at least a portion of the codons whose third base is cytosine contained in the pre-modification protein coding region may be substituted with codons encoding the same amino acid whose third base is guanine. In this case, in the modified protein coding region, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 100% of the codons whose third base is cytosine contained in the pre-modification protein coding region may be substituted with codons encoding the same amino acid whose third base is guanine.

[0036] For example, if codons encoding the same amino acid whose third base is cytosine are more frequently used in the target cells than codons whose third base is guanine, codons whose third base is cytosine may be selected. In this case, in the modified protein coding region, at least a portion of the codons whose third base is guanine contained in the pre-modification protein coding region may be substituted with codons encoding the same amino acid whose third base is cytosine. In this case, in the modified protein coding region, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 100% of the codons whose third base is guanine contained in the pre-modification protein coding region may be substituted with codons encoding the same amino acid whose third base is cytosine.

[0037] Codons that can be used in humans to satisfy conditions (i) to (iii) are shown in Table 4. When the target cell is a human cell, at least some of the codons contained in the modified protein coding region may be converted to the codons shown in Table 4 compared to the codons contained in the pre-modification protein coding region. When the target cell is a human cell, the codons shown in Table 4 may be used for 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 100% of the codons contained in the modified protein coding region. When the target cell is a human cell, preferably, the codons shown in Table 4 are used for all of the codons contained in the modified protein coding region.

[0038]

[0039] In one embodiment, the modified protein coding region may be obtained by codon-optimizing the pre-modification protein coding region for the cell into which it is to be introduced, and modifying the codon-optimized protein coding region to satisfy the above conditions (i) and (ii). For example, the modified protein coding region may include the following embodiments.

[0040] (a) A modified protein coding region, wherein the protein coding region before modification has been codon-optimized for the cell into which it is to be introduced, and at least some of the codons contained in the codon-optimized protein coding region have been substituted with codons encoding the same amino acid but with a lower proportion of uracil and adenine. (b) A modified protein coding region, wherein the protein coding region before modification has been codon-optimized for the cell into which it is to be introduced, and at least some of the codons contained in the codon-optimized protein coding region that have uracil as the first base have been substituted with codons encoding the same amino acid but with adenine as the first base. The modified protein coding region of (a) or (b), further comprising at least some of the codons that have guanine or cytosine as the third base have been substituted with codons encoding the same amino acid but with guanine or cytosine as the third base that are more frequently used in the cell.

[0041] In the above (a) to (c), "at least a portion" refers to 60% or more of all the relevant codons, or may be 70% or more, 80% or more, 90% or more, 95% or more, or 100% of all the relevant codons.

[0042] The modified nucleic acid comprises at least one open reading frame (ORF), and the modified protein coding region may be contained within the ORF.

[0043] As a specific example of the nucleotide sequence of a modified protein coding region, the nucleotide sequence of a modified protein coding region encoding human HGF is shown in SEQ ID NO: 1. In SEQ ID NO: 1, "T" represents a uracil residue in RNA and a thymine residue in DNA. "A" represents an adenine residue, "C" represents a cytosine residue, and "G" represents a guanine residue. The amino acid sequence of human HGF encoded by the modified protein coding region containing the nucleotide sequence set forth in SEQ ID NO: 1 is shown in SEQ ID NO: 2.

[0044] The amino acid sequence of human HGF is not limited to that set forth in SEQ ID NO: 2, and may include, for example, any of the following amino acid sequences (i) to (iii): (i) an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 2. (ii) an amino acid sequence in which one or several amino acids have been mutated in SEQ ID NO: 2. (iii) an amino acid sequence having 80% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 2.

[0045] In (i), the amino acid sequence may contain an amino acid sequence other than the amino acid sequence set forth in SEQ ID NO: 2 at either or both of the N-terminus and C-terminus. The length of the added amino acid sequence is not particularly limited, and examples include 1 to 100 amino acids, 1 to 50 amino acids, 1 to 30 amino acids, 1 to 20 amino acids, 1 to 10 amino acids, or 1 to 5 amino acids. In (ii), examples of "several" include 2 to 20, 2 to 15, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, or 2. The "mutation" may be any of deletion, substitution, addition, and insertion, or a combination thereof. In (iii) above, the sequence identity may be 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Preferably, the amino acid sequences of (i) to (iii) above are such that a protein consisting of the amino acid sequence is a functional HGF. "Functional HGF" refers to a protein that functions as an HGF. Preferably, functional HGF has HGF activity (e.g., hepatocyte proliferation-promoting activity) equivalent to or greater than that of a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2. In one embodiment, the modified protein coding region may encode any of the amino acid sequences of (i) to (iii) above.

[0046] <Other Regions> The modified nucleic acid may contain other regions in addition to the modified protein-coding region. For example, when the modified nucleic acid is mRNA, the other regions include a 5' Cap, a 5' untranslated region (5' UTR), a 3' untranslated region (3' UTR), and a poly(A) tail. When the modified nucleic acid is DNA, the other regions include a promoter, a terminator, etc.

[0047] Cap Structure: When the modified nucleic acid is mRNA, it may contain a 5' Cap at the 5' end. Examples of 5' Cap structures include Cap0 structure, Cap1 structure, and Cap2 structure. The cap structure is typically a 7-methylguanine ribonucleotide, attached via its 5'-triphosphate to the 5' position of the first nucleotide in the 5'-3' direction of the mRNA, i.e., the first cap-proximal nucleotide. In a Cap0 structure, the ribose of the first and second cap-proximal nucleotides of the mRNA both contain 2'-hydroxyl. In a Cap1 structure, the ribose of the first cap-proximal nucleotide of the mRNA contains 2'-methoxy, and the ribose of the second nucleotide contains 2'-hydroxyl (see Figure 1). In a Cap2 structure, the ribose of the first and second cap-proximal nucleotide of the mRNA both contain 2'-methoxy. Modified nucleic acids preferably contain a 5' Cap, and more preferably contain a Cap1 structure as the 5' Cap.

