Novel double-stranded sirna, conjugate thereof and use thereof

Abstract

The present invention provides a novel double-stranded siRNA, a conjugate thereof and a use thereof, wherein the double-stranded siRNA and the conjugate thereof can inhibit LAP gene expression, and can be used for preparing drugs for treating and / or preventing LAP-related diseases.

Description

A novel double-stranded siRNA, its conjugate and its use Technical Field

[0001] The present invention belongs to the field of small nucleic acid drugs and aims to provide a novel double-stranded siRNA, its conjugates, and uses thereof. The double-stranded siRNA and its conjugates of the present invention can be used to prepare drugs for treating and / or preventing LPA-related diseases. Background Art

[0002] Lipoprotein (a) (Lp(a)) is a heterogeneous low-density lipoprotein (LDL)-like particle containing a lipid core and apolipoprotein B (apoB-100) with a unique component, apolipoprotein (a) (apo(a)), linked to apoB-100 via a disulfide bond. The apo(a) gene (LPA) is primarily expressed in the liver, and expression is restricted to humans and non-human primates. Lp(a) levels in humans are genetically determined and do not significantly change with diet, exercise, or other lifestyle changes. The length of LPA varies depending on the number of Kringle KIV2 domains present, and its expression is inversely correlated with the number of domains present. Analysis of Lp(a) levels in multiple studies has implicated high Lp(a) levels as an independent risk factor for cardiovascular disease, stroke, and other related conditions, including atherosclerotic stenosis. In addition, genome-wide association studies have also implicated LPA as a genetic risk factor for diseases such as atherosclerotic stenosis. When therapeutic lipoprotein apheresis is used to lower Lp(a) and LDL levels in hyperlipidemic patients, a significant reduction in cardiovascular events has been observed. Therefore, there is a need for therapeutic agents and treatments related to these and other LPA-related diseases.

[0003] RNA interference (RNAi) refers to the highly conserved phenomenon of efficient and specific degradation of homologous mRNA induced by double-stranded small interfering RNA (siRNA). Research and development of novel LPA-specific RNA interference (RNAi) agents (also referred to herein as RNAi agents, RNAi triggers, or triggering agents), such as double-stranded RNAi agents, that can selectively and effectively inhibit LPA gene expression is of great significance. Summary of the Invention

[0004] The present invention provides a novel double-stranded siRNA, its conjugate, or salt, and uses thereof. These double-stranded siRNA, its conjugate, or salt can inhibit LPA gene expression and can be used to prepare a medicament for treating and / or preventing LPA-related diseases. The double-stranded siRNA, its conjugate, or salt described herein exhibits high in vivo delivery efficiency, excellent stability, high LPA gene expression inhibitory activity, and / or low toxicity.

[0005] In one aspect, the present invention provides a double-stranded siRNA, a conjugate or a salt thereof, comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises one of the nucleotide sequences shown in SEQ ID NO: 1 to SEQ ID NO: 28, or a nucleotide sequence having no more than 5 nucleotide differences therefrom; wherein, detailed information on the nucleotide sequences shown in SEQ ID NO: 1 to SEQ ID NO: 28 is shown in Table 1 of the present specification.

[0006] In some embodiments of the double-stranded siRNA, conjugates or salts thereof according to the present invention, the antisense strand of the present invention comprises one of the nucleotide sequences shown in SEQ ID NO: 29 to SEQ ID NO: 56, or a nucleotide sequence having no more than 5 nucleotide differences therefrom;

[0007] Detailed information on the nucleotide sequences shown in SEQ ID NO: 29 to SEQ ID NO: 56 can be found in Table 1 of the present specification.

[0008] In some embodiments of the present invention, the double-stranded siRNA, its conjugate or salt thereof is used to inhibit LPA gene expression.

[0009] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the double-stranded siRNA is a modified double-stranded siRNA.

[0010] In some embodiments of the double-stranded siRNA, its conjugate or salt described in the present invention, the double-stranded siRNA conjugate is formed by conjugating the double-stranded siRNA to a conjugating group.

[0011] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the length of the sense strand does not exceed 23 nucleotides, and the length of the antisense strand does not exceed 23 nucleotides.

[0012] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the sense strand and / or antisense strand comprises a 3' overhang and / or a 5' overhang, and the 3' overhang or 5' overhang comprises 1, 2 or 3 nucleotides.

[0013] In some embodiments of the double-stranded siRNA, its conjugate or salt described in the present invention, the double-stranded siRNA, its conjugate or salt comprises one of the double-stranded siRNAs shown in siRNA ID NO: 1 to siRNA ID NO: 28, wherein the information of the double-stranded siRNA shown in siRNA ID NO: 1 to siRNA ID NO: 28 is detailed in Table 1 of the specification of the present invention.

[0014] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the sense strand and / or the antisense strand comprises at least one modified nucleotide, and the modified nucleotide is independently selected from at least one of the following:

[0015] Non-natural base nucleotides, deoxynucleotides, 2'-fluoro modified nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-alkyl modified nucleotides, 2'-O-alkyl modified nucleotides (such as 2'-methoxy modified nucleotides), 2'-methoxyethyl modified nucleotides, locked nucleotides (LNA), unlocked nucleic acid modified nucleotides (UNA), 2'-allyl modified nucleotides, abasic nucleotides, invAb modified nucleotides, morpholino modified nucleotides, tetrahydropyran modified nucleotides, cyclohexenyl modified nucleotides, PEG modified nucleotides, 5'-phosphoramidate modified nucleotides, thiophosphate linked modified nucleotides (such as: 5'-thiophosphate linked modified nucleotides and / or the 3'-thiophosphate linked modified nucleotides), 5'-methylphosphonate modified nucleotides (such as 5'-( E)-VP modified nucleic acids), 5'-vinyl phosphate modified nucleic acids, 5'-phosphate mimetic modified nucleotides, TNA modified nucleotides, PNA modified nucleotides, D-FNA modified nucleotides, ANA modified nucleotides, HNA modified nucleotides, FANA modified nucleotides, bcDNA modified nucleotides, tcDNA modified nucleotides, S-MC modified nucleotides, N-MC modified nucleotides, 2'-F-NMC modified nucleotides, 5-methylcytosine modified nucleotides, 5-methyluracil modified nucleotides, 2,6-diamino modified adenine modified nucleotides and ethylene glycol nucleic acids (GNA), wherein the 2'- refers to the 2-position of ribose, such as a 2'-fluoro modified nucleotide refers to a nucleotide in which the 2-position of ribose is substituted by fluorine, and for example, a 2'-methoxy modified nucleotide refers to a nucleotide in which the 2-position of ribose is substituted by methoxy.

[0016] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the sense strand and / or the antisense strand comprises at least one modified nucleotide, and the modified nucleotide is independently selected from at least one of the following:

[0017] 2'-methoxy modified nucleotides, 2'-fluoro modified nucleotides, 2'-methoxyethyl modified nucleotides, 5'-phosphorothioate linked modified nucleotides, 3'-phosphorothioate linked modified nucleotides, 2'-deoxy modified nucleotides, 2'-amino modified nucleotides, 2'-hydroxy modified nucleotides, locked nucleic acid modified nucleotides, unlocked nucleic acid modified nucleotides (UNA), glycol nucleic acid (GNA), 5'-vinyl phosphate modified nucleotides, 5'-(E)-VP modified nucleic acids, invAb modified nucleotides, invdA modified nucleotides, i substituted nucleotides and Y substituted nucleotides, wherein the Y is The i is The 2'- refers to the 2-position of ribose, for example, a 2'-fluoro-modified nucleotide refers to a nucleotide in which the 2-position of ribose is replaced by fluorine, and a 2'-methoxy-modified nucleotide refers to a nucleotide in which the 2-position of ribose is replaced by methoxy.

[0018] In some embodiments of the double-stranded siRNA, its conjugates or salts described herein, the modified nucleotides are each independently present at one or more positions selected from the following:

[0019] The nucleotides at the 5' end of the sense strand are positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 and 21 of the starting point.

[0020] In some embodiments of the double-stranded siRNA, its conjugates or salts described herein, the modified nucleotides are each independently present at one or more positions selected from the following:

[0021] The nucleotides at the 5' end of the antisense strand are positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 and 23 of the starting point.

[0022] In some embodiments of the double-stranded siRNA, its conjugates or salts described herein, the 5'-phosphorothioate linkage modification or the 3'-phosphorothioate linkage modification are each independently present at one or more positions selected from the group consisting of:

[0023] The nucleotides at the 5' end of the sense strand are between positions 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, 14-15, 15-16, 16-17, 17-18, 18-19, 19-20 and 20-21 of the starting point.

[0024] In some embodiments of the double-stranded siRNA, its conjugates or salts described herein, the 5'-phosphorothioate linkage modification or the 3'-phosphorothioate linkage modification are each independently present at one or more positions selected from the group consisting of:

[0025] The nucleotides at the 5' end of the antisense strand are positions 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, 14-15, 15-16, 16-17, 17-18, between 18-19, 19-20, 20-21, 21-22, and between 22-23 of the starting point.

[0026] In some embodiments of the double-stranded siRNA, its conjugates or salts according to the present invention, the 2'-fluoro modified nucleotide, the 2'-methoxy modified nucleotide and Y are each independently optionally present at one or more positions selected from the following:

[0027] The nucleotides at the 5' end of the sense strand are positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 and 21 of the starting point.

[0028] In some embodiments of the double-stranded siRNA, its conjugates or salts according to the present invention, the 2'-fluoro modified nucleotide, the 2'-methoxy modified nucleotide and Y are each independently optionally present at one or more positions selected from the following:

[0029] The nucleotides at the 5' end of the antisense strand are positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 and 23 of the starting point.

[0030] In some embodiments of the double-stranded siRNA, its conjugates or salts described herein, the 2'-fluoro modified nucleotide is present at one or more positions selected from the group consisting of:

[0031] The nucleotides at the 5' end of the sense strand are the 5th, 7th, 8th, 9th, 10th and 11th nucleotides of the starting point.

[0032] In some embodiments of the double-stranded siRNA, its conjugates or salts described herein, the 2'-fluoro modified nucleotide is present at one or more positions selected from the group consisting of:

[0033] The nucleotides at the 5' end of the antisense strand are the 2nd, 6th, 8th, 9th, 12th, 14th and 16th nucleotides of the starting point.

[0034] In some embodiments of the double-stranded siRNA, its conjugates or salts described herein, the 5'-phosphorothioate linkage modification or the 3'-phosphorothioate linkage modification are each independently present at one or more positions selected from the group consisting of:

[0035] The nucleotides at the 5' end of the sense strand are between positions 1-2, 2-3 and 3-4 of the starting point.

[0036] In some embodiments of the double-stranded siRNA, its conjugates or salts described herein, the 5'-phosphorothioate linkage modification or the 3'-phosphorothioate linkage modification are each independently optionally present at one or more positions selected from the group consisting of:

[0037] The nucleotides at the 3' end of the sense strand are between positions 1-2, 2-3 and 3-4 of the starting point.

[0038] In some embodiments of the double-stranded siRNA, its conjugates or salts described herein, the 5'-phosphorothioate linkage modification or the 3'-phosphorothioate linkage modification are each independently present at one or more positions selected from the group consisting of:

[0039] The nucleotides at the 5' end of the antisense strand are between positions 1-2, 2-3 and 3-4 of the starting point.

[0040] In some embodiments of the double-stranded siRNA, its conjugates or salts described herein, the 5'-phosphorothioate linkage modification or the 3'-phosphorothioate linkage modification are each independently present at one or more positions selected from the group consisting of:

[0041] The nucleotides at the 3' end of the antisense strand are between positions 1-2, 2-3 and 3-4 of the starting point.

[0042] The term "each independently optionally exists in one or more positions selected from the following" or "one or more positions" in "each independently exists in one or more positions selected from the following" as used herein refers to the presence of modifications at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 positions, where "optionally" means that the modification may be present or there may be no modification, i.e., 0 modification.

[0043] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the double-stranded siRNA comprises at least one Y, wherein Y is

[0044] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the double-stranded region is 17-23 nucleotide pairs in length.

[0045] In some embodiments of the double-stranded siRNA, its conjugate or salt described in the present invention, the sense strand comprises one of the nucleotide sequences shown in SEQ ID NO: 57 to SEQ ID NO: 84 (i.e., one of the sense strands in Table 2 of the present specification), and the length of the sense strand does not exceed 21 nucleotides, wherein the detailed information of the nucleotide sequences of SEQ ID NO: 57 to SEQ ID NO: 84 is shown in Table 2 of the present specification.

[0046] In some embodiments of the double-stranded siRNA, its conjugate or salt described in the present invention, the antisense strand comprises one of the nucleotide sequences shown in SEQ ID NO:85 to SEQ ID NO:112 (i.e., one of the antisense strands in Table 2 of the present specification), and the length of the antisense strand does not exceed 23 nucleotides, wherein the detailed information of the nucleotide sequences of SEQ ID NO:85 to SEQ ID NO:112 is shown in Table 2 of the present specification.

[0047] In some embodiments of the double-stranded siRNA, conjugate, or salt thereof described herein, the double-stranded siRNA, conjugate, or salt thereof described herein comprises one of the double-stranded siRNAs shown in siRNA ID NO: 29 to siRNA ID NO: 56; wherein the sense strand is no longer than 21 nucleotides, and the antisense strand is no longer than 23 nucleotides. For details of the nucleotide information of the double-stranded siRNAs of siRNA ID NO: 29 to siRNA ID NO: 56, please refer to Table 2 of the present specification.

[0048] In some embodiments of the double-stranded siRNA, its conjugate or salt described in the present invention,

[0049] The sense strand comprises a nucleotide sequence of 5'-csasagcuUfgGfUfCfaucuausgsa-3', and the antisense strand comprises a nucleotide sequence of 5'-usCfsauaGfaugaccaAfgCfuugsgsc-3'; or

[0050] The sense strand comprises the nucleotide sequence of 5'-cscsacagAfaAfAfCfuacccasasa-3', and the antisense strand comprises the nucleotide sequence of 5'-usUfsuggGfuaguuuuCfuGfuggsusc-3'; or

[0051] The sense strand comprises a nucleotide sequence of 5'-cscsaaauGfcUfGfGfcuugauscsa-3', and the antisense strand comprises a nucleotide sequence of 5'-usGfsaucAfagccagcAfuUfuggsgsu-3'; or

[0052] The sense strand comprises a nucleotide sequence of 5'-gsusgcuaCfcAfUfGfguaaugsgsa-3', and the antisense strand comprises a nucleotide sequence of 5'-usCfscauUfaccauggUfaGfcacsusc-3'; or

[0053] The sense strand comprises the nucleotide sequence of 5'-csasaccuGfaCfAfCfaaugcuscsa-3', and the antisense strand comprises the nucleotide sequence of 5'-usGfsagcAfuugugucAfgGfuugscsa-3'; or

[0054] The positive chain comprises a nucleotide sequence of 5'-cscsuagaGfgCfUfCfcuucugsasa-3', and the antisense chain comprises a nucleotide sequence of 5'-

[0055] usUfscagAfaggagccUfcUfaggscsu-3'; or

[0056] The sense strand comprises the nucleotide sequence of 5'-gsasaaugUfcCfUfGfgaagcasusa-3', and the antisense strand comprises the nucleotide sequence of 5'-usAfsugcUfuccaggaCfaUfuucsusu-3'; or

[0057] The sense strand comprises a nucleotide sequence of 5'-csasugguAfaUfGfGfacagagsusa-3', and the antisense strand comprises a nucleotide sequence of 5'-usAfscucUfguccauuAfcCfaugsgsu-3'.

