Universal non-targeting siRNA compositions and methods of use thereof

Abstract

The present invention provides compositions, systems, and methods for modulating protein expression using an iRNA composition that achieves the cleavage of an RNA-induced silence complex (RISC)-mediated universal RNAi target sequence, and a REVERSIR compound that eliminates the activity of such an iRNA composition.

Description

[0001] Related Applications

[0002] This application claims the benefit of priority of U.S. Provisional Application No. 63 / 398,894, filed Aug. 18, 2022, the entire content of which is incorporated herein by reference.

[0003] Background

[0004] Adeno-associated virus (AAV) vectors have emerged as the dominant platform for most in vivo gene therapy applications, enabling the potential for several years after a single dose in disease treatment options that, if not life-long. 1 However, a key challenge that has emerged in recent systemic AAV-mediated gene therapy clinical trials is the wide inter-individual variability in therapeutic protein expression at the same vector dose, which can lead to phenotoxicity at supra-physiological transgene levels in some cases. 41,42 This, combined with the difficulty of extrapolating a therapeutically effective dose range in humans from preclinical data for AAV gene therapy modalities, underscores the need for clinically translatable methods to regulate transgene expression after AAV administration. 2-3,43 The ability to fine-tune transgene dose to a targeted therapeutic range or abrogate expression in the event of adverse events represents an important feature that can maximize the safety and utility of AAV-based therapies.

[0005] RNA interference (RNAi) is an evolutionarily conserved mechanism in which endogenous (microRNA) or exogenous (siRNA, shRNA) short non-coding RNAs downregulate gene expression of mRNA transcripts in a sequence-dependent manner. 4 As a natural pathway that harnesses an efficient cellular catalytic mechanism, RNAi can be used to achieve robust, persistent, and specific silencing of gene transcripts of interest. In recent years, several RNAi-based drugs have been successfully validated in clinical studies, demonstrating benefits at low doses and dosing frequencies (e.g., up to 6 months) compared to alternative gene silencing strategies. 44 Novel delivery regimens, along with highly chemically modified siRNAs, have improved potency, persistence, and safety, and have thus greatly expanded the reach of RNAi therapies, culminating in four approved drugs and several others in clinical development. 11-13 In the liver, occasional delivery of metabolically stable siRNAs conjugated to N-acetylgalactosamine (GalNAc) results in potent gene silencing that persists for several months in humans, with favorable safety and tolerability profiles. 14-16 Recent work has also expanded the scope of siRNA delivery to extrahepatic tissues, with conjugation of 2'-O-palmitoyl (C16) demonstrating broad distribution across cell types and persistent gene silencing in the central nervous system, eye, and lung. 17All of these advancements in the therapeutic silencing of endogenous disease-related genes using RNAi also maintain the potential for the on-demand regulation of exogenously delivered transgenes in a therapeutic setting.

[0006] Given the small footprint of RNAi elements, binding sites for fully or partially complementary interfering RNAs (typically 19 - 23 nucleotides) can be readily integrated into the viral genome in the 3'UTR of the vector-encoded transgene. AAV integration of binding sites for endogenous microRNAs has been exploited to improve the tissue specificity of gene targeting by selectively attenuating expression in undesired cell types. 24-26 Previously designed RNAi-based switches have fully utilized ligand binding to control the processing of delivered engineered interfering RNAs along with therapeutic transgenes, or to modulate the accessibility of endogenous microRNAs to their cognate binding sites on virus-delivered mRNAs. 5-10 However, their applicability has been hampered by the limitations imposed by endogenous microRNA expression levels, off-target related risks, and the lack of non-protein ligands or generalizable aptamers. 5 Conversely, exogenously provided chemically modified siRNAs overcome the dependence on endogenous microRNAs and provide precise and flexible dose control. As an alternative to the repeated but infrequent administration of siRNAs, RNAi via shRNAs stably introduced into AAV vectors in a gene therapy setting allows for the continuous regulation of cis-expressed transgenes as a single treatment.

[0007] While exogenous RNAi modalities can enable low basal expression of transgenes, the versatility of these systems would be greatly enhanced by the ability to reverse transgene silencing as a means of controlling therapeutic transgene expression in the on state. The use of short, synthetic single-stranded oligonucleotides (termed REVERSIR) has recently been reported. 21Highly potent and generalizable methods for in vivo control of RNAi pharmacology. REVERSIR acts by serving as a synthetic high-affinity decoy to compete with siRNA target mRNAs, thereby sequestering RNA-induced silencing complexes (RISCs) loaded with complementary siRNA antisense (guide) strands to functionally abrogate RNAi activity. REVERSIR stably binds to the seed region of the antisense strand, preventing RISC-mediated recognition and degradation of target mRNA transcripts and thus enhancing their translation. Through modular design and generalizable templates of length and chemical modifications, REVERSIR has been shown to effectively reverse in vivo gene silencing across several targets via multiple siRNA sequences. The development of REVERSIR as an RNAi activity antidote represents a valuable tool that can be co-opted to regulate the on-state of exogenously delivered transcripts by enabling transgenic expression from RNAi-regulated AAV vectors.

[0008] Accordingly, there is a need in the art for a composition, system, and method that combines RNAi-mediated knockdown and REVERSIR-driven rescue of gene silencing as a molecular rheostat or switch for AAV-delivered transcripts. Summary of the Invention

[0009] The present invention provides compositions, systems, and methods for using iRNA compositions and REVERSIR compounds for regulating protein expression, wherein the iRNA compositions effect cleavage of a common RNAi target sequence mediated by RNA-induced silencing complexes (RISCs), and the REVERSIR compounds abrogate the activity of such iRNA compositions. The present invention also provides controlled methods for fine-tuning the amount and timing of expression of a therapeutic transgene.

[0010] The common iRNAs of the present invention are designed to have favorable thermodynamic properties for RISC loading and RNAi functionality, as well as having little to no sequence complementarity to any annotated genes in the human, cynomolgus monkey, rat, and mouse transcriptomes. Such common iRNAs have proven to be potent RNAi triggers with high target specificity and minimal off-target gene disruption propensity. Additionally, and as described herein, these common dsRNA agents are shown to regulate expression in exogenous vector delivery systems without causing unwanted off-target silencing within the endogenous transcriptomes of human and mammalian preclinical models. Thus, the use of these common iRNAs, REVERSIR molecules that abrogate the activity of these common iRNAs, and systems comprising these common iRNAs and / or REVERSIR molecules allows for refinement of the transgene dose and induction timing of exogenous vector delivery systems (e.g., AAV vector delivery systems), thereby providing in vivo gene therapy methods that achieve long-term correction of gene deficiencies across a wide range of target organs following a single administration.

[0011] Thus, in one aspect, the present invention provides a general double-stranded ribonucleic acid (dsRNA) agent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22 or 23 consecutive nucleotides, and wherein no more than 3, e.g., 3, 2, 1 or 0 nucleotides are different from any of the antisense strand nucleotide sequences in Table 2.

[0012] In another aspect, the present invention provides a general double-stranded ribonucleic acid (dsRNA) agent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides, and wherein no more than 3, e.g., 3, 2, 1 or 0 nucleotides are different from any of the sense strand nucleotide sequences in Table 2, and wherein the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22 or 23 consecutive nucleotides, and wherein no more than 3, e.g., 3, 2, 1 or 0 nucleotides are different from any of the antisense strand nucleotide sequences in Table 2.

[0013] In one aspect, the present invention provides a general double-stranded ribonucleic acid (dsRNA) agent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a region complementary to any of the target nucleotide sequences in Table 2 or 3.

[0014] In one embodiment, the dsRNA agent comprises at least one modified nucleotide.

[0015] In one embodiment, substantially all of the nucleotides of the sense strand are modified nucleotides; substantially all of the nucleotides of the antisense strand are modified nucleotides; or substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides.

[0016] In one embodiment, all of the nucleotides of the sense strand are modified nucleotides; all of the nucleotides of the antisense strand are modified nucleotides; or all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides.

[0017] In one embodiment, at least one modified nucleotide is selected from the group consisting of: deoxynucleotides, 3'-terminal deoxythymidine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, unlocked nucleotides, conformationally restricted nucleotides, constrained ethyl nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-alkyl modified nucleotides, 2'-hydroxy modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides containing unnatural bases, tetrahydropyran modified nucleotides, 1,5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides containing phosphorothioate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphates, nucleotides containing 5'-phosphate mimics, thermally labile nucleotides, glycol modified nucleotides (GNA), nucleotides containing 2'-phosphates, and 2-O-(N-methylacetamide) modified nucleotides; and combinations thereof.

[0018] In another embodiment, the modification on the modified nucleotide is selected from the group consisting of: LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2'-hydroxy, and glycol; and combinations thereof.

[0019] In yet another embodiment, at least one modified nucleotide is selected from the group consisting of: deoxynucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, glycol modified nucleotides (GNA), nucleotides containing 2'-phosphates, and vinylphosphonate nucleotides; and combinations thereof.

[0020] In one embodiment, at least one modification on the modified nucleotide is a thermally labile nucleotide modification.

[0021] In one embodiment, the thermally labile nucleotide modification is selected from the group consisting of: abasic modification; mismatch with a relative nucleotide in a duplex; and labile sugar modification, 2'-deoxy modification, acyclic nucleotides, unlocked nucleic acids (UNA), and glycerol nucleic acids (GNA).

[0022] The length of the double-stranded region can be 19-30 nucleotide pairs; the length can be 19-25 nucleotide pairs; the length can be 19-23 nucleotide pairs; the length can be 23-27 nucleotide pairs; or the length can be 21-23 nucleotide pairs.

[0023] In one embodiment, the length of each strand is independently no more than 30 nucleotides.

[0024] In one embodiment, the sense strand has a length of 21 nucleotides and the antisense strand has a length of 23 nucleotides.

[0025] In one embodiment, the complementary region has a length of at least 17 nucleotides.

[0026] In one embodiment, at least one strand comprises a 3' overhang of at least 1 nucleotide.

[0027] In another embodiment, at least one strand comprises a 3' overhang of at least 2 nucleotides.

[0028] In one embodiment, the universal dsRNA agent further comprises a ligand.

[0029] In one embodiment, the ligand is conjugated to the 3' end of the sense strand of the dsRNA agent.

[0030] In one embodiment, the ligand is an N-acetylgalactosamine (GalNAc) derivative.

[0031] In one embodiment, the ligand is one or more GalNAc derivatives attached via a monovalent, divalent or trivalent branched linker.

[0032] In one embodiment, the ligand is

[0033]

[0034] In one embodiment, the dsRNA agent is conjugated to the ligand according to the following scheme

[0035]

[0036] and wherein X is O or S.

[0037] In one embodiment, X is O.

[0038] In one embodiment, wherein the dsRNA agent further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.

[0039] In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is located at the 3' end of one strand.

[0040] In one embodiment, the strand is the antisense strand.

[0041] In another embodiment, the strand is the sense strand.

[0042] In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is located at the 5' end of one strand.

[0043] In one embodiment, the strand is an antisense strand.

[0044] In another embodiment, the strand is a sense strand.

[0045] In one embodiment, phosphorothioate or methylphosphonate internucleotide linkages are located at both the 5'- and 3'-ends of a strand.

[0046] In one embodiment, the strand is an antisense strand.

[0047] In one embodiment, the base pair at position 1 of the 5'-end of the antisense strand of the duplex is an AU base pair.

[0048] The present invention further provides cells comprising the universal dsRNA agent of the present invention, and vectors comprising the universal dsRNA agent of the present invention.

[0049] In one embodiment, the vector is an expression vector.

[0050] In one embodiment, the vector is a viral vector.

[0051] In one embodiment, the viral vector is an adeno-associated (AAV) vector.

[0052] In one embodiment, the viral vector is a bicistronic vector.

[0053] In one embodiment, the vector of the present invention further comprises a transgene, such as a transgene.

[0054] The present invention also provides cells comprising the vector of the present invention.

[0055] The present invention further provides a pharmaceutical composition comprising the universal dsRNA agent of the present invention or the vector of the present invention and a pharmaceutically acceptable carrier.

[0056] In one embodiment, the dsRNA agent or vector is in a buffer-free solution.

[0057] In one embodiment, the buffer-free solution is saline or water.

[0058] In another embodiment, the dsRNA agent is in a buffer solution.

[0059] In one embodiment, the buffer solution comprises acetate, citrate, prolamine, carbonate or phosphate or any combination thereof.

[0060] In another embodiment, the buffer solution is phosphate buffered saline (PBS).

[0061] In one aspect, the present invention provides REVERSIR compounds that abolish the iRNA activity of the general dsRNA agents of the present invention.

[0062] In another aspect, the present invention provides REVERSIR compounds comprising a single-stranded oligonucleotide having a length of 6 to 30 nucleotides and comprising a nucleotide sequence that is at least about 90% complementary to any of the antisense strand nucleotide sequences in Table 2 or Table 3.

[0063] In one embodiment, the oligonucleotide is 100% complementary to any of the antisense strand nucleotide sequences in Table 2 or Table 3.

[0064] In one embodiment, the oligonucleotide comprises at least one modified nucleotide.

[0065] In one embodiment, substantially all nucleotides of the oligonucleotide are modified nucleotides.

[0066] In one embodiment, all nucleotides of the oligonucleotide are modified nucleotides.

[0067] In one embodiment, at least one modified nucleotide comprises a modified nucleobase.

[0068] In one embodiment, the modified nucleobase is 5'-methylcytosine.

[0069] In one embodiment, at least one modified nucleotide comprises a modified sugar.

[0070] In one embodiment, the modified sugar is selected from the group consisting of 2'-O-methoxyethyl-modified sugar, 2'-methoxy-modified sugar, 2'-O-alkyl-modified sugar, and bicyclic sugar.

[0071] In one embodiment, the oligonucleotide further comprises a ligand.

[0072] In one embodiment, the ligand is conjugated to the 3'-end of the oligonucleotide.

[0073] In one embodiment, the ligand is an N-acetylgalactosamine (GalNAc) derivative.

[0074] In one embodiment, the ligand is one or more GalNAc derivatives attached via a monovalent, divalent, or trivalent branched linker.

[0075] In one embodiment, the ligand is

[0076]

[0077] In one embodiment, the oligonucleotide is conjugated to the ligand according to the following scheme

[0078]

[0079] And, wherein X is O or S.

[0080] In one embodiment, X is O.

[0081] In one embodiment, the oligonucleotide further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.

[0082] In one embodiment, the oligonucleotide has a length of 6 - 15, 7 - 11 or 8 - 10 nucleotides.

[0083] In another embodiment, the oligonucleotide has a length of 15 - 25, 17 - 25, 19 - 25 or 21 - 25 nucleotides.

[0084] The present invention further provides a cell comprising the REVERSIR compound of the present invention.

[0085] In one aspect, the present invention provides a system for expressing a transgene on demand. The system includes an expression vector encoding a transgene and comprising a universal iRNA target site; a universal double-stranded ribonucleic acid (dsRNA) agent, which comprises a sense strand and an antisense strand forming a double-stranded region, the double-stranded region recognizing and binding to the universal iRNA target site so as to inhibit the expression of the transgene; and optionally, a REVERSIR compound, which eliminates the iRNA activity of the universal dsRNA agent so as to allow the expression of the transgene.

[0086] The universal iRNA target site may be located in the 5'-untranslated region or 3'-untranslated region (UTR) of the transgene.

[0087] In one embodiment, the universal dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises at least 15, such as 15, 16, 17, 18, 19, 20, 21, 22 or 23 consecutive nucleotides, and no more than 3, such as 3, 2, 1 or 0 nucleotides are different from any of the antisense strand nucleotide sequences in Table 2 or Table 3.

[0088] In one embodiment, the universal dsRNA agent comprises a sense strand and an antisense strand that form a double-stranded region, wherein the sense strand comprises at least 15, such as 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides, wherein no more than 3, such as 3, 2, 1 or 0 nucleotides are different from any of the sense strand nucleotide sequences in Table 2; and the antisense strand comprises at least 15, such as 15, 16, 17, 18, 19, 20, 21, 22 or 23 consecutive nucleotides, wherein no more than 3, such as 3, 2, 1 or 0 nucleotides are different from any of the antisense strand nucleotide sequences in any of Table 2 or Table 3.

[0089] In one embodiment, the universal dsRNA agent comprises a sense strand and an antisense strand that form a double-stranded region, wherein the antisense strand comprises a region complementary to any of the target nucleotide sequences in any of Table 3 or Table 4.

[0090] In one embodiment, the universal dsRNA agent comprises at least one modified nucleotide.

[0091] In one embodiment, substantially all of the nucleotides of the sense strand are modified nucleotides; substantially all of the nucleotides of the antisense strand are modified nucleotides; or substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides.

[0092] In one embodiment, all of the nucleotides of the sense strand are modified nucleotides; all of the nucleotides of the antisense strand are modified nucleotides; or all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides.

[0093] In one embodiment, at least one modified nucleotide is selected from the group consisting of: deoxynucleotides, 3'-terminal deoxythymidine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, unlocked nucleotides, conformationally restricted nucleotides, constrained ethyl nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-alkyl modified nucleotides, 2'-hydroxy modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides containing unnatural bases, tetrahydropyran modified nucleotides, 1,5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides containing phosphorothioate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphates, nucleotides containing 5'-phosphate mimics, thermally labile nucleotides, ethylene glycol modified nucleotides (GNA), nucleotides containing 2'-phosphates, and 2-O-(N-methylacetamide) modified nucleotides; and combinations thereof.

[0094] In another embodiment, the modification on the nucleotide is selected from the group consisting of: LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2'-hydroxy, and ethylene glycol; and combinations thereof.

[0095] In another embodiment, at least one modified nucleotide is selected from the group consisting of: deoxynucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, ethylene glycol modified nucleotides (GNA), nucleotides containing 2'-phosphate, and vinyl phosphonate nucleotides; and combinations thereof.

[0096] In another embodiment, at least one modification on the modified nucleotide is a thermally labile nucleotide modification.

[0097] In one embodiment, the thermally labile nucleotide modification is selected from the group consisting of: abasic modification; mismatch with a relative nucleotide in a duplex; and labile sugar modification; 2'-deoxy modification; acyclic nucleotides; unlocked nucleic acids (UNA), and glycerol nucleic acids (GNA).

[0098] The length of the double-stranded region can be 19 - 30 nucleotide pairs; the length can be 19 - 25 nucleotide pairs; the length can be 19 - 23 nucleotide pairs; the length can be 23 - 27 nucleotide pairs; or the length can be 21 - 23 nucleotide pairs.

[0099] In one embodiment, the length of each strand is independently no more than 30 nucleotides.

[0100] In one embodiment, the length of the sense strand is 21 nucleotides and the length of the antisense strand is 23 nucleotides.

[0101] In one embodiment, the length of the complementary region is at least 17 nucleotides.

[0102] In one embodiment, at least one strand contains a 3'-overhang of at least 1 nucleotide.

[0103] In another embodiment, at least one strand contains a 3'-overhang of at least 2 nucleotides.

[0104] In one embodiment, the universal dsRNA agent further comprises a ligand.

[0105] In one embodiment, the ligand is conjugated to the 3'-end of the sense strand of the universal dsRNA agent.

[0106] In one embodiment, the ligand is an N-acetylgalactosamine (GalNAc) derivative.

[0107] In one embodiment, the ligand is one or more GalNAc derivatives attached via a monovalent, divalent, or trivalent branched linker.

[0108] In one embodiment, the ligand is

[0109]

[0110] In one embodiment, the universal dsRNA agent is conjugated to the ligand according to the following scheme,

[0111]

[0112] and wherein X is O or S.

[0113] In one embodiment, X is O.

[0114] In one embodiment, the universal dsRNA agent further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.

[0115] In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is located at the 3'-end of one strand.

[0116] In one embodiment, the strand is the antisense strand.

[0117] In another embodiment, the strand is the sense strand.

[0118] In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is located at the 5'-end of one strand.

[0119] In one embodiment, the strand is the antisense strand.

[0120] In another embodiment, the strand is the sense strand.

[0121] In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is located at both the 5'- and 3'-ends of one strand.

[0122] In one embodiment, the strand is the antisense strand.

[0123] In one embodiment, the base pair at position 1 of the 5'-end of the antisense strand of the duplex is an AU base pair.

[0124] In another aspect, the present invention provides a system for expressing a transgene on demand. The system includes an expression vector encoding the transgene and a double-stranded ribonucleic acid (dsRNA) agent targeting the transgene; wherein the expression of the transgene is inhibited by expressing the dsRNA agent targeting the transgene; and optionally, a REVERSIR compound that eliminates the iRNA activity of the dsRNA agent, thereby allowing the expression of the transgene.

[0125] In one embodiment, the REVERSIR compound comprises a single-stranded oligonucleotide having a length of 6 - 30, such as 6 - 25, 6 - 20, 8 - 25, 8 - 20, 10 - 25, 10 - 20, 12 - 25, 15 - 25, 17 - 25, 19 - 25, 7 - 23, 19 - 23, such as 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, and a nucleotide sequence comprising at least about 90% complementarity to any of the antisense strand nucleotide sequences in Table 2 or Table 3.

[0126] In one embodiment, the oligonucleotide is 100% complementary to any of the antisense strand nucleotide sequences in Table 2 or Table 3.

[0127] In one embodiment, the oligonucleotide comprises at least one modified nucleotide.

[0128] In one embodiment, substantially all nucleotides of the oligonucleotide are modified nucleotides.

[0129] In one embodiment, all nucleotides of the oligonucleotide are modified nucleotides.

[0130] In one embodiment, at least one modified nucleotide comprises a modified nucleobase.

[0131] In one embodiment, the modified nucleobase is 5'-methylcytosine.

[0132] In one embodiment, at least one modified nucleotide comprises a modified sugar.

[0133] In one embodiment, the modified sugar is selected from the group consisting of: 2'-O-methoxyethyl modified sugar, 2'-methoxy modified sugar, 2'-O-alkyl modified sugar, and bicyclic sugar.

[0134] In one embodiment, the REVERSIR compound comprises a ligand.

[0135] In one embodiment, the ligand is coupled to the 3'-end of the oligonucleotide.

[0136] In one embodiment, the ligand is an N-acetylgalactosamine (GalNAc) derivative.

[0137] In one embodiment, the ligand is one or more GalNAc derivatives attached via a monovalent, divalent or trivalent branched linker.

[0138] In one embodiment, the ligand is

[0139]

[0140] In one embodiment, the oligonucleotide is conjugated to the ligand according to the following scheme

[0141]

[0142] and wherein X is O or S.

[0143] In one embodiment, X is O.

[0144] In one embodiment, the oligonucleotide further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.

[0145] In one embodiment, the oligonucleotide has a length of 6-15, 7-11 or 8-10 nucleotides.

[0146] In one embodiment, the oligonucleotide has a length of 15-25, 17-25, 19-25 or 21-25 nucleotides.

[0147] In one embodiment, the expression vector is a viral vector.

[0148] In one embodiment, the viral vector is an adeno-associated (AAV) vector.

[0149] In one embodiment, the viral vector is a bicistronic vector.

[0150] In one aspect, the present invention provides a method of modulating the expression of a transgene in a cell, the method comprising: contacting the cell with an expression vector encoding the transgene and comprising a universal iRNA target site; contacting the cell with a universal double-stranded ribonucleic acid (dsRNA) agent that recognizes and binds to the universal iRNA target site, thereby inhibiting the expression of the transgene; and optionally, further contacting the cell with a REVERSIR compound that abolishes the iRNA activity of the universal dsRNA agent thereby allowing the expression of the transgene.

[0151] In one embodiment, the cell is located within a subject.

[0152] In another aspect, the present invention provides a method of treating a subject in need thereof. The method comprises: contacting an expression vector encoding a therapeutic transgene and comprising a universal iRNA target site administered to the subject with a universal double-stranded ribonucleic acid (dsRNA) agent that recognizes and binds to the universal iRNA target site thereby inhibiting the expression of the transgene, thereby treating the subject.

[0153] In one embodiment, the universal dsRNA agent is further contacted with a REVERSIR compound that eliminates the iRNA activity of the universal dsRNA agent, thereby allowing the expression of the transgene.

[0154] In one aspect, the present invention provides a method of treating a subject in need thereof. The method comprises: contacting an expression vector encoding a therapeutic transgene administered to the subject and a double-stranded ribonucleic acid (dsRNA) agent targeting the transgene with a REVERSIR compound that eliminates the iRNA activity of the dsRNA agent thereby allowing the expression of the transgene, thereby treating the subject.

[0155] In another aspect, the present invention provides a method of treating a subject in need thereof. The method comprises: administering to the subject an expression vector encoding a transgene and comprising a universal iRNA target site; allowing the expression of the transgene until a desired expression level has been reached; and once the desired transgene expression level has been reached, administering to the subject a universal double-stranded ribonucleic acid (dsRNA) agent comprising a sense strand and an antisense strand that form a double-stranded region that recognizes and binds to the universal iRNA target site thereby inhibiting the expression of the transgene, thereby treating the subject.

[0156] In one embodiment, the method further comprises: once the level of the transgene has dropped below the desired expression level, administering to the subject a REVERSIR compound, wherein the REVERSIR compound eliminates the iRNA activity of the universal dsRNA agent, thereby allowing the expression of the transgene.

[0157] In one embodiment, the universal iRNA target site is located in the 5'-untranslated region (UTR) or the 3'-untranslated region (UTR) of the transgene.

[0158] In one embodiment, the universal dsRNA agent comprises a sense strand and an antisense strand that form a double-stranded region, wherein the antisense strand comprises at least 15, such as 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides, wherein no more than 3, such as 3, 2, 1, or 0 nucleotides are different from any of the antisense strand nucleotide sequences in any of Tables 2 or 3.

[0159] In one embodiment, the universal dsRNA agent comprises a sense strand and an antisense strand that form a double-stranded region, wherein the sense strand comprises at least 15, such as 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides, wherein no more than 3, such as 3, 2, 1 or 0 nucleotides are different from any of the sense strand nucleotide sequences in Table 2; and the antisense strand comprises at least 15, such as 15, 16, 17, 18, 19, 20, 21, 22 or 23 consecutive nucleotides, wherein no more than 3, such as 3, 2, 1 or 0 nucleotides are different from any of the antisense strand nucleotide sequences in any of Table 2 or Table 3.

[0160] In one embodiment, the universal dsRNA agent comprises a sense strand and an antisense strand that form a double-stranded region, wherein the antisense strand comprises a region complementary to any of the target nucleotide sequences in any of Table 3 or Table 4.

[0161] In one embodiment, the universal dsRNA agent comprises at least one modified nucleotide.

[0162] In one embodiment, substantially all of the nucleotides of the sense strand are modified nucleotides; substantially all of the nucleotides of the antisense strand are modified nucleotides; or substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides.

[0163] In another embodiment, all of the nucleotides of the sense strand are modified nucleotides; all of the nucleotides of the antisense strand are modified nucleotides; or all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides.

[0164] In one embodiment, at least one modified nucleotide is selected from the group consisting of: deoxynucleotides, 3'-terminal deoxythymidine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, unlocked nucleotides, conformationally restricted nucleotides, constrained ethyl nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-alkyl modified nucleotides, 2'-hydroxy modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides containing unnatural bases, tetrahydropyran modified nucleotides, 1,5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides containing phosphorothioate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphates, nucleotides containing 5'-phosphate mimics, thermolabile nucleotides, glycol modified nucleotides (GNA), nucleotides containing 2'-phosphates, and 2-O-(N-methylacetamide) modified nucleotides; and combinations thereof.

[0165] In another embodiment, the modification on the nucleotide is selected from the group consisting of: LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2'-hydroxy, and ethylene glycol; and combinations thereof.

[0166] In one embodiment, at least one modified nucleotide is selected from the group consisting of: deoxynucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, ethylene glycol modified nucleotides (GNA), nucleotides containing 2'-phosphate, and vinyl phosphonate nucleotides; and combinations thereof.

[0167] In another embodiment, at least one modification on the modified nucleotide is a thermally labile nucleotide modification.

[0168] In one embodiment, the thermally labile nucleotide modification is selected from the group consisting of: abasic modification; mismatch with a relative nucleotide in a duplex; and labile sugar modification, 2'-deoxy modification, acyclic nucleotides, unlocked nucleic acids (UNA), and glycerol nucleic acids (GNA).

[0169] The length of the double-stranded region can be 19-30 nucleotide pairs; the length can be 19-25 nucleotide pairs; the length can be 19-23 nucleotide pairs; the length can be 23-27 nucleotide pairs; or the length can be 21-23 nucleotide pairs.

[0170] In one embodiment, the length of each strand is independently no more than 30 nucleotides.

[0171] In one embodiment, the length of the sense strand is 21 nucleotides and the length of the antisense strand is 23 nucleotides.

[0172] In one embodiment, the length of the complementary region is at least 17 nucleotides.

[0173] In one embodiment, at least one strand contains a 3' overhang of at least 1 nucleotide.

[0174] In another embodiment, at least one strand contains a 3' overhang of at least 2 nucleotides.

[0175] In one embodiment, the universal dsRNA agent further comprises a ligand.

[0176] In one embodiment, the ligand is conjugated to the 3' end of the sense strand of the universal dsRNA agent.

[0177] In one embodiment, the ligand is an N-acetylgalactosamine (GalNAc) derivative.

[0178] In one embodiment, the ligand is one or more GalNAc derivatives attached via a monovalent, divalent or trivalent branching linker.

[0179] In one embodiment, the ligand is

[0180]

[0181] In one embodiment, the universal dsRNA agent is conjugated to the ligand according to the following scheme

[0182]

[0183] And wherein X is O or S.

[0184] In one embodiment, X is O.

[0185] In one embodiment, the universal dsRNA agent further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.

[0186] In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is located at the 3'-end of one strand.

[0187] In one embodiment, the strand is the antisense strand.

[0188] In another embodiment, the strand is the sense strand.

[0189] In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is located at the 5'-end of one strand.

[0190] In one embodiment, the strand is the antisense strand.

[0191] In one embodiment, the strand is the sense strand.

[0192] In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is located at both the 5'- and 3'-ends of one strand.

[0193] In one embodiment, the strand is the antisense strand.

[0194] In one embodiment, the base pair at position 1 of the 5'-end of the antisense strand of the duplex is an AU base pair.

[0195] In one embodiment, the REVERSIR compound comprises a single-stranded oligonucleotide acid having a length of 6 to 30, such as 6 to 25, 6 to 20, 8 to 25, 8 to 20, 10 to 25, 10 to 20, 12 to 25, 15 to 25, 17 to 25, 19 to 25, 7 to 23, 19 to 23, such as 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, and a nucleotide sequence comprising at least about 90% complementarity to any one of the antisense strand nucleotide sequences in any one of Table 2 or Table 3.

[0196] In one embodiment, the oligonucleotide is 100% complementary to any one of the antisense strand nucleotide sequences in any one of Table 2 or Table 3.

[0197] In one embodiment, the oligonucleotide comprises at least one modified nucleotide.

[0198] In one embodiment, substantially all nucleotides of the oligonucleotide are modified nucleotides.

[0199] In another embodiment, all nucleotides of the oligonucleotide are modified nucleotides.

[0200] In one embodiment, at least one modified nucleotide comprises a modified nucleobase.

[0201] In one embodiment, the modified nucleobase is 5'-methylcytosine.

[0202] In one embodiment, at least one modified nucleotide comprises a modified sugar.

[0203] In one embodiment, the modified sugar is selected from the group consisting of: 2'-O-methoxyethyl modified sugar, 2'-methoxy modified sugar, 2'-O-alkyl modified sugar, and bicyclic sugar.

[0204] In one embodiment, the REVERSIR compound comprises a ligand.

[0205] In one embodiment, the ligand is coupled to the 3'-end of the oligonucleotide.

[0206] In one embodiment, the ligand is an N-acetylgalactosamine (GalNAc) derivative.

[0207] In one embodiment, the ligand is one or more GalNAc derivatives attached via a monovalent, divalent or trivalent branched linker.

[0208] In one embodiment, the ligand is

[0209]

[0210] In one embodiment, the oligonucleotide is conjugated to a ligand according to the following scheme

[0211]

[0212] and wherein X is O or S.

[0213] In one embodiment, X is O.

[0214] In one embodiment, the oligonucleotide further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.

[0215] In one embodiment, the oligonucleotide is 6-15, 7-11 or 8-10 nucleotides in length.

[0216] In one embodiment, the oligonucleotide is 15-25, 17-25, 19-25 or 21-25 nucleotides in length.

[0217] In one embodiment, the expression vector is an adeno-associated (AAV) vector.

[0218] In one embodiment, the viral vector is a bicistronic vector.

[0219] BRIEF DESCRIPTION OF THE DRAWINGS

[0220] Figures 1A - 1I Depicts restoration of transgene expression from an shRNA-regulated self-silencing AAV vector using REVERSIR.

[0221] Figure 1A Is a schematic illustration that shows an AAV switch for transgene silencing using an intron-encoded shRNA and transgene induction using REVERSIR. The shRNA is expressed from a chimeric intron upstream of the viral transgene cassette. After intracellular processing of the shRNA, the RISC-loaded antisense strand binds to complementary target sites within the rAAV 3'UTR, resulting in sequential cleavage and degradation of the AAV-delivered transgene mRNA. Exogenously delivered REVERSIR blocks RISC activity, relieves the inhibition of transgene mRNA and induces protein expression.

[0222] Figure 1B Is a schematic of the viral genome of an ssAAV8 self-silencing GLuc reporter vector. The vector expresses an intron-encoded miR-33-embedded TTR shRNA and contains homologous (TTR-ts) or scrambled (NT-ts) target sites in the 3'UTR of the GLuc transgene.

[0223] Figure 1CIt is a graph depicting the time course of in vitro shRNA-mediated transgene silencing. HepG2 cells were transfected with the indicated AAV plasmids, and cell culture medium was collected at each time point to quantify the secreted GLuc levels. The medium was completely replaced at each collection, and each line corresponds to the GLuc accumulated in a single well since the previous time point.

[0224] Figure 1D It is a graph that depicts the validation of transgene self-inhibition with an AAV construct containing intronic miR-33 and induction using REVERSIR in HepG2 cells. 20 ng of total DNA (which consisted of the GLuc AAV construct described in 1B and the FLuc internal control plasmid at a 5:1 ratio) was co-transfected with the indicated concentrations of full-length TTR-REVERSIR or a chemically matched non-targeting (NT) control. 48 hours after transfection, GLuc and FLuc intensities were measured in the cell culture supernatant and lysate, respectively, and the GLuc / FLuc ratio was calculated to normalize transfection efficiency.

[0225] Figure 1E It is a graph that depicts the secreted GLuc levels measured in serum collected on the indicated days before and after treatment with equimolar doses of 9-mer (0.1 mg / kg) or 22-mer (0.2 mg / kg) TTR or NT REVERSIR on D0. Mice were injected with 2 X 10 11 genomic copies (GC) of shTTR miR-33 encoding the GLuc reporter miR-33 / TTR-ts or control shTTR

[0226] Figure 1F It is a graph that depicts a qRT-PCR analysis of GLuc transcript levels in liver tissue at the terminal day 47 time point, plotted relative to the endogenous Gapdh control and relative to the shTTR / NT-ts condition set as 100%.

