Sodium-glucose cotransporter 2 (SGLT2) IRNA compositions and methods of use thereof

Abstract

The present invention relates to RNAi agents, e.g., dsRNA agents, that target the sodium-glucose cotransporter 2 (SGLT2) gene. The present invention also relates to methods of using such RNAi agents to inhibit expression of the SGLT2 gene, and to methods of treating or preventing an SGLT2-related disease, such as, for example, gout or diabetes, in a subject.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 189,760, filed May 18, 2021, and U.S. Provisional Patent Application No. 63 / 320,278, filed March 16, 2022. The entire contents of each of the foregoing applications are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated by reference in its entirety. The ASCII copy was created on April 6, 2022, is named 121301_15820_SL.TXT, and is 192,240 bytes in size. [Background technology]

[0003] Diabetes mellitus is a group of metabolic diseases characterized by high blood glucose levels due to defects in insulin secretion or action, or both. There are two most common types of diabetes: type 1 diabetes and type 2 diabetes, both of which result from an inability to regulate insulin, a hormone released by the pancreas in response to increased levels of blood sugar (glucose).

[0004] Type 1 diabetes occurs when the pancreas produces too little insulin to properly regulate blood glucose levels. In contrast, in type 2 diabetes, the pancreas continues to manufacture insulin. However, the body responds poorly to insulin and develops resistance to its effects, resulting in too much sugar circulating in the bloodstream. Type 2 diabetes is an increasingly prevalent metabolic disease with frequent complications leading to a significant decrease in life expectancy. Type 2 diabetes is currently the most common cause of adult-onset vision loss, kidney failure, and amputation in industrialized societies. Furthermore, the presence of type 2 diabetes is associated with a two- to five-fold increased risk of cardiovascular disease.

[0005] Hyperinsulinemia or insulin resistance in diabetes is also associated with decreased renal clearance of uric acid (also referred to herein as urate) and elevated serum uric acid levels. Chronic elevated serum uric acid (chronic hyperuricemia), typically defined as serum uric acid levels above 6.8 mg / dL (greater than 360 mmol / L), a level above the physiological saturation threshold (Mandell, Cleve. Clin. Med. 75:S5-S8, 2008), can lead to many different diseases. Gout is characterized by repeated attacks of acute inflammatory arthritis, typically caused by insufficient renal clearance of uric acid or an inflammatory response to uric acid crystals in the joints due to excessive uric acid production. Elevated uric acid is also associated with nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), metabolic disorders, cardiovascular disease, and conditions related to oxidative stress, chronic low-grade inflammation, and insulin resistance (Xu et al., J. Hepatol. 62:1412-1419, 2015; Cardoso et al., J. Pediatr. 89:412-418, 2013; Sertoglu et al., Clin. Biochem., 47:383-388, 2014).

[0006] Plasma glucose is normally maintained within a relatively narrow range, requiring a delicate balance between glucose production (liver and kidney), intestinal absorption, renal reabsorption, and glucose utilization in tissues throughout the body. Glucose reabsorption across renal epithelial cells depends on a class of sodium-dependent glucose cotransporters (SGLTs) located in the brush border membrane of the tubules along the sodium gradient. There are at least three SGLT isoforms that differ in their expression patterns and physicochemical properties.

[0007] One of these cotransporters, SGLT2, also known as solute carrier family 5 member 2 (SLC5A2), is expressed in the convoluted tubules of the kidney. The SGLT2 transporter is the primary cotransporter involved in glucose reabsorption in the kidney, responsible for approximately 90% of filtered glucose reabsorption (Scheen AJ, Drugs. 2015 Jan;75(1):33-59).

[0008] SGLT2 inhibitors are a class of drugs known to treat type 2 diabetes or hyperglycemia. SGLT2 inhibitors prevent renal reabsorption of glucose, thereby promoting urinary glucose excretion and resulting in lower serum glucose levels. SGLT2 inhibitors are also effective in lowering serum uric acid levels. However, SGLT2 inhibitors can also cause side effects, including diabetic ketoacidosis and blood and kidney infections.

[0009] Therefore, there is an unmet need for effective treatments for diabetes and related diseases, such as gout, such as agents that can selectively and efficiently silence the SGLT2 gene using the cell's own RNAi machinery, which have both high biological activity and in vivo stability and can effectively inhibit the expression of the target SGLT2 gene. Summary of the Invention

[0010] The present disclosure provides an RNAi agent composition that performs RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the gene encoding sodium-glucose cotransporter 2 (SGLT2).The SGLT2 gene can be present in a cell, for example, in a subject, such as a human.The present disclosure also provides a method of using the RNAi agent composition of the present disclosure to inhibit the expression of the SGLT2 gene or to treat a subject who will benefit from inhibiting or reducing the expression of the SGLT2 gene, for example, a subject who has an SGLT2-related disorder, for example, a subject who has gout or diabetes, such as type II diabetes, or a subject who is at risk of developing gout or diabetes.

[0011] Thus, in one aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent that inhibits expression of sodium-glucose cotransporter 2 (SGLT2) in a cell, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, wherein the sense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, or 20 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO:1 by no more than 0, 1, 2, or 3 nucleotides, and wherein said antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, or 20 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO:6 by no more than 0, 1, 2, or 3 nucleotides, and wherein either the sense strand or the antisense strand is conjugated to one or more lipophilic moieties.

[0012] In another aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent that inhibits expression of sodium-glucose cotransporter 2 (SGLT2) in a cell, wherein the dsRNA agent comprises a sense strand and an antisense strand that form a double-stranded region, wherein the antisense strand comprises a region that is complementary to an mRNA encoding SGLT2, and wherein the complementary region comprises at least 15 contiguous nucleotides, e.g., 15, 16, 17, 18, 19, or 20, that differs by 0, 1, 2, or 3 nucleotides from any one of the antisense nucleotide sequences of any one of Tables 2-3, and wherein the sense strand or the antisense strand is conjugated to one or more lipophilic moieties.

[0013] In one embodiment, both the sense and antisense strands are conjugated to one or more lipophilic moieties.

[0014] In one embodiment, the lipophilicity of the lipophilic moiety is greater than 0, as measured by log Kow.

[0015] In one embodiment, the hydrophobicity of the double-stranded RNAi agent is greater than 0.2, as measured by the unbound fraction in a plasma protein binding assay of the double-stranded RNAi agent.

[0016] In one embodiment, the plasma protein binding assay is an electrophoretic mobility shift assay using human serum albumin protein.

[0017] In one embodiment, the dsRNA agent includes at least one modified nucleotide.

[0018] In some embodiments, substantially all of the nucleotides of the antisense strand are modified nucleotides.

[0019] In another embodiment, all of the nucleotides in the sense strand and all of the nucleotides in the antisense strand comprise a modification.

[0020] In one embodiment, at least one of the modified nucleotides is a deoxy-nucleotide, a 3'-terminal deoxythymidine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, an unlocked 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 nucleotides, cyclohexenyl modified nucleotides, nucleotides containing a 5'-phosphorothioate group, nucleotides containing a 5'-methylphosphonate group, nucleotides containing a 5' phosphate or a 5' phosphate mimic, nucleotides containing vinyl phosphonate, nucleotides containing adenosine-glycol nucleic acid (GNA), nucleotides containing thymidine-glycol nucleic acid (GNA) S-isomer, nucleotides containing 2-hydroxymethyl-tetrahydrofuran-5-phosphate, nucleotides containing 2'-deoxythymidine-3' phosphate, nucleotides containing 2'-deoxyguanosine-3'-phosphate, 2'-O-hexadecyl nucleotides, nucleotides containing 2'-phosphate, cytidine-2'-phosphate nucleotides, guanosine- The nucleotide is selected from the group consisting of 2'-phosphate nucleotides, 2'-O-hexadecyl-cytidine-3'-phosphate nucleotides, 2'-O-hexadecyl-adenosine-3'-phosphate nucleotides, 2'-O-hexadecyl-guanosine-3'-phosphate nucleotides, 2'-O-hexadecyl-uridine-3'-phosphate nucleotides, 5'-vinylphosphonate (VP), 2'-deoxyadenosine-3'-phosphate nucleotides, 2'-deoxycytidine-3'-phosphate nucleotides, 2'-deoxyguanosine-3'-phosphate nucleotides, 2'-deoxythymidine-3'-phosphate nucleotides, 2'-deoxyuridine nucleotides, and terminal nucleotides linked to cholesteryl derivatives and dodecanoic acid bisdecylamide groups, and combinations thereof.

[0021] In another embodiment, the modified nucleotide is selected from the group consisting of 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, 3'-terminal deoxythymidine nucleotides (dT), locked nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, 2'-O-methyl modified nucleotides, nucleosides including glycol nucleic acids (GNAs), morpholino nucleotides, phosphoramidates, and nucleotides containing unnatural bases.

[0022] In another embodiment, the modified nucleotides include a short sequence of 3'-terminal deoxythymidine nucleotides (dT).

[0023] In yet another embodiment, the modifications on the nucleotide are 2'-O-methyl modifications, 2'-deoxy-modifications, 2'fluoro-modifications, 5'-vinylphosphonate (VP) modifications, and 2'-O hexadecyl nucleotide modifications.

[0024] In certain embodiments, the double-stranded RNAi agent does not include an inverted abasic nucleotide.

[0025] In one embodiment, the dsRNA agent further comprises at least one phosphorothioate internucleotide linkage.

[0026] In one embodiment, the dsRNA agent includes 6 to 8 phosphorothioate internucleotide linkages.

[0027] In one embodiment, each strand is 30 nucleotides or less in length.

[0028] In one embodiment, at least one strand comprises a 3' overhang of at least 1 nucleotide.

[0029] In another embodiment, at least one strand comprises a 3' overhang of at least 2 nucleotides.

[0030] The double-stranded region can be 15 to 30 nucleotide pairs in length, 17 to 23 nucleotide pairs in length, 17 to 25 nucleotide pairs in length, 23 to 27 nucleotide pairs in length, 19 to 21 nucleotide pairs in length, or 21 to 23 nucleotide pairs in length.

[0031] Each strand of the dsRNA agent can be 15-30, 17-20, 19-30 nucleotides in length, 19-23 nucleotides in length, or 21-23 nucleotides in length, e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0032] In certain embodiments, the double-stranded RNAi agent further comprises a lipophilic ligand, e.g., a C16 ligand, conjugated to the 3' end of the sense strand via a monovalent or branched bivalent or trivalent linker.

[0033] In one embodiment, the ligand is conjugated at the 2'-position of a nucleotide or modified nucleotide in the sense or antisense strand. For example, a C16 ligand may be conjugated as shown in the following structure: [ka] , During the ceremony, * represents a bond to an adjacent nucleotide, and B is a nucleobase or nucleobase analog, optionally B is adenine, guanine, cytosine, thymine, or uracil.

[0034] In other embodiments, the agent further comprises a targeting ligand that targets liver tissue, e.g., one or more GalNAc derivatives, optionally conjugated to the double-stranded RNAi agent via a linker or carrier.

[0035] In yet other embodiments, the agent further comprises a lipophilic ligand, e.g., a C16 ligand, conjugated to the 3' end of the sense strand, e.g., via a monovalent or branched bivalent or trivalent linker, and a targeting ligand that targets liver tissue, such as one or more GalNAc derivatives, conjugated to the 3' end of the sense strand, e.g., via a monovalent or branched bivalent or trivalent linker.

[0036] In one embodiment, one or more lipophilic moieties are conjugated to one or more interior positions on at least one strand.

[0037] In one embodiment, one or more lipophilic moieties are conjugated via a linker or carrier to one or more interior positions on at least one chain.

[0038] In certain embodiments, the lipophilic moiety is not a cholesterol moiety.

[0039] In certain embodiments, the agent further comprises a targeting ligand that targets liver tissue, e.g., one or more GalNAc derivatives, optionally conjugated to the double-stranded RNAi agent via a linker or carrier.

[0040] In yet other embodiments, the agent further comprises one or more lipophilic moieties conjugated, optionally via a linker or carrier, to one or more internal nucleotide positions, and a targeting ligand that targets liver tissue, e.g., one or more GalNAc derivatives, optionally conjugated to the double-stranded RNAi agent via a linker or carrier.

[0041] In one embodiment, the interior positions include all but the two most distal positions on each end of at least one strand.

[0042] In another embodiment, the interior positions include all but the three most distal positions on each of at least one strand.

[0043] In another embodiment, the internal position excludes the cleavage site region of the sense strand.

[0044] In yet another embodiment, the internal positions include all positions except positions 9 to 12 counting from the 5' end of the sense strand. In certain embodiments, the sense strand is 21 nucleotides in length.

[0045] In one embodiment, the internal positions include all positions except positions 11-13 from the 3' end of the sense strand. Optionally, the internal positions exclude the cleavage site region of the antisense strand. In certain embodiments, the sense strand is 21 nucleotides in length.

[0046] In one embodiment, the internal position excludes the cleavage site region of the antisense strand.

[0047] In one embodiment, internal positions include all positions except positions 12 to 14 counting from the 5' end of the antisense strand. In a particular embodiment, the antisense strand is 23 nucleotides in length.

[0048] In one embodiment, internal positions include all positions except positions 11-13 on the sense strand, counting from the 3' end, and positions 12-14 on the antisense strand, counting from the 5' end. In a specific embodiment, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

[0049] In one embodiment, the one or more lipophilic moieties are conjugated to one or more of the internal positions selected from the group consisting of positions 4-8 and 13-18 on the sense strand and positions 6-10 and 15-18 on the antisense strand, counting from the 5' end of each strand.

[0050] In one embodiment, the one or more lipophilic moieties are conjugated to one or more internal positions selected from the group consisting of positions 5, 6, 7, 15, and 17 on the sense strand and positions 15 and 17 on the antisense strand, counting from the 5' end of each strand. In a particular embodiment, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

[0051] In one embodiment, the position in the double-stranded region excludes the cleavage site region of the sense strand.

[0052] In one embodiment, the sense strand is 21 nucleotides in length, the antisense strand is 23 nucleotides in length, and the lipophilic moiety is conjugated to position 21, 20, 15, 1, 7, 6, or 2 of the sense strand or position 16 of the antisense strand, counting from the 5' end of each strand.

[0053] In one embodiment, the lipophilic moiety is conjugated to position 21, 20, 15, 1, or 7 of the sense strand, counting from the 5' end of the strand.

[0054] In one embodiment, the lipophilic moiety is conjugated to position 21, 20, or 15 of the sense strand, counting from the 5' end of the strand.

[0055] In one embodiment, the lipophilic moiety is conjugated to position 20 or 15 of the sense strand, counting from the 5' end of the strand.

[0056] In one embodiment, the lipophilic moiety is conjugated to position 16 of the antisense strand, counting from the 5' end of the strand.

[0057] In one embodiment, the lipophilic moiety is an aliphatic, alicyclic, or polyalicyclic compound.

[0058] In one embodiment, the lipophilic moiety is selected from the group consisting of a lipid, cholesterol, retinoic acid, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine. In certain embodiments, the lipophilic moiety is not cholesterol.

[0059] In one embodiment, the lipophilic moiety contains a saturated or unsaturated C4-C30 hydrocarbon chain and an optional functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne.

[0060] In one embodiment, the lipophilic moiety contains a saturated or unsaturated C6 to C18 hydrocarbon chain.

[0061] In one embodiment, the lipophilic moiety contains a saturated or unsaturated C16 hydrocarbon chain.

[0062] In one embodiment, a saturated or unsaturated C16 hydrocarbon chain is conjugated to position 6, counting from the 5' end of one chain.

[0063] In one embodiment, the lipophilic moiety is conjugated via a carrier that replaces one or more nucleotides in an internal position or in the double-stranded region.

[0064] In one embodiment, the carrier is a cyclic group selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl, or is an acyclic portion of a serinol or diethanolamine backbone system.

[0065] In one embodiment, the lipophilic moiety is conjugated to the double-stranded iRNA agent via a linker that contains an ether, a thioether, a urea, a carbonate, an amine, an amide, a maleimide-thioether, a disulfide, a phosphodiester, a sulfonamide linkage, a product of a click reaction, or a carbamate.

[0066] In one embodiment, the lipophilic moiety is conjugated to a nucleobase, a sugar moiety, or an internucleoside linkage.

[0067] In one embodiment, the double-stranded RNAi agent further comprises a phosphate or a phosphate mimic at the 5'-end of the antisense strand. Optionally, the phosphate mimic is 5'-vinylphosphonate (VP). When the phosphate mimic is 5'-vinylphosphonate (VP), the 5'-terminal nucleotide may have the following structure: [ka] During the ceremony, * indicates the position of attachment to the 5'-position of the adjacent nucleotide; R is hydrogen, hydroxy, methoxy, fluoro, or another 2'-modification described herein (e.g., hydroxy or methoxy); B is a nucleobase or modified nucleobase; optionally, B is adenine, guanine, cytosine, thymine, or uracil.

[0068] In certain embodiments, the RNAi agent does not include an inverted abasic nucleotide.

[0069] In certain embodiments, the double-stranded RNAi agent does not comprise a targeting ligand.

[0070] In certain embodiments, double-stranded RNAi agent further comprises targeting ligand, which targets the receptor that mediates delivery to liver tissue, for example, lipophilic ligand.In certain embodiments, targeting ligand is C16 ligand.In certain embodiments, lipophilic ligand is not cholesterol moiety.

[0071] In one embodiment, the lipophilic moiety or targeting ligand is conjugated via a biocleavable linker selected from the group consisting of DNA, RNA, disulfide, amide, and functionalized mono- or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.

[0072] In one embodiment, the 3' end of the sense strand is protected via an end cap that is an amine-bearing cyclic group selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl.

[0073] In one embodiment, the dsRNA agent further comprises a targeting ligand that targets liver tissue.

[0074] In one embodiment, the targeting ligand is a GalNAc conjugate.

[0075] In one embodiment, the dsRNA agent further includes a terminal chiral modification that occurs in a first internucleotide linkage at the 3'-end of the antisense strand having a linking phosphorus atom in the Sp configuration, a terminal chiral modification that occurs in a first internucleotide linkage at the 5'-end of the antisense strand having a linking phosphorus atom in the Rp configuration, and a terminal chiral modification that occurs in a first internucleotide linkage at the 5'-end of the sense strand having a linking phosphorus atom in either the Rp or Sp configuration.

[0076] In one embodiment, the dsRNA agent further includes a terminal chiral modification occurring at the first and second internucleotide linkages at the 3'-end of the antisense strand, with the linking phosphorus atom in the Sp configuration; a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the antisense strand, with the linking phosphorus atom in the Rp configuration; and a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the sense strand, with the linking phosphorus atom in either the Rp or Sp configuration.

[0077] In one embodiment, the dsRNA agent further includes a terminal chiral modification occurring at the first, second, and third internucleotide linkages at the 3'-end of the antisense strand having a linking phosphorus atom in the Sp configuration, a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the antisense strand having a linking phosphorus atom in the Rp configuration, and a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the sense strand having a linking phosphorus atom in either the Rp or Sp configuration.

[0078] In one embodiment, the dsRNA agent further includes a terminal chiral modification occurring at the first and second internucleotide linkages at the 3'-end of the antisense strand, the terminal chiral modification having the linking phosphorus atom in the Sp configuration; a terminal chiral modification occurring at the third internucleotide linkage at the 3'-end of the antisense strand, the terminal chiral modification having the linking phosphorus atom in the Rp configuration; a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the antisense strand, the terminal chiral modification having the linking phosphorus atom in the Rp configuration; and a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the sense strand, the terminal chiral modification having the linking phosphorus atom in either the Rp or Sp configuration.

[0079] In one embodiment, the dsRNA agent further includes a terminal chiral modification occurring at the first and second internucleotide linkages at the 3'-end of the antisense strand, the first and second internucleotide linkages having the linking phosphorus atom in the Sp configuration, a terminal chiral modification occurring at the first and second internucleotide linkages at the 5'-end of the antisense strand, the first internucleotide linkages having the linking phosphorus atom in the Rp configuration, and a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the sense strand, the first internucleotide linkage having the linking phosphorus atom in either the Rp or Sp configuration.

[0080] In one embodiment, the dsRNA agent further comprises a phosphate or a phosphate mimic at the 5' end of the antisense strand.

[0081] In one embodiment, the phosphate mimic is a 5'-vinylphosphonate (VP).

[0082] In one embodiment, the base pair at one position of the 5'-end of the antisense strand of the duplex is an AU base pair.

[0083] In one embodiment, the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

[0084] In another embodiment, the RNAi agent is a pharmaceutically acceptable salt thereof. The "pharmaceutically acceptable salt" of each RNAi agent herein includes, but is not limited to, sodium, calcium, lithium, potassium, ammonium, magnesium, and mixtures thereof. Those skilled in the art will understand that when an RNAi agent is provided as a polycationic salt, it will have one cation per free acid group of the optionally modified phosphodiester backbone and / or any other acidic modifications (e.g., a phosphonate group at the 5' end). For example, an oligonucleotide "n" nucleotides in length contains n-1 optionally modified phosphodiesters, such that an oligonucleotide 21 nt in length can be provided as a salt with up to 20 cations (e.g., 20 sodium cations). Similarly, an RNAi agent having a 21 nt sense strand and a 23 nt antisense strand can be provided as a salt with up to 42 cations (e.g., 42 sodium cations). In the foregoing examples, the RNAi agent also includes a 5'-terminal phosphate group or a 5'-terminal vinylphosphonate group, and the RNAi agent can be provided as a salt with up to 44 cations (e.g., 44 sodium cations).

[0085] The present invention further provides cells, pharmaceutical compositions for inhibiting expression of the SGLT2 gene, and pharmaceutical compositions comprising lipid formulations comprising a dsRNA agent of the invention.

[0086] In one aspect, the present invention provides a method for inhibiting the expression of the SGLT2 gene in a cell, the method comprising contacting the cell with a dsRNA agent of the present invention or a pharmaceutical composition of the present invention, and maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the SGLT2 gene, thereby inhibiting the expression of the SGLT2 gene in the cell.

[0087] In one embodiment, the cell is in a subject, e.g., a human subject, e.g., a human subject with an SGLT2-associated disorder, such as gout or diabetes.

[0088] In one embodiment, expression of the SGLT2 gene is inhibited by at least about 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%.

[0089] In one aspect, the present invention provides a method for treating a subject having a disorder that would benefit from reduced SGLT2 expression, comprising administering to the subject a therapeutically effective amount of any of the dsRNAs of the present invention or any of the pharmaceutical compositions of the present invention, thereby treating the subject having a disorder that would benefit from reduced SGLT2 expression.

[0090] In another aspect, the present invention provides a method for preventing at least one symptom in a subject with a disorder that would benefit from reduced SGLT2 expression, comprising administering to the subject a prophylactically effective amount of any of the dsRNAs of the present invention or any of the pharmaceutical compositions of the present invention, thereby preventing at least one symptom in the subject with a disorder that would benefit from reduced SGLT2 expression.

[0091] In certain embodiments, the disorder is an SGLT2-associated disorder.

[0092] In some embodiments, the SGLT2-related disorder is gout.

[0093] In some embodiments, the SGLT2-related disorder is a carbohydrate disorder, e.g., diabetes, type I diabetes, type II diabetes, galactosemia, hereditary fructose intolerance, fructose 1,6-diphosphatase deficiency, glycogen storage disorders, congenital glycosylation disorders, insulin resistance, insulin deficiency, hyperinsulinemia, impaired glucose tolerance (IGT), abnormal glycogen metabolism; a disorder of amino acid metabolism, e.g., maple syrup urine disease (MSUD), or homocystinuria; a disorder of organic acid metabolism, e.g., methylmalonic aciduria, 3-methylglutaconic aciduria-Barth syndrome, glutaric aciduria, or 2-hydroxyglutaric aciduria-D and L forms; a disorder of fatty acid beta-oxidation, e.g., medium-chain acyl-CoA dehydrogenase deficiency (MCAD), long-chain 3-hydroxyacyl-CoA the metabolic disease is selected from the group consisting of: A dehydrogenase deficiency (LCHAD), very long-chain acyl-CoA dehydrogenase deficiency (VLCAD), disorders of lipid metabolism, such as GM1 gangliosidosis, Tay-Sachs disease, Sandhoff disease, Fabry disease, Gaucher disease, Niemann-Pick disease, Krabbe disease, mucolipidosis, or mucopolysaccharidosis; mitochondrial disorders, such as mitochondrial cardiomyopathy; Leigh disease; mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS); myoclonic epilepsy with ragged fibers (MERRF); neuropathy, ataxia, and retinitis pigmentosa (NARP); Barth syndrome; or peroxisomal disorders, such as Zellweger syndrome (cerebrohepatorenal syndrome), X-linked adrenoleukodystrophy, or Refsum disease.

[0094] In some embodiments, the SGLT2-associated disease is diabetes. In some embodiments, the SGLT2-associated disease is type II diabetes.

[0095] In one embodiment, the subject is a human.

[0096] In one embodiment, administration of the dsRNA agent to a subject causes a decrease in SGLT2 protein accumulation in the subject. In some embodiments, administration of the dsRNA agent results in a reduction in blood glucose and / or uric acid levels in the subject.

[0097] In one embodiment, the dsRNA agent is administered to the subject at a dose of about 0.01 mg / kg to about 50 mg / kg.

[0098] In some embodiments, the double-stranded RNAi agent is administered orally to the subject.

[0099] In some embodiments, the double-stranded RNAi agent is administered to the subject subcutaneously.

[0100] In one embodiment, the double-stranded RNAi agent is administered intravenously.

[0101] In certain embodiments, the RNAi agent is further taken up by one or more tissues or cell types, for example, the liver or kidney.

[0102] In one embodiment, the method further comprises determining the level of SGLT2 in a sample from the subject. In some embodiments, the SGLT2 level in the subject sample is the SGLT2 protein level in a blood or serum sample.

[0103] In one embodiment, the method further comprises administering to the subject an additional agent or therapy suitable for the treatment or prevention of an SGLT2-associated disorder.

[0104] In one embodiment, the additional therapeutic agent is selected from the group comprising an agent for treating diabetes mellitus, an agent for treating diabetic complications, an agent for treating cardiovascular disease, an antihyperlipidemic agent, an antihypertensive or antihypertensive agent, an antiobesity agent, an agent for treating nonalcoholic steatohepatitis (NASH), a chemotherapeutic agent, an immunotherapeutic agent, an immunosuppressant, an anti-inflammatory agent, an anti-steatotic agent, an antifibrotic agent, an immunomodulatory agent, a tyrosine kinase inhibitor, an antifibrotic agent, and a combination of any of the foregoing.

[0105] The present invention also provides a kit comprising any of the dsRNAs of the present invention or any of the pharmaceutical compositions of the present invention, and optionally instructions for use.In one embodiment, the present invention provides a kit for carrying out a method for inhibiting the expression of SGLT2 gene in cells by contacting cells with the double-stranded RNAi agent of the present invention in an amount effective to inhibit the expression of SGLT2 in cells.The kit comprises an RNAi agent and instructions for use, and optionally includes a means for administering the RNAi agent to a subject.

[0106] The present invention is further illustrated by the following detailed description. DETAILED DESCRIPTION OF THE INVENTION

[0107] The present invention provides iRNA compositions that cause RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of SGLT2 gene.The SGLT2 gene can be present in a cell, for example, in a subject cell, such as a human.The use of these iRNAs allows the targeted degradation of the mRNA of the corresponding gene (SGLT2 gene) in mammals.

[0108] The iRNAs of the present invention are designed to target the human SGLT2 gene, including portions of the gene that are conserved in SGLT2 orthologs of other mammalian species. Without intending to be limited by theory, it is believed that combinations or subcombinations of the above-mentioned features and specific target sites or specific modifications in these iRNAs improve the efficacy, stability, potency, durability, and safety of the iRNAs of the present invention.

