RNAI agents for inhibiting expression of ANGPTL3 and uses thereof

Abstract

An RNAi agent for inhibiting expression of the ANGPTL3 gene in a cell and a pharmaceutical composition thereof, and a therapeutic use of the RNAi agent.

Description

RNAi agents for inhibiting ANGPTL3 expression and their applications Technical Field

[0001] The present invention relates to an RNAi agent for inhibiting ANGPTL3 gene expression in cells and a pharmaceutical composition thereof, and also relates to therapeutic uses of the RNAi agent. Background Art

[0002] Angiopoietin-like 3 (ANGPTL3) is a member of the angiopoietin-like protein family involved in regulating lipid metabolism. The protein encoded by the ANGPTL3 gene is produced exclusively in the liver and secreted into the circulation. Within the liver, ANGPTL3 is exclusively produced by hepatocytes, and its expression is regulated by the hydroxysterol-activated liver X receptor (LXR). Studies have shown that ANGPTL3 exerts two biological effects: regulating angiogenesis and lipid metabolism. While its induction of angiogenesis is relatively weak, its regulation of lipid metabolism is more pronounced, including by inhibiting the activity of lipoprotein lipase (LPL) and endothelial lipase (EL). Dysregulation of lipid metabolism can lead to elevated levels of blood lipids, such as triglycerides or cholesterol. Elevated blood lipids are a significant risk factor for hypertension / cardiovascular disease, diabetes, and other pathological conditions.

[0003] Currently, there is only one monoclonal antibody drug targeting ANGPTL3, Evinacumab-dgnb (a product of Regeneron Pharmaceuticals), approved for marketing in the world for the treatment of homozygous familial hypercholesterolemia (HoFH) in children aged 12 years and above or adults. No siRNA drug with a similar target has been approved for marketing in the world.

[0004] Summary of the Invention

[0005] One aspect of the present invention provides an RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the RNAi agent inhibits the expression of ANGPTL3 in a cell, wherein the antisense strand is no longer than 23 nucleotides and comprises any one nucleotide sequence selected from SEQ ID NOs: 13, 14, 27 and 28.

[0006] In some embodiments, the sense strand is no longer than 21 nucleotides and comprises any one nucleotide sequence selected from SEQ ID NO: 5, 7, 8, 19, 20, and 22.

[0007] In some embodiments, the RNAi agent for inhibiting the expression of ANGPTL3 in cells provided by the present invention comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand is no longer than 23 nucleotides, and the sense strand is no longer than 21 nucleotides, wherein:

[0008] The sense strand comprises or is the sequence shown in SEQ ID NO: 5, and the antisense strand comprises or is the sequence shown in SEQ ID NO: 13;

[0009] The sense strand comprises or is the sequence shown in SEQ ID NO: 5, and the antisense strand comprises or is the sequence shown in SEQ ID NO: 14;

[0010] The sense strand comprises or is the sequence shown in SEQ ID NO:7, and the antisense strand comprises or is the sequence shown in SEQ ID NO:13;

[0011] The sense strand comprises or is the sequence shown in SEQ ID NO:8, and the antisense strand comprises or is the sequence shown in SEQ ID NO:13;

[0012] The sense strand comprises or is the sequence shown in SEQ ID NO: 19, and the antisense strand comprises or is the sequence shown in SEQ ID NO: 27;

[0013] The sense strand comprises or is the sequence shown in SEQ ID NO: 19, and the antisense strand comprises or is the sequence shown in SEQ ID NO: 28;

[0014] The sense strand comprises or is the sequence shown in SEQ ID NO: 20, and the antisense strand comprises or is the sequence shown in SEQ ID NO: 27;

[0015] The sense strand comprises or is the sequence shown in SEQ ID NO: 20, and the antisense strand comprises or is the sequence shown in SEQ ID NO: 28;

[0016] The sense strand comprises or is the sequence shown in SEQ ID NO: 22, and the antisense strand comprises or is the sequence shown in SEQ ID NO: 28;

[0017] The sense strand comprises or is the sequence shown in SEQ ID NO: 5, and the antisense strand comprises or is the sequence shown in SEQ ID NO: 18; or

[0018] The sense strand comprises or is the sequence shown in SEQ ID NO: 19, and the antisense strand comprises or is the sequence shown in SEQ ID NO: 32.

[0019] 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.

[0020] In some embodiments, the ligand comprises a structure as shown in formula (I):

[0021] in,

[0022] X is -C(O)-NH-, -NH-C(O)-, -OCH2-CH2O-, -S- or -SS-;

[0023] A or C is independently 0 or an integer between 1 and 5;

[0024] B is 0 or an integer between 1 and 3;

[0025] The wavy lines represent the location of attachment to the rest of the RNAi agent, wherein the ligand is attached to the 5' and / or 3' end of the sense and / or antisense strand; preferably, the ligand is attached to the 5' and / or 3' end of the sense strand; more preferably, the ligand is attached to the 3' end of the sense strand.

[0026] In some embodiments, the structure of the ligand is as shown in formula (II) or formula (III):

[0027] in,

[0028] X is -C(O)-NH-, -NH-C(O)-, -OCH2-CH2O-, -S- or -SS-;

[0029] Y1 is -C(O)-NR1-, -NH-C(O)-, -OCH2-CH2O-, -SS-, Wherein R1 is an alkyl group containing 1 to 6 carbon atoms or hydrogen;

[0030] W is -NH-, -O- or The wavy line on the right represents the position of connection with the sense strand or antisense strand;

[0031] The wavy line connected to W in Formula II represents the position of attachment to the sense strand or antisense strand;

[0032] A, C or F is 0 or an integer between 1 and 5;

[0033] B or E is 0 or an integer between 1 and 3;

[0034] D is an integer between 1 and 6;

[0035] in,

[0036] X is -C(O)-NH-, -NH-C(O)-, -OCH2-CH2O-, -S- or -SS-;

[0037] Y2 is -(CH2)p-(O-CH2-CH2)q-(CH2)j-Z3-, wherein Z3 is O, NH or C(O), p is an integer from 1 to 3, q ​​is an integer from 3 to 10; j is 0 or 1;

[0038] W does not exist or is The wavy line on the right indicates the position where it is connected to the sense strand or antisense strand;

[0039] The wavy line connected to W in Formula III represents the position of attachment to the sense strand or antisense strand;

[0040] A or C is 0 or an integer between 1 and 5;

[0041] B is 0 or an integer between 1 and 3.

[0042] In some embodiments, the structure of the ligand is as shown in Formula (IV) or Formula (V):

[0043] The wavy line represents the position of attachment to the sense strand or antisense strand, wherein the ligand is attached to the 5' and / or 3' end of the sense strand; preferably, the ligand is attached to the 3' end of the sense strand; wherein the targeting ligand is attached to the sense strand or antisense strand via a phosphate group, a phosphorothioate group, or a phosphonate group.

[0044] In some embodiments, the RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, the antisense strand is no more than 23 nucleotides in length, and the sense strand is no more than 21 nucleotides in length, wherein:

[0045] The sense strand comprises or is the sequence shown in SEQ ID NO:33, and the antisense strand comprises or is the sequence shown in SEQ ID NO:13;

[0046] The sense strand comprises or is the sequence shown in SEQ ID NO: 34, and the antisense strand comprises or is the sequence shown in SEQ ID NO: 13;

[0047] The sense strand comprises or is the sequence shown in SEQ ID NO: 38, and the antisense strand comprises or is the sequence shown in SEQ ID NO: 13;

[0048] The sense strand comprises or is the sequence shown in SEQ ID NO: 39, and the antisense strand comprises or is the sequence shown in SEQ ID NO: 13;

[0049] The sense strand comprises or is the sequence shown in SEQ ID NO:35, and the antisense strand comprises or is the sequence shown in SEQ ID NO:27;

[0050] The sense strand comprises or is the sequence shown in SEQ ID NO:36, and the antisense strand comprises or is the sequence shown in SEQ ID NO:27;

[0051] The sense strand comprises or is the sequence shown in SEQ ID NO:40, and the antisense strand comprises or is the sequence shown in SEQ ID NO:27;

[0052] The sense strand comprises or is the sequence shown in SEQ ID NO:41, and the antisense strand comprises or is the sequence shown in SEQ ID NO:27;

[0053] The sense strand comprises or is the sequence shown in SEQ ID NO:35, and the antisense strand comprises or is the sequence shown in SEQ ID NO:28;

[0054] The sense strand comprises or is the sequence shown in SEQ ID NO:36, and the antisense strand comprises or is the sequence shown in SEQ ID NO:28;

[0055] The sense strand comprises or is the sequence shown in SEQ ID NO:37, and the antisense strand comprises or is the sequence shown in SEQ ID NO:28;

[0056] The sense strand comprises or is the sequence shown in SEQ ID NO:40, and the antisense strand comprises or is the sequence shown in SEQ ID NO:28;

[0057] The sense strand comprises or is the sequence shown in SEQ ID NO:41, and the antisense strand comprises or is the sequence shown in SEQ ID NO:28; or

[0058] The sense strand comprises or is the sequence shown in SEQ ID NO:42, and the antisense strand comprises or is the sequence shown in SEQ ID NO:28.

[0059] In some particularly preferred embodiments, the present invention provides a double-stranded RNAi agent for inhibiting ANGPTL3 expression in a cell, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises SEQ ID NO. 19: 5'-mG*mA*mGmAfAmGfAfAfCmUmAmCmAmUmAmUmAmAmA-3' and has a length of no more than 21 nucleotides, and the antisense strand comprises SEQ ID NO. 27: 5'-mU*fU*mUmAfUmAfUmGmUmAmGmUmUfCmUfUmCmUmC*mA*mG-3' and has a length of no more than 23 nucleotides.

[0060] Wherein mA, mU, mG and mC are 2'-OMe A, 2'-OMe U, 2'-OMe G and 2'-OMe C respectively; fA, fU, fG and fC are 2'-FA, 2'-FU, 2'-FG and 2'-FC respectively; * is a phosphorothioate linkage;

[0061] The 3'-end of the sense chain is conjugated to a ligand targeting hepatocytes, preferably, the ligand comprises a galactose moiety, a galactosamine moiety or an N-acetylgalactosamine moiety, and further preferably, the ligand is a trivalent or tetravalent N-acetylgalactosamine moiety.

[0062] In the particularly preferred embodiment, the ligand is preferably a structure shown in Formula IV:

[0063] It is linked to the sense strand via a phosphate group or a phosphorothioate group.

[0064] In the particularly preferred embodiment, more preferably, the sense strand comprises SEQ ID NO.40: 5'-mG*mA*mGmAfAmGfAfAfCmUmAmCmAmUmAmUmAmAmA-YHZY12001-3' and the length of the sense strand is no more than 21 nucleotides, and the antisense strand comprises SEQ ID NO.27: 5'-mU*fU*mUmAfUmAfUmGmUmAmGmUmUfCmUfUmCmUmC*mA*mG-3' and the length is no more than 23 nucleotides,

[0065] Wherein mA, mU, mG and mC are 2'-OMe A, 2'-OMe U, 2'-OMe G and 2'-OMe C respectively; fA, fU, fG and fC are 2'-FA, 2'-FU, 2'-FG and 2'-FC respectively; * is a phosphorothioate linkage group,

[0066] Wherein YHZY12001 has the structure of Formula IV and is conjugated to the 3'-end of the sense strand as follows:

[0067] 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.

[0068] 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:

[0069] (i) an agent for reducing the expression level of ANGPTL3 in cells;

[0070] (ii) a drug for preventing or treating a disease caused by elevated ANGPTL3 expression levels; or

[0071] (iii) Medicaments for the prevention or treatment of metabolic diseases, such as hypertriglyceridemia, obesity, hyperlipidemia, abnormal lipid and / or cholesterol metabolism, atherosclerosis, type II diabetes, cardiovascular disease, coronary artery disease, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, homozygous or heterozygous familial hypercholesterolemia, statin-resistant hypercholesterolemia.

[0072] Accordingly, the present invention also provides a method for reducing the expression level of ANGPTL3 in a cell, comprising administering to a subject in need thereof a therapeutically effective amount of any RNAi agent of the present invention. The present invention also provides a method for preventing or treating a disease caused by elevated expression levels of ANGPTL3, comprising administering to a subject in need thereof a therapeutically effective amount of any RNAi agent of the present invention. The present invention also provides a method for preventing or treating a metabolic disease, such as one or more of hypertriglyceridemia, obesity, hyperlipidemia, abnormal lipid and / or cholesterol metabolism, atherosclerosis, type II diabetes, cardiovascular disease, coronary artery disease, nonalcoholic steatohepatitis, nonalcoholic fatty liver disease, homozygous or heterozygous familial hypercholesterolemia, and statin-resistant hypercholesterolemia, comprising administering to a subject in need thereof a therapeutically effective amount of any RNAi agent of the present invention.

[0073] Another aspect of the present invention provides an RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region, wherein, 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

[0074] From the 5' end to the 3' end, the 7th and 9th nucleotides of the sense strand are 2'-fluoro-modified nucleotides, one or two of the 5th, 8th and 11th nucleotides 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.

[0075] In some embodiments, the antisense strand of the RNAi agent of the present invention 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.

[0076] In some embodiments, the sense strand has at least one phosphorothioate internucleotide linkage; preferably, the phosphorothioate internucleotide linkage is present between (i) the first and second nucleotides at the 5' end of the sense strand; and / or (ii) the second and third nucleotides at the 5' end of the sense strand.

[0077] 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.

[0078] 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.

[0079] In some embodiments, the RNAi agent comprises one or two blunt ends, preferably one blunt end.

[0080] 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.

[0081] 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.

[0082] Other aspects of the present invention will become apparent from the detailed description of the specification which follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] FIG1 is a graph showing changes over time in the levels of ANGPTL3 protein, triglyceride, and total cholesterol in the plasma of mice administered with the RNAi agent of the present invention.

[0084] FIG2 shows that the serum ANGPTL3 protein level and triglyceride level of hyperlipidemic elderly cynomolgus monkeys administered with the RNAi agent of the present invention were significantly reduced. DETAILED DESCRIPTION

[0085] definition

[0086] ANGPTL3 herein refers to angiopoietin-like protein 3 or its encoding gene. As used herein, "ANGPTL3 mRNA" refers to ANGPTL3 messenger RNA, including allelic variants and splice variants. The NCBI reference sequence number for human ANGPTL3 mRNA is NM_014495.4.

[0087] As used herein, the term "RNAi agent" refers to an agent comprising an RNA molecule that, when introduced into a cell, is capable of downregulating the expression of a target gene (herein, the ANGPTL3 gene) through an RNA interference mechanism. The terms "RNAi agent of the present invention," "RNAi agent described herein," or similar expressions include 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 ANGPTL3 gene. RNA interference refers to the process by which a nucleic acid molecule induces the cleavage and degradation of a target RNA molecule (such as an mRNA molecule) in a sequence-specific manner, for example, through the 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 bonding (e.g., Watson-Crick hydrogen bonding, Wobble hydrogen bonding, Hoogsteen hydrogen bonding, or reversed Hoogsteen hydrogen bonding) 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.

[0088] 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" 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.

[0089] "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.

[0090] 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.

[0091] 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.

[0092] As used herein, the terms "blunt-ended" or "blunt-ended" with respect to dsRNA refer 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-ended, the dsRNA is said to be blunt-ended. It should be noted that a "blunt-ended" dsRNA is a dsRNA with both ends blunt-ended, 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.

[0093] The dsRNA molecule may include chemical modifications to ribonucleotides, including modifications to the ribose sugars, bases, or backbone components of ribonucleic acid, as described herein or modifications known in the art. Any such modifications, as used in double-stranded ribonucleic acid molecules (such as 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 core bases. Therefore, the term "modified nucleotides" includes substitutions, additions, or removals of, for example, functional groups or atoms of internucleoside linkages, sugar moieties, or core bases. Modifications suitable for use in the present invention include all types of modifications disclosed herein or known in the art. For example, the modified nucleotides are selected from 2'-deoxy-thymine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, unlocked nucleotides, conformationally restricted nucleotides, restricted ethyl nucleotides, 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-methoxyethyl modified nucleotides, abasic nucleotides, inverted abasic nucleotides, inverted nucleotides, morpholino nucleotides, phosphoramidates, tetrahydropyran modified nucleotides, 1,5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides containing phosphorothioate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphates, and nucleotides containing 5'-phosphate mimetics; preferably selected from 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, and nucleotides containing phosphorothioate internucleotide linkages.

[0094] 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, such as L96, attached via a bivalent or trivalent branched linker.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] Modified RNAi agents

[0100] One aspect of the present invention provides a modified RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region, wherein, 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.

[0101] In some embodiments, the present invention provides a modified RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region, wherein, 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.

[0102] In some embodiments, the present invention provides a modified RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region, wherein, 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.

[0103] In some embodiments, the present invention provides a modified RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region, 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, 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 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. 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.

[0104] The double-stranded region of RNAi agent should have enough length, to allow RNAi agent to enter RNA interference pathway, for example, by engaging Dicer enzyme and / or RISC complex.In some embodiments, the length of the double-stranded region of RNAi agent of the present invention is about 17 to about 23 base pairs.For example, the 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 double-stranded region is about 18 to about 21 base pairs in length. In other embodiments, the double-stranded region is about 19 base pairs in length.

