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

Abstract

To provide a nucleic acid having reduced cytotoxic activity and enhanced protein production, a pharmaceutical composition comprising the nucleic acid, and a method of producing the nucleic acid.SOLUTION: 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.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to nucleic acids, pharmaceutical compositions, and methods for producing nucleic acids.

Background Art

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

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] In nucleic acid drugs containing a protein-coding region, improvement in the production amount of the protein is required along with suppression of cytotoxic activity due to immunogenicity and the like.

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

Means for Solving the Problems

[0006] The present invention includes the following aspects. [1] A nucleic acid containing a modified protein-coding region, wherein the total ratio of uracil residues and adenine residues in the modified protein-coding region is decreased as compared to the protein-coding region before modification. [2] The nucleic acid according to [1], wherein at least a part of the codons contained in the protein-coding region before modification are codons encoding the same amino acid, and are replaced with codons having a lower ratio of uracil and adenine. [3] The nucleic acid according to [1] or [2], wherein at least a part of the codons among the codons contained in the protein-coding region before modification, in which the first base is uracil, are codons encoding the same amino acid, and are replaced with codons in which the first base is adenine. [4] The nucleic acid according to any one of [1] to [3], wherein at least a part of the codons contained in the protein-coding region before modification are codons encoding the same amino acid, and are replaced with codons having a higher usage frequency in the cells into which the nucleic acid is to be introduced. [5] The nucleic acid according to any one of [1] to [4], wherein at least a part of the uracil residues contained in the nucleic acid are residues derived from modified uracil nucleotides. [6] The modified uracil nucleotide is at least one selected from the group consisting of 5-methoxyuridine-5'-3 phosphate, 5-methyluridine-5'-3 phosphate, pseudouridine-5'-3 phosphate, N1-methylpseudouridine-5'-3 phosphate, N1-methyl-2'-O-methylpseudouridine-5'-3 phosphate, N1-methoxymethylpseudouridine-5'-3 phosphate, N1-propylpseudouridine-5'-3 phosphate, biotin-16-aminoallyluridine-5'-3 phosphate, 2'-O-methyluridine-5'-3 phosphate, 5-bromouridine-5'-3 phosphate, 5-iodouridine-5'-3 phosphate, 4-thiouridine-5'-3 phosphate, 1-thio-uridine-5'-3 phosphate, and the nucleic acid according to [5]. [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 ischemic diseases, comprising the nucleic acid according to [8] or [9].

[12] The pharmaceutical composition according to

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

[13] A method for producing a nucleic acid containing a protein coding region, comprising: (a) a step of determining the nucleotide sequence of the protein coding region according to the following guidelines (a1) to (a4): (a1) optimizing the codons of the protein coding region for the cells 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 the first base of the codon has options of uracil and adenine among the codons encoding the same amino acid, selecting a codon in which the first base of the codon is adenine; and (a4) when guanine and cytosine are included in the options of the third base of the codon among the codons encoding the same amino acid, selecting a codon in which the third base of the codon is guanine or cytosine and which has a higher usage frequency in the cells, and (b) a step of producing a nucleic acid containing the protein coding region of the nucleotide sequence determined in the 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 according to

[14] , wherein the modified uracil nucleotide is at least one selected from the group consisting of 5-methoxyuridine-5'-3 phosphate, 5-methyluridine-5'-3 phosphate, pseudouridine-5'-3 phosphate, N1-methylpseudouridine-5'-3 phosphate, N1-methyl-2'-O-methylpseudouridine-5'-3 phosphate, N1-methoxymethylpseudouridine-5'-3 phosphate, N1-propylpseudouridine-5'-3 phosphate, biotin-16-aminoallyluridine-5'-3 phosphate, 2'-O-methyluridine-5'-3 phosphate, 5-bromouridine-5'-3 phosphate, 5-iodouridine-5'-3 phosphate, 4-thiouridine-5'-3 phosphate, 1-thio-uridine-5'-3 phosphate.

Advantages of the Invention

[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 amount.

Brief Description of the Drawings

[0008]

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

Fig. 6

Fig. 7

Fig. 8

Fig. 9

Fig. 10

Fig. 11

Mode for Carrying Out the Invention

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

[0010] The terms "polypeptide", "peptide" and "protein" are used interchangeably with each other and refer to polymers of amino acids linked by amide bonds. A "polypeptide", "peptide" or "protein" may be a polymer of natural amino acids, a polymer of natural amino acids and non-natural amino acids (such as chemical analogs, modified derivatives, etc. of natural amino acids), or a polymer of non-natural amino acids. Unless otherwise specified, the amino acid sequences of "polypeptide", "peptide" or "protein" are described in the generally recognized one-letter code or three-letter code. Unless otherwise specified, the amino acid sequence is described from the N-terminal side to the C-terminal side.

[0011] The term "gene" refers to a polynucleotide containing at least one open reading frame (ORF) encoding a specific protein. A gene may contain both exons and introns.

[0012] The term "protein-coding region" refers to the region in a nucleic acid that is translated into a protein. More specifically, it refers to the region in mRNA that is translated into a protein and is flanked by a start codon and a stop codon, and the corresponding region in the DNA that serves as the template for the mRNA. The protein-coding region may be divided into multiple exons.