[0109] Thus, the present invention provides methods for treating and preventing SGLT2-related disorders, such as gout or diabetes, using iRNA compositions that result in RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the SGLT2 gene.

[0110] The iRNA of the present invention may have a length of up to about 30 nucleotides 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, 19-25, 19-26, 19-25, 19-28, 19-29, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25 ... The RNA strand (antisense strand) comprises an RNA strand having a region of 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, which region is substantially complementary to at least a portion of an mRNA transcript of the SGLT2 gene.

[0111] In certain embodiments, one or both strands of a double-stranded RNAi agent of the invention are up to 66 nucleotides in length, e.g., 36-66, 26-36, 25-36, 31-60, 22-43, 27-53 nucleotides in length, with a region of at least 19 contiguous nucleotides that is substantially complementary to at least a portion of the mRNA of the SGLT2 gene. In some embodiments, such iRNA agents with longer antisense strands can include a second RNA strand (sense strand) that is, for example, 20-60 nucleotides in length, where the sense and antisense strands form a duplex of 18-30 contiguous nucleotides.

[0112] The use of iRNA of the present invention allows for the targeted degradation of SGLT2 mRNA in mammals.Therefore, methods and compositions comprising these iRNAs are useful for treating subjects with SGLT2-related disorders, such as gout or diabetes (e.g., type II diabetes), or for treating subjects at risk of developing gout or diabetes.

[0113] Thus, the present invention provides methods and combination therapies for treating subjects with disorders that would benefit from inhibiting or reducing expression of the SGLT2 gene, e.g., SGLT2-associated diseases, e.g., gout and diabetes, including type II diabetes, using iRNA compositions that effect RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of the SGLT2 gene.

[0114] The present invention also provides methods for preventing at least one symptom in a subject with a disorder that would benefit from inhibiting or reducing expression of the SGLT2 gene, e.g., gout or diabetes, such as type II diabetes, e.g., a disorder associated with thrombosis.

[0115] In one aspect, the invention provides a RISC complex comprising a dsRNA agent according to any one of claims 1-70.

[0116] The following detailed description of the invention discloses methods of making and using compositions containing iRNA that inhibit expression of the SGLT2 gene, as well as compositions, uses, and methods of treating subjects who would benefit from inhibiting and / or reducing expression of the SGLT2 gene, e.g., subjects susceptible to or diagnosed with an SGLT2-related disorder.

[0117] I. Definition In order that the present invention may be more readily understood, certain terms are first defined. Additionally, it should be noted that whenever a value or range of values ​​for a parameter is listed, it is intended that values ​​and ranges intermediate to the listed values ​​are also intended to be part of the invention.

[0118] 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. For example, "an element" means one element or to more than one element, e.g., a plurality of elements.

[0119] The term "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to."

[0120] The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless context clearly indicates otherwise.

[0121] The term "about" is used herein to mean within a typical tolerance in the art. For example, "about" can be understood as about 2 standard deviations from the mean. In certain embodiments, about means ±10%. In certain embodiments, about means ±5%. When about precedes a series of numbers or ranges, it is understood that "about" can modify each of the consecutive numbers or ranges.

[0122] The terms "at least," "greater than," or "or more" preceding a number or series of numbers are understood to include the number adjacent to the term "at least," and all subsequent numbers or integers that may logically be included, if this is clear from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 18 nucleotides of a 21-nucleotide nucleic acid molecule" means that 18, 19, 20, or 21 nucleotides have the specified property. When at least precedes a series of numbers or ranges, it is understood that "at least" can modify each of the series of numbers or ranges.

[0123] As used herein, "less than" or "or less than" refers to the value adjacent to the phrase and, if logical from the context, to zero, any logically smaller value or integer than that value.For example, a duplex with an overhang of "2 nucleotides or less" has 2, 1, or 0 nucleotides overhang.When "less than" is used before a series of numbers or ranges, it is understood that "less than" can modify each of the series of numbers or ranges.As used herein, a range includes both upper and lower limits.

[0124] As used herein, a method of detection can include determining that the amount of analyte present is below the detection level of the method.

[0125] In the event of a conflict between a given target site and the nucleotide sequence for either the sense or antisense strand, the given sequence controls.

[0126] In the event of a discrepancy between a sequence on a transcript or other sequence and its indicated site, the nucleotide sequence listed herein takes precedence.

[0127] As used herein, the term "sodium-glucose cotransporter 2" ("SGLT2") refers to a well-known gene and polypeptide in the art, also known as solute carrier family 5 member 2, SLC5A2, and low-affinity sodium-glucose cotransporter. SGLT2 is a member of the sodium-glucose cotransporter family, which is a sodium-dependent glucose transport protein. SGLT2 is the primary cotransporter involved in glucose reabsorption in the kidney.

[0128] The term "SGLT2" includes human SGLT2, the amino acid and nucleotide sequence of which can be found, for example, at GenBank Accession No. NM_003041.4 (GI:1519244485, SEQ ID NO:1); mouse SGLT2, the amino acid and nucleotide sequence of which can be found, for example, at GenBank Accession No. NM_133254.5 (GI:1685839526, SEQ ID NO:2); and rat SGLT2, the amino acid and nucleotide sequence of which can be found, for example, at GenBank Accession No. NM_022590.2 (GI:78486543, SEQ ID NO:3).

[0129] The term "SGLT2" also includes Macaca mulatta SGLT2, the amino acid and nucleotide sequences of which can be found, for example, in GenBank Accession No. XM_015126028.2 (GI:1622915477, SEQ ID NO:4), and Macaca fascicularis SGLT2, the amino acid and nucleotide sequences of which can be found, for example, in GenBank Accession No. XM_005591767.2 (GI:982318456, SEQ ID NO:5).

[0130] Further examples of SGLT2 mRNA sequences are readily available using, for example, GenBank, UniProt, OMIM, and the Macaca Genome Project websites.

[0131] Exemplary SGLT2 nucleotide sequences can also be found in SEQ ID NOs: 1 to 10. SEQ ID NOs: 6 to 10 are the reverse complementary sequences of SEQ ID NOs: 1 to 5, respectively.

[0132] Further information on SGLT2 is provided in the NCBI gene database, e.g., https: / / www.ncbi.nlm.nih.gov / gene / 1803.

[0133] The entire contents of each of the foregoing GenBank Accession Numbers and Gene Database Numbers are incorporated herein by reference as of the filing date of this application.

[0134] As used herein, the terms "sodium-glucose cotransporter 2" and "SGLT2" also refer to naturally occurring DNA sequence variations of the SGLT2 gene. Numerous sequence variations within the SGLT2 gene have been identified and can be found, for example, in NCBI dbSNP and UniProt (see, for example, https: / / www.ncbi.nlm.nih.gov / snp / ?term=SGLT2), the entire contents of which are incorporated herein by reference as of the filing date of this application.

[0135] As used herein, "target sequence" refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of SGLT2 gene, such as the mRNA that is the product of RNA processing of a primary transcript.In one embodiment, the target portion of the sequence is at least long enough to serve as a substrate for RNAi-dependent cleavage at or near a portion of the nucleotide sequence of an mRNA molecule formed during the transcription of SGLT2 gene.In one embodiment, the target sequence is located in the protein coding region of SGLT2 gene.In another embodiment, the target sequence is located in the 3'UTR of SGLT2 gene.

[0136] The target sequence can be about 9 to 36 nucleotides in length, e.g., about 15 to 30 nucleotides in length. For example, the target sequence can be about 15 to 30 nucleotides in length, 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 The target sequence may be 9-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. In some embodiments, the target sequence is about 19 to about 30 nucleotides in length. In other embodiments, the target sequence is about 19 to about 25 nucleotides in length. In still other embodiments, the target sequence is about 19 to about 23 nucleotides in length. In some embodiments, the target sequence is about 21 to about 23 nucleotides in length. Ranges and lengths between the ranges and lengths listed above are also intended to be part of the present invention.

[0137] As used herein, the term "strand comprising a sequence" refers to an oligonucleotide comprising a chain of nucleotides described by a sequence referenced using standard nucleotide nomenclature.

[0138] Generally, "G", "C", "A", "T" and "U" respectively represent the nucleotide that contains guanine, cytosine, adenine, thymidine and uracil as a base. However, it is understood that the term "ribonucleotide" or "nucleotide" can also refer to modified nucleotides, as described in more detail below, or alternative replacement moieties (see, for example, Table 1). Those skilled in the art are well aware that guanine, cytosine, adenine and uracil can be substituted with other moieties without substantially changing the base pairing properties of the oligonucleotide that contains the nucleotide with such replacement moiety. When a cDNA sequence is provided, it is understood that the corresponding mRNA or RNAi agent contains U instead of T. For example, but not limited to, a nucleotide that contains inosine as its base can base pair with a nucleotide that contains adenine, cytosine or uracil. Therefore, a nucleotide that contains uracil, guanine or adenine can be substituted with a nucleotide that contains inosine, for example, in the nucleotide sequence of the dsRNA of the present invention. In another example, any adenine and cytosine in oligonucleotide can be replaced with guanine and uracil, respectively, to form GU wobble base pair with target mRNA.The sequence containing such replacement part is suitable for the compositions and methods of the present invention.Furthermore, those skilled in the art will understand that T is target gene sequence or its reverse complement, and will often be replaced with U in the RNAi agent of the present invention.

[0139] The terms "iRNA", "RNAi agent", "iRNA agent", "RNA interference agent", used interchangeably herein, refer to an agent that contains RNA as defined herein and mediates the targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway.RNA interference (RNAi) is the process that controls the sequence-specific degradation of mRNA.RNAi regulates, for example, inhibits, the expression of SGLT2 gene in cells, for example, in cells of a subject, such as a mammalian subject.

[0140] In one embodiment, the RNAi agent of the present disclosure comprises a single-stranded RNAi that interacts with a target RNA sequence, e.g., an SGLT2 mRNA sequence, to mediate cleavage of the target RNA. Without wishing to be bound by theory, it is believed that long double-stranded RNAs introduced into cells are degraded into double-stranded small interfering RNAs (siRNAs) comprising a sense strand and an antisense strand by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, an RNase III-like enzyme, processes these dsRNAs into 19-23 base pair small interfering RNAs with characteristic two-base 3' overhangs (Bernstein, et al., (2001) Nature 409:363). These siRNAs are then incorporated into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, thereby allowing complementary antisense strands 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 and induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188). That is, in one aspect, the present disclosure relates to a single-stranded RNA (ssRNA) (the antisense strand of the siRNA duplex) that is produced in cells and promotes the formation of a RISC complex to silence target genes. Therefore, the term "siRNA" is used herein to also refer to the RNAi described above.

[0141] In another embodiment, the RNAi agent can be a single-stranded RNA introduced into a cell or organism to inhibit target mRNA. The single-stranded RNAi agent binds 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 RNAs are described in U.S. Patent No. 8,101,348 and Lima et al., (2012) Cell 150:883-894, the entire contents of each of which are incorporated herein by reference. Any of the antisense nucleotide sequences described herein can be used as the single-stranded siRNA described herein or as the single-stranded siRNA that is chemically modified by the method described in Lima et al., (2012) Cell 150:883-894.

[0142] In another embodiment, the "RNAi agent" used in the compositions and methods of the present disclosure is double-stranded RNA, and is also referred to herein as "double-stranded RNAi agent," "double-stranded RNA (dsRNA) molecule," "dsRNA agent," or "dsRNA." The term "dsRNA" refers to a complex of ribonucleic acid molecules with a double-stranded structure, comprising two antiparallel, substantially complementary nucleic acid strands, which are referred to as having "sense" or "antisense" orientation with respect to the target RNA, i.e., the mRNA gene of SGLT2. In some embodiments of the present disclosure, double-stranded RNA (dsRNA) induces the degradation of target RNA, for example, mRNA, through a post-transcriptional gene silencing mechanism, herein referred to as RNA interference or RNAi.

[0143] Generally, dsRNA molecule can comprise ribonucleotide, but as described in detail herein, each strand or both strands can also comprise one or more non-ribonucleotides, such as deoxyribonucleotide, modified nucleotide.In addition, as used herein, " RNAi agent " can comprise the ribonucleotide with chemical modification, and RNAi agent can comprise the substantial modification in multiple nucleotides.

[0144] As used herein, the term "modified nucleotide" refers to the nucleotide that has independently modified sugar moiety, modified internucleotide linkage, or modified nucleobase.Therefore, the term modified nucleotide encompasses the substitution, addition, or removal of, for example, functional group or atom, etc., to internucleoside linkage, sugar moiety, or nucleobase.The modification suitable for use in the agent of the present disclosure includes all types of modification disclosed herein or known in the art.Any such modification used in siRNA type molecules is encompassed by "RNAi agent" for the purpose of this specification and claims.

[0145] In certain embodiments of the present disclosure, the inclusion of deoxy-nucleotides, which are recognized as naturally occurring forms of nucleotides, can be considered to constitute modified nucleotides when present in an RNAi agent.

[0146] The double-stranded region can be any length that allows for specific degradation of the desired target RNA via the RISC pathway, and can be about 9 to 36 base pairs in length, e.g., about 15 to 30 base pairs in length, e.g., 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, 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, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, The length may range from 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. Ranges and lengths between the ranges and lengths listed above are also intended to be part of the invention.

[0147] The two strands forming the double-stranded structure can be different portions of a single larger RNA molecule, or they can be separate RNA molecules. When the two strands are part of a single larger molecule and are therefore connected by an uninterrupted chain of nucleotides between the 3' end of one strand forming the double-stranded structure and the corresponding 5' end of the other strand, the connected RNA strands are called "hairpin loops." A hairpin loop can contain at least one unpaired nucleotide. In some embodiments, a hairpin loop can contain at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23, or more unpaired nucleotides or nucleotides not directed toward the target site of the dsRNA. In some embodiments, a hairpin loop can be 10 or fewer nucleotides. In some embodiments, a hairpin loop can be 8 or fewer unpaired nucleotides. In some embodiments, a hairpin loop can be 4 to 10 unpaired nucleotides. In some embodiments, a hairpin loop can be 4 to 8 nucleotides.

[0148] In certain embodiments, the two strands of a double-stranded oligomeric compound may be linked to each other. The two strands may be linked to each other at both ends or only one end. Linked at one end means that the 5' end of the first strand is linked to the 3' end of the second strand, or the 3' end of the first strand is linked to the 5' end of the second strand. When the two strands are linked to each other at both ends, the 5' end of the first strand is linked to the 3' end of the second strand, and the 3' end of the first strand is linked to the 5' end of the second strand. The two strands may be linked together by an oligonucleotide linker, including, but not limited to, (N)n, where N is independently a modified or unmodified nucleotide, and n is 3 to 23. In some embodiments, n is 3 to 10, e.g., 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the oligonucleotide linker is selected from the group consisting of GNRA, (G)4, (U)4, and (dT)4, where N is a modified or unmodified nucleotide, and R is a modified or unmodified purine nucleotide. Some of the nucleotides in the linker can participate in base pairing interactions with other nucleotides in the linker. The two strands can also be linked together by a non-nucleoside linker, for example, by a linker described herein. Those skilled in the art will understand that any chemical modification or variation of the oligonucleotides described herein can be used in the oligonucleotide linker.

[0149] Hairpin and dumbbell oligomeric compounds will have a double-stranded region of at least or equal to 14, 15, 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The double-stranded region can be up to 200, 100, or 50 nucleotide pairs in length. In some embodiments, the ranges for the double-stranded region are 15-30, 17-23, 19-23, and 19-21 nucleotide pairs in length.

[0150] Hairpin oligomeric compounds may have a single-stranded overhang or terminal unpaired region, in some embodiments at the 3' end, and in some embodiments on the antisense side of the hairpin. In some embodiments, the overhang is 1 to 4 nucleotides in length, more generally 2 to 3 nucleotides in length. Hairpin oligomeric compounds capable of inducing RNA interference are also referred to herein as "shRNAs."

[0151] When the two substantially complementary strands of dsRNA are composed of separate RNA molecules, these molecules can, but do not necessarily, be covalently linked.When the two strands are covalently linked by a means other than an uninterrupted chain of nucleotides between the 3'-end of one strand and the 5'-end of the corresponding other strand that form a double-stranded structure, this connecting 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 in the shortest strand of dsRNA minus any overhangs present in the double strand.In addition to the double-stranded structure, RNAi can also contain one or more nucleotide overhangs.

[0152] In one embodiment, the RNAi agent of the present invention is a dsRNA, each strand of which is 24-30 nucleotides in length and interacts with a target RNA sequence, e.g., the mRNA sequence of SGLT2, to induce cleavage of the target RNA. Without wishing to be bound by theory, 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, an RNase III-like enzyme, processes the dsRNA into 19-23 base pair small interfering RNAs with characteristic two-base 3' overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNA is then incorporated into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, thereby allowing the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188).

[0153] In one embodiment, the RNAi agents of the present invention are dsRNA agents, each strand of which contains 19-23 nucleotides that interact with the SGLT2 mRNA sequence to induce cleavage of the target RNA. Without wishing to be bound by theory, long double-stranded RNAs introduced into cells are degraded into siRNAs by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, an RNase III-like enzyme, processes the dsRNA into 19-23 base pair small interfering RNAs with characteristic two-base 3' overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNA is then incorporated into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, thereby allowing target recognition to be induced by the complementary antisense strand (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188). In one embodiment, the RNAi agent of the present invention is a 24-30 nucleotide dsRNA that interacts with the SGLT2 mRNA sequence to induce cleavage of the target RNA.

[0154] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that protrudes from the double-stranded structure of an RNAi agent, such as dsRNA.For example, a nucleotide overhang exists when the 3'-end of one strand of dsRNA extends beyond the 5'-end of the other strand, or vice versa.A dsRNA can comprise an overhang of at least one nucleotide, or the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more.A nucleotide overhang can comprise or consist of a nucleotide / nucleoside analog, such as deoxynucleotide / nucleoside.An overhang can be on the sense strand, on the antisense strand, or any combination thereof.Furthermore, a certain overhanging nucleotide can be present on the 5'-end, 3'-end, or both ends of either the antisense strand or the sense strand of dsRNA.

[0155] In one embodiment of dsRNA, at least one strand comprises a 3' overhang of at least 1 nucleotide.In another embodiment, at least one strand comprises a 3' overhang of at least 2 nucleotides, for example, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides.In other embodiments, at least one strand of RNAi agent comprises a 5' overhang of at least 1 nucleotide.In certain embodiments, at least one strand comprises a 5' overhang of at least 2 nucleotides, for example, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides.In still other embodiments, both the 3' end and the 5' end of one strand of RNAi agent comprise an overhang of at least 1 nucleotide.

[0156] In one embodiment, the antisense strand of the dsRNA has an overhang of 1 to 10 nucleotides at the 3' or 5' end, for example, 0 to 3, 1 to 3, 2 to 4, 2 to 5, 4 to 10, 5 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In one embodiment, the sense strand of the dsRNA has an overhang of 1 to 10 nucleotides at the 3' or 5' end, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In another embodiment, one or more of the nucleotides in the overhang are substituted with a nucleoside thiophosphate.

[0157] In certain embodiments, the overhang on the sense strand or the antisense strand, or both, can comprise an extended length greater than 10 nucleotides, e.g., a length of 1 to 30 nucleotides, 2 to 30 nucleotides, 10 to 30 nucleotides, or 10 to 15 nucleotides. In certain embodiments, the extended overhang is on the sense strand of the duplex. In certain embodiments, the extended overhang is on the 3'-end of the sense strand of the duplex. In certain embodiments, the extended overhang is on the 5'-end of the sense strand of the duplex. In certain embodiments, the extended overhang is on the antisense strand of the duplex. In certain embodiments, the extended overhang is on the 3'-end of the antisense strand of the duplex. In certain embodiments, the extended overhang is on the 5'-end of the antisense strand of the duplex. In certain embodiments, one or more of the nucleotides in the overhang are substituted with a nucleoside thiophosphate. In certain embodiments, the overhang comprises a self-complementary portion that enables the overhang to form a stable hairpin structure under physiological conditions.

[0158] The term "blunt" or "blunt-end" when used herein in relation to dsRNA means that there is no unpaired nucleotide or nucleotide analogue at the predetermined end of dsRNA, that is, there is no nucleotide overhang.One end or both ends of dsRNA can be blunt.When both ends of dsRNA are blunt, dsRNA is said to be blunt-ended.For clarity, "blunt-end" dsRNA is the dsRNA that has both ends blunt, that is, there is no nucleotide overhang at either end of the molecule.In most cases, this molecule will be double-stranded throughout its entire length.

[0159] 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., an SGLT2 mRNA sequence.

[0160] As used herein, term " complementary region " refers to the region on antisense strand that is substantially complementary to sequence, for example, target sequence, for example, SGLT2 nucleotide sequence, as defined herein.If complementary region is not completely complementary to target sequence, its mismatch can be in the inner region or terminal region of molecule.Generally, the most tolerable mismatch is in terminal region, for example, within 5, 4, 3 or 2 nucleotides of the 5'-end or 3'-end of RNAi agent.

[0161] In some embodiments, a double-stranded RNA agent of the invention comprises a nucleotide mismatch in the antisense strand. In some embodiments, the antisense strand of a double-stranded RNA agent of the invention comprises four or fewer mismatches with the target mRNA, e.g., the antisense strand comprises 4, 3, 2, 1, or 0 mismatches with the target mRNA. In some embodiments, the antisense strand of a double-stranded RNA agent of the invention comprises four or fewer mismatches with the sense strand, e.g., the antisense strand comprises 4, 3, 2, 1, or 0 mismatches with the sense strand. In some embodiments, a double-stranded RNA agent of the invention comprises a nucleotide mismatch in the sense strand. In some embodiments, the sense strand of a double-stranded RNA agent of the invention comprises four or fewer mismatches with the antisense strand, e.g., the sense strand comprises 4, 3, 2, 1, or 0 mismatches with the antisense strand. In some embodiments, the nucleotide mismatch is within, for example, 5, 4, or 3 nucleotides from the 3' end of the iRNA. In another embodiment, the nucleotide mismatch is within, for example, the 3' terminal nucleotide of the iRNA agent. In some embodiments, the mismatch is not in the seed region.

[0162] Therefore, the RNAi agents described herein may contain one or more mismatches to the target sequence. In one embodiment, the RNAi agents described herein contain three or fewer mismatches (i.e., three, two, one, or zero mismatches). In one embodiment, the RNAi agents described herein contain two or fewer mismatches. In one embodiment, the RNAi agents described herein contain one or fewer mismatches. In one embodiment, the RNAi agents described herein contain zero mismatches. In certain embodiments, when the antisense strand of an RNAi agent contains a mismatch to the target sequence, the mismatch can be optionally limited to be within the last five nucleotides from either the 5'-end or the 3'-end of the complementary region. For example, in such an embodiment, in the case of a 23-nucleotide RNAi agent, the strand complementary to a region of the SGLT2 gene generally does not contain any mismatches within the central 13 nucleotides. By using the methods described herein or known in the art, it can be determined whether an RNAi agent containing a mismatch to the target sequence is effective in inhibiting the expression of the SGLT2 gene. It is important to consider the efficacy of mismatched RNAi agents to inhibit SGLT2 expression, especially when specific regions of complementarity in the SGLT2 gene are known to be altered.

[0163] The term "sense strand" or "passenger strand," as used herein, refers to the strand of an RNAi agent that includes a region that is substantially complementary to a region of the antisense strand, as those terms are defined herein.

[0164] As used herein, "substantially all of the nucleotides are modified" means broadly but not entirely modified and may include no more than 5, 4, 3, 2, or 1 or unmodified nucleotides.

[0165] As used herein, the term "cleavage region" refers to the region located directly adjacent to the cleavage site. The cleavage site is the site on the target where cleavage occurs. In some embodiments, the cleavage region comprises three bases directly adjacent to either end of the cleavage site. In some embodiments, the cleavage region comprises two bases directly adjacent to either end 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 this cleavage region comprises nucleotides 11, 12, and 13.

[0166] As used herein, unless otherwise specified, the term "complementary," when used to describe a first nucleotide sequence in the context of a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising a first nucleotide sequence to hybridize to form a double-stranded structure with an oligonucleotide or polynucleotide comprising the second nucleotide sequence under specified conditions, as would be understood by one of ordinary skill in the art. Such conditions may be, for example, "stringent conditions," which 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 be encountered in an organism, may be applied. Those skilled in the art can determine the most appropriate set of conditions for testing the complementarity of two sequences depending on the ultimate use of the hybridized nucleotides.

[0167] The complementary sequence in an RNAi agent, for example, in the dsRNA described herein, comprises the base pairing of the oligonucleotide or polynucleotide comprising the first nucleotide sequence with the oligonucleotide or polynucleotide comprising the second nucleotide sequence throughout the entire length of one or both nucleotide sequences.Such sequences can be referred to herein as "fully complementary" to each other.However, when the first sequence is referred to herein as "substantially complementary" to the second sequence, these two sequences can be completely complementary, or they can form one or more, but generally not more than 5, 4, 3, or 2 mismatched base pairs when hybridizing a double strand of up to 30 base pairs, while maintaining the ability to hybridize under the conditions most relevant to its final use, for example, in vitro or in vivo, inhibiting gene expression.However, if two oligonucleotides are designed to form one or more single-stranded overhangs when hybridizing, these overhangs shall not be considered as mismatches when determining complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, where the longer oligonucleotide comprises a 21-nucleotide sequence that is perfectly complementary to the shorter oligonucleotide, may still be referred to as "fully complementary" for purposes described herein.

[0168] "Complementary" sequences, as used herein, may also include or be formed entirely of non-Watson-Crick base pairs or base pairs formed from non-naturally occurring modified nucleotides, such as, but not limited to, G:U wobble base pairs or Hoogsteen base pairs, so long as they satisfy the above requirements regarding their ability to hybridize.

[0169] The terms "complementary," "fully complementary," and "substantially complementary," as used herein, may be used in reference to base matching between two oligonucleotides or polynucleotides, such as between the sense and antisense strands of a dsRNA, or the antisense strand of an RNAi agent and a target sequence, as will be understood from the context of their use.

[0170] As used herein, a polynucleotide that is "substantially complementary to at least a portion of" a messenger RNA (mRNA) or target sequence refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA or target sequence of interest (e.g., an mRNA encoding SGLT2). For example, a polynucleotide is complementary to at least a portion of an SGLT2 mRNA if the sequence is substantially complementary to an uninterrupted portion of the mRNA encoding SGLT2.

[0171] Thus, in some embodiments, the antisense strand polynucleotides disclosed herein are perfectly complementary to the target SGLT2 sequence.

[0172] In other embodiments, the antisense strand polynucleotides disclosed herein are substantially complementary to the target SGLT2 sequence and comprise a contiguous nucleotide sequence that is at least about 80% complementary, e.g., about 85%, 86%, 87%, 88%, 89%, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or about 99% complementary to the equivalent region of the nucleotide sequence of SEQ ID NOs: 1-5 for SGLT2, or to a fragment of SEQ ID NOs: 1-5, over its entire length.

[0173] In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to a target SGLT2 sequence and comprise a contiguous nucleotide sequence 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 about 99% complementary over its entire length to any one of the sense strand nucleotide sequences in any one of Tables 2-3 or a fragment of any one of the sense strand nucleotide sequences in any one of Tables 2-3.

[0174] In one embodiment, an RNAi agent of the present disclosure comprises a sense strand that is substantially complementary to an antisense polynucleotide that is also identical to a target SGLT2 sequence, and the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least about 80% complementary, e.g., 86%, 87%, 88%, 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about or 99% complementary to the nucleotide sequence of SEQ ID NOs: 6-10 or an equivalent region of a fragment of any one of SEQ ID NOs: 6-10, throughout its entire length.