[0227] Figure 1G It is a graph that depicts the longitudinal quantification of serum GLuc levels in mice administered 0.1 mg / kg or 0.3 mg / kg of 9-mer tunable TTR REVERSIR (TTR REVERSIR 2) or NT REVERSIR on D0, followed by a second administration on D47.

[0228] Figure 1His a graph depicting serum EPO concentration measured by ELISA in mice treated with 0.1 mg / kg of 9-mer TTR or NT REVERSIR at D0 at the indicated time points. Mice were injected with 2x 10 11 GC of AAV8 virus, which encodes murine EPO transgene under the control of TTR shRNA with intact (TTR-ts) or non-targeting (NT-ts) binding sites in the 3'UTR.

[0229] Figure 1I is a graph depicting serum EPO concentration at the indicated time points in mice treated at D0 with increasing doses of tunable TTR REVERSIR (0.01, 0.03, 0.1 or 0.3 mg / kg of TTR REVERSIR 2) relative to 0.1 mg / kg of NT REVERSIR.

[0230] Figure 2A -G depicts in vivo regulation of an AAV-delivered reporter transgene by exogenous delivery of siRNA and homologous REVERSIR.

[0231] Figure 2A is a schematic depicting the exogenous siRNA approach for AAV transgene regulation. siRNA administration promotes inactivation or attenuation of AAV gene expression by RNAi-mediated degradation of viral transcripts containing the target site within the 3’UTR. Sequence-specific elimination of siRNA activity using REVERSIR results in derepression of viral mRNA transcripts and consequent increase in therapeutic protein expression.

[0232] Figure 2B is a schematic of an ssAAV serotype 8 vector carrying a bicistronic expression cassette encoding PMP-22 and GLuc reporter genes. The fully complementary binding site of TTR siRNA was inserted directly adjacent to the stop codon within the 3'UTR (left). Six-week-old female C57BL / 6 mice were injected intravenously with 2x10 10 genome copies (GC) of AAV. Two weeks after AAV administration, mice were injected subcutaneously (SC) with either vehicle or 9 mg / kg of TTR siRNA (D0). Two weeks later, a single molar equidose of full-length 22-mer (3 mg / kg) or 9-mer TTR REVERSIR (1.6 mg / kg) was injected and compared with vehicle or length-matched NT REVERSIR as a control (D14). Blood was collected as indicated and terminal liver tissue was harvested at D42 (right).

[0233] Figure 2Cis a graph depicting the quantification of serum GLuc levels at specified time points normalized to the pre-dose for each animal.

[0234] Figure 2D is a graph depicting the qRT-PCR analysis of GLuc transcript levels in terminal liver tissue at D42, normalized to Gapdh control and plotted relative to the PBS condition set as 100%.

[0235] Figure 2E is a graph depicting the serum GLuc levels in mice at D21 that were transduced with AAV as shown in (2B) of 2X10 11 GC and treated with TTR siRNA (9 mg / kg; D0), followed by various doses of 9-mer TTR REVERSIR or a high dose of NT REVERSIR alone as a control (D14).

[0236] Figure 2F depicts a schematic of an AAV vector for evaluating shRNA-based regulation of the AAV-hANGPTL3 transgene (left) and a graph of plasma hANGPTL3 protein concentration evaluated by ELISA over the specified time course (right). C57BL / 6 mice have received intravenous administration of 1.5x 10 11 GC of an AAV8 vector carrying the human ANGPTL3 coding region (with a target site for GLuc siRNA within the 3'UTR). Two weeks later, the mice were treated with 9 mg / kg GLuc siRNA for 14 days and then administered 1.5 mg / kg 9-mer GLuc REVERSIR or NT REVERSIR. Blood was drawn at the indicated time points.

[0237] Figure 2G depicts a schematic of an AAV vector for siRNA-mediated regulation of the human factor XII (hF12)-GLuc transgene (left) and a graph of serum GLuc intensity measured at the specified time points and plotted relative to pretreatment with siRNA (right). Mice were injected with 2x10 11 GC of a bicistronic vector encoding hF12 and GLuc (with a TTR siRNA binding site in the 3'UTR). The mice were administered 9 mg / kg TTR siRNA, and then two weeks later were given 156 mg / kg TTRREVERSIR or NT REVERSIR, with blood drawn as indicated.

[0238] Figures 3A - 3D depicts an in vitro characterization of the on-target and off-target activities of transgene regulator siRNA sequences.

[0239] Figure 3AIs a graph that depicts the on-target silencing potency (solid black line) of three major transgenic regulator siRNA sequences measured by co-transfecting serial titration doses of siRNA with a dual luciferase sensor containing perfectly matched binding sites. Seed-mediated off-target repression was similarly evaluated by the dose-response activity of siRNA in the presence of luciferase reporters containing target sites with 1 (medium gray dashed line) or 4 tandem (light gray dashed line) seed matches. The RLuc / FLuc ratio was normalized to a mock transfection control (no siRNA) condition set to 100% and plotted as the mean ± SEM of 3 - 6 replicates.

[0240] Figure 3B Is a Bland - Altman plot (MA plot) that depicts differential gene expression analysis of RNA-seq data obtained from transfected transgenic regulator siRNA in Hep3B cells (top; 10 nM dose, harvested at 24 h) and primary mouse hepatocytes (bottom; 50 nM dose, harvested at 48 h). Points represent individual transcripts, the average normalized read counts across replicates, and the log 2 Fold change relative to the mock transfection control. "Black" points represent genes with significantly differential expression (FDR < 0.05) but no canonical seed-matching sites (8-mer, 7-mer-m8, and 7-mer-A1) within their 3'UTR. "Dark gray" points represent genes that contain canonical 3'UTR seed-matching binding sites but are not significantly differentially expressed. (N = 4 technical replicates).

[0241] Figure 3C Is a table that shows differential gene expression analysis of in vitro RNAseq data from transfected transgenic regulator siRNA in mouse (primary mouse hepatocytes; top) and human (Hep3B; bottom) hepatocytes.

[0242] Figure 3D Is a graph that depicts serum alanine aminotransferase (ALT) and glutamate dehydrogenase (GLDH) in rats at necropsy (D16) that received 3 once-weekly injections (qw x 3) of the indicated transgenic regulator siRNA (TR-siRNA) at 30 or 100 mg / kg doses. N = 4 male rats per group (6 - 8 weeks old); qw is once-weekly dosing.

[0243] Figures 4A - 4E Depicts additional in vitro and in vivo analyses of an AAV regulatory switch that supports the full utilization of intron-expressed shRNA and REVERSIR.

[0244] Figure 4ASchematic of the marker construct and in vitro evaluation chart depicting reversal of miRNA-mediated shRNA REVERSIR-mediated target silencing in a dual-luciferase reporter assay. Cos7 cells were co-transfected for 48 hours with a luciferase reporter plasmid and a GFP-tagged construct expressing miR-30E-embedded TTR or NT shRNA, along with increasing concentrations of 22-mer TTR or a matching NT REVERSIR.

[0245] Figure 4B Schematic of the marker construct and in vitro evaluation chart depicting reversal of miRNA-mediated shRNA REVERSIR-mediated target silencing in a dual-luciferase reporter assay. Cos7 cells were co-transfected for 48 hours with a luciferase reporter plasmid and a GFP-tagged construct expressing miR-33-embedded TTR or NT shRNA, along with increasing concentrations of 22-mer TTR or a matching NT REVERSIR.

[0246] Figure 4C Chart showing validation of GLuc transgene inhibition using a self-silencing AAV construct containing intronic miR-30E-shRNA in HepG2 cells and subsequent induction using increasing doses of REVERSIR. The AAV construct was co-transfected with an FLuc control plasmid for normalization at a 5:1 molar ratio. GLuc and FLuc intensities were measured in cell culture supernatants and lysates, respectively, and the GLuc / FLuc ratio was expressed as a percentage relative to the AAV plasmid expressing shNT.

[0247] Figure 4D Chart depicting quantification of GLuc mRNA levels by qRT-PCR in HepG2 cells 48 h after transfection with a self-silencing AAV plasmid containing miR-33 and REVERSIR. GLuc transcript levels were normalized to FLucmRNA used as an internal control.

[0248] Figure 4E Chart showing miR-33 successful in vivo knockdown of endogenous TTR protein levels by intronic expression of shTTR, and subsequent restoration to baseline by exogenous administration of TTR REVERSIR but not NT REVERSIR.

[0249] Figures 5A - 5F Depicts additional in vitro and in vivo analyses of an AAV regulatory switch that enables the full utilization of exogenous siRNA and REVERSIR.

[0250] Figures 5A - 5C Chart depicting asFigure 2B – Data from individual animals or additional groups tested as part of the study shown in –D. AAV injection, test article administration times, and blood collections were performed as Figure 2B described. PBS and 9 mg / kg siRNA conditions were the same as those shown in the main figure.

[0251] Figures 5A - 5F is a graph that depicts data from individual animals or additional groups tested as part of the study shown in Figure 2B –D. AAV injection, test article administration times, and blood collections were performed as Figure 2B described. PBS and 9 mg / kg siRNA conditions were the same as those shown in the main figure.

[0252] Figure 5A is a graph that depicts the continuous dose-dependent knockdown of serum GLuc levels in AAV-injected mice treated with 1, 3, and 9 mg / kg TTR siRNA relative to a PBS control.

[0253] Figure 5B is a graph that depicts longitudinal measurements of serum GLuc levels in AAV-injected mice treated with 3 mg / kg TTR siRNA, followed by vehicle or a 1 mg / kg dose of the indicated REVERSIR (left). Figure 2C The mean data in are presented as a spaghetti graph that plots serum GLuc over time relative to pre-dose for each individual animal treated with 9 mg / kg TTR siRNA followed by 3 mg / kg of the indicated REVERSIR molecule (right).

[0254] Figure 5C is a graph depicting positive controls that demonstrate the expected silencing of endogenous TTR mRNA with 9 mg / kg TTR siRNA and complete reversal of knockdown with 3 mg / kg 22-mer and 9-mer TTR REVERSIR (but not the corresponding NT REVERSIR).

[0255] Figure 5D is a graph that depicts on-target silencing activity of GLuc siRNA in a dual luciferase reporter system (left). Normalized luciferase activity 48 h after co-transfection of Cos7 cells with 10 nM GLuc siRNA and increasing doses of 22-mer or 9-mer GLuc REVERSIR (right).

[0256] Figure 5E is a spaghetti graph that shows the responses of individual animals averaged by condition to generate Figure 2FThe graphs shown in

[0257] Figure 5F is a spaghetti plot that shows the responses of individual animals averaged by condition to generate Figure 2G the graphs shown in

[0258] Figures 6A - 6B depicts the lack of seed-mediated off-target effects from transgenic regulators siRNA.

[0259] Figure 6A depicts a cumulative distribution function (CDF) plot that shows transcriptional changes 24 h after transfection of transgenic regulator siRNA at 10 nM in Hep3B. Each line represents the cumulative distribution of expression changes between target genes with a designated seed match (8mer, 7mer-m8, and 7mer-A1) to the antisense (top) or sense (bottom) strand of the siRNA within the 3′UTR relative to genes without such a canonical seed match site (background). The black line represents background genes lacking the designated seed match, while the different gray lines represent genes with at least one seed match, segmented by the strength of the binding site (dark gray = 8mer, medium gray = 7mer-m8, light gray = m7mer-A1). The Δ value reflects the magnitude of the CDF change relative to the background. N = 4 technical replicates.

[0260] Figure 6B depicts a cumulative distribution function (CDF) plot that shows transcriptional changes 48 h after transfection of transgenic regulator siRNA at 50 nM in primary mouse hepatocytes. Each line represents the cumulative distribution of expression changes between target genes with a designated seed match (8mer, 7mer-m8, and 7mer-A1) to the antisense (top) or sense (bottom) strand of the siRNA within the 3′UTR relative to genes without such a canonical seed match site (background). The black line represents background genes lacking the designated seed match, while the different gray lines represent genes with at least one seed match, segmented by the strength of the binding site (dark gray = 8mer, medium gray = 7mer-m8, light gray = m7mer-A1). The Δ value reflects the magnitude of the CDF change relative to the background. N = 4 technical replicates.

[0261] Figure 7It is a graph that depicts the lack of elevation of liver function tests (LFTs) in a rat toxicity study of a transgenic regulator siRNA. In particular, the graph depicts the serum levels of aspartate aminotransferase (AST), albumin (ALB), alkaline phosphatase (ALP), and total protein (TP) at necropsy (D16) in rats that received 3 once-weekly injections (qw x 3) of the indicated transgenic regulator siRNA (TR-siRNA) at 30 or 100 mg / kg doses. N = 4 male rats per group (6 - 8 weeks old); qw is once-weekly dosing. DETAILED DESCRIPTION OF THE INVENTION

[0263] The present invention provides compositions, systems, and methods for regulating protein expression using iRNA compositions and REVERSIR compounds, wherein the iRNA compositions effect cleavage of a generally RNAi target sequence mRNA mediated by an RNA-induced silencing complex (RISC), and the REVERSIR compounds abrogate the activity of such iRNA compositions.

[0264] The general iRNAs of the present invention are designed to have favorable thermodynamic properties for RISC loading and RNAi functionality, as well as having little to no sequence complementarity to any annotated genes in the human, cynomolgus monkey, rat, and mouse transcriptomes. Such general iRNAs have proven to be potent RNAi triggers with high target specificity and minimal off-target gene disruption propensity. Additionally, and as described herein, these general dsRNA agents show regulation of expression in exogenous vector delivery systems without causing unwanted off-target silencing within the endogenous transcriptomes of human and mammalian preclinical models. Thus, use of these general iRNAs, REVERSIR molecules that abrogate the activity of these general iRNAs, and systems comprising these general iRNAs and / or REVERSIR molecules allows refinement of the transgenic dose and induction time of exogenous vector delivery systems (e.g., AAV vector delivery systems), thereby providing in vivo gene therapy methods that achieve long-term gene defect correction across a wide range of target organs following a single administration.

[0265] The iRNA of the present invention comprises an RNA strand (antisense strand) having a region up to about 30 nucleotides in length or shorter, for example, 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 nucleotides in length, the region of which is substantially complementary to at least a portion of the mRNA transcript of a universal target.

[0266] In certain embodiments, one or both strands of the double-stranded RNAi agent of the present invention are up to 66 nucleotides in length, for example, 36-66, 26-36, 25-36, 31-60, 22-43, 27-53 nucleotides in length, having a region of at least 19 contiguous nucleotides that is substantially complementary to at least a portion of the mRNA transcript of a universal target. In some embodiments, such iRNA agents having a longer length antisense strand may, for example, include a second RNA strand (sense strand) that is 20-60 nucleotides in length, wherein the sense strand and the antisense strand form a duplex of 18-30 contiguous nucleotides.

[0267] The following detailed description discloses how to prepare and use compositions comprising iRNA to inhibit the expression of a universal target sequence, REVERSIR compounds that abrogate the activity of such iRNA, and systems, uses and methods for treating a subject in need thereof.

[0268] I. Definitions

[0269] To make the present invention more readily understood, certain terms are first defined. In addition, it should be noted that whenever a value or range of values of a parameter is recited, it is intended that the recited values and intermediate ranges of values are also intended to be part of the present invention.

[0270] As used herein, the articles "a", "an" and "the" refer to one or more than one (i.e., to at least one) of the grammatical objects of the article. For example, "an element" refers to one element or more than one element, e.g., a plurality of elements.

[0271] As used herein, the term "comprising" means the phrase "comprising but not limited to" and may be used interchangeably with the phrase "comprising but not limited to".

[0272] As used herein, the term "or" means the term "and / or" and may be used interchangeably with the term "and / or" unless the context clearly indicates otherwise. For example, "sense strand or antisense strand" is understood to mean "sense strand or antisense strand or sense strand and antisense strand".

[0273] As used herein, the term "about" means within the typical tolerances in the art. For example, "about" can be understood to be about 2 standard deviations from the mean. In certain embodiments, about means ±10%. In certain embodiments, about means ±5%. When "about" appears before a series of numbers or a range, it is understood that "about" can modify each number in the series or range.

[0274] The terms "at least", "not less than", or "or more" before a number or a series of numbers are understood to include the number immediately following the term "at least", and all subsequent numbers or integers that are logically included as is clear from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 19 nucleotides of a 21 - nucleotide molecule" means that 19, 20, or 21 nucleotides have the indicated property. When "at least" appears before a series of numbers or a range, it is understood that "at least" can modify each number in the series or range.

[0275] As used herein, "not more than" or "or less than" is understood to be the value immediately following the phrase and the logically lower value or integer down to zero as is logically deduced from the context. For example, a duplex with a "not more than 2 - nucleotide overhang" has an overhang of 2, 1, or 0 nucleotides. When "not more than" appears before a series of numbers or a range, it is understood that "not more than" can modify each number in the series or range. As used herein, a range includes both the upper and lower limits.

[0276] As used herein, a detection method can include determining that the amount of analyte present is below the detection level of the method.

[0277] If there is a conflict between the indicated target site and the nucleotide sequence of the sense strand or antisense strand, the indicated sequence is given priority.

[0278] If there is a conflict between a sequence and its indicated site on a transcript or other sequence, the nucleotide sequence described in the specification is given priority.

[0279] As used herein, a "target sequence" refers to a continuous portion of the nucleotide sequence of an mRNA molecule (including an mRNA that is an RNA processing product of a primary transcript) formed during transcription of a universal target sequence. In one embodiment, the target portion of the sequence will be at least long enough to serve as a substrate for iRNA - directed cleavage at or near a portion of the nucleotide sequence of an mRNA molecule formed during transcription of the universal target sequence.

[0280] "Universal target sequence" is a nucleotide sequence that has favorable thermodynamic properties for RISC loading and RNAi functionality and has little to no sequence complementarity to any annotated genes in the human, cynomolgus monkey, rat, and mouse transcriptomes. Such universal iRNAs have been shown to be potent RNAi triggers with high target specificity and minimal off-target gene perturbation propensity.

[0281] The nucleotide sequences of exemplary universal target sequences are provided in Tables 3 and 4 below.

[0282] The length of the target sequence can be about 19 - 36 nucleotides. For example, the target sequence length can be about 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 nucleotides. In certain embodiments, the target sequence length is 19 - 23 nucleotides, optionally 21 - 23 nucleotides. Intermediate ranges and lengths of the ranges and lengths recited above are also considered to be part of the present disclosure.

[0283] As used herein, the term "strand comprising a sequence" refers to an oligonucleotide comprising a nucleotide strand described by the sequence referred to using standard nucleotide nomenclature.

[0284] "G", "C", "A", "T", and "U" generally each represent a nucleotide containing guanine, cytosine, adenine, thymine, and uracil as bases, respectively. However, it should be understood that the term "ribonucleotide" or "nucleotide" may also refer to a modified nucleotide, as further detailed below, or a substituted moiety (see, e.g., Table 1). Those skilled in the art are well aware that guanine, cytosine, adenine, and uracil can be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide containing nucleotides with such substituted moieties. For example, without limitation, a nucleotide containing inosine as its base can pair with a nucleotide containing adenine, cytosine, or uracil. Thus, a nucleotide containing uracil, guanine, or adenine in the nucleotide sequence of a dsRNA particularly described in the present invention can be replaced by a nucleotide containing, for example, inosine. In another example, adenine and cytosine anywhere in an oligonucleotide can be replaced by guanine and uracil, respectively, to form G - U wobble base pairing with the target mRNA. Sequences containing such substituted moieties are applicable to the compositions and methods particularly described in the present invention.

[0285] The terms "iRNA", "RNAi agent", "iRNA agent", "RNA interference agent" are used interchangeably herein and refer to an agent that contains RNA as defined in this term and mediates the targeted cleavage of an RNA transcript via the RNA-induced silencing complex (RISC) pathway. iRNA directs the sequence-specific degradation of mRNA through a process called RNA interference (RNAi). iRNA regulates, for example inhibits, the expression of a general target mRNA sequence in a cell, such as a cell of a subject (such as a mammalian subject).

[0286] In one embodiment, the RNAi agent of the present invention comprises single-stranded RNA that interacts with a target RNA sequence (e.g., a general target mRNA sequence) to direct the cleavage of the target RNA. Without wishing to be bound by theory, it is believed that long double-stranded RNA introduced into a cell is broken down into siRNA by a type III endonuclease called Dicer (Sharp et al., (2001) Genes Dev. 15:485). Dicer, an enzyme similar to ribonuclease III, processes dsRNA into short interfering RNAs of 19-23 base pairs 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, enabling the complementary antisense strand to guide target recognition (Nykanen et al., (2001) Cell 107:309). Once bound to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir et al., Genes Dev. 15:188). Thus, in one aspect, the present invention relates to single-stranded RNA (siRNA) generated intracellularly and which promotes the formation of the RISC complex to effect silencing of a target sequence (i.e., a general target sequence). Thus, the term "siRNA" is also used herein to refer to the iRNA described above.

[0287] In certain embodiments, the RNAi agent can be single-stranded siRNA (ssRNAi), which is introduced into a cell or an organism to inhibit a target mRNA. The single-stranded RNAi agent binds to the RISC endonuclease Argonaute 2, which then cleaves the target mRNA. Single-stranded siRNAs are typically 15-30 nucleotides in length and are chemically modified. The design and testing of single-stranded siRNAs are described in U.S. Patent No. 8,101,348 and Lima et al., (2012) Cell 150:883-894 (the entire contents of each of which are hereby incorporated by reference herein). Any antisense nucleotide sequence described herein can be used as the single-stranded siRNA described herein or can be chemically modified by the methods described in Lima et al., (2012) Cell 150:883-894.

[0288] In certain embodiments, the “iRNA” for the compositions, uses, and methods of the present invention is double-stranded RNA and is herein referred to as “double-stranded RNA agent”, “double-stranded RNA (dsRNA) molecule”, “dsRNA agent”, or “dsRNA”. The term “dsRNA” refers to a complex of ribonucleic acid molecules having a duplex structure, the duplex structure comprising two anti-parallel and substantially complementary nucleic acid strands, referred to as having “sense” and “antisense” orientations relative to the target RNA (i.e., a general target mRNA sequence). In certain embodiments of the present invention, the double-stranded RNA (dsRNA) triggers the degradation of the target RNA (e.g., mRNA) by a post-transcriptional gene silencing mechanism referred to herein as RNA interference or RNAi.

[0289] Typically, most of the nucleotides of each strand of the dsRNA molecule are ribonucleotides, but as described in detail herein, each strand or both strands can also include one or more non-ribonucleotides, such as deoxyribonucleotides or modified ribonucleotides. In addition, as used in this specification, “iRNA” can include ribonucleotides with chemical modifications; the iRNA can include substantial modifications at multiple nucleotides. As used herein, the term “modified nucleotide” refers to a nucleotide independently having a modified sugar moiety, a modified internucleotide linkage, or a modified nucleobase, or any combination thereof. Thus, the term modified nucleotide encompasses substitutions, additions, or removals to the internucleoside linkage, sugar moiety, or nucleobase, such as functional groups or atoms. Modifications applicable to the agents of the present invention include all types of modifications disclosed herein or known in the art. For the purposes of this specification and the claims, any such modification used in an siRNA-type molecule is encompassed by “iRNA” or “RNAi agent”.

[0290] In certain embodiments of the present disclosure, the inclusion of deoxynucleotides (if present within the RNAi agent) can be considered to constitute a modified nucleotide.

[0291] The length of the duplex region can be any length that permits specific degradation of the desired target RNA via the RISC pathway, and the length can range from about 19 to 36 base pairs, such as from about 19 to 30 base pairs, such as lengths of 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, such as lengths of about 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. In certain embodiments, the length of the duplex region is 19 - 21 base pairs, such as a length of 21 base pairs. Intermediate ranges and lengths of the ranges and lengths recited above are also considered to be part of the present disclosure.

[0292] The two strands forming the duplex structure can be different portions of a larger RNA molecule, or they can be separate RNA molecules. When the two strands are part of a larger molecule and are thus joined by an uninterrupted nucleotide chain between the 3' end of one strand forming the duplex structure and the 5' end of the corresponding other strand, the joined RNA strands are referred to as a "hairpin loop". The hairpin loop can contain at least one unpaired nucleotide. In some embodiments, the hairpin loop can contain at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 23, or more unpaired nucleotides. In some embodiments, the hairpin loop can be 10 or fewer nucleotides. In some embodiments, the hairpin loop can be 8 or fewer unpaired nucleotides. In some embodiments, the hairpin loop can be 4 - 10 unpaired nucleotides. In some embodiments, the hairpin loop can be 4 - 8 nucleotides.

[0293] When separate RNA molecules contain two substantially complementary strands of dsRNA, these molecules need not but may be covalently linked. When the two strands are covalently linked in a manner other than by an uninterrupted nucleotide chain between the 3' end of one strand forming the duplex structure and the 5' end of the corresponding other strand, the linked structure is called a "hairpin". The RNA strands may have the same or different numbers of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest dsRNA strand minus any overhangs present in the duplex. In addition to the duplex structure, the RNAi may contain one or more nucleotide overhangs. In one embodiment of the RNAi agent, at least one strand contains a 3' overhang of at least 1 nucleotide. In another embodiment, at least one strand contains a 3' overhang of at least 2 nucleotides (e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides). In other embodiments, at least one strand of the RNAi agent contains a 5' overhang of at least 1 nucleotide. In certain embodiments, at least one strand contains a 5' overhang of at least 2 nucleotides (e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides). In still other embodiments, both the 3' and 5' ends of one strand of the RNAi agent contain overhangs of at least 1 nucleotide.

[0294] In certain embodiments, the iRNA agent of the invention is dsRNA (each strand of which contains 19-23 nucleotides), which interacts with a target RNA sequence (e.g., a universal target mRNA sequence) to direct cleavage of the target RNA.

[0295] In some embodiments, the iRNA of the invention is dsRNA of 24-30 nucleotides, which interacts with a target RNA sequence (e.g., a universal target mRNA sequence) to direct cleavage of the target RNA.

[0296] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that protrudes from the duplex structure of a double-stranded iRNA. For example, a nucleotide overhang exists when the 3' end of one strand of the dsRNA extends beyond the 5' end of the other strand, or vice versa. The dsRNA may contain an overhang of at least one nucleotide; alternatively, the overhang may contain at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more. The nucleotide overhang may contain nucleotide / nucleoside analogs (including deoxynucleotides / deoxynucleosides) or consist of nucleotide / nucleoside analogs (including deoxynucleotides / deoxynucleosides). The overhang may be located on the sense strand, the antisense strand, or any combination thereof. Further, the overhanging nucleotides may be present at the 5' end, 3' end, or both ends of the antisense or sense strand of the dsRNA.

[0297] In one embodiment, the antisense strand of the dsRNA has a 1-10 nucleotide (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides) overhang at the 3'-end or 5'-end. In one embodiment, the sense strand of the dsRNA has a 1-10 nucleotide (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides) overhang at the 3'-end or 5'-end. In another embodiment, one or more nucleotides in the overhang are replaced with nucleoside phosphorothioates.

[0298] In certain embodiments, the antisense strand of the dsRNA has a 1-10 nucleotide (e.g., 0-3, 1-3, 2-4, 2-5, 4-10, 5-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides) overhang at the 3'-end or 5'-end. In one embodiment, the sense strand of the dsRNA has a 1-10 nucleotide (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides) overhang at the 3'-end or 5'-end. In another embodiment, one or more nucleotides in the overhang are replaced with nucleoside phosphorothioates.

[0299] In certain embodiments, the antisense strand of the dsRNA has a 1-10 nucleotide (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides) overhang at the 3'-end or 5'-end. In certain embodiments, the overhangs on either the sense strand or the antisense strand or both can include an extended length longer than 10 nucleotides (e.g., a length of 1-30 nucleotides, 2-30 nucleotides, 10-30 nucleotides, 10-25 nucleotides, 10-20 nucleotides, or 10-15 nucleotides). In certain embodiments, the extended overhang is on the sense strand of the duplex. In certain embodiments, the extended overhang is present at the 3'-end of the sense strand of the duplex. In certain embodiments, the extended overhang is present at 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 present at the 3'-end of the antisense strand of the duplex. In certain embodiments, the extended overhang is present at the 5'-end of the antisense strand of the duplex. In certain embodiments, one or more nucleotides in the extended overhang are replaced with nucleoside phosphorothioates. In certain embodiments, the overhang includes a self-complementary portion such that the overhang can form a stable hairpin structure under physiological conditions.

[0300] "Blunt" or "blunt end" means that this end of the double-stranded RNA agent has no unpaired nucleotides, i.e., no nucleotide overhang. A "blunt end" double-stranded RNA agent is double-stranded throughout its length, i.e., there are no nucleotide overhangs at either end of the molecule. The RNAi agents of the present invention include RNAi agents that have no nucleotide overhang at one end (i.e., agents having one overhang and one blunt end) or RNAi agents that have no nucleotide overhangs at either end. Most commonly, such molecules will be double-stranded throughout their length.

[0301] The term "antisense strand" or "guide strand" refers to the strand of an iRNA (e.g., dsRNA) that includes a region that is substantially complementary to a target sequence (e.g., a universal target mRNA).

[0302] As used herein, the term "complementary region" refers to a region on the antisense strand that is substantially complementary to a sequence (e.g., a target sequence, e.g., a universal target nucleotide sequence as defined herein). If the complementary region is not completely complementary to the target sequence, the mismatches can be in the internal or terminal regions of the molecule. Generally, the most tolerated mismatches are in the terminal regions, e.g., within 5, 4, or 3 nucleotides of the 5'- or 3'-end of the iRNA. In some embodiments, the double-stranded RNA agents of the present invention include nucleotide mismatches in the antisense strand. In some embodiments, the antisense strand of the double-stranded RNA agent of the present invention includes no more than 4 mismatches with the target mRNA, e.g., the antisense strand includes 4, 3, 2, 1, or 0 mismatches with the target mRNA. In some embodiments, the antisense strand of the double-stranded RNA agent of the present invention includes no more than 4 mismatches with the sense strand, e.g., the antisense strand includes 4, 3, 2, 1, or 0 mismatches with the sense strand. In some embodiments, the double-stranded RNA agents of the present invention include nucleotide mismatches in the sense strand. In some embodiments, the sense strand of the double-stranded RNA agent of the present invention includes no more than 4 mismatches with the antisense strand, e.g., the sense strand includes 4, 3, 2, 1, or 0 mismatches with the antisense strand. In some embodiments, the nucleotide mismatches are, for example, within 5, 4, 3 nucleotides of the 3'-end of the iRNA. In another embodiment, the nucleotide mismatches are, for example, in the 3'-terminal nucleotide of the iRNA agent. In some embodiments, the mismatches are not in the seed region.

[0303] Thus, an RNAi agent as described herein can include one or more mismatches with a target sequence. In one embodiment, an RNAi agent as described herein includes no more than 3 mismatches (i.e., 3, 2, 1, or 0 mismatches). In one embodiment, an RNAi agent as described herein includes no more than 2 mismatches. In one embodiment, an RNAi agent as described herein includes no more than 1 mismatch. In one embodiment, an RNAi agent as described herein includes 0 mismatches. In certain embodiments, if the antisense strand of the RNAi agent includes a mismatch with the target sequence, the mismatch can optionally be restricted to the last 5 nucleotides at the 5'- or 3'-end of the complementary region. For example, in such embodiments, for a 23-nucleotide RNAi agent, the strand complementary to the common target sequence region generally does not include any mismatches within the central 13 nucleotides. Methods described herein or known in the art can be used to determine whether an RNAi agent that includes a mismatch with the target sequence is effective in inhibiting the expression of a common target mRNA sequence. It is important to consider the potency of an RNAi agent with a mismatch in inhibiting the expression of a common target sequence, particularly if a specific complementary region within the common target sequence is known to have polymorphic sequence variations within a population.

[0304] As used herein, the term "sense strand" or "passenger strand" refers to the strand of the iRNA that includes a region that is substantially complementary to the region of the antisense strand as defined by that term herein.

[0305] As used herein, the term "substantially all nucleotides are modified" means that most but not all are modified and can include no more than 5, 4, 3, 2, or 1 unmodified nucleotide.

[0306] As used herein, the term "cleavage region" refers to the region located immediately adjacent to the cleavage site. The cleavage site is the site on the target where cleavage occurs. In some embodiments, the cleavage region includes three bases located on either side of and immediately adjacent to the cleavage site. In some embodiments, the cleavage region includes two bases located on either side of and immediately adjacent to the cleavage site. In some embodiments, cleavage specifically occurs at the site where the 10th and 11th nucleotides of the antisense strand bind, and the cleavage region includes the 11th, 12th, and 13th nucleotides.

[0307] As used herein, and unless otherwise specified, the term "complementary", when used to describe a first nucleotide sequence with respect to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide comprising the second nucleotide sequence under certain conditions and form a duplex structure, as will be understood by those skilled in the art. Such conditions can be, for example, stringent conditions, where stringent conditions can include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50 °C or 70 °C for 12 - 16 hours, followed by washing (see, for example, "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 vivo) may apply. Those skilled in the art will be able to determine the set of conditions most suitable for testing the complementarity of two sequences based on the ultimate application of the hybridizing nucleotides.

[0308] Complementary sequences within an iRNA (such as within a dsRNA described herein) include base pairing of an oligonucleotide or polynucleotide comprising a first nucleotide sequence with an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences may be referred to herein as "fully complementary" to each other. However, when a first sequence is referred to herein as "substantially complementary" to a second sequence, the two sequences may be fully complementary, or they may form one or more, but typically no more than 5, 4, 3, or 2 mismatched base pairs when hybridizing to form a duplex of up to 30 base pairs, while retaining the ability to hybridize under conditions most relevant to their ultimate application, such as inhibiting gene expression in vitro or in vivo. However, when two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs should not be considered mismatches when determining complementarity. For example, a dsRNA comprising an oligonucleotide of length 21 nucleotides and another oligonucleotide of length 23 nucleotides, where the longer oligonucleotide comprises a 21-nucleotide sequence that is fully complementary to the shorter oligonucleotide, may still be referred to as "fully complementary" for the purposes described herein.

[0309] As used herein, "complementary" sequences may also include or consist entirely of non-Watson-Crick base pairs or base pairs formed by non-natural and modified nucleotides, provided that the requirements regarding their hybridization ability as described above are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble or Hoogsteen base pairing.

[0310] As used herein, the terms "complementary", "fully complementary" and "substantially complementary" may be used with respect to base pairing between the sense and antisense strands of a dsRNA, or between two oligonucleotides or polynucleotides (such as the antisense strand of a double-stranded RNA agent and a target sequence), as will be understood from the context of their use.

[0311] As used herein, a polynucleotide that is "substantially complementary to at least a portion of messenger RNA (mRNA)" refers to a polynucleotide that is substantially complementary to a continuous portion of the mRNA of interest (e.g., the mRNA encoding a common target sequence). For example, if a sequence is substantially complementary to an uninterrupted portion of the mRNA encoding a common target sequence, the polynucleotide is complementary to at least a portion of the common target mRNA sequence.

