Lactate dehydrogenase A(LDHA)iRNA composition and method of use thereof

Abstract

To provide alternative treatments for subjects suffering from diseases, disorders, and conditions related to the oxalate pathway. [Solution] The present invention relates to a double-stranded ribonucleic acid (dsRNA) composition that targets the LDHA gene, a method for inhibiting LDHA expression, a method for inhibiting LDHA and HAO1 using such a dsRNA composition, and a method for treating subjects who may benefit from reduced LDHA expression, for example, subjects suffering from diseases, disorders or conditions related to the oxalate pathway.

Description

[Technical Field]

[0001] Related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 576,783, filed on 25 October 2017, and to U.S. Provisional Patent Application No. 62 / 532,020, filed on 13 July 2017. The entire contents of each of the aforementioned provisional applications are incorporated herein by reference.

[0002] Sequence List This application includes a sequence listing submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. The name of the ASCII copy made on 12 July 2018 is 121301-07520_SL.TXT, and its size is 1,154,808 bytes. [Background technology]

[0003] Oxalate (C2O4) 2- Oxalates are salt-forming ions of oxalic acid (C2H2O4), which are widely distributed in both plants and animals. Oxalates are an essential component of the human diet and a common component of plants and plant-derived foods. Oxalates can also be synthesized endogenously via metabolic pathways present in the liver. The dietary and endogenous contributions to urinary oxalate excretion are equivalent. Glyoxylates are the direct precursors of oxalates and are derived from the oxidation of glycolate by the enzyme glycolate oxidase (GO), also known and referred to herein as hydroxy acid oxidase (HAO1), or from the catabolism of hydroxyproline, a component of collagen. The aminotransfer of alanine and glyoxylate by the enzyme alanine / glyoxylate aminotransferase (AGT) results in the formation of pirubate and glycine. Excess glyoxylate is converted to oxalates by lactate dehydrogenase A (referred to herein as LDHA). The endogenous pathway for oxalate metabolism is shown in Figure 1A.

[0004] Lactate dehydrogenase is a protein found in all tissues. It consists of four subunits, the two most common being the LDH-M and LDH-H proteins. These proteins are encoded by the LDHA and LDHB genes, respectively. Various combinations of the LDH-M and LDH-H proteins result in five different isoforms of LDH. LDHA is the most important gene involved in the hepatic lactate dehydrogenase isoform. In particular, within the liver, LDHA is crucial as the final step in the endogenous production of oxalates by converting the precursor glyoxylate to oxalate. LDHA also plays a vital role in the Cori cycle and in the anaerobic stage of glycolysis, where LDHA converts lactate to pirubate and vice versa.

[0005] Oxalic acid can form oxalates with various cations, including sodium, potassium, magnesium, and calcium. Sodium oxalate, potassium oxalate, and magnesium oxalate are water-soluble, while calcium oxalate (CaOx) is nearly insoluble. Excretion of oxalates is mainly carried out by the kidneys via glomerular filtration and tubular secretion.

[0006] Oxalates bind with calcium in the kidneys, which can lead to urinary CaOx supersaturation, resulting in the formation and deposition of CaOx crystals in renal tissue or aggregates. These CaOx crystals contribute to the formation of extensive nephrocalcification (nephrocalcemia) and kidney stones (nephrolithiasis). Individuals with extensive nephrocalcification or non-obstructive stones are typically asymptomatic. However, obstructive stones can cause severe pain. Furthermore, over time, these CaOx crystals cause kidney damage and progressive inflammation, and if secondary complications such as obstruction are present, these CaOx crystals can lead to decreased renal function, and in severe cases, further to end-stage renal failure and the need for dialysis. In addition, systemic deposition of CaOx (systemic oxalic acidosis) can occur in extrarenal tissues, including soft tissues (such as the thyroid and breast), heart, nerves, joints, skin, and retina, and if left untreated, this can lead to premature death.

[0007] The most well-known disease associated with the oxalate pathway is primary hyperoxaluria, a genetic disorder characterized by increased endogenous oxalate synthesis and variable clinical phenotypes, among other kidney stone formation diseases. Currently, there are no treatments available to regulate oxalate synthesis, and very few treatment options exist for individuals with hereditary hyperoxaluria. Ultimately, some individuals with hereditary hyperoxaluria require kidney / liver transplantation. Other diseases, disorders, and conditions associated with the oxalate pathway include calcium oxalate tissue deposition diseases, disorders, and conditions.

[0008] Currently, many first-line treatments for diseases, disorders, and conditions associated with these oxalate pathways (e.g., those associated with kidney stones) involve increased fluid intake and dietary changes (e.g., reduced protein intake, reduced sodium intake, reduced ascorbic acid intake, moderate calcium intake, phosphate or magnesium supplementation, and pyridoxine treatment). However, patients are often unable to adhere to such lifestyle changes and do not receive significant benefits. Treatment for some other diseases, disorders, and conditions associated with the oxalate pathways, such as chronic kidney disease, involves the use of ACE inhibitors (angiotensin-inverting enzyme inhibitors) and ARBs (angiotensin II antagonists), which can slow disease progression. Nevertheless, patients with chronic kidney disease experience a gradual decline in renal function, progression, and the need for dialysis or kidney transplantation. For the majority of these diseases associated with the oxalate pathways, there is no cure, and currently, no oxalate-reducing treatments are available.

[0009] Furthermore, diseases, disorders, and conditions related to the oxalate pathway include those related to lactate dehydrogenase. For example, the role of lactate dehydrogenase in cancer (hepatocellular carcinoma) is well known, and its inhibition has been shown to reduce cancer growth. Other diseases, disorders, and conditions related to lactate dehydrogenase include fatty liver (steatosis), non-alcoholic steatohepatitis (NASH), cirrhosis, accumulation of fat in the liver, liver inflammation, hepatocyte necrosis, hepatic fibrosis, and non-alcoholic fatty liver disease (NAFLD). However, given the important role of LDH in glycolysis, treatment options are limited. [Overview of the Initiative] [Problems that the invention aims to solve]

[0010] Therefore, there is a need in the field for alternative therapies for subjects suffering from diseases, disorders, and conditions related to the oxalate pathway. [Means for solving the problem]

[0011] The present invention is at least in part based on the discovery that liver-specific and excellent LDHA and urinary oxalate reduction effects can be achieved by targeting LDHA using iRNA agents, compositions containing such agents, and methods disclosed herein.

[0012] Accordingly, the present invention provides an iRNA composition that results in RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the LDHA gene. The LDHA gene may be present in cells, for example, in subjects such as humans. The present invention also provides a method of using the iRNA composition of the present invention to inhibit the expression of the LDHA gene in order to treat subjects who may benefit from inhibiting or reducing the expression of the LDHA gene, for example, subjects who may benefit from reducing or inhibiting urinary oxalate production, for example, subjects suffering from or susceptible to diseases, disorders, or conditions related to the oxalate pathway, for example, diseases, disorders, or conditions related to oxalate formation, for example, kidney stone formation diseases, disorders, or conditions or calcium oxalate tissue deposition diseases, disorders, or conditions; or subjects suffering from or susceptible to diseases, disorders, or conditions related to LDHA.

[0013] The present invention also provides iRNA compositions that result in RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of the LDHA gene and the HAO1 gene. The LDHA gene and the HAO1 gene may be present in cells, for example, in subjects such as humans. The present invention also provides methods of using the iRNA compositions of the present invention to inhibit the expression of the LDHA gene and the HAO1 gene in order to treat subjects who may benefit from inhibiting or reducing the expression of the LDHA gene and the HAO1 gene, for example, subjects who may benefit from reducing or inhibiting urinary oxalate production, for example, oxalate-related diseases, disorders, or conditions, for example, kidney stone formation diseases, disorders, or conditions or calcium oxalate tissue deposition diseases, disorders, or conditions; or subjects who have or are susceptible to LDH-related diseases, disorders, or conditions.

[0014] Accordingly, in one embodiment, the present invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of lactate dehydrogenase A (LDHA) in cells, wherein the dsRNA agent comprises a sense strand and an antisense strand, and the antisense strand comprises a complementary region containing at least 15 consecutive nucleotides that differ by three or fewer nucleotides from any one of the antisense sequences listed in any one of Tables 2 to 5.

[0015] In one embodiment, the dsRNA agent comprises at least one modified nucleotide.

[0016] In other embodiments, substantially all of the nucleotides in the sense strand are modified; substantially all of the nucleotides in the antisense strand are modified; or substantially all of the nucleotides in both the sense strand and the antisense strand are modified.

[0017] In yet another embodiment, all nucleotides of the sense strand are modified; all nucleotides of the antisense strand are modified; or all nucleotides of the sense strand and all nucleotides of the antisense strand are modified.

[0018] In one embodiment, at least one of the modified nucleotides is selected from the group consisting of deoxy-nucleotides, 3'-terminal deoxythymine (dT) nucleotides, 2'-O-methyl-modified nucleotides, 2'-fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, fixed nucleotides, unfixed nucleotides, conformationally restricted nucleotides, restricted ethyl nucleotides, non-basic nucleotides, 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-hydroxyl-modified nucleotides, 2'-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, morpholino nucleotides, phosphoramides, nucleotides containing non-natural bases, tetrahydropyran-modified nucleotides, 1,5-anhydrohexitol-modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides containing a phosphorothioate group, nucleotides containing a methylphosphonate group, nucleotides containing a 5'-phosphate, nucleotides containing a 5'-phosphate mimetic, glycol-modified nucleotides, and 2-O-(N-methylacetamide)-modified nucleotides, and combinations thereof.

[0019] The complementary region may be at least 17 nucleotides long; 19-30 nucleotides long; 19-25 nucleotides long; or 21-23 nucleotides long.

[0020] Each strand of the dsRNA agent may be 30 nucleotides or less in length. Each strand of the dsRNA agent may independently be 19-30 nucleotides long; independently 19-25 nucleotides long; or independently 21-23 nucleotides long.

[0021] At least one strand of the dsRNA agent may contain a 3' overhang of at least one nucleotide; or at least one strand may contain a 3' overhang of at least two nucleotides.

[0022] In one embodiment, the dsRNA agent further comprises at least one phosphorothioate or methylphosphonate internucleotide bond.

[0023] A phosphorothioate or methylphosphonate internucleotide bond may be located at the 3' end of a single chain (e.g., an antisense chain; or a sense chain); or a phosphorothioate or methylphosphonate internucleotide bond may be located at the 5' end of a single chain (e.g., an antisense chain; or a sense chain); or a phosphorothioate or methylphosphonate internucleotide bond may be located at both the 5' and 3' ends of a single chain.

[0024] dsRNA agents may further contain ligands.

[0025] In one embodiment, the ligand is conjugated to the 3' end of the sense strand of the dsRNA agent.

[0026] In one embodiment, the ligand is one or more N-acetylgalactosamine (GalNAc) derivatives linked via monovalent, divalent, or trivalent branched linkers.

[0027] In another embodiment, the ligand is [ka] That is the case.

[0028] In one embodiment, the dsRNA agent is shown in the following schematic diagram. [ka] It is conjugated to the ligand shown in the formula, where X is O or S.

[0029] In one embodiment, X is O.

[0030] In one embodiment, the complementary region consists of one of the antisense sequences listed in any one of Tables 2 to 5.

[0031] In one embodiment, the sense strand and the antisense strand include a nucleotide sequence selected from the group consisting of any one nucleotide sequence of an agent listed in any one of Tables 2 to 5.

[0032] In another embodiment, the present invention provides a dual-targeted RNAi agent comprising: a first double-stranded ribonucleic acid (dsRNA) agent that inhibits the expression of lactate dehydrogenase A (LDHA) including a sense strand and an antisense strand; and a second double-stranded ribonucleic acid (dsRNA) agent that inhibits the expression of hydroxy acid oxidase 1 (glycolate oxidase) (HAO1) ​​including a sense strand and an antisense strand, wherein the first dsRNA agent and the second dsRNA agent are covalently linked.

[0033] In one embodiment, the sense strand of the first dsRNA agent comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 1 by three or fewer nucleotides, and the antisense strand of the first dsRNA agent comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 2 by three or fewer nucleotides.

[0034] In another embodiment, the antisense strand of the first dsRNA agent includes a complementary region comprising at least 15 consecutive nucleotides that differ by three or fewer nucleotides from any one of the antisense sequences listed in any one of Tables 2-5.

[0035] In one embodiment, the sense strand of the second dsRNA agent comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 21 by three or fewer nucleotides, and the antisense strand of the second dsRNA agent comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 22 by three or fewer nucleotides.

[0036] In another embodiment, the antisense strand of the second dsRNA agent includes a complementary region comprising at least 15 consecutive nucleotides that differ by three or fewer nucleotides from any one of the antisense sequences listed in any one of Tables 7-14.

[0037] In one embodiment, the first dsRNA agent and the second dsRNA agent each independently contain at least one modified nucleotide.

[0038] In another embodiment, substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand of the first dsRNA agent and substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand of the second dsRNA agent are modified nucleotides.

[0039] In one embodiment, at least one modified nucleotide of the first dsRNA agent and at least one modified nucleotide of the second dsRNA agent are, independently, deoxy-nucleotide, 3'-terminal deoxythymine (dT) nucleotide, 2'-O-methyl-modified nucleotide, 2'-fluoro-modified nucleotide, 2'-deoxy-modified nucleotide, fixed nucleotide, unfixed nucleotide, conformationally restricted nucleotide, restricted ethyl nucleotide, non-basic nucleotide, 2'-amino-modified nucleotide, 2'-O-allyl-modified nucleotide, 2'-C- The group is selected from the following: nucleotides containing lucyl-modified nucleotides, 2'-hydroxyl-modified nucleotides, 2'-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides containing unnatural bases, tetrahydropyran-modified nucleotides, 1,5-anhydrohexitol-modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides containing a phosphorothioate group, nucleotides containing a methylphosphonate group, nucleotides containing a 5'-phosphate, and nucleotides containing a 5'-phosphate mimetic.

[0040] In another embodiment, at least one modified nucleotide of the first dsRNA agent and at least one modified nucleotide of the second dsRNA agent are each independently selected from the group consisting of 2'-O-methyl and 2'-fluoro modifications.

[0041] The complementary region of the first dsRNA agent and / or the complementary region of the second dsRNA agent may each be independently 19 to 30 nucleotides long.

[0042] Each strand of the first dsRNA agent and each strand of the second dsRNA agent can independently be 19 to 30 nucleotides long.

[0043] In one embodiment, at least one strand of the first dsRNA agent and / or at least one strand of the second dsRNA agent each independently contains a 3' overhang of at least one nucleotide.

[0044] In one embodiment, the first dsRNA agent and / or the second dsRNA agent each independently further comprises at least one phosphorothioate or methylphosphonate internucleotide bond.

[0045] In one embodiment, the first dsRNA agent and / or the second dsRNA agent each independently further comprises at least one ligand.

[0046] In another embodiment, at least one ligand is conjugated to the sense strand of a first dsRNA agent and / or a second dsRNA agent.

[0047] In one embodiment, at least one ligand is conjugated to one of the 3' ends, 5' ends, or an internal position of the sense strand.

[0048] In another embodiment, at least one ligand is conjugated to the antisense strand of a first dsRNA agent and / or a second dsRNA agent.

[0049] In one embodiment, at least one ligand is conjugated to one of the 3' ends, 5' ends, or an internal position of the antisense chain.

[0050] In one embodiment, the ligand is an N-acetylgalactosamine (GalNAc) derivative.

[0051] In one embodiment, the ligand is one or more GalNAc derivatives bound via a monovalent, divalent, or trivalent branched linker.

[0052] In one embodiment, the ligand is [ka] That is the case.

[0053] In one embodiment, the first dsRNA agent and the second dsRNA agent are each independently shown in the following schematic diagram. [ka] It is conjugated to the ligand shown in the formula, where X is O or S.

[0054] In one embodiment, X is O.

[0055] In one embodiment, the first dsRNA agent and the second dsRNA agent are covalently linked via a covalent linker.

[0056] In one embodiment, the covalent linker is selected from the group consisting of single-stranded nucleic acid linkers, double-stranded nucleic acid linkers, partially single-stranded nucleic acid linkers, partially double-stranded nucleic acid linkers, carbohydrate sublinkers, and peptide linkers. In another embodiment, the covalent linker is a cleavable or non-cleavable linker. In one embodiment, the covalent linker links the sense strand of a first dsRNA agent to the sense strand of a second dsRNA agent. In another embodiment, the covalent linker links the antisense strand of a first dsRNA agent to the antisense strand of a second dsRNA agent.

[0057] In one embodiment, the covalent linker further comprises at least one ligand.

[0058] In one embodiment, contacting cells with the dual-targeted RNAi agent of the present invention inhibits the expression of the LDHA gene and the HAO1 gene to substantially the same level of inhibition as that obtained by contacting cells with each dsRNA agent individually. In another embodiment, contacting cells with the dual-targeted RNAi agent inhibits the expression of the LDHA gene and the HAO1 gene to a higher level than that obtained by contacting cells with each dsRNA agent individually.

[0059] In one embodiment, the level of inhibition of LDHA expression is at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, or about 100% higher than the level of inhibition of expression obtained by contacting the cells with both dsRNA agents individually.

[0060] In one embodiment, the level of inhibition of HAO1 expression is at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, or about 100% higher than the level of inhibition of expression obtained by contacting the cells with both dsRNA agents individually.

[0061] In one embodiment, contacting cells with a dual-targeted RNAi agent inhibits oxalate and / or glyoxylate protein production to a level lower than that achieved by contacting cells with both dsRNA agents individually. In another embodiment, contacting cells with a dual-targeted RNAi agent inhibits oxalate and / or glyoxylate protein production to a level lower than that achieved by contacting cells with both dsRNA agents individually.

[0062] The present invention also provides cells containing the dsRNA agent or dual-targeting RNAi agent of the present invention; and a vector encoding at least one strand of the dsRNA agent or dual-targeting RNAi agent of the present invention.

[0063] Furthermore, the present invention provides a pharmaceutical composition for inhibiting the expression of the lactate dehydrogenase A (LDHA) gene, comprising the dsRNA agent of the present invention; or a pharmaceutical composition for inhibiting the expression of the lactate dehydrogenase A (LDHA) gene and the hydroxy acid oxidase 1 (glycolate oxidase) (HAO1) ​​gene, comprising the dual-targeted RNAi agent of the present invention.

[0064] In one embodiment, the present invention provides a pharmaceutical composition comprising: a first double-stranded ribonucleic acid (dsRNA) agent that inhibits the expression of lactate dehydrogenase A (LDHA) including a sense strand and an antisense strand, wherein the sense strand comprises at least 15 consecutive nucleotides that differ by three or fewer nucleotides from the nucleotide sequence of SEQ ID NO: 1, and the antisense strand comprises at least 15 consecutive nucleotides that differ by three or fewer nucleotides from the nucleotide sequence of SEQ ID NO: 2; and a second double-stranded ribonucleic acid (dsRNA) agent that inhibits the expression of hydroxy acid oxidase 1 (glycolate oxidase) (HAO1) ​​including a sense strand and an antisense strand, wherein the sense strand comprises at least 15 consecutive nucleotides that differ by three or fewer nucleotides from the nucleotide sequence of SEQ ID NO: 21, and the antisense strand comprises at least 15 consecutive nucleotides that differ by three or fewer nucleotides from the nucleotide sequence of SEQ ID NO: 22.

[0065] In another embodiment, the present invention provides a pharmaceutical composition comprising: a first double-stranded ribonucleic acid (dsRNA) agent that inhibits the expression of lactate dehydrogenase A (LDHA) including a sense strand and an antisense strand, wherein the antisense strand comprises a complementary region comprising at least 15 consecutive nucleotides that differ by three or fewer nucleotides from any one of the antisense sequences listed in any one of Tables 2 to 5; and a second double-stranded ribonucleic acid (dsRNA) agent that inhibits the expression of hydroxy acid oxidase 1 (glycolate oxidase) (HAO1) ​​including a sense strand and an antisense strand, wherein the antisense strand comprises a complementary region comprising at least 15 consecutive nucleotides that differ by three or fewer nucleotides from any one of the antisense sequences listed in any one of Tables 7 to 14.

[0066] This agent may be formulated in a non-buffered solution such as physiological saline or water; or it may be formulated with a solution containing acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof; or with a buffer such as phosphate-buffered saline (PBS).

[0067] The present invention provides a method for inhibiting intracellular expression of lactate dehydrogenase A (LDHA). This method includes the step of contacting cells with the agent or pharmaceutical composition of the present invention, thereby inhibiting intracellular expression of LDHA.

[0068] The present invention also provides a method for inhibiting intracellular expression of lactate dehydrogenase A (LDHA) and hydroxy acid oxidase 1 (glycolate oxidase) (HAO1). This method includes contacting cells with a pharmaceutical composition containing the dual-targeting RNAi agent or the dual-targeting agent of the present invention, thereby inhibiting intracellular expression of LDHA and HAO1.

[0069] In one embodiment, the cells are present in a subject such as a human.

[0070] In one embodiment, LDHA expression is inhibited by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%, or to a level below the detection level of LDHA expression.

[0071] In one embodiment, HAO1 expression is inhibited by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%, or to a level below the detection level of HAO1 expression.

[0072] In one embodiment, the human subject suffers from a disease, disorder, or condition related to the oxalate pathway.

[0073] In one embodiment, a disease, disorder, or condition related to the oxalate pathway is a disease, disorder, or condition related to oxalate, or a disease, disorder, or condition related to lactate dehydrogenase.

[0074] In one embodiment, the disease, disorder, or condition related to oxalate is a disease, disorder, or condition of kidney stone formation, or a disease, disorder, or condition of calcium oxalate tissue deposition.

[0075] In one embodiment, the kidney stone formation disease, disorder, or condition is a calcium oxalate stone formation disease, disorder, or condition or a non-calcium oxalate stone formation disease, disorder, or condition.

[0076] In one embodiment, the calcium oxalate stone formation disease, disorder, or condition is a hyperoxaluria disease, disorder, or condition or a non-hyperoxaluria disease, disorder, or condition.

[0077] In one embodiment, the hyperoxaluria disease, disorder, or pathological condition is selected from the group consisting of primary hyperoxaluria, intestinal hyperoxaluria, dietary hyperoxaluria, and idiopathic hyperoxaluria.

[0078] In one embodiment, the non-hyperoxaluria stone-forming disease, disorder, or condition is hypercalciuria and / or hypocitric aciduria.

[0079] In one embodiment, the non-hyperoxaluria stone formation disease, disorder, or pathology is a calcium oxalate or non-calcium oxalate kidney stone formation disease.

[0080] In one embodiment, the calcium oxalate tissue deposition disease, disorder, or condition is selected from the group consisting of systemic calcium oxalate tissue deposition disease, disorder, or condition or tissue-specific calcium oxalate tissue deposition disease, disorder, or condition.

[0081] In one embodiment, the disease, disorder, or condition related to lactate dehydrogenase is selected from the group consisting of cancer, fatty liver (steatosis), non-alcoholic steatohepatitis (NASH), cirrhosis, accumulation of fat in the liver, inflammation of the liver, hepatocyte necrosis, hepatic fibrosis, and non-alcoholic fatty liver disease (NAFLD).

[0082] In one embodiment, the cells are hepatocytes.

[0083] In one embodiment, the present invention provides a method for inhibiting the expression of LDHA in a subject. This method comprises the step of administering a therapeutically effective amount of the agent or pharmaceutical composition of the present invention to a subject, thereby inhibiting the expression of LDHA in the subject.

[0084] In another embodiment, the present invention provides a method for inhibiting lactate dehydrogenase A (LDHA) expression and hydroxy acid oxidase 1 (glycolate oxidase) (HAO1) ​​expression in a subject. The method comprises administering a therapeutically effective amount of the dual-targeted RNAi agent of the present invention, or a pharmaceutical composition containing the dual-targeted RNAi agent of the present invention, to a subject, thereby inhibiting the expression of LDHA and HAO1 in the subject.

[0085] In one embodiment, the present invention provides a method for treating a subject suffering from a disorder in which a reduction in LDHA expression can be beneficial. The method comprises the step of administering a therapeutically effective amount of the agent or pharmaceutical composition of the present invention to the subject, thereby treating the subject.

[0086] In another embodiment, the present invention provides a method for preventing at least one symptom of a disease or disorder in which a reduction in LDHA gene expression may be beneficial. The method comprises administering a prophylactically effective amount of the agent or pharmaceutical composition of the present invention to a subject, thereby preventing at least one symptom of the subject.

[0087] In one embodiment, the disorder is a disease, disorder, or condition related to the oxalate pathway.

[0088] In one embodiment, the present invention provides a method for treating a subject suffering from a disease, disorder, or condition related to the oxalate pathway. The method comprises administering a therapeutically effective amount of the agent or pharmaceutical composition of the present invention to the subject, thereby treating the subject.

[0089] In another embodiment, the present invention provides a method for preventing at least one symptom of a subject suffering from a disease, disorder, or condition related to the oxalate pathway. The method comprises administering a prophylactically effective amount of the agent or pharmaceutical composition of the present invention to a subject, thereby preventing at least one symptom of the subject.

[0090] In one embodiment, administration of a dsRNA agent or pharmaceutical composition to a subject causes a decrease in 1 or urinary oxalates, tissue oxalates, plasma oxalates, decreased LDHA enzyme activity, decreased LDHA protein accumulation, and / or decreased HAO1 protein accumulation.

[0091] In one embodiment, a disease, disorder, or condition related to the oxalate pathway is a disease, disorder, or condition related to oxalate, or a disease, disorder, or condition related to lactate dehydrogenase.

[0092] In one embodiment, the disease, disorder, or condition related to oxalate is a disease, disorder, or condition of kidney stone formation, or a disease, disorder, or condition of calcium oxalate tissue deposition.

[0093] In one embodiment, the kidney stone formation disease, disorder, or condition is a calcium oxalate stone formation disease, disorder, or condition or a non-calcium oxalate stone formation disease, disorder, or condition.

[0094] In one embodiment, the calcium oxalate stone formation disease, disorder, or condition is a hyperoxaluria disease, disorder, or condition or a non-hyperoxaluria disease, disorder, or condition.

[0095] In one embodiment, the hyperoxaluria disease, disorder, or pathological condition is selected from the group consisting of primary hyperoxaluria, intestinal hyperoxaluria, dietary hyperoxaluria, and idiopathic hyperoxaluria.

[0096] In one embodiment, the non-hyperoxaluria stone-forming disease, disorder, or condition is hypercalciuria and / or hypocitric aciduria.

[0097] In one embodiment, the non-hyperoxaluria stone formation disease, disorder, or pathology is a calcium oxalate or non-calcium oxalate kidney stone formation disease.

[0098] In one embodiment, the calcium oxalate tissue deposition disease, disorder, or condition is selected from the group consisting of systemic calcium oxalate tissue deposition disease, disorder, or condition or tissue-specific calcium oxalate tissue deposition disease, disorder, or condition.

[0099] In one embodiment, the disease, disorder, or condition related to lactate dehydrogenase is selected from the group consisting of cancer, fatty liver (steatosis), non-alcoholic steatohepatitis (NASH), cirrhosis, accumulation of fat in the liver, inflammation of the liver, hepatocyte necrosis, hepatic fibrosis, and non-alcoholic fatty liver disease (NAFLD).

[0100] In one embodiment, the disease, disorder, or condition is primary hyperoxaluria 2 (PH2).

[0101] In one embodiment, the method further includes altering the diet in question (for example, reducing protein intake, reducing sodium intake, reducing ascorbic acid intake, suppressing calcium intake, supplementing phosphates, supplementing magnesium, and pyridoxine treatment; and any combination thereof).