[0058] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the length of the sense strand does not exceed 21, 20 or 19 nucleotides, and the length of the sense strand does not exceed 23, 22 or 21 nucleotides.

[0059] In some embodiments of the double-stranded siRNA, its conjugate or salt described in the present invention, the double-stranded siRNA conjugate is formed by conjugating the double-stranded siRNA to a conjugating group.

[0060] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, in the double-stranded siRNA conjugate, the 3' end or the 5' end of the sense strand of the double-stranded siRNA is conjugated to a conjugation group.

[0061] In some embodiments of the double-stranded siRNA, its conjugate or salt thereof according to the present invention, in the double-stranded siRNA conjugate, the 3' end or the 5' end of the antisense strand of the double-stranded siRNA is conjugated to a conjugation group.

[0062] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, preferably, the 3' end of the sense strand of the double-stranded siRNA is conjugated to the conjugate.

[0063] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the 3' end or the 5' end of the sense strand of the double-stranded siRNA is conjugated to the conjugation group through a phosphate bond or a phosphorothioate bond.

[0064] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the conjugated group comprises GalNAc or a derivative thereof.

[0065] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the conjugated group is GalNAc or a derivative thereof connected via a divalent, trivalent or tetravalent branched linker.

[0066] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the conjugated group is L-96, DAW40007-4, NAG37, NAG25 or a stereoisomer thereof, wherein the structures of the conjugated groups DAW40007-4, L-96, NAG37 and NAG25 are respectively:

[0067] In some embodiments of the double-stranded siRNA, its conjugate or salt, or double-stranded siRNA conjugate or salt thereof described in the present invention, the double-stranded siRNA or its conjugate or salt further includes (R)- and (S)-enantiomers, diastereomers, and / or racemic mixtures thereof.

[0068] In some embodiments of the double-stranded siRNA, its conjugate or salt, or double-stranded siRNA conjugate or salt thereof described in the present invention, the phosphorothioate portion of the double-stranded siRNA or its conjugate or salt includes (R)- and (S)-enantiomers, diastereomers, and / or racemic mixtures thereof.

[0069] In another aspect, the present invention provides a pharmaceutical composition comprising the double-stranded siRNA, its conjugate or salt thereof, the double-stranded siRNA conjugate or salt thereof, and a pharmaceutically acceptable carrier according to the present invention.

[0070] In another aspect, the present invention provides use of the double-stranded siRNA, its conjugate or salt thereof, and the pharmaceutical composition of the present invention in the preparation of a medicament for treating and / or preventing LPA-related diseases.

[0071] In some embodiments of the use described in the present invention, the LPA-related disease is a metabolic disease or a cardiovascular disease.

[0072] In some embodiments of the use described herein, the LPA-related disease is hypertriglyceridemia, obesity, dyslipidemia, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, hyperlipidemia, abnormal lipid and / or cholesterol metabolism, atherosclerosis, cardiovascular disease, coronary artery disease, hypertriglyceridemia-induced pancreatitis, metabolic syndrome, type II diabetes, familial chylomicronemia syndrome, chylomicronemia, multifactorial chylomicronemia, lipodystrophy syndrome, NAFLD (non-alcoholic fatty liver disease), NASH (non-alcoholic steatohepatitis) or familial partial lipodystrophy.

[0073] In some embodiments of the use of the present invention, the LPA-related disease is atherosclerosis, dyslipidemia, NAFLD or NASH.

[0074] In yet another aspect, the present invention provides the double-stranded siRNA, its conjugate and salt thereof, the double-stranded siRNA conjugate or its salt, and the pharmaceutical composition of the present invention for preventing, treating, curing or alleviating LPA-related diseases in patients.

[0075] In some embodiments of the double-stranded siRNA, its conjugate and salt thereof, the double-stranded siRNA conjugate or its salt, and the pharmaceutical composition of the present invention for preventing, treating, treating or alleviating LPA-related diseases in patients, the LPA-related diseases are dyslipidemia, hypertriglyceridemia, obesity, dyslipidemia, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, hyperlipidemia, abnormal lipid and / or cholesterol metabolism, atherosclerosis, cardiovascular disease, coronary artery disease, hypertriglyceridemia-induced pancreatitis, metabolic syndrome, type II diabetes, familial chylomicronemia syndrome, chylomicronemia, multifactorial chylomicronemia, lipodystrophy syndrome or familial partial lipodystrophy.

[0076] In yet another aspect, the present invention provides a method for preventing, treating, curing or alleviating an LPA-related disease in a patient, comprising administering to the patient an effective therapeutic amount of the double-stranded siRNA, its conjugate or salt thereof, or the pharmaceutical composition of the present invention.

[0077] In some embodiments of the methods described herein, the LPA-associated disease is dyslipidemia, hypertriglyceridemia, obesity, dyslipidemia, nonalcoholic steatohepatitis, nonalcoholic fatty liver disease, hyperlipidemia, abnormal lipid and / or cholesterol metabolism, atherosclerosis, cardiovascular disease, coronary artery disease, hypertriglyceridemia-induced pancreatitis, metabolic syndrome, type II diabetes, familial chylomicronemia syndrome, chylomicronemia, multifactorial chylomicronemia, lipodystrophy syndrome, or familial partial lipodystrophy.

[0078] Detailed description of the present invention

[0079] Definitions and General Terms

[0080] In the present invention, the term "comprise" or "include" is an open expression, that is, it includes the content specified in the present invention, but does not exclude other aspects of the content.

[0081] As used herein, the term "small interfering RNA (siRNA)" refers to a double-stranded RNA (17 to 30 nucleotides) comprising a sense strand and an antisense strand. siRNAs mediate the targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway by forming a silencing complex. Specifically, siRNAs direct the specific degradation of mRNA sequences through the well-known RNA interference (RNAi) process, inhibiting the translation of mRNA into amino acids and protein.

[0082] The sense strand and antisense strand typically form a double-stranded siRNA ("dsRNA"), also referred to as an "RNAi agent" in the present invention. The double-stranded region of an RNAi agent can be 12-30 nucleotide pairs long. For example, the duplex region can be 14-30 nucleotide pairs long, 17-30 nucleotide pairs long, 27-30 nucleotide pairs long, 17-23 nucleotide pairs long, 17-21 nucleotide pairs long, 17-19 nucleotide pairs long, 19-25 nucleotide pairs long, 19-23 nucleotide pairs long, 19-21 nucleotide pairs long, 21-25 nucleotide pairs long, or 21-23 nucleotide pairs long. In another embodiment, the duplex region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides long.

[0083] The terms "iRNA," "RNAi agent," "iRNA agent," and "RNA interfering agent" are used interchangeably herein to refer to RNA agents, as those terms are defined herein, that mediate targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway. iRNAs direct sequence-specific degradation of mRNAs through a process known as RNA interference (RNAi). iRNAs modulate, e.g., inhibit, expression of LPA in a cell, e.g., a cell of a subject, e.g., a mammalian subject.

[0084] In the present invention, the term "antisense strand (or guide strand)" includes a region that is substantially complementary to a target sequence. "Sense strand (or follower strand)" refers to an RNAi strand that is substantially complementary to the antisense strand. The term "substantially complementary" refers to complete complementarity or at least partial complementarity, for example, the antisense strand is completely complementary to the target sequence or at least partially complementary. In the case of partial complementarity, mismatches can exist within the interior or terminal regions of the molecule, wherein the most tolerated mismatches exist within the terminal regions, for example, within 5, 4, 3 or 2 nucleotides of the 5'- and / or 3'-end of the RNAi.

[0085] It should be noted that the "at least part of the antisense strand being substantially complementary to the mRNA" means that the antisense strand has a polynucleotide that is substantially complementary to a continuous part of the mRNA of interest.

[0086] The term "nucleotide overhang" or "overhang" refers to at least one unpaired nucleotide that overhangs the duplex structure of an iRNA (e.g., dsRNA). For example, a nucleotide overhang exists when the 3'-end of one strand of a dsRNA extends beyond the 5'-end of the other strand, or vice versa. A dsRNA can include an overhang of at least one nucleotide; alternatively, the overhang can include at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 or more nucleotides. The nucleotide overhang can include or consist of nucleotide / nucleoside analogs (including deoxynucleotides / nucleosides). One or more overhangs can be on the sense strand, the antisense strand, or any combination thereof. Additionally, the one or more nucleotides of the overhang can be present on the 5' end, the 3' end, or both ends of the antisense or sense strand of the dsRNA.

[0087] The conjugate groups described herein include pharmaceutically acceptable conjugate groups. Generally, pharmaceutically acceptable conjugate groups include a pharmaceutically acceptable targeting molecule and an optional linker. Exemplary conjugate groups, linkers, and targeting molecule types can be found in the disclosures of WO2015006740A2 and CN114555188A. Exemplary conjugate groups include, but are not limited to, L96, NAG37, NAG25, or DAW40007-4.

[0088] Unless otherwise indicated, "conjugation" refers to the covalent linkage of two or more chemical moieties, each with a specific function, to each other; accordingly, "conjugate" refers to a compound formed by covalent linkage of the chemical moieties.

[0089] The double-stranded siRNA conjugate of the present invention is a compound formed by connecting the double-stranded siRNA and a pharmaceutically acceptable conjugated group, and the double-stranded siRNA and the pharmaceutically acceptable conjugated group are covalently linked.

[0090] When used for the treatment of a disease, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of combining the active ingredient with an excipient that constitutes one or more adjunct ingredients. Typically, the composition is prepared by uniformly and thoroughly combining the active siRNA with a liquid excipient, a finely divided solid excipient, or both.

[0091] In the present invention, the term "pharmaceutically acceptable" means that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients of the formulation and / or the mammal to be treated therewith. Preferably, the "pharmaceutically acceptable" herein means approved by federal regulatory agencies or national governments or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia for use in animals, particularly humans.

[0092] In the present invention, the term "pharmaceutically acceptable carrier" may include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for the specific target dosage form. Except to the extent that any conventional carrier is incompatible with the RNAi (e.g., siRNA) of the present invention, for example, by producing any adverse biological effects or interacting in a deleterious manner with any other component of the pharmaceutically acceptable composition, their use is also contemplated by the present invention.

[0093] In addition to any conventional carriers, to the extent that they are incompatible with the RNAi (e.g., siRNA) of the present invention, for example, by producing any adverse biological effects or interacting in a deleterious manner with any other component of the pharmaceutically acceptable composition, their use is also contemplated by the present invention.

[0094] In some embodiments, according to the pharmaceutical composition of the present invention, the pharmaceutically acceptable carrier may be any of various carriers conventionally used in the art, for example, it may include at least one of a pH buffer, a protective agent, and an osmotic pressure regulator. The pH buffer may be acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof. The pH buffer may 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, preferably a phosphate buffer with a pH of 5.5-8.5. The protective agent may be at least one of inositol, sorbitol, and sucrose. Based on the total weight of the pharmaceutical composition, the content of the protective agent may be 0.01-30% by weight (e.g., 0.01% by weight, 0.05% by weight, 0.1% by weight, 0.5% by weight, 1% by weight, 5% by weight, 10% by weight, 15% by weight, 20% by weight, 25% by weight, 30% by weight, or any value between any two of the above values). The osmotic pressure regulator may be sodium chloride and / or potassium chloride. The content of the osmotic pressure regulator is such that the osmotic pressure of the pharmaceutical composition is 200-700 mOsmole / kg. According to the desired osmotic pressure, those skilled in the art can determine the content of the osmotic pressure regulator.

[0095] In the present invention, the term "treatment" refers to the use of a pharmacological and / or physiological effect to obtain the desired effect. The effect may be preventive in terms of complete or partial prevention of a disease or its symptoms, and / or therapeutic in terms of partial or complete cure of a disease and / or adverse effects caused by the disease. The "treatment" used in the present invention covers diseases in mammals, especially humans, including: (a) preventing the occurrence of a disease or condition in individuals who are susceptible to the disease but have not yet been diagnosed with the disease; (b) inhibiting the disease, such as blocking the development of the disease; or (c) alleviating the disease, such as alleviating symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a drug, RNAi agent or siRNA to an individual to treat, cure, alleviate, improve, reduce or inhibit the individual's disease, including but not limited to administering a drug containing the RNAi agent, siRNA or siRNA conjugate of the present invention to an individual in need.

[0096] As used herein, the term "RNAi agent" refers to an agent containing RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecules that are capable of degrading or inhibiting the transcription and translation of target messenger RNA (mRNA) in a sequence-specific manner. The RNAi agent herein may be manipulated via an RNA interference mechanism (i.e., by inducing RNA interference through interaction with the RNA interference pathway machinery of mammalian cells (RNA-induced silencing complex or RISC)), or may act via any other mechanism or pathway. RNAi agents include, but are not limited to, single-stranded oligonucleotides, single-stranded antisense oligonucleotides, short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), and Dicer substrates.

[0097] Unless otherwise specified, in the context of the present invention, capital letters C, G, U, and A represent bases of natural nucleotides; lowercase letters represent bases whose ribose position 2 is modified by a methoxy group, such as c, g, u, and a represent 2'-OMe (2'-O-methyl) C, 2'-OMe G, 2'-OMe U, and 2'-OMe A, respectively; the f to the right of the capital letter represents a base whose ribose position 2 is modified by a fluorine group, such as Cf, Gf, Uf, and Af represent 2'-F (2'-fluoro) C, 2'-FG, 2'-FU, and 2'-FA, respectively; "s" indicates that the two nucleotide residues adjacent to the left and right of "s" are connected by a thiophosphate group, for example, "gsu" indicates that the g and u residues are connected by a thiophosphate group; Tgn represents a thymine-diol nucleotide residue, whose structure is Y in the double-stranded siRNA or its conjugate represents invAb represents an inverted abasic deoxynucleoside residue having the structure shown below: invdA

[0098] The structures of TNA, PNA, D-FNA, ANA, HNA, FANA, bcDNA, tcDNA, S-MC, N-MC and 2'-F-NMC of the present invention are as follows:

[0099] Wherein, B is a base (including a natural base (A, U, G, C or T) or a modified base), and each TNA, PNA, D-FNA, HNA, FNA, bcDNA, tcDNA, S-MC, N-MC and 2'-F-NMC is independently linked to the remaining nucleosides or conjugated groups through a phosphate bond or a phosphorothioate bond.

