Universal non-targeting SIRNA compositions and methods of use thereof

Abstract

The present invention provides compositions, systems, and methods for regulating protein expression using iRNA compositions that carry out RNA-induced silencing complex (RISC)-mediated cleavage of universal target sequence RNAi target sequences, and REVERSIR compounds that inhibit the activity of such iRNA compositions.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 398,894, filed August 18, 2022, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Adeno-associated virus (AAV) vectors have emerged as the primary platform for most in vivo gene therapy applications, potentially enabling years, if not lifelong, treatment of disease with a single administration. 1 However, one of the key challenges that has emerged from recent clinical trials of AAV-mediated systemic gene therapy is the high interindividual variability in therapeutic protein expression at the same vector dose, which in some cases can lead to phenotoxicity at supraphysiological transgene levels. 41、42 This highlights the difficulty of extrapolating preclinical data to a therapeutically effective dose range in humans for AAV gene therapy and the need for clinically applicable approaches to modulate transgene expression after AAV administration. 2-3、43 The ability to adjust transgene dosage within a target therapeutic range or silence expression if adverse events occur represents a key feature that can maximize the safety and efficacy of AAV-based therapies.

[0003] RNA interference (RNAi) is an evolutionarily conserved mechanism by which endogenous (microRNAs) or exogenous (siRNAs, shRNAs) short non-coding RNAs downregulate gene expression of mRNA transcripts in a sequence-dependent manner. 4As a native pathway that exploits efficient cellular catalytic mechanisms, RNAi can be used to achieve robust, durable, and specific silencing of gene transcripts of interest. In recent years, several RNAi-based drugs have been successfully tested in clinical trials, demonstrating efficacy at lower doses and less frequent administration for up to 6 months compared to alternative gene silencing strategies. 44 Novel delivery solutions, along with highly chemically modified siRNA, have improved efficacy, durability, and safety, resulting in a significant expansion of the range of RNAi therapeutics, with four approved drugs and several others in clinical development. 11-13 In the liver, infrequent delivery of metabolically stabilized N-galactosamine (GalNAc)-conjugated siRNAs results in potent gene silencing that persists for months in humans, with a favorable safety and tolerability profile. 14-16 Recent studies have demonstrated that 2'-O-palmityl (C16) conjugates enable widespread distribution and sustained gene silencing in cell types in the central nervous system, eye, and lung, broadening the scope of siRNA delivery to extrahepatic tissues. 17 All these advances in RNAi-mediated therapeutic silencing of endogenous disease-associated genes also hold the potential for on-demand control of exogenously delivered transgenes in a therapeutic setting.

[0004] Due to the small footprint of RNAi elements, fully or partially complementary binding sites (usually 19–23 nucleotides) for interfering RNAs can be easily incorporated into the viral genome, usually within the 3′UTR of a vector-encoded transgene. AAV integration of binding sites for endogenous microRNAs has been exploited to improve tissue specificity of gene targeting by selectively attenuating expression in undesired cell types. 24-26Previous designs of RNAi-based on-switches have utilized ligand binding to control the processing of engineered interfering RNAs delivered along with therapeutic transgenes or to modulate the accessibility of endogenous microRNAs to their cognate binding sites on virally delivered mRNAs. 5-10 However, their applicability has been hindered by limitations imposed by endogenous microRNA expression levels, risks associated with off-targeting, and the lack of non-protein ligands or universal aptamers. 5 In contrast, exogenously delivering chemically modified siRNA overcomes the dependency on endogenous miRNAs and allows precise and flexible control of dosage. As an alternative to siRNA, which requires repeated administration at a less frequent rate, shRNA-mediated RNAi, which can be stably delivered in AAV vectors in gene therapy settings, can continuously regulate transgene expression in cis after a single treatment.

[0005] While exogenous RNAi modalities may enable low basal transgene expression, the versatility of these systems would be greatly enhanced if transgene silencing could be reversed as a means of controlling therapeutic transgene expression in the on state. A highly potent and generalizable approach for in vivo pharmacological control of RNAi using a short synthetic single-stranded oligonucleotide known as REVERSIR21 has recently been reported. REVERSIR functionally suppresses RNAi activity by acting as a synthetic high-affinity decoy that competes with and captures the RNA-induced silencing complex (RISC) loaded with a complementary siRNA antisense (guide) strand from the siRNA target mRNA. REVERSIR stably binds to the seed region of the antisense strand, thereby preventing RISC-mediated recognition and degradation of the target mRNA transcript, resulting in increased translation. Due to its modular design and versatile template for length and chemical modification, REVERSIR has been shown to potently reverse in vivo gene silencing by multiple siRNA sequences across multiple targets. The development of REVERSIR as an antidote to RNAi activity is a useful tool that can be used to control the on-state of exogenously delivered transcripts by enabling induction of transgene expression from RNAi-controlled AAV vectors.

[0006] Thus, there is a need in the art for compositions, systems, and methods that combine RNAi-mediated knockdown and rescue of gene silencing by REVERSIR as a molecular rheostat or switch for AAV-delivered transcripts. Summary of the Invention

[0007] The present invention provides compositions, systems, and methods for regulating protein expression using iRNA compositions that perform RNA-induced silencing complex (RISC)-mediated cleavage of universal target sequences (RNAi) and reversible inhibitor compounds that inhibit the activity of such iRNA compositions. The present invention also provides a controllable approach to fine-tune the magnitude and timing of therapeutic transgene expression.

[0008] The universal iRNAs of the present invention were designed to have favorable thermodynamic properties for RISC loading and RNAi function, and to have little sequence complementarity with any annotated genes in the transcriptomes of humans, cynomolgus monkeys, rats, and mice. Such universal iRNAs have been demonstrated to be potent RNAi triggers with high on-target specificity and a low tendency for off-target gene disruption. Additionally, as described herein, these universal dsRNA agents have been shown to regulate expression from exogenous vector delivery systems without causing undesired off-target silencing within the endogenous transcriptomes of humans and preclinical mammalian models. Therefore, the use of these universal iRNAs, REVERSIR molecules that inhibit the activity of these universal iRNAs, and systems containing these universal iRNAs and / or REVERSIR molecules can improve the dosage and timing of transgene induction from exogenous vector delivery systems, such as AAV vector delivery systems, thereby providing an in vivo gene therapy method that achieves long-term correction of genetic defects across a wide range of target organs with a single administration.

[0009] Thus, in one aspect, the invention provides a universal double-stranded ribonucleic acid (dsRNA) agent, comprising a sense strand and an antisense strand that form a duplex region, wherein the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ from any of the antisense strand nucleotide sequences in Table 2 by 3 or less, e.g., 3, 2, 1, or 0 nucleotides.

[0010] In another embodiment, the invention provides a universal double-stranded ribonucleic acid (dsRNA) agent, comprising a sense strand and an antisense strand which form a duplex region, wherein the sense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides that differ from any of the sense strand nucleotide sequences in Table 2 by no more than 3, e.g., 3, 2, 1, or 0 nucleotides, and the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ from any of the antisense strand nucleotide sequences in Table 2 by no more than 3, e.g., 3, 2, 1, or 0 nucleotides.

[0011] In one aspect, the invention provides a universal double-stranded ribonucleic acid (dsRNA) agent, comprising a sense strand and an antisense strand that form a duplex region, wherein the antisense strand comprises a region of complementarity to a target nucleotide sequence in any of Tables 2 or 3.

[0012] In one embodiment, the dsRNA agent includes at least one modified nucleotide.

[0013] In one embodiment, substantially all of the nucleotides in the sense strand are modified nucleotides and substantially all of the nucleotides in the antisense strand are modified nucleotides, or substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides.

[0014] In one embodiment, all nucleotides in the sense strand are modified nucleotides and all nucleotides in the antisense strand are modified nucleotides, or all nucleotides in the sense strand and all nucleotides in the antisense strand are modified nucleotides.

[0015] In one embodiment, at least one of the aforementioned modified nucleotides is a deoxy-nucleotide, a 3'-terminal deoxythymidine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, a non-locked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino modified nucleotide, a 2'-O-allyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2'-hydroxyl modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a The nucleotide is selected from the group consisting of a nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide containing a non-natural base, a tetrahydropyran-modified nucleotide, a 1,5-anhydrohexitol-modified nucleotide, a cyclohexenyl-modified nucleotide, a nucleotide containing a phosphorothioate group, a nucleotide containing a methylphosphonate group, a nucleotide containing a 5'-phosphate, a nucleotide containing a 5'-phosphate mimic, a thermally destabilized nucleotide, a glycol-modified nucleotide (GNA), a nucleotide containing a 2' phosphate, and a 2-O-(N-methylacetamido)-modified nucleotide, and combinations thereof.

[0016] In another embodiment, the modification of 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'-hydroxyl, and glycol, and combinations thereof.

[0017] In yet another embodiment, at least one of the modified nucleotides is selected from the group consisting of deoxynucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, glycol modified nucleotides (GNAs), 2' phosphate containing nucleotides, and vinyl phosphonate nucleotides, and combinations thereof.

[0018] In one embodiment, at least one of the modifications of the modified nucleotides is a thermally destabilizing nucleotide modification.

[0019] In one embodiment, the thermally destabilizing nucleotide modification is selected from the group consisting of an abasic modification, a mismatch with the opposite nucleotide in a duplex, and a destabilizing sugar modification, a 2'-deoxy modification, an acyclic nucleotide, a non-locked nucleic acid (UNA), and a glycerol nucleic acid (GNA).

[0020] In one embodiment, each strand is independently 30 nucleotides or less in length.

[0021] In one embodiment, the region of complementarity is at least 17 nucleotides in length.

[0022] In one embodiment, at least one strand comprises a 3' overhang of at least one nucleotide.

[0023] In another embodiment, at least one strand comprises a 3' overhang of at least two nucleotides.

[0024] In one embodiment, X is O.

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

[0026] In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the 3' end of one strand.

[0027] In one embodiment, the strand is the antisense strand.

[0028] In another embodiment, the strand is the sense strand.

[0029] In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the 5' end of one strand.

[0030] In one embodiment, the strand is the antisense strand.

[0031] In another embodiment, the strand is the sense strand.

[0032] In one embodiment, phosphorothioate or methylphosphonate internucleotide linkages are at both the 5' and 3' ends of one strand.

[0033] In one embodiment, the strand is the antisense strand.

[0034] The invention further provides cells comprising universal dsRNA agents of the invention as well as vectors comprising universal dsRNA agents of the invention.

[0035] In one embodiment, the vector is an expression vector.

[0036] In one embodiment, the vector is a viral vector.

[0037] In one embodiment, the viral vector is an adeno-associated viral (AAV) vector.

[0038] In one embodiment, the viral vector is a bicistronic vector.

[0039] In one embodiment, the vector of the invention further comprises a transgene, e.g., a transgene.

[0040] Also provided by the present invention are cells containing the vectors of the present invention.

[0041] The invention further provides a pharmaceutical composition comprising a universal dsRNA agent of the invention or a vector of the invention, and a pharmaceutically acceptable carrier.

[0042] In one embodiment, the dsRNA agent or vector is in an unbuffered solution.

[0043] In one embodiment, the unbuffered solution is saline or water.

[0044] In another embodiment, the buffer solution is phosphate buffered saline (PBS).

[0045] In one aspect, the invention provides REVERSIR compounds that inhibit the iRNA activity of a universal dsRNA agent of the invention.

[0046] In another embodiment, the present invention provides REVERSIR compounds comprising a single-stranded oligonucleotide 6-30 nucleotides in length, 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.

[0047] In one embodiment, the oligonucleotide is 100% complementary to any of the antisense strand nucleotide sequences in Table 2 or Table 3.

[0048] In one embodiment, the oligonucleotide comprises at least one modified nucleotide.

[0049] In one embodiment, substantially all of the nucleotides of the oligonucleotide are modified nucleotides.

[0050] In one embodiment, all of the nucleotides in the oligonucleotide are modified nucleotides.

[0051] In one embodiment, at least one of the modified nucleotides comprises a modified nucleobase.

[0052] In one embodiment, the modified nucleobase is a 5' methylcytosine.

[0053] In one embodiment, at least one of the modified nucleotides comprises a modified sugar.

[0054] In one embodiment, the modified sugar is selected from the group consisting of a 2'-O-methoxyethyl-modified sugar, a 2'-methoxy-modified sugar, a 2'-O-alkyl-modified sugar, and a bicyclic sugar.

[0055] In one embodiment, the oligonucleotide further comprises a ligand.

[0056] In one embodiment, the ligand is conjugated to the 3' end of the oligonucleotide.

[0057] In one embodiment, the ligand is an N-acetylgalactosamine (GalNAc) derivative.

[0058] In one embodiment, the ligand is one or more GalNAc derivatives attached via a monovalent, divalent, or trivalent branched linker.

[0059] In one embodiment, the ligand is: [ka]

[0060] In one embodiment, the universal dsRNA agent is conjugated to a ligand as shown in the following schematic, where X is O or S: [ka]

[0061] In one embodiment, X is O.

[0062] In one embodiment, the oligonucleotide further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.

[0063] In one embodiment, the oligonucleotide is 6-15, 7-11, or 8-10 nucleotides in length.

[0064] In another embodiment, the oligonucleotide is 15-25, 17-25, 19-25, or 21-25 nucleotides in length.

[0065] The present invention further provides cells comprising the REVERSIR compounds of the present invention.

[0066] In one aspect, the present invention provides a system for on-demand expression of a transgene, the system comprising an expression vector encoding the transgene, a universal iRNA target site, a universal double-stranded ribonucleic acid (dsRNA) agent, and a REVERSIR compound comprising a sense strand and an antisense strand that form a duplex region that recognizes and binds to the universal iRNA target site, thereby inhibiting expression of the transgene, and optionally suppressing the activity of the universal dsRNA agent iRNA, thereby enabling expression of the transgene.

[0067] The universal iRNA target site can be located in the 5' untranslated region or the 3' untranslated region (UTR) of the transgene.

[0068] In one embodiment, a universal double-stranded ribonucleic acid (dsRNA) agent includes a sense strand and an antisense strand that form a duplex region, wherein the antisense strand includes at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ by 3 or less, e.g., 3, 2, 1, or 0 nucleotides, from any antisense strand nucleotide sequence in either Table 2 or Table 3.

[0069] In one embodiment, a universal double-stranded ribonucleic acid (dsRNA) agent includes a sense strand and an antisense strand that form a duplex region, wherein the antisense strand includes at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ by 3 or less, e.g., 3, 2, 1, or 0 nucleotides, from any antisense strand nucleotide sequence in either Table 2 or Table 3.

[0070] In one embodiment, a universal dsRNA agent comprises a sense strand and an antisense strand that form a duplex region, and the antisense strand comprises a region of complementarity to any target nucleotide sequence in either Table 3 or Table 4.

[0071] In one embodiment, the universal dsRNA agent includes at least one modified nucleotide.

[0072] In one embodiment, substantially all of the nucleotides in the sense strand are modified nucleotides and substantially all of the nucleotides in the antisense strand are modified nucleotides, or substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides.

[0073] In one embodiment, substantially all of the nucleotides in the sense strand are modified nucleotides and substantially all of the nucleotides in the antisense strand are modified nucleotides, or substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides.

[0074] In one embodiment, at least one of the aforementioned modified nucleotides is a deoxy-nucleotide, a 3'-terminal deoxythymidine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, a non-locked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino modified nucleotide, a 2'-O-allyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2'-hydroxyl modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a The nucleotide is selected from the group consisting of a nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide containing a non-natural base, a tetrahydropyran-modified nucleotide, a 1,5-anhydrohexitol-modified nucleotide, a cyclohexenyl-modified nucleotide, a nucleotide containing a phosphorothioate group, a nucleotide containing a methylphosphonate group, a nucleotide containing a 5'-phosphate, a nucleotide containing a 5'-phosphate mimic, a thermally destabilized nucleotide, a glycol-modified nucleotide (GNA), a nucleotide containing a 2' phosphate, and a 2-O-(N-methylacetamido)-modified nucleotide, and combinations thereof.

[0075] In another embodiment, the modification of 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'-hydroxyl, and glycol, and combinations thereof.

[0076] In yet another embodiment, at least one of the modified nucleotides is selected from the group consisting of deoxynucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, glycol modified nucleotides (GNAs), 2' phosphate containing nucleotides, and vinyl phosphonate nucleotides, and combinations thereof.

[0077] In another embodiment, at least one of the modifications of the modified nucleotides is a thermally destabilizing nucleotide modification.

[0078] In one embodiment, the thermally destabilizing nucleotide modification is selected from the group consisting of an abasic modification, a mismatch with the opposite nucleotide in a duplex, and a destabilizing sugar modification, a 2'-deoxy modification, an acyclic nucleotide, a non-locked nucleic acid (UNA), and a glycerol nucleic acid (GNA).

[0079] The duplex region can be 19 to 30 nucleotide pairs in length, 19 to 25 nucleotide pairs in length, 19 to 23 nucleotide pairs in length, 23 to 27 nucleotide pairs in length, or 21 to 23 nucleotide pairs in length.

[0080] In one embodiment, each strand is independently 30 nucleotides or less in length.

[0081] In one embodiment, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

[0082] In one embodiment, the region of complementarity is at least 17 nucleotides in length.

[0083] In one embodiment, at least one strand comprises a 3' overhang of at least one nucleotide.

[0084] In another embodiment, at least one strand comprises a 3' overhang of at least two nucleotides.

[0085] In one embodiment, the universal dsRNA agent further comprises a ligand.

[0086] In one embodiment, the ligand is conjugated to the 3' end of the sense strand of the universal dsRNA agent.

[0087] In one embodiment, the ligand is an N-acetylgalactosamine (GalNAc) derivative.

[0088] In one embodiment, the ligand is one or more GalNAc derivatives attached via a monovalent, divalent, or trivalent branched linker.

[0089] In one embodiment, the ligand is: [ka]

[0090] In one embodiment, the universal dsRNA agent is conjugated to a ligand as shown in the following schematic, where X is O or S: [ka]

[0091] In one embodiment, X is O.

[0092] In one embodiment, the universal dsRNA agent further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.

[0093] In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the 3' end of one strand.

[0094] In one embodiment, the strand is the antisense strand.

[0095] In another embodiment, the strand is the sense strand.

[0096] In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the 5' end of one strand.

[0097] In one embodiment, the strand is the antisense strand.

[0098] In another embodiment, the strand is the sense strand.

[0099] In one embodiment, phosphorothioate or methylphosphonate internucleotide linkages are at both the 5' and 3' ends of one strand.

[0100] In one embodiment, the strand is the antisense strand.

[0101] 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.

[0102] In another aspect, the invention provides a system for on-demand expression of a transgene, the system including an expression vector encoding the transgene and a double-stranded ribonucleic acid (dsRNA) agent that targets the transgene, wherein expression of the transgene is inhibited by expression of the dsRNA agent that targets the transgene, and optionally a REVERSIR compound that suppresses iRNA activity of the dsRNA agent, thereby allowing expression of the transgene.

[0103] In one embodiment, the REVERSIR compound comprises a single-stranded oligonucleotide of 6 to 30, e.g., 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, e.g., 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 in length, 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.

[0104] In one embodiment, the oligonucleotide is 100% complementary to any of the antisense strand nucleotide sequences in Table 2 or Table 3.

[0105] In one embodiment, the oligonucleotide comprises at least one modified nucleotide.

[0106] In one embodiment, substantially all of the nucleotides of the oligonucleotide are modified nucleotides.

[0107] In one embodiment, all of the nucleotides in the oligonucleotide are modified nucleotides.

[0108] In one embodiment, at least one of the modified nucleotides comprises a modified nucleobase.

[0109] In one embodiment, the modified nucleobase is a 5' methylcytosine.

[0110] In one embodiment, at least one of the modified nucleotides comprises a modified sugar.

[0111] In one embodiment, the modified sugar is selected from the group consisting of a 2'-O-methoxyethyl-modified sugar, a 2'-methoxy-modified sugar, a 2'-O-alkyl-modified sugar, and a bicyclic sugar.

[0112] In one embodiment, the REVERSIR compound comprises a ligand.

[0113] In one embodiment, the ligand is conjugated to the 3' end of the oligonucleotide.

[0114] In one embodiment, the ligand is an N-acetylgalactosamine (GalNAc) derivative.

[0115] In one embodiment, the ligand is one or more GalNAc derivatives attached via a monovalent, divalent, or trivalent branched linker.

[0116] In one embodiment, the ligand is: [ka]

[0117] In one embodiment, the oligonucleotide is conjugated to a ligand as shown in the following scheme, where X is O or S: [ka]

[0118] In one embodiment, X is O.

[0119] In one embodiment, the oligonucleotide further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.

[0120] In one embodiment, the oligonucleotide is 6 to 15, 7 to 11, or 8 to 10 nucleotides in length.

[0121] In one embodiment, the oligonucleotide is 15-25, 17-25, 19-25, or 21-25 nucleotides in length.

[0122] In one embodiment, the expression vector is a viral vector.

[0123] In one embodiment, the viral vector is an adeno-associated viral (AAV) vector.

[0124] In one embodiment, the viral vector is a bicistronic vector.

[0125] In one aspect, the invention provides a method for regulating expression of a transgene in a cell, the method comprising contacting a 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 expression of the transgene, and optionally further contacting the cell with a REVERSIR compound that inhibits iRNA activity of the universal dsRNA agent, thereby allowing expression of the transgene.

[0126] In one embodiment, the cell is present in a subject.

[0127] In another aspect, the invention provides a method of treating a subject in need thereof, comprising contacting an expression vector encoding a therapeutic transgene and containing 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 expression of the transgene and thereby treating the subject.

[0128] In one embodiment, the universal dsRNA agent is further contacted with a REVERSIR compound that inhibits the iRNA activity of the universal dsRNA agent, thereby allowing expression of the transgene.

[0129] In one embodiment, the invention provides a method of treating a subject in need thereof, comprising contacting an expression vector encoding a therapeutic transgene and a double-stranded ribonucleic acid (dsRNA) agent targeting the transgene administered to the subject with a REVERSIR compound that inhibits iRNA activity of the dsRNA agent, thereby allowing expression of the transgene and thereby treating the subject.

[0130] In another aspect, the present invention provides a method of treating a subject in need thereof, comprising administering to the 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 is achieved, and, when the desired level of expression of the transgene is achieved, administering to the subject a universal double-stranded ribonucleic acid (dsRNA) agent comprising a sense strand and an antisense strand that form a duplex region that recognizes and binds to the universal iRNA target site, thereby inhibiting expression of the transgene and thereby treating the subject.

[0131] In one embodiment, the method further includes administering to the subject a REVERSIR compound when the level of the transgene falls below a desired level of expression, wherein the REVERSIR compound suppresses iRNA activity of the universal dsRNA agent, thereby allowing expression of the transgene.

[0132] In one embodiment, the universal iRNA target site is located in the 5' or 3' untranslated region (UTR) of the transgene.

[0133] In one embodiment, a universal double-stranded ribonucleic acid (dsRNA) agent includes a sense strand and an antisense strand that form a duplex region, wherein the antisense strand includes at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ by 3 or less, e.g., 3, 2, 1, or 0 nucleotides, from any antisense strand nucleotide sequence in either Table 2 or Table 3.

[0134] In one embodiment, a universal double-stranded ribonucleic acid (dsRNA) agent includes a sense strand and an antisense strand that form a duplex region, wherein the antisense strand includes at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ by 3 or less, e.g., 3, 2, 1, or 0 nucleotides, from any antisense strand nucleotide sequence in either Table 2 or Table 3.

[0135] In one embodiment, a universal dsRNA agent comprises a sense strand and an antisense strand that form a duplex region, and the antisense strand comprises a region of complementarity to any target nucleotide sequence in either Table 3 or Table 4.

[0136] In one embodiment, a universal dsRNA agent includes at least one modified nucleotide.

[0137] In one embodiment, substantially all of the nucleotides in the sense strand are modified nucleotides and substantially all of the nucleotides in the antisense strand are modified nucleotides, or substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides.

[0138] In another embodiment, all nucleotides in the sense strand are modified nucleotides and all nucleotides in the antisense strand are modified nucleotides, or all nucleotides in the sense strand and all nucleotides in the antisense strand are modified nucleotides.

[0139] In one embodiment, at least one of the aforementioned modified nucleotides is a deoxy-nucleotide, a 3'-terminal deoxythymidine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, a non-locked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino modified nucleotide, a 2'-O-allyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2'-hydroxyl modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a The nucleotide is selected from the group consisting of a nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide containing a non-natural base, a tetrahydropyran-modified nucleotide, a 1,5-anhydrohexitol-modified nucleotide, a cyclohexenyl-modified nucleotide, a nucleotide containing a phosphorothioate group, a nucleotide containing a methylphosphonate group, a nucleotide containing a 5'-phosphate, a nucleotide containing a 5'-phosphate mimic, a thermally destabilized nucleotide, a glycol-modified nucleotide (GNA), a nucleotide containing a 2' phosphate, and a 2-O-(N-methylacetamido)-modified nucleotide, and combinations thereof.

[0140] In another embodiment, the modification of 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'-hydroxyl, and glycol, and combinations thereof.

[0141] In one embodiment, at least one of the modified nucleotides is selected from the group consisting of deoxynucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, glycol modified nucleotides (GNAs), 2' phosphate containing nucleotides, and vinyl phosphonate nucleotides, and combinations thereof.

[0142] In another embodiment, at least one of the modifications of the modified nucleotides is a thermally destabilizing nucleotide modification.

[0143] In one embodiment, the thermally destabilizing nucleotide modification is selected from the group consisting of an abasic modification, a mismatch with the opposite nucleotide in a duplex, and a destabilizing sugar modification, a 2'-deoxy modification, an acyclic nucleotide, a non-locked nucleic acid (UNA), and a glycerol nucleic acid (GNA).

[0144] The duplex region can be 19 to 30 nucleotide pairs in length, 19 to 25 nucleotide pairs in length, 19 to 23 nucleotide pairs in length, 23 to 27 nucleotide pairs in length, or 21 to 23 nucleotide pairs in length.

[0145] In one embodiment, each strand is independently 30 nucleotides or less in length.

[0146] In one embodiment, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

[0147] In one embodiment, the region of complementarity is at least 17 nucleotides in length.

[0148] In one embodiment, at least one strand comprises a 3' overhang of at least one nucleotide.

[0149] In another embodiment, at least one strand comprises a 3' overhang of at least two nucleotides.

[0150] In one embodiment, the universal dsRNA agent further comprises a ligand.

[0151] In one embodiment, the ligand is conjugated to the 3' end of the sense strand of the universal dsRNA agent.

[0152] In one embodiment, the ligand is an N-acetylgalactosamine (GalNAc) derivative.

[0153] In one embodiment, the ligand is one or more GalNAc derivatives attached via a monovalent, divalent, or trivalent branched linker.

[0154] In one embodiment, the ligand is: [ka]

[0155] In one embodiment, the universal dsRNA agent is conjugated to a ligand as shown in the following scheme, where X is O or S: [ka]

[0156] In one embodiment, X is O.

[0157] In one embodiment, the universal dsRNA agent further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.

[0158] In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the 3' end of one strand.

[0159] In one embodiment, the strand is the antisense strand.

[0160] In another embodiment, the strand is the sense strand.

[0161] In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the 5' end of one strand.

[0162] In one embodiment, the strand is the antisense strand.

[0163] In another embodiment, the strand is the sense strand.

[0164] In one embodiment, phosphorothioate or methylphosphonate internucleotide linkages are at both the 5' and 3' ends of one strand.

[0165] In one embodiment, the strand is the antisense strand.

[0166] 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.

[0167] In one embodiment, the REVERSIR compound comprises a single-stranded oligonucleotide of 6 to 30, e.g., 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, e.g., 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 in length, 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.

[0168] In one embodiment, the oligonucleotide is 100% complementary to any antisense strand nucleotide sequence in either Table 2 or Table 3.