[0048] The cap structure can be incorporated into the 5' end of mRNA during transcription using known methods. For example, a Cap1 structure can be incorporated into mRNA by co-transcription using CleanCap™ AG (TriLink Biotechnologies). The cap structure may also be added to mRNA after transcription or to chemically synthesized RNA using a capping enzyme.

[0049] 5'UTR and 3'UTR: When the modified nucleic acid is an mRNA, the modified nucleic acid may contain either or both of a 5'UTR and a 3'UTR. The 5'UTR and 3'UTR may be those of the mRNA from which the original protein-coding region is derived, or may be those of a different mRNA. The nucleotide sequence of the 5'UTR may have 100% sequence identity with the nucleotide sequence of the wild-type 5'UTR, or may be at least partially modified. The nucleotide sequence of the 3'UTR may have 100% sequence identity with the nucleotide sequence of the wild-type 3'UTR, or may be at least partially modified. Examples of mRNAs from which the 5'UTR and 3'UTR are derived include, in addition to the mRNA from which the pre-modification protein coding region is derived, mRNAs such as globin (e.g., human alpha globin (HBA), human beta globin (HBB), Xenopus beta globin (XBG)), bovine growth hormone, cytomegalovirus (CMV), mouse Hba-a1, hydroxysteroid 17-beta dehydrogenase 4 (e.g., HSD17B4, HSD), albumin, heat shock protein 90 (Hsp90), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), beta-actin, alpha-tubulin, tumor protein (p53), and epidermal growth factor receptor (EGFR), but are not limited to these.

[0050] The modified nucleic acid may contain a Kozak sequence. The Kozak sequence can affect translation initiation and the total amount of protein produced from the mRNA. The Kozak sequence contains a methionine codon that can function as an initiation codon. A minimal Kozak sequence is NNNRUGN (N is any nucleotide residue, and R is a purine residue (A or G)). In the formula, the first N is preferably A or G, and the second N is preferably G. In one embodiment, the Kozak sequence is RNNRUGN, NNNRUGG, RNNRUGG, RNNAUGN, NNNAUGG, or RNNAUGG.

[0051] Poly(A) tail: When the modified nucleic acid is an mRNA, the modified nucleic acid may include a poly(A) tail at the 3' end. The poly(A) tail may include a sequence of at least 8 consecutive adenine nucleotides, but may also include one or more non-adenine nucleotide residues (e.g., G, C, U). The length of the poly(A) tail can be, for example, 10 to 500 nucleotides, 30 to 300 nucleotides, or 60 to 250 nucleotides.

[0052] Examples of mRNA structures include the following. In the following, the 5'UTR may contain only a Kozak sequence. (1) An mRNA containing, in order from the 5' end, a 5'Cap, a 5'UTR, a modified protein coding region (or an ORF containing a modified protein coding region), a 3'UTR, and a poly(A) tail. (2) An mRNA containing, in order from the 5' end, a 5'Cap, a 5'UTR, a modified protein coding region (or an ORF containing a modified protein coding region), and a poly(A) tail. (3) An mRNA containing, in order from the 5' end, a 5'UTR, a modified protein coding region (or an ORF containing a modified protein coding region), a 3'UTR, and a poly(A) tail. (4) An mRNA containing, in order from the 5' end, a 5'UTR, a disulfide bond-cleaving enzyme coding region, and a poly(A) tail. (5) An mRNA comprising, in order from the 5' end, a 5' Cap, a 5' UTR, a modified protein coding region (or an ORF comprising a modified protein coding region), and a 3' UTR. (6) An mRNA comprising, in order from the 5' end, a 5' Cap, a 5' UTR, and a modified protein coding region (or an ORF comprising a modified protein coding region). (7) An mRNA comprising, in order from the 5' end, a 5' UTR, a modified protein coding region (or an ORF comprising a modified protein coding region), and a 3' UTR. (8) An mRNA comprising, in order from the 5' end, a 5' UTR and a modified protein coding region (or an ORF comprising a modified protein coding region). (9) An mRNA comprising, in order from the 5' end, a 5' Cap, a modified protein coding region (or an ORF comprising a modified protein coding region), a 3' UTR, and a poly(A) tail. (10) An mRNA comprising, in order from the 5' end, a 5' Cap, a modified protein coding region (or an ORF containing a modified protein coding region), and a poly(A) tail.

[0053] <Modified Nucleotides> The modified nucleic acid may contain modified nucleotides. The term "modified nucleotide" refers to a nucleotide in which at least one site selected from the group consisting of the base, sugar, and phosphate group is modified. The modified nucleotide is not particularly limited, and known modified nucleotides can be used.

