Lactate dehydrogenase A (LDHA) iRNA compositions and methods of use

Abstract

To provide compositions and methods for treating subjects having an oxalate pathway-associated disease, disorder, or condition.SOLUTION: The present invention provides a double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of lactate dehydrogenase A (LDHA) in a cell, wherein the dsRNA agent comprises a sense strand and an antisense strand, the antisense strand comprising a region of complementarity which comprises at least 15 contiguous nucleotides differing by at most three nucleotides from any one of specific antisense sequences.SELECTED DRAWING: None

Description

[Technical Field]

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

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy, created on July 12, 2018, is titled 121301-07520_SL.TXT and is 1,154,808 bytes in size. [Background technology]

[0003] Oxalate (C2O4 2- Oxalate (CO₂) is a salt-forming ion of oxalic acid (C₂H₂O₄) that is widely distributed in both plants and animals. Oxalate is an inevitable component of the human diet and a common component of plants and plant-derived foods. Oxalate can also be endogenously synthesized through metabolic pathways present in the liver. Dietary and endogenous contributions to urinary oxalate excretion are comparable. Glyoxylate is the direct precursor of oxalate and is derived from the oxidation of glycolate by the enzyme glycolate oxidase (GO), also known and referred to herein as hydroxyacid oxidase (HAO1), or from the catabolism of hydroxyproline, a component of collagen. Transamination of alanine and glyoxylate by the enzyme alanine / glyoxylate aminotransferase (AGT) results in the formation of pyruvate and glycine. Excess glyoxylate is converted to oxalate 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 is composed of four subunits, the two most common of which are the LDH-M and LDH-H proteins. These proteins are encoded by the LDHA and LDHB genes, respectively. Various combinations of LDH-M and LDH-H proteins result in five different isoforms of LDH. LDHA is the most important gene responsible for hepatic lactate dehydrogenase isoforms. In particular, in the liver, LDHA is important as the final step in the endogenous production of oxalate by converting the precursor glyoxylate to oxalate. LDHA also plays an important role in the Cori cycle and in the anaerobic step of glycolysis, where LDHA converts lactate to pyruvate and vice versa.

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

[0006] Because oxalate binds calcium in the kidney, urinary CaOx supersaturation can occur, leading to the formation and deposition of CaOx crystals in renal tissues or the renal system. These CaOx crystals contribute to the formation of extensive kidney calcification (nephrocalcinosis) and stones (nephrolithiasis). Subjects with extensive kidney calcification or non-obstructive stones typically have no symptoms. However, obstructive stones can cause severe pain. Over time, these CaOx crystals cause kidney damage and progressive inflammation. In the presence of secondary complications such as obstruction, these CaOx crystals can lead to a decline in kidney function and, in severe cases, end-stage renal failure and the need for dialysis. Furthermore, systemic deposition of CaOx (systemic oxalosis) can occur in extrarenal tissues, including soft tissues (such as the thyroid and breast), heart, nerves, joints, skin, and retina, which, if left untreated, can lead to premature death.

[0007] Among the most well-known diseases related to the oxalate pathway, such as kidney stone formation diseases, there is primary hyperoxaluria, a genetic disease characterized by increased endogenous oxalate synthesis, with variable clinical phenotypes.Currently, no treatment modulating oxalate synthesis is available, and there are only a few treatment options for subjects suffering from hereditary hyperoxaluria.Ultimately, some subjects suffering from hereditary hyperoxaluria require kidney / liver transplantation.Other diseases, disorders, and pathologies related to the oxalate pathway include calcium oxalate tissue deposition diseases, disorders, and pathologies.

[0008] Currently, the primary treatment for many of these oxalate pathway-related diseases, disorders, and conditions (e.g., those associated with kidney stone disease) is 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, subjects often fail to comply with such lifestyle changes and do not achieve significant benefit. Treatments for some other oxalate pathway-related diseases, disorders, and conditions, such as chronic kidney disease, include the use of ACE inhibitors (angiotensin-converting enzyme inhibitors) and ARBs (angiotensin II antagonists), which can slow disease progression. Nevertheless, subjects with chronic kidney disease experience a gradual decline in kidney function, progressing to the need for dialysis or kidney transplantation. Most of these oxalate pathway-related diseases have no cure, and no oxalate-reducing treatments are currently available.

[0009] Furthermore, diseases, disorders, and pathologies related to the oxalate pathway include diseases, disorders, and pathologies 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 pathologies related to lactate dehydrogenase include fatty liver (steatosis), nonalcoholic steatohepatitis (NASH), cirrhosis, liver fat accumulation, liver inflammation, hepatocyte necrosis, liver fibrosis, and nonalcoholic fatty liver disease (NAFLD).However, considering the important role of LDH in glycolysis, treatment options are limited. Summary of the Invention [Problem to be solved by the invention]

[0010] Thus, there is a need in the art for alternative treatments for subjects suffering from diseases, disorders, and conditions associated with the oxalate pathway. [Means for solving the problem]

[0011] The present invention is based, at least in part, on the discovery that by targeting LDHA using iRNA agents, compositions comprising such agents, and methods disclosed herein, liver-specific and superior LDHA and urinary oxalate lowering effects can be achieved. The present disclosure may provide the following aspects. [Document name] Claims [Section 1] 1. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of lactate dehydrogenase A (LDHA) in a cell, the dsRNA agent comprising a sense strand and an antisense strand, the antisense strand comprising a region of complementarity comprising at least 15 contiguous nucleotides that differs by no more than 3 nucleotides from any one of the antisense sequences listed in any one of Tables 2-5. [Section 2] The dsRNA agent of Paragraph 1, wherein the dsRNA agent comprises at least one modified nucleotide. [Section 3] 3. The dsRNA agent of paragraph 1 or 2, wherein substantially all of the nucleotides of the sense strand include a modification; substantially all of the nucleotides of the antisense strand include a modification; or substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand include a modification. [Section 4] 4. The dsRNA agent of Paragraph 3, wherein all of the nucleotides of the sense strand include a modification; all of the nucleotides of the antisense strand include a modification; or all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand include a modification. [Section 5] At least one of the modified nucleotides is a deoxy-nucleotide, a 3'-terminal deoxy-thymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, a non-locked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino-modified nucleotide, a 2'-O-allyl-modified nucleotide, a 2'-C-alkyl-modified nucleotide, a 2'-hydroxyl-modified nucleotide, a 2'-methoxyethyl-modified nucleotide, a 2'-O-alkyl-modified nucleotide, a 2'-O-alkyl-modified nucleotide, a 2'-O-methyl-modified nucleotide, a 2'-fluoro-modified nucleotide, a 2'-deoxy-modified nucleotide, a 2'-O-methyl-modified nucleotide, a 2'-fluoro-modified nucleotide, a 2'-deoxy-modified nucleotide, a 2'-amino-modified nucleotide, a 2'-O-allyl-modified nucleotide, a 2'-C-alkyl-modified nucleotide, a 2'-hydroxyl-modified nucleotide, a 2'-methoxyethyl-modified nucleotide, a 2'-O-alkyl ... 5. The dsRNA agent of any one of Items 2 to 4, wherein the nucleotide is selected from the group consisting of nucleotides including nucleotides such as nucleotides containing 5'-phosphates, morpholino nucleotides, phosphoramidates, nucleotides containing unnatural bases, tetrahydropyran-modified nucleotides, 1,5-anhydrohexitol-modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides containing phosphorothioate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphates, nucleotides containing 5'-phosphate mimetics, glycol-modified nucleotides, and 2-O-(N-methylacetamido)-modified nucleotides, and combinations thereof. [Section 6] The dsRNA agent of paragraph 1, wherein the region of complementarity is at least 17 nucleotides in length. [Section 7] Item 10. The dsRNA agent of Item 1, wherein the region of complementarity is 19 to 30 nucleotides in length. [Section 8] 8. The dsRNA agent of Item 7, wherein the region of complementarity is 19 to 25 nucleotides in length. [Section 9] 8. The dsRNA agent of Item 7, wherein the region of complementarity is 21 to 23 nucleotides in length. [Section 10] Item 10. The dsRNA agent of any one of Items 1 to 9, wherein each strand is 30 nucleotides or less in length. [Section 11] 11. The dsRNA agent of any one of paragraphs 1 to 10, wherein each strand is independently 19 to 30 nucleotides in length. [Section 12] 12. The dsRNA agent of paragraph 11, wherein each strand is independently 19 to 25 nucleotides in length. [Section 13] 12. The dsRNA agent of paragraph 11, wherein each strand is independently 21 to 23 nucleotides in length. [Section 14] 14. The dsRNA agent of any one of paragraphs 1 to 13, wherein at least one strand comprises at least one nucleotide 3' overhang. [Section 15] 14. The dsRNA agent of any one of paragraphs 1 to 13, wherein at least one strand comprises at least a two nucleotide 3' overhang. [Section 16] 16. The dsRNA agent of any one of paragraphs 1 to 15, wherein the agent further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage. [Section 17] 17. The dsRNA agent of Paragraph 16, wherein the phosphorothioate or methylphosphonate internucleotide linkage is at the 3' end of one strand. [Section 18] 18. The dsRNA agent of Paragraph 17, wherein the strand is the antisense strand. [Section 19] 18. The dsRNA agent of Paragraph 17, wherein the strand is the sense strand. [Section 20] 17. The dsRNA agent of Paragraph 16, wherein the phosphorothioate or methylphosphonate internucleotide linkage is at the 5' end of one strand. [Section 21] 21. The dsRNA agent of Paragraph 20, wherein the strand is the antisense strand. [Section 22] 21. The dsRNA agent of Paragraph 20, wherein the strand is the sense strand. [Section 23] 17. The dsRNA agent of Paragraph 16, wherein the phosphorothioate or methylphosphonate internucleotide linkages are at both the 5' and 3' ends of one strand. [Section 24] Item 24. The dsRNA agent of any one of Items 1 to 23, further comprising a ligand. [Section 25] 25. The dsRNA agent of Paragraph 24, wherein the ligand is conjugated to the 3' end of the sense strand of the dsRNA agent. [Section 26] 26. The dsRNA agent of paragraph 24 or 25, wherein the ligand is one or more N-acetylgalactosamine (GalNAc) derivatives attached via a monovalent, divalent, or trivalent branched linker. [Section 27] The ligand is JPEG0007775254000001.jpg60161 27. The dsRNA agent of claim 26, wherein [Section 28] 10. The dsRNA agent according to claim 1, wherein the dsRNA agent is JPEG0007775254000002.jpg59161 28. The dsRNA agent of paragraph 27, wherein the dsRNA agent is conjugated to the ligand shown in [Section 29] 29. The dsRNA agent of paragraph 28, wherein X is O. [Section 30] Item 10. The dsRNA agent of Item 1, wherein the region of complementarity consists of one of the antisense sequences listed in any one of Tables 2-5. [Section 31] Item 1, wherein the sense strand and the antisense strand comprise a nucleotide sequence selected from the group consisting of the nucleotide sequences of any one of the agents listed in any one of Tables 2-5. [Section 32] a first double-stranded ribonucleic acid (dsRNA) agent that inhibits the expression of lactate dehydrogenase A (LDHA), the first double-stranded ribonucleic acid (dsRNA) agent comprising a sense strand and an antisense strand; a second double-stranded ribonucleic acid (dsRNA) agent that inhibits expression of hydroxyacid oxidase 1 (glycolate oxidase) (HAO1), comprising a sense strand and an antisense strand; A dual-targeting RNAi agent, wherein the first dsRNA agent and the second dsRNA agent are covalently linked. [Section 33] 33. The dual-targeting RNAi agent of Paragraph 32, wherein the sense strand of the first dsRNA agent comprises at least 15 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO:1 by no more than 3 nucleotides, and the antisense strand of the first dsRNA agent comprises at least 15 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO:2 by no more than 3 nucleotides. [Section 34] 33. The dual-targeting RNAi agent of Paragraph 32, wherein the antisense strand of the first dsRNA agent comprises a region of complementarity comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the antisense sequences listed in any one of Tables 2-5. [Section 35] 33. The dual-targeting RNAi agent of Paragraph 32, wherein the sense strand of the second dsRNA agent comprises at least 15 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO:21 by no more than 3 nucleotides, and the antisense strand of the second dsRNA agent comprises at least 15 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO:22 by no more than 3 nucleotides. [Section 36] 33. The dual-targeting RNAi agent of Paragraph 32, wherein the antisense strand of the second dsRNA agent comprises a region of complementarity comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the antisense sequences listed in any one of Tables 7-14. [Section 37] 37. The dual-targeting RNAi agent of any one of paragraphs 32-36, wherein the first dsRNA agent and the second dsRNA agent each independently comprise at least one modified nucleotide. [Section 38] 37. The dual-targeting RNAi agent of any one of Paragraphs 32-36, wherein 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. [Section 39] At least one of the modified nucleotides of the first dsRNA agent and at least one of the modified nucleotides of the second dsRNA agent are each, independently, a deoxy-nucleotide, a 3'-terminal deoxy-thymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, a non-locked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino-modified nucleotide, a 2'-O-allyl-modified nucleotide, a 2'-C-alkyl-modified nucleotide, a 2'-C-alkyl-modified nucleotide, a 2'-C-methyl ... 39. The dual-targeting RNAi agent of claim 38, wherein the nucleotide is selected from the group consisting of nucleotides comprising a 5'-phosphate, a 2'-hydroxyl-modified nucleotide, a 2'-methoxyethyl-modified nucleotide, a 2'-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide comprising a non-natural base, a tetrahydropyran-modified nucleotide, a 1,5-anhydrohexitol-modified nucleotide, a cyclohexenyl-modified nucleotide, a nucleotide comprising a phosphorothioate group, a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5'-phosphate, and a nucleotide comprising a 5'-phosphate mimic. [Section 40] 39. The dual-targeting RNAi agent of Paragraph 38, wherein at least one of the modified nucleotides of the first dsRNA agent and at least one of the modified nucleotides of the second dsRNA agent are each, independently, selected from the group consisting of a 2'-O-methyl and a 2' fluoro modification. [Section 41] 37. The dual-targeting RNAi agent of paragraph 34 or 36, wherein the region of complementarity of the first dsRNA agent and / or the region of complementarity of the second dsRNA agent are each, independently, 19 to 30 nucleotides in length. [Section 42] 37. The dual-targeting RNAi agent of any one of paragraphs 32-36, wherein each strand of the first dsRNA agent and each strand of the second dsRNA agent are each, independently, 19-30 nucleotides in length. [Section 43] 37. The dual-targeting RNAi agent of any one of paragraphs 32-36, wherein at least one strand of the first dsRNA agent and / or at least one strand of the second dsRNA agent each, independently, comprises a 3' overhang of at least one nucleotide. [Section 44] 37. The dual-targeting RNAi agent of any one of paragraphs 32-36, wherein the first dsRNA agent and / or the second dsRNA agent each, independently, further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage. [Section 45] 37. The dual-targeting RNAi agent of any one of paragraphs 32 to 36, wherein the first dsRNA agent and / or the second dsRNA agent each, independently, further comprise at least one ligand. [Section 46] 46. ​​The dual-targeting RNAi agent of Paragraph 45, wherein the at least one ligand is conjugated to the sense strand of the first dsRNA agent and / or the second dsRNA agent. [Section 47] 46. ​​The dual-targeting RNAi agent of paragraph 45, wherein the at least one ligand is conjugated to the 3' end, 5' end, or an internal position of one of the sense strands. [Section 48] 46. ​​The dual-targeting RNAi agent of Paragraph 45, wherein the at least one ligand is conjugated to the antisense strand of the first dsRNA agent and / or the second dsRNA agent. [Section 49] 46. ​​The dual-targeting RNAi agent of paragraph 45, wherein the at least one ligand is conjugated to the 3' end, 5' end, or an internal position of one of the antisense strands. [Section 50] 46. ​​The dual-targeting RNAi agent of paragraph 45, wherein the ligand is an N-acetylgalactosamine (GalNAc) derivative. [Section 51] 51. The dual-targeting RNAi agent of paragraph 50, wherein the ligand is one or more GalNAc derivatives attached via a monovalent, divalent, or trivalent branched linker. [Section 52] The ligand is JPEG0007775254000003.jpg59161 52. The dual-targeting RNAi agent of Paragraph 51, wherein [Section 53] the first dsRNA agent and the second dsRNA agent each independently represent a sequence as shown in the following schematic diagram: JPEG0007775254000004.jpg61161 53. The dual targeting RNAi agent of paragraph 52, conjugated to the ligand shown in formula: [Section 54] 54. The dual-targeting RNAi agent of paragraph 53, wherein said X is O. [Section 55] 37. The dual-targeting RNAi agent of any one of paragraphs 32 to 36, wherein the first dsRNA agent and the second dsRNA agent are covalently linked via a covalent linker. [Section 56] 56. The dual-targeting RNAi agent of paragraph 55, wherein the covalent linker is selected from the group consisting of a single-stranded nucleic acid linker, a double-stranded nucleic acid linker, a partially single-stranded nucleic acid linker, a partially double-stranded nucleic acid linker, a carbohydrate moiety linker, and a peptide linker. [Section 57] 56. The dual-targeting RNAi agent of paragraph 55, wherein the covalent linker is a cleavable linker or a non-cleavable linker. [Section 58] 56. The dual-targeting RNAi agent of Paragraph 55, wherein the covalent linker joins the sense strand of the first dsRNA agent to the sense strand of the second dsRNA agent. [Section 59] 56. The dual-targeting RNAi agent of Paragraph 55, wherein the covalent linker joins the antisense strand of the first dsRNA agent to the antisense strand of the second dsRNA agent. [Section 60] 56. The dual-targeting RNAi agent of paragraph 55, wherein the covalent linker further comprises at least one ligand. [Section 61] 37. The dual-targeting RNAi agent of any one of paragraphs 32 to 36, wherein contacting a cell with the dual-targeting RNAi agent inhibits expression of the LDHA gene and the HAO1 gene to substantially the same level of inhibition of expression as obtained by contacting a cell with both dsRNA agents individually. [Section 62] 37. The dual-targeting RNAi agent of any one of paragraphs 32 to 36, wherein contacting a cell with the dual-targeting RNAi agent inhibits expression of the LDHA gene and the HAO1 gene to a level greater than the level of inhibition of expression obtained by contacting a cell with both dsRNA agents individually. [Section 63] 62. The dual-targeting RNAi agent of paragraph 61, wherein 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% greater than the level of inhibition of expression obtained by contacting the cell with both dsRNA agents individually. [Section 64] 62. The dual-targeting RNAi agent of paragraph 61, wherein 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% greater than the level of inhibition of expression obtained by contacting the cell with both dsRNA agents individually. [Section 65] 37. The dual-targeting RNAi agent of any one of paragraphs 32-36, wherein contacting a cell with said dual-targeting RNAi agent inhibits oxalate and / or glyoxylate protein production to a level lower than the level of protein production obtained by contacting a cell with both dsRNA agents individually. [Section 66] 37. The dual-targeting RNAi agent of any one of paragraphs 32-36, wherein contacting a cell with said dual-targeting RNAi agent inhibits oxalate and / or glyoxylate protein production to a level lower than the level of protein production obtained by contacting a cell with both dsRNA agents individually. [Section 67] Item 32. A cell comprising the dsRNA agent of any one of items 1 to 31. [Section 68] 67. A cell comprising the dual-targeting RNAi agent of any one of paragraphs 32 to 66. [Section 69] 32. A vector encoding at least one strand of the dsRNA agent of any one of items 1-31. [Section 70] 67. A vector encoding at least one strand of the dual-targeting RNAi agent of any one of paragraphs 32-66. [Section 71] 32. A pharmaceutical composition for inhibiting the expression of lactate dehydrogenase A (LDHA) gene, comprising the agent according to any one of items 1 to 31. [Section 72] Item 67. A pharmaceutical composition for inhibiting the expression of lactate dehydrogenase A (LDHA) gene and hydroxyacid oxidase 1 (glycolic acid oxidase) (HAO1) ​​gene, comprising the dual-targeting RNAi agent according to any one of Items 32 to 66. [Section 73] a first double-stranded ribonucleic acid (dsRNA) agent that inhibits expression of lactate dehydrogenase A (LDHA), comprising a sense strand and an antisense strand, wherein the sense strand comprises at least 15 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO: 1 by no more than 3 nucleotides, and the antisense strand comprises at least 15 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO: 2 by no more than 3 nucleotides; a second double-stranded ribonucleic acid (dsRNA) agent that inhibits expression of hydroxyacid oxidase 1 (glycolic acid oxidase) (HAO1), comprising a sense strand and an antisense strand, wherein the sense strand comprises at least 15 contiguous nucleotides that differ by no more than three nucleotides from the nucleotide sequence of SEQ ID NO: 21, and the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than three nucleotides from the nucleotide sequence of SEQ ID NO: 22; 10. A pharmaceutical composition comprising: [Section 74] a first double-stranded ribonucleic acid (dsRNA) agent that inhibits expression of lactate dehydrogenase A (LDHA), comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementarity comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the antisense sequences listed in any one of Tables 2-5; a second double-stranded ribonucleic acid (dsRNA) agent that inhibits expression of hydroxyacid oxidase 1 (glycolic acid oxidase) (HAO1), comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementarity comprising at least 15 contiguous nucleotides that differ by no more than three nucleotides from any one of the antisense sequences listed in any one of Tables 7-14; 10. A pharmaceutical composition comprising: [Section 75] 75. The pharmaceutical composition according to any one of items 71 to 74, wherein the agent is formulated in a non-buffered solution. [Section 76] Item 76. The pharmaceutical composition according to Item 75, wherein the non-buffered solution is saline or water. [Section 77] Item 75. The pharmaceutical composition according to any one of items 71 to 74, wherein the agent is formulated in a buffer solution. [Section 78] 78. The pharmaceutical composition of claim 77, wherein the buffer comprises acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. [Section 79] Item 78. The pharmaceutical composition of Item 77, wherein the buffer is phosphate buffered saline (PBS). [Section 80] A method for inhibiting lactate dehydrogenase A (LDHA) expression in a cell, comprising the step of contacting the cell with the agent according to any one of Items 1 to 66 or the pharmaceutical composition according to any one of Items 71 to 74, thereby inhibiting the expression of LDHA in the cell. [Section 81] A method for inhibiting lactate dehydrogenase A (LDHA) expression and hydroxyacid oxidase 1 (glycolate oxidase) (HAO1) ​​expression in a cell, the method comprising the step of contacting the cell with the agent according to any one of Items 32 to 66 or the pharmaceutical composition according to any one of Items 72 to 74, thereby inhibiting the expression of LDHA and HAO1 in the cell. [Section 82] 82. The method of paragraph 80 or 81, wherein the cell is in a subject. [Section 83] 83. The method of paragraph 82, wherein the subject is a human. [Section 84] 84. The method of any one of paragraphs 80 to 83, wherein the LDHA expression is inhibited by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or to below the level of detection of LDHA expression. [Section 85] 84. The method of any one of paragraphs 81 to 83, wherein the HAO1 expression is inhibited by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or to below the level of detection of HAO1 expression. [Section 86] 84. The method of paragraph 83, wherein the human subject is suffering from a disease, disorder or condition associated with the oxalate pathway. [Section 87] 87. The method of paragraph 86, wherein the oxalate pathway-associated disease, disorder, or condition is an oxalate-associated disease, disorder, or condition, or a lactate dehydrogenase-associated disease, disorder, or condition. [Section 88] 88. The method of claim 87, wherein the oxalate-associated disease, disorder, or condition is a kidney stone formation disease, disorder, or condition, or a calcium oxalate tissue deposition disease, disorder, or condition. [Section 89] 89. The method of paragraph 88, wherein the kidney stone-forming disease, disorder, or condition is a calcium oxalate stone-forming disease, disorder, or condition or a non-calcium oxalate stone-forming disease, disorder, or condition. [Section 90] 90. The method of claim 89, wherein the calcium oxalate stone forming disease, disorder, or condition is a hyperoxaluric disease, disorder, or condition or a non-hyperoxaluric disease, disorder, or condition. [Section 91] 91. The method of paragraph 90, wherein the hyperoxaluric disease, disorder, or condition is selected from the group consisting of primary hyperoxaluria, enteric hyperoxaluria, dietary hyperoxaluria, and idiopathic hyperoxaluria. [Section 92] 91. The method of paragraph 90, wherein the non-hyperoxaluric stone-forming disease, disorder, or condition is hypercalciuria and / or hypocitriuria. [Section 93] 91. The method of paragraph 90, wherein the non-hyperoxaluric stone-forming disease, disorder, or condition is a calcium oxalate or non-calcium oxalate kidney stone-forming disease. [Section 94] 89. The method of claim 88, wherein the calcium oxalate tissue deposition disease, disorder, or condition is selected from the group consisting of a systemic calcium oxalate tissue deposition disease, disorder, or condition or a tissue-specific calcium oxalate tissue deposition disease, disorder, or condition. [Section 95] 89. The method of claim 88, wherein the lactate dehydrogenase-related disease, disorder, or condition 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, hepatocellular necrosis, liver fibrosis, and non-alcoholic fatty liver disease (NAFLD). [Section 96] Item 96. The method according to any one of Items 80 to 95, wherein the cells are hepatocytes. [Section 97] 1. A method for inhibiting expression of LDHA in a subject, comprising: A method comprising the step of administering to the subject a therapeutically effective amount of the agent according to any one of items 1 to 66 or the pharmaceutical composition according to any one of items 71 to 74, thereby inhibiting the expression of LDHA in the subject. [Section 98] 1. A method for inhibiting lactate dehydrogenase A (LDHA) expression and hydroxyacid oxidase 1 (glycolate oxidase) (HAO1) ​​expression in a subject, comprising: A method comprising the step of administering to the subject a therapeutically effective amount of the pharmaceutical composition described in any one of items 32 to 66 or any one of items 72 to 74, thereby inhibiting the expression of LDHA and HAO1 in the subject. [Section 99] 1. A method of treating a subject suffering from a disorder that would benefit from reduced LDHA expression, comprising: A method comprising the step of administering to the subject a therapeutically effective amount of the agent according to any one of items 1 to 66 or the pharmaceutical composition according to any one of items 71 to 74, thereby treating the subject. [Section 100] 1. A method for preventing at least one symptom in a subject suffering from a disease or disorder that would benefit from reduced expression of the LDHA gene, comprising: A method comprising the step of administering to the subject a prophylactically effective amount of the agent according to any one of Items 1 to 66 or the pharmaceutical composition according to any one of Items 71 to 74, thereby preventing at least one symptom in the subject. [Section 101] 101. The method of paragraph 99 or 100, wherein the disorder is a disease, disorder or condition associated with the oxalate pathway. [Section 102] 1. A method of treating a subject suffering from a disease, disorder, or condition associated with the oxalate pathway, comprising: A method comprising the step of administering to the subject a therapeutically effective amount of the agent according to any one of items 1 to 66 or the pharmaceutical composition according to any one of items 71 to 74, thereby treating the subject. [Section 103] 1. A method for preventing at least one symptom in a subject suffering from a disease, disorder, or condition associated with the oxalate pathway, comprising: A method comprising the step of administering to the subject a prophylactically effective amount of the agent according to any one of Items 1 to 66 or the pharmaceutical composition according to any one of Items 71 to 74, thereby preventing at least one symptom in the subject. [Section 104] 104. The method of any one of paragraphs 98 to 103, wherein administration of the dsRNA agent or the pharmaceutical composition to the subject results in decreased urinary oxalate, tissue oxalate, plasma oxalate, decreased LDHA enzyme activity, decreased LDHA protein accumulation, and / or decreased HAO1 protein accumulation. [Section 105] Item 105. The method according to any one of Items 101 to 104, wherein the disease, disorder, or condition associated with the oxalate pathway is an oxalate-associated disease, disorder, or condition, or a lactate dehydrogenase-associated disease, disorder, or condition. [Section 106] 106. The method of claim 105, wherein the oxalate-associated disease, disorder, or condition is a kidney stone formation disease, disorder, or condition, or a calcium oxalate tissue deposition disease, disorder, or condition. [Section 107] 107. The method of paragraph 106, wherein the kidney stone-forming disease, disorder, or condition is a calcium oxalate stone-forming disease, disorder, or condition or a non-calcium oxalate stone-forming disease, disorder, or condition. [Section 108] 108. The method of claim 107, wherein the calcium oxalate stone forming disease, disorder, or condition is a hyperoxaluric disease, disorder, or condition or a non-hyperoxaluric disease, disorder, or condition. [Section 109] 109. The method of claim 108, wherein the hyperoxaluric disease, disorder, or condition is selected from the group consisting of primary hyperoxaluria, enteric hyperoxaluria, dietary hyperoxaluria, and idiopathic hyperoxaluria. [Section 110] 110. The method of claim 109, wherein the non-hyperoxaluric stone-forming disease, disorder, or condition is hypercalciuria and / or hypocitriuria. [Section 111] 111. The method of paragraph 110, wherein the non-hyperoxaluric stone-forming disease, disorder, or condition is a calcium oxalate or non-calcium oxalate kidney stone-forming disease. [Section 112] 102. The method of claim 101, wherein the calcium oxalate tissue deposition disease, disorder, or condition is selected from the group consisting of a systemic calcium oxalate tissue deposition disease, disorder, or condition or a tissue-specific calcium oxalate tissue deposition disease, disorder, or condition. [Section 113] Item 105. The method according to any one of Items 101 to 104, wherein the lactate dehydrogenase-related disease, disorder, or condition is selected from the group consisting of cancer, fatty liver (steatosis), non-alcoholic steatohepatitis (NASH), cirrhosis, accumulation of fat in the liver, liver inflammation, hepatocellular necrosis, liver fibrosis, and non-alcoholic fatty liver disease (NAFLD). [Section 114] 99. The method of any one of items 97 to 99, wherein the disease, disorder or condition is primary hyperoxaluria 2 (PH2). [Section 115] 115. The method of claim 114, further comprising altering the diet of the subject. [Section 116] 116. The method of paragraph 114 or 115, wherein the subject is undergoing a kidney transplant. [Section 117] Item 117. The method according to any one of Items 98 to 116, wherein the subject is a human. [Section 118] Item 118. The method of any one of items 98 to 117, further comprising administering to the subject an additional therapeutic agent. [Section 119] Item 119. The method according to any one of Items 98 to 118, wherein the agent is administered to the subject at a dose of about 0.01 mg / kg to about 10 mg / kg or about 0.5 mg / kg to about 50 mg / kg. [Section 120] Item 120. The method of any one of items 98 to 119, wherein the agent is administered subcutaneously to the subject. [Section 121] Item 121. The method according to any one of Items 98 to 120, wherein the agent does not substantially inhibit the expression and / or activity of lactate dehydrogenase B (LDHB).

