Microtubule-associated protein tau (MAPT) iRNA agent compositions and methods of use thereof

Abstract

The present disclosure relates to double-stranded ribonucleic acid interference (dsRNAi) agents and compositions that target a hotspot within exon 10 of the microtubule-associated protein tau (MAPT) gene, and methods of using such dsRNAi agents and compositions to inhibit expression of 4R tau and to treat a subject having a 4R tauopathy, such as a 4R tauopathy, such as progressive supranuclear palsy (PSP), corticobasal syndrome (CBD), argyrophilic grain disease (AGD), multisystem tauopathy with presenile dementia (MSTD), globuloglial tauopathy (GGT), sporadic (spAD) Alzheimer's disease, or rapidly progressive (rpAD) Alzheimer's disease.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 309,399, filed February 11, 2022, and U.S. Provisional Patent Application No. 63 / 322,618, filed March 22, 2022, the entire contents of which are incorporated herein by reference.

[0002] Array List This application contains a Sequence Listing that has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML copy created on February 10, 2023 is named A108868_1580WO_SL_XML and is 4,839,728 bytes in size. [Background technology]

[0003] The microtubule-associated protein tau (MAPT) gene, which encodes the protein Microtubule-Associated Protein Tau (Mapt), a member of the microtubule-associated protein family, is located in chromosome region 17q21.31 (base pairs 45,894,382 to 46,028,334 on chromosome 17). The MAPT gene consists of 16 exons. Alternative mRNA splicing generates six MAPT isoforms with a total length of 352 to 441 amino acids. In three of the six MAPT isoforms, the microtubule-binding domain of MAPT contains three repeating segments (3R tau isoforms), whereas in the other three MAPT isoforms (4R tau isoforms), the corresponding domain contains four repeating segments.

[0004] MAPT transcripts are differentially expressed throughout the body, primarily in the central and peripheral nervous systems. Wild-type tau is involved in stabilizing microtubules in axons, maintaining dendritic spines, and regulating axonal transport, microtubule dynamics, and cell division. Pathogenic MAPT variants are found in approximately 10% of patients with primary tauopathies. These variants are primarily missense mutations, located in exons 9–13 (the microtubule-binding domain), and primarily affect alternative splicing of exon 10.

[0005] Tauopathies are a heterogeneous class of progressive neurodegenerative disorders pathologically characterized by the presence of tau aggregates in the brain. Phenotypically, tauopathies exhibit variable progression of motor, cognitive, and behavioral impairments. Humans typically express equal proportions of 3R tau and 4R tau. In some tauopathies, such as Alzheimer's disease, experimental evidence from postmortem brain tissue suggests that insoluble tau aggregates are composed of 3R tau and 4R tau. However, both sporadic (spAD) and rapidly progressive (rpAD) Alzheimer's disease have recently been characterized by aggregates enriched in 4R tau (see Kim et al. Science Translational Med. 2022 14, eabg025). In other 4R tauopathy diseases, such as progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD), 4R tau protein is the tau species that is prone to aggregation. The reasons underlying the differences in these aggregate types in the diseases are unknown. Summary of the Invention [Problem to be solved by the invention]

[0006] At present, there is no therapeutic treatment for 4R tauopathy, and treatment is only aimed at alleviating symptoms and improving the quality of life of patients.Therefore, there is a need for a drug that can effectively, preferably preferentially inhibit or regulate the expression of 4R tau, so as to effectively treat subjects with 4R tau-related disorders, such as progressive supranuclear palsy (PSP), corticobasal ganglia syndrome (CBD), argyrophilic grain disease (AGD), multisystem tauopathy with presenile dementia (MSTD), glioglobular tauopathy (GGT), sporadic (spAD) Alzheimer's disease, rapidly progressive (rpAD) Alzheimer's disease, or other 4R tauopathy. [Means for solving the problem]

[0007] The present disclosure provides an RNAi composition that targets specific hotspot regions within exon 10 of the MAPT gene, thereby carrying out RNA-induced silencing complex (RISC)-mediated cleavage of the 4R tau RNA transcript of the MAPT gene.The MAPT gene can be located within a cell, for example, within a subject, for example, a human cell.The use of these iRNAs allows the targeted degradation of the 4R tau mRNA of the corresponding gene (MAPT gene) in mammals.

[0008] The iRNAs of the present disclosure were designed to target a hotspot within exon 10 of the MAPT gene, e.g., a gene with a missense and / or deletion mutation and a combination of nucleotide modifications in the exon. The iRNAs of the present disclosure inhibit the expression of 4R tau transcripts by at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% relative to control levels, and reduce the levels of sense- and antisense-containing aggregates. Without intending to be limited by theory, it is believed that the combination or subcombination of the aforementioned properties and specific target sites or specific modifications in these iRNAs confers improved efficacy, stability, potency, durability, and safety to the iRNAs of the present disclosure. Combinations or subcombinations of the aforementioned properties and specific target sites or specific modifications in these iRNAs can also confer enhanced specificity to the iRNAs of the present disclosure for the 4R tau isoform of the MAPT gene over the 3R tau isoform.

[0009] In one aspect, the present disclosure provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of 4R tau, the dsRNA agent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides that differ by no more than three nucleotides from the nucleotide sequence of nucleotides 1009-1053 of SEQ ID NO: 3, and the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than three nucleotides from the nucleotide sequence of nucleotides 1007-1053 of SEQ ID NO: 4. In certain embodiments, the sense strand comprises at least 15 contiguous nucleotides of nucleotides 1009-1053 of SEQ ID NO: 3, and the antisense strand comprises at least 15 contiguous nucleotides of nucleotides 1007-1053 of SEQ ID NO: 4.

[0010] In another aspect, the present disclosure provides a dsRNA agent for inhibiting expression of 4R tau, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, the antisense strand comprising a region of complementarity to an mRNA encoding 4R tau, the region of complementarity comprising at least 15 contiguous nucleotides that differ by no more than three nucleotides from the nucleotide sequence of nucleotides 1007-1053 of SEQ ID NO: 4. In certain embodiments, the region of complementarity comprises at least 15 contiguous nucleotides of nucleotides 1007-1053 of SEQ ID NO: 4. The region of complementarity may reside entirely within nucleotides 1007-1053 of SEQ ID NO: 4.

[0011] In yet another aspect, the present disclosure provides a dsRNA agent for inhibiting expression of 4R tau, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, the antisense strand comprising a region of complementarity to an mRNA encoding 4R tau, the region of complementarity comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the antisense nucleotide sequences in Table 7 or 8. In certain embodiments, the nucleotide sequences of the sense and antisense strands comprise the sense and antisense strand nucleotide sequences of any one of the duplexes in Table 7 or 8.

[0012] In some embodiments of a dsRNA agent according to any one of the foregoing aspects, the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the nucleotide sequences of nucleotides 1009-1029, 1016-1036, 1018-1038, 1020-1040, 1021-1041, 1022-1042, 1024-1044, 1027-1047, 1028-1048, 1029-1049, 1030-1050, 1032-1052, 1033-1053, 1014-1048, and 1022-1048 of SEQ ID NO:3, and the antisense strand comprises at least 15 contiguous nucleotides from the corresponding nucleotide sequence of SEQ ID NO:4.

[0013] In some embodiments, the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the antisense strand nucleotide sequences of the duplex selected from the group consisting of AD-1423276, AD-1423283, AD-1423285, AD-1423287, AD-1423288, AD-1423289, AD-1423291, AD-1423294, AD-1423295, AD-1423296, AD-1423297, AD-1423299, and AD-1423300. In certain embodiments, the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the antisense strand nucleotide sequences of the duplex selected from the group consisting of AD-1423283, AD-1423291, AD-1423294, and AD-1423295.

[0014] In some embodiments, the dsRNA agent is capable of reducing mRNA expression of a 4R tau isoform by at least about 50%, 55%, 60%, 65%, 70%, or 75% in a cell expressing both 3R and 4R tau isoforms, while reducing mRNA expression of a 3R tau isoform by no more than about 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%.

[0015] In some embodiments, the dsRNA agent is capable of reducing mRNA expression of a 4R tau isoform by at least about 70% while reducing mRNA expression of a 3R tau isoform by no more than about 30% in a cell expressing both a 3R tau isoform and a 4R tau isoform.

[0016] In one embodiment, the sense strand, the antisense strand, or both the sense and antisense strands described herein are conjugated to one or more lipophilic moieties.

[0017] In one embodiment, the lipophilic moiety is conjugated to one or more internal positions in the double-stranded region of the dsRNA agent.

[0018] In one embodiment, the lipophilic moiety is conjugated via a linker or carrier.

[0019] In one embodiment, the lipophilicity of the lipophilic moiety, as measured by log Kow, is greater than zero.

[0020] In one embodiment, the hydrophobicity of the double-stranded RNA agent is greater than 0.2 as measured by the unbound fraction in a plasma protein binding assay of the double-stranded RNA agent.

[0021] In one embodiment, the plasma protein binding assay is an electrophoretic mobility shift assay using human serum albumin protein.

[0022] In some embodiments, the dsRNA agent includes at least one modified nucleotide.

[0023] In one embodiment, no more than five of the nucleotides in the sense strand and no more than five of the nucleotides in the antisense strand in a dsRNA agent of the present disclosure are unmodified nucleotides.

[0024] In one embodiment, all of the nucleotides in the sense strand and all of the nucleotides in the antisense strand in a dsRNA agent are modified nucleotides.

[0025] In some embodiments, at least one of the modified nucleotides of a dsRNA agent is a deoxynucleotide, 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 (hydroxly) modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide containing a non-natural base, a tetrahydropyran modified ...C-alkyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2'-amino modified nucleotide, a 2'-C-allyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2'-hydroxyl (hydroxly) modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide containing a non-natural base, a tetrahydropyran modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2' nucleotides, 1,5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides containing a 5'-phosphorothioate group, nucleotides containing a 5'-methylphosphonate group, nucleotides containing a 5' phosphate or a 5' phosphate mimic, nucleotides containing vinyl phosphonate, nucleotides containing adenosine-glycol nucleic acid (GNA), nucleotides containing thymidine-glycol nucleic acid (GNA) S-isomer, nucleotides containing 2-hydroxymethyl-tetrahydrofuran-5-phosphate, nucleotides containing 2'-deoxythymidine-3' phosphate, nucleotides containing 2'-deoxyguanosine-3'-phosphate, and terminal nucleotides linked to a cholesteryl derivative and a dodecanoic acid bisdecylamide group; and combinations thereof.

[0026] In one embodiment, the modified nucleotide of the dsRNA agent is selected from the group consisting of 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, 3' terminal deoxythymidine nucleotides (dT), locked nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, and nucleotides containing unnatural bases.

[0027] In one embodiment, the modified nucleotides of the dsRNA agent include a short sequence of 3' terminal deoxythymidine nucleotides (dT).

[0028] In one embodiment, modifications to nucleotides of a dsRNA agent are 2'-O-methyl, 3-RNA, and 2'-deoxy-2'-fluoro modifications.

[0029] In one embodiment, the modifications to the nucleotides of the dsRNA agent are selected from 2'-O-methyl, 2'-deoxy, GNA, and 2'-deoxy-2'-fluoro modifications.

[0030] In some embodiments, the dsRNA agent further comprises at least one phosphorothioate internucleotide linkage.

[0031] In one embodiment, the dsRNA agent includes 6 to 8 phosphorothioate internucleotide linkages.

[0032] In one embodiment, each strand of the dsRNA is 30 nucleotides or less in length.

[0033] In one embodiment, at least one strand of the dsRNA agent includes a 3' overhang of at least 1 nucleotide. In another embodiment, at least one strand of the dsRNA agent includes a 3' overhang of at least 2 nucleotides.

[0034] In some embodiments, the double-stranded region of a dsRNA agent can be 15 to 30 nucleotide pairs in length; 17 to 23 nucleotide pairs in length; 17 to 25 nucleotide pairs in length; 23 to 27 nucleotide pairs in length; 19 to 21 nucleotide pairs in length; or 21 to 23 nucleotide pairs in length.

[0035] In some embodiments, each strand of the dsRNA can have between 19 and 30 nucleotides; between 19 and 23 nucleotides; or between 21 and 23 nucleotides.

[0036] In one embodiment, one or more lipophilic moieties are conjugated to one or more internal positions on at least one chain, eg, via a linker or carrier.

[0037] In one embodiment, internal positions include all but the two most distal positions from each end of at least one strand.

[0038] In another embodiment, the internal positions include all but the last three positions from each end of at least one strand.

[0039] In one embodiment, the internal position excludes the cleavage site region of the sense strand.

[0040] In one embodiment, internal positions include all positions except positions 9-12 counting from the 5' end of the sense strand.

[0041] In another embodiment, internal positions include all positions except positions 11-13 counting from the 3' end of the sense strand.

[0042] In one embodiment, the internal position excludes the cleavage site region of the antisense strand.

[0043] In one embodiment, internal positions include all positions except positions 12-14 counting from the 5' end of the antisense strand.

[0044] In one embodiment, internal positions include all positions except positions 11-13 counting from the 3' end of the sense strand and positions 12-14 counting from the 5' end of the antisense strand.

[0045] In one embodiment, the one or more lipophilic moieties are conjugated to one or more internal positions selected from the group consisting of positions 4-8 and 13-18 in the sense strand and positions 6-10 and 15-18 in the antisense strand, counting from the 5' end of each strand.

[0046] In another embodiment, the one or more lipophilic moieties are conjugated to one or more internal positions selected from the group consisting of positions 5, 6, 7, 15, and 17 in the sense strand and positions 15 and 17 in the antisense strand, counting from the 5' end of each strand.

[0047] In one embodiment, the internal position in the double-stranded region excludes the cleavage site region of the sense strand.

[0048] In one embodiment, the sense strand is 21 nucleotides in length, the antisense strand is 23 nucleotides in length, and the lipophilic moiety is conjugated to position 21, position 20, position 15, position 1, position 7, position 6, or position 2 of the sense strand or position 16 of the antisense strand.

[0049] In one embodiment, the lipophilic moiety is conjugated to position 21, position 20, position 15, position 1, or position 7 of the sense strand.

[0050] In another embodiment, the lipophilic moiety is conjugated to position 21, 20, or 15 of the sense strand.

[0051] In yet another embodiment, the lipophilic moiety is conjugated to position 20 or 15 of the sense strand.

[0052] In one embodiment, the lipophilic moiety is conjugated to position 16 of the antisense strand.

[0053] In one embodiment, the lipophilic moiety is an aliphatic, alicyclic, or polyalicyclic compound.

[0054] In one embodiment, the lipophilic moiety is selected from the group consisting of a lipid, cholesterol, retinoic acid, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, a heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenoic acid, dimethoxytrityl, or phenoxazine.

[0055] In one embodiment, the lipophilic moiety contains a saturated or unsaturated C4-C30 hydrocarbon chain and an optional functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne.

[0056] In one embodiment, the lipophilic moiety contains a saturated or unsaturated C6 to C18 hydrocarbon chain.

[0057] In one embodiment, the lipophilic moiety contains a saturated or unsaturated C16 hydrocarbon chain.

[0058] In one embodiment, the saturated or unsaturated C16 hydrocarbon chain is conjugated to position 6, counting from the 5' end of the chain.

[0059] In one embodiment, modifications to the nucleotides of the dsRNA agent are selected from 2'-O-methyl, 2'-deoxy, 3-RNA, and 2'-deoxy-2'-fluoro modifications; and at least one nucleotide containing a lipophilic moiety (e.g., 2'-hexadecyloxy).

[0060] In one embodiment, modifications to nucleotides of the dsRNA agent are selected from 2'-O-methyl, 2'-deoxy, GNA, and 2'-deoxy-2'-fluoro modifications; and at least one nucleotide containing a lipophilic moiety (e.g., 2'-hexadecyloxy).

[0061] In one embodiment, the lipophilic moiety is conjugated via a carrier that replaces one or more nucleotides at an internal position or in the double-stranded region.

[0062] In one embodiment, the carrier is a cyclic group selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl; or an acyclic moiety based on a serinol or diethanolamine backbone.

[0063] In one embodiment, the lipophilic moiety is conjugated to the double-stranded iRNA agent via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide linkage, the product of a click reaction, or a carbamate.

[0064] In one embodiment, the lipophilic moiety is conjugated to a nucleobase, a sugar moiety, or an internucleoside linkage.

[0065] In one embodiment, the lipophilic moiety or targeting ligand is conjugated via a biological linker selected from the group consisting of DNA, RNA, disulfides, amides, and functionalized mono- or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.

[0066] In one embodiment, the 3' end of the sense strand is protected via an end cap that is an amine-bearing cyclic group selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl.

[0067] In one embodiment, the dsRNA agent further comprises a targeting ligand that targets a neuronal cell.

[0068] In one embodiment, the dsRNA agent further includes a terminal chiral modification that occurs at the first internucleotide linkage at the 3'-end of the antisense strand, with the linking phosphorus atom in the Sp configuration, a terminal chiral modification that occurs at the first internucleotide linkage at the 5'-end of the antisense strand, with the linking phosphorus atom in the Rp configuration, and a terminal chiral modification that occurs at the first internucleotide linkage at the 5'-end of the sense strand, with the linking phosphorus atom in either the Rp or Sp configuration.

[0069] In another embodiment, the dsRNA agent further includes a terminal chiral modification occurring at the first and second internucleotide linkages at the 3'-end of the antisense strand, with the linking phosphorus atom in the Sp configuration, a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the antisense strand, with the linking phosphorus atom in the Rp configuration, and a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the sense strand, with the linking phosphorus atom in either the Rp or Sp configuration.

[0070] In yet another embodiment, the dsRNA agent further includes a terminal chiral modification occurring at the first, second, and third internucleotide linkages at the 3'-end of the antisense strand, with the linking phosphorus atom in the Sp configuration, a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the antisense strand, with the linking phosphorus atom in the Rp configuration, and a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the sense strand, with the linking phosphorus atom in either the Rp or Sp configuration.

[0071] In another embodiment, the dsRNA agent further includes a terminal chiral modification occurring at the first and second internucleotide linkages at the 3'-end of the antisense strand having the linking phosphorus atom in the Sp configuration, a terminal chiral modification occurring at the third internucleotide linkage at the 3'-end of the antisense strand having the linking phosphorus atom in the Rp configuration, a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the antisense strand having the linking phosphorus atom in the Rp configuration, and a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the sense strand having the linking phosphorus atom in either the Rp or Sp configuration.

[0072] In another embodiment, the dsRNA agent further includes a terminal chiral modification occurring at the first and second internucleotide linkages at the 3'-end of the antisense strand, with the linking phosphorus atom in the Sp configuration, a terminal chiral modification occurring at the first and second internucleotide linkages at the 5'-end of the antisense strand, with the linking phosphorus atom in the Rp configuration, and a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the sense strand, with the linking phosphorus atom in either the Rp or Sp configuration.

[0073] In one embodiment, the dsRNA agent further comprises a phosphate or a phosphate mimetic at the 5'-end of the antisense strand.

[0074] In one embodiment, the phosphate mimic is 5'-vinylphosphonate (VP).

[0075] In one embodiment, the base pair at one position at the 5' end of the antisense strand of the duplex is an AU base pair.

[0076] In one embodiment, the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

[0077] The present disclosure also provides cells and pharmaceutical compositions comprising the dsRNA agents of the disclosure and lipid formulations.

[0078] The present disclosure also provides a pharmaceutical composition for inhibiting expression of a gene encoding 4R tau, comprising a dsRNA agent of the present disclosure.

[0079] The present disclosure also provides a pharmaceutical composition for reducing aberrant expression (eg, overexpression) of an exon 10-containing MAPT transcript, comprising a dsRNA agent of the present disclosure.

[0080] In one embodiment, the dsRNA agent is in an unbuffered solution, such as saline or water.

[0081] In another embodiment, the dsRNA agent is in a buffer solution, eg, a buffer solution comprising acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof; or phosphate buffered saline (PBS).

[0082] In one aspect, the present disclosure provides a method of inhibiting or reducing expression of 4R tau in a cell, the method comprising contacting the cell with a dsRNA agent of this disclosure or a pharmaceutical composition of this disclosure, thereby inhibiting expression of 4R tau in the cell.

[0083] In another aspect, the present disclosure provides a method for reducing aberrant expression (e.g., overexpression) of an exon 10-containing MAPT transcript in a cell, the method comprising contacting the cell with a dsRNA agent of this disclosure or a pharmaceutical composition of this disclosure, thereby degrading the exon 10-containing MAPT transcript in the cell.

[0084] In one embodiment, the cell is in a subject.

[0085] In one embodiment, the subject is a human.

[0086] In one embodiment, the subject has a 4R tau-associated disorder.

[0087] In one embodiment, the subject has a 4R tau-associated disorder that is a neurodegenerative disorder.

[0088] In one embodiment, the subject's neurodegenerative disorder is associated with an abnormality in the protein 4R tau, which is encoded by the MAPT gene.

[0089] In one embodiment, abnormalities in the protein 4R tau, encoded by the MAPT gene, cause aggregation of 4R tau in the brain of a subject.

[0090] In one embodiment, the neurodegenerative disorder is a familial disorder.

[0091] In one embodiment, the neurodegenerative disorder is a sporadic disorder.

[0092] In one embodiment, the 4R tau-related disorder is progressive supranuclear palsy (PSP), progressive supranuclear palsy-Richardson syndrome (PSP-RS), corticobasal ganglia syndrome (CBD), argyrophilic grain disease (AGD), frontotemporal lobar degeneration due to tau (FTLD-tau), FTLD-tau due to MAPT mutations (e.g., mutations selected from N279K, L284L, DN296, N296N, N296H, P301L P301S, S305N, and S305S), multisystem tauopathy with presenile dementia (MSTD), globuloglial tauopathy (GGT), Parkinsonism resembling Parkinson's disease, non-fluent variant primary progressive aphasia (nfvPPA), behavioral variant frontotemporal dementia (bvFTD), primary akinesia with freezing of gait and 4R tauopathy selected from the group consisting of primary gait freezing (PAGF), primary lateral sclerosis (PLS), familial multisystem tauopathy with presenile dementia (FMSTD), familial progressive subcortical gliosis (familial pSG), pallido-pontine-nigral degeneration (pPND), familial frontotemporal dementia (e.g., FTD-Kumamoto), and N279K tauopathy.

[0093] In one embodiment, the 4R tau-associated disorder is a 4R tauopathy that is rapidly progressive Alzheimer's disease (rpAD). In one embodiment, the 4R tau-associated disorder is a 4R tauopathy that is sporadic Alzheimer's disease (spAD).

[0094] In one embodiment, the 4R tau-related disorder is a 4R tauopathy that is progressive supranuclear palsy (PSP) or progressive supranuclear palsy-Richardson syndrome (PSP-RS).In another embodiment, the 4R tau-related disorder is a 4R tauopathy that is progressive supranuclear palsy (PSP).

[0095] In some embodiments, contacting a cell with a dsRNA agent inhibits the expression of 4R tau by at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% compared to control levels.In one embodiment, the dsRNA agent inhibits the expression of 4R tau by at least about 25%.

[0096] In some embodiments, inhibiting the expression of 4R tau reduces the 4R tau protein level in the serum of the subject by at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% compared to control level.In one embodiment, the dsRNA agent reduces the 4R tau protein level in the serum of the subject by at least about 25%.

[0097] In one aspect, the present disclosure provides a method of treating a subject having a disorder that would benefit from reduced 4R tau expression, comprising administering a therapeutically effective amount of a dsRNA agent of the disclosure or a pharmaceutical composition of the disclosure to the subject, thereby treating the subject having a disorder that would benefit from reduced 4R tau expression.

[0098] In another aspect, the present disclosure provides a method for preventing at least one symptom in a subject having a disorder that would benefit from reduced 4R tau expression, the method comprising administering to the subject a prophylactically effective amount of a dsRNA agent of the disclosure or a pharmaceutical composition of the disclosure, thereby preventing at least one symptom in the subject having a disorder that would benefit from reduced 4R tau expression.

[0099] In one embodiment, the disorder is a 4R tau-associated disorder.

[0100] In one embodiment, the disorder is associated with an abnormality in the protein 4R tau, which is encoded by the MAPT gene.

[0101] In one embodiment, abnormalities in the protein 4R tau, encoded by the MAPT gene, cause aggregation of 4R tau in the brain of a subject.

[0102] In one embodiment, the 4R tau-associated disorder is progressive supranuclear palsy (PSP), progressive supranuclear palsy-Richardson syndrome (PSP-RS), corticobasal ganglia syndrome (CBD), argyrophilic grain disease (AGD), frontotemporal lobar degeneration due to tau (FTLD-tau), FTLD-tau due to MAPT mutations (e.g., N279K, L284L, DN296, N296N, N296H, P301L, and N279K tauopathy.

[0103] In certain embodiments, the 4R tau-associated disorder is a 4R tauopathy, e.g., selected from the group consisting of progressive supranuclear palsy (PSP), corticobasal syndrome (CBD), argyrophilic grain disease (AGD), multisystem tauopathy with presenile dementia (MSTD), glioglobular tauopathy (GGT), sporadic (spAD) Alzheimer's disease, and rapidly progressive (rpAD) Alzheimer's disease.

[0104] In one embodiment, the subject is a human.

[0105] In one embodiment, administration of a dsRNA agent of this disclosure, or a pharmaceutical composition of this disclosure, results in a decrease in the aggregation of 4R tau in the brain of the subject.

[0106] In one embodiment, administration of the agent to the subject results in a decrease in 4R tau aggregation.

[0107] In one embodiment, the dsRNA agent is administered to the subject at a dose of about 0.01 mg / kg to about 50 mg / kg.

[0108] In another embodiment, the dsRNA agent is administered to the subject intrathecally (IT) or intracerebroventricularly (ICV).

[0109] In yet another embodiment, the dsRNA agent is administered to the subject intracisternally. Non-limiting exemplary intracisternal administration includes injection into the cisterna magna (cerebellomedullary cistern) via suboccipital puncture.

[0110] In one embodiment, the method of the present disclosure further comprises determining the level of 4R tau in a sample from the subject.

[0111] In one embodiment, the level of 4R tau in a subject sample is the level of 4R tau protein in a blood, serum, or cerebrospinal fluid sample.

[0112] In one embodiment, the methods of the present disclosure further comprise administering to the subject an additional therapeutic agent.

[0113] In one aspect, the present disclosure provides a kit comprising a dsRNA agent of this disclosure or a pharmaceutical composition of this disclosure.

[0114] In another aspect, the present disclosure provides a vial containing a dsRNA agent of this disclosure or a pharmaceutical composition of this disclosure.