[0102] In one embodiment, the subject undergoes a kidney transplant.

[0103] In one embodiment, the subject is a human.

[0104] In one embodiment, the method further includes the step of administering an additional therapeutic agent to the target.

[0105] In one embodiment, the RNAi agent is administered to the subject at a dose of approximately 0.01 mg / kg to approximately 10 mg / kg or approximately 0.5 mg / kg to approximately 50 mg / kg.

[0106] In one embodiment, the agent is administered subcutaneously to the subject.

[0107] In one embodiment, the agent does not substantially inhibit the expression and / or activity of lactate dehydrogenase B (LDHB). [Brief explanation of the drawing]

[0108] [Figure 1A] Figure 1A is a schematic diagram of the endogenous pathway for oxalate synthesis. [Figure 1B] Figure 1B is a schematic diagram of the metabolic pathways related to LDHA. [Figure 2] Figure 2 is a graph showing the levels of Ldha mRNA remaining in wild-type C57BL / 6J mice 10 days after administration of a single dose of AD-84788 at doses of 0.1 mg / kg, 0.3 mg / kg, 1.0 mg / kg, 3.0 mg / kg, or 10 mg / kg. [Figure 3] Figure 3 is a graph showing liver LDHA activity in adult male Agxt knockout mice 4 weeks after single subcutaneous administration of AD-84788 at doses of 0.3 mg / kg, 1 mg / kg, 3 mg / kg, or 10 mg / kg. Agxt knockout mice administered 0 mg / kg of AD-84788 were used as an untreated control. [Figure 4] Figure 4 is a schematic diagram of the test protocol described in Example 3 and shown in Figures 6-17B. [Figure 5]Figure 5 is a graph showing the amount of urinary oxalates (mg per gram of creatinine) excreted by Agxt knockout mice over a 24-hour period at 0, 1, 2, 3, 4, 6, 8, 9, and 10 weeks after a single subcutaneous administration of AD-84788 at doses of 0.3 mg / kg, 1 mg / kg, 3 mg / kg, or 10 mg / kg. Agxt knockout mice administered 0 mg / kg of AD-84788 were used as an untreated control. [Figure 6] Figure 6 is a graph showing the amount of oxalate (mg per gram of creatinine) excreted in the urine of Agxt knockout mice, wild-type mice, and Grhpr (glyoxylate reductase / hydroxypyruvate reductase) knockout mice 4 weeks after a single dose of 10 mg / kg of AD-84788. [Figure 7] Figure 7 is a graph showing the amount of oxalate (mg per g of creatinine) excreted in the urine of Agxt-deficient mice that were administered the dsRNA agent AD-84788 on day 0, before administration (baseline, i.e., days -6, -5, -4, and -3); 7–10 days after a single dose of 10 mg / kg of AD-84788; and 28–31 days after the last administration of four doses of 10 mg / kg of AD-84788 on days 0, 11, 18, and 25 (see Figure 4). [Figure 8]Figure 8A is a graph showing the enzymatic activity of LdhA in wild-type liver homogenates from untreated control mice and mice administered four doses of 10 mg / kg AD-84788 (see Figure 4) using lactate as a substrate. Absorbance increases as NAD is reduced to NADH by LDH enzymatic activity. An initial linear range was selected, and the absorbances at 1 and 6 minutes were used as Δabs over a Δtime of 5 minutes in the specific activity calculation. Figure 8B is a graph showing the mean specific activity of LdhA in wild-type liver homogenates from untreated control mice and mice administered four doses of 10 mg / kg AD-84788 (see Figure 4) using lactate as a substrate. Specific activity is expressed as μmol / min / g protein of NADH formed. Calculations were performed individually for all animals, and a t-test was performed to compare all specific activity data from both treatment groups. The mean specific activity of both treatment groups is shown. (p<0.001). [Figure 9] Figure 9A is a graph showing the enzymatic activity of LdhA in wild-type liver homogenates from untreated control mice and mice administered four doses of 10 mg / kg AD-84788 (see Figure 4) using glyoxylate as a substrate. Absorbance increases as NAD is reduced to NADH by LDH enzymatic activity. An initial linear range was selected, and the absorbances at 0 and 4 minutes were used as Δabs over Δtime of 4 minutes in the specific activity calculation. Figure 9B is a graph showing the mean specific activity of LdhA in wild-type liver homogenates from untreated control mice and mice administered four doses of 10 mg / kg AD-84788 (see Figure 4) using glyoxylate as a substrate. Specific activity is expressed as μmol / min / g protein of NADH formed. Calculations were performed individually for all animals, and a t-test was performed to compare all specific activity data from both treatment groups. The mean specific activity of both treatment groups is shown. (p<0.001). [Figure 10]Figure 10A is a graph showing the enzymatic activity of LdhA in Agxt-deficient liver homogenates from untreated control mice and mice administered four doses of 10 mg / kg AD-84788 (see Figure 4) using lactate as a substrate. Absorbance increases as NAD is reduced to NADH by LDH enzymatic activity. An initial linear range was selected, and the absorbances at 0 and 4 minutes were used as Δabs over Δtime of 4 minutes in the specific activity calculation. In the mean treatment group, the standard deviation (SD) is too small to be visualized. Figure 10B is a graph showing the mean specific activity of LdhA in Agxt-deficient liver homogenates from untreated control mice and mice administered four doses of 10 mg / kg AD-84788 (see Figure 4) using lactate as a substrate. Specific activity is expressed as μmol / min / g protein of NADH formed. The calculations were performed individually for all animals, and a t-test was conducted to compare all specific activity data from both treatment groups. The mean specific activity of both treatment groups is shown (p<0.001). [Figure 11] Figure 11A is a graph showing the enzymatic activity of LdhA in Agxt-deficient liver homogenates from untreated control mice and mice administered four doses of 10 mg / kg AD-84788 (see Figure 4) using glyoxylate as a substrate. Absorbance increases as NAD is reduced to NADH by LDH enzymatic activity. An initial linear range was selected, and the absorbances at 0 and 4 minutes were used as Δabs over Δtime of 4 minutes in the specific activity calculation. Figure 11B is a graph showing the mean specific activity of LdhA in Agxt-deficient liver homogenates from untreated control mice and mice administered four doses of 10 mg / kg AD-84788 (see Figure 4) using glyoxylate as a substrate. Specific activity is expressed as μmol / min / g protein of NADH formed. Calculations were performed individually for all animals, and a t-test was performed to compare all specific activity data from both treatment groups. The average specific activity of both treatment groups is shown (p<0.001). [Figure 12]Figure 12A is a graph showing the enzymatic activity of LdhA in wild-type cardiac homogenates from untreated control mice and mice administered four doses of 10 mg / kg AD-84788 (see Figure 4) using lactate as a substrate. Absorbance increases for both the control and treatment groups as NAD is reduced to NADH by LDH enzymatic activity. An initial linear range was selected, and the absorbances at 0 and 4 minutes were used as Δabs over Δtime (4 minutes) in the specific activity calculation. Figure 12B is a graph showing the mean specific activity of LdhA in wild-type cardiac homogenates from untreated control mice and mice administered four doses of 10 mg / kg AD-84788 (see Figure 4) using lactate as a substrate. Specific activity is expressed as μmol / min / g protein of NADH formed. Calculations were performed individually for all animals, and a t-test was performed to compare all specific activity data from both treatment groups. The mean specific activity of both treatment groups is shown. There is no significant difference. Figure 12C is a graph showing the enzymatic activity of LdhA in wild-type thigh muscle homogenate from untreated control mice and mice administered four doses of 10 mg / kg AD-84788 (see Figure 4) using lactate as a substrate. Absorbance increases for both the control and treatment groups as NAD is reduced to NADH by LDH enzymatic activity. An initial linear range was selected, and the absorbance at 0 and 4 minutes was used as Δabs over Δtime of 4 minutes in the specific activity calculation. Figure 12D is a graph showing the mean specific activity of LdhA in wild-type thigh muscle homogenate from untreated control mice and mice administered four doses of 10 mg / kg AD-84788 (see Figure 4) using lactate as a substrate. Specific activity is expressed as μmol / min / g protein of NADH formed. Calculations were performed individually for all animals, and a t-test was conducted to compare all specific activity data from both treatment groups. The mean specific activity of both treatment groups is shown. There was no significant difference. [Figure 13]Figure 13A is a graph showing the mean amount of lactate in wild-type liver homogenate of wild-type mice before administration of four 10 mg / kg doses of AD-84788 (baseline), and the mean amount of lactate in wild-type liver homogenate of wild-type mice four weeks after administration of four 10 mg / kg doses of AD-84788 (see Figure 4). Figure 13B is a graph showing the mean amount of pirubate in wild-type liver homogenate of wild-type mice before administration of four 10 mg / kg doses of AD-84788 (baseline), and the mean amount of pirubate in wild-type liver homogenate of wild-type mice four weeks after administration of four 10 mg / kg doses of AD-84788 (see Figure 4). [Figure 14] Figure 14A is a graph showing the average amount of lactate in Agxt-deficient liver homogenate of Agxt-deficient mice before administration of four 10 mg / kg doses of AD-84788 (baseline), and the average amount of lactate in Agxt-deficient liver homogenate of Agxt-deficient mice four weeks after administration of four 10 mg / kg doses of AD-84788 (see Figure 4). Figure 14B is a graph showing the average amount of pirubate in Agxt-deficient liver homogenate of Agxt-deficient mice before administration of four 10 mg / kg doses of AD-84788 (baseline), and the average amount of pirubate in Agxt-deficient liver homogenate of Agxt-deficient mice four weeks after administration of four 10 mg / kg doses of AD-84788 (see Figure 4). [Figure 15]Figure 15A is a graph showing the average amount of glyoxylate in wild-type liver homogenate of wild-type mice before administration of four 10 mg / kg doses of AD-84788 (baseline), and the average amount of glyoxylate in wild-type liver homogenate of wild-type mice four weeks after administration of four 10 mg / kg doses of AD-84788 (see Figure 4). Figure 15B is a graph showing the average amount of glyoxylate in Agxt-deficient liver homogenate of Agxt-deficient mice before administration of four 10 mg / kg doses of AD-84788 (baseline), and the average amount of glyoxylate in Agxt-deficient liver homogenate of Agxt-deficient mice four weeks after administration of four 10 mg / kg doses of AD-84788 (see Figure 4). [Figure 16] Figure 16A is a graph showing the mean body weight of wild-type mice before administration of four 10 mg / kg doses of AD-84788 (baseline) and the mean body weight of wild-type mice four weeks after administration of four 10 mg / kg doses of AD-84788 (see Figure 4). Figure 16B is a graph showing the mean body weight of Agxt-deficient mice before administration of four 10 mg / kg doses of AD-84788 (baseline) and the mean body weight of Agxt-deficient mice four weeks after administration of four 10 mg / kg doses of AD-84788 (see Figure 4). [Figure 17] Figure 17A is a graph showing the mean plasma lactate levels of wild-type mice before administration of four 10 mg / kg doses of AD-84788 (baseline) and the mean plasma lactate levels of wild-type mice four weeks after administration of four 10 mg / kg doses of AD-84788 (see Figure 4). Figure 17B is a graph showing the mean plasma lactate levels of Agxt-deficient mice before administration of four 10 mg / kg doses of AD-84788 (baseline) and the mean plasma lactate levels of Agxt-deficient mice four weeks after administration of four 10 mg / kg doses of AD-84788 (see Figure 4). [Figure 18-1]Figures 18A-18O show exemplary dual-targeting agents of the present invention. Figure 18A shows an exemplary dual-targeting agent of the present invention comprising a first dsRNA agent targeting LDHA and a second dsRNA agent targeting HAO1, wherein the first dsRNA agent comprises a first sense strand (S) and a first antisense strand (AS), and the second dsRNA agent comprises a second sense strand (S) and a second antisense strand, wherein the 3' end of the first sense strand is covalently linked to the 5' end of the second sense strand by a nucleotide linker containing a 2'OMe-modified nucleotide (uuu), the 3' end of the second sense strand contains a GalNAc ligand, and the two outermost 5' nucleotides of the first sense strand each independently contain a phosphorothioate linkage. Figure 18B shows an exemplary dual-targeting agent of the present invention, comprising a first dsRNA agent targeting LDHA and a second dsRNA agent targeting HAO1, wherein the first dsRNA agent comprises a first sense strand (S) and a first antisense strand (AS), and the second dsRNA agent comprises a second sense strand (S) and a second antisense strand (AS), wherein the 3' end of the first sense strand is covalently linked to the 5' end of the second sense strand by a nucleotide linker comprising a 2' fluoromodified nucleotide (GfAfAf), the 3' end of the second sense strand comprises a GalNAc ligand, and the two outermost 5' nucleotides of the first sense strand, the outermost 3' nucleotide of the first sense strand, and the outermost 5' nucleotide of the second sense strand each independently contain a phosphorothioate linkage.Figure 18C shows an exemplary dual-targeting agent of the present invention, comprising a first dsRNA agent targeting LDHA and a second dsRNA agent targeting HAO1, wherein the first dsRNA agent comprises a first sense strand (S) and a first antisense strand (AS), and the second dsRNA agent comprises a second sense strand (S) and a second antisense strand (AS), wherein the 3' end of the first sense strand is covalently linked to the 5' end of the second sense strand by a nucleotide linker comprising a 2' fluoromodified nucleotide (GfAfUf), the 3' end of the second sense strand comprises a GalNAc ligand, and the two outermost 5' nucleotides of the first sense strand, the outermost 3' nucleotide of the first sense strand, and the outermost 5' nucleotide of the second sense strand each independently contain a phosphorothioate linkage. Figure 18D shows an exemplary dual-targeting agent of the present invention, comprising a first dsRNA agent targeting LDHA and a second dsRNA agent targeting HAO1, wherein the first dsRNA agent comprises a first sense strand (S) and a first antisense strand (AS), and the second dsRNA agent comprises a second sense strand (S) and a second antisense strand (AS), wherein the 3' end of the first sense strand is covalently linked to the 5' end of the second sense strand by a nucleotide linker comprising a deoxyribonucleotide (dgdada), the 3' end of the second sense strand comprises a GalNAc ligand, and the two outermost 5' nucleotides of the first sense strand, the outermost 3' nucleotide of the first sense strand, and the outermost 5' nucleotide of the second sense strand each independently contain a phosphorothioate linkage.Figure 18E shows an exemplary dual-targeting agent of the present invention, comprising a first dsRNA agent targeting LDHA and a second dsRNA agent targeting HAO1, wherein the first dsRNA agent comprises a first sense strand (S) and a first antisense strand (AS), and the second dsRNA agent comprises a second sense strand (S) and a second antisense strand (AS), wherein the 3' end of the first sense strand is covalently linked to the 5' end of the second sense strand by a nucleotide linker comprising a deoxyribonucleotide (dgda), the 3' end of the second sense strand comprises a GalNAc ligand, and the two outermost 5' nucleotides of the first sense strand, the outermost 3' nucleotide of the first sense strand, and the outermost 5' nucleotide of the second sense strand each independently contain a phosphorothioate linkage. Figure 18F shows an exemplary dual-targeting agent of the present invention, comprising a first dsRNA agent targeting LDHA and a second dsRNA agent targeting HAO1, wherein the first dsRNA agent comprises a first sense strand (S) and a first antisense strand (AS), and the second dsRNA agent comprises a second sense strand (S) and a second antisense strand (AS), the 3' end of the first sense strand being directly (without a linker) bound to the 5' end of the second sense strand, the two outermost 5' nucleotides of the first sense strand and the two outermost 3' nucleotides of the second sense strand each independently containing a phosphorothioate bond, and the 3' end of the first sense strand containing a GalNAc ligand. Figure 18G shows an exemplary dual-targeting agent of the present invention comprising a first dsRNA agent targeting LDHA and a second dsRNA agent targeting HAO1, wherein the first dsRNA agent comprises a first sense strand (S) and a first antisense strand (AS), and the second dsRNA agent comprises a second sense strand (S) and a second antisense strand (AS), wherein the 5' end of the first antisense strand is covalently linked to the 3' end of the second antisense strand by a nucleotide linker comprising a 2'OMe-modified nucleotide (acu), the 3' end of the second sense strand comprises a GalNAc ligand, and the two outermost 3' nucleotides of the first antisense strand and the two outermost 5' nucleotides of the second antisense strand each independently contain a phosphorothioate linkage.Figure 18H shows an exemplary dual-targeting agent of the present invention comprising a first dsRNA agent targeting LDHA and a second dsRNA agent targeting HAO1, wherein the first dsRNA agent comprises a first sense strand (S) and a first antisense strand (AS), and the second dsRNA agent comprises a second sense strand (S) and a second antisense strand (AS), wherein the 5' end of the first antisense strand is covalently linked to the 3' end of the second antisense strand by a nucleotide linker comprising a 2' fluoromodified nucleotide (AfAfGf), the 3' end of the second sense strand comprises a GalNAc ligand, and the two outermost 3' nucleotides of the first antisense strand, the 5' nucleotide of the first antisense strand, the 3' nucleotide of the second antisense strand, and the two outermost 5' nucleotides of the second antisense strand each independently contain a phosphorothioate linkage. Figure 18I shows an exemplary dual-targeting agent of the present invention, comprising a first dsRNA agent targeting LDHA and a second dsRNA agent targeting HAO1, wherein the first dsRNA agent comprises a first sense strand (S) and a first antisense strand (AS), and the second dsRNA agent comprises a second sense strand (S) and a second antisense strand (AS), wherein the 5' end of the first antisense strand is directly (without a linker) bound to the 3' end of the second antisense strand, the 3' end of the second sense strand contains a GalNAc ligand, and the two outermost 3' nucleotides of the first antisense strand and the two outermost 5' nucleotides of the second antisense strand each independently contain a phosphorothioate bond.Figure 18J shows an exemplary dual-targeting agent of the present invention comprising a first dsRNA agent targeting LDHA and a second dsRNA agent targeting HAO1, wherein the first dsRNA agent comprises a first sense strand (S) and a first antisense strand (AS), and the second dsRNA agent comprises a second sense strand (S) and a second antisense strand (AS), wherein the 3' end of the first sense strand is covalently bonded to the 5' end of the second sense strand by a nucleotide linker containing a 2'OMe-modified nucleotide (uuu), the 5' end of the first sense strand and the 3' end of the second sense strand each independently contain a GalNAc ligand, and the 5' nucleotide of the first sense strand contains a phosphorothioate bond. Figure 18K shows an exemplary dual-targeting agent of the present invention, comprising a first dsRNA agent targeting LDHA and a second dsRNA agent targeting HAO1, wherein the first dsRNA agent comprises a first sense strand (S) and a first antisense strand (AS), and the second dsRNA agent comprises a second sense strand (S) and a second antisense strand (AS), wherein the 3' end of the first sense strand is covalently linked to the 5' end of the second sense strand by a nucleotide linker comprising a 2' fluoromodified nucleotide (GfAfAf), the 5' end of the first sense strand and the 3' end of the second sense strand each independently contain a GalNAc ligand, and the 5' nucleotide of the first sense strand, the 3' nucleotide of the first sense strand, and the 5' nucleotide of the second sense strand each independently contain a phosphorothioate bond. Figure 18L shows an exemplary dual-targeting agent of the present invention, comprising a first dsRNA agent targeting LDHA and a second dsRNA agent targeting HAO1, wherein the first dsRNA agent comprises a first sense strand (S) and a first antisense strand (AS), and the second dsRNA agent comprises a second sense strand (S) and a second antisense strand (AS), the 3' end of the first sense strand being directly (without a linker) bound to the 5' end of the second sense strand, the 3' ends of the first sense strand and the 3' ends of the second sense strand each independently containing a GalNAc ligand, and the two outermost 5' nucleotides of the first sense strand each independently containing a phosphorothioate bond.Figure 18M shows an exemplary dual-targeting agent of the present invention, comprising a first dsRNA agent targeting LDHA and a second dsRNA agent targeting HAO1, wherein the first dsRNA agent comprises a first sense strand (S) and a first antisense strand (AS), and the second dsRNA agent comprises a second sense strand (S) and a second antisense strand (AS), wherein the 5' end of the first antisense strand is covalently linked to the 3' end of the second antisense strand by a nucleotide linker comprising a 2'-O-Me modified nucleotide (acu), the 3' ends of the first antisense strand and the 3' ends of the second sense strand each independently contain a GalNAc ligand, and the two outermost 5' nucleotides of the second antisense strand each independently contain a phosphorothioate linkage. Figure 18N shows an exemplary dual-targeting agent of the present invention comprising a first dsRNA agent targeting LDHA and a second dsRNA agent targeting HAO1, wherein the first dsRNA agent comprises a first sense strand (S) and a first antisense strand (AS), and the second dsRNA agent comprises a second sense strand (S) and a second antisense strand (AS), wherein the 5' end of the first antisense strand is covalently linked to the 3' end of the second antisense strand by a nucleotide linker comprising a 2' fluoromodified nucleotide (AfAfGf), the 3' end of the first antisense strand and the 3' end of the second sense strand each independently contain a GalNAc ligand, and the 5' nucleotide of the first antisense strand, the 3' nucleotide of the second antisense strand, and the two outermost 5' nucleotides of the second antisense strand each independently contain a phosphorothioate linkage.Figure 18O shows an exemplary dual-targeting agent of the present invention comprising a first dsRNA agent targeting LDHA and a second dsRNA agent targeting HAO1, wherein the first dsRNA agent comprises a first sense strand (S) and a first antisense strand (AS), and the second dsRNA agent comprises a second sense strand (S) and a second antisense strand (AS), wherein the 5' end of the first antisense strand is directly (without a linker) bound to the 3' end of the second antisense strand, the 3' ends of the first antisense strand and the 3' ends of the second sense strand each independently contain a GalNAc ligand, and the two outermost 5' nucleotides of the second antisense strand each independently contain a phosphorothioate bond. [Figure 18-2] Same as above. [Figure 18-3] Same as above. [Figure 18-4] Same as above. [Figure 18-5] Same as above. [Modes for carrying out the invention]

[0109] The present invention provides an iRNA composition that induces RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the LDHA gene. The LDHA gene may be present in cells, for example, in subjects such as humans. The present invention also provides a method of using the iRNA composition of the present invention to inhibit the expression of the LDHA gene and to treat subjects who may benefit from inhibiting or reducing the expression of the LDHA gene, for example, subjects who may benefit from reduced or inhibited urinary oxalate production, for example, subjects suffering from or susceptible to diseases, disorders, or conditions related to the oxalate pathway, for example, diseases, disorders, or conditions related to oxalate formation, for example, kidney stone formation diseases, disorders, or conditions or calcium oxalate tissue deposition diseases, disorders, or conditions; or subjects suffering from or susceptible to diseases, disorders, or conditions related to LDH.

[0110] The present invention also provides a method of using the iRNA composition of the present invention to inhibit the expression of the LDHA gene and the HAO1 gene in order to treat subjects who may benefit from inhibiting or reducing the expression of the LDHA gene and the HAO1 gene, for example, subjects who may benefit from reducing or inhibiting urinary oxalate production, for example, subjects suffering from or susceptible to diseases, disorders, or conditions related to the oxalate pathway, for example, diseases, disorders, or conditions related to oxalate formation, for example, kidney stone formation diseases, disorders, or conditions or calcium oxalate tissue deposition diseases, disorders, or conditions; or subjects suffering from or susceptible to diseases, disorders, or conditions related to LDH.

[0111] The iRNAs of the present invention that target LDHA are approximately 30 nucleotides or less in length, for example, 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, 19-25, 1 The RNA strand (antisense strand) may include a region having a length of 9-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides, and this region is substantially complementary to at least a portion of the mRNA transcript of the LDHA gene.

[0112] The iRNAs of the present invention that target HAO1 are approximately 30 nucleotides or less in length, for example, 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, 19-25, 1 The RNA strand (antisense strand) may include a region having a length of 9-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides, and this region is substantially complementary to at least a portion of the mRNA transcript of the HAO1 gene.

[0113] If the RNAi agent is a dual-targeted RNAi agent as described herein, the LDHA-targeting agent may include an antisense strand containing a region complementary to LDHA, which is of the same length as or different from the complementary region of the antisense strand of the HAO1-targeting agent.

[0114] In one embodiment, one or both strands of the double-stranded RNAi agent of the present invention have a region of at least 19 consecutive nucleotides substantially complementary to at least a portion of the mRNA transcript of the LDHA gene, and are up to 66 nucleotides long, for example, 36-66, 26-36, 25-36, 31-60, 22-43, or 27-53 nucleotides long. In one embodiment, such an iRNA agent having a longer antisense strand may include a second RNA strand (sense strand) of 20-60 nucleotides long, where the sense and antisense strands form a double helix of 18-30 consecutive nucleotides.

[0115] In other embodiments, one or both strands of the double-stranded RNAi agent of the present invention have a region of at least 19 consecutive nucleotides substantially complementary to at least a portion of the mRNA transcript of the HAO1 gene, and are up to 66 nucleotides long, for example, 36-66, 26-36, 25-36, 31-60, 22-43, or 27-53 nucleotides long. In some embodiments, such an iRNA agent having a longer antisense strand may include a second RNA strand (sense strand) of 20-60 nucleotides long, where the sense and antisense strands form a double helix of 18-30 consecutive nucleotides.

[0116] In embodiments in which a first dsRNA agent targeting LDHA and a second dsRNA agent targeting HAO1 are covalently bonded, the double-strand lengths of the first agent and the second agent may be the same or different.

[0117] The use of these iRNA agents described herein enables targeted degradation of LDHA gene mRNA in mammals or targeted degradation of both the LDHA gene and the HAO1 gene in mammals.

[0118] Very low doses of iRNA can, in particular, specifically and efficiently mediate RNA interference (RNAi) to result in significant inhibition of LDHA gene expression or LDHA gene and HAO1 gene expression. Using cell-based and in vivo assays, we have demonstrated that LDHA-targeting iRNAs can mediate RNAi to result in significant inhibition of LDHA gene expression and significant inhibition of oxalate production. Therefore, methods and compositions containing these iRNAs are useful for treating subjects who may benefit from reduced or inhibited LDHA expression or LDHA and HAO1 expression, such as subjects suffering from or susceptible to diseases, disorders, or conditions related to the oxalate pathway.

[0119] The following detailed description discloses compositions containing iRNAs for the purpose of inhibiting the expression of the LDHA gene, the HAO1 gene, and both the LDHA gene and the HAO1 gene, as well as compositions and methods for treating subjects suffering from diseases and disorders in which the inhibition and / or reduction of the expression of these genes may be beneficial.

[0120] I. Definition To make the present invention easier to understand, several terms are first defined. Furthermore, it should be noted that whenever a variable value or range of values ​​is stated, intermediate values ​​and ranges of the stated values ​​are also intended to be part of the present invention.

[0121] The articles “a” and “an” are used herein to refer to one or more (i.e., at least one) grammatical objects of the article. For example, “an element” means one or more elements, e.g., multiple elements.

[0122] The term "including" is used herein to mean "including but not limited to," and is used synonymously with this term. The term "or" is used herein to mean "and / or," and is used synonymously with this term, unless the context clearly indicates otherwise.

[0123] The term "LDHA" (used synonymously with "Ldha" in this specification), also known as cell proliferation-inducing gene 19 protein, renal cancer antigen NY-REN-59, LDH muscle subunit, EC 1.1.1.27 4 61, LDH-A, LDH-M, epididymal secretory sperm-binding protein Li 133P, L-lactate dehydrogenase A chain, proliferation-inducing gene 19, lactate dehydrogenase M, HEL-S-133P, EC 1.1.1, GSD11, PIG19, and LDHM, refers, unless otherwise specified, to well-known genes encoding lactate dehydrogenase A from any vertebrate or mammalian source, including humans, cattle, chickens, rodents, mice, rats, pigs, sheep, primates, monkeys, and guinea pigs.

