Rnai agent for inhibiting lysophosphatidic acid receptor 1 (LPAR1) gene expression, and use thereof

Abstract

Disclosed in the present invention are an RNAi agent for inhibiting LPAR1 gene expression in cells, and a pharmaceutical composition thereof. The present invention further relates to the therapeutic use of the RNAi agent.

Description

RNAi agent for inhibiting lysophosphatidic acid receptor 1 (LPAR1) gene expression and its use Technical Field

[0001] The present invention relates to nucleic acids that inhibit the expression of lysophosphatidic acid receptor 1 (LPAR1) gene in cells, including RNAi agents (such as siRNA) and pharmaceutical compositions thereof. The present invention also relates to the medical use of these RNAi agents. Background Art

[0002] Lysophosphatidic acid (LPA) is a bioactive phospholipid molecule produced during cell membrane synthesis. It is present in all eukaryotic tissues and fluids (blood, cerebrospinal fluid, semen, urine, saliva, and aqueous humor) and can be detected under physiological and pathological conditions. LPA can be detected in conditions of neurological injury, in the serum of systemic sclerosis (SSc), in sepsis, in ascites associated with pancreatic cancer, and in the plasma of patients with chronic liver damage and obesity. LPA acts through specific LPA receptors (LPAR1 to LPAR6) coupled to G proteins and is associated with a variety of cellular responses (such as proliferation and migration) and the pathological processes of various diseases (including fibrosis, cancer, neuronal disorders, and bone metabolism).

[0003] The biological signaling of LPA is mediated by intracellular receptors (nuclear receptors, peroxisome proliferator-activated receptor γ (PPAR), actin-binding protein (ABP)) and extracellular receptors (LPAR1-6). Among them, LPA receptor subtypes (LPAR1-6) are divided into the endothelial differentiation gene (Edg) family of G protein-coupled receptors (GPCRs) and the non-Edg ​​family of purinergic receptors. The first three subtypes of LPA receptors (LPAR1-3) belong to the Edg family of GPCRs (Edg2, Edg4, Edg7), and the amino acid sequence composition of these three receptors is similar. LPAR4 (GPR23 / P2Y9), LPAR5 (GPR92) and LPAR6 (GPR87) belong to the non-Edg ​​subfamily cluster of purinergic GPCRs.

[0004] LPAR1 was first discovered in the developing brain in 1996. It is primarily enriched in the ventricular zone of the embryonic cerebral cortex, but has also been found in the superficial marginal zone and meninges, where it was identified as a receptor for LPA. LPAR1 is widely expressed in various human tissues and organs, with high mRNA levels in the brain, heart, colon, small intestine, and placenta, and relatively low levels in other organs and tissues. LPA-induced binding of the LPAR1 / Edg2 receptor to Gαi / o, Gαq / 11, and Gα12 / 13 forms a complex that transmits signals through molecules such as Ras homology (Rho) protein family GTPases, phospholipase C (PLC), diacylglycerol (DAG), mitogen-activated protein kinases (MAPKs), and phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt). LPA mediates multiple functions through G protein-coupled LPAR1, including cell survival, cell proliferation, cell adhesion, cell migration, cytoskeletal changes, Ca2+ mobilization, immune function, and myelination. For example, it promotes astrocyte proliferation and neuronal differentiation, oligodendrocyte and smooth muscle cell proliferation, Schwann cell migration and anti-apoptosis, and valvular interstitial cell (VIC) mineralization and osteogenic transformation. Studies have confirmed that abnormal LPA-LPAR1 signaling leads to a range of diseases, including neuropathic pain, neurodevelopmental, neuropsychiatric, and neurodegenerative disorders, cardiovascular disease, skeletal disease, fibrosis, cancer, infertility, and obesity. Pulmonary fibrosis is a common pathological process, with over 100 clinical diseases causing this pathological phenomenon. For example, idiopathic pulmonary fibrosis (IPF) is the most common and severe interstitial lung disease, which can be triggered by allergens, toxic chemicals, radiation, other persistent irritants, and unknown factors. Numerous studies have demonstrated that abnormally elevated LPA expression levels in the bronchoalveoli are associated with IPF. Studies have shown that bone marrow-derived cells are an important source of myofibroblasts in fibrotic organs. In a bleomycin-induced lung injury model, migrating bone marrow-derived mesenchymal stem cells (BMSCs) significantly contribute to the formation of smooth muscle actin (α-SMA)-positive myofibroblasts. LPAR1 antagonists can alleviate lung fibrosis by inhibiting BMSC differentiation into myofibroblasts and secretion of extracellular matrix (ECM).

[0005] Hepatic fibrosis (HF) is a chronic liver disease characterized by the repeated destruction and subsequent regeneration of hepatocytes, resulting in the diffuse, excessive deposition and abnormal distribution of extracellular matrix (ECM) such as collagen, glycoproteins, and proteoglycans in the liver. Severe fibrosis can lead to tissue disorganization and liver failure. Hepatic stellate cells (HSCs) are the primary source of ECM, which are activated into myofibroblasts upon liver injury. Activated myofibroblasts are primarily characterized by the expression of α-smooth muscle actin (α-SMA), playing a crucial role in the development of fibrosis. Studies have shown that LPA stimulates the proliferation of hepatocytes and HSCs. In chronic hepatitis C-induced liver fibrosis, LPA plasma concentrations are increased. Studies have shown that silymarin, caffeine, and their combination significantly reduce the degree of liver fibrosis by significantly reducing the expression of LPAR1, TGF-β1, CTGF, and α-SMA genes in the liver, thereby improving liver function. Furthermore, studies have shown that LPAR1 blockade inhibits liver fibrosis in rodent NASH models.

[0006] Renal fibrosis is often caused by an abnormal wound healing process. The wound healing response involves multiple cell types, ultimately leading to the expansion and activation of fibroblasts into myofibroblasts. Studies have shown that in a UUO (unilateral ureteral obstruction)-induced renal fibrosis model, LPAR1-specific LPA signaling induces the expression of connective tissue growth factor (CTGF) through the myocardium-related transcription factor (MRTF)-serum response factor (SRF) pathway, thereby mediating communication between pro-fibrotic epithelial cells and fibroblasts, driving fibroblast proliferation and myofibroblast differentiation, and causing renal fibrosis. LPAR1 gene knockout protects mice from UUO renal fibrosis. Disruption of the LPA-LPAR1 pathway significantly reduces the aggregation of fibroblasts and myofibroblasts and improves renal fibrosis.

[0007] Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiomyopathy. HCM patients are at risk for arrhythmias, heart failure, and stroke, and myocardial fibrosis is a major pathological feature of HCM. Studies have shown that in HCM mouse models, the Lpar1 gene is primarily expressed in lymphatic endothelial cells (LECs) and cardiac fibroblasts, and knocking out Lpar1 reduces hypertrophy and fibrosis.

[0008] Currently, there are no small nucleic acid drugs targeting this target on the market, and there is still a need to develop such drugs with better efficacy, long-term effectiveness, specific targeting and / or safety. Summary of the Invention

[0009] The present invention is dedicated to targeting the LPAR1 gene through RNAi agents, specifically and efficiently inhibiting target genes and proteins in target organs, and can block a variety of diseases caused by abnormal LPA-LPAR1 signaling pathways, such as fibrotic diseases (such as pulmonary fibrosis, liver fibrosis, and renal fibrosis), hypertrophic cardiomyopathy, diffuse scleroderma, adult sclerosis, cancer (such as hepatocellular carcinoma, ovarian cancer, and glioblastoma), diabetic nephropathy, atherosclerosis, rheumatoid arthritis, neuropathic pain, non-alcoholic steatohepatitis, benign prostatic hyperplasia, and demyelination.

[0010] One aspect of the present invention provides an RNAi agent for inhibiting the expression of the lysophosphatidic acid receptor 1 (LPAR1) gene in a cell, comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 or at least 21 consecutive nucleotides of equal length that are at least 80%, 85%, 90%, 95% or 100% complementary to a target region of an mRNA encoding LPAR1, or differs from it by no more than 3 nucleotides.

[0011] In some embodiments, the target region is 602-631, 793-825, 833-859, 1107-1143, 1167-1194, 1211-1288, 1326-1360, 1489-1523, 1777-1804, 2264-2290 or 2786-2813 of the nucleotide sequence shown in SEQ ID NO:1.

[0012] In some embodiments, the target region is 602-628, 604-630, 605-631, 793-819, 794-820, 795-821, 796-822, 797-823, 799-825, 833-859, 1107-1133, 1108-1134, 1116-1142, 1117-1143, 1167-1193, 1168-1194, 1211-1237, 1225-1251, 1229-125 5. 1238-1264, 1252-1278, 1258-1284, 1260-1286, 1262-1288, 1326-1352, 1328-1354, 1334-1360, 1489-1515, 1493-1519, 1494-1520, 1495-1521, 1497-1523, 1777-1803, 1778-1804, 2264-2290, 2786-2812 or 2787-2813.

[0013] In some embodiments, the target region is 605-625, 607-627, 608-628, 796-816, 797-817, 798-818, 799-819, 800-820, 802-822, 836-856, 1110-1130, 1111-1131, 1119-1139, 1120-1140, 1170-1190, 1171-1191, 1214-1234, 1228-1248, 1232-125 2. 1241-1261, 1255-1275, 1261-1281, 1263-1283, 1265-1285, 1329-1349, 1331-1351, 1337-1357, 1492-1512, 1496-1516, 1497-1517, 1498-1518, 1500-1520, 1780-1800, 1781-1801, 2267-2287, 2789-2809 or 2790-2810.

[0014] In some embodiments, the antisense strand comprises at least 15 consecutive nucleotides selected from SEQ ID NOs: 40 to 76 and nucleotide sequences having 1 to 3 nucleotide differences therefrom.

[0015] In some embodiments, the length of the double-stranded region is 17 to 23 base pairs, preferably 18 to 21 base pairs, and more preferably 19 base pairs.

[0016] In some embodiments, the sense strand and the antisense strand are each 17 to 23 nucleotides in length, preferably 19 to 21 nucleotides in length.

[0017] In some embodiments, the RNAi agent comprises one or two blunt ends, preferably one blunt end.

[0018] In some embodiments, the RNAi agent comprises one or two overhangs, preferably one overhang, each overhang having 1 to 4 unpaired nucleotides, preferably 2 unpaired nucleotides.

[0019] In some embodiments, the overhang is located at the 3' end of the sense strand, the 3' end of the antisense strand, or at both the 3' end of the sense strand and the 3' end of the antisense strand; preferably, the overhang is located at the 3' end of the antisense strand, and further preferably, the RNAi agent has a blunt end.

[0020] In some embodiments, the sense strand comprises at least 15 consecutive nucleotides selected from any one nucleotide sequence of SEQ ID NOs: 3 to 39 and nucleotide sequences having 1 to 3 nucleotide differences therefrom.

[0021] In some embodiments, the antisense strand has no more than 23 nucleotides and comprises a nucleotide sequence selected from SEQ ID NOs: 40 to 76; the sense strand has no more than 21 nucleotides and comprises a nucleotide sequence selected from SEQ ID NOs: 3 to 39.

[0022] In some embodiments, in the RNAi agent:

[0023] The sense strand comprises or is the sequence set forth in SEQ ID NO: 3, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO: 40, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0024] The sense strand comprises or is the sequence set forth in SEQ ID NO: 4, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO: 41, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0025] The sense strand comprises or is the sequence set forth in SEQ ID NO:5, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:42, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0026] The sense strand comprises or is the sequence set forth in SEQ ID NO:6, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:43, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0027] The sense strand comprises or is the sequence set forth in SEQ ID NO:7, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:44, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0028] The sense strand comprises or is the sequence set forth in SEQ ID NO:8, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:45, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0029] The sense strand comprises or is the sequence set forth in SEQ ID NO:9, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:46, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0030] The sense strand comprises or is the sequence set forth in SEQ ID NO: 10, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO: 47, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0031] The sense strand comprises or is the sequence set forth in SEQ ID NO: 11, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO: 48, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0032] The sense strand comprises or is the sequence set forth in SEQ ID NO: 12, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO: 49, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0033] The sense strand comprises or is the sequence set forth in SEQ ID NO: 13, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO: 50, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0034] The sense strand comprises or is the sequence set forth in SEQ ID NO: 14, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO: 51, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0035] The sense strand comprises or is the sequence set forth in SEQ ID NO: 15, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO: 52, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0036] The sense strand comprises or is the sequence set forth in SEQ ID NO: 16, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO: 53, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0037] The sense strand comprises or is the sequence set forth in SEQ ID NO: 17, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO: 54, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0038] The sense strand comprises or is the sequence set forth in SEQ ID NO: 18, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO: 55, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0039] The sense strand comprises or is the sequence set forth in SEQ ID NO: 19, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO: 56, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0040] The sense strand comprises or is the sequence set forth in SEQ ID NO:20, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:57, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0041] The sense strand comprises or is the sequence set forth in SEQ ID NO:21, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:58, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0042] The sense strand comprises or is the sequence set forth in SEQ ID NO:22, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:59, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0043] The sense strand comprises or is the sequence set forth in SEQ ID NO:23, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:60, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0044] The sense strand comprises or is the sequence set forth in SEQ ID NO:24, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:61, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0045] The sense strand comprises or is the sequence set forth in SEQ ID NO:25, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:62, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0046] The sense strand comprises or is the sequence set forth in SEQ ID NO:26, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:63, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0047] The sense strand comprises or is the sequence set forth in SEQ ID NO:27, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:64, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0048] The sense strand comprises or is the sequence set forth in SEQ ID NO:28, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:65, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0049] The sense strand comprises or is the sequence set forth in SEQ ID NO:29, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:66, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0050] The sense strand comprises or is the sequence set forth in SEQ ID NO:30, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:67, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0051] The sense strand comprises or is the sequence set forth in SEQ ID NO:31, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:68, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0052] The sense strand comprises or is the sequence set forth in SEQ ID NO:32, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:69, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0053] The sense strand comprises or is the sequence set forth in SEQ ID NO:33, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:70, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0054] The sense strand comprises or is the sequence set forth in SEQ ID NO:34, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:71, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0055] The sense strand comprises or is the sequence set forth in SEQ ID NO:35, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:72, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0056] The sense strand comprises or is the sequence set forth in SEQ ID NO:36, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:73, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0057] The sense strand comprises or is the sequence set forth in SEQ ID NO:37, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:74, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0058] The sense strand comprises or is SEQ ID NO:38, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is SEQ ID NO:75, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom; or

[0059] The sense strand comprises or is the sequence shown in SEQ ID NO: 39, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO: 76, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom.

[0060] In some embodiments, the RNAi agent comprises duplex 8033, 8034, 8035, 8064, 8065, 8066, 8067, 8068, 8069, 8070, 8088, 8089, 8090, 8091, 8105, 8106, 8108, 8110, 8111, 8112, 8113, 8114, 8115, 8116, 8120, 8121, 8122, 8139, 8140, 8141, 8142, 8143, 8163, 8164, 8290, 8425, or 8426.

[0061] In some embodiments, the sense strand and / or antisense strand of the RNAi agent comprises at least one modified nucleotide independently selected from 2'-deoxy-thymine (dT) nucleotides, 2'-O-methyl modified nucleotides (2'-OMe), 2'-fluorine modified nucleotides (2'-F), 2'-deoxy modified nucleotides, locked nucleic acids (LNA), open circle nucleic acids (UNA), bridge nucleic acids (BNA), glycol nucleic acids (GNA), athreose nucleic acids (TNA), conformationally restricted nucleotides, restricted ethyl nucleotides (cEt), 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-O-methoxyethyl modified nucleotides (2'-MOE), abasic nucleotides, inverted nucleotides, nucleotides, nucleotides containing 5'-phosphate groups, nucleotides covalently linked to cationic lipids, nucleotides containing 5'-phosphate mimetics, nucleotides containing 5'-vinylphosphonate (5'-VP) and combinations thereof; preferably selected from 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, nucleotides containing thiophosphate bonds between nucleotides and combinations thereof; and / or preferably, each nucleotide of the sense chain and / or antisense chain of the RNAi agent is modified.

