Novel double-stranded siRNA (small interfering ribonucleic acid) as well as conjugate and application thereof

Abstract

The present invention provides a novel double-stranded siRNA, a conjugate thereof and uses thereof, 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

Technical Field

[0001] The present invention belongs to the field of small nucleic acid drugs, and the purpose of the present invention is to provide a novel double-stranded siRNA, its conjugate and its use. The double-stranded siRNA and its conjugate 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) having 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 defined and do not change significantly 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 analyses have also implicated LPA as a genetic risk factor for diseases such as atherosclerotic stenosis. When therapeutic lipoprotein apheresis is used to reduce 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 a phenomenon that is highly conserved in the evolutionary process and is induced by double-stranded small interfering RNA (siRNA) to efficiently and specifically degrade homologous mRNA. It is of great significance to study and develop new LPA-specific RNA interference (RNAi) agents (also referred to as RNAi agents, RNAi triggers or triggers in the present invention), such as double-stranded RNAi agents, that can selectively and effectively inhibit the expression of LPA genes. Summary of the invention

[0004] The present invention provides a novel double-stranded siRNA, a conjugate or a salt thereof and uses thereof, wherein the double-stranded siRNA, a conjugate or a salt thereof can inhibit the expression of the LPA gene and can be used to prepare a drug for treating and / or preventing LPA-related diseases. The double-stranded siRNA, a conjugate or a salt thereof of the present invention has high in vivo delivery efficiency, good stability, high LPA gene expression inhibition activity and / or low toxicity.

[0005] On the one hand, the present invention provides a double-stranded siRNA, a conjugate or a salt thereof, which comprises 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 a difference of no more than 5 nucleotides 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 specification of the present invention.

[0006] In some embodiments of the double-stranded siRNA, its conjugate or salt 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 no more than 5 nucleotide differences therefrom;

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

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

[0009] In some embodiments of the double-stranded siRNA, its conjugate or salt described in 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 with a conjugation group.

[0011] In some embodiments of the double-stranded siRNA, its conjugate or salt thereof 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 thereof 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 thereof 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 acid (GNA), wherein the 2'- refers to the 2-position of ribose, such as 2'-fluoro modified nucleotides refer to nucleotides in which the 2-position of ribose is substituted by fluorine, and for example, 2'-methoxy modified nucleotides refer to nucleotides in which the 2-position of ribose is substituted by methoxy.

[0016] In some embodiments of the double-stranded siRNA, its conjugate or salt thereof 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 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 for example, 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 conjugate or salt according to the present invention, the modified nucleotides are each independently present in one or more positions selected from the following:

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

[0020] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the modified nucleotides are each independently present in one or more positions selected from the following:

[0021] The nucleotides at the 5' end of the antisense strand are 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 positions of the starting point.

[0022] In some embodiments of the double-stranded siRNA, its conjugate or salt thereof according to the present invention, the 5'-phosphorothioate linkage modification or the 3'-phosphorothioate linkage modification is each independently present at one or more positions selected from the following:

[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 conjugate or salt thereof according to the present invention, the 5'-phosphorothioate linkage modification or the 3'-phosphorothioate linkage modification is each independently present at one or more positions selected from the following:

[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 conjugate or salt thereof according to the present invention, the 2'-fluoro modified nucleotide, the 2'-methoxy modified nucleotide and Y are each independently and optionally present in one or more positions selected from the following:

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

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

[0029] The nucleotides at the 5' end of the antisense strand are 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 positions of the starting point.

[0030] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the 2'-fluoro modified nucleotide is present at one or more positions selected from the following:

[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 conjugate or salt according to the present invention, the 2'-fluoro modified nucleotide is present at one or more positions selected from the following:

[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 conjugate or salt thereof according to the present invention, the 5'-phosphorothioate linkage modification or the 3'-phosphorothioate linkage modification is each independently present at one or more positions selected from the following:

[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 conjugate or salt thereof according to the present invention, the 5'-phosphorothioate linkage modification or the 3'-phosphorothioate linkage modification is each independently and optionally present at one or more positions selected from the following:

[0037] The nucleotides at the 3' end of the positive strand are between the 1st and 2nd positions, the 2nd and 3rd positions, and the 3rd and 4th positions of the starting point.

[0038] In some embodiments of the double-stranded siRNA, its conjugate or salt thereof according to the present invention, the 5'-phosphorothioate linkage modification or the 3'-phosphorothioate linkage modification is each independently present at one or more positions selected from the following:

[0039] The nucleotides at the 5' 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.

[0040] In some embodiments of the double-stranded siRNA, its conjugate or salt thereof according to the present invention, the 5'-phosphorothioate linkage modification or the 3'-phosphorothioate linkage modification is each independently present at one or more positions selected from the following:

[0041] 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.

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

[0043] In some embodiments of the double-stranded siRNA, its conjugate or salt described in 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 described in 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 chain comprises one of the nucleotide sequences shown in SEQ ID NO:57 to SEQ ID NO:84 (i.e., one of the sense chains in Table 2 of the specification of the present invention), and the length of the sense chain 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 specification of the present invention.

[0046] In some embodiments of the double-stranded siRNA, its conjugate or salt described in the present invention, the antisense strand includes 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 specification of the present invention), 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 specification of the present invention.

[0047] In some embodiments of the double-stranded siRNA, its conjugate or salt of the present invention, the double-stranded siRNA, its conjugate or salt of the present invention 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 is no more than 21 nucleotides, and the length of the antisense strand is no more than 23 nucleotides. The nucleotide information of the double-stranded siRNA of siRNA ID NO: 29 to siRNA ID NO: 56 is detailed in 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 a nucleotide sequence of 5'-cscsacagAfaAfAfCfuacccasasa-3', and the antisense strand comprises a 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 a nucleotide sequence of 5'-csasaccuGfaCfAfCfaaugcuscsa-3', and the antisense strand comprises a nucleotide sequence of 5'-usGfsagcAfuugugucAfgGfuugscsa-3'; or

[0054] The sense strand comprises a nucleotide sequence of 5'-cscsuagaGfgCfUfCfcuucugsasa-3', and the antisense strand comprises a nucleotide sequence of 5'-usUfscagAfaggagccUfcUfaggscsu-3'; or

[0055] The sense strand comprises a nucleotide sequence of 5'-gsasaaugUfcCfUfGfgaagcasusa-3', and the antisense strand comprises a nucleotide sequence of 5'-usAfsugcUfuccaggaCfaUfuucsusu-3'; or

[0056] The sense strand comprises a nucleotide sequence of 5'-csasugguAfaUfGfGfacagagsusa-3', and the antisense strand comprises a nucleotide sequence of 5'-usAfscucUfguccauuAfcCfaugsgsu-3'.

[0057] In some embodiments of the double-stranded siRNA, its conjugate or salt thereof 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.

[0058] 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 with a conjugation group.

[0059] 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 sense strand of the double-stranded siRNA is conjugated to a conjugating group.

[0060] 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 conjugating group.

[0061] In some embodiments of the double-stranded siRNA, its conjugate or salt thereof according to the present invention, preferably, the 3' end of the sense strand of the double-stranded siRNA is conjugated to the conjugate.

[0062] In some embodiments of the double-stranded siRNA, its conjugate or salt thereof 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.

[0063] In some embodiments of the double-stranded siRNA, its conjugate or salt described in the present invention, the conjugated group includes GalNAc or its derivatives.

[0064] 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 by a divalent, trivalent or tetravalent branched linker.

[0065] 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:

[0066]

[0067]

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

[0069] In some embodiments of the double-stranded siRNA, its conjugate or salt, 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.

[0070] In another aspect, the present invention provides a pharmaceutical composition comprising the double-stranded siRNA, its conjugate or salt thereof, double-stranded siRNA conjugate or salt thereof, and a pharmaceutically acceptable carrier according to the present invention.