[0124] This term also refers to fragments and variants of natural LDHA that maintain at least one in vivo or in vitro activity of natural LDHA. This term encompasses the full-length unprocessed precursor form of LDHA, as well as the mature form obtained from post-translational cleavage of the signal peptide and the form obtained from proteolytic treatment.

[0125] The sequences of human LDHA mRNA transcripts can be found, for example, in GenBank registry numbers GI:207028493 (NM_001135239.1; SEQ ID NO: 1), GI:260099722 (NM_001165414.1; SEQ ID NO: 3), GI:260099724 (NM_001165415.1; SEQ ID NO: 5), GI:260099726 (NM_001165416.1; SEQ ID NO: 7), and GI:207028465 (NM_005566.3; SEQ ID NO: 9); mouse LDHA The mRNA transcript sequences can be found, for example, in GenBank registry number GI:257743038 (NM_001136069.2; SEQ ID NO: 11) and GenBank registry number GI:257743036 (NM_010699.2; SEQ ID NO: 13); the rat LDHA mRNA transcript sequence can be found, for example, in GenBank registry number GI:8393705 (NM_017025.1; SEQ ID NO: 15); and the monkey LDHA mRNA transcript sequence can be found, for example, in GenBank registry number GI:402766306 (NM_001257735.2; SEQ ID NO: 17) and GenBank registry number GI:545687102 (NM_001283551.1; SEQ ID NO: 19).

[0126] Further examples of LDHA mRNA sequences are readily available using publicly available databases, such as GenBank, UniProt, and OMIM.

[0127] As used herein, the term "LDHA" also refers to certain polypeptides expressed in cells due to innate DNA sequence mutations in the LDHA gene, such as single nucleotide polymorphisms in the LDHA gene. Many SNPs within the LDHA gene have been identified and can be found, for example, in NCBI dbSNP (see, for example, www.ncbi.nlm.nih.gov / snp).

[0128] As used herein, the term "HAO1" refers, unless otherwise specified, to a well-known gene encoding the enzyme hydroxy acid oxidase 1 from any vertebrate or mammalian source, including humans, cattle, chickens, rodents, mice, rats, pigs, sheep, primates, monkeys, and guinea pigs. Other gene names include GO, GOX, GOX1, HAO, and HAOX1. This protein is also known as glycolate oxidase and (S)-2-hydroxy acid oxidase.

[0129] This term also refers to fragments and variants of natural HAO1 that maintain at least one in vivo or in vitro activity of natural HAO1. This term encompasses the full-length unprocessing precursor form of HAO1, as well as the mature form obtained from post-translational cleavage of the signal peptide and the form obtained from proteolytic treatment. The sequence of the human HAO1 mRNA transcript can be found, for example, at GenBank registry number GI:11184232 (NM_017545.2; SEQ ID NO: 21); the sequence of the monkey HAO1 mRNA transcript can be found, for example, at GenBank registry number GI:544464345 (XM_005568381.1; SEQ ID NO: 23); the sequence of the mouse HAO1 mRNA transcript can be found, for example, at GenBank registry number GI:133893166 (NM_010403.2; SEQ ID NO: 25); and the sequence of the rat HAO1 mRNA transcript can be found, for example, at GenBank registry number GI:166157785 (NM_001107780.2; SEQ ID NO: 27).

[0130] As used herein, the term "HAO1" also refers to innate DNA sequence variations of the HAO1 gene, such as single nucleotide polymorphisms (SNPs) in the HAO1 gene. Exemplary SNPs can be found in the NCBI dbSNP Short Genetic Variations database, available at www.ncbi.nlm.nih.gov / projects / SNP.

[0131] As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of the LDHA gene or the HAO1 gene, including mRNA, which is the product of RNA processing of the primary transcript. In one embodiment, the target portion of the sequence would be long enough to serve as a substrate for iRNA-directed cleavage at or near the relevant portion of the nucleotide sequence of the mRNA molecule formed during the transcription of the LDHA gene. In another embodiment, the target portion of the sequence would be long enough to serve as a substrate for iRNA-directed cleavage at or near the relevant portion of the nucleotide sequence of the mRNA molecule formed during the transcription of the HAO1 gene.

[0132] The target sequence of the LDHA gene can be approximately 9-36 nucleotides long, for example, approximately 15-30 nucleotides long. For example, the target sequence can be approximately 15-30 nucleotides, 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, 1 The nucleotide lengths may be 9-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides. Intermediate ranges and lengths between those listed above are also considered to be part of the present invention.

[0133] The target sequence of the HAO1 gene can be approximately 9-36 nucleotides long, for example, approximately 15-30 nucleotides long. For example, the target sequence could be approximately 15-30 nucleotides, 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, 1 The nucleotide lengths may be 9-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides. Intermediate ranges and lengths within the above ranges and lengths are also considered to be part of the present invention.

[0134] In a configuration in which a first dsRNA agent targeting LDHA and a second dsRNA agent targeting HAO1 are covalently bonded (i.e., a dual-targeting RNAi agent), the length of the LDHA target sequence may be the same as or different from that of the HAO1 target sequence.

[0135] As used herein, the term “sequence-containing chain” refers to an oligonucleotide containing a chain of nucleotides represented by a sequence shown using standard nucleotide nomenclature.

[0136] "G," "C," "A," and "U" generally represent nucleotides containing guanine, cytosine, adenine, and uracil as bases, respectively. However, it will be understood that the terms "ribonucleotide" or "nucleotide" may also refer to modified nucleotides or surrogate replacement moieties, as will be further detailed below (see, for example, Table 1). Those skilled in the art will be well aware that guanine, cytosine, adenine, and uracil may be substituted by other moieties without significantly altering the base-pairing properties of oligonucleotides containing such substitution moieties. For example, but not limited to, nucleotides containing inosine as a base may base-pair with nucleotides containing adenine, cytosine, or uracil. Therefore, nucleotides containing uracil, guanine, or adenine may be substituted, for example, with nucleotides containing inosine in the nucleotide sequences of dsRNA characterized in the present invention. In another example, adenine and cytosine at any point in the oligonucleotide can be substituted with guanine and uracil, respectively, to form a GU fluctuation base pair with the target mRNA. Sequences containing such substitutions are suitable for the compositions and methods addressed in the present invention.

[0137] The terms “iRNA,” “RNAi agent,” “iRNA agent,” and “RNA interfering agent,” as used synonymously herein, refer to agents containing RNA and mediating targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway, as defined herein. iRNAs induce sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). iRNAs regulate (e.g., inhibit) the expression of LDHA and / or HAO1 genes in cells, for example, in mammalian subjects.

[0138] In one embodiment, the RNAi agent of the present invention comprises a single-stranded RNA that interacts with a target RNA sequence, such as an LDHA-targeted mRNA sequence and / or a HAO1-targeted mRNA sequence, to lead to the cleavage of the target RNA. Although we do not wish to be constrained by theory, it is thought that long double-stranded RNA introduced into the cell is degraded into siRNA by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease III-like enzyme, processes dsRNA into short interfering RNAs of 19-23 base pairs with characteristic two base 3' overhangs (Bernstein, et al., (2001) Nature 409:363). Next, the siRNA is incorporated into an RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA double strand, allowing the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to a suitable target mRNA, one or more endonucleases in the RISC cleave the target, inducing silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Herein, in one embodiment, the present invention relates to single-stranded RNA (sssiRNA) that is generated in cells and promotes the formation of a RISC complex resulting in the silencing of target genes, namely the LDHA gene and / or the HAO1 gene. Accordingly, the term "siRNA" is also used herein to refer to the above RNAi.

[0139] In another embodiment, the RNAi agent may be a single-stranded RNAi agent introduced into a cell or organism to inhibit a target mRNA. The single-stranded RNAi agent (ssRNAi) binds to the RISC endonuclease Argonaute 2, which then cleaves the target mRNA. Single-stranded siRNAs are generally 15 to 30 nucleotides and are 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 which are incorporated herein by reference. Any of the antisense nucleotide sequences described herein may be used as single-stranded siRNAs chemically modified as described herein or by the methods described in Lima et al., (2012) Cell 150:883-894.

[0140] In another embodiment, the “iRNA” for use in the compositions and methods of the present invention is double-stranded RNA, and is referred herein to as “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-stranded structure containing two antiparallel and substantially complementary nucleic acid strands, which are shown to have “sense” and “antisense” orientations toward the target RNA, i.e., the LDHA gene and / or HAO1 gene. In one embodiment of the present invention, double-stranded RNA (dsRNA) induces degradation of the target RNA, e.g., mRNA, by a post-transcriptional gene silencing mechanism referred herein to as RNA interference or RNAi.

[0141] In yet another embodiment, the “iRNA” for use in the compositions and methods of the present invention is a “dual-targeting RNAi agent.” The term “dual-targeting RNAi agent” refers to a molecule containing a first dsRNA agent, which contains a complex of ribonucleic acid molecules having a double-stranded structure containing two antiparallel and substantially complementary nucleic acid strands, shown to have “sense” and “antisense” orientations toward the HAO1 gene, and is covalently bonded to a molecule containing a second dsRNA agent, which contains a complex of ribonucleic acid molecules having a double-stranded structure containing two antiparallel and substantially complementary nucleic acid strands, shown to have “sense” and “antisense” orientations toward the LDHA gene. In one embodiment of the present invention, the dual-targeting RNAi agent causes degradation of the first and second target RNAs, e.g., mRNA, by a post-transcriptional gene silencing mechanism referred to herein as RNA interference or RNAi.

[0142] Generally, the majority of nucleotides in each strand of a dsRNA molecule are ribonucleotides, but as described in detail herein, each or both strands may also contain one or more non-ribonucleotides, such as deoxyribonucleotides and / or modified nucleotides. Furthermore, as used herein, “RNAi agent” may include ribonucleotides having chemical modifications; an RNAi agent may include substantial modifications in multiple nucleotides. As used herein, the term “modified nucleotide” independently refers to a nucleotide having a modified sugar moiety, a modified internucleotide bond, and / or a modified nucleic acid base. Thus, the term modified nucleotide includes, for example, the substitution, addition, or removal of functional groups or atoms to internucleoside bonds, sugar moieties, or nucleic acid bases. Modifications suitable for use in the agents of the present invention include any type of modification disclosed herein or known in the art. Any such modification when used in siRNA-type molecules is encompassed by “RNAi agent” for the purposes of this specification and the claims.

[0143] The double-stranded region may be of any length that allows for the specific degradation of the desired target RNA via the RISC pathway, and can range from approximately 9 to 36 base pairs in length, for example, in the range of approximately 15 to 30 base pairs, for example, approximately 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, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26 Lengths of approximately 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, such as 21-25, 21-24, 21-23, or 21-22 base pairs. Intermediate ranges and lengths between those described above are also considered to be part of the present invention.

[0144] In embodiments in which a first dsRNA agent targeting LDHA and a second dsRNA agent targeting HAO1 are covalently bonded (i.e., a dual-targeted RNAi agent), the lengths of the double-stranded regions of the first agent and the second agent may be the same or different.

[0145] The two strands forming the double-stranded structure may be different parts of one larger RNA molecule, or they may be separate RNA molecules. When the two strands are part of one larger molecule and are therefore joined by a contiguous chain of nucleotides between the 3' end of one strand and the 5' end of the other, the joining RNA strands are called a “hairpin loop.” A hairpin loop may contain at least one unpaired nucleotide. In some embodiments, a hairpin loop may contain at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least twenty, at least 23 or more unpaired nucleotides.

[0146] In embodiments in which a first dsRNA agent targeting LDHA and a second dsRNA agent targeting HAO1 are covalently bonded (i.e., a dual-targeting RNAi agent), the first dsRNA agent may contain a hairpin loop, the second dsRNA agent may contain a hairpin loop, or both the first and second dsRNA agents may independently contain a hairpin loop. Furthermore, in embodiments in which a first dsRNA agent targeting LDHA and a second dsRNA agent targeting HAO1 are covalently bonded (i.e., a dual-targeting RNAi agent), the first dsRNA agent may contain an unpaired nucleotide, the second dsRNA agent may contain an unpaired nucleotide, or both the first and second dsRNA agents may independently contain an unpaired nucleotide. When both the first and second dsRNA agents independently contain an unpaired nucleotide, the first and second dsRNA agents may contain the same or different numbers of unpaired nucleotides.

[0147] If the two substantially complementary strands of a dsRNA are composed of separate RNA molecules, these molecules may or may not be covalently linked. If the two strands are covalently linked by means other than a contiguous chain of nucleotides between the 3' end of one strand and the 5' end of the other, the linking structure is called 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 the overhangs present in the double helix. In addition to the double helix structure, RNAi agents may contain one or more nucleotide overhangs.

[0148] In one embodiment, the RNAi agent of the present invention is a dsRNA, each of which contains 19 to 23 nucleotides that interact with a target RNA sequence, for example, an LDHA target mRNA sequence, to lead to the cleavage of the target RNA. In another embodiment, the RNAi agent of the present invention is a dsRNA, each of which contains 19 to 23 nucleotides that interact with a target RNA sequence, for example, an HAO1 target mRNA sequence, to lead to the cleavage of the target RNA. In yet another embodiment, the RNAi agent of the present invention comprises a first dsRNA agent (each of which contains 19 to 23 nucleotides that interact with a target RNA sequence, for example, an LDHA target mRNA sequence, to lead to the cleavage of the target RNA) and a second dsRNA agent (each of which independently contains 19 to 23 nucleotides that interact with a target RNA sequence, for example, an HAO1 target mRNA sequence, to lead to the cleavage of the target RNA), wherein the first and second dsRNA agents are covalently bonded.

[0149] In embodiments in which a first dsRNA agent targeting LDHA and a second dsRNA agent targeting HAO1 are covalently bonded (i.e., a dual-targeting RNAi agent), the two strands of the first dsRNA agent may be covalently bonded by means other than a contiguous chain of nucleotides between the 3' end of one strand forming a double-stranded structure and the 5' end of the other strand; the two strands of the second dsRNA agent may be covalently bonded by means other than a contiguous chain of nucleotides between the 3' end of one strand forming a double-stranded structure and the 5' end of the other strand; or the two strands of the first dsRNA agent and the two strands of the second dsRNA agent may be independently covalently bonded by means other than a contiguous chain of nucleotides between the 3' end of one strand forming a double-stranded structure and the 5' end of the other strand.

[0150] As used herein, the term “nucleotide overhang” refers to at least one unpaired nucleotide that protrudes from the double-stranded structure of an iRNA, such as a dsRNA. For example, a nucleotide overhang exists if the 3' end of one strand of a dsRNA extends beyond the 5' end of the other strand, or vice versa. A dsRNA may contain an overhang of at least one nucleotide; or the overhang may contain at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more. A nucleotide overhang may contain or consist of a nucleotide / nucleoside analog containing a deoxynucleotide / nucleoside. The overhang may be the sense strand, the antisense strand, or any combination thereof. Furthermore, the nucleotides of the overhang may be located at the 5' end, the 3' end, or both ends of either the antisense strand or the sense strand of the dsRNA.

[0151] In embodiments in which a first dsRNA agent targeting LDHA and a second dsRNA agent targeting HAO1 are covalently bonded (i.e., a dual-targeting RNAi agent), the first agent may contain a nucleotide overhang, the second agent may contain a nucleotide overhang, or both the first and second agents may independently contain a nucleotide overhang, for example, the 5' end of the sense strand of the first agent may contain an overhang, the 3' end of the sense strand of the first agent may contain an overhang, the 5' end of the antisense strand of the first agent may contain an overhang, the 3' end of the antisense strand of the first agent may contain an overhang, and The 5' and 3' ends of the sense chain of agent 1 may include overhangs, the 5' and 3' ends of the antisense chain of the first agent may include overhangs, the 5' end of the sense chain of the second agent may include overhangs, the 3' end of the sense chain of the second agent may include overhangs, the 5' end of the antisense chain of the second agent may include overhangs, the 3' end of the antisense chain of the second agent may include overhangs, the 5' and 3' ends of the sense chain of the second agent may include overhangs, the 5' and 3' ends of the antisense chain of the second agent may include overhangs, or any combination thereof.

[0152] In embodiments in which a first dsRNA agent targeting LDHA and a second dsRNA agent targeting HAO1 are covalently bonded (i.e., a dual-targeting RNAi agent), the lengths of the overhangs of the first agent and the second agent may be the same or different.

[0153] In one embodiment, the antisense strand of the dsRNA has an overhang of 1 to 10 nucleotides at its 3' and / or 5' ends, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In another embodiment, one or more nucleotides in the overhang are substituted with nucleoside thiophosphates.

[0154] In certain embodiments, the overhang on the sense strand, the antisense strand, or both may include an extended length longer than 10 nucleotides, for example, 10-30 nucleotides, 10-25 nucleotides, 10-20 nucleotides, or 10-15 nucleotides. In certain embodiments, the extended overhang is located on the double-stranded sense strand. In certain embodiments, the extended overhang is located on the 3' end of the double-stranded sense strand. In certain embodiments, the extended overhang is located on the 5' end of the double-stranded sense strand. In certain embodiments, the extended overhang is located on the double-stranded antisense strand. In certain embodiments, the extended overhang is located on the 3' end of the double-stranded antisense strand. In certain embodiments, the extended overhang is located on the 5' end of the double-stranded antisense strand. In certain embodiments, one or more nucleotides in the extended overhang are substituted with a nucleoside thiophosphate.

[0155] In embodiments in which a first dsRNA agent targeting LDHA and a second dsRNA agent targeting HAO1 are covalently bonded (i.e., a dual-targeting RNAi agent), one and / or both strands of both the first and second dsRNA agents independently include overhangs, e.g., extended overhangs, the lengths of which may be the same or different, and / or, in some embodiments, one or more nucleotides in the overhangs in the first dsRNA agent and one or more nucleotides in the overhangs in the second dsRNA agent may independently be substituted with nucleoside thiophosphates.

[0156] As used herein in relation to dsRNA, the terms “blunt” or “blunt-ended” mean that there are no unpaired nucleotides or nucleotide analogs at a given end of the dsRNA; that is, there are no nucleotide overhangs. One or both ends of a dsRNA may be blunt. If both ends of a dsRNA are blunt, it is described as blunt-ended. For clarity, a “blunt-ended” dsRNA is one in which both ends are blunt, i.e., there are no nucleotide overhangs at either end of the molecule. Most such molecules are often double-stranded along their entire length.

[0157] In embodiments in which a first dsRNA agent targeting LDHA and a second dsRNA agent targeting HAO1 are covalently bonded (i.e., a dual-targeting RNAi agent), one or both of the dsRNA agents may independently contain blunt ends.

[0158] The terms "antisense strand" or "guide strand" refer to a strand of iRNA, such as dsRNA, that contains a region substantially complementary to the target sequence, such as LDHA mRNA or HAO1 mRNA.

[0159] As used herein, the term “complementary region” refers to a region of the antisense strand that is substantially complementary to a sequence, such as a target sequence, such as an LDHA nucleotide sequence or an HAO1 nucleotide sequence, as defined herein. If the complementary region is not perfectly complementary to the target sequence, mismatches may exist in the internal or terminal regions of the molecule. Generally, most acceptable mismatches are located in terminal regions, for example, in the 5, 4, 3, or 2 nucleotides of the 5' and / or 3' ends of an iRNA.

[0160] In embodiments in which a first dsRNA agent targeting LDHA and a second dsRNA agent targeting HAO1 are covalently bonded (i.e., a dual-targeting RNAi agent), one or both of the dsRNA agents may independently contain a mismatch.

[0161] As used herein, the terms “sense strand” or “passenger strand” refer to a strand of iRNA that contains a region substantially complementary to the antisense strand region, as defined herein.

[0162] As used herein, the term “cleavage region” refers to a region located directly adjacent to a cleavage site. A cleavage site is the site in the target where cleavage occurs. In some embodiments, the cleavage region includes three bases directly adjacent to the cleavage site at either end of the cleavage site. In some embodiments, the cleavage region includes two bases directly adjacent to the cleavage site at either end of the cleavage site. In some embodiments, the cleavage site occurs specifically at a site bound by nucleotides 10 and 11 of the antisense strand, and the cleavage region includes nucleotides 11, 12, and 13.

[0163] Where used herein, unless otherwise specified, the term “complementary” means, as understood by those skilled in the art, the ability of an oligonucleotide or polynucleotide containing a first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide containing a second nucleotide sequence under given conditions to form a double-stranded structure, when used to describe a first nucleotide sequence in relation to a second nucleotide sequence. Such conditions may be, for example, stringent conditions, which may include 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, e.g., “Molecular Cloning: A Laboratory Manual,” Sambrook, et al. (1989), Cold Spring Harbor Laboratory Press). Other conditions, such as physiologically relevant conditions that may occur within living organisms, may be applied. Those skilled in the art will be able to determine the most appropriate set of conditions for testing the complementarity of the two sequences, depending on the final use of the hybridized nucleotides.

[0164] In the iRNAs described herein, for example, complementary sequences in dsRNAs include base pairings of one or both nucleotide sequences over the full length of one or both nucleotide sequences of an oligonucleotide or polynucleotide containing a first nucleotide sequence to an oligonucleotide or polynucleotide containing a second nucleotide sequence. Such sequences may be referred to herein as “fully complementary” to each other. However, if the first sequence is referred to herein as “substantially complementary” to the second sequence, the two sequences may be fully complementary, or, when hybridized to double helixes of up to 30 base pairs, while retaining their ability to hybridize under optimal conditions for their final application, e.g., inhibition of gene expression via the RISC pathway, they may form one or more mismatched base pairs, but generally five or fewer, four or fewer, three or fewer, or two or fewer. However, if the two oligonucleotides are designed to form one or more single-stranded overhangs after hybridization, such overhangs shall not be considered mismatches for the purpose of determining complementarity. For example, a dsRNA comprising one oligonucleotide of 21 nucleotides and the other oligonucleotide of 23 nucleotides for the purposes described herein may be referred to as “fully complementary” if the longer oligonucleotide contains a 21-nucleotide sequence that is fully complementary to the shorter oligonucleotide.

[0165] As used herein, “complementary” sequences may include, or may be entirely formed from, non-Watson-Crick base pairs and / or non-natural and modified nucleotides, provided that the above requirements related to their hybridizing ability are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U fluctuations or Hoogsteen-type base pairs.

[0166] The terms “complementary,” “fully complementary,” and “substantially complementary” as used herein may be used in relation to matching bases between the sense strand and antisense strand of a dsRNA, or between the antisense strand of a dsRNA and a target sequence, as can be understood from the context in which they are used.

[0167] As used herein, a polynucleotide "substantially complementary to at least a portion of" messenger RNA (mRNA) means a polynucleotide substantially complementary to a contiguous portion of the mRNA of interest (e.g., mRNA encoding LDHA or mRNA encoding HAO1) ​​including the 5'UTR, open reading frame (ORF), or 3'UTR. For example, a polynucleotide is complementary to at least a portion of LDHA mRNA if its sequence is substantially complementary to a contiguous portion of LDHA mRNA.

[0168] Accordingly, in one embodiment, the antisense strand polynucleotide disclosed herein is fully complementary to the target LDHA sequence. In another embodiment, the antisense strand polynucleotide disclosed herein is substantially complementary to the target LDHA sequence and comprises a continuous nucleotide sequence that is at least about 80% complementary, for example, 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 to the corresponding region of the nucleotide sequence of SEQ ID NO: 1 or a fragment of SEQ ID NO: 1 over its entire length.

[0169] In one embodiment, the RNAi agent of the present invention comprises a sense strand substantially complementary to the antisense polynucleotide, and consequently complementary to the target LDHA sequence, wherein the sense strand polynucleotide comprises a continuous nucleotide sequence that, over its entire length, is at least about 80% complementary, for example, 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 to the corresponding region of the nucleotide sequence of SEQ ID NO: 2 or any one of the fragments of SEQ ID NO: 2.

[0170] In one embodiment, the iRNA of the present invention comprises an antisense strand substantially complementary to a target LDHA sequence, and throughout its entire length comprises a continuous nucleotide sequence that is at least about 80% complementary, for example, about about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementary, or 100% complementary, to the corresponding region of the nucleotide sequence of any one of the sense strands in any one of Tables 2-5, or a fragment of any one of the sense strands in any one of Tables 2-5.

[0171] Accordingly, in one embodiment, the antisense strand polynucleotide disclosed herein is fully complementary to the target HAO1 sequence. In another embodiment, the antisense strand polynucleotide disclosed herein is substantially complementary to the target HAO1 sequence and comprises a continuous nucleotide sequence that is at least about 80% complementary, for example, 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 to the corresponding region of the nucleotide sequence of SEQ ID NO: 21 or a fragment of SEQ ID NO: 21 over its entire length.

[0172] In one embodiment, the RNAi agent of the present invention comprises a sense strand substantially complementary to the antisense polynucleotide, and consequently complementary to the target HAO1 sequence, wherein the sense strand polynucleotide comprises a continuous nucleotide sequence that, over its entire length, is at least about 80% complementary, for example, 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 to the corresponding region of the nucleotide sequence of SEQ ID NO: 22 or any one fragment of SEQ ID NO: 22.

[0173] In one embodiment, the iRNA of the present invention comprises an antisense strand substantially complementary to a target HAO1 sequence, and throughout its entire length comprises a continuous nucleotide sequence that is at least about 80% complementary, for example, about about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementary, or 100% complementary, to the corresponding region of the nucleotide sequence of any one of the sense strands in any one of Tables 7-14, or a fragment of any one of the sense strands in any one of Tables 7-14.

[0174] As used herein, the term “inhibiting” is synonymous with “reducing,” “silencing,” “downregulating,” “suppressing,” and other similar terms, and includes any level of inhibition.

[0175] As used herein, the phrase "inhibit LDHA gene expression" includes inhibiting the expression of any LDHA gene (e.g., mouse LDHA gene, rat LDHA gene, monkey LDHA gene, or human LDHA gene) as well as variants or mutants of LDHA genes that encode LDHA protein.

[0176] "Inhibiting LDHA gene expression" includes at least partial suppression of LDHA gene expression, such as inhibition at any level of LDHA gene expression, e.g., 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%.

[0177] As used herein, the phrase "inhibit the expression of the HAO1 gene" includes inhibiting the expression of any HAO1 gene (e.g., mouse HAO1 gene, rat HAO1 gene, monkey HAO1 gene, or human HAO1 gene) as well as mutants or mutants of the HAO1 gene that encode the HAO1 protein.

[0178] "Inhibiting HAO1 gene expression" includes at least partial suppression of HAO1 gene expression, such as inhibition at any level of HAO1 gene expression, e.g., 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%.

[0179] In embodiments in which a first dsRNA agent targeting LDHA and a second dsRNA agent targeting HAO1 are covalently bonded, the inhibition of LDHA expression may be the same as or different from the inhibition of HAO1 expression.

[0180] The expression of the LDHA gene and / or HAO1 gene may be assessed based on the levels of any variable related to LDHA gene expression and / or HAO1 gene expression, e.g., LDHA and / or HAO1 mRNA levels or LDHA and / or HAO1 protein levels. LDHA gene and / or HAO1 gene expression may also be indirectly assessed based on the levels of oxalates or glycolates in urine, plasma, or tissue samples, or on the enzymatic activity of LDHA in tissue samples, e.g., liver samples, skeletal muscle samples, and / or cardiac samples. Inhibition may be assessed by a decrease in the absolute or relative levels of one or more of these variables compared to control levels. Control levels may be levels of any type of control used in the art, e.g., pre-dose baseline levels, or levels measured from similar subjects, cells, or samples that have been untreated or treated with a control (e.g., a buffer-only control or an inactive agent control).