[0062] In some embodiments, in the RNAi agent, in the 5' to 3' direction, the nucleotides at positions 2, 5, 7 and 14 of the antisense chain are 2'-fluoro-modified nucleotides, and one of the nucleotides at positions 12 and 16 of the antisense chain is a 2'-fluoro-modified nucleotide, and the nucleotides at the remaining positions of the antisense chain are all 2'-methoxy-modified nucleotides.

[0063] In some embodiments, in the RNAi agent, the antisense strand has at least one phosphorothioate internucleotide linkage; preferably, the phosphorothioate internucleotide linkage is present in one or more of the following: (i) between the first and second nucleotides at the 5' end of the antisense strand; (ii) between the second and third nucleotides at the 5' end of the antisense strand; (iii) between the first and second nucleotides at the 3' end of the antisense strand; and (iv) between the second and third nucleotides at the 3' end of the antisense strand.

[0064] In some embodiments, in the RNAi agent, from the 5' end to the 3' end, the nucleotides at positions 7 and 9 of the sense chain are 2'-fluoro-modified nucleotides, one or two of the nucleotides at positions 5, 8 and 11 of the sense chain are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions of the sense chain are all 2'-methoxy-modified nucleotides.

[0065] In some embodiments, the sense strand has at least one phosphorothioate internucleotide linkage; preferably, the phosphorothioate internucleotide linkage is present at one or more of the following positions: (i) between the first and second nucleotides at the 5' end of the sense strand; (ii) between the second and third nucleotides at the 5' end of the sense strand; (iii) between the first and second nucleotides at the 3' end of the sense strand; and (iv) between the second and third nucleotides at the 3' end of the sense strand.

[0066] In some embodiments, the modification is selected from one of STC, ESC, Advanced ESC, ESC+, AD1-3, AD5, and GalXC.

[0067] In some embodiments, the modification is that the 5'-terminal nucleotide of the antisense strand contains a phosphate or phosphate analog modification, preferably 5'-VP.

[0068] In some embodiments, the RNAi agent further comprises a ligand targeting hepatocytes, preferably, the ligand comprises a galactose moiety, a galactosamine moiety or an N-acetylgalactosamine moiety, further preferably, the ligand is a trivalent or tetravalent N-acetylgalactosamine moiety, and further preferably, the ligand targeting hepatocytes is L96, NAG25 or NAG37; or the RNAi agent further comprises a ligand targeting non-hepatocytes, preferably, the ligand is a lipophilic group, an integrin ligand or a transferrin receptor 1 ligand, and the lipophilic group is preferably selected from: lipids, vitamins, steroids, C5-C 30 Saturated or unsaturated fatty acids, C5-C 30alkyl, and a polypeptide comprising at least one positively charged amino acid residue; the lipophilic group is more preferably selected from cholesterol, C 16 Saturated or unsaturated fatty acids, C 16 Alkyl, C 22 Saturated or unsaturated fatty acids or C 22 alkyl.

[0069] Another aspect of the present invention provides a pharmaceutical composition comprising any RNAi agent of the present invention and a pharmaceutically acceptable carrier; preferably, the pharmaceutical composition is formulated as an intravenous or subcutaneous injection.

[0070] Another aspect of the present invention provides the use of any one of the RNAi agents of the present invention in the preparation of the following medicaments:

[0071] (i) a drug for reducing the expression level of LPAR1 in cells;

[0072] (ii) a drug for preventing or treating a disease mediated by abnormal LPAR1 expression levels; or

[0073] (iii) an agent for preventing or treating a disease selected from the group consisting of fibrotic diseases (e.g., pulmonary fibrosis, liver fibrosis, renal fibrosis), hypertrophic cardiomyopathy, diffuse scleroderma, adult sclerosis, cancer (e.g., hepatocellular carcinoma, ovarian cancer, glioblastoma), diabetic nephropathy, atherosclerosis, rheumatoid arthritis, neuropathic pain, non-alcoholic steatohepatitis, prostatic hyperplasia, and demyelination.

[0074] Another aspect of the present invention provides the use of an agent that reduces the expression of the gene encoding lysophosphatidic acid receptor 1 (LPAR1) in the preparation of a medicament for treating a disease or condition treatable by LPAR1 knockdown. Preferably, the agent is an RNAi agent. Preferably, the disease or condition is selected from fibrotic diseases (e.g., pulmonary fibrosis, liver fibrosis, renal fibrosis), hypertrophic cardiomyopathy, diffuse scleroderma, adult sclerosis, cancer (e.g., hepatocellular carcinoma, ovarian cancer, glioblastoma), diabetic nephropathy, atherosclerosis, rheumatoid arthritis, neuropathic pain, non-alcoholic steatohepatitis, benign prostatic hyperplasia, and demyelination.

[0075] Accordingly, the present invention also provides a method for reducing the expression level of LPAR1 in a cell, the method comprising administering to a subject in need thereof a therapeutically effective amount of any one of the RNAi agents of the present invention. The present invention also provides a method for preventing or treating a disease mediated by abnormal LPAR1 expression levels (e.g., caused by increased levels), the method comprising administering to a subject in need thereof a therapeutically effective amount of any one of the RNAi agents of the present invention. The present invention also provides a method for preventing or treating a disease selected from fibrotic diseases (e.g., pulmonary fibrosis, liver fibrosis, renal fibrosis), hypertrophic cardiomyopathy, diffuse scleroderma, adult sclerosis, cancer (e.g., hepatocellular carcinoma, ovarian cancer, glioblastoma), diabetic nephropathy, atherosclerosis, rheumatoid arthritis, neuropathic pain, non-alcoholic steatohepatitis, benign prostatic hyperplasia, and demyelination, the method comprising administering to a subject in need thereof a therapeutically effective amount of any one of the RNAi agents of the present invention. The present invention also provides the use of any one of the RNAi agents of the present invention in the preparation of a medicament for preventing or treating the above-mentioned diseases. The present invention also provides any one of the RNAi agents of the present invention for preventing or treating the above-mentioned diseases.

[0076] Other aspects of the present invention will become apparent from the detailed description of the specification which follows. DETAILED DESCRIPTION

[0077] definition

[0078] Lysophosphatidic acid receptor 1 (LPAR1), referred to herein as the lysophosphatidic acid receptor 1 protein or its encoding gene, is also known as EDG2, LPA1, VZG1, edg-2, vzg-1, Gpcr26, Mrec1.3, or rec.1.3, with Gene ID: 1902. The NCBI accession number for the human full-length LPAR1 mRNA transcript is NM_001351411.2. The NCBI accession number for the cynomolgus macaque LPAR1 mRNA transcript is XM_005581086.3.

[0079] As used herein, "oligonucleotide" refers to a nucleotide sequence formed by internucleotide linkages to form a nucleotide chain, wherein each nucleoside and internucleotide linkage may be modified or unmodified. Unless otherwise indicated, an oligonucleotide consists of 12-30 linked nucleotides.

[0080] "Internucleotide linkage" means a covalent linkage between adjacent nucleotides in an oligonucleotide. As used herein, "modified internucleotide linkage" means any internucleotide linkage other than a phosphodiester internucleotide linkage. A "phosphorothioate internucleotide linkage" is a modified internucleotide linkage in which one of the non-bridging oxygen atoms of the phosphodiester internucleotide linkage is replaced by a sulfur atom.

[0081] As used herein, the term "RNAi agent" refers to an agent comprising an RNA molecule that is capable of downregulating the expression of a target gene (herein, the LPAR1 gene) through an RNA interference mechanism when introduced into a cell. The term "RNAi agent of the present invention," "RNAi agent described herein," or similar expressions includes both modified RNAi agents of the present invention, regardless of sequence and target gene, and RNAi agents of the present invention having a specific sequence for interfering with the LPAR1 gene. RNA interference refers to a process in which a nucleic acid molecule induces the cutting and degradation of a target RNA molecule (such as an mRNA molecule) in a sequence-specific manner, such as through an RNA-induced silencing complex (RISC) pathway. RNAi agents herein include siRNA, shRNA, and DNA / RNA hybrid molecules, sometimes also collectively referred to herein as double-stranded RNA (dsRNA), which comprise two antiparallel continuous nucleotide chains that are sufficiently complementary to each other to hybridize to form a double-stranded region. "Hybridization" refers to the pairing of complementary polynucleotides, typically through hydrogen bonds (e.g., Watson-Crick hydrogen bonds, Wobble hydrogen bonds, Hoogsteen hydrogen bonds, or reversed Hoogsteen hydrogen bonds) between complementary bases in the two polynucleotides. "Double-stranded region" refers to a region in two complementary or substantially complementary polynucleotides that hybridize to form base pairs, thereby forming a double strand between the two polynucleotide chains.

[0082] The term "antisense strand" refers to the strand of a dsRNA that contains a region that is substantially complementary to the target sequence. The term "sense strand" or "sense strand" refers to the strand of a dsRNA that contains a region that is substantially complementary to the antisense strand region as defined herein. The term "substantially complementary region" refers to a region that is fully complementary or incompletely complementary. When the complementary region is not fully complementary to the target sequence, mismatches may be located in the interior or terminal regions of the molecule. Typically, the most tolerable mismatches are located in the terminal regions, for example, 5, 4, 3, or 2 at the 5' and / or 3' ends of the dsRNA.

[0083] "siRNA" refers to a nucleic acid that forms double-stranded RNA that has the ability to reduce or inhibit the expression of a target gene when the siRNA and the target gene are present in the same cell. siRNAs are typically about 15 to about 30 base pairs in length, most typically about 19 to 25 base pairs in length, e.g., 19, 20, 21, 22, 23, 24, or 25 nucleotide pairs in length.

[0084] shRNA refers to a short hairpin RNA that includes two short inverted repeats and an intermediate stem-loop structure connecting the two. The stem-loop may contain at least one unpaired nucleotide, for example, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides. The stem-loop may be 10 or fewer nucleotides. The stem-loop may be 8 or fewer unpaired nucleotides. The stem-loop may be 4 to 10 unpaired nucleotides. The stem-loop may be 4 to 8 nucleotides.

[0085] The two substantially complementary chains of a dsRNA need not but may also be covalently linked. The maximum number of base pairs is the number of nucleotides in the shortest chain of the dsRNA minus any overhangs present in the duplex. In addition to the duplex structure, the dsRNA may also comprise one or more nucleotide overhangs. Overhanging nucleotides refer to one or more unpaired nucleotides that extend beyond the double-stranded region at the end of the chain. When the 3' end of a chain extends beyond the 5' end of the other chain, or when the 5' end of a line extends beyond the 3' end of the other line, nucleotide overhangs are usually produced. For example, at least one chain comprises a 3' overhang of at least 1 nucleotide, for example, 1 to 4 nucleotides overhang. For another example, at least one chain comprises a 5' overhang of at least 1 nucleotide, for example, 1 to 4 nucleotides overhang. In other embodiments, both the 3' end and the 5' end of a chain of the dsRNA comprise an overhang of at least 1 nucleotide.

[0086] As used herein, the term "blunt end" or "blunt end" with respect to dsRNA refers to the absence of unpaired nucleotides or nucleotide analogs at a given end of the dsRNA, i.e., the absence of nucleotide overhangs. One or both ends of a dsRNA may be blunt. If both ends of a dsRNA are blunt, the dsRNA is said to be blunt-ended. It should be noted that a "blunt-ended" dsRNA is a dsRNA with both ends blunt, i.e., there are no nucleotide overhangs at either end of the molecule. In most cases, such molecules are double-stranded throughout their entire length. As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that protrudes from the duplex structure of a dsRNA. For example, a nucleotide overhang is present when the 3' end of one strand of a dsRNA extends beyond the 5' end of the other strand, or vice versa. Nucleotide overhangs may comprise or consist of nucleotide / nucleoside analogs, including deoxynucleotides / nucleosides. The overhangs may be on the sense strand, the antisense strand, or any combination thereof. Furthermore, overhanging nucleotides may be present at the 5' end, the 3' end, or both ends of the antisense or sense strand of the dsRNA.

[0087] The dsRNA molecule may include chemical modifications to ribonucleotides, including modifications to the ribose, bases, or backbone components of the ribonucleic acid, as described herein or known in the art. Any such modifications, as used in double-stranded ribonucleic acid molecules (e.g., siRNA, shRNA, etc.), are encompassed by the term "dsRNA" for the purposes of this disclosure. "Modified" nucleotides refer to nucleotides that independently have modified sugar moieties, modified internucleotide linkages, and / or modified nucleobases. Thus, the term "modified nucleotides" includes substitutions, additions, or removals of, for example, functional groups or atoms of internucleoside linkages, sugar moieties, or nucleobases.

[0088] The term "ligand" refers to a cell or tissue targeting agent that binds to a specified cell type (e.g., hepatocytes), such as a lectin, glycoprotein, lipid, or protein (e.g., an antibody). Exemplary targeting agents include thyrotropin, melanocyte-stimulating hormone, lectin, glycoprotein, surfactant protein A, mucin carbohydrates, multivalent lactose, multivalent galactose, N-acetylgalactosamine (GalNAc), multivalent (e.g., divalent or trivalent) GalNAc, N-acetylglucosamine, multivalent mannose, multivalent trehalose, glycosylated polyamino acids, multivalent galactose, transferrin, bisphosphonates, polyglutamate, polyaspartate, cholesterol, steroids, bile acid, folate, vitamin B12, biotin, RGD peptide, and RGD peptide mimetics. In a preferred embodiment, the ligand is a carbohydrate, such as a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, or polysaccharide. For example, the ligand can be a derivative comprising GalNAc. In a preferred embodiment, the ligand comprises one or more N-acetylgalactosamine derivatives attached via a bivalent or trivalent branched linker.

[0089] The term "therapeutically effective amount" refers to an amount of a RNAi agent of the invention or composition thereof effective to produce some desired therapeutic effect in at least a subpopulation of cells in an animal, at a reasonable benefit / risk ratio applicable to any medical treatment.

[0090] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0091] The term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle that is involved in carrying or delivering a RNAi agent from one organ or part of the body to another organ or part of the body, such as a liquid or solid filler, diluent, excipient, manufacturing aid, or solvent encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not injurious to the patient.

[0092] The term "treatment" encompasses prevention, therapy, and cure. The patient receiving such treatment is generally any animal in need thereof, including primates (particularly humans) and other mammals such as horses, cattle, pigs, sheep, poultry, and pets.

[0093] RNAi agents for inhibiting LPAR1 gene expression

[0094] One aspect of the present invention provides an RNAi agent for inhibiting LPAR1 gene expression, comprising a sense strand and an antisense strand forming a complementary double-stranded region, wherein the antisense strand comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 or at least 21 consecutive nucleotides of equal length that are at least 80%, 85%, 90%, 95% or 100% complementary to a target region of an mRNA encoding LPAR1, or differs from it by no more than 3 nucleotides.

[0095] In some embodiments, the target region is 602-631, 793-825, 833-859, 1107-1143, 1167-1194, 1211-1288, 1326-1360, 1489-1523, 1777-1804, 2264-2290 or 2786-2813 of the nucleotide sequence shown in SEQ ID NO: 1. In a preferred embodiment, the target region is SEQ ID NO: 602-628, 604-630, 605-631, 793-819, 794-820, 795-821, 796-822, 797-823, 799-825, 833-859, 1107-1133, 1108-1134, 1116-1142, 1117-1143, 1167-1193, 1168-1194, 1211-1237, 1225-1251, 1229-125 5, 1238-1264, 1252-1278, 1258-1284, 1260-1286, 1262-1288, 1326-1352, 1328-1354, 1334-1360, 1489-1515, 1493-1519, 1494-1520, 1495-1521, 1497-1523, 1777-1803, 1778-1804, 2264-2290, 2786-2812 or 2787-2813. In a preferred embodiment, wherein the target region is SEQ ID 605-625, 607-627, 608-628, 796-816, 797-817, 798-818, 799-819, 800-820, 802-822, 836-856, 1110-1130, 1111-1131, 1119-1139, 1120-1140, 1170-1190, 1171-1191, 1214-1234, 1228-1248, 1232-125 2. 1241-1261, 1255-1275, 1261-1281, 1263-1283, 1265-1285, 1329-1349, 1331-1351, 1337-1357, 1492-1512, 1496-1516, 1497-1517, 1498-1518, 1500-1520, 1780-1800, 1781-1801, 2267-2287, 2789-2809 or 2790-2810.

