Human chromosome 9 open reading frame 72 (c9orf72) irna agent compositions and methods of use thereof

Abstract

To provide an agent that can selectively and effectively inhibit expression of the C9orf72 gene for treatment of a subject having a C9orf72-related disorder.SOLUTION: Provided is a double-stranded ribonucleic acid (dsRNA) agent or a salt thereof for inhibiting expression of C9orf72, the dsRNA agent or the salt thereof comprising a sense strand and an antisense strand forming a double-stranded region, the antisense strand comprising at least 15 consecutive nucleotides from the complement of nucleotides 230-261 of a specific sequence, all nucleotides of the sense strand and all nucleotides of the antisense strand containing nucleotide modifications, and the dsRNA agent comprising at least one phosphorothioate internucleotide linkage.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 947,605, filed December 13, 2019, and U.S. Provisional Patent Application No. 62 / 947,768, filed December 13, 2019, the entire contents of each of which are incorporated herein by reference.

[0002] Array List This application contains a Sequence Listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy created on XXX, 2020, is named 121301_10220_SL.txt and is XXX bytes in size. [Background technology]

[0003] Human chromosome 9 open reading frame 72 (C9orf72) is a protein encoded by the c9orf72 gene. C9orf72 is found in many regions of the brain, including the cerebral cortex, the cytoplasm of neurons, and presynaptic terminals.

[0004] The differential use of alternative start and stop sites generates three RNA transcripts from C9orf72 DNA, encoding two protein isoforms: a long, approximately 54 kDa isoform (isoform A) derived from variants 2 (NM_018325.4) and 3 (NM_001256054.2), and a short, approximately 24 kDa isoform (isoform B) derived from variant 1 (NM_145005.6) (see, for example, Figure 1 in Barker, et al. (2017) Frontiers Cell Neurosci 11:1-15).

[0005] The two alternatively used first exons of the C9orf72 gene are exons 1a and 1b (see, for example, Figure 1 in Barker et al., supra). Large GGGGCC (G4C2) hexanucleotide repeat expansions (approximately 2–22 copies to 700–1600 copies) in the first intron of the C9orf72 gene between exons 1a and 1b have been shown to interfere with the function of the C9orf72 protein and be pathogenic, resulting in several neurodegenerative diseases with distinct clinical features but common pathological features and genetic causes (Ling, et al. (2013) Neuron 79:416–438). In particular, the presence of hexanucleotide repeat expansions in the C9orf72 gene is the most common genetic cause of familial and sporadic amyotrophic lateral sclerosis (ALS), a devastating degenerative disease of motor neurons in the brain and spinal cord. Indeed, C9orf72 mutational hexanucleotide repeat expansions are present in nearly 40% of familial ALS cases and 8-10% of sporadic ALS cases. Hexanucleotide repeat expansions in the C9orf72 gene are also the most common familial cause of frontotemporal dementia (FTD), the second most common form of presenile dementia after Alzheimer's disease, characterized by behavioral and language deficits and pathologically manifested by neuronal atrophy in the frontal and anterior temporal lobes of the brain. C9orf72-induced Huntington's-like syndrome, characterized by movement disorders including dystonia, chorea, myoclonus, tremor, and rigidity, cognitive and memory impairments, early psychiatric disability, and behavioral problems, has also been associated with hexanucleotide repeat expansions in the C9orf72 gene.

[0006] While the function of the C9orf72 protein is still under investigation, C9orf72 has been shown to interact with and activate Rab proteins, which are involved in regulating the cytoskeleton, autophagy, and intracellular trafficking. Furthermore, numerous cellular pathways have been demonstrated to be misregulated in neurodegenerative diseases associated with C9orf72 hexanucleotide repeat expansions. For example, altered RNA processing has consistently emerged at the forefront of C9orf72 disease research. These include bidirectional transcription of repeat sequences, accumulation of repeat RNAs in nuclear aggregates that capture specific RNA-binding proteins (RBPs), and translation of RNA repeats into dipeptide repeat proteins (DPRs) via repeat-associated non-AUG (RAN)-initiated translation. Furthermore, disruption of C9orf72 RNA release from RNA polymerase II, cytoplasmic translation, and denaturation has been shown to be disrupted by C9orf72 hexanucleotide repeat expansions. Furthermore, several changes in the processing of the C9orf72 RNA itself have been identified with respect to its transcription, splicing, and localization (see, eg, Barker, et al., supra).

[0007] Regardless of the mechanism, several groups have identified the presence of sense and antisense C9orf72-containing aggregates, as well as aberrant dipeptide repeat (DPR) proteins (poly(GA), poly(GR), poly(GP), poly(PA), and poly(PR)) produced through repeat-associated non-AUG-dependent (RAN) translation from all reading frames of sense- or antisense-repeat-containing C9orf72 RNA, in several cell types of the nervous system of subjects with C9orf72-related diseases (Lagier-Tourenne, et al. (2013) Proc Natl Acad Sci USA doi / 10.1073 / pnas.1318835110; Jiang, et al. (2016) Neuron 90:535-550). Furthermore, mice with one C9orf72 allele inactivated did not develop disease, whereas mice with both C9orf72 alleles inactivated exhibited splenomegaly, enlarged lymph nodes, and mild social interaction defects, but no motor dysfunction. Furthermore, mice expressing human C9orf72 RNA with up to 450 GGGGCC repeats showed that the hexanucleotide expansion induced age-, repeat-length-, and expression-level-dependent accumulation of sense- and antisense-RNA-containing aggregates and dipeptide repeat proteins synthesized by AUG-independent translation, accompanied by hippocampal neuron loss, increased anxiety, and impaired cognitive function (Jiang, et al. (2016) Neuron 90:535-550).

[0008] Currently, there is no cure for subjects with a C9orf72-associated disease, such as C9orf72 amyotrophic lateral sclerosis, C9orf72 frontotemporal dementia, or Huntington's disease, e.g., a Huntington-like syndrome caused by a C9orf72 expansion, and treatments are only aimed at alleviating symptoms and improving the patient's quality of life as the disease progresses.

[0009] Thus, there is a need in the art for agents that can selectively and effectively inhibit the expression of the C9orf72 gene, for example, for the treatment of subjects with C9orf72-associated disorders. Summary of the Invention [Means for solving the problem]

[0010] The present disclosure provides an RNAi composition that carries out RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of C9orf72 gene, for example, C9orf72 gene with expanded GGGGCC (G4C2) repeats.C9orf72 gene can be present in cells, for example, in cells of a subject, for example, a human.The use of these iRNAs allows the targeted degradation of the mRNA of corresponding gene (C9orf72 gene) in mammals.

[0011] The iRNA of the present invention is designed to target the C9orf72 gene, for example, the C9orf72 gene that has an extended GGGGCC hexanucleotide repeat in the intron of the gene and has a combination of nucleotide modifications.The agent can target mature C9orf72 mRNA (mRNA from which the intron has been spliced ​​out) or C9orf72 RNA containing hexanucleotide repeats (RNA containing C9orf72 intron 1A).The iRNA of the present invention can reduce the level of C9orf72 mature mRNA rather than reducing the level of C9orf72 RNA containing hexanucleotide repeats. For example, iRNAs of the invention can reduce levels of C9orf72 mature mRNA by about 50% or less, can reduce levels of sense- and antisense-containing aggregates and aberrant dipeptide repeat (DPR) proteins (poly(GA), poly(GR), poly(GP), poly(PA), and poly(PR)), and / or can reduce levels of hexanucleotide repeat-containing C9orf72 RNA by more than about 50%, and can reduce levels of sense- and antisense-containing aggregates and aberrant dipeptide repeat (DPR) proteins (poly(GA), poly(GR), poly(GP), poly(PA), and poly(PR)). Without intending to be limited by theory, it is believed that a 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 invention.

[0012] In one aspect, the present invention provides a double-stranded ribonucleic acid (dsRNA) agent for knocking down a C9orf72 target RNA in a cell.

[0013] In one embodiment, the dsRNA agent targets a region of the C9orf72 target RNA that contains a hexanucleotide repeat.

[0014] In one embodiment, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of C9orf72, e.g., a C9orf72 RNA containing hexanucleotide repeats, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, the antisense strand comprising at least 15 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO: 125 by no more than 3 nucleotides, and the sense strand, the antisense strand, or both the sense and antisense strands are conjugated to one or more lipophilic moieties.

[0015] In another aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of C9orf72, e.g., a C9orf72 RNA containing hexanucleotide repeats, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, the sense strand comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from nucleotides 200-290 of SEQ ID NO: 133, and the antisense strand comprising at least 15 contiguous nucleotides from the corresponding nucleotide sequence of SEQ ID NO: 134, wherein the sense strand, the antisense strand, or both the sense and antisense strands are conjugated to one or more lipophilic moieties.

[0016] In one aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of C9orf72, e.g., a C9orf72 RNA containing hexanucleotide repeats, wherein the dsRNA agent comprises a sense strand and an antisense strand that form a double-stranded region, and the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the antisense nucleotide sequences set forth in any one of Tables 10A, 10B, 12-15, 19, 20, or 21.

[0017] In one embodiment, the nucleotide sequences of the sense and antisense strands comprise any one of the nucleotide sequences of the sense and antisense strands set forth in any one of Tables 12 or 13. In one embodiment, the nucleotide sequence of the antisense strand comprises any one of the nucleotide sequences of the antisense strand set forth in any one of Tables 12 or 13.

[0018] In one embodiment, 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 230-270, 233-262, 800-840, 800-830, 802-828, 1240-1290, 1240-1280, 1247-1288, 1590-1645, 1590-1620, and 1594-1642 of SEQ ID NO: 121, and the antisense strand comprises at least 15 contiguous nucleotides from the corresponding nucleotide sequence of SEQ ID NO: 125.

[0019] In one embodiment, 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 1594-1616, 802-824, 239-261, 1308-1330, 233-255, 1595-1617, 240-262, 1532-1554, 237-259, 3268-3290, 806-828, 1620-1642, 526-548, 1169-1191, 1266-1288, 1247-1269, 586-608, 1257-1279, and 400-422 of SEQ ID NO: 121, and the antisense strand comprises at least 15 contiguous nucleotides from the corresponding nucleotide sequence of SEQ ID NO: 125.

[0020] In one embodiment, the antisense strand is selected from the group consisting of AD-348904.1, AD-348136.1, AD-347612.1, AD-348639.1, AD-347606.1, AD-348905.1, AD-347613.1, AD-348842.1, AD-347610.1, AD-350329.1, AD-348140.1, AD-348930.1, It comprises at least 15 consecutive nucleotides that differ by no more than three nucleotides from any one of the nucleotide sequences of the antisense strand of a duplex selected from the group consisting of AD-347863.1, AD-348500.1, AD-348597.1, AD-348578.1, AD-347923.1, AD-348588.1, and AD-347773.1.

[0021] In one embodiment, the nucleotide sequences of the sense and antisense strands comprise any one of the nucleotide sequences of the sense and antisense strands set forth in any one of Tables 14, 15, 19, 20, or 21.

[0022] In one embodiment, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for selectively inhibiting expression of C9orf72, the dsRNA agent comprising a hexanucleotide repeat comprising multiple contiguous copies of SEQ ID NO:1, wherein the dsRNA agent comprises a sense strand and an antisense strand that form a double-stranded region, and the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the antisense nucleotide sequences set forth in any one of Tables 14, 15, and 21.

[0023] In one embodiment, the antisense strand comprises at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from any one of the nucleotide sequences of the antisense strand of the duplex selected from the group consisting of AD-463863.1, AD-463862.1, AD-463869.1, AD-463873.1, AD-463872.1, and AD-463860.1.

[0024] In one embodiment, the antisense strand comprises at least 15, 16, 17, 18, 19, or 20 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the nucleotide sequences of the antisense strand of the duplex selected from the group consisting of AD-463863.1, AD-463862.1, AD-463869.1, AD-463873.1, AD-463872.1, and AD-463860.1. In some embodiments, the sense and antisense strands are each independently 19, 20, 21, 23, 23, 24, or 25 nucleotides in length.