[0071] 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.

[0072] In some embodiments of the use described in the present invention, the LPA-related disease is a metabolic disease or a cardiovascular disease.

[0073] In some embodiments of the use described in the present invention, 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.

[0074] In some embodiments of the use described in the present invention, the LPA-related disease is atherosclerosis, dyslipidemia, NAFLD or NASH.

[0075] Detailed description of the invention

[0076] Definitions and general terms

[0077] In the present invention, the terms "comprise" or "include" are open expressions, that is, including the contents specified in the present invention, but not excluding other contents.

[0078] In the present invention, the term "small interfering RNA (siRNA)" is a double-stranded RNA of 17 to 30 nucleotides in length, comprising a sense strand and an antisense strand. siRNA mediates the targeted cleavage of RNA transcripts in the RISC pathway by forming a silencing complex (RISC). Specifically, siRNA guides the specific degradation of mRNA sequences through the known RNA interference (RNAi) process, inhibiting the translation of mRNA into amino acids and proteins.

[0079] The sense strand and the 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.

[0080] The terms "iRNA", "RNAi agent", "iRNA agent", "RNA interfering agent" are used interchangeably herein to refer to RNA agents as defined herein and mediate targeted cleavage of RNA transcripts through 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, such as a cell of a subject, such as a mammalian subject.

[0081] 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 in the interior or terminal regions of the molecule, wherein the most tolerated mismatches exist in the terminal regions, for example, within 5, 4, 3 or 2 nucleotides of the 5'- and / or 3'-end of the RNAi.

[0082] It should be noted that "at least a portion of the antisense strand is substantially complementary to the mRNA" means that the antisense strand has a polynucleotide that is substantially complementary to a continuous portion of the mRNA of interest.

[0083] The term "nucleotide overhang" or "overhang" refers to at least one unpaired nucleotide that overhangs from 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 may include an overhang of at least one nucleotide; alternatively, the overhang may include at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 or more nucleotides. The nucleotide overhang may include or consist of nucleotide / nucleoside analogs (including deoxynucleotides / nucleosides). One or more overhangs may be on the sense strand, the antisense strand, or any combination thereof. In addition, one or more nucleotides of the overhang may be present at the 5' end, the 3' end, or both ends of the antisense or sense strand of the dsRNA.

[0084] The conjugated groups of the present invention include pharmaceutically acceptable conjugated groups. Generally speaking, pharmaceutically acceptable conjugated groups include pharmaceutically acceptable targeting molecules and optional linkers. Exemplary conjugated groups, linkers, and targeting molecules can be found in the disclosures of WO2015006740A2 and CN114555188A. Exemplary conjugated groups include, but are not limited to, L96, NAG37, NAG25, or DAW40007-4.

[0085] Unless otherwise specified, "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.

[0086] The double-stranded siRNA conjugate of the present invention is a compound formed by connecting the double-stranded siRNA and a pharmaceutically acceptable conjugation group, and the double-stranded siRNA and the pharmaceutically acceptable conjugation group are covalently linked.

[0087] When used for the treatment of diseases, 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 field. All methods include the step of combining the active ingredient with an excipient that constitutes one or more accessory ingredients. Generally, the composition is prepared by uniformly and fully combining the active siRNA with a liquid excipient, a finely divided solid excipient, or both.

[0088] 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 mammals treated therewith. Preferably, the "pharmaceutically acceptable" of the present invention refers to those approved by federal regulatory agencies or national governments or listed in the United States Pharmacopoeia or other generally recognized pharmacopeias for use in animals, especially humans.

[0089] In the present invention, the term "pharmaceutically acceptable carrier" may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for a specific target dosage form. Except for any conventional carrier incompatible with the RNAi (such as siRNA) of the present invention, such as any adverse biological effect produced or interaction with any other component of the pharmaceutically acceptable composition in a harmful manner, their use is also within the scope of the present invention.

[0090] In addition to any conventional carriers, to the extent that they are incompatible with the RNAi (such as siRNA) of the present invention, such as any adverse biological effects produced or interactions with any other components of the pharmaceutically acceptable composition in a deleterious manner, their use is also contemplated by the present invention.

[0091] In some embodiments, according to the pharmaceutical composition of the present invention, wherein the pharmaceutically acceptable carrier can be various carriers conventionally used in the art, for example, it can include at least one of a pH buffer, a protective agent and an osmotic pressure regulator. The pH buffer can be acetate, citrate, prolamin, carbonate or phosphate or any combination thereof. The pH buffer can be a tris(hydroxymethyl)aminomethane hydrochloride buffer with a pH value of 7.5-8.5 and / or a phosphate buffer with a pH value of 5.5-8.5, preferably a phosphate buffer with a pH value of 5.5-8.5. The protective agent can be at least one of inositol, sorbitol and sucrose. Based on the gross weight of the pharmaceutical composition, the content of the protective agent can be 0.01-30% by weight (such as 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 can 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, a person skilled in the art can determine the content of the osmotic pressure regulator.

[0092] In the present invention, the term "treatment" refers to the use of drugs to obtain the desired pharmacological and / or physiological effects. The effect can be preventive in terms of complete or partial prevention of a disease or its symptoms, and / or can be 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 diseases or conditions 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.

[0093] The "RNAi agent" described in the present invention refers to an agent containing RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecules that can degrade or inhibit the transcription and translation of target messenger RNA (mRNA) in a sequence-specific manner. The RNAi agent in the present invention can be manipulated by an RNA interference mechanism (i.e., inducing RNA interference by interacting with the RNA interference pathway-forming mechanism of mammalian cells (RNA-induced silencing complex or RISC)), or act through 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.

[0094] 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 modified by methoxy at the 2nd position of the ribose of the nucleotide, 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; f to the right of the capital letter represents a base modified by fluorine at the 2nd position of the ribose of the nucleotide, such as Cf, Gf, Uf, and Af represent 2'-F (2'-fluoro) C, 2'-FG, 2'-FU, and 2'-FA, respectively; "s" represents that the two nucleotide residues adjacent to the left and right of "s" are connected by thiophosphate groups, for example, "gsu" represents that the g and u residues are connected by thiophosphate groups; Tgn represents a thymine-diol nucleotide residue, and its 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

[0095]

[0096] 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:

[0097]

[0098] 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.

[0099] The structure of the unblocked nucleic acid modified nucleoside (UNA) 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).

[0100] 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.

[0101] In the present invention, "phosphate group", "phosphoester group", "phosphoester bond" can be used interchangeably, including phosphate monoester, phosphodiester or phosphotriester. "Phosphate group" in "phosphorothioate group" also has the same meaning. Unless otherwise specified, the phosphate group between natural nucleotides is a phosphodiester group.

[0102] In the present invention, "deoxynucleotide" refers to a nucleotide in which the hydroxyl group in the pentose of the nucleotide is deoxygenated, and the position of deoxygenation can be 2'-OH or 3'-OH.

[0103] In some optional embodiments of the present invention, the deoxynucleotides include 3'-deoxy modified nucleotides and 2'-deoxy modified nucleotides.

[0104] In the present invention, "2'-deoxy modification" means that the hydroxyl group (2'-OH) in the pentose nucleotide is deoxygenated to hydrogen (2'-H), and "3'-deoxy modification" means that the hydroxyl group (3'-OH) in the pentose nucleotide is deoxygenated to hydrogen (3'-H).