[0096] Therefore, in some embodiments, the present invention provides an RNAi agent for inhibiting LPAR1 gene expression, comprising a sense strand and an antisense strand forming complementary double-stranded regions, wherein the antisense strand comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 consecutive nucleotides that are at least 80%, 85%, 90%, 95%, or 100% complementary to, or differ from, the nucleotide sequence of SEQ ID NO: 1 by no more than 3 nucleotides.

[0097] In a preferred embodiment, the present invention provides an RNAi agent for inhibiting LPAR1 gene expression, comprising a sense strand and an antisense strand forming a complementary double-stranded region, wherein the antisense strand comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 or at least 21 sequences identical to those of SEQ ID NO: 602-628, 604-630, 605-631, 793-819, 794-820, 795-821, 796-822, 797-823, 799-825, 833-859, 1107-1133, 1108-1134, 1116-1142, 1117-1143, 1167-1193, 1168-1194, 1211-1237, 1225-1251, 1229-1255, 1238-1264, 1252-1278, 1258 or 2787-2813, or an equal length of consecutive nucleotides that is at least 80%, 85%, 90%, 95% or 100% complementary thereto, or nucleotides that differ by no more than 3 nucleotides.

[0098] In a preferred embodiment, the present invention provides an RNAi agent for inhibiting LPAR1 gene expression, comprising a sense strand and an antisense strand forming a complementary double-stranded region, wherein the antisense strand comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 or at least 21 sequences identical to those of SEQ ID NO: 605-625, 607-627, 608-628, 796-816, 797-817, 798-818, 799-819, 800-820, 802-822, 836-856, 1110-1130, 1111-1131, 1119-1139, 1120-1140, 1170-1190, 1171-1191, 1214-1234, 1228-1248, 1232-1252, 1241-1261, 1255-1275, 1261 or 2790-2810, or an equal length of consecutive nucleotides that is at least 80%, 85%, 90%, 95% or 100% complementary thereto, or nucleotides that differ by no more than 3 nucleotides.

[0099] In some embodiments, the antisense strand comprises at least 15 (e.g., 15, 16, 17, 18, 19, 20, or 21) consecutive nucleotides selected from SEQ ID NOs: 40 to 76, and nucleotide sequences that differ from each of them by 1, 2, or 3 nucleotides.

[0100] In some embodiments, the present invention provides an RNAi agent for inhibiting LPAR1 gene expression, comprising a sense strand and an antisense strand forming complementary double-stranded regions, wherein the antisense strand comprises at least 15 (e.g., 15, 16, 17, 18, 19, 20, or 21) consecutive nucleotides of any one nucleotide sequence selected from SEQ ID NOs: 40 to 76.

[0101] In some embodiments, the present invention provides an RNAi agent for inhibiting LPAR1 gene expression, comprising a sense strand and an antisense strand forming complementary double-stranded regions, wherein the antisense strand comprises at least 15 (e.g., 15, 16, 17, 18, 19, 20, or 21) consecutive nucleotides selected from a nucleotide sequence having 1, 2, or 3 nucleotide differences from any one of SEQ ID NOs: 40 to 76.

[0102] In the present invention, when referring to 1, 2, or 3 nucleotide differences, the 1, 2, or 3 nucleotide differences may be located on the sense strand and / or antisense strand outside the double-stranded region. In some embodiments, the 1, 2, or 3 nucleotide differences are located on the sense strand and / or antisense strand within the double-stranded region. In some embodiments, a portion of the 1, 2, or 3 nucleotide differences are located on the sense strand and / or antisense strand within the double-stranded region, and another portion is located on the sense strand and / or antisense strand outside the double-stranded region. For the antisense strand, in some embodiments, the 1, 2, or 3 nucleotide differences are located at the 3'-most end or the 5'-most end of the antisense strand. In some embodiments, the 1, 2, or 3 nucleotide differences are located between the 3'-most end and the 5'-most end of the antisense strand. In some embodiments, 1 or 2 of the 1, 2, or 3 nucleotide differences are located at the 3'-most end or the 5'-most end of the antisense strand, and the other 1 or 2 are located between the 3'-most end and the 5'-most end of the antisense strand. For the sense strand, in some embodiments, the 1, 2, or 3 nucleotide differences are located at the 3'-most end or the 5'-most end of the sense strand. In some embodiments, the 1, 2, or 3 nucleotide differences are located between the 3'-most end and the 5'-most end of the sense strand. In some embodiments, one or two of the 1, 2, or 3 nucleotide differences are located at the 3'-most end or the 5'-most end of the sense strand, and the other one or two are located between the 3'-most end and the 5'-most end of the sense strand.

[0103] In some embodiments, the present invention provides an RNAi agent for inhibiting LPAR1 gene expression, comprising a sense strand and an antisense strand forming a complementary double-stranded region, wherein the antisense strand comprises at least 15 (e.g., 15, 16, 17, 18, 19, 20, or 21) consecutive nucleotides selected from a nucleotide sequence having one nucleotide difference with any one of SEQ ID NOs: 40 to 76, and the one nucleotide difference is located at the extreme 3' end of the antisense strand. In some embodiments, the present invention provides an RNAi agent for inhibiting LPAR1 gene expression, comprising a sense strand and an antisense strand forming a complementary double-stranded region, wherein the antisense strand comprises at least 15 (e.g., 15, 16, 17, 18, 19, 20, or 21) consecutive nucleotides selected from a nucleotide sequence having two nucleotide differences with any one of SEQ ID NOs: 40 to 76, and the two nucleotide differences are consecutively located at the extreme 3' end of the antisense strand.

[0104] In some embodiments, the present invention provides an RNAi agent for inhibiting LPAR1 gene expression, comprising a sense strand and an antisense strand forming complementary double-stranded regions, wherein the antisense strand is no longer than 23 nucleotides and comprises at least 15 (e.g., 15, 16, 17, 18, 19, 20, or 21) consecutive nucleotides of any one nucleotide sequence selected from SEQ ID NOs: 40 to 76.

[0105] In some embodiments, the present invention provides an RNAi agent for inhibiting LPAR1 gene expression, comprising a sense strand and an antisense strand forming complementary double-stranded regions, wherein the antisense strand is no more than 23 nucleotides and comprises at least 15 (e.g., 15, 16, 17, 18, 19, 20, or 21) consecutive nucleotides selected from a nucleotide sequence having 1, 2, or 3 nucleotide differences from any one of SEQ ID NOs: 40 to 76.

[0106] One aspect of the present invention provides an RNAi agent for inhibiting LPAR1 gene expression, comprising an antisense strand, wherein the antisense strand comprises at least 15 (e.g., 15, 16, 17, 18, 19, 20 or 21) consecutive nucleotides selected from SEQ ID NOs: 40 to 76 and nucleotide sequences having 1, 2 or 3 nucleotide differences therefrom.

[0107] In some embodiments, the present invention provides an RNAi agent for inhibiting LPAR1 gene expression, comprising an antisense strand, wherein the antisense strand comprises at least 15 (e.g., 15, 16, 17, 18, 19, 20, or 21) consecutive nucleotides of any one nucleotide sequence selected from SEQ ID NOs: 40 to 76.

[0108] In some embodiments, the present invention provides an RNAi agent for inhibiting LPAR1 gene expression, comprising an antisense strand, wherein the antisense strand comprises at least 15 (e.g., 15, 16, 17, 18, 19, 20, or 21) consecutive nucleotides selected from a nucleotide sequence having 1, 2, or 3 nucleotide differences from any one of SEQ ID NOs: 40 to 76.

[0109] In some embodiments, the length of the double-stranded region is about 17 to about 23 base pairs. For example, a double-stranded region of suitable length is about 17 to about 22 base pairs, about 17 to about 21 base pairs, about 17 to about 20 base pairs, about 17 to about 19 base pairs, about 17 to about 18 base pairs, about 18 to about 23 base pairs, about 18 to about 22 base pairs, about 18 to about 21 base pairs, about 18 to about 20 base pairs, about 19 to 23 base pairs, about 19 to 22 base pairs, about 19 to 21 base pairs, about 20 to 23 base pairs, about 20 to 22 base pairs, or about 21 to 23 base pairs. In certain embodiments, the length of the double-stranded region is about 18 to about 21 base pairs. In other embodiments, the length of the double-stranded region is about 19 base pairs.

[0110] Therefore, in some embodiments of the present invention, an RNAi agent for inhibiting LPAR1 gene expression is provided, which comprises a sense strand and an antisense strand forming a complementary double-stranded region, the double-stranded region being about 17 to about 23 base pairs in length, and the antisense strand being no longer than 23 nucleotides and comprising at least 15 consecutive nucleotides selected from SEQ ID NOs: 40 to 76 and nucleotide sequences having 1 to 3 nucleotide differences therefrom.

[0111] In some embodiments of the present invention, an RNAi agent for inhibiting LPAR1 gene expression is provided, comprising a sense strand and an antisense strand forming a complementary double-stranded region, the double-stranded region being about 18 to about 21 base pairs in length, and the antisense strand being no longer than 23 nucleotides and comprising at least 15 consecutive nucleotides selected from SEQ ID NOs: 40 to 76 and nucleotide sequences having 1 to 3 nucleotide differences therefrom.

[0112] In some embodiments of the present invention, an RNAi agent for inhibiting LPAR1 gene expression is provided, comprising a sense strand and an antisense strand forming a complementary double-stranded region, wherein the double-stranded region is about 19 base pairs in length, and the antisense strand is no more than 23 nucleotides in length and comprises at least 15 (e.g., 15, 16, 17, 18, 19, 20, or 21) consecutive nucleotides selected from SEQ ID NOs: 40 to 76 and nucleotide sequences having 1, 2, or 3 nucleotide differences therefrom.

[0113] In some embodiments, the sense and antisense strands in the RNAi agents of the invention are each independently about 17 to about 23 nucleotides in length, e.g., about 18 to about 23 nucleotides, about 19 to about 23 nucleotides, about 20 to about 23 nucleotides, about 21 to about 23 nucleotides, about 17 to about 22 nucleotides, about 17 to about 21 nucleotides, about 17 to about 20 nucleotides, about 17 to about 19 nucleotides, about 18 to about 22 nucleotides, about 18 to about 21 nucleotides, about 18 to about 20 nucleotides, about 19 to about 22 nucleotides, about 19 to about 21 nucleotides, or about 20 to about 22 nucleotides. In certain embodiments, the sense and antisense strands are each independently about 17, about 18, about 19, about 20, about 21, about 22, or about 23 nucleotides in length.

[0114] In some embodiments, the sense strand and the antisense strand have the same length, but form a double-stranded region that is shorter than the strand, such that the RNAi agent for inhibiting LPAR1 gene expression has two nucleotide overhangs. For example, in one embodiment, the RNAi agent for inhibiting LPAR1 gene expression comprises (i) a sense strand and an antisense strand that are each 21 nucleotides in length, (ii) a double-stranded region that is 19 base pairs in length, and (iii) a nucleotide overhang of one unpaired nucleotide at the 3' end of the sense strand and the 3' end of the antisense strand. In another embodiment, the RNAi agent for inhibiting LPAR1 gene expression comprises (i) a sense strand and an antisense strand that are each 23 nucleotides in length, (ii) a double-stranded region that is 21 base pairs in length, and (iii) a nucleotide overhang of one unpaired nucleotide at the 3' end of the sense strand and the 3' end of the antisense strand.

[0115] In other embodiments, the sense and antisense strands are of the same length and form a double-stranded region over their entire length such that no nucleotides protrude from either end of the double-stranded molecule. In one such embodiment, the RNAi agent for inhibiting LPAR1 gene expression is blunt-ended and comprises (i) a sense and antisense strand each having a length of 21 nucleotides, and (ii) a double-stranded region of 21 base pairs in length. In another such embodiment, the RNAi agent for inhibiting LPAR1 gene expression is blunt-ended and comprises (i) a sense and antisense strand each having a length of 23 nucleotides, and (ii) a double-stranded region of 23 base pairs in length. In another such embodiment, the RNAi agent for inhibiting LPAR1 gene expression is blunt-ended and comprises (i) a sense and antisense strand each having a length of 19 nucleotides, and (ii) a double-stranded region of 19 base pairs in length.

[0116] In other embodiments, the sense strand or the antisense strand is longer than the other strand, and the two strands form a double-stranded region with a length equal to the length of the short strand, so that the RNAi agent for inhibiting LPAR1 gene expression includes at least one nucleotide overhang. For example, in some embodiments, the sense strand is 1 to 4 nucleotides longer than the antisense strand, and the double-stranded region formed by the two strands is equal to the length of the antisense strand, so that the sense strand forms an overhang with 1 to 4 unpaired nucleotides. In other embodiments, the antisense strand is 1 to 4 nucleotides longer than the sense strand, and the double-stranded region formed by the two strands is equal to the length of the sense strand, so that the antisense strand forms an overhang with 1 to 4 unpaired nucleotides. In some embodiments, the length of the nucleotide overhang is 1, 2, 3 or 4 nucleotides. In a specific embodiment, the overhang includes 2 nucleotides. In certain embodiments, the overhang includes a single nucleotide.

[0117] The nucleotides that protrude can be ribonucleotides or modified nucleotides as described herein. In some embodiments, the nucleotides that protrude are 2'-modified nucleotides (e.g., 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides) or combinations thereof. For example, in one embodiment, the nucleotides that protrude are deoxyribonucleotides, such as deoxythymidine. In another embodiment, the nucleotides that protrude are 2'-O-methyl-modified nucleotides, 2'-fluoro-modified nucleotides, 2'-methoxyethyl-modified nucleotides, abasic nucleotides, inverted abasic nucleotides, inverted nucleotides or combinations thereof. In other embodiments, the protrusion comprises a 5'-uridine-uridine-3' (5'-UU-3') dinucleotide. In such embodiments, the UU dinucleotide can include a ribonucleotide or modified nucleotide, such as a 2'-modified nucleotide. In other embodiments, the protrusion comprises a 5'-deoxythymidine-deoxythymidine-3' (5'-dTdT-3') dinucleotide. When there is a nucleotide overhang in the antisense strand, the nucleotides in the overhang may be complementary to the target gene sequence, form a mismatch with the target gene sequence, or contain some other sequence (such as UU, TT, AA, GG, etc.).

[0118] The nucleotide overhang may be at the 5' or 3' end of one or both strands. For example, in one embodiment, a RNAi agent for inhibiting LPAR1 gene expression comprises nucleotide overhangs at both the 5' and 3' ends of the antisense strand. In another embodiment, a RNAi agent for inhibiting LPAR1 gene expression comprises nucleotide overhangs at both the 5' and 3' ends of the sense strand. In some embodiments, a RNAi agent for inhibiting LPAR1 gene expression includes nucleotide overhangs at both the 5' end of the sense strand and the 5' end of the antisense strand. In other embodiments, a RNAi agent for inhibiting LPAR1 gene expression includes nucleotide overhangs at both the 3' end of the sense strand and the 3' end of the antisense strand. In some embodiments, a RNAi agent for inhibiting LPAR1 gene expression includes only a nucleotide overhang at the 5' end of the sense strand. In some embodiments, a RNAi agent for inhibiting LPAR1 gene expression includes only a nucleotide overhang at the 3' end of the sense strand. In some embodiments, a RNAi agent for inhibiting LPAR1 gene expression includes only a nucleotide overhang at the 3' end of the antisense strand. In some embodiments, the RNAi agent for inhibiting LPAR1 gene expression comprises only nucleotide overhangs at the 5' end of the antisense strand. In some embodiments, the RNAi agent for inhibiting LPAR1 gene expression comprises only nucleotide overhangs at the 5' end of the sense strand.