[0181] In one embodiment, at least partial suppression of LDHA gene expression is assessed by a decrease in the amount of LDHA mRNA that can be isolated from or detected in a first cell or cell group that has been transcribed and treated to inhibit LDHA gene expression, compared to a second cell or cell group (control cells) that is substantially identical to the first cell or cell group except that it has not been treated in this way.

[0182] In one embodiment, at least partial suppression of HAO1 gene expression is assessed by a decrease in the amount of HAO1 mRNA that can be isolated from or detected in a first cell or cell group that has been transcribed and treated to inhibit HAO1 gene expression, compared to a second cell or cell group (control cell) that is substantially identical to the first cell or cell group except that it has not been treated in this way.

[0183] In one embodiment, at least partial suppression of LDHA gene and HAO1 gene expression is evaluated by a decrease in the amounts of LDHA mRNA and HAO1 mRNA that can be isolated from or detected in a first cell or cell group that has been transcribed and treated to inhibit the expression of LDHA gene and HAO1 gene, compared to a second cell or cell group (control cell) that is substantially identical to the first cell or cell group except that it has not been treated in this way.

[0184] The degree of inhibition can be expressed by the following formula.

number

[0185] As used herein, the phrase "contacting cells with an RNAi agent (such as dsRNA)" includes the step of contacting cells by any possible means. The step of contacting cells with an RNAi agent includes the step of contacting cells with iRNA in vitro or the step of contacting cells with iRNA in vivo. Contact may be direct or indirect. For example, an RNAi agent may be physically contacted with cells by performing the method individually, or the RNAi agent may be made available for subsequent contact with cells or placed in a situation that allows for subsequent contact with cells.

[0186] In the method of the present invention in which a first dsRNA agent targeting LDHA and a second dsRNA agent targeting HAO1 are covalently bonded (i.e., a dual-targeted RNAi agent), the step of contacting cells may include a step of contacting cells with the first agent at the same time as or at a different time than the step of contacting cells with the second agent.

[0187] The process of contacting cells in vitro may be carried out, for example, by incubating the cells with an RNAi agent. The process of contacting cells in vivo may be carried out, 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 region, for example, the bloodstream or subcutaneous space, so that the RNAi agent subsequently reaches the tissue where the cells being contacted are located. For example, the RNAi agent may contain and / or be bound to a ligand that directs the RNAi agent to a target site, for example, the liver, such as GalNAc3. Combinations of in vitro and in vivo contact methods are also possible. For example, cells may also be contacted with an RNAi agent in vitro and then transplanted into a subject.

[0188] In one embodiment, the step of contacting cells with iRNA includes the step of “introducing” or “delivering iRNA into cells” by promoting or performing uptake or absorption into the cells. Absorption or uptake of iRNA may be carried out by unaided diffusive or active cellular processes, or by auxiliary drugs or devices. Introduction of iRNA into cells may be in vitro and / or in vivo. For example, in the case of in vivo introduction, iRNA may be injected into a tissue site or administered systemically. In vivo delivery may also be carried out by β-glucan delivery systems such as those described in U.S. Patent Nos. 5,032,401 and 5,607,677 and U.S. Patent Application Publication 2005 / 0281781, the entire contents of which are incorporated herein by reference. In vitro introduction into cells includes methods known in the art, such as electroporation and lipofection. Further methods are described later in this specification and / or are known in the art.

[0189] The terms “lipid nanoparticles” or “LNPs” refer to vesicles containing a lipid layer that encapsulates a nucleic acid molecule, such as iRNA or a plasmid on which iRNA is transcribed, which is pharmaceutically effective. LNPs are described, for example, in U.S. Patents No. 6,858,225, No. 6,815,432, No. 8,158,601, and No. 8,058,069, which are incorporated herein by reference in their entirety.

[0190] As used herein, “Subject” refers to animals such as primates (humans, non-human primates, e.g., monkeys and chimpanzees), mammals including non-primates (cattle, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, mice, horses and whales), or birds (e.g., ducks or geese).

[0191] In one embodiment, the subjects are people who are being treated or evaluated for a disease, disorder or condition for which a reduction in LDHA expression may be beneficial, as described herein; people at risk of a disease, disorder or condition for which a reduction in LDHA expression may be beneficial; people suffering from a disease, disorder or condition for which a reduction in LDHA expression may be beneficial; and / or people who are being treated for a disease, disorder or condition for which a reduction in LDHA expression may be beneficial.

[0192] It should be understood that a person being treated or evaluated for a disease, disorder, or condition in which a reduction in LDHA expression may be beneficial includes a person being treated or evaluated for a disease, disorder, or condition in which a reduction in LDHA and HAO1 expression may be beneficial; a person at risk of a disease, disorder, or condition in which a reduction in LDHA expression may be beneficial includes a person at risk of a disease, disorder, or condition in which a reduction in LDHA and HAO1 expression may be beneficial; a person suffering from a disease, disorder, or condition in which a reduction in LDHA expression may be beneficial includes a person at risk of a disease, disorder, or condition in which a reduction in LDHA and HAO1 expression may be beneficial; and a person being treated for a disease, disorder, or condition in which a reduction in LDHA expression may be beneficial includes a person being treated for a disease, disorder, or condition in which a reduction in LDHA and HAO1 expression may be beneficial as described herein.

[0193] As used herein, the terms “to treat” or “treatment” refer to a beneficial or desired outcome, such as reducing the urinary excretion level of oxalates in the subject. The terms “to treat” or “treatment” also include, but are not limited to, the alleviation or improvement of one or more symptoms of diseases, disorders, or conditions related to the oxalate pathway, such as slowing disease progression; reducing the severity of later-developing diseases; reducing edema of the limbs, face, larynx, upper respiratory tract, abdomen, torso, and / or genitals; prodromal symptoms, laryngeal edema, non-pruritic rash, nausea, vomiting, and / or abdominal pain; reducing the progression of liver disease to cirrhosis or hepatocellular carcinoma; stabilizing the current lithotripsy load; reducing the recurrence of lithotripsy formation; and / or preventing further oxalate tissue deposition. “Treatment” may also mean an extension of survival compared to the predicted survival time without treatment.

[0194] The term “reduce” in relation to disease markers or symptoms refers to a statistically significant reduction of such levels. The reduction may 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, and is preferably reduced to a level that is considered within the normal range for individuals without such impairment.

[0195] As used herein, “prevention” or “prevention” means, when used in relation to a disease, disorder, or condition for which a reduction in LDHA gene expression may be beneficial, a reduction in the likelihood of developing symptoms associated with such disease, disorder, or condition, such as lithotripsy. For example, the likelihood of lithotripsy is reduced to the extent that, for example, an individual with one or more risk factors for lithotripsy does not develop lithotripsy, or develops milder lithotripsy compared to a population with the same risk factors but who does not receive the treatment described herein. Effective prevention is considered to be the absence of disease, disorder, or condition, or a reduction in the occurrence of symptoms associated with such disease, disorder, or condition (e.g., at least about 10% on a clinically recognized scale of the disease or disorder), or a delay in the appearance of symptoms (e.g., only a few days, weeks, months, or years).

[0196] There are many disorders, conditions, or pathologies related to the oxalate pathway that may benefit from reduced expression of the LDHA gene.

[0197] As used herein, the term “disease, disorder, or condition related to the oxalate pathway” means a disease, disorder, or condition related to, or caused by, a disruption of lactate dehydrogenase production and / or urinary oxalate production, for example, for which lactate dehydrogenase knockdown is known or predicted to be therapeutically effective or otherwise beneficial.

[0198] In one embodiment, “a disease, disorder, or condition related to the oxalate pathway” is “a disease, disorder, or condition related to lactate dehydrogenase.” As used herein, “a disease, disorder, or condition related to lactate dehydrogenase” includes any disease, disorder, or condition in which a reduction in lactate dehydrogenase gene expression, replication, or protein activity may be beneficial. Exemplary diseases, disorders, and conditions related to lactate dehydrogenase include, for example, fatty liver (steatosis), non-alcoholic steatohepatitis (NASH), cirrhosis, accumulation of fat in the liver, inflammation of the liver, hepatocyte necrosis, hepatic fibrosis, obesity, non-alcoholic fatty liver disease (NAFLD), and cancer, such as hepatocellular carcinoma.

[0199] In another embodiment, “oxalate pathway-related disease, disorder, or condition” is “oxalate-related disease, disorder, or condition.” As used herein, “oxalate-related disease, disorder, or condition” includes any disease, disorder, or condition that may benefit from reduced lactate dehydrogenase gene expression, replication, or protein activity. The term “oxalate-related disease, disorder, or condition” refers to hereditary or induced or acquired diseases. Exemplary “oxalate-related disease, disorder, or condition” include “kidney stone formation disease, disorder, and condition” and “calcium oxalate tissue deposition disease, disorder, and condition.”

[0200] Exemplary kidney stone formation diseases, disorders, and conditions include "calcium oxalate stone formation diseases, disorders, and conditions" and "non-calcium oxalate stone formation diseases, disorders, and conditions."

[0201] Non-limiting examples of “calcium oxalate stone formation diseases, disorders, and conditions” include hyperoxaluria (e.g., primary hyperoxaluria such as primary hyperoxaluria 1 (PH1), primary hyperoxaluria 2 (PH2), primary hyperoxaluria 3 (PH3), and non-PH1 / PH2 / PH3; intestinal hyperoxaluria; dietary hyperoxaluria; and idiopathic hyperoxaluria) and non-hyperoxaluria disorders (e.g., hypercalciuria such as primary hyperparathyroidism, Dent's disease, absorptive hypercalciuria, and renal hypercalciuria; and hypocitrateuria).

[0202] Non-extended examples of “non-calcium oxalate stone formation diseases, disorders, and conditions” include subjects with kidney stones composed of less than approximately 50%, less than approximately 45%, less than approximately 40%, less than approximately 35%, less than approximately 30%, less than approximately 25%, less than approximately 20%, less than approximately 15%, or less than approximately 10% oxalates, and more than approximately 50% non-oxalates, such as calcium phosphate, uric acid, struvite, cystinuria, or other components.

[0203] Exemplary “calcium oxalate tissue deposition diseases, disorders, and conditions” include systemic calcium oxalate tissue deposition diseases, disorders, and conditions, e.g., calcium oxalate tissue deposition resulting from end-stage renal disease, sarcoidosis, or arthritis; and tissue-specific calcium oxalate deposition diseases, disorders, and conditions resulting from organ transplantation, such as kidney transplantation, e.g., in the kidney (e.g., resulting from nephrocalcemia or spongiform kidney), in the thyroid gland, in the breast, in the bone, in the heart, in the vascular system, or in any soft tissue.

[0204] As used herein, “therapeutic dose” is intended to include an amount of RNAi agent sufficient to treat a disease, disorder, or condition associated with the oxalate pathway when administered to a subject with such a condition (for example, by reducing, improving, or maintaining the symptoms of an existing disease or one or more of the symptoms of the disease). “Therapeutic dose” may vary depending on the RNAi agent, the method of administration, the disease and its severity, and the subject's medical history, age, weight, family history, genetic makeup, any previous or concomitant treatments, and other individual characteristics.

[0205] As used herein, “a prophylactically effective dose” is intended to contain an amount of iRNA sufficient to prevent or improve a disease or one or more symptoms of a disease, disorder, or condition associated with the oxalate pathway when administered to a subject. Improvement of the disease includes delaying the course of the disease or reducing the severity of any subsequent symptoms. “A prophylactically effective dose” may vary depending on the iRNA, the method of drug administration, the risk of developing the disease, and the patient’s medical history, age, weight, family history, genetic makeup, any previous or concomitant treatments, and other individual characteristics.

[0206] The “therapeutic effective dose” or “prophylactically effective dose” also includes the amount of RNAi agent that produces the desired local or systemic effect, which is a reasonable benefit-risk ratio applicable to any treatment. The iRNA used in the method of the present invention may be administered in an amount sufficient to obtain a reasonable benefit-risk ratio applicable to such treatment.

[0207] In a method of the present invention, comprising the step of administering a pharmaceutical composition comprising a first dsRNA agent targeting LDHA and a second dsRNA agent targeting HAO1, the therapeutically effective dose of the first dsRNA agent may be the same as or different from the therapeutically effective dose of the second dsRNA agent. Similarly, in a method of the present invention, comprising the step of administering a pharmaceutical composition comprising a first dsRNA agent targeting LDHA and a second dsRNA agent targeting HAO1, the prophylactically effective dose of the first dsRNA agent may be the same as or different from the prophylactically effective dose of the second dsRNA agent.

[0208] The phrase "pharmaceutically acceptable" is used herein to mean compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with the tissues of human and animal subjects within reasonable medical judgment and in proportion to a reasonable benefit-risk ratio, without causing excessive toxicity, irritation, allergic reactions, or other problems or complications.

[0209] As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc, magnesium stearate, calcium stearate, or zinc stearate, or stearic acid), or solvent encapsulating material, that is involved in transporting or carrying a compound to a subject from one organ or part of the body to another organ or part of the body. Each carrier must be “acceptable” in the sense that it is compatible with the other components of the formulation and is not harmful 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) celluloses such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate, and their derivatives; (4) tragacanth powder; (5) malt; (6) gelatin; (7) lubricants such as magnesium state, sodium lauryl sulfate, and talc; (8) excipients such as cocoa butter and suppository wax; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) propylene glycol Examples include glycols such as (11) polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) distilled water from which pyrogenic substances have been removed; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffer; (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 non-toxic, suitable substances used in pharmaceutical preparations.

[0210] As used herein, the term “sample” includes similar bodily fluids, cells, or tissues isolated from a subject, as well as aggregates of bodily fluids, cells, or tissues present in the subject. Examples of bodily fluids include blood, serum and serous fluid, plasma, cerebrospinal fluid, ocular fluid, lymph, urine, and saliva. Tissue samples may include samples derived from tissue, organ, or local area. For example, a sample may be derived from a specific organ, a part of an organ, or bodily fluids or cells within those organs. In certain embodiments, a sample may be derived from the liver (e.g., the whole liver or a specific part of the liver, or a specific type of cell in the liver, such as hepatocytes). In some embodiments, “sample derived from subject” refers to blood or plasma obtained from the subject.

[0211] II. The iRNA of the present invention iRNAs that inhibit the expression of target genes are described herein. In one embodiment, the iRNA inhibits the expression of the LDHA gene. In one embodiment, the iRNA agent comprises a double-stranded ribonucleic acid (dsRNA) molecule for inhibiting the expression of the LDHA gene in hepatocytes in cells, e.g., hepatocytes, e.g., subjects suffering from diseases, disorders, or conditions related to the oxalate pathway, e.g., kidney stone formation diseases, disorders, or conditions, e.g., mammals, e.g., humans. In another embodiment, the iRNA inhibits the expression of the HAO1 gene. In one embodiment, the iRNA agent comprises a double-stranded ribonucleic acid (dsRNA) molecule for inhibiting the expression of the HAO1 gene in cells, e.g., hepatocytes, e.g., subjects suffering from diseases, disorders, or conditions related to the oxalate pathway, e.g., oxalate-related diseases, disorders, or conditions, e.g., kidney stone formation diseases, disorders, or conditions, or calcium oxalate tissue deposition diseases, disorders, or conditions; or LDH-related diseases, disorders, or conditions, e.g., mammals, e.g., humans.

[0212] iRNAs that inhibit the expression of two target genes, known as dual-targeting RNAi agents, are also provided herein. In one embodiment, the dual-targeting RNAi agent comprises a first double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of the LDHA gene in cells (hepatocytes, e.g., hepatocytes in the subject) covalently bound to a second double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of the HAO1 gene in cells (hepatocytes, e.g., hepatocytes in the subject) for a subject suffering from, for example, a disease, disorder, or condition related to the oxalate pathway, e.g., a disease, disorder, or condition related to oxalate, e.g., a disease, disorder, or condition related to kidney stone formation, e.g., a disease, disorder, or condition related to LDH, e.g., a mammal, e.g., human).

[0213] dsRNA contains an antisense strand having a complementary region that is complementary to at least a portion of the mRNA formed during the expression of the LDHA gene or HAO1 gene. The complementary region is approximately 30 nucleotides or less in length (e.g., approximately 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 nucleotides or less). When in contact with cells expressing the target gene, iRNA inhibits the expression of the target gene (e.g., human, primate, non-primate, or avian target gene) by at least approximately 10% when assayed by methods such as PCR or branched DNA (bDNA) based methods, or by protein-based methods such as immunofluorescence analysis using Western blotting or flow cytometry.

[0214] A dsRNA contains two RNA strands, which are complementary and hybridize to form a double-stranded structure under the conditions in which the dsRNA is used. One strand of the dsRNA (the antisense strand) contains a region of complementarity that is substantially complementary to the target sequence, generally fully complementary. The target sequence may be derived from the mRNA sequence formed during the expression of the LDHA gene or the HAO1 gene. The other strand (the sense strand) contains a region complementary to the antisense strand, and the two strands hybridize to form a double-stranded structure when combined under suitable conditions. As described elsewhere in this specification and as is known in the art, the complementary sequence of the dsRNA may also be included as a self-complementary region of a single nucleic acid molecule, rather than on a separate oligonucleotide.

[0215] Generally, double-stranded structures are 15-30 base pairs long, 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, 19-2 The lengths are 9, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. Intermediate ranges and lengths within the above ranges and lengths are also considered to be part of the present invention.

[0216] Similarly, the complementary region of the target sequence is 15-30 nucleotides long, e.g., 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, 1 The nucleotide lengths are 9-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides. Intermediate ranges and lengths within the above ranges and lengths are also considered to be part of the present invention.

[0217] In some embodiments, the dsRNA is about 15–23 nucleotides long, or about 25–30 nucleotides long. Generally, dsRNA is long enough to serve as a substrate for the Dicer enzyme. For example, it is well known in the art that dsRNA longer than about 21–23 nucleotides can serve as a substrate for Dicer. As those skilled in the art will also recognize, the RNA region targeted for cleavage is in most cases part of a larger RNA molecule (often an mRNA molecule). Where applicable, the “part” of the mRNA target is a contiguous sequence of mRNA targets long enough to be a substrate for RNAi-directed cleavage (i.e., cleavage via the RISC pathway).

[0218] The double-stranded region is the primary functional part of dsRNA, for example, approximately 9-36 base pairs, for example, approximately 10-36, 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, 14-34, 15-34, 9-3 3, 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, 15-2 4, 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, 19-25, 19-24, 19-23, 1 Those skilled in the art will also recognize that the double-stranded region is 9-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. Herein, in one embodiment, a complex of an RNA molecule or an RNA molecule having a double-stranded region of more than 30 base pairs is dsRNA to the extent that it is processed into a functional double-strand of, for example, 15-30 base pairs that targets the desired RNA for cleavage. Thus, those skilled in the art will recognize that in one embodiment, miRNA is dsRNA. In another embodiment, dsRNA is not a natural miRNA. In another embodiment, iRNA agents useful for targeting LDHA expression or LDHA and HAO1 expression are not generated in target cells by cleavage of larger dsRNAs.

[0219] The dsRNAs described herein may further include one or more single-stranded nucleotide overhangs, e.g., 1, 2, 3, or 4 nucleotides. dsRNAs having at least one nucleotide overhang may have unexpectedly superior inhibitory properties compared to their blunt-ended counterparts. Nucleotide overhangs may contain or consist of nucleotide / nucleoside analogs, including deoxynucleotides / nucleosides. Overhangs may be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the nucleotides of the overhang may be located on the 5' end, 3' end, or both ends of either the antisense strand or the sense strand of the dsRNA.

[0220] dsRNA can be synthesized by standard methods known in the art, for example, by using an automated DNA synthesizer (such as those commercially available from Biosearch, Applied Biosystems, Inc.), as will be further described later.

[0221] The iRNA compounds of the present invention can be prepared using a two-step procedure. First, the individual strands of a 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, solid-phase organic synthesis, or both. Organic synthesis offers the advantage that oligonucleotide strands containing non-natural or modified nucleotides can be readily prepared. The single-stranded oligonucleotides of the present invention can be prepared using solution-phase, solid-phase organic synthesis, or both.

[0222] In one embodiment, 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 shown in any one of Tables 2 to 5, and the nucleotide sequence corresponding to the antisense strand of the sense strand is selected from the group of sequences shown in any one of Tables 2 to 5. In this embodiment, one of the two sequences is complementary to the other of the two sequences, and one of the sequences is substantially complementary to the mRNA sequence produced during the expression of the LDHA gene. Thus, in this embodiment, the dsRNA comprises two oligonucleotides, where one oligonucleotide is represented as the sense strand (passenger strand) in any one of Tables 2 to 5, and the second oligonucleotide is represented as the corresponding antisense strand (guide strand) of the sense strand in any one of Tables 2 to 5. In one embodiment, the substantially complementary sequences of the dsRNA are contained in separate oligonucleotides. In another embodiment, the substantially complementary sequences of the dsRNA are contained in a single oligonucleotide.

[0223] In another embodiment, the dsRNA of the present invention targets the HAO1 gene and 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 any one of Tables 7 to 14, and the corresponding nucleotide sequence of the antisense strand of the sense strand is selected from the group of sequences in any one of Tables 7 to 14. In this embodiment, one of the two sequences is complementary to the other of the two sequences, and one of the sequences is substantially complementary to the sequence of mRNA produced in the expression of the HAO1 gene. Thus, in this embodiment, the dsRNA comprises two oligonucleotides, where one oligonucleotide is represented as the sense strand (passenger strand) in any one of Tables 7 to 14, and the second oligonucleotide is represented as the corresponding antisense strand (guide strand) of the sense strand in any one of Tables 7 to 14. In one embodiment, the substantially complementary sequence of the dsRNA is contained in another oligonucleotide. In another embodiment, the substantially complementary sequence of the dsRNA is contained in a single oligonucleotide.

[0224] The sequences in Tables 2-5 and 7-14 are represented as modified, unmodified, unconjugated, and / or conjugated sequences, but it will be understood that the RNA of the present invention, for example, the dsRNA of the present invention, may, unlike those described herein, include any one of the sequences described in any one of Tables 2-5 and 7-14, which are unmodified, unconjugated, and / or modified and / or conjugated.

[0225] Those skilled in the art are well aware that dsRNAs having a double-stranded structure of approximately 20–23 base pairs, for example, 21 base pairs, have been shown to be particularly effective in inducing RNA interference (Elbashir et al., (2001) EMBO J.,: 6877-6888). However, other those skilled in the art have found that shorter or longer RNA double-stranded structures may also be effective (Chu and Rana (2007) RNA 14: 1714-1719; Kim et al. (2005) Nat Biotech 23: 222-226). In the embodiments described herein, the properties of the oligonucleotide sequences described herein may include at least one strand of a minimum length of 21 nucleotides. It can be naturally predicted that shorter double-stranded structures, with a very small number of nucleotides subtracted from one or both ends, may be equally effective compared to the dsRNAs described herein. Therefore, dsRNAs having at least 15, 16, 17, 18, 19, 20, or more consecutive nucleotide sequences from one of the sequences shown herein, wherein the ability to inhibit the expression of the LDHA gene or HAO1 gene differs from that of a dsRNA containing the complete sequence by approximately 5, 10, 15, 20, 25, or 30% or less, are considered to be within the scope of the present invention.

[0226] Furthermore, the RNAs listed in any one of Tables 2-5 identify sites in the LDHA transcript that are susceptible to RISC-mediated cleavage, and the RNAs listed in any one of Tables 7-14 identify sites in the HAO1 transcript that are susceptible to RISC-mediated cleavage. Thus, the present invention further features iRNAs that target these sites. When used herein, an iRNA is said to target a specific site within the RNA transcript if it induces cleavage of the transcript at any location within its particular site. Such an iRNA would generally consist of at least about 15 consecutive nucleotides from one of the sequences shown herein, bound to a further nucleotide sequence taken from a region adjacent to a selected sequence in the gene.

[0227] Target sequences are generally about 15–30 nucleotides long, but the suitability of specific sequences within this range to guide the cleavage of any given target RNA varies. While the various software packages and guidelines described herein provide guidance for identifying the optimal target sequence for any given gene target, empirical methods can also be employed, where a “window” or “mask” of a given size (21 nucleotides as an example) is placed literally or figuratively (including in silico) on the target RNA sequence to identify sequences within a size range in which they can act as target sequences. The next potential target sequence can be identified by gradually shifting the sequence “window” one nucleotide upstream or downstream of the initial target sequence position until a complete set of possible sequences is identified for any given target size of choice. This process, along with the systematic synthesis and testing of identified sequences (using assays described herein or known in the art) to identify the optimally functioning sequence, can identify the RNA sequence that best mediates the inhibition of target gene expression when targeted with an iRNA agent. Therefore, for example, while the sequences identified herein represent effective target sequences, further optimization of inhibition efficiency can be achieved by gradually "shifting the window" one nucleotide upstream or downstream of a given sequence to identify sequences with equivalent or better inhibitory properties.

[0228] Furthermore, for example, further optimization may be achieved by systematically adding or removing nucleotides to any sequence identified herein to generate longer or shorter sequences, and by testing the resulting sequences by shifting the longer or shorter size window upward or downward from that point towards the target RNA. In addition, the efficiency of inhibition may be further improved by combining this method for generating novel candidate targets in inhibition assays known in the art and / or described herein with testing the efficacy of iRNAs based on those target sequences. Moreover, such optimized sequences may be modified, for example, by introducing modified nucleotides described herein or known in the art, adding or changing overhangs, or other modifications known in the art and / or described herein to further optimize the molecule as an expression inhibitor (e.g., increased serum stability or circulating half-life, increased thermal stability, improved membrane permeable delivery, targeting to a specific location or cell type, increased interaction with silencing pathway enzymes, increased release from endosomes).

[0229] The iRNA agents described herein may contain one or more mismatches with the target sequence. In one embodiment, the iRNA described herein contains three or fewer mismatches. When the antisense strand of the iRNA contains a mismatch with the target sequence, it is preferable that the mismatch region is not located in the center of the complementary region. When the antisense strand of the iRNA contains a mismatch with the target sequence, it is preferable that the mismatch is limited to the last five nucleotides from either the 5' or 3' end of the complementary region. For example, for a 23-nucleotide iRNA agent, the strand complementary to the LDHA gene or HAO1 gene region generally contains no mismatches in the central 13 nucleotides. Using the methods described herein or methods known in the art, it can be determined whether an iRNA containing a mismatch with the target sequence is effective in inhibiting the expression of the LDHA gene and / or HAO1 gene. Considering the effectiveness of mismatched iRNAs in inhibiting the expression of the LDHA gene and / or HAO1 gene is particularly important when specific complementary regions in the LDHA gene and / or HAO1 gene are known to have polymorphic sequence variations within the population.

[0230] The dual-targeting RNAi agent of the present invention, comprising two dsRNA agents, is covalently linked, for example, via a covalent linker. Covalent linkers are well known in the art and include, for example, nucleic acid linkers, peptide linkers, and carbohydrate linkers. The covalent linker may contain RNA and / or DNA and / or peptides. The linker may be single-stranded, double-stranded, partially single-stranded, or partially double-stranded. Modified nucleotides or mixtures of nucleotides may also be present in the nucleic acid linker.