[0100] The structure of the nucleoside (UNA) modified by unlocking nucleic acid of the present invention is The structure of the glycol nucleic acid modified nucleoside (GNA) is B is a base (including a natural base (A, U, G, C or T) or a modified base).

[0101] In the present invention, A, U, G, C or T is a base or a nucleoside containing a base. Whether it is a base or a nucleoside containing a base should be understood according to common knowledge in the art. For example, in the present invention, Wherein B is a base (A, U, G, C or T) or a modified base, wherein A, U, G, C or T should be understood as a base A, U, G, C or T without sugar. For example, A, U, G, C or T in Table 1 of the present invention should be understood as a nucleoside containing the base A, U, G, C or T.

[0102] In the present invention, "phosphate group," "phosphate group," and "phosphate bond" are used interchangeably to refer to monoesters, phosphodiesters, or phosphotriesters. The term "phosphate group" in "phosphorothioate group" also has the same meaning. Unless otherwise specified, a natural internucleotide phosphate group is a phosphodiester group.

[0103] In the present invention, "deoxynucleotide" refers to a nucleotide obtained by deoxygenating the hydroxyl group in the pentose of the nucleotide, and the deoxygenation position can be 2'-OH or 3'-OH.

[0104] In some optional embodiments of the present invention, the deoxynucleotides include 3'-deoxy-modified nucleotides and 2'-deoxy-modified nucleotides.

[0105] In the present invention, "2'-deoxy modification" refers to the deoxygenation of the hydroxyl group (2'-OH) in the pentose nucleotide to hydrogen (2'-H), and "3'-deoxy modification" refers to the deoxygenation of the hydroxyl group (3'-OH) in the pentose nucleotide to hydrogen (3'-H).

[0106] In the present invention, "2'-X modification" refers to the replacement of the hydroxyl group (2'-OH) in the pentose of a nucleotide with X (2'-X). For example: "2'-fluoro modification" refers to the replacement of the hydroxyl group (2'-OH) in the nucleotide pentose by fluorine (2'-F), "2'-amino modification" refers to the replacement of the hydroxyl group (2'-OH) in the nucleotide pentose by fluorine (2'-NH2), "2'-O-allyl modification" refers to the replacement of the hydroxyl group (2'-OH) in the nucleotide pentose by an allyloxy group (2'-OCH2CH=CH2), "2'-alkyl modification" refers to the replacement of the hydroxyl group (2'-OH) in the nucleotide pentose by an alkyl group (2'-alkyl), "2'-O-alkyl modification" refers to the replacement of the hydroxyl group (2'-OH) in the nucleotide pentose by an alkoxy group (2'-alkoxy), "2'-methoxy modification" refers to the replacement of the hydroxyl group (2'-OH) in the nucleotide pentose by a methoxy group (2'-OCH3), and "2'-methoxyethyl modification" refers to the replacement of the hydroxyl group (2'-OH) in the nucleotide pentose by a methoxyethyl group (2'-OCH2CH2OCH3).

[0107] In the present invention, "locked nucleotide" refers to a nucleotide obtained by modifying the 2' and 4' carbons of the pentose sugar of the nucleotide to be linked together.

[0108] In the present invention, "5'-X modification" refers to the replacement of a phosphate group (5'-PO(OH)2) in a nucleotide pentose by an X(5'-X). For example, "5'-phosphoramidate modification" refers to the replacement of a phosphate group (5'-PO(OH)2) in a nucleotide pentose by a phosphoramidate group, "5'-phosphorothioate modification" refers to the replacement of a phosphate group (5'-PO(OH)2) in a nucleotide pentose by a phosphorothioate group, "5'-methylphosphonate modification" refers to the replacement of a phosphate group (5'-PO(OH)2) in a nucleotide pentose by a methylphosphonate group, and "5'-phosphate mimetic modification" refers to the replacement of a phosphate group (5'-PO(OH)2) in a nucleotide pentose by a phosphate mimetic.

[0109] In the present invention, “5′-methylated cytosine modification” or “5-methylcytosine modification” refers to methylation of the 5th carbon atom of cytosine; “5′-methylated uracil modification” or “5-methyluracil modification” refers to methylation of the 5th carbon atom of uracil.

[0110] In the present invention, the "2'" in 2'-fluoro modified nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-alkyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-methoxyethyl modified nucleotides, and 2'-allyl modified nucleotides means that the 2-position of the ribose is modified by the corresponding group.

[0111] As used herein, "chemically modified" or "modification" refers to a structure that is chemically different when compared to its naturally occurring counterpart, including all changes by chemical means, such as the addition or removal of a chemical moiety, or the substitution of one chemical moiety for another.

[0112] The compounds of the present invention may be asymmetric, for example, having one or more stereoisomers. Unless otherwise indicated, all stereoisomers are included, such as enantiomers and diastereomers. Compounds of the present invention containing asymmetric carbon atoms can be isolated in optically pure or racemic forms. Optically pure forms can be resolved from racemic mixtures or synthesized by using chiral starting materials or chiral reagents.

[0113] Optically active (R)- and (S)-isomers, as well as D and L isomers, can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the invention is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide the pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), diastereomeric salts are formed with an appropriate optically active acid or base, followed by diastereomeric resolution by conventional methods known in the art, and the pure enantiomers are recovered. In addition, separation of enantiomers and diastereomers is typically accomplished by using chromatography using a chiral stationary phase, optionally combined with chemical derivatization (e.g., carbamate formation from an amine).

[0114] The present invention also includes isotopically labeled compounds of the present invention that are identical to those described herein, but in which one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as 2 H. 3 H. 11 C, 13C, 14 C. 13 N. 15 N. 15 O. 17 O. 18 O. 31 P. 32 P. 35 S. 18 F. 123 I. 125 I and 36 Cl et al.

[0115] Unless otherwise stated, when a position is particularly designated as deuterium (D), the position should be understood to have at least 1000 times the abundance of deuterium greater than the natural abundance of deuterium (which is 0.015%) (i.e., at least 10% deuterium incorporation). In the example, the compound has at least 1000 times the abundance of deuterium, at least 2000 times the abundance of deuterium, at least 3000 times the abundance of deuterium, at least 4000 times the abundance of deuterium, at least 5000 times the abundance of deuterium, at least 6000 times the abundance of deuterium or more abundant deuterium. The present invention also includes various deuterated forms of formula (I) compounds. Each available hydrogen atom connected to a carbon atom can be independently replaced by a deuterium atom. Those skilled in the art can synthesize deuterated forms of formula (I) compounds with reference to relevant literature. In preparing deuterated forms of compounds of formula (I), commercially available deuterated starting materials may be used, or they may be synthesized using conventional techniques using deuterated reagents, including but not limited to deuterated borane, trideuterated borane in tetrahydrofuran, deuterated lithium aluminum hydride, deuterated iodoethane, deuterated iodomethane, and the like.

[0116] The conjugated groups of the present invention can enhance the delivery of therapeutic agents to specific target locations (e.g., specific organs or tissues) within a subject such as a human or animal. In some embodiments of the present invention, the conjugated groups can enhance the targeted delivery of expression-inhibitory oligonucleotides. In some embodiments of the present invention, the conjugated groups can enhance the delivery of expression-inhibitory oligonucleotides to the liver.

[0117] The conjugated groups of the present invention can be directly or indirectly connected to a compound, such as a therapeutic agent, for example, an expression inhibitory oligonucleotide, for example, the 3' or 5' end of an expression inhibitory oligonucleotide. In some embodiments of the present invention, the expression inhibitory oligonucleotide comprises one or more modified nucleotides. In some embodiments of the present invention, the expression inhibitory oligonucleotide is an RNAi agent, such as a double-stranded RNAi agent comprising a sense strand and an antisense strand. In some embodiments of the present invention, the conjugated groups disclosed in the present invention are connected to the 3' end of the sense strand of the double-stranded RNAi agent. In some embodiments, the conjugated groups disclosed in the present invention are connected to the expression inhibitory oligonucleotide agent at the 3' end of the sense strand of the double-stranded RNAi agent via a phosphate, phosphorothioate or phosphonate group.

[0118] The definitions and conventions of stereochemistry used herein are generally those of S.P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds", John Wiley & Sons, Inc., New York, 1994. The compounds of the present invention may contain asymmetric centers or chiral centers and therefore exist as different stereoisomers. All stereoisomeric forms of the compounds of the present invention, including but not limited to diastereomers, enantiomers, atropisomers, and mixtures thereof, such as racemic mixtures, form part of the present invention. Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane-polarized light. When describing an optically active compound, the prefix D, L or R, S is used to indicate the absolute configuration of the molecule about its chiral center. The prefixes d, l, or (+), (-) are used to designate the sign of rotation of plane-polarized light in a compound. (-) or l means the compound is levorotatory, and the prefix (+) or d means the compound is dextrorotatory. These stereoisomers have the same chemical structure, but their stereostructures are different. Specific stereoisomers can be enantiomers, and a mixture of isomers is often called an enantiomeric mixture. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which may result in a lack of stereoselectivity or stereospecificity during chemical reactions. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomers that lacks optical activity.

[0119] The term "tautomer" or "tautomeric form" refers to structural isomers of different energies that are interconvertible via a low energy barrier. For example, proton tautomers (i.e., prototropic tautomers) include interconversions via proton migration, such as keto-enol and imine-enamine isomerizations.

[0120] The term "composition" refers to a mixture of one or more siRNAs, siRNA conjugates, or physiologically acceptable salts or precursors thereof described herein, along with other chemical components, as well as other components such as physiologically acceptable carriers and excipients. The purpose of a composition is to facilitate administration to an organism, facilitating absorption of the active ingredient and thereby exerting its biological activity.

[0121] The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable carrier" includes, but is not limited to, any adjuvant, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavoring agent, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier that has been approved by the U.S. Food and Drug Administration for use by humans or domestic animals.

[0122] Unless otherwise specified, the "compound," "ligand," "nucleic acid conjugate," "double-stranded siRNA conjugate," "double-stranded siRNA," and "nucleic acid" of the present invention may independently exist in the form of a salt, a mixed salt, or a non-salt form (e.g., a free acid or a free base). When present in the form of a salt or a mixed salt, it may be a pharmaceutically acceptable salt.

[0123] The term "acceptable salt" includes acceptable acid addition salts and pharmaceutically acceptable base addition salts. "Acceptable acid addition salts" refers to salts formed with inorganic or organic acids that retain the biological effectiveness of the free base without other side effects. Inorganic acid salts include, but are not limited to, hydrochlorides, hydrobromides, sulfates, nitrates, and phosphates; organic acid salts include, but are not limited to, formates, acetates, 2,2-dichloroacetates, trifluoroacetates, propionates, caproates, octanoates, decanoates, undecylenates, glycolates, gluconates, lactates, sebacates, adipates, glutarates, malonates, oxalates, maleates, succinates, fumarates, tartrates, citrates, palmitates, stearates, oleates, cinnamates, laurates, malates, glutamate, pyroglutamate, aspartate, benzoates, methanesulfonates, benzenesulfonates, p-toluenesulfonates, alginate, ascorbate, salicylates, 4-aminosalicylates, and naphthalene disulfonates. These salts can be prepared by methods known in the art.

[0124] "Pharmaceutically acceptable base addition salts" refer to salts formed with inorganic or organic bases that retain the biological effectiveness of the free acid without other side effects. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, aluminum salts, and the like. Preferred inorganic salts are ammonium salts, sodium salts, potassium salts, calcium salts, and magnesium salts, with sodium salts being preferred. Salts derived from organic bases include, but are not limited to, following salts: primary amines, secondary amines and tertiary amines, substituted amines, including natural substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine. These salts can be prepared by methods known in the art.

[0125] As described herein, the compounds of the present invention may optionally be substituted with one or more substituents, as described above for the general formula compounds, or as described in the specific examples, subclasses, and classes of compounds encompassed by the present invention. Generally, the term "substituted" refers to the replacement of one or more hydrogen atoms in a given structure with a specified substituent. Unless otherwise indicated, an optionally substituted group may have a substituent at each substitutable position of the group. When more than one position in a given formula can be substituted with one or more substituents selected from a specified group, the substituents may be the same or different at each position.

[0126] The term "hydroxy protecting group" refers to an unstable chemical moiety that protects the hydroxyl group from undesirable reactions during one or more synthesis procedures. After the one or more synthesis procedures, the hydroxy protecting group can be selectively removed. Hydroxy protecting groups as known in the art are generally described in TH Greene and PGM Wuts, Protective Groups in Organic Synthesis, 3rd edition, John Wiley & Sons, New York (1999). Examples of hydroxy protecting groups of the present invention include, but are not limited to, C 1-10alkylmethyl, benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4-bromobenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, methoxycarbonyl, tert-butoxycarbonyl, isopropyloxycarbonyl, biphenylmethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl, 2-furfuryloxycarbonyl, allyloxycarbonyl, acetyl (Ac or -C(O)CH3), formyl, chloroacetyl, trifluoroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl (Bz or -C(O)C6H5), C 1-10 Alkyl (methyl, tert-butyl, etc.), 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 1,1-dimethyl-2-propenyl, 3-methyl-3-butenyl, allyl, C 6-10 Aryl C 1-4 Alkyl (such as benzyl, phenethyl, etc.), p-methoxybenzyldiphenylmethyl, triphenylmethyl (triphenylmethyl or trityl), tetrahydrofuranyl, methoxymethyl, methylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, methanesulfonyl, p-toluenesulfonyl, C 1-10 Alkylsilyl (such as trimethylsilyl (TMS or -Si(CH3)3)), triethylsilyl, triisopropylsilyl, MMTr, DMTr or 4',4',4'-trimethoxytrityl, etc.

[0127] The term "amino protecting group" refers to an unstable chemical moiety that protects the amino group from undesirable reaction during the building-up sequence. After the one or more building-up sequences, the amino protecting group as described herein can be selectively removed. Amino protecting groups as known in the art are generally described in TH Greene and PGM Woods, Protective Groups in Organic Synthesis, the 3rd edition, John Wiley & Sons, New York (1999). The example of amino protecting groups includes but is not limited to acetyl, tert-butyloxycarbonyl, 9-fluorenylmethoxycarbonyl and benzyloxycarbonyl etc.