[0115] In yet another aspect, the present disclosure provides a syringe comprising a dsRNA agent of this disclosure or a pharmaceutical composition of this disclosure.

[0116] In another aspect, the present disclosure provides an intrathecal pump comprising a dsRNA agent of this disclosure, or a pharmaceutical composition of this disclosure. [Brief explanation of the drawings]

[0117] [Figure 1] FIG. 1 shows the % RNA knockdown as measured by 4R tau and mCherry TaqMan assays for 65 candidate 4R tau siRNAs. [Figure 2] Figure 2 shows the relative 4R tau and mCherry expression for 13 4R-preferring siRNAs (1 nM and 0.1 nM). Lipofectamine was used as a negative control. Two total tau siRNAs were also used as controls. [Figure 3] Figure 3 shows TaqMan qPCR data for the expression levels of 4R tau and mCherry (a surrogate for 3R tau) after treatment of HEK293-2N4R-YFP+2N3R-mCherry dual reporter cells with 65 different GalNAc-conjugated siRNAs (1 nM) generated by walking through exon 10 of the MAPT gene. Lipofectamine was used as a negative control. Total tau siRNA was also used as a control. [Figure 4] Figure 4 shows the locations within exon 10 of the MAPT gene where 13 4R-preferential siRNAs bind. The bottom panel illustrates the sequence homology of the R2 domain with other repeat domains (R1, R3, and R4), where asterisks indicate nucleotide homology, and the boxed regions are identified hotspots where 13 siRNAs appear to have preferential activity against 4R tau. [Figure 5] Figure 5 shows a summary of IC50 (potency) and knockdown (%) at 100 pM concentration of siRNA. The dose of siRNA treatment was an 8-point dose curve, ranging from 0.01 pM to 10 nM depending on the experiment. The table is sorted by the 4R tau column. [Figure 6]Figures 6A-6B show IC50 graphs for the top four siRNAs (AD-1423291.2, AD-1423283.2, AD-1423294.2, and AD-1423295.2) selected for in vivo testing. siRNAs were tested at eight different concentrations in HEK293 dual reporter cells, HEK293-2N3R-mCherry-only cells, and HEK293-2N4R-YFP-only cells. [Figure 7] Figure 7 shows confirmation of 4R-preferential knockdown in duplexes using protein dot blotting. The left panel shows confirmation of the specificity of a commercially available total tau antibody (mouse monoclonal; clone Tau12), a 4R tau antibody (mouse monoclonal, clone 7D12.1), and a 3R tau antibody (mouse monoclonal, clone 8E6\C11) by blotting with recombinant 1N3R or 1N4R protein. The right panel shows the use of the same antibodies in HEK293 dual reporter cells treated with vehicle control or siRNA (AD-1091966 = total tau-reducing siRNA; AD-1423291.2, AD-1423283.2, and AD-1423294.2 = 4R-preferential siRNA; AD-1423295.2 = mixed-selectivity siRNA). The experimental setup is shown at the top. [Figure 8] Figure 8 shows confirmation of 4R-preferential knockdown with siRNA duplexes using a protein ELISA-type assay. In panel A, total soluble tau was measured using a commercially available Total Tau ALPHALISA (Perkin Elmer, catalog no. AL271C) according to the manufacturer's instructions. In panel B, 4R tau protein was measured using a PathScan ELISA kit from Cell Signaling Technologies (catalog no. 29443). The experimental setup is shown at the top. [Figure 9]Figure 9 shows in vivo validation of tau knockdown using 4R tau siRNA. The table shows the C16-conjugated duplexes for in vivo administration used in the RT-qPCR assay, the corresponding GalNAc-conjugated duplexes for in vitro experiments, and the number of mouse samples. The graph shows RT-qPCR data for total tau and 4R tau expression levels in RNA extracted from the brains of mice treated with 4R tau siRNA. Values ​​are graphed as relative expression (½ΔΔCt) to the untreated group, normalized to mouse GAPDH. [Figure 10] FIG. 10 shows dose-response graphs for the three TaqMan assays performed (mCherry, YFP, 4R tau) in a dual reporter cell line (HEK293-2N4R-YFP+2N3R-mCherry). [Figure 11] FIG. 11 shows dose-response graphs in a 3R-only reporter cell line (HEK293-2N3R-mCherry) for the three TaqMan assays performed (mCherry, YFP, 4R tau). [Figure 12] FIG. 12 shows dose-response graphs in a 4R-only reporter cell line (HEK293-2N4R-YFP) for the three TaqMan assays performed (mCherry, YFP, 4R tau). DETAILED DESCRIPTION OF THE INVENTION

[0118] The present disclosure provides RNAi compositions targeting the "hotspot" region of exon 10 of the MAPT gene (located at nucleotides 1007-1053 of NM_005910.6), which executes RNA-induced silencing complex (RISC)-mediated cleavage of the 4R tau RNA transcript of the MAPT gene. The MAPT gene can be located within a cell, e.g., within a subject, such as a human. Use of these iRNAs allows for targeted degradation of the 4R tau mRNA of the corresponding gene (MAPT gene) in a mammal.

[0119] The iRNAs of the present disclosure were designed to target hotspot regions of exon 10 of the MAPT gene, for example, with or without nucleotide modifications. The iRNAs of the present disclosure inhibit the expression of 4R tau isoforms by at least about 25% and reduce the levels of sense- and antisense-containing aggregates. Without intending to be limited by theory, it is believed that the combination or subcombination of the aforementioned properties with specific target sites or specific modifications of these iRNAs confers improved efficiency, stability, potency, durability, and safety to the iRNAs of the present disclosure.

[0120] Therefore, the present disclosure also provides the method of using the RNAi composition of the present disclosure for inhibiting the expression of 4R tau isoforms or for treating the subject with disorders that inhibiting or reducing the expression of 4R tau isoforms will be beneficial, such as 4R tau-related diseases, for example, 4R tauopathy, for example, PSP, CBD, AGD, MSTD, GGT, spAD or rpAD.In certain embodiments, the present disclosure provides the method of using the RNAi composition of the present disclosure for preferentially inhibiting the expression of 4R tau isoforms over inhibiting 3R tau isoforms.

[0121] RNAi agents of the disclosure can be about 30 nucleotides in length or less, e.g., 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-25 ...6, 19-25, 19-26, 19-25, 19-26, 19-25, 19-26, 19-25, 19 The RNAi agent of the present disclosure comprises an RNA strand (antisense strand) having a region that is 9-23, 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 a hotspot within exon 10 of an mRNA transcript of the MAPT gene. In certain embodiments, the RNAi agent of the present disclosure comprises an RNA strand (antisense strand) having a region that is about 21-23 nucleotides in length, which region is substantially complementary to a hotspot within exon 10 of an mRNA transcript of the MAPT gene.

[0122] In certain embodiments, the RNAi agents of the present disclosure comprise an RNA strand (antisense strand) having a region of at least 19 contiguous nucleotides that is substantially complementary to a hotspot within exon 10 of the mRNA transcript of the MAPT gene, and can comprise a longer length, e.g., up to 66 nucleotides, e.g., 36-66, 26-36, 25-36, 31-60, 22-43, 27-53 nucleotides in length. These RNAi agents with longer antisense strand lengths preferably comprise a second RNA strand (sense strand) of 20-60 nucleotides in length, in which case the sense and antisense strands form a duplex of 18-30 contiguous nucleotides.

[0123] The use of these RNAi agents allows the targeted degradation and / or inhibition of the mRNA of MAPT gene in mammals.Therefore, the method and composition comprising these RNAi agents are useful for treating the subject who will benefit from the reduction of 4R tau level or activity, for example, the subject who has 4R tau-related disease, for example, 4R tauopathy, for example, PSP, CBD, AGD, MSTD, GGT, spAD or rpAD.In certain embodiments, the method and composition comprising these RNAi agents are useful for treating the subject who will benefit from the preferential reduction of 4R tau level or activity over 3R tau.

[0124] The detailed description below discloses methods for making and using compositions containing RNAi agents to inhibit expression of the 4R tau isoform of the MAPT gene, as well as compositions and methods for treating subjects with diseases and disorders in which inhibiting or reducing expression of the gene would be beneficial.

[0125] I. Definition In order that this disclosure 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 this disclosure.

[0126] 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.

[0127] The term "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to." The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless context clearly indicates otherwise.

[0128] 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.

[0129] The term "at least" before a number or a series of numbers, if clear from the context, is understood to include the number adjacent to the term "at least" and all subsequent numbers or integers that may be logically included. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 18 nucleotides of a 21-nucleotide nucleic acid molecule" means that 18, 19, 20, or 21 nucleotides have the specified property. When the term "at least" is present before a series of numbers or a range, it is understood that "at least" can modify each of the numbers and ranges in the series.

[0130] As used herein, "less than" or "less than" shall be understood as 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.

[0131] As used herein, the term "at least about," when referring to a measurable value, e.g., a parameter, amount, etc., is meant to encompass a deviation of + / - 20%, preferably + / - 10%, more preferably + / - 5%, and even more preferably + / - 1% from the specified value, provided that such deviation is appropriate for functioning in the disclosed disclosure. For example, "at least about 25%" inhibition of expression of the MAPT gene means that inhibition of expression of the MAPT gene can be measured to any value within + / - 20% of the specified 25%, i.e., 20%, 30%, or any intermediate value between 20% and 30%.

[0132] As used herein, "control level" refers to the expression level of a gene, or the expression level of an RNA molecule, or the expression level of one or more proteins or protein subunits in an unregulated cell, tissue, or system that is the same as the cell, tissue, or system in which the RNAi agent described herein is expressed.The cell, tissue, or system in which the RNAi agent is expressed has at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, or more expression of the gene, RNA, and / or protein described herein compared to that observed in the absence of the RNAi agent.The percentage and / or fold difference relative to the control level can be, for example, [Expression with RNAi agent - Expression without RNAi agent] Difference (%)=-----------------------×100 Expression without RNAi agent It can be calculated as follows.

[0133] As used herein, a method of detection can include determining that the amount of analyte present is below the detection level of the method.

[0134] 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.

[0135] If the chemical structure and chemical name do not agree, the chemical structure takes precedence.

[0136] The term "MAPT" gene, also known as "DDPAC," "FTDP-17," "MAPTL," "MSTD," "MTBT1," "MTBT2," "PPND," "PPP1R103," "TAU," and "microtubule-associated protein tau," refers to the gene encoding the protein called microtubule-associated protein tau (MAPT).

[0137] MAPT mRNA is expressed throughout the body, primarily in the central nervous system (i.e., brain and spinal cord) and peripheral nervous system. Wild-type tau is involved in stabilizing microtubules in neuronal axons, regulating axonal transport and microtubule dynamics, maintaining dendritic spines, and contributing to genomic DNA integrity.

[0138] Tauopathies are a heterogeneous class of progressive neurodegenerative disorders pathologically characterized by the presence of tau aggregates in the brain. Intracellular and extracellular neuronal tau aggregates cause microtubule dissociation and axonal degeneration, impaired synaptic vesicle release, and prion-like interneuronal propagation of tau aggregates, termed "seeding."

[0139] Tauopathies exhibit a diverse progression of motor, cognitive, and behavioral disorders. Examples of tauopathies include, but are not limited to, Alzheimer's disease (the most common form of presenile dementia, which begins with selective memory impairment and is associated with degeneration of the frontal, temporal (including the hippocampus), and parietal lobes of the brain); frontotemporal dementia (FTD) (the second most common form of presenile dementia, which is associated with atrophy of neurons in the frontal and temporal lobes and presents with a wide range of behavioral, language, and motor disorders); and progressive supranuclear palsy (PSP) (a brainstem and basal ganglia degeneration presenting with gaze disturbances, extrapyramidal symptoms (parkinsonism symptoms including limb apraxia, akinesia / bradykinesia, rigidity, and abnormal muscle tone), and cognitive impairment; which affects approximately 20,000 people in the United States).

[0140] Further FTDs include behavioral frontotemporal dementia (bvFTD) (pathologically associated with progressive atrophy in the prefrontal and anterior temporal lobes and clinically associated with complex thinking, personality, and behavioral changes, affecting approximately 30,000 people in the United States); semantic primary progressive aphasia (PPA-S) (frontal and temporal lobe degeneration associated with difficulty in understanding words and difficulty naming); non-fluent primary progressive aphasia (nfvPPA) (involving degeneration of the left posterior frontal and insular lobes, presenting with agrammatism and inability to understand complex sentences, affecting approximately 1,000 people in the United States); and logopenic primary progressive aphasia (PPA-L) (left posterior / superior temporal and medial parietal lobes associated with difficulty in word repetition and frequent interruptions). These include, but are not limited to, frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) (pathologically associated with frontal and temporal lobe degeneration and clinically associated with speech and movement disorders); Pick's disease (PiD) (frontal and temporal lobe degeneration associated with language and thinking difficulties and behavioral changes); FTD with motor neuron disease (involving cortical and motor neuron degeneration); and corticobasal syndrome (CBS) [degeneration of the posterior frontal and temporal lobes and basal ganglia (i.e., corticobasal degeneration (CBD)) presenting with extrapyramidal symptoms (similar to those in Parkinson's disease and PSP) and cognitive impairment; affecting approximately 2,000 people in the United States]. MAPT mutations have been reported in approximately 10% of patients with bvFTD, nfvPPA, CBS, and PSP, respectively. MAPT is a major component of neurofibrillary tangles in neuronal cytoplasm, a hallmark of Alzheimer's disease. MAPT aggregates and deposits have also been observed in approximately 50% of brains of patients with Parkinson's disease.Tau involvement has been shown in the pathogenesis of other diseases, including, but not limited to, argyrophilic grain disease (AGD), multisystem tauopathy with presenile dementia (MSTD), white matter tauopathy with globular glial inclusions (FTLD with GGI), FTLD with MAPT mutations, neurofibrillary tangle (NFT) dementia, FTD with motor neuron disease, amyotrophic lateral sclerosis (ALS), postencephalitic parkinsonism, Niemann-Pick disease, Huntington's disease, myotonic dystrophy type 1, and Down syndrome (DS).

[0141] The MAPT gene consists of 16 exons (E1-E16). Alternative mRNA splicing of E2, E3, and E10 generates six tau isoforms (352-441 amino acids). E1, E4, E5, E7, E9, E11, E12, and E13 are constitutively spliced ​​exons. E6 and E8 are not transcribed in the human brain. E4a is expressed only in the peripheral nervous system. E0 (part of the promoter) and E14 are non-coding exons.

[0142] Pathogenic variants in MAPT are found in approximately 10% of patients with primary tauopathies. These variants are primarily missense and located in exons 9–13 (microtubule-binding domain), often affecting alternative splicing of exon 10. Examples of mutations in the coding region include R5H and R5L in E1 of the MAPT gene; K257T, I260V, L266V, G272V, and G273R in E9; and N279K, K280del, L284L, ΔN296, N296N, N296H, ΔN298, P301L, P301S, P301T, G303V, and G304S in E10. These include S305I, S305N, and S305S; L315R, K317M, S320F, and P332S in E11; G335S, G335V, Q336R, V337M, E342V, S352L, S356T, V363I, P364S, G366R, and K369I in E12; and G389R, R406W, and T427M in E13. MAPT (tau) null (- / -) individuals are unlikely to survive. MAPT heterozygotes (+ / -) have unclear or unknown phenotypes. MAPT overexpression (+ / + / +) individuals are associated with early-onset dementia, FTD, PSP, and CBD.

[0143] Each of the six isoforms of MAPT (tau) protein contains three or four repeating segments (R1, R2, R3, and R4) in its microtubule-binding domain. Each repeat is 31 or 32 amino acids long. Splicing of E9, E10, E11, and E12 generates the repeating segments R1, R2, R3, and R4, respectively, in the microtubule-binding domain of MAPT. The three MAPT (tau) isoforms with E10 spliced ​​in ("4R tau isoforms") contain four repeating segments (4R), whereas the other three MAPT isoforms with E10 spliced ​​out ("3R tau isoforms") contain three repeating segments (3R).

[0144] Translation of E2 and E3 produces the N1 and N2 segments, respectively. Alternative splicing of E2 and E3 produces tau isoforms 0N (E2 and E3 are spliced ​​out, resulting in no N segment), 1N (E2 is spliced ​​in and E3 is spliced ​​out, resulting in one N segment), and 2N (E2 and E3 are spliced ​​in, resulting in two N segments). Thus, the six MAPT (tau) isoforms resulting from alternative splicing are 2N4R, 1N4R, 0N4R, 2N3R, 1N3R, and 0N3R.

[0145] In healthy individuals, the 3R and 4R MAPT transcript isoforms exist in a 1:1 ratio. The 3R / 4R isoform ratio is distorted in pathological conditions, and this ratio predicts the type of tau aggregates. For example, certain intronic and coding region mutations within exon 10 of MAPT (N279K, L284L, DN296, N296N, N296H, S305N, and S305S) can increase the splicing of exon 10, thereby changing the ratio between the 3-repeat and 4-repeat isoforms and resulting in the overproduction of 4-repeat tau.

[0146] Assembly of 4-repeat tau into fibrils is characteristic of PSP, CBD, argyrophilic grain disease (AGD), multisystem tauopathy with presenile dementia (MSTD), and white matter tauopathy with globular glial inclusions (FTD with GGI or "globular glial tauopathy" (GGT)), which belong to the FTD spectrum (4R tauopathies).

[0147] In contrast, in Pick's disease, three-repeat tau predominates in neuronal inclusions (3R tauopathy).

[0148] In Alzheimer's disease and other neurodegenerative diseases with neurofibrillary tangles (NFT dementia), both 3-repeat and 4-repeat tau isoforms can constitute neurofibrillary lesions (3 / 4R tauopathy). However, in certain Alzheimer's disease pathologies, the 4-repeat tau isoform may be the predominant isoform within the neurofibrillary lesions. FTLD with MAPT mutations can be 3R, 4R, or 3 / 4R tauopathy.

[0149] FTD with motor neuron disease is associated with FTLD-TDP43 and FTLD-FUS pathology, which is associated with genetic mutations in C9ORF72, FUS, TARDBP, and VCP.

[0150] bvFTD is associated with FTLD-Tau(3R) and FTLD-TDP43 pathology. 10 percent of cases involve MAPT mutations. It is associated with gene mutations in C9ORF72, GRN, and VCP.

[0151] PPA-S can be sporadic. It is associated with FTLD-TDP43 pathology.

[0152] nfvPPA has been associated with, in order of significance, the pathologies of FTLD-Tau(4R), Alzheimer's disease, and FTLD-TDP43. Ten percent of cases involve mutations in MAPT. nfvPPA is further associated with mutations in GRN.

[0153] PPA-L can be sporadic. It is associated, in order of significance, with the pathology of Alzheimer's disease and FTLD-Tau.

[0154] CBS is associated with, in order of significance, the pathology of FTLD-Tau(4R) and Alzheimer's disease. Ten percent of cases are associated with mutations in MAPT. The remaining cases may be sporadic.

[0155] PSP is involved in the pathology of FTLD-Tau(4R). Ten percent of cases are associated with MAPT mutations. The remainder of cases may be sporadic.

[0156] Tauopathies typically begin between the ages of 60 and 80, affecting life expectancy by 6 to 10 years. Tauopathies are phenotypically heterogeneous and involve a variety of motor, cognitive, and behavioral disorders. The progression of motor symptoms, in particular, is variable.

[0157] Currently, there are no approved disease-modifying therapies for tauopathies. Available treatments are only aimed at alleviating symptoms and improving patients' quality of life as the disease progresses. Drugs in preclinical or clinical development include active and passive immunotherapies; inhibitors of O-deglycosylation, aggregation, kinase, acetylation, caspase, or tau expression; phosphatase activators; microtubule stabilizers; and modulators of autophagy or proteosomal degradation. Biomarkers and tests used in clinical trials to assess tauopathies include tau protein phosphorylated at threonine 181 (pTau), total tau protein (tTau), neurofilament light chain (NfL), volumetric MRI (vMRI), and 18 F diagnostic PET label (e.g., 18 F-flortaucipir (AV-1451, TAUVID™).

[0158] Exemplary nucleotide and amino acid sequences of MAPT are found, for example, in GenBank Accession No. NM_016841.4 [Homo sapiens MAPT variant 4, SEQ ID NO: 1, reverse complement, SEQ ID NO: 2]; GenBank Accession No. NM_005910 (Human MAPT variant 2, SEQ ID NO: 3, reverse complement, SEQ ID NO: 4); GenBank Accession No. NM_001038609.2 [Mus musculus MAPT, SEQ ID NO: 5; reverse complement, SEQ ID NO: 6]; GenBank Accession No.: XM_005584540.1 [Macaca fascicularis MAPT variant X13, SEQ ID NO: 7, reverse complement, SEQ ID NO: 8]; GenBank Accession No.: XM_008768277.2 [Rat (Rattus norvegicus MAPT, variant X7, SEQ ID NO:9, reverse complement SEQ ID NO:10], and GenBank Accession No: XM_005624183.3 [gray wolf (Canis lupus) MAPT variant X23, SEQ ID NO:11, reverse complement SEQ ID NO:12].

[0159] The nucleotide sequence of the genomic region of a human chromosome containing the MAPT gene can be found, for example, in Genome Reference Consortium Human Build 38 (also referred to as human genome build 38 or GRCh38), available at GenBank. The nucleotide sequence of the genomic region of human chromosome 17 containing the MAPT gene can also be found, for example, in GenBank accession number NC_000017.11, which corresponds to nucleotides 45894382 to 46028334 of human chromosome 17. The nucleotide sequence of the human MAPT gene can be found, for example, in GenBank accession number NG_007398.2.

[0160] Further examples of MAPT sequences can be found in publicly available databases such as GenBank, OMIM, and UniProt.

[0161] Additional information regarding MAPT can be found, for example, at the NCBI website, which references gene 100128977. The term MAPT, as used herein, also refers to variations of the MAPT gene, including, for example, variants provided in clinical variant databases, for example, variants provided at the NCBI clinical variant website, which references the term mapt.

[0162] The entire contents of each of the foregoing GenBank Accession Numbers and Gene Database Numbers are incorporated herein by reference as of the filing date of this application.

[0163] As used herein, "target sequence" refers to a contiguous portion of a nucleotide sequence, such as exon 10 (SEQ ID NO: 13) within a 4R tau mRNA molecule formed upon transcription of the MAPT gene, e.g., an mRNA that is a product of RNA processing of a primary transcript (e.g., a MAPT mRNA resulting from alternative splicing). 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 MAPT gene.

[0164] The target sequence is about 15-30 nucleotides in length. For example, the target sequence can be about 15-30 nucleotides in length, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 1 The target sequence may be 9-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. In certain embodiments, the target sequence is 19-23 nucleotides in length, and, where appropriate, 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.

[0165] 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.

[0166] " G ", " C ", " A ", " T " and " U " each generally represent the nucleotide that contains guanine, cytosine, adenine, thymidine and uracil as a base, respectively, in relation to modified or unmodified nucleotide.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 2).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 this disclosure.

[0167] The terms " iRNA ", " RNAi agent ", " iRNA agent ", " RNA interference agent " are used interchangeably herein and refer to the agent that contains the RNA as defined herein and mediates the targeted cleavage in RNA transcription via the RNA-induced silencing complex (RISC) pathway.RNA interference (RNAi) is the process that directs the sequence-specific degradation of mRNA.RNAi modulates, for example, inhibits the expression of 4R tau in cells, for example, in cells of a subject, for example, a mammalian subject.

[0168] In one embodiment, the RNAi agent of the present disclosure comprises a single-stranded RNAi that interacts with a target RNA sequence, such as a 4R tau target mRNA sequence, to direct 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 double-stranded small interfering RNAs (siRNAs) containing a sense strand and an antisense strand by a type III endonuclease known as Dicer [Sharp et al. (2001) Genes Dev. 15:485]. Dicer, an RNase III-like enzyme, processes this dsRNA into 19-23 base pair small interfering RNAs with a characteristic two-base 3' overhang [Bernstein, et al., (2001) Nature 409:363]. These siRNAs are then introduced into the 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]. Upon binding to the appropriate target mRNA, one or more endonucleases in RISC cleave the target to induce silencing [Elbashir, et al., (2001) Genes Dev. 15:188]. Thus, in one aspect, the present disclosure relates to a single-stranded RNA (ssRNA) (the antisense strand of the siRNA duplex) that is produced in cells, promotes the formation of a RISC complex, and thereby silences the target gene, i.e., the MAPT gene. Therefore, the term "siRNA" is used herein to also mean the RNAi described above.

[0169] In another embodiment, the RNAi agent can be a single-stranded RNA introduced into a cell or organism to inhibit target mRNA. The single-stranded RNAi agent binds to the RISC endonuclease Argonaute 2 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 RNA is described in U.S. Patent No. 8,101,348 and Lima et al., (2012) Cell 150:883-894, the entire contents of each of which are incorporated herein by reference. Any of the antisense nucleotide sequences described herein can be used as 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.

[0170] In another embodiment, the "RNAi 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 RNAi agent," a "double-stranded RNA (dsRNA) molecule," a "dsRNA agent," or a "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 the target RNA, i.e., the MAPT gene. In some embodiments of the present disclosure, the double-stranded RNA (dsRNA) induces the degradation of the target RNA, for example, mRNA, by a post-transcriptional gene silencing mechanism, referred to herein as RNA interference or RNAi.

[0171] Generally, dsRNA molecules can comprise ribonucleotides, but 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, " RNAi agent " can comprise ribonucleotides with chemical modification; RNAi agent can comprise substantial modifications in multiple nucleotides.As used herein, the term " modified nucleotide " refers to the nucleotide that independently has modified sugar moiety, modified internucleotide linkage, or modified nucleobase.Therefore, the term modified nucleotide encompasses the substitution, addition, or removal of, for example, functional group or atom, etc., in internucleoside linkage, sugar moiety, or nucleobase.The modifications suitable for use in the agent of the present disclosure encompass all types of modifications disclosed herein or known in the art.Any such modifications used in siRNA type molecules are encompassed by " RNAi agent " for the purpose of this specification and claims.

[0172] In certain embodiments of the present disclosure, the inclusion of deoxynucleotides can be considered to constitute modified nucleotides when present within an RNAi agent.

[0173] The duplex region can be any length that allows for specific degradation of the desired target RNA by the RISC pathway, as well as about 15 to 36 base pairs in length, e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length, e.g., about 15 to 30, 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 31, 18 to 32, 18 to 33, 18 to 34, 18 to 35, 18 to 36, or 36 base pairs in length. 28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. In certain embodiments, the duplex region is 19-21 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.

