siRNA FOR SUPPRESSING EXPRESSION OF TRANSFERRIN RECEPTOR-2

Abstract

For example, the present invention provides a new siRNA that has a gene expression suppressing action and / or an RNA interference action with respect to mRNA encoding TfR2. The present invention pertains to: an oligonucleotide that has a knockdown action with respect to mRNA of transferrin receptor-2 and that includes an antisense strand region including a target corresponding sequence that is substantially complementary to a target sequence in mRNA encoding TfR2, and a sense strand region including a nucleotide sequence substantially complementary to the target corresponding sequence in the antisense strand region; a pharmaceutically acceptable salt of the oligonucleotide; and the like.

Description

siRNA that suppresses the expression of transferrin receptor 2

[0001] The present invention relates to siRNAs having an RNA interference effect and / or a gene expression-suppressing effect on mRNA encoding transferrin receptor 2 (TfR2), uses of the siRNAs, methods for suppressing TfR2 gene expression using the siRNAs, and pharmaceuticals containing the siRNAs.

[0002] siRNA against mRNA encoding the TfR2 protein can suppress TfR2 mRNA expression and inhibit the production of hepcidin, which plays a central role in iron metabolism. As a result, it is expected that activating iron metabolism in the body will promote iron bioavailability by erythroid progenitor cells and treat anemia. Therefore, studies are being conducted to use siRNA to suppress TfR2 mRNA expression by targeting the inhibition of hepcidin production.

[0003] Hepcidin is a peptide hormone consisting of 25 amino acids that is mainly produced in the liver and secreted into the blood, and plays a central role in iron metabolism in the body. Hepcidin binds to the iron transporter ferroportin (SLC40A1) and promotes the proteolysis of ferroportin, thereby suppressing iron absorption from the intestinal tract and iron release from macrophages, thereby negatively regulating iron metabolism. It has been reported that elevated blood hepcidin levels inhibit iron metabolism, thereby limiting iron bioavailability in erythroid progenitor cells, and contributing to various anemia diseases (Non-Patent Document 1).

[0004] Examples of anemic diseases in which elevated blood hepcidin levels are observed include anemia associated with chronic kidney disease, anemia associated with cancer, anemia induced by chemotherapy, iron refractory iron deficiency anemia (IRIDA), anemia associated with chronic inflammation (ACD) such as rheumatoid arthritis and Castleman's disease, Diamond-Blackfan anemia, aplastic anemia, β-thalassemia, sickle cell disease, paroxysmal nocturnal hemoglobinuria, autoimmune hemolytic anemia, anemia associated with myelodysplastic syndrome, chronic myelomonocytic leukemia, and anemia associated with myelofibrosis. Furthermore, patients with myelofibrosis may exhibit elevated blood hepcidin levels in correlation with the iron load associated with red blood cell transfusions and the chronic increase in inflammatory cytokines (Non-Patent Document 2). However, in anemia caused primarily by a lack of red blood cell production capacity due to hematopoietic dysfunction in the bone marrow, such as in myelofibrosis, the contribution of iron utilization disorders associated with elevated blood hepcidin levels to the pathology is thought to be low, and no therapeutic effect of inhibiting hepcidin production has been reported to date.Currently, drugs such as ESAs (erythropoiesis stimulating agents) are sometimes used as therapeutic agents for anemia associated with bone marrow dysfunction, such as myelofibrosis, but their therapeutic effect is limited.

[0005] Patent Document 1 discloses that administration of TfR2 siRNA AD-52590 to monkeys resulted in a decrease in TfR2 mRNA expression in the liver, a decrease in hepcidin mRNA expression, a decrease in hepcidin concentration in the blood, and an increase in blood iron concentration. It also discloses that administration of AD-47882 to rats exhibiting inflammatory anemia resulted in a decrease in TfR2 mRNA expression in the liver, a decrease in hepcidin concentration in the blood, an increase in blood iron concentration, and an increase in hemoglobin levels. However, Patent Document 1 only shows the results of administering siRNA encapsulated in lipid nanoparticles (LNPs), and the administration route is limited to intravenous administration. Furthermore, the therapeutic effect of TfR2 siRNA disclosed in Patent Document 1 is limited to the therapeutic effect on anemia associated with inflammation, and does not disclose the therapeutic effect on other anemias, such as anemia associated with bone marrow dysfunction.

[0006] One method for inhibiting the expression of a target gene in a cell, tissue, or individual involves introducing double-stranded RNA into the cell, tissue, or individual. The introduction of double-stranded RNA degrades mRNA with a homologous sequence, inhibiting the expression of the target gene. This effect is called "RNA interference" or "RNAi." Small interfering RNA (siRNA), a double-stranded RNA consisting of 21 nucleotides in each of the sense and antisense strands and a two-nucleotide overhang at the 3' end, has been reported to have RNA interference effects in cultured vertebrate cells (see, for example, Non-Patent Document 3).

[0007] Although siRNA is useful for identifying gene functions, screening cell lines suitable for producing useful substances, and regulating genes involved in diseases, it has the drawbacks of being easily degraded by RNases, making it difficult to maintain its activity in the body, and requiring high synthesis costs. Therefore, various chemical modifications have been attempted to develop oligonucleotides that are highly stable against RNases, can be produced inexpensively, and retain RNAi activity.

[0008] Phosphorothioate (PS) bonds, in which the non-bridging oxygen atom of the phosphate group of a phosphodiester bond in an oligonucleotide is replaced with a sulfur atom, are known to be resistant to nucleases. However, it has been reported that increasing the number of PS bonds in an oligonucleotide is undesirable because it leads to thermodynamic instability of the double-stranded RNA and binding to nonspecific proteins (see, for example, Non-Patent Document 4).

[0009] Furthermore, siRNAs employing sugar-modified nucleosides such as DNA, 2'-O-methylnucleosides, 2'-deoxy-2'-fluoronucleosides, and 2'-O,4'-C bridged nucleosides as nucleosides are disclosed in, for example, Patent Documents 2, 3, and 4.

[0010] Attempts have also been made to obtain stable siRNA by replacing natural RNA with modified RNA. For example, numerous derivatives have been reported in which the 2'-OH group of RNA is alkylated to form 2'-O-alkylnucleosides, which prevent the siRNA from becoming a substrate for RNase. 2'-O-methyl nucleotides are naturally occurring modified nucleotides that are also found in tRNA, and have been studied since the early days of antisense research (see, for example, Non-Patent Document 5).

[0011] It has been reported that RNAi activity is weakened or completely lost when all RNA in either or both of the sense and antisense strands of siRNA is substituted with 2'-O-methyl nucleotides (see, for example, Non-Patent Documents 6, 7, 8, and 9). It has also been reported that when three consecutive 2'-O-methyl nucleotides are introduced into the sense strand, no decrease in activity is observed, but when introduced into the antisense strand, a decrease in activity is observed, and that the activity is particularly marked when introduced at the 5' end of the sense strand (see, for example, Non-Patent Document 10).

[0012] Oligonucleotides containing 2'-deoxy-2'-fluoronucleotides (2'-F or 2'-F RNA), which are artificially synthesized modified RNAs, preferentially form the same N-type conformation as ribonucleotides and have high affinity with RNA. However, oligonucleotides containing phosphodiester bonds are not nuclease resistant, so they must be made nuclease resistant by using phosphorothioate bonds (see, for example, Non-Patent Document 11).

[0013] ENA (2'-O,4'-C-ethylene-bridged nucleic acids) is a modified nucleic acid that is stable against nucleases. However, it has been reported that when ENA is introduced into the two nucleotides in the overhanging region at the 3' end of one or both of the sense and antisense strands of siRNA, RNAi activity is reduced (see, for example, Non-Patent Document 12).

[0014] There have been many reports on siRNAs that combine multiple modified nucleic acids. For example, it has been reported that by alternately introducing 2'-O-methyl nucleotides and 2'-F into the sense and antisense strands of siRNA, RNAi activity comparable to or greater than that of unmodified siRNA was obtained, and the siRNA was relatively stable in serum (see, for example, Non-Patent Document 13).

[0015] Double-stranded oligonucleotides combining DNA, 2'-O-methyl RNA, and 2'-F have been reported, and in particular, siRNAs in which the 14th position from the 5' end of the antisense strand is 2'-F have been reported (see, for example, Patent Documents 5 and 6). Double-stranded oligonucleotides combining DNA, 2'-O-methyl RNA, 2'-F RNA, and LNA (2'-O,4'-C-methylene-bridged nucleic acids) have also been reported, and in particular, the activity of siRNAs in which the 2nd or 14th position from the 5' end of the antisense strand is 2'-F, DNA, or LNA has been reported (see, for example, Patent Documents 5 and 7).

[0016] International Publication No. WO 2012 / 177921, U.S. Pat. No. 9,399,775, U.S. Pat. No. 9,796,974, U.S. Pat. No. 10,612,024, International Publication No. WO 2012 / 058210, International Publication No. WO 2013 / 074974, and U.S. Pat. No. 9,399,775, U.S. Pat. No. 9,796,974, International Publication No. WO 2016 / 028649, U.S. Pat. No. 10,612,024

[0017] Pharmaceuticals 2019, 12, 170 Am. J. Hematol. 88:312-316, 2013. Nature, 2001, Vol. 411, pp. 494-498 Antisense Nucleic Acid Drug Development, 2000, Vol. 10, pp. 117-121 Nucleic Acids Research, 1987, Vol. 15, pp. 6131-6148 EMBO Journal, 2001, Vol. 20, pp. 6877-6888 RNA, Vol. 9, 2003, p. 1034-1048 Biochemistry, 2003, Vol. 42, pp. 7967-7975 Nucleic Acids Research, 2003, Vol. 31, pp. 2705-2716 Journal of Medicinal Chemistry, 2005, Vol. 48, pp. 4247-4253 Journal of Medicinal Chemistry, 1993, Vol. 36, pp. 831-841 Antisense and Nucleic Acid Drug Development, 2002, Vol. 12, pp. 301-309 Journal of Medicinal Chemistry., 2005, Vol. 48, pp. 901-904

[0018] There is a need for TfR2 siRNAs with high pharmacological activity that can prevent, improve, and / or treat anemia, as well as TfR2 siRNAs with low toxicity to cells and excellent stability and drug delivery in the blood.

[0019] When elevated hepcidin levels are observed in anemia associated with chronic inflammation, inhibiting hepcidin production in the blood can provide a therapeutic effect. However, this is primarily due to impaired iron utilization caused by elevated hepcidin levels, rather than abnormal red blood cell production. However, anemia associated with bone marrow dysfunction such as myelofibrosis is often caused by reduced red blood cell production, so even when elevated hepcidin levels are observed, inhibiting hepcidin production is not expected to provide a therapeutic effect, and there have been no reports of such an effect. Therefore, there is a particular need for novel, effective therapeutic agents for anemia associated with bone marrow dysfunction such as myelofibrosis. Furthermore, in the inventors' research, sequence-specific cytotoxicity was confirmed for the target sequence of the TfR2 siRNA in Patent Document 1, raising safety concerns about the siRNAs of the prior art.

[0020] An object of the present invention is to provide a novel siRNA that has RNA interference activity and / or gene expression inhibitory activity (these activities are also referred to as knockdown activity) against mRNA encoding TfR2. Another object of the present invention is to provide a novel TfR2 siRNA that has improved cytotoxicity, stability, and / or drug deliverability. A further object of the present invention is to provide a novel pharmaceutical composition for treating or preventing diseases that can be treated or prevented by inhibiting the expression of TfR2 mRNA.

[0021] The present inventors conducted extensive research and found that TfR2 siRNA targeting a base sequence at a specific position in TfR2 mRNA exhibits RNA interference and / or gene expression suppression, and that the cytotoxicity observed with the target sequence of Patent Document 1 was not observed with the target sequence of the present invention. Furthermore, the present inventors found that siRNAs containing oligonucleotides appropriately combining DNA, RNA, 2'-O-methyl RNA, 2'-F RNA, LNA, ENA, and 3'-O-methyl RNA exhibit RNA interference and / or gene expression suppression and are stable in vivo. Furthermore, the present inventors found that adding a transport unit such as GalNAc to siRNA improves drug delivery. Furthermore, the present inventors found that suppressing TfR2 mRNA expression using TfR2 siRNA improves anemia symptoms not only in a mouse model of anemia associated with inflammation but also in a mouse model of anemia associated with bone marrow dysfunction, thereby completing the present invention.

[0022] That is, the present invention provides the following: [1] An oligonucleotide or a pharmaceutically acceptable salt thereof, which has a knockdown effect on transferrin receptor 2 mRNA and comprises the following antisense strand region (A) and sense strand region (B): (A) an antisense strand region consisting of a target corresponding sequence substantially complementary to a target sequence consisting of 18 to 21 consecutive bases in the region between nucleotide numbers 2845 to 2867 or nucleotide numbers 1534 to 1556 of SEQ ID NO: 1, wherein the 5'-terminal nucleoside of the target corresponding sequence is a nucleoside substantially complementary to the 3'-terminal nucleoside of the target sequence, a nucleoside having adenine, or a nucleoside having uracil, and an overhang structure of 5 bases or less may be added to the 5'-terminal and / or 3'-terminal of the target corresponding sequence; (B) a sense strand region consisting of 18 to 31 bases in length, which contains a nucleotide sequence substantially complementary to the target-corresponding sequence of the antisense strand region, and which may further comprise an overhang structure of 5 bases or less at the 5'-end and / or 3'-end of the nucleotide sequence. [2] The oligonucleotide or a pharmaceutically acceptable salt thereof according to [1], wherein the target sequence is a 19-base nucleotide sequence consisting of nucleotides 2847 to 2865 or 1536 to 1554 of SEQ ID NO: 1. [3] The oligonucleotide or a pharmaceutically acceptable salt thereof according to [1] or [2], wherein the 5'-terminal nucleoside in the target-corresponding sequence of the antisense strand region is a nucleoside having adenine or uracil. [4] The oligonucleotide or pharmaceutically acceptable salt thereof according to any one of [1] to [3], wherein the target-corresponding sequence of the antisense strand region is a nucleotide sequence that is completely complementary to a target sequence consisting of 19 bases, nucleotide numbers 2847 to 2865 or nucleotide numbers 1536 to 1554 of SEQ ID NO: 1. [5] The oligonucleotide or pharmaceutically acceptable salt thereof according to any one of [1] to [4], wherein at least one of the sugars and / or phosphodiester bonds constituting the oligonucleotide is modified.[6] The oligonucleotide or a pharmaceutically acceptable salt thereof according to [5], wherein the sugar constituting the oligonucleotide is D-ribofuranose and the sugar modification is modification of the hydroxyl group at the 2'-position of D-ribofuranose. [7] The oligonucleotide or a pharmaceutically acceptable salt thereof according to [6], wherein the sugar modification is 2'-deoxygenation, 2'-O-alkylation, 2'-O-alkoxyalkylation, 2'-halogenation, and / or 2'-O,4'-C-alkylenation of D-ribofuranose. [8] The oligonucleotide or a pharmaceutically acceptable salt thereof according to [7], wherein the 2'-O-alkylation is 2'-O-methylation, the 2'-O-alkoxyalkylation is 2'-O-methoxyethylation, the 2'-halogenation is 2'-fluorolation, and the 2'-O,4'-C-alkylenation is 2'-O,4'-C-methylenation and / or 2'-O,4'-C-ethylenation. [9] The oligonucleotide or pharmaceutically acceptable salt thereof according to any one of [5] to [8], wherein the phosphodiester bond modification is phosphorothioate.

[10] The oligonucleotide or pharmaceutically acceptable salt thereof according to any one of [5] to [9], wherein the D-ribofuranose in the 3'-terminal nucleoside of the sense strand region and / or antisense strand region is 3'-O-alkylated.

[11] The oligonucleotide or pharmaceutically acceptable salt thereof according to

[10] , wherein the 3'-O-alkylation is 3'-O-methylation.

[12] The oligonucleotide or pharmaceutically acceptable salt thereof according to any one of [1] to

[11] , wherein an overhang structure of 3 bases or less is added to the 5'-end and / or 3'-end of the sense strand region and / or antisense strand region.

[13] The oligonucleotide or pharmaceutically acceptable salt thereof according to

[12] , wherein the overhang structure is 2 bases.

[14] The oligonucleotide or pharmaceutically acceptable salt thereof according to

[13] , wherein the overhang structure is a nucleoside containing two consecutive thymines or a nucleoside containing two consecutive uracils.

[15] The oligonucleotide or pharmaceutically acceptable salt thereof according to

[14] , wherein the overhang structure is two consecutive U (M) added to the 3'-end of the antisense strand region.

[16] The oligonucleotide according to [1], or a pharmaceutically acceptable salt thereof, wherein the sense strand region consists of an oligonucleotide represented by the following formula (I), the antisense strand region consists of an oligonucleotide represented by the following formula (II), and further has the following characteristics (a) to (g): Sense strand region: 5' S. Ｏ５ -S ａ -S １９ -S １８ -S １７ -S １６ -S １５ -S １４ -S １３ -S １２ -S １１ -S １０ -S ９ -S ８ -S ７ -S ６ -S ５ -S ４ -S ３ -S ２ -S １ -S Ｏ３ 3'(I) Antisense strand region: 5' A Ｏ５ -A １ -A ２ -A ３ -A ４ -A ５ -A ６ -A ７ -A ８ -A ９ -A １０ -A １１ -A １２ -A １３ -A １４ -A １５ -A １６ -A １７ -A １８ -A １９ -A ａ -A Ｏ３ 3'(II) (a)S １ is a 3'-modified nucleoside, and S ２ ~S １９ represents one nucleoside, each independently being 2'-OMe RNA, 2'-F RNA or DNA; S ａrepresents 0 to 3 nucleosides, each nucleoside independently representing 2'-OMe RNA, 2'-F RNA, or DNA; S Ｏ３ and S Ｏ５ each independently represent 0 to 3 nucleosides, each of which independently represents 2'-OMe RNA, 2'-F RNA, DNA, or a 2'-O,4'-C-bridged modified nucleoside. The bond between each nucleoside represents a phosphodiester bond which may be chemically modified; (b) A １１ , A １２ , A １３ , A １４ and A １５ represents one nucleoside, at least one of which is DNA, RNA, 2'-O,4'-C-bridged modified nucleoside or 2'-MOE RNA, and the others are 2'-OMe RNA or 2'-F RNA; A １ ~A １０ and A １６ ~A １９ represents one nucleoside, each independently representing 2'-OMe RNA, 2'-F RNA, or DNA; A ａ represents 0 to 3 nucleosides, each nucleoside independently representing 2'-OMe RNA, 2'-F RNA, or DNA; A Ｏ３ and A Ｏ５ each independently represent 0 to 3 nucleosides, each of which independently represents 2'-OMe RNA, 2'-F RNA, DNA, or a 2'-O,4'-C-bridged modified nucleoside. The bond between each nucleoside represents a phosphodiester bond which may be chemically modified; (c) A ２ ~A ａ The nucleotide sequence between A and B consists of a nucleotide sequence substantially complementary to the target sequence, １ is a nucleoside having a base complementary to the corresponding nucleoside of the target sequence, a nucleoside having adenine, a nucleoside having thymine, or a nucleoside having uracil; A Ｏ３ and A Ｏ５(d) S １ ~S ａ and the nucleotide sequence between A １ ~A ａ The nucleotide sequences between are substantially complementary nucleotide sequences and form a double-stranded structure; (e) S Ｏ５ and A Ｏ３ If both exist, S Ｏ５ and A Ｏ３ are nucleotide sequences that are not complementary to each other; (f) S Ｏ３ and A Ｏ５ If both exist, S Ｏ３ and A Ｏ５ are nucleotide sequences that are not complementary to each other; and (g) the 5'-position of the 5'-terminal nucleoside and / or the 3'-position of the 3'-terminal nucleoside in the sense strand region and / or the 2'-position of the 3'-terminal nucleoside when the 3'-terminal nucleoside is a 3'-modified nucleoside may be chemically modified in the sense strand region and / or the antisense strand region.

[17] In formula (II), S １

[18] The oligonucleotide or a pharmaceutically acceptable salt thereof according to

[16] or

[17] , wherein the 2'-O,4'-C-bridged modified nucleoside is LNA or ENA.

[19] The oligonucleotide or a pharmaceutically acceptable salt thereof according to

[16] or

[17] , wherein in formula (II), A １１ , A １２ , A １３ , A １４ and A １５

[20] The oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of

[16] to

[18] , wherein two or more of A may be the same or different, and are DNA, RNA, a 2'-O,4'-C-bridged modified nucleoside, or a 2'-MOE RNA. １４

[21] The oligonucleotide or a pharmaceutically acceptable salt thereof according to

[19] , wherein A is an RNA or a 2'-O,4'-C-bridged modified nucleoside. １３is a 2'-O,4'-C-bridged modified nucleoside or a 2'-OMe RNA, and A １４

[22] The oligonucleotide or a pharmaceutically acceptable salt thereof according to

[20] , wherein A is RNA. １１ -A １２ -A １３ -A １４ -A １５ is one of the following: A １１ (2'-OMe RNA)-A １２ (2'-OMe RNA)-A １３ (ENA)-A １４ (RNA)-A １５ (2'-OMe RNA), A １１ (2'-F RNA)-A １２ (2'-OMe RNA)-A １３ (ENA)-A １４ (RNA)-A １５ (2'-OMe RNA), A １１ (2'-OMe RNA)-A １２ (2'-F RNA)-A １３ (ENA)-A １４ (RNA)-A １５ (2'-OMe RNA), A １１ (2'-OMe RNA)-A １２ (2'-OMe RNA)-A １３ (LNA)-A １４ (RNA)-A １５ (2'-OMe RNA), A １１ (2'-F RNA)-A １２ (2'-OMe RNA)-A １３ (LNA)-A １４ (RNA)-A １５ (2'-OMe RNA), A １１ (2'-OMe RNA)-A １２ (2'-F RNA)-A １３ (LNA)-A １４ (RNA)-A １５ (2'-OMe RNA), A １１ (2'-F RNA)-A １２ (2'-OMe RNA)-A １３ (2'-OMe RNA)-A１４ (RNA)-A １５ (2'-OMe RNA), or A １１ (2'-OMe RNA)-A １２ (2'-F RNA)-A １３ (2'-OMe RNA)-A １４ (RNA)-A １５ (2'-OMe RNA), or a pharmaceutically acceptable salt thereof.

[23] In formula (II), A １２ is DNA, and A １４

[24] The oligonucleotide or a pharmaceutically acceptable salt thereof according to

[20] , wherein A is a 2'-O,4'-C-bridged modified nucleoside. １１ -A １２ -A １３ -A １４ -A １５ is one of the following: A １１ (2'-F RNA)-A １２ (DNA)-A １３ (2'-OMe RNA)-A １４ (ENA)-A １５ (2'-OMe RNA), A １１ (2'-OMe RNA)-A １２ (DNA)-A １３ (2'-OMe RNA)-A １４ (ENA)-A １５ (2'-OMe RNA), A １１ (2'-F RNA)-A １２ (DNA)-A １３ (2'-OMe RNA)-A １４ (LNA)-A １５ (2'-OMe RNA), or A １１ (2'-OMe RNA)-A １２ (DNA)-A １３ (2'-OMe RNA)-A １４ (LNA)-A １５ (2'-OMe RNA), or a pharmaceutically acceptable salt thereof. ２ , A ６ and A １６

[26] The oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of

[16] to

[24] , wherein A is 2'-F RNA. ８ , A ９ and A １０ one or two nucleosides selected from the group consisting of: １ , A ３ ~A ５ , A ７ , A １７ ~A １９ and A ａ

[27] The oligonucleotide or a pharmaceutically acceptable salt thereof according to

[25] , wherein the nucleoside represented by the formula (II) is 2'-OMe RNA. ２ , A ６ , A ８ , A １０ and A １６ is 2'-F RNA, and A １ , A ３ ~A ５ , A ７ , A ９ , A １７ ~A １９ and A ａ

[28] The oligonucleotide or a pharmaceutically acceptable salt thereof according to

[26] , wherein the nucleoside represented by the formula (I) is 2'-OMe RNA. １１ , S １２ , S １３ and S １５

[29] The oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of

[16] to

[27] , wherein S is 2'-F RNA. ２ ~S １０ , S １４ , S １６ ~S １９ and S ａ

[30] The oligonucleotide or pharmaceutically acceptable salt thereof according to any one of

[16] to

[29] , wherein when RNA is employed in formula (II), the bond between the RNA and the nucleoside adjacent to it on the 3' side is a phosphorothioate bond.

[31] The oligonucleotide or pharmaceutically acceptable salt thereof according to any one of

[16] to

[30] , wherein each internucleoside bond in 2 to 5 nucleotides from the 5' end and 3' end of the oligonucleotide is a phosphorothioate bond.

[32] S Ｏ３ , S Ｏ５ , A Ｏ５ and A Ｏ３

[33] The oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of

[16] to

[31] , wherein the number of nucleosides is 0 to 2. Ｏ３

[34] The oligonucleotide or a pharmaceutically acceptable salt thereof according to

[32] , wherein the number of nucleosides is 0. Ｏ３ , S Ｏ５ and A Ｏ５ The number of nucleosides in A is 0, Ｏ３

[35] The oligonucleotide or pharmaceutically acceptable salt thereof according to any one of [1] to

[34] , wherein the antisense strand region and the sense strand region each form a double-stranded oligonucleotide as independent oligonucleotides.

[36] The oligonucleotide or pharmaceutically acceptable salt thereof according to any one of [1] to

[34] , wherein the 5'-terminal nucleoside of the antisense strand region and the 3'-terminal nucleoside of the sense strand region are linked by a linker structure.

[37] The oligonucleotide or pharmaceutically acceptable salt thereof according to any one of [1] to

[34] , wherein the linker structure is represented by the following formula:

[0023]

[0024] [wherein the dashed line represents a bond, the oxygen atom bonded to the phenyl group represents a phosphodiester bond or a phosphorothioate bond with the 5'-phosphate group of the 5'-terminal nucleotide of the adjacent antisense strand region, and the methylene group at the other end represents a phosphodiester bond or a phosphorothioate bond with the 3'-phosphate group (the 2'-phosphate group in the case of a nucleotide modified at the 3'-position) of the 3'-terminal nucleotide of the adjacent sense strand region.]

[38] The oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of [1] to

[37] , wherein the 5'-position of the 5'-terminal nucleoside and / or the 3'-position of the 3'-terminal nucleoside of the oligonucleotide, or the 2'-position of the 3'-terminal nucleoside when the 3'-terminal nucleoside is a 3'-modified nucleoside, is chemically modified with a GalNAc unit.

[39] The oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of [1] to

[37] , wherein the GalNAc unit is

[0025]

[0026] [In the formula, the dashed line represents a phosphodiester bond with the 5'-terminal phosphate group and / or the 3'-terminal phosphate group (the 2'-terminal phosphate group in the case of a nucleotide modified at the 3'-position) of an adjacent nucleotide.]

[40] The oligonucleotide or a pharmaceutically acceptable salt thereof according to [1], wherein the sense strand region excluding the overhang structure is represented by the following formula (I-1), and the antisense strand region excluding the overhang structure is represented by any of the following formulas (II-1) to (II-10), and the sense strand region and the antisense strand region each form a double-stranded oligonucleotide as independent oligonucleotides, and the sense strand region and the antisense strand region each independently optionally contain an overhang structure. (Formula) 5' C(M)G(M)U(M)G(M)G(F)A(M)G(F)U(F)U(F)U(M)C(M)A(M)A(M)U(M)A(M)U(M)C(M)A(M)A(3M) 3' (I-1) 5' U(M)U(F)G(M)A(M)U(M)A(F)U(M)U(F)G(F)A(M)A(M)A(D)C(M)T(E)C(M)C(F)A(M)C(M)G(M) 3' (II-1) 5' U(M)U(F)G(M)A(F)U(M)A(F)U(M)U(M)G(F)A(M)A(M)A(D)C(M)T(E)C(M)C(F)A(M)C(M)G(M) 3' (II-2) 5' U(M)U(F)G(M)A(M)U(M)A(F)U(M)U(M)G(F)A(F)A(M)A(D)C(M)T(E)C(M)C(F)A(M)C(M)G(M) 3' (II-3) 5' U(M)U(F)G(M)A(M)U(M)A(F)U(M)U(M)G(F)A(M)A(M)A(D)C(M)T(E)C(M)C(F)A(M)C(F)G(M) 3' (II-4) 5' U(M)U(F)G(M)A(M)U(M)A(F)U(M)U(F)G(F)A(M)A(M)A(M)C(E)U(R)^C(M)C(F)A(M)C(M)G(M) 3' (II-5)5' U(M)U(F)G(M)A(M)U(M)A(F)U(M)U(M)G(F)A(F)A(M)A(M)C(E)U(R)^C(M)C(F)A(M)C(M)G(M) 3' (II-6) 5' U(M)U(F)G(M)A(M)U(M)A(F)U(M)U(M)G(F)A(M)A(F)A(M)C(E)U(R)^C(M)C(F)A(M)C(M)G(M) 3' (II-7) 5' U(M)U(F)G(M)A(M)U(M)A(F)U(M)U(M)G(F)A(M)A(M)A(F)C(E)U(R)^C(M)C(F)A(M)C(M)G(M) 3' (II-8) 5' U(M)U(F)G(M)A(M)U(M)A(F)U(M)U(M)G(F)A(M)A(F)A(M)C(M)U(R)^C(M)C(F)A(M)C(M)G(M) 3' (II-9) 5' U(M)U(F)G(M)A(M)U(M)A(F)U(M)U(M)G(F)A(M)A(M)A(F)C(M)U(R)^C(M)C(F)A(M)C(M)G(M) 3' (II-10) [Regarding the nucleic acid bases, A represents adenine, U represents uracil, T represents thymine, G represents guanine, and C represents cytosine (however, when C is ENA, it represents 2'-O,4'-C-ethylene-bridged-5-methylcytidine). Regarding nucleic acids, (R) represents RNA, (D) represents DNA, (M) represents 2'-OMe RNA, (3M) represents 3'-OMe RNA, (F) represents 2'-F RNA, and (E) represents ENA. In the formula, "^" indicates that the internucleoside bond is a phosphorothioate bond (-P(=S)(OH)-), and unless otherwise specified, indicates that the internucleoside bond is a phosphodiester bond (-P(=O)(OH)-), but even if otherwise specified, two internucleoside bonds in the three nucleosides from the 5'-end and 3'-end of the sense strand region, and two internucleoside bonds in the three nucleosides from the 5'-end and three internucleoside bonds in the four nucleosides from the 3'-end of the antisense strand region are phosphorothioate bonds.]