[0105] Therefore, in some embodiments, the present invention provides a modified RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region of about 17 to about 23 base pairs in length, preferably about 18 to about 21 base pairs, and more preferably about 19 base pairs, wherein, 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.

[0106] In some embodiments, the present invention provides a modified RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region of about 17 to about 23 base pairs in length, preferably about 18 to about 21 base pairs, and more preferably about 19 base pairs, 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, 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 bond between nucleotides.

[0107] In some embodiments, the present invention provides a modified RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region of about 17 to about 23 base pairs in length, preferably about 18 to about 21 base pairs, and more preferably about 19 base pairs in length, 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, 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, wherein the phosphorothioate internucleotide linkage is present 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.

[0108] In some embodiments, the sense and antisense strands in the modified RNAi agents provided herein 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.

[0109] In some embodiments, the sense and antisense strands are of the same length but form a double-stranded region that is shorter than the strand, such that the RNAi agent has two nucleotide overhangs. For example, in one embodiment, the RNAi agent comprises (i) a sense and antisense strand that are 21 nucleotides in length, (ii) a double-stranded region that is 19 base pairs in length, and (iii) a nucleotide overhang with 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 comprises (i) a sense and antisense strand that are 23 nucleotides in length, (ii) a double-stranded region that is 21 base pairs in length, and (iii) a nucleotide overhang with one unpaired nucleotide at the 3' end of the sense strand and the 3' end of the antisense strand.

[0110] In other embodiments, the sense strand and the antisense strand have the same length and form a double-stranded region over their entire length so that no nucleotides protrude from either end of the double-stranded molecule. In one such embodiment, the RNAi agent is flat-ended and includes (i) a sense strand and an antisense strand each having a length of 21 nucleotides, and (ii) a double-stranded region having a length of 21 base pairs. In another such embodiment, the RNAi agent is flat-ended and includes (i) a sense strand and an antisense strand each having a length of 23 nucleotides, and (ii) a double-stranded region having a length of 23 base pairs. In another such embodiment, the RNAi agent is flat-ended and includes (i) a sense strand and an antisense strand each having a length of 19 nucleotides, and (ii) a double-stranded region having a length of 19 base pairs.

[0111] 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, such that the RNAi agent 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, such 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, such 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.

[0112] 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.).

[0113] The nucleotide overhang may be at the 5' end or the 3' end of one or both strands. For example, in one embodiment, the RNAi agent comprises nucleotide overhangs at both the 5' end and the 3' end of the antisense strand. In another embodiment, the RNAi agent comprises nucleotide overhangs at both the 5' end and the 3' end of the sense strand. In some embodiments, the RNAi agent comprises nucleotide overhangs at both the 5' end of the sense strand and the 5' end of the antisense strand. In other embodiments, the RNAi agent comprises nucleotide overhangs at both the 3' end of the sense strand and the 3' end of the antisense strand. In some embodiments, the RNAi agent comprises only a nucleotide overhang at the 5' end of the sense strand. In some embodiments, the RNAi agent comprises only a nucleotide overhang at the 3' end of the sense strand. In some embodiments, the RNAi agent comprises only a nucleotide overhang at the 3' end of the antisense strand. In some embodiments, the RNAi agent comprises only a nucleotide overhang at the 5' end of the antisense strand. In some embodiments, the RNAi agent comprises only a nucleotide overhang at the 5' end of the sense strand.

[0114] The RNAi agent 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 and antisense strands are fully base-paired at the ends of the molecule and that no unpaired nucleotides extend beyond the double-stranded region. In some embodiments, the RNAi agent 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 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.

[0115] Specifically, for example, in one embodiment, the RNAi agent 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 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.

[0116] In some embodiments, 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.

[0117] For example, from the 5' end to the 3' end, the nucleotides at positions 5, 7, 8 and 9 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; from the 5' end to the 3' end, the nucleotides at positions 7, 8 and 9 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; from the 5' end to the 3' end, the nucleotides at positions 5, 7 and 9 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; or, from the 5' end to the 3' end, the nucleotides at positions 7, 9 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.

[0118] In a preferred embodiment, the sense strand has at least one phosphorothioate internucleotide linkage. In a preferred embodiment, the phosphorothioate internucleotide linkage is present between the first and second nucleotides at the 5' end of the sense strand; and / or between the second and third nucleotides at the 5' 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.

[0119] Therefore, in some embodiments, in the 5' to 3' direction, the 7th and 9th nucleotides of the sense strand are 2'-fluoro-modified nucleotides, one or two of the 5th, 8th and 11th nucleotides of the sense strand are 2'-fluoro-modified nucleotides, the nucleotides at the remaining positions of the sense strand are all 2'-methoxy-modified nucleotides, and the sense strand has at least one phosphorothioate internucleotide linkage, and the phosphorothioate internucleotide linkage is present between the first and second nucleotides at the 5' end of the sense strand; and / or between the second and third nucleotides at the 5' end of the sense strand.

[0120] In some embodiments, in the 5' to 3' direction, the 7th and 9th nucleotides of the sense strand are 2'-fluoro-modified nucleotides, one or two of the 5th, 8th and 11th nucleotides of the sense strand are 2'-fluoro-modified nucleotides, the nucleotides at the remaining positions of the sense strand are all 2'-methoxy-modified nucleotides, and the sense strand has at least one phosphorothioate internucleotide linkage, and 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.

[0121] In some embodiments, in the 5' to 3' direction, the nucleotides at positions 5, 7, 8, and 9 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; in the 5' to 3' direction, the nucleotides at positions 7, 8, and 9 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; in the 5' to 3' direction, the nucleotides at positions 5, 7, and 9 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. The nucleotides at the remaining positions of the sense strand are all 2'-methoxy-modified nucleotides; or, from the 5' end to the 3' end, the nucleotides at positions 7, 9 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, and the sense strand has at least one phosphorothioate internucleotide linkage, wherein the phosphorothioate internucleotide linkage exists between the first nucleotide and the second nucleotide at the 5' end of the sense strand; and between the second nucleotide and the third nucleotide at the 5' end of the sense strand.

[0122] In a preferred embodiment, the present invention provides a modified RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region, wherein, 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 wherein, 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.

[0123] In a preferred embodiment, the present invention provides a modified RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region, wherein, 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 wherein, from the 5' end to the 3' end, the nucleotides at positions 5, 7, 8, and 9 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. Oxy-modified nucleotides; from the 5' end to the 3' end, the nucleotides at positions 7, 8 and 9 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; from the 5' end to the 3' end, the nucleotides at positions 5, 7 and 9 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; or, from the 5' end to the 3' end, the nucleotides at positions 7, 9 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.

[0124] In a preferred embodiment, the present invention provides a modified RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region, wherein, 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 wherein, from the 5' end to the 3' end, the nucleotides at positions 5, 7, 8, and 9 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; from the 5' end to the 3' end direction, the nucleotides at positions 7, 8 and 9 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; from the 5' end to the 3' end, the nucleotides at positions 5, 7 and 9 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; or, from the 5' end to the 3' end, the nucleotides at positions 7, 9 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; and the antisense strand and the sense strand each have at least one phosphorothioate bond between nucleotides. In a preferred embodiment, the phosphorothioate internucleotide linkages of the antisense strand are present 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; the phosphorothioate internucleotide linkages of the sense strand are present between the first and second nucleotides at the 5' end of the sense strand; and / or between the second and third nucleotides at the 5' end of the sense strand.

[0125] In a preferred embodiment, the present invention provides a modified RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region, wherein, 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 wherein, from the 5' end to the 3' end, , the nucleotides at positions 5, 7, 8, and 9 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; from the 5' end to the 3' end, the nucleotides at positions 7, 8, and 9 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; from the 5' end to the 3' end, the nucleotides at positions 5, 7, and 9 of the sense strand are 2'-fluoro-modified nucleotides nucleotides, the nucleotides at the remaining positions of the sense strand are all 2'-methoxy-modified nucleotides; or, in the 5' to 3' direction, the nucleotides at positions 7, 9 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; and the antisense strand and the sense strand each have at least one phosphorothioate internucleotide linkage, wherein the phosphorothioate internucleotide linkage of the antisense strand 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; the phosphorothioate internucleotide linkage of the sense strand exists 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.

[0126] In a preferred embodiment, the present invention provides a modified RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region of about 17 to about 23 base pairs, preferably about 18 to about 21 base pairs, more preferably about 19 base pairs in length, 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'-fluoro-modified nucleotides. The nucleotides are all 2'-methoxy modified nucleotides; and wherein, from the 5' end to the 3' end, the nucleotides at positions 5, 7, 8 and 9 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; from the 5' end to the 3' end, the nucleotides at positions 7, 8 and 9 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; from the 5' end to the 3' end, the sense strand The nucleotides at positions 5, 7, and 9 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; or, from the 5' end to the 3' end, the nucleotides at positions 7, 9, 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; and the antisense strand and the sense strand each have at least one phosphorothioate internucleotide bond, wherein the phosphorothioate internucleotide bond of the antisense strand The 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; the phosphorothioate internucleotide linkage of the sense strand exists 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.

[0127] In a preferred embodiment, the present invention provides a modified RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region of about 17 to about 23 base pairs in length, preferably about 18 to about 21 base pairs, more preferably about 19 base pairs, and the length of the sense strand and the antisense strand are each independently about 17 to about 23 nucleotides, wherein the RNAi agent comprises a nucleotide overhang at the 3' end of the antisense strand and blunt ends at the 5' end of the antisense strand and the 3' end of the sense strand, wherein, in the 5' to 3' direction, nucleotides 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 wherein, from the 5' end to the 3' end, the nucleotides at positions 5, 7, 8 and 9 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; from the 5' end to the 3' end, the nucleotides at positions 7, 8 and 9 of the sense strand are 2'-fluoro-modified nucleotides, The nucleotides at positions 5, 7, and 9 of the sense strand are 2'-fluoro-modified nucleotides, and the nucleotides at positions 5, 7, and 9 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; or, the nucleotides at positions 7, 9, 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, and the antisense strand and the sense strand each have at least one phosphorothioate internucleotide linkage. The invention relates to a method for preparing a nucleic acid sequence of the present invention to a nucleic acid sequence of the present invention, wherein the phosphorothioate internucleotide linkage of the antisense strand exists between the first nucleotide and the second nucleotide at the 5' end of the antisense strand; 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 strand; and between the second nucleotide and the third nucleotide at the 3' end of the antisense strand; the phosphorothioate internucleotide linkage of the sense strand exists between the first nucleotide and the second nucleotide at the 5' end of the sense strand; and between the second nucleotide and the third nucleotide at the 5' end of the sense strand.

[0128] In a preferred embodiment, the present invention provides a modified RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region of about 19 base pairs in length, the sense strand being about 19 nucleotides in length and the antisense strand being about 21 nucleotides in length, wherein the RNAi agent comprises an overhang of about two nucleotides at the 3' end of the antisense strand and blunt ends at the 5' end of the antisense strand and the 3' end of the sense strand, wherein, in the 5' to 3' direction, nucleotides at positions 2, 5, 7, and 14 of the antisense strand are 2'-fluoro-modified nucleotides, and nucleotides at positions 1 and 2 of the antisense strand are fluoro-modified nucleotides. One of the nucleotides at positions 2 and 16 is a 2'-fluoro-modified nucleotide, and the nucleotides at the remaining positions of the antisense strand are all 2'-methoxy-modified nucleotides; and wherein, from the 5' end to the 3' end, the nucleotides at positions 5, 7, 8 and 9 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; from the 5' end to the 3' end, the nucleotides at positions 7, 8 and 9 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 2'-fluoro-modified nucleotides; from the 5' end to the 3' end, the nucleotides at positions 5, 7 and 9 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; or, from the 5' end to the 3' end, the nucleotides at positions 7, 9 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; and the antisense strand and the sense strand each have at least one phosphorothioate bond internucleotide connection, wherein the antisense strand The phosphorothioate internucleotide linkages of the sense strand exist 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; the phosphorothioate internucleotide linkages of the sense strand exist 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.

[0129] In a preferred embodiment, the present invention provides a modified RNAi agent that inhibits the expression of the ANGPTL3 gene and comprises a sense strand and an antisense strand forming a double-stranded region of about 19 base pairs in length, the sense strand being about 19 nucleotides in length and the antisense strand being about 21 nucleotides in length, wherein the RNAi agent comprises an overhang of about two nucleotides at the 3' end of the antisense strand and blunt ends at the 5' end of the antisense strand and the 3' end of the sense strand, wherein, in the 5' to 3' direction, the 2nd, 5th, 7th and 14th nucleotides of the antisense strand are The nucleotide at position 5 of the antisense strand is a 2'-fluoro-modified nucleotide, 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 wherein, from the 5' end to the 3' end, the nucleotides at positions 5, 7, 8 and 9 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; from the 5' end to the 3' end, the nucleotides at positions 7, 8 and 9 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; from the 5' end to the 3' end, the nucleotides at positions 5, 7 and 9 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; or, From the 5' end to the 3' end, the nucleotides at positions 7, 9 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; and the antisense strand and the sense strand each have at least one phosphorothioate internucleotide connection, wherein the phosphorothioate internucleotide connection of the antisense strand 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; the phosphorothioate internucleotide connection of the sense strand exists 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.

[0130] RNAi agents for inhibiting ANGPTL3 gene expression

[0131] Another aspect of the present invention provides an RNAi agent for inhibiting ANGPTL3 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 any one nucleotide sequence selected from SEQ ID NOs: 13, 14, 27, and 28.

[0132] 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.

[0133] Therefore, in some embodiments of the present invention, an RNAi agent for inhibiting ANGPTL3 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 17 to about 23 base pairs in length, and the antisense strand is no longer than 23 nucleotides and comprises any one nucleotide sequence selected from SEQ ID NOs: 13, 14, 27 and 28.

[0134] In some embodiments of the present invention, an RNAi agent for inhibiting ANGPTL3 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 18 to about 21 base pairs in length, and the antisense strand is no longer than 23 nucleotides and comprises any one nucleotide sequence selected from SEQ ID NOs: 13, 14, 27, and 28.

[0135] In some embodiments of the present invention, an RNAi agent for inhibiting ANGPTL3 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 longer than 23 nucleotides and comprises any one nucleotide sequence selected from SEQ ID NOs: 13, 14, 27, and 28.

[0136] In some embodiments, the length of the sense strand and the antisense strand in the RNAi agent of the invention is each independently about 17 to about 23 nucleotides, such as 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 length of the sense strand and the antisense strand is each independently about 17, about 18, about 19, about 20, about 21, about 22, or about 23 nucleotides.

[0137] In some embodiments, the sense and antisense strands are of the same length but form a double-stranded region that is shorter than the strand, such that the RNAi agent for inhibiting ANGPTL3 gene expression has a two-nucleotide overhang. For example, in one embodiment, the RNAi agent for inhibiting ANGPTL3 gene expression comprises (i) a sense and antisense strand each having a length of 21 nucleotides, (ii) a double-stranded region of 19 base pairs, 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 ANGPTL3 gene expression comprises (i) a sense and antisense strand each having a length of 23 nucleotides, (ii) a double-stranded region of 21 base pairs, 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.

[0138] In other embodiments, the sense and antisense strands are of the same length and form a double-stranded region along their entire length, such that no nucleotides overhang at either end of the double-stranded molecule. In one such embodiment, the RNAi agent for inhibiting expression of the ANGPTL3 gene is blunt-ended and comprises (i) a sense and antisense strand each having a length of 21 nucleotides, and (ii) a double-stranded region having a length of 21 base pairs. In another such embodiment, the RNAi agent for inhibiting expression of the ANGPTL3 gene is blunt-ended and comprises (i) a sense and antisense strand each having a length of 23 nucleotides, and (ii) a double-stranded region having a length of 23 base pairs. In another such embodiment, the RNAi agent for inhibiting expression of the ANGPTL3 gene is blunt-ended and comprises (i) a sense and antisense strand each having a length of 19 nucleotides, and (ii) a double-stranded region having a length of 19 base pairs.

[0139] In other embodiments, one of the sense strands or the antisense strand is longer than the other strand, and the two strands form a double-stranded region equal in length to the shorter strand, such that the RNAi agent for inhibiting ANGPTL3 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 in length to the antisense strand, such 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 in length to the sense strand, such that the antisense strand forms an overhang with 1 to 4 unpaired nucleotides. In some embodiments, the nucleotide overhang is 1, 2, 3, or 4 nucleotides in length. In a specific embodiment, the overhang comprises 2 nucleotides. In certain embodiments, the overhang comprises a single nucleotide.

[0140] 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.).

[0141] 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 the expression of an ANGPTL3 gene comprises nucleotide overhangs at both the 5' and 3' ends of the antisense strand. In another embodiment, a RNAi agent for inhibiting the expression of an ANGPTL3 gene comprises nucleotide overhangs at both the 5' and 3' ends of the sense strand. In some embodiments, a RNAi agent for inhibiting the expression of an ANGPTL3 gene comprises 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 the expression of an ANGPTL3 gene comprises 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 the expression of an ANGPTL3 gene comprises only a nucleotide overhang at the 5' end of the sense strand. In some embodiments, a RNAi agent for inhibiting the expression of an ANGPTL3 gene comprises only a nucleotide overhang at the 3' end of the sense strand. In some embodiments, a RNAi agent for inhibiting the expression of an ANGPTL3 gene comprises only a nucleotide overhang at the 3' end of the sense strand. In some embodiments, a RNAi agent for inhibiting the expression of an ANGPTL3 gene comprises only a nucleotide overhang at the 3' end of the antisense strand. In some embodiments, the RNAi agent for inhibiting expression of the ANGPTL3 gene includes only a nucleotide overhang at the 5' end of the antisense strand. In some embodiments, the RNAi agent for inhibiting expression of the ANGPTL3 gene includes only a nucleotide overhang at the 5' end of the sense strand.