[0169] In one embodiment, the oligonucleotide comprises at least one modified nucleotide.

[0170] In one embodiment, substantially all of the nucleotides of the oligonucleotide are modified nucleotides.

[0171] In another embodiment, all nucleotides of the oligonucleotide are modified nucleotides.

[0172] In one embodiment, at least one of the modified nucleotides comprises a modified nucleobase.

[0173] In one embodiment, the modified nucleobase is a 5' methylcytosine.

[0174] In one embodiment, at least one of the modified nucleotides comprises a modified sugar.

[0175] In one embodiment, the modified sugar is selected from the group consisting of a 2'-O-methoxyethyl-modified sugar, a 2'-methoxy-modified sugar, a 2'-O-alkyl-modified sugar, and a bicyclic sugar.

[0176] In one embodiment, the REVERSIR compound comprises a ligand.

[0177] In one embodiment, the ligand is conjugated to the 3' end of the oligonucleotide.

[0178] In one embodiment, the ligand is an N-acetylgalactosamine (GalNAc) derivative.

[0179] In one embodiment, the ligand is one or more GalNAc derivatives attached via a monovalent, divalent, or trivalent branched linker.

[0180] In one embodiment, the ligand is: [ka]

[0181] In one embodiment, the oligonucleotide is conjugated to a ligand as shown in the following scheme, where X is O or S: [ka]

[0182] In one embodiment, X is O.

[0183] In one embodiment, the oligonucleotide further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.

[0184] In one embodiment, the oligonucleotide is 6-15, 7-11, or 8-10 nucleotides in length.

[0185] In one embodiment, the oligonucleotide is 15-25, 17-25, 19-25, or 21-25 nucleotides in length.

[0186] In one embodiment, the expression vector is a viral vector.

[0187] In one embodiment, the viral vector is an adeno-associated viral (AAV) vector.

[0188] In one embodiment, the viral vector is a bicistronic vector. [Brief explanation of the drawings]

[0189] Figures 1A-1I show the restoration of transgene expression from an shRNA-controlled self-silencing AAV vector using REVERSIR. [Figure 1A]Figure 1A is a schematic diagram illustrating AAV switching, which uses an intron-encoded shRNA for transgene silencing and a REVERSIR for transgene induction. The shRNA is expressed from a chimeric intron preceding the viral transgene cassette. After intracellular processing of the shRNA, the RISC-loaded antisense binds to a complementary target site within the rAAV 3'UTR, triggering constitutive cleavage and degradation of the AAV-delivered transgene mRNA. Exogenous delivery of a REVERSIR blocks RISC activity, derepressing the transgene mRNA and inducing protein expression. [Figure 1B] Figure 1B shows a schematic representation of the viral genome of the ssAAV8 self-silencing GLuc reporter vector, which expresses a TTR shRNA incorporating an intron-encoded miR-33 and harbors a cognate (TTR-ts) or engineered (NT-ts) target site in the 3'UTR of the GLuc transgene. [Figure 1C] Figure 1C shows the in vitro time course of shRNA-mediated transgene silencing. HepG2 cells expressed the indicated AAV plasmids, and cell culture medium was harvested at each time point to quantify secreted GLuc levels. The medium was completely replaced at each harvest, and each line corresponds to the GLuc accumulated in one well from the previous time point. [Figure 1D] Figure 1D is a graph showing the validation of transgene autosilencing and REVERSIR induction in HepG2 cells using an intronic miR-33-containing AAV construct. 20 ng of total DNA consisting of the GLuc AAV construct described in (1B) and a FLuc internal control plasmid at a 5:1 ratio was cotransfected with the indicated concentrations of full-length TTR-REVERSIR or a chemically matched non-targeting (NT) control. Forty-eight hours after transfection, the intensities of GLuc and FLuc were assayed in cell culture supernatants and lysates, respectively, and the GLuc / FLuc ratio was calculated to normalize for transfection efficiency. [Figure 1E]Figure 1E is a graph showing secreted GLuc levels measured in serum collected on the indicated days before and after molar equivalent administration of 9-mer (0.1 mg / kg) or 22-mer (0.2 mg / kg) TTR or NT reverser on D0. Two weeks before reverser compound administration, mice were injected with 2 x 10 genome copies (GC) of shTTRmiR-33 / TTR-ts or control shTTRmiR-33 / NT-ts AAV8 vectors encoding a GLuc reporter. [Figure 1F] Figure 1F is a graph showing qRT-PCR analysis of GLuc transcript levels in liver tissue at the final 47-day time point, plotted relative to the endogenous Gapdh control and the shTTR / NT-ts condition, which was set at 100%. [Figure 1G] Figure 1G is a graph showing longitudinal quantification of serum GLuc levels in mice administered 0.1 mg / kg or 0.3 mg / kg of a 9-mer tunable TTR REVERSIR (TTR REVERSIR 2) or NT REVERSIR on D0, followed by a second dose on D47. [Figure 1H] Figure 1H is a graph showing serum EPO concentrations measured by ELISA at the indicated time points in mice treated with 0.1 mg / kg of 9-mer TTR or NT reverser on DO. Mice were injected with 2x10 GC AAV8 viruses encoding a murine EPO transgene under the control of a TTR shRNA with an intact (TTR-ts) or non-targeting (NT-ts) binding site in the 3'UTR. [Figure 1I] FIG. 1I is a graph showing serum EPO concentrations at the indicated time points in mice treated with increasing doses of a tunable TTR REVERSIR (TTR REVERSIR 2 at 0.01, 0.03, 0.1, or 0.3 mg / kg).

[0190] Figures 2A-G show in vivo regulation of an AAV-delivered reporter transgene by exogenous delivery of siRNA and cognate REVERSIR. [Figure 2A] Figure 2A is a schematic diagram illustrating the exogenous siRNA approach for AAV transgene regulation. Administration of siRNA promotes AAV gene inactivation or silencing through RNAi-mediated degradation of viral transcripts with target sites within the 3'UTR. Sequence-specific inhibition of siRNA activity by reverse transcription factors derepresses viral mRNA transcripts, resulting in increased expression of therapeutic proteins. [Figure 2B] Figure 2B is a schematic diagram of the ssAAV serotype 8 vector carrying a bicistronic expression cassette encoding PMP-22 and GLuc reporter genes. A perfectly complementary binding site for TTR siRNA was inserted directly adjacent to the stop codon within the 3'UTR (left). Six-week-old female C57BL / 6 mice were intravenously injected with 2x10 genome copies (GC) of AAV. Two weeks after AAV administration, mice were subcutaneously injected with vehicle or TTR siRNA at 9 mg / kg (D0). Two weeks later, mice were administered a molar equivalent of either a full-length 22-mer (3 mg / kg) or a 9-mer TTR reversir (1.6 mg / kg) compared with vehicle or a length-matched NT reversir (D14) as a control. Blood was collected as indicated, and liver tissue samples were collected at D42 (right). [Figure 2C] FIG. 2C is a graph showing quantification of serum GLuc levels at the indicated time points, normalized to pre-dose for each animal. [Figure 2D] Figure 2D is a graph showing qRT-PCR analysis of GLuc transcript levels in terminal liver tissue at D42, normalized to the Gapdh control and plotted against the PBS condition, which was set at 100%. [Figure 2E] Figure 2E is a graph showing serum GLuc levels at D21 in mice transduced with 2x1011GC of the AAVs shown in (2B) and treated with TTR siRNA (9 mg / kg; D0), followed by varying doses of a high dose of the 9-mer TTR reversir alone or NT reversir (D14) as a control. [Figure 2F]Figure 2F shows a schematic diagram of the AAV vector used to evaluate shRNA-based modulation of the AAV-hANGPTL3 transgene (left) and a graph of plasma hANGPTL3 protein concentrations assessed by ELISA over the indicated time course (right). C57BL / 6 mice were intravenously administered 1.5 x 10 GC of an AAV8 vector carrying the human ANGPTL3 coding region with a GLuc siRNA target site within the 3'UTR. Two weeks later, mice were treated with 9 mg / kg GLuc siRNA for 14 days, followed by administration of 1.5 mg / kg of the 9-mer GLuc reverser or NT reverser. Blood samples were collected at the indicated time points. [Figure 2G] Figure 2G shows a schematic diagram of the AAV vector for siRNA-mediated regulation of the human factor XII (hF12)-GLuc transgene (left) and a graph of serum GLuc levels measured at the indicated time points, plotted relative to siRNA pretreatment (right). Mice were injected with 2 x 10 GC of an IRES-containing bicistronic vector encoding hF12 and GLuc, with a TTR siRNA binding site in the 3'UTR. Mice were administered 9 mg / kg TTR siRNA and, 2 weeks later, 156 mg / kg TTR reverser or NT reverser. Blood samples were collected as indicated.

[0191] Figures 3A-3D show in vitro characterization of on-target and off-target activity of transgene regulator siRNA sequences. [Figure 3A]Figure 3A shows the on-target silencing efficacy (black solid lines) of three lead transgene regulator siRNA sequences, assayed by cotransfection of a dual luciferase sensor containing perfectly matched binding sites with serially increasing doses of siRNA. Seed-mediated off-target suppression was also assessed by dose-response activity of the siRNA in the presence of a luciferase reporter. Seeds matched one (medium gray dashed line) or four (light gray dashed line) target sites. RLuc / FLuc ratios were normalized to the mock-transfected control (no siRNA) condition, which was set at 100%, and plotted as the mean ± SEM of 3–6 replicates. [Figure 3B] Figure 3B shows a Bland-Altman plot (MA plot) of differential gene expression analysis of RNA-seq data obtained from transfection of transgene regulator siRNA in Hep3B cells (top; 10 nM dose, harvested 24 hours later) and primary mouse hepatocytes (bottom; 50 nM dose, harvested 48 hours later). Dots represent individual transcripts, average normalized read counts across replicates, and log2 fold change relative to mock-transfected controls. "Black" dots indicate genes with significant differential expression (FDR < 0.05) but lacking a canonical seed match site within their 3' UTR (8-mer, 7-mer-m8, and 7-mer-A1). "Dark gray" dots represent genes containing a canonical 3' UTR seed match binding site but without significant differential expression (N = 4 replicates). [Figure 3C] Figure 3C is a table showing differential gene expression analysis of in vitro RNAseq data obtained from transfection of transgene regulator siRNAs in mouse (primary mouse hepatocytes; top) and human (Hep3B; bottom) hepatocytes. [Figure 3D]Figure 3D is a graph showing serum alanine aminotransferase (ALT) and glutamate dehydrogenase (GLDH) levels at necropsy (D16) in rats injected three times per week (qw × 3) with the indicated transgene regulator siRNA (TR-siRNA) at 30 or 100 mg / kg doses. N = 4 males (6-8 weeks old) were injected once per week.

[0192] Figures 4A-4E show additional in vitro and in vivo analyses supporting an AAV regulatory switch utilizing intronically expressed shRNA and REVERSIR. [Figure 4A] Figure 4A shows a schematic diagram of the marker constructs and a graph depicting in vitro evaluation of REVERSIR-mediated reversal of target silencing by miRNA-mediated shRNA in a dual-luciferase reporter assay. Cos7 cells were cotransfected with luciferase reporter plasmids expressing miR-30E-incorporated TTR or NT shRNAs and GFP marker constructs along with increasing concentrations of 22-mer TTR or the corresponding NT REVERSIR for 48 hours. [Figure 4B] Figure 4B shows a schematic diagram of the marker constructs and a graph demonstrating in vitro evaluation of REVERSIR-mediated reversal of target silencing by miRNA-mediated shRNA in a dual-luciferase reporter assay. Cos7 cells were cotransfected with a luciferase reporter plasmid and GFP marker construct expressing miR-33-incorporated TTR or NT shRNA, along with increasing concentrations of the 22-mer TTR or corresponding NT REVERSIR for 48 hours. [Figure 4C]Figure 4C shows the validation of GLuc transgene suppression with an intronic miR-30E-shRNA-containing self-silencing AAV construct in HepG2 cells, followed by induction with increasing doses of REVERSIR. The AAV construct was co-transfected with a FLuc control plasmid at a 5:1 molar ratio and normalized. GLuc and FLuc intensities were measured in cell culture supernatants and lysates, respectively, and the GLuc / FLuc ratio was expressed relative to the shNT-expressing AAV plasmid. [Figure 4D] Figure 4D is a graph showing quantification of GLuc mRNA levels by qRT-PCR in HepG2 cells 48 hours after transfection with miR-33-containing self-silencing AAV plasmid and REVERSIR. GLuc transcription levels were normalized to Fluc mRNA as an internal control. [Figure 4E] Figure 4E is a graph showing successful in vivo knockdown of endogenous TTR protein levels by intronic expression of shTTRmiR-33 and subsequent recovery to baseline by exogenous administration of TTR REVERSIR, but not NT REVERSIR.

[0193] Figures 5A-5F show additional in vitro and in vivo analyses supporting the AAV-controlled switch utilizing exogenous siRNA and REVERSIR. Figures 5A-5C are graphs showing data from individual animals or additional groups tested as part of the study shown in Figures 2B-D. AAV injections, timing of test article administration, and blood collection were performed as described in Figure 2B. PBS and 9 mg / kg siRNA conditions were the same as in this figure. Figures 5A-5F are graphs showing data from individual animals or additional groups tested as part of the study shown in Figures 2B-D. AAV injections, timing of test article administration, and blood collection were performed as described in Figure 2B. PBS and 9 mg / kg siRNA conditions were the same as in this figure. [Figure 5A]FIG. 5A is a graph showing sustained dose-dependent knockdown of serum GLuc levels in AAV-injected mice treated with 1, 3, and 9 mg / kg TTR siRNA compared to PBS controls. [Figure 5B] Figure 5B is a graph showing longitudinal measurements of serum GLuc levels in AAV-injected mice administered 3 mg / kg TTR siRNA followed by vehicle or a 1 mg / kg dose of the indicated REVERSIR (left). The average data from Figure 2C is presented as a spaghetti graph plotting the change in serum GLuc over time relative to pre-dose for each individual animal treated with 9 mg / kg TTR siRNA and 3 mg / kg of the specified REVERSIR molecule (right). [Figure 5C] Figure 5C is a graph showing the positive controls demonstrating 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 REVERSIRs, but not the corresponding NT REVERSIRs. [Figure 5D] Figure 5D is a graph showing the on-target silencing activity of GLuc siRNA in a dual-luciferase reporter system (left). Cos7 cells were co-transfected with 10 nM GLuc siRNA and increasing doses of 22-mer or 9-mer GLuc REVERSIR, and luciferase activity was normalized 48 hours later (right). [Figure 5E] Figure 5E is a spaghetti plot showing the responses of individual animals averaged by condition to generate the graph shown in Figure 2F. The graph on the right shows the time course of hANGPTL3 concentrations in one animal that was identified as a significant outlier by the Grubb test due to low AAV transduction levels and was omitted from the main figure. [Figure 5F] Figure 5F is a spaghetti plot showing the responses of individual animals averaged by condition to generate the graph shown in Figure 2G.

[0194] Figures 6A-6B show the lack of seed-mediated off-target effects with transgene regulator siRNA. [Figure 6A] Figure 6A shows cumulative distribution function (CDF) plots depicting transcriptional changes after 24-hour transfection of Hep3B cells with 10 nM transgene regulator siRNA. Each line represents the cumulative distribution of expression changes for target genes with a specified seed match (8mer, 7mer-m8, and 7mer-A1) in the 3'UTR of the siRNA antisense strand (top) or sense strand (bottom) compared to genes without such a canonical seed match (background). Black lines represent background genes lacking the specified seed match, while various gray lines represent genes with at least one seed match, separated by binding site strength (dark gray = 8mer, medium gray = 7mer-m8, light gray = m7mer-A1). Delta values ​​reflect the magnitude of the CDF shift relative to the background. N = 4 replicates. [Figure 6B] Figure 6B shows cumulative distribution function (CDF) plots depicting transcriptional changes following 48-hour transfection of mouse primary hepatocytes with 50 nM transgene regulator siRNA. Each line represents the cumulative distribution of expression changes for target genes with a specified seed match (8mer, 7mer-m8, and 7mer-A1) in the siRNA antisense strand (top) or sense strand (bottom) within the 3'UTR, compared to genes without such a canonical seed match site (background). Black lines represent background genes lacking the specified seed match, while various gray lines represent genes with at least one seed match, separated by binding site strength (dark gray = 8mer, medium gray = 7mer-m8, light gray = m7mer-A1). Delta values ​​reflect the magnitude of the CDF shift relative to the background. N = 4 replicates.

[0195] [Figure 7]Figure 7 shows the lack of elevated liver function tests (LFTs) in a rat toxicity study of transgene regulator siRNA. Specifically, serum levels of aspartate aminotransferase (AST), albumin (ALB), alkaline phosphatase (ALP), and total protein (TP) are shown at the time of rat necropsy (D16). Rats received three weekly injections (qw x 3) of the indicated transgene regulator siRNA (TR-siRNA) at 30 mg / kg or 100 mg / kg. N = 4 male rats (6-8 weeks old) were injected once weekly.

[0196] Detailed Description of the Invention The present invention provides compositions, systems, and methods for regulating protein expression using iRNA compositions that carry out RNA-induced silencing complex (RISC)-mediated cleavage of universal target sequence RNAi target sequences, and REVERSIR compounds that inhibit the activity of such iRNA compositions.

[0197] The universal iRNAs of the present invention were designed to have favorable thermodynamic properties for RISC loading and RNAi function, and to have little sequence complementarity with any annotated genes in the transcriptomes of humans, cynomolgus monkeys, rats, and mice. Such universal iRNAs have been demonstrated to be potent RNAi triggers with high on-target specificity and a low tendency for off-target gene disruption. Additionally, as described herein, these universal dsRNA agents have been shown to regulate expression from exogenous vector delivery systems without causing undesired off-target silencing within the endogenous transcriptomes of humans and preclinical mammalian models. Therefore, the use of these universal iRNAs, REVERSIR molecules that inhibit the activity of these universal iRNAs, and systems containing these universal iRNAs and / or REVERSIR molecules can improve the dosage and timing of transgene induction from exogenous vector delivery systems, such as AAV vector delivery systems, thereby providing an in vivo gene therapy method that achieves long-term correction of genetic defects across a wide range of target organs with a single administration.

[0198] The iRNAs of the present invention comprise an RNA strand (antisense strand) having a region that is up to about 30 nucleotides in length, e.g., 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, which region is substantially complementary to at least a portion of an mRNA transcript of a universal target.

[0199] In certain embodiments, one or both strands of a double-stranded RNAi agent of the invention are up to 66 nucleotides in length, e.g., 36-66, 26-36, 25-36, 31-60, 22-43, 27-53 nucleotides in length, with a region of at least 19 contiguous nucleotides that is substantially complementary to at least a portion of an mRNA transcript of a universal target. In some embodiments, such iRNA agents with longer antisense strands can include a second RNA strand (sense strand) that is, e.g., 20-60 nucleotides in length, where the sense and antisense strands form a duplex of 18-30 contiguous nucleotides.

[0200] The following detailed description discloses methods of making and using compositions containing iRNA agents to inhibit expression of universal target sequences, REVERSIR compounds that suppress the activity of such iRNAs, and systems, uses, and methods for treating subjects in need thereof.

[0201] L. Definition In order that the present invention may be more readily understood, certain terms are first defined. Additionally, whenever a value or range of values ​​for a parameter is listed, it is intended that values ​​and ranges intermediate to the listed values ​​are also intended to be part of the invention.

[0202] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element, e.g., a plurality of elements.

[0203] The term "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to."

[0204] The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless the context clearly indicates otherwise. For example, "the sense strand or the antisense strand" is understood as "the sense strand or the antisense strand, or the sense strand and the antisense strand."

[0205] The term "about" is used herein to mean within a range that is typical in the art. For example, "about" can be understood as about 2 standard deviations from the mean. In certain embodiments, about means ±10%. In certain embodiments, about means ±5%. When about is present before a series of numbers or ranges, it will be understood that "about" can modify each number or range in the series.

[0206] The terms "at least," "no less than," and "or more" before a number or series of numbers are understood to include the number adjacent to the term "at least," and all subsequent numbers or integers that may logically be included, if 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 nucleic acid molecule" means that 19, 20, or 21 nucleotides have the specified property. When the term at least appears before a series of numbers or ranges, it will be understood that "at least" can modify each of the numbers and ranges in the series.

[0207] As used herein, "less than" or "less than" shall be understood as meaning from the value adjacent to the phrase and its logically smaller value or integer, if logical from the context, to zero.For example, a duplex with an overhang of "2 nucleotides or less" has an overhang of 2, 1, or 0 nucleotides.When "less than" is present before a series of numbers or ranges, it shall be understood that "less than" can modify each of the numbers or ranges in the series.As used herein, a range includes upper and lower limits.

[0208] As used herein, a method of detection can include determining that the amount of analyte present is below the detection level of the method.

[0209] In the event of a discrepancy between the nucleotide sequence for a given target site and the sense or antisense strand, the given sequence prevails.

[0210] In the event of a discrepancy between a sequence and that site on the transcript or other sequences shown, the nucleotide sequence set forth herein shall prevail.

[0211] As used herein, "target sequence" refers to a contiguous portion of a nucleotide sequence, such as an mRNA molecule formed upon transcription of a universal target sequence, e.g., an mRNA that is the product of RNA processing of a primary transcript. In one embodiment, the target portion of the sequence will be at least sufficiently long to serve as a substrate for RNAi-dependent cleavage at or near a portion of the nucleotide sequence of the mRNA molecule formed upon transcription of the universal target sequence.

[0212] A "universal target sequence" is a nucleotide sequence that has favorable thermodynamic properties for RISC loading and RNAi function and shares little or no sequence complementarity with any annotated genes in the human, cynomolgus monkey, rat, and mouse transcriptomes. Such universal iRNAs have been demonstrated to be potent RNAi triggers with high on-target specificity and minimal propensity for off-target gene disruption.

[0213] Nucleotide sequences of exemplary universal target sequences are provided in Tables 3 and 4 below.

[0214] The target sequence is about 19-36 nucleotides in length. For example, the target sequence 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 length. In certain embodiments, the target sequence is 19-23 nucleotides in length, and optionally may be 21-23 nucleotides in length. Ranges and lengths intermediate to the above-listed ranges and lengths are also contemplated as part of this disclosure.

[0215] As used herein, the term "strand comprising a sequence" means an oligonucleotide comprising a chain of nucleotides described by a sequence referenced using standard nucleotide nomenclature.

[0216] " G ", " C ", " A ", " T " and " U " respectively represent the nucleotide that contains guanine, cytosine, adenine and uracil as a base. However, it should be understood that the term "ribonucleotide" or "nucleotide" can also refer to modified nucleotides, as described in more detail below, or substitute replacement moieties (see, for example, Table 1). Those skilled in the art are well aware that guanine, cytosine, adenine, thymidine and uracil can be replaced with other moieties without substantially changing the base pairing properties of the oligonucleotide that contains the nucleotide with such replacement moieties. For example, but not limited to, the nucleotide that contains inosine as its base can form base pairs with the nucleotide that contains adenine, cytosine or uracil. Therefore, the nucleotide that contains uracil, guanine or adenine can be replaced with the nucleotide that contains inosine, for example, in the nucleotide sequence of the dsRNA that is featured in the present disclosure. In another example, adenine and cytosine in any of the oligonucleotides can be substituted with guanine and uracil, respectively, to form G-U Wobble base pairs with the target mRNA. Sequences containing such substituted moieties are suitable for the compositions and methods featured in the present invention.

[0217] The terms "iRNA," "RNAi agent," "iRNA agent," and "RNA interference agent," used interchangeably herein, refer to agents that contain RNA, as defined herein, and mediate targeted cleavage in RNA transcription via the RNA-induced silencing complex (RISC) pathway. iRNA is involved in the sequence-specific degradation of mRNA through a process also known as RNA interference. iRNA modulates, for example, inhibits, the expression of universal target mRNA sequences in cells, for example, in cells within a subject, for example, a mammalian subject.

[0218] In one embodiment, the RNAi agent of the present invention comprises a single-stranded RNAi that interacts with a target RNA sequence, such as a universal 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 cells is degraded into siRNA by a type III endonuclease known as Dicer [Sharp et al. (2001) Genes Dev. 15:485]. Dicer, an RNase III-like enzyme, processes this dsRNA into 19-23 base pair small interfering RNAs with characteristic two-base 3' overhangs [Bernstein, et al., (2001) Nature 409:363]. These siRNAs are then introduced into RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, allowing complementary antisense strands to induce target recognition [Nykanen, et al., (2001) Cell 107:309].When binding to appropriate target mRNA, one or more endonucleases in RISC cleave the target to induce silencing [Elbashir, et al., (2001) Genes Dev. 15:188].Therefore, in one aspect, the present invention relates to the single-stranded RNA (siRNA) that is produced in cells, promotes the formation of RISC complex, and thereby silences target sequence, i.e., universal target sequence.Therefore, the term " siRNA " is used herein to also mean the RNAi described above.

[0219] In some embodiments, the RNAi agent can be a single-stranded siRNA (ssRNAi) introduced into a cell or organism to inhibit target mRNA. The single-stranded RNAi agent binds to the RISC endonuclease Argonaute 2 and then cleaves the target mRNA. The single-stranded siRNA is generally 15-30 nucleotides and chemically modified. The design and testing of single-stranded RNAs are described in U.S. Patent No. 8,101,348 and Lima et al., (2012) Cell 150:883-894, the entire contents of each of which are incorporated herein by reference. Any of the antisense nucleotide sequences described herein can be used as the single-stranded siRNA described herein or as the single-stranded siRNA chemically modified by the method described in Lima et al., (2012) Cell 150:883-894.

[0220] In certain embodiments, an "iRNA agent" for use in the compositions and methods of the present disclosure is double-stranded RNA and is referred to herein as a "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 comprising two antiparallel, substantially complementary nucleic acid strands, said to have a "sense" or "antisense" orientation with respect to a target RNA, i.e., a universal target sequence. In some embodiments of the present invention, the double-stranded RNA (dsRNA) induces degradation of the target RNA, e.g., mRNA, by a post-transcriptional gene silencing mechanism referred to herein as RNA interference or RNAi.

[0221] Generally, the majority of the nucleotides in each strand of dsRNA molecule can comprise ribonucleotides; however, as described in detail herein, each strand or both strands can also comprise one or more ribonucleotides, such as deoxyribonucleotides, modified nucleotides.In addition, as used herein, " iRNA " can comprise ribonucleotides with chemical modifications; RNAi agents can comprise substantial modifications in multiple nucleotides.As used herein, the term "modified nucleotide" refers to a nucleotide that independently has a modified sugar moiety, a modified internucleotide linkage, or a modified nucleobase, or any combination thereof.Therefore, the term modified nucleotide encompasses the substitution, addition, or removal of, for example, functional groups or atoms, etc., in internucleoside linkages, sugar moieties, or nucleobases.The modifications suitable for use in the agent of the present invention encompass all types of modifications disclosed herein or known in the art.Any such modifications used in siRNA-type molecules are encompassed by " iRNA " or " RNAi agent " for the purpose of this specification and claims.

[0222] In certain embodiments of the present disclosure, the inclusion of deoxynucleotides can be considered to constitute modified nucleotides when present within an RNAi agent.

[0223] The duplex region can be of any length that allows for specific degradation of the desired target RNA by the RISC pathway, and can be about 19 to 36 base pairs in length, e.g., about 19 to 30 base pairs in length, e.g., about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length, e.g., about 19 to 30, 1 The duplex region may range in length from 9 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 30, 20 to 29, 20 to 28, 20 to 27, 20 to 26, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, or 21 to 22 base pairs. In certain embodiments, the duplex region is 19 to 21 base pairs in length, e.g., 21 base pairs in length. Ranges and lengths intermediate to the above-listed ranges and lengths are also contemplated as part of the present disclosure.

