Double-stranded ribonucleic acid for inhibiting GPR146 gene expression, conjugate, and use

Abstract

A double-stranded ribonucleic acid for inhibiting GPR146 gene expression, a conjugate, and use. The double-stranded ribonucleic acid can bind in cells to form an RNA-induced silencing complex (RISC), which cleaves mRNA transcribed by a GPR146 gene. Therefore, the double-stranded ribonucleic acid can selectively and efficiently inhibit the GPR146 gene expression and thereby be used in the treatment of diseases mediated by the GPR146 gene, demonstrating significant application prospects in disease treatment in clinic.

Description

Double-stranded RNA, conjugates, and applications for inhibiting GPR146 gene expression Technical Field

[0001] This disclosure pertains to the field of biomedicine. Specifically, this disclosure relates to a double-stranded ribonucleic acid, a double-stranded ribonucleic acid conjugate or a prodrug thereof, a pharmaceutical composition and its use for inhibiting GPR146 gene expression, and a method for inhibiting intracellular GPR146 gene expression in vivo or in vitro. Background Technology

[0002] G protein-coupled receptor 146 (GPR146) induces hepatic sterol regulatory element-binding protein 2 (SREBP2) by activating extracellular signal-regulated kinase 1 / 2 (ERK1 / 2) signaling, thereby leading to hepatic very low-density lipoprotein (VLDL) secretion. GPR146 deficiency in mice has been shown to reduce hepatic secretion of VLDL, the precursor of low-density lipoprotein (LDL), and prevent diet-induced atherosclerosis in LDL receptor knockout mice. This research suggests that GPR146 may be associated with the development of atherosclerosis (AS) and familial hypercholesterolemia (HoFH).

[0003] Atherosclerosis (AS) remains a leading cause of coronary artery disease, cerebral infarction, and peripheral vascular disease, imposing a global burden on the public. Studies have found that lipids are the primary deposits under the aortic intima, participating in the formation of atherosclerotic plaques and thus contributing to AS. Simultaneously, dysregulation of plasma lipids and lipoproteins, such as elevated plasma low-density lipoprotein cholesterol (LDL-C) and triglycerides (TGs), is a major pathological basis for AS progression. It is also noteworthy that holoprosclerotic fibrosis (HoFH) is a common inherited metabolic disorder characterized by severely elevated LDL-C levels, leading to early cardiovascular morbidity and mortality. Studies have shown that over 95% of HoFH patients exhibit loss-of-function mutations in both LDL receptor (LDLR) alleles. Therefore, inhibiting GPR146 may be an effective strategy for regulating cholesterol levels in the treatment of AS and HoFH. Meanwhile, identifying novel therapeutic targets for LDL-lowering drugs independent of LDLR activity in HoFH is urgently needed and of great significance (Lan She et al. GPR146: an emerging therapeutic target for hypercholesterolemia and atherosclerosis. Acta Biochim Biophys Sin (Shanghai). 2020 Aug 5; 52(8) 914-915.; Antoine Rimbert et al., Variants in the GPR146 Gene Are Associated With a Favorable Cardiometabolic Risk Profile. Arteriosclerosis, Thrombosis, and Vascular Biology. Volume 42, Number 10).

[0004] Furthermore, nonspecific orbital inflammation (NSOI) is a chronic idiopathic disease characterized by widespread polymorphic lymphoid infiltration in the orbital region. Zixuan Wu et al. analyzed the correlation between GPR146 gene expression and immune cell infiltration, thereby elucidating the immunological characteristics of GPR146 in the context of NSOI and deriving a therapeutic strategy targeting the immune metabolic pathway of GPR146 in NSOI (Zixuan Wu et al., Elucidating the multifaceted roles of GPR146 in non-specific orbital inflammation a concerted analytical approach through the prisms of bioinformatics and machine learning, Front Med (Lausanne). 2024 Jun 5:11:1309510.).

[0005] In summary, inhibiting GPR146 gene expression can prevent and / or treat nonspecific orbital inflammation, hypercholesterolemia, atherosclerosis, and coronary artery disease. Therefore, developing drugs targeting GPR146 is of significant value. Summary of the Invention

[0006] The problem the invention aims to solve

[0007] Given the problems existing in the prior art, such as the need to develop GPR146 inhibitors for the prevention and / or treatment of GPR146-related diseases, including nonspecific orbital inflammation, hypercholesterolemia, atherosclerosis, and coronary artery disease, as well as other unidentified related conditions, pathologies, or syndromes, this disclosure aims to provide a series of double-stranded ribonucleic acids, double-stranded ribonucleic acid conjugates, or prodrugs thereof, and pharmaceutical compositions for inhibiting GPR146 gene expression. These compounds are effectively targeted at RNA-induced silencing complex (RISC)-mediated cleavage of the GPR146 gene RNA transcript, thereby selectively and effectively inhibiting GPR146 gene expression, and have significant application prospects in clinical disease treatment.

[0008] Solution for solving the problem

[0009] In a first aspect, this disclosure provides a double-stranded ribonucleic acid (BRNA) for inhibiting GPR146 gene expression, comprising a sense strand and an antisense strand, wherein each nucleotide in the BRNA is independently modified or unmodified, the sense strand contains nucleotide sequence I, the antisense strand contains nucleotide sequence II, and nucleotide sequence I and nucleotide sequence II are at least partially anticomplementary to form a double-stranded region, wherein nucleotide sequence I and nucleotide sequence II are selected from the following sequences:

[0010] (1) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:1, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:2:

[0011] 5'-GGUCCUCACCAGAGCUCUA-3'(SEQ ID NO:1)

[0012] 5'-UAGAGCUCUGGUGAGGACC-3' (SEQ ID NO: 2);

[0013] (2) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:3, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:4:

[0014] 5'-CAGAUCCCCUUCAAUGUGU-3'(SEQ ID NO:3)

[0015] 5'-ACACAUUGAAGGGGAUCUG-3' (SEQ ID NO: 4);

[0016] (3) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:7, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:8:

[0017] 5'-CUCCCAAACACGCAGCUCA-3'(SEQ ID NO:7)

[0018] 5'-UGAGCUGCGUGUUUGGGAG-3' (SEQ ID NO:8);

[0019] (4) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:9, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:10:

[0020] 5'-GAGCCAGUAUUUAUACUUU-3'(SEQ ID NO:9)

[0021] 5'-AAAGUAUAAAUACUGGCUC-3' (SEQ ID NO: 10);

[0022] (5) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:11, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:12:

[0023] 5'-ACAGAUGUUUCCUAGAAAA-3'(SEQ ID NO:11)

[0024] 5'-UUUUCUAGGAAACAUCUGU-3' (SEQ ID NO: 12);

[0025] (6) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:13, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:14:

[0026] 5'-GAGGUUUUUUUCAGUAUGA-3'(SEQ ID NO:13)

[0027] 5'-UCAUACUGAAAAAAACCUC-3' (SEQ ID NO: 14);

[0028] (7) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:15, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:16:

[0029] 5'-GUCCUAAAUCAAUUCCUCA-3'(SEQ ID NO:15)

[0030] 5'-UGAGGAAUUGAUUUAGGAC-3' (SEQ ID NO: 16);

[0031] (8) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:17, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:18:

[0032] 5'-GCAUUCAGUUUGUCAAU-3'(SEQ ID NO:17)

[0033] 5'-AUUGACAAACUGAAUGC-3' (SEQ ID NO: 18);

[0034] (9) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:19, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:20:

[0035] 5'-GGUUAAAAUA-3'(SEQ ID NO:19)

[0036] 5'-UAUUUUAACC-3' (SEQ ID NO:20);

[0037] (10) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:21, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:22:

[0038] 5'-CCCCUUGUUUGUUU-3'(SEQ ID NO:21)

[0039] 5'-AAACAAACAAGGGG-3' (SEQ ID NO: 22);

[0040] (11) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:23, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:24:

[0041] 5'-CACAAAACUAAA-3'(SEQ ID NO:23)

[0042] 5'-UUUAGUUUUGUG-3' (SEQ ID NO: 24).

[0043] In some embodiments, the sense strand further contains nucleotide sequence III, and the antisense strand further contains nucleotide sequence IV, wherein nucleotide sequence III and nucleotide sequence IV are each independently 0-9 nucleotides in length (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides), wherein nucleotide sequence III is attached to the 5' end of nucleotide sequence I, and nucleotide sequence IV is attached to the 3' end of nucleotide sequence II, wherein nucleotide sequence III and nucleotide sequence IV are of equal length and are substantially anticomplementary or completely anticomplementary; substantially anticomplementary means that there is no more than one base mismatch between the two nucleotide sequences; completely anticomplementary means that there is no mismatch between the two nucleotide sequences.

[0044] In some embodiments, nucleotide sequence III is attached to the 3' end of nucleotide sequence I, and nucleotide sequence IV is attached to the 5' end of nucleotide sequence II. Nucleotide sequence III and nucleotide sequence IV are of equal length and are substantially anticomplementary or completely anticomplementary. Substantially anticomplementary means that there is no more than one base mismatch between the two nucleotide sequences. Completely anticomplementary means that there is no mismatch between the two nucleotide sequences.

[0045] In some embodiments, the nucleotide sequences I and III are at least partially anticomplementary to the nucleotide sequences II and IV to form a double-stranded region.

[0046] In some embodiments, the positive strand further contains nucleotide sequence V and / or the antisense strand further contains nucleotide sequence VI, wherein the length of nucleotide sequence V and nucleotide sequence VI are each independently 0-2 nucleotides (e.g., 0, 1, or 2 nucleotides), wherein nucleotide sequence V is attached to the 3' end of the positive strand to form a 3' overhang of the positive strand and / or nucleotide sequence VI is attached to the 3' end of the antisense strand to form a 3' overhang of the antisense strand; preferably, the length of nucleotide sequence V or nucleotide sequence VI is each 2 nucleotides; more preferably, along the 5' end to the 3' end direction, the nucleotide sequence V or nucleotide sequence VI is UU, AU, UA, AG, AA, AC, CU, or CA.

[0047] In some embodiments, the nucleotide sequence V is identical to or differs from the nucleotide at the corresponding position of the target mRNA, or the nucleotide sequence VI is mismatched or complementary to the nucleotide at the corresponding position of the target mRNA.

[0048] In some specific embodiments, the double-stranded ribonucleic acid comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand are selected from the following sequences:

[0049] The sense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:33, and the antisense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:57;

[0050] The sense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:47, and the antisense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:58; or

[0051] The sense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:55, and the antisense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:59.

[0052] In some embodiments, the length of the double-stranded region is 10-30 nucleotide pairs, preferably 17-25 nucleotide pairs, more preferably 19-23 nucleotide pairs, such as 19, 20, 21, 22 or 23 nucleotide pairs; even more preferably 19, 20, 21 or 23 nucleotide pairs.

[0053] In some embodiments, the sense strand or the antisense strand has 15-30 nucleotides, preferably 17-25 nucleotides, more preferably 19-23 nucleotides, such as 19, 20, 21, 22 or 23 nucleotides; and even more preferably 19, 21, 22 or 23 nucleotides.

[0054] In some implementations, each nucleotide in the positive strand is independently a modified nucleotide or an unmodified nucleotide.

[0055] In some implementations, each nucleotide in the antisense strand is independently a modified nucleotide or an unmodified nucleotide.

[0056] In some implementations, any two nucleotides linked together in the positive chain are connected by a phosphodiester bond or a thiophosphate group.

[0057] In some implementations, any two linked nucleotides in the antisense strand are connected by a phosphodiester bond or a thiophosphate group.

[0058] In some embodiments, the 5' terminal nucleotide of the antisense strand is linked to a 5' phosphate group or a 5' phosphate-derived group.

[0059] In some embodiments, the 5' terminal nucleotide of the antisense strand is not linked to a 5' phosphate group or a 5' phosphate-derived group.

[0060] In some implementations, the positive chain may contain one or more end-cap residues or portions, or the positive chain may not contain end-cap residues or portions.

[0061] In some preferred embodiments, the capping residue is an inverse debased deoxyribose residue (invAb).

[0062] In some preferred embodiments, the 3' terminal nucleotide of the positive strand is linked to one or more reverse debased deoxyribose residues.

[0063] In some preferred embodiments, the 5' terminal nucleotide of the positive strand is linked to one or more reverse debased deoxyribose residues.

[0064] In some preferred embodiments, the 3' terminal nucleotide and 5' terminal nucleotide of the positive strand are each linked to one or more reverse debased deoxyribose residues.

[0065] In some preferred embodiments, the reverse debased deoxyribose residue is linked to the 3' terminal nucleotide and / or 5' terminal nucleotide of the positive strand via a phosphodiester bond, a thiophosphate bond, or other nucleoside bonds.

[0066] In some implementations, the double-stranded ribonucleic acid is siRNA.

[0067] In some embodiments, the modified nucleotide is selected from 2'-fluoro-modified nucleotides, 2'-alkoxy-modified nucleotides, 2'-substituted alkoxy-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-substituted alkyl-modified nucleotides, 2'-deoxy-modified nucleotides, 2'-amino-modified nucleotides, 2'-substituted amino-modified nucleotides, nucleotide analogs, or any combination of two or more thereof.

[0068] In some embodiments, the modified nucleotide is selected from 2'-fluoro-modified nucleotides, 2'-methoxy-modified nucleotides, 2'-O-CH2-CH2-O-CH3-modified nucleotides, 2'-O-CH2-CH=CH2-modified nucleotides, 2'-CH2-CH2-CH=CH2-modified nucleotides, 2'-deoxy-modified nucleotides, nucleotide analogs, or any combination of two or more of these.

[0069] In some preferred embodiments, the nucleotide analogue is selected from isonucleotides, LNA, ENA, cET-BNA, UNA, or GNA.

[0070] In some implementations, each nucleotide in the sense strand and the antisense strand is independently a 2'-fluorinated nucleotide or a non-fluorinated nucleotide.

[0071] In some embodiments, in the positive strand, along the direction from the 3' end to the 5' end, 2'-fluorinated nucleotides are located at positions 9, 10, 11, and 13, with the remaining positions being non-fluorinated nucleotides; or, 2'-fluorinated nucleotides are located at positions 11, 12, 13, and 15, with the remaining positions being non-fluorinated nucleotides; or, 2'-fluorinated nucleotides are located at positions 11, 12, 13, and 17, with the remaining positions being non-fluorinated nucleotides; or, 2'-fluorinated nucleotides are located at positions 9, 11, 13, and 17, with the remaining positions being non-fluorinated nucleotides; or, 2'-fluorinated nucleotides are located at positions 6, 11, and 13, with the remaining positions being non-fluorinated nucleotides.

[0072] In some embodiments, in the antisense strand, 2'-fluorinated nucleotides are located at positions 2, 6, 14, and 16 along the 5' end to the 3' end, with the remaining positions being non-fluorinated nucleotides; or, 2'-fluorinated nucleotides are located at positions 2, 7, 10, and 14, with the remaining positions being non-fluorinated nucleotides; or, 2'-fluorinated nucleotides are located at positions 2, 3, 5, 7, 10, 12, and 14, with the remaining positions being non-fluorinated nucleotides.

[0073] In some preferred embodiments, along the 3' end to the 5' end, 2'-fluorinated nucleotides are located at positions 9, 10, 11, and 13 of the sense strand, with the remaining positions being non-fluorinated nucleotides; along the 5' end to the 3' end, 2'-fluorinated nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand, with the remaining positions being non-fluorinated nucleotides.

[0074] In some preferred embodiments, along the 3' end to the 5' end, 2'-fluorinated nucleotides are located at positions 11, 12, 13, and 15 of the sense strand, with the remaining positions being non-fluorinated nucleotides; along the 5' end to the 3' end, 2'-fluorinated nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand, with the remaining positions being non-fluorinated nucleotides.

[0075] In some preferred embodiments, along the 3' end to the 5' end, 2'-fluorinated nucleotides are located at positions 11, 12, 13, and 17 of the sense strand, with the remaining positions being non-fluorinated nucleotides; along the 5' end to the 3' end, 2'-fluorinated nucleotides are located at positions 2, 7, 10, and 14 of the antisense strand, with the remaining positions being non-fluorinated nucleotides.

[0076] In some preferred embodiments, along the 3' end to the 5' end, 2'-fluorinated nucleotides are located at positions 9, 11, 13, and 17 of the sense strand, with the remaining positions being non-fluorinated nucleotides; along the 5' end to the 3' end, 2'-fluorinated nucleotides are located at positions 2, 7, 10, and 14 of the antisense strand, with the remaining positions being non-fluorinated nucleotides.

[0077] In some preferred embodiments, along the 3' end to the 5' end, 2'-fluorinated nucleotides are located at positions 6, 11, and 13 of the sense strand, with the remaining positions being non-fluorinated nucleotides; along the 5' end to the 3' end, 2'-fluorinated nucleotides are located at positions 2, 7, 10, and 14 of the antisense strand, with the remaining positions being non-fluorinated nucleotides.

[0078] In some preferred embodiments, along the 3' end to the 5' end, 2'-fluorinated nucleotides are located at positions 9, 11, 13, and 17 of the sense strand, with the remaining positions being non-fluorinated nucleotides; along the 5' end to the 3' end, 2'-fluorinated nucleotides are located at positions 2, 3, 5, 7, 10, 12, and 14 of the antisense strand, with the remaining positions being non-fluorinated nucleotides.

[0079] In some preferred embodiments, each non-fluorinated nucleotide is a 2'-methoxylated nucleotide.

[0080] In some preferred embodiments, each nucleotide in the sense strand and the antisense strand is independently a 2'-fluoro-modified nucleotide, a 2'-methoxy-modified nucleotide, and / or a GNA-modified nucleotide.

[0081] In some embodiments, in the positive strand, along the direction from the 3' end to the 5' end, 2'-fluoromodified nucleotides are located at positions 9, 10, 11, and 13, with the remaining positions being 2'-methoxymodified nucleotides; or, 2'-fluoromodified nucleotides are located at positions 11, 12, 13, and 15, with the remaining positions being 2'-methoxymodified nucleotides; or, 2'-fluoromodified nucleotides are located at positions 11, 12, 13, and 17, with the remaining positions being 2'-methoxymodified nucleotides; or, 2'-fluoromodified nucleotides are located at positions 9, 11, 13, and 17, with the remaining positions being 2'-methoxymodified nucleotides; or, 2'-fluoromodified nucleotides are located at positions 6, 11, and 13, with the remaining positions being 2'-methoxymodified nucleotides.

[0082] In some embodiments, in the antisense strand, along the 5' end to the 3' end, 2'-fluoromodified nucleotides are located at positions 2, 6, 14, and 16, with the remaining positions being 2'-methoxymodified nucleotides; or, 2'-fluoromodified nucleotides are located at positions 2, 7, 10, and 14, with the remaining positions being 2'-methoxymodified nucleotides; or, 2'-fluoromodified nucleotides are located at positions 2, 3, 5, 7, 10, 12, and 14, and GNA-modified nucleotides are located at position 6 of the antisense strand, with the remaining positions being 2'-methoxymodified nucleotides.

[0083] In some preferred embodiments, along the 3' end to the 5' end, 2'-fluorinated nucleotides are located at positions 9, 10, 11, and 13 of the sense strand, with the remaining positions being 2'-methoxylated nucleotides; along the 5' end to the 3' end, 2'-fluorinated nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand, with the remaining positions being 2'-methoxylated nucleotides.

[0084] In some preferred embodiments, along the 3' end to the 5' end, 2'-fluorinated nucleotides are located at positions 11, 12, 13, and 15 of the sense strand, with the remaining positions being 2'-methoxylated nucleotides; along the 5' end to the 3' end, 2'-fluorinated nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand, with the remaining positions being 2'-methoxylated nucleotides.