[41] The oligonucleotide or a pharmaceutically acceptable salt thereof according to

[40] , wherein an overhang structure of 3 bases or less is added to the 5'-end and / or 3'-end of the sense strand region and / or the antisense strand region.

[42] The oligonucleotide or pharmaceutically acceptable salt thereof according to

[41] , wherein the overhang structure is two bases.

[43] The oligonucleotide or pharmaceutically acceptable salt thereof according to

[42] , wherein the overhang structure is a nucleoside containing two consecutive thymines or a nucleoside containing two consecutive uracils.

[44] The oligonucleotide or pharmaceutically acceptable salt thereof according to

[43] , wherein the overhang structure is two consecutive U (M) added to the 3'-end of the antisense strand region.

[45] The oligonucleotide or pharmaceutically acceptable salt thereof according to

[43] , wherein the 5'-end of the sense strand region is represented by the following formula:

[0027]

[0028] [In the formula, the dashed line represents a phosphodiester bond with the 5'-phosphate group of the 5'-terminal nucleotide of the adjacent sense strand region.]

[46] The oligonucleotide or a pharmaceutically acceptable salt thereof according to [1], wherein the sense strand region is represented by the following formula (I-2), the antisense strand region is represented by any of the following formulas (II-11) to (II-20), and the sense strand region and the antisense strand region each serve as independent oligonucleotides to form a double-stranded oligonucleotide. (Formula) 5' GNC(M)^G(M)^U(M)G(M)G(F)A(M)G(F)U(F)U(F)U(M)C(M)A(M)A(M)U(M)A(M)U(M)C(M)^A(M)^A(3M) 3' (I-2) 5' U(M)^U(F)^G(M)A(M)U(M)A(F)U(M)U(F)G(F)A(M)A(M)A(D)C(M)T(E)C(M)C(F)A(M)C(M)^G(M)^U(M)^U(M) 3' (II-11) 5' U(M)^U(F)^G(M)A(F)U(M)A(F)U(M)U(M)G(F)A(M)A(M)A(D)C(M)T(E)C(M)C(F)A(M)C(M)^G(M)^U(M)^U(M) 3' (II-12) 5' U(M)^U(F)^G(M)A(M)U(M)A(F)U(M)U(M)G(F)A(F)A(M)A(D)C(M)T(E)C(M)C(F)A(M)C(M)^G(M)^U(M)^U(M) 3' (II-13) 5' U(M)^U(F)^G(M)A(M)U(M)A(F)U(M)U(M)G(F)A(M)A(M)A(D)C(M)T(E)C(M)C(F)A(M)C(F)^G(M)^U(M)^U(M) 3' (II-14) 5' U(M)^U(F)^G(M)A(M)U(M)A(F)U(M)U(F)G(F)A(M)A(M)A(M)C(E)U(R)^C(M)C(F)A(M)C(M)^G(M)^U(M)^U(M) 3' (II-15)5' U(M)^U(F)^G(M)A(M)U(M)A(F)U(M)U(M)G(F)A(F)A(M)A(M)C(E)U(R)^C(M)C(F)A(M)C(M)^G(M)^U(M)^U'M) : (II!)) ))))5' U(M)^U(F)^G(M)A(M)U(M)A(F)U(M)U(M)G(F)A(M)A(F)A(M)C(E)U(R)^C(M)()C()M(^M)^MA(^M) 3' (I). U(M)^U(F)^G(M)A(M)U(M)A(F)U(M)U(M)G(F)A(M)A(M)A(F)C(E)U(R)^C(M)C(F)A(M)C(M)^G(M)^U(M)^U(M) !!?,' . 5' U(M)^U(F)^G(M)A(M)U(M)A(F)U(M)U(M)G(F)A(M)A(F)A(M)C(M)U(R)^C(M)C(F)A(M)C(M)^G(M)^U(M)^U'M) : (II!!9) ())5' U(M)^U(F)^G(M)A(M)U(M)A(F)U(M)U(M)G(F)A(M)A(M)A(F)C(M)U(R)^C(M)(G)C()M(^M)^MA(^M) 3' (IB!0) (In the formula, the dashed line indicates a phosphodiester bond to the 5'-phosphate group of the 5'-terminal nucleotide of the adjacent sense strand region.) Regarding the nucleic acid bases, A represents adenine, U represents uracil, T represents thymine, G represents guanine, and C represents cytosine (however, when C is ENA, it represents 2'-O,4'-C-ethylene-bridged-5-methylcytidine). Regarding the nucleic acids, (R) represents RNA, (D) represents DNA, (M) represents 2'-OMe RNA, (3M) represents 3'-OMe RNA, (F) represents 2'-F RNA, and (E) represents ENA. In addition, in the formula, "^" indicates that the internucleoside bond is a phosphorothioate bond (-P(=S)(OH)-), and unless otherwise specified, indicates that the internucleoside bond is a phosphodiester bond (-P(=O)(OH)-). The 3'-position of the 3'-terminal nucleotide of the sense strand region, the 5'-position of the 5'-terminal nucleotide of the antisense strand region, and the 3'-position of the 3'-terminal nucleotide of the antisense strand region are all hydroxyl groups.]

[47] A pharmaceutical composition comprising, as an active ingredient, the oligonucleotide according to any one of [1] to

[46] or a pharmaceutically acceptable salt thereof.

[48] The pharmaceutical composition according to

[47] , for treating or preventing a disease that can be treated or prevented by suppressing the expression of transferrin receptor 2.

[49] The pharmaceutical composition according to

[48] , for preventing or treating anemia.

[50] The pharmaceutical composition according to

[49] , wherein the anemia is anemia associated with chronic inflammation or anemia associated with bone marrow failure.

[51] The pharmaceutical composition according to

[50] , wherein the anemia associated with chronic inflammation is anemia associated with autoimmune disease, anemia associated with infection, anemia associated with inflammatory bowel disease, anemia associated with heart failure, anemia associated with chronic kidney disease, anemia associated with cancer, or anemia associated with Castleman's disease.

[52] The pharmaceutical composition according to

[50] , wherein the anemia associated with bone marrow failure is anemia associated with myelofibrosis, anemia associated with myelodysplastic syndrome, anemia associated with chronic myelomonocytic leukemia, or anemia associated with bone marrow suppression due to chemotherapy.

[53] The pharmaceutical composition according to

[47] to

[52] , wherein the other drug is a drug for treating anemia, a drug for treating a disease that causes anemia, or a drug for treating iron overload.

[55] The pharmaceutical composition of

[54] , wherein the anemia therapeutic agent is an erythropoiesis-stimulating factor preparation, a HIFPHD (hypoxia inducible factor prolyl hydroxylase) inhibitor, an oral iron preparation, an intravenous iron preparation, luspatercept, or danazol.

[56] The pharmaceutical composition of

[55] , wherein the erythropoiesis-stimulating factor preparation is epoetin alfa, epoetin beta, epoetin beta pegol, epoetin kappa, or darbepoetin alfa.

[57] The pharmaceutical composition of

[55] , wherein the HIFPHD (hypoxia inducible factor prolyl hydroxylase) inhibitor is roxadustat, vadadustat, daprodustat, enarodustat, or molidustat.

[58] The pharmaceutical composition of

[55] , wherein the oral iron preparation is ferrous citrate, ferric citrate, ferrous fumarate, soluble ferric pyrophosphate, or dry ferrous sulfate.

[59] The pharmaceutical composition of

[55] , wherein the oral iron preparation is an intravenous iron preparation, ferric carboxymaltose, or saccharified ferric oxide.

[60] The pharmaceutical composition of

[54] , wherein the therapeutic agent for a disease that causes anemia is a therapeutic agent for myelofibrosis, a therapeutic agent for myelodysplastic syndrome, a therapeutic agent for chronic kidney disease, or an anticancer agent.

[61] The pharmaceutical composition of

[60] , wherein the therapeutic agent for myelofibrosis is a JAK2 inhibitor.

[62] The pharmaceutical composition of

[61] , wherein the JAK2 inhibitor is ruxolitinib, fedratinib, pacritinib, or momelotinib.

[63] The pharmaceutical composition of

[60] , wherein the myelodysplastic syndrome therapeutic agent is azacitidine or lenalidomide.

[64] The pharmaceutical composition of

[60] , wherein the chronic kidney disease therapeutic agent is an SGLT2 (sodium glucose cotransporter 2) inhibitor, a renin-angiotensin system inhibitor, a calcium channel blocker, a diuretic, a diabetes therapeutic agent, a hyperlipidemia therapeutic agent, a steroid, or an immunosuppressant.

[65] The pharmaceutical composition of

[64] , wherein the renin-angiotensin system inhibitor is an angiotensin II receptor antagonist or an angiotensin-converting enzyme inhibitor.

[66] The pharmaceutical composition of

[60] , wherein the anticancer agent is a chemotherapeutic agent.

[67] The pharmaceutical composition of

[66] , wherein the chemotherapeutic agent is cisplatin, carboplatin, doxorubicin, paclitaxel, or amrubicin.

[68] The pharmaceutical composition of

[54] , wherein the iron overload treatment agent is an iron chelator.

[69] The pharmaceutical composition of

[68] , wherein the iron chelator is deferasirox, deferoxamine, or deferiprone.

[70] A method for treating or preventing anemia, comprising administering to a subject an effective amount of the oligonucleotide of any one of [1] to

[46] or a pharmaceutically acceptable salt thereof.

[71] A method for treating or preventing anemia, comprising administering to a subject the pharmaceutical composition of any one of

[47] to

[69] .

[72] The oligonucleotide of any one of [1] to

[46] or a pharmaceutically acceptable salt thereof for use in treating or preventing anemia.

[73] The oligonucleotide according to any one of [1] to

[46] or a pharmaceutically acceptable salt thereof for use as an active ingredient in the pharmaceutical composition according to any one of

[47] to

[69] .

[74] Use of the oligonucleotide according to any one of [1] to

[46] or a pharmaceutically acceptable salt thereof in the manufacture of the pharmaceutical composition according to any one of

[47] to

[69] .

[0029] In one embodiment, the present invention provides the following:

[75] A pharmaceutical composition for treating or preventing anemia associated with bone marrow failure, comprising as an active ingredient a compound having the effect of knocking down the transferrin receptor 2 gene.

[76] The pharmaceutical composition according to

[75] , wherein the compound is an RNAi-oligonucleotide.

[0030] The siRNA of the present invention can suppress the expression of TfR2 mRNA. Furthermore, the siRNA of the present invention can suppress the expression of TfR2 mRNA in the liver in vivo, thereby preventing, ameliorating, and / or treating anemia.

[0031] [Correction based on Rule 91 03.04.2023] This figure shows the modification patterns of double-stranded siRNA. In the figure, filled circles represent 2'-O-methyl RNA, open circles represent 2'-deoxy-2'-fluoro RNA, filled triangles represent DNA, open triangles represent 3'-O-methyl RNA, open diamonds represent ENA, filled diamonds represent RNA, open circles with vertical bars represent LNA, open circles with horizontal bars represent 2'-O-methoxyethyl RNA, the lines connecting the marks represent phosphodiester bonds, and lines connecting the marks with an "s" represent phosphorothioate bonds. In the figure, the top strand represents the sense strand (or passenger strand), with the left end of the sense strand representing the 5' end and the right end representing the 3' end. The bottom strand represents the antisense strand (or guide strand), with the right end of the antisense strand representing the 5' end and the left end representing the 3' end. Each siRNA is represented by NNN-xxx, and the numbers and letters following NNN-xxx represent the abbreviations for the modification patterns shown in Figures 1 to 6. １４ This figure shows the modification patterns when the A of the antisense strand in the double-stranded siRNA is DNA or RNA. Each symbol, position, modification, etc. is displayed in the same format as in Figure 1. ８ and A ９ This figure shows the modification pattern when A of the antisense strand in the double-stranded siRNA is 2'-deoxy-2'-fluoro RNA. Each symbol, position, modification, etc. are displayed in the same format as in Figure 1. １４ This figure shows the modification pattern when A of the antisense strand in the double-stranded siRNA is ENA. Each symbol, position, modification, etc. are displayed in the same format as in Figure 1. ８ and A ９ is 2'-deoxy-2'-fluoro RNA, and A of the antisense strand １３ This figure shows the modification pattern when A of the antisense strand in the double-stranded siRNA is ENA. Each symbol, position, modification, etc. are displayed in the same format as in Figure 1. ８ is 2'-O-methyl RNA, and A ９is a diagram showing the modification pattern when is 2'-deoxy-2'-fluoro RNA. Each symbol, position, modification, etc. are displayed in the same format as in Figure 1. Figure 7A is a diagram showing the change in plasma iron concentration following treatment with nucleic acid-lipid particles encapsulating TfR2 siRNA in a mouse model of anemia associated with bone marrow decline. Figure 7B is a diagram showing the change in plasma hepcidin concentration following treatment with LNP-encapsulated TfR2 siRNA in a mouse model of anemia associated with bone marrow decline. Figure 7C is a diagram showing the change in hemoglobin (HGB) levels following treatment with LNP-encapsulated TfR2 siRNA in a mouse model of anemia associated with bone marrow decline. Figure 7D is a diagram showing the mean corpuscular hemoglobin (MCH) levels following treatment with LNP-encapsulated TfR2 siRNA in a mouse model of anemia associated with bone marrow decline. Figure 7E is a graph showing hepatic Tfr2 (mTfr2) mRNA expression levels in anemia model mice associated with bone marrow hypofunction following treatment with LNP-encapsulated TfR2 siRNA. Figure 7F is a graph showing hepcidin (mHepcidin) mRNA expression levels in anemia model mice associated with bone marrow hypofunction following treatment with LNP-encapsulated TfR2 siRNA. Figure 8A is a graph showing the time course of HGB levels in anemia model mice associated with bone marrow hypofunction following treatment with GalNAc-conjugated TfR2 siRNA. Figure 8B is a graph showing HGB levels 3 days after drug treatment in anemia model mice associated with bone marrow hypofunction. Figure 8C is a graph showing hepatic mTfR2 mRNA expression levels in anemia model mice associated with bone marrow hypofunction following treatment with GalNAc-conjugated TfR2 siRNA. Figure 8D shows HGB values ​​after treatment with GalNAc-conjugated TfR2 siRNA in a mouse model of anemia associated with bone marrow hypofunction, Figure 8E shows MCH values ​​after treatment with GalNAc-conjugated TfR2 siRNA in a mouse model of anemia associated with bone marrow hypofunction, and Figure 8F shows hematocrit (HCT) values ​​after treatment with GalNAc-conjugated TfR2 siRNA in a mouse model of anemia associated with bone marrow hypofunction.Figure 8G shows the number of red blood cells (RBCs) in a mouse model of anemia associated with bone marrow hypofunction following treatment with GalNAc-conjugated TfR2 siRNA. Figure 9A shows the plasma iron concentration in a mouse model of anemia associated with inflammation following treatment with GalNAc-conjugated TfR2 siRNA. Figure 9B shows the plasma hepcidin concentration in a mouse model of anemia associated with inflammation following treatment with GalNAc-conjugated TfR2 siRNA. Figure 9C shows the HGB level in a mouse model of anemia associated with inflammation following treatment with GalNAc-conjugated TfR2 siRNA. Figure 9D shows the MCH level in a mouse model of anemia associated with inflammation following treatment with GalNAc-conjugated TfR2 siRNA. Figure 9E shows the number of RBCs in a mouse model of anemia associated with inflammation following treatment with GalNAc-conjugated TfR2 siRNA. Figure 9F shows the hepatic mTfR2 mRNA expression level in an inflammation-associated anemia model mouse following treatment with GalNAc-conjugated TfR2 siRNA. Figure 10A shows the plasma iron concentration in an inflammation-associated anemia model mouse following treatment with GalNAc-conjugated TfR2 siRNA. Figure 10B shows the HGB value in an inflammation-associated anemia model mouse following treatment with GalNAc-conjugated TfR2 siRNA. Figure 10C shows the MCH value in an inflammation-associated anemia model mouse following treatment with GalNAc-conjugated TfR2 siRNA. Figure 10D shows the RBC count in an inflammation-associated anemia model mouse following treatment with GalNAc-conjugated TfR2 siRNA. Figure 10E shows the hepatic mTfR2 mRNA expression level in an inflammation-associated anemia model mouse following treatment with GalNAc-conjugated TfR2 siRNA. This figure shows the results of evaluating the cytotoxicity of siRNA in HepG2 cells. 1 shows the hTfR2 knockdown activity of siRNA in HepG2 cells. 2 shows the change in plasma iron concentration in normal mice treated with GalNAc-conjugated TfR2 siRNA.Figure 14A shows HGB levels in a mouse model of anemia associated with bone marrow decline before treatment with GalNAc-conjugated TfR2 siRNA. Figure 14B shows plasma iron levels in a mouse model of anemia associated with bone marrow decline before treatment with GalNAc-conjugated TfR2 siRNA. Figure 14C shows plasma iron levels 8 days after treatment with GalNAc-conjugated TfR2 siRNA in a mouse model of anemia associated with bone marrow decline. Figure 14D shows HGB levels 9 days after treatment with GalNAc-conjugated TfR2 siRNA in a mouse model of anemia associated with bone marrow decline. Figure 14E shows MCH levels 9 days after treatment with GalNAc-conjugated TfR2 siRNA in a mouse model of anemia associated with bone marrow decline. Figure 14F shows RBC counts 9 days after treatment with GalNAc-conjugated TfR2 siRNA in a mouse model of anemia associated with bone marrow decline. Figure 15A shows the change in plasma iron concentration over time due to treatment with GalNAc-conjugated TfR2 siRNA in an inflammatory anemia model mouse. Figure 15B shows HGB levels before treatment with GalNAc-conjugated TfR2 siRNA in an inflammatory anemia model mouse. Figure 15C shows HGB levels 8 days after treatment with GalNAc-conjugated TfR2 siRNA in an inflammatory anemia model mouse. Figure 15D shows MCH levels 8 days after treatment with GalNAc-conjugated TfR2 siRNA in an inflammatory anemia model mouse. Figure 15E shows the RBC count 8 days after treatment with GalNAc-conjugated TfR2 siRNA in an inflammatory anemia model mouse. Figure 15F shows the expression level of hepatic mTfR2 mRNA in an inflammatory anemia model mouse 8 days after treatment with GalNAc-conjugated TfR2 siRNA. 16A and 16B show changes in plasma iron concentration and HGB levels in MDS model mice treated with GalNAc-conjugated TfR2 siRNA, respectively.Figure 16C shows HGB values ​​in MDS model mice 8 weeks after initiation of GalNAc-conjugated TfR2 siRNA treatment. Figure 16D shows MCH values ​​in MDS model mice 8 weeks after initiation of GalNAc-conjugated TfR2 siRNA treatment. Figure 16E shows RBC counts in MDS model mice 8 weeks after initiation of GalNAc-conjugated TfR2 siRNA treatment. Figure 16F shows hepatic mTfR2 mRNA expression levels in MDS model mice 8 weeks after initiation of GalNAc-conjugated TfR2 siRNA treatment. Figure 17A shows plasma iron levels before GalNAc-conjugated TfR2 siRNA treatment in a mouse model of anemia associated with chronic kidney disease. Figure 17B shows HGB levels in a mouse model of anemia associated with chronic kidney disease before treatment with GalNAc-conjugated TfR2 siRNA. Figure 17C shows plasma iron concentrations 6 weeks after treatment with GalNAc-conjugated TfR2 siRNA in a mouse model of anemia associated with chronic kidney disease. Figure 17D shows HGB levels 6 weeks after treatment with GalNAc-conjugated TfR2 siRNA in a mouse model of anemia associated with chronic kidney disease. Figure 17E shows MCH levels 6 weeks after treatment with GalNAc-conjugated TfR2 siRNA in a mouse model of anemia associated with chronic kidney disease. Figure 17F shows RBC counts 6 weeks after treatment with GalNAc-conjugated TfR2 siRNA in a mouse model of anemia associated with chronic kidney disease. Figure 17G shows hepatic mTfR2 mRNA expression levels in a mouse model of anemia associated with chronic kidney disease 6 weeks after treatment with GalNAc-conjugated TfR2 siRNA. Figure 18A shows HGB levels in a mouse model of anemia induced by Ruxolitinib before treatment with GalNAc-conjugated TfR2 siRNA. Figure 18B shows plasma iron levels in a mouse model of anemia induced by Ruxolitinib 3 weeks after treatment with GalNAc-conjugated TfR2 siRNA.Figure 18C shows HGB values ​​6 weeks after initiation of GalNAc-conjugated TfR2 siRNA treatment in Ruxolitinib-induced anemia model mice. Figure 18D shows MCH values ​​6 weeks after initiation of GalNAc-conjugated TfR2 siRNA treatment in Ruxolitinib-induced anemia model mice. Figure 18E shows RBC counts 6 weeks after initiation of GalNAc-conjugated TfR2 siRNA treatment in Ruxolitinib-induced anemia model mice. Figure 18F shows hepatic mTfR2 mRNA expression levels 6 weeks after initiation of GalNAc-conjugated TfR2 siRNA treatment in Ruxolitinib-induced anemia model mice. 21A is a graph showing the hepatic hTfR2 mRNA expression level in hTfR2 Tg mice 7 days after treatment with GalNAc-conjugated TfR2 siRNA. 21B is a graph showing the hTfR2 mRNA expression level in human primary liver cells after treatment with GalNAc-conjugated TfR2 siRNA. 21A is a graph showing the hepcidin mRNA expression inhibitory activity of TfR2-019 and TfR2-039 in HepG2 cells. 21B is a graph showing the hepcidin mRNA expression inhibitory activity of TfR2-019.94DUG and TfR2-039.23DUG in HepG2 cells. 21B is a graph showing the nucleotide sequence (SEQ ID NO: 1) of the mRNA of the gene encoding human transferrin receptor 2 (hTfR2). The underlined sequence (sequence from bases 2847 to 2865) represents the target sequence of TfR2-019, and the double-underlined sequence (sequence from bases 1536 to 1554) represents the target sequence of TfR2-039.

[0032] <1. Explanation of Terms> As used herein, the term "target gene" refers to mRNA for a gene encoding transferrin receptor 2 (TfR2), and may be immature mRNA before undergoing RNA processing (also referred to as pre-mRNA or pre-mRNA), or may be mature mRNA after undergoing RNA processing. RNA processing includes, for example, RNA splicing, formation of a cap structure at the 5' end, and formation of a polyA tail at the 3' end.

[0033] TfR2 is a type 2 membrane protein primarily expressed in the liver and is known to consist of an intracellular domain, a transmembrane domain, and an extracellular domain (The International Journal of Biochemistry & Cell Biology 35 (2003) 292-296). TfR2 is known to play a central role in the production of hepcidin, which plays an important role in iron metabolism, and in the regulation of iron metabolism (Front. Pharmacol., 06 March 2014, Haematologica 2011;96(4):500-506, Blood. 2011;117(10):2960-2966).

[0034] Anemia refers to a condition in which the concentration of hemoglobin, which is present in red blood cells and has the ability to carry oxygen, is reduced. Anemia is associated with decreased physical activity and increased mortality (Ann N Y Acad Sci. 2019 August; 1450(1): 15-31.). Anemia is classified in various ways based on its cause, clinical features, red blood cell indices such as mean corpuscular volume (MCV) (Japanese Journal of Internal Medicine, Vol. 104, No. 7: 1375-1382, Clinical Laboratory Guidelines, JSLM 2015: 175-180). Anemia is thought to be caused primarily by insufficient red blood cell production, excessive destruction of red blood cells, iron deficiency, etc., and is known to involve a variety of complex pathologies, such as iron utilization disorders associated with chronic inflammation and bone marrow failure (Ann N Y Acad Sci. 2019 August; 1450(1): 15-31.). In anemia associated with chronic inflammation, chronic elevation of inflammatory cytokines such as interleukin 6 (IL6) increases the blood concentration of hepcidin, which plays a major role in iron metabolism, causing iron utilization disorders and resulting in a decline in hematopoietic function (Med Clin North Am. 2017 March; 101(2): 285-296.). Examples of causes of such chronic inflammation include chronic diseases such as autoimmune diseases, infectious diseases, inflammatory bowel diseases, heart failure, chronic kidney disease, and cancer. Examples of anemia associated with chronic inflammation include anemia associated with autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, and autoimmune hemolytic anemia, anemia associated with infectious diseases, anemia associated with macrophage activation syndrome, anemia associated with Castleman's disease, anemia associated with inflammatory bowel diseases such as inflammatory bowel disease, anemia associated with heart failure, anemia associated with chronic kidney disease, and cancer-related anemia. Cancer-related anemia includes anemia associated with cancers such as multiple myeloma, acute leukemia, chronic leukemia, lung cancer, and breast cancer. Anemia associated with bone marrow failure occurs when red blood cell production is significantly impaired due to some factor.Examples of anemia associated with bone marrow failure include anemia associated with myelodysplastic syndrome, anemia associated with myelofibrosis, anemia associated with chronic myelomonocytic leukemia (CMML), anemia associated with chemotherapy-induced bone marrow suppression, aplastic anemia, Diamond-Blackfan anemia, Fanconi anemia, etc. Myelofibrosis is a disease in which acquired genetic mutations such as JAK2V617F occur in hematopoietic stem cells, causing the proliferation of clones derived from abnormal hematopoietic stem cells, leading to chronic increased inflammation and the progression of bone marrow fibrosis, resulting in decreased bone marrow function, and is known to present with severe anemia, splenomegaly, etc. (Am J Hematol. 2021; 96: 145-162.).

[0035] As used herein, nucleic acid or nucleic acid molecule is a general term for nucleosides, nucleotides, oligonucleotides, and polynucleotides.

[0036] "Nucleoside" refers to a chemical structural moiety that combines a base moiety that is important for genetic information with a sugar moiety that is important for the physicochemical properties of the molecule.

[0037] A "nucleotide" is a compound in which the hydroxyl group at the 3'-position (or the 2'-position if the 3'-position is modified) of the sugar moiety of a nucleoside forms an ester with a phosphate group.

[0038] An "oligonucleotide" refers to an oligomer composed of two or more nucleotides, each of which is formed by bonding the phosphate group of one nucleotide to the hydroxyl group at the 5' position of the sugar moiety of another nucleotide (phosphodiester bond). However, the 3' position of the 3'-terminal nucleotide (or the 2' position in a nucleotide modified at the 3' position) and / or the 5' position of the 5'-terminal nucleotide may be a hydroxyl group or a phosphate group, or these may be chemically modified. Furthermore, the phosphodiester bond between nucleosides may be chemically modified. In this specification, the terms "oligonucleotide" and "polynucleotide" are used interchangeably.

[0039] The base moieties of nucleic acids are adenine (A), guanine (G), cytosine (C), uracil (U), and thymine (T). A and U or T, and G and C, respectively, form Watson-Crick base pairs through hydrogen bonds, and the relationship between bases that form a base pair is called "complementary." Each base moiety may be chemically modified, but even modified bases usually exhibit the same base pairing ability as the original base. Known representative modified bases include 5-methylcytosine (5C) and 5-methyluracil (5U), and 5U has the same structure as T. In the sequence listing attached to this specification, 5C is represented as C, and 5U is represented as T or U.

[0040] In this specification, nucleic acids are sometimes represented as "base symbols (sugar moiety symbols)." The sugar moiety symbols are explained in the sections describing various nucleosides below.

[0041] As used herein, the "modification pattern" of an oligonucleotide refers to the arrangement of the modification mode of the sugar moiety of each nucleoside constituting the oligonucleotide, regardless of the base sequence, unless otherwise specified. Furthermore, when specifically mentioned, it also refers to the modification pattern combined with the bond mode between each nucleoside. The structure of the sugar moiety and the internucleoside bond are factors that affect the stability of the molecule and the binding performance resulting from that stability, independent of the target sequence. Therefore, it is highly likely that the effect of a modification pattern confirmed for a certain target sequence will also be similar when the same modification pattern is applied to another target sequence (see, for example, Mol Ther. (2018) 26:708-717).

[0042] As used herein, a "natural nucleoside" refers to a 2'-deoxynucleoside or a ribonucleoside. 2'-deoxynucleosides (also referred to as "DNAs"; the sugar moiety is represented by (D), and the base moiety may be represented by a lowercase letter) corresponding to each base have a structure represented by the following formula, where 2'-deoxyadenosine is sometimes represented as A(D) or a, 2'-deoxyguanosine is sometimes represented as G(D) or g, 2'-deoxycytidine is sometimes represented as C(D) or c, thymidine is sometimes represented as T(D) or t, 2'-deoxy-5-methylcytidine is sometimes represented as 5C(D) or 5c, and 2'-deoxyuridine is sometimes represented as U(D) or u. 2'-deoxynucleosides that do not specify a base may sometimes be represented as N(D).

[0043] (In the above formulas, dashed lines represent bonds.)

[0044] The ribonucleosides (also called "RNA"; the symbol for the sugar moiety is (R) and the base moiety is sometimes written in capital letters) corresponding to each base have the structure shown in the following formula, where adenosine is sometimes written as A(R) or A, guanosine as G(R) or G, cytidine as C(R) or C, and uridine as U(R) or U. Ribonucleosides that do not specify a base are sometimes written as N(R).