[0224] 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 can therefore be connected by an uninterrupted chain of nucleotides between the 3' end of one strand and the 5' end of the other strand forming the duplex structure, the connecting RNA strands are called "hairpin loops." A hairpin loop can contain at least one unpaired nucleotide. In some embodiments, a hairpin loop can contain at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 23, or more unpaired nucleotides or nucleotides not targeted to the target site of the dsRNA. In some embodiments, a hairpin loop can be 10 or fewer nucleotides. In some embodiments, a hairpin loop can be 8 or fewer unpaired nucleotides. In some embodiments, a hairpin loop can be 4 to 10 unpaired nucleotides. In some embodiments, a hairpin loop can be 4 to 8 unpaired nucleotides.

[0225] The two substantially complementary strands of dsRNA are contained in separate RNA molecules, and these molecules can, but do not necessarily, be covalently linked. When the two strands are covalently linked by means other than an uninterrupted chain of nucleotides between the 3' end of one strand and the 5' end of each other strand that forms a duplex structure, the connecting structure is called a "linker." The RNA strands can have the same or different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus all overhangs present in the duplex. In addition to the duplex structure, the RNAi can also contain one or more nucleotide overhangs. In one embodiment of an RNAi agent, at least one strand contains a 3' overhang of at least one nucleotide. In another embodiment, at least one strand contains a 3' overhang of at least two nucleotides, for example, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments, at least one strand of the RNAi agent comprises a 5' overhang of at least one nucleotide. In certain embodiments, at least one strand comprises a 5' overhang of at least two nucleotides, for example, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In yet other embodiments, both the 3' and 5' ends of one strand of the RNAi agent comprise an overhang of at least one nucleotide.

[0226] In certain embodiments, an iRNA agent of the invention is a dsRNA, each strand of which independently comprises 19-23 nucleotides that interact with a target RNA sequence, e.g., a universal target mRNA sequence, to direct cleavage of the target RNA.

[0227] In some embodiments, an iRNA of the invention is a 24-30 nucleotide dsRNA that interacts with a target RNA sequence, eg, a universal targeting mRNA sequence, to direct cleavage of the target RNA.

[0228] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that protrudes from the double-stranded structure of an RNAi agent, double-stranded iRNA.For example, if the 3'-end of one strand of dsRNA extends beyond the 5'-end of the other strand, or vice versa, there is a nucleotide overhang.A dsRNA can comprise an overhang of at least one nucleotide; alternatively, the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more nucleotides.A nucleotide overhang can comprise or consist of nucleotide / nucleoside analogs, such as deoxynucleotides / nucleosides.An overhang can be on the sense strand, antisense strand, or any combination thereof.Moreover, the nucleotide of the overhang can be present at the 5'-end, 3'-end, or both of the antisense strand or sense strand of dsRNA.

[0229] In one embodiment, the antisense strand of the dsRNA has a 1-10 nucleotide overhang at the 3'-end or 5'-end, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide overhang. In one embodiment, the sense strand of the dsRNA has a 1-10 nucleotide overhang at the 3'-end or 5'-end, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide overhang. In another embodiment, one or more of the nucleotides in the overhang are replaced with nucleoside thiophosphate.

[0230] In certain embodiments, the antisense strand of dsRNA has an overhang of 1 to 10 nucleotides at the 3'-end or 5'-end, for example, 0 to 3, 1 to 3, 2 to 4, 2 to 5, 4 to 10, 5 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In one embodiment, the sense strand of dsRNA has an overhang of 1 to 10 nucleotides at the 3'-end or 5'-end, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In another embodiment, one or more of the nucleotides in the overhang are replaced with nucleoside thiophosphate.

[0231] In certain embodiments, the antisense strand of the dsRNA has an overhang of 1 to 10 nucleotides at its 3' end, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In certain embodiments, the overhang on the sense strand, the antisense strand, or both may comprise an extended length of more than 10 nucleotides, e.g., 1 to 30 nucleotides, 2 to 30 nucleotides, 10 to 30 nucleotides, 10 to 25 nucleotides, 10 to 20 nucleotides, or 10 to 15 nucleotides. In certain embodiments, the extended overhang is on the sense strand of the duplex. In certain embodiments, the extended overhang is on the 3' end of the sense strand of the duplex. In certain embodiments, the extended overhang is on the 5' end of the sense strand of the duplex. In certain embodiments, the extended overhang is on the antisense strand of the duplex. In certain embodiments, the extended overhang is on the 3' end of the antisense strand of the duplex. In certain embodiments, the extended overhang is present at the 5'-end of the antisense strand of the double strand.In certain embodiments, one or more of the nucleotides in the extended overhang are replaced with nucleoside thiophosphate.In certain embodiments, the overhang comprises a self-complementary portion, so that the overhang can form a stable hairpin structure under physiological conditions.

[0232] "Blunt" or "blunt-ended" means that there are no unpaired nucleotides at that end of a double-stranded RNA, i.e., there are no nucleotide overhangs. A "blunt-ended" double-stranded RNA agent is double-stranded throughout its entire length, i.e., there are no nucleotide overhangs. RNAi agents of the present invention include RNAi agents that have no nucleotide overhangs at one end (i.e., one overhang, one blunt end) or no nucleotide overhangs at either end. In most cases, such molecules will be double-stranded throughout their entire length.

[0233] The term "antisense strand" or "guide strand" refers to the strand of an iRNA, e.g., a dsRNA, that includes a region that is substantially complementary to a target sequence, e.g., a universal target mRNA.

[0234] As used herein, the term "region of complementarity," as defined herein, 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. If the region of complementarity is not perfectly complementary to the target sequence, the mismatch may be in an internal or terminal region of the molecule. Generally, the most tolerable mismatch is in the terminal region, e.g., within 5, 4, or 3 nucleotides of the 5' or 3' end of the iRNA. In some embodiments, a double-stranded RNA agent of the present invention contains nucleotide mismatches in the antisense strand. In some embodiments, the antisense strand of a double-stranded RNA agent of the present invention contains four or fewer mismatches with the target mRNA, e.g., the antisense strand contains four, three, two, one, or zero mismatches with the target mRNA. In some embodiments, the antisense strand of a double-stranded RNA agent of the present invention contains four or fewer mismatches with the sense strand, e.g., the antisense strand contains four, three, two, one, or zero mismatches with the sense strand. In some embodiments, the double-stranded RNA agent of the present invention contains nucleotide mismatches in the sense strand. In some embodiments, the sense strand of the double-stranded RNA agent of the present invention contains four or fewer mismatches with the antisense strand, for example, the sense strand contains four, three, two, one, or zero mismatches with the antisense strand. In some embodiments, the nucleotide mismatch is, for example, within 5, 4, or 3 nucleotides from the 3' end of the iRNA. In another embodiment, the nucleotide mismatch is, for example, at the 3' terminal nucleotide of the iRNA agent. In some embodiments, the mismatch is not in the seed region.

[0235] Thus, the RNAi agents described herein can contain one or more mismatches to the target sequence. In one embodiment, the RNAi agents described herein contain three or fewer mismatches (i.e., three, two, one, or zero mismatches). In one embodiment, the RNAi agents described herein contain two or fewer mismatches. In one embodiment, the RNAi agents described herein contain one or fewer mismatches. In one embodiment, the RNAi agents described herein contain zero mismatches. In certain embodiments, when the antisense strand of an RNAi agent contains a mismatch to the target sequence, the mismatch can be limited to within the last five nucleotides from the 5' or 3' end of the region of complementarity, as appropriate. For example, in such an embodiment, for a 23-nucleotide RNAi agent, the strand complementary to a region of the universal target sequence generally does not contain any mismatches within the central 13 nucleotides. Using the methods described herein or known in the art, it can be determined whether an RNAi agent containing a mismatch to the target sequence is effective in inhibiting expression of the universal target. It is important to consider the efficacy of mismatched RNAi agents in inhibiting expression of the universal target, especially when the specific region of complementarity in the universal target is known to have polymorphic sequence variation within the population.

[0236] As used herein, the term "sense strand" or "passenger strand" refers to the strand of an iRNA that includes a region that is substantially complementary to a region of the antisense strand, as that term is defined herein.

[0237] "Substantially all of the nucleotides are modified," as used herein, means that most, but not all, are modified, including no more than 5, 4, 3, 2, or 1 unmodified nucleotides.

[0238] As used herein, the term "cleavage region" refers to the region located directly adjacent to the cleavage site.Cleavage site is the site on the target where cleavage occurs.In some embodiments, the cleavage region comprises three bases that are directly adjacent to either end of the cleavage site.In some embodiments, the cleavage region comprises two bases that are directly adjacent to either end of the cleavage site.In some embodiments, specifically, the cleavage site occurs at the site that is bound by nucleotides 10 and 11 of the antisense strand, and the cleavage region comprises nucleotides 11, 12, and 13.

[0239] As used herein, unless otherwise specified, the term "complementary," when used to describe a first nucleotide sequence in the context of a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising a first nucleotide sequence to hybridize to form a duplex with an oligonucleotide or polynucleotide comprising the second nucleotide sequence under certain conditions, as understood by those of skill in the art. Such conditions may be, for example, stringent conditions, such as 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, at 50°C or 70°C for 12-16 hours, followed by washing (see, e.g., "Molecular Cloning: A Laboratory Manual," Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). Other conditions, such as physiologically relevant conditions that may be encountered inside an organism, may also be applied. Those skilled in the art will be able to determine the most appropriate set of conditions for testing the complementarity of two sequences according to the ultimate application of the hybridized nucleotides.

[0240] A complementary sequence in an iRNA, such as a dsRNA described herein, includes base pairing of an oligonucleotide or polynucleotide comprising a first nucleotide sequence with an oligonucleotide or polynucleotide comprising a second nucleotide sequence across the entire length of one or both nucleotide sequences. Such sequences can be referred to herein as "fully complementary" to each other. However, when a first sequence is considered "substantially complementary" to a second sequence, the two sequences can be fully complementary, or they can form one or more, but generally no more than 5, 4, 3, or 2 mismatched base pairs during hybridization in a duplex of up to 30 base pairs, while retaining the ability to hybridize under conditions most relevant to the final application, such as inhibiting gene expression in vitro or in vivo. However, if two oligonucleotides are designed to form one or more single-stranded overhangs during hybridization, these overhangs are not considered mismatches when determining complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, where the longer oligonucleotide comprises a 21 nucleotide sequence that is perfectly complementary to the shorter oligonucleotide, can still be considered "perfectly complementary" for the purposes described herein.

[0241] "Complementary" sequences, as used herein, may also include or be formed entirely of non-Watson-Crick base pairs, or base pairs formed from non-naturally occurring modified nucleotides, so long as the above requirements regarding their ability to hybridize are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble or Hoogsteen base pairing.

[0242] The terms "complementary," "fully complementary," and "substantially complementary" may be used herein, as understood in connection with their use, in reference to base matching between two oligonucleotides or polynucleotides, such as between the sense and antisense strands of a dsRNA, or between the antisense strand of a double-stranded RNA and a target sequence.

[0243] As used herein, a polynucleotide that is "substantially complementary to at least a portion of" a messenger RNA (mRNA) means a polynucleotide that is substantially complementary to a continuous portion of an mRNA of interest (e.g., an mRNA encoding a universal target sequence). For example, a polynucleotide is complementary to at least a portion of a universal target mRNA sequence if the sequence is substantially complementary to an uninterrupted portion of the mRNA encoding the universal target sequence.

[0244] Therefore, in some embodiments, the antisense polynucleotide disclosed herein is completely complementary to the target universal sequence.In other embodiments, the antisense polynucleotide disclosed herein is substantially complementary to the target universal sequence, and comprises a continuous nucleotide sequence that is at least 80%, for example, 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 Tables 3 and 4 or a fragment of any one of Tables 3 and 4 over its entire length.

[0245] In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to a target universal sequence, and the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least 80%, e.g., 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 any one of the sense strand nucleotide sequences in either Table 2 or 3, or a fragment of any one of the sense strand nucleotide sequences in Table 2 or 3, over its entire length.

[0246] In some embodiments, an iRNA of the invention comprises a sense strand that is substantially complementary to an antisense polynucleotide that is complementary to a target universal sequence, wherein the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least 80%, e.g., 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 over its entire length to any one of the antisense strand nucleotide sequences in any of Tables 2 or 3, or a fragment of any one of the antisense strand nucleotide sequences in any of Tables 2 or 3.

[0247] Generally, "iRNA" comprises chemically modified ribonucleotides. Such modifications can include all types of modifications disclosed herein or known in the art. Any such modifications used in dsRNA molecules are encompassed by "iRNA" for the purposes of this specification and claims.

[0248] In certain embodiments of the present disclosure, the inclusion of deoxynucleotides can be considered to constitute modified nucleotides when present in an RNAi agent.

[0249] In one embodiment of the present invention, the agent for use in the methods and compositions of the present 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. Single-stranded antisense oligonucleotides can inhibit translation stoichiometrically by base pairing to the mRNA and physically interfering with the translation mechanism; see Dias, N. et al., (2002) Mol Cancer Ther 1:347-355. The single-stranded antisense oligonucleotide molecule may be about 14 to about 30 nucleotides in length and may have a sequence complementary to the target sequence. For example, the single-stranded antisense oligonucleotide molecule may comprise a sequence that is at least about 14, 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from any one of the antisense sequences described herein.

[0250] As used herein, the term "REVERSIR compound" refers to an oligomeric compound that is complementary to and can hybridize to (target) at least one strand of a conjugated or unconjugated universal dsRNA agent.

[0251] A "REVERSIR compound" reduces or inhibits the intensity and / or duration of activity of a universal dsRNA agent resulting from hybridization of the REVERSIR compound to one of the strands of the universal dsRNA agent.

[0252] The REVERSIR compounds disclosed herein are particularly effective in reducing siRNA activity. For example, the REVERSIR compounds disclosed herein can reduce siRNA activity 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 even 100% (i.e., to a non-existent level compared to a reference sample), or by any reduction between 50% and 100% compared to the baseline level. The baseline level can be siRNA activity in the absence of the REVERSIR compound.

[0253] In some embodiments, the REVERSIR compounds described herein can reduce the activity of universal dsRNA agents by at least 75%, for example, 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 baseline level.

[0254] The phrase "contacting cells with iRNA" such as dsRNA as used herein includes contacting cells by any possible means.Contacting cells with RNAi agents includes contacting cells with iRNA in vitro or contacting cells with iRNA in vivo.Contacting can be carried out directly or indirectly.Therefore, for example, iRNA can be physically contacted with cells by separately carrying out a method, or iRNA can be placed in a situation that can allow or cause it to contact cells later.

[0255] Contacting cells in vitro can be achieved, for example, by incubating cells with iRNA.Contacting cells in vivo (in vivo) can be achieved, for example, by injecting iRNA into the tissue where cells are located or nearby, or by injecting iRNA into another area, for example, bloodstream or subcutaneous space, so that the agent can then reach the tissue where the cells to be contacted are located.For example, RNAi agent can contain or be coupled to a ligand, such as GalNAc, that directs iRNA to the target site, for example, liver.Combination of in vitro and in vivo methods for contacting is also possible.For example, cell can be contacted with iRNA in vitro, and then transferred to a subject.

[0256] In certain embodiments, contacting a cell with an iRNA includes "introducing" or "delivering the iRNA into the cell" by promoting or effecting uptake or absorption into the cell. Absorption or uptake of the iRNA can occur through spontaneous diffusive or active cellular processes, or by auxiliary agents or devices. Introduction of the RNAi agent into the cell can be in vitro or in vivo. For example, in the case of in vivo introduction, the iRNA can be injected into a tissue site or administered systemically. In vitro introduction into the cell includes methods known in the art, such as electroporation and lipofection. Additional approaches are described herein below or known in the art.

[0257] The term "lipid nanoparticle" or "LNP" refers to a vesicle comprising a lipid layer that encapsulates a pharmaceutically active molecule, such as a nucleic acid molecule, e.g., an iRNA or a plasmid from which the iRNA is transcribed. LNPs are described, for example, in U.S. Patent Nos. 6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents of which are incorporated herein by reference.

[0258] As used herein, a "subject" is an animal, e.g., a mammal, e.g., a primate (e.g., a human, a non-human primate, e.g., a monkey or chimpanzee), a non-primate (e.g., a cow, pig, horse, goat, rabbit, sheep, hamster, guinea pig, cat, dog, rat, or mouse), or an avian that expresses an endogenous or heterologous universal taget sequence. In one embodiment, the subject is a human. In some embodiments, the subject is a human female. In other embodiments, the subject is a human male. In one embodiment, the subject is a human adult. In another embodiment, the subject is a human child.

[0259] As used herein, the term "treating" or "treatment" refers to a beneficial or desired result, such as the reduction of at least one sign or symptom of a disorder or the amelioration of at least one sign or symptom of a disease or condition in a subject.

[0260] "Treatment" can also mean prolonging survival compared to expected survival if no treatment is administered. The term "lower" refers to a statistically significant decrease in such level. The decrease can be, for example, at least 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more. In certain embodiments, the decrease is at least 50%. In certain embodiments, there is at least about a 50% decrease in the level of a disease marker, e.g., protein or gene expression. "Reducing" preferably refers to a reduction to a level that is accepted as being within the normal range in individuals without such a disorder. In certain embodiments, "reducing" refers to a reduction in the difference between the level of a marker or symptom in a subject suffering from a disease and a level that is accepted within the normal range in an individual, e.g., a reduction in the level of weight loss between an obese individual and an individual with a weight that is accepted within the normal range.

[0261] As used herein, "prevention" or "preventing," when used in reference to a disease, disorder, or condition, refers to reducing the likelihood that a subject will develop symptoms or signs associated with such disease, disorder, or condition, such as disease signs. Not developing a disease, disorder, or condition, or reducing the onset of symptoms associated with such disease, disorder, or condition (e.g., a reduction of at least about 10% of the clinically acceptable magnitude for the disease or disorder), or delaying the onset of symptoms (e.g., a delay of several days, weeks, months, or years) is considered effective prevention.

[0262] As used herein, a "therapeutically effective amount" is intended to include an amount of an RNAi agent or compound sufficient to treat a disease (e.g., by reducing, improving, or maintaining an existing disease or one or more symptoms of the disease) when administered to a subject. A "therapeutically effective amount" may vary depending on the RNAi agent or compound, how the agent is administered, the disease and its severity, and other individual characteristics of the subject being treated, such as medical history, age, weight, family history, genetic makeup, type of previous or concurrent treatment, if any.

[0263] As used herein, a "prophylactically effective amount" is intended to include an amount of an agent or compound sufficient to prevent or ameliorate a disease or one or more symptoms of a disease when administered to a subject. Amelioration of a disease includes slowing the progression of the disease or reducing the severity of subsequent disease development. A "prophylactically effective amount" may vary depending on how the agent or compound is administered, the degree of risk of the disease, and other individual characteristics of the patient being treated, such as medical history, age, weight, family history, genetic makeup, type of previous or concurrent treatment, if any.

[0264] A "therapeutically effective amount" or a "prophylactically effective amount" also encompasses that amount of an agent or compound that produces some desired effect at a reasonable benefit / risk ratio applicable to any treatment. The agents or compounds used in the methods of the invention can be administered in amounts sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0265] The phrase "pharmaceutically acceptable" is used herein to mean compounds, materials (including salts), compositions, or dosage forms that are suitable for use in contact with the tissues of human and animal subjects, within the scope of sound medical judgment, without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0266] The phrase "pharmaceutically acceptable carrier" as used herein refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, a diluent, an excipient, a manufacturing aid (e.g., lubricant, magnesium talc, calcium or zinc stearate, or stearic acid), or a solvent encapsulating material, that is involved in the transport or transportation of a compound of interest from one organ or part of the body to another organ, e.g., part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the subject being treated. Such carriers are known in the art. Pharmaceutically acceptable carriers include carriers that are administered by injection.

[0267] The term "sample," as used herein, encompasses similar bodily fluids, cells, or tissues isolated from a subject, as well as collections of bodily fluids, cells, or tissues present within a subject. Examples of biological fluids include blood, serum, serous fluid, plasma, cerebrospinal fluid, ocular fluid, lymphatic fluid, urine, saliva, and the like. Tissue samples can include samples from tissues, organs, or localized regions. For example, samples can be obtained from specific organs, parts of organs, or bodily fluids or cells within those organs. In certain embodiments, samples can be obtained from the liver (e.g., the entire liver or a specific segment of the liver, or a specific type of cell in the liver, such as hepatocytes). In some embodiments, "a sample obtained from a subject" refers to urine obtained from a subject. "A sample obtained from a subject" can refer to blood obtained from a subject, or blood obtained from plasma or serum.

[0268] II. Universal iRNAs of the Invention The present invention provides an iRNA that inhibits expression of a universal target sequence. In certain embodiments, the iRNA comprises a double-stranded ribonucleic acid (dsRNA) molecule for inhibiting expression of the ABLIM3 gene in a cell, e.g., a cell in a subject (e.g., a mammal, e.g., a human in need of treatment). The dsRNAi agent comprises an antisense strand having a region of complementarity that is complementary to at least a portion of the mRNA formed upon expression of the universal target sequence. The region of complementarity is about 19-30 nucleotides in length (e.g., about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, or 19 nucleotides in length).

[0269] Upon contact with cells expressing the universal target sequence, the iRNA inhibits expression of the universal target sequence by at least 50%, and gene expression can be assayed, for example, by PCR or branched DNA (bDNA)-based methods, or by protein-based methods, such as immunofluorescence analysis using Western blotting or flow cytometry techniques. In certain embodiments, inhibition of expression is determined in a suitable biological cell line provided herein using, for example, a 10 nM concentration of siRNA, by the qPCR method provided in the Examples herein. In certain embodiments, inhibition of in vivo expression is determined by knockdown of human universal target sequence mRNA in rodents expressing human mRNA, such as mice or AAV-infected mice expressing human universal target mRNA, for example, administered as a single dose, for example, at 3 mg / kg with minimal RNA expression.

[0270] dsRNA comprises two RNA strands, which are complementary and hybridize to form a duplex structure under the conditions when dsRNA is used.One strand of dsRNA (antisense strand) comprises a complementary region that is substantially complementary to the universal target sequence, and generally completely complementary.The target sequence can be obtained from the sequence of the hotspot of mRNA formed during the expression of the universal target sequence.The other strand (sense strand) comprises a complementary region to the antisense strand, so that when the two strands are combined under suitable conditions, they hybridize to form a duplex structure.As described elsewhere herein and known in the art, the complementary sequence of dsRNA can also be contained as a self-complementary region of a single nucleic acid molecule, so as to be opposite on separate oligonucleotides.

[0271] Generally, the duplex structure is 15 to 30 base pairs in length, e.g., 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-22, 19-23, 19-24, 19-25, 19-26, 19-27, 19-28, 19-29, 19-30, 19-31, 19-32, 19-33, 19-34, 19-35, 19-36, 19-37, 19-38, 19-39, 19-40, 19-41, 19-42, 19-43, 19-44, 19-45, 19-46, 19-47, 19-48, 19-49, 19-50, 19-51, 19-52, 19-53, 19-54, 19-55, 19-56, 19-57, 19-58, 19-59, 19-60, 19-61, 19-62, 19-63, 19-64, 19-65, 19- 9, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. In certain embodiments, the duplex structure is 18 to 25 base pairs in length, e.g., 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 in length, e.g., 19-21 base pairs in length. Ranges and lengths intermediate to the above-listed ranges and lengths are also contemplated as part of this disclosure.

[0272] Similarly, the region of complementarity to the target sequence may be 15 to 30 nucleotides in length, e.g., 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19- 27, 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, e.g., 19-23 nucleotides in length or 21-23 nucleotides in length. Ranges and lengths intermediate to the above-listed ranges and lengths are also contemplated as part of this disclosure.

[0273] In some embodiments, the duplex structure is 19 to 30 base pairs in length. Similarly, the region of complementarity to the target sequence is 19 to 30 nucleotides in length.

[0274] In some embodiments, the dsRNA is about 19 to about 23 nucleotides long, or about 25 to about 30 nucleotides long. Generally, the dsRNA is long enough to function as a substrate for Dicer enzyme. For example, it is well known in the art that dsRNA longer than about 21-23 nucleotides can function as a substrate for Dicer. As those skilled in the art will recognize, the region of RNA targeted for cleavage is almost always a part of a longer RNA molecule, often an mRNA molecule. In relevant cases, the "part" of the mRNA target is a continuous sequence of the mRNA target that is long enough to be a substrate for RNAi-dependent cleavage (i.e., cleavage by the RISC pathway).

[0275] Those skilled in the art will also recognize that a duplex region is a primary functional portion of a dsRNA, e.g., a duplex region of about 19 to about 30 base pairs, e.g., 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. Thus, in one embodiment, an RNA molecule or complex of RNA molecules having a duplex region of more than 30 base pairs is dsRNA, as long as it is processed into a functional duplex of, for example, 15-30 base pairs, that targets the desired RNA for cleavage.Thus, those skilled in the art will recognize that in one embodiment, miRNA is dsRNA.In another embodiment, the dsRNA is not a naturally occurring miRNA.In another embodiment, iRNA agents useful for targeting universal target sequences are not generated in target cells by cleavage of larger dsRNAs.

[0276] The dsRNA described herein can further comprise one or more single-stranded nucleotide overhangs, for example, 1-4, 2-4, 1-3, 2-31, 2, 3, or 4 nucleotides. A dsRNA with at least one nucleotide overhang may have superior inhibitory properties compared to its blunt-end counterpart. The nucleotide overhang may comprise or consist of a nucleotide / nucleoside analog, such as a deoxynucleotide / nucleoside. The overhang may be on the sense strand, the antisense strand, or any combination thereof. Moreover, the overhanging nucleotide may be present on the 5'-end, the 3'-end, or both of the antisense or sense strand of the dsRNA.

[0277] dsRNA can be synthesized by standard methods known in the art.Double-stranded RNAi compounds of the present invention can be prepared using a two-step method.First, each strand of double-stranded RNA molecules is prepared separately.Then, the strands of the components are annealed.The individual strands of siRNA compounds can be prepared using solution phase or solid phase organic synthesis or both.Organic synthesis has the advantage that it can easily prepare oligonucleotide strands that contain unnatural nucleotides or modified nucleotides.Similarly, single-stranded oligonucleotides of the present invention can be prepared using solution phase or solid phase organic synthesis or both.

[0278] In one embodiment, the dsRNA of the present invention comprises at least two nucleotide sequences, i.e., a sense strand and an antisense strand.The sense strand is selected from the group of sequences provided in any of Tables 2-3, and the corresponding antisense strand of the sense strand is selected from the group of sequences provided in any of Tables 2-3.In this embodiment, one of the two sequences is complementary to the other of the two sequences, and in this case, one of the sequences is substantially complementary to the sequence of the mRNA generated upon expression of the universal target sequence.Therefore, in this embodiment, the dsRNA will comprise two oligonucleotides, one oligonucleotide being described as the sense strand in any of Tables 2-3, and the second oligonucleotide being described as the corresponding antisense strand to the sense strand in any of Tables 2-3.

[0279] In certain embodiments, the sequences substantially complementary to the dsRNA are contained in separate oligonucleotides, hi other embodiments, the sequences substantially complementary to the dsRNA are contained in a single oligonucleotide.

[0280] In one embodiment, the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, or 20, contiguous nucleotides that differ by no more than 0, 1, 2, or 3 nucleotides from any of the antisense strand nucleotide sequences of any of Tables 2-3.

[0281] For example, although the sequences in Table 2 are not listed as modified or conjugated, it is understood that the RNA of the iRNA of the invention, e.g., the dsRNA of the invention, can include any of the sequences listed in Tables 2-3 that are unmodified, unconjugated, or modified or conjugated differently than those listed therein. In other words, the present invention encompasses dsRNAs of Tables 2-3 that are unmodified, unconjugated, modified, or conjugated as described herein.