[0312] Thus, in some embodiments, the antisense polynucleotides disclosed herein are fully complementary to a target common sequence. In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to a target common sequence and comprise a continuous nucleotide sequence that is at least 80% complementary, such as about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% complementary, to the nucleotide sequence of any one of the target nucleotide sequences in any of Tables 3 and 4, or a fragment of any one of the target nucleotide sequences in any of Tables 3 and 4, over its entire length.

[0313] In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to a target common sequence and comprise a continuous nucleotide sequence that is at least about 80% complementary, such as about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% complementary, to the nucleotide sequence of any one of the sense strand nucleotide sequences in any of Tables 2 and 3, or a fragment of any one of the sense strand nucleotide sequences in any of Tables 2 and 3, over its entire length.

[0314] In some embodiments, the iRNA of the invention includes a sense strand that is substantially complementary to an antisense polynucleotide, which in turn is complementary to a target common sequence, and wherein the sense strand polynucleotide comprises a continuous nucleotide sequence that is at least about 80% complementary, such as about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% complementary, to the nucleotide sequence of any one of the antisense strand nucleotide sequences in any of Tables 2 and 3, or a fragment of any one of the antisense strand nucleotide sequences in any of Tables 2 and 3, over its entire length.

[0315] Generally, "iRNA" includes ribonucleotides with chemical modifications. Such modifications can include all types of modifications disclosed herein or known in the art. Any such modification, as used in a dsRNA molecule, is encompassed by "iRNA" for the purposes of this specification and claims.

[0316] In certain embodiments of the present disclosure, if present within an RNAi agent, the inclusion of deoxynucleotides can be considered to constitute a modified nucleotide.

[0317] In one aspect of the invention, the agent for use in the methods and compositions of the invention is a single-stranded antisense oligonucleotide molecule that inhibits a target mRNA via an antisense inhibition mechanism. The single-stranded antisense oligonucleotide molecule is complementary to a sequence within the target mRNA. The single-stranded antisense oligonucleotide can inhibit translation stoichiometrically by base-pairing with the mRNA and physically hindering the translation machinery, see Dias, N. et al., (2002) Mol Cancer Ther 1:347 - 355. The length of the single-stranded antisense oligonucleotide molecule can be from about 14 to about 30 nucleotides and has a sequence complementary to the target sequence. For example, the single-stranded antisense oligonucleotide molecule can comprise a sequence of at least about 14, 15, 16, 17, 18, 19, 20 or more consecutive nucleotides from any one of the antisense sequences described herein.

[0318] As used herein, the term "REVERSIR compound" refers to an oligomeric compound that is complementary to and capable of hybridizing (targeting) at least one strand of a conjugated or unconjugated general dsRNA agent.

[0319] Since the REVERSIR compound hybridizes to one strand of the general dsRNA agent, the "REVERSIR compound" reduces or eliminates the activity intensity and / or duration of the general dsRNA agent.

[0320] The REVERSIR compounds disclosed herein are particularly effective in reducing the activity of siRNA. For example, the REVERSIR compounds disclosed herein can reduce the activity of siRNA by at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% or up to and including a 100% reduction (i.e., compared to the absence level of a reference sample), or any reduction between 50 - 100% compared to a reference sample. The reference level can be the siRNA activity in the absence of the REVERSIR compound.

[0321] In some embodiments, the REVERSIR compounds described herein can reduce the activity of a generic dsRNA agent by at least 75%, such as reducing siRNA activity by 80%, 85%, 90%, 95% or more, and up to and including complete reduction or inhibition of siRNA activity. Complete reduction of siRNA activity means a reduction of at least 80% of siRNA activity relative to a reference level.

[0322] As used herein, the phrase "contacting a cell with an iRNA (such as dsRNA)" includes contacting the cell by any possible means. Contacting a cell with an iRNA includes contacting the cell with the iRNA in vitro or contacting the cell with the iRNA in vivo. The contact can be direct or indirect. Thus, for example, the iRNA can be placed in physical contact with the cell by an individual performing the method, or the iRNA can be placed in a situation that will allow or cause its subsequent contact with the cell.

[0323] Contacting a cell in vitro can be performed, for example, by incubating the cell with the iRNA. Contacting a cell in vivo can be performed, for example, by injecting the iRNA into the tissue in which the cell is located or near the tissue in which the cell is located, or by injecting the iRNA into another area, such as the bloodstream or the subcutaneous space, such that the agent subsequently reaches the tissue in which the cell to be contacted is located. For example, the iRNA can comprise or be conjugated to a ligand, such as GalNAc, that directs the iRNA to the site of interest, such as the liver. Combinations of in vitro and in vivo contact methods are also possible. For example, a cell can be contacted with an iRNA in vitro and then transplanted into a subject.

[0324] In certain embodiments, contacting a cell with an iRNA includes "introducing" or "delivering the iRNA into the cell" by facilitating or effecting uptake or absorption into the cell. Uptake or absorption of the iRNA can occur by passive diffusion or active cellular processes, or by an assisting agent or device. Introducing the iRNA into the cell can be in vitro or in vivo. For example, for in vivo introduction, the iRNA can be injected into a tissue site or systemically administered. In vitro introduction into the cell includes methods known in the art, such as electroporation and liposome transfection. Further methods are described hereinbelow or are known in the art.

[0325] As used herein, the term "lipid nanoparticle" or "LNP" refers to a vesicle comprising a lipid layer encapsulating a pharmaceutically active molecule, such as a nucleic acid molecule (e.g., an iRNA or a plasmid from which an iRNA is transcribed). LNPs are described, for example, in U.S. Patent Nos. 6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents of which are incorporated herein by reference.

[0326] As used herein, "subject" refers to an animal, such as a mammal, including a primate (such as a human, non-human primate, e.g., monkey and chimpanzee), 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 common target sequence either endogenously or heterologously. In one embodiment, the subject is a human. 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.

[0327] As used herein, the terms "treating" or "treatment" refer to a beneficial or desired result, such as reducing at least one sign or symptom of a disorder in a subject, or improving at least one sign or symptom of a disease or condition.

[0328] "Treatment" may also refer to prolonging survival as compared to an expected survival in the absence of treatment. The term "reducing" refers to a statistically significant reduction of such levels. The reduction 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 reduction is at least 20%. In certain embodiments, the reduction of a disease marker (such as a protein or gene expression level) is at least 50%. "Reducing" also includes reducing to a level accepted within the normal range of an individual without such a disorder. In certain embodiments, "reducing" refers to reducing the difference between the marker or symptom level of an affected subject and the level accepted within the normal range of an individual, e.g., the level of weight reduction between an obese individual and an individual with a weight within the accepted normal range.

[0329] As used herein, "prevention" or "preventing", when used with respect to a disease, disorder or condition, refers to reducing the likelihood that a subject will develop symptoms associated with such disease, disorder or condition (e.g., symptoms of a disease). Failure to develop a disease, disorder or condition, or a reduction in the development of symptoms associated with such disease, disorder or condition (e.g., a reduction of at least about 10% on a clinically accepted scale for such disease or disorder), or a delay in the manifestation of symptoms (e.g., a delay of days, weeks, months or years) is considered effective prevention.

[0330] As used herein, "therapeutically effective amount" means an amount of an iRNA agent or compound that, when administered to a subject, is sufficient to effect the treatment of a disease (e.g., by alleviating, ameliorating or maintaining an existing disease or one or more symptoms of a disease). A "therapeutically effective amount" can vary depending on the agent or compound, how the agent is administered, the disease and its severity, and the history, age, weight, family history, genetic makeup, type of prior or concurrent treatment (if any), and other individual characteristics of the subject to be treated.

[0331] As used herein, "prophylactically effective amount" means an amount of an agent or compound that, when administered to a subject, is sufficient to prevent or ameliorate a disease or one or more symptoms of a disease. Ameliorating a disease includes slowing the progression of the disease or reducing the severity of the disease upon later development. A "prophylactically effective amount" can vary depending on the agent or compound, how the agent is administered, the degree of disease risk, the history, age, weight, family history, genetic makeup, type of prior or concurrent treatment (if any), and other individual characteristics of the patient to be treated.

[0332] A "therapeutically effective amount" or "prophylactically effective amount" also includes an amount of an agent or compound that produces some desired effect at a reasonable benefit / risk ratio applicable to any treatment. The agent or compound employed in the methods of the invention can be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0333] The phrase "pharmaceutically acceptable" as used herein refers to those compounds, materials (including salts), compositions, or dosage forms that, within the scope of reasonable medical judgment, are suitable for use in contact with the tissues of human and animal subjects without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0334] As used herein, the phrase "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, magnesium stearate, calcium stearate or zinc stearate, or stearic acid), or solvent encapsulating material involved in carrying or transporting the subject compound from one organ or portion of the body to another organ or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject to be treated. Such carriers are known in the art. Pharmaceutically acceptable carriers include carriers for administration by injection.

[0335] As used herein, the term "sample" includes a liquid, cell, or tissue similar to that isolated from a subject, as well as a collection of liquids, cells, or tissues present within a subject. Examples of biological liquids include blood, serum, and serous fluids, plasma, cerebrospinal fluid, ocular fluid, lymphatic fluid, urine, saliva, and the like. Tissue samples can include samples from a tissue, organ, or local area. For example, a sample can be derived from a specific organ, a part of an organ, or the liquids or cells within those organs. In certain embodiments, a sample can be derived from the liver (e.g., the whole liver or certain segments of the liver or certain types of cells in the liver, such as, for example, hepatocytes). In some embodiments, a "sample derived from a subject" refers to urine obtained from a subject. A "sample derived from a subject" can refer to blood or blood-derived serum or plasma from a subject.

[0336] II. General iRNA of the Invention

[0337] The invention provides iRNAs that inhibit the expression of a general target sequence. In certain embodiments, the iRNA includes a double-stranded ribonucleic acid (dsRNA) molecule for inhibiting the expression of a general target sequence in a cell (e.g., a cell within a subject, such as a mammal (e.g., a human in need of treatment)). The dsRNA agent includes an antisense strand having a complementary region that is complementary to at least a portion of the mRNA formed in the expression of the general target sequence. The length of the complementary region is about 19 - 30 nucleotides (e.g., a length of about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, or 19 nucleotides).

[0338] Once in contact with a cell expressing the general target sequence, the iRNA inhibits the expression of the general target sequence by at least about 50%, as determined, for example, by PCR or a branched DNA (bDNA)-based method, or by a protein-based method (such as immunofluorescence analysis using, for example, Western blotting or flow cytometry techniques). In certain embodiments, in an appropriate somatic cell line provided herein, the inhibition of expression is determined by the qPCR method provided in the examples herein using, for example, a concentration of 10 nM of siRNA. In certain embodiments, the inhibition of in vivo expression is determined by, for example, knocking down the human general target sequence mRNA in a rodent (e.g., a mouse or an AAV-infected mouse expressing the human general target mRNA) when administered at a single dose (e.g., at 3 mg / kg at the lowest point of RNA expression).

[0339] dsRNA includes two complementary RNA strands and hybridizes to form a duplex structure under the conditions in which the dsRNA is to be used. One strand (antisense strand) of the dsRNA includes a complementary region that is substantially complementary and typically fully complementary to a common target sequence. The target sequence can be derived from an mRNA sequence formed during the expression of the common target sequence. The other strand (sense strand) includes a region complementary to the antisense strand such that the two strands hybridize and form a duplex structure when bound under suitable conditions. As described elsewhere herein and known in the art, the complementary sequences of the dsRNA can also include self-complementary regions as a single nucleic acid molecule rather than on separate oligonucleotides.

[0340] Typically, the length of the duplex structure is from 15 to 30 base pairs, such as a length of 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. In certain embodiments, the length of the duplex structure is from 18 to 25 base pairs, such as a length of 18 - 25, 18 - 24, 18 - 23, 18 - 22, 18 - 21, 18 - 20, 19 - 25, 19 - 24, 19 - 23, 19 - 22, 19 - 21, 19 - 20, 20 - 25, 20 - 24, 20 - 23, 20 - 22, 20 - 21, 21 - 25, 21 - 24, 21 - 23, 21 - 22, 22 - 25, 22 - 24, 22 - 23, 23 - 25, 23 - 24 or 24 - 25 base pairs, such as a length of 19 - 21 base pairs. Intermediate ranges and lengths of the ranges and lengths described above are also considered to be part of the present disclosure.

[0341] Similarly, the length of the region complementary to the target sequence is from 15 to 30 nucleotides, such as from 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 nucleotides, such as from 19-23 nucleotides or from 21-23 nucleotides. Intermediate ranges and lengths of the ranges and lengths described above are also considered to be part of the present disclosure.

[0342] In some embodiments, the length of the duplex structure is from 19 to 30 base pairs. Similarly, the length of the region complementary to the target sequence is from 19 to 30 nucleotides.

[0343] In some embodiments, the dsRNA has a length of from about 19 to about 23 nucleotides, or a length of from about 25 to about 30 nucleotides. Generally, the dsRNA is long enough to be a substrate for the Dicer enzyme. For example, it is well known in the art that dsRNA longer than about 21-23 nucleotides in length can be a substrate for Dicer. As would also be recognized by one of ordinary skill in the art, the region of RNA targeted for cleavage is most commonly part of a larger RNA molecule, typically an mRNA molecule. In the relevant context, a "portion" of the mRNA target is a continuous sequence of the mRNA target that is long enough to be a substrate for RNAi-guided cleavage (i.e., cleavage via the RISC pathway).

[0344] Those skilled in the art will also recognize that the duplex region is the main functional part of dsRNA, for example, about 19 to about 30 base pairs, such as about 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 pair duplex regions. Thus, in one embodiment, an RNA molecule or RNA molecule complex having a duplex region greater than 30 base pairs is dsRNA to the extent that it is processed into a functional duplex (e.g., 15-30 base pairs) that targets the desired RNA for cleavage. Thus, those of ordinary skill in the art will recognize that in one embodiment, miRNA is dsRNA. In another embodiment, dsRNA is not a naturally occurring miRNA. In another embodiment, the iRNA agent for targeting the expression of a general target sequence is not generated by cleavage of a larger dsRNA in the target cell.

[0345] The dsRNA described herein may further include one or more single-stranded nucleotide overhangs, such as 1-4, 2-4, 1-3, 2-3, 1, 2, 3 or 4 nucleotides. dsRNAs having at least one nucleotide overhang may have superior inhibitory properties relative to their blunt-ended counterparts. The nucleotide overhangs may comprise nucleotide / nucleoside analogs (including deoxynucleotide / nucleosides) or consist of nucleotide / nucleoside analogs (including deoxynucleotide / nucleosides). The overhangs may be located on the sense strand, the antisense strand, or any combination thereof. Further, the overhanging nucleotides may be present at the 5'-end, 3'-end, or both ends of the antisense or sense strand of the dsRNA.

[0346] dsRNA can be synthesized by standard methods known in the art. The double-stranded RNAi 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. The advantage of organic synthesis is that oligonucleotide strands containing non-natural or modified nucleotides can be easily prepared. Similarly, the single-stranded oligonucleotides of the present invention can be prepared using solution-phase or solid-phase organic synthesis or both.

[0347] In one aspect, the dsRNA of the present invention comprises at least two nucleotide sequences, namely a sense sequence and an antisense sequence. The sense strand is selected from any of the sequence groups provided in Tables 2-3, and the corresponding antisense strand of the sense strand is selected from any of the sequence groups in Tables 2-3. In this aspect, one of the two sequences is complementary to the other of the two sequences, wherein one of the sequences is substantially complementary to the mRNA sequence generated in the expression of the common target sequence. Thus, in this aspect, the dsRNA will comprise two oligonucleotides, wherein one oligonucleotide is described as the sense strand in any of Tables 2-3, and the second oligonucleotide is described as the corresponding antisense strand of the sense strand in any of Tables 2-3.

[0348] In certain embodiments, the substantially complementary sequences of the dsRNA are contained on separate oligonucleotides. In other embodiments, the substantially complementary sequences of the dsRNA are contained on a single oligonucleotide.

[0349] In one embodiment, the antisense strand comprises at least 15, such as 15, 16, 17, 18, 19 or 20 consecutive nucleotides, wherein no more than 0, 1, 2 or 3 nucleotides differ from any of the antisense strand nucleotide sequences in any of Tables 2-3.

[0350] It should be understood that although the sequences in Table 2, for example, are not described as modified or conjugated sequences, the RNA of the iRNA of the present invention, such as the dsRNA of the present invention, may comprise any of the sequences listed in any of Tables 2-3, which are unmodified, unconjugated, or modified or conjugated in a manner different from that described herein. In other words, the present invention encompasses dsRNAs that are unmodified, unconjugated, modified or conjugated as described in Tables 2-3 herein.

[0351] It is well known to those skilled in the art that dsRNAs having a duplex structure of about 20 to 23 base pairs (e.g., 21 base pairs) have been heralded as being particularly effective in inducing RNA interference (Elbashir et al., Elbashir). However, others have found that shorter or longer RNA duplex structures can also be effective (Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226). In the above-described embodiments, due to the nature of any of the oligonucleotide sequences provided in Tables 2-3, the dsRNAs described herein can include at least one strand that is at least 21 nucleotides in length. It is reasonably expected that shorter duplexes having any of the sequences in any of Tables 2-3, minus only a few nucleotides at one or both ends, can be similarly effective compared to the above-described dsRNAs. Accordingly, dsRNAs having a sequence of at least 19, 20, or more contiguous nucleotides derived from any of the sequences in any of Tables 2-3 and whose ability to inhibit the expression of a common target sequence differs from that of the dsRNA containing the full sequence by no more than about 5, 10, 15, 20, 25, or 30% inhibition are contemplated within the scope of the present invention.

[0352] In addition, the RNAs provided in Tables 2-3 recognize sites within the common target sequence transcript that are susceptible to RISC-mediated cleavage. Accordingly, the present invention further features iRNAs that target one of these sites. As used herein, an iRNA is considered to target within a particular site of an RNA transcript if the iRNA promotes cleavage of the transcript anywhere within that particular site. Such iRNAs generally include at least about 19 contiguous nucleotides from any of the sequences provided in any of Tables 2-3, which contiguous nucleotides are coupled to an additional nucleotide sequence from a region adjacent to a selected sequence within the common target sequence.

[0353] III. Modified Common iRNAs of the Present Invention

[0354] In certain embodiments, the common iRNAs of the present invention, such as dsRNAs, are unmodified and do not include, for example, chemical modifications or conjugations known in the art and described herein. In other embodiments, the common iRNAs of the present invention, such as dsRNAs, are chemically modified to enhance stability or other beneficial characteristics. In certain embodiments of the present invention, substantially all of the nucleotides of the common iRNA of the present invention are modified, i.e., there are no more than 5, 4, 3, 2, or 1 unmodified nucleotides present in the iRNA strand. In other embodiments of the present invention, all of the nucleotides of the common iRNA are modified.

[0355] The nucleic acids specifically described in the present invention can be synthesized or modified by methods widely recognized in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S.L. et al. (eds.), John Wiley & Sons, Inc., New York, NY, USA (which is incorporated herein by reference). Modifications include, for example, end modifications such as 5′-end modifications (phosphorylation, conjugation, inverted linkage) or 3′-end modifications (conjugation, DNA nucleotides, inverted linkage, etc.); base modifications such as replacement with stabilizing bases, destabilizing bases, or bases with an expanded repertoire of base pairing, removal of bases (abasic nucleotides), or conjugated bases; sugar modifications (e.g., at the 2′- or 4′-position) or replacement of the sugar; or backbone modifications, which include modification or replacement of the phosphodiester bond. Examples of specific iRNA compounds useful in the embodiments described herein include, but are not limited to, RNAs containing a modified backbone or lacking a native internucleoside linkage. RNAs with a modified backbone particularly include those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referred to in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone may also be considered oligonucleotides. In some embodiments, the modified iRNA will have a phosphorus atom in its internucleoside backbone.

[0356] Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, dithiophosphates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates (including 3'-alkylene phosphonates and chiral phosphonates), phosphinates, phosphoramidates (including 3'-aminophosphoramidates and aminoalkyl phosphoramidates), phosphorothioamidates, thioalkyl phosphonates, thioalkyl phosphotriesters, and boranophosphates (with normal 3'-5' linkages), 2'-5'-linked analogs thereof, and those having inverted polarity (wherein adjacent nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'). Also included are various salt, mixed salt, and free acid forms. In some embodiments of the invention, the dsRNA agent of the invention is in free acid form. In other embodiments of the invention, the dsRNA agent of the invention is in salt form. In one embodiment, the dsRNA agent of the invention is in sodium salt form. In certain embodiments, when the dsRNA agent of the invention is in sodium salt form, sodium ions are present in the agent as counterions for substantially all of the phosphodiester and / or phosphorothioate groups. Agents in which substantially all of the phosphodiester and / or phosphorothioate linkages have sodium counterions include no more than 5, 4, 3, 2, or 1 phosphodiester and / or phosphorothioate linkages without sodium counterions. In some embodiments, when the dsRNA agent of the invention is in sodium salt form, sodium ions are present in the agent as counterions for all of the phosphodiester and / or phosphorothioate groups.

[0357] Representative U.S. patents that teach the preparation of the above phosphorus-linked include, but are not limited to, U.S. Patent Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Patent RE39464, the entire contents of each of which are hereby incorporated by reference herein.

[0358] Modified RNA backbones that do not contain phosphorus atoms 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. These include those having a morpholine linkage (partially formed from the sugar moiety of the nucleoside); a siloxane backbone; sulfide, sulfoxide, and sulfone backbones; formyl and thioformyl backbones; methyleneformyl and thioformyl backbones; olefin-containing backbones; sulfamate backbones; methyleneimine and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and those having a mixture of N, O, S, and CH 2 of the other of the component parts.

[0359] Representative U.S. patents that teach the preparation of the above oligonucleotides include, but are not limited to, U.S. Patent Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 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 hereby incorporated herein by reference.

[0360] Consider the suitable RNA mimetics for iRNA provided herein, in which both the sugar and the internucleoside linkage (i.e., the backbone) of the nucleotide unit are replaced by novel groups. The base units remain hybridized to the appropriate nucleic acid target compound. One such oligomeric compound that has been shown to have excellent hybridization properties is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced by an amide-containing backbone (specifically an aminoethylglycine backbone). The nucleobases are retained and are directly or indirectly bound to the azanitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Patent Nos. 5,539,082; 5,714,331 and 5,719,262, the entire contents of each of which are hereby incorporated herein by reference. Other PNA compounds suitable for the iRNA of the present invention are described, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0361] Certain embodiments specifically described in the present invention include oligonucleotides having a phosphorothioate backbone and having a heteroatom backbone (and in particular the --CH 2 --NH--CH 2 -, --CH 2 --N(CH 3 )--O--CH 2 -- (referred to as methylene(methylimino) or MMI backbone), --CH 2 --O--N(CH 3 )--CH 2 --, --CH 2 --N(CH 3 )--N(CH 3 )--CH 2—and --N(CH 3 )--CH 2 --CH 2 -- and the amide backbone of U.S. Patent No. 5,602,240 mentioned above). In some embodiments, the RNA specifically described in the present invention has the morpholino backbone structure of U.S. Patent No. 5,034,506 mentioned above. The natural phosphodiester backbone can be represented as O-P(O)(OH)-OCH2-.

[0362] The modified RNA may also include one or more substituted sugar moieties. The iRNA specifically described herein, such as dsRNA, may 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, wherein the alkyl, alkenyl, and alkynyl may be substituted or unsubstituted C 1 to C 10 alkyl or C 2 to C 10 alkenyl and alkynyl. Exemplary suitable modifications include O[(CH 2 ) n O] m CH 3 、O(CH 2 ). n OCH 3 、O(CH 2 ) n NH 2 、O(CH 2 ) n CH 3 、O(CH 2 ) n ONH 2 and O(CH 2 ) n ON[(CH 2 ) n CH 3 )] 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: C 1 to C 10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 、OCN、Cl、Br、CN、CF 3 、OCF 3 、SOCH 3 、SO 2 CH 3 、ONO 2 、NO 2 、N3 , NH 2 , heterocyclic alkyl, heterocyclic alkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, intercalator, group that improves the pharmacokinetic properties of iRNA, group that improves the pharmacodynamic properties of iRNA, or other substituents with similar properties. In some embodiments, the modification includes 2'-methoxyethoxy (2'-O--CH 2 CH 2 OCH 3 , also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), that is, alkoxy-alkoxy. Another exemplary modification is 2'-dimethylaminooxyethoxy, that is, O(CH 2 ) 2 ON(CH 3 ) 2 group, also known as 2'-DMAOE (as described in the following examples herein) and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), that is, 2'-O--CH 2 --O--CH 2 --N(CH 3 ) 2 . Further exemplary modifications include: 5’-Me-2’-F nucleotide, 5’-Me-2’-OMe nucleotide, 5’-Me-2’-deoxynucleotide (both R and S isomers in these three families); 2’-alkoxyalkyl; and 2’-NMA (N-methylacetamide).

[0363] Other modifications include 2'-methoxy (2'-OCH 3 ), 2'-aminopropoxy (2'-OCH 2 CH 2 CH 2 NH 2) and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA of the iRNA, particularly at the 3'-position of the 3'-terminal nucleotide or at the 3'-position in a 2'-5'-linked dsRNA and at the 5'-position of the 5'-terminal nucleotide. The iRNA can also have sugar mimetics, such as replacement of the pentafuranose with a cyclobutyl moiety. Representative U.S. patents teaching the preparation of such modified sugar structures include, but are not limited to, U.S. Patent Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, some of which are commonly owned with this application. The entire contents of each of the foregoing are hereby incorporated by reference herein.

[0364] The general iRNA may also include modified or substituted nucleobases (commonly simply referred to as "bases" in the art). As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include other synthetic and natural nucleobases, such as deoxythymidine (dT), 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-azauracil, cytosine, and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thio, 8-thioalkyl, 8-hydroxy, 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-deazaadenine, and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Patent No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P., ed., Wiley-VCH, 2008; and those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J.L., ed., John Wiley & Sons, 1990; those disclosed in Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; and those disclosed in Sanghvi, Y.S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S.T. and Lebleu, B., eds., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds specifically described in the present invention. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, which include 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine.5-Methylcytosine substitution has 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 is an exemplary base substitution, even more particularly when combined with 2'-O-methoxyethyl sugar modification.

[0365] Representative U.S. patents that teach the preparation of certain of the above - indicated modified nucleobases and other modified nucleobases include, but are not limited to, the above - indicated U.S. Patent Nos. 3,687,808; 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121; 5,596,091; 5,614,617; 5,681,941; 5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, the entire contents of each of which are hereby incorporated by reference herein.

[0366] In some embodiments, the RNAi agents of the present disclosure may also be modified to include one or more bicyclic sugar moieties. A "bicyclic sugar" is a furanose ring modified by a ring formed by bridging two carbons (whether adjacent or non - adjacent). A "bicyclic nucleoside" ("BNA") is a nucleoside having a sugar moiety that contains a ring formed by bridging two carbons (whether adjacent or non - adjacent) of the sugar ring, thereby forming a bicyclic ring system. In certain embodiments, the bridge optionally connects the 4'-carbon and the 2'-carbon of the sugar ring via a 2'-deoxy atom. Thus, in some embodiments, the agents of the invention may include one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleoside having a modified ribose moiety (wherein the ribose moiety contains an additional bridge connecting the 2' and 4' carbons). In other words, an LNA is a nucleoside containing 4'-CH 2Nucleosides having a bicyclic sugar moiety bridged at the 2'-position of the ribose. This structure effectively "locks" the ribose in the C3'-endo conformation. The addition of locked nucleic acids to siRNA has been shown to increase the stability of siRNA in serum and reduce off-target effects (Elmen, J. et al., (2005), Nucleic Acids Research 33(1):439-447; Mook, O.R. et al., (2007), Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003), Nucleic Acids Research 31(12):3185-3193). Examples of the bicyclic nucleosides of the polynucleotides of the present invention include, but are not limited to, nucleosides having a bridge between the 4'- and 2'-ribose ring atoms. In certain embodiments, the antisense polynucleotide agents of the present invention include one or more bicyclic nucleosides having a 4'- to 2'-bridge.

[0367] The locked nucleoside can be represented by the following structure (stereochemistry omitted):

[0368]

[0369] wherein B is a nucleobase or a modified nucleobase, and L is a linking group that links the 2'-carbon to the 4'-carbon of the ribose ring. Examples of such 4'- to 2'-bridged bicyclic nucleosides include, but are not limited to, 4'-(CH 2 )—O-2′ (LNA); 4'-(CH 2 )—S-2′; 4'-(CH 2 ) 2 —O-2′ (ENA); 4'-CH(CH 3 )—O-2′ (also referred to as "constrained ethyl" or "cEt") and 4'-CH(CH 2 OCH 3 )—O-2′ (and its analogs; see, for example, U.S. Patent No. 7,399,845); 4'-C(CH 3 )(CH 3 )—O-2′ (and its analogs; see, for example, U.S. Patent No. 8,278,283); 4'-CH 2 —N(OCH 3 )-2′ (and its analogs; see, for example, U.S. Patent No. 8,278,425); 4'-CH 2 —O—N(CH 3 )-2′ (see, for example, U.S. Patent Publication No. 2004 / 0171570); 4'-CH 2 —N(R)—O-2′, wherein R is H, C1-C12 alkyl or a nitrogen protecting group (see, for example, U.S. Patent No. 7,427,672); 4'-CH2 —C(H)(CH 3 )-2′(see, e.g., Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118 - 134); and 4′-CH 2 —C(═CH 2 )-2′(and its analogs; see, e.g., U.S. Patent No. 8,278,426). The entire content of each of the foregoing is hereby incorporated herein by reference.

[0370] Additional representative U.S. patents and U.S. patent publications that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Patent Nos. 6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207; 7,034,133; 7,084,125; 7,399,845; 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 content of each of which is hereby incorporated herein by reference.

[0371] Any of the foregoing bicyclic nucleosides can be prepared to have one or more stereochemical sugar configurations, including, for example, α-L-ribofuranose and β-D-ribofuranose (see WO 99 / 14226).

[0372] The iRNA of the present invention can also be modified to include one or more constrained ethyl nucleotides. As used herein, "constrained ethyl nucleotide" or "cEt" is a locked nucleic acid that includes a bicyclic sugar moiety containing a 4'-CH(CH 3 )-O-2' bridging (i.e., L in the foregoing structure). In one embodiment, the constrained ethyl nucleotide is in the S conformation referred to herein as "S-cEt".

[0373] The iRNA of the present invention can also include one or more "conformationally restricted nucleotides" ("CRN"). CRN is a nucleotide analog having a linker connecting the C2' and C4' carbons of ribose or the C3 and -C5′ carbons of ribose. CRN locks the ribose ring into a stable conformation and increases the hybridization affinity for mRNA. The linker has a sufficient length to place oxygen in the optimal position for stability and affinity, which results in less ribose ring folding.

[0374] Representative publications that teach the preparation of certain CRNs noted above include, but are not limited to, U.S. Patent Publication No. 2013 / 0190383; and PCT Publication WO 2013 / 036868, the entire content of each of which is incorporated herein by reference.

[0375] In some embodiments, the iRNAs of the invention include one or more monomers that are UNA (unlocked nucleic acid) nucleotides. UNA is unlocked propanoyl nucleic acid, in which any bond of the sugar has been removed to form an unlocked "sugar" residue. In one example, UNA also encompasses monomers in which the bond between C1'-C4' (i.e., the covalent carbon-oxygen-carbon bond between the C1' and C4' carbons) has been removed. In another example, the C2'-C3' bond of the sugar (i.e., the covalent carbon-carbon bond between the C2' and C3' carbons) has been removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039, which are incorporated herein by reference).

[0376] Representative U.S. publications that teach the preparation of UNA include, but are not limited to, U.S. Patent No. 8,314,227; and U.S. Patent Publication Nos. 2013 / 0096289; 2013 / 0011922; and 2011 / 0313020, the entire content of each of which is incorporated herein by reference.

[0377] In certain embodiments, the compositions and methods of the present disclosure include vinyl phosphonate (VP) modifications of the RNAi agents described herein. In an exemplary embodiment, the 5'-vinyl phosphonate-modified nucleotide of the present disclosure has the following structure:

[0378]

[0379] wherein X is O or S.

[0380] R is hydrogen, hydroxy, fluoro, or C 1-20 alkoxy (e.g., methoxy or n-hexadecyloxy);

[0381] R 5’ =C(H)-P(O)(OH) 2 and the double bond between the C5' carbon and R 5’ is in the E or Z orientation (e.g., the E orientation); and

[0382] B is a nucleobase or a modified nucleobase, optionally wherein B is adenine, guanine, cytosine, thymine, or uracil.

[0383] The vinyl phosphonates of the present disclosure can be attached to the antisense or sense strand of the dsRNA of the present disclosure. In certain embodiments, the vinyl phosphonates disclosed herein are attached to the antisense strand of the dsRNA, optionally at the 5' end of the antisense strand of the dsRNA.

[0384] Vinyl phosphonate modifications for the compositions and methods of the present disclosure are also contemplated. Exemplary vinyl phosphonate structures include the foregoing structures, where R5' = C(H)-OP(O)(OH) 2 , and the double bond between the C5' carbon and R5' is in the E or Z orientation (e.g., the E orientation).

[0385] Potential stabilizing modifications at the ends of RNA molecules can include N-(acetylaminohexanoyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(hexanoyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-O-deoxythymidine (ether), N-(aminohexanoyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosyl-uridine-3'-phosphate, inverted 2'-deoxy modified ribonucleotides, such as inverted dT (idT), inverted dA (idA), and inverted abasic 2'-deoxyribonucleotides (iAb), etc. The disclosure of such modifications can be found in WO 2011 / 005861.

[0386] In one example, the 3' or 5' end of the oligonucleotide is linked to an inverted 2'-deoxy modified ribonucleotide, such as inverted dT (idT), inverted dA (idA), or inverted abasic 2'-deoxyribonucleotide (iAb). In a specific 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 bond or a 3'-3'-thio-phosphodiester bond.

[0387] In another example, the 3' end of the sense strand is linked to an inverted abasic ribonucleotide (iAb) via a 3'-3'-thio-phosphodiester bond. In another example, the 3' end of the sense strand is linked to inverted dA (idA) via a 3'-3'-thio-phosphodiester bond.

[0388] In a specific 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 bond or a 3'-3'-thio-phosphodiester bond.

[0389] In another instance, the 3' terminal nucleotide of the sense strand is an inverted dA (idA) and is linked to the preceding nucleotide via a 3'-3'-bond (e.g., a 3'-3'-thio-phosphate bond).

[0390] Other modifications of the nucleotides of the iRNA of the present invention include 5'-phosphates or 5'-phosphate mimetics, such as a 5'-terminal phosphate or phosphate mimetic on the antisense strand of the iRNA. Suitable phosphate mimetics are disclosed, for example, in U.S. Patent Publication No. 2012 / 0157511, the entire content of which is incorporated herein by reference.