[0025] In one embodiment, the antisense strand comprises at least 15, 16, 17, 18, 19, or 20 contiguous nucleotides that differ by no more than two nucleotides from any one of the nucleotide sequences of the antisense strand of the duplex selected from the group consisting of AD-463863.1, AD-463862.1, AD-463869.1, AD-463873.1, AD-463872.1, and AD-463860.1. In some embodiments, the sense and antisense strands are each independently 19, 20, 21, 23, 23, 24, or 25 nucleotides in length.

[0026] In one embodiment, the antisense strand comprises at least 15, 16, 17, 18, 19, or 20 contiguous nucleotides that differ by no more than one nucleotide from any one of the nucleotide sequences of the antisense strand of the duplex selected from the group consisting of AD-463863.1, AD-463862.1, AD-463869.1, AD-463873.1, AD-463872.1, and AD-463860.1. In some embodiments, the sense and antisense strands are each independently 19, 20, 21, 23, 2324, or 25 nucleotides in length.

[0027] In one embodiment, the antisense strand comprises at least 15, 16, 17, 18, 19, or 20 contiguous nucleotides from any one of the nucleotide sequences of the antisense strand of the duplex selected from the group consisting of AD-463863.1, AD-463862.1, AD-463869.1, AD-463873.1, AD-463872.1, and AD-463860.1. In some embodiments, the sense and antisense strands are each independently 19, 20, 21, 23, 23, 24, or 25 nucleotides in length.

[0028] In one embodiment, the C9orf72 target RNA comprises a hexanucleotide repeat comprising multiple contiguous copies of SEQ ID NO:1, and the dsRNA agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementarity to the C9orf72 target RNA, wherein the region of complementarity comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the reverse complement of the nucleotide sequence between the start of exon 1A and the start of exon 2 of the C9orf72 target RNA.

[0029] In one embodiment, the C9orf72 target RNA comprises a hexanucleotide repeat comprising multiple contiguous copies of SEQ ID NO:1, and the dsRNA agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a sequence of 15-25 contiguous nucleotides that has at least 80% complementarity to a sequence of 15-25 contiguous nucleotides present in a sequence between the start of exon 1A and the start of exon 2 of the C9orf72 target RNA.

[0030] In one embodiment, the region of complementarity comprises the reverse complement of at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence between exon 1A and exon 1B of the C9orf72 target RNA.

[0031] In one embodiment, the region of complementarity comprises the reverse complement of at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence between exon 1A of the C9orf72 target RNA and the hexanucleotide repeat.

[0032] In one embodiment, the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:115, and the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:116.

[0033] In one embodiment, the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:117, and the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:118.

[0034] In one embodiment, the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the antisense strand sequences in SEQ ID NOs: 3, 5, 7, 9, 11, and 13, and / or the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the sense strand sequences in SEQ ID NOs: 2, 4, 6, 8, 10, and 12.

[0035] In one embodiment, the nucleotide sequence between the start of exon 1A and the start of exon 2 of the C9orf72 target RNA is not present in the messenger RNA of mature C9orf72.

[0036] In one embodiment, the sense strand, the antisense strand, or both the sense and antisense strands are conjugated to one or more lipophilic moieties.

[0037] In one embodiment, the lipophilic moiety is conjugated to one or more internal positions in the double-stranded region of the dsRNA agent.

[0038] In one embodiment, the lipophilic moiety is conjugated via a linker or carrier.

[0039] In one embodiment, the lipophilicity of the lipophilic moiety, as measured by log Kow, is greater than zero.

[0040] In one embodiment, the hydrophobicity of the double-stranded RNAi agent is greater than 0.2 as measured by the unbound fraction in a plasma protein binding assay of the double-stranded RNAi agent.

[0041] In one embodiment, the plasma protein binding assay is an electrophoretic mobility shift assay using human serum albumin protein.

[0042] In some embodiments, the dsRNA agent includes at least one modified nucleotide.

[0043] In one embodiment, no more than 5 of the sense strand nucleotides and no more than 5 of the nucleotides in the antisense strand are unmodified nucleotides.

[0044] In one embodiment, all of the nucleotides in the sense strand and all of the nucleotides in the antisense strand are modified nucleotides.

[0045] In one embodiment, at least one of the modified nucleotides is a deoxy-nucleotide, a 3' terminal deoxy-thymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, a non-locked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino modified nucleotide, a 2'-O-allyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2'-hydroxyl (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 nucleotide, or a nucleotide containing a non-natural base. nucleotides containing 5'-phosphate, 1,5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides containing 5'-phosphorothioate groups, nucleotides containing 5'-methylphosphonate groups, nucleotides containing 5' phosphate or 5' phosphate mimics, nucleotides containing vinylphosphonates, nucleotides containing adenosine-glycol nucleic acid (GNA), nucleotides containing thymidine-glycol nucleic acid (GNA) S-isomers, nucleotides containing 2-hydroxymethyl-tetrahydrofuran-5-phosphate, nucleotides containing 2'-deoxythymidine-3' phosphate, nucleotides containing 2'-deoxyguanosine-3'-phosphate, and terminal nucleotides linked to cholesteryl derivatives and dodecanoic acid bisdecylamide groups; and combinations thereof.

[0046] In one embodiment, the modified nucleotide is selected from the group consisting of 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, 3'-terminal deoxythymine nucleotides (dT), locked nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, and nucleotides containing unnatural bases.

[0047] In one embodiment, the modified nucleotides include a short sequence of 3' terminal deoxythymine nucleotides (dT).

[0048] In one embodiment, the modifications to the nucleotides are 2'-O-methyl, GNA and 2' fluoro modifications.

[0049] In one embodiment, substantially all of the nucleotides of the sense strand comprise a modification selected from the group consisting of a 2'-O-methyl modification and a 2'-fluoro modification. In some embodiments, all of the nucleotides of the sense strand comprise a modification selected from the group consisting of a 2'-O-methyl modification and a 2'-fluoro modification.

[0050] In one embodiment, substantially all of the nucleotides of the antisense strand comprise a modification selected from the group consisting of a 2'-O-methyl modification and a 2'-fluoro modification. In some embodiments, all of the nucleotides of the antisense strand comprise a modification selected from the group consisting of a 2'-O-methyl modification and a 2'-fluoro modification.

[0051] In one embodiment, substantially all of the nucleotides of the sense strand comprise a modification selected from the group consisting of a 2'-O-methyl modification, a 2'-fluoro modification, and a glycol nucleic acid (GNA) modification. In some embodiments, all of the nucleotides of the sense strand comprise a modification selected from the group consisting of a 2'-O-methyl modification, a 2'-fluoro modification, and a glycol nucleic acid (GNA) modification.

[0052] In one embodiment, substantially all of the nucleotides of the antisense strand comprise a modification selected from the group consisting of a 2'-O-methyl modification, a 2'-fluoro modification, and a glycol nucleic acid (GNA) modification. In some embodiments, all of the nucleotides of the antisense strand comprise a modification selected from the group consisting of a 2'-O-methyl modification, a 2'-fluoro modification, and a glycol nucleic acid (GNA) modification.

[0053] In some embodiments, the dsRNA agent further comprises at least one phosphorothioate internucleotide linkage.

[0054] In one embodiment, the dsRNA agent includes 6 to 8 phosphorothioate internucleotide linkages.

[0055] In one embodiment, the sense strand comprises at least one phosphorothioate or methylphosphonate internucleotide linkage and the antisense strand comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.

[0056] In one embodiment, the sense strand comprises at least two phosphorothioate or methylphosphonate internucleotide linkages.

[0057] In one embodiment, the antisense strand comprises at least two, at least three, or at least four phosphorothioate or methylphosphonate internucleotide linkages.

[0058] In one embodiment, at least one phosphorothioate or methylphosphonate internucleotide linkage is at the 5'-end of one strand, at the 3'-end of one strand, or at both the 5'-end and the 3'-end of one strand.

[0059] In one embodiment, at least one phosphorothioate or methylphosphonate internucleotide linkage is at the 5'-end of the sense strand. In some embodiments, the sense strand comprises two phosphorothioate internucleotide linkages at the 5'-end.

[0060] In one embodiment, at least one phosphorothioate or methylphosphonate internucleotide linkage is present at both the 5'- and 3'-ends of the antisense strand. In some embodiments, the antisense strand comprises two phosphorothioate internucleotide linkages at the 5'-end and two phosphorothioate internucleotide linkages at the 3'-end.

[0061] In one embodiment, all of the nucleotides of the sense strand comprise a modification selected from the group consisting of a 2'-O-methyl modification and a 2'-fluoro modification, all of the nucleotides of the antisense strand comprise a modification selected from the group consisting of a 2'-O-methyl modification and a 2'-fluoro modification, the sense strand comprises two phosphorothioate internucleotide linkages at the 5'-terminus, and the antisense strand comprises two phosphorothioate internucleotide linkages at the 5'-terminus and two phosphorothioate internucleotide linkages at the 3'-terminus.

[0062] In one embodiment, (I) the sense strand comprises (a) 2'-O-methyl modifications at a plurality of nucleotides; (b) 2'-fluoro modifications at a plurality of nucleotides; and (c) phosphorothioate internucleotide linkages at a plurality of nucleotides; (II) the antisense strand comprises (a) 2'-O-methyl modifications at a plurality of nucleotides; (c) 2'-fluoro modifications at a plurality of nucleotides; and (d) phosphorothioate internucleotide linkages between a plurality of nucleotides. Optionally, the dsRNA agent comprises an overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0063] In one embodiment, (I) the sense strand comprises (a) a length of 21 nucleotides; (b) 2'-O-methyl modifications at nucleotides 1 to 6, 8, and 12 to 21, counting from the 5' end; (c) 2'-fluoro modifications at nucleotides 7 and 9 to 11, counting from the 5' end; and (d) phosphorothioate internucleotide linkages at nucleotides between 1 and 2 and between 2 and 3, counting from the 5' end; and (II) the antisense strand comprises (a) a length of 23 nucleotides; (b) 2'-O-methyl modifications at nucleotides 1, 3 to 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 8 (c) 2'-O-methyl modifications at positions 10-13, 15, and 17-23; (c) 2'-fluoro modifications at positions 2, 6, 8, 9, 14, and 16, counting from the 5' end; and (d) phosphorothioate internucleotide linkages at nucleotides between positions 1 and 2, between positions 2 and 3, between positions 21 and 22, and between positions 22 and 23 (counting from the 5' end), wherein the dsRNA agent comprises a two-nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0064] In one embodiment, the antisense strand comprises any one of the antisense strand sequences in SEQ ID NOs: 3, 5, 7, 9, 11, and 13, and / or the sense strand comprises any one of the sense strand sequences in SEQ ID NOs: 2, 4, 6, 8, 10, and 12.

[0065] In one embodiment, the sense strand is 30 nucleotides or less in length. In another embodiment, the antisense strand is 30 nucleotides or less in length. In one embodiment, the sense strand and the antisense strand are each independently 30 nucleotides or less in length.

[0066] In one embodiment, at least one strand comprises a 3'-overhang of at least 1 nucleotide. In another embodiment, at least one strand comprises a 3'-overhang of at least 2 nucleotides. In one embodiment, the antisense strand comprises a 3'-overhang.

[0067] The double-stranded region may 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; 21 to 23 nucleotide pairs in length, or 21 nucleotide pairs in length.

[0068] The sense and antisense strands can each independently be 19 to 30 nucleotides; 19 to 25 nucleotides; 19 to 23 nucleotides; or 21 to 23 nucleotides in length, or 21 nucleotides in length.

[0069] In one embodiment, the region of complementarity is at least 17 nucleotides in length, hi other embodiments, the region of complementarity is 19-30 nucleotides in length; 19-25 nucleotides in length; or 21-23 nucleotides in length.

[0070] In one embodiment, the region of complementarity is at least 17 nucleotides in length, hi other embodiments, the region of complementarity is 19-30 nucleotides in length; 19-25 nucleotides in length; or 21-23 nucleotides in length.

[0071] In one embodiment, the region of complementarity is at least 85% complementary to the sequence between the start of exon 1A and the start of exon 2 of the C9orf72 gene. In some embodiments, the antisense strand comprises a sequence of 15-25 contiguous nucleotides that is at least 85% complementary to a sequence of 15-25 contiguous nucleotides present in the sequence between the start of exon 1A and the start of exon 2 of the C9orf72 target RNA. In other embodiments, the region of complementarity is at least 90% complementary to the sequence between the start of exon 1A and the start of exon 2 of the C9orf72 target RNA. In one embodiment, the region of complementarity is at least 95% complementary to the sequence between the start of exon 1A and the start of exon 2 of the C9orf72 target RNA. In some embodiments, the region of complementarity is 100% complementary to the sequence between the start of exon 1A and the start of exon 2 of the C9orf72 target RNA. In some embodiments, the region of complementarity is 100% complementary to the sequence between the end of exon 1A of the C9orf72 target RNA and the start of the hexanucleotide repeat region.