[0174] The two strands forming the duplex structure can be different portions of a larger RNA molecule, or they can be separate RNA molecules. When the two strands are part of a larger molecule and are therefore 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 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23, or more unpaired nucleotides or nucleotides not 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.

[0175] The two substantially complementary strands of dsRNA are contained in separate RNA molecules, and these molecules can, but do not necessarily, be covalently linked. In certain embodiments, where the two strands are covalently linked between the 3' end of one strand and the 5' end of each other strand forming a duplex structure by means other than an uninterrupted chain of nucleotides, the connecting structure is called a "linker" (although certain other structures defined elsewhere herein may also be called "linkers"). The RNA strands can have the same or different numbers of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus all overhangs present in the duplex. In addition to the duplex structure, the RNAi can 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.

[0176] In one embodiment, an RNAi agent of the present disclosure is a dsRNA, each strand of which independently comprises 19-23 nucleotides that interact with a target RNA sequence, e.g., a MAPT target mRNA sequence, to induce cleavage of the target RNA.

[0177] In some embodiments, an iRNA of the present disclosure is a 24-30 nucleotide dsRNA that interacts with a target RNA sequence, for example, a MAPT-targeting mRNA sequence, to direct cleavage of the target RNA.

[0178] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that protrudes from the double-stranded structure of an RNAi agent, such as dsRNA.For example, when the 3'-end of one strand of dsRNA extends beyond the 5'-end of the other strand, and vice versa, there is a nucleotide overhang.DsRNA can comprise at least one nucleotide overhang; alternatively, the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more nucleotides.The nucleotide overhang can comprise or consist of nucleotide / nucleoside analogs, such as deoxynucleotides / nucleosides.The overhang can be on the sense strand, antisense strand, or any combination thereof.In addition, 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.

[0179] In one embodiment, the antisense strand of the dsRNA has a 1-10 nucleotide overhang at the 3' 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' 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 thiophosphates.

[0180] In certain embodiments, the antisense strand of dsRNA has 1 to 10 nucleotide overhangs at the 3' or 5' end, for example, 0 to 3, 1 to 3, 2 to 4, 2 to 5, 4 to 10, or 5 to 10 nucleotide overhangs, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide overhangs. In one embodiment, the sense strand of dsRNA has 1 to 10 nucleotide overhangs at the 3' or 5' end, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide overhangs. In another embodiment, one or more of the nucleotides in the overhang are replaced with nucleoside thiophosphates.

[0181] In certain embodiments, the overhang on the sense strand or the antisense strand can comprise an extended length of more than 10 nucleotides, e.g., 1 to 30 nucleotides, 2 to 30 nucleotides, 10 to 30 nucleotides, or 10 to 15 nucleotides. In certain embodiments, the extended overhang is on the sense strand of the duplex. In certain embodiments, the extended overhang is at the 3'-end of the sense strand of the duplex. In certain embodiments, the extended overhang is at the 5'-end of the sense strand of the duplex. In certain embodiments, the extended overhang is on the antisense strand of the duplex. In certain embodiments, the extended overhang is at the 3'-end of the antisense strand of the duplex. In certain embodiments, the extended overhang is at the 5'-end of the antisense strand of the duplex. In certain embodiments, one or more of the nucleotides in the overhang are replaced with a nucleoside thiophosphate. In certain embodiments, the overhang comprises a self-complementary portion that enables the overhang to form a stable hairpin structure under physiological conditions.

[0182] The term "blunt" or "blunt-ended" as used herein with respect to dsRNA means that there are no unpaired nucleotides or nucleotide analogs at a given end of the dsRNA, i.e., there are no nucleotide overhangs. One or both ends of the dsRNA can be blunt. When both ends of a dsRNA are blunt, the dsRNA is said to be blunt-ended. For clarity, a "blunt-ended" dsRNA is a dsRNA that is blunt at both ends, i.e., there are no nucleotide overhangs at either end of the molecule. In most cases, such a molecule will be double-stranded throughout its entire length.

[0183] The term "antisense strand" or "guide strand" refers to the strand of an RNAi agent, eg, a dsRNA, that includes a region that is substantially complementary to a target sequence, eg, MAPT mRNA.

[0184] As used herein, the term "region of complementarity" refers to the region on the antisense strand that is substantially complementary to a sequence, for example, a target sequence, for example, a MAPT nucleotide sequence, as defined herein.If the region of complementarity is not completely complementary to the target sequence, the mismatch can be in the internal region or terminal region of the molecule.Generally, the most tolerable mismatch is in the terminal region, for example, within 5, 4, 3, or 2 nucleotides of the 5'-end or 3'-end of the RNAi agent.In some embodiments, the double-stranded RNA agent of the present disclosure comprises nucleotide mismatches in the antisense strand.In some embodiments, the antisense strand of the double-stranded RNA agent of the present disclosure comprises four or fewer mismatches with the target mRNA, for example, the antisense strand comprises 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 disclosure 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, a double-stranded RNA agent of the present disclosure contains a nucleotide mismatch in the sense strand. In some embodiments, the sense strand of a double-stranded RNA agent of the present disclosure contains four or fewer mismatches with the antisense strand, e.g., 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.

[0185] 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 also 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, in the case of a 23-nucleotide RNAi agent, the strand complementary to a region of the MAPT gene 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 a 4R tau isoform. For example, Jackson et al. (Nat. Biotechnol. 2003;21:635-637) described an expression profiling study showing that the expression of a small set of genes with only 12-18 nt of sequence identity to a MAPK14 siRNA on the sense strand was downregulated with kinetics similar to MAPK14. Similarly, Lin et al. (Nucleic Acids Res. 2005;33(14):4527-4535) used qPCR and reporter assays to show that 7 nt of complementarity between the siRNA and the target was sufficient to trigger target mRNA degradation. It is important to consider the efficacy of mismatched RNAi agents to inhibit the expression of 4R tau isoforms, especially when the specific region of complementarity in the MAPK14 gene is known to have polymorphic sequence variation within the population.

[0186] "Substantially all of the nucleotides are modified," as used herein, means that most, but not all, are modified, including, for example, no more than 5, 4, 3, 2, or 1 unmodified nucleotides.

[0187] The term "sense strand" or "passenger strand," as used herein, refers to the strand of an RNAi agent that includes a region that is substantially complementary to a region of the antisense strand, as those terms are defined herein.

[0188] 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.

[0189] 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," including, but not limited to, 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, for example, "Molecular Cloning: A Laboratory Manual," Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press.) As used herein, "stringent conditions" or "stringent hybridization conditions" refer to conditions under which an antisense compound hybridizes to its target sequence but to a minimal number of other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances, and the "stringent conditions" that antisense compounds hybridize with target sequence are determined by the nature and composition of antisense compounds and the assay they are investigated.Other conditions, such as physiologically relevant conditions that can be encountered inside living organisms, can also be applied.Those skilled in the art will be able to determine the most suitable set of conditions for testing the complementarity of two sequences according to the final application of hybridized nucleotide.

[0190] The complementary sequence in RNAi agent, for example, in the dsRNA described herein, comprises the base pairing of the oligonucleotide or polynucleotide comprising the first nucleotide sequence with the oligonucleotide or polynucleotide comprising the second nucleotide sequence throughout the entire length of one or both nucleotide sequences.Such sequences can be referred to herein as " completely complementary " with respect to each other.However, when the first sequence is considered herein as " substantially complementary " with the second sequence, the two sequences can be completely complementary, or they can form one or more, but generally not more than 5, 4, 3 or 2 mismatched base pairs during hybridization, in the case of a double strand of up to 30 base pairs. In some embodiments, the "substantially complementary" sequence disclosed herein comprises a contiguous nucleotide sequence that is at least about 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 corresponding region of the target exon 10 MAPT sequence over its entire length.However, if two oligonucleotides are designed to form one or more single-stranded overhangs during hybridization, such overhangs are not considered mismatches when determining complementarity.For example, a dsRNA comprising one oligonucleotide of 21 nucleotides in length and another oligonucleotide of 23 nucleotides in length, where the longer oligonucleotide comprises a 21-nucleotide sequence that is completely complementary to the shorter oligonucleotide, can still be considered "completely complementary" for the purposes described herein.

[0191] "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 Hoogstein base pairing.

[0192] The terms "complementary," "fully complementary," and "substantially complementary" can be used herein in reference to base matching between the sense and antisense strands of a dsRNA or between the antisense strand of an RNAi agent and a target sequence, as understood in connection with their use.

[0193] As used herein, a polynucleotide that is "substantially complementary to at least a portion of" a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a continuous portion of an mRNA of interest (e.g., an mRNA encoding tau). For example, a polynucleotide is complementary to at least a portion of MAPT mRNA if the sequence is substantially complementary to an uninterrupted portion of an mRNA encoding tau.

[0194] Thus, in some embodiments, the antisense polynucleotides disclosed herein are fully complementary to the target MAPT sequence. In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to the target MAPT sequence and comprise 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%, or about 99% complementary to the equivalent region of the nucleotide sequence of the hotspot of exon 10 (located at nucleotides 1007-1053 of SEQ ID NO: 3) or a fragment thereof, over its entire length.

[0195] In some embodiments, the antisense polynucleotides disclosed herein are substantially complementary to a fragment of the target MAPT sequence, and include nucleotides 1009-1029, 1010-1030, 1011-1031, 1012-1032, 1013-1033, 1014-1034, 1015-1035, 1016-1036, 1017-1037, 1018-1038, 1019-1039, 1020-1040, 1021-1041, 1022-1042, 1023-1043, 1024-1044 of SEQ ID NO:3. , 1025-1045, 1026-1046, 1027-1047, 1028-1048, 1029-1049, 1030-1050, 1031-1051, 1032-1052, 1033-1053, 1014-1048, and 1022-1048. Ranges between the above-listed ranges are also contemplated as part of the present disclosure. In certain embodiments, the antisense polynucleotides disclosed herein are substantially complementary to a fragment of the target MAPT sequence, spanning its entire length, including nucleotides 1009-1029, 1016-1036, 1018-1038, 1020-1040, 1021-1041, 1022-1042, 1024-1044, 1027-1047, 1028-1048, 1029-1050, 1030-1031, 1032-1033, 1034-1035, 1036-1037, 1038-1049, 1040-1041, 1042-1043, 1044-1045, 1046-1047, 1048-1049, 1050-1051, 1052-1053, 1054-1055, 1056-1057, 1058-1059, 1060-1061, 1062-1063, 1064-1065, 1066-1067, 1068-1069, 1069-1070, 1071-1072, 1073-1074, 1075-1076, 1077-1078, 1079-1080, 1081-1082, 1083-1084, 1085-1086, 1087-1088, 1089-1090, 1091-1092, 1093-1094, 109 The fragment comprises a contiguous nucleotide sequence that is at least 80% complementary, e.g., 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 a fragment of SEQ ID NO: 3 selected from the group consisting of 1029-1049, 1030-1050, 1032-1052, 1033-1053, 1014-1048, and 1022-1048.

[0196] In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to the target MAPT sequence and comprise 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 Table 7 or 8, or a fragment of any one of the sense strand nucleotide sequences in Table 7 or 8, over its entire length.

[0197] In one embodiment, an RNAi agent of the present disclosure comprises a sense strand that is substantially complementary to an antisense polynucleotide that is complementary to a target MAPT sequence, wherein the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least about 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 nucleotides 1007-1053 of SEQ ID NO: 4, or an equivalent region of a fragment thereof, over its entire length. In some embodiments, an iRNA of the present disclosure comprises a sense strand that is substantially complementary to an antisense polynucleotide that is complementary to a target MAPT 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 to any one of the antisense strand nucleotide sequences in Table 7 or 8, or a fragment of any one of the antisense strand nucleotide sequences in Table 7 or 8, over its entire length.

[0198] In certain embodiments, the sense and antisense strands are duplexed as follows: AD-1423276, AD-1423277, AD-1423278, AD-1423279, AD-1423280, AD-1423281, AD-1423282, AD-1423283, AD-1423284, AD-1423285, AD-1423286, AD-1423287 , AD-1423288, AD-1423289, AD-1423290, AD-1423291, AD-1423292, AD-1423293, AD-1423294, AD-1423295, AD-1423296, AD-1423297, AD-1423298, AD-1423299, and AD-1423300. In certain embodiments, the sense and antisense strands are selected from any one of the duplexes AD-1423276, AD-1423283, AD-1423285, AD-1423287, AD-1423288, AD-1423289, AD-1423291, AD-1423294, AD-1423295, AD-1423296, AD-1423297, AD-1423299, and AD-1423300. In further specific embodiments, the sense and antisense strands are selected from any one of the duplexes AD-1423283, AD-1423291, AD-1423294, and AD-1423295.

[0199] In one embodiment, at least partial suppression of expression of 4R tau isoforms is assessed by a reduction in the amount of 4R tau mRNA, e.g., sense mRNA, antisense mRNA, total 4R tau mRNA, which can be isolated from or detected in a first cell or group of cells in which the MAPT gene is transcribed and which has been treated or has been treated to inhibit expression of 4R tau isoforms, and compared with a second cell or group of cells (control cells) that is substantially identical to the first cell or group of cells but has been or has not been so treated. The degree of inhibition can be expressed by:

[0200]

number

[0201] As used herein, the phrase " contacting cell with RNAi agent " such as dsRNA includes contacting cell by any possible means.Contacting cell with RNAi agent includes contacting cell with RNAi agent in vitro or contacting cell with RNAi agent in vivo.Contacting can be carried out directly or indirectly.Therefore, for example, RNAi agent can be physically contacted with cell by carrying out a method separately, or RNAi agent can be placed in a situation that can allow or cause it to contact with cell afterwards.

[0202] Contacting cells in vitro can be achieved, for example, by incubating cells with an RNAi agent. Contacting cells in vivo can be achieved, for example, by injecting an RNAi agent into or near the tissue where the cells are located, or by injecting the RNAi agent into another region, for example, the central nervous system (CNS), by intrathecal injection, intravitreal injection, intracisternal injection, or other injection, as appropriate, or by injecting the RNAi agent into the bloodstream or subcutaneous space, so that the agent then reaches the tissue where the cells to be contacted are located. For example, the RNAi agent can contain or be coupled to a ligand that directs or stabilizes the RNAi agent to the target site, for example, the CNS, for example, a lipophilic moiety, as described below and further detailed in, for example, PCT / US2019 / 031170, which is incorporated herein by reference. A combination of in vitro and in vivo contacting methods is also possible. For example, cells may be contacted with an RNAi agent in vitro and then transferred into a subject.

[0203] In one embodiment, contacting a cell with an RNAi agent includes "introducing" or "delivering an RNAi agent into a cell" by promoting or performing uptake or absorption into the cell. The absorption or uptake of an RNAi agent can occur by spontaneous diffusive or active cellular processes, or by auxiliary agents or devices. The introduction of an RNAi agent into a cell can be in vitro or in vivo. For example, in the case of in vivo introduction, the RNAi agent can be injected into a tissue site or administered systemically. In vitro introduction into a cell includes methods known in the art, such as electroporation and lipofection. Further approaches are described herein below or known in the art.

[0204] The terms "lipophilic" or "lipophilic moiety" refer broadly to any compound or chemical moiety that has an affinity for lipids. One way to characterize the lipophilicity of a lipophilic moiety is by the octanol-water partition coefficient, log K ow In this case, K ow is the ratio of the concentration of a chemical in the octanol phase to the concentration of the chemical in the aqueous phase in a two-phase system at equilibrium. The octanol-water partition coefficient is a laboratory-measured property of a substance. However, it can also be predicted by using coefficients attributable to the structural components of a chemical calculated using first principles or empirical methods [see, e.g., Tetko et al., J. Chem. Inf. Comput. Sci. 41:1407-21 (2001), incorporated herein by reference in its entirety]. It provides a thermodynamic measure of a substance's tendency to prefer a non-aqueous or oily environment rather than water (i.e., hydrophilic / lipophilic balance). In principle, a chemical can be expressed as a function of the log K ow is greater than 0, it is lipophilic in nature. Typically, a lipophilic moiety has a log K ow For example, the log K of 6-aminohexanol owis expected to be approximately 0.7. Using the same method, the log K ow is expected to be 10.7.

[0205] The lipophilicity of a molecule can be modified with respect to the functional groups it carries. For example, adding a hydroxyl or amine group to the end of the lipophilic moiety can increase the partition coefficient (e.g., logK ow ) value can be increased or decreased.

[0206] Alternatively, the hydrophobicity of the double-stranded RNAi agent that is conjugated with one or more lipophilic moieties can be measured by its protein binding properties.For example, in certain embodiments, the unbound fraction of the plasma protein binding assay of double-stranded RNAi agent can be determined to be positively correlated with the relative hydrophobicity of double-stranded RNAi agent, which can be positively correlated with the silencing activity of double-stranded RNAi agent.

[0207] In one embodiment, the plasma protein binding assay to be determined is an electrophoretic mobility shift assay (EMSA) using human serum albumin protein. An exemplary protocol for this binding assay is described in detail, for example, in PCT / US2019 / 031170. Briefly, the duplex was incubated with human serum albumin and the unbound fraction was determined. An exemplary assay protocol involves diluting a 10 μM stock concentration of the duplex to a final concentration of 0.5 μM (total volume of 20 μL) in 1×PBS containing 0, 20, or 90% serum. The samples may be mixed, centrifuged for 30 seconds, and then incubated at room temperature for 10 minutes. After the incubation step is complete, 4 μL of 6×EMSA gel-loading solution may be added to each sample, centrifuged for 30 seconds, and 12 μL of each sample may be loaded onto a 26-well BioRad 10% PAGE (polyacrylamide gel electrophoresis) plate. The gel may be run at 100 volts for 1 hour. After completion of the run, the gel may be removed from the casing and washed in 50 mL of 10% TBE (Tris base, boric acid, and EDTA). Once the wash is complete, 5 μL of SYBR Gold may be added to the gel, which may then be incubated at room temperature for 10 minutes, and the gel may be washed again in 50 mL of 10% TBE. In this exemplary assay, the gel may be read using a Gel Doc XR+ gel documentation system using the following parameters: imaging application set to SYBR Gold, size set to Bio-Rad reference gel, exposure set to automatic for intense bands, highlight saturated pixels may be set to 1, and color set to gray. Detection, molecular weight analysis, and output may all be disabled. Once a clear picture of the gel is obtained, the image may be processed using Image Lab 5.2. Lanes and bands may be manually configured and band intensities may be measured. Band intensities for each sample may be normalized to PBS to obtain the unbound siRNA fraction. From this measurement, relative hydrophobicity may be determined.The hydrophobicity of the double-stranded RNAi agent, as measured by the fraction of unbound siRNA in a binding assay, is greater than 0.15, greater than 0.2, greater than 0.25, greater than 0.3, greater than 0.35, greater than 0.4, greater than 0.45, or greater than 0.5 for enhanced in vivo delivery of siRNA.

[0208] Thus, conjugating a lipophilic moiety to an internal position of a double-stranded RNAi agent provides optimal hydrophobicity in siRNA for enhanced in vivo delivery.

[0209] 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, such as an RNAi agent or a plasmid from which the RNAi agent is transcribed. LNPs are described, for example, in U.S. Patent Nos. 6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents of which are incorporated herein by reference.

[0210] As used herein, a "subject" refers to an animal, for example, a mammal, such as a primate (e.g., a human, a non-human primate, such as a monkey or chimpanzee), or a non-primate (e.g., a rat or a mouse). In a preferred embodiment, the subject is a human, for example, a human being treated or evaluated for a disease, disorder, or condition in which a reduction in 4R tau expression would be beneficial; a human being at risk for a disease, disorder, or condition in which a reduction in 4R tau expression would be beneficial; a human being with a disease, disorder, or condition in which a reduction in 4R tau expression would be beneficial; or a human being treated for a disease, disorder, or condition as described herein in which a reduction in 4R tau expression would be beneficial. 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 one embodiment, the subject is a human child. In another embodiment, the subject is a human juvenile, i.e., a subject under the age of 20.

[0211] As used herein, the term "treating" or "treatment" refers to a beneficial or desired result, including, but not limited to, alleviating or ameliorating one or more signs or symptoms associated with 4R tau expression or production in a 4R tau-associated disease, e.g., a 4R tauopathy, e.g., PSP, CBD, AGD, MSTD, GGT, spAD, or rpAD. "Treatment" can also mean prolonging survival as compared to expected survival if no treatment is administered.

[0212] The term "lower" in relation to the level of 4R tau or a disease marker or symptom in a subject 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 20%. In certain embodiments, the decrease is at least a 50% decrease in the level of a disease marker, e.g., sense- or antisense-containing aggregates, and / or the level of an abnormal dipeptide repeat protein, e.g., a 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more. In some embodiments, the reduction is at least about 25% in the disease marker, for example, 4R tau protein and / or gene expression levels are reduced, for example, by at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%. When referring to the level of 4R tau in a subject, "reducing" preferably refers to reducing 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 the level that an individual would consider to be within the normal range, for example, the level of weight reduction between an obese individual and an individual with a weight that is accepted to be within the normal range.

[0213] As used herein, "prevention" or "preventing," when used in reference to a disease, disorder, or condition for which a reduction in the expression of 4R tau isoforms or the production of 4R tau would be beneficial, refers to a reduction in the likelihood that a subject will develop symptoms associated with such disease, disorder, or condition, e.g., symptoms of a 4R tau-associated disease. Not developing the disease, disorder, or condition, or a reduction in the onset of symptoms associated with such disease, disorder, or condition (e.g., a reduction of at least about 10% on a scale clinically acceptable for the disease or disorder), or a delay in the onset of symptoms (e.g., a delay of days, weeks, months, or years) is considered effective prevention.

[0214] As used herein, the term "4R tau-related disease" or "4R tau-related disorder" includes any disease or disorder that would benefit from reducing the expression and / or activity of 4R tau isoforms.Exemplary 4R tau diseases include 4R tauopathies.As used herein, "4R tauopathies" refer to tauopathies characterized by the aggregation of tau protein, in which 4R tau isoforms are the main or predominant tau isoforms.4R tauopathies include, but are not limited to, progressive supranuclear palsy (PSP), corticobasal ganglia syndrome (CBD), argyrophilic grain disease (AGD), multisystem tauopathy with presenile dementia (MSTD), glioglobular tauopathy (GGT), sporadic (spAD) Alzheimer's disease, and rapidly progressive (rpAD) Alzheimer's disease.

[0215] A "therapeutically effective amount," as used herein, is intended to include an amount of an RNA agent sufficient to treat the disease (e.g., by reducing, ameliorating, or maintaining an existing disease or one or more symptoms of the disease) when administered to a subject with a 4R tau-associated disease. A "therapeutically effective amount" may vary depending on the RNAi agent, how the agent is administered, the disease and its severity, as well as medical history, age, weight, family history, genetic makeup, type of prior or concurrent treatment, if any, and other individual characteristics of the subject being treated.

[0216] As used herein, a "prophylactically effective amount" is intended to include an amount of an RNAi agent sufficient to prevent or ameliorate the disease or one or more symptoms of the disease when administered to a subject with a 4R tau-related disease. Amelioration of the disease includes slowing the progression of the disease or reducing the severity of subsequent disease. A "prophylactically effective amount" may vary depending on the RNAi agent, how the agent is administered, the degree of risk of the disease, as well as the patient's medical history, age, weight, family history, genetic makeup, type of previous or concurrent treatment, if any, and other individual characteristics of the patient being treated.

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

[0218] The phrase "pharmaceutically acceptable" is used herein to mean compounds, materials, 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.

[0219] The phrase "pharmaceutically acceptable carrier," as used herein, refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, magnesium talc, calcium or zinc stearate, or stearic acid), or solvent encapsulating material, 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 deleterious to the subject being treated. Some examples of materials that can function as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants, such as magnesium stearate, sodium lauryl sulfate, and talc; (8) excipients, such as cocoa butter and suppository wax; and (9) oils, such as peanut oil, cottonseed oil, safflower oil, and sesame oil. , olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffer solutions; (21) polyesters, polycarbonates, or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids; (23) serum components, such as serum albumin, HDL, and LDL; and (22) other non-toxic affinity substances used in pharmaceutical formulations.

[0220] The term "sample," as used herein, encompasses similar fluids, cells, or tissues isolated from a subject, as well as collections of fluids, cells, or tissues present within a subject. Examples of biological fluids include blood, serum, and serous fluid, plasma, 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 fluids or cells within those organs. In certain embodiments, samples can be obtained from the brain (e.g., the entire brain or a segment thereof, e.g., the striatum, or a type of cell in the brain, e.g., neurons and glial cells (astrocytes, oligodendrocytes, microglia)). In some embodiments, "a sample obtained from a subject" refers to blood obtained from a subject or plasma or serum obtained therefrom. In further embodiments, "a sample obtained from a subject" refers to brain tissue (or a subcomponent thereof) or retinal tissue (or a subcomponent thereof) obtained from a subject.

[0221] The term "substituted" refers to one or more hydrogen radicals in a given structure being replaced with the radical of a specified substituent. These include, but are not limited to, alkyl, alkenyl, alkynyl, aryl, heterocyclyl, halo, thiol, alkylthio, arylthio, alkylthioalkyl, arylthioalkyl, alkylsulfonyl, alkylsulfonylalkyl, arylsulfonylalkyl, alkoxy, aryloxy, aralkoxy, aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, alkoxycarbonyl, aryloxycarbonyl, haloalkyl, amino, trifluoromethyl, cyano, nitro, alkylamino, arylamino, alkylaminoalkyl, arylaminoalkyl, aminoalkylamino, hydroxy, alkoxyalkyl, carboxyalkyl, alkoxycarbonylalkyl, aminocarbonylalkyl, acyl, aralkoxycarbonyl, carboxylic acid, sulfonic acid, sulfonyl, phosphonic acid, aryl, heteroaryl, heterocyclic, and aliphatic. It is understood that the substituent can be further substituted.

[0222] The term "alkyl" refers to saturated and unsaturated non-aromatic hydrocarbon chains (including, but not limited to, propyl, allyl, or propargyl) that may be straight or branched and contain the indicated number of carbon atoms, and which may be interrupted by N, O, or S. For example, "(C1-C6) alkyl" refers to a radical having 1 to 6 carbon atoms in a linear or branched arrangement. "(C1-C6) alkyl" includes, for example, methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, pentyl, and hexyl. In certain embodiments, lipophilic moieties of the present disclosure may comprise a C6 to C18 alkyl hydrocarbon chain.