[0012] Therefore, 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 can be present in a cell, for example, in a cell of a subject, such as a human. The present invention also provides a method for using the iRNA composition of the present invention to inhibit the expression of the LDHA gene, for treating subjects who can benefit from inhibiting or reducing the expression of the LDHA gene, for example, subjects who can benefit from reducing or inhibiting urinary oxalate production, for example, subjects suffering from or susceptible to diseases, disorders, or conditions associated with the oxalate pathway, for example, oxalate-related diseases, disorders, or conditions, such as kidney stone formation diseases, disorders, or conditions, or calcium oxalate tissue deposition diseases, disorders, or conditions; or subjects suffering from or susceptible to LDHA-related diseases, disorders, or conditions.

[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 can be present in a cell, for example, a cell in a subject, such as a human. 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 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 an oxalate-related disease, disorder, or condition, such as a kidney stone-forming disease, disorder, or condition or a calcium oxalate tissue deposition disease, disorder, or condition; or an LDH-related disease, disorder, or condition.

[0014] Accordingly, in one aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of lactate dehydrogenase A (LDHA) in a cell, the dsRNA agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementarity comprising at least 15 contiguous nucleotides that differs by no more than 3 nucleotides from any one of the antisense sequences listed in any one of Tables 2-5.

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

[0016] In other embodiments, substantially all of the nucleotides in the sense strand include a modification; substantially all of the nucleotides in the antisense strand include a modification; or substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand include a modification.

[0017] In yet other embodiments, all of the nucleotides in the sense strand include a modification; all of the nucleotides in the antisense strand include a modification; or all of the nucleotides in the sense strand and all of the nucleotides in the antisense strand include a modification.

[0018] In one embodiment, at least one of the modified nucleotides is selected from the group consisting of a deoxy-nucleotide, a 3'-terminal deoxy-thymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, a non-locked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino-modified nucleotide, a 2'-O-allyl-modified nucleotide, a 2'-C-alkyl-modified nucleotide, a 2'-hydroxyl-modified nucleotide, a 2'-methoxyethyl-modified nucleotide, a 2'-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide containing a non-natural base, a tetrahydropyran-modified nucleotide, a 1,5-anhydrohexitol-modified nucleotide, a cyclohexenyl-modified nucleotide, a nucleotide containing a phosphorothioate group, a nucleotide containing a methylphosphonate group, a nucleotide containing a 5'-phosphate, a nucleotide containing a 5'-phosphate mimic, a glycol-modified nucleotide, and a 2-O-(N-methylacetamido)-modified nucleotide, and combinations thereof.

[0019] The region of complementarity can be at least 17 nucleotides in length; 19-30 nucleotides in length; 19-25 nucleotides in length; or 21-23 nucleotides in length.

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

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

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

[0023] The phosphorothioate or methylphosphonate internucleotide linkage can be at the 3'-end of one strand (e.g., the antisense strand; or the sense strand); or the phosphorothioate or methylphosphonate internucleotide linkage can be at the 5'-end of one strand (e.g., the antisense strand; or the sense strand); or the phosphorothioate or methylphosphonate internucleotide linkage can be at both the 5'-end and the 3'-end of one strand.

[0024] The dsRNA agent can further comprise a ligand.

[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 attached via a monovalent, divalent, or trivalent branched linker.

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

[0028] In one embodiment, the dsRNA agent is represented by the following schematic diagram: [ka] wherein X is O or S.

[0029] In one embodiment, X is O.

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

[0031] In one embodiment, the sense strand and the antisense strand comprise a nucleotide sequence selected from the group consisting of the nucleotide sequence of any one of the agents listed in any one of Tables 2-5.

[0032] In another aspect, the invention provides a dual-targeting RNAi agent comprising: a first double-stranded ribonucleic acid (dsRNA) agent that inhibits expression of lactate dehydrogenase A (LDHA), comprising a sense strand and an antisense strand; and a second double-stranded ribonucleic acid (dsRNA) agent that inhibits expression of hydroxyacid oxidase 1 (glycolate oxidase) (HAO1), comprising 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 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO:1 by no more than 3 nucleotides, and the antisense strand of the first dsRNA agent comprises at least 15 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO:2 by no more than 3 nucleotides.

[0034] In another embodiment, the antisense strand of the first dsRNA agent comprises a region of complementarity comprising at least 15 contiguous nucleotides that differ by no more than 3 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 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO:21 by no more than 3 nucleotides, and the antisense strand of the second dsRNA agent comprises at least 15 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO:22 by no more than 3 nucleotides.

[0036] In another embodiment, the antisense strand of the second dsRNA agent comprises a region of complementarity comprising at least 15 contiguous nucleotides that differ by no more than 3 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 include at least one modified nucleotide.

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

[0039] In one embodiment, at least one of the modified nucleotides of the first dsRNA agent and at least one of the modified nucleotides of the second dsRNA agent are each, independently, a deoxy-nucleotide, a 3'-terminal deoxy-thymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, a non-locked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino-modified nucleotide, a 2'-O-allyl-modified nucleotide, a 2'-C-allyl ... The nucleotides are selected from the group consisting of alkyl-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 phosphorothioate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphates, and nucleotides containing 5'-phosphate mimetics.

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

[0041] The region of complementarity of the first dsRNA agent and / or the region of complementarity of the second dsRNA agent can each, independently, be 19-30 nucleotides in length.

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

[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 includes 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 comprise at least one phosphorothioate or methylphosphonate internucleotide linkage.

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

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

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

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

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

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

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

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

[0053] In one embodiment, the first dsRNA agent and the second dsRNA agent each independently have a structure similar to that shown in the following schematic diagram: [ka] wherein 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 a single-stranded nucleic acid linker, a double-stranded nucleic acid linker, a partially single-stranded nucleic acid linker, a partially double-stranded nucleic acid linker, a carbohydrate moiety linker, and a peptide linker. In another embodiment, the covalent linker is a cleavable linker or a non-cleavable linker. In one embodiment, the covalent linker connects the sense strand of a first dsRNA agent to the sense strand of a second dsRNA agent. In another embodiment, the covalent linker connects 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 a cell with a dual-targeting RNAi agent of the present invention inhibits expression of the LDHA gene and the HAO1 gene to a level substantially the same as the level of inhibition of expression obtained by contacting the cell with both dsRNA agents individually. In another embodiment, contacting a cell with a dual-targeting RNAi agent inhibits expression of the LDHA gene and the HAO1 gene to a level greater than the level of inhibition of expression obtained by contacting the cell with both dsRNA agents 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% greater than the level of inhibition of expression obtained by contacting the cell 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% greater than the level of inhibition of expression obtained by contacting the cell with both dsRNA agents individually.

[0061] In one embodiment, contacting a cell with a dual-targeting RNAi agent inhibits oxalate and / or glyoxylate protein production to a level lower than the level of protein production obtained by contacting the cell with both dsRNA agents individually. In another embodiment, contacting a cell with a dual-targeting RNAi agent inhibits oxalate and / or glyoxylate protein production to a level lower than the level of protein production obtained by contacting the cell with both dsRNA agents individually.

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

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

[0064] In one aspect, the present invention provides a pharmaceutical composition comprising: a first double-stranded ribonucleic acid (dsRNA) agent that inhibits expression of lactate dehydrogenase A (LDHA), the first double-stranded ribonucleic acid (dsRNA) agent comprising a sense strand and an antisense strand, wherein the sense strand comprises at least 15 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO:1 by no more than 3 nucleotides and the antisense strand comprises at least 15 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO:2 by no more than 3 nucleotides; and a second double-stranded ribonucleic acid (dsRNA) agent that inhibits expression of hydroxyacid oxidase 1 (glycolic acid oxidase) (HAO1), the second double-stranded ribonucleic acid (dsRNA) agent comprising a sense strand and an antisense strand, the sense strand comprises at least 15 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO:21 by no more than 3 nucleotides and the antisense strand comprises at least 15 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO:22 by no more than 3 nucleotides.

[0065] In another aspect, the present invention provides a pharmaceutical composition comprising: a first double-stranded ribonucleic acid (dsRNA) agent that inhibits expression of lactate dehydrogenase A (LDHA), the first double-stranded ribonucleic acid (dsRNA) agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementarity comprising at least 15 contiguous nucleotides that differs by no more than 3 nucleotides from any one of the antisense sequences listed in any one of Tables 2-5; and a second double-stranded ribonucleic acid (dsRNA) agent that inhibits expression of hydroxyacid oxidase 1 (glycolic acid oxidase) (HAO1), the second double-stranded ribonucleic acid (dsRNA) agent comprising a sense strand and an antisense strand, the antisense strand comprising a region of complementarity comprising at least 15 contiguous nucleotides that differs by no more than 3 nucleotides from any one of the antisense sequences listed in any one of Tables 7-14.

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

[0067] The present invention provides a method for inhibiting lactate dehydrogenase A (LDHA) expression in a cell, comprising the step of contacting the cell with an agent or pharmaceutical composition of the present invention, thereby inhibiting the expression of LDHA in the cell.