[0391] A. Modified iRNAs Containing Motifs of the Present Invention

[0392] In certain aspects of the present invention, the double-stranded RNA agents of the present invention include agents having chemical modifications as disclosed in, for example, WO2013 / 075035 (the entire content of each of which is incorporated herein by reference). As shown herein and in WO2013 / 075035, one or more motifs of three identical modifications on three consecutive nucleotides can be introduced into the sense or antisense strand of the dsRNAi agent, particularly at or near the cleavage site. In some embodiments, the sense and antisense strands of the dsRNA agent can be otherwise fully modified. Introduction of these motifs disrupts the modification pattern of the sense or antisense strand, if any. The dsRNAi agent can be optionally conjugated with a GalNAc derivative ligand, for example, on the sense strand.

[0393] More specifically, gene silencing activity of the dsRNAi agent is observed when the sense and antisense strands of the double-stranded RNA agent are fully modified to have one or more motifs of three identical modifications on three consecutive nucleotides at or near the cleavage site of at least one strand of the dsRNAi agent.

[0394] Accordingly, the present invention provides double-stranded RNA agents capable of inhibiting the expression of a general target sequence in vivo. The RNAi agent includes a sense strand and an antisense strand. Each strand of the RNAi agent can be, for example, 17-30 nucleotides in length, 25-30 nucleotides in length, 27-30 nucleotides in length, 19-25 nucleotides in length, 19-23 nucleotides in length, 19-21 nucleotides in length, 21-25 nucleotides in length, or 21-23 nucleotides in length.

[0395] The sense and antisense strands typically form a double-stranded double-stranded RNA (“dsRNA”), also referred to herein as a “dsRNAi agent”. The double-stranded region of the dsRNAi agent can be, for example, a double-stranded region that is 27-30 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 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides in length.

[0396] In certain embodiments, the dsRNAi agent can include one or more overhang regions or cap groups at the 3′ end, 5′ end, or both ends of one or both strands. The overhang region can independently be 1-6 nucleotides in length, such as 2-6 nucleotides in length, 1-5 nucleotides in length, 2-5 nucleotides in length, 1-4 nucleotides in length, 2-4 nucleotides in length, 1-3 nucleotides in length, 2-3 nucleotides in length, or 1-2 nucleotides in length. In certain embodiments, the overhang region can include an extended overhang region as provided above. The overhang region can be the result of one strand being longer than the other, or the result of misalignment of two strands of the same length. The overhang region can form a mismatch with the target mRNA, or it can be complementary to the gene sequence being targeted or can be another sequence. The first and second strands can also be joined, for example, by additional bases to form a hairpin or by other non-base linkers.

[0397] In certain embodiments, the nucleotides in the overhang region of the dsRNAi agent can each independently be a modified or unmodified nucleotide, including but not limited to 2′-sugar modifications such as 2′-F, 2′-O-methyl, thymine (T), 2′-O-methoxyethyl-5-methyluridine (Teo), 2′-O-methoxyethyladenosine (Aco), 2′-O-methoxyethyl-5-methylcytidine (m5Cco), and any combination thereof.

[0398] For example, TT can be an overhang sequence at either end of either strand. The overhang can form a mismatch with the target mRNA, or it can be complementary to the gene sequence being targeted or can be another sequence.

[0399] The 5′- or 3′-overhang of the sense strand, antisense strand, or both strands of the dsRNAi agent can be phosphorylated. In some embodiments, the overhang region contains two nucleotides having a phosphorothioate between the two nucleotides, where the two nucleotides can be the same or different. In some embodiments, the overhang is present at the 3′ end of the sense strand, antisense strand, or both strands. In some embodiments, the 3′-overhang is present in the antisense strand. In some embodiments, the 3′-overhang is present in the sense strand.

[0400] The dsRNAi agent may contain only a single overhang, which can enhance the interfering 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 alternatively at the 3' end of the antisense strand. RNAi can also have blunt ends, which are 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 the dsRNAi agent has a nucleotide overhang at the 3' end and a blunt end at the 5' end. Although not wishing to be bound by theory, the asymmetric blunt end at the 5' end of the antisense strand and the overhang at the 3' end of the antisense strand are beneficial for guiding the strand loading into the RISC process.

[0401] In certain embodiments, the dsRNAi agent is a double blunt end of 19 nucleotides in length, wherein 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.

[0402] In other embodiments, the dsRNAi agent is a double blunt end of 20 nucleotides in length, wherein 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.

[0403] In still other embodiments, the dsRNAi agent is a double blunt end of 21 nucleotides in length, 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. 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.

[0404] In certain embodiments, the dsRNAi agent includes 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; 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, wherein one end of the RNAi agent is a blunt end and the other end contains a 2-nucleotide overhang. In one embodiment, the 2-nucleotide overhang region is located at the 3' end of the antisense strand.

[0405] When two nucleotide overhangs are located at the 3'-end of the antisense strand, there may be two phosphorothioate internucleotide linkages between the terminal three nucleotides, where two of the three nucleotides are overhang nucleotides and the third nucleotide is the paired nucleotide next to the overhang nucleotide. In one embodiment, the RNAi agent additionally has two phosphorothioate internucleotide linkages between the terminal three nucleotides at both the 5'-end of the sense strand and the 5'-end of the antisense strand. In certain embodiments, each nucleotide (including nucleotides that are part of a motif) in the sense and antisense strands of the dsRNAi agent is a modified nucleotide. In certain embodiments, each residue is independently modified with 2'-O-methyl or 3'-fluoro, for example in an alternating motif. Optionally, the dsRNAi agent further comprises a ligand (such as GalNAc 3 ).

[0406] In certain embodiments, the dsRNAi agent comprises a sense strand and an antisense strand, wherein the sense strand is 25-30 nucleotide residues in length, and wherein starting from the 5'-terminal nucleotide (position 1), positions 1 to 23 of the first strand comprise at least 8 ribonucleotides; the antisense strand is 36-66 nucleotide residues in length and, starting from the 3'-terminal nucleotide, comprises at least 8 ribonucleotides at positions paired with positions 1-23 of the sense strand to form a duplex; wherein at least the 3'-terminal nucleotide of the antisense strand is unpaired with the sense strand and up to 6 consecutive 3'-terminal nucleotides are unpaired with the sense strand, thereby forming a 3'-single-stranded overhang of 1-6 nucleotides; wherein the 5'-end of the antisense strand comprises 10-30 consecutive nucleotides that are unpaired with the sense strand, thereby forming a 10-30 nucleotide single-stranded 5'-overhang; wherein when the sense and antisense strands are aligned for maximum complementarity, at least the 5'-terminal and 3'-terminal nucleotides of the sense strand base pair with the nucleobases of the antisense strand, thereby forming a substantially duplex region between the sense and antisense strands; and when the double-stranded nucleic acid is introduced into a mammalian cell, at least 19 ribonucleotides along the length of the antisense strand are sufficiently complementary to the target RNA to reduce expression of a common target site; and wherein the sense strand comprises at least one motif of 3 2'-F modifications on three consecutive nucleotides, wherein at least one motif occurs at or near the cleavage site. The antisense strand contains at least one motif of 3 2'-O-methyl modifications on three consecutive nucleotides at or near the cleavage site.

[0407] In certain embodiments, the dsRNAi agent comprises a sense strand and an antisense strand, wherein the dsRNAi agent comprises a first strand having a length of at least 25 and no more than 29 nucleotides and a second strand having a length of at most 30 nucleotides, wherein there is at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end; wherein the 3' end of the first strand and the 5' end of the second strand form blunt ends, and the second strand is 1-4 nucleotides longer than the first strand at its 3' end, wherein the length of the duplex region is at least 25 nucleotides, and the second strand is sufficiently complementary to the target mRNA along at least 19 nucleotides of its length to reduce the expression of the common target site when the RNAi agent is introduced into mammalian cells, and wherein Dicer cleavage of the dsRNAi agent results in an siRNA comprising the 3' end of the second strand, thereby reducing the expression of the common target site in mammals. Optionally, the dsRNAi agent further comprises a ligand.

[0408] In certain embodiments, the sense strand of the dsRNAi agent contains at least one motif of three identical modifications on three consecutive nucleotides, wherein one motif is present at the cleavage site in the sense strand.

[0409] In certain embodiments, the antisense strand of the dsRNAi agent may further comprise at least one motif of three identical modifications on three consecutive nucleotides, wherein one motif is present at or near the cleavage site in the antisense strand.

[0410] For a dsRNAi agent having a duplex region with a length of 19-23 nucleotides, the cleavage site of the antisense strand is typically near positions 10, 11, and 12 from the 5' end. Thus, the motif of three identical modifications may be present at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, counted from the first nucleotide of the 5' end of the antisense strand, or, counted from the first paired nucleotide within the duplex region of the 5' end of the antisense strand. The cleavage site of the antisense strand may also vary according to the length of the duplex region of the dsRNAi agent from the 5' end.

[0411] The sense strand of the dsRNAi agent may comprise at least one motif of three identical modifications on three consecutive nucleotides at the cleavage site of the strand; and the antisense strand may have at least one motif of three identical modifications on three consecutive nucleotides at or near the cleavage site of the strand. When the sense strand and the antisense strand form a dsRNA duplex, the sense strand and the antisense strand may be aligned such that a motif of three nucleotides on the sense strand and a motif of three nucleotides on the antisense strand have at least one nucleotide overlap, i.e., at least one of the three nucleotides of the motif in the sense strand forms a base pair with at least one of the three nucleotides of the motif in the antisense strand. Alternatively, at least two nucleotides may overlap, or all three nucleotides may overlap.

[0412] In some embodiments, the sense strand of the dsRNAi agent may comprise more than one motif of three identical modifications on three consecutive nucleotides. The first motif may occur at or near the cleavage site of the strand, and the other motifs may be wing modifications. As used herein, the term "wing modification" refers to a motif that occurs at another part of the strand, which is separated from the motif at or near the cleavage site of the same strand. The wing modification is adjacent to the first motif or separated by at least one or more nucleotides. When the motifs are adjacent to each other, the chemical properties of the motifs are different from each other, and when the motifs are separated by one or more nucleotides, the chemical properties may be the same or different. There may be two or more wing modifications. For example, when there are two wing modifications, each wing modification may occur at one end relative to the first motif, which is located at or near the cleavage site, or on either side of the leading motif.

[0413] Like the sense strand, the antisense strand of the dsRNAi agent may comprise more than one motif of three identical modifications on three consecutive nucleotides, where at least one motif occurs at or near the cleavage site of the strand. The antisense strand may also comprise one or more wing modifications, which are aligned similarly to the wing modifications that may be present on the sense strand.

[0414] In some embodiments, the wing modifications on the sense strand or the antisense strand of the dsRNAi agent generally do not include the one or two terminal nucleotides at the 3' end, 5' end, or both ends of the strand.

[0415] In other embodiments, the wing modifications on the sense strand or the antisense strand of the dsRNAi agent generally do not include the one or two paired nucleotides within the duplex region at the 3' end, 5' end, or both ends of the strand.

[0416] When the sense strand and the antisense strand of the dsRNAi agent each contain at least one wing modification, the wing modifications may fall on the same end of the duplex region and have an overlap of one, two, or three nucleotides.

[0417] When the sense and antisense strands of a dsRNAi agent each contain at least two wing modifications, the sense and antisense strands can be aligned such that two modifications from each of one strand fall at one end of the duplex region, with an overlap of one, two, or three nucleotides; two modifications from each of one strand fall at the other end of the duplex region, with an overlap of one, two, or three nucleotides; and two modifications of one strand fall on each side of the leader motif, with an overlap of one, two, or three nucleotides in the duplex region.

[0418] In some embodiments, each nucleotide in the sense and antisense strands of a dsRNAi agent (including nucleotides that are part of a motif) can be modified. Each nucleotide can be modified with the same or different modifications, which can include one or more alterations of non-linking phosphate oxygens or one or more linking phosphate oxygens; alterations of the components of the ribose sugar, such as alterations of the 2'-hydroxyl on the ribose sugar; wholesale replacement of the phosphate moiety with a "dephospho" linker; modification or replacement of naturally occurring bases; and replacement or modification of the ribose-phosphate backbone.

[0419] Since nucleic acids are polymers of subunits, many modifications occur at repetitive positions within the nucleic acid, such as modification of bases, or of the phosphate moiety or non-linking O of the phosphate moiety. In some cases, the modification will occur at all such positions in the nucleic acid, but in many cases it will not. By way of example, the modification can occur only at 3'- or 5'-terminal positions, can occur only in terminal regions, e.g., at positions on the terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of the strand. The modification can occur in double-stranded regions, single-stranded regions, or both. The modification can occur only in the double-stranded region of the RNA or can occur only in the single-stranded region of the RNA. For example, a phosphorothioate modification of the non-linking O position can occur only at one or both termini, can occur only in terminal regions, e.g., at positions on the terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of the strand, or can occur in both double-stranded and single-stranded regions, particularly at the termini. One or more 5' ends can be phosphorylated.

[0420] For example, it may be possible to enhance stability by including specific bases in the overhang, or by including modified nucleotides or nucleotide replacements in single-stranded overhangs (e.g., in 5'- or 3'-overhangs, or in both). For example, it may be desirable to include purine nucleotides in the overhang. In some embodiments, all or some of the bases in a 3'- or 5'-overhang can be modified, e.g., using the modifications described herein. Modifications can include, for example, using modifications known in the art (e.g., replacing the ribose of a nucleobase with a deoxyribonucleotide, 2'-deoxy-2'-fluoro (2'-F), or 2'-O-methyl modification) and modifications in the phosphate group (e.g., phosphorothioate modification) to modify the 2' position of the ribose sugar. The overhang need not be homologous to the target sequence.

[0421] In some embodiments, each residue of the sense strand and the antisense strand is independently modified with LNA, CRN, cET, UNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-hydroxy or 2'-fluoro. The strand may contain more than one modification. In one embodiment, each residue of the sense strand and the antisense strand is independently modified with 2'-O-methyl or 2'-fluoro.

[0422] There are typically at least two different modifications present on the sense strand and the antisense strand. These two modifications can be 2'-O-methyl or 2'-fluoro modifications, or others.

[0423] In certain embodiments, N a or N b contains an alternating pattern of modifications. As used herein, the term "alternating motif" refers to a motif having one or more modifications, each modification occurring on alternating nucleotides of a strand. Alternating nucleotides can refer to every other nucleotide or every third nucleotide, or a similar pattern. For example, if A, B, and C each represent a type of modification to a nucleotide, the alternating motif can be "ABABABABABAB…", "AABBAABBAABB…", "AABAABAABAAB…", "AAABAAABAAAB…", "AAABBBAAABBB…" or "ABCABCABCABC…" etc.

[0424] The types of modifications contained within the alternating motif can be the same or different. For example, if A, B, C, D each represent a type of modification to a nucleotide, the alternating pattern (i.e., modification on every other nucleotide) can be the same, but each sense strand or antisense strand can be selected from several modification possibilities within the alternating motif, such as "ABABAB…", "ACACAC…", "BDBDBD…" or "CDCDCD…" etc.

[0425] In some embodiments, the dsRNAi agents of the present invention comprise a pattern of modifications of alternating motifs on the sense strand that is shifted relative to the pattern of modifications of alternating motifs on the antisense strand. The shift can cause the modified nucleotide sets of the sense strand to correspond to different modified nucleotide sets of the antisense strand, and vice versa. For example, within the duplex region, when the sense strand pairs with the antisense strand in the dsRNA duplex, the alternating motif in the sense strand can start with "ABABAB" from the 5' to the 3' of the strand, and the alternating motif in the antisense strand can start with "BABABA" from the 5' to the 3' of the strand. As another example, within the duplex region, the alternating motif in the sense strand can start with "AABBAABB" from the 5' to the 3' of the strand, and the alternating motif in the antisense strand can start with "BBAABBAA" from the 5' to the 3' of the strand, such that the pattern of modifications between the sense and antisense strands is completely or partially shifted.

[0426] In some embodiments, the dsRNAi agent comprises a pattern of alternating motifs of 2'-O-methyl modifications and 2'-F modifications on the sense strand that is initially offset relative to the pattern of alternating motifs of 2'-O-methyl modifications and 2'-F modifications on the antisense strand, i.e., the 2'-O-methyl modified nucleotides on the sense strand pair with 2'-F modified nucleobases on the antisense strand, and vice versa. The 1-position of the sense strand can start with a 2'-F modification, and the 1-position of the antisense strand can start with a 2'-O-methyl modification.

[0427] Introducing one or more motifs of three identical modifications on three consecutive nucleotides into the sense or antisense strand disrupts the initial modification pattern present in the sense or antisense strand. Disrupting the modification pattern of the sense or antisense strand by introducing one or more motifs of three identical modifications on three consecutive nucleotides can enhance gene silencing activity against a common target sequence.

[0428] In some embodiments, when a motif of three identical modifications on three consecutive nucleotides is introduced into any strand, the modification of the nucleotide adjacent to the motif is a different modification from the modification of the motif. For example, the sequence portion containing the motif is "...N a YYYN b ...", where "Y" represents the modification of the motif of three identical modifications on three consecutive nucleotides, and "N a " and "N b " represent the modifications of the nucleotides adjacent to the motif "YYY" that are different from the modification of Y, and where N a and Nb can be the same or different modifications. Alternatively, when wing modifications are present, N a or N b can be present or absent.

[0429] The iRNA may further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage. The phosphorothioate or methylphosphonate internucleotide linkage modification may occur on the sense strand, the antisense strand, or any nucleotide of both strands at any position of the strand. For example, the internucleotide linkage modification may occur on each nucleotide of the sense strand or the antisense strand; each internucleotide linkage modification may occur in an alternating pattern on the sense strand or the antisense strand; or the sense strand or the antisense strand may comprise two internucleotide linkage modifications in an alternating pattern. The alternating pattern of the internucleotide linkage modification on the sense strand may be the same as or different from that on the antisense strand, and the alternating pattern of the internucleotide linkage modification on the sense strand may be offset relative to the alternating pattern of the internucleotide modification on the antisense strand. In one embodiment, the double-stranded RNAi agent comprises 6-8 phosphorothioate internucleotide linkages. In some embodiments, the antisense strand comprises two phosphorothioate internucleotide linkages at the 5' end and two phosphorothioate internucleotide linkages at the 3' end, and the sense strand comprises at least two phosphorothioate internucleotide linkages at one of the 5' end or the 3' end.

[0430] In some embodiments, the dsRNAi agent comprises a phosphorothioate or methylphosphonate internucleotide linkage modification in the overhang region. For example, the overhang region may comprise two nucleotides having a phosphorothioate or methylphosphonate internucleotide linkage therebetween. Internucleotide linkage modification may also be performed to link the overhanging nucleotides to the terminal paired nucleotides within the duplex region. For example, at least 2, 3, 4, or all of the overhanging nucleotides may be linked by a phosphorothioate or methylphosphonate internucleotide linkage, and optionally, additional phosphorothioate or methylphosphonate internucleotide linkages may be present that link the overhanging nucleotides to the paired nucleotides adjacent to the overhanging nucleotides. For example, there may be at least two phosphorothioate internucleotide linkages between the terminal three nucleotides, two of the three nucleotides are overhanging nucleotides, and the third is the paired nucleotide adjacent to the overhanging nucleotide. These three terminal nucleotides may be located at the 3' end of the antisense strand, the 3' end of the sense strand, the 5' end of the antisense strand, or the 5' end of the antisense strand.

[0431] In some embodiments, two nucleotide overhangs are located at the 3' end of the antisense strand, and there are two phosphorothioate internucleotide linkages between the terminal three nucleotides, two of the three nucleotides are overhanging nucleotides, and the third nucleotide is the paired nucleotide adjacent to the overhanging end nucleotide. Optionally, the dsRNAi agent may additionally have two phosphorothioate internucleotide linkages between the terminal three nucleotides at both the 5' end of the sense strand and the 5' end of the antisense strand.

[0432] In one embodiment, the dsRNAi agent comprises a mismatch to the target, a mismatch within the duplex, or a combination thereof. The mismatch can occur in the overhang region or the duplex region. Base pairs can be ranked based on their tendency to promote dissociation or melting (e.g., based on the free energy of association or dissociation of specific pairings, and the simplest approach is to examine pairings on a single-pair basis, although adjacent or analogous analyses can also be used). In terms of promoting dissociation: A:U is superior to G:C; G:U is superior to G:C; and I:C is superior to G:C (I = inosine). Mismatches, such as non-canonical pairings or pairings other than canonical pairings (as described elsewhere herein) are superior to canonical (A:T, A:U, G:C) pairings; and pairings including universal bases are superior to canonical pairings.

[0433] In certain embodiments, the dsRNAi agent comprises at least one of the first 1, 2, 3, 4, or 5 base pairs within the duplex region from the 5'-end of the antisense strand, said base pairs being independently selected from the group consisting of: A:U, G:U, I:C, and mismatched pairs, such as non-canonical pairs or pairs other than canonical pairs or pairs including universal bases, to promote dissociation of the antisense strand at the 5'-end of the duplex.

[0434] In certain embodiments, the nucleotide at position 1 within the duplex region from the 5'-end in the antisense strand is selected from A, dA, dU, U, and dT. Alternatively, at least one of the first 1, 2, or 3 base pairs within the duplex region from the 5'-end of the antisense strand is an AU base pair. For example, the first base pair within the duplex region from the 5'-end of the antisense strand is an AU base pair.

[0435] In other embodiments, the nucleotide at the 3'-end of the sense strand is deoxythymidine (dT), or the nucleotide at the 3'-end of the antisense strand is deoxythymidine (dT). For example, there is a short deoxythymidine nucleotide sequence at the 3'-end of the sense strand, the antisense strand, or both strands, such as two dT nucleotides.

[0436] In certain embodiments, the sequence of the sense strand can be represented by formula (I):

[0437] 5'n p -N a -(X X X) i -N b -Y Y Y-N b -(Z Z Z) j -N a -n q 3' (I)

[0438] Wherein:

[0439] i and j are each independently 0 or 1;

[0440] p and q are each independently 0 - 6;

[0441] Each N a independently represents an oligonucleotide sequence comprising 0 - 25 modified nucleotides, each sequence comprising at least two different modified nucleotides;

[0442] Each N b independently represents an oligonucleotide sequence comprising 0 - 10 modified nucleotides;

[0443] Each n p and n q independently represent overhanging nucleotides;

[0444] wherein Nb and Y do not have the same modification; and

[0445] XXX, YYY, and ZZZ each independently represent a motif of three identical modifications on three consecutive nucleotides. In one embodiment, YYY is all 2'-F modified nucleotides.

[0446] In some embodiments, N a or N b comprises an alternating pattern of modifications.

[0447] In some embodiments, the YYY motif occurs at or near the cleavage site of the sense strand. For example, when the dsRNAi agent has a duplex region of 17 - 23 nucleotides in length, the YYY motif can occur at or near the cleavage site of the sense strand (e.g., can occur at positions 6, 7, 8; 7, 8, 9; 8, 9, 10; 9, 10, 11; 10, 11, 12; or 11, 12, 13), counting from the first nucleotide at the 5' end; or optionally, counting from the first paired nucleotide within the duplex region at the 5' end.

[0448] In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 1. The sense strand can thus be represented by the formula:

[0449] 5'n p -N a -YYY-N b -ZZZ-N a -n q 3'(Ib);

[0450] 5'n p -N a -XXX-N b -YYY-N a -n q 3'(Ic); or

[0451] 5'n p -Na -XXX-N b -YYY-N b -ZZZ-N a -n q 3'(Id).

[0452] When the sense strand is represented by formula (Ib), N b represents an oligonucleotide sequence comprising 0 - 10, 0 - 7, 0 - 5, 0 - 4, 0 - 2 or 0 modified nucleotides. Each N a independently may represent an oligonucleotide sequence comprising 2 - 20, 2 - 15 or 2 - 10 modified nucleotides.

[0453] When the sense strand is represented by formula (Ic), N b represents an oligonucleotide sequence comprising 0 - 10, 0 - 7, 0 - 10, 0 - 7, 0 - 5, 0 - 4, 0 - 2 or 0 modified nucleotides. Each N a may independently represent an oligonucleotide sequence comprising 2 - 20, 2 - 15 or 2 - 10 modified nucleotides.

[0454] When the sense strand is represented by formula (Id), each N b independently represents an oligonucleotide sequence comprising 0 - 10, 0 - 7, 0 - 5, 0 - 4, 0 - 2 or 0 modified nucleotides. In one embodiment, N b is 0, 1, 2, 3, 4, 5 or 6. Each N a may independently represent an oligonucleotide sequence comprising 2 - 20, 2 - 15 or 2 - 10 modified nucleotides.

[0455] Each X, Y and Z may be the same as or different from each other.

[0456] In other embodiments, i is 0 and j is 0, and the sense strand may be represented by the following formula:

[0457] 5'n p -N a -YYY-N a -n q 3'(Ia).

[0458] When the sense strand is represented by formula (Ia), each N a may independently represent an oligonucleotide sequence comprising 2 - 20, 2 - 15 or 2 - 10 modified nucleotides.

[0459] In one embodiment, the antisense strand sequence of RNAi may be represented by formula (II):

[0460] 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)

[0461] Wherein:

[0462] k and l are each independently 0 or 1;

[0463] p’ and q’ are each independently 0 - 6;

[0464] Each N a ′ independently represents an oligonucleotide sequence comprising 0 - 25 modified nucleotides, each sequence comprising at least two different modified nucleotides;

[0465] Each N b ′ independently represents an oligonucleotide sequence comprising 0 - 10 modified nucleotides;

[0466] Each n p ′ and n q ′ independently represent overhanging nucleotides;

[0467] Wherein N b ’ and Y’ do not have the same modification; and

[0468] X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent a motif of three identical modifications on three consecutive nucleotides.

[0469] In some embodiments, N a ’ or N b ’ contains an alternating pattern of modifications.

[0470] The Y′Y′Y′ motif occurs at or near the cleavage site of the antisense strand. For example, when the dsRNAi agent has a duplex 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 counting starts from the first nucleotide at the 5’ end; or optionally, from the first paired nucleotide within the duplex region at the 5’ end. In one embodiment, the Y′Y′Y′ motif occurs at positions 11, 12, 13.

[0471] In certain embodiments, the Y'Y'Y' motif is all 2’-OMe modified nucleotides.

[0472] In certain embodiments, k is 1 and l is 0, or k is 0 and l is 1, or both k and l are 1.

[0473] The antisense strand can thus be represented by the formula:

[0474] 5'n q’ -N a ′-Z′Z′Z′-Nb′-Y′Y′Y′-N a ′-n p’ 3'(IIb);

[0475] 5'n q’ -N a ′-Y′Y′Y′-N b ′-X′X′X′-n p’ 3'(IIc); or

[0476] 5'n q’ -N a ′-Z′Z′Z′-Nb′-Y′Y′Y′-Nb′-X′X′X′-N a ′-n p’ 3'(IId).

[0477] When the antisense strand is represented by formula (IIb), N b ’ represents an oligonucleotide sequence comprising 0 - 10, 0 - 7, 0 - 10, 0 - 7, 0 - 5, 0 - 4, 0 - 2 or 0 modified nucleotides. Each N a ’ independently represents an oligonucleotide sequence comprising 2 - 20, 2 - 15 or 2 - 10 modified nucleotides.

[0478] When the antisense strand is represented by formula (IIc), N b ’ represents an oligonucleotide sequence comprising 0 - 10, 0 - 7, 0 - 10, 0 - 7, 0 - 5, 0 - 4, 0 - 2 or 0 modified nucleotides. Each N a ’ independently represents an oligonucleotide sequence comprising 2 - 20, 2 - 15 or 2 - 10 modified nucleotides.

[0479] When the antisense strand is represented by formula (IId), each N b ’ independently represents an oligonucleotide sequence comprising 0 - 10, 0 - 7, 0 - 10, 0 - 7, 0 - 5, 0 - 4, 0 - 2 or 0 modified nucleotides. Each N a ’ independently represents an oligonucleotide sequence comprising 2 - 20, 2 - 15 or 2 - 10 modified nucleotides. In one embodiment, N b is 0, 1, 2, 3, 4, 5 or 6.

[0480] In other embodiments, k is 0 and l is 0, and the antisense strand can be represented by the following formula:

[0481] 5'n p’ -N a’ -Y’Y’Y’-N a’ -n q’ 3'(Ia).

[0482] When the antisense strand is represented by formula (IIa), each N a ’ independently represents an oligonucleotide sequence comprising 2-20, 2-15 or 2-10 modified nucleotides.

[0483] Each X', Y' and Z' can be the same as or different from each other.

[0484] Each nucleotide of the sense strand and the antisense strand can be independently modified with LNA, CRN, UNA, cEt, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-hydroxy or 2'-fluoro. For example, each nucleotide of the sense strand and the antisense strand is independently modified with 2'-O-methyl or 2'-fluoro. Specifically, each X, Y, Z, X′, Y′ and Z′ can represent a 2'-O-methyl modification or a 2'-fluoro modification.

[0485] In some embodiments, the sense strand of the dsRNAi agent can comprise a YYY motif that, when the duplex region is 21 nt, occurs at positions 9, 10 and 11 of the strand, counting from the first nucleotide at the 5' end, or optionally from the first paired nucleotide within the duplex region at the 5' end; and Y represents a 2'-F modification. The sense strand can additionally comprise an XXX motif or a ZZZ motif as a wing modification at the opposite ends of the duplex region; and XXX and ZZZ each independently represent a 2'-OMe modification or a 2'-F modification.

[0486] In some embodiments, the antisense strand can comprise a Y′Y′Y′ motif located at positions 11, 12, 13 of the strand, counting from the first nucleotide at the 5' end, or optionally from the first paired nucleotide within the duplex region at the 5' end; and Y′ represents a 2'-O-methyl modification. The antisense strand can further comprise an X′X′X′ motif or a Z′Z′Z′ motif as a wing modification at the opposite ends of the duplex region; and X′X′X′ and Z′Z′Z′ each independently represent a 2'-OMe modification or a 2'-F modification.

[0487] The sense strand represented by any one of the above formulas (Ia), (Ib), (Ic) and (Id) forms a duplex with an antisense strand represented by any one of formulas (IIa), (IIb), (IIc) and (IId).

[0488] Thus, the dsRNAi agent for use in the method of the present invention may comprise a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, and the iRNA duplex is represented by formula (III):

[0489] Sense strand: 5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3'

[0490] Antisense strand: 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'

[0491] (III)

[0492] Wherein:

[0493] i, j, k, and l are each independently 0 or 1;

[0494] p, p', q, and q' are each independently 0 - 6;

[0495] Each N a and N a ’ independently represents an oligonucleotide sequence comprising 0 - 25 modified nucleotides, each sequence comprising at least two different modified nucleotides;

[0496] Each N b and N b ’ independently represents an oligonucleotide sequence comprising 0 - 10 modified nucleotides;

[0497] Wherein each n p ’、n p 、n q ’ and n q , each of which may or may not be present, independently represents a protruding nucleotide; and

[0498] XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent a motif of three identical modifications on three consecutive nucleotides.

[0499] In one embodiment, i is 0 and j is 0; or i is 1 and j is 0; or i is 0 and j is 1; or both i and j are 0; or both i and j are 1. In another embodiment, k is 0 and l is 0; or k is 1 and l is 0; k is 0 and l is 1; or both k and l are 0; or both k and l are 1.

[0500] Exemplary combinations of the sense and antisense strands forming the iRNA duplex include the following formula:

[0501] 5'n p -N a -Y Y Y-N a -n q 3'

[0502] 3'n p ’-N a ’-Y′Y′Y′-N a ’n q ’5'

[0503] (IIIa)

[0504] 5'n p -N a -Y Y Y-N b -Z Z Z-N a -n q 3'

[0505] 3'n p ’-N a ’-Y′Y′Y′-N b ’-Z′Z′Z′-N a ’n q ’5'

[0506] (IIIb)

[0507] 5'n p -N a -X X X-N b -Y Y Y-N a -n q 3'

[0508] 3'n p ’-N a ’-X′X′X′-N b ’-Y′Y′Y′-N a ’-n q ’5'

[0509] (IIIc)

[0510] 5'n p -N a -X X X-Nb -YYY-N b -ZZZ-N a -n q 3'

[0511] 3'n p ’-N a ’-X′X′X′-N b ’-Y′Y′Y′-N b ’-Z′Z′Z′-N a -n q ’5'

[0512] (IIId)

[0513] When the dsRNAi agent is represented by formula (IIIa), each N a independently represents an oligonucleotide sequence comprising 2-20, 2-15 or 2-10 modified nucleotides.

[0514] When the dsRNAi agent is represented by formula (IIIb), each N b independently represents an oligonucleotide sequence comprising 1-10, 1-7, 1-5 or 1-4 modified nucleotides. Each N a independently represents an oligonucleotide sequence comprising 2-20, 2-15 or 2-10 modified nucleotides.

[0515] When the dsRNAi agent is represented by formula (IIIc), each N b , N b independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N a independently represents an oligonucleotide sequence comprising 2-20, 2-15 or 2-10 modified nucleotides.

[0516] When the dsRNAi agent is represented by formula (IIId), each N b , N b ’ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N a , N a ’ independently represents an oligonucleotide sequence comprising 2-20, 2-15 or 2-10 modified nucleotides. Each N a , N b , N b and N b ’ independently comprises an alternating pattern of modifications.

[0517] In Formulas (III), (IIIa), (IIIb), (IIIc) and (IIId), each X, Y and Z can be the same as or different from each other.

[0518] When the dsRNAi agent is represented by Formulas (III), (IIIa), (IIIb), (IIIc) and (IIId), at least one Y nucleotide can form a base pair with one of the Y' nucleotides. Alternatively, at least two Y nucleotides form base pairs with the corresponding Y' nucleotides; or all three Y nucleotides form base pairs with the corresponding Y' nucleotides.

[0519] When the dsRNAi agent is represented by Formula (IIIb) or (IIId), at least one Z nucleotide can form a base pair with one of the Z' nucleotides. Alternatively, at least two Z nucleotides form base pairs with the corresponding Z' nucleotides; or all three Z nucleotides form base pairs with the corresponding Z' nucleotides.

[0520] When the dsRNAi agent is represented by Formula (IIIc) or (IIId), at least one X nucleotide can form a base pair with one of the X' nucleotides. Alternatively, at least two X nucleotides form base pairs with the corresponding X' nucleotides; or all three X nucleotides form base pairs with the corresponding X' nucleotides.

[0521] In certain embodiments, the modification on the Y nucleotide is different from the modification on the Y' nucleotide, the modification on the Z nucleotide is different from the modification on the Z' nucleotide, or the modification on the X nucleotide is different from the modification on the X' nucleotide.

