dsRNA molecules for regulating CIDEB mRNA expression

Abstract

The present invention provides a double-stranded RNA for suppressing the expression of cell death-inducing DFFA-like effector B (CIDEB) in cells, cells containing the double-stranded RNA, and a method for treating diseases or symptoms mediated by or related to CIDEB expression in a target using the dsRNA or the cells.

Description

[Technical Field]

[0001] This disclosure relates to the field of RNA interference. [Background technology]

[0002] Cell death-inducing DFFA-like effector B (CIDEB) is a member of the cell death-inducing DFFA-like effector (CIDE) protein family. CIDEB is highly expressed in the liver and kidneys, and low in the small intestine, white adipose tissue, and colon (Li, JZet al. 2007. Diabetes. 56:2523-2532). CIDEB is present in lipid droplets and smooth endoplasmic reticulum and can directly interact with apoB100 and apoB48 (Ye et al. 2009 Cell Metab. 9:177-190). As a member of the CIDE family, overexpression of the CIDEB protein induces cell death.

[0003] The physiological functions of CIDEB are associated with various lipid metabolic pathways, particularly the VLDL pathway. For example, CIDEB mediates the lipidization and maturation of VLDL through its interaction with ApoB. Furthermore, CIDEB is required for the biosynthesis of VLDL transport vesicles and the lipidization of intestinal chylomicrons. CIDEB transcription is regulated by the transport of nuclear factors in stem cells. According to Li et al. (Ye et al. 2009. Cell Metab. 9:177-190; Li JWet al. 2010. Biochim. Biophys. Acta. 1801:577-586), CideB knockout mice exhibited different lipid metabolic levels compared to wild-type mice. In addition, previous studies have shown that CIDEB is also required for the formation of hepatitis C virus particles.

[0004] Non-alcoholic fatty liver disease (NAFLD) / non-alcoholic steatohepatitis (NASH) is a symptom of metabolic syndrome affecting the liver. Due to changes in diet and lifestyle, obesity and metabolic syndrome are widespread in many countries, resulting in a significant increase in the incidence of NAFLD. NASH is a more advanced stage of simple fatty liver disease, exhibiting pathological findings such as lipid deposition, inflammatory cell infiltration, liver tissue necrosis, and fibrotic lesions, and can progress to severe cirrhosis and hepatocellular carcinoma (HCC). NAFLD not only affects the hepatobiliary system but is also closely associated with insulin resistance, dyslipidemia, arteriosclerosis, fat embolism, and hematological disorders (Friedman SL et al., Nat Med, 2018, 24:908-22). NAFLD has a high prevalence of approximately 15% to 40% in developed countries and regions, and 10% to 20% of NAFLD patients progress to non-alcoholic steatohepatitis (NASH). It is estimated that the global incidence of NASH is 5% to 7%, rising to 22% in diabetic patients, with approximately 15% to 25% of NASH patients developing cirrhosis.

[0005] Therefore, in this field, there is a need for compositions and methods for treating diseases, disorders, and conditions associated with the activity of cell death-inducing DFFA-like effector B (CIDEB).

[0006] Suppressing CIDEB expression with double-stranded RNA (dsRNA), particularly small interfering RNA (siRNA), based on RNA interference mechanisms, represents a novel approach to treating diseases, disorders, and conditions associated with the activity of cell death-inducing DFFA-like effector B (CIDEB). [Overview of the project] [Means for solving the problem]

[0007] This disclosure provides a novel double-stranded RNA (dsRNA) and cells for suppressing the expression of cell death-inducing DFFA-like effector B (CIDEB) in cells, a pharmaceutical composition and kit containing the same, and a method for suppressing or reducing the gene expression of cell death-inducing DFFA-like effector B (CIDEB), or a method for treating a disease or disorder in which a reduction in the expression of cell death-inducing DFFA-like effector B (CIDEB) is beneficial, using the dsRNA, cells, pharmaceutical composition and kit.

[0008] In a first embodiment, the disclosure provides a double-stranded RNA (dsRNA) for suppressing the expression of cell death-inducing DFFA-like effector B (CIDEB) in cells, comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand and the antisense strand are each independently 15 to 30 nucleotides long, and the antisense strand contains a nucleotide sequence of at least 15 adjacent nucleotides from the nucleotide sequence described in any one of SEQ ID NOs. 62 to 122. In some embodiments, the sense strand contains a nucleotide sequence of at least 15 adjacent nucleotides from the nucleotide sequence described in any one of SEQ ID NOs. 1 to 61.

[0009] In some embodiments, the sense chain and the antisense chain are each independently 15 to 27 nucleotides long, preferably 18 to 25 nucleotides long, and more preferably 19 to 21 nucleotides long. In some embodiments, the sense chain is 15 to 27 nucleotides long, preferably 17 to 25 nucleotides long, more preferably 18 to 23 nucleotides long, more preferably 19 to 21 nucleotides long, and most preferably 19 amino acids long. In some embodiments, the antisense chain is 15 to 27 nucleotides long, preferably 17 to 25 nucleotides long, more preferably 18 to 23 nucleotides long, more preferably 19 to 22 nucleotides long, and most preferably 21 amino acids long.

[0010] In some embodiments, a hairpin loop is formed between the sense strand and the antisense strand of the dsRNA. In other embodiments, the dsRNA is siRNA.

[0011] In some embodiments, the length of the double-stranded region is 15 to 25 nucleotide pairs, preferably 16 to 23 nucleotide pairs, and more preferably 18 to 20 nucleotide pairs.

[0012] In some embodiments, one or both of the sense strand and the antisense strand include a 3' overhang and / or a 5' overhang having at least one nucleotide. For example, one or both of the sense strand and the antisense strand include a 3' overhang and / or a 5' overhang having at least two nucleotides. In some embodiments, the antisense strand includes a 3' overhang and / or a 5' overhang of at least one nucleotide, preferably the antisense strand includes a 3' overhang and / or a 5' overhang of two nucleotides. In some specific embodiments, the dsRNA has a two-nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end.

[0013] In some embodiments, the antisense strand comprises a nucleotide sequence of at least 16 adjacent nucleotides, a nucleotide sequence of at least 17 adjacent nucleotides, a nucleotide sequence of at least 18 adjacent nucleotides, a nucleotide sequence of at least 19 adjacent nucleotides, or a nucleotide sequence of at least 20 adjacent nucleotides from any one of the nucleotide sequences described in SEQ ID NOs.62 to 122, and preferably the antisense strand comprises a nucleotide sequence described in any one of SEQ ID NOs.62 to 122. In some embodiments, the sense strand comprises a nucleotide sequence of at least 16 adjacent nucleotides, a nucleotide sequence of at least 17 adjacent nucleotides, or a nucleotide sequence of at least 18 adjacent nucleotides from any one of the nucleotide sequences described in SEQ ID NOs.1 to 61, and preferably the sense strand comprises a nucleotide sequence described in any one of SEQ ID NOs.1 to 61.

[0014] In some embodiments, the dsRNA includes one of the paired sense strand sequences and antisense strand sequences shown in Table 3.

[0015] In some embodiments, substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, or all nucleotides of the sense strand and all nucleotides of the antisense strand are modified nucleotides.

[0016] In some embodiments, the sense strand and the antisense strand are independently SCP-modified nucleotides, 2'-O-alkyl-modified nucleotides (such as 2'-O-methyl-modified nucleotides), 2'-methoxyethyl-modified nucleotides, 2'-fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, inosine ribonucleotides, debasalized nucleotides, reverse debasalized deoxyribonucleotides, phosphorothioate internucleotide bond modifications, vinyl phosphonate-modified nucleotides, locked nucleotides, unlocked nucleotides, 2'-amino-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-O-allyl-modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides containing non-natural bases, cholesteryl derivatives or terminal nucleotides bonded to a bisdecylamide dodecanoate group, deoxyribonucleotides, 3'-terminal deoxythymine (dT) nucleotides, stereostructure-restricted nucleotides, and restricted ethyl nucleotides. The sensor strand and the antisense strand each independently contain one or more nucleotide modifications selected from the group consisting of nucleotides, 2'-hydroxy-modified nucleotides, nucleotides containing a methylphosphonate group, nucleotides containing 5'-phosphate, nucleotides containing a 5'-phosphate mimetic, glycol-modified nucleotides (GNAs), and 2-O-(N-methylacetamide)-modified nucleotides.

[0017] In some embodiments, the sense strand and / or the antisense strand contains at least two 2'-fluoromodified nucleotides. In some embodiments, the sense strand and / or the antisense strand contains at least eight 2'-O-methylmodified nucleotides. In some embodiments, the 3' and / or 5' ends of the sense strand and / or the antisense strand contain 1 to 5 phosphorothioate nucleotide interlinks, preferably 2 to 3 phosphorothioate nucleotide interlinks.

[0018] In some embodiments, the 3' and / or 5' ends of the sense strand and / or antisense strand contain 1 to 5 phosphorothioate nucleotide interbonds, preferably 2 to 3 phosphorothioate nucleotide interbonds.

[0019] In some embodiments, the antisense strand of the dsRNA is 21 nucleotides long, and (i) 2'-O-methyl modified nucleotides at positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21, and 2'-fluoro modified nucleotides at positions 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 (counted from the 5' end); and / or (ii) Internucleotide links of phosphorothioate between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21 (counted from the 5' end) It holds.

[0020] In some embodiments, the antisense strand of the dsRNA is 21 nucleotides long, and (i) 2'-O-methyl modified nucleotides at positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 20, and 21, and 2'-fluoro modified nucleotides at positions 2, 4, 6, 8, 10, 12, 14, 16, and 18 (counted from the 5' end); and / or (ii) Internucleotide links of phosphorothioate between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21 (counted from the 5' end) It holds.

[0021] In some embodiments, the antisense strand of the dsRNA is 21 nucleotides long, and (i) 2'-deoxyribochemical modifications at one or more nucleotide positions; (ii) SCP modifications at one or more nucleotide positions; (iii) 2'-fluoromodifications at one or more nucleotide positions; (iv) 2'-O-methyl modifications at positions other than those having the 2'-deoxy, SCP, and 2'-fluoro modifications described in (i), (ii), and (iii); and / or (v) Internucleotide links of phosphorothioate between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21 (counted from the 5' end) It holds.

[0022] In some embodiments, the antisense strand of the dsRNA is 21 nucleotides long, and (i) 2'-deoxy modifications at positions 2, 5, 7, and 12 (counted from the 5' end); (ii) The SCP modification at position 1 (counted from the 5' end); (iii) 2'-fluoro modification at position 14 (counted from the 5' end); (iv) 2'-O-methyl modifications at positions 3, 4, 6, 8, 9, 10, 11, 13, 15, 16, 17, 18, 19, 20, and 21 (counted from the 5' end); and / or (v) Internucleotide links of phosphorothioate between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21 (counted from the 5' end) It holds.

[0023] In some embodiments, the antisense strand of the dsRNA is 23 nucleotides long, and (i) The SCP modification at position 1 (counted from the 5' end); (ii) 2'-fluoromodifications at positions 2, 4, 6, 8, 10, 12, 14, 16, and 18 (counted from the 5' end); (iii) 2'-O-methyl modifications at positions 3, 5, 7, 9, 11, 13, 15, 17, 19, 20, 21, 22, and 23 (counted from the 5' end); and / or (iv) Internucleotide links of phosphorothioate between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counted from the 5' end) It holds.

[0024] In some embodiments, the sense strand of the dsRNA is 19 nucleotides long, and (i) 2'-O-methyl modified nucleotides at positions 1-6 and 10-19, and 2'-fluoro modified nucleotides at positions 7-9 (counted from the 5' end); and / or (ii) Phosphothioate internucleotide bonds between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counted from the 5' end) It holds.

[0025] In some embodiments, the sense strand of the dsRNA is 19 nucleotides long, and (i) 2'-O-methyl modified nucleotides at positions 1-6 and 10-19, and 2'-fluoro modified nucleotides at positions 7-9 (counted from the 5' end); and / or (ii) Internucleotide links between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between positions 17 and 18, and between positions 18 and 19 (counted from the 5' end) It holds.

[0026] In some embodiments, the sense strand of the dsRNA is 21 nucleotides long, and (i) 2'-fluoromodifications at positions 9, 10, and 11 (counted from the 5' end); (ii) 2'-O-methyl modifications at positions 1, 2, 3, 4, 5, 6, 7, 8, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 (counted from the 5' end); and / or (iii) Phosphothioate internucleotide bonds (counted from the 5' end) between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, and between nucleotide positions 20 and 21. It holds.

[0027] In some embodiments, the dsRNA is further bound to a ligand moiety containing N-acetylgalactosamine, preferably the sense strand of the dsRNA is bound to the ligand moiety. In some embodiments, the 3' end of the sense strand is bound to the ligand moiety. In other embodiments, the 5' end of the sense strand is bound to the ligand moiety. In some specific embodiments, the sense strand of the dsRNA includes a phosphorothioate internucleotide bond (counted from the 3' end) between nucleotide positions 1 and 2, and is bound to the ligand moiety at the 3' end of the sense strand via the phosphorothioate.

[0028] In some embodiments, the ligand portion includes a binding group of formula (X'). [ka] During the ceremony, [ka] The symbol represents the point that connects to the above dsRNA; Q is independent of H, [ka] and; L1 is a bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O-, or -NHC(O)-(CH2NHC(O)) , 1-2 , -; L2 is a bond or -CH2CH2C(O)-; L3 is a bond, -(NHCH2CH2) b -, -(NHCH2CH2CH2) b -, or -C(O)CH2-; L4 is -(OCH2CH2) c -, -(OCH2CH2CH2) c -, -(OCH2CH2CH2CH2) c -, -(OCH2CH2CH2CH2CH2) c -, or -NHC(O)-(CH2) d -; a = 0, 1, 2, or 3; b = 1, 2, 3, 4, or 5; c = 1, 2, 3, 4, or 5; d = 1, 2, 3, 4, 5, 6, 7, or 8; L is a bond, -CH2O-, or -NHC(O)-; L’ is a bond, -C(O)NH-, -NHC(O)-, or -O(CH2CH2O) e -; e = 1, 2, 3, 4, or 5; T is a bond, -CH2-, -C(O)-, -M-, -CH2-M-, or -C(O)-M-; M is

Chemical formula

[0029] In some embodiments, the binding ligand targets the asialoglycoprotein receptor (ASGPR).

[0030] In some preferred embodiments, the above-mentioned bonding group is selected from Table 1. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0031] In some preferred embodiments, the above-mentioned bonding group is selected from Table 2. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5]

[0032] In some embodiments, the ligand moiety in the above dsRNA has the following structure: [ka] (In the formula, [ka] (This indicates a point that is connected to the sense strand of the dsRNA (preferably the 3' end of the sense strand) via a phosphate ester group or a phosphorothioate group.)

[0033] In some embodiments, the ligand moiety in the above dsRNA has the following structure: [ka] (In the formula, [ka] (This indicates a point that is connected to the sense strand of the dsRNA (preferably the 3' end of the sense strand) via a phosphate ester group or a phosphorothioate group.)

[0034] In some embodiments, the ligand portion of the dsRNA of this disclosure has the following structure: [ka] (In the formula, [ka] (This indicates a point that connects to the above dsRNA via a phosphate ester group or a phosphorothioate group.)

[0035] In some embodiments, the ligand portion of the dsRNA of this disclosure has the following structure: [ka] (In the formula, [ka] (This indicates a point that is connected to the sense strand of the dsRNA (preferably the 3' end of the sense strand) via a phosphate ester group or a phosphorothioate group.)

[0036] In some embodiments, the antisense strand contains one of the modified nucleotide sequences shown in Table 5, and / or the sense strand contains one of the modified nucleotide sequences shown in Table 4. In some embodiments, the dsRNA contains one of the paired modified sense strand sequences and modified antisense strand sequences shown in Table 6.