[0068] The present invention also provides a method for inhibiting lactate dehydrogenase A (LDHA) expression and hydroxyacid oxidase 1 (glycolate oxidase) (HAO1) ​​expression in a cell, comprising contacting the cell with a dual-targeting RNAi agent of the invention or a pharmaceutical composition comprising a dual-targeting agent of the invention, thereby inhibiting expression of LDHA and HAO1 in the cell.

[0069] In one embodiment, the cell is 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%, 95%, or to below the level of detection of LDHA expression.

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

[0072] In one embodiment, the human subject is suffering from a disease, disorder or condition associated with the oxalate pathway.

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

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

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

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

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

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

[0079] In one embodiment, the non-hyperoxaluric stone-forming disease, disorder, or condition is a calcium oxalate or non-calcium oxalate kidney stone-forming disease.

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

[0081] In one embodiment, the lactate dehydrogenase-associated disease, disorder, or condition 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, hepatocellular necrosis, liver fibrosis, and non-alcoholic fatty liver disease (NAFLD).

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

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

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

[0085] In one aspect, the present invention provides a method for treating a subject suffering from a disorder that may benefit from reduced LDHA expression, comprising administering a therapeutically effective amount of an agent or pharmaceutical composition of the present invention to the subject, thereby treating the subject.

[0086] In another aspect, the present invention provides a method for preventing at least one symptom in a subject suffering from a disease or disorder that can benefit from reduced expression of the LDHA gene, comprising administering a prophylactically effective amount of an agent or pharmaceutical composition of the present invention to the subject, thereby preventing at least one symptom in the subject.

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

[0088] In one aspect, the present invention provides a method of treating a subject suffering from a disease, disorder, or condition associated with the oxalate pathway, comprising administering to the subject a therapeutically effective amount of an agent or pharmaceutical composition of the present invention, thereby treating the subject.

[0089] In another aspect, the present invention provides a method for preventing at least one symptom in a subject suffering from a disease, disorder, or condition associated with the oxalate pathway, comprising administering to the subject a prophylactically effective amount of an agent or pharmaceutical composition of the present invention, thereby preventing at least one symptom in the subject.

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

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

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

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

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

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

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

[0097] In one embodiment, the non-hyperoxaluric stone-forming disease, disorder, or condition is a calcium oxalate or non-calcium oxalate kidney stone-forming disease.

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

[0099] In one embodiment, the lactate dehydrogenase-associated disease, disorder, or condition 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, hepatocellular necrosis, liver 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 comprises altering the subject's diet (e.g., reducing protein intake, reducing sodium intake, reducing ascorbic acid intake, reducing calcium intake, phosphate supplementation, magnesium supplementation, and pyridoxine treatment; and any combination of the above).

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

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

[0104] In one embodiment, the method further comprises administering to the subject an additional therapeutic agent.

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

[0108] [Figure 1A] FIG. 1A is a schematic diagram of the endogenous pathway for oxalate synthesis. [Figure 1B] FIG. 1B is a schematic diagram of the metabolic pathways associated with 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 0.1 mg / kg, 0.3 mg / kg, 1.0 mg / kg, 3.0 mg / kg, or 10 mg / kg dose of AD-84788. [Figure 3] 3 is a graph showing hepatic LDHA activity in adult male Agxt knockout mice 4 weeks after subcutaneous administration of a single dose of AD-84788 at 0.3 mg / kg, 1 mg / kg, 3 mg / kg, or 10 mg / kg. Agxt knockout mice administered 0 mg / kg AD-84788 served as untreated controls. [Figure 4] FIG. 4 is a schematic diagram of the test protocol described in Example 3 and shown in FIGS. 6-17B. [Figure 5]5 is a graph showing the amount of urinary oxalate (mg per g 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 subcutaneous administration of a single 0.3 mg / kg, 1 mg / kg, 3 mg / kg, or 10 mg / kg dose of AD-84788. Agxt knockout mice administered 0 mg / kg AD-84788 served as untreated controls. [Figure 6] FIG. 6 is a graph showing the amount of oxalate (mg per g of creatinine) excreted in the urine of Agxt knockout, wild-type, and Grhpr (glyoxylate reductase / hydroxypyruvate reductase) knockout mice after 4 weeks of a single 10 mg / kg dose 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 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 10 mg / kg dose of AD-84788; and 28-31 days after the last administration of four 10 mg / kg doses of AD-84788 on days 0, 11, 18, and 25 (see Figure 4). [Figure 8]Figure 8A is a graph showing the enzyme activity of LdhA in wild-type liver homogenates from untreated control mice and mice administered four 10 mg / kg doses of AD-84788 (see Figure 4) using lactate as a substrate. Absorbance increases as NAD is reduced to NADH by LDH enzyme activity. An initial linear range was selected, and absorbances at 1 and 6 minutes were used as the Δabs over a 5-minute Δtime in specific activity calculations. Figure 8B is a graph showing the average specific activity of LdhA in wild-type liver homogenates from untreated control mice and mice administered four 10 mg / kg doses of AD-84788 (see Figure 4) using lactate as a substrate. Specific activity is expressed as μmol of NADH formed / min / g protein. Calculations were performed separately for all animals, and t-tests were performed to compare all specific activity data from both treatment groups. The average specific activity for both treatment groups is shown. (p<0.001). [Figure 9] Figure 9A is a graph showing the enzyme activity of LdhA in wild-type liver homogenates from untreated control mice and mice administered four 10 mg / kg doses of AD-84788 (see Figure 4) using glyoxylate as a substrate. Absorbance increases as NAD is reduced to NADH by LDH enzyme activity. An initial linear range was selected, and absorbances at 0 and 4 minutes were used as the Δabs over a 4-minute Δtime in specific activity calculations. Figure 9B is a graph showing the average specific activity of LdhA in wild-type liver homogenates from untreated control mice and mice administered four 10 mg / kg doses of AD-84788 (see Figure 4) using glyoxylate as a substrate. Specific activity is expressed as μmol of NADH formed / min / g protein. Calculations were performed separately for all animals, and t-tests were performed to compare all specific activity data from both treatment groups. The average specific activity for 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 10 mg / kg doses of AD-84788 (see Figure 4) using lactate as a substrate. Absorbance increases as NAD is reduced to NADH by LDH enzyme activity. An initial linear range was selected, and absorbances at 0 and 4 minutes were used as the Δabs over a Δtime of 4 minutes in the specific activity calculation. The SD for the average treatment group was too small to be visualized. Figure 10B is a graph showing the average specific activity of LdhA in Agxt-deficient liver homogenates from untreated control mice and mice administered four 10 mg / kg doses of AD-84788 (see Figure 4) using lactate as a substrate. Specific activity is expressed as μmol of NADH formed / min / g protein. Calculations were performed separately for all animals and t-tests were performed comparing all specific activity data from both treatment groups. The mean specific activity for 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 10 mg / kg doses of AD-84788 (see Figure 4) using glyoxylate as a substrate. Absorbance increases as NAD is reduced to NADH by LDH enzyme activity. An initial linear range was selected, and absorbances at 0 and 4 minutes were used as the Δabs over a 4-minute Δtime in specific activity calculations. Figure 11B is a graph showing the average specific activity of LdhA in Agxt-deficient liver homogenates from untreated control mice and mice administered four 10 mg / kg doses of AD-84788 (see Figure 4) using glyoxylate as a substrate. Specific activity is expressed as μmol of NADH formed / min / g protein. Calculations were performed separately for all animals, and t-tests were 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 12]Figure 12A is a graph showing the enzymatic activity of LdhA in wild-type heart homogenates from untreated control mice and mice administered four 10 mg / kg doses of AD-84788 (see Figure 4) using lactate as a substrate. The absorbance for both the control and treatment groups increases as NAD is reduced to NADH by LDH enzyme activity. An initial linear range was selected, and the absorbance at 0 and 4 minutes was used as the Δabs over a 4-minute Δtime in the specific activity calculation. Figure 12B is a graph showing the average specific activity of LdhA in wild-type heart homogenates from untreated control mice and mice administered four 10 mg / kg doses of AD-84788 (see Figure 4) using lactate as a substrate. Specific activity is expressed as μmol of NADH formed / min / g protein. Calculations were performed separately for all animals, and t-tests were performed to compare all specific activity data from both treatment groups. The average specific activity of both treatment groups is shown. There is no significant difference. Figure 12C is a graph showing the enzyme activity of LdhA in wild-type thigh muscle homogenates from untreated control mice and mice administered four 10 mg / kg doses of AD-84788 (see Figure 4) using lactate as a substrate. As NAD is reduced to NADH by LDH enzyme activity, the absorbance increases for both the control and treatment groups. An initial linear range was selected, and the absorbance at 0 and 4 minutes was used as the Δabs over a Δtime of 4 minutes in the specific activity calculation. Figure 12D is a graph showing the average specific activity of LdhA in wild-type thigh muscle homogenates from untreated control mice and mice administered four 10 mg / kg doses of AD-84788 (see Figure 4) using lactate as a substrate. Specific activity is expressed as μmol of NADH formed / min / g protein. Calculations were performed for all animals individually and t-tests were performed comparing all specific activity data from both treatment groups. The mean specific activity for both treatment groups is shown. There are no significant differences. [Figure 13]Figure 13A is a graph showing the average amount of lactate in wild-type liver homogenates of wild-type mice before administration of four 10 mg / kg doses of AD-84788 (baseline) and four weeks after administration of four 10 mg / kg doses of AD-84788 (see Figure 4). Figure 13B is a graph showing the average amount of pyruvate in wild-type liver homogenates of wild-type mice before administration of four 10 mg / kg doses of AD-84788 (baseline) and 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 homogenates of Agxt-deficient mice before administration of four 10 mg / kg doses of AD-84788 (baseline) and 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 pyruvate in Agxt-deficient liver homogenates of Agxt-deficient mice before administration of four 10 mg / kg doses of AD-84788 (baseline) and 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 homogenates of wild-type mice before administration of four 10 mg / kg doses of AD-84788 (baseline) and 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 homogenates of Agxt-deficient mice before administration of four 10 mg / kg doses of AD-84788 (baseline) and 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 average body weight of wild-type mice before administration of four 10 mg / kg doses of AD-84788 (baseline) and four weeks after administration of four 10 mg / kg doses of AD-84788 (see Figure 4). Figure 16B is a graph showing the average body weight of Agxt-deficient mice before administration of four 10 mg / kg doses of AD-84788 (baseline) and 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 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 four weeks after administration of four 10 mg / kg doses of AD-84788 (see Figure 4). [Figure 18-1]18A-18O show exemplary dual targeting agents of the 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 comprising a 2'OMe-modified nucleotide (uuu), the 3'-end of the second sense strand comprises a GalNAc ligand, and the two 5'-most nucleotides of the first sense strand each independently comprise a phosphorothioate linkage. 18B shows an exemplary dual-targeting agent of the invention comprising a first dsRNA agent targeted to LDHA and a second dsRNA agent targeted to 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 joined to the 5'-end of the second sense strand by a nucleotide linker comprising a 2' fluoro-modified nucleotide (GfAfAf), the 3'-end of the second sense strand comprises a GalNAc ligand, and wherein the two 5'-most nucleotides of the first sense strand, the 3'-most nucleotide of the first sense strand, and the 5'-most nucleotide of the second sense strand each, independently, comprise a phosphorothioate linkage.18C shows an exemplary dual-targeting agent of the invention comprising a first dsRNA agent targeted to LDHA and a second dsRNA agent targeted to HAO1, wherein the first dsRNA agent comprises a first sense strand (S) and a first antisense strand (AS); the second dsRNA agent comprises a second sense strand (S) and a second antisense strand (AS); 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' fluoro-modified nucleotide (GfAfUf); the 3'-end of the second sense strand comprises a GalNAc ligand; and the two 5'-most nucleotides of the first sense strand, the 3'-most nucleotide of the first sense strand, and the 5'-most nucleotide of the second sense strand each, independently, comprise a phosphorothioate linkage. FIG. 18D shows an exemplary dual-targeting agent of the invention comprising a first dsRNA agent targeted to LDHA and a second dsRNA agent targeted to HAO1, wherein the first dsRNA agent comprises a first sense strand (S) and a first antisense strand (AS); the second dsRNA agent comprises a second sense strand (S) and a second antisense strand (AS); 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 deoxyribonucleotides (dgdada); the 3'-end of the second sense strand comprises a GalNAc ligand; and the two 5'-most nucleotides of the first sense strand, the 3'-most nucleotide of the first sense strand, and the 5'-most nucleotide of the second sense strand each, independently, comprise a phosphorothioate linkage.FIG. 18E shows an exemplary dual-targeting agent of the invention comprising a first dsRNA agent targeted to LDHA and a second dsRNA agent targeted to 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 joined 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 wherein the two 5'-most nucleotides of the first sense strand, the 3'-most nucleotide of the first sense strand, and the 5'-most nucleotide of the second sense strand each, independently, comprise a phosphorothioate linkage. Figure 18F shows an exemplary dual-targeting agent of the invention comprising a first dsRNA agent targeted to LDHA and a second dsRNA agent targeted to HAO1, wherein the first dsRNA agent comprises a first sense strand (S) and a first antisense strand (AS), the second dsRNA agent comprises a second sense strand (S) and a second antisense strand (AS), the 3' end of the first sense strand is joined directly (without a linker) to the 5' end of the second sense strand, the two 5'-most nucleotides of the first sense strand and the two 3'-most nucleotides of the second sense strand each, independently, comprise a phosphorothioate linkage, and the 3' end of the first sense strand comprises a GalNAc ligand. FIG. 18G shows an exemplary dual-targeting agent of the 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); the second dsRNA agent comprises a second sense strand (S) and a second antisense strand (AS); 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 3'-most nucleotides of the first antisense strand and the two 5'-most nucleotides of the second antisense strand each, independently, comprise a phosphorothioate linkage.FIG. 18H shows an exemplary dual-targeting agent of the 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); the second dsRNA agent comprises a second sense strand (S) and a second antisense strand (AS); 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 2' fluoro-modified nucleotides (AfAfGf); the 3'-end of the second sense strand comprises a GalNAc ligand; and the two 3'-most 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 5'-most nucleotides of the second antisense strand each independently comprise a phosphorothioate linkage. FIG. 18I shows an exemplary dual-targeting agent of the 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 attached directly (without a linker) to the 3'-end of the second antisense strand, the 3'-end of the second sense strand comprises a GalNAc ligand, and the two 3'-most nucleotides of the first antisense strand and the two 5'-most nucleotides of the second antisense strand each, independently, comprise a phosphorothioate linkage.FIG. 18J shows an exemplary dual-targeting agent of the 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'OMe-modified nucleotide (uuu), wherein the 5' end of the first sense strand and the 3' end of the second sense strand each independently comprise a GalNAc ligand, and wherein the 5' nucleotide of the first sense strand comprises a phosphorothioate linkage. FIG. 18K shows an exemplary dual-targeting agent of the 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' fluoro-modified nucleotide (GfAfAf), the 5' end of the first sense strand and the 3' end of the second sense strand each independently comprise 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 comprise a phosphorothioate linkage. Figure 18L shows an exemplary dual-targeting agent of the invention comprising a first dsRNA agent targeted to LDHA and a second dsRNA agent targeted to HAO1, wherein the first dsRNA agent comprises a first sense strand (S) and a first antisense strand (AS), the second dsRNA agent comprises a second sense strand (S) and a second antisense strand (AS), the 3' end of the first sense strand is joined directly (without a linker) to the 5' end of the second sense strand, the 3' end of the first sense strand and the 3' end of the second sense strand each independently comprise a GalNAc ligand, and the two 5'-most nucleotides of the first sense strand each independently comprise a phosphorothioate linkage.Figure 18M shows an exemplary dual-targeting agent of the 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), wherein the 3'-end of the first antisense strand and the 3'-end of the second sense strand each independently comprise a GalNAc ligand, and wherein the two 5'-most nucleotides of the second antisense strand each independently comprise a phosphorothioate linkage. FIG. 18N shows an exemplary dual-targeting agent of the 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 2' fluoro-modified nucleotides (AfAfGf), wherein the 3'-end of the first antisense strand and the 3'-end of the second sense strand each independently comprise a GalNAc ligand, and wherein the 5'-nucleotide of the first antisense strand, the 3'-nucleotide of the second antisense strand, and the two 5'-most nucleotides of the second antisense strand each independently comprise a phosphorothioate linkage.FIG. 18O shows an exemplary dual-targeting agent of the 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) attached to the 3' end of the second antisense strand, the 3' end of the first antisense strand and the 3' end of the second sense strand each independently comprise a GalNAc ligand, and the two 5'-most nucleotides of the second antisense strand each independently comprise a phosphorothioate linkage. [Figure 18-2] Same as above. [Figure 18-3] Same as above. [Figure 18-4] Same as above. [Figure 18-5] Same as above. DETAILED DESCRIPTION OF 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 can be present in a cell, for example, a cell in a subject, such as a human. 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, such as subjects who may benefit from reducing or inhibiting urinary oxalate production, for example, subjects suffering from or susceptible to diseases, disorders, or conditions associated with the oxalate pathway, such as oxalate-related diseases, disorders, or conditions, such as kidney stone formation diseases, disorders, or conditions, or calcium oxalate tissue deposition diseases, disorders, or conditions; or subjects suffering from or susceptible to LDH-related diseases, disorders, or conditions.

[0110] The present invention also provides methods of using iRNA compositions of the present invention to inhibit expression of the LDHA gene and the HAO1 gene to treat subjects who may benefit from inhibiting or reducing expression of the LDHA gene and the HAO1 gene, e.g., subjects who may benefit from a reduction or inhibition of urinary oxalate production, e.g., subjects suffering from or susceptible to a disease, disorder, or condition associated with the oxalate pathway, e.g., an oxalate-associated disease, disorder, or condition, e.g., a kidney stone formation disease, disorder, or condition or a calcium oxalate tissue deposition disease, disorder, or condition; or a LDH-associated disease, disorder, or condition.

[0111] The iRNA of the present invention targeting LDHA is about 30 nucleotides in length or less, for example, 15 to 30, 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 26, 19 to 27, 19 to 28, 19 to 29, 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 26, 19 to 26, 19 to 27, 19 to 28, 19 to 29, 19 to 26, 19 to 25, 19 to 26, 19 to 26, 19 to 26, 19 to 26, 19 to 27, 19 to 26 ... The LDHA gene may comprise an RNA strand (antisense strand) having a region that is 9 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 30, 20 to 29, 20 to 28, 20 to 27, 20 to 26, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, or 21 to 22 nucleotides in length, which region is substantially complementary to at least a portion of an mRNA transcript of the LDHA gene.

[0112] The iRNA of the present invention targeting HAO1 may be about 30 nucleotides in length or less, 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, 19-26, 19-27, 19-28, 19-29 ... The HAO1 gene may comprise an RNA strand (antisense strand) having a region that is 9 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 30, 20 to 29, 20 to 28, 20 to 27, 20 to 26, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, or 21 to 22 nucleotides in length, which region is substantially complementary to at least a portion of an mRNA transcript of the HAO1 gene.

[0113] When the RNAi agent is a dual-targeting RNAi agent as described herein, the agent targeting LDHA may include an antisense strand that includes a region of complementarity to LDHA that is the same length as or a different length from the region of complementarity of the antisense strand of the agent targeting HAO1.

[0114] In certain embodiments, one or both strands of a double-stranded RNAi agent of the invention have a region of at least 19 contiguous nucleotides substantially complementary to at least a portion of an mRNA transcript of the LDHA gene and are up to 66 nucleotides in length, e.g., 36-66, 26-36, 25-36, 31-60, 22-43, 27-53 nucleotides in length. In certain embodiments, such iRNA agents having a longer length antisense strand can include a second RNA strand (sense strand) that is 20-60 nucleotides in length, where the sense and antisense strands form a duplex of 18-30 contiguous nucleotides.

[0115] In other embodiments, one or both strands of a double-stranded RNAi agent of the invention have a region of at least 19 contiguous nucleotides substantially complementary to at least a portion of an mRNA transcript of the HAO1 gene and are up to 66 nucleotides in length, e.g., 36-66, 26-36, 25-36, 31-60, 22-43, 27-53 nucleotides in length. In certain embodiments, such iRNA agents having a longer antisense strand length can include a second RNA strand (sense strand) that is 20-60 nucleotides in length, where the sense and antisense strands form a duplex of 18-30 contiguous nucleotides.