[0085] In some preferred embodiments, along the 3' end to the 5' end, 2'-fluorinated nucleotides are located at positions 11, 12, 13, and 17 of the sense strand, with the remaining positions being 2'-methoxylated nucleotides; along the 5' end to the 3' end, 2'-fluorinated nucleotides are located at positions 2, 7, 10, and 14 of the antisense strand, with the remaining positions being 2'-methoxylated nucleotides.

[0086] In some preferred embodiments, along the 3' end to the 5' end, 2'-fluorinated nucleotides are located at positions 9, 11, 13, and 17 of the sense strand, with the remaining positions being 2'-methoxylated nucleotides; along the 5' end to the 3' end, 2'-fluorinated nucleotides are located at positions 2, 7, 10, and 14 of the antisense strand, with the remaining positions being 2'-methoxylated nucleotides.

[0087] In some preferred embodiments, along the 3' end to the 5' end, 2'-fluorinated nucleotides are located at positions 6, 11, and 13 of the sense strand, with the remaining positions being 2'-methoxylated nucleotides; along the 5' end to the 3' end, 2'-fluorinated nucleotides are located at positions 2, 7, 10, and 14 of the antisense strand, with the remaining positions being 2'-methoxylated nucleotides.

[0088] In some preferred embodiments, along the 3' end to the 5' end, 2'-fluorinated nucleotides are located at positions 9, 11, 13, and 17 of the sense strand, with the remaining positions being 2'-methoxylated nucleotides; along the 5' end to the 3' end, 2'-fluorinated nucleotides are located at positions 2, 3, 5, 7, 10, 12, and 14 of the antisense strand, GNA-modified nucleotides are located at position 6 of the antisense strand, with the remaining positions being 2'-methoxylated nucleotides.

[0089] In some embodiments, the positive strand of the double-stranded ribonucleic acid contains phosphate thioester groups located at the following positions, in a 5' to 3' orientation:

[0090] Between the first and second nucleotides starting at the 5' end of the positive strand;

[0091] Between the second and third nucleotides starting at the 5' end of the positive strand;

[0092] or,

[0093] The positive chain contains thiophosphate groups located at the following positions:

[0094] Between the first nucleotide starting at the 5' end of the positive strand and the reverse debased deoxyribose residue;

[0095] Between the first and second nucleotides starting at the 5' end of the positive strand;

[0096] Between the first nucleotide starting at the 3' end of the positive strand and the reverse debased deoxyribose residue;

[0097] or,

[0098] The positive chain contains thiophosphate groups located at the following positions:

[0099] Between the first and second nucleotides starting at the 5' end of the positive strand;

[0100] Between the second and third nucleotides starting at the 5' end of the positive strand;

[0101] The first nucleotide starting at the 3' end of the positive strand is between the reverse debased deoxyribose residue.

[0102] In some embodiments, the antisense strand of the double-stranded ribonucleic acid contains phosphate thioester groups located at the following positions, in a 5' to 3' orientation:

[0103] Between the first and second nucleotides starting at the 5' end of the antisense strand;

[0104] Between the second and third nucleotides starting at the 5' end of the antisense strand;

[0105] Between the first and second nucleotides starting at the 3' end of the antisense strand;

[0106] Between the second and third nucleotides starting at the 3' end of the antisense strand.

[0107] In some preferred embodiments, along the 3' end to the 5' end, 2'-fluorinated nucleotides are located at positions 9, 10, 11, and 13 of the sense strand, with the remaining positions being 2'-methoxylated nucleotides; along the 5' end to the 3' end, 2'-fluorinated nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand, with the remaining positions being 2'-methoxylated nucleotides, wherein the 5'-terminal nucleotide of the antisense strand is linked to a 5' phosphate group.

[0108] In some preferred embodiments, along the 3' end to the 5' end, 2'-fluorinated nucleotides are located at positions 9, 10, 11, and 13 of the sense strand, with the remaining positions being 2'-methoxylated nucleotides; along the 5' end to the 3' end, 2'-fluorinated nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand, with the remaining positions being 2'-methoxylated nucleotides, wherein the 5'-terminal nucleotide of the antisense strand is not linked to a 5' phosphate group or a 5' phosphate-derived group.

[0109] In some preferred embodiments, along the 3' end to the 5' end, 2'-fluorinated nucleotides are located at positions 9, 10, 11, and 13 of the sense strand, with the remaining positions being 2'-methoxylated nucleotides; along the 5' end to the 3' end, 2'-fluorinated nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand, with the remaining positions being 2'-methoxylated nucleotides, wherein the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0110] In some preferred embodiments, along the 3' end to the 5' end, 2'-fluorinated nucleotides are located at positions 11, 12, 13, and 15 of the sense strand, with the remaining positions being 2'-methoxylated nucleotides; along the 5' end to the 3' end, 2'-fluorinated nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand, with the remaining positions being 2'-methoxylated nucleotides, wherein the 5'-terminal nucleotide of the antisense strand is not linked to a 5' phosphate group or a 5' phosphate-derived group.

[0111] In some preferred embodiments, along the 3' end to the 5' end, 2'-fluorinated nucleotides are located at positions 11, 12, 13, and 15 of the sense strand, with the remaining positions being 2'-methoxylated nucleotides; along the 5' end to the 3' end, 2'-fluorinated nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand, with the remaining positions being 2'-methoxylated nucleotides, wherein the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0112] In some preferred embodiments, along the 3' end to the 5' end, 2'-fluorinated nucleotides are located at positions 11, 12, 13, and 17 of the sense strand, with the remaining positions being 2'-methoxylated nucleotides; along the 5' end to the 3' end, 2'-fluorinated nucleotides are located at positions 2, 7, 10, and 14 of the antisense strand, with the remaining positions being 2'-methoxylated nucleotides, wherein the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0113] In some preferred embodiments, along the 3' end to the 5' end, 2'-fluorinated nucleotides are located at positions 9, 11, 13, and 17 of the sense strand, with the remaining positions being 2'-methoxylated nucleotides; along the 5' end to the 3' end, 2'-fluorinated nucleotides are located at positions 2, 7, 10, and 14 of the antisense strand, with the remaining positions being 2'-methoxylated nucleotides, wherein the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0114] In some preferred embodiments, along the 3' end to the 5' end, 2'-fluorinated nucleotides are located at positions 6, 11, and 13 of the sense strand, with the remaining positions being 2'-methoxylated nucleotides; along the 5' end to the 3' end, 2'-fluorinated nucleotides are located at positions 2, 7, 10, and 14 of the antisense strand, with the remaining positions being 2'-methoxylated nucleotides, wherein the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0115] In some preferred embodiments, along the 3' end to the 5' end, 2'-fluorinated nucleotides are located at positions 9, 11, 13, and 17 of the sense strand, with the remaining positions being 2'-methoxylated nucleotides, and the first nucleotide at the 3' end is linked to a reverse debased deoxyribose residue; along the 5' end to the 3' end, 2'-fluorinated nucleotides are located at positions 2, 7, 10, and 14 of the antisense strand, with the remaining positions being 2'-methoxylated nucleotides, and the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0116] In some preferred embodiments, along the 3' end to the 5' end, 2'-fluorinated nucleotides are located at positions 9, 11, 13, and 17 of the sense strand, with the remaining positions being 2'-methoxylated nucleotides; along the 5' end to the 3' end, 2'-fluorinated nucleotides are located at positions 2, 3, 5, 7, 10, 12, and 14 of the antisense strand, GNA-modified nucleotides are located at position 6 of the antisense strand, with the remaining positions being 2'-methoxylated nucleotides, wherein the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0117] In some preferred embodiments, along the 3' end to the 5' end, 2'-fluorinated nucleotides are located at positions 11, 12, 13, and 17 of the sense strand, with the remaining positions being 2'-methoxylated nucleotides. The first nucleotide at the 5' end and the first nucleotide at the 3' end are respectively linked to reverse debased deoxyribose residues. Along the 5' end to the 3' end, 2'-fluorinated nucleotides are located at positions 2, 7, 10, and 14 of the antisense strand, with the remaining positions being 2'-methoxylated nucleotides. The 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0118] In some preferred embodiments, along the 3' end to the 5' end, 2'-fluorinated nucleotides are located at positions 11, 12, 13, and 17 of the sense strand, with the remaining positions being 2'-methoxylated nucleotides, and the first nucleotide at the 3' end is linked to a reverse debased deoxyribose residue; along the 5' end to the 3' end, 2'-fluorinated nucleotides are located at positions 2, 7, 10, and 14 of the antisense strand, with the remaining positions being 2'-methoxylated nucleotides, and the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0119] In some preferred embodiments, along the 3' end to the 5' end, 2'-fluorinated nucleotides are located at positions 9, 10, 11, and 13 of the sense strand, with the remaining positions being 2'-methoxylated nucleotides, and the first nucleotide at the 3' end is linked to a reverse debased deoxyribose residue; along the 5' end to the 3' end, 2'-fluorinated nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand, with the remaining positions being 2'-methoxylated nucleotides, and the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0120] In some preferred embodiments, the sense strand and the antisense strand are selected from the following combinations: the sense strand includes the sense strand of any one of the siRNAs shown in Tables 1, 1-1 and 1-2 of this document, and the antisense strand includes the antisense strand of the corresponding siRNA.

[0121] In some preferred embodiments, the siRNA is selected from N-ER-FY033131, N-ER-FY033131M44, N-ER-FY033131M48, N-ER-FY033131M49, N-ER-FY033131M50, N-ER-FY033147, N-ER-FY033147M44, N-ER-FY033147M49, N-ER-FY033147M50, N-ER-FY033151, N-ER-FY033151M44, N-ER-FY033151M49, and N-ER-FY033151M50.

[0122] Secondly, this disclosure provides a double-stranded ribonucleic acid conjugate or a prodrug thereof, wherein the double-stranded ribonucleic acid conjugate comprises a double-stranded ribonucleic acid as provided herein, and a conjugating group conjugated to the double-stranded ribonucleic acid.

[0123] In some embodiments, the conjugation group is attached to the 3' end of the positive chain.

[0124] In some preferred embodiments, the conjugating group is conjugated to the 3' end of the positive chain via a phosphodiester bond.

[0125] In some preferred embodiments, the conjugation group is conjugated to a reverse debased deoxyribose residue at the 3' end of the positive chain via a thiophosphate group.

[0126] In some embodiments, the conjugating group is selected from any one of the group consisting of formulas (I) to (VIII):

[0127] In some embodiments, the double-stranded ribonucleic acid conjugate has a structure as shown in Formula (IX):

[0128] The double helix structure is a double-stranded ribonucleic acid.

[0129] In some embodiments, the double-stranded ribonucleic acid conjugate is an siRNA conjugate.

[0130] In some preferred embodiments, the double-stranded ribonucleic acid conjugate is formed by linking any one of the siRNAs shown in Tables 1, 1-1 and 1-2 to a conjugate group.

[0131] In some preferred embodiments, the sense strand and the antisense strand in the double-stranded RNA conjugate are selected from the following combinations: the sense strand comprises the sense strand of any of the siRNA conjugates shown in Table 2 of this document, and the antisense strand comprises the antisense strand of the corresponding siRNA conjugate.

[0132] In some preferred embodiments, the siRNA conjugate is selected from N-ER-FY033131M44L96, N-ER-FY033131M48L96, N-ER-FY033131M49L96, N-ER-FY033131M50L96, N-ER-FY033147M44L96, N-ER-FY033147M49L96, N-ER-FY033147M50L96, N-ER-FY033151M44L96, N-ER-FY033151M49L96, and N-ER-FY033151M50L96.

[0133] Thirdly, this disclosure provides a pharmaceutical composition comprising the double-stranded ribonucleic acid or the double-stranded ribonucleic acid conjugate or prodrug described herein.

[0134] In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, and optionally includes one or more additional therapeutic agents.

[0135] Fourthly, this disclosure also provides the use of the double-stranded ribonucleic acid, the double-stranded ribonucleic acid conjugate or its prodrug, or the pharmaceutical composition in at least one of the following:

[0136] (1) Inhibit GPR146 gene expression in vivo or in vitro, or prepare a drug for inhibiting GPR146 gene expression;

[0137] (2) To prevent or treat diseases associated with abnormal expression of the GPR146 gene, or to prepare drugs for the prevention or treatment of diseases associated with abnormal expression of the GPR146 gene;

[0138] (3) A medicine for treating subjects with a disease in which they would benefit from reduced GPR146 gene expression, or a medicine for treating subjects with a disease in which they would benefit from reduced GPR146 gene expression.

[0139] In some implementations, the diseases associated with abnormal GPR146 gene expression are selected from the group consisting of: nonspecific orbital inflammation, hypercholesterolemia, atherosclerosis, and coronary artery disease.

[0140] Fifthly, this disclosure also provides a method for inhibiting intracellular GPR146 gene expression in vivo or in vitro, comprising contacting the cells with the double-stranded ribonucleic acid, the double-stranded ribonucleic acid conjugate or its prodrug provided herein, or the pharmaceutical composition provided herein.

[0141] In some implementations, the cells are in vivo cells or in vitro cells.

[0142] In some implementations, the cells are in the subject's body.

[0143] In some implementations, the subject is a mammal, preferably a human.

[0144] In some preferred embodiments, the subject has at least one of the following characteristics:

[0145] Abnormal expression of the GPR146 gene in vivo, more specifically, abnormally high expression of the GPR146 gene;

[0146] Suffering from a disease associated with abnormal expression of the GPR146 gene;

[0147] Having a disease that would benefit from reduced GPR146 gene expression.

[0148] Sixthly, this disclosure also provides double-stranded ribonucleic acid, double-stranded ribonucleic acid conjugates or prodrugs thereof as described herein, or pharmaceutical compositions as described herein, for use as pharmaceuticals.

[0149] In a seventh aspect, this disclosure also provides double-stranded ribonucleic acid, double-stranded ribonucleic acid conjugates or prodrugs thereof as described herein, or pharmaceutical compositions as described herein for treatment.

[0150] Eighthly, this disclosure also provides a method for preventing and / or treating a disease, comprising administering to a subject in need a therapeutically effective amount of the double-stranded ribonucleic acid, the double-stranded ribonucleic acid conjugate or its prodrug as provided herein, or the pharmaceutical composition as provided herein.

[0151] In some implementations, the disease is selected from the group consisting of: nonspecific orbital inflammation, hypercholesterolemia, atherosclerosis, and coronary artery disease.

[0152] Ninthly, this disclosure also provides a kit comprising the double-stranded ribonucleic acid as provided herein, the double-stranded ribonucleic acid conjugate or its prodrug as provided herein, or the pharmaceutical composition as provided herein.

[0153] The effects of the invention

[0154] In some embodiments, the double-stranded ribonucleic acid provided in this disclosure can bind within cells to form an RNA-induced silencing complex (RISC), cleaving the mRNA transcribed from the GPR146 gene, thereby efficiently and specifically inhibiting the expression of the GPR146 gene for the treatment of conditions including nonspecific orbital inflammation, hypercholesterolemia, atherosclerosis, and coronary artery disease.

[0155] Furthermore, the double-stranded ribonucleic acid provided in this disclosure is siRNA. The siRNA targets and binds to and degrades the mRNA of the transcription product of the GPR146 gene, exerting the effect of RNA interference and inhibiting the expression of GPR146 protein. It is a GPR146 inhibitor with high inhibition rate and good specificity.

[0156] In some implementations, the double-stranded ribonucleic acid provided in this disclosure is a modified double-stranded ribonucleic acid, which has the advantage of high stability and is suitable for use in in vivo disease treatment.

[0157] Furthermore, the double-stranded ribonucleic acid provided in this disclosure is a modified siRNA, which has high stability and good inhibitory activity.

[0158] In some embodiments, this disclosure involves attaching a conjugating group to a double-stranded ribonucleic acid (BRNA) to obtain a BRNA conjugate, which can be used for highly efficient targeted delivery to tissues and cells, reducing the impact of BRNA on non-targeted normal tissues and cells, and improving its safety in the treatment of clinical diseases.

[0159] Furthermore, the double-stranded ribonucleic acid conjugate is an siRNA conjugate, which, while maintaining the inhibitory activity and stability of siRNA, also has organ or tissue targeting properties. This can reduce the impact on other tissues or organs and reduce the amount of siRNA molecules used, thereby achieving the goals of reducing toxicity and lowering costs.

[0160] Furthermore, the conjugating group in this disclosure is a group with the structure shown in formula (I) (GalNAc). GalNAc can be used for targeted delivery into liver cells and tissues to efficiently inhibit the expression of the GPR146 gene. In addition, the siRNA conjugate of this disclosure has low toxicity and an excellent drug safety window. Attached Figure Description

[0161] Figure 1 shows the free uptake inhibition rate of siRNA conjugates in human hepatocytes.

[0162] Figure 2 shows the differential gene results of N-ER-FY033131M50L96 after sequencing at a concentration of 5 nM, which conforms to |log2(FoldChange)|>1&padj<0.05.

[0163] Figure 3 shows the differential gene results of N-ER-FY033131M50L96 after sequencing at 50 nM concentration, which conforms to |log2(FoldChange)|>1&padj<0.05.

[0164] Figure 4 shows the differential gene results of N-ER-FY033151M50L96 after sequencing at 100 nM concentration, which conforms to |log2(FoldChange)|>1&padj<0.05.

[0165] Figure 5 shows the inhibition rate of free uptake of the differentially expressed gene SLC16A7 in PHH cells in vitro.

[0166] Figure 6 shows the inhibition rate of free uptake of differentially expressed genes TNRC6A and KIF5B in PHH cells in vitro. Detailed Implementation

[0167] definition

[0168] Unless otherwise stated, the terms used in this disclosure have the following meanings.

[0169] In the claims and / or specification of this disclosure, the words “a”, “an”, or “the” may mean “one”, but may also mean “one or more”, “at least one”, and “one or more”.

[0170] As used in the claims and specification, the words “comprising,” “having,” “including,” or “containing” mean included or open-ended and do not exclude additional, uncited elements or method steps.

[0171] Throughout this application, the term "about" means that a value includes the standard deviation of the error of the apparatus or method used to determine that value. The numerical ranges and parameters used to define this disclosure are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains a standard deviation due to the aforementioned test methods or apparatus. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified by the term "about." Here, "about" generally means an actual value within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range.

[0172] As used in the context of this disclosure, the term "GPR146" refers to G protein-coupled receptor 146, which induces hepatosterol regulatory element-binding protein 2 (SREBP2) by activating extracellular signal-regulated kinase 1 / 2 (ERK1 / 2) signal transduction, thereby leading to the secretion of low-extreme-density lipoprotein (VLDL) in the liver. In this document, the term "GPR146" will include the gene and its mRNA product as well as the corresponding protein product. In some embodiments, the term "GPR146" may also refer to the GPR146 gene (NM_001303474.2) encoding the protein. Repression of GPR146 can refer to the inhibition of the GPR146 protein, the inhibition of the GPR146 gene at the transcriptional level, and / or the inhibition of the enzymatic activity of GPR146.

[0173] The terms "peptide" and "protein" are used interchangeably to refer to a string of at least two amino acid residues linked together by covalent bonds (e.g., peptide bonds), and can be recombinant, natural, or synthetic peptides. Peptides can be linear or branched, can contain modified amino acids, and can be separated by non-amino acid segments. The term also includes amino acid polymers that have been modified (e.g., through disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with labeled components).