[0231] Suitable linkers for use in the dual-targeting agent of the present invention include those described in U.S. Patent No. 9,187,746, which is incorporated herein by reference in its entirety.

[0232] In one embodiment, the linker includes a disulfide bond. The linker may be cleavable or incleavable.

[0233] The linker is, for example, dTsdTuu=(5'-2'deoxythymidyl-3'-thiophosphate-5'-2'deoxythymidyl-3'-phosphate-5'-uridyl-3'-phosphate-5'-uridyl-3'-phosphate);rUsrU(thiophosphate linker: 5'-uridyl-3'-thiophosphate-5'-uridyl-3'-phosphate);rUrU linker;dTsdTaa(aadTsdT, 5'-2'deoxythymidyl-3'-thiophosphate-5'-2' Deoxythymidyl-3'-phosphate-5'-adenyle-3'-phosphate-5'-adenyle-3'-phosphate);dTsdT(5'-2'deoxythymidyl-3'-thiophosphate-5'-2'deoxythymidyl-3'-phosphate);dTsdTuu=uudTsdT=5'-2'deoxythymidyl-3'-thiophosphate-5'-2'deoxythymidyl-3'-phosphate-5'-uridyl-3'-phosphate-5'-uridyl-3'-phosphate.

[0234] The linker may be poly(5'-adenyl-3'-phosphate-AAAAAAAA) or poly(5'-cytidyl-3'-phosphate-5'-uridyl-3'-phosphate-CUCUCUCU)), for example, a polyRNA such as an Xn single-stranded polyRNA linker, where n is an integer between 2 and 50, preferably between 4 and 15, and most preferably between 7 and 8. Modified nucleotides or mixtures of nucleotides may also be present in the polyRNA linker. The covalent linker may be polyDNA such as poly(5'-2'deoxythymidyl-3'-phosphate-TTTTTTTT), for example, where n is an integer between 2 and 50, preferably between 4 and 15, and most preferably between 7 and 8. Modified nucleotides or mixtures of nucleotides may also be present in the polyDNA linker, single-stranded polyDNA linker, where n is an integer between 7 and 8, preferably between 7 and 50, more preferably between 4. Modified nucleotides or mixtures of nucleotides may also be present in the polyDNA linker.

[0235] The linker may include a disulfide bond, optionally a bis-hexyl-disulfide linker. In one embodiment, the disulfide linker is [ka] That is the case.

[0236] The linker may contain peptide bonds, for example, amino acids. In one embodiment, the covalent linker is preferably a linker 1 to 10 amino acid long, containing 4 to 5 amino acids, optionally X-Gly-Phe-Gly-Y, where X and Y represent any amino acids.

[0237] The linker may include HEG and hexaethylene glycol linker.

[0238] The covalent linker may bind the sense strand of the first dsRNA agent to the sense strand of the second dsRNA agent; bind the antisense strand of the first dsRNA agent to the antisense strand of the second dsRNA agent; bind the sense strand of the first dsRNA agent to the antisense strand of the second dsRNA agent; or bind the antisense strand of the first dsRNA agent to the sense strand of the second dsRNA agent.

[0239] In one embodiment, the covalent linker further comprises at least one ligand as described below.

[0240] III. Modified iRNA of the present invention In one embodiment, the RNA of the iRNA of the present invention, e.g., dsRNA, is unmodified and does not contain, for example, any chemical modifications and / or conjugates known in the art and described herein. In another embodiment, the RNA of the iRNA of the present invention, e.g., dsRNA, is chemically modified to improve stability or other beneficial properties. In a particular embodiment of the present invention, substantially all of the nucleotides of the iRNA of the present invention are modified. In another embodiment of the present invention, all of the nucleotides of the iRNA of the present invention are modified. The iRNA of the present invention that "substantially all of its nucleotides are modified" is mostly modified but not completely modified and may contain five or fewer, four or fewer, three or fewer, two or fewer, or one or fewer unmodified nucleotides.

[0241] In embodiments in which a first dsRNA agent targeting LDHA and a second dsRNA agent targeting HAO1 are covalently bonded (i.e., a dual-targeting RNAi agent), substantially all of the nucleotides of the first agent and substantially all of the nucleotides of the second agent may be independently modified; all of the nucleotides of the first agent may be modified and all of the nucleotides of the second agent may be independently modified; substantially all of the nucleotides of the first agent and all of the nucleotides of the second agent may be independently modified; or all of the nucleotides of the first agent may be modified and substantially all of the nucleotides of the second agent may be independently modified.

[0242] In one embodiment of the present invention, substantially all of the nucleotides of the iRNA of the present invention are modified, and the iRNA agent contains 10 or fewer nucleotides including 2'-fluoro modifications (e.g., 9 or fewer 2'-fluoro modifications, 8 or fewer 2'-fluoro modifications, 7 or fewer 2'-fluoro modifications, 6 or fewer 2'-fluoro modifications, 5 or fewer 2'-fluoro modifications, 4 or fewer 2'-fluoro modifications, 5 or fewer 2'-fluoro modifications, 3 or fewer 2'-fluoro modifications, or 2 or fewer 2'-fluoro modifications). For example, in one embodiment, the sense strand contains 4 or fewer nucleotides including 2'-fluoro modifications (e.g., 3 or fewer 2'-fluoro modifications, or 2 or fewer 2'-fluoro modifications). In another embodiment, the antisense strand contains 6 or fewer nucleotides including 2'-fluoro modifications (e.g., 5 or fewer 2'-fluoro modifications, 4 or fewer 2'-fluoro modifications, or 2 or fewer 2'-fluoro modifications).

[0243] In embodiments in which a first dsRNA agent targeting LDHA and a second dsRNA agent targeting HAO1 are covalently bonded (i.e., a dual-targeting RNAi agent), substantially all of the nucleotides of the first agent and / or substantially all of the nucleotides of the second agent may be independently modified, and the first and second agents may independently contain 10 or fewer nucleotides including 2'-fluoro modifications.

[0244] In another aspect of the present invention, all nucleotides of the iRNA of the present invention are modified, and the iRNA agent contains 10 or fewer nucleotides including 2'-fluoro modifications (for example, 9 or fewer 2'-fluoro modifications, 8 or fewer 2'-fluoro modifications, 7 or fewer 2'-fluoro modifications, 6 or fewer 2'-fluoro modifications, 5 or fewer 2'-fluoro modifications, 4 or fewer 2'-fluoro modifications, 5 or fewer 2'-fluoro modifications, 4 or fewer 2'-fluoro modifications, 3 or fewer 2'-fluoro modifications, or 2 or fewer 2'-fluoro modifications).

[0245] In embodiments in which a first dsRNA agent targeting LDHA and a second dsRNA agent targeting HAO1 are covalently bonded (i.e., a dual-targeting RNAi agent), all nucleotides of the first agent and / or all nucleotides of the second agent may be independently modified, and the first and second agents may independently contain 10 or fewer nucleotides including 2'-fluoro modifications.

[0246] In one embodiment, the double-stranded RNAi agent of the present invention further comprises a 5'-phosphate or a 5'-phosphate mimetic in the 5' nucleotide of the antisense strand. In another embodiment, the double-stranded RNAi agent further comprises a 5'-phosphate mimetic in the 5' nucleotide of the antisense strand. In a particular embodiment, the 5'-phosphate mimetic is 5'-vinyl phosphate (5'-VP).

[0247] In embodiments in which a first dsRNA agent targeting LDHA and a second dsRNA agent targeting HAO1 are covalently bonded (i.e., a dual-targeting RNAi agent), the first agent may further contain a 5'-phosphate or a 5'-phosphate mimetic at the 5' nucleotide of the antisense strand; the second agent may further contain a 5'-phosphate or a 5'-phosphate mimetic at the 5' nucleotide of the antisense strand; or the first agent and the second agent may independently further contain a 5'-phosphate or a 5'-phosphate mimetic at the 5' nucleotide of the antisense strand.

[0248] Nucleic acids addressed in the present invention may be synthesized and / or modified by methods well established in the art, such as those described herein by reference in “Current protocols in nucleic acid chemistry,” Beaucage, S. Let al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA. Modifications include, for example, terminal modifications, e.g., 5'-terminal modifications (phosphorylation, conjugate, inverted linkage) or 3'-terminal modifications (conjugate, DNA nucleotide, inverted linkage, etc.); base modifications, e.g., substitution with stable bases, unstable bases, or bases that base-pair with a wide range of partners, base removal (non-basic nucleotides), or conjugated bases; sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions; and / or skeletal modifications, including modifications or substitutions of phosphodiester bonds. Specific examples of iRNA compounds useful in the embodiments described herein include, but are not limited to, RNAs containing a modified skeleton or lacking natural internucleoside bonds. RNAs having a modified skeleton include, in particular, those that do not have a phosphorus atom in their skeleton. For the purposes of this specification and as is sometimes referred to in the art, modified RNAs that do not have a phosphorus atom in their internucleoside skeleton can also be considered as oligonucleosides. In some embodiments, the modified iRNA has a phosphorus atom in its internucleoside skeleton.

[0249] Examples of modified RNA backbones include phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methylphosphonates, and other alkylphosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having the usual 3'-5' linkage, their 2'-5' linkage analogs, and those with inverted polarity where adjacent pairs of nucleoside units are linked to 3'-5'~5'-3' or 2'-5'~5'-2'. Various salts, mixed salts, and free acid forms are also included.

[0250] Representative U.S. patents teaching the preparation of the phosphorus-containing bond described above include, but are not limited to, U.S. Patent No. 3,687,808; No. 4,469,863; No. 4,476,301; No. 5,023,243; No. 5,177,195; No. 5,188,897; No. 5,264,423; No. 5,276,019; No. 5,278,302; No. 5,286,717; and No. 5,32 Specification No. 1,131; Specification No. 5,399,676; Specification No. 5,405,939; Specification No. 5,453,496; Specification No. 5,455,233; Specification No. 5,466,677; Specification No. 5,476,925 Specification No. 5,519,126; Specification No. 5,536,821; Specification No. 5,541,316; Specification No. 5,550,111; Specification No. 5,563,253; Specification No. 5,571,799; Specification No. 5,587 ,361 Specification; 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, Examples include U.S. Patent No. 639; No. 6,608,035; No. 6,683,167; No. 6,858,715; No. 6,867,294; No. 6,878,805; No. 7,015,315; No. 7,041,816; No. 7,273,933; No. 7,321,029; and U.S. Reissue Patent No. RE39464, the entire contents of each of these are incorporated herein by reference.

[0251] Modified RNA skeletons that do not contain phosphorus atoms internally have skeletons formed by short alkyl or cycloalkyl nucleoside bonds, mixed heteroatoms and alkyl or cycloalkyl nucleoside bonds, or one or more short heteroatoms or heterocyclic nucleoside bonds. These include those having morpholino bonds (partially formed from the sugar portion of a nucleoside); siloxane skeletons; sulfide, sulfoxide, and sulfone skeletons; formacetyl and thioformacetyl skeletons; methyleneformacetyl and thioformacetyl skeletons; alkene-containing skeletons; sulfamate skeletons; methyleneimino and methylenehydrazino skeletons; sulfonate and sulfonamide skeletons; amide skeletons; and others having mixed N, O, S, and CH2 constituent parts.

[0252] Representative U.S. patents teaching the preparation of the above-mentioned oligonucleotides include, but are not limited to, U.S. Patent No. 5,034,506; No. 5,166,315; No. 5,185,444; No. 5,214,134; No. 5,216,141; No. 5,235,033; No. 5,64,562; No. 5,264,564; No. 5,405,938; No. 5,434,257; No. 5,466,677; No. 5,470,967; and No. 5 Examples include the specifications No. 489,677; No. 5,541,307; No. 5,561,225; No. 5,596,086; No. 5,602,240; No. 5,608,046; No. 5,610,289; No. 5,618,704; No. 5,623,070; No. 5,663,312; No. 5,633,360; No. 5,677,437; and No. 5,677,439, the entire contents of each of these specifications being incorporated herein by reference.

[0253] In other embodiments, suitable RNA mimetic compounds are considered for use in iRNA, where both the sugar and nucleoside bonds, i.e., the nucleotide unit backbone, are replaced with novel groups. The base units are maintained for hybridization with suitable nucleic acid target compounds. One such oligomeric compound, an RNA mimetic compound, that has been shown to have excellent hybridization properties is called a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleic acid bases are retained and bonded directly or indirectly to the aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents teaching the preparation of PNA compounds include, but are not limited to, U.S. Patent No. 5,539,082; U.S. Patent No. 5,714,331; and U.S. Patent No. 5,719,262, the entire contents of which are incorporated herein by reference. Further PNA compounds suitable for use in the iRNA of the present invention are described, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0254] Some embodiments of the present invention include RNA having a phosphorothioate skeleton and oligonucleosides having a heteroatom skeleton, in particular including the --CH2--NH--CH2-, --CH2--N(CH3)--O--CH2-- [known as the methylene (methylimino) or MMI skeleton], --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2-- and --N(CH3)--CH2--CH2-- [wherein the natural phosphodiester skeleton is represented as --O--P--O--CH2--], and the amide skeleton of the aforementioned U.S. Patent No. 5,602,240. In some embodiments, the RNA described herein has the morpholino skeleton structure of the aforementioned U.S. Patent No. 5,034,506.

[0255] Modified RNAs can also contain one or more substituted sugar moieties. The iRNAs taken up herein, for example, dsRNAs, can contain at the 2'-position one of OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, where alkyl, alkenyl and alkynyl are substituted or unsubstituted C1-C 10 alkyl or C2-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, where n and m are from 1 to about 10. In other embodiments, the dsRNA is at the 2'-position C1-C 10The modifications include lower alkyl groups, substituted lower alkyl groups, alkali groups, aralkyl groups, O-alkaryl or O-aralkyl groups, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl groups, heterocycloalkaryl groups, aminoalkylamino groups, polyalkylamino groups, substituted silyl groups, RNA cleavage groups, reporter groups, intercalator groups, groups that improve the pharmacodynamic properties of iRNA, or groups that improve the pharmacokinetic properties of iRNA, and one of other substituents having similar properties. In some embodiments, the modifications include 2'-methoxyethoxy (2'-O-(2-methoxyethyl) or 2'-MOE, also known as 2'-O--CH2CH2OCH3) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., an alkoxy-alkoxy group. Other exemplary modifications are 2'-dimethylaminooxyethoxy, also known as 2'-DMAOE, i.e., the O(CH2)2ON(CH3)2 group, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O--CH2--O--CH2--N(CH2)2, as described in the following examples herein. Further exemplary modifications include 5'-Me-2'-F nucleotide, 5'-Me-2'-OMe nucleotide, 5'-Me-2'-deoxyribonucleotide (both R and S isomers in these three families); 2'-alkoxyalkyl; and 2'-NMA (N-methylacetamide).

[0256] Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2), and 2'-fluoro (2'-F). Similar modifications can also occur at other positions in the iRNA's RNA, particularly on the 3' terminal nucleotide or at the 3' and 5' positions of the sugar in the 2'-5' linked dsRNA. The iRNA may also have sugar mimetic molecules such as cyclobutyl moieties instead of pentofuranosyl sugars. Representative U.S. patents teaching the preparation of such modified sugar structures include, but are not limited to, U.S. Patent Nos. 4,981,957; Nos. 5,118,800; Nos. 5,319,080; Nos. 5,359,044; Nos. 5,393,878; Nos. 5,446,137; Nos. 5,466,786; Nos. 5,514,785; Nos. 5,519,134; and Nos. 5,567,81 Examples include Specification No. 1; Specification No. 5,576,427; Specification No. 5,591,722; Specification No. 5,597,909; Specification No. 5,610,300; Specification No. 5,627,053; Specification No. 5,639,873; Specification No. 5,646,265; Specification No. 5,658,873; Specification No. 5,670,633; and Specification No. 5,700,920, some of which are owned by the same person as the present application. The entire contents of each of the above are incorporated herein by reference.

[0257] The iRNAs of the present invention may also include modifications or substitutions of nucleic acid bases (often simply referred to as "bases" in the art). As used herein, "unmodified" or "natural" nucleic acid bases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleic acid bases include 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-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudracil), 4- This includes thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, especially 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-daazaadenine, and other synthetic and natural nucleic acid bases such as 3-deazaguanine and 3-deazaadenine.Further nucleic acid bases include those disclosed in U.S. Patent No. 3,687,808; those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in 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., Ed., CRC Press, 1993. Some of these nucleic acid bases are particularly useful for increasing the binding affinity of the oligomeric compounds addressed 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. The 5-methylcytosine substituent has been shown to increase nucleic acid double-strand 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 is an exemplary base substitution, especially when combined with 2'-O-methoxyethyl sugar modification.

[0258] Representative U.S. patents teaching the above-mentioned modified nucleic acid bases and some other modified nucleic acid base preparations include, but are not limited to, 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; and 5,594,1 Examples include Specification No. 21, No. 5,596,091; No. 5,614,617; No. 5,681,941; No. 5,750,692; No. 6,015,886; No. 6,147,200; No. 6,166,197; No. 6,222,025; No. 6,235,887; No. 6,380,368; No. 6,528,640; No. 6,639,062; No. 6,617,438; No. 7,045,610; No. 7,427,672; and No. 7,495,088, the entire contents of each of these are incorporated herein by reference.

[0259] The iRNA of the present invention may also be modified to include one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide having a modified ribose moiety, the ribose moiety containing additional crosslinks connecting the 2' and 4' carbons. This structure effectively "locks" the ribose in its 3'-endo structural 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, OR. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).

[0260] The iRNA of the present invention may also be modified to include one or more bicyclic sugar moieties. A “bicyclic sugar” is a furanosyl ring modified by a bridge of two atoms. A “bicyclic nucleoside” (“BNA”) is a nucleoside having a sugar moiety that includes a bridge that connects two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In certain embodiments, the bridge connects the 4'-carbon and 2'-carbon of the sugar ring. Thus, in certain embodiments, the agent of the present invention may include one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide having a modified ribose moiety, the ribose moiety including an additional bridge that connects the 2' and 4' carbons. In other words, an LNA is a nucleotide containing a bicyclic sugar moiety that includes a 4'-CH2-O-2' bridge. This structure effectively “locks” the ribose in its 3'-endo structural configuration. The addition of locked nucleic acids to siRNA has been shown to increase the stability of siRNA in serum and reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, OR. 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 containing a bridge between the 4' and 2' ribosyl ring atoms. In certain embodiments, the antisense polynucleotide agent of the present invention may be one or more bicyclic nucleosides containing a 4'-2' bridge.Examples of such 4'-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 "restricted ethyl" or "cEt") and 4'-CH(CH2OCH3)-O-2' (and its analogues; see, for example, U.S. Patent No. 7,399,845); 4'-C(CH3)(CH3)-O-2' (and its analogues; see, for example, U.S. Patent No. 8,278,2 Examples include: (see Patent No. 83); 4'-CH2-N(OCH3)-2' (and its analogues; see, e.g., U.S. Patent No. 8,278,425); 4'-CH2-ON(CH3)-2' (see, e.g., U.S. Patent Application Publication No. 2004 / 0171570); 4'-CH2-N(R)-O-2' (where R is H, C1-C12 alkyl), or protecting groups (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 its analogues; see, e.g., U.S. Patent No. 8,278,426). The entire contents of each of these are incorporated herein by reference.

[0261] Further representative U.S. patents and publications teaching the preparation of locked nucleic acid nucleotides include, but are not limited to, 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; 7,399,845; and Examples include U.S. Patent Application Publication 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; U.S. Patent Application Publication No. 2008 / 0039618; and U.S. Patent Application Publication No. 2009 / 0012281, the entire contents of each of these are incorporated herein by reference.

[0262] For example, any of the above bicyclic nucleosides having one or more stereochemical sugar configurations, including α-L-ribofuranose and β-D-ribofuranose, can be prepared (see International Publication No. 99 / 14226).

[0263] The iRNA of the present invention may also be modified to include one or more restricted ethyl nucleotides. As used herein, “restricted ethyl nucleotide” or “cEt” is a restricted nucleic acid comprising a bicyclic sugar moiety including a 4'-CH(CH3)-O-2' bridge. In one embodiment, the restricted ethyl nucleotide is in an S configuration referred herein to as “S-cEt”.

[0264] The iRNA of the present invention may also include one or more “contourally restricted nucleotides” (“CRNs”). A CRN is a nucleotide analog having a linker that connects the C2' and C4' carbons or the C3 and -C5' carbons of ribose. CRNs fix the ribose ring to a stable configuration and increase its hybridization affinity to mRNA. The linker is long enough to position oxygen in an optimal location for stability and affinity, and reduces puckering of the ribose ring.

[0265] Representative publications teaching some of the above preparations of CRN include, but are not limited to, U.S. Patent Application Publication No. 2013 / 0190383 and PCT Publication No. 2013 / 036868, the entire contents of which are incorporated herein by reference.

[0266] In one embodiment, the iRNA of the present invention comprises one or more monomers that are UNA (unlocked nucleic acid) nucleotides. UNA is an unlocked acyclic nucleic acid in which one of the sugar bonds has been removed to form an unlocked "sugar" residue. In one example, UNA also includes monomers in which the bond between C1' and C4' (i.e., the covalent carbon-oxygen-carbon bond between the C1' and C4' carbons) has been removed. In another example, the C2'-C3' bond of the sugar (i.e., the covalent carbon-carbon bond between the 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, as incorporated herein by reference).

[0267] Representative U.S. patent publications teaching the preparation of UNA include, but are not limited to, U.S. Patent No. 8,314,227; and U.S. Patent Application Publications 2013 / 0096289; 2013 / 0011922; and 2011 / 0313020, the entire contents of which are incorporated herein by reference.

[0268] Potentially stable modifications to the ends of RNA molecules may include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl 4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyluridine-3”-phosphate, and inverted base dT (idT). Disclosure of these modifications can be found in PCT publication number International Publication No. 2011 / 005861.

[0269] Other modifications of the iRNA of the present invention include a 5' phosphate or 5' phosphate mimetic in the antisense strand of the RNAi agent, for example, a 5' terminal phosphate or phosphate mimetic. Suitable phosphate mimetics are disclosed, for example, in U.S. Patent Application Publication No. 2012 / 0157511, which is incorporated herein by reference in its entirety.

[0270] In some specific embodiments, the RNAi agent of the present invention is an agent that inhibits the expression of the LDHA gene, selected from the group of agents listed in any one of Tables 2 to 5. In other embodiments, the RNAi agent of the present invention is a dual-targeted iRNA agent that inhibits the expression of the LDHA gene and HAO1, where a first dsRNA inhibits the expression of the LDHA gene and is selected from the group of agents listed in any one of Tables 2 to 5, and a second dsRNA inhibits the expression of the HAO1 gene and is selected from the group of agents listed in any one of Tables 7 to 14. Any of these agents may further contain a ligand.

[0271] A. Modified iRNA containing the motif of the present invention In particular embodiments of the present invention, examples of double-stranded RNAi agents of the present invention include chemically modified agents disclosed in International Publication No. 2013 / 075035, filed on November 16, 2012, which are incorporated herein by reference in their entirety.

[0272] In embodiments in which a first dsRNA agent targeting LDHA and a second dsRNA agent targeting HAO1 are covalently bonded (i.e., a dual-targeting RNAi agent), it should be understood that the first agent may contain one or more of the motifs described below, the second agent may contain one or more of the motifs described below, or both the first and second agents may independently contain one or more of the motifs described below.

[0273] Accordingly, the present invention provides a double-stranded RNAi agent capable of inhibiting the expression of a target gene (i.e., the LDHA gene or the LDHA gene and the HAO1 gene) in vivo. The RNAi agent comprises a sense strand and an antisense strand. Each strand of the RNAi agent may be in the range of 12 to 30 nucleotides in length. For example, each strand may be 14 to 30 nucleotides, 17 to 30 nucleotides, 25 to 30 nucleotides, 27 to 30 nucleotides, 17 to 23 nucleotides, 17 to 21 nucleotides, 17 to 19 nucleotides, 19 to 25 nucleotides, 19 to 23 nucleotides, 19 to 21 nucleotides, 21 to 25 nucleotides, or 21 to 23 nucleotides in length.

[0274] The sense strand and antisense strand typically form a double-stranded RNA ("dsRNA"), also referred to herein as the "RNAi agent." The double-stranded region of the RNAi agent may be 12–30 nucleotide pairs long. For example, the double-stranded region may be 14–30 nucleotide pairs long, 17–30 nucleotide pairs long, 27–30 nucleotide pairs long, 17–23 nucleotide pairs long, 17–21 nucleotide pairs long, 17–19 nucleotide pairs long, 19–25 nucleotide pairs long, 19–23 nucleotide pairs long, 19–21 nucleotide pairs long, 21–25 nucleotide pairs long, or 21–23 nucleotide pairs long. In another example, the double-stranded region may be selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotide lengths.

[0275] In one embodiment, the RNAi agent may contain one or more overhang regions and / or capping groups at the 3' end, 5' end, or both ends of one or both strands. The overhang may be 1 to 6 nucleotides long, e.g., 2 to 6 nucleotides, 1 to 5 nucleotides, 2 to 5 nucleotides, 1 to 4 nucleotides, 2 to 4 nucleotides, 1 to 3 nucleotides, 2 to 3 nucleotides, or 1 to 2 nucleotides. The overhang may result from one strand being longer than the other, or from two strands of equal length being staggered. The overhang may form a mismatch with the target mRNA, or it may be complementary to or different from the target gene sequence. The first and second strands may also be joined by additional bases or other non-base linkers, for example, to form a hairpin.

[0276] In one embodiment, each nucleotide in the overhang region of the RNAi agent may independently be a modified or unmodified nucleotide, including, but not limited to, 2-F, 2'-O-methyl, thymidine (T), 2'-O-methoxyethyl-5-methyluridine (Teo), 2'-O-methoxyethyl adenosine (Aeo), 2'-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combination thereof, including 2'-sugar modifications. For example, TT may be an overhang sequence for any end on any strand. The overhang may form a mismatch with the target mRNA, or it may be complementary to the targeted gene sequence, or it may be a different sequence.

[0277] 5'- or 3'-overhangs in the sense strand, antisense strand, or both strands of an RNAi agent can be phosphorylated. In one embodiment, the overhang region comprises two nucleotides having a phosphorothioate between them, where the two nucleotides may be the same or different. In one embodiment, the overhang is located at the 3' end of the sense strand, antisense strand, or both strands. In one embodiment, this 3'-overhang is located in the antisense strand. In one embodiment, this 3'-overhang is located in the sense strand.

[0278] RNAi agents may contain only one overhang that can enhance RNAi interference activity without affecting their overall stability. For example, a single-stranded overhang may be located at the 3' end of the sense strand or the 3' end of the antisense strand. RNAi may also have a blunt end located at the 5' end of the antisense strand (or the 3' end of the sense strand) or vice versa. Generally, the antisense strand of RNAi has a nucleotide overhang at the 3' end and a blunt end at the 5' end. While we do not wish to be constrained by theory, asymmetric blunt ends at the 5' end and 3' end overhangs of the antisense strand are favorable for introducing guide strands into RISC processes.

[0279] In one embodiment, the RNAi agent is a 19-nucleotide long double-ended bluntmer, and the sense strand contains at least one motif of three 2'-F modifications in three consecutive nucleotides from the 5' end at positions 7, 8, and 9. The antisense strand contains at least one motif of three 2'-O-methyl modifications in three consecutive nucleotides from the 5' end at positions 11, 12, and 13.