[0119] The RNAi agent used to inhibit LPAR1 gene expression can include a nucleotide overhang at one end of the double-stranded RNA molecule and a blunt end at the other end. "Blunt end" means that the sense strand and the antisense strand are completely base-paired at the ends of the molecule, and no unpaired nucleotides extend beyond the double-stranded region. In some embodiments, the RNAi agent used to inhibit LPAR1 gene expression includes a nucleotide overhang at the 3' end of the sense strand and a blunt end at the 5' end of the sense strand and the 3' end of the antisense strand. In other embodiments, the RNAi agent used to inhibit LPAR1 gene expression includes a nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand and the 3' end of the sense strand.

[0120] Specifically, for example, in one embodiment, the RNAi agent for inhibiting LPAR1 gene expression comprises (i) a sense strand of 19 nucleotides in length, (ii) an antisense strand of 21 nucleotides in length, and the two strands form a double-stranded region whose length is equal to the length of the sense strand. In another embodiment, the RNAi agent for inhibiting LPAR1 gene expression comprises (i) a sense strand of 21 nucleotides in length, (ii) an antisense strand of 23 nucleotides in length, and the two strands form a double-stranded region whose length is equal to the length of the sense strand.

[0121] In some embodiments, an RNAi agent for inhibiting LPAR1 gene expression in a cell is provided, comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand is no longer than 23 nucleotides and comprises at least 15 (e.g., 15, 16, 17, 18, 19, 20, or 21) consecutive nucleotides selected from SEQ ID NOs: 40 to 76, and nucleotide sequences having 1, 2, or 3 nucleotide differences therefrom, and the RNAi agent comprises an overhang and a blunt end, and the overhang preferably has 2 unpaired nucleotides.

[0122] In some embodiments, an RNAi agent for inhibiting LPAR1 gene expression in a cell is provided, comprising a sense strand and an antisense strand forming a double-stranded region, the antisense strand being no longer than 23 nucleotides and comprising at least 15 (e.g., 15, 16, 17, 18, 19, 20, or 21) consecutive nucleotides selected from SEQ ID NOs: 40 to 76, and nucleotide sequences differing therefrom by 1, 2, or 3 nucleotides, and the RNAi agent comprising an overhang and a blunt end, the overhang preferably having 2 unpaired nucleotides, wherein the overhang is formed at the 3' end of the antisense strand, and the blunt end is formed at the 3' end of the sense strand and the 5' end of the antisense strand.

[0123] In some embodiments, an RNAi agent for inhibiting LPAR1 gene expression in a cell is provided, comprising a sense strand and an antisense strand forming a double-stranded region, wherein the double-stranded region is 19 base pairs in length, the antisense strand is no longer than 23 nucleotides and comprises at least 15 (e.g., 15, 16, 17, 18, 19, 20, or 21) consecutive nucleotides selected from SEQ ID NOs: 40 to 76, and nucleotide sequences differing therefrom by 1, 2, or 3 nucleotides, and the RNAi agent comprises an overhang and a blunt end, the overhang preferably having 2 unpaired nucleotides, wherein the overhang is formed at the 3' end of the antisense strand, and the blunt end is formed at the 3' end of the sense strand and the 5' end of the antisense strand.

[0124] In some embodiments, an RNAi agent for inhibiting LPAR1 gene expression in a cell is provided, comprising a sense strand and an antisense strand forming a double-stranded region, wherein the double-stranded region is 19 base pairs in length, the antisense strand is no longer than 23 nucleotides and comprises at least 15 (e.g., 15, 16, 17, 18, 19, 20, or 21) consecutive nucleotides selected from SEQ ID NOs: 40 to 76, and nucleotide sequences differing therefrom by 1, 2, or 3 nucleotides, the sense strand is 19 to 21 nucleotides in length, and the RNAi agent includes an overhang and a blunt end, the overhang preferably having 2 unpaired nucleotides, wherein the overhang is formed at the 3' end of the antisense strand, and the blunt ends are formed at the 3' end of the sense strand and the 5' end of the antisense strand.

[0125] In some embodiments, an RNAi agent for inhibiting LPAR1 gene expression in a cell is provided, comprising a sense strand and an antisense strand forming a double-stranded region, wherein the double-stranded region is 19 base pairs in length, the antisense strand is 21 nucleotides in length and is at least 15 (e.g., 15, 16, 17, 18, 19, 20, or 21) consecutive nucleotides selected from SEQ ID NOs: 40 to 76, and nucleotide sequences having 1, 2, or 3 nucleotide differences therefrom, the sense strand is 19 nucleotides in length, and the RNAi agent includes an overhang and a blunt end, the overhang preferably having 2 unpaired nucleotides, wherein the overhang is formed at the 3' end of the antisense strand, and the blunt ends are formed at the 3' end of the sense strand and the 5' end of the antisense strand.

[0126] In some embodiments, the present invention provides an RNAi agent for inhibiting LPAR1 gene expression in a cell, comprising a sense strand and an antisense strand forming a double-stranded region, wherein the double-stranded region is 19 base pairs in length, the antisense strand is 21 nucleotides in length and is any one nucleotide sequence selected from SEQ ID NOs: 40 to 76, the sense strand is 19 nucleotides in length, and the RNAi agent includes an overhang and a blunt end, the overhang preferably having 2 unpaired nucleotides, wherein the overhang is formed at the 3' end of the antisense strand, and the blunt ends are formed at the 3' end of the sense strand and the 5' end of the antisense strand.

[0127] In a preferred embodiment, the present invention provides an RNAi agent for inhibiting LPAR1 gene expression in a cell, comprising a sense strand and an antisense strand forming a double-stranded region, the antisense strand comprising at least 15 (e.g., 15, 16, 17, 18, 19, 20, or 21) consecutive nucleotides selected from SEQ ID NOs: 40 to 76, and nucleotide sequences having 1, 2, or 3 nucleotide differences therefrom, and the sense strand comprising at least 15 (e.g., 15, 16, 17, 18, or 19) consecutive nucleotides selected from any one of SEQ ID NOs: 3 to 39, and nucleotide sequences having 1, 2, or 3 nucleotide differences therefrom.

[0128] In a preferred embodiment, the present invention provides an RNAi agent for inhibiting LPAR1 gene expression in a cell, comprising a sense strand and an antisense strand forming a double-stranded region, the antisense strand having no more than 23 nucleotides and comprising at least 15 (e.g., 15, 16, 17, 18, 19, 20, or 21) consecutive nucleotides selected from SEQ ID NOs: 40 to 76, and nucleotide sequences having 1, 2, or 3 nucleotide differences therefrom, and the sense strand having no more than 21 nucleotides and comprising at least 15 (e.g., 15, 16, 17, 18, or 19) consecutive nucleotides selected from any one of SEQ ID NOs: 3 to 39, and nucleotide sequences having 1, 2, or 3 nucleotide differences therefrom.

[0129] In a preferred embodiment, the present invention provides an RNAi agent for inhibiting LPAR1 gene expression in a cell, comprising a sense strand and an antisense strand forming a double-stranded region, the antisense strand having no more than 23 nucleotides and comprising a nucleotide sequence selected from any one of SEQ ID NOs: 40 to 76, and the sense strand having no more than 21 nucleotides and comprising a nucleotide sequence selected from any one of SEQ ID NOs: 3 to 39.

[0130] In a preferred embodiment, the present invention provides an RNAi agent for inhibiting LPAR1 gene expression in a cell, comprising a sense strand and an antisense strand forming a double-stranded region, wherein:

[0131] The sense strand comprises or is the sequence set forth in SEQ ID NO: 3, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO: 40, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0132] The sense strand comprises or is the sequence set forth in SEQ ID NO: 4, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO: 41, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0133] The sense strand comprises or is the sequence set forth in SEQ ID NO:5, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:42, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0134] The sense strand comprises or is the sequence set forth in SEQ ID NO:6, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:43, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0135] The sense strand comprises or is the sequence set forth in SEQ ID NO:7, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:44, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0136] The sense strand comprises or is the sequence set forth in SEQ ID NO:8, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:45, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0137] The sense strand comprises or is the sequence set forth in SEQ ID NO:9, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:46, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0138] The sense strand comprises or is the sequence set forth in SEQ ID NO: 10, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO: 47, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0139] The sense strand comprises or is the sequence set forth in SEQ ID NO: 11, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO: 48, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0140] The sense strand comprises or is the sequence set forth in SEQ ID NO: 12, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO: 49, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0141] The sense strand comprises or is the sequence set forth in SEQ ID NO: 13, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO: 50, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0142] The sense strand comprises or is the sequence set forth in SEQ ID NO: 14, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO: 51, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0143] The sense strand comprises or is the sequence set forth in SEQ ID NO: 15, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO: 52, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0144] The sense strand comprises or is the sequence set forth in SEQ ID NO: 16, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO: 53, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0145] The sense strand comprises or is the sequence set forth in SEQ ID NO: 17, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO: 54, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0146] The sense strand comprises or is the sequence set forth in SEQ ID NO: 18, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO: 55, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0147] The sense strand comprises or is the sequence set forth in SEQ ID NO: 19, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO: 56, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0148] The sense strand comprises or is the sequence set forth in SEQ ID NO:20, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:57, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0149] The sense strand comprises or is the sequence set forth in SEQ ID NO:21, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:58, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0150] The sense strand comprises or is the sequence set forth in SEQ ID NO:22, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:59, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0151] The sense strand comprises or is the sequence set forth in SEQ ID NO:23, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:60, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0152] The sense strand comprises or is the sequence set forth in SEQ ID NO:24, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:61, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0153] The sense strand comprises or is the sequence set forth in SEQ ID NO:25, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:62, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0154] The sense strand comprises or is the sequence set forth in SEQ ID NO:26, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:63, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0155] The sense strand comprises or is the sequence set forth in SEQ ID NO:27, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:64, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0156] The sense strand comprises or is the sequence set forth in SEQ ID NO:28, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:65, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0157] The sense strand comprises or is the sequence set forth in SEQ ID NO:29, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:66, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0158] The sense strand comprises or is the sequence set forth in SEQ ID NO:30, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:67, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0159] The sense strand comprises or is the sequence set forth in SEQ ID NO:31, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:68, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0160] The sense strand comprises or is the sequence set forth in SEQ ID NO:32, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:69, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0161] The sense strand comprises or is the sequence set forth in SEQ ID NO:33, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:70, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0162] The sense strand comprises or is the sequence set forth in SEQ ID NO:34, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:71, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0163] The sense strand comprises or is the sequence set forth in SEQ ID NO:35, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:72, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0164] The sense strand comprises or is the sequence set forth in SEQ ID NO:36, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:73, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0165] The sense strand comprises or is the sequence set forth in SEQ ID NO:37, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:74, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0166] The sense strand comprises or is SEQ ID NO:38, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is SEQ ID NO:75, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom; or

[0167] The sense strand comprises or is the sequence shown in SEQ ID NO: 39, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO: 76, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom.

[0168] In a preferred embodiment, the present invention provides an RNAi agent for inhibiting LPAR1 gene expression, which comprises a sense strand and an antisense strand forming complementary double-stranded regions, wherein the sense strand and the antisense strand pair to form any one of duplexes 8033, 8034, 8035, 8064, 8065, 8066, 8067, 8068, 8069, 8070, 8088, 8089, 8090, 8091, 8105, 8106, 8108, 8110, 8111, 8112, 8113, 8114, 8115, 8116, 8120, 8121, 8122, 8139, 8140, 8141, 8142, 8143, 8163, 8164, 8290, 8425 or 8426 as described herein.

[0169] Nucleotide-modified RNAi agents

[0170] For any of the embodiments of the RNAi agent of the present invention described in the section "RNAi agent for inhibiting LPAR1 gene expression", the sense strand and / or antisense strand of the RNAi agent may comprise at least one modified nucleotide.

[0171] In some embodiments, the sense strand and the antisense strand of the RNAi agent for inhibiting LPAR1 gene expression provided by the present invention each contain at least one modified nucleotide.

[0172] In some embodiments, each nucleotide in the sense strand and the antisense strand of the RNAi agent for inhibiting LPAR1 gene expression provided by the present invention is modified.

[0173] In any of the above embodiments, the modified nucleotides are independently selected from 2'-deoxy-thymine (dT) nucleotides, 2'-O-methyl modified nucleotides (2'-OMe), 2'-fluorine modified nucleotides (2'-F), 2'-deoxy modified nucleotides, locked nucleic acids (LNA), open ring nucleic acids (UNA), bridge nucleic acids (BNA), glycol nucleic acids (GNA), athreose nucleic acids (TNA), conformationally restricted nucleotides, restricted ethyl nucleotides (cEt), 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-O -methoxyethyl modified nucleotides (2'-MOE), abasic nucleotides, inverted abasic nucleotides, inverted nucleotides, morpholino nucleotides (MOP), phosphoramidates (PN), tetrahydropyran modified nucleotides (THP), 1,5-anhydrohexitol modified (HNA) nucleotides, cyclohexenyl modified nucleotides, nucleotides containing phosphorothioate groups (PS), nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphate, nucleotides containing 5'-phosphate mimetics, nucleotides covalently linked to cationic lipids, nucleotides containing 5'-vinylphosphonate (5'-VP), and combinations thereof.

[0174] In a preferred embodiment, the modified nucleotides are independently selected from 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, nucleotides comprising thiophosphate internucleotide linkages, and combinations thereof. In a preferred embodiment, each nucleotide of the sense strand and / or antisense strand of the RNAi agent is modified. In a preferred embodiment, each nucleotide of the sense strand and / or antisense strand of the RNAi agent is modified, and the modified nucleotides are independently selected from 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, nucleotides comprising thiophosphate internucleotide linkages, and combinations thereof.

[0175] In a preferred embodiment, each nucleotide of the sense strand and the antisense strand of the RNAi agent for inhibiting LPAR1 gene expression provided by the present invention is modified, and the modification method is selected from one of STC (Alnylam), ESC (Alnylam), Advanced ESC (Alnylam), ESC+ (Alnylam), AD1-3 (Arrowhead), AD5 (Arrowhead) and GalXC (Dicerna) (see, for example, Hu B, Zhong L, Weng Y, et al. Therapeutic siRNA: state of the art. Signal Transduct Target Ther. 2020; 5(1): 101).

[0176] In a preferred embodiment, the 5'-terminal nucleotide of the antisense strand of the RNAi agent for inhibiting LPAR1 gene expression provided by the present invention contains a phosphate or phosphate analog modification, preferably 5'-VP (see Parmar R, Willoughby JL, Liu J, et al. 5'-I-Vinylphosphonate: A Stable Phosphate Mimic Can Improve the RNAi Activity of siRNA-GalNAc Conjugates. Chembiochem. 2016; 17(11): 985-989).

[0177] In some embodiments, the modified nucleobase can be a nucleobase covalently attached to a cationic lipid. By introducing a modified nucleobase with a cationic lipid covalently attached thereto or its equivalent, antisense oligonucleotides can be made highly permeable to mammalian cell membranes. Suitable covalent attachment of cationic lipid modifications can be those described in WO2019022434A1, the entire contents of which are incorporated herein by reference.

[0178] In some embodiments, each nucleotide of the antisense strand of the RNAi agent for inhibiting LPAR1 gene expression provided by the present invention is modified, and from the 5' end to the 3' end, the nucleotides at positions 2, 5, 7 and 14 of the antisense strand are 2'-fluoro-modified nucleotides, and one of the nucleotides at positions 12 and 16 of the antisense strand is a 2'-fluoro-modified nucleotide, and the nucleotides at the remaining positions of the antisense strand are all 2'-methoxy-modified nucleotides.