[0142] RNAi agents for inhibiting ANGPTL3 gene expression may 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 and antisense strands are fully base-paired at the ends of the molecule and no unpaired nucleotides extend beyond the double-stranded region. In some embodiments, the RNAi agent for inhibiting ANGPTL3 gene expression includes a nucleotide overhang at the 3' end of the sense strand and blunt ends at the 5' end of the sense strand and the 3' end of the antisense strand. In other embodiments, the RNAi agent for inhibiting ANGPTL3 gene expression includes a nucleotide overhang at the 3' end of the antisense strand and blunt ends at the 5' end of the antisense strand and the 3' end of the sense strand.

[0143] Specifically, for example, in one embodiment, the RNAi agent for inhibiting the expression of the ANGPTL3 gene comprises (i) a sense strand having a length of 19 nucleotides, and (ii) an antisense strand having a length of 21 nucleotides, the two strands forming a double-stranded region having a length equal to the length of the sense strand. In another embodiment, the RNAi agent for inhibiting the expression of the ANGPTL3 gene comprises (i) a sense strand having a length of 21 nucleotides, and (ii) an antisense strand having a length of 23 nucleotides, the two strands forming a double-stranded region having a length equal to the length of the sense strand.

[0144] In some embodiments, an RNAi agent for inhibiting the expression of an ANGPTL3 gene 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 any one nucleotide sequence selected from SEQ ID NOs: 13, 14, 27, and 28, and the RNAi agent includes an overhang and a blunt end, and the overhang preferably has two unpaired nucleotides.

[0145] In some embodiments, an RNAi agent for inhibiting the expression of an ANGPTL3 gene 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 any one nucleotide sequence selected from SEQ ID NOs: 13, 14, 27, and 28, and the RNAi agent includes an overhang and a blunt end, wherein the overhang preferably has two 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.

[0146] In some embodiments, an RNAi agent for inhibiting ANGPTL3 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 any one nucleotide sequence selected from SEQ ID NOs: 13, 14, 27, and 28, and the RNAi agent includes an overhang and a blunt end, wherein the overhang preferably has two 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.

[0147] In some embodiments, an RNAi agent for inhibiting ANGPTL3 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 any one nucleotide sequence selected from SEQ ID NOs: 13, 14, 27, and 28, the sense strand is 19 to 21 nucleotides in length, and the RNAi agent includes an overhang and a blunt end, wherein the overhang preferably has two 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.

[0148] In some embodiments, an RNAi agent for inhibiting ANGPTL3 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 any one nucleotide sequence selected from SEQ ID NOs: 13, 14, 27, and 28, the sense strand is 19 nucleotides in length, and the RNAi agent includes an overhang and a blunt end, wherein the overhang preferably has two 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.

[0149] In some embodiments, from the 5' end to the 3' end, the nucleotides at positions 7 and 9 of the sense strand of the RNAi agent for inhibiting the expression of the ANGPTL3 gene in a cell 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.

[0150] For example, in the RNAi agent for inhibiting the expression of the ANGPTL3 gene in a cell, in the 5' to 3' direction, the nucleotides at positions 5, 7, 8, and 9 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; in the 5' to 3' direction, the nucleotides at positions 7, 8, and 9 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; in the 5' to 3' direction, the nucleotides at positions 5, 7, and 9 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; or, in the 5' to 3' direction, the nucleotides at positions 7, 9, 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.

[0151] In a preferred embodiment, the sense strand has at least one phosphorothioate internucleotide linkage. In a preferred embodiment, the phosphorothioate internucleotide linkage is present between the first and second nucleotides at the 5' end of the sense strand; and / or between the second and third nucleotides at the 5' 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.

[0152] Therefore, in some embodiments, in the RNAi agent for inhibiting the expression of the ANGPTL3 gene in a cell, in the 5' to 3' direction, the 7th and 9th nucleotides of the sense strand are 2'-fluoro-modified nucleotides, one or two of the 5th, 8th and 11th nucleotides 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, and the sense strand has at least one phosphorothioate internucleotide linkage, and the phosphorothioate internucleotide linkage exists between the first and second nucleotides at the 5' end of the sense strand; and / or between the second and third nucleotides at the 5' end of the sense strand.

[0153] In some embodiments, in the RNAi agent for inhibiting the expression of the ANGPTL3 gene in a cell, 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 all 2'-methoxy-modified nucleotides, and the sense strand has at least one phosphorothioate internucleotide linkage, wherein the phosphorothioate internucleotide linkage exists 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.

[0154] In some embodiments, in the RNAi agent for inhibiting the expression of the ANGPTL3 gene in a cell, from the 5' end to the 3' end, the nucleotides at positions 5, 7, 8, and 9 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; from the 5' end to the 3' end, the nucleotides at positions 7, 8, and 9 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; from the 5' end to the 3' end, the nucleotides at positions 5, 7, and 9 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. '-fluoro-modified nucleotides, and the nucleotides at the remaining positions of the sense strand are all 2'-methoxy-modified nucleotides; or, from the 5' end to the 3' end, the nucleotides at positions 7, 9 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, and the sense 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 sense strand; and between the second nucleotide and the third nucleotide at the 5' end of the sense strand.

[0155] In a preferred embodiment, the sense strand is no more than 21 nucleotides in length and comprises any one nucleotide sequence selected from SEQ ID NOs: 5, 7, 8, 19, 20, and 22. In a preferred embodiment, the sense strand is 20 nucleotides in length and comprises any one nucleotide sequence selected from SEQ ID NOs: 5, 7, 8, 19, 20, and 22. In a preferred embodiment, the sense strand is any one nucleotide sequence selected from SEQ ID NOs: 5, 7, 8, 19, 20, and 22.

[0156] In some embodiments, an RNAi agent for inhibiting ANGPTL3 gene expression is provided, comprising a sense strand and an antisense strand forming complementary double-stranded regions, the antisense strand being no longer than 23 nucleotides and comprising any one nucleotide sequence selected from SEQ ID NOs: 13, 14, 27, and 28, and the sense strand being no longer than 21 nucleotides and comprising any one nucleotide sequence selected from SEQ ID NOs: 5, 7, 8, 19, 20, and 22.

[0157] In some embodiments, an RNAi agent for inhibiting ANGPTL3 gene expression is provided, comprising a sense strand and an antisense strand forming complementary double-stranded regions, the antisense strand being any one nucleotide sequence selected from SEQ ID NOs: 13, 14, 27, and 28, and the sense strand being any one nucleotide sequence selected from SEQ ID NOs: 5, 7, 8, 19, 20, and 22.

[0158] In a preferred embodiment, an RNAi agent for inhibiting ANGPTL3 gene expression is provided, comprising a sense strand and an antisense strand forming complementary double-stranded regions, the sense strand and the antisense strand each having a length of no more than 23 nucleotides, and wherein:

[0159] The sense strand comprises or is the sequence shown in SEQ ID NO: 5, and the antisense strand comprises or is the sequence shown in SEQ ID NO: 13;

[0160] The sense strand comprises or is the sequence shown in SEQ ID NO: 5, and the antisense strand comprises or is the sequence shown in SEQ ID NO: 14;

[0161] The sense strand comprises or is the sequence shown in SEQ ID NO:7, and the antisense strand comprises or is the sequence shown in SEQ ID NO:13;

[0162] The sense strand comprises or is the sequence shown in SEQ ID NO:8, and the antisense strand comprises or is the sequence shown in SEQ ID NO:13;

[0163] The sense strand comprises or is the sequence shown in SEQ ID NO: 19, and the antisense strand comprises or is the sequence shown in SEQ ID NO: 27;

[0164] The sense strand comprises or is the sequence shown in SEQ ID NO: 19, and the antisense strand comprises or is the sequence shown in SEQ ID NO: 28;

[0165] The sense strand comprises or is the sequence shown in SEQ ID NO: 20, and the antisense strand comprises or is the sequence shown in SEQ ID NO: 27;

[0166] The sense strand comprises or is the sequence shown in SEQ ID NO: 20, and the antisense strand comprises or is the sequence shown in SEQ ID NO: 28;

[0167] The sense strand comprises or is the sequence shown in SEQ ID NO: 22, and the antisense strand comprises or is the sequence shown in SEQ ID NO: 28;

[0168] The sense strand comprises or is the sequence shown in SEQ ID NO: 5, and the antisense strand comprises or is the sequence shown in SEQ ID NO: 18; or

[0169] The sense strand comprises or is the sequence shown in SEQ ID NO: 19, and the antisense strand comprises or is the sequence shown in SEQ ID NO: 32.

[0170] In some embodiments, an RNAi agent for inhibiting ANGPTL3 gene expression is provided, comprising 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 the duplex siRNAs described herein: 2002-010, 2002-011, 2002-018, 2002-034, 2008-010, 2008-011, 2008-018, 2008-019, and 2008-035.

[0171] The RNAi agent of the present invention may include a ligand. As used herein, a "ligand" refers to any compound or molecule that can interact 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 change 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.

[0172] The ANGPTL3 gene is primarily expressed in the liver. Therefore, in certain embodiments, it is desirable to deliver the RNAi agents of the present invention specifically to hepatocytes. Thus, in certain embodiments, ligands are targeted to specifically deliver RNAi agents to hepatocytes using various methods described in more detail below. In certain embodiments, RNAi agents are targeted to hepatocytes using ligands that bind to surface-expressed asialoglycoprotein receptors (ASGRs) or components thereof (e.g., ASGR1, ASGR2).

[0173] 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.

[0174] 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.

[0175] 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.

[0176] 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.

[0177] 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 linker. 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.

[0178] In some embodiments, the ligand can be attached to the 3' or 5' end of the sense 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 at 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 certain such embodiments, the ligand is attached at 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.

[0179] In certain embodiments, the ligand is connected to the sense strand or antisense strand by a joint." joint " refers to an atom or a group of atoms by which a ligand is covalently connected to a polynucleotide component of an 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 a compound of interest (such as the sense or antisense strand of an 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.

[0180] 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 10 Alkyl, substituted or unsubstituted C2-C 10 Alkenyl or substituted or unsubstituted C2-C 10 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.

[0181] 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.

[0182] 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.

[0183] 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.

[0184] 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.

[0185] In certain 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 multivalent GalNAc. In some embodiments, the multivalent GalNAc moiety is a trivalent GalNAc and is attached to the 3' end of the sense strand. In other embodiments, the multivalent GalNAc moiety is a trivalent GalNAc and is attached to the 5' end of the sense strand. In other embodiments, the multivalent GalNAc moiety is a tetravalent GalNAc moiety and is attached to the 3' end of the sense strand. In other embodiments, the multivalent GalNAc moiety is a tetravalent GalNAc moiety and is attached to the 5' end of the sense strand.

[0186] In some embodiments, the RNAi agent of the present invention comprises a ligand comprising a structure as shown in Formula I:

[0187] in,

[0188] X is -C(O)-NH-, -NH-C(O)-, -OCH2-CH2O-, -S- or -SS-;

[0189] A or C is independently 0 or an integer between 1 and 5;

[0190] B is 0 or an integer between 1 and 3;

[0191] The wavy lines represent the locations of attachment to the rest of the RNAi agent; wherein the ligand is attached to the 5' and / or 3' end of the sense strand and / or antisense strand; preferably, the ligand is attached to the 5' and / or 3' end of the sense strand; more preferably, the ligand is attached to the 3' end of the sense strand.

[0192] In a preferred embodiment, the ligand is covalently attached to the 5' and / or 3' end of the sense strand via a linker. In one embodiment, the linker is a PEG linker.

[0193] In a preferred embodiment, the structure of the ligand is as shown in formula (II) or formula (III):

[0194] in,

[0195] X is -C(O)-NH-, -NH-C(O)-, -OCH2-CH2O-, -S- or -SS-;

[0196] Y1 is -C(O)-NR1-, -NH-C(O)-, -OCH2-CH2O-, -SS-, Wherein R1 is an alkyl group containing 1 to 6 carbon atoms or hydrogen;

[0197] W is -NH-, -O- or The wavy line on the right represents the position of connection with the sense strand or antisense strand;

[0198] The wavy line connected to W in Formula II represents the position of attachment to the sense strand or antisense strand;

[0199] A, C or F is 0 or an integer between 1 and 5;

[0200] B or E is 0 or an integer between 1 and 3;

[0201] D is an integer between 1 and 6;

[0202] in,

[0203] X is -C(O)-NH-, -NH-C(O)-, -OCH2-CH2O-, -S- or -SS-;

[0204] Y2 is -(CH2)p-(O-CH2-CH2)q-(CH2)j-Z3-, wherein Z3 is O, NH or C(O), p is an integer from 1 to 3, q ​​is an integer from 3 to 10; j is 0 or 1;

[0205] W does not exist or is The wavy line on the right represents the position of connection with the sense strand or antisense strand;

[0206] The wavy line connected to W in Formula III represents the position of attachment to the sense strand or antisense strand;

[0207] A or C is 0 or an integer between 1 and 5;

[0208] B is 0 or an integer between 1 and 3.

[0209] Preferably, exemplary trivalent and / or tetravalent GalNAc ligands that can be linked to double-stranded RNA molecules in the RNAi agents of the present invention are provided in Structural Formula IV or V below:

[0210] Wherein, the wavy line represents the position of connection to the sense strand or antisense strand; preferably, the ligand is connected to the 5' and / or 3' end of the sense strand; more preferably, the ligand is connected to the 3' end of the sense strand; wherein, the targeting ligand is connected to the sense strand or antisense strand via a phosphate group, a thiophosphate group or a phosphonate group.

[0211] In some embodiments, the RNAi agent of the present invention further complexes a metal cation, preferably a monovalent metal cation, preferably Na + , K + 、Ag + ; divalent metal cation, preferably Ca 2+ Mg 2+ 、Cu 2+ 、Fe 2+ 、Zn 2+ 、Mn 2+ ; or a trivalent metal cation, preferably Fe 3+ 、Al 3+ In some embodiments, the metal cation is complexed with the nitrogen heterocyclic structure in any one of the structures shown in Formula II to Formula V.

[0212] For example, in one embodiment, the RNAi agent provided by the present invention has the structure shown below, wherein K+ is complexed to the nitrogen heterocyclic structure of the ligand, wherein represents a duplex portion of an RNAi agent for ANGPTL3 gene expression,

[0213] For ease of description, the ligand represented by formula (IV) is referred to herein as YHZY12001, and the ligand represented by formula (V) is referred to herein as YHZY12002. RNAi agents having a ligand represented by formula (I), (II), or (III) linked to the 5' and / or 3' end of the sense strand are referred to herein as MCPP-siRNA.

[0214] In some embodiments, the present invention provides an RNAi agent for inhibiting ANGPTL3 gene expression, wherein:

[0215] The sense strand comprises or is the sequence shown in SEQ ID NO:33, and the antisense strand comprises or is the sequence shown in SEQ ID NO:13;

[0216] The sense strand comprises or is the sequence shown in SEQ ID NO: 34, and the antisense strand comprises or is the sequence shown in SEQ ID NO: 13;

[0217] The sense strand comprises or is the sequence shown in SEQ ID NO: 38, and the antisense strand comprises or is the sequence shown in SEQ ID NO: 13;

[0218] The sense strand comprises or is the sequence shown in SEQ ID NO: 39, and the antisense strand comprises or is the sequence shown in SEQ ID NO: 13;

[0219] The sense strand comprises or is the sequence shown in SEQ ID NO:35, and the antisense strand comprises or is the sequence shown in SEQ ID NO:27;

[0220] The sense strand comprises or is the sequence shown in SEQ ID NO:36, and the antisense strand comprises or is the sequence shown in SEQ ID NO:27;

[0221] The sense strand comprises or is the sequence shown in SEQ ID NO:40, and the antisense strand comprises or is the sequence shown in SEQ ID NO:27;

[0222] The sense strand comprises or is the sequence shown in SEQ ID NO:41, and the antisense strand comprises or is the sequence shown in SEQ ID NO:27;

[0223] The sense strand comprises or is the sequence shown in SEQ ID NO:35, and the antisense strand comprises or is the sequence shown in SEQ ID NO:28;

[0224] The sense strand comprises or is the sequence shown in SEQ ID NO:36, and the antisense strand comprises or is the sequence shown in SEQ ID NO:28;

[0225] The sense strand comprises or is the sequence shown in SEQ ID NO:37, and the antisense strand comprises or is the sequence shown in SEQ ID NO:28;

[0226] The sense strand comprises or is the sequence shown in SEQ ID NO:40, and the antisense strand comprises or is the sequence shown in SEQ ID NO:28;

[0227] The sense strand comprises or is the sequence shown in SEQ ID NO:41, and the antisense strand comprises or is the sequence shown in SEQ ID NO:28; or

[0228] The sense strand comprises or is the sequence shown in SEQ ID NO:42, and the antisense strand comprises or is the sequence shown in SEQ ID NO:28.