[0072] In one embodiment, the region of complementarity is at least 85% complementary to the sequence between the end of exon 1A and the start of the hexanucleotide repeat in intron 1A of the C9orf72 gene. In some embodiments, the antisense strand comprises a sequence of 15-25 contiguous nucleotides that is at least 85% complementary to a sequence of 15-25 contiguous nucleotides present in the sequence between the end of exon 1A and the start of the hexanucleotide repeat in intron 1A of the C9orf72 target RNA. In other embodiments, the region of complementarity is at least 90% complementary to the sequence between the end of exon 1A and the start of the hexanucleotide repeat in intron 1A of the C9orf72 target RNA. In one embodiment, the region of complementarity is at least 95% complementary to the sequence between the end of exon 1A and the start of the hexanucleotide repeat in intron 1A of the C9orf72 target RNA. In some embodiments, the region of complementarity is 100% complementary to the sequence between the end of exon 1A and the start of the hexanucleotide repeat in intron 1A of the C9orf72 target RNA.

[0073] 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.

[0074] In one embodiment, internal positions include all but the two most distal positions from each end of at least one strand.

[0075] In another embodiment, the internal positions include all but the last three positions from each end of at least one strand.

[0076] In one embodiment, the internal position excludes the cleavage site region of the sense strand.

[0077] In one embodiment, internal positions include all positions except positions 9-12 counting from the 5' end of the sense strand.

[0078] In another embodiment, internal positions include all positions except positions 11-13 counting from the 3' end of the sense strand.

[0079] In one embodiment, the internal position excludes the cleavage site region of the antisense strand.

[0080] In one embodiment, internal positions include all positions except positions 12-14 counting from the 5' end of the antisense strand.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] In one embodiment, the internal position in the double-stranded region excludes the cleavage site region of the sense strand.

[0085] 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.

[0086] In one embodiment, the lipophilic moiety is conjugated to position 21, position 20, position 15, position 1, or position 7 of the sense strand.

[0087] In another embodiment, the lipophilic moiety is conjugated to position 21, 20, or 15 of the sense strand.

[0088] In yet another embodiment, the lipophilic moiety is conjugated to position 20 or 15 of the sense strand.

[0089] In one embodiment, the lipophilic moiety is conjugated to position 16 of the antisense strand.

[0090] In one embodiment, the lipophilic moiety is an aliphatic, alicyclic, or polyalicyclic compound.

[0091] In one embodiment, the lipophilic moiety is 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 group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenoic acid, dimethoxytrityl, or phenoxazine.

[0092] 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.

[0093] In one embodiment, the lipophilic moiety contains a saturated or unsaturated C6 to C18 hydrocarbon chain.

[0094] In one embodiment, the lipophilic moiety contains a saturated or unsaturated C16 hydrocarbon chain.

[0095] In one embodiment, the saturated or unsaturated C16 hydrocarbon chain is conjugated to position 6, counting from the 5' end of the chain.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] In one embodiment, the lipophilic moiety is conjugated to a nucleobase, a sugar moiety, or an internucleoside linkage.

[0100] In one embodiment, the lipophilic moiety or targeting ligand is conjugated via a biocleavable linker selected from the group consisting of DNA, RNA, disulfide, amide, and functionalized mono- or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.

[0101] 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.

[0102] In one embodiment, the dsRNA agent further comprises a targeting ligand that targets liver tissue.

[0103] In one embodiment, the targeting ligand is a GalNAc conjugate.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] In another embodiment, the dsRNA agent further comprises 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.

[0108] 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, the first and second internucleotide linkages having the linking phosphorus atom in the Sp configuration, a terminal chiral modification occurring at the first and second internucleotide linkages at the 5'-end of the antisense strand, the first internucleotide linkages having the linking phosphorus atom in the Rp configuration, and a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the sense strand, the first internucleotide linkage having the linking phosphorus atom in either the Rp or Sp configuration.

[0109] In one embodiment, the dsRNA agent further comprises a phosphate or a phosphate mimetic at the 5'-end of the antisense strand.

[0110] In one embodiment, the phosphate mimic is 5'-vinylphosphonate (VP).

[0111] 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.

[0112] In one embodiment, the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

[0113] In one embodiment, the dsRNA agent inhibits expression of a C9orf72 target RNA that includes a hexanucleotide repeat by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% within 24 to 48 hours after administration to a cell that expresses the C9orf72 target RNA that includes the hexanucleotide repeat.

[0114] In one embodiment, the dsRNA agent selectively inhibits expression of a C9orf72 target RNA that includes a hexanucleotide repeat relative to expression of the mature C9orf72 messenger RNA.

[0115] In one embodiment, the dsRNA agent inhibits expression of mature C9orf72 messenger RNA by less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% within 24 to 48 hours after administration to a cell expressing mature C9orf72 messenger RNA.

[0116] In one embodiment, the dsRNA agent reduces dipeptide repeat protein synthesis within 24-48 hours after administration to a cell expressing a C9orf72 target RNA containing a hexanucleotide repeat. In some embodiments, the dsRNA agent reduces dipeptide repeat protein synthesis by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% within 24-48 hours after administration to the cell.

[0117] The present invention also provides cells and pharmaceutical compositions for inhibiting expression of the gene encoding C9orf72 comprising a dsRNA agent of the invention.

[0118] In one embodiment, the dsRNA agent is in an unbuffered solution, such as saline or water.

[0119] 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).

[0120] In one aspect, the invention provides a method of inhibiting expression of C9orf72, e.g., a C9orf72 RNA containing hexanucleotide repeats, e.g., a C9orf72 gene comprising multiple consecutive copies of hexanucleotide repeats, in a cell, e.g., in a neuron, e.g., a motor neuron, comprising contacting the cell with a dsRNA agent of the invention or a pharmaceutical composition of the invention, thereby inhibiting expression of the C9orf72 gene in the cell.

[0121] In another aspect, the present invention provides a method for reducing dipeptide repeat protein synthesis or dipeptide repeat protein aggregation in cells.The method comprises introducing a dsRNA agent of the present invention into cells, thereby reducing dipeptide repeat protein synthesis or dipeptide repeat protein aggregation in cells.

[0122] In another embodiment, the present invention provides a method for reducing the sense and / or antisense C9orf72 RNA aggregation in the nucleus and / or cytoplasm of a cell.The method includes introducing the dsRNA agent of the present invention into a cell, thereby reducing the sense and / or antisense C9orf72 RNA aggregation in the nucleus and / or cytoplasm of the cell.

[0123] In one embodiment, the cell is in a subject.

[0124] In one embodiment, the subject is a human.

[0125] In one embodiment, the subject has or is at risk of developing a C9orf72-associated disorder, eg, a disease, condition, or disorder associated with a C9orf72 hexanucleotide repeat expansion.

[0126] In one embodiment, the C9orf72-associated disorder is selected from the group consisting of C9orf72 amyotrophic lateral sclerosis / frontotemporal dementia or Huntington-like syndrome caused by C9orf72 expansion.

[0127] In one embodiment, contacting the cell with the dsRNA agent inhibits expression of C9orf72 by 50%.

[0128] In one embodiment, the dsRNA agent inhibits expression of a C9orf72-targeting mRNA that includes the hexanucleotide repeat by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% within 24 to 48 hours after administration to a cell expressing the C9orf72-targeting mRNA that includes the hexanucleotide repeat.

[0129] In some embodiments, the dsRNA agent selectively inhibits expression of a C9orf72 target RNA that includes a hexanucleotide repeat relative to expression of mature C9orf72 messenger RNA. In other embodiments, the dsRNA agent inhibits expression of mature C9orf72 messenger RNA by less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% within 24 to 48 hours after administration to cells expressing mature C9orf72 messenger RNA.

[0130] In some embodiments, the dsRNA agent reduces dipeptide repeat protein synthesis or dipeptide repeat protein aggregation in a cell.

[0131] In some embodiments, the dsRNA agent reduces nuclear and / or cytoplasmic sense and / or antisense C9orf72 RNA aggregation in the cell.

[0132] In one embodiment, inhibiting expression of C9orf72 reduces C9orf72 protein levels in the serum of the subject by 50% or less.

[0133] In some embodiments, the dsRNA agent reduces dipeptide repeat protein synthesis or dipeptide repeat protein aggregation by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% within 24 to 48 hours after administration to a cell.

[0134] In one aspect, the invention provides a method of treating a subject having a disorder that would benefit from knocking down a target C9orf72 RNA, e.g., a disease, condition, or disorder associated with a C9orf72 hexanucleotide repeat expansion, comprising administering to the subject a therapeutically effective amount of a dsRNA agent of the invention or a pharmaceutical composition of the invention, thereby treating the subject having a disorder that would benefit from reduced C9orf72 expression.

[0135] In another aspect, the invention provides a method for preventing at least one symptom in a subject having a disorder that would benefit from reduced C9orf72 expression, e.g., a disease, condition, or disorder associated with a C9orf72 hexanucleotide repeat expansion, comprising administering to the subject a prophylactically effective amount of a dsRNA agent of the invention or a pharmaceutical composition of the invention, thereby preventing at least one symptom in a subject having a disorder that would benefit from reduced C9orf72 expression.

[0136] In one embodiment, the disorder is a C9orf72-associated disorder.

[0137] In one embodiment, the C9orf723-associated disorder is selected from the group consisting of C9orf72 amyotrophic lateral sclerosis / frontotemporal dementia or Huntington-like syndrome caused by C9orf72 expansion.

[0138] In one embodiment, the subject is a human.

[0139] In one embodiment, administration of the agent to the subject results in a decrease in C9orf72 protein accumulation.

[0140] In some embodiments, the method reduces dipeptide repeat protein synthesis or reduces dipeptide repeat protein aggregation in a subject. In some embodiments, the method reduces expression of a C9orf72 target RNA comprising a hexanucleotide repeat comprising multiple consecutive copies of SEQ ID NO: 1 in a subject.

[0141] 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.

[0142] In one embodiment, the dsRNA agent is administered to the subject subcutaneously.

[0143] In another embodiment, the dsRNA agent is administered to the subject intrathecally.

[0144] In one embodiment, the method of the invention further comprises determining the level of C9orf72 in a sample from the subject.

[0145] In one embodiment, the level of C9orf72 in the subject sample is the C9orf72 protein level in a blood, serum, or cerebrospinal fluid sample.

[0146] In one embodiment, the methods of the invention further comprise administering to the subject an additional therapeutic agent.

[0147] In one aspect, the invention provides a kit comprising a dsRNA agent of the invention or a pharmaceutical composition of the invention.

[0148] In another embodiment, the invention provides a vial containing a dsRNA agent of the invention or a pharmaceutical composition of the invention.

