17 beta-hydroxysteroid dehydrogenase type 13 (HSD17B13) iRNA compositions and methods of use thereof

Abstract

The present invention relates to methods of treating an individual who would benefit from reduced expression of HSD17B13, such as an individual with a HSD17B13-related disease, disorder or condition, such as non-alcoholic steatohepatitis (NASH), using a double stranded ribonucleic acid (dsRNA) composition targeting the HSD17B13 gene. The invention also provides methods for preventing at least one symptom in an individual suffering from a HSD17B13-related disease, disorder or condition (e.g., NASH).

Description

[0001] Related applications

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 406,894, filed on September 15, 2022, U.S. Provisional Application No. 63 / 434,581, filed on December 22, 2022, and U.S. Provisional Application No. 63 / 521,752, filed on June 19, 2023. The entire contents of each of the foregoing applications are incorporated herein by reference. Background Art

[0003] Type 13 17β-hydroxysteroid dehydrogenase (HSD17B13) is a member of the 17β-hydroxysteroid dehydrogenase (HSD17B) family of enzymes, whose members have diverse functions, including, for example, the in vivo reduction or oxidation of sex hormones, fatty acids, and bile acids (Moeller and Adamski (2009) Mol Cell Endocrinol 301:7). Members of the HSD17B family vary in tissue distribution, subcellular localization, catalytic preferences, and have varying substrate specificities due to their ability to catalyze the conversion of substrates other than steroids, such as lipids and retinoids (Marchais-Oberwinkler et al. (2011) J Steroid Biochem Mol Biol 125(1-2):66-82). HSD17B13 has been shown to enhance hepatic lipogenesis in normal mouse liver and in cultured human hepatocytes (Su et al. (2014) Proc Natl Acad Sci USA 111:11437).

[0004] Hepatocytes form the parenchymal tissue of the liver and are responsible for mobilizing lipids for energy and storing excess lipids in the form of lipid droplets (LDs), making the liver the primary organ responsible for lipid homeostasis.

[0005] LD is now recognized as a biologically active organelle involved in lipid metabolism, membrane trafficking and signal transduction. LD is generally composed of a core of neutral lipids (such as triglycerides (TG) and cholesterol esters) and a phospholipid / cholesterol monolayer surrounding the core. Many LD-specific proteins are associated with the LD membrane and play a role, for example, in controlling the flux of molecules into and out of the LD. The major hepatocyte LD-associated proteins are members of the perilipin family, but non-perilipin proteins, such as hypoxia-inducible protein 2 (HIG2), patanin-like phospholipase domain-containing protein 3 (PNPLA3) and HSD17B13, have also been identified as LD-associated proteins (Carr and Ahima (2016) Exp Cell Res 15:187; Su et al., (2014) Proc Natl Acad Sci USA 111:11437).

[0006] Increased LD accumulation is associated with many metabolic diseases and chronic fibro-inflammatory liver diseases, such as liver fibrosis, NASH, and NAFLD. HSD17B13 has been identified as one of the most abundantly expressed LD proteins, specifically localized on the surface of LDs in humans and mice with NAFLD. HSD17B13 expression has also been shown to be upregulated in the livers of patients and mice with NAFLD. Overexpression of HSD17B13 leads to an increase in the number and size of LDs. Hepatic overexpression of HSD17B13 in C57BL / 6 mice significantly increases liver lipogenesis and triglyceride content, leading to a fatty liver phenotype.

[0007] Currently, there is no treatment for chronic fibroinflammatory liver disease. The current standard of care for individuals with chronic fibroinflammatory liver disease includes lifestyle modifications and management of associated comorbidities, such as hypertension, hyperlipidemia, diabetes, obesity, etc. Therefore, because the prevalence of chronic fibroinflammatory liver disease has gradually increased over the past 10 years and is expected to continue to increase, there is a need in the art for alternative treatments for individuals with chronic fibroinflammatory liver disease. Summary of the Invention

[0008] The present invention is based at least in part on the discovery that administering (e.g., subcutaneously) a RNAi agent targeting the HSD17B13 gene (e.g., AD-288996) to individuals with NASH reduces the level of HSD17B13 mRNA and reduces liver enzymes and biopsy-derived non-alcoholic fatty liver disease (NAFLD) activity score (NAS) within six months.

[0009] Thus, in one aspect, the present invention provides a method for reducing HSD17B13 mRNA levels in a subject. The method comprises administering to the subject a therapeutically effective amount (e.g., a dose of about 25 mg to about 800 mg) of a dsRNA agent or a pharmaceutical composition thereof, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises the nucleotide sequence 5'-AUGCUUUUGCAUGGACUAUCU-3' (SEQ ID NO: 26) and the antisense strand comprises the nucleotide sequence 5'-AGAUAGTCCAUGCAAAAGCAUUC-3' (SEQ ID NO: 27), thereby reducing HSD17B13 mRNA levels in the subject. In some embodiments, the subject suffers from an HSD17B13-associated disease, disorder, or condition. Furthermore, in some embodiments, the subject suffers from at least one condition selected from the group consisting of chronic fibroinflammatory liver disease, chronic fibroinflammatory liver disease associated with accumulation and / or expansion of lipid droplets in the liver, liver inflammation, liver fibrosis, fatty liver disease (steatosis), non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), cirrhosis, alcoholic steatohepatitis (ASH), alcoholic liver disease (ALD), HCV-related cirrhosis, drug-induced liver injury, hepatocellular necrosis, and obesity.

[0010] In another aspect, the present invention provides a method for treating a subject suffering from a disease, disorder, or condition associated with HSD17B13, such as non-alcoholic steatohepatitis (NASH). The method comprises administering to the subject a therapeutically effective amount (e.g., a dose of about 25 mg to about 800 mg) of a dsRNA agent or a pharmaceutical composition thereof, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises the nucleotide sequence 5'-AUGCUUUUGCAUGGACUAUCU-3' (SEQ ID NO: 26) and the antisense strand comprises the nucleotide sequence 5'-AGAUAGTCCAUGCAAAAGCAUUC-3' (SEQ ID NO: 27), thereby treating the subject suffering from a disease, disorder, or condition associated with HSD17B13, such as non-alcoholic steatohepatitis (NASH).

[0011] In one embodiment, after 6 months of treatment, HSD17B13 mRNA levels are reduced to at least about 70%, 65%, 60%, 55% or 50% of baseline levels.

[0012] In another aspect, the present invention provides a method for preventing at least one symptom in a subject suffering from a disease, disorder, or condition that would benefit from reduced expression of the HSD17B13 gene, such as nonalcoholic steatohepatitis (NASH). The method comprises administering to the subject a prophylactically effective amount (e.g., a dose of about 25 mg to about 800 mg) of a dsRNA agent or a pharmaceutical composition thereof, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises the nucleotide sequence 5'-AUGCUUUUGCAUGGACUAUCU-3' (SEQ ID NO: 26) and the antisense strand comprises the nucleotide sequence 5'-AGAUAGTCCAUGCAAAAGCAUUC-3' (SEQ ID NO: 27), thereby preventing at least one symptom in a subject suffering from a disease, disorder, or condition that would benefit from reduced expression of the HSD17B13 gene, such as nonalcoholic steatohepatitis (NASH).

[0013] In another aspect, the present invention provides a method for reducing the risk of a subject with steatosis developing chronic liver disease. The method comprises administering to the subject a therapeutically effective amount (e.g., a dose of about 25 mg to about 800 mg) of a dsRNA agent or a pharmaceutical composition thereof, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises the nucleotide sequence 5'-AUGCUUUUGCAUGGACUAUCU-3' (SEQ ID NO: 26) and the antisense strand comprises the nucleotide sequence 5'-AGAUAGTCCAUGCAAAAGCAUUC-3' (SEQ ID NO: 27), thereby reducing the risk of a subject with steatosis developing chronic liver disease.

[0014] In yet another aspect, the present invention provides a method for inhibiting the progression of steatosis to steatohepatitis in a subject suffering from steatosis. The method comprises administering to the subject a therapeutically effective amount (e.g., a dose of about 25 mg to about 800 mg) of a dsRNA agent or a pharmaceutical composition thereof, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises the nucleotide sequence 5'-AUGCUUUUGCAUGGACUAUCU-3' (SEQ ID NO: 26) and the antisense strand comprises the nucleotide sequence 5'-AGAUAGTCCAUGCAAAAGCAUUC-3' (SEQ ID NO: 27), thereby inhibiting the progression of steatosis to steatohepatitis in the subject.

[0015] In one aspect, the present invention provides a method of inhibiting lipid droplet accumulation in the liver of a subject suffering from an HSD17B13-associated disease, disorder, or condition, such as nonalcoholic steatohepatitis (NASH). The method comprises administering to the subject a therapeutically effective amount (e.g., a dose of about 25 mg to about 800 mg) of a dsRNA agent or a pharmaceutical composition thereof, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises the nucleotide sequence 5'-AUGCUUUUGCAUGGACUAUCU-3' (SEQ ID NO: 26) and the antisense strand comprises the nucleotide sequence 5'-AGAUAGTCCAUGCAAAAGCAUUC-3' (SEQ ID NO: 27), thereby inhibiting fat accumulation in the liver of a subject suffering from an HSD17B13-associated disease, disorder, or condition, such as nonalcoholic steatohepatitis (NASH).

[0016] In one embodiment, administering the dsRNA agent or the pharmaceutical composition to a subject results in a decrease in HSD17B13 enzyme activity, a decrease in HSD17B13 protein accumulation, and / or a decrease in fat accumulation and / or lipid droplet expansion in the subject's liver.

[0017] In one embodiment, the HSD17B13-associated disease, disorder or condition is chronic fibroinflammatory liver disease.

[0018] In one embodiment, the chronic fibroinflammatory liver disease is selected from the group consisting of fat accumulation in the liver, liver inflammation, liver fibrosis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), cirrhosis, alcoholic steatohepatitis (ASH), alcoholic liver disease (ALD), HCV-related cirrhosis, drug-induced liver injury and hepatocellular necrosis.

[0019] In one embodiment, the chronic fibroinflammatory liver disease is nonalcoholic steatohepatitis (NASH).

[0020] In one embodiment, the subject is a human subject.

[0021] In some embodiments, the individual is obese.

[0022] In some embodiments, the dsRNA agent comprises at least one modified nucleotide.

[0023] In some embodiments, substantially all nucleotides of the sense strand comprise modifications. In some embodiments, substantially all nucleotides of the antisense strand comprise modifications. In some embodiments, substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand comprise modifications.

[0024] In some embodiments, at least one of the modified nucleotides is selected from the group consisting of deoxynucleotides, 3'-terminal deoxythymidine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, unlocked nucleotides, conformationally constrained nucleotides, constrained ethyl nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-alkyl modified nucleotides, 2'-hydroxy modified nucleotides, 2' -methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, N-morpholino nucleotides, phosphoramidates, nucleotides containing unnatural bases, tetrahydropyran modified nucleotides, 1,5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides containing phosphorothioate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphates, nucleotides containing 5'-phosphate mimetics, diol modified nucleotides, and 2-O-(N-methylacetamide) modified nucleotides, and combinations thereof.

[0025] In some embodiments, the nucleotide modification is a 2'-O-methyl and / or 2'-fluoro modification.

[0026] In some embodiments, the length of each strand of the dsRNA agent does not exceed 30 nucleotides. In some embodiments, the length of each strand of the dsRNA agent is independently 19 to 30 nucleotides. In some embodiments, the length of each strand of the dsRNA agent is independently 19 to 25 nucleotides. In some embodiments, the length of each strand of the dsRNA agent is independently 21 to 23 nucleotides.

[0027] In some embodiments, at least one strand of the dsRNA agent comprises a 3' overhang of at least 1 nucleotide. In some embodiments, at least one strand of the dsRNA agent comprises a 3' overhang of at least 2 nucleotides.

[0028] In some embodiments, the dsRNA agent further comprises a ligand. In some embodiments, the ligand is conjugated to the 3' end of the sense strand of the dsRNA agent.

[0029] In some embodiments, the ligand is an N-acetylgalactosamine (GalNAc) derivative.

[0030] In some embodiments, the ligand is

[0031]

[0032] In some embodiments, the dsRNA agent is conjugated to a ligand as shown in the following schematic diagram

[0033]

[0034] wherein X is O or S.

[0035] In some embodiments, X is O.

[0036] In some embodiments, the sense strand comprises the nucleotide sequence 5'-asusgcuuUfuGfCfAfuggacuaucu-3' (SEQ ID NO: 24) and the antisense strand comprises the nucleotide sequence 5'-asGfsauag(Tgn)ccaugcAfaAfagcaususc-3' (SEQ ID NO: 25), wherein Af is 2'-fluoroadenosine-3'-phosphate; Cf is 2'-fluorocytidine-3'-phosphate; Gf is 2'-fluoroguanosine-3'-phosphate; Gfs is 2'-fluoroguanosine-3'-thiophosphate; U is uridine-3'-phosphate; Uf is 2'-fluorouridine-3'-phosphate; a is 2'-O-methyladenosine-3'-phosphate; as is 2'-O-methyladenosine-3'-thiophosphate; c is 2'-O-methylcytidine-3'-phosphate; cs is 2'-O-methylcytidine-3'-phosphorothioate; g is 2'-O-methylguanosine-3'-phosphate; gs is 2'-O-methylguanosine-3'-phosphorothioate; u is 2'-O-methyluridine-3'-phosphate; us is 2'-O-methyluridine-3'-phosphorothioate; Tgn is the thymidine diol nucleic acid (GNA) S isomer; and s is a phosphorothioate linkage.

[0037] In some embodiments, the dsRNA agent is conjugated to a ligand as shown in the following schematic diagram

[0038]

[0039] And wherein X is O.

[0040] In one aspect, the present invention provides a method for treating a subject suffering from non-alcoholic steatohepatitis (NASH), the method comprising administering to the subject a dose of about 25 mg to about 800 mg of a double-stranded ribonucleic acid (dsRNA) agent targeting the HSD17B13 gene, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises the nucleotide sequence 5'-asusgcuuUfuGfCfAfuggacuaucu-3' (SEQ ID NO: 24) and the antisense strand comprises the nucleotide sequence 5'-asGfsauag(Tgn)ccaugcAfaAfagcaususc-3' (SEQ ID NO: NO:25), wherein Af is 2'-fluoroadenosine-3'-phosphate; Cf is 2'-fluorocytidine-3'-phosphate; Gf is 2'-fluoroguanosine-3'-phosphate; Gfs is 2'-fluoroguanosine-3'-thiophosphate; U is uridine-3'-phosphate; Uf is 2'-fluorouridine-3'-phosphate; a is 2'-O-methyladenosine-3'-phosphate; as is 2'-O-methyladenosine-3'-thiophosphate; c is 2'-O-methylcytidine-3'-phosphate. '-phosphate; cs is 2'-O-methylcytidine-3'-phosphorothioate; g is 2'-O-methylguanosine-3'-phosphate; gs is 2'-O-methylguanosine-3'-phosphorothioate; u is 2'-O-methyluridine-3'-phosphate; us is 2'-O-methyluridine-3'-phosphorothioate; Tgn is thymidine-diol nucleic acid (GNA) S isomer; and s is a phosphorothioate linkage; wherein the dsRNA agent is conjugated to a ligand having the structure

[0041] and wherein the ligand is conjugated to the 3' end of the sense strand, as shown in the following schematic diagram

[0042] Where X is O.

[0043] In one aspect, the present invention provides a method for preventing at least one symptom in a subject having nonalcoholic steatohepatitis (NASH), the method comprising administering to the subject a dose of about 25 mg to about 800 mg of a double-stranded ribonucleic acid (dsRNA) agent targeting the HSD17B13 gene, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises the nucleotide sequence 5'-asusgcuuUfuGfCfAfuggacuaucu-3' (SEQ ID NO: 24) and the antisense strand comprises the nucleotide sequence 5'-asGfsauag(Tgn)ccaugcAfaAfagcaususc-3' (SEQ ID NO: NO:25), wherein Af is 2'-fluoroadenosine-3'-phosphate; Cf is 2'-fluorocytidine-3'-phosphate; Gf is 2'-fluoroguanosine-3'-phosphate; Gfs is 2'-fluoroguanosine-3'-thiophosphate; U is uridine-3'-phosphate; Uf is 2'-fluorouridine-3'-phosphate; a is 2'-O-methyladenosine-3'-phosphate; as is 2'-O-methyladenosine-3'-thiophosphate; c is 2'-O-methylcytidine-3'-phosphate. '-phosphate; cs is 2'-O-methylcytidine-3'-phosphorothioate; g is 2'-O-methylguanosine-3'-phosphate; gs is 2'-O-methylguanosine-3'-phosphorothioate; u is 2'-O-methyluridine-3'-phosphate; us is 2'-O-methyluridine-3'-phosphorothioate; Tgn is thymidine-diol nucleic acid (GNA) S isomer; and s is a phosphorothioate linkage; wherein the dsRNA agent is conjugated to a ligand having the structure

[0044] and wherein the ligand is conjugated to the 3' end of the sense strand, as shown in the following schematic diagram

[0045] Where X is O.

[0046] In one aspect, the present invention provides a method for reducing the risk of a subject with steatosis developing chronic liver disease, the method comprising administering to the subject a dose of about 25 mg to about 800 mg of a double-stranded ribonucleic acid (dsRNA) agent targeting the HSD17B13 gene, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises the nucleotide sequence 5'-asusgcuuUfuGfCfAfuggacuaucu-3' (SEQ ID NO: 24) and the antisense strand comprises the nucleotide sequence 5'-asGfsauag(Tgn)ccaugcAfaAfagcaususc-3' (SEQ ID NO: NO:25), wherein Af is 2'-fluoroadenosine-3'-phosphate; Cf is 2'-fluorocytidine-3'-phosphate; Gf is 2'-fluoroguanosine-3'-phosphate; Gfs is 2'-fluoroguanosine-3'-thiophosphate; U is uridine-3'-phosphate; Uf is 2'-fluorouridine-3'-phosphate; a is 2'-O-methyladenosine-3'-phosphate; as is 2'-O-methyladenosine-3'-thiophosphate; c is 2'-O-methylcytidine-3'-phosphate. '-phosphate; cs is 2'-O-methylcytidine-3'-phosphorothioate; g is 2'-O-methylguanosine-3'-phosphate; gs is 2'-O-methylguanosine-3'-phosphorothioate; u is 2'-O-methyluridine-3'-phosphate; us is 2'-O-methyluridine-3'-phosphorothioate; Tgn is thymidine-diol nucleic acid (GNA) S isomer; and s is a phosphorothioate linkage; wherein the dsRNA agent is conjugated to a ligand having the structure

[0047] and wherein the ligand is conjugated to the 3' end of the sense strand, as shown in the following schematic diagram

[0048] Where X is O.

[0049] In one aspect, the present invention provides a method for inhibiting the progression of steatosis to steatohepatitis in a subject suffering from steatosis, the method comprising administering to the subject a dose of about 25 mg to about 800 mg of a double-stranded ribonucleic acid (dsRNA) agent targeting the HSD17B13 gene, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises the nucleotide sequence 5'-asusgcuuUfuGfCfAfuggacuaucu-3' (SEQ ID NO: 24) and the antisense strand comprises the nucleotide sequence 5'-asGfsauag(Tgn)ccaugcAfaAfagcaususc-3' (SEQ ID NO: NO:25), wherein Af is 2'-fluoroadenosine-3'-phosphate; Cf is 2'-fluorocytidine-3'-phosphate; Gf is 2'-fluoroguanosine-3'-phosphate; Gfs is 2'-fluoroguanosine-3'-thiophosphate; U is uridine-3'-phosphate; Uf is 2'-fluorouridine-3'-phosphate; a is 2'-O-methyladenosine-3'-phosphate; as is 2'-O-methyladenosine-3'-thiophosphate; c is 2'-O-methylcytidine-3'-phosphate. '-phosphate; cs is 2'-O-methylcytidine-3'-phosphorothioate; g is 2'-O-methylguanosine-3'-phosphate; gs is 2'-O-methylguanosine-3'-phosphorothioate; u is 2'-O-methyluridine-3'-phosphate; us is 2'-O-methyluridine-3'-phosphorothioate; Tgn is thymidine-diol nucleic acid (GNA) S isomer; and s is a phosphorothioate linkage; wherein the dsRNA agent is conjugated to a ligand having the structure

[0050] and wherein the ligand is conjugated to the 3' end of the sense strand, as shown in the following schematic diagram

[0051] Where X is O.

[0052] In one aspect, the present invention provides a method of inhibiting lipid droplet accumulation in the liver of a subject suffering from non-alcoholic steatohepatitis (NASH), the method comprising administering to the subject a dose of about 25 mg to about 800 mg of a double-stranded ribonucleic acid (dsRNA) agent targeting the HSD17B13 gene, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises the nucleotide sequence 5'-asusgcuuUfuGfCfAfuggacuaucu-3' (SEQ ID NO: 24) and the antisense strand comprises the nucleotide sequence 5'-asGfsauag(Tgn)ccaugcAfaAfagcaususc-3' (SEQ ID NO: NO:25), wherein Af is 2'-fluoroadenosine-3'-phosphate; Cf is 2'-fluorocytidine-3'-phosphate; Gf is 2'-fluoroguanosine-3'-phosphate; Gfs is 2'-fluoroguanosine-3'-thiophosphate; U is uridine-3'-phosphate; Uf is 2'-fluorouridine-3'-phosphate; a is 2'-O-methyladenosine-3'-phosphate; as is 2'-O-methyladenosine-3'-thiophosphate; c is 2'-O-methylcytidine-3'-phosphate. '-phosphate; cs is 2'-O-methylcytidine-3'-phosphorothioate; g is 2'-O-methylguanosine-3'-phosphate; gs is 2'-O-methylguanosine-3'-phosphorothioate; u is 2'-O-methyluridine-3'-phosphate; us is 2'-O-methyluridine-3'-phosphorothioate; Tgn is thymidine-diol nucleic acid (GNA) S isomer; and s is a phosphorothioate linkage; wherein the dsRNA agent is conjugated to a ligand having the structure

[0053] and wherein the ligand is conjugated to the 3' end of the sense strand, as shown in the following schematic diagram

[0054] Where X is O.

[0055] In one embodiment, the methods of the present invention further comprise administering to the individual an additional therapeutic agent.

[0056] In some embodiments, the methods of the present invention further comprise determining a NAFLD activity score (NAS), a ballooning score, a lobular inflammation score, a steatosis score, and / or a fibrosis score for the individual.

[0057] In some embodiments, the dsRNA agent is administered to a subject at a dose of about 25 mg, about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, or about 800 mg.

[0058] In some embodiments, the dsRNA agent is administered to the individual every month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, or every 12 months.

[0059] In some embodiments, the dsRNA agent is administered to an individual at a dosage of about 25 mg per month. In some embodiments, the dsRNA agent is administered to an individual at a dosage of about 200 mg per month. In some embodiments, the dsRNA agent is administered to an individual at a dosage of about 400 mg per month.

[0060] In some embodiments, the dsRNA agent is administered to an individual at a dosage of about 25 mg every three months. In some embodiments, the dsRNA agent is administered to an individual at a dosage of about 200 mg every three months. In some embodiments, the dsRNA agent is administered to an individual at a dosage of about 400 mg every three months.

[0061] In some embodiments, the dsRNA agent is administered to the individual intravenously, intramuscularly, or subcutaneously. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 Phase I study design Part A of AD-288996 (ALN-HSD) is schematically depicted.

[0064] a The cohorts were selected in order. b Including a dedicated Japanese cohort.

[0065] Abbreviations: AE, adverse event; AUC, area under the concentration-time curve; BMI, body mass index; C max , maximum plasma concentration; ECG, electrocardiogram; PK, pharmacokinetic; QTcF, Fridericia-corrected QT interval; SAD, single ascending dose; SC, subcutaneous.

[0066] Figure 2 A table is provided providing baseline demographics for individuals enrolled in Part A of the Phase I study of AD-288996 (ALN-HSD).

[0067] a Normal range of ALT: 10-35U / L (female), 10-50U / L (male). b Normal range of AST: 0-31U / L (female), 0-37U / L (male).

[0068] Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; SD, standard deviation.

[0069] Figure 3Schematic depiction of the Phase I study design, Part B, of AD-288996 (ALN-HSD).

[0070] a Cohorts were enrolled sequentially. Patients receiving placebo underwent biopsy at 6 or 12 months. b Biopsy was performed at 12 months instead of 6 months. c One patient discontinued due to elevated liver function tests (due to hepatitis E). d Main reason for study withdrawal: loss to follow-up. NCT04565717.

[0071] Abbreviations: AE, adverse event; BMI, body mass index; CRN, clinical research network; M, month; mRNA, messenger RNA; NAFLD, nonalcoholic fatty liver disease; NAS, NAFLD activity score; NASH, nonalcoholic steatohepatitis; PK, pharmacokinetic; Q12W, every 12 weeks; SC, subcutaneous.

[0072] Figure 4 A table is provided providing baseline demographics for individuals enrolled in Part B of the Phase I study of AD-288996 (ALN-HSD).

[0073] a Normal range of ALT: ≤33U / L (female), ≤41U / L (male). b Normal range of AST: ≤31U / L (female), ≤37U / L (male). c Alleles used: rs62305723:A, rs72613567:TA, and rs80182459:G.

[0074] Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; NAFLD, nonalcoholic fatty liver disease; SD, standard deviation.

[0075] Figure 5 Graph depicting mean plasma concentrations of ALN-HSD at indicated time points following a single subcutaneous dose of 25 mg, 100 mg, 200 mg, 400 mg, or 800 mg of ALN-HSD.

[0076] Figure 6A Figure 1. Graphically depicts trends in improvement in alanine aminotransferase (ALT) levels at designated time points in subjects with NASH who were administered placebo or AD-288996 at doses of 25 mg every 12 weeks (Q12w) x 2, 200 mg Q12w x 2, or 400 mg Q12w x 2 in a Phase 1 study.

[0077] Abbreviations: ALT, alanine aminotransferase; BL, baseline; D, day; SEM, standard error of the mean; W-7 / PRE, within 7 days / before dosing.

[0078] Figure 6B Graphically depicts trends in improvement in aspartate aminotransferase (AST) levels at designated time points in subjects administered placebo or AD-288996 in the Phase I study.

[0079] Figure 7 Table depicting reduction in HSD17B13 mRNA expression in the liver of NASH subjects administered AD-288996 subcutaneously at doses of 25 mg once every 12 weeks (Q12w) x 2, 200 mg Q12w x 2, or 400 mg Q12w x 2, 6 months after the first dose.

[0080] a The percent change from baseline observed for patients who received only the first ALN-HSD dose was -53.7%. b No assessment was performed at month 12. No patient had homozygous protective alleles for any of the three HSD17B13 variants.

[0081] Abbreviations: M, month; mRNA, messenger RNA; N / A, not applicable; SD, standard deviation.

[0082] Figure 8A and Figure 8B Figure 2 is a graph depicting categorical changes from baseline in histological parameters for subjects administered ALN-HSD.

[0083] Figure 8A To characterize the effects of ALN-HSD on the a Figure 3. Numerical reduction in the NAFLD activity score obtained by biopsy at 4 hr post-surgery compared with placebo.

[0084] a A 12-month biopsy was performed in only one of the 200 mg cohorts. b Subcomponents of the total NAFLD activity score. c One of 36 patients in the ALN-HSD group had a second biopsy visit canceled.

[0085] Worsening: change from baseline > 0. Improvement: change from baseline < 0. Abbreviation: NAFLD, nonalcoholic fatty liver disease.

[0086] Figure 8B To characterize the effects of ALN-HSD on the a Figure 3. Graph showing the numerical reduction in fibrosis stage obtained by biopsy at 4 hr relative to placebo.

[0087] a A 12-month biopsy was performed in only one of the 200 mg cohorts. b One of 36 patients in the ALN-HSD group had a second biopsy visit canceled.

[0088] Worsening: Change from baseline > 0. Improvement: Change from baseline < 0. Detailed Description of the Invention

[0090] The present invention is based at least in part on the discovery that administering (e.g., subcutaneously) a RNAi agent targeting the HSD17B13 gene (e.g., AD-288996) to individuals with NASH reduces the level of HSD17B13 mRNA and reduces liver enzymes and biopsy-derived non-alcoholic fatty liver disease (NAFLD) activity score (NAS) within six months.

[0091] The present invention provides iRNA compositions that achieve RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of the HSD17B13 gene. The HSD17B13 gene can be in a cell, for example, in a cell within an individual (such as a human). The present invention also provides methods of using the iRNA compositions of the present invention to inhibit HSD17B13 gene expression and to treat an individual who would benefit from inhibition or reduction of HSD17B13 gene expression, such as an individual who would benefit from reduced liver inflammation, for example, an individual suffering from or susceptible to an HSD17B13-related disease, disorder, or condition, such as an individual suffering from or susceptible to non-alcoholic steatohepatitis (NASH).

[0092] The following detailed description discloses how to make and use compositions containing iRNA to inhibit the expression of the HSD17B13 gene, as well as compositions and methods for treating individuals suffering from diseases and disorders that would benefit from inhibition and / or reduction of expression of this gene.

[0093] I. Definition

[0094] In order that the present invention may be more readily understood, certain terms are first defined. In addition, it should be noted that whenever a value or range of values ​​for a parameter is recited, it is intended that the values ​​and ranges between the recited values ​​are also intended to be part of the present invention.

[0095] "A" and "an" are used herein to refer to one or more than one (ie, at least one) of the grammatical objects of the article. For example, "an element" means one element or more than one element, eg, a plurality of elements.

[0096] The term "comprising" is used herein to mean the phrase "including but not limited to", and they are used interchangeably with each other.

[0097] Unless the context clearly indicates otherwise, the term "or" is used herein to mean the term "and / or" and are used interchangeably with each other.

[0098] The term "about" is used herein to mean within a typical tolerance range in the art. For example, "about" can be understood as about 2 standard deviations from the mean. In certain embodiments, about means ±10%. In certain embodiments, about means ±5%. When about precedes a series of numbers or a range, it is understood that "about" modifies each number in the series or range.