[0037] In some embodiments, the dsRNA in this disclosure (a) The sense chain includes UmsAmsCmUmCmAmGfGfUfCmAmGmUmAmUmCmUmAmsAms-GL6 (SEQ ID NO: 149), and the antisense chain includes UmsUfsAmGfAmUfAmCfUmGfAmCfCmUfGmAfGmUfAmsAfsGm (SEQ ID NO: 259); (b) The sense chain includes CmsAmsCmCmAmUmGfGfAfGmUmAmCmCmUmCmUmCmsAms-GL6 (SEQ ID NO: 150), and the antisense chain includes UmsGfsAmGfAmGfGmUfAmCfUmCfCmAfUmGfGmUfGmsGfsAm (SEQ ID NO: 257); (c) The sense chain described above includes AmsGmsGmUmCmAmGfUfAfUmCmUmAmAmUmAmUmAmsAms-GL6 (SEQ ID NO: 151), and the antisense chain described above includes UmsUfsAmUfAmUfUmAfGmAfUmAfCmUfGmAfCmCfUmsGfsAm (SEQ ID NO: 260); (d) The sense chain includes CmsUmsCmUmAmUmGfAfGfUmUmGmUmGmAmCmUmUmsUms-GL6 (SEQ ID NO: 152), and the antisense chain includes AmsAfsAmGfUmCfAmCfAmAfCmUfCmAfUmAfGmAfGmsUfsAm (SEQ ID NO: 263); (e) The sense chain includes AmsGmsAmCmUmAmUfGfAfCmAmGmCmAmUmCmAmAmsAms-GL6 (SEQ ID NO: 153), and the antisense chain includes UmsUfsUmGfAmUfGmCfUmGfUmCfAmUfAmGfUmCfUmsUfsUm (SEQ ID NO: 267); (f) The sense chain includes GmsAmsCmUmAmUmGfAfCfAmGmCmAmUmCmAmAmAmsUms-GL6 (SEQ ID NO: 154), and the antisense chain includes AmsUfsUmUfGmAfUmGfCmUfGmUfCmAfUmAfGmUfCmsUfsUm (SEQ ID NO: 268); (g) The sense strand includes AmsCmsUmAmUmGmAfCfAfGmCmAmUmCmAmAmAmUmsUms-GL6 (SEQ ID NO: 155), and the antisense strand includes AmsAfsUmUfUmGfAmUfGmCfUmGfUmCfAmUfAmGfUmsCfsUm (SEQ ID NO: 269); (h) The sense chain described above includes CmsUmsAmUmGmAmCfAfGfCmAmUmCmAmAmAmUmUmsUms-GL6 (SEQ ID NO: 156), and the antisense chain described above includes AmsAfsAmUfUmUfGmAfUmGfCmUfGmUfCmAfUmAfGmsUfsCm (SEQ ID NO: 270); (i) The sense chain includes AmsUmsGmAmCmAmGfCfAfUmCmAmAmAmUmUmUmCmsAms-GL6 (SEQ ID NO: 157), and the antisense chain includes UmsGfsAmAfAmUfUmUfGmAfUmGfCmUfGmUfCmAfUmsAfsGm (SEQ ID NO: 271); (j) The sense chain described above includes UmsGmsAmCmAmGmCfAfUfCmAmAmAmUmUmUmCmAmsAms-GL6 (SEQ ID NO: 158), and the antisense chain described above includes UmsUfsGmAfAmAfUmUfUmGfAmUfGmCfUmGfUmCfAmsUfsAm (SEQ ID NO: 272); (k) The sense chain includes CmsAmsGmAmCmAmGfUfAfCmAmGmGmCmUmAmGmAmsUms-GL6 (SEQ ID NO: 159), and the antisense chain includes AmsUfsCmUfAmGfCmCfUmGfUmAfCmUfGmUfCmUfGmsCfsAm (SEQ ID NO: 273); (l) The sense chain above includes AmsGmsAmCmAmGmUfAfCfAmGmGmCmUmAmGmAmUmsAms-GL6 (SEQ ID NO: 160), and the antisense chain above includes UmsAfsUmCfUmAfGmCfCmUfGmUfAmCfUmGfUmCfUmsGfsCm (SEQ ID NO: 274); (m) The sense chain above includes GmsAmsCmAmGmUmAfCfAfGmGmCmUmAmGmAmUmAmsAms-GL6 (SEQ ID NO: 161), and the antisense chain above includes UmsUfsAmUfCmUfAmGfCmCfUmGfUmAfCmUfGmUfCmsUfsGm (SEQ ID NO: 275); (n) The sense chain above includes AmsAmsAmCmAmUmUfUfCfCmAmAmUmAmAmAmAmAmsUms-GL6 (SEQ ID NO: 162), and the antisense chain above includes AmsUfsUmUfUmUfAmUfUmGfGmAfAmAfUmGfUmUfUmsUfsUm (SEQ ID NO: 276); (o) The sense chain above includes AmsCmsAmUmUmUmCfCfAfAmUmAmAmAmAmAmAmUmAmsUms-GL6 (SEQ ID NO: 163), and the antisense chain above includes AmsUfsAmUfUmUfUmUfAmUfUmGfGmAfAmAfUmGfUmsUfsUm (SEQ ID NO: 277); (p) The sense chain above includes CmsCmsCmUmAmAmAfCfUfCmCmCmCmAmGmCmAmUmsAms-GL6 (SEQ ID NO: 164), and the antisense chain above includes UmsAfsUmGfCmUfGmGfGmGfAmGfUmUfUmAfGmGfGmsAfsCm (SEQ ID NO: 281); (q) The sense chain described above includes UmsAmsCmUmCmAmGfGfUfCmAmGmUmAmUmCmUmAmsAms-GL6 (SEQ ID NO: 149), and the antisense chain described above includes (SCP-U)sdTsAmGmdAUmdACmUmGmAmdCCmUfGmAmGmUmAmsAmsGm (SEQ ID NO: 283); (r) The sense chain described above includes AmsGmsGmUmCmAmGfUfAfUmCmUmAmAmUmAmUmAmsAms-GL6 (SEQ ID NO: 151), and the antisense chain described above includes (SCP-U)sdTsAmUmdAUmdTAmGmAmUmdACmUfGmAmCmCmUmsGmsAm (SEQ ID NO: 284); (s) The sense chain described above includes AmsGmsAmCmUmAmUfGfAfCmAmGmCmAmUmCmAmAmsAms-GL6 (SEQ ID NO: 153), and the antisense chain described above includes (SCP-U)sdTsUmGmdAUmdGCmUmGmUmdCAmUfAmGmUmCmUmsUmsUm (SEQ ID NO: 285); (t) The sense chain described above includes GmsAmsCmUmAmUmGfAfCfAmGmCmAmUmCmAmAmAmsAms-GL6 (SEQ ID NO: 165), and the antisense chain described above includes (SCP-U)sdTsUmUmdGAmdTGmCmUmGmdTCmAfUmAmGmUmCmsUmsUm (SEQ ID NO: 286); (u) The sense chain described above includes UmsGmsAmCmAmGmCfAfUfCmAmAmAmUmUmUmCmAmsAms-GL6 (SEQ ID NO: 158), and the antisense chain described above includes (SCP-U)sdTsGmAmdAAmdTUmUmGmAmdTGmCfUmGmUmCmAmsUmsAm (SEQ ID NO: 288); (v) The sense chain described above includes AmsGmsAmCmAmGmUfAfCfAmGmGmCmUmAmGmAmUmsAms-GL6 (SEQ ID NO: 160), and the antisense chain described above includes (SCP-U)sdAsUmCmdTAmdGCmCmUmGmdTAmCfUmGmUmCmUmsGmsCm (SEQ ID NO: 289); (w) The above sense chain includes GmsAmsCmAmGmUmAfCfAfGmGmCmUmAmGmAmUmAmsAms-GL6 (SEQ ID NO: 161), and the above antisense chain includes (SCP-U)sdTsAmUmdCUmdAGmCmCmUmdGUmAfCmUmGmUmCmsUmsGm (SEQ ID NO: 290); (x) The sense chain above includes AmsAmsAmCmAmUmUfUfCfCmAmAmUmAmAmAmAmAmsAms-GL6 (SEQ ID NO: 166), and the antisense chain above includes (SCP-U)sdTsUmUmdTUmdAUmUmGmGmdAAmAfUmGmUmUmUmsUmsUm (SEQ ID NO: 291); (y) The sense chain described above includes AmsCmsAmUmUmUmCfCfAfAmUmAmAmAmAmAmAmUmAmsAms-GL6 (SEQ ID NO: 167), and the antisense chain described above includes (SCP-U)sdTsAmUmdTUmdTUmAmUmUmdGGmAfAmAmUmGmUmsUmsUm (SEQ ID NO: 292); (z) The sense chain described above includes GmsCmsUmCmUmGmAfAfCfCmCmCmAmGmUmGmAmCmsAms-GL6 (SEQ ID NO: 168), and the antisense chain described above includes (SCP-U)sdGsUmCmdACmdTGmGmGmGmdTUmCfAmGmAmGmCmsUmsGm (SEQ ID NO: 293); (ab) The sense chain described above includes GmsCmsCmAmGmGmAfGfCfUmGmCmUmAmGmCmCmAmsAms-GL6 (SEQ ID NO: 169), and the antisense chain described above includes (SCP-U)sdTsGmGmdCUmdAGmCmAmGmdCUmCfCmUmGmGmCmsUmsGm (SEQ ID NO: 294); (ac) The sense chain above includes CmsCmsUmUmUmGmAfCfGfUmGmUmAmCmAmAmGmCmsAms-GL6 (SEQ ID NO: 170), and the antisense chain above includes (SCP-U)sdGsCmUmdTGmdTAmCmAmCmdGUmCfAmAmAmGmGmsUmsGm (SEQ ID NO: 295); (ad) The above sense chain includes GmsCmsCmAmUmAmUfGfUfUmGmCmUmGmGmGmAmAmsAms-GL6 (SEQ ID NO: 171), and the above antisense chain includes (SCP-U)sdTsUmCmdCCmdAGmCmAmAmdCAmUfAmUmGmGmCmsUmsCm (SEQ ID NO: 296); (ae) The above sense chain includes CmsCmsGmCmCmUmCfCfAfUmUmCmCmUmAmCmUmAmsAms-GL6 (SEQ ID NO: 172), and the above antisense chain includes (SCP-U)sdTsAmGmdTAmdGGmAmAmUmdGGmAfGmGmCmGmGmsUmsCm (SEQ ID NO: 297); (af) The sense chain above includes GmsCmsAmAmAmGmAfCfUfAmUmGmAmCmAmGmCmAmsAms-GL6 (SEQ ID NO: 173), and the antisense chain above includes (SCP-U)sdTsGmCmdTGmdTCmAmUmAmdGUmCfUmUmUmGmCmsAmsGm (SEQ ID NO: 298); (ag) The sense chain above includes CmsUmsCmUmGmAmAfCfCfCmCmAmGmUmGmAmCmUmsAms-GL6 (SEQ ID NO: 174), and the antisense chain above includes (SCP-U)sdAsGmUmdCAmdCUmGmGmGmdGUmUfCmAmGmAmGmsCmsUm (SEQ ID NO: 299); (ah) The above sense chain includes CmsCmsUmCmAmCmAfUfCfCmCmAmAmGmUmCmUmAmsAms-GL6 (SEQ ID NO: 176), and the above antisense chain includes (SCP-U)sdTsAmGmdACmdTUmGmGmGmdAUmGfUmGmAmGmGmsCmsGm (SEQ ID NO: 301); (ai) The above sense chain includes AmsUmsUmUmCmCmAfAfUfAmAmAmAmAmUmAmUmCmsAms-GL6 (SEQ ID NO: 177), and the above antisense chain includes (SCP-U)sdGsAmUmdAUmdTUmUmUmAmdTUmGfGmAmAmAmUmsGmsUm (SEQ ID NO: 302); (aj) The above sense chain includes AmsAmsAmAmAmCmAmUmUfUfCfCmAmAmUmAmAmAmAmAmsAms-GL6 (SEQ ID NO: 178), and the above antisense chain includes (SCP-U)sUfsUmUfUmUfAmUfUmGfGmAfAmAfUmGfUmUfUmUmUmsGmsUm (SEQ ID NO: 303); (ak) The above sense chain includes AmsAmsAmCmAmUmUmUmCfCfAfAmUmAmAmAmAmAmAmUmAmsAms-GL6 (Sequence ID 179), and the above antisense chain includes (SCP-U)sUfsAmUfUmUfUmUfAmUfUmGfGmAfAmAfUmGfUmUmUmsUmsUm (Sequence ID 304), GL6 is [ka] (In the formula, [ka] (This indicates a point that connects to the 3' end of the sense strand of the dsRNA via a phosphate ester group or a phosphorothioate group.)

[0038] In a second embodiment, the Disclosure provides cells comprising the dsRNA of the Disclosure.

[0039] In a third aspect, the Disclosure provides a pharmaceutical composition comprising the dsRNA or cells of the Disclosure and, optionally, a pharmaceutically acceptable carrier or excipient.

[0040] In a fourth embodiment, the Disclosure provides a kit comprising the dsRNA, cells, or pharmaceutical composition of the Disclosure.

[0041] In a fifth aspect, the Disclosure provides a method for suppressing cell death-inducing DFFA-like effector B (CIDEB) in a subject, comprising the step of administering the dsRNA, cells, or pharmaceutical composition of the Disclosure to the subject. The Disclosure also provides a method for treating a disease or disorder in which a reduction in the expression of cell death-inducing DFFA-like effector B (CIDEB) is beneficial, comprising the step of administering the dsRNA, cells, or pharmaceutical composition of the Disclosure to the subject. The Disclosure also provides a method for preventing at least one symptom in a subject having a disease or disorder in which a reduction in the expression of cell death-inducing DFFA-like effector B (CIDEB) is beneficial, comprising the step of administering the dsRNA, cells, or pharmaceutical composition of the Disclosure to the subject.

[0042] In some embodiments, diseases or disorders in which a reduction in the expression of the above-mentioned cell death-inducing DFFA-like effector B (CIDEB) is beneficial are CIDEB-mediated diseases or CIDEB-related diseases.

[0043] In some embodiments, the CIDEB-mediated disease or CIDEB-related disease includes liver diseases (fatty liver, hepatitis (steatohepatitis, non-alcoholic steatohepatitis, alcoholic steatohepatitis, and viral hepatitis, etc.), non-alcoholic fatty liver disease, alcoholic fatty liver disease, hepatic fibrosis, cirrhosis, and hepatic failure, etc.), cholangitis (primary biliary cholangitis, primary sclerosing cholangitis), endocrine disorders, urinary tract diseases, metabolic diseases (metabolic syndrome, etc.), hepatobiliary diseases, fibrotic diseases, and cardiovascular diseases (hypertension, endothelial cell dysfunction). The following conditions are selected from among arteriosclerosis, atherosclerosis, coronary artery disease, myocardial infarction, ischemic stroke, and other heart diseases, etc., immunoinflammatory diseases, central nervous system diseases, digestive diseases, hyperproliferative diseases (cancer, etc.), dyslipidemia (hyperlipidemia, hypercholesterolemia, hypertriglyceridemia, high LDL cholesterol, low HDL cholesterol, postprandial hypertriglyceridemia, etc.), impaired blood glucose control (insulin resistance, type 2 diabetes, etc.), adipocyte dysfunction, visceral fat accumulation, obesity, eating disorders, and excessive cravings for sugar. In some embodiments, the CIDEB-mediated disease or CIDEB-related disease is selected from liver diseases (fatty liver, hepatitis (steatohepatitis, non-alcoholic steatohepatitis, alcoholic steatohepatitis, and viral hepatitis, etc.), non-alcoholic fatty liver disease, alcoholic fatty liver disease, hepatic fibrosis, cirrhosis, and hepatic failure, etc.), cholangitis (primary biliary cholangitis, primary sclerosing cholangitis), metabolic diseases (metabolic syndrome, etc.), autoimmune diseases, cardiovascular diseases (hypertension, endothelial cell dysfunction, arteriosclerosis, atherosclerosis, coronary artery disease, myocardial infarction, ischemic stroke, and other heart diseases, etc.), and dyslipidemia (hyperlipidemia, hypercholesterolemia, hypertriglyceridemia, high LDL cholesterol, low HDL cholesterol, and postprandial hypertriglyceridemia, etc.).

[0044] In some embodiments, the present invention provides a method for treating a disease or disorder in which a reduction in the expression of cell death-inducing DFFA-like effector B (CIDEB) is beneficial, a method for preventing at least one symptom in a patient having a disease or disorder in which a reduction in the expression of cell death-inducing DFFA-like effector B (CIDEB) is beneficial, or a method for reducing cell death-inducing DFFA-like effector B (CIDEB) in a subject, comprising the step of subcutaneously, topically, or intravenously administering the above-mentioned dsRNA, the above-mentioned cells, or the above-mentioned pharmaceutical composition to the above-mentioned subject. In some embodiments, the above-mentioned subject is a human patient. [Modes for carrying out the invention]

[0045] Embodiments of the present invention will be described below through specific examples. Those skilled in the art will readily understand other advantages and effects of the present invention from the disclosures herein. The present invention can also be implemented or applied through various other specific embodiments. Various details herein can be modified or altered in various ways based on various viewpoints and uses without departing from the spirit of the invention.

[0046] It should be understood that the scope of protection of the present invention is not limited to the following specific embodiments. Furthermore, it should be understood that the terminology used in the embodiments of the present invention is intended to describe specific embodiments, not to limit the scope of protection of the present invention.

[0047] In the specification and claims of the present invention, the singular forms "a," "an," and "the" include the plural form unless otherwise explicitly stated in the context.

[0048] Where numerical ranges are given in embodiments, it should be understood that, unless otherwise specifically stated in the present invention, the values ​​at both ends of each numerical range and any values ​​between those ends can be selected. Unless otherwise specifically defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. In addition to the specific methods, apparatus, and materials used in embodiments, those skilled in the art can also implement the present invention using similar or equivalent prior art methods, apparatus, and materials to those described in embodiments of the present invention, based on their understanding of the prior art and the specification of the present invention, and these are all within the scope of protection of the present invention. Embodiments of the present invention are described in more detail below.

[0049] definition In this specification, “double-stranded region” means a region comprising two nucleic acid strands that are antiparallel and complementary or substantially complementary to each other.

[0050] In this specification, "double-stranded RNA" or "dsRNA" refers to a ribonucleic acid molecule or a complex of ribonucleic acid molecules containing the double-stranded region as defined above. The two parts forming the double-stranded region may be two different parts of a larger RNA molecule, or they may be separate RNA molecules.

[0051] When the two parts are separate RNA molecules, dsRNA as used herein refers to small interfering RNA or short interfering RNA, and is abbreviated as siRNA.

[0052] When two parts are two distinct parts of a larger molecule, that is, when the 3' end of one part is joined to the 5' end of the other part via one or more uninterrupted nucleotides to form a double-stranded region, the uninterrupted nucleotides used in this joining are called a "hairpin loop." When two parts are covalently joined by means other than a hairpin loop to form a double-stranded region, the joining structure is called a "linker." When such dsRNA is introduced into a cell, it can be cleaved by an endribonuclease called a dicer enzyme within the cell to become siRNA.

[0053] In this specification, “siRNA” is a class of dsRNA molecules comprising a sense strand and an antisense strand that can mediate the silencing of a target RNA (e.g., mRNA, e.g., a transcript of a protein-coding gene) that is complementary or substantially complementary to the antisense strand. siRNA is generally double-stranded and comprises an antisense strand complementary to its target RNA and a sense strand complementary or substantially complementary to the antisense strand. For convenience, such mRNA is also referred to as silencing target mRNA, and such a gene is also referred to as the target gene. Typically, silencing target RNA is an endogenous gene or a pathogen gene. In addition, RNA other than mRNA (e.g., tRNA) and viral RNA can also be targeted.

[0054] In this specification, “antisense strand” means a strand in dsRNA (particularly siRNA) that includes a region that is completely or substantially complementary to its target sequence.

[0055] In this specification, “complementary region” refers to a region on the antisense strand that is completely or substantially complementary to its target mRNA sequence. If the complementary region is not completely complementary to its target sequence, mismatches may be located in the internal or terminal regions of the molecule. Typically, the most acceptable mismatches are located in terminal regions such as within 5, 4, 3, 2, or 1 nucleotide at the 5' and / or 3' ends. The region of the antisense strand most sensitive to mismatches is called the “seed region.” For example, in an siRNA containing a 19nt strand, position 19 (5' to 3') may tolerate several mismatches.

[0056] In this specification, “complementary” refers to the ability of a first polynucleotide to hybridize with a second polynucleotide under certain conditions, such as stringent conditions. For example, stringent conditions include 400 mM NaCl, 40 mM PIPES, pH 6.4, 1 mM EDTA, and 12–16 hours at 50°C or 70°C.

[0057] In this specification, a “complementary” sequence for satisfying the above requirements regarding its hybridizing ability may include, and may consist solely of, non-Watson-Crick base pairs and / or base pairs formed with non-natural and modified nucleotides. Examples of such non-Watson-Crick base pairs include, but are not limited to, G:U fluctuation base pairs or Hoogsteen base pairs.

[0058] In this specification, a polynucleotide that is "at least partially complementary" or "substantially complementary" to messenger RNA (mRNA) means a polynucleotide that is substantially complementary to the sequence of the mRNA of interest (such as the mRNA encoding CIDEB). For example, a polynucleotide is at least partially complementary to the mRNA encoding CIDEB if its sequence is substantially complementary to the uninterrupted portion of the CIDEB mRNA.

[0059] In this specification, “complementary,” “fully complementary,” and “substantially complementary” may be used with respect to base pairs between the sense strand and antisense strand of dsRNA, particularly siRNA, or between the antisense strand and target sequence of dsRNA, particularly siRNA.

[0060] In this specification, “sense strand” means one strand of siRNA that contains a region substantially complementary to the antisense strand region as defined herein.

[0061] A "nucleoside" is a compound containing two components: one is a purine or pyrimidine base, and the other is ribose or deoxyribose. A "nucleotide" is a compound containing three components: one is a purine or pyrimidine base, another is ribose or deoxyribose, and the third is phosphate. An "oligonucleotide" refers to a nucleic acid molecule (RNA or DNA) that is, for example, less than 100, 200, 300, or 400 nucleotides in length.

[0062] A "base" is the fundamental building block of nucleosides, nucleotides, and nucleic acids, and is also called a "nitrogen base" because it always contains nitrogen. Unless otherwise specified, capital letters in this specification, namely A, U, T, G, and C, indicate the bases of nucleotides, representing adenine, uracil, thymine, guanine, and cytosine, respectively.

[0063] In this specification, “nucleotide overhang” means at least one unpaired nucleotide that protrudes from the double-stranded region of an siRNA. For example, a nucleotide overhang exists when the 3' end of one strand of an siRNA extends beyond the 5' end of the other strand, or vice versa. An siRNA may contain an overhang having at least one nucleotide, at least two nucleotides, at least three nucleotides, at least four nucleotides, or at least five or more nucleotides. A nucleotide overhang may contain or be composed of nucleotides / modified nucleotides (including deoxyribonucleotides / nucleosides). One or more overhangs may be on the sense strand or the antisense strand, or a combination thereof. An overhang having one or more nucleotides may be located at the 5' end, 3' end, or both ends of the antisense strand or sense strand of an siRNA.

[0064] "Bluish end" means that there are no unpaired nucleotides, or nucleotide overhangs, at the end of a double-stranded siRNA. "Bluish-end siRNA" is siRNA that is double-stranded throughout its entire length, meaning that there are no nucleotide overhangs at any end of the molecule.

[0065] The dsRNA, particularly siRNA, of the present invention has substantially all nucleotides modified. For example, substantially all nucleotides of the sense strand are modified nucleotides, or substantially all nucleotides of the antisense strand are modified nucleotides, or substantially all nucleotides of both the sense and antisense strands are modified nucleotides. In other embodiments of the present invention, all nucleotides of the dsRNA, particularly siRNA, of the present invention are modified nucleotides. For example, all nucleotides of the sense strand are modified nucleotides, or all nucleotides of the antisense strand are modified nucleotides, or all nucleotides of both the sense and antisense strands are modified nucleotides. In this specification, “substantially all nucleotides are modified” means that the majority (but not necessarily all) of the nucleotides of the dsRNA, particularly siRNA, of the present invention are modified and may contain 5, 4, 3, 2, or 1 or fewer unmodified nucleotides.