[0149] In yet another embodiment, the invention provides a syringe comprising a dsRNA agent of the invention or a pharmaceutical composition of the invention. [Brief explanation of the drawings]

[0150] [Figure 1] Figure 1 (not to scale) is a schematic diagram of the wild-type mouse C9orf72 locus, the wild-type human C9orf72 locus, and the humanized mouse C9orf72 locus (MAID8029a) containing 92 repeats of the hexanucleotide sequence set forth in SEQ ID NO: 1 (GGGGCC). The mouse sequence is indicated by a box with a dashed line and a dash, and the human sequence is indicated by a solid black line. Exons are represented by boxes. The positions of the hexanucleotide repeat sequences are indicated. [Figure 2] FIG. 2 (not to scale) is a schematic diagram of the concept of using a nuclease agent to introduce a double-stranded break near a repeat sequence (e.g., the hexanucleotide sequence set forth in SEQ ID NO: 1 in the C9orf72 locus) in order to extend the repeat sequence in a target genomic locus using the recombination machinery. [Figure 3] FIG. 3 (not to scale) is a schematic diagram with the locations of eight guide RNA target sequences located near the ends of the hexanucleotide repeat expansion sequences in C9orf72 humanized 92x repeat-containing ES cells (MAID8029a). [Figure 4] Figure 4 depicts the cleavage efficiency of eight guide RNAs evaluated in a cell-free system using a humanized 92x repeat-containing allele (MAID8029a) and a plasmid containing the same sequence (8028 Stvec). [Figure 5]Figure 5 (not to scale) is a schematic diagram of a scheme for extending the 92x repeat by introducing double-stranded breaks near the 5' end (Box 2), near the 3' end (Box 3), or near both the 5' and 3' ends (Box 1) of the 92x repeat extension sequence. Black boxes indicate the endogenous murine sequence; white boxes indicate the humanized region. [Figure 6] Figure 6A (not to scale) is a schematic diagram of a conventional two-primer PCR used to assess the number of instances of the hexanucleotide sequence set forth in SEQ ID NO: 1 in endogenous C9orf72 ES cell clones. Figure 6B (not to scale) is a schematic diagram of a primed PCR using three primers to assess the number of instances of the hexanucleotide sequence set forth in SEQ ID NO: 1 in endogenous C9orf72 ES cell clones. [Figure 7] Figure 7A shows the results of conventional PCR of the C9orf72 locus to assess the size of the repeat section after truncation near the 5' end of the 92x repeat extension sequence. The extended repeat is marked with an asterisk. Figure 7B shows the results of conventional PCR of the C9orf72 locus to assess the size of the repeat section after truncation near the 3' end of the 92x repeat extension sequence. The extended repeat is marked with an asterisk. [Figure 8] Figure 8 shows the results of primed PCR to confirm the number of repeats in one of the clones from Figure 7A and one of the clones from Figure 7B. The parental 8029a (92x repeats) clone was used as a control. Figure 8 shows the results from capillary electrophoresis. Signal intensity is shown on the Y-axis and PCR product size on the X-axis. The readout is the number of peaks. [Figure 9] FIG. 9 shows the results of primed PCR to confirm the number of repeats in one of the clones from FIG. 7A and one of the clones from FIG. 7B. [Figure 10]Figure 10 shows the results of conventional PCR of the C9orf72 locus to assess the size of the repeat section after truncation near the 5' end of the 92x repeat extension sequence, truncation near the 3' end of the 92x repeat extension sequence, or truncation near both the 5' and 3' ends of the 92x repeat extension sequence. The expanded repeat is marked by an arrow. [Figure 11-1] Figure 11A (not to scale) shows a schematic diagram of a scheme for extending 92x repeats in mouse ES cells by first introducing a double-stranded break near the 5' end of the 92x repeat extension sequence to generate a first extended clone, and then introducing a double-stranded break near the 3' end of the repeat extension sequence in the first extended clone to generate a second extended clone. The humanized region is indicated by a label and a white box. The locations of the 5' and 3' DSBs are shown, along with the locations of primers for detection by PCR. [Figure 11-2] Figure 11B shows the results of conventional PCR of the C9orf72 locus to assess the size of the repeat section after the second extension after excision of the 250x repeat extension sequence clone near the 3' end of the 250x repeat extension sequence. The extended repeat and parent repeat are marked by labeled arrows. [Figure 12-1] 12A-12D are bar graphs showing the expression levels (y-axis) of transcripts from the C9orf72 locus [determined by TAQMAN® quantitative reverse transcription-linked PCR (RT-qPCR) assay shown in the depiction of the C9orf72 locus above each figure] that are exon 1A-exon 2 spliced ​​transcripts (FIG. 12A), exon 1B-exon 2 spliced ​​transcripts (FIG. 12B), contain intronic sequences near exon 1A (FIG. 12C), and retain intronic sequences near exon 1B (FIG. 12D) in embryonic stem cell-derived motor neurons (ESMN) heterozygous for a modified C9orf72 locus containing 3, 92, 250, or 300 repeats of the hexanucleotide sequence set forth as SEQ ID NO: 1 compared to ESMN containing three repeats of the hexanucleotide sequence set forth as SEQ ID NO: 1. [Figure 12-2] Same as above. [Figure 12-3] Figures 12E-12H are bar graphs showing the expression levels (y-axis) of transcripts from the C9orf72 locus [determined by TAQMAN® quantitative reverse transcription-linked PCR (RT-qPCR) assay shown in the depiction of the C9orf72 locus above each figure] that are exon 1A-exon 2 spliced ​​transcripts (Figure 12E), exon 1B-exon 2 spliced ​​transcripts (Figure 12F), contain intronic sequences near exon 1A (Figure 12G), and retain intronic sequences near exon 1B (Figure 12H) in embryonic stem cell-derived motor neurons (ESMN) heterozygous for modified C9orf72 loci containing 3, 500, or 600 repeats of the hexanucleotide sequence set forth as SEQ ID NO: 1 compared with ESMN containing three repeats of the hexanucleotide sequence set forth as SEQ ID NO: 1. [Figure 12-4] Same as above. [Figure 13] Figure 13 (top) shows Western slot blot images of lysates from embryonic stem cell-derived motor neurons (ESMN) heterozygous for modified C9orf72 loci containing 3, 92, 300, 500, or 600 repeats of the hexanucleotide sequence set forth as SEQ ID NO: 1. Lysates containing 0 μg, 1.25 μg, 2.5 μg, 5 μg, 10 μg, or 20 μg of total protein were blotted with anti-poly-GlyPro or anti-poly-GlyAla antibodies. Figure 13 (bottom) shows quantification of the Western slot blot in the upper part of the figure. [Figure 14]14A-14B show the C9orf72 locus in embryonic stem cell-derived motor neurons (ESMN) heterozygous for a modified C9orf72 locus containing 3, 92, 300, 500, or 600 repeats of the hexanucleotide sequence set forth as SEQ ID NO: 1 compared to ESMN containing three repeats of the hexanucleotide sequence set forth as SEQ ID NO: 1 (FIG. 14A), or in brainstem and spinal cord samples from mice containing three repeats of the hexanucleotide sequence set forth as SEQ ID NO: 1 compared to ESMN. 14A and 14B are bar graphs showing the expression levels (y-axis) of transcripts from the C9orf72 locus containing intronic sequences near exon 1A (as determined by TAQMAN® quantitative reverse transcription-linked PCR (RT-qPCR) assays shown in the depiction of the C9orf72 locus above each figure) in brainstem and spinal cord samples from mice heterozygous for a modified (humanized) C9orf72 locus containing 3 or 300 repeats of the hexanucleotide sequence depicted as 1 (FIG. 14B). [Figure 15-1] 15A-15D are bar graphs showing the expression levels (y-axis) of transcripts from the C9orf72 locus [determined by TAQMAN® quantitative reverse transcription-linked PCR (RT-qPCR) assays shown in the depiction of the C9orf72 locus above each figure] that are exon 1A-exon 2 spliced ​​transcripts (FIG. 15A), exon 1B-exon 2 spliced ​​transcripts (FIG. 15B), contain intronic sequences near exon 1A (FIG. 15C), and retain intronic sequences near exon 1B (FIG. 15D) in embryonic stem cell-derived motor neurons (ESMN) that are hypaxial-like or limb-like motor neurons and are heterozygous for a modified C9orf72 locus containing 3, 92, 300, 500, or 600 repeats of the hexanucleotide sequence set forth as SEQ ID NO: 1 compared to ESMN that contain three repeats of the hexanucleotide sequence set forth as SEQ ID NO: 1. [Figure 15-2] Same as above. [Figure 15-3]Figures 15E-15F are bar graphs showing the expression levels (y-axis) of transcripts from the C9orf72 locus [determined by TAQMAN® quantitative reverse transcription-linked PCR (RT-qPCR) assays shown in the depiction of the C9orf72 locus above each figure], which are either unspliced ​​precursor transcripts (Figure 15E) or spliced ​​C9orf72 mRNA (Figure 15F), in embryonic stem cell-derived motor neurons (ESMN) that are hypaxial-like or limb-like motor neurons and are heterozygous for a modified C9orf72 locus containing 3, 92, 300, 500, or 600 repeats of the hexanucleotide sequence set forth as SEQ ID NO:1, compared to ESMN that contain three repeats of the hexanucleotide sequence set forth as SEQ ID NO:1. [Figure 16] 16A-16B show a mutant GAPDH gene that is heterozygous for a modified C9orf72 locus containing 300 repeats of the hexanucleotide sequence set forth as SEQ ID NO: 1 and is mutated using siRNA targeting intron 1 of C9orf72, siRNA targeting spliced ​​C9orf72 mRNA [exon 11 (siRNA 1), exon 5 (siRNAs 2 and 6), or exon 2 (siRNAs 3 and 5), or exon 9 (siRNA 4)], or a control GAPDH gene. 16A and 16B are bar graphs showing the expression levels (y-axis) of transcripts from the C9orf72 locus (determined by TAQMAN® quantitative reverse transcription-linked PCR (RT-qPCR) assays shown in the depiction of the C9orf72 locus above each figure) that are exon 2-3 spliced ​​transcripts (FIG. 16A) or contain intronic sequences near exon 1A (FIG. 16B) in embryonic stem cell-derived motor neurons (ESMN) treated with siRNA at 24 and 48 hours after treatment. Asterisks indicate which samples correspond to those in the Western blots of FIG. 17A and FIG. 17B. [Figure 17]Figure 17A shows Western slot blot images of lysates from embryonic stem cell-derived motor neurons (ESMN) heterozygous for a modified C9orf72 locus containing 300 repeats of the hexanucleotide sequence set forth as SEQ ID NO: 1 and treated with siRNA targeting intron 1 of C9orf72, siRNA targeting spliced ​​C9orf72 mRNA [exon 11 (siRNA 1), exon 5 (siRNA 2), or exon 2 (siRNA 3)], or control GAPDH siRNA at 48 hours post-treatment. Lysates containing 1.25 μg or 2.5 μg of total protein were blotted with an anti-poly-GlyAla antibody. Figure 17B shows quantification of the Western slot blot in Figure 17A. [Figure 18-1] FIG. 18A is a schematic diagram (not to scale) of siRNA targeting intron 1A of C9orf72. [Figure 18-2] Figure 18B is a schematic diagram (not to scale) of TAQMAN® qualitative PCR assays A, B, C, D, E, F, G, H, I, and J across different sections of the C9orf72 pre-mRNA. Primer and probe sequences are shown in Table 8. [Figure 19-1]19A-19C show the expression of 300 repeats of the hexanucleotide sequence set forth as SEQ ID NO:1 in mouse embryonic stem cells containing a modified C9orf72 locus containing exon 1A (siRNA E1A #1, E1A #2, and E1A #3), intron 1A upstream of the hexanucleotide repeats (siRNA I1A #2, I1A #4, I1A #6, and I1A #10), or intron 1B downstream of the hexanucleotide repeats and downstream of exon 1B (siRNA I1B #1 and I1B #2). 19A and 19B are bar graphs showing the relative expression levels (y-axis) of transcripts from the C9orf72 locus (as determined by TAQMAN® quantitative reverse transcription-linked PCR (RT-qPCR) assay) that contain the C9orf72 intron 1A sequence (FIG. 19A; Assay F), are C9orf72 exon 2-3 spliced ​​transcripts (FIG. 19B), or are C9orf72 exon 1a-2 spliced ​​transcripts (FIG. 19C; Assay B) 24 hours after transfection of siRNA targeting Gapdh siRNA (Gapdh siRNA #6). Gapdh siRNA was used as a control, and mock was used as a negative control. [Figure 19-2] Same as above. [Figure 20-1]Figures 20A-20C show the expression of 300 repeats of the hexanucleotide sequence set forth as SEQ ID NO:1 in mouse embryonic stem cells containing a modified C9orf72 locus containing exon 1A (siRNA E1A #1, E1A #2, and E1A #3), intron 1A upstream of the hexanucleotide repeats (siRNA I1A #2, I1A #4, I1A #6, and I1A #10), or intron 1B downstream of the hexanucleotide repeats and downstream of exon 1B (siRNA I1B #1 and I1B #2). 20A ; Assay F); C9orf72 exon 2-3 spliced ​​transcript (FIG. 20B); or C9orf72 exon 1a-2 spliced ​​transcript (FIG. 20C; Assay B). 48 hours after transfection of siRNA targeting Gapdh siRNA (Gapdh siRNA #6), relative expression levels (y-axis) of transcripts from the C9orf72 locus (as determined by TAQMAN® quantitative reverse transcription-linked PCR (RT-qPCR) assay) containing C9orf72 intron 1A (FIG. 20A ; Assay F), C9orf72 exon 2-3 spliced ​​transcript (FIG. 20B), or C9orf72 exon 1a-2 spliced ​​transcript (FIG. 20C; Assay B). Gapdh siRNA was used as a control, and mock was used as a negative control. [Figure 20-2] Same as above. [Figure 21-1]21A-21C show the expression of 300 repeats of the hexanucleotide sequence set forth as SEQ ID NO:1 in mouse embryonic stem cells containing a modified C9orf72 locus containing exon 1A (siRNA E1A #1, E1A #2, and E1A #3), intron 1A upstream of the hexanucleotide repeats (siRNA I1A #2, I1A #4, I1A #6, and I1A #10), or intron 1B downstream of the hexanucleotide repeats and downstream of exon 1B (siRNA I1B #1 and I1B #2). 21A ; Assay F); C9orf72 exon 2-3 spliced ​​transcript (FIG. 21B); or C9orf72 exon 1a-2 spliced ​​transcript (FIG. 21C; Assay B). 72 hours after transfection of siRNA targeting Gapdh siRNA (Gapdh siRNA #6), relative expression levels (y-axis) of transcripts from the C9orf72 locus (as determined by TAQMAN® quantitative reverse transcription-linked PCR (RT-qPCR) assay) containing C9orf72 intron 1A (FIG. 21A ; Assay F), C9orf72 exon 2-3 spliced ​​transcript (FIG. 21B), or C9orf72 exon 1a-2 spliced ​​transcript (FIG. 21C; Assay B). Gapdh siRNA was used as a control, and mock was used as a negative control. [Figure 21-2] Same as above. [Figure 22-1]Figures 22A-22E show the expression of mature C9orf72 mRNA (siRNA #1) or intron 1A upstream of the hexanucleotide repeats (siRNA #2) in mouse embryonic stem cells containing a modified C9orf72 locus containing 300 repeats of the hexanucleotide sequence set forth as SEQ ID NO: 1. Figure 22B is a bar graph showing the relative expression levels (y-axis) of transcripts from the C9orf72 locus (as determined by TAQMAN® quantitative reverse transcription-linked PCR (RT-qPCR) assay) containing the C9orf72 intron 1A sequence (Figure 22A; Assay F), the C9orf72 intron 1A sequence linked to exon 1A (Figure 22B; Assay E), the C9orf72 exon 1a-2 spliced ​​transcript (Figure 22C; Assay B), the C9orf72 exon 1b-2 spliced ​​transcript (Figure 22D; Assay C), or the C9orf72 exon 5-6 spliced ​​transcript (Figure 22E; Assay D) 24 hours after electroporation of siRNA targeting Gapdh (Gapdh siRNA #12). Gapdh siRNA and mock samples were used as negative controls. [Figure 22-2] Same as above. [Figure 22-3] Same as above. [Figure 23-1]Figures 23A-23E show the expression of mature C9orf72 mRNA (siRNA #1) or intron 1A upstream of the hexanucleotide repeats (siRNA #2) in mouse embryonic stem cells containing a modified C9orf72 locus containing 300 repeats of the hexanucleotide sequence set forth as SEQ ID NO: 1. Figure 23B is a bar graph showing the relative expression levels (y-axis) of transcripts from the C9orf72 locus (as determined by TAQMAN® quantitative reverse transcription-linked PCR (RT-qPCR) assay) containing the C9orf72 intron 1A sequence (Figure 23A; Assay F), the C9orf72 intron 1A sequence linked to exon 1A (Figure 23B; Assay E), the C9orf72 exon 1a-2 spliced ​​transcript (Figure 23C; Assay B), the C9orf72 exon 1b-2 spliced ​​transcript (Figure 23D; Assay C), or the C9orf72 exon 5-6 spliced ​​transcript (Figure 23E; Assay D) 48 hours after electroporation of siRNA targeting Gapdh siRNA and mock samples. Gapdh siRNA and mock samples were used as negative controls. [Figure 23-2] Same as above. [Figure 23-3] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0151] The present disclosure provides an RNAi composition that carries out RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of C9orf72 gene, for example, C9orf72 gene with expanded GGGGCC (G4C2) repeats.C9orf72 gene can be in a cell, for example, in a cell of a subject such as human.The use of these iRNAs allows the targeted degradation of the mRNA of corresponding gene (C9orf72 gene) in mammals.