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

[0014] The term "uracil residue" refers to a residue derived from a uracil nucleotide or a modified uracil nucleotide. The term "adenine residue" refers to a residue derived from an adenine nucleotide or a modified adenine nucleotide. The term "cytosine residue" refers to a residue derived from a cytosine nucleotide or a modified cytosine nucleotide. The term "guanine residue" refers to a residue derived from a guanine nucleotide or a modified guanine nucleotide. The term "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] "Modified uracil nucleotide" refers to a nucleotide containing uracil or modified uracil as a base, wherein at least one site selected from the group consisting of a base (uracil), a sugar (ribose), and a phosphate group (triphosphate) is modified. "Modified adenine nucleotide" refers to a nucleotide containing adenine or modified adenine as a base, wherein at least one site selected from the group consisting of a base (adenine), a sugar (ribose or deoxyribose), and a phosphate group (triphosphate) is modified. "Modified cytosine nucleotide" refers to a nucleotide containing cytosine or modified cytosine as a base, wherein at least one site selected from the group consisting of a base (cytosine), a sugar (ribose or deoxyribose), and a phosphate group (triphosphate) is modified. "Modified guanine nucleotide" refers to a nucleotide containing guanine or modified guanine as a base, wherein at least one site selected from the group consisting of a base (guanine), a sugar (ribose or deoxyribose), and a phosphate group (triphosphate) is modified. "Modified thymine nucleotide" refers to a nucleotide containing thymine or modified thymine as a base, wherein at least one site selected from the group consisting of a base (thymine), a sugar (deoxyribose), and a phosphate group (triphosphate) is modified. In a nucleic acid, a residue derived from a modified uracil nucleotide has a function similar to that of a residue derived from uridine 3-phosphate. In a nucleic acid, a residue derived from a modified adenine nucleotide has a function similar to that of a residue derived from adenosine 3-phosphate or deoxyadenosine 3-phosphate. In a nucleic acid, a residue derived from a modified cytosine nucleotide has a function similar to that of a residue derived from cytidine 3-phosphate or deoxycytidine 3-phosphate. In a nucleic acid, a residue derived from a modified guanine nucleotide has a function similar to that of a residue derived from guanosine 3-phosphate or deoxyguanosine 3-phosphate. A residue derived from a modified thymine nucleotide has a function similar to that of a residue derived from deoxythymidine 3-phosphate. As the modified uracil nucleotide, modified adenine nucleotide, modified cytosine nucleotide, modified guanine nucleotide, and modified thymine nucleotide, those that are publicly known can be used without particular limitation.

[0017] "Wild type" refers to what exists in nature (naturally). For example, a wild-type nucleic acid refers to a nucleic acid that exists in nature. For example, examples of wild-type nucleic acids include nucleic acids isolated from cells or viruses of organisms that exist in nature. For example, a wild-type gene may be a gene isolated from a wild-type nucleic acid. For example, examples of wild-type proteins include proteins isolated from cells or viruses of organisms that exist in nature.

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

[0019] (Nucleic acid) A first aspect of the present invention is a nucleic acid (hereinafter also referred to as "modified nucleic acid") containing a modified protein-coding region (hereinafter also referred to as "modified protein-coding region"). In one embodiment, the modified nucleic acid has a lower ratio of the total 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 "pre-modification protein-coding region").

[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 read as thymine (T). Alternatively, the mRNA transcribed from the DNA may contain the modified protein-coding region described below.

[0021] <Modified protein-coding region> The modified nucleic acid contains at least one modified protein coding region. The nucleotide sequence of the modified protein coding region is modified from that of the pre-modification protein coding region. The pre-modification protein coding region may be the protein coding region of the wild-type nucleic acid or may be a region in which a change has been made to the protein coding region of the wild-type nucleic acid. 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 in which the wild-type protein has been modified. The protein may be a protein derived from any organism (including animals, plants, protists, bacteria, viruses, etc.) and may be any type of protein. As an example of the protein encoded by the pre-modification protein coding region, hepatocyte growth factor (HGF) can be mentioned. 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. Preferably, the modified protein coding region encodes the same amino acid sequence as the pre-modification protein coding region.

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

[0024] The modified protein coding region satisfies the following condition (i). Also, preferably, the modified protein coding region satisfies one or more of the following conditions (ii) to (iii). (i) The total ratio of uracil residues and adenine residues is decreased compared to the pre-modification protein coding region. (ii) Among the codons included in the protein coding region before modification, at least a part of the codons in which the first base is uracil are codons encoding the same amino acid and are substituted with codons in which the first base is adenine. (iii) At least a part of the codons included in the protein coding region before modification are codons encoding the same amino acid and are substituted with codons having a higher usage frequency in the cells into which the modified nucleic acid is introduced.

[0025] Condition (i): The modified protein coding region is modified so that the total ratio of uracil residues and adenine residues decreases as compared with the protein coding region before modification. The modified protein coding sequence has, for example, a decrease in the total ratio of uracil residues and adenine residues by, for example, 5% or more, 10% or more, 15% or more, or 20% or more as compared with the protein coding region before modification.

[0026] The above-described modification can be achieved, for example, by substituting at least a part of the codons included in the wild-type protein coding region with codons having a lower ratio of uracil and adenine. 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 containing either or both of uracil and adenine among the codons included in the protein coding region before modification are substituted with codons having a lower ratio of uracil and adenine.

[0027] The codon table is shown in Table 1.

[0028]

Table 1

[0029] Examples of codons with low ratios of uracil and adenine include, for example, the codons shown in Table 2. In one embodiment, at least a part of the codons included in the modified protein coding region is converted into the codons shown in Table 2, compared to the codons included in the pre-modification protein coding region. In one embodiment, among the codons included 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 use the codons shown in Table 2. Preferably, all of the codons included in the modified protein coding region use the codons shown in Table 2.