[0175] In some embodiments, the iRNA of the invention comprises a sense strand that is substantially complementary to an antisense polynucleotide that is also complementary to a target SGLT2 sequence, and the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least about 80% complementary, e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% complementary, over its entire length, to any one of the antisense strand nucleotide sequences in any one of Tables 2-3, or a fragment of any one of the antisense strand nucleotide sequences in any one of Tables 2-3.

[0176] In some embodiments, the double-stranded region of a double-stranded iRNA agent is equal to or at least equal to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotide pairs in length.

[0177] In some embodiments, the antisense strand of the double-stranded iRNA agent is equal to or at least equal to 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0178] In some embodiments, the sense strand of a double-stranded iRNA agent is equal to or at least equal to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0179] In one embodiment, the sense and antisense strands of the double-stranded iRNA agent are each independently 15 to 30 nucleotides in length.

[0180] In one embodiment, the sense and antisense strands of the double-stranded iRNA agent are each independently 19 to 25 nucleotides in length.

[0181] In one embodiment, the sense and antisense strands of the double-stranded iRNA agent are each independently 21 to 23 nucleotides in length.

[0182] In one embodiment, the sense strand of the iRNA agent is 21 nucleotides in length and the antisense strand is 23 nucleotides in length, the strands forming a double-stranded region of 21 contiguous base pairs with a 2-nucleotide long single-stranded overhang at the 3' end.

[0183] In one embodiment of the present invention, an agent for use in the methods and compositions of the present invention is a single-stranded antisense nucleic acid molecule that inhibits a target mRNA via an antisense inhibition mechanism. The single-stranded antisense RNA molecule is complementary to a sequence within the target mRNA. Single-stranded antisense oligonucleotides can inhibit translation stoichiometrically by base pairing with the mRNA and physically interfering with the translation machinery. See Dias, N. et al., (2002) Mol Cancer Ther 1:347-355. The single-stranded antisense RNA molecule may be about 15 to about 30 nucleotides in length and may have a sequence complementary to the target sequence. For example, the single-stranded antisense RNA molecule may comprise a sequence that is at least about 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from any one of the antisense sequences described herein.

[0184] In one embodiment, at least partial suppression of SGLT2 gene expression is assessed by a decrease in the amount of SGLT2 mRNA that can be isolated from or detected in a first cell or group of cells in which the SGLT2 gene is transcribed and that have been treated to inhibit expression of the SGLT2 gene, compared to a second cell or group of cells (control cells) that are substantially identical to the first cell or group of cells but have not been treated like the first cell or group of cells. The degree of inhibition can be expressed as follows:

number

[0185] In one embodiment, inhibition of expression is determined by the dual luciferase method in which the RNAi agent is present at 10 nM.

[0186] As used herein, the phrase " contacting cell with RNAi agent " such as dsRNA includes contacting cell by any possible means.Contacting cell with RNAi agent includes contacting cell with RNAi agent in vitro or contacting cell with RNAi agent in vivo.Contacting can be carried out directly or indirectly.Therefore, for example, RNAi agent can be physically contacted with cell by carrying out method separately, or RNAi agent can be placed in the situation that can allow or cause it to contact with cell afterwards.

[0187] Contacting cells in vitro can be carried out, for example, by incubating cells with RNAi agent.Contacting cells in vivo can be carried out, for example, by injecting RNAi agent into or near the tissue where the cells are present, or by injecting RNAi agent into another region, or into the bloodstream or subcutaneous cavity, so that the agent then reaches the tissue where the cells to be contacted are present.For example, RNAi agent can comprise or be coupled to a ligand that directs or otherwise stabilizes the RNAi agent to a target site, for example, the liver or kidney, for example, a lipophilic moiety as described below and further described in, for example, PCT Publication No. WO2019 / 217459, the entire contents of which are incorporated herein by reference.In some embodiments, RNAi agent can contain or be coupled to a ligand that directs or otherwise stabilizes the RNAi agent to a target site, for example, the liver, for example, one or more GalNAc derivatives as described below. In other embodiments, RNAi agent can contain or be bound to lipophilic moiety or one or more GalNAc derivatives.It is also possible to combine in vitro and in vivo contact methods.For example, cell can be contacted with RNAi agent in vitro, and then transferred to subject.

[0188] In one embodiment, contacting a cell with an RNAi agent includes "introducing" or "delivering an RNAi agent into a cell" by promoting or performing uptake or absorption into the cell. The absorption or uptake of an RNAi agent can occur by spontaneous diffusive or active cellular processes, or by auxiliary agents or devices. The introduction of an RNAi agent into a cell can be in vitro or in vivo. For example, in the case of in vivo introduction, the RNAi agent can be injected into a tissue site or administered systemically. In vitro introduction into a cell can include methods known in the art, such as electroporation and lipofection. Further approaches are described herein below or known in the art.

[0189] The terms "lipid-soluble" or "lipophilic moiety" refer broadly to any compound or chemical moiety that has an affinity for lipids. One way to characterize the lipophilicity of a lipophilic moiety is by the octanol-water partition coefficient, log K ow In this case, K ow is the ratio of the concentration of a chemical in the octanol phase to the concentration of the chemical in the aqueous phase of a two-phase system at equilibrium. The octanol-water partition coefficient is a laboratory-measured property of a substance. However, it can also be predicted by using coefficients attributable to the structural components of a chemical calculated using first principles or empirical methods (see, e.g., Tetko et al., J. Chem. Inf. Comput. Sci. 41:1407-21 (2001), incorporated herein by reference in its entirety). It provides a thermodynamic measure of a substance's tendency to prefer a non-aqueous or oily environment rather than water (i.e., its hydrophilic / lipophilic balance). In principle, a chemical can be expressed as its logK ow is greater than 0, the molecule is lipophilic in nature. Typically, a lipophilic moiety has a logK ow For example, the log K of 6-aminohexanol owFor example, the log K of cholesteryl N-(hexan-6-ol)carbamate is predicted to be approximately 0.7. ow is predicted to be 10.7.

[0190] The lipophilicity of a molecule can be changed depending on the functional groups it contains. For example, adding a hydroxyl or amine group to the end of the lipophilic moiety can increase the partition coefficient (e.g., logK ow ) may increase or decrease in value.

[0191] Alternatively, the hydrophobicity of the double-stranded RNAi agent that is conjugated with one or more lipophilic moieties can be measured by its protein binding property.For example, in certain embodiments, if the unbound fraction of the plasma protein binding assay of double-stranded RNAi agent is determined to be positively correlated with the relative hydrophobicity of double-stranded RNAi agent, then it will be positively correlated with the silencing activity of double-stranded RNAi agent.

[0192] In one embodiment, the plasma protein binding assay that is determined is electrophoretic mobility shift assay (EMSA) that uses human serum albumin protein.The exemplary protocol of this binding assay is described in detail, for example, in PCT Publication No. WO2019 / 217459.The hydrophobicity of double-stranded RNAi agent measured by the unbound fraction of siRNA in binding assay is greater than 0.15, greater than 0.2, greater than 0.25, greater than 0.3, greater than 0.35, greater than 0.4, greater than 0.45 or greater than 0.5 in the case of enhanced siRNA in vivo delivery.

[0193] Thus, conjugating a lipophilic moiety to an internal position of a double-stranded RNAi agent provides optimal hydrophobicity for enhanced in vivo delivery in siRNA.

[0194] The term "lipid nanoparticle" or "LNP" refers to a vesicle comprising a lipid layer that encapsulates a pharmaceutically active molecule, such as a nucleic acid molecule, e.g., an RNAi agent or a plasmid from which an RNAi agent 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.

[0195] As used herein, a "subject" refers to an animal such as a mammal, including a primate (such as a human, a non-human primate such as a monkey, or a chimpanzee), or a non-primate (such as a cow, pig, horse, goat, rabbit, sheep, hamster, guinea pig, cat, dog, rat, or mouse), or a bird that expresses a target gene endogenously or heterologously. In one embodiment, the subject is a human, such as a human being treated or evaluated for a disease, disorder, or condition that would benefit from reduced SGLT2 expression, a human being at risk for a disease, disorder, or condition that would benefit from reduced SGLT2 expression, a human being with a disease, disorder, or condition that would benefit from reduced SGLT2 expression, or a human being treated for a disease, disorder, or condition that would benefit from reduced SGLT2 expression as described herein. In some embodiments, the subject is a female human. In other embodiments, the subject is a male human. In one embodiment, the subject is an adult subject. In another embodiment, the subject is a pediatric subject.

[0196] As used herein, the term "treating" or "treatment" refers to a beneficial, desired result, including, but not limited to, reducing or ameliorating one or more signs or symptoms associated with SGLT2 expression or SGLT2 protein production, such as an SGLT2-related disease, e.g., diabetes, such as gout or type II diabetes, or a condition associated with unwanted SGLT2 expression; reducing the degree of unwanted SGLT2 activation or stabilization; improving or alleviating unwanted SGLT2 activation or stabilization. "Treatment" can also mean prolonging survival compared to expected survival if no treatment is administered.

[0197] The term "reducing" in the context of SGLT2 levels or disease markers or symptoms in a subject refers to a statistically significant decrease in such levels. The decrease can be, for example, at least 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more. In certain embodiments, the decrease is at least 20%. In certain embodiments, the decrease is at least 50% in disease markers, such as protein levels or gene expression levels. "Reducing" in the context of SGLT2 levels in a subject preferably refers to a decrease to a level that is accepted as within the normal range in individuals without the disorder. In certain embodiments, the expression of a target is normalized, i.e., decreased toward or to a level that is accepted as within the normal range for individuals without such a disorder, such as blood glucose levels, blood uric acid levels, blood lipid levels, blood oxygen levels, white blood cell count, kidney function, spleen function, and liver function. For example, chronic hyperuricemia is defined as a serum uric acid level above 6.8 mg / dL (greater than 360 mmol / L), a level that exceeds the physiological saturation threshold (Mandell, Cleve. Clin. Med. 75:S5-S8, 2008). As used herein, "reducing" in a subject can refer to reducing gene expression or protein production in the subject's cells, but does not require reducing expression in all cells or tissues of the subject. For example, as used herein, reducing in a subject can include reducing gene expression or protein production in the subject.

[0198] The term "reduce" can also be used in reference to normalizing a disease symptom or pathology, i.e., reducing the difference between the level in a subject suffering from an SGLT2-related disease and the level in a normal subject not suffering from an SGLT2-related disease toward or to the level of a normal subject not suffering from an SGLT2-related disease. As used herein, if the disease involves an elevated value for a symptom, "normal" refers to the upper limit of normal. If the disease involves a decreased value for a symptom, "normal" refers to the lower limit of normal.

[0199] As used herein, "prevention" or "preventing", when used in reference to a disease, disorder, or condition that would benefit from a reduction in SGLT2 gene expression or SGLT2 protein production, refers to a reduction in the likelihood that a subject will develop such a disease, disorder, or condition, for example, a symptom of an SGLT2-related disease, for example, a symptom associated with diabetes, such as gout or type II diabetes.Not developing a disease, disorder, or condition, or reducing the onset of symptoms associated with such a disease, disorder, or condition (for example, by at least about 10% of a clinically acceptable magnitude for the disease or disorder), or delaying the onset of delayed symptoms (for example, by several days, weeks, months, or years), or reducing or maintaining serum uric acid levels at or below 6.8 mg / dl in subjects prone to elevated serum uric acid levels, is considered effective prevention.

[0200] As used herein, the term "SGLT2-associated disease" refers to a disease or disorder that would benefit from reduced SGLT2 expression or activity. The term "SGLT2-associated disease" refers to a disease or disorder caused by or associated with SGLT2 expression or SGLT2 protein production. The term "SGLT2-associated disease" includes diseases, disorders, or conditions that would benefit from reduced SGLT2 expression or SGLT2 protein activity. For example, as described above and in the Examples of the present application, SGLT2 inhibitors used to treat type 2 diabetes or hyperglycemia reduce serum glucose levels and lower serum uric acid levels, and predicted loss-of-function and predicted missense variants of SGLT2 are significantly associated with reduced urate levels and possibly reduced gout diagnoses (see Example 3). Thus, an "SGLT2-associated disease" refers to a disease associated with elevated serum glucose and / or serum uric acid levels. Non-limiting examples of SGLT2-associated diseases include, for example, gout, diabetes (type I or type II diabetes), or other metabolic diseases.

[0201] As used herein, "gout" refers to a type of arthritis that causes severe pain, swelling, and stiffness in joints, most frequently the thumb. Gout occurs when uric acid or uric acid crystals accumulate in joints or surrounding tissues, causing inflammation and the severe pain of gout attacks. Uric acid crystals can form when there are high levels of uric acid in the blood. Gout can be diagnosed by a joint fluid test, in which fluid is extracted from the affected joint with a needle to check for the presence of any uric acid crystals, or by a blood test to measure the level of uric acid in the blood. Ultrasound scans can also be performed to detect uric acid crystals around the joint. Currently available treatments for gout are contraindicated or ineffective in many subjects. Allopurinol, a common first-line treatment for lowering uric acid levels in subjects with gout, is contraindicated in many populations, especially those with impaired renal function. Furthermore, many subjects fail treatment with allopurinol, for example, suffering from gout flares despite treatment or from allopurinol-related rashes or hypersensitivity reactions.

[0202] As used herein, "metabolic disease" refers to any disease or disorder that disrupts normal metabolism, the process that converts food into energy at the cellular level. Metabolic diseases affect the ability of cells to carry out important biochemical reactions that involve the processing or transport of proteins (amino acids), carbohydrates (sugars and starches), or lipids (fatty acids). Non-limiting examples of metabolic diseases include carbohydrate disorders, e.g., diabetes, type I diabetes, type II diabetes, galactosemia, hereditary fructose intolerance, fructose 1,6-diphosphatase deficiency, glycogen storage disorders, congenital glycosylation disorders, insulin resistance, insulin deficiency, hyperinsulinemia, impaired glucose tolerance (IGT), abnormal glycogen metabolism; disorders of amino acid metabolism, e.g., maple syrup urine disease (MSUD), or homocystinuria; disorders of organic acid metabolism, e.g., methylmalonic aciduria, 3-methylglutaconic aciduria-Barth syndrome, glutaric aciduria, or 2-hydroxyglutaric aciduria-D and L forms; disorders of fatty acid beta-oxidation, e.g., medium-chain acyl-CoA dehydrogenase deficiency (MCAD), long-chain 3-hydroxyaciduria, and disorders of fatty acid beta-oxidation, e.g., medium-chain acyl-CoA dehydrogenase deficiency (MCAD), long-chain 3-hydroxyaciduria. These disorders include acetyl-CoA dehydrogenase deficiency (LCHAD), very long-chain acyl-CoA dehydrogenase deficiency (VLCAD), disorders of lipid metabolism such as GM1 gangliosidosis, Tay-Sachs disease, Sandhoff disease, Fabry disease, Gaucher disease, Niemann-Pick disease, Krabbe disease, mucolipidosis, or mucopolysaccharidoses; mitochondrial disorders such as mitochondrial cardiomyopathy; Leigh disease; mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS); myoclonic epilepsy with ragged fibers (MERRF); neuropathy, ataxia, and retinitis pigmentosa (NARP); Barth syndrome; or peroxisomal disorders such as Zellweger syndrome (cerebrohepatorenal syndrome), X-linked adrenoleukodystrophy, or Refsum disease.

[0203] As used herein, the term "diabetes" refers to a group of metabolic diseases characterized by high blood sugar (glucose) levels resulting from defects in insulin secretion or action, or both. There are two most common types of diabetes: type 1 diabetes and type 2 diabetes, both of which result from the body's inability to regulate insulin. Insulin is a hormone released by the pancreas in response to increased blood sugar (glucose) levels in the blood.

[0204] As used herein, the term "type 1 diabetes" refers to a chronic disease that occurs when the pancreas produces too little insulin to properly regulate blood glucose levels. Type 1 diabetes is also known as insulin-dependent diabetes mellitus, IDDM, and juvenile-onset diabetes. People with type 1 diabetes (insulin-dependent diabetes) produce little or no insulin. Approximately 6 percent of the U.S. population has some form of diabetes, but only about 10 percent of all diabetic patients have type 1 disease. Most people with type 1 diabetes develop the disease before the age of 30. Type 1 diabetes represents the result of progressive autoimmune destruction of pancreatic beta cells with subsequent insulin deficiency. More than 90% of the pancreas' insulin-producing cells (beta cells) are permanently destroyed. The resulting insulin deficiency is severe, and to survive, type 1 diabetic patients must receive regular insulin injections.

[0205] In type II diabetes (also called non-insulin-dependent diabetes mellitus, or NDDM), the pancreas continues to produce insulin, sometimes at higher-than-normal levels. However, the body develops resistance to its effects, resulting in relative insulin deficiency. Type II diabetes can develop in children and adolescents but usually begins after age 30 and becomes progressively more common with age; approximately 15% of people over 70 have type II diabetes. Obesity is a risk factor for type II diabetes; 80–90% of people with this disorder are obese.

[0206] In some embodiments, diabetes includes prediabetes. Prediabetes refers to one or more early diabetic conditions, including impaired glucose utilization, abnormal or impaired fasting glucose levels, impaired glucose tolerance, impaired insulin sensitivity, and insulin resistance. Prediabetes is a major risk factor for the development of type 2 diabetes mellitus, cardiovascular disease, and death. Much attention has been focused on developing therapeutic interventions that effectively treat prediabetes to prevent the onset of type 2 diabetes.

[0207] Diabetes can be diagnosed by performing a glucose tolerance test. Clinically, diabetes is often divided into several basic categories. Primary examples of these categories include autoimmune diabetes mellitus, non-insulin-dependent diabetes mellitus (NDDM type 1), insulin-dependent diabetes mellitus (IDDM type 2), non-autoimmune diabetes mellitus, non-insulin-dependent diabetes mellitus (NIDDM type 2), and maturity-onset diabetes of the young (MODY). A further category, often referred to as secondary, refers to diabetes resulting from some identifiable condition that causes or enables the development of a diabetic syndrome. Examples of secondary categories include diabetes caused by pancreatic disease, diabetes induced by hormonal abnormalities, drugs, or chemicals, diabetes caused by insulin receptor abnormalities, diabetes associated with genetic syndromes, and diabetes of other causes (see, e.g., Harrison's (1996) 14th ed., New York, McGraw-Hill).

[0208] As used herein, "disorder of lipid metabolism" or "disturbance of lipid metabolism" refers to any disorder related to or caused by disturbance of lipid metabolism. This term also includes any disorder, disease, or condition that can lead to hyperlipidemia, or a condition characterized by abnormally elevated levels of any or all of lipids and / or lipoproteins in the blood. This term refers to induced or acquired disorders, such as genetic disorders such as familial hypertriglyceridemia, familial partial lipodystrophy type 1 (FPLD1), or disorders induced or acquired as a result of a disease, disorder, or condition (e.g., renal failure), diet, or certain drugs (e.g., as a result of highly active antiretroviral therapy (HAART) used to treat AIDS or HIV).

[0209] Additional examples of disorders of lipid metabolism include, but are not limited to, atherosclerosis, dyslipidemia, hypertriglyceridemia (drug-induced hypertriglyceridemia, diuretic-induced hypertriglyceridemia, alcoholic hypertriglyceridemia, beta-adrenergic blocker-induced hypertriglyceridemia, estrogen-induced hypertriglyceridemia, glucocorticoid-induced hypertriglyceridemia, retinoid-induced hypertriglyceridemia, cimetidine-induced hypertriglyceridemia, and familial hypertriglyceridemia. These include lysitolemia, acute pancreatitis with hypertriglyceridemia, chylomicron syndrome, familial chylomicronemia, Apo-E deficiency or resistance, LPL deficiency or hypoactivity, hyperlipidemia (including familial combined hyperlipidemia), hypercholesterolemia, gout associated with hypercholesterolemia, xanthomatosis (subcutaneous cholesterol deposits), hyperlipidemia with heterogeneous LPL deficiency, and hyperlipidemia with hyperlipidemia and heterogeneous LPL deficiency, fatty liver disease, or nonalcoholic steatohepatitis (NASH).

[0210] Cardiovascular diseases, as defined herein, are also considered "metabolic disorders." These diseases can include coronary artery disease (also called ischemic heart disease), hypertension, inflammation associated with coronary artery disease, restenosis, peripheral vascular disease, and stroke.

[0211] Weight-related disorders are also considered "metabolic disorders" as defined herein. Such disorders can include obesity, metabolic syndrome, including its independent components (e.g., central obesity, FBG / prediabetes / diabetes, hypercholesterolemia, hypertriglyceridemia, and hypertension), hypometabolic states, hypothyroidism, uric acidosis, and other conditions associated with weight gain (including rapid weight gain), weight loss, weight loss maintenance, or risk of weight loss after weight loss.

[0212] Blood glucose disorders, as defined herein, are also considered "metabolic disorders." Such disorders may include diabetes, hypertension, and polycystic ovarian syndrome associated with insulin resistance. Other exemplary metabolic disorders may include kidney transplantation, nephrotic syndrome, Cushing's syndrome, acromegaly, systemic lupus erythematosus, dysglobulinemia, lipodystrophy, glycogenosis type I, and Addison's disease.

[0213] Additional diseases or conditions associated with metabolic disorders will be apparent to one of skill in the art and are within the scope of the present disclosure.

[0214] Symptoms of an SGLT2-associated disease, e.g., gout or diabetes, include, for example, insulin resistance, an inability to regulate blood sugar, high blood uric acid levels, loss of fat-free or lean muscle mass, excess fat mass, a slower metabolic rate, weight gain, and / or an increased body mass index. Further details regarding the signs and symptoms of various diseases or conditions are provided herein and are well known in the art.

[0215] A "therapeutically effective amount," as used herein, is intended to include an amount of an RNAi agent that, when administered to a subject with an SGLT2-related disease, is sufficient to treat the disease (e.g., by reducing, ameliorating, or maintaining an existing disease or one or more symptoms of the disease). A "therapeutically effective amount" may vary depending on the RNAi agent, the method of administration of the agent, the disease and its severity and medical history, age, weight, family history, genetic makeup, type of prior or concomitant treatment, if any, and other personal characteristics of the subject to be treated.

[0216] A "prophylactically effective amount," as used herein, is intended to include an amount of an RNAi agent that, when administered to a subject with an SGLT2-related disorder, such as gout or diabetes, is sufficient to prevent or ameliorate the disease or one or more symptoms of the disease. Ameliorating the disease includes slowing the course of the disease or reducing the severity of any disease that develops. A "prophylactically effective amount" can vary depending on the RNAi agent, the method of administration of the agent, the degree of risk of the disease, and the medical history, age, weight, family history, genetic makeup, type of prior or concomitant treatment, if any, and other personal characteristics of the patient to be treated.

[0217] A "therapeutically effective amount" or a "prophylactically effective amount" also encompasses the amount of an RNAi agent that produces some desired local or systemic effect at a reasonable benefit / risk ratio applicable to any treatment. The RNAi agent used in the methods of the present disclosure can be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0218] The phrase "pharmaceutically acceptable" is used herein to refer to those compounds, materials (including salts), compositions or dosage forms that are suitable for use in contact with the tissues of human and animal subjects without excessive toxicity, irritation, allergic response, or other problem or complication, within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio.

[0219] The phrase "pharmaceutically acceptable carrier," as used herein, means a pharmaceutically acceptable material, composition, or vehicle involved in the transport or transfer of a subject compound from one organ or part of the body to another, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, magnesium talc, calcium or zinc stearate, or stearic acid), or solvent encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the subject being treated. Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose, 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; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar. (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide, (15) alginic acid, (16) pyrogen-free water, (17) isotonic saline, (18) Ringer's solution, (19) ethyl alcohol, (20) pH buffer solutions, (21) polyesters, polycarbonates, 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 substances employed in pharmaceutical formulations.Pharmaceutically acceptable carriers for pulmonary delivery are known in the art and vary depending on the desired location for deposition of the drug, e.g., upper or lower respiratory system, and the type of device used for delivery, e.g., sprayer, nebulizer, dry powder inhaler.

[0220] The term "sample" as used herein includes similar fluids, cells, or tissues isolated from a subject, as well as collections of fluids, cells, or tissues present within a subject. Examples of biological fluids include blood, serum, and serous fluid, plasma, bronchial fluid, sputum, cerebrospinal fluid, ocular fluid, lymphatic fluid, urine, saliva, sputum, etc. Tissue samples can include samples from tissues, organs, or localized areas. For example, samples can be derived from specific organs, parts of organs, or fluids or cells within those organs.

[0221] II. RNAi Agents of the Disclosure Described herein is an RNAi agent that inhibits the expression of SGLT2 gene. In one embodiment, the RNAi agent comprises a double-stranded ribonucleic acid (dsRNA) molecule for inhibiting the expression of SGLT2 gene in a cell, such as a cell in a subject, for example, in a mammal, for example, a human, who has an SGLT2-related disorder, such as gout or diabetes, or is at risk of an SGLT2-related disease.

[0222] dsRNAi comprises an antisense strand having a complementary region that is complementary to at least a portion of a target RNA, e.g., mRNA, formed upon expression of the SGLT2 gene. The region of complementarity is no more than about 15-30 nucleotides in length. When contacted with a cell expressing the SGLT2 gene, the RNAi agent inhibits expression of the SGLT2 gene (e.g., human, primate, or non-primate gene) by at least 50%, as assayed, for example, by PCR or branched DNA (bDNA)-based methods, or by protein-based methods, such as immunofluorescence, e.g., using Western blotting or flow cytometry techniques. In certain embodiments, inhibition of expression is at least 50%, as assayed by the Dual-Glo luciferase assay of Example 1, where the siRNA is at a concentration of 10 nM.

[0223] 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 complementary region that is substantially complementary, and generally completely complementary, to the target sequence. For example, the target sequence can be derived from the sequence of the mRNA formed during the expression of the SGLT2 gene. The other strand (sense strand) comprises a region that is 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 sequences of dsRNA can also be contained as self-complementary regions of a single nucleic acid molecule, such that they are opposite each other on separate oligonucleotides.

[0224] 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 9, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. In certain embodiments, the double-stranded structure is 18 to 25 base pairs in length, e.g., 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 25, 21 to 24, 21 to 23, 21 to 22, 22 to 25, 22 to 24, 22 to 23, 23 to 25, 23 to 24, or 24 to 25 base pairs in length, e.g., 19 to 21 base pairs in length. Ranges and lengths between the ranges and lengths listed above are also intended to be part of this disclosure.

[0225] 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, 19-29, 19-28, 19-27 ...30, 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, 19-66, 19-67, 19-68, 19-69, 19-7 21-23, or 21-22 nucleotides in length, e.g., 19-23 nucleotides in length or 21-23 nucleotides in length. Ranges and lengths between the above-listed ranges and lengths are also intended to be part of this disclosure.

[0226] In some embodiments, the dsRNA is 15-23 nucleotides in length or 25-30 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 dsRNA longer than about 21-23 nucleotides can serve as a substrate for Dicer. As those skilled in the art will recognize, the region of RNA targeted for cleavage will most often be a portion of a longer RNA molecule, often an mRNA molecule. In relevant cases, a "portion" of an mRNA target is a contiguous sequence of the mRNA target that is long enough to allow it to serve as a substrate for RNAi-dependent cleavage (i.e., cleavage via the RISC pathway).

[0227] Those skilled in the art will appreciate that the double-stranded region is a primary functional portion of the dsRNA, e.g., about 15-36 base pairs, e.g., 15-36, 15-35, 15-34, 15-33, 15-32, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21 , 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs, for example, 19-21 base pairs. That is, in one embodiment, an RNA molecule or complex of RNA molecules having a double-stranded region of more than 30 base pairs is a dsRNA, as long as it is processed into a functional double-strand of, for example, 15-30 base pairs that targets the desired RNA for cleavage. Thus, those skilled in the art will recognize that, in one embodiment, miRNA is a dsRNA. In another embodiment, the dsRNA is not a naturally occurring miRNA. In another embodiment, the RNAi agent useful for targeting SGLT2 expression is not generated in target cells by cleavage of a larger dsRNA.