[0152] The iRNAs of the present invention are designed to target C9orf72 target RNAs, such as C9orf72 target RNAs that have an expanded GGGGCC hexanucleotide repeat in an intron of a gene and have a combination of nucleotide modifications. The agents can target mature C9orf72 mRNA (mRNA from which the intron has been spliced ​​out) or C9orf7 pre-mRNA (mRNA containing an intron). The iRNAs of the invention can inhibit expression of the C9orf7 gene (e.g., mature mRNA) by about 50% or less, reduce the levels of sense- and antisense-containing aggregates and aberrant dipeptide repeat (DPR) proteins (poly(GA), poly(GR), poly(GP), poly(PA), and poly(PR)), and / or inhibit expression of the C9orf7 gene (e.g., precursor mRNA) by more than about 50%, reduce the levels of sense- and antisense-containing aggregates and aberrant dipeptide repeat (DPR) proteins (poly(GA), poly(GR), poly(GP), poly(PA), and poly(PR)). Without intending to be limited by theory, it is believed that the combination or subcombination of the foregoing properties with specific target sites or specific modifications of these iRNAs confers improved efficiency, stability, potency, durability, and safety to the iRNAs of the invention.

[0153] Thus, the present disclosure also provides methods of using the RNAi compositions of the present disclosure to inhibit expression of the C9orf72 gene or to treat a subject having a disorder that is expected to benefit from inhibiting or reducing expression of the C9orf72 gene, e.g., a C9orf72-associated disease, e.g., a disease associated with an expanded GGGGCC hexanucleotide repeat in an intron of the C9orf72 gene, e.g., C9orf72 amyotrophic lateral sclerosis / frontotemporal dementia, or Huntington's disease, e.g., a Huntington-like syndrome caused by a C9orf72 expansion.

[0154] RNAi agents of the disclosure can be about 30 nucleotides or less in length, 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 ... The RNA strand (antisense strand) comprises an RNA strand having a region of 3, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, which region is substantially complementary to at least a portion of a target RNA transcript of the C9orf72 gene, for example, a C9orf72 intron. In certain embodiments, an RNAi agent of the present disclosure comprises an RNA strand (antisense strand) having a region that is about 21-23 nucleotides in length, the region being substantially complementary to a target RNA transcript of a C9orf72 gene, e.g., at least a portion of a C9orf72 intron.

[0155] 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 at least a portion of an mRNA transcript of the C9orf72 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, or 27-53 nucleotides in length. These RNAi agents with longer antisense strand lengths preferably include 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.

[0156] The use of these RNAi agents allows the targeted degradation of the target RNA of C9orf72 gene in mammals.Therefore, the method and composition comprising these RNAi agents are useful for treating the subjects who are expected to benefit from knocking down target C9orf72 RNA, reducing normal C9orf72 protein, and / or reducing the pathogenic dipeptide repeat protein that results from pathogenic hexanucleotide repeat expansion, for example, the subjects who have C9orf72-related diseases, such as C9orf72 amyotrophic lateral sclerosis / frontotemporal dementia or Huntington's disease, for example, the Huntington-like syndrome caused by C9orf72 expansion.

[0157] The detailed description below discloses methods for making and using compositions containing RNAi agents to inhibit expression of the C9orf72 gene, as well as compositions or methods for treating subjects with diseases or disorders that would benefit from inhibiting or reducing expression of the gene.

[0158] 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.

[0159] 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.

[0160] 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.

[0161] 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.

[0162] 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.

[0163] 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.

[0164] As used herein, a method of detection can include determining that the amount of analyte present is below the detection level of the method.

[0165] 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.

[0166] If the chemical structure and chemical name do not agree, the chemical structure takes precedence.

[0167] A composition or method that "comprises" or "includes" one or more recited elements may include other elements not specifically recited. For example, a composition that "comprises" or "includes" a protein may contain the protein alone or in combination with other components. The transitional phrase "consisting essentially of" means that the claim should be interpreted to include the specified elements recited in the claim plus elements that do not significantly affect the novel characteristics underlying the claimed invention. Thus, it is not intended that the term "consisting essentially of" be interpreted as equivalent to "comprises" when used in the claims of this invention.

[0168] "Optionally" or "as needed" means that the subsequently described event or circumstance may or may not occur, and the description includes instances in which the event or circumstance occurs and instances in which the event or circumstance does not occur.

[0169] The term "C9orf72" gene, also known as "C9orf72-SMCR8 complex subunit," "guanine nucleotide exchange C9orf72," "chromosome 9 open reading frame 72," "protein C9orf72," "DENNL72," "FTDALS1," "ALSFTD," and "FTDALS," refers to C9orf72, a gene encoding a known protein involved in regulating endosomal trafficking. The C9orf72 protein has been shown to interact with Rab proteins, which are involved in autophagy and endocytic transport. Expansion of GGGGCC repeats from approximately 2 to approximately 22 copies between alternating 5' exons in the intronic sequence to approximately 700 to approximately 1600 copies is associated with C9orf72 amyotrophic lateral sclerosis / frontotemporal dementia and Huntington's disease, a Huntington-like syndrome caused by C9orf72 expansion. Alternative splicing results in multiple transcript variants encoding different isoforms.

[0170] Exemplary nucleotide and amino acid sequences for C9orf72 can be found, for example, in GenBank Accession No. NM_001256054.2 (human C9orf72, SEQ ID NO:121, reverse complement SEQ ID NO:125); GenBank Accession No.: XM_005581570.2 (cynomolgus monkey (Macaca fascicularis) C9orf72, SEQ ID NO:122, reverse complement SEQ ID NO:126); GenBank Accession No.: NM_001081343.2 (house mouse (Mus musculus) C9orf72, SEQ ID NO:123, reverse complement SEQ ID NO:127); and GenBank Accession No.: NM_001007702.1 (brown rat (Rattus norvegicus) C9orf72, SEQ ID NO:124, reverse complement SEQ ID NO:128).

[0171] Additional nucleotide and amino acid sequences for human C9orf72 can be found, for example, in GenBank Accession Nos. NM_145005.6, transcript variant 1 (SEQ ID NO: 129, reverse complement SEQ ID NO: 130); and NM_018325.5, transcript variant 2 (SEQ ID NO: 131, reverse complement SEQ ID NO: 132).

[0172] The nucleotide sequence of the genomic region of human chromosome 9 containing the C9orf72 gene can be found, for example, in Genome Reference Consortium Human Build 38 (also referred to as human genome build38 or GRCh38), available at GenBank. The nucleotide sequence of the genomic region of human chromosome 9 containing the C9orf72 gene can also be found, for example, in GenBank accession number NC_000009.12 (SEQ ID NO: 133, reverse complement SEQ ID NO: 134), which corresponds to nucleotides 27546545 to 27573866 of human chromosome 9. The nucleotide sequence of the human c9orf72 gene can be found, for example, in GenBank accession number NG_031977.1.

[0173] Further examples of C9orf72 sequences can be found in publicly available databases such as GenBank, OMIM, and UniProt.

[0174] Additional information regarding C9orf72 can be found, for example, at www.ncbi.nlm.nih.gov / gene / 203228. The term C9orf72 as used herein also refers to variations of the C9orf72 gene, such as those provided in the clinical variant database at www.ncbi.nlm.nih.gov / clinvar / ?term=NM_001256054.2.

[0175] 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.

[0176] As used herein, " target sequence " refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of the C9orf72 gene, such as the mRNA that is the product of RNA processing of the primary transcript.In one embodiment, the target portion of the sequence will be at least sufficiently long to serve as a substrate for RNAi-dependent cleavage at or near a portion of the nucleotide sequence of the mRNA molecule formed during the transcription of the C9orf72 gene.