[0030]

Table 2

[0031] Condition (ii): In the modified protein coding region, at least a part of the codons among those included in the pre-modification protein coding region, where the first base is uracil, may be replaced with codons encoding the same amino acid and having an 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 among those included in the wild-type protein coding region, where the first base is uracil, are replaced with codons encoding the same amino acid and having an adenine as the first base.

[0032] Table 3 shows codons that can be used to satisfy conditions (i) and (ii). In one embodiment, at least a part of the codons included in the modified protein coding region is converted into the codons shown in Table 3, compared to the codons included in the pre-modification protein coding region. In one embodiment, among the codons included 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 use the codons shown in Table 3. Preferably, all of the codons included in the modified protein coding region use the codons shown in Table 3.

[0033]

Table 3

[0034] Condition (iii): In the modified protein coding region, at least a part of the codons included in the protein coding region before modification may be replaced with codons that encode the same amino acid and have a higher usage frequency in the cell into which the modified nucleic acid is to be introduced (hereinafter also referred to as the "target cell for introduction"). For example, when the target cell for introducing the modified nucleic acid is a human cell, in the modified protein coding region, at least a part of the codons included in the protein coding region before modification may be replaced with codons that have a higher usage frequency in humans. The codon usage frequency table in the target cell for introduction is easily obtainable, for example, from the "Codon Usage Database" (www.kazusa.or.jp / codon / ) provided by the Kazusa DNA Research Institute, a public interest incorporated foundation.

[0035] For example, among codons encoding the same amino acid, if a codon with guanine as the third base has a higher usage frequency in the target cell for introduction than a codon with cytosine as the third base, a codon with guanine as the third base may be selected. In this case, in the modified protein coding region, at least a part of the codons included in the protein coding region before modification, in which the third base is cytosine, may be replaced with codons that encode the same amino acid and have guanine as the third base. 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 included in the protein coding region before modification, in which the third base is cytosine, may be replaced with codons that encode the same amino acid and have guanine as the third base.

[0036] For example, among codons encoding the same amino acid, if the codon with cytosine as the third base has a higher usage frequency in the target cells to be introduced than the codon with guanine as the third base, the codon with cytosine as the third base may be selected. In this case, in the modified protein coding region, at least a part of the codons contained in the pre-modified protein coding region that have guanine as the third base may be replaced with codons encoding the same amino acid and having cytosine as the third base. 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 contained in the pre-modified protein coding region that have guanine as the third base may be replaced with codons encoding the same amino acid and having cytosine as the third base.

[0037] Table 4 shows codons that can be used in humans to satisfy conditions (i) to (iii). When the target cells to be introduced are human cells, at least a part of the codons contained in the modified protein coding region may be converted into the codons shown in Table 4. When the target cells to be introduced are human cells, 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 may use the codons shown in Table 4. When the target cells to be introduced are human cells, preferably, all of the codons contained in the modified protein coding region use the codons shown in Table 4.

[0038]

Table 4

[0039] In one embodiment, the modified protein coding region may be one in which the protein coding region before modification is codon-optimized for the target cell to be introduced, and the codon-optimized protein coding region is modified 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 in which the protein coding region before modification is codon-optimized for the target cell to be introduced, and at least a part of the codons included in the codon-optimized protein coding region are substituted with codons that encode the same amino acid and have a lower ratio of uracil and adenine. (b) A modified protein coding region in which the protein coding region before modification is codon-optimized for the target cell to be introduced, and at least a part of the codons among the codons included in the codon-optimized protein coding region whose first base is uracil are substituted with codons that encode the same amino acid and whose first base is adenine. In the modified protein coding region of (a) or (b), further, at least a part of the codons whose third base is guanine or cytosine are substituted with codons that encode the same amino acid and whose third base is guanine or cytosine and are more frequently used in the cell.

[0041] In the above (a) to (c), "at least a part" includes 60% or more of the entire corresponding codons, and may be 70% or more, 80% or more, 90% or more, 95% or more, or 100% of the entire corresponding codons.

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

[0043] As a specific example of the nucleotide sequence of the modified protein coding region, the nucleotide sequence of the modified protein coding region encoding human HGF is shown in SEQ ID NO: 1. In SEQ ID NO: 1, "T" indicates a uracil residue in RNA and a thymine residue in DNA. "A" indicates an adenine residue, "C" indicates a cytosine residue, and "G" indicates 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, for example, include any of the following amino acid sequences (i) to (iii). (i) An amino acid sequence containing the amino acid sequence set forth in SEQ ID NO: 2. (ii) An amino acid sequence in which one or several amino acids are mutated in the sequence set forth 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 the above (i), the amino acid sequence may contain an amino acid sequence other than at either or both of the N-terminus and C-terminus of the amino acid sequence set forth in SEQ ID NO: 2. 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 the above (ii), "several" includes 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. "Mutation" may be any of deletion, substitution, addition, and insertion, or a combination thereof. In the above (iii), 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. The amino acid sequences of (i) to (iii) above are preferably such that the protein consisting of the amino acid sequences is functional HGF. "Functional HGF" refers to a protein that functions as HGF. The functional HGF preferably has an HGF activity (such as hepatocyte growth promoting activity) equal to or higher than that of the 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 region> In addition to the modified protein coding region, the modified nucleic acid may include other regions. For example, when the modified nucleic acid is mRNA, examples of 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, examples of other regions include a promoter and a terminator.