[0282] Those skilled in the art are well aware that dsRNAs with duplex structures of approximately 20 to 23 base pairs, e.g., 21 base pairs, are hailed as being particularly effective in inducing RNA interference [Elbashir et al., (2001) EMBO J., 20:6877-6888]. However, others have found that shorter or longer RNA duplex structures can also be effective [Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226]. In the above-described embodiments, due to the nature of the oligonucleotide sequences provided herein, the dsRNAs described in any of Tables 2-3 can contain at least one strand with a minimum length of 21 nucleotides. It can be reasonably expected that shorter duplexes having any of the sequences in any of Tables 2-3, minus a few nucleotides at one or both ends, can be similarly effective compared to the dsRNAs described above. Thus, dsRNAs having a sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides derived from one of the sequences provided herein and that differ by no more than about 5, 10, 15, 20, 25, or 30% inhibitory percent from a dsRNA comprising a full-length sequence, as provided in the Examples herein, are contemplated to be within the scope of the present invention.

[0283] Additionally, the RNAs provided in Tables 2-3 identify sites in the universal target sequence transcript that are susceptible to RISC-mediated cleavage. Therefore, the present invention further features iRNAs that target within one of these sites. As used herein, an iRNA is said to target within a specific site of an mRNA transcript if it promotes cleavage of the mRNA transcript anywhere within that specific site. Such iRNAs will generally comprise at least about 19 nucleotides from any of the sequences provided in any of Tables 2-3 coupled to additional nucleotide sequences taken from regions adjacent to the selected sequence in the universal target sequence.

[0284] III. Modified Universal iRNAs of the Invention In certain embodiments, the universal iRNA of the present invention, for example, dsRNA, is unmodified, for example, does not contain chemical modifications or conjugations known in the art and described herein.In other embodiments, the universal iRNA of the present invention, for example, dsRNA, is chemically modified to enhance stability or other beneficial characteristics.In certain embodiments of the present invention, substantially all of the nucleotides of the universal iRNA of the present invention are modified, i.e., there are 5, 4, 3, 2, or 1 or less unmodified nucleotides in the chain of iRNA.In other embodiments of the present invention, all of the nucleotides of the universal iRNA are modified.

[0285] Nucleic acids featured in the present invention can be synthesized or modified by methods well established 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, incorporated herein by reference. Modifications include, for example, terminal modifications, such as 5'-end modifications (phosphorylation, conjugation, reverse ligation) or 3'-end modifications (conjugation, DNA nucleotides, reverse ligation, etc.), base modifications, such as replacement with a stabilizing base, a destabilizing base, or a base that base-pairs with an expanded repertoire partner, removal of a base (abasic nucleotide) or a conjugated base, sugar modifications (e.g., at the 2' or 4' position) or sugar replacement, or backbone modifications, including modification or replacement of a phosphodiester bond. Specific examples of iRNA compounds useful in the embodiments described herein include, but are not limited to, RNAs containing modified backbones or lacking natural internucleoside linkages. Among the RNAs with modified backbones, those that do not have phosphorus atoms in backbones can be considered.For the purpose of this specification, as sometimes referred to in the art, the modified RNA that does not have phosphorus atoms in its internucleoside backbone can also be considered as oligonucleoside.In some embodiments, modified iRNA has phosphorus atoms in its internucleoside backbone.

[0286] Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methylphosphonates, and other alkyl phosphonates, including 3'-alkylene phosphonates and chiral phosphonates; phosphinates; phosphoramidates, including 3'-aminophosphoramidates and aminoalkylphosphoramidates; thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates with normal 3'-5' linkages, their analogs linked 2'-5', and those with reverse polarity, in which adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included. In some embodiments of the present invention, the dsRNA agents of the present invention are in free acid form. In other embodiments of the present invention, the dsRNA agents of the present invention are in salt form. In one embodiment, the dsRNA agents of the present invention are in sodium salt form. In certain embodiments, when the dsRNA agent of the present invention is in sodium salt form, sodium ion exists in the agent as the counterion for substantially all of the phosphodiester and / or phosphorothioate groups present in the agent.The agent in which substantially all of the phosphodiester and / or phosphorothioate linkages have sodium counterion comprises 5, 4, 3, 2 or 1 or less phosphodiester and / or phosphorothioate linkages that do not have sodium counterion.In some embodiments, when the dsRNA agent of the present invention is in sodium salt form, sodium ion exists as the counterion for all of the phosphodiester and / or phosphorothioate groups present in the agent.

[0287] Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Patent Nos. 3,687,808, 4,469,863, 4,476,301, 5,023,243, 5,177,195, 5,188,897, 5,264,423, 5,276,019, 5,278,302, 5,286,711, and 5,286,712. No. 7, No. 5,321,131, No. 5,399,676, No. 5,405,939, No. 5,453,496, No. 5,455,233, No. 5,466,677, No. 5,476 , No. 925, No. 5,519,126, No. 5,536,821, No. 5,541,316, No. 5,550,111, No. 5,563,253, No. 5,571,799, No. 5,5 87,361, 5,625,050, 6,028,188, 6,124,445, 6,160,109, 6,169,170, 6,172,209, No. 6,239,265, No. 6,277,603, No. 6,326,199, No. 6,346,614, No. 6,444,423, No. 6,531,590, No. 6,534,639 , 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. Reissue Patent No. RE39464, the entire contents of each of which are incorporated herein by reference.

[0288] 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, including morpholino linkages (some formed from the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and others with mixed N, O, S, and CH2 constituent moieties.

[0289] Representative United States patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Patent Nos. 5,034,506, 5,166,315, 5,185,444, 5,214,134, 5,216,141, 5,235,033, 5,64,562, 5,264,564, 5,405,938, 5,434,257, 5,466,677, 5,470,967, Nos. 5,489,677, 5,541,307, 5,561,225, 5,596,086, 5,602,240, 5,608,046, 5,610,289, 5,618,704, 5,623,070, 5,663,312, 5,633,360, 5,677,437 and 5,677,439, the entire contents of each of which are incorporated herein by reference.

[0290] RNA mimetics suitable for use in the iRNAs provided herein are contemplated, in which both the sugar and internucleoside linkages, i.e., the backbone, of nucleotide units are replaced with novel groups. The base units are maintained for hybridization with appropriate nucleic acid target compounds. One such oligomeric compound, an RNA mimetic known to have excellent hybridization properties, is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleobases are retained and are directly or indirectly linked to the aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents teaching the preparation of PNA compounds include, but are not limited to, U.S. Patent Nos. 5,539,082, 5,714,331, and 5,719,262, the entire contents of each of which are incorporated herein by reference. Further suitable PNA compounds for use in the iRNA of the present invention are described, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0291] Some embodiments featured in the present invention include RNAs with phosphorothioate backbones and heteroatom backbones, particularly --CH2--NH--CH2-, --CH2--N(CH3)--O--CH2-- (known as methylene (methylimino) or MMI backbones), --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2--, and --N(CH3)--CH2--CH2-- of the above-referenced U.S. Pat. No. 5,489,677, and oligonucleosides with amide backbones of the above-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAs featured herein have the morpholino backbone structure of the above-referenced U.S. Pat. No. 5,034,506. The natural phosphodiester backbone can be represented as --OP(O)(OH)--OCH2--.

[0292] Modified RNAs may also contain one or more substituted sugar moieties. The iRNAs, e.g., dsRNAs, featured herein may contain one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl, or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C6. 10 Alkyl or C2-C 10 It can be alkenyl and alkynyl. Exemplary suitable modifications include O[(CH) n O] m CH3, O(CH2). n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2 and O(CH2) n ON[(CH2) n CH3)]2, where n and m are from 1 to about 10. In other embodiments, the dsRNA has at the 2' position one of the following: C1 to C 10The modification may include one of the following: lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH, OCN, Cl, Br, CN, CF, OCF, SOCH, SOCH, ONO, NO, N, NH, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving group, reporter group, interfering substance, group for improving the pharmacokinetic properties of iRNA or group for improving the pharmacodynamic properties of iRNA, and other substituents with similar properties. In some embodiments, the modification includes 2'-methoxyethoxy (2'-O--CHCHOCH, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., an alkoxy-alkoxy group. Another exemplary modification is the 2'-dimethylaminooxyethoxy, i.e., O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, as described herein below in the Examples, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O--CH2--O--CH2--N(CH3)2. Further exemplary modifications include 5'-Me-2'-F nucleotides, 5'-Me-2'-OMe nucleotides, 5'-Me-2'-deoxynucleotides (both R and S isomers in these three families), 2'-alkoxyalkyl, and 2'-NMA (N-methylacetamide).

[0293] Other modifications include 2'-methoxy (2'-OCH), 2'-aminopropoxy (2'-OCHCHCHNH), 2'-O-hexadecyl, and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA of an iRNA, particularly the 3' position of the sugar on the 3'-terminal nucleotide or in a 2'-5'-linked dsRNA and the 5' position of the 5'-terminal nucleotide. An iRNA can also have a sugar mimetic, such as a cyclobutyl moiety in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Patent Nos. 4,981,957, 5,118,800, 5,319,080, 5,359,044, 5,393,878, 5,446,137, 5,466,786, 5,514,785, 5,519,134, and 5,56 Nos. 7,811, 5,576,427, 5,591,722, 5,597,909, 5,610,300, 5,627,053, 5,639,873, 5,646,265, 5,658,873, 5,670,633, and 5,700,920, certain of which are commonly owned with the present application, the entire contents of each of which are incorporated herein by reference.

[0294] Universal iRNAs may also contain nucleobase (often simply referred to in the art as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include 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-azouracil, cytosine, and thymine. , 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, particularly 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.Additional nucleobases include those disclosed in U.S. Patent No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008, those disclosed in The Concise Encyclopedia of Polymer Science and Engineering, pages 858-859, Kroschwitz, J. L. ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., (1991) Angewandte Chemie, International Edition, 30:613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the present invention. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-Methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, YS, Crooke, ST, and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), and are an exemplary base substitution, even more particularly when combined with a 2'-O-methoxyethyl sugar modification.

[0295] Representative United States patents that teach the preparation of certain of the above-mentioned modified nucleobases, as well as other modified nucleobases, include, but are not limited to, the above-mentioned U.S. Patent Nos. 3,687,808, 4,845,205, 5,130,30, 5,134,066, 5,175,273, 5,367,066, 5,432,272, 5,457,187, 5,459,255, 5,484,908, 5,502,177, 5,525,711, 5,552,540, 5,587,469, Nos. 5,594,121, 5,596,091, 5,614,617, 5,681,941, 5,750,692, 6,015,886, 6,147,200, 6,166,197, 6,222,025, 6,235,887, 6,380,368, 6,528,640, 6,639,062, 6,617,438, 7,045,610, 7,427,672 and 7,495,088, the entire contents of each of which are incorporated herein by reference.

[0296] In some embodiments, the RNAi agents of the present disclosure can also be modified to include one or more bicyclic sugar moieties. A "bicyclic sugar" is a furanosyl ring modified with a ring formed by a bridge between two carbon atoms, whether adjacent or non-adjacent. A "bicyclic nucleoside" ("BNA") is a nucleoside having a sugar moiety containing a ring formed by bridging two carbon atoms, whether adjacent or non-adjacent, of the sugar ring, thereby forming a bicyclic ring structure. In certain embodiments, the bridge connects the 4'-carbon and 2'-carbon of the sugar ring, as appropriate, through the 2' acyclic carbon atom. Thus, in some embodiments, the agents of the present invention can include one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety includes an additional bridge connecting the 2' and 4' carbons. In other words, an LNA is a nucleotide containing a bicyclic sugar moiety containing a 4'-CH2-O-2' bridge. This structure effectively "locks" the ribose into a 3'-endo conformation. The addition of locked nucleic acids to siRNA has been shown to increase siRNA stability in serum and reduce off-target effects [Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, OR. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193]. Examples of bicyclic nucleosides for use in the polynucleotides of the present disclosure include, but are not limited to, nucleosides containing a bridge between the 4' and 2' ribosyl ring atoms. In certain embodiments, the antisense polynucleotide agent of the present disclosure includes one or more bicyclic nucleosides containing a 4' to 2' bridge.

[0297] Locked nucleic acids can be represented by the following structure (stereochemistry omitted): [ka] B is a nucleobase or modified nucleobase, and L is a linking group connecting the 2'-carbon and 4'-carbon of the ribose ring. Examples of such 4' to 2' bridged bicyclic nucleosides include, but are not limited to, 4'-(CH2)-O-2' (LNA), 4'-(CH2)-S-2', 4'-(CH2)2-O-2' (ENA), 4'-CH(CH3)-O-2' (also known as "constrained ethyl" or "cEt"), and 4'-CH(CHOCH3)-O-2' (and analogs thereof, see e.g., U.S. Pat. No. 7,399,845), 4'-C(CH3)(CH3)-O-2' (and analogs thereof, see e.g., U.S. Pat. No. 8,222,222). No. 78,283), 4'-CH2-N(OCH3)-2' (and analogs thereof, see, e.g., U.S. Pat. No. 8,278,425), 4'-CH2-ON(CH3)-2' (see, e.g., U.S. Patent Publication No. 2004 / 0171570), 4'-CH2-N(R)-O-2' (wherein R is H, C1-C12 alkyl, or a nitrogen protecting group) (see, e.g., U.S. Pat. No. 7,427,672), 4'-CH2-C(H)(CH3)-2' (see, e.g., Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134) and 4'-CH2-C(-CH2)-2' (and analogs thereof, see, e.g., U.S. Pat. No. 8,278,426), the entire contents of each of which are incorporated herein by reference.

[0298] Additional representative U.S. patents and publications that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Patent Nos. 6,268,490, 6,525,191, 6,670,461, 6,770,748, 6,794,499, 6,998,484, 7,053,207, 7,034,133, 7,084,125, Nos. 7,399,845, 7,427,672, 7,569,686, 7,741,457, 8,022,193, 8,030,467, 8,278,425, 8,278,426, 8,278,283, US2008 / 0039618 and US2009 / 0012281, the entire contents of each of which are incorporated herein by reference.

[0299] For example, any of the above bicyclic nucleosides can be prepared with one or more stereochemical sugar configurations including α-L-ribofuranose and β-D-ribofuranose (see WO 99 / 14226).

[0300] The iRNAs of the present invention can also be modified to contain one or more constrained ethyl nucleotides. As used herein, a "constrained ethyl nucleotide" or "cEt" is a locked nucleic acid containing a bicyclic sugar moiety containing a 4'-CH(CH3)-0-2' bridge (i.e., L in the structure above). In one embodiment, the constrained ethyl nucleotide is in the S conformation and is referred to herein as an "S-cEt."

[0301] The iRNA of the present invention may also contain one or more "conformation-restricting nucleotides" ("CRNs"). A CRN is a nucleotide analogue with a linker connecting the C2' and C4' carbons of ribose or the C3 and C5' carbons of ribose. The CRN locks the ribose ring into a stable conformation, increasing hybridization affinity to mRNA. The linker is of sufficient length to position the oxygen in an optimal position for stability and affinity, resulting in less ribose ring puckering.

[0302] Representative publications that teach the preparation of certain of the above CRNs include, but are not limited to, U.S. Patent Publication No. 2013 / 0190383 and PCT Publication WO2013 / 036868, the entire contents of each of which are incorporated herein by reference.

[0303] In some embodiments, the iRNA of the present invention includes one or more monomers that are UNA (non-locked nucleic acid) nucleotides. UNAs are non-locked acyclic nucleic acids in which any of the sugar linkages have been removed to form non-locked "sugar" residues. In one example, UNAs also encompass monomers in which the C1'-C4' bond (i.e., the covalent carbon-oxygen-carbon bond between the C1' and C4' carbons) has been removed. In another example, the C2'-C3' bond (i.e., the covalent carbon-carbon bond between the C2' and C3' carbons) of the sugar has been removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039, which are incorporated herein by reference).

[0304] Representative U.S. publications that teach the preparation of UNAs 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 contents of each of which are incorporated herein by reference.

[0305] In certain embodiments, the compositions and methods of the present disclosure include vinyl phosphonate (VP) modifications of RNAi agents as described herein. In an exemplary embodiment, a 5' vinyl phosphonate modified nucleotide of the present disclosure has the following structure: [ka] X is O or S; R is hydrogen, hydroxy, fluoro, or C1~20 alkoxy (e.g., methoxy or n-hexadecyloxy); R 5’ is =C(H)-P(O)(OH)2, and the double bond between the C5' carbon and R5' is in the E or Z configuration (e.g., the E configuration); B is a nucleobase or a modified nucleobase, where B may be adenine, guanine, cytosine, thymine, or uracil.

[0306] The vinyl phosphonate of the present disclosure can be attached to either the antisense or sense strand of the dsRNA of the present disclosure.In certain embodiments, the vinyl phosphonate of the present disclosure is attached to the antisense strand of dsRNA at the 5' end of the antisense strand of dsRNA as appropriate.The dsRNA agent can comprise a phosphorus-containing group at the 5' end of the sense strand or the antisense strand.

[0307] Vinyl phosphonate modifications are also contemplated in the compositions and methods of the present disclosure. Exemplary vinyl phosphonate modifications are included in the structures above, R 5’ is =C(H)-P(O)(OH)2, and the double bond between the C5' carbon and R5' is in the E or Z configuration (e.g., the E configuration).

[0308] Potentially stabilizing modifications to the ends of RNA molecules include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3'-phosphate, reverse dT (idT), reverse dA (idA), reverse abasic 2' deoxyribonucleotide (iAb), and others. Disclosure of this modification can be found in WO2011 / 005861.

[0309] In one example, the 3' or 5' end of the oligonucleotide is linked to a reverse 2'-deoxy modified ribonucleotide, such as, for example, reverse dT (idT), reverse dA (idA), or reverse abasic 2'-deoxyribonucleotide (iAb). In certain examples, the reverse 2'-deoxy modified ribonucleotide is linked to the 3' end of the oligonucleotide, such as the 3' end of the sense strand described herein, and this linkage is via a 3'-3' phosphodiester linkage or a 3'-3' phosphorothioate linkage.

[0310] In another example, the 3'-end of the sense strand is linked to an inverted abasic ribonucleotide (iAb) via a 3'-3'-phosphorothioate linkage. In another example, the 3'-end of the sense strand is linked to an inverted dA (idA) via a 3'-3'-phosphorothioate linkage.

[0311] In certain instances, the reverse 2'-deoxy modified ribonucleotide is linked to the 3' end of the oligonucleotide, such as the 3' end of the sense strand described herein, via a 3'-3' phosphodiester linkage or a 3'-3' phosphorothioate linkage.

[0312] In another example, the 3'-terminal nucleotide of the sense strand is an inverted dA (idA) and is linked to the preceding nucleotide via a 3'-3'-linkage (eg, a 3'-3'-phosphorothioate linkage).

[0313] Other modifications of the nucleotides of the iRNAs of the present invention include 5' phosphates or 5' phosphate mimics, such as 5' terminal phosphates or phosphate mimics on the antisense strand of the iRNA. Suitable phosphate mimics are disclosed, for example, in U.S. Patent Publication No. 2012 / 0157511, the entire contents of which are incorporated herein by reference.

[0314] A. Modified iRNAs Containing Motifs of the Invention In certain embodiments of the present invention, the double-stranded iRNA of the present invention comprises an agent with chemical modifications, such as those disclosed in WO2013 / 075035, the entire contents of which are each 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 strand or antisense strand of dsRNAi agent at or near the cleavage site.In some embodiments, the sense strand and antisense strand of dsRNAi agent can be otherwise completely modified.The introduction of these motifs will disrupt the modification pattern of the sense or antisense strand if present.The dsRNAi agent can also be conjugated with GalNAc derivative ligand, for example, C16 ligand on the sense strand.

[0315] More specifically, gene silencing activity of a dsRNAi agent was observed when the sense and antisense strands of the double-stranded RNA agent were fully modified to have one or more identical triple-modification motifs in three consecutive bases at or near the cleavage site of at least one strand of the dsRNAi agent.

[0316] Thus, the present invention provides double-stranded RNA agents capable of inhibiting expression of universal target genes in vivo. The RNAi agents include a sense strand and an antisense strand. Each strand of the RNAi agent can be, for example, 17-30 nucleotides, 25-30 nucleotides, 27-30 nucleotides, 19-25 nucleotides, 19-23 nucleotides, 19-21 nucleotides, 21-25 nucleotides, or 21-23 nucleotides in length.

[0317] The sense strand and antisense strand typically form a duplex, double-stranded RNA ("dsRNA"), also referred to herein as a "dsRNAi agent." The duplex region of a dsRNAi agent can be, for example, 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 duplex region is selected from 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides in length.

[0318] In certain embodiments, a dsRNAi agent may contain one or more overhang regions or capping groups at the 3'-end, 5'-end, or both ends of one or both strands. The overhangs may be 1 to 6 nucleotides in length, e.g., 2 to 6 nucleotides, 1 to 5 nucleotides, 2 to 5 nucleotides, 1 to 4 nucleotides, 2 to 4 nucleotides, 1 to 3 nucleotides, 2 to 3 nucleotides, or 1 to 2 nucleotides in length. In certain embodiments, the overhang region may include an extended overhang region as provided above. The overhang may be the result of one strand being longer than the other or the result of two strands of the same length being staggered. The overhang may form a mismatch with the target mRNA, be complementary to the targeted gene sequence, or be a different sequence. The first and second strands may also be connected by additional bases, for example, to form a hairpin, or by other non-basic linkers.

[0319] In certain embodiments, the nucleotides in the overhang region of a dsRNAi agent can each independently be a modified or unmodified nucleotide, including, but not limited to, a 2'-sugar modified, e.g., 2'-F, 2'-O-methyl, thymidine (T), 2'-O-methoxyethyl-5-methyluridine (Teo), 2'-O-methoxyethyladenosine (Aeo), 2'-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combination thereof.

[0320] For example, TT can be an overhang sequence at either end on either strand. The overhang can form a mismatch with the target mRNA, or can be complementary to the targeted gene sequence, or can be another sequence.

[0321] The 5'- or 3'-overhang of the sense strand, antisense strand, or both strands of dsRNAi agent can be phosphorylated.In some embodiments, the overhang region(s) contain two nucleotides with phosphorothioate between them, and 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, this 3'-overhang is present in the antisense strand.In some embodiments, this 3'-overhang is present in the sense strand.

[0322] dsRNAi agent can contain only a single overhang, which can enhance the interference activity of RNAi without affecting its overall stability.For example, the single-stranded overhang can be located at the 3' end of the sense strand or at the 3' end of the antisense strand.RNAi can also have a blunt end 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 dsRNAi agent has a nucleotide overhang at the 3' end, and the 5' end is blunt.Without wishing to be bound by theory, the blunt end at the 5' end of the asymmetric antisense strand and the 3' end overhang of the antisense strand are favorable for the guide strand loading into RISC process.

[0323] In certain embodiments, the dsRNAi agent is blunt-ended at both ends and is 19 nucleotides in length, and the sense strand contains at least one motif with 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 with three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0324] In other embodiments, the dsRNAi agent is blunt-ended at both ends and is 20 nucleotides in length, and the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 8, 9, and 10 from the 5' end. The antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0325] In yet other embodiments, the dsRNAi agent is blunt-ended at both ends and is 21 nucleotides in length, and the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 9, 10, and 11 from the 5' end. The antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0326] In one embodiment, the dsRNAi agent comprises a 21-nucleotide sense strand and a 23-nucleotide antisense strand, wherein the sense strand comprises at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 9, 10, and 11 from the 5' end, and the antisense strand comprises at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end, and one end of the RNAi agent is blunt, and the other end comprises a 2-nucleotide overhang.Preferably, the 2-nucleotide overhang is at the 3' end of the antisense strand.

[0327] When there is a 2-nucleotide overhang at the 3'-end of antisense strand, there may be two phosphorothioate internucleotide linkages between the three nucleotides at the end, two of which are overhanging nucleotides, and the third nucleotide is the next paired nucleotide of the overhanging nucleotide.In certain embodiments, the dsRNAi agent further has two phosphorothioate internucleotide linkages between the three nucleotides at the 5'-end of sense strand and the 5'-end of antisense strand.In certain embodiments, every nucleotide in the sense strand and antisense strand of the RNAi agent, including the nucleotide that is part of the motif, is a modified nucleotide.In one embodiment, each residue is independently modified with 2'-O-methyl or 3'-fluoro, for example, in the alternating motif.The dsRNAi agent may further comprise a ligand (for example, GalNAc3).

[0328] In certain embodiments, a dsRNAi agent comprises a sense and an antisense strand, wherein the sense strand is 25-30 nucleotide residues in length, and starting from the 5'-terminal nucleotide (position 1), positions 1-23 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 that pair with positions 1-23 of the sense strand to form a duplex; at least the 3'-terminal nucleotide of the antisense strand is not paired with the sense strand, and up to six consecutive 3'-terminal nucleotides are not paired with the sense strand, thereby forming a 3' single-stranded overhang of 1-6 nucleotides; and the 5' end of the antisense strand comprises 10-30 consecutive ribonucleotides that are not paired with the sense strand. The double-stranded nucleic acid comprises at least one nucleotide sequence consisting of three 2'-F modifications on three consecutive nucleotides, thereby forming a 10-30 nucleotide single-stranded 5' overhang, at least the 5'- and 3'-terminal nucleotides of the sense strand being base-paired with nucleotides in the antisense strand when the sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially double-stranded region between the sense and antisense strands, the antisense strand being sufficiently complementary to the target RNA along at least 19 ribonucleotides of the length of the antisense strand, such that expression of the universal target site is reduced when the double-stranded nucleic acid is introduced into a mammalian cell, the sense strand containing at least one motif of three 2'-F modifications on three consecutive nucleotides, at least one of the motifs occurring at or near the cleavage site, and the antisense strand containing at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at or near the cleavage site.

[0329] In certain embodiments, the dsRNAi agent comprises a sense and an antisense strand, the dsRNAi agent comprising a first strand having a length of at least 25 and at most 29 nucleotides, and a second strand having a length of at most 30 nucleotides and having at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end, the 3' end of the first strand and the 5' end of the second strand forming a blunt end, the second strand being 1 to 4 nucleotides longer at its 3' end than the first strand, the duplex region being at least 25 nucleotides long, the second strand being sufficiently complementary to the target mRNA along the length of the second strand of at least 19 nucleotides, the RNAi agent reducing expression of the universal target site when introduced into a mammalian cell, and Dicer cleavage of the dsRNAi agent preferentially producing siRNAs comprising the 3' end of the second strand, thereby reducing expression of the universal target site in the mammal. Optionally, the dsRNAi agent may further comprise a ligand.

[0330] In certain embodiments, the sense strand of the dsRNAi agent contains at least one motif of three identical modifications on three consecutive nucleotides, one of the motifs occurring at the cleavage site in the sense strand.

[0331] In certain embodiments, the antisense strand of the dsRNAi agent can also contain at least one motif of three identical modifications on three consecutive nucleotides, one of the motifs occurring at or near the cleavage site in the antisense strand.

[0332] For RNAi agents with duplex regions 19-23 nucleotides long, the cleavage sites in the antisense strand are typically approximately positions 10, 11, and 12 from the 5' end. Thus, three identical modification motifs may occur in the antisense strand at positions 9, 10, and 11; positions 10, 11, and 12; positions 11, 12, and 13; positions 12, 13, and 14; or positions 13, 14, and 15, with the numbers starting from the first nucleotide from the 5' end of the antisense strand, or the numbers starting from the first paired nucleotide in the duplex region from the 5' end of the antisense strand. The cleavage site in the antisense strand may also vary depending on the length of the duplex region of the dsRNAi agent from the 5' end.