[0177] C9orf72 mRNA (target C9orf72 RNA) is RNA transcribed from the C9orf72 gene. C9orf72 mRNA includes C9orf72 mature mRNA, C9orf72 precursor mRNA, or any part thereof (e.g., spliced-out intron region or alternatively spliced ​​RNA). C9orf72 mature mRNA is C9orf72 mRNA from which introns have been removed (spliced-out) and from which C9orf72 protein is translated. C9orf72 precursor mRNA is C9orf72 mRNA from which at least one intron, particularly the first intron (intron 1), has been removed.

[0178] C9orf72 proteins include any protein expressed from C9orf72 mRNA, including proteins expressed from C9orf72 mature mRNA, as well as dipeptide repeat proteins (e.g., poly(glycine-alanine), poly(glycine-proline), poly(glycine-arginine), poly(alanine-proline), and poly(proline-arginine)) that result from repeat-associated non-AUG (AUG) translation from C9orf72 RNA containing hexanucleotide repeats.

[0179] The C9orf72 target RNA can include a hexanucleotide repeat comprising multiple contiguous copies of SEQ ID NO: 1. The C9orf72 target RNA can be, for example, one that has a pathogenic hexanucleotide repeat expansion (e.g., one that has at least about 30, at least about 35, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 100, at least about 200, at least about 300, at least about 400, or at least about 500 copies of the hexanucleotide repeat).

[0180] The target sequence can be 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.

[0181] 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.

[0182] " 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 the modified nucleotide or substitute replacement part (see, for example, Table 11) as described in more detail below.Those skilled in the art are well aware that guanine, cytosine, adenine, thymidine and uracil can be replaced with other parts without substantially changing the base pairing properties of the oligonucleotide that contains the nucleotide with such replacement part.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.

[0183] The terms " iRNA ", " RNAi agent ", " iRNA agent ", " RNA interference agent ", used interchangeably herein, refer to an agent that contains RNA as defined herein and mediates 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 C9orf72, C9orf72-related transcription, or C9orf72-related peptide in cells, for example, in cells of a subject, for example, a mammalian subject.

[0184] 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 C9orf72 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 these dsRNAs into 19-23 base pair small interfering RNAs with characteristic two-base 3' overhangs [Bernstein, et al., (2001) Nature 409:363]. These siRNAs are then introduced into RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, allowing complementary antisense strands to induce target recognition [Nykanen, et al., (2001) Cell 107:309]. 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 RISC complex, and thereby silences the target gene, i.e., C9orf72 gene. Therefore, the term "siRNA" is used herein to also mean the RNAi described above.

[0185] 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.

[0186] 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 C9orf72 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.

[0187] The dsRNA agents described herein can be distinct from (ie, do not include) antisense oligonucleotides (ASOs) or gapmer antisense oligonucleotides (ASOs).

[0188] Generally, dsRNA molecules can comprise ribonucleotides, but as described in detail herein, each 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 has independently modified sugar moiety, modified internucleotide linkage, or modified nucleobase.Therefore, the term modified nucleotide encompasses the substitution, addition, or removal of, for example, functional group or atom, etc., 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.

[0189] In certain embodiments of the present disclosure, the inclusion of deoxy-nucleotides can be considered to constitute modified nucleotides when present within an RNAi agent.

[0190] 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.

[0191] 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.

[0192] 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 number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus all overhangs present in the duplex. In addition to the duplex structure, the RNAi can 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 still other embodiments, both the 3' and 5' ends of one strand of the RNAi agent comprise an overhang of at least one nucleotide.

[0193] 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 C9orf72 target mRNA sequence, to induce cleavage of the target RNA.

[0194] In some embodiments, the iRNA of the invention is a 24-30 nucleotide dsRNA that interacts with a target RNA sequence, eg, a C9orf72-targeted mRNA sequence, to direct cleavage of the target RNA.

[0195] 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.

[0196] 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 thiophosphate.

[0197] In certain embodiments, the antisense strand of dsRNA has 1-10 nucleotide overhangs at the 3'-end or 5'-end, for example, 0-3, 1-3, 2-4, 2-5, 4-10, or 5-10 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 1-10 nucleotide overhangs at the 3'-end 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.

[0198] 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 such that the overhang can form a stable hairpin structure under physiological conditions.

[0199] In certain embodiments, at least one end of at least one chain is extended beyond double-stranded targeting region, such as the structure where one of the chains comprises thermodynamically stable tetraloop structure (see, for example, U.S. Patent Nos. 8,513,207 and 8,927,705, and WO2010033225, each of which is incorporated herein by reference in its entirety).Such structure can comprise single-stranded extension (at one or both ends of molecule) and double-stranded extension.

[0200] In certain embodiments, the 3' end of the sense strand and the 5' end of the antisense strand are joined by a polynucleotide sequence comprising ribonucleotides, deoxyribonucleotides, or both, and optionally, the polynucleotide sequence may comprise a tetraloop sequence. In certain embodiments, the sense strand is 25-35 nucleotides in length.

[0201] The tetraloop can contain ribonucleotides, deoxyribonucleotides, modified nucleotides, and combinations thereof. Typically, a tetraloop has 4 to 5 nucleotides. In some embodiments, the loop comprises a sequence described as GAAA. In some embodiments, at least one of the nucleotides (GAAA) of the loop comprises a nucleotide modification. In some embodiments, the modified nucleotide comprises a 2' modification. In some embodiments, the 2' modification is a modification selected from the group consisting of 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl, 2'-aminodiethoxymethanol, 2'-adem, and 2'-deoxy-2'-phiolo-d-arabinonucleic acid. In some embodiments, all nucleotides of the loop are modified. In some embodiments, the G in the GAAA sequence comprises a 2'-OH. In some embodiments, each nucleotide in the GAAA sequence comprises a 2'-O-methyl modification. In some embodiments, each A in the GAAA sequence comprises a 2'-OH and the G in the GAAA sequence comprises a 2'-O-methyl modification. In a preferred embodiment, in some embodiments, each A in the GAAA sequence comprises a 2'-O-methoxyethyl (MOE) modification, and the G in the GAAA sequence comprises a 2'-O-methyl modification; or each A in the GAAA sequence comprises a 2'-adem modification, and the G in the GAAA sequence comprises a 2'-O-methyl modification. For example, see PCT Publication No. WO 2020 / 206350, the entire contents of which are incorporated herein by reference.

[0202] Exemplary 2'adem modified nucleotides are shown below.

[0203] [ka]

[0204] The term "blunt" or "blunt-ended" as used herein in reference 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 the 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.

[0205] The term "antisense strand" or "guide strand" refers to the strand of an RNAi agent, e.g., a dsRNA, that includes a region that is substantially complementary to a target sequence, e.g., C9orf72 mRNA.

[0206] 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 C9orf72 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' or 3' end of the RNAi agent.In some embodiments, the double-stranded RNA agent of the present invention comprises nucleotide mismatches in the antisense strand.In some embodiments, the antisense strand of the double-stranded RNA agent of the present invention 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 invention contains four or fewer mismatches with the sense strand, e.g., the antisense strand contains four, three, two, one, or zero mismatches with the sense strand. In some embodiments, a double-stranded RNA agent of the invention contains a nucleotide mismatch in the sense strand. In some embodiments, the sense strand of a double-stranded RNA agent of the invention 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.

[0207] 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, for a 23-nucleotide RNAi agent, the strand complementary to a region of the C9orf72 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 the C9orf72 gene. It is important to consider the efficacy of mismatched RNAi agents in inhibiting C9orf72 expression, particularly when specific regions of complementarity in the C9orf72 gene are known to have polymorphic sequence variation within the population.

[0208] "Substantially all of the nucleotides are modified," as used herein, means that most, but not all, are modified, including no more than 5, 4, 3, 2, or 1 unmodified nucleotides.

[0209] 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.

[0210] 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.

[0211] As used herein, unless otherwise specified, the term "complementary," when used to describe a first nucleotide sequence in the context of a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising a first nucleotide sequence to hybridize to form a duplex with an oligonucleotide or polynucleotide comprising the second nucleotide sequence under certain conditions, as understood by those of skill in the art. Such conditions may be, for example, stringent conditions, such as 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, at 50°C or 70°C for 12-16 hours, followed by washing (see, e.g., "Molecular Cloning: A Laboratory Manual," Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). Other conditions, such as physiologically relevant conditions that may be encountered inside an organism, may also be applied. Those skilled in the art will be able to determine the most appropriate set of conditions for testing the complementarity of two sequences according to the ultimate application of the hybridized nucleotides.

[0212] The complementary sequence in an RNAi agent, for example, in the dsRNA described herein, comprises the base pairing of the oligonucleotide or polynucleotide comprising the first nucleotide sequence with the oligonucleotide or polynucleotide comprising the second nucleotide sequence throughout the entire length of one or both nucleotide sequences.Such sequences can be referred to herein as " completely complementary " with respect to each other.However, in the present specification, when a first sequence is considered to be " substantially complementary " with a 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, while maintaining the ability to hybridize under the conditions most suitable for their final use, for example, the inhibition of gene expression by the RISC pathway, in the case of a duplex of up to 30 base pairs.However, when two oligonucleotides are designed to form one or more single-stranded overhangs during hybridization, these overhangs are not considered as mismatches when determining complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, where the longer oligonucleotide comprises a 21-nucleotide sequence that is perfectly complementary to the shorter oligonucleotide, can still be considered "perfectly complementary" for the purposes described herein.

[0213] "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.

[0214] 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.

[0215] 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 C9orf72). For example, a polynucleotide is complementary to at least a portion of a C9orf72 mRNA if the sequence is substantially complementary to an uninterrupted portion of an mRNA encoding C9orf72.

[0216] Thus, in some embodiments, the antisense polynucleotides disclosed herein are fully complementary to the target C9orf72 sequence. In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to the target C9orf72 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 any of the nucleotide sequences of SEQ ID NOs: 121-124 and 133 or the equivalent regions of SEQ ID NOs: 121-124 and 133 throughout its entire length.

[0217] In some embodiments, the antisense polynucleotides disclosed herein are substantially complementary to a fragment of the target C9orf72 sequence, spanning its entire length, including nucleotides 1594-1616, 802-824, 239-261, 1308-1330, 233-255, 1595-1617, 240-262, 1532-1554, 237-259, 3268-3290, 806-828, 162-1632, 162-1634, 162-1636, 162-1638, 162-1639 ... The fragment of SEQ ID NO: 121 selected from the group consisting of 0-1642, 526-548, 1169-1191, 1266-1288, 1247-1269, 586-608, 1257-1279, and 400-422 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: 121 selected from the group consisting of 0-1642, 526-548, 1169-1191, 1266-1288, 1247-1269, 586-608, 1257-1279, and 400-422.

[0218] In some embodiments, the antisense polynucleotides disclosed herein are substantially complementary to a fragment of a target C9orf72 sequence, e.g., a fragment of SEQ ID NO: 133, over its entire length, e.g., nucleotides 200-290, 200-280, 200-270, 200-260, 200-250, 200-240, 200-230, 200-225, 210-290, 210-280, 210-270, 210-260, 210-250, 210-240, 210-235, 220-290, 220-280, 220-270, , 220-260, 220-250, 220-245, 225-250, 225-245, 230-290, 230-280, 230-270, 230-260, 230-255, 235-260, 240-265, 240-290, 240-280, 240-270, 240-265, 250-290, 250-280, 250-275, 260-290, 260-285, or a contiguous nucleotide sequence that is at least 80% complementary, 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 sequences of ... Ranges between the above recited ranges are also contemplated as being part of the disclosure.

[0219] In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to a target C9orf72 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 over its entire length to any one of the sense strand nucleotide sequences in any one of any one of Tables 10A, 10B, 12-15, 19, or 20, or a fragment of any one of the sense strand nucleotide sequences in any one of Tables 10A, 10B, 12-15, 19, or 20.

[0220] In one embodiment, an RNAi agent of the present disclosure comprises a sense strand that is substantially complementary to an antisense polynucleotide that is identical to a target C9orf72 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 the equivalent region of SEQ ID NOs: 125-128 and 134 throughout its entire length.

[0221] In some embodiments, an iRNA of the invention comprises a sense strand that is substantially complementary to an antisense polynucleotide that is complementary to a target C9orf72 sequence, wherein the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least 80%, e.g., about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100%, complementary over its entire length to any one of the antisense strand nucleotide sequences in any one of Tables 10A, 10B, 12-15, 19, or 20, or a fragment of any one of the antisense strand nucleotide sequences in any one of Tables 10A, 10B, 12-25, 19, or 20.