[0223] The term "alkylene" refers to an optionally substituted saturated aliphatic branched or straight-chain divalent hydrocarbon radical having the specified number of carbon atoms. For example, "(C1-C6) alkylene" refers to a divalent saturated aliphatic radical having 1 to 6 carbon atoms arranged in a linear fashion, e.g., [(CH2) n ], where n is an integer from 1 to 6. "(C1-C6) alkylene" includes methylene, ethylene, propylene, butylene, pentylene, and hexylene. Alternatively, "(C1-C6) alkylene" refers to a divalent saturated radical having from 1 to 6 carbon atoms arranged in a branched configuration, such as: [(CH2CH2CH2CH2CH(CH3)], [(CH2CH2CH2CH2C(CH3)2], [(CH2C(CH3)2CH(CH3))], and the like. The term "alkylenedioxo" refers to a divalent species of the structure -ORO-, where R represents alkylene.

[0224] The term "mercapto" refers to an -SH radical. The term "thioalkoxy" refers to an -S-alkyl radical.

[0225] The term "halo" refers to any radical of fluorine, chlorine, bromine, or iodine. "Halogen" and "halo" are used interchangeably herein.

[0226] As used herein, unless otherwise specified, the term "cycloalkyl" refers to a saturated or unsaturated non-aromatic hydrocarbon ring group having 3 to 14 carbon atoms. For example, "(C3-C10)cycloalkyl" refers to a hydrocarbon radical of a (3-10)-membered saturated aliphatic cyclic hydrocarbon ring. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, methylcyclopropyl, 2,2-dimethylcyclobutyl, 2-ethylcyclopentyl, cyclohexyl, and the like. Cycloalkyl groups may include multiple spiro or fused rings. Cycloalkyl groups may be mono-, di-, tri-, tetra-, or penta-substituted at any position, as permitted by normal valence.

[0227] As used herein, the term "alkenyl" refers to a straight-chain or branched, non-aromatic hydrocarbon radical containing at least one carbon-carbon double bond and, unless otherwise specified, having 2 to 10 carbon atoms. Up to five carbon-carbon double bonds may be present in such a group. For example, a "C2-C6" alkenyl is defined as an alkenyl radical having 2 to 6 carbon atoms. Examples of alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, and cyclohexenyl. The straight-chain, branched, or cyclic portions of an alkenyl group may contain double bonds and may be mono-, di-, tri-, tetra-, or penta-substituted at any position, as allowed by the normal valence. The term "cycloalkenyl" refers to a monocyclic hydrocarbon group having the specified number of carbon atoms and at least one carbon-carbon double bond.

[0228] As used herein, the term "alkynyl," unless otherwise specified, refers to a straight-chain or branched hydrocarbon radical containing 2 to 10 carbon atoms and at least one carbon-carbon triple bond. Up to five carbon-carbon triple bonds may be present. Thus, "C2-C6 alkynyl" refers to an alkynyl radical having 2 to 6 carbon atoms. Examples of alkynyl groups include, but are not limited to, ethynyl, 2-propynyl, and 2-butynyl. The straight-chain or branched portion of an alkynyl group may contain triple bonds as permitted by normal valence and may be mono-, di-, tri-, tetra-, or penta-substituted at any position as permitted by normal valence.

[0229] As used herein, "alkoxyl" or "alkoxy" refers to an alkyl group, as defined above, with the indicated number of carbon atoms attached through an oxygen bridge. For example, "(C-C)alkoxy" includes methoxy, ethoxy, and propoxy. For example, "(C-C)alkoxy" is intended to include C, C, C, C, C, C, C, C, and C alkoxy groups. For example, "(C-C)alkoxy" is intended to include C, C, C, C, C, C, C, C, and C alkoxy groups. Examples of alkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, s-butoxy, t-butoxy, n-pentoxy, s-pentoxy, n-heptoxy, and n-octoxy. "Alkylthio" refers to an alkyl radical attached through a sulfur linking atom. The term "alkylamino" or "aminoalkyl" refers to an alkyl radical linked through an N-H linkage. "Dialkylamino" refers to two alkyl radicals linked through a nitrogen linking atom. The amino group can be unsubstituted, monosubstituted, or disubstituted. In some embodiments, the two alkyl radicals are the same (e.g., N,N-dimethylamino). In some embodiments, the two alkyl radicals are different (e.g., N-ethyl-N-methylamino).

[0230] As used herein, "aryl" or "aromatic" refers to any stable monocyclic or polycyclic carbon ring having up to seven atoms in each ring, with at least one ring being aromatic. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl, tetrahydronaphthyl, indanyl, and biphenyl. When the aryl substituent is bicyclic and one ring is non-aromatic, it is understood that the bond is via the aromatic ring. The aryl group may be mono-, di-, tri-, tetra-, or penta-substituted at any position, as permitted by normal valences. The term "arylalkyl" or "aralkyl" refers to an alkyl substituted with an aryl. The term "arylalkoxy" refers to an alkoxy substituted with an aryl.

[0231] "Hetero" refers to at least one carbon atom in a ring structure replaced with at least one heteroatom selected from N, S, and O. "Hetero" also refers to at least one carbon atom in a non-ring structure replaced. A heterocyclic or heteroacyclic structure can have, for example, 1, 2, or 3 carbon atoms replaced by heteroatoms.

[0232] As used herein, the term "heteroaryl" refers to a stable monocyclic or polycyclic ring having up to seven atoms in each ring, wherein at least one ring is aromatic and contains from 1 to 4 heteroatoms selected from the group consisting of O, N, and S. Examples of heteroaryl groups include, but are not limited to, acridinyl, carbazolyl, cinnolinyl, quinoxalinyl, pyrazolyl, indolyl, benzotriazolyl, furanyl, thienyl, benzothienyl, benzofuranyl, benzimidazolonyl, benzoxazolonyl, quinolinyl, isoquinolinyl, dihydroisoindolonyl, imidazopyridinyl, isoindolonyl, indazolyl, oxazolyl, oxadiazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl, and tetrahydroquinoline. It is understood that "heteroaryl" includes the N-oxide derivative of any nitrogen-containing heteroaryl. If the heteroaryl substituent is bicyclic and one ring is non-aromatic or does not contain a heteroatom, it is understood that attachment is through the aromatic ring or through the ring containing the heteroatom. Heteroaryl groups may be mono-, di-, tri-, tetra-, or penta-substituted at any position, as permitted by normal valences.

[0233] As used herein, the term "heterocycle," "heterocyclic," or "heterocyclyl" refers to a 3- to 14-membered aromatic or non-aromatic heterocycle, including polycyclic groups, containing 1 to 4 heteroatoms selected from the group consisting of O, N, and S. As used herein, the term "heterocyclic" is also considered synonymous with the terms "heterocycle" and "heterocyclyl," which are also understood to have the same definition as set forth herein. "Heterocyclyl" includes the heteroaryls described above, as well as dihydro and tetrahydro analogs thereof.Examples of heterocyclyl groups include azetidinyl, benzimidazolyl, benzofuranyl, benzofurazanyl, benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl, cinnolinyl, furanyl, imidazolyl, indolinyl, indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthopyridinyl, oxadiazolyl, and oxooxazolidinyl. , oxazolyl, oxazoline, oxopiperazinyl, oxopyrrolidinyl, oxomorpholinyl, isoxazoline, oxetanyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl, pyridinonyl, pyrimidyl, pyrimidinonyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl, tetrahydropyranyl, tetrahydrofuranyl, tetrahydrothiopyranyl, tetrahydroisoquinolinyl, tetrazolyl, tetrazolopyridyl, thiadiazinyl Zolyl, thiazolyl, thienyl, triazolyl, 1,4-dioxanyl, hexahydroazepinyl, piperazinyl, piperidinyl, pyridin-2-onyl, pyrrolidinyl, morpholinyl, thiomorpholinyl, dihydrobenzimidazolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, dihydrobenzoxazolyl, dihydrofuranyl, dihydroimidazolyl, dihydroindolyl, dihydroisoxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydroo Heterocyclyl groups include, but are not limited to, oxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl, dioxidothiomorpholinyl, methylenedioxybenzoyl, tetrahydrofuranyl, and tetrahydrothienyl, and their N-oxides. Attachment of heterocyclyl substituents can occur through a carbon atom or through a heteroatom. Heterocyclyl groups may be mono-, di-, tri-, tetra-, or penta-substituted at any position, as permitted by normal valences.

[0234] "Heterocycloalkyl" refers to a cycloalkyl residue in which one to four of the carbons are replaced by a heteroatom such as oxygen, nitrogen, or sulfur. Examples of heterocycles in which the radical is a heterocyclyl group include tetrahydropyran, morpholine, pyrrolidine, piperidine, thiazolidine, oxazole, oxazoline, isoxazole, dioxane, tetrahydrofuran, and the like.

[0235] The term "heteroaryl" refers to an aromatic 5- to 8-membered monocyclic, 8- to 12-membered bicyclic, or 11- to 14-membered tricyclic ring system having 1 to 3 heteroatoms if monocyclic, 1 to 6 heteroatoms if bicyclic, or 1 to 9 heteroatoms if tricyclic, selected from O, N, or S (e.g., carbon atoms and 1 to 3, 1 to 6, or 1 to 9 N, O, or S heteroatoms if monocyclic, bicyclic, or tricyclic, respectively), in which 0, 1, 2, 3, or 4 atoms in each ring are optionally substituted. Examples of heteroaryl groups include pyridyl, furyl or furanyl, imidazolyl, benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, quinolinyl, indolyl, thiazolyl, and the like. The term "heteroarylalkyl" or "heteroaralkyl" refers to an alkyl substituted with a heteroaryl. The term "heteroarylalkoxy" refers to an alkoxy substituted with a heteroaryl.

[0236] The term "cycloalkyl," as used herein, includes saturated and partially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons, e.g., 3 to 8 carbons, e.g., 3 to 6 carbons, which cycloalkyl groups may be further optionally substituted. Cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.

[0237] The term "acyl" refers to an alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl, heterocyclylcarbonyl, or heteroarylcarbonyl substituent, any of which may be further substituted by substituents.

[0238] As used herein, "keto" refers to any alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclyl, heteroaryl, or aryl group as defined herein attached through a carbonyl bridge.

[0239] Examples of keto groups include, but are not limited to, alkanoyl (e.g., acetyl, propionyl, butanoyl, pentanoyl, hexanoyl), alkenoyl (e.g., acryloyl), alkynoyl (e.g., ethinoyl, propynoyl, butynoyl, pentinoyl, hexynoyl), aryloyl (e.g., benzoyl), heteroaryloyl (e.g., pyrrolyl, imidazoloyl, quinolinoyl, pyridinoyl).

[0240] As used herein, "alkoxycarbonyl" refers to any alkoxy group as defined above attached through a carbonyl bridge (i.e., -C(O)O-alkyl). Examples of alkoxycarbonyl groups include, but are not limited to, methoxycarbonyl, ethoxycarbonyl, iso-propoxycarbonyl, n-propoxycarbonyl, t-butoxycarbonyl, benzyloxycarbonyl, or n-pentoxycarbonyl.

[0241] As used herein, "aryloxycarbonyl" refers to any aryl group, as defined herein, attached through an oxycarbonyl bridge (i.e., -C(O)O-aryl). Examples of aryloxycarbonyl groups include, but are not limited to, phenoxycarbonyl and naphthyloxycarbonyl.

[0242] As used herein, "heteroaryloxycarbonyl" refers to any heteroaryl group as defined herein attached through an oxycarbonyl bridge (i.e., -C(O)O-heteroaryl). Examples of heteroaryloxycarbonyl groups include, but are not limited to, 2-pyridyloxycarbonyl, 2-oxazolyloxycarbonyl, 4-thiazolyloxycarbonyl, or pyrimidinyloxycarbonyl.

[0243] The term "oxo" refers to an oxygen atom which forms a carbonyl when attached to carbon, an N-oxide when attached to nitrogen, or a sulfoxide or sulfone when attached to sulfur.

[0244] Those skilled in the art will readily understand and appreciate that the compounds and compositions disclosed herein may have certain atoms (e.g., N, O, or S atoms) in a protonated or deprotonated state depending on the environment in which the compound or composition is placed. Thus, as used herein, it is assumed that the structures disclosed herein may have certain functional groups, such as OH, SH, or NH, in a protonated or deprotonated state. The disclosure of the present invention is intended to cover the disclosed compounds and compositions regardless of their protonation state based on the pH of the environment, as will be readily understood by those skilled in the art.

[0245] II. RNAi Agents of the Disclosure Described herein are RNAi agents that inhibit the expression of 4R tau isoforms. In one embodiment, the RNAi agent comprises a double-stranded ribonucleic acid (dsRNA) molecule for inhibiting the expression of 4R tau in a cell, e.g., a cell in a subject (e.g., a mammal, e.g., a human with a 4R tau-associated disease, e.g., a 4R tauopathy, e.g., PSP, CBD, AGD, MSTD, GGT, spAD, or rpAD). The dsRNA comprises an antisense strand having a region of complementarity that is complementary to at least a portion of a hotspot within exon 10 in the mRNA formed upon expression of the 4R tau isoform. The region of complementarity is no more than about 15-30 nucleotides in length. Upon contact with a cell expressing a 4R tau isoform, the RNAi agent inhibits expression of the 4R tau isoform (e.g., expressed by a human gene, a primate gene, or a non-primate gene) by at least 25% or more as described herein, compared to similar cells not contacted with the RNAi agent or an RNAi agent that is not complementary to the MAPT gene. Expression of 4R tau isoforms 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 one embodiment, the level of knockdown is assayed in BE(2)-C cells using the assay method provided in Example 1 below. In some embodiments, the level of knockdown is assayed in primary mouse hepatocytes. In some embodiments, the level of knockdown is assayed in Neuro-2a cells.

[0246] dsRNA comprises two RNA strands, which are complementary and hybridize to form a duplex structure under the conditions in which dsRNA is used.One strand of dsRNA (antisense strand) comprises a complementary region that is substantially complementary, and generally completely complementary to the target sequence.The target sequence can be obtained from the sequence of the hotspot in exon 10 of mRNA formed during the expression of 4R tau isoform.The other strand (sense strand) comprises a region complementary 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 sequences of dsRNA can also be contained as self-complementary regions of a single nucleic acid molecule, so as to be opposite each other on separate oligonucleotides.

[0247] 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 preferred 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.

[0248] 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.

[0249] 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.

[0250] In some embodiments, the dsRNA is 15 to 23 nucleotides long, 19 to 23 nucleotides long, or 25 to 30 nucleotides long. Generally, the dsRNA is long enough to function as a substrate for the Dicer enzyme. For example, it is well known in the art that dsRNA longer than about 21 to 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 portion of a longer RNA molecule, often an mRNA molecule. In relevant cases, a "portion" 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).

[0251] Those skilled in the art will appreciate that the duplex region may be a primary functional portion of a dsRNA, e.g., 15 to 36 base pairs, e.g., 15-36, 15-35, 15-34, 15-33, 15-32, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-22, 18-23, 18-24, 18-25, 18-24, 18-23, 18-22, 18-21, 18-25, 18-26, 18-27, 18-28, 18-29, 18-30, 18-31, 18-32, 18-33, 18-34, 18-35, 18-36, 18-37, 18-38, 18-39, 18-40, 18-41, 18-42, 18-43, 18-44, 18-45, 18-46, 18-47, 18-48, 18-49, 18-50, 18-51, 18-52, 18- It will also be appreciated that the duplex region may be 8-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs, e.g., 19-21 base pairs. Therefore, in one embodiment, an RNA molecule or a 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, which targets the desired RNA for cleavage.Therefore, 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, the RNAi agent useful for targeting 4R tau expression is not generated in target cells by cleavage of larger dsRNA.

[0252] dsRNA described herein can further comprise one or more single-stranded nucleotide overhangs, for example, 1, 2, 3 or 4 nucleotides.Nucleotide overhangs can comprise or consist of nucleotide / nucleoside analogs such as deoxynucleotide / nucleoside.Overhangs can be on sense strand, antisense strand, or any combination thereof.In addition, the nucleotide of overhang can be on the 5' end, 3' end, or both of the antisense strand or sense strand of dsRNA.

[0253] dsRNA can be synthesized by standard methods known in the art.The double-stranded RNAi compounds of the present disclosure can be prepared using a two-step method.First, the individual strands of double-stranded RNA molecules are 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 the oligonucleotide strands that contain unnatural nucleotides or modified nucleotides.Similarly, the single-stranded oligonucleotides of the present disclosure can be prepared using solution phase or solid phase organic synthesis or both.

[0254] In one embodiment, the dsRNA of the present disclosure comprises at least two nucleotide sequences, i.e., a sense strand and an antisense strand.The sense strand sequence for MAPT can be selected from the group of sequences provided in Table 7 or 8, and the corresponding nucleotides of the sense strand and the antisense strand can be selected from the group of sequences in Table 7 or 8. 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 during the expression of 4R tau isoform.Therefore, in this embodiment, the dsRNA will comprise two oligonucleotides, one oligonucleotide being described as the sense strand (passenger strand) in Table 7 or 8, and the second oligonucleotide being described as the corresponding antisense strand (guide strand) to the sense strand in Table 7 or 8.

[0255] In one embodiment, the sense strand comprises nucleotides 1009-1029, 1010-1030, 1011-1031, 1012-1032, 1013-1033, 1014-1034, 1015-1035, 1016-1036, 1017-1037, 1018-1038, 1019-1039, 1020-1040, 1021-1041, 1022-1042, 1023-1043, 1024-1044, 1025-1045, 1026-1047, 1027-1048, 1028-1049, 1030-1031, 1032-1033, 1034-1035, 1036-1037, 1038-1039, 1040-1041, 1042-1043, 1044-1045, 1046-1047, 1048-1049, 1050-1051, 1052-1053, 1054-1055, 1056-1057, 1058-1059, 1060-1061, 1062-1063, 1063-1064, 1064-1065, 1065-1066, 1066-1067, 1067-1068, 1068-1069, 1070-1071, 1072-1073, 1074 and the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than three nucleotides from any one of the nucleotide sequences of SEQ ID NO:4, 1046, 1027-1047, 1028-1048, 1029-1049, 1030-1050, 1031-1051, 1032-1052, 1033-1053, 1014-1048, and 1022-1048, and the antisense strand comprises at least 15 contiguous nucleotides from the corresponding nucleotide sequence of SEQ ID NO:4. In certain embodiments, the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the nucleotide sequences of nucleotides 1009-1029, 1016-1036, 1018-1038, 1020-1040, 1021-1041, 1022-1042, 1024-1044, 1027-1047, 1028-1048, 1029-1049, 1030-1050, 1032-1052, 1033-1053, 1014-1048, and 1022-1048 of SEQ ID NO:3, and the antisense strand comprises at least 15 contiguous nucleotides from the corresponding nucleotide sequence of SEQ ID NO:4.

[0256] In certain embodiments, the antisense polynucleotides disclosed herein are substantially complementary to a fragment of the target MAPT sequence and comprise a contiguous nucleotide sequence that is at least 80% complementary to a fragment of SEQ ID NO:3 over its entire length, wherein the sense strand is at least 80% complementary to nucleotides 1009-1029, 1010-1030, 1011-1031, 1012-1032, 1013-1033, 1014-1034, 1015-1035, 1016-1036, 1017-1037, 1018-1038, 1019-1039, 1020-1040, 1021-1041, 1022-1042, 1023-1043, 1024-1044, 1025-1045, 1026-1046, 1027-1047, 1028-1048, 1029-1050, 1031-1032, 1033-1034, 1035-1036, 1037-1038, 1039-1049, 1040-1051, 1041-1052, 1042-1053, 1043-1054, 1044-1055, 1045-1056, 1046-1057, 1047-1058, 1048-1059, 1050-1060, 1051-1061, 1052-1062, 1053-1063, 1054-10 and the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the nucleotide sequences of SEQ ID NO: 1-1041, 1022-1042, 1023-1043, 1024-1044, 1025-1045, 1026-1046, 1027-1047, 1028-1048, 1029-1049, 1030-1050, 1031-1051, 1032-1052, 1033-1053, 1014-1048, and 1022-1048, and the antisense strand comprises at least 15 contiguous nucleotides from the corresponding nucleotide sequence of SEQ ID NO: 4. In some embodiments, the antisense polynucleotides disclosed herein are substantially complementary to a fragment of a target MAPT sequence and comprise a contiguous nucleotide sequence that is complementary to a fragment of SEQ ID NO:3 over its entire length, wherein the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the nucleotide sequences of nucleotides 1009-1029, 1016-1036, 1018-1038, 1020-1040, 1021-1041, 1022-1042, 1024-1044, 1027-1047, 1028-1048, 1029-1049, 1030-1050, 1032-1052, 1033-1053, 1014-1048, and 1022-1048 of SEQ ID NO:3, and the antisense strand comprises at least 15 contiguous nucleotides from the corresponding nucleotide sequence of SEQ ID NO:4.

[0257] In one embodiment, the antisense strand is selected from the group consisting of AD-1423276, AD-1423277, AD-1423278, AD-1423279, AD-1423280, AD-1423281, AD-1423282, AD-1423283, AD-1423284, AD-1423285, AD-1423286, AD-1423287, AD-1423288, AD-1423289, AD-1423290, AD and AD-1423291, AD-1423292, AD-1423293, AD-1423294, AD-1423295, AD-1423296, AD-1423297, AD-1423298, AD-1423299, and AD-1423300. In certain embodiments, the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the antisense strand nucleotide sequences of the duplex selected from the group consisting of AD-1423276, AD-1423283, AD-1423285, AD-1423287, AD-1423288, AD-1423289, AD-1423291, AD-1423294, AD-1423295, AD-1423296, AD-1423297, AD-1423299, and AD-1423300.

[0258] In some embodiments, the disclosure provides a dsRNA agent for inhibiting expression of 4R tau, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, the antisense strand comprising a region of complementarity to an mRNA encoding 4R tau, the region of complementarity comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the antisense nucleotide sequences set forth in Tables 7 or 8.

[0259] In one embodiment, the sequences substantially complementary to the dsRNA are contained in separate oligonucleotides, hi another embodiment, the sequences substantially complementary to the dsRNA are contained in a single oligonucleotide.

[0260] Although the sequences in Tables 7 and 8 are described as modified or conjugated sequences, it is understood that the RNA of an RNAi agent of the present disclosure, e.g., a dsRNA of the present disclosure, can comprise any one of the sequences described in Tables 7 and 8 unmodified, unconjugated, or modified or conjugated differently than described. For example, the sense strand of an agent of the present disclosure may be conjugated to a GalNAc ligand, but these agents may also be conjugated to a moiety that directs delivery to the CNS, such as a C16 ligand as described herein. In one embodiment, the lipophilic moiety contains a saturated or unsaturated C16 hydrocarbon chain (e.g., a linear C16 alkyl or alkenyl). The lipophilic ligand may be included in any of the positions provided herein. In some embodiments, the lipophilic moiety is conjugated to the nucleobase, sugar moiety, or internucleoside linkage of a double-stranded iRNA agent. For example, a C16 ligand may be conjugated via the 2'-oxygen of a ribonucleotide, as shown in the following structure:

[0261] [ka] (In the formula, * represents a bond to an adjacent nucleotide, and B is a nucleobase or nucleobase analog, where B may be adenine, guanine, cytosine, thymine, or uracil. The design and synthesis of the ligands and monomers provided herein are described, for example, in PCT Publication Nos. WO2019 / 217459, WO2020 / 132227, and WO2020 / 257194, the entireties of which are incorporated herein by reference.

[0262] In some embodiments, the double-stranded iRNA agent further comprises a phosphate or phosphate mimic at the 5'-end of the antisense strand. In one embodiment, the phosphate mimic is 5'-vinyl phosphonate (VP). In one embodiment, the phosphate mimic is 5'-cyclopropyl phosphonate (VP). In some embodiments, the 5'-end of the antisense strand of the double-stranded iRNA agent does not contain a 5'-vinyl phosphonate (VP).

[0263] 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), e.g., Ggn, Cgn, Tgn, or Agn, nucleotides having a 2' phosphate, e.g., G2p, C2p, A2p, or U2p, and vinyl phosphonate nucleotides; and combinations thereof.

[0264] In other embodiments, each of the duplexes in Tables 7 and 8 may be individually modified to provide another double-stranded iRNA agent of the present disclosure. In one example, the 3' end of each sense duplex may be modified by removing the 3'-terminal L96 ligand and replacing the two phosphodiester internucleotide linkages between the three 3'-terminal nucleotides with phosphorothioate internucleotide linkages, i.e., a duplex of the following formula: 5'-N1-...-N n-2 N n-1 N n L96 3' The three 3' terminal nucleotides (N) of the sense sequence of 5'-N1-...-N n-2 sN n-1 sN n 3' may be replaced by That is, for example, in the case of AD-1423297, the sense sequence is: asasgga(Uhd)aaUfAfUfcaaacacguaL96 teeth, asasgga(Uhd)aaUfAfUfcaaacacgsusa Alternatively, the antisense sequence may be left unchanged to provide another double-stranded iRNA agent of the present disclosure. In another example, the sense strand of each of the following duplexes is modified as described above to provide the duplexes of the present disclosure: AD-1423277, AD-1423278, AD-1423279, AD-1423280, AD-1423281, AD-1423282, AD-1423283, AD-1423284, AD-1423285, AD-1423286, AD-1423287, AD-1423288, AD-1423289, AD-1423290, AD-1423291, AD-1423292, AD-1423293, AD-1423294, AD-1423295, AD-1423296, AD-1423297, AD-1423298, AD-1423299, and AD-1423300.

[0265] Those skilled in the art are well aware that dsRNAs with duplex structures of about 20 to 23 base pairs, for example, 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 embodiment, due to the nature of the oligonucleotide sequences provided herein, the dsRNAs described herein can comprise at least one strand with a minimum length of 21 nucleotides. It can be reasonably expected that shorter duplexes, minus a few nucleotides at one or both ends, can be similarly effective compared to the dsRNAs described above. Therefore, it is contemplated that the dsRNA that has a sequence of at least 15, 16, 17, 18, 19, 20 or more consecutive nucleotides from one of the sequences provided herein, and when using, for example, A549 cells and 10nM concentration of RNA agent in vitro assay and PCR assay as provided in the examples herein, their ability to inhibit the expression of 4R tau isoform by at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 95% compared to control level is different from the dsRNA that comprises full-length sequence.In some embodiments, the inhibition from the dsRNA that comprises full-length sequence is measured using the in vitro assay that uses primary mouse hepatocytes.

[0266] In addition, the RNA described herein specifies the site of MAPT transcript that is susceptible to RISC-mediated cleavage.Therefore, the present disclosure further features the RNAi agent that targets within this site.As used herein, if an RNAi agent promotes the cleavage of the transcript at any of the specific sites, it is said to target within the specific site of RNA transcript.Such RNAi agent will generally comprise at least about 15 nucleotides, preferably at least 19 nucleotides, from one of the sequences provided herein, coupled with additional nucleotide sequences taken from the region adjacent to the selected sequence in MAPT gene.