[0229] In a particularly preferred embodiment, the present invention provides a double-stranded RNAi agent for inhibiting ANGPTL3 expression in a cell, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises SEQ ID NO. 19: 5'-mG*mA*mGmAfAmGfAfAfCmUmAmCmAmUmAmUmAmAmA-3' and has a length of no more than 21 nucleotides, and the antisense strand comprises SEQ ID NO. 27: 5'-mU*fU*mUmAfUmAfUmGmUmAmGmUmUfCmUfUmCmUmC*mA*mG-3' and has a length of no more than 23 nucleotides.

[0230] Wherein mA, mU, mG and mC are 2'-OMe A, 2'-OMe U, 2'-OMe G and 2'-OMe C respectively; fA, fU, fG and fC are 2'-FA, 2'-FU, 2'-FG and 2'-FC respectively; * is a phosphorothioate linkage;

[0231] The 3'-end of the sense chain is conjugated to a ligand targeting hepatocytes, preferably, the ligand comprises a galactose moiety, a galactosamine moiety or an N-acetylgalactosamine moiety, and further preferably, the ligand is a trivalent or tetravalent N-acetylgalactosamine moiety.

[0232] In the above particularly preferred embodiment, the ligand may be a structure shown in Formula IV:

[0233] It is linked to the sense strand via a phosphate group or a phosphorothioate group.

[0234] In a particularly preferred embodiment, the present invention provides a double-stranded RNAi agent for inhibiting ANGPTL3 expression in a cell, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises SEQ ID NO. 40: 5'-mG*mA*mGmAfAmGfAfAfCmUmAmCmAmUmAmUmAmAmA-YHZY12001-3' and has a length of no more than 21 nucleotides, and the antisense strand comprises SEQ ID NO. 27: 5'-mU*fU*mUmAfUmAfUmGmUmAmGmUmUfCmUfUmCmUmC*mA*mG-3' and has a length of no more than 23 nucleotides.

[0235] Wherein mA, mU, mG and mC are 2'-OMe A, 2'-OMe U, 2'-OMe G and 2'-OMe C respectively; fA, fU, fG and fC are 2'-FA, 2'-FU, 2'-FG and 2'-FC respectively; * is a phosphorothioate linkage.

[0236] Wherein YHZY12001 has the structure of Formula IV and is conjugated to the 3'-end of the sense strand as follows:

[0237] Pharmaceutical composition

[0238] 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 ANGPTL3 gene in patients in need thereof. With clinical applications in mind, pharmaceutical compositions and formulations will be prepared in a form suitable for the intended application. Typically, this will require preparing compositions that are substantially free of pyrogens and other impurities that may be harmful to humans or animals.

[0239] 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).

[0240] In some embodiments, the pharmaceutical composition comprises an effective amount of a 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 the expression of the ANGPTL3 gene in a particular tissue or cell type (e.g., liver or hepatocytes) of a patient. An effective amount of a 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.

[0241] 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 route. These routes include but are not limited to parenteral (e.g., subcutaneous, intramuscular, intraperitoneal or intravenous), oral, nasal, oral, intradermal, transdermal and sublingual routes, or by direct injection into liver tissue or delivered by 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.

[0242] 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 delivering nucleic acids of the present invention include (Baxter International Inc.), (Abbott Pharmaceuticals), II (Hospira), III (Hospire), Nutrilipid (B.Braun Medical Inc.) and other similar fat emulsions. The preferred colloidal system used as a delivery vehicle in vivo is a liposome (i.e., an artificial membrane vesicle). The RNAi agent of the present invention can be encapsulated in 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, glycol tetramethylaminopropyl (DOTAP) and glycol phosphatidylethanolamine (DOTMA).

[0243] 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.

[0244] 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.

[0245] 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.

[0246] 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.

[0247] 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.

[0248] Treatment methods and uses

[0249] The present invention provides a method for reducing or inhibiting ANGPTL3 gene expression in cells (e.g., hepatocytes) by contacting the cells with any of the RNAi agents described herein. The cells can be in vitro or in vivo. ANGPTL3 gene expression can be assessed by measuring the amount or level of ANGPTL3 mRNA or another biomarker associated with ANGPTL3 expression (e.g., serum ANGPTL3 protein, triglycerides, LDL-C, or HDL-C). Reduction of ANGPTL3 expression in cells or animals treated with the RNAi agents of the present invention can be determined relative to ANGPTL3 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 ANGPTL3 expression is assessed by (a) measuring the amount or level of ANGPTL3 mRNA in hepatocytes treated with a RNAi agent of the invention, (b) measuring the amount or level of ANGPTL3 mRNA in hepatocytes treated with a control RNAi agent (e.g., an RNAi agent directed against an RNA molecule not expressed in hepatocytes or an RNAi agent having a nonsense or scrambled sequence) or no RNAi agent, and (c) comparing the ANGPTL3 mRNA level measured in the treated cells in (a) with the ANGPTL3 mRNA level in the control cells in (b). Prior to comparison, the ANGPTL3 mRNA levels in the treated and control cells can be normalized to the RNA level of a control gene (e.g., 18S ribosomal RNA or a housekeeping gene). ANGPTL3 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.

[0250] In some embodiments, the method for assessing ANGPTL3 expression levels is performed in vitro in cells that naturally express the ANGPTL3 gene (e.g., hepatocytes) or cells that have been engineered to express ANGPTL3. In certain embodiments, the method is performed in hepatocytes in vitro. Suitable hepatocytes include, but are not limited to, primary hepatocytes (e.g., human or non-human primate hepatocytes), HepAD38 cells, HuH-6 cells, HuH-7 cells, HuH-5-2 cells, BNLCL2 cells, Hep3B cells, or HepG2 cells. In one embodiment, the hepatocytes are HuH-7 cells. In another embodiment, the hepatocytes are primary human hepatocytes.

[0251] In other embodiments, the method of assessing ANGPTL3 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 ANGPTL3 gene), and ANGPTL3 mRNA or serum ANGPTL3 protein levels can be assessed in liver tissue harvested from the animal after treatment. Alternatively or additionally, biomarkers or functional phenotypes associated with ANGPTL3 expression can be assessed in the treated animal. For example, ANGPTL3 protein is an expression product of ANGPTL3 mRNA in serum or plasma. Therefore, serum or plasma levels of ANGPTL3 protein can be measured in animals treated with the RNAi agents of the invention to assess the functional efficacy of reducing ANGPTL3 expression.

[0252] In certain embodiments, ANGPTL3 expression in hepatocytes is reduced by at least 40%, at least 45%, or at least 50% by a RNAi agent of the invention. In some embodiments, ANGPTL3 expression in hepatocytes 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 invention. In other embodiments, ANGPTL3 expression in hepatocytes is reduced by about 90% or more, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, by a RNAi agent of the invention. The percentage reduction in ANGPTL3 expression can be measured by any of the methods described herein, as well as other methods known in the art.

[0253] The present invention provides methods for reducing or inhibiting the expression of the ANGPTL3 gene, thereby reducing or inhibiting the production of ANGPTL3 protein, in a patient in need thereof, as well as methods for treating or preventing diseases or conditions associated with ANGPTL3 expression or activity. A "disease or condition associated with ANGPTL3 expression" refers to a disease or condition in which ANGPTL3 expression levels are altered, or a condition in which elevated ANGPTL3 expression levels are associated with an increased risk of developing the disease or condition. Diseases or conditions associated with ANGPTL3 expression may also include those caused by abnormal changes in lipoprotein metabolism, such as changes that result in abnormal or elevated levels of ANGPTL3 protein, cholesterol, lipids, triglycerides, etc., or impaired clearance of these molecules. In certain embodiments, the RNAi agents of the present invention are particularly useful for treating or preventing metabolic diseases and reducing circulating levels of ANGPTL3.

[0254] Diseases and conditions associated with ANGPTL3 expression that can be treated or prevented according to the methods of the present invention include, but are not limited to, metabolic diseases, such as hypertriglyceridemia, obesity, hyperlipidemia, abnormal lipid and / or cholesterol metabolism, atherosclerosis, type II diabetes, cardiovascular disease, coronary artery disease, nonalcoholic steatohepatitis, nonalcoholic fatty liver disease, homozygous or heterozygous familial hypercholesterolemia, and statin-resistant hypercholesterolemia.

[0255] In certain embodiments, the present invention provides a method for reducing ANGPTL3 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 level of ANGPTL3 expression in the patient's hepatocytes is reduced compared to the level of ANGPTL3 expression in the patient not receiving the RNAi agent, or compared to the level of ANGPTL3 expression in the patient before administration of the RNAi agent. In some embodiments, after administration of the RNAi agent of the invention, ANGPTL3 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 percentage reduction in ANGPTL3 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 ANGPTL3 expression is determined by assessing ANGPTL3 protein levels in the patient's serum or plasma according to the methods described herein.

[0256] In some embodiments, following administration of the RNAi agent, the patient's serum or plasma triglyceride (TG) levels are reduced by at least about 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% compared to ANGPTL3 expression levels in patients who did not receive the RNAi agent, or compared to ANGPTL3 expression levels in the patient before administration of the RNAi agent.

[0257] In some embodiments, following administration of the RNAi agent, the patient's total cholesterol level is reduced by at least about 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% compared to ANGPTL3 expression levels in a patient who did not receive the RNAi agent, or compared to ANGPTL3 expression levels in the patient before administration of the RNAi agent.

[0258] In some embodiments, following administration of the RNAi agent, the patient's low-density lipoprotein cholesterol (LDL-c) level is reduced by at least about 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% compared to ANGPTL3 expression levels in a patient who did not receive the RNAi agent, or compared to ANGPTL3 expression levels in the patient before administration of the RNAi agent.

[0259] In certain embodiments, the patient in need of reduced ANGPTL3 expression is a patient diagnosed with or at risk for a metabolic disease. Thus, the present invention includes a method of treating or preventing a metabolic disease 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 metabolic disease in a patient in need thereof. In other embodiments, the present invention provides an ANGPTL3-targeting RNAi agent for use in a method of treating or preventing a metabolic disease in a patient in need thereof. Metabolic diseases include, but are not limited to, hypertriglyceridemia, obesity, hyperlipidemia, abnormal lipid and / or cholesterol metabolism, atherosclerosis, type II diabetes, cardiovascular disease, coronary artery disease, nonalcoholic steatohepatitis, nonalcoholic fatty liver disease, homozygous or heterozygous familial hypercholesterolemia, and statin-resistant hypercholesterolemia. In some embodiments, the disease to be treated or prevented according to the methods of the present invention is hypertriglyceridemia. In other embodiments, the disease to be treated or prevented according to the methods of the present invention is obesity. In other embodiments, the disease to be treated or prevented according to the methods of the present invention is hyperlipidemia. In other embodiments, the disease to be treated or prevented according to the methods of the present invention is abnormal lipid metabolism. In other embodiments, the disease to be treated or prevented according to the methods of the present invention is abnormal cholesterol metabolism. In other embodiments, the disease to be treated or prevented according to the methods of the present invention is abnormal lipid and cholesterol metabolism. In other embodiments, the disease to be treated or prevented according to the methods of the present invention is atherosclerosis. In other embodiments, the disease to be treated or prevented according to the methods of the present invention is type II diabetes. In other embodiments, the disease to be treated or prevented according to the methods of the present invention is cardiovascular disease. In other embodiments, the disease to be treated or prevented according to the methods of the present invention is coronary artery disease. In other embodiments, the disease to be treated or prevented according to the methods of the present invention is non-alcoholic steatohepatitis. In other embodiments, the disease to be treated or prevented according to the methods of the present invention is non-alcoholic fatty liver disease. In other embodiments, the disease to be treated or prevented according to the methods of the present invention is homozygous or heterozygous familial hypercholesterolemia. In other embodiments, the disease to be treated or prevented according to the methods of the present invention is statin-resistant hypercholesterolemia.

[0260] In certain embodiments, administration of the RNAi agents described herein reduces the risk of non-fatal myocardial infarction, fatal and non-fatal stroke, certain types of cardiac surgery (e.g., angioplasty, bypass surgery), hospitalization for heart failure, chest pain in patients with heart disease, and / or cardiovascular events in patients with established heart disease (e.g., previous myocardial infarction, previous cardiac surgery, and / or chest pain with evidence of arterial blockage). In some embodiments, administration of the RNAi agents described herein according to the methods of the invention can be used to reduce the risk of recurrent cardiovascular events.

[0261] In certain other embodiments, the patient in need of reduced ANGPTL3 expression is a patient with elevated circulating ANGPTL3 levels. Thus, in some embodiments, the present invention provides a method of reducing serum or plasma levels of an ANGPTL3 protein 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 serum or plasma levels of an ANGPTL3 protein in a patient in need thereof. In other embodiments, the present invention provides an ANGPTL3-targeting RNAi agent for use in a method of reducing serum or plasma levels of an ANGPTL3 protein in a patient in need thereof.

[0262] Sequence Listing

[0263] Table 1. ANGPTL3 siRNA without nucleotide modification

[0264] Table 2. Nucleotide-modified ANGPTL3 siRNA

[0265] Table 3. GalNAc-conjugated ANGPTL3 siRNA

[0266] Table 4. Exemplary ANGPTL3 siRNA duplexes (without ligand)

[0267] Where m represents 2'-O-methyl modification, f represents 2'-fluoro modification, and * represents phosphorothioate modification;

[0268] Example

[0269] Experimental methods and materials

[0270] Target sequence screening

[0271] siRNAs were designed based on the full-length ANGPTL3 mRNA sequence (NM_014495.4). All sequences were obtained from the NCBI gene database. All siRNAs were designed to ensure homology to human (Gene ID: 27329, NM_014495.4) and cynomolgus macaque (Gene ID: 102136264, XM_005543185.3) sequences.

[0272] GalNAc resin synthesis

[0273] GalNAc is a terminally reactive compound. Examples include Gal-C7, Gal-C6, Gal-C5, Gal-C4, and Gal-C3, which react with carboxylic acids, and Gal-C7-NH2, Gal-C6-NH2, Gal-C5-NH2, Gal-C4-NH2, and Gal-C3-NH2, which react with amino groups. These compounds were synthesized according to reported methods, and detailed information is provided in Table 5.

[0274] Table 5: Chemical structures, nomenclature, and synthesis methods of compounds containing terminal reactive groups (GalNAc)

[0275] Synthesis of key intermediate compound A

[0276] Synthesis of compound A-2:

[0277] Compound A-1 (3.0 g, 5.24 mmol, 1.0 equiv.) was dissolved in DMF (20 mL), and HBTU (3.9 g, 10.5 mmol, 2.0 equiv.) and DIPEA (1.73 mL, 2.0 equiv.) were added sequentially. The mixture was stirred at room temperature for 5 minutes. 1,9-Diaminononane (993 mg, 6.28 mmol) dissolved in DMF (10 mL) was then added, and the mixture was stirred at room temperature for 5 hours. After the reaction was complete, the reaction solution was quenched with water (30 mL) and extracted with ethyl acetate (20 mL). The organic phase was washed sequentially with 5% sodium bicarbonate solution (20 mL) and saturated brine (10 mL), dried over anhydrous sodium sulfate, and concentrated to give the crude product A-2 (3.65 g). The crude product was used directly in the next step. LCMS: (ESI) m / z = 714 [M+H] + .

[0278] Synthesis of compound A-3:

[0279] The crude product A-2 (3.65 g, 5.13 mmol) obtained in the previous step was dissolved in THF (10 mL), and Cbz-OSu (1.28 g, 5.13 mmol) and saturated sodium bicarbonate solution (10 mL) were added sequentially. The mixture was stirred at room temperature for 2 hours. After the reaction was completed, the reaction mixture was diluted with water (20 mL) and extracted with ethyl acetate (3×20 mL). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was separated and purified by preparative chromatography to obtain A-3 (1.1 g, yield: 25.6%) as a white solid. LCMS: (ESI) m / z = 847.5 [M+H] + .

[0280] HPLC purification conditions: Column: Xbridge C18, 19×250mm, 10μm, Mobile phase: A: water (containing 0.05% TFA), B: acetonitrile (containing 0.05% TFA); gradient: from 5% B to 95% B in 20 minutes.

[0281] Synthesis of compound A:

[0282] Intermediate A-3 (1.1 g, 1.30 mmol) was dissolved in DCM (5 mL) and cooled to -10°C using a cryogenic reactor. A mixture of TFA and DCM (15 mL, v:v = 1:1) was slowly added. The reaction was stirred at -10°C for 15 hours. LCMS confirmed the reaction was complete. Cold methyl tert-butyl ether (40 mL) was added, resulting in a white turbidity. The mixture was centrifuged (3200 rpm) for 3 minutes, and the supernatant was discarded. Methyl tert-butyl ether (40 mL) was added to the solid residue, and the mixture was sonicated for 3 minutes. The reaction was centrifuged (3200 rpm) for 3 minutes, and the supernatant was discarded. The solid residue was dried to afford A (660 mg, yield: 74.9%) as a white solid. LCMS: (ESI) m / z = 679 [M+H] + .