[0228] The dsRNA described herein can further comprise one or more single-stranded nucleotide overhangs, for example, 1, 2, 3 or 4 nucleotides.Nucleotide overhangs can comprise or consist of nucleotide / nucleoside analogs, such as deoxynucleotides / nucleosides.Overhangs can be on sense strand, antisense strand, or any combination thereof.Furthermore, the nucleotide of a certain overhang can be present on the 5'-end, 3'-end, or both ends of either the antisense strand or the sense strand of dsRNA.In certain embodiments, longer extended overhangs are possible.

[0229] dsRNA can be synthesized by standard methods known in the art, for example, using an automated DNA synthesizer, such as those commercially available from Biosearch, Applied Biosystems, Inc., as discussed further below.

[0230] The iRNA compounds of the present invention can be prepared using a two-step method. 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 has the advantage that it is easy to prepare oligonucleotide strands containing unnatural nucleotides or modified nucleotides. The single-stranded oligonucleotides of the present invention can be prepared using solution phase or solid phase organic synthesis or both.

[0231] siRNAs can be produced, e.g., in bulk, by a variety of methods. Exemplary methods include organic synthesis and RNA cleavage, e.g., in vitro cleavage.

[0232] siRNAs can be produced by separately synthesizing a single-stranded RNA molecule, or each strand of a double-stranded RNA molecule, and then annealing the component strands.

[0233] Large-scale bioreactors, such as the OligoPilot II from Pharmacia Biotec AB (Uppsala, Sweden), can be used to produce large quantities of specific RNA strands for a given siRNA. The OligoPilot II reactor can efficiently attach nucleotides using only a 1.5 molar excess of phosphoramidite nucleotides. Ribonucleotide amidites are used to create the RNA strands. Using standard cycles of monomer addition, 21- to 23-nucleotide strands of siRNA can be synthesized. Typically, the two complementary strands are generated separately and then annealed, for example, after release from the solid support and deprotection.

[0234] Organic synthesis can be used to produce individual siRNA species.The complementarity of the species to SGLT2 gene can be precisely specified.For example, the species can be complementary to the region containing polymorphism, for example, single nucleotide polymorphism.Furthermore, the position of polymorphism can be precisely specified.In some embodiments, the polymorphism is located in the internal region, for example, within at least 4, 5, 7 or 9 nucleotides from one or both ends.

[0235] In one embodiment, the RNA produced is carefully purified, and end siRNA is removed, and for example, using Dicer or equivalent RNAse III-based activity to be cut into siRNA in vitro.For example, dsiRNA can be incubated in the extract from Drosophila in vitro, or with purified components, for example, purified RNAse or RISC complex (RNA-induced silencing complex).For example, see Ketting et al.Genes Dev 2001 Oct 15;15(20):2654-9 and Hammond Science 2001 Aug 10;293(5532):1146-50.

[0236] dsiRNA cleavage typically generates multiple siRNA species, each a specific 21-23 nucleotide fragment of the source dsiRNA molecule. For example, there may be siRNAs that contain sequences complementary to overlapping and flanking regions of the source dsiRNA molecule.

[0237] Regardless of synthesis method, siRNA preparation can be prepared in a solution (for example, aqueous solution or organic solution) that is suitable for formulation.For example, siRNA preparation can be precipitated, redissolved in pure double distilled water, and lyophilized.Then, dried siRNA can be resuspended in a solution that is suitable for the intended formulation process.

[0238] In one embodiment, the dsRNA of the present disclosure comprises at least two nucleotide sequences, a sense strand and an antisense strand. The sense strand sequence for SGLT2 can be selected from the sequences provided in any one of Tables 2-3, and the corresponding nucleotide sequence of the antisense strand of the sense strand can be selected from the sequences provided in any one of Tables 2-3. In this embodiment, one of the two sequences is complementary to the other of the two sequences, where one of the sequences is substantially complementary to the sequence of mRNA produced upon expression of the SGLT2 gene. Thus, in this embodiment, the dsRNA comprises two oligonucleotides, one of which is described in any one of Tables 2-3 as the sense strand (passenger strand) and the second of which is described in any one of Tables 2-3 for SGLT2 as the corresponding antisense strand (guide strand).

[0239] In one embodiment, the substantially complementary sequences of the dsRNA are contained on separate oligonucleotides, hi another embodiment, the substantially complementary sequences of the dsRNA are contained on a single oligonucleotide.

[0240] Although the sequences provided herein are described as modified or conjugated sequences, it will be understood that the RNA of the RNAi agents of this disclosure, e.g., the dsRNA of this disclosure, can comprise any one of the sequences set forth in any one of Tables 2-3, unmodified, unconjugated, or modified or conjugated differently from those set forth therein. One or more lipophilic ligands or one or more GalNAc ligands can be included at any position in the RNAi agents provided in this application.

[0241] Those skilled in the art are well aware that dsRNAs having a duplex structure of approximately 20-23 base pairs, e.g., 21 base pairs, have been hailed as particularly effective in inducing RNA interference (Elbashir et al., (2001) EMBO J., 20:6877-6888). However, others have discovered 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-described embodiments, due to the nature of the oligonucleotide sequences provided herein, the dsRNAs described herein can comprise at least one strand that is a minimum of 21 nucleotides in length. It can be reasonably predicted that shorter duplexes, minus only a few nucleotides at one or both ends, can be similarly effective compared to the dsRNAs described above. Thus, dsRNAs that have a sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides derived from one of the sequences provided herein and that differ in their ability to inhibit expression of the SGLT2 gene by no more than 10, 15, 20, 25, or 30% inhibition from dsRNAs containing the entire sequence using in vitro assays with Cos7 and 10 nM concentrations of the RNA agent and the PCR assays provided in the Examples herein are intended to be within the scope of this disclosure.

[0242] In addition, the RNA described herein identifies the site in SGLT2 transcript that is susceptible to RISC-mediated cleavage.Therefore, the present disclosure also features an RNAi agent that targets within this site.As used herein, if an RNAi agent promotes the cleavage of a transcript at any of the specific sites, the RNAi agent is said to target within the specific site of an RNA transcript.This RNAi agent will generally comprise at least about 15 consecutive nucleotides, for example, at least 19 nucleotides, from one of the sequences provided herein, which is combined with additional nucleotide sequences taken from the region adjacent to the selected sequence in SGLT2 gene.

[0243] The RNAi agents described herein may contain one or more mismatches to the target sequence. In one embodiment, the RNAi agents described herein contain three or fewer mismatches (i.e., three, two, one, or zero mismatches). In one embodiment, the RNAi agents described herein contain two or fewer mismatches. In one embodiment, the RNAi agents described herein contain one or fewer mismatches. In one embodiment, the RNAi agents described herein contain zero mismatches. In certain embodiments, when the antisense strand of an RNAi agent contains a mismatch to the target sequence, the mismatch can be optionally limited to be within the last five nucleotides from either the 5'-end or the 3'-end of the complementary region. For example, in such an embodiment, in the case of a 23-nucleotide RNAi agent, the strand complementary to a region of the SGLT2 gene generally does not contain any mismatches within the central 13 nucleotides. By using the methods described herein or known in the art, it can be determined whether an RNAi agent containing a mismatch to the target sequence is effective in inhibiting the expression of the SGLT2 gene. It is important to consider the efficacy of mismatched RNAi agents to inhibit SGLT2 expression, especially if the specific region of complementarity in the SGLT2 gene is known to be mutated.

[0244] III. Modified RNAi Agents of the Present Disclosure In one embodiment, the RNA of the RNAi agent of the present disclosure, e.g., dsRNA, is unmodified and does not contain, for example, chemical modifications or conjugations known in the art and described herein. In certain embodiments, the RNA of the RNAi agent of the present disclosure, e.g., dsRNA, is chemically modified to enhance stability or other beneficial characteristics. In certain embodiments of the present disclosure, substantially all of the nucleotides of the RNAi agent of the present disclosure are modified. In other embodiments of the present disclosure, all of the nucleotides of the RNAi agent of the present disclosure are modified. An RNAi agent of the present disclosure in which "substantially all of the nucleotides are modified" may be widely modified but not entirely modified, and may contain 5, 4, 3, 2, or 1 or less unmodified nucleotides. In yet other embodiments of the present disclosure, an RNAi agent of the present disclosure may contain 5, 4, 3, 2, or 1 or less modified nucleotides.

[0245] Nucleic acids featured in this disclosure can be synthesized or modified by methods well established in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, SLet 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 nucleotides, inverted linkage, etc.), base modifications, such as substitution with stabilizing bases, destabilizing bases, or bases that base-pair with partners in an extended repertoire, base removal (abasic nucleotides) or conjugated bases, sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions, or backbone modifications, including phosphodiester bond modifications or substitutions. Specific examples of RNAi agents useful in the embodiments described herein include, but are not limited to, RNAs containing modified backbones or lacking natural internucleoside linkages. Among the RNAs with modified backbone, those that do not have phosphorus atom in backbone are included.For the purpose of this specification, and as sometimes referred to in the art, the modified RNAs that do not have phosphorus atom in their internucleoside backbone can also be considered as oligonucleoside.In some embodiments, modified RNAi agent will have phosphorus atom in its internucleoside backbone.

[0246] Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl 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, their 2'-5' linked analogs, and those with reversed polarity, where adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'.Various salts, such as sodium salts, mixed salts, and free acid forms, are also included.

[0247] Representative U.S. 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, 5,286,711, and the like. No. 7, No. 5,321,131, No. 5,399,676, No. 5,405,939, No. 5,453,496, No. 5,455,233, No. 5,466,677, No. 5,47 No. 6,925, No. 5,519,126, No. 5,536,821, No. 5,541,316, No. 5,550,111, No. 5,563,253, No. 5,571,799, No. 5 , 587,361, 5,625,050, 6,028,188, 6,124,445, 6,160,109, 6,169,170, 6,172,209 No. 6,239,265, No. 6,277,603, No. 6,326,199, No. 6,346,614, No. 6,444,423, No. 6,531,590, No. 6,534, Nos. 6,639, 6,608,035, 6,683,167, 6,858,715, 6,867,294, 6,878,805, 7,015,315, 7,041,816, 7,273,933, 7,321,029, and U.S. Patent No. RE39464, the contents of each of which are incorporated herein by reference in their entirety.

[0248] Modified RNA backbones that do not contain a phosphorus atom in the backbone have backbones formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatom or heterocyclic internucleoside linkages, including 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.

[0249] 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.

[0250] In another embodiment, RNA mimics suitable for use in RNAi agents are considered, in which both the sugar and internucleoside linkages of nucleotide units, i.e., the backbone, 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 known 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 directly or indirectly linked to the aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents teaching the preparation of PNA compounds include, but are not limited to, U.S. Patent 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 RNAi agents of the present disclosure are described, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0251] Some embodiments featured in this disclosure include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, particularly --CH2--NH--CH2-, --CH2--N(CH3)--O--CH2-- (known as methylene (methylimino) or MMI backbones), --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2--, and --N(CH3)--CH2--CH2-- of the above-referenced U.S. Patent No. 5,489,677, and the amide backbones of the above-referenced U.S. Patent No. 5,602,240. In some embodiments, RNAs featured herein have the morpholino backbone structure of the above-referenced U.S. Patent No. 5,034,506. The natural phosphodiester backbone can be represented as OP(O)(OH)-OCH2-.

[0252] Modified RNAs can also contain one or more substituted sugar moieties. The RNAi agents featured herein, e.g., dsRNAs, 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 or 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 includes one of the following at the 2' position: C1 to C 10The substituents include 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 groups, reporter groups, interfering substances, groups for improving the pharmacokinetic properties of RNAi agents or groups for improving the pharmacodynamic properties of RNAi agents, and other substituents with similar properties. In some 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 the 2'-dimethylaminooxyethoxy, i.e., O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, as described herein below in the Examples, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O-CH2-O-CH2-N(CH3)2. Further exemplary modifications include 5'-Me-2'-F nucleotides, 5'-Me-2'-OMe nucleotides, 5'-Me-2'-deoxynucleotides (both R and S isomers within these three families), 2'-alkoxyalkyl, and 2'-NMA (N-methylacetamide).

[0253] Other modifications include 2'-methoxy (2'-OCH), 2'-aminopropoxy (2'-OCHCHNH), 2'-O-hexadecyl, and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA of an RNAi agent, 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. RNAi agents can also have sugar mimetics, such as a cyclobutyl moiety 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, and 5,56 Nos. 7,811, 5,576,427, 5,591,722, 5,597,909, 5,610,300, 5,627,053, 5,639,873, 5,646,265, 5,658,873, 5,670,633, and 5,700,920, some of which are commonly owned with this application, the entire contents of each of the foregoing being incorporated herein by reference.

[0254] The RNAi agent of the present disclosure can also comprise modification or substitution of nucleobase (often simply referred to in the art as "base").As used herein, "unmodified" or "natural" nucleobase includes purine bases adenine (A) and guanine (G), and pyrimidine bases thymine (T), cytosine (C) and uracil (U).Modified nucleobases include other synthetic nucleobases and natural nucleobases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, These include 5-uracil (pseudouracil), 4-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.Additional nucleobases include those disclosed in United States Patent No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P.ed.Wiley-VCH, 2008, those disclosed in The Concise Encyclopedia of Polymer Science and Engineering, pages 858-859, Kroschwitz, JL, ed.John Wiley&Sons, 1990, those disclosed in Englisch et al., (1991) Angewandte Chemie, International Edition, 30:613, and those disclosed in Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 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 disclosure. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and 0-6 substituted purines, such as 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), and are exemplary base substitutions, especially when combined with 2'-O-methoxyethyl sugar modifications.

[0255] Representative United States patents that teach the preparation of certain of the above modified nucleobases, as well as other modified nucleobases, include, but are not limited to, the above-referenced 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, Nos. 5,594,121, 5,596,091, 5,614,617, 5,681,941, 5,750,692, 6,015,886, 6,147,200, 6,166,197, 6,222,025, 6,235,887, 6,380,368, 6,528,640, 6,639,062, 6,617,438, 7,045,610, 7,427,672, and 7,495,088, the entire contents of each of which are incorporated herein by reference.

[0256] The RNAi agents of the present disclosure can also be modified to include one or more bicyclic sugar moieties. A "bicyclic sugar" is a furanosyl ring modified by a ring formed by a bridge between two carbon atoms, whether adjacent or non-adjacent. A "bicyclic nucleoside" ("BNA") is a nucleoside having a sugar moiety that includes a ring formed by bridging two carbon atoms of the sugar ring, whether adjacent or non-adjacent, thereby forming a bicyclic ring system. In certain embodiments, a bridge connects the 4'-carbon and 2'-carbon of the sugar ring, optionally via a 2'-acyclic oxygen atom. Thus, in some embodiments, the agents of the present disclosure can include one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety includes an additional bridge connecting the 2' and 4' carbons. In other words, an LNA is a nucleotide containing a bicyclic sugar moiety that includes a 4'-CH2-O-2' bridge. This structure effectively "locks" the ribose in the 3'-endo conformation. The addition of a locking 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 the polynucleotides of the present disclosure include, but are not limited to, nucleosides comprising a bridge between the 4' and 2' ribosyl ring atoms. In certain embodiments, the antisense polynucleotide agent of the present disclosure includes one or more bicyclic nucleosides comprising a 4' to 2' bridge.

[0257] A locked nucleoside can be represented by this structure (stereochemistry omitted): [ka] wherein B is a nucleobase or modified nucleobase, and L is a linking group connecting the 2'-carbon and the 4'-carbon of the ribose ring. Examples of such 4' to 2' bridged bicyclic nucleosides include, but are not limited to, 4'-(CH2)-O-2' (LNA), 4'-(CH2)-S-2', 4'-(CH2)2-O-2' (ENA), 4'-CH(CH3)-O-2' (also referred to 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, e.g., U.S. Pat. No. 7,399,845). , U.S. Pat. No. 8,278,283), 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 nitrogen 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 the foregoing are incorporated herein by reference.

[0258] Additional representative U.S. patents and publications that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Patent Nos. 6,268,490, 6,525,191, 6,670,461, 6,770,748, 6,794,499, 6,998,484, 7,053,207, 7,034,133, 7,084,125, Nos. 7,399,845, 7,427,672, 7,569,686, 7,741,457, 8,022,193, 8,030,467, 8,278,425, 8,278,426, 8,278,283, US2008 / 0039618, and US2009 / 0012281, the entire contents of each of which are incorporated herein by reference.

[0259] Any of the foregoing bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations, including, for example, α-L-ribofuranose and β-D-ribofuranose (see WO 99 / 14226).

[0260] The RNAi agents of the present disclosure can also be modified to include one or more constrained ethyl nucleotides. As used herein, a "constrained ethyl nucleotide" or "cEt" is a locked nucleic acid that includes a bicyclic sugar moiety containing a 4'-CH(CH3)-O-2' bridge (L in the preceding structure). In one embodiment, the constrained ethyl nucleotide is in the S conformation and is referred to herein as an "S-cEt."

[0261] The RNAi agent of the present disclosure may also contain one or more "conformation-restricting nucleotides" ("CRNs"). A CRN is a nucleotide analogue with a linker connecting the C2' and C4' carbons of ribose or the C3' and C5' carbons of ribose. The CRN locks the ribose ring into a stable conformation, increasing hybridization affinity to mRNA. The linker is long enough to position the oxygen at an optimal position for stability and affinity, thereby reducing puckering of the ribose ring.

[0262] Representative publications that teach the preparation of certain of the above CRNs include, but are not limited to, US2013 / 0190383 and WO2013 / 036868, the entire contents of each of which are incorporated herein by reference.

[0263] In some embodiments, the RNAi agent of the present disclosure includes one or more monomers that are UNA (unlocked nucleic acid) nucleotides. UNAs are unlocked acyclic nucleic acids in which any of their sugar linkages have been removed to form an unlocked "sugar" residue. In one example, UNAs also encompass monomers in which the C1'-C4' bond has been removed (i.e., a covalent carbon-oxygen-carbon bond between the C1' and C4' carbons). In another example, the C2'-C3' bond of the sugar (i.e., a covalent carbon-carbon bond between the C2' and C3' carbons) has been removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039, which are incorporated herein by reference).

[0264] Representative U.S. 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.

[0265] Potential stabilizing 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'-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3'-phosphate, inverted 2'-deoxy-modified ribonucleotides such as inverted dT (idT) and inverted dA (idA), and inverted abasic 2'-deoxyribonucleotides (iAb), among others. Disclosure of these modifications can be found in WO 2011 / 005861.

[0266] In one example, the 3' or 5' end of the oligonucleotide is linked to an inverted 2'-deoxy modified ribonucleotide, such as an inverted dT (idT), an inverted dA (idA), or an inverted abasic 2'-deoxyribonucleotide (iAb). In one particular example, the inverted 2'-deoxy-modified ribonucleotide is linked to the 3' end of the oligonucleotide, such as the 3' end of the sense strand described herein, where the linkage is via a 3'-3' phosphodiester linkage or a 3'-3'-phosphorothioate linkage.

[0267] In another embodiment, the 3' end of the sense strand is linked to an inverted abasic ribonucleotide (iAb) via a 3'-3'-phosphorothioate linkage. In another embodiment, the 3' end of the sense strand is linked to an inverted dA (idA) via a 3'-3'-phosphorothioate linkage.

[0268] In one particular example, the inverted 2'-deoxy-modified ribonucleotide is linked to the 3' end of an oligonucleotide, such as the 3' end of the sense strand described herein, wherein the linkage is via a 3'-3' phosphodiester linkage or a 3'-3'-phosphorothioate linkage.

[0269] In another example, the 3'-terminal nucleotide of the sense strand is an inverted dA (idA) and is joined to the preceding nucleotide via a 3'-3'-linkage (eg, a 3'-3'-phosphorothioate linkage).

[0270] Other modifications of the RNAi agent of the present disclosure include 5' phosphate or 5' phosphate mimic, for example, the 5' terminal phosphate or phosphate mimic on the antisense strand of RNAi agent.Suitable phosphate mimic is disclosed, for example, in US2012 / 0157511, the entire content of which is incorporated herein by reference.

[0271] A. Modified RNAi Agents Comprising Motifs of the Disclosure In certain aspects of the present disclosure, the double-stranded RNAi agent of the present disclosure includes an agent having chemical modifications, such as those disclosed in International Publication No. 2013 / 075035, the entire contents of which are incorporated herein by reference. As shown herein and in International Publication No. 2013 / 075035, one or more motifs of three identical modifications on three consecutive nucleotides can be introduced into the sense or antisense strand of the RNAi agent, particularly at or near the cleavage site. In some embodiments, the sense and antisense strands of the RNAi agent can be otherwise completely modified. The introduction of these motifs interrupts the modification pattern of the sense or antisense strand, if present. The RNAi agent can optionally be conjugated with a lipophilic ligand, for example, a C16 ligand, for example, on the sense strand. The RNAi agent can optionally be modified with an (S)-glycol nucleic acid (GNA) modification, for example, at one or more residues of the antisense strand.

[0272] Thus, the present disclosure provides a double-stranded RNAi agent capable of inhibiting expression of a target genome or gene (i.e., the SGLT2 gene) in vivo. The RNAi agent comprises a sense strand and an antisense strand. Each strand of the RNAi agent can be 15 to 30 nucleotides in length. Each strand can be, for example, 16 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. In certain embodiments, each strand is 19 to 23 nucleotides in length.

[0273] The sense and antisense strands typically form a double-stranded double-stranded RNA ("dsRNA"), also referred to herein as an "RNAi agent." The double-stranded region of an RNAi agent can be 15-30 nucleotide pairs in length. For example, the double-stranded region can be 16-30 nucleotide pairs in length, 17-30 nucleotide pairs in length, 27-30 nucleotide pairs in length, 17-23 nucleotide pairs in length, 17-21 nucleotide pairs in length, 17-19 nucleotide pairs in length, 19-25 nucleotide pairs in length, 19-23 nucleotide pairs in length, 19-21 nucleotide pairs in length, 21-25 nucleotide pairs in length, or 21-23 nucleotide pairs in length. In another 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. In certain embodiments, the double-stranded region is 19 to 21 nucleotide pairs in length.

[0274] In one embodiment, the RNAi agent may contain one or more overhang regions or capping groups at the 3'-end, 5'-end, or both ends of one or both strands. The overhangs may be 1 to 6 nucleotides in length, e.g., 2 to 6 nucleotides in length, 1 to 5 nucleotides in length, 2 to 5 nucleotides in length, 1 to 4 nucleotides in length, 2 to 4 nucleotides in length, 1 to 3 nucleotides in length, 2 to 3 nucleotides in length, or 1 to 2 nucleotides in length. In certain embodiments, the nucleotide overhang region is 2 nucleotides in length. The overhang may be the result of one strand being longer than the other, or the result of two strands of the same length being twisted. The overhang may form a mismatch with the target mRNA, be complementary to the targeted gene sequence, or be a different sequence. The first and second strands may also be linked by additional bases, for example, to form a hairpin, or by other non-basic linkers.

[0275] In one embodiment, the nucleotides in the overhang region of an RNAi agent can each independently be modified or unmodified nucleotides, including, but not limited to, 2'-sugar modifications, such as 2-F, 2'-O-methyl, thymidine (T), and any combination thereof.

[0276] For example, TT can be an overhang sequence for either end on either strand, which can form a mismatch with the target mRNA, or can be complementary to the targeted gene sequence, or can be another sequence.

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

[0278] RNAi agent can only contain a single 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 at the 3'-end of the antisense strand.RNAi can also have a blunt end, which is located at the 5'-end of the antisense strand (i.e., 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 its 5'-end is blunt.Without wishing to be bound by theory, the asymmetry between the blunt end at the 5'-end of the antisense strand and the 3'-end overhang of the antisense strand is favorable for the insertion of guide strand into RISC process.

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

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

[0281] In yet another embodiment, the RNAi agent is 21 nucleotides in length and double-blunt ended, and the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 9, 10, and 11 from the 5' end. The antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at 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 contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 9, 10, and 11 from the 5' end, and the antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end, and one end of the RNAi agent is blunt and the other end contains a two-nucleotide overhang. In one example, the two-nucleotide overhang is at the 3' end of the antisense strand. When the two-nucleotide overhang is at the 3' end of the antisense strand, there may be two phosphorothioate internucleotide linkages between the three terminal nucleotides, two of which are overhanging nucleotides, and the third nucleotide is a paired nucleotide adjacent to the overhanging nucleotide. In one embodiment, the RNAi agent further comprises two phosphorothioate internucleotide linkages between the three terminal nucleotides at both the 5'-end of the sense strand and the 5'-end of the antisense strand.In one embodiment, every nucleotide in the sense strand and antisense strand of the RNAi agent, including the nucleotide that is part of a motif, is a modified nucleotide.In one embodiment, each residue is independently modified with 2'-O-methyl or 2'-fluoro, for example, within an alternating motif.Optionally, the RNAi agent further comprises a ligand (e.g., a lipophilic ligand, optionally a C16 ligand).

[0283] 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), comprises at least 8 ribonucleotides at positions 1 to 23 of the first strand; and 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 1 to 23 of the sense strand to form a duplex; wherein at least the 3'-terminal nucleotide of the antisense strand is unpaired with the sense strand, and up to 6 consecutive 3'-terminal nucleotides are unpaired with the sense strand, thereby forming a 3' single-stranded overhang of 1 to 6 nucleotides; and the 5'-end of the antisense strand comprises 10 to 30 consecutive ribonucleotides that are not paired with the sense strand. the sense strand comprises nucleotides corresponding to the 2'-F modification on three consecutive nucleotides, thereby forming a single-stranded 5' overhang of 10 to 30 nucleotides, at least the 5'- and 3'-terminal nucleotides of the sense strand base-pair with nucleotides of the antisense strand when the sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially double-stranded region between the sense and antisense strands, and the antisense strand is sufficiently complementary to the target RNA along at least 19 ribonucleotides of the antisense strand length, such that the double-stranded nucleic acid reduces target gene expression when introduced into a mammalian cell, and the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides, at least one of the motifs occurring at or near the cleavage site. The antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at or near the cleavage site.

[0284] 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 nucleotides and at most 29 nucleotides, and a second strand having a length of at most 30 nucleotides with at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end, the 3' end of the first strand and the 5' end of the second strand forming a blunt end, the second strand being 1 to 4 nucleotides longer at its 3' end than the first strand, the double-stranded region being at least 25 nucleotides in length, the second strand being sufficiently complementary to a target mRNA along at least 19 nucleotides of the length of the second strand, the RNAi agent reducing target gene expression when introduced into a mammalian cell, and Dicer cleavage of the RNAi agent resulting in an siRNA comprising the 3' end of the second strand, thereby reducing target gene expression in the mammalian cell. Optionally, the RNAi agent further comprises a ligand.

[0285] In one embodiment, the sense strand of the RNAi agent contains at least one motif of three identical modifications on three consecutive nucleotides, one of which motifs occurs at the cleavage site within the sense strand.

[0286] In one embodiment, the antisense strand of the RNAi agent can also contain at least one motif of three identical modifications on three consecutive nucleotides, one of which occurs at or near the cleavage site in the antisense strand.

[0287] For RNAi agents with a double-stranded region of 17 to 23 nucleotides in length, the cleavage site of the antisense strand is typically approximately 10, 11, and 12 positions from the 5' end. Thus, three identical modification motifs can occur at positions 9, 10, and 11, 10, 11, and 12, 11, 12, and 13, 12, 13, and 14, or 13, 14, and 15 of the antisense strand, starting from the first nucleotide from the 5' end of the antisense strand, or starting from the first paired nucleotide within the double-stranded region from the 5' end of the antisense strand. The cleavage site within the antisense strand can also vary depending on the length of the double-stranded region of the RNAi from the 5' end.