[0066] In this specification, "modified nucleotide" is not particularly limited to, but includes SCP-modified nucleotides, 2'-O-alkyl-modified nucleotides (such as 2'-O-methyl-modified nucleotides or 2'-methoxyethyl-modified nucleotides), 2'-fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, inosine ribonucleotides, debased nucleotides, reverse debased deoxyribonucleotides, phosphorothioate internucleotide bond modifications, vinyl phosphonate-modified nucleotides, locked nucleotides, unlocked nucleotides, 2'-amino-modified nucleotides, 2'-C-alkyl-modified nucleotides, and 2'-O-allyl nucleotides. This includes modified nucleotides, morpholinonucleotides, phosphoramidates, nucleotides containing unnatural bases, cholesteryl derivatives or terminal nucleotides bonded to a bisdecylamide dodecanoate group, deoxyribonucleotides, 3'-terminal deoxythymine (dT) nucleotides, sterically restricted nucleotides, restrictive ethyl nucleotides, 2'-hydroxy-modified nucleotides, nucleotides containing a methylphosphonate group, nucleotides containing 5'-phosphate, nucleotides containing a 5'-phosphate mimetic, glycol-modified nucleotides (GNAs), and 2-O-(N-methylacetamide)-modified nucleotides, etc.

[0067] A "2'-fluoromodified nucleotide" refers to a nucleotide in which the hydroxyl atom at the 2' position of the ribosyl group is replaced with a fluorine atom.

[0068] A "2'-O-methyl modified nucleotide" refers to a nucleotide in which the 2'-hydroxyl group of the ribosyl group is replaced with a methoxyl group.

[0069] "Phosphozoate internucleotide bond modification" refers to a nucleotide in which one or more oxygen atoms on the phosphate group are replaced by sulfur atoms. "Phosphozoate internucleotide bond modification" refers to a modification in which two adjacent nucleotides are joined by a phosphorothioate.

[0070] "SCP-modified nucleotides" have the following structure: [ka] (wherein Base is independently selected from H, a modified or unmodified base, or a leaving group) a modified nucleotide. Preferably, Base is an unmodified base, such as an adenine base, a guanine base, a uracil base, and a cytosine base. In other embodiments, Base is a modified base.

[0071] In some embodiments, the sense strand of the dsRNA, particularly siRNA, of the disclosed herein has two phosphorothioate nucleotide linkage modifications at positions 1-5 (counted from the 5' end) and / or two phosphorothioate nucleotide linkage modifications at positions 1-5 (counted from the 3' end), and / or the antisense strand of the dsRNA, particularly siRNA, of the disclosed herein has two phosphorothioate nucleotide linkage modifications at positions 1-5 (counted from the 5' end) and / or two phosphorothioate nucleotide linkage modifications at positions 1-5 (counted from the 3' end).

[0072] In this specification, the terms “ligand moiety” or “ligand” as used interchangeably refer to a chemical component bound to dsRNA, particularly siRNA, which can alter the distribution, targeting, or lifespan of dsRNA, particularly siRNA. In some embodiments, the ligand moiety enhances affinity to selected targets, such as molecules, cells or cell types, and compartments (compartments of cells or organs, tissues, organs, or regions of the body, etc.), compared to siRNA without the ligand moiety. In some embodiments, the ligand moiety targets the asialocrycoprotein receptor (ASGPR) on hepatocytes. When the ligand moiety binds to ASGPR, internalization by clathrin-coated vesicles is mediated. As the endosome matures, the lysosomal pH decreases, promoting the dissociation of the ligand-receptor complex and releasing the dsRNA, particularly siRNA. Binding of the ligand moiety targeting the asialocrycoprotein receptor (ASGPR) on hepatocytes provides efficacy and stability of dsRNA, particularly siRNA, in vivo or intracellularly. This facilitates subcutaneous administration of dsRNA, particularly siRNA.

[0073] In this specification, “suppression” is used interchangeably with similar terms such as “reduction,” “silencing,” and “downward control,” and encompasses any level of suppression.

[0074] "Suppressing the expression of cell death-inducing DFFA-like effector B (CIDEB)" refers to suppressing the expression of the CIDEB gene or a variant or mutant of the CIDEB gene. Therefore, in the context of genetically modified cells, cell populations, or living organisms, the CIDEB gene may be the wild-type CIDEB gene, the mutant CIDEB gene, or the transgenic CIDEB gene.

[0075] "Suppression of CIDEB gene expression" encompasses any level of suppression of the CIDEB gene, including at least partial suppression of CIDEB gene expression. CIDEB gene expression can be assessed based on the level of any variable related to CIDEB gene expression (e.g., CIDEB mRNA level or CIDEB protein level) or a change in such level. This level may be assessed in a single cell or in a cell population (e.g., including a sample derived from the subject).

[0076] Suppression can be assessed by a decrease in the absolute or relative level of one or more variables related to CIDEB expression compared to the control level. The control level may be any type of control level used in the art, such as the baseline level before administration or the level determined in subjects, cells, or samples treated with similar untreated or controlled (such as a buffer-only control or an inactive control).

[0077] A "hydroxyl protecting group" refers to a group that can prevent a hydroxyl molecule from undergoing a chemical reaction and can be removed under certain conditions to restore the hydroxyl molecule. Examples of hydroxyl protecting groups mainly include silane, acyl, or ether-based protecting groups, preferably trimethylsilyl (TMS), triethylsilyl (TES), dimethylisopropylsilyl (DMIPS), diethylisopropylsilyl (DEIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), acetyl (Ac), chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl (TFA), benzoyl, p-methoxybenzoyl, and 9-fluorenylmethoxycarboxylate. Examples include vonyl (Fmoc), allyloxycarbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), benzyl (Bn), p-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), di-p-methoxytrityl (DMTr), methoxymethyl (MOM), benzyloxymethyl (BOM), 2,2,2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), and p-methoxybenzyloxymethyl (PMBM).

[0078] "Halo" or "halogen" refers to substitutions by fluorine (F), chlorine (Cl), bromine (Br), and iodine (I).

[0079] "C 1-6 "Haloalkyl" refers to the above "C 1-6 This refers to a case where "alkyl" is substituted with one or more halogen groups. In some embodiments, C 1-4 Haloalkyl is particularly preferred, C 1-2Haloalkyls are even more preferred. Examples of haloalkyls, though not limited to them, include -CF3, -CH2F, -CHF2, -CHFCH2F, -CH2CHF2, -CF2CF3, -CCl3, -CH2Cl, -CHCl2, and 2,2,2-trifluoro-1,1-dimethylethyl. Haloalkyls may be substituted at any substitutable bond point, such as 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0080] "C 1-6 "Alkylene" refers to C 1-6 This refers to a divalent group formed by removing another hydrogen atom from an alkyl group, which may be substituted or unsubstituted. In some embodiments, C 1-4 Alkilen, C 2-4 Alkylene and C 1-2 Alkylenes are preferred. Unsubstituted alkylenes are not particularly limited, but include methylene groups (-CH2-), ethylene groups (-CH2CH2-), propylene groups (-CH2CH2CH2-), butylene groups (-CH2CH2CH2CH2-), pentylene groups (-CH2CH2CH2CH2CH2-), and hexylene groups (-CH2CH2CH2CH2CH2CH2-). Examples of the above-mentioned substituted alkylenes, such as alkylenes substituted with one or more alkyl(methyl) groups, are not particularly limited, but include substituted methylene (-CH(CH3)- and -C(CH3)2-), substituted ethylene (-CH(CH3)CH2-, -CH2CH(CH3)-, -C(CH3)2CH2-, and -CH2C(CH3)2-), substituted propylene (-CH(CH3)CH2CH2-, -CH2CH(CH3)CH2-, -CH2CH2CH(CH3)-, -C(CH3)2CH2CH2-, -CH2C(CH3)2CH2-, -CH2CH2C(CH3)2-), etc.

[0081] In this specification, “to treat” and “treatment,” etc., refer to administering a drug or performing a procedure to obtain an effect. The effect may be preventive in that it completely or partially prevents a disease or its symptoms, and / or therapeutic in that it affects the partial or complete cure of the disease and / or its symptoms. In this specification, “to treat” may include treating a disease or disorder (such as cancer) in a mammal, in particular a human, and may include (a) preventing the onset of the disease or its symptoms (e.g., diseases that may be related to or cause the underlying disease) in a subject susceptible to the disease but not diagnosed with the disease; (b) suppressing the disease, i.e., preventing its progression; and (c) alleviating the disease, i.e., causing it to regress. Treatment may refer to any reference to the success of treating, improving or preventing cancer, and may include objective or subjective parameters such as disappearance; remission; reduction of symptoms or a disease state that is tolerable to the patient; delay of exacerbation or decline; or reduction of weakness in the exacerbation endpoint. Treatment or improvement of symptoms is based on one or more objective or subjective parameters, including the results of a medical examination. Accordingly, “treatment” includes administering the antibodies, compositions, or conjugates disclosed herein to prevent, delay, alleviate, halt, or suppress the onset of symptoms or conditions associated with a disease (such as cancer). “Therapeutic effect” means reducing, eliminating, or preventing a disease, its symptoms, or its side effects in a subject.

[0082] In this specification, “effective dose” means an amount sufficient to achieve treatment of a disease when administered to a subject for the treatment of that disease.

[0083] In this specification, “subject” means a mammal subject for which diagnosis, cure, or treatment is desired. “Mammal” for therapeutic purposes means any animal classified as a mammal, including humans, livestock, laboratory animals, zoo animals, sport animals, or pet animals (such as dogs, horses, cattle, cattle, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, and monkeys).

[0084] I.dsRNA The present invention provides a double-stranded RNA (dsRNA) for suppressing the expression of cell death-inducing DFFA-like effector B (CIDEB) in cells, comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand and the antisense strand are each independently 15 to 30 nucleotides long, and the antisense strand contains the nucleotide sequence of at least 15 adjacent nucleotides from the nucleotide sequence described in any one of Sequence IDs 62 to 122.

[0085] In some embodiments, the double-stranded region formed by the sense strand and the antisense strand is completely complementary. In other embodiments, the double-stranded region formed by the sense strand and the antisense strand is substantially complementary and may contain one, two, three, four, or five non-complementary sites.

[0086] In some specific embodiments, the sense strand contains a nucleotide sequence of at least 15 adjacent nucleotides from the nucleotide sequences described in any one of Sequence IDs 1 to 61.

[0087] In some embodiments, the sense strand and the antisense strand are each independently 15 to 27 nucleotides long, preferably 18 to 25 nucleotides long, and more preferably 19 to 21 nucleotides long.

[0088] In some embodiments, the double-stranded region is 15 to 25 base pairs long, preferably 16 to 23 base pairs, and more preferably 18 to 20 base pairs long.

[0089] In some embodiments, the dsRNA of the present invention is siRNA. In some other embodiments, a hairpin loop is formed between the sense strand and the antisense strand of the dsRNA of the present invention.

[0090] One or both of the sense strand and the antisense strand include the 3' overhang and / or 5' overhang of at least one nucleotide. For example, one or both of the sense strand and the antisense strand include the 3' overhang and / or 5' overhang of at least one nucleotide. In some preferred embodiments, the antisense strand includes the 3' overhang and / or 5' overhang of at least two nucleotides, preferably the antisense strand includes the 3' overhang and / or 5' overhang of two nucleotides. In some embodiments, the sense strand and the antisense strand are of the same length. In some embodiments, the total length of the sense strand is complementary to the total length of the antisense strand, forming a double helix, i.e., having blunt ends. In some other embodiments, the sense strand and the antisense strand are of the same length, and a portion of the sense strand is complementary to a portion of the antisense strand, i.e., both the sense strand and the antisense strand have 5' overhangs. In some embodiments, the sense strand and the antisense strand are of different lengths. In a preferred embodiment, the 5' end of the antisense strand has an overhang of at least one nucleotide, more preferably two or three nucleotides.

[0091] The dsRNAs of this disclosure include dsRNAs having a nucleotide overhang at one end (i.e., a substance having one overhang and one blunt end), or dsRNAs having nucleotide overhangs at both ends. For example, the 5' end of the sense strand of a dsRNA includes an overhang having one or more nucleotides, and the 3' end of the sense strand includes an overhang having one or more nucleotides. For example, the 5' end of the antisense strand of a dsRNA includes an overhang having one or more nucleotides, and the 3' end of the antisense strand includes an overhang having one or more nucleotides. For example, the 5' end of the sense strand of a dsRNA includes an overhang having one or more nucleotides, and the 5' end of the antisense strand includes an overhang having one or more nucleotides. For example, the 3' end of the sense strand of a dsRNA includes an overhang having one or more nucleotides, and the 3' end of the antisense strand includes an overhang having one or more nucleotides. For example, the 5' end of the sense strand of dsRNA contains an overhang with one or more nucleotides, and the 3' end of the sense strand contains a blunt end. For example, the 3' end of the sense strand of dsRNA contains an overhang with one or more nucleotides, and the 3' end of the sense strand contains a blunt end. For example, the 5' end of the antisense strand of dsRNA contains an overhang with one or more nucleotides, and the 3' end of the antisense strand contains a blunt end. For example, the 3' end of the antisense strand of dsRNA contains an overhang with one or more nucleotides, and the 5' end of the antisense strand contains a blunt end.

[0092] In some embodiments, the antisense strand comprises a nucleotide sequence of at least 16 adjacent nucleotides, a nucleotide sequence of at least 17 adjacent nucleotides, a nucleotide sequence of at least 18 adjacent nucleotides, a nucleotide sequence of at least 19 adjacent nucleotides, or a nucleotide sequence of at least 20 adjacent nucleotides from any one of the nucleotide sequences described in SEQ ID NOs. 62 to 122, and preferably, the antisense strand comprises a nucleotide sequence described in any one of SEQ ID NOs. 62 to 122.

[0093] In some embodiments, the sense strand comprises a nucleotide sequence of at least 16 adjacent nucleotides from any one of the nucleotide sequences described in SEQ ID NOs: 1 to 61, a nucleotide sequence of at least 17 adjacent nucleotides, or a nucleotide sequence of at least 18 adjacent nucleotides, and preferably the sense strand comprises a nucleotide sequence described in any one of SEQ ID NOs: 1 to 61.

[0094] In some embodiments, the dsRNA includes one of the paired sense strand sequences and antisense strand sequences shown in Table 3.

[0095] II. Modification of nucleotides In some embodiments, substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides. In some embodiments, at least 80%, at least 85%, at least 90%, at least 92%, or at least 95% of the nucleotides of the sense strand are modified nucleotides, and / or at least 80%, at least 85%, at least 90%, at least 92%, or at least 95% of the nucleotides of the antisense strand are modified nucleotides.

[0096] In some embodiments, all nucleotides in the sense strand are modified nucleotides, and / or all nucleotides in the antisense strand are modified nucleotides.

[0097] The nucleotide modifications described in the present invention may be modifications to the phosphate ester group, ribose group, and / or base group of the nucleotide.

[0098] In some specific embodiments, the sense strand and antisense strand are independently SCP-modified nucleotides, 2'-O-alkyl-modified nucleotides (such as 2'-O-methyl-modified nucleotides), 2'-methoxyethyl-modified nucleotides, 2'-fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, inosine ribonucleotides, debasalized nucleotides, reverse debasalized deoxyribonucleotides, phosphorothioate internucleotide bond modifications, vinyl phosphonate-modified nucleotides, locked nucleotides, unlocked nucleotides, 2'-amino-modified nucleotides, 2'-C-alkyl-modified nucleotides, and 2'-O-allyl-modified nucleotides. The nucleotide modification comprises one or more nucleotide modifications selected from the group consisting of nucleotides, morpholinonucleotides, phosphoramidates, nucleotides containing unnatural bases, cholesteryl derivatives or terminal nucleotides bonded to a dodecanoate bisdecylamide group, deoxyribonucleotides, 3'-terminal deoxythymine (dT) nucleotides, stereostructure-restricted nucleotides, restrictive ethyl nucleotides, 2'-hydroxy-modified nucleotides, nucleotides containing a methylphosphonate group, nucleotides containing 5'-phosphate, nucleotides containing a 5'-phosphate mimetic, glycol-modified nucleotides (GNAs), and 2-O-(N-methylacetamide)-modified nucleotides.

[0099] In some preferred embodiments, the sense strand and the antisense strand each independently contain one or more nucleotide modifications selected from the group consisting of 2'-O-methyl-modified nucleotides, 2'-fluoro-modified nucleotides, and phosphorothioate nucleotide interlinking modifications. In some preferred embodiments, the sense strand and / or antisense strand contains at least two 2'-fluoro-modified nucleotides. In some preferred embodiments, the sense strand and / or antisense strand contains at least eight 2'-O-methyl-modified nucleotides. In some preferred embodiments, the 3' and / or 5' ends of the sense strand and / or antisense strand contain 1 to 5 phosphorothioate nucleotide interlinks, preferably 2 to 3 phosphorothioate nucleotide interlinks. In some embodiments, the sense strand and / or antisense strand contains adenine deoxyribonucleotide, thymine deoxyribonucleotide, guanine deoxyribonucleotide, and / or cytosine deoxyribonucleotide. In a more preferred embodiment, the sense strand and / or antisense strand contains thymine deoxyribonucleotide. In the most preferred embodiment, the sense strand comprises a thymine deoxyribonucleotide.

[0100] In some preferred embodiments, the antisense strand comprises one of the modified nucleotide sequences shown in Table 5, and / or the sense strand comprises one of the modified nucleotide sequences shown in Table 4. In some preferred embodiments, the dsRNA comprises one of the paired modified sense strand sequences and modified antisense strand sequences shown in any one of Table 6.

[0101] III. Ligand part The dsRNA of the present invention is further bound to a ligand moiety containing N-acetylgalactosamine. In preferred embodiments, the sense strand of the dsRNA is bound to the ligand moiety. In some embodiments, the 3' end of the sense strand is bound to the ligand moiety. In some other embodiments, the 5' end of the sense strand is bound to the ligand moiety.