[0047] Cap structure: When the modified nucleic acid is mRNA, it may contain a 5' Cap at the 5' end. Examples of the 5' Cap include structures such as a Cap0 structure, a Cap1 structure, and a Cap2 structure. The cap structure is usually a 7-methylguanosine ribonucleotide, which is linked via a 5'-3 phosphate to the 5' position of the first nucleotide of the mRNA 5'-3' strand, i.e., the first cap-proximal nucleotide. In the Cap0 structure, the riboses of the first and second cap-proximal nucleotides of the mRNA both contain a 2'-hydroxyl. In the Cap1 structure, the ribose of the first cap-proximal nucleotide of the mRNA contains a 2'-methoxy, and the ribose of the second nucleotide contains a 2'-hydroxyl (see Figure 1). In the Cap2 structure, the riboses of the first and second cap-proximal nucleotides of the mRNA both contain a 2'-methoxy. The modified nucleic acid preferably contains a 5' Cap, and more preferably contains a Cap1 structure as the 5' Cap.

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

[0049] 5’UTR and 3’UTR: When the modified nucleic acid is mRNA, the modified nucleic acid may contain either or both of the 5’UTR and the 3’UTR. The 5’UTR and the 3’UTR may be those of the mRNA from which the pre-modification protein coding region is derived, or 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 at least a part thereof may be modified. The nucleotide sequence of the 3’UTR may have 100% sequence identity with the nucleotide sequence of the wild-type 3’UTR, or at least a part thereof may be modified. Examples of the mRNA from which the 5’UTR and the 3’UTR are derived include, in addition to the mRNA from which the pre-modification protein coding region is derived, for example, 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), epidermal growth factor receptor (EGFR), etc., but are not limited thereto.

[0050] The modified nucleic acid may contain a Kozak sequence. The Kozak sequence can affect translation initiation and the total production amount of the protein translated from the mRNA. The Kozak sequence contains a methionine codon that can function as a start codon. The minimal Kozak sequence is NNNRUGN (N is any nucleotide residue, and R is a purine residue (A or G)). In the above 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 mRNA, the modified nucleic acid may contain a poly(A) tail at the 3'-end. The poly(A) tail may contain at least 8 consecutive adenine nucleotide sequences, but may also contain one or more non-adenine nucleotide residues (e.g., G, C, U). Examples of the length of the poly(A) tail include 10 - 500 nucleotides, 30 - 300 nucleotides, and 60 - 250 nucleotides.

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

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

[0054] It is preferable that at least a part of the uracil residues of the modified nucleic acid is a residue derived from a modified uracil nucleotide. By using the modified uracil nucleotide, 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 a modified uracil nucleotide. Preferably, all of the uracil residues contained in the modified nucleic acid are residues derived from a modified uracil nucleotide. The modified uracil nucleotides are not particularly limited, and known ones can be used. Specific examples of the modified uracil nucleotides include 5-methoxyuridine-5'-3 phosphate, 5-methyluridine-5'-3 phosphate, pseudouridine-5'-3 phosphate, N1-methylpseudouridine-5'-3 phosphate, N1-methyl-2'-O-methylpseudouridine-5'-3 phosphate, N1-methoxymethylpseudouridine-5'-3 phosphate, N1-propylpseudouridine-5'-3 phosphate, biotin-16-aminoallyluridine-5'-3 phosphate, 2'-O-methyluridine-5'-3 phosphate, 5-bromouridine-5'-3 phosphate, 5-iodouridine-5'-3 phosphate, 4-thiouridine-5'-3 phosphate, and 1-thio-uridine-5'-3 phosphate, but are not limited thereto. In one embodiment, the modified uracil nucleotide is one in which the base (uracil) is modified (modified uracil). In one embodiment, the modified uracil nucleotide can be selected from the group consisting of 5-methoxyuridine-5'-3 phosphate, 5-methyluridine-5'-3 phosphate, pseudouridine-5'-3 phosphate, N1-methylpseudouridine-5'-3 phosphate, N1-methyl-2'-O-methylpseudouridine-5'-3 phosphate, N1-methoxymethylpseudouridine-5'-3 phosphate, N1-propylpseudouridine-5'-3 phosphate, biotin-16-aminoallyluridine-5'-3 phosphate, 5-bromouridine-5'-3 phosphate, 5-iodouridine-5'-3 phosphate, and 4-thiouridine-5'-3 phosphate. In one embodiment, the modified uracil nucleotide is 5-methoxyuridine-5'-3 phosphate. The modified uracil nucleotides may be used alone or in combination of two or more.

[0055] By having the above characteristics, the modified nucleic acid has reduced cytotoxic activity and improved protein production amount when introduced into cells as compared with the nucleic acid before modification. Therefore, it can be suitably used as an agent for producing a desired protein in target cells.

[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 described above.

[0057] In one embodiment, the pharmaceutical composition may contain a pharmaceutically acceptable carrier. A "pharmaceutically acceptable carrier" means a carrier that does not inhibit the biological activity of the active ingredient and does not exhibit substantial toxicity to the administration subject. "Not exhibiting substantial toxicity" means that the component does not show toxicity to the administration subject at the dosage 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 does not exhibit substantial toxicity to the administration subject. Pharmaceutically acceptable carriers typically include all known pharmaceutically acceptable components that are considered inactive ingredients. Pharmaceutically acceptable carriers are not particularly limited, and examples include solvents, diluents, vehicles, excipients, flow promoters, binders, granulating agents, dispersants, suspending agents, wetting agents, lubricants, disintegrants, solubilizers, stabilizers, emulsifiers, fillers, etc. A pharmaceutically acceptable carrier may be used alone or in combination of two or more. Specific examples of pharmaceutically acceptable carriers include excipients such as sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate; binders such as cellulose, methylcellulose, hydroxypropylcellulose, gelatin, gum arabic, polyethylene glycol, sucrose, starch; disintegrants such as starch, carboxymethylcellulose, hydroxypropyl starch, sodium starch glycolate, sodium hydrogen carbonate, calcium phosphate, calcium citrate; lubricants such as magnesium stearate, aerosil, talc, sodium lauryl sulfate; fragrances such as citric acid, menthol, glycyrrhizin ammonium salt, glycine, orange powder; preservatives such as sodium benzoate, sodium bisulfite, methyl paraben, propyl paraben; stabilizers such as citric acid, sodium citrate, acetic acid; suspending agents such as methylcellulose, polyvinylpyrrolidone, aluminum stearate; dispersants such as surfactants; diluents such as water, physiological saline; base waxes such as cocoa butter, polyethylene glycol, white mineral oil, but are not limited thereto.