[0116] In embodiments in which a first dsRNA agent targeting LDHA and a second dsRNA agent targeting HAO1 are covalently linked, the duplex lengths of the first and second agents can be the same or different.

[0117] The use of these iRNA agents described herein allows for the targeted degradation of the mRNA of the LDHA gene in a mammal or the targeted degradation of the LDHA gene and the HAO1 gene in a mammal.

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

[0119] The following detailed description discloses methods for making and using compositions containing iRNA to inhibit 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 that may benefit from inhibiting and / or reducing the expression of these genes.

[0120] I. Definition So that the present invention may be more readily understood, several terms are first defined. Furthermore, it should be noted that whenever a value or range of values ​​for a variable is recited, all values ​​and ranges intermediate to the recited values ​​are also intended to be part of the invention.

[0121] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element, e.g., a plurality of elements.

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

[0123] The term "LDHA" (used interchangeably herein with the term "Ldha"), also known as cell proliferation-inducing gene 19 protein, kidney 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 the well-known gene encoding lactate dehydrogenase A from any vertebrate or mammalian source, including, but not limited to, human, bovine, chicken, rodent, mouse, rat, pig, sheep, primate, monkey, and guinea pig.

[0124] The term also refers to fragments and variants of native LDHA that maintain at least one in vivo or in vitro activity of native LDHA. The term encompasses full-length unprocessed precursor forms of LDHA as well as mature forms resulting from post-translational cleavage of the signal peptide and forms resulting from proteolytic processing.

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

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

[0127] As used herein, the term "LDHA" also refers to the specific polypeptide that is expressed in cells due to the natural DNA sequence variation of the LDHA gene, such as single nucleotide polymorphism in the LDHA gene.Many SNPs in 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, unless otherwise specified, the term "HAO1" refers to the well-known gene encoding the enzyme hydroxyacid oxidase 1 from any vertebrate or mammalian source, including, but not limited to, human, cow, chicken, rodent, mouse, rat, pig, sheep, primate, monkey, and guinea pig. Other gene names include GO, GOX, GOX1, HAO, and HAOX1. This protein is also known as glycolate oxidase and (S)-2-hydroxyacid oxidase.

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

[0130] As used herein, the term "HAO1" also refers to natural 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 upon transcription of the LDHA gene or HAO1 gene, including mRNAs that are the product of RNA processing of a primary transcript. In one embodiment, the target portion of the sequence will be at least long enough to serve as a substrate for iRNA-directed cleavage at or near that portion of the nucleotide sequence of an mRNA molecule formed upon transcription of the LDHA gene. In another embodiment, the target portion of the sequence will be at least long enough to serve as a substrate for iRNA-directed cleavage at or near that portion of the nucleotide sequence of an mRNA molecule formed upon transcription of the HAO1 gene.

[0132] The target sequence of the LDHA gene can be about 9 to 36 nucleotides in length, for example, about 15 to 30 nucleotides in length. For example, the target sequence can be about 15 to 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 It can 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 in length. Ranges and lengths intermediate to the above-listed ranges and lengths are also considered part of the invention.

[0133] The target sequence of the HAO1 gene can be about 9 to 36 nucleotides in length, for example, about 15 to 30 nucleotides in length. For example, the target sequence can be about 15 to 30 nucleotides, 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to 29, 1 It can 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 in length. Ranges and lengths intermediate to the above ranges and lengths are also contemplated as part of the invention.

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

[0135] As used herein, the term "strand comprising a sequence" refers to an oligonucleotide comprising a strand of nucleotides represented by a sequence given 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 term "ribonucleotide" or "nucleotide" can also refer to modified nucleotides or surrogate replacement moieties, as described in more detail below (see, for example, Table 1). Those skilled in the art will appreciate that guanine, cytosine, adenine, and uracil may be substituted by other moieties without significantly changing the base pairing properties of oligonucleotides containing nucleotides with such replacement moieties. For example, but not limited to, a nucleotide containing inosine as its base can base pair with a nucleotide containing adenine, cytosine, or uracil. Thus, a nucleotide containing uracil, guanine, or adenine may be substituted, for example, by a nucleotide containing inosine in the nucleotide sequence of a dsRNA characterized in the present invention. In another example, adenine and cytosine anywhere in an oligonucleotide can be substituted with guanine and uracil, respectively, to form a GU wobble base pair with the target mRNA. Sequences containing such substitutions are suitable for the compositions and methods featured in the present invention.

[0137] The terms "iRNA," "RNAi agent," "iRNA agent," and "RNA interfering agent," as used interchangeably herein, refer to an agent that contains RNA and mediates targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway, as those terms are defined herein. iRNA directs sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). iRNA regulates (e.g., inhibits) expression of the LDHA and / or HAO1 gene in a cell, e.g., a cell in a subject, such as a mammalian subject.

[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 target mRNA sequence and / or an HAO1 target mRNA sequence, and guides cleavage of the target RNA. Without wishing to be bound by theory, it is believed that long double-stranded RNA introduced into cells is degraded into siRNAs 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 19-23 base pair short interfering RNAs with characteristic two-base 3' overhangs (Bernstein, et al., (2001) Nature 409:363). Then, siRNA is incorporated into RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, allowing the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). When bound to the appropriate target mRNA, one or more endonucleases in RISC cleave the target, inducing silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Here, in one aspect, the present invention relates to a single-stranded RNA (sssiRNA) that is produced in cells and promotes the formation of a RISC complex that leads to the silencing of target genes, i.e., LDHA gene and / or HAO1 gene. Therefore, the term "siRNA" is also used herein to refer to the above-mentioned RNAi.

[0139] In another embodiment, the RNAi agent can be a single-stranded RNAi agent introduced into a cell or organism to inhibit target mRNA. Single-stranded RNAi agents (ssRNAi) bind to the RISC endonuclease Argonaute 2, which then cleaves the target mRNA. Single-stranded siRNAs are generally 15-30 nucleotides and chemically modified. The design and testing of single-stranded RNAi agents is described in U.S. Patent No. 8,101,348 and Lima et al., (2012) Cell 150:883-894, the entire contents of each of which are incorporated herein by reference. Any of the antisense nucleotide sequences described herein can 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 to herein as a "double-stranded RNAi agent," "double-stranded RNA (dsRNA) molecule," "dsRNA agent," or "dsRNA." The term "dsRNA" refers to a complex of ribonucleic acid molecules having a double-stranded structure comprising two antiparallel and substantially complementary nucleic acid strands, which are shown to have "sense" and "antisense" orientations relative to the target RNA, i.e., the LDHA gene and / or HAO1 gene. In one embodiment of the present invention, the double-stranded RNA (dsRNA) causes degradation of the target RNA, e.g., mRNA, by a post-transcriptional gene silencing mechanism, referred to herein 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 comprising a first target RNA, i.e., a first dsRNA agent comprising a complex of ribonucleic acid molecules having a double-stranded structure comprising two antiparallel and substantially complementary nucleic acid strands, shown to have a "sense" and "antisense" orientation relative to the LDHA gene, covalently linked to a molecule comprising a second target RNA, i.e., a second dsRNA agent comprising a complex of ribonucleic acid molecules having a double-stranded structure comprising two antiparallel and substantially complementary nucleic acid strands, shown to have a "sense" and "antisense" orientation relative to the HAO1 gene. In one embodiment of the present invention, the dual-targeting RNAi agent causes degradation of the first and second target RNAs, e.g., mRNAs, 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; however, 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 with chemical modifications; RNAi agents may contain substantial modifications in multiple nucleotides. As used herein, the term "modified nucleotide" refers to a nucleotide that independently has a modified sugar moiety, a modified internucleotide bond, and / or a modified nucleobase. Thus, the term modified nucleotide encompasses, for example, the substitution, addition, or removal of a functional group or atom in the internucleoside bond, sugar moiety, or nucleobase. 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 modifications when used in siRNA-type molecules are encompassed by "RNAi agent" for the purposes of this specification and claims.

[0143] The double-stranded region can be of any length that allows for specific degradation of the desired target RNA via the RISC pathway, and can range from about 9 to 36 base pairs in length, e.g., about 15 to 30 base pairs in length, e.g., about 15 to 30, 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to 29, 19 to 28, 19 to 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 The nucleic acid sequence may be about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length, such as 21-25, 21-24, 21-23, or 21-22 base pairs in length. Ranges and lengths intermediate to the above-listed ranges and lengths are also contemplated as part of the invention.

[0144] In embodiments in which a first dsRNA agent targeting LDHA and a second dsRNA agent targeting HAO1 are covalently linked (i.e., a dual-targeting RNAi agent), the lengths of the double-stranded regions of the first and second agents can 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 connected by a continuous chain of nucleotides between the 3' end of one strand and the 5' end of the other strand that form the double-stranded structure, the connected RNA strands are called "hairpin loops." 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 20, 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 linked (i.e., a dual-targeting RNAi agent), the first dsRNA agent may comprise a hairpin loop, the second dsRNA agent may comprise a hairpin loop, or both the first and second dsRNA agents may independently comprise a hairpin loop. Further, in embodiments in which a first dsRNA agent targeting LDHA and a second dsRNA agent targeting HAO1 are covalently linked (i.e., a dual-targeting RNAi agent), the first dsRNA agent may comprise an unpaired nucleotide, the second dsRNA agent may comprise an unpaired nucleotide, or both the first and second dsRNA agents may independently comprise unpaired nucleotides. When both the first and second dsRNA agents independently comprise unpaired nucleotides, the first dsRNA agent and the second dsRNA agent may comprise the same or different numbers of unpaired nucleotides.

[0147] When the two substantially complementary strands of dsRNA are composed of separate RNA molecules, these molecules can be covalently linked, but they do not have to be.When the two strands are covalently linked by means other than a continuous chain of nucleotides between the 3'-end of one strand and the 5'-end of the other strand that form a double-stranded structure, the linked structure is called a "linker".The RNA strands can have the same or different number of nucleotides.The maximum number of base pairs is the number of nucleotides of the shortest strand of dsRNA minus the overhang that exists in the double strand.In addition to the double-stranded structure, the RNAi agent can include one or more nucleotide overhangs.

[0148] In one embodiment, an RNAi agent of the invention is a dsRNA, each strand of which comprises 19-23 nucleotides that interact with a target RNA sequence, e.g., an LDHA target mRNA sequence, to direct cleavage of the target RNA. In another embodiment, an RNAi agent of the invention is a dsRNA, each strand of which comprises 19-23 nucleotides that interact with a target RNA sequence, e.g., an HAO1 target mRNA sequence, to direct cleavage of the target RNA. In yet another embodiment, an RNAi agent of the invention comprises a first dsRNA agent, each strand of which comprises 19-23 nucleotides that interact with a target RNA sequence, e.g., an LDHA target mRNA sequence, to direct cleavage of the target RNA, and a second dsRNA agent, each strand of which independently comprises 19-23 nucleotides that interact with a target RNA sequence, e.g., an HAO1 target mRNA sequence, to direct cleavage of the target RNA, wherein the first and second dsRNA agents are covalently linked.

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

[0150] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide protruding from the double-stranded structure of an iRNA, such as a dsRNA. For example, a nucleotide overhang exists when the 3'-end of one strand of a dsRNA extends beyond the 5'-end of the other strand, or vice versa. A dsRNA may contain an overhang of at least one nucleotide; alternatively, 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 comprise or consist of nucleotide / nucleoside analogs, including deoxynucleotides / nucleosides. The overhang may be in the sense strand, the antisense strand, or any combination thereof. Furthermore, the overhanging nucleotide may be present at the 5'-end, the 3'-end, or both ends of either the antisense strand or the sense strand of a dsRNA.

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

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

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

[0154] In certain embodiments, the overhang on the sense strand or the antisense strand, or both, may comprise an extended length greater than 10 nucleotides, e.g., 10-30 nucleotides, 10-25 nucleotides, 10-20 nucleotides, or 10-15 nucleotides in length. In certain embodiments, the extended overhang is present on the sense strand of the duplex. In certain embodiments, the extended overhang is present on the 3'-end of the sense strand of the duplex. In certain embodiments, the extended overhang is present on the 5'-end of the sense strand of the duplex. In certain embodiments, the extended overhang is present on the antisense strand of the duplex. In certain embodiments, the extended overhang is present on the 3'-end of the antisense strand of the duplex. In certain embodiments, the extended overhang is present on the 5'-end of the antisense strand of the duplex. In certain embodiments, one or more of the 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 linked (i.e., a dual-targeting RNAi agent), one and / or both strands of both the first and second dsRNA agents independently comprise an overhang, e.g., an extended overhang, the lengths of the overhangs may be the same or different, and / or in certain embodiments, one or more of the nucleotides in the overhang in the first dsRNA agent and one or more nucleotides in the overhang in the second dsRNA agent may independently be substituted with a nucleoside thiophosphate.

[0156] The term "blunt" or "blunt-ended" used herein in relation to dsRNA means that there are no unpaired nucleotides or nucleotide analogs at a given end of dsRNA, i.e., there are no nucleotide overhangs.One or both ends of dsRNA can be blunt.If both ends of dsRNA are blunt, the dsRNA is described as blunt-ended.For clarity, "blunt-ended" dsRNA is a dsRNA whose both ends are blunt, i.e., there are no nucleotide overhangs at either end of the molecule.Most of these molecules are often double-stranded throughout their entire length.

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

[0158] The term "antisense strand" or "guide strand" refers to the strand of an iRNA, e.g., a dsRNA, that includes a region that is substantially complementary to a target sequence, e.g., LDHA mRNA or HAO1 mRNA.

[0159] As used herein, the term "region of complementarity," as defined herein, refers to a region of the antisense strand that is substantially complementary to a sequence, e.g., a target sequence, e.g., an LDHA nucleotide sequence or an HAO1 nucleotide sequence. If the region of complementarity is not completely complementary to the target sequence, mismatches may exist in internal or terminal regions of the molecule. Generally, the most tolerated mismatches are found in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides at the 5' and / or 3' ends of the iRNA.

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

[0161] The term "sense strand," or "passenger strand," as used herein, refers to the strand of an iRNA that includes a region that is substantially complementary to a region of the antisense strand, as those terms are defined herein.

[0162] As used herein, the term "cleavage region" refers to a region located immediately adjacent to the cleavage site. The cleavage site is the site in the target where cleavage occurs. In some embodiments, the cleavage region comprises three bases immediately adjacent to the cleavage site on either side of the cleavage site. In some embodiments, the cleavage region comprises two bases immediately adjacent to the cleavage site on either side of the cleavage site. In some embodiments, the cleavage site specifically occurs at the site bound by nucleotides 10 and 11 of the antisense strand, and the cleavage region comprises nucleotides 11, 12, and 13.

[0163] As used herein, unless otherwise indicated, the term "complementary," when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize to an oligonucleotide or polynucleotide comprising the second nucleotide sequence and form a double-stranded structure under defined conditions, as understood by those of skill in the art. Such conditions may be, for example, stringent conditions, where stringent conditions may include 400 mM NaCl, 40 mM PIPES (pH 6.4), 1 mM EDTA, at 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 inside an organism, may be applied. Those skilled in the art will be able to determine the most appropriate set of conditions for testing the complementarity of two sequences depending on the ultimate use of the hybridized nucleotides.

[0164] A complementary sequence in an iRNA, e.g., a dsRNA, described herein includes base pairing across the entire length of one or both nucleotide sequences of an oligonucleotide or polynucleotide comprising a first nucleotide sequence to an oligonucleotide or polynucleotide comprising a second nucleotide sequence. Such sequences may be referred to herein as "fully complementary" to each other. However, when a first sequence is referred to herein as "substantially complementary" to a second sequence, the two sequences may be perfectly complementary, or they may form one or more, but generally no more than five, four, three, or two mismatched base pairs upon hybridization to a duplex of up to 30 base pairs while retaining the ability to hybridize under conditions optimal for their ultimate application, e.g., inhibiting gene expression via the RISC pathway. However, if two oligonucleotides are designed to form one or more single-stranded overhangs after hybridization, such overhangs shall not be considered mismatches in determining complementarity. For example, for purposes described herein, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length may be referred to as "fully complementary" if the longer oligonucleotide comprises a 21 nucleotide sequence that is perfectly complementary to the shorter oligonucleotide.

[0165] As used herein, "complementary" sequences can also include, or be formed entirely of, non-Watson-Crick base pairs and / or base pairs formed from unnatural and modified nucleotides, so long as the above requirements related to their ability to hybridize are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble or Hoogsteen base pairs.

[0166] The terms "complementary," "fully complementary," and "substantially complementary" herein may be used in reference to matching bases between the sense and antisense strands of a dsRNA, or between the antisense strand of a dsRNA and a target sequence, as understood from the context of their use.

[0167] As used herein, a polynucleotide "substantially complementary to at least a portion of" a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of an mRNA of interest (e.g., an mRNA encoding LDHA or an 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 an LDHA mRNA if its sequence is substantially complementary to a contiguous portion of an mRNA encoding LDHA.

[0168] Thus, in some embodiments, the antisense strand polynucleotides disclosed herein are fully complementary to the target LDHA sequence. In other embodiments, the antisense strand polynucleotides disclosed herein are substantially complementary to the target LDHA sequence, and include a contiguous 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 that is substantially complementary to the antisense polynucleotide and thus complementary to the target LDHA sequence, wherein the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least about 80% complementary, e.g., 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 fragments of SEQ ID NO: 2, over its entire length.

[0170] In one embodiment, an iRNA of the invention comprises an antisense strand that is substantially complementary to a target LDHA sequence and comprises a contiguous nucleotide sequence over its entire length that is at least about 80% complementary, e.g., about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementary, or 100% complementary, to a 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] Thus, in certain embodiments, the antisense strand polynucleotides disclosed herein are fully complementary to the target HAO1 sequence. In other embodiments, the antisense strand polynucleotides disclosed herein are substantially complementary to the target HAO1 sequence, and comprise a contiguous nucleotide sequence over its entire length that is at least about 80% complementary, e.g., 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.

[0172] In one embodiment, the RNAi agent of the present invention comprises a sense strand that is substantially complementary to the antisense polynucleotide and thus complementary to the target HAO1 sequence, and the sense strand polynucleotide comprises a contiguous nucleotide sequence over its entire length that is at least about 80% complementary, e.g., 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 fragment of SEQ ID NO:22.

[0173] In one embodiment, an iRNA of the invention comprises an antisense strand that is substantially complementary to a target HAO1 sequence and comprises a contiguous nucleotide sequence over its entire length that is at least about 80% complementary, e.g., about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementary, or 100% complementary, to a 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 used interchangeably with "reducing," "silencing," "downregulating," "suppressing," and other similar terms, and includes any level of inhibition.

[0175] As used herein, the phrase "inhibiting expression of the LDHA gene" includes inhibition of 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 the LDHA gene that encode the LDHA protein.

[0176] "Inhibiting expression of the LDHA gene" includes any level of inhibition of the LDHA gene, e.g., at least partial suppression of expression of the LDHA gene, such as at least about 20% inhibition. In certain embodiments, inhibition is by 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 "inhibiting expression of the HAO1 gene" includes inhibition of expression of any HAO1 gene (e.g., mouse HAO1 gene, rat HAO1 gene, monkey HAO1 gene, or human HAO1 gene, etc.) as well as variants or mutants of the HAO1 gene that encode the HAO1 protein.

[0178] "Inhibiting expression of the HAO1 gene" includes any level of inhibition of the HAO1 gene, e.g., at least partial suppression of expression of the HAO1 gene, such as at least about 20% inhibition. In certain embodiments, inhibition is by 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 linked, the inhibition of expression of LDHA can be the same as or different from the inhibition of HAO1 expression.