[0099] The term "HSD17B13" is also referred to as "hydroxysteroid 17-beta dehydrogenase 13", "short-chain dehydrogenase / reductase family 16C member", "short-chain dehydrogenase / reductase 9", "17-beta-HSD 13", "17beta-HSD13", "SDR16C3", "SCDR9", "short-chain dehydrogenase / reductase family 16C, member 3", "hydroxysteroid (17-beta) dehydrogenase 13", "17-beta-hydroxysteroid dehydrogenase 13", "17-beta hydroxysteroid dehydrogenase", "HMFN0376" and "NIIL497", which refers to the well-known gene encoding type 13 17beta-hydroxysteroid dehydrogenase protein from any vertebrate or mammalian source, including but not limited to humans, cows, chickens, rodents, mice, rats, pigs, sheep, primates, monkeys and guinea pigs, unless otherwise indicated.

[0100] The term also refers to fragments and variants of native HSD17B13 that retain at least one in vivo or in vitro activity of native HSD17B13. The term encompasses the full-length unprocessed precursor form of HSD17B13 as well as the mature form resulting from post-translational cleavage of the signal peptide and forms resulting from proteolytic processing.

[0101] Two variants of the human HSD17B13 gene have been previously identified: variant A (or transcript A) and variant B (or transcript B). Transcript A includes all seven exons of the HSD17B13 gene, while exon 2 is skipped in transcript B. The nucleotide and amino acid sequences of human HSD17B13 variant A can be found, for example, in GenBank reference sequence: NM_178135.4; SEQ ID NO: 1); and the nucleotide and amino acid sequences of human HSD17B13 variant B can be found, for example, in GenBank reference sequence: NM_001136230.2; SEQ ID NO: 2. As described in U.S. Patent Application No. 15 / 875,514, filed January 19, 2018, and PCT Application No. PCT / US2018 / 014357, filed January 19, 2018, the entire contents of each of which are incorporated herein by reference, six additional expressed HSD17B13 transcripts (C to H, SEQ ID NOs: 17, 18, 19, 20, 21, and 22, respectively) have been identified. In transcript C, exon 6 is skipped compared to transcript A. In transcript D, there is an insertion of a guanine 3' to exon 6 compared to transcript A, resulting in a frameshift and premature truncation of exon 7. In transcript E, there is an additional exon between exons 3 and 4 compared to transcript A. In transcript F, which is expressed only in HSD17B13 rs72613567 variant vectors, there is readthrough from exon 6 to intron 6 compared to transcript A. In transcript G, exon 2 is skipped compared to transcript A, and there is an insertion 3' to a guanine in exon 6, resulting in a frameshift and premature truncation of exon 7. In transcript H, there is an additional exon between exons 3 and 4 compared to transcript A, and there is an insertion 3' to a guanine in exon 6, resulting in a frameshift and premature truncation of exon 7.

[0102] Another HSD17B13 transcript (F', SEQ ID NO: 23) was also identified that was expressed at a low level. Like transcript F, transcript F' also includes readthrough from exon 6 to intron 6 compared to transcript A, but unlike transcript F, this readthrough does not include the inserted thymidine present in the HSD17B13 rs72613567 variant gene. The nucleotide positions of the exons within the HSD17B13 gene for each transcript are provided below.

[0103] SEQ ID NO:15 is the nucleotide sequence of the HSD17B13 wild-type genomic sequence (human genome assembly GRCh38), and SEQ ID NO:16 is the nucleotide sequence of the HSD17B13 genomic sequence variant: T inserted at position 12666 (human genome assembly GRCh38; rs72613567-chr4: T inserted at 87310241-87310240).

[0104] The nucleotide positions of exons of the HSD17B13 transcript that are more prevalent in individuals homozygous for the wild-type HSD17B13 gene are in SEQ ID NO: 15.

[0105] Transcript A Transcript B Transcript E Transcript F' Exon 1 1-275 1-275 1-275 1-275 Exon 2 4471-4578 jump over 4471-4578 4471-4578 Exon 3 5684-5815 5684-5815 5684-5815 5684-5815 Exon 3' does not exist does not exist 6210-6281 does not exist Exon 4 7308-7414 7308-7414 7308-7414 7308-7414 Exon 5 8947-9084 8947-9084 8947-9084 8947-9084 Exon 6 12548-12664 12548-12664 12548-12664 12548-13501* Exon 7 17599-19118 17599-19118 17599-19118 jump over

[0106] *Includes readthrough from exon 6 to intron 6; readthrough = positions 12665-13501

[0107] The nucleotide position of the exon of the HSD17B13 transcript that is more prevalent in individuals homozygous for the rs72613567 HSD17B13 variant gene (T insertion at position 12666) is in SEQ ID NO: 16.

[0108]

[0109] ^Includes additional residues 12665 ​​at the 3' end compared to transcript A

[0110] *Includes readthrough from exon 6 to intron 6; readthrough = positions 12665-13502

[0111] There are two mouse HSD17B13 gene variants. The nucleotide and amino acid sequences of mouse Hsd17b13 transcript variant 1 can be found, for example, in GenBank Reference Sequence: NM_001163486.1 (SEQ ID NO: 3), and the nucleotide and amino acid sequences of mouse Hsd17b13 transcript variant 2 can be found, for example, in GenBank Reference Sequence: NM_198030.2 (SEQ ID NO: 4). The nucleotide and amino acid sequences of the rat Hsd17b13 gene can be found, for example, in GenBank Reference Sequence: NM_001009684.1 (SEQ ID NO: 5). The nucleotide and amino acid sequences of the rhesus macaque (Macaca mulatta) HSD17B13 gene can be found, for example, in GenBank Reference Sequence: XM_015138766.1 (SEQ ID NO: 6). The nucleotide and amino acid sequences of the cynomolgus macaque ( Macaca fascicularis ) HSD17B13 gene can be found, for example, in GenBank reference sequence: XM_005555367.2; SEQ ID NO: 7).

[0112] Other examples of HSD17B13 mRNA sequences are readily available using publicly available databases such as GenBank, UniProt, and OMIM.

[0113] As used herein, the term "HSD17B13" also refers to a specific polypeptide expressed in a cell by naturally occurring DNA sequence variations in the HSD17B13 gene, such as single nucleotide polymorphisms in the HSD17B13 gene. Many SNPs within the HSD17B13 gene have been identified and can be found, for example, in NCBI dbSNP (see, for example, www.ncbi.nlm.nih.gov / snp).

[0114] As used herein, "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during transcription of the HSD17B13 gene, including mRNAs that are products of RNA processing of the primary transcript. In one embodiment, the target portion of the sequence will be at least long enough to serve as a substrate for iRNA-directed cleavage at or near that portion of the nucleotide sequence of the mRNA molecule formed during transcription of the HSD17B13 gene.

[0115] The target sequence of the HSD17B13 gene may be about 9 to 36 nucleotides in length, such as about 15 to 30 nucleotides in length. For example, the target sequence may be about 15 to 30 nucleotides in length, 15 to 29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 1 Intermediate ranges and lengths between the above recited ranges and lengths are also contemplated as part of the invention.

[0116] As used herein, the term "strand comprising a sequence" refers to an oligonucleotide comprising a chain of nucleotides described by a sequence designated using standard nucleotide nomenclature.

[0117] "G," "C," "A," "T," and "U" each generally represent a nucleotide containing guanine, cytosine, adenine, thymidine, and uracil as a base, respectively. However, it should be understood that the term "ribonucleotide" or "nucleotide" may also refer to a modified nucleotide, as further described below, or an alternative replacement moiety (see, for example, Table 1). It is well known to those skilled in the art that guanine, cytosine, adenine, and uracil can be replaced with other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such a replacement moiety. For example, but not limited to, a nucleotide comprising inosine as its base can form base pairs with a nucleotide containing adenine, cytosine, or uracil. Thus, a nucleotide containing uracil, guanine, or adenine can be replaced with a nucleotide containing, for example, inosine in a dsRNA nucleotide sequence characterized in the present invention. In another example, an adenine and a cytosine at any position in an oligonucleotide can be replaced with a guanine and a uracil, respectively, to form a GU wavy base pair with a target mRNA. Sequences containing such replacement moieties are suitable for use in the compositions and methods featured in the invention.

[0118] The terms "iRNA," "RNAi agent," "iRNA agent," and "RNA interfering agent," as used interchangeably herein, refer to agents that contain RNA, as such terms are defined herein, and that mediate targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway. iRNAs direct sequence-specific degradation of mRNAs via a process known as RNA interference (RNAi). iRNAs modulate, for example, inhibit expression of the HSD17B13 gene in cells, for example, in cells within an individual (such as a mammalian individual).

[0119] In one embodiment, the RNAi agent of the present invention comprises a single-stranded RNA that interacts with a target RNA sequence (e.g., an HSD17B13 target mRNA sequence) to guide the cleavage of the target RNA. Without wishing to be bound by theory, it is believed that long double-stranded RNA introduced into cells is broken down into siRNA by a type III endonuclease called Dicer (Sharp et al., (2001) Genes Dev. 15:485). Dicer, an enzyme similar to ribonuclease III, processes dsRNA into 19-23 base pair short interfering RNAs with a characteristic two-base 3' overhang (Bernstein et al., (2001) Nature 409:363). The siRNA is then incorporated into an RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA double helix, enabling the complementary antisense strand to guide target recognition (Nykanen et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within RISC cleave the target to induce silencing (Elbashir et al., (2001) Genes Dev. 15:188). Thus, in one aspect, the present invention relates to single-stranded RNA (sssiRNA) produced within a cell that promotes the formation of the RISC complex to achieve silencing of a target gene (also, the HSD17B13 gene). Thus, the term "siRNA" is also used herein to refer to RNAi as described above.

[0120] In another embodiment, the RNAi agent may be a single-stranded RNAi agent introduced into a cell or organism to inhibit a target mRNA. Single-stranded RNAi agents (ssRNAi) bind to the RISC endonuclease Argonaute 2, which then cleaves the target mRNA. Single-stranded siRNAs are generally 15 to 30 nucleotides and chemically modified. The design and testing of single-stranded RNAi agents are described in U.S. Patent No. 8,101,348 and Lima et al., (2012) Cell 150:883-894, the entire contents of each of which are hereby incorporated herein by reference. Any of the antisense nucleotide sequences described herein can be used as a single-stranded siRNA as described herein, or chemically modified as described by Lima et al., (2012) Cell 150:883-894.

[0121] In another embodiment, the "iRNA" used in the compositions and methods of the present invention is double-stranded RNA and is referred to herein as a "double-stranded RNAi agent," "double-stranded RNA (dsRNA) molecule," "dsRNA agent," or "dsRNA." The term "dsRNA" refers to a complex of ribonucleic acid molecules having a double helix structure comprising two antiparallel and substantially complementary nucleic acid strands that are referred to as having "sense" and "antisense" orientations relative to the target RNA (i.e., the HSD17B13 gene). In some embodiments of the present invention, double-stranded RNA (dsRNA) triggers degradation of a target RNA (e.g., mRNA) via a post-transcriptional gene silencing mechanism, referred to herein as RNA interference or RNAi.

[0122] In general, most of the nucleotides in each chain of the dsRNA molecule are ribonucleotides, but as described in detail herein, each chain or both chains may also include one or more non-ribonucleotides, such as deoxyribonucleotides and / or modified nucleotides. In addition, as used in this specification, "RNAi agents" may include ribonucleotides with chemical modifications; RNAi agents may include substantial modifications in multiple nucleotides. As used herein, the term "modified nucleotides" refers to nucleotides with independently modified sugar moieties, modified internucleotide linkages and / or modified nucleobases. Therefore, the term modified nucleotides encompasses substitutions, additions or removals of, for example, functional groups or atoms, of internucleoside bonds, sugar moieties or nucleobases. Modifications suitable for use in the agents of the present invention include all types of modifications disclosed herein or known in the art. Any such modifications as used in siRNA-type molecules are encompassed in "RNAi agents" for the purposes of this specification and the scope of the claims.

[0123] The duplex region can be of any length that allows for specific degradation of the desired target RNA via the RISC pathway, and can be in the range of about 9 to 36 base pairs in length, for example, about 15 to 30 base pairs in length, for example, about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length, such as about 15 to 30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-29, 15-2 7, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-2 21-24, 21 to 23 or 21 to 22 base pairs. Intermediate ranges and lengths between the above-recited ranges and lengths are also contemplated as part of the invention.

[0124] The two strands forming the double helix structure may be different parts of a larger RNA molecule, or they may be separate RNA molecules. Where the two strands are part of a larger molecule and are therefore connected without interruption by nucleotides between the 3' end of one strand and the 5' end of the corresponding other strand forming the double helix structure, the connected RNA strands are referred to as "hairpin loops". The hairpin loop may include at least one unpaired nucleotide. In some embodiments, the hairpin loop may include 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.

[0125] Where the two substantially complementary strands of a dsRNA are composed of separate RNA molecules, those molecules need not be but may be covalently linked. The connection between the two strands is by means of covalent linkage, rather than by nucleotides on one strand from the 3'-end of one strand to the 5'-end of the other strand forming a double helix structure without interrupting the strands. This connection structure is referred to as a "linker." RNA strands may have the same or different numbers of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs present in the double helix. In addition to the double helix structure, RNAi may include one or more nucleotide overhangs.

[0126] In one embodiment, the RNAi agent of the present invention is a dsRNA, wherein each chain comprises less than 30 nucleotides, such as 17 to 27, 19 to 27, 17 to 25, 19 to 25, or 19 to 23 nucleotides, which interact with a target RNA sequence (e.g., a HSD17B13 target mRNA sequence) to guide the cleavage of the target RNA. In another embodiment, the RNAi agent of the present invention is a dsRNA, wherein each chain comprises 19 to 23 nucleotides, which interact with a target RNA sequence (e.g., a HSD17B13 target mRNA sequence) to guide the cleavage of the target RNA. In one embodiment, the sense strand is 21 nucleotides in length. In another embodiment, the antisense strand is 23 nucleotides in length.

[0127] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that protrudes beyond the duplex structure of an iRNA, such as 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. A dsRNA may comprise an overhang of at least one nucleotide; alternatively, the overhang may comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more. The nucleotide overhang may comprise or consist of nucleotide / nucleoside analogs, including deoxynucleotides / nucleosides. The multiple overhangs may be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the multiple nucleotides of the overhang may be present on the 5' end, the 3' end, or both ends of the antisense strand or the sense strand of the dsRNA.

[0128] In one embodiment, the antisense strand of the dsRNA has 1-10 nucleotides, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, overhanging at the 3' end and / or the 5' end. In one embodiment, the sense strand of the dsRNA has 1 to 10 nucleotides, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, overhanging at the 3' end and / or the 5' end. In another embodiment, one or more of the nucleotides in the overhang are substituted with phosphorothioate nucleosides.

[0129] In certain embodiments, the overhang on the sense strand or the antisense strand or both may comprise an extension length greater than 10 nucleotides, for example, 10-30 nucleotides, 10-25 nucleotides, 10-20 nucleotides, or 10-15 nucleotides in length. In certain embodiments, the extension overhang is located on the sense strand of the duplex. In certain embodiments, the extension overhang is present at the 3' end of the sense strand of the duplex. In certain embodiments, the extension overhang is present at the 5' end of the sense strand of the duplex. In certain embodiments, the extension overhang is on the antisense strand of the duplex. In certain embodiments, the extension overhang is present at the 3' end of the antisense strand of the duplex. In certain embodiments, the extension overhang is present at the 5' end of the antisense strand of the duplex. In certain embodiments, one or more of the nucleotides in the extension overhang are substituted with phosphorothioate nucleosides.

[0130] As used herein with respect to dsRNA, the term "blunt" or "blunt ended" refers to the absence of unpaired nucleotides or nucleotide analogs at a given end of the dsRNA, i.e., the absence of nucleotide overhangs. One or both ends of a dsRNA may be blunt. Where both ends of a dsRNA are blunt, the dsRNA is referred to as blunt-ended. For clarity, a "blunt-ended" dsRNA is a dsRNA that is blunt at both ends, i.e., there are no nucleotide overhangs at either end of the molecule. Typically, such molecules will be double-stranded throughout their entire length.

[0131] The term "antisense strand" or "guide strand" refers to the strand of an iRNA (eg, dsRNA) that includes a region that is substantially complementary to a target sequence, eg, HSD17B13 mRNA.

[0132] As used herein, the term "complementarity region" refers to a region on the antisense strand that is substantially complementary to a sequence as defined herein (e.g., a target sequence, such as an HSD17B13 nucleotide sequence). When the complementary region is not completely complementary to the target sequence, the mismatch may be in an internal or terminal region of the molecule. Generally, the most tolerated mismatch is in the terminal region, for example, within 5, 4, 3, or 2 nucleotides of the 5' and / or 3' end of the iRNA.

[0133] As used herein, the term "sense strand" or "passenger strand" refers to the strand of an iRNA that comprises a region that is substantially complementary to a region of the antisense strand as defined herein.

[0134] As used herein, the term "cleavage zone" refers to a region located adjacent to a cleavage site. A cleavage site is a site on a target where cleavage occurs. In some embodiments, the cleavage zone includes three bases located on either end of the cleavage site and adjacent to the cleavage site. In some embodiments, the cleavage zone includes two bases located at either end of the cleavage site and adjacent to the cleavage site. In some embodiments, the cleavage site specifically occurs at the site where nucleotides 10 and 11 of the antisense strand bind, and the cleavage zone includes nucleotides 11, 12, and 13.

[0135] As used herein, unless otherwise indicated, the term "complementary" when used to describe a first nucleotide sequence relative to a second nucleotide sequence, as will be understood by the skilled artisan, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure with an oligonucleotide or polynucleotide comprising the second nucleotide sequence under specific conditions. Such conditions may be, for example, stringent conditions, wherein stringent conditions may comprise: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. for 12-16 hours, followed by washing (see, for example, Molecular Cloning: A Laboratory Manual, Sambrook et al. (1989) Cold Spring Harbor Laboratory Press). Other conditions may be applicable, such as physiologically relevant conditions that may be encountered inside an organism. One skilled in the art will be able to determine a set of conditions that are most suitable for testing the complementarity of two sequences, depending on the ultimate application of the hybridizing nucleotides.

[0136] Complementary sequences within an iRNA, such as within a dsRNA as described herein, comprise base pairing an oligonucleotide or polynucleotide comprising a first nucleotide sequence to an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the full length of one or both nucleotide sequences. Such sequences may be referred to herein as "fully complementary" to one another. However, when a first sequence is referred to herein as "substantially complementary" to a second sequence, the two sequences may be fully complementary, or they may form one or more, but typically no more than 5, 4, 3, or 2, mismatched base pairs when a duplex of up to 30 base pairs hybridizes, while retaining the ability to hybridize under conditions most relevant to its ultimate application, such as inhibition of gene expression via the RISC pathway. However, when two oligonucleotides are designed to form one or more single-stranded overhangs after hybridization, such overhangs should not be considered mismatches for purposes of complementarity determination. For example, for the purposes described herein, a dsRNA comprising an oligonucleotide of 21 nucleotides in length and a dsRNA comprising another oligonucleotide of 23 nucleotides in length, wherein the longer oligonucleotide comprises a 21 nucleotide sequence that is fully complementary to the shorter oligonucleotide, may still be referred to as "fully complementary."

[0137] As used herein, "complementary" sequences may also include, or be formed entirely of, non-Watson-Crick base pairs and / or base pairs formed by non-natural and modified nucleotides, as long as the above requirements regarding their hybridization ability are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble or Hoostein base pairing.

[0138] As will be understood from the context of their use, the terms "complementary," "fully complementary," and "substantially complementary" can be used with respect to base matching between the sense and antisense strands of a dsRNA, or between the antisense strand of an iRNA agent and a target sequence.

[0139] As used herein, a polynucleotide that is "substantially complementary to at least a portion of a messenger RNA (mRNA)" refers to a polynucleotide that is substantially complementary to a contiguous portion of an mRNA of interest (e.g., an mRNA encoding HSD17B13). For example, if a polynucleotide is substantially complementary to an uninterrupted portion of an mRNA encoding HSD17B13, then the sequence is complementary to at least a portion of the HSD17B13 mRNA.

[0140] Thus, in some embodiments, the antisense polynucleotides disclosed herein are fully complementary to the target HSD17B13 sequence. In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to the target HSD17B13 sequence and comprise a contiguous nucleotide sequence that is at least about 80% complementary to the equivalent region of the nucleotide sequence of SEQ ID NO: 1 or a fragment of SEQ ID NO: 1, such as about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary over its entire length.

[0141] In one embodiment, the RNAi agent of the invention comprises a sense strand that is substantially complementary to an antisense polynucleotide that is in turn complementary to a target HSD17B13 sequence, and wherein the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least about 80% complementary over its entire length to an equivalent region of the nucleotide sequence of SEQ ID NO: 8 or any fragment of SEQ ID NO: 8, such as about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary.

[0142] In some embodiments, the iRNA of the invention includes an antisense strand that is substantially complementary to a target HSD17B13 sequence and comprises a contiguous nucleotide sequence that is at least about 80% complementary to the equivalent region of the nucleotide sequence of any sense strand in Table 2 or a fragment of any sense strand in Table 2, such as about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementary, or 100% complementary over its entire length.

[0143] As used herein, the term "inhibiting" is used interchangeably with "reducing," "silencing," "downregulating," "suppressing," and other similar terms, and encompasses any degree of inhibition.

[0144] As used herein, the phrase "inhibiting the expression of the HSD17B13 gene" includes inhibiting the expression of any HSD17B13 gene (such as a mouse HSD17B13 gene, a rat HSD17B13 gene, a monkey HSD17B13 gene, or a human HSD17B13 gene) and HSD17B13 gene variants or mutants encoding HSD17B13 proteins.

[0145] "Inhibiting the expression of the HSD17B13 gene" includes any degree of inhibition of the HSD17B13 gene, for example, at least partial suppression of the expression of the HSD17B13 gene, such as inhibition of at least about 20%. In certain embodiments, inhibition is at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0146] The expression of the HSD17B13 gene can be assessed based on the level of any variable associated with HSD17B13 gene expression, such as HSD17B13 mRNA levels or HSD17B13 protein levels. The expression of the HSD17B13 gene can also be assessed indirectly based on, for example, the level of circulating alanine aminotransferase (ALT) in a tissue sample (such as a liver sample) or the enzymatic activity of HSD17B13. Inhibition can be assessed by a decrease in the absolute or relative level of one or more of these variables compared to a control level. The control level can be any type of control level used in the art, for example, a pre-dose baseline level, or a level determined by a similar individual, cell, or sample that has not been treated or that has been treated with a control (such as, for example, a buffer-only control or an inactive agent control).

[0147] In one embodiment, at least partial suppression of HSD17B13 gene expression is assessed by a decrease in the amount of HSD17B13 mRNA isolatable from or detectable in a first cell or cell population in which the HSD17B13 gene is transcribed and which has been treated such that expression of the HSD17B13 gene is inhibited, compared to a second cell or cell population that is substantially identical to the first cell or cell population but has not been treated (control cells).

[0148] The degree of inhibition can be expressed as follows:

[0149]

[0150] As used herein, the phrase "contacting a cell with an RNAi agent," such as dsRNA, includes contacting the cell by any possible means. Contacting the cell with the RNAi agent includes contacting the cell with the iRNA in vitro or contacting the cell with the iRNA in vivo. Contact can be performed directly or indirectly. Thus, for example, the RNAi agent can be physically contacted with the cell by the individual performing the method, or the RNAi agent can be placed in a situation that allows or enables subsequent contact with the cell.

[0151] Cells can be contacted in vitro, for example, by incubating the cells with an RNAi agent. Cells can be contacted in vivo, for example, by injecting the RNAi agent into or near the tissue where the cells are located, or by injecting the RNAi agent into another area, such as the bloodstream or subcutaneous space, so that the agent will then reach the tissue where the cells are to be contacted. For example, the RNAi agent can contain and / or be coupled to a ligand, such as GalNAc3, which directs the RNAi agent to the site of interest, such as the liver. Combinations of in vitro and in vivo contact methods are also possible. For example, cells can also be contacted with an RNAi agent in vitro and then transplanted into an individual.

[0152] In one embodiment, contacting a cell with an iRNA comprises "introducing" or "delivering the iRNA into a cell" by promoting or influencing its uptake or absorption by the cell. The absorption or uptake of the iRNA can occur via spontaneous diffusion or active cellular processes, or by an adjuvant or device. The iRNA can be introduced into the cell in vitro and / or in vivo. For example, for in vivo introduction, the iRNA can be injected into a tissue site or administered systemically. In vivo delivery can also be performed by a β-glucan delivery system, such as those described in U.S. Patents 5,032,401 and 5,607,677 and U.S. Publication No. 2005 / 0281781, the entire contents of which are hereby incorporated herein by reference. In vitro introduction into cells includes methods known in the art, such as electroporation and lipofection. Other methods are described below and / or are methods known in the art.

[0153] The term "lipid nanoparticle" or "LNP" refers to a vesicle comprising a lipid layer that encapsulates a pharmaceutically active molecule, such as a nucleic acid molecule, e.g., an iRNA or a plasmid from which an iRNA is transcribed. LNPs are described, for example, in U.S. Patent Nos. 6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents of which are hereby incorporated by reference herein.

[0154] As used herein, a "subject" is an animal, such as a mammal, including primates (such as humans, non-human primates, e.g., monkeys and chimpanzees), non-primates (such as cows, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, mice, horses, and whales), or birds (e.g., ducks or geese).

[0155] In one embodiment, the subject is a human, such as a human being treated or evaluated for a disease, disorder, or condition that would benefit from reduced expression of HSD17B13; a human at risk for a disease, disorder, or condition that would benefit from reduced expression of HSD17B13; a human suffering from a disease, disorder, or condition that would benefit from reduced expression of HSD17B13; and / or a human being treated for a disease, disorder, or condition that would benefit from reduced expression of HSD17B13, as described herein.

[0156] In one embodiment, the individual is homozygous for a gene encoding a functional HSD17B13 protein. In another embodiment, the individual is heterozygous for a gene encoding a functional HSD17B13 protein. In yet another embodiment, the individual is heterozygous for a gene encoding a functional HSD17B13 protein and a gene encoding a loss-of-function variant of HSD17B13. In another embodiment, the individual is not a carrier of the HSD17B13 rs72613567 variant, e.g., HSD17B13 rs72613567:TA.

[0157] As used herein, the term "treating" or "treatment" refers to beneficial or desired results, including but not limited to alleviating or improving one or more symptoms associated with HSD17B13 gene expression and / or HSD17B13 protein production, for example, HSD17B13-related diseases, such as chronic fibroinflammatory liver diseases, for example, liver inflammation, liver fibrosis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), cirrhosis, alcoholic steatohepatitis (ASH), alcoholic liver disease (ALD), HCV-related cirrhosis, drug-induced liver damage, hepatocellular necrosis and / or hepatocellular carcinoma. "Treatment" can also mean prolonging survival compared to expected survival in the absence of treatment.

[0158] In the case of an HSD17B13-associated disease, the term "lower" refers to a statistically significant decrease in such levels. The decrease can be, for example, at least 10%, at least 15%, at least 20%, at least 25%, 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%, at least 90%, at least 95%, or more. In certain embodiments, the decrease is at least 20%. In the case of HSD17B13 levels in an individual, "lower" preferably refers to a decrease in levels that are within the normal range for individuals without such a disorder.

[0159] As used herein, "prevention" or "preventing" when used with respect to a disease, disorder, or condition that would benefit from reduced HSD17B13 gene expression means reducing the likelihood that a subject will develop symptoms associated with such a disease, disorder, or condition, e.g., symptoms of HSD17B13 gene expression, such as liver inflammation, liver fibrosis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), cirrhosis, alcoholic steatohepatitis (ASH), alcoholic liver disease (ALD), HCV-related cirrhosis, drug-induced liver damage, hepatocellular necrosis, and / or hepatocellular carcinoma. Failure to develop a disease, disorder, or condition, or a reduction in the development of symptoms associated with such a disease, disorder, or condition (e.g., a reduction of at least about 10% according to a clinically recognized scale for the disease or condition), or a delay in the onset of symptoms (e.g., a reduction in lipid accumulation in the liver and / or fat droplet expansion in the liver) for a period of time (e.g., days, weeks, months, or years) is considered effective prevention.

[0160] As used herein, the term "HSD17B13-related disease" refers to a disease or condition caused by or associated with HSD17B13 gene expression or HSD17B13 protein production. The term "HSD17B13-related disease" includes diseases, disorders, or conditions that would benefit from reduced HSD17B13 gene expression or protein activity.

[0161] In one embodiment, an "HSD17B13-associated disease" is a chronic fibroinflammatory liver disease. A "chronic fibroinflammatory liver disease" is any disease, disorder, or condition associated with chronic hepatitis and / or fibrosis. Non-limiting examples of chronic fibroinflammatory liver diseases include, for example, liver inflammation, liver fibrosis, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), cirrhosis, alcoholic steatohepatitis (ASH), alcoholic liver disease (ALD), HCV-related cirrhosis, drug-induced liver damage, hepatocellular necrosis, and / or hepatocellular carcinoma.

[0162] As used herein, "therapeutically effective amount" is intended to include an amount of a RNAi agent that, when administered to an individual suffering from a disease, disorder, or symptom associated with HSD17B13, is sufficient to effectively treat the disease (e.g., by slowing, ameliorating, or maintaining an existing disease or one or more disease symptoms). The "therapeutically effective amount" may vary depending on the RNAi agent, how the agent is administered, the disease and its severity, and the medical history, age, weight, family history, genetic makeup, type of previous or concomitant treatment (if any), and other individual characteristics of the individual to be treated.

[0163] As used herein, a "prophylactically effective amount" is intended to include an amount of an iRNA that, when administered to an individual suffering from an HSD17B13-associated disease, disorder, or symptom, is sufficient to prevent or ameliorate the disease or one or more symptoms of the disease. Amelioration of a disease includes slowing the course of the disease or reducing the severity of a disease that subsequently develops. A "prophylactically effective amount" may vary depending on the iRNA, how the agent is administered, the degree of disease risk, medical history, age, weight, family history, genetic makeup, type of previous or concomitant treatment, if any, and other individual characteristics of the patient to be treated.