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

[0059] The pharmaceutical composition may contain a nucleic acid transfection reagent in order to promote the introduction of the modified nucleic acid into cells. Examples of the nucleic acid transfection reagent include, but are not limited to, cationic lipids, cationic liposomes, cationic polymers, etc. Specific examples of the reagent for nucleic acid transfection include Lipofectin (trade name, Invitrogen), Lipofectamine (trade name, Invitrogen), Transfectam (trade name, Promega), DOTAP (trade name, Roche Applied Science), dioctadecylamidoglycyl spermine (DOGS), L-dioleoyl phosphatidylethanolamine (DOPE), dimethyldioctadecylammonium bromide (DDAB), N,N-di-n-hexadecyl-N,N-dihydroxyethylammonium bromide (DHDEAB), N-n-hexadecyl-N,N-dihydroxyethylammonium bromide (HDEAB), polybrene, poly(ethyleneimine) (PEI), etc., but are not limited to these.

[0060] The dosage form of the pharmaceutical composition is not particularly limited and can be a dosage form commonly used as a pharmaceutical preparation. The pharmaceutical composition of the present embodiment may be an oral preparation or a parenteral preparation, but a parenteral preparation is preferred. Examples of oral preparations include tablets, coated tablets, pills, powders, granules, capsules, syrups, fine granules, liquids, drops, emulsions, etc. Examples of parenteral preparations include injections, suppositories, nasal drops, enteral preparations, inhalants, etc. The pharmaceutical composition of these dosage forms can be formulated according to a standard method (for example, the method described in the Japanese Pharmacopoeia). The pharmaceutical composition is preferably a parenteral preparation, and more preferably an injection.

[0061] The administration route of the pharmaceutical composition of the present embodiment is not particularly limited and can be administered orally or parenterally, but parenteral administration is preferred. Examples of the administration route of parenteral administration include intravenous administration, intranasal administration, subcutaneous administration, intradermal administration, intramuscular administration, intraperitoneal administration, enteral administration, etc. The administration route is preferably subcutaneous administration, intradermal administration, or intramuscular administration.

[0062] The pharmaceutical composition can administer a therapeutically effective amount of the modified nucleic acid. The "therapeutically effective amount" means the amount of a drug effective for the treatment or prevention of the target disease. For example, the therapeutically effective amount of the modified nucleic acid can be an amount effective for the production of an effective amount of the protein encoded by the modified nucleic acid in the cell. The therapeutically effective amount may be appropriately determined according to the patient's symptoms, weight, age, gender, etc., as well as the dosage form and administration method of the pharmaceutical composition. For example, the pharmaceutical composition can have a single dose of the modified nucleic acid of 0.01 to 1000 mg per 1 kg of the body weight of the administration subject. The above dosage may be 0.05 to 500 μg, may be 0.1 to 200 μg, may be 0.5 to 100 μg, or may be 1 to 50 μg.

[0063] The pharmaceutical composition may be administered once or repeatedly. In the case of repeated administration, the dosing interval may be appropriately determined according to the patient's symptoms, body weight, age, gender, etc., as well as the type of modified nucleic acid, dosage form of the pharmaceutical composition, and administration method, etc. The dosing 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 pharmaceutical composition can select the applicable disease according to 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 for the treatment or prevention of the 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, for example, chronic arterial occlusion, chronic obstructive pulmonary disease, interstitial pneumonia, acute lung injury, ophthalmic diseases, optic nerve injury diseases, and intractable skin ulcers, etc.

[0065] The administration target of the pharmaceutical composition is not particularly limited. The administration target of the pharmaceutical composition is preferably a mammal, which may be a human or a non-human mammal. Examples of non-human mammals include, for example, primates (such as monkeys, chimpanzees, gorillas, etc.), rodents (such as mice, hamsters, rats, etc.), rabbits, dogs, cats, cows, goats, sheep, horses, etc., but are not limited thereto. The administration target is preferably the 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 administered to humans.

[0066] (Method for producing nucleic acid) The third aspect of the present invention is a method for producing a nucleic acid containing a protein coding region. The method of this embodiment includes the following steps (a) and (b). (a) A step of determining the nucleotide sequence of the protein coding region according to the following guidelines: (a1) Optimize the protein coding region for the cells into which the nucleic acid is to be introduced; (a2) Select 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 the codon are uracil and adenine among the codons encoding the same amino acid, select the codon in which the first base of the codon is adenine, and (b) A step of producing a nucleic acid containing a protein coding region of the nucleotide sequence determined in the step (a).

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

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

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

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

[0071] When the target cell for introduction is a human, the codons listed in Table 4 can be selected as the codons according to the guidelines of (a1) to (a3).