[0180] The expression of the LDHA gene and / or the HAO1 gene can be evaluated based on the level of any variable associated with LDHA gene expression and / or HAO1 gene expression, such as the LDHA and / or HAO1 mRNA level or the LDHA and / or HAO1 protein level. The expression of the LDHA gene and / or the HAO1 gene can also be evaluated indirectly based on the level of oxalate or glycolate in urine, plasma, or tissue samples, or the enzymatic activity of LDHA in tissue samples, such as liver, skeletal muscle, and / or heart samples. Inhibition can be evaluated by a decrease in the absolute or relative level of one or more of these variables compared to the control level. The control level can be any type of control level used in the art, such as a pre-dose baseline level, or a level measured from a similar subject, cell, or sample that is 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 expression of the LDHA gene is assessed by a reduction in the amount of LDHA mRNA that can be isolated from or detected in a first cell or group of cells in which the LDHA gene is transcribed and that have been treated to inhibit expression of the LDHA gene, compared to a second cell or group of cells (control cells) that is substantially identical to the first cell or group of cells but has not been so treated.

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

[0183] In one embodiment, at least partial suppression of expression of the LDHA gene and HAO1 gene is assessed by a reduction in the amount of LDHA mRNA and HAO1 mRNA that can be isolated from or detected in a first cell or cell group in which the LDHA gene and HAO1 gene are transcribed and that has been treated to inhibit expression of the LDHA gene and HAO1 gene, 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 so treated.

[0184] The degree of inhibition can be expressed in the following formula:

number

[0185] As used herein, the phrase "contacting a cell with an RNAi agent (such as dsRNA)" includes contacting a cell by any possible means. Contacting a cell with an RNAi agent includes contacting a cell with an iRNA in vitro or contacting a cell with an iRNA in vivo. Contact can be performed directly or indirectly. Thus, for example, an RNAi agent can be physically contacted with a cell by performing a method individually, or the RNAi agent can be placed in a situation that allows it to contact or allows it to contact a cell later.

[0186] In methods of the invention in which a first dsRNA agent targeting LDHA and a second dsRNA agent targeting HAO1 are covalently linked (i.e., a dual-targeting RNAi agent), the step of contacting the cell can include contacting the cell with the first agent at the same time as or at different times from contacting the cell with the second agent.

[0187] The step of contacting cells in vitro can be carried out, for example, by incubating cells with an RNAi agent.The step of contacting cells in vivo can 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 area, for example, the bloodstream or subcutaneous cavity, so that the RNAi agent can reach the tissue where the contacted cells are located later.For example, the RNAi agent can contain and / or be bound to a ligand, such as GalNAc3, that directs the RNAi agent to a target site, for example, the liver.A combination of in vitro and in vivo contacting methods is also possible.For example, cells can also be contacted with an RNAi agent in vitro and then transplanted into a subject.

[0188] In one embodiment, contacting a cell with an iRNA includes "introducing" or "delivering the iRNA into a cell" by promoting or effecting uptake or absorption into the cell. Absorption or uptake of the iRNA can occur by unassisted diffusion or active cellular processes, or by auxiliary agents or devices. Introduction of the iRNA into a cell can be in vitro and / or in vivo. For example, for in vivo introduction, the iRNA can be injected into a tissue site or administered systemically. In vivo delivery can also be achieved by β-glucan delivery systems, such as those described in U.S. Pat. Nos. 5,032,401 and 5,607,677 and U.S. Patent Application Publication No. 2005 / 0281781, the entire contents of which are incorporated herein by reference. In vitro introduction into a cell includes methods known in the art, such as electroporation and lipofection. Additional techniques are described herein below and / or are known in the art.

[0189] The term "lipid nanoparticle" or "LNP" refers to a vesicle comprising a lipid layer that encapsulates a pharmaceutically effective molecule, such as a nucleic acid molecule, e.g., an iRNA or a plasmid into which the iRNA is transcribed. LNPs are described, for example, in U.S. Patent Nos. 6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents of which are incorporated herein by reference.

[0190] As used herein, a "subject" is an animal such as a mammal, including a primate (such as a human or a non-human primate, e.g., monkey, or chimpanzee), a non-primate (such as a cow, pig, camel, llama, horse, goat, rabbit, sheep, hamster, guinea pig, cat, dog, rat, mouse, horse, and whale), or a bird (e.g., a duck or goose).

[0191] In one embodiment, the subject is a human, such as a human being treated or evaluated for a disease, disorder, or condition that may benefit from reduced LDHA expression, as described herein; a human being at risk for a disease, disorder, or condition that may benefit from reduced LDHA expression; a human suffering from a disease, disorder, or condition that may benefit from reduced LDHA expression; and / or a human being being treated for a disease, disorder, or condition that may benefit from reduced LDHA expression.

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

[0193] As used herein, the term "treat" or "treatment" refers to a beneficial or desired result, such as reducing the urinary excretion level of oxalate in a subject. The term "treat" or "treatment" also includes, but is not limited to, alleviating or ameliorating one or more symptoms of a disease, disorder, or condition associated with the oxalate pathway, such as, for example, slowing the progression of the disease; reducing the severity of late-developing disease; reducing swelling of the limbs, face, larynx, upper respiratory tract, abdomen, trunk, and / or genitalia, 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 current stone burden; reducing the recurrence of stone formation; and / or preventing further oxalate tissue deposition. "Treatment" can also mean prolonging survival compared to the expected survival time without treatment.

[0194] The term "reducing," in reference to a disease marker or symptom, refers to a statistically significant decrease in such level, which may be, for example, by 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, preferably to a level considered to be within the normal range for individuals without such disorder.

[0195] As used herein, "prevention" or "preventing," when used in connection with a disease, disorder, or condition that may benefit from reduced expression of the LDHA gene, refers to a reduction in the likelihood that a subject will develop symptoms associated with such a disease, disorder, or condition, such as stone formation. For example, the likelihood of stone formation is reduced when, for example, an individual with one or more risk factors for stone formation does not develop stones, or develops milder stones than a group with the same risk factors who does not receive the treatment described herein. Effective prevention is considered to be a lack of disease, disorder, or condition, or a reduction in the occurrence of symptoms associated with such a disease, disorder, or condition (e.g., at least about 10% on a clinically recognized scale for the disease or disorder), or a delayed onset of symptoms (e.g., by only a few days, weeks, months, or years).

[0196] There are many disorders that could benefit from reduced expression of the LDHA gene, such as diseases, disorders, or conditions associated with the oxalate pathway.

[0197] As used herein, the term "oxalate pathway-associated disease, disorder, or condition" refers to a disease, disorder, or condition, e.g., associated with or caused by disruption of lactate dehydrogenase production and / or urinary oxalate production, for which lactate dehydrogenase knockdown is known or predicted to be therapeutically or otherwise beneficial.

[0198] In one embodiment, the "oxalate pathway-associated disease, disorder, or condition" is a "lactate dehydrogenase-associated disease, disorder, or condition." As used herein, a "lactate dehydrogenase-associated disease, disorder, or condition" includes any disease, disorder, or condition that would benefit from a decrease in lactate dehydrogenase gene expression, replication, or protein activity. Exemplary lactate dehydrogenase-associated diseases, disorders, and conditions include, for example, fatty liver (steatosis), non-alcoholic steatohepatitis (NASH), cirrhosis, accumulation of fat in the liver, liver inflammation, hepatocellular necrosis, liver fibrosis, obesity, non-alcoholic fatty liver disease (NAFLD), and cancer, e.g., hepatocellular carcinoma.

[0199] In another embodiment, an "oxalate pathway-associated disease, disorder, or condition" is an "oxalate-associated disease, disorder, or condition." As used herein, an "oxalate-associated disease, disorder, or condition" includes any disease, disorder, or condition that would benefit from a decrease in lactate dehydrogenase gene expression, replication, or protein activity. The term "oxalate-associated disease, disorder, or condition" refers to an inherited disease, or an induced or acquired disease. Exemplary "oxalate-associated diseases, disorders, or conditions" include "kidney stone formation diseases, disorders, and conditions" and "calcium oxalate tissue deposition diseases, disorders, and conditions."

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

[0201] Non-limiting examples of "calcium oxalate stone-forming 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; enteric hyperoxaluria; dietary hyperoxaluria; and idiopathic hyperoxaluria) and non-hyperoxaluric disorders (e.g., hypercalciuria, such as primary hyperparathyroidism, Dent's disease, absorptive hypercalciuria, and nephrogenic hypercalciuria; and hypocitraturia).

[0202] Non-limiting examples of "non-calcium oxalate stone-forming diseases, disorders, and conditions" include subjects with kidney stones that are composed of less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, or less than about 10% oxalate and more than about 50% non-oxalate, e.g., 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 due to end-stage renal disease, sarcoidosis, or arthritis; and tissue-specific calcium oxalate deposition diseases, disorders, and conditions due to organ transplantation, such as kidney transplantation, e.g., in the kidney (e.g., due to nephrocalcinosis, or sponge kidney), in the thyroid gland, in the breast, in the bone, in the heart, in the vasculature, or in any soft tissue.

[0204] As used herein, a "therapeutically effective amount" is intended to include the amount of an RNAi agent that, when administered to a subject suffering from a disease, disorder, or condition associated with the oxalate pathway, is sufficient to treat the disease (e.g., by reducing, ameliorating, or maintaining one or more symptoms of an existing disease or condition). A "therapeutically effective amount" may vary depending on the RNAi agent, the manner in which the agent is administered, the disease and its severity, and the medical history, age, weight, family history, genetic makeup, type of previous or concurrent treatment, if any, and other individual characteristics of the subject being treated.

[0205] As used herein, a "prophylactically effective amount" is intended to include an amount of iRNA sufficient to prevent or ameliorate the disease or one or more symptoms of the disease when administered to a subject suffering from a disease, disorder, or condition associated with the oxalate pathway. Amelioration of the disease includes slowing the progression of the disease or reducing the severity of subsequent disease. A "prophylactically effective amount" may vary depending on the iRNA, the method by which the agent is administered, the risk of developing the disease, and the patient's medical history, age, weight, family history, genetic makeup, type of previous or concurrent treatment, if any, and other individual characteristics.

[0206] A "therapeutically effective amount" or "prophylactically effective amount" also includes an amount of an RNAi agent that produces a desired local or systemic effect at a reasonable benefit-to-risk ratio applicable to any treatment. The iRNAs used in the methods of the invention can be administered in amounts sufficient to obtain a reasonable benefit-to-risk ratio applicable to such treatment.

[0207] In the methods of the present invention, which comprise administering to a subject a pharmaceutical composition comprising a first dsRNA agent targeting LDHA and a second dsRNA agent targeting HAO1, the therapeutically effective amount of the first dsRNA agent can be the same as or different from the therapeutically effective amount of the second dsRNA agent.Similarly, in the methods of the present invention, which comprise administering to a subject a pharmaceutical composition comprising a first dsRNA agent targeting LDHA and a second dsRNA agent targeting HAO1, the prophylactically effective amount of the first dsRNA agent can be the same as or different from the prophylactically effective amount of the second dsRNA agent.

[0208] The phrase "pharmaceutically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are suitable, within the scope of sound medical judgment, for use in contact with the tissues of human and animal subjects without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit-risk ratio.

[0209] As used herein, the phrase "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc, magnesium, calcium, or zinc stearate, or stearic acid), or solvent encapsulating material, that is involved in carrying or transporting 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 of being compatible with the other ingredients of the formulation and 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 and their derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (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; and (10) propylene glycol. (11) polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free distilled water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffers; (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, compatible materials used in pharmaceutical formulations.

[0210] As used herein, the term "sample" includes similar bodily fluids, cells, or tissues isolated from a subject, as well as collections of bodily fluids, cells, or tissues present in a subject. Examples of biological fluids include blood, serum and serous fluid, plasma, cerebrospinal fluid, ocular fluid, lymph, urine, saliva, etc. Tissue samples can include samples derived from tissues, organs, or localized regions. For example, samples can be derived from specific organs, parts of organs, or bodily fluids or cells within those organs. In certain embodiments, samples can be derived from the liver (e.g., the whole liver or specific parts of the liver, or specific types of cells in the liver, such as hepatocytes). In certain embodiments, a "sample derived from a subject" refers to blood or plasma obtained from a subject.

[0211] II. iRNAs of the 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 a cell, e.g., a hepatocyte, e.g., a hepatocyte in a subject, e.g., a mammal, e.g., a human, suffering from a disease, disorder, or condition associated with the oxalate pathway, e.g., a stone-forming disease, disorder, or condition. 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 a cell, e.g., a hepatocyte in a subject, e.g., a mammal, e.g., a human, suffering from a disease, disorder, or condition associated with the oxalate pathway, e.g., an oxalate-associated disease, disorder, or condition, e.g., a kidney stone-forming disease, disorder, or condition, or a calcium oxalate tissue deposition disease, disorder, or condition; or a disease, disorder, or condition associated with LDH.

[0212] Also provided herein are iRNAs that inhibit the expression of two target genes, referred to as dual-targeting RNAi agents. 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 a cell (such as a liver cell, for example, a liver cell in a subject), covalently linked to a second double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of the HAO1 gene in a cell (such as a liver cell, for example, a liver cell in a subject) in a subject, for example, a mammal, for example, a human, suffering from a disease, disorder, or condition associated with the oxalate pathway, for example, an oxalate-related disease, disorder, or condition, such as a kidney stone formation disease, disorder, or condition or a calcium oxalate tissue deposition disease, disorder, or condition; or an LDH-related disease, disorder, or condition.

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

[0214] dsRNA comprises 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 (antisense strand) comprises a region of complementarity that is substantially complementary to the target sequence, generally completely complementary. The target sequence can be derived from the sequence of mRNA formed during the expression of the LDHA gene or HAO1 gene. The other strand (sense strand) comprises a region complementary to the antisense strand, so that the two strands hybridize to form a double-stranded structure when combined under suitable conditions. As described elsewhere herein and known in the art, the complementary sequence of the dsRNA can also be contained as a self-complementary region of a single nucleic acid molecule, rather than being on separate oligonucleotides.

[0215] Generally, the double-stranded structure is 15-30 base pairs in length, 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, 19-22, 19-23, 19-24, 19-25, 19-26, 19-27, 19-28, 19-29, 19-30, 19-31, 19-32, 19-33, 19-34, 19-35, 19-36, 19-37, 19-38, 19-39, 19-40, 19-41, 19-42, 19-43, 19-44, 19-45, 19-46, 19-47, 19-48, 19-49, 19-50, 19-51, 19-52, 19-53, 19-54, 19-55, 19-56, 19-57, 19-58, 19-59, 19-60, 19-61, 19-62, 19-63, 19-64, 19-65, 1 21-23, or 21-22 base pairs in length. Ranges and lengths intermediate to the above ranges and lengths are also contemplated as part of the present invention.

[0216] Similarly, the region of complementarity to the target sequence may be 15-30 nucleotides in length, 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, 19-40, 19-50, 19-60, 19-70, 19-80, 19-90, 19-100, 19-110, 19-120, 19-130, 19-140, 19-150, 19-210, 19-220, 19-230, 19-240, 19-250, 19-260, 19-270, 19-280, 19-290, 19-300, 19-310, 19-320, 19-330, 19-340, 19-350, 19-360, 19-370, 19-380, 19-410, 19-420, 19-430, 19-440, 19-450, 19-460, 19-470, 19-480, 19-510, 21-23, or 21-22 nucleotides in length. Ranges and lengths intermediate to the above ranges and lengths are also contemplated as part of the present invention.

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

[0218] The double-stranded region may be a primary functional portion of the dsRNA, e.g., about 9-36 base pairs, e.g., about 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 a double-stranded region of 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 is also a dsRNA. In one embodiment, an RNA molecule or a complex of RNA molecules having a double-stranded region of more than 30 base pairs is a dsRNA, to the extent that it is processed into a functional double-strand of, for example, 15-30 base pairs that targets a desired RNA for cleavage. Thus, those skilled in the art will recognize that, in one embodiment, an miRNA is a dsRNA. In another embodiment, the dsRNA is not a naturally occurring miRNA. In another embodiment, an iRNA agent useful for targeting expression of LDHA or expression of LDHA and HAO1 is not produced in the target cell by cleavage of a larger dsRNA.

[0219] The dsRNA described herein may further comprise one or more single-stranded nucleotide overhangs, for example, 1, 2, 3, or 4 nucleotides. dsRNAs with at least one nucleotide overhang may have unexpectedly superior inhibitory properties compared to their blunt-ended counterparts. The nucleotide overhangs may comprise or consist of nucleotide / nucleoside analogs, including deoxynucleotides / nucleosides. The overhangs may be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the overhanging nucleotides may be present 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 use of an automated DNA synthesizer (such as those commercially available from Biosearch, Applied Biosystems, Inc.), as further described below.

[0221] The iRNA compounds of the present invention can be prepared using a two-step procedure. First, the individual strands of the double-stranded RNA molecule are prepared separately. Then, the component strands are annealed. The individual strands of the siRNA compounds can be prepared using solution phase or solid phase organic synthesis, or both. Organic synthesis offers the advantage that oligonucleotide strands containing unnatural or modified nucleotides can be easily prepared. The single-stranded oligonucleotides of the present invention can be prepared using solution phase or 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 sequence of mRNA produced during 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 antisense strand (guide strand) corresponding to 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 7-14, and the corresponding nucleotide sequence of the antisense strand of the sense strand is selected from the group of sequences provided in any one of Tables 7-14. 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 upon expression of the HAO1 gene. Thus, in this embodiment, the dsRNA comprises two oligonucleotides, wherein one oligonucleotide is represented in any one of Tables 7-14 as the sense strand (passenger strand) and the second oligonucleotide is represented in any one of Tables 7-14 as the corresponding antisense strand (guide strand) of the sense strand. In one embodiment, the substantially complementary sequences of the dsRNA are comprised in separate oligonucleotides. In another embodiment, the substantially complementary sequences of the dsRNA are comprised in a single oligonucleotide.

[0224] Although the sequences in Tables 2-5 and 7-14 are represented as modified, unmodified, unconjugated, and / or conjugated sequences, it will be understood that an iRNA of the invention, e.g., a dsRNA of the invention, can comprise any one of the sequences set forth in any one of Tables 2-5 and 7-14 unmodified, unconjugated, and / or modified and / or conjugated, other than as described herein.

[0225] Those skilled in the art are well aware that dsRNAs having a duplex structure of about 20-23 base pairs, for example, 21 base pairs, have been found to be particularly effective in inducing RNA interference (Elbashir et al., (2001) EMBO J.,:6877-6888). However, others skilled in the art have found that shorter or longer RNA duplex structures can also be effective (Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226). In the above embodiment, due to the nature of the oligonucleotide sequences shown herein, the dsRNAs described herein may contain at least one strand with a minimum length of 21 nucleotides. It can be reasonably expected that shorter duplexes, minus a small number of nucleotides at one or both ends, may be similarly effective compared to the above dsRNAs. Thus, dsRNA having at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotide sequences from one of the sequences set forth herein, and whose ability to inhibit expression of the LDHA gene or HAO1 gene differs from a dsRNA containing the entire sequence by no more than about 5, 10, 15, 20, 25, or 30% inhibition, is considered to be within the scope of the present invention.

[0226] Furthermore, RNAs set forth in any one of Tables 2-5 identify sites in the LDHA transcript that are susceptible to RISC-mediated cleavage, and RNAs set forth in any one of Tables 7-14 identify sites in the HAO1 transcript that are susceptible to RISC-mediated cleavage. Accordingly, the present invention further features iRNAs that target within these sites. As used herein, an iRNA is said to target within a specific site in an RNA transcript if the iRNA promotes cleavage of the transcript anywhere within that specific site. Such iRNAs will generally comprise at least about 15 contiguous nucleotides from one of the sequences set forth herein linked to additional nucleotide sequences taken from regions adjacent to the selected sequence in the gene.