[0164] "Therapeutically effective amount" or "prophylactically effective amount" also includes the amount of RNAi agent that produces some desired local or systemic effect at a reasonable benefit / risk ratio applicable to any treatment. The iRNA used in the methods of the present invention can be administered in sufficient amount to produce a reasonable benefit / risk ratio applicable to such treatment.

[0165] As used herein, the phrase "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 subjects and animal subjects without excessive toxicity, irritation, allergic response, or other problems or complications, consistent with a reasonable benefit / risk ratio.

[0166] As used herein, the phrase "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, magnesium talc, calcium or zinc stearate or stearic acid), or solvent for an encapsulating material, which participates in carrying the compound of the invention from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject being treated. Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose, and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants such as magnesium sulfate, sodium lauryl sulfate, and talc; (8) excipients such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols such as propylene glycol; (11) 1) polyols, such as glycerol, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) pH buffered solutions; (21) polyesters, polycarbonates, and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids; (23) serum components, such as serum albumin, HDL, and LDL; and (22) other nontoxic, compatible substances used in pharmaceutical formulations.

[0167] As used herein, the term "sample" includes a group of similar fluids, cells or tissues separated from an individual, as well as fluids, cells or tissues present in an individual. Examples of biological fluids include blood, serum and semen, plasma, cerebrospinal fluid, ocular fluid, lymph, urine, saliva and the like. Tissue samples may include samples from tissues, organs or local areas. For example, a sample may be derived from a specific organ, part of an organ or the fluid or cells within these organs. In certain embodiments, a sample may be derived from the liver (e.g., the entire liver or certain liver segments or certain types of cells in the liver, such as, for example, hepatocytes). In some embodiments, a "sample derived from an individual" refers to blood or plasma extracted from an individual.

[0168] II. Method of the Invention

[0169] The present invention provides methods for treating or preventing at least one symptom in an individual suffering from a disorder that would benefit from reduced HSD17B13 expression, such as an HSD17B13-associated disease, such as a chronic fibroinflammatory disease, such as nonalcoholic steatohepatitis (NASH).

[0170] The present invention also provides methods for reducing the risk of developing chronic liver disease in an individual with steatosis, methods for inhibiting the progression of steatosis to steatohepatitis in an individual with steatosis, or methods for inhibiting lipid droplet accumulation in the liver of an individual with nonalcoholic steatohepatitis (NASH).

[0171] The method comprises administering to the individual a dose of about 25 mg to about 800 mg of a dsRNA agent of the invention. In some embodiments, the dsRNA agent is administered to the individual at a dose of about 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700 or 800 mg.

[0172] In one embodiment, the HSD17B13-associated disease, disorder or condition is a chronic fibroinflammatory liver disease. Non-limiting examples of chronic fibroinflammatory liver diseases include cancer (e.g., cancer such as hepatocellular carcinoma), non-alcoholic steatohepatitis (NASH), cirrhosis, liver inflammation, hepatocellular necrosis, liver fibrosis and non-alcoholic fatty liver disease (NAFLD).

[0173] In one aspect, the present invention also provides the use of an iRNA agent (e.g., dsRNA) or a pharmaceutical composition comprising an iRNA agent that inhibits HSD17B13 expression of the present invention, in combination with a dsRNA targeting the PNPLA3 gene or a pharmaceutical composition comprising such an agent, for treating a subject, e.g., a subject who would benefit from reduced and / or inhibition of HSD17B13 expression, e.g., a subject with an HSD17B13-associated disease, e.g., a subject with a chronic fibroinflammatory disease, e.g., a subject with non-alcoholic steatohepatitis (NASH).

[0174] In one aspect, the present invention also provides the use of an iRNA agent (e.g., dsRNA) targeting the HSD17B13 gene of the present invention, or a pharmaceutical composition comprising an iRNA agent targeting the HSD17B13 gene, in combination with a dsRNA targeting the PNPLA3 gene, or a pharmaceutical composition comprising such an agent, for preventing at least one symptom in an individual suffering from a disorder that would benefit from reduced HSD17B13 expression, such as a chronic fibroinflammatory disease, such as non-alcoholic steatohepatitis (NASH).

[0175] The combination methods of the present invention for treating individuals (e.g., human individuals) with HSD17B13-associated diseases, disorders, or conditions, such as chronic fibroinflammatory liver diseases, e.g., NASH, can be used to treat such individuals because silencing PNPLA3 reduces steatosis (i.e., liver fat), while silencing HSD17B13 reduces inflammation and fibrosis. For example, genome-wide association studies have demonstrated that silencing PNPLA3 and HSD17B13 has an additive effect in reducing NASH pathology. Indeed, in individuals with pathogenic PNPLA3 alleles, protective loss-of-function HSD17B13 alleles were found to be associated with a lower prevalence of NASH. In individuals with a wild-type PNPLA3 allele, who are at lower risk for NASH, increasing the presence of the loss-of-function HSD17B13 allele conferred even greater protection.

[0176] Thus, in one aspect, the present invention provides methods for treating an individual having a condition that would benefit from reduced HSD17B13 expression, e.g., an HSD17B13-associated disease, such as a chronic fibroinflammatory liver disease, e.g., nonalcoholic steatohepatitis (NASH), cancer (e.g., hepatocellular carcinoma), cirrhosis, liver inflammation, hepatocellular necrosis, liver fibrosis, and nonalcoholic fatty liver disease (NAFLD). In one embodiment, the chronic fibroinflammatory liver disease is NASH.

[0177] The combination treatment methods (and uses) of the present invention comprise administering to a subject (e.g., a human subject) a therapeutically effective amount of a dsRNA agent that inhibits HSD17B13 expression or a pharmaceutical composition comprising a dsRNA that inhibits HSD17B13 expression and a dsRNA agent that inhibits PNPLA3 expression or a pharmaceutical composition comprising a dsRNA that inhibits PNPLA3 expression, thereby treating the subject.

[0178] In one aspect, the present invention provides a method for preventing at least one symptom in a subject suffering from a disorder that would benefit from reduced HSD17B13 expression, such as a chronic fibroinflammatory disease, such as NASH. The method comprises administering to the subject a prophylactically effective amount of a dsRNA agent that inhibits HSD17B13 expression, or a pharmaceutical composition comprising a dsRNA that inhibits HSD17B13 expression, and a dsRNA agent that inhibits PNPLA3 expression, or a pharmaceutical composition comprising a dsRNA that inhibits PNPLA3 expression, thereby preventing at least one symptom in the subject.

[0179] Additionally, the present invention provides methods for inhibiting lipid droplet accumulation and / or expansion in cells, such as cells of a subject, for example, hepatocytes. The methods comprise contacting the cells with an RNAi agent or a pharmaceutical composition comprising an iRNA agent of the present invention and an iRNA agent targeting the PNPLA3 gene and / or a pharmaceutical composition comprising an iRNA agent targeting PNPLA3. In some embodiments, the cells are maintained for a sufficient period of time to achieve degradation of mRNA transcripts of the HSD17B13 and PNPLA3 genes.

[0180] Suitable agents targeting the PNPLA3 gene are described, for example, in U.S. Patent Publication No. 2017 / 0340661, the entire contents of which are incorporated herein by reference.

[0181] In the methods of the invention, cells may be contacted in vitro or in vivo, that is, the cells may be within an individual.

[0182] Cells suitable for treatment using the methods of the present invention can be any cell that expresses the HSD17B13 gene (and, in some embodiments, the PNPLA3 gene). Cells suitable for use in the methods of the present invention can be mammalian cells, for example, primate cells (such as human cells or non-human primate cells, for example, monkey cells or chimpanzee cells), non-primate cells (such as bovine cells, porcine cells, camel cells, llama cells, horse cells, goat cells, rabbit cells, sheep cells, hamster cells, guinea pig cells, cat cells, dog cells, rat cells, mouse cells, lion cells, tiger cells, bear cells, or buffalo cells), bird cells (for example, duck cells or goose cells), or whale cells. In one embodiment, the cell is a human cell, for example, a human hepatocyte.

[0183] HSD17B13 expression in the cells is inhibited by at least about 5%, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 1, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or about 100%. In preferred embodiments, HSD17B13 expression is inhibited by at least 20%.

[0184] In some embodiments, PNPLA3 expression in the cells is also inhibited by at least about 5%, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83 99%, or about 100%. In preferred embodiments, PNPLA3 expression is inhibited by at least 20%.

[0185] In one embodiment, the in vivo methods of the present invention can include administering to a subject a composition comprising an iRNA, wherein the iRNA comprises a nucleotide sequence complementary to at least a portion of an RNA transcript of an HSD17B13 gene of a mammal to be treated.

[0186] In another embodiment, the in vivo methods of the invention can comprise administering to a subject a composition comprising a first iRNA agent and a second iRNA agent, wherein the first iRNA comprises a nucleotide sequence complementary to at least a portion of an RNA transcript of an HSD17B13 gene of a mammal to be treated, and the second iRNA comprises a nucleotide sequence complementary to at least a portion of an RNA transcript of a PNPLA3 gene of a mammal to be treated.

[0187] When the organism to be treated is a mammal such as a human, the composition can be administered by any means known in the art, including but not limited to oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal, and intrathecal), intravenous, intramuscular, subcutaneous, transdermal, respiratory (aerosol), nasal, rectal, and topical (including buccal and sublingual) administration. In certain embodiments, the composition is administered by intravenous infusion or injection. In certain embodiments, the composition is administered by subcutaneous injection.

[0188] In some embodiments, administration is via depot injection. Depot injection can release the iRNA in a consistent manner over a long period of time. Thus, depot injection can reduce the frequency of dosing required to achieve a desired effect, such as desired inhibition of HSD17B13, or a therapeutic or preventive effect. Depot injection can also provide more consistent serum concentrations. Depot injection can comprise subcutaneous injection or intramuscular injection. In a preferred embodiment, the depot injection is subcutaneous injection.

[0189] In some embodiments, administration is via a pump. The pump can be an external pump or a surgically implanted pump. In certain embodiments, the pump is an osmotic pump that is implanted subcutaneously. In other embodiments, the pump is an infusion pump. Infusion pumps can be used for intravenous, subcutaneous, intraarterial, or epidural infusion. In preferred embodiments, the infusion pump is a subcutaneous infusion pump. In other embodiments, the pump is a surgically implanted pump that delivers the iRNA to the liver.

[0190] The iRNA of the present invention may be present in a pharmaceutical composition, such as in a suitable buffer solution. The buffer solution may include acetate, citrate, propylamine, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate buffered saline (PBS). The pH and osmotic pressure of the buffer solution containing the iRNA can be adjusted to make it suitable for administration to a subject.

[0191] Alternatively, the iRNA of the invention can be administered as a pharmaceutical composition, such as a dsRNA liposomal formulation.

[0192] The mode of administration can be selected based on whether local or systemic treatment is desired and based on the area to be treated. The route and site of administration can be selected to enhance targeting.

[0193] In one aspect, the present invention also provides a method for inhibiting the expression of the HSD17B13 gene in a mammal, comprising administering to the mammal a composition comprising a dsRNA targeting the HSD17B13 gene in a cell of the mammal, thereby inhibiting the expression of the HSD17B13 gene in the cell.

[0194] In some embodiments, the method comprises administering to a mammal a composition comprising a dsRNA targeting an HSD17B13 gene in a mammalian cell, thereby inhibiting the expression of the HSD17B13 gene in the cell. In another embodiment, the method comprises administering to a mammal a pharmaceutical composition comprising a dsRNA agent targeting an HSD17B13 gene in a mammalian cell.

[0195] Reduction in gene expression can be assessed by any method known in the art and by methods described herein (e.g., qRT-PCR). Reduction in protein production can be assessed by any method known in the art and by methods described herein (e.g., ELISA, enzyme activity). For example, reduction in HSD17B13 expression can be determined by measuring HSD17B13 mRNA expression using methods conventional to those skilled in the art, such as Northern blotting or qRT-PCR; or by measuring HSD17B13 protein levels using methods conventional to those skilled in the art, such as Western blotting or immunoassays. Reduction in HSD17B13 expression can also be assessed indirectly by measuring a reduction in HSD17B13 biological activity in plasma or tissue samples, such as a reduction in HSD17B13 enzymatic activity and / or a reduction in one or more of lipids, triglycerides, cholesterol (including LDL-C, HDL-C, VLDL-C, IDL-C, and total cholesterol), or free fatty acids, and / or a reduction in fat accumulation and / or lipid droplet expansion in the liver.

[0196] In some embodiments, the method further comprises determining the HSD17B13 and / or PNPLA3 genotype of the individual.

[0197] In one embodiment, the individual is heterozygous for the gene encoding the patatin-like phospholipase domain-containing protein 3 (PNPLA3) I148M variant or the PNPLA3:rs738409:G allele. In another embodiment, the individual is homozygous for the gene encoding the PNPLA3 I148M variant or the PNPLA3:rs738409:G allele. In one embodiment, the individual is heterozygous for the gene encoding the patatin-like phospholipase domain-containing protein 3 (PNPLA3) I144M variant. In another embodiment, the individual is homozygous for the gene encoding the PNPLA3 I144M variant.

[0198] In one embodiment, the individual is homozygous for the gene encoding a functional HSD17B13 protein. In another embodiment, the individual is heterozygous for the gene encoding a functional HSD17B13 protein. In yet another embodiment, the individual is heterozygous for the gene encoding a functional HSD17B13 protein and the gene encoding a loss-of-function variant of HSD17B13. In another embodiment, the individual is not a carrier of the HSD17B13 rs72613567 variant. In some embodiments, the individual is homozygous for the HSD17B13:rs72613567:T allele, the HSD17B13:rs62305723:G allele, and / or the HSD17B13:rs80182459:GG allele. In some embodiments, the individual is heterozygous for the HSD17B13:rs72613567:T allele, the HSD17B13:rs62305723:G allele, and / or the HSD17B13:rs80182459:GG allele.

[0199] In certain embodiments of the present invention, the methods can include identifying individuals who would benefit from reduced HSD17B13 expression. The methods generally include determining whether a sample from the individual contains a nucleic acid encoding the PNPLA3 Ile148Met variant or the PNPLA3 Ile144Met variant. The methods can also include classifying the individual as a candidate for treatment or inhibition of liver disease by inhibiting HSD17B13 expression by: determining whether the sample from the individual contains a first nucleic acid encoding a PNPLA3 protein comprising the I148M variant and a second nucleic acid encoding a functional HSD17B13 protein, and / or a PNPLA3 protein comprising the I148M variant and a functional HSD17B13 protein; and classifying the individual as a candidate for treatment or inhibition of liver disease by inhibiting HSD17B13 when both the first and second nucleic acids are detected and / or when both proteins are detected.

[0200] The variant PNPLA3 Ile148Met variant or PNPLA3 Ile144Met variant can be any of the PNPLA3 Ile148Met variant and PNPLA3 Ile144Met variant described herein. The PNPLA3 Ile148Met variant or PNPLA3 Ile144Met variant can be detected by any suitable means, such as ELISA analysis, RT-PCR, or sequencing.

[0201] In some embodiments, the method further comprises determining whether the individual is homozygous or heterozygous for the PNPLA3 Ile148Met variant or the PNPLA3 Ile144Met variant. In some embodiments, the individual is homozygous for the PNPLA3 Ile148Met variant or the PNPLA3 Ile144Met variant. In some embodiments, the individual is heterozygous for the PNPLA3 Ile148Met variant or the PNPLA3 Ile144Met variant. In some embodiments, the individual is homozygous for the PNPLA3 Ile148Met variant. In some embodiments, the individual is heterozygous for the PNPLA3 Ile148Met variant. In some embodiments, the individual is homozygous for the PNPLA3 Ile144Met variant. In some embodiments, the individual is heterozygous for the PNPLA3 Ile144Met variant.

[0202] In some embodiments, the individual does not contain any gene encoding a loss-of-function variant of the HSD17B13 protein. Loss-of-function variants of the HSD17B13 protein, including those described herein and in U.S. Provisional Application No. 62 / 570,985, filed October 11, 2017, are believed to confer protection against liver disease, and this protection is further believed to be enhanced in the presence of the variant PNPLA3 Ile148M variant.

[0203] In some embodiments, the method further comprises determining whether the individual is obese. In some embodiments, if the individual's body mass index (BMI) exceeds 30 kg / m 2 , then the individual is obese. Obesity can be a characteristic of an individual with liver disease or at risk of developing liver disease. In some embodiments, the method further comprises determining whether the individual has fatty liver disease. Fatty liver disease can be a characteristic of an individual with liver disease or at risk of developing liver disease. In some embodiments, the method further comprises determining whether the individual is obese and has fatty liver disease.

[0204] In some embodiments, the method further comprises determining a NAFLD activity score (NAS), a ballooning score, a lobular inflammation score, a steatosis score, and / or a fibrosis score for the individual. The NAFLD activity score (NAS), a ballooning score, a lobular inflammation score, a steatosis score, and / or a fibrosis score can be determined by any method known in the art and the methods described herein.

[0205] As used herein, "non-alcoholic fatty liver disease" is used interchangeably with the term "NAFLD" and refers to a disease defined by the presence of macrovascular steatosis in the presence of less than 20 gm of alcohol per day. NAFLD is the most common liver disease in the United States and is often associated with insulin resistance / type 2 diabetes and obesity. NAFLD manifests as steatosis, steatohepatitis, cirrhosis, and sometimes hepatocellular carcinoma. For a review of NAFLD, see Tolman and Dalpiaz (2007) Ther. Clin. Risk. Manag., 3(6): 1153-1163, the entire contents of which are incorporated herein by reference.

[0206] As used herein, the terms "steatosis," "hepatic steatosis," and "fatty liver disease" refer to the accumulation of triglycerides and other fats in liver cells.

[0207] As used herein, the term "non-alcoholic steatohepatitis" or "NASH" refers to liver inflammation and damage caused by a buildup of fat in the liver. NASH is part of a group of conditions known as non-alcoholic fatty liver disease (NAFLD). NASH is similar to alcoholic liver disease, but occurs in people who drink little or no alcohol. The main characteristics of NASH are fat in the liver, as well as inflammation and damage. Most people with NASH feel well and are unaware that they have liver problems. However, NASH can be serious and can lead to cirrhosis, in which the liver is permanently damaged and scarred and no longer able to function properly. NASH is usually first suspected in a person when elevated liver test results are found, which are included in a routine blood test panel, such as alanine aminotransferase (ALT) or aspartate aminotransferase (AST). NASH is suspected when further evaluation shows no obvious cause of liver disease (such as medications, viral hepatitis, or excessive alcohol consumption) and when x-rays or imaging studies of the liver show fat. The only way to confirm the diagnosis of NASH and distinguish it from simple fatty liver disease is a liver biopsy.

[0208] As used herein, the term "cirrhosis" is defined histologically as a diffuse hepatic process characterized by fibrosis and transformation of the normal liver architecture into architecturally abnormal nodules.

[0209] As used herein, the term "serum lipids" refers to any major lipid present in the blood. Serum lipids may be present in the blood in free form or as part of a protein complex (e.g., a lipoprotein complex). Non-limiting examples of serum lipids may include triglycerides (TG), cholesterol, such as total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), very low-density lipoprotein cholesterol (VLDL-C), and intermediate-density lipoprotein cholesterol (IDL-C).

[0210] In one embodiment, individuals who would benefit from reduced expression of HSD17B13 (and in some embodiments, PNPLA3) are, for example, individuals with type 2 diabetes, prediabetes, or obesity; individuals with elevated levels of fats (such as cholesterol) in the blood or high blood pressure; individuals with certain metabolic disorders, including metabolic syndrome; individuals experiencing rapid weight loss; individuals with certain infections, such as hepatitis C infection; or individuals who have been exposed to certain toxins. In one embodiment, individuals who would benefit from reduced expression of HSD17B13 (and in some embodiments, PNPLA3) are, for example, middle-aged or older individuals; individuals who are Hispanic, non-Hispanic white, or African American; and individuals taking certain medications, such as corticosteroids and cancer medications.

[0211] In the methods (and uses) of the present invention, which comprise administering to a subject a first dsRNA agent targeting HSD17B13 and a second dsRNA agent targeting PNPLA3, the first and second dsRNA agents may be formulated in the same composition or different compositions and may be administered to the subject in the same composition or separate compositions.

[0212] In one embodiment, the "iRNA" used in the methods of the present invention is a "dual-targeting RNAi agent." The term "dual-targeting RNAi agent" refers to a covalent linkage of a molecule comprising a first dsRNA agent comprising a complex of ribonucleic acid molecules having a double helix structure comprising two antiparallel and substantially complementary nucleic acid strands, which are referred to as having "sense" and "antisense" orientations relative to a first target RNA (also, i.e., the HSD17B13 gene); and a molecule comprising a second dsRNA agent comprising a complex of ribonucleic acid molecules having a double helix structure comprising two antiparallel and substantially complementary nucleic acid strands, which are referred to as having "sense" and "antisense" orientations relative to a second target RNA (also, i.e., the PNPLA3 gene). In some embodiments of the present invention, the dual-targeting RNAi agent triggers degradation of the first and second target RNAs (e.g., mRNAs) via a post-transcriptional gene silencing mechanism, referred to herein as RNA interference or RNAi.

[0213] iRNA can be administered by any method known in the art. In some embodiments, iRNA is administered to a subject intravenously, intramuscularly, or subcutaneously.

[0214] The iRNA can be administered regularly over a period of time by intravenous infusion. In certain embodiments, after the initial treatment regimen, the treatment can be administered less frequently.

[0215] Administration of the iRNA can, for example, reduce the level of HSD17B13 in a patient's cells, tissues, blood, urine, or other compartment by at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118 , 47, 48, 39, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98%, or at least about 99% or more. In a preferred embodiment, administration of the iRNA can reduce the level of HSD17B13 in, for example, cells, tissues, blood, urine, or other compartments of the patient by at least 20%.

[0216] Administration of the iRNA can, for example, reduce the level of PNPLA3 in a patient's cells, tissues, blood, urine, or other compartment by at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 99%, or more. In a preferred embodiment, administration of the iRNA reduces the level of PNPLA3 in, for example, a cell, tissue, blood, urine, or other compartment of a patient by at least 20%.

[0217] Before administering a full dose of iRNA, a smaller dose, such as a 5% infusion reaction, can be administered to the patient and adverse effects, such as allergic reactions, can be monitored. In another example, the patient can be monitored for undesired immune stimulation, such as an increase in cytokine (e.g., TNF-α or INF-α) levels.

[0218] Alternatively, the iRNA may be administered subcutaneously, i.e., by subcutaneous injection. One or more injections may be used to deliver a daily dose of the iRNA to an individual. The injections may be repeated for a period of time. The administration may be repeated regularly. In certain embodiments, after the initial treatment regimen, the treatment may be administered less frequently. A repeated dosing regimen may comprise administering a therapeutic amount of the iRNA on a regular basis, such as once every other day or once a year. In certain embodiments, the iRNA is administered approximately once a week, once every 7 to 10 days, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks, once every 10 weeks, once every 11 weeks, once every 12 weeks, once a month, once every 2 months, once every 3 months (once every quarter), once every 4 months, once every 5 months, or once every 6 months. The method of any one of claims 1 to 45, wherein the dsRNA agent is administered to the individual monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, or every 12 months.

[0219] In some embodiments, the dsRNA agent is administered to an individual at a dosage of about 25 mg per month. In some embodiments, the dsRNA agent is administered to an individual at a dosage of about 200 mg per month. In some embodiments, the dsRNA agent is administered to an individual at a dosage of about 400 mg per month.

[0220] In some embodiments, the dsRNA agent is administered to an individual at a dosage of about 25 mg every three months. In some embodiments, the dsRNA agent is administered to an individual at a dosage of about 200 mg every three months. In some embodiments, the dsRNA agent is administered to an individual at a dosage of about 400 mg every three months.

[0221] In one embodiment, the method comprises administering a composition characterized herein such that expression of the target HSD17B13 gene is reduced, such as for about 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 18, 24 hours, 28, 32, or about 36 hours. In one embodiment, expression of the target HSD17B13 gene is reduced for an extended duration, such as at least about two, three, four, or more days, for example, about one, two, three, or four weeks, or longer.

[0222] In another embodiment, the method comprises administering a composition characterized herein such that expression of the target PNPLA3 gene is reduced, such as for about 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 18, 24 hours, 28, 32, or about 36 hours. In one embodiment, expression of the target PNPLA3 gene is reduced for an extended duration, such as at least about two, three, four, or more days, for example, about one, two, three, or four weeks, or longer.

[0223] Preferably, iRNAs suitable for use in the methods and compositions characterized herein specifically target RNA (primary RNA or processed RNA) of the target HSD17B13 gene (and in some embodiments, the PNPLA3 gene). Compositions and methods for inhibiting the expression of such genes using iRNAs can be prepared and performed as described herein.

[0224] Administration of dsRNA according to the methods of the present invention can result in a reduction in the severity, signs, symptoms, and / or markers of such diseases or conditions in patients with lipid metabolism disorders. In this context, "reduction" means a statistically significant reduction in such levels. The reduction can be, for example, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100%.

[0225] The efficacy of treating or preventing a disease can be assessed, for example, by measuring disease progression, disease remission, symptom severity, pain relief, quality of life, the dosage of the drug required to maintain the therapeutic effect, the level of a disease marker, or any other measurable parameter suitable for treating or targeting a given disease for prevention. Those skilled in the art are well-versed in the ability to monitor the efficacy of treatment or prevention by measuring any one or any combination of such parameters. For example, the efficacy of treatment of a lipid metabolism disorder can be assessed, for example, by periodically monitoring one or more serum lipid levels, such as triglyceride levels. Subsequent readings are compared with initial readings to provide the physician with an indication of whether the treatment is effective. Those skilled in the art are well-versed in the ability to monitor the efficacy of treatment or prevention by measuring any one or any combination of such parameters. With respect to the administration of an iRNA or its pharmaceutical composition, "effective against" a lipid metabolism disorder means that administration in a clinically appropriate manner produces a beneficial effect on at least a statistically significant fraction of patients, such as improving symptoms, curing, alleviating the disease, prolonging life, improving quality of life, or other effects generally considered positive by physicians familiar with the treatment of lipid metabolism disorders and related causes.

[0226] A therapeutic or preventive effect is evident when there is a statistically significant improvement in one or more parameters of the disease state, or by the absence of worsening or development of otherwise expected symptoms. As an example, a favorable change of at least 10% in a measurable disease parameter can indicate effective treatment, and preferably at least 20%, 30%, 40%, 50%, or more. The efficacy of a given iRNA drug or formulation of such a drug can also be determined using experimental animal models of a given disease, as known in the art.

[0227] The present invention further provides methods of using the iRNA agents or pharmaceutical compositions of the present invention, for example, to treat individuals who would benefit from HSD17B13 expression or reduction and / or inhibition of HSD17B13, e.g., individuals with HSD17B13-associated diseases, disorders, or conditions, in combination with other drugs and / or other treatments, e.g., with known drugs and / or known treatments, such as those currently used to treat such disorders. In some embodiments, the present invention provides methods of using the iRNA agents or pharmaceutical compositions of the present invention and an iRNA agent targeting PNPLA3, for example, to treat individuals who would benefit from HSD17B13 expression and reduction and / or inhibition of PNPLA3 expression, e.g., individuals with HSD17B13-associated diseases, disorders, or conditions (e.g., NASH), in combination with other drugs and / or other treatments, e.g., with known drugs and / or known treatments, such as those currently used to treat such disorders. For example, in certain embodiments, the iRNA agent or pharmaceutical composition of the invention is administered in combination with, for example, pyridoxine, an ACE inhibitor (angiotensin-converting enzyme inhibitor), such as benazepril, an agent that lowers blood pressure, such as a diuretic, a beta-blocker, an ACE inhibitor, an angiotensin II receptor blocker, a calcium channel blocker, an alpha blocker, an alpha-2 receptor antagonist, a combined alpha and beta blocker, a central agonist, a peripheral adrenergic inhibitor, and a vasodilator; or an agent that lowers cholesterol, such as a statin, a selective cholesterol absorption inhibitor, a resin; lipid-lowering therapy; an insulin sensitizer, such as the PPARγ agonist pioglitazone; a glp-1r agonist, such as liraglutatide; vitamin E; an SGLT2 inhibitor; or a DPPIV inhibitor; or a combination of any of the foregoing. In one embodiment, the iRNA agent or pharmaceutical composition of the present invention is administered in combination with an agent that inhibits the expression and / or activity of the transmembrane 6 superfamily, member 2 (TM6SF2) gene, such as an RNAi agent that inhibits the expression of the TM6SF2 gene.

[0228] The iRNA agent and the additional therapeutic agent and / or treatment can be administered simultaneously and / or in the same combination, e.g., subcutaneously, or the additional therapeutic agent can be administered as part of a separate composition or at a separate time and / or by another method known in the art or described herein.

[0229] III. Delivery of the iRNA of the Invention

[0230] Delivery of the iRNA of the present invention to cells can be achieved in a variety of different ways, such as cells within an individual, such as a human individual (e.g., an individual in need, such as an individual suffering from a lipid metabolism disorder). For example, delivery can be carried out by contacting the cell with the iRNA of the present invention in vitro or in vivo. In vivo delivery can also be carried out directly by administering a composition comprising an iRNA (e.g., dsRNA) to the individual. Alternatively, in vivo delivery can be carried out indirectly by administering one or more vectors that encode and guide the expression of the iRNA. These alternatives will be discussed further below.