[0267] III. Modified RNAi Agents of the Present Disclosure In one embodiment, the RNA of the RNAi agent of the present disclosure, for example, dsRNA, is unmodified, and does not contain, for example, chemical modifications or conjugations known in the art and described herein.In a preferred embodiment, the RNA of the RNAi agent of the present disclosure, for example, dsRNA, is chemically modified to enhance stability or other beneficial characteristics.In certain embodiments of the present disclosure, substantially all of the nucleotides of the RNAi agent of the present disclosure are modified.In other embodiments of the present disclosure, all of the nucleotides of the RNAi agent of the present disclosure are modified.The RNAi agent of the present disclosure in which "substantially all of the nucleotides are modified" is mostly, but not entirely, modified, and may contain 5, 4, 3, 2, or unmodified nucleotides.In yet other embodiments of the present disclosure, the RNAi agent of the present disclosure may contain 5, 4, 3, 2, or 1 modified nucleotide.

[0268] Nucleic acids featured in this disclosure can be synthesized or modified by methods well established in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, 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 RNAi agents useful in the embodiments described herein include, but are not limited to, RNAs containing modified backbones or that do not contain natural internucleoside linkages. Among the RNAs with modified backbone, those that do not have phosphorus atom in backbone include.For the purpose of this specification, as sometimes referred to in the art, the modified RNA that does not have phosphorus atom in its internucleoside backbone can also be considered as oligonucleoside.In some embodiments, modified RNAi agent has phosphorus atom in its internucleoside backbone.

[0269] 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 by 2'-5' linkages, and those with reverse polarity, where adjacent pairs of nucleoside units are linked by 3'-5' to 5'-3' or 2'-5' to 5'-2'. Also included are various salts, mixed salts, and free acid forms. In some embodiments of the present disclosure, the dsRNA agent of the present disclosure is in free acid form. In other embodiments of the present disclosure, the dsRNA agent of the present disclosure is in salt form. In one embodiment, the dsRNA agent of the present disclosure is in sodium salt form.In certain embodiments, when the dsRNA agent of the present disclosure 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 disclosure is in sodium salt form, sodium ion exists as the counterion for all of the phosphodiester and / or phosphorothioate groups present in the agent.

[0270] 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.

[0271] Modified RNA backbones that do not contain phosphorus atoms have backbones formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatom or heterocyclic internucleoside linkages. These include morpholino linkages (formed in part from the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and others with mixed N, O, S, and CH2 constituent moieties.

[0272] 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.

[0273] In another embodiment, RNA mimetics suitable for use in RNAi agents are contemplated, in which both the sugar and internucleoside linkages, i.e., backbone, of nucleotide units are replaced with alternative groups. 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. Nucleic acid bases are retained and are directly or indirectly linked to the aza nitrogen atom of the amide portion of the backbone. Representative U.S. patents teaching the preparation of PNA compounds include, but are not limited to, U.S. Patent Nos. 5,539,082, 5,714,331, and 5,719,262, the entire contents of each of which are incorporated herein by reference. Additional PNA compounds suitable for use in the RNAi agents of the present disclosure are described, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0274] Some embodiments featured in this disclosure include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, particularly --CH2--NH--CH2-, --CH2--N(CH3)--O--CH2-- (known as the methylene (methylimino) or MMI backbone), --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2--, and --N(CH3)--CH2-- (the natural phosphodiester backbone is represented as --O--P--O--CH2--) of the above-referenced U.S. Patent No. 5,489,677, and oligonucleosides with amide backbones of the above-referenced U.S. Patent No. 5,602,240. In some embodiments, the RNAs featured herein have the morpholino backbone structure of the above-referenced U.S. Patent No. 5,034,506.

[0275] Modified RNAs may also contain one or more substituted sugar moieties. The RNAi agents, 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 RNAi agents or group for improving the pharmacodynamic properties of RNAi agents, and other substituents with similar properties. In some embodiments, the modification includes 2'-methoxyethoxy (2'-O--CHCHOCH, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., an alkoxy-alkoxy group. Another exemplary modification is the 2'-dimethylaminooxyethoxy, i.e., O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, as described herein below in the Examples, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O--CH2--O--CH2--N(CH2)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).

[0276] Other modifications include 2'-methoxy (2'-OCH), 2'-aminopropoxy (2'-OCHCHNH), 2'-O-hexadecyl, and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA of an RNAi agent, particularly at the 3'-position of the sugar on the 3'-terminal nucleotide or in a 2'-5'-linked dsRNA and at the 5'-position of the 5'-terminal nucleotide. RNAi agents can also have sugar mimetics, such as cyclobutyl moieties instead of pentofuranosyl sugars. 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.

[0277] The RNAi agent of the present disclosure may also include nucleobase (often simply referred to in the art as "base") modifications or substitutions.As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), the pyrimidine bases thymine (T), cytosine (C) and uracil (U).Modified nucleobases include other synthetic and natural nucleobases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil ... These include 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and adenine, 8-azaguanine and adenine, 7-deazaguanine and adenine, and 3-deazaguanine and adenine.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 disclosure. 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 exemplary base substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar modifications.

[0278] 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.

[0279] The RNAi agent of the present disclosure can also be modified to include one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide with a modified ribose moiety, which includes an additional bridge connecting the 2' and 4' carbons of the ribose moiety. This structure effectively "locks" the ribose in 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 anc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193].

[0280] The RNAi agents of the present disclosure can also be modified to include one or more bicyclic sugar moieties. A "bicyclic sugar" is a furanosyl ring modified by bridging two atoms. A "bicyclic nucleoside" ("BNA") is a nucleoside having a sugar moiety containing a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic ring structure. In certain embodiments, the bridge connects the 4'-carbon and 2'-carbon of the sugar ring. Thus, in some embodiments, the agents of the present disclosure can include one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety contains an additional bridge connecting the 2' and 4' carbons. In other words, an LNA is a nucleotide containing a bicyclic sugar moiety containing a 4'-CH2-O-2' bridge. This structure effectively "locks" the ribose into a 3'-endo conformation. The addition of a locked nucleic acid to siRNA has been shown to increase siRNA stability in serum and reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, OR. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). Examples of bicyclic nucleosides for use in the polynucleotides of the present disclosure include, but are not limited to, nucleosides comprising a bridge between the 4' and 2' ribosyl ring atoms. In certain embodiments, the antisense polynucleotide agent of the present disclosure includes one or more bicyclic nucleosides comprising a 4' to 2' bridge.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, see e.g., U.S. Pat. No. 7,399,845), 4'-C(CH3)(CH3)-O-2' (and analogs, see e.g., U.S. Pat. No. 8,399,845). 278,283), 4'-CH2-N(OCH3)-2' (and analogs thereof, see, e.g., U.S. Pat. No. 8,278,425), 4'-CH2-ON(CH3)-2' (see, e.g., U.S. Patent Publication No. 2004 / 0171570), 4'-CH2-N(R)-O-2' (wherein R is H, C1-C12 alkyl, or a protecting group) (see, e.g., U.S. Pat. No. 7,427,672), 4'-CH2-C(H)(CH3)-2' (see, e.g., Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134) and 4'-CH2-C(-CH2)-2' (and analogs thereof, see, e.g., U.S. Pat. No. 8,278,426), the entire contents of each of which are incorporated herein by reference.

[0281] 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.

[0282] 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).

[0283] RNAi agents of the present disclosure can also be modified to include one or more constrained ethyl nucleotides. As used herein, a "constrained ethyl nucleotide" or "cEt" is a locked nucleic acid containing a bicyclic sugar moiety containing a 4'-CH(CH3)-0 to 2' bridge. In one embodiment, the constrained ethyl nucleotide is in the S conformation and is referred to herein as an "S-cEt."

[0284] The RNAi agents of the present disclosure may also contain one or more "conformation-restricting nucleotides" ("CRNs"). A CRN is a nucleotide analogue with a linker connecting the C2' and C4' carbons of ribose or the C3 and C5' carbons of ribose. The CRN locks the ribose ring into a stable conformation and increases 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.

[0285] Representative publications that teach the preparation of certain of the above CRNs include, but are not limited to, US2013 / 0190383 and WO2013 / 036868, the entire contents of each of which are incorporated herein by reference.

[0286] In some embodiments, the RNAi agent of the present disclosure comprises one or more monomers that are UNA (non-locked nucleic acid) nucleotides. UNA is a non-locked acyclic nucleic acid, and any of the sugar bonds have been removed to form a non-locked "sugar" residue. In one example, UNA also encompasses a monomer in which the bond between C1'-C4' (i.e., the covalent carbon-oxygen-carbon bond between the C1' and C4' carbons) has been removed. In another example, the C2'-C3' bond of the sugar (i.e., the covalent carbon-carbon bond between the C2' and C3' carbons) has been removed [see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039, which are incorporated herein by reference].

[0287] Representative U.S. publications that teach the preparation of UNAs include, but are not limited to, U.S. Pat. Nos. 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.

[0288] The RNAi agent of the present disclosure may also contain one or more "cyclohexene nucleic acids" or ("CeNA"). CeNA is a nucleotide analog in which the furanose moiety of DNA is replaced with a cyclohexene ring. Incorporating cyclohexenyl nucleosides into a DNA strand increases the stability of DNA / RNA hybrids. CeNA is stable against degradation in serum, and CeNA / RNA hybrids can activate Escherichia coli (E. coli) RNase H, thereby cleaving the RNA strand. (See Wang et al., Am. Chem. Soc. 2000, 122, 36, 8595-8602, incorporated herein by reference.)

[0289] 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'-0-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3"-phosphate, inverted base dT (idT), and others. Disclosure of this modification can be found in WO2011 / 005861.

[0290] Other modifications of the RNAi agent of the present disclosure include 5' phosphate or 5' phosphate mimic, for example, the 5' terminal phosphate or phosphate mimic on the antisense strand of RNAi agent.Suitable phosphate mimic is disclosed in, for example, US2012 / 0157511, the entire content of which is incorporated herein by reference.

[0291] A. Modified RNAi Agents Containing Motifs of the Disclosure In certain aspects of the present disclosure, the double-stranded RNAi agent of the present disclosure includes an agent having chemical modifications, for example, as disclosed in WO2013 / 075035, the entire contents of which are incorporated herein by reference.As shown herein and in WO2013 / 075035, excellent results can be obtained by introducing one or more motifs of three identical modifications on three consecutive nucleotides into the sense or antisense strand of the RNAi agent at or near the cleavage site.In some embodiments, the sense and antisense strands of the RNAi agent can be otherwise completely modified.The introduction of these motifs disrupts the modification pattern of the sense or antisense strand, if present.The RNAi agent can also be conjugated with a lipophilic ligand, for example, a C16 ligand on the sense strand.The RNAi agent can also be modified, for example, with (S)-glycol nucleic acid (GNA) modification at one or more residues of the antisense strand.The resulting RNAi agent exhibits excellent gene silencing activity.

[0292] Thus, the present disclosure provides double-stranded RNAi agents capable of inhibiting the expression of a target gene (i.e., the MAPT gene) in vivo. The RNAi agent comprises a sense strand and an antisense strand. Each strand of the RNAi agent can be 15 to 30 nucleotides in length. For example, each strand can be 16 to 30 nucleotides in length, 17 to 30 nucleotides in length, 25 to 30 nucleotides in length, 27 to 30 nucleotides in length, 17 to 23 nucleotides in length, 17 to 21 nucleotides in length, 17 to 19 nucleotides in length, 19 to 25 nucleotides in length, 19 to 23 nucleotides in length, 19 to 21 nucleotides in length, 21 to 25 nucleotides in length, or 21 to 23 nucleotides in length. In certain embodiments, each strand is 19 to 23 nucleotides in length.

[0293] The sense and antisense strands typically form a duplex, double-stranded RNA ("dsRNA"), also referred to herein as an "RNAi agent." The duplex region of an RNAi agent can be 15-30 nucleotide pairs in length. For example, the duplex region can be 16-30 nucleotide pairs in length, 17-30 nucleotide pairs in length, 27-30 nucleotide pairs in length, 17-23 nucleotide pairs in length, 17-21 nucleotide pairs in length, 17-19 nucleotide pairs in length, 19-25 nucleotide pairs in length, 19-23 nucleotide pairs in length, 19-21 nucleotide pairs in length, 21-25 nucleotide pairs in length, or 21-23 nucleotide pairs in length. In another example, the duplex region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides in length. In a preferred embodiment, the duplex region is 19-21 nucleotide pairs in length.

[0294] In one embodiment, the RNAi agent may contain one or more overhang regions or capping groups at the 3', 5', 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 a preferred embodiment, the nucleotide overhang region is 2 nucleotides in length. The overhang may be the result of one strand being longer than the other or the result of two strands of the same length being 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 joined by additional bases, for example, to form a hairpin, or by other non-basic linkers.

[0295] In one embodiment, the nucleotides in the overhang region of an RNAi 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), and any combination thereof.

[0296] 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.

[0297] The 5'- or 3'-overhang of the sense strand, antisense strand, or both strands of the RNAi agent can be phosphorylated. In some embodiments, the overhang region(s) contain two nucleotides with phosphorothioate between them, and the two nucleotides can be the same or different. In one embodiment, the overhang is present at the 3'-end of the sense strand, antisense strand, or both strands. In one embodiment, the 3'-overhang is present in the antisense strand. In one embodiment, the 3'-overhang is present in the sense strand.

[0298] RNAi 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 (or the 3' end of the sense strand), or vice versa.Generally, the antisense strand of RNAi has a nucleotide overhang at the 3' end, and the 5' end is blunt.Without wishing to be bound by theory, the 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 the RISC process.

[0299] In one embodiment, the RNAi 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.

[0300] In another embodiment, the RNAi 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.

[0301] In yet another embodiment, the RNAi 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.

[0302] In one embodiment, the RNAi agent comprises a 21-nucleotide sense strand and a 23-nucleotide antisense strand, wherein the sense strand comprises at least one motif of three 2'-F modifications 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 two-nucleotide overhang. Preferably, the two-nucleotide overhang is at the 3' end of the antisense strand. When the two-nucleotide overhang is at the 3' end of the antisense strand, there may be two phosphorothioate internucleotide linkages between the three terminal nucleotides, two of which are overhanging nucleotides, and the third nucleotide is the next paired nucleotide after the overhanging nucleotide. In one embodiment, the RNAi agent further comprises two phosphorothioate internucleotide linkages between the three terminal nucleotides at both the 5'-end of the sense strand and the 5'-end of the antisense strand.In one embodiment, every nucleotide in the sense strand and the antisense strand of the RNAi agent, including the nucleotide that is part of a motif, is a modified nucleotide.In one embodiment, each residue is independently modified with 2'-O-methyl or 3'-fluoro, for example, in the alternating motif.The RNAi agent may further comprise a ligand (for example, a lipophilic ligand, optionally a C16 ligand).

[0303] In one embodiment, the RNAi 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; and 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, wherein 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. 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 when the double-stranded nucleic acid is introduced into a mammalian cell, target gene expression is reduced, the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides, at least one of the motifs occurring at or near the cleavage site, and the antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at or near the cleavage site.

[0304] In one embodiment, the RNAi agent comprises a sense and an antisense strand, the RNAi 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 a target mRNA along the length of at least 19 nucleotides of the second strand, the RNAi agent reducing target gene expression when introduced into a mammalian cell, and Dicer cleavage of the RNAi agent preferentially producing siRNAs comprising the 3' end of the second strand, thereby reducing target gene expression in the mammal. Optionally, the RNAi agent may further comprise a ligand.

[0305] In one embodiment, the sense strand of the RNAi agent contains at least one motif of three identical modifications on three consecutive nucleotides, one of the motifs occurring at the cleavage site in the sense strand.

[0306] In one embodiment, the antisense strand of the RNAi agent can also contain at least one motif of three identical modifications on three consecutive nucleotides, one of the motifs occurring at or near the cleavage site in the antisense strand.

[0307] For RNAi agents with a duplex region 17-23 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 RNAi agent from the 5' end.

[0308] The sense strand of RNAi agent can contain at least one motif of three identical modifications on three consecutive nucleotides at the site of strand breakage, and antisense strand can have at least one motif of three identical modifications on three consecutive nucleotides at or near the site of strand breakage.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.

[0309] In one embodiment, the sense strand of an RNAi 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, away from a motif at or near the cleavage site of the same strand. The wing modifications may be adjacent to the first motif or separated by at least one or more nucleotides. When the motifs are immediately adjacent to each other, the 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 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.

[0310] Similar to the sense strand, the antisense strand of an RNAi agent may contain two or more motifs of three identical modifications on three consecutive nucleotides, with at least one of the motifs occurring at or near the site of strand cleavage.The antisense strand may also contain one or more wing modifications in the same sequence as the wing modifications that may be present on the sense strand.

[0311] In one embodiment, wing modifications on the sense or antisense strand of an RNAi agent typically do not include the first one or two terminal nucleotides at the 3' end, 5' end, or both ends of the strand.

[0312] In another embodiment, wing modifications on the sense or antisense strand of an RNAi agent typically do not include the first one or two paired nucleotides in the duplex region at the 3' end, 5' end, or both ends of the strand.

[0313] When the sense and antisense strands of an RNAi 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.

[0314] When the sense or antisense strand of an RNAi 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 from one strand occupy either side of the lead motif, with an overlap of 1, 2, or 3 nucleotides in the duplex region.

[0315] In one embodiment, the RNAi agent contains mismatch(es) or combinations thereof within the double strand with the target. Mismatches can occur in overhang regions or double-stranded regions. Base pairs can be ranked based on their tendency to promote dissociation or melting (e.g., the free energy of association or dissociation of a particular 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 preferred over G:C, G:U is preferred over G:C, and I:C is preferred over G:C (I=inosine). Mismatches, such as non-canonical pairings or those other than canonical pairings (as described elsewhere herein), are preferred over canonical (A:T, A:U, G:C) pairings, and pairings involving universal bases are preferred over canonical pairings.

[0316] In one embodiment, the RNAi 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.

[0317] In one embodiment, the nucleotide at position 1 in the double-stranded region from the 5' end of the antisense strand is selected from the group consisting of A, dA, dU, U and dT.Alternatively, at least one of the first 1, 2 or 3 base pairs in the double-stranded region from the 5' end of the antisense strand is an AU base pair.For example, the first base pair in the double-stranded region from the 5' end of the antisense strand is an AU base pair.

[0318] In another embodiment, the nucleotide at the 3' end of the sense strand is deoxythymine (dT). In another embodiment, the nucleotide at the 3' end of the antisense strand is deoxythymine (dT). In one embodiment, there is a short sequence of deoxythymine nucleotides, for example, two dT nucleotides, at the 3' end of either the sense or antisense strand.

[0319] In one embodiment, the sense strand sequence has formula (I): 5'n p -N a -(XX) i -N b -YY -N b -(Z) 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 bindependently 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. Preferably, all of YYY are 2'-F modified nucleotides.

[0320] In one embodiment, N a or N b includes alternating pattern modifications.

[0321] In one embodiment, the YYY motif occurs at or near the cleavage site of the sense strand. For example, if the RNAi agent has a duplex region 17-23 nucleotides in length, the YYY motif can occur at or near the cleavage site of the sense strand (e.g., can occur at positions 6, 7, 8, 7, 8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12, or 11, 12, 13), with the number starting from the first nucleotide from the 5' end, or, optionally, the number starting from the first paired nucleotide within the duplex region from the 5' end.

[0322] 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 -Na -XXX-N b -YYY-N b -ZZZ-N a -n q 3' (Id) It can be expressed as:

[0323] 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.

[0324] each N a can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0325] 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 containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0326] When the sense strand is represented by formula (Id), each N b independently represent an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. b is 0, 1, 2, 3, 4, 5 or 6. Each N a may independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0327] Each of X, Y and Z may be the same as or different from one another.

[0328] In other embodiments, i is 0, j is 0, and the sense strand has the formula: 5' n p -Na -YYY-N a -n q 3' (Ia) It can be expressed as:

[0329] When the sense strand is represented by formula (Ia), each N a may independently comprise an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0330] In one embodiment, the antisense strand sequence of the RNAi has formula (Ie): 5' n q’ -N a '-(Z'Z'Z') k -N b '-Y'Y'Y'-N b '-(X'X'X') l -N' a -n p ' 3' (IE) [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 comprising 0 to 25 modified nucleotides, each sequence comprising 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:

[0331] In one embodiment, N a ' or N b' includes alternating pattern modifications.

[0332] The Y'Y'Y' motif occurs at or near the cleavage site of the sense strand. For example, if the RNAi agent has a duplex region 17-23 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, where appropriate, the numbers starting from the first paired nucleotide in the duplex region from the 5' end. Preferably, the Y'Y'Y' motif occurs at positions 11, 12, 13.

[0333] In one embodiment, the Y'Y'Y' motif is all 2'-OMe modified nucleotides.

[0334] In one embodiment, k is 1 and l is 0, or k is 0 and l is 1, or k and l are both 1.

[0335] 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' (If), 5' n q’ -N a '-Y'Y'Y'-N b '-X'X'X'-n p’ 3' (Ig), or 5' n q’ -N a '-Z'Z'Z'-N b '-Y'Y'Y'-N b '-X'X'X'-N a '-n p’ 3' (Ih) It can be expressed as:

[0336] When the antisense strand is represented by formula (If), Nb ’ 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.

[0337] When the antisense strand is represented by formula (Ig), N b ' represents an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. a ' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0338] When the antisense strand is represented by formula (Ih), 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 ' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. b is 0, 1, 2, 3, 4, 5 or 6.

[0339] 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:

[0340] When the antisense strand is represented by formula (Ie), each N a ' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0341] Each of X', Y' and Z' may be the same as or different from one another.

[0342] Each nucleotide of sense strand and antisense strand can be independently modified with LNA, 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.

[0343] In one embodiment, the sense strand of the RNAi 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, where appropriate, the numbers may start from the 5' end with the first paired nucleotide in the duplex region, 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.

[0344] In one embodiment, 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 the opposite end of the duplex region, where X'X'X' and Z'Z'Z' each independently represent a 2'-OMe modification or a 2'-F modification.

[0345] 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 formulas (Ie), (If), (Ig) and (Ih), respectively.

[0346] Thus, an RNAi agent for use in the methods of the disclosure can include a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, and the RNAi duplex can have the formula (Ii): Sense: 5' n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3' Antisense: 3' n p ’ -N a ’ -(X'X'X') k -N b ’ -Y'Y'Y'-N b ’ -(Z'Z'Z') l -N a ’ -n q ’ 5' (II) [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

[0347] 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.

[0348] An exemplary combination of sense and antisense strands that form an RNAi duplex has 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' (Ij) 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' (Ik) 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' (Il) 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' (Im) Includes.

[0349] When the RNAi agent is represented by formula (Ij), each N a independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0350] When the RNAi agent is represented by formula (Ik), 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.

[0351] When the RNAi agent is represented by formula (II), each N b , N bEach N' independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0352] When the RNAi agent is represented by formula (Im), 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.

[0353] In one embodiment, when the RNAi agent is represented by formula (Im), N a The modification is a 2'-O-methyl or a 2'-fluoro modification. In another embodiment, when the RNAi agent is represented by formula (Im), 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 (Im), 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 pIn another embodiment, when the RNAi agent is represented by formula (Im), N' is linked to adjacent nucleotides via phosphorothioate linkages, and the sense strand is conjugated to one or more C16 (or related) moieties 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 ' are 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 lipophilic, e.g., C16 (or related) moieties, which may be attached by a divalent or trivalent branched linker.

[0354] In one embodiment, when the RNAi agent is represented by formula (Ij), N a The modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n p ' are 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 lipophilic, e.g., C16 (or related) moieties attached by a divalent or trivalent branched linker.

[0355] In one embodiment, the RNAi agent is a multimer containing at least two duplexes represented by formula (Ii), (Ij), (Ik), (Il) and (Im), and the duplexes are connected by a linker. The linker may be cleavable or non-cleavable. The multimer may further comprise a ligand. Each duplex may target the same gene, or may target two different genes, or each duplex may target the same gene at two different target sites.

[0356] In one embodiment, the RNAi agent is a multimer that contains 3, 4, 5, 6 or more double strands represented by formula (Ii), (Ij), (Ik), (Il) and (Im), and the double strands are connected by a linker. The linker may be cleavable or non-cleavable. The multimer may further comprise a ligand. Each double strand may target the same gene, or may target two different genes, or each double strand may target the same gene at two different target sites.

[0357] In one embodiment, two RNAi agents represented by formula (Ii), (Ij), (Ik), (Il) and (Im) are linked to each other at the 5' end, and one or both of the 3' ends may be conjugated to a ligand. Each of the agents may target the same gene, or may target two different genes, or each of the agents may target the same gene at two different target sites.

[0358] Various publications describe the multimeric RNAi agent that can be used in the method of the present disclosure.Such publications include WO2007 / 091269, WO2010 / 141511, WO2007 / 117686, WO2009 / 014887 and WO2011 / 031520 and US7858769, each of whose entire contents is incorporated herein by reference.

[0359] In certain embodiments, the compositions and methods of the present disclosure include vinyl phosphonate (VP) modifications of RNAi agents as described herein. In exemplary embodiments, the 5'-vinyl phosphonate modified nucleotides of the present disclosure have the following structure:

[0360] [ka] wherein X is O or S; R is hydrogen, hydroxy, fluoro, or C 1~20alkoxy (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. It has.

[0361] In one embodiment, R 5’ is ═C(H)—P(O)(OH) and the double bond between the C5′ carbon and R5′ is in the E configuration. In another embodiment, R is methoxy and R 5’ is ═C(H)—P(O)(OH) 2 and the double bond between the C5′ carbon and R5′ is in the E configuration. In another embodiment, X is S, R is methoxy, and R 5’ is =C(H)-P(O)(OH)2, and the double bond between the C5' carbon and R5' is in the E configuration.

[0362] 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 preferred embodiments, the vinyl phosphonate of the present disclosure is attached to the antisense strand of the dsRNA at the 5' end of the antisense strand of the dsRNA, as appropriate.

[0363] Vinyl phosphate modifications are also contemplated for the compositions and methods of the present disclosure. Exemplary vinyl phosphate structures include those shown above, where R 5’ is =C(H)-OP(O)(OH)2, and the double bond between the C5' carbon and R5' is in the E or Z configuration (e.g., the E configuration).