[0054] In the modified nucleic acid, at least a portion of the uracil residues are preferably residues derived from modified uracil nucleotides. By using modified uracil nucleotides, the immunogenicity of the modified nucleic acid can be suppressed. In one embodiment, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 100% of the uracil residues contained in the modified nucleic acid are residues derived from modified uracil nucleotides. Preferably, all of the uracil residues contained in the modified nucleic acid are residues derived from modified uracil nucleotides. The modified uracil nucleotide is not particularly limited, and known uracil nucleotides can be used. Specific examples of modified uracil nucleotides include, but are not limited to, 5-methoxyuridine-5'-triphosphate, 5-methyluridine-5'-triphosphate, pseudouridine-5'-triphosphate, N1-methylpseudouridine-5'-triphosphate, N1-methyl-2'-O-methylpseudouridine-5'-triphosphate, N1-methoxymethylpseudouridine-5'-triphosphate, N1-propylpseudouridine-5'-triphosphate, biotin-16-aminoallyluridine-5'-triphosphate, 2'-O-methyluridine-5'-triphosphate, 5-bromouridine-5'-triphosphate, 5-iodouridine-5'-triphosphate, 4-thiouridine-5'-triphosphate, and 1-thio-uridine-5'-triphosphate. In one embodiment, the modified uracil nucleotide has a modified base (uracil) (modified uracil). In one embodiment, the modified uracil nucleotide can be selected from the group consisting of 5-methoxyuridine-5'-triphosphate, 5-methyluridine-5'-triphosphate, pseudouridine-5'-triphosphate, N1-methylpseudouridine-5'-triphosphate, N1-methyl-2'-O-methylpseudouridine-5'-triphosphate, N1-methoxymethylpseudouridine-5'-triphosphate, N1-propylpseudouridine-5'-triphosphate, biotin-16-aminoallyluridine-5'-triphosphate, 5-bromouridine-5'-triphosphate, 5-iodouridine-5'-triphosphate, and 4-thiouridine-5'-triphosphate. In one embodiment, the modified uracil nucleotide is 5-methoxyuridine-5'-triphosphate.The modified uracil nucleotides may be used singly or in combination of two or more.

[0055] The modified nucleic acid has the above-mentioned characteristics, and thus has reduced cytotoxic activity compared to the unmodified nucleic acid, and when introduced into cells, increases the amount of protein produced, making it suitable for use as a drug for causing target cells to produce a desired protein.

[0056] (Pharmaceutical Composition) A second aspect of the present invention is a pharmaceutical composition. In one embodiment, the pharmaceutical composition comprises the modified nucleic acid of the first aspect.

[0057] In one embodiment, the pharmaceutical composition may contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier that does not inhibit the physiological activity of the active ingredient and is not substantially toxic to the recipient. The term "not substantially toxic" refers to a carrier that is not toxic to the recipient at a dose typically used. In the pharmaceutical composition of this embodiment, the pharmaceutically acceptable carrier is a carrier that does not inhibit the function of the modified nucleic acid according to the first aspect and is not substantially toxic to the recipient. Pharmaceutically acceptable carriers include any known pharmaceutically acceptable ingredient that is typically considered an inactive ingredient. Pharmaceutically acceptable carriers include, but are not limited to, solvents, diluents, vehicles, excipients, glidants, binders, granulating agents, dispersing agents, suspending agents, wetting agents, lubricants, disintegrants, solubilizers, stabilizers, emulsifiers, fillers, etc. Pharmaceutically acceptable carriers may be used alone or in combination. Specific examples of pharmaceutically acceptable carriers include, but are not limited to, excipients such as sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, and calcium carbonate; binders such as cellulose, methylcellulose, hydroxypropyl cellulose, gelatin, gum arabic, polyethylene glycol, sucrose, and starch; disintegrants such as starch, carboxymethylcellulose, hydroxypropyl starch, sodium starch glycolate, sodium bicarbonate, calcium phosphate, and calcium citrate; lubricants such as magnesium stearate, aerosil, talc, and sodium lauryl sulfate; flavorings such as citric acid, menthol, glycyrrhizin ammonium salt, glycine, and orange powder; preservatives such as sodium benzoate, sodium bisulfite, methylparaben, and propylparaben; stabilizers such as citric acid, sodium citrate, and acetic acid; suspending agents such as methylcellulose, polyvinylpyrrolidone, and aluminum stearate; dispersing agents such as surfactants; diluents such as water and physiological saline; and base waxes such as cocoa butter, polyethylene glycol, and white kerosene.

[0058] The pharmaceutical composition may contain other ingredients in addition to the above-mentioned ingredients. The other ingredients are not particularly limited, and those commonly used in the pharmaceutical field can be used without particular limitation. Examples of other ingredients include pharmaceutical additives other than those mentioned above. Examples of pharmaceutical additives include, but are not limited to, preservatives (e.g., antioxidants), chelating agents, flavoring agents, sweeteners, thickeners, buffers, colorants, etc. The pharmaceutical composition may contain an active ingredient other than the modified nucleic acid. Examples of active ingredients include, but are not limited to, antiviral agents, antibiotics, anti-inflammatory agents, antipyretics, analgesics, etc. The other ingredients may be used alone or in combination of two or more.

[0059] The pharmaceutical composition may contain a nucleic acid transfer reagent to facilitate the transfer of the modified nucleic acid into cells, including, but not limited to, cationic lipids, cationic liposomes, cationic polymers, etc. Specific examples of nucleic acid transfer reagents include Lipofectin (trade name, Invitrogen), Lipofectamine (trade name, Invitrogen), Transfectam (trade name, Promega), DOTAP (trade name, Roche Applied Science), dioctadecylamidoglycyl spermine (DOGS), L-dioleoyl phosphatidyl-ethanolamine (DOPE), dimethyldioctadecyl-ammonium bromide (DDAB), N,N-di-n-hexadecyl-N,N-dihydroxyethylammonium bromide (DHDEAB), N-n-hexadecyl-N,N-dihydroxyethylammonium bromide (HDEAB), polybrene, poly(ethyleneimine) (PEI), and the like are not limited thereto.