[0105] In the present invention, "2'-X modification" means that the hydroxyl group (2'-OH) in the pentose of the nucleotide is replaced by X (2'-X). For example, "2'-fluoro modification" means that the hydroxyl group (2'-OH) in the pentose of the nucleotide is replaced by fluorine (2'-F), and "2'-amino modification" means that the hydroxyl group (2'-OH) in the pentose of the nucleotide is replaced by fluorine (2'-NH 2 ) is replaced by an allyloxy group (2'-OCH 2 CH=CH 2 ), "2'-alkyl modification" refers to the replacement of the hydroxyl group (2'-OH) in the pentose nucleotide by an alkyl group (2'-alkyl), "2'-O-alkyl modification" refers to the replacement of the hydroxyl group (2'-OH) in the pentose nucleotide by an alkoxy group (2'-alkoxy), and "2'-methoxy modification" refers to the replacement of the hydroxyl group (2'-OH) in the pentose nucleotide by a methoxy group (2'-OCH 3 ) is replaced by a methoxyethyl group (2'-OCH 2 CH 2 OCH 3 )replace.

[0106] In the present invention, "locked nucleotide" refers to a nucleotide obtained by modifying the 2' and 4' carbons on the pentose of the nucleotide to be linked together.

[0107] In the present invention, "5'-X modification" refers to the phosphate (5'-PO(OH) 2 ) is replaced by X (5'-X). For example, "5'-phosphoramidate modification" refers to the phosphate group (5'-PO(OH) 2 ) is replaced by a phosphoramidate group, and "5'-phosphorothioate modification" refers to the phosphate group (5'-PO(OH) 2 ) is replaced by a thiophosphate group, and "5'-methylphosphonate modification" refers to the phosphate group (5'-PO(OH) 2 ) is replaced by a methyl phosphate group, and "5'-phosphate mimetic modification" refers to the phosphate group (5'-PO(OH) 2 ) were replaced by phosphate mimetics.

[0108] 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.

[0109] 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.

[0110] As used herein, "chemically modified" or "modification" means a structure that is chemically different when compared to a 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.

[0111] 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. The compounds of the present invention containing asymmetric carbon atoms can be isolated in optically pure forms or racemic forms. Optically pure forms can be resolved from racemic mixtures or synthesized by using chiral starting materials or chiral reagents.

[0112] 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 then the pure enantiomer is recovered. In addition, separation of enantiomers and diastereomers is usually accomplished by using chromatography, which employs a chiral stationary phase and is optionally combined with chemical derivatization (e.g., carbamate formation from an amine).

[0113] 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 atoms having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that may be incorporated into 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.

[0114] Unless otherwise indicated, when a position is specifically designated as deuterium (D), the position is understood to have deuterium (i.e., at least 10% deuterium incorporation) at least 1000 times greater than the natural abundance of deuterium (which is 0.015%). The natural abundance of the compound in the example may be at least 1000 times greater than deuterium, at least 2000 times greater than deuterium, at least 3000 times greater than deuterium, at least 4000 times greater than deuterium, at least 5000 times greater than deuterium, at least 6000 times greater than deuterium or more. The present invention also includes various deuterated forms of the formula (I) compound. Each available hydrogen atom connected to a carbon atom may be independently replaced by a deuterium atom. Those skilled in the art can synthesize deuterated forms of the formula (I) compound with reference to the relevant literature. In preparing the deuterated form of the compound 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 and deuterated iodomethane.

[0115] The conjugated groups of the present invention can enhance the delivery of therapeutic agents to specific target locations (e.g., specific organs or tissues) in objects such as humans or animals. 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.

[0116] 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, a phosphorothioate or a phosphonate group.

[0117] The definitions and conventions of stereochemistry used in this invention are generally based on the following references: SP 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 in 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, constitute a part of the present invention. Many organic compounds exist in optically active forms, that is, they have the ability to rotate the plane of plane polarized light. When describing optically active compounds, the prefixes D, L or R, S are used to indicate the absolute configuration of the chiral center of the molecule. The prefixes d, l or (+), (-) are used to name the sign of rotation of plane polarized light of a compound. (-) or l means that the compound is levorotatory, and the prefix (+) or d means that the compound is dextrorotatory. The chemical structures of these stereoisomers are identical, 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 no stereoselectivity or stereospecificity during chemical reactions. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomers that lacks optical activity.

[0118] The term "tautomer" or "tautomeric form" refers to structural isomers of different energies that can be interconverted 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.

[0119] The term "composition" refers to a mixture of drugs and other chemical components containing one or more siRAN, siRNA conjugates or physiologically acceptable salts or precursors thereof described in the present invention, as well as other components such as physiologically acceptable carriers and excipients. The purpose of the composition is to facilitate administration to an organism, facilitate the absorption of the active ingredients, and thus exert biological activity.

[0120] 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 approved by the U.S. Food and Drug Administration for use by humans or domestic animals.

[0121] 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 (e.g., a free acid or a free base). When it exists in the form of a salt or a mixed salt, it may be a pharmaceutically acceptable salt.

[0122] The term "acceptable salt" includes acceptable acid addition salts and pharmaceutically acceptable base addition salts. "Acceptable acid addition salt" refers to salts formed with inorganic or organic acids that can retain the biological effectiveness of free bases without other side effects. Inorganic acid salts include, but are not limited to, hydrochlorides, hydrobromides, sulfates, nitrates, phosphates, etc.; organic acid salts include, but are not limited to, formates, acetates, 2,2-dichloroacetates, trifluoroacetates, propionates, caproates, caprylates, decanoates, undecylenates, glycolates, gluconates, lactates, sebacates, adipates, glutarates, malonates, oxalates, maleates, succinates, fumarates, tartrates, citrates, palmitates, stearates, oleates, cinnamates, laurates, malates, glutamates, pyroglutamates, aspartates, benzoates, methanesulfonates, benzenesulfonates, p-toluenesulfonates, alginate, ascorbates, salicylates, 4-aminosalicylates, naphthalene disulfonates, etc. These salts can be prepared by methods known in the art.

[0123] "Pharmaceutically acceptable base addition salt" refers to a salt formed with an inorganic base or an organic base that can maintain 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, preferably sodium salts. The salt derived from organic base includes but is not limited to the following salt: 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, methylglucosamine, 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.

[0124] As described herein, the compounds of the present invention may optionally be substituted with one or more substituents, such as the general formula compounds above, or as specific examples in the embodiments, subclasses, and classes of compounds encompassed by the present invention. In general, the term "substituted" means that one or more hydrogen atoms in a given structure are replaced by a specific substituent. Unless otherwise indicated, an optional substituted group may have a substituent substituted at each substitutable position of the group. When more than one position in a given structural formula can be substituted with one or more substituents selected from a specific group, the substituents may be substituted at each position in the same or different manner.

[0125] The term "hydroxy protecting group" refers to an unstable chemical moiety that protects the hydroxyl group from undesirable reactions during one or more synthetic procedures. After the one or more synthetic procedures, the hydroxy protecting group can be selectively removed. Hydroxy protecting groups 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, isopropoxycarbonyl, biphenylmethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl, 2-furfuryloxycarbonyl, allyloxycarbonyl, acetyl (Ac or -C(O)CH 3 ), formyl, chloroacetyl, trifluoroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl (Bz or -C(O)C 6 H 5 ), 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(CH 3 ) 3 )), triethylsilyl, triisopropylsilyl, MMTr, DMTr or 4',4',4'-trimethoxytrityl, etc.

[0126] The term "amino protecting group" refers to an unstable chemical moiety that protects the amino group from undesirable reactions during the synthesis procedure. After the one or more synthesis procedures, 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 Wuts, Protective Groups in Organic Synthesis, the 3rd edition, John Wiley & Sons, New York (1999). The example of amino protecting group includes but is not limited to acetyl, tert-butyloxycarbonyl, 9-fluorenylmethoxycarbonyl and benzyloxycarbonyl etc.