[0333] The sense strand of dsRNAi agent can contain at least one motif of three identical modifications on three consecutive nucleotides at the breakpoint of strand, and antisense strand can have at least one motif of three identical modifications on three consecutive nucleotides at or near the breakpoint of strand.When sense strand and antisense strand form dsRNA duplex, sense strand and antisense strand can be arranged so that one motif of three nucleotides on sense strand and one motif of three nucleotides on antisense strand have at least one nucleotide overlap, that is, at least one of the three nucleotides of the motif in sense strand and at least one of the three nucleotides of the motif in antisense strand form base pairs.Alternatively, at least two nucleotides can overlap, or all three nucleotides can overlap.

[0334] In some embodiments, the sense strand of a dsRNAi agent may contain two or more motifs 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 occurring in another part of the strand that is distant from the motif at or near the cleavage site of the same strand. The wing modifications are adjacent to the first motif or are separated by at least one or more nucleotides. When the motifs are immediately adjacent to each other, the chemistry of the motifs is distinct from each other, and when the motifs are separated by one or more nucleotides, the chemistry may be the same or different. Two or more wing modifications may also be present. For example, when two wing modifications are present, each wing modification may occur at one end of the first motif at or near the cleavage site, or on either side of the lead motif.

[0335] Similar to sense strand, antisense strand of dsRNAi agent can contain two or more motifs of three identical modifications on three consecutive nucleotides, and at least one of the motifs occurs at or near the break site of strand.This antisense strand can also contain one or more wing modifications with the same sequence as the wing modifications that can be present on sense strand.

[0336] In some embodiments, wing modifications on the sense or antisense strand of a dsRNAi agent typically do not include the first one or two terminal nucleotides at the 3' end, 5' end, or both ends of the strand.

[0337] In other embodiments, wing modifications on the sense or antisense strand of a dsRNAi agent typically do not include the first one or two paired nucleotides in the duplex region at the 3' end, 5' end, or both ends of the strand.

[0338] When the sense and antisense strands of a dsRNAi agent each contain at least one wing modification, the wing modifications can fall at the same end of the duplex region and have an overlap of 1, 2, or 3 nucleotides.

[0339] When the sense or antisense strand of a dsRNAi agent each contains at least two wing modifications, the sense and antisense strands can be arranged such that two modifications from one strand each occupy one end of the duplex region, with an overlap of 1, 2, or 3 nucleotides; two modifications from one strand each occupy the other end of the duplex region, with an overlap of 1, 2, or 3 nucleotides; and two modifications one strand occupy each side of the lead motif, with an overlap of 1, 2, or 3 nucleotides in the duplex region.

[0340] In some embodiments, any nucleotide in the sense strand and antisense strand of dsRNA agent, including the nucleotide that is part of motif, can be modified.Each nucleotide can be modified with the same or different modifications, which can include one or more of the non-linked phosphate oxygen or one or more of the linking phosphate oxygen, one or both of the components of ribose sugar, for example, the 2' hydroxyl on ribose sugar, the large-scale replacement of phosphate moiety with " dephosphorylation " linker, the modification or replacement of naturally occurring base, and the replacement or modification of ribose-phosphate backbone.

[0341] Because nucleic acids are polymers of subunits, many modifications occur at positions that are repeated within nucleic acids, such as modifications of bases or phosphate moieties or non-linked Os of phosphate moieties. In some cases, modifications occur at all target positions in nucleic acids, but in many cases, they do not occur. For example, modifications may occur only at the 3'- or 5'-terminal position, or only in terminal regions, for example, at the terminal nucleotide position of the chain, or in the last 2, 3, 4, 5, or 10 nucleotides. Modifications may occur in double-stranded regions, single-stranded regions, or both. Modifications may occur only in the double-stranded region of RNA, or only in the single-stranded region of RNA. For example, phosphorothioate modifications at non-linked O positions may occur only at one or both ends, or only in terminal regions, for example, at the terminal nucleotide position of the chain, or in the last 2, 3, 4, 5, or 10 nucleotides, or in double-stranded and single-stranded regions, especially at the ends. The 5'-end or both ends can be phosphorylated.

[0342] For example, it may be possible to enhance stability, include specific bases in the overhang, or include modified nucleotides or nucleotide substitutes in the single-stranded overhang, for example, in the 5'- or 3'-overhang, or both. For example, it may be desirable to include purine nucleotides in the overhang. In some embodiments, all or some of the bases in the 3'- or 5'-overhang can be modified, for example, with the modifications described herein. Modifications can include, for example, the use of modifications at the 2' position of the ribose sugar with modifications known in the art, for example, the use of 2'-deoxy-2'-fluoro (2'-F) or 2'-O-methyl modified deoxyribonucleotides instead of the ribosugar of the nucleobase, and modifications at the phosphate group, for example, phosphorothioate modifications. The overhang does not need to be homologous to the target sequence.

[0343] 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'-hydroxyl, or 2'-fluoro. The strand can contain two or more modifications. In one embodiment, each residue of the sense strand and the antisense strand is independently modified with 2'-O-methyl or 2'-fluoro.

[0344] Usually, there are at least two different modifications in the sense and antisense strands, which can be 2'-O-methyl or 2'-fluoro modifications, or other modifications.

[0345] In certain embodiments, N a or N b includes an alternating pattern of modifications. The term "alternating motif," as used herein, refers to a motif having one or more modifications, each modification occurring at alternating nucleotides in a strand. Alternating nucleotides may refer to one every other nucleotide or one every third nucleotide, or similar patterns. For example, if A, B, and C each represent one type of modification to a nucleotide, the alternating motif could be "ABABABABABAB...," "AABBAABBAABB...," "AABAABAABAAB...," "AAABAAABAAAB...," "AABBBAAABBB...," or "ABCABCABCABC...," etc.

[0346] The types of modifications contained within the alternating motifs can be the same or different. For example, if A, B, C, and D each represent one type of modification on a nucleotide, the alternating turns, i.e., the modifications on every other nucleotide, can be the same, but each of the sense or antisense strands can be selected from several possibilities for modifications within the alternating motif, such as "ABABAB...", "ACACAC...", "BDBDBD..." or "CDCDCD...".

[0347] In some embodiments, the dsRNAi agent of the present invention comprises an alternating motif modification pattern on the sense strand that is shifted relative to the alternating motif modification pattern on the antisense strand. The shift can be such that the modified groups of the nucleotides of the sense strand correspond to the differently modified groups of the nucleotides of the antisense strand, or vice versa. For example, when the sense strand is paired with the antisense strand in a dsRNA duplex, the alternating motif in the sense strand may start with "ABABAB" from the 5'-3' of the strand, and the alternating motif in the antisense strand may start with "BABABA" from the 5'-3' of the strand in the duplex region. As another example, the alternating motif in the sense strand may start with "AABBAABB" from the 5'-3' of the strand, and the alternating motif in the antisense strand may start with "BBAABBAA" from the 5'-3' of the strand in the duplex region, resulting in a complete or partial shift in the modification pattern between the sense strand and the antisense strand.

[0348] In some embodiments, dsRNAi agent is firstly shifted to the pattern of alternating motifs of 2'-O-methyl modification and 2'-F modification on sense strand firstly relative to the pattern of alternating motifs of 2'-O-methyl modification and 2'-F modification on antisense strand, that is, the 2'-O-methyl modified nucleotide on sense strand comprises base pair with the 2'-F modified nucleotide on antisense strand, and vice versa.Position 1 of sense strand can start with 2'-F modification, and position 1 of antisense strand can start with 2'-O-methyl modification.

[0349] By introducing one or more motifs of three identical modifications on three consecutive nucleotides into sense strand or antisense strand, the initial modification pattern that exists in sense strand or antisense strand is interrupted.By introducing one or more motifs of three identical modifications on three consecutive nucleotides into sense strand or antisense strand, this interruption of the modification pattern of sense strand or antisense strand can enhance the gene silencing activity against universal target sequence.

[0350] In some embodiments, when a motif of three identical modifications on three consecutive nucleotides is introduced on either strand, the modification of the nucleotide next to the motif is a different modification than the modification of the motif. For example, the portion of the sequence containing the motif may be described as "...N a YYYN b ...', where 'Y' represents a modification of a motif of three identical modifications on three consecutive nucleotides, and 'N a " and "N b " represents a modification of the nucleotide next to the motif "YYY" that is different from the modification of Y, and where N a and N b may be the same or different modifications. a or N b may or may not be present if wing modifications are present.

[0351] The iRNA may further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage. The phosphorothioate or methylphosphonate internucleotide linkage modification may occur at any nucleotide in the sense strand, the antisense strand, or both strands at any position along the strand. For example, the internucleotide linkage modification may occur at any nucleotide on the sense strand or the antisense strand, and 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 contain both internucleotide linkage modifications in an alternating pattern. The alternating pattern of internucleotide linkage modifications on the sense strand may be the same as or different from that of the antisense strand, and the alternating pattern of internucleotide linkage modifications on the sense strand may have a shift relative to the alternating pattern of internucleotide linkage modifications on the antisense strand. In one embodiment, the double-stranded RNAi agent comprises 6 to 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 either the 5'-end or the 3'-end.

[0352] In some embodiments, dsRNA agent comprises phosphorothioate or methylphosphonate internucleotide linkage modification in overhang region.For example, overhang region can comprise two nucleotides with phosphorothioate or methylphosphonate internucleotide linkage between two nucleotides.Internucleotide linkage modification can also be carried out so that overhang nucleotide is linked to the nucleotide paired at the end in double-stranded region.For example, at least 2, 3, 4 or all overhang nucleotides can be linked by phosphorothioate or methylphosphonate internucleotide linkage, and there can be additional phosphorothioate or methylphosphonate internucleotide linkage that connects overhang nucleotide to the nucleotide paired adjacent to overhang nucleotide.For example, there can be at least two phosphorothioate internucleotide linkages between the three nucleotides at the end, where two of the three nucleotides are overhang nucleotides, and the third is the nucleotide paired adjacent to overhang nucleotide. These terminal three nucleotides can be 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.

[0353] In some embodiments, when a 2-nucleotide overhang is at the 3'-end of the antisense strand, there are two phosphorothioate internucleotide linkages between the terminal three nucleotides, two of which are overhanging nucleotides, and the third nucleotide is the next paired nucleotide after the overhanging nucleotide.Optionally, the dsRNAi agent can further 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.

[0354] In one embodiment, dsRNAi agent comprises mismatch(es) or combinations thereof in the double strand with target. Mismatch can occur in overhang region or double-stranded region. Base pair can be ranked based on its tendency to promote dissociation or melting (for example, the free energy of association or dissociation of specific pairing; the simplest approach is to examine pairs on an individual basis, but next-neighbor analysis or similar analysis can also be used). In terms of promoting dissociation: A:U is more preferred than G:C, G:U is more preferred than G:C, and I:C is more preferred than G:C (I=inosine). Mismatch, for example, non-canonical pairing or other than canonical pairing (as described elsewhere herein) is more preferred than canonical (A:T, A:U, G:C) pairing, and pairing involving universal bases is more preferred than canonical pairing.

[0355] 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 independently selected from the group of A:U, G:U, I:C, and mismatch pairs, e.g., non-canonical pairings or other than canonical pairings or pairings including universal bases, to promote dissociation of the antisense strand at the 5' end of the duplex.

[0356] In certain embodiments, the nucleotide at position 1 in the double-stranded region from the 5' end of antisense strand is selected from A, dA, dU, U and dT.Alternatively, at least one of the first 1, 2 or 3 base pairs in the double-stranded region from the 5' end of antisense strand is AU base pair.For example, the first base pair in the double-stranded region from the 5' end of antisense strand is AU base pair.

[0357] In other embodiments, the 3'-terminal nucleotide of the sense strand is deoxythymidine (dT) or the 3'-terminal nucleotide of the antisense strand is deoxythymidine (dT), for example, there is a short sequence of deoxythymidine nucleotides, e.g., two dT nucleotides at the 3'-end of the sense strand, the antisense strand, or both strands.

[0358] In certain embodiments, the sense strand sequence has formula (I): 5'n p -N a -(XXX) i -N b -YYY -N b -(ZZZ) j -N a -n q 3' (I) [In the formula, i and j each independently represent 0 or 1; p and q each independently represent 0 to 6; each N a independently represent oligonucleotide sequences comprising 0 to 25 modified nucleotides, each sequence comprising at least two differently modified nucleotides; each N b independently represent an oligonucleotide sequence comprising 0 to 10 modified nucleotides; each n p and n q independently represent an overhanging nucleotide; Nb and Y do not have the same modification, and XXX, YYY and ZZZ each independently represent one motif of three identical modifications on three consecutive nucleotides. In one embodiment, YYY are all 2'-F modified nucleotides.

[0359] In some embodiments, N a or N b includes alternating pattern modifications.

[0360] 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 to 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), and the number can start from the first nucleotide from the 5' end, or optionally, the number can start from the first paired nucleotide in the duplex region from the 5' end.

[0361] In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or i and j are both 1. Thus, the sense strand has the following formula: 5' n p -N a -YYY-N b -ZZZ-N a -n q 3' (Ib); 5' n p -N a -XXX-N b -YYY-N a -n q 3' (Ic); or 5' n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3' (Id) It can be expressed as:

[0362] When the sense strand is represented by formula (Ib), N b represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. a can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0363] When the sense strand is represented by formula (Ic), N b represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a may independently represent an oligonucleotide sequence that includes 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0364] When the sense strand is represented by formula (Id), each N b independently represent an oligonucleotide sequence comprising 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. b is 0, 1, 2, 3, 4, 5 or 6. Each N a may independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0365] Each of X, Y and Z may be the same as or different from one another.

[0366] In other embodiments, i is 0, j is 0, and the sense strand has the formula: 5' n p -N a -YYY-N a -n q 3' (Ia) It can be expressed as:

[0367] When the sense strand is represented by formula (Ia), each N a can independently comprise an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0368] In one embodiment, the antisense strand sequence of the RNAi has formula (II): 5' n q’ -N a '-(Z'Z'Z') k -N b '-Y'Y'Y'-Nb '-(X'X'X') l -N' a -n p '3' (II) [In the formula, k and l each independently represent 0 or 1; p' and q' each independently represent 0 to 6; each N a each independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides; each N b independently represents an oligonucleotide sequence comprising 0 to 10 modified nucleotides; each n p ' and n q ' independently represent an overhanging nucleotide; N b ' and Y' do not have the same modification, X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides. It can be expressed as:

[0369] In some embodiments, N a ' or N b ' includes alternating pattern modifications.

[0370] The Y'Y'Y' motif occurs at or near the cleavage site of the sense strand. For example, if the dsRNAi agent has a duplex region 17 to 23 nucleotides long, 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, the numbers starting from the first nucleotide from the 5' end, or, optionally, the numbers starting from the first paired nucleotide in the duplex region from the 5' end. In one embodiment, the Y'Y'Y' motif occurs at positions 11, 12, 13.

[0371] In certain embodiments, the Y'Y'Y' motif is all 2'-OMe modified nucleotides.

[0372] In certain embodiments, k is 1 and l is 0, or k is 0 and l is 1, or k and l are both 1.

[0373] Thus, the antisense strand has the formula: 5' n q’ -N a '-Z'Z'Z'-N b '-Y'Y'Y'-N a '-n p’ 3' (IIb), 5' n q’ -N a '-Y'Y'Y'-N b '-X'X'X'-n p’ 3' (IIc), or 5' n q’ -N a '-Z'Z'Z'-N b '-Y'Y'Y'-N b '-X'X'X'-N a '-n p’ 3' (IId) It can be expressed as:

[0374] When the antisense strand is represented by formula (IIb), N b ' represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a ' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0375] When the antisense strand is represented by formula (IIc), N b ’ represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0376] When the antisense strand is represented by formula (IId), each N b Each N' independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a In one embodiment, N' independently represents an oligonucleotide sequence comprising 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. b is 0, 1, 2, 3, 4, 5 or 6.

[0377] In other embodiments, k is 0, l is 0, and the antisense strand has the formula: 5' n p’ -N a’ -Y'Y'Y'- N a’ -n q’ 3' (Ia) It can be expressed as:

[0378] When the antisense strand is represented by formula (IIa), each N a ' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0379] Each of X', Y' and Z' may be the same as or different from one another.

[0380] Each nucleotide of sense strand and antisense strand can be independently modified with LNA, CRN, UNA, cEt, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-hydroxyl or 2'-fluoro.For example, each nucleotide of sense strand and antisense strand is independently modified with 2'-O-methyl or 2'-fluoro.Each X, Y, Z, X', Y' and Z' can specifically represent 2'-O-methyl modification or 2'-fluoro modification.

[0381] In some embodiments, the sense strand of a dsRNAi agent may contain a YYY motif occurring at positions 9, 10, and 11 of the strand when the duplex region is 21 nt, the numbers starting from the first nucleotide from the 5' end, or optionally the numbers starting from the first paired nucleotide in the duplex region from the 5' end, and Y represents a 2'-F modification. The sense strand may further contain a XXX motif or a ZZZ motif as a wing modification at the opposite end of the duplex region, where XXX and ZZZ each independently represent a 2'-OMe modification or a 2'-F modification.

[0382] In some embodiments, the antisense strand may contain a Y'Y'Y' motif occurring at positions 11, 12, 13 of the strand, where the number starts from the first nucleotide from the 5' end, or, where appropriate, the number may start from the 5' end with the first paired nucleotide in the duplex region, and Y' represents a 2'-O-methyl modification. The antisense strand may further contain an X'X'X' motif or a Z'Z'Z' motif as a wing modification at opposite ends of the duplex region, where X'X'X' and Z'Z'Z' each independently represent a 2'-OMe modification or a 2'-F modification.

[0383] The sense strand represented by any one of the above formulas (Ia), (Ib), (Ic) and (Id) forms a duplex with the antisense strand represented by any one of the formulas (IIa), (IIb), (IIc) and (IId), respectively.

[0384] Thus, a dsRNAi agent for use in the methods of the invention can include a sense strand and an antisense strand, each strand having 14-30 nucleotides, and the iRNA duplex can have the formula (III): Sense: 5' n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3' Antisense: 3' np ’ -N a ’ -(X'X'X') k -N b ’ -Y'Y'Y'-N b ’ -(Z'Z'Z') l -N a ’ -n q ’ 5' (III) [In the formula, i, j, k, and l each independently represent 0 or 1; p, p', q and q' each independently represent 0 to 6; each N a and N a ’ independently represent oligonucleotide sequences comprising 0 to 25 modified nucleotides, each sequence comprising at least two differently modified nucleotides; each N b and N b ’ independently represent an oligonucleotide sequence comprising 0 to 10 modified nucleotides; each n p ',n p , n q ' and n q independently represent overhanging nucleotides, each of which may or may not be present; XXX, YYY, ZZZ, X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides. is expressed by

[0385] In one embodiment, i is 0 and j is 0, or i is 1 and j is 0, or i is 0 and j is 1, or i and j are both 0, or i and j are both 1. In another embodiment, k is 0 and l is 0, or k is 1 and l is 0 and k is 0 and l is 1, or k and l are both 0, or k and l are both 1.

[0386] Exemplary combinations of sense and antisense strands that form iRNA duplexes have the following formula: 5' n p -N a -YYY -N a -n q 3' 3' n p ’ -N a ’ -Y'Y'Y'-N a ’ n q ’ 5' (IIIa) 5' n p -N a -YYY -N b -ZZZ -N a -n q 3' 3' n p ’ -N a ’ -Y'Y'Y'-N b ’ -Z'Z'Z'-N a ’ n q ’ 5' (IIIb) 5' n p -N a -XXX -N b -YYY -N a -n q 3' 3' n p ’ -N a ’ -X'X'X'-N b ’ -Y'Y'Y'-N a ’ -n q ’ 5' (IIIc) 5' n p -N a -XXX -N b -YYY-N b - ZZZ -N a -n q 3' 3' n p ’ -N a ’ -X'X'X'-N b ’ -Y'Y'Y'-N b ’ -Z'Z'Z'-N a -n q ’ 5' (IIId) Includes.

[0387] When the dsRNAi agent is represented by formula (IIIa), each N a independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0388] When the dsRNAi agent is represented by formula (IIIb), each N b independently represent an oligonucleotide sequence containing 1 to 10, 1 to 7, 1 to 5, or 1 to 4 modified nucleotides. a independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0389] When the dsRNAi agent is represented by formula (IIIc), each N b , N b Each N' independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0390] When the dsRNAi agent is represented by formula (IIId), each N b , N b Each N' independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a , N a ’independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. a , N a ', N b and N b ’ each independently comprises an alternating pattern of modifications.

[0391] Each of X, Y and Z in formulas (III), (IIIa), (IIIb), (IIIc) and (IIId) may be the same as or different from one another.

[0392] When the dsRNAi agent is represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), at least one of the Y nucleotides can be base-paired with one of the Y' nucleotides. Alternatively, at least two of the Y nucleotides are base-paired with the corresponding Y' nucleotide; or all three of the Y nucleotides are base-paired with the corresponding Y' nucleotide.

[0393] When the dsRNAi agent is represented by formula (IIIb) or (IIId), at least one of the Z nucleotides can be base-paired with one of the Z' nucleotides. Alternatively, at least two of the Z nucleotides are base-paired with the corresponding Z' nucleotide, or all three of the Z nucleotides are base-paired with the corresponding Z' nucleotide.

[0394] When the dsRNAi agent is represented by formula (IIIc) or (IIId), at least one of the X nucleotides can be base-paired with one of the X' nucleotides. Alternatively, at least two of the X nucleotides are base-paired with the corresponding X' nucleotide, or all three of the X nucleotides are base-paired with the corresponding X' nucleotide.

[0395] In certain embodiments, the modification on a Y nucleotide is different from the modification on a Y' nucleotide, the modification on a Z nucleotide is different from the modification on a Z' nucleotide, or the modification on an X nucleotide is different from the modification on an X' nucleotide.

[0396] In certain embodiments, when the dsRNAi agent is represented by formula (IIId), N a The modification is a 2'-O-methyl or a 2'-fluoro modification. In other embodiments, when the RNAi agent is represented by formula (IIId), N a The modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n p In yet another embodiment, when the RNAi agent is represented by formula (IIId), N' is linked to the adjacent nucleotide via a phosphorothioate linkage. a The modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n p In another embodiment, when the RNAi agent is represented by formula (IIId), N' is linked to adjacent nucleotides via phosphorothioate linkages, and the sense strand is conjugated to one or more GalNAc derivatives attached by a bivalent or trivalent branched linker (described below). a The modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n p ' is linked to adjacent nucleotides via phosphorothioate linkages, and the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives, which may be attached by a bivalent or trivalent branched linker.

[0397] In some embodiments, when the dsRNAi agent is represented by Formula (IIIa), N a The modifications are 2'-O-methyl or 2'-fluoro modifications, n p'>0 and at least one n p ' is linked to adjacent nucleotides via phosphorothioate linkages, and the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives attached by a bivalent or trivalent branched linker.

[0398] In certain embodiments, the RNAi agent of the present invention may contain a small number of nucleotides containing 2'-fluoro modifications, for example, the number of nucleotides containing 2'-fluoro modifications may be 10 or less. For example, the RNAi agent may contain 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 nucleotides containing 2'-fluoro modifications. In a specific embodiment, the RNAi agent of the present invention contains 10 nucleotides containing 2'-fluoro modifications, for example, 4 nucleotides containing 2'-fluoro modifications in the sense strand and 6 nucleotides containing 2'-fluoro modifications in the antisense strand. In another specific embodiment, the RNAi agent of the present invention contains 6 nucleotides containing 2'-fluoro modifications, for example, 4 nucleotides containing 2'-fluoro modifications in the sense strand and 2 nucleotides containing 2'-fluoro modifications in the antisense strand.

[0399] In other embodiments, the RNAi agent of the present invention has very few nucleotides containing 2'-fluoro modifications, for example, the number of nucleotides containing 2'-fluoro modifications is two or less. For example, the RNAi agent can contain two, one, or zero nucleotides with 2'-fluoro modifications. In a specific embodiment, the RNAi agent can contain two nucleotides with 2'-fluoro modifications, for example, zero nucleotides with 2'-fluoro modifications in the sense strand and two nucleotides with 2'-fluoro modifications in the antisense strand.

[0400] As described in more detail below, iRNAs containing one or more carbohydrate moieties conjugated to the iRNA can optimize one or more properties of the iRNA. Often, the carbohydrate moiety is attached to a modified subunit of the iRNA. For example, the ribose sugar of one or more ribonucleotide subunits of the iRNA can be replaced with another moiety, such as a non-carbohydrate (e.g., cyclic) carrier to which a carbohydrate ligand is attached. Ribonucleotide subunits in which the ribose sugar of the subunit has been so replaced are referred to herein as ribose-replacement modified subunits (RRMS). The cyclic carrier can be a carbocyclic ring system, i.e., all ring atoms are carbon atoms, or a heterocyclic ring structure, i.e., one or more ring atoms can be a heteroatom, such as nitrogen, oxygen, or sulfur. The cyclic carrier can be a monocyclic ring structure or can contain two or more rings, e.g., fused rings. The cyclic carrier can be a fully saturated ring structure or can contain one or more double bonds.

[0401] Ligands can be attached to polynucleotides via carriers. The carriers include (i) at least one "backbone attachment point," e.g., two "backbone attachment points," and (ii) at least one "tethering attachment point." As used herein, "backbone attachment point" refers to a functional group, e.g., a hydroxyl group, or generally a bond available and suitable for incorporation of the carrier into a backbone, e.g., a phosphate or modified phosphate, e.g., sulfur-containing backbone, of a ribonucleic acid. "Tethering attachment point" (TAP) refers, in some embodiments, to a constituent ring atom, e.g., a carbon atom or heteroatom (separate from the atom providing the backbone attachment point), of a cyclic carrier that connects the selected moiety. The moiety can be, for example, a carbohydrate, e.g., a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide. The selected moiety may be connected to the cyclic carrier by an intervening tether. Thus, cyclic carriers often contain a functional group, e.g., an amino group, or generally provide a bond suitable for incorporation or tethering another chemical entity, e.g., a ligand, to the constituent ring.

[0402] The iRNA may be conjugated to the ligand via a carrier, and the carrier may be a cyclic or 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, pyridazinonyl, tetrahydrofuryl, and decalin.In one embodiment, the acyclic group is a serinol skeleton or a diethanolamine skeleton.

[0403] B. Thermal destabilization modification In certain embodiments, dsRNA molecules can be optimized for RNA interference by incorporating thermally destabilizing modifications into the seed region of antisense strand.As used herein, " seed region " refers to the 2-9 position of the 5' end of the strand that is referred to or the 2-8 position of the 5' end of the strand that is referred to.For example, thermally destabilizing modifications can be incorporated into the seed region of antisense strand to reduce or inhibit off-target gene silencing.

[0404] The term "thermally destabilizing modification(s)" refers to a modification that is lower than the melting temperature (T m ) than the overall melting temperature (T m For example, thermally destabilizing modification(s) may result in a dsRNA having a T m can be decreased by 1-4° C., for example, 1, 2, 3, or 4 degrees Celsius. Also, the term "thermally destabilized nucleotide" refers to a nucleotide that contains one or more thermally destabilizing modifications.

[0405] It has been discovered that dsRNAs having an antisense strand containing at least one duplex thermally destabilizing modification within the first 9 nucleotide positions counting from the 5' end of the antisense strand have reduced off-target gene silencing activity. Thus, in some embodiments, the antisense strand contains at least one (e.g., 1, 2, 3, 4, 5, or more) duplex thermally destabilizing modification within the first 9 nucleotide positions of the 5' region of the antisense strand. In some embodiments, one or more duplex thermally destabilizing modifications are located in positions 2-9, e.g., positions 4-8, from the 5' end of the antisense strand. In some further embodiments, the duplex thermally destabilizing modification(s) are located in positions 6, 7, or 8 from the 5' end of the antisense strand. In yet some further embodiments, the duplex thermally destabilizing modification is located in position 7 from the 5' end of the antisense strand. In some embodiments, the duplex thermally destabilizing modification is located in positions 2, 3, 4, 5, or 9 from the 5' end of the antisense strand.