[0288] The sense strand of RNAi agent can contain at least one motif of three identical modifications on three consecutive nucleotides at the break site of its strand, and antisense strand can have at least one motif of three identical modifications on 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 so that one motif of three nucleotides on sense strand and one motif of three nucleotides on 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 pairing.Alternatively, at least two nucleotides can overlap, or all three nucleotides can overlap.

[0289] In one embodiment, the sense strand of an RNAi agent may contain multiple motifs of three identical modifications on three consecutive nucleotides. The first motif may occur at or near the site of strand cleavage, and the other motifs may be wing modifications. As used herein, the term "wing modification" refers to a motif that occurs in another portion of the strand separated from a motif at or near the site of cleavage of the same strand. The wing modifications may be adjacent to the first motif or separated by at least one or more nucleotides. When the motifs are immediately adjacent to each other, the chemical nature of the motifs is distinct from each other, and when the motifs are separated by one or more nucleotides, the chemical nature 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 occur at one end of the first motif at or near the cleavage site, or on either side of the lead motif.

[0290] Like the sense strand, the antisense strand of an RNAi agent may contain multiple motifs of three identical modifications on three consecutive nucleotides, at least one of which occurs at or near the cleavage site of that strand. The antisense strand may also contain one or more wing modifications in the same alignment as the wing modifications that may be present on the sense strand.

[0291] 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.

[0292] 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 in the double-stranded region at the 3' end, 5' end, or both ends of the strand.

[0293] When the sense and antisense strands of an RNAi agent each contain at least one wing modification, the wing modifications can be at the same end of the double-stranded region and have an overlap of one, two, or three nucleotides.

[0294] When the sense and antisense strands of an RNAi agent each contain at least two wing modifications, the sense and antisense strands can be aligned such that the two modifications from each single strand are at one end of a double-stranded region with one, two, or three nucleotide overlap, the two modifications from each single strand are at the other end of the double-stranded region with one, two, or three nucleotide overlap, and the two modifications from the single strand are on either side of a lead motif with one, two, or three nucleotide overlap within the double-stranded region.

[0295] In one embodiment, the RNAi agent contains mismatches or combinations thereof in the double strand with the target. Mismatches can occur in the overhang region or in the double-stranded region. Base pairs can be ranked based on their tendency to promote dissociation or melting (for example, based on the free energy of association or dissociation of a particular pairing; the simplest approach is to examine each pair individually, but adjacent or similar analysis can also be used). In terms of promoting dissociation, A:U is preferred to G:C, G:U is preferred to G:C, and I:C is preferred to G:C (I=inosine). Mismatches, such as non-standard pairings or non-standard pairings (described elsewhere herein), are preferred to standard pairings (A:T, A:U, G:C), and pairings containing universal bases are preferred to standard pairings.

[0296] In one embodiment, the RNAi agent comprises the first 1, 2, 3, 4, or 5 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 at least one mismatch pair, e.g., a non-canonical pairing or a non-canonical pairing or a pairing containing a universal base, to promote dissociation of the antisense strand at the 5' end of the duplex.

[0297] 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 one, two, or three 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.

[0298] In another embodiment, the nucleotide at the 3' end of the sense strand is deoxythymidine (dT). In another embodiment, the nucleotide at the 3' end of the antisense strand is deoxythymidine (dT). In one embodiment, there is a short sequence of deoxythymidine nucleotides, for example, two dT nucleotides on the 3' end of either the sense strand or the antisense strand.

[0299] 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), During the ceremony, i and j each independently represent 0 or 1; p and q each independently represent 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, and XXX, YYY and ZZZ each independently represent one motif of three identical modifications on three consecutive nucleotides. In one embodiment, YYY are all 2'-F modified nucleotides.

[0300] In one embodiment, N a or N b includes alternating pattern modifications.

[0301] In one embodiment, YYY motif occurs at or near the cleavage site of sense strand.For example, when RNAi agent has a double-stranded region of 17-23 nucleotides in length, YYY motif can occur at or near the cleavage site of sense strand (for example, can occur at position 6, 7, 8, 7, 8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12, or 11, 12, 13), and this number starts from the first nucleotide from the 5' end, or optionally this number starts from the first paired nucleotide in double-stranded region from the 5' end.

[0302] 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 has 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), which may be represented as:

[0303] 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.

[0304] each N a can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0305] When the sense strand is represented by formula (Ic), 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 can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0306] When the sense strand is represented by formula (Id), each N b independently represent an oligonucleotide sequence comprising 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. b is 0, 1, 2, 3, 4, 5, or 6. Each N a can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0307] Each of X, Y and Z may be the same or different from one another.

[0308] In other embodiments, i is 0 and j is 0 and the sense strand has the formula: 5'n p -N a -YYY-N a -nq 3'(Ia) It can be expressed as:

[0309] When the sense strand is represented by formula (Ia), each N a can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0310] In one embodiment, the antisense strand sequence of the RNAi has the 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), It can be expressed as During the ceremony, k and l each independently represent 0 or 1; p' and q' each independently represent 0 to 6; each N a each independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides; each N b ' independently represents an oligonucleotide sequence comprising 0 to 10 modified nucleotides; each n p ' and n q ' independently represent an overhanging nucleotide; In the formula, N b ' and Y' do not have the same modification, and X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides.

[0311] In one embodiment, N a ' or N b ' includes alternating pattern modifications.

[0312] The Y'Y'Y' motif occurs at or near the cleavage site of the antisense strand.For example, when the RNAi agent has a double-stranded region of 17-23 nucleotides in length, the Y'Y'Y' motif can occur at positions 9, 10, 11, 10, 11, 12, 11, 12, 13, 12, 13, 14, or 13, 14, 15 of the antisense strand, where the numbers start from the first nucleotide from the 5' end, or optionally, the numbers start from the first paired nucleotide in the double-stranded region from the 5' end.In one embodiment, the Y'Y'Y' motif occurs at positions 11, 12, and 13.

[0313] In one embodiment, the Y'Y'Y' motif is all 2'-OMe modified nucleotides.

[0314] In one embodiment, k is 1 and l is 0, or k is 0 and l is 1, or k and l are both 1.

[0315] Thus, the antisense strand has 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), It can be expressed as:

[0316] 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 represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0317] 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 represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0318] When the antisense strand is represented by formula (IId), each N b Each 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 In one embodiment, N' 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.

[0319] In other embodiments, k is 0 and l is 0, and the antisense strand has the formula: 5'n p’ -N a’ -Y'Y'Y'-N a’ -n q’ 3'(Ia), It can be expressed as:

[0320] When the antisense strand is represented by formula (IIa), each N a ' independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0321] Each of X', Y' and Z' may be the same as or different from one another.

[0322] Each nucleotide of sense strand and antisense strand can be independently modified with LNA, glycol nucleic acid (GNA), hexitol nucleic acid (HNA), 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-hydroxyl or 2'-fluoro.For example, each nucleotide of sense strand and antisense strand is independently modified with 2'-O-methyl or 2'-fluoro.Each X, Y, Z, X', Y' and Z' can specifically represent 2'-O-methyl modification or 2'-fluoro modification.

[0323] In one embodiment, the sense strand of the RNAi agent can contain a YYY motif occurring at positions 9, 10, and 11 of the strand when the double-stranded region is 21 nt, the numbers starting from the first nucleotide from the 5' end, or optionally, the numbers starting from the 5' end with the first paired nucleotide in the double-stranded region, and Y represents a 2'-F modification. The sense strand can further contain a XXX motif or a ZZZ motif as a wing modification at the opposite end of the double-stranded region, and XXX and ZZZ each independently represent a 2'-OMe modification or a 2'-F modification.

[0324] In one embodiment, the antisense strand can contain a Y'Y'Y' motif occurring at positions 11, 12, or 13 of the strand, where the numbers start from the first nucleotide from the 5' end, or optionally, the numbers start from the 5' end with the first paired nucleotide in the double-stranded region, and Y' represents a 2'-O-methyl modification. The antisense strand can further contain an X'X'X' motif or a Z'Z'Z' motif as a wing modification at opposite ends of the double-stranded region, and X'X'X' and Z'Z'Z' each independently represent a 2'-OMe modification or a 2'-F modification.

[0325] 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).

[0326] Thus, the RNAi agent used in the methods of the present disclosure can include a sense strand and an antisense strand, each strand having 14-30 nucleotides, and the RNAi duplex can have the formula (III): Sense:5'n p -N a -(XXX) i -N b -YY YN 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) During the ceremony, i, j, k, and l each independently represent 0 or 1; p, p', q and q' each independently represent 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; During the ceremony, each n p ',n p , n q ' and n q each of which may or may not be present independently represents an overhanging nucleotide; 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.

[0327] In one embodiment, i is 0 and j is 0, or i is 1 and j is 0, or i is 0 and j is 1, or i and j are both 0, or i and j are both 1. In another embodiment, k is 0 and l is 0, or k is 1 and l is 0, or k is 0 and l is 1, or k and l are both 0, or k and l are both 1.

[0328] An exemplary combination of sense and antisense strands that form an RNAi duplex is of the following formula: 5'n p -N a -YYY-N a -n q 3' 3'n p ’ -N a ’ -Y'Y'Y'-N a ’ n q ’ 5' (IIIa) 5'n p -N a -YYY-N b -ZZZ-N a -n q 3' 3'n p ’ -N a ’ -Y'Y'Y'-N b ’ -Z'Z'Z'-N a ’ n q ’ 5' (IIIb) 5'n p -N a -XXX-N b -YYY-N a -nq 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'n p ’ -N a ’ -X'X'X'-N b ’ -Y'Y'Y'-N b ’ -Z'Z'Z'-N a -n q ’ 5' (IIId).

[0329] When the RNAi agent is represented by formula (IIIa), each N a independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0330] When the RNAi agent is represented by formula (IIIb), each N b independently represent an oligonucleotide sequence containing 1 to 10, 1 to 7, 1 to 5, or 1 to 4 modified nucleotides. a ' independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0331] When the RNAi agent is represented by formula (IIIc), each N b , 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 represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0332] When the RNAi agent is represented by formula (IIId), each N b , N b Each 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 , N a ’ independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. a , N a ', N b , and N b ’ each independently comprises an alternating pattern of modifications.

[0333] In one embodiment, when the RNAi agent has formula (IIId), N a The modification is a 2'-O-methyl modification or a 2'-fluoro modification. In another embodiment, when the RNAi agent has formula (IIId), N a The modifications are 2'-O-methyl or 2'-fluoro modifications, and p '>0 and at least one n p In yet another embodiment, when the RNAi agent has formula (IIId), N' is linked to the adjacent nucleotide via a phosphorothioate linkage. a The modification is 2'-O-methyl or 2'-fluoro modification, p '>0 and at least one n p In another embodiment, when the RNAi agent has formula (IIId), N' is linked to adjacent nucleotides via phosphorothioate linkages, and the sense strand is conjugated to one or more C16 (or related thereto) moieties attached via a divalent or trivalent branched linker (described below).a The modification is 2-O-methyl or 2-fluoro modification, n p '>0 and at least one n p ' are linked to adjacent nucleotides via phosphorothioate linkages, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more lipophilic moieties, e.g., C16 (or related) moieties, which may optionally be attached via a divalent or trivalent branched linker.

[0334] In one embodiment, when the RNAi agent has formula (IIIa), N a The modification is 2-O-methyl or 2-fluoro modification, n p '>0 and at least one n p ' are linked to adjacent nucleotides via phosphorothioate linkages, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more lipophilic moieties, e.g., C16 (or related) moieties, which may be attached via a divalent or trivalent branched linker.

[0335] In one embodiment, the RNAi agent is a multimer containing at least two double strands represented by formula (III), (IIIa), (IIIb), (IIIc) and (IIId), and these double strands are connected by a linker. The linker may be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the double strands can target the same gene or two different genes, or each of the double strands can target the same gene at two different target sites.

[0336] In one embodiment, the RNAi agent is a multimer that contains three, four, five, six or more double strands represented by formula (III), (IIIa), (IIIb), (IIIc) and (IIId), and the double strands are connected by a linker. The linker may be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each double strand can target the same gene or two different genes, or each double strand can target the same gene at two different target sites.

[0337] In one embodiment, two RNAi agents represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId) are linked to each other at their 5' ends and optionally conjugated to a ligand at one or both of their 3' ends. The agents can each target the same gene or two different genes, or the agents can each target the same gene at two different target sites.

[0338] Various publications describe the multimeric RNAi agent that can be used in the method of the present disclosure.These publications include International Publication No. 2007 / 091269, International Publication No. 2010 / 141511, International Publication No. 2007 / 117686, International Publication No. 2009 / 014887 and International Publication No. 2011 / 031520 and United States Patent No. 7858769, each of whose entire contents is incorporated herein by reference.

[0339] In certain embodiments, the compositions and methods of the present disclosure include vinyl phosphonate (VP) modifications of RNAi agents as described herein. In exemplary embodiments, a 5'-vinyl phosphonate modified nucleotide of the present disclosure has the following structure: [ka] wherein X is O or S; R is hydrogen, hydroxy, fluoro, or C 1-20 alkoxy (e.g., methoxy or n-hexadecyloxy); R 5’ is =C(H)-P(O)(OH)2, and the C5' carbon and R 5’ and the double bond between is in the E or Z configuration (e.g., E configuration), and B is a nucleobase or modified nucleobase, optionally B is adenine, guanine, cytosine, thymine, or uracil.

[0340] In one embodiment, R is ═C(H)—P(O)(OH) and the double bond between the C carbon and R is in the E configuration. In another embodiment, R is methoxy and R is ═C(H)—P(O)(OH) and the double bond between the C carbon and R is in the E configuration. In another embodiment, X is S, R is methoxy and R is ═C(H)—P(O)(OH) and the double bond between the C carbon and R is in the E configuration.

[0341] The vinyl phosphonate of the present disclosure can be attached to either the antisense or sense strand of the dsRNA of the present disclosure. In certain embodiments, the vinyl phosphonate of the present disclosure is attached to the antisense strand of the dsRNA, optionally at the 5' end of the antisense strand of the dsRNA.

[0342] Vinyl phosphate modifications are also contemplated for the compositions and methods of the present disclosure. Exemplary vinyl phosphate structures include those described above, where R5' is =C(H)-OP(O)(OH)2, and the double bond between the C5' carbon and R5' is in the E or Z configuration (e.g., E configuration).

[0343] Thermal destabilization modification In certain embodiments, dsRNA molecules can be optimized for RNA interference by incorporating thermostabilizing modifications into the seed region of antisense strand.As used herein, "seed region" refers to the 2-9 positions of the 5' end of the referenced strand.For example, thermostabilizing modifications can be incorporated into the seed region of antisense strand to reduce or inhibit off-target gene silencing.

[0344] The term "thermally destabilizing modification" includes modifications that result in a dsRNA having an overall melting temperature (Tm) that is lower than the Tm of a dsRNA that does not have such a modification. For example, a thermally destabilizing modification can decrease the Tm of a dsRNA by 1-4°C, e.g., one, two, three, or four degrees Celsius. Additionally, the term "thermally destabilizing nucleotide" refers to a nucleotide that contains one or more thermally destabilizing modifications.

[0345] It has been discovered that dsRNAs having an antisense strand containing at least one thermally destabilizing modification of the duplex within the first 9 nucleotide positions counting from the 5' end of the antisense strand have reduced off-target gene silencing activity. Thus, in some embodiments, the antisense strand contains at least one (e.g., one, two, three, four, five, or more) thermally destabilizing modification of the duplex within the first 9 nucleotide positions of the 5' region of the antisense strand. In some embodiments, the one or more thermally destabilizing modifications of the duplex are located at positions 2 to 9, e.g., positions 4 to 8, from the 5' end of the antisense strand. In some further embodiments, the thermally destabilizing modification of the duplex is located at positions 6, 7, or 8 from the 5' end of the antisense strand. In still some further embodiments, the thermally destabilizing modification of the duplex is located at position 7 from the 5' end of the antisense strand. In some embodiments, the thermally destabilizing modification of the duplex is located at positions 2, 3, 4, 5, or 9 from the 5' end of the antisense strand.

[0346] Thermally destabilizing modifications can include, but are not limited to, abasic modifications, mismatches with the opposing nucleotide in the opposing strand, and sugar modifications such as 2'-deoxy modifications, acyclic nucleotides such as unlocked nucleic acids (UNAs) or glycol nucleic acids (GNAs), and 2'-5'-linked ribonucleotides ("3'-RNAs").

[0347] Exemplary abasic modifications include, but are not limited to, the following: [ka] wherein R=H, Me, Et or OMe, R'=H, Me, Et or OMe, R"=H, Me, Et or OMe [ka] wherein B is a modified or unmodified nucleobase.

[0348] Exemplary sugar modifications include, but are not limited to, the following: [ka] wherein B is a modified or unmodified nucleobase.

[0349] In some embodiments, the thermally destabilizing modification of the duplex is selected from the group consisting of: [ka] where B is a modified or unmodified nucleobase, and the asterisk on each structure represents either R, S, or racemic.

[0350] In some embodiments, the thermally destabilizing modification of the duplex is selected from the group consisting of: [ka] wherein B is a modified or unmodified nucleobase and the asterisk represents either R, S or racemic (e.g., S).

[0351] The term "acyclic nucleotide" refers to any nucleotide having an acyclic ribose sugar, e.g., one in which any of the bonds of the ribose carbon ring (e.g., C1'-C2', C2'-C3', C3'-C4', C4'-O4', or C1'-O4') is absent, or at least one of the ribose carbons or oxygens (e.g., C1', C2', C3', C4', or O4'), independently or in combination, is absent from the nucleotide.

[0352] In some embodiments, the acyclic nucleotide is [ka] B is a modified or unmodified nucleobase; R 1 and R 2 are independently H, halogen, OR3, or alkyl, and R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar. The term "UNA" refers to an acyclic unlocked nucleic acid in which any of the sugar bonds has been removed to form an unlocked "sugar" residue. In one example, UNA also encompasses monomers in which the C1'-C4' bond has been removed (i.e., a carbon-oxygen-carbon covalent bond between the C1' and C4' carbons). In another example, the C2'-C3' bond of the sugar has been removed (i.e., a carbon-carbon covalent bond between the C2' and C3' carbons) (see Mikhailov et al., Tetrahedron Letters, 26(17):2059 (1985), and Fluiter et al., Mol. Biosyst., 10:1039 (2009), which are incorporated herein by reference in their entireties). Acyclic derivatives offer greater backbone flexibility without affecting Watson-Crick pairing. Acyclic nucleotides can be linked via 2'-5' or 3'-5' linkages.

[0353] The term "GNA" refers to glycol nucleic acid, which is a polymer similar to DNA or RNA, but differs in the composition of its "backbone" in that it is made up of repeating glycerol units linked by phosphodiester bonds. [ka]

[0354] The thermally destabilizing modification of the double strand can be a mismatch (i.e., non-complementary base pair) between the thermally destabilizing nucleotide and the opposite nucleotide in the opposite strand of the dsRNA duplex.Exemplary mismatch base pairs include G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T, U:T, or a combination thereof.Other mismatch base pairings known in the art are also contemplated by the present invention.Mismatches can occur between nucleotides that are either naturally occurring or modified nucleotides, i.e., mismatch base pairings can occur between the nucleobases derived from each nucleotide independently of the modification on the ribose sugar of the nucleotide.In certain embodiments, the dsRNA molecule contains at least one nucleobase in the mismatch pairing that is a 2'-deoxynucleobase, for example, the 2'-deoxynucleobase is in the sense strand.

[0355] In some embodiments, the thermally destabilizing modifications of the duplex in the seed region of the antisense strand include nucleotides, such as modified nucleobases, that have impaired Watson-Crick hydrogen bonding (WC H-bonding) with complementary bases on the target mRNA. [ka]

[0356] Many examples of abasic nucleotides, acyclic nucleotide modifications (including UNA and GNA) and mismatch modifications are described in detail in WO 2011 / 133876, which is incorporated herein by reference in its entirety.

[0357] Thermally destabilizing modifications can also include universal base and phosphate modifications that have reduced or eliminated ability to form hydrogen bonds with opposing bases.

[0358] In some embodiments, the thermal destabilizing modification of double strand comprises the nucleotide with non-standard base, for example but not limited to, the nucleobase modification that the ability to form hydrogen bond with the base in opposite strand is impaired or completely lost.These nucleobase modifications have been evaluated for the destabilization of the central region of dsRNA double strand, as described in International Publication No. 2010 / 0011895, the entire contents of which are incorporated herein by reference.Exemplary nucleobase modifications include: [ka]

[0359] In some embodiments, the duplex thermally destabilizing modifications in the seed region of the antisense strand include one or more α-nucleotides that are complementary to bases on the target mRNA, such as: [ka] wherein R is H, OH, OCH3, F, NH2, NHMe, NMe2 or O-alkyl.

[0360] Exemplary phosphate modifications that have been shown to decrease the thermal stability of dsRNA duplexes, relative to natural phosphodiester linkages, include the following: [ka]

[0361] The alkyl R group can be a C1-C6 alkyl. Specific alkyl R groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, pentyl, and hexyl.

[0362] As those skilled in the art will recognize, considering that the functional role of nucleobase defines the specificity of the RNAi agent of the present disclosure, nucleobase modification can be carried out in various ways as described herein, for example, for the purpose of enhancing on-target effect against off-target effect, for example, introducing destabilizing modification into the RNAi agent of the present disclosure, but generally the range of modification that exists on the RNAi agent of the present disclosure tends to be much larger than the non-nucleobase modification, for example, the modification of the sugar group or phosphate backbone of polyribonucleotide.Such modification will be described in more detail in other sections of the present disclosure, and is expressly intended for the RNAi agent of the present disclosure that has either natural nucleobase or modified nucleobase as described above or elsewhere herein.

[0363] In addition to the antisense strand containing a thermally destabilizing modification, the dsRNA may also contain one or more stabilizing modifications. For example, the dsRNA may contain at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) stabilizing modifications. Without limitation, all of the stabilizing modifications may be present in one strand. In some embodiments, both the sense and antisense strands contain at least two stabilizing modifications. The stabilizing modifications may occur at any nucleotide in either the sense strand or the antisense strand. For example, the stabilizing modifications may occur at any nucleotide in the sense strand or the antisense strand, and each stabilizing modification may occur in an alternating pattern on the sense strand or the antisense strand, or the sense strand or the antisense strand may contain both stabilizing modifications in an alternating pattern. The alternating pattern of stabilizing modifications on the sense strand may be the same as or different from that of the antisense strand, and the alternating pattern of stabilizing modifications on the sense strand may be shifted relative to the alternating pattern of stabilizing modifications on the antisense strand.

[0364] In some embodiments, the antisense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) stabilizing modifications. Without limitation, the stabilizing modifications within the antisense strand can be located at any position. In some embodiments, the antisense strand comprises stabilizing modifications at positions 2, 6, 8, 9, 14, and 16 from the 5' end. In some other embodiments, the antisense strand comprises stabilizing modifications at positions 2, 6, 14, and 16 from the 5' end. In yet some other embodiments, the antisense strand comprises stabilizing modifications at positions 2, 14, and 16 from the 5' end.

[0365] In some embodiments, the antisense strand comprises at least one stabilizing modification adjacent to the destabilizing modification.For example, the stabilizing modification can be at the 5'-end or 3'-end of the destabilizing modification, i.e., at the -1 or +1 position from the position of the destabilizing modification, nucleotide.In some embodiments, the antisense strand comprises a stabilizing modification at each of the 5'-end and 3'-end of the destabilizing modification, i.e., at the -1 and +1 positions from the position of the destabilizing modification.

[0366] In some embodiments, the antisense strand comprises at least two stabilizing modifications at the 3' end of the destabilizing modification, ie, at positions +1 and +2 from the position of the destabilizing modification.

[0367] In some embodiments, the sense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) stabilizing modifications. Without limitation, the stabilizing modifications within the sense strand can be located at any position. In some embodiments, the sense strand comprises stabilizing modifications at positions 7, 10, and 11 from the 5' end. In some other embodiments, the sense strand comprises stabilizing modifications at positions 7, 9, 10, and 11 from the 5' end. In some embodiments, the sense strand comprises stabilizing modifications at positions opposite or complementary to positions 11, 12, and 15 of the antisense strand, counting from the 5' end of the antisense strand. In some other embodiments, the sense strand comprises stabilizing modifications at positions opposite or complementary to positions 11, 12, 13, and 15 of the antisense strand, counting from the 5' end of the antisense strand. In some embodiments, the sense strand comprises blocks of two, three, or four stabilizing modifications.

[0368] In some embodiments, the sense strand does not contain a stabilizing modification at a position opposite or complementary to a thermally destabilizing modification of the duplex in the antisense strand.

[0369] Exemplary thermally stabilizing modifications include, but are not limited to, 2'-fluoro modifications. Other thermally stabilizing modifications include, but are not limited to, LNA.

[0370] In some embodiments, the dsRNA of the present disclosure contains at least four (e.g., four, five, six, seven, eight, nine, ten or more) 2'-fluoro nucleotides. Without limitation, all of the 2'-fluoro nucleotides can be present in one strand. In some embodiments, both the sense and antisense strands contain at least two 2'-fluoro nucleotides. The 2'-fluoro modification can occur on any nucleotide in either the sense strand or the antisense strand. For example, the 2'-fluoro modification can occur on any nucleotide in the sense strand or the antisense strand, and each 2'-fluoro modification can occur in an alternating pattern on the sense strand or the antisense strand, or both the sense strand and the antisense strand contain 2'-fluoro modifications in an alternating pattern. The alternating pattern of 2'-fluoro modifications on the sense strand can be the same as or different from that of the antisense strand, and the alternating pattern of 2'-fluoro modifications on the sense strand can be shifted relative to the alternating pattern of 2'-fluoro modifications on the antisense strand.

[0371] In some embodiments, the antisense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) 2'-fluoro nucleotides. Without limitation, the 2'-fluoro modifications in the antisense strand can be present at any position. In some embodiments, the antisense strand comprises 2'-fluoro nucleotides at positions 2, 6, 8, 9, 14, and 16 from the 5' end. In some other embodiments, the antisense strand comprises 2'-fluoro nucleotides at positions 2, 6, 14, and 16 from the 5' end. In yet some other embodiments, the antisense strand comprises 2'-fluoro nucleotides at positions 2, 14, and 16 from the 5' end.

[0372] In some embodiments, antisense strand comprises at least one 2'-fluoro nucleotide adjacent to destabilizing modification.For example, 2'-fluoro nucleotide can be at the 5'-end or 3'-end of destabilizing modification, that is, at the -1 position or +1 position from the position of destabilizing modification, nucleotide.In some embodiments, antisense strand comprises 2'-fluoro nucleotide at each of the 5'-end and 3'-end of destabilizing modification, that is, at the -1 and +1 positions from the position of destabilizing modification.

[0373] In some embodiments, the antisense strand comprises at least two 2'-fluoro nucleotides at the 3' end of the destabilizing modification, ie, at positions +1 and +2 from the position of the destabilizing modification.

[0374] In some embodiments, the sense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) 2'-fluoro nucleotides. Without limitation, the 2'-fluoro modifications within the sense strand can be present at any position. In some embodiments, the antisense strand comprises 2'-fluoro nucleotides at positions 7, 10, and 11 from the 5'-end. In some other embodiments, the sense strand comprises 2'-fluoro nucleotides at positions 7, 9, 10, and 11 from the 5'-end. In some embodiments, the sense strand comprises 2'-fluoro nucleotides at positions opposite or complementary to positions 11, 12, and 15 of the antisense strand, counting from the 5'-end of the antisense strand. In some other embodiments, the sense strand comprises 2'-fluoro nucleotides at positions opposite or complementary to positions 11, 12, 13, and 15 of the antisense strand, counting from the 5'-end of the antisense strand. In some embodiments, the sense strand comprises two, three, or four blocks of 2'-fluoro nucleotides.