[0231] In general, the iRNAs of the present invention can be delivered using any method for delivering nucleic acid molecules (in vitro or in vivo) (see, for example, Akhtar S. and Julian RL., (1992) Trends Cell. Biol. 2(5):139-144 and WO94 / 02595, the entire disclosures of which are incorporated herein by reference). When used for in vivo delivery, considerations for delivering iRNA molecules include, for example, the biostability of the delivered molecule, preventing nonspecific effects and accumulation of the delivered molecule in the target tissue. Nonspecific effects of iRNA can be minimized by local administration, for example, by direct injection or implantation into the tissue or by local administration of the formulation. Local administration to the treatment site allows for maximum local concentration of the agent, limits exposure of the agent to systemic tissues that may be damaged and degraded by the agent, and reduces the total dose of the iRNA molecule administered. Several studies have shown successful blockade of gene products when iRNA is administered locally. For example, intraocular delivery of VEGF dsRNA by intravitreal injection in macaques (Tolentino, MJ. et al. (2004) Retina 24:132-138) and subretinal injection in mice (Reich, SJ. et al. (2003) Mol. Vis. 9:210-216) has been shown to prevent neovascularization in experimental models of age-related macular degeneration. Furthermore, direct intratumoral injection of dsRNA in mice reduced tumor volume (Pille, J. et al. (2005) Mol. Ther. 11:267-274) and prolonged the survival of tumor-bearing mice (Kim, WJ. et al. (2006) Mol. Ther. 14:343-350; Li, S. et al. (2007) Mol. Ther. 15:515-523).RNA interference has also been successfully delivered locally to the CNS by direct injection (Dorn, G. et al. (2004) Nucleic Acids 32:e49; Tan, PH. et al. (2005) Gene Ther. 12:59-66; Makimura, H. et al. (2002) BMC Neurosci. 3:18; Shishkina, GT. et al. (2004) Neuroscience 129:521-528; Thakker, ER. et al. (2004) Proc. Natl. Acad. Sci. USA 101:17270-17275; Akaneya, Y. et al. (2005) J. Neurophysiol. 93:594-602) and locally delivered to the lungs by intranasal administration (Howard, KA. et al. (2006) Mol. Ther. 14:476-484; Zhang, X. et al. (2004) J. Biol. Chem. 279:10677-10684; Bitko, V. et al. (2005) Nat. Med. 11:50-55). When iRNA is administered systemically for disease treatment, the RNA can be modified or delivered using a drug delivery system; both approaches prevent rapid in vivo degradation of dsRNA by endo- and exonucleases. Modifying RNA or pharmaceutical carriers also allows iRNA compositions to be targeted to target tissues and avoid undesirable off-target effects. iRNA molecules can be modified by chemical conjugation with lipophilic groups, such as cholesterol, to enhance cellular uptake and prevent degradation. For example, systemic injection of an iRNA targeting ApoB conjugated to a lipophilic cholesterol moiety into mice resulted in the knockdown of apoB mRNA in both the liver and jejunum (Soutschek, J. et al., (2004) Nature 432:173-178). In a mouse model of prostate cancer, conjugation of iRNA to an aptamer has been shown to inhibit tumor growth and mediate tumor regression (McNamara, J. O. et al., (2006) Nat. Biotechnol. 24:1005-1015). In an alternative embodiment, the iRNA can be delivered using a drug delivery system, such as nanoparticles, dendrimers, polymers, liposomes, or cationic delivery systems. Positively charged cationic delivery systems can promote the binding of iRNA molecules (negatively charged) and also enhance interactions on negatively charged cell membranes to enable efficient cell uptake of the iRNA. Cationic lipids, dendrimers, or polymers can bind to the iRNA or be induced to form vesicles or micelles that encapsulate the iRNA (see, e.g., Kim SH. et al., (2008) Journal of Controlled Release 129(2):107-116).When administered systemically, the formation of vesicles or micelles also prevents iRNA degradation. Methods for making and administering cation-iRNA complexes are well within the capabilities of those skilled in the art (see, e.g., Sorensen, DR. et al., (2003) J. Mol. Biol 327:761-766; Verma, UN. et al., (2003) Clin. Cancer Res. 9:1291-1300; Arnold, AS et al., (2007) J. Hypertens. 25:197-205, which are incorporated herein by reference in their entirety). Some non-limiting examples of drug delivery systems suitable for systemic delivery of iRNA include DOTAP (Sorensen, DR. et al. (2003), supra; Verma, UN. et al. (2003), supra), Oligofectamine, "solid nucleic acid lipid particles" (Zimmermann, TS. et al. (2006) Nature 441: 111-114), cardiolipin (Chien, PY. et al. (2005) Cancer Gene Ther. 12: 321-328; Pal, A. et al. (2005) Int J. Oncol. 26: 1087-1091), polyethyleneimine (Bonnet ME, et al., (2008) Pharm. Res., August 16, electronic advance edition; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines (Tomalia, D. A., et al., (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., et al., (1999) Pharm. Res. 16:1799-1804). In some embodiments, iRNA and cyclodextrin form a complex for systemic administration. Methods for administering iRNA and cyclodextrin and pharmaceutical compositions can be found in U.S. Patent No. 7,427,605, the entire disclosure of which is incorporated herein by reference.

[0232] A. Vector-Encoded iRNA of the Invention

[0233] iRNAs targeting the HSD17B13 gene can be expressed from transcription units inserted into DNA or RNA vectors (see, for example, Couture, A. et al., TIG. (1996), 12: 5-10; Skillern, A. et al., International PCT Publication No. WO 00 / 22113, Conrad, International PCT Publication No. WO 00 / 22114, and Conrad, U.S. Pat. No. 6,054,299). Expression can be transient (hours to weeks) or sustained (weeks to months or longer), depending on the specific construct used and the target tissue or cell type. These transgenes can be introduced as linear constructs, circular plasmids, or viral vectors, which can be integrating or non-integrating vectors. These transgenes can also be constructed so that they are inherited as extrachromosomal plastids (Gassmann et al., (1995) Proc. Natl. Acad. Sci. USA 92: 1292).

[0234] Individual or multiple strands of an iRNA can be transcribed from a promoter on an expression vector. When it is desired that the two individual strands be expressed to produce, for example, dsRNA, two separate expression vectors can be co-introduced (e.g., transfected or infected) into a target cell. Alternatively, the individual strands of the dsRNA can be transcribed by two promoters located on the same expression plasmid. In one embodiment, the dsRNA is expressed as an inverted repeat polynucleotide joined by a linker polynucleotide sequence, resulting in the dsRNA having a stem and loop structure.

[0235] iRNA expression vectors are typically DNA plasmids or viral vectors. Expression vectors compatible with eukaryotic cells, preferably those compatible with vertebrate cells, can be used to produce recombinant constructs for expressing iRNA as described herein. Eukaryotic cell expression vectors are well known in the art and can be obtained from many commercial sources. Typically, such vectors are provided, containing convenient restriction sites for inserting the desired nucleic acid segment. The delivery of the iRNA expression vector can be systemic, such as by intravenous or intramuscular administration, by administration to the target cells transplanted from the patient, subsequently reintroduced into the patient, or by any other means allowing introduction into the desired target cells.

[0236] Viral vector systems that can be utilized with the methods and compositions described herein include, but are not limited to, (a) adenoviral vectors; (b) retroviral vectors, including, but not limited to, lentiviral vectors, Moloney leukemia virus, and the like; (c) adeno-associated viral vectors; (d) herpes simplex viral vectors; (e) SV 40 vectors; (f) polyoma viral vectors; (g) papilloma viral vectors; (h) picorna viral vectors; (i) pox viral vectors, such as orthopox, e.g., vaccinia virus vectors or avian pox, e.g., canarypox or fowlpox; and (j) helper virus-dependent or enteroadenoviruses. Replication-defective viruses may also be advantageous. Different vectors may or may not be incorporated into the genome of the cell. If desired, the construct may comprise viral sequences for transfection. Alternatively, the construct may be incorporated into a vector capable of episomal replication, such as EPV and EDV vectors. Constructs for recombinant expression of iRNA will typically require regulatory elements, such as promoters, enhancers, and the like, to ensure expression of the iRNA in the target cell. Other considerations for vectors and constructs are known in the art.

[0237] IV. iRNAs used in the methods of the invention

[0238] Described herein are iRNAs for use in the methods of the present invention. iRNA is a double-stranded ribonucleic acid (dsRNA) molecule. In one embodiment, the dsRNA agent targets the HSD17B13 gene and inhibits the expression of the HSD17B13 gene. In one embodiment, the iRNA agent includes a dsRNA molecule for inhibiting the expression of the HSD17B13 gene in a cell, such as a hepatocyte, such as a hepatocyte in an individual, such as a mammal, such as a human suffering from a chronic fibrotic inflammatory liver disease, disorder or condition, such as non-alcoholic steatohepatitis (NASH) or a disease, disorder or condition associated with non-alcoholic steatohepatitis (NASH), such as accumulation and / or expansion of lipid droplets in the liver and / or fibrosis of the liver.

[0239] The dsRNA used in the methods of the present invention includes an antisense strand having a complementary region that is complementary to at least a portion of the mRNA formed during expression of the HSD17B13 gene. The complementary region is about 30 nucleotides or less in length (e.g., about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 nucleotides or less in length). Upon contact with cells expressing the target gene, the iRNA inhibits expression of the target gene (e.g., a human, primate, non-primate, or avian target gene) by at least about 10%, as analyzed by, for example, PCR-based or branched DNA (bDNA) methods, or by protein-based methods, such as immunofluorescence analysis, Western blotting, or flow cytometry.

[0240] dsRNA comprises two complementary and hybridizing RNA chains to form a double helix structure under the conditions in which the dsRNA will be used. One chain of the dsRNA (antisense strand) comprises a complementary region that is substantially complementary to the target sequence and is roughly completely complementary. The target sequence can be derived from the sequence of the mRNA formed during the expression of the HSD17B13 gene. The other chain (sense strand) comprises a region complementary to the antisense strand so that when combined under appropriate conditions, the two chains hybridize and form a double helix structure. As described elsewhere herein and as known in the art, the complementary sequence of the dsRNA can also contain a self-complementary region that is a single nucleic acid molecule, rather than being located on a separate oligonucleotide.

[0241] Generally, the length of the double helix is ​​between 15 and 30 base pairs, for example, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30 Intermediate ranges and lengths between the above recited ranges and lengths are also contemplated as part of the invention.

[0242] Similarly, the complementary region of the target sequence is between 15 and 30 nucleotides in length, for example, 15 to 29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-2 ... Intermediate ranges and lengths between the above recited ranges and lengths are also contemplated as part of the invention.

[0243] In some embodiments, the sense and antisense strands of the dsRNA are each independently about 15 to about 30 nucleotides in length, or about 25 to about 30 nucleotides in length, for example, each strand is independently 15 to 29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-2 In some embodiments, the dsRNA is between about 15 and about 23 nucleotides in length, or between about 25 and about 30 nucleotides in length. In general, dsRNA is long enough to serve as a substrate for the Dicer enzyme. For example, it is well known in the art that dsRNAs longer than about 21-23 nucleotides can serve as substrates for Dicer. As will also be appreciated by those of ordinary skill, the region of RNA targeted for cleavage will most often be a portion of a larger RNA molecule, typically an mRNA molecule. In relevant cases, a "portion" of an mRNA target is a contiguous sequence of an mRNA target that is of sufficient length to allow it to serve as a substrate for RNAi-guided cleavage (i.e., cleavage via a RISC pathway).

[0244] Those skilled in the art will also recognize that the duplex region is the major functional portion of the dsRNA, e.g., about 9 to 36 base pairs, e.g., about 10 to 36, 11-36, 12-36, 13-36, 14-36, 15-36, 9-35, 10-35, 11-35, 12-35, 13-35, 14-35, 15-35, 9-34, 10-34, 11-34, 12-34, 13-34, 14-35. 4, 15-34, 9-33, 10-33, 11-33, 12-33, 13-33, 14-33, 15-33, 9-32, 10-32, 11-32, 12-32, 13-32, 14-32, 15-32, 9-31, 10-31, 11-31, 12-31, 13-32, 14-31, 15-31, 15-30, 15-29, 15-28, 15-27, 15 -26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19 In one embodiment, a miRNA or RNA molecule complex having a duplex region of more than 30 base pairs is a dsRNA to the extent that it is processed into a functional duplex of, for example, 15 to 30 base pairs that targets a desired RNA for cleavage. Thus, one of ordinary skill will recognize that in one embodiment, a miRNA is a dsRNA. In another embodiment, a dsRNA is not a naturally occurring miRNA. In another embodiment, iRNA agents useful for targeting HSD17B13 expression are not produced in the target cell by cleavage of a larger dsRNA.

[0245] DsRNA as described herein can also include one or more single-stranded nucleotide overhangs, for example 1,2,3 or 4 nucleotides. The dsRNA with at least one nucleotide overhang can have unexpectedly good inhibitory properties relative to their blunt end counterparts. The nucleotide overhangs can include nucleotides / nucleoside analogs or be made up of nucleotides / nucleoside analogs, including deoxynucleotides / nucleosides. A plurality of overhangs can be on sense strand, antisense strand or its any combination. In addition, a plurality of nucleotides of the overhang can be present in the 5' end, 3' end or both ends of the antisense strand or sense strand of the dsRNA.

[0246] dsRNA can be synthesized by standard methods known in the art as discussed further below, for example by using an automated DNA synthesizer, such as those commercially available from, for example, Biosearch, Applied Biosystems.

[0247] The iRNA compounds of the present invention can be prepared using a two-step process. First, the individual strands of the double-stranded RNA molecule are prepared separately. Next, the component strands are annealed. The individual strands of the siRNA compound can be prepared using solution phase or solid phase organic synthesis, or both. Organic synthesis offers the advantage of easily preparing oligonucleotide strands containing non-natural or modified nucleotides. The single-stranded oligonucleotides of the present invention can be prepared using solution phase or solid phase organic synthesis, or both.

[0248] In one aspect, the dsRNA of the present invention comprises at least two nucleotide sequences, a sense sequence and an antisense sequence. The sense strand sequence is selected from the group of sequences provided in Table 2, and the corresponding antisense strand of the sense strand is selected from the group of sequences in Table 2. In this respect, one of the two sequences is complementary to another of the two sequences, wherein one of the sequences is substantially complementary to the sequence of the mRNA produced in HSD17B13 gene expression. Therefore, in this respect, the dsRNA will include two oligonucleotides, wherein one oligonucleotide is described as the sense strand (passenger strand) in Table 2, and the second oligonucleotide is described as the corresponding antisense strand (guide strand) of the sense strand in Table 2. In one embodiment, the substantially complementary sequences of the dsRNA are contained in the oligonucleotides separated. In another embodiment, the substantially complementary sequences of the dsRNA are contained in a single oligonucleotide.

[0249] It will be understood that although the sequences in Table 2 are described as modified, unmodified, unconjugated, and / or conjugated sequences, the RNA of an iRNA of the invention (e.g., a dsRNA of the invention) can comprise any of the sequences set forth in Table 2 that are unmodified, unconjugated, and / or modified and / or conjugated differently than described therein.

[0250] It is well known to those skilled in the art that dsRNAs having a duplex structure between about 20 and 23 base pairs (e.g., 21 base pairs) have been hailed as being particularly effective in inducing RNA interference (Elbashir et al., (2001) EMBO J., 20:6877-6888). However, others have found that shorter or longer RNA duplex structures can also be effective (Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226). In the embodiments described above, by the nature of the oligonucleotide sequences provided herein, the dsRNAs described herein can comprise at least one strand of at least 21 nucleotides in length. It is reasonable to expect that shorter duplexes minus only a few nucleotides at one or both ends can be equally effective compared to the dsRNAs described above. Thus, within the scope of the present invention are dsRNAs having a sequence of at least 15, 16, 17, 18, 19, 20 or more consecutive nucleotides derived from any one of the sequences provided herein, and whose ability to inhibit HSD17B13 gene expression differs by no more than about 5%, 10%, 15%, 20%, 25% or 30% inhibition compared to a dsRNA comprising the entire sequence.

[0251] In addition, the RNAs described in Table 2 identify sites in the HSD17B13 transcript that are susceptible to RISC-mediated cleavage. Thus, the present invention is further characterized by iRNAs that target these multiple sites. As used herein, an iRNA is said to target a specific site in an RNA transcript if it promotes cleavage of the transcript at any one of the specific sites. Such iRNAs will typically comprise at least about 15 consecutive nucleotides from one of the sequences provided herein, coupled to additional nucleotide sequences obtained from a region adjacent to the selected sequence in the gene.

[0252] Although the length of the target sequence is generally about 15-30 nucleotides, the suitability of a specific sequence for guiding the cleavage of any given target RNA within this range varies widely. The various software suites and guides described herein provide guidance for identifying the optimal target sequence for any given gene target, but an empirical approach can also be employed, wherein a "window" or "screen" of a given size (as a non-limiting example, 21 nucleotides) is placed on the target RNA sequence literally or symbolically (including, for example, performed in a computer) to identify sequences within a size range that can serve as target sequences. By gradually moving the sequence "window" one nucleotide upstream or downstream of the initial target sequence position, the next potential target sequence can be identified until a complete set of possible sequences is identified for any given target size selected. This process is coupled with systematic synthesis and testing of the identified sequences (using analysis as described herein or as known in the art) to identify those sequences that are optimally expressed and that can identify these RNA sequences, mediating optimal inhibition of target gene expression when the iRNA agent targets the sequence. Thus, while the sequences identified herein represent effective target sequences, it is contemplated that further optimization of inhibition efficiency can be achieved by progressively "moving the window" one nucleotide upstream or downstream of a given sequence to identify sequences with equal or better inhibition characteristics.

[0253] In addition, it is expected that for any sequence identified herein, further optimization can be achieved by systematically adding or removing nucleotides to generate longer or shorter sequences, and testing those sequences generated by moving the window of longer or shorter size up or down the target RNA from that point. Again, coupling the method of generating new candidate targets with the effectiveness testing of iRNAs based on these target sequences in inhibition assays as known in the art and / or as described herein can lead to further improvements in inhibition efficiency. In addition, such optimized sequences can be adjusted by, for example, introducing modified nucleotides as described herein or as known in the art, additions or changes in overhangs, or other modifications as known in the art and / or discussed herein to further optimize the molecule (e.g., to increase serum stability or circulation half-life, increase thermal stability, enhance transmembrane delivery, target specific locations or cell types, increase interaction with silencing pathway enzymes, increase endosome release) as an expression inhibitor.

[0254] iRNA agents as described herein may contain one or more mismatches with the target sequence. In one embodiment, an iRNA as described herein contains no more than three mismatches. If the antisense strand of the iRNA contains a mismatch with the target sequence, it is best not to locate the mismatch region in the center of the complementary region. If the antisense strand of the iRNA contains a mismatch with the target sequence, the mismatch is preferably limited to the last five nucleotides from the 5' or 3' end of the complementary region. For example, for a 23-nucleotide iRNA agent, the strand complementary to the region of the HSD17B13 gene typically does not contain any mismatches within the central 13 nucleotides. The methods described herein or methods known in the art can be used to determine whether an iRNA containing a mismatch with the target sequence effectively inhibits the expression of the HSD17B13 gene. It is important to consider the efficacy of iRNAs with mismatches in inhibiting the expression of the HSD17B13 gene, especially when it is known that a specific complementary region in the HSD17B13 gene has polymorphic sequence variation within a population.

[0255] In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises the nucleotide sequence 5'-AUGCUUUUGCAUGGACUAUCU-3' (SEQ ID NO: 26) and the antisense strand comprises the nucleotide sequence 5'-AGAUAGTCCAUGCAAAAGCAUUC-3' (SEQ ID NO: 27).

[0256] In some embodiments, the dsRNA comprises any of the dsRNA agents described, for example, in International Patent Application No. PCT / US2019 / 023079, the entire contents of which are incorporated herein by reference.

[0257] i. Modified iRNA of the present invention

[0258] In one embodiment, the iRNA (e.g., dsRNA) used in the methods of the present invention is unmodified and does not comprise chemical modifications and / or conjugations such as those known in the art and described herein. In another embodiment, the iRNA (e.g., dsRNA) used in the methods of the present invention is chemically modified to enhance stability or other beneficial characteristics. In certain embodiments of the present invention, substantially all nucleotides of the iRNA of the present invention are modified. In other embodiments of the present invention, all nucleotides of the iRNA of the present invention are modified. The iRNA of the present invention wherein "substantially all nucleotides are modified" is primarily but not completely modified and may comprise no more than 5, 4, 3, 2, or 1 unmodified nucleotides.

[0259] In some aspects of the invention, substantially all nucleotides of the iRNA of the invention are modified and the iRNA agent comprises no more than 10 nucleotides comprising a 2'-fluoro modification (e.g., no more than 9 2'-fluoro modifications, no more than 8 2'-fluoro modifications, no more than 7 2'-fluoro modifications, no more than 6 2'-fluoro modifications, no more than 5 2'-fluoro modifications, no more than 4 2'-fluoro modifications, no more than 5 2'-fluoro modifications, no more than 4 2'-fluoro modifications, no more than 3 2'-fluoro modifications, or no more than 2 2'-fluoro modifications). For example, in some embodiments, the sense strand comprises no more than 4 nucleotides comprising a 2'-fluoro modification (e.g., no more than 3 2'-fluoro modifications, or no more than 2 2'-fluoro modifications). In other embodiments, the antisense strand comprises no more than 6 nucleotides comprising a 2'-fluoro modification (e.g., no more than 5 2'-fluoro modifications, no more than 4 2'-fluoro modifications, no more than 4 2'-fluoro modifications, or no more than 2 2'-fluoro modifications).

[0260] In other aspects of the invention, all nucleotides of an iRNA of the invention are modified and the iRNA agent includes no more than 10 nucleotides that include a 2'-fluoro modification (e.g., no more than 9 2'-fluoro modifications, no more than 8 2'-fluoro modifications, no more than 7 2'-fluoro modifications, no more than 6 2'-fluoro modifications, no more than 5 2'-fluoro modifications, no more than 4 2'-fluoro modifications, no more than 5 2'-fluoro modifications, no more than 4 2'-fluoro modifications, no more than 3 2'-fluoro modifications, or no more than 2 2'-fluoro modifications).

[0261] In one embodiment, the double-stranded RNAi agent of the present invention further comprises a 5'-phosphate or a 5'-phosphate mimic at the 5' nucleotide of the antisense strand. In another embodiment, the double-stranded RNAi agent further comprises a 5'-phosphate mimic at the 5' nucleotide of the antisense strand. In a specific embodiment, the 5'-phosphate mimic is 5'-vinyl phosphate (5'-VP).

[0262] The nucleic acids featured in the present invention can be synthesized and / or modified by methods well known in the art, such as those described in "Current protocols in nucleic acid chemistry", Beaucage, SL et al. (eds.), John Wiley & Sons, New York, NY, USA, which is incorporated herein by reference. Modifications include, for example, terminal modifications, such as 5'-terminal modifications (phosphorylation, conjugation, reverse linkage) or 3'-terminal modifications (conjugation, DNA nucleotides, inverted linkages, etc.); base modifications, such as substitution with a stabilizing base, base destabilization, or base pairing with an extended partner, removal of a base (abasic nucleotides), or a conjugate base; sugar modifications (e.g., at the 2'-position or 4'-position) or sugar replacement; and / or backbone modifications, including modification or replacement of phosphodiester bonds. Specific examples of iRNA compounds suitable for use in the embodiments described herein include, but are not limited to, RNAs containing modified backbones or lacking natural internucleoside linkages. RNAs with modified backbones particularly include RNAs that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referred to in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbones can also be considered oligonucleotides. In some embodiments, modified iRNAs will have a phosphorus atom in their internucleoside backbones.

[0263] Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphates, including 3'-alkylene phosphates and chiral phosphates, phosphates, phosphates including 3'-aminophosphoramidic acid and aminoalkylphosphoramidic acid, sulfinyl phosphates, sulfinyl phosphates, sulfinyl phosphotriesters, and borane phosphates with normal 3'-5' linkages, 2'-5'-linked analogs of those, and those with opposite polarity, wherein adjacent nucleoside unit pairs are connected with 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included. In some embodiments of the present invention, the dsRNA agent of the present invention is in free acid form. In other embodiments of the present invention, the dsRNA agent of the present invention is in salt form. In one embodiment, the dsRNA agent of the present invention is in sodium salt form. In certain embodiments, when the dsRNA agents of the invention are in the form of sodium salts, sodium ions are present in the agent as counterions for substantially all phosphodiester and / or phosphorothioate groups present in the agent. Substantially all phosphodiester and / or phosphorothioate linkages in the agent have sodium counterions, including no more than 5, 4, 3, 2, or 1 phosphodiester and / or phosphorothioate linkages in the absence of sodium counterions. In some embodiments, when the dsRNA agents of the invention are in the form of sodium salts, sodium ions are present in the agent as counterions for all phosphodiester and / or phosphorothioate groups present in the agent.

[0264] Representative U.S. patents that teach the preparation of the above-mentioned phosphorus-containing linkages include, but are not limited to: U.S. Patent Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717 ; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5, 587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534 ,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; No. 7,321,029; and U.S. Patent RE39464, the entire contents of each of which are hereby incorporated herein by reference.

[0265] Modified RNA backbones that do not contain a phosphorus atom have backbones formed from short-chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short-chain heteroatom or heterocyclic internucleoside linkages. Examples include those having morpholino linkages (formed in part by the sugar portion of the nucleoside); siloxane backbones; sulfide, sulfoxide, and sulfone backbones; formyl and thioformyl backbones; methyleneformyl and thioformyl backbones; olefin-containing backbones; sulfonate backbones; methyleneimino and methylenehydrazinyl backbones; sulfonate and sulfonamide backbones; amide backbones; and other moieties having mixed N, O, S, and CH2 components.

[0266] Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to: U.S. Patent Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,4 and 5,677,439, the entire contents of each of which are hereby incorporated herein by reference.

[0267] In other embodiments, suitable RNA mimics are expected to be used in iRNA, wherein the sugar and internucleoside linkages (i.e., main chains) of the nucleotide units are replaced by new groups. The base units are maintained to hybridize with appropriate nucleic acid target compounds. One such oligomeric compound, which is an RNA mimic that has been shown to have excellent hybridization properties, is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced by a main chain containing amides, specifically an aminoethylglycine main chain. The core base is retained and is directly or indirectly bound to the hetero-nitrogen atom of the amide portion of the main chain. Representative U.S. patents for teaching the preparation of PNA compounds include, but are not limited to, U.S. Patents Nos. 5,539,082, 5,714,331, and 5,719,262, the entire contents of each of which are hereby incorporated herein by reference. Additional PNA compounds suitable for iRNA of the present invention are described in, for example, Nielsen et al., Science, 1991, 254, 1497-1500.

[0268] Some embodiments characterized herein include RNAs having phosphorothioate backbones and oligonucleotides having heteroatom backbones, and specifically, --CH2--NH--CH2-, --CH2--N(CH3)--O--CH2-- [known as a methylene (methylimino) or MMI backbone], --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2--, and --N(CH3)--CH2--CH2-- [where the natural phosphodiester backbone is represented as --O--P--O--CH2-- in the aforementioned cited U.S. Patent No. 5,489,677], and amide backbones in the aforementioned cited U.S. Patent No. 5,602,240. In some embodiments, the RNAs characterized herein have a morpholino backbone structure as described in the aforementioned cited U.S. Patent No. 5,034,506.

[0269] The modified RNA may also contain one or more substituted sugar moieties. At the 2'-position, the iRNAs (e.g., dsRNAs) characterized herein may comprise one of the following: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, wherein alkyl, alkenyl, and alkynyl groups may be substituted or unsubstituted C1 to C 10 Alkyl or C2 to C 10 Alkenyl and alkynyl. Exemplary suitable modifications include O[(CH2) n O] m CH3, O(CH2). n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2, and O(CH2) n ON[(CH2) n CH3)]2, wherein n and m are from 1 to about 10. In other embodiments, the dsRNA comprises one of the following at the 2' position: C1 to C 10Lower alkyl, substituted lower alkyl, alkane, aralkyl, O-alkane or O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , SO 2 CH 3 , ONO 2 , NO 2 , N 3 , NH 2 , heterocycloalkyl, heterocycloalkane, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, intercalator, group for improving the pharmacokinetic properties of iRNA or group for improving the pharmacodynamic properties of iRNA, and other substituents with similar properties. In some embodiments, the modification comprises 2'-methoxyethoxy (2'-O--CH 2 C H 2 OCH 3 , also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78: 486-504), also known as alkoxy-alkoxy. Another exemplary modification is 2'-dimethylaminooxyethoxy, also known as O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, as described in the Examples below, and 2'-dimethylaminoethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), also known as 2'-O--CH2--O--CH2--N(CH2)2. Other exemplary modifications include: 5'-Me-2'-F nucleotides, 5'-Me-2'-OMe nucleotides, 5'-Me-2'-deoxynucleotides (both R and S isomers in these three families); 2'-alkoxyalkyl; and 2'-NMA (N-methylacetamide).

[0270] Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2) and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA of the iRNA, specifically, at the 3' position of the sugar on the 3' terminal nucleotide or on the 2'-5' linked dsRNA and in the 5' position of the 5' terminal nucleotide. The iRNA can also have sugar mimetics, such as a cyclobutyl moiety in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Patent Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, some of which are commonly owned with the present application. The entire contents of each of the foregoing are hereby incorporated herein by reference.

[0271] The iRNA of the present invention may also comprise modifications or substitutions of nucleobases (often referred to in the art as "bases"). As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thioimide and 2-thiocytosine, 5-fluorouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-sulfanyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halo, in particular 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazacytosine and 3-deazaadenine. Other nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P., ed., Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pp. 858-859, Kroschwitz, JL, ed., John Wiley & Sons, 1990, those disclosed by Englisch et al., (1991) Angewandte Chemie, International Edition, 30: 613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pp. 289-302, Crooke, ST and Lebleu, B., eds., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the present invention. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine.5-Methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, YS, Crooke, ST, and Lebleu, B., eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar modifications.

[0272] Representative U.S. patents that teach the preparation of some of the modified nucleobases mentioned above, as well as other modified nucleobases, include, but are not limited to, the above-mentioned U.S. Patent Nos. 3,687,808; 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5 ,594,121,5,596,091;5,614,617;5,681,941;5,750,692;6,015,886;6,147,200;6,166,197;6,222,025;6,235,887;6,380,368;6,528,640;6,639,062;6,617,438;7,045,610; No. 7,427,672; and No. 7,495,088, the entire contents of each of which are hereby incorporated herein by reference.