[0072] <Step (b)> In Step (b), a nucleic acid containing a protein-coding region consisting of the nucleotide sequence determined in Step (a) is produced. The method for producing the nucleic acid is not particularly limited, and known methods can be used. In addition to the protein-coding region, the nucleic acid may contain other regions. When the nucleic acid is mRNA, it may contain at least one 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 structure of mRNA are the same as those described in the above item “(Nucleic acid)”.

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

[0074] When producing RNA, modified nucleotides may be used as the nucleotides for the transcription reaction. In one embodiment, in the transcription reaction, it is preferable to use a modified uracil nucleotide instead of a uracil nucleotide. Examples of the modified uracil nucleotide include those exemplified above. In one embodiment, a modified uracil nucleotide in which the base (uracil) is modified (modified uracil) can be used. In one embodiment, 5-methoxyuridine-5′-3 phosphate can be used as the modified uracil nucleotide. The modified uracil nucleotides may be used alone or in combination of two or more.

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

[0076] After synthesizing the nucleic acid by step (b), the nucleic acid may be purified. When the nucleic acid is RNA, purification may be performed by DNase treatment, oligo dT purification etc.

[0077] By the method of this embodiment, the nucleic acid of the first aspect can be produced.

[0078] This disclosure includes the following aspects. [1] A nucleic acid comprising a modified protein coding region, wherein the total ratio of uracil residues and adenine residues in the modified protein coding region is decreased as compared with the protein coding region before modification. [2] The nucleic acid according to [1], wherein at least a part of the codons included in the protein coding region before modification are codons encoding the same amino acid and are substituted with codons having a lower ratio of uracil and adenine. [3] The nucleic acid according to [1] or [2], wherein at least a part of the codons included in the protein coding region before modification, in which the first base is uracil, are codons encoding the same amino acid and are substituted with codons in which the first base is adenine. [4] The modified nucleic acid according to any one of [1] to [3], wherein at least a part of the codons included in the protein coding region before modification, in which the third base is guanine, are codons encoding the same amino acid and are substituted with codons in which the third base is cytosine. [5] Among the codons included in the protein coding region before the modification, at least a part of the codons in which the third base is cytosine are replaced with codons that encode the same amino acid and in which the third base is guanine, the nucleic acid according to any one of [1] to [4]. [6] At least a part of the codons included in the protein coding region before the modification are replaced with codons that encode the same amino acid and are codons with a higher frequency of use in the cells into which the nucleic acid is to be introduced, the nucleic acid according to any one of [1] to [5]. [7] The modified protein coding region is codon-optimized for the cells into which the nucleic acid is to be introduced, and at least a part of the codons included in the codon-optimized protein coding region are replaced with codons that encode the same amino acid and have a low proportion of uracil and adenine, the nucleic acid according to [1]. [8] Among the codons included in the codon-optimized protein coding region, at least a part of the codons in which the first base is uracil are replaced with codons that encode the same amino acid and in which the first base is adenine, the nucleic acid according to [7]. [9] Among the codons included in the codon-optimized protein coding region, at least a part of the codons in which the third base is guanine or cytosine are replaced with codons that encode the same amino acid and are codons with a higher frequency of use in the cells among the codons in which the third base is guanine or cytosine, the nucleic acid according to [7] or [8].

[10] At least a part of the uracil residues included in the nucleic acid are residues derived from modified uracil nucleotides, the nucleic acid according to any one of [1] to [9].

[11] All of the uracil residues included in the nucleic acid are residues derived from modified uracil nucleotides, the nucleic acid according to

[10] .

[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'-3 phosphate, 5-methyluridine-5'-3 phosphate, pseudouridine-5'-3 phosphate, N1-methylpseudouridine-5'-3 phosphate, N1-methyl-2'-O-methylpseudouridine-5'-3 phosphate, N1-methoxymethylpseudouridine-5'-3 phosphate, N1-propylpseudouridine-5'-3 phosphate, biotin-16-aminoallyluridine-5'-3 phosphate, 2'-O-methyluridine-5'-3 phosphate, 5-bromouridine-5'-3 phosphate, 5-iodouridine-5'-3 phosphate, 4-thiouridine-5'-3 phosphate, and 1-thio-uridine-5'-3 phosphate.

[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 is a Cap1 structure.

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

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

[17] The nucleic acid according to

[16] , wherein the hepatocyte growth factor is a 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 in which one or several amino acids are mutated in the sequence set forth in SEQ ID NO: 2; and (iii) An amino acid sequence having 80% or more sequence identity with 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] . A pharmaceutical composition for treating or preventing an ischemic disease, comprising the nucleic acid according to any one of

[21] to

[19] .

[22] The pharmaceutical composition according to

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

[23] A method for producing a nucleic acid containing a protein coding region, comprising: (a) A step of determining the nucleotide sequence of the protein coding region according to the following guidelines: (a1) Optimize the codons of the protein coding region for the cells into which the nucleic acid is to be introduced. (a2) Select codons so as to reduce the total proportion of uracil residues and adenine residues in the protein coding region. (a3) When the first base option of the codon is uracil and adenine among codons encoding the same amino acid, select the codon whose first base is adenine; and (a4) When the third base option of the codon includes guanine and cytosine among codons encoding the same amino acid, select the codon whose third base is guanine or cytosine and which is more frequently used in the cells, and (b) A step of producing a nucleic acid containing the protein coding region of the nucleotide sequence determined in the step (a). A method comprising the above steps.