[0375] In some embodiments, the sense strand does not contain a 2'-fluoro nucleotide at a position opposite or complementary to a thermally destabilizing modification of the duplex in the antisense strand.

[0376] In some embodiments, a dsRNA molecule of the disclosure comprises a 21-nucleotide (nt) sense strand and a 23-nucleotide (nt) antisense strand, wherein the antisense strand contains at least one thermally destabilized nucleotide, wherein the at least one thermally destabilized nucleotide occurs within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), one end of the dsRNA is blunt while the other end comprises a 2-nt overhang, and the dsRNA optionally comprises at least one (e.g., one, two, three, four, five, six, or all seven) of the following characteristics: The antisense strand further comprises the following features: (i) the antisense strand comprises 2, 3, 4, 5, or 6 2'-fluoro modifications, (ii) the antisense strand comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages, (iii) the sense strand is conjugated with a ligand, (iv) the sense strand comprises 2, 3, 4, or 5 2'-fluoro modifications, (v) the sense strand comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages, (vi) the dsRNA comprises at least four 2'-fluoro modifications, and (vii) the dsRNA comprises a blunt end at the 5' end of the antisense strand. In one embodiment, the two nucleotide overhangs are located at the 3' end of the antisense strand.

[0377] In some embodiments, a dsRNA molecule of the present disclosure 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), comprises at least 8 ribonucleotides at positions 1 to 23 of the sense strand; the antisense strand is 36 to 66 nucleotide residues in length and, starting from the 3'-terminal nucleotide, at least 8 ribonucleotides at these positions are 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 1-6 nucleotide 3' single-stranded overhang; and the 5'-end of the antisense strand is composed of 10 to 30 consecutive nucleotides that are not paired with the sense strand. the antisense strand is composed of at least one ribonucleotide, thereby forming a single-stranded 5' overhang of 10 to 30 nucleotides, at least the 5'- and 3'-terminal nucleotides of the sense strand base-pair with nucleotides of the antisense strand when the sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially double-stranded region between the sense and antisense strands, and the antisense strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the length of the antisense strand to reduce target gene expression when the double-stranded nucleic acid is introduced into a mammalian cell, and the antisense strand contains at least one thermally destabilizing nucleotide, the at least one thermally destabilizing nucleotide being within the seed region of the antisense strand (i.e., at positions 2-9 of the 5'-end of the antisense strand).For example, the thermally destabilizing nucleotide occurs between positions 14-17 opposite or complementary to positions 14-17 of the 5' end of the sense strand, and the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six, or all seven) of the following features: (i) the antisense strand contains two, three, four, five, or six 2'-fluoro modifications; (ii) the antisense strand contains one, two, three, four, or five phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated to a ligand; (iv) the sense strand contains two, three, four, or five 2'-fluoro modifications; (v) the sense strand contains one, two, three, four, or five phosphorothioate internucleotide linkages; and (vi) the dsRNA contains at least four 2'-fluoro modifications; and (vii) the dsRNA contains a double-stranded region 12-30 nucleotide pairs in length.

[0378] In some embodiments, a dsRNA molecule of the present disclosure comprises a sense strand and an antisense strand, the dsRNA molecule comprising a sense strand having a length of at least 25 nucleotides and at most 29 nucleotides, and an antisense strand having a length of at most 30 nucleotides, the sense strand comprising a modified nucleotide at position 11 from the 5' end that is susceptible to enzymatic degradation, the 3' end of the sense strand and the 5' end of the antisense strand forming a blunt end, and the antisense strand is 1 to 4 nucleotides longer at its 3' end than the sense strand, the double-stranded region being at least 25 nucleotides in length, and the antisense strand being sufficiently complementary to a target mRNA along at least 19 nt of the antisense strand, such that the dsRNA molecule reduces target gene expression when introduced into a mammalian cell, and Dicer cleavage of the dsRNA results in an siRNA comprising the 3' end of the antisense strand, thereby inhibiting the expression of the target gene in the mammalian cell. and wherein the antisense strand contains at least one thermally destabilized nucleotide, the at least one thermally destabilized nucleotide being located within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), and the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six, or all seven) of the following features: (i) the antisense strand contains two, three, four, five, or six 2'-fluoro modifications; (ii) the antisense strand contains at least one 2'-fluoro modification; (iii) the sense strand is conjugated to a ligand; (iv) the sense strand contains two, three, four, or five 2'-fluoro modifications; (v) the sense strand contains one, two, three, four, or five phosphorothioate internucleotide linkages; and (vi) the dsRNA contains at least four 2'-fluoro modifications; and (vii) the dsRNA has a double-stranded region 12 to 29 nucleotide pairs in length.

[0379] In some embodiments, any nucleotide in the sense strand and antisense strand of dsRNA molecule can be modified.Each nucleotide can be modified with the same or different modifications, and these modifications can include one or both of non-linked phosphate oxygen or one or more of linked phosphate oxygen, modifying the ribose sugar component, for example, modifying the 2' hydroxyl on the ribose sugar, replacing phosphate moiety extensively with " dephosphorylation " linker, modifying or replacing naturally occurring base, and replacing or replacing ribose phosphate backbone.

[0380] Because nucleic acids are polymers of subunits, many modifications occur at positions that are repeated within nucleic acids, such as modifications of bases or phosphate moieties, or non-linked Os of phosphate moieties.In some cases, modifications occur at all target positions in nucleic acids, but in many cases, they do not occur.For example, modifications can occur only at the 3'-end or 5'-end positions, or only in terminal regions, such as at the terminal nucleotide position of the chain or in 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 the double-stranded regions of RNA, or only in the single-stranded regions of RNA, for example, phosphorothioate modification 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 of the chain or in the last 2, 3, 4, 5, or 10 nucleotides, or in double-stranded and single-stranded regions, especially at the ends. The 5' end can be phosphorylated.

[0381] This may, for example, enhance stability, include specific bases in the overhang, or include modified nucleotides or nucleotide substitutes in the single-stranded overhang, for example, in the 5' overhang or 3' overhang, or in both overhangs.For example, it may be desirable to include purine nucleotides in the overhang.In some embodiments, all or part of the bases in the 3' overhang or 5' overhang can be modified, for example, with the modifications described herein.Modifications may include, for example, the use of modifications at the 2' position of the ribose sugar with modifications known in the art, for example, the use of deoxyribonucleotides, 2'-deoxy-2'-fluoro (2'-F) or 2'-O-methyl, instead of the ribosugar of the nucleic acid base, and modifications at the phosphate group, for example, phosphorothioate modifications.The overhang does not need to be homologous to the target sequence.

[0382] In some embodiments, each residue of the sense strand and the antisense strand is independently modified with LNA, glycol nucleic acid (GNA), hexitol nucleic acid (HNA), 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, or 2'-fluoro. These strands may contain two or more modifications. In some embodiments, each residue of the sense strand and the antisense strand is independently modified with 2'-O-methyl or 2'-fluoro. It should be understood that these modifications are in addition to at least one thermally destabilizing modification of the duplex present in the antisense strand.

[0383] At least two different modifications are typically present on the sense strand and the antisense strand. These two modifications may be 2'-deoxy, 2'-O-methyl, or 2'-fluoro modifications, acyclic nucleotides, etc. In some embodiments, the sense strand and the antisense strand each contain two differently modified nucleotides selected from 2'-O-methyl or 2'-deoxy. In some embodiments, each residue in the sense strand and the antisense strand is independently modified with a 2'-O-methyl nucleotide, a 2'-deoxy nucleotide, a 2'-deoxy-2'-fluoro nucleotide, a 2'-ON-methylacetamide (2'-O-NMA, 2'O-CHC(O)N(Me)H) nucleotide, a 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) nucleotide, a 2'-O-aminopropyl (2'-O-AP) nucleotide, or a 2'-ara-F nucleotide. Again, it is understood that these modifications are in addition to at least one thermally destabilizing modification of the duplex present in the antisense strand.

[0384] In some embodiments, the dsRNA molecules of the present disclosure comprise an alternating pattern of modifications. The term "alternating motif" or "alternating pattern," as used herein, refers to a motif with one or more modifications, each modification occurring on alternating nucleotides in a single strand. The alternating nucleotides may refer to one every other nucleotide, one every three nucleotides, or similar patterns. For example, if A, B, and C each represent a type of modification to a nucleotide, the alternating motif may be "ABABABABABAB...," "AABBAABBAABB...," "AABAABAABAAB...," "AAABAAABAAAB...," "AAABBBAAABBB...," or "ABCABCABCABC...," etc.

[0385] The types of modifications contained within an 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 on every other nucleotide, can be the same, but each of the sense or antisense strands can be selected from several possible modifications within the alternating motif, such as "ABABAB...," "ACACAC...," "BDBDBD...," or "CDCDCD...," etc.

[0386] In some embodiments, dsRNA molecules of the present disclosure include an alternating motif modification pattern on the sense strand that is shifted relative to the alternating motif modification pattern on the antisense strand. The shift can be such that modified groups on nucleotides in the sense strand correspond to differently modified groups on nucleotides in 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 begin with "ABABAB" from the 5'-3' end of the strand, and the alternating motif in the antisense strand can begin with "BABABA" from the 3'-5' end of the strand in the double-stranded region. As another example, the alternating motif in the sense strand can begin with "AABBAABB" from the 5'-3' end of the strand, and the alternating motif in the antisense strand can begin with "BBAABBAA" from the 3'-5' end of the strand in the double-stranded region, resulting in a complete or partial shift in the modification pattern between the sense and antisense strands.

[0387] In one particular example, the alternating motif in the sense strand is "ABABAB" from 5'-3' of the strand, where each A is an unmodified ribonucleotide and each B is a 2'-O methyl modified nucleotide.

[0388] In one particular example, the alternating motif in the sense strand is "ABABAB" from 5'-3' of the strand, where each A is a 2'-deoxy-2'-fluoro modified nucleotide and each B is a 2'-O methyl modified nucleotide.

[0389] In another particular example, the alternating motif in the antisense strand is "BABABA" from 3'-5' of the strand, where each A is a 2'-deoxy-2'-fluoro modified nucleotide and each B is a 2'-O methyl modified nucleotide.

[0390] In one particular example, the alternating motif in the sense strand is "ABABAB" from 5'-3' of the strand and the alternating motif in the antisense strand is "BABABA" from 3'-5' of the strand, where each A is an unmodified ribonucleotide and each B is a 2'-O methyl modified nucleotide.

[0391] In one particular example, the alternating motif in the sense strand is "ABABAB" from 5'-3' of the strand and the alternating motif in the antisense strand is "BABABA" from 3'-5' of the strand, where each A is a 2'-deoxy-2'-fluoro modified nucleotide and each B is a 2'-O methyl modified nucleotide.

[0392] The dsRNA molecule of the present disclosure can further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage.Phosphorothioate or methylphosphonate internucleotide linkage modification can occur on the nucleotide of either sense strand or antisense strand or both strands at any position of the strand.For example, internucleotide linkage modification can occur on any nucleotide on sense strand or antisense strand, each internucleotide linkage modification can occur in an alternating pattern on sense strand or antisense strand, or sense strand or antisense strand contains both internucleotide linkage modifications in an alternating pattern.The alternating pattern of internucleotide linkage modification on sense strand can be the same or different from that of antisense strand, and the alternating pattern of internucleotide linkage modification on sense strand can be shifted relative to the alternating pattern of internucleotide linkage modification on antisense strand.

[0393] In some embodiments, the dsRNA molecule comprises phosphorothioate or methylphosphonate internucleotide linkage modification in the overhang region.For example, the overhang region comprises two nucleotides, and the two nucleotides have phosphorothioate or methylphosphonate internucleotide linkage between the two nucleotides.Internucleotide linkage modification can also be made to link the overhang nucleotide to 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 linkage, and optionally there can be an additional phosphorothioate or methylphosphonate internucleotide linkage connecting the overhang nucleotide to the paired nucleotide adjacent to the overhang nucleotide.For example, there can be at least two phosphorothioate internucleotide linkages between the terminal three nucleotides, two of which are overhang nucleotides and the third is the paired nucleotide adjacent to the overhang nucleotide.In one embodiment, these terminal three nucleotides can be at the 3'-end of the antisense strand.

[0394] In some embodiments, the sense strand of the dsRNA molecule comprises 1 to 10 blocks of two to ten phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate internucleotide linkages, one of which is positioned at any position within the oligonucleotide sequence, and the sense strand is paired with an antisense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0395] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of two phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is positioned at any position within the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0396] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of three phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate internucleotide linkages, one of the phosphorothioate or methylphosphonate internucleotide linkages being positioned at any position within the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0397] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of four phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 phosphate internucleotide linkages, one of which is positioned at any position within the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0398] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of five phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 phosphate internucleotide linkages, one of which is positioned at any position within the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0399] In some embodiments, the antisense strand of the dsRNA molecule contains two blocks of six phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphate internucleotide linkages, one of which is positioned at any position within the oligonucleotide sequence, and the antisense strand is paired with a sense strand containing any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or with an antisense strand containing either phosphorothioate or methylphosphonate or phosphate linkages.

[0400] In some embodiments, the antisense strand of the dsRNA molecule contains two blocks of seven phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, or 8 phosphate internucleotide linkages, one of which is positioned at any position within the oligonucleotide sequence, and the antisense strand is paired with a sense strand containing any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or with an antisense strand containing either phosphorothioate or methylphosphonate or phosphate linkages.

[0401] In some embodiments, the antisense strand of a dsRNA molecule comprises two blocks of eight phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, or 6 phosphate internucleotide linkages, one of which is positioned at any position within the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0402] In some embodiments, the antisense strand of a dsRNA molecule comprises two blocks of nine phosphorothioate or methylphosphonate internucleotide linkages separated by one, two, three, or four phosphate internucleotide linkages, one of which is positioned at any position within the oligonucleotide sequence, and the antisense strand is paired with a sense strand containing any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or with an antisense strand containing either phosphorothioate or methylphosphonate or phosphate linkages.

[0403] In some embodiments, the dsRNA molecules of this disclosure further comprise one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within 1 to 10 nucleotides at the termini of the sense or antisense strand, for example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides can be linked via phosphorothioate or methylphosphonate internucleotide linkages at one or both termini of the sense or antisense strand.

[0404] In some embodiments, the dsRNA molecules of the present disclosure further comprise one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within 1 to 10 nucleotides of the inner region of each double strand of the sense or antisense strand. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides can be linked via phosphorothioate or methylphosphonate internucleotide linkage modifications at positions 8 to 16 of the double-stranded region, counting from the 5' end of the sense strand, and the dsRNA molecule can optionally further comprise one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 1 to 10 of that end.

[0405] In some embodiments, dsRNA molecules of the disclosure further comprise one to five phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) and one to five phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 18-23 of the sense strand, and one to two phosphorothioate or methylphosphonate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and one to five within positions 18-23 of the antisense strand.

[0406] In some embodiments, dsRNA molecules of the disclosure further comprise one phosphorothioate internucleotide linkage modification within the range of positions 1-5 (counting from the 5' end) and one phosphorothioate or methylphosphonate internucleotide linkage modification within the range of positions 18-23 of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 or 2 (counting from the 5' end) and two phosphorothioate or methylphosphonate internucleotide linkage modifications within the range of positions 18-23 of the antisense strand.

[0407] In some embodiments, dsRNA molecules of the disclosure further comprise two phosphorothioate internucleotide linkage modifications within the range of positions 1-5 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within the range of positions 18-23 of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within the range of positions 18-23 of the antisense strand.

[0408] In some embodiments, dsRNA molecules of the disclosure further comprise two phosphorothioate internucleotide linkage modifications within the range of positions 1 to 5 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within the range of positions 18 to 23 of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within the range of positions 18 to 23 of the antisense strand.

[0409] In some embodiments, dsRNA molecules of the disclosure further comprise two phosphorothioate internucleotide linkage modifications within the range of positions 1-5 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within the range of positions 18-23 of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within the range of positions 18-23 of the antisense strand (counting from the 5' end).

[0410] In some embodiments, dsRNA molecules of the disclosure further comprise one phosphorothioate internucleotide linkage modification within the range of positions 1-5 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within the range of positions 18-23 of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within the range of positions 18-23 of the antisense strand (counting from the 5' end).

[0411] In some embodiments, dsRNA molecules of the disclosure further comprise one phosphorothioate internucleotide linkage modification within the range of positions 1-5 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within the range of positions 18-23 of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within the range of positions 18-23 of the antisense strand.

[0412] In some embodiments, dsRNA molecules of the present disclosure further comprise one phosphorothioate internucleotide linkage modification within the range of positions 1 to 5 (counting from the 5' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within the range of positions 18 to 23 (counting from the 5' end) of the antisense strand.

[0413] In some embodiments, dsRNA molecules of the present disclosure further comprise two phosphorothioate internucleotide linkage modifications within the range of positions 1 to 5 (counting from the 5' end) of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within the range of positions 18 to 23 (counting from the 5' end) of the antisense strand.

[0414] In some embodiments, dsRNA molecules of the disclosure further comprise two phosphorothioate internucleotide linkage modifications within the range of positions 1 to 5 (counting from the 5' end) and one within the range of positions 18 to 23 of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within the range of positions 18 to 23 of the antisense strand.

[0415] In some embodiments, dsRNA molecules of the disclosure further comprise two phosphorothioate internucleotide linkage modifications within the range of positions 1 to 5 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within the range of positions 18 to 23 of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within the range of positions 18 to 23 of the antisense strand (counting from the 5' end).

[0416] In some embodiments, dsRNA molecules of the disclosure further comprise two phosphorothioate internucleotide linkage modifications within the range of positions 1-5 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within the range of positions 18-23 of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within the range of positions 18-23 of the antisense strand.

[0417] In some embodiments, the dsRNA molecules of this disclosure further comprise two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications at positions 20 and 21 of the sense strand, and one phosphorothioate internucleotide linkage modification at position 1 and one at position 21 (counting from the 5' end) of the antisense strand.

[0418] In some embodiments, dsRNA molecules of the disclosure further comprise one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 20 and 21 (counting from the 5' end) of the antisense strand.

[0419] In some embodiments, dsRNA molecules of the disclosure further comprise two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications at positions 21 and 22 of the sense strand, and one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the antisense strand.

[0420] In some embodiments, dsRNA molecules of the disclosure further comprise one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 21 and 22 (counting from the 5' end) of the antisense strand.

[0421] In some embodiments, dsRNA molecules of the disclosure further comprise two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications at positions 22 and 23 of the sense strand, and one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the antisense strand.

[0422] In some embodiments, the dsRNA molecules of this disclosure further comprise one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 23 and 23 (counting from the 5' end) of the antisense strand.

[0423] In some embodiments, the compounds of the present disclosure comprise a pattern of backbone chiral centers. In some embodiments, the regular pattern of backbone chiral centers comprises at least 5 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 6 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 7 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 8 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 9 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 10 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 11 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 12 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 13 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 14 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 15 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 16 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 17 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 18 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 19 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises 8 or fewer internucleotide linkages in the Rp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises 7 or fewer internucleotide linkages in the Rp configuration.In some embodiments, the regular pattern of backbone chiral centers comprises 6 or fewer internucleotide linkages in the Rp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises 5 or fewer internucleotide linkages in the Rp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises 4 or fewer internucleotide linkages in the Rp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises 3 or fewer internucleotide linkages in the Rp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises 2 or fewer internucleotide linkages in the Rp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises 1 or fewer internucleotide linkages in the Rp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises 8 or fewer non-chiral internucleotide linkages (phosphodiesters as a non-limiting example). In some embodiments, the regular pattern of backbone chiral centers comprises 7 or fewer non-chiral internucleotide linkages. In some embodiments, the regular pattern of backbone chiral centers comprises 6 or fewer non-chiral internucleotide linkages. In some embodiments, the regular pattern of backbone chiral centers comprises 5 or fewer non-chiral internucleotide linkages. In some embodiments, the regular pattern of backbone chiral centers comprises 4 or fewer non-chiral internucleotide linkages. In some embodiments, the regular pattern of backbone chiral centers comprises 3 or fewer non-chiral internucleotide linkages. In some embodiments, the regular pattern of backbone chiral centers comprises 2 or fewer non-chiral internucleotide linkages. In some embodiments, the regular pattern of backbone chiral centers comprises 1 or fewer non-chiral internucleotide linkages. In some embodiments, the regular pattern of backbone chiral centers comprises at least 10 internucleotide linkages in the Sp configuration and 8 or fewer non-chiral internucleotide linkages. In some embodiments, the regular pattern of backbone chiral centers comprises at least 11 internucleotide linkages in the Sp configuration and 7 or fewer non-chiral internucleotide linkages. In some embodiments, the regular pattern of backbone chiral centers comprises at least 12 internucleotide linkages in the Sp configuration and 6 or fewer non-chiral internucleotide linkages.In some embodiments, the regular pattern of backbone chiral centers comprises at least 13 internucleotide linkages in the Sp configuration and no more than 6 non-chiral internucleotide linkages. In some embodiments, the regular pattern of backbone chiral centers comprises at least 14 internucleotide linkages in the Sp configuration and no more than 5 non-chiral internucleotide linkages. In some embodiments, the regular pattern of backbone chiral centers comprises at least 15 internucleotide linkages in the Sp configuration and no more than 4 non-chiral internucleotide linkages. In some embodiments, the internucleotide linkages in the Sp configuration may be contiguous or non-contiguous. In some embodiments, the internucleotide linkages in the Rp configuration may be contiguous or non-contiguous. In some embodiments, the non-chiral internucleotide linkages may be contiguous or non-contiguous.

[0424] In some embodiments, compounds of the present disclosure include blocks that are stereochemical blocks. In some embodiments, the blocks are Rp blocks, in that each internucleotide linkage of the block is Rp. In some embodiments, the 5'-block is an Rp block. In some embodiments, the 3'-block is an Rp block. In some embodiments, the blocks are Sp blocks, in that each internucleotide linkage of the block is Sp. In some embodiments, the 5'-block is an Sp block. In some embodiments, the 3'-block is an Sp block. In some embodiments, provided oligonucleotides include both Rp and Sp blocks. In some embodiments, provided oligonucleotides include one or more Rp blocks but no Sp blocks. In some embodiments, provided oligonucleotides include one or more Sp blocks but no Rp blocks. In some embodiments, provided oligonucleotides include one or more PO blocks, where each internucleotide linkage is a natural phosphate linkage.

[0425] In some embodiments, compounds of the present disclosure include a 5'-block that is an Sp block in which each sugar moiety comprises a 2'-F modification. In some embodiments, the 5'-block is an Sp block in which each internucleotide linkage is a modified internucleotide linkage and each sugar moiety comprises a 2'-F modification. In some embodiments, the 5'-block is an Sp block in which each internucleotide linkage is a phosphorothioate linkage and each sugar moiety comprises a 2'-F modification. In some embodiments, the 5'-block comprises four or more nucleoside units. In some embodiments, the 5'-block comprises five or more nucleoside units. In some embodiments, the 5'-block comprises six or more nucleoside units. In some embodiments, the 5'-block comprises seven or more nucleoside units. In some embodiments, the 3'-block is an Sp block in which each sugar moiety comprises a 2'-F modification. In some embodiments, the 3'-block is an Sp block in which each internucleotide linkage is a modified internucleotide linkage and each sugar moiety comprises a 2'-F modification. In some embodiments, the 3'-block is an Sp block, in which each of the internucleotide linkages is a phosphorothioate linkage and each sugar moiety comprises a 2'-F modification. In some embodiments, the 3'-block comprises 4 or more nucleoside units. In some embodiments, the 3'-block comprises 5 or more nucleoside units. In some embodiments, the 3'-block comprises 6 or more nucleoside units. In some embodiments, the 3'-block comprises 7 or more nucleoside units.

[0426] In some embodiments, compounds of the disclosure include a region of nucleosides or oligonucleotides followed by a particular type of internucleotide linkage, such as a natural phosphate linkage, a modified internucleotide linkage, an Rp chiral internucleotide linkage, an Sp chiral internucleotide linkage, etc. In some embodiments, A is followed by Sp. In some embodiments, A is followed by Rp. In some embodiments, A is followed by a natural phosphate linkage (PO). In some embodiments, U is followed by Sp. In some embodiments, U is followed by Rp. In some embodiments, U is followed by a natural phosphate linkage (PO). In some embodiments, C is followed by Sp. In some embodiments, C is followed by Rp. In some embodiments, C is followed by a natural phosphate linkage (PO). In some embodiments, G is followed by Sp. In some embodiments, G is followed by Rp. In some embodiments, G is followed by a natural phosphate linkage (PO). In some embodiments, C and U are followed by Sp. In some embodiments, C and U are followed by Rp. In some embodiments, C and U are followed by a natural phosphate linkage (PO). In some embodiments, A and G are followed by Sp. In some embodiments, A and G are followed by Rp.

[0427] In some embodiments, the antisense strand comprises phosphorothioate internucleotide linkages between nucleotides 21 and 22 and between nucleotides 22 and 23, the antisense strand contains at least one thermally destabilizing modification of the duplex located within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), and the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six, seven, or all eight) of the following features: (i) the antisense strand contains two, three, four, five, or six 2'-fluoro modifications; (ii) the antisense strand comprises three, four, or five phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated to a ligand; (iv) the sense strand comprises two, three, four, or five 2'-fluoro modifications; (v) the sense strand comprises one, two, three, four, or five phosphorothioate internucleotide linkages; (vi) the dsRNA comprises at least four 2'-fluoro modifications; (vii) the dsRNA comprises a double-stranded region 12 to 40 nucleotide pairs in length; and (viii) the dsRNA has a blunt end at the 5' end of the antisense strand.

[0428] In some embodiments, the antisense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23, the antisense strand contains at least one thermally destabilizing modification of the duplex located within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), and the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six, seven, or all eight) of the following features: (i) the antisense strand comprises two, (ii) the sense strand is conjugated to a ligand; (iii) the sense strand contains two, three, four, or five 2'-fluoro modifications; (iv) the sense strand contains one, two, three, four, or five phosphorothioate internucleotide linkages; (v) the dsRNA contains at least four 2'-fluoro modifications; (vi) the dsRNA contains a double-stranded region between 12 and 40 nucleotide pairs in length; (vii) the dsRNA contains a double-stranded region between 12 and 40 nucleotide pairs in length; and (viii) the dsRNA has a blunt end at the 5' end of the antisense strand.

[0429] In some embodiments, the sense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3, and the antisense strand contains at least one thermally destabilizing modification of the duplex located within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), and the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six, seven, or all eight) of the following features: (i) the antisense strand comprises two, three, four, five, or six 2'-fluoro modifications; (ii) the antisense strand comprises one, two, three, four, or five phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated to a ligand; (iv) the sense strand comprises two, three, four, or five 2'-fluoro modifications; (v) the sense strand comprises three, four, or five phosphorothioate internucleotide linkages; (vi) the dsRNA comprises at least four 2'-fluoro modifications; (vii) the dsRNA comprises a double-stranded region 12 to 40 nucleotide pairs in length; and (viii) the dsRNA has a blunt end at the 5' end of the antisense strand.