[0060] The dosage form of the pharmaceutical composition is not particularly limited and can be any dosage form commonly used for pharmaceutical preparations. The pharmaceutical composition of this embodiment may be an oral or parenteral formulation, with parenteral formulations being preferred. Oral formulations include, for example, tablets, coated tablets, pills, powders, granules, capsules, syrups, fine granules, liquids, drops, and emulsions. Parenteral formulations include, for example, injections, suppositories, nasal drops, enteral preparations, and inhalants. Pharmaceutical compositions of these dosage forms can be formulated according to standard methods (e.g., methods described in the Japanese Pharmacopoeia). The pharmaceutical composition is preferably a parenteral formulation, and more preferably an injection.

[0061] The administration route of the pharmaceutical composition of this embodiment is not particularly limited, and can be oral or parenteral, with parenteral administration being preferred. Examples of parenteral administration routes include intravenous administration, intranasal administration, subcutaneous administration, intradermal administration, intramuscular administration, intraperitoneal administration, and enteral administration. The preferred administration route is subcutaneous administration, intradermal administration, or intramuscular administration.

[0062] The pharmaceutical composition can be administered in a therapeutically effective amount of the modified nucleic acid. A "therapeutically effective amount" refers to an amount of drug effective for treating or preventing a target disease. For example, a therapeutically effective amount of the modified nucleic acid can be an amount effective for producing an effective amount of the protein encoded by the modified nucleic acid in cells. The therapeutically effective amount can be determined appropriately based on the patient's symptoms, body weight, age, and sex, as well as the dosage form and administration method of the pharmaceutical composition. For example, the pharmaceutical composition can be administered in a single dose of 0.01 to 1000 mg of modified nucleic acid per kg of body weight of the subject. The dose can be 0.05 to 500 μg, 0.1 to 200 μg, 0.5 to 100 μg, or 1 to 50 μg.

[0063] The pharmaceutical composition may be administered in a single dose or repeatedly. In the case of repeated administration, the administration interval may be appropriately determined depending on the patient's symptoms, body weight, age, sex, etc., as well as the type of modified nucleic acid, the dosage form of the pharmaceutical composition, the administration method, etc. The administration interval may be, for example, every few hours, 2 to 3 times a day, once every 1 to 5 days, once a week, once a month, once every few months, etc.

[0064] The disease to which the pharmaceutical composition is applied can be selected depending on the type of protein encoded by the modified protein coding region of the modified nucleic acid. In one embodiment, the modified protein coding region of the modified nucleic acid encodes a protein effective in treating or preventing a target disease. When the modified protein coding region encodes HGF, the pharmaceutical composition can be used as a pharmaceutical composition for treating or preventing ischemic diseases. Examples of ischemic diseases include chronic arterial occlusion, chronic obstructive pulmonary disease, interstitial pneumonia, acute lung injury, ophthalmological diseases, optic nerve damage diseases, and intractable skin ulcers.

[0065] The recipient of the pharmaceutical composition is not particularly limited. The recipient of the pharmaceutical composition is preferably a mammal, and may be a human or a non-human mammal. Non-human mammals include, but are not limited to, primates (monkeys, chimpanzees, gorillas, etc.), rodents (mice, hamsters, rats, etc.), rabbits, dogs, cats, cows, goats, sheep, and horses. The recipient is preferably a biological species from which the protein encoded by the modified protein coding region of the modified nucleic acid is derived. For example, when the modified protein coding region encodes a human protein, the pharmaceutical composition is preferably intended for administration to humans.

[0066] (Method for Producing Nucleic Acid) A third aspect of the present invention is a method for producing a nucleic acid comprising a protein-coding region. The method of this embodiment comprises the following steps (a) and (b): (a) determining the nucleotide sequence of the protein-coding region according to the following principles: (a1) codon-optimizing the protein-coding region for a cell into which the nucleic acid is to be introduced; (a2) selecting codons so as to reduce the total proportion of uracil residues and adenine residues in the protein-coding region; and (a3) ​​when the options for the first base of a codon are uracil and adenine for codons encoding the same amino acid, selecting a codon whose first base is adenine; and (b) producing a nucleic acid comprising the protein-coding region of the nucleotide sequence determined in step (a).

[0067] <Step (a)> In step (a), the nucleotide sequence of the protein-coding region is determined according to the above-mentioned guidelines (a1) to (a3). A desired protein can be selected as the protein encoded by the protein-coding region. Based on the amino acid sequence of the protein, the nucleotide sequence of the protein-coding region is determined according to the above-mentioned guidelines (a1) to (a3).

[0068] In the policy (a1), codons can be selected as explained in the condition (iii) in the "<Modified protein coding region>" section of the "(Nucleic acid)" section above.

[0069] In policy (a2), codons can be selected as described in condition (i) in the "<Modified protein coding region>" section of the "(Nucleic acid)" section above. For example, codons listed in Table 2 can be selected.

[0070] In policy (a3), codons can be selected as described in condition (ii) in the "<Modified protein coding region>" section of the "(Nucleic acid)" section above. For example, codons listed in Table 3 can be selected.

[0071] When the target cells are human, the codons listed in Table 4 can be selected in accordance with the principles of (a1) to (a3).