[0128] Term " solid phase support " specifically represents any particle, pearl or the surface on which oligonucleotide synthesis can occur.For example inorganic solid phase support and organic solid phase support, all can be selected for use in embodiments of the present invention.Inorganic solid phase support is preferably selected from silica gel and controlled pore glass beads (Controlled-pore glass is called for short CPG).Organic solid phase support is a resin, preferably macroporous resin, more preferably highly cross-linked polystyrene, Tentagel (graft copolymer made up of low cross-linked polystyrene matrix, polyethylene glycol (PEG or POE) is grafted thereon), polyvinyl acetate (PVA), copolymer, aminopolyethylene glycol and cellulose etc. of Bo Luosi (Poros)-polystyrene / divinylbenzene.Preferred embodiments of the present invention utilize solid phase support based on CPG.Many other purchasable solid phase supports all belong to the present invention.

[0129] Detailed description of the compounds of the present invention

[0130] The present invention provides a novel double-stranded siRNA and its conjugate, which can achieve RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of the LPA gene. The LPA gene can be within a cell, for example, within a cell in a subject, such as a human. The present invention also provides the use of the double-stranded siRNA and its conjugate in the preparation of a medicament for treating and / or preventing LPA-related diseases (such as dyslipidemia). The double-stranded siRNA and its conjugate can inhibit or reduce LPA gene expression, and are used to inhibit LPA gene expression by RNA-induced silencing complex (RISC)-mediated cleavage of LPA gene RNA transcripts. The siRNA and its conjugate of the present invention have good LPA inhibitory activity both in vivo and in vitro, have high in vivo delivery efficiency and good stability, and have high gene expression inhibitory activity and / or low toxicity against LPA.

[0131] In one aspect, the present invention provides a double-stranded siRNA, a conjugate or a salt thereof, comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises one of the nucleotide sequences shown in SEQ ID NO: 1 to SEQ ID NO: 28, or a nucleotide sequence having 0, 1, 2, 3, 4 or 5 nucleotide differences therefrom; wherein the detailed information of the nucleotide sequences shown in SEQ ID NO: 1 to SEQ ID NO: 28 is shown in Table 1 of the present specification.

[0132] In some embodiments of the double-stranded siRNA, conjugates or salts thereof according to the present invention, wherein the antisense strand of the present invention comprises one of the nucleotide sequences shown in SEQ ID NO:29 to SEQ ID NO:56, or a nucleotide sequence having 0, 1, 2, 3, 4 or 5 nucleotide differences therefrom;

[0133] Detailed information on the nucleotide sequences shown in SEQ ID NO: 29 to SEQ ID NO: 56 can be found in Table 1 of the present specification.

[0134] In some embodiments of the present invention, the double-stranded siRNA, its conjugate or salt thereof is used to inhibit LPA gene expression.

[0135] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the double-stranded siRNA is a modified double-stranded siRNA.

[0136] In some embodiments of the double-stranded siRNA, its conjugate or salt described in the present invention, the double-stranded siRNA conjugate is formed by conjugating the double-stranded siRNA to a conjugating group.

[0137] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the sense strand is no longer than 23 nucleotides, and the antisense strand is no longer than 23 nucleotides.

[0138] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the sense strand is 19, 20, 21, 22 or 23 nucleotides long, and the length of the sense strand does not exceed 23 nucleotides.

[0139] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the antisense strand is 21, 22 or 23 nucleotides long.

[0140] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the length of the sense strand does not exceed 23 nucleotides. The length of the sense strand does not exceed 23 nucleotides.

[0141] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the sense strand comprises a 3' overhang and / or a 5' overhang, and the 3' overhang or 5' overhang comprises 1, 2 or 3 nucleotides.

[0142] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the antisense strand comprises a 3' overhang and / or a 5' overhang, and the 3' overhang or 5' overhang comprises 1, 2 or 3 nucleotides.

[0143] In some embodiments of the double-stranded siRNA, its conjugate or salt described in the present invention, the double-stranded siRNA, its conjugate or salt comprises one of the double-stranded siRNAs shown in siRNA ID NO: 1 to siRNA ID NO: 29, wherein the information of the double-stranded siRNA shown in siRNA ID NO: 1 to siRNA ID NO: 29 is detailed in Table 1 of the specification of the present invention.

[0144] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the sense strand and / or the antisense strand comprises at least one modified nucleotide, and the modified nucleotide is independently and optionally selected from at least one of the following:

[0145] Non-natural base nucleotides, deoxynucleotides, 2'-fluoro modified nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-alkyl modified nucleotides, 2'-O-alkyl modified nucleotides (such as 2'-methoxy modified nucleotides), 2'-methoxyethyl modified nucleotides, locked nucleotides (LNA), unlocked nucleic acid modified nucleotides (UNA), 2'-allyl modified nucleotides, abasic nucleotides, invAb modified nucleotides, morpholino modified nucleotides, tetrahydropyran modified nucleotides, cyclohexenyl modified nucleotides, PEG modified nucleotides, 5'-phosphoramidate modified nucleotides, thiophosphate linked modified nucleotides (such as: 5'-thiophosphate linked modified nucleotides and / or the 3'-thiophosphate linked modified nucleotides), 5'-methylphosphonate modified nucleotides (such as 5'-( E)-VP modified nucleic acids), 5'-vinyl phosphate modified nucleic acids, 5'-phosphate mimetic modified nucleotides, TNA modified nucleotides, PNA modified nucleotides, D-FNA modified nucleotides, ANA modified nucleotides, HNA modified nucleotides, FANA modified nucleotides, bcDNA modified nucleotides, tcDNA modified nucleotides, S-MC modified nucleotides, N-MC modified nucleotides, 2'-F-NMC modified nucleotides, 5-methylcytosine modified nucleotides, 5-methyluracil modified nucleotides, 2,6-diamino modified adenine modified nucleotides and ethylene glycol nucleic acids (GNA), wherein the 2'- refers to the 2-position of ribose, such as a 2'-fluoro modified nucleotide refers to a nucleotide in which the 2-position of ribose is substituted by fluorine, and for example, a 2'-methoxy modified nucleotide refers to a nucleotide in which the 2-position of ribose is substituted by methoxy. In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the sense strand and / or the antisense strand comprises at least one modified nucleotide, and the modified nucleotide is independently and optionally selected from at least one of the following:

[0146] 2'-methoxy modified nucleotides, 2'-fluoro modified nucleotides, 2'-methoxyethyl modified nucleotides, 5'-phosphorothioate linked modified nucleotides, 3'-phosphorothioate linked modified nucleotides, 2'-deoxy modified nucleotides, 2'-amino modified nucleotides, 2'-hydroxy modified nucleotides, locked nucleic acid modified nucleotides, unlocked nucleic acid modified nucleotides (UNA), glycol nucleic acid (GNA), 5'-vinyl phosphate modified nucleotides, 5'-(E)-VP modified nucleic acids, invAb modified nucleotides, invdA modified nucleotides, i substituted nucleotides and Y substituted nucleotides, wherein Y is The i is The 2'- refers to the 2-position of ribose, for example, a 2'-fluoro-modified nucleotide refers to a nucleotide in which the 2-position of ribose is replaced by fluorine, and a 2'-methoxy-modified nucleotide refers to a nucleotide in which the 2-position of ribose is replaced by methoxy.

[0147] In some embodiments of the double-stranded siRNA, its conjugates or salts described herein, the modified nucleotides are each independently present at one or more positions selected from the following:

[0148] The nucleotides at the 5' end of the sense strand are positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 and 21 of the starting point.

[0149] In some embodiments of the double-stranded siRNA, its conjugates or salts described herein, the modified nucleotides are each independently present at one or more positions selected from the following:

[0150] The nucleotides at the 5' end of the antisense strand are positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 and 23 of the starting point.

[0151] In some embodiments of the double-stranded siRNA, its conjugates or salts described herein, the 5'-phosphorothioate linkage modification or the 3'-phosphorothioate linkage modification are each independently present at one or more positions selected from the group consisting of:

[0152] The nucleotides at the 5' end of the sense strand are between positions 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, 14-15, 15-16, 16-17, 17-18, 18-19, 19-20 and 20-21 of the starting point.

[0153] In some embodiments of the double-stranded siRNA, its conjugates or salts described herein, the 5'-phosphorothioate linkage modification or the 3'-phosphorothioate linkage modification are each independently present at one or more positions selected from the group consisting of:

[0154] The nucleotides at the 5' end of the antisense strand are positions 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, 14-15, 15-16, 16-17, 17-18, between 18-19, 19-20, 20-21, 21-22, and between 22-23 of the starting point.

[0155] In some embodiments of the double-stranded siRNA, its conjugates or salts according to the present invention, the 2'-fluoro modified nucleotide, the 2'-methoxy modified nucleotide and Y are each independently optionally present at one or more positions selected from the following:

[0156] The nucleotides at the 5' end of the sense strand are positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 and 21 of the starting point.

[0157] In some embodiments of the double-stranded siRNA, its conjugates or salts according to the present invention, the 2'-fluoro modified nucleotide, the 2'-methoxy modified nucleotide and Y are each independently optionally present at one or more positions selected from the following:

[0158] The nucleotides at the 5' end of the antisense strand are positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 and 23 of the starting point.

[0159] In some embodiments of the double-stranded siRNA, its conjugates or salts described herein, the 2'-fluoro modified nucleotide is present at one or more positions selected from the group consisting of:

[0160] The nucleotides at the 5' end of the sense strand are the 5th, 7th, 8th, 9th, 10th and 11th nucleotides of the starting point.

[0161] In some embodiments of the double-stranded siRNA, its conjugates or salts described herein, the 2'-fluoro modified nucleotide is present at one or more positions selected from the group consisting of:

[0162] The nucleotides at the 5' end of the antisense strand are the 2nd, 6th, 8th, 9th, 12th, 14th and 16th nucleotides of the starting point.

[0163] In some embodiments of the double-stranded siRNA, its conjugates or salts described herein, the 5'-phosphorothioate linkage modification or the 3'-phosphorothioate linkage modification are each independently present at one or more positions selected from the group consisting of:

[0164] The nucleotides at the 5' end of the sense strand are between positions 1-2, 2-3 and 3-4 of the starting point.

[0165] In some embodiments of the double-stranded siRNA, its conjugates or salts described herein, the 5'-phosphorothioate linkage modification or the 3'-phosphorothioate linkage modification are each independently optionally present at one or more positions selected from the group consisting of:

[0166] The nucleotides at the 3' end of the sense strand are between positions 1-2, 2-3 and 3-4 of the starting point.

[0167] In some embodiments of the double-stranded siRNA, its conjugates or salts described herein, the 5'-phosphorothioate linkage modification or the 3'-phosphorothioate linkage modification are each independently present at one or more positions selected from the group consisting of:

[0168] The nucleotides at the 5' end of the antisense strand are between positions 1-2, 2-3 and 3-4 of the starting point.

[0169] In some embodiments of the double-stranded siRNA, its conjugates or salts described herein, the 5'-phosphorothioate linkage modification or the 3'-phosphorothioate linkage modification are each independently present at one or more positions selected from the group consisting of:

[0170] The nucleotides at the 3' end of the antisense strand are between positions 1-2, 2-3 and 3-4 of the starting point.

[0171] The “at least one modified nucleotide” described in the present invention refers to the double-stranded siRNA, its conjugate or salt described in the present invention, wherein the sense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 modified nucleotides, and / or the antisense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 modified nucleotides.

[0172] The "one or more positions" in "one or more positions selected from the following" or "one or more positions selected from the following" as described in the present invention refers to the presence of modifications at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 positions, wherein "optionally" means that the modification may be or may not be present, that is, 0 modification. In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the double-stranded siRNA comprises at least one Y, and Y is

[0173] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the double-stranded siRNA comprises 2 Ys.

[0174] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the double-stranded siRNA comprises three Ys.

[0175] In some embodiments of the double-stranded siRNA, its conjugate or salt described in the present invention, the sense strand comprises one of the nucleotide sequences shown in SEQ ID NO: 57 to SEQ ID NO: 84 (i.e., one of the sense strands in Table 2 of the present specification), and the length of the sense strand does not exceed 21 nucleotides, wherein the detailed information of the nucleotide sequences of SEQ ID NO: 57 to SEQ ID NO: 84 is shown in Table 2 of the present specification.

[0176] In some embodiments of the double-stranded siRNA, its conjugate or salt described in the present invention, the antisense strand comprises one of the nucleotide sequences shown in SEQ ID NO:85 to SEQ ID NO:112 (i.e., one of the antisense strands in Table 2 of the present specification), and the length of the antisense strand does not exceed 23 nucleotides, wherein the detailed information of the nucleotide sequences of SEQ ID NO:85 to SEQ ID NO:112 is shown in Table 2 of the present specification.

[0177] In some embodiments of the double-stranded siRNA, conjugate, or salt thereof described herein, the double-stranded siRNA, conjugate, or salt thereof described herein comprises one of the double-stranded siRNAs shown in siRNA ID NO: 29 to siRNA ID NO: 56; wherein the sense strand is no longer than 21 nucleotides, and the antisense strand is no longer than 23 nucleotides. For details of the nucleotide information of the double-stranded siRNAs of siRNA ID NO: 29 to siRNA ID NO: 56, please refer to Table 2 of the present specification.

[0178] In some embodiments of the double-stranded siRNA, its conjugate or salt described in the present invention,

[0179] The sense strand comprises a nucleotide sequence of 5'-csasagcuUfgGfUfCfaucuausgsa-3', and the antisense strand comprises a nucleotide sequence of 5'-usCfsauaGfaugaccaAfgCfuugsgsc-3'; or

[0180] The sense strand comprises the nucleotide sequence of 5'-cscsacagAfaAfAfCfuacccasasa-3', and the antisense strand comprises the nucleotide sequence of 5'-usUfsuggGfuaguuuuCfuGfuggsusc-3'; or

[0181] The sense strand comprises a nucleotide sequence of 5'-cscsaaauGfcUfGfGfcuugauscsa-3', and the antisense strand comprises a nucleotide sequence of 5'-usGfsaucAfagccagcAfuUfuggsgsu-3'; or

[0182] The sense strand comprises a nucleotide sequence of 5'-gsusgcuaCfcAfUfGfguaaugsgsa-3', and the antisense strand comprises a nucleotide sequence of 5'-usCfscauUfaccauggUfaGfcacsusc-3'; or

[0183] The sense strand comprises the nucleotide sequence of 5'-csasaccuGfaCfAfCfaaugcuscsa-3', and the antisense strand comprises the nucleotide sequence of 5'-usGfsagcAfuugugucAfgGfuugscsa-3'; or

[0184] The positive chain comprises a nucleotide sequence of 5'-cscsuagaGfgCfUfCfcuucugsasa-3', and the antisense chain comprises a nucleotide sequence of 5'-

[0185] usUfscagAfaggagccUfcUfaggscsu-3'; or

[0186] The sense strand comprises the nucleotide sequence of 5'-gsasaaugUfcCfUfGfgaagcasusa-3', and the antisense strand comprises the nucleotide sequence of 5'-usAfsugcUfuccaggaCfaUfuucsusu-3'; or

[0187] The sense strand comprises a nucleotide sequence of 5'-csasugguAfaUfGfGfacagagsusa-3', and the antisense strand comprises a nucleotide sequence of 5'-usAfscucUfguccauuAfcCfaugsgsu-3'.