[0045] (In the above formulas, dashed lines represent bonds.)

[0046] As used herein, the terms "modified nucleoside" and "modified nucleotide" refer to a nucleoside or nucleotide that contains at least one chemically modified structure in the base moiety, sugar moiety, or phosphate diester moiety of a natural nucleoside.

[0047] As used herein, the term "optionally chemically modified phosphodiester bond" refers to a phosphodiester bond used in natural internucleoside bonds or a chemically modified phosphodiester bond. A natural internucleoside bond is a phosphodiester bond represented by the following formula (P). In representing base sequences herein, no letters or symbols are inserted between nucleosides, or between a nucleoside and an adjacent structural unit (e.g., a GalNAc unit), a chemically modified structure (e.g., a structure represented by "2" described below), or a chemical linker or oligonucleotide linker (e.g., a structure represented by "Z" described below). Furthermore, as used herein, the term "chemically modified phosphodiester bond" refers to a bond in which the phosphorus atom in the phosphodiester bond is chemically modified. Examples of chemically modified phosphodiester bonds include a phosphorothioate bond represented by the following formula (PS), a phosphoramidate bond which is a modified bond in which a nitrogen atom is added to the phosphorus atom, an alkylphosphonate bond which is a modified bond in which a substitutable alkyl group is added to the phosphorus atom (for example, in the case of a methyl group, it becomes a methylphosphonate bond), and a phosphotriester bond which is a modified bond in which a substitutable alkyl group is added to the non-covalently bonded oxygen atom of a phosphate group. When a phosphorothioate bond is used, in the representation of the base sequence herein, a "^" is displayed between nucleosides, or between a nucleoside and its adjacent structural unit (e.g., a GalNAc unit), a chemically modified structure (e.g., a structure represented by "2" described below), or a chemical linker or oligonucleotide linker (e.g., a structure represented by "Z" described below).

[0048]

[0049] (In each of the above formulas, the dashed lines represent bonds, one of which represents an ester bond with the oxygen atom of the 3'-hydroxyl group of the adjacent nucleoside on the 5' side (the oxygen atom at the 2'-position in a nucleoside modified at the 3'-position), an adjacent structural unit, or a chemical linker or oligonucleotide linker; and the other bond represents an ester bond with the oxygen atom of the 5'-hydroxyl group of the adjacent nucleoside on the 3' side, a structural unit, or a chemical linker or oligonucleotide linker.)

[0050] As used herein, the term "sugar-modified nucleoside" refers to a nucleoside in which the sugar moiety of the nucleoside has been modified. Sugar-modified nucleosides include all types of sugar modifications known in the technical field to which the present invention pertains. Examples of sugar-modified nucleosides include 2'-modified nucleosides, 3'-modified nucleosides, 4'-thio-modified nucleosides, 4'-thio-2'-modified nucleosides, and 2'-O,4'-C-bridged modified nucleosides. Preferred examples of sugar modifications include 2'-O-alkylation (methylation, ethylation, propylation, isopropylation, butylation, etc.), 2'-O-alkoxyalkylation (methoxyethylation, methoxypropylation, methoxybutylation, etc.), 2'-halogenation (chlorination, fluorination, etc.), 3'-O-alkylation (methylation, ethylation, propylation, isopropylation, butylation, etc.), 3'-O-alkoxyalkylation (methoxyethylation, methoxypropylation, methoxybutylation, etc.), 3'-halogenation (chlorination, fluorination, etc.), and 2'-O,4'-C-bridging (alkylenation bridge, etc.).

[0051] As used herein, the term "2'-modified nucleoside" refers to a nucleoside in which the 2'-position of the ribofuranose moiety is chemically modified. Examples of such modifications include 2'-O-alkylated (methylated, ethylated, propylated, isopropylated, butylated, etc.) nucleosides, 2'-O-alkoxyalkylated (methoxyethylated, methoxypropylated, methoxybutyl, etc.) nucleosides, 2'-halogenated (chlorinated, fluorinated, etc.) nucleosides, 2'-allylated nucleosides, 2'-aminated nucleosides, 2'-azido nucleosides, 2'-O-allylated nucleosides, 2'-OCF ３ Nucleosides, 2'-O(CH ２ ) ２ SCH ３ Nucleosides, 2'-O-(CH ２ )2-O-N(R ｍ ) (R ｎ ) nucleoside, or 2'-O-CH ２ -C(=O)-N(R ｍ ) (R ｎ ) nucleosides (each R ｍ and R ｎ are individually H, an amino-protecting group, or a substituted or unsubstituted C1-C10 alkyl. Preferred 2'-modified nucleosides are 2'-O-alkylated nucleosides, 2'-O-alkoxyalkylated nucleosides, or 2'-halogenated nucleosides.

[0052] Among sugar-modified nucleosides, examples of 2'-O-alkylated modifications include 2'-O-methylnucleosides (sometimes referred to as "2'-OMe RNA" or "2'-O-methyl RNA." The symbol representing the sugar moiety is (M)). 2'-O-methylnucleosides corresponding to each base have the structure represented by the following formula, and are sometimes represented as follows: 2'-O-methyladenosine as A(M), 2'-O-methylguanosine as G(M), 2'-O-methylcytidine as C(M), 2'-O-methyl-5-methylcytidine as 5C(M), 2'-O-methyluridine as U(M), and 2'-O-methyl-5-methyluridine as T(M). 2'-O-methylnucleosides that do not specify a base are sometimes represented as N(M).

[0053] (In the above formulas, dashed lines represent bonds.)

[0054] Examples of 2'-O-alkoxyalkylated modifications include 2'-O-methoxyethyl nucleosides (sometimes referred to as "2'-MOE RNA," "2'-O-methoxyethyl RNA," or "2'-methoxyethoxy RNA." The symbol representing the sugar moiety is (m)). 2'-O-Methoxyethyl nucleosides corresponding to each base have the structures represented by the following formulas, where 2'-O-methoxyethyl adenosine is represented as A(m), 2'-O-methoxyethyl guanosine as G(m), 2'-O-methoxyethyl-5-methylcytidine as C(m) (although the base structure is 5C, it is represented as C(m) for convenience. It can also be used interchangeably with 2'-O-methoxyethylcytidine), 2'-O-methoxyethyluridine as U(m), and 2'-O-methoxyethyl-5-methyluridine as T(m). Furthermore, 2'-O-methoxyethyl nucleosides that do not specify a base may be represented as N(m).

[0055] (In the above formulas, dashed lines represent bonds.)

[0056] Furthermore, an example of a 2'-halogenated modification is 2'-deoxy-2'-fluoronucleoside (sometimes referred to as "2'-F RNA" or "2'-deoxy-2'-fluoro RNA." The symbol representing the sugar moiety is (F)). 2'-Deoxy-2'-fluoronucleosides corresponding to each base have the structure represented by the following formula, and are sometimes represented as follows: 2'-deoxy-2'-fluoroadenosine as A(F), 2'-deoxy-2'-fluoroguanosine as G(F), 2'-deoxy-2'-fluorocytidine as C(F), 2'-deoxy-2'-fluoro-5-methylcytidine as 5mC(F), 2'-deoxy-2'-fluorouridine as U(F), and 2'-deoxy-2'-fluoro-5-methyluridine as T(F). Furthermore, 2'-deoxy-2'-fluoronucleosides that do not specify a base may be represented as N(F).

[0057] (In the above formulas, dashed lines represent bonds.)

[0058] As used herein, the term "3'-modified nucleoside" refers to a nucleoside in which the 3'-position of the ribofuranose moiety contained therein has been chemically modified. The 2'-hydroxyl group in a 3'-modified nucleoside may be modified. Examples of such modifications include 3'-O-alkylated (methylated, ethylated, propylated, isopropylated, butylated, etc.) nucleosides, 3'-O-alkoxyalkylated (methoxyethylated, methoxypropylated, methoxybutylated, etc.) nucleosides, 3'-halogenated (chlorinated, fluorinated, etc.) nucleosides, 3'-allylated nucleosides, 3'-aminated nucleosides, 3'-azido nucleosides, 3'-O-allylated nucleosides, and 3'-OCF ３ Nucleosides, 3'-O(CH ２ ) ２ SCH ３ Nucleosides, 3'-O-(CH ２ )2-O-N(R ｍ ) (R ｎ ) nucleoside, or 3'-O-CH ２ -C(=O)-N(R ｍ ) (R ｎ ) nucleosides (each Rｍ and R ｎ are individually H, an amino-protecting group, or a substituted or unsubstituted C1-C10 alkyl). Preferred 3'-modified nucleosides are 3'-O-alkylated nucleosides, 3'-O-alkoxyalkylated nucleosides, or 3'-halogenated nucleosides.

[0059] Among 3'-modified nucleosides, an example of a 3'-O-alkylated modification is 3'-O-methylnucleoside (sometimes referred to as "3'-OMe RNA." The symbol representing the sugar moiety is (3M)). 3'-O-methylnucleosides corresponding to each base have the structure represented by the following formula, and are sometimes represented as follows: 3'-O-methyladenosine as A(3M), 3'-O-methylguanosine as G(3M), 3'-O-methylcytidine as C(3M), 3'-O-methyl-5-methylcytidine as 5mC(3M), 3'-O-methyluridine as U(3M), and 3'-O-methyl-5-methyluridine as T(3M). Furthermore, 3'-O-methylnucleosides that do not specify a base are sometimes represented as N(3M).

[0060] (In the above formulas, dashed lines represent bonds.)

[0061] An example of a 3'-O-alkoxyalkylation modification is 3'-O-methoxyethyl nucleoside (sometimes referred to as "3'-MOE RNA," "3'-O-methoxyethyl RNA," or "3'-methoxyethoxy RNA." The symbol representing the sugar moiety is (3m).) The 3'-O-methoxyethyl nucleoside corresponding to each base has a structure in which a methoxyethyl group is bonded in place of the methyl group bonded to the oxygen atom at the 3' position in the structural diagram of the 3'-O-methyl nucleoside corresponding to each base. The 3'-O-methoxyethyl nucleosides corresponding to each base are sometimes represented as follows: 3'-O-methoxyethyl adenosine as A(3m), 3'-O-methoxyethyl guanosine as G(3m), 3'-O-methoxyethyl-5-methylcytidine as C(3m) (although the base structure is 5C, it is represented as C(3m) for convenience. It can also be used in place of 3'-O-methoxyethyl cytidine), 3'-O-methoxyethyl uridine as U(3m), and 3'-O-methoxyethyl-5-methyluridine as T(3m). Also, 3'-O-methoxyethyl nucleosides that do not specify a base may be represented as N(3m).

[0062] An example of a 3'-halogenated modification is 3'-deoxy-3'-fluoronucleoside (sometimes referred to as "3'-F RNA" or "3'-deoxy-3'-fluoro RNA." The symbol representing the sugar moiety is (3F).) The 3'-deoxy-3'-fluoronucleoside corresponding to each base has a structure in which a fluorine atom is bonded in place of the methoxy group bonded to the carbon atom at the 3' position in the above structural diagram of the 3'-O-methylnucleoside corresponding to each base. The 3'-deoxy-3'-fluoronucleosides corresponding to each base are sometimes represented as follows: 3'-deoxy-3'-fluoroadenosine as A(3F), 3'-deoxy-3'-fluoroguanosine as G(3F), 3'-deoxy-3'-fluorocytidine as C(3F), 3'-deoxy-3'-fluoro-5-methylcytidine as 5mC(3F), 3'-deoxy-3'-fluorouridine as U(3F), and 3'-deoxy-3'-fluoro-5-methyluridine as T(3F). Also, 3'-deoxy-3'-fluoronucleosides that do not specify a base may be represented as N(3F).

[0063] As used herein, the term "4'-thio-modified nucleoside" refers to a nucleoside in which the oxygen atom at the 4'-position of the ribofuranose moiety is replaced with a sulfur atom. Examples of 4'-thio-modified nucleosides include β-D-ribonucleosides in which the 4'-oxygen atom is replaced with a sulfur atom (Hoshika, S. et al. FEBS Lett. 579, pp. 3115-3118, (2005); Dande, P. et al. J. Med. Chem. 49, pp. 1624-1634 (2006); Hoshika, S. et al. ChemBioChem. 8, pp. 2133-2138, (2007)).

[0064] As used herein, the term "4'-thio-2'-modified nucleoside" refers to a nucleoside in which the 2'-position of the ribofuranose moiety is chemically modified and the oxygen atom at the 4'-position of the ribofuranose moiety is replaced with a sulfur atom. Examples of 4'-thio-2'-modified nucleosides include 4'-thio-2'-modified nucleosides that retain 2'-H or 2'-O-methyl (Matsugami, et al. Nucleic Acids Res. 36, 1805 (2008)).

[0065] Among sugar-modified nucleosides, examples of 2'-O,4'-C-bridged modifications include 2'-O,4'-C-ethylene-bridged nucleosides (sometimes referred to as "ENA" or "ENA unit." The symbol representing the sugar moiety is (E)) (Morita, K. et al. Bioorg. Med. Chem. Lett., 12, p. 73 (2002); Morita, K. et al. Bioorg. Med. Chem., 11, p. 2211 (2003)). The 2'-O,4'-C-ethylene-bridged nucleosides corresponding to each base have the structures represented by the following formulas, where 2'-O,4'-C-ethylene-bridged adenosine is sometimes represented as A(E), 2'-O,4'-C-ethylene-bridged guanosine as G(E), 2'-O,4'-C-ethylene-bridged-5-methylcytidine as C(E) (although the base structure is 5C, it is represented as C(E) for convenience. It can also be used in place of 2'-O,4'-C-ethylene-bridged cytidine), 2'-O,4'-C-ethylene-bridged uridine as U(E), and 2'-O,4'-C-ethylene-bridged-5-methyluridine as T(E). Furthermore, 2'-O,4'-C-ethylene-bridged nucleosides that do not specify a base are sometimes represented as N(E).

[0066] (In the above formulas, dashed lines represent bonds.)

[0067] Another example of the 2'-O,4'-C-bridged modification is, for example, 2'-O,4'-C-methylene-bridged nucleoside (sometimes referred to as "LNA" or "LNA unit", the symbol representing the sugar moiety is (L)) (Obika, S. et al. Tetrahedron Lett., 38, p. 8735- (1997); Obika, S. et al., Tetrahedron Lett., 39, p. 5401- (1998); A. A. Koshkin, A. A. et al. Tetrahedron, 54, p. 3607 (1998); Obika, S., Bioorg. Med. Chem., 9, p. 1001 (2001).) 2'-O,4'-C-methylene-bridged nucleosides corresponding to each base have structures represented by the following formulas, where 2'-O,4'-C-methylene-bridged adenosine is represented as A(L), 2'-O,4'-C-methylene-bridged guanosine as G(L), 2'-O,4'-C-methylene-bridged-5-methylcytidine as C(L) (although the base structure is 5C, it is represented as C(L) for convenience. It can also be used interchangeably with 2'-O,4'-C-methylene-bridged cytidine), 2'-O,4'-C-methylene-bridged uridine as U(L), and 2'-O,4'-C-methylene-bridged-5-methyluridine as T(L). Furthermore, 2'-O,4'-C-methylene-bridged nucleosides that do not specify a base may be represented as N(L).

[0068] (In the above formulas, dashed lines represent bonds.)

[0069] The oligonucleotide or salt thereof of the present invention has an antisense strand region (sometimes simply referred to as the antisense strand) having a nucleotide sequence substantially complementary to the nucleotide sequence of a target sequence, and a sense strand region (sometimes simply referred to as the sense strand) having a nucleotide sequence substantially complementary to the nucleotide sequence in the complementary region of the antisense strand region. The sense strand region and the antisense strand region form a double-stranded structure in the nucleotide sequence of the complementary region, but each may form a double-stranded oligonucleotide as an independent oligonucleotide, or may form a single-stranded oligonucleotide by linking the 5' position of the 5'-terminal nucleotide of one to the 3' position of the 3'-terminal nucleotide of the other (the 2' position in the case of a nucleotide modified at the 3' position). That is, the oligonucleotide of the present invention may be an oligonucleotide of two molecules or an oligonucleotide of one molecule. When the oligonucleotide of the present invention is a single molecule of oligonucleotide, the linking mode of the sense strand region and the antisense strand region is not particularly limited as long as the oligonucleotide of the present invention has RNA interference activity and / or gene expression suppression activity, but for example, the 5' position of the 5'-terminal nucleotide of one side and the 3' position of the 3'-terminal nucleotide of the other side (the 2' position in the case of a nucleotide whose 3' position is modified) can be linked by a desired chemical linker or oligonucleotide linker. Such linkers include, for example, the linkers described in WO2012 / 074038, and preferably include the linker represented by the following formula (Z). The linker represented by the following formula (Z) can be appropriately prepared according to WO2012 / 074038.

[0070]

[0071] (In the above formula, the dashed lines represent bonds, and the oxygen atom bonded to the phenyl group represents a phosphodiester bond with the 5'-phosphate group of the 5'-terminal nucleotide in the adjacent antisense strand region, while the methylene group at the other end represents a phosphodiester bond with the 3'-phosphate group (or the 2'-phosphate group in a nucleotide modified at the 3' position) of the 3'-terminal nucleotide in the adjacent sense strand region, and each phosphodiester bond formed may be a chemically modified phosphodiester bond such as a phosphorothioate bond.)

[0072] In the oligonucleotide of the present invention, the 3'-position of the 3'-terminal nucleotide (2'-position in the case of a nucleotide modified at the 3'-position) and / or the 5'-position of the 5'-terminal nucleotide may be a hydroxyl group or a phosphate group, or may have a chemically modified structure. Examples of the chemically modified structure include the structure shown in the following formula (2).

[0073]

[0074] (In the above formula, the dashed lines represent bonds, one of which represents a bond to an adjacent nucleotide, forming a phosphodiester bond with the 5'-phosphate group when bonding to the 5'-terminal nucleotide, or with the 3'-phosphate group (the 2'-phosphate group in a nucleotide modified at the 3'-position) when bonding to the 3'-terminal nucleotide; the other bond represents a bond to a hydrogen atom to form a hydroxyl group, and the phosphodiester bond formed may be a chemically modified phosphodiester bond such as a phosphorothioate bond.)

[0075] As used herein, "substantially identical nucleotide sequences" refers to nucleotide sequences consisting entirely of identical bases relative to the target nucleotide sequence, as well as nucleotide sequences with 70% or greater sequence identity, and nucleotide sequences containing one or more non-identical bases but capable of performing the desired function as an oligonucleotide. Here, "identical bases" include not only bases identical to the original base, but also bases with base pairing ability equivalent to or greater than that of the original base. Examples of such bases include chemically modified bases with base pairing ability equivalent to or greater than that of the original base (e.g., C and 5C, U and T (5U)). Sequence identity refers to a sequence with preferably 80% or greater identity, more preferably 90% or greater, and even more preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity relative to the target nucleotide sequence, or a sequence with 3-, 2-, or 1-base mismatch. Nucleotide sequence identity can be calculated using known genetic analysis software such as BLAST (registered trademark).

[0076] As used herein, the term "substantially complementary nucleotide sequence" refers not only to a nucleotide sequence consisting entirely of nucleotides complementary to a target nucleotide sequence, but also to a nucleotide sequence in which one or several (e.g., five, four, three, or two) nucleotides are not complementary nucleotides but form base pairs between oligonucleotides. Furthermore, as used herein, the term "complementary base" refers to a base that can form a Watson-Crick base pair with a target base, and may be an unmodified base or a chemically modified base. For example, 5-methyluracil and adenine are complementary bases, and 5-methylcytosine and guanine are complementary bases.

[0077] As used herein, the term "double-stranded structure" of an oligonucleotide refers to a double-stranded structure formed by the formation of Watson-Crick base pairs between substantially complementary nucleotide sequences. The nucleotide sequences forming the double-stranded structure may be substantially complementary nucleotide sequences between two different oligonucleotide molecules, or may be substantially complementary sequences within a single-stranded oligonucleotide.

[0078] As used herein, the term "structural unit" refers to a chemical structural unit that imparts a desired function to an oligonucleotide. Examples of the desired function imparted to an oligonucleotide include the function of transporting the oligonucleotide to a target tissue or target cell (also referred to as a target tissue, etc.), the function of improving the pharmacokinetics of the oligonucleotide, and the like. The structural unit can be attached to an oligonucleotide by known methods. For example, by having an amidite structure in the structural unit, the structural unit can be attached to the 3'-position of the 3'-terminal nucleotide (the 2'-position in a nucleotide modified at the 3'-position) or the 5'-position of the 5'-terminal nucleotide of the oligonucleotide. Furthermore, by having a structure in the structural unit that promotes the transport of a ligand or the like to a target tissue, etc., the oligonucleotide can be imparted with transportability to the target tissue, etc. Examples of such structures include a GalNAc unit for transport to hepatocytes, a fatty acid unit for transport to liver or muscle tissue, etc. In the present specification, such a structural unit for imparting transportability to a target tissue, etc., is sometimes referred to as a "transport unit."

[0079] 2. RNAi Oligonucleotides As used herein, an "RNAi oligonucleotide" refers to an oligonucleotide that includes a complementary region in which substantially complementary nucleotides contained in the sense strand region and the antisense strand region form Watson-Crick base pairs to form a double-stranded structure, and that has an RNA interference effect and / or gene expression suppression effect on a target gene. As long as the RNAi oligonucleotide has an RNA interference effect and / or gene expression suppression effect on a target gene, not all bases in the complementary region contained in the RNAi oligonucleotide need form Watson-Crick base pairs. As used herein, RNAi oligonucleotides may also be referred to as "siRNA."

[0080] As used herein, the term "a nucleotide sequence substantially identical to a target sequence" refers to a nucleotide sequence identical to the target sequence, but includes not only a completely identical sequence but also a substantially identical nucleotide sequence as long as the RNAi-oligonucleotide has an RNA interference effect and / or a gene expression suppression effect on the target gene.

[0081] As used herein, the term "nucleotide sequence substantially complementary to a target sequence" refers to a nucleotide sequence complementary to a target sequence, and includes not only completely complementary sequences but also substantially complementary nucleotide sequences as long as the RNAi-oligonucleotide has an RNA interference effect and / or a gene expression suppression effect on a target gene.

[0082] As used herein, an oligonucleotide that contains a nucleotide sequence substantially complementary to a target sequence and has an RNA interference effect and / or gene expression suppression effect on a target gene is referred to as an RNAi-oligonucleotide for a target gene.

[0083] The nucleotide sequence of an RNAi-oligonucleotide for a target gene is not particularly limited as long as it has an RNA interference effect and / or a gene expression suppressing effect on the target gene, and can be determined, for example, based on a sequence predicted to have an RNA interference effect and / or a gene expression suppressing effect on the target gene using computer software (e.g., GENETYX (registered trademark): manufactured by GENETYX COORPORATION, etc.). Furthermore, the RNA interference effect and / or the gene expression suppressing effect of an RNAi-oligonucleotide prepared based on a selected sequence can also be determined by confirming the effect.

[0084] As used herein, the term "gene expression inhibitory effect" includes not only the effect of completely suppressing gene expression but also the effect of reducing gene expression compared to a control, and gene silencing is also included in the term "gene expression inhibitory effect." Furthermore, as used herein, the terms "gene expression inhibitory effect" and "gene expression inhibitory activity" are used interchangeably.

[0085] The RNA interference effect and / or gene expression suppression effect can be confirmed by methods commonly used by those skilled in the art, for example, by administering an RNAi-oligonucleotide against the target gene to cells in which the target gene is expressed, quantifying the protein that is the translation product of the target gene after a certain period of time using Western blot analysis, and comparing the expression level of the protein with that of a control. Alternatively, it can be confirmed by measuring the expression level of the target gene in real time after administering an RNAi-oligonucleotide against the target gene using real-time PCR techniques.

[0086] As used herein, the term "target sequence" refers to a sequence of 18 or more nucleotides, which may be any portion of the nucleotide sequence of mRNA for the gene encoding transferrin receptor 2, which is the target gene, and which is targeted by the RNAi-oligonucleotide of the present invention. Furthermore, if SNPs or the like are known in the target sequence, sequences containing these mutations are also included in the target sequence. The RNAi-oligonucleotide of the present invention contains a sequence substantially complementary to the target sequence (hereinafter referred to as a "target-corresponding sequence") in at least a portion of the antisense strand region.

[0087] As used herein, the term "complementary region" refers to a region in which the nucleotide sequences of the sense strand region and the antisense strand region contained in an RNAi oligonucleotide are substantially complementary to each other. The sense strand region and the antisense strand region do not necessarily have to be completely complementary in their complementary region, but at least the terminal bases in the complementary region are complementary to each other. In the RNAi oligonucleotide of the present invention, the complementary region of the antisense strand region includes the target-corresponding sequence, but may maintain complementarity with the sense strand region in a region further extended at the 5' end and / or 3' end of the complementary region.

[0088] The length of the target-corresponding sequence in the antisense strand region constituting the RNAi-oligonucleotide of the present invention is not particularly limited as long as it has RNA interference activity and / or gene expression inhibitory activity, and may be any length from 18 nucleotides to the full length of the open reading frame (ORF) of the target gene. For example, the length of the target-corresponding sequence can be 18 to 29 nucleotides, preferably 18 to 24 nucleotides, 18 to 23 nucleotides, or 18 to 22 nucleotides, more preferably 18 to 21 nucleotides, and even more preferably 18 or 19 nucleotides.

[0089] The length of each complementary region in the sense strand and antisense strand constituting the RNAi-oligonucleotide of the present invention is not particularly limited as long as it has an RNA interference effect and / or a gene expression inhibitory effect, and may be any length. For example, the length of the complementary region may be 19 to 29 nucleotides, preferably 19 to 24 nucleotides, 19 to 23 nucleotides, or 19 to 22 nucleotides, more preferably 19 to 21 nucleotides, and even more preferably 19 nucleotides.

[0090] The chain lengths of the sense strand region and antisense strand region constituting the RNAi-oligonucleotide of the present invention are not particularly limited as long as they have an RNA interference effect and / or a gene expression inhibitory effect, and may be any length. The chain length of the sense strand region can be, for example, 19 to 39 nucleotides, preferably 19 to 29 nucleotides, more preferably 19 to 24 nucleotides, 19 to 23 nucleotides, or 19 to 22 nucleotides, even more preferably 19 to 21 nucleotides, and most preferably 19 nucleotides. The chain length of the antisense strand region can be, for example, 19 to 39 nucleotides, preferably 19 to 29 nucleotides, more preferably 19 to 24 nucleotides, 19 to 23 nucleotides, or 19 to 22 nucleotides, even more preferably 21 to 23 nucleotides, and most preferably 21 nucleotides.

[0091] In the RNAi-oligonucleotide of the present invention, when the sense strand region and the antisense strand region are present in different oligonucleotide molecules, the entire structure does not need to be a double-stranded structure, and the 5'- and / or 3'-terminal nucleosides may have an overhang structure in which some of the nucleosides protrude, or which do not form a double-stranded structure. Examples of such an overhang structure include a structure consisting of 1 to 5 nucleosides, preferably 1 to 3 nucleosides, and more preferably 2 nucleosides. Furthermore, the overhang structure may be present at all ends of the double-stranded oligonucleotide, or may be only a part of it. In this specification, the 5'-end of the sense strand region is represented by S Ｏ５, 3' end is S Ｏ３ , the 5' end of the antisense strand region is A Ｏ５ , 3' end is A Ｏ３ The base sequence of the overhang structure is not particularly limited, but oligo T or oligo U can be used.

[0092] The RNAi-oligonucleotide has at least one property selected from the following (i) to (iv): (i) it has an RNA interference effect and / or a gene expression-inhibiting effect on a target gene; (ii) it is stable against RNases and has an RNA interference effect and / or a gene expression-inhibiting effect on a target gene; (iii) it is stable against exonucleases and has an RNA interference effect and / or a gene expression-inhibiting effect on a target gene; (iv) it is stable against RNases and exonucleases and has an RNA interference effect and / or a gene expression-inhibiting effect on a target gene;

[0093] An example of an RNAi oligonucleotide is derived from a double-stranded oligonucleotide having an antisense strand region for a target gene and a sense strand region having a nucleotide sequence substantially complementary to the antisense strand region, and the 5'-position of the 5'-terminal nucleotide of the antisense strand region and the 3'-position of the 3'-terminal nucleotide of the sense strand region (or the 2'-position in the case of a nucleotide modified at the 3'-position) may each be linked by a linker forming a phosphodiester structure, which may be chemically modified. Such an oligonucleotide may have the structure oligonucleotide 3'-P(=O)(OH)-[linker]-P(=O)(OH)-5'-oligonucleotide (where "oligonucleotide 3'" refers to a structure in which the 3'-terminal nucleotide of the oligonucleotide does not have a hydrogen atom on the hydroxyl group at the 3'-position (or the 2'-position in the case of a nucleotide modified at the 3'-position), and "5'-oligonucleotide" refers to a structure in which the 5'-terminal nucleotide of the oligonucleotide does not have a hydrogen atom on the hydroxyl group at the 5'-position). Such linkers are described, for example, in WO2012 / 074038 and can be synthesized by referring to the descriptions in these documents.

[0094] The nucleosides constituting the RNAi oligonucleotide may be natural nucleosides or sugar-modified nucleosides. The internucleoside bond is a phosphodiester bond which may be chemically modified, such as a phosphodiester bond or a phosphorothioate bond.