[0072] <Step (b)> In step (b), a nucleic acid is produced that contains a protein-coding region consisting of the nucleotide sequence determined in step (a). The method for producing the nucleic acid is not particularly limited, and known methods can be used. The nucleic acid may contain other regions in addition to the protein-coding region. When the nucleic acid is mRNA, it may contain at least one or more selected from the group consisting of 5'Cap, 5'UTR, 3'UTR, and poly(A) tail. Specific examples of these and specific examples of the mRNA structure are the same as those described above in the section "(Nucleic Acid)."

[0073] When the nucleic acid is RNA, the RNA can be produced by a transcription reaction using DNA containing the nucleotide sequence of the RNA as a sense strand as a template with RNA polymerase. The template DNA can be obtained, for example, by chemically synthesizing DNA of about 100 to 300 nucleotides and linking them together using a method for DNA assembly.

[0074] When producing RNA, modified nucleotides may be used as nucleotides in the transcription reaction. In one embodiment, it is preferable to use modified uracil nucleotides instead of uracil nucleotides in the transcription reaction. Examples of modified uracil nucleotides include those exemplified above. In one embodiment, modified uracil nucleotides in which the base (uracil) is modified (modified uracil) can be used. In one embodiment, 5-methoxyuridine-5'-triphosphate can be used as the modified uracil nucleotide. One type of modified uracil nucleotide may be used alone, or two or more types may be used in combination.

[0075] When the nucleic acid is mRNA, the 5' Cap may be added by co-transcriptional capping or post-transcriptional capping. When a Cap1 structure is added as the 5' Cap in the co-transcriptional method, CleanCap (registered trademark) AG (TriLink Biotechnologies) or the like can be used.

[0076] After the nucleic acid is synthesized in step (b), the nucleic acid may be purified. When the nucleic acid is RNA, the nucleic acid may be purified by DNase treatment, oligo dT purification, or the like.

[0077] The method of this embodiment makes it possible to produce the nucleic acid of the first aspect.

[0078] The present disclosure includes the following aspects: [1] A nucleic acid comprising a modified protein-coding region, wherein the total proportion of uracil and adenine residues in the modified protein-coding region is reduced compared to the protein-coding region before modification. [2] The nucleic acid according to [1], wherein at least a portion of the codons contained in the protein-coding region before modification have been substituted with codons encoding the same amino acid but with a lower proportion of uracil and adenine. [3] The nucleic acid according to [1] or [2], wherein, among the codons contained in the protein-coding region before modification, at least a portion of the codons whose first base is uracil have been substituted with codons encoding the same amino acid but with adenine as the first base. [4] The modified nucleic acid according to any one of [1] to [3], wherein, among the codons contained in the protein-coding region before modification, at least a portion of the codons whose third base is guanine have been substituted with codons encoding the same amino acid but with a cytosine as the third base. [5] The nucleic acid according to any one of [1] to [4], wherein at least a portion of the codons contained in the protein-coding region before modification that have a cytosine third base have been substituted with codons encoding the same amino acid but with a guanine third base. [6] The nucleic acid according to any one of [1] to [5], wherein at least a portion of the codons contained in the protein-coding region before modification have been substituted with codons encoding the same amino acid but with a higher frequency of use in a cell into which the nucleic acid is to be introduced. [7] The nucleic acid according to [1], wherein the modified protein-coding region has been codon-optimized for a cell into which the nucleic acid is to be introduced, and at least a portion of the codons contained in the codon-optimized protein-coding region have been substituted with codons encoding the same amino acid but with a low proportion of uracil and adenine. [8] The nucleic acid according to [7], wherein at least a portion of the codons contained in the codon-optimized protein-coding region that have a uracil first base have been substituted with codons encoding the same amino acid but with adenine first base.[9] The nucleic acid according to [7] or [8], wherein at least some of the codons contained in the codon-optimized protein-coding region, which have a guanine or cytosine third base, are substituted with codons encoding the same amino acid but having a guanine or cytosine third base that are more frequently used in the cell.

[10] The nucleic acid according to any one of [1] to [9], wherein at least some of the uracil residues contained in the nucleic acid are residues derived from modified uracil nucleotides.

[11] The nucleic acid according to

[10] , wherein all of the uracil residues contained in the nucleic acid are residues derived from modified uracil nucleotides.

[12] The nucleic acid according to

[11] , wherein the modified uracil nucleotide is at least one selected from the group consisting of 5-methoxyuridine-5'-triphosphate, 5-methyluridine-5'-triphosphate, pseudouridine-5'-triphosphate, N1-methylpseudouridine-5'-triphosphate, N1-methyl-2'-O-methylpseudouridine-5'-triphosphate, N1-methoxymethylpseudouridine-5'-triphosphate, N1-propylpseudouridine-5'-triphosphate, biotin-16-aminoallyluridine-5'-triphosphate, 2'-O-methyluridine-5'-triphosphate, 5-bromouridine-5'-triphosphate, 5-iodouridine-5'-triphosphate, 4-thiouridine-5'-triphosphate, and 1-thiouridine-5'-triphosphate.

[13] The nucleic acid according to any one of [1] to

[12] , wherein the nucleic acid is mRNA.

[14] The nucleic acid according to

[13] , further comprising a 5' Cap, a 5' untranslated region, and a 3' untranslated region.

[15] The nucleic acid according to

[14] , wherein the 5' Cap has a Cap1 structure.

[16] The nucleic acid according to any one of [1] to

[15] , wherein the protein coding region encodes hepatocyte growth factor.

[17] The nucleic acid according to

[16] , wherein the hepatocyte growth factor is human hepatocyte growth factor.