[0102] In some embodiments, the ligand moiety includes a binding group of formula (X'). [ka] During the ceremony, [ka] The symbol represents the point that connects to the dsRNA; Q is independent of H, [ka] and; L1 represents a bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O-, or -NHC(O)-(CH2NHC(O)). a -and; L2 is a bond or -CH2CH2C(O)-; L3 is a bond, -(NHCH2CH2) b -,-(NHCH2CH2CH2) b -, or -C(O)CH2-; L4 is -(OCH2CH2) c -,-(OCH2CH2CH2) c -,-(OCH2CH2CH2CH2) c -,-(OCH2CH2CH2CH2CH2) c -, or -NHC(O)-(CH2) d -and; a = 0, 1, 2, or 3; b = 1, 2, 3, 4, or 5; c = 1, 2, 3, 4, or 5; d = 1, 2, 3, 4, 5, 6, 7, or 8; L is a bond, -CH2O-, or -NHC(O)-; L' represents a bond, -C(O)NH-, -NHC(O)-, or -O(CH2CH2O) e -and; e is 1, 2, 3, 4, or 5; T is a bond, -CH2-, -C(O)-, -M-, -CH2-M-, or -C(O)-M-; M is [ka] and; R1 and R2 both form -CH2CH2O- or -CH2CH(R)-O- and R3 is H; or R1 and R3 both form -C 1-2 It forms an alkylene group, and R2 is H; R is -OR', -CH2OR', or -CH2CH2OR'; R' is H, a hydroxy protecting group, or a solid support, where the hydroxy protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl; m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0103] In some embodiments, the bonding group is as shown in formula (I'). [ka] During the ceremony, [ka] The symbol represents the point that connects to the dsRNA; Q is independent of H, [ka] and; L1 represents a bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O-, or -NHC(O)-(CH2NHC(O)). a -and; L2 is a bond or -CH2CH2C(O)-; L3 is a bond, -(NHCH2CH2) b -,-(NHCH2CH2CH2) b-, or -C(O)CH2-; L4 is -(OCH2CH2) c -,-(OCH2CH2CH2) c -,-(OCH2CH2CH2CH2) c -,-(OCH2CH2CH2CH2CH2) c -, or -NHC(O)-(CH2) d -and; a = 0, 1, 2, or 3; b = 1, 2, 3, 4, or 5; c = 1, 2, 3, 4, or 5; d = 1, 2, 3, 4, 5, 6, 7, or 8; L is either -CH2O- or -NHC(O)-; L' is a bond, -C(O)NH-, or -NHC(O)-; R1 and R2 both form -CH2CH2O- or -CH2CH(R)-O- and R3 is H; or R1 and R3 both form -C 1-2 It forms an alkylene group, and R2 is H; R is -OR', -CH2OR', or -CH2CH2OR'; R' is H, a hydroxy protecting group, or a solid support, where the hydroxy protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl; m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0104] In some specific embodiments, During the ceremony, Q is independent of H or [ka] and; L1 is either -CH2O- or -NHC(O)- (CH2NHC(O)). a -and; L2 is -CH2CH2C(O)-; L3 is -(NHCH2CH2)b -or-(NHCH2CH2CH2) b -and; L4 is -(OCH2CH2) c - or -NHC(O)-(CH2) d -and; a = 0, 1, 2, or 3; b = 1, 2, 3, 4, or 5; c = 1, 2, 3, 4, or 5; d = 1, 2, 3, 4, 5, 6, 7, or 8; L is -CH2O-; L' is a combination; R1 and R2 both form -CH2CH2O- or -CH2CH(R)-O- and R3 is H; or R1 and R3 both form -C 1-2 It forms an alkylene group, and R2 is H; R is -OR', -CH2OR', or -CH2CH2OR'; R' is H, a hydroxy protecting group, or a solid support, where the hydroxy protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl; m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0105] In some embodiments, the bonding group is as shown in formula (I'-1), formula (I'-2), or formula (I'-3). [ka] During the ceremony, [ka] The symbol represents the point that connects to the dsRNA; Q is [ka] and; L1 is -CH2O- or -NHC(O)-; L2 is -CH2CH2C(O)-; L3 is -(NHCH2CH2) b -or-(NHCH2CH2CH2) b -and; L4 is -(OCH2CH2) c - or -NHC(O)-(CH2) d -and; b = 1, 2, 3, 4, or 5; c = 1, 2, 3, 4, or 5; d = 1, 2, 3, 4, 5, 6, 7, or 8; L is -CH2O-; R' is H, a hydroxy protecting group, or a solid support, and the hydroxy protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl; n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0106] In some specific embodiments, in the formula, Q is independent of H, [ka] and; L1 is -CH2O-, -CH2O-CH2CH2O-, or -NHC(O)-(CH2NHC(O)) a -and; L2 is -CH2CH2C(O)-; L3 is -(NHCH2CH2) b -,-(NHCH2CH2CH2) b -, or -C(O)CH2-; L4 is -(OCH2CH2) c - or -NHC(O)-(CH2) d -and; a = 0, 1, 2, or 3; b = 1, 2, 3, 4, or 5; c = 1, 2, 3, 4, or 5; d = 1, 2, 3, 4, 5, 6, 7, or 8; L is either -CH2O- or -NHC(O)-; L' is a bond or -C(O)NH-; R1 and R2 both form -CH2CH2O- or -CH2CH(R)-O- and R3 is H; or R1 and R3 both form -C 1-2 It forms an alkylene group, and R2 is H; R is -OR', -CH2OR', or -CH2CH2OR'; R' is H, a hydroxy protecting group, or a solid support, where the hydroxy protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl; m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0107] In some embodiments, the bonding group is as shown in formula (II'-1) or formula (II'-2). [ka] During the ceremony, [ka] The symbol represents the point that connects to the dsRNA; Q is independent, [ka] and; L1 is -CH2O- or -CH2O-CH2CH2O-; L3 is -(NHCH2CH2) b -,-(NHCH2CH2CH2) b -, or -C(O)CH2-; L4 is -(OCH2CH2) c - or -NHC(O)-(CH2) d -and; b = 1, 2, 3, 4, or 5; c = 1, 2, 3, 4, or 5; d = 1, 2, 3, 4, 5, 6, 7, or 8; L is -NHC(O)-; L' is a bond or -C(O)NH-; R' is H, a hydroxy protecting group, or a solid support, and the hydroxy protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl; m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0108] In some specific embodiments, in the formula, Q is independent of H, [ka] and; L1 is -CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O-, or -NHC(O)-(CH2NHC(O)) a -and; L2 is a connection; L3 is -(NHCH2CH2) b -,-(NHCH2CH2CH2) b -, or -C(O)CH2-; L4 is -(OCH2CH2) c - or -NHC(O)-(CH2) d -and; a = 0, 1, 2, or 3; b = 1, 2, 3, 4, or 5; c = 1, 2, 3, 4, or 5; d = 1, 2, 3, 4, 5, 6, 7, or 8; L is either -CH2O- or -NHC(O)-; L' is a bond or -C(O)NH-; R1 and R2 both form -CH2CH2O- or -CH2CH(R)-O- and R3 is H; or R1 and R3 both form -C 1-2 It forms an alkylene group, and R2 is H; R is -OR’, -CH2OR’, or -CH2CH2OR’; R’ is H, a hydroxy protecting group, or a solid support, and the hydroxy protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4’-dimethoxytrityl; m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0109] In some embodiments, the linking group is as represented by formula (II’-2).

Chemical formula

Chemical formula

Chemical formula

[0110] In some specific embodiments, in the formula, Q is independently H,

Chemical formula

[0111] In some specific embodiments, in the formula, T is -M-, -CH2-M-, or -C(O)-M-, and M is

Chemical formula

[0112] In some specific embodiments, in the formula, Q is independently H or

Chemical formula

[0113] In some embodiments, the bonding group is as shown in formula (III'-1), formula (III'-2), or formula (III'-3). [ka] During the ceremony, Q is [ka] and; L1 is -CH2O- or -NHC(O)-; L2 is -CH2CH2C(O)-; L3 is -(NHCH2CH2) b -or-(NHCH2CH2CH2) b -and; L4 is -(OCH2CH2) c - or -NHC(O)-(CH2) d -and; b = 1, 2, 3, 4, or 5; c = 1, 2, 3, 4, or 5; d = 1, 2, 3, 4, 5, 6, 7, or 8; L is a bond or -CH2O-; R’ is H, a hydroxy protecting group, or a solid support, and the hydroxy protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl; n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; T is as defined in the above embodiments.

[0114] In some specific embodiments, in the formula, Q is independently H,

Chemical formula

[0115] In some embodiments, the bonding group is as shown in formula (IV-1) or formula (IV-2). [ka] During the ceremony, Q is independent, [ka] and; L1 is -CH2-, -CH2O-, or -C(O)-; L3 is -(NHCH2CH2) b -,-(NHCH2CH2CH2) b -, or -C(O)CH2-; L4 is -(OCH2CH2) c - or -NHC(O)-(CH2) d -and; b = 1, 2, 3, 4, or 5; c = 1, 2, 3, 4, or 5; d = 1, 2, 3, 4, 5, 6, 7, or 8; L is a bond or -NHC(O)-; L' is a combination; R' is H, a hydroxy protecting group, or a solid support, and the hydroxy protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl; m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; T is as defined in the above embodiment.

[0116] In some specific embodiments, in the formula, Q is independent of H, [ka] and; L1 is a bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O-, or -NHC(O)-(CH2NHC(O)) a -; L2 is a bond or -CH2CH2C(O)-; L3 is a bond, -(NHCH2CH2) b -, -(NHCH2CH2CH2) b -, or -C(O)CH2-; L4 is -(OCH2CH2) c -, -(OCH2CH2CH2) c -, -(OCH2CH2CH2CH2) c -, -(OCH2CH2CH2CH2CH2) c -, or -NHC(O)-(CH2) d -; a = 0, 1, 2, or 3; b = 1, 2, 3, 4, or 5; c = 1, 2, 3, 4, or 5; d = 1, 2, 3, 4, 5, 6, 7, or 8; L is a bond, -CH2O-, or -NHC(O)-; L’ is -O(CH2CH2O) e -; e = 1, 2, 3, 4, or 5; T is a bond, -CH2-, -C(O)-, -M-, -CH2-M-, or -C(O)-M-; M is

Chemical formula

[0117] In some preferred embodiments, the bonding group is selected from Tables 1 and 2.

[0118] In some embodiments, the ligand targets the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand targets the asialoglycoprotein receptor (ASGPR) on hepatocytes.

[0119] In some embodiments, the ligand has the following structure. [ka] During the ceremony, [ka] This indicates a point that connects to the sense strand of dsRNA (preferably the 3' end of the sense strand) via a phosphate ester group or a phosphorothioate group.

[0120] In some embodiments, the ligand has the following structure. [ka] During the ceremony, [ka] This indicates a point that connects to the sense strand of dsRNA (preferably the 3' end of the sense strand) via a phosphate ester group or a phosphorothioate group.

[0121] In some embodiments, the ligand has the following structure. [ka] During the ceremony, [ka] This indicates a point that connects to the sense strand of dsRNA (preferably the 3' end of the sense strand) via a phosphate ester group or a phosphorothioate group.

[0122] In some embodiments, the ligand has the following structure. [ka] During the ceremony, [ka] This indicates a point that connects to the sense strand of dsRNA (preferably the 3' end of the sense strand) via a phosphate ester group or a phosphorothioate group.

[0123] IV. Repression of APOC3 gene expression The dsRNA of the present invention can suppress CIDEB gene expression by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0124] Suppression of CIDEB gene expression may be indicated by a decrease in mRNA levels expressed in a first cell or cell population (such cells may, for example, be present in a sample derived from a subject). In these cells, the CIDEB gene is transcribed, and these cells are treated (for example, by contacting them with the dsRNA of the present invention, or by administering the dsRNA of the present invention to a subject in which the cells are present or previously present, thereby suppressing CIDEB gene expression compared to a second cell or cell population (one or more control cells) that is substantially the same as the first cell or cell population but untreated.

[0125] In a preferred embodiment, suppression is evaluated by expressing the mRNA level in treated cells as a percentage of the mRNA level in control cells using the following formula. In some specific embodiments, 2 -△△Ct The value is calculated and the difference between the experimental group and the control group is compared. Here, △△Ct=[(Ct 標的遺伝子,実験群 -Ct 参照遺伝子,実験群 )-(Ct 標的遺伝子,コントロール群 -Ct 参照遺伝子,コントロール群 )]

[0126] Alternatively, the suppression of CIDEB gene expression, such as CIDEB protein expression, may be evaluated in terms of a decrease in parameters functionally related to CIDEB gene expression, such as lipid levels and cholesterol levels (LDLc levels, etc.). CIDEB gene silencing may be measured by any assay known in the art in any cell that constitutively or by genomically engineered CIDEB. The liver and kidneys are primary sites of CIDEB expression, and other important expression sites include the small intestine, white adipose tissue, and colon.

[0127] Suppression of CIDEB protein expression can be indicated by a decrease in the level of CIDEB protein expressed in cells or cell populations (e.g., the level of protein expressed in a sample derived from the target). As explained above regarding the evaluation of mRNA suppression, the suppression of protein expression levels in treated cells or cell populations may also be expressed as a percentage of the protein level in control cells or cell populations.

[0128] Examples of control cells or cell populations that can be used to evaluate the suppression of CIDEB gene expression include cells or cell populations that have not been exposed to the dsRNA of the present invention. For example, the control cells or cell population may be derived from individual subjects (human or animal subjects, etc.) before they are treated with the dsRNA.

[0129] V. Cell This invention provides cells containing the dsRNA of the present invention.

[0130] VI. Pharmaceutical Compositions The present invention provides pharmaceutical compositions comprising dsRNA or cells as disclosed herein and, optionally, pharmaceutically acceptable carriers or excipients.

[0131] In this specification, “pharmaceutically acceptable” means a compound, material, composition, and / or dosage form that is suitable for contact with human and animal tissues, within the bounds of appropriate medical judgment, without causing excessive toxicity, irritation, allergic reactions, or other problems or complications, and that provides a reasonable benefit-risk ratio.

[0132] In this specification, a pharmaceutically acceptable carrier refers to a pharmaceutical carrier that facilitates the administration of dsRNA or cells containing dsRNA to the human body and / or promotes their absorption or action. Examples include diluents, excipients (such as water), and fillers (such as starch and sucrose); binders (such as cellulose derivatives, alginates, gelatin, and polyvinylpyrrolidone); humectants (such as glycerin); disintegrants (such as agar, calcium carbonate, and sodium bicarbonate); absorption enhancers (such as quaternary ammonium compounds); surfactants (such as cetyl alcohol); adsorbents (such as kaolin and bentonite); and lubricants (such as talc, calcium / magnesium stearate, and polyethylene glycol). Other adjuvants such as fragrances and sweeteners may also be added to the composition.

[0133] The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable diluent or a sustained-release substrate, and the dsRNA or vector of the present invention is incorporated into the sustained-release substrate.

[0134] The pharmaceutical compositions of this disclosure may include a drug delivery system for delivering dsRNA. The drug delivery systems of this disclosure are not limited to but include nanoparticles (lipid nanoparticles, polymer nanoparticles, etc.), polymers, PEG, or cation delivery systems, polylactic acid (PLA) microspheres, poly(lactic acid-co-glycolic acid) (PLGA) microspheres, liposomes, micelles, reverse micelles, lipid helices (cochleates), or lipid microtubules, cholesterol, PEG lipids such as PEG-2000-C-DMG and PEG-2000-DMG(Moderna), ALC-0159, and DSPC.

[0135] In some embodiments, the siRNA in the pharmaceutical composition of the present invention may be contained within a polymer or polymer-based nanoparticles.

[0136] In some specific embodiments, the polymer is a polymer based on poly(lactic acid-coglycolic acid) (abbreviated as PLGA). In some specific embodiments, the PLGA-based polymer is modified to include independent cationic groups.

[0137] In some specific embodiments, the polymer contains amine groups that can become cationic, such as polyethyleneimine (PEI) and poly(L-lysine) (PLL), and can form complexes with siRNA through electrostatic interactions to deliver siRNA into cells. In some embodiments, chemical modification of PEG and PLL improves in vivo efficacy and tolerability.

[0138] In some embodiments, the siRNA in the pharmaceutical composition of the present disclosure can be delivered via a cationic polymer, poly(β-aminoester) (PBAE).

[0139] VII. Kit The present invention provides a kit comprising the dsRNA or cells of the present invention.

[0140] The present invention also provides a kit for using the dsRNA of the present invention and / or for carrying out the method of the present invention. The kit comprises one or more of the dsRNA of the present invention or cells, and may further include instructions for use. The instructions for use may contain instructions for suppressing CIDEB expression in cells by contacting the cells with an amount of the dsRNA of the present invention effective in suppressing CIDEB expression.

[0141] When the dsRNA of the present invention is brought into contact with cells in vitro, the kit of the present invention may further include, as necessary, means for bringing cells into contact with the dsRNA of the present invention (such as an injection device), or means for measuring CIDEB inhibition (such as a device for measuring the inhibition of CIDEB mRNA or protein). Such a device for measuring CIDEB inhibition may include a device for obtaining a sample (such as a plasma sample) from the subject.

[0142] When administering the dsRNA of the present invention, or cells into which the dsRNA has been introduced in vitro, into the body, the kit of the present invention may further include, as necessary, a device for administering the dsRNA or cells of the present invention, or a device for determining a therapeutic or prophylactic dose.

[0143] VIII. Treatment Methods and Pharmaceutical Uses This disclosure provides a method for reducing cell death-inducing DFFA-like effector B (CIDEB) in a subject, comprising the step of administering the subject with the dsRNA, cells, or pharmaceutical composition of this disclosure.

[0144] This disclosure provides a method for treating, preventing, suppressing, or mitigating a disease or disorder in which a reduction in the expression of cell death-inducible DFFA-like effector B (CIDEB) is beneficial, the method comprising the step of administering the dsRNA, cells, or pharmaceutical composition of this disclosure to the subject. This disclosure also provides a method for treating, preventing, suppressing, or mitigating at least one symptom in a patient having a disease or disorder in which a reduction in the expression of cell death-inducible DFFA-like effector B (CIDEB) is beneficial.

[0145] In some embodiments, CIDEB-related diseases are those in which a reduction in the expression of cell death-inducing DFFA-like effector B (CIDEB) is beneficial.

[0146] In some embodiments, the present invention's methods for reducing cell death-inducing DFFA-like effector B (CIDEB) in a subject, the methods for treating, preventing, suppressing, or alleviating a disease or disorder in which a reduction in the expression of cell death-inducing DFFA-like effector B (CIDEB) is beneficial in a subject, or the methods for treating, preventing, suppressing, or alleviating at least one symptom in a patient having a disease or disorder in which a reduction in the expression of cell death-inducing DFFA-like effector B (CIDEB) is beneficial, include administering dsRNA, cells, or a pharmaceutical composition to a subject subcutaneously, topically, or intravenously. In some embodiments, the subject is a human patient.

[0147] The present invention also relates to dsRNA, cells, or pharmaceutical compositions used to treat diseases or conditions associated with CIDEB expression in a subject.

[0148] The present invention also relates to the use of the dsRNA, cells, or pharmaceutical compositions of the present invention in the manufacture of pharmaceuticals for treating, preventing, or alleviating diseases or disorders in which a reduction in the expression of cell death-inducible DFFA-like effector B (CIDEB) is beneficial in a subject. The disclosure also relates to the use of the dsRNA, cells, or pharmaceutical compositions of the present disclosure in the manufacture of pharmaceuticals for reducing cell death-inducible DFFA-like effector B (CIDEB) in a subject. The disclosure also relates to the use of the dsRNA, cells, or pharmaceutical compositions of the present disclosure in the manufacture of pharmaceuticals for treating, preventing, suppressing, or alleviating at least one symptom in patients with diseases or disorders in which a reduction in the expression of cell death-inducible DFFA-like effector B (CIDEB) is beneficial.

[0149] array The RNA sequence provided by this invention targets the human CIDEB gene (or target gene, target mRNA sequence, or target sequence). The target CIDEB mRNA sequence is the gene represented by Genbank accession number NM_001393338.1. [Table 3-1] [Table 3-2] [Table 3-3]

[0150] Tables 4 and 5 below represent the modified RNA sequences used in this invention, respectively.

[0151] The meanings of the abbreviations used in this specification are as follows:

[0152] A, U, G, and C represent natural adenine ribonucleotide, uracil ribonucleotide, guanine ribonucleotide, and cytosine ribonucleotide, respectively.

[0153] The letter 'd' indicates that the nucleotide immediately to its right is a deoxyribonucleotide (i.e., 2'-deoxy modified). For example, dA, dT, dG, and dC represent adenine deoxyribonucleotide, thymine deoxyribonucleotide, guanine deoxyribonucleotide, and cytosine deoxyribonucleotide, respectively.

[0154] The letter 'm' indicates that the nucleotide immediately to its left is a 2'-OCH3 modified nucleotide. For example, Am, Um, Gm, and Cm represent 2'-OCH3 modified A, U, G, and C, respectively.

[0155] The letter 'f' indicates that the nucleotide on the left is a 2'-fluoro-modified nucleotide. For example, Af, Uf, Gf, and Cf represent 2'-fluoro-modified A, U, G, and C, respectively.

[0156] The letter "s" indicates that two adjacent nucleotides are linked by a phosphorothioate.

[0157] The "s-" indicates that the nucleotide immediately to its left and the delivery portion immediately to its right are linked by a phosphorothioate bond.

[0158] VP indicates that the nucleotide on the right is a vinyl phosphonate-modified nucleotide, a well-known modification in this field. See, for example, PCT publication numbers WO2011139702, WO2013033230, and WO2019105419.

[0159] "SCP-modified nucleotides" have the following structure: [ka] (wherein Base is independently selected from H, a modified or unmodified base, or a leaving group) a modified nucleotide. Preferably, Base is an unmodified base, such as an adenine base, a guanine base, a uracil base, and a cytosine base. In other embodiments, Base is a modified base.