[0227] Target sequences are generally approximately 15-30 nucleotides in length, although the suitability of specific sequences within this range for directing cleavage of any given target RNA varies. While the various software packages and guidelines described herein provide guidance for identifying optimal target sequences for any given gene target, an empirical approach can also be taken in which a "window" or "mask" of a given size (21 nucleotides, as a non-limiting example) is placed literally or figuratively (including, for example, in silico) over the target RNA sequence to identify sequences within a size range that could serve 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 has been identified for any given target size selected. This process, along with systematic synthesis and testing of identified sequences (using assays described herein or known in the art) to identify optimally functioning sequences, can identify RNA sequences that mediate the best inhibition of target gene expression when targeted with an iRNA agent. Thus, for example, while the sequences identified herein represent effective target sequences, it is believed that further optimization of inhibitory efficiency may be achieved by gradually "moving the window" one nucleotide upstream or downstream of the given sequence to identify sequences with equivalent or better inhibitory properties.

[0228] Furthermore, it is contemplated that further optimization can be achieved, for example, by systematically adding or removing nucleotides to generate longer or shorter sequences for any sequence identified herein, and testing the resulting sequences by shifting the longer or shorter size window up or down the target RNA from that point. Furthermore, coupling this approach to generating novel candidate targets with testing the effectiveness of iRNAs based on those target sequences in inhibition assays known in the art and / or described herein can further improve the efficiency of inhibition. Furthermore, such optimized sequences can be adjusted, for example, by introducing modified nucleotides described herein or known in the art, adding or altering overhangs, or other modifications known in the art and / or described herein to further optimize the molecule as an expression inhibitor (e.g., increasing serum stability or circulating half-life, increasing thermostability, improving transmembrane delivery, targeting to specific locations or cell types, increasing interaction with silencing pathway enzymes, increasing release from endosomes).

[0229] The iRNA agents described herein can contain one or more mismatches with the target sequence. In one embodiment, the iRNAs described herein contain three or fewer mismatches. If the antisense strand of the iRNA contains mismatches with the target sequence, it is preferred that the region of mismatch is not located in the center of the region of complementarity. If the antisense strand of the iRNA contains mismatches with the target sequence, it is preferred that the mismatches be limited to within the last five nucleotides from either the 5' or 3' end of the region of complementarity. For example, for a 23-nucleotide iRNA agent, the strand complementary to a region of the LDHA or HAO1 gene generally does not contain any mismatches within the central 13 nucleotides. Methods described herein or known in the art can be used to determine whether an iRNA containing mismatches with the target sequence is effective in inhibiting expression of the LDHA and / or HAO1 gene. Consideration of the effectiveness of iRNAs with mismatches in inhibiting expression of the LDHA gene and / or HAO1 gene is important, especially when the particular region of complementarity in the LDHA gene and / or HAO1 gene is known to have polymorphic sequence variation within the population.

[0230] The dual-targeting RNAi agent of the present invention, which comprises 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, carbohydrate linkers, etc.Covalent linkers can comprise RNA and / or DNA and / or peptide.Linkers can be single-stranded, double-stranded, partially single-stranded, or partially double-stranded.Modified nucleotides or a mixture of nucleotides can also be present in nucleic acid linkers.

[0231] Suitable linkers for use in the dual targeting agents of the present invention include those described in US Pat. No. 9,187,746, the entire contents of which are incorporated herein by reference.

[0232] In certain embodiments, the linker comprises a disulfide bond. The linker can be cleavable or non-cleavable.

[0233] Linkers include, 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' It can be deoxythymidyl-3'-phosphate-5'-adenyI-3'-phosphate-5'-adenyI-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 can be polyRNA, such as poly(5'-adenyI-3'-phosphate-AAAAAAAA) or poly(5'-cytidyl-3'-phosphate-5'-uridyl-3'-phosphate-CUCUCUCU)), e.g., 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 can also be present in the polyRNA linker. The covalent linker can be polyDNA, such as poly(5'-2'deoxythymidyl-3'-phosphate-TTTTTTTT), e.g., where n is an integer between 2 and 50, preferably between 4 and 15, and most preferably between 7 and 8. A modified nucleotide or a mixture of nucleotides may also be present in the polyDNA linker, a single-stranded polyDNA linker, where n is an integer between 2 and 50, preferably including 4, and most preferably between 7 and 8. A modified nucleotide or a mixture of nucleotides may also be present in the polyDNA linker.

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

[0236] The linker may comprise a peptide bond, e.g., an amino acid. In one embodiment, the covalent linker is preferably a 1-10 amino acid long linker comprising 4-5 amino acids, optionally X-Gly-Phe-Gly-Y, where X and Y represent any amino acid.

[0237] The linker may include HEG, a hexaethylene glycol linker.

[0238] The covalent linker can join the sense strand of a first dsRNA agent to the sense strand of a second dsRNA agent; the antisense strand of a first dsRNA agent to the antisense strand of a second dsRNA agent; the sense strand of a first dsRNA agent to the antisense strand of a second dsRNA agent; or the antisense strand of a first dsRNA agent to the sense strand of a second dsRNA agent.

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

[0240] III. Modified iRNAs of the Invention In one embodiment, the RNA, e.g., dsRNA, of an iRNA of the invention is unmodified, e.g., does not contain chemical modifications and / or conjugates known in the art and described herein. In another embodiment, the RNA, e.g., dsRNA, of an iRNA of the invention is chemically modified to improve stability or other beneficial properties. In certain embodiments of the invention, substantially all of the nucleotides of an iRNA of the invention are modified. In other embodiments of the invention, all of the nucleotides of an iRNA of the invention are modified. An iRNA of the invention in which "substantially all of the nucleotides are modified" is mostly, but not completely, modified and may contain no more than five, no more than four, no more than three, no more than two, or no more than one unmodified nucleotide.

[0241] In embodiments in which a first dsRNA agent targeting LDHA and a second dsRNA agent targeting HAO1 are covalently linked (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 certain aspects of the invention, substantially all of the nucleotides of an iRNA of the invention are modified, and the iRNA agent includes 10 or fewer nucleotides that contain 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, 4 or fewer 2'-fluoro modifications, 3 or fewer 2'-fluoro modifications, or 2 or fewer 2'-fluoro modifications). For example, in certain embodiments, the sense strand includes 4 or fewer nucleotides that contain 2'-fluoro modifications (e.g., 3 or fewer 2'-fluoro modifications, or 2 or fewer 2'-fluoro modifications). In other embodiments, the antisense strand includes 6 or fewer nucleotides that contain 2'-fluoro modifications (e.g., 5 or fewer 2'-fluoro modifications, 4 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 linked (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 comprise 10 or fewer nucleotides that include 2'-fluoro modifications.

[0244] In other embodiments of the invention, all of the nucleotides of an iRNA of the invention are modified, and the iRNA agent includes no more than 10 nucleotides that contain 2'-fluoro modifications (e.g., no more than 9 2'-fluoro modifications, no more than 8 2'-fluoro modifications, no more than 7 2'-fluoro modifications, no more than 6 2'-fluoro modifications, no more than 5 2'-fluoro modifications, no more than 4 2'-fluoro modifications, no more than 5 2'-fluoro modifications, no more than 4 2'-fluoro modifications, no more than 3 2'-fluoro modifications, or no more than 2 2'-fluoro modifications).

[0245] In embodiments in which a first dsRNA agent targeting LDHA and a second dsRNA agent targeting HAO1 are covalently linked (i.e., a dual-targeting RNAi agent), all of the nucleotides of the first agent and / or all of the nucleotides of the second agent may be independently modified, and the first and second agents may independently include no more than 10 nucleotides that include 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 mimic at the 5' nucleotide of the antisense strand. In another embodiment, the double-stranded RNAi agent further comprises a 5'-phosphate mimic at the 5' nucleotide of the antisense strand. In a specific embodiment, the 5'-phosphate mimic is 5'-vinyl phosphate (5'-VP).

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

[0248] Nucleic acids featured in the present invention can be synthesized and / or modified by methods well established in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, S. Lett. et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference. Modifications include, for example, terminal modifications, such as 5'-end modifications (phosphorylation, conjugation, inverted linkage) or 3'-end modifications (conjugation, DNA nucleotide, inverted linkage, etc.); base modifications, such as substitution with a stable base, an unstable base, or a base that base-pairs with a wide range of partners, base removal (abasic nucleotide), or conjugated base; sugar modifications (e.g., at the 2' or 4' position) or sugar substitution; and / or backbone modifications, including modification or substitution of phosphodiester linkages. Specific examples of iRNA compounds useful in the embodiments described herein include, but are not limited to, RNAs containing modified backbones or that do not contain natural internucleoside linkages. The RNA with modified backbone particularly includes those that do not have a phosphorus atom in the backbone.For the purpose of this specification and as sometimes referred to in the art, the modified RNA that does not have a phosphorus atom in the internucleoside backbone can also be considered as oligonucleoside.In some embodiments, the modified iRNA has a phosphorus atom in its internucleoside backbone.

[0249] Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methylphosphonates, and other alkyl phosphonates, including 3'-alkylene phosphonates and chiral phosphonates; phosphinates; phosphoramidates, including 3'-aminophosphoramidates and aminoalkylphosphoramidates; thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters; and boranophosphates with normal 3'-5' linkages, 2'-5' linked analogs thereof, and those with reversed polarity, in which adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included.

[0250] Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Patent Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,32 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, Nos. 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Reissue Patent No. RE39464, the entire contents of each of which are incorporated herein by reference.

[0251] Modified RNA backbones that do not contain internal phosphorus atoms have backbones formed by short alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short heteroatom or heterocyclic internucleoside linkages. These include those with morpholino linkages (formed in part from the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide, and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and others with mixed N, O, S, and CH2 constituent moieties.

[0252] Representative United States patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Patent Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; Nos. 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, the entire contents of each of which are incorporated herein by reference.

[0253] In other embodiments, suitable RNA mimics are contemplated for use in iRNA, in which both the sugar and internucleoside linkages, i.e., the backbone of the nucleotide units, are replaced with novel groups. The base units are maintained for hybridization with appropriate nucleic acid target compounds. One such oligomeric compound, an RNA mimic, that has been shown to have excellent hybridization properties is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleobases are retained and are directly or indirectly linked to the aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, the entire contents of each of which are incorporated herein by reference. Additional 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] Certain embodiments featured in the present invention include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, particularly those described in U.S. Pat. No. 5,489,677, such as --CH2--NH--CH2-, --CH2--N(CH3)--O--CH2-- (also known as the methylene (methylimino) or MMI backbone), --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2--, and --N(CH3)--CH2--CH2-- (where the natural phosphodiester backbone is represented as --O--P--O--CH2--) and those described in U.S. Pat. No. 5,602,240, such as the amide backbones described in U.S. Pat. In certain embodiments, RNAs featured herein have the morpholino backbone structures described in U.S. Pat. No. 5,034,506, such as the morpholino backbone structures described in U.S. Pat.

[0255] Modified RNAs may also contain one or more substituted sugar moieties. The iRNAs, e.g., dsRNAs, featured herein, can include one of the following at the 2' position: 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-C6. 10 Alkyl or C2-C 10 It can be alkenyl and alkynyl. Exemplary suitable modifications include O[(CH) 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 comprises at the 2' position a C1 to C 10The modification may include one of the following: lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH, OCN, Cl, Br, CN, CF, OCF, SOCH, SOCH, ONO, NO, N, NH, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving group, reporter group, intercalator, group that improves the pharmacodynamic properties of iRNA, or group that improves the pharmacokinetic properties of iRNA, and other substituents with similar properties. In certain embodiments, the modification includes 2'-methoxyethoxy (2'-O--CHCHOCH, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., an alkoxy-alkoxy group. Another exemplary modification is 2'-dimethylaminooxyethoxy, also known as 2'-DMAOE, i.e., O(CH)ON(CH), and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O-CH-O-CH-N(CH). Further exemplary modifications include 5'-Me-2'-F nucleotides, 5'-Me-2'-OMe nucleotides, 5'-Me-2'-deoxyribonucleotides (both R and S isomers in these three families); 2'-alkoxyalkyl; and 2'-NMA (N-methylacetamide), described herein in the Examples below.

[0256] Other modifications include 2'-methoxy (2'-OCH), 2'-aminopropoxy (2'-OCHCHCHNH), and 2'-fluoro (2'-F). Similar modifications can also be made at other positions in the RNA of an iRNA, particularly the 3' position of the sugar on the 3'-terminal nucleotide or in a 2'-5'-linked dsRNA and the 5' position of the 5'-terminal nucleotide. iRNAs can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Patent Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,81 Nos. 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, several of which are co-owned with the present application, the entire contents of each of which are incorporated herein by reference.

[0257] The iRNAs of the present invention may also include nucleobase (often simply referred to in the art as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include 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 (pseudouracil), 4- Included are other synthetic and natural nucleobases such as 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 3-deazaguanine and 3-deazaadenine.Further nucleobases 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 nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the present invention. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-Methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, Y.S., Crooke, S.T., and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), making them exemplary base substitutions, especially when combined with 2'-O-methoxyethyl sugar modifications.

[0258] Representative United States patents that teach the preparation of some of the above-mentioned modified nucleobases, as well as other modified nucleobases, include, but are not limited to, the above-mentioned U.S. Patent Nos. 3,687,808; 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,1 Nos. 21, 5,596,091; 5,614,617; 5,681,941; 5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, the entire contents of each of which are incorporated herein by reference.

[0259] The iRNA of the present invention can also be modified to contain one or more locked nucleic acids (LNAs). Locked nucleic acids are nucleotides with a modified ribose moiety, which contains an additional bridge connecting the 2' and 4' carbons. This structure effectively "locks" the ribose in a 3'-endo structural configuration. The addition of 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] iRNAs of the present invention can also be modified to contain one or more bicyclic sugar moieties. A "bicyclic sugar" is a furanosyl ring modified by a two-atom bridge. A "bicyclic nucleoside" ("BNA") is a nucleoside having a sugar moiety containing a bridge connecting 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, an agent of the present invention can contain one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide with a modified ribose moiety, in which the ribose moiety contains an additional bridge connecting the 2' and 4' carbons. In other words, an LNA is a nucleotide containing a bicyclic sugar moiety containing a 4'-CH2-O-2' bridge. This structure effectively "locks" the ribose in the 3'-endo structural configuration. The addition of a locked nucleic acid to siRNA has been shown to increase siRNA stability in serum and reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, 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 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 includes 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 "constrained ethyl" or "cEt") and 4'-CH(CHOCH3)-O-2' (and analogs thereof; see, e.g., U.S. Pat. No. 7,399,845); 4'-C(CH3)(CH3)-O-2' (and analogs thereof; see, e.g., U.S. Pat. No. 8,278,282). 83); 4'-CH2-N(OCH3)-2' (and analogs thereof; see, e.g., U.S. Pat. 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 a protecting group (see, e.g., U.S. Pat. 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 analogs thereof; see, e.g., U.S. Pat. No. 8,278,426). The entire contents of each of these are incorporated herein by reference.

[0261] Further representative U.S. patents and U.S. patent publications that teach 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; Nos. 7,427,672; 7,569,686; 7,741,457; 8,022,193; 8,030,467; 8,278,425; 8,278,426; 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 which are incorporated herein by reference.

[0262] For example, any of the bicyclic nucleosides described above can be prepared with one or more stereochemical sugar configurations including α-L-ribofuranose and β-D-ribofuranose (see WO 99 / 14226).

[0263] The iRNAs of the invention can also be modified to contain one or more constrained ethyl nucleotides. As used herein, a "constrained ethyl nucleotide" or "cEt" is a locked nucleic acid containing a bicyclic sugar moiety containing a 4'-CH(CH3)-0-2' bridge. In one embodiment, the constrained ethyl nucleotide is in the S configuration, referred to herein as an "S-cEt."

[0264] The iRNA of the present invention may also contain one or more "conformationally restricted nucleotides" ("CRNs"). CRNs are nucleotide analogs with a linker connecting the C2' and C4' carbons of ribose or the C3 and C5' carbons of ribose. The CRNs lock the ribose ring into a stable conformation, increasing hybridization affinity for mRNA. The linker is long enough to position the oxygen in an optimal position for stability and affinity, reducing puckering of the ribose ring.

[0265] Representative publications that teach the preparation of some of the above-described CRNs include, but are not limited to, U.S. Patent Application Publication No. 2013 / 0190383; and PCT Publication No. WO 2013 / 036868, the entire contents of each of which are incorporated herein by reference.

[0266] In some embodiments, the iRNA of the present invention includes one or more monomers that are UNA (unlocked nucleic acid) nucleotides. UNAs are unlocked acyclic nucleic acids in which one of the sugar linkages has been removed to form an unlocked "sugar" residue. In one example, UNAs also include monomers in which the C1'-C4' bond (i.e., the covalent carbon-oxygen-carbon bond between the C1' and C4' carbons) has been removed. In another example, the C2'-C3' bond (i.e., the covalent carbon-carbon bond between the C2' and C3' carbons) of the sugar has been removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039, which are incorporated herein by reference).

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

[0268] Potentially stable modifications to the ends of RNA molecules can include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl 4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-0-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3"-phosphate, inverted base dT (idT), and the like. Disclosure of this modification can be found in PCT Publication No. WO 2011 / 005861.

[0269] Other modifications of the iRNAs of the invention include a 5' phosphate or 5' phosphate mimic, such as a 5' terminal phosphate or phosphate mimic, on the antisense strand of the RNAi agent. Suitable phosphate mimics are disclosed, for example, in U.S. Patent Application Publication No. 2012 / 0157511, the entire contents of which are incorporated herein by reference.

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

[0271] A. Modified iRNAs Containing Motifs of the Invention In certain embodiments of the invention, double-stranded RNAi agents of the invention include, for example, agents having chemical modifications disclosed in WO 2013 / 075035, filed November 16, 2012, the entire contents of which are incorporated herein by reference.

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

[0273] Thus, the present invention provides double-stranded RNAi agents 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 can be in the range of 12 to 30 nucleotides in length. For example, each strand can be 14 to 30 nucleotides in length, 17 to 30 nucleotides in length, 25 to 30 nucleotides in length, 27 to 30 nucleotides in length, 17 to 23 nucleotides in length, 17 to 21 nucleotides in length, 17 to 19 nucleotides in length, 19 to 25 nucleotides in length, 19 to 23 nucleotides in length, 19 to 21 nucleotides in length, 21 to 25 nucleotides in length, or 21 to 23 nucleotides in length.

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

[0275] In one embodiment, an RNAi agent can include one or more overhang regions and / or capping groups at the 3'-end, 5'-end, or both ends of one or both strands. The overhangs can be 1 to 6 nucleotides in length, 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 in length. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. The overhangs can form mismatches with the target mRNA, or the overhangs can be complementary to the targeted gene sequence or can be another sequence. The first and second strands can also be joined by additional bases or other non-basic linkers, e.g., to form a hairpin.

[0276] In one embodiment, each nucleotide in the overhang region of an RNAi agent can independently be a modified or unmodified nucleotide, including, but not limited to, 2'-sugar modifications such as 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. For example, TT can be an overhang sequence for either end on either strand. The overhang can form a mismatch with the target mRNA, or the overhang can be complementary to the targeted gene sequence, or it can be another sequence.

[0277] The 5'- or 3'-overhang on the sense strand, antisense strand, or both strands of an RNAi agent can be phosphorylated. In some embodiments, the overhang region comprises two nucleotides with a phosphorothioate between them, wherein the two nucleotides can be the same or different. In one embodiment, the overhang is present at the 3'-end of the sense strand, the antisense strand, or both strands. In one embodiment, the 3'-overhang is present in the antisense strand. In one embodiment, the 3'-overhang is present in the sense strand.

[0278] RNAi agent can have only one overhang, which can enhance the interference activity of RNAi without affecting its overall stability.For example, the single-stranded overhang can be located at the 3'-end of the sense strand or the 3'-end of the antisense strand.RNAi can 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 the 5'-end is blunt.Without wishing to be bound by theory, the asymmetric blunt ends at the 5'-end of the antisense strand and the 3'-end overhang of the antisense strand favor the introduction of the guide strand into the RISC process.