[0283] Synthesis of Class IA molecules

[0284] 3.1 Synthesis of I-A10-5FAM

[0285] Synthesis of intermediate I-A10-A

[0286] 1-tert-Butyloxycarbonyl-ethylenediamine (71 mg, 0.442 mmol, 5.0 equiv.) and Intermediate A (60 mg, 0.0884 mmol) were dissolved in DMF (3 mL). EDCI (68 mg, 0.354 mmol), HOAt (48 mg, 0.354 mmol), and DIPEA (151 μl, 0.884 mmol) were added sequentially. The reaction mixture was stirred at room temperature overnight. The reaction mixture was directly purified by preparative chromatography to obtain I-A10-A as a white solid (68 mg, yield: 68.0%). LCMS: (ESI) m / z = 1127.7 [M+Na] + ;1105.7[M+H] + ; 553.5[M+2H] / 2 + .

[0287] HPLC purification conditions: Column: Welch Topsil C18, 21.2×250mm, 5μm, Mobile phase: A: water (containing 0.05% TFA), B: acetonitrile (containing 0.05% TFA); gradient: from 20% B to 70% B in 20 minutes.

[0288] Synthesis of intermediate I-A10-B

[0289] I-A10-A (68 mg, 0.0435 mmol, 1.0 equiv.) was dissolved in DCM (3 mL), cooled to -5°C in an ice-salt bath, and TFA (3 mL) was slowly added dropwise. The mixture was stirred for 2 hours. The reaction solution was concentrated to dryness to afford the trifluoroacetate salt of intermediate I-A10-B as a colorless, transparent liquid (80 mg, yield >100%). LCMS: (ESI) m / z = 805.7 [M+H] + ; 403.5[M+2H] / 2 + .

[0290] Synthesis of intermediate I-A10-C

[0291] Intermediate I-A10-B (80 mg, 0.0435 mmol) and Gal-C4 (94 mg, 0.218 mmol) were dissolved in DMF (2 mL), and HOAt (30 mg, 0.218 mmol), EDCI (42 mg, 0.218 mmol) and DIPEA (74 μL, 0.435 mmol) were added in sequence. The reaction solution was stirred at room temperature overnight. After the reaction monitoring was substantially completed, the reaction solution was directly purified by preparative chromatography to obtain I-A10-C as a white solid (56 mg, yield: 62.7%). LCMS: (ESI) m / z = 1026.6 [M+2H] / 2 +; 684.5[M+3H] / 3 + .

[0292] HPLC purification conditions: Column: Xbridge C18 21.2mm×250mm, 10μm, Mobile phase: A: water (containing 10 mmol / L NH4HCO3), B: acetonitrile; gradient: from 38% B to 48% B in 20 minutes.

[0293] Synthesis of intermediate I-A10-E

[0294] I-A10-C (25 mg, 0.0122 mmol) was dissolved in methanol (3 mL), and potassium carbonate (10 mg) and Pd / C (10% w / w adsorbed on activated carbon, 5 mg) were added sequentially. The mixture was stirred under hydrogen for 2 hours, and the reaction was monitored by LC-MS. The reaction solution was filtered, and approximately 2 g of dry ice was added to the filtrate, stirred for 5 minutes, and concentrated to obtain the crude product I-A10-E, which was used directly in the next reaction. LCMS: (ESI) m / z = 770.2 [M+2H] / 2 + ; 668.5[M+2H-204] / 2 + .

[0295] Synthesis of compound I-A10-5FAM

[0296] The crude product I-A10-E (0.0122 mmol) obtained in the previous step was dissolved in water (2 mL) and acetonitrile (0.5 mL). 5-FAM-OSu (8 mg, 0.0165 mmol) and saturated sodium bicarbonate solution (0.5 mL) were added sequentially, and the reaction was stirred at room temperature for 1 hour. The reaction solution was directly purified by preparative chromatography to obtain I-A10-5FAM (10.0 mg, two-step yield: 43.3%) as an orange solid. LCMS: (ESI) m / z = 967.8 [M+H+K] / 2 + ; 949.3[M+2H] / 2 + ;645.8[M+2H+K] / 3 + ; 633.3[M+3H] / 3 + .

[0297] HPLC purification conditions: Column: Gemini C18 21.2mm×250mm, 10μm, Mobile phase: A: water (containing 10 mmol / L NH4HCO3), B: acetonitrile; gradient: from 14% B to 24% B in 20 minutes.

[0298] HPLC: 91.1578% (214nm), RT=13.049min; 7.2549% (214nm), RT=13.406min.

[0299] Mobile phase: A: water (0.05% TFA), B: acetonitrile (0.05% TFA)

[0300] Gradient: 5% B equilibration for 3 min, increasing to 65% B in 20 min, increasing to 95% B in 5 min.

[0301] Flow rate: 1.0 mL / min.

[0302] Chromatographic column: XBridge peptide BEH column C18, 4.6×150mm, 3.5μm,

[0303] Column temperature: 60℃.

[0304] 3.2 Synthesis of I-A11-5FAM

[0305] Synthesis of intermediate I-A11-A

[0306] Following the method for synthesizing intermediate I-A10-A, 60 mg of A (0.0884 mmol) was reacted with N-tert-butyloxycarbonyl-propylenediamine (73 mg, 0.442 mmol) in DMF (3 mL). After purification, I-A11-A (70 mg, yield: 68.0%) was obtained as a white solid. LCMS: (ESI) m / z = 1147.8 [M+H] + .

[0307] Synthesis of intermediate I-A11-B

[0308] Following the method for synthesizing intermediate I-A10-B, I-A11-B (80 mg, yield: quantitative) was obtained from 70 mg of I-A11-A (0.0436 mmol) as a colorless oily liquid. LCMS: (ESI) m / z = 847.7 [M+H] + .

[0309] Synthesis of intermediate I-A11-C

[0310] Following the method for synthesizing intermediate I-A10-C, I-A11-C (32 mg, yield: 35.1%) was obtained from the crude product I-A11-B in the previous step as a white solid. LCMS: (ESI) m / z = 1047.7 [M+2H] / 2 + .

[0311] HPLCC purification conditions: Column: Gemini C18 21.2mm×250mm, 10μm, Mobile phase: A: water (containing 10 mmol / L NH4HCO3), B: acetonitrile; gradient: from 41% B to 51% B in 20 minutes.

[0312] Synthesis of intermediate I-A11-E

[0313] According to the method for synthesizing intermediate I-A10-E, I-A11-e (40 mg, quantitative) was obtained from 32 mg of I-A11-C (0.0156 mmol) as a white solid. LCMS: (ESI) m / z = 791.0 [M+2H] / 2 + .

[0314] Synthesis of compound I-A11-5FAM

[0315] According to the method for synthesizing I-A10-5FAM, I-A11-5FAM (6.3 mg, yield: 20.8%) was obtained from 40 mg of I-A11-C as an orange solid. LCMS: (ESI) m / z = 989.2 [M+H+K] / 2 + .

[0316] HPLC purification conditions: Column: Gemini C18 21.2mm×250mm, 10μm, Mobile phase: A: water (containing 10 mmol / L NH4HCO3), B: acetonitrile; gradient: from 13% B to 23% B in 20 minutes.

[0317] HPLC purity: 60.0611% (214 nm), RT = 12.894 min; 37.2783% (214 nm), RT = 13.256 min.

[0318] The analytical conditions were the same as those for I-A10-5FAM.

[0319] Synthesis of I-A12-5FAM

[0320] Synthesis of intermediate I-A12-A

[0321] Following the method for synthesizing intermediate I-A10-A, 60 mg of A (0.0884 mmol) was reacted with N-tert-butyloxycarbonyl-butanediamine (75 mg, 0.396 mmol) in DMF (2 mL). After purification, I-A12-A (73 mg, yield: 69.5%) was obtained as a white solid. LCMS: (ESI) m / z = 595.5 [M+2H] / 2+ .

[0322] HPLC purification conditions: Column: Xbridge C18, 19×250mm, 10μm, Mobile phase: A: water (containing 0.05% TFA), B: acetonitrile (containing 0.05% TFA); gradient: from 20% B to 80% B in 20 minutes.

[0323] Synthesis of intermediate I-A12-B

[0324] I-A12-A (73 mg, 0.061 mmol) was dissolved in DCM (2 mL) and TFA (2 mL) was slowly added dropwise. The mixture was allowed to react at room temperature for 1 hour. The reaction solution was concentrated to dryness to obtain I-A12-B (53 mg, yield: quantitative) as a colorless oil. LCMS: (ESI) m / z = 911.7 [M+Na] + ; 445.4[M+2H] / 2 + .

[0325] Synthesis of intermediate I-A12-C

[0326] Gal-C4 (1.2 g, 2.77 mmol, 5.0 eq.) was dissolved in DMF (20 mL), and I-A12-B (900 mg, 0.553 mmol), EDCI (530 mg, 2.77 mmol), HOAt (376 mg, 2.77 mmol), and DIPEA (944 μl, 5.53 mmol) were added sequentially. The mixture was stirred at room temperature for 3 hours. LCMS confirmed the reaction was complete, and the reaction solution was directly purified by preparative chromatography to obtain I-A12-C (900 mg, yield: 76%). LCMS: (ESI) m / z = 1068.8 [M+2H] / 2 + ;903.8[M+2H-330] / 2;712.8[M+3H] / 3 + . 1H NMR (400MHz, DMSO) δ8.07-7.71(m,10H,10NH),7.39-7.19(m,6H,Bn-5H+1NH),5.20(d,J=3.2Hz,3H,3Gal-OCHO),5.00(s,2H,Ph- CH2),4.96(dd,J=3.2,11.2Hz,3H,3Gal-OCHC),4.48(d,J=8.4Hz,3H,3Gal-OCHC),4.02(brs,9H),3.92-3.80(m,3H,3Gal-OCHC) ,3.70-3.63(m,3H,3Gal-OCHC),3.47-3.39(m,3H,3Gal-OCHC),3.131-2.90(m,26H),2.72-2.52(m,14H),2.16-2.03(m,6H),2.1 0(s,9H,3CH3),1.99(s,9H,3CH3),1.89(s,9H,3CH3),1.76(s,9H,3CH3),1.74-1.61(m,6H),1.47-1.30(m,16H),1.23(brs,10H).

[0327] HPLC purification conditions: Column: Xbridge C18, 19×250mm, 10μm, Mobile phase: A: water (containing 10 mmol / L NH4HCO3), B: acetonitrile; gradient: from 39% B to 49% B in 10 minutes.

[0328] Synthesis of intermediate I-A12-E

[0329] According to the method for synthesizing intermediate I-A10-E, I-A12-E (33 mg, quantitative) was obtained from 30 mg of I-A12-C (0.0140 mmol) as a white solid. LCMS: (ESI) m / z = 812 [M+2H] / 2 + .

[0330] Synthesis of compound I-A12-5FAM

[0331] According to the method for synthesizing I-A10-5FAM, I-A12-5FAM (8.5 mg, yield: 21.5%) was obtained as an orange solid from 33 mg of I-A12-E (0.020 mmol). LCMS: (ESI) m / z = 991.0 [M+H+K] / 2 + ;674.0[M+2H+K] / 3 + .

[0332] HPLC purification conditions: Column: Gemini C18 21.2mm×250mm, 10μm, Mobile phase: A: water (containing 10 mmol / L NH4HCO3), B: acetonitrile; gradient: from 5% B to 35% B in 10 minutes.

[0333] HPLC purity: 9.5195% (214 nm), RT = 13.588 min; 87.8220% (214 nm), RT = 13.008 min.

[0334] The analytical conditions were the same as those for I-A10-5FAM.

[0335] 3.4 Synthesis of I-A12-Cy5

[0336] According to the method for synthesizing I-A12-5FAM, I-A12-Cy5 (8.0 mg, yield: 26.5%) was obtained from 30 mg of I-A12-E (0.020 mmol) as an orange solid. LCMS: (ESI) m / z = 1044.5 [M+2H] / 2 + ,709.5[M+3H+K] / 3 + ,696.8[M+3H] / 3 + .

[0337] HPLC purification conditions: Column: Gemini C18 21.2mm×250mm, 10μm, Mobile phase: A: water (containing 10 mmol / L NH4HCO3), B: acetonitrile; gradient: from 5% B to 35% B in 10 minutes.

[0338] HPLC purity: 84.9% (214 nm), RT = 17.535 min; 12.1% (214 nm), RT = 17.945 min.

[0339] 3.5 Synthesis of Compound I-A12-Ac-5FAM

[0340] I-A12-C (40 mg, 0.0187 mmol) was dissolved in methanol (5 mL) and Pd / C (10% w / w adsorbed on activated carbon, 8 mg) was added. The mixture was stirred under hydrogen for 13 hours and monitored by LC-MS. The reaction solution was filtered, concentrated, and purified to afford 5 mg of I-A12-Ac (yield: 13.3%), which was used directly in the next reaction. LCMS: (ESI) m / z = 1001.7 [M+2H] / 2 + ;680.7[M+2H+K] / 3+ .

[0341] I-A12-Ac (5 mg, 0.00250 mmol) obtained in the previous step was dissolved in water (2 mL) and acetonitrile (0.5 mL). 5-FAM-OSu (1.2 mg, 0.0165 mmol) and saturated sodium bicarbonate solution (0.5 mL) were added sequentially, and the reaction was stirred at room temperature for 1 hour. The reaction solution was directly purified by preparative chromatography to obtain I-A12-Ac-5FAM (3.5 mg, yield: 59.3%) as an orange solid. LCMS: (ESI) m / z = 800.4 [M+2H+K] / 3 + ;787.5[M+3H] / 3 + .

[0342] HPLC purification conditions: HPLC preparation column: Gemini C18 21.2mm×250mm, 10μm, Mobile phase: A: water (containing 10 mmol / L NH4HCO3), B: acetonitrile; gradient: from 35% B to 55% B in 20 minutes.

[0343] HPLC purity: 75.08% (214 nm), RT = 17.237 min; 18.82% (214 nm), RT = 17.738 min.

[0344] The analytical conditions were the same as those for I-A10-5FAM.

[0345] 3.6 Synthesis of I-A13-5FAM

[0346] Synthesis of intermediate I-A13-A

[0347] Following the method for synthesizing intermediate I-A10-A, 60 mg of A (0.0884 mmol) was reacted with N-tert-butyloxycarbonyl-pentanediamine (89 mg, 0.442 mmol) in DMF (2 mL) at room temperature for 2 hours. The reaction solution was directly purified to obtain I-A13-A (72 mg, yield: 66.7%) as a white solid. LCMS: (ESI) m / z = 1231.7 [M+H] + ; 616.3[M+2H] / 2 + .

[0348] HPLC purification conditions: HPLC preparative column: Xbridge C18, 19×250 mm, 10 μm, Mobile phase: A: water (containing 10 mmol / L NH4HCO3), B: acetonitrile; gradient: from 33% B to 43% B in 20 minutes.

[0349] Synthesis of intermediate I-A13-B

[0350] Following the method for synthesizing intermediate I-A12-B, I-A13-B (65 mg, yield: quantitative) was obtained from 72 mg of I-A13-A (0.058 mmol) as a colorless oily liquid. LCMS: (ESI) m / z = 953.8 [M+Na] + ;931.7[M+H] + ; 466.5[M+2H] / 2 + .

[0351] Synthesis of intermediate I-A13-C

[0352] Following the method for synthesizing intermediate I-A10-C, crude product I-A13-B from the previous step was reacted to obtain I-A13-C (20 mg, yield: 13.4%) as a white solid. LCMS: (ESI) m / z = 1089.4 [M+2H] / 2 + ;924[M+2H-330] / 2 + .

[0353] HPLC purification conditions: HPLC preparative column: Xbridge C18, 19×250 mm, 10 μm, Mobile phase: A: water (containing 10 mmol / L NH4HCO3), B: acetonitrile; gradient: from 43% B to 53% B in 10 minutes.

[0354] Synthesis of intermediate I-A13-E

[0355] According to the method for synthesizing intermediate I-A10-E, I-A13-E (20 mg, quantitative) was obtained from 20 mg of I-A13-C (0.0090 mmol) as a white solid. LCMS: (ESI) m / z = 833.0 [M+2H] / 2 + ;731.4[M+2H-204] / 2 + .

[0356] Synthesis of intermediate I-A13-5FAM

[0357] According to the method for synthesizing I-A10-5FAM, I-A13-5FAM (10.0 mg, yield: 29.4%) was obtained as an orange solid from 20 mg of I-A13-E (0.0090 mmol). LCMS: (ESI) m / z = 1031 [M+H+K] / 2 + ;1012.7[M+2H] / 2 + ;687.0[M+2H+K] / 3 + ; 675.3[M+3H] / 3 + .

[0358] HPLC purification conditions: HPLC preparation column: Gemini C18 21.2mm×250mm, 10μm, Mobile phase: A: water (containing 10 mmol / L NH4HCO3), B: acetonitrile; gradient: from 5% B to 35% B in 10 minutes.

[0359] HPLC purity: 24.2020% (214 nm), RT = 14.195 min; 71.6996% (214 nm), RT = 13.440 min.

[0360] The analytical conditions were the same as those for I-A10-5FAM.