[0127] The term "solid support" specifically refers to any particle, bead or surface on which oligonucleotide synthesis can occur. For example, inorganic solid supports and organic solid supports can be selected for use in embodiments of the present invention. Inorganic solid supports are preferably selected from silica gel and controlled-pore glass beads (Controlled-pore glass, CPG for short). Organic solid supports are resins, preferably macroporous resins, more preferably highly cross-linked polystyrene, Tentagel (graft copolymers composed of low cross-linked polystyrene matrix, polyethylene glycol (PEG or POE) grafted thereon), polyvinyl acetate (PVA), copolymers of Poros (Poros)-polystyrene / divinylbenzene, aminopolyethylene glycol and cellulose etc. Preferred embodiments of the present invention utilize solid supports based on CPG. Many other purchasable solid supports all belong to the present invention.

[0128] Detailed description of the compounds of the present invention

[0129] 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 LPA genes. The LPA gene can be in a cell, for example, in a cell in a subject, such as a person. The present invention also provides the use of the double-stranded siRNA and its conjugate in the preparation of a drug for treating and / or preventing LPA-related diseases (such as dyslipidemia), and the double-stranded siRNA and its conjugate can inhibit or reduce LPA gene expression, which is used to inhibit LPA gene expression by RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of LPA genes. 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 to LPA.

[0130] On the one hand, the present invention provides a double-stranded siRNA, a conjugate or a salt thereof, which comprises 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 specification of the present invention.

[0131] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, wherein the antisense strand according to 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;

[0132] The detailed information of the nucleotide sequences shown in SEQ ID NO:29 to SEQ ID NO:56 can be found in Table 1 of the specification of the present invention.

[0133] In some embodiments described in the present invention, the double-stranded siRNA, its conjugate or salt described in the present invention is used to inhibit LPA gene expression.

[0134] In some embodiments of the double-stranded siRNA, its conjugate or salt described in the present invention, the double-stranded siRNA is a modified double-stranded siRNA.

[0135] 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 with a conjugation group.

[0136] In some embodiments of the double-stranded siRNA, its conjugate or salt thereof according to the present invention, the length of the sense strand is no more than 23 nucleotides, and the length of the antisense strand is no more than 23 nucleotides.

[0137] In some embodiments of the double-stranded siRNA, its conjugate or salt thereof according to the present invention, the length of the sense strand is 19, 20, 21, 22 or 23 nucleotides in length, and the length of the sense strand does not exceed 23 nucleotides.

[0138] In some embodiments of the double-stranded siRNA, its conjugate or salt thereof according to the present invention, the antisense strand is 21, 22 or 23 nucleotides long.

[0139] In some embodiments of the double-stranded siRNA, its conjugate or salt thereof according to the present invention, the length of the sense strand does not exceed 23 nucleotides, and the length of the sense strand does not exceed 23 nucleotides.

[0140] In some embodiments of the double-stranded siRNA, its conjugate or salt thereof according to the present invention, the sense strand comprises a 3' overhang and / or a 5' overhang, and the 3' overhang or the 5' overhang comprises 1, 2 or 3 nucleotides.

[0141] In some embodiments of the double-stranded siRNA, its conjugate or salt thereof according to the present invention, the 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.

[0142] 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.

[0143] In some embodiments of the double-stranded siRNA, its conjugate or salt thereof 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:

[0144] 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 acid (GNA), wherein the 2'- refers to the 2-position of ribose, such as 2'-fluoro modified nucleotides refer to nucleotides in which the 2-position of ribose is substituted by fluorine, and for example, 2'-methoxy modified nucleotides refer to nucleotides in which the 2-position of ribose is substituted by methoxy. In some embodiments of the double-stranded siRNA, its conjugate or salt thereof 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] 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 acids (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 for example, a 2'-methoxy-modified nucleotide refers to a nucleotide in which the 2-position of ribose is replaced by methoxy.

[0146] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the modified nucleotides are each independently present in one or more positions selected from the following:

[0147] The nucleotides at the 5' end of the sense strand are the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th and 21st nucleotides from the starting point.

[0148] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the modified nucleotides are each independently present in one or more positions selected from the following:

[0149] The nucleotides at the 5' end of the antisense strand are 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 positions of the starting point.

[0150] In some embodiments of the double-stranded siRNA, its conjugate or salt thereof according to the present invention, the 5'-phosphorothioate linkage modification or the 3'-phosphorothioate linkage modification is each independently present at one or more positions selected from the following:

[0151] 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.

[0152] In some embodiments of the double-stranded siRNA, its conjugate or salt thereof according to the present invention, the 5'-phosphorothioate linkage modification or the 3'-phosphorothioate linkage modification is each independently present at one or more positions selected from the following:

[0153] 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.

[0154] In some embodiments of the double-stranded siRNA, its conjugate or salt thereof according to the present invention, the 2'-fluoro modified nucleotide, the 2'-methoxy modified nucleotide and Y are each independently and optionally present in one or more positions selected from the following:

[0155] The nucleotides at the 5' end of the sense strand are the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th and 21st nucleotides from the starting point.

[0156] In some embodiments of the double-stranded siRNA, its conjugate or salt thereof according to the present invention, the 2'-fluoro modified nucleotide, the 2'-methoxy modified nucleotide and Y are each independently and optionally present in one or more positions selected from the following:

[0157] The nucleotides at the 5' end of the antisense strand are 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 positions of the starting point.

[0158] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the 2'-fluoro modified nucleotide is present at one or more positions selected from the following:

[0159] The nucleotides at the 5' end of the sense strand are the 5th, 7th, 8th, 9th, 10th and 11th nucleotides of the starting point.

[0160] In some embodiments of the double-stranded siRNA, its conjugate or salt according to the present invention, the 2'-fluoro modified nucleotide is present at one or more positions selected from the following:

[0161] 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.

[0162] In some embodiments of the double-stranded siRNA, its conjugate or salt thereof according to the present invention, the 5'-phosphorothioate linkage modification or the 3'-phosphorothioate linkage modification is each independently present at one or more positions selected from the following:

[0163] The nucleotides at the 5' end of the sense strand are between positions 1-2, 2-3 and 3-4 of the starting point.

[0164] In some embodiments of the double-stranded siRNA, its conjugate or salt thereof according to the present invention, the 5'-phosphorothioate linkage modification or the 3'-phosphorothioate linkage modification is each independently and optionally present at one or more positions selected from the following:

[0165] The nucleotides at the 3' end of the positive strand are between the 1st and 2nd positions, the 2nd and 3rd positions, and the 3rd and 4th positions of the starting point.

[0166] In some embodiments of the double-stranded siRNA, its conjugate or salt thereof according to the present invention, the 5'-phosphorothioate linkage modification or the 3'-phosphorothioate linkage modification is each independently present at one or more positions selected from the following:

[0167] The nucleotides at the 5' 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.

[0168] In some embodiments of the double-stranded siRNA, its conjugate or salt thereof according to the present invention, the 5'-phosphorothioate linkage modification or the 3'-phosphorothioate linkage modification is each independently present at one or more positions selected from the following:

[0169] 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.

[0170] 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, 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.

[0171] The "one or more positions" in "one or more positions selected from the following" or "one or more positions selected from the following" in the present invention refers to the presence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 modifications, wherein "optionally" means that the modification may be present or there may not be any modification, i.e., 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

[0172] In some embodiments of the double-stranded siRNA, its conjugate or salt described in the present invention, the double-stranded siRNA comprises 2 Ys.

[0173] In some embodiments of the double-stranded siRNA, its conjugate or salt described in the present invention, the double-stranded siRNA comprises 3 Ys.

[0174] In some embodiments of the double-stranded siRNA, its conjugate or salt described in the present invention, the sense chain comprises one of the nucleotide sequences shown in SEQ ID NO:57 to SEQ ID NO:84 (i.e., one of the sense chains in Table 2 of the specification of the present invention), and the length of the sense chain 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 specification of the present invention.