[0160] L96 represents the GalNAc delivery portion of the following structure, which is well known in the field, in the formula, [ka] This indicates a point that connects to dsRNA via a phosphate ester group or a phosphorothioate group. See, for example, PCT publication numbers WO2009073809 and WO2009082607. [ka]

[0161] GL6 represents the GalNAc delivery portion of the following structure, in the formula, [ka] This indicates a point that connects to dsRNA via a phosphate ester group or a phosphorothioate group. [ka]

[0162] [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5]

[0163] [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5]

[0164] [Table 6-1] [Table 6-2] [Table 6-3]

[0165] The contents of this disclosure will be further explained below with reference to examples. Please understand that the following examples are illustrative and should not be construed as limiting the scope of the present invention. [Examples]

[0166] Unless otherwise specified, the sources of the materials used in the examples are as follows: Huh7 cell line: Nanjing Cobioer Biotechnology, Cat#CBP60202; PHH cell line: Shanghai Xuanyi Biotechnology, Cat#QYLF-HPMC; PCH cell line: Milestone Biotechnologies, Cat#cmTCSC; Hep3B cell line: Nanjing Cobioer Biotechnology, Cat#CBP60197

[0167] Example 1: Preparation of Compound E7 1. Preparation of intermediates 3-4 1.1 Preparation of Compound 2 [ka] At 15°C, benzyl(2,5-dioxopyrrolidine-1-yl) carbonate (600g, 2.40mol) was slowly added to a solution of compound 1 (300g, 2.01mol) in DCM (1.80L), and TEA (203g, 2.01mol, 280mL) was added dropwise. The mixture was then stirred at 25°C for 16 hours. TLC (dichloromethane:methanol = 10:1) confirmed that reaction product 1 was retained (R f =0.32) and prominent spots (R f The detection of compound 2 (=0.52) was confirmed. The reaction mixture was washed with saturated sodium bicarbonate solution (1.00 L x 2). The organic phase was washed with brine (1.00 L), dried over anhydrous Na2SO4, and concentrated under vacuum. Compound 2 (approximately 385 g) was obtained as a yellow oily substance without further purification.

[0168] 1.2 Preparation of Compound 2A [ka] At 0-15°C, DMAP (19.8g, 162 mmol) was added all at once to a pyridine (1.75L) solution of compound 4 (350g, 1.62mol, HCl) and Ac2O (994g, 9.74mol, 912mL), and TEA (164g, 1.62mol, 226mL) was added dropwise. The mixture was stirred at 25°C for 16 hours. LC-MS (product: RT=0.687 min) confirmed that the starting reaction products had been completely consumed. At 25°C, HCl (1.40L) was added to the mixture and stirred for 30 minutes. The resulting mixture was then filtered, and the filtered cake was washed with HCl (300mL). The filtered cake was pulverized with water (1.45L) at 25°C for 30 minutes. The mixture was filtered, and the filtered cake was washed with water (175mL x 3). The filtered cake was recovered, and compound 2A (approximately 580g) was obtained as a white solid.

[0169] 1.3 Preparation of Compound 2B [ka] Three reactions were carried out in parallel.

[0170] At 10-15°C, TMSOTf (137g, 616mmol, 111mL) was added dropwise over 0.5 hours to a solution of compound 2A (200g, 514mmol) in DCM (800mL). The mixture was then stirred at 25°C for 3 hours. Compound 2A (R) was analyzed by TLC (dichloromethane:methanol = 20:1). f =0.54) has been completely consumed and a new spot (R f It was confirmed that compound 2B (=0.24) was formed. The three reactions were combined. The mixture was cooled to 0-15°C and slowly poured into NaHCO3 (300g dissolved in 3.00L of water) at 0-5°C. The organic phase was separated and the aqueous phase was extracted with DCM (1.00L x 3). The combined organic phase was dried with Na2SO4, filtered, and vacuum concentrated. Compound 2B (approximately 507g) for use in the next step was obtained as a yellow oil without further purification.

[0171] 1.4 Preparation of Compound 3 [ka] At 0-10°C, TMSOTf (84.4g, 380mmol, 69.0mL) was added dropwise to a mixture of compound 2B (250g, 759mmol) and compound 2 (151g, 531mmol) in DCM (1.00L). The mixture was stirred at 20°C for 12 hours. Compound 2(R) was analyzed by TLC (dichloromethane:methanol = 20:1). f =0.33) has been completely consumed and a new spot (R f The formation of compound 3 (=0.03) was confirmed. After cooling the combined reaction mixture to 0-5°C, it was poured into NaHCO3 (100g in 1L of water) and stirred at 5-10°C for 10 minutes to separate the phases. The aqueous phase was extracted with DCM (500mL x 2). The combined organic phase was dried with Na2SO4, filtered, and vacuum concentrated. Compound 3 (approximately 360g) was obtained as a yellow oil without further purification. 1 1H NMR: (400 MHz, DMSO). δ=7.79-7.37 (m, 1H), 7.35-7.26 (m, 5H), 5.21-5.20 (m, 1H), 5.00-4.95 (m, 3H), 4.55-4.53 (m, 1H), 4.03-3.86 (m, 3H), 3.61-3.59 (m, 1H), 3.59-3.57 (m, 1H), 3.48-3.40 (m, 6H), 3.39-3.31 (m, 2H), 3.14-3.13 (m, 2H), 2.09 (s, 3H), 1.99 (s, 3H), 1.88 (s, 3H), 1.76-1.74 (m, 3H).

[0172] 1.5 Preparation of intermediates 3-4 (TFA salts) [ka] Three reactions were carried out in parallel.

[0173] Compound 3 (180 g, 293 mmol, 21.8 mL) and TFA (33.5 g, 293 mmol, 21.8 mL) were added to a Pd / C mixture (18.0 g, 16.3 mmol, 10% content) in THF (1.80 L) under an argon atmosphere. The suspension was degassed and purged three times with hydrogen. The mixture was stirred under H2 (50 Psi) at 30°C for 2 hours. LC-MS (product: RT=0.697 min) confirmed that compound 3 had been consumed and a product peak was detected. The three reactions were combined. The mixture was filtered through Celite, and the filtrate was concentrated under reduced pressure to remove the solvent. Without further purification, intermediate 3-4 (TFA salt) (393 g, 660 mmol, yield 74.8%, purity 99.6%, TFA) was obtained as a yellow solid. 1 1H NMR: (400 MHz, DMSO-d6) δ = 7.92 (d, J = 9.1 Hz, 4H), 5.27-5.17 (m, 1H), 5.03-4.91 (m, 1H), 4.60-4.50 (m, 1H), 4.09-3.97 (m, 4H), 3.85 (s, 2H), 3.65-3.46 (m, 10H), 3.04-2.92 (m, 2H), 2.10 (s, 3H), 2.00 (s, 3H), 1.94-1.86 (m, 3H), 1.82-1.71 (m, 4H).

[0174] 2. Preparation of intermediate 3-3 2.1 Preparation of Compound 5 [ka] At 25°C, DIEA (30.3g, 234mmol, 40.8mL, 6.60eq) was added all at once to a 1.00L DCM (1.00L) solution of compound 4B (10.0g, 35.5mmol, 1.00eq) and compound 3-4 (46.3g, 78.2mmol, 2.20eq, TFA) prepared above. The mixture was stirred at 25°C for 30 minutes. HBTU (30.3g, 234mmol, 40.8mL, 6.60eq) was added to the mixture. The mixture was stirred at 25°C for 16 hours. The completion of the reaction was confirmed by LCMS (product: RT=0.681 min). The mixture was concentrated under vacuum. The mixture was added to 0.50N HCl (200mL x 2) at 20°C, and then extracted with DCM (3 x 500mL). The combined organic phase was washed with saturated NaHCO3 (3 × 800 mL) to pH=8, then washed with brine (3 × 500 mL), dried over Na2SO4, and vacuum concentrated. The residue was purified by column chromatography (SiO2, DCM:MeOH = 50:1-15:1). The residue was vacuum concentrated at 40°C and purified by preparative MPLC (column: 800 g Agela C18; mobile phase: [water-ACN]; 15-45%, 25 min; 45%, 10 min). By vacuum drying, compound 5 (approximately 180 g + 75.0 g + 87.0 g + 40.0 g + 38.0 g) was obtained as a yellow solid.

[0175] 417.0g of compound 3-4 was divided into 9 batches and converted to compound 5.

[0176] 2.2 Preparation of Intermediate 3-3 [ka] Under an argon atmosphere, compound 5 (73.0 g, 61.7 mmol, 1.00 eq) and TFA (7.04 g, 61.7 mmol, 4.57 mL, 1.00 eq) were added to Pd / C (3.00 g, 10% content) in THF (300 mL). The suspension was degassed and purged three times with hydrogen. The mixture was stirred under H2 (20 Psi) at 20°C for 16 hours. TLC (dichloromethane:methanol = 8:1, R) fThe reaction was confirmed to be complete by the test (=0.0). The mixture was filtered through Celite, and the filtrate was concentrated under pressure to remove the solvent, yielding compound 3-3 (approximately 33.4g + 129g + 75.0g) as a white solid. 1 1H NMR: (400 MHz, DMSO) δ=8.53 (t, J = 5.2 Hz, 1H), 8.18 (d, J = 2.4 Hz, 3H), 8.03 (t, J = 5.2 Hz, 1H), 7.84 (dd, J = 3.6 Hz, 2H), 5.22 (d, J = 3.2 Hz, 2H), 4.96 (dd, J = 3.2 Hz, 2H), 4.55 (d, J =8.4 Hz, 2H), 4.02 (t, J =8.8 Hz, 6H), 3.77-3.59 (m, 5H), 3.58-3.45 (m, 21H), 3.40-3.20 (m, 4H), 2.18 (t, J = 7.6 Hz, 2H), 2.17 (d, J =8.0 Hz, 6H), 2.10 (s, 6H), 1.99 (s, 6H), 1.90-1.80 (m, 8H), 1.77 (s, 6H).

[0177] 3. Preparation of compound E7 3.1 Preparation of Compound 3 [ka] Compound 1 (2.00 g, 1.87 mmol, prepared according to the method of intermediate 3-3 described above) was dissolved in DCM (20.0 mL) at room temperature. DIEA (0.135 mL, 0.814 mmol) and Compound 2 (0.550 g, 0.814 mmol) were sequentially added to this solution, and the mixture was purged with nitrogen three times. The reaction mixture was stirred at 25°C for 16 hours. The MS response of the product was detected by liquid chromatography / tandem mass spectrometry (LC-MS / MS). Thin-layer chromatography (dichloromethane / methanol = 5 / 1) confirmed the disappearance of the starting material and the formation of new spots. The reaction mixture was concentrated under reduced pressure. The resulting crude product was purified by column chromatography (dichloromethane / methanol = 5 / 1) to obtain Compound 3 (approximately 780 mg) as a white solid. 1 1H NMR (400 MHz, CD3OD) δ=7.28-7.42 (m, 5H), 5.30-5.34 (m, 4H), 5.04-5.14 (m, 6H), 4.63-4.67 (m, 4H), 4.36-4.44 (m, 2H), 4.00-4.20 (m, 23H), 3.91-3.95 (m, 4H), 3.69-3.77 (m, 9H), 3.52-3.67 (m, 32H), 3.34-3.43 (m, 9H), 2.29-2.31 (m, 4H), 2.14 (s, 12H), 2.03 (s, 12H), 1.92-1.96 (m, 24H). LCMS: m / z = 1221.6 (M / 2+H) + .

[0178] 3.2 Preparation of Compound 4 [ka] Compound 3 (1.10 g, 0.451 mmol) was dissolved in MeOH (10.0 mL) at room temperature, and 10% mass fraction wet Pd / C (0.050 g, 0.451 mmol) was added to this solution. The reaction mixture was purged with hydrogen three times and then stirred at 25°C for 18 hours under a hydrogen atmosphere (14.696 psi). The MS response of the product was detected by LC-MS / MS. Furthermore, TLC (dichloromethane / methanol = 10 / 1, color development: phosphomolybdic acid) confirmed that the starting material had been completely consumed and that new spots had been formed. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain compound 4 (approximately 840 mg) as a white solid.

[0179] 1 1H NMR (400 MHz, CD3OD) δ=5.32-5.34 (m 4H), 5.06-5.10 (m, 4H), 4.63-4.65 (m, 4H), 4.38-4.40 (m, 2H), 3.99-4.20 (m, 20H), 3.90-3.97 (m, 4H), 3.69-3.76 (m, 6H), 3.50-3.68 (m, 36H), 3.35-3.44 (m, 11H), 2.28-2.38 (m, 4H), 2.15 (s, 12H), 2.03 (s, 12H), 1.90-1.94 (m, 24H). LCMS: m / z = 1154.7 (M / 2+H) + .

[0180] 3.3 Preparation of Compound 6 [ka] Compound 5 (232 mg, 0.364 mmol) was dissolved in DCM (10.0 mL) at room temperature. HBTU (207 mg, 0.546 mmol), DIEA (0.181 mL, 1.09 mmol), and Compound 4 (840 mg, 0.364 mmol) were added sequentially to this solution, and the mixture was purged with nitrogen three times. The reaction mixture was stirred at 25°C for 1 hour. LC-MS / MS detected the disappearance of the starting material. TLC (dichloromethane / methanol = 5 / 1) confirmed the burning of the starting material and the formation of new spots. The reaction mixture was concentrated under reduced pressure. The resulting crude product was purified by column chromatography (dichloromethane / methanol = 8 / 1-5 / 1) to obtain Compound 6 (approximately 620 mg) as a white solid. 1 1H NMR (400 MHz, CD3OD) δ=7.41-7.43 (m, 2H), 7.23-7.34 (m, 7H), 6.83-6.90 (m, 4H), 5.31-5.35 (m, 4H), 5.01-5.12 (m, 4H), 4.63-4.65 (m, 4H), 4.41-4.45 (m, 2H), 4.31-4.33 (m, 1H), 3.99-4.22 (m, 22H), 3.87-3.97 (m, 6H), 3.58-3.81 (m, 45H), 3.34-3.43 (m, 10H), 2.19-2.40 (m, 10H), 2.14 (s, 12H), 2.02 (s, 12H), 1.92-1.96 (mz, 24H), 1.48-1.63 (m, 4H), 1.28-1.38 (m, 8H). LCMS: m / z = 1460.0 (M / 2+H) + .

[0181] 4. Preparation of compound E7 [ka] Compound 6 (300 mg, 0.103 mmol) was dissolved in DCM (10.0 mL) at room temperature. DIEA (0.102 mL, 0.618 mmol), compound 7 (10.3 mg, 0.103 mmol), and DMAP (12.6 mg, 0.103 mmol) were sequentially added to this solution, and the mixture was purged with nitrogen three times. The reaction mixture was stirred at 25°C for 2 hours. Disappearance of the starting materials was detected by LC-MS / MS. The reaction mixture was concentrated under reduced pressure. The resulting crude product was separated by preparative MPLC (prep-HPLC, column: Waters Xbridge BEH C18 100*30 mm*10 μm; mobile phase: water-ACN; B%: 17%-57%, 5 min) to obtain compound E7 (53.0 mg, yield 17.08%, purity 78.94%) as a white solid. 1 1H NMR (400 MHz, CD3OD) δ=7.41-7.45 (m, 2H), 7.17-7.34 (m, 7H), 6.85-6.89 (m, 4H), 5.32-5.36 (m, 4H), 5.03-5.13 (m, 4H), 4.63-4.67 (m, 4H), 4.38-4.47 (m, 2H), 4.32-4.34 (m, 1H), 4.01-4.26 (m, 22H), 3.88-4.00 (m, 6H), 3.77-3.81 (m, 7H), 3.49-3.76 (m, 45H), 3.33-3.47 (m, 10H), 2.56-2.62 (m, 2H), 2.45-2.55 (m, 3H), 2.21-2.38 (m, 7H), 2.14 (s, 12H), 2.05-2.11 (m, 2H), 2.02 (s, 12H), 1.92-1.96 (m, 24H), 1.47-1.68 (m, 4H), 1.28-1.34 (m, 8H) MS: m / z = 3022.36 (M+H) + .

[0182] Example 2 Preparation of compounds E1-1 and E1-2 [ka] 1. Preparation of compound 1b [ka] Compound 1a (200 g, 1.33 mol, 1.00 eq) was suspended in a mixed solution of anhydrous acetone (1.00 L) and anhydrous methanol (1.00 L). Concentrated sulfuric acid (20.0 mL, 0.27 eq) was added dropwise, and the mixture was reacted at 25°C for 24 hours. The reaction mixture was neutralized with saturated sodium bicarbonate and concentrated. The resulting residue was dissolved in ethyl acetate and washed three times with saturated brine (500 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated to obtain compound 1b (242 g, 88.9%). 1 H NMR: 400 MHz CDCl3δ 4.97 (s, 1H), 4.84 (d, J = 6.0 Hz, 1H), 4.59 (d, J = 6.0 Hz, 1H), 4.44-4.43 (m, 1H), 3.72-3.59 (m, 2H), 3.44 (s, 3H), 1.49 (s, 3H), 1.32 (s, 3H).

[0183] 2. Preparation of compound 1c [ka] Compound 1b (310 g, 1.52 mol, 1.00 eq), imidazole (206 g, 3.02 mol, 2.00 eq), and triphenylphosphine (476 g, 1.82 mol, 1.20 eq) were dissolved in toluene (2.1 L), and elemental iodine (446 g, 1.76 mmol, 1.16 eq) was added in several portions at room temperature. The mixture was heated to 70°C and stirred for 1.5 hours. TLC confirmed that the reaction was complete. The reaction mixture was quenched with methanol (60 mL), cooled, and saturated aqueous solution of sodium thiosulfate (2.1 L) was added. The organic phase was separated and recovered, washed twice with saturated brine (1.5 L), dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was pulped with methyl tert-butyl ether and filtered. The filtrate was concentrated, and the resulting crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate) to obtain compound 1c (401 g, 1.27 mol, 91.1%). 1 H NMR: 400 MHz CDCl3δ 4.98 (s, 1H), 4.69 (d, J = 6.0 Hz, 1H), 4.56 (d, J = 6.0 Hz, 1H), 4.39-4.35 (m, 1H), 3.30 (s, 3H), 3.24-3.20 (m, 1H), 3.09 (t, J = 10.0 Hz, 1H), 1.41 (s, 3H), 1.26 (s, 3H).

[0184] 3. Preparation of compound 1d [ka] Compound 1c (203 g, 646 mmol, 1.00 eq) was dissolved in tetrahydrofuran (2.03 L), and potassium tert-butoxide (145 g, 1.29 mol, 1.29 eq) was added in several portions at 0°C. The mixture was stirred at 25°C for 16 hours, then cooled to 5°C and quenched with ice water (1.1 L). The mixture was extracted with methyl tert-butyl ether (2.0 L), washed with saturated brine (2.0 L), dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate) to obtain compound 1d (160 g, 91.3%). 1 H NMR: 400 MHz CDCl3δ 5.11 (s, 1H), 5.02 (d, J = 5.6 Hz, 1H), 4.06 (d, J = 1.6 Hz, 1H), 4.50 (d, J = 5.6 Hz, 1H), 4.39 (d, J = 1.6 Hz, 1H), 3.41 (s, 3H), 3.24-3.20 (m, 1H), 3.09 (t, J = 10.0 Hz, 1H), 1.47 (s, 3H), 1.35 (s, 3H).

[0185] 4. Preparation of Compound 1e [ka] A zinc-copper couple (90.0 g, 1.37 mol, 5.50 eq) was dispersed in diethyl ether (200 mL). Compound 1d (60 g, 241 mmol, 1.00 eq) was added at 25°C, and a solution of trichloroacetyl chloride (61.5 g, 338 mmol, 1.40 eq) in diethyl ether (200 mL) was added dropwise. The mixture was stirred for a further 1 hour. After filtration, the filtrate was rinsed with methyl tert-butyl ether (500 mL), and the filtrate was poured into saturated sodium bicarbonate aqueous solution (2.50 L) and filtered. The filtrate was washed three times with saturated brine (500 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain crude compound 1e (71.8 g). 1H NMR: 400 MHz CDCl3δ 5.12 (d, J = 5.6 Hz, 1H), 5.09 (s, 1H), 4.70 (d, J = 6.0 Hz, 1H), 3.70-3.55 (m, 2H), 3.54 (s, 3H), 1.45 (s, 3H), 1.36 (s, 3H).

[0186] 5. Preparation of compound 1f [ka] Compound 1e (71.8 g, 242 mmol, 1.00 eq) was dissolved in tetrahydrofuran (1.60 L). Glacial acetic acid (69.1 mL, 1.20 mol, 5.00 eq) was added, and zinc powder (142 g, 2.17 mol, 9.00 eq) was added in several portions. The mixture was stirred at 25°C for 18 hours. After the reaction was complete, the mixture was filtered, and the filtrate was concentrated to obtain the crude product. The crude product was dissolved in methyl tert-butyl ether, poured into saturated sodium bicarbonate aqueous solution (2.0 L), and filtered. The filtrate was washed twice with saturated brine (500 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate / dichloromethane) to obtain compound 1f (31 g, 2-step yield 56.3%). 1 H NMR: 400 MHz CDCl3δ 4.97 (s, 1H), 4.70-4.67 (m, 2H), 3.54-3.48 (m, 2H), 3.37-3.31 (m, 4H), 3.16-3.10 (m, 1H), 3.09-3.03 (m, 1H), 1.43 (s, 3H), 1.34 (s, 3H).