[0361] Synthesis of class IB molecules

[0362] 4.1 Synthesis of I-B11-5FAM

[0363] Synthesis of intermediate A-4

[0364] 5-Hydroxypentylamine (5.0 g, 23.9 mmol) was dissolved in DMF (50 mL), and N-benzyloxycarbonyl-glycine (2.96 g, 28.7 mmol), HOBt (4.84 g, 35.9 mmol), EDCI (6.9 g, 35.9 mmol), and DIPEA (14.7 mL, 81.1 mmol) were added sequentially. The mixture was stirred at room temperature for 3 hours. The reaction solution was quenched with 50 mL of water and extracted with ethyl acetate (60 mL). The organic phase was washed with 5% NaHCO₃ (40 mL), dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by reverse phase chromatography to give compound A-4 (6.3 g, yield: 90%) as a white solid. LCMS: (ESI) m / z = 295.1 [M+H] + .

[0365] Purification conditions: Chromatographic column: 120 g C18 column; Mobile phase: A: water (containing 10 mmol / L NH4HCO3), B: acetonitrile; Gradient: from 5% B to 95% B in 20 minutes.

[0366] Synthesis of intermediate A-5

[0367] Add the activated Molecular sieves (3.0 g), freshly prepared GalNAc-1 (2.0 g, 6.079 mmol), and intermediate A-4 (1.96 g, 6.687 mmol) were added and the atmosphere was replaced with nitrogen three times. Anhydrous 1,2-dichloroethane (20 mL) was added and the mixture was stirred at room temperature for 10 minutes. TMSOTf (110 μl, 0.608 mmol) was added and the reaction was stirred at room temperature for 3 hours. The reaction mixture was filtered to remove the molecular sieves, and the filtrate was quenched with saturated aqueous NaHCO₃ (15 mL). The organic phase was separated and the aqueous phase was extracted twice with DCM (20 mL). The combined organic phases were dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by reverse phase chromatography to afford intermediate A-5 (400 mg, yield: 10.5%) as a white solid. LCMS: (ESI) m / z = 624.1 [M+H] + .

[0368] Purification conditions: Chromatographic column: 120 g C18 column; Mobile phase: A: water (containing 10 mmol / L NH4HCO3), B: acetonitrile; Gradient: from 20% B to 60% B in 20 minutes.

[0369] Synthesis of intermediate A-6

[0370] A 50 mL round-bottom flask was charged with intermediate A-5 (400 mg, 0.642 mmol) and Pd / C (10%, 40 mg). The reaction system was purged with hydrogen twice, and THF (5 mL) was added. The mixture was stirred at room temperature under hydrogen for 90 minutes. The reaction mixture was filtered and concentrated to give the crude product A-6 (320 mg) as a light black oily liquid, which was used directly in the next reaction. LCMS: (ESI) m / z = 490.1 [M+H] + .

[0371] Synthesis of intermediate I-B11-A

[0372] Compound A (50 mg, 0.074 mmol) was dissolved in DMF (2 mL), and HOBt (35 mg, 0.258 mmol), EDCI (45 mg, 0.258 mmol), DIPEA (88 μl, 0.516 mmol), and intermediate A-6 (126 mg, 0.258 mmol) were added sequentially. The mixture was stirred at room temperature for 3 hours. The reaction solution was directly purified by reverse phase preparative chromatography to obtain I-B11-A (50 mg, yield: 32.3%) as a white solid. LCMS: (ESI) m / z = 1047.0 [M+2H] / 2 + .

[0373] Preparation conditions: HPLC preparation column: Xbridge C18, 19×250mm, 10μm, Mobile phase: A: water (containing 10 mmol / L NH4HCO3), B: acetonitrile; gradient: from 40% B to 50% B in 10 minutes.

[0374] Synthesis of intermediate I-B11-C

[0375] I-B11-A (50 mg, 0.024 mmol), potassium carbonate (10 mg, 0.150 mmol), and Pd / C (10%, 12 mg) were added to a 50 mL round-bottom flask, followed by methanol (5 mL). The mixture was stirred under hydrogen for 3 hours. The reaction mixture was filtered and concentrated to afford I-B11-C (40 mg, crude product) as a white solid. LCMS: (ESI) m / z = 791.0 [M+2H] / 2 + .

[0376] Synthesis of compound I-B11-5FAM

[0377] The crude product I-B11-C (40 mg, 0.024 mmol) obtained in the previous step was dissolved in a mixture of water (2 mL) and acetonitrile (0.5 mL). 5FAM-OSu (14 mg, 0.030 mmol) and saturated NaHCO3 solution (0.5 mL) were added sequentially. The mixture was stirred at room temperature for 60 minutes. The reaction solution was directly separated and purified by preparative chromatography to obtain I-B11-5FAM (9.5 mg, yield: 19.8%) as an orange solid. LCMS: (ESI) m / z = 989 [M+H+K] / 2 + .

[0378] Preparation conditions: HPLC preparation column: Gemini C18 21.2mm×250mm, 10μm, Mobile phase: A: water (containing 10 mmol / L NH4HCO3), B: acetonitrile; gradient: from 17% B to 27% B in 20 minutes.

[0379] HPLC purity: 9.5962% (214 nm), RT = 13.531 min; 88.8773% (214 nm), RT = 13.301 min.

[0380] The analytical conditions were the same as those for I-A10-5FAM.

[0381] 4.2 Synthesis of I-B12-5FAM

[0382] Synthesis of intermediate A-7

[0383] 5-Hydroxypentylamine (5.0 g, 22.4 mmol) was dissolved in DMF (50 mL), and N-benzyloxycarbonyl-β-alanine (2.77 g, 26.9 mmol), HOBt (4.54 g, 33.6 mmol), EDCI (6.46 g, 33.6 mmol), and DIPEA (18.3 mL, 101 mmol) were added in sequence. The mixture was stirred at room temperature for 3 hours. The reaction solution was quenched with 50 mL of water and extracted with ethyl acetate (60 mL). The organic phase was washed with 5% NaHCO3 (40 mL), dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by reverse phase chromatography to give compound A-7 (7.0 g, yield: 92%) as a white solid. LCMS: (ESI) m / z = 309.1 [M+H] + .

[0384] Purification conditions: Chromatographic column: 120 g C18 column; Mobile phase: A: water (containing 10 mmol / L NH4HCO3), B: acetonitrile; Gradient: from 5% B to 95% B in 20 minutes.

[0385] Synthesis of intermediate A-8

[0386] Add the activated Molecular sieves (3.0 g) and DCM (20 mL) were added to freshly prepared GalNAc-1 (2.0 g, 6.08 mmol) and intermediate A-7 (2.0 g, 6.69 mmol), and stirred at room temperature for 10 minutes. TMSOTf (110 μl, 0.608 mmol) was added, and the reaction was stirred at room temperature for 3 hours. The reaction solution was filtered to remove the molecular sieves, and the filtrate was quenched with saturated aqueous NaHCO3 (15 mL). The organic phase was separated, and the aqueous phase was extracted twice with DCM (20 mL). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by reverse phase chromatography to obtain intermediate A-8 (2.0 g, yield: 51.7%) as a white solid. LCMS: (ESI) m / z = 638.2 [M+H] + .

[0387] Purification conditions: Chromatographic column: 120 g C18 column; Mobile phase: A: water (containing 10 mmol / L NH4HCO3), B: acetonitrile; Gradient: from 5% B to 65% B in 20 minutes.

[0388] Synthesis of intermediate A-9

[0389] A 50 mL round-bottom flask was charged with intermediate A-8 (400 mg, 0.628 mmol) and Pd / C (10%, 40 mg). The reaction system was purged with hydrogen twice, and THF (5 mL) was added. The mixture was stirred at room temperature under hydrogen for 90 minutes. The reaction mixture was filtered and concentrated to give the crude product A-9 (320 mg) as a light black oily liquid, which was used directly in the next reaction. LCMS: (ESI) m / z = 504.1 [M+H] + .

[0390] Synthesis of intermediate I-B12-A

[0391] Compound A (45 mg, 0.066 mmol) was dissolved in DMF (2 mL), and HOBt (31 mg, 0.232 mmol), EDCI (45 mg, 0.232 mmol), DIPEA (79 μl, 0.464 mmol), and intermediate A-9 (117 mg, 0.232 mmol) were added sequentially. The mixture was stirred at room temperature for 3 hours. The reaction solution was directly purified by reverse phase preparative chromatography to obtain I-B12-A (72 mg, yield: 51.1%) as a white solid. LCMS: (ESI) m / z = 1068.4 [M+2H] / 2 + .

[0392] Preparation conditions: HPLC preparation column: Xbridge C18, 19×250mm, 10μm, Mobile phase: A: water (containing 10 mmol / L NH4HCO3), B: acetonitrile; gradient: from 39% B to 49% B in 10 minutes.

[0393] Synthesis of intermediate I-B12-C

[0394] I-B12-A (72 mg, 0.034 mmol), potassium carbonate (28 mg, 0.202 mmol), and Pd / C (10%, 5 mg) were added to a 50 mL round-bottom flask, followed by methanol (2 mL). The mixture was stirred under hydrogen for 3 hours. The reaction mixture was filtered and concentrated to afford I-B11-C (60 mg, crude product) as a white solid. LCMS: (ESI) m / z = 812.1 [M+2H] / 2 + ;711.0[M+2H] / 2 + ; 541.8[M+3H] / 3 + .

[0395] Synthesis of compound I-B12-5FAM

[0396] The crude product I-B12-C (40 mg, 0.025 mmol) obtained in the previous step was dissolved in a mixture of water (1.2 mL) and acetonitrile (0.3 mL). 5FAM-OSu (14 mg, 0.029 mmol) and saturated NaHCO3 solution (0.3 mL) were added sequentially. The mixture was stirred at room temperature for 60 minutes. The reaction solution was directly separated and purified by preparative chromatography to obtain I-B11-5FAM (12.5 mg, yield: 25.3%) as an orange solid. LCMS: (ESI) m / z = 1010.3 [M+H+K] / 2 + ;991.0[M+2H] / 2 + ;673.7[M+2H+K] / 3 + ; 661.3[M+3H] / 3 + .

[0397] Preparation conditions: HPLC preparation column: Gemini C18 21.2mm×250mm, 10μm, Mobile phase: A: water (containing 10 mmol / L NH4HCO3), B: acetonitrile; gradient: from 17% B to 27% B in 20 minutes.

[0398] HPLC purity: 93.1525% (214 nm), RT = 13.435 min; 4.4401% (214 nm), RT = 13.806 min.

[0399] The analytical conditions were the same as those for I-A10-5FAM.

[0400] 4.3 Synthesis of I-B12-Cy5

[0401] According to the method for synthesizing I-A12-Cy5, I-B12-Cy5 (5.0 mg, yield: 19.2%) was obtained from I-B12-E (0.0124 mmol) as a dark blue solid. LCMS: (ESI) m / z = 1045.3 [M + +H] / 2 + ;697.0[M + +2H] / 3 + .

[0402] HPLC purification conditions: Column: Xbridge C18, 19×250mm, 10μm, Mobile phase: A: water (containing 0.05% TFA), B: acetonitrile (containing 0.05% TFA); gradient: from 31% B to 41% B in 20 minutes.

[0403] HPLC purity: 90.9250% (214 nm), RT = 17.723 min; 8.5560% (214 nm), RT = 17.878 min.

[0404] Synthesis of IC molecules

[0405] 5.1 Synthesis of Compound I-C10-5FAM

[0406] Synthesis of intermediate I-C10-A

[0407] Compound A (100 mg, 0.147 mmol) was dissolved in DMF (3 mL), and HOBt (138 mg, 1.029 mmol), EDCI (198 mg, 1.029 mmol), and GalNAc-C7-NH2 (408 mg, 0.884 mmol) were added sequentially. The reaction was stirred at room temperature for 16 hours. The reaction was quenched by adding water (5 mL), extracted with ethyl acetate (8 mL), and the organic phase was washed with 5% aqueous sodium bicarbonate solution (10 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was separated and purified by preparative chromatography to obtain I-C10-A (170 mg, yield: 57.6%) as a white solid. LCMS: (ESI) m / z = 1003.0 [M+2H] / 2 + .

[0408] Preparation conditions: HPLC preparation column: Xbridge C18, 19×250mm, 10μm, Mobile phase: A: water (containing 10 mmol / L NH4HCO3), B: acetonitrile; gradient: from 20% B to 80% B in 20 minutes.

[0409] Synthesis of intermediate I-C10-C

[0410] I-C10-A (50 mg, 0.025 mmol), potassium carbonate (21 mg, 0.150 mmol), and Pd / C (10%, 12 mg) were added to a 50 mL round-bottom flask. Methanol (5 mL) was then added and the mixture was stirred under hydrogen for 3 hours. The reaction mixture was filtered and concentrated to afford I-C10-C (50 mg, crude product) as a white solid. LCMS: (ESI) m / z = 766.5 [M+H+K] / 2 + .511.6[M+2H+K] / 3 + .

[0411] Synthesis of compound I-C10-5FAM

[0412] The crude product I-C10-C (50 mg, 0.025 mmol) obtained in the previous step was dissolved in a saturated NaHCO3 solution in CH3CN / H2O (1.8 mL, v / v = 1:4:4), followed by the addition of 5-FAM-OSu (19 mg, 0.040 mmol). The mixture was stirred at room temperature for 1 hour. The reaction solution was directly separated and purified by preparative chromatography to obtain I-C10-5FAM (23 mg, yield: 37.7%) as an orange solid. LCMS: (ESI) m / z = 946 [M+H+K] / 2 + .

[0413] Preparation conditions: HPLC preparation column: Gemini C18 21.2mm×250mm, 10μm, Mobile phase: A: water (containing 10 mmol / L NH4HCO3), B: acetonitrile; gradient: from 22% B to 32% B in 20 minutes.

[0414] HPLC purity: 7.4271% (214 nm), RT = 14.574 min; 91.5431% (214 nm), RT = 15.705 min.

[0415] Synthesis of ID molecules

[0416] 6.1 Synthesis of Compound I-D11-5FAM

[0417] Synthesis of GalNAc ligand-loaded resin for solid-phase oligonucleotide synthesis:

[0418] 7.1 Synthesis of Intermediate A-13

[0419] Compound A-10 (3.2 g, 5.0 mmol, 1.0 eq.) was dissolved in DCM (50 mL), and benzyl succinate (1.25 g, 6.0 mmol, 1.2 equiv.), DCC (1.24 g, 6.0 mmol, 1.2 equiv.) and DMAP (122 mg, 1 mmol, 0.2 equiv.) were added in sequence. The reaction solution was stirred at room temperature overnight and concentrated. DCM (20 mL) was added to the residue, stirred and dissolved, and the insoluble white solid was filtered off. The filtrate was concentrated and dried to give the crude product A-11 as a light yellow oily liquid, which was directly used in the next reaction.

[0420] The crude intermediate A-11 from the previous step was dissolved in DCM (25 mL), and diethylamine (25 mL) was added. The reaction mixture was stirred at room temperature for 3 hours. After reaction monitoring, the reaction mixture was concentrated and dried to obtain the crude product A-12 as a light yellow oil, which was used directly in the next reaction.

[0421] The crude intermediate A-12 from the previous step was dissolved in DCM (100 mL), and succinic anhydride (1.5 g, 15 mmol), triethylamine (2.1 mL, 15 mmol), and DMAP (122 mg) were added sequentially. The reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated, and the residue was purified by reverse-phase column chromatography to obtain intermediate A-13 (2.95 g, total yield for 3 steps: 83%) as a white solid. LCMS: (ESI) m / z = 708.2 [MH] - .

[0422] Reverse phase purification conditions: chromatographic column: 120 g C18 column; mobile phase: A: water (containing 10 mmol / L NH4HCO3), B: acetonitrile; gradient: from 20% B to 60% B in 20 minutes.

[0423] 7.2 Synthesis of Resin I-A12-H-PS

[0424] Synthesis of intermediate I-A12-D

[0425] Intermediate I-A12-C (900 mg, 0.422 mmol, 1.0 equiv.) was dissolved in tetrahydrofuran (40 mL) and methanol (5 mL). Pd / C (10%, 200 mg) was added and the mixture was stirred under hydrogen for 3 hours. The reaction mixture was filtered and concentrated to afford crude product I-A12-D, which was used directly in the next reaction. LCMS: (ESI) m / z = 1001.7 [M+2H] / 2 +; 681.1[M+3H] / 3 + .

[0426] Synthesis of intermediate I-A12-G

[0427] Intermediate A-13 (359 mg, 0.506 mmol, 1.2 eq.) was dissolved in DMF (10 mL), and I-A12-D (863 mg, 0.422 mmol), EDCI (97 mg, 0.506 mmol), HOAt (69 mg, 0.506 mmol), and DIPEA (430 μl, 2.52 mmol) were added sequentially. The mixture was stirred at room temperature for 3 hours. LCMS confirmed the reaction was complete, and the reaction solution was directly purified by preparative chromatography to obtain I-A12-G (420 mg, yield: 37%) as a white solid. LCMS: (ESI) m / z = 1196.1 [M+2H] / 2 + ;797.7[M+3H] / 3 + . 1 H NMR(400MHz,DMSO)δ8.08-7.73(m,11H,11NH),7.37-7.15(m,14H,DMTr+Bn),6.88(d,J=7.2Hz,4H,DMTr),5.38-5.31(m,1H,proline-CH-OH), 5.21(d,J=3.2Hz,3H,GalNAc-OCHO-), 5.09(s,2H,Ph-CH2-), 4.96(dd,J=11.6,3.2Hz,3H,GalNAc), 4.48(d,J=7.6Hz, 3H,GalNAc),4.25-4.07(m,2H),4.02(brs,9H),3.93-3.70(m,5H),3.73(brs,6H),3.70-3.53(m,4H),3.49-3.37(m,5H ),3.23-2.87(m,28H),2.70-2.53(m,17H),2.46-2.02(m,5H),2.10(s,9H,GalNAc-OCOCH3),1.99(s,9H,GalNAc-OCOC H3),1.89(s,9H,GalNAc-OCOCH3),1.78(s,9H,GalNAc-NHCOCH3),1.74-1.61(m,7H),1.36(brs,17H),1.21(brs,11H).