[0522] In certain embodiments, when the dsRNAi agent is represented by Formula (IIId), N a is modified with a 2'-O-methyl or 2'-fluoro modification. In other embodiments, when the RNAi agent is represented by Formula (IIId), N a is modified with a 2'-O-methyl or 2'-fluoro modification, and n p '>0 and at least one n p ' is linked to an adjacent nucleotide via a phosphorothioate bond. In still other embodiments, when the RNAi agent is represented by Formula (IIId), N a is modified with a 2'-O-methyl or 2'-fluoro modification, n p '>0 and at least one n p ' is linked to an adjacent nucleotide via a phosphorothioate bond, and the sense strand is coupled to one or more attached GalNAc derivatives via a divalent or trivalent branching linker (as described below). In other embodiments, when the RNAi agent is represented by Formula (IIId), N a is modified with a 2'-O-methyl or 2'-fluoro modification, n p '>0 and at least one np ′ is linked to an adjacent nucleotide via a phosphorothioate bond, the sense strand comprises at least one phosphorothioate bond, and the sense strand is coupled to one or more attached GalNAc derivatives via a divalent or trivalent branching linker.

[0523] In some embodiments, when the dsRNAi agent is represented by formula (IIIa), N a is modified to 2′-O-methyl or 2′-fluoro modification, n p ′>0 and at least one n p ′ is linked to an adjacent nucleotide via a phosphorothioate bond, the sense strand comprises at least one phosphorothioate bond, and the sense strand is coupled to one or more attached GalNAc derivatives via a divalent or trivalent branching linker.

[0524] In certain embodiments, the RNAi agents of the present invention may contain a small amount of nucleotides containing 2′-fluoro modification, such as 10 or fewer nucleotides having 2′-fluoro modification. For example, the RNAi agent may contain 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 nucleotides having 2′-fluoro modification. In a particular embodiment, the RNAi agent of the present invention contains 10 nucleotides having 2′-fluoro modification. For example, 4 nucleotides having 2′-fluoro modification are in the sense strand, and 6 nucleotides having 2′-fluoro modification are in the antisense strand. In another specific embodiment, the RNAi agent of the present invention contains 6 nucleotides having 2′-fluoro modification. For example, 4 nucleotides having 2′-fluoro modification are in the sense strand, and 2 nucleotides having 2′-fluoro modification are in the antisense strand.

[0525] In other embodiments, the RNAi agents of the present invention may contain an extremely small amount of nucleotides containing 2′-fluoro modification, such as 2 or fewer nucleotides containing 2′-fluoro modification. For example, the RNAi agent may contain 2, 1, or 0 nucleotides having 2′-fluoro modification. In a particular embodiment, the RNAi agent may contain 2 nucleotides having 2′-fluoro modification. For example, 0 nucleotides having 2′-fluoro modification are in the sense strand, and 2 nucleotides having 2′-fluoro modification are in the antisense strand.

[0526] As described in more detail below, iRNAs that include one or more carbohydrate moieties conjugated to the iRNA can optimize one or more properties of the iRNA. In many cases, the carbohydrate moiety will be attached to a modified subunit of the iRNA. For example, the ribose sugars of one or more ribonucleotide subunits of the iRNA can be replaced with another moiety, such as a non-carbohydrate (such as, cyclic) carrier to which a carbohydrate ligand is attached. A ribonucleotide subunit whose ribose sugar has been so replaced is referred to herein as a ribose replacement modified subunit (RRMS). The cyclic carrier can be a carbocyclic 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, sulfur. The cyclic carrier can be a monocyclic system, or can include two or more rings, such as fused rings. The cyclic carrier can be a fully saturated ring system, or it can include one or more double bonds.

[0527] The ligand can be attached to the polynucleotide via a carrier. The carrier includes (i) at least one "backbone attachment point", such as two "backbone attachment points" and (ii) at least one "tether attachment point". As used herein, a "backbone attachment point" refers to a functional group, such as a hydroxyl group, or generally a bond that can be used and is suitable for incorporating the carrier into a backbone, such as a phosphate or modified phosphate (such as sulfur-containing) backbone of a ribonucleic acid. In some embodiments, a "tether attachment point" (TAP) refers to a constituent ring atom of the cyclic carrier, such as a carbon atom or heteroatom (different from the atom providing the backbone attachment point), that is linked to a selected moiety. The selected moiety can be, for example, a carbohydrate, such as a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide or polysaccharide. Optionally, the selected moiety is linked to the cyclic carrier via an intervening tether. Thus, the cyclic carrier generally includes a functional group, such as an amino group, or generally provides a bond suitable for incorporating or tethering another chemical entity, such as a ligand, to the constituent ring.

[0528] The iRNA can be conjugated to a ligand via a carrier, where the carrier can be a cyclic group or an acyclic group. In one embodiment, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinone, tetrahydrofuranyl and decalin. In one embodiment, the acyclic group is a serine alcohol backbone or a diethanolamine backbone.

[0529] B. Thermally labile modifications

[0530] In certain embodiments, the dsRNA molecule can be optimized for RNA interference by introducing thermally labile modifications in the seed region of the antisense strand. As used herein, "seed region" refers to positions 2-9 or positions 2-8 at the 5' end of the reference strand. For example, thermally labile modifications can be introduced in the seed region of the antisense strand to reduce or inhibit off-target gene silencing.

[0531] The term "thermally labile modification" includes modifications that result in an overall melting temperature (T m ) of the dsRNA that is lower than the T m of a dsRNA that does not have such modifications. For example, a thermally labile modification can lower the T m of the dsRNA by 1–4 °C, such as one, two, three, or four degrees Celsius. And the term "thermally labile nucleotide" refers to a nucleotide that contains one or more thermally labile modifications.

[0532] It has been found that a dsRNA having an antisense strand that contains at least one thermally labile modification of the duplex within the first 9 nucleotide positions counted from the 5' end of the antisense strand has 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 labile modification of the duplex within the first 9 nucleotide positions of the 5' region of the antisense strand. In some embodiments, one or more thermally labile modifications of the duplex are located at positions 2-9, such as positions 4-8, from the 5' end of the antisense strand. In some further embodiments, the thermally labile modification of the duplex is located at position 6, 7, or 8 from the 5' end of the antisense strand. In still some further embodiments, the thermally labile modification of the duplex is located at position 7 at the 5' end of the antisense strand. In some embodiments, the thermally labile modification of the duplex is located at positions 2, 3, 4, 5, or 9 from the 5' end of the antisense strand.

[0533] The RNAi agent comprises a sense strand and an antisense strand, each strand having from 14 to 40 nucleotides. The RNAi agent can be represented by formula (L):

[0534]

[0535] In formula (L), B1, B2, B3, B1', B2', B3' and B4' are each independently a modified nucleotide selected from the group consisting of: 2'-O-alkyl, 2'-substituted alkoxy, 2'-substituted alkyl, 2'-halogen, ENA, and BNA / LNA. In one embodiment, B1, B2, B3, B1', B2', B3' and B4' each contain a 2'-OMe modification. In one embodiment, B1, B2, B3, B1', B2', B3' and B4' each contain a 2'-OMe or 2'-F modification. In one embodiment, at least one of B1, B2, B3, B1', B2', B3' and B4' contains a 2'-O-N-methylacetamido (2'-O-NMA, 2'O-CH2C(O)N(Me)H) modification.

[0536] C1 is a thermally labile nucleotide located at a site opposite the seed region of the antisense strand (i.e., at positions 2-8 of the 5'-end of the antisense strand, or at positions 2-9 of the 5'-end of the antisense strand). For example, C1 is located at the position of the sense strand that pairs with nucleotides at positions 2-8 of the 5'-end of the antisense strand. In one example, C1 is located at position 15 from the 5'-end of the sense strand. The C1 nucleotide bears a thermally labile modification, which may include abasic modification; a mismatch with the opposing nucleotide in the duplex; and sugar modifications such as 2'-deoxy modification or acyclic nucleotides, such as unlocked nucleic acid (UNA) or glycerol nucleic acid (GNA), or 2'-5'-linked ribonucleotides ("3'-RNA"). In one embodiment, C1 has a thermally labile modification selected from the group consisting of: i) a mismatch with the opposing nucleotide in the antisense strand; ii) abasic modifications selected from the group consisting of:

[0537] and iii) sugar modifications selected from the group consisting of: where B is a modified or unmodified nucleobase, R 1 and R 2 are independently H, halogen, OR 3 or alkyl; and R 3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar. In one embodiment, the thermally destabilizing modification in C1 is a mismatch selected from the group consisting of G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T, and U:T and U:T; and optionally, at least one of the nucleobases in the mismatch pair is a 2'-deoxynucleobase. In one example, the thermally destabilizing modification in C1 is GNA or

[0538] T1, T1’, T2’ and T3’ each independently represent a nucleotide comprising a modification that provides a steric volume less than or equal to that of a 2’-OMe modification. Steric volume refers to the sum of the steric effects of the modification. Methods for determining the steric effects of modifications of nucleotides are known to those skilled in the art. The modification can be located at the 2’ position of the ribose sugar of the nucleotide, or can be a modification to a non-ribonucleotide, acyclic nucleotide or the backbone of the nucleotide that is similar or equivalent to the 2’ position of the ribose sugar and provides a steric volume less than or equal to that of a 2’-OMe modification. For example, T1, T1’, T2’ and T3’ are each independently selected from DNA, RNA, LNA, 2’-F and 2’-F-5’-methyl. In one embodiment, T1 is DNA. In one embodiment, T1’ is DNA, RNA or LNA. In one embodiment, T2’ is DNA or RNA. In one embodiment, T3’ is DNA or RNA.

[0539] n 1 , n 3 and q 1 are each independently 4 to 15 nucleotides in length.

[0540] n 5 , q 3 and q 7 are each independently 1 to 6 nucleotides in length.

[0541] n 4 , q 2 and q 6 are each independently 1 to 3 nucleotides in length; or, n 4 is 0.

[0542] q 5 is 0 to 10 nucleotides in length.

[0543] n 2 and q 4 are each independently 0 to 3 nucleotides in length.

[0544] Or, n 4 is 0 to 3 nucleotides in length.

[0545] In one embodiment, n 4 can be 0. In one instance, n 4 is 0, and q 2 and q 6 is 1. In another instance, n 4 is 0, and q 2 and q 6is 1, having two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) of the antisense strand, and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end of the antisense strand) of the antisense strand.

[0546] In one embodiment, n 4 , q 2 and q 6 are each 1.

[0547] In one embodiment, n 2 , n 4 , q 2 , q 4 and q 6 are each 1.

[0548] In one embodiment, when the length of the sense strand is 19-22 nucleotides, C1 is located at positions 14-17 at the 5' end of the sense strand, and n 4 is 1. In one embodiment, C1 is located at position 15 at the 5' end of the sense strand.

[0549] In one embodiment, T3' starts at position 2 from the 5' end of the antisense strand. In one instance, T3' is located at position 2 from the 5' end of the antisense strand, and q 6 equals 1.

[0550] In one embodiment, T1' starts at position 14 from the 5' end of the antisense strand. In one instance, T1' is located at position 14 from the 5' end of the antisense strand, and q 2 equals 1.

[0551] In an exemplary embodiment, T3' starts at position 2 from the 5' end of the antisense strand, and T1' starts at position 14 from the 5' end of the antisense strand. In one instance, T3' starts at position 2 from the 5' end of the antisense strand, and q 6 equals 1, and T1' starts at position 14 from the 5' end of the antisense strand, and q 2 equals 1.

[0552] In one embodiment, T1' and T3' are separated by a length of 11 nucleotides (i.e., not counting the T1' and T3' nucleotides).

[0553] In one embodiment, T1' is located at position 14 from the 5' end of the antisense strand. In one instance, T1' is located at position 14 from the 5' end of the antisense strand, and q 2is equal to 1, and a modification at one or more 2'-positions of the non-ribose, acyclic or backbone provides a smaller steric volume than 2'-OMe ribose.

[0554] In one embodiment, T3' is located at the 2-position of the 5'-end of the antisense strand. In one instance, T3' is located at the 2-position of the 5'-end of the antisense strand, and q 6 is equal to 1, and a modification at one or more 2'-positions of the non-ribose, acyclic or backbone provides a steric hindrance less than or equal to the steric volume of 2'-OMe ribose.

[0555] In one embodiment, T1 is located at the cleavage site of the sense strand. In one instance, when the length of the sense strand is 19-22 nucleotides, T1 is located at the 11-position of the 5'-end of the sense strand, and n 2 is 1. In one exemplary embodiment, when the length of the sense strand is 19-22 nucleotides, T1 is located at the cleavage site of the sense strand, and the cleavage site is located at the 11-position from the 5'-end of the sense strand, and n 2 is 1.

[0556] In one embodiment, T2' starts from the 6-position of the 5'-end of the antisense strand. In one instance, T2' is located at the 6-10 positions from the 5'-end of the antisense strand, and q 4 is 1.

[0557] In one exemplary embodiment, T1 is located at the cleavage site of the sense strand (e.g., when the length of the sense strand is 19-22 nucleotides, T1 is located at the 11-position from the 5'-end of the sense strand), and n 2 is 1; T1' is located at the 14-position of the 5'-end of the antisense strand, and q 2 is equal to 1, and the modification of T1' is located at the 2'-position of the ribose sugar or at a non-ribose, acyclic or backbone position that provides a steric hindrance less than 2'-OMe ribose; T2' is located at the 6-10 positions from the 5'-end of the antisense strand, and q 4 is 1; and T3' is located at the 2-position from the 5'-end of the antisense strand, and q 6 is equal to 1, and the modification of T3' is located at the 2'-position or at a non-ribose, acyclic or backbone position that provides a steric hindrance less than or equal to 2'-OMe ribose.

[0558] In one embodiment, T2' starts from the 8-position of the 5'-end of the antisense strand. In one instance, T2' starts from the 8-position of the 5'-end of the antisense strand, and q 4 is 2.

[0559] In one embodiment, T2' starts from the 9-position of the 5'-end of the antisense strand. In one instance, T2' is located at the 9-position of the 5'-end of the antisense strand, and q4 is 1.

[0560] In one embodiment, B1’ is 2’-OMe or 2’-F, q 1 is 9, T1’ is 2’-F, q 2 is 1, B2’ is 2’-OMe or 2’-F, q 3 is 4, T2’ is 2’-F, q 4 is 1, B3’ is 2’-OMe or 2’-F, q 5 is 6, T3’ is 2’-F, q 6 is 1, B4’ is 2’-OMe, and q 7 is 1; wherein there are two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5’ end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5’ end of the antisense strand) of the antisense strand, and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5’ end of the antisense strand) of the antisense strand.

[0561] In one embodiment, n 4 is 0, B3 is 2’-OMe, n 5 is 3, B1’ is 2’-OMe or 2’-F, q 1 is 9, T1’ is 2’-F, q 2 is 1, B2’ is 2’-OMe or 2’-F, q 3 is 4, T2’ is 2’-F, q 4 is 1, B3’ is 2’-OMe or 2’-F, q 5 is 6, T3’ is 2’-F, q 6 is 1, B4’ is 2’-OMe, and q 7 is 1; wherein there are two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5’ end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5’ end of the antisense strand) of the antisense strand, and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5’ end of the antisense strand) of the antisense strand.

[0562] In one embodiment, B1 is 2’-OMe or 2’-F, n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, n 3 is 7, n 4 is 0, B3 is 2’OMe, n 5 is 3, B1’ is 2’-OMe or 2’-F, q 1 is 9, T1’ is 2’-F, q2 is 1, B2’ is 2’-OMe or 2’-F, q 3 is 4, T2’ is 2’-F, q 4 is 2, B3’ is 2’-OMe or 2’-F, q 5 is 5, T3’ is 2’-F, q 6 is 1, B4’ is 2’-OMe, and q 7 is 1.

[0563] In one embodiment, B1 is 2’-OMe or 2’-F, n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, n 3 is 7, n 4 is 0, B3 is 2’-OMe, n 5 is 3, B1’ is 2’-OMe or 2’-F, q 1 is 9, T1’ is 2’-F, q 2 is 1, B2’ is 2’-OMe or 2’-F, q 3 is 4, T2’ is 2’-F, q 4 is 2, B3’ is 2’-OMe or 2’-F, q 5 is 5, T3’ is 2’-F, q 6 is 1, B4’ is 2’-OMe, and q 7 is 1; wherein there are two internucleotide phosphorothioate link modification within positions 1-5 (counting from the 5’ end of the sense strand) of the sense strand, and two internucleotide phosphorothioate link modifications at positions 1 and 2 (counting from the 5’ end of the antisense strand) of the antisense strand, and two internucleotide phosphorothioate link modifications within positions 18-23 (counting from the 5’ end of the antisense strand) of the antisense strand.

[0564] In one embodiment, B1 is 2’-OMe or 2’-F, n 1 is 6, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, n 3 is 7, n 4 is 0, B3 is 2’OMe, n 5 is 3, B1’ is 2’-OMe or 2’-F, q 1 is 7, T1’ is 2’-F, q 2 is 1, B2’ is 2’-OMe or 2’-F, q 3 is 4, T2’ is 2’-F, q 4 is 2, B3’ is 2’-OMe or 2’-F, q 5 is 5, T3’ is 2’-F, q 6is 1, B4’ is 2’-OMe, and q 7 is 1.

[0565] In one embodiment, B1 is 2’-OMe or 2’-F, n 1 is 6, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, n 3 is 7, n 4 is 0, B3 is 2’-OMe, n 5 is 3, B1’ is 2’-OMe or 2’-F, q 1 is 7, T1’ is 2’-F, q 2 is 1, B2’ is 2’-OMe or 2’-F, q 3 is 4, T2’ is 2’-F, q 4 is 2, B3’ is 2’-OMe or 2’-F, q 5 is 5, T3’ is 2’-F, q 6 is 1, B4’ is 2’-OMe, and q 7 is 1; wherein there are two internucleotide phosphorothioate link modifications within positions 1-5 (counting from the 5’ end of the sense strand) of the sense strand, and two internucleotide phosphorothioate link modifications at positions 1 and 2 (counting from the 5’ end of the antisense strand) of the antisense strand, and two internucleotide phosphorothioate link modifications within positions 18-23 (counting from the 5’ end of the antisense strand) of the antisense strand.

[0566] In one embodiment, B1 is 2’-OMe or 2’-F, n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, n 3 is 7, n 4 is 0, B3 is 2’OMe, n 5 is 3, B1’ is 2’-OMe or 2’-F, q 1 is 9, T1’ is 2’-F, q 2 is 1, B2’ is 2’-OMe or 2’-F, q 3 is 4, T2’ is 2’-F, q 4 is 1, B3’ is 2’-OMe or 2’-F, q 5 is 6, T3’ is 2’-F, q 6 is 1, B4’ is 2’-OMe, and q 7 is 1.

[0567] In one embodiment, B1 is 2’-OMe or 2’-F, n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, n3 is 7, n 4 is 0, B3 is 2’-OMe, n 5 is 3, B1’ is 2’-OMe or 2’-F, q 1 is 9, T1’ is 2’-F, q 2 is 1, B2’ is 2’-OMe or 2’-F, q 3 is 4, T2’ is 2’-F, q 4 is 1, B3’ is 2’-OMe or 2’-F, q 5 is 6, T3’ is 2’-F, q 6 is 1, B4’ is 2’-OMe, and q 7 is 1; wherein there are two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5’ end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5’ end of the antisense strand) of the antisense strand, and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5’ end of the antisense strand) of the antisense strand.

[0568] In one embodiment, B1 is 2’-OMe or 2’-F, n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, n 3 is 7, n 4 is 0, B3 is 2’OMe, n 5 is 3, B1’ is 2’-OMe or 2’-F, q 1 is 9, T1’ is 2’-F, q 2 is 1, B2’ is 2’-OMe or 2’-F, q 3 is 5, T2’ is 2’-F, q 4 is 1, B3’ is 2’-OMe or 2’-F, q 5 is 5, T3’ is 2’-F, q 6 is 1, B4’ is 2’-OMe, and q 7 is 1; optionally having at least 2 additional TT at the 3’ end of the antisense strand.

[0569] In one embodiment, B1 is 2’-OMe or 2’-F, n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, n 3 is 7, n 4 is 0, B3 is 2’-OMe, n 5 is 3, B1’ is 2’-OMe or 2’-F, q 1 is 9, T1’ is 2’-F, q 2is 1, B2’ is 2’-OMe or 2’-F, q 3 is 5, T2’ is 2’-F, q 4 is 1, B3’ is 2’-OMe or 2’-F, q 5 is 5, T3’ is 2’-F, q 6 is 1, B4’ is 2’-OMe, and q 7 is 1; optionally having at least 2 additional TT at the 3’ end of the antisense strand; wherein there are two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5’ end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5’ end of the antisense strand) of the antisense strand, and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5’ end of the antisense strand) of the antisense strand.

[0570] In one embodiment, B1 is 2’-OMe or 2’-F, n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, n 3 is 7, n 4 is 0, B3 is 2’-OMe, n 5 is 3, B1’ is 2’-OMe or 2’-F, q 1 is 9, T1’ is 2’-F, q 2 is 1, B2’ is 2’-OMe or 2’-F, q 3 is 4, q 4 is 0, B3’ is 2’-OMe or 2’-F, q 5 is 7, T3’ is 2’-F, q 6 is 1, B4’ is 2’-OMe, and q 7 is 1.

[0571] In one embodiment, B1 is 2’-OMe or 2’-F, n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, n 3 is 7, n 4 is 0, B3 is 2’-OMe, n 5 is 3, B1’ is 2’-OMe or 2’-F, q 1 is 9, T1’ is 2’-F, q 2 is 1, B2’ is 2’-OMe or 2’-F, q 3 is 4, q 4 is 0, B3’ is 2’-OMe or 2’-F, q 5 is 7, T3’ is 2’-F, q 6is 1, B4’ is 2’-OMe, and q 7 is 1; wherein there are two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5’ end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5’ end) of the antisense strand, and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5’ end) of the antisense strand.

[0572] In one embodiment, B1 is 2’-OMe or 2’-F, n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, n 3 is 7, n 4 is 0, B3 is 2’OMe, n 5 is 3, B1’ is 2’-OMe or 2’-F, q 1 is 9, T1’ is 2’-F, q 2 is 1, B2’ is 2’-OMe or 2’-F, q 3 is 4, T2’ is 2’-F, q 4 is 2, B3’ is 2’-OMe or 2’-F, q 5 is 5, T3’ is 2’-F, q 6 is 1, B4’ is 2’-F, and q 7 is 1.

[0573] In one embodiment, B1 is 2’-OMe or 2’-F, n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, n 3 is 7, n 4 is 0, B3 is 2’-OMe, n 5 is 3, B1’ is 2’-OMe or 2’-F, q 1 is 9, T1’ is 2’-F, q 2 is 1, B2’ is 2’-OMe or 2’-F, q 3 is 4, T2’ is 2’-F, q 4 is 2, B3’ is 2’-OMe or 2’-F, q 5 is 5, T3’ is 2’-F, q 6 is 1, B4’ is 2’-F, and q 7is 1; wherein there are two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand).

[0574] In one embodiment, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, q 4 is 0, B3' is 2'-OMe or 2'-F, q 5 is 7, T3' is 2'-F, q 6 is 1, B4' is 2'-F, and q 7 is 1.

[0575] In one embodiment, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, q 4 is 0, B3' is 2'-OMe or 2'-F, q 5 is 7, T3' is 2'-F, q 6 is 1, B4' is 2'-F, and q 7 is 1; wherein there are two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand).

[0576] The RNAi agent may comprise a phosphorus-containing group at the 5'-end of the sense strand or the antisense strand. The 5'-end phosphorus-containing group may be a 5'-end phosphate (5'-P), a 5'-end phosphorothioate (5'-PS), a 5'-end dithiophosphate (5'-PS 2 ), a 5'-end vinylphosphonate (5'-VP), a 5'-end methylphosphonate (MePhos), or 5'-deoxy-5'-C-malonyl When the 5'-end phosphorus-containing group is a 5'-end vinylphosphonate (5'-VP), the 5'-VP may be a 5'-E-VP isomer (i.e., trans-vinylphosphonate, ), a 5'-Z-VP isomer (i.e., cis-vinylphosphonate, ), or a mixture thereof.

[0577] In one embodiment, the RNAi agent comprises a phosphorus-containing group at the 5'-end of the sense strand. In one embodiment, the RNAi agent comprises a phosphorus-containing group at the 5'-end of the antisense strand.

[0578] In one embodiment, the RNAi agent comprises 5'-P. In one embodiment, the RNAi agent comprises 5'-P in the antisense strand.

[0579] In one embodiment, the RNAi agent comprises 5'-PS. In one embodiment, the RNAi agent comprises 5'-PS in the antisense strand.

[0580] In one embodiment, the RNAi agent comprises 5'-VP. In one embodiment, the RNAi agent comprises 5'-VP in the antisense strand. In one embodiment, the RNAi agent comprises 5'-E-VP in the antisense strand. In one embodiment, the RNAi agent comprises 5'-Z-VP in the antisense strand.

[0581] In one embodiment, the RNAi agent comprises 5'-PS 2 . In one embodiment, the RNAi agent comprises 5'-PS in the antisense strand 2 .

[0582] In one embodiment, the RNAi agent comprises 5'-PS 2 . In one embodiment, the RNAi agent comprises 5'-deoxy-5'-C-malonyl in the antisense strand.

[0583] In one embodiment, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'OMe, n 5is 3, B1’ is 2’-OMe or 2’-F, q 1 is 9, T1’ is 2’-F, q 2 is 1, B2’ is 2’-OMe or 2’-F, q 3 is 4, T2’ is 2’-F, q 4 is 2, B3’ is 2’-OMe or 2’-F, q 5 is 5, T3’ is 2’-F, q 6 is 1, B4’ is 2’-OMe, and q 7 is 1. The RNAi agent further comprises 5’-PS.

[0584] In one embodiment, B1 is 2’-OMe or 2’-F, n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, n 3 is 7, n 4 is 0, B3 is 2’OMe, n 5 is 3, B1’ is 2’-OMe or 2’-F, q 1 is 9, T1’ is 2’-F, q 2 is 1, B2’ is 2’-OMe or 2’-F, q 3 is 4, T2’ is 2’-F, q 4 is 2, B3’ is 2’-OMe or 2’-F, q 5 is 5, T3’ is 2’-F, q 6 is 1, B4’ is 2’-OMe, and q 7 is 1. The RNAi agent further comprises an agent further comprising 5’-P.

[0585] In one embodiment, B1 is 2’-OMe or 2’-F, n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, n 3 is 7, n 4 is 0, B3 is 2’OMe, n 5 is 3, B1’ is 2’-OMe or 2’-F, q 1 is 9, T1’ is 2’-F, q 2 is 1, B2’ is 2’-OMe or 2’-F, q 3 is 4, T2’ is 2’-F, q 4 is 2, B3’ is 2’-OMe or 2’-F, q 5 is 5, T3’ is 2’-F, q 6 is 1, B4’ is 2’-OMe, and q 7is 1. The RNAi agent also contains 5'-VP. 5'-VP can be 5'-E-VP, 5'-Z-VP, or a combination thereof.

[0586] In one embodiment, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, T2' is 2'-F, q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The RNAi agent also contains 5'-PS 2 .

[0587] In one embodiment, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, T2' is 2'-F, q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The RNAi agent also contains 5'-deoxy-5'-C-malonyl.

[0588] In one embodiment, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5is 3, B1’ is 2’-OMe or 2’-F, q 1 is 9, T1’ is 2’-F, q 2 is 1, B2’ is 2’-OMe or 2’-F, q 3 is 4, T2’ is 2’-F, q 4 is 2, B3’ is 2’-OMe or 2’-F, q 5 is 5, T3’ is 2’-F, q 6 is 1, B4’ is 2’-OMe, and q 7 is 1; wherein there are two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5’ end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5’ end of the antisense strand) of the antisense strand, and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5’ end of the antisense strand) of the antisense strand. The RNAi agent further comprises 5’-P.

[0589] In one embodiment, B1 is 2’-OMe or 2’-F, n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, n 3 is 7, n 4 is 0, B3 is 2’-OMe, n 5 is 3, B1’ is 2’-OMe or 2’-F, q 1 is 9, T1’ is 2’-F, q 2 is 1, B2’ is 2’-OMe or 2’-F, q 3 is 4, T2’ is 2’-F, q 4 is 2, B3’ is 2’-OMe or 2’-F, q 5 is 5, T3’ is 2’-F, q 6 is 1, B4’ is 2’-OMe, and q 7 is 1; wherein there are two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5’ end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5’ end of the antisense strand) of the antisense strand, and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5’ end of the antisense strand) of the antisense strand. The RNAi agent further comprises 5’-PS.

[0590] In one embodiment, B1 is 2’-OMe or 2’-F, n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, n 3 is 7, n 4is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, T2' is 2'-F, q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7 is 1; wherein there are two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5'-end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5'-end of the antisense strand) of the antisense strand, and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5'-end of the antisense strand) of the antisense strand. The RNAi agent further comprises 5'-VP. 5'-VP can be 5'-E-VP, 5'-Z-VP or a combination thereof.

[0591] In one embodiment, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, T2' is 2'-F, q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7 is 1; wherein there are two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5'-end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5'-end of the antisense strand) of the antisense strand, and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5'-end of the antisense strand) of the antisense strand. The RNAi agent further comprises 5'-PS 2 .

[0592] In one embodiment, B1 is 2'-OMe or 2'-F, n 1is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, n 3 is 7, n 4 is 0, B3 is 2’-OMe, n 5 is 3, B1’ is 2’-OMe or 2’-F, q 1 is 9, T1’ is 2’-F, q 2 is 1, B2’ is 2’-OMe or 2’-F, q 3 is 4, T2’ is 2’-F, q 4 is 2, B3’ is 2’-OMe or 2’-F, q 5 is 5, T3’ is 2’-F, q 6 is 1, B4’ is 2’-OMe, and q 7 is 1; wherein there are two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5’ end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5’ end of the antisense strand) of the antisense strand, and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5’ end of the antisense strand) of the antisense strand. The RNAi agent further comprises 5’-deoxy-5’-C-malonyl.

[0593] In one embodiment, B1 is 2’-OMe or 2’-F, n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, n 3 is 7, n 4 is 0, B3 is 2’-OMe, n 5 is 3, B1’ is 2’-OMe or 2’-F, q 1 is 9, T1’ is 2’-F, q 2 is 1, B2’ is 2’-OMe or 2’-F, q 3 is 4, q 4 is 0, B3’ is 2’-OMe or 2’-F, q 5 is 7, T3’ is 2’-F, q 6 is 1, B4’ is 2’-OMe and q 7 is 1. The RNAi agent further comprises 5’-P.

[0594] In one embodiment, B1 is 2’-OMe or 2’-F, n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, n 3 is 7, n 4 is 0, B3 is 2’-OMe, n 5is 3, B1’ is 2’-OMe or 2’-F, q 1 is 9, T1’ is 2’-F, q 2 is 1, B2’ is 2’-OMe or 2’-F, q 3 is 4, q 4 is 0, B3’ is 2’-OMe or 2’-F, q 5 is 7, T3’ is 2’-F, q 6 is 1, B4’ is 2’-OMe, and q 7 is 1. The dsRNA agent further comprises 5’-PS.

[0595] In one embodiment, B1 is 2’-OMe or 2’-F, n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, n 3 is 7, n 4 is 0, B3 is 2’-OMe, n 5 is 3, B1’ is 2’-OMe or 2’-F, q 1 is 9, T1’ is 2’-F, q 2 is 1, B2’ is 2’-OMe or 2’-F, q 3 is 4, q 4 is 0, B3’ is 2’-OMe or 2’-F, q 5 is 7, T3’ is 2’-F, q 6 is 1, B4’ is 2’-OMe, and q 7 is 1. The RNAi agent further comprises 5’-VP. 5’-VP can be 5’-E-VP, 5’-Z-VP or a combination thereof.

[0596] In one embodiment, B1 is 2’-OMe or 2’-F, n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, n 3 is 7, n 4 is 0, B3 is 2’-OMe, n 5 is 3, B1’ is 2’-OMe or 2’-F, q 1 is 9, T1’ is 2’-F, q 2 is 1, B2’ is 2’-OMe or 2’-F, q 3 is 4, q 4 is 0, B3’ is 2’-OMe or 2’-F, q 5 is 7, T3’ is 2’-F, q 6 is 1, B4’ is 2’-OMe, and q 7 is 1. The RNAi agent further comprises 5’-PS 2。

[0597] In one embodiment, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, q 4 is 0, B3' is 2'-OMe or 2'-F, q 5 is 7, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The RNAi agent further comprises 5'-deoxy-5'-C-malonyl.

[0598] In one embodiment, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, q 4 is 0, B3' is 2'-OMe or 2'-F, q 5 is 7, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7 is 1; wherein there are two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) of the antisense strand, and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) of the antisense strand. The RNAi agent further comprises 5'-P.

[0599] In one embodiment, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, q 4 is 0, B3' is 2'-OMe or 2'-F, q 5 is 7, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7 is 1; wherein there are two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5'-end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5'-end) of the antisense strand, and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5'-end) of the antisense strand. The RNAi agent further comprises 5'-PS.

[0600] In one embodiment, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, q 4 is 0, B3' is 2'-OMe or 2'-F, q 5 is 7, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7 is 1; wherein there are two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5'-end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5'-end) of the antisense strand, and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5'-end) of the antisense strand. The RNAi agent further comprises 5'-VP. 5'-VP can be 5'-E-VP, 5'-Z-VP or a combination thereof.

[0601] In one embodiment, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, q 4 is 0, B3' is 2'-OMe or 2'-F, q 5 is 7, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7 is 1; wherein there are two phosphorothioate internucleotide linkage modifications within positions 1 - 5 (counting from the 5' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) of the antisense strand, and two phosphorothioate internucleotide linkage modifications within positions 18 - 23 (counting from the 5' end) of the antisense strand. The RNAi agent further comprises 5'-PS 2 .

[0602] In one embodiment, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, q 4 is 0, B3' is 2'-OMe or 2'-F, q 5 is 7, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7 is 1; wherein there are two phosphorothioate internucleotide linkage modifications within positions 1 - 5 (counting from the 5' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) of the antisense strand, and two phosphorothioate internucleotide linkage modifications within positions 18 - 23 (counting from the 5' end) of the antisense strand. The RNAi agent further comprises 5'-deoxy-5'-C-malonyl

[0603] In one embodiment, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3is 7, n 4 is 0, B3 is 2’-OMe, n 5 is 3, B1’ is 2’-OMe or 2’-F, q 1 is 9, T1’ is 2’-F, q 2 is 1, B2’ is 2’-OMe or 2’-F, q 3 is 4, T2’ is 2’-F, q 4 is 2, B3’ is 2’-OMe or 2’-F, q 5 is 5, T3’ is 2’-F, q 6 is 1, B4’ is 2’-F, and q 7 is 1. The RNAi agent further comprises 5’-P.

[0604] In one embodiment, B1 is 2’-OMe or 2’-F, n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, n 3 is 7, n 4 is 0, B3 is 2’OMe, n 5 is 3, B1’ is 2’-OMe or 2’-F, q 1 is 9, T1’ is 2’-F, q 2 is 1, B2’ is 2’-OMe or 2’-F, q 3 is 4, T2’ is 2’-F, q 4 is 2, B3’ is 2’-OMe or 2’-F, q 5 is 5, T3’ is 2’-F, q 6 is 1, B4’ is 2’-F, and q 7 is 1. The RNAi agent further comprises 5’-PS.