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

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

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

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

[0283] When two nucleotide overhangs are at the 3'-end of the antisense strand, there may be two phosphorothioate internucleotide linkages between the terminal three nucleotides, two of which are overhanging nucleotides, and the third nucleotide is the paired nucleotide adjacent to the overhanging nucleotide. In one embodiment, the RNAi agent further has two phosphorothioate internucleotide linkages between the terminal three 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 with 2'-O-methyl or 3'-fluoro, for example, 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, wherein the sense strand is 25 to 30 nucleotide residues in length, and starting from the 5'-terminal nucleotide (position 1), positions 1 to 23 of the first strand comprise at least 8 ribonucleotides; the antisense strand is 36 to 66 nucleotide residues in length, and starting from the 3'-terminal nucleotide, comprises at least 8 ribonucleotides at positions paired with positions 1 to 23 of the sense strand to form a duplex; at least the 3'-terminal nucleotide of the antisense strand is not paired with the sense strand, and up to 6 consecutive 3'-terminal nucleotides are not paired with the sense strand, thereby forming a 3' single-stranded overhang of 1 to 6 nucleotides; and the 5' end of the antisense strand is 10 to 30 consecutive nucleotides that are not paired with the sense strand. The antisense strand comprises at least one ribonucleotide at the 5'-end of the sense strand, thereby forming a 10-30 nucleotide single-stranded 5' overhang; at least the 5'- and 3'-terminal nucleotides of the sense strand are base-paired with nucleotides in 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 expression of the target gene when the double-stranded nucleic acid is introduced into a mammalian cell; the sense strand comprises at least one motif of three 2'-F modifications in three consecutive nucleotides, where at least one of the motifs is located at or near the cleavage site; and the antisense strand comprises at least one motif of three 2'-O-methyl modifications in three consecutive nucleotides 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 length of at least 25 and no more than 29 nucleotides, and a second strand having a length of no more than 30 nucleotides, the second strand comprising at least one motif of three 2'-O-methyl modifications at three consecutive nucleotides, 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 a blunt end, the second strand is 1 to 4 nucleotides longer than the first strand at its 3' end, the double-stranded region is at least 25 nucleotides long, the second strand is sufficiently complementary to a target mRNA along at least 19 nucleotides of the second strand length such that the RNAi agent reduces expression of the target gene when introduced into a mammalian cell, and dicer cleavage of the RNAi agent preferentially yields siRNA comprising the 3' end of the second strand, thereby reducing expression of the target gene in a mammal. Optionally, the RNAi agent further comprises a ligand.

[0286] In one embodiment, the sense strand of the RNAi agent contains at least one motif of three identical modifications in three consecutive nucleotides, one of the motifs being at the cleavage site of the sense strand.

[0287] In one embodiment, the antisense strand of the RNAi agent can also contain at least one motif of three identical modifications in three consecutive nucleotides, one of the motifs being at or near the cleavage site on the antisense strand.

[0288] In RNAi agents having a double-stranded region 17-23 nucleotides in length, the cleavage sites in the antisense strand are typically near positions 10, 11, and 12 from the 5' end. Thus, 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, counting from the first nucleotide from the 5' end of the antisense strand, or from the first paired nucleotide in the double-stranded region from the 5' end of the antisense strand. The cleavage site 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 RNAi agent can comprise at least one motif of three identical modifications in three consecutive nucleotides at the break site of strand; antisense strand can have at least one motif of three identical modifications in three consecutive nucleotides at or near the break site of strand.When sense strand and antisense strand form dsRNA duplex, sense strand and antisense strand can be aligned such that one motif of three nucleotides in sense strand and one motif of three nucleotides in antisense strand have at least one nucleotide overlap, that is, at least one of the three nucleotides of the motif in sense strand and at least one of the three nucleotides of the motif in antisense strand form base pairs.Alternatively, at least two nucleotides can overlap, or all three nucleotides can overlap.

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

[0291] Like the sense strand, the antisense strand of an RNAi agent may contain two or more motifs of three identical modifications in three consecutive nucleotides, with at least one of the motifs occurring at or near the site of strand cleavage. The antisense strand may also contain one or more wing modifications in the same sequence as the wing modifications that may be present in the sense strand.

[0292] In one embodiment, wing modifications on the sense or antisense strand of an RNAi agent typically do 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, wing modifications on the sense or antisense strand of an RNAi agent typically do not include 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] When the sense and antisense strands of an RNAi agent each include at least one wing modification, the wing modifications may be located at the same end of the double-stranded region and may have an overlap of 1, 2, or 3 nucleotides.

[0295] When the sense and antisense strands of an RNAi agent each contain at least two wing modifications, the sense and antisense strands can be arranged such that two modifications from one strand are each located at one end of the double-stranded region and have an overlap of one, two, or three nucleotides; two modifications from one strand are each located at the other end of the double-stranded region and have an overlap of one, two, or three nucleotides; or two modifications from one strand are located on either side of the lead motif and have an overlap of one, two, or three nucleotides 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, can be modified. Each nucleotide can be modified with the same or different modifications, and these modifications can include one or more changes to one or both of the non-linked phosphate oxygen and / or one or more linking phosphate oxygens; changes to components of the ribose sugar, such as the 2' hydroxyl of the ribose sugar; large-scale replacement of the phosphate moiety with a "dephosphorylation" linker; modifications or replacement of natural bases; and replacement or modification of the ribose-phosphate backbone.

[0297] Because nucleic acids are polymers of subunits, many modifications, such as modifications of bases, phosphate moieties, or non-linked Os in phosphate moieties, occur at repeated positions within the nucleic acid. In some cases, modifications can occur at all of the intended positions in the nucleic acid, but often this is not the case. For example, modifications can occur only at the 3' or 5' terminal positions, or only in terminal regions, such as at the terminal nucleotide position or the last 2, 3, 4, 5, or 10 nucleotides of the chain. Modifications can occur in double-stranded regions, single-stranded regions, or both. Modifications can occur only in double-stranded regions of RNA, or only in single-stranded regions of RNA. For example, phosphorothioate modifications at non-linked O positions can occur only at one or both ends, or only in terminal regions, such as at the terminal nucleotide position or the last 2, 3, 4, 5, or 10 nucleotides of the chain, or in double-stranded and single-stranded regions, especially at the ends. The 5' or both ends can be phosphorylated.

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

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

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

[0301] In one embodiment, N a and / or N b includes an alternating pattern of modifications. As used herein, the term "alternating motif" refers to a motif having one or more modifications, each modification occurring at alternating nucleotides in a strand. The alternating nucleotides can refer to one at every other nucleotide or one at every third nucleotide, or a similar pattern. For example, if A, B, and C each represent one type of modification to a nucleotide, the alternating motif could be "ABABABABABAB...", "AABBAABBAABB...", "AABAABAABAAB...", "AAABAAABAAAB...", "AAABBBAAABBB...", or "ABCABCABCABC...", etc.

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

[0303] In one embodiment, the RNAi agent of the present invention comprises an alternating motif modification pattern in the sense strand that is shifted relative to the alternating motif modification pattern in the antisense strand. This shift can be such that the modification group of the nucleotide of the sense strand corresponds to a different modification group of the nucleotide of the antisense strand, or vice versa. For example, when the sense strand is paired with the antisense strand in a dsRNA duplex, the alternating motif in the sense strand can start with "ABABAB" from 5' to 3' of the strand, and the alternating motif in the antisense strand can start with "BABABA" from 5' to 3' of the strand in the double-stranded region. As another example, the alternating motif in the sense strand can start with "AABBAABB" from 5' to 3' of the strand, and the alternating motif in the antisense strand can start with "BBAABBAA" from 5' to 3' of the strand in the double-stranded region, thereby resulting in a complete or partial shift in the modification pattern between the sense strand and the antisense strand.

[0304] In one embodiment, the RNAi agent comprises a pattern of alternating motifs of 2'-O-methyl and 2'-F modifications in the sense strand, and this pattern has a first shift with respect to the pattern of alternating motifs of 2'-O-methyl and 2'-F modifications in the antisense strand, i.e., the 2'-O-methyl modified nucleotides in the sense strand form base pairs with the 2'-F modified nucleotides in the antisense strand, and vice versa. Position 1 of the sense strand may start with a 2'-F modification, and position 1 of the antisense strand may start with a 2'-O-methyl modification.

[0305] The introduction of one or more motifs of three identical modifications on three consecutive nucleotides into the sense strand and / or antisense strand interrupts the original modification pattern present in the sense strand and / or antisense strand. This interruption of the modification pattern of the sense strand and / or antisense strand by introducing one or more motifs of three identical modifications on three consecutive nucleotides into the sense strand and / or antisense strand unexpectedly enhances the gene silencing activity against the target gene.

[0306] In one embodiment, when a motif of three identical modifications on three consecutive nucleotides is introduced into either strand, the modifications of the nucleotides adjacent to the motif are different from the modification of the motif. For example, a portion of a sequence containing a motif may be represented by "...N a YYYN b ...", where "Y" represents a modification of a motif of three identical modifications in three consecutive nucleotides, and "N a " and "N b " represents a modification of the nucleotide adjacent to the motif "YYY" that is different from the modification of Y, and N a and N b may be the same or different modifications. a and / or N b may or may not be present if wing modifications are present.

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

[0308] In one embodiment, the RNAi comprises a phosphorothioate or methylphosphonate internucleotide bond modification in the overhang region. For example, the overhang region can comprise two nucleotides with a phosphorothioate or methylphosphonate internucleotide bond between the two nucleotides. The internucleotide bond modification can also be formed to link the overhang nucleotide with the terminal paired nucleotide in the double-stranded region. For example, at least 2, 3, 4, or all of the overhang nucleotides can be linked by phosphorothioate or methylphosphonate internucleotide bonds, and optionally, there can be additional phosphorothioate or methylphosphonate internucleotide bonds that link the overhang nucleotide with the paired nucleotide adjacent to the overhang nucleotide. For example, there can be at least two phosphorothioate internucleotide bonds between the terminal three nucleotides, two of the three nucleotides being overhang nucleotides, and the third nucleotide being the paired nucleotide adjacent to the overhang nucleotide. These terminal three nucleotides can be at the 3' end of the antisense strand, the 3' end of the sense strand, the 5' end of the antisense strand, and / or the 5' end of the antisense strand.

[0309] In one embodiment, the two nucleotide overhangs are at the 3'-end of the antisense strand, and there are two phosphorothioate internucleotide bonds between the terminal three nucleotides, two of which are overhanging nucleotides, and the third nucleotide is a paired nucleotide adjacent to the overhanging nucleotide. Optionally, the RNAi agent can further have two phosphorothioate internucleotide bonds between the terminal three nucleotides at both the 5'-end of the sense strand and the 5'-end of the antisense strand.

[0310] The RNAi agent contains mismatches with the target, mismatches within the duplex, or a combination thereof. Mismatches can occur in the overhang region or the duplex region. Base pairs can be evaluated based on their tendency to promote dissociation or melting (e.g., for the free energy of binding or dissociation of a particular pairing; the simplest approach is to examine each pair individually, but similar or equivalent analyses can also be used). With regard to promoting dissociation: A:U is preferred over G:C; G:U is preferred over G:C; and I:C is preferred over G:C (I=inosine). Mismatches, such as non-canonical or non-canonical pairings (described elsewhere herein), are preferred over canonical (A:T, A:U, G:C) pairings; and pairings involving universal bases are preferred over canonical pairings.

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

[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, there is a short sequence of deoxythymine nucleotides, e.g., two dT nucleotides, at the 3' end of the sense and / or antisense strands.

[0314] In one embodiment, the sense strand sequence has 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 each independently 0 or 1; p and q are each independently 0 to 6; each N a independently represent oligonucleotide sequences containing 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides; each N b independently represent an oligonucleotide sequence comprising 0 to 10 modified nucleotides; each n p and n q independently represent overhanging nucleotides; wherein Nb and Y do not have the same modification; XXX, YYY and ZZZ each independently represent one motif of three identical modifications in three consecutive nucleotides. Preferably, all of YYY are 2'-F modified nucleotides.

[0315] In one embodiment, N a and / or N b contains alternating patterns of modifications.

[0316] In one embodiment, the YYY motif is located at or near the cleavage site of the sense strand. For example, if the RNAi agent has a double-stranded region 17 to 23 nucleotides in length, the YYY motif can be located at or near the cleavage site of the sense strand, counting from the first nucleotide from the 5' end; or optionally, counting from the first paired nucleotide in the double-stranded region from the 5' end (e.g., 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 strand may 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] When the sense strand is represented by formula (Ib), N b represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a can independently represent an oligonucleotide sequence that includes 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0319] When the sense strand is represented by formula (Ic), N brepresents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a can independently represent an oligonucleotide sequence that includes 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0320] When the sense strand is represented as formula (Id), each N b independently represent an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. b is 0, 1, 2, 3, 4, 5 or 6. Each N a can independently represent an oligonucleotide sequence that includes 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0321] Each of X, Y and Z can be the same or different from each other.

[0322] In other embodiments, i is 0, j is 0, and the sense strand may be represented by the formula: 5'n p -N a -YYY-N a -n q 3'(Ia).

[0323] When the sense strand is represented by formula (Ia), each N a can independently represent an oligonucleotide sequence that includes 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0324] In one embodiment, the antisense strand sequence of the RNAi has 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 each independently 0 or 1; p' and q' are each independently 0 to 6; each N a ' independently represent oligonucleotide sequences containing 0 to 25 modified nucleotides, each sequence containing at least two differently 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; where N b ' and Y' do not have the same modification; X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent one motif of three identical modifications in three consecutive nucleotides. 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 present at or near the cleavage site of the antisense strand. For example, if the RNAi agent has a double-stranded region 17 to 23 nucleotides in length, the Y'Y'Y' motif can be present at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, counting from the first nucleotide from the 5' end; or optionally, counting from the first paired nucleotide in the double-stranded region from the 5' end. Preferably, the Y'Y'Y' motif is present at positions 11, 12, or 13.

[0327] In one embodiment, the Y'Y'Y' motif is all 2'-OMe modified nucleotides.

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

[0329] Thus, 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 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a ' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

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

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

[0333] In other embodiments, k is 0, l is 0, and the antisense strand may be represented by the formula: 5'n p’ -N a’ -Y'Y'Y'-N a’ -n q’ 3'(Ia).

[0334] When the antisense strand is represented by formula (IIa), each N a ' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0335] Each of X', Y' and Z' can be the same or different from each other.

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

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

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

[0339] The sense strand represented by any one of the above formulas (Ia), (Ib), (Ic), and (Id) forms a duplex with the antisense strand represented by any one of the above formulas (IIa), (IIb), (IIc), and (IId).

[0340] Thus, an RNAi agent for use in the methods of the invention may comprise a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, and the RNAi duplex has the 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 0 or 1; p, p', q, and q' are each independently 0 to 6; each N a and N a ’ independently represent oligonucleotide sequences containing 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides; each N b and N b ’ independently represent an oligonucleotide sequence comprising 0 to 10 modified nucleotides; where: Each of np', np, nq', and n may or may not be present. q independently represent overhanging nucleotides; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides. is expressed by

[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; or k is 0 and l is 1; or both k and l are 0; or both k and l are 1.

[0342] Exemplary combinations of sense and antisense strands that form RNAi duplexes include the following formulas: 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] When the RNAi agent is represented by formula (IIIa), each N a represents an oligonucleotide sequence that independently contains 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0344] When the RNAi agent is represented by formula (IIIb), each N b represents an oligonucleotide sequence containing, independently, 1 to 10, 1 to 7, 1 to 5, or 1 to 4 modified nucleotides. a represents an oligonucleotide sequence that independently contains 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

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

[0346] When an RNAi agent is represented as formula (IIId), each Nb, Nb' independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. Each Na, Na' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. Each of Na, Na', Nb, and Nb' independently comprises an alternating pattern of modifications.

[0347] Each of X, Y and Z in formulas (III), (IIIa), (IIIb), (IIIc), and (IIId) can be the same as or different from each other.

[0348] When an RNAi agent is represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), at least one of the Y nucleotides can be base-paired with one of the Y' nucleotides, alternatively, at least two of the Y nucleotides are base-paired with the corresponding Y' nucleotide; or all three of the Y nucleotides are base-paired with the corresponding Y' nucleotide.

[0349] When the RNAi agent is represented by formula (IIIb) or (IIId), at least one of the Z nucleotides can be base-paired with one of the Z' nucleotides, or at least two of the Z nucleotides can be base-paired with a corresponding Z' nucleotide; or all three of the Z nucleotides can be base-paired with a corresponding Z' nucleotide.

[0350] When an RNAi agent is represented as formula (IIIc) or (IIId), at least one of the X nucleotides can be base-paired with one of the X' nucleotides, or at least two of the X nucleotides can be base-paired with a corresponding X' nucleotide; or all three of the X nucleotides can be base-paired with a corresponding X' nucleotide.

[0351] In one embodiment, the modification on a Y nucleotide is different from the modification on a Y' nucleotide, the modification on a Z nucleotide is different from the modification on a Z' nucleotide, and / or the modification on an X nucleotide is different from the modification on an X' nucleotide.

[0352] In one embodiment, when the RNAi agent is represented by formula (IIId), 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 linked 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 linked to an adjacent nucleotide via a phosphorothioate bond, and the sense strand is conjugated to one or more GalNAc derivatives linked via a bivalent or trivalent branched linker (described below). In another embodiment, when the RNAi agent is represented by formula (IIId), the Na modification is a 2'-O-methyl or 2'-fluoro modification, np'>0, and at least one np' is linked to an adjacent nucleotide via a phosphorothioate linkage, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives linked via a divalent or trivalent branched linker.

[0353] In one embodiment, when 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 linked to an adjacent nucleotide via a phosphorothioate linkage, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives linked via a divalent or trivalent branched linker.

[0354] In one embodiment, the RNAi agent is a multimer comprising at least two duplexes represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), wherein the duplexes are linked by a linker. The linker may be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the duplexes can target the same gene or two different genes; or each of the duplexes can 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 duplexes represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId), the duplexes being linked by a linker. The linker may be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the duplexes can target the same gene or two different genes; or each of the duplexes can target the same gene at two different target sites.

[0356] In one embodiment, two RNAi agents represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId) are linked to each other at one or both of the 5' and 3' ends, and are optionally conjugated to a ligand. Each of the RNAi agents can target the same gene or two different genes; or each of the RNAi agents can 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 containing 2'-fluoro modifications, for example, 10 or fewer nucleotides with 2'-fluoro modifications. For example, the RNAi agent may contain 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 nucleotides with 2'-fluoro modifications. In certain embodiments, the RNAi agent of the present invention contains 10 nucleotides with 2'-fluoro modifications, for example, four nucleotides with 2'-fluoro modifications in the sense strand and six nucleotides with 2'-fluoro modifications in the antisense strand. In another specific embodiment, the RNAi agent of the present invention contains six nucleotides with 2'-fluoro modifications, for example, four nucleotides with 2'-fluoro modifications in the sense strand and two nucleotides with 2'-fluoro modifications in the antisense strand.

[0358] In other embodiments, the RNAi agent of the present invention may contain only a 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 with 2'-fluoro modifications. In certain embodiments, the RNAi agent may contain two nucleotides with 2'-fluoro modifications, for example, zero nucleotides with 2'-fluoro modifications in the sense strand and two nucleotides with 2'-fluoro modifications in the antisense strand.

[0359] Various publications describe the multimeric RNAi agent that can be used in the method of the present invention.Such publications include WO2007 / 091269, US Patent No. 7858769, WO2010 / 141511, WO2007 / 117686, WO2009 / 014887 and WO2011 / 031520, each of which is incorporated herein by reference in its entirety.