[0280] In another embodiment, the RNAi agent is a 20-nucleotide blunt-ended double strand, the sense strand containing at least one motif of three 2'-F modifications in three consecutive nucleotides from the 5' end at positions 8, 9, and 10. The antisense strand contains at least one motif of three 2'-O-methyl modifications in three consecutive nucleotides from the 5' end at positions 11, 12, and 13.

[0281] In yet another embodiment, the RNAi agent is a 21-nucleotide blunt-ended double strand, the sense strand containing at least one motif of three 2'-F modifications in three consecutive nucleotides from the 5' end at positions 9, 10, and 11. The antisense strand contains at least one motif of three 2'-O-methyl modifications in three consecutive nucleotides from the 5' end at positions 11, 12, and 13.

[0282] 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 in three consecutive nucleotides from the 5' end to positions 9, 10, and 11; and the antisense strand comprises at least one motif of three 2'-O-methyl modifications in three consecutive nucleotides from the 5' end to positions 11, 12, and 13, with one end of the RNAi agent being blunt and the other end comprising two nucleotide overhangs. Preferably, the two nucleotide overhangs are located at the 3' end of the antisense strand.

[0283] If two nucleotide overhangs are located at the 3' end of the antisense strand, there may be two phosphorothioate nucleotide interbonds between the three terminal nucleotides, where two of the three nucleotides are overhang nucleotides and the third nucleotide is a paired nucleotide adjacent to the overhang nucleotides. In one embodiment, the RNAi agent further has two phosphorothioate nucleotide interbonds between the three terminal nucleotides at both the 5' end of the sense strand and the 5' end of the antisense strand. In one embodiment, all nucleotides in the sense and antisense strands of the RNAi agent, including nucleotides that are part of a motif, are modified nucleotides. In one embodiment, each residue is independently modified, for example, with 2'-O-methyl or 3'-fluoro in alternating motifs. Optionally, the RNAi agent further comprises a ligand (preferably GalNAc3).

[0284] In one embodiment, the RNAi agent comprises a sense strand and an antisense strand, the sense strand being 25-30 nucleotides long, with positions 1-23 of the first strand containing at least 8 ribonucleotides starting from the 5' terminal nucleotide (position 1); the antisense strand being 36-66 nucleotides long, with at least 8 ribonucleotides starting from the 3' terminal nucleotide, forming a double helix with positions 1-23 of the sense strand paired with the sense strand; at least 3' terminal nucleotides of the antisense strand not paired with the sense strand, with up to 6 consecutive 3' terminal nucleotides not paired with the sense strand, thereby forming a 3' single-stranded overhang of 1-6 nucleotides; and 10-30 consecutive nucleotides of the 5' end of the antisense strand not paired with the sense strand. It contains an oside, thereby forming a single-stranded 5' overhang of 10–30 nucleotides; at least the 5' and 3' terminal nucleotides of the sense strand are bases that pair with the nucleotides of the antisense strand when the sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially double-stranded region between the sense and antisense strands; the antisense strand is sufficiently complementary to the target RNA along at least 19 ribonucleotides of the antisense strand length so as to reduce the expression of the target gene when the double-stranded nucleic acid is introduced into mammalian cells; the sense strand contains at least one motif of three 2'-F modifications in a triple nucleotide, where at least one of the motifs is located at or near the cleavage site; the antisense strand contains at least one motif of three 2'-O-methyl modifications in a triple nucleotide at or near the cleavage site.

[0285] In one embodiment, the RNAi agent comprises a sense strand and an antisense strand, the RNAi agent comprising a first strand having a nucleotide length of at least 25 and no more than 29, and a second strand having a length of no more than 30 nucleotides, comprising at least one motif of three 2'-O-methyl modifications in three consecutive nucleotides at positions 11, 12, and 13 from the 5' end; the 3' end of the first strand and the 5' end of the second strand form blunt ends, the second strand is 1 to 4 nucleotides longer than the first strand at its 3' end, and the double-stranded region is at least 25 nucleotides long, the second strand is sufficiently complementary to the target mRNA along at least 19 nucleotides of the length of the second strand so that when the RNAi agent is introduced into mammalian cells, the expression of the target gene is reduced, and dicer cleavage of the RNAi agent preferentially yields the siRNA including the 3' end of the second strand, thereby reducing the expression of the target gene in mammals. Optionally, RNAi agents may further contain ligands.

[0286] In one embodiment, the sense strand of the RNAi agent includes at least one motif of three identical modifications in a triple nucleotide sequence, one of which is located at a cleavage site on the sense strand.

[0287] In one embodiment, the antisense strand of the RNAi agent may also contain at least one motif of three identical modifications in a triple nucleotide sequence, one of which is located at or near the cleavage site of the antisense strand.

[0288] In RNAi agents with a double-stranded region of 17–23 nucleotides in length, the cleavage sites on the antisense strand are typically located near positions 10, 11, and 12 from the 5' end. Therefore, the three identical modification motifs can be located at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, starting from the first nucleotide from the 5' end of the antisense strand, or starting from the first paired nucleotide in the double-stranded region from the 5' end of the antisense strand. The cleavage sites in the antisense strand can also vary depending on the length of the double-stranded region of the RNAi from the 5' end.

[0289] The sense strand of an RNAi agent may contain at least one motif of three identical modifications in a triple nucleotide sequence at the cleavage site; the antisense strand may have at least one motif of three identical modifications in a triple nucleotide sequence at or near the cleavage site. When the sense and antisense strands form a dsRNA double helix, the sense and antisense strands may be aligned such that one motif of three nucleotides in the sense strand and one motif of three nucleotides in the antisense strand have at least one nucleotide duplication, 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 be duplicated, or all three nucleotides may be duplicated.

[0290] In one embodiment, the sense strand of an RNAi agent may contain two or more motifs of three identical modifications in a triple nucleotide sequence. The first motif may be located at or near a cleavage site on the strand, and the other motifs may be wing modifications. The term “wing modification” as used herein refers to a motif located on a different part of the strand, away from the motif at or near the cleavage site on the same strand. The wing modifications are adjacent to the first motif or separated by at least one or more nucleotides. If the motifs are directly adjacent to each other, their chemical structures are different; if the motifs are separated by one or more nucleotides, their chemical structures may be the same or different. Two or more wing modifications may be present. For example, if two wing modifications are present, each wing modification may be located at one end relative to the first motif at or near the cleavage site, or on either side of the lead motif.

[0291] Similar to the sense strand, the antisense strand of an RNAi agent may contain two or more motifs of three identical modifications in a triple nucleotide sequence, with at least one of the motifs located at or near the cleavage site of the strand. This antisense strand may also contain one or more wing modifications in a sequence similar to those present in the sense strand.

[0292] In one embodiment, the wing modification in the sense or antisense strand of the RNAi agent typically does not include the first one or two terminal nucleotides at the 3' end, 5' end, or both ends of the strand.

[0293] In another embodiment, the wing modification in the sense or antisense strand of the RNAi agent typically does not contain the first one or two paired nucleotides within the double-stranded region at the 3' end, 5' end, or both ends of the strand.

[0294] If the sense strand and antisense strand of the RNAi agent each contain at least one wing modification, the wing modification may be located at the same end of the double-stranded region and may have one, two, or three nucleotide duplicates.

[0295] If the sense strand and antisense strand of an RNAi agent each contain at least two wing modifications, the sense strand and antisense strand may be aligned such that two modifications from one strand are each located at one end of the double-stranded region and have one, two, or three nucleotide duplicates; two modifications from one strand are each located at the other end of the double-stranded region and have one, two, or three nucleotide duplicates; and two modifications from one strand are located on each side of the read motif and have one, two, or three nucleotide duplicates in the double-stranded region.

[0296] In one embodiment, all nucleotides in the sense and antisense strands of an RNAi agent, including nucleotides that are part of a motif, may be modified. Each nucleotide may be modified with the same or different modifications, which may include changes to one or more of the unbound phosphate oxygens and / or bound phosphate oxygens; changes to components of the ribose sugar, e.g., the 2' hydroxyl of the ribose sugar; large-scale substitution of the phosphate moiety by a "dephospho" linker; modification or substitution of native bases; and substitution or modification of the ribose-phosphate backbone.

[0297] Because nucleic acids are polymers of subunits, many modifications, such as modifications to bases, phosphate moieties, or unbound oxygen atoms of phosphate moieties, are located at repeating positions within the nucleic acid. In some cases, modifications may be present at all desired positions in the nucleic acid, but often this is not the case. For example, modifications may be present only at the 3' or 5' end, or only in the terminal region, e.g., at a position on the terminal nucleotide or at the last 2, 3, 4, 5, or 10 nucleotides of the strand. Modifications may be present in the double-stranded region, the single-stranded region, or both. Modifications may be present only in the double-stranded region of RNA, or only in the single-stranded region of RNA. For example, phosphorothioate modifications at unbound oxygen positions may be present at only one or both ends, or only in the terminal region, e.g., at a position on the terminal nucleotide or at the last 2, 3, 4, 5, or 10 nucleotides of the strand, or in both the double-stranded and single-stranded regions, particularly at the ends. The 5' end or both ends may be phosphorylated.

[0298] For example, it may be possible to enhance stability, include specific bases in the overhang, or include modified nucleotides or nucleotide substitutes (surrogates) in single-stranded overhangs, e.g., 5' or 3' overhangs, or both. For example, it may be desirable to include purine nucleotides in the overhang. In some embodiments, all or some of the bases in the 3' or 5' overhang may be modified, for example, with modifications described herein. Modifications may include, for example, the use of modifications at the 2' position of ribose sugars by modifications known in the art, e.g., the use of deoxyribonucleotides, 2'-deoxy-2'-fluoro(2'-F), or 2'-O-methyl modifications instead of ribosaccharides in nucleic acid bases, and modifications of phosphate groups, e.g., phosphorothioate modifications. The overhang does not need to be homologous to the target sequence.

[0299] In one embodiment, each residue in the sense and antisense chains 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 chains may contain two or more modifications. In one embodiment, each residue in the sense and antisense chains is independently modified with 2'-O-methyl or 2'-fluoro.

[0300] At least two distinct modifications are typically present in the sense and antisense chains. These two modifications may be 2'-O-methyl or 2'-fluoro modifications, or others.

[0301] In one embodiment, N a and / or N b This includes alternating pattern modifications. As used herein, the term “alternating motif” refers to a motif having one or more modifications, where each modification is present in alternating nucleotides on a single chain. Alternating nucleotides may refer to one modification on every other nucleotide, one modification on every three nucleotides, or similar patterns. For example, if A, B, and C each represent one type of modification to a nucleotide, the alternating motifs may be “ABABABABABAB···”, “AABBAABBAABB···”, “AABAABAABAAB···”, “AAABAAABAAAB···”, “AAABBBAAABBB···”, or “ABCABCABCABC···”.

[0302] The types of modifications included in alternating motifs can be the same or different. For example, if A, B, C, and D each represent one type of modification on a nucleotide, the alternating pattern, i.e., the modifications on every other nucleotide, may be the same, but each of the sense strand or antisense strand may be selected from several possibilities of modifications within the alternating motif, such as "ABABAB...", "ACACAC...", "BDBDBD...", or "CDCDCD...".

[0303] In one embodiment, the RNAi agent of the present invention includes a modification pattern of alternating motifs in the sense strand that is shifted relative to the modification pattern of alternating motifs in the antisense strand. This shift may be such that the modification groups of the nucleotides in the sense strand correspond to different modification groups of the nucleotides in the antisense strand, or vice versa. For example, when the sense strand is paired with an antisense strand in a dsRNA double strand, the alternating motifs in the sense strand may begin with "ABABAB" from 5' to 3' of the strand, and the alternating motifs in the antisense strand may begin with "BABABA" from 5' to 3' of the strand within the double-stranded region. As another example, the alternating motifs in the sense strand may begin with "AABBAABB" from 5' to 3' of the strand, and the alternating motifs in the antisense strand may begin with "BBAABBAA" from 5' to 3' of the strand within the double-stranded region, thereby resulting in a complete or partial shift of the modification patterns between the sense strand and the antisense strand.

[0304] In one embodiment, the RNAi agent comprises a pattern of alternating 2'-O-methyl and 2'-F modifications in the sense strand, which initially has a shift relative to the pattern of alternating 2'-O-methyl and 2'-F modifications in the antisense strand, i.e., a 2'-O-methyl modified nucleotide in the sense strand base-pairs with a 2'-F modified nucleotide in the antisense strand, and vice versa. The sense strand may begin at position 1 with a 2'-F modification, and the antisense strand may begin at position 1 with a 2'-O-methyl modification.

[0305] The introduction of one or more motifs of three identical modifications on a triple nucleotide sequence into the sense strand and / or antisense strand disrupts the initial modification pattern present in the sense strand and / or antisense strand. This disruption of the modification pattern in the sense strand and / or antisense strand, by introducing one or more motifs of three identical modifications on a triple nucleotide sequence into the sense strand and / or antisense strand, unexpectedly enhances the gene silencing activity against the target gene.

[0306] In one embodiment, when three identical modification motifs on three consecutive nucleotides are introduced into any of the chains, the modifications of nucleotides adjacent to the motif are different from the modifications of the motif. For example, a portion of the sequence containing the motif is "···N a YYYN b ..." where "Y" represents the modification of three identical modification motifs in three consecutive nucleotides, and "N a " and "N b " represents a modification of a nucleotide adjacent to the motif "YYY", which is different from the modification of Y, and N a and N b These can be the same or different modifications. Or, N a and / or N b This may or may not exist if a wing modifier is present.

[0307] The RNAi agent may further contain at least one phosphorothioate or methylphosphonate internucleotide bond. The modification of the phosphorothioate or methylphosphonate internucleotide bond may be present on any nucleotide in the sense strand, antisense strand, or both strands, at any position in the strand. For example, the modification of the internucleotide bond may be present on all nucleotides in the sense strand and / or antisense strand; each modification of the internucleotide bond may be present in an alternating pattern in the sense strand and / or antisense strand; or the sense strand or antisense strand may contain modifications of both internucleotide bonds in an alternating pattern. The alternating pattern of the internucleotide bond modifications in the sense strand may be the same as or different from that in the antisense strand, and the alternating pattern of the internucleotide bond modifications in the sense strand may have a shift relative to the alternating pattern of the internucleotide bond modifications in the antisense strand. In one embodiment, the double-stranded RNAi agent contains 6 to 8 phosphorothioate internucleotide bonds. In one embodiment, the antisense strand includes two phosphorothioate nucleotide interlinks at its 5' end and two phosphorothioate nucleotide interlinks at its 3' end, and the sense strand includes at least two phosphorothioate nucleotide interlinks at either its 5' or 3' end.

[0308] In one embodiment, RNAi includes a modification of phosphorothioate or methylphosphonate internucleotide bonds in the overhang region. For example, the overhang region may include two nucleotides having a phosphorothioate or methylphosphonate internucleotide bond between them. The internucleotide bond modification may also be formed to bind the overhang nucleotide to a terminal paired nucleotide in the double-stranded region. For example, at least 2, 3, 4, or all of the overhang nucleotides may be bound by phosphorothioate or methylphosphonate internucleotide bonds, and optionally, there may be further phosphorothioate or methylphosphonate internucleotide bonds that bind the overhang nucleotide to a paired nucleotide adjacent to the overhang nucleotide. For example, there may be at least two phosphorothioate internucleotide bonds between three terminal nucleotides, where two of the three nucleotides are overhang nucleotides and the third nucleotide is a paired nucleotide adjacent to the overhang nucleotide. These three terminal nucleotides may be located 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.

[0309] In one embodiment, two nucleotide overhangs are located at the 3' end of the antisense strand, with two phosphorothioate nucleotide interbonds between the three terminal nucleotides, two of which are overhang nucleotides, and the third nucleotide is a paired nucleotide adjacent to the overhang nucleotides. Optionally, the RNAi agent may further have two phosphorothioate nucleotide interbonds between the three terminal nucleotides at both the 5' end of the sense strand and the 5' end of the antisense strand.

[0310] RNAi agents include mismatches with targets, intra-double-strand mismatches, or combinations thereof. Mismatches may occur in overhang regions or double-strand regions. Base pairs may be evaluated based on their tendency to promote dissociation or dissolution (e.g., with respect to the free energy of bonding or dissociation of a particular pair; the simplest method is to examine each pair individually, although similar or equivalent analyses may also be used). With respect to promoting dissociation: A:U is preferred over G:C; G:U is preferred over G:C; I:C is preferred over G:C (I = inosine). Mismatches, e.g., non-canonical or non-canonical pairings (as described elsewhere in this specification), are preferred over canonical (A:T, A:U, G:C) pairings; pairings containing universal bases are preferred over canonical pairings.

[0311] In one embodiment, the RNAi agent comprises at least one of the first one, two, three, four, or five base pairs in the double-stranded region from the 5' end of the antisense strand, independently selected from the group A:U, G:U, and I:C, and a mismatch pair to facilitate the dissociation of the antisense strand at the 5' end of the double-stranded region, such as a non-canonical or non-canonical pair or a pair containing a universal base.

[0312] In one embodiment, the nucleotide at position 1 in the double-stranded region from 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 double-stranded region from the 5' end of the antisense strand is an AU base pair. For example, the first base pair in the double-stranded region from the 5' end of the antisense strand is an AU base pair.

[0313] In another embodiment, the nucleotide at the 3' end of the sense strand is deoxythymine (dT). In another embodiment, the nucleotide at the 3' end of the antisense strand is deoxythymine (dT). In one embodiment, the deoxythymine nucleotide, for example, the sense and / or antisense strands have a short sequence of two dT nucleotides at their 3' ends.

[0314] In one embodiment, the sense strand sequence is given by formula (I): 5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3'(I) (In the formula: i and j are independently either 0 or 1; p and q are independently between 0 and 6; each N a However, each independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, where each sequence contains at least two different modified nucleotides; each N b However, independently, it represents an oligonucleotide sequence containing 0 to 10 modified nucleotides; each n p and n q However, independently, it represents an overhanging nucleotide; Here, Nb and Y do not have the same modifications; (XXX, YYY, and ZZZ each independently represent one motif of three identical modifications in a triple nucleotide sequence.) This can be represented by [formula]. Preferably, all YYY are 2'-F modified nucleotides.

[0315] In one embodiment, N a and / or N b This includes alternating pattern modifications.

[0316] In one embodiment, the YYY motif is located at or near the sense strand cleavage site. For example, if the RNAi agent has a double-stranded region of 17-23 nucleotides in length, the YYY motif may be located at or near the sense strand cleavage site, starting from the first nucleotide from the 5' end; or optionally, starting from the 5' end and counting from the first paired nucleotide in the double-stranded region (e.g., it may be located at positions 6, 7, 8, 7, 8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12 or 11, 12, 13).

[0317] 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. Thus, the sense chain can be represented by the following formula: 5'n p -N a -YYY-N b -ZZZ-N a -n q 3'(Ib); 5'n p -N a -XXX-N b -YYY-N a -n q 3'(Ic); or 5'n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3'(Id).

[0318] If the sense chain is represented by formula (Ib), then N b This represents an oligonucleotide sequence containing modified nucleotides of 0-10, 0-7, 0-5, 0-4, 0-2, or 0. a This can independently represent oligonucleotide sequences containing 2-20, 2-15, or 2-10 modified nucleotides.

[0319] If the sense chain is expressed as equation (Ic), then N bThis represents an oligonucleotide sequence containing modified nucleotides of 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0. Each N a This can independently represent oligonucleotide sequences containing 2-20, 2-15, or 2-10 modified nucleotides.

[0320] If the sense chain is represented as expression (Id), then each N b This independently represents an oligonucleotide sequence containing modified nucleotides of 0-10, 0-7, 0-5, 0-4, 0-2, or 0. Preferably, N b is 0, 1, 2, 3, 4, 5, or 6. Each N a This can independently represent oligonucleotide sequences containing 2-20, 2-15, or 2-10 modified nucleotides.

[0321] X, Y, and Z can each be the same or different from one another.

[0322] In another embodiment, i is 0, j is 0, and the sense chain can be represented by the following equation: 5'n p -N a -YYY-N a -n q 3'(Ia).

[0323] If the sense chain is represented by equation (Ia), then each N a This can independently represent oligonucleotide sequences containing 2-20, 2-15, or 2-10 modified nucleotides.

[0324] In one embodiment, the antisense strand sequence of RNAi is given by formula (II): 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) (In the formula: k and l are independently either 0 or 1; p' and q' are each independently between 0 and 6; each N a ' independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, where each sequence contains at least two different modified nucleotides; each N b ' independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides; each n p 'and n q ' independently represents an overhanging nucleotide; Here, N b 'and Y' do not have the same modifier; X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent a single motif of three identical modifications in a triple nucleotide sequence. It can be represented by:

[0325] In one embodiment, N a 'and / or N b ' includes alternating pattern modifications.

[0326] The Y'Y'Y' motif is located at or near the cleavage site of the antisense strand. For example, if the RNAi agent has a double-stranded region of 17-23 nucleotides in length, the Y'Y'Y' motif may be located at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, starting from the first nucleotide from the 5' end; or optionally, starting from the first paired nucleotide in the double-stranded region from the 5' end. Preferably, the Y'Y'Y' motif is located at positions 11, 12, and 13.

[0327] In one embodiment, all Y'Y'Y' motifs are 2'-OMe modified nucleotides.

[0328] In one embodiment, k is 1, l is 0, or k is 0, l is 1, or both k and l are 1.

[0329] Therefore, the antisense strand can be represented by the following formula: 5’n q’ -N a ’-Z’Z’Z’-N b ’-Y’Y’Y’-N a ’-n p’ 3’(IIb); 5’n q’ -N a ’-Y’Y’Y’-N b ’-X’X’X’-n p’ 3’(IIc); or 5’n q’ -N a ’-Z’Z’Z’-N b ’-Y’Y’Y’-N b ’-X’X’X’-N a ’-n p’ 3’(IId).

[0330] When the antisense strand is represented by formula (IIb), N b ’ represents an oligonucleotide sequence containing modified nucleotides of 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2 or 0. Each N a ’ independently represents an oligonucleotide sequence containing modified nucleotides of 2 to 20, 2 to 15, or 2 to 10.

[0331] When the antisense strand is represented by formula (IIc), N b ’ represents an oligonucleotide sequence containing modified nucleotides of 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2 or 0. Each N a ’ independently represents an oligonucleotide sequence containing modified nucleotides of 2 to 20, 2 to 15, or 2 to 10.

[0332] When the antisense strand is represented by formula (IId), each N b' independently represents oligonucleotide sequences containing modified nucleotides of 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0. Each N a ' independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides. Preferably, N b is 0, 1, 2, 3, 4, 5, or 6.

[0333] In another embodiment, when k is 0 and l is 0, the antisense chain can be represented by the following formula: 5'n p’ -N a’ -Y'Y'Y'-N a’ -n q’ 3'(Ia).

[0334] If the antisense chain is represented by equation (IIa), then each N a ' independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

[0335] X', Y', and Z' can each be the same or different from one another.

[0336] Each nucleotide in 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 in the sense and antisense strands can be independently modified with 2'-O-methyl or 2'-fluoro. Each X, Y, Z, X', Y', and Z' can, in particular, represent a 2'-O-methyl modification or a 2'-fluoro modification.

[0337] In one embodiment, the sense strand of the RNAi agent may, if the double-stranded region has 21 nucleotides, include YYY motifs located at positions 9, 10, and 11 of the strand, starting from the first nucleotide from the 5' end; or optionally, starting from the 5' end, starting from the first paired nucleotide in the double-stranded region; where Y represents a 2'-F modification. The sense strand may further include XXX or ZZZ motifs as wing modifications at the opposite end of the double-stranded region; where XXX and ZZZ independently represent a 2'-OMe modification or a 2'-F modification, respectively.

[0338] In one embodiment, the antisense strand may contain a Y'Y'Y' motif located at positions 11, 12, and 13 of the strand, starting from the first nucleotide from the 5' end; or optionally, starting from the first paired nucleotide in the double-stranded region from the 5' end; where Y' represents a 2'-O-methyl modification. The antisense strand may further contain an X'X'X' motif or a Z'Z'Z' motif as a wing modification at the opposite end of the double-stranded region; where X'X'X' and Z'Z'Z' independently represent a 2'-OMe modification or a 2'-F modification.

[0339] Each sense strand represented by any one of the above equations (Ia), (Ib), (Ic), and (Id) forms a double helix with an antisense strand represented by any one of the above equations (IIa), (IIb), (IIc), and (IId).

[0340] Therefore, the RNAi agent for use in the method of the present invention may include a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, and the RNAi double strand is given by formula (III): Sense: 5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3' Antisense: 3'n p’ -N a ’ -(X'X'X') k -N b ’ -Y'Y'Y'-N b ’ -(Z'Z'Z') l -N a ’ -n q ’ 5' (III) (In the formula: i, j, k, and l are each independently either 0 or 1; p, p', q, and q' are each independently between 0 and 6; each N a and N a ’ However, each independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, where each sequence contains at least two different modified nucleotides; each N b and N b ’ However, independently, it represents an oligonucleotide sequence containing 0 to 10 modified nucleotides; Here, Each of the np', np, nq', and n may or may not exist. q However, independently, it represents an overhanging nucleotide; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent a single motif of three identical modifications on three consecutive nucleotides. It is represented by [this].

[0341] 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 both i and j are 0; or both i and j are 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 both k and l are 0; or both k and l are 1.

[0342] An exemplary combination of sense and antisense strands that form an RNAi double helix includes the following formula: 5'n p -N a -YYY-N a -n q 3' 3'n p ’ -N a ’ -Y'Y'Y'-N a ’ n q ’ 5' (IIIa) 5'n p -N a -YYY-N b -ZZZ-N a -n q 3' 3'n p ’ -N a ’ -Y'Y'Y'-N b ’ -Z'Z'Z'-N a ’ n q ’ 5' (IIIb) 5'n p -N a -XXX-N b -YYY-N a -n q 3' 3'n p ’ -N a ’ -X'X'X'-N b ’ -Y'Y'Y'-N a ’ -n q ’ 5' (IIIc) 5'n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3' 3'np ’ -N a ’ -X'X'X'-N b ’ -Y'Y'Y'-N b ’ -Z'Z'Z'-N a -n q ’ 5' (IIId)

[0343] If an RNAi agent is represented by formula (IIIa), then each N a Each independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

[0344] If an RNAi agent is represented by formula (IIIb), then each N b Each N independently represents an oligonucleotide sequence containing 1-10, 1-7, 1-5, or 1-4 modified nucleotides. a Each independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

[0345] When an RNAi agent is represented by formula (IIIc), each Nb and Nb' independently represents an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each Na independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

[0346] When an RNAi agent is represented by formula (IIId), each Nb and Nb' independently represents an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each Na and Na' independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides. Each of Na, Na', Nb, and Nb' independently contains alternating modification patterns.

[0347] In equations (III), (IIIa), (IIIb), (IIIc), and (IIId), X, Y, and Z may be the same as or different from each other.

[0348] If an RNAi agent is represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId), then at least one Y nucleotide may form a base pair with one of the Y' nucleotides; or at least two Y nucleotides may form base pairs with the corresponding Y' nucleotides; or all three Y nucleotides may form base pairs with the corresponding Y' nucleotides.

[0349] If the RNAi agent is represented by formula (IIIb) or (IIId), then at least one Z nucleotide may form a base pair with one of the Z' nucleotides; or at least two Z nucleotides may form base pairs with the corresponding Z' nucleotides; or all three Z nucleotides may form base pairs with the corresponding Z' nucleotides.