[0605] In one embodiment, B1 is 2’-OMe or 2’-F, n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, n 3 is 7, n 4 is 0, B3 is 2’OMe, n 5 is 3, B1’ is 2’-OMe or 2’-F, q 1 is 9, T1’ is 2’-F, q 2 is 1, B2’ is 2’-OMe or 2’-F, q 3 is 4, T2’ is 2’-F, q 4 is 2, B3’ is 2’-OMe or 2’-F, q 5 is 5, T3’ is 2’-F, q 6 is 1, B4’ is 2’-F, and q7 is 1. The RNAi agent further comprises 5'-VP. The 5'-VP can be 5'-E-VP, 5'-Z-VP or a combination thereof.

[0606] In one embodiment, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, T2' is 2'-F, q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6 is 1, B4' is 2'-F, and q 7 is 1. The dsRNAi RNA agent further comprises 5'-PS 2 .

[0607] In one embodiment, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, T2' is 2'-F, q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6 is 1, B4' is 2'-F, and q 7 is 1. The RNAi agent further comprises 5'-deoxy-5'-C-malonyl.

[0608] In one embodiment, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5is 3, B1’ is 2’-OMe or 2’-F, q 1 is 9, T1’ is 2’-F, q 2 is 1, B2’ is 2’-OMe or 2’-F, q 3 is 4, T2’ is 2’-F, q 4 is 2, B3’ is 2’-OMe or 2’-F, q 5 is 5, T3’ is 2’-F, q 6 is 1, B4’ is 2’-F, and q 7 is 1; wherein there are two phosphorothioate internucleotide linkage modifications within positions 1 - 5 (counting from the 5’ end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5’ end of the antisense strand) of the antisense strand, and two phosphorothioate internucleotide linkage modifications within positions 18 - 23 (counting from the 5’ end of the antisense strand) of the antisense strand. The RNAi agent further comprises 5’-P.

[0609] In one embodiment, B1 is 2’-OMe or 2’-F, n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, n 3 is 7, n 4 is 0, B3 is 2’-OMe, n 5 is 3, B1’ is 2’-OMe or 2’-F, q 1 is 9, T1’ is 2’-F, q 2 is 1, B2’ is 2’-OMe or 2’-F, q 3 is 4, T2’ is 2’-F, q 4 is 2, B3’ is 2’-OMe or 2’-F, q 5 is 5, T3’ is 2’-F, q 6 is 1, B4’ is 2’-F, and q 7 is 1; wherein there are two phosphorothioate internucleotide linkage modifications within positions 1 - 5 (counting from the 5’ end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5’ end of the antisense strand) of the antisense strand, and two phosphorothioate internucleotide linkage modifications within positions 18 - 23 (counting from the 5’ end of the antisense strand) of the antisense strand. The RNAi agent further comprises 5’-PS.

[0610] In one embodiment, B1 is 2’-OMe or 2’-F, n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, n 3 is 7, n 4is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, T2' is 2'-F, q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6 is 1, B4' is 2'-F, and q 7 is 1; wherein there are two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5'-end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5'-end of the antisense strand) of the antisense strand, and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5'-end of the antisense strand) of the antisense strand. The RNAi agent further comprises 5'-VP. 5'-VP can be 5'-E-VP, 5'-Z-VP or a combination thereof.

[0611] In one embodiment, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, T2' is 2'-F, q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6 is 1, B4' is 2'-F, and q 7 is 1; wherein there are two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5'-end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5'-end of the antisense strand) of the antisense strand, and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5'-end of the antisense strand) of the antisense strand. The RNAi agent further comprises 5'-PS 2 .

[0612] In one embodiment, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, T2' is 2'-F, q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6 is 1, B4' is 2'-F, and q 7 is 1; wherein there are two phosphorothioate internucleotide linkage modifications within positions 1 - 5 (counting from the 5'-end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5'-end of the antisense strand) of the antisense strand, and two phosphorothioate internucleotide linkage modifications within positions 18 - 23 (counting from the 5'-end of the antisense strand) of the antisense strand. The RNAi agent further comprises 5'-deoxy-5'-C-malonyl.

[0613] In one embodiment, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, q 4 is 0, B3' is 2'-OMe or 2'-F, q 5 is 7, T3' is 2'-F, q 6 is 1, B4' is 2'-F, and q 7 is 1. The RNAi agent further comprises 5'-P.

[0614] In one embodiment, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1is 9, T1’ is 2’-F, q 2 is 1, B2’ is 2’-OMe or 2’-F, q 3 is 4, q 4 is 0, B3’ is 2’-OMe or 2’-F, q 5 is 7, T3’ is 2’-F, q 6 is 1, B4’ is 2’-F, and q 7 is 1. The RNAi agent further comprises 5’-PS.

[0615] In one embodiment, B1 is 2’-OMe or 2’-F, n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, n 3 is 7, n 4 is 0, B3 is 2’-OMe, n 5 is 3, B1’ is 2’-OMe or 2’-F, q 1 is 9, T1’ is 2’-F, q 2 is 1, B2’ is 2’-OMe or 2’-F, q 3 is 4, q 4 is 0, B3’ is 2’-OMe or 2’-F, q 5 is 7, T3’ is 2’-F, q 6 is 1, B4’ is 2’-F, and q 7 is 1. The RNAi agent further comprises 5’-VP. 5’-VP can be 5’-E-VP, 5’-Z-VP or a combination thereof.

[0616] In one embodiment, B1 is 2’-OMe or 2’-F, n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, n 3 is 7, n 4 is 0, B3 is 2’-OMe, n 5 is 3, B1’ is 2’-OMe or 2’-F, q 1 is 9, T1’ is 2’-F, q 2 is 1, B2’ is 2’-OMe or 2’-F, q 3 is 4, q 4 is 0, B3’ is 2’-OMe or 2’-F, q 5 is 7, T3’ is 2’-F, q 6 is 1, B4’ is 2’-F, and q 7 is 1. The RNAi agent further comprises 5’-PS 2 .

[0617] In one embodiment, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, q 4 is 0, B3' is 2'-OMe or 2'-F, q 5 is 7, T3' is 2'-F, q 6 is 1, B4' is 2'-F, and q 7 is 1. The RNAi agent further comprises 5'-deoxy-5'-C-propionyl.

[0618] In one embodiment, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, q 4 is 0, B3' is 2'-OMe or 2'-F, q 5 is 7, T3' is 2'-F, q 6 is 1, B4' is 2'-F, and q 7 is 1; wherein there are two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5'-end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5'-end of the antisense strand) of the antisense strand, and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5'-end of the antisense strand) of the antisense strand. The RNAi agent further comprises 5'-P.

[0619] In one embodiment, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n5 is 3, B1’ is 2’-OMe or 2’-F, q 1 is 9, T1’ is 2’-F, q 2 is 1, B2’ is 2’-OMe or 2’-F, q 3 is 4, q 4 is 0, B3’ is 2’-OMe or 2’-F, q 5 is 7, T3’ is 2’-F, q 6 is 1, B4’ is 2’-F, and q 7 is 1; wherein there are two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5’ end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5’ end of the antisense strand) of the antisense strand, and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5’ end of the antisense strand) of the antisense strand. The RNAi agent further comprises 5’-PS.

[0620] In one embodiment, B1 is 2’-OMe or 2’-F, n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, n 3 is 7, n 4 is 0, B3 is 2’-OMe, n 5 is 3, B1’ is 2’-OMe or 2’-F, q 1 is 9, T1’ is 2’-F, q 2 is 1, B2’ is 2’-OMe or 2’-F, q 3 is 4, q 4 is 0, B3’ is 2’-OMe or 2’-F, q 5 is 7, T3’ is 2’-F, q 6 is 1, B4’ is 2’-F, and q 7 is 1; wherein there are two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5’ end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5’ end of the antisense strand) of the antisense strand, and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5’ end of the antisense strand) of the antisense strand. The RNAi agent further comprises 5’-VP. 5’-VP can be 5’-E-VP, 5’-Z-VP or a combination thereof.

[0621] In one embodiment, B1 is 2’-OMe or 2’-F, n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, n 3 is 7, n4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, q 4 is 0, B3' is 2'-OMe or 2'-F, q 5 is 7, T3' is 2'-F, q 6 is 1, B4' is 2'-F, and q 7 is 1; wherein there are two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and two phosphorothioate internucleotide linkages within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The modified RNAi agent further comprises 5'-PS 2 .

[0622] In one embodiment, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, q 4 is 0, B3' is 2'-OMe or 2'-F, q 5 is 7, T3' is 2'-F, q 6 is 1, B4' is 2'-F, and q 7 is 1; wherein there are two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and two phosphorothioate internucleotide linkages within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent further comprises 5'-deoxy-5'-C-malonyl

[0623] In one embodiment, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, T2' is 2'-F, q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7 is 1; wherein there are two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5'-end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5'-end of the antisense strand) of the antisense strand, and two phosphorothioate internucleotide linkages within positions 18-23 (counting from the 5'-end of the antisense strand) of the antisense strand. The RNAi agent further comprises 5'-P and a targeting ligand. In one embodiment, 5'-P is located at the 5'-end of the antisense strand, and the targeting ligand is located at the 3'-end of the sense strand.

[0624] In one embodiment, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, T2' is 2'-F, q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7 is 1; wherein there are two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5'-end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5'-end of the antisense strand) of the antisense strand, and two phosphorothioate internucleotide linkages within positions 18-23 (counting from the 5'-end of the antisense strand) of the antisense strand. The RNAi agent further comprises 5'-PS and a targeting ligand. In one embodiment, 5'-PS is located at the 5'-end of the antisense strand, and the targeting ligand is located at the 3'-end of the sense strand.

[0625] In one embodiment, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, T2' is 2'-F, q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7 is 1; wherein there are two internucleotide phosphorothioate modifications within positions 1-5 (counting from the 5'-end of the sense strand) of the sense strand, and two internucleotide phosphorothioate modifications at positions 1 and 2 (counting from the 5'-end of the antisense strand) of the antisense strand, and two internucleotide phosphorothioates within positions 18-23 (counting from the 5'-end of the antisense strand). The RNAi agent further comprises 5'-VP (e.g., 5'-E-VP, 5'-Z-VP or a combination thereof) and a targeting ligand.

[0626] In one embodiment, 5'-VP is located at the 5'-end of the antisense strand, and the targeting ligand is located at the 3'-end of the sense strand.

[0627] In one embodiment, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, T2' is 2'-F, q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7is 1; wherein there are two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) of the antisense strand, and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end of the antisense strand) of the antisense strand. The RNAi agent further comprises 5'-PS 2 and a targeting ligand. In one embodiment, 5'-PS 2 is located at the 5' end of the antisense strand, and the targeting ligand is located at the 3' end of the sense strand.

[0628] In one embodiment, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, T2' is 2'-F, q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7 is 1; wherein there are two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) of the antisense strand, and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end of the antisense strand) of the antisense strand. The RNAi agent further comprises 5'-deoxy-5'-C-malonyl and a targeting ligand. In one embodiment, 5'-deoxy-5'-C-malonyl is located at the 5' end of the antisense strand, and the targeting ligand is located at the 3' end of the sense strand.

[0629] In one embodiment, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q2 is 1, B2’ is 2’-OMe or 2’-F, q 3 is 4, q 4 is 0, B3’ is 2’-OMe or 2’-F, q 5 is 7, T3’ is 2’-F, q 6 is 1, B4’ is 2’-OMe, and q 7 is 1; wherein there are two internucleotide phosphorothioate link modifications within positions 1-5 (counting from the 5’ end of the sense strand) of the sense strand, and two internucleotide phosphorothioate link modifications at positions 1 and 2 (counting from the 5’ end) of the antisense strand, and two internucleotide phosphorothioate link modifications within positions 18-23 (counting from the 5’ end) of the antisense strand. The RNAi agent further comprises 5’-P and a targeting ligand. In one embodiment, 5’-P is located at the 5’ end of the antisense strand, and the targeting ligand is located at the 3’ end of the sense strand.

[0630] In one embodiment, B1 is 2’-OMe or 2’-F, n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, n 3 is 7, n 4 is 0, B3 is 2’-OMe, n 5 is 3, B1’ is 2’-OMe or 2’-F, q 1 is 9, T1’ is 2’-F, q 2 is 1, B2’ is 2’-OMe or 2’-F, q 3 is 4, q 4 is 0, B3’ is 2’-OMe or 2’-F, q 5 is 7, T3’ is 2’-F, q 6 is 1, B4’ is 2’-OMe, and q 7 is 1; wherein there are two internucleotide phosphorothioate link modifications within positions 1-5 (counting from the 5’ end of the sense strand) of the sense strand, and two internucleotide phosphorothioate link modifications at positions 1 and 2 (counting from the 5’ end) of the antisense strand, and two internucleotide phosphorothioate link modifications within positions 18-23 (counting from the 5’ end) of the antisense strand. The RNAi agent further comprises 5’-PS and a targeting ligand. In one embodiment, 5’-PS is located at the 5’ end of the antisense strand, and the targeting ligand is located at the 3’ end of the sense strand.

[0631] In one embodiment, B1 is 2’-OMe or 2’-F, n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, n 3 is 7, n 4is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, q 4 is 0, B3' is 2'-OMe or 2'-F, q 5 is 7, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7 is 1; wherein there are two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5'-end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5'-end) of the antisense strand, and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5'-end) of the antisense strand. The RNAi agent further comprises 5'-VP (e.g., 5'-E-VP, 5'-Z-VP or a combination thereof) and a targeting ligand. In one embodiment, 5'-VP is located at the 5'-end of the antisense strand, and the targeting ligand is located at the 3'-end of the sense strand.

[0632] In one embodiment, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, q 4 is 0, B3' is 2'-OMe or 2'-F, q 5 is 7, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7 is 1; wherein there are two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5'-end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5'-end) of the antisense strand, and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5'-end) of the antisense strand. The RNAi agent further comprises 5'-PS 2 and a targeting ligand. In one embodiment, 5'-PS 2 is located at the 5'-end of the antisense strand, and the targeting ligand is located at the 3'-end of the sense strand.

[0633] In one embodiment, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, q 4 is 0, B3' is 2'-OMe or 2'-F, q 5 is 7, T3' is 2'-F, q 6 is 1, B4' is 2'-OMe, and q 7 is 1; wherein there are two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5'-end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5'-end) of the antisense strand, and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5'-end) of the antisense strand. The RNAi agent further comprises a 5'-deoxy-5'-C-malonyl and a targeting ligand. In one embodiment, the 5'-deoxy-5'-C-malonyl is located at the 5'-end of the antisense strand, and the targeting ligand is located at the 3'-end of the sense strand.

[0634] In one embodiment, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, T2' is 2'-F, q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6 is 1, B4' is 2'-F, and q 7is 1; wherein there are two internucleotide phosphorothioate link modifications within positions 1 - 5 (counting from the 5' end of the sense strand) of the sense strand, and two internucleotide phosphorothioate link modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) of the antisense strand, and two internucleotide phosphorothioate link modifications within positions 18 - 23 (counting from the 5' end of the antisense strand) of the antisense strand. The RNAi agent further comprises 5'-P and a targeting ligand. In one embodiment, 5'-P is located at the 5' end of the antisense strand, and the targeting ligand is located at the 3' end of the sense strand.

[0635] In one embodiment, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, T2' is 2'-F, q 4 is 2, B3' is 2'-OMe or 2'-F, q 5 is 5, T3' is 2'-F, q 6 is 1, B4' is 2'-F, and q 7 is 1; wherein there are two internucleotide phosphorothioate link modifications within positions 1 - 5 (counting from the 5' end of the sense strand) of the sense strand, and two internucleotide phosphorothioate link modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) of the antisense strand, and two internucleotide phosphorothioate link modifications within positions 18 - 23 (counting from the 5' end of the antisense strand) of the antisense strand. The RNAi agent further comprises 5'-PS and a targeting ligand. In one embodiment, 5'-PS is located at the 5' end of the antisense strand, and the targeting ligand is located at the 3' end of the sense strand.

[0636] In one embodiment, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3is 4, T2’ is 2’-F, q 4 is 2, B3’ is 2’-OMe or 2’-F, q 5 is 5, T3’ is 2’-F, q 6 is 1, B4’ is 2’-F, and q 7 is 1; wherein there are two internucleotide phosphorothioate linkages within positions 1-5 (counting from the 5’ end of the sense strand) of the sense strand, and two internucleotide phosphorothioate linkages at positions 1 and 2 (counting from the 5’ end of the antisense strand) of the antisense strand, and two internucleotide phosphorothioate linkages within positions 18-23 (counting from the 5’ end of the antisense strand) of the antisense strand. The RNAi agent further comprises 5'-VP (e.g., 5’-E-VP, 5’-Z-VP or a combination thereof) and a targeting ligand. In one embodiment, the 5’-VP is located at the 5’ end of the antisense strand and the targeting ligand is located at the 3’ end of the sense strand.

[0637] In one embodiment, B1 is 2’-OMe or 2’-F, n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, n 3 is 7, n 4 is 0, B3 is 2’-OMe, n 5 is 3, B1’ is 2’-OMe or 2’-F, q 1 is 9, T1’ is 2’-F, q 2 is 1, B2’ is 2’-OMe or 2’-F, q 3 is 4, T2’ is 2’-F, q 4 is 2, B3’ is 2’-OMe or 2’-F, q 5 is 5, T3’ is 2’-F, q 6 is 1, B4’ is 2’-F, and q 7 is 1; wherein there are two internucleotide phosphorothioate linkages within positions 1-5 (counting from the 5’ end of the sense strand) of the sense strand, and two internucleotide phosphorothioate linkages at positions 1 and 2 (counting from the 5’ end of the antisense strand) of the antisense strand, and two internucleotide phosphorothioate linkages within positions 18-23 (counting from the 5’ end of the antisense strand) of the antisense strand. The RNAi agent further comprises 5’-PS 2 and a targeting ligand. In one embodiment, the 5’-PS 2 is located at the 5’ end of the antisense strand and the targeting ligand is located at the 3’ end of the sense strand.

[0638] In one embodiment, B1 is 2’-OMe or 2’-F, n 1 is 8, T1 is 2’F, n 2For 3, B2 is 2'-OMe, n 3 For 7, n 4 For 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 For 9, T1' is 2'-F, q 2 For 1, B2' is 2'-OMe or 2'-F, q 3 For 4, T2' is 2'-F, q 4 For 2, B3' is 2'-OMe or 2'-F, q 5 For 5, T3' is 2'-F, q 6 For 1, B4' is 2'-F, and q 7 For 1; wherein there are two phosphorothioate internucleotide linkage modifications within positions 1 - 5 (counting from the 5' end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) of the antisense strand, and two phosphorothioate internucleotide linkage modifications within positions 18 - 23 (counting from the 5' end of the antisense strand) of the antisense strand. The RNAi agent further comprises 5'-deoxy-5'-C-malonyl and a targeting ligand. In one embodiment, the 5'-deoxy-5'-C-malonyl is located at the 5' end of the antisense strand, and the targeting ligand is located at the 3' end of the sense strand.

[0639] In one embodiment, B1 is 2'-OMe or 2'-F, n 1 For 8, T1 is 2'F, n 2 For 3, B2 is 2'-OMe, n 3 For 7, n 4 For 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 For 9, T1' is 2'-F, q 2 For 1, B2' is 2'-OMe or 2'-F, q 3 For 4, q 4 For 0, B3' is 2'-OMe or 2'-F, q 5 For 7, T3' is 2'-F, q 6 For 1, B4' is 2'-F, and q 7is 1; wherein there are two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5'-end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5'-end of the antisense strand) of the antisense strand, and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5'-end of the antisense strand) of the antisense strand. The RNAi agent further comprises 5'-P and a targeting ligand. In one embodiment, 5'-P is located at the 5'-end of the antisense strand, and the targeting ligand is located at the 3'-end of the sense strand.

[0640] In one embodiment, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, q 4 is 0, B3' is 2'-OMe or 2'-F, q 5 is 7, T3' is 2'-F, q 6 is 1, B4' is 2'-F, and q 7 is 1; wherein there are two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5'-end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5'-end of the antisense strand) of the antisense strand, and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5'-end of the antisense strand) of the antisense strand. The RNAi agent further comprises 5'-PS and a targeting ligand. In one embodiment, 5'-PS is located at the 5'-end of the antisense strand, and the targeting ligand is located at the 3'-end of the sense strand.

[0641] In one embodiment, B1 is 2'-OMe or 2'-F, n 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, q 4is 0, B3’ is 2’-OMe or 2’-F, q 5 is 7, T3’ is 2’-F, q 6 is 1, B4’ is 2’-F, and q 7 is 1; wherein there are two internucleotide phosphorothioate link modifications within positions 1-5 of the sense strand (counting from the 5’ end of the sense strand), and two internucleotide phosphorothioate link modifications at positions 1 and 2 of the antisense strand (counting from the 5’ end of the antisense strand), and two internucleotide phosphorothioate link modifications within positions 18-23 of the antisense strand (counting from the 5’ end of the antisense strand). The RNAi agent further comprises 5'-VP (e.g., 5’-E-VP, 5’-Z-VP or a combination thereof) and a targeting ligand. In one embodiment, the 5’-VP is located at the 5' end of the antisense strand, and the targeting ligand is located at the 3' end of the sense strand.

[0642] In one embodiment, B1 is 2’-OMe or 2’-F, n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, n 3 is 7, n 4 is 0, B3 is 2’-OMe, n 5 is 3, B1’ is 2’-OMe or 2’-F, q 1 is 9, T1’ is 2’-F, q 2 is 1, B2’ is 2’-OMe or 2’-F, q 3 is 4, q 4 is 0, B3’ is 2’-OMe or 2’-F, q 5 is 7, T3’ is 2’-F, q 6 is 1, B4’ is 2’-F, and q 7 is 1; wherein there are two internucleotide phosphorothioate link modifications within positions 1-5 of the sense strand (counting from the 5’ end of the sense strand), and two internucleotide phosphorothioate link modifications at positions 1 and 2 of the antisense strand (counting from the 5’ end of the antisense strand), and two internucleotide phosphorothioate link modifications within positions 18-23 of the antisense strand (counting from the 5’ end of the antisense strand). The RNAi agent further comprises 5’-PS 2 and a targeting ligand. In one embodiment, the 5’-PS 2 is located at the 5’ end of the antisense strand, and the targeting ligand is located at the 3’ end of the sense strand.

[0643] In one embodiment, B1 is 2’-OMe or 2’-F, n 1 is 8, T1 is 2’F, n 2 is 3, B2 is 2’-OMe, n 3 is 7, n 4is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, q 1 is 9, T1' is 2'-F, q 2 is 1, B2' is 2'-OMe or 2'-F, q 3 is 4, q 4 is 0, B3' is 2'-OMe or 2'-F, q 5 is 7, T3' is 2'-F, q 6 is 1, B4' is 2'-F, and q 7 is 1; wherein there are two phosphorothioate internucleotide linkage modifications within positions 1 - 5 (counting from the 5'-end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5'-end of the antisense strand) of the antisense strand, and two phosphorothioate internucleotide linkage modifications within positions 18 - 23 (counting from the 5'-end of the antisense strand) of the antisense strand. The RNAi agent further comprises 5'-deoxy-5'-C-malonyl and a targeting ligand. In one embodiment, the 5'-deoxy-5'-C-malonyl is located at the 5'-end of the antisense strand, and the targeting ligand is located at the 3'-end of the sense strand.

[0644] In certain embodiments, the RNAi agent of the present invention comprises

[0645] (a) A sense strand having the following:

[0646] (i) A length of 21 nucleotides;

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

[0648] (iii) 2'-F modifications at positions 1, 3, 5, 7, 9 to 11, 13, 17, 19 and 21, and 2'-OMe modifications at positions 2, 4, 6, 8, 12, 14 to 16, 18 and 20 (counting from the 5'-end);

[0649] and

[0650] (b) An antisense strand having the following:

[0651] (i) A length of 23 nucleotides;

[0652] (ii) 2'-OMe modifications at positions 1, 3, 5, 9, 11 to 13, 15, 17, 19, 21 and 23, and 2'F modifications at positions 2, 4, 6 to 8, 10, 14, 16, 18, 20 and 22 (counting from the 5'-end); and;

[0653] (iii) phosphorothioate internucleotide linkages between the 21st and 22nd nucleotides and between the 22nd and 23rd nucleotides (counting from the 5' end);

[0654] wherein the dsRNA agent has two nucleotide overhangs at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0655] In another specific embodiment, the RNAi agent of the present invention comprises:

[0656] (a) a sense strand having:

[0657] (i) a length of 21 nucleotides;

[0658] (ii) an ASGPR ligand attached to the 3' end, wherein the ASGPR ligand comprises three GalNAc derivatives attached via a trivalent branched linker;

[0659] (iii) 2'-F modifications at positions 1, 3, 5, 7, 9 to 11, 13, 15, 17, 19 and 21, and 2'-OMe modifications at positions 2, 4, 6, 8, 12, 14, 16, 18 and 20 (counting from the 5' end);

[0660] and

[0661] (iv) phosphorothioate internucleotide linkages between the 1st and 2nd nucleotides and between the 2nd and 3rd nucleotides (counting from the 5' end);

[0662] and

[0663] (b) an antisense strand having:

[0664] (i) a length of 23 nucleotides;

[0665] (ii) 2'-OMe modifications at positions 1, 3, 5, 7, 9, 11 to 13, 15, 17, 19 and 21 to 23, and 2'F modifications at positions 2, 4, 6, 8, 10, 14, 16, 18 and 20 (counting from the 5' end); and

[0666] (iii) phosphorothioate internucleotide linkages between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 21st and 22nd nucleotides and between the 22nd and 23rd nucleotides (counting from the 5' end); wherein the RNAi agent has two nucleotide overhangs at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0667] In another specific embodiment, the RNAi agent of the present invention comprises:

[0668] (a) a sense strand having:

[0669] (i) a length of 21 nucleotides;

[0670] (ii) an ASGPR ligand attached to the 3'-end, wherein the ASGPR ligand comprises three GalNAc derivatives attached via a trivalent branched linker;

[0671] (iii) 2'-OMe modifications at positions 1 to 6, 8, 10, and 12 to 21, 2'-F modifications at positions 7 and 9, and a deoxynucleotide (e.g., dT) at position 11 (counting from the 5'-end); and

[0672] (iv) phosphorothioate internucleotide linkages between the nucleotides at positions 1 and 2 and between the nucleotides at positions 2 and 3 (counting from the 5'-end);

[0673] and

[0674] (b) an antisense strand having:

[0675] (i) a length of 23 nucleotides;

[0676] (ii) 2'-OMe modifications at positions 1, 3, 7, 9, 11, 13, 15, 17, and 19 to 23, and 2'-F modifications at positions 2, 4 to 6, 8, 10, 12, 14, 16, and 18 (counting from the 5'-end); and

[0677] (iii) phosphorothioate internucleotide linkages between the nucleotides at positions 1 and 2, between the nucleotides at positions 2 and 3, between the nucleotides at positions 21 and 22, and between the nucleotides at positions 22 and 23 (counting from the 5'-end);

[0678] wherein the RNAi agent has two nucleotide overhangs at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand.

[0679] In another specific embodiment, the RNAi agent of the present invention comprises:

[0680] (a) a sense strand having:

[0681] (i) a length of 21 nucleotides;

[0682] (ii) an ASGPR ligand attached to the 3'-end, wherein the ASGPR ligand comprises three GalNAc derivatives attached via a trivalent branched linker;

[0683] (iii) 2'-OMe modifications at positions 1 to 6, 8, 10, 12, 14, and 16 to 21, and 2'-F modifications at positions 7,

[0684] 9, 11, 13, and 15; and

[0685] (iv) phosphorothioate internucleotide linkages between the nucleotide at position 1 and the nucleotide at position 2 and between the nucleotide at position 2 and the nucleotide at position 3 (counting from the 5'-end);

[0686] and

[0687] (b) an antisense strand having:

[0688] (i) a length of 23 nucleotides;

[0689] (ii) 2'-OMe modifications at positions 1, 5, 7, 9, 11, 13, 15, 17, 19 and 21 to 23, and 2'-F modifications at positions 2 to 4, 6, 8, 10, 12, 14, 16, 18 and 20 (counting from the 5'-end); and

[0690] (iii) phosphorothioate internucleotide linkages between the nucleotide at position 1 and the nucleotide at position 2, between the nucleotide at position 2 and the nucleotide at position 3, between the nucleotide at position 21 and the nucleotide at position 22, and between the nucleotide at position 22 and the nucleotide at position 23 (counting from the 5'-end);

[0691] wherein the RNAi agent has two nucleotide overhangs at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand.

[0692] In another specific embodiment, the RNAi agent of the present invention comprises:

[0693] (a) a sense strand having:

[0694] (i) a length of 21 nucleotides;

[0695] (ii) an ASGPR ligand attached to the 3'-end, wherein the ASGPR ligand comprises three GalNAc derivatives attached via a trivalent branched linker;

[0696] (iii) 2'-OMe modifications at positions 1 to 9 and 12 to 21, and 2'-F modifications at positions 10 and 11; and

[0697] (iv) phosphorothioate internucleotide linkages between the nucleotide at position 1 and the nucleotide at position 2 and between the nucleotide at position 2 and the nucleotide at position 3 (counting from the 5'-end);

[0698] and

[0699] (b) an antisense strand having:

[0700] (i) a length of 23 nucleotides;

[0701] (ii) 2'-OMe modifications at positions 1, 3, 5, 7, 9, 11 to 13, 15, 17, 19 and 21 to 23, and 2'-F modifications at positions 2, 4, 6, 8, 10, 14, 16, 18 and 20 (counting from the 5' end); and

[0702] (iii) phosphorothioate internucleotide linkages between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 21st and 22nd nucleotides, and between the 22nd and 23rd nucleotides (counting from the 5' end);

[0703] wherein the RNAi agent has two nucleotide overhangs at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0704] In another specific embodiment, the RNAi agent of the present invention comprises:

[0705] (a) a sense strand having:

[0706] (i) a length of 21 nucleotides;

[0707] (ii) an ASGPR ligand attached to the 3' end, wherein the ASGPR ligand comprises three GalNAc derivatives attached via a trivalent branched linker;

[0708] (iii) 2'-F modifications at positions 1, 3, 5, 7, 9 to 11 and 13, and 2'-OMe modifications at positions 2, 4, 6, 8, 12 and 14 to 21; and

[0709] (iv) phosphorothioate internucleotide linkages between the 1st and 2nd nucleotides and between the 2nd and 3rd nucleotides (counting from the 5' end);

[0710] and

[0711] (b) an antisense strand having:

[0712] (i) a length of 23 nucleotides;

[0713] (ii) 2'-OMe modifications at positions 1, 3, 5 to 7, 9, 11 to 13, 15, 17 to 19 and 21 to 23, and 2'-F modifications at positions 2, 4, 8, 10, 14, 16 and 20 (counting from the 5' end);

[0714] and

[0715] (iii) phosphorothioate internucleotide linkages between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 21st and 22nd nucleotides, and between the 22nd and 23rd nucleotides (counting from the 5' end);

[0716] Among them, the RNAi agent has two nucleotide overhangs at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand.

[0717] In another specific embodiment, the RNAi agent of the present invention comprises:

[0718] (a) A sense strand having the following:

[0719] (i) A length of 21 nucleotides;

[0720] (ii) An ASGPR ligand attached to the 3'-end, wherein the ASGPR ligand comprises three GalNAc derivatives attached via a trivalent branched linker;

[0721] (iii) 2'-OMe modifications at positions 1, 2, 4, 6, 8, 12, 14, 15, 17 and 19 to 21, and 2'-F modifications at positions 3, 5, 7, 9 to 11, 13, 16 and 18; and

[0722] (iv) Phosphorothioate internucleotide linkages between nucleotides at positions 1 and 2 and between nucleotides at positions 2 and 3 (counting from the 5'-end);

[0723] And

[0724] (b) An antisense strand having the following:

[0725] (i) A length of 25 nucleotides;

[0726] (ii) 2'-OMe modifications at positions 1, 4, 6, 7, 9, 11 to 13, 15, 17 and 19 to 23, 2'-F modifications at positions 2, 3, 5, 8, 10, 14, 16 and 18, and deoxynucleotides (such as dT) at positions 24 and 25 (counting from the 5'-end); and

[0727] (iii) Phosphorothioate internucleotide linkages between nucleotides at positions 1 and 2, between nucleotides at positions 2 and 3, between nucleotides at positions 21 and 22, and between nucleotides at positions 22 and 23 (counting from the 5'-end);

[0728] Among them, the RNAi agent has four nucleotide overhangs at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand.

[0729] In another specific embodiment, the RNAi agent of the present invention comprises:

[0730] (a) A sense strand having the following:

[0731] (i) A length of 21 nucleotides;

[0732] (ii) An ASGPR ligand attached to the 3'-end, wherein the ASGPR ligand comprises three GalNAc derivatives attached via a trivalent branched linker;

[0733] (iii) 2'-OMe modifications at positions 1 to 6, 8, and 12 to 21, and 2'-F modifications at positions 7 and 9 to 11; and

[0734] (iv) Phosphorothioate internucleotide linkages between the nucleotides at positions 1 and 2 and between the nucleotides at positions 2 and 3 (counting from the 5'-end);

[0735] and

[0736] (b) An antisense strand having the following:

[0737] (i) A length of 23 nucleotides;

[0738] (ii) 2'-OMe modifications at positions 1, 3 to 5, 7, 8, 10 to 13, 15, and 17 to 23, and 2'-F modifications at positions 2, 6, 9, 14, and 16 (counting from the 5'-end); and

[0739] (iii) Phosphorothioate internucleotide linkages between the nucleotides at positions 1 and 2, between the nucleotides at positions 2 and 3, between the nucleotides at positions 21 and 22, and between the nucleotides at positions 22 and 23 (counting from the 5'-end); wherein the RNAi agent has two nucleotide overhangs at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand.