[0179] In some embodiments, each nucleotide of the antisense strand of the RNAi agent for inhibiting LPAR1 gene expression provided by the present invention is modified, and from the 5' end to the 3' end, the nucleotides at positions 2, 5, 7, 12 and 14 of the antisense strand are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions of the antisense strand are all 2'-methoxy-modified nucleotides.

[0180] In some embodiments, each nucleotide of the antisense strand of the RNAi agent for inhibiting LPAR1 gene expression provided by the present invention is modified, and from the 5' end to the 3' end, the nucleotides at positions 2, 5, 7, 14 and 16 of the antisense strand are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions of the antisense strand are all 2'-methoxy-modified nucleotides.

[0181] In some embodiments, each nucleotide of the antisense strand of the RNAi agent for inhibiting LPAR1 gene expression provided by the present invention is modified, and from the 5' end to the 3' end, the nucleotides at positions 2, 5, 7, and 14 of the antisense strand are 2'-fluoro-modified nucleotides, and one of the nucleotides at positions 12 and 16 of the antisense strand is a 2'-fluoro-modified nucleotide, and the nucleotides at the remaining positions of the antisense strand are all 2'-methoxy-modified nucleotides, and the antisense strand has at least one phosphorothioate internucleotide linkage. In a preferred embodiment, the phosphorothioate internucleotide linkage exists at one or more of the following: between the first and second nucleotides at the 5' end of the antisense strand; between the second and third nucleotides at the 5' end of the antisense strand; between the first and second nucleotides at the 3' end of the antisense strand; and between the second and third nucleotides at the 3' end of the antisense strand. In a preferred embodiment, the phosphorothioate internucleotide linkage exists between the first and second nucleotides at the 5' end of the antisense strand; between the second and third nucleotides at the 5' end of the antisense strand; between the first and second nucleotides at the 3' end of the antisense strand; and between the second and third nucleotides at the 3' end of the antisense strand.

[0182] In some embodiments, each nucleotide of the sense strand and antisense strand of the RNAi agent for inhibiting LPAR1 gene expression provided by the present invention is modified, and from the 5' end to the 3' end, the nucleotides at positions 2, 5, 7 and 14 of the antisense strand are 2'-fluoro-modified nucleotides, and one of the nucleotides at positions 12 and 16 of the antisense strand is a 2'-fluoro-modified nucleotide, and the nucleotides at the remaining positions of the antisense strand are all 2'-methoxy-modified nucleotides; from the 5' end to the 3' end, the nucleotides at positions 7 and 9 of the sense strand are 2'-fluoro-modified nucleotides, one or two of the nucleotides at positions 5, 8 and 11 of the sense strand are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions of the sense strand are all 2'-methoxy-modified nucleotides.

[0183] For example, each nucleotide of the sense chain and antisense chain of the RNAi agent for inhibiting LPAR1 gene expression provided by the present invention is modified, and from the 5' end to the 3' end, the nucleotides at positions 2, 5, 7 and 14 of the antisense chain are 2'-fluoro-modified nucleotides, and one of the nucleotides at positions 12 and 16 of the antisense chain is a 2'-fluoro-modified nucleotide, and the nucleotides at the remaining positions of the antisense chain are all 2'-methoxy-modified nucleotides; from the 5' end to the 3' end, the nucleotides at positions 5, 7, 8 and 9 of the sense chain are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions of the sense chain are all 2'-methoxy-modified nucleotides.

[0184] For example, each nucleotide of the sense chain and antisense chain of the RNAi agent for inhibiting LPAR1 gene expression provided by the present invention is modified, and from the 5' end to the 3' end, the nucleotides at positions 2, 5, 7 and 14 of the antisense chain are 2'-fluoro-modified nucleotides, and one of the nucleotides at positions 12 and 16 of the antisense chain is a 2'-fluoro-modified nucleotide, and the nucleotides at the remaining positions of the antisense chain are all 2'-methoxy-modified nucleotides; from the 5' end to the 3' end, the nucleotides at positions 7, 8 and 9 of the sense chain are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions of the sense chain are all 2'-methoxy-modified nucleotides.

[0185] For example, each nucleotide of the sense chain and antisense chain of the RNAi agent for inhibiting LPAR1 gene expression provided by the present invention is modified, and from the 5' end to the 3' end, the nucleotides at positions 2, 5, 7 and 14 of the antisense chain are 2'-fluoro-modified nucleotides, and one of the nucleotides at positions 12 and 16 of the antisense chain is a 2'-fluoro-modified nucleotide, and the nucleotides at the remaining positions of the antisense chain are all 2'-methoxy-modified nucleotides; from the 5' end to the 3' end, the nucleotides at positions 5, 7 and 9 of the sense chain are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions of the sense chain are all 2'-methoxy-modified nucleotides.

[0186] For example, each nucleotide of the sense chain and antisense chain of the RNAi agent for inhibiting LPAR1 gene expression provided by the present invention is modified, and from the 5' end to the 3' end, the nucleotides at positions 2, 5, 7 and 14 of the antisense chain are 2'-fluoro-modified nucleotides, and one of the nucleotides at positions 12 and 16 of the antisense chain is a 2'-fluoro-modified nucleotide, and the nucleotides at the remaining positions of the antisense chain are all 2'-methoxy-modified nucleotides; from the 5' end to the 3' end, the nucleotides at positions 7, 9 and 11 of the sense chain are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions of the sense chain are all 2'-methoxy-modified nucleotides.

[0187] In a preferred embodiment, the sense strand has at least one phosphorothioate internucleotide linkage. In a preferred embodiment, the phosphorothioate internucleotide linkage is present at one or more of the following positions: (i) between the first and second nucleotides at the 5' end of the sense strand; (ii) between the second and third nucleotides at the 5' end of the sense strand; (iii) between the first and second nucleotides at the 3' end of the sense strand; and (iv) between the second and third nucleotides at the 3' end of the sense strand. In a more preferred embodiment, the phosphorothioate internucleotide linkage is present between the first and second nucleotides at the 5' end of the sense strand; and between the second and third nucleotides at the 5' end of the sense strand. In a more preferred embodiment, the phosphorothioate internucleotide linkage exists between the first and second nucleotides at the 5' end of the sense strand; between the second and third nucleotides at the 5' end of the sense strand; between the first and second nucleotides at the 3' end of the sense strand; and between the second and third nucleotides at the 3' end of the sense strand.

[0188] In a preferred embodiment, the antisense strand has at least one phosphorothioate internucleotide linkage. Preferably, the phosphorothioate internucleotide linkage is present in one or more of the following: between the first and second nucleotides at the 5' end of the antisense strand; between the second and third nucleotides at the 5' end of the antisense strand; between the first and second nucleotides at the 3' end of the antisense strand; and between the second and third nucleotides at the 3' end of the antisense strand.

[0189] Therefore, in some embodiments, each nucleotide of the sense strand and the antisense strand of the RNAi agent for inhibiting LPAR1 gene expression provided by the present invention is modified, and in the 5' to 3' direction, the nucleotides at positions 2, 5, 7, and 14 of the antisense strand are 2'-fluoro-modified nucleotides, and one of the nucleotides at positions 12 and 16 of the antisense strand is a 2'-fluoro-modified nucleotide, the nucleotides at the remaining positions of the antisense strand are all 2'-methoxy-modified nucleotides, and the antisense strand has at least one phosphorothioate internucleotide linkage, and the phosphorothioate internucleotide linkage is present in one or more of the following: between the first and second nucleotides at the 5' end of the antisense strand; between the second and third nucleotides at the 5' end of the antisense strand; 3 nucleotides; between the 1st nucleotide and the 2nd nucleotide at the 3' end of the antisense chain; and between the 2nd nucleotide and the 3rd nucleotide at the 3' end of the antisense chain; in the 5' to 3' direction, the 7th and 9th nucleotides of the sense chain are 2'-fluoro-modified nucleotides, one or two of the 5th, 8th and 11th nucleotides of the sense chain are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions of the sense chain are all 2'-methoxy-modified nucleotides, and the sense chain has at least one phosphorothioate bond internucleotide connection, and the phosphorothioate bond internucleotide connection exists between the 1st nucleotide and the 2nd nucleotide at the 5' end of the sense chain and / or between the 2nd nucleotide and the 3rd nucleotide at the 5' end of the sense chain.

[0190] In some embodiments, each nucleotide of the sense strand and antisense strand of the RNAi agent for inhibiting LPAR1 gene expression provided by the present invention is modified, and from the 5' end to the 3' end, the nucleotides at positions 2, 5, 7, and 14 of the antisense strand are 2'-fluoro-modified nucleotides, and one of the nucleotides at positions 12 and 16 of the antisense strand is a 2'-fluoro-modified nucleotide, the nucleotides at the remaining positions of the antisense strand are all 2'-methoxy-modified nucleotides, and the antisense strand has at least one phosphorothioate internucleotide linkage, and the phosphorothioate internucleotide linkage exists between: the first nucleotide and the second nucleotide at the 5' end of the antisense strand, and between the second nucleotide and the third nucleotide at the 5' end of the antisense strand. , between the first nucleotide and the second nucleotide at the 3' end of the antisense chain, and between the second nucleotide and the third nucleotide at the 3' end of the antisense chain; in the direction from 5' to 3', the nucleotides at positions 7 and 9 of the sense chain are 2'-fluoro-modified nucleotides, one or two of the nucleotides at positions 5, 8 and 11 of the sense chain are 2'-fluoro-modified nucleotides, the nucleotides at the remaining positions of the sense chain are all 2'-methoxy-modified nucleotides, and the sense chain has at least one phosphorothioate bond internucleotide connection, and the phosphorothioate bond internucleotide connection exists between the first nucleotide and the second nucleotide at the 5' end of the sense chain and between the second nucleotide and the third nucleotide at the 5' end of the sense chain.

[0191] RNAi agents comprising ligands

[0192] For any of the embodiments of the RNAi agent of the present invention described in the above sections "RNAi agents for inhibiting LPAR1 gene expression" and "Nucleotide-modified RNAi agents", the RNAi agent may comprise a ligand. As used herein, "ligand" refers to any compound or molecule that is capable of interacting directly or indirectly with another compound or molecule. The interaction of a ligand with another compound or molecule may trigger a biological response (e.g., initiating a signal transduction cascade, inducing receptor-mediated endocytosis), or it may simply be a physical connection. The ligand may alter one or more properties of the attached double-stranded RNA molecule, such as the pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, cellular uptake, charge, and / or clearance of the RNA molecule.

[0193] The LPAR1 gene is expressed in a variety of cells and tissues. Therefore, in certain embodiments, it is desirable to specifically deliver the RNAi agent of the present invention to hepatocytes. Therefore, in certain embodiments, the ligand is targeted to specifically deliver the RNAi agent to hepatocytes using various methods described in more detail below. In certain embodiments, the RNAi agent is targeted to hepatocytes using a ligand that binds to the surface-expressed asialoglycoprotein receptor (ASGR) or its components (e.g., ASGR1, ASGR2).

[0194] In some embodiments, RNAi agents can be specifically targeted to the liver by using ligands that bind to or interact with proteins expressed on the surface of hepatocytes. For example, in certain embodiments, the ligand may include an antigen binding protein (e.g., an antibody or its binding fragment (e.g., Fab, scFv)) that specifically binds to receptors expressed on hepatocytes, such as asialoglycoprotein receptors and LDL receptors. In a specific embodiment, the ligand includes an antibody or its binding fragment that specifically binds to ASGR1 and / or ASGR2. In another embodiment, the ligand includes a Fab fragment of an antibody that specifically binds to ASGR1 and / or ASGR2. In another embodiment, the ligand includes a single-chain variable antibody fragment (scFv fragment) of an antibody that specifically binds to ASGR1 and / or ASGR2. Exemplary antibodies and their binding fragments that can be used as ligands for targeting the RNAi agents of the present invention to the liver are described in WO 2017 / 058944, which is incorporated herein by reference in its entirety. Other antibodies or their binding fragments that specifically bind to ASGR1, LDL receptor, or other liver-surface expressed proteins suitable for use as ligands in the RNAi agents of the present invention are purchased from commercial sources.

[0195] In some embodiments, when used for extrahepatic delivery, including but not limited to, the central nervous system (CNS) (e.g., brain, spine, or eye), muscle, lung, or fat, the RNAi agent may comprise one or more lipophilic groups conjugated to one or more positions of the oligonucleotide or one or more positions of the RNAi agent antisense strand and / or sense strand. For example, a lipophilic group may be connected to the antisense strand and / or sense strand of the RNAi agent via a nucleobase, a sugar moiety, or an internucleoside bond. In some embodiments, a lipophilic group having a phosphoramidite group is coupled to the 3' end or 5' end of the sense strand or antisense strand in the final synthesis cycle. In some embodiments, the lipophilic group has an octanol-water partition coefficient greater than 0, 1, 1.5, 2, 3, 4, 5, or 10. In some embodiments, the ligand is a lipophilic group, preferably selected from the group consisting of lipids, vitamins, steroids, C5-C 30 Saturated or unsaturated fatty acids, C5-C 30alkyl, and a polypeptide comprising at least one positively charged amino acid residue; the lipophilic group is more preferably selected from cholesterol, C 16 Saturated or unsaturated fatty acids, C 16 Alkyl, C 22 Saturated or unsaturated fatty acids or C 22 Suitable lipophilic groups may be aliphatic, alicyclic, polyalicyclic, steroid, straight chain or branched aliphatic hydrocarbon.

[0196] Exemplary lipophilic groups are, for example, lipophilic groups Y132 to Y135, Y158, Y165 to Y168, L10, L57, L321, L322, Q361 to Q367, Q361s to Q367s, Q370, Q377 to Q379, Q383, etc., described in WO 2021 / 092371. Other ligands that can be linked to the RNAi agents of the present invention can be found in WO2017053995, WO2019217459, WO2021092371, WO2017053995, WO2010039548, WO2023064530, WO2022213118, and WO2019079386, the entire contents of each of which are incorporated herein by reference.

[0197] In certain embodiments, part includes carbohydrate." carbohydrate " refers to the compound that is made up of one or more monosaccharide units with at least 6 carbon atoms (can be straight chain, branched or cyclic), and oxygen, nitrogen or sulphur atom is connected on each carbon atom. Carbohydrate includes but is not limited to sugar (for example, monosaccharide, disaccharide, trisaccharide, tetrasaccharide and oligosaccharide containing about 4,5,6,7,8 or 9 monosaccharide units) and polysaccharide (such as starch, glycogen, cellulose and polysaccharide glue). In some embodiments, the carbohydrate that is incorporated into the part is disaccharide and trisaccharide selected from pentose, hexose or heptose and comprises such monosaccharide unit. In other embodiments, the carbohydrate that is incorporated into the part is amino sugar, for example galactosamine, glucosamine, N-acetylgalactosamine and N-acetylglucosamine.

[0198] In some embodiments, the ligand comprises a hexose or a hexosamine. The hexose can be selected from glucose, galactose, mannose, fucose or fructose. The hexosamine can be selected from fructosamine, galactosamine, glucosamine or mannosamine. In certain embodiments, the ligand comprises glucose, galactose, galactosamine or glucosamine. In one embodiment, the ligand comprises glucose, glucosamine or N-acetylglucosamine. In another embodiment, the ligand comprises galactose, galactosamine or N-acetylgalactosamine. In specific embodiments, ligands comprising glucose, galactose and N-acetylgalactosamine (GalNAc) are particularly effective in targeting RNA to hepatocytes because these ligands bind to ASGR expressed on the surface of hepatocytes. Examples of GalNAc- or galactose-containing ligands that can be incorporated into the RNAi agents of the invention are described in USP 7,491,805, 8,106,022, and 8,877,917; US Patent Publication No. US20030130186; and WIPO Publication No. WO 2013 / 166155, all of which are incorporated herein by reference in their entirety.