[24] The method according to

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

[25] The method according to

[24] , wherein the modified uracil nucleotide is at least one selected from the group consisting of 5-methoxyuridine-5'-3 phosphate, 5-methyluridine-5'-3 phosphate, pseudouridine-5'-3 phosphate, N1-methylpseudouridine-5'-3 phosphate, N1-methyl-2'-O-methylpseudouridine-5'-3 phosphate, N1-methoxymethylpseudouridine-5'-3 phosphate, N1-propylpseudouridine-5'-3 phosphate, biotin-16-aminoallyluridine-5'-3 phosphate, 2'-O-methyluridine-5'-3 phosphate, 5-bromouridine-5'-3 phosphate, 5-iodouridine-5'-3 phosphate, 4-thiouridine-5'-3 phosphate, 1-thio-uridine-5'-3 phosphate.

Example

[0079] Hereinafter, the present invention will be described by way of examples, but the present invention is not limited to the following examples.

[0080] (Design of the nucleotide sequence of the human HGF coding region) Based on the following guidelines, nucleotide sequence modifications were made from the nucleotide sequence (SEQ ID NO: 3) of the cDNA of human hepatocyte growth factor (HGF). (a) The amino acid sequence of human HGF (SEQ ID NO: 2) is not changed. (b) The codons in the HGF coding region were changed to the codons with the highest usage frequency in humans (codon optimization for humans). (c) Codons containing uracil and adenine were changed, as much as possible, to codons encoding the same amino acid and having a low proportion of uracil and adenine. (d) When there are codons with uracil as the first base and codons with adenine as the first base among codons encoding the same amino acid, the codons with adenine as the first base are selected. (e) When there are codons with guanine as the third base and codons with cytosine as the third base among codons encoding the same amino acid, the codon with the higher usage frequency in humans is selected.

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

[0082] (Synthesis of mRNA) Using the HGF coding region with the nucleotide sequence determined as described above as an ORF, TriLink BioTechnologies was commissioned to synthesize mRNA. For the synthesis of mRNA, 5-methoxyuridine triphosphate was used as the uracil nucleotide. The synthesis of mRNA was performed by the short oligo priming method. For capping of the mRNA, CleanCap (registered trademark) AG (TriLink BioTechnologies) was used to synthesize mRNA (see Figure 2) with a Cap1 structure (see Figure 1) added. Purification of the mRNA 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 transported from the commissioned party by dry ice transport.

[0083] (Evaluation of mRNA) As a control for the above-synthesized mRNA (hereinafter referred to as "AG-mRNA"), BioCap TM HGF mRNA (hereinafter referred to as "BioCap", phaRNA), Human HGF mRNA (hereinafter referred to as "BioGene", Creative BioGene), collagen which is plasmid DNA (hereinafter referred to as "HGF Plasmid", Anges Inc.), and Naked HGF plasmid (hereinafter referred to as "Naked HGF Plasmid") were used for a comparative test.

[0084] When introducing nucleic acids into cultured cells, when the introduced nucleic acid is mRNA, Lipofectamine Messenger MAX from Thermo Fisher Scientific was used and performed according to the attached protocol. When the introduced nucleic acid is plasmid DNA, Lipofectamine LTX from Thermo Fisher Scientific was used and performed according to the attached protocol. Naked plasmid in Figures 3 and 5 indicates the case where plasmid DNA was introduced as it is without using an introducing reagent.

[0085] HGF production amount: IMR-90 (10 5 cells / mL) after AG-mRNA introduction was cultured at 37 °C for 48 hours using Minimum Essential Media from Thermo Fisher Scientific. After culturing, the culture solution was centrifuged and the supernatant was collected. The amount of HGF in the supernatant was measured using the HGF Human ELISA kit Quantikine from R&D Systems according to the attached protocol.

[0086] The results are shown in Figure 3. For all mRNAs, much higher HGF production was confirmed compared to HGF Plasmid and Naked HGF plasmid. Among the mRNAs, the highest HGF production was confirmed for AG-mRNA. The maximum HGF production amount of AG-mRNA (about 700 ng / mL) was confirmed at an introduction amount of 2 μg. For BioCap and BioGene, the maximum HGF production amount was confirmed at an introduction amount of 0.5 μg, both being about 400 ng / mL.

[0087] Figure 4 shows the dose-response regression line created from the introduction amount of AG-mRNA and the HGF production amount. From the dose-response regression line, the introduction amount of AG-mRNA having the same activity as 1 μg of HGF plasmid was 0.0130 μg. From this, it was confirmed that AG-mRNA has approximately 80 times the activity of HGF plasmid.

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

[0089] The results are shown in Figure 5. For AG-mRNA, compared with BioGene and BioCap, the cell viability was high and the cytotoxic activity was low. For AG-mRNA and HGF Plasmid, it was considered that the cell viability was high and the cytotoxic activity was low.

[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 cells / mL) was cultured at 37 °C using Minimum Essential Media from Thermo Fisher Scientific. At 48 hours, 72 hours, 96 hours, and 120 hours after the start of culturing, samples of the culture medium were taken. The sampled culture medium was centrifuged to obtain the culture supernatant and cells respectively. For each of the obtained culture supernatant and cells, the HGF concentration was measured using the HGF Human ELISA kit Quantikine from R&D Systems according to the attached protocol (see Figure 6).

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

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

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

[0094] When using AG-mRNA, the intracellular HGF concentration was higher compared to when using other mRNAs and HGF Plasmid. In the cells using AG-mRNA, the HGF concentration tended to gradually decrease until 120 hours. In the cells using AG-mRNA, a high HGF concentration was maintained even at 120 hours (Figure 8).