[0095] The RNAi-oligonucleotides of the present invention can suppress the expression of TfR2 mRNA. Furthermore, the RNAi-oligonucleotides of the present invention have low toxicity to cells and can be used as highly safe pharmaceuticals. Furthermore, by suppressing the expression of TfR2 mRNA, the RNAi-oligonucleotides of the present invention can suppress the production of hepcidin, which plays a central role in iron metabolism. Furthermore, by suppressing the expression of TfR2 mRNA, the RNAi-oligonucleotides of the present invention can increase plasma iron concentrations and / or HGB levels. While the mechanism of action is not particularly limited, the RNAi-oligonucleotides of the present invention can be used to treat or prevent diseases that can be treated or prevented by suppressing the expression of TfR2 mRNA and / or the production of hepcidin. Examples of such diseases include anemia. While the mechanism of action is not particularly limited, the RNAi-oligonucleotides of the present invention can treat or prevent anemia, for example, by increasing HGB levels. Furthermore, the RNAi-oligonucleotides of the present invention can reduce the frequency and / or amount of blood cell transfusions in anemic patients who require blood cell transfusions. For example, the frequency and / or volume of blood cell transfusions for an anemic patient over a period of time (e.g., 1 day, 1 week, 2 weeks, 4 weeks, 8 weeks, 12 weeks, 1 month, 2 months, 3 months, 6 months, or 1 year) may be reduced by about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%.

[0096] The present invention provides inventions for medicinal uses, such as a pharmaceutical composition containing the RNAi-oligonucleotide of the present invention as an active ingredient and used for treating or preventing anemia, a method for treating or preventing anemia comprising administering an effective amount of the oligonucleotide, the oligonucleotide for use in treating or preventing anemia, and use of the RNAi-oligonucleotide of the present invention in the manufacture of a pharmaceutical composition for treating or preventing anemia. Examples of anemia that can be treated or prevented by the RNAi-oligonucleotides of the present invention include anemia of chronic inflammation (ACD), anemia associated with bone marrow failure, iron deficiency anemia, iron refractory iron deficiency anemia (IRIDA), anemia resistant to erythropoietin stimulating agents (ESA), anemia induced by chemotherapy, aplastic anemia, Diamond-Blackfan anemia, and hereditary hemoglobin disorders such as β-thalassemia or sickle cell disease. Examples of anemia associated with chronic inflammation include anemia associated with autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, or autoimmune hemolytic anemia, anemia associated with infectious diseases, anemia associated with inflammatory bowel diseases such as inflammatory colitis, anemia associated with heart failure, anemia associated with chronic kidney disease, cancer-related anemia, anemia associated with macrophage activation syndrome, and anemia associated with Castleman's disease.Examples of anemia associated with chronic kidney disease include kidney disease associated with diabetes, kidney disease associated with hypertension, nephrotic syndrome (e.g., Finnish-type congenital nephrotic syndrome, diffuse mesangial sclerosis, minimal change nephrotic syndrome, focal segmental glomerulosclerosis, membranous nephropathy, Galloway-Mowat syndrome, etc.), kidney disease associated with chronic glomerulonephritis (e.g., IgA nephropathy, mesangial proliferative glomerulonephritis, membranoproliferative glomerulonephritis, purpura nephritis, anti-glomerular basement membrane nephritis (Goodpasture syndrome), etc.), and kidney disease associated with chronic glomerulonephritis (e.g., IgA nephropathy, mesangial proliferative glomerulonephritis, membranoproliferative glomerulonephritis, purpura nephritis, anti-glomerular basement membrane nephritis (Goodpasture syndrome)). Examples of anemia include anemia associated with chronic kidney diseases such as idiopathic pulmonary fibrosis (IPF), Alport syndrome, Epstein syndrome, lupus nephritis, microscopic polyangiitis, atypical hemolytic uremic syndrome, Nail-Patella syndrome, fibronectin nephropathy, and lipoprotein glomerulopathy, kidney disease with chronic tubulointerstitial nephritis, kidney disease with chronic pyelonephritis, kidney disease with amyloid deposition, autosomal dominant tubulointerstitial kidney disease, kidney disease with nephronophthisis, and kidney disease with renal malformation (including, for example, polycystic kidney disease). Examples of anemia associated with cancer include anemia associated with cancers such as multiple myeloma, acute leukemia, chronic leukemia, lung cancer, and breast cancer. Examples of anemia associated with bone marrow failure include anemia associated with myelofibrosis, anemia associated with myelodysplastic syndrome, anemia associated with chronic myelomonocytic leukemia (CMML), anemia associated with bone marrow suppression due to chemotherapy, aplastic anemia, Diamond-Blackfan anemia, and Fanconi anemia.

[0097] The RNAi-oligonucleotides of the present invention can be preferably used for the treatment or prevention of anemia associated with chronic inflammation and anemia associated with bone marrow failure, and more preferably for the treatment or prevention of anemia associated with autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, or autoimmune hemolytic anemia, anemia associated with infectious diseases, anemia associated with inflammatory bowel diseases such as inflammatory bowel disease, anemia associated with heart failure, anemia associated with chronic kidney disease, anemia due to cancer, anemia associated with Castleman's disease, anemia associated with myelofibrosis, anemia associated with myelodysplastic syndrome, anemia associated with chronic myelomonocytic leukemia, and anemia associated with bone marrow suppression due to chemotherapy.

[0098] As used herein, the term "combination use" refers to the simultaneous, separate, or sequential administration of two or more different drugs. The present invention provides a method for treating or preventing anemia, comprising the combined use of the RNAi-oligonucleotide of the present invention with one or more other drugs. The RNAi-oligonucleotide of the present invention may be used in combination with one or more other drugs, as long as the effects of the present invention are achieved. Preferably, a greater therapeutic effect can be achieved by combining the RNAi-oligonucleotide of the present invention with another drug that has a different mechanism of action on anemia symptoms. For example, for myelodysplastic syndrome (MDS), drugs such as ESAs (erythropoiesis stimulating agents), which have a mechanism of action to treat anemia by promoting red blood cell proliferation, luspatercept, which has a mechanism of action to treat anemia by promoting red blood cell differentiation, and azacitidine, which has a mechanism of action to suppress DNA methylation and kill abnormal cells, may be prescribed. However, these drugs have different mechanisms of action from TfR2 siRNA. Therefore, the combined use of these drugs with TfR2 siRNA can provide greater therapeutic effects for MDS than either drug alone. In addition, for anemia associated with chronic kidney disease, drugs such as ESAs and HIFPHD (hypoxia inducible factor prolyl hydroxylase) inhibitors, which have a mechanism of action that treats anemia by promoting red blood cell proliferation, may be prescribed, but these drugs have a different mechanism of action from TfR2 siRNA. Therefore, the combination of these drugs with TfR2 siRNA can provide greater therapeutic effects for anemia associated with chronic kidney disease than single drugs. In addition, ruxolitinib is known to be effective in treating myelofibrosis, mainly symptoms such as splenomegaly (N Engl J Med 2012; 366:799-807). However, it is known that the effect on anemia symptoms is limited or ineffective, or that it worsens anemia (Journal of Hematology & Oncology 2013, 6:79).The mechanism by which Ruxolitinib worsens anemia is thought to be that JAK2 inhibition suppresses EPO signaling, which is necessary for the proliferation of red blood cells. However, since Ruxolitinib has demonstrated a certain therapeutic effect on symptoms of myelofibrosis other than anemia, the combination of Ruxolitinib with TfR2 siRNA can provide a useful therapeutic effect on anemia in myelofibrosis. Similarly, the combination of Ruxolitinib with other JAK2 inhibitors other than Ruxolitinib can also provide a useful therapeutic effect on anemia in myelofibrosis. Examples of other JAK2 inhibitors include fedratinib, pacritinib, and momelotinib.

[0099] When the RNAi-oligonucleotide of the present invention is used in combination with other drugs, the order of administration of the RNAi-oligonucleotide of the present invention is not particularly limited as long as the effects of the present invention are achieved. The RNAi-oligonucleotide of the present invention may be administered simultaneously with the other drug, or before or after the other drug. Examples of drugs that can be used in combination with the RNAi-oligonucleotide of the present invention include other anemia therapeutic drugs or therapeutic drugs for diseases that cause anemia. Examples of other anemia therapeutic drugs include ESAs, HIFPHD (hypoxia inducible factor prolyl hydroxylase) inhibitors, oral iron preparations, intravenous iron preparations, danazol, Luspatercept, and the like. Examples of ESAs include epoetin alpha, epoetin beta, epoetin beta pegol, epoetin kappa, darbepoetin alpha, and the like. Examples of HIFPHD inhibitors include roxadustat, vadadustat, daprodustat, enarodustat, molidustat, etc. Examples of oral iron preparations include ferrous citrate, ferric citrate, ferrous fumarate, soluble ferric pyrophosphate, dried ferrous sulfate, etc. Examples of intravenous iron preparations include ferric carboxymaltose, saccharified ferric oxide, etc. Furthermore, examples of therapeutic agents for diseases that cause anemia include, for example, myelofibrosis therapeutic agents, myelodysplastic syndrome therapeutic agents, chronic kidney disease therapeutic agents, anticancer agents, etc. Examples of therapeutic agents for myelofibrosis include JAK2 inhibitors. Examples of JAK2 inhibitors include ruxolitinib, fedratinib, pacritinib, momelotinib, etc., and combination use with ruxolitinib is preferred. Examples of therapeutic agents for myelodysplastic syndrome include azacitidine, lenalidomide, etc., and combination use with azacitidine or lenalidomide is preferred.Examples of therapeutic agents for chronic kidney disease include SGLT2 (sodium glucose cotransporter 2) inhibitors, renin-angiotensin system inhibitors (e.g., angiotensin II receptor blockers (ARBs), angiotensin-converting enzyme (ACE) inhibitors, etc.), calcium channel blockers, diuretics, antidiabetic drugs, antihyperlipidemic drugs, steroids, immunosuppressants, etc. Anticancer agents include chemotherapeutic agents, such as cisplatin, carboplatin, doxorubicin, paclitaxel, amrubicin, etc.

[0100] Furthermore, the RNAi-oligonucleotide of the present invention may be used in combination with a therapeutic agent for iron overload. When blood cell transfusions are performed as a treatment for anemia, iron overload may develop due to chronic blood cell transfusions. Iron overload is a pathological condition caused by the accumulation of iron derived from transfused blood cells in tissues such as the liver and heart, and may present with liver failure, heart failure, diabetes, joint pain, hypothyroidism, hypopituitarism, sexual dysfunction, and the like. Therefore, in the treatment of anemia, therapeutic agents for iron overload may be used to treat or prevent iron overload, and may be suitably used in combination with the RNAi-oligonucleotide of the present invention. Examples of therapeutic agents for iron overload include iron chelators, such as deferasirox, deferoxamine, and deferiprone.

[0101] <3. S3M-oligonucleotide> The present invention provides an RNAi-oligonucleotide (hereinafter referred to as "S3M-oligonucleotide") or a pharmaceutically acceptable salt thereof, wherein the sense strand region consists of an oligonucleotide represented by the following formula (I), the antisense strand region consists of an oligonucleotide represented by the following formula (II), and further has the following characteristics (a) to (g): Sense strand region: 5' S Ｏ５ -S ａ -S １９ -S １８ -S １７ -S １６ -S １５ -S １４ -S １３ -S１２ -S １１ -S １０ -S ９ -S ８ -S ７ -S ６ -S ５ -S ４ -S ３ -S ２ -S １ -S Ｏ３ 3' (I) Antisense strand region: 5' A Ｏ５ -A １ -A ２ -A ３ -A ４ -A ５ -A ６ -A ７ -A ８ -A ９ -A １０ -A １１ -A １２ -A １３ -A １４ -A １５ -A １６ -A １７ -A １８ -A １９ -A ａ -A Ｏ３ 3' (II) (a)S １ ~S １９ each represents one nucleoside, and S １ is a 3'-modified nucleoside. ａ is 0 to 10 nucleosides, preferably 0 to 5 or 0 to 4, more preferably 0 to 3, 0 to 2, or 0 to 1 nucleosides. Ｏ３ and S Ｏ５ are each independently 0 to 5, preferably 0 to 4, more preferably 0 to 3, 0 to 2, or 0 to 1 nucleosides. The bond between each nucleoside represents a phosphodiester bond which may be chemically modified; (b) A １ ~A １９ each represents one nucleoside, and A １１ , A １２ , A １３ , A １４ and A １５At least one of the is DNA, RNA, 2'-O,4'-C-bridged nucleoside or 2'-MOE RNA. ａ A is 0 to 10 nucleosides, preferably 0 to 5 or 0 to 4, more preferably 0 to 3, 0 to 2, or 0 to 1 nucleosides. Ｏ３ and A Ｏ５ are each independently 0 to 5, preferably 0 to 4, more preferably 0 to 3, 0 to 2, or 0 to 1 nucleosides. The bond between each nucleoside represents a phosphodiester bond which may be chemically modified; (c) A ２ ~A ａ The nucleotide sequence between A and B consists of a nucleotide sequence substantially complementary to the target sequence (target-corresponding sequence), １ is a nucleoside having a base complementary to the corresponding nucleoside of the target sequence, a nucleoside having adenine, a nucleoside having thymine, or a nucleoside having uracil; A Ｏ３ and A Ｏ５ (d) S １ ~S ａ The nucleotide sequence between １ ~A ａ The nucleotide sequence between is a complementary region consisting of nucleotide sequences that are substantially complementary to each other and form a double-stranded structure; (e) S Ｏ５ and A Ｏ３ If both exist, S Ｏ５ and A Ｏ３ are nucleosides that are not complementary to each other; (f) S Ｏ３ and A Ｏ５ If both exist, S Ｏ３ and A Ｏ５ are nucleosides that are not complementary to each other; and (g) the 5'-position of the 5'-terminal nucleoside and / or the 3'-position of the 3'-terminal nucleoside in the sense strand region and / or the 2'-position of the 3'-terminal nucleoside when the 3'-terminal nucleoside is a 3'-modified nucleoside may be chemically modified.

[0102] <3-1. Sense strand region> The sense strand region of the S3M-oligonucleotide is the region represented by the above formula (I), i.e., S Ｏ５ ~S Ｏ３ The complementary region in the sense strand region of the S3M-oligonucleotide is numbered S starting from the 3'-terminal nucleoside. １ ~S １９ and S located on the 5' side of the complementary region ａ That is, the complementary region in the sense strand of the S3M-oligonucleotide is represented by S in the above formula (I). １ ~S ａ This is the area of ​​S １ ~S １９ each represents one nucleoside, S ａ represents 0 to 5, preferably 0 to 4, more preferably 0 to 3, 0 to 2, or 0 to 1 nucleosides. Ｏ５ , and those added to the 3' end are S Ｏ３ and each represents 0 to 5, preferably 0 to 4, more preferably 0 to 3, 0 to 2, or 0 to 1 nucleosides.

[0103] The S3M-oligonucleotide is a nucleoside located at the 3' end of the complementary region in the sense strand region (i.e., S in the above formula (I)). １ ) is a 3'-modified nucleoside. Such a 3'-modified nucleoside is not particularly limited as long as the S3M-oligonucleotide has an RNA interference activity and / or a gene expression inhibitory activity, but is preferably 3'-deoxy-3'-fluoroRNA (3'-F RNA), 3'-OMe RNA, or 3'-MOE RNA, and more preferably 3'-OMe RNA. Furthermore, the 2'-position of this 3'-modified nucleoside may be a hydroxyl group, an optionally modified alkoxy group (preferably a methoxy group, an ethoxy group, or a methoxyethoxy group), or an optionally modified phosphate group (preferably a thiophosphate group, a dithiophosphate group, a methylphosphate group, or an ethylphosphate group), and an overhang structure (i.e., S in the above formula (I)) may be Ｏ３The 2'-position is preferably a hydroxyl group.

[0104] S3M - S of the complementary region in the sense strand region of the oligonucleotide １ The nucleosides other than the above are not particularly limited, and various natural nucleosides or sugar-modified nucleosides can be employed, but preferably, they are each independently 2'-OMe RNA, 2'-F RNA or DNA.

[0105] One embodiment of the complementary region in the sense strand region of the S3M-oligonucleotide is the 11th, 12th, 13th and 15th nucleosides from the 3' end (i.e., S in the above formula (I)). １１ , S １２ , S １３ and S １５ ) at least one of the nucleosides is 2'-F RNA, and the other nucleosides are 2'-OMe RNA, 2'-F RNA, DNA, or a combination thereof. １１ , S １２ , S １３ and S １５ are all 2'-F RNAs.

[0106] One preferred embodiment of the complementary region in the sense strand region of the S3M-oligonucleotide is ２ ~S １０ , S １４ , S １６ ~S １９ and S ａ are each independently 2'-OMe RNA, 2'-F RNA or DNA, or a modified pattern in which two types selected from them are alternately arranged, more preferably, are each independently 2'-OMe RNA or DNA, or a modified pattern in which they are alternately arranged, and even more preferably, all of them are 2'-OMe RNA. ａ The number of nucleosides can be appropriately selected from 0 to 10, and if necessary, from the 3' side, ２０ , S ２１ , S ２２ , S ２３, S ２４ , S ２５ , S ２６ , S ２７ , S ２８ , and S ２９ It may be displayed as.

[0107] The nucleotide sequence of the complementary region in the sense strand region of the S3M-oligonucleotide (i.e., S in the above formula (I) １ ~S ａ ) is a complementary region in the antisense region (i.e., A in the above formula (II) １ ~A ａ ) is a nucleotide sequence substantially complementary to

[0108] The sense strand region of the S3M-oligonucleotide has an overhang structure (i.e., S in the above formula (I)) at the 3' end and / or 5' end of its complementary region. Ｏ３ and S Ｏ５ ) may be included. Ｏ３ and S Ｏ５ each independently represent 0 to 5 (preferably 4, 3, 2, 1 or 0) nucleosides, each of which is independently 2'-OMe RNA, 2'-F RNA, DNA or a 2'-O,4'-C-bridged modified nucleoside. Ｏ３ , and S Ｏ５ does not exist, and

[0109] S Ｏ３ and S Ｏ５ When present, the base is not particularly limited as long as it functions as an overhang, but is preferably oligo-U or oligo-T.

[0110] The modification patterns of the sense strand region of the S3M-oligonucleotide are exemplified by the following formulas (Ia) to (Ic): 5' S Ｏ５ (N(M))-S ａ (N(M)-N(M))-S １９ (N(M))-S １８ (N(M))-S １７ (N(M))-S １６ (N(M))-S １５ (N(F))-S １４(N(M))-S １３ (N(F))-S １２ (N(F))-S １１ (N(F))-S １０ (N(M))-S ９ (N(M))-S ８ (N(M))-S ７ (N(M))-S ６ (N(M))-S ５ (N(M))-S ４ (N(M))-S ３ (N(M))-S ２ (N(M))-S １ (N(3M))-2’H 3’(Ia) 5’ S ａ (N(M))-S １９ (N(M))-S １８ (N(M))-S １７ (N(M))-S １６ (N(M))-S １５ (N(F))-S １４ (N(M))-S １３ (N(F))-S １２ (N(F))-S １１ (N(F))-S １０ (N(M))-S ９ (N(M))-S ８ (N(M))-S ７ (N(M))-S ６ (N(M))-S ５ (N(M))-S ４ (N(M))-S ３ (N(M))-S ２ (N(M))-S １ (N(3M))-2’H 3’(Ib) 5’ S １９ (N(M))-S １８ (N(M))-S １７ (N(M))-S １６ (N(M))-S １５ (N(F))-S １４ (N(M))-S １３ (N(F))-S １２ (N(F))-S １１ (N(F))-S １０ (N(M))-S ９ (N(M))-S ８ (N(M))-S ７(N(M))-S ６ (N(M))-S ５ (N(M))-S ４ (N(M))-S ３ (N(M))-S ２ (N(M))-S １ (N(3M))-2'H 3'(Ic) [In the above formula, (N(M)) represents 2'-OMe RNA, (N(F)) represents 2'-F RNA, and (N(3M)) represents 3'-OMe RNA. S Ｏ５ (N(M)) and S ａ (N(M)) represents 0 to 3 nucleosides consisting of 2'-OMe RNA. Two internucleoside bonds between the three nucleosides from the 5' and 3' ends of each oligonucleotide are phosphorothioate bonds, and the remaining internucleoside bonds are phosphodiester bonds. In other words, each oligonucleotide has two phosphorothioate bonds at each of the 3' and 5' ends, for a total of four.]

[0111] <3-2. Antisense strand region> The antisense strand region of the S3M-oligonucleotide is the region represented by the above formula (II), i.e., A Ｏ５ ~A Ｏ３ The complementary region in the antisense strand of the S3M-oligonucleotide is numbered from the 5'-terminal nucleoside to the A １ ~A １９ and A located on the 3' side of the complementary region ａ That is, the complementary region of the antisense strand is represented by A in the above formula (II). １ ~A ａ The nucleotide sequence of the complementary region in the antisense strand region of the S3M-oligonucleotide may be a nucleotide sequence that is substantially complementary to the target sequence, and may contain, for example, 5, 4, 3, 2, or 1 base mismatches with the target sequence, but is preferably completely complementary. １ ~A １９ each represents one nucleoside, A ａ represents 0 to 5 nucleosides. The overhang structure of the antisense strand region is added to the 5' end as A Ｏ５, and the one added to the 3' end is A Ｏ３ and displays:

[0112] A １ may be a base substantially complementary to the corresponding base in the target sequence, or may be adenine, uracil, or thymine, independent of the target sequence. Patent US10093923 and other publications have shown that when the base at the 3' end of the complementary region of the sense strand of an siRNA is adenine or uracil, regardless of the target sequence, it exhibits good RNA interference activity. Furthermore, a literature (Nature 465, 818-822 (2010)) has shown that the nucleoside at the 5' end of the antisense strand binds to the Ago2 protein, which is involved in RNA interference activity, but does not bind to the target mRNA. It has also been shown that when the nucleoside at the 5' end of the antisense strand is an adenine or uracil nucleoside, it binds more strongly to the Ago2 protein than when it is guanine or cytosine, so it is preferable that the 5' end of the antisense strand be an adenine or uracil nucleoside.

[0113] A of the complementary region in the antisense strand region of the S3M-oligonucleotide １１ , A １２ , A １３ , A １４ and A １５ Among these, at least one (preferably two or more) is selected from the group consisting of DNA, RNA, 2'-O,4'-C-bridged modified nucleosides, and 2'-MOE RNA. The 2'-O,4'-C-bridged modified nucleosides used herein are not particularly limited, but are preferably LNA or ENA. In the antisense strand complementary region, nucleosides other than those identified above are not particularly limited, and various natural nucleosides or sugar-modified nucleosides can be used, but are preferably each independently 2'-OMe RNA, 2'-F RNA, or DNA.

[0114] In one example of the present invention, the complementary region A in the antisense strand region of the S3M-oligonucleotide １４However, the oligonucleotide is RNA. Examples of the modification patterns of such oligonucleotides include, but are not limited to, types 56 and 57 in Figure 2, types 61, 62, 66, and 67 in Figure 3, types 75 to 83 in Figure 5, and types 91 to 95 in Figure 6.

[0115] A １４ A preferred embodiment of the antisense strand region of the oligonucleotide is RNA is １３ is a 2'-O,4'-C-bridged modified nucleoside, 2'-OMe RNA or 2'-F RNA, more preferably a 2'-O,4'-C-bridged modified nucleoside, even more preferably ENA or LNA (Types 76 to 83 in Figure 5 and Types 91 to 93 in Figure 6). １１ -A １２ -A １３ -A １４ -A １５ The modification pattern of is preferably any one of the following formulas (IIIa) to (IIIh): １１ (2'-OMe RNA)-A １２ (2'-OMe RNA)-A １３ (ENA)-A １４ (RNA)-A １５ (2'-OMe RNA) (IIIa) A １１ (2'-F RNA)-A １２ (2'-OMe RNA)-A １３ (ENA)-A １４ (RNA)-A １５ (2'-OMe RNA) (IIIb) A １１ (2'-OMe RNA)-A １２ (2'-F RNA)-A １３ (ENA)-A １４ (RNA)-A １５ (2'-OMe RNA) (IIIc) A １１ (2'-OMe RNA)-A １２ (2'-OMe RNA)-A １３ (LNA)-A １４ (RNA)-A １５ (2'-OMe RNA) (IIId) A１１ (2'-F RNA)-A １２ (2'-OMe RNA)-A １３ (LNA)-A １４ (RNA)-A １５ (2'-OMe RNA) (IIIe) A １１ (2'-OMe RNA)-A １２ (2'-F RNA)-A １３ (LNA)-A １４ (RNA)-A １５ (2'-OMe RNA) (IIIf) A １１ (2'-F RNA)-A １２ (2'-OMe RNA)-A １３ (2'-OMe RNA)-A １４ (RNA)-A １５ (2'-OMe RNA) (IIIg) A １１ (2'-OMe RNA)-A １２ (2'-F RNA)-A １３ (2'-OMe RNA)-A １４ (RNA)-A １５ (2'-OMe RNA) (IIIh)

[0116] In one example of the present invention, the complementary region A in the antisense strand region of the S3M-oligonucleotide １４ is a 2'-O,4'-C-bridged modified nucleoside. The 2'-O,4'-C-bridged modified nucleoside is preferably ENA or LNA. Modification patterns of such oligonucleotides include, but are not limited to, those shown in Types 70 to 74 in Figure 4 and Types 88 to 90 in Figure 6.

[0117] A １４ A preferred embodiment of the oligonucleotide in which is a 2'-O,4'-C-bridged modified nucleoside is １２ In such an S3M-oligonucleotide, the A of the antisense strand complementary region is １１ -A １２ -A １３ -A １４ -A １５The modification pattern of is preferably any one of the following formulae (IIIi) to (IIIl): １１ (2'-F RNA)-A １２ (DNA) -A １３ (2'-OMe RNA)-A １４ (ENA)-A １５ (2'-OMe RNA) (IIIi) A １１ (2'-OMe RNA)-A １２ (DNA)-A １３ (2'-OMe RNA)-A １４ (ENA)-A １５ (2'-OMe RNA) (IIIj) A １１ (2'-F RNA)-A １２ (DNA)-A １３ (2'-OMe RNA)-A １４ (LNA)-A １５ (2'-OMe RNA) (IIIk) A １１ (2'-OMe RNA)-A １２ (DNA)-A １３ (2'-OMe RNA)-A １４ (LNA)-A １５ (2'-OMe RNA) (IIIl)

[0118] In one example of the present invention, the complementary region A in the antisense strand region of the S3M-oligonucleotide １４ The present invention provides an oligonucleotide in which the nucleotide sequence is DNA. Examples of the modification patterns of such oligonucleotides include, but are not limited to, those shown in types 58 and 59 in Figure 2, and types 63, 64, 68, and 69 in Figure 3.

[0119] In one example of the present invention, the antisense strand region of the S3M-oligonucleotide is A of formula (II). １５ is 2'-MOE RNA. Examples of the modification pattern of such an oligonucleotide include, but are not limited to, those shown in types 79 and 83 in Figure 5.

[0120] In one example of the present invention, the 11th to 13th nucleosides from the 5' end of the complementary region in the antisense strand region of the S3M-oligonucleotide (A of formula (II)) １１ , A １２ , A １３ ) is a 2'-O,4'-C-bridged modified nucleoside. Examples of modification patterns of such oligonucleotides include, but are not limited to, ENA types 43 and 45 in Figure 1, types 75 to 84 in Figure 4, and types 85, 86, and 91 to 93 in Figure 6, and LNA types 44 and 46 in Figure 1, and types 67 and 69 in Figure 3.

[0121] In one example of the present invention, the 11th to 13th nucleosides from the 5' end of the complementary region in the antisense strand region of the S3M-oligonucleotide (A of formula (II)) １１ , A １２ , A １３ ) is DNA. Examples of modification patterns of such oligonucleotides include, but are not limited to, those shown in types 41 and 42 in Figure 1, type 71 in Figure 4, and types 87 to 90 in Figure 6.

[0122] In one example of the present invention, the 11th to 13th nucleosides from the 5' end of the complementary region in the antisense strand region of the S3M-oligonucleotide (A of formula (II)) １１ , A １２ , A １３ ) is 2'-MOE RNA. Examples of modification patterns of such oligonucleotides include, but are not limited to, those shown in types 47 and 48 in Figure 1 and type 74 in Figure 4.

[0123] In the complementary region of the antisense strand region of the S3M-oligonucleotide of the present invention, various types of nucleosides other than the nucleosides specified above can be appropriately selected. ２ , A ６ and A １６At least one (preferably all) nucleoside of the antisense strand is 2'-F RNA. ２ , A ６ and A １６ In addition, A ８ , A ９ and A １０ One or two nucleosides selected from the group consisting of are 2'-F RNA, and nucleosides other than those specified above (e.g., A １ , A ３ ~A ５ , A ７ , A １７～ A １９ and A ａ ) is 2'-OMe RNA or DNA.

[0124] In the complementary region of the antisense strand region of the S3M-oligonucleotide of the present invention, A １１ ~A １５ Preferred modification patterns for regions other than the above are exemplified in, for example, Tables A-1 and A-2 below.

[0125]

[0126]

[0127] [In the above table, (M) is 2'-OMe RNA and (F) is 2'-F RNA. ａ A is 0 to 5 nucleosides, and all nucleosides other than 0 are the same. Ｏ３ represents 0 to 5 nucleosides, and all nucleosides other than 0 are the same. The internucleoside bond is appropriately selected from a phosphodiester bond or a phosphorothioate bond.]

[0128] The antisense strand region contained in the S3M-oligonucleotide has an overhang structure (i.e., A in the above formula (II)) at the 3' end and / or 5' end of its complementary region. Ｏ３ and A and A Ｏ５ ) may be present. Ｏ３ and A Ｏ５each independently represent 0 to 5 (preferably 3, 2, 1 or 0) nucleosides, each of which is independently 2'-OMe RNA, 2'-F RNA, DNA or a 2'-O,4'-C-bridged modified nucleoside. Ｏ３ is an overhang consisting of two or three 2'-OMe RNAs, or an overhang consisting of one or two 2'-OMe RNAs and one or two 2'-O,4'-C-bridged modified nucleosides, and A Ｏ５ does not exist.