[0174] In this art, “G”, “C”, “A”, “T” and “U” generally represent the bases of guanine, cytosine, adenine, thymine and uracil, respectively. However, it is also generally known in the art that each of “G”, “C”, “A”, “T” and “U” generally also represents a nucleotide containing guanine, cytosine, adenine, thymine and uracil as a base, respectively. This is a common practice in representing deoxyribonucleic acid sequences and / or ribonucleic acid sequences. Therefore, in the context of this disclosure, the meanings of “G”, “C”, “A”, “T” and “U” include all the above-mentioned possible cases. However, it should be understood that the term “ribonucleotide” or “nucleotide” can also refer to a modified nucleotide (as further detailed below) or an alternative substitution. In this document, “ribonucleotide”, “ribonucleic acid” and “nucleotide” are used interchangeably, and “deoxyribonucleotide” and “2'-deoxyribonucleotide” are used interchangeably. Those skilled in the art will recognize that guanine, cytosine, adenine, and uracil can be substituted with other moieties without substantially altering the base-pairing properties of an oligonucleotide (including a nucleotide having such a substitution moiety). For example, without limitation, a nucleotide including inosine as its base can base-pair with a nucleotide including adenine, cytosine, or uracil. Therefore, a nucleotide containing uracil, guanine, or adenine can be substituted in the nucleotide sequence of the dsRNA characterized in this disclosure with a nucleotide containing, for example, inosine. In another example, adenine and cytosine anywhere in an oligonucleotide can be substituted with guanine and uracil, respectively, to form a GU swing base pairing with the target mRNA. Sequences containing such substitution moieties are suitable for the compositions and methods characterized in this disclosure.

[0175] In this disclosure, lowercase letters a, u, c, g: represent nucleotides modified with 2'-methoxy groups; Af, Gf, Cf, Uf: represent nucleotides modified with 2'-fluoride groups; lowercase letter s: indicates that the two nucleotides adjacent to the letter s on the left and right are linked by thiophosphate groups; P1: indicates that the nucleotide adjacent to the right of P1 is a 5'-phosphate nucleotide; EVP: indicates that the nucleotide adjacent to the right of EVP is a 5'-trans-vinylphosphonate nucleotide (i.e., a 5'-trans-vinylphosphonate group); (Underlined + Bold + Italic): indicates GNA-modified nucleotides; "(invAb)" indicates reverse debasing deoxyribose residues.

[0176] In this application, "5'-nucleotide" refers to a nucleotide in which the phosphate group is attached to the 5' carbon of a pentose sugar, and it is the main type of nucleotide that exists freely in organisms. "3'-nucleotide" refers to a nucleotide in which the phosphate group is attached to the 3' carbon of a pentose sugar, and may include, for example, adenosine-3'-phosphate, guanosine-3'-phosphate, cytidine-3'-phosphate, uridine-3'-phosphate, 2'-deoxythymidine-3'-phosphate, 2'-O-methyladenosine-3'-phosphate, 2'-O-methyladenosine-3'-thiophosphate, 2'-fluoroadenosine-3'-phosphate, 2'-fluoroadenosine-3'-thiophosphate, 2'-O-methylguanosine-3'-phosphate, and 2'-O-methylguanosine-3'-phosphate. The terms glycoside-3'-thiophosphate, 2'-fluoroguanosine-3'-phosphate, 2'-fluoroguanosine-3'-thiophosphate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methylcytidine-3'-thiophosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluorocytidine-3'-thiophosphate, 2'-O-methyluridine-3'-phosphate, 2'-O-methyluridine-3'-thiophosphate, 2'-fluorouridine-3'-phosphate, 2'-fluorouridine-3'-thiophosphate, and 2'-deoxythymidine-3'-thiophosphate are used. This definition can be applied to modified or unmodified nucleoside phosphoramidamide monomers.

[0177] As used in the context of this disclosure, the terms "double-stranded ribonucleic acid," "double-stranded RNA (dsRNA) molecule," and "dsRNA" are used interchangeably. The term "dsRNA" refers to a complex of ribonucleic acid molecules having a double-stranded structure containing two antiparallel and substantially complementary nucleic acid strands, described as having "sense" and "antisense" orientation relative to a target gene. In some embodiments, double-stranded ribonucleic acid (dsRNA) triggers the degradation of target RNA, such as mRNA, through a post-transcriptional gene silencing mechanism (referred to herein as RNA interference or RNAi).

[0178] Typically, the majority of nucleotides in each strand of a dsRNA molecule are ribonucleotides; however, as detailed herein, each or both strands may also include one or more non-ribonucleotides, such as deoxyribonucleotides and / or modified nucleotides. Additionally, as used herein, “double-stranded ribonucleic acid” can include chemically modified ribonucleotides, phosphate backbones, etc. These modifications can include all types of modifications disclosed herein or known in the art.

[0179] As used in the context of this disclosure, the term "isonucleotide" refers to a compound formed by altering the position of a base on the ribose ring, for example, a compound formed by attaching a base to the 2' or 3' position of the ribose ring instead of the 1' position.

[0180] In some embodiments, the double-stranded RNA disclosed herein is siRNA, which interacts with the mRNA sequence transcribed from the target gene to guide the cleavage of the target RNA. Not wishing to be bound by theory, long double-stranded RNA introduced into the cell is broken down into siRNA by a type III endonuclease called Dicer (Sharp et al., Genes Dev. 2001, 15:485). Dicer (a ribonuclease III-like enzyme) processes dsRNA into short 19–23 base pairs of interfering RNA with a characteristic dibase 3' overhang (Bernstein et al., (2001) Nature 409:363). These siRNAs are then incorporated into the RNA-inducible silencing complex (RISC), where one or more helicases unwind the siRNA duplex, enabling complementary antisense strands to guide target recognition (Nykanen et al., (2001) Cell 107:309). Once bound to a suitable target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir et al., (2001) Genes Dev. 15:188).

[0181] In the foregoing and hereinafter, "2'-fluorinated nucleotide" refers to a nucleotide in which the hydroxyl group at the 2' position of the ribosyl group is replaced by a fluorine group. "Non-fluorinated nucleotide" refers to a nucleotide in which the hydroxyl group at the 2' position of the ribosyl group is replaced by a non-fluorinated group, or, alternatively, a nucleotide analog. In some embodiments, each non-fluorinated nucleotide is independently selected from a nucleotide or nucleotide analog in which the hydroxyl group at the 2' position of the ribosyl group is replaced by a non-fluorinated group. These nucleotides in which the hydroxyl group at the 2' position of the ribosyl group is replaced by a non-fluorinated group are well known to those skilled in the art, and may be selected from 2'-alkoxy-modified nucleotides, 2'-substituted alkoxy-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-substituted alkyl-modified nucleotides, 2'-amino-modified nucleotides, 2'-substituted amino-modified nucleotides, and 2'-deoxy-modified nucleotides.

[0182] "Alkyl" includes straight-chain, branched, or cyclic saturated alkyl groups. For example, alkyl groups include, but are not limited to, methyl, ethyl, propyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, cyclobutyl, n-pentyl, cyclohexyl, and similar groups. For example, "C 1-6 The "C" in "alkyl" 1-6 "" refers to a group consisting of 1, 2, 3, 4, 5 or 6 carbon atoms arranged in a straight chain, branched chain or cyclic form.

[0183] "Alkoxy" herein refers to an alkyl group that is attached to the remainder of a molecule by an oxygen atom (-O-alkyl), wherein the alkyl group is as defined herein. Non-limiting examples of alkoxy groups include methoxy, ethoxy, trifluoromethoxy, difluoromethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, n-pentoxy, etc.

[0184] "Nucleotide analogues" refer to groups that can replace nucleotides in nucleic acids, but whose structure differs from that of adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, or thymine deoxyribonucleotides. Examples include isonucleotides, bridged nucleic acids (BNAs), or acyclic nucleotides.

[0185] BNA refers to a restricted or inaccessible nucleotide. BNA can contain a five-membered, six-membered, or seven-membered ring with a "fixed" C3'-endoglycan condensation bridging structure. This bridge is typically incorporated into the 2'-, 4'-position of the ribose to provide a 2',4'-BNA nucleotide, such as LNA, ENA, cET BNA, etc., where LNA is shown in formula (1), ENA in formula (2), and cET BNA in formula (3).

[0186] Base represents a base.

[0187] Acyclic nucleotides are a class of nucleotides formed by opening the sugar ring of a nucleotide, such as unopened nucleic acids (UNA) or glycerol nucleic acids (GNA). UNA is shown in formula (4), and GNA is shown in formula (5).

[0188] In formulas (4) and (5) above, Base represents a base, and R is selected from H, OH or alkoxy (O-alkyl).

[0189] Heteronucleotides are compounds formed by changing the position of the bases in the ribose ring of a nucleotide. For example, compounds formed by moving the bases from the 1'-position to the 2'-position or 3'-position of the ribose ring, as shown in formula (6) or (7):

[0190] In the compounds of formulas (6) and (7) above, Base represents a base, such as A, U, G, C or T; R is selected from H, OH, F or non-fluorine groups as described above.

[0191] In some embodiments, the nucleotide analogue is selected from one of the following: isonucleotides, LNA, ENA, cET BNA, UNA, and GNA. In some embodiments, each non-fluorinated nucleotide is a 2'-methoxy-modified nucleotide, a GNA-modified nucleotide, or any combination of two or more thereof. In some preferred embodiments, each non-fluorinated nucleotide is a 2'-methoxy-modified nucleotide, wherein, as stated above and below, the 2'-methoxy-modified nucleotide refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribosome with a methoxy group.

[0192] The term "thiophosphate group" refers to a thiophosphate group formed when an oxygen atom in the phosphate diester bond is replaced by a sulfur atom.

[0193] The "thiophosphate group" refers to the following formula:

[0194] The "5'-phosphate nucleotide" refers to the structure of the following formula:

[0195] In the context of this disclosure, the terms "complementary" and "reverse complementary" are used interchangeably and have the meaning known to those skilled in the art: in a double-stranded nucleic acid molecule, the bases of one strand are paired complementaryly with the bases of the other strand. In DNA, the purine base adenine (A) always pairs with the pyrimidine base thymine (T) (or uracil (U) in RNA); the purine base guanine (G) always pairs with the pyrimidine base cytosine (C). Each base pair consists of one purine and one pyrimidine. When adenine on one strand always pairs with thymine (or uracil) on the other strand, and guanine always pairs with cytosine, the two strands are considered complementary, and the sequence of the complementary strand can be inferred from its sequence. Correspondingly, "mismatch" in the art means, in a double-stranded nucleic acid molecule, that the bases at corresponding positions are not paired complementaryly.

[0196] In the preceding and following text, a "nucleotide difference" between two nucleotide sequences refers to a change in the type of bases at the same position of the nucleotides compared to the latter. For example, if a nucleotide base in the latter is A, and the corresponding nucleotide base at the same position in the former is U, C, G, or T, then a nucleotide difference at that position is considered to exist between the two nucleotide sequences. In some embodiments, replacing the nucleotide at the original position with a baseless nucleotide or its equivalent can also be considered a nucleotide difference at that position.

[0197] Unless otherwise specified above and below, "substantially anticomplementary" means that there are no more than 3 base mismatches between the two nucleotide sequences involved; "substantially anticomplementary" means that there are no more than 1 base mismatch between the two nucleotide sequences; and "completely anticomplementary" means that there are no base mismatches between the two nucleotide sequences.

[0198] In this context, a "protruding end" refers to one or more unpaired nucleotides that protrude from the double-stranded structure of a double-stranded RNA (e.g., siRNA) when one 3' end of one strand extends beyond the 5' end of the other strand, or vice versa. A "flat-ended" or "knock-off" term means that there are no unpaired nucleotides at that end of the double-stranded RNA, i.e., no nucleotide protrusions. A "flat-ended double-stranded RNA" is a double-stranded RNA that is double-stranded throughout its entire length, i.e., without nucleotide protrusions at either end of the molecule. A "double-stranded region" refers to the complementary formation of the sense and antisense strands of the siRNA to form its double-stranded region.

[0199] In the context of this disclosure, particularly in describing methods for preparing siRNA, pharmaceutical compositions, or siRNA conjugates, unless otherwise specified, the nucleoside monomer refers to a modified or unmodified nucleoside phosphorus amide monomer used in solid-phase phosphorus amide synthesis, depending on the type and sequence of nucleotides in the desired siRNA or siRNA conjugate. Solid-phase phosphorus amide synthesis is a method known to those skilled in the art for RNA synthesis. All nucleoside monomers used in this disclosure are commercially available.

[0200] In the context of this disclosure, unless otherwise stated, "conjugation" refers to the covalent connection between two or more chemical parts, each having a specific function; correspondingly, "conjugated compound" refers to a compound formed by the covalent connection of these chemical parts. Further, "double-stranded ribonucleic acid conjugate" or "siRNA conjugate" refers to a compound formed by the covalent attachment of one or more chemical parts having a specific function to double-stranded ribonucleic acid or siRNA. Double-stranded ribonucleic acid conjugate or siRNA conjugate should be understood, depending on the context, as a collective term for multiple double-stranded ribonucleic acid conjugates or siRNA conjugates, or a double-stranded ribonucleic acid conjugate or siRNA conjugate represented by a specific chemical formula. In the context of this disclosure, "conjugated molecule" should be understood as a specific compound that can be reactively conjugated to double-stranded ribonucleic acid or siRNA to ultimately form the double-stranded ribonucleic acid conjugate or siRNA conjugate of this disclosure.

[0201] The double-stranded RNA conjugates or siRNA conjugates provided in this disclosure also exist in prodrug form. The prodrugs of the double-stranded RNA conjugates or siRNA conjugates described herein readily undergo chemical changes under physiological conditions to convert into the double-stranded RNA conjugates or siRNA conjugates disclosed herein. Any substance that can be converted in vivo to provide a bioactive substance (i.e., the double-stranded RNA conjugates or siRNA conjugates provided in this disclosure) is a prodrug within the scope and intent of this disclosure. For example, the siRNA modifier or conjugate with the M6 ​​pattern modification in this application is a prodrug of the siRNA modifier or conjugate with the M2 pattern modification, because the difference between the M2 pattern modification and the M6 ​​pattern modification is the presence or absence of P1 at the 5' end of the antisense strand; therefore, double-stranded RNA in this document includes its corresponding prodrug.

[0202] Various hydroxyl protecting groups may be used in this disclosure. Generally, protecting groups insensitize chemical functional groups to specific reaction conditions and can be added to and removed from the functional group in a molecule without substantially impairing the rest of the molecule. In some embodiments, the protecting group is stable under basic conditions but can be removed under acidic conditions. In some embodiments, non-exclusive examples of hydroxyl protecting groups that may be used in this disclosure include monomethoxytriphenylmethyl, 9-phenylxanthine-9-yl (Pixyl), and 9-(p-methoxyphenyl)xanthine-9-yl (Mox). In some embodiments, non-exclusive examples of hydroxyl protecting groups that may be used in this disclosure include Tr (triphenylmethyl), MMTr (4-methoxytriphenylmethyl), DMTr (4,4'-dimethoxytriphenylmethyl), and TMTr (4,4',4”-trimethoxytriphenylmethyl).

[0203] As used herein, “optional” or “optionally” means that the event or condition described thereafter may or may not occur, and the description includes both the possibility that the event or condition may occur and the possibility that it may not occur.

[0204] The term “inhibition” can be used interchangeably with “reduction,” “silence,” “downregulation,” “suppression,” and other similar terms, and includes any level of inhibition.

[0205] The term "suppressing GPR146 gene expression" includes suppressing the expression of any gene encoding GPR146, as well as variants (e.g., naturally occurring variants) or mutants of the GPR146-encoding gene. Therefore, the gene encoding GPR146 can be the gene encoding wild-type GPR146, the gene encoding mutant GPR146, or, in the case of genetically manipulated cells, cell groups, or organisms, a transgenic GPR146-encoding gene.

[0206] "Inhibition of GPR146 gene expression" includes inhibition of the GPR146 gene at any level, such as at least partial inhibition of GPR146 gene expression, such as inhibition of 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%.

[0207] The term "treatment" refers to the process of exposing a subject to (e.g., administering) double-stranded RNA, a double-stranded RNA conjugate, or a prodrug or pharmaceutical composition after the onset of a disease, thereby reducing the symptoms of the disease compared to when the subject is not exposed. It does not imply the complete suppression of the disease symptoms. Having a disease means that the body exhibits symptoms of a disease.

[0208] The term "prevention" means that, prior to the onset of a disease, by exposing (e.g., administering) a subject to the double-stranded RNA, double-stranded RNA conjugate, or a prodrug or pharmaceutical composition disclosed herein, the symptoms of the disease are reduced compared to when the subject is not exposed, without implying the necessity of completely suppressing the disease.

[0209] The term "effective amount" refers to the quantity or dose of the double-stranded RNA, double-stranded RNA conjugate, or prodrug or pharmaceutical composition of this disclosure, which, when administered to a patient in a single or multiple doses, produces the intended effect in a patient requiring treatment or prevention. The effective amount can be readily determined by an attending physician skilled in the art by considering a variety of factors, such as: the species of the mammal; its size, age, and general health; the specific disease involved; the degree or severity of the disease; the individual patient's response; the specific antibody administered; the administration method; the bioavailability characteristics of the administered formulation; the chosen dosing regimen; and the use of any concomitant therapies.

[0210] The term "diseases associated with abnormal GPR146 gene expression" includes diseases, disorders, or conditions that would benefit from reduced GPR146 gene expression. In some implementations, diseases associated with abnormal GPR146 gene expression are selected from the group consisting of: nonspecific orbital inflammation, hypercholesterolemia, atherosclerosis, and coronary artery disease. By way of example, reference is made to the following scientific literature, but not limited to this: Lan She et al. Profile. Arteriosclerosis, Thrombosis, and Vascular Biology. Volume 42, Number 10; Zixuan Wu et al., Elucidating the multifaceted roles of GPR146 in non-specific orbital inflammation a concerted analytical approach through the prisms of bioinformatics and machine learning, Front Med(Lausanne). 2024Jun 5:11:1309510.

[0211] The terms "pharmaceuticalally acceptable excipient" or "pharmaceuticalally acceptable carrier" refer to excipient materials widely used in the pharmaceutical manufacturing industry. The primary purpose of using excipients is to provide a pharmaceutical composition that is safe to use, stable in nature, and / or has specific functionalities, and also to provide a method for the active ingredient to dissolve at a desired rate or to promote the effective absorption of the active ingredient in the body of the administered subject after administration of the drug. Pharmaceutically acceptable excipients can be inert fillers or functional ingredients that provide a function to the pharmaceutical composition (e.g., stabilizing the overall pH of the composition or preventing the degradation of the active ingredient in the composition). Non-limiting examples of pharmaceutically acceptable excipients include, but are not limited to, binders, suspending agents, emulsifiers, diluents (or fillers), granulating agents, adhesives, disintegrants, lubricants, anti-adhesion agents, flow aids, wetting agents, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, sweeteners, etc.

[0212] The pharmaceutical compositions disclosed herein can be prepared using any method known to those skilled in the art. For example, conventional mixing, dissolving, granulation, emulsification, grinding, encapsulation, embedding, and / or lyophilization processes.

[0213] In this disclosure, the application method can be varied or modified in any applicable manner to meet the needs of the properties of the drug, the convenience of patients and medical personnel, and other relevant factors.