[0360] As described in more detail below, RNAi agents that include one or more carbohydrate moieties conjugated to the RNAi agent can optimize one or more properties of the RNAi agent. Often, the carbohydrate moiety is attached to a modified subunit of the RNAi agent. For example, the ribose sugar of one or more ribonucleotide subunits of a dsRNA agent can be replaced with another moiety, such as a non-carbohydrate (preferably cyclic) carrier to which a carbohydrate ligand is attached. A ribonucleotide subunit in which the ribose sugar of the subunit has been replaced in this manner is referred to herein as a ribose-replacement modified subunit (RRMS). The cyclic carrier can be a carbocyclic ring system, i.e., all ring atoms are carbon atoms, or a heterocyclic ring system, i.e., one or more ring atoms can be a heteroatom, such as nitrogen, oxygen, or sulfur. The cyclic carrier can be a monocyclic ring system or can contain two or more rings, such as fused rings. The cyclic carrier can be a fully saturated ring system or can contain one or more double bonds.

[0361] The ligand can be attached to the polynucleotide via a carrier. The carrier comprises (i) at least one "backbone attachment point," preferably two "backbone attachment points," and (ii) at least one "tether attachment point." As used herein, "backbone attachment point" refers to a bond available and suitable for incorporation of the carrier into the backbone of a ribonucleic acid, containing a functional group, e.g., a hydroxyl group, or generally a backbone, e.g., a phosphate, or a modified phosphate, e.g., sulfur. In certain embodiments, a "tether attachment point" (TAP) refers to a ring atom, e.g., a carbon atom or heteroatom (different from the atom providing the backbone attachment point), of the cyclic carrier to which the selected moiety is attached. This moiety can be, for example, a carbohydrate, e.g., a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide. Optionally, the selected moiety is attached to the cyclic carrier by an intervening tether. Thus, cyclic carriers often contain functional groups, such as amino groups, or generally provide bonds suitable for the incorporation or tethering of another chemical moiety, 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 serinol backbone or a diethanolamine backbone.

[0363] In another embodiment of the invention, the iRNA agent comprises a sense strand and an antisense strand, each strand having between 14 and 40 nucleotides. The RNAi agent has the formula (L): [ka] It can be represented by:

[0364] In Formula (L), B1, B2, B3, B1', B2', B3', and B4' are each independently a nucleotide 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 destabilizing nucleotide located at a site 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 in the sense strand at a position that pairs with 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 has a thermally destabilizing modification that can include a non-basic modification; a mismatch with the opposing nucleotide in the duplex; and a sugar modification such as a 2'-deoxy modification or an acyclic nucleotide, e.g., unlocked nucleic acid (UNA) or glycerol nucleic acid (GNA). In one embodiment, C1 i) mismatches with the opposing nucleotide in the antisense strand; and ii) a non-basic modification selected from the group consisting of: [ka] and iii) a sugar modification selected from the group consisting of: [ka] wherein B is a modified or unmodified nucleobase; and R 1 and R 2are independently H, halogen, OR3, or alkyl; and R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar. In one embodiment, the thermally destabilizing 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 nucleobase in the mismatch pair is a 2'-deoxynucleobase. In one example, the thermally destabilizing modification in C1 is GNA or [ka] is.

[0366] T1, T1', T2', and T3' each independently represent a nucleotide containing a modification that confers steric bulk to the nucleotide equal to or less than the steric bulk of the 2'-OMe modification. Steric bulk refers to the total steric effect of the modification. Methods for determining the steric effect of a nucleotide modification are known to those skilled in the art. The modification can be at the 2'-position of the ribose sugar of the nucleotide, or can be a non-ribose nucleotide, an acyclic nucleotide, or a modification to the backbone of the nucleotide similar or equivalent to the 2'-position of the ribose sugar, which confers steric bulk to the nucleotide equal to or less than the steric bulk of the 2'-OMe modification. For example, T1, T1', T2', and T3' are each independently selected from DNA, RNA, LNA, 2'-F, and 2'-F-5'-methyl. In 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 are independently 4 to 15 nucleotides in length.

[0368] n 5 , q 3 , and q 7are independently 1 to 6 nucleotides in length.

[0369] n 4 , q 2 , and q 6 are independently 1 to 3 nucleotides in length; or 4 is 0.

[0370] q 5 are independently 0 to 10 nucleotides in length.

[0371] n 2 and q 4 are independently 0 to 3 nucleotides in length.

[0372] Or, n 4 but are 0 to 3 nucleotides in length.

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

[0374] In one embodiment, n 4 , q 2 , and q 6 are each 1.

[0375] In one embodiment, n 2 , n 4 , q 2 , q 4 , and q 6 are each 1.

[0376] In one embodiment, when the sense strand is 19 to 22 nucleotides in length, C1 is at positions 14 to 17 of the 5' end of the sense strand, and n 4 is 1. In one embodiment, C1 is at position 15 of the 5' end of the sense strand.

[0377] In one embodiment, T3' begins at position 2 from the 5' end of the antisense strand. 6 is equal to 1.

[0378] In one embodiment, T1' begins at position 14 from the 5' end of the antisense strand. 2 is equal to 1.

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

[0380] In one embodiment, T1' and T3' are separated by 11 nucleotides (ie, not counting the T1' and T3' nucleotides).

[0381] In one embodiment, T1' is at position 14 from the 5' end of the antisense strand. 2 is equal to 1 and the modification is at the 2' position or at a non-ribose, acyclic or backbone position that confers less steric bulk than 2'-OMe ribose.

[0382] In one embodiment, T3' is at position 2 from the 5' end of the antisense strand. 6 is equal to 1 and the modification is at the 2' position or at a non-ribose, acyclic or backbone position that confers steric bulk less than 2'-OMe ribose.

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

[0384] In one embodiment, T2' begins at position 6 from the 5' end of the antisense strand. In one example, T2' is at positions 6-10 from the 5' end of the antisense strand, and q 4 is 1.

[0385] In an exemplary embodiment, when the sense strand is 19-22 nucleotides in length, T1 is at the cleavage site of the sense strand, e.g., at position 11 from the 5' end of the sense strand, and n 2 is 1; T1' is at position 14 from the 5' end of the antisense strand; and q 2 is equal to 1, the modification to T1' is at the 2' position of the ribose sugar or at a non-ribose, acyclic, or backbone position that confers less steric bulk than 2'-OMe ribose; T2' is at positions 6-10 from the 5' end of the antisense strand; and q 4 is 1; T3' is at the second position from the 5' end of the antisense strand; and q 6 is equal to 1 and the modification to T3' is at the 2' position or at a non-ribose, acyclic or backbone position that confers steric bulk less than 2'-OMe ribose.

[0386] In one embodiment, T2' starts at position 8 from the 5' end of the antisense strand. 4 is 2.

[0387] In one embodiment, T2' begins at position 9 from the 5' end of the antisense strand. 4 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 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand.

[0389] In one embodiment, 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, 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 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 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 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, T2' is 2'-F, and q 4 is 2, 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 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 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, T2' is 2'-F, and q 4is 2, 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 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 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 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 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, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 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 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 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, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 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 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, 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 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 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, 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 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 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 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 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 is 1; and optionally has at least two additional TTs at the 3' end of the antisense strand.

[0397] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1is 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 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 is 1; optionally having at least two additional TTs at the 3'-end of the antisense strand; two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5'-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5'-end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 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 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'-OMe, and q7 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 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'-OMe, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within positions 18 to 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 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, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 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 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, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 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 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 q4 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.

[0403] 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; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand.

[0404] The RNAi agent can include a phosphorus-containing group at the 5'-end of the sense or antisense strand, such as 5'-terminal phosphate (5'-P), 5'-terminal phosphorothioate (5'-PS), 5'-terminal phosphorodithioate (5'-PS2), 5'-terminal vinylphosphonate (5'-VP), 5'-terminal methylphosphonate (MePhos), or 5'-deoxy-5'-C-malonyl. [ka] When the 5'-terminal phosphorus-containing group is a 5'-terminal vinyl phosphonate (5'-VP), the 5'-VP may be a 5'-E-VP isomer (i.e., trans-vinyl phosphate, [ka] ), 5'-Z-VP isomer (i.e., cis-vinyl phosphate, [ka] ), or a mixture thereof.

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

[0406] In one embodiment, the RNAi agent comprises a 5'-P. In one embodiment, the RNAi agent comprises a 5'-P on the antisense strand.

[0407] In one embodiment, the RNAi agent comprises a 5'-PS. In one embodiment, the RNAi agent comprises a 5'-PS on the antisense strand.

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

[0409] In one embodiment, the RNAi agent comprises a 5'-PS2. In one embodiment, the RNAi agent comprises a 5'-PS2 on the antisense strand.

[0410] In one embodiment, the RNAi agent comprises a 5'-PS2. In one embodiment, the RNAi agent comprises a 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 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, T2' is 2'-F, and q 4 is 2, 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 is 1. The RNAi agent also contains a 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 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, T2' is 2'-F, and q 4 is 2, 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 is 1. The RNAi agent also contains a 5'-P.

[0413] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1is 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, T2' is 2'-F, and q 4 is 2, 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 is 1. The RNAi agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 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 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, T2' is 2'-F, and q 4 is 2, 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 is 1. The RNAi agent also includes a 5'-PS2.

[0415] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1is 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, T2' is 2'-F, and q 4 is 2, 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 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 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, T2' is 2'-F, and q 4 is 2, 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 7is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also includes a 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 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, T2' is 2'-F, and q 4 is 2, 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 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also includes a 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 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, 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 2, 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 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 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 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, T2' is 2'-F, and q 4 is 2, 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 7is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also includes a 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 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, T2' is 2'-F, and q 4 is 2, 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 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also includes 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 is 3, B2 is 2'-OMe, and n 3 is 7 and n 4 is 0, B3 is 2'-OMe, and n5 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'-OMe, and q 7 is 1. The RNAi agent also contains a 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 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'-OMe, and q 7 is 1. The dsRNA agent also includes a 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 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 q2 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'-OMe, and q 7 is 1. The RNAi agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 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 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'-OMe, and q 7 is 1. The RNAi agent also includes a 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 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 2is 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'-OMe, and q 7 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 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'-OMe, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also includes a 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 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, 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'-OMe, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also includes a 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 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'-OMe, and q 7is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 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 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'-OMe, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also includes a 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 is 3, B2 is 2'-OMe, and n 3 is 7 and n 4 is 0, B3 is 2'-OMe, and n5 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'-OMe, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also includes 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 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, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The RNAi agent also contains a 5'-P.

[0432] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n2 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, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The RNAi agent also contains a 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 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, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The RNAi agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 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 2is 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, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The dsRNAi RNA agent also includes a 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 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, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 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 is 3, B2 is 2'-OMe, and n 3 is 7 and n4 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, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also includes a 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 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, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also includes a 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 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, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 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 is 3, B2 is 2'-OMe, and n 3 is 7 and n4 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, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also includes a 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 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, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also includes 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 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. The RNAi agent also contains a 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 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 4is 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. The RNAi agent also contains a 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 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. The RNAi agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 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 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 5is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The RNAi agent also includes a 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 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. 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 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 7is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also includes a 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 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; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also includes a 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 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, 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; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 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 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 7is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also includes a 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; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also includes 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, 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 2, 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 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also includes a 5'-P and a targeting ligand. In one embodiment, the 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 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, T2' is 2'-F, and q 4 is 2, 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 7is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also includes a 5'-PS and a targeting ligand. In one embodiment, the 5'-PS is at the 5' end of the antisense strand and the targeting ligand is 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 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, T2' is 2'-F, and q 4 is 2, 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 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5'-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5'-end of the antisense strand) and two phosphorothioate internucleotide 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 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, T2' is 2'-F, and q 4 is 2, 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 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also includes a 5'-PS2 and a targeting ligand. In one embodiment, the 5'-PS2 is at the 5' end of the antisense strand and the targeting ligand is 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 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, T2' is 2'-F, and q 4is 2, 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 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5'-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5'-end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also includes a 5'-deoxy-5'-C-malonyl and a targeting ligand. In one embodiment, the 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, 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'-OMe, and q 7is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also includes a 5'-P and a targeting ligand. In one embodiment, the 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 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'-OMe, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand. The RNAi agent also includes a 5'-PS and a targeting ligand. In one embodiment, the 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 2is 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...

Claims

1. A double-stranded ribonucleic acid (dsRNA) agent or a salt thereof for inhibiting the expression of lactate dehydrogenase A (LDHA) in a cell, the dsRNA agent or salt thereof comprises a sense strand and an antisense strand forming a double-stranded region; the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length; the sense strand and the antisense strand a) 5'-UGCCAUCAGUAUCUUAAUGAA-3' of SEQ ID NO: 3263 and 5'-UUCAUUAAGAUACUGAUGGCACA-3' of SEQ ID NO: 3449; b) 5'-GGCCUGUGCCAUCAGUAUCUU-3' of SEQ ID NO: 3278 and 5'-AAGAUACUGAUGGCACAGGCCAU-3' of SEQ ID NO: 3464; c) 5'-GCCAUCAGUAUCUUAAUGAAA-3' of SEQ ID NO: 3347 and 5'-UUUCAUUAAGAUACUGAUGGCAC-3' of SEQ ID NO: 3533; and d) 5'-GCCUGUGCCAUCAGUAUCUUA-3' of SEQ ID NO: 3367 and 5'-UAAGAUACUGAUGGCACAGGCCA-3' of SEQ ID NO: 3553; and comprising at least 19 contiguous nucleotides from any one of the sense and antisense nucleotide sequences selected from the group consisting of: All of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are deoxy-nucleotide modified, 3'-terminal deoxy-thymine (dT) nucleotide modified, 2'-O-methyl nucleotide modified, 2'-fluoro nucleotide modified, 2'-deoxy-nucleotide modified, locked nucleotide modified, non-locked nucleotide modified, conformationally restricted nucleotide modified, constrained ethyl nucleotide modified, abasic nucleotide modified, 2'-amino-nucleotide modified, 2'-O-allyl-nucleotide modified, 2'-C-alkyl-nucleotide modified, 2'-hydroxyl-nucleotide modified, 2'-methoxyethyl nucleotide modified, 2' -O-alkyl-nucleotide modifications, morpholino nucleotide modifications, phosphoramidate nucleotide modifications, nucleotide modifications containing unnatural bases, tetrahydropyran nucleotide modifications, 1,5-anhydrohexitol nucleotide modifications, cyclohexenyl nucleotide modifications, nucleotide modifications containing phosphorothioate groups, nucleotide modifications containing methylphosphonate groups, nucleotide modifications containing 5'-phosphates, nucleotide modifications containing 5'-phosphate mimetics, glycol nucleotide modifications, and 2-O-(N-methylacetamido) nucleotide modifications, and combinations thereof; A dsRNA agent, or a salt thereof, wherein the dsRNA agent further comprises 6 to 8 phosphorothioate internucleotide linkages, and wherein a ligand comprising one or more N-acetylgalactosamine (GalNAc) derivatives is conjugated to at least one strand.

2. 2. The dsRNA agent, or a salt thereof, of claim 1, wherein the nucleotide modification is selected from the group consisting of a 2'-O-methyl nucleotide modification and a 2'-fluoro nucleotide modification.

3. 10. The dsRNA agent or salt thereof of claim 1, wherein the ligand is conjugated to the 3' end of the sense strand of the dsRNA agent or salt thereof.

4. 10. The dsRNA agent or salt thereof of claim 1 or 3, wherein the ligand is conjugated to the dsRNA agent or salt thereof via a monovalent, divalent, or trivalent linker.

5. The ligand is 【Chemistry 1】 5. The dsRNA agent or salt thereof of any one of claims 1, 3 and 4, wherein:

6. Schematic diagram below: 【Chemistry 2】 6. The dsRNA agent or salt thereof of claim 5, wherein the dsRNA agent or salt thereof is conjugated to a ligand as shown in

7. 7. The dsRNA agent of claim 6, or a salt thereof, wherein X is O.

8. 8. The dsRNA agent or salt thereof of any one of claims 1-7, wherein the antisense strand comprises two phosphorothioate internucleotide linkages at the 5'-end and two phosphorothioate internucleotide linkages at the 3'-end, and the sense strand comprises at least two phosphorothioate internucleotide linkages at the 5'-end or the 3'-end, or at both the 5'-end and the 3'-end.

9. The dsRNA agent or salt thereof according to any one of claims 1 to 8, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 3263, 5'-UGCCAUCAGUAUCUUAAUGAA-3', and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 3449, 5'-UUCAUUAAGAUACUGAUGGCACA-3'.

10. 9. The dsRNA agent of any one of claims 1-8, or a salt thereof, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 3278, 5'-GGCCUGUGCCAUCAGUAUCUU-3', and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 3464, 5'-AAGAUACUGAUGGCACAGGCCAU-3'.

11. 9. The dsRNA agent of any one of claims 1 to 8, or a salt thereof, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 3347, 5'-GCCAUCAGUAUCUUAAUGAAA-3', and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 3533, 5'-UUUCAUUAAGAUACUGAUGGCAC-3'.

12. 9. The dsRNA agent of any one of claims 1 to 8, or a salt thereof, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 3367, 5'-GCCUGUGCCAUCAGUAUCUUA-3', and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 3553, 5'-UAAGAUACUGAUGGCAACAGGCCA-3.

13. 13. An isolated cell comprising the dsRNA agent or salt thereof of any one of claims 1 to 12.

14. A pharmaceutical composition for inhibiting the expression of lactate dehydrogenase A (LDHA) gene, comprising the dsRNA agent or salt thereof according to any one of claims 1 to 12.

15. 16. An in vitro method of inhibiting lactate dehydrogenase A (LDHA) expression in a cell, comprising contacting the cell with the dsRNA agent or salt thereof of any one of claims 1-12, or the pharmaceutical composition of claim 14, thereby inhibiting expression of LDHA in the cell.

16. 16. The method of claim 15, wherein LDHA expression is inhibited by at least 30%.

17. 15. A pharmaceutical composition comprising the dsRNA agent or salt thereof of any one of claims 1 to 12, or the pharmaceutical composition of claim 14, for use in a method of inhibiting LDHA expression in a subject.

18. 15. A pharmaceutical composition comprising the dsRNA agent or salt thereof of any one of claims 1-12, or the pharmaceutical composition of claim 14, for use in a method of treating a subject having a disorder that could benefit from reduced LDHA expression.

19. 19. The pharmaceutical composition of claim 18, wherein the disorder is a disease, disorder or condition associated with the oxalate pathway.

20. 20. The pharmaceutical composition of claim 19, wherein the disease, disorder, or condition associated with the oxalate pathway is an oxalate-associated disease, disorder, or condition, or a lactate dehydrogenase-associated disease, disorder, or condition.

21. 21. The pharmaceutical composition of claim 20, wherein the oxalate-associated disease, disorder, or condition is a kidney stone formation disease, disorder, or condition, or a calcium oxalate tissue deposition disease, disorder, or condition.

22. 21. The pharmaceutical composition of claim 20, wherein the lactate dehydrogenase-associated disease, disorder, or condition 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, hepatocellular necrosis, liver fibrosis, and non-alcoholic fatty liver disease (NAFLD).

23. 20. The pharmaceutical composition of claim 19, wherein the disease, disorder or condition associated with the oxalate pathway is primary hyperoxaluria type 2 (PH2).

24. The pharmaceutical composition according to any one of claims 17 to 23, wherein the subject is a human.

25. The pharmaceutical composition of any one of claims 17 to 24, wherein the method further comprises administering to the subject an additional therapeutic agent.

26. 26. The pharmaceutical composition of any one of claims 17-25, wherein the dsRNA agent is administered to the subject at a dose of 0.01 mg / kg to 10 mg / kg.

27. The pharmaceutical composition of any one of claims 17 to 25, wherein the dsRNA agent is administered to a subject at a dose of 0.5 mg / kg to 50 mg / kg.

28. 28. The pharmaceutical composition of any one of claims 17-27, wherein the dsRNA agent is administered subcutaneously to the subject.

29. 29. The pharmaceutical composition of any one of claims 17 to 28, wherein administration of the pharmaceutical composition to a subject results in decreased urinary oxalate, tissue oxalate, plasma oxalate, decreased LDHA enzyme activity, decreased LDHA protein accumulation, and / or decreased HAO1 protein accumulation.

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