[0350] If an RNAi agent is represented by formula (IIIc) or (IIId), then at least one X nucleotide may form a base pair with one of the X' nucleotides; or at least two X nucleotides may form a base pair with the corresponding X' nucleotide; or all three X nucleotides may form a base pair with the corresponding X' nucleotide.

[0351] In one embodiment, modifications on the Y nucleotide differ from modifications on the Y' nucleotide, modifications on the Z nucleotide differ from modifications on the Z' nucleotide, and / or modifications on the X nucleotide differ from modifications on the X' nucleotide.

[0352] In one embodiment, if the RNAi agent is represented by formula (IIId), then N aThe modification is a 2'-O-methyl or 2'-fluoro modification. 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 bound to an adjacent nucleotide via a phosphorothioate bond. In yet 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 bound to an adjacent nucleotide via a phosphorothioate bond, and the sense strand is conjugated to one or more GalNAc derivatives linked via a divalent or trivalent branched linker (described later). In another embodiment, if 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 attached to an adjacent nucleotide via a phosphorothioate bond, the sense strand comprises at least one phosphorothioate bond, and the sense strand is conjugated to one or more GalNAc derivatives linked via a divalent or trivalent branched linker.

[0353] In one embodiment, if the RNAi agent is represented by formula (IIIa), the Na modification is a 2'-O-methyl or 2'-fluoro modification, np'>0, and at least one np' is attached to an adjacent nucleotide via a phosphorothioate bond, the sense strand comprises at least one phosphorothioate bond, and the sense strand is conjugated to one or more GalNAc derivatives linked via a divalent or trivalent branched linker.

[0354] In one embodiment, the RNAi agent is a multimer comprising at least two double strands represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId), the double strands being joined by a linker. The linker may be cleavable or incleavable. Optionally, the multimer further comprises a ligand. Each double strand may target the same gene or two different genes; or each double strand may target the same gene at two different target sites.

[0355] In one embodiment, the RNAi agent is a multimer comprising three, four, five, six or more double strands represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId), where the double strands are linked by linkers. The linkers may be cleavable or incleavable. Optionally, the multimer further comprises ligands. Each double strand may target the same gene or two different genes; or each double strand may target the same gene at two different target sites.

[0356] In one embodiment, two RNAi agents represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId) are bound to each other at one or both of their 5' and 3' ends and optionally conjugated to a ligand. Each RNAi agent may target the same gene or two different genes; or each RNAi agent may target the same gene at two different target sites.

[0357] In certain embodiments, the RNAi agent of the present invention contains a small number of nucleotides having 2'-fluoro modifications, for example, 10 or fewer nucleotides having 2'-fluoro modifications. For example, the RNAi agent may contain 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 nucleotides having 2'-fluoro modifications. In certain embodiments, the RNAi agent of the present invention contains 10 nucleotides having 2'-fluoro modifications, for example, 4 nucleotides having 2'-fluoro modifications on the sense strand and 6 nucleotides having 2'-fluoro modifications on the antisense strand. In another particular embodiment, the RNAi agent of the present invention contains 6 nucleotides having 2'-fluoro modifications, for example, 4 nucleotides having 2'-fluoro modifications on the sense strand and 2 nucleotides having 2'-fluoro modifications on the antisense strand.

[0358] In other embodiments, the RNAi agent of the present invention may contain a very small number of nucleotides containing 2'-fluoro modifications, for example, two or fewer nucleotides containing 2'-fluoro modifications. For example, the RNAi agent may contain two, one, or zero nucleotides having 2'-fluoro modifications. In a particular embodiment, the RNAi agent may contain two nucleotides having 2'-fluoro modifications, for example, zero nucleotides having 2'-fluoro modifications on the sense strand and two nucleotides having 2'-fluoro modifications on the antisense strand.

[0359] Various publications describe multimeric RNAi agents that can be used in the methods of the present invention. Such publications include International Publication No. 2007 / 091269, U.S. Patent No. 7858769, International Publication No. 2010 / 141511, International Publication No. 2007 / 117686, International Publication No. 2009 / 014887, and International Publication No. 2011 / 031520, the entire contents of which are incorporated herein by reference.

[0360] As will be described in more detail below, RNAi agents containing conjugations of one or more carbohydrate moieties can optimize one or more properties of the RNAi agent. Often, the carbohydrate moiety is conjugated to a modified subunit of the RNAi agent. For example, the ribose sugar of one or more ribonucleotide subunits of a dsRNA agent may be replaced by another moiety, e.g., a non-carbohydrate (preferably cyclic) carrier to which a carbohydrate ligand is conjugated. A ribonucleotide subunit in which the ribose sugar of the subunit is thus substituted is referred herein to a ribose-substituted modified subunit (RRMS). The cyclic carrier may be a carbocyclic system, i.e., all ring atoms are carbon atoms, or a heterocyclic system, i.e., one or more ring atoms are heteroatoms, e.g., nitrogen, oxygen, sulfur. The cyclic carrier may be a monocyclic system, or may contain two or more rings, e.g., a fused ring. The cyclic carrier may be a fully saturated ring system, or may contain one or more double bonds.

[0361] Ligands can be bound to polynucleotides via a carrier. The carrier comprises (i) at least one “skeletal attachment point,” preferably two “skeletal attachment points,” and (ii) at least one “tethering attachment point.” As used herein, “skeletal attachment point” refers to a bond available for and suitable for incorporating the carrier into the ribonucleic acid skeleton, which may contain a functional group, e.g., a hydroxyl group, or generally a skeleton, e.g., a phosphate, or a modified phosphate, e.g., sulfur. A “tethering attachment point” (TAP) refers, in some embodiments, to a ring atom of a cyclic carrier connecting a selected moiety, e.g., a carbon atom or a heteroatom (different from the atoms providing the skeletal attachment points). This moiety may be, for example, a carbohydrate, e.g., monosaccharides, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides, and polysaccharides. Optionally, the selected moiety is connected to the cyclic carrier by an intervening tether. Therefore, cyclic carriers often contain functional groups, such as amino groups, or generally provide a bond suitable for the incorporation or tethering of another chemical component, such as a ligand, to the constituent ring.

[0362] The RNAi agent may be conjugated to the ligand via a carrier, which may be a cyclic group or a 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, pyridazinonyl, tetrahydrofuryl, and decalin; preferably, the cyclic group is selected from a selinol skeleton or a diethanolamine skeleton.

[0363] 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 is of formula (L): [ka] It can be represented by:

[0364] In formula (L), B1, B2, B3, B1', B2', B3', and B4' are each independently nucleotides containing a modification selected from the group consisting of 2'-O-alkyl, 2'-substituted alkoxy, 2'-substituted alkyl, 2'-halo, ENA, and BNA / LNA. In one embodiment, B1, B2, B3, B1', B2', B3', and B4' each contain a 2'-OMe modification. In one embodiment, B1, B2, B3, B1', B2', B3', and B4' each contain a 2'-OMe or 2'-F modification. In one embodiment, at least one of B1, B2, B3, B1', B2', B3', and B4' contains a 2'-ON-methylacetamide (2'-O-NMA) modification.

[0365] C1 is a thermally destabilized nucleotide located opposite the seed region of the antisense strand (i.e., positions 2-8 of the 5' end of the antisense strand). For example, C1 is located on the sense strand opposite the nucleotide at positions 2-8 of the 5' end of the antisense strand. In one example, C1 is located at position 15 from the 5' end of the sense strand. The C1 nucleotide may have thermally destabilized modifications, including nonbasic modifications; mismatches with opposing nucleotides in the double helix; and 2'-deoxy modifications or sugar modifications such as acyclic nucleotides, e.g., unlocked nucleic acid (UNA) or glycerol nucleic acid (GNA). In one embodiment, C1 has i) a mismatch with opposing nucleotides in the antisense strand; and ii) a nonbasic modification selected from the group consisting of: [ka] ; and iii) Sugar modifications selected from the group consisting of the following: [ka] It has a thermal destabilization modification selected from the group consisting of, where B is a modified or unmodified nucleic acid base, and R 1 and R 2R3 is independently H, halogen, OR3, or alkyl; R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar. In one embodiment, the thermal destabilization 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; optionally, at least one nucleic acid base in the mismatch pair is a 2'-deoxynucleotide base. In one example, the thermal destabilization modification in C1 is GNA or [ka] That is the case.

[0366] T1, T1', T2', and T3' each independently represent a nucleotide containing a modification that imparts a nucleotide a steric bulk less than or equal to the steric bulk of the 2'-OMe modification. Steric bulk 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 it may be a modification of a non-ribose nucleotide, an acyclic nucleotide, or a nucleotide skeleton similar to or equivalent to the 2' position of the ribose sugar, imparting a nucleotide a steric bulk less than or equal to the steric bulk of the 2'-OMe modification. For example, T1, T1', T2', and T3' each independently are selected from DNA, RNA, LNA, 2'-F, and 2'-F-5'-methyl. In one embodiment, T1 is DNA. In one embodiment, T1' is DNA, RNA, or LNA. In one embodiment, T2' is DNA or RNA. In one embodiment, T3' is DNA or RNA.

[0367] n 1 , n 3 , and q 1 However, they are independent and have a length of 4 to 15 nucleotides.

[0368] n 5 , q 3 , and q 7However, they are independent and have a length of 1 to 6 nucleotides.

[0369] n 4 , q 2 , and q 6 However, they are independently 1-3 nucleotides long; or, n 4 It is 0.

[0370] q 5 However, they are independent and have a length of 0 to 10 nucleotides.

[0371] n 2 and q 4 However, they are independent and have a length of 0 to 3 nucleotides.

[0372] Or, n 4 However, they are 0 to 3 nucleotides long.

[0373] In one embodiment, n 4 n can be 0. In one example, n 4 0 and q 2 and q 6 In another example, n 4 0 and q 2 and q 6 The configuration is 1, and has two phosphorothioate nucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate nucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand), and two phosphorothioate nucleotide linkage modifications within positions 18-23 of the antisense strand.

[0374] In one embodiment, n 4 , q 2 , and q 6 Each of these values ​​is 1.

[0375] In one embodiment, n 2 , n 4 , q 2 , q 4 , and q 6 Each of these values ​​is 1.

[0376] In one embodiment, when the sense strand is 19-22 nucleotides long, C1 is located at the 14-17 position of the 5' end of the sense strand, n 4 In one embodiment, C1 is located at position 15 of the 5' end of the sense strand.

[0377] In one embodiment, T3' starts at position 2 from the 5' end of the antisense chain. In one example, T3' is at position 2 from the 5' end of the antisense chain, q 6 It is equal to 1.

[0378] In one embodiment, T1' starts at position 14 from the 5' end of the antisense chain. In one example, T1' is located at position 14 from the 5' end of the antisense chain, q 2 It is equal to 1.

[0379] In an exemplary embodiment, T3' starts at position 2 from the 5' end of the antisense chain, and T1' starts at position 14 from the 5' end of the antisense chain. In one example, T3' starts at position 2 from the 5' end of the antisense chain, and q 6 is equal to 1, T1' starts from position 14 from the 5' end of the antisense chain, q 2 It is equal to 1.

[0380] In one embodiment, T1' and T3' are separated by 11 nucleotides (i.e., T1' and T3' nucleotides are not counted).

[0381] In one embodiment, T1' is located at position 14 from the 5' end of the antisense chain. In one example, T1' is located at position 14 from the 5' end of the antisense chain, q 2 The value is equal to 1, and the modification is located at the 2' position, or at a non-ribose, acyclic, or skeletal position that gives a stereobulb smaller than 2'-OMe ribose.

[0382] In one embodiment, T3' is located at position 2 from the 5' end of the antisense chain. In one example, T3' is located at position 2 from the 5' end of the antisense chain, q 6 The value is equal to 1, and the modification is located at the 2' position, or at a non-ribose, acyclic, or skeletal position that gives a stereobulk of 2'-OMe ribose or less.

[0383] In one embodiment, T1 is located at the cleavage site of the sense strand. In one example, if the sense strand is 19-22 nucleotides long, T1 is located at position 11 from the 5' end of the sense strand, n 2 is 1. In an exemplary embodiment, when the sense strand is 19-22 nucleotides long, T1 is located at the sense strand cleavage site from the 5' end to position 11, n 2 The value is 1.

[0384] In one embodiment, T2' starts at position 6 from the 5' end of the antisense chain. In one example, T2' is located at positions 6-10 from the 5' end of the antisense chain, q 4 The value is 1.

[0385] In an exemplary embodiment, if the sense strand is 19-22 nucleotides long, T1 is located at the sense strand cleavage site, for example, at position 11 from the 5' end of the sense strand, n 2 is 1; T1' is at position 14 from the 5' end of the antisense chain, q 2 is equal to 1, and the modification to T1' is at the 2' position of the ribose sugar, or at a position in the non-ribose, acyclic or skeletal structure that gives a smaller stereobulb than 2'-OMe-ribose; T2' is at the 6-10 position from the 5' end of the antisense chain, q 4 is 1; T3' is at position 2 from the 5' end of the antisense chain, q 6 The value is equal to 1, and the modification to T3' is at the 2' position, or at a non-ribose, acyclic, or skeletal position that gives a stereobulk of 2'-OMe-ribose or less.

[0386] In one embodiment, T2' starts at position 8 from the 5' end of the antisense chain. In one example, T2' starts at position 8 from the 5' end of the antisense chain, and q 4 The answer is 2.

[0387] In one embodiment, T2' starts at position 9 from the 5' end of the antisense chain. In one example, T2' is located at position 9 from the 5' end of the antisense chain, q 4 The value is 1.

[0388] In one embodiment, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 1, B3' is 2'-OMe or 2'-F, and q 5 is 6, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The value is 1; the sense strand has two phosphorothioate nucleotide linkage modifications within positions 1-5 (counting from the 5' end of the sense strand), and the antisense strand has two phosphorothioate nucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) and two phosphorothioate nucleotide linkage modifications within positions 18-23 of the antisense strand.

[0389] In one embodiment, n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 1, B3' is 2'-OMe or 2'-F, and q 5 is 6, T3' is 2'-F, and q 6is 1, B4' is 2'-OMe, and q 7 The value is 1; the sense strand has two phosphorothioate nucleotide linkage modifications within positions 1-5 (counting from the 5' end of the sense strand), and the antisense strand has two phosphorothioate nucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) and two phosphorothioate nucleotide linkage modifications within positions 18-23 of the antisense strand.

[0390] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The value is 1.

[0391] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The value is 1; the sense strand has two phosphorothioate nucleotide linkage modifications within positions 1-5 (counting from the 5' end of the sense strand), and the antisense strand has two phosphorothioate nucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) and two phosphorothioate nucleotide linkage modifications within positions 18-23 of the antisense strand.

[0392] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 6, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 7, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The value is 1.

[0393] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 6, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 7, T1' is 2'-F, and q 2is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The value is 1; the sense strand has two phosphorothioate nucleotide linkage modifications within positions 1-5 (counting from the 5' end of the sense strand), and the antisense strand has two phosphorothioate nucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) and two phosphorothioate nucleotide linkage modifications within positions 18-23 of the antisense strand.

[0394] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 1, B3' is 2'-OMe or 2'-F, and q 5 is 6, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The value is 1.

[0395] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 1, B3' is 2'-OMe or 2'-F, and q 5 is 6, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The value is 1; the sense strand has two phosphorothioate nucleotide linkage modifications within positions 1-5 (counting from the 5' end of the sense strand), and the antisense strand has two phosphorothioate nucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) and two phosphorothioate nucleotide linkage modifications within positions 18-23 of the antisense strand.

[0396] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 5, T2' is 2'-F, and q 4 is 1, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The value is 1; optionally, the antisense chain has at least two further TTs at its 3' end.

[0397] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 5, T2' is 2'-F, and q 4 is 1, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The antisense strand is 1; optionally, has at least two additional TTs at the 3' end of the antisense strand; has two phosphorothioate nucleotide bond modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), as well as two phosphorothioate nucleotide bond modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) and two phosphorothioate nucleotide bond modifications within positions 18-23 of the antisense strand.

[0398] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q7 The value is 1.

[0399] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The value is 1; the sense strand has two phosphorothioate nucleotide linkage modifications within positions 1-5 (counting from the 5' end), and two phosphorothioate nucleotide linkage modifications at positions 1 and 2 (counting from the 5' end), and two phosphorothioate nucleotide linkage modifications within positions 18-23 of the antisense strand.

[0400] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The value is 1.

[0401] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The value is 1; the sense strand has two phosphorothioate nucleotide linkage modifications within positions 1-5 (counting from the 5' end of the sense strand), and the antisense strand has two phosphorothioate nucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) and two phosphorothioate nucleotide linkage modifications within positions 18-23 of the antisense strand.

[0402] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 4 and q4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The value is 1.

[0403] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The value is 1; the sense strand has two phosphorothioate nucleotide linkage modifications within positions 1-5 (counting from the 5' end of the sense strand), and the antisense strand has two phosphorothioate nucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) and two phosphorothioate nucleotide linkage modifications within positions 18-23 of the antisense strand.

[0404] RNAi agents may contain a phosphorus-containing group at the 5' end of the sense or 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 vinyl phosphonate (5'-VP), a 5'-terminal methylphosphonate (MePhos), or 5'-deoxy-5'-C-malonyl [ka] It is possible. If the 5'-terminal phosphorus-containing group is a 5'-terminal vinyl phosphonate (5'-VP), then 5'-VP is a 5'-E-VP isomer (i.e., trans-vinyl phosphate). [ka] ), 5'-Z-VP isomer (i.e., cis-vinyl phosphate, [ka] ), or a mixture thereof.

[0405] In one embodiment, the RNAi agent contains a phosphorus-containing group at the 5' end of the sense strand. In another embodiment, the RNAi agent contains a phosphorus-containing group at the 5' end of the antisense strand.

[0406] In one embodiment, the RNAi agent contains 5'-P. In one embodiment, the RNAi agent contains 5'-P in the antisense strand.

[0407] In one embodiment, the RNAi agent contains 5'-PS. In one embodiment, the RNAi agent contains 5'-PS in the antisense strand.

[0408] In one embodiment, the RNAi agent contains 5'-VP. In one embodiment, the RNAi agent contains 5'-VP in the antisense strand. In one embodiment, the RNAi agent contains 5'-E-VP in the antisense strand. In one embodiment, the RNAi agent contains 5'-Z-VP in the antisense strand.

[0409] In one embodiment, the RNAi agent contains 5'-PS2. In one embodiment, the RNAi agent contains 5'-PS2 in the antisense strand.

[0410] In one embodiment, the RNAi agent contains 5'-PS2. In one embodiment, the RNAi agent contains 5'-deoxy-5'-C-malonyl in the antisense strand.

[0411] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The value is 1. RNAi agents also include 5'-PS.

[0412] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The value is 1. RNAi agents also include 5'-P.

[0413] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The value is 1. RNAi agents also include 5'-VP. 5'-VP can be 5'-E-VP, 5'-Z-VP, or a combination thereof.

[0414] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The value is 1. RNAi agents also include 5'-PS2.

[0415] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The value is 1. RNAi agents also include 5'-deoxy-5'-C-malonyl.

[0416] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7The RNAi agent has 1; two phosphorothioate nucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate nucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand), and two phosphorothioate nucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also contains 5'-P.

[0417] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The function is 1; the sense strand has two phosphorothioate nucleotide linkage modifications within positions 1-5 (counting from the 5' end of the sense strand), and the antisense strand has two phosphorothioate nucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) and two phosphorothioate nucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also contains 5'-PS.

[0418] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The RNAi agent is 1; it has two phosphorothioate nucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate nucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand), and two phosphorothioate nucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also includes 5'-VP. 5'-VP may be 5'-E-VP, 5'-Z-VP, or a combination thereof.

[0419] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7The function is 1; the sense strand has two phosphorothioate nucleotide linkage modifications within positions 1-5 (counting from the 5' end of the sense strand), and the antisense strand has two phosphorothioate nucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) and two phosphorothioate nucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also contains 5'-PS2.

[0420] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The RNAi agent has 1; two phosphorothioate nucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate nucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand), and two phosphorothioate nucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also contains 5'-deoxy-5'-C-malonyl.

[0421] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The value is 1. RNAi agents also include 5'-P.

[0422] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The value is 1. dsRNA agents also include 5'-PS.

[0423] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q2 is 1, B2' is 2'-OMe or 2'-F, and q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The value is 1. RNAi agents also include 5'-VP. 5'-VP can be 5'-E-VP, 5'-Z-VP, or a combination thereof.

[0424] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The value is 1. RNAi agents also include 5'-PS2.

[0425] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2is 1, B2' is 2'-OMe or 2'-F, and q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The value is 1. RNAi agents also include 5'-deoxy-5'-C-malonyl.

[0426] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The RNAi agent has 1; two phosphorothioate nucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end), two phosphorothioate nucleotide linkage modifications at positions 1 and 2 (counting from the 5' end), and two phosphorothioate nucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also contains 5'-P.

[0427] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The function is 1; it has two phosphorothioate nucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end), two phosphorothioate nucleotide linkage modifications at positions 1 and 2 (counting from the 5' end), and two phosphorothioate nucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also includes 5'-PS.

[0428] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7The RNAi agent is 1; it has two phosphorothioate nucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end), and two phosphorothioate nucleotide linkage modifications at positions 1 and 2 (counting from the 5' end), and two phosphorothioate nucleotide linkage modifications within positions 18-23 of the antisense strand. RNAi agents also include 5'-VP. 5'-VP may be 5'-E-VP, 5'-Z-VP, or a combination thereof.

[0429] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The function is 1; it has two phosphorothioate nucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end), two phosphorothioate nucleotide linkage modifications at positions 1 and 2 (counting from the 5' end), and two phosphorothioate nucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also contains 5'-PS2.

[0430] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The RNAi agent is 1; it has two phosphorothioate internucleotide bond modifications within positions 1-5 of the sense strand (counting from the 5' end), two phosphorothioate internucleotide bond modifications at positions 1 and 2 (counting from the 5' end), and two phosphorothioate internucleotide bond modifications within positions 18-23 of the antisense strand. The RNAi agent also contains 5'-deoxy-5'-C-malonyl.

[0431] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The value is 1. RNAi agents also include 5'-P.

[0432] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The value is 1. RNAi agents also include 5'-PS.

[0433] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The value is 1. RNAi agents also include 5'-VP. 5'-VP can be 5'-E-VP, 5'-Z-VP, or a combination thereof.

[0434] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The value is 1. dsRNAi RNA agents also contain 5'-PS2.

[0435] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The value is 1. RNAi agents also include 5'-deoxy-5'-C-malonyl.

[0436] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The RNAi agent has 1; two phosphorothioate nucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate nucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand), and two phosphorothioate nucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also contains 5'-P.

[0437] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7The function is 1; the sense strand has two phosphorothioate nucleotide linkage modifications within positions 1-5 (counting from the 5' end of the sense strand), and the antisense strand has two phosphorothioate nucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) and two phosphorothioate nucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also contains 5'-PS.

[0438] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The RNAi agent is 1; it has two phosphorothioate nucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate nucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand), and two phosphorothioate nucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also includes 5'-VP. 5'-VP may be 5'-E-VP, 5'-Z-VP, or a combination thereof.

[0439] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The function is 1; the sense strand has two phosphorothioate nucleotide linkage modifications within positions 1-5 (counting from the 5' end of the sense strand), and the antisense strand has two phosphorothioate nucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) and two phosphorothioate nucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also contains 5'-PS2.

[0440] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7The RNAi agent has 1; two phosphorothioate nucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate nucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand), and two phosphorothioate nucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also contains 5'-deoxy-5'-C-malonyl.

[0441] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The value is 1. RNAi agents also include 5'-P.

[0442] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 4 and q 4If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The value is 1. RNAi agents also include 5'-PS.

[0443] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The value is 1. RNAi agents also include 5'-VP. 5'-VP can be 5'-E-VP, 5'-Z-VP, or a combination thereof.

[0444] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The value is 1. RNAi agents also include 5'-PS2.

[0445] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The value is 1. RNAi agents also include 5'-deoxy-5'-C-malonyl.

[0446] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7The RNAi agent has 1; two phosphorothioate nucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate nucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand), and two phosphorothioate nucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also contains 5'-P.

[0447] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The function is 1; the sense strand has two phosphorothioate nucleotide linkage modifications within positions 1-5 (counting from the 5' end of the sense strand), and the antisense strand has two phosphorothioate nucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) and two phosphorothioate nucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also contains 5'-PS.

[0448] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The RNAi agent is 1; it has two phosphorothioate nucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate nucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand), and two phosphorothioate nucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also includes 5'-VP. 5'-VP may be 5'-E-VP, 5'-Z-VP, or a combination thereof.

[0449] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7is 1; has modifications of two phosphorothioate nucleotide internucleotide linkages within positions 1 to 5 of the sense strand (counting from the 5'-end of the sense strand), and modifications of two phosphorothioate nucleotide internucleotide linkages at positions 1 and 2 and within positions 18 to 23 of the antisense strand (counting from the 5'-end of the antisense strand). The RNAi agent also contains 5'-PS2.

[0450] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; has modifications of two phosphorothioate nucleotide internucleotide linkages within positions 1 to 5 of the sense strand (counting from the 5'-end of the sense strand), and modifications of two phosphorothioate nucleotide internucleotide linkages at positions 1 and 2 and within positions 18 to 23 of the antisense strand (counting from the 5'-end of the antisense strand). The RNAi agent also contains 5'-deoxy-5'-C-malonyl.

[0451] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The RNAi agent is 1; it has two phosphorothioate nucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate nucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) and two phosphorothioate nucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also includes 5'-P and a targeting ligand. In one embodiment, 5'-P is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0452] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7The RNAi agent is 1; and has two phosphorothioate nucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate nucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) and two phosphorothioate nucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also includes 5'-PS and a targeting ligand. In one embodiment, 5'-PS is located at the 5' end of the antisense strand, and the targeting ligand is located at the 3' end of the sense strand.

[0453] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The RNAi agent is 1; it has two phosphorothioate nucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate nucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) and two phosphorothioate nucleotide linkage modifications within positions 18-23 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. In one embodiment, the 5'-VP is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0454] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The RNAi agent is 1; it has two phosphorothioate nucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate nucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) and two phosphorothioate nucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also includes 5'-PS2 and a targeting ligand. In one embodiment, 5'-PS2 is located at the 5' end of the antisense strand, and the targeting ligand is located at the 3' end of the sense strand.

[0455] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and 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, q 7 is 1; within positions 1 to 5 of the sense strand (counting from the 5’ end of the sense strand), there are modifications of two phosphorothioate internucleotide linkages, and within positions 1 and 2 (counting from the 5’ end of the antisense strand) and within positions 18 to 23 of the antisense strand, there are modifications of two phosphorothioate internucleotide linkages. The RNAi agent also includes 5’-deoxy-5’-C-malonyl and a targeting ligand. In one embodiment, 5’-deoxy-5’-C-malonyl is at the 5’ end of the antisense strand, and the targeting ligand is at the 3’ end of the sense strand.