[0406] An iRNA agent includes a sense strand and an antisense strand, each strand having 14 to 40 nucleotides. The RNAi agent can be represented by the formula (L): [ka]

[0407] In Formula (L), B1, B2, B3, B1', B2', B3', and B4' are each independently a nucleotide containing a modification selected from the group consisting of 2'-O-alkyl, 2'-substituted alkoxy, 2'-substituted alkyl, 2'-halo, ENA, and BNA / LNA. In one embodiment, B1, B2, B3, B1', B2', B3', and B4' each contain a 2'-OMe modification. In one embodiment, B1, B2, B3, B1', B2', B3', and B4' each contain a 2'-OMe or 2'-F modification. In one embodiment, at least one of B1, B2, B3, B1', B2', B3', and B4' contains a 2'-ON-methylacetamide (2'-O-NMA, 2'O-CHC(O)N(Me)H) modification.

[0408] C1 is a thermally destabilizing nucleotide located at the site opposite the seed region of the antisense strand (i.e., positions 2-8 of the 5' end of the antisense strand or positions 2-9 of the 5' end of the antisense strand). For example, C1 is located in the sense strand at a position that pairs with the nucleotide at positions 2-8 of the 5' end of the antisense strand. In one example, C1 is located at position 15 from the 5' end of the sense strand. The C1 nucleotide has a thermally destabilizing modification, which can include an abasic modification, a mismatch with the opposing nucleotide in the opposing duplex, and a sugar modification, such as a 2'-deoxy modification or an acyclic nucleotide, e.g., a non-locked nucleic acid (UNA), or a 2'-5'-linked ribonucleotide ("3'-RNA"). In one embodiment, C1 has a thermally destabilizing modification selected from the group consisting of: i) a mismatch with the opposing nucleotide in the antisense strand; ii) an abasic modification selected from the group consisting of: [ka] and iii) a sugar modification selected from the group consisting of: [ka] wherein B is a modified or unmodified nucleobase, R and R are independently H, halogen, OR, or alkyl; and R is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar. In one embodiment, the thermally destabilizing modification of C is a mismatch selected from the group consisting of G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T, and U:T, and optionally at least one nucleobase of the mismatch pair is a 2'-deoxynucleobase. In one example, the thermally destabilizing modification of C is GNA or [ka]

[0409] T1, T1', T2', and T3' each independently represent a nucleotide containing a modification that provides the nucleotide with steric bulk that is equal to or less than that of a 2'-OMe modification. Steric bulk refers to the sum of the steric effects of the modifications. Methods for determining the steric effect of a nucleotide modification are known to those skilled in the art. The modification may be at the 2' position of the ribose sugar of the nucleotide, or may be a non-ribose nucleotide, an acyclic nucleotide, or a modification to the backbone of the nucleotide that is similar or equivalent to the 2' position of the ribose sugar and provides the nucleotide with steric bulk that is equal to or less than that of the 2'-OMe modification. For example, T1, T1', T2', and T3' are each independently selected from DNA, RNA, LNA, 2'-F, and 2'-F-5'-methyl. In one embodiment, T1 is DNA. In one embodiment, T1' is DNA, RNA, or LNA. In one embodiment, T2' is DNA or RNA. In one embodiment, T3' is DNA or RNA.

[0410] n 1 , n 3 , and q 1 are independently 4 to 15 nucleotides in length.

[0411] n 5 , q 3 , and q 7 are independently 1 to 6 nucleotides in length.

[0412] n 4 , q 2 , and q 6 are independently 1 to 3 nucleotides in length, or n 4 is 0.

[0413] q 5 are independently 0 to 10 nucleotides in length.

[0414] n 2 and q 4 are independently 0 to 3 nucleotides in length.

[0415] Or, n4 is 0 to 3 nucleotides in length.

[0416] In one embodiment, n 4 may be 0. In one embodiment, n 4 is 0, and q 2 and q 6 is 1. In another embodiment, n 4 is 0, and q 2 and q 6 is 1 and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 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).

[0417] In one embodiment, n 4 , q 2 , and q 6 are each 1.

[0418] In one embodiment, n 2 , n 4 , q 2 , q 4 , and q 6 are each 1.

[0419] In one embodiment, the sense strand is 19 to 22 nucleotides in length, 4 When C1 is 1, C1 is at positions 14 to 17 of the 5'-end of the sense strand. In one embodiment, C1 is at position 15 of the 5'-end of the sense strand.

[0420] In one embodiment, T3' begins at the second position from the 5' end of the antisense strand. In one example, T3' is at the second position from the 5' end of the antisense strand, and q 6 is equal to 1.

[0421] In one embodiment, T1' begins at position 14 from the 5' end of the antisense strand. 2 is equal to 1.

[0422] In an exemplary embodiment, T3' starts at the second position from the 5' end of the antisense strand, and T1' starts at the 14th position from the 5' end of the antisense strand. In one example, T3' starts at the second position from the 5' end of the antisense strand, and q 6 is equal to 1, T1' starts at the 14th position from the 5' end of the antisense strand, and q 2 is equal to 1.

[0423] In one embodiment, T1' and T3' are separated by a length of 11 nucleotides (ie, not counting the nucleotides of T1' and T3').

[0424] In one embodiment, T1' is the 14th position from the 5' end of the antisense strand. 2 is equal to 1, and the modification is at the 2' position or at a non-ribose, acyclic, or backbone position that provides less steric bulk than 2'-OMe ribose.

[0425] In one embodiment, T3' is the second position from the 5' end of the antisense strand. 6 is equal to 1 and is a modification at the 2' position or at a non-ribose, acyclic, or backbone position that provides less steric bulk than 2'-OMe ribose.

[0426] In one embodiment, T1 is at the cleavage site of the sense strand. In one example, the sense strand is 19-22 nucleotides in length, 2 When n is 1, T1 is at the 11th position from the 5' end of the sense strand. In an exemplary embodiment, the sense strand is 19-22 nucleotides in length, and n 2When T1 is 1, T1 is at the cleavage site of the sense strand at position 11 from the 5' end of the sense strand.

[0427] In one embodiment, T2' begins at position 6 from the 5' end of the antisense strand. In one example, T2' is located at positions 6-10 from the 5' end of the antisense strand, and q 4 is 1.

[0428] In an exemplary embodiment, T1 is at the cleavage site of the sense strand, e.g., at position 11 from the 5' end of the sense strand, and when the sense strand is 19-22 nucleotides long, n 2 is 1; T1' is at the 14th position from the 5' end of the antisense strand, and q 2 is equal to 1, and the modification to T1' is at the 2' position of the ribose sugar or at a non-ribose, acyclic, or backbone position that has less steric bulk than 2'-OMe ribose; T2' is at positions 6–10 from the 5' end of the antisense strand, and q 4 is 1; and T3' is at the second position from the 5' end of the antisense strand, and q 6 is equal to 1, and the modification to T3' is at the 2' position of the 2'-OMe ribose or at a non-ribose, acyclic, or backbone position that provides less steric bulk.

[0429] In one embodiment, T2' starts at the 8th position from the 5' end of the antisense strand. 4 is 2.

[0430] In one embodiment, T2' begins at the 9th position from the 5' end of the antisense strand. 4 is 1.

[0431] In one embodiment, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 1, B3' is 2'-OMe or 2'-F, and q 5 is 6, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1 and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand).

[0432] In one embodiment, n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 1, B3' is 2'-OMe or 2'-F, and q 5 is 6, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1 and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 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).

[0433] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1.

[0434] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1 and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 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).

[0435] In one embodiment, B1 is 2'-OMe or 2'-F, and n1 is 6, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 7, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1.

[0436] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 6, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 7, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7is 1 and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 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).

[0437] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 1, B3' is 2'-OMe or 2'-F, and q 5 is 6, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1.

[0438] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 1, B3' is 2'-OMe or 2'-F, and q5 is 6, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1 and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand).

[0439] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 5, T2' is 2'-F, and q 4 is 1, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1.

[0440] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q3 is 5, T2' is 2'-F, and q 4 is 1, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1, optionally with at least two additional TTs at the 3'-end of the antisense strand and with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5'-end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5'-end of the antisense strand).

[0441] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'OMe, and q 7 is 1.

[0442] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'OMe, and q 7 is 1, which has two phosphorothioate internucleotide linkage modifications in positions 1-5 of the sense strand (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications in positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (counting from the 5' end).

[0443] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1.

[0444] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1 and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 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).

[0445] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1.

[0446] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'F, and q 7 is 1 and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 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).

[0447] The RNAi agent can include a phosphorus-containing group at the 5'-end of the sense or antisense strand. The 5'-terminal phosphorus-containing group can be 5'-terminal phosphate (5'-P), 5'-terminal phosphorothioate (5'-PS), 5'-terminal phosphorodithioate (5'-PS2), 5'-terminal vinylphosphonate (5'-VP), 5'-terminal methylphosphonate (MePhos), or 5'-deoxy-5'-C-malonyl ( [ka] When the 5'-terminal phosphorus-containing group is a 5'-terminal vinyl phosphonate (5'-VP), the 5'-VP may be a 5'-E-VP isomer (i.e., trans-vinyl phosphonate, [ka] ), 5'-Z-VP isomer (i.e., cis-vinyl phosphonate, [ka] ), or a mixture thereof.

[0448] 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.

[0449] In one embodiment, the RNAi agent comprises a 5'-P. In one embodiment, the RNAi agent comprises a 5'-P in the antisense strand.

[0450] In one embodiment, the RNAi agent comprises a 5'-PS. In one embodiment, the RNAi agent comprises a 5'-P. In one embodiment, the RNAi agent comprises a 5'-PS in the antisense strand.

[0451] 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.

[0452] In one embodiment, the RNAi agent comprises a 5'-PS2. In one embodiment, the RNAi agent comprises a 5'-PS2 on the antisense strand.

[0453] In one embodiment, the RNAi agent comprises a 5'-PS2. In one embodiment, the RNAi agent comprises a 5'-deoxy-5'-C-malonyl.

[0454] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The RNAi agent also contains a 5'-PS.

[0455] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The RNAi agent also contains a 5'-P.

[0456] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The RNAi agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0457] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The RNAi agent also contains a 5'-PS2.

[0458] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. RNAi agents also include 5'-deoxy-5'-C-malonyl.

[0459] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1 and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 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). The RNAi agent also includes a 5'-P.

[0460] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1 and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 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). The RNAi agent also includes a 5'-PS.

[0461] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7is 1 and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 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). The RNAi agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0462] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1 and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 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). The RNAi agent also includes a 5'-PS2.

[0463] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1 and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 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). The RNAi agent also includes 5'-deoxy-5'-C-malonyl.

[0464] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'OMe, and q 7 is 1. The RNAi agent also contains a 5'-P.

[0465] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The RNAi agent also contains a 5'-PS.

[0466] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The RNAi agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0467] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The RNAi agent also contains a 5'-PS2.

[0468] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The RNAi agent also includes 5'-VP. The RNAi agent also includes 5'-deoxy-5'-C-malonyl.

[0469] In one embodiment, B1 is 2'-OMe or 2'-F, and n1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1 and has two phosphorothioate internucleotide linkage modifications in positions 1-5 of the sense strand (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications in positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (counting from the 5' end). The RNAi agent also contains a 5'-P.

[0470] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7is 1 and has two phosphorothioate internucleotide linkage modifications in positions 1-5 of the sense strand (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications in positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications in positions 18-23 of the antisense strand (counting from the 5' end). The RNAi agent also contains a 5'-PS.

[0471] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1 and has two phosphorothioate internucleotide linkage modifications (counting from the 5' end) in positions 1-5 of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications (counting from the 5' end) in positions 18-23 of the antisense strand. The RNAi agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0472] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1 and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end). The RNAi agent also includes a 5'-PS2.

[0473] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7is 1 and has two phosphorothioate internucleotide linkage modifications (counting from the 5' end) within positions 1-5 of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications (counting from the 5' end) within positions 18-23 of the antisense strand. The RNAi agent also includes 5'-deoxy-5'-C-malonyl.

[0474] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The RNAi agent also contains a 5'-P.

[0475] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The RNAi agent also contains a 5'-PS.

[0476] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The RNAi agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0477] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The dsRNAiRNA agent also includes a 5'-PS2.

[0478] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. RNAi agents also include 5'-deoxy-5'-C-malonyl.

[0479] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The RNAi agent has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-P.

[0480] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1 and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 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). The RNAi agent also includes a 5'-PS.

[0481] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1 and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 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). The RNAi agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0482] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q7 is 1 and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 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). The RNAi agent also includes a 5'-PS2.

[0483] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1 and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 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). The RNAi agent also includes 5'-deoxy-5'-C-malonyl.

[0484] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The RNAi agent also contains a 5'-P.

[0485] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The RNAi agent also contains a 5'-PS.

[0486] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The RNAi agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0487] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The RNAi agent also contains a 5'-PS2.

[0488] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. RNAi agents also include 5'-deoxy-5'-C-malonyl.

[0489] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The RNAi agent has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-P.

[0490] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1 and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 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). The RNAi agent also includes a 5'-PS.

[0491] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7is 1 and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 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). The RNAi agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0492] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'F, and q 7 is 1 and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 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). The RNAi agent also includes a 5'-PS2.

[0493] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1 and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 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). The RNAi agent also includes 5'-deoxy-5'-C-malonyl.

[0494] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7is 1 and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 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). The RNAi agent also includes a 5'-P and a targeting ligand. In one embodiment, the 5'-P is at the 5'-end of the antisense strand and the targeting ligand is at the 3'-end of the sense strand.

[0495] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1 and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 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). The RNAi agent also includes a 5'-PS and a targeting ligand. In one embodiment, the 5'-PS is at the 5'-end of the antisense strand and the targeting ligand is at the 3'-end of the sense strand.

[0496] In one embodiment, B1 is 2'-OMe or 2'-F, and n1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1 and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 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). The RNAi agent also includes a 5'-VP (e.g., 5'-E-VP, 5'-Z-VP, or a combination thereof) and a targeting ligand.

[0497] In one embodiment, the 5'-VP is at the 5'-end of the antisense strand and the targeting ligand is at the 3'-end of the sense strand.

[0498] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1 and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 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). The RNAi agent also includes a 5'-PS2 and a targeting ligand. In one embodiment, the 5'-PS2 is at the 5'-end of the antisense strand and the targeting ligand is at the 3'-end of the sense strand.

[0499] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7is 1 and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 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). The RNAi agent also includes a 5'-deoxy-5'-C-malonyl and a targeting ligand. In one embodiment, the 5'-deoxy-5'-C-malonyl is at the 5'-end of the antisense strand and the targeting ligand is at the 3'-end of the sense strand.

[0500] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1 and has two phosphorothioate internucleotide linkage modifications (counting from the 5' end) in positions 1-5 of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications (counting from the 5' end) in positions 18-23 of the antisense strand. In one embodiment, the 5'-P is at the 5'-end of the antisense strand and the targeting ligand is at the 3'-end of the sense strand.

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

[0502] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6is 1, B4' is 2'-OMe, and q 7 is 1 and has two phosphorothioate internucleotide linkage modifications (counting from the 5' end) within positions 1-5 of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications (counting from the 5' end) within positions 18-23 of the antisense strand. The RNAi agent also includes a 5'-VP (e.g., 5'-E-VP, 5'-Z-VP, or a combination thereof) and a targeting ligand. In one embodiment, the 5'-VP is at the 5'-end of the antisense strand and the targeting ligand is at the 3'-end of the sense strand.

[0503] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1 and has two phosphorothioate internucleotide linkage modifications (counting from the 5' end) in positions 1-5 of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications (counting from the 5' end) in positions 18-23 of the antisense strand. The RNAi agent also includes a 5'-PS2 and a targeting ligand. In one embodiment, the 5'-PS2 is at the 5'-end of the antisense strand and the targeting ligand is at the 3'-end of the sense strand.

[0504] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1 and has two phosphorothioate internucleotide linkage modifications (counting from the 5' end) within positions 1-5 of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications (counting from the 5' end) within positions 18-23 of the antisense strand. The RNAi agent also includes a 5'-deoxy-5'-C-malonyl and a targeting ligand. In one embodiment, the 5'-deoxy-5'-C-malonyl is at the 5'-end of the antisense strand and the targeting ligand is at the 3'-end of the sense strand.

[0505] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1 and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 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). The RNAi agent also includes a 5'-P and a targeting ligand. In one embodiment, the 5'-P is at the 5'-end of the antisense strand and the targeting ligand is at the 3'-end of the sense strand.

[0506] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7is 1 and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 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). The RNAi agent also includes a 5'-PS and a targeting ligand. In one embodiment, the 5'-PS is at the 5'-end of the antisense strand and the targeting ligand is at the 3'-end of the sense strand.

[0507] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1 and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 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). The RNAi agent also includes a 5'-VP (e.g., 5'-E-VP, 5'-Z-VP, or a combination thereof) and a targeting ligand. In one embodiment, the 5'-VP is at the 5'-end of the antisense strand and the targeting ligand is at the 3'-end of the sense strand.

[0508] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1 and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 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). The RNAi agent also includes a 5'-PS2 and a targeting ligand. In one embodiment, the 5'-VP2 is at the 5'-end of the antisense strand and the targeting ligand is at the 3'-end of the sense strand.

[0509] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1 and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 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). The RNAi agent also comprises a 5'-deoxy-5'-C-malonyl and a targeting ligand. The RNAi agent also comprises a 5'-deoxy-5'-C-malonyl and a targeting ligand. In one embodiment, the 5'-deoxy-5'-C-malonyl is at the 5'-end of the antisense strand and the targeting ligand is at the 3'-end of the sense strand.

[0510] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'F, and q 7is 1 and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 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). The RNAi agent also includes a 5'-P and a targeting ligand. In one embodiment, the 5'-P is at the 5'-end of the antisense strand and the targeting ligand is at the 3'-end of the sense strand.

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

[0512] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'F, and q 7 is 1 and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 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). The RNAi agent also includes a 5'-VP (e.g., 5'-E-VP, 5'-Z-VP, or a combination thereof) and a targeting ligand. In one embodiment, the 5'-VP is at the 5'-end of the antisense strand and the targeting ligand is at the 3'-end of the sense strand.

[0513] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'F, and q 7 is 1 and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 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). The RNAi agent also includes a 5'-PS2 and a targeting ligand. In one embodiment, the 5'-PS2 is at the 5'-end of the antisense strand and the targeting ligand is at the 3'-end of the sense strand.

[0514] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'F, and q 7is 1 and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 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). The RNAi agent also includes a 5'-deoxy-5'-C-malonyl and a targeting ligand. In one embodiment, the 5'-deoxy-5'-C-malonyl is at the 5'-end of the antisense strand and the targeting ligand is at the 3'-end of the sense strand.

[0515] In certain embodiments, the RNAi agent of the invention comprises: (a) a sense strand having: (i) 21 nucleotides long; (ii) an ASGPR ligand attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; and (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); (b) an antisense strand having: (i) 23 nucleotides long; (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); (iii) phosphorothioate internucleotide linkages between nucleotide positions 21 and 22 and between nucleotide positions 22 and 23 (counting from the 5′ end); Here, the dsRNA agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0516] In another specific embodiment, the RNAi agent of the invention comprises: (a) a sense strand having: (i) 21 nucleotides long; (ii) an ASGPR ligand attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; and (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); (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5' end); and (b) an antisense strand having: (i) 23 nucleotides long; (ii) 2′-OMe modifications at positions 1, 3, 5, 7, 9, 11 to 13, 15, 17, 19, 21 to 23, and 2′F modifications at positions 2, 4, 6 to 8, 10, 14, 16, 18, and 20 (counting from the 5′ end); (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5′ end); Here, the iRNA agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0517] In another specific embodiment, the RNAi agent of the invention comprises: (a) a sense strand having: (i) 21 nucleotides long; (ii) an ASGPR ligand attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; and (iii) 2′-OMe modifications at positions 1 to 6, 8, 10, and 12 to 21, and 2′-F modifications at positions 7 and 9, and a deoxynucleotide (e.g., dT) at position 11 (counting from the 5′ end); (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5' end); and (b) an antisense strand having: (i) 23 nucleotides long; (ii) 2'-OMe modifications at positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 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 (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5′ end); Here, the iRNA agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0518] In another specific embodiment, the RNAi agent of the invention comprises: (a) a sense strand having: (i) 21 nucleotides long; (ii) an ASGPR ligand attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; and (iii) 2′-OMe modifications at positions 1 to 6, 8, 10, 12, 14, and 16 to 21, and 2′-F modifications at positions 7, 9, 11, 13, and 15; (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5' end); and (b) an antisense strand having: (i) 23 nucleotides long; (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); (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5′ end); Here, the iRNA agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0519] In another specific embodiment, the RNAi agent of the invention comprises: (a) a sense strand having: (i) 21 nucleotides long; (ii) an ASGPR ligand attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; and (iii) 2′-OMe modifications at positions 1 to 9, 12 to 21, and 2′-F modifications at positions 10 and 11; (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5' end); and (b) an antisense strand having: (i) 23 nucleotides long; (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, 12, 14, 16, 18, and 20 (counting from the 5′ end); (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5′ end); Here, the iRNA agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0520] In another specific embodiment, the RNAi agent of the invention comprises: (a) a sense strand having: (i) 21 nucleotides long; (ii) an ASGPR ligand attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; and (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; (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5' end); and (b) an antisense strand having: (i) 23 nucleotides long; (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); (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5′ end); Here, the iRNA agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0521] In another specific embodiment, the RNAi agent of the invention comprises: (a) a sense strand having: (i) 21 nucleotides long; (ii) an ASGPR ligand attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; and (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; (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5' end); and (b) an antisense strand having: (i) 23 nucleotides long; (ii) 2′-OMe modifications at positions 1, 4, 6, 7, 9, 11 to 13, 15, 17, and 19 to 23, and 2′F modifications at positions 2, 3, 5, 8, 10, 14, 16, to 18, and a deoxynucleotide (e.g., dT) at position 11 (counting from the 5′ end); (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5′ end); Here, the iRNA agent has a four nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0522] In another specific embodiment, the RNAi agent of the invention comprises: (a) a sense strand having: (i) 21 nucleotides long; (ii) an ASGPR ligand attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; and (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; (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5' end); and (b) an antisense strand having: (i) 23 nucleotides long; (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); (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5′ end); Here, the iRNA agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0523] In another specific embodiment, the RNAi agent of the invention comprises: (a) a sense strand having: (i) 21 nucleotides long; (ii) an ASGPR ligand attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; and (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; (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5' end); and (b) an antisense strand having: (i) 23 nucleotides long; (ii) 2′-OMe modifications at positions 1, 3 to 5, 7, 10 to 13, 15, and 17 to 23, and 2′F modifications at positions 2, 6, 8, 9, 14, and 16 (counting from the 5′ end); (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5′ end); Here, the iRNA agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0524] In another specific embodiment, the RNAi agent of the invention comprises: (a) a sense strand having: (i) 19 nucleotides long; (ii) an ASGPR ligand attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; and (iii) 2′-OMe modifications at positions 1 to 4, 6, and 10 to 19, and 2′-F modifications at positions 5, and 7 to 9; (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5' end); and (b) an antisense strand having: (i) 21 nucleotides long; (ii) 2′-OMe modifications at positions 1, 3 to 5, 7, 10 to 13, 15, and 17 to 21, and 2′F modifications at positions 2, 6, 8, 9, 14, and 16 (counting from the 5′ end); (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21 (counting from the 5′ end); Here, the iRNA agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0525] In certain embodiments, the iRNA for use in the methods of the invention is an agent selected from the agents listed in any one of Tables 2-3. These agents may further comprise a ligand.

[0526] IV. REVERSIR COMPOUNDS OF THE INVENTION The present invention also provides REVERSIR compounds that suppress the activity of the universal dsRNA agents of the present invention. The design, synthesis, and suitable modifications of REVERSIR compounds are disclosed in WO2016 / 100716, WO2019 / 036612, and US2017 / 369872, the entire contents of each of which are incorporated herein by reference.

[0527] Generally, the REVERSIR compounds of the invention are single-stranded oligonucleotides (oligomers) between 6 and 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 to any one of the antisense strand nucleotide sequences in Tables 2 or 3.

[0528] Exemplary REVERSIR compounds of the present invention are presented in Table 4 herein.

[0529] In certain embodiments, REVERSIR compounds are oligomeric compounds that are chemically modified compared to naturally occurring oligomers such as DNA or RNA.

[0530] Thus, in certain embodiments, the REVERSIR compounds of the present 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 yet other embodiments, all of the nucleotides of the oligonucleotide are modified nucleotides.

[0531] In certain embodiments, oligomeric compounds comprise one or more modified monomers. In certain embodiments, REVERSIR compounds of the present invention comprise one or more high-affinity monomers. In certain embodiments, such high-affinity monomers are selected from monomers (e.g., nucleosides and nucleotides) containing 2'-modified sugars, including, but not limited to, BNAs and monomers (e.g., nucleosides and nucleotides) having 2'-substituents, such as allyl, amino, azido, thio, O-allyl, O-C1-C2 10 alkyl, -OCF3-, O-(CH2)2-O-CH3, 2'-O(CH2)2SCH3, O-(CH2)2-ON(Rm)(Rn), or O-CH2-C(=O)-N(Rm)(Rn), where each Rm and Rn is independently H or a substituted or unsubstituted C1-C 10 It is alkyl.

[0532] In certain embodiments, REVERSIR compounds of the invention comprise one or more β-D-methyleneoxy(4′-CH 2 —O-2′) LNA monomers.

[0533] In certain embodiments, REVERSIR compounds of the invention comprise one or more α-D-methyleneoxy (4′-CH 2 —O-2′) LNA monomers.

[0534] In certain embodiments, the REVERSIR compounds of the invention comprise one or more (S)-cEt monomers.

[0535] In certain embodiments, the REVERSIR compounds of the invention comprise one or more high affinity monomers, provided that the oligomeric compounds are 2'-O(CH2) n It does not contain nucleotides containing H (n is 1 to 6).

[0536] In certain embodiments, REVERSIR compounds of the invention comprise one or more high affinity monomers, with the proviso that the oligomeric compound does not comprise a nucleotide containing 2'-OCH3 or 2'-O(CH2)2OCH3.

[0537] In certain embodiments, REVERSIR compounds of the 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, with the proviso that the oligomeric compound does not comprise α-L-methyleneoxy(4′-CH2-O-2′) LNA.

[0538] In certain embodiments, the REVERSIR compounds of the invention comprise one or more high affinity monomers, with the proviso that the oligomeric compound does not comprise β-D-methyleneoxy(4′-CH 2 —O-2′) LNA.

[0539] In certain embodiments, REVERSIR compounds of the invention comprise one or more high affinity monomers, with the proviso that the oligomeric compound does not comprise α-L-methyleneoxy(4'-CH2-O-2')LNA or β-D-methyleneoxy(4'-CH2-O-2')LNA.

[0540] The naturally occurring base moiety of a nucleoside is typically a heterocyclic base. The two most common classes of such heterocyclic bases are purines and pyrimidines. In nucleosides containing a pentofuranosyl sugar, the phosphate group can be linked to the 2', 3', or 5' hydroxyl moiety of the sugar. When forming oligonucleotides, these phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. Within oligonucleotides, the phosphate groups are generally referred to as forming the internucleoside or internucleotide backbone of the oligonucleotide. The naturally occurring linkage or backbone of RNA and DNA is the 3'-5' phosphodiester bond.