[0428] Preparation conditions: HPLC preparation column: Xbridge C18, 19×250mm, 10μm, Mobile phase: A: water (containing 10 mmol / L NH4HCO3), B: acetonitrile; gradient: from 51% B to 61% B in 20 minutes.

[0429] Synthesis of intermediate I-A12-H

[0430] I-A12-G (400 mg, 0.149 mmol, 1.0 eq.) was dissolved in tetrahydrofuran (12 mL) and methanol (4.0 mL). Pd / C (10%, 100 mg) was added and the mixture was stirred under hydrogen for 3 hours. The reaction mixture was filtered and concentrated, and the crude product was purified by preparative chromatography to afford I-A12-H ​​(350 mg, yield: 90%) as a white solid. LCMS: (ESI) m / z = 1321.2 [M+H+K] / 2 + ,1150.7[M+2H-DMTr] / 2 + . 1 H NMR(400MHz,DMSO)δ8.08-7.73(m,11H,11NH),7.34-7.15(m,9H,DMTr),6.88(d,J=8.8Hz,4H,DMTr),5.38-5.31(m,1H,proline-CH-OH),5.21(d,J =3.2Hz,3H,GalNAc-OCHO-),4.95(dd,J=11.2,3.2Hz,3H,GalNAc),4.48(d,J=8.0Hz,3H,GalNAc),4.25-4.07(m,2H),4.02(brs,9H),3.93-3.70(m ,5H),3.73(brs,6H),3.70-3.53(m,5H),3.49-3.37(m,5H),3.23-2.87(m ,28H),2.70-2.39(m,19H),2.46-2.02(m,5H),2.10(s,9H,GalNAc-OCOCH 3),1.99(s,9H,GalNAc-OCOCH3),1.89(s,9H,GalNAc-OCOCH3),1.78(s,9H,GalNAc-NHCOCH3),1.74-1.61(m,7H),1.37(brs,17H),1.22(brs,11H).

[0431] HPLC preparation conditions: Column: Xbridge C18, 19×250mm, 10μm, Mobile phase: A: water (containing 10 mmol / L NH4HCO3), B: acetonitrile; gradient: from 39% B to 49% B in 20 minutes.

[0432] HPLC purity: >99% (214 nm), RT=14.992 min.

[0433] Mobile phase: A: water (10 mM NH4HCO3), B: acetonitrile.

[0434] Gradient: 5% B equilibration for 1 min, increase to 95% B in 20 min, increase to 95% in 5 min.

[0435] Flow rate: 1.0 mL / min.

[0436] HPLC analytical column: XBridge peptide BEH column C18, 4.6×150mm, 3.5μm,

[0437] Column temperature: 45℃.

[0438] Synthesis of Resin I-A12-H-PS

[0439] Macroporous aminomethyl resin (250 mg, 0.4 mmol / g, 2.17 equiv.) was placed in a 5 mL plastic syringe with a filter membrane and soaked in DCM (3 mL) for 30 minutes. The resin was then washed with DCM (3 mL × 3) and then DMF (3 mL × 3) by aspiration, shaking, and extrusion. I-A12-H ​​(125 mg, 0.0461 mmol, 1.0 equiv.), DIPEA (39 μL, 0.231 mol, 5 equiv.), and PyAOP (52 mg, 0.101 mmol, 2.2 equiv.) were dissolved in DMF (3 mL) one by one. The clear solution was pipetted into the washed resin and shaken at 25°C for 24 hours. A slightly blue color was detected by Kaiser assay. The resin was washed with DMF (3 mL × 6). Unreacted amino groups on the resin were capped with a mixture of pyridine and acetic anhydride (3 mL, 2:1) by shaking for 3 hours. The resin was then washed with DMF (3 mL x 6), and Kaiser assay revealed a colorless product. The resin was then washed with DCM (3 mL x 3) and MTBE (3 mL x 3), and vacuum dried overnight to yield an off-white resin, I-A12-H-PS (415 mg).

[0440] Test resin loading

[0441] To 18.2 mg of the resin, 8.0 mL of a 0.48 N HCl / MeCN solution was accurately added. The mixture was gently shaken for 10 minutes. 2.0 mL of the clear solution was collected and examined by LCMS. The absorption peak area was 1340.29. The loading was calculated as 164.8 μmol / g using the following formula.

[0442] Cutting resin

[0443] The residual resin after DMTr removal was washed with acetonitrile (2 mL x 3) and 2 mL of concentrated aqueous ammonia was added. The mixture was shaken at 60°C for 3 hours, filtered, and the filtrate was analyzed by HPLC to confirm that the resin was loaded with high-purity GalNAc ligand (I-A12-I). LCMS: (ESI) m / z = 931.0 [M+H+K] / 2 + ;911.7[M+2H] / 2 + ; 608.3[M+3H] / 3 + HPLC purity: 87.80% (214 nm), RT = 9.588 min; 12.20% (214 nm), RT = 10.398 min.

[0444] Mobile phase: A: water (10 mM NH4HCO3), B: acetonitrile.

[0445] Gradient: 5% B equilibration for 3 minutes, ramp to 65% B in 20 minutes, equilibration for 2 minutes.

[0446] Flow rate: 1.0 mL / min.

[0447] HPLC analytical column: XBridge peptide BEH column C18, 4.6×150mm, 3.5μm,

[0448] Column temperature: 60℃.

[0449] 7.3 Synthesis of Resin I-B12-F-PS

[0450] Synthesis of intermediate I-B12-B

[0451] Intermediate I-B12-A (400 mg, 0.187 mmol, 1.0 equiv.) was dissolved in tetrahydrofuran (10 mL) and methanol (1 mL). Pd / C (10%, 100 mg) was added and the mixture was stirred under hydrogen for 16 hours. The reaction mixture was filtered and concentrated to afford I-B12-B as a crude product, which was used directly in the next reaction. LCMS: (ESI) m / z = 1001.7 [M+2H] / 2 + ; 668.2[M+3H] / 3 + .

[0452] Synthesis of intermediate I-B12-E

[0453] Intermediate A-13 (153 mg, 0.22 mmol, 1.2 equiv.) was dissolved in DMF (10 mL), and I-B12-B (used directly from the previous step), EDCI (42 mg, 0.22 mmol), HOAt (30 mg, 0.22 mmol), and DIPEA (92 μl, 0.54 mmol) were added sequentially. The mixture was stirred at room temperature for 3 hours. LCMS confirmed the reaction was complete, and the reaction solution was directly purified by preparative chromatography to yield I-B12-E (350 mg, yield: 72%) as a white solid. LCMS: (ESI) m / z = 1196.1 [M+2H] / 2 + .

[0454] HPLC preparation conditions: Column: Xbridge C18, 19×250mm, 10μm, Mobile phase: A: water (containing 10 mmol / L NH4HCO3), B: acetonitrile; gradient: from 54% B to 64% B in 20 minutes.

[0455] Synthesis of intermediate I-B12-F

[0456] I-B12-E (200 mg, 0.074 mmol, 1.0 equiv.) was dissolved in tetrahydrofuran (6 mL) and methanol (2.0 mL). Pd / C (10%, 40 mg) was added and the mixture was stirred under hydrogen for 6 hours. The reaction mixture was filtered and concentrated. The crude product was purified by HPLC to obtain I-B12-F (150 mg, yield: 78%) as a white solid. LCMS: (ESI) m / z = 1321.2 [M+H+K] / 2 + ;1169.7[M+2H-DMTr] / 2 + .

[0457] HPLC preparation conditions: Column: Xbridge C18, 19×250mm, 10μm, Mobile phase: A: water (containing 10 mmol / L NH4HCO3); B: acetonitrile; gradient: from 39% B to 49% B in 20 minutes.

[0458] HPLC purity: >99% (214 nm), RT = 15.734 min

[0459] The analytical conditions were the same as those for I-A12-H.

[0460] Synthesis of Resin I-B12-F-PS

[0461] I-B12-F-PS was synthesized similarly to I-A12-H-PS resin. The loading value was 118.0 μmol / g as determined by the same method.

[0462] The residual resin after the DMTr group removal was washed with acetonitrile (2 mL x 3), and 2 mL of concentrated aqueous ammonia was added. The mixture was shaken at 60°C for 3 hours, filtered, and the filtrate was analyzed by HPLC to confirm that the resin was loaded with a high-purity peptide. LCMS: (ESI) m / z = 930.8 [M+H+K] / 2 + 911.8[M+2H] / 2 + ; 608.3[M+3H] / 3 + .

[0463] GalNAc ligand-loaded resin-synthesized oligonucleotide conjugates

[0464] GalNAc-siRNA-SS-3' was synthesized on a DNA / RNA synthesizer using the following method:

[0465] The siRNA synthesis method is similar to the conventional phosphoramidite solid-phase synthesis method (all synthesized by Suzhou Beixin Biotechnology Co., Ltd.), including four steps: deprotection, coupling, capping, and oxidation or sulfurization. When synthesizing the sense strand with GalNAc delivery, the aforementioned synthesized amino resin support (I-A12-H-PS resin, I-B12-F-PS resin, etc.) with GalNAc attached is used instead of the conventional Universal-CPG support. Using the solid-phase support as the starting cycle, nucleoside monomers are attached one by one from the 3' to the 5' direction. The raw materials for the nucleoside phosphoramidite monomers, such as 2'-F RNA and 2'-OMe RNA, were purchased from Shanghai Zhaowei Technology Development Co., Ltd. After the solid-phase synthesis is completed, the solid-phase support is transferred to a centrifuge tube and soaked in a 3:1 solution of 28% ammonia and ethanol at 50°C for 16 hours to cleave the oligonucleotides from the solid-phase support into the solution. The supernatant was transferred to another centrifuge tube, concentrated and evaporated to dryness, then redissolved in deionized water and purified using a C18 reverse-phase column with a mobile phase of 0.1 M TEAA and acetonitrile. The target oligonucleotide was collected and lyophilized, identified as the target product by LC-MS, and quantified by UV (260 nm). The resulting sense and antisense strands were annealed at a fixed molar ratio to yield complementary double-stranded siRNA, which was then adjusted to the desired concentration.

[0466] Huh7 transfection

[0467] On the first day, a mixture of RNAiMAX transfection reagent and Opti-MEM was prepared as needed (RNAiMAX transfection reagent: Opti-MEM = 1.5:48.5) and incubated at room temperature for 15 minutes. For each well of cells, a predetermined amount of the diluted compound was added to an equal volume of RNAiMAX Opti-MEM mixture, mixed, and incubated at room temperature for 15 minutes. Huh7 cells were washed with DPBS and then trypsinized to adjust the cell density to 2*10^5 / mL. Simultaneously with seeding the cells, 20 μL of the Opti-MEM RNAiMAX and compound mixture was added to a cell culture plate and seeded into a 96-well plate at a density of 20,000 cells per well (100 μL / well). The final volume of culture medium per well was 120 μL. The cells were cultured in a 5% CO2, 37°C incubator for 24 hours.

[0468] PHH free intake

[0469] PHH cells were revived and adjusted to a cell density of 6 x 10^5 / mL. 10 μL of compound was added from the dilution plate to a cell culture plate, with final compound concentrations of 100, 40, 12.5, 5, and 2 nM. Cells were seeded into 96-well plates at a density of 54,000 cells per well (90 μL / well), with a final volume of 100 μL per well. Cells were cultured in a 5% CO2, 37°C incubator for 72 hours.

[0470] PCH free uptake

[0471] PCH cells were first revived in 10% FBS InvitroGRO™ HT Medium and then adjusted to 3 x 10^5 cells / mL in 10% FBS InvitroGRO™ CP Medium. 10 μL of compound was added from the dilution plate to a cell culture plate at final concentrations of 25, 5, and 1 nM. Cells were seeded into 96-well plates at a density of 27,000 cells per well (90 μL / well), with a final volume of 100 μL per well. Cells were cultured in a 5% CO2, 37°C incubator for 72 hours.

[0472] RNA extraction and reverse transcription

[0473] RNA was extracted from Huh7 cells in the previous step according to the instructions of the RNA extraction kit (Qiagen, 74182), and reverse transcribed into cDNA according to the instructions of the FastKing cDNA First Strand Synthesis Kit (TianGen, KR116-02).

[0474] QPCR detection and data analysis

[0475] Target gene cDNA was detected using qPCR. The qPCR reaction system was prepared as shown in Table 6. GAPDH was used as an internal reference gene. qPCR was performed in a 384-well plate. The qPCR reaction program was as follows: heating at 95°C for 10 minutes, followed by cycling at 95°C for 15 seconds, followed by 60°C for 1 minute, for a total of 40 cycles.

[0476] Table 6. RT-PCR reaction system

[0477] Data Analysis: The ΔΔCT method (CT difference comparison method) requires the introduction of the internal reference gene GAPDH. This internal reference gene is expressed in all cells, its product is essential for cell survival, and its expression level or genomic copy number in cells is constant, making it less susceptible to environmental influences. After qRT-PCR, the CT value of the internal reference is recorded and is referred to as Ct(GAPDH). The CT value of the sample is referred to as Ct(sample).

[0478] ΔCt(sample)=Ct(sample)-Ct(GAPDH)

[0479] ΔCt(control)=Ct(control)-Ct(GAPDH)

[0480] ΔΔCt = ΔCt(sample) - ΔCt(control)

[0481] Relative gene expression = 2^-ΔΔCt

[0482] Inhibition rate % = (1-relative expression level of sample / average relative expression level of control group) * 100

[0483] ELISA detection and data analysis

[0484] The supernatant of PHH and PCH cells in the above step was aspirated, transferred to a new 96V well plate and centrifuged. Human Angiopoietin-like 3 protein (hANGPTL3 protein; DANL30) ELISA test was performed according to the instructions of the HANGPTL 3 protein kit.

[0485] Data Analysis: Four Parameter Logistic Method: This method constructs a standard curve using a plot with the average absorbance of the standard sample on the y-axis and the concentration on the x-axis. A best-fit curve is drawn at each point on the plot. Regression analysis is used to determine the optimal fit line between the OD value of the test sample, the logarithm of the HANGPTL 3 protein concentration, and the logarithm of the optimal concentration.

[0486] Inhibition rate % = (average content of control group - sample content) / average content of control group * 100

[0487] Example 1. Study on the hepatocyte endocytic activity of poly-GalNAc ligands with macrocyclic skeleton structures

[0488] Flow cytometry was used to evaluate the uptake efficiency of FAM fluorescently labeled test products in HepG2 cells.

[0489] Human hepatocellular carcinoma cell line (HepG2) was purchased from ATCC (USA) and cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco, ThermoFisher Scientific, USA) containing 10% fetal bovine serum (ExCell Bio, South America), 100 units / mL penicillin, and 100 units / mL streptomycin in a 37°C, 5% CO2 incubator.

[0490] On day 0, the HepG2 cell suspension was adjusted to an appropriate density (1.5E+05 / wells) and the cells were seeded into 48-well plates. On day 1, the diluted fluorescently labeled test products were added. The test products were diluted to different concentrations, 1.6, 8, 40, 200 and 1000 nM. The GalNAc ligand was used as a positive control for Givosirna, and 5FAM was used as a negative control. After incubation of the test products with the cells for 4 hours or 24 hours, all liquids were removed, and the cells were washed twice with PBS and collected. After digestion and fixation, the cells were analyzed by flow cytometry (BD FACSCanto TM The corresponding fluorescence in HepG2 cells was detected using a ELISA kit (Becton Dickinson, USA). The mean fluorescence intensity (MFI) of the FAM of the test compounds was compared with the mean fluorescence intensity of the positive compounds. Table 7 shows the data at the 4-hour time point, and Table 8 shows the data at the 24-hour time point.

[0491] Table 7. Ratio of MFI of GalNAc ligand endocytosis in hepatocytes to MFI of positive compounds (FAM fluorescence channel, 4 hours time point)

[0492] Table 8. Ratio of MFI of GalNAc ligand endocytosis in hepatocytes to MFI of positive compounds (FAM fluorescence channel, 24 h time point)

[0493] Example 2. Screening of modified siRNA sequences

[0494] In order to evaluate the in vitro inhibitory activity of the modified siRNA against ANGPTL3 mRNA in the Huh7 cell line, the designed siRNA was subjected to in vitro functional evaluation. The results are shown in Table 9.

[0495] Table 9. In vitro inhibitory activity of modified siRNA against ANGPTL3 mRNA in Huh7 cell line

[0496] The results showed that the siRNA of the present invention can effectively reduce the ANGPTL3 mRNA level in Huh7 cells, and some of the modification schemes are comparable to those of Alnylam Advanced ESC (2002-050, 2008-050, see Foster DJ, Brown CR, Shaikh S, et al. Advanced siRNA Designs Further Improve In Vivo Performance of GalNAc-siRNA Conjugates. Mol Ther. 2018; 26(3):708-717.doi:10.1016 / j.ymthe.2017.12.021).