[0273] The iRNA of the present invention can also be modified to include one or more locked nucleic acids (LNAs). Locked nucleic acids are nucleotides with modified ribose moieties, wherein the ribose moiety includes an additional bridge connecting the 2' and 4' carbons. This structure effectively "locks" the ribose in a 3'-endo configuration. Adding locked nucleic acids to siRNA has been shown to increase siRNA stability in serum and reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O. R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).

[0274] iRNA of the present invention can also be modified to include one or more bicyclic sugar moieties." Bicyclic sugar " is a furanoyl ring modified by bridging two atoms. " Bicyclic nucleoside " (" BNA ") is a nucleoside with a sugar moiety, and this sugar moiety includes a bridge connecting two carbon atoms of a sugar ring, thereby forming a bicyclic system. In certain embodiments, the bridge connects the 4'-carbon and 2'-carbon of the sugar ring. Therefore, in some embodiments, the medicament of the present invention can include one or more locked nucleic acids (LNA). Locked nucleic acid is a nucleotide with a modified ribose moiety, wherein the ribose moiety includes an extra bridge connecting 2' and 4' carbon. In other words, LNA is a nucleotide including a bicyclic sugar moiety, and this bicyclic sugar moiety includes a 4'-CH2-O-2' bridge. This structure effectively "locks" ribose in a 3'-endostructure configuration. The addition of locked nucleic acid to siRNA has been shown to increase siRNA stability in serum and reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O. R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). Examples of bicyclic nucleosides for use in the polynucleotides of the present invention include, but are not limited to, nucleosides comprising a bridge between the 4' and 2' ribosyl ring atoms. In certain embodiments, the antisense polynucleotides of the present invention comprise one or more bicyclic nucleosides comprising a 4' to 2' bridge.Examples of such 4' to 2' bridged bicyclic nucleosides include, but are not limited to, 4'-(CH2)-O-2' (LNA); 4'-(CH2)-S-2'; 4'-(CH2)2-O-2' (ENA); 4'-CH(CH3)-O-2' (also known as "constrained ethyl" or "cEt") and 4'-CH(CH2OCH3)-O-2' (and analogs thereof; see, e.g., U.S. Patent No. 7,399,845); 4'-C(CH3)(CH3)-O-2' (and analogs thereof; see, e.g., U.S. Patent No. 8,278,283); 4'-CH2-N(OCH3)-2' (and analogs thereof; see, e.g., U.S. Patent No. 8,278,283); 8,278,425); 4'-CH2-O-N(CH3)-2' (see, e.g., U.S. Patent Publication No. 2004 / 0171570); 4'-CH2-N(R)-O-2', wherein R is H, C1-C12 alkyl, or a protecting group (see, e.g., U.S. Patent No. 7,427,672); 4'-CH2-C(H)(CH3)-2' (see, e.g., Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH2-C(═CH2)-2' (and the like; see, e.g., U.S. Patent No. 8,278,426). The entire contents of each of the foregoing are hereby incorporated by reference herein.

[0275] Additional representative U.S. patents and U.S. patent publications that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Patent Nos. 6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207; 7,034,133; 7,084,125; 7,399, No. 845; No. 7,427,672; No. 7,569,686; No. 7,741,457; No. 8,022,193; No. 8,030,467; No. 8,278,425; No. 8,278,426; No. 8,278,283; US2008 / 0039618 and US2009 / 0012281, the entire contents of each of the foregoing are hereby incorporated by reference herein.

[0276] Any of the foregoing bicyclic nucleosides can be prepared with one or more stereochemical sugar configurations, including, for example, α-L-furanose and β-D-furanose (see WO 99 / 14226).

[0277] iRNA of the present invention can also be modified to include one or more restricted ethyl nucleotides. As used herein, "restricted ethyl nucleotides" or "cEt" are locked nucleic acids that include a bicyclic sugar moiety that includes a 4'-CH(CH3)-O-2' bridge. In one embodiment, the restricted ethyl nucleotides are in the S configuration referred to herein as "S-cEt."

[0278] The iRNAs of the present invention may also comprise one or more "conformationally constrained nucleotides ("CRNs")." CRNs are nucleotide analogs that have a linker attached to the C2' and C4' carbons of the ribose sugar or to the C3 and -C5' carbons of the ribose sugar. The CRNs lock the ribose ring into a stable conformation and increase hybridization affinity for the mRNA. The linker is of sufficient length to place the oxygen in the optimal position for stability and affinity, resulting in less ribose ring wrinkling.

[0279] Representative publications teaching the preparation of certain CRNs referred to above include, but are not limited to, US Patent Publication No. 2013 / 0190383; and PCT Publication WO 2013 / 036868, each of which is hereby incorporated by reference in its entirety.

[0280] In some embodiments, the iRNA of the present invention includes one or more monomers that are UNA (unlocked nucleic acid) nucleotides. UNA is an unlocked non-cyclic nucleic acid in which any bond of the sugar has been removed to form an unlocked "sugar" residue. In one example, UNA also encompasses monomers having a bond between C1'-C4' (i.e., a covalent carbon-oxygen-carbon bond between C1' and C4' carbons) that has been removed. In another example, the C2'-C3' bond of the sugar (i.e., a covalent carbon-carbon bond between C2' and C3' carbons) has been removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039, incorporated herein by reference).

[0281] Representative U.S. publications that teach the preparation of UNA include, but are not limited to, U.S. Patent No. 8,314,227; and U.S. Patent Publication Nos. 2013 / 0096289; 2013 / 0011922; and 2011 / 0313020, each of which is incorporated herein by reference in its entirety.

[0282] Potential stabilizing modifications to the ends of RNA molecules may include N-(acetamidohexanoyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(hexanoyl)-4-hydroxyprolinol (Hyp-C6), N-(acetyl)-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-O-deoxythymidine (ether), N-(aminohexanoyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-eicosanoyl-uridine-3"-phosphate, inverted base dT (idT), and others. Disclosure of such modifications can be found in PCT Publication No. WO 2011 / 005861.

[0283] Other modifications of the iRNAs of the invention include a 5' phosphate or a 5' phosphate mimetic, such as a 5' terminal phosphate or phosphate mimetic on the antisense strand of the RNAi agent. Suitable phosphate mimetics are disclosed, for example, in U.S. Patent Publication No. 2012 / 0157511, the entire contents of which are incorporated herein by reference.

[0284] In certain specific embodiments, the RNAi agent of the present invention is an agent that inhibits the expression of the HSD17B13 gene, which is selected from the group of agents listed in Table 2 or disclosed in PCT / US2019 / 023079. Any of these agents may further comprise a ligand.

[0285] A. Modified iRNAs Including Motifs of the Invention

[0286] In certain aspects of the invention, the double-stranded RNAi agents of the invention comprise agents with chemical modifications, such as those disclosed in WO 2013 / 075035, filed November 16, 2012, the entire contents of which are incorporated herein by reference.

[0287] Therefore, the present invention provides double-stranded RNAi agents that can inhibit the expression of target genes (i.e., HSD17B13 genes) in vivo. RNAi agents include sense strands and antisense strands. The length of each chain of RNAi agents can be within the range of 12-30 nucleotides. For example, the length of each chain can be between 14-30 nucleotides, 17-30 nucleotides in length, 25-30 nucleotides in length, 27-30 nucleotides in length, 17-23 nucleotides in length, 17-21 nucleotides in length, 17-19 nucleotides in length, 19-25 nucleotides in length, 19-23 nucleotides in length, 19-21 nucleotides in length, 21-25 nucleotides in length, or 21-23 nucleotides in length. In one embodiment, the sense strand is 21 nucleotides in length. In one embodiment, the antisense strand is 23 nucleotides in length.

[0288] The sense strand and the antisense strand typically form double-stranded RNA ("dsRNA"), also referred to herein as an "RNAi agent". The length of the duplex region of an RNAi agent may be 12-30 nucleotide pairs. For example, the length of the duplex region may be between 14-30 nucleotide pairs, 17-30 nucleotide pairs in length, 27-30 nucleotide pairs in length, 17-23 nucleotide pairs in length, 17-21 nucleotide pairs in length, 17-19 nucleotide pairs in length, 19-25 nucleotide pairs in length, 19-23 nucleotide pairs in length, 19-21 nucleotide pairs in length, 21-25 nucleotide pairs in length, or 21-23 nucleotide pairs in length. In another embodiment, the length of the duplex region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides.

[0289] In one embodiment, the RNAi agent may contain one or more overhang regions and / or blocking groups at the 3' end, 5' end or both ends of one or both chains. The length of the overhang may be 1-6 nucleotides, for example, 2-6 nucleotides in length, 1-5 nucleotides in length, 2-5 nucleotides in length, 1-4 nucleotides in length, 2-4 nucleotides in length, 1-3 nucleotides in length, 2-3 nucleotides in length or 1-2 nucleotides in length. The overhang may be the result of one chain being longer than the other, or it may be the result of two chains of the same length being intertwined with each other. The overhang may form a mismatch with the target mRNA, or it may be complementary to the targeted gene sequence or may be another sequence. The first and second chains may also be joined, for example, by additional bases to form a hairpin, or by other non-alkaline linkers.

[0290] In one embodiment, the nucleotides in the overhang region of the RNAi agent can each independently be a modified or unmodified nucleotide, including but not limited to 2'-sugar modified nucleotides such as 2-F, 2'-O methyl, thymidine (T), 2'-O-methoxyethyl-5-methyluridine (Teo), 2'-O-methoxyethyladenosine (Aeo), 2'-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combination thereof. For example, TT can be an overhang sequence at either end of either strand. The overhang can form a mismatch with the target mRNA, or it can be complementary to the targeted gene sequence or another sequence.

[0291] The 5'- or 3'-overhangs at the sense strand, antisense strand, or both strands of the RNAi agent may be phosphorylated. In some embodiments, multiple overhang regions contain two nucleotides with a phosphorothioate between the two nucleotides, wherein the two nucleotides may be the same or different. In one embodiment, the overhang is present at the 3' end of the sense strand, antisense strand, or both strands. In one embodiment, the 3'-overhang is present in the antisense strand. In one embodiment, the 3'-overhang is present in the sense strand.

[0292] RNAi agents may contain only a single overhang, which can enhance the interference activity of RNAi without affecting its overall stability. For example, a single-stranded overhang may be located at the 3' end of the sense strand, or at the 3' end of the antisense strand. RNAi may also have a blunt end, which is located at the 5' end of the antisense strand (or the 3' end of the sense strand), or vice versa. In general, the antisense strand of RNAi has a nucleotide overhang at the 3' end, and its 5' end is a blunt end. Although not wishing to be bound by theory, the asymmetric blunt ends at the 5' end of the antisense strand and the 3' end overhang of the antisense strand are conducive to loading the guide strand into the RISC process.

[0293] In one embodiment, the RNAi agent is a double-ended blunt-end polymer of 19 nucleotides in length, wherein the sense strand contains at least one motif of three 2'-F modifications at three consecutive nucleotides at positions 7, 8, and 9 from the 5' end. The antisense strand contains at least one motif of three 2'-O-methyl modifications at three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0294] In another embodiment, the RNAi agent is a double-ended blunt-end polymer of 20 nucleotides in length, wherein the sense strand contains at least one motif of three 2'-F modifications at three consecutive nucleotides at positions 8, 9, and 10 from the 5' end. The antisense strand contains at least one motif of three 2'-O-methyl modifications at three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0295] In yet another embodiment, the RNAi agent is a double-ended blunt-end polymer of 21 nucleotides in length, wherein the sense strand contains at least one motif of three 2'-F modifications at three consecutive nucleotides at positions 9, 10, and 11 from the 5' end. The antisense strand contains at least one motif of three 2'-O-methyl modifications at three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0296] In one embodiment, the RNAi agent comprises a 21-nucleotide sense strand and a 23-nucleotide antisense strand, wherein the sense strand comprises at least one motif of three 2'-F modifications at three consecutive nucleotides at positions 9, 10, and 11 from the 5' end; and the antisense strand comprises at least one motif of three 2'-O-methyl modifications at three consecutive nucleotides at positions 11, 12, and 13 from the 5' end, wherein one end of the RNAi agent is blunt and the other end comprises a 2-nucleotide overhang. Preferably, the 2-nucleotide overhang is located at the 3' end of the antisense strand.

[0297] When two nucleotide overhangs are located at the 3' end of the antisense strand, there may be two phosphorothioate internucleotide linkages between the three nucleotides at the end, wherein two of the three nucleotides are protruding nucleotides, and the third nucleotide is a pairing nucleotide near the protruding nucleotide. In one embodiment, the RNAi agent additionally has two phosphorothioate internucleotide linkages between the three nucleotides at the 5' end of the sense strand and the 5' end of the antisense strand. In one embodiment, each nucleotide in the sense strand and the antisense strand of the RNAi agent, comprising a nucleotide as part of a motif, is a modified nucleotide. In one embodiment, each residue is independently modified with 2'-O-methyl or 3'-fluoro, for example, with an alternating motif. Optionally, the RNAi agent further comprises a ligand (preferably GalNAc3).

[0298] In one embodiment, the RNAi agent comprises a sense strand and an antisense strand, wherein the sense strand is 25-30 nucleotide residues in length and comprises at least 8 ribonucleotides starting from positions 1 to 23 of the 5'-terminal nucleotide (position 1) of the first strand; the antisense strand is 36-66 nucleotide residues in length and comprises at least 8 ribonucleotides starting from the 3'-terminal nucleotide at positions that pair with positions 1 to 23 of the sense strand to form a duplex; wherein at least the 3'-terminal nucleotide of the antisense strand is unpaired with the sense strand, and up to 6 consecutive 3'-terminal nucleotides are unpaired with the sense strand, thereby forming a 3' single-stranded overhang of 1-6 nucleotides; wherein the 5' end of the antisense strand comprises a ribonucleotide that is paired with the sense strand. The invention relates to a method for preparing a double-stranded nucleic acid comprising: a first nucleic acid sequence comprising 10 to 30 consecutive unpaired nucleotides, thereby forming a 10 to 30 nucleotide single-stranded 5' overhang; wherein when the sense and antisense strands are aligned for maximum complementarity, at least the 5'-terminal and 3'-terminal nucleotides of the sense strand are base-paired with nucleotides of the antisense strand, thereby forming a substantially double-helical region between the sense and antisense strands; and the antisense strand is sufficiently complementary to the target RNA along at least 19 ribonucleotides of the antisense strand length to reduce target gene expression when the double-stranded nucleic acid is introduced into a mammalian cell; and wherein the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides, wherein at least one of the motifs occurs at or near the cleavage site. The antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at or near the cleavage site.

[0299] In one embodiment, the RNAi agent comprises a sense strand and an antisense strand, wherein the RNAi agent comprises a first strand of at least 25 and at most 29 nucleotides in length and a second strand of at most 30 nucleotides in length, having at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end; wherein the 3' end of the first strand and the 5' end of the second strand form a blunt end, and the second strand is 1 to 4 nucleotides longer than the first strand at its 3' end, wherein the duplex region is at least 25 nucleotides in length, and when the RNAi agent is introduced into a mammalian cell, the second strand is sufficiently complementary to a target mRNA along at least 19 nucleotides of the second strand length to reduce target gene expression, and wherein dicer cleavage of the RNAi agent preferably produces an siRNA comprising the 3' end of the second strand, thereby reducing target gene expression in the mammal. Optionally, the RNAi agent further comprises a ligand.

[0300] In one embodiment, the sense strand of the RNAi agent contains at least one motif of three identical modifications on three consecutive nucleotides, wherein one of the motifs occurs at the cleavage site in the sense strand.

[0301] In one embodiment, the antisense strand of the RNAi agent may also contain at least one motif of three identical modifications on three consecutive nucleotides, wherein one of the motifs occurs at or near the cleavage site in the antisense strand.

[0302] For RNAi agents with a duplex region of 17-23 nucleotides in length, the cleavage sites of the antisense strand are typically located near positions 10, 11, and 12 from the 5' end. Thus, three identical modified motifs may occur at positions 9, 10, and 11; positions 10, 11, and 12; positions 11, 12, and 13; positions 12, 13, and 14; or positions 13, 14, and 15 of the antisense strand, counting from the first nucleotide at the 5' end of the antisense strand, or from the first paired nucleotide within the duplex region at the 5' end of the antisense strand. The cleavage site in the antisense strand may also vary depending on the length of the RNAi duplex region from the 5' end.

[0303] The sense strand of the RNAi agent may contain at least one motif of three identical modifications on three consecutive nucleotides at the cleavage site of the strand; and the antisense strand may have at least one motif of three identical modifications on three consecutive nucleotides at or near the cleavage site of the strand. When the sense strand and the antisense strand form a dsRNA duplex, the sense strand and the antisense strand may be aligned such that one motif in the three nucleotides on the sense strand and one motif in the three nucleotides on the antisense strand have at least one nucleotide overlap, i.e., at least one of the three nucleotides of the motif in the sense strand forms a base pair with at least one of the three nucleotides of the motif in the antisense strand. Alternatively, at least two nucleotides may overlap, or all three nucleotides may overlap.

[0304] In one embodiment, the sense strand of an RNAi agent may contain more than one motif with three identical modifications on three consecutive nucleotides. The first motif may occur at or near the cleavage site of the chain, and the other motifs may be wing modifications. The term "wing modification" herein refers to a motif that occurs at another part of the chain, which is separated from the motif at or near the cleavage site of the same chain. The wing modification is adjacent to the first motif or is separated by at least one or more nucleotides. When the motifs are adjacent to each other, the chemical properties of the motifs are different from each other, and when the motifs are separated by one or more nucleotides, the chemical properties may be the same or different. Two or more wing modifications may be present. For example, when there are two wing modifications, each wing modification may occur relative to the first motif at or near the cleavage site or at one end on either side of the guide motif.

[0305] As with the sense strand, the antisense strand of the RNAi agent may contain more than one motif having three identical modifications on three consecutive nucleotides, wherein at least one of the motifs occurs at or near the cleavage site of the strand. This antisense strand may also contain one or more wing modifications, which are presented in an alignment similar to the wing modifications that may be present on the sense strand.

[0306] In one embodiment, the wing modifications on the sense or antisense strand of the RNAi agent typically do not include one or both terminal nucleotides preceding the 3' end, the 5' end, or both ends of the strand.

[0307] In another embodiment, the wing modifications on the sense or antisense strand of the RNAi agent typically do not include the first one or two paired nucleotides within the duplex region at the 3' end, the 5' end, or both ends of the strand.

[0308] When the sense and antisense strands of the RNAi agent each contain at least one wing modification, the wing modifications can fall on the same end of the duplex region and have one, two, or three nucleotide overlaps.

[0309] When the sense and antisense strands of the RNAi agent each contain at least two wing modifications, the sense and antisense strands can be aligned such that two modifications from each strand each fall on one end of the duplex region with one, two, or three nucleotides overlapping; two modifications from each strand each fall on the other end of the duplex region with one, two, or three nucleotides overlapping; and two modifications from each strand fall on each side of the guide motif with one, two, or three nucleotides overlapping.

[0310] In one embodiment, each nucleotide in the sense and antisense strands of the RNAi agent, including nucleotides that are part of the motif, can be modified. Each nucleotide can be modified with the same or different modifications, which can include one or more of the following: changes in one or both of the non-linked phosphate oxygens and / or one or more of the linked phosphate oxygens; changes in the sugar composition of the ribose, for example, the composition of the 2' hydroxyl group on the ribose sugar; complete replacement of the phosphate moiety with a "dephospho" linker; modification or replacement of naturally occurring bases; and replacement or modification of the ribose-phosphate backbone.

[0311] Since nucleic acid is a polymer of subunits, many modifications occur at repeated positions within the nucleic acid, such as modifications of bases or phosphate moieties, or non-connected oxygens of phosphate moieties. In some cases, modification will occur at all target positions in the nucleic acid, but will not occur in many cases. For example, modification may occur only at the 3' or 5' end position, may occur only in the terminal region, such as at the position on the terminal nucleotide or in the last 2, 3, 4, 5 or 10 nucleotides of the chain. Modification may occur in double-stranded regions, single-stranded regions or both. Modification may occur only in the double-stranded regions of RNA or may occur only in the single-stranded regions of RNA. For example, phosphorothioate modification at non-connected oxygen positions may occur only at one or both ends, may occur only in the terminal region, such as at the position on the terminal nucleotide or in the last 2, 3, 4, 5 or 10 nucleotides of the chain, or may appear in double-stranded and single-stranded regions, especially in the terminal region. The 5' end or end may be phosphorylated.

[0312] It may, for example, enhance stability, include specific bases in the overhang, or include modified nucleotides or nucleotide substitutes, in the single-stranded overhang (e.g., in the 5' or 3' overhang, or in both). For example, it may be necessary to include purine nucleotides in the overhang. In certain embodiments, all or some bases in the 3' or 5' overhang may be modified, for example, with modifications described herein. Modifications may include, for example, modifications at the 2' position using ribose and modifications known in the art, such as using modified deoxyribonucleotides, 2'-deoxy-2'-fluoro (2'-F) or 2'-O-methyl to replace the ribose of the core base, and modifications of phosphate groups, such as phosphorothioate modifications. The overhang does not need to be homologous to the target sequence.

[0313] In one embodiment, each residue of the sense and antisense strands is independently modified with LNA, CRN, cET, UNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-hydroxyl, or 2'-fluoro. The strands may contain more than one modification. In one embodiment, each residue of the sense and antisense strands is independently modified with 2'-O-methyl or 2'-fluoro.

[0314] There are usually at least two different modifications on the sense strand and the antisense strand. These two modifications can be 2'-O-methyl or 2'-fluoro modifications or other modifications.

[0315] In one embodiment, N a and / or N bModifications including alternating patterns. As used herein, the term "alternating motif" refers to a motif having one or more modifications, each modification occurring at alternating nucleotides in a chain. Alternating nucleotides may refer to one every other nucleotide or every three nucleotides, or a similar pattern. For example, if A, B, and C each represent a type of nucleotide modification, the alternating motif may be "ABABABABABAB...", "AABBAABBAABB...", "AABAABAABAAB...", "AAABAAABAAAB...", "AAABBBAAABBB...", or "ABCABCABCABC...", etc.

[0316] The types of modifications contained in the alternating motif can be the same or different. For example, if A, B, C, and D each represent a type of modification on a nucleotide, the alternating pattern, i.e., the modification on every other nucleotide, can be the same, but each of the sense and antisense strands can be selected from several possibilities for modifications within the alternating motif, such as "ABABAB...", "ACACAC...", "BDBDBD...", or "CDCDCD...", etc.

[0317] In one embodiment, the RNAi agent of the present invention includes an alternating motif modification pattern on the sense strand that is offset relative to the alternating motif modification pattern on the antisense strand. The offset can be such that the modified nucleotide groups of the sense strand correspond to differently modified nucleotide groups of the antisense strand, and vice versa. For example, when the sense strand is paired with the antisense strand in a dsRNA duplex, the alternating motif in the sense strand can start from 5'-3' "ABABAB" of the strand, and the alternating motif in the antisense strand can start from 5'-3' "BABABA" of the strand within the duplex region. As another example, the alternating motif in the sense strand can start from 5'-3' "AABBAABB" of the strand, and the alternating motif in the antisense strand can start from 5'-3' "BBAABBAA" of the strand within the duplex region, such that there is a complete or partial offset in the modification pattern between the sense and antisense strands.

[0318] In one embodiment, the RNAi agent comprises a pattern of alternating 2'-O-methyl and 2'-F modifications on the sense strand, initially offset relative to the pattern of alternating 2'-O-methyl and 2'-F modifications on the antisense strand, i.e., a 2'-O-methyl modified nucleotide on the sense strand base pairs with a 2'-F modified nucleotide on the antisense strand, and vice versa. Position 1 of the sense strand may begin with a 2'-F modification, and position 1 of the antisense strand may begin with a 2'-O-methyl modification.

[0319] The introduction of one or more motifs of three identical modifications on three consecutive nucleotides of the sense and / or antisense strands interrupts the initial modification pattern present in the sense and / or antisense strands. This interruption of the modification pattern of the sense and / or antisense strands by introducing one or more motifs of three identical modifications on three consecutive nucleotides into the sense and / or antisense strands unexpectedly promotes gene silencing activity on the target gene.

[0320] In one embodiment, when a motif of three identical modifications on three consecutive nucleotides is introduced into any one of the strands, the modifications of the nucleotides next to the motif are different from the modification of the motif. For example, the portion of the sequence containing the motif is "... N a YYYN b …”, where “Y” represents a modification of a motif of three identical modifications on three consecutive nucleotides, and “N a ” and “N b " indicates a modification of the nucleotide next to the motif "YYY" that is different from the modification of Y, and wherein N a and N b Can be the same or different modifications. Alternatively, when a wing modification is present, N a and / or N b May be present or absent.

[0321] The RNAi agent may also include at least one phosphorothioate or methylphosphonate internucleotide linkage. The phosphorothioate or methylphosphonate internucleotide linkage modification may occur on any nucleotide of the sense strand or antisense strand or on both strands at any position of the strand. For example, the internucleotide linkage modification may occur on each nucleotide on the sense strand and / or antisense strand; each internucleotide linkage modification may occur in an alternating pattern on the sense strand and / or antisense strand; or the sense strand or antisense strand may contain two internucleotide linkage modifications in an alternating pattern. The alternating pattern of the internucleotide linkage modification on the sense strand may be the same or different from the antisense strand, and the alternating pattern of the internucleotide linkage modification on the sense strand may have an offset relative to the alternating pattern of the internucleotide linkage modification on the antisense strand. In one embodiment, the double-stranded RNAi agent comprises 6 to 8 phosphorothioate internucleotide linkages. In one embodiment, the antisense strand comprises two phosphorothioate internucleotide linkages at the 5' end and two phosphorothioate internucleotide linkages at the 3' end, and the sense strand comprises at least two phosphorothioate internucleotide linkages at either the 5' end or the 3' end.

[0322] In one embodiment, RNAi includes phosphorothioate or methylphosphonate internucleotide linkage modification in the overhang region. For example, the overhang region may contain two nucleotides, and the nucleotides have a phosphorothioate or methylphosphonate internucleotide linkage between the two nucleotides. Internucleotide linkage modification can also be performed to connect the overhanging nucleotide to the terminal paired nucleotide in the double helix region. For example, at least 2, 3, 4 or all of the overhanging nucleotides can be connected via phosphorothioate or methylphosphonate internucleotide linkage, and optionally there may be additional phosphorothioate or methylphosphonate internucleotide linkages connecting the overhanging nucleotide to the paired nucleotide of the next overhanging nucleotide. For example, there may be at least two phosphorothioate internucleotide linkages between the three nucleotides at the end, two of the three nucleotides being overhanging nucleotides, and the third being the paired nucleotide next to the overhanging nucleotide. These three terminal nucleotides may be at the 3' end of the antisense strand, the 3' end of the sense strand, the 5' end of the antisense strand, and / or the 5' end of the antisense strand.

[0323] In one embodiment, a two-nucleotide overhang is located at the 3' end of the antisense strand, and there are two phosphorothioate internucleotide linkages between the terminal three nucleotides, wherein two of the three nucleotides are overhang nucleotides and the third nucleotide is a paired nucleotide near the overhang nucleotide. Optionally, the RNAi agent may additionally have two phosphorothioate internucleotide linkages between the terminal three nucleotides at the 5' end of the sense strand and at the 5' end of the antisense strand.

[0324] In one embodiment, the RNAi agent includes a mismatch with the target, within the duplex, or a combination thereof. Mismatches may occur in overhang regions or duplex regions. Base pairs can be rated based on their tendency to promote dissociation or melting (e.g., based on the free energy of association or dissociation of a particular pair, the simplest approach is to examine the pair based on individual pairs, although next-adjacent or similar analyses may also be used). In terms of promoting dissociation: A:U is better than G:C; G:U is better than G:C; and I:C is better than G:C (I = inosine). Mismatches, such as atypical or in addition to atypical pairings (as described elsewhere herein), are better than typical (A:T, A:U, G:C) pairings; and pairings comprising universal bases are better than typical pairings.

[0325] In one embodiment, the RNAi agent comprises at least one of the first 1, 2, 3, 4, or 5 base pairs within the duplex region from the 5' end of the antisense strand independently selected from the group consisting of: A:U, G:U, I:C, and mismatched pairs, such as atypical or in addition to atypical pairs or pairs comprising universal bases, to promote dissociation of the antisense strand at the 5' end of the duplex.

[0326] In one embodiment, the nucleotide at position 1 in the duplex region at the 5' end of the antisense strand is selected from the group consisting of A, dA, dU, U, and dT. Alternatively, at least one of the first 1, 2, or 3 base pairs in the duplex region from the 5' end of the antisense strand is an AU base pair. For example, the first base pair in the duplex region from the 5' end of the antisense strand is an AU base pair.

[0327] In another embodiment, the nucleotide at the 3' end of the sense strand is deoxythymidine (dT). In another embodiment, the nucleotide at the 3' end of the antisense strand is deoxythymidine (dT). In one embodiment, there is a short sequence of deoxy-thymidine nucleotides, for example, two dT nucleotides at the 3' end of the sense strand and / or the antisense strand.

[0328] In one embodiment, the sense strand sequence can be represented by formula (I):

[0329] 5'n p -N a -(XXX) i -N b -YY YN b -(ZZZ) j -N a -n q 3' (I)

[0330] in:

[0331] i and j are each independently 0 or 1;

[0332] p and q are each independently 0-6;

[0333] Each N a independently represent an oligonucleotide sequence comprising 0 to 25 modified nucleotides, each sequence comprising at least two different modified nucleotides;

[0334] Each N b independently represents an oligonucleotide sequence comprising 0 to 10 modified nucleotides;

[0335] Each n p and n q Overhanging nucleotides are represented independently;

[0336] wherein Nb and Y do not have the same modification; and

[0337] XXX, YYY and ZZZ each independently represent a motif of three identical modifications on three consecutive nucleotides. Preferably, YYY represents all 2'-F modified nucleotides.

[0338] In one embodiment, N a and / or Nb Includes modifications in alternating patterns.