[0430] In some embodiments, the sense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3, and the antisense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23, the antisense strand containing at least one thermally destabilizing modification of the duplex located within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), and the dsRNA optionally has at least one of the following features (e.g., (e.g., one, two, three, four, five, six, or all seven): (i) the antisense strand comprises two, three, four, five, or six 2'-fluoro modifications; (ii) the sense strand is conjugated to a ligand; (iii) the sense strand comprises two, three, four, or five 2'-fluoro modifications; (iv) the sense strand comprises three, four, or five phosphorothioate internucleotide linkages; (v) the dsRNA comprises at least two 2'-fluoro modifications; (vi) the dsRNA comprises a double-stranded region 12 to 40 nucleotide pairs in length; and (vii) the dsRNA has a blunt end at the 5' end of the antisense strand.

[0431] In some embodiments, the dsRNA molecule of the present disclosure comprises mismatches in the double strand with the target, or a combination thereof. Mismatches can occur in the overhang region or in the double-stranded region. Base pairs can be ranked based on their tendency to promote dissociation or melting (for example, based on the free energy of association or dissociation of a particular pairing; the simplest approach is to examine each pair individually, but then adjacent or similar analysis can also be used). In terms of promoting dissociation, A:U is more preferable than G:C, G:U is more preferable than G:C, and I:C is more preferable than G:C (I=inosine). Mismatches, such as non-standard pairings or non-standard pairings (described elsewhere herein), are more preferable than standard pairings (A:T, A:U, G:C), and pairings that include universal bases are more preferable than standard pairings.

[0432] In some embodiments, the dsRNA molecules of the present disclosure comprise at least one mismatch pair within the duplex region from the 5' end of the antisense strand, which may be independently selected from the group of A:U, G:U, I:C, and a non-canonical or non-standard or universal base pair, for example, a mismatch pair that promotes dissociation of the antisense strand at the 5' end of the duplex.

[0433] In some embodiments, 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 one, two, or three 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.

[0434] It has been found that the introduction of a 4'- or 5'-modified nucleotide at the 3' end of a dinucleotide phosphodiester (PO), phosphorothioate (PS) or phosphorodithioate (PS2) linkage at any position in a single- or double-stranded oligonucleotide can exert a steric effect on the internucleotide linkage, thereby protecting or stabilizing it against nucleases.

[0435] In some embodiments, 5'-modified nucleotide is introduced at the 3' end of dinucleotide at any position in single-stranded or double-stranded siRNA.For example, 5'-alkylated nucleotide can be introduced at the 3' end of dinucleotide at any position in single-stranded or double-stranded siRNA.The alkyl group at the 5' position of ribose sugar can be racemic or chiral pure R or S isomer.An exemplary 5'-alkylated nucleotide is 5'-methyl nucleoside.5'-methyl can be racemic or chiral pure R or S isomer.

[0436] In some embodiments, 4'-modified nucleotides are introduced at the 3' end of dinucleotides at any position in single-stranded or double-stranded siRNA. For example, 4'-alkylated nucleotides can be introduced at the 3' end of dinucleotides at any position in single-stranded or double-stranded siRNA. The alkyl group at the 4' position of ribose sugar can be racemic or chirally pure R- or S-isomer. An exemplary 4'-alkylated nucleotide is 4'-methyl nucleotide. 4'-methyl can be racemic or chirally pure R- or S-isomer. Alternatively, 4'-O-alkylated nucleotides can be introduced at the 3' end of dinucleotides at any position in single-stranded or double-stranded siRNA. The 4'-O-alkyl of ribose sugar can be racemic or chirally pure R- or S-isomer. An exemplary 4'-O-alkylated nucleotide includes 4'-O-methyl nucleotide. The 4'-O-methyl can be either racemic or chirally pure R or S isomers.

[0437] In some embodiments, 5'-alkylated nucleotide is introduced at any position of the sense strand or antisense strand of dsRNA, and this modification maintains or improves the efficacy of dsRNA.5'-alkyl can be either racemic or chiral pure R or S isomer.Exemplary 5'-alkylated nucleotide includes 5'-methyl nucleotide.5'-methyl can be either racemic or chiral pure R or S isomer.

[0438] In some embodiments, 4'-alkylated nucleotide is introduced at any position of the sense strand or antisense strand of dsRNA, and this modification maintains or improves the efficacy of dsRNA.4'-alkyl can be either racemic or chiral pure R or S isomer.Exemplary 4'-alkylated nucleotide is 4'-methyl nucleotide.4'-methyl can be either racemic or chiral pure R or S isomer.

[0439] In some embodiments, 4'-O-alkylated nucleotide is introduced at any position of the sense strand or antisense strand of dsRNA, and this modification maintains or improves the efficacy of dsRNA.5'-Alkyl can be either racemic or chiral pure R or S isomer.Exemplary 4'-O-alkylated nucleotide includes 4'-O-methyl nucleotide.4'-O-methyl can be either racemic or chiral pure R or S isomer.

[0440] In some embodiments, the dsRNA molecules of the present disclosure can comprise 2'-5' linkages (having 2'-H, 2'-OH and 2'-OMe, and also having P=O or P=S). For example, 2'-5' linkage modifications can be used to promote nuclease resistance, or to inhibit the binding of the sense strand to the antisense strand, or can be used at the 5' end of the sense strand to prevent the activation of the sense strand by RISC.

[0441] In another embodiment, the dsRNA molecule of the present disclosure can comprise L sugar (for example, L-ribose, L-arabinose, with 2'-H, 2'-OH and 2'-OMe).For example, these L sugar modifications can be used to promote nuclease resistance, or to inhibit the binding of sense strand to antisense strand, or can be used at the 5' end of sense strand to prevent the activation of sense strand by RISC.

[0442] Multimeric siRNA has been described in various publications, and all of them can be used with the dsRNA of the present disclosure.These publications include WO2007 / 091269, US Patent No. 7858769, WO2010 / 141511, WO2007 / 117686, WO2009 / 014887 and WO2011 / 031520, and each of them is incorporated herein by reference in its entirety.

[0443] As described in more detail below, RNAi agents containing one or more carbohydrate moieties conjugated to them can optimize one or more properties of the RNAi agent. In many cases, the carbohydrate moiety will be 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 so replaced is referred to herein as a ribose-replacement modified subunit (PRMS). The cyclic carrier can be a carbon-cyclic system, i.e., all ring atoms are carbon atoms, or a heterocyclic system, i.e., one or more ring atoms can be heteroatoms, such as nitrogen, oxygen, or sulfur. The cyclic carrier can be a monocyclic ring system or can contain two or more rings, e.g., fused rings. The cyclic carrier can be a fully saturated ring system or can contain one or more double bonds.

[0444] The ligand can be attached to the polynucleotide via a carrier. The carrier comprises (i) at least one "backbone attachment point," e.g., two "backbone attachment points," and (ii) at least one "tethering attachment point." As used herein, "backbone attachment point" refers to a functional group, e.g., a hydroxyl group, or generally a bond that is available and suitable for incorporating the carrier into the backbone of a ribonucleic acid, e.g., a phosphate, or a modified phosphate backbone, e.g., sulfur-containing. In some embodiments, a "tethering attachment point" (TAP) refers to a ring atom, e.g., a carbon atom or heteroatom (other than the atom that provides 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, e.g., amino groups, or generally bonds, that provide a linkage suitable for the incorporation or tethering of another chemical entity, e.g., a ligand, to the constituent ring.

[0445] RNAi agent can be conjugated to ligand through carrier, and said carrier can be cyclic or acyclic group.In one embodiment, 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.In another embodiment, acyclic group is selected from serinol skeleton or diethanolamine skeleton.

[0446] In certain embodiments, the RNAi agent used in the methods of the disclosure is an agent selected from the group of agents listed in any one of Tables 2-3. These agents may further comprise a ligand, such as one or more lipophilic moieties, one or more GalNAc derivatives, or both one or more lipophilic moieties and one or more GalNAc derivatives.

[0447] IV. iRNA conjugated to a ligand Another modification of the iRNA of the invention involves chemically linking the iRNA to one or more ligands, moieties, or conjugates that enhance the activity, cellular distribution, or cellular uptake of the iRNA. Such moieties include, but are not limited to, lipid moieties such as cholesterol moieties (Letsinger et al., Proc. Natl. Acids Sci. USA, 1989, 86:6553-6556), cholic acid (Manoharan et al., Bior. Med. Chem. Let., 1994, 4:1053-1060), thioethers such as beryl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306-309; Manoharan et al., Bior. Med. Chem. Let., 1993, 3:2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), fatty chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), phospholipids such as dihexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), palmityl moieties (Mishra et al., Biochim. Biophys.Acta, 1995, 1264:229-237), or octadecylamine or hexylamino-carbonyloxycholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0448] In certain embodiments, ligand changes the distribution, targeting or life span of the iRNA agent that it is incorporated into.In some embodiments, ligand brings about enhanced affinity to selected target, for example, molecule, cell or cell type, compartment, tissue, organ or region of body, for example, cell or organ compartment, when compared with the species that do not have such ligand.Normal ligand does not participate in double-stranded pairing in double-stranded nucleic acid.

[0449] Ligands can include naturally occurring substances such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL) or globulins), carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid), or lipids. Ligands can also be recombinant or synthetic molecules, such as synthetic polymers, e.g., synthetic polyamino acids. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryl acid), N-isopropylacrylamide polymer, or polyphosphazine. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimeric polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or α-helical peptides.

[0450] The ligand can also include a targeting group, such as a cell or tissue targeting agent, such as a lectin, a glycoprotein, lipid, or protein, such as an antibody, that binds to a specific cell type, such as a kidney cell.The targeting group can be thyroid stimulating hormone, melanocyte stimulating hormone, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine polyvalent mannose, polyvalent fucose, glycosylated polyamino acid, polyvalent galactose, transferrin, bisphosphonate, polyglutamic acid, polyaspartic acid, lipid, cholesterol, steroid, bile acid, folic acid, vitamin B12, biotin, or RGD peptide or RGD peptide mimic.In certain embodiments, the ligand is a polyvalent galactose, such as N-acetyl-galactosamine.

[0451] Other examples of ligands include dyes, intercalating agents (e.g., acridine), crosslinkers (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules, such as cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutanoic acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithophosphate, and the like. Examples of suitable cleavage inhibitors include oleic acid, O3-(oleoyl)cholenoic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bis-imidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

[0452] Ligands can be proteins, e.g., glycoproteins or peptides, e.g., molecules with specific affinity for a co-ligand, or antibodies, e.g., antibodies that bind to specific cell types, such as cancer cells, endothelial cells, or bone cells. Ligands can also include hormones and hormone receptors. They can also include non-peptide species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, multivalent mannose, or multivalent fucose. Ligands can be, for example, lipopolysaccharides, activators of p38 MAP kinase, or activators of NF-κB.

[0453] The ligand can be a substance, e.g., a drug, that can increase the uptake of an iRNA agent into a cell, for example, by disrupting the cytoskeleton of the cell, e.g., by disrupting the cell's microtubules, microfilaments, or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, jasplakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

[0454] In some embodiments, the ligands binding to the iRNAs described herein act as pharmacokinetic modulators (PK modulators). PK modulators include lipophilic substances, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, and the like. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, and the like. Oligonucleotides containing several phosphorothioate linkages have also been shown to bind to serum proteins, and therefore short oligonucleotides containing multiple phosphorothioate linkages in the backbone, e.g., oligonucleotides of about 5, 10, 15, or 20 bases, are also suitable as ligands (e.g., as PK-modulating ligands) for the present invention. Furthermore, aptamers that bind serum components (e.g., serum proteins) are also suitable for use as PK-modulating ligands in the embodiments described herein.

[0455] The ligand-conjugated iRNAs of the invention can be synthesized by using oligonucleotides bearing reactive functional pendant side chains, such as those derived from the attachment of a linking molecule onto the oligonucleotide (described below). The reactive oligonucleotides can be reacted directly with commercially available ligands, synthesized ligands bearing any of a variety of protecting groups, or ligands bearing a linking moiety attached thereto.

[0456] The oligonucleotides used in the conjugates of the present invention can be conveniently and routinely prepared by the well-known technique of solid-phase synthesis. Equipment for such synthesis is sold by several vendors, including, for example, Applied Biosystems® (Foster City, California). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is also known to use similar techniques to prepare other oligonucleotides, such as phosphorothioates and alkylated derivatives.

[0457] In the ligand-conjugated oligonucleotides and sequence-specific linked nucleosides bearing ligand molecules of the present invention, the oligonucleotides and oligonucleosides can be assembled on a suitable DNA synthesizer utilizing standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors already bearing a linking moiety, ligand-nucleotide or ligand-nucleoside conjugate precursors already bearing a ligand molecule, or non-nucleoside ligand-bearing building blocks.

[0458] When using a nucleotide-conjugate precursor that already possesses a linking moiety, typically, synthesis of the sequence-specifically linked nucleoside is completed, and then a ligand molecule is reacted with the linking moiety to form the ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or linked nucleosides of the invention are synthesized by automated synthesizers using phosphoramidites derived from ligand-nucleoside conjugates, in addition to commercially available standard and non-standard phosphoramidites routinely used in oligonucleotide synthesis.

[0459] A. Lipid Conjugates In certain embodiments, the ligand or conjugate is lipid or lipid-based molecule.This lipid or lipid-based molecule can usually bind serum protein, for example, human serum albumin (HSA).HSA-binding ligand allows the distribution of conjugate to target tissue in the body, for example, to target tissue other than the kidney.For example, the target tissue can be the liver, including the parenchymal cells of the liver.Other molecules that can bind HSA can also be used as ligand.For example, naproxen or aspirin can be used.Lipid or lipid-based ligand can (a) increase the resistance of conjugate to degradation, (b) increase targeting or transport into target cell or cell membrane, or (c) be used to adjust the binding to serum protein, for example, HSA.

[0460] Lipid-based ligands can be used to regulate, for example, control (for example, inhibit) the binding of conjugate to target tissue.For example, lipid or lipid-based ligands that bind more strongly to HSA are less likely to target the kidney, and therefore less likely to be eliminated from the body.A lipid or lipid-based ligand that binds less strongly to HSA can be used so that the conjugate targets the kidney.

[0461] In certain embodiments, the lipid-based ligand binds HSA. For example, the ligand can bind to HSA with sufficient affinity, thereby enhancing the distribution of the conjugate to non-renal tissues. However, this affinity is usually not so strong that the HSA-ligand binding is irreversible.

[0462] In certain embodiments, the lipid-based ligand binds weakly or not at all to HSA, thereby enhancing distribution of the conjugate to the kidney. Other moieties that target kidney cells can be used instead of or in addition to the lipid-based ligand.

[0463] In another aspect, the ligand is a moiety, for example, a vitamin, that is taken up by target cells, for example, proliferating cells.These are particularly useful for treating disorders characterized by unwanted cell proliferation, for example, malignant or non-malignant, for example, cancer cells.Exemplary vitamins include vitamins A, E and K.Other exemplary vitamins include vitamin B, for example, folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients that are taken up by cancer cells.Also included are HSA and low-density lipoprotein (LDL).

[0464] B. Cell-penetrating agents In another aspect, the ligand is a cell-penetrating agent, such as a helical cell-penetrating agent. In certain embodiments, the cell-penetrating agent is amphipathic. Exemplary cell-penetrating agents include peptides such as tat or antennapedia. When the cell-penetrating agent is a peptide, it can be modified, including peptidyl mimics, invertomers, non-peptide or pseudopeptide linkages, and the use of D-amino acids. Helical agents are usually α-helical agents, and can have lipophilic and lipophobic phases.

[0465] The ligand can be a peptide or peptidomimetic. Peptidomimetics (also referred to herein as oligopeptidomimetics) are molecules that can fold into defined three-dimensional structures similar to natural peptides. Attachment of peptides and peptidomimetics to iRNA agents can affect the pharmacokinetic distribution of iRNAs, such as by enhancing cellular recognition and uptake. The peptide or peptidomimetic portion can be about 5 to 50 amino acids in length, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

[0466] The peptide or peptidomimetic can be, for example, a cell-penetrating peptide, a cationic peptide, an amphipathic peptide, or a hydrophobic peptide (e.g., composed primarily of Tyr, Trp, or Phe). The peptide moiety can be a dendrimeric peptide, a constrained peptide, or a cross-linked peptide. Alternatively, the peptide moiety can contain a hydrophobic membrane transport sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF, which has the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 11). An RFGF analog containing a hydrophobic MTS (e.g., the amino acid sequence AALLPVLLAAP (SEQ ID NO: 12)) can also be a targeting moiety. The peptide moiety can be a "delivery" peptide, capable of transporting large polar molecules, including peptides, oligonucleotides, and proteins, across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 13)) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 14)) have been shown to function as delivery peptides. Peptides or peptidomimetics can be encoded by random sequences of DNA, such as peptides identified from phage display libraries or one-bead-one-compound (OBOC) combinatorial libraries (Lam et al., Nature, 354:82-84, 1991). Peptides or peptidomimetics that are typically tethered to dsRNA agents via incorporated monomer units include cell-targeting peptides, such as arginine-glycine-aspartic acid (RGD)-peptides or RGD mimetics. The peptide portion can range in length from about 5 amino acids to about 40 amino acids. The peptide portion can have structural modifications, such as those that increase stability or affect conformational properties. Any of the structural modifications described below can be utilized.

[0467] The RGD peptides used in the compositions and methods of the present invention can be linear or cyclic, and can be modified, for example, glycosylated or methylated, to facilitate targeting to specific tissues.RGD-containing peptides and peptidiomimetics can contain D-amino acids and synthetic RGD mimics.In addition to RGD, other moieties can be used to target integrin ligands, such as PECAM-1 or VEGF.

[0468] RGD peptide moieties can be used to target specific cell types, such as tumor cells, e.g., endothelial tumor cells or breast cancer tumor cells (Zitzmann et al., Cancer Res., 62:5139-43, 2002). RGD peptides can promote targeting of dsRNA agents to tumors in various other tissues, including the lung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy 8:783-787, 2001). Typically, RGD peptides promote targeting of iRNA agents to the kidney. RGD peptides can be linear or cyclic, and can be modified to promote targeting to specific tissues, e.g., glycosylated or methylated. For example, glycosylated RGD peptides can be used to target α V iRNA agents can be delivered to tumor cells that express β3 (Haubner et al., Jour. Nucl. Med., 42:326-336, 2001).

[0469] A "cell-penetrating peptide" is capable of penetrating cells, such as microbial cells, e.g., bacterial or fungal cells, or mammalian cells, e.g., human cells. Microbial cell-penetrating peptides can be, for example, α-helical linear peptides (e.g., LL-37 or Ceropin P1), disulfide bond-containing peptides (e.g., α-defensins, β-defensins, or bactenecins), or peptides containing only one or two dominant amino acids (e.g., PR-39 or indolicidin). Cell-penetrating peptides can also contain a nuclear localization signal (NLS). For example, a cell-penetrating peptide can be a bipartite amphipathic peptide, such as MPG, derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

[0470] C. Carbohydrate conjugates In some embodiments of the compositions and methods of the present invention, the iRNA further comprises a carbohydrate. Carbohydrate-conjugated iRNAs are advantageous for in vivo delivery of nucleic acids, as described herein, and are suitable for in vivo therapeutic use. As used herein, "carbohydrate" refers to a compound that is either a carbohydrate itself (which may be linear, branched, or cyclic) composed of one or more monosaccharide units having at least six carbon atoms with an oxygen, nitrogen, or sulfur atom attached to each carbon atom, or a compound having as part thereof a carbohydrate moiety (which may be linear, branched, or cyclic) composed of one or more monosaccharide units, each having at least six carbon atoms with an oxygen, nitrogen, or sulfur atom attached to each carbon atom. Exemplary carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about 4, 5, 6, 7, 8, or 9 monosaccharide units) and polysaccharides, such as starch, glycogen, cellulose, and polysaccharide resins. Particular monosaccharides include C5 and above (e.g., C5, C6, C7 or C8) sugars, and disaccharides and trisaccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7 or C8).

[0471] In certain embodiments, the carbohydrate conjugate comprises a monosaccharide.

[0472] In certain embodiments, the monosaccharide is N-acetylgalactosamine (GalNAc). GalNAc conjugates containing one or more N-acetylgalactosamine (GalNAc) derivatives are described, for example, in U.S. Patent No. 8,106,022, the entire contents of which are incorporated herein by reference. In some embodiments, the GalNAc conjugate functions as a ligand that targets iRNA to specific cells. In some embodiments, the GalNAc conjugate targets iRNA to liver cells, for example, by functioning as a ligand for the asialoglycoprotein receptor of liver cells (e.g., hepatocytes).

[0473] In some embodiments, the carbohydrate conjugate comprises one or more GalNAc derivatives. The GalNAc derivatives can be attached via a linker, e.g., via a bivalent or trivalent branched linker. In some embodiments, the GalNAc conjugate is conjugated to the 3' end of the sense strand. In some embodiments, the GalNAc conjugate is conjugated to the iRNA agent (e.g., to the 3' end of the sense strand) via a linker, e.g., via a linker described herein. In some embodiments, the GalNAc conjugate is conjugated to the 5' end of the sense strand. In some embodiments, the GalNAc conjugate is conjugated to the iRNA agent (e.g., to the 5' end of the sense strand) via a linker, e.g., via a linker described herein.

[0474] In certain embodiments of the invention, GalNAc or GalNAc derivatives are linked to iRNA agents of the invention via a monovalent linker. In some embodiments, GalNAc or GalNAc derivatives are linked to iRNA agents of the invention via a bivalent linker. In yet other embodiments of the invention, GalNAc or GalNAc derivatives are linked to iRNA agents of the invention via a trivalent linker. In other embodiments of the invention, GalNAc or GalNAc derivatives are linked to iRNA agents of the invention via a tetravalent linker.

[0475] In certain embodiments, a double-stranded RNAi agent of the invention comprises one GalNAc or GalNAc derivative attached to an iRNA agent, hi certain embodiments, a double-stranded RNAi agent of the invention comprises multiple (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAc derivatives, each of which is independently attached to multiple nucleotides of the double-stranded RNAi agent via multiple monovalent linkers.

[0476] In some embodiments, for example, when the two strands of an iRNA agent of the invention are part of a larger molecule connected by an uninterrupted chain of nucleotides between the 3' end of one strand and the 5' end of the corresponding other strand, forming a hairpin loop containing multiple unpaired nucleotides, each unpaired nucleotide in the hairpin loop can independently comprise a GalNAc or GalNAc derivative linked via a monovalent linker. This hairpin loop can also be formed by an extended overhang in one of the strands of the duplex.

[0477] In some embodiments, for example, when the two strands of an iRNA agent of the invention are part of a larger molecule connected by an uninterrupted chain of nucleotides between the 3' end of one strand and the 5' end of the corresponding other strand, forming a hairpin loop containing multiple unpaired nucleotides, each unpaired nucleotide in the hairpin loop can independently comprise a GalNAc or GalNAc derivative linked via a monovalent linker. This hairpin loop can also be formed by an extended overhang in one of the strands of the duplex.

[0478] In some embodiments, the GalNAc conjugate is [ka] Formula II is.

[0479] In some embodiments, the RNAi agent is attached to the carbohydrate conjugate via a linker as shown in the following schematic diagram, where X is O or S: [ka]

[0480] In some embodiments, the RNAi agent is conjugated to L96, as defined in Table 1 and shown below. [ka]

[0481] In certain embodiments, the carbohydrate conjugate for use in the compositions and methods of the present invention is selected from the group consisting of: [ka] Formula II, [ka] Formula III, [ka] Formula IV, [ka] Formula V, [ka] Formula VI, [ka] Formula VII, [ka] Formula VIII, [ka] Formula IX, [ka] Formula X, [ka] Formula XI, [ka] Formula XII, [ka] Formula XIII, [ka] Formula XIV, [ka] Formula XV, [ka] Formula XVI, [ka] Formula XVII, [ka] Formula XVIII, [ka] Formula XIX, [ka] Formula XX, [ka] Formula XXI, [ka] Formula XXII, [ka] Formula XXIII, [ka] , wherein Y is O or S and n is 3 to 6 (Formula XXIV), [ka] , wherein Y is O or S and n is 3 to 6 (Formula XXV), [ka] Formula XXVI, [ka] , wherein X is O or S (Formula XXVII), [ka] Formula XXVII, Formula XXIX, [ka] Formula XXX, Formula XXXI, [ka] Formula XXXII, Formula XXXIII, [ka] Formula XXXIV.

[0482] In certain embodiments, the carbohydrate conjugate used in the compositions and methods of the present invention is a monosaccharide. In certain embodiments, the monosaccharide is N-acetylgalactosamine, e.g., [ka] Formula II is.

[0483] Other exemplary carbohydrate conjugates for use in the embodiments described herein include, but are not limited to: [ka] (formula XXXVI),

[0484] When one of X or Y is an oligonucleotide, the other is hydrogen.

[0485] In some embodiments, suitable ligands are those disclosed in WO 2019 / 055633, the entire contents of which are incorporated herein by reference. In one embodiment, the ligand comprises the following structure: [ka]

[0486] In certain embodiments, an RNAi agent of the present disclosure may comprise a GalNAc ligand.

[0487] In certain embodiments of the invention, GalNAc or GalNAc derivatives are linked to iRNA agents of the invention via a monovalent linker. In some embodiments, GalNAc or GalNAc derivatives are linked to iRNA agents of the invention via a bivalent linker. In yet other embodiments of the invention, GalNAc or GalNAc derivatives are linked to iRNA agents of the invention via a trivalent linker. In other embodiments of the invention, GalNAc or GalNAc derivatives are linked to iRNA agents of the invention via a tetravalent linker.

[0488] In certain embodiments, a double-stranded RNAi agent of the invention comprises one GalNAc or GalNAc derivative attached to an iRNA agent, e.g., at the 5' end of the sense strand of a dsRNA agent, or at the 5' end of one or both sense strands of a dual-targeting RNAi agent described herein. In certain embodiments, a double-stranded RNAi agent of the invention comprises multiple (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAc derivatives, each of which is independently attached to multiple nucleotides of the double-stranded RNAi agent via multiple monovalent linkers.

[0489] In some embodiments, for example, when the two strands of an iRNA agent of the invention are part of a single larger molecule connected by an uninterrupted chain of nucleotides between the 3' end of one strand and the corresponding 5' end of the other strand, forming a hairpin loop containing multiple unpaired nucleotides, each unpaired nucleotide in the hairpin loop can independently comprise a GalNAc or GalNAc derivative attached via a monovalent linker.

[0490] In some embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, such as, but not limited to, a PK modulator or a cell-penetrating peptide.

[0491] Further carbohydrate conjugates and linkers suitable for use in the present invention include those described in WO 2014 / 179620 and WO 2014 / 179627, the entire contents of each of which are incorporated herein by reference.

[0492] D. Linker In some embodiments, the conjugates or ligands described herein can be attached to the iRNA oligonucleotide using a variety of linkers, which may or may not be cleavable.

[0493] The term "linker" or "linking group" means an organic moiety that connects two parts of a compound, e.g., covalently bonds the two parts of a compound.Typically, a linker is a direct bond or an atom such as oxygen or sulfur, a unit such as NR, C(O), C(O)NH, SO, SO, SONH, or a chain of atoms, including, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylhetero ...alkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkynylarylalkynyl, alkylheteroarylalkynyl, alkyl alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclyl and the like, wherein one or more methylenes can be interrupted or terminated by O, S, S(O), SO, N(R), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle, where R is hydrogen, acyl, aliphatic, or substituted aliphatic.In certain embodiments, the linker is about 1 to 24 atoms, 2 to 24, 3 to 24, 4 to 24, 5 to 24, 6 to 24, 6 to 18, 7 to 18, 8 to 18 atoms, 7 to 17, 8 to 17, 6 to 16, 7 to 16, or 8 to 16 atoms.