[0740] In another specific embodiment, the RNAi agent of the present invention comprises:

[0741] (a) A sense strand having the following:

[0742] (i) A length of 21 nucleotides;

[0743] (ii) An ASGPR ligand attached to the 3'-end, wherein the ASGPR ligand comprises three GalNAc derivatives attached via a trivalent branched linker;

[0744] (iii) 2'-OMe modifications at positions 1 to 6, 8, and 12 to 21, and 2'-F modifications at positions 7 and 9 to 11; and

[0745] (iv) Phosphorothioate internucleotide linkages between the nucleotides at positions 1 and 2 and between the nucleotides at positions 2 and 3 (counting from the 5'-end);

[0746] and

[0747] (b) An antisense strand having the following:

[0748] (i) a length of 23 nucleotides;

[0749] (ii) 2'-OMe modifications at positions 1, 3 to 5, 7, 10 to 13, 15 and 17 to 23, and 2'-F modifications at positions

[0750] 2, 6, 8, 9, 14 and 16 (counting from the 5' end); and

[0751] (iii) phosphorothioate internucleotide linkages between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 21st and 22nd nucleotides, and between the 22nd and 23rd nucleotides (counting from the 5' end);

[0752] wherein the RNAi agent has two nucleotide overhangs at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0753] In another specific embodiment, the RNAi agent of the present invention comprises:

[0754] (a) a sense strand having the following:

[0755] (i) a length of 19 nucleotides;

[0756] (ii) an ASGPR ligand attached to the 3' end, wherein the ASGPR ligand comprises three GalNAc derivatives attached via a trivalent branched linker;

[0757] (iii) 2'-OMe modifications at positions 1 to 4, 6 and 10 to 19, and 2'-F modifications at positions

[0758] 5 and 7 to 9; and

[0759] (iv) phosphorothioate internucleotide linkages between the 1st and 2nd nucleotides and between the 2nd and 3rd nucleotides (counting from the 5' end);

[0760] and

[0761] (b) an antisense strand having the following:

[0762] (i) a length of 21 nucleotides;

[0763] (ii) 2'-OMe modifications at positions 1, 3 to 5, 7, 10 to 13, 15 and 17 to 21, and 2'-F modifications at positions

[0764] 2, 6, 8, 9, 14 and 16 (counting from the 5' end); and

[0765] (iii) phosphorothioate internucleotide linkages between nucleotides 1 and 2, between nucleotides 2 and 3, between nucleotides 19 and 20, and between nucleotides 20 and 21 (counting from the 5' end);

[0766] wherein the RNAi agent has two nucleotide overhangs at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0767] In certain embodiments, the iRNA used in the methods of the invention is an agent selected from the agents listed in any of Tables 2-3. These agents may further comprise a ligand.

[0768] IV. REVERSIR Compounds of the Invention

[0769] The invention also provides REVERSIR compounds that abrogate the activity of the general dsRNA agents of the invention. The design, synthesis, and appropriate modification of REVERSIR compounds are disclosed in WO 2016 / 100716, WO 2019 / 036612, and U.S. 2017 / 369872, each of which is incorporated herein by reference.

[0770] Generally, the REVERSIR compounds of the invention are single-stranded oligonucleotides (oligomers) that are 6-30 nucleotides in length. The nucleotide sequence of the oligonucleotide can be at least about 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary) complementary to any of the antisense strand nucleotide sequences in Table 2 or Table 3.

[0771] Exemplary REVERSIR compounds of the invention are presented in Table 4 herein.

[0772] In certain embodiments, the REVERSIR compounds are chemically modified oligomeric compounds as compared to naturally occurring oligomers such as DNA or RNA.

[0773] Thus, in certain embodiments, the REVERSIR compounds of the invention comprise at least one modified nucleotide, i.e., at least one modified monomer. In other embodiments, substantially all of the nucleotides of the oligonucleotide are modified nucleotides. In still other embodiments, all of the nucleotides of the oligonucleotide are modified nucleotides.

[0774] In certain such embodiments, the REVERSIR compounds of the invention comprise one or more high-affinity monomers. In certain embodiments, such high-affinity monomers are selected from monomers (e.g., nucleosides and nucleotides) comprising 2'-modified sugars, including but not limited to: BNA and monomers (e.g., nucleosides and nucleotides) having a 2'-substituent, such as allyl, amino, azido, thio, O-allyl, O—C 1 -C 10 alkyl, —OCF 3 、O—(CH 2 ) 2 -O—CH 3 、2′-O(CH 2 ) 2 SCH 3 、O—(CH 2 ) 2 -O—N(Rm)(Rn) or O—CH 2 -C(═O)—N(Rm)(Rn), where each Rm and Rn is independently H or substituted or unsubstituted C 1 -C 10 alkyl.

[0775] In certain embodiments, the REVERSIR compounds of the invention comprise one or more β-D-methyleneoxy (4′-CH 2 -O-2′) LNA monomers.

[0776] In certain embodiments, the REVERSIR compounds of the invention comprise one or more α-D-methyleneoxy (4′-CH 2 -O-2′) LNA monomers.

[0777] In certain embodiments, the REVERSIR compounds of the invention comprise one or more (S)-cEt monomers.

[0778] In certain embodiments, the REVERSIR compounds of the invention comprise one or more high-affinity monomers, provided that the compound does not comprise a nucleotide containing 2′-O(CH 2 ) n H, where n is from one to six.

[0779] In certain embodiments, the REVERSIR compounds of the invention comprise one or more high-affinity monomers, provided that the compound does not comprise a nucleotide containing 2′-OCH 3 or 2′-O(CH 2 ) 2 OCH 3 .

[0780] In certain embodiments, the REVERSIR compounds of the present invention comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) high-affinity monomers, provided that the compound does not comprise α-L-methylenoxy (4′-CH 2 -O-2′) LNA.

[0781] In certain embodiments, the REVERSIR compounds of the present invention comprise one or more high-affinity monomers, provided that the compound does not comprise β-D-methylenoxy (4′-CH 2 -O-2′) LNA.

[0782] In certain embodiments, the REVERSIR compounds of the present invention comprise one or more high-affinity monomers, provided that the compound does not comprise α-L-methylenoxy (4′-CH 2 -O-2′) LNA or β-D-methylenoxy (4′-CH 2 -O-2′) LNA.

[0783] The naturally occurring base moieties of nucleosides are generally heterocyclic bases. The two most common types of such heterocyclic bases are purines and pyrimidines. For nucleosides containing a pentofuranose, the phosphate group can be linked to the 2′, 3′ or 5′ hydroxyl moiety of the sugar. In forming an oligonucleotide, these phosphate groups covalently link adjacent nucleosides to each other to form a linear polymeric compound. Within an oligonucleotide, the phosphate groups generally refer to the internucleoside or inter-nucleotide backbone that forms the oligonucleotide. The naturally occurring bond or backbone of RNA and DNA is a 3′ to 5′ phosphodiester bond.

[0784] In addition to "unmodified" or "natural" nucleobases such as the purine nucleobases adenine (A) and guanine (G), and the pyrimidine nucleobases thymine (T), cytosine (C), and uracil (U), many modified nucleobases or nucleobase analogs known to those skilled in the art are also suitable for the compounds described herein. Unmodified or natural nucleobases can be modified or replaced to provide oligonucleotides with improved properties. For example, nuclease-resistant oligonucleotides can be prepared using these bases or with synthetic and natural nucleobases (e.g., inosine, xanthine, hypoxanthine, nubularine, isoguanisine, or tubercidin) and any of the oligomers described herein. Alternatively, substitution or modified analogs of any of the above bases and "universal bases" can be employed. When natural bases are replaced with unnatural and / or universal bases, the nucleotides are said to contain modified nucleobases and / or nucleobase modifications as described herein. Modified nucleobases and / or nucleobase modifications also include natural, unnatural, and universal bases that contain a coupling moiety, such as a ligand described herein. Preferred coupling moieties for coupling to a nucleobase include a cationic amino group that is coupled to the nucleobase via a suitable alkyl, alkenyl, or linker having an amide bond.

[0785] The REVERSIR compounds described herein may also include nucleobase (commonly referred to in the art as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Exemplary modified nucleobases include, but are not limited to, other synthetic and natural nucleobases such as inosine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidin, 2-(halo)adenine, 2-(alkyl)adenine, 2-(propyl)adenine, 2-(amino)adenine, 2-(aminoalkyl)adenine, 2-(aminopropyl)adenine, 2-(methylthio)-N 6 -(isopentenyl)adenine, 6-(alkyl)adenine, 6-(methyl)adenine, 7-(deaza)adenine, 8-(alkenyl)adenine, 8-(alkyl)adenine, 8-(alkynyl)adenine, 8-(amino)adenine, 8-(halo)adenine, 8-(hydroxy)adenine, 8-(thioalkyl)adenine, 8-(thiol)adenine, N 6 -(isopentyl)adenine, N 6 -(methyl)adenine, N 6 ,N 6-(dimethyl)adenine, 2-(alkyl)guanine, 2-(propyl)guanine, 6-(alkyl)guanine, 6-(methyl)guanine, 7-(alkyl)guanine, 7-(methyl)guanine, 7-(deazaguanine), 8-(alkyl)guanine, 8-(alkenyl)guanine, 8-(alkynyl)guanine, 8-(amino)guanine, 8-(halo)guanine, 8-(hydroxy)guanine, 8-(thioalkyl)guanine, 8-(thiol)guanine, N-(methyl)guanine, 2-(thio)cytosine, 3-(deaza)-5-(aza)cytosine, 3-(alkyl)cytosine, 3-(methyl)cytosine, 5-(alkyl)cytosine, 5-(alkynyl)cytosine, 5-(halo)cytosine, 5-(methyl)cytosine, 5-(propynyl)cytosine, 5-(propynyl)cytosine, 5-(trifluoromethyl)cytosine, 6-(azo)cytosine, N 4 -(acetyl)cytosine, 3-(3-amino-3-carboxypropyl)uracil, 2-(thio)uracil, 5-(methyl)-2-(thio)uracil, 5-(methylaminomethyl)-2-(thio)uracil, 4-(thio)uracil, 5-(methyl)-4-(thio)uracil, 5-(methylaminomethyl)-4-(thio)uracil, 5-(methyl)-2,4-(dithio)uracil, 5-(methylaminomethyl)-2,4-(dithio)uracil, 5-(2-aminopropyl)uracil, 5-(alkyl)uracil, 5-(alkynyl)uracil, 5-(allylamino)uracil, 5-(aminoallyl)uracil, 5-(aminoalkyl)uracil, 5-(guanidinoalkyl)uracil, 5-(1,3-diazol-1-ylalkyl)uracil, 5-(cyanoalkyl)uracil, 5-(dialkylaminoalkyl)uracil, 5-(dimethylaminoalkyl)uracil, 5-(halo)uracil, 5-(methoxy)uracil, uracil-5-hydroxyacetic acid, 5-(methoxycarbonylmethyl-2-(thio)uracil, 5-(methoxycarbonyl-methyl)uracil, 5-(propynyl)uracil, 5-(propynyl)uracil, 5-(trifluoromethyl)uracil, 6-(azo)uracil, dihydrouracil, N 3-(methyl)uracil, 5-uracil (i.e., pseudouracil), 2-(thio)pseudouracil, 4-(thio)pseudouracil, 2,4-(dithio)pseudouracil, 5-(alkyl)pseudouracil, 5-(methyl)pseudouracil, 5-(alkyl)-2-(thio)pseudouracil, 5-(methyl)-2-(thio)pseudouracil, 5-(alkyl)-4-(thio)pseudouracil, 5-(methyl)-4-(thio)pseudouracil, 5-(alkyl)-2,4-(dithio)pseudouracil, 5-(methyl)-2,4-(dithio)pseudouracil, 1-substituted pseudouracil, 1-substituted 2(thio)-pseudouracil, 1-substituted 4-(thio)pseudouracil, 1-substituted 2,4-(dithio)pseudouracil, 1-(aminocarbonylvinyl)-pseudouracil, 1-(aminocarbonylvinyl)-2(thio)-pseudouracil, 1-(aminocarbonylvinyl)-4-(thio)pseudouracil, 1-(aminocarbonylvinyl)-2,4-(dithio)pseudouracil, 1-(aminoalkylaminocarbonylvinyl)-pseudouracil, 1-(aminoalkylamino-carbonylvinyl)-2(thio)-pseudouracil, 1-(aminoalkylaminocarbonylvinyl)-4-(thio)pseudouracil, 1-(aminoalkylaminocarbonylvinyl)-2,4-(dithio)pseudouracil, 1,3-(diazido)-2-(oxo)-phenoxazin-1-yl, 1-(azido)-2-(thio)-3-(azido)-phenoxazin-1-yl, 1,3-(diazido)-2-(oxo)-phenothiazin-1-yl, 1-(azido)-2-(thio)-3-(azido)-phenothiazin-1-yl, 7-substituted 1,3-(diazido)-2-(oxo)-phenoxazin-1-yl, 7-substituted 1-(azido)-2-(thio)-3-(azido)-phenoxazin-1-yl, 7-substituted 1,3-(diazido)-2-(oxo)-phenothiazin-1-yl, 7-substituted 1-(azido)-2-(thio)-3-(azido)-phenothiazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diazido)-2-(oxo)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1-(azido)-2-(thio)-3-(azido)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diazido)-2-(oxo)-phenothiazin-1-yl, 7-(aminoalkylhydroxy)-1-(azido)-2-(thio)-3-(azido)-phenothiazin-1-yl, 7-(guanidylhydroxy)-1,3-(diazido)-2-(oxo)-phenoxazin-1-yl, 7-(guanidylhydroxy)-1-(azido)-2-(thio)-3-(azido)-phenoxazin-1-yl, 7-(guanidyl-hydroxy)-1,3-(diazido)-2-(oxo)-phenothiazin-1-yl, 7-(guanidylhydroxy)-1-(azido)-2-(thio)-3-(azido)-phenothiazin-1-yl, 1,3,5-(triaza)-2,6-(dioxo)naphthalene, inosine, xanthine, hypoxanthine, nubularine, tubercidin, isoguanosine, inosinyl, 2-aza-inosinyl, 7-deaza-inosinyl, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl, nitroindazolyl, aminoindolyl, pyrrolopyrimidinyl, 3-(methyl)isoquinolinone, 5-(methyl)isoquinolinone, 3-(methyl)-7-(propargyl)isoquinolinone, 7-(aza)indolyl, 6-(methyl)-7-(aza)indolyl, imidazopyridinyl, 9-(methyl)-imidazopyridinyl, pyrrolopyrazinyl, isoquinolinone, 7-(propargyl)isoquinolinone, propargyl-7-(aza)indolyl, 2,4,5-(trimethyl)phenyl, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl, phenyl, naphthyl, anthryl, phenanthryl, pyrenyl, stilbenyl, tetracenyl, pentacenyl, difluorotoluene, 4-(fluoro)-6-(methyl)benzimidazole, 4-(methyl)benzimidazole, 6-(azo)thymine, 2-pyridone, 5-nitroindole, 3-nitropyrrole, 6-(aza)pyrimidine, 2-(amino)purine, 2,6-(diamino)purine, 5-substituted pyrimidine, N, 2 -substituted purine, N 6 -substituted purine, O 6 -substituted purine, substituted 1,2,4-triazole, pyrrolo-pyrimidin-2-one-3-yl, 6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, para-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, bis-ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, bis-ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, pyridopyrimidin-3-yl, 2-oxo-7-amino-pyridopyrimidin-3-yl, 2-oxo-pyridopyrimidin-3-yl, or any O-alkylated or N-alkylated derivative thereof. Alternatively, substituted or modified analogs of any of the above bases and "universal bases" may be employed.

[0786] As used herein, a universal nucleobase is any nucleobase that can base pair with all four naturally occurring nucleobases without substantially affecting melting behavior, recognition by intracellular enzymes, or the activity of an oligonucleotide duplex. Some exemplary universal nucleobases include, but are not limited to, 2,4-difluorotoluene, nitropyrrolyl, nitroindolyl, 8-aza-7-deazaguanine, 4-fluoro-6-methylbenzimidazole, 4-methylbenzimidazole, 3-methylisoquinolinone, 5-methylisoquinolinone, 3-methyl-7-propynylisoquinolinone, 7-azaindolyl, 6-methyl-7-azaindolyl, imidazopyridinyl, 9-methyl-imidazopyridinyl, pyrrolopyrazinyl, isoquinolinone, 7-propynylisoquinolinone, propynyl-7-azaindolyl, 2,4,5-trimethylphenyl, 4-methylindolyl, 4,6-dimethylindolyl, phenyl, naphthyl, anthracenyl, phenanthrenyl, pyrenyl, stilbenyl, tetracene, pentacene, and their structural derivatives (see, e.g., Loakes, 2001, Nucleic Acids Research, 29, 2437-2447).

[0787] Further nucleobases include those disclosed in U.S. Patent Publication No. 3,687,808; those disclosed in International Application No. PCT / US09 / 038425, filed Mar. 26, 2009; those disclosed in Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J.I., ed., John Wiley & Sons, 1990; those disclosed by English et al., Angewandte Chemie, International Edition, 1991, 30, 613; those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijin, P., ed., Wiley-VCH, 2008; and those disclosed by Sanghvi, Y.S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S.T. and Lebleu, B., eds., CRC Press, 1993. All of the foregoing are hereby incorporated by reference.

[0788] In certain embodiments, the modified nucleobases are nucleobases that are fairly similar in structure to the parent nucleobase, such as, for example, 7-deazapurines, 5-methylcytosine, or G-clamps. In certain embodiments, nucleobase analogs include more complex structures, such as, for example, tricyclic phenoxazine nucleobase analogs. Methods for preparing the above-mentioned modified nucleobases are well known to those skilled in the art.

[0789] In certain embodiments, the REVERSIR compounds of the present invention include at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) G-clamp nucleobases selected from the following:

[0790]

[0791] where n is 0, 1, 2, 3, 4, 5, or 6.

[0792] The REVERSIR compounds provided herein may comprise one or more monomers, including nucleosides or nucleotides having modified sugar moieties. For example, the furanose ring of a nucleoside can be modified in various ways, including but not limited to adding substituents, bridging two non-bicyclic atoms to form locked nucleic acid or bicyclic nucleic acid. In certain embodiments, the compound comprises one or more monomers that are LNA.

[0793] In certain embodiments of locked nucleic acid, the 2'-position of the furanone group is linked to the 4'-position by a linker independently selected from the following: –[C(R1)(R2)] n –, –[C(R1)(R2)] n –O–, –[C(R1)(R2)] n -N(R1)–, –[C(R1)(R2)] n -N(R1)–O-, —[C(R1R2)] n -O-N(R1)—, –C(R1)═C(R2)–O–, –C(R1)═N–, –C(R1)═N–O-, —C(═NR1)-, —C(═NR1)-O-, —C(═O)—, —C(═O)O—, —C(═S)—, —C(═S)O—, —C(═S)S—, —O—, —Si(R1)2-, —S(═O) x - and —N(R1)-;

[0794] where:

[0795] x is 0, 1, or 2;

[0796] n is 1, 2, 3, or 4;

[0797] Each R1 and R2 is independently H, a protecting group, a hydroxyl group, a C1-C12 alkyl group, a substituted C1-C12 alkyl group, a C2-C12 alkenyl group, a substituted C2-C12 alkenyl group, a C2-C12 alkynyl group, a substituted C2-C12 alkynyl group, a C5-C20 aryl group, a substituted C5-C20 aryl group, a heterocyclic radical, a substituted heterocyclic radical, a heteroaryl group, a substituted heteroaryl group, a C5-C7 cycloaliphatic radical, a substituted C5-C7 cycloaliphatic radical, a halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, an acyl group (C(═O)—H), a substituted acyl group, CN, a sulfonyl group (S(═O)2-J1) or a sulfinyl group (S(═O)-J1); and

[0798] Each J1 and J2 is independently H, a C1-C12 alkyl group, a substituted C1-C12 alkyl group, a C2-C12 alkenyl group, a substituted C2-C12 alkenyl group, a C2-C12 alkynyl group, a substituted C2-C12 alkynyl group, a C5-C20 aryl group, a substituted C5-C20 aryl group, an acyl group (C(═O)—H), a substituted acyl group, a heterocyclic radical, a substituted heterocyclic radical, a C1-C12 aminoalkyl group, a substituted C1-C12 aminoalkyl group or a protecting group.

[0799] In one embodiment, each linker of the LNA compound is independently —[C(R1)(R2)]n-, —[C(R1)(R2)]n-O—, —C(R1R2)-N(R1)-O— or —C(R1R2)-O—N(R1)-. In another embodiment, each said linker is independently 4′-CH 2 -2′, 4′-(CH 2 ) 2 -2′, 4′-(CH 2 ) 3 -2′, 4′-CH 2 -O-2′, 4′-(CH 2 ) 2 -O-2′, 4′-CH 2 -O—N(R1)-2′ and 4′-CH 2 -N(R1)-O-2′-, where each R1 is independently H, a protecting group or a C1-C12 alkyl group.

[0800] Certain LNAs have been prepared and disclosed in the patent literature as well as in the scientific literature (Singh et al., Chem. Commun., 1998, 4, 455 - 456; Koshkin et al., Tetrahedron, 1998, 54, 3607 - 3630; Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633 - 5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219 - 2222; WO 94 / 14226; WO 2005 / 021570; Singh et al., J. Org. Chem., 1998, 63, 10035 - 10039; Examples of issued U.S. patents and published applications that disclose LNA include, for example, U.S. Patent Nos. 7,053,207; 6,268,490; 6,770,748; 6,794,499; 7,034,133; and 6,525,191; and; U.S. Pre - grant Publication Nos. 2004 - 0171570; 2004 - 0219565; 2004 - 0014959; 2003 - 0207841; 2004 - 0143114; and 20030082807.

[0801] Also provided herein are LNAs in which the 2′ - hydroxy group of the ribose sugar ring is linked to the 4′ - carbon atom of the sugar ring, thereby forming a methyleneoxy (4′ - CH 2 -O - 2′) linkage to form a bicyclic sugar moiety (see Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558 - 561; Braasch et al., Chem. Biol., 2001, 8 1 - 7; and Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239 - 243; see also U.S. Patent Nos. 6,268,490 and 6,670,461). The linkage can be a methylene (—CH 2 -) group that bridges the 2′ - oxygen atom and the 4′ - carbon atom, for which the bicyclic moiety is termed methyleneoxy (4′ - CH 2 -O - 2′) LNA; in the case where an ethylene group is in this position, the term ethyleneoxy (4′ - CH 2 CH 2 -O - 2′) LNA is used (Singh et al., Chem. Commun., 1998, 4, 455 - 456; Morita et al., Bioorganic Medicinal Chemistry, 2003, 11, 2211 - 2226). Methyleneoxy (4′ - CH 2-O-2′) LNA and other bicyclic sugar analogues exhibit very high duplex thermal stability with complementary DNA and RNA (Tm = +3 to +10 °C), stability against 3′-exonuclease degradation, and good solubility. Potent and non-toxic antisense oligonucleotides containing BNA have been described (Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638).

[0802] The methyleneoxy (4′-CH 2 -O-2′) LNA isomer is α-L-methyleneoxy (4′-CH 2 -O-2′) LNA, which has shown excellent stability against 3′-exonuclease. α-L-methyleneoxy (4′-CH 2 -O-2′) LNA has been incorporated into antisense gapmers and chimeras that show potent antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).

[0803] Methyleneoxy (4′-CH 2 -O-2′) The synthesis and preparation of LNA monomers of adenine, cytosine, guanine, 5-methylcytosine, thymine, and uracil, as well as their oligomerization and nucleic acid recognition properties, have been described (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). BNA and its preparation are also described in WO 98 / 39352 and WO 99 / 14226.

[0804] Methyleneoxy (4′-CH 2 -O-2′) LNA, phosphorothioate-methyleneoxy (4′-CH 2 -O-2′) LNA and analogues of 2′-thio-LNA have also been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). The preparation of locked nucleic acid analogues containing oligodeoxynucleotide duplexes as nucleic acid polymerase substrates has also been described (Wengel et al., WO 99 / 14226). In addition, the synthesis of 2′-amino-LNA (a novel conformationally restricted high-affinity oligonucleotide analogue) has also been described in the art (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). Additionally, 2′-amino- and 2′-methylamino-LNA have been prepared, and the thermal stability of their duplexes with complementary RNA and DNA strands has been previously reported.

[0805] Modified sugar moieties are well known and can be used to alter (usually increase) the affinity of an antisense compound for its target and / or increase nuclease resistance. Representative lists of preferred modified sugars include, but are not limited to, bicyclic modified sugars, including methyleneoxy (4′-CH 2 -O-2′) LNA and ethyleneoxy (4′-(CH 2 ) 2 -O-2′ bridge) ENA; substituted sugars, especially 2′-substituted sugars having 2′-F, 2′-OCH 3 or 2′-O(CH 2 ) 2 -OCH 3 substituents; and 4′-thio modified sugars. The sugar can also be replaced with a sugar mimetic group, etc. Methods for preparing modified sugars are well known to those skilled in the art. Some representative patents and publications teaching the preparation of such modified sugars include, but are not limited to, U.S. Patent Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; 5,700,920; 6,531,584; and 6,600,032; and WO2005 / 121371.

[0806] Examples of "oxygen"-modified 2′-hydroxy groups include alkoxy or aryloxy (OR, e.g., R = H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar); polyethylene glycol (PEG), O(CH 2 CH 2 O) n CH 2 CH 2 OR, n = 1 - 50; "locked" nucleic acid (LNA), in which the furanose moiety of the nucleoside includes a bridge connecting two carbon atoms on the furanose ring, thus forming a bicyclic system; O-AMINE or O-(CH 2 ) n AMINE (n = 1 - 10, AMINE = NH 2 ; alkylamino, dialkylamino, heterocyclic, arylamino, diarylamino, heteroarylamino, diheteroarylamino, ethylenediamine, or polyamine); and O-CH 2 CH 2 (NCH 2 CH 2NMe 2 ) 2 。

[0807] "Deoxy" modifications include hydrogen (i.e., deoxyribose sugars, which are particularly relevant to single-stranded overhangs); halogens (e.g., fluorine); amino groups (e.g., NH 2 ; alkylamino, dialkylamino, heterocyclic, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); NH(CH 2 CH 2 NH) n CH 2 CH 2 -AMINE (AMINE = NH 2 ; alkylamino, dialkylamino, heterocyclic, arylamino, diarylamino, heteroarylamino, or diheteroarylamino); -NHC(O)R (R = alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar); cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; thioalkyl; alkyl; cycloalkyl; aryl; alkenyl, and alkynyl, which may optionally be substituted with, for example, amino functionality.

[0808] Other suitable 2'-modifications, such as modified MOE, are described in U.S. Patent Application Publication No. 20130130378, the content of which is incorporated herein by reference.

[0809] Modifications at the 2'-position may be present in the arabinose configuration. The term "arabinose configuration" refers to placing a substituent on C2' of ribose in the same configuration as the 2'-OH in arabinose.

[0810] The sugar may contain two different modifications on the same carbon in the sugar, such as dual modifications. The sugar moiety may also contain one or more carbons with a stereochemical configuration opposite to the corresponding carbon in ribose. Thus, REVERSIR compounds may include one or more monomers containing, for example, arabinose as the sugar. The monomer may have an α-linkage at the 1'-position of the sugar, such as an α-nucleoside. The monomer may also have an opposite configuration at the 4'-position, such as C5' and H4' or the substituents replacing them are exchanged with each other. When C5' and H4' or the substituents replacing them are exchanged with each other, the sugar is considered to be modified at the 4'-position.

[0811] The REVERSIR compounds of the present invention may also include abasic sugars, i.e., sugars lacking a nucleobase at C-1′ or having other chemical groups replacing the nucleobase at C1'. See, for example, U.S. Patent No. 5,998,203, the content of which is incorporated herein in its entirety. These abasic sugars may further contain modifications at one or more of the constituent sugar atoms. The REVERSIR compounds may also contain one or more sugars that are L-isomers, such as L-nucleosides. Modifications to the sugar moiety may also include replacing with sulfur, optionally substituted nitrogen, or CH2 Group replacement of 4'-O. In some embodiments, the linkage between C1' and the nucleobase is in the α configuration.

[0812] Sugar modifications may also include acyclic nucleotides, where the C-C bonds between ribose carbons (e.g., C1'-C2', C2'-C3', C3'-C4', C4'-O4', C1'-O4') are absent and / or at least one ribose carbon or oxygen (e.g., C1', C2', C3', C4' or O4') is independently or combinatorially deleted from the nucleotide. In some embodiments, the acyclic nucleotide is where B is a modified or unmodified nucleobase, R 1 and R 2 are independently H, halogen, OR 3 or alkyl; R 3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar.

[0813] In some embodiments, the sugar modifications are selected from the group consisting of: 2'-H, 2′-O-Me (2′-O-methyl), 2′-O-MOE (2′-O-methoxyethyl), 2'-F, 2′-O-[2-(methylamino)-2-oxoethyl] (2′-O-NMA), 2'-S-methyl, 2'-O-CH 2 -(4'-C) (LNA), 2'-O-CH 2 CH 2 -(4'-C) (ENA), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) and gem 2’-OMe / 2’F‘ of 2’-O-Me in the arabinose configuration.

[0814] It should be understood that when a particular nucleotide is linked to the next nucleotide through its 2'-position, the sugar modifications described herein may be at the 3'-position of that particular nucleotide (e.g., the nucleotide linked through its 2'-position is placed on the sugar). The modification at the 3'-position may be in the xylose configuration. The term "xylose configuration" refers to placing a substituent on C3’ of ribose in the same configuration as the 3'-OH in xylose.

[0815] The hydrogen attached to C4’ and / or C1’ can be replaced by a straight-chain or branched-chain optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, where the backbone of the alkyl, alkenyl and alkynyl may contain one or more O, S, S(O), SO 2, N(R’), C(O), N(R’)C(O)O, OC(O)N(R’), CH(Z’), a phosphorus linkage, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted heterocycle or an optionally substituted cycloalkyl, where R’ is hydrogen, acyl or an optionally substituted aliphatic group, and Z’ is selected from the group consisting of: OR 11 , COR 11 , CO 2 R 11 , NR 21 R 31 , CONR 21 R 31 , CON(H)NR 21 R 31 , ONR 21 R 31 , CON(H)N=CR 41 R 51 , N(R 21 ), C(=NR 31 ), NR 21 R 31 , N(R 21 ), C(O)NR 21 R 31 , N(R 21 ), C(S)NR 21 R 31 , OC(O)NR 21 R 31 , SC(O)NR 21 R 31 , N(R 21 ), C(S)OR 11 , N(R 21 ), C(O)OR 11 , N(R 21 ), C(O)SR 11 , N(R 21 ), N=CR 41 R 51 , ON=CR 41 R 51 , SO 2 R 11 , SOR 11 , SR 11 and a substituted or unsubstituted heterocycle; each occurrence of R 21 and R 31 is independently hydrogen, acyl, unsubstituted or substituted aliphatic, aryl, heteroaryl, heterocycle, OR 11 , COR 11 , CO 2 R 11 or NR11 R 11 ’; or R 21 and R 31 together with the atom to which they are attached form a heterocycle; each occurrence of R 41 and R 51 independently is hydrogen, acyl, unsubstituted or substituted aliphatic, aryl, heteroaryl, heterocycle, OR 11 , COR 11 or CO 2 R 11 or NR 11 R 11 ’; and R 11 and R 11 ’ independently is hydrogen, an aliphatic group, a substituted aliphatic group, aryl, heteroaryl or heterocycle. In some embodiments, the hydrogen attached to C4' of the 5'-terminal nucleotide is replaced.

[0816] In some embodiments, C4' and C5' together form an optionally substituted heterocycle, preferably containing at least one -PX(Y)-, where X is H, OH, OM, SH, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted alkylamino or optionally substituted dialkylamino, where each occurrence of M independently is an alkali metal or a transition metal, where the total charge is +1; and Y is O, S or NR', where R' is hydrogen or optionally substituted aliphatic. Preferably such modification is located at the 5'-terminus of the oligonucleotide.

[0817] In certain embodiments, LNA includes a bicyclic nucleotide having the following formula:

[0818]

[0819] wherein:

[0820] Bx is a heterocyclic base moiety;

[0821] T1 is hydrogen or a hydroxyl protecting group;

[0822] T2 is hydrogen, a hydroxyl protecting group or a reactive phosphorus group;

[0823] Z is C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted C1-C6 alkyl, substituted C2-C6 alkenyl, substituted C2-C6 alkynyl, acyl, substituted acyl or substituted amide.

[0824] In one embodiment, each substituent group is independently mono- or multi-substituted with an optionally protected substituent independently selected from the following: halogen, oxo, hydroxy, OJ1, NJ1J2, SJ1, N3, OC(═X)J1, OC(═X)NJ1J2, NJ3C(═X)NJ1J2, and CN, where each J1, J2, and J3 is independently hydrogen or C1-C6 alkyl, and X is O, S, or NJ1.

[0825] In certain such embodiments, each substituent group is independently mono- or multi-substituted with a substituent independently selected from the following: halogen, oxo, hydroxy, OJ1, NJ1J2, SJ1, N3, OC(═X)J1, and NJ3C(═X)NJ1J2, where each J1, J2, and J3 is independently hydrogen, C1-C6 alkyl, or substituted C1-C6 alkyl, and X is O or NJ1.

[0826] In certain embodiments, the Z group is a C1-C6 alkyl substituted with one or more Xx, where each Xx is independently OJ1, NJ1J2, SJ1, N3, OC(═X)J1, OC(═X)NJ1J2, NJ3C(═X)NJ1J2, or CN; where each J1, J2, and J3 is independently H or C1-C6 alkyl, and X is O, S, or NJ1. In another embodiment, the Z group is a C1-C6 alkyl substituted with one or more Xx, where each Xx is independently halogen (e.g., fluorine), hydroxy, alkoxy (e.g., CH3O-), substituted alkoxy, or azide.

[0827] In certain embodiments, the Z group is —CH2Xx, where Xx is OJ1, NJ1J2, SJ1, N3, OC(═X)J1, OC(═X)NJ1J2, NJ3C(═X)NJ1J2, or CN; where each J1, J2, and J3 is independently H or C1-C6 alkyl, and X is O, S, or NJ1. In another embodiment, the Z group is —CH2Xx, where Xx is halogen (e.g., fluorine), hydroxy, alkoxy (e.g., CH3O—), or azide.

[0828] In certain such embodiments, the Z group is of the (R)-configuration:

[0829]

[0830] In certain such embodiments, the Z group is of the (S)-configuration:

[0831]

[0832] In certain embodiments, each of T1 and T2 is a hydroxyl protecting group. Preferred lists of hydroxyl protecting groups include benzyl, benzoyl, 2,6-dichlorobenzyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, mesylate, tosylate, dimethoxytrityl (DMT), 9-phenylxanthin-9-yl (Pixyl), and 9-(p-methoxyphenyl)xanthin-9-yl (MOX). In certain embodiments, T1 is a hydroxyl protecting group selected from acetyl, benzyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, and dimethoxytrityl, with the more preferred hydroxyl protecting group T1 being 4,4'-dimethoxytrityl.

[0833] In certain embodiments, T2 is a reactive phosphorus group, with preferred reactive phosphorus groups including diisopropylcyanoethoxy phosphoramidite and H-phosphonate. In certain embodiments, T1 is 4,4'-dimethoxytrityl and T2 is diisopropylcyanoethoxy phosphoramidite.

[0834] In certain embodiments, the REVERSIR compound has at least one monomer having the formula:

[0835]

[0836] or having the formula:

[0837]

[0838] or having the formula:

[0839]

[0840] wherein

[0841] Bx is a heterocyclic base moiety;

[0842] T3 is H, a hydroxyl protecting group, a linked coupling group, or a internucleoside linking group attached to a nucleoside, nucleotide, oligonucleoside, oligonucleotide, monomer subunit, or oligomeric compound;

[0843] T4 is H, a hydroxyl protecting group, a linked coupling group, or a internucleoside linking group attached to a nucleoside, nucleotide, oligonucleoside, oligonucleotide, monomer subunit, or oligomeric compound;

[0844] wherein at least one of T3 and T4 is an internucleoside linking group attached to a nucleoside, nucleotide, oligonucleoside, oligonucleotide, monomer subunit, or oligomeric compound; and

[0845] Z is C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted C1-C6 alkyl, substituted C2-C6 alkenyl, substituted C2-C6 alkynyl, acyl, substituted acyl, or substituted amide.