[0214] As used in the context of this disclosure, the terms “individual,” “patient,” or “subject” include mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0215] As used in the context of this disclosure, the term "corresponding siRNA" refers to the same siRNA mentioned above. For example, when referring to "the positive strand comprising the positive strand of any of the siRNAs shown in Tables 1, 1-1, and 1-2 herein, and the antisense strand comprising the antisense strand of the corresponding siRNA," it means that the included positive and antisense strands are from the same siRNA shown in Tables 1, 1-1, and 1-2 herein. For example, when the positive strand comprises 5'-CUUUGUGGUUAAAAUACUU-3' (SEQ ID NO:33), the antisense strand comprises 5'-AAGUAUUUUAACCACAAAGUA-3' (SEQ ID NO:34). Similarly, the term "corresponding siRNA conjugate" refers to the same siRNA conjugate mentioned above. For example, when referring to "the positive strand comprising the positive strand of any of the siRNA conjugates shown in Table 2 herein, and the antisense strand comprising the antisense strand of the corresponding siRNA conjugate," it means that the included positive and antisense strands are from the same siRNA conjugate shown in Table 2 herein. Additionally, in these contexts, "contains" includes cases where these sequences are composed of each other.

[0216] Unless otherwise defined or clearly indicated by the context, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0217] Double-stranded RNA

[0218] The first aspect of this disclosure provides a double-stranded ribonucleic acid (dsRNA) for inhibiting GPR146 gene expression. One strand of the dsRNA is an antisense strand, which is complementary to the mRNA sequence formed during the expression of the target gene (i.e., the GPR146 gene) to guide the cleavage of the target mRNA. The other strand of the dsRNA includes a double-stranded region that is partially or completely complementary to the antisense strand.

[0219] In some embodiments, double-stranded ribonucleic acid (dsRNA) is used as a substrate of a digestion enzyme (Dicer) and cleaved into small fragments of dsRNA, i.e., siRNA. In some embodiments, the double-stranded ribonucleic acid is siRNA. siRNA inhibits GPR146 gene expression by assembling to form an RNA-induced silencing complex (RISC) that cleaves the target mRNA. The siRNA disclosed herein contains nucleotide groups as basic structural units, which, as is known to those skilled in the art, contain phosphate groups, ribose groups, and bases. Typically, active, i.e., functional siRNAs are approximately 12-40 nucleotides in length, and in some embodiments, approximately 15-30 nucleotides.

[0220] The double-stranded ribonucleic acid disclosed herein comprises a sense strand and an antisense strand, wherein each nucleotide in the siRNA is independently modified or unmodified, wherein the sense strand contains a nucleotide sequence I, and the antisense strand contains a nucleotide sequence II, wherein nucleotide sequence I and nucleotide sequence II are at least partially anticomplementary to form a double-stranded region. In some embodiments, the length of the double-stranded region is 10-30 nucleotide pairs. For example, the length of the double-stranded region is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotide pairs.

[0221] Preferably, the length of the double-stranded region is 17-25 nucleotides. More preferably, the length of the double-stranded region is 19-23 nucleotides. Even more preferably, the length of the double-stranded region is 19, 20, 21, 22 or 23 nucleotides.

[0222] In some embodiments, the sense strand further contains nucleotide sequence III, and the antisense strand further contains nucleotide sequence IV, each nucleotide sequence III and nucleotide sequence IV being independently 0-9 nucleotides in length. Nucleotide sequence III is attached to the 5' end of nucleotide sequence I, and nucleotide sequence IV is attached to the 3' end of nucleotide sequence II. Nucleotide sequence III and nucleotide sequence IV are of equal length and are substantially anticomplementary or completely anticomplementary. Substantially anticomplementary means that there is no more than one base mismatch between the two nucleotide sequences; completely anticomplementary means that there is no mismatch between the two nucleotide sequences.

[0223] In some embodiments, the sense strand further contains nucleotide sequence III, and the antisense strand further contains nucleotide sequence IV, each nucleotide sequence III and nucleotide sequence IV being 0-9 nucleotides in length independently. Nucleotide sequence III is attached to the 3' end of nucleotide sequence I, and nucleotide sequence IV is attached to the 5' end of nucleotide sequence II. Nucleotide sequence III and nucleotide sequence IV are of equal length and are substantially anticomplementary or completely anticomplementary. Substantially anticomplementary means that there is no more than one base mismatch between the two nucleotide sequences; completely anticomplementary means that there is no mismatch between the two nucleotide sequences.

[0224] In some embodiments, the sense strand further contains nucleotide sequence III, and the antisense strand further contains nucleotide sequence IV, each of which is independently 0-9 nucleotides in length. Nucleotide sequence III is attached to the 5' end of nucleotide sequence I, and nucleotide sequence IV is attached to the 3' end of nucleotide sequence II. Nucleotide sequences III and IV are of equal length and are substantially anticomplementary or completely anticomplementary. Alternatively, nucleotide sequence III is attached to the 3' end of nucleotide sequence I, and nucleotide sequence IV is attached to the 5' end of nucleotide sequence II. Nucleotide sequences III and IV are of equal length and are substantially anticomplementary or completely anticomplementary. "Substantially anticomplementary" means that there is no more than one base mismatch between the two nucleotide sequences; "completely anticomplementary" means that there is no mismatch between the two nucleotide sequences.

[0225] In some embodiments, the positive strand further contains nucleotide sequence V and / or the antisense strand further contains nucleotide sequence VI, wherein nucleotide sequences V and VI are each independently 0-2 nucleotides in length, with nucleotide sequence V attached to the 3' end of the positive strand to form a 3' overhang, and / or nucleotide sequence VI attached to the 3' end of the antisense strand to form a 3' overhang. In some embodiments, the length of nucleotide sequence V or VI is each 2 nucleotides. In other embodiments, along the 5' end to the 3' end, the nucleotide sequence V or VI is UU, AU, UA, AG, AA, AC, CU, or CA. In still other embodiments, the nucleotide sequence V is identical to or differs from the nucleotide at the corresponding position on the target mRNA, or the nucleotide sequence VI is mismatched or complementary to the nucleotide at the corresponding position on the target mRNA.

[0226] The sense and antisense strands disclosed herein may be of the same or different lengths. In some embodiments, the sense or antisense strand has 15-30 nucleotides. In other embodiments, the sense or antisense strand has 17-25 nucleotides. In still other embodiments, the sense or antisense strand has 19-23 nucleotides. More preferably, the sense or antisense strand has 19, 20, 21, 22, or 23 nucleotides. The length ratio of the sense and antisense strands of the siRNA disclosed herein can be 15 / 15, 16 / 16, 17 / 17, 18 / 18, 19 / 19, 19 / 20, 19 / 21, 19 / 22, 19 / 23, 20 / 19, 20 / 20, 20 / 21, 20 / 22, 20 / 23, 21 / 19, 21 / 20, 21 / 21, 21 / 22, 21 / 23. Examples of possible siRNA length ratios include 22 / 19, 22 / 20, 22 / 21, 22 / 22, 22 / 23, 23 / 19, 23 / 20, 23 / 21, 23 / 22, 23 / 23, 24 / 24, 25 / 25, 26 / 26, 27 / 27, 28 / 28, 29 / 29, 30 / 30, 22 / 24, 22 / 25, 22 / 26, 23 / 24, 23 / 25, or 23 / 26. In some embodiments, the length ratio of the sense strand to the antisense strand is 19 / 19, 21 / 21, 19 / 21, 21 / 23, or 23 / 23, in which case the siRNA of this disclosure exhibits better cellular mRNA silencing activity.

[0227] Studies have found that different modification strategies can have drastically different effects on indicators such as the stability, biological activity, and cytotoxicity of double-stranded RNA. For example, CN102140458B investigated various chemical modification strategies for siRNA, confirming seven effective modification methods. Compared with unmodified siRNA, one of the modification methods resulted in siRNA that improved blood stability while maintaining inhibitory activity essentially equivalent to that of unmodified siRNA.

[0228] The nucleotides in the double-stranded RNA disclosed herein are each independently modified or unmodified nucleotides. In some embodiments, each nucleotide in the double-stranded RNA of this disclosure is an unmodified nucleotide; in other embodiments, some or all of the nucleotides in the double-stranded RNA of this disclosure are modified nucleotides, and these modifications on the nucleotide groups do not result in a significant weakening or loss of the function of the double-stranded RNA of this disclosure in inhibiting GPR146 gene expression.

[0229] In some embodiments, the double-stranded ribonucleic acid of this disclosure contains at least one modified nucleotide. In the context of this disclosure, the term "modified nucleotide" refers to a nucleotide or nucleotide analog formed by replacing the 2' hydroxyl group of the ribosyl group with another group, or a nucleotide having a modified base. The modified nucleotide does not cause a significant reduction or loss of the double-stranded ribonucleic acid's function in suppressing gene expression. For example, the modified nucleotide disclosed in JKWatts, G.F. Deleavey, and MJDamha, Chemically Modified siRNA: Tools and Applications. Drug Discov Today, 2008, 13(19-20):842-55 may be selected.

[0230] In some embodiments, at least one nucleotide in the sense strand or antisense strand of the double-stranded ribonucleic acid provided in this disclosure is a modified nucleotide, and / or at least one phosphate ester group is a phosphate ester group with a modifying group; in other words, at least a portion of the phosphate ester group and / or ribosome in the phosphate-sugar backbone of at least one single strand of the sense strand and the antisense strand is a phosphate ester group with a modifying group and / or a ribosome with a modifying group. In some embodiments, the phosphate ester group containing the modifying group is a thiophosphate ester group formed by replacing an oxygen atom in the phosphodiester bond of the phosphate ester group with a sulfur atom.

[0231] In some implementations, when the nucleotide sequences of the sense and antisense strands are complementary to form a double-stranded region, and there is no protruding nucleotide at the 3' end of the sense strand, a nucleotide sequence V is added to the 3' end of the sense strand as the protruding nucleotide. Then, after the nucleotide sequence formed by linking nucleotide sequence V to the 3' end of the sense strand is chemically modified, nucleotide sequence V is excluded, and correspondingly, the sense strand of the double-stranded ribonucleic acid forms a blunt end.

[0232] In some implementations, when the nucleotide sequences of the sense strand and the antisense strand are complementary to form a double-stranded region, and the 3' end of the sense strand has a protruding nucleotide extending out of the double-stranded region, the protruding nucleotide at the 3' end of the sense strand is excluded and the nucleotide sequence of the sense strand is obtained. Accordingly, the sense strand of the double-stranded ribonucleic acid forms a blunt end.

[0233] In some embodiments, the 5' terminal nucleotide of the antisense strand is linked to a 5' phosphate group or a 5' phosphate-derived group.

[0234] When the 5' terminal nucleotide of the antisense strand is attached to a 5' phosphate group or a 5' phosphate derivative, the following structure is formed:

[0235] Formula (8) shows a structure formed by linking a 5' nucleotide to a 5' phosphate group, Formula (9) shows a structure formed by linking a 5' nucleotide to a 5' phosphate-derived group (EVP), and Formula (10) shows a structure formed by linking a 5' nucleotide to a 5' phosphate-derived group (5' methylene phosphate group); where Base represents a base, such as A, U, G, C, or T. R' is a hydroxyl group or is substituted by various groups known to those skilled in the art, for example, the substituted modified nucleotide can be a 2'-fluoro(2'-F) modified nucleotide, a 2'-alkoxy modified nucleotide, a 2'-substituted alkoxy modified nucleotide, a 2'-alkyl modified nucleotide, a 2'-substituted alkyl modified nucleotide, a 2'-amino modified nucleotide, a 2'-substituted amino modified nucleotide, or a 2'-deoxy modified nucleotide.

[0236] In some embodiments, the 5' terminal nucleotide of the antisense strand is not linked to a 5' phosphate group, a 5' phosphate-derived group, or a reverse debased deoxyribose residue (invAb), and its structure is shown in formula (X):

[0237] Wherein, Base represents a base, such as A, U, G, C, or T. R' is a hydroxyl group or is substituted by various groups known to those skilled in the art. For example, the substituted modified nucleotide can be a 2'-fluoro (2'-F) modified nucleotide, a 2'-alkoxy modified nucleotide, a 2'-substituted alkoxy modified nucleotide, a 2'-alkyl modified nucleotide, a 2'-substituted alkyl modified nucleotide, a 2'-amino modified nucleotide, a 2'-substituted amino modified nucleotide, or a 2'-deoxy modified nucleotide.

[0238] In some specific embodiments, the double-stranded ribonucleic acid is selected from any siRNA shown in Tables 1, 1-1, and 1-2. In some specific embodiments, the siRNA of this disclosure has an inhibition rate of at least about 10% against the GPR146 gene, and may be at least about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or any value or range between these values.

[0239] The double-stranded ribonucleic acid disclosed herein has high specificity in binding to target mRNA and good target mRNA silencing activity. It can significantly inhibit the expression of the GPR146 gene and can be used to treat conditions including nonspecific orbital inflammation, hypercholesterolemia, atherosclerosis, and coronary artery disease.

[0240] In some embodiments, this disclosure provides an siRNA composition comprising any one or more of the siRNAs shown in Tables 1, 1-1 and 1-2.

[0241] In some embodiments, each nucleotide of the sense strand is independently a modified or unmodified nucleotide. In some embodiments, each nucleotide of the antisense strand is independently a modified or unmodified nucleotide.

[0242] In some embodiments, any two nucleotides linked in the sense strand are connected by a phosphodiester bond or a thiophosphate group. In some embodiments, any two nucleotides linked in the antisense strand are connected by a phosphodiester bond or a thiophosphate group.

[0243] The double-stranded ribonucleic acid disclosed herein includes a modification of at least one nucleotide. In some embodiments, the nucleotide-modified siRNA can improve the stability of the siRNA while maintaining high GPR146 mRNA repressive activity.

[0244] In some embodiments, the nucleotide modification is selected from at least one of ribose group modification and base modification. In some embodiments, "nucleotide modification" refers to a nucleotide or nucleotide analog formed by replacing the 2' hydroxyl group of the ribose group with another group, or a nucleotide in which the bases on the nucleotide are modified bases. The nucleotide modification does not result in a significant weakening or loss of the siRNA's ability to suppress gene expression. For example, modified nucleotides disclosed in JKWatts, G.F. Deleavey, and MJDamha, Chemically Modified siRNA: Tools and Applications. Drug Discov Today, 2008, 13(19-20):842-55 can be selected. Nucleotide modification can improve the stability of siRNA and maintain its high repressive efficiency against the GPR146 gene.

[0245] For example, the modified nucleotide has the following structure:

[0246] Wherein, Base represents a base, such as A, U, G, C, or T. The hydroxyl group at the 2' position of the ribosome is replaced by R. These hydroxyl groups at the 2' position of the ribosome can be replaced by various groups known to those skilled in the art, for example, the substituted modified nucleotide can be a 2'-fluoro(2'-F) modified nucleotide, a 2'-alkoxy modified nucleotide, a 2'-substituted alkoxy modified nucleotide, a 2'-alkyl modified nucleotide, a 2'-substituted alkyl modified nucleotide, or a 2'-deoxyribonucleotide.

[0247] In some implementations, the 2'-alkoxy modified nucleotide is a 2'-methoxy (2'-OMe, 2'-O-CH3) modified nucleotide, etc.

[0248] In some embodiments, the 2'-substituted alkoxy modified nucleotide is a nucleotide modified with 2'-methoxyethoxy (2'-O-CH2-CH2-O-CH3), a nucleotide modified with 2'-O-CH2-CH=CH2, etc.

[0249] In some embodiments, the 2'-substituted alkyl-modified nucleotide is a 2'-CH2-CH2-CH=CH2 modified nucleotide, etc.

[0250] In some embodiments, the modification of the nucleotide is a modification of the bases. Base modifications can be of various types known to those skilled in the art. Exemplarily, base modifications include, but are not limited to, m 6 A、Ψ、m 1 A、m 5 A, ms 2 i 6 A、i 6 A、m 3 C, m 5 C、ac 4 C, m 7 G, m 2,2 G, m 2 G, m 1 G, Q, m 5 U、mcm 5 U、ncm 5 U、ncm 5 Um, D, mcm 5 s 2 U, Inosine(I), hm 5 C, s 4 U、s 2 U, azobenzene, Cm, Um, Gm, t 6 A, yW, ms 2 t 6 A or its derivatives.

[0251] In some implementations, nucleotide analog modification refers to the replacement of nucleotides in nucleic acids with nucleotide analogs. Exemplarily, nucleotide analogs are selected from isonucleotides, LNA, ENA, cET-BNA, UNA, or GNA. For example, "GNA-modified nucleotide" means that the nucleotide is replaced with GNA.

[0252] In some embodiments, the nucleotides in the nucleic acid are replaced with isonucleotides, also referred to as isonucleotide modification in the context of this disclosure. In some embodiments, isonucleotide modification includes incorporating an isonucleotide at one or more sites on the sense and / or antisense strands of the siRNA to be modified, in place of the native nucleoside for coupling at the corresponding position.

[0253] In some embodiments, the isonucleoside modification is D-isonucleoside modification. In other embodiments, the isonucleoside modification is L-isonucleoside modification. In still other embodiments, the isonucleoside modification is a combination of D-isonucleoside and L-isonucleoside modification.

[0254] In some implementations, the double-stranded ribonucleic acid includes at least one of the following chemical modifications:

[0255] (1) Modification of at least one nucleotide in the positive strand,

[0256] (2) Modification of the phosphodiester bond at at least one position in the positive chain.

[0257] (3) The first nucleotide at the 5' end and / or the first nucleotide at the 3' end of the positive strand are linked to a reverse debased deoxyribose residue.

[0258] (4) Modification of at least one nucleotide in the antisense strand,

[0259] (5) Modification of the phosphodiester bond at at least one position in the antisense chain.

[0260] Furthermore, the double-stranded ribonucleic acid is a siRNA containing at least one of the chemical modifications in (1)-(5).

[0261] In some implementations, all nucleotides in the sense strand and / or the antisense strand are modified nucleotides.

[0262] In some embodiments, each nucleotide in the sense strand and the antisense strand is independently a 2'-fluoro-modified nucleotide or a non-fluoro-modified nucleotide. In some embodiments, each non-fluoro-modified nucleotide is a 2'-methoxy-modified nucleotide or a GNA-modified nucleotide, wherein the 2'-methoxy-modified nucleotide refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribosome with a methoxy group.

[0263] In some embodiments, at least one of the following nucleotide linkages in the double-stranded ribonucleic acid is a phosphate thioester linkage:

[0264] The connection between the first and second nucleotides starting at the 5' end of the positive strand;

[0265] The connection between the second and third nucleotides starting at the 5' end of the positive strand;

[0266] The link between the first nucleotide starting at the 5' end of the positive strand and the reverse debased deoxyribose residue;

[0267] The link between the first nucleotide starting at the 3' end of the positive strand and the reverse debased deoxyribose residue;

[0268] The link between the first and second nucleotides starting at the 5' end of the antisense strand;

[0269] The link between the second and third nucleotides starting at the 5' end of the antisense strand;

[0270] The link between the first and second nucleotides starting at the 3' end of the antisense strand;

[0271] The link between the second and third nucleotides starting at the 3' end of the antisense strand.

[0272] In some alternative implementations, the justice chain includes or consists of any of the justice chains shown in Tables 1, 1-1 and 1-2, and the antisense chain includes or consists of any of the antisense chains shown in Tables 1, 1-1 and 1-2.

[0273] In some implementations, double-stranded ribonucleic acid includes, but is not limited to, siRNAs as shown in Tables 1, 1-1 and 1-2.

[0274] In some specific embodiments, the siRNA disclosed herein has an inhibition rate of at least about 10% against the GPR146 gene, and may be at least about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or any value or range between these values.