[0188] In some embodiments of the double-stranded siRNA, its conjugates or salts described herein, the length of the sense strand is no more than 21, 20 or 19 nucleotides, and the length of the sense strand is no more than 23, 22 or 21 nucleotides. In some embodiments of the double-stranded siRNA, its conjugates or salts described herein, the sense strand has one of the following modification patterns:

[0189] Sense strand (5'-3'): mBsmBsmBmBmBmBfBmBfBfBfBmBmBmBmBmBmBmBmB; and

[0190] Sense strand (5'-3'): mBsmBsmBmBmBmBfBmBfBfBfBmBmBmBmBmBmYmBmB;

[0191] wherein each B is independently a nucleoside of base A, base U, base G or base C, m is a 2'-methoxy modified ribose, s is a phosphorothioate bond, f is a 2'-F modified ribose, and if there is no s between the bases, it means that the nucleosides are connected by a phosphate bond, wherein Y is The 3' end of the positive strand is connected to L96 or DAW4007 via a phosphate bond or phosphorothioate.

[0192] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, its antisense strand has one of the following modification patterns:

[0193] Antisense strand (5'-3'): mBsfBsmBmBmBfBmBmBmBmBmBmBmBfBmBfBmBmBmBmBsmB;

[0194] Antisense strand (5'-3'): mBsfBsmBmBmBfBmBmBmBmBmBmBmBfBmBfBmBmBmBmBsmB;

[0195] Antisense strand (5'-3'): mBsfBsmBmBmBfBYmBmBmBmBmBmBfBmBfBmBmBmBsmBsmB;

[0196] Antisense strand (5'-3'): mBsfBsmBmBmBYmBmBmBmBmBmBmBfBmBfBmBmBmBmBsmBsmB;

[0197] Antisense strand (5'-3'): mBsfBsmBmBmBYmBmBmBmBmBmBmBfBmBfBmBmBmBmBsmB; and

[0198] Antisense strand (5'-3'): mBsfBsmBmBmBfBYmBmBmBmBmBmBfBmBfBmBmBmBsmBsmB;

[0199] Wherein, each B is independently a nucleoside of A, U, G or C, m is a 2'-methoxy modified ribose, s is a phosphorothioate bond, f is a 2'-F modified ribose, and if there is no s between the bases, it means that the nucleosides are connected by a phosphate bond, and Y is

[0200] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the double-stranded region is 17-23 nucleotide pairs in length.

[0201] In some embodiments of the double-stranded siRNA, conjugates or salts thereof according to the present invention, the double-stranded region is 17, 18, 19, 20, 21, 22 or 23 nucleotide pairs in length.

[0202] In some embodiments of the double-stranded siRNA, its conjugate or salt thereof according to the present invention, in the double-stranded siRNA conjugate, the 3' end or the 5' end of the antisense strand of the double-stranded siRNA is conjugated to a conjugation group.

[0203] In some embodiments of the double-stranded siRNA, its conjugate or salt thereof according to the present invention, in the double-stranded siRNA conjugate, the 3' end or the 5' end of the antisense strand of the double-stranded siRNA is conjugated to a conjugation group.

[0204] In some embodiments of the double-stranded siRNA, its conjugate or salt thereof according to the present invention, the 3' end of the sense strand of the double-stranded siRNA is conjugated to the conjugate.

[0205] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the 3' end or the 5' end of the sense strand of the double-stranded siRNA is conjugated to the conjugation group through a phosphate bond or a phosphorothioate bond.

[0206] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the conjugated group comprises GalNAc or a derivative thereof.

[0207] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the conjugated group is GalNAc or a derivative thereof connected via a divalent, trivalent or tetravalent branched linker.

[0208] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the conjugated group is DAW40007-4, L-96, NAG37, NAG25 or a stereoisomer thereof, wherein the structures of the conjugated groups DAW40007-4, L-96, NAG37 and NAG25 are respectively:

[0209] In some embodiments of the double-stranded siRNA, its conjugate or salt, or double-stranded siRNA conjugate or salt thereof described in the present invention, the double-stranded siRNA or its conjugate or salt further includes (R)- and (S)-enantiomers, diastereomers, and / or racemic mixtures thereof.

[0210] In some embodiments of the double-stranded siRNA, its conjugate or salt, or double-stranded siRNA conjugate or salt thereof described in the present invention, the phosphorothioate portion of the double-stranded siRNA or its conjugate or salt includes (R)- and (S)-enantiomers, diastereomers, and / or racemic mixtures thereof.

[0211] In another aspect, the present invention provides a pharmaceutical composition comprising the double-stranded siRNA, its conjugate or salt thereof, the double-stranded siRNA conjugate or salt thereof, and a pharmaceutically acceptable carrier according to the present invention.

[0212] In some embodiments, the pharmaceutical composition of the present invention may be an injection.

[0213] In some embodiments, the injection solution of the present invention can be used for subcutaneous, intramuscular or intravenous injection.

[0214] On the other hand, the present invention also provides a method for inhibiting the expression of the LPA gene in a patient, which comprises administering to the patient a double-stranded siRNA and a double-stranded siRNA conjugate of the present invention or a composition thereof (i.e., a double-stranded RNAi agent), wherein the nucleic acid ligand conjugate or the composition thereof may be in a therapeutically effective amount.

[0215] In one embodiment, administration of the double-stranded siRNA and double-stranded siRNA conjugates of the present invention or compositions thereof to the subject results in a decrease in blood lipid, triglyceride, cholesterol and / or free fatty acid levels.

[0216] In some embodiments, the double-stranded RNAi agent is administered at a dose of 0.01 mg / kg to 10 mg / kg or 0.5 mg / kg to 50 mg / kg, or at a dose of 10 mg / kg to 30 mg / kg, or at a dose of 3 mg / kg, or at a dose of 10 mg / kg.

[0217] In some embodiments, the double-stranded RNAi agent is administered at a dose of 0.5 mg / kg twice a week, or at a dose of 10 mg / kg every other week, or at a dose of 0.5-1 mg / kg once a week.

[0218] In some embodiments, the double-stranded RNAi agent is administered subcutaneously or intravenously.

[0219] In some embodiments, the double-stranded RNAi agent is administered in two or more doses.

[0220] In another aspect, the present invention provides use of the double-stranded siRNA, its conjugate or salt thereof, and the pharmaceutical composition of the present invention in the preparation of a medicament for treating and / or preventing LPA-related diseases.

[0221] In some embodiments of the use described in the present invention, the LPA-related disease is a metabolic disease or a cardiovascular disease.

[0222] In some embodiments of the use described herein, the LPA-related disease is hypertriglyceridemia, obesity, dyslipidemia, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, hyperlipidemia, abnormal lipid and / or cholesterol metabolism, atherosclerosis, cardiovascular disease, coronary artery disease, hypertriglyceridemia-induced pancreatitis, metabolic syndrome, type II diabetes, familial chylomicronemia syndrome, chylomicronemia, multifactorial chylomicronemia, lipodystrophy syndrome, NAFLD (non-alcoholic fatty liver disease), NASH (non-alcoholic steatohepatitis) or familial partial lipodystrophy.

[0223] In some embodiments of the use of the present invention, the LPA-related disease is atherosclerosis, dyslipidemia, NAFLD or NASH.

[0224] Pharmaceutical compositions, preparations, administration and disease treatment methods of the nucleic acid conjugates of the present invention

[0225] The effective amount of the nucleic acid conjugates (e.g., siRNA conjugates or pharmaceutical compositions) of the present invention may vary depending on the mode of administration and the severity of the disease to be treated. The preferred effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to, the pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration.

[0226] The pharmaceutical compositions or medicaments of the present invention comprise a pharmacologically effective amount of at least one LPA RNAi agent and one or more pharmaceutically acceptable excipients. A pharmaceutically acceptable excipient is a substance, other than the active pharmaceutical ingredient (API, therapeutic product, such as the LPA RNAi agent of the present invention), that is intentionally included in a drug delivery system. An excipient does not exert or is not intended to exert a therapeutic effect at the intended dose. Excipients may function to a) aid in the processing of the drug delivery system during manufacturing, b) protect, support, or enhance the stability, bioavailability, or patient acceptability of the API, c) aid in product identification, and / or d) enhance any other properties of the overall safety and efficacy of API delivery during storage or use. Pharmaceutically acceptable excipients may or may not be inert substances.

[0227] Excipients may include, but are not limited to, absorption enhancers, antiadherents, antifoaming agents, antioxidants, binders, buffers, carriers, coatings, colorants, delivery enhancers, delivery polymers, dextran, dextrose, diluents, disintegrants, emulsifiers, extenders, fillers, flavorings, glidants, humectants, lubricants, oils, polymers, preservatives, saline, salts, solvents, sugars, suspending agents, sustained-release matrices, sweeteners, thickeners, tonicity agents, vehicles, waterproofing agents, and wetting agents.

[0228] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (water soluble). For subcutaneous or intravenous administration, suitable carriers may include physiological saline, antibacterial water, The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol) and a suitable mixture thereof.

[0229] Sterile injectable solutions can be prepared by mixing the required amount of the active compound with one or a combination of the ingredients listed above (if necessary) in an appropriate solvent, followed by filtered sterilization. Typically, dispersions are prepared by mixing the active compound into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those listed above.

[0230] LPA RNAi agents can be formulated into compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect, in association with the required pharmaceutical carrier.

[0231] In some embodiments, LPA is administered at a dose of RNAi agent: about 0.01 mg / kg to about 10 mg / kg, e.g., about 0.05 mg / kg to about 5 mg / kg, about 0.05 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 5 mg / kg, about 0.1 mg / kg to about 10 mg / kg, about 0.2 mg / kg to about 5 mg / kg, about 0.2 mg / kg to about 10 mg / kg, about 0.3 mg / kg to about 5 mg / kg, about 0.3 mg / kg to about 10 mg / kg, about 0.4 mg / kg to about 5 mg / kg, about 0.4 mg / kg to about 10 mg / kg, about 0.5 mg / kg to about 5 mg / kg, about 0.5 mg / kg to about 10 mg / kg, about 1 mg / kg to about 5 mg / kg, about 1 mg / kg to about 10 mg / kg, about 2 mg / kg to about 5 mg / kg, about 3 mg / kg to about 10 mg / kg.

[0014] In some embodiments, the present invention provides an oral dosage form of at least 2.5 mg / kg, about 2 mg / kg to about 10 mg / kg, about 3 mg / kg to about 5 mg / kg, about 3 mg / kg to about 10 mg / kg, about 3.5 mg / kg to about 5 mg / kg, about 4 mg / kg to about 5 mg / kg, about 4.5 mg / kg to about 5 mg / kg, about 4 mg / kg to about 10 mg / kg, about 4.5 mg / kg to about 10 mg / kg, about 5 mg / kg to about 10 mg / kg, about 5.5 mg / kg to about 10 mg / kg, about 6 mg / kg to about 10 mg / kg, about 6.5 mg / kg to about 10 mg / kg, about 7 mg / kg to about 10 mg / kg, about 7.5 mg / kg to about 10 mg / kg, about 8 mg / kg to about 10 mg / kg, about 8.5 mg / kg to about 10 mg / kg, about 9 mg / kg to about 10 mg / kg, or about 9.5 mg / kg to about 10 mMg / kg. Values ​​and ranges intermediate to the recited values ​​are also intended to be part of the present invention.

[0232] It should be understood that in some cases, the initial dose administered may be increased above the upper limit level to quickly reach the desired blood level or tissue level, or in some cases, the initial dose may be less than the optimal value. For example, in some embodiments, an initial dose or first dose of about 1 mg to about 100 mg of an LPA RNAi drug substance is administered, followed by a second dose of about 1 to 100 mg of an LPA RNAi drug substance about 1 month later, and additional doses are administered every three months thereafter (e.g., once per calendar quarter or once every 12 weeks (q12w)).

[0233] The drug can be administered to a subject by any suitable route known in the art, including but not limited to oral or parenteral routes, including intravenous administration, intramuscular administration, subcutaneous administration, transdermal administration, airway administration (aerosol), pulmonary administration, nasal administration, rectal administration and topical administration (including buccal administration and sublingual administration), preferably intravenous administration and subcutaneous administration.

[0234] In certain embodiments, the pharmaceutical composition can be administered by intravenous infusion over a period of time, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21, 22, 23, 24, or about 25 minutes. For example, it is possible to regularly repeat administration, such as weekly, two weeks (i.e., every two weeks), for one month, two months, three months, four months, or longer. After the initial treatment regimen, treatment can be given at a lower frequency. For example, after weekly or two weeks of administration for three months, administration can be repeated once a month for six months or one year or longer.

[0235] In certain embodiments, pharmaceutical composition can be used by subcutaneous administration.Pharmaceutical composition can be used once a day, or iRNA can be used as two, three or more subdose with suitable intervals in one day, or even use continuous infusion or send by controlled release formulation.In this case, the iRNA contained in each subdose must be correspondingly less, to obtain total daily dose.Also compound dosage unit can be used for sending in a few days, for example, using conventional sustained release formulation, it provides lasting iRNA release in the time period of a few days.Sustained release formulation is well known in the art and is particularly useful for delivering medicament in a specific portion, as can be used together with medicament of the present invention.In this embodiment, dosage unit contains corresponding multiple daily doses.Initially can use higher dose (that is, loading dose), then use lower dose in the persistent period.

[0236] In certain embodiments, a single dose of the pharmaceutical composition can be long-acting, such that subsequent dosages are administered at intervals of no more than 3, 4, or 5 days, or no more than 1, 2, 3, or 4 weeks. In some embodiments of the invention, a single dose of the pharmaceutical composition of the invention is administered once a week. In other embodiments of the invention, a single dose of the pharmaceutical composition of the invention is administered every two months. In specific embodiments, the iRNA is administered from about once a month to about once per quarter (i.e., about once every three months).