[0222] In certain embodiments, the sense and antisense strands are selected from any one of the duplexes AD-348904.1, AD-348136.1, AD-347612.1, AD-348639.1, AD-347606.1, AD-348905.1, AD-347613.1, AD-348842.1, AD-347610.1, AD-350329.1, AD-348140.1, AD-348930.1, AD-347863.1, AD-348500.1, AD-348597.1, AD-348578.1, AD-347923.1, AD-348588.1, AD-347773.1, and AD-348136.1.

[0223] In certain embodiments, the sense and antisense strands are selected from any one of the duplexes AD-463863.1, AD-463862.1, AD-463869.1, AD-463873.1, AD-463872.1, and AD-463860.1.

[0224] In one embodiment, at least partial suppression of expression of the C9orf72 gene is assessed by a reduction in the amount of C9orf72 mRNA, e.g., sense mRNA, antisense mRNA, total C9orf72 mRNA, mRNA containing a sense C9orf72 repeat, and / or mRNA containing an antisense C9orf72 repeat, which mRNA can be isolated from or detected in a first cell or group of cells in which the C9orf72 gene is transcribed and which has been treated or has been treated to inhibit expression of the C9orf72 gene, and compared to a second cell or group of cells (control cells) that is substantially identical to the first cell or group of cells but has been or has not been so treated. The degree of inhibition can be expressed in the following units:

[0225]

number

[0226] 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 cell afterwards.

[0227] 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, or other injection, as appropriate, or by injecting the RNAi agent into the bloodstream or subcutaneous space so that the agent subsequently 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 desired 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 can be contacted with an RNAi agent in vitro and then transferred to a subject.

[0228] 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 the 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.

[0229] The term "lipophilic" or "lipophilic moiety" broadly refers 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 Kow, where Kow is the ratio of the concentration of a chemical in the octanol phase to the concentration of the chemical in the aqueous phase in an equilibrium two-phase system. The octanol-water partition coefficient is a laboratory-measured property of a substance. However, it can also be predicted by using coefficients attributed to the structural components of a chemical calculated using first principles or empirical methods [see, for example, 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 is lipophilic in nature if its log Kow is greater than 0. Typically, lipophilic moieties have a log Kow greater than 1, greater than 1.5, greater than 2, greater than 3, greater than 4, greater than 5, or greater than 10. For example, the log Kow of 6-aminohexanol is predicted to be approximately 0.7. Using the same method, the log Kow of cholesteryl N-(hexan-6-ol) carbamate is predicted to be 10.7.

[0230] The lipophilicity of a molecule can be altered with respect to the functional groups it carries, for example, adding hydroxyl or amine groups to the terminus of the lipophilic moiety can increase or decrease the partition coefficient (e.g., log Kow) value of the lipophilic moiety.

[0231] 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.

[0232] In one embodiment, the plasma protein binding assay that is determined is electrophoretic mobility shift assay (EMSA) using human serum albumin protein.The exemplary protocol of this binding assay is described in detail in, for example, PCT / US2019 / 031170.The hydrophobicity of double-stranded RNAi agent measured by the fraction of unbound siRNA in binding assay is greater than 0.15, greater than 0.2, greater than 0.25, greater than 0.3, greater than 0.35, greater than 0.4, greater than 0.45 or greater than 0.5 for enhanced in vivo delivery of siRNA.

[0233] 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.

[0234] 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.

[0235] As used herein, a "subject" refers to an animal, e.g., a mammal, e.g., a primate (e.g., a human, a non-human primate, e.g., a monkey or chimpanzee), or a non-primate (e.g., a rat or a mouse). In a preferred embodiment, the subject is a human, e.g., a human being treated or evaluated for a disease, disorder, or condition that is expected to benefit from reduced C9orf72 expression; a human being at risk for a disease, disorder, or condition that is expected to benefit from reduced levels of target C9orf72 RNA; a human being with a disease, disorder, or condition that is expected to benefit from reduced C9orf72 expression; or a human being treated for a disease, disorder, or condition that is expected to benefit from reduced C9orf72 expression as described herein. 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.

[0236] As used herein, the terms "treating" or "treatment" refer to a beneficial or desired result, for example, but not limited to, alleviating or ameliorating one or more signs or symptoms associated with C9orf72 gene expression or C9orf72 protein production, such as a C9orf72-associated disease. "Treatment" can also mean prolonging survival as compared to expected survival if no treatment is administered.

[0237] The term "lower" in relation to the level of C9orf72 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 a 50% or less reduction in disease marker, e.g., C9orf72 protein and / or gene expression levels, for example, a 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or less reduction. When referring to the level of C9orf72 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 accept as being within the normal range, for example, a reduction in the level of weight between an obese individual and an individual with a weight that is accepted as being within the normal range.

[0238] As used herein, "prevention" or "preventing," when used in reference to a disease, disorder, or condition that would benefit from reduced expression of the C9orf72 gene or C9orf72 protein production, refers to a reduction in the likelihood that a subject will develop symptoms associated with such disease, disorder, or condition, such as symptoms of a C9orf72-associated disease. Not developing a disease, disorder, or condition, or a reduction in the onset of symptoms associated with such a 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 several days, weeks, months, or years) is considered effective prevention.

[0239] The term "C9orf72-associated disease" or "C9orf72-associated disorder," as used herein, includes any disease or disorder that would benefit from reduced expression and / or activity of C9orf72. Exemplary C9orf72-associated diseases include diseases in which a subject has a hexanucleotide repeat (GGGCC) expansion in the intron between exon 1a and exon 1b in the C9orf72 gene, such as amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD), and Huntington's-like syndrome caused by a C9orf72 expansion.

[0240] Normal G4C2 repeats are approximately 25 units or less, while high-penetrance disease alleles typically range from approximately 60 repeat units to over 4,000 units; rarely, familial cases of the disease segregate between 47 and 60 repeats. To detect smaller expansions (<80), repeat-primed PCR assays are typically used, but larger repeats that are precisely sized require other techniques (e.g., Southern blot hybridization) that provide length estimates.

[0241] Subjects with a GGGGCC (or G4C2) hexanucleotide expansion in an intron of the C9orf72 gene may present as amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD), even within the same family; therefore, the neurodegeneration associated with this expansion is referred to herein as "C9orf72 amyotrophic lateral sclerosis / frontotemporal dementia" or "C9orf72ALS / FTD." This is an autosomal dominant disease and is the most common form of familial ALS, accounting for one-third of ALS families and 5-10% of sporadic cases in ALS clinics. It is also a common cause of FTD, accounting for approximately one-quarter of familial FTD cases. The age of onset of symptoms ranges from 30 to 70 years, with the average age of onset being in the late 50s. C9orf72-mediated ALS most often resembles typical ALS, can have bulbar or limb onset, can (but does not always) progress rapidly, and may be accompanied by subsequent cognitive symptoms. Therefore, C9orf72-mediated ALS is evaluated and treated exactly like any other ALS patient. The most common pattern of C9orf72-mediated FTD is behavioral variant FTD, with a full range of behavioral and cognitive symptoms, including disinhibition, apathy, and higher brain dysfunction. Less commonly, C9orf72-mediated FTD presents with semantic variant primary progressive aphasia (PPA) or nonfluent variant PPA. Very rarely, it may resemble a corticobasal syndrome, progressive supranuclear palsy, or HD-like syndrome. In some cases, Parkinson's disease features are seen in C9orf72-mediated ALS or FTD.

[0242] Subjects may present with frontotemporal lobar degeneration (FTLD), characterized by progressive behavioral changes, higher brain dysfunction, and / or language impairment. Among the three FTLD clinical syndromes, behavioral-type FTD (bvFTD) is most often, but not always, present. This is characterized by progressive behavioral disturbances and decline in executive function, accompanied by predominant frontal lobe atrophy on brain MRI. Motor neuron disease, such as upper or lower motor neuron dysfunction (or both), may also be present, which may or may not meet the criteria for a full ALS phenotype. Many individuals with C9orf72-associated bvFTD have some degree of parkinsonism, typically characterized by a tremor-free, akinetic-rigid type that is unresponsive to levodopa.

[0243] Huntington's disease-like syndromes (HD-like syndromes, or HDL syndromes) are a group of inherited neurodegenerative disorders that closely resemble Huntington's disease (HD) in that they typically result in a combination of chorea, cognitive decline or dementia, and behavioral or psychiatric problems.

[0244] Subjects with Huntington's disease-like syndrome caused by C9orf72 expansion are characterized by movement disorders such as dystonia, chorea, myoclonus, tremor, and rigidity. Related features include cognitive and memory impairment, early psychiatric disorders, and behavioral problems. The average age at onset is approximately 43 years (range 8-60 years). Early psychiatric and behavioral problems (e.g., depression, apathy, obsessive-compulsive behavior, and psychosis) are common. Cognitive symptoms manifest as higher brain dysfunction. Motor disturbances are prominent, and Parkinson's disease features and pyramidal tract features may also be present. As used herein, "therapeutically effective amount" is intended to include an amount of an RNAi agent sufficient, when administered to a subject with a C9orf72-associated disease, to treat the disease (e.g., by reducing, ameliorating, or maintaining an existing disease or one or more symptoms of the disease). A "therapeutically effective amount" may vary depending on the RNAi agent, how the agent is administered, the disease and its severity, and other individual characteristics of the subject being treated, such as medical history, age, weight, family history, genetic makeup, type of prior or concurrent treatment, if any.

[0245] 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 C9orf72-associated disease. Amelioration of the disease includes slowing the course 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.

[0246] 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.

[0247] 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.

[0248] 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.

[0249] 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 in the brain, 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) obtained from a subject. ) or retinal tissue (or a subcomponent thereof).

[0250] II. RNAi Agents of the Disclosure As described elsewhere herein, mutations in C9orf72 have been shown to be associated with familial frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). The mutation is the result of a G4C2 (SEQ ID NO: 1) hexanucleotide repeat expansion located within an intron between exon 1A and exon 1B of the C9orf72 gene. The hexanucleotide repeat may be translated via a non-AUG initiation mechanism. The accumulation of RNA (targeting RNA) containing the repeat expansion or the translation of the repeat sequence may cause or contribute to FTD and / or ALS or disease symptoms associated with FTD and / or ALS.

[0251] Therefore, the present invention provides a dsRNA RNAi agent that selectively and efficiently reduces the expression of C9orf72-related expression products, RNAs associated with hexanucleotide repeat expansions, and / or translated polypeptides.In some embodiments, the dsRNA agent targets (e.g., selectively targets) RNAs containing hexanucleotide repeats (target RNAs) and knocks down the polypeptides expressed from the target RNAs and RNAs containing hexanucleotide repeats.The dsRNA agent can be used in methods for therapeutic treatment and / or prevention of signs or symptoms associated with FTD and / or ALS, including but not limited to, the formation of repeat-length-dependent RNA aggregates, the capture of specific RNA-binding proteins, and the accumulation and aggregation of dipeptide repeat proteins (e.g., poly(glycine-alanine), poly(glycine-proline), poly(glycine-arginine), poly(alanine-proline), and poly(proline-arginine)) resulting from repeat-associated non-AUG (AUG) translation in neurons. dsRNA agents can be used in methods for the therapeutic treatment and / or prevention of signs or symptoms related to FTD and / or ALS, including but not limited to the signs and symptoms of motor neuron disease and the signs and symptoms of dementia.Signs and symptoms of motor neuron disease can include, for example, tripping, dropping things, abnormal fatigue in arms and / or legs, slurred speech, muscle spasms and twitching, inability to control laughing or crying, and difficulty breathing.Signs and symptoms of dementia can include, for example, behavioral changes, personality changes, speech and language problems, and movement-related problems.Such methods include administering one or more dsRNA agents described herein to subjects (for example, human or animal subjects).

[0252] The dsRNA agents described herein can stop or reduce the accumulation of repeat-containing C9orf72 RNA (e.g., assayed as RNA aggregates), thereby preventing RNA translation from synthesizing dipeptide repeat proteins.