[0364] i.Thermal destabilization modification In certain embodiments, dsRNA molecules can be optimized for RNA interference by incorporating a thermally destabilizing modification into the seed region of the antisense strand (i.e., positions 2-9 at the 5' end of the antisense strand) to reduce or inhibit off-target gene silencing. It has been discovered that dsRNAs having an antisense strand containing at least one thermally destabilizing duplex 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) thermally destabilizing duplex modification within the first 9 nucleotide positions of the 5' region of the antisense strand. In some embodiments, one or more thermally destabilizing duplex modifications are located in positions 2-9, or preferably positions 4-8, from the 5' end of the antisense strand. In some further embodiments, the thermally destabilizing duplex modification(s) are located at positions 6, 7, or 8 from the 5' end of the antisense strand. In still some further embodiments, the thermally destabilizing modification of the duplex is located at position 7 from the 5' end of the antisense strand. The term "thermally destabilizing modification(s)" includes modification(s) that will result in a dsRNA having a lower overall melting temperature (Tm) (preferably 1, 2, 3, or 4 degrees lower than the Tm of a dsRNA without such modification(s). In some embodiments, the thermally destabilizing modification of the duplex is located at position 2, 3, 4, 5, or 9 from the 5' end of the antisense strand.

[0365] Thermally destabilizing modifications can include, but are not limited to, abasic modifications, mismatches with opposing nucleotides in the opposing strand, and sugar modifications such as 2'-deoxy modifications or acyclic nucleotides such as non-locked nucleic acids (UNAs) or glycol nucleic acids (GNAs).

[0366] Exemplary abasic modifications include, but are not limited to, the following:

[0367] [ka] wherein R=H, Me, Et, or OMe; R′=H, Me, Et, or OMe; and R″=H, Me, Et, or Ome.

[0368] [ka] wherein B is a modified or unmodified nucleobase. Examples include:

[0369] Exemplary sugar modifications include, but are not limited to, the following:

[0370] [ka] wherein B is a modified or unmodified nucleobase. Examples include:

[0371] In some embodiments, the thermally destabilizing modification of the duplex is one of the following:

[0372] [ka] where B is a modified or unmodified nucleobase and the asterisk on each structure represents either R, S or racemic. is selected from the group consisting of:

[0373] In some embodiments, the thermally destabilizing modification of the duplex is one of the following:

[0374] [ka] wherein B is a modified or unmodified nucleobase and the asterisk represents either R, S or racemic (e.g., S). is selected from the group consisting of:

[0375] The term "acyclic nucleotide" refers to any nucleotide having an acyclic ribose sugar, e.g., in which any of the bonds between the ribose carbons (e.g., C1'-C2', C2'-C3', C3'-C4', C4'-O4', or C1'-O4') are absent, or at least one of the ribose carbons or oxygens (e.g., C1', C2', C3', C4', or O4'), independently or in combination, is absent from the nucleotide. In some embodiments, an acyclic nucleotide is

[0376] [ka] wherein B is a modified or unmodified nucleobase and R 1 and R 2 are independently H, halogen, OR3, or alkyl; and R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar. Acyclic derivatives offer greater backbone flexibility without affecting Watson-Crick pairing. Acyclic nucleotides can be linked via 2'-5' or 3'-5' linkages.

[0377] The term "GNA" refers to glycol nucleic acid, a polymer similar to DNA or RNA, but differing in the composition of its "backbone" in that it is made up of repeating glycerol units linked by phosphodiester bonds:

[0378] [ka]

[0379] The thermally destabilizing modification of the double strand can be a mismatch (i.e., non-complementary base pair) between the thermally destabilizing nucleotide and the opposite nucleotide in the opposite strand of the dsRNA duplex.Exemplary mismatch base pairs include G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T, U:T, or combinations thereof.Other mismatch base pairings known in the art are also suitable for the present disclosure.Mismatches can occur between nucleotides that are either naturally occurring or modified nucleotides, that is, mismatch base pairing can occur between the nucleobases derived from each nucleotide independently of the modification on the ribose sugar of the nucleotide.In certain embodiments, the dsRNA molecule contains at least one nucleobase in mismatch pairing that is a 2'-deoxynucleobase, for example, the 2'-deoxynucleobase is in the sense strand.

[0380] In some embodiments, the duplex thermally destabilizing modification in the seed region of the antisense strand is a nucleotide that has impaired WCH bonding with a complementary base on the target mRNA, such as:

[0381] [ka] Includes.

[0382] Many more examples of abasic nucleotides, acyclic nucleotide modifications (including UNA and GNA) and mismatch modifications are described in detail in WO2011 / 133876, which is incorporated herein by reference in its entirety.

[0383] Thermally destabilizing modifications can also include universal base and phosphate modifications that have reduced or eliminated ability to form hydrogen bonds with opposing bases.

[0384] In some embodiments, the thermal destabilizing modification of duplex comprises the nucleotide with non-canonical base, for example but not limited to, the nucleobase modification that the ability to form hydrogen bond with the base in opposite strand is impaired or completely lost.These nucleobase modifications have been evaluated for the destabilization of the central region of dsRNA duplex, as described in WO2010 / 0011895, the entirety of which is incorporated herein by reference.Exemplary nucleobase modifications include:

[0385] [ka] There is.

[0386] In some embodiments, the duplex thermally destabilizing modifications in the seed region of the antisense strand include one or more α-nucleotides that are complementary to bases on the target mRNA, such as the following:

[0387] [ka] wherein R is H, OH, OCH, F, NH, NHMe, NMe, or O-alkyl. Includes:

[0388] Exemplary phosphate modifications known to reduce the thermal stability of dsRNA duplexes compared to native phosphodiester linkages include:

[0389] [ka] There is.

[0390] The alkyl R group can be a C1-C6 alkyl. Particular alkyl R groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, pentyl, and hexyl.

[0391] As those skilled in the art will recognize, considering that the functional role of nucleobase defines the specificity of the RNAi agent of the present disclosure, nucleobase modification can be carried out in various ways as described herein, for example, for the purpose of enhancing on-target effect against off-target effect, for example, for introducing destabilizing modifications into the RNAi agent of the present disclosure, but the range of modifications available and generally present on the RNAi agent of the present disclosure tends to be greater for non-nucleobase modifications, for example, modifications to the sugar group or phosphate backbone of polyribonucleotide.Such modifications are described in more detail in other sections of this disclosure, and are expressly intended for the RNAi agent of the present disclosure that has either natural nucleobase or modified nucleobase, as described above or elsewhere herein.

[0392] In addition to the antisense strand that contains thermal destabilizing modifications, dsRNA can also contain one or more stabilizing modifications.For example, dsRNA can contain at least two (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) stabilizing modifications.Without being limited, all stabilizing modifications can be present in one strand.In some embodiments, both sense and antisense strands contain at least two stabilizing modifications.Stabilizing modifications can occur at any nucleotide of sense strand or antisense strand.For example, stabilizing modifications can occur at any nucleotide on sense strand or antisense strand, and each stabilizing modification can occur in an alternating pattern on sense strand or antisense strand, or both sense strand and antisense strand contain stabilizing modifications in an alternating pattern.The alternating pattern of stabilizing modifications on sense strand can be the same or different from that of antisense strand, and the alternating pattern of stabilizing modifications on sense strand can have a shift compared to the alternating pattern of stabilizing modifications on antisense strand.

[0393] In some embodiments, the antisense strand comprises at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) stabilizing modifications. Without limitation, the stabilizing modifications in the antisense strand can be located at any position. In some embodiments, the antisense strand comprises stabilizing modifications at positions 2, 6, 8, 9, 14, and 16 from the 5' end. In some other embodiments, the antisense strand comprises stabilizing modifications at positions 2, 6, 14, and 16 from the 5' end. In yet some other embodiments, the antisense strand comprises stabilizing modifications at positions 2, 14, and 16 from the 5' end.

[0394] In some embodiments, antisense strand comprises at least one stabilizing modification adjacent to destabilizing modification.For example, stabilizing modification can be at the 5'-end or 3'-end of destabilizing modification, that is, at the nucleotide of position -1 or +1 from the position of destabilizing modification.In some embodiments, antisense strand comprises stabilizing modification at each of the 5'-end and 3'-end of destabilizing modification, that is, at the nucleotide of position -1 and +1 from the position of destabilizing modification.

[0395] In some embodiments, the antisense strand comprises at least two stabilizing modifications at the 3' end of the destabilizing modification, ie, at positions +1 and +2 from the position of the destabilizing modification.

[0396] In some embodiments, the sense strand comprises at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) stabilizing modifications. Without limitation, the stabilizing modifications in the sense strand can be located at any position. In some embodiments, the sense strand comprises stabilizing modifications at positions 7, 10, and 11 from the 5' end. In some other embodiments, the sense strand comprises stabilizing modifications at positions 7, 9, 10, and 11 from the 5' end. In some embodiments, the sense strand comprises stabilizing modifications at positions opposite or complementary to positions 11, 12, and 15 of the antisense strand, counting from the 5' end of the antisense strand. In some other embodiments, the sense strand comprises stabilizing modifications at positions opposite or complementary to positions 11, 12, 13, and 15 of the antisense strand, counting from the 5' end of the antisense strand. In some embodiments, the sense strand comprises blocks of two, three, or four stabilizing modifications.

[0397] In some embodiments, the sense strand does not contain a stabilizing modification at a position opposite or complementary to a thermally destabilizing modification of the duplex in the antisense strand.

[0398] Exemplary thermally stabilizing modifications include, but are not limited to, 2'-fluoro modifications. Other thermally stabilizing modifications include, but are not limited to, LNA.

[0399] In some embodiments, the dsRNA of the present disclosure comprises at least four (for example, 4, 5, 6, 7, 8, 9, 10 or more) 2'-fluoro nucleotides. Without being limited thereto, all 2'-fluoro nucleotides may be present in one strand. In some embodiments, both sense and antisense strands comprise at least two 2'-fluoro nucleotides. 2'-fluoro modification can occur at any nucleotide of sense strand or antisense strand. For example, 2'-fluoro modification can occur at any nucleotide on sense strand or antisense strand, and each 2'-fluoro modification can occur in an alternating pattern on sense strand or antisense strand, or both sense strand and antisense strand contain 2'-fluoro modification in an alternating pattern. The alternating pattern of 2'-fluoro modification on sense strand can be the same or different from that of antisense strand, and the alternating pattern of 2'-fluoro modification on sense strand can have a shift compared to the alternating pattern of 2'-fluoro modification on antisense strand.

[0400] In some embodiments, the antisense strand comprises at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-fluoro nucleotides. Without limitation, the 2'-fluoro modifications in the antisense strand can be present at any position. In some embodiments, the antisense strand comprises 2'-fluoro nucleotides at positions 2, 6, 8, 9, 14 and 16 from the 5' end. In some other embodiments, the antisense strand comprises 2'-fluoro nucleotides at positions 2, 6, 14 and 16 from the 5' end. In yet some other embodiments, the antisense strand comprises 2'-fluoro nucleotides at positions 2, 14 and 16 from the 5' end.

[0401] In some embodiments, antisense strand comprises at least one 2'-fluoro nucleotide adjacent to destabilizing modification.For example, 2'-fluoro nucleotide can be at the 5' end or 3' end of destabilizing modification, that is, at the nucleotide of position -1 or +1 from the position of destabilizing modification.In some embodiments, antisense strand comprises 2'-fluoro nucleotide at each of the 5' end and 3' end of destabilizing modification, that is, at position -1 and +1 from the position of destabilizing modification.

[0402] In some embodiments, the antisense strand comprises at least two 2'-fluoro nucleotides at the 3' end of the destabilizing modification, ie, at positions +1 and +2 from the position of the destabilizing modification.

[0403] In some embodiments, the sense strand comprises at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-fluoro nucleotides. Without limitation, the 2'-fluoro modifications in the sense strand can be present at any position. In some embodiments, the antisense strand comprises 2'-fluoro nucleotides at positions 7, 10 and 11 from the 5' end. In some other embodiments, the sense strand comprises 2'-fluoro nucleotides at positions 7, 9, 10 and 11 from the 5' end. In some embodiments, the sense strand comprises 2'-fluoro nucleotides at positions opposite or complementary to positions 11, 12 and 15 of the antisense strand, counting from the 5' end of the antisense strand. In some other embodiments, the sense strand comprises 2'-fluoro nucleotides at positions opposite or complementary to positions 11, 12, 13 and 15 of the antisense strand, counting from the 5' end of the antisense strand. In some embodiments, the sense strand comprises 2'-fluoro nucleotides at positions opposite or complementary to positions 11, 12, 13 and 15 of the antisense strand, counting from the 5' end of the antisense strand. In some embodiments, the sense strand comprises 2, 3 or 4 blocks of 2'-fluoro nucleotides.

[0404] In some embodiments, the sense strand does not contain 2'-fluoro nucleotides at positions opposite or complementary to thermally destabilizing modifications of the duplex in the antisense strand.

[0405] In some embodiments, a dsRNA molecule of the present disclosure comprises a 21-nucleotide (nt) sense strand and a 23-nucleotide (nt) antisense strand, wherein the antisense strand contains at least one thermally destabilized nucleotide, wherein the at least one thermally destabilized nucleotide occurs in the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), one end of the dsRNA is blunt and the other end comprises a 2-nt overhang, and the dsRNA further has at least one (e.g., 1, 2, 3, 4, 5, 6, or all 7) of the following features: The dsRNA may have: (i) two, three, four, five, or six 2'-fluoro modifications in the antisense strand; (ii) one, two, three, four, or five phosphorothioate internucleotide linkages in the antisense strand; (iii) the sense strand is conjugated with a ligand; (iv) the sense strand has two, three, four, or five 2'-fluoro modifications; (v) the sense strand has one, two, three, four, or five phosphorothioate internucleotide linkages; (vi) the dsRNA has at least four 2'-fluoro modifications; and (vii) the dsRNA has a blunt end at the 5' end of the antisense strand. Preferably, a 2-nt overhang is at the 3' end of the antisense strand.

[0406] In some embodiments, a dsRNA molecule of the present disclosure comprises a sense and an antisense strand, wherein the sense strand is 25 to 30 nucleotide residues in length, and starting from the 5'-terminal nucleotide (position 1), positions 1 to 23 of the sense strand comprise at least 8 ribonucleotides; the antisense strand is 36 to 66 nucleotide residues in length, and starting from the 3'-terminal nucleotide, at least 8 ribonucleotides in positions paired with positions 1 to 23 of the sense strand form a duplex; at least the 3'-terminal nucleotide of the antisense strand is not paired with the sense strand; and up to 6 consecutive 3'-terminal nucleotides are not paired with the sense strand, thereby forming a 3' single-stranded overhang of 1 to 6 nucleotides; and the 5' end of the antisense strand is 10 to 30 consecutive nucleotides that are not paired with the sense strand. wherein at least the 5'- and 3'-terminal nucleotides of the sense strand are base-paired with nucleotides in the antisense strand when the sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially double-stranded region between the sense and antisense strands; the antisense strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the length of the antisense strand, such that when the double-stranded nucleic acid is introduced into a mammalian cell, target gene expression is reduced; and the antisense strand contains at least one thermally destabilizing nucleotide, the at least one thermally destabilizing nucleotide being in the seed region of the antisense strand (i.e., at positions 2-9 of the 5'-end of the antisense strand).For example, the thermally destabilizing nucleotide occurs between positions 14-17 at the 5' end of the sense strand and the positions opposite or complementary to them, and the dsRNA may further have at least one (e.g., one, two, three, four, five, six, or all seven) of the following features: (i) the antisense strand contains two, three, four, five, or six 2'-fluoro modifications; (ii) the antisense strand contains one, two, three, four, or five phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated to a ligand; (iv) the sense strand contains two, three, four, or five 2'-fluoro modifications; (v) the sense strand contains one, two, three, four, or five phosphorothioate internucleotide linkages; (vi) the dsRNA contains at least four 2'-fluoro modifications; and (vii) the dsRNA contains a duplex region 12-30 nucleotide pairs in length.

[0407] In some embodiments, a dsRNA molecule of the present disclosure comprises a sense and an antisense strand, wherein the dsRNA molecule comprises a sense strand having a length of at least 25 and at most 29 nucleotides and an antisense strand having a length of at most 30 nucleotides, wherein the sense strand comprises a modified nucleotide at position 11 from its 5' end that is susceptible to enzymatic degradation, wherein the 3' end of the sense strand and the 5' end of the antisense strand form a blunt end, wherein the antisense strand is 1-4 nucleotides longer at its 3' end than the sense strand, wherein the duplex region is at least 25 nucleotides in length, and wherein the antisense strand is sufficiently complementary to a target mRNA along at least 19 nucleotides of the length of the antisense strand, wherein the dsRNA molecule reduces target gene expression when introduced into a mammalian cell, wherein Dicer cleavage of the dsRNA preferentially yields siRNA comprising the 3' end of the antisense strand, thereby reducing target gene expression in the mammal, and wherein the antisense strand wherein at least one thermally destabilized nucleotide is in the seed region of the antisense strand (i.e., positions 2-9 at the 5' end of the antisense strand), and the dsRNA may further have at least one (e.g., one, two, three, four, five, six, or all seven) of the following features: (i) the antisense contains two, three, four, five, or six 2'-fluoro modifications; (ii) the antisense contains two, three, four, five, or six 2'-fluoro modifications; (ii) the antisense strand contains 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated to a ligand; (iv) the sense strand contains 2, 3, 4, or 5 2'-fluoro modifications; (v) the sense strand contains 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (vi) the dsRNA contains at least four 2'-fluoro modifications; and (vii) the dsRNA has a duplex region 12 to 29 nucleotide pairs in length.

[0408] In some embodiments, any nucleotide in the sense strand and antisense strand of dsRNA molecule can be modified.Each nucleotide can be modified with the same or different modifications, which can include one or more of non-linked phosphate oxygen or one or more of linking phosphate oxygen, one or both of the modifications of ribose sugar components, 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.

[0409] 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 at phosphate moieties. In some cases, modifications occur at all target positions in nucleic acids, but in many cases, they do not occur. For example, modifications can occur only at the 3' or 5' terminal position, or only in the terminal region, for example, at the terminal nucleotide position of the chain, or at the last 2, 3, 4, 5, or 10 nucleotides. Modifications can occur in double-stranded regions, single-stranded regions, or both. Modifications can 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 can occur only at one or both ends, or only in the terminal region, for example, at the terminal nucleotide position of the chain, or at 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.

[0410] 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.

[0411] In some embodiments, each residue of the sense strand and the antisense strand is independently modified with locked nucleic acid (LNA), non-locked nucleic acid (UNA), cyclohexene nucleic acid (CeNA), 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, or 2'-fluoro. Strands can contain two or more modifications. In some embodiments, each residue of the sense strand and the antisense strand is independently modified with 2'-O-methyl or 2'-fluoro. It should be understood that these modifications are in addition to at least one thermally destabilizing modification of the duplex present in the antisense strand.

[0412] At least two different modifications are usually present on the sense strand and the antisense strand. These two modifications can be 2'-deoxy, 2'-O-methyl or 2'-fluoro modifications, acyclic nucleotides, etc. In some embodiments, the sense strand and the antisense strand each contain two differently modified nucleotides selected from 2'-O-methyl or 2'-deoxy. In some embodiments, each residue of the sense strand and the antisense strand is independently modified with 2'-O-methyl nucleotides, 2'-deoxy nucleotides, 2'-deoxy-2'-fluoro nucleotides, 2'-ON-methylacetamide (2'-O-NMA) nucleotides, 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) nucleotides, 2'-O-aminopropyl (2'-O-AP) nucleotides, or 2'-ara-F nucleotides. It should be understood that these modifications are in addition to at least one thermally destabilizing modification of the duplex present in the antisense strand.

[0413] In some embodiments, the dsRNA molecule of the present disclosure comprises alternating pattern modifications, particularly in the B1, B2, B3, B1', B2', B3', and B4' regions.The term "alternating motif" or "alternating pattern" as used herein refers to a motif with one or more modifications, where each modification occurs at alternating nucleotides in a single strand.Alternating nucleotides can refer to one every other nucleotide or one every three nucleotides, or similar patterns.For example, if A, B, and C each represent one type of modification to nucleotide, the alternating motif can be "ABABABABABAB...", "AABBAABBAABB...", "AABAABAABAAB...", "AAABAAABAAAB...", "AABBBAAABBB..." or "ABCABCABCABC...", etc.

[0414] 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...".

[0415] In some embodiments, the dsRNA molecules of the present disclosure comprise 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' end of the strand, and the alternating motif in the antisense strand may start with "BABABA" from the 3'-5' end of the strand within the duplex region. As another example, the alternating motif in the sense strand may start with "AABBAABB" from the 5'-3' end of the strand, and the alternating motif in the antisense strand may start with "BBAABBAA" from the 3'-5' end of the strand within the duplex region, resulting in a complete or partial shift in the modification pattern between the sense strand and the antisense strand.

[0416] The dsRNA molecule of the present disclosure can further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage.Phosphorothioate or methylphosphonate internucleotide linkage modification can occur at any nucleotide of sense strand or antisense strand or both at any position of chain.For example, internucleotide linkage modification can occur at any nucleotide on sense strand or antisense strand, and each internucleotide linkage modification can occur in an alternating pattern on sense strand or antisense strand, or sense strand or antisense strand contains both internucleotide linkage modifications in an alternating pattern.The alternating pattern of internucleotide linkage modification on sense strand can be the same or different from that of antisense strand, and the alternating pattern of internucleotide linkage modification on sense strand can have a shift with respect to the alternating pattern of internucleotide linkage modification on antisense strand.

[0417] In some embodiments, dsRNA molecule comprises phosphorothioate or methylphosphonate internucleotide linkage modification in overhang region.For example, overhang region comprises two nucleotides with phosphorothioate or methylphosphonate internucleotide linkage between the two nucleotides.Internucleotide linkage modification can also be performed to connect overhang nucleotide with the terminal pairing nucleotide in duplex 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 with the pairing nucleotide adjacent to overhang nucleotide.For example, there can be at least two phosphorothioate internucleotide linkages between the terminal three nucleotides, two of which are overhang nucleotides, and the third is the pairing nucleotide adjacent to overhang nucleotide.Preferably, these terminal three nucleotides can be the 3' end of antisense strand.

[0418] In some embodiments, the sense strand of the dsRNA molecule comprises 1 to 10 blocks of 2 to 10 phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is positioned at any position in the oligonucleotide sequence, and the sense strand is paired with an antisense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0419] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of two phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages and an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0420] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of three phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate internucleotide linkages, one of the phosphorothioate or methylphosphonate internucleotide linkages being located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0421] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of four phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 phosphate internucleotide linkages, one of the phosphorothioate or methylphosphonate internucleotide linkages being located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0422] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of five phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 phosphate internucleotide linkages, one of the phosphorothioate or methylphosphonate internucleotide linkages being located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0423] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of six phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphate internucleotide linkages, one of the phosphorothioate or methylphosphonate internucleotide linkages being located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0424] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of seven phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, or 8 phosphate internucleotide linkages, one of the phosphorothioate or methylphosphonate internucleotide linkages being located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0425] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of eight phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, or 6 phosphate internucleotide linkages, one of the phosphorothioate or methylphosphonate internucleotide linkages being located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0426] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of nine phosphorothioate or methylphosphonate internucleotide linkages separated by one, two, three, or four phosphate internucleotide linkages, one of the phosphorothioate or methylphosphonate internucleotide linkages being located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0427] In some embodiments, the dsRNA molecules of this disclosure further comprise one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within the 1-10 terminal positions of the sense or antisense strand. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides can be linked by phosphorothioate or methylphosphonate internucleotide linkages at one or both termini of the sense or antisense strand.

[0428] In some embodiments, dsRNA molecules of the present disclosure further comprise one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 1-10 of the internal region of the duplex of each of the sense or antisense strands. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides can be linked by phosphorothioate methylphosphonate internucleotide linkages at positions 8-16 of the duplex region, counting from the 5' end of the sense strand. The dsRNA molecule may further comprise one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 1-10 of the terminal positions.

[0429] In some embodiments, dsRNA molecules of this disclosure further comprise one to five phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 1-5 of the sense strand and one to five phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) and one to five phosphorothioate or methylphosphonate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and one to five within positions 18-23 (counting from the 5' end).

[0430] In some embodiments, dsRNA molecules of this disclosure further comprise one phosphorothioate internucleotide linkage modification within positions 1-5 and one phosphorothioate or methylphosphonate internucleotide linkage modification within positions 18-23 (counting from the 5' end) of the sense strand and one phosphorothioate internucleotide linkage modification at position 1 or 2 and two phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) of the antisense strand.

[0431] In some embodiments, dsRNA molecules of this disclosure further comprise two phosphorothioate internucleotide linkage modifications within positions 1-5 and one phosphorothioate internucleotide linkage modification within positions 18-23 (counting from the 5' end) of the sense strand and one phosphorothioate internucleotide linkage modification at position 1 or 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) of the antisense strand.

[0432] In some embodiments, dsRNA molecules of this disclosure further comprise two phosphorothioate internucleotide linkage modifications within positions 1-5 and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) of the sense strand and one phosphorothioate internucleotide linkage modification at position 1 or 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) of the antisense strand.

[0433] In some embodiments, dsRNA molecules of this disclosure further comprise two phosphorothioate internucleotide linkage modifications within positions 1-5 and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) of the sense strand and one phosphorothioate internucleotide linkage modification at position 1 or 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 (counting from the 5' end) of the antisense strand.

[0434] In some embodiments, dsRNA molecules of this disclosure further comprise one phosphorothioate internucleotide linkage modification within positions 1-5 and one phosphorothioate internucleotide linkage modification within positions 18-23 (counting from the 5' end) of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) of the antisense strand.

[0435] In some embodiments, dsRNA molecules of this disclosure further comprise one phosphorothioate internucleotide linkage modification within positions 1-5 and one within positions 18-23 (counting from the 5' end) of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 (counting from the 5' end) of the antisense strand.

[0436] In some embodiments, dsRNA molecules of this disclosure further comprise one phosphorothioate internucleotide linkage modification 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 one phosphorothioate internucleotide linkage modification within positions 18-23 (counting from the 5' end).

[0437] In some embodiments, dsRNA molecules of this disclosure further comprise two phosphorothioate internucleotide linkage modifications (counting from the 5' end) within positions 1-5 of the sense strand and one phosphorothioate internucleotide linkage modification at position 1 or 2 and two phosphorothioate internucleotide linkage modifications (counting from the 5' end) within positions 18-23 of the antisense strand.