[0275] Double-stranded ribonucleic acid conjugates or their prodrugs

[0276] This disclosure also provides a double-stranded ribonucleic acid conjugate, which is obtained by conjugating and linking the double-stranded ribonucleic acid provided in this disclosure with a conjugating group.

[0277] In this disclosure, the sense strand and antisense strand of the double-stranded ribonucleic acid conjugate form the double-stranded region of the double-stranded ribonucleic acid conjugate.

[0278] In some preferred embodiments, the double-stranded ribonucleic acid conjugate is an siRNA conjugate, which is obtained by conjugating siRNA with a conjugating group. Specifically, the sense and antisense strands of the siRNA are complementary to form the double-stranded region of the siRNA, and the conjugating group is conjugated to the 3' end of the sense strand to form the siRNA conjugate.

[0279] In some preferred embodiments, the 3' end of the positive strand of the siRNA has a protruding nucleotide extending into a double-stranded region. The sequence with a blunt 3' end formed after excluding the protruding nucleotide at the 3' end of the positive strand is used as the nucleotide sequence for attaching the conjugate group. The conjugate group is attached to the blunt 3' end of the positive strand to form an siRNA conjugate.

[0280] In this disclosure, "the conjugation group is attached to the 3' end of the motif-sensing chain" includes the case where the 3' end of the motif-sensing chain originally has a blunt end, and the case where the 3' end of the motif-sensing chain has a protruding end, and the protruding end is excluded to form a blunt end.

[0281] In some preferred embodiments, when the nucleotide sequences of the sense and antisense strands are complementary to form a double-stranded region, and there is no protruding nucleotide at the 3' end of the sense strand, a nucleotide sequence V is added to the 3' end of the sense strand as the protruding nucleotide. The sequence with a blunt 3' end formed after excluding the protruding nucleotide at the 3' end of the sense strand is used as the nucleotide sequence for attaching the conjugate group. The conjugate group is attached to the blunt 3' end of the sense strand to form an siRNA conjugate.

[0282] In some implementations, when the nucleotide sequences of the sense and antisense strands are complementary to form a double-stranded region, and the 3' end of the sense strand has a protruding nucleotide extending out of the double-stranded region, the sequence with a blunt 3' end formed after excluding the protruding nucleotide at the 3' end of the sense strand is used as the nucleotide sequence for connecting the conjugate group. The conjugate group is then connected to the blunt 3' end of the sense strand to form an siRNA conjugate.

[0283] In some preferred embodiments, the double-stranded ribonucleic acid conjugate is an siRNA conjugate, wherein the siRNA molecule linked to the conjugate group in the siRNA conjugate can be unmodified siRNA or modified siRNA. The siRNA molecule linked to the conjugate group maintains high inhibitory activity and stability while also exhibiting good tissue and organ targeting and the ability to promote endocytosis, thus reducing the impact on other tissues or organs and decreasing the amount of siRNA molecule used, thereby achieving the goals of reducing toxicity and lowering costs. Optionally, any one of the siRNA molecules shown in Tables 1, 1-1, and 1-2 can be linked to the conjugate group to obtain the double-stranded ribonucleic acid conjugate.

[0284] Generally, the conjugation group comprises at least one pharmaceutically acceptable target group, or further comprises a linker, and the siRNA, the linker, and the target group are sequentially linked. In some embodiments, there are 1-6 target groups. In some embodiments, there are 2-4 target groups. The siRNA molecule can be non-covalently or covalently conjugated to the conjugation group, for example, it can be covalently conjugated to the conjugation group. The conjugation site of the siRNA and the conjugation group can be at the 3' end or 5' end of the siRNA's sense strand, at the 5' end of the antisense strand, or within the siRNA's internal sequence. In some embodiments, the conjugation site of the siRNA and the conjugation group is at the 3' end of the siRNA's sense strand.

[0285] In some embodiments, the conjugation group may be attached to a phosphate group, a 2'-hydroxyl group, or a base of a nucleotide. In some embodiments, the conjugation group may also be attached to a 3'-hydroxyl group, in which case the nucleotides are linked by a 2',5'-phosphodiester bond. When the conjugation group is attached to the end of the siRNA chain, it is usually attached to a phosphate group of the nucleotide; when the conjugation group is attached to the inner sequence of the siRNA, it is usually attached to a ribose ring or a base. In some embodiments, the first nucleotide at the 3' end of the positive strand is attached to a reverse debased deoxyribose residue, and the 3' end of the reverse debased deoxyribose residue is attached to the conjugation group. Various connection methods can be found in the reference: Muthiah Manoharan et al. siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo in hepatocytes. ACS Chemical biology, 2015, 10(5): 1181-7.

[0286] In some embodiments, the positive strand may contain one or more capping residues or portions, sometimes referred to in the art as a “cap,” “terminal cap,” or “capping residue.” As used herein, a “capping residue” is a nonnucleotide compound or other portion that may be incorporated at one or more ends of the nucleotide sequence of the siRNA disclosed herein. In some cases, capping residues may provide certain beneficial properties to the siRNA, such as protection against exonuclease degradation. In some embodiments, an inverse debased deoxyribose residue (invAb) is added as a capping residue. In some embodiments, the capping residue appears at the 5' end, the 3' end, or both the 5' and 3' ends of the positive strand.

[0287] In some embodiments, one or more inverted debased deoxyribose residues (invAb) are added to the 3' end of the positive strand. In some embodiments, one or more inverted debased deoxyribose residues (invAb) are added to the 5' end of the positive strand. In some embodiments, one or more inverted debased deoxyribose residues (invAb) are added to both the 5' end and the 3' end of the positive strand. The inverted debased deoxyribose residues may be linked via phosphodiester bonds, thiophosphate groups, or other nucleoside internucleotide bonds. When describing modification sites in modification methods, (invAb) is not counted as the first site of the sequence. In some embodiments, inverted debased deoxyribose residues (invAb) (also referred to in the art as "inverted debasing sites") may be added. Wherein, when (invAb) is located at the 3' end of the siRNA, its structure is Formula B; when (invAb) is located at the 5' end of the siRNA, its structure is Formula C:

[0288] In some embodiments, the siRNA and the conjugate group are linked by acid-labile or reducible chemical bonds. These bonds are degradable in the acidic environment of the endosomes, thus freeing the siRNA. For non-degradable conjugates, the conjugate group can be attached to the positive and negative strands of the siRNA to minimize the impact of the conjugate on the siRNA's activity.

[0289] In some embodiments, the conjugation group may be a ligand commonly used in the field of siRNA drug delivery, such as the various ligands described in WO2009082607A2, which are incorporated herein by reference in their entirety.

[0290] In some embodiments, the conjugation group may be selected from one or more ligands formed from the following targeting molecules or their derivatives: lipophilic molecules, such as cholesterol, bile acids, vitamins (e.g., vitamin E), lipid molecules of different chain lengths; polymers, such as polyethylene glycol; polypeptides, such as transmembrane peptides; aptamers; antibodies; quantum dots; carbohydrates, such as lactose, polylactose, mannose, galactose, N-acetylgalactosamine (GalNAc); folic acid; receptor ligands expressed by hepatocytes, such as desialyl glycoprotein, desialyl sugar residues, lipoproteins (e.g., high-density lipoprotein, low-density lipoprotein, etc.), glucagon, neurotransmitters (e.g., adrenaline), growth factors, transferrin, etc.

[0291] In some embodiments, each ligand is independently selected from a ligand capable of binding to a cell surface receptor. In some embodiments, at least one ligand is capable of binding to a hepatocyte surface receptor. In some embodiments, at least one ligand is capable of binding to a mammalian cell surface receptor. In some embodiments, at least one ligand is capable of binding to a human hepatocyte surface receptor. In some embodiments, at least one ligand is capable of binding to the liver surface desialylate glycoprotein receptor (ASGPR). The types of these ligands are well known to those skilled in the art, and their function is generally to bind to specific receptors on the surface of target cells, mediating the delivery of ligand-linked siRNA to the target cells.

[0292] In some embodiments, the pharmaceutically acceptable targeting group can be any ligand that binds to the desialyl glycoprotein receptor (ASGPR) on the surface of mammalian hepatocytes. In some embodiments, each ligand is independently a desialyl glycoprotein, such as asialolesomucoid (ASOR) or asialofetin (ASF). In some embodiments, the ligand is a sugar or a sugar derivative.

[0293] In some embodiments, at least one ligand is a sugar. In some embodiments, each ligand is a sugar. In some embodiments, at least one ligand is a monosaccharide, polysaccharide, modified monosaccharide, modified polysaccharide, or sugar derivative. In some embodiments, at least one of the ligands may be a monosaccharide, disaccharide, or trisaccharide. In some embodiments, at least one ligand is a modified sugar. In some embodiments, each ligand is a modified sugar. In some embodiments, each ligand is independently selected from polysaccharides, modified polysaccharides, monosaccharides, modified monosaccharides, polysaccharide derivatives, or monosaccharide derivatives. In some embodiments, each or at least one ligand is selected from the group consisting of glucose and its derivatives, mannan and its derivatives, galactose and its derivatives, xylose and its derivatives, ribose and its derivatives, fucose and its derivatives, lactose and its derivatives, maltose and its derivatives, arabinose and its derivatives, fructose and its derivatives, and sialic acid.

[0294] In some embodiments, each of the ligands may be independently selected from D-mannose, L-mannose, D-arabinose, D-xylfuranose, L-xylfuranose, D-glucose, L-glucose, D-galactose, L-galactose, α-D-mannose, β-D-mannose, α-D-mannose, β-D-mannose, α-D-glucose, β-D-glucose, α-D-glucose, β-D-glucose Sugars, α-D-furanose glucose, α-D-furanose glucose, α-D-furanose fructose, α-D-fructose pyranose, α-D-galactopyranose, β-D-galactopyranose, α-D-furanose galactopyranose, β-D-furanose galactopyranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-butyrylgalactosamine, N-isobutyrylgalactosamine 2-Amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-carboxamido-2,3-di-O-methyl-D-mannpyranose, 2-deoxy-2-sulfonamido-D-glucopyranose, N-ethanolyl-α-neuraminic acid, 5-thio-β-D-glucopyranose, 2, 3,4-Tri-O-acetyl-1-thio-6-O-triphenylmethyl-α-D-glucopyranoside methyl ester, 4-thio-β-D-galactopyranose, 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-glucopyranoside ethyl ester, 2,5-dehydrated-D-aloxonitrile, ribose, D-ribose, D-4-thioribose, L-ribose, or L-4-thioribose. Other options for the ligands may be found, for example, in CN105378082A, which is incorporated herein by reference in its entirety.

[0295] The targeting group can be linked to the siRNA molecule via a suitable adapter. Those skilled in the art can select a suitable adapter based on the specific type of the targeting group. For details on these adapters, the types of targeting groups, and the connection methods with siRNA, please refer to the disclosure of WO2015006740A2, which is incorporated herein by reference in its entirety.

[0296] In some specific embodiments, the conjugation group is selected from any one of the group consisting of formulas (I) to (VIII) above.

[0297] Among them, the conjugation group shown in formula (I) is GalNAc. GalNAc has liver targeting and can deliver siRNA molecules to liver tissue with high specificity, specifically inhibiting the high expression of GPR146 gene in the liver.

[0298] In some specific implementations, GalNAc is conjugated to the 3' end of the positive strand via a phosphodiester bond to obtain a double-stranded ribonucleic acid conjugate as shown in formula (IX):

[0299] The double helix structure is a double-stranded ribonucleic acid, preferably unmodified siRNA or modified siRNA.

[0300] In some implementations, the double-stranded ribonucleic acid conjugates include, but are not limited to, siRNA conjugates as shown in Table 2.

[0301] In some specific embodiments, the siRNA conjugate of this disclosure has an inhibition rate of at least about 10% against the GPR146 gene, and may be at least about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or any value or range between these values.

[0302] siRNA synthesis methods

[0303] Nucleoside monomers are sequentially linked from the 3'-5' direction according to the nucleotide arrangement using the conventional solid-phase phosphoramide method. Each linkage of a nucleoside monomer involves four steps: deprotection, coupling, oxidation or sulfidation, and capping. Specifically, when two nucleotides are linked by a phosphodiester bond, the linkage of the subsequent nucleoside monomer involves these four steps. When two nucleotides are linked by a thiophosphate group, the linkage of the subsequent nucleoside monomer involves these four steps.

[0304] This invention selects nucleotide monomers based on the target sequence for synthesis. The selected nucleotide monomers are those commonly used by those skilled in the art. For example, the nucleotide monomer for synthesizing A can be, but is not limited to, adenosine-3-phosphate. It should be understood that these monomers, when present in oligonucleotides, are interconnected via 5′-3′ phosphodiester bonds or 5′-3′ thiophosphate groups. When, for example, the 3′ position of the last nucleotide in the 5′ to 3′ direction is a hydroxyl group, this is achieved using conventional methods in the art.

[0305] For example, the synthesis conditions for the siRNA disclosed herein can be as follows:

[0306] The nucleoside monomer was provided as a 0.1 M acetonitrile solution, and the deprotection reaction conditions were identical for each step. The deprotection conditions included: a reaction temperature of 25 °C, a reaction time of 70 seconds, a deprotecting agent selected from a dichloromethane solution of dichloroacetic acid (3% v / v), and a molar ratio of the deprotecting agent to the 4,4'-dimethoxytriphenylmethyl protecting group on the solid support of 5:1.

[0307] The coupling reaction conditions were identical for each step. The coupling reaction conditions included: a reaction temperature of 25°C, a molar ratio of nucleic acid sequence to nucleoside monomer linked on the solid-phase support of 1:10, a molar ratio of nucleic acid sequence to coupling reagent linked on the solid-phase support of 1:65, a reaction time of 600 seconds, and the coupling reagent being a 0.5M acetonitrile solution of 5-ethylthio-1H-tetrazole (ETT).

[0308] The oxidation reaction conditions were identical for each step. These conditions included a reaction temperature of 25°C, a reaction time of 15 seconds, and the oxidizing agent being 0.05M iodine solution. The molar ratio of iodine to the nucleic acid sequence linked on the solid-phase support in the coupling step was 30:1. The reaction was carried out in a mixed solvent of tetrahydrofuran:water:pyridine = 3:1:1.

[0309] The conditions for each step of the sulfidation reaction are identical. These conditions include: a reaction temperature of 25°C, a reaction time of 300 seconds, the sulfidation reagent being selected from hydroflavin, and a molar ratio of the sulfidation reagent to the nucleic acid sequence linked on the solid-phase support in the coupling step of 120:1. The reaction is carried out in a mixed solvent of acetonitrile:pyridine = 1:1.

[0310] The capping conditions were identical for each step. The capping reaction conditions included: a reaction temperature of 25°C, a reaction time of 15 seconds, and the capping reagent being a 1:1 mixture of CapA (10% acetic anhydride / acetonitrile solution) and CapB (10% N-methylimidazolium pyridine / acetonitrile solution). The molar ratio of the capping reagent to the nucleic acid sequence linked on the solid-phase support was acetic anhydride:N-methylimidazolium:the nucleic acid sequence linked on the solid-phase support was 1:1:1.

[0311] After the last nucleoside monomer is ligated, the nucleic acid sequence ligated on the solid-phase carrier is subjected to ammonolysis, purification, and desalting, followed by freeze-drying to obtain the siRNA sense and antisense strands. Finally, the two strands are heated and annealed to obtain the product.

[0312] Methods for ammonolysis, purification, desalting, and annealing are well known in the art. For example, ammonolysis is performed by contacting the nucleotide sequence linked to a solid-phase support with concentrated ammonia; purification is performed by chromatography; desalting is performed by reversed-phase chromatography; and cooling is performed by gradually cooling after mixing sense and antisense strands in equimolar ratios under different stringent conditions.

[0313] Taking L96, the conjugating group shown in formula (I), as an example, the siRNA conjugation synthesis method is as follows:

[0314] The first step involves reacting DMTr-L96 with succinic anhydride to obtain compound L96-A:

[0315] Preparation process: DMTr-L96, succinic anhydride, 4-dimethylaminopyridine, and diisopropylethylamine were added to dichloromethane and stirred at 25°C for 24 hours. The reaction solution was then washed with 0.5M triethylamine phosphate, and the aqueous phase was washed three times with dichloromethane. The combined organic phases were evaporated to dryness under reduced pressure to obtain the crude product. Then, column chromatography was used to purify the product to obtain pure L96-A.

[0316] The second step involves reacting L96-A with NH2-SPS to obtain L96-B:

[0317] Preparation process: L96-A, O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU), and diisopropylethylamine (DIPEA) were mixed and dissolved in acetonitrile. The mixture was stirred at room temperature for 5 minutes to obtain a homogeneous solution. Aminomethyl resin (NH2-SPS, 100-200 mesh) was added to the reaction solution, and the reaction was initiated at 25°C on a shaker. After 18 hours of reaction, the mixture was filtered. The filter cake was washed successively with dichloromethane and acetonitrile to obtain the filter cake. The obtained filter cake was subjected to a capping reaction with a CapA / CapB mixed solution to obtain L96-B, which is the solid-phase support containing the conjugated molecules.

[0318] The third step is to prepare siRNA conjugates.

[0319] Using L96-B as a solid-phase carrier, the siRNA was synthesized and linked to the siRNA sense strand according to the siRNA synthesis method described above. The siRNA antisense strand was synthesized using the siRNA molecule synthesis method described above, and the siRNA conjugate was annealed to produce the disclosed siRNA conjugate.

[0320] Pharmaceutical Composition

[0321] This disclosure also provides a pharmaceutical composition comprising one or more of the double-stranded ribonucleic acid, the double-stranded ribonucleic acid conjugate, or the prodrug thereof provided in this disclosure.

[0322] In some embodiments, the pharmaceutical composition contains siRNA as an active ingredient and a pharmaceutically acceptable carrier as described above. In some embodiments, the pharmaceutical composition also contains one or more additional therapeutic agents, such as those beneficial for the prevention or treatment of diseases, conditions, or symptoms at least partially mediated by GPR146 gene expression. In this disclosure, the purpose of using the pharmaceutical composition is to facilitate administration to an organism, to facilitate the absorption of the active ingredient, and thereby to exert its biological activity. The pharmaceutical compositions of this disclosure can be administered in any form, including by injection (intra-arterial, intravenous, intramuscular, intraperitoneal, subcutaneous), mucosal, oral (oral solid dosage form, oral liquid dosage form), rectal, inhalation, implantation, topical (e.g., ocular) administration, etc. Non-limiting examples of oral solid dosage forms include, but are not limited to, powders, capsules, lozenges, granules, tablets, etc. Non-limiting examples of oral or mucosal liquid dosage forms include, but are not limited to, suspensions, tinctures, elixirs, solutions, etc. Non-limiting examples of topical dosage forms include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops, or serum preparations. Non-limiting examples of parenteral administration formulations include, but are not limited to, solutions for injection, dry powders for injection, suspensions for injection, and emulsions for injection. The pharmaceutical compositions disclosed herein can also be formulated into controlled-release or delayed-release dosage forms (e.g., liposomes or microspheres). In some embodiments, the pharmaceutical composition may be a liquid formulation, such as an injection solution; or it may be a lyophilized powder for injection, formulated into a liquid formulation by mixing with liquid excipients for administration. The liquid formulation may be used, but is not limited to, for subcutaneous, intramuscular, or intravenous administration, and may also be used, but is not limited to, for administration via a spray to the lungs, or via a spray to other organs or tissues (such as the liver). In some embodiments, the pharmaceutical composition is used for intravenous administration.