[0237] Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions can be produced from a variety of components, including, but not limited to, preformed liquids, self-emulsifying solids, and self-emulsifying semisolids. Particularly preferred are formulations that target the liver when treating liver disorders (e.g., liver cancer).

[0238] The pharmaceutical preparations of the present invention (which may conveniently be in unit dosage form) can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the steps of combining the active ingredients with the pharmaceutical carrier(s) or excipient(s). Generally speaking, the preparations are prepared by uniformly and finely combining the active ingredients with a liquid carrier or a finely dispersed solid carrier or both, and, if desired, shaping the product.

[0239] Compositions of the present invention can be formulated as any one of many possible dosage forms, such as but not limited to tablets, capsules, gel capsules, liquid syrups, soft capsules, suppositories and enemas. Compositions of the present invention can also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions can further comprise materials that increase the viscosity of the suspension, such materials comprising for example sodium carboxymethylcellulose, sorbitol and / or dextran. The suspension can also comprise a stabilizing agent.

[0240] The pharmaceutical compositions disclosed herein include formulations suitable for parenteral administration. The formulations can conveniently be presented in unit dosage form and can be prepared by any method known in the pharmaceutical art. The amount of active ingredient that can be combined with excipients to prepare a single dose is generally the amount of siRNA that will produce a therapeutic effect. Generally, in percent units, this amount is from about 1% to about 99% of the active ingredient, preferably from about 5% to about 70%, and most preferably from about 10% to about 30%.

[0241] The siRNA conjugates of the present invention or their pharmaceutical compositions provide a significant effect of reducing the amount or level of LPA expression, LPA mRNA, apo(a) protein, apo(a) activity, OxPL, LDL-C, apoB-100, TG, LDL cholesterol, or any combination thereof, and are used to treat LPA-related diseases and conditions, such as hypertriglyceridemia-induced pancreatitis, metabolic syndrome, type II diabetes, familial chylomicronemia syndrome (FCS), chylomicronemia, multifactorial chylomicronemia, lipodystrophy syndrome including familial partial lipodystrophy, obesity, dyslipidemia, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, hyperlipidemia, hypertriglyceridemia, abnormal lipid and / or cholesterol metabolism, atherosclerosis, cardiovascular disease, coronary artery disease and other dyslipidemias, NAFLD, NASH, and metabolic-related conditions and diseases.

[0242] The present invention provides methods for contacting or delivering an effective amount of any of the oligonucleotides of the present invention (e.g., double-stranded oligonucleotides) to a cell or cell population to achieve the purpose of reducing LPA expression. In some embodiments, the reduction in LPA expression is determined by measuring a decrease in the amount or level of LPA mRNA, apo(a) protein, or apo(a) activity in the cell. The methods of the present invention can be used with any appropriate cell type. In some embodiments, LPA expression in a cell or cell population is determined at least about 4 hours, about 8 hours, about 12 hours, about 18 hours, about 24 hours after contacting or delivering the oligonucleotide to the cell or cell population; or at least about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 21 days, about 28 days, about 35 days, about 42 days, about 49 days, about 56 days, about 63 days, about 70 days, about 77 days, or about 84 days or more. In some embodiments, LPA expression in a cell or cell population is determined at least about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months or more after contacting or delivering the oligonucleotide to the cell or cell population.

[0243] General synthesis methods of the compounds, double-stranded siRNA, and double-stranded siRNA conjugates of the present invention

[0244] Generally, the compounds and nucleic acid conjugates of the present invention can be prepared by the methods described in the present invention. The following reaction schemes and examples are provided to further illustrate the present invention.

[0245] In the examples described below, all temperatures are in degrees Celsius (°C) unless otherwise indicated. Silica gel (200-300 mesh) was purchased from Qingdao Ocean Chemical Plant, and NH2CPG was purchased from Hebei Dinaxingke. Nuclear magnetic resonance spectra were obtained using CDC13, DMSO-d6, CD3OD, or acetone-d6 as solvents (in ppm), with TMS (0 ppm) or chloroform (7.25 ppm) as the reference standard. When multiple peaks are present, the following abbreviations are used: s (singlet), d (doublet), t (triplet), m (multiplet), br (broadened), dd (doublet of doublets), dt (doublet of triplets), br.s (broadened singlet), and q (quartet). Coupling constants, J, are expressed in Hertz (Hz).

[0246] Low-resolution mass spectrometry (MS) data were measured by an Agilent 6320 series LC-MS spectrometer equipped with a G1312A binary pump and a G1316A TCC (column temperature maintained at 30 °C). A G1329A autosampler and a G1315B DAD detector were used for analysis, and an ESI source was applied to the LC-MS spectrometer.

[0247] High-resolution mass spectrometry (MS) data were determined by an Agilent 6130 series LC-MS spectrometer equipped with a G1311A quaternary pump and a G1316A TCC (column temperature maintained at 30 °C). A G1329A autosampler and a G1315D DAD detector were used for analysis, and an ESI source was applied to the HR-MS spectrometer.

[0248] The following abbreviations are used throughout this disclosure: DETAILED DESCRIPTION

[0249] The scheme of the present invention will be explained below with reference to the examples. Those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. In particular, the synthesis of small nucleic acids and the synthesis of nucleic acid conjugates can be synthesized according to the embodiments of the present invention or routine adjustments in the art. If specific techniques or conditions are not specified in the examples, they are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

[0250] Preparation Example

[0251] In the following preparation examples, the inventors describe in detail the preparation process of the compounds of the present invention using some of the compounds of the present invention as examples, wherein: For CPG.

[0252] Example 1: Synthesis of Nucleoside Monomer Y

[0253] Step 1: Synthesis of compound 1-2

[0254] (2R,3R,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-carbonitrile (i.e., compound 1-1) (1.0 g, 3.43 mmol) was added to pyridine (15 mL). The mixture was protected by nitrogen and cooled to -5°C. 1,3-Dichloro-1,1,3,3-tetraisopropyldisiloxane (1.13 mL, 3.53 mmol) was then added dropwise. After the addition was complete, the reaction mixture was heated to 25°C and stirred for 20 hours. The reaction solution was concentrated under reduced pressure to remove the solvent, and the residue was purified by silica gel column chromatography (eluent: PE / EA (v / v) = 2 / 3) to obtain compound 1-2 (1.38 g, 75.3%) as a white solid. MS (ESI, pos. ion) m / z: 534.3 [M+H] + .

[0255] Step 2: Synthesis of Compound 1-3

[0256] Compound 1-2 (1.08 g, 2.02 mmol) and N,N-dimethylformamide dimethyl acetal (0.48 g, 4.04 mmol) were added to toluene (15 mL) and stirred at 50°C for 4 h. After the reaction was complete, the mixture was concentrated under reduced pressure to obtain compound 1-3 as a white foamy solid (1.19 g, 100%). MS (ESI, pos. ion) m / z: 589.3 [M+H] + ; 1 H NMR(599MHz,DMSO-d6)δ8.95(s,1H),8.15(s,1H),6.90(d,J=4.4Hz,1H),6.80(d ,J=4.5Hz,1H),6.49(d,J=5.7Hz,1H),4.58(dd,J=5.7,4.4Hz,1H),4.23–4.12(m ,3H),3.92(dd,J=13.2,2.5Hz,1H),3.25(s,3H),3.19(s,3H),1.06–1.04(m,7H) ,1.01(dd,J=7.2,2.0Hz,6H),0.96(dd,J=8.8,7.0Hz,8H),0.89(t,J=7.6Hz,7H).

[0257] Step 3: Synthesis of Compound 1-4

[0258] Compound 1-3 (0.84 g, 1.43 mmol) was added to N,N-dimethylformamide (10 mL), cooled to 0°C, and then iodomethane (0.41 g, 2.86 mmol) and sodium hydride (0.11 g, 2.86 mmol, 60%) were added sequentially. The reaction was stirred at 0°C for 20 min. After the reaction was complete, the reaction solution was poured into saturated NH4Cl solution and extracted with EA. The organic phase was concentrated, and the residue was purified by silica gel column chromatography (EA / PE (v / v) = 1 / 1) to obtain compound 1-4 as a white solid (0.71 g, 83%). MS (ESI, pos. ion) m / z: 603.40 [M+H] + .

[0259] Step 4: Synthesis of Compound 1-5

[0260] Compound 1-4 (5.1 g, 8.46 mmol), tetrahydrofuran (50 mL), and a tetrabutylammonium fluoride solution in tetrahydrofuran (9.48 mL, 9.48 mmol, 1 M) were mixed and stirred at room temperature. After completion of the reaction, the mixture was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (MeOH / DCM (v / v) = 1 / 20) to obtain compound 1-5 (2.4 g, 79%) as a white solid. MS (ESI, pos. ion) m / z: 361.3 [M+H] + .

[0261] Step 5: Synthesis of Compound 1-6

[0262] Compound 1-5 (2.4 g, 6.66 mmol), dichloromethane (50 mL), triethylamine (2.02 g, 19.98 mmol), and 4-dimethylaminopyridine (0.081 g, 0.67 mmol) were mixed and then cooled to 0°C. 4,4'-bismethoxytrityl chloride (2.71 g, 7.99 mmol) was added portionwise. The resulting reaction mixture was warmed to room temperature and stirred for 3 h. The temperature was then lowered to 0°C, and 4,4'-bismethoxytrityl chloride (1.35 g) was added in two portions. The reaction mixture was stirred at 0°C for another 2 h. The reaction system was diluted with MeOH (1 mL) and DCM (100 mL), washed sequentially with saturated sodium bicarbonate solution and saturated sodium chloride solution. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting residue was purified on a silica gel column (EA / PE (v / v) = 2 / 1) to afford compound 1-6 (4.0 g, 91%) as a light yellow solid. MS (ESI, pos.ion) m / z: 663.7 [M+H] + .

[0263] Step 6: Synthesis of Compound 1

[0264] Compound 1-6 (2.0 g, 3.02 mmol) and 1H-tetrazole (0.25 g, 3.62 mmol) were added to dichloromethane (40 mL) under nitrogen atmosphere. Bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.92 mL, 6.04 mmol) was then added dropwise. The mixture was stirred at room temperature for 4 h. After the reaction, DCM (20 mL) and saturated sodium bicarbonate solution (10 mL) were added. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (EA / PE (v / v) = 4 / 1) to yield compound 1 (2.13 g, 82%). MS (ESI, pos. ion) m / z: 864.9 [M+H] + ; 1 H NMR(400MHz, CDCl3)δ8.84(s,1H),8.00(d,J=12.0Hz,1H),7.45–7.39(m,2H),7.34–7.28(m,4H),7.22(qd,J=7.6,6.4,3.5Hz, 3H),7.02(dd,J=6.8,4.5Hz,1H),6.92(dd,J=4.6,3.1Hz,1H),6.78(td,J=8.5,4.4Hz,4H),4.93(dd,J=25.0,5.0Hz,1H),4.64 –4.43(m,2H),4.05–3.88(m,2H),3.79(dd,J=3.7,2.4Hz,6H),3.60(d,J=6.3Hz,4H),3.48(dd,J=10.5,3.6Hz,1H),3.27(s,3H ),3.24(s,3H),2.66(d,J=6.4Hz,1H),2.06(s,1H),1.28(t,J=7.1Hz,2H),1.19(dd,J=6.8,4.1Hz,8H),1.04(d,J=6.8Hz,4H); 31 P NMR (162MHz, CDCl3) δ150.01,149.80.

[0265] According to technical knowledge in this field, compound 1 is deprotected after nucleic acid synthesis to obtain a nucleic acid residue embedded group Y.

[0266] Example 2: Synthesis of compound DAW40007-3

[0267] Step 1: Synthesis of compound 2-2

[0268] Compound 2-1 (2.50 g, 28.05 mmol) and triethylamine (7.8 mL, 56.1 mmol) were dissolved in DCM (120 mL). Benzyl chloroformate (9.57 g, 56.1 mmol) was added dropwise at 0°C. After the addition, the reaction mixture was warmed to room temperature and stirred for 20 h. Saturated ammonium chloride solution (50 mL) was added for dilution. The layers were separated, the aqueous phase was discarded, and the organic phase was concentrated. The resulting residue was purified by silica gel column chromatography (MeOH / DCM (V / V) = 1 / 30) to obtain compound 2-2 (2.96 g, 47.2%) as a white solid. MS (ESI, pos. ion) m / z: 224.2 [M+H] + ; 1 H NMR(400MHz, CDCl3)δ7.37(d,J=4.2Hz,5H),5.11(s,2H),5.00(s,1H),4.19–4.14(m,1H),3.70–3.64(m, 2H), 3.24 (t, J=6.4Hz, 2H), 3.15 (q, J=4.6Hz, 1H), 1.90 (dq, J=14.2, 5.0, 4.3Hz, 2H), 1.77–1.71 (m, 1H).

[0269] Step 2: Synthesis of Compound 2-4

[0270] Compound 2-3 (1.5 g, 4.56 mmol) and compound 2-2 (1.22 g, 5.47 mmol) were dissolved in 1,2-dichloroethane (30 mL), and 3A molecular sieves (2.0 g) were added. The mixture was stirred at room temperature for 10 min. TMSOTf (0.51 g, 2.28 mmol) was then added, and the reaction mixture was stirred at room temperature for 18 h. The reaction mixture was poured into saturated sodium bicarbonate solution (100 mL), extracted with DCM (100 mL), washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, and the solvent evaporated under reduced pressure. The resulting residue was purified by silica gel column chromatography (PE / EA (V / V) = 2 / 1 to 0 / 1) to afford compound 2-4 (1.8 g, 71.51%) as a light brown oil. MS (ESI, pos. ion) m / z: 553.3 [M+H] + .

[0271] Step 3: Synthesis of Compound 2-5

[0272] Compound 2-4 (0.57 g, 1.03 mmol) and palladium carbon (0.11 g, 0.1 mmol, 10%) were added to THF (10 mL), followed by the addition of TFA (0.12 g, 1.03 mmol). The mixture was then replaced with hydrogen three times and stirred at room temperature under a hydrogen atmosphere for 19 h. After the reaction was complete, the mixture was filtered through celite and the solvent was evaporated under reduced pressure to obtain compound 2-5 (0.55 g, 100.32%) as a light brown oil. MS (ESI, pos. ion) m / z: 419.3 [M-TFA+H] + .