[0438] In some embodiments, dsRNA molecules of this disclosure further comprise two phosphorothioate internucleotide linkage modifications within positions 1-5 and one within positions 18-23 (counting from the 5' end) of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 (counting from the 5' end) of the antisense strand.

[0439] In some embodiments, dsRNA molecules of this disclosure further comprise two phosphorothioate internucleotide linkage modifications within positions 1-5 and one phosphorothioate internucleotide linkage modification within positions 18-23 (counting from the 5' end) of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) of the antisense strand.

[0440] In some embodiments, dsRNA molecules of this disclosure further comprise two phosphorothioate internucleotide linkage modifications within positions 1-5 and one phosphorothioate internucleotide linkage modification within positions 18-23 (counting from the 5' end) of the sense strand and one phosphorothioate internucleotide linkage modification at position 1 or 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) of the antisense strand.

[0441] In some embodiments, a dsRNA molecule of the present disclosure further comprises two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 20 and 21 (counting from the 5' end) of the sense strand and one phosphorothioate internucleotide linkage modification at position 1 and one at position 21 (counting from the 5' end) of the antisense strand.

[0442] In some embodiments, dsRNA molecules of the present disclosure further comprise one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 20 and 21 (counting from the 5' end) of the antisense strand.

[0443] In some embodiments, dsRNA molecules of the present disclosure further comprise two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 21 and 22 (counting from the 5' end) of the sense strand and one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the antisense strand.

[0444] In some embodiments, dsRNA molecules of the present disclosure further comprise one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 21 and 22 (counting from the 5' end) of the antisense strand.

[0445] In some embodiments, dsRNA molecules of the present disclosure further comprise two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 22 and 23 (counting from the 5' end) of the sense strand and one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the antisense strand.

[0446] In some embodiments, dsRNA molecules of the present disclosure further comprise one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 23 and 23 (counting from the 5' end) of the antisense strand.

[0447] In some embodiments, the compounds of the present disclosure comprise a pattern of backbone chiral centers. In some embodiments, the general pattern of backbone chiral centers comprises at least 5 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 6 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 7 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 8 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 9 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 10 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 11 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 12 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 13 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 14 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 15 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 16 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 17 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 18 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 19 internucleotide linkages in the Sp configuration.In some embodiments, the general pattern of backbone chiral centers comprises eight or fewer internucleotide linkages in the Rp configuration. In some embodiments, the general pattern of backbone chiral centers comprises seven or fewer internucleotide linkages in the Rp configuration. In some embodiments, the general pattern of backbone chiral centers comprises six or fewer internucleotide linkages in the Rp configuration. In some embodiments, the general pattern of backbone chiral centers comprises five or fewer internucleotide linkages in the Rp configuration. In some embodiments, the general pattern of backbone chiral centers comprises four or fewer internucleotide linkages in the Rp configuration. In some embodiments, the general pattern of backbone chiral centers comprises three or fewer internucleotide linkages in the Rp configuration. In some embodiments, the general pattern of backbone chiral centers comprises two or fewer internucleotide linkages in the Rp configuration. In some embodiments, the general pattern of backbone chiral centers comprises one or fewer internucleotide linkages in the Rp configuration. In some embodiments, the general pattern of backbone chiral centers comprises eight or fewer non-chiral internucleotide linkages (phosphodiesters, as a non-limiting example). In some embodiments, the general pattern of backbone chiral centers comprises seven or fewer non-chiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers comprises six or fewer non-chiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers comprises five or fewer non-chiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers comprises four or fewer non-chiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers comprises three or fewer non-chiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers comprises two or fewer non-chiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers comprises one or fewer non-chiral internucleotide linkages.In some embodiments, the general pattern of backbone chiral centers comprises at least 10 internucleotide linkages in the Sp configuration and no more than 8 nonchiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers comprises at least 11 internucleotide linkages in the Sp configuration and no more than 7 nonchiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers comprises at least 12 internucleotide linkages in the Sp configuration and no more than 6 nonchiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers comprises at least 13 internucleotide linkages in the Sp configuration and no more than 6 nonchiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers comprises at least 14 internucleotide linkages in the Sp configuration and no more than 5 nonchiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers comprises at least 15 internucleotide linkages in the Sp configuration and no more than 4 nonchiral internucleotide linkages. In some embodiments, the internucleotide linkages in the Sp configuration may be contiguous or non-contiguous. In some embodiments, the internucleotide linkages in the Rp configuration may or may not be contiguous. In some embodiments, the non-chiral internucleotide linkages may or may not be contiguous.

[0448] In some embodiments, compounds of the present disclosure include blocks that are stereochemical blocks. In some embodiments, a block is an Rp block, in that each internucleotide linkage of the block is Rp. In some embodiments, the 5'-block is an Rp block. In some embodiments, the 3'-block is an Rp block. In some embodiments, a block is an Sp block, in that each internucleotide linkage of the block is Sp. In some embodiments, the 5'-block is an Sp block. In some embodiments, the 3'-block is an Sp block. In some embodiments, provided oligonucleotides include both Rp and Sp blocks. In some embodiments, provided oligonucleotides include one or more Rp blocks but do not include Sp blocks. In some embodiments, provided oligonucleotides include one or more Sp blocks but do not include Rp blocks. In some embodiments, provided oligonucleotides include one or more PO blocks in which each internucleotide linkage is a natural phosphate linkage.

[0449] In some embodiments, compounds of the present disclosure include a 5'-block that is an Sp block in which each sugar moiety comprises a 2'-F modification. In some embodiments, the 5'-block is an Sp block in which each internucleotide linkage is a modified internucleotide linkage and each sugar moiety comprises a 2'-F modification. In some embodiments, the 5'-block is an Sp block in which each internucleotide linkage is a phosphorothioate linkage and each sugar moiety comprises a 2'-F modification. In some embodiments, the 5'-block comprises four or more nucleoside units. In some embodiments, the 5'-block comprises five or more nucleoside units. In some embodiments, the 5'-block comprises six or more nucleoside units. In some embodiments, the 5'-block comprises seven or more nucleoside units. In some embodiments, the 3'-block is an Sp block in which each sugar moiety comprises a 2'-F modification. In some embodiments, the 3'-block is an Sp block in which each internucleotide linkage is a modified internucleotide linkage and each sugar moiety comprises a 2'-F modification. In some embodiments, the 3'-block is an Sp block in which each of the internucleotide linkages is a phosphorothioate linkage and each sugar moiety includes a 2'-F modification. In some embodiments, the 3'-block includes 4 or more nucleoside units. In some embodiments, the 3'-block includes 5 or more nucleoside units. In some embodiments, the 3'-block includes 6 or more nucleoside units. In some embodiments, the 3'-block includes 7 or more nucleoside units.

[0450] In some embodiments, compounds of the disclosure include a certain type of nucleoside in a region, or an oligonucleotide is followed by a particular type of internucleotide linkage, such as a natural phosphate linkage, a modified internucleotide linkage, an Rp chiral internucleotide linkage, an Sp chiral internucleotide linkage, etc. In some embodiments, A is followed by Sp. In some embodiments, A is followed by Rp. In some embodiments, A is followed by a natural phosphate linkage (PO). In some embodiments, U is followed by Sp. In some embodiments, U is followed by Rp. In some embodiments, U is followed by a natural phosphate linkage (PO). In some embodiments, C is followed by Sp. In some embodiments, C is followed by Rp. In some embodiments, C is followed by a natural phosphate linkage (PO). In some embodiments, G is followed by Sp. In some embodiments, G is followed by Rp. In some embodiments, G is followed by a natural phosphate linkage (PO). In some embodiments, C and U are followed by Sp. In some embodiments, C and U are followed by Rp. In some embodiments, C and U are followed by a natural phosphate linkage (PO). In some embodiments, A and G are followed by Sp. In some embodiments, A and G are followed by Rp.

[0451] In some embodiments, the antisense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 21 and 22 and between nucleotide positions 22 and 23, the antisense strand contains at least one thermally destabilizing modification of the duplex located in the seed region of the antisense strand (i.e., at positions 2-9 at the 5' end of the antisense strand), and the dsRNA may further have at least one (e.g., one, two, three, four, five, six, seven, or all eight) of the following features: (i) the antisense strand comprises two, three, four, five, or six 2'-fluoro modifications; (i) the antisense strand contains 3, 4, or 5 phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated to a ligand; (iv) the sense strand contains 2, 3, 4, or 5 2'-fluoro modifications; (v) the sense strand contains 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (vi) the dsRNA contains at least four 2'-fluoro modifications; (vii) the dsRNA contains a duplex region 12 to 40 nucleotide pairs in length; and (viii) the dsRNA has a blunt end at the 5' end of the antisense strand.

[0452] In some embodiments, the antisense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23, the antisense strand contains at least one thermally destabilizing modification of the duplex located in the seed region of the antisense strand (i.e., at positions 2-9 at the 5' end of the antisense strand), and the dsRNA may further have at least one (e.g., 1, 2, 3, 4, 5, 6, 7, or all 8) of the following features: (i) the antisense strand has 2, 3, 4, 5, or 6 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; (ii) the sense strand is conjugated to a ligand; (iii) the sense strand comprises two, three, four, or five 2'-fluoro modifications; (iv) the sense strand comprises one, two, three, four, or five phosphorothioate internucleotide linkages; (v) the dsRNA comprises at least four 2'-fluoro modifications; (vi) the dsRNA comprises a duplex region 12 to 40 nucleotide pairs in length; (vii) the dsRNA comprises a duplex region 12 to 40 nucleotide pairs in length; and (viii) the dsRNA has a blunt end at the 5' end of the antisense strand.

[0453] In some embodiments, the sense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3, the antisense strand contains at least one thermally destabilizing modification of the duplex located in the seed region of the antisense strand (i.e., positions 2-9 at the 5' end of the antisense strand), and the dsRNA may further have at least one (e.g., one, two, three, four, five, six, seven, or all eight) of the following features: (i) the antisense strand comprises two, three, four, five, or six 2'-fluoro modifications; (ii) the antisense strand comprises two, three, four, five, or six 2'-fluoro modifications; (iii) the sense strand contains 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (iv) the sense strand contains 2, 3, 4, or 5 2'-fluoro modifications; (v) the sense strand contains 3, 4, or 5 phosphorothioate internucleotide linkages; (vi) the dsRNA contains at least four 2'-fluoro modifications; (vii) the dsRNA contains a duplex region 12 to 40 nucleotide pairs in length; and (viii) the dsRNA has a blunt end at the 5' end of the antisense strand.

[0454] In some embodiments, the sense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3, the antisense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23, the antisense strand contains at least one thermally destabilizing modification of the duplex located in the seed region of the antisense strand (i.e., at positions 2-9 at the 5' end of the antisense strand), and the dsRNA has at least one of the following characteristics (e.g., 1, (ii) the sense strand is conjugated to a ligand; (iii) the sense strand comprises two, three, four, or five 2'-fluoro modifications; (iv) the sense strand comprises three, four, or five phosphorothioate internucleotide linkages; (v) the dsRNA comprises at least four 2'-fluoro modifications; (vi) the dsRNA comprises a duplex region 12 to 40 nucleotide pairs in length; and (vii) the dsRNA has a blunt end at the 5' end of the antisense strand.

[0455] In some embodiments, the dsRNA molecule of the present disclosure comprises mismatch(es) or combinations thereof in the double strand with the target. Mismatches can occur in overhang regions or double-stranded regions. Base pairs can be ranked based on their tendency to promote dissociation or melting (for example, the free energy of association or dissociation of a particular 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 preferred over G:C, G:U is preferred over G:C, and I:C is preferred over G:C (I=inosine). Mismatches, such as non-canonical pairings or those other than canonical pairings (as described elsewhere herein), are preferred over canonical (A:T, A:U, G:C) pairings, and pairings involving universal bases are preferred over canonical pairings.

[0456] In some embodiments, the dsRNA molecules of the present disclosure comprise 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, which can be independently selected from the group of A:U, G:U, I:C, and mismatch pairs, e.g., non-canonical or other than canonical pairings or pairings including universal bases, to promote dissociation of the antisense strand at the 5' end of the duplex.

[0457] In some embodiments, the nucleotide at position 1 in the double-stranded region from the 5' end of the antisense strand is selected from the group consisting of A, dA, dU, U and dT.Alternatively, at least one of the first 1, 2 or 3 base pairs in the double-stranded region from the 5' end of the antisense strand is an AU base pair.For example, the first base pair in the double-stranded region from the 5' end of the antisense strand is an AU base pair.

[0458] It has been found that introducing a 4'- or 5'-modified nucleotide into the 3'-end of a dinucleotide phosphodiester (PO), phosphorothioate (PS), or phosphorodithioate (PS2) linkage at any position in a single-stranded or double-stranded oligonucleotide can exert a steric effect on the internucleotide linkage, thereby protecting it from nucleases and stabilizing it. In some embodiments, introducing a 4'- or 5'-modified nucleotide into the 3'-end of a dinucleotide PO, PS, or PS2 linkage modifies the second nucleotide in a dinucleotide pair. In other embodiments, introducing a 4'- or 5'-modified nucleotide into the 3'-end of a dinucleotide PO, PS, or PS2 linkage modifies the nucleotide at the 3'-end of a dinucleotide pair.

[0459] In some embodiments, 5'-modified nucleoside is introduced at the 3' end of dinucleotide at any position in single-stranded or double-stranded siRNA.For example, 5'-alkylated nucleoside can be introduced at the 3' end of dinucleotide at any position in single-stranded or double-stranded siRNA.The alkyl group at the 5' position of ribose sugar can be racemic or chirally pure R or S isomer.Exemplary 5'-alkylated nucleoside is 5'-methyl nucleoside.5'-methyl can be racemic or chirally pure R or S isomer.

[0460] In some embodiments, 4'-modified nucleosides are introduced at the 3' end of dinucleotides at any position in single-stranded or double-stranded siRNA. For example, 4'-alkylated nucleosides can be introduced at the 3' end of dinucleotides at any position in single-stranded or double-stranded siRNA. The alkyl group at the 5' position of the ribose sugar can be racemic or chirally pure R or S isomer. An exemplary 4'-alkylated nucleoside is 4'-methyl nucleoside. 4'-methyl can be racemic or chirally pure R or S isomer. Alternatively, 4'-O-alkylated nucleosides can be introduced at the 3' end of dinucleotides at any position in single-stranded or double-stranded siRNA. The 4'-O-alkyl group at the ribose sugar can be racemic or chirally pure R or S isomer. Exemplary 4'-O-alkylated nucleosides include 4'-O-methyl nucleosides, which can be either racemic or chirally pure R or S isomers.

[0461] In some embodiments, 5'-alkylated nucleoside is introduced at any position of the sense strand or antisense strand of dsRNA, and this modification maintains or improves the efficacy of dsRNA.5'-alkyl can be either racemic or chirally pure R or S isomer.Exemplary 5'-alkylated nucleoside is 5'-methyl nucleoside.5'-methyl can be either racemic or chirally pure R or S isomer.

[0462] In some embodiments, 4'-alkylated nucleoside is introduced at any position of the sense strand or antisense strand of dsRNA, and this modification maintains or improves the efficacy of dsRNA.4'-alkyl can be either racemic or chirally pure R or S isomer.Exemplary 4'-alkylated nucleoside is 4'-methyl nucleoside.4'-methyl can be either racemic or chirally pure R or S isomer.

[0463] In some embodiments, 4'-O-alkylated nucleosides are introduced at any position of the sense strand or antisense strand of dsRNA, and such modifications maintain or improve the efficacy of dsRNA. 5'-Alkyl can be either racemic or chirally pure R or S isomer. Exemplary 4'-O-alkylated nucleosides include 4'-O-methyl nucleosides. 4'-O-methyl can be either racemic or chirally pure R or S isomer.

[0464] In some embodiments, the dsRNA molecules of the present disclosure may contain 2'-5' linkages (having 2'-H, 2'-OH, and 2'-OMe, with P=O or P=S). For example, 2'-5' linkage modifications can be used to promote nuclease resistance or to inhibit binding of the sense strand to the antisense strand, or at the 5' end of the sense strand to prevent sense strand activation by RISC.

[0465] In another embodiment, the dsRNA molecules of the present disclosure can contain L sugars (e.g., L-ribose, L-arabinose, with 2'-H, 2'-OH, and 2'-OMe).For example, these L sugar modifications can be used to promote nuclease resistance or inhibit binding of the sense strand to the antisense strand, or can be used at the 5' end of the sense strand to prevent sense strand activation by RISC.

[0466] Various publications have described multimeric siRNA, and all of them can be used with the dsRNA of the present disclosure.Such publications include WO2007 / 091269, US7858769, WO2010 / 141511, WO2007 / 117686, WO2009 / 014887 and WO2011 / 031520, all of which are incorporated herein in their entirety.

[0467] As described in more detail below, RNAi agents containing one or more carbohydrate moieties conjugated to the RNAi agent can optimize one or more properties of the RNAi agent. In many cases, the carbohydrate moiety is attached to a modified subunit of the RNAi agent. For example, the ribose sugar of one or more ribonucleotide subunits of a dsRNA agent can be replaced with another moiety, such as a non-carbohydrate (preferably cyclic) carrier to which a carbohydrate ligand is attached. A ribonucleotide subunit in which the ribose sugar of the subunit has been so replaced is referred to herein as a ribose-replacement modified subunit (RRMS). The cyclic carrier can be a carbocyclic ring system, i.e., all ring atoms are carbon atoms, or a heterocyclic ring structure, i.e., one or more ring atoms can be heteroatoms, such as nitrogen, oxygen, or sulfur. The cyclic carrier can be a monocyclic ring structure or can contain two or more rings, such as fused rings. The cyclic carrier can be a fully saturated ring structure or can contain one or more double bonds.

[0468] Ligands can be attached to polynucleotides via carriers. Carriers include (i) at least one "backbone attachment point," preferably two "backbone attachment points," and (ii) at least one "tethering attachment point." As used herein, "backbone attachment point" refers to a functional group, such as a hydroxyl group, or generally a bond available and suitable for incorporation of a carrier into a backbone, such as a phosphate or modified phosphate, e.g., sulfur-containing backbone, of a ribonucleic acid. In some embodiments, a "tethering attachment point" (TAP) refers to a ring atom, such as a carbon atom or heteroatom (other than the atom providing the backbone attachment point), of a cyclic carrier that connects a selected moiety. The moiety can be, for example, a carbohydrate, such as 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, such as an amino group, or generally provide a bond suitable for incorporation or tethering another chemical entity, such as a ligand, to the ring.

[0469] The RNAi agent may be conjugated to the ligand via a carrier, which can be a cyclic or acyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl, and decalin; preferably, the acyclic group is selected from a serinol backbone or a diethanolamine backbone.

[0470] In certain specific embodiments, an RNAi agent for use in the methods of the present disclosure is an agent selected from the group of agents listed in any one of Tables 2-5, 7 or 8.

[0471] IV. Ligand-Conjugated iRNA Another modification of the iRNA of the present disclosure includes chemically linking the iRNA to one or more ligands, moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the iRNA, for example, into cells. Such moieties include, but are not limited to, lipid moieties such as cholesterol moieties (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), thioethers, e.g., beryl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), and the like. 20:533-538), fatty chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), phospholipids such as di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973) or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0472] In certain embodiments, a ligand alters the distribution, targeting, or lifespan of the iRNA agent into which it is incorporated. In some embodiments, a ligand provides enhanced affinity for a selected target, such as a molecule, cell or cell type, compartment, such as a cellular or organ compartment, tissue, organ, or region of the body, for example, compared to a species in which such a ligand is not present. Conventional ligands do not participate in duplex pairing in double-stranded nucleic acids.

[0473] Ligands can include naturally occurring substances such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL) or globulins), carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid), or lipids. Ligands can also be recombinant or synthetic molecules, such as synthetic polymers, e.g., synthetic polyamino acids. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolized) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylamide), N-isopropylacrylamide polymer, or polyphosphazine. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimeric polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or alpha-helical peptides.

[0474] Ligands can also include targeting groups, such as cell or tissue targeting agents, such as lectins, glycoproteins, lipids, or proteins, for example, antibodies that bind to specific cell types, such as kidney cells.Targeting groups can be thyroid-stimulating hormone, melanocyte-stimulating hormone, lectins, glycoproteins, surfactant protein A, mucin carbohydrates, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, multivalent fucose, glycosylated polyamino acids, multivalent galactose, transferrin, bisphosphonates, polyglutamic acid, polyaspartic acid, lipids, cholesterol steroids, bile acid, folic acid, vitamin B12, biotin, or RGD peptide or RGD peptide mimetics.In certain embodiments, the ligand is multivalent galactose, such as N-acetyl-galactosamine.

[0475] Other examples of ligands include dyes, intercalating agents (e.g., acridine), crosslinkers (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules such as cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithium, and the like. Examples of suitable cleavage inhibitors include cleavage inhibitors (e.g., oleic acid, O3-(oleoyl)cholenoic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bis-imidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

[0476] The ligand can be a protein, such as a glycoprotein or peptide, a molecule with specific affinity for the co-ligand, or an antibody, such as an antibody that binds to a specific cell type, such as cancer cells, endothelial cells, or bone cells. Ligands can also include hormones and hormone receptors. They can also include non-peptide species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, multivalent mannose, or multivalent fucose. Ligands can be, for example, lipopolysaccharides, p38 MAP kinase activators, or NF-κB activators.

[0477] The ligand can be, for example, a substance, e.g., a drug, that can increase cellular uptake of an iRNA agent by, for example, disrupting the cytoskeleton of a cell, e.g., by disrupting cellular microtubules, microfilaments, or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, jasplakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

[0478] In some embodiments, the ligand attached to an iRNA as described herein acts as a pharmacokinetic modulator (PK modulator). PK modulators include lipophiles, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, and the like. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, and the like. Oligonucleotides containing several phosphorothioate linkages are also known to bind to serum proteins. Therefore, short oligonucleotides, e.g., oligonucleotides of about 5, 10, 15, or 20 bases containing multiple phosphorothioate linkages in the backbone, are also suitable as ligands (e.g., as PK-modulating ligands) for the present disclosure. Furthermore, aptamers that bind to serum components (e.g., serum proteins) are also suitable for use as PK-modulating ligands in the embodiments described herein.

[0479] Ligand-conjugated iRNAs of the present disclosure can be synthesized by using oligonucleotides bearing pendant reactive functionalities, e.g., derived from the attachment of a linking molecule onto an oligonucleotide (described below). This reactive oligonucleotide can be reacted directly with commercially available ligands, ligands that have been synthesized with any of a variety of protecting groups, or ligands that have a linking moiety attached to them.

[0480] The oligonucleotides used in the conjugates disclosed in the present invention can be conveniently and routinely produced by known techniques of solid phase synthesis.The equipment for such synthesis is sold by several vendors, including, for example, Applied Biosystems® (Foster City, California).Any other means for such synthesis known in the art can also or alternatively be used.It is also known to use similar techniques to prepare other oligonucleotides, for example, phosphorothioates and alkylated derivatives.

[0481] For the ligand-conjugated oligonucleotides and molecules having ligand-sequence-specific linked nucleosides of the present disclosure, the oligonucleotides and oligonucleosides can be assembled on a suitable DNA synthesizer using standard nucleotide or nucleoside precursors or nucleotide or nucleoside conjugate precursors that already have a linking moiety, ligand-nucleotide or nucleoside conjugate precursors that already have a ligand molecule, or non-nucleoside ligand-containing building blocks.

[0482] When using a nucleotide-conjugate precursor that already has a linking moiety, synthesis of the sequence-specific linked nucleoside is usually completed, and then a ligand molecule is reacted with the linking moiety to form the ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or linked nucleosides of the present disclosure are synthesized by automated synthesizers using phosphoramidites derived from ligand-nucleoside conjugates, in addition to standard and non-standard phosphoramidites that are commercially available and routinely used in oligonucleotide synthesis.

[0483] A. Lipid Conjugates In certain embodiments, the ligand or conjugate is lipid or lipid-based molecule.This lipid or lipid-based molecule can usually bind to serum protein, for example, human serum albumin (HSA).HSA binding ligand allows the distribution of conjugate to target tissue in the body, for example, non-renal target tissue.For example, the target tissue can be the liver, including liver parenchymal cells.Other molecules that can bind to HSA can also be used as ligand.For example, naproxen or aspirin can be used.Lipid or lipid-based ligand can (a) increase the resistance of conjugate to degradation, (b) increase targeting or transport into target cell or cell membrane, or (c) adjust the binding to serum protein, for example, HSA.

[0484] The lipid-based ligand can be used to modulate, e.g., manage (e.g., inhibit), the binding of the conjugate to the target tissue. For example, a lipid or lipid-based ligand that binds more strongly to HSA is less likely to be targeted to the kidney and therefore less likely to be eliminated from the body. A lipid or lipid-based ligand that binds less strongly to HSA can be used to target the conjugate to the kidney.

[0485] In certain embodiments, lipid-based ligand binds to HSA.For example, the ligand can bind to HSA with sufficient affinity, so that the distribution of conjugate to non-renal tissue is enhanced.However, the affinity is usually not so strong that HSA-ligand binding cannot be reversed.

[0486] In certain embodiments, the lipid-based ligand binds weakly or not at all to HSA, thereby enhancing distribution of the conjugate to the kidney. Other moieties that target kidney cells can be used instead of or in addition to the lipid-based ligand.

[0487] In another embodiment, the ligand is a moiety, such as a vitamin, that is taken up by target cells, for example, proliferating cells. These are particularly useful for treating disorders characterized by unwanted cell proliferation, for example, malignant or non-malignant types, such as cancer cells. Exemplary vitamins include vitamins A, E, and K. Other exemplary vitamins include B vitamins, such as folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients taken up by cancer cells. Also included are HSA and low-density lipoprotein (LDL).

[0488] B. Cell-penetrating agents In another aspect, the ligand is a cell-penetrating agent, for example, a helical cell-penetrating agent. In certain embodiments, these cell-penetrating agents are amphipathic. Exemplary cell-penetrating agents include peptides, such as tat or antennopedia. When the agent is a peptide, it may be modified, including peptidyl mimetics, invertomers, non-peptide or pseudopeptide linkages, and the use of D-amino acids. Helical agents are usually α-helical agents and may have a lipophilic and lipophobic phase.