[0323] In some embodiments, the pharmaceutical composition may be in the form of a liposomal formulation. In some embodiments, the pharmaceutically acceptable carrier used in the liposomal formulation comprises an amine-containing transfection compound (hereinafter also referred to as an organic amine), a cofactor lipid, and / or a polyethylene glycol-modified lipid.

[0324] The pharmaceutically acceptable carrier can be a carrier commonly used in the field of siRNA delivery, such as, but not limited to, lipid nanoparticles (LNP), magnetic nanoparticles (e.g., Fe3O4 or Fe2O3-based nanoparticles), carbon nanotubes, mesoporous silicon, calcium phosphate nanoparticles, polyethylenimine (PEI), polyamidoamine (PAMAM) dendrimer, poly(L-lysine) (PLL), chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), poly(D&L-lactic / glycolic acid) copolymer (PLGA), and poly(2-aminoethyl ethylene phosphate). One or more of the following: phosphate), PPEEA, and poly(2-dimethylaminoethyl methacrylate), PDMAEMA, and their derivatives.

[0325] The pharmaceutical composition does not have specific requirements for the content of siRNA and pharmaceutically acceptable carriers; the content of each component can be the conventional content.

[0326] In some embodiments, the pharmaceutical composition may further comprise other pharmaceutically acceptable excipients, which may be one or more of a variety of formulations or compounds conventionally used in the art. For example, the other pharmaceutically acceptable excipients may include at least one of pH buffers, protectants, and osmotic pressure regulators.

[0327] The pH buffer solution can be a tris(hydroxymethyl)aminomethane hydrochloride buffer with a pH of 7.5-8.5 and / or a phosphate buffer with a pH of 5.5-8.5, for example, a phosphate buffer with a pH of 5.5-8.5.

[0328] The protective agent may be at least one selected from inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose, and glucose. Based on the total weight of the pharmaceutical composition, the content of the protective agent may be 0.01-30% by weight.

[0329] The osmotic pressure regulator may be sodium chloride and / or potassium chloride. The content of the osmotic pressure regulator results in an osmotic pressure of 200-700 milliosm / kg (mOsm / kg) for the pharmaceutical composition. The content of the osmotic pressure regulator can be readily determined by those skilled in the art based on the desired osmotic pressure.

[0330] In this disclosure, the application method can be varied or modified in any applicable manner to meet the needs of the properties of the drug, the convenience of patients and medical personnel, and other relevant factors.

[0331] Medical Use

[0332] This disclosure also provides at least one use of double-stranded ribonucleic acid, double-stranded ribonucleic acid conjugates or their prodrugs, and pharmaceutical compositions as follows:

[0333] (1) Inhibit GPR146 gene expression in vivo or in vitro, or prepare a drug for inhibiting GPR146 gene expression;

[0334] (2) To prevent or treat diseases associated with abnormal expression of the GPR146 gene, or to prepare drugs for the prevention or treatment of diseases associated with abnormal expression of the GPR146 gene;

[0335] (3) A medicine for treating subjects with a disease in which they would benefit from reduced GPR146 gene expression, or a medicine for treating subjects with a disease in which they would benefit from reduced GPR146 gene expression.

[0336] This disclosure further provides the use of siRNA molecules (including unmodified siRNA, siRNA containing at least one modified siRNA, siRNA conjugates or prodrugs thereof) or pharmaceutical compositions in at least one of (1)-(3) above.

[0337] In this disclosure, abnormal expression of the GPR146 gene leads to one or more diseases associated with abnormal GPR146 gene expression, including nonspecific orbital inflammation, hypercholesterolemia, atherosclerosis, and coronary artery disease.

[0338] The siRNA molecule causes the expression of the GPR146 gene to be suppressed 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%, thereby achieving the treatment of diseases related to abnormal expression of the GPR146 gene.

[0339] In some embodiments, this disclosure provides a method for inhibiting intracellular GPR146 gene expression in vivo or in vitro, comprising contacting cells with double-stranded ribonucleic acid, a double-stranded ribonucleic acid conjugate or a prodrug thereof, or a pharmaceutical composition.

[0340] Furthermore, a method for inhibiting intracellular GPR146 gene expression is to introduce siRNA molecules (including unmodified siRNA, siRNA containing at least one modified siRNA, siRNA conjugates or their prodrugs) or drug compositions into the cells.

[0341] In some embodiments, the cells are in vivo or in vitro cells. In some specific embodiments, the cells are in the body of the subject.

[0342] In some embodiments, this disclosure provides methods for preventing or treating a disease, including administering a therapeutically effective amount of double-stranded ribonucleic acid, a double-stranded ribonucleic acid conjugate or a prodrug thereof, or a pharmaceutical composition to a subject in need.

[0343] Furthermore, methods for preventing or treating diseases include administering siRNA molecules (including unmodified siRNA, siRNA containing at least one modified siRNA, siRNA conjugates or their prodrugs) or pharmaceutical compositions to subjects or patients.

[0344] In this disclosure, "subject" includes either a human or a non-human animal, preferably a vertebrate, and more preferably a mammal. The subject may include a transgenic organism. Most preferably, the subject is a human. Further, the subject has at least one of the following characteristics:

[0345] (1) Abnormal expression of GPR146 gene in vivo, more specifically, abnormally high expression of GPR146 gene;

[0346] (2) Suffering from a disease associated with abnormal expression of the GPR146 gene;

[0347] (3) People with diseases that would benefit from reduced GPR146 gene expression. Such as people who have or are predisposed to diseases associated with abnormal GPR146 gene expression.

[0348] The dosage of the siRNA molecule (including unmodified siRNA, siRNA containing at least one modified siRNA, siRNA conjugate or prodrug thereof) or pharmaceutical composition disclosed herein can be determined based on the patient's weight, age, sex, disease severity, etc. Based on the amount of double-stranded ribonucleic acid contained therein, the dosage of the siRNA molecule (including unmodified siRNA, siRNA containing at least one modified siRNA, siRNA conjugate or prodrug thereof) or pharmaceutical composition disclosed herein is approximately 1-300 mg / kg body weight.

[0349] The dosing frequency can be daily, weekly, every two weeks, every three weeks, every one month, every two months, every three months, every four months, every five months, every six months, every seven months, every eight months, every nine months, every ten months, every eleven months, or annually, once or more.

[0350] The total number of times the siRNA molecule (including unmodified siRNA, siRNA containing at least one modified siRNA, siRNA conjugate or prodrug thereof) or pharmaceutical composition of this disclosure is applied may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 times. For example, the siRNA molecule (including unmodified siRNA, siRNA containing at least one modified siRNA, siRNA conjugate or prodrug thereof) or pharmaceutical composition of this disclosure may be applied about 1, 2, 3 or 4 times.

[0351] In some embodiments, the siRNA molecules of this disclosure (including unmodified siRNA, siRNA containing at least one modified siRNA, siRNA conjugates, or prodrugs thereof), pharmaceutical compositions, and optionally other therapeutic agents may be packaged in a kit, wherein the siRNA molecules (including unmodified siRNA, siRNA containing at least one modified siRNA, siRNA conjugates, or prodrugs thereof), pharmaceutically acceptable carriers, and optionally other therapeutic agents may be provided in liquid or dry form. In some embodiments, the kit includes instructions on how to mix the siRNA molecules (including unmodified siRNA, siRNA containing at least one modified siRNA, siRNA conjugates, or prodrugs thereof) with a pharmaceutically acceptable carrier or other components.

[0352] Table 1 siRNA sequence information

[0353] Table 1-1 siRNA sequence information

[0354] Table 1-2 siRNA sequence information

[0355] Table 2 siRNA conjugates

[0356] In Tables 1, 1-1, 1-2, and 2, the uppercase letters “G”, “C”, “A”, “T”, and “U” typically represent nucleotides containing guanine, cytosine, adenine, thymine, and uracil as bases, respectively; the lowercase letters a, u, c, and g indicate nucleotides modified with 2'-methoxy groups; Af, Gf, Cf, and Uf indicate nucleotides modified with 2'-fluorine groups; the lowercase letter s indicates that the two nucleotides adjacent to it are linked by thiophosphate groups; P1 indicates that the nucleotide adjacent to the right of P1 is a 5'-phosphate nucleotide; EVP indicates that the nucleotide adjacent to the right of EVP is a 5'-trans-vinylphosphonate nucleotide. (Underlined + Bold + Italic): indicates GNA-modified nucleotides; "(invAb)" indicates reverse debasing deoxyribose residues.

[0357] In Table 2, L96 is also the conjugate group GalNAc shown in formula (I). L96 is connected to the 3' end of the positive chain in Table 1, Table 1-1 and Table 1-2 via a phosphodiester bond.

[0358] In Tables 1, 1-1, 1-2, and 2, if the left side of the 5' terminal nucleotide of the positive strand, the modified positive strand, and the modified positive strand with a conjugated group is not marked with P1, EVP, or (invAb), it means that the 5' terminal nucleotide is not connected to a 5' phosphate group, a 5' phosphate-derived group, or (invAb) (i.e., the ribonucleotide of the 5' terminal nucleotide is a 5' hydroxyl group), and its structure is shown in Formula X:

[0359] Wherein, Base represents a base, such as A, U, G, C or T; R is a hydroxyl group or hydrogen or is substituted by various groups known to those skilled in the art, for example, R can be fluorine, alkoxy, substituted alkoxy, alkyl, substituted alkyl, amino, substituted amino.

[0360] In Tables 1, 1-1, 1-2 and 2, if the left side of the 5' terminal nucleotide of the antisense strand and the modified antisense strand is not marked with P1 or EVP, it means that the ribosome group of the 5' terminal nucleotide is 5' hydroxyl, and its structure is as shown in Formula X.

[0361] In Tables 1, 1-1, 1-2, and 2, when the 3' ends of the sense strand and the modified sense strand are not connected (invAb), the 3' position of the nucleotide at the 3' end of the sense strand and the modified sense strand is a hydroxyl group; in Tables 1, 1-1, 1-2, and 2, the 3' position of the nucleotide at the 3' end of the antisense strand and the modified antisense strand is a hydroxyl group.

[0362] Example

[0363] Other objects, features, and advantages of this disclosure will become apparent from the following detailed description. However, it should be understood that the detailed description and specific embodiments (although illustrating specific implementations of this disclosure) are given for illustrative purposes only, as various changes and modifications that can be made within the spirit and scope of this disclosure will become apparent to those skilled in the art upon reading this detailed description.

[0364] Unless otherwise specified, the experimental techniques and methods used in this embodiment are conventional techniques and methods. For example, experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in the embodiments can be obtained through legitimate commercial channels.

[0365] The siRNAs and siRNA conjugates involved in the following examples were synthesized by Tianlin Biotechnology (Shanghai) Co., Ltd.

[0366] Example 1: Preparation of siRNA

[0367] 1.1 siRNA sequence design

[0368] Based on the mRNA sequence (NM_001303474.2) of the Homo sapiens GPR146 gene, multiple pairs of siRNAs were designed at different sites. All individual siRNAs designed can target all transcripts of the target gene. These multiple pairs of siRNAs showed the lowest homology with all other non-target gene sequences after sequence similarity comparison with sequence similarity software.

[0369] 1.2 Synthesis of siRNA

[0370] The siRNA disclosed herein is synthesized according to the siRNA synthesis method described above.

[0371] The synthesized siRNAs are shown in Tables 1, 1-1, and 1-2.

[0372] Example 2: Synthesis of siRNA conjugate (GalNAc-siRNA)

[0373] 2.1 The siRNA conjugate has the structure shown in formula (IX):

[0374] The double helix structure is a double-stranded ribonucleic acid.

[0375] 2.2 Synthesis process of siRNA conjugates

[0376] The siRNA conjugates disclosed herein are synthesized according to the synthesis method of the siRNA conjugates described above.

[0377] The synthesized siRNA conjugates are shown in Table 2.

[0378] Example 3: siRNA inhibits GPR146 gene expression

[0379] 3.1 Experimental Materials:

[0380] Huh7 cells, provided by Shanghai WuXi AppTec Co., Ltd.

[0381] RNAiMAX transfection reagent, purchased from Invitrogen, catalog number 13778-150;

[0382] FUGENE HD Transfection, purchased from Promega, item number E2311;

[0383] DMEM medium, purchased from ATCC, catalog number 302003;

[0384] Dual-Glo luciferase Assay System( Luciferase reagent, purchased from Promega, catalog number E2940;

[0385] Opti-MEM( RNAiMAX medium, purchased from Gibco, catalog number 31985-070;

[0386] The GPR146-X4_psiCHECK(TM)-2 vector plasmid was synthesized using GenScript.

[0387] 3.2 Experimental Methods:

[0388] 3.2.1 On day 0, the GPR146-X4_psiCHECK(TM)-2 plasmid was transfected into Huh7 cells.

[0389] (1) Transfer Opti-MEM medium and GPR146-X4_psiCHECK(TM)-2 vector plasmid (100μL:1μg) into RNase-free Eppendorf tubes;

[0390] (2) Add trypsin to dissociate Huh7 cells and adjust the cell density to 1×10⁻⁶. 5 cells / mL;

[0391] (3) Add FuGENE HD transfection reagent to the solution mixture in step (1) according to the ratio of plamisd (μg): Opti-MEM: FuGENE = 1 μg: 100 μL: 3 μL, mix the resulting solution and incubate for 10 minutes;

[0392] (4) Add the solution from step (3) to the cell suspension from step (2), mix, and distribute the suspension into 96-well plates (100 μL / well). Incubate Huh7 cells overnight in a 5% CO2, 37°C incubator.

[0393] 3.2.2 Day 1: Treatment with siRNA compounds

[0394] (1) Dilute in Opti-MEM medium at a ratio of 48.5:1.5 RNAiMAX transfection reagent, incubate at room temperature for 15 minutes;

[0395] (2) Dilute siRNA with PBS to prepare a 12-fold stock solution (e.g., if the final test concentration is 1 nM, prepare this stock solution to 12 nM; the final concentrations measured in this example are 1 nM and 0.1 nM).

[0396] (3) Mix equal volumes of dilution RNAiMAX transfection reagent and siRNA (V:V = 1:1). Incubate the mixture at room temperature for 15 minutes to form a complex;

[0397] (4) 60 μL / pore compound Add the RNAiMAX (Opti-MEM) mixture to 300 μL / well of fresh DMEM medium, discard the supernatant in the analysis plate, add 120 μL / well of the above mixture to a 96-well plate, and incubate the 96-well plate in a CO2 cell culture incubator for 48 hours.

[0398] 3.3 Day 2: Genetic Testing Report

[0399] Observe the cell state under a microscope, discard the cell supernatant, and add 75 μL to each well. Add luciferase reagent and 75 μL of fresh 10% FBS (fetal bovine serum) DMEM medium, and shake in the dark for 10 minutes. Transfer 80 μL of the above sample to a 96-well pure white detection plate and detect the luminescence value (Firefly lum) of firefly luciferase under a multi-functional microplate reader. Then add 40 μL of [unspecified reagent] to each well. The luciferase reagent was shaken in a light-protected environment for 10 minutes, and the luminescence value of the Renilla luciferase was measured.

[0400] 3.4 Calculate the relative expression

[0401] The ratio of the fluorescence value of Renal luciferase to that of firefly luciferase is denoted as α, and the formula is:

[0402] α = (average Renilla lum in test wells - average Renilla lum in the blank group) / (average Firefly lum in test wells - average Firefly lum in the blank group);

[0403] The experimental group ratio calculated according to the above ratio formula is denoted as α(experimental group), and the control group ratio is denoted as α(control group).

[0404] The inhibition rate of siRNA in inhibiting the expression of the target gene GPR146 was calculated using the following formula:

[0405] Inhibition rate (%) = [1 - α(average of experimental group) / α(average of control group)] × 100%

[0406] The inhibition rates of each siRNA are shown in Table 3.

[0407] Table 3 Inhibition rate of each siRNA Note: "--" indicates that the result is not shown.

[0408] As shown in Table 3, in the Huh7 cell experiment, the siRNA disclosed in this paper showed a good inhibition rate of the GPR146 gene at a concentration of 1 nM. The concentration of 1 nM generally showed a higher inhibition of the GPR146 gene than the concentration of 0.1 nM, showing a dose-dependent effect.

[0409] Example 4: PHH experiment of siRNA conjugates

[0410] 4.1 Experimental Materials:

[0411] RNAiMAX transfection reagent, purchased from Invitrogen, catalog number 13778-150;

[0412] AceQ Universal U+Probe Master Mix V2, purchased from Vazyme, item number Q513-03;

[0413] 96Kit(12), purchased from Qiagen, item number 74182;

[0414] InvitroGRO CP Medium, purchased from BIOIVT, item number S03316;

[0415] HiScript III RT SuperMix for qPCR (+gDNAwiper), purchased from Vazyme, catalog number R323-01;

[0416] Fastking RT Kit (With gDNase), purchased from TIANGEN, item number KR116-01;

[0417] TaqMan Gene Expression Assay (GAPDH), purchased from Thermo, catalog number ID-Hs02786624_g1;

[0418] The GPR146 primer and probe were synthesized by Sangon Biotech.

[0419] 4.2 Experimental Methods:

[0420] 4.2.1

[0421] siRNA conjugates (final concentrations of 200 nM and 10 nM, in duplicate) were introduced into PHH cells via free uptake, as described below: Frozen PHH cells were harvested, thawed, counted, and adjusted to a cell volume of 6 × 10⁶ cells / well. 5 The kit was administered at a density of 54,000 cells / mL, with siRNA conjugate added, and seeded into 96-well plates at a density of 100 μL of culture medium per well. Cells were incubated in a 5% CO2, 37°C incubator. After 48 hours, the culture medium was removed and cells were collected for total RNA extraction. Use according to the kit instructions. Total RNA was extracted using 96Kit(12).

[0422] 4.2.2 The extracted total RNA was reverse transcribed into cDNA using a Fastking RT Kit (With gDNase), following these steps:

[0423] a) Remove gDNA using gDNAase according to Table 4 below;

[0424] Table 4

[0425] 42℃, 2 min; 4℃, stand.

[0426] b) Add the reagents shown in Table 5 below to the system obtained in step a) and perform reverse transcription:

[0427] Table 5

[0428] 37℃, 15min; 85℃, 5s.

[0429] c) Store the reverse transcription product obtained in step b) at -20°C for real-time PCR analysis.

[0430] 4.2.3 Perform real-time PCR analysis

[0431] (1) Prepare the qPCR reaction mixture as shown in Tables 6 and 7 below. All reagents were kept on ice throughout the operation.

[0432] Table 6

[0433] Table 7

[0434] (2) Perform the qPCR procedure as follows:

[0435] 95℃, 10 minutes;

[0436] 95℃, 15 seconds, 60℃, 1 minute (this operation is repeated 40 times).

[0437] 4.3 Results Analysis

[0438] a) Use Quant Studio 7 software with default settings to automatically calculate the Ct value;

[0439] b) Calculate the relative expression level of the gene using the following formula:

[0440] ΔCt=Ct(GPR146 gene)–Ct(GAPDH)

[0441] ΔCt = ΔCt(sample group) - ΔCt(mock group)

[0442] mRNA expression relative to the Mock group = 2 -ΔΔCt .

[0443] Mock group: The group without siRNA conjugates compared to the test sample group.

[0444] Inhibition rate (%) = (Relative expression level of mRNA in the Mock group – Relative expression level of mRNA in the test sample group) / Relative expression level of mRNA in the Mock group × 100%

[0445] The experimental results are shown in Table 8 below.

[0446] Table 8

[0447] As shown in Table 8, the siRNA conjugate disclosed herein exhibits a strong inhibitory effect on the GPR146 gene when freely taken into PHH cells at concentrations of 200 nM and 10 nM.