[0187] 6. Preparation of 1 g of compound [ka] Compound 1f (50 g, 219 mmol, 1.00 eq) and diethyl phosphite (33.2 g, 240 mmol, 1.20 eq) were dissolved in dichloromethane (100 mL), and 1,8-diazabicycloundeca-7-ene (DBU, 6.67 g, 43.8 mmol, 0.20 eq) was added at 0°C. The mixture was stirred at 25°C for 18 hours. The reaction mixture was washed three times with saturated NH4Cl (100 mL) and once with saturated brine (100 mL), dried over anhydrous sodium sulfate, and concentrated. The resulting crude product was purified by silica gel column chromatography (eluent: petroleum ether / dichloromethane) to obtain compound 1 g (70 g, 87.2%). 1 H NMR: 400 MHz CDCl3δ 5.27 (s, 1H), 5.01 (brs, 1H), 4.78 (s, 1H), 4.65 (d, J = 5.6 Hz, 1H), 4.49 (d, J = 6.0 Hz, 1H), 4.20-4.05 (m, 5H), 3.31 (s, 1H), 3.29-3.05 (m, 1H), 2.75-2.68 (m, 1H), 2.50-2.40 (m, 1H), 2.35-2.25 (m, 1H), 1.36-1.25 (m, 12H).

[0188] 7. Preparation of compound 1h [ka] 1 g of the compound (80.2 g, 219 mmol, 1.00 eq) and DMAP (40.1 g, 328 mmol, 1.50 eq) were dissolved in acetonitrile (562 mL). Methyl oxalyl chloride (40.2 g, 328 mmol, 1.50 eq) was added at 5°C. The mixture was stirred at 25°C for 30 minutes. The reaction mixture was concentrated. The resulting crude product was dissolved in ethyl acetate, washed five times with saturated ammonium chloride (300 mL), once with water (200 mL), and once with brine (200 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 1 h (99 g) of the crude compound. 1H NMR: 400 MHz CDCl3δ 4.88-4.80 (m, 2H), 4.67-4.53 (m, 2H), 4.24-4.12 (m, 4H), 3.89 (s, 3H), 3.51-3.43 (m, 1H), 3.35-3.33 (m, 3H), 3.15-3.07 (m, 1H), 2.92-2.80 (m, 1H), 2.69-2.61 (m, 1H), 1.39-1.29 (m, 12H).

[0189] 8. Preparation of compound 1i [ka] Compound 1h (99 g, 218 mmol, 1.00 eq) was dissolved in anhydrous toluene (1.00 L). Tri-n-butyltin hydride (76.4 g, 262 mmol, 1.20 eq) and azobisisobutyronitrile (AIBN, 1.08 g, 6.56 mmol, 0.03 eq) were added. The mixture was refluxed for 2 hours. The reaction mixture was concentrated. The resulting crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate) to obtain crude compound 1i (146 g). 1 H NMR: 400 MHz CDCl3δ 4.75-4.49 (m, 3H), 4.05-3.95 (m, 4H), 3.26-3.24 (m, 3H), 2.76-2.07 (m, 5H), 1.39-1.29 (m, 12H).

[0190] 9. Preparation of compound 1j [ka] Compound 1i (141 g, 201 mmol, 1.00 eq) was dissolved in methanol (1.41 L). An aqueous HCl solution (704 mL, 1.40 mol, 2 M, 7.00 eq) was added, and the reaction mixture was stirred at 60°C for 1 hour. The reaction mixture was extracted with a methyl tert-butyl ether / petroleum ether mixture. The aqueous phase was collected, adjusted to pH 8 with saturated sodium bicarbonate, and concentrated. Tetrahydrofuran was added to the resulting residue and filtered. The filtrate was concentrated to obtain crude compound 1j (62.4 g, 201 mmol).

[0191] 10. Preparation of compound 1k [ka] Compound 1j (62.4 g, 201 mmol, 1.00 eq) was dissolved in pyridine (300 mL). Acetic anhydride (47.4 mL, 502 mmol, 2.50 eq) was added, and the mixture was stirred at 25°C for 12 hours. The reaction mixture was diluted with saturated sodium bicarbonate (1.00 L), extracted twice with ethyl acetate (600 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate) to obtain compound 1k (74 g, 93.3%). 1 H NMR: 400 MHz CDCl3δ 5.38-5.10 (m, 3H), 4.07-4.03 (m, 4H), 3.36-3.32 (m, 3H), 2.41-2.05 (m, 5H), 2.04-1.98 (m, 9H), 1.27-1.23 (m, 6H).

[0192] 11. Preparation of 1 liter of compound [ka] 1 k (79.3 g, 201 mmol, 1.00 eq) of compound was dissolved in ethyl acetate (476 mL). Acetic anhydride (62.6 mL, 663 mmol, 3.30 eq) and concentrated sulfuric acid (5.38 mL, 100 mmol, 0.50 eq) were added, and the mixture was stirred at 25°C for 3 hours. The reaction mixture was neutralized with saturated sodium bicarbonate aqueous solution and extracted twice with ethyl acetate (1.00 L). The combined organic phase was washed twice with saturated brine (500 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography to obtain 1 L (43 g, 50.6%) of compound. 1 H NMR: 400 MHz CDCl3δ 6.11 (d, J = 2.8 Hz, 1H), 5.44-5.39 (m, 1H), 5.33-5.32 (m, 1H), 4.10-4.05 (m, 5H), 2.80-2.35 (m, 4H), 2.12-2.02 (m, 9H), 1.32-1.23 (m, 6H).

[0193] 12. Preparation of compound 1n [ka] 1 ml of compound (8.55 g, 76.2 mmol, 2.30 eq) and bis(trimethylsilyl)acetamide (BSA, 34.3 g, 169 mmol, 5.10 eq) were suspended in acetonitrile (200 mL) and stirred at 85°C for 1 hour. After cooling, 1 l of compound (14.0 g, 33.1 mmol, 1.00 eq) in acetonitrile (50.0 mL) solution was added, followed by tin tetrachloride (37.1 g, 142 mmol, 4.30 eq). The mixture was stirred at 25°C for 15 minutes, then stirred in an oil bath at 85°C for 45 minutes. After cooling, the reaction mixture was poured into saturated sodium bicarbonate aqueous solution (1.50 L) and extracted three times with dichloromethane (700 mL). The combined organic phase was washed with saturated brine (500 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol) to obtain compound 1n (14.4 g, 84.3%). 1 H NMR: 400 MHz CDCl3δ 9.70 (brs, 0.46H), 9.60 (brs, 0.60H), 7.17 (d, J = 8.0 Hz, 0.60H), 7.12 (d, J = 8.0 Hz, 0.49H), 5.98 (d, J = 6.4 Hz, 0.43H), 5.93 (d, J = 5.2 Hz, 0.55H), 5.78 (dt, J1 = 8.0 Hz, J2 = 2.0 Hz, 1H), 5.50-5.45 (m, 2H), 4.45-4.02 (m, 5H), 2.77-2.42 (m, 6H), 2.18 (s, 1.22H), 2.16 (s, 1.74H), 2.04 (s, 1.31H), 2.02 (s, 1.67H), 1.32-1.28 (m, 6H).

[0194] 13. Preparation of Compound 10 [ka] Compound 1n (8.30 g, 17.4 mmol, 1.00 eq) was dissolved in DMF (40.0 mL). DBU (2.93 g, 19.2 mmol, 1.10 eq) was added, and after cooling, benzyl chloromethyl ether (BOMCl, 3.01 g, 19.2 mmol, 1.10 eq) was added. The mixture was stirred at 0-5°C for 2.5 hours. The reaction mixture was diluted with saturated ammonium chloride (160 mL) and extracted twice with dichloromethane (80.0 mL). The combined organic phase was washed with saturated brine (80 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol) to obtain compound 1o (12.0 g, 92.3%). 1H NMR: 400 MHz CDCl3δ 7.36-7.24 (m, 5H), 7.15-7.09 (m, 1H), 5.96-5.92 (m, 1H), 5.81-5.79 (m, 1H), 5.52-5.42 (m, 4H), 4.67 (s, 2H), 4.15-4.05 (m, 4H), 2.80-2.45 (m, 5H), 2.19-2.17 (m, 3H), 2.05-2.03 (m, 3H), 1.34-1.30 (m, 6H).

[0195] 14. Preparation of compound 1p [ka] Compound 1o (10.4 g, 17.4 mmol, 1.00 eq) was dissolved in ammonia-methanol solution (29.9 mL, 7 M) and stirred at 21°C for 1 hour. The reaction mixture was concentrated to obtain the crude product. Subsequently, this was purified by silica gel column chromatography (eluent: dichloromethane / methanol) to obtain compound 1p (8.00 g, 62.7%). 1 H NMR: 400 MHz CDCl3δ 7.34-7.06 (m, 5H), 5.83 (s, 0.52H), 5.71 (d, J = 8.4 Hz, 0.41H), 5.661 (d, J = 8.4 Hz, 0.53H), 5.60 (d, J = 2.0 Hz, 0.59H), 5.53 (d, J = 5.2 Hz, 0.41H), 5.42-5.36 (m, 2H), 4.62 (s, 2H), 4.51 (d, J = 3.6 Hz, 0.45H), 3.72 (d, J = 2.8 Hz, 0.36H), 3.54 (d, J = 2.0 Hz, 0.48H), 2.93-2.40 (m, 5H), 2.24-2.13 (m, 1H), 1.27-1.23 (m, 6H).

[0196] 15. Preparation of compound 1q [ka] Compound 1p (1 g, 1.95 mmol, 1.00 eq) was dissolved in acetone (19.6 mL). Silver oxide (3.63 g, 15.6 mmol, 10.0 eq) and methyl iodide (1.22 mL, 19.5 mmol, 10.0 eq) were added, and the mixture was stirred at 25°C for 24 hours. The reaction mixture was filtered and concentrated to obtain crude compound 1q (1.36 g). 1 H NMR: 400 MHz CDCl3δ 5.11 (s, 1H), 5.02 (d, J = 5.6 Hz, 1H), 4.06 (d, J = 1.6 Hz, 1H), 4.50 (d, J = 5.6 Hz, 1H), 4.39 (d, J = 1.6 Hz, 1H), 3.41 (s, 3H), 3.24-3.20 (m, 1H), 3.09 (t, J = 10.0 Hz, 1H), 1.47 (s, 3H), 1.35 (s, 3H).

[0197] 16. Preparation of compounds 1q-1 and 1q-2 [ka] Crude compound 1q (10.9 g, 20.7 mmol, 1.00 eq) was purified by C18 reversed-phase column chromatography (ammonium bicarbonate aqueous solution / acetonitrile) to obtain compound 1q-1 (1.50 g, 11.5%) and compound 1q-2 (1.50 g, 11.5%).

[0198] Compound 1q-1: m / z: ES+ [M+H]+ 525.2 HPLC: Retention time 0.820 min (Column: XBridge C18 2.1*50mm,5μm; Mobile phase: A: 10mM NH4HCO3 aqueous solution B: Acetonitrile; Gradient: 0-0.01 min 5% B, 0.01-0.7 min 5-95% B, 0.7-1.16 min 95% B, 1.16-1.5min, 95%-5% B; Flow rate: 1.5 mL / min; Column temp.: 40°C) 1 H NMR: 400 MHz CDCl3 δ 7.37-7.08 (m, 5H), 7.09 (d, J = 8.0 Hz, 1H), 5.76-5.74 (m, 2H), 5.49-5.44 (m, 2H), 4.73-4.67 (m, 2H), 4.15-4.06 (m, 4H), 4.02 (d, J = 4.8 Hz, 1H), 3.87-3.85 (m, 1H), 3.56 (s, 3H), 2.81-2.64 (m, 2H), 2.50-2.39 (m, 2H), 2.36-2.27 (m, 1H), 1.34-1.30 (m, 6H).

[0199] Compound 1q-2 m / z: ES+ [M+H]+ 525.2 HPLC: Retention time 0.836 min (Column: XBridge C18 2.1*50mm, 5μm; Mobile phase: A: 10mM NH4HCO3 aqueous solution B: Acetonitrile; Gradient: 0-0.01 min 5% B, 0.01-0.7 min 5-95% B, 0.7-1.16 min 95% B, 1.16-1.5min, 95%-5% B; Flow rate: 1.5 mL / min; Column temp.: 40°C) 1H NMR: 400 MHz CDCl3δ 7.37-7.27 (m, 5H), 7.08 (d, J = 8.0 Hz, 1H), 5.75-5.73 (m, 2H), 5.50-5.45 (m, 2H), 4.73-4.67 (m, 2H), 4.18-4.08 (m, 4H), 4.06 (d, J = 4.8 Hz, 1H), 3.88-3.86 (m, 1H), 3.56 (s, 3H), 3.06-2.95 (m, 1H), 2.83-2.41 (m, 5H), 1.34-1.31 (m, 6H).

[0200] 17. Preparation of compound 1r-1 [ka] Compound 1q-1 (1.00 g, 1.90 mmol, 1.00 eq) was dissolved in anhydrous dichloromethane (DCM, 20.0 mL) and cooled to -60°C. Boron trichloride (7.62 mL, 7.62 mmol, 1 M in dichloromethane, 4.00 eq) was added, and the mixture was stirred at -20°C for 1 hour. The reaction mixture was quenched with anhydrous ethanol (10.0 mL), neutralized with concentrated ammonia until the pH was approximately 7, and concentrated. The crude product was added to a mixed solution of dichloromethane and ethanol, and then filtered. The filtrate was concentrated, and the resulting residue was purified by C18 reversed-phase column chromatography (aqueous solution of ammonium bicarbonate / acetonitrile) to obtain compound 1r-1 (400 mg, 51.9%). m / z: ES+ [M+H]+ 405.3 1H NMR: 400 MHz CDCl3δ 8.51-8.43 (m, 1H), 7.15 (d, J = 8.4 Hz, 1H), 5.78 (d, J = 3.2 Hz, 1H), 5.76 (dd, J1 = 8.0 Hz, J2 = 2.4 Hz, 1H), 4.16-4.07 (m, 5H), 3.94 (dd, J1 = 4.8 Hz, J2 = 3.2 Hz, 1H), 3.55 (s, 3H), 2.96-2.94 (m, 1H),2.79-2.66 (m, 2H), 2.52-2.41 (m, 2H), 2.35-2.26 (m, 1H), 1.34-1.31 (m, 6H).

[0201] 18. Preparation of compound 1r-2 [ka] Compound 1r-2 was obtained using the same synthesis method as compound 1r-1. 1 H NMR: 400 MHz CDCl3δ 9.16-8.81 (m, 1H), 7.15 (d, J = 8.0 Hz, 1H), 5.78 (d, J = 2.8 Hz, 1H), 5.76 (dd, J1 = 8.0 Hz, J2 = 2.0 Hz, 1H), 4.65 (brs, 1H), 4.18-4.08 (m, 5H), 3.96 (dd, J1 = 4.8 Hz, J2 = 3.2 Hz, 1H), 3.53 (s, 3H), 3.05-2.95 (m, 1H), 2.81-2.41 (m, 2H), 1.34-1.30 (m, 6H).

[0202] 19. Preparation of Compound E1-1 [ka] Compound 1r-1 (500 mg, 1.23 mmol, 1.00 eq) was dissolved in dichloromethane (5.00 mL). Compound 1s (0.59 mL, 1.85 mmol, 1.50 eq) was added, and after cooling to 0°C, 4,5-dicyanoimidazole (160 mg, 1.36 mmol, 1.10 eq) was added, and the mixture was stirred at 15°C for 4 hours. The reaction mixture was quenched with saturated sodium bicarbonate (10.0 mL) and extracted twice with dichloromethane (10.0 mL). After concentration to obtain the crude product, compound E1-1 (530 mg, 70.9%) was obtained by purification by silica gel column chromatography (eluent: dichloromethane / acetone). 1 H NMR: 400 MHz CDCl3δ 9.17 (brs, 1H), 7.24 (d, J = 8.4 Hz, 1H), 5.83-5.79 (m, 1H), 5.64-5.61 (m, 1H), 4.43-4.33 (m, 1H), 4.09-3.62 (m, 9H), 3.43-3.38 (m, 3H), 2.86-2.66 (m, 3H), 2.57-2.21 (m, 4H), 1.28-1.18 (m, 18H).

[0203] 20. Preparation of Compound E1-2 [ka] Compound 1r-2 (500 mg, 1.23 mmol, 1.00 eq) was dissolved in dichloromethane (5.00 mL). Compound 1s (0.59 mL, 1.85 mmol, 1.50 eq) was added, and after cooling to 0°C, 4,5-dicyanoimidazole (160 mg, 1.36 mmol, 1.10 eq) was added, and the mixture was stirred at 15°C for 4 hours. The reaction mixture was quenched with saturated sodium bicarbonate (10.0 mL) and extracted twice with dichloromethane (10.0 mL). After concentration to obtain the crude product, compound E1-2 (480 mg, 64.0%) was obtained by purification by silica gel column chromatography (eluent: dichloromethane / acetone). 1H NMR: 400 MHz CDCl3δ 9.06 (brs, 1H), 7.33-7.31 (m, 1H), 5.89-5.88 (m, 1H), 5.66-5.64 (m, 1H), 4.49-4.40 (m, 1H), 4.08-3.64 (m, 9H), 3.40-3.36 (m, 3H), 3.00-2.19 (m, 7H), 1.29-1.18 (m, 18H).

[0204] Example 3 Preparation of siRNA The siRNA of the present invention was prepared using the solid-phase phosphoramidite method, which is well known in this field. For specific details, see, for example, PCT publication numbers WO2016081444 and WO2019105419, but a brief explanation is provided below.

[0205] 1. Preparation of siRNA without the use of bound ligands 1.1 Synthesis of Sense Chains (SS) The solid-phase phosphoramidite method was employed, using a blank CPG solid support as the starting cycle. Nucleoside monomers were attached one by one from 3' to 5' according to the composition of the sense chain nucleotide. Each nucleoside monomer attachment involved four steps: deprotection, coupling, capping, and oxidation or thiolation. The synthesis conditions for synthesizing 5 μmol of oligonucleotides were as follows:

[0206] Commercially available phosphoramidites modified with 2'-F and 2'-O-methyl were used. Nucleoside monomers were supplied as a 0.05 mol / L acetonitrile solution. The conditions for each step were the same: temperature 25°C; deprotection three times with 3% trichloroacetic acid-dichloromethane solution; coupling twice with 0.25 mol / L ETT-acetonitrile solution as an activator; capping twice with 10% anhydrous acetobiliary and pyridine / N-methylimidazole / acetonitrile (10:14:76, v / v / v); oxidation twice with 0.05 mol / L iodine in tetrahydrofuran / pyridine / water (70:20:10, v / v / v) solution; and thiolation twice with 0.2 mol / L PADS in acetonitrile / 3-methylpyridine (1:1, v / v) solution.

[0207] 1.2 Synthesis of antisense chains (AS) A solid-phase phosphoramidite method was employed, using a blank CPG solid support as the starting cycle. Nucleoside monomers were attached one by one from 3' to 5' according to the composition of the antisense chain nucleotide. Each nucleoside monomer attachment involved four steps: deprotection, coupling, capping, and oxidation or thiolation. The conditions for synthesizing 5 μmol of oligonucleotide as the antisense chain were the same as those for the sense chain.

[0208] 1.3 Purification and annealing of oligonucleotides 1.3.1 Ammonolithesis The synthesized solid support (sense chain or antisense chain) was transferred to a 5 mL centrifuge tube, and 3% diethylamine / ammonia (v / v) was added. The mixture was reacted in a constant temperature water bath at 35°C for 16 hours (or at 55°C for 8 hours), and then filtered. The solid support was washed three times with 1 mL of ethanol / water each time. The filtrate was concentrated by centrifugation, and the crude product was purified.