[18] The nucleic acid according to

[17] , wherein the human hepatocyte growth factor comprises an amino acid sequence selected from the group consisting of the following (i) to (iii): (i) the amino acid sequence set forth in SEQ ID NO: 2; (ii) an amino acid sequence set forth in SEQ ID NO: 2 in which one or several amino acids have been mutated; and (iii) an amino acid sequence having 80% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 2.

[19] The modified nucleic acid according to

[17] , wherein the nucleotide sequence of the protein-coding region is the nucleotide sequence set forth in SEQ ID NO: 1.

[20] A pharmaceutical composition comprising the nucleic acid according to any one of [1] to

[19] .

[21] A pharmaceutical composition for treating or preventing an ischemic disease, comprising the nucleic acid according to any one of

[16] to

[19] .

[22] The pharmaceutical composition according to

[21] , wherein the ischemic disease is chronic arterial occlusion disease, chronic obstructive pulmonary disease, interstitial pneumonia, acute lung injury, ophthalmological disease, optic nerve injury disease, or intractable skin ulcer.

[23] A method for producing a nucleic acid comprising a protein-coding region, comprising: (a) determining the nucleotide sequence of the protein-coding region in accordance with the following principles: (a1) codon-optimizing the protein-coding region for a cell into which the nucleic acid is to be introduced; (a2) selecting codons so as to reduce the total proportion of uracil residues and adenine residues in the protein-coding region; (a3) ​​when the options for the first base of a codon encoding the same amino acid are uracil and adenine, selecting a codon whose first base is adenine; and (a4) when the options for the third base of a codon encoding the same amino acid include guanine and cytosine, selecting a codon whose third base is guanine or cytosine and which is more frequently used in the cell; and (b) producing a nucleic acid comprising the protein-coding region of the nucleotide sequence determined in step (a).

[24] The method of

[23] , wherein a modified uracil nucleotide is used as the uracil nucleotide in step (b).

[25] The method of

[24] , wherein the modified uracil nucleotide is at least one selected from the group consisting of 5-methoxyuridine-5'-triphosphate, 5-methyluridine-5'-triphosphate, pseudouridine-5'-triphosphate, N1-methylpseudouridine-5'-triphosphate, N1-methyl-2'-O-methylpseudouridine-5'-triphosphate, N1-methoxymethylpseudouridine-5'-triphosphate, N1-propylpseudouridine-5'-triphosphate, biotin-16-aminoallyluridine-5'-triphosphate, 2'-O-methyluridine-5'-triphosphate, 5-bromouridine-5'-triphosphate, 5-iodouridine-5'-triphosphate, 4-thiouridine-5'-triphosphate, and 1-thiouridine-5'-triphosphate.

[0079] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0080] (Design of Nucleotide Sequence of Human HGF Coding Region) The nucleotide sequence of the cDNA nucleotide sequence of human hepatocyte growth factor (HGF) (SEQ ID NO: 3) was modified based on the following guidelines: (a) The amino acid sequence of human HGF (SEQ ID NO: 2) was left unchanged. (b) Codons in the HGF coding region were changed to those most frequently used in humans (human codon optimization). (c) Codons containing uracil and adenine were changed, whenever possible, to codons that encode the same amino acid and have a low ratio of uracil and adenine. (d) When codons encoding the same amino acid exist, one with uracil as the first base and the other with adenine as the first base, the codon with adenine as the first base was selected. (e) When codons encoding the same amino acid exist, one with guanine as the third base and the other with cytosine as the third base, the codon most frequently used in humans was selected.

[0081] The nucleotide sequence of the modified human HGF obtained was the nucleotide sequence set forth in SEQ ID NO: 1. In SEQ ID NO: 1, "T" represents a uracil residue in RNA and a thymine residue in DNA.

[0082] (mRNA synthesis) Using the HGF coding region whose nucleotide sequence was determined as described above as the ORF, mRNA synthesis was outsourced to TriLink BioTechnologies. 5-Methoxyuridine triphosphate was used as the uracil nucleotide for mRNA synthesis. mRNA synthesis was performed using the short oligo priming method. CleanCap® AG (TriLink BioTechnologies) was used for mRNA capping, and mRNA (see Figure 2) with the Cap1 structure (see Figure 1) added was synthesized. mRNA purification was performed by DNase treatment and oligo dT purification. The purified mRNA was dissolved in DNase-free and RNase-free water at a concentration of approximately 1 mg / mL and dispensed in 500 μg aliquots. This was stored at -80°C and shipped from the supplier on dry ice.

[0083] (Evaluation of mRNA) As a control for the synthesized mRNA (hereinafter referred to as "AG-mRNA"), BioCap ＴＭ A comparative test was conducted using HGF mRNA (hereinafter referred to as "BioCap", phaRNA), human HGF mRNA (hereinafter referred to as "BioGene", Creative BioGene), a plasmid DNA called Collategene (hereinafter referred to as "HGF Plasmid", Anges Inc.), and naked HGF plasmid (hereinafter referred to as "Naked HGF Plasmid").

[0084] When the nucleic acid to be introduced was mRNA, the introduction of nucleic acid into cultured cells was carried out using Thermo Fisher Scientific's Lipofectamine Messenger MAX according to the attached protocol. When the nucleic acid to be introduced was plasmid DNA, the introduction was carried out using Thermo Fisher Scientific's Lipofectamine LTX according to the attached protocol. In Figures 3 and 5, "Naked plasmid" refers to the case where the plasmid DNA was introduced directly without using an introduction reagent.