[0273] Step 4: Synthesis of Compound 2-7

[0274] Compound 2-6 (0.46 g, 2.29 mmol, purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd.) and compound 2-5 (1.16 g, 2.18 mmol) were dissolved in DCM (30 mL). HOBT (0.46 g, 3.44 mmol), HBTU (1.30 g, 3.44 mmol), and DIPEA (2.66 mL, 16.03 mmol) were added sequentially. The reaction mixture was allowed to react at room temperature for 16 h. After completion of the reaction, water (20 mL) and DCM (50 mL x 2) were added sequentially. The organic phase was washed sequentially with saturated sodium bicarbonate solution (30 mL) and saturated brine (20 mL). The solvent was evaporated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (MeOH / EA (v / v) = 1 / 20) to obtain 2-7 (1.0 g, 72.7% yield) as a white solid. MS (ESI, pos. ion) m / z: 602.3 [M+H] + ; 1 H NMR (400MHz, CD3OD) δ5.39–5.33(m,1H),5.07(dd,J=11.2,3.4Hz,1H),4.58(d,J= 8.4Hz,1H),4.21–4.09(m,3H),4.04(t,J=6.7Hz,1H),3.89(dt,J=10.4,5.1Hz,1H ),3.60–3.50(m,1H),3.30–3.13(m,2H),2.16(s,3H),2.04(s,3H),1.97(s,3H),1 .94(s,3H),1.67–1.56(m,4H),1.48(s,9H),1.41–1.37(m,2H),1.02–0.98(m,2H).

[0275] Step 5: Synthesis of compound 2-8

[0276] Compound 2-7 (0.72 g, 1.17 mmol) was dissolved in DCM (8 mL), and then TFA (0.87 mL, 11.7 mmol) was added. The reaction mixture was stirred at 25 °C for 16 h, and the solvent was concentrated to give brown oily compound 2-8 (0.74 g, 103.1%).

[0277] MS (ESI, pos.ion) m / z: 502.2 [M+H] + ; 1 H NMR (400MHz, CD3OD) δ5.36 (d, J=3.3Hz, 1H), 5.08 (dd, J=11.3, 3.3Hz, 1H), 4.5 8(d,J=8.4Hz,1H),4.16–4.10(m,3H),4.08–4.02(m,1H),3.92–3.85(m,1H),3 .58–3.50(m,1H),3.26–3.20(m,2H),2.16(s,3H),2.04(d,J=5.2Hz,6H),1.98 (s,3H),1.97(s,3H),1.61–1.56(m,4H),1.54–1.52(m,2H),1.42–1.38(m,2H).

[0278] Step 6: Synthesis of Compound 2-10

[0279] Compound 2-9 (0.19 g, 0.30 mmol) and compound 2-8 (0.50 g, 0.99 mmol) were dissolved in DCM (30 mL), and HOBT (0.17 g, 1.26 mmol), HBTU (0.48 g, 1.26 mmol), and DIPEA (0.5 mL, 3.0 mmol) were added sequentially. The reaction mixture was stirred at 30°C for 3 h. After completion of the reaction, water (20 mL) was added to quench the reaction, and the mixture was extracted with DCM (100 mL × 2). The organic phases were combined and washed sequentially with saturated sodium bicarbonate solution (40 mL) and saturated sodium chloride solution (40 mL), dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The resulting residue was purified by silica gel column chromatography (DCM / MeOH (v / v) = 10 / 1) to obtain compound 2-10 (0.23 g, yield 37%) as a white solid. MS(ESI,pos.ion)m / z:1046.3[M / 2+H] + ; 1H NMR (400MHz, CD3OD) δ7.78(t,J=5.8Hz,2H),7.38–7.36(m,3H),5.36(d,J=3.4Hz,3H),5.13(s,2H),5.08(dd,J=11.2, 3.4Hz,3H),4.58(d,J=8.4Hz,3H),4.21–4.08(m,9H),4.04(t,J=6.7Hz,3H),3.93–3.82(m,3H),3.73–3.66(m,12H),3. 60–3.52(m,3H),3.29–3.18(m,6H),2.52(t,J=6.0Hz,6H),2.38(t,J=7.4Hz,2H),2.20(t,J=7.7Hz,2H),2.16(s,9H), 2.04(s,9H),1.97(s,9H),1.95(s,9H),1.62–1.56(m,12H),1.50–1.42(m,6H),1.35–1.27(m,16H),1.04–0.97(m,6H).

[0280] Step 7: Synthesis of compound 2-11

[0281] Compound 2-10 (0.20 g, 0.094 mmol) was dissolved in methanol (10 mL), and then Pd / C (10 mg, 10%) was added. The atmosphere was replaced with hydrogen three times, and the reaction mixture was stirred at room temperature for 11 h under a hydrogen atmosphere. After the reaction was complete, the reaction mixture was filtered through celite, and the filtrate was evaporated to dryness under reduced pressure to obtain compound 2-11 (0.19 g, 100%) as a white solid. MS (ESI, pos. ion) m / z: 1001.1 [M / 2+H] + .

[0282] Step 8: Synthesis of compound DAW40007-1

[0283] Compound 2-11 (0.28 g, 0.14 mmol) was dissolved in DCM (20 mL), and HOBT (0.038 g, 0.28 mmol), HBTU (0.080 g, 0.21 mmol), DIPEA (0.054 g, 0.42 mmol) and compound 13 (0.068 g, 0.16 mmol) were added in sequence. The reaction mixture was stirred at room temperature for 13 h. After the reaction was completed, water (10 mL) was added to quench the reaction, and the mixture was extracted with DCM (20 mL). The organic phase was washed with saturated sodium bicarbonate solution (10 mL) and the mixture was evaporated under reduced pressure. The solvent was evaporated to dryness, and the resulting residue was dissolved in acetonitrile (10 mL) and separated on a reverse-phase preparative column (acetonitrile / water solution (v / v) = 43% to 60%, 50 min). Salt was added to the product-containing solution after preparative separation to saturate the solution. The organic phase was separated, and the aqueous phase was extracted with acetonitrile (100 mL x 2). The organic phases were combined and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the residue was added to acetonitrile (30 mL), dried over sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain compound DAW40007-1 (0.080 g, 24% yield) as a light yellow solid. MS (ESI, negative ion) m / z: 2400.18 [MH] - ; 1 H NMR (400MHz, DMSO-d6) δ8.31 (s, 3H), 7.81 (d, J = 9.2Hz, 3H), 7.57 (t, J = 6.0 Hz,3H),7.35–7.26(m,4H),7.20(td,J=8.9,3.0Hz,5H),6.95(s,1H),6.88( ddd,J=8.8,5.8,2.2Hz,4H),5.22(d,J=3.4Hz,3H),4.97(dd,J=11.2,3.5H z,4H),4.49(d,J=8.5Hz,3H),4.40(d,J=4.8Hz,1H),4.15(s,1H),4.07–4.0 0(m,9H),3.88(dt,J=11.2,8.8Hz,3H),3.74(s,9H),3.59–3.48(m,12H),3 .17(dd,J=8.8,5.0Hz,1H),3.10–2.95(m,8H),2.35(t,J=6.3Hz,6H),2.10( s,9H),2.08(s,3H),2.04(d,J=4.7Hz,2H),2.00(s,9H),1.89(s,9H),1.78( s,9H),1.40(d,J=12.2Hz,17H),1.31–1.16(m,18H),0.79(q,J=3.2Hz,6H).

[0284] Step 9: Synthesis of compound DAW40007-2

[0285] Compound DAW40007-1 (0.080 g, 0.033 mmol) was dissolved in DCM (10 mL), and DIPEA (0.029 mL, 0.17 mmol), succinic anhydride (0.008 g, 0.083 mmol) and DMAP (0.014 g, 0.12 mmol) were added. The mixture was stirred at 40°C for 5 h, and succinic anhydride (10 mg) was added. After the reaction was continued for 16 h, DCM (10 mL), succinic anhydride (10 mg) and DIPEA (0.05 mL) were added. The mixture was stirred for another 9 h, and then succinic anhydride (10 mg) was added. After the reaction was continued for another 10 h, DCM (20 mL) was added to dilute the mixture, and the mixture was washed with saturated sodium bicarbonate solution (10 mL). The aqueous phase was discarded, and the organic phase was dried over anhydrous sodium sulfate. The solvent was concentrated under reduced pressure to obtain compound DAW40007-2 (0.08 g, 96.07%) as a white solid. MS(ESI,neg.ion)m / z:2500.12[MH] - .

[0286] Step 10: Synthesis of compound DAW40007-3

[0287] Compound DAW40007-2 (0.08 g, 0.032 mmol), HBTU (0.015 g, 0.04 mmol), and DIPEA (0.011 mL, 0.064 mmol) were dissolved in ACN (5 mL) and stirred at room temperature for 5 min. The mixture was then transferred to a solid-phase synthesizer containing 0.35 g of H2N-CPG (purchased from Hebei Dinaxingke) and shaken for 22.5 h. The filter cake was filtered, rinsed with DCM / MeOH (V / V = 9 / 1, 10 mL) and DCM (10 mL), and dried. The resulting filter cake was diluted in 25% Ac2O / Py solution (5 mL) and stirred for 3 h. The mixture was filtered, and the filter cake was rinsed sequentially with DCM / MeOH (V / V = 9 / 1, 10 mL) and DCM (10 mL). The mixture was dried under reduced pressure to obtain DAW40007-3 (0.357 g), a white solid with a measured loading of 14.95 μmol / g. Compound L96-DMTr-CPG was conjugated to an oligonucleotide and then deprotected to obtain L96. Compound DAW40007-3 was conjugated to an oligonucleotide and then deprotected to obtain DAW40007-4.

[0288] Synthesis of compound L96-DMTr-CPG: Compound L96-DMTr-CPG was prepared according to the method described in patent application WO2014025805A1.

[0289] Synthesis of compound 38: Compound 38 was prepared according to the method described in patent application WO2018044350A1.

[0290] The conjugated group of the present invention can be connected (conjugated) to the siRNA molecule according to methods known in the art, such as linking compound 38 and siRNA and removing the protecting group to form a conjugated group with NAG37 structure.

[0291] Example 3: Synthesis of double-stranded siRNA and double-stranded siRNA conjugates

[0292] 1. Synthesis of double-stranded siRNA without conjugation groups

[0293] The steps for synthesizing the siRNA sense strand and antisense strand of the present invention are as follows:

[0294] The synthesis was completed according to the theoretical yield of 1umol. Weigh 1umol of solid support CPG (purchased from Hebei Dina Xingke). All 2'-modified RNA phosphoramidite monomers and auxiliary reagents were commercially obtained, and all phosphoramidite monomers were provided in 0.1M anhydrous acetonitrile solution. For oligonucleotides with phosphate backbone thiolation modification, 0.1M DDTT solution was used as the thiolation reagent. 5-Ethylthio-1H-tetrazole acetonitrile solution (0.25M) was used as an activator (purchased from Suzhou Kelema), 0.02M iodine pyridine / water solution was used as an oxidant, and 3% trichloroacetic acid in dichloromethane was used as a deprotection reagent. They were placed in the designated reagent position corresponding to the KA-H8 model DNA / RNA automatic synthesizer. Set the synthesis program and enter the specified oligonucleotide base sequence. After checking that everything is correct, start the cyclic oligonucleotide synthesis. The coupling time for each step is 6 minutes, and the thiolation time is 6 minutes. After automatic circulation, an oligonucleotide containing solid support CPG is obtained.

[0295] The CPG-containing nucleotides obtained above were blown dry with dry argon, then transferred to a 2 mL EP tube and 28% ammonia solution (1.8 mL) was added and heated at 55°C for 5 to 18 hours. Filter, wash the filter cake with water (0.5 mL), combine the filtrates, and concentrate under reduced pressure to obtain a white or yellow colloidal solid. After reverse phase preparative purification, the prepared solution was concentrated and passed through a gel column to remove excess salt to obtain the oligonucleotide. The concentration of the obtained oligonucleotide was determined by a micro-UV spectrophotometer (SPECTRO stat Nano). Mass spectrometry detection and analysis were performed on an Agilent 6530LC-MS Q-Tof system. After primary scanning, the nucleic acid molecular weight was calculated after deconvolution.

[0296] Annealing step:

[0297] The double-stranded siRNA sense strand synthesized above was mixed with the antisense strand synthesized above in equimolar amounts, heated to 95°C, maintained at this temperature for 10 minutes, and then slowly cooled to room temperature. The target double-stranded siRNA was then lyophilized.

[0298] 2. Synthesis of siRNA conjugates:

[0299] Synthesis of antisense strand: refer to the above synthesis method.

[0300] Synthesis of the sense strand: The universal solid support CPG is replaced with the GalNAc solid support prepared in the present invention (such as compound DAW40007-3 or L96-DMr-CPG), and the sense strand connected to the conjugated group in the double-stranded siRNA conjugate of the present invention is prepared according to the above-mentioned synthesis method.

[0301] Annealing step:

[0302] The synthesized sense strand was mixed with the synthesized antisense strand in equimolar amounts, heated to 95°C, maintained at this temperature for 10 minutes, and then slowly cooled to room temperature. The target siRNA conjugate was then lyophilized.

[0303] The above synthesis schemes list general experimental steps for preparing the double-stranded siRNA and its conjugates of the present invention. Those skilled in the art can prepare the double-stranded siRNA and its conjugates of the present invention by making appropriate modifications to the methods or adjusting the raw materials according to actual circumstances. Unless otherwise specified, the double-stranded siRNA and its conjugates of the present invention can be prepared using the methods described in the above synthesis schemes.

[0304] The double-stranded siRNA synthesized by the present invention is shown in Table 1, and the double-stranded siRNA synthesized and modified by the present invention is shown in Table 2.

[0305] Table 1: Unmodified double-stranded siRNA synthesized by the present invention

[0306] Table 2: Modified double-stranded siRNA synthesized by the present invention

[0307] Unless otherwise specified, in the context of the present invention, capital letters C, G, U, and A represent bases of natural nucleotides; lowercase letters represent bases whose ribose position 2 is modified by a methoxy group, such as c, g, u, and a represent 2'-OMe (2'-O-methyl) C, 2'-OMe G, 2'-OMe U, and 2'-OMe A, respectively; the f to the right of the capital letter represents a base whose ribose position 2 is modified by a fluorine group, such as Cf, Gf, Uf, and Af represent 2'-F (2'-fluoro) C, 2'-FG, 2'-FU, and 2'-FA, respectively; "s" indicates that the two nucleotide residues adjacent to the left and right of "s" are connected by a thiophosphate group, for example, "gsu" indicates that the g and u residues are connected by a thiophosphate group.