[0175] In some embodiments of the double-stranded siRNA, its conjugate or salt described in the present invention, the antisense strand includes 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 specification of the present invention), 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 specification of the present invention.

[0176] In some embodiments of the double-stranded siRNA, its conjugate or salt of the present invention, the double-stranded siRNA, its conjugate or salt of the present invention 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 is no more than 21 nucleotides, and the length of the antisense strand is no more than 23 nucleotides. The nucleotide information of the double-stranded siRNA of siRNA ID NO: 29 to siRNA ID NO: 56 is detailed in Table 2 of the present specification.

[0177] In some embodiments of the double-stranded siRNA, its conjugate or salt described in the present invention,

[0178] The sense strand comprises a nucleotide sequence of 5'-csasagcuUfgGfUfCfaucuausgsa-3', and the antisense strand comprises a nucleotide sequence of 5'-usCfsauaGfaugaccaAfgCfuugsgsc-3'; or

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

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

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

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

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

[0184] The sense strand comprises a nucleotide sequence of 5'-gsasaaugUfcCfUfGfgaagcasusa-3', and the antisense strand comprises a nucleotide sequence of 5'-usAfsugcUfuccaggaCfaUfuucsusu-3'; or

[0185] The sense strand comprises a nucleotide sequence of 5'-csasugguAfaUfGfGfacagagsusa-3', and the antisense strand comprises a nucleotide sequence of 5'-usAfscucUfguccauuAfcCfaugsgsu-3'.

[0186] In some embodiments of the double-stranded siRNA, its conjugate or salt of the present invention, 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 conjugate or salt of the present invention, its sense strand has one of the following modification patterns:

[0187] Sense strand (5'-3'): mBsmBsmBmBmBmBfBmBfBfBfBmBmBmBmBmBmBmBmB; and

[0188] Sense strand (5'-3'): mBsmBsmBmBmBmBfBmBfBfBfBmBmBmBmBmBmYmBmB;

[0189] 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.

[0190] In some embodiments of the double-stranded siRNA, its conjugate or salt described in the present invention, its antisense strand has one of the following modification patterns:

[0191] Antisense strand (5'-3'): mBsfBsmBmBmBfBmBmBmBmBmBmBmBfBmBmBmBmBsmBsmB;

[0192] Antisense strand (5'-3'): mBsfBsmBmBmBfBmBmBmBmBmBmBmBfBmBmBmBmBsmBsmB;

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

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

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

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

[0197] 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

[0198] In some embodiments of the double-stranded siRNA, its conjugate or salt described in the present invention, the double-stranded region is 17-23 nucleotide pairs in length.

[0199] In some embodiments of the double-stranded siRNA, its conjugate or salt thereof according to the present invention, the double-stranded region is 17, 18, 19, 20, 21, 22 or 23 nucleotide pairs in length.

[0200] 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 conjugating group.

[0201] 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 conjugating group.

[0202] In some embodiments of the double-stranded siRNA, its conjugate or salt described in the present invention, the 3' end of the sense strand of the double-stranded siRNA is conjugated to a conjugate.

[0203] In some embodiments of the double-stranded siRNA, its conjugate or salt thereof 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.

[0204] In some embodiments of the double-stranded siRNA, its conjugate or salt described in the present invention, the conjugated group includes GalNAc or its derivatives.

[0205] 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 by a divalent, trivalent or tetravalent branched linker.

[0206] 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:

[0207]

[0208]

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

[0210] In some embodiments of the double-stranded siRNA, its conjugate or salt, 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, 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 LPA gene in a patient, which comprises administering to the patient the double-stranded siRNA and double-stranded siRNA conjugate or a composition thereof (i.e., double-stranded RNAi agent) of the present invention, wherein the nucleic acid ligand conjugate or the composition thereof may be a therapeutically effective amount.

[0215] In one embodiment, administering the double-stranded siRNA and double-stranded siRNA conjugates of the present invention or a composition 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 in the present invention, 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 described in the present invention, the LPA-related disease is atherosclerosis, dyslipidemia, NAFLD or NASH.

[0224] Pharmaceutical composition, preparation, administration and disease treatment method of the nucleic acid conjugate of the present invention

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

[0226] The pharmaceutical composition or medicament of the present invention comprises 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 that is intentionally included in a drug delivery system in addition to an active pharmaceutical ingredient (API, therapeutic product, such as an LPA RNAi agent of the present invention). 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, effectiveness of API delivery during storage or use. A pharmaceutically acceptable excipient may or may not be an inert substance.

[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, flavoring agents, 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, ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). It should be stable under the conditions of production and storage, and should prevent the contamination of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol) and suitable mixtures 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 that contains a basic dispersion medium and the required other ingredients from the above list.

[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 suitable 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, for example, 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 1.5 mg / kg to about 10 mg / kg, about 2 mg / kg to about 5 mg / kg bout 2.5mg / kg, about 2mg / kg to about 10mg / kg, about 3mg / kg to about 5mg / kg, about 3mg / kg to about 10mg / kg, about 3.5mg / kg to about 5mg / kg, about 4mg / kg to about 5mg / kg, about 4.5mg / kg to about 5mg / kg, about 4mg / kg to about 10mg / kg, about 4.5mg / kg to about 10mg / kg, about 5mg / kg to about 10mg / kg, about 5.5mg / kg to about 10mg / kg, about 6mg / kg to about 10mg / kg, about 6.5mg / kg to about 10mg / kg, about 7mg / kg to about 10mg / kg, about 7.5mg / kg to about 10mg / kg, about 8mg / kg to about 10mg / kg, about 8.5mg / kg to about 10mg / kg, about 9mg / kg to about 10mg / kg, or about 9.5mg / kg to about 10mMg / 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 LPA RNAi drug substance is administered, followed by a second dose of about 1 to 100 mg of LPA RNAi drug substance administered about 1 month later, and additional doses are administered once 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 injection and subcutaneous administration.

[0234] In certain embodiments, the pharmaceutical composition can be administered by intravenous infusion within a period of time, such as within 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 can be regularly repeated, 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 with a lower frequency. For example, after three months of weekly or two weeks of administration, 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 daily, or iRNA can be used as two, three or more sub-doses at suitable intervals in one day, or even use continuous infusion or send by controlled release formulation.In this case, the iRNA contained in each sub-dosage must be correspondingly less, to obtain total daily dose.Also composite dosage unit can be used for sending in a few days, for example, using conventional sustained release formulation, it provides continuous 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 part, as can be used together with medicament of the present invention.In this embodiment, dosage unit contains corresponding multiple daily doses.Higher dose (that is, loading dose) can be used initially, then lower dose is used in the duration period.

[0236] In some embodiments, a single dose of the pharmaceutical composition can be long-acting, so that subsequent doses 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 a specific embodiment, the iRNA is administered about once a month to about once a 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) may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the following steps: combining the active ingredients with the pharmaceutical carrier(s) or excipient(s). Generally speaking, these preparations are prepared by uniformly and finely combining the active ingredients with liquid carriers or finely dispersed solid carriers 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 media or mixed media. Aqueous suspensions can further include materials that increase the viscosity of the suspension, such materials include, for example, sodium carboxymethylcellulose, sorbitol and / or dextran. The suspension can also include a stabilizing agent.

[0240] The pharmaceutical composition disclosed in the present invention includes preparations suitable for parenteral administration. The preparation can be conveniently present in unit dosage form and can be prepared by any method known in the pharmaceutical field. The amount of active ingredient that can be combined with auxiliary materials to prepare a single dose form is generally the amount of siRNA that produces a therapeutic effect. Generally speaking, in units of one percent, the amount is about 1% to about 99% active ingredient, preferably about 5% to about 70%, and most preferably 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 dyslipidemia, NAFLD, NASH and metabolic-related conditions and diseases.