[0846] In one embodiment, each substituent group is independently mono- or polysubstituted with an optionally protected substituent independently selected from the group consisting of: halogen, oxo, hydroxy, OJ1, NJ1J2, SJ1, N3, OC(═X)J1, OC(═X)NJ1J2, NJ3C(═X)NJ1J2, and CN, where each J1, J2, and J3 is independently H or C1-C6 alkyl, and X is O, S, or NJ1.

[0847] In one embodiment, each substituent group is independently mono- or polysubstituted with a substituent group independently selected from the group consisting of: halogen, oxo, hydroxy, OJ1, NJ1J2, SJ1, N3, OC(═X)J1, and NJ3C(═X)NJ1J2, where each J1, J2, and J3 is independently H or C1-C6 alkyl, and X is O or NJ1.

[0848] In certain such embodiments, at least one Z is C1-C6 alkyl or substituted C1-C6 alkyl. In certain embodiments, each Z is independently C1-C6 alkyl or substituted C1-C6 alkyl. In certain embodiments, at least one Z is C1-C6 alkyl. In certain embodiments, each Z is independently C1-C6 alkyl. In certain embodiments, at least one Z is methyl. In certain embodiments, each Z is methyl. In certain embodiments, at least one Z is ethyl. In certain embodiments, each Z is ethyl. In certain embodiments, at least one Z is substituted C1-C6 alkyl. In certain embodiments, each Z is independently substituted C1-C6 alkyl. In certain embodiments, at least one Z is substituted methyl. In certain embodiments, each Z is substituted methyl. In certain embodiments, at least one Z is substituted ethyl. In certain embodiments, each Z is substituted ethyl.

[0849] In certain embodiments, at least one substituent group is C1-C6 alkoxy (e.g., at least one Z is C1-C6 alkyl substituted with one or more C1-C6 alkoxy groups). In another embodiment, each substituent group is independently C1-C6 alkoxy (e.g., each Z is independently C1-C6 alkyl substituted with one or more C1-C6 alkoxy groups).

[0850] In certain embodiments, at least one C1-C6 alkoxy substituent is CH3O— (e.g., at least one Z is CH 3 OCH 2 -). In another embodiment, each C1-C6 alkoxy substituent is CH 3 O— (e.g., each Z is CH 3 OCH 2 -).

[0851] In certain embodiments, at least one substituent is a halogen (e.g., at least one Z is a C1-C6 alkyl group substituted with one or more halogens). In certain embodiments, each substituent is independently a halogen (e.g., each Z is independently a C1-C6 alkyl group substituted with one or more halogens). In certain embodiments, at least one halogen substituent is fluorine (e.g., at least one Z is CH 2 FCH 2 -, CHF 2 CH 2 -, or CF 3 CH 2 -). In certain embodiments, each halogen substituent is fluorine (e.g., each Z ...

Claims

1. A general double-stranded ribonucleic acid (dsRNA) agent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises at least 15 consecutive nucleotides, and no more than 3 nucleotides are different from any of the antisense strand nucleotide sequences in Table 2.

2. A general double-stranded ribonucleic acid (dsRNA) agent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises at least 15 consecutive nucleotides, no more than 3 nucleotides are different from any of the sense strand nucleotide sequences in Table 2, and the antisense strand comprises at least 15 consecutive nucleotides, and no more than 3 nucleotides are different from any of the antisense strand nucleotide sequences in Table 2.

3. A general double-stranded ribonucleic acid (dsRNA) agent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a region complementary to any of the target nucleotide sequences in Table 3.

4. The general dsRNA agent according to any one of claims 1-3, wherein the dsRNA agent comprises at least one modified nucleotide.

5. The general dsRNA agent according to any one of claims 1-4, wherein substantially all nucleotides of the sense strand are modified nucleotides; substantially all nucleotides of the antisense strand are modified nucleotides; or substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides.

6. The general dsRNA agent according to any one of claims 1-5, wherein all nucleotides of the sense strand are modified nucleotides; all nucleotides of the antisense strand are modified nucleotides; or all nucleotides of the sense strand and all nucleotides of the antisense strand are modified nucleotides.

7. The general dsRNA agent according to any one of claims 4-6, wherein at least one modified nucleotide is selected from the group consisting of: deoxynucleotide, 3'-terminal deoxythymidine (dT) nucleotide, 2'-O-methyl modified nucleotide, 2'-fluoro modified nucleotide, 2'-deoxy modified nucleotide, locked nucleic acid, unlocked nucleic acid, conformationally restricted nucleotide, constrained ethyl nucleotide, abasic nucleotide, 2'-amino modified nucleotide, 2'-O-allyl modified nucleotide, 2'-C-alkyl modified nucleotide, 2'-hydroxy modified nucleotide, 2'-methoxyethyl modified nucleotide, 2'-O-alkyl modified nucleotide, morpholino nucleotide, phosphoramidate, nucleotide containing unnatural base, tetrahydropyran modified nucleotide, 1,5-anhydrohexitol modified nucleotide, cyclohexenyl modified nucleotide, nucleotide containing phosphorothioate group, nucleotide containing methylphosphonate group, nucleotide containing 5'-phosphate, nucleotide containing 5'-phosphate mimetic, thermally labile nucleotide, ethylene glycol modified nucleotide (GNA), nucleotide containing 2'-phosphate and 2-O-(N-methylacetamide) modified nucleotide; and combinations thereof.

8. The general dsRNA agent according to any one of claims 4-6, wherein the modification on the modified nucleotide is selected from the group consisting of: LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2'-hydroxy and ethylene glycol; and combinations thereof.

9. The general dsRNA agent according to any one of claims 4-6, wherein at least one modified nucleotide is selected from the group consisting of: deoxynucleotide, 2'-O-methyl modified nucleotide, 2'-fluoro modified nucleotide, 2'-deoxy modified nucleotide, ethylene glycol modified nucleotide (GNA), nucleotide containing 2'-phosphate, and vinyl phosphonate nucleotide; and combinations thereof.

10. The general dsRNA agent according to any one of claims 4-6, wherein at least one modification on the modified nucleotide is a thermally labile nucleotide modification.

11. The general dsRNA agent according to claim 10, wherein the thermally labile nucleotide modification is selected from the group consisting of: abasic modification, mismatch with the opposing nucleotide in the duplex, and labile sugar modification, 2'-deoxy modification, acyclic nucleotide, unlocked nucleic acid (UNA), and glycerol nucleic acid (GNA).

12. The general dsRNA agent according to any one of claims 1-11, wherein the length of the double-stranded region is 19-30 nucleotide pairs.

13. The general dsRNA agent according to claim 12, wherein the length of the double-stranded region is 19-25 nucleotide pairs.

14. The general dsRNA agent according to claim 12, wherein the length of the double-stranded region is 19-23 nucleotide pairs.

15. The general dsRNA agent according to claim 12, wherein the length of the double-stranded region is 23-27 nucleotide pairs.

16. The general dsRNA agent according to claim 12, wherein the length of the double-stranded region is 21-23 nucleotide pairs.

17. The general dsRNA agent according to any one of claims 1-16, wherein the length of each strand is independently not more than 30 nucleotides.

18. The general dsRNA agent according to any one of claims 1-17, wherein the length of the sense strand is 21 nucleotides and the length of the antisense strand is 23 nucleotides.

19. The general dsRNA agent according to any one of claims 3-18, wherein the length of the complementary region is at least 17 nucleotides.

20. The general dsRNA agent according to any one of claims 1-19, wherein at least one strand comprises a 3' overhang of at least 1 nucleotide.

21. The general dsRNA agent according to any one of claims 1-19, wherein at least one strand comprises a 3' overhang of at least 2 nucleotides.

22. The general dsRNA agent according to any one of claims 1-21, which further comprises a ligand.

23. The general dsRNA agent according to claim 22, wherein the ligand is conjugated to the 3' end of the sense strand of the dsRNA agent.

24. The general dsRNA agent according to claim 22 or 23, wherein the ligand is an N-acetylgalactosamine (GalNAc) derivative.

25. The general dsRNA agent according to any one of claims 22-24, wherein the ligand is one or more GalNAc derivatives attached via a monovalent, divalent or trivalent branched linker.

26. The general dsRNA agent according to claim 24 or 25, wherein the ligand is 27. The general dsRNA agent according to claim 26, wherein the dsRNA agent is conjugated to the ligand as shown in the following scheme and, wherein X is O or S.

28. The general dsRNA agent according to claim 27, wherein X is O.

29. The general dsRNA agent according to any one of claims 1-28, wherein the dsRNA agent further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.

30. The general dsRNA agent according to claim 29, wherein the phosphorothioate or methylphosphonate internucleotide linkage is located at the 3' end of one strand.

31. The general dsRNA agent according to claim 30, wherein the strand is the antisense strand.

32. The general dsRNA agent according to claim 30, wherein the strand is the sense strand.

33. The general dsRNA agent according to claim 29, wherein the phosphorothioate or methylphosphonate internucleotide linkage is located at the 5' terminus of one strand.

34. The general dsRNA agent according to claim 33, wherein the strand is the antisense strand.

35. The general dsRNA agent according to claim 33, wherein the strand is the sense strand.

36. The general dsRNA agent according to claim 29, wherein the phosphorothioate or methylphosphonate internucleotide linkage is located at both the 5' and 3' termini of one strand.

37. The general dsRNA agent according to claim 36, wherein the strand is the antisense strand.

38. The general dsRNA agent according to any one of claims 1-37, wherein the base pair at the 5' end position 1 of the antisense strand of the duplex is an AU base pair.

39. A cell comprising the general dsRNA agent according to any one of claims 1-38.

40. A vector comprising the general dsRNA agent according to any one of claims 1-38.

41. The vector according to claim 40, which is an expression vector.

42. The vector according to claim 40 or 41, which is a viral vector.

43. The vector according to claim 42, wherein the viral vector is an adeno-associated (AAV) vector.

44. The vector according to claim 42 or 43, wherein the viral vector is a bicistronic vector.

45. The vector according to any one of claims 40-44, which further comprises a transgene.

46. A cell comprising the vector according to any one of claims 40-45.

47. A pharmaceutical composition comprising the general dsRNA agent according to any one of claims 1-38 or the carrier according to any one of claims 40-45 and a pharmaceutically acceptable carrier.

48. The pharmaceutical composition according to claim 47, wherein the dsRNA agent or the carrier is in a buffer-free solution.

49. The pharmaceutical composition according to claim 48, wherein the buffer-free solution is saline or water.

50. The pharmaceutical composition according to claim 47, wherein the dsRNA agent or the carrier is in a buffered solution.

51. The pharmaceutical composition according to claim 50, wherein the buffered solution comprises acetate, citrate, prolamine, carbonate or phosphate or any combination thereof.

52. The pharmaceutical composition according to claim 51, wherein the buffered solution is phosphate buffered saline (PBS).

53. A REVERSIR compound that eliminates the iRNA activity of the general dsRNA agent according to any one of claims 1-38.

54. A REVERSIR compound comprising a single-stranded oligonucleotide having a length of 6-30 nucleotides and a nucleotide sequence comprising at least about 90% complementarity to the nucleotide sequence of any one of the antisense strands in Table 2 or Table 3.

55. The REVERSIR compound according to claim 54, wherein the oligonucleotide is 100% complementary to the nucleotide sequence of any one of the antisense strands in Table 2 or Table 3.

56. The REVERSIR compound according to claim 54 or 55, wherein the oligonucleotide comprises at least one modified nucleotide.

57. The REVERSIR compound according to any one of claims 54-56, wherein substantially all nucleotides of the oligonucleotide are modified nucleotides.

58. The REVERSIR compound according to claim 57, wherein all nucleotides of the oligonucleotide are modified nucleotides.

59. The REVERSIR compound according to any one of claims 56-58, wherein at least one modified nucleotide comprises a modified nucleobase.

60. The REVERSIR compound according to claim 59, wherein the modified nucleobase is 5'-methylcytosine.

61. The REVERSIR compound according to any one of claims 56-60, wherein at least one modified nucleotide comprises a modified sugar.

62. The REVERSIR compound according to claim 61, wherein the modified sugar is selected from the group consisting of 2'-O-methoxyethyl modified sugar, 2'-methoxy modified sugar, 2'-O-alkyl modified sugar and bicyclic sugar.

63. The REVERSIR compound according to any one of claims 54-62, further comprising a ligand.

64. The REVERSIR compound according to claim 63, wherein the ligand is coupled to the 3'-end of the oligonucleotide.

65. The REVERSIR compound according to claim 63 or 64, wherein the ligand is an N-acetylgalactosamine (GalNAc) derivative.

66. The REVERSIR compound according to any one of claims 63 - 65, wherein the ligand is one or more GalNAc derivatives attached via a monovalent, divalent or trivalent branched linker.

67. The REVERSIR compound according to claim 65 or 66, wherein the ligand is 68. The REVERSIR compound according to claim 67, wherein the oligonucleotide is conjugated to the ligand as shown in the following scheme and, wherein X is O or S.

69. The REVERSIR compound according to claim 68, wherein X is O.

70. The REVERSIR compound according to any one of claims 54 - 69, wherein the oligonucleotide further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.

71. The REVERSIR compound according to any one of claims 54 - 70, wherein the oligonucleotide has a length of 6 - 15, 7 - 11 or 8 - 10 nucleotides.

72. The REVERSIR compound according to any one of claims 54 - 70, wherein the oligonucleotide has a length of 15 - 25, 17 - 25, 19 - 25 or 21 - 25 nucleotides.

73. A cell comprising the REVERSIR compound according to any one of claims 54 - 72.

74. A system for inducible expression of a transgene, the system comprising an expression vector encoding the transgene and comprising a universal iRNA target site; a universal double-stranded ribonucleic acid (dsRNA) agent comprising a sense strand and an antisense strand that form a double-stranded region that recognizes and binds to the universal iRNA target site to inhibit expression of the transgene; and optionally, a REVERSIR compound that abolishes the iRNA activity of the universal dsRNA agent to permit expression of the transgene.

75. The system according to claim 74, wherein the universal iRNA target site is located in the 5'-untranslated region (UTR) or 3'-untranslated region (UTR) of the transgene.

76. The system according to claim 74 or 75, wherein the universal dsRNA agent comprises a sense strand and an antisense strand that form a double-stranded region, wherein the antisense strand comprises at least 15 contiguous nucleotides, and wherein no more than 3 nucleotides differ from any one of the antisense strand nucleotide sequences in Table 2 or Table 3.

77. The system according to claim 74 or 75, wherein the universal dsRNA agent comprises a sense strand and an antisense strand that form a double-stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides, wherein no more than 3 nucleotides differ from any one of the sense strand nucleotide sequences in Table 2, and the antisense strand comprises at least 15 contiguous nucleotides, wherein no more than 3 nucleotides differ from any one of the antisense strand nucleotide sequences in Table 2 or Table 3.

78. The system according to claim 74 or 75, wherein the universal dsRNA agent comprises a sense strand and an antisense strand that form a double-stranded region, wherein the antisense strand comprises a region complementary to any one of the target nucleotide sequences in Table 3 or Table 4.

79. The system according to any one of claims 74 - 78, wherein the general dsRNA agent comprises at least one modified nucleotide.

80. The system according to any one of claims 74 - 79, wherein substantially all nucleotides of the sense strand are modified nucleotides; substantially all nucleotides of the antisense strand are modified nucleotides; or substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides.

81. The system according to any one of claims 74 - 80, wherein all nucleotides of the sense strand are modified nucleotides; all nucleotides of the antisense strand are modified nucleotides; or all nucleotides of the sense strand and all nucleotides of the antisense strand are modified nucleotides.

82. The system according to any one of claims 79 - 81, wherein the at least one modified nucleotide is selected from the group consisting of: deoxynucleotide, 3'-terminal deoxythymidine (dT) nucleotide, 2'-O-methyl modified nucleotide, 2'-fluoro modified nucleotide, 2'-deoxy modified nucleotide, locked nucleic acid, unlocked nucleic acid, conformationally restricted nucleotide, constrained ethyl nucleotide, abasic nucleotide, 2'-amino modified nucleotide, 2'-O-allyl modified nucleotide, 2'-C-alkyl modified nucleotide, 2'-hydroxy modified nucleotide, 2'-methoxyethyl modified nucleotide, 2'-O-alkyl modified nucleotide, morpholino nucleotide, phosphoramidate, nucleotide containing unnatural base, tetrahydropyran modified nucleotide, 1,5-anhydrohexitol modified nucleotide, cyclohexenyl modified nucleotide, nucleotide containing phosphorothioate group, nucleotide containing methylphosphonate group, nucleotide containing 5'-phosphate, nucleotide containing 5'-phosphate mimic, thermolabile nucleotide, ethylene glycol modified nucleotide (GNA), nucleotide containing 2'-phosphate, and 2-O-(N-methylacetamide) modified nucleotide; and combinations thereof.

83. The system according to any one of claims 79 - 81, wherein the modification on the nucleotide is selected from the group consisting of: LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2'-hydroxy, and ethylene glycol; and combinations thereof.

84. The system according to any one of claims 79 - 81, wherein the at least one modified nucleotide is selected from the group consisting of: deoxynucleotide, 2'-O-methyl modified nucleotide, 2'-fluoro modified nucleotide, 2'-deoxy modified nucleotide, ethylene glycol modified nucleotide (GNA), nucleotide containing 2'-phosphate, and vinyl phosphonate nucleotide; and combinations thereof.

85. The system according to any one of claims 79 - 81, wherein at least one modification on the modified nucleotide is a thermolabile nucleotide modification.

86. The system according to claim 85, wherein the thermally labile nucleotide modification is selected from the group consisting of: abasic modification, mismatch with a relative nucleotide in a duplex, and labile sugar modification, 2'-deoxy modification, acyclic nucleotides, unlocked nucleic acids (UNA), and glycerol nucleic acids (GNA).

87. The system according to any one of claims 74-86, wherein the length of the double-stranded region is 19-30 nucleotide pairs.

88. The system according to claim 86, wherein the length of the double-stranded region is 19-25 nucleotide pairs.

89. The system according to claim 86, wherein the length of the double-stranded region is 19-23 nucleotide pairs.

90. The system according to claim 86, wherein the length of the double-stranded region is 23-27 nucleotide pairs.

91. The system according to claim 86, wherein the length of the double-stranded region is 21-23 nucleotide pairs.

92. The system according to any one of claims 74-91, wherein the length of each strand is independently no more than 30 nucleotides.

93. The system according to any one of claims 74-92, wherein the length of the sense strand is 21 nucleotides and the length of the antisense strand is 23 nucleotides.

94. The system according to any one of claims 78-93, wherein the length of the complementary region is at least 17 nucleotides.

95. The system according to any one of claims 74-94, wherein at least one strand comprises a 3' overhang of at least 1 nucleotide.

96. The system according to any one of claims 74-95, wherein at least one strand comprises a 3' overhang of at least 2 nucleotides.

97. The system according to any one of claims 74-96, wherein the universal dsRNA agent further comprises a ligand.

98. The system according to claim 97, wherein the ligand is conjugated to the 3' end of the sense strand of the universal dsRNA agent.

99. The system according to claim 97 or 98, wherein the ligand is an N-acetylgalactosamine (GalNAc) derivative.

100. The system according to any one of claims 97-99, wherein the ligand is one or more GalNAc derivatives attached via a monovalent, divalent, or trivalent branched linker.

101. The system according to claim 99 or 100, wherein the ligand is 102. The system according to claim 101, wherein the universal dsRNA agent is conjugated to the ligand as shown in the following scheme and, wherein X is O or S.

103. The system according to claim 102, wherein X is O.

104. The system according to any one of claims 74-103, wherein the universal dsRNA agent further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.

105. The system according to claim 104, wherein the phosphorothioate or methylphosphonate internucleotide linkage is located at the 3' terminus of one strand.

106. The system according to claim 105, wherein the strand is the antisense strand.

107. The system according to claim 105, wherein the strand is the sense strand.

108. The system according to claim 104, wherein the phosphorothioate or methylphosphonate internucleotide bond is located at the 5'-end of a strand.

109. The system according to claim 108, wherein the strand is an antisense strand.

110. The system according to claim 108, wherein the strand is a sense strand.

111. The system according to claim 104, wherein the phosphorothioate or methylphosphonate internucleotide linkage is located at both the 5'- and 3'-ends of a strand.

112. The system according to claim 111, wherein the strand is an antisense strand.

113. The system according to any one of claims 74-112, wherein the base pair at the 5'-end position 1 of the antisense strand of the duplex is an AU base pair.

114. A system for the on-demand expression of a transgene, the system comprising an expression vector encoding the transgene and a double-stranded ribonucleic acid (dsRNA) agent targeting the transgene; wherein the expression of the transgene is inhibited by the expression of the dsRNA agent targeting the transgene; and optionally, a REVERSIR compound that eliminates the iRNA activity of the dsRNA agent thereby allowing the expression of the transgene.

115. The system according to any one of claims 74-114, wherein the REVERSIR compound comprises a single-stranded oligonucleotide having a length of 6-30 nucleotides and comprises a nucleotide sequence that is at least about 90% complementary to any one of the antisense strand nucleotide sequences in any one of Table 2 or Table 3.

116. The system according to claim 115, wherein the oligonucleotide is 100% complementary to any one of the antisense strand nucleotide sequences in any one of Table 2 or Table 3.

117. The system according to claim 115 or 116, wherein the oligonucleotide comprises at least one modified nucleotide.

118. The system according to any one of claims 115-117, wherein substantially all nucleotides of the oligonucleotide are modified nucleotides.

119. The system according to claim 118, wherein all nucleotides of the oligonucleotide are modified nucleotides.

120. The system according to any one of claims 117-119, wherein at least one modified nucleotide comprises a modified nucleobase.

121. The system according to claim 120, wherein the modified nucleobase is 5'-methylcytosine.

122. The system according to any one of claims 117-121, wherein at least one modified nucleotide comprises a modified sugar.

123. The system according to claim 122, wherein the modified sugar is selected from the group consisting of: 2'-O-methoxyethyl modified sugar, 2'-methoxy modified sugar, 2'-O-alkyl modified sugar, and bicyclic sugar.

124. The system according to any one of claims 114-123, wherein the REVERSIR compound comprises a ligand.

125. The system according to claim 124, wherein the ligand is coupled to the 3'-end of the oligonucleotide.

126. The system according to claim 124 or 125, wherein the ligand is an N-acetylgalactosamine (GalNAc) derivative.

127. The system according to any one of claims 124-126, wherein the ligand is one or more GalNAc derivatives attached via a monovalent, divalent or trivalent branched linker.

128. The system according to claim 126 or 127, wherein the ligand is:

129. The system according to claim 128, wherein the oligonucleotide is coupled to the ligand as shown in the following scheme and, wherein X is O or S.

130. The system according to claim 129, wherein X is O.

131. The system according to any one of claims 115-130, wherein the oligonucleotide further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.

132. The system according to any one of claims 115-131, wherein the oligonucleotide has a length of 6-15, 7-11 or 8-10 nucleotides.

133. The system according to any one of claims 115-131, wherein the oligonucleotide has a length of 15-25, 17-25, 19-25 or 21-25 nucleotides.

134. The system according to any one of claims 74-133, wherein the expression vector is a viral vector.

135. The system according to claim 134, wherein the viral vector is an adeno-associated (AAV) vector.

136. The system according to claim 134 or 135, wherein the viral vector is a bicistronic vector.

137. A method of regulating the expression of a transgene in a cell, the method comprising contacting the cell with an expression vector encoding the transgene and comprising a universal iRNA target site; contacting the cell with a universal double-stranded ribonucleic acid (dsRNA) agent that recognizes and binds to the universal iRNA target site to inhibit the expression of the transgene, thereby inhibiting the expression of the transgene; and, optionally further contacting the cell with a REVERSIR compound that abrogates the iRNA activity of the universal dsRNA agent to permit the expression of the transgene.

138. The method according to claim 137, wherein the cell is located within a subject.

139. A method of treating a subject in need thereof, the method comprising contacting an expression vector encoding a therapeutic transgene and comprising a universal iRNA target site administered to the subject with a universal double-stranded ribonucleic acid (dsRNA) agent that recognizes and binds to the universal iRNA target site to inhibit the expression of the transgene, thereby treating the subject.

140. The method according to claim 139, wherein the universal dsRNA agent is further contacted with a REVERSIR compound that abrogates the iRNA activity of the universal dsRNA agent to permit the expression of the transgene.

141. A method of treating a subject in need thereof, the method comprising contacting an expression vector encoding a therapeutic transgene administered to the subject and a double-stranded ribonucleic acid (dsRNA) agent targeting the transgene with a REVERSIR compound that abrogates the iRNA activity of the dsRNA agent to permit the expression of the transgene, thereby treating the subject.

142. A method of treating a subject in need thereof, the method comprising Administering to a subject an expression vector encoding a transgene and comprising a universal iRNA target site; allowing expression of the transgene until a desired level of expression has been reached; and once the desired level of transgene expression has been reached, administering to the subject a universal double-stranded ribonucleic acid (dsRNA) agent comprising a sense strand and an antisense strand that form a double-stranded region, thereby treating the subject, wherein the double-stranded region recognizes and binds to the universal iRNA target site to inhibit expression of the transgene.

143. The method of claim 142, the method further comprising administering to the subject a REVERSIR compound once the level of the transgene has dropped below the desired level of expression, wherein the REVERSIR compound abrogates the iRNA activity of the universal dsRNA agent, thereby allowing expression of the transgene.

144. The method of any one of claims 137-140, 142, and 143, wherein the universal iRNA target site is located in the 5'-untranslated region (UTR) or the 3'-untranslated region (UTR) of the transgene.

145. The method of any one of claims 137-140 and 142-144, wherein the universal dsRNA agent comprises a sense strand and an antisense strand that form a double-stranded region, wherein the antisense strand comprises at least 15 contiguous nucleotides, wherein no more than 3 nucleotides differ from any of the antisense strand nucleotide sequences in Table 2 or Table 3.

146. The method of any one of claims 137-140 and 142-144, wherein the universal dsRNA agent comprises a sense strand and an antisense strand that form a double-stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides, wherein no more than 3 nucleotides differ from any of the sense strand nucleotide sequences in Table 2; and the antisense strand comprises at least 15 contiguous nucleotides, wherein no more than 3 nucleotides differ from any of the antisense strand nucleotide sequences in Table 2 or Table 3.

147. The method of any one of claims 137-140 and 142-144, wherein the universal dsRNA agent comprises a sense strand and an antisense strand that form a double-stranded region, wherein the antisense strand comprises a region complementary to any of the target nucleotide sequences in Table 3 or Table 4.

148. The method of any one of claims 137-140 and 142-147, wherein the universal dsRNA agent comprises at least one modified nucleotide.

149. The method of any one of claims 137-140 and 142-148, wherein substantially all of the nucleotides of the sense strand are modified nucleotides; substantially all of the nucleotides of the antisense strand are modified nucleotides; or substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides.

150. The method according to any one of claims 137 - 140 and 142 - 149, wherein all nucleotides of the sense strand are modified nucleotides; all nucleotides of the antisense strand are modified nucleotides; or all nucleotides of the sense strand and all nucleotides of the antisense strand are modified nucleotides.

151. The method according to any one of claims 148 - 150, wherein at least one modified nucleotide is selected from the group consisting of: deoxynucleotide, 3'-terminal deoxythymidine (dT) nucleotide, 2'-O-methyl modified nucleotide, 2'-fluoro modified nucleotide, 2'-deoxy modified nucleotide, locked nucleic acid, unlocked nucleic acid, conformationally restricted nucleotide, constrained ethyl nucleotide, abasic nucleotide, 2'-amino modified nucleotide, 2'-O-allyl modified nucleotide, 2'-C-alkyl modified nucleotide, 2'-hydroxy modified nucleotide, 2'-methoxyethyl modified nucleotide, 2'-O-alkyl modified nucleotide, morpholino nucleotide, phosphoramide, nucleotide containing unnatural base, tetrahydropyran modified nucleotide, 1,5-anhydrohexitol modified nucleotide, cyclohexenyl modified nucleotide, nucleotide containing thiophosphate group, nucleotide containing methylphosphonate group, nucleotide containing 5'-phosphate, nucleotide containing 5'-phosphate mimic, thermally labile nucleotide, glycol modified nucleotide (GNA), nucleotide containing 2'-phosphate, and 2-O-(N-methylacetamide) modified nucleotide; and combinations thereof.

152. The method according to any one of claims 148 - 150, wherein the modification on the nucleotide is selected from the group consisting of: LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2'-hydroxy, and glycol; and combinations thereof.

153. The method according to any one of claims 148 - 150, wherein at least one modified nucleotide is selected from the group consisting of: deoxynucleotide, 2'-O-methyl modified nucleotide, 2'-fluoro modified nucleotide, 2'-deoxy modified nucleotide, glycol modified nucleotide (GNA), nucleotide containing 2'-phosphate, and vinyl phosphonate nucleotide; and combinations thereof.

154. The method according to any one of claims 148 - 150, wherein at least one modification on the modified nucleotide is a thermally labile nucleotide modification.

155. The method according to claim 154, wherein the thermally labile nucleotide modification is selected from the group consisting of: abasic modification; mismatch with the opposing nucleotide in the duplex; and labile sugar modification; 2'-deoxy modification; acyclic nucleotide; unlocked nucleic acid (UNA), and glycerol nucleic acid (GNA).

156. The method according to any one of claims 137 - 140 and 142 - 155, wherein the length of the double-stranded region is 19 - 30 nucleotide pairs.

157. The method according to claim 156, wherein the length of the double-stranded region is 19-25 nucleotide pairs.

158. The method according to claim 156, wherein the length of the double-stranded region is 19-23 nucleotide pairs.

159. The method according to claim 156, wherein the length of the double-stranded region is 23-27 nucleotide pairs.

160. The method according to claim 156, wherein the length of the double-stranded region is 21-23 nucleotide pairs.

161. The method according to any one of claims 137-140 and 142-160, wherein the length of each strand is independently no more than 30 nucleotides.

162. The method according to any one of claims 137-140 and 142-161, wherein the sense strand has a length of 21 nucleotides and the antisense strand has a length of 23 nucleotides.

163. The method according to any one of claims 137-140 and 142-162, wherein the length of the complementary region is at least 17 nucleotides.

164. The method according to any one of claims 137-140 and 142-163, wherein at least one strand comprises a 3' overhang of at least 1 nucleotide.

165. The method according to any one of claims 137-140 and 142-163, wherein at least one strand comprises a 3' overhang of at least 2 nucleotides.

166. The method according to any one of claims 137-140 and 142-165, wherein the universal dsRNA agent further comprises a ligand.

167. The method according to claim 166, wherein the ligand is conjugated to the 3' end of the sense strand of the universal dsRNA agent.

168. The method according to claim 166 or 167, wherein the ligand is an N-acetylgalactosamine (GalNAc) derivative.

169. The method according to any one of claims 166-168, wherein the ligand is one or more GalNAc derivatives attached via a monovalent, divalent or trivalent branched linker.

170. The method according to claim 168 or 169, wherein the ligand is:

171. The method according to claim 170, wherein the universal dsRNA agent is conjugated to the ligand as shown in the following scheme and, wherein X is O or S.

172. The method according to claim 171, wherein X is O.

173. The method according to any one of claims 137-140 and 142-172, wherein the universal dsRNA agent further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.

174. The method according to claim 173, wherein the phosphorothioate or methylphosphonate internucleotide linkage is located at the 3' terminus of one strand.

175. The method according to claim 174, wherein the strand is the antisense strand.

176. The method according to claim 174, wherein the strand is the sense strand.

177. The method according to claim 173, wherein the phosphorothioate or methylphosphonate internucleotide linkage is located at the 5' terminus of one strand.

178. The method according to claim 177, wherein the strand is an antisense strand.

179. The method according to claim 177, wherein the strand is a sense strand.

180. The method according to claim 173, wherein the phosphorothioate or methylphosphonate internucleotide linkage is located at both the 5' and 3' termini of a strand.

181. The system according to claim 180, wherein the strand is an antisense strand.

182. The method according to any one of claims 137 - 140 and 142 - 181, wherein the base pair at position 1 of the 5' end of the antisense strand of the duplex is an AU base pair.

183. The method according to any one of claims 137, 138, 140, 141 and 143 - 182, wherein the REVERSIR compound comprises a single - stranded oligonucleotide having a length of 6 - 30 nucleotides and comprises a nucleotide sequence that is at least 90% complementary to any one of the antisense strand nucleotide sequences in any one of Table 2 or Table 3.

184. The method according to claim 183, wherein the oligonucleotide is 100% complementary to any one of the antisense strand nucleotide sequences in any one of Table 2 or Table 3.

185. The method according to claim 183 or 184, wherein the oligonucleotide comprises at least one modified nucleotide.

186. The method according to any one of claims 183 - 185, wherein substantially all nucleotides of the oligonucleotide are modified nucleotides.

187. The method according to claim 186, wherein all nucleotides of the oligonucleotide are modified nucleotides.

188. The method according to any one of claims 183 - 187, wherein at least one modified nucleotide comprises a modified nucleobase.

189. The method according to claim 188, wherein the modified nucleobase is 5'-methylcytosine.

190. The method according to any one of claims 183 - 189, wherein at least one modified nucleotide comprises a modified sugar.

191. The method according to claim 190, wherein the modified sugar is selected from the group consisting of: 2'-O-methoxyethyl - modified sugar, 2'-methoxy - modified sugar, 2'-O-alkyl - modified sugar and bicyclic sugar.

192. The method according to any one of claims 183 - 191, wherein the REVERSIR compound comprises a ligand.

193. The method according to claim 192, wherein the ligand is coupled to the 3' end of the oligonucleotide.

194. The method according to claim 192 or 193, wherein the ligand is an N - acetylgalactosamine (GalNAc) derivative.

195. The method according to any one of claims 192 - 194, wherein the ligand is one or more GalNAc derivatives attached via a monovalent, divalent or trivalent branched linker.

196. The method according to claim 194 or 195, wherein the ligand is 197. The method according to claim 196, wherein the oligonucleotide is conjugated to the ligand as described in the following protocol And, wherein X is O or S.

198. The method according to claim 197, wherein X is O.

199. The method according to any one of claims 183 - 198, wherein the oligonucleotide further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.

200. The method according to any one of claims 183-199, wherein the oligonucleotide has a length of 6-15, 7-11 or 8-10 nucleotides.

201. The method according to any one of claims 183-200, wherein the oligonucleotide has a length of 15-25, 17-25, 19-25 or 21-25 nucleotides.

202. The method according to any one of claims 137-201, wherein the expression vector is a viral vector.

203. The method according to claim 202, wherein the viral vector is an adeno-associated (AAV) vector.

204. The method according to claim 202 or 203, wherein the viral vector is a bicistronic vector.

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