[0489] The ligand can be a peptide or peptidomimetic. Peptidomimetics (also referred to herein as oligopeptidomimetics) are molecules capable of folding into defined three-dimensional structures similar to natural peptides. Attachment of peptides and peptidomimetics to iRNA agents can affect the pharmacokinetic distribution of iRNAs, such as by enhancing cellular recognition and uptake. The peptide or peptidomimetic moiety can be about 5 to 50 amino acids in length, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

[0490] The peptide or peptidomimetic can be, for example, a cell-penetrating peptide, a cationic peptide, an amphipathic peptide, or a hydrophobic peptide (e.g., composed primarily of Tyr, Trp, or Phe). The peptide moiety can be a dendrimeric peptide, a constrained peptide, or a cross-linked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF, which has the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 14). An RFGF analog containing a hydrophobic MTS (e.g., the amino acid sequence AALLPVLLAAP (SEQ ID NO: 15)) can also be a targeting moiety. The peptide moiety can be a "delivery" peptide, capable of transporting large polar molecules, including peptides, oligonucleotides, and proteins, across cell membranes. For example, the sequence derived from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 16)) and the sequence derived from the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 17)) have been found to function as delivery peptides. Peptides or peptidomimetics can be encoded by random sequences of DNA, such as peptides identified from phage display libraries or one-bead-one-compound (OBOC) combinatorial libraries (Lam et al., Nature, 354:82-84, 1991). Typically, peptides or peptidomimetics tethered to dsRNA agents via incorporated monomer units include cell-targeting peptides, such as arginine-glycine-aspartic acid (RGD) peptides or RGD mimics. The peptide portion can range in length from about 5 amino acids to about 40 amino acids. The peptide portion can have structural modifications, for example, to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized.

[0491] The RGD peptide for use in the compositions and methods of the present disclosure can be linear or cyclic, and can be modified, for example, glycosylated or methylated, to facilitate targeting to specific tissue(s).RGD-containing peptides and peptidomimetics can contain D-amino acids and synthetic RGD mimics.In addition to RGD, other moieties can be used to target integrin ligands.Preferred conjugates of this ligand target PECAM-1 or VEGF.

[0492] RGD peptide moieties can be used to target specific cell types, such as tumor cells, e.g., endothelial tumor cells or breast cancer tumor cells (Zitzmann et al., Cancer Res., 62:5139-43, 2002). RGD peptides can facilitate targeting of dsRNA agents to tumors in various other tissues, including the lung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy 8:783-787, 2001). Typically, RGD peptides facilitate targeting of iRNA agents to the kidney. RGD peptides can be linear or cyclic, and can be modified, e.g., glycosylated or methylated, to facilitate targeting to a specific tissue(s). For example, glycosylated RGD peptides can facilitate targeting of iRNA agents to α- V It can be delivered to tumor cells that express β3 (Haubner et al., Jour. Nucl. Med., 42:326-336, 2001).

[0493] A "cell-penetrating peptide" is capable of penetrating cells, such as microbial cells, e.g., bacterial or fungal cells, or mammalian cells, e.g., human cells. Microbial cell-penetrating peptides can be, for example, α-helical linear peptides (e.g., LL-37 or seropin P1), disulfide bond-containing peptides (e.g., α-defensins, β-defensins, or bactenecins), or peptides containing only one or two dominant amino acids (e.g., PR-39 or indolicidin). Cell-penetrating peptides can also contain a nuclear localization signal (NLS). For example, a cell-penetrating peptide can be a bisected amphipathic peptide such as MPG, derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

[0494] C. Carbohydrate conjugates In some embodiments of the compositions and methods of the present disclosure, the iRNA further comprises a carbohydrate. Carbohydrate-conjugated iRNA is advantageous for in vivo delivery of nucleic acids and compositions suitable for in vivo therapeutic use, as described herein. As used herein, "carbohydrate" refers to a compound that is either a carbohydrate itself, composed of one or more monosaccharide units (which may be linear, branched, or cyclic) having at least six carbon atoms, with an oxygen, nitrogen, or sulfur atom attached to each carbon atom, or a compound that has as part thereof a carbohydrate moiety composed of one or more monosaccharide units, each having at least six carbon atoms (which may be linear, branched, or cyclic), with an oxygen, nitrogen, or sulfur atom attached to each carbon atom. Representative carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about 4, 5, 6, 7, 8, or 9 monosaccharide units) and polysaccharides, such as starch, glycogen, cellulose, and polysaccharide gums. Particular monosaccharides include C5, and above (e.g., C5, C6, C7, or C8) sugars, disaccharides, and trisaccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7, or C8).

[0495] In certain embodiments, the carbohydrate conjugate comprises a monosaccharide.

[0496] In certain embodiments, the monosaccharide is N-acetylgalactosamine (GalNAc).GalNAc conjugates containing one or more N-acetylgalactosamine (GalNAc) derivatives are described, for example, in US 8,106,022, the entire contents of which are incorporated herein by reference.In some embodiments, the GalNAc conjugate serves as a ligand that targets iRNA to specific cells.In some embodiments, the GalNAc conjugate targets iRNA to liver cells, for example, by serving as a ligand for the asialoglycoprotein receptor of liver cells (e.g., hepatocytes).

[0497] In some embodiments, the carbohydrate conjugate comprises one or more GalNAc derivatives. The GalNAc derivatives can be attached via a linker, e.g., a bivalent or trivalent branched linker. In some embodiments, the GalNAc conjugate is conjugated to the 3' end of the sense strand. In some embodiments, the GalNAc conjugate is conjugated to the iRNA agent (e.g., to the 3' end of the sense strand) via a linker, e.g., a linker as described herein. In some embodiments, the GalNAc conjugate is conjugated to the 5' end of the sense strand. In some embodiments, the GalNAc conjugate is conjugated to the iRNA agent (e.g., to the 5' end of the sense strand) via a linker, e.g., a linker as described herein.

[0498] In certain embodiments of the present disclosure, GalNAc or a GalNAc derivative is attached to an iRNA agent of the present disclosure via a monovalent linker. In some embodiments, GalNAc or a GalNAc derivative is attached to an iRNA agent of the present disclosure via a bivalent linker. In yet other embodiments of the present disclosure, GalNAc or a GalNAc derivative is attached to an iRNA agent of the present disclosure via a trivalent linker. In other embodiments of the present disclosure, GalNAc or a GalNAc derivative is attached to an iRNA agent of the present disclosure via a tetravalent linker.

[0499] In certain embodiments, the double-stranded RNAi agent of the present disclosure comprises one GalNAc or GalNAc derivative attached to the iRNA agent. In certain embodiments, the double-stranded RNAi agent of the present disclosure comprises multiple (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAc derivatives, each independently attached to multiple nucleotides of the double-stranded RNAi agent via multiple monovalent linkers.

[0500] In some embodiments, for example, when the two strands of an iRNA agent of the present disclosure are part of a single larger molecule connected by an uninterrupted chain of nucleotides between the 3' end of one strand and the 5' end of the other strand, forming a hairpin loop containing multiple unpaired nucleotides, each unpaired nucleotide in the hairpin loop can independently comprise a GalNAc or GalNAc derivative attached via a monovalent linker. Hairpin loops can also be formed by an extended overhang on one strand of the duplex.

[0501] In some embodiments, for example, when the two strands of an iRNA agent of the present disclosure are part of a single larger molecule connected by an uninterrupted chain of nucleotides between the 3' end of one strand and the 5' end of the other strand, forming a hairpin loop containing multiple unpaired nucleotides, each unpaired nucleotide in the hairpin loop can independently comprise a GalNAc or GalNAc derivative attached via a monovalent linker. Hairpin loops can also be formed by an extended overhang on one strand of the duplex.

[0502] In some embodiments, the GalNAc conjugate is

[0503] [ka] is.

[0504] In some embodiments, the RNAi agent is attached to the carbohydrate conjugate via a linker as shown in the following schematic diagram, where X is O or S:

[0505] [ka]

[0506] In some embodiments, the RNAi agent is conjugated to L96 as defined in Table 2 and shown below:

[0507] [ka]

[0508] In certain embodiments, the carbohydrate conjugate for use in the compositions and methods of the present disclosure is selected from the group consisting of:

[0509] [ka] [ka] [ka] [ka] [ka] [wherein Y is O or S, and n is 3 to 6] (Formula XXIV);

[0510] [ka] [wherein Y is O or S, and n is 3 to 6] (Formula XXV);

[0511] [ka] [wherein X is O or S] (Formula XXVII);

[0512] [ka] [ka]

[0513] In certain embodiments, the carbohydrate conjugate for use in the compositions and methods of the present disclosure is a monosaccharide. In certain embodiments, the monosaccharide is N-acetylgalactosamine, e.g.,

[0514] [ka] is.

[0515] Further exemplary carbohydrate conjugates for use in the embodiments described herein include, but are not limited to:

[0516] [ka] [When one of X or Y is an oligonucleotide, the other is hydrogen] Examples include:

[0517] In some embodiments, suitable ligands are those disclosed in WO2019 / 055633, the entire contents of which are incorporated herein by reference. In one embodiment, the ligand has the following structure:

[0518] [ka] Includes.

[0519] In certain embodiments, RNAi agents of the present disclosure may include GalNAc ligands, even though such GalNAc ligands are currently predicted to be of limited value for the preferred intrathecal / CNS delivery route(s) of the present disclosure.

[0520] In certain embodiments of the present disclosure, GalNAc or GalNAc derivatives are attached to the iRNA agent of the present disclosure via a monovalent linker.In some embodiments, GalNAc or GalNAc derivatives are attached to the iRNA agent of the present disclosure via a bivalent linker.In yet other embodiments of the present disclosure, GalNAc or GalNAc derivatives are attached to the iRNA agent of the present disclosure via a trivalent linker.

[0521] In one embodiment, the double-stranded RNAi agent of the present disclosure includes one or more GalNAc or GalNAc derivatives attached to the iRNA agent. GalNAc can be attached to any nucleotide via a linker on the sense strand or antisense strand. GalNAc can be attached to the 5' end of the sense strand, the 3' end of the sense strand, the 5' end of the antisense strand, or the 3' end of the antisense strand. In one embodiment, GalNAc is attached to the 3' end of the sense strand, for example, via a trivalent linker.

[0522] In other embodiments, a double-stranded RNAi agent of the present disclosure comprises multiple (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAc derivatives independently attached to each of multiple nucleotides of the double-stranded RNAi agent via multiple linkers, e.g., monovalent linkers.

[0523] In some embodiments, for example, when the two strands of an iRNA agent of the present disclosure are part of one larger molecule connected by an uninterrupted chain of nucleotides between the 3' end of one strand and the 5' end of each of the other strands, forming a hairpin loop containing multiple unpaired nucleotides, each unpaired nucleotide in the hairpin loop can independently comprise a GalNAc or GalNAc derivative attached via a monovalent linker.

[0524] In some embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, such as, but not limited to, a PK modulator or a cell-penetrating peptide.

[0525] Additional carbohydrate conjugates and linkers suitable for use in the present disclosure include those described in WO2014 / 179620 and WO2014 / 179627, the contents of each of which are incorporated herein by reference in their entirety.

[0526] D. Linker In some embodiments, the conjugates or ligands described herein can be attached to the iRNA oligonucleotide using a variety of linkers, which may or may not be cleavable.

[0527] The term "linker" or "linking group" means an organic moiety that connects two parts of a compound, e.g., attaches two parts of a compound by a covalent bond.A linker is typically a direct bond or an atom such as oxygen or sulfur, a unit such as NR, C(O), C(O)NH, SO, SO, SONH, or a substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, or heteroarylalkynyl, where one or more methylenes may be interrupted or terminated by O, S, S(O), SO, N(R), C(O). ynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkynyl

[0044] In some embodiments, R8 is substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, etc. In some embodiments, R8 is substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, etc. In some embodiments, R8 is substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, etc. In some embodiments, R8 is substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, etc. In some embodiments, R8 is substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, etc. In some embodiments, R8 is substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, etc. In some embodiments, R8 is substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, etc.In certain embodiments, the linker is about 1 to 24 atoms, 2 to 24, 3 to 24, 4 to 24, 5 to 24, 6 to 24, 6 to 18, 7 to 18, 8 to 18 atoms, 7 to 17, 8 to 17, 6 to 16, 7 to 16, or 8 to 16 atoms.

[0528] A cleavable linking group is one that is sufficiently stable outside a cell, but that, upon entry into a target cell, is cleaved to release the two moieties held together by the linker. In preferred embodiments, the cleavable linking group is cleaved at least about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or more, or at least about 100-fold faster in the target cell or under a first reference condition (which may, for example, be selected to mimic or represent intracellular conditions) than in the subject's blood or under a second reference condition (which may be selected to mimic or represent conditions found in blood or serum).

[0529] Cleavable linking groups are susceptible to the influence of cleaving agents, such as pH, redox potential, or the presence of degradable molecules.Generally, cleaving agents are more common or found at higher levels or activity inside cells than in serum or blood.Examples of such degrading agents include redox agents that are selected for specific substrates or do not have substrate specificity, such as oxidizing enzymes or reductases or reducing agents present in cells, such as mercaptans, which can degrade redox-cleavable linking groups by reduction, esterases, endosomes, or agents that can create an acidic environment, such as those that produce a pH of 5 or less, general acids, peptidases (can be substrate-specific), and enzymes that can hydrolyze or degrade acid-cleavable linking groups by acting as phosphatases.

[0530] Cleavable linking groups, such as disulfide bonds, can be pH-sensitive. While the pH of human serum is 7.4, the average intracellular pH is slightly lower, ranging from approximately 7.1 to 7.3. Endosomes have a more acidic pH, ranging from 5.5 to 6.0, and lysosomes have an even more acidic pH of approximately 5.0. Some linkers will have a cleavable linking group that is cleaved at a preferred pH, thereby releasing the cationic lipid from the ligand inside the cell into the desired compartment of the cell.

[0531] Linker can comprise a cleavable linking group that can be cleaved by specific enzyme.The type of cleavable linking group incorporated into linker can vary depending on the cell to be targeted.For example, liver targeting ligand can be linked to cationic lipid via a linker that comprises ester group.Liver cell is rich in esterase, therefore, linker is cleaved more efficiently in liver cell than in cell types that are not rich in esterase.Other cell types that are rich in esterase include lung, renal cortex and testis cell.

[0532] When targeting cell types that are rich in peptidases, such as liver cells and synovial cells, linkers containing peptide bonds can be used.

[0533] Generally, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degrading agent (or condition) to cleave the candidate linking group. It may also be desirable to test the candidate cleavable linking group for its ability to resist cleavage when in blood or in contact with other non-target tissues. Thus, the relative susceptibility to cleavage between a first and a second condition can be determined, with the first being selected to exhibit cleavage in target cells, and the second being selected to exhibit cleavage in other tissues or biological fluids, such as blood or serum. Evaluation can be carried out in a cell-free system, in cells, in cell culture, in organs or tissue culture, or in whole animals. It may be useful to perform initial evaluation in cell-free o...

Claims

1. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of 4R tau, the dsRNA agent comprising a sense strand and an antisense strand forming a double-stranded region; the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of nucleotides 1009-1053 of SEQ ID NO:3, and the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of nucleotides 1007-1053 of SEQ ID NO:4; or the antisense strand comprises a region of complementarity to an mRNA encoding 4R tau, wherein the region of complementarity comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of nucleotides 1007 to 1053 of SEQ ID NO: 4; or The antisense strand comprises a region of complementarity to an mRNA encoding 4R tau, wherein the region of complementarity is any one of the antisense nucleotide sequences set forth in SEQ ID NOs: 144, 136, 147, or 148, or any one of the antisense nucleotide sequences set forth in the table below. Table 1 Table 2 and at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from any one of the antisense nucleotide sequences in dsRNA agents.

2. 2. The dsRNA agent of claim 1, wherein the sense strand comprises at least 15 contiguous nucleotides from nucleotides 1009 to 1053 of SEQ ID NO:3, and the antisense strand comprises at least 15 contiguous nucleotides from nucleotides 1007 to 1053 of SEQ ID NO:

4.

3. 2. The dsRNA agent of claim 1, wherein the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the nucleotide sequences of nucleotides 1024-1044, 1016-1036, 1027-1047, 1028-1048, 1009-1029, 1018-1038, 1020-1040, 1021-1041, 1022-1042, 1029-1049, 1030-1050, 1032-1052, 1033-1053, 1014-1048, and 1022-1048 of SEQ ID NO:3, and the antisense strand comprises at least 15 contiguous nucleotides from the corresponding nucleotide sequence of SEQ ID NO:

4.

4. The antisense strand is selected from the group consisting of (a) AD-1423291, AD-1423283, AD-1423294, AD-1423295, AD-1423276, AD-1423285, AD-1423287, AD-1423288, AD-1423289, AD-1423296, AD-1423297, AD-1423299, and AD-1423300; or (b) AD-2204877, AD-1597146, AD-2204883, AD- 2204885, AD-2204886, AD-1597147, AD-1597148, AD-2126092, AD-2126093, AD-2204890, AD-2204891, AD-1597149, and AD-2204893.

5. 10. The dsRNA agent of claim 1, wherein the sense strand, the antisense strand, or both the sense and antisense strands are conjugated to one or more lipophilic moieties.

6. The dsRNA agent of claim 5, wherein one or more lipophilic moieties are conjugated to one or more internal positions within the double-stranded region of the dsRNA agent.

7. The dsRNA agent of claim 5, wherein one or more lipophilic moieties are conjugated via a linker or carrier.

8. 10. The dsRNA agent of claim 1, wherein the dsRNA agent comprises at least one modified nucleotide.

9. 9. The dsRNA agent of claim 8, wherein all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides.

10. The method according to claim 10, wherein the at least one modified nucleotide is a deoxynucleotide, a 3'-terminal deoxythymidine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-deoxy-2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, a non-locked nucleotide, a 2'-5' linked nucleotide (3'-RNA), 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 (hydroxyly) modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide containing a non-natural base, a tetrahydropyran modified nucleotide ...

9. The dsRNA agent of claim 8, wherein the dsRNA agent is selected from the group consisting of a nucleotide containing a 5'-phosphorothioate group, a nucleotide containing a 5'-methylphosphonate group, a nucleotide containing a 5' phosphate or a 5' phosphate mimic, a nucleotide containing a vinyl phosphonate, a nucleotide containing a 5'-vinyl phosphonate, a glycol nucleic acid (GNA), a glycol nucleic acid S-isomer (S-GNA), a nucleotide containing 2-hydroxymethyl-tetrahydrofuran-5-phosphate, a nucleotide containing 2'-deoxythymidine-3' phosphate, a nucleotide containing 2'-deoxyguanosine-3'-phosphate, and a terminal nucleotide linked to a cholesteryl derivative and a dodecanoic acid bisdecylamide group; and combinations thereof.

11. The dsRNA agent of claim 10, wherein at least one modified nucleotide comprises a short sequence of 3'-terminal deoxythymidine nucleotides (dT).

12. The dsRNA agent of claim 10, wherein the at least one modified nucleotide is selected from a 2'-O-methyl modified nucleotide, a 2'-deoxy modified nucleotide, a GNA modified nucleotide, and a 2'-deoxy-2'-fluoro modified nucleotide.

13. 10. The dsRNA agent of claim 1, further comprising at least one phosphorothioate internucleotide linkage.

14. 14. The dsRNA agent of claim 13, comprising 6 to 8 phosphorothioate internucleotide linkages.

15. 10. The dsRNA agent of claim 1, wherein at least one strand comprises a 3' overhang of at least 1 nucleotide, or at least one strand comprises a 3' overhang of at least 2 nucleotides.

16. 10. The dsRNA agent of claim 1, wherein the double-stranded region is 15 to 30 nucleotide pairs in length.

17. 17. The dsRNA agent of claim 16, wherein the double-stranded region is 19 to 21 nucleotide pairs in length.

18. The dsRNA agent of claim 1, wherein each strand has 19 to 30 nucleotides.

19. 10. The dsRNA agent of claim 1, wherein each strand has 19 to 23 nucleotides.

20. 7. The dsRNA agent of claim 6, wherein the one or more lipophilic moieties are conjugated to one or more internal positions in at least one strand.

21. 21. The dsRNA agent of claim 20, wherein the one or more lipophilic moieties are conjugated to one or more internal positions in at least one strand via a linker or carrier.

22. 22. The dsRNA agent of claim 21, wherein the internal positions include all but two positions from each end of at least one strand, or all but three positions from each end of at least one strand, and the internal positions exclude the cleavage site region of the sense strand.

23. 7. The dsRNA agent of claim 6, wherein the one or more lipophilic moieties are conjugated to one or more internal positions selected from the group consisting of positions 4-8 and 13-18 in the sense strand, and positions 6-10 and 15-18 in the antisense strand, counting from the 5' end of each strand.

24. 6. The dsRNA agent of claim 5, wherein the sense strand is 21 nucleotides in length, the antisense strand is 23 nucleotides in length, and the one or more lipophilic moieties are conjugated to position 21, 20, 15, 1, 7, 6, or 2 of the sense strand or position 16 of the antisense strand.

25. The dsRNA agent of claim 5, wherein one or more lipophilic moieties are aliphatic compounds, alicyclic compounds, or polyalicyclic compounds.

26. The dsRNA agent of claim 25, wherein the one or more lipophilic moieties are selected from the group consisting of lipids, cholesterol, retinoic acid, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl groups, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenoic acid, dimethoxytrityl, or phenoxazine.

27. The dsRNA agent of claim 25, wherein the one or more lipophilic moieties contain a saturated or unsaturated C4-C30 hydrocarbon chain and an appropriate functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne.

28. The dsRNA agent of claim 27, wherein one or more lipophilic moieties contain a saturated or unsaturated C6-C18 hydrocarbon chain.

29. The dsRNA agent of claim 27, wherein one or more lipophilic moieties contain a saturated or unsaturated C16 hydrocarbon chain.

30. 30. The dsRNA agent of claim 28, wherein the saturated or unsaturated C16 hydrocarbon chain is conjugated to position 6, counting from the 5' end of the chain.

31. The dsRNA agent of claim 5, wherein the one or more lipophilic moieties are conjugated to the double-stranded iRNA agent via a linker containing an ether, a thioether, a urea, a carbonate, an amine, an amide, a maleimide-thioether, a disulfide, a phosphodiester, a sulfonamide linkage, a product of a click reaction, or a carbamate.

32. The double-stranded iRNA agent of claim 15, wherein one or more lipophilic moieties are conjugated to a nucleobase, a sugar moiety, or an internucleoside linkage.

33. The dsRNA agent of claim 5, wherein the one or more lipophilic moieties are conjugated via a biocleavable linker selected from the group consisting of DNA, RNA, disulfides, amides, functionalized mono- or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.

34. 6. The dsRNA agent of claim 5, wherein the 3' end of the sense strand is protected through an end cap that is a cyclic group having an amine, and the cyclic group is selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl.

35. The dsRNA agent of claim 5, further comprising a phosphate or phosphate mimetic at the 5'-end of the antisense strand.

36. 36. The dsRNA agent of claim 35, wherein the phosphate mimetic is a 5'-vinylphosphonate (VP).

37. 2. The dsRNA agent of claim 1, wherein the base pair at position 1 of the 5' end of the antisense strand of the duplex is an AU base pair.

38. 2. The dsRNA agent of claim 1, wherein the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

39. (a) the sense strand comprises the sequence of SEQ ID NO:221 and all modifications, and the antisense strand comprises the sequence of SEQ ID NO:298 and all modifications; (b) the sense strand comprises the sequence of SEQ ID NO: 213 and all modifications, and the antisense strand comprises the sequence of SEQ ID NO: 290 and all modifications; (c) the sense strand comprises the sequence of SEQ ID NO: 224 and all modifications, and the antisense strand comprises the sequence of SEQ ID NO: 301 and all modifications; or (d) the sense strand comprises the sequence of SEQ ID NO: 225 and all modifications, and the antisense strand comprises the sequence of SEQ ID NO: 302 and all modifications; The dsRNA agent of claim 1.

40. 40. A pharmaceutical composition for inhibiting expression of a gene encoding 4R tau, comprising the dsRNA agent of any one of claims 1 to 39.

41. A pharmaceutical composition for use in a method for inhibiting expression of 4R tau in a cell, or for use in a method for reducing aberrant expression of exon 10-containing MAPT transcripts in a cell, comprising the dsRNA agent of any one of claims 1 to 39.

42. 42. The pharmaceutical composition of claim 41, wherein the cell is in a human subject with a 4R tau-associated neurodegenerative disorder.

43. The disorders include progressive supranuclear palsy (PSP), progressive supranuclear palsy-Richardson syndrome (PSP-RS), corticobasal ganglia syndrome (CBD), argyrophilic grain disease (AGD), frontotemporal lobar degeneration due to tau (FTLD-tau), FTLD-tau due to MAPT mutations, multisystem tauopathy with presenile dementia (MSTD), glioglobular tauopathy (GGT), Parkinson's disease-like parkinsonism, non-fluent variant primary progressive aphasia (nfvPPA), behavioral frontotemporal dementia (bvFTD) ), pure akinesia with freezing of gait (PAGF), primary lateral sclerosis (PLS), familial multisystem tauopathy with presenile dementia (FMSTD), familial progressive subcortical gliosis (familial pSG), pallido-pontine-nigral degeneration (pPND), familial frontotemporal dementia (e.g., FTD-Kumamoto), N279K tauopathy, rapidly progressive Alzheimer's disease (rpAD), and sporadic Alzheimer's disease (spAD).

44. A pharmaceutical composition comprising the dsRNA agent of any one of claims 1 to 39 for use in a method of treating a human subject having a disorder in which reduced 4R tau expression would be beneficial, or for use in a method of preventing at least one symptom in a human subject having a disorder in which reduced 4R tau expression would be beneficial.

45. The disorders include progressive supranuclear palsy (PSP), progressive supranuclear palsy-Richardson syndrome (PSP-RS), corticobasal ganglia syndrome (CBD), argyrophilic grain disease (AGD), frontotemporal lobar degeneration due to tau (FTLD-tau), FTLD-tau due to MAPT mutations, multisystem tauopathy with presenile dementia (MSTD), glioglobular tauopathy (GGT), Parkinson's disease-like parkinsonism, non-fluent variant primary progressive aphasia (nfvPPA), behavioral frontotemporal dementia (bvFTD) ), pure akinesia with freezing of gait (PAGF), primary lateral sclerosis (PLS), familial multisystem tauopathy with presenile dementia (FMSTD), familial progressive subcortical gliosis (familial pSG), pallido-pontine-nigral degeneration (pPND), familial frontotemporal dementia (e.g., FTD-Kumamoto), N279K tauopathy, rapidly progressive Alzheimer's disease (rpAD), and sporadic Alzheimer's disease (spAD).

46. 45. The pharmaceutical composition of claim 44, wherein the dsRNA agent is administered to the subject intrathecally (IT) or intracerebroventricularly (ICV).

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