[0308] Example 4: Cellular activity and cytotoxicity test of siRNA or its conjugates of the present invention

[0309] In vitro knockdown activity screening:

[0310] HEK293 cells were cultured in DMEM medium containing 10% fetal bovine serum in a 5% CO2, 37°C incubator. When the cells were in the logarithmic growth phase and in good condition (70% confluence), they were transfected with a transfection reagent. The cell concentration was adjusted to 2.5×10 5 / mL, and 1*10 4 Cells were transfected with psiCHECK2 plasmid containing the full-length LPA fragment and different concentrations of siRNA, positive control and negative control according to the instructions of lipo2000 transfection reagent. The plasmid was 20 ng per well. After incubation at 37°C, 5% CO2 for 48 h, the cells were collected and detected using a dual-luciferase assay kit (Promega E1980) to detect firefly fluorescence and Renilla fluorescence signals, respectively.

[0311] The experimental results show that the siRNA or its conjugates of the present invention have good inhibitory activity against LPA. The results of the inhibitory activity of some double-stranded siRNAs of the present invention against LPA are shown in Table A.

[0312] Table A: Experimental results of the inhibitory activity of some double-stranded siRNAs of the present invention on LPA

[0313] According to the technical knowledge in the art, the siRNA conjugates of the present invention are obtained by conjugating siRNA to a GalNAc or its derivative conjugation group, wherein the conjugation group primarily functions as a delivery agent. If the siRNA exhibits good activity, those skilled in the art can expect that the siRNA conjugate to which the conjugation group is attached will exhibit similar or superior gene suppression / silencing activity as the siRNA. Furthermore, those skilled in the art can select suitable conjugation groups for the siRNA conjugates based on the technical knowledge in the art, such as GalNAc or its derivatives, such as L96 and DAW40007-4.

[0314] Example 5: Cytotoxicity Assay

[0315] HepG2 cells were cultured in DMEM medium containing 10% fetal bovine serum in a 5% CO2, 37°C constant temperature incubator. When the cells were in the logarithmic growth phase and in good condition (70% confluence), they were transfected with transfection reagent. The cell concentration was adjusted to 2.5×10 5 / mL, and 1*10 4 Cells were transfected with various concentrations of siRNA and a negative control according to the RNAiMAX transfection reagent instructions. After incubation at 37°C, 5% CO₂ for 48 hours, 11 μl of CCK-8 was added to each well and incubated in a 37°C incubator for 1.5-3 hours. Absorbance at 450 nm was measured using a microplate reader, and data were processed using Excel and graphed using GraphPad Prism.

[0316] Experimental results show that the siRNA and siRNA conjugates of the present invention have low cytotoxicity.

[0317] Example 6: Lysosome stability assay

[0318] 20 μM of the conjugate was added to lysosomes (final concentration 0.2 mU / μL) and mixed with sodium citrate solution (pH 5.0). No reference reagent was added to lysosomes. The mixture was incubated at 37°C. 5 μL of the sample was removed at 1, 2, 5, 8, 24, and 48 hours and denatured in 15 μL of 9 M urea. 4 μL of 6* loading buffer was then added, and the reaction was immediately terminated at -80°C. 10 μL of the sample was loaded onto a 16% non-denaturing polyacrylamide gel. After electrophoresis, the gel was stained with Gelred on a shaker for 10 minutes. The gel was then imaged and photographed.

[0319] The experimental results show that the siRNA and siRNA conjugates of the present invention have good stability in lysosomes.

[0320] Example 7: Activity detection using human primary cells

[0321] Primary human hepatocytes were cultured in DMEM medium supplemented with 10% fetal bovine serum in a 5% CO2, 37°C incubator. When the cells were in logarithmic growth phase and in good condition, they were seeded. Various concentrations of siRNA, siRNA conjugates, positive controls, and negative controls were added to the cells. After incubation at 37°C, 5% CO2 for 48 hours, the cells were harvested and the mRNA expression levels of the target gene LPA and the internal control GAPDH were measured using the QuickEasy Cell Direct RT-qPCR kit (Taqman).

[0322] The experimental results show that the siRNA or its conjugate of the present invention has good inhibitory activity against LPA.

[0323] Example 8: Evaluation of LPA siRNA conjugate knockdown activity using hLPA transgenic mice

[0324] To evaluate the in vivo activity of LPA siRNA, a humanized LPA transgenic mouse model (5-8 mice per group) was used. Baseline serum body weight, ALT, and LPA protein concentrations were collected before administration and grouped. A single dose of 0.5 mg / kg of GalNAc-siRNA or saline (the vehicle) was administered subcutaneously at the nape of the neck on day 0. Blood samples were collected on days 0, 4, 7, 14, 21, 28, 35, and 49, and human LPA protein concentrations were measured using a human Lipoprotein A ELISA kit (ab212165). Percent knockdown was calculated by comparing LPA protein levels in the siRNA and vehicle groups.

[0325] The experimental results show that the siRNA or its conjugates of the present invention have good knockdown activity against LPA in mice

[0326] Although the present invention has been described in detail above using general explanations, specific embodiments, and experiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A double-stranded siRNA, a conjugate or a salt thereof, comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises one of the nucleotide sequences shown in SEQ ID NO: 1 to SEQ ID NO: 28, or a nucleotide sequence having no more than 5 nucleotide differences therefrom.

2. The double-stranded siRNA, its conjugate or salt according to claim 1, characterized in that The antisense strand includes one of the nucleotide sequences shown in SEQ ID NO: 29 to SEQ ID NO: 56, or a nucleotide sequence having no more than 5 nucleotide differences therefrom.

3. The double-stranded siRNA, its conjugate or salt according to claim 1 or 2, characterized in that: The length of the sense strand does not exceed 23 nucleotides, and the length of the antisense strand does not exceed 23 nucleotides.

4. The double-stranded siRNA, its conjugate or salt according to any one of claims 1 to 3, characterized in that The sense strand and / or antisense strand comprises a 3' overhang and / or a 5' overhang, and the 3' overhang or the 5' overhang comprises 1, 2 or 3 nucleotides.

5. The double-stranded siRNA, its conjugate or salt according to any one of claims 1 to 4, characterized in that It contains one of the double-stranded siRNAs shown in siRNA ID NO:1 to siRNA ID NO:

28.

6. The double-stranded siRNA, its conjugate or salt according to any one of claims 1 to 5, characterized in that The sense strand and / or the antisense strand comprises at least one modified nucleotide, and the modified nucleotide is independently selected from at least one of the following: Non-natural base nucleotides, deoxynucleotides, 2'-fluoro modified nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-alkyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-methoxyethyl modified nucleotides, locked nucleotides, unlocked nucleic acid modified nucleotides, 2'-allyl modified nucleotides, abasic nucleotides, invAb modified nucleotides, morpholino modified nucleotides, tetrahydropyran modified nucleotides, cyclohexenyl modified nucleotides, PEG modified nucleotides, 5'-phosphoramidate modified nucleotides, phosphorothioate linked modified nucleotides, 5'-methyl Phosphonate-modified nucleotides, 5'-vinyl phosphate-modified nucleic acids, 5'-phosphate mimetic-modified nucleotides, TNA-modified nucleotides, PNA-modified nucleotides, D-FNA-modified nucleotides, ANA-modified nucleotides, HNA-modified nucleotides, FANA-modified nucleotides, bcDNA-modified nucleotides, tcDNA-modified nucleotides, S-MC-modified nucleotides, N-MC-modified nucleotides, 2'-F-NMC-modified nucleotides, 5-methylcytosine-modified nucleotides, 5-methyluracil-modified nucleotides, 2,6-diamino-modified adenine-modified nucleotides, and ethylene glycol nucleic acids.

7. The double-stranded siRNA, its conjugate or salt according to any one of claims 1 to 6, characterized in that The sense strand and / or the antisense strand comprises at least one modified nucleotide, and the modified nucleotide is independently selected from at least one of the following: 2'-methoxy modified nucleotides, 2'-fluoro modified nucleotides, 2'-methoxyethyl modified nucleotides, 5'-phosphorothioate linked modified nucleotides, 3'-phosphorothioate linked modified nucleotides, 2'-deoxy modified nucleotides, 2'-amino modified nucleotides, 2'-hydroxy modified nucleotides, locked nucleic acid modified nucleotides, unlocked nucleic acid modified nucleotides, ethylene glycol nucleic acids, 5'-vinyl phosphate modified nucleotides, 5'-(E)-VP modified nucleic acids, invAb modified nucleotides, invdA modified nucleotides, i substituted nucleotides and Y substituted nucleotides, wherein Y is The i is 8. The double-stranded siRNA, its conjugate or salt according to claim 6 or 7, characterized in that: The modified nucleotides are each independently present in one or more positions selected from the following: The nucleotide at the 5' end of the sense strand is the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th and 21st nucleotide of the starting point; and / or The nucleotide at the 5' end of the antisense strand is the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd and 23rd nucleotide of the starting point; and / or The 5'-phosphorothioate linkage modification or the 3'-phosphorothioate linkage modification is each independently present at one or more positions selected from the group consisting of: The nucleotides at the 5' end of the sense strand are between positions 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, 14-15, 15-16, 16-17, 17-18, 18-19, 19-20 and 20-21 of the starting point; and / or The nucleotides at the 5' end of the antisense strand are positions 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, 14-15, 15-16, 16-17, 17-18, between 18-19, 19-20, 20-21, 21-22 and between 22-23 of the starting point.

9. The double-stranded siRNA, its conjugate or salt according to any one of claims 6 to 8, characterized in that: The 2'-fluoro modified nucleotide is present at one or more positions selected from the group consisting of: The nucleotides at the 5' end of the sense strand are the 5th, 7th, 8th, 9th, 10th and 11th nucleotides of the starting point; and / or The nucleotides at the 5' end of the antisense strand are the 2nd, 6th, 8th, 9th, 12th, 14th and 16th positions of the starting point; Optionally, the 5'-phosphorothioate linkage modification or the 3'-phosphorothioate linkage modification is each independently present at one or more positions selected from the group consisting of: The nucleotides at the 5' end of the sense strand are between positions 1-2, 2-3 and 3-4 of the starting point; and / or The nucleotides at the 5' end of the antisense strand are between positions 1-2, 2-3 and 3-4 of the starting point; and / or The nucleotides at the 3' end of the antisense strand are between the 1st and 2nd positions, the 2nd and 3rd positions, and the 3rd and 4th positions of the starting point.

10. The double-stranded siRNA, its conjugate or salt according to any one of claims 1 to 9, characterized in that: The double-stranded region is 17-23 nucleotide pairs in length.

11. The double-stranded siRNA, conjugate or salt thereof according to any one of claims 1 to 10, wherein the sense strand comprises one of the nucleotide sequences shown in SEQ ID NO: 57 to SEQ ID NO: 84, and the length of the sense strand does not exceed 21 nucleotides; Optionally, the antisense strand comprises one of the nucleotide sequences shown in SEQ ID NO: 85 to SEQ ID NO: 112, and the length of the antisense strand does not exceed 23 nucleotides.

12. The double-stranded siRNA, conjugate or salt thereof according to any one of claims 1 to 11, characterized in that: It comprises one of the double-stranded siRNAs shown in siRNA ID NO: 29 to siRNA ID NO: 56; wherein the length of the sense strand does not exceed 21 nucleotides, and the length of the antisense strand does not exceed 23 nucleotides.

13. The double-stranded siRNA, conjugate or salt thereof according to any one of claims 1 to 12, characterized in that: The double-stranded siRNA conjugate is formed by conjugating the double-stranded siRNA with a conjugation group; preferably, in the double-stranded siRNA conjugate, the 3' end or 5' end of the sense strand or antisense strand of the double-stranded siRNA is conjugated to the conjugation group; preferably, the 3' end of the sense strand of the double-stranded siRNA is conjugated to the conjugation group; optionally, the 3' end or 5' end of the sense strand of the double-stranded siRNA is conjugated to the conjugation group via a phosphate bond or a phosphorothioate bond.

14. The double-stranded siRNA, its conjugate or salt according to claim 13, characterized in that: The conjugated group includes GalNAc or its derivatives; preferably, the conjugated group is GalNAc or its derivatives connected by a divalent, trivalent or tetravalent branched linker; more preferably, the conjugated group is DAW40007-4, L-96, NAG37, NAG25 or a stereoisomer thereof, wherein the structures of the conjugated groups DAW40007-4, L-96, NAG37 and NAG25 are respectively:

15. A pharmaceutical composition comprising the double-stranded siRNA, its conjugate or salt according to any one of claims 1 to 14, and a pharmaceutically acceptable carrier.

16. Use of the double-stranded siRNA, its conjugate or salt according to any one of claims 1 to 14, or the pharmaceutical composition according to claim 15 in the preparation of a medicament for treating and / or preventing LPA-related diseases.

17. The use according to claim 16, wherein The LPA-related disease is dyslipidemia, hypertriglyceridemia, obesity, dyslipidemia, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, hyperlipidemia, abnormal lipid and / or cholesterol metabolism, atherosclerosis, cardiovascular disease, coronary artery disease, hypertriglyceridemia-induced pancreatitis, metabolic syndrome, type II diabetes, familial chylomicronemia syndrome, chylomicronemia, multifactorial chylomicronemia, lipodystrophy syndrome or familial partial lipodystrophy.

18. The double-stranded siRNA, its conjugate and salt thereof according to any one of claims 1 to 14, or the pharmaceutical composition according to claim 15 is used for preventing, treating, curing or alleviating LPA-related diseases in patients.

19. The double-stranded siRNA, its conjugate or salt thereof, or pharmaceutical composition according to claim 18, wherein: The LPA-related disease is dyslipidemia, hypertriglyceridemia, obesity, dyslipidemia, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, hyperlipidemia, abnormal lipid and / or cholesterol metabolism, atherosclerosis, cardiovascular disease, coronary artery disease, hypertriglyceridemia-induced pancreatitis, metabolic syndrome, type II diabetes, familial chylomicronemia syndrome, chylomicronemia, multifactorial chylomicronemia, lipodystrophy syndrome or familial partial lipodystrophy.

20. A method for preventing, treating, curing or alleviating an LPA-related disease in a patient, comprising administering to the patient an effective therapeutic amount of the double-stranded siRNA, its conjugate or salt thereof according to any one of claims 1 to 14, or the pharmaceutical composition according to claim 15.

21. The method according to claim 20, wherein: The LPA-related disease is dyslipidemia, hypertriglyceridemia, obesity, dyslipidemia, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, hyperlipidemia, abnormal lipid and / or cholesterol metabolism, atherosclerosis, cardiovascular disease, coronary artery disease, hypertriglyceridemia-induced pancreatitis, metabolic syndrome, type II diabetes, familial chylomicronemia syndrome, chylomicronemia, multifactorial chylomicronemia, lipodystrophy syndrome or familial partial lipodystrophy.

Images