[0242] The present invention provides methods for contacting or delivering an effective amount of any oligonucleotide of the present invention (e.g., a double-stranded oligonucleotide) 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 the reduction 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 for any appropriate cell type. In some embodiments, the expression of LPA 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 the oligonucleotide is contacted or delivered 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, the expression of LPA 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 the oligonucleotide is contacted or delivered to the cell or cell population.

[0243] General synthesis method of the compound, double-stranded siRNA and double-stranded siRNA conjugate 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 used to further illustrate the content of the present invention.

[0245] In the following examples, all temperatures are in degrees Celsius (°C) unless otherwise indicated. The chromatographic column used was a silica gel column, and the silica gel (200-300 mesh) was purchased from Qingdao Ocean Chemical Plant, NH 2 CPG was purchased from Hebei Dinaxingke. Nuclear magnetic resonance spectroscopy was performed with CDC1 3 DMSO-d 6 , CD 3 OD or acetone-d 6is the solvent (in ppm), TMS (0 ppm) or chloroform (7.25 ppm) is used as the reference standard. When multiple peaks appear, 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), q (quartet). The coupling constant J is expressed in Hertz (Hz).

[0246] Low-resolution mass spectrometry (MS) data were determined by an Agilent 6320 series LC-MS spectrometer equipped with a G1312A binary pump and a G1316A TCC (column temperature was 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 was 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 invention:

[0249] DCM Dichloromethane DMTrCl 4,4'-Bismethoxytrityl chloride TFA Trifluoroacetic acidTFA Trifluoroacetic acid

[0250] Py Pyridine i-Pr Isopropyl

[0251] PE Petroleum ether EA Ethyl acetate

[0252] TBAF Tetrabutylammonium fluoride in tetrahydrofuran HOBT 1-Hydroxybenzotriazole

[0253] ACN Acetonitrile DMAP 4-Dimethylaminopyridine

[0254] Ac 2 O Acetic anhydride MsCl Methanesulfonyl chloride

[0255] Boc tert-ButyloxycarbonylTMsOTf Trimethylsilyl trifluoromethanesulfonate MeOH Methanol THF Tetrahydrofuran

[0256] DMSO Dimethyl sulfoxide mL milliliter

[0257] DMF N,N-Dimethylformamide min minutes

[0258] DCM dichloromethane M,N,mol / L mole / liter DIPEA N,N-Diisopropylethylamine h hours

[0259] TiPDSCl 2 1,3-Dichloro-1,1,3,3-tetraisopropyldisiloxane RT, rt Room temperature

[0260] HBTU Benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate DETAILED DESCRIPTION

[0261] The scheme of the present invention will be explained below in conjunction with 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 the routine adjustments in the field. If the specific techniques or conditions are not indicated in the examples, they are carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. The reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

[0262] Preparation Example

[0263] In the following preparation examples, the inventors describe in detail the preparation process of the compounds of the present invention by taking some of the compounds of the present invention as examples, wherein: For CPG.

[0264] Example 1: Synthesis of Nucleoside Monomer Y

[0265]

[0266] Step 1: Synthesis of compound 1-2

[0267] (2R,3R,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazine-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, cooled to -5°C, and then 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (1.13 mL, 3.53 mmol) was added dropwise. After the addition, 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 separated and purified by silica gel column chromatography (eluent: PE / EA (v / v) = 2 / 3) to obtain white solid compound 1-2 (1.38 g, 75.3%), MS (ESI, pos.ion) m / z: 534.3 [M+H] + .

[0268] Step 2: Synthesis of Compound 1-3

[0269] 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), heated to 50°C and stirred for 4 h. After the reaction, 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 (599 MHz, DMSO-d 6 )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).

[0270] Step 3: Synthesis of Compound 1-4

[0271] 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 in sequence, and stirred at 0°C for 20 min. After the reaction was completed, the reaction solution was poured into saturated NH 4 Cl solution, extracted with EA, concentrated the organic phase, and purified the residue 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] + .

[0272] Step 4: Synthesis of Compound 1-5

[0273] Compound 1-4 (5.1 g, 8.46 mmol), tetrahydrofuran (50 mL) and tetrabutylammonium fluoride tetrahydrofuran solution (9.48 mL, 9.48 mmol, 1 M) were mixed and stirred at room temperature for reaction. After the reaction was completed, the mixture was concentrated under reduced pressure and the residue was purified by silica gel column chromatography (MeOH / DCM (v / v) = 1 / 20) to obtain white solid compound 1-5 (2.4 g, 79%). MS (ESI, pos.ion) m / z: 361.3 [M+H] + .

[0274] Step 5: Synthesis of Compound 1-6

[0275] 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, then cooled to 0°C, and 4,4'-bismethoxytrityl chloride (2.71 g, 7.99 mmol) was added in batches. The resulting reaction mixture was heated to room temperature and stirred for 3 h. The temperature was lowered to 0°C, and 4,4'-bismethoxytrityl chloride (1.35 g) was added twice, and the reaction was continued to stir at 0°C for 2 h. The reaction system was diluted with MeOH (1 mL) and DCM (100 mL), and washed with saturated sodium bicarbonate solution and saturated sodium chloride solution in turn. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column (EA / PE (v / v) = 2 / 1) to obtain a light yellow solid compound 1-6 (4.0 g, 91%). MS (ESI, pos.ion) m / z: 663.7 [M+H] + .

[0276] Step 6: Synthesis of Compound 1

[0277] Compound 1-6 (2.0 g, 3.02 mmol) and 1H-tetrazole (0.25 g, 3.62 mmol) were added to dichloromethane (40 mL), and bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.92 mL, 6.04 mmol) was added dropwise under nitrogen protection, and 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, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (EA / PE (v / v) = 4 / 1) to obtain compound 1 (2.13 g, 82%). MS (ESI, pos.ion) m / z: 864.9 [M+H] + ; 1 H NMR (400 MHz, CDCl 3 )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 (162 MHz, CDCl 3 )δ150.01,149.80.

[0278] According to technical knowledge in the art, compound 1 is deprotected after nucleic acid synthesis to obtain a nucleic acid residue embedded group Y.

[0279] Example 2: Synthesis of compound DAW40007-3

[0280]

[0281]

[0282] Step 1: Synthesis of compound 2-2

[0283] Compound 2-1 (2.50 g, 28.05 mmol) and triethylamine (7.8 mL, 56.1 mmol) were dissolved in DCM (120 mL), and benzyl chloroformate (9.57 g, 56.1 mmol) was added dropwise at 0°C. After the addition, the reaction mixture was heated to room temperature and stirred for 20 h, and saturated ammonium chloride solution (50 mL) was added for dilution, the liquids were separated, the aqueous phase was discarded, the organic phase was concentrated, and the residue was purified by silica gel column chromatography (MeOH / DCM (V / V) = 1 / 30) to obtain a white solid compound 2-2 (2.96 g, 47.2%). MS (ESI, pos.ion) m / z: 224.2 [M+H] + ; 1 H NMR (400 MHz, CDCl 3 )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).

[0284] Step 2: Synthesis of compound 2-4

[0285] 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 and stirred at room temperature for 10 min. TMSOTf (0.51 g, 2.28 mmol) was added and the reaction mixture was stirred at room temperature for 18 h. The reaction solution was poured into a saturated sodium bicarbonate solution (100 mL), then extracted with DCM (100 mL), washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (PE / EA (V / V) = 2 / 1 to 0 / 1) to obtain a light brown oil compound 2-4 (1.8 g, 71.51%). MS (ESI, pos.ion) m / z: 553.3 [M+H] + .