[0541] 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 mimics known to those skilled in the art can be used in the compounds described herein. Unmodified or natural nucleobases can be modified or substituted to provide oligonucleotides with improved properties. For example, nuclease-resistant oligonucleotides can be prepared using these bases or synthetic and natural nucleobases (e.g., inosine, xanthine, hypoxanthine, nubularine, isoguanisine, or tubercidin) and any one of the oligomeric modifications described herein. Alternatively, substituted or modified analogs of any of the above bases and "universal bases" can be used. When a natural base is replaced with a non-natural and / or universal base, the nucleotide is said herein to comprise a modified nucleobase and / or nucleobase modification. Modified nucleobases and / or nucleobase modifications also include natural, non-natural, and universal bases containing conjugate moieties, such as the ligands described herein. Preferred conjugate moieties for conjugation to nucleobases include cationic amino groups, which can be conjugated to the nucleobases via linkers having suitable alkyl, alkenyl or amide bonds.

[0542] The REVERSIR compounds described herein can also include nucleobase (often simply referred to in the art as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G) and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Exemplary modified nucleobases include, but are not limited to, inosine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidin, 2-(halo)adenine, 2-(alkyl)adenine, 2-(propyl)adenine, 2-(amino)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-(Hydroxyl)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-(deaza)guanine, 8-(alkyl)guanine, 8-(alkenyl)guanine, 8-(alkynyl)guanine, 8-(amino)guanine, 8-(halo)guanine, 8-(hydroxyl)guanine, 8-(thioalkyl)guanine cytosine, 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-(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-(methylaminomethylethyl)-2,4-(dithio)uracil, 5-(2-aminopropyl)uracil, 5-(alkyl)uracil, 5-(alkynyl)uracil, 5-(allylamino)uracil, 5-(aminoallyl)uracil uracil, 5-(aminoalkyl)uracil, 5-(guanidiniumalkyl)uracil, 5-(1,3-diazole-1-alkyl)uracil, 5-(cyanoalkyl)uracil, 5-(dialkylaminoalkyl)uracil, 5-(dimethylaminoalkyl)uracil, 5-(halo)uracil, 5-(methoxy)uracil, uracil-5-oxyacetic acid, 5-(methoxycarbonylmethyl)-2-(thio)uracil, 5-(methoxycarbonylmethyl)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 Douracil, 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-(aminocarbonylethylenyl)-pseudouracil, 1-(aminocarbonylethylenyl)-2(thio)-pseudouracil, 1-(aminocarbonylethylenyl)-4-(thio)-pseudouracil, 1-(aminocarbonylethylenyl)-2,4-(dithio)-pseudouracil pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-2(thio)-pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-4-(thio)-pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)-pseudouracil, 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1, ,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl (hydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(guanidinium alkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-(guanidinium alkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 1,3,5-(triaza)-2,6-(dioxa-naphthalene, inosine, xanthine, hypoxanthine, nubularine, tubercidin, isoguanisine, inosinyl, 2-aza-inosinyl, 7-deazaino Indolyl, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl, nitroindazolyl, aminoindolyl, pyrrolopyrimidinyl, 3-(methyl)isocarbostyrilyl, 5-(methyl)isocarbostyrilyl, 3-(methyl)-7-(propynyl)isocarbostyrilyl, 7-(aza)indolyl, 6-(methyl)-7-(aza)indolyl, imidizopyridinyl, 9-(methyl)-imidizopyridinyl, pyrrolopyridinyl, isocarbostyrilyl, 7-(propynyl)isocarbostyrilyl, propynyl-7-(aza)indolyl allyl, 2,4,5-(trimethyl)phenyl, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl, phenyl, naphthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stivenyl, tetracenyl, pentacenyl, difluorotolyl, 4-(fluoro)-6-(methyl)benzimidazole, 4-(methyl)benzimidazole, 6-(azo)thymine, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 6-(aza)pyrimidine, 2-(amino)purine, 2,6-(diamino)purine, 5-substituted pyrimidines, N, 2 -substituted purines, N6 -substituted purines, O 6 -substituted purines, substituted 1,2,4-triazoles, 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- Other synthetic or natural nucleic acid bases include 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- or N-alkylated derivative thereof. Alternatively, substituted or modified analogs of any of the above-listed bases and "universal bases" can also be used.

[0543] As used herein, a universal nucleobase is capable of base pairing with all four naturally occurring nucleobases without substantially affecting the melting behavior, recognition by intracellular enzymes, or activity of the oligonucleoside duplex. Some exemplary universal nucleobases include, but are not limited to, 2,4-difluorotoluene, nitropyrrolyl, nitroindolyl, 8-aza-7-deazaadenine, 4-fluoro-6-methylbenzimidazur, 4-methylbenzimidazur, 3-methylisocarbostyrilyl, 5-methylisocarbostyrilyl, 3-methyl-7-propynylisocarbostyrilyl, 7-azaindolyl, 6-methyl-7-azaindolyl, imidizopyridinyl, Examples include 9-methyl-imidizopyridinyl, pyrrolopyridinyl, isocarbostyrilyl, 7-propynylisocarbostyrilyl, propynyl-7-azaindolyl, 2,4,5-trimethylphenyl, 4-methylindolyl, 4,6-dimethylindolyl, phenyl, naphthalenyl, anthracenyl, phenathracenyl, pyrenyl, stilbenyl, tetracenyl, pentacenyl, and structural derivatives thereof (see, for example, Loakes, 2001, Nucleic Acids Research, 29, 2437-2447).

[0544] Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808; those disclosed in International Application PCT / US Patent Application No. 09 / 038425, filed March 26, 2009; those disclosed in Concise Encyclopedia of Polymer Science and Engineering, pages 858-859, Kroschwitz, JI, 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, Herdewijn, p. Ed. Wiley-VCH, 2008; and those disclosed in Sanghvi, YS, Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., Eds., CRC Press, 1993, the contents of all of which are incorporated herein by reference.

[0545] In some embodiments, the modified nucleobase is a nucleobase that is substantially similar in structure to the parent nucleobase, such as, for example, a 7-deazapurine, 5-methylcytosine, or G-clamp. In some embodiments, the nucleobase mimic comprises a more complex structure, such as, for example, a tricyclic phenoxazine nucleobase mimic. Methods for preparing the aforementioned modified nucleobases are well known to those skilled in the art.

[0546] In some embodiments, the REVERSIR compounds of the present invention are [ka] (n is 0, 1, 2, 3, 4, 5 or 6) and at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) G-clamp nucleobase selected from:

[0547] The REVERSIR compounds provided herein may contain one or more monomers, such as nucleosides or nucleotides, having modified sugar moieties. For example, the furanosyl sugar ring of a nucleoside can be modified in several ways, including, but not limited to, the addition of a substituent or bridging of two non-geminal ring atoms to form a locked nucleic acid or bicyclic nucleic acid. In some embodiments, the compound contains one or more monomers that are LNAs.

[0548] In some embodiments of the locked nucleic acid, the 2' position of the furnaosyl is -[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 -ON(R1)-, -C(R1)=C(R2)-O-, -C(R1)=N-, -C(R1)=NO-, -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)-; During the ceremony, x is 0, 1 or 2; n is 1, 2, 3 or 4; each R1 and R2 is independently H, a protecting group, hydroxyl, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, heterocyclic radical, substituted heterocyclic radical, heteroaryl, substituted heteroaryl, C5-C7 cycloaliphatic radical, substituted C5-C7 cycloaliphatic radical, halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C(=O)-H), substituted acyl, CN, sulfonyl (S(=O)2-J1), or sulfoxyl (S(=O)-J1); and Each J1 and J2 is independently H, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, acyl (C(=O)-H), substituted acyl, a heterocyclic radical, a substituted heterocyclic radical, C1-C12 aminoalkyl, substituted C1-C12 aminoalkyl, or a protecting group.

[0549] In one embodiment, each of the linkers of the LNA compound is independently -[C(R1)(R2)] n -, -[C(R1)(R2)] n In another embodiment, each of the linkers is independently 4'-CH2-2', 4'-(CH2)2-2', 4'-(CH2)3-2', 4'-CH2-O-2', 4'-(CH2)2-O-2', 4'-CH2-ON(R1)-2', and 4'-CH2-N(R1)-O-2'-, where each R1 is independently H, a protecting group, or a C1-C12 alkyl.

[0550] Certain LNAs have been prepared and disclosed in the patent and 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. USA, 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; WO 94 / 14226; WO 2005 / 021570; Singh ... al., J. Org. Chem., 1998, 63, 10035-10039; examples of issued U.S. patents and published applications disclosing LNAs include, for example, U.S. Pat. Nos. 7,053,207; 6,268,490; 6,770,748; 6,794,499; 7,034,133; and 6,525,191; and U.S. Patent Application Publication Nos. 2004-0171570; 2004-0219565; 2004-0014959; 2003-0207841; 2004-0143114; and 20030082807.

[0551] Additionally, LNAs have been described in which the 2'-hydroxyl group of the ribosyl sugar ring is linked to the 4' carbon atom of the sugar ring, thereby forming a methyleneoxy (4'-CH2-O-2') bond to form a bicyclic sugar moiety (discussed in 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. Pat. Nos. 6,268,490 and 6,670,461). The linkage can be a methylene (-CH2-) group bridging the 2' oxygen atom and the 4' carbon atom, in which case the term methyleneoxy(4'-CH2-O-2')LNA is used for the bicyclic moiety; if there is an ethylene group at this position, the term ethyleneoxy(4'-CH2CH2-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'-CH2-O-2')LNA and other bicyclic sugar analogs 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, including BNAs, have been described (Wahlestedt et al., Proc. Natl. Acad. Sci. USA, 2000, 97, 5633-5638).

[0552] An isomer of methyleneoxy(4'-CH2-O-2')LNA that has also been discussed is α-L-methyleneoxy(4'-CH2-O-2')LNA, which has been shown to have excellent stability against 3'-exonucleases. α-L-methyleneoxy(4'-CH2-O-2')LNA has been incorporated into antisense gapmers and chimeras, which have shown potent antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).

[0553] The synthesis and preparation of methyleneoxy (4'-CH2-O-2') LNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine, and uracil have been described, along with their oligomerization and nucleic acid recognition properties (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). BNAs and their preparation are also described in WO 98 / 39352 and WO 99 / 14226.

[0554] Furthermore, analogs of methyleneoxy(4'-CH2-O-2')LNA, phosphorothioate-methyleneoxy(4'-CH2-O-2')LNA and 2'-thio-LNA have also been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). The preparation of locked nucleoside analogs containing oligodeoxynucleotide duplexes as substrates for nucleic acid polymerases has also been described (Wengel et al., WO 99 / 14226). Furthermore, the synthesis of a novel conformationally restricted, high-affinity oligonucleotide analog, 2'-amino-LNA, has also been described in the art (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). Furthermore, 2'-amino- and 2'-methylamino-LNAs have been prepared and the thermal stability of their duplexes with complementary RNA and DNA strands has been reported.

[0555] Modified sugar moieties are well known and can be used to alter, typically increase, the affinity of an antisense compound for its target and / or increase nuclease resistance. A representative list of preferred modified sugars includes, but is not limited to, bicyclic modified sugars such as methyleneoxy (4'-CH2-O-2') LNA and ethyleneoxy (4'-(CH2)2-O-2'-bridged) ENA; substituted sugars, particularly 2'-substituted sugars having a 2'-F, 2'-OCH3, or 2'-O(CH2)2-OCH3 substituent; and 4'-thio-modified sugars. Sugars can also be substituted with sugar mimetic groups, among others. Methods for preparing modified sugars are well known to those of skill in the art. Some representative patents and publications that teach 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; Nos. 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 WO 2005 / 121371.

[0556] Examples of "oxy"-2' hydroxyl group modifications include alkoxy or aryloxy (OR, e.g., R=H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar); polyethylene glycol (PEG), O(CH2CHO) nCH2CH2OR, n=1-50; "locked" nucleic acids (LNA) in which the furanose portion of the nucleoside contains a bridge connecting two carbon atoms on the furanose ring, thereby forming a bicyclic ring system; O-AMINE or O-(CH2) n AMINE (n=1 to 10, AMINE=NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, ethylenediamine or polyamino); and O-CH2CH2(NCH2CH2NMe2)2.

[0557] "Deoxy" modifications include hydrogen (i.e., the deoxyribose sugar particularly associated with overhanging single strands); halo (e.g., fluoro); amino (e.g., NH; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); NH(CHCHNH) n Included are CH2CH2-AMINE (AMINE = NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino or diarylamino); -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 can be optionally substituted, for example with an amino function.

[0558] Other suitable 2'-modifications, such as modified MOEs, are described in US Patent Application Publication No. 20130130378, the contents of which are incorporated herein by reference.

[0559] The modification at the 2' position can be in the arabinose configuration. The term "arabinose configuration" refers to the placement of the substituent on C2' of the ribose in the same configuration as the 2'-OH in arabinose.

[0560] A sugar can contain two different modifications, e.g., gem modifications, at the same carbon of the sugar. The sugar group can also contain one or more carbons with the opposite stereochemical configuration to that of the corresponding one in ribose. Thus, a REVERSIR compound can include one or more monomers containing, for example, arabinose as the sugar. The monomer can have an α-linkage, e.g., an α-nucleotide, at the 1-position of the sugar. The monomer can also have the opposite stereoconfiguration at the 4'-position, e.g., C5' and H4', or the substituents replacing them, are swapped. When C5' and H4', or the substituents replacing them, are swapped, the sugar is said to be 4'-modified.

[0561] The REVERSIR compounds of the present invention may also contain abasic sugars, i.e., sugars lacking a nucleobase at C-1' or having another chemical group in place of the nucleobase at C1'. See, e.g., U.S. Pat. No. 5,998,203, the contents of which are incorporated herein by reference in their entirety. These abasic sugars may also contain modifications to one or more of the constituent sugar atoms. REVERSIR compounds may also contain one or more sugars that are L-isomers, e.g., L-nucleosides. Additionally, modifications to the sugar group may include replacement of the 4'-O with a sulfur, an optionally substituted nitrogen, or a CH2 group. In some embodiments, the linkage between C1' and the nucleobase is in the α-configuration.

[0562] Sugar modifications can also include acyclic nucleotides in which a C-C bond between ribose carbons (e.g., C1'-C2', C2'-C3', C3'-C4', C4'-O4', C1'-O4') is absent and / or at least one of the ribose carbons or oxygens (e.g., C1', C2', C3', C4', or O4'), independently or in combination, is absent from the nucleotide. In some embodiments, an acyclic nucleotide is [ka] wherein B is a modified or unmodified nucleobase, R and R are independently H, halogen, OR, or alkyl; and R is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar.

[0563] In some embodiments, the sugar modification is 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-CH2-(4'-C) (LNA), 2'-O-CH2CH2-(4'-C) (ENA), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), and gem 2'-OMe / 2'F with 2'-O-Me in the arabinose configuration.

[0564] It should be understood that when a particular nucleotide is linked to the next nucleotide through its 2' position, the sugar modifications described herein can be located at the 3' position of the sugar for that particular nucleotide, e.g., a nucleotide linked to the next nucleotide through its 2' position. The 3' modification can be in the xylose configuration. The term "xylose configuration" refers to the placement of the substituent on the C3' of the ribose in the same configuration as the 3'-OH of the xylose sugar.

[0565] The hydrogen attached to C4' and / or C1' can be replaced by a straight or branched, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl, and the backbone of the alkyl, alkenyl, and alkynyl can contain one or more of O, S, S(O), SO2, N(R'), C(O), N(R')C(O)O, OC(O)N(R'), CH(Z'), a phosphorus-containing bond, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclic, or optionally substituted cycloalkyl, where R' is hydrogen, acyl, or optionally substituted aliphatic, and Z' is OR 11 , C.O.R. 11 , CO2R 11 , [ka] , N.R. 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 R51 , SO2R 11 , SOR 11 , S.R. 11 and substituted or unsubstituted heterocyclic; R 21 and R 31 is, for each occurrence, independently hydrogen, acyl, unsubstituted or substituted aliphatic, aryl, heteroaryl, heterocyclic, OR 11 , C.O.R. 11 , CO2R 11 or NR 11 R 11 ' or R 21 and R 31 together with the atoms to which they are attached form a heterocyclic ring; R 41 and R 51 is, for each occurrence, independently hydrogen, acyl, unsubstituted or substituted aliphatic, aryl, heteroaryl, heterocyclic, OR 11 , C.O.R. 11 or CO2R 11 or NR 11 R 11 ' and ;R 11 and R 11 is independently hydrogen, aliphatic, substituted aliphatic, aryl, heteroaryl, or heterocyclic. In some embodiments, the hydrogen attached to C4' of the 5'-terminal nucleotide is substituted.

[0566] In some embodiments, C4' and C5' together form an optionally substituted heterocyclic ring, which contains 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 M, for each occurrence, is independently an alkyl metal or transition metal with a total charge of +1; Y is O, S, or NR'; where R' is hydrogen or an optionally substituted aliphatic. This modification is preferably at the 5' end of the oligonucleotide.

[0567] In some embodiments, the LNA has the formula: [ka] and a bicyclic nucleotide having the formula: During the ceremony, Bx is a heterocyclic base moiety; T1 is H or a hydroxyl protecting group; T2 is H, a hydroxyl protecting group or a reactive phosphorus group; Z is C1 to C6 alkyl, C2 to C6 alkenyl, C2 to C6 alkynyl, substituted C1 to C6 alkyl, substituted C2 to C6 alkenyl, substituted C2 to C6 alkynyl, acyl, substituted acyl, or substituted amido.

[0568] In one embodiment, each of the substituted groups is mono- or polysubstituted with optionally protected substituents independently selected from halogen, oxo, hydroxyl, OJ1, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2 and CN (each J1, J2 and J3 is independently H or C1-C6 alkyl and X is O, S or NJ1).

[0569] In some such embodiments, each of the substituted groups is mono- or polysubstituted with substituents independently selected from halogen, oxo, hydroxyl, OJ1, NJ1J2, SJ1, N3, OC(=X)J1, and NJ3C(=X)NJ1J2 (each J1, J2, and J3 is independently H, C1-C6 alkyl, or substituted C1-C6 alkyl, and X is O or NJ1).

[0570] In some embodiments, the Z group is C-C alkyl substituted with one or more X, where each X is independently OJ, NJJ, SJ, N, OC(=X)J, OC(=X)NJJ, NJC(=X)NJJ, or CN (each J, J, and J is independently H or C-C alkyl and X is O, S, or NJ). In other embodiments, the Z group is C-C alkyl substituted with one or more X, where each X is independently halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CH—), substituted alkoxy, or azido.

[0571] In some embodiments, the Z group is -CH2Xx, where Xx is selected from OJ1, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2, or CN (each J1, J2, and J3 is independently H or C1-C6 alkyl and X is O, S, or NJ1). In other embodiments, the Z group is -CH2Xx, where Xx is halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CH3-), or azido.

[0572] In some embodiments, the Z group is in the (R)-configuration: [ka] is.

[0573] In some embodiments, the Z group is in the (S)-configuration: [ka] is.

[0574] In some embodiments, each of T1 and T2 is a hydroxyl protecting group. A preferred list of hydroxyl protecting groups includes benzyl, benzoyl, 2,6-dichlorobenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, mesylate, tosylate, dimethoxytrityl (DMT), 9-phenylxanthin-9-yl (Pixyl), and 9-(p-methoxyphenyl)xanthin-9-yl (MOX). In some embodiments, T1 is a hydroxyl protecting group selected from acetyl, benzyl, t-butyldimethylsilyl, t-butyldimethylsilyl, and dimethoxytrityl, and a more preferred hydroxyl protecting group is T1 which is 4,4'-dimethoxytrityl.

[0575] In some embodiments, T2 is a reactive phosphorus group, and preferred reactive phosphorus groups include diisopropylcyanoethoxyphosphoramidite and H-phosphate. In some embodiments, T1 is 4,4'-dimethoxytrityl and T2 is diisopropylcyanoethoxyphosphoramidite.

[0576] In some embodiments, the REVERSIR compound has the formula: [ka] or the expression: [ka] or the expression: [ka] and having at least one monomer of During the ceremony, Bx is a heterocyclic base moiety; T3 is H, a hydroxyl protecting group, a linked conjugate group, or an internucleoside linking group attached to a nucleoside, nucleotide, oligonucleoside, oligonucleotide, monomeric subunit, or oligomeric compound; T4 is H, a hydroxyl protecting group, a linked conjugate group, or an internucleoside linking group attached to a nucleoside, nucleotide, oligonucleoside, oligonucleotide, monomeric subunit, or oligomeric compound; wherein at least one of T3 and T4 is an internucleoside linking group attached to a nucleoside, nucleotide, oligonucleoside, oligonucleotide, monomeric subunit, or oligomeric compound; Z is C1 to C6 alkyl, C2 to C6 alkenyl, C2 to C6 alkynyl, substituted C1 to C6 alkyl, substituted C2 to C6 alkenyl, substituted C2 to C6 alkynyl, acyl, substituted acyl, or substituted amido.

[0577] In one embodiment, each of the substituted groups is independently mono- or polysubstituted with optionally protected substituents independently selected from halogen, oxo, hydroxyl, OJ1, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2 and CN (each J1, J2 and J3 is independently H or C1-C6 alkyl and X is O, S or NJ1).

[0578] In one embodiment, each of the substituted groups is mono- or polysubstituted with substituents independently selected from halogen, oxo, hydroxyl, OJ1, NJ1J2, SJ1, N3, OC(=X)J1, and NJ3C(=X)NJ1J2 (each J1, J2, and J3 is independently H or C1-C6 alkyl, and X is O or NJ1).

[0579] In some such embodiments, at least one Z is C1-C6 alkyl or substituted C1-C6 alkyl. In some embodiments, each Z is independently C1-C6 alkyl or substituted C1-C6 alkyl. In some embodiments, at least one Z is C1-C6 alkyl. In some embodiments, each Z is independently C1-C6 alkyl. In some embodiments, at least one Z is methyl. In some embodiments, each Z is methyl. In some embodiments, at least one Z is ethyl. In some embodiments, each Z is ethyl. In some embodiments, at least one Z is substituted C1-C6 alkyl. In some embodiments, each Z is independently substituted C1-C6 alkyl. In some embodiments, at least one Z is substituted methyl. In some embodiments, each Z is substituted methyl. In some embodiments, at least one Z is substituted ethyl. In some embodiments, each Z is substituted ethyl.

[0580] In some embodiments, at least one substituent is C1-C6 alkoxy (e.g., at least one Z is C1-C6 alkyl substituted with one or more C1-C6 alkoxy). In other embodiments, each substituent is independently C1-C6 alkoxy (e.g., each Z is independently C1-C6 alkyl substituted with one or more C1-C6 alkoxy).

[0581] In some embodiments, at least one C1-C6 alkoxy substituent is CHO- (e.g., at least one Z is CHOCH-). In other embodiments, each C1-C6 alkoxy substituent is CHO- (e.g., each Z is CHOCH-).

[0582] In some embodiments, at least one substituent is halogen (e.g., at least one Z is C1-C6 alkyl substituted with one or more halogens). In some embodiments, each substituent is independently halogen (e.g., each Z is independently C1-C6 alkyl substituted with one or more halogens). In some embodiments, at least one substituent is fluoro (e.g., at least one Z is CH2FCH2-, CHF2CH2-, or CF3CH2-). In some embodiments, each halo substituent is fluoro (e.g., each Z is CH2FCH2-, CHF2CH2-, or CF3CH2-).

[0583] In some embodiments, at least one substituent is hydroxyl (e.g., at least one Z is C1-C6 alkyl substituted with one or more hydroxyl). In some embodiments, each substituent is independently hydroxyl (e.g., each Z is independently C1-C6 alkyl substituted with one or more hydroxyl). In some embodiments, at least one Z is HOCH2-. In other embodiments, each Z is HOCH2-.

[0584] In some embodiments, at least one Z is CH3-, CH3CH2-, CH2OCH3-, CH2F-, or HOCH2-. In some embodiments, each Z is CH3-, CH3CH2-, CH2OCH3-, CH2F-, or HOCH2-.

[0585] In some embodiments, at least one Z group is C-C alkyl substituted with one or more X, where each X is independently OJ, NJJ, SJ, N, OC(=X)J, OC(=X)NJJ, NJC(=X)NJJ, or CN; each J, J, and J is independently H or C-C alkyl, and X is O, S, or NJ. In other embodiments, at least one Z group is C-C alkyl substituted with one or more X, where each X is independently halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CHO—), or azido.

[0586] In some embodiments, each Z group is independently C-C alkyl substituted with one or more X, where each X is independently OJ, NJJ, SJ, N, OC(=X)J, OC(=X)NJJ, NJC(=X)NJJ, or CN; each J, J, and J is independently H or C-C alkyl, and X is O, S, or NJ. In other embodiments, each Z group is independently C-C alkyl substituted with one or more X, where each X is independently halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CHO—), or azido.

[0587] In some embodiments, at least one Z group is -CH2Xx, where each Xx is OJ1, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2, or CN; each J1, J2, and J3 is independently H or C1-C6 alkyl, and X is O, S, or NJ1. In some embodiments, at least one Z group is -CH2Xx, where each Xx is halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CHO-), or azido.

[0588] In some embodiments, each Z group is independently -CH2Xx, where each Xx is independently OJ1, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2, or CN; each J1, J2, and J3 is independently H or C1-C6 alkyl, and X is O, S, or NJ1. In other embodiments, each Z group is independently -CH2Xx, where each Xx is halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CHO-), or azido.

[0589] In some embodiments, at least one Z group is CH3-. In other embodiments, each Z group is CH3-.

[0590] In some embodiments, the Z group of at least one monomer has the formula: [ka] or the expression: [ka] or the expression: [ka] It has an (R)-configuration represented by

[0591] In some embodiments, the Z group of each monomer of the above formula is in the (R)-configuration.

[0592] In some embodiments, the Z group of at least one monomer has the formula: [ka] or the expression: [ka] or the expression: [ka] The (S)-configuration is represented by

[0593] In certain embodiments, the Z group of each monomer of the above formula is in the (S)-configuration.

[0594] In certain embodiments, T3 is H or a hydroxyl protecting group. In some embodiments, T4 is H or a hydroxyl protecting group. In other embodiments, T3 is an internucleo...

Claims

1. A universal double-stranded ribonucleic acid (dsRNA) agent, comprising a sense strand and an antisense strand that form a duplex region; A universal dsRNA agent wherein the antisense strand comprises at least 15 contiguous nucleotides that differ from any of the antisense strand nucleotide sequences in Table 2 by no more than 3 nucleotides.

2. A universal double-stranded ribonucleic acid (dsRNA) agent, comprising a sense strand and an antisense strand that form a duplex region; the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any sense strand nucleotide sequence in Table 2; A universal dsRNA agent wherein the antisense strand comprises at least 15 contiguous nucleotides that differ from any of the antisense strand nucleotide sequences in Table 2 by no more than 3 nucleotides.

3. A universal double-stranded ribonucleic acid (dsRNA) agent, comprising a sense strand and an antisense strand that form a duplex region; A universal dsRNA agent wherein the antisense strand comprises a region of complementarity to any of the target nucleotide sequences in Table 3.

4. The universal dsRNA agent of any of claims 1 to 3, comprising at least one modified nucleotide.

5. 5. The universal dsRNA agent of claim 1, A universal dsRNA agent, wherein substantially all of the nucleotides in the sense strand are modified nucleotides; substantially all of the nucleotides in the antisense strand are modified nucleotides; or substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides.

6. 6. A universal dsRNA agent according to any one of claims 1 to 5, A universal dsRNA agent in which all nucleotides in the sense strand are modified nucleotides; all nucleotides in the antisense strand are modified nucleotides; or all nucleotides in the sense strand and all nucleotides in the antisense strand are modified nucleotides.

7. 7. The universal dsRNA agent of claim 4, At least one of the modified nucleotides is a deoxy-nucleotide, a 3'-terminal deoxythymidine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, a non-locked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino modified nucleotide, a 2'-O-allyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2'-hydroxyl modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a morpholino ... a universal dsRNA agent selected from the group consisting of a nucleotide, a nucleotide containing a 2' phosphate, a 2-O-(N-methylacetamido) modified nucleotide, a nucleotide containing a 2' phosphate ...