[0209] 1.3.2 Purification Methods for purification and desalting are well known to those skilled in the art. For example, elution and purification may be performed using a strongly anion-packed column and a sodium chloride-sodium hydroxide system. The product can be recovered in a tube and desalted using a gel-packed purification column with purified water as the eluent.

[0210] 1.3.3 Annealing According to Table 6, sense chains (SS) and antisense chains (AS) were mixed in a molar ratio (SS / AS = 1 / 1.05). The mixture was heated in a water bath to 70–95°C, held for 3–5 minutes, and then allowed to cool naturally to room temperature. The product was obtained by freeze-drying the system.

[0211] 2. Preparation of siRNA using a ligand-bound sense strand 2.1 Ligand binding to CPG carrier Binding of compound E7 to the CPG support Compound E7 (53 mg, 0.018 mmol) and HBTU (13.3 mg, 0.035 mmol) were mixed and shaken to dissolve in acetonitrile (5 mL). Next, DIEA (9.0 mg, 0.07 mmol) and DMAP (2.1 mg, 0.018 mmol) were added and shaken until dissolved. Blank support resin (550 mg, CPG pore size 1000 Å) was weighed and added to the reaction mixture, and the mixture was left in a shaker at 20°C overnight. A sample was taken and monitored, and thin-layer chromatography (TLC) (developing solvent: DCM / methanol = 4 / 1; color development: phosphomolybdic acid) was performed to confirm that the reaction was complete. The reaction mixture was filtered through a sand-core funnel. The filtration cake was washed with anhydrous acetonitrile (20 mL * 5) and collected. 530 mg of off-white solid was obtained by suction filtration under reduced pressure using an oil pump for 6 hours.

[0212] The concentrated product (530 mg) was transferred to a 50 mL round-bottom flask. CapC (DMAP / acetonitrile), CapB (N-methylimidazole / pyridine / acetonitrile), and CapA (acetic anhydride / acetonitrile) were added sequentially. The mixture was then left in a shaker at room temperature overnight. After filtration, the filtration cake was washed with acetonitrile (20 mL x 4) and collected. 200 mg of an off-white solid was obtained by suction filtration under reduced pressure using an oil pump for 8 hours, and this solid was used for solid-phase synthesis.

[0213] 2.2 Synthesis of Sense Chains (SS) The solid-phase phosphoramidite method was employed, and the E7 solid support prepared above was used as the starting cycle. Nucleoside monomers were attached one by one from 3' to 5' according to the composition of the sense chain nucleotide. Each nucleoside monomer attachment involved four steps: deprotection, coupling, capping, and oxidation or thiolation. The synthesis conditions for synthesizing 5 μmol of oligonucleotides were as follows.

[0214] Nucleoside monomers were supplied as a 0.05 mol / L acetonitrile solution. The conditions for each step were the same: temperature 25°C; deprotection three times with 3% trichloroacetic acid-dichloromethane solution; coupling twice with 0.25 mol / L ETT-acetonitrile solution as an activator; capping twice with 10% anhydrous acetobiliary and pyridine / N-methylimidazole / acetonitrile (10:14:76, v / v / v); oxidation twice with 0.05 mol / L iodine in tetrahydrofuran / pyridine / water (70:20:10, v / v / v) solution; and thiolation twice with 0.2 mol / L PADS in acetonitrile / 3-methylpyridine (1:1, v / v) solution.

[0215] 2.3 Synthesis of antisense chains (AS) A solid-phase phosphoramidite method was employed, using a blank CPG solid support as the starting cycle. Nucleoside monomers were attached one by one from 3' to 5' according to the composition of the antisense chain nucleotide. Each nucleoside monomer attachment involved four steps: deprotection, coupling, capping, and oxidation or thiolation. The conditions for synthesizing 5 μmol of oligonucleotide as the antisense chain were the same as those for the sense chain.

[0216] Finally, the antisense strands of the siRNAs listed in Table 5 were synthesized. Of these, the modified nucleotide SCP-U of the antisense strand was synthesized using compound E1-1 from Example 2 as the nucleoside monomer.

[0217] 2.4 Purification and annealing of oligonucleotides 2.4.1 Ammonolithesis The synthesized solid support (sense chain or antisense chain) was transferred to a 5 mL centrifuge tube, and 3% diethylamine / ammonia (v / v) was added. The mixture was reacted in a constant temperature water bath at 35°C for 16 hours (or at 55°C for 8 hours), and then filtered. The solid support was washed three times with 1 mL of ethanol / water each time. The filtrate was concentrated by centrifugation, and the crude product was purified.

[0218] 2.4.2 Purification

[0219] Methods for purification and desalting are well known to those skilled in the art. For example, elution and purification may be performed using a strongly anion-packed column and a sodium chloride-sodium hydroxide system. The product can be recovered in a tube and desalted using a gel-packed purification column with purified water as the eluent.

[0220] 2.4.3 Annealing

[0221] According to Table 6, sense chains (SS) and antisense chains (AS) were mixed in a molar ratio (SS / AS = 1 / 1.05). The mixture was heated in a water bath to 70–95°C, held for 3–5 minutes, and then allowed to cool naturally to room temperature. The product was obtained by freeze-drying the system.

[0222] Example 4: Activity screening in Huh7 cell line Cell transfection On day 1, human hepatocellular carcinoma Huh7 cell line was digested, resuspended, and counted. The resuspended cells were placed in a 96-well plate at a rate of 100 μL / well (1 × 10⁶ cells). 4 Cells were seeded in wells and transfection was performed 18 hours later.

[0223] On the second day, the 20 μM siRNA stock solution was diluted with Opti-MEM. 198 μL of Opti-MEM was added to 2 μL of the 20 μM siRNA stock solution to obtain the final siRNA concentration shown below. The mixture was thoroughly mixed by pipetting and set aside for later use.

[0224] On day 2, 0.9 μL of RNAiMAX (Thermo, 13778150) was diluted with 14.1 μL of Opti-MEM, gently mixed by pipetting, and allowed to stand at room temperature for 5 minutes. Then, 15 μL of the prepared RNAi-MAX mixture and 15 μL of diluted siRNA were gently mixed by pipetting (to avoid creating air bubbles) and allowed to stand at room temperature for 10 minutes. The mixture was transferred to a 96-well plate at a rate of 10 μL / well and incubated in a 5% CO2 incubator at 37°C for 24 hours (siRNA was not added to the control group).

[0225] RNA extraction Cellular RNA was extracted using a nucleic acid extraction device (Hangzhou Allsheng, Auto-pure96) according to the protocol of a high-throughput cell RNA extraction kit (FireGen, FG0417-L).

[0226] RNA reverse transcription PrimeScript TMReferring to the II 1st Strand cDNA Synthesis Kit (Takara, 6210B), a denaturation reaction mixture was prepared using 1 μL of oligo dT primer, 1 μL of dNTP mixture, and 12.5 μL of template RNA per single well. The mixture was incubated at 65°C for 5 minutes using a conventional PCR instrument, and then rapidly cooled on ice for 2 minutes.

[0227] PrimeScript TM Reverse transcription reaction mixtures were prepared according to the Prime Script II 1st Strand cDNA Synthesis Kit (Takara, 6210B). Each well contained 4 μL of 5× Prime Script II Buffer, 0.5 μL of RNase Inhibitor, and 1 μL of PrimeScript II RTase.

[0228] The denaturation reaction mixture (14.5 μL) was gently mixed with the reverse transcription reaction mixture. Reverse transcription was performed by incubation at 42°C for 45 minutes using a conventional PCR instrument, and then the enzyme was inactivated by incubation at 95°C for 5 minutes. Subsequently, the reverse transcript (cDNA) was cooled at 4°C.

[0229] After reverse transcription, 30 μL of DNase / RNase-free distilled water was added to each well's cDNA sample.

[0230] Fluorescence Quantitative PCR TaqMan TM A fluorescence quantitative PCR reaction was performed in a 20 μL system (ABI, QuantStudio3) following the instructions for Fast Advanced Master Mix (ABI, 4444965). The reaction program was as follows: (50°C, 2 min) x 1 cycle; (95°C, 20 sec) x 1 cycle; (95°C, 1 sec; 60°C, 24 sec) x 40 cycles.

[0231] [Table 7]

[0232] Data Statistics 2- △△Ct The value was calculated and converted to a percentage to obtain the residual suppression rate. △△Ct=[(Ct 標的遺伝子,実験群 -Ct 参照遺伝子,実験群 )-(Ct 標的遺伝子,コントロール群 -Ct 参照遺伝子,コントロール群 )]

[0233] The target gene was hCIDEB, and the reference gene was hACTB.

[0234] siRNA compounds were screened at a final siRNA concentration of 10 nM to determine their activity in cell lines. The screening results are shown in Table 8.

[0235] [Table 8]

[0236] Example 5 Activity screening in primary human hepatocytes (PHH) Cell transfection 1.4 mL of rat tail collagen solution (Sigma, C3867) was added to 40.6 mL of DNase / RNase-free distilled water and thoroughly mixed. The mixture was added to 96-well culture plates at a rate of 40 μL / well and coated overnight at 4°C. The coating medium was removed the following day.

[0237] On the second day, prior to use, the coated cell plates were rinsed with DPBS and then aspirated. PHH (Shanghai Xuanyi Biotechnology, Cat#: QYLF-HPMC) was recovered at 37°C and transferred to recovery medium, then centrifuged, resuspended, and counted. PHH was added to 96-well plates at a rate of 90 μL / well (2 × 10⁶). 4 Cells were seeded in wells. The complete culture medium was changed after 4 hours, and transfection was performed after 18 hours.

[0238] On the third day, the 20 μM siRNA stock solution was diluted with Opti-MEM. 198 μL of Opti-MEM was added to 2 μL of the 20 μM siRNA stock solution and thoroughly mixed by pipetting to obtain the first concentration point. Serial dilutions were performed as required by experiment. siRNA was not added to the control group.

[0239] On day 3, 0.9 μL of RNAiMAX (Thermo, 13778150) was diluted with 14.1 μL of Opti-MEM, gently mixed by pipetting, and allowed to stand at room temperature for 5 minutes. Then, 15 μL of the prepared RNAi-MAX mixture and 15 μL of the diluted compound were gently mixed by pipetting (avoiding the formation of air bubbles) and allowed to stand at room temperature for 10 minutes. The mixture was transferred to a 96-well plate at a rate of 10 μL / well. After incubation in a 5% CO2 incubator at 37°C for 24 hours, RNA was extracted.

[0240] RNA extraction Cellular RNA was extracted using a nucleic acid extraction device (Hangzhou Allsheng, Auto-pure96) according to the protocol of a high-throughput cell RNA extraction kit (FireGen, FG0412).

[0241] RNA reverse transcription PrimeScript TM The denaturation reaction mixture was prepared according to the 1st Strand cDNA Synthesis Kit (Takara, 6210B). Each well contained 1 μL of Oligo dT Primer, 1 μL of dNTP Mixture, and 12.5 μL of template RNA. The denaturation reaction was carried out by incubation at 65°C for 5 minutes in a conventional PCR instrument. The mixture was rapidly cooled on ice for 2 minutes.

[0242] PrimeScript TMReverse transcription reaction mixtures were prepared according to the Prime Script II 1st Strand cDNA Synthesis Kit (Takara, 6210B). Each well contained 4 μL of 5× Prime Script II Buffer, 0.5 μL of RNase Inhibitor, and 1 μL of PrimeScript II RTase.

[0243] The denaturation reaction mixture (14.5 μL) was gently mixed with the reverse transcription reaction mixture. Reverse transcription was performed by incubation at 42°C for 45 minutes using a conventional PCR instrument, and then the enzyme was inactivated by incubation at 95°C for 5 minutes. Subsequently, the reverse transcript (cDNA) was cooled at 4°C.

[0244] After reverse transcription, 30 μL of DNase / RNase-free distilled water was added to each well's cDNA sample.

[0245] Fluorescence Quantitative PCR TaqMan TM A fluorescence quantitative PCR reaction was performed in a 20 μL system (ABI, QuantStudio3) following the instructions for Fast Advanced Master Mix (ABI, 4444965). The reaction program was as follows: (50°C, 2 min) × 1 cycle; (95°C, 20 sec) × 1 cycle; (95°C, 1 sec; 60°C, 24 sec) × 40 cycles.

[0246] [Table 9]

[0247] Data Statistics 2- △△Ct The value was calculated and converted to a percentage to obtain the residual suppression rate. △△Ct=[(Ct 標的遺伝子,実験群 -Ct 参照遺伝子,実験群 )-(Ct 標的遺伝子,コントロール群 -Ct 参照遺伝子,コントロール群 )]

[0248] The target gene was hCIDEB, and the reference gene was hACTB.

[0249] The initial concentration of siRNA was set at 10 nM, and 47 siRNAs were screened for activity in human primary hepatocytes at five concentration points (10 nM, 1 nM, 0.1 nM, 0.01 nM, 0.001 nM) obtained by 10-fold serial dilution. The screening results are shown in Table 10, where columns 2-6 represent the residual inhibition rate and column 7 represents the IC50 value.

[0250] [Table 10]

[0251] Example 6: Activity screening in Hep3B cell lines Referring to the protocol of Example 4, the human hepatocellular carcinoma cell line Hep3B (Nanjing Cobioer Biotechnology, Cat#:CBP60197) was used. The siRNA compound was screened for activity in the cell line at five concentration points (10nM, 1nM, 0.1nM, 0.01nM, 0.001nM) obtained by 10-fold serial dilution, with the initial concentration of the siRNA compound being 10nM. The experimental results are shown in Table 11.

[0252] [Table 11]

[0253] Example 7: Activity screening in PCH cell lines Following the protocol of Example 4, a primary monkey hepatocyte PCH cell line (Milestone Biotechnologies, Cat#cmTCSC) was used. The siRNA compound was screened for activity in the cell line at 11 concentration points (10 nM, 3.33 nM, 1.11 nM, 0.37 nM, 0.123 nM, 0.041 nM, 0.0136 nM, 0.0045 nM, 0.00152 nM, 0.000508 nM, 0.000169 nM) obtained by 3-fold serial dilutions, with the initial concentration of the siRNA compound being 10 nM. The experimental results are shown in Table 12.

[0254] [Table 12]

[0255] Example 8. Detection of activity in an HDI mouse model 1. HDI Animal Modeling: Using tail vein hyperbaric injection, a dual gene stable transfection system was transfected in vivo into 6-8 week old male Balb / c mice. The TransIT system contained a Piggy-Bac transposon plasmid with the cDNA sequence of the target gene encoding cell death-inducing DFFA-like effector B, and a helper plasmid in a 1:1 mass ratio. (R) -QR Delivery Solution (total volume: 10% of the animal's body weight, Mirusbio-MIR5240) was injected into mice via the tail vein within 5-7 seconds using a 27-gauge needle. After injection, the mice were returned to their cages and observed for 30 minutes. Modeling day was designated as day 0, and SEAP expression levels were detected on days 7 (D7), 14 (D14), 21 (D21), and 28 (D28) after modeling.

[0256] 2. Detection of SEAP expression Kit (Phospha-Light) TM The standard sample included with the SEAP Reporter Gene Assay System (Invitrogen, T1016) was diluted 2-fold from 15 mU / mL to obtain a total of seven concentration points.

[0257] A reaction mixture was prepared by mixing the CSPD substrate with Reaction Buffer Diluent in a 1:20 ratio.

[0258] 5× Dilution Buffer was diluted to 1× Dilution Buffer with DNase RNase-Free Distilled Water.

[0259] Sample diluents were prepared by mixing serum with 1× Dilution Buffer in a centrifuge tube, incubated at 65°C for 30 minutes, and then cooled to room temperature.

[0260] 50 μL of sample diluent was added to a 96-well plate, followed by 50 μL of Assay Buffer per well, and incubated at room temperature for 5 minutes.

[0261] 50 μL of reaction solution was added to each well and incubated at room temperature for 20 minutes. SEAP chemiluminescence values ​​were read using a microplate reader (Tecan, Infinite200).

[0262] In this experiment, modeling was performed using a total of 100 μg of plasmid. On day 15 after modeling, each mouse was subcutaneously administered either 200 μl of physiological saline containing 3 mg / kg (mpk) of siRNA reagent or 200 μl of physiological saline without siRNA reagent (used as a blank control). The screening results of the HDI model experiment are shown in Table 13.

[0263] [Table 13-1] [Table 13-2]

[0264] Example 9: Activity screening in Huh7 cell line The Huh7 cell line was used, referring to the protocol of Example 4. siRNA compounds were screened for activity in the cell line at concentrations of 10 nM and 1 nM. The experimental results are shown in Table 14.

[0265] [Table 14-1] [Table 14-2]

Claims

1. A double-stranded nucleotide (dsRNA) for suppressing the expression of cell death-inducing DFFA-like effector B (CIDEB) in cells, comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand and the antisense strand are each independently 15 to 30 nucleotides long, and the antisense strand contains the nucleotide sequence of at least 15 adjacent nucleotides from the nucleotide sequence described in any one of SEQ ID NOs. 62 to 122.

2. The dsRNA according to claim 1, wherein the sense strand contains a nucleotide sequence of at least 15 adjacent nucleotides from the nucleotide sequences described in any one of Sequence IDs 1 to 61.

3. The dsRNA according to claim 1 or 2, wherein the dsRNA is siRNA.

4. The dsRNA according to any one of claims 1 to 3, wherein the sense strand and the antisense strand are each independently 15 to 27 nucleotides long, preferably 18 to 25 nucleotides long, and more preferably 19 to 21 nucleotides long.

5. The antisense strand comprises a nucleotide sequence of at least 16 adjacent nucleotides, a nucleotide sequence of at least 17 adjacent nucleotides, a nucleotide sequence of at least 18 adjacent nucleotides, a nucleotide sequence of at least 19 adjacent nucleotides, or a nucleotide sequence of at least 20 adjacent nucleotides from the nucleotide sequence described in any one of SEQ ID NOs: 62 to 122, and preferably comprises a nucleotide sequence described in any one of SEQ ID NOs: 62 to 122, the dsRNA according to any one of claims 1 to 4.

6. The sense strand comprises at least 16 adjacent nucleotides, a nucleotide sequence of at least 17 adjacent nucleotides, or a nucleotide sequence of at least 18 adjacent nucleotides from the nucleotide sequence described in any one of SEQ ID NOs: 1 to 61, and preferably comprises the nucleotide sequence described in any one of SEQ ID NOs: 1 to 61, the dsRNA according to any one of claims 1 to 5.

7. The above-mentioned siRNA comprises either a pair of sense strand sequences and antisense strand sequences as shown in Table 3 of the specification, wherein the dsRNA is according to any one of claims 1 to 6.

8. The dsRNA according to any one of claims 1 to 7, wherein substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, or all nucleotides of the sense strand and all nucleotides of the antisense strand are modified nucleotides.

9. The dsRNA according to claim 8, wherein the sense strand and the antisense strand each independently contain one or more nucleotide modifications selected from the group consisting of 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, SCP modified nucleotides, and phosphorothioate nucleotide inter-nucleotide bond modifications.

10. The dsRNA according to claim 8 or 9, wherein the 3' and / or 5' ends of the sense strand and / or antisense strand contain 1 to 5 phosphorothioate nucleotide interbonds, preferably 2 to 3 phosphorothioate nucleotide interbonds.

11. The above antisense strand is 21 nucleotides long, and (i) 2'-O-methyl modified nucleotides at positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21, and 2'-fluoro modified nucleotides at positions 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 (counted from the 5' end); and / or (ii) Inter-phosphorothioate nucleotide bonds (counted from the 5' end) between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21. A dsRNA according to any one of claims 1 to 10, having the characteristics of the dsRNA.

12. The above antisense strand is 21 nucleotides long, and (i) 2'-O-methyl modified nucleotides at positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 20, and 21, and 2'-fluoro modified nucleotides at positions 2, 4, 6, 8, 10, 12, 14, 16, and 18 (counted from the 5' end); and / or (ii) Inter-phosphorothioate nucleotide bonds (counted from the 5' end) between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21. A dsRNA according to any one of claims 1 to 10, having the characteristics of the dsRNA.