[0448] Example 5: siRNA inhibits GPR146 gene expression

[0449] Following the same method as in Example 3, the inhibition rate of siRNA was measured, and the results are shown in Table 9.

[0450] Table 9

[0451] As shown in Table 9, in the Huh7 cell experiment, the siRNA disclosed in this paper showed a good inhibition rate of the GPR146 gene at a concentration of 1 nM. The concentration of 1 nM generally showed a higher inhibition of the GPR146 gene than the concentration of 0.1 nM, showing a dose-dependent effect.

[0452] Example 6: PHH experiment of siRNA conjugates

[0453] Following the same method as in Example 4, the inhibition rate of the siRNA conjugate was determined, and the results are shown in Table 10.

[0454] Table 10

[0455] As shown in Table 10, the siRNA conjugate disclosed herein exhibits a strong inhibitory effect on the GPR146 gene when freely taken into PHH cells at concentrations of 200 nM and 10 nM.

[0456] Example 7: In vitro stability test of rat liver homogenate

[0457] 7.1 Experimental reagents and consumables

[0458] 7.2 Experimental Procedure

[0459] 7.2.1 Preparation of liver homogenate

[0460] 7.2.1.1 Grinding Fluid Preparation

[0461] 7.2.1.2 Tissue Homogenization

[0462] Rat liver tissue (collected from SD rats, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) was mixed with homogenizing solution at a ratio of 100mg:5mL to prepare liver homogenate (concentration of 20mg / mL). After preparation, the homogenate was added to a homogenizer and the homogenization parameters were set as follows.

[0463] 7.2.2 Sample Preparation

[0464] The siRNA conjugate sample was prepared into a 1 mg / mL solution using enzyme-free water and set aside for use. The internal standard sample was prepared into a 0.125 mg / mL solution using enzyme-free water.

[0465] 7.2.3 Incubation of biological samples

[0466] (1) Add 250 μL of prepared liver homogenate to a 2 mL enzyme-free tube;

[0467] (2) Add 50 μL of siRNA conjugate sample solution based on step (1);

[0468] (3) The system is a 300 μL biological sample solution. Vortex and let stand for 5 min.

[0469] (4) Divide into 2 tubes, each containing 100 μL;

[0470] (5) The system was incubated at 37°C for 48 hours.

[0471] 7.2.4 Biological Sample Processing

[0472] Vortex each 100 μL biological sample system, mix well, add 300 μL of Clarity OTX Lysis-loading Buffer (purchased from Agilent-FinnoMed, catalog number AL0-8579), vortex, let stand for 30 min, add 100 μL of internal standard solution, vortex, centrifuge at low speed for 1 min, and set aside (total sample volume approximately 500 μL).

[0473] 7.2.5 Solid-phase extraction:

[0474] (1) Preparation of solid phase extraction reagent

[0475] Activator: Add 200 mL of methanol to the mobile phase bottle and label it as activator;

[0476] Equilibrium buffer: Prepare 1M phosphate buffer solution [877mL sodium dihydrogen phosphate (1.56g / L) + 123mL disodium hydrogen phosphate (3.58g / L)], dilute 100 times, adjust pH to 5.5 with phosphate, and label as equilibrium buffer;

[0477] Rinse solution: Take 500 mL of equilibrium solution into a 1 L mobile phase bottle, add 500 mL of acetonitrile, mix well, and label as rinsing solution;

[0478] Eluent: Weigh 7.9g of ammonium bicarbonate into a 1L mobile phase bottle, add 1L of water, adjust the pH to 9 using sodium hydroxide, take 500mL of the adjusted ammonium bicarbonate solution into a 1L mobile phase bottle, add 500mL of acetonitrile, mix well, and label it as eluent.

[0479] (2) The extraction steps are as follows:

[0480] 7.2.6 Post-processing

[0481] Take the eluent (600 μL each time, totaling 1800 μL) in three portions and place it in a 2 mL EP tube. Concentrate under vacuum (or dry under nitrogen at 40 °C) for 10 hours at 1800 rpm. Reconstitute the concentrated sample with 100 μL of mobile phase (initial ratio) and centrifuge at 12000 rpm for 20 min at 15 °C. Inject 10 μL of the supernatant into a high-resolution mass spectrometer. The antisense strand ratio of the siRNA conjugate disclosed in this invention was semi-quantitatively determined using LC-MS / MS. The calculation formula is: AS remaining percentage % = AS MS intensity The sum of the / AS chain and all related degradation products was calculated as MS intensity × 100%, where MS intensity is the mass spectrometry signal value. The metabolic results after in vitro incubation in rat liver homogenate for 48 hours are shown in Table 11 below.

[0482] Table 11. Remaining percentage of antisense strand in siRNA conjugates

[0483] Where AS represents the antisense strand of the siRNA conjugate, a higher percentage of remaining AS indicates better drug stability and longer-lasting effect. Table 11 shows that the siRNA conjugate disclosed in this invention exhibits excellent in vitro stability and long-lasting effect in rat liver homogenate.

[0484] Example 8: Silent effect of siRNA conjugates on mice expressing the human GPR146 (hGPR146) gene

[0485] 8.1 AAV-based mouse model overexpressing hGPR146 gene

[0486] Six- to eight-week-old male C57BL / 6 mice (provided by Beijing Vital River Laboratory Animal Technology Co., Ltd.) were introduced into the facility. After acclimatization for 3-5 days, a single tail vein injection of adeno-associated virus (AAV) containing the hGPR146 gene (pAAV[Exp]-CBh>SEAP(ns):T2A:{GPR146CDS+UTR}, virus provided by Yunzhou Biotechnology (Guangzhou) Co., Ltd.) was administered to induce target gene overexpression. The administration volume was 100 μL (5 × 10⁻⁶ mcg). 11 vg) / animal, then fed with regular feed.

[0487] 8.2 In vivo efficacy study of siRNA silencing in hGPR146 mouse model

[0488] Fourteen days after AAV virus injection, mice were divided into groups of five. Mice were subcutaneously administered a single 3 mg / kg dose of the disclosed siRNA conjugate at a volume of 5 μL / g in RNase-free sterile PBS. The control group received the same volume of RNase-free sterile PBS. Serum was collected from mice on days 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 91, and 98 post-administration. The serum solutions were stored at -80°C for extended periods. Before testing, the solutions were thawed on ice, centrifuged, and the supernatant was used for protein content analysis using Phospha-Light. TM SEAP reporter gene assay system (Invitrogen) TM (Purchased from Thermo Fisher Scientific, catalog number T1017) SEAP protein expression assay (reflecting hGPR146 protein expression). The inhibition rate (%) of the siRNA conjugate in mice expressing the human GPR146 (hGPR146) gene was calculated according to the formula: inhibition rate % = (1 - mean protein expression level in the treatment group / mean protein expression level in the control group) × 100%, as shown in Table 12.

[0489] Table 12 Note: "--" indicates that the current node of this group of mice was not detected.

[0490] As can be seen from Table 12, the siRNA conjugate disclosed herein has high inhibitory activity against the hGPR146 gene in vivo, and can reduce the expression level of hGPR146 for a long time, thus exhibiting long-term effects.

[0491] Table 13 Sequences of Yang-Shen Conjugates

[0492] Example 9: Silent effect of siRNA conjugates on mice expressing the human GPR146 (hGPR146) gene

[0493] 9.1 Constructing a mouse model overexpressing the hGPR146 gene using AAV

[0494] Six- to eight-week-old male C57BL / 6 mice (provided by Beijing Vital River Laboratory Animal Technology Co., Ltd.) were introduced into the facility. A single tail vein injection of adeno-associated virus (AAV) containing the hGPR146 gene (pAAV[Exp]-CBh>SEAP(ns):T2A:{GPR146CDS+UTR}, virus provided by Yunzhou Biotechnology (Guangzhou) Co., Ltd.) was administered to induce target gene overexpression. The injection volume was 100 μL (5 × 10⁻⁶ mcg). 11 vg) / animal, then fed with regular feed.

[0495] 9.2 In vivo efficacy study of siRNA silencing in hGPR146 mouse model

[0496] Fourteen days after AAV virus injection, mice were divided into groups of five. Each group received a subcutaneous dose of 3 mg / kg of the disclosed siRNA conjugates (N-ER-FY033131M50L96, N-ER-FY033131M56L96, N-ER-FY033147M50L96, N-ER-FY033151M49L96, N-ER-FY033151M50L96, and N-ER-FY033151M56L96) at a volume of 5 μL / g in RNase-free sterile PBS. The control group received the same volume of RNase-free sterile PBS. Serum was collected weekly after administration. The inhibition rate (%) of the siRNA conjugates in mice expressing the human GPR146 (hGPR146) gene was calculated using the same method as in Example 8. The siRNA conjugate disclosed herein exhibits high inhibitory activity against the hGPR146 gene in vivo, and can reduce hGPR146 expression levels for a long time, achieving an inhibition rate of over 80% even on day 63, demonstrating long-lasting efficacy.

[0497] Example 10: In vitro off-target experiment

[0498] 10.1 Main Reagents and Consumables

[0499] InvitroGRO CPMedium, serum-free, purchased from Bioreclamation, product number IVT-S03316.

[0500] Fetal bovine serum was purchased from ExCellBio, catalog number: FSP500.

[0501] Penicillin / streptomycin was purchased from Hyclone, catalog number: SV30010.

[0502] AO / PI staining solution was purchased from Countstar, catalog number: RE010212.

[0503] Nuclease-free water was purchased from Invitrogen, product number: AM9932.

[0504] The ribonucleic acid extraction kit (miRNeasy Tissue / Cells Advanced Mini Kit) was purchased from Qiagen, catalog number: 217604.

[0505] The Agilent RNA 6000 Nano Kit was purchased from Agilent, catalog number 5067-1511.

[0506] Transcriptome library construction kit The Universal V10RNA-seq Library Prep Kit for Illumina was purchased from Vazyme, catalog number: NR606-02.

[0507] The high-sensitivity double-stranded deoxyribonucleic acid concentration assay kit (Qubit 1X dsDNA HS Assay Kit) was purchased from Thermo Fisher Scientific, catalog number: Q33231.

[0508] The High Sensitivity D1000 ScreenTape pre-fabricated deoxyribonucleic acid electrophoresis strip was purchased from Agilent, catalog number: 5067-5584.

[0509] The NovaSeq 6000S4 Reagent Kit v1.5 (300 cycles) was purchased from Illumina, catalog number: 20028312.

[0510] Human primary hepatocytes (PHH) (batch: LGI) were provided by Shanghai WuXi AppTec New Drug Development Co., Ltd.

[0511] 10.2 Main Instruments

[0512] Biosafety cabinet (Lixin (Shanghai) Instrument Co., Ltd., Hfsafe-1800LCA2), micro-volume UV-Vis spectrophotometer (NanoDrop One, Thermo Fisher Scientific), Agilent 2100 Bioanalyzer (Agilent), thermal cycler (T100 thermal cycler, Bio-Rad), fluorescence meter (Qubit 4fluorometer, Thermo Fisher Scientific), Agilent 4200 automated electrophoresis system (Agilent 4200 TapeStation), sequencer (NovaSeq 6000 (Illumina)), fully automated cell fluorescence analyzer (Rigel S2, Countstar).

[0513] 10.3 Culture medium formulation

[0514] The cell culture medium preparation system containing 10% fetal bovine serum in 20 mL consists of: 17.8 mL of serum-free in vitro culture medium InvitroGRO CP Medium (Bioreclamation IVT-S03316), 2 mL of fetal bovine serum (ExCellBio-FSP500), and 0.2 mL of penicillin / streptomycin (Hyclone-SV30010).

[0515] 10.4 Experimental Procedure

[0516] 10.4.1 Cell Plating

[0517] Conjugates N-ER-FY033131M50L96 and N-ER-FY033151M50L96 enter human primary hepatocytes via free uptake, as described below:

[0518] (1) Dilute the siRNA conjugate with nuclease-free water to 10 times the final concentration (final concentration is 5nM, 50nM or 100nM).

[0519] (2) Remove one vial of cryopreserved human primary hepatocytes (PHH) from the liquid nitrogen tank, gently agitate in a water bath until the cryopreservation solution thaws, transfer the cells to a cell culture medium containing 10% fetal bovine serum, and adjust the final cell density to 6.7 × 10⁻⁶. 5 cells / mL.

[0520] (3) Take 50 μL of the conjugate from step (1) into a collagen-coated 24-well cell plate, and then add 450 μL of cell suspension from step (2) to achieve a final cell density of 300,000 per well. The final concentration of the siRNA conjugate is 5 nM, 50 nM, or 100 nM. Each conjugate is tested in triplicate, and the wells without the conjugate are used as the control group.

[0521] (4) After culturing for 72 hours, each well was washed with PBS and then 260 μL of lysis buffer was added to lyse the cells for subsequent experiments.

[0522] 10.4.2 Next-Generation Sequencing

[0523] (1) Purify total RNA from cells according to the manufacturer’s (Qiagen-217604_miRNeasy Tissue / Cells Advanced Mini Kit) instructions.

[0524] (2) The concentration of total RNA was detected using NanoDrop One, and RNA integrity was analyzed according to the manufacturer’s (Agilent 2100 Bioanalyzer\Agilent-5067-1511_Agilent RNA 6000Nano Kit) guidelines.

[0525] (3) According to the manufacturer ( Library preparation was performed using the Universal V10RNA-seq Library Prep Kit for Illumina.

[0526] (4) Detect the library concentration according to the manufacturer's (Thermo Fisher Scientific-Qubit 4fluorometer\Thermo Fisher Scientific-Q33231_Qubit 1X dsDNA HS Assay Kit) guidelines, and analyze the library fragment size according to the manufacturer's (Agilent 4200 TapeStation\Agilent-5067-5584_High Sensitivity D1000 ScreenTape) guidelines.

[0527] (5) Perform library sequencing according to the manufacturer's (Illumina-NovaSeq 6000\Illumina-20028312_NovaSeq 6000S4 Reagent Kit v1.5(300cycles)) guidelines.

[0528] This study, based on next-generation sequencing technology, investigated the effects of free uptake of test samples (N-ER-FY033131M50L96 and N-ER-FY033151M50L96) by human hepatocytes on transcriptome expression. |log2(FoldChange)|>1 & padj.<0.05 was set as the screening criterion for differentially expressed genes.

[0529] IC50 in 10.5 PHH cells 50 test

[0530] The concentration ranges (nM) for the siRNA conjugate assay were set as follows: 500, 125, 31.25, 7.8125, 1.9531, 0.4883, 0.1221, and 0.03052. IC50 assays were then performed using a method similar to that in Example 4. 50 The free uptake in PHH cells was determined, and the IC50 values ​​of N-ER-FY033131M50L96 and N-ER-FY033151M50L96 in human hepatocytes were obtained. 50 The concentrations were 0.25 nM and 0.20 nM, respectively. The inhibition rates at each concentration are shown in Figure 1.

[0531] Sequencing of N-ER-FY033131M50L96 at concentrations of 5 nM and 50 nM revealed 0 and 2 differentially expressed genes (SLC16A7 and NRARP) at the two concentrations, respectively, that met the criteria of |log2(FoldChange)|>1&padj<0.05 (as shown in Figures 2 and 3). In vitro PHH cell uptake validation was performed on differentially expressed genes such as SLC16A7, showing an inhibition rate of approximately 60% at the maximum concentration of 500 nM, with an IC50 value of [missing information]. 50 The value was 9.53 nM (as shown in Figure 5), while the IC50 of N-ER-FY033131M50L96 in PHH cells was... 50 The concentration was 0.25 nM, indicating that the conjugate has a safety window of more than 38 times for SLC16A7, with a low off-target risk. Furthermore, no differentially expressed genes were observed at 5 nM, suggesting that the conjugate has a safety window of at least 20 times, thus indicating a low off-target risk in vivo, good safety profile, and good drug-like properties.

[0532] Sequencing of N-ER-FY033151M50L96 at a concentration of 100 nM revealed two differentially expressed genes (TNRC6A and KIF5B) that met the criteria of |log2(FoldChange)|>1&padj<0.05 (as shown in Figure 4). In vitro PHH cell uptake validation was performed on these differentially expressed genes, TNRC6A and KIF5B. The results showed that TNRC6A exhibited an inhibition rate of less than 50% at all tested concentrations, while KIF5B showed an inhibition rate of approximately 50% at the maximum concentration of 500 nM. IC50... 50 The value was 252.60 nM (as shown in Figure 6), indicating that N-ER-FY033151M50L96 has a low off-target risk for TNRC6A and KIF5B. The IC50 value of N-ER-FY033151M50L96 in PHH cells... 50 The concentration was 0.20 nM, and the selected concentration for off-target testing was IC50. 50 The value is 500 times higher than that of the conjugate. In addition, the low off-target risk of differentially expressed genes at 100 nM indicates that the safety window of the conjugate is at least 500 times higher than that of the conjugate. Therefore, the off-target risk in vivo is low, the safety is good, and it has good drug-like properties.

[0533] We also conducted in vitro off-target experiments on multiple siRNA conjugates. The results showed that the safety windows of the siRNA conjugates N-ER-FY033131M49L96, N-ER-FY033131M56L96, N-ER-FY033151M49L96, and N-ER-FY033151M56L96 were at least 20 times, with low off-target risk in vivo, good safety profile, and good drug-like properties.

[0534] The embodiments described above are merely examples for clearly illustrating the present disclosure and are not intended to limit the implementation of the present disclosure. Those skilled in the art can make other variations or modifications based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this disclosure should be included within the scope of protection of the claims of this disclosure.