[0129] A Ｏ３ and A Ｏ５ When present, the base is not particularly limited as long as it functions as an overhang, but is preferably oligo-U or oligo-T.

[0130] <3-3. Internucleoside Bonds> The bond between each nucleoside of the S3M-oligonucleotide of the present invention can be appropriately selected from optionally chemically modified phosphodiester bonds, and is preferably a phosphodiester bond or a phosphorothioate bond, either alone or in combination. Preferably, a combination of a phosphodiester bond or a phosphorothioate bond is used, and the content of phosphorothioate bonds in the internucleoside bond in each of the sense strand region and the antisense strand region is 50% or less, 40% or less, 30% or less, 29% or less, 28% or less, 27% or less, 26% or less, or 25% or less.

[0131] When RNA and / or 2'-F RNA is used, the bond between the nucleoside and its adjacent nucleoside on the 3' side may be a phosphorothioate bond. Preferably, when RNA is used, the bond between the nucleoside and its adjacent nucleoside on the 3' side is a phosphorothioate bond.

[0132] In the S3M-oligonucleotide of the present invention, the first, second, third, fourth, or fifth internucleoside bond (1, 2, 3, or 4 internucleoside bonds) from the 5'-end and / or 3'-end of the oligonucleotide is preferably a phosphorothioate bond. In the oligonucleotide of the present invention, when the sense strand region and the antisense strand region are contained in different oligonucleotides, the internucleoside bond between the first, second, third, fourth, or fifth nucleotide from the 5'-end and 3'-end of the sense strand region, and the internucleoside bond between the first, second, third, fourth, or fifth nucleotide from the 5'-end and 3'-end of the antisense strand region may be a phosphorothioate bond, and preferably, the internucleoside bond between the first, second, and third nucleotides from the 5'-end and 3'-end of the sense strand region (two internucleoside bonds), the internucleoside bond between the first, second, and third nucleotides from the 5'-end of the antisense strand region (two internucleoside bonds), and the internucleoside bond between the first, second, and third nucleotides from the 3'-end of the antisense strand region (three internucleoside bonds) are phosphorothioate bonds.

[0133] 4. Oligonucleotide Synthesis Method The method for preparing the oligonucleotide of the present invention is not particularly limited as long as it allows the synthesis of the desired oligonucleotide, and known chemical synthesis methods (such as the phosphate triester method, phosphoramidite method, or H-phosphonate method) can be used. For example, the oligonucleotide can be synthesized using a commercially available nucleic acid synthesizer and commercially available reagents used for DNA / RNA synthesis.

[0134] By the conventional phosphoramidite method, an oligonucleotide having a desired nucleotide sequence can be synthesized using a DNA synthesizer, such as Perkin-Elmer's Model 392 phosphoramidite method, according to the method described in the literature (Nucleic Acids Research, 12, 4539 (1984)).

[0135] Amidite reagents corresponding to various nucleosides may be purchased commercially or may be synthesized according to known methods.

[0136] The oligonucleotides of the present invention can be synthesized using a commercially available synthesizer (e.g., Perkin-Elmer Model 392 using the phosphoramidite method) according to the method described in the literature (Nucleic Acids Research, 12, 4539 (1984)). Regarding the phosphoramidite reagents used, commercially available phosphoramidite reagents can be used for natural nucleosides and 2'-O-methylnucleosides (i.e., 2'-O-methylguanosine, 2'-O-methyladenosine, 2'-O-methylcytidine, and 2'-O-methyluridine). The following phosphoramidite reagents are used for 2'-O-alkylguanosine, 2'-O-alkyladenosine, 2'-O-alkylcytidine, and 2'-O-alkyluridine, each of which has a 2- to 6-carbon atom 2'-O-alkyl group.

[0137] 2'-O-aminoethylguanosine, 2'-O-aminoethyladenosine, 2'-O-aminoethylcytidine, and 2'-O-aminoethyluridine can be synthesized by synthesizing the corresponding phosphoramidite reagents according to the literature (Blommers et al., Biochemistry (1998), 37, 17714-17725).

[0138] For 2'-O-propylguanosine, 2'-O-propyladenosine, 2'-O-propylcytidine, and 2'-O-propyluridine, the corresponding phosphoramidite reagents can be synthesized according to the literature (Lesnik, EA et al., Biochemistry (1993), 32, 7832-7838).

[0139] Commercially available phosphoramidite reagents can be used for 2'-O-allylguanosine, 2'-O-allyl adenosine, 2'-O-allyl cytidine, and 2'-O-allyl uridine.

[0140] For 2'-MOE RNA, 2'-O-methoxyethylguanosine, 2'-O-methoxyethyladenosine, 2'-O-methoxyethyl-5-methylcytidine, and 2'-O-methoxyethyl-5-methyluridine can be synthesized using the corresponding phosphoramidite reagents according to a patent (US6261840) or the literature (Martin, P. Helv. Chim. Acta. (1995) 78, 486-504.), or commercially available phosphoramidite reagents can also be used.

[0141] 2'-O-butylguanosine, 2'-O-butyladenosine, 2'-O-butylcytidine, and 2'-O-butyluridine can be synthesized as corresponding phosphoramidite reagents according to the literature (Lesnik, EA et al., Biochemistry (1993), 32, 7832-7838).

[0142] For 2'-O-pentylguanosine, 2'-O-pentyladenosine, 2'-O-pentylcytidine, and 2'-O-pentyluridine, the corresponding phosphoramidite reagents can be synthesized according to the literature (Lesnik, EA et al., Biochemistry (1993), 32, 7832-7838).

[0143] Commercially available phosphoramidite reagents can be used for 2'-O-propargylguanosine, 2'-O-propargyladenosine, 2'-O-propargylcytidine, and 2'-O-propargyluridine.

[0144] For 2'-F RNA, 2'-deoxy-2'-fluoroguanosine, 2'-deoxy-2'-fluoroadenosine, 2'-deoxy-2'-fluorocytidine, and 2'-deoxy-2'-fluorouridine can be synthesized using the corresponding phosphoramidite reagents according to the literature (J. Med. Chem. 36, 831 (1993)), or commercially available phosphoramidite reagents can also be used.

[0145] For the 3'-modified nucleoside 3'-F RNA, the corresponding phosphoramidite reagents can be synthesized according to WO2017027645 for 3'-deoxy-3'-fluoroguanosine, WO2018198076 for 3'-deoxy-3'-fluoroadenosine, and US2011 / 0196141 for 3'-deoxy-3'-fluorouridine. 3'-Deoxy-3'-fluorocytidine can be synthesized by reference to known methods such as those described in the above-mentioned literature.

[0146] Regarding 3'-modified nucleosides, 3'-OMe RNA, 3'-O-methylguanosine, 3'-O-methyladenosine, 3'-O-methylcytidine, and 3'-O-methyluridine can be synthesized using commercially available phosphoramidite reagents.

[0147] For 3'-modified nucleosides such as 3'-MOE RNA, 3'-O-methoxyethylguanosine, 3'-O-methoxyethyladenosine, 3'-O-methoxyethyl-5-methylcytidine, and 3'-O-methoxyethyl-5-methyluridine, the corresponding phosphoramidite reagents can be synthesized according to US2003 / 0096979.

[0148] For LNAs, 2'-O,4'-C-methyleneguanosine, 2'-O,4'-C-methyleneadenosine, 2'-O,4'-C-methylenecytidine, 2'-O,4'-C-methylene-5-methylcytidine, and 2'-O,4'-C-methylene-5-methyluridine, the corresponding phosphoramidite reagents can be prepared according to the method described in WO99 / 14226.

[0149] For 2'-O,4'-C-alkyleneguanosine, 2'-O,4'-C-alkyleneadenosine, 2'-O,4'-C-alkylenecytidine, 2'-O,4'-C-alkylene-5-methylcytidine, and 2'-O,4'-C-alkylene-5-methyluridine, each of which has 2 to 5 carbon atoms in the alkylene group of the 2'-O,4'-C-bridged moiety, the corresponding phosphoramidite reagents can be prepared according to the method described in WO 00 / 47599.

[0150] The 2'-deoxy-2'-C,4'-C-methyleneoxymethylenated nucleoside of D-ribofuranose can be synthesized into the corresponding phosphoramidite reagent according to the literature (Wang, G. et al. Tetrahedron (1999), 55, 7707-7724).

[0151] S-cEt (constrained ethyl) can be synthesized into the corresponding phosphoramidite reagent according to the literature (Seth, PP et al. J. Org. Chem (2010), 75, 1569-1581.).

[0152] AmNA can be synthesized by synthesizing the corresponding phosphoramidite reagent according to the literature (Yahara, A. et al. ChemBioChem (2012), 13, 2513-2516.) or WO2014 / 109384.

[0153] Antisense oligonucleotides having phosphorothioate bonds can be synthesized by coupling a phosphoramidite reagent followed by reaction with a reagent such as sulfur, tetraethylthiuram disulfide (TETD, Applied Biosystems), Beaucage reagent (Glen Research), or xanthan hydride (Tetrahedron Letters, 32, 3005 (1991), J. Am. Chem. Soc. 112, 1253 (1990), PCT / WO98 / 54198).

[0154] The solid phase supports used in the synthesizer, such as controlled pore glass (CPG) and polystyrene, to which 2'-O-methylnucleosides and 3'-O-methylnucleosides are bound, can be commercially available. Nucleosides produced according to the method described in WO 99 / 14226 can be linked to CPG for 2'-O,4'-C-methyleneguanosine, 2'-O,4'-C-methyleneadenosine, 2'-O,4'-C-methylene-5-methylcytidine, and 2'-O,4'-C-methylene-5-methyluridine, each of which has an alkylene group of 2 to 5 carbon atoms, using nucleosides produced according to the method described in WO 00 / 47599 (Oligonucleotide Synthesis, Edited by M.J. Gait, Oxford University Press, 1984). Alternatively, oligonucleotides can be synthesized by coupling the aforementioned phosphoramidite reagent using a universal resin. Furthermore, modified CPG (described in Example 12b of JP-A-7-87982) can be used to synthesize oligonucleotides with a 2-hydroxyethyl phosphate group attached to the 3' end. Furthermore, 3'-amino-Modifier C3 CPG, 3'-amino-Modifier C7 CPG, Glyceryl CPG (Glen Research), 3'-specer C3 SynBase CPG 1000, or 3'-specer C9 SynBase CPG 1000 (Link Technologies) can be used to synthesize oligonucleotides with a hydroxyalkyl phosphate group or an aminoalkyl phosphate group attached to the 3' end.

[0155] Furthermore, when thioating an oligonucleotide analogue, a thioate derivative can be obtained in accordance with the method described in the literature (Tetarhedron Letters, 32, 3005 (1991), J. Am. Chem. Soc., 112, 1253 (1990)) using a reagent such as tetraethylthiuram disulfide (TETD, Applied Biosystems), Beaucage reagent, or phenylacetyl disulfide / pyridine-acetonitrile (1:1 v / v) solution (Ravikumar, V.T. et al. Bioorg.Med.Chem.Lett. (2006) 16, pp. 2513-2517) in addition to sulfur.

[0156] When a silyl protecting group such as t-butyldimethylsilyl (TBS) is used as the protecting group for the 2'-hydroxyl group of RNA, the protecting group can be deprotected using tetrabutylammonium fluoride (TBAF), HF-pyridine, HF-triethylamine, or the like.

[0157] The acyl group at the base moiety and the cyanoethyl group at the phosphate moiety can be deprotected using concentrated aqueous ammonia, methanolic ammonia, ethanolic ammonia, a mixture of concentrated aqueous ammonia and ethanol (3:1 V / V), a mixture of concentrated aqueous ammonia and 40% aqueous methylamine (1:1 V / V), methylamine, 0.5 M aqueous LiOH, a mixture of 3.5 M triethylamine / methanol (1:10 V / V), 0.05 M potassium carbonate in methanol, or the like, with concentrated aqueous ammonia and a mixture of concentrated aqueous ammonia and ethanol (3:1 V / V) being preferred.

[0158] The oligonucleotide of the present invention can be obtained by purifying it using a purification procedure typically used for purifying nucleic acids, such as various types of chromatography, such as reverse phase chromatography and ion exchange chromatography (including high performance liquid chromatography).

[0159] The oligonucleotides of the present invention include oligonucleotides conjugated with a transport unit for transporting the oligonucleotide to a target tissue or target cell. Examples of oligonucleotides conjugated with such a transport unit include oligonucleotides in which a unit having an aminoalkyl phosphate group (e.g., an alkyl having 3 to 9 carbon atoms) introduced into a desired chemical structure is conjugated to the 5' position of the 5'-terminal nucleotide and / or the 3' position of the 3'-terminal nucleotide (the 2' position for nucleotides modified at the 3' position). Examples of such transport units include GalNAc units that bind to the asialoglucosyl receptor (ASGRP) of liver parenchymal cells, and fatty acid units (e.g., myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, etc.) for transport to liver or muscle tissue (see, for example, Nucleic Acids Res. (2020) 47, 6029-6044, WO2017 / 19267, etc.).

[0160] A GalNAc unit is a structural unit containing N-acetyl-D-galactosamine (GalNAc) that can be bound to ASGRP and can be used to deliver the oligonucleotide of the present invention to the liver. The GalNAc unit may have a linear or branched linker structure, for example, a linear, two or more-branched, three or more-branched, or four or more-branched, four or less-branched, three or less-branched, or two or less-branched linker structure. The GalNAc unit may also contain a phosphate group or a thiophosphate group for binding to an oligonucleotide. As long as the ability to bind to ASGRP is maintained, there is no limit to the number of GalNAc units contained in one GalNAc unit, and the GalNAc structure may be modified. Examples of GalNAc units that can be used in the oligonucleotides of the present invention include those described in WO2021 / 049504, WO2009 / 073809, WO2014 / 076196, WO2014 / 179620, WO2015 / 006740, WO2015 / 105083, WO2016 / 0556012017 / 023817, WO2017 / 084987, WO2017 / 131236, Methods in Enzymology, 1999, Vol. 313, pp. 297-321, and Bioorganic & Medicinal Chemistry Letters 26 (2016). Known GalNAc units such as 3690-3693 can be appropriately employed.

[0161] For example, a GalNAc unit that can be used includes the following formula, which can be appropriately prepared according to the method described in WO2021 / 049504.

[0162]

[0163] (In the above formula, the dashed line represents a bond, and the oxygen atom of the bond represents bonding to an adjacent nucleotide; when bonding to the 5'-terminal nucleotide, it represents forming a phosphodiester bond with the 5'-phosphate group, and when bonding to the 3'-terminal nucleotide, it represents forming a phosphodiester bond with the 3'-phosphate group (the 2'-phosphate group in a nucleotide modified at the 3'-position). The phosphodiester bond formed may be a chemically modified phosphodiester bond such as a phosphorothioate bond.)

[0164] The oligonucleotide of the present invention includes oligonucleotides into which a cholesterol unit, a lipid unit, or a vitamin E unit has been introduced (e.g., Lorenz, C. et al. Bioorg. Med. Chem. Lett., 14, pp. 4975-4977 (2004); Soutschek, J., et al. Nature, 432, pp. 173-178, (2004); Wolfrum, C. et al. Nature Biotech. 25, pp. 1149-1157, (2007)), Kubo, T. et al. Oligonucleotides, 17, pp. 1-20, (2007); Kubo, T., et al. Biochem. Biophys. Res. Comm. 365, pp. 54-61, (2008); Nishina, K., et al., Mol. Ther. 16, pp. 734-740, (2008). ) and oligonucleotides having an aptamer, a nucleic acid molecule that binds to proteins, attached to the end of the oligonucleotide.

[0165] The oligonucleotides of the present invention also include those bound to a monoclonal antibody (or a suitable binding portion thereof) or a protein (or a suitable oligopeptide fragment thereof) (see, for example, Song, et al. Nature Biotech. 23, pp. 709-717 (2005); Xia et al. Pharm. Res. 24, pp. 2309-2316 (2007); Kumar, et al. Nature, 448, pp. 39-43 (2007)). The oligonucleotides of the present invention also include those which are positively charged complexes formed by adding a cationic polymer to the oligonucleotide (see Leng et al. J. Gen. Med. 7, pp. 977-986 (2005), Baigude et al. 2, pp. 237-241, ACS Chem. Biol. (2007), Yadava et al. Oligonucleotide 17, pp. 213-222 (2007) for examples in which distribution to organs and cells has been achieved).

[0166] The oligonucleotides of the present invention include any pharmaceutically acceptable salts, esters, or salts of such esters of the above-mentioned oligonucleotides. Preferred pharmaceutically acceptable salts of the oligonucleotides of the present invention include metal salts such as alkali metal salts (e.g., sodium salt, potassium salt, and lithium salt), alkaline earth metal salts (e.g., calcium salt and magnesium salt), aluminum salt, iron salt, zinc salt, copper salt, nickel salt, and cobalt salt; inorganic salts (e.g., ammonium salt), t-octylamine salt, dibenzylamine salt, morpholine salt, glucosamine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt, pipette salt, and the like. Examples of the salt include amine salts, such as organic salts like azine salts, tetramethylammonium salts, and tris(hydroxymethyl)aminomethane salts; inorganic acid salts like hydrohalogen salts like hydrofluoride, hydrochloride, hydrobromide, and hydroiodide, nitrate, perchlorate, sulfate, and phosphate; organic acid salts like lower alkanesulfonates like methanesulfonate, trifluoromethanesulfonate, and ethanesulfonate, arylsulfonates like benzenesulfonate and p-toluenesulfonate, acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, and maleate; and amino acid salts like glycine salt, lysine salt, arginine salt, ornithine salt, glutamate, and aspartate.

[0167] The oligonucleotide of the present invention or a pharmaceutically acceptable salt thereof can suppress the expression of TfR2 mRNA. Furthermore, the oligonucleotide of the present invention or a pharmaceutically acceptable salt thereof can suppress the production of hepcidin, which plays a central role in iron metabolism, by suppressing the expression of TfR2 mRNA. While the mechanism of action is not particularly limited, the oligonucleotide of the present invention or a pharmaceutically acceptable salt thereof can be used to treat or prevent diseases that can be treated or prevented by suppressing the expression of TfR2 mRNA and / or the production of hepcidin. In a preferred embodiment, when administered to an anemic patient, the oligonucleotide can suppress the expression of TfR2 mRNA, and an effect of improving anemia symptoms can be expected. Furthermore, the oligonucleotide of the present invention or a pharmaceutically acceptable salt thereof can also be used as a pharmaceutical composition containing the oligonucleotide or a pharmaceutically acceptable salt thereof as an active ingredient.

[0168] Pharmaceutical compositions containing the oligonucleotides of the present invention may be mixed, encapsulated, or conjugated with other molecules, molecular structures, or compound mixtures, for example, as liposomes, receptor-targeting molecules, orally, rectally, topically, or other formulations to aid uptake, distribution, and / or absorption. When the oligonucleotides of the present invention are used as prophylactic or therapeutic agents for diseases, the oligonucleotides or pharmaceutically acceptable salts thereof may be administered orally as tablets, capsules, granules, powders, syrups, or parenterally as injections, suppositories, patches, or topical preparations.

[0169] These preparations contain excipients (e.g., sugar derivatives such as lactose, sucrose, glucose, mannitol, and sorbitol; starch derivatives such as corn starch, potato starch, alpha starch, and dextrin; cellulose derivatives such as crystalline cellulose; gum arabic; dextran; organic excipients such as pullulan; and silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, calcium silicate, and magnesium aluminometasilicate; phosphates such as calcium hydrogen phosphate; carbonates such as calcium carbonate; and calcium sulfate). Examples of suitable excipients include inorganic excipients such as sulfates, lubricants (e.g., metal stearates such as stearic acid, calcium stearate, and magnesium stearate; talc; colloidal silica; waxes such as beeswax and Gay's wax; boric acid; adipic acid; sulfates such as sodium sulfate; glycol; fumaric acid; sodium benzoate; DL-leucine; lauryl sulfates such as sodium lauryl sulfate and magnesium lauryl sulfate; silicic acids such as anhydrous silicic acid and hydrated silicic acid; and the above-mentioned starch derivatives. Examples of the additives include: binders (for example, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, polyvinylpyrrolidone, macrogol, and compounds similar to the excipients described above); disintegrants (for example, cellulose derivatives such as low-substituted hydroxypropyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose calcium, and internally cross-linked sodium carboxymethyl cellulose; and chemically modified starches and celluloses such as carboxymethyl starch, carboxymethyl starch sodium, and cross-linked polyvinylpyrrolidone); emulsifiers (for example, colloidal clays such as bentonite and Veegum; metal hydroxides such as magnesium hydroxide and aluminum hydroxide; anionic surfactants such as sodium lauryl sulfate and calcium stearate; cationic surfactants such as benzalkonium chloride; and nonionic surfactants such as polyoxyethylene alkyl ethers, polyoxyethylene sorbitan fatty acid esters, and sucrose fatty acid esters.The composition is produced by a well-known method using additives such as stabilizers (including paraoxybenzoic acid esters such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol, and phenylethyl alcohol; benzalkonium chloride; phenols such as phenol and cresol; thimerosal; dehydroacetic acid; and sorbic acid), flavoring agents (including, for example, commonly used sweeteners, acidulants, fragrances, etc.), and diluents.

[0170] The recipient into which the oligonucleotide prepared as described above is introduced is not particularly limited as long as the target gene can be transcribed into RNA within the cell. The recipient refers to a cell, tissue, or individual.

[0171] The cells into which the oligonucleotides of the present invention are introduced may be any of germline cells, somatic cells, totipotent cells, pluripotent cells, dividing cells, non-dividing cells, parenchymal cells, epithelial cells, immortalized cells, transformed cells, neural cells, and immune cells.

[0172] Tissues include single-cell embryos or constitutive cells, or multi-cell embryos, fetal tissues, etc. Furthermore, examples of the differentiated cells include adipocytes, fibroblasts, muscle cells, cardiac muscle cells, endothelial cells, nerve cells, glia, blood cells, megakaryocytes, lymphocytes, macrophages, neutrophils, eosinophils, basophils, mast cells, leukocytes, granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts, hepatocytes, and cells of endocrine or exocrine glands. Examples of such cells that are preferably used include CHO-K1 cells (RIKEN Cell bank), Drosophila S2 cells (Schneider, I. et al., J. Embryol. Exp. Morph., 27, pp. 353-365 (1972)), human HeLa cells (ATCC: CCL-2), and human HEK293 cells (ATCC: CRL-1573).

[0173] Furthermore, specific examples of individuals to be used as recipients of the oligonucleotides of the present invention include plants, animals, protozoa, viruses, bacteria, and fungi. Plants may be monocotyledonous, dicotyledonous, or gymnosperms, and animals may be vertebrates or invertebrates. Preferred vertebrates are mammals, including mice, rats, monkeys, dogs, and humans.

[0174] When the recipient is a cell or tissue, the oligonucleotide of the present invention can be introduced into the recipient by calcium phosphate, electroporation, lipofection, viral infection, immersion in a 3L5-oligonucleotide solution, transformation, or the like. Methods for introducing the oligonucleotide into an embryo include microinjection, electroporation, viral infection, and the like. When the recipient is a plant, injection, perfusion, or spraying into the body cavity or interstitial cells of the plant body can be used. When the recipient is an animal, systemic introduction can be achieved by oral, topical, subcutaneous, intramuscular, or intravenous administration, parenteral, vaginal, rectal, nasal, ocular, or intramembrane administration, or by electroporation or viral infection. Oral introduction can also be achieved by directly mixing the oligonucleotide of the present invention with the food of the organism.

[0175] In addition to the above, a colloidal dispersion system can be used to deliver the oligonucleotide of the present invention to a patient. Colloidal dispersion systems are expected to enhance the stability of compounds in vivo and to efficiently transport compounds to specific organs, tissues, or cells. The colloidal dispersion system is not limited to a commonly used one, and examples thereof include lipid-based dispersion systems including polymer complexes, nanocapsules, microspheres, beads, and oil-in-water emulsions, micelles, mixed micelles, and liposomes. Preferably, the colloidal dispersion system is a plurality of liposomes or artificial membrane vesicles, which are effective in efficiently transporting compounds to specific organs, tissues, or cells (Mannino et al., Biotechniques, 1988, 6, p. 682-; Blume and Cevc, Biochem. et Biophys. Acta, 1990, 1029, p. 91-; Lappalainen et al., Antiviral Res., 1994, 23, p. 119-; Chonn and Cullis, Current Op. Biotech., 1995, 6, p. 698-). Unilamellar liposomes with a size range of 0.2-0.4 μm can encapsulate a significant proportion of aqueous buffer containing macromolecules, and the compounds are encapsulated in this aqueous inner membrane and transported to brain cells in a biologically active form (Fraley et al., Trends Biochem. Sci., 1981, 6, p. 77-).

[0176] Liposome compositions are typically lipids, particularly phospholipids, especially high-phase-transition-temperature phospholipids, usually complexed with one or more steroids, especially cholesterol. Examples of lipids useful in liposome production include phosphatidyl compounds such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, sphingolipids, phosphatidylethanolamine, cerebrosides, and gangliosides.

[0177] Particularly useful are diacylphosphatidylglycerols, in which the lipid moiety contains 14-18 carbon atoms, particularly 16-18 carbon atoms, and is saturated (lacking a double bond within the 14-18 carbon atom chain).

[0178] Representative phospholipids include phosphatidylcholine, dipalmitoylphosphatidylcholine, and distearoylphosphatidylcholine.

[0179] Targeting of colloidal dispersion systems, including liposomes, can be either passive or active.

[0180] Passive targeting is achieved by taking advantage of the natural tendency of liposomes to distribute to reticuloendothelial cells of organs containing sinusoidal capillaries.

[0181] On the other hand, active targeting can be achieved by, for example, attaching specific ligands to liposomes, such as viral protein coats (Morishita et al., Proc. Natl. Acad. Sci. (U.S.A.), 1993, 90, p. 8474-), monoclonal antibodies (or suitable binding portions thereof), sugars, glycolipids, or proteins (or suitable oligopeptide fragments thereof), or by modifying liposomes by changing their composition to achieve distribution to organs and cell types other than their naturally occurring localization site.

[0182] The surface of targeted colloidal dispersion systems can be modified in various ways. In liposome-targeted delivery systems, lipid groups can be incorporated into the lipid bilayer of the liposome to maintain the targeting ligand in close association with the lipid bilayer. Various linking groups can be used to link the lipid chains to the targeting ligand.

[0183] The targeting ligand that binds to a specific cell surface molecule found predominantly on cells to which delivery of the oligonucleotides of the invention is desired can be, for example, (1) a hormone, growth factor, or appropriate oligopeptide fragment thereof, that binds to a specific cellular receptor that is predominantly expressed by the cells to which delivery is desired, or (2) a polyclonal or monoclonal antibody, or appropriate fragment thereof (e.g., Fab; F(ab')2), that specifically binds to an antigenic epitope found predominantly on the target cells. Two or more bioactive agents can also be combined within a single liposome and administered.

[0184] Agents that enhance the intracellular stability and / or targeting of the contents can also be added to the colloidal dispersion system.

[0185] The dosage of the oligonucleotide of the present invention or a pharmaceutically acceptable salt thereof varies depending on symptoms, age, etc., but it is desirable to administer the following to an adult 1 to 3 times per day depending on symptoms: oral administration: a lower limit of 1 mg (preferably 30 mg) and an upper limit of 2000 mg (preferably 1500 mg); intravenous or subcutaneous administration: a lower limit of 0.5 mg (preferably 5 mg) and an upper limit of 1000 mg (preferably 250 mg); intratracheal administration: a lower limit of 0.5 mg (preferably 5 mg) and an upper limit of 500 mg (preferably 250 mg); and intraocular administration: a lower limit of 0.05 mg (preferably 0.5 mg) and an upper limit of 10 mg (preferably 5 mg). In addition, in the case of a drug with enhanced stability, it is desirable to administer it 1 to 3 times a week, and in the case of a drug with even enhanced stability, it is desirable to administer it 1 to 3 times every 1 month, 3 months, 6 months, 12 months, 18 months, or 24 months, depending on the symptoms.

[0186] Pharmaceutical compositions and formulations for topical administration include transdermal patches, ointments, lotions, creams, gels, lozenges, suppositories, sprays, liquids and powders.

[0187] The oligonucleotides of the present invention can suppress the expression of TfR2 mRNA. Furthermore, the oligonucleotides of the present invention have low toxicity to cells and can be used as highly safe pharmaceuticals. Furthermore, by suppressing the expression of TfR2 mRNA, the oligonucleotides of the present invention can suppress the production of hepcidin, which plays a central role in iron metabolism. Furthermore, by suppressing the expression of TfR2 mRNA, the oligonucleotides of the present invention can increase plasma iron concentration and / or HGB levels. While the mechanism of action is not particularly limited, the oligonucleotides of the present invention can be used to treat or prevent diseases that can be treated or prevented by suppressing the expression of TfR2 mRNA and / or the production of hepcidin. Examples of such diseases include anemia. While the mechanism of action is not particularly limited, the RNAi-oligonucleotides of the present invention can treat or prevent anemia, for example, by increasing HGB levels. Furthermore, the RNAi-oligonucleotides of the present invention can reduce the frequency and / or amount of blood cell transfusions in anemic patients who require blood cell transfusions. For example, the frequency and / or volume of blood cell transfusions for an anemic patient over a period of time (e.g., 1 day, 1 week, 2 weeks, 4 weeks, 8 weeks, 12 weeks, 1 month, 2 months, 3 months, 6 months, or 1 year) may be reduced by about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%.