[0494] A cleavable linking group is one that is sufficiently stable outside a cell, but that, once inside a target cell, is cleaved to release the two moieties held together by the linker. In one embodiment, the cleavable linking group is cleaved at a rate that is at least about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or more, or at least about 100-fold faster in the target cell or under first reference conditions (which may, for example, be selected to mimic or represent intracellular conditions) than in the subject's blood or under second reference conditions (which may, for example, be selected to mimic or represent conditions found in blood or serum).

[0495] Cleavable linking groups are susceptible to cleaving agents, such as pH, redox potential, or the presence of degradable molecules. Cleavage agents are generally more widespread or found at higher levels or activity inside cells than in serum or blood. Examples of such degrading agents include redox agents that are selective for a specific substrate or have no substrate specificity, e.g., oxidases or reductases or reducing agents present in cells, such as mercaptans, that can degrade redox-cleavable linking groups by reduction; esterases; reagents that can create an endosomal or acidic environment, e.g., reagents that result in a pH of 5 or less; and enzymes that can hydrolyze or degrade acid-cleavable linking groups by acting as general acids, peptidases (which may be substrate-specific), and phosphatases.

[0496] Cleavable linking groups, such as disulfide bonds, can be pH-sensitive. While the pH of human serum is 7.4, the average intracellular pH is slightly lower, ranging from about 7.1 to 7.3. Endosomes have a more acidic pH, ranging from 5.5 to 6.0, and lysosomes have an even more acidic pH of approximately 5.0. Some linkers have cleavable linking groups that are cleaved at a selected pH, thereby releasing the cationic lipid from the ligand inside the cell or into a desired compartment of the cell.

[0497] Linker can contain a cleavable linking group that can be cleaved by specific enzyme.The type of cleavable linking group incorporated into linker can depend on the target cell.For example, liver targeting ligand can be linked to cationic lipid through a linker that contains ester group.Hepatocytes are rich in esterase, so linker will be cleaved more efficiently in hepatocytes than in cell types that are not rich in esterase.Other cell types that are rich in esterase include lung, renal cortex and testicular cells.

[0498] When targeting cell types rich in peptidases, such as hepatocytes and synoviocytes, linkers containing peptide bonds can be used.

[0499] Generally, the suitability of a candidate cleavable linking group can be evaluated by testing the ability (or conditions) of a degrading agent to cleave the candidate linking group. It may also be desirable to test the candidate cleavable linking group for its ability to resist cleavage in blood or when in contact with other non-target tissues. Thus, the relative susceptibility to cleavage between first and second conditions can be determined, where the first condition is selected to exhibit cleavage in target cells, and the second condition is selected to exhibit cleavage in other tissues or biological fluids, such as blood or serum. Evaluation can be performed in a cell-free system, in cells, in cell culture, in organ or tissue culture, or in whole animals. It may be useful to perform initial evaluation in cell-free or culture conditions and confirm by further evaluation in whole animals. In certain embodiments, useful candidate compounds are cleaved at a rate that is at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0500] i. Redox-cleavable linking group In certain embodiments, the cleavable linking group is a redox-cleavable linking group that is cleaved upon reduction or oxidation. An example of a reductively cleavable linking group is a disulfide linking group (-SS-). To determine whether a candidate cleavable linking group is a suitable "reductively cleavable linking group" or is suitable for use with, for example, a particular iRNA moiety and a particular targeting agent, one can refer to the methods described herein. For example, candidates can be evaluated in cells by incubating with dithiothreitol (DTT) or other reducing agents using reagents known in the art that mimic the cleavage rate that would be observed in target cells. Candidates can also be evaluated under conditions selected to mimic blood or serum conditions. In some conditions, the candidate compound is cleaved at a maximum of about 10% in blood. In other embodiments, useful candidate compounds are degraded at a rate at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of the candidate compound can be determined using standard enzyme kinetic assays under conditions selected to mimic intracellular media and compared to conditions selected to mimic extracellular media.

[0501] ii. Phosphate-based cleavable linkers In certain embodiments, the cleavable linker comprises a phosphate-based cleavable linking group. The phosphate-based cleavable linking group is cleaved by a reagent that decomposes or hydrolyzes the phosphate group. An example of a reagent that cleaves the phosphate group within a cell is an enzyme such as a phosphatase within the cell. Examples of phosphate-based linking groups include -OP(O)(ORk)-O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S-, -OP(S)(Rk)-S, where Rk in each occurrence can independently be C1-C20 alkyl, C1-C20 haloalkyl, C6-C10 aryl, or C7-C12 aralkyl. Exemplary embodiments include -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O-, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O, -SP(S)(H)-O-, -SP(O)(H)-S-, and -OP(S)(H)-S-. In certain embodiments, the phosphate-based linking group is -OP(O)(OH)-O. These candidates can be evaluated using methods similar to those described above.

[0502] iii. Acid-cleavable linking group In certain embodiments, the cleavable linker comprises an acid-cleavable linking group. An acid-cleavable linking group is a linking group that is cleaved under acidic conditions. In other embodiments, the acid-cleavable linking group is cleaved in an acidic environment with a pH of about 6.5 or less (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0 or less), or by an agent, such as an enzyme, that can act as a general acid. Within a cell, certain low-pH organelles, such as endosomes and lysosomes, can provide a cleavage environment for the acid-cleavable linking group. Examples of acid-cleavable linking groups include, but are not limited to, hydrazones, esters, and esters of amino acids. Acid-cleavable groups can have the general formula -C=NN-, C(O)O, or -OC(O). Exemplary embodiments include, when the carbon is bonded to the oxygen of the ester (alkoxy group), an aryl group, a substituted alkyl group, or a tertiary alkyl group, such as dimethylpentyl or t-butyl. These candidates can be evaluated using methods similar to those described above.

[0503] iv. Ester-based cleavable linking groups In certain embodiments, the cleavable linker comprises an ester-based cleavable linking group. Ester-based cleavable linking groups are cleaved in cells by enzymes such as esterases and amidases. Examples of ester-based cleavable linking groups include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. Ester cleavable linking groups have the general formula -C(O)O- or -OC(O)-. These candidates can be evaluated using methods similar to those described above.

[0504] v. Peptide-based cleavable linking groups In yet another embodiment, the cleavable linker comprises a peptide-based cleavable linking group. Peptide-based cleavable linking groups are cleaved intracellularly by enzymes such as peptidases and proteases. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable groups do not include amide groups (—C(O)NH—). Amide groups can be formed between any alkylene, alkenylene, or alkynylene. A peptide bond is a special type of amide bond formed between amino acids to yield peptides and proteins. Peptide-based cleaving groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids to yield peptides and proteins, and do not include all amide functionalities. Peptide-based cleavable linking groups have the general formula —NHCHRAC(O)NHCHRBC(O)—, where R and R are the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

[0505] In some embodiments, the iRNA of the present invention is conjugated to a carbohydrate via a linker. Non-limiting examples of iRNA carbohydrate conjugates with linkers of the compositions and methods of the present invention include, but are not limited to: [ka] (formula XXXVII), [ka] (formula XXXVIII), [ka] (formula XXXIX), [ka] (formula XL), [ka] (formula XLI), [ka] (formula XLII), [ka] (Formula XLIII), and [ka] (Formula XLIV), and when one of X or Y is an oligonucleotide, the other is hydrogen.

[0506] In certain embodiments of the compositions and methods of the present invention, the ligand is one or more "GalNAc" (N-acetylgalactosamine) derivatives attached via a bivalent or trivalent branched linker.

[0507] In certain embodiments, the dsRNA of the invention is conjugated to a bivalent or trivalent branched linker selected from the group of structures shown in any of formulas (XLV)-(XLVI). [ka] During the ceremony, q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B, and q5C each independently represent 0 to 20, and the repeat units may be the same or different; P 2A , P 2B , P 3A , P 3B , P 4A , P 4B , P 5A , P 5B , P 5C , T 2A , T 2B , T 3A , T 3B , T 4A , T 4B , T 4A , T 5B , T 5Cis, independently at each occurrence, absent, CO, NH, O, S, OC(O), NHC(O), CH, CHNH, or CHO; Q 2A , Q 2B , Q 3A , Q 3B , Q 4A , Q 4B , Q 5A , Q 5B , Q 5C is independently at each occurrence absent, alkylene, or substituted alkylene, wherein one or more methylenes are selected from O, S, S(O), SO, N(R N ), C(R')=C(R''), C≡C or C(O), R 2A , R 2B , R 3A , R 3B , R 4A , R 4B , R 5A , R 5B , R 5C are each independently absent, NH, O, S, CH2, C(O)O, C(O)NH, NHCH(R a )C(O), -C(O)-CH(R a )-NH-, CO, CH=NO, [ka] or heterocyclyl, L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B , and L 5C represents a ligand, i.e., each occurrence independently represents a monosaccharide (e.g., GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; and R a is H or an amino acid side chain. Trivalent conjugated GalNAc derivatives are useful in part for use with RNAi agents to inhibit expression of target genes, such as those of formula (XLIX): [ka] In the formula, L 5A , L 5B , and L 5C represents a monosaccharide, such as a GalNAc derivative.

[0508] Examples of bivalent and trivalent branched linker groups for conjugating GalNAc derivatives include, but are not limited to, the structures listed above, such as Formulas II, VII, XI, X, and XIII.

[0509] Representative United States patents that teach the preparation of RNA conjugates include, but are not limited to, U.S. Patent Nos. 4,828,979, 4,948,882, 5,218,105, 5,525,465, 5,541,313, 5,545,730, 5,552,538, 5,578,717, 5,580,731, 5,591,584, 5,109,124, 5,118,802, 5,138,045, 5,414,077, and 5,414,078. No. 7, No. 5,486,603, No. 5,512,439, No. 5,578,718, No. 5,608,046, No. 4,587,044, No. 4,605,735, No. 4,667,025, No. 4,762,779, No. 4,789,73 No. 7, No. 4,824,941, No. 4,835,263, No. 4,876,335, No. 4,904,582, No. 4,958,013, No. 5,082,830, No. 5,112,963, No. 5,214,136, No. 5,082,830 No. 5,112,963, No. 5,214,136, No. 5,245,022, No. 5,254,469, No. 5,258,506, No. 5,262,536, No. 5,272,250, No. 5,292,873, No. 5,317,098 No. 5,371,241, No. 5,391,723, No. 5,416,203, No. 5,451,463, No. 5,510,475, No. 5,512,667, No. 5,514,785, No. 5,565,552, No. 5,567,810 Nos. 5,574,142, 5,585,481, 5,587,371, 5,595,726, 5,597,696, 5,599,923, 5,599,928, 5,688,941, 6,294,664, 6,320,017, 6,576,752, 6,783,931, 6,900,297, 7,037,646, and 8,106,022, the entire contents of each of which are incorporated herein by reference.

[0510] It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the foregoing modifications can be incorporated within a single compound, or even at a single nucleoside within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds.

[0511] A "chimeric" iRNA compound or "chimera," in the context of this invention, is an iRNA compound, e.g., a dsRNA agent, that contains two or more chemically distinct regions, each of which is composed of at least one monomer unit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAs typically contain at least one region in which the RNA is modified to confer on the iRNA increased resistance to nuclease degradation, increased cellular uptake, or increased binding affinity for the target nucleic acid. Additional regions of the iRNA may serve as substrates for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. Thus, activation of RNase H results in cleavage of the RNA target, thereby greatly enhancing the efficiency of iRNA inhibition of gene expression. Consequently, comparable results can often be obtained with shorter iRNA when using chimeric dsRNA compared to the phosphorothioate deoxydsRNA hybridized to the same target region.Cleavage of RNA target can be routinely detected by gel electrophoresis, and if necessary, by related nucleic acid hybridization techniques known in the art.

[0512] In certain instances, the RNA of an iRNA can be modified with a non-ligand group. Several non-ligand molecules have been conjugated to iRNAs to enhance their activity, cellular distribution, or cellular uptake, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties include lipid moieties such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), thioethers such as hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 3:2765), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), fatty chains, e.g., dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), phospholipids, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), polyamines or polyethylene glycol chains (Manoharan et al. al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), palmityl moieties (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative U.S. patents teaching the preparation of such RNA conjugates are listed above. A typical conjugation protocol involves the synthesis of RNA bearing an amino linker at one or more positions in the sequence. The amino group is then reacted with the molecule being conjugated using an appropriate coupling or activating reagent. The conjugation reaction can be performed while the RNA is still attached to the solid support or after cleavage of the RNA in solution phase. Purification of the RNA conjugate by HPLC typically yields a pure conjugate.

[0513] V. Delivery of RNAi Agents of the Present Disclosure Delivery of an RNAi agent of the present disclosure to a cell within a human subject (e.g., a subject in need thereof, e.g., a subject having an SGLT2-related disorder such as gout or diabetes, e.g., a subject having or at risk of developing gout or diabetes, or a subject at risk of having gout or diabetes) can be achieved in many different ways. For example, delivery can be carried out by contacting a cell with an RNAi agent of the present disclosure either in vitro or in vivo. In vivo delivery can be carried out directly by administering to the subject a composition comprising the RNAi agent, e.g., a dsRNA. Alternatively, in vivo delivery can be carried out indirectly by administering one or more vectors that encode and induce expression of the RNAi agent. These options are described further below.

[0514] Generally, any method of delivering nucleic acid molecules (in vitro or in vivo) can be adapted to use the RNAi agent of the present disclosure (see, for example, Akhtar S. and Julian RL., (1992) Trends Cell.Biol.2(5):139-144 and International Publication No. 94 / 02595, which are incorporated herein by reference in their entirety). For in vivo delivery, factors to consider for delivering RNAi agents include, for example, the biological stability of the delivered agent, the prevention of non-specific effects, and the accumulation of the delivered agent in target tissue. The non-specific effects of RNAi agents can be minimized by local administration, for example, by direct injection or implantation into tissue or by local administration of preparations. Local administration to the treatment site maximizes the local concentration of the agent, limiting the exposure of the agent to systemic tissues that may otherwise be harmed by the agent or decompose the agent, and also allows for a smaller total dosage of the RNAi agent to be administered. Some studies have shown that RNAi agents can successfully knock down gene products when administered locally.For example, pulmonary delivery, such as inhalation of dsRNA (e.g., SOD1), has been shown to effectively knock down gene and protein expression in lung tissue, and there is excellent uptake of dsRNA by the bronchioles and alveoli of the lungs.Intraocular delivery of VEGF dsRNA by intravitreal injection in cynomolgus monkeys (Tolentino, MJ. et al., (2004) Retina 24:132-138) and subretinal injection in mice (Reich, SJ. et al. (2003) Mol.Vis.9:210-216) have also shown to prevent neovascularization in experimental models of age-related macular degeneration. In addition, direct intratumoral injection of dsRNA in mice can reduce tumor volume (Pille, J. et al. (2005) Mol. Ther. 11:267-274) and prolong the survival of tumor-bearing mice (Kim, WJ. et al., (2006) Mol. Ther. 14:343-350, Li, S. et al., (2007) Mol. Ther. 15:515-523).RNA interference has been shown to be successful when delivered locally to the CNS by direct injection (Dorn, G. et al., (2004) Nucleic Acids 32:e49; Tan, P. H. et al. (2005) Gene Ther. 12:59-66; Makimura, H. et al. (2002) BMC Neurosci. 3:18; Shishkina, G. T. et al. (2004) Neuroscience 129:521-528; Thakker, E. R. et al. (2004) Proc. Natl. Acad. Sci. USA 101:17270-17275; Akaneya, Y. et al. (2005) J. Neurophysiol. 93:594-602), and to the lungs by intranasal administration (Howard, K. A. et al. (e.g., Zhang, X. et al., (2006) Mol. Ther. 14: 476-484; Zhang, X. et al., (2004) J. Biol. Chem. 279: 10677-10684; Bitko, V. et al., (2005) Nat. Med. 11: 50-55). When RNAi agents are administered systemically to treat disease, the RNA can be modified or alternatively delivered using a drug delivery system, either of which functions to prevent rapid degradation of dsRNA by endonucleases and exonucleases in vivo. Modification of RNA or pharmaceutical carriers can also enable targeting of RNAi agents to target tissues and avoid undesirable off-target effects (for example, without wishing to be bound by theory, the use of GNAs described herein has been determined to destabilize the seed region of dsRNA, thereby increasing the preference for on-target effects over off-target effects of the dsRNA, thereby significantly reducing off-target effects due to the destabilization of the seed region). RNAi agents can be modified by chemical conjugation to lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation.For example, systemic injection of ApoB-directed RNAi agents conjugated to lipophilic cholesterol moieties into mice resulted in knockdown of apoB mRNA in both the liver and jejunum (Soutschek, J. et al., (2004) Nature 432:173-178). Conjugation of RNAi agents to aptamers has been shown to inhibit tumor growth and mediate tumor regression in mouse models of prostate cancer (McNamara, JO. et al., (2006) Nat. Biotechnol. 24:1005-1015). In alternative embodiments, RNAi agents can be delivered using drug delivery systems such as nanoparticles, dendrimers, polymers, liposomes, or cationic delivery systems. Positively charged cationic delivery systems promote the binding of molecular RNAi agents (negatively charged) and also improve interaction with negatively charged cell membranes, thereby enabling efficient uptake of RNAi agents by cells. Cationic lipids, dendrimers, or polymers can be bound to RNAi agents, or can be induced to form vesicles or micelles that encapsulate RNAi agents (see, for example, Kim SH. et al., (2008) Journal of Controlled Release 129(2):107-116). The formation of vesicles or micelles further prevents the degradation of RNAi when administered systemically. The method of creating and administering cationic RNAi agent complexes is well within the capabilities of those skilled in the art (see, for example, Sorensen, DR., et al. (2003) J. Mol. Biol 327:761-766; Verma, UN. et al., (2003) C...

Claims

1. A double-stranded ribonucleic acid (dsRNA) agent or a pharma- ceutical acceptable salt thereof for inhibiting expression of sodium-glucose cotransporter 2 (SGLT2) in a cell, comprising: said dsRNA agent or a pharma- ceutically acceptable salt thereof comprising a sense strand and an antisense strand which form a double-stranded region; one or more lipophilic moieties are conjugated to one or more interior positions on at least one chain; (a) the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence 5'-UCGAAGAGCGCAUUCCACUCGAA-3' of SEQ ID NO: 175; (b) 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:6 by no more than 3 nucleotides; or (c) the antisense strand is selected from the group consisting of the following Tables 1 to 15 【Table 1】 【Table 2】 【Table 3】 【Table 4】 【Table 5】 【Table 6】 【Table 7】 【Table 8】 【Table 9】 【Table 10】 【Table 11】 【Table 12】 【Table 13】 【Table 14】 【Table 15】 comprising at least 15 consecutive nucleotides that differ by 3 nucleotides or less from any one of the antisense nucleotide sequences in any one of A dsRNA agent or a pharma- ceutically acceptable salt thereof.

2. The dsRNA agent or pharma- ceutically acceptable salt thereof of claim 1, wherein one or more lipophilic moieties are conjugated to one or more interior positions on at least one strand via a linker or carrier.

3. The dsRNA agent or pharma- ceutically acceptable salt thereof of claim 1, wherein the dsRNA agent or pharma- ceutically acceptable salt thereof comprises a nucleotide modification.

4. 4. The dsRNA agent of claim 3, or a pharma- ceutically acceptable salt thereof, wherein every nucleotide of the sense strand and every nucleotide of the antisense strand comprises a nucleotide modification.

5. At least one of the nucleotide modifications is a deoxy-nucleotide modification, a 3'-terminal deoxy-thymidine (dT) nucleotide modification, a 2'-O-methyl nucleotide modification, a 2'-fluoro nucleotide modification, a 2'-deoxy-nucleotide modification, a 2'-5'-linked ribonucleotide (3'-RNA) modification, a locked nucleotide modification, an unlocked nucleotide modification, a conformationally restricted nucleotide modification, a constrained ethyl nucleotide modification, an abasic nucleotide modification, a 2'-amino-nucleotide modification, a 2'-O-allyl-nucleotide modification, a 2'-C-alkyl-nucleotide modification, a 2'-methoxyethyl nucleotide modification, a 2'-O-alkyl-nucleotide modification, a morpholino nucleotide modification, a phosphoramidate modification, a nucleotide modification comprising a non-natural base, a tetrahydrofuran nucleotide modification ...

4. The dsRNA agent of claim 3, or a pharma- ceutically acceptable salt thereof, selected from the group consisting of pyran nucleotide modifications, 1,5-anhydrohexitol nucleotide modifications, cyclohexenyl nucleotide modifications, nucleotide modifications comprising 5' phosphate or 5' phosphate mimics, nucleotide modifications comprising vinyl phosphonates, glycol nucleic acid (GNA) modifications, glycol nucleic acid S isomer (S-GNA) modifications, nucleotide modifications comprising 2-hydroxymethyl-tetrahydrofuran-5-phosphate, nucleotide modifications comprising 2'-deoxythymidine-3' phosphate, nucleotide modifications comprising 2'-deoxyguanosine-3'-phosphate, and terminal nucleotide modifications linked to cholesteryl derivatives and dodecanoic acid bisdecylamide groups, and combinations thereof.

6. 2. The dsRNA agent of claim 1, or a pharma- ceutically acceptable salt thereof, further comprising at least one phosphorothioate internucleotide linkage.

7. 7. The dsRNA agent of claim 6, or a pharma- ceutically acceptable salt thereof, wherein said dsRNA agent comprises 6-8 phosphorothioate internucleotide linkages.

8. 2. The dsRNA agent of claim 1, or a pharma- ceutically acceptable salt thereof, wherein each strand is 30 nucleotides or less in length.

9. 2. The dsRNA agent of claim 1, or a pharma- ceutically acceptable salt thereof, wherein at least one strand comprises a 3' overhang of at least 1 nucleotide or at least 2 nucleotides.

10. 2. The dsRNA agent of claim 1, or a pharma- ceutically acceptable salt thereof, wherein said double-stranded region is 15 to 30 nucleotide pairs in length.

11. 2. The dsRNA agent of claim 1, or a pharma- ceutically acceptable salt thereof, wherein each strand is 19-30 nucleotides in length.

12. 2. The dsRNA agent of claim 1, or a pharma- ceutically acceptable salt thereof, wherein said one or more lipophilic moieties are conjugated to one or more of internal positions selected from the group consisting of positions 4-8 and 13-18 on the sense strand and positions 6-10 and 15-18 on the antisense strand, counting from the 5' end of each strand.

13. 2. The dsRNA agent of claim 1, or a pharma- ceutically acceptable salt thereof, wherein said lipophilic moiety is an aliphatic, alicyclic, or polyalicyclic compound.

14. 14. The dsRNA agent of claim 13, or a pharma- ceutically acceptable salt thereof, wherein said lipophilic moiety contains a saturated or unsaturated C4-C30 hydrocarbon chain and any functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne.

15. 15. The dsRNA agent of claim 14, or a pharma- ceutically acceptable salt thereof, wherein said lipophilic moiety contains a saturated or unsaturated C6-C18 hydrocarbon chain.

16. 15. The dsRNA agent of claim 14, or a pharma- ceutically acceptable salt thereof, wherein said lipophilic moiety contains a saturated or unsaturated C16 hydrocarbon chain.

17. 17. The dsRNA agent of claim 16, or a pharma- ceutically acceptable salt thereof, wherein the saturated or unsaturated C16 hydrocarbon chain is conjugated to position 6, counting from the 5' end of the sense strand.

18. 2. The dsRNA agent of claim 1, or a pharma- ceutically acceptable salt thereof, wherein said lipophilic moiety is conjugated via a carrier that replaces one or more nucleotides at an internal position or in said double-stranded region.

19. 2. The dsRNA agent of claim 1, or a pharma- ceutically acceptable salt thereof, wherein the lipophilic moiety is conjugated via a linker that contains an ether, a thioether, a urea, a carbonate, an amine, an amide, a maleimide-thioether, a disulfide, a phosphodiester, a sulfonamide linkage, a product of a click reaction, or a carbamate.

20. 2. The dsRNA agent of claim 1, or a pharma- ceutically acceptable salt thereof, wherein said lipophilic moiety is conjugated to a nucleobase, a sugar moiety, or an internucleoside linkage.

21. 2. The dsRNA agent of claim 1, or a pharma- ceutically acceptable salt thereof, wherein said lipophilic moiety is conjugated via a biocleavable linker selected from the group consisting of DNA, RNA, disulfides, amides, and functionalized mono- or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.

22. 2. The dsRNA agent of claim 1, or a pharma- ceutically acceptable salt thereof, wherein the 3' end of the sense strand is protected via an end cap that is a cyclic group having an amine, the cyclic group being selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl.

23. 2. The dsRNA agent of claim 1, or a pharma- ceutically acceptable salt thereof, further comprising a phosphate or a phosphate mimetic at the 5'-end of the antisense strand.

24. 24. The dsRNA agent of claim 23, or a pharma- ceutically acceptable salt thereof, wherein said phosphate mimetic is a 5'-vinylphosphonate (VP).

25. 13. A cell comprising the dsRNA agent of claim 1 or a pharma- ceutically acceptable salt thereof.

26. 10. A pharmaceutical composition for inhibiting expression of the SGLT2 gene, comprising the dsRNA agent of claim 1 or a pharma- ceutically acceptable salt thereof.

27. 1. An in vitro method for inhibiting expression of the SGLT2 gene in a cell, the method comprising: (a) contacting said cell with the dsRNA agent of claim 1 or a pharma- ceutically acceptable salt thereof, or the pharmaceutical composition of claim 26; (b) maintaining the cells produced in step (a) for a time sufficient to obtain degradation of the SGLT2 gene, thereby inhibiting expression of the SGLT2 gene in the cells.

28. 28. The method of claim 27, wherein the expression of the SGLT2 gene is inhibited by at least 50%, 60%, 70%, 80%, 90%, or 95%.

29. A pharmaceutical composition for treating a subject having a disorder that would benefit from reduced expression of sodium-glucose cotransporter 2 (SGLT2), comprising a therapeutically effective amount of the dsRNA agent of claim 1 or a pharma- ceutically acceptable salt thereof.

30. A pharmaceutical composition for preventing at least one symptom in a subject having a disorder that would benefit from reduced expression of sodium-glucose cotransporter 2 (SGLT2), comprising a prophylactically effective amount of the dsRNA agent of claim 1 or a pharma- ceutically acceptable salt thereof.

31. 31. The pharmaceutical composition of claim 29 or 30, wherein the disorder is an SGLT2-associated disorder.

32. 32. The pharmaceutical composition of claim 31, wherein the SGLT2-related disorder is gout.

33. 32. The pharmaceutical composition of claim 31, wherein the SGLT2-related disease is diabetes.

34. The pharmaceutical composition of claim 31 , wherein the SGLT2-related disease is type II diabetes.

35. 31. The pharmaceutical composition of claim 29 or 30, wherein the subject is a human.

36. 31. The pharmaceutical composition of claim 29 or 30, wherein the pharmaceutical composition is for pulmonary or subcutaneous administration.

37. 31. The pharmaceutical composition of claim 29 or 30, further comprising administering to the subject an additional drug or therapy suitable for the treatment or prevention of an SGLT2-related disorder.

38. 38. The pharmaceutical composition of claim 37, wherein the additional therapeutic agent is selected from the group consisting of an agent for treating diabetes mellitus, an agent for treating diabetic complications, an agent for treating cardiovascular disease, an antihyperlipidemic agent, an antihypertensive or antihypertensive agent, an antiobesity agent, an agent for treating nonalcoholic steatohepatitis (NASH), a chemotherapeutic agent, an immunotherapeutic agent, an immunosuppressant, an anti-inflammatory agent, an antisteatosis agent, an antiviral agent, an antifibrotic agent, an immunomodulatory agent, a tyrosine kinase inhibitor, an antifibrotic agent, and any combination of the foregoing.

39. 27. A kit, vial or syringe comprising the dsRNA agent of claim 1 or a pharma- ceutically acceptable salt thereof or the pharmaceutical composition of claim 26.

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