[0286] Step 3: Synthesis of Compound 2-5

[0287] 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), and then TFA (0.12 g, 1.03 mmol) was added, followed by hydrogen replacement three times, and then stirred at room temperature for 19 h in a hydrogen atmosphere. After the reaction was completed, diatomaceous earth was filtered, and the solvent was evaporated under reduced pressure to obtain a light brown oily compound 2-5 (0.55 g, 100.32%). MS (ESI, pos.ion) m / z: 419.3 [M-TFA+H] + .

[0288] Step 4: Synthesis of Compound 2-7

[0289] 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), and HOBT (0.46 g, 3.44 mmol), HBTU (1.30 g, 3.44 mmol) and DIPEA (2.66 mL, 16.03 mmol) were added in sequence. The reaction mixture was reacted at room temperature for 16 h. After the reaction, water (20 mL) and DCM (50 mL × 2) were added in sequence, and the organic phase was washed with saturated sodium bicarbonate solution (30 mL) and saturated brine (20 mL) in sequence. The solvent was evaporated under reduced pressure, and the obtained residue was separated and purified by silica gel column chromatography (MeOH / EA (v / v) = 1 / 20) to obtain a white solid 2-7 (1.0 g, yield 72.7%). MS (ESI, pos.ion) m / z: 602.3 [M+H] + ; 1 H NMR (400MHz, CD 3 OD)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).

[0290] Step 5: Synthesis of Compound 2-8

[0291] 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%).

[0292] MS (ESI, pos.ion) m / z: 502.2 [M+H] + ; 1 H NMR (400MHz, CD 3 OD)δ5.36(d,J=3.3Hz,1H),5.08(dd,J=11.3,3.3Hz,1H),4.58(d,J=8.4H z,1H),4.16–4.10(m,3H),4.08–4.02(m,1H),3.92–3.85(m,1H),3.58–3.5 0(m,1H),3.26–3.20(m,2H),2.16(s,3H),2.04(d,J=5.2Hz,6H),1.98(s,3 H),1.97(s,3H),1.61–1.56(m,4H),1.54–1.52(m,2H),1.42–1.38(m,2H).

[0293] Step 6: Synthesis of Compound 2-10

[0294] 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 then HOBT (0.17 g, 1.26 mmol), HBTU (0.48 g, 1.26 mmol) and DIPEA (0.5 mL, 3.0 mmol) were added in sequence, and the reaction mixture was stirred at 30 ° C for 3 h. After the reaction was completed, water (20 mL) was added to quench the reaction, and then extracted with DCM (100 mL × 2), and the organic phases were combined, and the organic phases were washed with saturated sodium bicarbonate solution (40 mL) and saturated sodium chloride solution (40 mL) in sequence, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was separated and purified by silica gel column chromatography (DCM / MeOH (v / v) = 10 / 1) to obtain white solid compound 2-10 (0.23 g, yield 37%). MS(ESI,pos.ion)m / z:1046.3[M / 2+H] + ; 1 H NMR (400MHz, CD 3OD)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).

[0295] Step 7: Synthesis of Compound 2-11

[0296] 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 hydrogen was replaced three times, and the reaction mixture was stirred at room temperature for 11 h in a hydrogen atmosphere. After the reaction was completed, the reaction mixture was filtered through diatomaceous earth, and the obtained filtrate was evaporated under reduced pressure to dry the solvent to obtain a white solid compound 2-11 (0.19 g, 100%). MS (ESI, pos.ion) m / z: 1001.1 [M / 2+H] + .

[0297] Step 8: Synthesis of compound DAW40007-1

[0298] 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 heated to 40 ℃. The solvent was evaporated, and the obtained residue was dissolved in acetonitrile (10 mL), and separated by reverse phase preparative column (acetonitrile / water solution (v / v) = 43% to 60%, 50 min). Salt was added to the solution containing the product after preparative separation to saturate the solution, and the organic phase was separated. The aqueous phase was extracted with acetonitrile (100 mL×2), and the organic phases were combined. The combined organic phases were dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. Acetonitrile (30 mL) was added to the residue, and then dried over sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a light yellow solid compound DAW40007-1 (0.080 g, yield 24%). MS (ESI, neg.ion) m / z: 2400.18 [MH] - ; 1 H NMR (400 MHz, DMSO-d 6 )δ8.31(s,3H),7.81(d,J=9.2Hz,3H),7.57(t,J=6.0Hz,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=11.2,3.5Hz,4H),4.49(d,J=8.5Hz,3H),4.40(d,J=4.8Hz,1H),4.15(s,1H),4.07–4.00(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).

[0299] Step 9: Synthesis of compound DAW40007-2

[0300] 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 for dilution, 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 a white solid compound DAW40007-2 (0.08 g, 96.07%). MS(ESI,neg.ion)m / z:2500.12[MH] - .

[0301] Step 10: Synthesis of compound DAW40007-3

[0302] 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. Then the mixture was transferred to a flask containing 0.35 g H 2 N-CPG (purchased from Hebei Dinaxingke) was placed in a solid phase synthesizer and shaken for 22.5 hours. The filter cake was filtered and washed with DCM / MeOH (V / V=9 / 1, 10 mL) and DCM (10 mL), and then dried. The filter cake was diluted with 25% Ac 2 The mixture was stirred in O / Py solution (5 mL) for 3 h, filtered, and the filter cake was rinsed with DCM / MeOH (V / V=9 / 1, 10 mL) and DCM (10 mL) in turn, and dried under reduced pressure to obtain white solid DAW40007-3 (0.357 g), with a measured loading of 14.95 μmol / g. Compound L96-DMTr-CPG was conjugated with oligonucleotides and then deprotected to obtain L96, and compound DAW40007-3 was conjugated with oligonucleotides and then deprotected to obtain DAW40007-4.

[0303] Synthesis of compound L96-DMTr-CPG: Compound L96-DMTr-CPG was prepared according to the method described in patent application WO2014025805A1.

[0304]

[0305] Synthesis of compound 38: Compound 38 was prepared according to the method described in patent application WO2018044350A1.

[0306]

[0307] 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 a NAG37 structure.

[0308] Example 3: Synthesis of double-stranded siRNA and double-stranded siRNA conjugates

[0309] 1. Synthesis of double-stranded siRNA without conjugation group

[0310] The synthesis steps of the siRNA sense strand and antisense strand of the present invention are as follows:

[0311] The synthesis was completed according to the theoretical yield of 1umol. Weigh 1umol of solid support CPG (purchased from Hebei Dinaxingke). All 2'-modified RNA phosphoramidite monomers and auxiliary reagents were commercially available, 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, which was placed in the reagent designated 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.

[0312] The nucleotide containing CPG on the solid support obtained above was blown dry with dry argon, then transferred to a 2mL EP tube, and 28% ammonia solution (1.8mL) was added and heated at 55°C for 5 to 18 hours. Filter, wash the filter cake with water (0.5mL), combine the filtrate, and concentrate under reduced pressure to obtain a white or yellow colloidal solid. After reverse phase preparation and purification, the preparation solution was concentrated, passed through a gel column, and excess salt was removed to obtain an oligonucleotide. The concentration of the obtained oligonucleotide was determined by a micro-ultraviolet spectrophotometer (SPECTRO statNano). Mass spectrometry detection and analysis was completed on the Agilent 6530LC-MS Q-Tof system. After primary scanning, the molecular weight of the nucleic acid was calculated after deconvolution.

[0313] Annealing steps:

[0314] The double-stranded siRNA sense strand synthesized above was mixed with the anti-sense strand synthesized above in equimolar amounts, heated to 95° C., maintained at the temperature for 10 minutes, and then slowly cooled to room temperature, and then freeze-dried to obtain the target double-stranded siRNA.