[0188] The present invention provides inventions for medicinal uses, such as a pharmaceutical composition containing the oligonucleotide of the present invention as an active ingredient and used for treating or preventing anemia, a method for treating or preventing anemia comprising administering an effective amount of the oligonucleotide, the oligonucleotide for use in treating or preventing anemia, and use of the oligonucleotide of the present invention in the manufacture of a pharmaceutical composition for treating or preventing anemia. Examples of anemia that can be treated or prevented by the oligonucleotides of the present invention include anemia of chronic inflammation (ACD), anemia associated with bone marrow failure, iron deficiency anemia, iron refractory iron deficiency anemia (IRIDA), anemia resistant to erythropoietin stimulating agents (ESAs), chemotherapy-induced anemia, aplastic anemia, Diamond-Blackfan anemia, and hereditary hemoglobin disorders such as β-thalassemia or sickle cell disease. Examples of anemia associated with chronic inflammation include anemia associated with autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, or autoimmune hemolytic anemia, anemia associated with infectious diseases, anemia associated with inflammatory bowel diseases such as inflammatory colitis, anemia associated with heart failure, anemia associated with chronic kidney disease, cancer-related anemia, anemia associated with macrophage activation syndrome, and anemia associated with Castleman's disease.Examples of anemia associated with chronic kidney disease include kidney disease associated with diabetes, kidney disease associated with hypertension, nephrotic syndrome (e.g., Finnish-type congenital nephrotic syndrome, diffuse mesangial sclerosis, minimal change nephrotic syndrome, focal segmental glomerulosclerosis, membranous nephropathy, Galloway-Mowat syndrome, etc.), kidney disease associated with chronic glomerulonephritis (e.g., IgA nephropathy, mesangial proliferative glomerulonephritis, membranoproliferative glomerulonephritis, purpura nephritis, anti-glomerular basement membrane nephritis (Goodpasture syndrome), etc.), and kidney disease associated with chronic glomerulonephritis (e.g., IgA nephropathy, mesangial proliferative glomerulonephritis, membranoproliferative glomerulonephritis, purpura nephritis, anti-glomerular basement membrane nephritis (Goodpasture syndrome)). Examples of anemia include anemia associated with chronic kidney diseases such as idiopathic pulmonary fibrosis (IPF), Alport syndrome, Epstein syndrome, lupus nephritis, microscopic polyangiitis, atypical hemolytic uremic syndrome, Nail-Patella syndrome, fibronectin nephropathy, and lipoprotein glomerulopathy, kidney disease with chronic tubulointerstitial nephritis, kidney disease with chronic pyelonephritis, kidney disease with amyloid deposition, autosomal dominant tubulointerstitial kidney disease, kidney disease with nephronophthisis, and kidney disease with renal malformation (including, for example, polycystic kidney disease). Examples of anemia associated with cancer include anemia associated with cancers such as multiple myeloma, acute leukemia, chronic leukemia, lung cancer, and breast cancer. Examples of anemia associated with bone marrow failure include anemia associated with myelofibrosis, anemia associated with myelodysplastic syndrome, anemia associated with chronic myelomonocytic leukemia (CMML), anemia associated with bone marrow suppression due to chemotherapy, aplastic anemia, Diamond-Blackfan anemia, and Fanconi anemia.

[0189] The oligonucleotides of the present invention can be preferably used for the treatment or prevention of anemia associated with chronic inflammation and anemia associated with bone marrow failure, and more preferably for the treatment or prevention of anemia associated with autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, or autoimmune hemolytic anemia, anemia associated with infectious diseases, anemia associated with inflammatory bowel diseases such as inflammatory bowel disease, anemia associated with heart failure, anemia associated with chronic kidney disease, anemia due to cancer, anemia associated with Castleman's disease, anemia associated with myelofibrosis, anemia associated with myelodysplastic syndrome, anemia associated with chronic myelomonocytic leukemia, and anemia associated with bone marrow suppression due to chemotherapy.

[0190] The present invention provides a method for treating or preventing anemia, comprising using the oligonucleotide of the present invention in combination with one or more other drugs. The oligonucleotide of the present invention may be used in combination with one or more other drugs as long as the effects of the present invention are achieved. Preferably, the oligonucleotide of the present invention can be used in combination with other drugs that have a different mechanism of action against anemic symptoms to provide a greater therapeutic effect. For example, for myelodysplastic syndrome (MDS), drugs such as ESAs (erythropoiesis stimulating agents) that treat anemia by promoting red blood cell proliferation, luspatercept that treats anemia by promoting red blood cell differentiation, and azacitidine that inhibits DNA methylation and kills abnormal cells are sometimes prescribed, but these drugs have a different mechanism of action from TfR2 siRNA. Therefore, the combination of these drugs with TfR2 siRNA can provide greater therapeutic effects against MDS than single drugs. Furthermore, for anemia associated with chronic kidney disease, drugs such as ESAs and HIFPHD (hypoxia inducible factor prolyl hydroxylase) inhibitors, which have a mechanism of action that treats anemia by promoting red blood cell proliferation, are sometimes prescribed, but these drugs have a different mechanism of action from TfR2 siRNA. Therefore, the combination of these drugs with TfR2 siRNA can provide greater therapeutic effects against anemia associated with chronic kidney disease than single drugs. Furthermore, ruxolitinib is known to be effective against myelofibrosis, primarily for symptoms such as splenomegaly (N Engl J Med 2012; 366:799-807). However, its effects on anemia symptoms are limited or ineffective, or it is known to worsen anemia (Journal of Hematology & Oncology 2013, 6:79). The mechanism by which ruxolitinib worsens anemia is thought to be that inhibiting JAK2 suppresses the EPO signaling required for red blood cell proliferation.However, since Ruxolitinib has demonstrated a certain therapeutic effect on symptoms of myelofibrosis other than anemia, a combination of Ruxolitinib and TfR2 siRNA may provide a useful therapeutic effect on anemia in myelofibrosis. Similarly, a combination of Ruxolitinib with other JAK2 inhibitors other than Ruxolitinib may also provide a useful therapeutic effect on anemia in myelofibrosis. Examples of other JAK2 inhibitors include fedratinib, pacritinib, and momelotinib.

[0191] When the oligonucleotide of the present invention is used in combination with other drugs, the order of administration of the RNAi-oligonucleotide of the present invention is not particularly limited as long as the effects of the present invention are achieved. The RNAi-oligonucleotide of the present invention may be administered simultaneously with the other drug, or before or after the other drug. Examples of drugs that can be used in combination with the RNAi-oligonucleotide of the present invention include other anemia therapeutic drugs or therapeutic drugs for diseases that cause anemia. Examples of other anemia therapeutic drugs include ESAs, HIFPHD (hypoxia inducible factor prolyl hydroxylase) inhibitors, oral iron preparations, intravenous iron preparations, danazol, and Luspatercept. Examples of ESAs include epoetin alpha, epoetin beta, epoetin beta pegol, epoetin kappa, and darbepoetin alpha. Examples of HIFPHD inhibitors include roxadustat, vadadustat, daprodustat, enarodustat, molidustat, etc. Examples of oral iron preparations include ferrous citrate, ferric citrate, ferrous fumarate, soluble ferric pyrophosphate, dried ferrous sulfate, etc. Examples of intravenous iron preparations include ferric carboxymaltose, saccharified ferric oxide, etc. Furthermore, examples of therapeutic agents for diseases that cause anemia include, for example, myelofibrosis therapeutic agents, myelodysplastic syndrome therapeutic agents, chronic kidney disease therapeutic agents, anticancer agents, etc. Examples of therapeutic agents for myelofibrosis include JAK2 inhibitors. Examples of JAK2 inhibitors include ruxolitinib, fedratinib, pacritinib, momelotinib, etc., and combination use with ruxolitinib is preferred. Examples of therapeutic agents for myelodysplastic syndrome include azacitidine, lenalidomide, etc., and combination use with azacitidine or lenalidomide is preferred.Examples of therapeutic agents for chronic kidney disease include SGLT2 (sodium glucose cotransporter 2) inhibitors, renin-angiotensin system inhibitors (e.g., angiotensin II receptor blockers (ARBs), angiotensin-converting enzyme (ACE) inhibitors, etc.), calcium channel blockers, diuretics, antidiabetic drugs, antihyperlipidemic drugs, steroids, immunosuppressants, etc. Anticancer agents include chemotherapeutic agents, such as cisplatin, carboplatin, doxorubicin, paclitaxel, amrubicin, etc.

[0192] In addition, the oligonucleotide of the present invention may be used in combination with a therapeutic agent for iron overload. When blood cell transfusion is performed to treat anemia, iron overload may occur due to chronic blood cell transfusion. Iron overload is a pathological condition caused by the accumulation of iron derived from transfused blood cells in tissues such as the liver and heart, and may present with liver failure, heart failure, diabetes, arthralgia, hypothyroidism, hypopituitarism, sexual dysfunction, etc. Therefore, in the treatment of anemia, a therapeutic agent for iron overload may be used to treat or prevent iron overload, and may be suitably used in combination with the oligonucleotide of the present invention. Examples of therapeutic agents for iron overload include iron chelators, such as deferasirox, deferoxamine, and deferiprone.

[0193] (Examples 1 to 82) The compounds of Examples 1 to 82 shown in Table 1 are double-stranded oligonucleotides composed of a sense strand region and an antisense strand region, each of which is an independent oligonucleotide, and the complementary regions of the sense strand region and the antisense strand region are all composed of natural RNA (i.e., A(R), G(R), C(R), and U(R)). In addition, each internucleoside bond is a chemically unmodified phosphodiester bond. The antisense strand region in the compound of each Example has a complementary region consisting of a sequence completely complementary to each target sequence (the base sequence of the region sandwiched between the start position and the end position) listed in Table 1, and is an oligonucleotide in which two consecutive Ts (Ds) are bound as an overhang structure to the 3' end of the complementary region. In addition, the sense strand region in the compound of each Example has a complementary region consisting of a sequence completely complementary to the nucleotide sequence in the complementary region of the corresponding antisense strand region, and is an oligonucleotide in which two consecutive Ts (Ds) are bound as an overhang structure to the 3' end of the complementary region. The compound of Example 61 is an siRNA against human TfR2 (hTfR2) described in WO2012 / 177921, AD-47826, in which all 19 base pairs are natural RNA, and two consecutive Ts (Ds) are bound to the 3'-end of each of the sense strand and antisense strand regions as an overhang structure.

[0194] Each compound in Table 1 was synthesized using the phosphoramidite method (Nucleic Acids Research, 12, 4539 (1984), Nature Communications 6, Article number: 6317 (2015)). The compounds of each example were identified by negative ion ESI mass spectrometry, and the results are shown in Table 1. Furthermore, for each compound of each example, equimolar amounts of the sense strand region and antisense strand region, which were individually synthesized, were placed in one tube and annealed, and it was confirmed that they formed double-stranded oligonucleotides in a native gel.

[0195]

[0196]

[0197]

[0198] In the table, the "start position of target sequence" and "end position of target sequence" each indicate the position of a base in the base sequence of human TfR2 mRNA shown in SEQ ID NO: 1. The compound of each Example is a double-stranded oligonucleotide whose target sequence is the base sequence sandwiched between these two. That is, the antisense strand region of each Example has a sequence complementary to the base sequence sandwiched between the start and end positions of the target sequence, and the sense strand region has a sequence complementary to the antisense strand region. For example, the sequence of the complementary region of the sense strand in the compound of Example 19 (TfR2-019) is 5' CGUGGAGUUUCAAUAUCAA 3' (SEQ ID NO: 43), and the sequence of the complementary region of the antisense strand is 5' UUGAUAUUGAAACUCCACG 3' (SEQ ID NO: 44). The sequence of the complementary region of the sense strand in the compound of Example 39 (TfR2-039) is 5' ACCUCAAAGCCGUAGUGUA 3' (SEQ ID NO: 45), and the sequence of the complementary region of the antisense strand is 5' UACACUACGGCUUUGAGGU 3' (SEQ ID NO: 46). Two consecutive Ts (Ds) are attached to the 3' end of each complementary region in the sense strand and antisense strand of each compound of each Example in Table 1, forming an overhang structure. The 5' position of the 5'-terminal nucleotide and the 3' position of the 3'-terminal nucleotide of each oligonucleotide are hydroxyl groups, with hydrogen atoms bonded to the oxygen atoms in the structural diagram of each nucleoside described above. The "molecular weight" in the table indicates the actual value measured by negative ion electron spin-on ion mass spectrometry.

[0199] (Examples 83 to 184) The compounds of Examples 83 to 133 shown in Table 2 are single-stranded oligonucleotides in which the 3'-position (2'-position in the case of a nucleotide modified at the 3'-position) of the 3'-terminal nucleotide of the sense strand region and the 5'-position of the 5'-terminal nucleotide of the antisense strand region are linked by a linker represented by the above-mentioned formula (Z), and a double-stranded structure is formed by the complementary region of the sense strand region and the complementary region of the antisense strand region. Furthermore, the compounds of Examples 134 to 184 shown in Table 3 are double-stranded oligonucleotides having a sense strand region and an antisense strand region which are independent oligonucleotides.

[0200] The compounds of Example 180 (TfR2-019.94DUG.s1) and Example 181 (TfR2-019.94DUG.s2) are siRNAs in which the overhang structure at the 3' end of the antisense strand region of the compound of Example 169 (TfR2-019.94DUG) is either one nucleotide long or absent, respectively. The compounds of Example 182 (TfR2-019.94DUG.e1) and Example 183 (TfR2-019.94DUG.e2) are siRNAs whose target sequences are the same as the target sequence of the compound of Example 19 (TfR2-019) (i.e., the sequence from bases 2847 to 2865 of SEQ ID NO: 1) but extended by one or two nucleotides from the 5' end (i.e., the sequence from bases 2846 to 2865 of SEQ ID NO: 1 and the sequence from bases 2845 to 2865 of SEQ ID NO: 1, respectively), and the lengths of the complementary regions are 20 bp and 21 bp, respectively. The compound of Example 184 (TfR2-019.94DUG.e3) is an siRNA in which the overhang structure at the 3' end of the antisense strand region of the compound of Example 169 (TfR2-019.94DUG) is G(M)U(M).

[0201] The compounds of Examples 83 to 184 shown in Tables 2 and 3 were synthesized in the same manner as in Examples 1 to 82. In the sequences, the "2" portion was synthesized using DMT-ethane-Diol phosphoramidite (ChemGene, catalog number: CLP-2250), and the "Z" portion was synthesized using the method described in Example 12 of WO2012 / 074038. The "2'-O-methylnucleoside" portion was synthesized using the phosphoramidite described in Nucleic Acids Research 17, 3373 (1989). The "DNA" portion was synthesized using the phosphoramidite described in Nucleic Acids Research 11, 4539 (1984). The "3'-O-methylnucleoside" moiety was synthesized using 3'-O-methyl adenosine (n-bz) CED phosphoramidite (ChemGene, Catalog No.: ANP-2901), 3'-O-methyl cytidine (n-bz) CED phosphoramidite (ChemGene, Catalog No.: ANP-2902), 3'-O-methyl guanosine (n-ibu) CED phosphoramidite (ChemGene, Catalog No.: ANP-2903), or 3'-O-methyl uridine CED phosphoramidite (ChemGene, Catalog No.: ANP-2904). The "2'-O,4'-C-methylenenucleoside" moiety was synthesized using the phosphoramidites described in WO99 / 14226. The "2'-deoxy-2'-fluoronucleoside" portion was synthesized using the phosphoramidite described in J. Med. Chem. 36, 831 (1993). The "2'-O-methoxyethylnucleoside" portion was synthesized using the phosphoramidite described in Helv. Chim. Acta 78, 486-504 (1995). The "GN" portion was synthesized using the phosphoramidite described in Reference Example 41 of WO2019 / 172286.For the compounds of Examples 134 to 179, the base sequences of the sense strand regions are shown in Tables 3-1 to 3-5, and the base sequences of the antisense strand regions are shown in Tables 3-6 to 3-10. For the compounds of Examples 180 to 184, the base sequences of the sense strand regions are shown in Table 3-11, and the base sequences of the antisense strand regions are shown in Table 3-12.

[0202] The compounds of Examples 134 to 184 were prepared by placing equimolar amounts of the sense strand region and the antisense strand region in one tube and annealing them, and then using a native gel or HPLC, it was confirmed that they formed double-stranded oligonucleotides.

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222]

[0223]

[0224]

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235] The "molecular weight" in the table indicates the actual measured value by negative ion ESI mass spectrometry. In the "base sequence" in the table, N(R) indicates RNA, N(D) indicates DNA, N(M) indicates 2'-OMe RNA, N(m) indicates 2'-MOE RNA, N(3M) indicates 3'-OMe RNA, N(F) indicates 2'-F RNA, N(L) indicates LNA, and N(E) indicates ENA. The structure of each nucleoside is represented by the structural formula described above.

[0236] In the table, "GN" represents a GalNAc unit represented by the formula (GN) described above. In the table, "2" represents -O(CH2)2O- (represented by the formula (2) described above), and "Z" represents -O(CH2)8NHC(=O)(CH2)2PhO- (represented by the formula (Z) described above). Regarding the internucleoside bond or the bond between a nucleoside and the GalNAc unit, "2" or "Z," "^" in the table represents a phosphorothioate bond (-P(=S)(OH)-, represented by the internucleoside bond structural diagram (PS) described above). Unless otherwise specified, this represents a phosphodiester bond (-P(=O)(OH)-, represented by the internucleoside bond structural diagram (P) described above).

[0237] The 5' position of the 5'-terminal nucleotide and the 3' position of the 3'-terminal nucleotide (2' position for nucleotides with a modified 3' position) of each oligonucleotide have a hydrogen atom bonded to an oxygen atom in the structural diagram of each nucleoside shown above, forming a hydroxyl group. When "2" is the terminal, in the structural diagram of "2" shown above, a hydrogen atom is bonded to the oxygen atom on the side of the bond that is not bonded to the oligonucleotide. When "^" is the terminal, in the structural diagram of "PS" shown above, a hydroxyl group is bonded to the phosphorus atom. However, when "GN" is the terminal, no hydrogen atom bond is required.

[0238] Test Example 1: Screening of TfR2 siRNA in HepG2 cells by forward transfection The following experiment was performed using the compounds of Examples 1 to 82 as TfR2 siRNA. The day before transfection, 2.5 x 10^4 HepG2 cells per well were seeded into a 96-well plate (Corning) in 100 μL of DMEM medium (Gibco) supplemented with 10% FBS (Gibco). On the day of transfection, two wells of each type of siRNA sample, two wells of a negative control (NT) containing distilled water instead of siRNA, and two wells of TfR2-061 (Example 61) as a positive control were prepared. To prepare the reagent for transfection of one siRNA sample, 50 μL of Opti-MEM (Life Technology) and 3 μL of RNAiMAX (Thermo Fisher Scientific) were mixed, to which 50 μL of Opti-MEM and 1 μL of siRNA (concentration: 10 μM) were added and incubated at room temperature for 15 minutes. Then, 1 mL of DMEM containing 10% FBS, which had been prepared in advance and warmed to 37°C, was added. 110 μL of this mixture was added to a 96-well plate seeded with HepG2 cells after removing the medium. The final concentration of siRNA was 10 nM in all samples. To prepare the negative control reagent, 50 μL of Opti-MEM (Life Technology) and 3 μL of RNAiMAX (Thermo Fisher Scientific) were mixed, to which 50 μL of Opti-MEM and 1 μL of distilled water were added, and the mixture was incubated at room temperature for 15 minutes.

[0239] Two days after transfection, the cells were washed once with PBS (Thermo Fisher Scientific) and RNA was extracted. RNA was extracted using the RNeasy Mini Kit (Quagen) according to the kit's protocol. 300 ng of the resulting RNA was reverse transcribed using the High Capacity RNA to cDNA kit (Applied Biosystems) to synthesize cDNA. The resulting cDNA was used to measure the amount of endogenous hTfR2 mRNA by quantitative PCR. The amount of human 18S ribosomal RNA (h18S) mRNA was measured as a housekeeping gene.

[0240] Quantitative PCR was performed using IDT PrimeTime Gene Expression Master Mix (Integrated DNA Technologies) as follows: The obtained cDNA was diluted 10-fold with distilled water (15 μL of cDNA and 135 μL of distilled water were mixed). Per well of a 384-well PCR plate (Applied Biosystems), 5 μL of IDT PrimeTime Gene Expression Master Mix, 0.5 μL of hTfR2 primer (Hs.PT.58.20599434, Integrated DNA Technologies), 0.5 μL of h18S primer (Hs.PT.39a.22214856.g, Integrated DNA Technologies), 2 μL of distilled water, 2 μL of cDNA were mixed (total 10 μL), and two wells were prepared for each cDNA, and quantitative PCR was performed. hTfR2 and h18S mRNA expression levels were measured as the average value between two wells. The hTfR2 mRNA expression level was normalized to the mRNA expression level of the housekeeping gene h18S. The relative hTfR2 mRNA expression level of each siRNA-treated sample was calculated and expressed as a relative value when the hTfR2 mRNA expression level in the negative control was set to 1.0. The results are shown in Table 4 below.

[0241]

[0242]

[0243]

[0244] Test Example 1-1: Evaluation of cytotoxicity using HepG2 cells Cytotoxicity was evaluated 4 days after siRNA was introduced into HepG2 cells using the reverse transfection method in the same manner as in Test Example 2. TfR2-061 (Example 61), TfR2-019 (Example 19), and TfR2-039 (Example 39) were used as siRNAs.

[0245] The cytotoxicity of siRNA against liver cells was tested using Cell Counting Kit-8 (CCK8, Dojindo Laboratories). After removing the culture supernatant, a solution prepared by dissolving 10 μL of CCK8 reagent in 100 μL of DMEM containing 10% FBS was added at 110 μL / well. After that, the cells were incubated at 37°C for 30 minutes in CO ２ The cells were incubated in an incubator. The absorbance at OD450 nm was then measured using a microplate reader. The OD450 nm values ​​in the negative control (NT) wells were set at 1.0, and the OD450 nm values ​​in the wells treated with each siRNA were calculated and expressed as relative values. The results are shown in Figure 11.

[0246] This result indicates that TfR2-019 and TfR2-039 in particular have low toxicity to liver cells.

[0247] Test Example 2: Screening of TfR2 siRNA in HepG2 cells by reverse transfection The following experiment was carried out using the compounds of each example as TfR2 siRNA. siRNA screening was carried out by reverse transfection as follows. On the day of transfection, two wells of each type of siRNA sample, two wells of a negative control (NT) in which distilled water was used instead of siRNA, and two wells of TfR2-061 (Example 61) or siRNA against hTfR2 (AD-52590) described in WO2012 / 177921 were prepared as positive controls.

[0248] 5 μL of siRNA was added to a mixture of 14.8 μL of Opti-MEM and 0.2 μL of RNAiMAX per well and incubated at room temperature for 20 minutes. For negative control reagent preparation, 5 μL of distilled water was added to the mixture of Opti-MEM and RNAiMAX instead of siRNA. For positive control reagent preparation, 5 μL of TfR2-061 (Example 61) or AD-52590 was added to the mixture of Opti-MEM and RNAiMAX. Next, 20 μL of the mixture containing siRNA, negative control, or positive control was added to a 96-well cell culture plate (SUMIRON). HepG2 cells (2 x 10^4 cells / mL) prepared using DMEM containing 10% FBS pre-warmed to 37°C were seeded into the 96-well plate at 80 μL per well. The final concentration of siRNA was adjusted to a common ratio of 10 or 50 times from 10 nM.

[0249] One or two days after transfection, cells were washed once with PBS. RNA was extracted and reverse-transcribed using the Superprep Cell Lysis RT Kit for qPCR (Takara Bio Inc.) according to the kit's protocol to synthesize cDNA. Endogenous hTfR2 mRNA expression was measured by quantitative PCR using the resulting cDNA. h18S mRNA expression was measured as a housekeeping gene.

[0250] Quantitative PCR was performed using IDT PrimeTime Gene Expression Master Mix (Integrated DNA Technologies) as follows: The obtained cDNA was used as a stock solution. 5 μL of IDT PrimeTime Gene Expression Master Mix, 0.25 μL of hTfR2 primer (Hs.PT.58.20599434, Integrated DNA Technologies) or 0.25 μL of h18S primer (Hs.PT.39a.22214856.g, Integrated DNA Technologies), 2.75 μL of distilled water, and 2 μL of cDNA were mixed per well of a 384-well PCR plate (Applied Biosystems) (total 10 μL), and two wells were prepared for each cDNA, and quantitative PCR was performed. The mRNA expression levels of hTfR2 and h18S were measured as the average value between two wells. The mRNA expression level of hTfR2 was normalized by the mRNA expression level of h18S, a housekeeping gene. The relative hTfR2 mRNA expression level of the samples treated with each siRNA was calculated and expressed as a relative value when the hTfR2 mRNA expression level in the negative control was set to 1.0. The results are shown in Tables 5 to 20 below.

[0251]

[0252]

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261]

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282] Test Example 2-1: hTfR2 knockdown activity using HepG2 cells The level of hTfR2 mRNA expression was evaluated 2 days after siRNA was introduced into HepG2 cells using the reverse transfection method in the same manner as in Test Example 2. TfR2-019 (Example 19) and TfR2-019.94DUG (Example 169) were used as siRNAs.

[0283] The hTfR2 mRNA expression level in the negative control (NT) was set to 1.0, and the relative hTfR2 mRNA expression level in the samples treated with each siRNA was calculated and expressed as a value relative to that in the negative control (NT). The results are shown in Figure 12.

[0284] This result indicates that TfR2-019.94DUG in particular has high knockdown activity against hTfR2 mRNA.

[0285] Test Example 3: Single-dose administration test of TfR2 siRNA to male cynomolgus monkeys Plasma iron and hepcidin concentrations were measured after subcutaneous administration of TfR2-039.5G (Example 127) or TfR2-039.23DUG (Example 144) at a dose of 30 mg / kg. Plasma iron concentrations were measured using a Metalloassay iron measurement kit (Metallogenics). Plasma hepcidin concentrations were measured using LC / MS. The results of administration of TfR2-039.5G (Example 127) are shown in Table 21, and the results of administration of TfR2-039.23DUG (Example 144) are shown in Table 22.

[0286]

[0287]

[0288] Test Example 4: Preparation of siRNA-encapsulated nucleic acid lipid particles using TfR2 siRNA (1) Preparation of siRNA-encapsulated nucleic acid lipid particles Distearoylphosphatidylcholine (1,2-Distearoyl-sn-glycero-3-phosphocholine: hereinafter referred to as DSPC, NOF CORPORATION), cholesterol (Cholesterol: hereinafter referred to as Chol, Sigma-Aldrich, Inc.), (7R,9Z)-18-({[3-(dimethylamino)propyloxy]carbonyl}oxy)octacos-9-en-7-yl acetate (the compound described in Example 28 of WO2015 / 005253) (hereinafter referred to as LP), and 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol having a molecular weight of about 2000 were used. Glycol (1,2-Dimyristoyl-sn-Glycero-3-Methoxypolyethylene Glycol, hereinafter referred to as PEG-DMG, NOF CORPORATION) was dissolved in ethanol at a molar ratio of 10:48:40:2 to a total lipid concentration of 10 mM.

[0289] On the other hand, AD-47882 (an siRNA against mouse TfR2 described in WO2012 / 177921) was diluted with citrate buffer (20 mM citrate buffer, pH 4.0) as mouse TfR2 siRNA.

[0290] The lipid solution and siRNA solution were mixed in a microchannel using a NanoAssembler BenchTop (Precision Nanosystems Inc.) so that the total lipid weight ratio to siRNA was 11 and the volume ratio was 1:3, to obtain a crude dispersion of nucleic acid-lipid particles. The nucleic acid-lipid particle dispersion was dialyzed (Float-A-Lyzer G2, MWCO: 1,000 kD, Spectra / Por) for 12 to 18 hours against approximately 25 to 50 times the amount of phosphate buffer, thereby removing ethanol and obtaining a dispersion of purified siRNA-encapsulated nucleic acid-lipid particles. LP was synthesized according to the method described in Example 28 of WO2015 / 005253.

[0291] (2) Characterization of siRNA-Encapsulated Nucleic Acid-Lipid Particles The dispersion containing the nucleic acid-lipid particles prepared in (1) was subjected to the following characterization. (2-1) Encapsulation Rate of siRNA The encapsulation rate of siRNA was measured using a Quant-iT RiboGreen RNA Assay kit (Invitrogen) in accordance with the package insert. That is, the amount of siRNA in the dispersion of nucleic acid-lipid particles was quantified in the presence and absence of 0.015% Triton X-100 surfactant, and the encapsulation rate was calculated using the following formula: Encapsulation rate (%) = {([Amount of siRNA in the presence of surfactant] - [Amount of siRNA in the absence of surfactant]) / [Amount of siRNA in the presence of surfactant]} x 100 (%)

[0292] (2-2) Ratio of siRNA to Lipid The nucleic acid-lipid particle dispersion was diluted with 1.0% Triton X-100 and the amount of siRNA in the nucleic acid-lipid particle dispersion was measured by ion exchange chromatography (System: Agilent 1260 series, Column: Dionex BioLC DNAPac PA200 (8 μm, 4.0 mm × 250 mm) (ThermoFisher Scientific) Buffer A: 10 mM Tris-HCl buffer, 25 mM sodium perchlorate, 20% ethanol (pH 7.0) Buffer B: 10 mM Tris-HCl buffer, 250 mM sodium perchlorate, 20% ethanol (pH 7.0) (B%): 20-70% (15 min) Flow Rate: 0.5 mL / min, Temperature: 40°C, Detection: 260 nm).