13. The above antisense strand is 21 nucleotides long, and (i) 2'-deoxyribochemical modifications at one or more nucleotide positions; (ii) SCP modifications at one or more nucleotide positions; (iii) 2'-fluoromodifications at one or more nucleotide positions; (iv) 2'-O-methyl modifications at positions other than those having 2'-deoxy modifications, SCP modifications, and 2'-fluoro modifications as defined in (i), (ii), and (iii); and / or (v) Phosphothioate internucleotide bonds (counted from the 5' end) between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21 A dsRNA according to any one of claims 1 to 10, having the characteristics of the dsRNA.

14. The above antisense strand is 21 nucleotides long, and (i) 2'-deoxy modifications at positions 2, 5, 7, and 12 (counted from the 5' end); (ii) SCP modification at position 1 (counted from the 5' end); (iii) 2'-fluoro modification at position 14 (counted from the 5' end); (iv) 2'-O-methyl modifications at positions 3, 4, 6, 8, 9, 10, 11, 13, 15, 16, 17, 18, 19, 20, and 21 (counted from the 5' end); and / or (v) Phosphothioate internucleotide bonds (counted from the 5' end) between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21 The dsRNA according to claim 13, having the following characteristics.

15. The above antisense strand is 23 nucleotides long, and (i) SCP modification at position 1 (counted from the 5' end); (ii) 2'-fluoro modifications at positions 2, 4, 6, 8, 10, 12, 14, 16, and 18 (counted from the 5' end); (iii) 2'-O-methyl modifications at positions 3, 5, 7, 9, 11, 13, 15, 17, 19, 20, 21, 22, and 23 (counted from the 5' end); and / or (iv) Phosphothioate internucleotide bonds between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counted from the 5' end) A dsRNA according to any one of claims 1 to 10, having the characteristics of the dsRNA.

16. The sense strand described above is 19 nucleotides long, and (i) 2'-O-methyl modified nucleotides at positions 1-6 and 10-19, and 2'-fluoro modified nucleotides at positions 7-9 (counted from the 5' end); and / or (ii) Phosphothioate internucleotide bonds between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counted from the 5' end) The dsRNA according to any one of claims 1 to 15, having the above characteristics.

17. The sense strand described above is 19 nucleotides long, and (i) 2'-O-methyl modified nucleotides at positions 1-6 and 10-19, and 2'-fluoro modified nucleotides at positions 7-9 (counted from the 5' end); and / or (ii) Inter-phosphorothioate nucleotide bonds (counted from the 5' end) between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between positions 17 and 18, and between positions 18 and 19. The dsRNA according to any one of claims 1 to 15, having the above characteristics.

18. The sense strand described above is 21 nucleotides long, and (i) 2'-fluoro modifications at positions 9, 10, and 11 (counted from the 5' end); (ii) 2'-O-methyl modifications at positions 1, 2, 3, 4, 5, 6, 7, 8, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 (counted from the 5' end); and / or (iii) Phosphothioate internucleotide bonds between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, and between nucleotide positions 20 and 21 (counted from the 5' end) The dsRNA according to any one of claims 1 to 15, having the above characteristics.

19. The dsRNA according to any one of claims 1 to 18, wherein the dsRNA is further bound to a ligand portion containing N-acetylgalactosamine, and preferably the sense strand is bound to the ligand portion.

20. The dsRNA according to claim 19, wherein the sense strand of the dsRNA includes a phosphorothioate internucleotide bond (counted from the 3' end) between nucleotide positions 1 and 2, and is bound to the ligand portion at the 3' end of the sense strand via the phosphorothioate.

21. The above ligand has the following structure: 【Chemistry 1】 (In the formula, 【Chemistry 2】 The dsRNA according to claim 19 or 20, wherein (where represents a point that is connected to the sense strand (preferably the 3' end of the sense strand) of the dsRNA via a phosphate ester group or a phosphorothioate group).

22. The dsRNA according to any one of claims 1 to 21, wherein the antisense strand comprises one of the modified nucleotide sequences shown in Table 5 of the specification, and / or the sense strand comprises one of the modified nucleotide sequences shown in Table 4 of the specification.

23. The dsRNA according to any one of claims 1 to 22, wherein the dsRNA includes either a pair of modified sense strand sequences and modified antisense strand sequences shown in Table 6 of the specification.

24. (a) The sense chain includes UmsAmsCmUmCmAmGfGfUfCmAmGmUmAmUmCmUmAmsAms-GL6 (SEQ ID NO: 149), and the antisense chain includes UmsUfsAmGfAmUfAmCfUmGfAmCfCmUfGmAfGmUfAmsAfsGm (SEQ ID NO: 259); (b) The sense chain comprises CmsAmsCmCmAmUmGfGfAfGmUmAmCmCmUmCmUmCmsAms-GL6 (SEQ ID NO: 150), and the antisense chain comprises UmsGfsAmGfAmGfGmUfAmCfUmCfCmAfUmGfGmUfGmsGfsAm (SEQ ID NO: 257); (c) The sense chain includes AmsGmsGmUmCmAmGfUfAfUmCmUmAmAmUmAmUmAmsAms-GL6 (SEQ ID NO: 151), and the antisense chain includes UmsUfsAmUfAmUfUmAfGmAfUmAfCmUfGmAfCmCfUmsGfsAm (SEQ ID NO: 260); (d) The sense chain includes CmsUmsCmUmAmUmGfAfGfUmUmGmUmGmAmCmUmUmsUms-GL6 (SEQ ID NO: 152), and the antisense chain includes AmsAfsAmGfUmCfAmCfAmAfCmUfCmAfUmAfGmAfGmsUfsAm (SEQ ID NO: 263); (e) The sense chain includes AmsGmsAmCmUmAmUfGfAfCmAmGmCmAmUmCmAmAmSAms-GL6 (SEQ ID NO: 153), and the antisense chain includes UmsUfsUmGfAmUfGmCfUmGfUmCfAmUfAmGfUmCfUmsUfsUm (SEQ ID NO: 267); (f) The sense chain includes GmsAmsCmUmAmUmGfAfCfAmGmCmAmUmCmAmAmAmsUms-GL6 (SEQ ID NO: 154), and the antisense chain includes AmsUfsUmUfGmAfUmGfCmUfGmUfCmAfUmAfGmUfCmsUfsUm (SEQ ID NO: 268); (g) The sense strand comprises AmsCmsUmAmUmGmAfCfAfGmCmAmUmCmAmAmUmsUms-GL6 (SEQ ID NO: 155), and the antisense strand comprises AmsAfsUmUfUmGfAmUfGmCfUmGfUmCfAmUfAmGfUmsCfsUm (SEQ ID NO: 269); (h) The sense chain includes CmsUmsAmUmGmAmCfAfGfCmAmUmCmAmAmUmUmsUms-GL6 (SEQ ID NO: 156), and the antisense chain includes AmsAfsAmUfUmUfGmAfUmGfCmUfGmUfCmAfUmAfGmsUfsCm (SEQ ID NO: 270); (i) The sense chain includes AmsUmsGmAmCmAmGfCfAfUmCmAmAmUmUmUmCmAms-GL6 (SEQ ID NO: 157), and the antisense chain includes UmsGfsAmAfAmUfUmUfGmAfUmGfCmUfGmUfCmAfUmsAfsGm (SEQ ID NO: 271); (j) The sense chain includes UmsGmsAmCmAmGmCfAfUfCmAmAmUmUmUmCmAmsAms-GL6 (SEQ ID NO: 158), and the antisense chain includes UmsUfsGmAfAmAfUmUfUmGfAmUfGmCfUmGfUmCfAmsUfsAm (SEQ ID NO: 272); (k) The sense strand includes CmsAmsGmAmCmAmGfUfAfCmAmGmGmCmUmAmGmAmsUms-GL6 (SEQ ID NO: 159), and the antisense strand includes AmsUfsCmUfAmGfCmCfUmGfUmAfCmUfGmUfCmUfGmsCfsAm (SEQ ID NO: 273); (l) The sense chain includes AmsGmsAmCmAmGmUfAfCfAmGmGmCmUmAmGmAmUmsAms-GL6 (SEQ ID NO: 160), and the antisense chain includes UmsAfsUmCfUmAfGmCfCmUfGmUfAmCfUmGfUmCfUmsGfsCm (SEQ ID NO: 274); (m) The sense chain includes GmsAmsCmAmGmUmAfCfAfGmGmCmUmAmGmAmUmAmsAms-GL6 (SEQ ID NO: 161), and the antisense chain includes UmsUfsAmUfCmUfAmGfCmCfUmGfUmAfCmUfGmUfCmSUfsGm (SEQ ID NO: 275); (n) The sense chain includes AmsAmsAmCmAmUmUfUfCfCmAmAmUmAmAmAmAmAmsUms-GL6 (SEQ ID NO: 162), and the antisense chain includes AmsUfsUmUfUmUfAmUfUmGfGmAfAmAfUmGfUmUfUmsUfsUm (SEQ ID NO: 276); (o) The sense chain includes AmsCmsAmUmUmUmCfCfAfAmUmAmAmAmAmUmAmsUms-GL6 (SEQ ID NO: 163), and the antisense chain includes AmsUfsAmUfUmUfUmUfAmUfUmGfGmAfAmAfUmGfUmsUfsUm (SEQ ID NO: 277); (p) The sense strand includes CmsCmsCmUmAmAmAfCfUfCmCmCmCmAmGmCmAmUmsAms-GL6 (SEQ ID NO: 164), and the antisense strand includes UmsAfsUmGfCmUfGmGfGmGfAmGfUmUfUmAfGmGfGmsAfsCm (SEQ ID NO: 281); (q) The sense strand includes UmsAmsCmUmCmAmGfGfUfCmAmGmUmAmUmCmUmAmsAms-GL6 (SEQ ID NO: 149), and the antisense strand includes (SCP-U)sdTsAmGmdAUmdACmUmGmAmdCCmUfGmAmGmUmAmsAmsGm (SEQ ID NO: 283); (r) The sense strand includes AmsGmsGmUmCmAmGfUfAfUmCmUmAmAmUmAmUmAmS-GL6 (SEQ ID NO: 151), and the antisense strand includes (SCP-U)sdTsAmUmdAUmdTAmGmAmUmdACmUfGmAmCmCmUmsGmsAm (SEQ ID NO: 284); (s) The sense strand includes AmsGmsAmCmUmAmUfGfAfCmAmGmCmAmUmCmAmAmSAms-GL6 (SEQ ID NO: 153), and the antisense strand includes (SCP-U)sdTsUmGmdAUmdGCmUmGmUmdCAmUfAmGmUmCmUmsUmsUm (SEQ ID NO: 285); (t) The sense strand includes GmsAmsCmUmAmUmGfAfCfAmGmCmAmUmCmAmAmAmsAms-GL6 (SEQ ID NO: 165), and the antisense strand includes (SCP-U)sdTsUmUmdGAmdTGmCmUmGmdTCmAfUmAmGmUmCmsUmsUm (SEQ ID NO: 286); (u) The sense strand includes UmsGmsAmCmAmGmCfAfUfCmAmAmAmUmUmUmCmAmsAms-GL6 (SEQ ID NO: 158), and the antisense strand includes (SCP-U)sdTsGmAmdAAmdTUmUmGmAmdTGmCfUmGmUmCmAmsUmsAm (SEQ ID NO: 288); (v) The sense strand includes AmsGmsAmCmAmGmUfAfCfAmGmGmCmUmAmGmAmUmsAms-GL6 (SEQ ID NO: 160), and the antisense strand includes (SCP-U)sdAsUmCmdTAmdGCmCmUmGmdTAmCfUmGmUmCmUmsGmsCm (SEQ ID NO: 289); (w) The sense strand above includes GmsAmsCmAmGmUmAfCfAfGmGmCmUmAmGmAmUmAmsAms-GL6 (SEQ ID NO: 161), and the antisense strand above includes (SCP-U)sdTsAmUmdCUmdAGmCmCmUmdGUmAfCmUmGmUmCmUmsGm (SEQ ID NO: 290); (x) The sense chain includes AmsAmsAmCmAmUmUfUfCfCmAmAmUmAmAmAmAmAmsAms-GL6 (SEQ ID NO: 166), and the antisense chain includes (SCP-U)sdTsUmUmdTUmdAUmUmGmGmdAAmAfUmGmUmUmUmsUmsUm (SEQ ID NO: 291); (y) The sense chain includes AmsCmsAmUmUmUmCfCfAfAmUmAmAmAmAmUmAmsAms-GL6 (SEQ ID NO: 167), and the antisense chain includes (SCP-U)sdTsAmUmdTUmdTUmAmUmUmdGGmAfAmAmUmGmUmsUmsUm (SEQ ID NO: 292); (z) The sense strand includes GmsCmsUmCmUmGmAfAfCfCmCmCmAmGmUmGmAmCmsAms-GL6 (SEQ ID NO: 168), and the antisense strand includes (SCP-U)sdGsUmCmdACmdTGmGmGmGmdTUmCfAmGmAmGmCmsUmsGm (SEQ ID NO: 293); (ab) The sense strand contains GmsCmsCmAmGmGmAfGfCfUmGmCmUmAmGmCmCmAmsAms-GL6 (SEQ ID NO: 169), and the antisense strand contains (SCP-U)sdTsGmGmdCUmdAGmCmAmGmdCUmCfCmUmGmGmCmSUmsGm (SEQ ID NO: 294); (ac) The sense strand above includes CmsCmsUmUmUmGmAfCfGfUmGmUmAmCmAmAmGmCmsAms-GL6 (SEQ ID NO: 170), and the antisense strand above includes (SCP-U)sdGsCmUmdTGmdTAmCmAmCmdGUmCfAmAmAmGmGmsUmsGm (SEQ ID NO: 295); (ad) The sense strand includes GmsCmsCmAmUmAmUfGfUfUmGmCmUmGmGmGmAmAmSAms-GL6 (SEQ ID NO: 171), and the antisense strand includes (SCP-U)sdTsUmCmdCCmdAGmCmAmAmdCAmUfAmUmGmGmCmUmSCm (SEQ ID NO: 296); (ae) The sense strand includes CmsCmsGmCmCmUmCfCfAfUmUmCmCmUmAmCmUmAmsAms-GL6 (SEQ ID NO: 172), and the antisense strand includes (SCP-U)sdTsAmGmdTAmdGGmAmAmUmdGGmAfGmGmCmGmGmsUmsCm (SEQ ID NO: 297); (af) The sense strand includes GmsCmsAmAmAmGmAfCfUfAmUmGmAmCmAmGmCmAmsAms-GL6 (SEQ ID NO: 173), and the antisense strand includes (SCP-U)sdTsGmCmdTGmdTCmAmUmAmdGUmCfUmUmUmGmCmsAmsGm (SEQ ID NO: 298); (ag) The sense strand contains CmsUmsCmUmGmAmAfCfCfCmCmAmGmUmGmAmCmUmsAms-GL6 (SEQ ID NO: 174), and the antisense strand contains (SCP-U)sdAsGmUmdCAmdCUmGmGmGmdGUmUfCmAmGmAmGmsCmsUm (SEQ ID NO: 299); (ah) The sense strand includes CmsCmsUmCmAmCmAfUfCfCmCmAmAmGmUmCmUmAmsAms-GL6 (SEQ ID NO: 176), and the antisense strand includes (SCP-U)sdTsAmGmdACmdTUmGmGmGmdAUmGfUmGmAmGmGmsCmsGm (SEQ ID NO: 301); (ai) The sense chain above includes AmsUmsUmUmCmCmAfAfUfAmAmAmAmUmAmUmCmsAms-GL6 (SEQ ID NO: 177), and the antisense chain above includes (SCP-U)sdGsAmUmdAUmdTUmUmUmAmdTUmGfGmAmAmAmUmsGmsUm (SEQ ID NO: 302); (aj) The sense chain above includes AmsAmsAmAmAmCmAmUmUfUfCfCmAmAmUmAmAmAmAmAmsAms-GL6 (SEQ ID NO: 178), and the antisense chain above includes (SCP-U)sUfsUmUfUmUfAmUfUmGfGmAfAmAfUmGfUmUfUmUmUmsGmsUm (SEQ ID NO: 303); (ak) The sense chain above includes AmsAmsAmCmAmUmUmUmCfCfAfAmUmAmAmAmAmUmAmsAms-GL6 (SEQ ID NO: 179), and the antisense chain above includes (SCP-U)sUfsAmUfUmUfUmUfAmUfUmGfGmAfAmAfUmGfUmUmUmsUmsUm (SEQ ID NO: 304), GL6 is 【Transformation 3】 (In the formula, 【Chemistry 4】 The dsRNA according to claim 23, wherein (where represents a point that is connected to the 3' end of the sense strand of the dsRNA via a phosphate ester group or a phosphorothioate group).

25. A cell containing the dsRNA described in any one of claims 1 to 24.

26. A pharmaceutical composition comprising dsRNA according to any one of claims 1 to 24 or cells according to claim 25, and optionally a pharmaceutically acceptable carrier or excipient.

27. A kit comprising dsRNA according to any one of claims 1 to 24, cells according to claim 25, or a pharmaceutical composition according to claim 26.

28. A method for treating a disease or disorder in which a reduction in the expression of cell death-inducing DFFA-like effector B (CIDEB) is beneficial, comprising the step of administering to the subject a dsRNA according to any one of claims 1 to 24, a cell according to claim 25, or a pharmaceutical composition according to claim 26.

29. A method for preventing at least one symptom in a subject having a disease or disorder in which a reduction in the expression of cell death-inducing DFFA-like effector B (CIDEB) is beneficial, comprising the step of administering to the subject a dsRNA according to any one of claims 1 to 24, a cell according to claim 25, or a pharmaceutical composition according to claim 26.

30. Diseases or disorders in which a reduction in the expression of the above-mentioned cell death-inducing DFFA-like effector B (CIDEB) is beneficial are CIDEB-mediated diseases or CIDEB-related diseases, preferably the above-mentioned CIDEB-mediated diseases or CIDEB-related diseases are liver diseases (fatty liver, hepatitis (steatohepatitis, non-alcoholic steatohepatitis, and viral hepatitis, etc.), non-alcoholic fatty liver disease, alcoholic fatty liver disease, hepatic fibrosis, cirrhosis, and hepatic failure, etc.), cholangitis (primary biliary cholangitis, primary sclerosing cholangitis), Autoimmune diseases, endocrine disorders, urinary tract diseases, metabolic diseases (metabolic syndrome, etc.), hepatobiliary diseases, fibrous diseases, cardiovascular diseases (hypertension, endothelial cell dysfunction, arteriosclerosis, atherosclerosis, coronary artery disease, myocardial infarction, ischemic stroke, and other heart diseases, etc.), immunoinflammatory diseases, central nervous system diseases, digestive diseases, hyperproliferative diseases (cancer, etc.), dyslipidemia (hyperlipidemia, hypercholesterolemia, hypertriglyceridemia, high LDL cholesterol, low HDL cholesterol, and The CIDEB-mediated disease or CIDEB-related disease is selected from among postprandial hypertriglyceridemia, impaired blood glucose control (insulin resistance, type 2 diabetes, etc.), adipocyte dysfunction, visceral fat accumulation, obesity, eating disorders, and excessive sugar cravings, and more preferably the above CIDEB-mediated disease or CIDEB-related disease is liver disease (fatty liver, hepatitis (steatohepatitis, non-alcoholic steatohepatitis, alcoholic steatohepatitis, and viral hepatitis, etc.), non-alcoholic fatty liver disease, alcoholic fatty liver disease, hepatic fibrosis, cirrhosis, and hepatic failure, etc.) The method according to claim 28 or 29, selected from ), cholangitis (primary biliary cholangitis, primary sclerosing cholangitis, etc.), metabolic diseases (metabolic syndrome, etc.), cardiovascular diseases (hypertension, endothelial cell dysfunction, arteriosclerosis, atherosclerosis, coronary artery disease, myocardial infarction, ischemic stroke, and other heart diseases, etc.), and dyslipidemia (hyperlipidemia, hypercholesterolemia, hypertriglyceridemia, high LDL cholesterol, low HDL cholesterol, and postprandial hypertriglyceridemia, etc.).

31. A method for reducing the level of cell death-inducing DFFA-like effector B (CIDEB) in a subject, comprising the step of administering to the subject a dsRNA according to any one of claims 1 to 24, a cell according to claim 25, or a pharmaceutical composition according to claim 26.

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