[0199] In certain embodiments, the ligand comprises a multivalent carbohydrate moiety. As used herein, a "multivalent carbohydrate moiety" refers to a moiety comprising two or more carbohydrate units that are capable of independently binding to or interacting with other molecules. For example, a multivalent carbohydrate moiety comprises two or more binding domains composed of carbohydrates that can bind to two or more different molecules or two or more different sites on the same molecule. The "valency" of a carbohydrate moiety refers to the number of individual binding domains within the carbohydrate moiety. For example, the terms "monovalent," "divalent," "trivalent," and "tetravalent" refer to carbohydrate moieties having one, two, three, and four binding domains, respectively, with respect to a carbohydrate moiety. The multivalent carbohydrate moiety can include a multivalent lactose moiety, a multivalent galactose moiety, a multivalent glucose moiety, a multivalent N-acetylgalactosamine moiety, a multivalent N-acetylglucosamine moiety, a multivalent mannose moiety, or a multivalent fucose moiety. In some embodiments, the ligand comprises a multivalent galactose moiety. In other embodiments, the ligand comprises a multivalent N-acetyl-galactosamine moiety. In these and other embodiments, the multivalent carbohydrate moiety can be divalent, trivalent, or tetravalent. In such embodiments, the multivalent carbohydrate moiety can be bivalent or trivalent. In a specific embodiment, the multivalent N-acetylgalactosamine moiety is trivalent or tetravalent. In another specific embodiment, the multivalent galactose moiety is trivalent or tetravalent. Exemplary trivalent and tetravalent GalNAc-containing ligands for incorporation into the RNAi agents of the present invention are described in detail below.

[0200] The ligand can also include an integrin ligand, such as a ligand that specifically binds to an integrin (including but not limited to αVβ6). Suitable integrin ligands are, for example, those mentioned in WO2019089765A1 or WO2022056286A1, the entire contents of which are incorporated herein by reference.

[0201] The ligand may also include a transferrin receptor 1 (TfR1) ligand, such as an anti-TfR1 antibody or polypeptide. Suitable TfR1 ligands are, for example, the TfR1 antibodies described in WO2022147209A1 or WO2021154476A1, or the bicyclic peptide ligand described in WO2022101633A1. The entire contents of these patent documents are incorporated herein by reference.

[0202] The ligand can be directly or indirectly connected or conjugated to the RNA molecule of the RNAi agent.For example, in some embodiments, the ligand is directly covalently connected to the sense strand or antisense strand of the RNAi agent.In other embodiments, the ligand is covalently connected to the sense strand or antisense strand of the RNAi agent through a joint.The ligand can be connected to the core base, sugar moiety or internucleotide junction of the sense strand or antisense strand of the RNAi agent of the present invention.

[0203] In some embodiments, the ligand can be connected to the 3' or 5' end of the sense strand or antisense strand. In certain embodiments, the ligand is covalently attached to the 5' end of the sense strand. In such embodiments, the ligand is attached to the 5'-terminal nucleotide of the sense strand. In these and other embodiments, the ligand is attached to the 5'-position of the 5'-terminal nucleotide of the sense strand. In other embodiments, the ligand is covalently attached to the 3' end of the sense strand. For example, in some embodiments, the ligand is attached to the 3'-terminal nucleotide of the sense strand. In some such embodiments, the ligand is attached to the 3'-position of the 3'-terminal nucleotide of the sense strand. In alternative embodiments, the ligand is attached near the 3' end of the sense strand, but before one or more terminal nucleotides (i.e., before 1, 2, 3, or 4 terminal nucleotides). In some embodiments, the ligand is attached to the 2'-position of the sugar of the 3'-terminal nucleotide of the sense strand. In other embodiments, the ligand is attached to the 2'-position of the sugar of the 5'-terminal nucleotide of the sense strand.

[0204] In certain embodiments, the ligand is connected to the sense strand or antisense strand by a joint." joint " refers to the atom or a group of atoms by which the ligand is covalently connected to the polynucleotide component of the RNAi agent. The length of the joint can be about 1 to about 30 atoms, about 2 to about 28 atoms, about 3 to about 26 atoms, about 4 to about 24 atoms, about 6 to about 20 atoms, about 7 to about 20 atoms, about 8 to about 20 atoms, about 8 to about 18 atoms, and about 12 to about 18 atoms. In some embodiments, the joint can include a bifunctional linking portion, which generally includes an alkyl portion with two functional groups. One of the functional groups is selected to be combined with the compound of interest (such as the sense or antisense strand of the RNAi agent chain), while the other functional group is selected to be combined with substantially any selected group, such as a ligand as described herein. In certain embodiments, the joint includes an oligomer of a chain structure or a repeating unit, such as ethylene glycol or an amino acid unit. The example of the functional group typically used in the bifunctional linking portion includes but is not limited to an electrophilic reagent for reacting with a nucleophilic group and a nucleophilic reagent for reacting with an electrophilic group. In some embodiments, the bifunctional linking moiety includes an amino group, a hydroxyl group, a carboxylic acid, a thiol group, an unsaturated bond (eg, a double bond or a triple bond), and the like.

[0205] Linkers that can be used to attach a ligand to the sense or antisense strand of the RNAi agent of the invention include, but are not limited to, pyrrolidine, 8-amino-3,6-dioxooctanoic acid, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid, 6-aminohexanoic acid, substituted C1-C 20 Alkyl, substituted or unsubstituted C2-C 20 Alkenyl or substituted or unsubstituted C2-C 20 Preferred substituents for such linkers include, but are not limited to, hydroxy, amino, alkoxy, carboxyl, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl.

[0206] In certain embodiments, joint is cleavable.Cleaving joint is a kind of enough stable outside cell but is cracked to release two parts that combine together by joint when entering target cell.In some embodiments, cleavable joint is in target cell or under the first reference condition (it can for example be selected as simulation or represent cell condition) than cracking in experimenter's blood fast at least 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or more, or at least 100 times.

[0207] Cleavable linkers are susceptible to the effects of cleavage agents, such as pH, redox potential, or the presence of degrading molecules. Generally, cleavage agents are more prevalent in cells than in serum or blood, or are found at higher levels or activity in cells. Examples of such cleavage agents include: redox agents selected for specific substrates or having no substrate specificity, including, for example, oxidases or reductases present in cells; esterases; endosomes or reagents that can produce an acidic environment, such as those that result in a pH of 5 or less; enzymes that can hydrolyze or degrade acid-cleavable linkers as general acids, peptidases (which can be substrate-specific), and phosphatases.

[0208] Cleavable linkers can include pH-sensitive moieties. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1 to 7.3. Endosomes have a more acidic pH, in the range of 5.5 to 6.0, and lysosomes have a more acidic pH (about 5.0). Some linkers will have a cleavable group that is cleaved at a preferred pH, thereby releasing the RNA molecule from the ligand in the cell, or releasing it into the desired organelle of the cell. The linker can include a cleavable group that can be cleaved by a specific enzyme. The type of cleavable group incorporated into the linker can depend on the cell to be targeted. For example, a liver targeting ligand can be connected to the RNA molecule by a linker that includes an ester group. Hepatocytes are rich in esterases, so the linker is more effectively cleaved in hepatocytes than in cell types that are not rich in esterases. Other types of cells rich in esterases include cells of the lung, renal cortex, and testis. When targeting cells rich in peptidases, such as hepatocytes and synovial cells, a linker containing a peptide bond can be used.

[0209] Other types of linkers suitable for attaching ligands to the sense or antisense strands in the RNAi agents of the invention are known in the art, such as those described in U.S. Patents 7,723,509, 8,017,762, 8,828,956, 8,877,917, and 9,181,551, all of which are incorporated herein by reference in their entirety.

[0210] In some embodiments, the ligand covalently attached to the sense strand or antisense strand of the RNAi agent of the present invention includes a GalNAc moiety, such as a polyvalent GalNAc. In some embodiments, the polyvalent GalNAc moiety is a trivalent GalNAc and is attached to the 3' end of the sense strand. In other embodiments, the polyvalent GalNAc moiety is a trivalent GalNAc and is attached to the 5' end of the sense strand. In other embodiments, the polyvalent GalNAc moiety is a tetravalent GalNAc moiety and is attached to the 3' end of the sense strand. In other embodiments, the polyvalent GalNAc moiety is a tetravalent GalNAc moiety and is attached to the 5' end of the sense strand.

[0211] In some embodiments, the ligand is L96, NAG25, and NAG37, each having the structural formula shown below, wherein the wavy line represents the position of attachment to the sense or antisense strand of the RNAi agent.

[0212] Pharmaceutical composition

[0213] The present invention also includes pharmaceutical compositions and formulations comprising the RNAi agents described herein and a pharmaceutically acceptable carrier, excipient, or diluent. Such compositions and formulations can be used to reduce the expression of the LPAR1 gene in patients in need thereof. Where clinical applications are contemplated, pharmaceutical compositions and formulations will be prepared in a form suitable for the intended application. Typically, this will require the preparation of a composition that is substantially free of pyrogens and other impurities that may be harmful to humans or animals.

[0214] The composition and method for preparing the pharmaceutical composition depend on many standards, including but not limited to route of administration, the type and degree of the disease to be treated or the condition or the dosage to be administered. In some embodiments, the pharmaceutical composition is prepared based on the expected route of delivery. For example, in certain embodiments, the pharmaceutical composition is formulated for parenteral delivery. Parenteral administration forms include intravenous, intraarterial, subcutaneous, intrathecal, intraperitoneal or intramuscular injection or infusion. In one embodiment, the pharmaceutical composition is formulated for intravenous delivery. In such an embodiment, the pharmaceutical composition may include a lipid-based delivery vehicle. In another embodiment, the pharmaceutical composition is formulated for subcutaneous delivery. In such an embodiment, the pharmaceutical composition may include a targeting ligand (such as a ligand containing GalNAc as described herein or containing an antibody).

[0215] In some embodiments, the pharmaceutical composition comprises an effective amount of the RNAi agent described herein. An "effective amount" refers to an amount sufficient to produce a beneficial or desired clinical outcome. In some embodiments, an effective amount is an amount sufficient to reduce LPAR1 gene expression in a patient's specific tissue or cell type (e.g., liver or hepatocytes). The effective amount of the RNAi agent of the present invention can be from about 0.01 mg / kg body weight to about 100 mg / kg body weight, and can be administered daily, weekly, monthly, or at longer intervals. Accurately determining a specific effective dosage and dosing frequency may be based on several factors, including the patient's size, age, and general condition, the type of disease to be treated (e.g., myocardial infarction, coronary artery disease, peripheral artery disease, stroke), the specific RNAi agent used, and the route of administration.

[0216] The administration of the pharmaceutical composition of the present invention can be carried out by any common route, as long as the target tissue can be obtained by the approach. These approaches include but are not limited to parenteral (for example, subcutaneous, intramuscular, intraperitoneal or intravenous), oral, nasal, oral, intradermal, transdermal and sublingual routes, or by direct injection into liver tissue or by delivery through the portal vein. In some embodiments, the pharmaceutical composition is parenteral. For example, in certain embodiments, the pharmaceutical composition is administered intravenously. In other embodiments, the pharmaceutical composition is administered subcutaneously. In certain embodiments, the pharmaceutical composition is administered via pulmonary or intranasal administration, for example, by inhalation, atomization or spray administration.

[0217] Colloidal dispersion systems can be used as delivery vehicles for the RNAi agents of the present invention, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Commercially available fat emulsions suitable for transporting nucleic acids of the present invention include Intralipid (Baxter International Inc.), Liposyn (Abbott Pharmaceuticals), Lipsyn II (Hospira), Liposyn III (Hospire), Nutrilipid (B.Braun Medical Inc.), and other similar fat emulsions. A preferred colloidal system for use as an in vivo delivery vehicle is a liposome (i.e., an artificial membrane vesicle). The RNAi agent of the present invention can be encapsulated within a liposome, or can form a complex therewith, particularly with a cationic liposome. Alternatively, the RNAi agent of the present invention can be complexed with lipids, particularly with cationic lipids. Suitable cationic lipids are, for example, diol tetramethylaminopropyl (DOTAP) and diol phosphatidylethanolamine (DOTMA).

[0218] Liposomal formulations are particularly well-suited for topical administration, and liposomes offer several advantages over other formulations. These advantages include reduced side effects associated with high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer RNAi agents to the skin. In some embodiments, liposomes are used to deliver RNAi agents to epidermal cells and also enhance the penetration of RNAi agents into dermal tissue, such as the skin.

[0219] RNAi agent of the present invention can be fully encapsulated in lipid formulations, such as LNP or other nucleic acid-lipid particles. As used herein, term " LNP " refers to stable nucleic acid-lipid particles. LNP generally comprises cationic lipids, non-cationic lipids and the lipid (such as PEG-lipid conjugate) that prevents particle aggregation. LNP is very useful for systemic application because they show prolonged circulation time after intravenous (iv) injection, and accumulate in distal sites (such as the position physically separated from the administration site). LNP includes " pSPLP ", which includes the condensing agent-nucleic acid complex of the encapsulation described in WO00 / 03683. LNP particles of the present invention generally have an average diameter of about 50nm to about 150nm, more typically about 60nm to about 130nm, more typically about 70nm to about 110nm, most typically about 70nm to about 90nm, and substantially nontoxic. In addition, when nucleic acid is present in nucleic acid-lipid particles of the present invention, it has resistance to the degradation of nuclease in aqueous solution. Nucleic acid-lipid particles and methods for preparing the same are disclosed in, for example, U.S. Patent Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; U.S. Publication Nos. 2010 / 0324120 and WO 96 / 40964. In one embodiment, the lipid to drug ratio (mass / mass ratio) (e.g., lipid to RNAi agent ratio) will be in the range of about 1:1 to about 50:1, about 1:1 to about 25:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1.

[0220] The cationic lipid can be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DLenDMA). Dioleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dihydroxypropoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dihydroxypropoxy-3-morpholinopropane (DLin-MA), 1,2-dilinoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride (DLin-TMA.Cl), 1,2-di Linoleoyl-3-trimethylaminopropane chloride (DLin-TAP.Cl), 1,2-dilinoleoyloxy-3-(N-methylpiperazino)propane (DLin-MPZ) or 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxy-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-dilinolenoyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin -K-DMA) or its analogues, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriacontac-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (MC3), 1,1'-(2-(4-(2-((2-(bis(2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazadiyl)didecadecan-2-ol (Tech G1) or mixtures thereof.The cationic lipid may comprise from about 20 mol% to about 50 mol%, or about 40 mol% of the total lipid present in the particle.

[0221] In some embodiments, the compound 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used to prepare lipid-siRNA nanoparticles. In some embodiments, the lipid-siRNA particles comprise 40% 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40% cholesterol: 10% PEG-C-DOMG (molar percentage), have a particle size of 63.0±20 nm, and a siRNA / lipid ratio of 0.027.

[0222] The ionizable / non-cationic lipids can be anionic lipids or neutral lipids, including but not limited to distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoyl DP-acylglyceroglycerophosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), oleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), oleoylphosphatidylcholine (POPC), palmitoylphosphatidylcholine ...DPPC), palmitoylphosphatidylcholine (DPPC), palmitoylphosphatidylcholine (DPPC), palmitoylphosphatidylcholine (DPPC), palmitoylphosphatidylcholine (DPPC), palmitoylphosphatidylcholine (DPPC), palmitoylphosphatidylcholine (DPPC), palmitoylphosphatidylcholine (DPPC), palmitoylphosphatidylcholine (DPPC), palmitoylphosphatidylcholine (DPPC), palmitoylphosphatidylcholine (DPPC The non-cationic lipid may be present in an amount of about 5 mol % to about 90 mol %, about 10 mol %, or about 58 mol % (if cholesterol is included) of the total lipids present in the particle.

[0223] In some embodiments, the nucleic acid-lipid particle further comprises cholesterol, for example, 10 mol% to 20 mol% of the total lipid present in the particle, or about 2 mol%. In some embodiments, the nucleic acid-lipid particle further comprises cholesterol, for example, 10 mol% to 60 mol% of the total lipid present in the particle, or about 48 mol%.