[0095] HGF production in the 48-hour exposure test: In the presence of 1 μg or 0.5 μg of AG-mRNA, IMR-90 (10 5 cells / mL) was cultured at 37 °C for 48 hours using Minimum Essential Media from Thermo Fisher Scientific. Then, the same amount of the same medium without AG-mRNA was added to the removed medium, and the culture was continued. Sampling of the culture solution was performed at 48 hours, 72 hours, 96 hours, and 120 hours after the start of the culture. The sampled culture solution was used with the HGF Human ELISA kit Quantikine from R&D Systems, and the concentration of HGF was measured according to the attached protocol (see Figure 9).

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

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

[0098] When using AG-mRNA, the amount of HGF released from cells to the culture supernatant was higher compared to when using other mRNAs and HGF Plasmid (Figs. 10, 11). When using AG-mRNA, although the amount of HGF released from cells to the culture supernatant decreased from 48 hours to 120 hours, HGF release continued even at the 120-hour time point (Figs. 10, 11). The cumulative HGF release amount (48 → 120 hours) when using AG-mRNA was approximately 10 times that of the HGF Plasmid at an introduced amount of 1 μg and approximately 7 times at an introduced amount of 0.5 μg. At 72 hours (3 days) after the introduction of AG-mRNA, the maximum amount of HGF per day was released extracellularly. The HGF release amount gradually decreased thereafter, but HGF was still released extracellularly even at the 120-hour (5 days) time point.

[0099] From the above results, it was confirmed that AG-mRNA has low cytotoxic activity and high HGF-producing ability compared to other HGF nucleic acids. It was confirmed that AG-mRNA shows a concentration-dependent increase in HGF production up to an introduced amount of 2 μg. AG-mRNA showed the maximum HGF-producing ability (700 ng / mL) at an introduced amount of 2 μg among the tested introduced amounts. The minimum introduced amount at which HGF production was confirmed was 0.0130 μg (Figs. 3, 4). No significant decrease in cell viability was confirmed for AG-mRNA up to an introduced amount of 2 μg. On the other hand, for the control mRNAs of BioCap and Biogene, cell viability began to decrease from an introduced amount of 0.5 μg, and further decreased at an introduced amount of 1 μg (Fig. 5).

[0100] It was confirmed that the extracellular release of HGF produced intracellularly by AG-mRNA introduction lasted for 120 hours or more. On the other hand, with the HGF Plasmid, the duration was about 72 hours (Figs. 10, 11). It was confirmed that a certain amount of HGF was released from intracellular to extracellular until 72 hours after AG-mRNA introduction (Figs. 10, 11).

Industrial Applicability

[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 amount.

Claims

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. Nucleic acid.

2. The nucleic acid according to claim 1, wherein at least a part of the codons included in the protein coding region before modification are codons encoding the same amino acid and are replaced with codons having a lower proportion of uracil and adenine.

3. The nucleic acid according to claim 1, wherein at least a part of the codons in which the first base is uracil among the codons included in the protein coding region before modification are codons encoding the same amino acid and are replaced with codons in which the first base is adenine.

4. The nucleic acid according to claim 1, wherein at least a part of the codons included in the protein coding region before modification are codons encoding the same amino acid and are replaced with codons having a higher usage frequency in the cells into which the nucleic acid is to be introduced.

5. The nucleic acid according to claim 1, wherein at least a part of the uracil residues included in the nucleic acid are residues derived from modified uracil nucleotides.

6. The modified uracil nucleotide is at least one selected from the group consisting of 5-methoxyuridine-5'-3 phosphate, 5-methyluridine-5'-3 phosphate, pseudouridine-5'-3 phosphate, N1-methylpseudouridine-5'-3 phosphate, N1-methyl-2'-O-methylpseudouridine-5'-3 phosphate, N1-methoxymethylpseudouridine-5'-3 phosphate, N1-propylpseudouridine-5'-3 phosphate, biotin-16-aminoallyluridine-5'-3 phosphate, 2'-O-methyluridine-5'-3 phosphate, 5-bromouridine-5'-3 phosphate, 5-iodouridine-5'-3 phosphate, 4-thiouridine-5'-3 phosphate, 1-thio-uridine-5'-3 phosphate, and the nucleic acid according to claim 5.

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

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

9. The nucleic acid according to 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 injury, an ophthalmic disease, an optic nerve injury disease, or a refractory skin ulcer.

13. A method for producing a nucleic acid containing a protein coding region, comprising: (a) determining the nucleotide sequence of the protein coding region according to the following guidelines (a1) to (a4): (a1) optimizing the codons of the protein coding region for the cells 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 the first base option of a codon encoding the same amino acid is uracil and adenine, selecting a codon in which the first base of the codon is adenine; and (a4) when the third base option of a codon encoding the same amino acid includes guanine and cytosine, selecting a codon in which the third base of the codon is guanine or cytosine and which is used more frequently in the cells, and (b) producing a nucleic acid containing the protein coding region of the nucleotide sequence determined in the step (a), the method comprising.

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

15. The method according to claim 14, wherein the modified uracil nucleotide is at least one selected from the group consisting of 5-methoxyuridine-5'-3 phosphate, 5-methyluridine-5'-3 phosphate, pseudouridine-5'-3 phosphate, N1-methylpseudouridine-5'-3 phosphate, N1-methyl-2'-O-methylpseudouridine-5'-3 phosphate, N1-methoxymethylpseudouridine-5'-3 phosphate, N1-propylpseudouridine-5'-3 phosphate, biotin-16-aminoallyluridine-5'-3 phosphate, 2'-O-methyluridine-5'-3 phosphate, 5-bromouridine-5'-3 phosphate, 5-iodouridine-5'-3 phosphate, 4-thiouridine-5'-3 phosphate, 1-thio-uridine-5'-3 phosphate.

Images