[0339] In one embodiment, the YYY motif occurs at or near the cleavage site of the sense strand. For example, when the RNAi agent has a duplex region of 17 to 23 nucleotides in length, the YYY motif may occur at or near the cleavage site of the sense strand (e.g., may occur at positions 6, 7, 8; 7, 8, 9; 8, 9, 10; 9, 10, 11; 10, 11, 12; or 11, 12, 13), counting from the first nucleotide from the 5' end; or optionally, counting from the first paired nucleotide in the duplex region from the 5' end.

[0340] In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 1. The sense strand can thus be represented by the formula:

[0341] 5'n p -N a -YYY-N b -ZZZ-N a -n q 3' (Ib);

[0342] 5'n p -N a -XXX-N b -YYY-N a -n q 3'(Ic); or

[0343] 5'n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3'(Id).

[0344] When the sense strand is represented by formula (Ib), N b represents an oligonucleotide sequence comprising 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2 or 0 modified nucleotides. a Independently may refer to an oligonucleotide sequence comprising 2 to 20, 2 to 15 or 2 to 10 modified nucleotides.

[0345] When the sense strand is represented by formula (Ic), N b = represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. a An oligonucleotide sequence comprising 2 to 20, 2 to 15 or 2 to 10 modified nucleotides may independently be represented.

[0346] When the sense strand is represented by formula (Id), each N b independently represents an oligonucleotide sequence comprising 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2 or 0 modified nucleotides. b is 0, 1, 2, 3, 4, 5 or 6. Each N a An oligonucleotide sequence comprising 2 to 20, 2 to 15 or 2 to 10 modified nucleotides may independently be represented.

[0347] Each of X, Y, and Z may be the same as or different from each other.

[0348] In other embodiments, i is 0 and j is 0, and the sense strand can be represented by the formula:

[0349] 5'n p -N a -YYY-N a -n q 3' (Ia).

[0350] When the sense strand is represented by formula (Ia), each N a Independently may refer to an oligonucleotide sequence comprising 2 to 20, 2 to 15 or 2 to 10 modified nucleotides.

[0351] In one embodiment, the antisense strand sequence of RNAi can be represented by formula (II):

[0352] 5'n q' -N a '-(Z'Z'Z') k -N b '-Y'Y'Y'-N b '-(X'X'X') l -N' a -n p '3' (II)

[0353] in:

[0354] k and l are each independently 0 or 1;

[0355] p' and q' are each independently 0-6;

[0356] Each N a ' independently represents an oligonucleotide sequence comprising 0 to 25 modified nucleotides, each sequence comprising at least two different modified nucleotides;

[0357] Each N b ' independently represents an oligonucleotide sequence comprising 0 to 10 modified nucleotides;

[0358] Each n p ' and n q ' independently represent overhanging nucleotides;

[0359] where N b ' and Y' do not have the same modification; and

[0360] X'X'X', Y'Y' and Z'Z'Z' each independently represent a motif of three identical modifications on three consecutive nucleotides.

[0361] In one embodiment, N a ' and / or N b 'Includes modifications of alternating patterns.

[0362] The Y'Y'Y' motif occurs at or near the cleavage site of the antisense strand. For example, when the RNAi agent has a duplex region of 17 to 23 nucleotides in length, the Y'Y'Y' motif can occur at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, counting from the first nucleotide from the 5' end; or, as appropriate, from the first paired nucleotide in the duplex region from the 5' end. Preferably, the Y'Y'Y' motif occurs at positions 11, 12, and 13.

[0363] In one embodiment, the Y'Y'Y' motif is all 2'-OMe modified nucleotides.

[0364] In one embodiment, k is 1 and l is 0, or k is 0 and l is 1, or both k and l are 1.

[0365] The antisense strand can therefore be represented by the following formula:

[0366] 5'n q' -N a '-Z'Z'Z'-N b '-Y'Y'Y'-N a '-n p' 3' (IIb);

[0367] 5'n q' -N a '-Y'Y'Y'-N b '-X'X'X'-n p' 3' (IIc); or

[0368] 5'n q' -N a '-Z'Z'Z'-N b '-Y'Y'Y'-N b '-X'X'X'-N a'-n p' 3' (IId).

[0369] When the antisense strand is represented by formula (IIb), N b ' represents an oligonucleotide sequence comprising 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2 or 0 modified nucleotides. Each N a ' independently refers to an oligonucleotide sequence comprising 2 to 20, 2 to 15 or 2 to 10 modified nucleotides.

[0370] When the antisense strand is represented by formula (IIc), N b ' represents an oligonucleotide sequence comprising 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2 or 0 modified nucleotides. Each N a ' independently refers to an oligonucleotide sequence comprising 2 to 20, 2 to 15 or 2 to 10 modified nucleotides.

[0371] When the antisense strand is represented by formula (IId), each N b ' independently represents an oligonucleotide sequence comprising 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. Each N a ' independently represents an oligonucleotide sequence comprising 2 to 20, 2 to 15 or 2 to 10 modified nucleotides. Preferably, N b is 0, 1, 2, 3, 4, 5, or 6.

[0372] In other embodiments, k is 0 and l is 0, and the antisense strand can be represented by the formula:

[0373] 5'n p' -N a' -Y'Y'Y'-N a' -n q' 3' (Ia).

[0374] When the antisense strand is represented by formula (IIa), each N a ' independently refers to an oligonucleotide sequence comprising 2 to 20, 2 to 15 or 2-10 modified nucleotides.

[0375] Each of X', Y', and Z' may be the same as or different from each other.

[0376] Each nucleotide of the sense and antisense strands can be independently modified with LNA, CRN, UNA, cEt, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-hydroxyl, or 2'-fluoro. For example, each nucleotide of the sense and antisense strands can be independently modified with 2'-O-methyl or 2'-fluoro. In particular, each of X, Y, Z, X', Y', and Z' can represent a 2'-O-methyl modification or a 2'-fluoro modification.

[0377] In one embodiment, when the duplex region is 21 nt, the sense strand of the RNAi agent may contain a YYY motif occurring at positions 9, 10, and 11 of the strand, counting from the first nucleotide at the 5' end, or optionally, from the first paired nucleotide within the duplex region at the 5' end; and Y represents a 2'-F modification. The sense strand may additionally contain an XXX motif or a ZZZ motif as wing modifications at opposite ends of the duplex region; and XXX and ZZZ each independently represent a 2'-OMe modification or a 2'-F modification.

[0378] In one embodiment, the antisense strand may contain a Y'Y'Y' motif occurring at strand positions 11, 12, and 13, counting from the first nucleotide at the 5' end, or alternatively, from the first paired nucleotide in the duplex region at the 5' end; and Y' represents a 2'-O-methyl modification. The antisense strand may additionally contain an X'X'X' motif or a Z'Z'Z' motif as wing modifications at opposite ends of the duplex region; and X'X'X' and Z'Z'Z' each independently represent a 2'-OMe modification or a 2'-F modification.

[0379] The sense strand represented by any one of the above formulae (Ia), (Ib), (Ic) and (Id) forms a duplex with the antisense strand represented by any one of the formulae (IIa), (IIb), (IIc) and (IId).

[0380] Thus, the RNAi agent used in the methods of the present invention may comprise a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the RNAi duplex being represented by formula (III):

[0381] Meaningful: 5'np-Na-(XXX)i-Nb-Y Y Y-Nb-(ZZZ)j-Na-nq 3'

[0382] Antonym: 3'np'-Na'-(X'X'X')k-Nb'-Y'Y'Y'-Nb'-(Z'Z'Z')l-Na'-nq'5'

[0383] (III)

[0384] in:

[0385] i, j, k and l are each independently 0 or 1;

[0386] p, p', q and q' are each independently 0 to 6;

[0387] each Na and Na' independently represents an oligonucleotide sequence comprising 0 to 25 modified nucleotides, each sequence comprising at least two different modified nucleotides;

[0388] Each Nb and Nb' independently represents an oligonucleotide sequence comprising 0 to 10 modified nucleotides;

[0389] wherein each of np', np, nq' and nq, each of which may be present or absent, independently represents an overhanging nucleotide; and

[0390] XXX, YYY, ZZZ, X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent a motif of three identical modifications on three consecutive nucleotides.

[0391] In one embodiment, i is 0 and j is 0; or i is 1 and j is 0; or i is 0 and j is 1; or i and j are both 0; or i and j are both 1. In another embodiment, k is 0 and l is 0; or k is 1 and l is 0; k is 0 and l is 1; or k and l are both 0; or k and l are both 1.

[0392] Exemplary combinations of sense and antisense strands that form an RNAi duplex include the following:

[0393] 5'np-Na-Y Y Y-Na-nq 3'

[0394] 3'np'-Na'-Y'Y'Y'-Na'nq'5'

[0395] (IIIa)

[0396] 5'np-Na-Y Y Y-Nb-Z Z Z-Na-nq 3'

[0397] 3'np'-Na'-Y'Y'Y'-Nb'-Z'Z'Z'-Na'nq'5'

[0398] (IIIb)

[0399] 5'np-Na-X X X-Nb-Y Y Y-Na-nq 3'

[0400] 3'np'-Na'-X'X'X'-Nb'-Y'Y'Y'-Na'-nq'5'

[0401] (IIIc)

[0402] 5'np-Na-X X X-Nb-Y Y Y-Nb-Z Z Z-Na-nq 3'

[0403] 3'np'-Na'-X'X'X'-Nb'-Y'Y'Y'-Nb'-Z'Z'Z'-Na-nq'5'

[0404] (IIId)

[0405] When the RNAi agent is represented by formula (IIIa), each Na independently represents an oligonucleotide sequence comprising 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0406] When the RNAi agent is represented by formula (IIIb), each Nb independently represents an oligonucleotide sequence comprising 1 to 10, 1 to 7, 1 to 5, or 1 to 4 modified nucleotides. Each Na independently represents an oligonucleotide sequence comprising 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0407] When the RNAi agent is represented by formula (IIIc), each Nb, Nb' independently represents an oligonucleotide sequence comprising 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. Each Na independently represents an oligonucleotide sequence comprising 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0408] When the RNAi agent is represented by Formula (IIId), each Nb, Nb' independently represents an oligonucleotide sequence comprising 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. Each Na, Na' independently represents an oligonucleotide sequence comprising 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. Each of Na, Na', Nb, and Nb' independently comprises an alternating pattern of modifications.

[0409] Each of X, Y, and Z in Formulas (III), (IIIa), (IIIb), (IIIc), and (IIId) may be the same as or different from each other.

[0410] When the RNAi agent is represented by Formula (III), (IIIa), (IIIb), (IIIc), and (IIId), at least one of the Y nucleotides can form a base pair with one of the Y' nucleotides. Alternatively, at least two of the Y nucleotides form a base pair with the corresponding Y' nucleotide; or all three of the Y nucleotides form a base pair with the corresponding Y' nucleotide.

[0411] When the RNAi agent is represented by Formula (IIIb) or (IIId), at least one of the Z nucleotides can form a base pair with one of the Z' nucleotides. Alternatively, at least two of the Z nucleotides form a base pair with the corresponding Z' nucleotide; or all three of the Z nucleotides form base pairs with the corresponding Z' nucleotide.

[0412] When the RNAi agent is represented by Formula (IIIc) or (IIId), at least one of the X nucleotides can form a base pair with one of the X' nucleotides. Alternatively, at least two of the X nucleotides form a base pair with the corresponding X' nucleotide; or all three of the X nucleotides form base pairs with the corresponding X' nucleotide.

[0413] In one embodiment, the modification on the Y nucleotide is different from the modification on the Y' nucleotide, the modification on the Z nucleotide is different from the modification on the Z' nucleotide, and / or the modification on the X nucleotide is different from the modification on the X' nucleotide.

[0414] In one embodiment, when the RNAi agent is represented by formula (IIId), Na is modified to 2'-O-methyl or 2'-fluoro modification. In another embodiment, when the RNAi agent is represented by formula (IIId), Na is modified to 2'-O-methyl or 2'-fluoro modification and np'>0 and at least one np' is connected to the adjacent nucleotide via a phosphorothioate bond. In another embodiment, when the RNAi agent is represented by formula (IIId), Na is modified to 2'-O-methyl or 2'-fluoro modification, np'>0 and at least one np' is connected to the adjacent nucleotide via a phosphorothioate bond, and the sense strand is conjugated to one or more GalNAc derivatives connected via a divalent or trivalent branched linker (described below). In another embodiment, when the RNAi agent is represented by formula (IIId), the Na modification is a 2'-O-methyl or 2'-fluoro modification, np'>0 and at least one np' is linked to the adjacent nucleotide via a phosphorothioate linkage, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives linked via a divalent or trivalent branched linker.

[0415] In one embodiment, when the RNAi agent is represented by formula (IIIa), Na is modified to 2'-O-methyl or 2'-fluoro, np'>0 and at least one np' is linked to an adjacent nucleotide via a phosphorothioate linkage, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives linked via a divalent or trivalent branched linker.

[0416] In one embodiment, the RNAi agent is a polymer containing at least two double helices represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), wherein the double helices are connected by a linker. The linker can be cleavable or non-cleavable. Optionally, the polymer further includes a ligand. Each of the double helices can target the same gene or two different genes; or each of the double helices can target the same gene at two different target sites.

[0417] In one embodiment, the RNAi agent is a polymer containing three, four, five, six or more double helices represented by formula (III), (IIIa), (IIIb), (IIIc) and (IIId), wherein the double helices are connected by a linker. The linker may be cleavable or non-cleavable. Optionally, the polymer further comprises a ligand. Each of the double helices can target the same gene or two different genes; or each of the double helices can target the same gene at two different target sites.

[0418] In one embodiment, two RNAi agents represented by Formula (III), (IIIa), (IIIb), (IIIc), and (IIId) are linked to each other at the 5' end, and one or both 3' ends are optionally conjugated to a ligand. Each of the agents can target the same gene or two different genes; or each of the agents can target the same gene at two different target sites.

[0419] In certain embodiments, the RNAi agent of the present invention may contain a small amount of 2'-fluoro-modified nucleotides, such as 10 or fewer nucleotides with 2'-fluoro modifications. For example, the RNAi agent may contain 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 nucleotides with 2'-fluoro modifications. In a specific embodiment, the RNAi agent of the present invention contains 10 nucleotides with 2'-fluoro modifications, such as 4 nucleotides with 2'-fluoro modifications in the sense strand and 6 nucleotides with 2'-fluoro modifications in the antisense strand. In another specific embodiment, the RNAi agent of the present invention contains 6 nucleotides with 2'-fluoro modifications, such as 4 nucleotides with 2'-fluoro modifications in the sense strand and 2 nucleotides with 2'-fluoro modifications in the antisense strand.

[0420] In other embodiments, the RNAi agent of the present invention may contain an ultra-low number of nucleotides containing a 2'-fluoro modification, such as 2 or fewer nucleotides containing a 2'-fluoro modification. For example, the RNAi agent may contain 2, 1, or 0 nucleotides containing a 2'-fluoro modification. In a specific embodiment, the RNAi agent may contain 2 nucleotides containing a 2'-fluoro modification, such as 0 nucleotides containing a 2'-fluoro modification in the sense strand and 2 nucleotides containing a 2'-fluoro modification in the antisense strand.

[0421] Various publications describe poly RNAi agents that can be used in the methods of the present invention. Such publications include WO2007 / 091269, U.S. Patent No. 7,858,769, WO2010 / 141511, WO2007 / 117686, WO2009 / 014887, and WO2011 / 031520, the entire contents of each of which are hereby incorporated herein by reference.

[0422] As described in more detail below, RNAi agents with conjugates containing one or more carbohydrate moieties can optimize one or more properties of the RNAi agent. In many cases, the carbohydrate moiety will be attached to the modified subunit of the RNAi agent. For example, the sugar of the ribose of one or more ribonucleotide subunits of the dsRNA agent can be replaced by another part, such as a non-carbohydrate (preferably cyclic) carrier attached to a carbohydrate ligand. Ribonucleotide subunits in which the ribose of the subunit has been replaced in this way are referred to herein as ribose replacement modified subunits (RRMS). The cyclic carrier can be a carbocyclic ring system, i.e., all ring atoms are carbon atoms, or a heterocyclic ring system, i.e., one or more ring atoms can be heteroatoms, such as nitrogen, oxygen, sulfur. The cyclic carrier can be a monocyclic ring system, or can contain two or more rings, such as a fused ring. The cyclic carrier can be a fully saturated ring system, or it can contain one or more double bonds.

[0423] The ligand can be attached to the polynucleotide via the carrier. The carrier comprises (i) at least one "backbone attachment point", preferably two "backbone attachment points" and (ii) at least one "tethered attachment point". As used herein, a "backbone attachment point" refers to a functional group, such as a hydroxyl group, or generally, a bond that can be used and adapted to incorporate the carrier into the backbone, such as a phosphate, or a modified phosphate, such as a sulfur-containing nucleic acid backbone. In some embodiments, a "tethered attachment point" (TAP) refers to a constituent ring atom of the cyclic carrier to which a selected portion is attached, such as a carbon atom or a heteroatom (different from the atom that provides the backbone attachment point). The portion can be, for example, a carbohydrate, such as a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, and polysaccharide. Alternatively, the selected portion is connected to the cyclic carrier by an intervening linker. Thus, the cyclic carrier will generally include a functional group, such as an amino group, or generally provide a bond suitable for incorporating or tethering another chemical entity, such as a ligand to a constituent ring.

[0424] The RNAi agent can be conjugated to the ligand via a carrier, wherein the carrier can be a cyclic group or a non-cyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinone, tetrahydrofuranyl and decahydronaphthalene; preferably, the non-cyclic group is selected from a serinol backbone or a diethanolamine backbone.

[0425] In another embodiment of the present invention, the iRNA agent comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides. The RNAi agent can be represented by formula (L):

[0426]

[0427] In Formula (L), B1, B2, B3, B1', B2', B3', and B4' are each independently a modified nucleotide containing a group selected from the group consisting of 2'-O alkyl, 2'-substituted alkoxy, 2'-substituted alkyl, 2'-halo, NA, and BNA / LNA. In certain embodiments, B1, B2, B3, B1', B2', B3', and B4' each contain a 2'-OMe modification. In certain embodiments, B1, B2, B3, B1', B2', B3', and B4' each contain a 2'-OMe or 2'-F modification. In certain embodiments, at least one of B1, B2, B3, B1', B2', B3', and B4' contains a 2'-ON-methylacetamido (2'-O-NMA) modification.

[0428] C1 is a thermolabile nucleotide placed at a site opposite to the seed region of the antisense strand (i.e., at positions 2-8 from the 5' end of the antisense strand). For example, C1 is located at a position on the sense strand that pairs with the nucleotides at positions 2-8 from the 5' end of the antisense strand. In one example, C1 is at position 15 from the 5' end of the sense strand. The C1 nucleotide carries a thermolabile modification, which may include an abasic modification; a mismatch with the relative nucleotide in the duplex; and a sugar modification, such as a 2'-deoxy modification or acyclic nucleotide, such as an unlocked nucleic acid (UNA) or a glycerol nucleic acid (GNA). In certain embodiments, C1 has a thermolabile modification selected from the group consisting of: i) a mismatch with the relative nucleotide in the antisense strand; ii) an abasic modification selected from the group consisting of:

[0429] and iii) sugar modifications selected from the group consisting of:

[0430] and Wherein B is a modified or unmodified nucleobase, R 1 and R2 are independently H, halogen, OR3 or alkyl; and R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar. In certain embodiments, the thermolytically stable modification in C1 is a mismatch selected from the group consisting of G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T and U:T; and optionally at least one of the nucleobases in the mismatch is a 2'-deoxynucleobase. In one example, the thermolabile modification in C1 is GNA or

[0431] T1, T1', T2' and T3' each independently represent a nucleotide comprising a modification that provides a steric volume of the nucleotide that is less than or equal to the steric volume of the 2'-OMe modification. Steric volume refers to the sum of the steric effects of the modifications. Methods for determining the steric effects of nucleotide modifications are known to those skilled in the art. The modification may be at the 2' position of the ribose sugar of the nucleotide, or a modification of a non-ribonucleotide, a non-cyclic nucleotide, or the backbone of the nucleotide that is similar or equivalent to the 2' position of the ribose sugar, and provides the nucleotide with a steric volume that is less than or equal to the steric volume of the 2'-OMe modification. For example, T1, T1', T2' and T3' are each independently selected from DNA, RNA, LNA, 2'-F and 2'-F-5'-methyl. In certain embodiments, T1 is DNA. In certain embodiments, T1' is DNA, RNA or LNA. In certain embodiments, T2' is DNA or RNA. In certain embodiments, T3' is DNA or RNA.

[0432] n 1 、n 3 and q 1 The length of is independently 4 to 15 nucleotides.

[0433] n 5 ,q 3 and q 7 The length of is independently 1-6 nucleotides.

[0434] n 4 ,q 2 and q 6 The length of n is independently 1-3 nucleotides; or, n 4 is 0.

[0435] q 5 The length of is independently 0-10 nucleotides.

[0436] n 2 and q 4 The length of is independently 0-3 nucleotides.

[0437] Or, 4The length is 0-3 nucleotides.

[0438] In certain embodiments, n 4 Can be 0. In one embodiment, n 4 is 0, and q 2 and q 6 In another example, n 4 is 0, and q 2 and q 6 is 1, with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand, and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand).

[0439] In certain embodiments, n 4 ,q 2 and q 6 Each is 1.

[0440] In certain embodiments, n 2 、n 4 ,q 2 ,q 4 and q 6 Each is 1.

[0441] In certain embodiments, when the sense strand is 19-22 nucleotides in length and n 4 When C1 is 1, C1 is at position 14-17 from the 5' end of the sense strand. In certain embodiments, C1 is at position 15 from the 5' end of the sense strand.

[0442] In certain embodiments, T3' begins at position 2 from the 5' end of the antisense strand. In one example, T3' begins at position 2 from the 5' end of the antisense strand, and q 6 Equal to 1.

[0443] In certain embodiments, T1' begins at position 14 from the 5' end of the antisense strand. In one example, T1' is at position 14 from the 5' end of the antisense strand, and q 2 Equal to 1.

[0444] In an exemplary embodiment, T3' starts at position 2 of the 5' end of the antisense strand, and T1' starts at position 14 of the 5' end of the antisense strand. In one example, T3' starts at position 2 of the 5' end of the antisense strand and 6 is equal to 1, and T1' starts at position 14 of the 5' end of the antisense strand and q 2 Equal to 1.

[0445] In certain embodiments, T1' and T3' are separated by 11 nucleotides (ie, T1' and T3' nucleotides are not counted).

[0446] In certain embodiments, T1' is at position 14 from the 5' end of the antisense strand. In one example, T1' is at position 14 from the 5' end of the antisense strand and q 2 =1, and the modification is at the 2' position or at a non-ribose, non-cyclic or backbone position that provides less steric bulk than 2'-OMe ribose.

[0447] In certain embodiments, T3' is at position 2 from the 5' end of the antisense strand. In one example, T3' is at position 2 from the 5' end of the antisense strand and q 6 Equal to 1, and modification at the 2' position or positions that are non-ribose, non-cyclic, or in the backbone provides a steric bulk that is less than or equal to that of 2'-OMe ribose.

[0448] In certain embodiments, T1 is at the cleavage site of the sense strand. In one example, when the sense strand is 19-22 nucleotides long and n 2 When n is 1, T1 is at position 11 from the 5' end of the sense strand. In an exemplary embodiment, when the length of the sense strand is 19-22 nucleotides and n is 1, T1 is at position 11 from the 5' end of the sense strand. 2 When T1 is 1, the cleavage site of the sense strand is located at position 11 from the 5' end of the sense strand.

[0449] In certain embodiments, T2' begins at position 6 from the 5' end of the antisense strand. In one example, T2' is at position 6-10 from the 5' end of the antisense strand, and q 4 is 1.

[0450] In an exemplary embodiment, T1 is located at the cleavage site of the sense strand, for example, at position 11 from the 5' end of the sense strand, when the sense strand is 19-22 nucleotides long, and n 2 is 1; T1' is at position 14 from the 5' end of the antisense strand, and q 2 =1, and the modification to T1' is at the 2' position of the sugar of ribose or at a non-ribose, non-cyclic, or backbone position that provides less steric bulk than 2'-OMe ribose; T2' is at positions 6-10 from the 5' end of the antisense strand, and q 4 is 1; and T3' is located at position 2 from the 5' end of the antisense strand, and q 6 =1, and the modification to T3' is at the 2' position or at a non-ribose, non-cyclic, or backbone position that provides less or equal steric bulk than 2'-OMe ribose.

[0451] In certain embodiments, T2' begins at position 8 of the 5' end of the antisense strand. In one example, T2' begins at position 8 of the 5' end of the antisense strand, and q 4 is 2.

[0452] In certain embodiments, T2' begins at position 9 of the 5' end of the antisense strand. In one example, T2' is at position 9 of the 5' end of the antisense strand, and q 4 is 1.

[0453] In certain embodiments, B1' is 2'-OMe or 2'-F,q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, T2' is 2'-F, q 4 is 1, B3' is 2'-OMe or 2'-F, q 5 is 6, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7 is 1; has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand).

[0454] In certain embodiments, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, T2' is 2'-F, q 4 is 1, B3' is 2'-OMe or 2'-F, q 5 is 6, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7 is 1; has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand).

[0455] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, T2' is 2'-F, q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7 is 1.

[0456] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, T2' is 2'-F, q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7 is 1; has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand).

[0457] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 6, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 7, T1' is 2'-F, q 2is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, T2' is 2'-F, q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7 is 1.

[0458] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 6, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 7, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, T2' is 2'-F, q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7 is 1; has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand).

[0459] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, T2' is 2'-F, q 4 is 1, B3' is 2'-OMe or 2'-F, q 5 is 6, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7is 1.

[0460] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, T2' is 2'-F, q 4 is 1, B3' is 2'-OMe or 2'-F, q 5 is 6, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7 is 1; has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand).

[0461] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 5, T2' is 2'-F, q 4 is 1, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7 is 1; optionally with at least 2 additional TTs at the 3' end of the antisense strand.

[0462] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 5, T2' is 2'-F, q 4 is 1, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7 1; optionally having at least 2 additional TT at the 3' end of the antisense strand; two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand).

[0463] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, q 4 is 0, B3' is 2'-OMe or 2'-F, q 5 is 7, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7 is 1.

[0464] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, q4 is 0, B3' is 2'-OMe or 2'-F, q 5 is 7, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7 is 1; has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end).

[0465] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, T2' is 2'-F, q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6 is 1, B4' is 2'-F, and q 7 is 1.

[0466] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, T2' is 2'-F, q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6 is 1, B4' is 2'-F, and q 7is 1; has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand).

[0467] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, q 4 is 0, B3' is 2'-OMe or 2'-F, q 5 is 7, T3' is 2'-F, q 6 is 1, B4' is 2'-F, and q 7 is 1.

[0468] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, q 4 is 0, B3' is 2'-OMe or 2'-F, q 5 is 7, T3' is 2'-F, q 6 is 1, B4' is 2'-F, and q 7 is 1; has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand).

[0469] The RNAi agent may include a phosphorus-containing group at the 5' end of the sense strand or the antisense strand. The 5'-terminal phosphorus-containing group may be a 5'-terminal phosphate (5'-P), a 5'-terminal phosphorothioate (5'-PS), a 5'-terminal phosphorodithioate (5'-PS2), a 5'-terminal vinylphosphonate (5'-VP), a 5'-terminal methylphosphonate (MePhos), or a 5'-deoxy-5'-C-malonyl ( When the 5'-terminal phosphorus-containing group is a 5'-terminal vinyl phosphonate (5'-VP), the 5'-VP may be a 5'-E-VP isomer (i.e., trans-vinyl phosphate). ), 5'-Z-VP isomer (also known as cis vinyl phosphate ) or a mixture thereof.

[0470] In certain embodiments, the RNAi agent comprises a phosphorus-containing group at the 5' end of the sense strand. In certain embodiments, the RNAi agent comprises a phosphorus-containing group at the 5' end of the antisense strand.

[0471] In certain embodiments, the RNAi agent comprises a 5'-P. In certain embodiments, the RNAi agent comprises a 5'-P in the antisense strand.

[0472] In certain embodiments, the RNAi agent comprises a 5'-PS. In certain embodiments, the RNAi agent comprises a 5'-PS in the antisense strand.

[0473] In certain embodiments, the RNAi agent comprises a 5'-VP. In certain embodiments, the RNAi agent comprises a 5'-VP in the antisense strand. In certain embodiments, the RNAi agent comprises a 5'-E-VP in the antisense strand. In certain embodiments, the RNAi agent comprises a 5'-Z-VP in the antisense strand.

[0474] In certain embodiments, the RNAi agent comprises a 5'-PS2. In certain embodiments, the RNAi agent comprises a 5'-PS2 in the antisense strand.

[0475] In certain embodiments, the RNAi agent comprises 5'-PS2. In certain embodiments, the RNAi agent comprises a 5'-deoxy-5'-C-malonyl group in the antisense strand.

[0476] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, T2' is 2'-F, q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6 is 1, B4 is 2'-OMe, and q 7 is 1. RNAi agents also include 5'-PS.

[0477] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, T2' is 2'-F, q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7 is 1. RNAi agents also include 5'-P.

[0478] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, T2' is 2'-F, q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7 The RNAi agent further comprises a 5'-VP. The 5'-VP may be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0479] In certain embodiments, B1 is 2'-OMe or 2'-F, n1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, T2' is 2'-F, q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7 is 1. RNAi agents also include 5'-PS2.

[0480] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, T2' is 2'-F, q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The RNAi agent also includes a 5'-deoxy-5'-C-malonyl group.

[0481] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, T2' is 2'-F, q 4is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7 is 1; has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counted from the 5' end of the sense strand), and has two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counted from the 5' end of the antisense strand). The RNAi agent also includes a 5'-P.

[0482] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, T2' is 2'-F, q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7 1; two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counted from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counted from the 5' end of the antisense strand). RNAi agents also include 5'-PS.

[0483] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, T2' is 2'-F, q 4is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7 1; two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counted from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counted from the 5' end of the antisense strand). The RNAi agent further comprises a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0484] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, T2' is 2'-F, q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7 1; two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counted from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counted from the 5' end of the antisense strand). RNAi agents also include 5'-PS2.