[0224] In one embodiment, lipidoid ND98·4HCl (molecular weight 1487) (see U.S. Patent Application No. 12 / 056,230, incorporated herein by reference), cholesterol (Sigma-Aldrich), and PEG-ceramide C16 (Avanti Polar Lipids) can be used to prepare lipid-dsRNA nanoparticles (i.e., LNP01 particles). Each stock solution in ethanol can be prepared as follows: ND98, 133 mg / ml; cholesterol, 25 mg / ml; PEG-ceramide C16, 100 mg / ml. The stock solutions of ND98, cholesterol, and PEG-ceramide C16 can then be mixed in a molar ratio of, for example, 42:48:10. The combined lipid solution can be mixed with aqueous siRNA (e.g., in sodium acetate at pH 5) to a final ethanol concentration of approximately 35-45% and a final sodium acetate concentration of approximately 100-300 mM. Lipid-siRNA nanoparticles typically form spontaneously upon mixing.

[0225] Other exemplary lipid-siRNA formulations can be found in, for example, WO2009 / 127060 (SNALP), PCT / US2010 / 022614 (XTC), US2010 / 0324120 (MC3), PCT / US09 / 63933 (ALNY-100), and WO2010 / 129709 (C12-200).

[0226] In some embodiments, the RNAi formulations of the present invention can be linked to a cell penetrating peptide (CPP), which can include at least one positively charged amino acid or ionizable amino acid, such as arginine, to form a peptide-oligonucleotide conjugate. Suitable CPPs for forming peptide-oligonucleotide conjugates can be found, for example, in WO2022213118A1 or WO2019079386A1, the entire contents of which are incorporated herein by reference.

[0227] Pharmaceutical compositions suitable for injection include, for example, sterile aqueous solutions or dispersions and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. In general, these preparations are sterile and, to a certain extent, fluid and easy to inject. The preparation should remain stable under production and storage conditions and should be preserved to prevent contamination by microorganisms such as bacteria and fungi. Suitable solvents or dispersion media can include, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. For example, suitable fluidity can be maintained by using a coating such as lecithin, by maintaining the desired particle size in the case of dispersion, and by using a surfactant. The effects of microorganisms can be prevented by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is preferred to include isotonic agents, such as sugar or sodium chloride. Prolonged absorption of injectable compositions can be achieved by using agents that delay absorption in the composition, such as aluminum monostearate and gelatin.

[0228] Sterile injectable solutions can be prepared by adding an appropriate amount of the active compound to a solvent along with any other ingredients (e.g., those listed above) and then sterilizing by filtration. Typically, dispersions are prepared by adding the various sterilized active ingredients to a dispersion medium containing an alkaline dispersion medium and the desired other ingredients, e.g., as described above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred preparation methods include vacuum drying and freeze drying techniques, which produce a powder of the active ingredient and any additional desired ingredients from a previously sterile-filtered solution thereof.

[0229] The compositions of the present invention can generally be formulated in neutral form or salt form. Pharmaceutically acceptable salts include, for example, acid addition salts (formed by free amino groups) derived from inorganic acids (such as hydrochloric acid or phosphoric acid) or organic acids (such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc.). Salts formed with free carboxyl groups can also be derived from inorganic bases (such as sodium, potassium, ammonium, calcium or iron oxide) or organic bases (such as isopropylamine, trimethylamine, histidine, procaine, etc.). In some embodiments, the RNAi agent of the present invention is formulated as a sodium salt.

[0230] For example, for parenteral administration in the form of an aqueous solution, the solution is generally appropriately buffered, and the liquid diluent is first made isotonic with, for example, enough saline or glucose. Such an aqueous solution can be used for, for example, intravenous, intramuscular, subcutaneous, and intraperitoneal administration. Preferably, a sterile aqueous medium is used. For example, a single dose can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous infusion liquid, or injected at the infusion site of the suggestion. For human administration, the preparation should meet the sterility, pyrogenicity, general safety, and purity standards required by the local Food and Drug Administration. In certain embodiments, the pharmaceutical composition of the present invention comprises sterile saline solution and RNAi agent as described herein or consists of the two. In other embodiments, the pharmaceutical composition of the present invention comprises RNAi agent as described herein and sterile water (e.g., water for injection, WFI) or consists of the two. In other embodiments, the pharmaceutical composition of the present invention comprises RNAi agent as described herein and phosphate buffered saline (PBS) or consists of it.

[0231] In some embodiments, the pharmaceutical compositions of the present invention are packaged with or stored within a drug delivery device. Devices for injecting formulations include, but are not limited to, injection ports, prefilled syringes, autoinjectors, syringe pumps, intracorporeal syringes, and injection pens. Devices for aerosolizing or powdered formulations include, but are not limited to, inhalers, insufflators, aspirators, and the like. Thus, the present invention includes a drug delivery device containing a pharmaceutical composition of the present invention for use in treating or preventing one or more diseases or conditions described herein.

[0232] Treatment methods and uses

[0233] The present invention provides a method for reducing or inhibiting LPAR1 gene expression in a cell by contacting the cell with any of the RNAi agents described herein. The cell can be in vitro or in vivo. LPAR1 gene expression can be assessed by measuring the amount or level of LPAR1 mRNA or LPAR1-C protein. Reduction of LPAR1 expression in cells or animals treated with the RNAi agents of the present invention can be determined relative to LPAR1 expression in cells and animals not treated with the RNAi agent or treated with a control RNAi agent. For example, in some embodiments, reduction of LPAR1 expression is assessed by (a) measuring the amount or level of LPAR1 mRNA in cells treated with the RNAi agent of the present invention, (b) measuring the amount or level of LPAR1 mRNA in cells treated with a control RNAi agent (e.g., an RNAi agent directed against an RNA molecule not expressed in the cell or an RNAi agent having a nonsense or scrambled sequence) or no RNAi agent, and (c) comparing the LPAR1 mRNA level measured in the treated cells in (a) with the LPAR1 mRNA level in the control cells in (b). Prior to comparison, LPAR1 mRNA levels in treated and control cells can be normalized to the RNA level of a control gene (e.g., 18S ribosomal RNA or a housekeeping gene). LPAR1 mRNA levels can be measured by a variety of methods, including Northern blot analysis, nuclease protection assays, fluorescence in situ hybridization (FISH), reverse transcriptase (RT)-PCR, real-time RT-PCR, quantitative PCR, droplet digital PCR, and the like.

[0234] In some embodiments, the method of assessing LPAR1 expression levels is performed in vitro in cells that naturally express the LPAR1 gene or in cells that have been engineered to express LPAR1.

[0235] In other embodiments, the method for assessing LPAR1 expression levels is performed in vivo. The RNAi agent and any control RNAi agent can be administered to an animal (e.g., a transgenic animal or non-human primate expressing an LPAR1 gene), and LPAR1 mRNA or LPAR1 protein levels can be assessed in various tissues harvested from the animal after treatment. Alternatively or additionally, biomarkers or functional phenotypes associated with LPAR1 expression can be assessed in the treated animal. For example, LPAR1 protein is an expression product of LPAR1 mRNA. Therefore, LPAR1 protein levels can be measured in animals treated with the RNAi agents of the present invention to assess the functional efficacy of reducing LPAR1 expression.

[0236] In certain embodiments, the expression of LPAR1 in a cell is reduced by at least 40%, at least 45%, or at least 50% by a RNAi agent of the present invention. In some embodiments, the expression of LPAR1 in a cell is reduced by at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% by a RNAi agent of the present invention. In other embodiments, the expression of LPAR1 in a cell is reduced by about 90% or more, e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, by a RNAi agent of the present invention. The percentage reduction in LPAR1 expression can be measured by any of the methods described herein, as well as other methods known in the art.

[0237] The present invention provides methods for reducing or inhibiting the expression of the LPAR1 gene, thereby reducing or inhibiting the production of LPAR1 protein, in a patient in need thereof, as well as methods for treating or preventing diseases or conditions associated with LPAR1 expression or activity. A "disease or condition mediated by aberrant LPAR1 expression" refers to a disease or condition in which LPAR1 expression levels are altered, or a condition in which elevated LPAR1 expression levels are associated with an increased risk of developing the disease or condition. In certain embodiments, the RNAi agents of the present invention are particularly useful for reducing LPAR1 levels.

[0238] Diseases and conditions associated with LPAR1 expression that can be treated or prevented according to the methods of the present invention include, but are not limited to, fibrotic diseases (e.g., pulmonary fibrosis, liver fibrosis, renal fibrosis), hypertrophic cardiomyopathy, diffuse scleroderma, adult sclerosis, cancer (e.g., hepatocellular carcinoma, ovarian cancer, glioblastoma), diabetic nephropathy, atherosclerosis, rheumatoid arthritis, neuropathic pain, non-alcoholic steatohepatitis, prostatic hyperplasia, and demyelinating diseases.

[0239] In certain embodiments, the present invention provides a method for reducing LPAR1 expression in a patient in need thereof, comprising administering to the patient any of the RNAi agents described herein. Preferably, after administration of the RNAi agent, the expression level of LPAR1 in the patient's cells is reduced compared to the level of LPAR1 expression in the patient who did not receive the RNAi agent, or compared to the level of LPAR1 expression in the patient before administration of the RNAi agent. In some embodiments, after administration of the RNAi agent of the present invention, LPAR1 expression in the patient is reduced by at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The percent reduction in LPAR1 expression can be measured by any of the methods described herein, as well as other methods known in the art. In certain embodiments, the percent reduction in LPAR1 expression is determined by assessing the patient's LPAR1 protein level according to the methods described herein.

[0240] In certain embodiments, the patient in need of reduced LPAR1 expression is a patient diagnosed with or at risk for a disease mediated by abnormal LPAR1 expression levels. Therefore, the present invention includes a method for treating or preventing a disease mediated by abnormal LPAR1 expression levels in a patient in need thereof by administering any of the RNAi agents of the present invention. In some embodiments, the present invention includes the use of any of the RNAi agents described herein in the preparation of a medicament for treating or preventing a disease mediated by abnormal LPAR1 expression levels in a patient in need thereof. In other embodiments, the present invention provides an LPAR1-targeting RNAi agent for use in a method for treating or preventing a disease mediated by abnormal LPAR1 expression levels in a patient in need thereof. As described above, the disease mediated by abnormal LPAR1 expression levels is, for example, a fibrotic disease (e.g., pulmonary fibrosis, liver fibrosis, renal fibrosis), hypertrophic cardiomyopathy, diffuse scleroderma, adult sclerosis, cancer (e.g., hepatocellular carcinoma, ovarian cancer, glioblastoma), diabetic nephropathy, atherosclerosis, rheumatoid arthritis, neuropathic pain, non-alcoholic steatohepatitis, prostatic hyperplasia, and demyelination.

[0241] In certain other embodiments, the patient in need of reduced LPAR1 expression is a patient with elevated LPAR1 levels. Thus, in some embodiments, the present invention provides a method for reducing LPAR1 protein levels in a patient in need thereof by administering to the patient any of the RNAi agents described herein. In some embodiments, the present invention includes the use of any of the RNAi agents described herein in the preparation of a medicament for reducing LPAR1 protein levels in a patient in need thereof. In other embodiments, the present invention provides an LPAR1-targeting RNAi agent for use in a method for reducing LPAR1 protein levels in a patient in need thereof.

[0242] In some embodiments of the present invention, the present invention provides the use of an agent that reduces expression of the gene encoding lysophosphatidic acid receptor 1 (LPAR1) in the preparation of a medicament for treating a disease or condition treatable by LPAR1 knockdown. In a preferred embodiment, the agent is an RNAi agent. In a preferred embodiment, the disease or condition is selected from fibrotic diseases (e.g., pulmonary fibrosis, liver fibrosis, renal fibrosis), hypertrophic cardiomyopathy, diffuse scleroderma, adult sclerosis, cancer (e.g., hepatocellular carcinoma, ovarian cancer, glioblastoma), diabetic nephropathy, atherosclerosis, rheumatoid arthritis, neuropathic pain, non-alcoholic steatohepatitis, benign prostatic hyperplasia, and demyelination.

[0243] Therefore, in some embodiments, the present invention provides a method for treating a disease or condition selected from the group consisting of a fibrotic disease (e.g., pulmonary fibrosis, liver fibrosis, renal fibrosis), hypertrophic cardiomyopathy, diffuse scleroderma, adult sclerosis, cancer (e.g., hepatocellular carcinoma, ovarian cancer, glioblastoma), diabetic nephropathy, atherosclerosis, rheumatoid arthritis, neuropathic pain, non-alcoholic steatohepatitis, prostatic hyperplasia, and demyelination.

[0244] Sequence Listing

[0245] Human LPAR1, mRNA (SEQ ID NO. 1, NCBI accession number NM_001351411.2)

[0246] Cynomolgus monkey LPAR1 mRNA (SEQ ID NO. 2, NCBI accession number XM_005581086.3) Example

[0247] Experimental methods and materials

[0248] Target sequence screening

[0249] siRNAs were designed based on the full-length LPAR1 mRNA sequence (NM_001351411.2). All sequences were obtained from the NCBI gene database. Homology to human (Gene ID: 1902, NM_001351411.2, SEQ ID NO. 1) and cynomolgus macaque (Gene ID: 101864894, XM_005581086.3, SEQ ID NO. 2) sequences was ensured during design.

[0250] siRNA synthesis

[0251] The synthesis method of siRNA is the same as the conventional phosphoramidite solid phase synthesis method (all synthesized by Suzhou Beixin Biotechnology Co., Ltd.). According to the scale, an Oligo-48 multi-channel nucleic acid synthesizer was used. Unless otherwise specified, a solid support made of commercially available controlled pore glass (CPG, ) was synthesized on . All phosphoramidite monomers were purchased from Shanghai Zhaowei. The phosphoramidite monomers used were prepared in 0.1M acetonitrile solution. Oligonucleotide synthesis was completed from 3'-5' by deprotection (3% DCA), coupling (5-ethylthiotetrazole, ETT as activator, 0.6M acetonitrile solution), capping (acetic anhydride, N-methylimidazole, pyridine and acetonitrile mixed reagent), oxidation (iodine / water / pyridine) or sulfurization (2% PADS in 2,6-lutidine / acetonitrile solution). Purification was performed using a C18 reverse chromatography column with a mobile phase of 0.1M TEAA and acetonitrile. The target oligonucleotides were collected and lyophilized, and identified as the target product by LC-MS and then quantified by UV (260nm). The obtained sense chain and antisense chain were annealed according to the molar ratio to obtain complementary paired double-stranded siRNA and adjusted to the required concentration.

[0252] In vitro screening of the inhibitory activity of unmodified siRNA against LPAR1 mRNA in A549 cell line

[0253] Dissolution of compounds

[0254] Place the compound powder tube in a centrifuge and centrifuge at 12,000 rpm for 5 min. Add an appropriate amount of RNase-free deionized water and mix well to prepare a 20 μM stock solution. Each compound is dispensed into 3 tubes, 10 μl / tube, and stored in a -20°C refrigerator.

[0255] Reverse transfection

[0256] Compound dilution: 20 μM compound stock solution was diluted with Opti-MEM reduced serum medium to 12 times the working concentration and allowed to stand for use.

[0257] Dilute the transfection reagent with Opti-MEM Reduced Serum Medium at a ratio of 98.5:1.5, mix gently, and let stand at room temperature for 15 minutes.

[0258] To prepare the transfection complex, mix equal volumes of the diluted compound and transfection reagent and let it stand at room temperature for 15 minutes. Add 20 μl of transfection complex to each well of a 96-well cell plate.

[0259] For seeding, select A549 cells at approximately 80% confluency, trypsinize, resuspend in complete medium without penicillin and streptomycin, and count the cells. Prepare a cell suspension at a concentration of 100,000 cells / ml and add 100 μl of this suspension to each well of the cell plate containing the transfection complex.

[0260] The cells were cultured in a 37°C 5% CO2 incubator for 24 hrs.