Claims

A double-stranded ribonucleic acid (GRNA) for inhibiting GPR146 gene expression comprises a sense strand and an antisense strand, wherein each nucleotide in the double-stranded ribonucleic acid is independently modified or unmodified, the sense strand contains nucleotide sequence I, and the antisense strand contains nucleotide sequence II, wherein nucleotide sequence I and nucleotide sequence II are at least partially anticomplementary to form a double-stranded region. The nucleotide sequence I and the nucleotide sequence II are selected from the following sequences: (1) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:1, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:2: 5'-GGUCCUCACCAGAGCUCUA-3'(SEQ ID NO:1) 5'-UAGAGCUCUGGUGAGGACC-3' (SEQ ID NO: 2); (2) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:3, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:4: 5'-CAGAUCCCCUUCAAUGUGU-3'(SEQ ID NO:3) 5'-ACACAUUGAAGGGGAUCUG-3' (SEQ ID NO: 4); (3) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:7, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:8: 5'-CUCCCAAACACGCAGCUCA-3'(SEQ ID NO:7) 5'-UGAGCUGCGUGUUUGGGAG-3' (SEQ ID NO:8); (4) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:9, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:10: 5'-GAGCCAGUAUUUAUACUUU-3'(SEQ ID NO:9) 5'-AAAGUAUAAAUACUGGCUC-3' (SEQ ID NO: 10); (5) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:11, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:12: 5'-ACAGAUGUUUCCUAGAAAA-3'(SEQ ID NO:11) 5'-UUUUCUAGGAAACAUCUGU-3' (SEQ ID NO: 12); (6) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:13, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:14: 5'-GAGGUUUUUUUCAGUAUGA-3'(SEQ ID NO:13) 5'-UCAUACUGAAAAAAACCUC-3' (SEQ ID NO: 14); (7) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:15, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:16: 5'-GUCCUAAAUCAAUUCCUCA-3'(SEQ ID NO:15) 5'-UGAGGAAUUGAUUUAGGAC-3' (SEQ ID NO: 16); (8) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:17, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:18: 5'-GCAUUCAGUUUGUCAAU-3'(SEQ ID NO:17) 5'-AUUGACAAACUGAAUGC-3' (SEQ ID NO: 18); (9) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:19, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:20: 5'-GGUUAAAAUA-3'(SEQ ID NO:19) 5'-UAUUUUAACC-3' (SEQ ID NO:20); (10) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:21, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:22: 5'-CCCCUUGUUUGUUU-3'(SEQ ID NO:21) 5'-AAACAAACAAGGGG-3' (SEQ ID NO: 22); (11) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:23, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:24: 5'-CACAAAACUAAA-3'(SEQ ID NO:23) 5'-UUUAGUUUUGUG-3' (SEQ ID NO: 24). According to claim 1, the double-stranded ribonucleic acid, wherein, The sense strand further contains nucleotide sequence III, and the antisense strand further contains nucleotide sequence IV. Nucleotide sequence III and nucleotide sequence IV are each independently 0-9 nucleotides in length. Nucleotide sequence III is attached to the 5' end of nucleotide sequence I, and nucleotide sequence IV is attached to the 3' end of nucleotide sequence II. Nucleotide sequences III and IV are of equal length and are substantially anticomplementary or completely anticomplementary. Substantially anticomplementary means that there is no more than one base mismatch between the two nucleotide sequences; completely anticomplementary means that there is no mismatch between the two nucleotide sequences; and / or The nucleotide sequence III is attached to the 3' end of the nucleotide sequence I, and the nucleotide sequence IV is attached to the 5' end of the nucleotide sequence II. The nucleotide sequences III and IV are of equal length and are substantially anticomplementary or completely anticomplementary. Substantially anticomplementary means that there is no more than one base mismatch between the two nucleotide sequences. Completely anticomplementary means that there is no mismatch between the two nucleotide sequences. According to claim 1 or 2, the double-stranded ribonucleic acid, wherein, The positive strand further contains nucleotide sequence V and / or the antisense strand further contains nucleotide sequence VI, wherein the length of nucleotide sequence V and nucleotide sequence VI are each independently 0-2 nucleotides, wherein nucleotide sequence V is attached to the 3' end of the positive strand to form a 3' overhang of the positive strand and / or nucleotide sequence VI is attached to the 3' end of the antisense strand to form a 3' overhang of the antisense strand; preferably, the length of nucleotide sequence V or nucleotide sequence VI is each 2 nucleotides; more preferably, along the direction from the 5' end to the 3' end, nucleotide sequence V or nucleotide sequence VI is UU, AU, UA, AG, AA, AC, CU or CA; Alternatively, the nucleotide sequence V may be identical to or differ from the nucleotide at the corresponding position of the target mRNA, or the nucleotide sequence VI may be mismatched or complementary to the nucleotide at the corresponding position of the target mRNA. The double-stranded ribonucleic acid according to any one of claims 1-3, wherein the double-stranded ribonucleic acid comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand are selected from the following sequences: The sense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:33, and the antisense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:57; The sense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:47, and the antisense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:58; or The sense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:55, and the antisense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:

59. The double-stranded ribonucleic acid according to any one of claims 1-4, wherein, The length of the double-stranded region is 10-30 nucleotide pairs, preferably 17-25 nucleotide pairs, more preferably 19-23 nucleotide pairs, and even more preferably 19, 20, 21, 22 or 23 nucleotide pairs; and / or The sense strand or the antisense strand has 15-30 nucleotides, preferably 17-25 nucleotides, more preferably 19-23 nucleotides, and even more preferably 19, 20, 21, 22 or 23 nucleotides. The double-stranded ribonucleic acid according to any one of claims 1-5, wherein, Each nucleotide in the sense strand is independently a modified or unmodified nucleotide, and / or each nucleotide in the antisense strand is independently a modified or unmodified nucleotide; Preferably, the modified nucleotide is selected from 2'-fluoro-modified nucleotides, 2'-alkoxy-modified nucleotides, 2'-substituted alkoxy-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-substituted alkyl-modified nucleotides, 2'-deoxy-modified nucleotides, 2'-amino-modified nucleotides, 2'-substituted amino-modified nucleotides, nucleotide analogs, or any combination of two or more thereof. More preferably, the modified nucleotide is selected from 2'-fluoromodified nucleotides, 2'-methoxymodified nucleotides, 2'-O-CH2-CH2-O-CH3modified nucleotides, 2'-O-CH2-CH=CH2modified nucleotides, 2'-CH2-CH2-CH=CH2modified nucleotides, 2'-deoxymodified nucleotides, nucleotide analogs, or any combination of two or more thereof; More preferably, the nucleotide analogue is selected from isonucleotides, LNA, ENA, cET-BNA, UNA, or GNA. The double-stranded ribonucleic acid according to any one of claims 1-6, wherein, Any two linked nucleotides in the positive strand are connected by a phosphodiester bond or a thiophosphate group; and / or In the antisense strand, any two linked nucleotides are connected by a phosphodiester bond or a thiophosphate group; and / or The 5' terminal nucleotide of the antisense strand is connected to a 5' phosphate group or a 5' phosphate derivative group, or the 5' terminal nucleotide of the antisense strand is not connected to a 5' phosphate group or a 5' phosphate derivative group. The double-stranded ribonucleic acid according to any one of claims 1-7, wherein, The positive chain contains one or more end-cap residues or portions, or the positive chain does not contain end-cap residues or portions; Preferably, the capping residue is a reverse debasing deoxyribose residue; More preferably, the 3' terminal nucleotide and / or 5' terminal nucleotide of the positive strand are linked to one or more of the reverse debased deoxyribose residues; More preferably, the 3' terminal nucleotide of the positive strand is linked to one or more of the reverse debased deoxyribose residues; More preferably, the reverse debased deoxyribose residue is linked to the 3' terminal nucleotide and / or 5' terminal nucleotide of the positive strand via a phosphodiester bond, a thiophosphate bond, or other nucleoside bonds. The double-stranded ribonucleic acid according to any one of claims 1-8, wherein, The double-stranded ribonucleic acid is siRNA. Double-stranded ribonucleic acid according to any one of claims 1-9, wherein, Each nucleotide in the sense strand and the antisense strand is independently a 2'-fluoro-modified nucleotide or a non-fluoro-modified nucleotide. Preferably, in the positive strand, along the direction from the 3' end to the 5' end, 2'-fluorinated nucleotides are located at positions 9, 10, 11, and 13, with the remaining positions being non-fluorinated nucleotides; or, 2'-fluorinated nucleotides are located at positions 11, 12, 13, and 15, with the remaining positions being non-fluorinated nucleotides; or, 2'-fluorinated nucleotides are located at positions 11, 12, 13, and 17, with the remaining positions being non-fluorinated nucleotides; or, 2'-fluorinated nucleotides are located at positions 9, 11, 13, and 17, with the remaining positions being non-fluorinated nucleotides; or, 2'-fluorinated nucleotides are located at positions 6, 11, and 13, with the remaining positions being non-fluorinated nucleotides; and / or In the antisense strand, along the direction from the 5' end to the 3' end, 2'-fluorinated nucleotides are located at positions 2, 6, 14, and 16, with the remaining positions being non-fluorinated nucleotides; or, 2'-fluorinated nucleotides are located at positions 2, 7, 10, and 14, with the remaining positions being non-fluorinated nucleotides; or, 2'-fluorinated nucleotides are located at positions 2, 3, 5, 7, 10, 12, and 14, with the remaining positions being non-fluorinated nucleotides. The double-stranded ribonucleic acid according to any one of claims 1-10, wherein, Each nucleotide in the sense strand and the antisense strand is independently a 2'-fluoromodified nucleotide, a 2'-methoxymodified nucleotide, and / or a GNA-modified nucleotide; Preferably, in the positive strand, along the direction from the 3' end to the 5' end, 2'-fluorinated nucleotides are located at positions 9, 10, 11, and 13, with the remaining positions being 2'-methoxyinated nucleotides; or, 2'-fluorinated nucleotides are located at positions 11, 12, 13, and 15, with the remaining positions being 2'-methoxyinated nucleotides; or, 2'-fluorinated nucleotides are located at positions 11, 12, 13, and 17, with the remaining positions being 2'-methoxyinated nucleotides; or, 2'-fluorinated nucleotides are located at positions 9, 11, 13, and 17, with the remaining positions being 2'-methoxyinated nucleotides; or, 2'-fluorinated nucleotides are located at positions 6, 11, and 13, with the remaining positions being 2'-methoxyinated nucleotides; and / or In the antisense strand, along the direction from the 5' end to the 3' end, 2'-fluoromodified nucleotides are located at positions 2, 6, 14, and 16, with the remaining positions being 2'-methoxymodified nucleotides; or, 2'-fluoromodified nucleotides are located at positions 2, 7, 10, and 14, with the remaining positions being 2'-methoxymodified nucleotides; or, 2'-fluoromodified nucleotides are located at positions 2, 3, 5, 7, 10, 12, and 14, and GNA-modified nucleotides are located at position 6 of the antisense strand, with the remaining positions being 2'-methoxymodified nucleotides. According to any one of claims 1-11, the double-stranded ribonucleic acid, in the 5' to 3' orientation, comprises phosphate thioester groups located at the following positions: Between the first and second nucleotides starting at the 5' end of the positive strand; Between the second and third nucleotides starting at the 5' end of the positive strand; or, The positive chain contains thiophosphate groups located at the following positions: Between the first nucleotide starting at the 5' end of the positive strand and the reverse debased deoxyribose residue; Between the first and second nucleotides starting at the 5' end of the positive strand; Between the first nucleotide starting at the 3' end of the positive strand and the reverse debased deoxyribose residue; or, The positive chain contains thiophosphate groups located at the following positions: Between the first and second nucleotides starting at the 5' end of the positive strand; Between the second and third nucleotides starting at the 5' end of the positive strand; The first nucleotide starting at the 3' end of the positive strand is between the reverse debased deoxyribose residue. The double-stranded ribonucleic acid according to any one of claims 1-12, wherein the antisense strand comprises phosphate thioester groups located at the following positions, in the 5' to 3' orientation: Between the first and second nucleotides starting at the 5' end of the antisense strand; Between the second and third nucleotides starting at the 5' end of the antisense strand; Between the first and second nucleotides starting at the 3' end of the antisense strand; Between the second and third nucleotides starting at the 3' end of the antisense strand. The double-stranded ribonucleic acid according to any one of claims 1-13, wherein, Along the 3' end to the 5' end, 2'-fluorinated nucleotides are located at positions 9, 10, 11, and 13 of the sense strand, with the remaining positions being 2'-methoxylated nucleotides; along the 5' end to the 3' end, 2'-fluorinated nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand, with the remaining positions being 2'-methoxylated nucleotides, wherein the 5'-terminal nucleotide of the antisense strand is linked to a 5' phosphate group; Alternatively, along the 3' end to the 5' end, 2'-fluorinated nucleotides are located at positions 9, 10, 11, and 13 of the sense strand, with the remaining positions being 2'-methoxylated nucleotides; along the 5' end to the 3' end, 2'-fluorinated nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand, with the remaining positions being 2'-methoxylated nucleotides, wherein the 5'-terminal nucleotide of the antisense strand is not linked to a 5' phosphate group or a 5' phosphate-derived group; Alternatively, along the 3' end to the 5' end, 2'-fluorinated nucleotides are located at positions 9, 10, 11, and 13 of the sense strand, with the remaining positions being 2'-methoxylated nucleotides; along the 5' end to the 3' end, 2'-fluorinated nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand, with the remaining positions being 2'-methoxylated nucleotides, wherein the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group; Alternatively, along the 3' end to the 5' end, 2'-fluorinated nucleotides are located at positions 11, 12, 13, and 15 of the sense strand, with the remaining positions being 2'-methoxylated nucleotides; along the 5' end to the 3' end, 2'-fluorinated nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand, with the remaining positions being 2'-methoxylated nucleotides, wherein the 5'-terminal nucleotide of the antisense strand is not connected to a 5' phosphate group or a 5' phosphate-derived group; Alternatively, along the 3' end to the 5' end, 2'-fluorinated nucleotides are located at positions 11, 12, 13, and 15 of the sense strand, with the remaining positions being 2'-methoxylated nucleotides; along the 5' end to the 3' end, 2'-fluorinated nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand, with the remaining positions being 2'-methoxylated nucleotides, wherein the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group; Alternatively, along the 3' end to the 5' end, 2'-fluorinated nucleotides are located at positions 11, 12, 13, and 17 of the sense strand, with the remaining positions being 2'-methoxylated nucleotides; along the 5' end to the 3' end, 2'-fluorinated nucleotides are located at positions 2, 7, 10, and 14 of the antisense strand, with the remaining positions being 2'-methoxylated nucleotides, wherein the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group; Alternatively, along the 3' end to the 5' end, 2'-fluorinated nucleotides are located at positions 9, 11, 13, and 17 of the sense strand, with the remaining positions being 2'-methoxylated nucleotides; along the 5' end to the 3' end, 2'-fluorinated nucleotides are located at positions 2, 7, 10, and 14 of the antisense strand, with the remaining positions being 2'-methoxylated nucleotides, wherein the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group; Alternatively, along the 3' end to the 5' end, 2'-fluorinated nucleotides are located at positions 6, 11, and 13 of the sense strand, with the remaining positions being 2'-methoxylated nucleotides; along the 5' end to the 3' end, 2'-fluorinated nucleotides are located at positions 2, 7, 10, and 14 of the antisense strand, with the remaining positions being 2'-methoxylated nucleotides, wherein the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group; Alternatively, along the 3' end to the 5' end, 2'-fluorinated nucleotides are located at positions 9, 11, 13, and 17 of the sense strand, with the remaining positions being 2'-methoxylated nucleotides; along the 5' end to the 3' end, 2'-fluorinated nucleotides are located at positions 2, 3, 5, 7, 10, 12, and 14 of the antisense strand, GNA-modified nucleotides are located at position 6 of the antisense strand, with the remaining positions being 2'-methoxylated nucleotides, wherein the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group. The double-stranded ribonucleic acid according to any one of claims 1-14, wherein, The justice chain and the antisense chain are selected from the following combinations: The sense strand includes the sense strand of any one of the siRNAs shown in Tables 1, 1-1 and 1-2 of this document, and the antisense strand includes the antisense strand of the corresponding siRNA. Preferably, the siRNA is selected from N-ER-FY033131, N-ER-FY033131M44, N-ER-FY033131M48, N-ER-FY033131M49, N-ER-FY033131M50, N-ER-FY033147, N-ER-FY033147M44, N-ER-FY033147M49, N-ER-FY033147M50, N-ER-FY033151, N-ER-FY033151M44, N-ER-FY033151M49, and N-ER-FY033151M50. A double-stranded ribonucleic acid conjugate or a prodrug thereof, wherein, The double-stranded ribonucleic acid conjugate comprises the double-stranded ribonucleic acid as described in any one of claims 1-15, and a conjugating group conjugated to the double-stranded ribonucleic acid. According to claim 16, the double-stranded ribonucleic acid conjugate or its prodrug, wherein, The conjugation group is attached to the 3' end of the positive chain; Preferably, the conjugating group is conjugated to the 3' end of the positive chain via a phosphodiester bond; or, the conjugating group is conjugated to the reverse debased deoxyribose residue at the 3' end of the positive chain via a thiophosphate group. According to claim 16 or 17, the double-stranded ribonucleic acid conjugate or its prodrug, wherein, The conjugating group is selected from any one of the group consisting of formulas (I) to (VIII): The double-stranded ribonucleic acid conjugate or its prodrug according to any one of claims 16-18, wherein, The double-stranded ribonucleic acid conjugate has the structure shown in Formula (IX): The double helix structure is a double-stranded ribonucleic acid. The double-stranded ribonucleic acid conjugate or its prodrug according to any one of claims 16-19, wherein, The double-stranded ribonucleic acid conjugate is an siRNA conjugate. The double-stranded ribonucleic acid conjugate or its prodrug according to any one of claims 16-20, wherein, The double-stranded ribonucleic acid conjugate is formed by linking any one of the siRNAs shown in Tables 1, 1-1 and 1-2 of this document with a conjugation group; Preferably, in the double-stranded ribonucleic acid conjugate, the sense strand and the antisense strand are selected from the following combinations: The sense strand comprises the sense strand of any of the siRNA conjugates shown in Table 2 of this document, and the antisense strand comprises the antisense strand of the corresponding siRNA conjugate; more preferably, the siRNA conjugate is selected from N-ER-FY033131M44L96, N-ER-FY033131M48L96, N-ER-FY033131M49L96, N-ER-FY033131M50L96, N-ER-FY033147M44L96, N-ER-FY033147M49L96, N-ER-FY033147M50L96, N-ER-FY033151M44L96, N-ER-FY033151M49L96, and N-ER-FY033151M50L96. A pharmaceutical composition comprising the double-stranded ribonucleic acid of any one of claims 1-15 or the double-stranded ribonucleic acid conjugate or its prodrug of any one of claims 16-21. The pharmaceutical composition according to claim 22, wherein, The pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, and optionally includes one or more additional therapeutic agents. Use of the double-stranded ribonucleic acid of any one of claims 1-15, the double-stranded ribonucleic acid conjugate or its prodrug of any one of claims 16-21, or the pharmaceutical composition of claim 22 or 23 in at least one of the following: (1) Inhibit GPR146 gene expression in vivo or in vitro, or prepare a drug for inhibiting GPR146 gene expression; (2) To prevent or treat diseases associated with abnormal expression of the GPR146 gene, or to prepare drugs for the prevention or treatment of diseases associated with abnormal expression of the GPR146 gene; (3) A medicine for treating subjects with a disease in which they would benefit from reduced GPR146 gene expression, or a medicine for treating subjects with a disease in which they would benefit from reduced GPR146 gene expression. The use according to claim 24, wherein, The diseases associated with abnormal GPR146 gene expression are selected from the group consisting of the following diseases: nonspecific orbital inflammation, hypercholesterolemia, atherosclerosis, and coronary artery disease. A method for inhibiting intracellular GPR146 gene expression in vivo or in vitro, comprising contacting the cells with a double-stranded ribonucleic acid (GRNA) according to any one of claims 1-15, a GRNA conjugate or prodrug according to any one of claims 16-21, or a pharmaceutical composition according to claim 22 or 23. The method according to claim 26, wherein, The cells are either in vivo cells or in vitro cells; Alternatively, the cells may be present in the subject's body. The method according to claim 26, wherein, The subjects are mammals, preferably humans; More preferably, the subject has at least one of the following characteristics: Abnormal expression of the GPR146 gene in vivo, more specifically, abnormally high expression of the GPR146 gene; Suffering from a disease associated with abnormal expression of the GPR146 gene; Having a disease that would benefit from reduced GPR146 gene expression. The double-stranded ribonucleic acid of any one of claims 1-15, the double-stranded ribonucleic acid conjugate or its prodrug of any one of claims 16-21, or the pharmaceutical composition of claim 22 or 23, are used as a drug or for treatment. A method for preventing and / or treating a disease, comprising administering to a subject in need a therapeutically effective amount of any one of claims 1-15, any one of claims 16-21, a double-stranded ribonucleic acid conjugate or its prodrug, or the pharmaceutical composition of claim 22 or 23. The method according to claim 30, wherein, The diseases mentioned are selected from the group consisting of the following diseases: nonspecific orbital inflammation, hypercholesterolemia, atherosclerosis, and coronary artery disease. A kit comprising the double-stranded ribonucleic acid of any one of claims 1-15, the double-stranded ribonucleic acid conjugate or its prodrug of any one of claims 16-21, or the pharmaceutical composition of claim 22 or 23.

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