[0456] In one embodiment, 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, q 7The RNAi agent is 1; it has two phosphorothioate nucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end), and two phosphorothioate nucleotide linkage modifications at positions 1 and 2 (counting from the 5' end), and two phosphorothioate nucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also includes 5'-P and a targeting ligand. In one embodiment, 5'-P is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0457] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The RNAi agent is 1; it has two phosphorothioate nucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end), and two phosphorothioate nucleotide linkage modifications at positions 1 and 2 (counting from the 5' end), and two phosphorothioate nucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also includes 5'-PS and a targeting ligand. In one embodiment, 5'-PS is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0458] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The RNAi agent is 1; it has two phosphorothioate nucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end), and two phosphorothioate nucleotide linkage modifications at positions 1 and 2 (counting from the 5' end), and two phosphorothioate nucleotide linkage modifications within positions 18-23 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. In one embodiment, the 5'-VP is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0459] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6is 1, B4' is 2'-OMe, and q 7 The RNAi agent is 1; it has two phosphorothioate nucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end), and two phosphorothioate nucleotide linkage modifications at positions 1 and 2 (counting from the 5' end), and two phosphorothioate nucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also includes 5'-PS2 and a targeting ligand. In one embodiment, 5'-PS2 is located at the 5' end of the antisense strand, and the targeting ligand is located at the 3' end of the sense strand.

[0460] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The RNAi agent is 1; it has two phosphorothioate nucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end), and two phosphorothioate nucleotide linkage modifications at positions 1 and 2 (counting from the 5' end), and two phosphorothioate nucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also includes 5'-deoxy-5'-C-malonyl and a targeting ligand. In one embodiment, 5'-deoxy-5'-C-malonyl is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0461] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The RNAi agent is 1; it has two phosphorothioate nucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate nucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) and two phosphorothioate nucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also includes 5'-P and a targeting ligand. In one embodiment, 5'-P is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0462] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The RNAi agent is 1; and has two phosphorothioate nucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate nucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) and two phosphorothioate nucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also includes 5'-PS and a targeting ligand. In one embodiment, 5'-PS is located at the 5' end of the antisense strand, and the targeting ligand is located at the 3' end of the sense strand.

[0463] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7The RNAi agent is 1; it has two phosphorothioate nucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate nucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) and two phosphorothioate nucleotide linkage modifications within positions 18-23 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. In one embodiment, the 5'-VP is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0464] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The RNAi agent is 1; it has two phosphorothioate nucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate nucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) and two phosphorothioate nucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also includes 5'-PS2 and a targeting ligand. In one embodiment, 5'-PS2 is located at the 5' end of the antisense strand, and the targeting ligand is located at the 3' end of the sense strand.

[0465] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The RNAi agent is 1; it has two phosphorothioate nucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate nucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) and two phosphorothioate nucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also includes 5'-deoxy-5'-C-malonyl and a targeting ligand. In one embodiment, 5'-deoxy-5'-C-malonyl is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0466] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 4 and q4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The RNAi agent is 1; it has two phosphorothioate nucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate nucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) and two phosphorothioate nucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also includes 5'-P and a targeting ligand. In one embodiment, 5'-P is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0467] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7The RNAi agent is 1; and has two phosphorothioate nucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate nucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) and two phosphorothioate nucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also includes 5'-PS and a targeting ligand. In one embodiment, 5'-PS is located at the 5' end of the antisense strand, and the targeting ligand is located at the 3' end of the sense strand.

[0468] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The RNAi agent is 1; it has two phosphorothioate nucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate nucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) and two phosphorothioate nucleotide linkage modifications within positions 18-23 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. In one embodiment, the 5'-VP is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0469] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The RNAi agent is 1; it has two phosphorothioate nucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate nucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) and two phosphorothioate nucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also includes 5'-PS2 and a targeting ligand. In one embodiment, 5'-PS2 is located at the 5' end of the antisense strand, and the targeting ligand is located at the 3' end of the sense strand.

[0470] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 Since is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 If is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 4 and q 4 If 0, B3' is 2'-OMe or 2'-F, and q5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The RNAi agent is 1; it has two phosphorothioate nucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate nucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) and two phosphorothioate nucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also includes 5'-deoxy-5'-C-malonyl and a targeting ligand. In one embodiment, 5'-deoxy-5'-C-malonyl is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0471] In a particular embodiment, the RNAi agent of the present invention is (a) sense chain having the following: (i) Length of 21 nucleotides; (ii) an ASGPR ligand bound to the 3' end (wherein the ASGPR ligand comprises three GalNAc derivatives linked via a trivalent branched linker); and (iii) 2'-F modification at positions 1, 3, 5, 7, 9-11, 13, 17, 19, and 21 (counting from the 5' end), and 2'-OMe modification at positions 2, 4, 6, 8, 12, 14-16, 18, and 20; and (b) Antisense chain having the following: (i) Length of 23 nucleotides; (ii) 2'-OMe modification at positions 1, 3, 5, 9, 11-13, 15, 17, 19, 21 and 23 (counting from the 5' end), and 2'F modification at positions 2, 4, 6-8, 10, 14, 16, 18, 20 and 22; and (iii) Phosphothioate internucleotide binding between nucleotide positions 21 and 22 (counting from the 5' end) and between nucleotide positions 22 and 23. Includes; Here, the dsRNA agent has two nucleotide overhangs at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0472] In another specific embodiment, the RNAi agent of the present invention is (a) sense chain having the following: (i) Length of 21 nucleotides; (ii) An ASGPR ligand attached to the 3' end (wherein the ASGPR ligand comprises three GalNAc derivatives attached via a trivalent branched linker); (iii) 2'-F modification at positions 1, 3, 5, 7, 9-11, 13, 15, 17, 19, and 21 (counting from the 5' end), and 2'-OMe modification at positions 2, 4, 6, 8, 12, 14, 16, 18, and 20; and (iv) Phosphothioate internucleotide bonding between nucleotide positions 1 and 2 (counting from the 5' end), and between nucleotide positions 2 and 3; and (b) Antisense chain having the following: (i) Length of 23 nucleotides; (ii) 2'-OMe modification at positions 1, 3, 5, 7, 9, 11-13, 15, 17, 19 and 21-23 (counting from the 5' end), and 2'F modification at positions 2, 4, 6, 8, 10, 14, 16, 18 and 20; and (iii) Phosphothioate internucleotide bonds 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). Includes; Here, the RNAi agent has two nucleotide overhangs at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0473] In another specific embodiment, the RNAi agent of the present invention is (a) sense chain having the following: (i) Length of 21 nucleotides; (ii) An ASGPR ligand attached to the 3' end (wherein the ASGPR ligand comprises three GalNAc derivatives attached via a trivalent branched linker); (iii) 2'-OMe modifications at positions 1-6, 8, 10, and 12-21 (counting from the 5' end), 2'-F modifications at positions 7 and 9, and a deoxyribonucleotide (e.g., dT) at position 11; and (iv) Phosphothioate internucleotide bonding between nucleotide positions 1 and 2 (counting from the 5' end), and between nucleotide positions 2 and 3; and (b) Antisense chain having the following: (i) Length of 23 nucleotides; (ii) 2'-OMe modification at positions 1, 3, 7, 9, 11, 13, 15, 17 and 19-23 (counting from the 5' end), and 2'-F modification at positions 2, 4-6, 8, 10, 12, 14, 16 and 18; and (iii) Phosphothioate internucleotide bonds 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). Includes; Here, the RNAi agent has two nucleotide overhangs at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0474] In another specific embodiment, the RNAi agent of the present invention is (a) sense chain having the following: (i) Length of 21 nucleotides; (ii) An ASGPR ligand attached to the 3' end (wherein the ASGPR ligand comprises three GalNAc derivatives attached via a trivalent branched linker); (iii) 2'-OMe modification at positions 1-6, 8, 10, 12, 14, and 16-21, and 2'-F modification at positions 7, 9, 11, 13, and 15; and (iv) Phosphothioate internucleotide bonding between nucleotide positions 1 and 2 (counting from the 5' end), and between nucleotide positions 2 and 3; and (b) Antisense chain having the following: (i) Length of 23 nucleotides; (ii) 2'-OMe modification at positions 1, 5, 7, 9, 11, 13, 15, 17, 19 and 21-23 (counting from the 5' end), and 2'-F modification at positions 2-4, 6, 8, 10, 12, 14, 16, 18 and 20; and (iii) Phosphothioate internucleotide bonds 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). Includes; Here, the RNAi agent has two nucleotide overhangs at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0475] In another specific embodiment, the RNAi agent of the present invention is (a) sense chain having the following: (i) Length of 21 nucleotides; (ii) An ASGPR ligand attached to the 3' end (wherein the ASGPR ligand comprises three GalNAc derivatives attached via a trivalent branched linker); (iii) 2'-OMe modification at positions 1-9 and 12-21, and 2'-F modification at positions 10 and 11; and (iv) Phosphothioate internucleotide bonding between nucleotide positions 1 and 2 (counting from the 5' end), and between nucleotide positions 2 and 3; and (b) Antisense chain having the following: (i) Length of 23 nucleotides; (ii) 2'-OMe modification at positions 1, 3, 5, 7, 9, 11-13, 15, 17, 19 and 21-23 (counting from the 5' end), and 2'-F modification at positions 2, 4, 6, 8, 10, 14, 16, 18 and 20; and (iii) Phosphothioate internucleotide bonds 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). Includes; Here, the RNAi agent has two nucleotide overhangs at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0476] In another specific embodiment, the RNAi agent of the present invention is (a) sense chain having the following: (i) Length of 21 nucleotides; (ii) An ASGPR ligand attached to the 3' end (wherein the ASGPR ligand comprises three GalNAc derivatives attached via a trivalent branched linker); (iii) 2'-F modification at positions 1, 3, 5, 7, 9-11 and 13, and 2'-OMe modification at positions 2, 4, 6, 8, 12 and 14-21; and (iv) Phosphothioate internucleotide bonding between nucleotide positions 1 and 2 (counting from the 5' end), and between nucleotide positions 2 and 3; and (b) Antisense chain having the following: (i) Length of 23 nucleotides; (ii) 2'-OMe modification at positions 1, 3, 5-7, 9, 11-13, 15, 17-19, and 21-23 (counting from the 5' end), and 2'-F modification at positions 2, 4, 8, 10, 14, 16, and 20; and (iii) Phosphothioate internucleotide bonds 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). Includes; Here, the RNAi agent has two nucleotide overhangs at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0477] In another specific embodiment, the RNAi agent of the present invention is (a) sense chain having the following: (i) Length of 21 nucleotides; (ii) An ASGPR ligand attached to the 3' end (wherein the ASGPR ligand comprises three GalNAc derivatives attached via a trivalent branched linker); (iii) 2'-OMe modification at positions 1, 2, 4, 6, 8, 12, 14, 15, 17, and 19-21, and 2'-F modification at positions 3, 5, 7, 9-11, 13, 16, and 18; and (iv) Phosphothioate internucleotide bonding between nucleotide positions 1 and 2 (counting from the 5' end), and between nucleotide positions 2 and 3; and (b) Antisense chain having the following: (i) Length of 25 nucleotides; (ii) 2'-OMe modifications at positions 1, 4, 6, 7, 9, 11-13, 15, 17, and 19-23 (counting from the 5' end), 2'-F modifications at positions 2, 3, 5, 8, 10, 14, 16, and 18, and deoxyribonucleotides (e.g., dT) at positions 24 and 25; and (iii) Phosphothioate internucleotide bonds 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). Includes; Here, the RNAi agent has four nucleotide overhangs at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0478] In another specific embodiment, the RNAi agent of the present invention is (a) sense chain having the following: (i) Length of 21 nucleotides; (ii) An ASGPR ligand attached to the 3' end (wherein the ASGPR ligand comprises three GalNAc derivatives attached via a trivalent branched linker); (iii) 2'-OMe modification at positions 1-6, 8, and 12-21, and 2'-F modification at positions 7 and 9-11; and (iv) Phosphothioate internucleotide bonding between nucleotide positions 1 and 2 (counting from the 5' end), and between nucleotide positions 2 and 3; and (b) Antisense chain having the following: (i) Length of 23 nucleotides; (ii) 2'-OMe modifications at positions 1, 3-5, 7, 8, 10-13, 15, and 17-23 (counting from the 5' end), and 2'-F modifications at positions 2, 6, 9, 14, and 16; and (iii) Phosphothioate internucleotide bonds 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). Includes; Here, the RNAi agent has two nucleotide overhangs at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0479] In another specific embodiment, the RNAi agent of the present invention is (a) sense chain having the following: (i) Length of 21 nucleotides; (ii) An ASGPR ligand attached to the 3' end (wherein the ASGPR ligand comprises three GalNAc derivatives attached via a trivalent branched linker); (iii) 2'-OMe modification at positions 1-6, 8, and 12-21, and 2'-F modification at positions 7 and 9-11; and (iv) Phosphothioate internucleotide bonding between nucleotide positions 1 and 2 (counting from the 5' end), and between nucleotide positions 2 and 3; and (b) Antisense chain having the following: (i) Length of 23 nucleotides; (ii) 2'-OMe modifications at positions 1, 3-5, 7, 10-13, 15, and 17-23 (counting from the 5' end), and 2'-F modifications at positions 2, 6, 8, 9, 14, and 16; and (iii) Phosphothioate internucleotide bonds 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). Includes; Here, the RNAi agent has two nucleotide overhangs at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0480] In another specific embodiment, the RNAi agent of the present invention is (a) sense chain having the following: (i) Length of 19 nucleotides; (ii) An ASGPR ligand attached to the 3' end (wherein the ASGPR ligand comprises three GalNAc derivatives attached via a trivalent branched linker); (iii) 2'-OMe modification at positions 1-4, 6, and 10-19, and 2'-F modification at positions 5 and 7-9; and (iv) Phosphothioate internucleotide bonding between nucleotide positions 1 and 2 (counting from the 5' end), and between nucleotide positions 2 and 3; and (b) Antisense chain having the following: (i) Length of 21 nucleotides; (ii) 2'-OMe modifications at positions 1, 3-5, 7, 10-13, 15, and 17-21 (counting from the 5' end), and 2'-F modifications at positions 2, 6, 8, 9, 14, and 16; and (iii) Phosphothioate internucleotide bonds 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). Includes; Here, the RNAi agent has two nucleotide overhangs at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0481] IV. iRNA conjugated to a ligand Another modification of the iRNA of the present invention involves chemically attaching one or more ligands, portions, or conjugates to the RNA to improve the activity, cell distribution, or cell uptake of the iRNA. These parts are not limited to, but include the cholesterol portion (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, for example, beryl-S-tritylthiol (Manoharan et al., (1992) Ann. NYAcad. Sci., 660:306-309; Manoharan et al., (1993) Biorg. Med. Chem. Let., 3:2765-2770), and thiocholesterol (Oberhauser et al., (1992) Nucl. Acids Res.,20:533-538), aliphatic chains, e.g., 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, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al.,(1995)Tetrahedron Lett.,36:3651-3654; Shea et al.,(1990)Nucl.Acids Res., 18:3777-3783), polyamine or polyethylene glycol chain (Manoharan et al., (1995) Nucleosides & Nucleotides, 14:969-973), or adamantane acetate (Manoharan et al., (1995) Tetrahedron Lett., 36:3651-3654), palmityl moiety (Mishra et al., (1995) Biochim. Biophys.Examples include lipid moieties such as Acta, 1264:229-237, or octadecylamine or hexylamino-carbonyloxycholesterol moieties (Crooke et al., (1996) J. Pharmacol. Exp. Ther., 277:923-937).

[0482] In embodiments in which a first dsRNA agent targeting LDHA and a second dsRNA agent targeting HAO1 are covalently bonded (i.e., dual-targeted RNAi agents as described herein), one or both of the dsRNA agents may independently comprise one or more ligands.

[0483] In one embodiment, the ligand alters the distribution, targeting, or lifespan of the iRNA agent into which it is incorporated. In a preferred embodiment, the ligand provides, for example, improved affinity to selected targets (e.g., molecules, cells, or cell types), compartments (e.g., compartments of cells or organs), body tissues, organs, or regions compared to species without such ligands. Preferred ligands do not participate in double-strand pairing in double-stranded nucleic acids.

[0484] Ligands may include natural substances such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylglucosamine, N-acetylgalactosamine, or hyaluronic acid); or lipids. Ligands may also be synthetic polymers, such as recombinant or synthetic molecules including synthetic polyamino acids. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-coglycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphatidine. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudo-peptide polyamines, peptide-mimicking polyamines, dendrimer polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or α-helix peptides.

[0485] The ligand may also include a target group, e.g., a cell or tissue targeting agent, e.g., lectins, glycoproteins, lipids or proteins, or antibodies that bind to a specific cell type, such as kidney cells. The target group may be thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, polyhydric lactose, polyhydric galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyhydric mannose, polyhydric fucose, glycosylated polyamino acids, polyhydric galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, lipid, cholesterol, steroid, bile acid, folate, vitamin B12, vitamin A, biotin, or RGD peptide, or RGD peptide mimetic or aptamer.

[0486] Other examples of ligands include dyes, inserts (e.g., acridine), crosslinking agents (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphylline, saffrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases or chelating agents (e.g., EDTA), lipophilic molecules such as cholesterol, cholic acid, adamantane acetate, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid (cholenic acid Examples include acids, dimethoxytrityl, or phenoxazine, and peptide complexes (e.g., Antennapedia peptide, Tat peptide), alkylating agents, phosphates, amino acids, mercaptos, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino acids, alkyls, substituted alkyls, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole cluster, acridine-imidazole complex, Eu3+ tetraaza macrocyclic complex), dinitrophenyl, HRP, or AP.

[0487] Ligands can be proteins, such as glycoproteins, or peptides, such as coligands, or antibodies, such as molecules that have specific affinity for antibodies that bind to specific cell types, such as hepatocytes. Ligands may also include hormones a...

Claims

1. A double-stranded ribonucleic acid (dsRNA) agent or salt thereof for inhibiting the expression of lactate dehydrogenase A (LDHA) in cells, The dsRNA agent or a salt thereof comprises a sense strand and an antisense strand that form a double-stranded region, The aforementioned antisense chain, a) Sequence ID 3426, 5'-UUGUUCUAAGGAAAAAGCUGCCA-3'; b) Sequence ID 3415 5'-UGUGUGUUCUAAGGAAAAAGGCUGCC-3'; c) Sequence ID 3580'-UGGUGUUCUAAGGAAAGGCUGC-3'; d) Sequence ID 3411'5'-UAAUCUUUGGUGUUCUUCUAAGGAAAA-3'; e) Sequence ID 3451'-AGACAAUCUUUGGUGUUCUAAGG-3'; f) Sequence ID 3406 5'-UAGAGACAAUCUUUGGUGUUCUA-3'; and g) Sequence ID 3407 5'-UCAGAGACAAUCUUUGGUGUUCU-3', It comprises at least 17 consecutive nucleotides from any one of the antisense nucleotide sequences selected from the group consisting of the following: A dsRNA agent or a salt thereof, wherein all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand include nucleotide modifications, and a ligand is conjugated to at least one strand.

2. The dsRNA agent or salt thereof according to claim 1, wherein at least one nucleotide modification is selected from the group consisting of deoxy-nucleotide modification, 3'-terminal deoxythymine (dT) nucleotide modification, 2'-O-methyl nucleotide modification, 2'-fluoro nucleotide modification, 2'-deoxy-nucleotide modification, fixed nucleotide modification, unfixed nucleotide modification, conformationally restricted nucleotide modification, restricted ethyl nucleotide modification, non-basic nucleotide modification, 2'-amino-nucleotide modification, 2'-O-allyl-nucleotide modification, 2'-C-alkyl-nucleotide modification, 2'-hydroxyl-nucleotide modification, 2'-methoxyethyl nucleotide modification, 2'-O-alkyl-nucleotide modification, morpholino nucleotide modification, phosphoramide nucleotide modification, nucleotide modification containing a non-natural base, tetrahydropyran nucleotide modification, 1,5-anhydrohexitol nucleotide modification, cyclohexenyl nucleotide modification, glycol nucleotide modification, and 2-O-(N-methylacetamide) nucleotide modification, and combinations thereof.

3. The dsRNA agent or salt thereof according to claim 1 or 2, wherein the nucleotide modification is selected from the group consisting of 2'-O-methylnucleotide modification and 2'-fluoronucleotide modification.

4. A dsRNA agent or salt thereof according to any one of claims 1 to 3, wherein each chain has a length of 30 nucleotides or less.

5. A dsRNA agent or salt thereof according to any one of claims 1 to 4, wherein each chain is independently 19 to 30 nucleotides long.

6. The dsRNA agent or salt thereof according to claim 5, wherein each chain is independently 19 to 25 nucleotides long.

7. The dsRNA agent or salt thereof according to claim 5, wherein each chain is independently 21 to 23 nucleotides long.

8. A dsRNA agent or salt thereof according to any one of claims 1 to 7, wherein at least one strand comprises a 3' overhang of at least one nucleotide.

9. A dsRNA agent or salt thereof according to any one of claims 1 to 8, wherein at least one strand comprises a 3' overhang of at least two nucleotides.

10. The dsRNA agent or salt thereof according to any one of claims 1 to 9, wherein the ligand is conjugated to the 3' end of the sense strand of the dsRNA agent or salt thereof.

11. A dsRNA agent or salt thereof according to any one of claims 1 to 10, wherein the ligand is an N-acetylgalactosamine (GalNAc) derivative.

12. The dsRNA agent or salt thereof according to any one of claims 1 to 11, wherein the ligand is conjugated to the dsRNA agent or salt thereof via a monovalent, divalent, or trivalent linker.

13. The ligand is 【Chemistry 1】 The dsRNA agent or salt thereof according to any one of claims 1 to 12.

14. The following is a schematic diagram: 【Chemistry 2】 As shown, the dsRNA agent or salt thereof is conjugated to a ligand, wherein X is O or S, according to claim 13.

15. The dsRNA agent or salt thereof according to claim 14, wherein X is O in the formula.

16. The dsRNA agent or salt thereof according to any one of claims 1 to 15, wherein the dsRNA agent or salt thereof further comprises at least one phosphorothioate or methylphosphonate internucleotide bond.

17. The dsRNA agent or salt thereof according to claim 16, wherein the sense strand comprises at least one phosphorothioate nucleotide internucleotide bond at its 3' end.

18. The dsRNA agent or salt thereof according to claim 16 or 17, wherein the antisense strand comprises at least one phosphorothioate nucleotide bond at its 3' end.

19. A dsRNA agent or salt thereof according to any one of claims 1 to 18, wherein the antisense strand comprises two phosphorothioate nucleotide interbonds at its 5' end and two phosphorothioate nucleotide interbonds at its 3' end, and the sense strand comprises at least two phosphorothioate nucleotide interbonds at its 5' end or 3' end, or at both its 5' and 3' ends.

20. The antisense chain, a) Sequence ID 3426, 5'-UUGUUCUAAGGAAAAAGCUGCCA-3'; b) Sequence ID 3415 5'-UGUGUGUUCUAAGGAAAAAGGCUGCC-3'; c) Sequence ID 3580'-UGGUGUUCUAAGGAAAGGCUGC-3'; d) Sequence ID 3411'5'-UAAUCUUUGGUGUUCUUCUAAGGAAAA-3'; e) Sequence ID 3451'-AGACAAUCUUUGGUGUUCUAAGG-3'; f) Sequence ID 3406 5'-UAGAGACAAUCUUUGGUGUUCUA-3'; and g) Sequence ID 3407 5'-UCAGAGACAAUCUUUGGUGUUCU-3', A dsRNA agent or salt thereof according to any one of claims 1 to 19, comprising at least 19 consecutive nucleotides from any one antisense nucleotide sequence selected from the group consisting of the following.

21. The antisense chain, a) Sequence ID 3426, 5'-UUGUUCUAAGGAAAAAGCUGCCA-3'; b) Sequence ID 3415 5'-UGUGUGUUCUAAGGAAAAAGGCUGCC-3'; c) Sequence ID 3580'-UGGUGUUCUAAGGAAAGGCUGC-3'; d) Sequence ID 3411'5'-UAAUCUUUGGUGUUCUUCUAAGGAAAA-3'; e) Sequence ID 3451'-AGACAAUCUUUGGUGUUCUAAGG-3'; f) Sequence ID 3406 5'-UAGAGACAAUCUUUGGUGUUCUA-3'; and g) Sequence ID 3407 5'-UCAGAGACAAUCUUUGGUGUUCU-3', A dsRNA agent or salt thereof according to any one of claims 1 to 20, comprising one antisense nucleotide sequence selected from the group consisting of the following.

22. The sense chain, a) Sequence ID 3240, 5'-GCAGCCCUUUUCCUUAGAACAA-3'; b) Sequence ID 3229 5'-CAGCCUUUUCCUUAGAACACA-3'; c) Sequence ID 3394 5'-AGCCCUUUUCCUUAGAACACCA-3'; d) Sequence ID 3225'-UCCUUAGAACACCAAAAGAUUA-3'; e) Sequence ID 3265'-UUAGAACACCAAAAGAUUGUCU-3'; f) Sequence ID 3220 5'-GAACACCAAGAUUGUCUCUA-3'; and g) Sequence ID 3221 5'-AACACCAAAAGAUUGUCUCUGA-3', A dsRNA agent or salt thereof according to any one of claims 1 to 21, comprising at least 17 consecutive nucleotides from any one of the sense nucleotide sequences selected from the group consisting of the following.

23. Isolated cells comprising a dsRNA agent or a salt thereof according to any one of claims 1 to 22.

24. A pharmaceutical composition for inhibiting the expression of the lactate dehydrogenase A (LDHA) gene, comprising a dsRNA agent or a salt thereof as described in any one of claims 1 to 22.

25. An in vitro method for inhibiting intracellular lactate dehydrogenase A (LDHA) expression, comprising the step of contacting the cells with a dsRNA agent or salt thereof according to any one of claims 1 to 22, or the pharmaceutical composition according to claim 24, thereby inhibiting intracellular LDHA expression.

26. A pharmaceutical composition comprising a dsRNA agent or a salt thereof according to any one of claims 1 to 22, or the pharmaceutical composition according to claim 24, for use in a method for inhibiting LDHA expression in a target.

27. A pharmaceutical composition comprising a dsRNA agent or a salt thereof according to any one of claims 1 to 22, or the pharmaceutical composition according to claim 24, for use in a method of treating a subject having a disorder for which a reduction in LDHA expression can be beneficial.

28. The pharmaceutical composition according to claim 27, wherein the disorder is a disease, disorder, or condition related to the oxalate pathway.

29. The pharmaceutical composition according to claim 28, wherein the disease, disorder, or condition related to the oxalate pathway is a disease, disorder, or condition related to oxalates, or a disease, disorder, or condition related to lactate dehydrogenase.

30. The pharmaceutical composition according to claim 29, wherein the disease, disorder, or condition related to oxalate is a disease, disorder, or condition of kidney stone formation, or a disease, disorder, or condition of calcium oxalate tissue deposition.

31. The pharmaceutical composition according to claim 29, wherein the disease, disorder, or pathological condition related to lactate dehydrogenase is selected from the group consisting of cancer, fatty liver (steatosis), non-alcoholic steatohepatitis (NASH), cirrhosis, accumulation of fat in the liver, inflammation of the liver, hepatocyte necrosis, hepatic fibrosis, and non-alcoholic fatty liver disease (NAFLD).

32. The pharmaceutical composition according to claim 29, wherein the disease, disorder, or condition related to the oxalate pathway is primary hyperoxaluria type 2 (PH2).

33. A pharmaceutical composition according to any one of claims 26 to 32, wherein the target is a human.

34. The pharmaceutical composition according to any one of claims 26 to 33, wherein the method further comprises the step of administering a further therapeutic agent to a target.

35. A pharmaceutical composition according to any one of claims 26 to 34, wherein a dsRNA agent is administered subcutaneously to the target.

Images