[0085] HGF production: IMR-90 (10５ The cells (1000 cells / mL) were cultured at 37°C for 48 hours using Thermo Fisher Scientific's Minimum Essential Media. After the culture, the culture medium was centrifuged and the supernatant was collected. The amount of HGF in the supernatant was measured using the HGF Human ELISA kit Quantikine (R&D Systems) according to the attached protocol.

[0086] The results are shown in Figure 3. Both mRNAs were confirmed to produce much higher levels of HGF than HGF Plasmid and Naked HGF Plasmid. Among the mRNAs, AG-mRNA produced the highest HGF. The maximum HGF production level (approximately 700 ng / mL) with AG-mRNA was confirmed at an introduction amount of 2 µg. With BioCap and BioGene, the maximum HGF production level was confirmed at an introduction amount of 0.5 µg, and both were approximately 400 ng / mL.

[0087] Figure 4 shows a dose-response regression line created from the amount of AG-mRNA introduced and the amount of HGF produced. From the dose-response regression line, the amount of AG-mRNA introduced that had the same activity as 1 μg of HGF plasmid was 0.0130 μg. This confirmed that AG-mRNA had approximately 80 times the activity of HGF plasmid.

[0088] Cell viability: IMR-90 (10 ５ The cells (1000 cells / mL) were cultured at 37°C for 48 hours using Minimum Essential Media from Thermo Fisher Scientific. After culture, the cells were collected, and the viability of each cell after nucleic acid transfection was measured using Takara Bio's Premix WST-1 Cell Proliferation Assay System according to the attached protocol.

[0089] The results are shown in Figure 5. Compared with BioGene and BioCap, AG-mRNA had higher cell viability and lower cytotoxic activity. AG-mRNA and HGF Plasmid appeared to have higher cell viability and lower cytotoxic activity.

[0090] <Evaluation of duration of action> HGF production in continuous exposure test: In the presence of 1 μg or 0.5 μg of AG-mRNA, IMR-90 (10 ５ The cells (2000 x 1000 cells / mL) were cultured at 37°C using Minimum Essential Media (Thermo Fisher Scientific). The culture medium was sampled 48, 72, 96, and 120 hours after the start of culture. The sampled culture medium was centrifuged to obtain the culture supernatant and cells, respectively. The HGF concentration of the obtained culture supernatant and cells was measured using the HGF Human ELISA kit Quantikine (R&D Systems) according to the attached protocol (see Figure 6).

[0091] As a control, a similar test was performed using 0.5 μg each of BioCap and Biogene and 100 μg of HGF Plasmid. As a negative control, IMR-90 was cultured without adding any mRNA or plasmid.

[0092] The results are shown in Figures 7 and 8. Figure 7 shows the HGF concentration in the culture supernatant, and Figure 8 shows the intracellular HGF concentration.

[0093] When AG-mRNA was used, the HGF concentration in the culture supernatant was higher than when other mRNAs or HGF Plasmid were used. In the culture supernatant using AG-mRNA, the HGF concentration increased gradually up to 120 hours. In the culture supernatant using AG-mRNA, HGF was present at a concentration of 70% or more of the concentration at 120 hours at 48 hours (Figure 7).

[0094] When AG-mRNA was used, the intracellular HGF concentration was higher than when other mRNAs or HGF Plasmid were used. In cells using AG-mRNA, the HGF concentration tended to gradually decrease up to 120 hours. In cells using AG-mRNA, high HGF concentrations were maintained even at 120 hours (Figure 8).

[0095] HGF production in 48-hour exposure test: In the presence of 1 μg or 0.5 μg of AG-mRNA, IMR-90 (10５ The cells (2000 cells / mL) were cultured at 37°C for 48 hours using Minimum Essential Media (from Thermo Fisher Scientific). Next, the same medium without AG-mRNA was added in the same volume as the removed medium, and the culture was continued. The culture medium was sampled 48 hours, 72 hours, 96 hours, and 120 hours after the start of culture. The HGF concentration in the sampled culture medium was measured using the HGF Human ELISA kit Quantikine (from R&D Systems) according to the attached protocol (see Figure 9).

[0096] As a control, a similar test was performed using 0.5 μg each of BioCap and BioGene and 1 μg of HGF Plasmid. As a negative control, IMR-90 was cultured without adding any mRNA or plasmid.

[0097] The results are shown in Figures 10 and 11. Figure 10 shows the amount of HGF released from the cells into the culture supernatant per day, calculated from the HGF concentration in the culture supernatant. Figure 11 shows the cumulative amount of HGF released from the cells into the culture supernatant from 48 to 120 hours of culture.

[0098] When AG-mRNA was used, the amount of HGF released from cells into the culture supernatant was higher than when other mRNAs or HGF Plasmid were used (Figures 10 and 11). When AG-mRNA was used, the amount of HGF released from cells into the culture supernatant decreased from 48 to 120 hours, but HGF release continued even at 120 hours (Figures 10 and 11). The cumulative HGF release (48 to 120 hours) when AG-mRNA was used was approximately 10-fold greater at 1 μg and approximately 7-fold greater at 0.5 μg compared to HGF Plasmid. After AG-mRNA introduction, the maximum amount of HGF released per day was achieved at 72 hours (3 days). The amount of HGF released gradually decreased thereafter, but HGF continued to be released extracellularly even at 120 hours (5 days).