[0293] The nucleic acid-lipid particle dispersion was diluted with ethanol, and the amount of each lipid in the nucleic acid-lipid particle dispersion was measured by reverse phase chromatography (System: DIONEX UltiMate 3000, Column: XSelect CSH C18 (130 Å, 3.5 μm, 3.0 mm × 150 mm) (Waters catalog # 186005263), Buffer A: 0.2% formic acid, Buffer B: 0.2% formic acid, methanol, (B%): 75-95% (15 min), 95% (2 min), Flow Rate: 0.45 mL / min, Temperature: 50°C, Detection: Corona CAD (Charged Aerosol) The ratio of the total lipid amount to the siRNA was calculated using the following formula: Total lipid amount to siRNA (wt / wt) = [total lipid concentration] / [siRNA concentration] (wt / wt)

[0294] (2-3) Average particle size The particle size of the nucleic acid-lipid particles was measured using a Zeta Potential / Particle Sizer NICOMP™ 380ZLS (PARTICLE SIZING SYSTEMS). The average particle size in the table represents the volume average particle size, and ± indicates deviation. The results of each characteristic evaluation are shown in Table 23.

[0295]

[0296] From the above results, it was revealed that these nucleic acid-lipid particles had approximately 95% of the siRNA encapsulated within the lipid particles and had an average particle size of approximately 90 nm.

[0297] Test Example 5: Efficacy evaluation test of nucleic acid lipid particles encapsulating TfR2 siRNA in a mouse model of anemia associated with bone marrow dysfunction The following experiment was performed using nucleic acid lipid particles encapsulating AD-47882 (an siRNA against mouse TfR2 described in WO 2012 / 177921) as nucleic acid lipid particles encapsulating mouse TfR2 siRNA. The nucleic acid lipid particles were prepared in the same manner as in Test Example 4. To induce bone marrow dysfunction, 100 mg / kg of carboplatin (Tokyo Chemical Industry Co., Ltd.) was administered intraperitoneally (i.p.) to male C57BL / 6NJcl mice (CLEA Japan, Inc.). As a control group (n=5) in which bone marrow dysfunction was not induced, PBS was administered i.p. instead of carboplatin. Four days after Carboplatin administration, the animals were grouped based on plasma iron concentration and HGB values, and then drug treatment was performed (n=5 / group). The drugs were AD-47882, administered intravenously (i.v.) once as nucleic acid-lipid particles at 0.5 mg / kg (0.5 mpk) and 0.25 mg / kg (0.25 mpk). The vehicle control group received PBS i.v. instead of the drug. Blood was collected from the tail vein before administration of AD-47882 (Day 0), and 3 days (Day 3), 7 days (Day 7), 10 days (Day 10), and 11 days (Day 11) after administration of AD-47882, and HGB levels were measured using a QuantiChrom Whole Blood HB kit (Bioassay Systems). The HGB level results are shown in Figure 7C. Plasma iron and hepcidin levels were measured before administration of AD-47882 (Day 0), 3 days (Day 3), and 7 days (Day 7) after administration. Plasma iron levels were measured using a Metalloassay iron measurement kit (Metalogenix). Plasma hepcidin concentrations were measured using LC / MS. The results of plasma iron and plasma hepcidin concentrations are shown in Figures 7A and 7B, respectively.Eleven days after AD-47882 treatment (Day 11), blood was collected from the abdominal vena cava under isoflurane anesthesia, and mean corpuscular hemoglobin (MCH) values ​​were measured using an XT2000 automated hematology analyzer (Sysmex Corporation). The MCH values ​​are shown in Figure 7D.

[0298] Mice were euthanized by exsanguination, and their livers were collected to measure the mRNA expression levels of mouse TfR2 (mTfR2) and mouse Hepcidin (mHepcidin). RNA was extracted using the RNeasy Mini Kit (QUAGEN) according to the kit's protocol. 5,000 ng of the resulting RNA was reverse-transcribed using the High Capacity RNA to cDNA kit (Applied Biosystems). The cDNA obtained was then used to measure the mRNA levels of endogenous mouse TfR2 and Hepcidin by quantitative PCR.

[0299] Quantitative PCR was performed using IDT PrimeTime Gene Expression Master Mix (Integrated DNA Technologies) as follows: The obtained cDNA was diluted 10-fold with distilled water (15 μL of cDNA and 135 μL of distilled water were mixed). 5 μL of IDT PrimeTime Gene Expression Master Mix, 0.25 μL of mouse TfR2 (mTfR2) primer (Mm.PT.58.7860185, Integrated DNA Technologies), or 0.25 μL of mouse Hepcidin (mHepcidin) primer (Mm.PT.58.43563393.g, Integrated DNA Technologies) were placed per well of a 384-well PCR plate (Applied Biosystems). The cDNA was mixed with 2.75 μL of distilled water and 2 μL of cDNA (total 10 μL), and two wells were prepared for quantitative PCR. Mouse β-actin (mActb) primers (Mm.PT.39a.22214843.g, Integrated DNA Technologies) were used as the housekeeping gene. The primers were mixed at the ratios described above, and two wells were prepared for quantitative PCR. The mRNA levels of mTfR2, mHepcidin, and mActb were measured as the average between two wells. The mRNA expression levels of mTfR2 and mHepcidin were normalized to the mRNA expression level of the housekeeping gene mActb. The relative mRNA levels in the vehicle and AD-47882-treated groups were calculated and expressed as mean ± standard error (SEM) relative to the mRNA level in the control group, which had not been induced to have bone marrow dysfunction. The results for the mRNA expression levels of mTfR2 and mHepcidin in the liver are shown in Figures 7E and 7F, respectively.

[0300] These results suggest that suppressing TfR2 mRNA expression through TfR2 siRNA treatment may increase HGB levels and provide a therapeutic effect against anemic diseases such as anemia associated with bone marrow failure, e.g., anemia associated with myelofibrosis.

[0301] Test Example 6: Efficacy Evaluation of GalNAc-Conjugated TfR2 siRNA in a Mouse Model of Anemia Associated with Bone Marrow Decline The following experiment was performed using AD47882.23DUG (Example 171) as the GalNAc-conjugated TfR2 siRNA. To induce bone marrow decline, 100 mg / kg of carboplatin was administered intraperitoneally (i.p.). As a control group (n=5) in which bone marrow decline was not induced, PBS was administered i.p. instead of carboplatin. Four days after carboplatin administration, mice were divided into groups based on HGB values ​​and then treated with the drug (n=9 or 10 / group). The group treated with GalNAc-conjugated TfR2 siRNA received a single subcutaneous dose of 3 mg / kg (3 mpk). For the positive control group, Fc-conjugated ActRIIB (ActRIIB-Fc), a drug that traps activin receptor type IIB (ActRIIB) ligands, was subcutaneously administered at a dose of 10 mg / kg every 3 or 4 days for a total of three times. ActRIIB-Fc is a protein preparation in which the extracellular domain of Luspatercept (ACE-536) is bound to the Fc domain of mouse IgG2a (WO2016090188, Blood. 2014; 123 (25): 3864-3872), and is known to promote erythroid differentiation by suppressing Smad2 / 3 signaling and to exhibit therapeutic effects on beta thalassemia model mice and myelodysplastic syndrome model mice (Blood. 2014; 123 (25): 3864-3872, Nature Medicine volume 20, pages 408-414 (2014)). In the vehicle group, PBS was administered subcutaneously a total of three times every 3 to 4 days. For the group treated with a combination of GalNAc-conjugated TfR2 siRNA and ActRIIB-Fc, siRNA was administered subcutaneously once and ActRIIB-Fc was administered subcutaneously a total of three times as described above.

[0302] Blood was collected from the tail vein before drug treatment (Day 0), 3 days after treatment (Day 3), and 8 days after treatment (Day 8), and HGB levels were measured using a QuantiChrom Whole Blood HB kit (Bioassay Systems). The HGB results are shown in Figure 8A (mean ± standard error). Figure 8B shows the HGB levels 3 days after drug treatment, demonstrating the combined effect of GalNAc-conjugated TfR2 siRNA and ActRIIB-Fc. Nine days after drug treatment (Day 9), blood was collected from the abdominal vena cava under isoflurane anesthesia, and HGB, MCH, hematocrit (HCT), and red blood cell (RBC) counts were measured using an XT2000 automated hematology analyzer (Sysmex Corporation). The results are shown in Figures 8D to 8G (mean ± standard error). After euthanasia, the mice were exsanguinated, and their livers were collected to measure mTfR2 mRNA expression levels. RNA extraction and quantitative PCR were performed as described in Test Example 5. mTfR2 mRNA expression levels were normalized to the mRNA expression levels of the housekeeping gene mActb. The results for mTfR2 mRNA expression levels are shown in Figure 8C. The mRNA amount in the control group in which bone marrow function was not induced to be depressed was set at 1.0, and the relative mRNA amounts in the other groups were calculated and expressed as mean values ​​± standard error.

[0303] These results suggest that treatment with GalNAc-conjugated TfR2 siRNA may increase HGB levels by suppressing TfR2 mRNA expression, thereby providing a therapeutic effect for anemia associated with bone marrow failure, such as anemia associated with myelofibrosis. Furthermore, the combined administration of GalNAc-conjugated TfR2 siRNA and Luspatercept may provide a greater therapeutic effect than either agent alone for anemia associated with myelofibrosis.

[0304] Test Example 7: Efficacy evaluation test of GalNAc-conjugated TfR2 siRNA in an inflammation-associated anemia model mouse. The following experiment was performed using the compounds AD47882.5G (Example 131), AD47882.23G (Example 132), AD47882.23sG (Example 133), AD47882.23DUG (Example 171), and AD47882.23sDUG (Example 172) as GalNAc-conjugated TfR2 siRNA. Each GalNAc-conjugated TfR2 siRNA was administered once (Day 0) to male C57BL / 6NJcl mice (manufactured by CLEA Japan, Inc.). PBS was administered subcutaneously to the vehicle group. Five days after treatment with siRNA or PBS (Day 5), turpentine was subcutaneously administered at 150 μL per mouse to induce inflammation. A control group (not induced with inflammation) received PBS instead of turpentine sc. In this study, n = 5 per group. Two days after the first turpentine administration (Day 7), plasma iron and hepcidin concentrations were measured. Plasma iron concentrations were measured using a Metalloassay iron measurement kit (Metalogenix). Plasma hepcidin concentrations were measured using LC / MS. The results are shown in Figures 9A and 9B, respectively.

[0305] Six days after the first turpentine administration (Day 11), turpentine was administered subcutaneously again at 150 μL / mouse. Two days after the second turpentine administration (Day 13), blood was collected from the abdominal vena cava under isoflurane anesthesia, and HGB values, MCH values, and RBC counts were measured using an XT2000 automated hematology analyzer (Sysmex Corporation). The results are shown in Figures 9C to 9E, respectively. After euthanasia, the mice were exsanguinated, and their livers were collected to measure mTfR2 mRNA expression levels. RNA extraction and quantitative PCR were performed as described in Test Example 5. mTfR2 mRNA expression levels were normalized to the mRNA expression levels of the housekeeping gene mActb. The results for mTfR2 mRNA expression levels are shown in Figure 9F. The mRNA amount in the control group in which no inflammation was induced was set to 1.0, and the relative mRNA amounts in the other groups were calculated and expressed as mean values ​​± standard error.

[0306] These results suggest that suppressing TfR2 mRNA expression levels through treatment with GalNAc-conjugated TfR2 siRNA may increase HGB levels and provide therapeutic effects against anemia associated with inflammation, such as anemia associated with chronic kidney disease and anemia associated with cancer.

[0307] Test Example 8: Efficacy evaluation test of GalNAc-conjugated TfR2 siRNA in an inflammation-associated anemia mouse model The following experiment was performed using GalNAc-conjugated TfR2 siRNAs: AD47882.41DUG (Example 173), AD47882.88DUG (Example 174), AD47882.89DUG (Example 175), AD47882.91DUG (Example 176), AD47882.92DUG (Example 177), and AD47882.93DUG (Example 178). Each GalNAc-conjugated TfR2 siRNA was administered once at a dose of 1 mg / kg (1 mpk) to male C57BL / 6NJcl mice (CLEA Japan, Inc.) (Day 0). PBS was administered subcutaneously to the vehicle group. Four days after treatment with siRNA or PBS (Day 4), turpentine was administered subcutaneously at 150 μL per mouse to induce inflammation. PBS was administered sc instead of turpentine to the control group (control) in which inflammation was not induced. In this study, n = 5 per group. Plasma iron concentrations were measured two days after the first turpentine administration (Day 6). Plasma iron concentrations were measured using a Metalloassay iron measurement kit (Metalogenix). The results are shown in Figure 10A.

[0308] Seven days after the first turpentine administration (Day 11), turpentine was administered subcutaneously again at 150 μL / mouse. Three days after the second turpentine administration (Day 14), blood was collected from the abdominal vena cava under isoflurane anesthesia, and HGB, MCH, and RBC counts were measured using an XT2000 automated hematology analyzer (Sysmex Corporation). The results are shown in Figures 10B to 10D, respectively. After euthanasia, the mice were exsanguinated, and their livers were collected to measure mTfR2 mRNA expression levels. RNA extraction and quantitative PCR were performed as described in Test Example 5. mTfR2 mRNA expression levels were normalized to the mRNA expression levels of the housekeeping gene mActb. The results for mTfR2 mRNA expression levels are shown in Figure 10E. The mRNA amount in the control group in which no inflammation was induced was set to 1.0, and the relative mRNA amounts in the other groups were calculated and expressed as mean values ​​± standard error.

[0309] These results suggest that suppressing TfR2 mRNA expression levels through treatment with GalNAc-conjugated TfR2 siRNA may increase HGB levels and provide therapeutic effects against anemia associated with inflammation, such as anemia associated with chronic kidney disease and anemia associated with cancer.

[0310] Test Example 9: Screening of HepG2 cells by reverse transfection of TfR2 siRNA In a similar manner to Test Example 2, the siRNAs TfR2-019.94DUG (Example 169), TfR2-019.94DUG.s1 (Example 180), TfR2-019.94DUG.s2 (Example 181), TfR2-019.94DUG.e1 (Example 182), TfR2-019.94DUG.e2 (Example 183), and TfR2-019.94DUG.e3 (Example 184) were used to test the expression level of hTfR2 mRNA. The relative hTfR2 mRNA expression level of the samples treated with each siRNA was calculated and expressed as a relative value when the hTfR2 mRNA expression level in the negative control (NT) was set to 1.0. The results are shown in Table 24 below.

[0311]

[0312]

[0313] Test Example 10: Efficacy evaluation test of GalNAc-conjugated TfR2 siRNA in normal mice The following experiment was performed using AD47882.5G (Example 131) and AD47882.94DUG (Example 179) as GalNAc-conjugated siRNA. AD47882.5G as GalNAc-conjugated siRNA was subcutaneously administered (s.c.) to male C57BL / 6NJcl mice (CLEA Japan) at a dose of 10 mg / kg (10 mpk), 3 mg / kg (3 mpk), or 1 mg / kg (1 mpk) (n=5 / group). PBS was administered subcutaneously to the vehicle group (n=5 / group). Blood samples were collected from the tail vein before treatment with siRNA or PBS (Day 0), and 7, 21, and 28 days after treatment (Day 7, Day 21, and Day 28), and plasma iron concentrations were measured using a Metalloassay iron measurement kit (Metalogenix, Inc.). The results are shown in Figure 13A (mean ± standard error).

[0314] AD47882.94DUG, a GalNAc-conjugated siRNA, was administered subcutaneously (s.c.) to male C57BL / 6NJcl mice (CLEA Japan) at doses of 3 mg / kg (3 mpk), 1 mg / kg (1 mpk), or 0.3 mg / kg (0.3 mpk) (n = 5 / group). PBS was administered subcutaneously to the vehicle group (n = 5 / group). Blood samples were collected from the tail vein before siRNA or PBS treatment (Day 0), and 7, 21, and 28 days after treatment (Day 21 and Day 28). Plasma iron concentrations were measured using a Metalloassay Iron Kit (Metalogenix). The plasma iron concentration results are shown in Figure 13B (mean ± standard error).

[0315] These results indicate that AD47882.94DUG, in particular, stably suppresses the expression level of TfR2 mRNA in vivo and increases the plasma iron concentration. Similarly, the compounds AD47882.88DUG (compound of Example 174), AD47882.89DUG (compound of Example 175), AD47882.91DUG (compound of Example 176), AD47882.92DUG (compound of Example 177), AD47882.93DUG (compound of Example 178), and AD47882.71DUG (compound of AD47882.88DUG) exhibited the 71-type modification pattern. In another study in which normal mice were treated with AD47882.80DUG (AD47882.88DUG compound modified with an 80-type modification pattern), or AD47882.90DUG (AD47882.88DUG compound modified with a 90-type modification pattern), plasma iron levels were confirmed to be elevated 28 days after treatment. These results suggest that by modifying TfR2 siRNA with a different target sequence with the same modification pattern as these compounds, it is possible to obtain siRNA that can stably suppress TfR2 mRNA expression levels and increase plasma iron levels.

[0316] Test Example 11: Efficacy Evaluation of GalNAc-Conjugated TfR2 siRNA in a Mouse Model of Anemia Associated with Bone Marrow Depression The following experiment was conducted using AD47882.23DUG (Example 171) and AD47882.94DUG (Example 179) as GalNAc-conjugated TfR2 siRNA. To induce bone marrow depression, 100 mg / kg of carboplatin was administered intraperitoneally (i.p.) to male C57BL / 6NJcl mice (CLEA Japan, Inc.). Four days after carboplatin administration, mice were grouped based on HGB values ​​and plasma iron concentrations, and then treated with the drug (n = 6 / group). The GalNAc-conjugated TfR2 siRNA treatment group received a single subcutaneous (s.c.) administration of 3 mg / kg (3 mpk). In the vehicle group, PBS was administered subcutaneously once.

[0317] Prior to drug treatment (Day 0), blood was collected from the tail vein, and HGB levels were measured using a QuantiChrom Whole Blood HB kit (Bioassay Systems). The results for HGB levels before drug treatment are shown in Figure 14A (mean ± standard error). Plasma iron levels were measured using a Metalloassay iron measurement kit (Metalogenix). The results for plasma iron levels before drug treatment are shown in Figure 14B (mean ± standard error). Figure 14C shows the results for plasma iron levels 8 days after drug treatment (Day 8). Nine days after drug treatment (Day 9), blood was collected from the abdominal vena cava under isoflurane anesthesia, and HGB levels, MCH levels, and RBC counts were measured using an XT2000 automated hematology analyzer (Sysmex Corporation). The respective results are shown in Figures 14D to 14F (mean values ​​± standard error).

[0318] These results suggest that treatment with GalNAc-conjugated TfR2 siRNA may increase plasma iron levels, thereby increasing HGB levels and providing a therapeutic effect against anemia associated with bone marrow failure, e.g., anemia associated with myelofibrosis.

[0319] Test Example 12: Efficacy evaluation test of GalNAc-conjugated TfR2 siRNA in an inflammation-associated anemia mouse model The following experiment was performed using AD47882.92DUG (Example 177) and AD47882.94DUG (Example 179) as GalNAc-conjugated TfR2 siRNA. To induce inflammation, turpentine was subcutaneously administered (s.c.) to male C57BL / 6NJcl mice (CLEA Japan) at 150 μL / mouse three times a week. PBS was administered subcutaneously instead of turpentine to a control group (control) in which no inflammation was induced. Seven days after the first turpentine administration (Day 0), blood was collected from the tail vein, and plasma iron concentration and HGB level were measured. Plasma iron concentrations were measured using a Metalloassay iron measurement kit (Metalogenix). HGB levels were measured using a QuantiChrom Whole Blood HB kit (Bioassay Systems). The results are shown in Figures 15A and 15B (mean ± standard error). The drug was administered to male C57BL / 6NJcl mice (CLEA Japan) (n = 5 / group). The GalNAc-conjugated TfR2 siRNA treatment group received a single subcutaneous dose of 3 mg / kg (3 mpk). The vehicle group received a single subcutaneous dose of PBS.

[0320] Blood samples were collected from the tail vein four days (Day 4) and eight days (Day 8) after drug treatment, and plasma iron concentrations were measured using a Metalloassay iron measurement kit (Metalogenix). The results are shown in Figure 15A (mean ± standard error). Eight days after drug treatment, blood was collected from the abdominal vena cava under isoflurane anesthesia, and HGB levels, MCH levels, and RBC counts were measured using an XT2000 automated hematology analyzer (Sysmex Corporation). The results are shown in Figures 15C to 15E (mean ± standard error). Mice were euthanized by exsanguination under isoflurane anesthesia, and livers were collected to measure mTfR2 mRNA expression levels. RNA extraction and quantitative PCR were performed as described in Test Example 5. mTfR2 mRNA expression levels were normalized to the mRNA expression levels of the housekeeping gene mActb. The results of mTfR2 mRNA expression levels are shown in Figure 15F. The mRNA level in the control group, in which inflammation was not induced, was set to 1.0, and the relative mRNA levels in the other groups were calculated and expressed as mean values ​​± standard error.

[0321] These results suggest that suppressing TfR2 mRNA expression levels through treatment with GalNAc-conjugated TfR2 siRNA may increase HGB levels and provide therapeutic effects against anemia associated with inflammation, such as anemia associated with chronic kidney disease and anemia associated with cancer.

[0322] Test Example 13: Efficacy evaluation test of GalNAc-conjugated TfR2 siRNA in NUP98-HOXD13 mice NUP98-HOXD13 mice (C57BL / 6-Tg(Vav1-NUP98 / HOXD13)G2Apla / J) are known as a mouse model of myelodysplastic syndrome (MDS), which is characterized by ineffective hematopoiesis and insufficient erythroid differentiation and maturation (Nature Medicine, volume 20, pages 408-414 (2014)). To verify the therapeutic effect of TfR2 siRNA on MDS, a test was conducted in which GalNAc-conjugated TfR2 siRNA was administered to NUP98-HOXD13 mice. The compound used was AD47882.23DUG (Example 171).

[0323] NUP98-HOXD13 mice were purchased from Jackson Laboratory Japan, Inc. and used in the experiment. Four-month-old NUP98-HOXD13 mice were grouped based on plasma iron concentration, HGB value, and body weight, and then treated with drugs (n = 8 / group). The GalNAc-conjugated TfR2 siRNA treatment group received a subcutaneous (s.c.) administration of 5 mg / kg (5 mpk) once a week. The vehicle group received a subcutaneous administration of PBS once a week.

[0324] Two, four, and eight weeks after the start of drug treatment, blood was collected from the tail vein, and plasma iron concentrations were measured using a Metalloassay iron measurement kit (Metalogenix). The results are shown in Figure 16A (mean ± standard error). Blood was also collected from the tail vein two, four, six, and eight weeks after the start of drug treatment, and HGB levels were measured using a QuantiChrom Whole Blood HB kit (Bioassay Systems). The results are shown in Figure 16B (mean ± standard error). Eight weeks after the start of drug treatment, blood was collected from the abdominal vena cava under isoflurane anesthesia, and HGB levels, MCH levels, and RBC counts were measured using an XT2000 automated hematology analyzer (Sysmex Corporation). The results are shown in Figures 16C to 16E (mean ± standard error). Mice were euthanized by exsanguination under isoflurane anesthesia, and their livers were collected to measure mTfR2 mRNA expression levels. RNA extraction and quantitative PCR were performed as described in Test Example 5. mTfR2 mRNA expression levels were normalized to the mRNA expression level of the housekeeping gene mActb. The results for mTfR2 mRNA expression levels are shown in Figure 16F. The relative mRNA levels in the other groups were calculated and expressed as mean values ​​± standard error, relative to the mRNA level in the vehicle group, which was set to 1.0.

[0325] These results suggest that treatment with GalNAc-conjugated TfR2 siRNA may be effective in treating MDS, an anemic disease associated with decreased bone marrow function, by suppressing TfR2 mRNA expression and increasing plasma iron concentration, thereby increasing HGB levels.

[0326] Test Example 14: Efficacy evaluation test of GalNAc-conjugated TfR2 siRNA in a mouse model of anemia associated with chronic kidney disease The following experiment was performed using the compound AD47882.94DUG (Example 179) as the GalNAc-conjugated TfR2 siRNA. To induce chronic kidney disease and associated anemia, male C57BL / 6NJcl mice (manufactured by CLEA Japan) were fed a diet containing 0.2% adenine (product of WAKO Corporation, 010-11513). Two weeks after the 0.2% adenine diet, blood was collected from the tail vein, and plasma iron concentration and HGB level were measured. Plasma iron concentration was measured using a Metalloassay iron measurement kit (manufactured by Metallogenics). HGB levels were measured using a QuantiChrom Whole Blood HB kit (Bioassay Systems). The results are shown in Figures 17A and 17B (mean ± standard error). Animals were grouped based on plasma iron concentration, HGB levels, and body weight, and then treated with drugs (n = 6 / group). GalNAc-conjugated TfR2 siRNA treatment groups received subcutaneous administration of 3 mg / kg (3 mpk), 1 mg / kg (1 mpk), or 0.3 mg / kg (0.3 mpk) once a week. PBS was administered subcutaneously to the vehicle group once a week. A control group (control) without induced anemia associated with chronic kidney disease received a diet without 0.2% adenine.

[0327] Six weeks after the start of drug treatment, blood was collected from the tail vein and plasma iron concentrations were measured using a Metalloassay iron measurement kit (Metalogenix). The results are shown in Figure 17C (mean ± standard error). Six weeks after the start of drug treatment, blood was collected from the abdominal vena cava under isoflurane anesthesia and used to measure HGB levels, MCH levels, and RBC counts using an XT2000 automated hematology analyzer (Sysmex Corporation). The results are shown in Figures 17D to 17F (mean ± standard error). Mice were euthanized by exsanguination under isoflurane anesthesia, and livers were collected to measure mTfR2 mRNA expression levels. RNA extraction and quantitative PCR were performed as described in Test Example 5. mTfR2 mRNA expression levels were normalized to the mRNA expression levels of the housekeeping gene mActb. The results for mTfR2 mRNA expression levels are shown in Figure 17G. The mRNA amount in the vehicle group was set to 1.0, and the relative mRNA amounts in the other groups were calculated and expressed as mean values ​​± standard error.

[0328] These results suggest that suppressing TfR2 mRNA expression through treatment with GalNAc-conjugated TfR2 siRNA may increase plasma iron and HGB levels, thereby providing a therapeutic effect for anemia associated with chronic kidney disease.

[0329] Test Example 15: Efficacy evaluation test of GalNAc-conjugated TfR2 siRNA in a mouse model of anemia caused by administration of Ruxolitinib (JAK1 / JAK2 inhibitor) In order to verify the effect of TfR2 siRNA on anemia caused by administration of Ruxolitinib, the following experiment was carried out using the compound AD47882.23DUG (Example 171) as the GalNAc-conjugated TfR2 siRNA.

[0330] To induce anemia by Ruxolitinib administration, male C57BL / 6NJcl mice (manufactured by CLEA Japan) were fed a diet containing 0.1% Ruxolitinib (product R-6688, LC Laboratories). Six weeks after the 0.1% Ruxolitinib diet, blood was collected from the tail vein and HGB values ​​were measured using an XT2000 automated hematology analyzer (manufactured by Sysmex Corporation). The results are shown in Figure 18A (mean ± standard error). Based on these HGB values ​​and body weight values, mice were grouped and treated with drugs (n = 5 / group). The GalNAc-conjugated TfR2 siRNA treatment group received a subcutaneous injection (s.c.) of 3 mg / kg (3 mpk) once weekly. The vehicle group received PBS subcutaneously once weekly. As a control group in which anemia was not induced by Ruxolitinib, rats were given food that did not contain 0.1% Ruxolitinib.

[0331] Three weeks after the start of drug treatment, blood was collected from the tail vein and plasma iron concentrations were measured using a Metalloassay iron measurement kit (Metalogenix). The results are shown in Figure 18B (mean ± standard error). Six weeks after the start of drug treatment, blood was collected from the abdominal vena cava under isoflurane anesthesia and measured for HGB, MCH, and RBC counts using an XT2000 automated hematology analyzer (Sysmex Corporation). The results are shown in Figures 18C to 18E (mean ± standard error). Mice were euthanized by exsanguination under isoflurane anesthesia, and livers were collected to measure mTfR2 mRNA expression levels. RNA extraction and quantitative PCR were performed as described in Test Example 5. mTfR2 mRNA expression levels were normalized to the mRNA expression levels of the housekeeping gene mActb. The results for mTfR2 mRNA expression levels are shown in Figure 18F. The mRNA amount in the control group not fed with 0.1% Ruxolitinib-containing food was set at 1.0, and the relative mRNA amounts in the other groups were calculated and expressed as mean values ​​± standard error.

[0332] These results suggest that a combination of a JAK2 inhibitor such as Ruxolitinib and TfR2 siRNA may be effective in treating myelofibrosis.

[0333] Test Example 16: Efficacy evaluation test of GalNAc-conjugated TfR2 siRNA in human TfR2-expressing mice The following experiment was carried out to verify the effect of GalNAc-conjugated TfR2 siRNA on human TfR2 mRNA using male or female TfR2-humanized mice carrying the human TfR2 locus, which were produced by the Institute of Specialized Immunology, Inc. The TfR2-humanized mice were produced using a BAC (bacterial artificial chromosome) clone (RP11-264N5) containing the human TfR2 locus. As GalNAc-conjugated TfR2 siRNAs, TfR2-019.88DUG (compound of Example 162), TfR2-019.92DUG (compound of Example 167), TfR2-019.93DUG (compound of Example 168), and TfR2-019.94DUG (compound of Example 169) were used.

[0334] The GalNAc-conjugated TfR2 siRNA treatment group was administered subcutaneously (s.c.) once at a dose of 3 mg / kg (3 mpk) or 0.3 mg / kg (0.3 mpk) (n=3 / group).The vehicle group was administered PBS subcutaneously once (n=4 / group).

[0335] Seven days after the start of drug treatment, mice were euthanized by exsanguination under isoflurane anesthesia, and livers were collected to measure hTfR2 mRNA expression levels. RNA extraction and quantitative PCR were performed as described in Test Example 5. hTfR2 mRNA expression levels were normalized to the mRNA expression level of the housekeeping gene mActb. Primers for hTfR2 were used as described in Test Examples 1 and 2. The results for hTfR2 mRNA expression levels are shown in Figure 19. The relative mRNA levels in the other groups were calculated and expressed as mean values ​​± standard error, relative to the mRNA level in the vehicle group, which was set to 1.0.