[0497] Example 3. MCPP-siRNA screening

[0498] The in vitro inhibitory activity of ANGPTL3 Protein was evaluated in primary human hepatocytes (PHH) and primary cynomolgus monkey hepatocytes (PCH) for the sequences with good activity in Table 8. The results are shown in Tables 10 and 11.

[0499] Table 10. In vitro inhibitory activity of MCPP-siRNA against ANGPTL3 mRNA in PHH cell lines

[0500] The results showed that the MCPP-siRNA of the present invention can effectively reduce the ANGPTL3 protein level in PHH cells, and the partial modification scheme is equivalent to L96 (2002-050-L96).

[0501] Table 11. In vitro inhibitory activity of MCPP-siRNA against ANGPTL3 mRNA in PCH cell lines

[0502] The results showed that the MCPP-siRNA of the present invention can effectively reduce the ANGPTL3 protein level in PCH cells.

[0503] Example 4. In vivo efficacy of ANGPTL3 GalNAc-siRNA in hANGPTL3 humanized mice

[0504] To evaluate the in vivo efficacy of siRNA against ANGPTL3 in mice, female ANGPTL3 humanized mice (purchased from Shanghai Model Organisms Science Co., Ltd.) were used for in vivo efficacy testing. The mice were randomly divided into five groups, each containing five mice: G1, a negative control group (PBS); G2, a 2002-034-001 (3 mg / kg) treatment group; G3, a 2008-010-001 (3 mg / kg) treatment group; G4, a 2008-019-001 (3 mg / kg) treatment group; and G5, a 2008-019-002 (3 mg / kg) treatment group.

[0505] Pre-dose plasma samples were obtained on days -7 and -1 after a 4-hour fast. The siRNA conjugates were diluted with saline and administered subcutaneously on day 0 according to mouse body weight. Plasma was collected on days 7, 14, 21, and 28 after a 4-hour fast. ANGPTL3 protein levels in plasma were measured by ELISA (R&D Systems). Plasma triglycerides (TG), total cholesterol (CHO), high-density lipoprotein cholesterol (HDL-c), low-density lipoprotein cholesterol (LDL-c), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were assayed by Shanghai Bikeway Biotechnology Co., Ltd.

[0506] To evaluate the in vivo efficacy of siRNA against ANGPTL3 in mice, female ANGPTL3 humanized mice (purchased from Shanghai Model Organisms Science Co., Ltd.) were used for in vivo efficacy testing. The mice were randomly divided into 5 groups, 5 mice in each group, namely:

[0507] G1, Control (PBS) negative control group;

[0508] G2, 2002-034-001 (3 mg / kg) treatment group;

[0509] G3, 2008-010-001 (3 mg / kg) treatment group;

[0510] G4, 2008-019-001 (3 mg / kg) treatment group;

[0511] G5, 2008-019-002 (3 mg / kg) treatment group;

[0512] The day of the first administration is designated as Day 0, the day before administration is designated as Day -1, the day after administration is designated as Day 1, and so on.

[0513] On day -7, all mice were fasted for 4 hours, and submandibular blood was collected for plasma analysis of hANGPTL3 protein. Plasma preparation was performed by collecting whole blood into sodium heparin-coated anticoagulant tubes, centrifuging at 7000 g for 10 minutes at 4°C, and collecting the supernatant. 20 μL of the supernatant was transferred to an in vitro laboratory for ELISA analysis of hANGPTL3 protein expression in plasma (R&D Systems), and the remaining 50 μL was sent to Shanghai Biochem Biotech for blood biochemical analysis of triglyceride (TG) and total cholesterol (CHO). Based on the test results of the day -7 plasma samples and mouse body weight, 25 mice were randomly divided into five groups.

[0514] On day -1, after a 4-hour fast, a pre-dose plasma sample was obtained. The siRNA conjugates were diluted in normal saline and administered subcutaneously on day 0 according to mouse body weight. Plasma was collected after a 4-hour fast on days 7, 14, 21, and 28. ANGPTL3 protein levels in plasma were measured by ELISA (R&D Systems). Plasma triglycerides (TG) and total cholesterol (CHO) were measured by Shanghai Bikeway Biotechnology Co., Ltd.

[0515] Plasma ANGPTL3 protein, triglyceride (TG), and total cholesterol (CHO) levels in humanized mice were normalized to day -1 levels. Data at other time points were processed by dividing the plasma ANGPTL3 protein, triglyceride, and total cholesterol levels of each mouse at a given time point by the expression levels on day -1 before dosing.

[0516] The results are shown in FIG1 , indicating that the siRNA conjugates tested in 2008-010 were able to significantly reduce the levels of ANGPTL3 protein, triglyceride (TG), and total cholesterol (CHO) in the plasma of humanized mice compared to the control.

[0517] Example 5. In vivo efficacy of ANGPTL3 GalNAc-siRNA in hyperlipidemic cynomolgus monkeys

[0518] To evaluate the in vivo activity of siRNA against ANGPTL3, six cynomolgus macaques induced by a high-fat diet (HFD) were used. Pre-dose serum samples were obtained on days -7 and -14 after a 4-hour fast. siRNA conjugates were diluted in saline and administered subcutaneously on day 0 according to the experimental protocol. A blank saline solution was used as a negative control. Plasma was collected after a 4-hour fast on days 0, 7, 14, 28, 42, 49, and 56. Serum triglycerides were measured using an automated biochemical analyzer.

[0519] ANGPTL3 protein levels and triglyceride levels for each animal were normalized. For normalization, the ANGPTL3 protein and triglyceride levels for each animal at a time point were divided by the animal's pre-treatment expression level (the mean of the pre-experimental samples for that group) to determine the change "normalized to pre-treatment."

[0520] The results are shown in Figure 2. A single subcutaneous injection of YH2008-010-001 in cynomolgus monkeys can significantly inhibit the serum ANGPTL3 protein level in HFD-induced hyperlipidemic elderly cynomolgus monkeys and significantly reduce the level of triglycerides.

Claims

1. An RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the RNAi agent inhibits the expression of ANGPTL3 in a cell, wherein the antisense strand has a length of no more than 23 nucleotides and comprises any one nucleotide sequence selected from SEQ ID NO: 13, 14, 27 and 28.

2. The RNAi agent of claim 1, wherein the sense strand is no longer than 21 nucleotides and comprises any one nucleotide sequence selected from SEQ ID NO: 5, 7, 8, 19, 20 and 22.

3. The RNAi agent according to any one of claims 1 or 2, wherein: (a1) the sense strand comprises or is the sequence shown in SEQ ID NO:5, and the antisense strand comprises or is the sequence shown in SEQ ID NO:13; (a2) the sense strand comprises or is the sequence shown in SEQ ID NO:5, and the antisense strand comprises or is the sequence shown in SEQ ID NO:14; (a3) the sense strand comprises or is the sequence shown in SEQ ID NO:7, and the antisense strand comprises or is the sequence shown in SEQ ID NO:13; (a4) the sense strand comprises or is the sequence shown in SEQ ID NO:8, and the antisense strand comprises or is the sequence shown in SEQ ID NO:13; (a5) the sense strand comprises or is the sequence shown in SEQ ID NO: 19, and the antisense strand comprises or is the sequence shown in SEQ ID NO: 27; (a6) the sense strand comprises or is the sequence shown in SEQ ID NO: 19, and the antisense strand comprises or is the sequence shown in SEQ ID NO: 28; (a7) the sense strand comprises or is the sequence shown in SEQ ID NO:20, and the antisense strand comprises or is the sequence shown in SEQ ID NO:27; (a8) the sense strand comprises or is the sequence shown in SEQ ID NO:20, and the antisense strand comprises or is the sequence shown in SEQ ID NO:28; (a9) the sense strand comprises or is the sequence shown in SEQ ID NO:22, and the antisense strand comprises or is the sequence shown in SEQ ID NO:28; (a10) the sense strand comprises or is the sequence shown in SEQ ID NO:5, and the antisense strand comprises or is the sequence shown in SEQ ID NO:18; or (a11) the sense strand comprises or is the sequence shown in SEQ ID NO:19, and the antisense strand comprises or is the sequence shown in SEQ ID NO:

32.

4. The RNAi agent according to any one of claims 1 to 3, 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, and further preferably, the ligand is a trivalent or tetravalent N-acetylgalactosamine moiety.

5. The RNAi agent according to claim 4, wherein the ligand comprises a structure as shown in formula (I): in, X is -C(O)-NH-, -NH-C(O)-, -OCH2-CH2O-, -S- or -SS-; A or C is independently 0 or an integer between 1 and 5; B is 0 or an integer between 1 and 3; The wavy lines represent locations where the rest of the RNAi agent is attached.

6. The RNAi agent according to claim 4, wherein the structure of the ligand is as shown in formula (II) or formula (III): in, X is -C(O)-NH-, -NH-C(O)-, -OCH2-CH2O-, -S- or -SS-; Y1 is -C(O)-NR1-, -NH-C(O)-, -OCH2-CH2O-, -SS-, Wherein R1 is an alkyl group containing 1 to 6 carbon atoms or hydrogen; W is -NH-, -O- or The wavy line on the right represents the position of connection with the sense strand or antisense strand; The wavy line connected to W in Formula II represents the position of attachment to the sense strand or antisense strand, wherein the ligand is attached to the 5' and / or 3' end of the sense strand and / or antisense strand. A, C or F is 0 or an integer between 1 and 5; B or E is 0 or an integer between 1 and 3; D is an integer between 1 and 6; in, X is -C(O)-NH-, -NH-C(O)-, -OCH2-CH2O-, -S- or -SS-; Y2 is -(CH2)p-(O-CH2-CH2)q-(CH2)j-Z3-, wherein Z3 is O, NH or C(O), p is an integer from 1 to 3, q ​​is an integer from 3 to 10; j is 0 or 1; W does not exist or is The wavy line on the right is The location where the sense or antisense strand is attached; The wavy line connected to W in Formula III represents the position of attachment to the sense strand or antisense strand, wherein the ligand is attached to the 5' and / or 3' end of the sense strand and / or antisense strand. A or C is 0 or an integer between 1 and 5; B is 0 or an integer between 1-3.

7. The RNAi agent according to claim 5 or 6, wherein the ligand structure is as shown in formula (IV) or formula (V): in, The wavy line represents the position of connection to the sense strand or antisense strand, and the ligand is connected to the 5' and / or 3' end of the sense strand; preferably, the ligand is connected to the 3' end of the sense strand; preferably, wherein the ligand is connected to the sense strand or antisense strand via a phosphate group, a thiophosphate group or a phosphonate group.

8. The RNAi agent according to any one of claims 5 to 7, wherein: (b1) the sense strand comprises or is the sequence shown in SEQ ID NO:33, and the antisense strand comprises or is the sequence shown in SEQ ID NO:13; (b2) the sense strand comprises or is the sequence shown in SEQ ID NO:34, and the antisense strand comprises or is the sequence shown in SEQ ID NO:13; (b3) the sense strand comprises or is the sequence shown in SEQ ID NO:38, and the antisense strand comprises or is the sequence shown in SEQ ID NO:13; (b4) the sense strand comprises or is the sequence shown in SEQ ID NO:39, and the antisense strand comprises or is the sequence shown in SEQ ID NO:13; (b5) the sense strand comprises or is the sequence shown in SEQ ID NO:35, and the antisense strand comprises or is the sequence shown in SEQ ID NO:27; (b6) the sense strand comprises or is the sequence shown in SEQ ID NO:36, and the antisense strand comprises or is the sequence shown in SEQ ID NO:27; (b7) the sense strand comprises or is the sequence shown in SEQ ID NO:40, and the antisense strand comprises or is the sequence shown in SEQ ID NO:27; (b8) the sense strand comprises or is the sequence shown in SEQ ID NO:41, and the antisense strand comprises or is the sequence shown in SEQ ID NO:27; (b9) the sense strand comprises or is the sequence shown in SEQ ID NO:35, and the antisense strand comprises or is the sequence shown in SEQ ID NO:28; (b10) the sense strand comprises or is the sequence shown in SEQ ID NO:36, and the antisense strand comprises or is the sequence shown in SEQ ID NO:28; (b11) the sense strand comprises or is the sequence shown in SEQ ID NO:37, and the antisense strand comprises or is the sequence shown in SEQ ID NO:28; (b12) the sense strand comprises or is the sequence shown in SEQ ID NO:40, and the antisense strand comprises or is the sequence shown in SEQ ID NO:28; (b13) the sense strand comprises or is the sequence shown in SEQ ID NO:41, and the antisense strand comprises or is the sequence shown in SEQ ID NO:28; or (b14) the sense strand comprises or is the sequence shown in SEQ ID NO:42, and the antisense strand comprises or is the sequence shown in SEQ ID NO:

28.

9. A double-stranded RNAi agent for inhibiting ANGPTL3 expression in a cell, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises SEQ ID NO.19: 5'-mG*mA*mGmAfAmGfAfAfCmUmAmCmAmUmAmUmAmAmA-3' and has a length of no more than 21 nucleotides, and the antisense strand comprises SEQ ID NO.27: 5'-mU*fU*mUmAfUmAfUmGmUmAmGmUmUfCmUfUmCmUmC*mA*mG-3' and has a length of no more than 23 nucleotides, Wherein mA, mU, mG and mC are 2'-OMe A, 2'-OMe U, 2'-OMe G, 2'-OMe C, respectively; fA, fU, fG and fC are 2'-FA, 2'-FU, 2'-FG, 2'-FC, respectively; * is a phosphorothioate linkage; wherein the 3'-end of the sense chain is conjugated to a ligand targeting hepatocytes, preferably, the ligand includes a galactose portion, a galactosamine portion or an N-acetylgalactosamine portion, and further preferably, the ligand is a trivalent or tetravalent N-acetylgalactosamine portion.

10. The RNAi agent according to claim 9, wherein the ligand is a structure shown in Formula IV: It is linked to the sense strand via a phosphate group or a phosphorothioate group.

11. The RNAi agent of claim 9 or 10, wherein the sense strand comprises SEQ ID NO.40: 5'-mG*mA*mGmAfAmGfAfAfCmUmAmCmAmUmAmUmAmAmA-YHZY12001-3' and the length of the sense strand is no more than 21 nucleotides, and the antisense strand comprises SEQ ID NO.27: 5'-mU*fU*mUmAfUmAfUmGmUmAmGmUmUfCmUfUmCmUmC*mA*mG-3' and the length is no more than 23 nucleotides, Wherein mA, mU, mG and mC are 2'-OMe A, 2'-OMe U, 2'-OMe G, 2'-OMe C, respectively; fA, fU, fG and fC are 2'-FA, 2'-FU, 2'-FG, 2'-FC, respectively; * is a phosphorothioate linkage, wherein YHZY12001 has a structure of Formula IV and is conjugated to the 3'-end of the sense strand as follows:

12. A pharmaceutical composition comprising the RNAi agent according to any one of claims 1 to 11 and a pharmaceutically acceptable carrier; preferably, the pharmaceutical composition is formulated as an intravenous or subcutaneous injection.

13. Use of the RNAi agent according to any one of claims 1 to 11 in the preparation of the following medicines: (i) an agent for reducing the expression level of ANGPTL3 in cells; (ii) a drug for preventing or treating a disease caused by increased ANGPTL3 expression level; or Drugs for the prevention or treatment of metabolic diseases, such as hypertriglyceridemia, obesity, hyperlipidemia, abnormal lipid and / or cholesterol metabolism, atherosclerosis, type II diabetes, cardiovascular disease, coronary artery disease, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, homozygous or heterozygous familial hypercholesterolemia, statin-resistant hypercholesterolemia.

14. A RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region, wherein: 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; and 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.

15. The RNAi agent of claim 14, wherein 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 nucleotide and the second nucleotide at the 5' end of the antisense strand; (ii) between the second nucleotide and the third nucleotide at the 5' end of the antisense strand; (iii) between the first nucleotide and the second nucleotide at the 3' end of the antisense strand; and (iv) between the second nucleotide and the third nucleotide from the 3' end of the antisense strand.

16. The RNAi agent of claim 15, wherein the sense strand has at least one phosphorothioate internucleotide linkage; preferably, the phosphorothioate internucleotide linkage is present between (i) the first nucleotide and the second nucleotide at the 5' end of the sense strand; and / or (ii) the second nucleotide and the third nucleotide at the 5' end of the sense strand.

17. The RNAi agent according to any one of claims 14 to 16, wherein 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.

18. The RNAi agent according to any one of claims 14 to 17, wherein the sense strand and the antisense strand are each 17 to 23 nucleotides in length, preferably 19 to 21 nucleotides in length.

19. The RNAi agent according to any one of claims 14 to 18, wherein the RNAi agent comprises one or two blunt ends, preferably one blunt end.

20. The RNAi agent according to any one of claims 14 to 19, wherein the RNAi agent comprises one or two overhangs, preferably one overhang, each overhang having 1 to 4 unpaired nucleotides, preferably 2 unpaired nucleotides.

21. The RNAi agent of claim 20, wherein 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.

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