[0261] Cell lysis

[0262] Prepare cell lysis buffer according to the following table

[0263] Remove the cell culture plate, discard the culture medium, and wash the cells once with pre-chilled DPBS. Add 50 μl of cell lysis buffer to each well, pipette up and down 5-10 times to mix thoroughly, and let stand at room temperature for 10 minutes. Then, add 5 μl of cell lysis stop buffer to each well, pipette up and down 5-10 times to mix thoroughly, and wait at room temperature for 2 minutes to terminate the reaction. Place the cell plate on ice until ready to use.

[0264] Real-time fluorescence quantitative PCR detection

[0265] Quantitative PCR reaction system

[0266] Prepare a quantitative PCR reaction system according to the table above and add 9 μl to a 384-well plate. Add 1 μl of cell lysate as template. Complete the PCR amplification reaction according to the protocol in the table below.

[0267] Data Analysis

[0268] The relative quantitative PCR method (2 -ΔΔCT ) to analyze the relative expression of genes.

[0269] In order to evaluate the in vitro inhibitory activity of unmodified siRNA on LPAR1 mRNA in A549 cell line, the designed siRNA was subjected to in vitro functional evaluation. The results are shown in Table 2.

[0270] Table 2. In vitro inhibitory activity of unmodified siRNA against LPAR1 mRNA in A549 cells

[0271] In order to evaluate the in vitro inhibitory activity of unmodified siRNA on LPAR1 mRNA in A549 cell line, the designed siRNA was subjected to in vitro functional evaluation. The results are shown in Table 3.

[0272] Table 3. In vitro inhibitory activity of unmodified siRNA against LPAR1 mRNA in A549 cell line

[0273] In order to evaluate the in vitro inhibitory activity of unmodified siRNA on LPAR1 mRNA in A549 cell line, the designed siRNA was subjected to in vitro functional evaluation. The results are shown in Table 4.

[0274] Table 4. In vitro inhibitory activity of unmodified siRNA against LPAR1 mRNA in A549 cell line

[0275] The results showed that the siRNA of the present invention can effectively reduce the LPAR1 mRNA level in A549 cells.

Claims

1. An RNAi agent for inhibiting the expression of lysophosphatidic acid receptor 1 (LPAR1) gene in cells, comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 or at least 21 consecutive nucleotides that are at least 80%, 85%, 90%, 95% or 100% complementary to the target region of the mRNA encoding LPAR1 or that differ from it by no more than 3 nucleotides.

2. The RNAi agent according to claim 1, wherein the target region is 602-631, 793-825, 833-859, 1107-1143, 1167-1194, 1211-1288, 1326-1360, 1489-1523, 1777-1804, 2264-2290 or 2786-2813 of the nucleotide sequence shown in SEQ ID NO:

1.

3. The RNAi agent according to claim 1 or 2, wherein the antisense strand comprises at least 15 consecutive nucleotides selected from SEQ ID NO:40 to 76 and nucleotide sequences having 1 to 3 nucleotide differences from them.

4. The RNAi agent according to any one of claims 1 to 3, wherein the length of the double-stranded region is 17 to 23 base pairs, preferably 18 to 21 base pairs, more preferably 19 base pairs.

5. The RNAi agent according to any one of claims 1 to 4, wherein the length of each of the sense strand and the antisense strand is 17 to 23 nucleotides, preferably 19 to 21 nucleotides.

6. The RNAi agent according to any one of claims 1 to 5, wherein the RNAi agent comprises one or two blunt ends, preferably one blunt end.

7. The RNAi agent according to any one of claims 1 to 6, wherein the RNAi agent comprises one or two overhangs, preferably one overhang, and each overhang has 1 to 4 unpaired nucleotides, preferably 2 unpaired nucleotides.

8. The RNAi agent according to claim 7, wherein the overhang is located at the 3'-end of the sense strand, the 3'-end of the antisense strand or both at the 3'-ends of the sense strand and the antisense strand; preferably, the overhang is located at the 3'-end of the antisense strand, and further preferably, the RNAi agent has one blunt end.

9. The RNAi agent according to any one of claims 1 to 8, wherein the sense strand comprises at least 15 consecutive nucleotides selected from any one of the nucleotide sequences of SEQ ID NO:3 to 39 and nucleotide sequences having 1 to 3 nucleotide differences from them.

10. The RNAi agent according to any one of claims 1 to 9, wherein the antisense strand has no more than 23 nucleotides and comprises a nucleotide sequence selected from SEQ ID NO:40 to 76; the sense strand has no more than 21 nucleotides and comprises the nucleotide sequence of SEQ ID NO:3 to 39.

11. The RNAi agent according to claim 10, wherein: the sense strand comprises or is the sequence shown in SEQ ID NO:3 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO:40 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; the sense strand comprises or is the sequence shown in SEQ ID NO:4 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO:41 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; the sense strand comprises or is the sequence shown in SEQ ID NO:5 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO:42 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; the sense strand comprises or is the sequence shown in SEQ ID NO:6 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO:43 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; the sense strand comprises or is the sequence shown in SEQ ID NO:7 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO:44 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; the sense strand comprises or is the sequence shown in SEQ ID NO:8 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO:45 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; the sense strand comprises or is the sequence shown in SEQ ID NO:9 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO:46 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; the sense strand comprises or is the sequence shown in SEQ ID NO:10 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO:47 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; the sense strand comprises or is the sequence shown in SEQ ID NO:11 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO:48 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; the sense strand comprises or is the sequence shown in SEQ ID NO:12 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO:49 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; The sense strand comprises or is a nucleotide sequence as set forth in SEQ ID NO:13 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is a nucleotide sequence as set forth in SEQ ID NO:50 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; The sense strand comprises or is a nucleotide sequence as set forth in SEQ ID NO:14 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is a nucleotide sequence as set forth in SEQ ID NO:51 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; The sense strand comprises or is a nucleotide sequence as set forth in SEQ ID NO:15 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is a nucleotide sequence as set forth in SEQ ID NO:52 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; The sense strand comprises or is a nucleotide sequence as set forth in SEQ ID NO:16 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is a nucleotide sequence as set forth in SEQ ID NO:53 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; The sense strand comprises or is a nucleotide sequence as set forth in SEQ ID NO:17 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is a nucleotide sequence as set forth in SEQ ID NO:54 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; The sense strand comprises or is a nucleotide sequence as set forth in SEQ ID NO:18 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is a nucleotide sequence as set forth in SEQ ID NO:55 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; The sense strand comprises or is a nucleotide sequence as set forth in SEQ ID NO:19 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is a nucleotide sequence as set forth in SEQ ID NO:56 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; The sense strand comprises or is a nucleotide sequence as set forth in SEQ ID NO:20 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is a nucleotide sequence as set forth in SEQ ID NO:57 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; The sense strand comprises or is a nucleotide sequence as set forth in SEQ ID NO:21 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is a nucleotide sequence as set forth in SEQ ID NO:58 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; The sense strand comprises or is a nucleotide sequence as set forth in SEQ ID NO:22 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is a nucleotide sequence as set forth in SEQ ID NO:59 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; The sense strand comprises or is the sequence shown in SEQ ID NO:23 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO:60 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom; The sense strand comprises or is the sequence shown in SEQ ID NO:24 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO:61 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom; The sense strand comprises or is the sequence shown in SEQ ID NO:25 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO:62 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom; The sense strand comprises or is the sequence shown in SEQ ID NO:26 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO:63 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom; The sense strand comprises or is the sequence shown in SEQ ID NO:27 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO:64 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom; The sense strand comprises or is the sequence shown in SEQ ID NO:28 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO:65 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom; The sense strand comprises or is the sequence shown in SEQ ID NO:29 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO:66 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom; The sense strand comprises or is the sequence shown in SEQ ID NO:30 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO:67 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom; The sense strand comprises or is the sequence shown in SEQ ID NO:31 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO:68 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom; The sense strand comprises or is the sequence shown in SEQ ID NO:32 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO:69 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom; The sense strand comprises or is a nucleotide sequence as shown in SEQ ID NO: 33 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is a nucleotide sequence as shown in SEQ ID NO: 70 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; The sense strand comprises or is a nucleotide sequence as shown in SEQ ID NO: 34 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is a nucleotide sequence as shown in SEQ ID NO: 71 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; The sense strand comprises or is a nucleotide sequence as shown in SEQ ID NO: 35 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is a nucleotide sequence as shown in SEQ ID NO: 72 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; The sense strand comprises or is a nucleotide sequence as shown in SEQ ID NO: 36 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is a nucleotide sequence as shown in SEQ ID NO: 73 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; The sense strand comprises or is a nucleotide sequence as shown in SEQ ID NO: 37 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is a nucleotide sequence as shown in SEQ ID NO: 74 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; The sense strand comprises or is a nucleotide sequence as shown in SEQ ID NO: 38 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is a nucleotide sequence as shown in SEQ ID NO: 75 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; or The sense strand comprises or is a nucleotide sequence as shown in SEQ ID NO: 39 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is a nucleotide sequence as shown in SEQ ID NO: 76 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom.

12. The RNAi agent according to claim 11, wherein the RNAi agent comprises duplexes 8033, 8034, 8035, 8064, 8065, 8066, 8067, 8068, 8069, 8070, 8088, 8089, 8090, 8091, 8105, 8106, 8108, 8110, 8111, 8112, 8113, 8114, 8115, 8116, 8120, 8121, 8122, 8139, 8140, 8141, 8142, 8143, 8163, 8164, 8290, 8425 or 8426.

13. The RNAi agent according to any one of claims 1 to 12, wherein the sense strand and / or the antisense strand of the RNAi agent comprises at least one modified nucleotide, and the modified nucleotides are independently selected from 2'-deoxy-thymine (dT) nucleotides, 2'-O-methyl modified nucleotides (2'-OMe), 2'-fluoro modified nucleotides (2'-F), 2'-deoxy modified nucleotides, locked nucleic acids (LNA), unlocked nucleic acids (UNA), bridged nucleic acids (BNA), glycol nucleic acids (GNA), threose nucleic acids (TNA), conformationally restricted nucleotides, constrained ethyl nucleotides (cEt), 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-O-methoxyethyl modified nucleotides (2'-MOE), abasic nucleotides, inverted abasic nucleotides, inverted nucleotides, morpholino nucleotides (MOP), aminophosphates (PN), tetrahydropyran modified nucleotides (THP), 1,5-anhydrohexitol modified (HNA) nucleotides, cyclohexenyl modified nucleotides, nucleotides containing phosphorothioate linkages (PS), nucleotides containing methylphosphonate linkages, nucleotides containing 5'-phosphates, nucleotides containing 5'-phosphate mimics, cationic lipid covalently linked nucleotides, nucleotides containing 5'-vinylphosphonates (5'-VP), and combinations thereof; preferably selected from 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, nucleotides with phosphorothioate bond internucleotide linkages, and combinations thereof; and / or preferably, each nucleotide of the sense strand and / or the antisense strand of the RNAi agent is modified.

14. The RNAi agent according to claim 13, wherein, in the 5' to 3' direction, the nucleotides at positions 2, 5, 7, and 14 of the antisense strand are 2'-fluoro modified nucleotides, and one of the nucleotides at positions 12 and 16 of the antisense strand is a 2'-fluoro modified nucleotide, and the nucleotides at the remaining positions of the antisense strand are 2'-methoxy modified nucleotides.

15. The RNAi agent according to claim 13 or 14, wherein the antisense strand has at least one phosphorothioate bond internucleotide linkage; preferably, the phosphorothioate bond internucleotide linkage is present in one or more of the following: (i) between the 1st nucleotide and the 2nd nucleotide at the 5' end of the antisense strand; (ii) between the 2nd nucleotide and the 3rd nucleotide at the 5' end of the antisense strand; (iii) between the 1st nucleotide and the 2nd nucleotide at the 3' end of the antisense strand; and (iv) between the 2nd nucleotide and the 3rd nucleotide at the 3' end of the antisense strand.

16. The RNAi agent according to any one of claims 13 to 15, wherein, in the 5'-to-3' direction, the nucleotides at positions 7 and 9 of the sense strand are 2'-fluoro-modified nucleotides, one or two of the nucleotides at positions 5, 8, and 11 of the sense strand are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions of the sense strand are 2'-methoxy-modified nucleotides.

17. The RNAi agent according to any one of claims 13 to 16, wherein the sense strand has at least one phosphorothioate internucleotide linkage; preferably, the phosphorothioate internucleotide linkage is present in one or more of the following positions: (i) between the 1st nucleotide and the 2nd nucleotide at the 5'-end of the sense strand; (ii) between the 2nd nucleotide and the 3rd nucleotide at the 5'-end of the sense strand; (iii) between the 1st nucleotide and the 2nd nucleotide at the 3'-end of the sense strand; and (iv) between the 2nd nucleotide and the 3rd nucleotide at the 3'-end of the sense strand.

18. The RNAi agent according to claim 13, wherein the modification is selected from one of STC, ESC, Advanced ESC, ESC+, AD1-3, AD5, and GalXC.

19. The RNAi agent according to any one of claims 13 to 18, wherein the 5'-terminal nucleotide of the antisense strand contains a phosphate or phosphate analogue modification, preferably 5'-VP.

20. The RNAi agent according to any one of claims 13 to 19, wherein the RNAi agent further comprises a ligand targeting hepatocytes, preferably, the ligand comprises a galactose moiety, a galactosamine moiety or an N-acetylgalactosamine moiety, more preferably, the ligand is a trivalent or tetravalent N-acetylgalactosamine moiety, still more preferably, the ligand targeting hepatocytes is L96, NAG25 or NAG37; or the RNAi agent further comprises a ligand targeting non-hepatocytes, preferably the ligand is a lipophilic group, an integrin ligand or a transferrin receptor 1 ligand, and the lipophilic group is preferably selected from: lipids, vitamins, steroids, C5-C 30 saturated or unsaturated fatty acids, C5-C 30 alkyls, and polypeptides comprising at least one positively charged amino acid residue; the lipophilic group is more preferably selected from cholesterol, C 16 saturated or unsaturated fatty acids, C 16 alkyls, C 22 saturated or unsaturated fatty acids or C 22 alkyls.

21. A pharmaceutical composition comprising the RNAi agent according to any one of claims 1 to 20; and a pharmaceutically acceptable carrier; preferably, the pharmaceutical composition is formulated as an intravenous or subcutaneous injection, or the pharmaceutical composition is formulated as a pulmonary or intranasal administration preparation.

22. Use of the RNAi agent according to any one of claims 1 to 20, or the pharmaceutical composition according to claim 21, in the preparation of the following drugs: (i) A drug for reducing the expression level of LPAR1 in cells; (ii) A drug for preventing or treating a disease mediated by abnormal LPAR1 expression level; or (iii) A drug for preventing or treating a disease selected from fibrotic diseases (such as pulmonary fibrosis, liver fibrosis, renal fibrosis), hypertrophic cardiomyopathy, diffuse scleroderma, adult scleroderma, cancer (such as hepatocellular carcinoma, ovarian cancer, glioblastoma), diabetic nephropathy, atherosclerosis, rheumatoid arthritis, neuropathic pain, non-alcoholic steatohepatitis, prostatic hyperplasia, and demyelinating diseases.

23. Use of an agent that reduces the expression of the lysophosphatidic acid receptor 1 (LPAR1) gene in the preparation of a medicament for treating a disease or disorder treatable by LPAR1 knockdown; preferably, wherein, The reagent is an RNAi agent; preferably, the disease or disorder is selected from fibrotic diseases (such as pulmonary fibrosis, liver fibrosis, renal fibrosis), hypertrophic cardiomyopathy, diffuse scleroderma, adult scleroderma, cancer (such as hepatocellular carcinoma, ovarian cancer, glioblastoma), diabetic nephropathy, atherosclerosis, rheumatoid arthritis, neuropathic pain, non-alcoholic steatohepatitis, prostatic hyperplasia, and demyelination.

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