[0336] These results demonstrate that treatment with GalNAc-conjugated TfR2 siRNA can suppress the expression level of human TfR2 mRNA. Furthermore, suppression of human TfR2 mRNA expression can increase HGB levels, suggesting the possibility of providing therapeutic effects for anemia associated with bone marrow failure, such as anemia associated with myelofibrosis, and anemia associated with inflammation, such as anemia associated with chronic kidney disease. In another study in which mice expressing human TfR2 were further knocked out of endogenous mouse TfR2 and treated with TfR2-019.94DUG (the compound of Example 169), human TfR2 gene expression was suppressed even after 42 days, confirming the stable suppression of human TfR2 gene expression by TfR2-019.94DUG.

[0337] Test Example 17: Efficacy evaluation test of GalNAc-conjugated TfR2 siRNA using human primary hepatocytes GalNAc-conjugated siRNA is known to be taken up into the liver via ASGPR (asialoglycoprotein receptor 1) expressed in hepatic hepatocytes. Therefore, to verify the effect of GalNAc-conjugated TfR2 siRNA under conditions without using a transfection reagent, a drug efficacy evaluation test of GalNAc-conjugated TfR2 siRNA was carried out using human primary hepatocytes. The following compounds were used as GalNAc-conjugated TfR2 siRNAs: TfR2-019.88DUG (Example 162), TfR2-019.89DUG (Example 163), TfR2-019.90DUG (Example 164), TfR2-019.91DUG (Example 166), TfR2-019.92DUG (Example 167), TfR2-019.93DUG (Example 168), TfR2-019.94DUG (Example 169), and TfR2-019.95DUG (Example 170).

[0338] (Culturing of Human Primary Hepatocytes) Human primary hepatocytes (LIFE TECHNOLOGIES, HMCPIS) stored in a liquid nitrogen tank were thawed in a 37°C hot bath and poured into 10 mL of Cryopreserved Hepatocyte Recovery Medium (CHRM) (LIFE TECHNOLOGIES, CM7000) preheated to 37°C, and mixed by inversion. The cells were then centrifuged (100g, 10 minutes), the supernatant was removed, and a cell suspension was prepared using 5 mL of cell seeding medium preheated to 37°C. Cell seeding medium was prepared by adding Hepatocyte Plating Supplement Pack (LIFE TECHNOLOGIES, CM3000) to William's Medium E (LIFE TECHNOLOGIES, A1217601). The viable cell count was then calculated, and the cells were prepared using cell seeding medium to achieve a concentration of 5x10^5 cells / mL. The cells were seeded at 5x10^4 cells per well of a collagen-coated 96-well plate, cultured in a CO2 incubator for 4-6 hours, and confirmed to have adhered to the plate using a microscope. Then, an experiment was performed in which GalNAc-conjugated TfR2 siRNA was added.

[0339] (Addition of GalNAc-conjugated TfR2 siRNA) Cell culture medium pre-warmed to 37°C was used to prepare medium containing GalNAc-conjugated TfR2 siRNA. Two wells of each siRNA sample were prepared, and two wells of a negative control (NT) containing distilled water instead of siRNA were prepared. The final siRNA concentration ranged from 10,000 nM to 4.8 nM, adjusted to a 5-fold common ratio. Cell culture medium was prepared by adding Hepatocyte Maintenance Supplement Pack (LIFE TECHNOLOGIES, CM4000) to William's Medium E (LIFE TECHNOLOGIES, A1217601). After removing the cell seeding medium from each well of human primary hepatocytes seeded in a 96-well plate by aspiration, 100 μL of the medium containing the prepared GalNAc-conjugated TfR2 siRNA was added to each well. Three days after the addition of the compound, the cells were washed once with PBS and subjected to RNA extraction.

[0340] (RNA extraction, qPCR) RNA extraction and quantitative PCR were performed as described in Test Example 1. The mRNA expression levels of hTfR2 and h18S were measured as the average between two wells. The mRNA expression level of hTfR2 was normalized by the mRNA expression level of h18S, a housekeeping gene. The relative hTfR2 mRNA expression level of the samples treated with each siRNA was calculated and expressed as mean ± standard error, relative to the hTfR2 mRNA expression level in the negative control (NT) set at 1.0. The results are shown in Figures 20A to 20C, respectively.

[0341] These results indicate that GalNAc-conjugated TfR2 siRNA can be taken up by human liver hepatocytes and suppress the expression of human TfR2 mRNA. Furthermore, suppressing the expression of human TfR2 mRNA increases HGB levels, potentially providing therapeutic benefits for anemia associated with bone marrow failure, such as anemia associated with myelofibrosis, and anemia associated with inflammation, such as anemia associated with chronic kidney disease.

[0342] Test Example 18: Effect of TfR2 siRNA on Hepcidin Gene Expression Using HepG2 Cells

[0111] Using the same method as in Test Example 2, siRNA was introduced into HepG2 cells using the reverse transfection method, and the expression level of Hepcidin mRNA was evaluated two days later. Hs00221783_m1 (Thermo Fisher Scientific) was used as the primer for Hepcidin. The siRNAs used were TfR2-019 (Example 19), TfR2-039 (Example 39), TfR2-019.94DUG (Example 169), and TfR2-039.23DUG (Example 144).

[0343] The expression level of hepcidin mRNA in the negative control (NT) was set to 1.0, and the relative expression level of hepcidin mRNA in the samples treated with each siRNA was calculated and expressed as a relative value. The results are shown in Figure 21.

[0344] The siRNA of the present invention can suppress the expression of TfR2 mRNA and further suppress the production of hepcidin, which plays a central role in iron metabolism. The siRNA of the present invention is useful for treating or preventing diseases that can be treated or prevented by suppressing the expression of TfR2 mRNA and / or the production of hepcidin.

[0345] SEQ ID NO: 1: nucleotide sequence of hTfR2 mRNA SEQ ID NOs: 2 to 24: nucleotide sequences of single-stranded oligonucleotides SEQ ID NOs: 25 to 28: nucleotide sequences of the sense strand region of the double-stranded oligonucleotides SEQ ID NOs: 29 to 35: nucleotide sequences of the antisense strand region of the double-stranded oligonucleotides SEQ ID NOs: 36, 37: nucleotide sequences of the sense strand region of the double-stranded oligonucleotides SEQ ID NOs: 38 to 42: nucleotide sequences of the antisense strand region of the double-stranded oligonucleotides SEQ ID NO: 43: nucleotide sequence of the complementary region of the sense strand region in TfR2-019 SEQ ID NO: 44: nucleotide sequence of the complementary region of the antisense strand region in TfR2-019 SEQ ID NO: 45: nucleotide sequence of the complementary region of the sense strand region in TfR2-039 SEQ ID NO: 46: nucleotide sequence of the complementary region of the antisense strand region in TfR2-039 SEQ ID NO: 47: nucleotide sequence of the sense strand region of the double-stranded oligonucleotide

Claims

1. Oligonucleotides or pharmaceutically acceptable salts thereof that have a knockdown effect on transferrin receptor 2 mRNA, comprising the antisense strand region of (A) and the sense strand region of (B) below; (A) An antisense chain region consisting of 18 to 31 bases in length, comprising a target-corresponding sequence substantially complementary to a target sequence consisting of 18 to 21 consecutive bases in the region between nucleotide numbers 2845 to 2867 or nucleotide numbers 1534 to 1556 of Sequence ID No. 1, wherein the nucleotide at the 5' end of the target-corresponding sequence is a nucleotide substantially complementary to the nucleotide at the 3' end of the target sequence, a nucleotide containing adenine, or a nucleotide containing uracil, and an overhang structure of 5 bases or less may be added to the 5' and / or 3' ends of the target-corresponding sequence, (B) A sense strand region having a length of 18 to 31 nucleotides, which contains a nucleotide sequence substantially complementary to the target corresponding sequence of the antisense strand region, and which may further have an overhang structure of 5 nucleotides or less attached to the 5' and / or 3' ends of the nucleotide sequence.

2. The oligonucleotide according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the target sequence is a nucleotide sequence consisting of 19 bases, nucleotide numbers 2847 to 2865 or nucleotide numbers 1536 to 1554 of SEQ ID NO:

1.

3. The oligonucleotide according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the nucleotide at the 5' end of the target corresponding sequence in the antisense chain region is a nucleotide having adenine or uracil.

4. The oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 1, wherein the target-corresponding sequence of the antisense strand region is a nucleotide sequence that is completely complementary to the target sequence consisting of 19 bases with nucleotide numbers 2847 to 2865 or nucleotide numbers 1536 to 1554 of SEQ ID NO:

1.

5. The oligonucleotide according to claim 1, or a pharmaceutically acceptable salt thereof, wherein at least one of the sugar and / or phosphate diester bonds constituting the oligonucleotide is modified.

6. The sugar constituting the oligonucleotide is D-ribofuranose, and the sugar modification is the modification of the hydroxyl group at the 2' position of D-ribofuranose. Preferably, the sugar modification is 2'-deoxy, 2'-O-alkylation, 2'-O-alkoxyalkylation, 2'-halogenation, and / or 2'-O,4'-C-alkyleneation of D-ribofuranose. More preferably, the oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 5, wherein the 2'-O-alkylation is 2'-O-methylation, the 2'-O-alkoxyalkylation is 2'-O-methoxyethylation, the 2'-halogenation is 2'-fluoration, and the 2'-O,4'-C-alkylenation is 2'-O,4'-C-methyleneation and / or 2'-O,4'-C-ethyleneation.

7. The oligonucleotide according to claim 5, or a pharmaceutically acceptable salt thereof, wherein the modification of the phosphate diester bond is a phosphorothioate.

8. It is characterized in that the D-ribofuranose at the 3'-terminal nucleoside of the sense chain region and / or antisense chain region is 3'-O-alkylated. Preferably, the oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 5, wherein the 3'-O-alkylation is 3'-O-methylation.

9. An oligonucleotide according to claim 1, or a pharmaceutically acceptable salt thereof, wherein an overhang structure of three bases or less is added to the 5' and / or 3' ends of the sense strand region and / or antisense strand region.

10. The overhang structure consists of two bases. Preferably, the overhang structure is a nucleotide containing two consecutive thymine molecules, or a nucleotide containing two consecutive uracil molecules. More preferably, the oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 9, wherein the overhang structure is two consecutive U(M) added to the 3' end of the antisense chain region.

11. The oligonucleotide according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the sense chain region consists of an oligonucleotide shown in the following formula (I), the antisense chain region consists of an oligonucleotide shown in the following formula (II), and further has the characteristics shown in (a) to (g) below: Sense strand region: 5’ S Ｏ５ -S ａ -S １９ -S １８ -S １７ -S １６ -S １５ -S １４ -S １３ -S １２ -S １１ -S １０ -S ９ -S ８ -S ７ -S ６ -S ５ -S ４ -S ３ -S ２ -S １ -S Ｏ３ 3’ (I) Antisense strand region: 5' A Ｏ５ -A １ -A ２ -A ３ -A ４ -A ５ -A ６ -A ７ -A ８ -A ９ -A １０ -A １１ -A １２ -A １３ -A １４ -A １５ -A １６ -A １７ -A １８ -A １９ -A ａ -A Ｏ３ 3' (II) (a) S １ It is a 3'-modified nucleoside, and S ２ ~S １９ Each represents a single nucleoside, which independently is 2'-OMe RNA, 2'-F RNA, or DNA, and S ａ This represents 0 to 3 nucleosides, and each nucleoside independently represents 2'-OMe RNA, 2'-F RNA, or DNA, S Ｏ３ and S Ｏ５ Each independently represents 0 to 3 nucleosides, and each nucleoside independently represents 2'-OMe RNA, 2'-F RNA, DNA, or a 2'-O,4'-C-crosslinked nucleoside. The bonds between each nucleoside represent phosphate diester bonds, which may be chemically modified; (b) A １１ A １２ A １３ A １４ and A １５ represents one nucleoside, of which at least one is DNA, RNA, a 2'-O,4'-C-crosslinked nucleoside, or 2'-MOE RNA, and the others are 2'-OMe RNA or 2'-F RNA, A １ ~A １０ and A １６ ~A １９ Each represents a single nucleoside, independently representing 2'-OMe RNA, 2'-F RNA, or DNA, and A ａ represents 0 to 3 nucleosides, and each nucleoside independently represents 2'-OMe RNA, 2'-F RNA, or DNA, A Ｏ３ and A Ｏ５ Each independently represents 0 to 3 nucleosides, and each nucleoside independently represents 2'-OMe RNA, 2'-F RNA, DNA, or a 2'-O,4'-C-crosslinked nucleoside. The bonds between each nucleoside represent phosphate diester bonds, which may be chemically modified; (c) A ２ ~A ａ The nucleotide sequence between them consists of a nucleotide sequence that is substantially complementary to the target sequence, A １ A is a nucleoside having a base complementary to the corresponding nucleoside of the target sequence, a nucleoside having adenine, a nucleoside having thymine, or a nucleoside having uracil. Ｏ３ and A Ｏ５ If present, each nucleotide sequence is independently selected, independently of the corresponding nucleoside of the target sequence; (d) S １ ~S ａ The nucleotide sequence between and A １ ~A ａ The nucleotide sequences between them are substantially complementary to each other and form a double-stranded structure; (e) S Ｏ５ and A Ｏ３ If both exist, S Ｏ５ and A Ｏ３ These are nucleotide sequences that are not complementary to one another; (f) S Ｏ３ and A Ｏ５ If both exist, S Ｏ３ and A Ｏ５ These are nucleotide sequences that are not complementary to each other; and, (g) The sense chain region and / or the antisense chain region may be chemically modified at the 5' position of the 5'-terminal nucleoside and / or at the 3' position of the 3'-terminal nucleoside, or at the 2' position if the 3'-terminal nucleoside is a 3'-modified nucleoside.

12. In equation (I), S １ is 3'-OMe RNA, and / or The oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 11, wherein the 2'-O,4'-C-crosslinked nucleoside is LNA or ENA.

13. In equation (II), A １１ A １２ A １３ A １４ and A １５ The oligonucleotide or pharmaceutically acceptable salt thereof according to claim 11, wherein two or more of the following may be the same or different, and are DNA, RNA, a 2'-O,4'-C-crosslinked nucleoside, or 2'-MOE RNA.

14. In equation (II), A １４ The oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 13, wherein is RNA or a 2'-O,4'-C-crosslinked nucleoside.

15. In equation (II), A １３ is a 2'-O,4'-C-crosslinked nucleoside or 2'-OMe RNA, and A １４ It is RNA, Preferably, in formula (II), A11 - A12 - A13 - A14 - A15 is one of the following: A11 (2'-OMe RNA)-A12 (2'-OMe RNA)-A13 (ENA)-A14 (RNA)-A15 (2'-OMe RNA), A11 (2'-F RNA)-A12 (2'-OMe RNA)-A13 (ENA)-A14 (RNA)-A15 (2'-OMe RNA), A11 (2'-OMe RNA)-A12 (2'-F RNA)-A13 (ENA)-A14 (RNA)-A15 (2'-OMe RNA), A11 (2'-OMe RNA)-A12 (2'-OMe RNA)-A13 (LNA)-A14 (RNA)-A15 (2'-OMe RNA), A11 (2'-F RNA)-A12 (2'-OMe RNA)-A13 (LNA)-A14 (RNA)-A15 (2'-OMe RNA), A11 (2'-OMe RNA)-A12 (2'-F RNA)-A13 (LNA)-A14 (RNA)-A15 (2'-OMe RNA), A 11 (2'-F RNA)-A 12 (2'-OMe RNA)-A 13 (2'-OMe RNA)-A 14 (RNA)-A 15 (2'-OMe RNA), or A11 (2'-OMe RNA)-A12 (2'-F RNA)-A13 (2'-OMe RNA)-A14 (RNA)-A15 (2'-OMe RNA), The oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 14.

16. In formula (II), A １２ is DNA, and A １４ is a 2'-O,4'-C-bridged modified nucleoside, Preferably, in formula (II), A11 - A12 - A13 - A14 - A15 is one of the following: A11 (2'-F RNA)-A12 (DNA)-A13 (2'-OMe RNA)-A14 (ENA)-A15 (2'-OMe RNA), A11 (2'-OMe RNA)-A12 (DNA)-A13 (2'-OMe RNA)-A14 (ENA)-A15 (2'-OMe RNA), A 11 (2'-F RNA)-A 12 (DNA)-A 13 (2'-OMe RNA)-A 14 (LNA)-A 15 (2'-OMe RNA), or A11 (2'-OMe RNA)-A12 (DNA)-A13 (2'-OMe RNA)-A14 (LNA)-A15 (2'-OMe RNA), The oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 14.

17. In equation (II), A ２ A ６ and A １６ This is 2'-F RNA, Preferably, in formula (II), one or two nucleosides selected from the group consisting of A8, A9, and A10 are 2'-F RNA, and the nucleosides A1, A3 to A5, A7, A17 to A19, and Aa are 2'-OMe RNA. More preferably, in formula (II), A2, A6, A8, A10, and A16 are 2'-F RNA, and the nucleosides of A1, A3 to A5, A7, A9, A17 to A19, and Aa are 2'-OMe RNA, according to claim 11 or a pharmaceutically acceptable salt thereof.

18. In formula (I), S １１ , S １２ , S １３ and S １５ are 2'-F RNA, Preferably, the oligonucleotide according to claim 11 or a pharmaceutically acceptable salt thereof, wherein in formula (I), S2 to S10, S14, S16 to S19 and Sa are 2'-OMe RNA.

19. The oligonucleotide according to claim 11 or a pharmaceutically acceptable salt thereof, characterized in that, when RNA is used in formula (II), the bond between the RNA and the nucleoside adjacent to its 3' end is a phosphorothioate bond.

20. The oligonucleotide according to claim 11, or a pharmaceutically acceptable salt thereof, characterized in that, in 2 to 5 nucleotides from the 5' and 3' ends of the oligonucleotide, each nucleoside bond is a phosphorothioate bond.

21. S Ｏ３ S Ｏ５ A Ｏ５ and A Ｏ３ The number of nucleosides is 0 to 2. Preferably, the nucleoside number of S O 3 is 0, More preferably, the oligonucleotide or pharmaceutically acceptable salt thereof according to claim 11, wherein the nucleoside numbers of S₂O₃, S₂O₅, and A₂O₅ are 0, and the nucleoside number of A₂O₃ is 2.

22. The oligonucleotide according to claim 1, or a pharmaceutically acceptable salt thereof, characterized in that the antisense chain region and the sense chain region each form a double-stranded oligonucleotide as independent oligonucleotides.

23. The antisense strand region's 5' terminal nucleotide and the sense strand region's 3' terminal nucleotide are linked by a linker structure. Preferably, the linker structure is the following formula 【Chemistry 1】 [In the formula, dashed lines indicate bonding bonds. The oxygen atom bonded to the phenyl group is linked to the 5'-phosphate group of the nucleotide at the 5' end of the adjacent antisense chain region via a phosphate diester bond or phosphorothioate bond. The methylene group at the other end is linked to the 3'-phosphate group (or the phosphate group at the 2' position in nucleotides modified at the 3' position) of the nucleotide at the 3' end of the adjacent sense chain region via a phosphate diester bond or phosphorothioate bond.] An oligonucleotide according to claim 1 or a pharmaceutically acceptable salt thereof, having a structure represented by .

24. The oligonucleotide is characterized by the fact that the 5' position of the 5' terminal nucleotide and / or the 3' position of the 3' terminal nucleotide, or the 2' position if the 3' terminal nucleotide has a 3'-modified nucleoside, are chemically modified with a GalNAc unit. Preferably, the GalNAc unit is 【Chemistry 2】 [In the formula, the dashed line indicates a phosphate diester bond with the phosphate group at the 5' end and / or the phosphate group at the 3' end (or the phosphate group at the 2' position in nucleotides modified at the 3' position) of an adjacent nucleotide.] The oligonucleotide according to claim 1, or a pharmaceutically acceptable salt thereof, characterized in that it is a unit represented by .

25. The oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 1, wherein the sense chain region excluding the overhang structure is represented by the following formula (I-1), the antisense chain region excluding the overhang structure is represented by any of the following formulas (II-1) to (II-10), the sense chain region and the antisense chain region each form a double-stranded oligonucleotide as independent oligonucleotides, and the sense chain region and the antisense chain region may each independently contain an overhang structure. (formula) 5' C(M)G(M)U(M)G(M)G(F)A(M)G(F)U(F)U(F)U(M)C(M)A(M)A(M)U(M)A(M)U(M)C(M)A(M)A(3M) 3' (I-1) 5' U(M)U(F)G(M)A(M)U(M)A(F)U(M)U(F)G(F)A(M)A(M)A(D)C(M)T(E)C(M)C(F)A(M)C(M)G(M) 3' (II-1) 5' U(M)U(F)G(M)A(F)U(M)A(F)U(M)U(M)G(F)A(M)A(M)A(D)C(M)T(E)C(M)C(F)A(M)C(M)G(M) 3' (II-2) 5' U(M)U(F)G(M)A(M)U(M)A(F)U(M)U(M)G(F)A(F)A(M)A(D)C(M)T(E)C(M)C(F)A(M)C(M)G(M) 3' (II-3) 5' U(M)U(F)G(M)A(M)U(M)A(F)U(M)U(M)G(F)A(M)A(M)A(D)C(M)T(E)C(M)C(F)A(M)C(F)G(M) 3' (II-4) 5' U(M)U(F)G(M)A(M)U(M)A(F)U(M)U(F)G(F)A(M)A(M)A(M)C(E)U(R)^C(M)C(F)A(M)C(M)G(M) 3' (II-5) 5' U(M)U(F)G(M)A(M)U(M)A(F)U(M)U(M)G(F)A(F)A(M)A(M)C(E)U(R)^C(M)C(F)A(M)C(M)G(M) 3' (II-6) 5' U(M)U(F)G(M)A(M)U(M)A(F)U(M)U(M)G(F)A(M)A(F)A(M)C(E)U(R)^C(M)C(F)A(M)C(M)G(M) 3' (II-7) 5' U(M)U(F)G(M)A(M)U(M)A(F)U(M)U(M)G(F)A(M)A(M)A(F)C(E)U(R)^C(M)C(F)A(M)C(M)G(M) 3' (II-8) 5' U(M)U(F)G(M)A(M)U(M)A(F)U(M)U(M)G(F)A(M)A(F)A(M)C(M)U(R)^C(M)C(F)A(M)C(M)G(M) 3' (II-9) 5' U(M)U(F)G(M)A(M)U(M)A(F)U(M)U(M)G(F)A(M)A(M)A(F)C(M)U(R)^C(M)C(F)A(M)C(M)G(M) 3' (II-10) [In the formula, for nucleic acid bases, A represents adenine, U represents uracil, T represents thymine, G represents guanine, and C represents cytosine (however, when C is ENA, it represents 2'-O,4'-C-ethylene-bridged-5-methylcytidine), and for nucleosides, (R) represents RNA, (D) represents DNA, (M) represents 2'-OMe RNA, (3M) represents 3'-OMe RNA, (F) represents 2'-F RNA, and (E) represents ENA. Furthermore, in the formula, "^" indicates that the bond between nucleosides is a phosphorothioate bond (-P(=S)(OH)-), and unless otherwise specified, it indicates that the bond between nucleosides is a phosphate diester bond (-P(=O)(OH)-). However, even unless otherwise specified, the two internucleoside bonds in the 5' and 3' ends of the sense chain region, and the two internucleoside bonds in the 5' end of the antisense chain region and the three internucleoside bonds in the 3' end of the nucleosides are phosphorothioate bonds.

26. An overhang structure of three bases or less is added to the 5' and / or 3' ends of the sense strand region and / or antisense strand region. Preferably, the overhang structure has two bases. More preferably, the overhang structure is a nucleotide containing two consecutive thymines, or a nucleotide containing two consecutive uracils. More preferably, the oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 25, wherein the overhang structure is two consecutive U(M) added to the 3' end of the antisense chain region.

27. The 5' end of the sense chain region is given by the following formula 【Transformation 3】 [In the formula, the dashed line indicates a phosphate diester bond with the 5'-phosphate group of the nucleotide at the 5' end of the adjacent sense strand region.] The oligonucleotide according to claim 25, or a pharmaceutically acceptable salt thereof, characterized by being chemically modified with a GalNAc unit represented by .

28. The oligonucleotide according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the sense chain region is of the following formula (I-2), the antisense chain region is of any of the following formulas (II-11) to (II-20), and the sense chain region and the antisense chain region each form a double-stranded oligonucleotide as independent oligonucleotides. (formula) 5' GNC(M)^G(M)^U(M)G(M)G(F)A(M)G(F)U(F)U(F)U(M)C(M)A(M)A(M)U(M)A(M)U(M)C(M)^A(M)^A(3M) 3' (I-2) 5' U(M)^U(F)^G(M)A(M)U(M)A(F)U(M)U(F)G(F)A(M)A(M)A(D)C(M)T(E)C(M)C(F)A(M)C(M)^G(M)^U(M)^U(M) 3' (II-11) 5' U(M)^U(F)^G(M)A(F)U(M)A(F)U(M)U(M)G(F)A(M)A(M)A(D)C(M)T(E)C(M)C(F)A(M)C(M)^G(M)^U(M)^U(M) 3' (II-12) 5' U(M)^U(F)^G(M)A(M)U(M)A(F)U(M)U(M)G(F)A(F)A(M)A(D)C(M)T(E)C(M)C(F)A(M)C(M)^G(M)^U(M)^U(M) 3' (II-13) 5' U(M)^U(F)^G(M)A(M)U(M)A(F)U(M)U(M)G(F)A(M)A(M)A(D)C(M)T(E)C(M)C(F)A(M)C(F)^G(M)^U(M)^U(M) 3' (II-14) 5' U(M)^U(F)^G(M)A(M)U(M)A(F)U(M)U(F)G(F)A(M)A(M)A(M)C(E)U(R)^C(M)C(F)A(M)C(M)^G(M)^U(M)^U(M) 3' (II-15) 5' U(M)^U(F)^G(M)A(M)U(M)A(F)U(M)U(M)G(F)A(F)A(M)A(M)C(E)U(R)^C(M)C(F)A(M)C(M)^G(M)^U(M)^U(M) 3' (II-16) 5' U(M)^U(F)^G(M)A(M)U(M)A(F)U(M)U(M)G(F)A(M)A(F)A(M)C(E)U(R)^C(M)C(F)A(M)C(M)^G(M)^U(M)^U(M) 3' (II-17) 5' U(M)^U(F)^G(M)A(M)U(M)A(F)U(M)U(M)G(F)A(M)A(M)A(F)C(E)U(R)^C(M)C(F)A(M)C(M)^G(M)^U(M)^U(M) 3' (II-18) 5' U(M)^U(F)^G(M)A(M)U(M)A(F)U(M)U(M)G(F)A(M)A(F)A(M)C(M)U(R)^C(M)C(F)A(M)C(M)^G(M)^U(M)^U(M) 3' (II-19) 5' U(M)^U(F)^G(M)A(M)U(M)A(F)U(M)U(M)G(F)A(M)A(M)A(F)C(M)U(R)^C(M)C(F)A(M)C(M)^G(M)^U(M)^U(M) 3' (II-20) [In the formula, "GN" is the formula below] 【Chemistry 4】 The formula shows a GalNAc unit represented by (In the formula, the dashed line indicates a phosphate diester bond with the 5'-phosphate group of the nucleotide at the 5' end of the adjacent sense strand region.) For nucleic acid bases, A represents adenine, U represents uracil, T represents thymine, G represents guanine, and C represents cytosine (however, when C is ENA, it represents 2'-O,4'-C-ethylene-bridged-5-methylcytidine). For nucleosides, (R) represents RNA, (D) represents DNA, (M) represents 2'-OMe RNA, (3M) represents 3'-OMe RNA, (F) represents 2'-F RNA, and (E) represents ENA. In addition, in the formula, "^" indicates that the bond between nucleosides is a phosphorothioate bond (-P(=S)(OH)-), and unless otherwise specified, it indicates that the bond between nucleosides is a phosphate diester bond (-P(=O)(OH)-). The 3' position of the nucleotide at the 3' end of the sense strand, the 5' position of the nucleotide at the 5' end of the antisense strand, and the 3' position of the nucleotide at the 3' end of the antisense strand are all hydroxyl groups.

29. A pharmaceutical composition containing an oligonucleotide or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 28 as an active ingredient.

30. The pharmaceutical composition according to claim 29, for the treatment or prevention of a disease that can be treated or prevented by suppressing the expression of transferrin receptor 2.

31. The pharmaceutical composition according to claim 30 for the prevention or treatment of anemia.

32. Anemia can be anemia associated with chronic inflammation or anemia associated with bone marrow dysfunction. Preferably, the anemia associated with chronic inflammation is anemia associated with autoimmune disease, anemia associated with infection, anemia associated with inflammatory bowel disease, anemia associated with heart failure, anemia associated with chronic kidney disease, cancer anemia, or anemia associated with Castleman disease, or the anemia associated with bone marrow dysfunction is anemia associated with myelofibrosis, anemia associated with myelodysplastic syndrome, or anemia associated with chronic myelomonocytic leukemia, or anemia associated with bone marrow suppression due to chemotherapy, according to claim 31.

33. This program targets patients receiving treatment for anemia, diseases causing anemia, or iron overload. Preferably, the pharmaceutical composition according to claim 29, wherein the anemia treatment drug, the drug for treating a disease causing anemia, or the iron overload treatment drug is ruspatercept or ruxolitinib.

34. Use of an oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 28 in the manufacture of a pharmaceutical composition.

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