[0485] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3is 4, T2' is 2'-F, q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7 is 1; has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counted from the 5' end of the sense strand), and has two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counted from the 5' end of the antisense strand). The RNAi agent also includes a 5'-deoxy-5'-C-malonyl group.

[0486] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, q 4 is 0, B3' is 2'-OMe or 2'-F, q 5 is 7, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7 is 1. RNAi agents also include 5'-P.

[0487] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, q 4 is 0, B3' is 2'-OMe or 2'-F, q 5 is 7, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7is 1. The dsRNA agent also includes 5'-PS.

[0488] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, q 4 is 0, B3' is 2'-OMe or 2'-F, q 5 is 7, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7 The RNAi agent further comprises a 5'-VP. The 5'-VP may be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0489] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, q 4 is 0, B3' is 2'-OMe or 2'-F, q 5 is 7, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7 is 1. RNAi agents also include 5'-PS2.

[0490] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, q 4 is 0, B3' is 2'-OMe or 2'-F, q 5 is 7, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The RNAi agent also includes a 5'-deoxy-5'-C-malonyl group.

[0491] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, q 4 is 0, B3' is 2'-OMe or 2'-F, q 5 is 7, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7 is 1; has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counted from the 5' end), and has two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counted from the 5' end). The RNAi agent also includes a 5'-P.

[0492] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, q 4 is 0, B3' is 2'-OMe or 2'-F, q 5 is 7, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q7 is 1; has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counted from the 5' end), and has two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counted from the 5' end). The RNAi agent also includes 5'-PS.

[0493] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, q 4 is 0, B3' is 2'-OMe or 2'-F, q 5 is 7, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7 The RNAi agent further comprises a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0494] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, q 4 is 0, B3' is 2'-OMe or 2'-F, q 5 is 7, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 71; two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counted from the 5' end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counted from the 5' end). The RNAi agent also includes 5'-PS2.

[0495] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, q 4 is 0, B3' is 2'-OMe or 2'-F, q 5 is 7, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7 is 1; has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counted from the 5' end), and has two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counted from the 5' end). The RNAi agent also includes a 5'-deoxy-5'-C-malonyl group.

[0496] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, T2' is 2'-F, q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6 is 1, B4' is 2'-F, and q 7 is 1. RNAi agents also include 5'-P.

[0497] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, T2' is 2'-F, q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6 is 1, B4' is 2'-F, and q 7 is 1. RNAi agents also include 5'-PS.

[0498] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, T2' is 2'-F, q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6 is 1, B4' is 2'-F, and q 7 The RNAi agent further comprises a 5'-VP. The 5'-VP may be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0499] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, T2' is 2'-F, q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6 is 1, B4' is 2'-F, and q 7 1. The dsRNA RNA agent also includes 5'-PS2.

[0500] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, T2' is 2'-F, q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6 is 1, B4' is 2'-F, and q 7 is 1. The RNAi agent also includes a 5'-deoxy-5'-C-malonyl group.

[0501] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, T2' is 2'-F, q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6 is 1, B4' is 2'-F, and q 7is 1; has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counted from the 5' end of the sense strand), and has two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counted from the 5' end of the antisense strand). The RNAi agent also includes a 5'-P.

[0502] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, T2' is 2'-F, q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6 is 1, B4' is 2'-F, and q 7 1; two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counted from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counted from the 5' end of the antisense strand). RNAi agents also include 5'-PS.

[0503] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, T2' is 2'-F, q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6 is 1, B4' is 2'-F, and q 71; two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counted from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counted from the 5' end of the antisense strand). The RNAi agent further comprises a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0504] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, T2' is 2'-F, q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6 is 1, B4' is 2'-F, and q 7 1; two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counted from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counted from the 5' end of the antisense strand). RNAi agents also include 5'-PS2.

[0505] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, T2' is 2'-F, q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6is 1, B4' is 2'-F, and q 7 is 1; has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counted from the 5' end of the sense strand), and has two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counted from the 5' end of the antisense strand). The RNAi agent also includes a 5'-deoxy-5'-C-malonyl group.

[0506] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, q 4 is 0, B3' is 2'-OMe or 2'-F, q 5 is 7, T3' is 2'-F, q 6 is 1, B4' is 2'-F, and q 7 is 1. RNAi agents also include 5'-P.

[0507] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, q 4 is 0, B3' is 2'-OMe or 2'-F, q 5 is 7, T3' is 2'-F, q 6 is 1, B4' is 2'-F, and q 7 is 1. RNAi agents also include 5'-PS.

[0508] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, q 4 is 0, B3' is 2'-OMe or 2'-F, q 5 is 7, T3' is 2'-F, q 6 is 1, B4' is 2'-F, and q 7 The RNAi agent further comprises a 5'-VP. The 5'-VP may be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0509] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, q 4 is 0, B3' is 2'-OMe or 2'-F, q 5 is 7, T3' is 2'-F, q 6 is 1, B4' is 2'-F, and q 7 is 1. RNAi agents also include 5'-PS2.

[0510] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, q 4 is 0, B3' is 2'-OMe or 2'-F, q 5 is 7, T3' is 2'-F, q6 is 1, B4' is 2'-F, and q 7 is 1. The RNAi agent also includes a 5'-deoxy-5'-C-malonyl group.

[0511] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, q 4 is 0, B3' is 2'-OMe or 2'-F, q 5 is 7, T3' is 2'-F, q 6 is 1, B4' is 2'-F, and q 7 is 1; has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counted from the 5' end of the sense strand), and has two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counted from the 5' end of the antisense strand). The RNAi agent also includes a 5'-P.

[0512] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, q 4 is 0, B3' is 2'-OMe or 2'-F, q 5 is 7, T3' is 2'-F, q 6 is 1, B4' is 2'-F, and q 71; two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counted from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counted from the 5' end of the antisense strand). RNAi agents also include 5'-PS.

[0513] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, q 4 is 0, B3' is 2'-OMe or 2'-F, q 5 is 7, T3' is 2'-F, q 6 is 1, B4' is 2'-F, and q 7 1; two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counted from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counted from the 5' end of the antisense strand). The RNAi agent further comprises a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0514] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, q 4 is 0, B3' is 2'-OMe or 2'-F, q 5 is 7, T3' is 2'-F, q 6 is 1, B4' is 2'-F, and q7 1; two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counted from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counted from the 5' end of the antisense strand). RNAi agents also include 5'-PS2.

[0515] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, q 4 is 0, B3' is 2'-OMe or 2'-F, q 5 is 7, T3' is 2'-F, q 6 is 1, B4' is 2'-F, and q 7 is 1; has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counted from the 5' end of the sense strand), and has two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counted from the 5' end of the antisense strand). The RNAi agent also includes a 5'-deoxy-5'-C-malonyl group.

[0516] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, T2' is 2'-F, q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6 is 1, B4 is 2'-OMe, and q7 In some embodiments, the RNAi agent comprises a 5'-P and a target ligand. In some embodiments, the 5'-P is at the 5' end of the antisense strand, and the target ligand is at the 3' end of the sense strand.

[0517] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, T2' is 2'-F, q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6 is 1, B4 is 2'-OMe, and q 7 In certain embodiments, the RNAi agent comprises a 5'-PS and a target ligand. In certain embodiments, the 5'-PS is at the 5' end of the antisense strand, and the target ligand is at the 3' end of the sense strand.

[0518] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, T2' is 2'-F, q4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7 is 1; has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counted from the 5' end of the sense strand), and has two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counted from the 5' end of the antisense strand). The RNAi agent also includes a 5'-VP (e.g., 5'-E-VP, 5'-Z-VP, or a combination thereof) and a targeting ligand.

[0519] In certain embodiments, the 5'-VP is located at the 5' end of the antisense strand and the ligand of interest is located at the 3' end of the sense strand.

[0520] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, T2' is 2'-F, q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7 In certain embodiments, the RNAi agent comprises a 5'-PS2 and a target ligand. In certain embodiments, the 5'-PS2 is located at the 5' end of the antisense strand, and the target ligand is located at the 3' end of the sense strand.

[0521] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, T2' is 2'-F, q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7 is 1; there are two phosphorothioate internucleotide linkage modifications in positions 1-5 of the sense strand (counted from the 5' end of the sense strand), and there are two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (counted from the 5' end of the antisense strand). The RNAi agent also includes a 5'-deoxy-5'-C-malonyl group and a target ligand. In certain embodiments, the 5'-deoxy-5'-C-malonyl group is located at the 5' end of the antisense strand, and the target ligand is located at the 3' end of the sense strand.

[0522] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, q 4 is 0, B3' is 2'-OMe or 2'-F, q 5 is 7, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7 is 1; there are two phosphorothioate internucleotide linkage modifications in positions 1-5 of the sense strand (counted from the 5' end), and there are two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (counted from the 5' end). The RNAi agent also includes 5'-P and a target ligand. In certain embodiments, the 5'-P is located at the 5' end of the antisense strand, and the target ligand is located at the 3' end of the sense strand.

[0523] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, q 4 is 0, B3' is 2'-OMe or 2'-F, q 5 is 7, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7 1; There are two phosphorothioate internucleotide linkage modifications in positions 1-5 of the sense strand (counted from the 5' end), and there are two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (counted from the 5' end). The RNAi agent also includes a 5'-PS and a target ligand. In certain embodiments, the 5'-PS is at the 5' end of the antisense strand, and the target ligand is at the 3' end of the sense strand.

[0524] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, q 4 is 0, B3' is 2'-OMe or 2'-F, q 5 is 7, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7is 1; has two phosphorothioate internucleotide linkage modifications in positions 1-5 of the sense strand (counted from the 5' end), and has two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and has two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (counted from the 5' end). The RNAi agent further includes a 5'-VP (e.g., 5'-E-VP, 5'-Z-VP, or a combination thereof) and a target ligand. In certain embodiments, the 5'-VP is located at the 5' end of the antisense strand, and the target ligand is located at the 3' end of the sense strand.

[0525] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, q 4 is 0, B3' is 2'-OMe or 2'-F, q 5 is 7, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7 In some embodiments, the RNAi agent comprises a 5'-PS2 and a target ligand. In some embodiments, the 5'-PS2 is located at the 5' end of the antisense strand, and the target ligand is located at the 3' end of the sense strand.

[0526] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, q 4is 0, B3' is 2'-OMe or 2'-F, q 5 is 7, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7 is 1; has two phosphorothioate internucleotide linkage modifications in positions 1-5 of the sense strand (counted from the 5' end), and has two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and has two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (counted from the 5' end). The RNAi agent also includes a 5'-deoxy-5'-C-malonyl group and a target ligand. In certain embodiments, the 5'-deoxy-5'-C-malonyl group is located at the 5' end of the antisense strand, and the target ligand is located at the 3' end of the sense strand.

[0527] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, T2' is 2'-F, q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6 is 1, B4' is 2'-F, and q 7 In some embodiments, the RNAi agent comprises a 5'-P and a target ligand. In some embodiments, the 5'-P is at the 5' end of the antisense strand, and the target ligand is at the 3' end of the sense strand.

[0528] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, T2' is 2'-F, q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6 is 1, B4' is 2'-F, and q 7 In certain embodiments, the RNAi agent comprises a 5'-PS and a target ligand. In certain embodiments, the 5'-PS is at the 5' end of the antisense strand, and the target ligand is at the 3' end of the sense strand.

[0529] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, T2' is 2'-F, q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6 is 1, B4' is 2'-F, and q 7 is 1; there are two phosphorothioate internucleotide linkage modifications in positions 1-5 of the sense strand (counted from the 5' end of the sense strand), and there are two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (counted from the 5' end of the antisense strand). The RNAi agent also includes a 5'-VP (e.g., 5'-E-VP, 5'-Z-VP, or a combination thereof) and a target ligand. In certain embodiments, the 5'-VP is located at the 5' end of the antisense strand, and the target ligand is located at the 3' end of the sense strand.

[0530] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, T2' is 2'-F, q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6 is 1, B4' is 2'-F, and q 7 In certain embodiments, the RNAi agent comprises a 5'-PS2 and a target ligand. In certain embodiments, the 5'-PS2 is located at the 5' end of the antisense strand, and the target ligand is located at the 3' end of the sense strand.

[0531] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, T2' is 2'-F, q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6 is 1, B4' is 2'-F, and q 7is 1; there are two phosphorothioate internucleotide linkage modifications in positions 1-5 of the sense strand (counted from the 5' end of the sense strand), and there are two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (counted from the 5' end of the antisense strand). The RNAi agent also includes a 5'-deoxy-5'-C-malonyl group and a target ligand. In certain embodiments, the 5'-deoxy-5'-C-malonyl group is located at the 5' end of the antisense strand, and the target ligand is located at the 3' end of the sense strand.

[0532] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, q 4 is 0, B3' is 2'-OMe or 2'-F, q 5 is 7, T3' is 2'-F, q 6 is 1, B4' is 2'-F, and q 7 In some embodiments, the RNAi agent comprises a 5'-P and a target ligand. In some embodiments, the 5'-P is at the 5' end of the antisense strand, and the target ligand is at the 3' end of the sense strand.

[0533] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, q 4is 0, B3' is 2'-OMe or 2'-F, q 5 is 7, T3' is 2'-F, q 6 is 1, B4' is 2'-F, and q 7 In certain embodiments, the RNAi agent comprises a 5'-PS and a target ligand. In certain embodiments, the 5'-PS is at the 5' end of the antisense strand, and the target ligand is at the 3' end of the sense strand.

[0534] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, q 4 is 0, B3' is 2'-OMe or 2'-F, q 5 is 7, T3' is 2'-F, q 6 is 1, B4' is 2'-F, and q 7 is 1; there are two phosphorothioate internucleotide linkage modifications in positions 1-5 of the sense strand (counted from the 5' end of the sense strand), and there are two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (counted from the 5' end of the antisense strand). The RNAi agent also includes a 5'-VP (e.g., 5'-E-VP, 5'-Z-VP, or a combination thereof) and a target ligand. In certain embodiments, the 5'-VP is located at the 5' end of the antisense strand, and the target ligand is located at the 3' end of the sense strand.

[0535] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, q 4 is 0, B3' is 2'-OMe or 2'-F, q 5 is 7, T3' is 2'-F, q 6 is 1, B4' is 2'-F, and q 7 In certain embodiments, the RNAi agent comprises a 5'-PS2 and a target ligand. In certain embodiments, the 5'-PS2 is located at the 5' end of the antisense strand, and the target ligand is located at the 3' end of the sense strand.

[0536] In certain embodiments, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, q 4 is 0, B3' is 2'-OMe or 2'-F, q 5 is 7, T3' is 2'-F, q 6 is 1, B4' is 2'-F, and q 7 is 1; there are two phosphorothioate internucleotide linkage modifications in positions 1-5 of the sense strand (counted from the 5' end of the sense strand), and there are two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (counted from the 5' end of the antisense strand). The RNAi agent also includes a 5'-deoxy-5'-C-malonyl group and a target ligand. In certain embodiments, the 5'-deoxy-5'-C-malonyl group is located at the 5' end of the antisense strand, and the target ligand is located at the 3' end of the sense strand.

[0537] In a specific embodiment, the RNAi agent of the present invention comprises:

[0538] (a) having a sense strand as follows:

[0539] (i) 21 nucleotides in length;

[0540] (ii) an ASGPR ligand attached to the 3′ end, wherein the ASGPR ligand comprises three GalNAc derivatives attached via a trivalent branched linker; and

[0541] (iii) 2'-F modifications at positions 1, 3, 5, 7, 9 to 11, 13, 17, 19, and 21, and 2'-OMe modifications at positions 2, 4, 6, 8, 12, 14 to 16, 18, and 20 (counting from the 5' end);

[0542] and

[0543] (b) having an antisense strand of:

[0544] (i) 23 nucleotides in length;

[0545] (ii) 2'-OMe modifications at positions 1, 3, 5, 9, 11 to 13, 15, 17, 19, 21, and 23, and 2'F modifications at positions 2, 4, 6 to 8, 10, 14, 16, 18, 20, and 22 (counting from the 5' end); and

[0546] (iii) phosphorothioate internucleotide linkages between nucleotide positions 21 and 22 and between nucleotide positions 22 and 23 (counting from the 5' end);

[0547] wherein the dsRNA agent has a dinucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0548] In another specific embodiment, the RNAi agent of the invention comprises:

[0549] (a) having a sense strand as follows:

[0550] (i) 21 nucleotides in length;

[0551] (ii) an ASGPR ligand attached to the 3′ end, wherein the ASGPR ligand comprises three GalNAc derivatives attached via a trivalent branched linker;

[0552] (iii) 2'-F modifications at positions 1, 3, 5, 7, 9 to 11, 13, 15, 17, 19, and 21, and 2'-OMe modifications at positions 2, 4, 6, 8, 12, 14, 16, 18, and 20 (counting from the 5' end); and

[0553] (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counted from the 5' end);

[0554] and

[0555] (b) having an antisense strand of:

[0556] (i) 23 nucleotides in length;

[0557] (ii) 2'-OMe modifications at positions 1, 3, 5, 7, 9, 11 to 13, 15, 17, 19, and 21 to 23, and 2'F modifications at positions 2, 4, 6, 8, 10, 14, 16, 18, and 20 (counting from the 5' end); and

[0558] (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end);

[0559] wherein the RNAi agent has a dinucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0560] In another specific embodiment, the RNAi agent of the invention comprises:

[0561] (a) having a sense strand as follows:

[0562] (i) 21 nucleotides in length;

[0563] (ii) an ASGPR ligand attached to the 3′ end, wherein the ASGPR ligand comprises three GalNAc derivatives attached via a trivalent branched linker;

[0564] (iii) 2'-OMe modifications at positions 1 to 6, 8, 10, and 12 to 21, 2'-F modifications at positions 7 and 9, and a deoxynucleotide (e.g., dT) at position 11 (counting from the 5' end); and

[0565] (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counted from the 5' end);

[0566] and

[0567] (b) having an antisense strand of:

[0568] (i) 23 nucleotides in length;

[0569] (ii) 2'-OMe modifications at positions 1, 3, 7, 9, 11, 13, 15, 17, and 19 to 23, and 2'-F modifications at positions 2, 4 to 6, 8, 10, 12, 14, 16, and 18 (counting from the 5' end); and

[0570] (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end);

[0571] wherein the RNAi agent has a dinucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0572] In another specific embodiment, the RNAi agent of the invention comprises:

[0573] (a) having a sense strand as follows:

[0574] (i) 21 nucleotides in length;

[0575] (ii) an ASGPR ligand attached to the 3′ end, wherein the ASGPR ligand comprises three GalNAc derivatives attached via a trivalent branched linker;

[0576] (iii) 2'-OMe modifications at positions 1 to 6, 8, 10, 12, 14, and 16 to 21, and 2'-F modifications at positions 7, 9, 11, 13, and 15; and

[0577] (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counted from the 5' end);

[0578] and

[0579] (b) having an antisense strand of:

[0580] (i) 23 nucleotides in length;

[0581] (ii) 2'-OMe modifications at positions 1, 5, 7, 9, 11, 13, 15, 17, 19, and 21 to 23, and 2'-F modifications at positions 2 to 4, 6, 8, 10, 12, 14, 16, 18, and 20 (counting from the 5' end); and

[0582] (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end);

[0583] wherein the RNAi agent has a dinucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0584] In another specific embodiment, the RNAi agent of the invention comprises:

[0585] (a) having a sense strand as follows:

[0586] (i) 21 nucleotides in length;

[0587] (ii) an ASGPR ligand attached to the 3′ end, wherein the ASGPR ligand comprises three GalNAc derivatives attached via a trivalent branched linker;

[0588] (iii) 2'-OMe modifications at positions 1 to 9 and 12 to 21, and 2'-F modifications at positions 10 and 11; and

[0589] (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counted from the 5' end);

[0590] and

[0591] (b) having an antisense strand of:

[0592] (i) 23 nucleotides in length;

[0593] (ii) 2'-OMe modifications at positions 1, 3, 5, 7, 9, 11-13, 15, 17, 19, and 21-23, and 2'-F modifications at positions 2, 4, 6, 8, 10, 14, 16, 18, and 20 (counting from the 5' end); and

[0594] (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end);

[0595] wherein the RNAi agent has a dinucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0596] In another specific embodiment, the RNAi agent of the invention comprises:

[0597] (a) having a sense strand as follows:

[0598] (i) 21 nucleotides in length;

[0599] (ii) an ASGPR ligand attached to the 3′ end, wherein the ASGPR ligand comprises three GalNAc derivatives attached via a trivalent branched linker;

[0600] (iii) 2'-F modifications at positions 1, 3, 5, 7, 9 to 11, and 13, and 2'-OMe modifications at positions 2, 4, 6, 8, 12, and 14 to 21; and

[0601] (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counted from the 5' end);

[0602] and

[0603] (b) having an antisense strand of:

[0604] (i) 23 nucleotides in length;

[0605] (ii) 2'-OMe modifications at positions 1, 3, 5 to 7, 9, 11 to 13, 15, 17 to 19, and 21 to 23, and 2'-F modifications at positions 2, 4, 8, 10, 14, 16, and 20 (counting from the 5' end); and

[0606] (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end);

[0607] wherein the RNAi agent has a dinucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0608] In another specific embodiment, the RNAi agent of the invention comprises:

[0609] (a) having a sense strand as follows:

[0610] (i) 21 nucleotides in length;

[0611] (ii) an ASGPR ligand attached to the 3′ end, wherein the ASGPR ligand comprises three GalNAc derivatives attached via a trivalent branched linker;

[0612] (iii) 2'-OMe modifications at positions 1, 2, 4, 6, 8, 12, 14, 15, 17, and 19 to 21, and 2'-F modifications at positions 3, 5, 7, 9 to 11, 13, 16, and 18; and

[0613] (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counted from the 5' end);

[0614] and

[0615] (b) having an antisense strand of:

[0616] (i) 25 nucleotides in length;

[0617] (ii) 2'-OMe modifications at positions 1, 4, 6, 7, 9, 11 to 13, 15, 17, and 19 to 23, 2'-F modifications at positions 2, 3, 5, 8, 10, 14, 16, and 18, and deoxynucleotides (e.g., dT) at positions 24 and 25 (counting from the 5' end); and

[0618] (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end);

[0619] wherein the RNAi agent has a tetranucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0620] In another specific embodiment, the RNAi agent of the invention comprises:

[0621] (a) having a sense strand as follows:

[0622] (i) 21 nucleotides in length;

[0623] (ii) an ASGPR ligand attached to the 3′ end, wherein the ASGPR ligand comprises three GalNAc derivatives attached via a trivalent branched linker;

[0624] (iii) 2'-OMe modifications at positions 1 to 6, 8, and 12 to 21, and 2'-F modifications at positions 7 and 9 to 11; and

[0625] (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counted from the 5' end);

[0626] and

[0627] (b) having an antisense strand of:

[0628] (i) 23 nucleotides in length;

[0629] (ii) 2'-OMe modifications at positions 1, 3 to 5, 7, 8, 10 to 13, 15, and 17 to 23, and 2'-F modifications at positions 2, 6, 9, 14, and 16 (counting from the 5' end); and

[0630] (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end);

[0631] wherein the RNAi agent has a dinucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0632] In another specific embodiment, the RNAi agent of the invention comprises:

[0633] (a) having a sense strand as follows:

[0634] (i) 21 nucleotides in length;

[0635] (ii) an ASGPR ligand attached to the 3′ end, wherein the ASGPR ligand comprises three GalNAc derivatives attached via a trivalent branched linker;

[0636] (iii) 2'-OMe modifications at positions 1 to 6, 8, and 12 to 21, and 2'-F modifications at positions 7 and 9 to 11; and

[0637] (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counted from the 5' end);

[0638] and

[0639] (b) having an antisense strand of:

[0640] (i) 23 nucleotides in length;

[0641] (ii) 2'-OMe modifications at positions 1, 3 to 5, 7, 10 to 13, 15, and 17 to 23, and 2'-F modifications at positions 2, 6, 8, 9, 14, and 16 (counting from the 5' end); and

[0642] (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end);

[0643] wherein the RNAi agent has a dinucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0644] In another specific embodiment, the RNAi agent of the invention comprises:

[0645] (a) having a sense strand as follows:

[0646] (i) 19 nucleotides in length;

[0647] (ii) an ASGPR ligand attached to the 3′ end, wherein the ASGPR ligand comprises three GalNAc derivatives attached via a trivalent branched linker;

[0648] (iii) 2'-OMe modifications at positions 1 to 4, 6, and 10 to 19, and 2'-F modifications at positions 5 and 7 to 9; and

[0649] (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counted from the 5' end);

[0650] and

[0651] (b) having an antisense strand of:

[0652] (i) 21 nucleotides in length;

[0653] (ii) 2'-OMe modifications at positions 1, 3 to 5, 7, 10 to 13, 15, and 17 to 21, and 2'-F modifications at positions 2, 6, 8, 9, 14, and 16 (counting from the 5' end); and

[0654] (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21 (counting from the 5' end);

[0655] wherein the RNAi agent has a dinucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0656] In certain embodiments, the iRNA used in the methods of the present invention is an agent selected from the group consisting of those listed in Table 2 or disclosed in PCT / US2019 / 023079. In one embodiment, the agent is AD-288996. Such agents may further include a ligand.

[0657] V. iRNA conjugated to ligand

[0658] Another modification of the iRNA used in the methods of the invention involves chemically attaching to the RNA one or more ligands, moieties, or conjugates that enhance the activity, cellular distribution, or cellular uptake of the iRNA. Such moieties include, but are not limited to, lipid moieties, such as cholesterol moieties (Letsinger et al., (1989) Proc. Natl. Acid. Sci. USA, 86:6553-6556), cholic acid (Manoharan et al., (1994) Biorg. Med. Chem. Let., 4:1053-1060), thioethers, e.g., hexyl-S-tritylthiol (Manoharan et al., (1992) Ann. NY Acad. Sci., 660:306-309; Manoharan et al., (1993) Biorg. Med. Chem. Let., 3:2765-2770), thiocholesterol (Oberhauser et al., (1992) Nucl. Acids Res., 20:533-538), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., (1991) EMBO J, 10: 1111-1118; Kabanov et al., (1990) FEBS Lett., 259: 327-330; Svinarchuk et al., (1993) Biochimie, 75: 49-54), phospholipids, such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glyceryl-3-H-phosphonate (Manoharan et al., (1995) Tetrahedron Lett., 36: 3651-3654; Shea et al., (1990) Nucl. Acids Res., 18:3777-3783), a polyamine or polyethylene glycol chain (Manoharan et al., (1995) Nucleosides & Nucleotides, 14:969-973), or adamantane acetic acid (Manoharan et al., (1995) Tetrahedron Lett., 36:3651-3654), a palmityl moiety (Mishra et al., (1995) Biochim. Biophys. Acta, 1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., (1996) J. Pharmacol. Exp. Ther., 277:923-937).

[0659] In one embodiment, the ligand alters the distribution, targeting, or lifespan of the iRNA agent into which it is incorporated. In preferred embodiments, the ligand provides enhanced affinity for a selected target (e.g., a molecule, cell or cell type, compartment), such as a cell or organ compartment, tissue, organ, or region of the body, compared to a species in which such a ligand is not present. Preferably, the ligand will not participate in duplex pairing in duplex nucleic acids.

[0660] The ligand may comprise a naturally occurring substance such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL) or globulin); a carbohydrate (e.g., dextran, pullulan, chitin, chitosan, insulin, cyclodextrin, N-acetylglucosamine, N-acetylgalactosamine or hyaluronic acid); or a lipid. The ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly-(L-lactide-co-glycolide) copolymer, divinyl ether maleic anhydride copolymer, N-(2-hydroxypropyl) methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly-(2-ethyl acrylic acid), N-isopropylacrylamide polymer or polyphosphazene. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimer polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or alpha helical peptides.

[0661] The ligand may also comprise a targeting group that binds to a specific cell type (such as kidney cells), for example, a cell or tissue targeting agent, for example, a lectin, a glycoprotein, a lipid or a protein, such as an antibody. The targeting group may be thyrotropin, melanocyte stimulating hormone, agglutinin, a glycoprotein, surfactant protein A, a glycomucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, multivalent fructose, glycosylated polyamino acids, multivalent galactose, transferrin, bisphosphonates, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, vitamin A, biotin, or an RGD peptide or RGD peptide mimetic.

[0662] Other examples of ligands include dyes, intercalating agents (e.g., acridines), cross-linking agents (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., morphine, dihydromorphine), synthetic endonucleases or chelating agents (e.g., EDTA), lipophilic molecules such as cholesterol, bile acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propylene glycol, heptadecyl group, palmitic acid, myristic acid, acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or morphine and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphates, amino groups, sulfhydryl groups, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino groups, alkyl groups, substituted alkyl groups, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption aids (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, diimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, tetraazamacrocyclic Eu3+ complexes), dinitrophenyl groups, HRP, or AP.

[0663] The ligand can be a protein that binds to a specific cell type (such as a hepatocyte), for example, a glycoprotein or peptide, for example, a molecule with specific affinity for a co-ligand or antibody, for example, an antibody. The ligand can also include hormones and hormone receptors. It can also include non-peptide species such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, multivalent mannose, or multivalent fucose. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB.

[0664] The ligand can be a substance, such as a drug, that increases the uptake of the iRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, for example, by disrupting the cell's microtubules, microfilaments, and / or intermediate filaments. The drug can be, for example, taxol, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, erythromycin A, asparagine, muscarine, bryocin A, indanoxin, or myersin.

[0665] In certain embodiments, the part attached to iRNA as described herein is used as a pharmacokinetic modulator (PK modulator).PK modulators include lipophilicity, cholic acid, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, etc. Exemplary PK modulators include but are not limited to cholesterol, fatty acids, cholic acid, lithocholic acid, dialkyl glycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, etc. It is also known that the oligonucleotide binding serum protein including multiple thiophosphate linkages, therefore including the short oligonucleotides (such as oligonucleotides of about 5 bases, 10 bases, 15 bases or 20 bases) of multiple thiophosphate linkages in the main chain are also suitable for the present invention as part (such as as PK regulating part). In addition, the fit of serum components (such as serum protein) is also applicable to be used as PK regulating part in the embodiments described herein.