[0315] 2. Synthesis of siRNA conjugates:

[0316] Synthesis of antisense strand: refer to the above synthesis method.

[0317] Synthesis of the sense strand: Replace the universal solid support CPG with the GalNAc solid support prepared in the present invention (such as compound DAW40007-3 or L96-DMr-CPG), and prepare the sense strand connected to the conjugation group in the double-stranded siRNA conjugate of the present invention by referring to the above-mentioned synthesis method.

[0318] Annealing steps:

[0319] The sense strand synthesized above was mixed with the antisense strand synthesized above in equimolar amounts, heated to 95° C., maintained at the temperature for 10 min, and then slowly cooled to room temperature, and then freeze-dried to obtain the target siRNA conjugate.

[0320] The above synthesis scheme lists the general synthetic experimental steps for preparing the double-stranded siRNA and its conjugates in the present invention, including that those skilled in the art can make appropriate method modifications or raw material adjustments according to actual conditions to prepare the double-stranded siRNA and its conjugates of the present invention. Unless otherwise specified, the double-stranded siRNA and its conjugates of the present invention can be prepared by the method described in the above synthesis scheme.

[0321] 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.

[0322] Table 1: Unmodified double-stranded siRNA synthesized by the present invention

[0323]

[0324]

[0325] Table 2: Modified double-stranded siRNA synthesized by the present invention

[0326]

[0327] 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 modified by methoxy at the 2nd position of the ribose sugar of the nucleotide, 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 on the right side of the capital letter represents a base modified by fluorine at the 2nd position of the ribose sugar of the nucleotide, 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 thiophosphate groups, for example, "gsu" indicates that the g and u residues are connected by thiophosphate groups.

[0328] Example 4: Cellular activity and cytotoxicity test of siRNA or its conjugates of the present invention

[0329] In vitro knockdown activity screening:

[0330] HEK293 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 Each well was transfected with psiCHECK2 plasmid containing the full-length fragment of LPA 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 and 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.

[0331] The experimental results show that the siRNA or its conjugate of the present invention has good inhibitory activity against LPA. The results of the inhibitory activity of some double-stranded siRNA of the present invention against LPA are shown in Table A.

[0332] Table A: Experimental results of partial double-stranded siRNA of the present invention on LPA inhibition activity

[0333]

[0334] According to the technical knowledge in the art, the siRNA conjugate of the present invention is obtained by conjugating siRNA with GalNAc or its derivatives, wherein the main function of the conjugated group is the delivery function. In the case where the siRNA has good activity, the skilled person in the art can expect that the siRNA conjugate connected with the conjugated group has similar or better gene inhibition / silencing activity than the siRNA. In addition, for the conjugated group on the siRNA conjugate, the skilled person in the art can select a suitable conjugated group according to the technical knowledge in the art, such as GalNAc or its derivatives such as L96 and DAW40007-4.

[0335] Example 5: Cytotoxicity Assay

[0336] HepG2 cells were cultured in DMEM medium containing 10% fetal bovine serum in an atmosphere of 5% CO 2 , 37°C constant temperature incubator. When the cells are in logarithmic growth phase and in good condition (70% confluence), transfection is performed using transfection reagent. The cell concentration is adjusted to 2.5×10 5 / mL, and 1*10 4 cells. According to the instructions of RNAiMAX transfection reagent, different concentrations of siRNA and negative control were transfected. After incubation at 37°C and 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. The absorbance at 450 nm was measured with an ELISA reader, and data were processed using Excel and graphed using GraphPad Prism.

[0337] The experimental results show that the siRNA and siRNA conjugates of the present invention have low cytotoxicity.

[0338] Example 6: Lysosome stability assay

[0339] Take 20μM of the conjugate and lysosome (final concentration 0.2mU / μL), mix with sodium citrate solution (PH5.0), do not add lysosome to the reference reagent, incubate at 37 degrees, take 5μL of samples at 1 / 2 / 5 / 8 / 24 / 48h, add to 15μL 9M urea for denaturation, then add 4μL 6*loading buffer, and immediately terminate the reaction at -80 degrees. Take 10μL of sample and load it on 16% non-denaturing polyacrylamide gel. After the electrophoresis, the gel is stained with Gelred on a shaker for 10 minutes, and the gel is imaged and photographed.

[0340] The experimental results show that the siRNA and siRNA conjugates of the present invention have good stability in lysosomes.

[0341] Example 7: Activity detection using human primary cells

[0342] Human primary hepatocytes were cultured in DMEM medium containing 10% fetal bovine serum in an atmosphere of 5% CO 2 When the cells are in the logarithmic growth phase and in good condition, inoculate the cells, add different concentrations of siRNA, siRNA conjugates, positive controls and negative controls to the cells, and incubate at 37°C and 5% CO 2 After incubation for 48 h, the cells were collected and the mRNA expression levels of the target gene LPA and the internal reference GAPDH were detected using the QuickEasy CellDirect RT-qPCR kit (Taqman).

[0343] The experimental results show that the siRNA or its conjugate of the present invention has good inhibitory activity against LPA.

[0344] Example 8: Evaluation of LPA siRNA conjugate knockdown activity using hLPA transgenic mice

[0345] To evaluate the in vivo activity of LPA siRNA, an LPA humanized transgenic mouse model (5-8 mice per group) was used. Baseline serum weight, ALT and LPA protein concentrations of mice in each group were measured before administration and grouped. A single dose of 0.5 mg / kg of GalNAc-siRNA or saline was subcutaneously administered at the back of the neck on D0. Blood samples were collected on D0 / 4 / 7 / 14 / 21 / 28 / 35 / 49, and human LPA protein concentration was determined using a human Lipoprotein A ELISA kit (ab212165). The knockdown percentage was calculated by comparing the LPA protein levels in the siRNA group and the vehicle group.

[0346] The experimental results show that the siRNA or its conjugate of the present invention has good knockdown activity against LPA in mice

[0347] Although the present invention has been described in detail above by means of general description, specific implementation methods and tests, it is obvious to those skilled in the art that some modifications or improvements may be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

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; Optionally, the antisense strand 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; Optionally, the sense strand is no longer than 23 nucleotides and the antisense strand is no longer than 23 nucleotides.

2. The double-stranded siRNA, its conjugate or salt according to claim 1, 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.

3. The double-stranded siRNA, its conjugate or salt according to any one of claims 1 to 2, characterized in that: It contains one of the double-stranded siRNAs shown in siRNA ID NO: 1 to siRNA ID NO:

28.

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 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.

5. The double-stranded siRNA, its conjugate or salt according to any one of claims 1 to 4, 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 6. The double-stranded siRNA, its conjugate or salt according to claim 4 or 5, 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.

7. The double-stranded siRNA, its conjugate or salt according to any one of claims 4 to 6, 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.

8. The double-stranded siRNA, its conjugate or salt according to any one of claims 1 to 7, characterized in that: The double-stranded region is 17-23 nucleotide pairs in length.

9. The double-stranded siRNA, conjugate or salt thereof according to any one of claims 1 to 8, 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.

10. The double-stranded siRNA, its conjugate or salt according to any one of claims 1 to 9, 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.

11. The double-stranded siRNA, conjugate or salt thereof according to any one of claims 1 to 10, 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.

12. The double-stranded siRNA, its conjugate or salt according to claim 11, 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:

13. A pharmaceutical composition comprising the double-stranded siRNA, its conjugate or salt according to any one of claims 1 to 12, and a pharmaceutically acceptable carrier.

14. Use of the double-stranded siRNA, its conjugate or salt according to any one of claims 1 to 12, or the pharmaceutical composition according to claim 13 in the preparation of a medicament for treating and / or preventing LPA-related diseases.

15. The use according to claim 14, 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.

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