[0099] These results confirmed that AG-mRNA has lower cytotoxicity and higher HGF production capacity compared to other HGF nucleic acids. AG-mRNA was confirmed to exhibit a concentration-dependent increase in HGF production up to an introduction amount of 2 μg. Of the tested introduction amounts, AG-mRNA exhibited maximum HGF production capacity (700 ng / mL) at an introduction amount of 2 μg. The minimum introduction amount at which HGF production was confirmed was 0.0130 μg (Figures 3 and 4). No significant decrease in cell viability was observed with AG-mRNA up to an introduction amount of 2 μg. On the other hand, with the control mRNAs BioCap and Biogene, cell viability began to decrease at an introduction amount of 0.5 μg, and further decreased at an introduction amount of 1 μg (Figure 5).

[0100] It was confirmed that the extracellular release of HGF produced within the cells was sustained for more than 120 hours after AG-mRNA introduction. In contrast, the release was sustained for approximately 72 hours after HGF Plasmid introduction (Figs. 10 and 11). It was confirmed that a constant amount of HGF was released from within the cells up to 72 hours after AG-mRNA introduction (Figs. 10 and 11).

[0101] According to the present invention, there are provided a nucleic acid, a pharmaceutical composition containing the nucleic acid, and a method for producing the nucleic acid, which have low cytotoxic activity and improved protein production.

[0102] Although the preferred embodiments of the present invention have been described above, the present invention is not limited to these embodiments. Addition, omission, substitution, and other modifications of the configuration are possible within the scope of the spirit of the present invention. The present invention is not limited by the above description, but is limited only by the scope of the appended claims.

Claims

1. A nucleic acid comprising a modified protein coding region, wherein the total percentage of uracil and adenine residues in the modified protein coding region is reduced compared to the protein coding region before modification.

2. The nucleic acid according to claim 1, wherein at least a portion of the codons contained in the protein coding region before modification have been replaced with codons encoding the same amino acid but with a lower proportion of uracil and adenine.

3. The nucleic acid according to claim 1, wherein at least a portion of the codons whose first base is uracil among the codons contained in the protein coding region before the modification have been replaced with codons whose first base is adenine and which encode the same amino acid.

4. The nucleic acid according to claim 1, wherein at least a portion of the codons contained in the protein coding region before the modification have been replaced with codons encoding the same amino acid that are more frequently used in a cell into which the nucleic acid is introduced.

5. The nucleic acid of claim 1, wherein at least a portion of the uracil residues contained in the nucleic acid are derived from modified uracil nucleotides.

6. The nucleic acid according to claim 5, wherein the modified uracil nucleotide is at least one selected from the group consisting of 5-methoxyuridine-5'-triphosphate, 5-methyluridine-5'-triphosphate, pseudouridine-5'-triphosphate, N1-methylpseudouridine-5'-triphosphate, N1-methyl-2'-O-methylpseudouridine-5'-triphosphate, N1-methoxymethylpseudouridine-5'-triphosphate, N1-propylpseudouridine-5'-triphosphate, biotin-16-aminoallyluridine-5'-triphosphate, 2'-O-methyluridine-5'-triphosphate, 5-bromouridine-5'-triphosphate, 5-iodouridine-5'-triphosphate, 4-thiouridine-5'-triphosphate and 1-thio-uridine-5'-triphosphate.

7. The nucleic acid of claim 1, wherein the nucleic acid is mRNA.

8. The nucleic acid of claim 1, wherein the protein coding region encodes hepatocyte growth factor.

9. The nucleic acid of claim 8, wherein the nucleotide sequence of the protein coding region is the nucleotide sequence set forth in SEQ ID NO:

1.

10. A pharmaceutical composition comprising the nucleic acid according to any one of claims 1 to 9.

11. A pharmaceutical composition for treating or preventing an ischemic disease, comprising the nucleic acid according to claim 8 or 9.

12. The pharmaceutical composition according to claim 11, wherein the ischemic disease is chronic arterial occlusion, chronic obstructive pulmonary disease, interstitial pneumonia, acute lung disorder, ophthalmologic disease, optic nerve injury disease, or intractable skin ulcer.

13. A method for producing a nucleic acid comprising a protein coding region, comprising: (a) determining a nucleotide sequence of the protein coding region in accordance with the following guidelines (a1) to (a4): (a1) codon-optimizing the protein coding region for a cell into which the nucleic acid is to be introduced; (a2) selecting codons so as to reduce the total ratio of uracil residues and adenine residues in the protein coding region; (a3) ​​when options for the first base of a codon encoding the same amino acid include uracil and adenine, selecting a codon whose first base is adenine; and (a4) when options for the third base of a codon encoding the same amino acid include guanine and cytosine, selecting a codon whose third base is guanine or cytosine and which is more frequently used in the cell; and (b) producing a nucleic acid comprising a protein coding region of the nucleotide sequence determined in step (a).

14. The method according to claim 13, wherein in step (b), a modified uracil nucleotide is used as the uracil nucleotide.

15. The method of claim 14, wherein the modified uracil nucleotide is at least one selected from the group consisting of 5-methoxyuridine-5'-triphosphate, 5-methyluridine-5'-triphosphate, pseudouridine-5'-triphosphate, N1-methylpseudouridine-5'-triphosphate, N1-methyl-2'-O-methylpseudouridine-5'-triphosphate, N1-methoxymethylpseudouridine-5'-triphosphate, N1-propylpseudouridine-5'-triphosphate, biotin-16-aminoallyluridine-5'-triphosphate, 2'-O-methyluridine-5'-triphosphate, 5-bromouridine-5'-triphosphate, 5-iodouridine-5'-triphosphate, 4-thiouridine-5'-triphosphate, and 1-thio-uridine-5'-triphosphate.

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