[0666] The ligand-conjugated oligonucleotides of the present invention can be synthesized by using oligonucleotides with pendant reactive functionalities, such as oligonucleotides derived from linker molecules attached to the oligonucleotide (described below). This reactive oligonucleotide can react directly with commercially available ligands, synthetic ligands carrying any of a variety of protecting groups, or ligands having a linker moiety attached thereto.

[0667] Oligonucleotides used in the conjugates of the present invention can be conveniently and routinely manufactured via well-known solid phase synthesis techniques. Equipment for such synthesis is sold by several suppliers, including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may be additionally or alternatively implemented. It is also known to use similar techniques to prepare other oligonucleotides, such as phosphorothioates and alkylated derivatives.

[0668] In the ligand-conjugated oligonucleotides and ligand molecules with sequence-specifically linked nucleosides of the present invention, the oligonucleotides and oligonucleosides can be assembled on a suitable DNA synthesizer using standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors already bearing the linking moiety, ligand-nucleotide or nucleoside-conjugate precursors already bearing the ligand molecule, or building blocks with non-nucleoside ligands.

[0669] When using a nucleotide conjugate precursor that already carries a linking moiety, the synthesis of sequence-specific linked nucleosides is typically completed, and the ligand molecule is then reacted with the linking moiety to form a ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or linked nucleosides of the present invention are synthesized by an automated synthesizer using phosphoramidites derived from ligand-nucleoside conjugates in addition to standard phosphoramidites and non-standard phosphoramidites that are commercially available and routinely used for oligonucleotide synthesis.

[0670] A. Lipid Conjugates

[0671] In one embodiment, the ligand or conjugate is a lipid or lipid-based molecule. Such lipid or lipid-based molecules preferably bind to serum proteins, such as human serum albumin (HSA). HSA binding ligands allow the conjugate to be distributed to target tissues, for example, non-kidney target tissues of the body. For example, the target tissue can be the liver, including the parenchymal cells of the liver. Other molecules that can bind to HSA can also be used as ligands. For example, proline or aspirin can be used. The lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport to target cells or cell membranes, and / or (c) be used to modulate binding to serum proteins such as HSA.

[0672] Lipid-based ligands can be used to inhibit, e.g., control, the binding of a conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds more strongly to HSA is less likely to be targeted to the kidneys and, therefore, less likely to be cleared from the body. A lipid or lipid-based ligand that binds less strongly to HSA can be used to target a conjugate to the kidneys.

[0673] In a preferred embodiment, the lipid-based ligand binds to HSA. Preferably, it binds to HSA with sufficient affinity so that the conjugate will preferentially distribute to non-renal tissues. However, preferably, its affinity is not so strong that HSA ligand binding is irreversible.

[0674] In another preferred embodiment, the lipid-based ligand binds weakly or barely at all to HSA, allowing the conjugate to be better distributed to the kidney.Other moieties that target kidney cells may also be used instead of or in addition to the lipid-based ligand.

[0675] In another aspect, the ligand is a portion of, for example, a vitamin that is taken up by target cells, such as proliferating cells. These are particularly useful for treating conditions characterized by unwanted cell proliferation, such as malignant or non-malignant types, such as cancer cells. Exemplary vitamins include vitamins A, E, and K. Other exemplary vitamins include B vitamins, such as folic acid, B12, riboflavin, biotin, pyridine oxide, or other vitamins or nutrients that are taken up by target cells, such as hepatocytes. HSA and low-density lipoprotein (LDL) are also included.

[0676] B. Cell Permeabilization Agents

[0677] In another aspect, the ligand is a cell permeating agent, preferably a helical cell permeating agent. Preferably, the agent is amphiphilic. Exemplary agents are peptides, such as tat or antennapedia peptides. If the agent is a peptide, it may be modified to include peptide mimetics, reverse isomers, non-peptide or pseudopeptide linkages, and the use of D-amino acids. The helical agent is preferably an alpha helical agent, which preferably has lipophilic and hydrophobic phases.

[0678] The ligand may be a peptide or peptide mimetic. Peptide mimetics (also referred to herein as oligopeptide mimetics) are molecules that can fold into a defined three-dimensional structure similar to a natural peptide. Attachment of peptides and peptide mimetics to iRNA agents can affect the pharmacokinetic profile of the iRNA, such as by enhancing cellular recognition and uptake. The peptide or peptide mimetic portion may be about 5-50 amino acids long, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

[0679] The peptide or peptide mimetic can be, for example, a cell penetrating peptide, a cationic peptide, an amphipathic peptide, or a hydrophobic peptide (e.g., consisting primarily of Tyr, Trp, or Phe). The peptide portion can be a dendrimer peptide, a restricted peptide, or a cross-linked peptide. In another alternative, the peptide portion can comprise a hydrophobic membrane translocation sequence (MTS). An exemplary peptide containing a hydrophobic MTS is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 28). An RFGF analog containing a hydrophobic MTS (e.g., the amino acid sequence AALLPVLLAAP (SEQ ID NO: 29)) can also be a targeting moiety. The peptide portion can be a "delivery" peptide that can carry large polar molecules, including peptides, oligonucleotides, and proteins, on the cell membrane. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 30)) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 31)) have been found to function as delivery peptides. Peptides or peptide mimetics can be encoded by random DNA sequences, such as peptides identified from phage display libraries or one-bead-one-compound (OBOC) combinatorial libraries (Lam et al., Nature, 354: 82-84, 1991). An example of a peptide or peptide mimetic that is tethered to a dsRNA agent via an incorporated monomeric unit for cellular targeting purposes is an arginine-glycine-aspartic acid (RGD)-peptide or RGD mimetic. The length of the peptide portion can range from about 5 amino acids to about 40 amino acids. The peptide portion can have structural modifications, such as to increase stability or direct conformational properties. Any of the following structural modifications can be used.

[0680] The RGD peptides used in the compositions and methods of the present invention can be linear or cyclic and can be modified, for example, by glycosylation or methylation, to facilitate targeting to various specific tissues. RGD-containing peptides and peptide mimetics can include D-amino acids as well as synthetic RGD mimetics. In addition to RGD, other moieties of integrin-targeting ligands can be used. Preferred conjugates of this ligand target PECAM-1 or VEGF.

[0681] "Cell penetrating peptides" are capable of penetrating cells, for example, microbial cells, such as bacterial or fungal cells, or mammalian cells, such as human cells. Peptides that can penetrate microbial cells can be, for example, α-helical linear peptides (e.g., LL-37 or Ceropin P1), peptides comprising disulfide bonds (e.g., α-defensins, β-defensins, or bacteriostatins), or peptides comprising only one or two primary amino acids (e.g., PR-39 or indole antibiotic peptides). Cell penetrating peptides can also comprise a nuclear localization signal (NLS). For example, a cell penetrating peptide can be a two-part amphipathic peptide, such as MPG, which is derived from the fused peptide domains of the NLS of HIV-1 gp41 and SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31: 2717-2724, 2003).

[0682] C. Carbohydrate conjugates

[0683] In some embodiments of the compositions and methods of the present invention, the iRNA oligonucleotide further comprises a carbohydrate. As described herein, carbohydrate-conjugated iRNAs facilitate in vivo delivery of nucleic acids, and compositions suitable for in vivo therapeutic use. As used herein, "carbohydrate" refers to a compound that is itself a carbohydrate composed of one or more monosaccharide units having at least 6 carbon atoms (which may be linear, branched, or cyclic) and oxygen, nitrogen, or sulfur atoms bonded to each carbon atom; or a compound having a carbohydrate composed of one or more monosaccharide units having at least 6 carbon atoms (which may be linear, branched, or cyclic) and oxygen, nitrogen, or sulfur atoms bonded to each carbon atom as part of it. Representative carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about 4, 5, 6, 7, 8, or 9 monosaccharide units), and polysaccharides such as starch, glycogen, cellulose, and polysaccharide gums. Particular monosaccharides include C5 and above (eg, C5, C6, C7, or C8) sugars; disaccharides and trisaccharides include sugars having two or three monosaccharide units (eg, C5, C6, C7, or C8).

[0684] In one embodiment, the carbohydrate conjugate used in the compositions and methods of the present invention is selected from the group consisting of:

[0685]

[0686]

[0687]

[0688]

[0689]

[0690] wherein Y is O or S and n is 3-6 (Formula XXIV);

[0691] wherein Y is O or S and n is 3-6 (Formula XXV);

[0692]

[0693] wherein X is O or S (Formula XXVII);

[0694]

[0695]

[0696] and

[0697]

[0698]

[0699] In another embodiment, the carbohydrate conjugate used in the compositions and methods of the present invention is a monosaccharide. In one embodiment, the monosaccharide is N-acetylgalactosamine, such as

[0700]

[0701] Another representative carbohydrate conjugate for use in the embodiments described herein includes, but is not limited to:

[0702]

[0703] When one of X or Y is an oligonucleotide, the other is hydrogen.

[0704] In certain embodiments of the invention, GalNAc or a GalNAc derivative is attached to an iRNA agent of the invention via a monovalent linker. In some embodiments, GalNAc or a GalNAc derivative is attached to an iRNA agent of the invention via a divalent linker. In yet other embodiments of the invention, GalNAc or a GalNAc derivative is connected to an iRNA agent of the invention via a trivalent linker.

[0705] In one embodiment, the double-stranded RNAi agent of the invention comprises GalNAc or a GalNAc derivative linked to the 3' or 5' end of the sense strand of an iRNA agent, e.g., a dsRNA agent as described herein. In another embodiment, the double-stranded RNAi agent of the invention comprises a plurality (e.g., 2, 3, 4, 5, or 6) of GalNAc or a GalNAc derivative, each of which is independently linked to a plurality of nucleotides of the double-stranded RNAi agent via a plurality of monovalent linkers.

[0706] In some embodiments, for example, when the two strands of an iRNA agent of the invention are part of a larger molecule connected by an uninterrupted nucleotide chain between the 3' end of one strand and the 5' end of the respective other strand forming a hairpin loop, the plurality of unpaired nucleotides within the hairpin loop can each independently comprise GalNAc or a GalNAc derivative attached via a monovalent linker.

[0707] In some embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, such as, but not limited to, a PK modifier and / or a cell penetrating peptide.

[0708] Additional carbohydrate conjugates (and linkers) suitable for use in the present invention include those described in PCT Publication Nos. WO 2014 / 179620 and WO 2014 / 179627, each of which is incorporated herein by reference in its entirety.

[0709] D.Connector

[0710] In some embodiments, the conjugates or ligands described herein can be linked to the iRNA oligonucleotide via various linkers, either cleavable or non-cleavable.

[0711] The term "linker" or "linking group" refers to an organic moiety that connects two parts of a compound, for example, covalently connects two parts of a compound. Linkers typically include a direct bond or an atom (such as oxygen or sulfur), a unit (such as NR8, C(O), C(O)NH, SO, SO2, SO2NH, or a chain of atoms), such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl wherein one or more methylene groups are interrupted or terminated by O, S, S(O), SO2, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle; wherein R8 is hydrogen, acyl, aliphatic or substituted aliphatic. In one embodiment, the linker is between about 1 to 24 atoms, 2 to 24 atoms, 3 to 24 atoms, 4 to 24 atoms, 5 to 24 atoms, 6 to 24 atoms, 6 to 18 atoms, 7 to 18 atoms, 8 to 18 atoms, 7 to 17 atoms, 8 to 17 atoms, 6 to 16 atoms, 7 to 17 atoms, or 8 to 16 atoms.

[0712] A cleavable linker is a linker that is sufficiently stable outside of a cell but cleaves upon entry into a target cell to release the two moieties to which it co-immobilizes. In a preferred embodiment, the cleavable linker is cleaved at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, or more times faster, or at least about 100 times faster, in a target cell or under first reference conditions (which can be, for example, selected to mimic or represent conditions within a cell) than in a subject's blood or under second reference conditions (which can be, for example, selected to mimic or represent conditions in blood or serum).

[0713] Cleavable linking groups are susceptible to the effects of cleavage agents, such as pH, redox potential, or the presence of degrading molecules. In general, cleavage agents are more prevalent or found at higher levels or activity within cell...

Claims

1. A method of reducing HSD17B13 mRNA levels in an individual, the method comprising administering to the individual a dose of about 25 mg to about 800 mg of a double-stranded ribonucleic acid (dsRNA) agent targeting the HSD17B13 gene, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises the nucleotide sequence 5'-AUGCUUUUGCAUGGACUAUCU-3' (SEQ ID NO: 26) and the antisense strand comprises the nucleotide sequence 5'-AGAUAGTCCAUGCAAAAGCAUUC-3' (SEQ ID NO: 27), thereby reducing the level of HSD17B13 mRNA in the subject.

2. A method for treating an individual suffering from non-alcoholic steatohepatitis (NASH), the method comprising administering to the individual a dose of about 25 mg to about 800 mg of a double-stranded ribonucleic acid (dsRNA) agent targeting the HSD17B13 gene, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises the nucleotide sequence 5'-AUGCUUUUGCAUGGACUAUCU-3' (SEQ ID NO:26) and the antisense strand comprises the nucleotide sequence 5'-AGAUAGTCCAUGCAAAAGCAUUC-3' (SEQ ID NO:27), thereby treating the individual suffering from non-alcoholic steatohepatitis (NASH).

3. A method of preventing at least one symptom in an individual with non-alcoholic steatohepatitis (NASH), the method comprising administering to the individual a dose of about 25 mg to about 800 mg of a double-stranded ribonucleic acid (dsRNA) agent targeting the HSD17B13 gene, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises the nucleotide sequence 5'-AUGCUUUUGCAUGGACUAUCU-3' (SEQ ID NO:26) and the antisense strand comprises the nucleotide sequence 5'-AGAUAGTCCAUGCAAAAGCAUUC-3' (SEQ ID NO:27), thereby preventing at least one symptom in a subject suffering from non-alcoholic steatohepatitis (NASH).

4. A method for reducing the risk of an individual with steatosis developing chronic liver disease, the method comprising administering to the individual a dose of about 25 mg to about 800 mg of a double-stranded ribonucleic acid (dsRNA) agent targeting the HSD17B13 gene, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises the nucleotide sequence 5'-AUGCUUUUGCAUGGACUAUCU-3' (SEQ ID NO:26) and the antisense strand comprises the nucleotide sequence 5'-AGAUAGTCCAUGCAAAAGCAUUC-3' (SEQ ID NO:27), thereby reducing the risk of the individual with steatosis developing chronic liver disease.

5. A method for inhibiting the progression of steatosis to steatohepatitis in an individual suffering from steatosis, the method comprising administering to the individual a dose of about 25 mg to about 800 mg of a double-stranded ribonucleic acid (dsRNA) agent targeting the HSD17B13 gene, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises the nucleotide sequence 5'-AUGCUUUUGCAUGGACUAUCU-3' (SEQ ID NO:26) and the antisense strand comprises the nucleotide sequence 5'-AGAUAGTCCAUGCAAAAGCAUUC-3' (SEQ ID NO:27), thereby inhibiting the progression of steatosis to steatohepatitis in the subject.

6. A method of inhibiting lipid droplet accumulation in the liver of an individual suffering from non-alcoholic steatohepatitis (NASH), the method comprising administering to the individual a dose of about 25 mg to about 800 mg of a double-stranded ribonucleic acid (dsRNA) agent targeting the HSD17B13 gene, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises the nucleotide sequence 5'-AUGCUUUUGCAUGGACUAUCU-3' (SEQ ID NO:26) and the antisense strand comprises the nucleotide sequence 5'-AGAUAGTCCAUGCAAAAGCAUUC-3' (SEQ ID NO:27), thereby inhibiting fat accumulation in the liver of the individual suffering from non-alcoholic steatohepatitis (NASH).

7. The method of any one of claims 1 to 6, wherein the subject's HSD17B13 mRNA levels are reduced to at least about 70%, 65%, 60%, 55% or 50% of baseline levels 6 months after the first administration of the dsRNA agent.

8. The method of any one of claims 1 to 6, wherein administering the dsRNA agent to the individual results in reduced HSD17B13 enzyme activity, reduced HSD17B13 protein accumulation, and / or reduced fat accumulation and / or fat droplet expansion in the liver of the individual.

9. The method of any one of claims 1 to 8, wherein the subject is a human subject.

10. The method of any one of claims 1 to 9, wherein the individual is obese.

11. The method of any one of claims 1 to 10, wherein the dsRNA agent comprises at least one modified nucleotide.

12. The method of any one of claims 1 to 11, wherein substantially all nucleotides of the sense strand comprise modifications.

13. The method of any one of claims 1 to 12, wherein substantially all nucleotides of the antisense strand comprise modifications.

14. The method of any one of claims 1 to 13, wherein substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand comprise a modification.

15. The method of any one of claims 11 to 14, wherein at least one of the modified nucleotides is selected from the group consisting of deoxynucleotides, 3'-terminal deoxythymine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, unlocked nucleotides, conformationally constrained nucleotides, constrained ethyl nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-alkyl modified nucleotides, 2'-hydroxy modified nucleotides. The invention relates to nucleotides comprising a 5'-phosphate group, a 5'-phosphate group, a 5'-phosphate mimetic group, a diol-modified nucleotide, a 2'-O-(N-methylacetamide)-modified nucleotide, a 2'-methoxyethyl-modified nucleotide, a 2'-O-alkyl-modified nucleotide, an N-morpholino nucleotide, a phosphoramidate, a nucleotide comprising a non-natural base, a tetrahydropyran-modified nucleotide, a 1,5-anhydrohexitol-modified nucleotide, a cyclohexenyl-modified nucleotide, a nucleotide comprising a phosphorothioate group, a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5'-phosphate group, a nucleotide comprising a 5'-phosphate mimetic, a diol-modified nucleotide, and a 2-O-(N-methylacetamide)-modified nucleotide, and combinations thereof. The method of claim 15 , wherein the nucleotide modification is 2′-O-methyl and / or 2′-fluoro modification.

17. The method of any one of claims 1 to 16, wherein each strand of the dsRNA agent is no more than 30 nucleotides in length.

18. The method of any one of claims 1 to 17, wherein each strand of the dsRNA agent is independently 19 to 30 nucleotides in length.

19. The method of claim 18, wherein each strand of the dsRNA agent is independently 19 to 25 nucleotides in length.

20. The method of claim 19, wherein each strand of the dsRNA agent is independently 21 to 23 nucleotides in length.

21. The method of any one of claims 1 to 20, wherein at least one strand of the dsRNA agent comprises a 3' overhang of at least 1 nucleotide.

22. The method of any one of claims 1 to 21, wherein at least one strand of the dsRNA agent comprises a 3' overhang of at least 2 nucleotides.

23. The method of any one of claims 1 to 22, wherein the dsRNA agent further comprises a ligand.

24. The method of claim 23, wherein the ligand is conjugated to the 3' end of the sense strand of the dsRNA agent.

25. The method of claim 23 or 24, wherein the ligand is an N-acetylgalactosamine (GalNAc) derivative.

26. The method of any one of claims 23 to 25, wherein the ligand is 27. The method of any one of claims 23 to 26, wherein the dsRNA agent is conjugated to the ligand as shown in the following schematic diagram wherein X is O or S.

28. The method of claim 27, wherein X is O.

29. The method of any one of claims 1 to 28, wherein the sense strand comprises the nucleotide sequence 5'-asusgcuuUfuGfCfAfuggacuaucu-3' (SEQ ID NO: 24) and the antisense strand comprises the nucleotide sequence 5'-asGfsauag(Tgn)ccaugcAfaAfagcaususc-3' (SEQ ID NO: 25), Af is 2'-fluoroadenosine-3'-phosphate; Cf is 2'-fluorocytidine-3'-phosphate; Gf is 2'-fluoroguanosine-3'-phosphate; Gfs is 2'-fluoroguanosine-3'-thiophosphate; U is uridine-3'-phosphate; Uf is 2'-fluorouridine-3'-phosphate; a is 2'-O-methyladenosine-3'-phosphate; as is 2'-O-methyladenosine-3'-thiophosphate; c is 2'-O -methylcytidine-3'-phosphate; cs is 2'-O-methylcytidine-3'-phosphorothioate; g is 2'-O-methylguanosine-3'-phosphate; gs is 2'-O-methylguanosine-3'-phosphorothioate; u is 2'-O-methyluridine-3'-phosphate; us is 2'-O-methyluridine-3'-phosphorothioate; Tgn is thymidine-diol nucleic acid (GNA) S isomer; and s is a phosphorothioate linkage.

30. The method of claim 29, wherein the dsRNA agent is conjugated to the ligand as shown in the following schematic diagram And wherein X is O.

31. A method of treating an individual suffering from non-alcoholic steatohepatitis (NASH), the method comprising administering to the individual a dose of about 25 mg to about 800 mg of a double-stranded ribonucleic acid (dsRNA) agent targeting the HSD17B13 gene, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises the nucleotide sequence 5'-asusgcuuUfuGfCfAfuggacuaucu-3' (SEQ ID NO: 24) and the antisense strand comprises the nucleotide sequence 5'-asGfsauag(Tgn)ccaugcAfaAfagcaususc-3' (SEQ ID NO: 25), Af is 2'-fluoroadenosine-3'-phosphate; Cf is 2'-fluorocytidine-3'-phosphate; Gf is 2'-fluoroguanosine-3'-phosphate; Gfs is 2'-fluoroguanosine-3'-thiophosphate; U is uridine-3'-phosphate; Uf is 2'-fluorouridine-3'-phosphate; a is 2'-O-methyladenosine-3'-phosphate; as is 2'-O-methyladenosine-3'-thiophosphate; c is 2'-O -methylcytidine-3'-phosphate; cs is 2'-O-methylcytidine-3'-phosphorothioate; g is 2'-O-methylguanosine-3'-phosphate; gs is 2'-O-methylguanosine-3'-phosphorothioate; u is 2'-O-methyluridine-3'-phosphate; us is 2'-O-methyluridine-3'-phosphorothioate; Tgn is the S isomer of thymidine-diol nucleic acid (GNA); and s is a phosphorothioate linkage; wherein the dsRNA agent is conjugated to a ligand having the structure and Wherein the ligand is conjugated to the 3' end of the sense strand, as shown in the following schematic diagram Where X is O.

32. A method of preventing at least one symptom in an individual with non-alcoholic steatohepatitis (NASH), the method comprising administering to the individual a dose of about 25 mg to about 800 mg of a double-stranded ribonucleic acid (dsRNA) agent targeting the HSD17B13 gene, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises the nucleotide sequence 5'-asusgcuuUfuGfCfAfuggacuaucu-3' (SEQ ID NO: 24) and the antisense strand comprises the nucleotide sequence 5'-asGfsauag(Tgn)ccaugcAfaAfagcaususc-3' (SEQ ID NO: 25), Af is 2'-fluoroadenosine-3'-phosphate; Cf is 2'-fluorocytidine-3'-phosphate; Gf is 2'-fluoroguanosine-3'-phosphate; Gfs is 2'-fluoroguanosine-3'-thiophosphate; U is uridine-3'-phosphate; Uf is 2'-fluorouridine-3'-phosphate; a is 2'-O-methyladenosine-3'-phosphate; as is 2'-O-methyladenosine-3'-thiophosphate; c is 2'-O -methylcytidine-3'-phosphate; cs is 2'-O-methylcytidine-3'-phosphorothioate; g is 2'-O-methylguanosine-3'-phosphate; gs is 2'-O-methylguanosine-3'-phosphorothioate; u is 2'-O-methyluridine-3'-phosphate; us is 2'-O-methyluridine-3'-phosphorothioate; Tgn is the S isomer of thymidine-diol nucleic acid (GNA); and s is a phosphorothioate linkage; wherein the dsRNA agent is conjugated to a ligand having the structure and Wherein the ligand is conjugated to the 3' end of the sense strand, as shown in the following schematic diagram Where X is O.

33. A method of reducing the risk of an individual with steatosis developing chronic liver disease, the method comprising administering to the individual a dose of about 25 mg to about 800 mg of a double-stranded ribonucleic acid (dsRNA) agent targeting the HSD17B13 gene, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises the nucleotide sequence 5'-asusgcuuUfuGfCfAfuggacuaucu-3' (SEQ ID NO: 24) and the antisense strand comprises the nucleotide sequence 5'-asGfsauag(Tgn)ccaugcAfaAfagcaususc-3' (SEQ ID NO: 25), Af is 2'-fluoroadenosine-3'-phosphate; Cf is 2'-fluorocytidine-3'-phosphate; Gf is 2'-fluoroguanosine-3'-phosphate; Gfs is 2'-fluoroguanosine-3'-thiophosphate; U is uridine-3'-phosphate; Uf is 2'-fluorouridine-3'-phosphate; a is 2'-O-methyladenosine-3'-phosphate; as is 2'-O-methyladenosine-3'-thiophosphate; c is 2'-O -methylcytidine-3'-phosphate; cs is 2'-O-methylcytidine-3'-phosphorothioate; g is 2'-O-methylguanosine-3'-phosphate; gs is 2'-O-methylguanosine-3'-phosphorothioate; u is 2'-O-methyluridine-3'-phosphate; us is 2'-O-methyluridine-3'-phosphorothioate; Tgn is the S isomer of thymidine-diol nucleic acid (GNA); and s is a phosphorothioate linkage; wherein the dsRNA agent is conjugated to a ligand having the structure and Wherein the ligand is conjugated to the 3' end of the sense strand, as shown in the following schematic diagram Where X is O.

34. A method of inhibiting the progression of steatosis to steatohepatitis in an individual suffering from steatosis, the method comprising administering to the individual a dose of about 25 mg to about 800 mg of a double-stranded ribonucleic acid (dsRNA) agent targeting the HSD17B13 gene, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises the nucleotide sequence 5'-asusgcuuUfuGfCfAfuggacuaucu-3' (SEQ ID NO: 24) and the antisense strand comprises the nucleotide sequence 5'-asGfsauag(Tgn)ccaugcAfaAfagcaususc-3' (SEQ ID NO: 25), Af is 2'-fluoroadenosine-3'-phosphate; Cf is 2'-fluorocytidine-3'-phosphate; Gf is 2'-fluoroguanosine-3'-phosphate; Gfs is 2'-fluoroguanosine-3'-thiophosphate; U is uridine-3'-phosphate; Uf is 2'-fluorouridine-3'-phosphate; a is 2'-O-methyladenosine-3'-phosphate; as is 2'-O-methyladenosine-3'-thiophosphate; c is 2'-O -methylcytidine-3'-phosphate; cs is 2'-O-methylcytidine-3'-phosphorothioate; g is 2'-O-methylguanosine-3'-phosphate; gs is 2'-O-methylguanosine-3'-phosphorothioate; u is 2'-O-methyluridine-3'-phosphate; us is 2'-O-methyluridine-3'-phosphorothioate; Tgn is the S isomer of thymidine-diol nucleic acid (GNA); and s is a phosphorothioate linkage; wherein the dsRNA agent is conjugated to a ligand having the structure and Wherein the ligand is conjugated to the 3' end of the sense strand, as shown in the following schematic diagram Where X is O.

35. A method of inhibiting lipid droplet accumulation in the liver of an individual suffering from non-alcoholic steatohepatitis (NASH), the method comprising administering to the individual a dose of about 25 mg to about 800 mg of a double-stranded ribonucleic acid (dsRNA) agent targeting the HSD17B13 gene, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises the nucleotide sequence 5'-asusgcuuUfuGfCfAfuggacuaucu-3' (SEQ ID NO: 24) and the antisense strand comprises the nucleotide sequence 5'-asGfsauag(Tgn)ccaugcAfaAfagcaususc-3' (SEQ ID NO: 25), Af is 2'-fluoroadenosine-3'-phosphate; Cf is 2'-fluorocytidine-3'-phosphate; Gf is 2'-fluoroguanosine-3'-phosphate; Gfs is 2'-fluoroguanosine-3'-thiophosphate; U is uridine-3'-phosphate; Uf is 2'-fluorouridine-3'-phosphate; a is 2'-O-methyladenosine-3'-phosphate; as is 2'-O-methyladenosine-3'-thiophosphate; c is 2'-O -methylcytidine-3'-phosphate; cs is 2'-O-methylcytidine-3'-phosphorothioate; g is 2'-O-methylguanosine-3'-phosphate; gs is 2'-O-methylguanosine-3'-phosphorothioate; u is 2'-O-methyluridine-3'-phosphate; us is 2'-O-methyluridine-3'-phosphorothioate; Tgn is the S isomer of thymidine-diol nucleic acid (GNA); and s is a phosphorothioate linkage; wherein the dsRNA agent is conjugated to a ligand having the structure and Wherein the ligand is conjugated to the 3' end of the sense strand, as shown in the following schematic diagram Where X is O.

36. The method of any one of claims 1 to 35, further comprising administering to the individual an additional therapeutic agent.

37. The method of any one of claims 1 to 36, further comprising determining a NAFLD activity score (NAS), a ballooning score, a lobular inflammation score, a steatosis score, and / or a fibrosis score for the subject.

38. The method of any one of claims 1 to 37, wherein the dsRNA agent is administered to the individual at a dose of about 25 mg, about 100 mg, about 200 mg, about 400 mg, or about 800 mg.

39. The method of any one of claims 1 to 38, wherein the dsRNA agent is administered to the individual every month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, or every 12 months.

40. The method of any one of claims 1 to 39, wherein the dsRNA agent is administered to the individual at a dose of about 25 mg per month.

41. The method of any one of claims 1 to 39, wherein the dsRNA agent is administered to the individual at a dose of about 200 mg per month.

42. The method of any one of claims 1 to 39, wherein the dsRNA agent is administered to the individual at a dose of about 400 mg per month.

43. The method of any one of claims 1 to 39, wherein the dsRNA agent is administered to the individual at a dose of about 25 mg every three months.

44. The method of any one of claims 1 to 39, wherein the dsRNA agent is administered to the individual at a dose of about 200 mg every three months.

45. The method of any one of claims 1 to 39, wherein the dsRNA agent is administered to the individual at a dose of about 400 mg every three months.

46. ​​The method of any one of claims 1 to 45, wherein the dsRNA agent is administered to the individual intravenously, intramuscularly, or subcutaneously.

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