8. 7. The universal dsRNA agent of any of claims 4-6, wherein the modification of 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'-hydroxyl, and glycol, and combinations thereof.

9. 7. The universal dsRNA agent of any of claims 4-6, wherein at least one of the modified nucleotides is selected from the group consisting of deoxynucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, glycol-modified nucleotides (GNAs), nucleotides containing a 2' phosphate, and vinyl phosphonate nucleotides, and combinations thereof.

10. The universal dsRNA agent of any of claims 4-6, wherein at least one of the modifications of the modified nucleotides is a thermally destabilizing nucleotide modification.

11. 11. The universal dsRNA agent of claim 10, wherein the thermally destabilizing nucleotide modification is selected from the group consisting of an abasic modification, a mismatch with the opposite nucleotide in the duplex, and a destabilizing sugar modification, a 2'-deoxy modification, an acyclic nucleotide, a non-locked nucleic acid (UNA), and a glycerol nucleic acid (GNA).

12. The universal dsRNA agent of any of claims 1-11, wherein the duplex region is 19-30 nucleotide pairs in length.

13. 13. The universal dsRNA agent of claim 12, wherein the duplex region is 19 to 25 nucleotide pairs in length.

14. 13. The universal dsRNA agent of claim 12, wherein the duplex region is 19 to 23 nucleotide pairs in length.

15. 13. The universal dsRNA agent of claim 12, wherein the duplex region is 23 to 27 nucleotide pairs in length.

16. 13. The universal dsRNA agent of claim 12, wherein the duplex region is 21 to 23 nucleotide pairs in length.

17. The universal dsRNA agent of any of claims 1-16, wherein each strand is independently 30 nucleotides or less in length.

18. The universal dsRNA agent of any of claims 1-17, wherein the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

19. The universal dsRNA agent of any of claims 3-18, wherein the region of complementarity is at least 17 nucleotides in length.

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

21. The universal dsRNA agent of any of claims 1-19, wherein at least one strand comprises a 3' overhang of at least two nucleotides.

22. The universal dsRNA agent of any of claims 1-21, further comprising a ligand.

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

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

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

26. 26. The universal dsRNA agent of claim 24 or 25, wherein the ligand is: 【Chemical 1】 A universal dsRNA agent,

27. 27. The universal dsRNA agent of claim 26, conjugated to a ligand as shown in the following schematic diagram: 【Chemistry 2】 A universal dsRNA agent wherein X is O or S.

28. 28. The universal dsRNA agent of claim 27, wherein X is O.

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

30. 30. The universal dsRNA agent of claim 29, wherein the phosphorothioate or methylphosphonate internucleotide linkage is at the 3'-end of one strand.

31. 31. The universal dsRNA agent of claim 30, wherein the strand is the antisense strand.

32. 31. The universal dsRNA agent of claim 30, wherein the strand is the sense strand.

33. 30. The universal dsRNA agent of claim 29, wherein the phosphorothioate or methylphosphonate internucleotide linkage is at the 5'-end of one strand.

34. 34. The universal dsRNA agent of claim 33, wherein the strand is the antisense strand.

35. 34. The universal dsRNA agent of claim 33, wherein the strand is the sense strand.

36. 30. The universal dsRNA agent of claim 29, wherein the phosphorothioate or methylphosphonate internucleotide linkages are at both the 5'-end and the 3'-end of one strand.

37. 37. The universal dsRNA agent of claim 36, wherein the strand is the antisense strand.

38. 38. The universal dsRNA agent of any of claims 1-37, wherein the base pair at position 1 of the 5'-end of the antisense strand of the duplex is an AU base pair.

39. 39. A cell containing the universal dsRNA agent of any of claims 1-38.

40. 39. A vector comprising the universal dsRNA agent of any of claims 1-38.

41. 41. The vector of claim 40, which is an expression vector.

42. 42. The vector of claim 40 or 41, which is a viral vector.

43. 43. The vector of claim 42, wherein the viral vector is an adeno-associated viral (AAV) vector.

44. 44. The vector of claim 42 or 43, wherein the viral vector is a bicistronic vector.

45. The vector according to any one of claims 40 to 44, further comprising a transgene.

46. A cell containing the vector according to any one of claims 40 to 45.

47. A pharmaceutical composition comprising a universal dsRNA agent of any of claims 1-38 or a vector of any of claims 40-45, and a pharmaceutically acceptable carrier.

48. 48. The pharmaceutical composition of claim 47, wherein the dsRNA agent or vector is in an unbuffered solution.

49. 49. The pharmaceutical composition of claim 48, wherein the unbuffered solution is saline or water.

50. 48. The pharmaceutical composition of claim 47, wherein the dsRNA agent or vector is in a buffer solution.

51. 51. The pharmaceutical composition of claim 50, wherein the buffer solution comprises acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof.

52. 52. The pharmaceutical composition of claim 51, wherein the buffer solution is phosphate buffered saline (PBS).

53. A REVERSIR compound that inhibits the iRNA activity of the universal dsRNA agent of any of claims 1-38.

54. A REVERSIR compound comprising a single-stranded oligonucleotide of 6-30 nucleotides in length, the nucleotide sequence being at least about 90% complementary to any of the antisense strand nucleotide sequences in Table 2 or Table 3.

55. 55. The REVERSIR compound of claim 54, wherein the oligonucleotide is 100% complementary to any of the antisense strand nucleotide sequences in Table 2 or Table 3.

56. 56. The REVERSIR compound of claim 54 or 55, wherein the oligonucleotide comprises at least one modified nucleotide.

57. 57. The REVERSIR compound of any of claims 54-56, wherein substantially all of the nucleotides of the oligonucleotide are modified nucleotides.

58. 58. The REVERSIR compound of claim 57, wherein all nucleotides of the oligonucleotide are modified nucleotides.

59. 59. The REVERSIR compound of any of claims 56-58, wherein at least one of the modified nucleotides comprises a modified nucleobase.

60. 60. The REVERSIR compound of claim 59, wherein the modified nucleobase is a 5'-methylcytosine.

61. 61. The REVERSIR compound of any of claims 56-60, wherein at least one of the modified nucleotides comprises a modified sugar.

62. 62. The REVERSIR compound of claim 61, wherein the modified sugar is selected from the group consisting of a 2'-O-methoxyethyl-modified sugar, a 2'-methoxy-modified sugar, a 2'-O-alkyl-modified sugar, and a bicyclic sugar.

63. 63. The REVERSIR compound of any of claims 54-62, further comprising a ligand.

64. 64. The REVERSIR compound of claim 63, wherein the ligand is conjugated to the 3' end of the oligonucleotide.

65. 65. The REVERSIR compound of claim 63 or 64, wherein the ligand is an N-acetylgalactosamine (GalNAc) derivative.

66. 66. The REVERSIR compound of any of claims 63-65, wherein the ligand is one or more GalNAc derivatives attached via a monovalent, divalent, or trivalent branched linker.

67. 67. The REVERSIR compound of claim 65 or 66, wherein the ligand is 【Chemistry 3】 The REVERSIR compound is

68. 68. The REVERSIR compound of claim 67, wherein the oligonucleotide is conjugated to a ligand as shown in the following schematic diagram: 【Chemistry 4】 ReverSir compounds wherein X is O or S.

69. 69. The REVERSIR compound of claim 68, wherein X is O.

70. 70. The REVERSIR compound of any of claims 54-69, wherein the oligonucleotide further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.

71. 71. The REVERSIR compound of any of claims 54-70, wherein the oligonucleotide is 6-15, 7-11, or 8-10 nucleotides in length.

72. 71. The REVERSIR compound of any of claims 54-70, wherein the oligonucleotide is 15-25, 17-25, 19-25, or 21-25 nucleotides in length.

73. A cell containing the REVERSIR compound of any one of claims 54-72.

74. 1. A system for on-demand expression of a transgene, comprising: an expression vector encoding a transgene, a universal iRNA target moiety, a universal double-stranded ribonucleic acid (dsRNA) agent; It comprises a sense strand and an antisense strand that form a duplex region that recognizes and binds to a universal iRNA target site, thereby inhibiting expression of the transgene, and optionally A system comprising a REVERSIR compound that inhibits iRNA activity of a universal dsRNA agent, thereby allowing expression of a transgene.

75. 75. The system of claim 74, wherein the universal iRNA target site is located in the 5'-untranslated region (UTR) or the 3'-untranslated region (UTR) of the transgene.

76. 76. A system according to claim 74 or 75, comprising: the universal dsRNA agent comprises a sense strand and an antisense strand which form a duplex region; The system wherein the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any antisense strand nucleotide sequence in either Table 2 or Table 3.

77. 76. A system according to claim 74 or 75, comprising: the universal dsRNA agent comprises a sense strand and an antisense strand which form a duplex region; the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any sense strand nucleotide sequence in Table 2; The system wherein the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any antisense strand nucleotide sequence in either Table 2 or Table 3.

78. the universal dsRNA agent comprises a sense strand and an antisense strand which form a duplex region; 76. The system of claim 74 or 75, wherein the antisense strand comprises a region of complementarity to any target nucleotide sequence in either Table 3 or Table 4.

79. 79. The system of any of claims 74-78, wherein the universal dsRNA agent comprises at least one modified nucleotide.

80. A system according to any one of claims 74 to 79, comprising: A system wherein substantially all of the nucleotides in the sense strand are modified nucleotides, substantially all of the nucleotides in the antisense strand are modified nucleotides, or substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides.

81. A system according to any one of claims 74 to 80, comprising: A system in which all nucleotides in the sense strand are modified nucleotides and all nucleotides in the antisense strand are modified nucleotides, or all nucleotides in the sense strand and all nucleotides in the antisense strand are modified nucleotides.

82. A system according to any one of claims 79 to 81, comprising: At least one of the modified nucleotides is a deoxy-nucleotide, a 3'-terminal deoxythymidine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, a non-locked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino modified nucleotide, a 2'-O-allyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2'-hydroxyl modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a morpho the nucleotide is selected from the group consisting of linonucleotides, phosphoramidates, nucleotides containing unnatural bases, tetrahydropyran-modified nucleotides, 1,5-anhydrohexitol-modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides containing phosphorothioate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphates, nucleotides containing 5'-phosphate mimetics, thermally destabilized nucleotides, glycol-modified nucleotides (GNAs), nucleotides containing 2' phosphates, and 2-O-(N-methylacetamido)-modified nucleotides, and combinations thereof.

83. 82. The system of any one of claims 79 to 81, wherein the modification of 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'-hydroxyl, and glycol, and combinations thereof.

84. 82. The system of any of claims 79 to 81, wherein at least one of the modified nucleotides is selected from the group consisting of deoxynucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, glycol-modified nucleotides (GNAs), nucleotides containing a 2' phosphate, and vinyl phosphonate nucleotides, and combinations thereof.

85. 82. The system of any one of claims 79 to 81, wherein at least one of the modifications of the modified nucleotides is a thermally destabilizing nucleotide modification.

86. 86. The system of claim 85, wherein the thermally destabilizing nucleotide modification is selected from the group consisting of an abasic modification, a mismatch with the opposite nucleotide in a duplex, and a destabilizing sugar modification, a 2'-deoxy modification, an acyclic nucleotide, a non-locked nucleic acid (UNA), and a glycerol nucleic acid (GNA).

87. 87. The system of any one of claims 74 to 86, wherein the duplex region is 19 to 30 nucleotide pairs in length.

88. 87. The system of claim 86, wherein the duplex region is 19 to 25 nucleotide pairs in length.

89. 87. The system of claim 86, wherein the duplex region is 19 to 23 nucleotide pairs in length.

90. 87. The system of claim 86, wherein the duplex region is 23 to 27 nucleotide pairs in length.

91. 87. The system of claim 86, wherein the duplex region is 21 to 23 nucleotide pairs in length.

92. 92. The system of any of claims 74-91, wherein each strand is independently 30 nucleotides or less in length.

93. 93. The system of any one of claims 74 to 92, wherein the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

94. 94. The system of any one of claims 78 to 93, wherein the region of complementarity is at least 17 nucleotides in length.

95. 95. The system of any one of claims 74 to 94, wherein at least one strand comprises a 3' overhang of at least one nucleotide.

96. 96. The system of any of claims 74 to 95, wherein at least one strand comprises a 3' overhang of at least two nucleotides.

97. 97. The system of any of claims 74-96, wherein the universal dsRNA agent further comprises a ligand.

98. 98. The system of claim 97, wherein the ligand is conjugated to the 3' end of the sense strand of the universal dsRNA agent.

99. The system of claim 97 or 98, wherein the ligand is an N-acetylgalactosamine (GalNAc) derivative.

100. 100. The system of any one of claims 97 to 99, wherein the ligand is one or more GalNAc derivatives attached via a monovalent, divalent, or trivalent branched linker.

101. 101. The system of claim 99 or 100, wherein the ligand is: 【Chemistry 5】 That is, the system.

102. 102. The system of claim 101, wherein the universal dsRNA agent is conjugated to a ligand as shown in the following schematic diagram: 【Chemistry 6】 wherein X is O or S.

103. 103. The system of claim 102, wherein X is O.

104. 104. The system of any of claims 74-103, wherein the universal dsRNA agent further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.

105. 105. The system of claim 104, wherein the phosphorothioate or methylphosphonate internucleotide linkage is at the 3'-end of one strand.

106. The system of claim 105, wherein the strand is an antisense strand.

107. 106. The system of claim 105, wherein the strand is the sense strand.

108. 105. The system of claim 104, wherein the phosphorothioate or methylphosphonate internucleotide linkage is at the 5'-end of one strand.

109. The system of claim 108, wherein the strand is an antisense strand.

110. 109. The system of claim 108, wherein the strand is the sense strand.

111. 105. The system of claim 104, wherein the phosphorothioate or methylphosphonate internucleotide linkages are at both the 5'-end and the 3'-end of one strand.

112. The system of claim 111, wherein the strand is an antisense strand.

113. The system according to any one of claims 74 to 112, wherein the base pair at position 1 of the 5'-end of the antisense strand of the duplex is an AU base pair.

114. 1. A system for on-demand expression of a transgene, comprising: an expression vector encoding a transgene and a double-stranded ribonucleic acid (dsRNA) agent that targets the transgene, wherein expression of the transgene is inhibited by expression of the dsRNA agent that targets the transgene; Optionally, the system includes a REVERSIR compound that inhibits iRNA activity of the dsRNA agent, thereby allowing expression of the transgene.

115. 115. The system of any of claims 74-114, wherein the REVERSIR compound comprises a single-stranded oligonucleotide 6-30 nucleotides in length and comprises a nucleotide sequence that is at least about 90% complementary to any antisense strand nucleotide sequence in either Table 2 or Table 3.

116. 116. The system of claim 115, wherein the oligonucleotide is 100% complementary to any antisense strand nucleotide sequence in either Table 2 or Table 3.

117. 117. The system of claim 115 or 116, wherein the oligonucleotide comprises at least one modified nucleotide.

118. The system of any of claims 115 to 117, wherein substantially all of the nucleotides of the oligonucleotide are modified nucleotides.

119. The system of claim 118, wherein all nucleotides of the oligonucleotide are modified nucleotides.

120. 120. The system of any one of claims 117 to 119, wherein at least one of the modified nucleotides comprises a modified nucleobase.

121. 121. The system of claim 120, wherein the modified nucleobase is 5'-methylcytosine.

122. 122. The system of any one of claims 117 to 121, wherein at least one of the modified nucleotides comprises a modified sugar.

123. 123. The system of claim 122, wherein the modified sugar is selected from the group consisting of a 2'-O-methoxyethyl-modified sugar, a 2'-methoxy-modified sugar, a 2'-O-alkyl-modified sugar, and a bicyclic sugar.

124. The system of any of claims 114-123, wherein the REVERSIR compound comprises a ligand.

125. 125. The system of claim 124, wherein the ligand is conjugated to the 3' end of the oligonucleotide.

126. The system of claim 124 or 125, wherein the ligand is an N-acetylgalactosamine (GalNAc) derivative.

127. The system of any one of claims 124 to 126, wherein the ligand is one or more GalNAc derivatives attached via a monovalent, divalent, or trivalent branched linker.

128. 128. The system of claim 126 or 127, wherein the ligand is: 【Chemistry 7】 That is, the system.

129. 129. The system of claim 128, wherein the oligonucleotide is conjugated to a ligand as shown in the following schematic diagram: 【Chemistry 8】 wherein X is O or S.

130. 130. The system of claim 129, wherein X is O.

131. 131. The system of any of claims 115-130, wherein the oligonucleotide further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.

132. 132. The system of any of claims 115-131, wherein the oligonucleotides are 6-15, 7-11, or 8-10 nucleotides in length.

133. 132. The system of any of claims 115-131, wherein the oligonucleotides are 15-25, 17-25, 19-25, or 21-25 nucleotides in length.

134. The system of any one of claims 74 to 133, wherein the expression vector is a viral vector.

135. The system of claim 134, wherein the viral vector is an adeno-associated viral (AAV) vector.

136. The system of claim 134 or 135, wherein the viral vector is a bicistronic vector.

137. 1. A method for regulating expression of a transgene in a cell, comprising: contacting the cell with an expression vector encoding a 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 a universal iRNA target site, thereby inhibiting expression of the transgene, and optionally, and contacting the cells with a REVERSIR compound that inhibits the iRNA activity of the universal dsRNA agent, thereby allowing expression of the transgene. A method comprising:

138. The method of claim 137, wherein the cell is present in a subject.

139. 1. A method of treating a subject in need thereof, comprising: A method comprising contacting an expression vector encoding a therapeutic transgene and comprising a universal iRNA target site administered to a subject with a universal double-stranded ribonucleic acid (dsRNA) agent that recognizes and binds to the universal iRNA target site, thereby inhibiting expression of the transgene, thereby treating the subject.

140. 140. The method of claim 139, wherein the universal dsRNA agent is further contacted with a REVERSIR compound that inhibits iRNA activity of the universal dsRNA agent, thereby allowing expression of the transgene.

141. 1. A method of treating a subject in need thereof, comprising: A method comprising contacting an expression vector encoding a therapeutic transgene and a double-stranded ribonucleic acid (dsRNA) agent that targets the transgene, which is administered to a subject, with a REVERSIR compound that inhibits iRNA activity of the dsRNA agent, thereby allowing expression of the transgene, thereby treating the subject.

142. 1. A method of treating a subject in need thereof, comprising: administering to the 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 is achieved; and When a desired level of expression of the transgene is achieved, administering to the subject a universal double-stranded ribonucleic acid (dsRNA) agent comprising a sense strand and an antisense strand that form a duplex region, which recognizes and binds to a universal iRNA target site, thereby inhibiting expression of the transgene, thereby treating the subject; A method comprising:

143. 143. The method of claim 142, further comprising administering to said subject a REVERSIR compound when the level of the transgene falls below a desired level of expression, wherein the REVERSIR compound suppresses iRNA activity of the universal dsRNA agent, thereby allowing expression of the transgene.

144. 144. The method of any 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. A method according to any one of claims 137 to 140 and 142 to 144, comprising: A method wherein the universal dsRNA agent comprises a sense strand and an antisense strand that form a duplex region, and wherein the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any antisense strand nucleotide sequence in either Table 2 or Table 3.

146. A method according to any one of claims 137 to 140 and 142 to 144, comprising: the universal dsRNA agent comprises a sense strand and an antisense strand which form a duplex region; the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any sense strand nucleotide sequence in Table 2; The method, wherein the antisense strand comprises at least 15 contiguous nucleotides that differ from any antisense strand nucleotide sequence in either Table 2 or Table 3 by no more than 3 nucleotides.

147. the universal dsRNA agent comprises a sense strand and an antisense strand which form a duplex region; The method of any of claims 137-140 and 142-144, wherein the antisense strand comprises a region of complementarity to any target nucleotide sequence in either Table 3 or Table 4.

148. 148. The method of any of claims 137-140 and 142-147, wherein the universal dsRNA agent comprises at least one modified nucleotide.

149. A method according to any one of claims 137 to 140 and 142 to 148, comprising: The method, wherein substantially all of the nucleotides in the sense strand are modified nucleotides; substantially all of the nucleotides in the antisense strand are modified nucleotides; or substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides.

150. A method according to any one of claims 137 to 140 and 142 to 149, comprising: A method wherein all nucleotides in the sense strand are modified nucleotides; all nucleotides in the antisense strand are modified nucleotides; or all nucleotides in the sense strand and all nucleotides in the antisense strand are modified nucleotides.

151. 151. The method of any one of claims 148 to 150, At least one of the modified nucleotides is a deoxy-nucleotide, a 3'-terminal deoxythymidine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, a non-locked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino modified nucleotide, a 2'-O-allyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2'-hydroxyl modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a mol the nucleotide is selected from the group consisting of a phosphononucleotide, a phosphoramidate, a nucleotide containing a non-natural base, a tetrahydropyran-modified nucleotide, a 1,5-anhydrohexitol-modified nucleotide, a cyclohexenyl-modified nucleotide, a nucleotide containing a phosphorothioate group, a nucleotide containing a methylphosphonate group, a nucleotide containing a 5'-phosphate, a nucleotide containing a 5'-phosphate mimic, a thermally destabilized nucleotide, a glycol-modified nucleotide (GNA), a nucleotide containing a 2' phosphate, and a 2-O-(N-methylacetamido)-modified nucleotide, and combinations thereof.

152. 151. The method of any of claims 148 to 150, wherein the modification of 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'-hydroxyl, and glycol, and combinations thereof.

153. 151. The method of any of claims 148 to 150, wherein at least one of the modified nucleotides is selected from the group consisting of deoxynucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, glycol modified nucleotides (GNAs), 2' phosphate containing nucleotides, and vinyl phosphonate nucleotides, and combinations thereof.

154. 151. The method of any of claims 148 to 150, wherein at least one of the modifications of the modified nucleotides is a thermally destabilizing nucleotide modification.

155. 155. The method of claim 154, wherein the thermally destabilizing nucleotide modification is selected from the group consisting of an abasic modification, a mismatch with the opposite nucleotide in a duplex, and a destabilizing sugar modification, a 2'-deoxy modification, an acyclic nucleotide, a non-locked nucleic acid (UNA), and a glycerol nucleic acid (GNA).

156. 156. The method of any one of claims 137-140 and 142-155, wherein the duplex region is 19-30 nucleotide pairs in length.

157. 157. The method of claim 156, wherein the duplex region is 19 to 25 nucleotide pairs in length.

158. 157. The method of claim 156, wherein the duplex region is 19 to 23 nucleotide pairs in length.

159. 157. The method of claim 156, wherein the duplex region is 23 to 27 nucleotide pairs in length.

160. 157. The method of claim 156, wherein the duplex region is 21 to 23 nucleotide pairs in length.

161. 161. The method of any of claims 137-140 and 142-160, wherein each strand is independently 30 nucleotides or less in length.

162. 162. The method of any one of claims 137 to 140 and 142 to 161, wherein the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

163. 163. The method of any of claims 137-140 and 142-162, wherein the region of complementarity is at least 17 nucleotides in length.

164. 164. The method of any of claims 137-140 and 142-163, wherein at least one strand comprises a 3' overhang of at least one nucleotide.

165. 164. The method of any of claims 137-140 and 142-163, wherein at least one strand comprises a 3' overhang of at least two nucleotides.

166. 166. The method of any of claims 137-140 and 142-165, wherein the universal dsRNA agent further comprises a ligand.

167. 167. The method of claim 166, wherein the ligand is conjugated to the 3' end of the sense strand of the universal dsRNA agent.

168. The method of claim 166 or 167, wherein the ligand is an N-acetylgalactosamine (GalNAc) derivative.

169. 169. The method of any of claims 166-168, wherein the ligand is one or more GalNAc derivatives attached via a monovalent, divalent, or trivalent branched linker.

170. 170. The method of claim 168 or 169, wherein the ligand is 【Chemistry 9】 That's the method.

171. 171. The method of claim 170, wherein the universal dsRNA agent is conjugated to a ligand as shown in the following schematic diagram: 【Chemistry 10】 wherein X is O or S.

172. 172. The method of claim 171, wherein X is O.

173. 173. The method of any of claims 137-140 and 142-172, wherein the universal dsRNA agent further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.

174. 174. The method of claim 173, wherein the phosphorothioate or methylphosphonate internucleotide linkage is at the 3'-end of one strand.

175. 175. The method of claim 174, wherein the strand is the antisense strand.

176. 175. The method of claim 174, wherein the strand is the sense strand.

177. 174. The method of claim 173, wherein the phosphorothioate or methylphosphonate internucleotide linkage is at the 5'-end of one strand.

178. 178. The method of claim 177, wherein the strand is an antisense strand.

179. 178. The method of claim 177, wherein the strand is the sense strand.

180. 174. The method of claim 173, wherein the phosphorothioate or methylphosphonate internucleotide linkages are at both the 5'-end and the 3'-end of one strand.

181. The system of claim 180, wherein the strand is an antisense strand.

182. The method of any one of claims 137 to 140 and 142 to 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. 183. The method of any of claims 137, 138, 140, 141, and 143-182, wherein the REVERSIR compound comprises a single-stranded oligonucleotide 6-30 nucleotides in length and comprises a nucleotide sequence that is at least about 90% complementary to any antisense strand nucleotide sequence in either Table 2 or Table 3.

184. 184. The method of claim 183, wherein the oligonucleotide is 100% complementary to any antisense strand nucleotide sequence in either Table 2 or Table 3.

185. 185. The method of claim 183 or 184, wherein the oligonucleotide comprises at least one modified nucleotide.

186. 186. The method of any of claims 183 to 185, wherein substantially all of the nucleotides of the oligonucleotide are modified nucleotides.

187. 187. The method of claim 186, wherein all nucleotides of the oligonucleotide are modified nucleotides.

188. 188. The method of any of claims 183 to 187, wherein at least one of the modified nucleotides comprises a modified nucleobase.

189. 189. The method of claim 188, wherein the modified nucleobase is 5'-methylcytosine.

190. 190. The method of any of claims 183-189, wherein at least one of the modified nucleotides comprises a modified sugar.

191. 191. The method of claim 190, wherein the modified sugar is selected from the group consisting of a 2'-O-methoxyethyl-modified sugar, a 2'-methoxy-modified sugar, a 2'-O-alkyl-modified sugar, and a bicyclic sugar.

192. 192. The method of any one of claims 183-191, wherein the REVERSIR compound comprises a ligand.

193. 193. The method of claim 192, wherein the ligand is conjugated to the 3' end of the oligonucleotide.

194. 194. The method of claim 192 or 193, wherein the ligand is an N-acetylgalactosamine (GalNAc) derivative.

195. 195. The method of any of claims 192-194, wherein the ligand is one or more GalNAc derivatives attached via a monovalent, divalent, or trivalent branched linker.

196. 196. The method of claim 194 or 195, wherein the ligand is 【Chemistry 11】 That's the method.

197. 200. The method of claim 196, wherein the oligonucleotide is conjugated to a ligand as shown in the following schematic diagram: 【Chemistry 12】 wherein X is O or S.

198. 198. The method of claim 197, wherein X is O.

199. 199. The method of any of claims 183-198, wherein the oligonucleotide further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.

200. 200. The method of any of claims 183-199, wherein the oligonucleotide is 6-15, 7-11, or 8-10 nucleotides in length.

201. 201. The method of any of claims 183-200, wherein the oligonucleotide is 15-25, 17-25, 19-25, or 21-25 nucleotides in length.

202. The method of any one of claims 137 to 201, wherein the expression vector is a viral vector.

203. 203. The method of claim 202, wherein the viral vector is an adeno-associated viral (AAV) vector.

204. The method of claim 202 or 203, wherein the viral vector is a bicistronic vector.

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