[0253] In some embodiments, the dsRNA agent of the present invention targets mature C9orf72 mRNA (i.e., mRNA from which the intron has been spliced ​​out). In other embodiments, the dsRNA agent of the present invention targets C9orf72 RNA that contains an intron, such as intron 1A (i.e., mRNA from which the intron has not been spliced ​​out, an RNA region spliced ​​out of a pre-mRNA, or an alternatively spliced ​​RNA).

[0254] In some embodiments, the dsRNA agent of the present invention targets the C9orf72 transcript that starts at non-coding exon 1A upstream of repeat.C9orf72 repeat expansion is a dominant mutation that essentially always exists as heterozygous mutation.Therefore, in some embodiments, the dsRNA agent described herein targets the transcript that starts at exon 1A, and mainly affects only pathogenic alleles; normal alleles almost exclusively start transcription at exon 1B downstream of repeat.Therefore, normal (non-pathogenic) transcripts are avoided, and therefore the dsRNA agent does not eliminate all C9orf72 protein production. In one embodiment, the RNAi agent comprises a double-stranded ribonucleic acid (dsRNA) molecule for inhibiting expression of C9orf72 in a cell, e.g., a cell in a subject, e.g., a mammal, e.g., a human with a C9orf72-associated disorder, such as a C9orf72-associated disorder. The dsRNA comprises an antisense strand having a region of complementarity that is complementary to at least a portion of an mRNA formed upon expression of the C9orf72 gene. The region of complementarity is about 15-30 nucleotides in length or less. Upon contact with a cell expressing the C9orf72 gene, the RNAi agent inhibits expression of the C9orf72 gene (e.g., a human gene, a primate gene, a non-primate gene) by at least 50%, as assayed, e.g., by PCR or branched DNA (bDNA)-based methods, or by protein-based methods, e.g., immunofluorescence analysis using Western blotting or flow cytometry techniques. In a preferred embodiment, the level of knockdown is assayed in human neuroblastoma BE(2)C cells using the dual luciferase assay method provided in Example 1 below.

[0255] In some embodiments, the dsRNA agent described herein is designed to stop or reduce the accumulation of repeat-containing C9orf72 RNA (e.g., assayed as RNA aggregates), thereby preventing the synthesis of dipeptide repeat proteins by RNA translation.To achieve this, in certain embodiments, the dsRNA agent targets the C9orf72 mRNA protein-coding sequence (see, for example, Tables 12 and 13).In other embodiments, the dsRNA agent targets the C9orf72 transcript that starts in the non-coding exon 1A upstream of the repeat.Transcripts that start downstream of the repeat are not expected to contain pathogenic RNA.C9orf72 repeat expansion is a dominant mutation that essentially always exists as a heterozygous mutation. Thus, when the dsRNA agents described herein target transcripts that initiate in exon 1A, they therefore primarily affect only the pathogenic allele; normal alleles almost exclusively initiate transcription in exon 1B downstream of the repeat, thus sparing normal (non-pathogenic) transcripts.

[0256] dsRNA comprises two RNA strands, which are complementary and hybridize to form a duplex structure under the conditions that dsRNA is used.One strand (antisense strand) of dsRNA comprises a complementary region that is substantially complementary, and generally completely complementary to target sequence.Target sequence can be obtained from the sequence of mRNA formed during the expression of C9orf72 gene.The other strand (sense strand) comprises a region that is complementary to antisense strand, so that when the two strands are combined under suitable conditions, they hybridize to form a duplex structure.As described elsewhere herein and known in the art, the complementary sequence of dsRNA can also be contained as a self-complementary region of a single nucleic acid molecule, so as to be opposite on separate oligonucleotides.

[0257] 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 the present disclosure.

[0258] 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.

[0259] 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.

[0260] 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 most often 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 long enough to be a substrate for RNAi-dependent cleavage (i.e., cleavage by the RISC pathway).

[0261] 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 with 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, dsRNA is not naturally occurring miRNA.In another embodiment, the RNAi agent useful for targeting C9orf72 expression is not generated in target cells by cleavage of larger dsRNA.

[0262] 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.

[0263] dsRNA can be synthesized by standard methods known in the art.Double-stranded RNAi compounds of the present invention can be prepared using a two-step method.First, each strand of double-stranded RNA molecules is prepared separately.Then, the strands of the components are annealed.The individual strands of siRNA compounds can be prepared using solution phase or solid phase organic synthesis or both.Organic synthesis has the advantage that it can easily prepare oligonucleotide strands that contain unnatural nucleotides or modified nucleotides.Similarly, single-stranded oligonucleotides of the present invention can be prepared using solution phase or solid phase organic synthesis or both.

[0264] In certain embodiments, a dsRNA agent of the invention targets a C9orf72 target RNA that includes a hexanucleotide repeat that includes multiple contiguous copies of SEQ ID NO:1, e.g., a C9orf72 target RNA that has a pathogenic hexanucleotide repeat expansion (e.g., having at least about 30, at least about 35, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 100, at least about 200, at least about 300, at least about 400, or at least about 500 copies of the hexanucleotide repeat).

[0265] The dsRNA agents described herein can target any region of C9orf72 RNA (C9orf72 target RNA) containing hexanucleotide repeats. In certain embodiments, the dsRNA agents described herein can target the nucleotide sequence between the start of exon 1A and the start of exon 2 of C9orf72 RNA (i.e., including exon 1A but not including exon 2) (i.e., corresponding to the region from the start of exon 1A to the start of exon 2 in the corresponding C9orf72 gene). In certain embodiments, the dsRNA agents described herein can target the nucleotide sequence between the start of exon 1A and the end of exon 1B of C9orf72 RNA (i.e., including exon 1A and exon 1B). In certain embodiments, the dsRNA agents described herein can target the nucleotide sequence between the start of exon 1A and the start of exon 1B of C9orf72 RNA (i.e., including exon 1A but not including exon 1B). In certain embodiments, the dsRNA agent described herein can target the nucleotide sequence between the start of exon 1A of C9orf72 RNA and the end of the hexanucleotide repeat sequence (i.e., including exon 1A and the hexanucleotide repeat sequence). In certain embodiments, the dsRNA agent described herein can target the nucleotide sequence between the start of exon 1A of C9orf72 RNA and the start of the hexanucleotide repeat sequence (i.e., including exon 1A but not including the hexanucleotide repeat sequence). In certain embodiments, the dsRNA agent described herein can target the nucleotide sequence between the start of exon 1A of C9orf72 RNA and the end of exon 1A.

[0266] In certain embodiments, a dsRNA agent described herein can target a nucleotide sequence between the end of exon 1A and the start of exon 2 of a C9orf72 RNA (i.e., excluding exon 1A and excluding exon 2). In certain embodiments, a dsRNA agent described herein can target a nucleotide sequence between the end of exon 1A and the end of exon 1B of a C9orf72 RNA (i.e., including exon 1A but including exon 1B). In certain embodiments, a dsRNA agent described herein can target a nucleotide sequence between the end of exon 1A and the start of exon 1B of a C9orf72 RNA (i.e., excluding exon 1A and including exon 1B). In certain embodiments, a dsRNA agent described herein can target a nucleotide sequence between the end of exon 1A and the end of the hexanucleotide repeat sequence of a C9orf72 RNA (i.e., excluding exon 1A but including the hexanucleotide repeat sequence). In certain embodiments, a dsRNA agent described herein can target the nucleotide sequence between the end of exon 1A of C9orf72 RNA and the start of the hexanucleotide repeat sequence (i.e., not including exon 1A and not including the hexanucleotide repeat sequence).

[0267] In certain embodiments, a dsRNA agent described herein can target a nucleotide sequence between the start of the hexanucleotide repeat sequence and the start of exon 2 of a C9orf72 RNA (i.e., including the hexanucleotide repeat sequence but not including exon 2). In certain embodiments, a dsRNA agent described herein can target a nucleotide sequence between the start of the hexanucleotide repeat sequence and the end of exon 1B of a C9orf72 RNA (i.e., including the hexanucleotide repeat sequence and exon 1B). In certain embodiments, a dsRNA agent described herein can target a nucleotide sequence between the start of the hexanucleotide repeat sequence and the start of exon 1B of a C9orf72 RNA (i.e., including the hexanucleotide repeat sequence but not including exon 1B). In certain embodiments, a dsRNA agent described herein can target a nucleotide sequence between the start of the hexanucleotide repeat sequence and the end of the hexanucleotide repeat sequence of a C9orf72 RNA.

[0268] In certain embodiments, a dsRNA agent described herein can target a nucleotide sequence between the end of the hexanucleotide repeat sequence and the start of exon 2 of a C9orf72 RNA (i.e., not including the hexanucleotide repeat sequence and not including exon 2). In certain embodiments, a dsRNA agent described herein can target a nucleotide sequence between the end of the hexanucleotide repeat sequence and the end of exon 1B of a C9orf72 RNA (i.e., not including the hexanucleotide repeat sequence but including exon 1B). In certain embodiments, a dsRNA agent described herein can target a nucleotide sequence between the end of the hexanucleotide repeat sequence and the start of exon 1B of a C9orf72 RNA (i.e., not including the hexanucleotide repeat sequence and not including exon 1B).

[0269] In certain embodiments, a dsRNA agent described herein can target a nucleotide sequence between the beginning of the end of exon 1B and the beginning of exon 2 of C9orf72 RNA (i.e., including exon 1B, but not including exon 2).

[0270] In certain embodiments, a dsRNA agent described herein can target a nucleotide sequence between the start of the end of exon 1B and the end of exon 1B of a C9orf72 RNA.

[0271] In certain embodiments, a dsRNA agent described herein can target a nucleotide sequence between the end of exon 1B and the start of exon 2 of C9orf72 RNA (i.e., excluding exon 1B and excluding exon 2).

[0272] Some dsRNA agents target the intron region of C9orf72 RNA. For example, the dsRNA agent described herein can target the intron between exon 1A and exon 1B of the C9orf72 gene (i.e., the intron between the end of exon 1A and the beginning of exon 1B of the C9orf72 target RNA). The region targeted by the dsRNA agent can be upstream (5') or downstream (3') of the hexanucleotide repeat in the intron sequence between exon 1A and the hexanucleotide repeat sequence of the C9orf72 target RNA. In a specific example, the dsRNA agent targets the region upstream (5') of the hexanucleotide repeat in the C9orf72 target RNA. For example, the dsRNA agent can target the intron sequence between the end of exon 1A and the beginning of the hexanucleotide repeat sequence.

[0273] In one embodiment, a dsRNA agent targets a region in a C9orf72 target RNA corresponding to a region of the human C9orf72 gene between human genome assembly GRCh38 / hg38 coordinates chr9:27,567,165-27,573,866 (corresponding to the region from the start of exon 1A to the start of exon 2 (i.e., the end of the intron before exon 2); an exemplary RNA sequence having three GGGGCC repeats transcribed from this region is set forth in SEQ ID NO:111, and the reverse complement is set forth in SEQ ID NO:112). In another specific example, a dsRNA agent targets a region in a C9orf72 target RNA corresponding to a region of the human C9orf72 gene between human genome assembly GRCh38 / hg38 coordinates chr9:27,573,494-27,573,708 (corresponding to the region of intron sequence between exon 1A and exon 1B (i.e., between the end of exon 1A and the start of exon 1B); an exemplary RNA sequence having three GGGGCC repeats transcribed from this region is set forth in SEQ ID NO:113, and the reverse complement is set forth in SEQ ID NO:114). In another specific example, a dsRNA agent targets a region in a C9orf72 target RNA corresponding to a region of the human C9orf72 gene between human genome assembly GRCh38 / hg38 coordinates chr9:27,573,547-27,573,708 (corresponding to the region of intron sequence between exon 1A and the start of the hexanucleotide repeat sequence (i.e., between the end of exon 1A and the start of the hexanucleotide repeat sequence); an exemplary RNA sequence transcribed from this region is set forth in SEQ ID NO:115, and the reverse complement is set forth in SEQ ID NO:116). In another specific example, a dsRNA agent targets a region in a C9orf72 target RNA corresponding to a region of the human C9orf72 gene between human genome assembly GRCh38 / hg38 coordinates chr9:27,573,605-27,573,640 (corresponding to a fragment from the region of intron sequence between exon 1A and the start of the hexanucleotide repeat sequence; an exemplary RNA sequence transcribed from this region is set forth in SEQ ID NO:117, and the reverse complement is set forth in SEQ ID NO:118).

[0274] In certain embodiments, a dsRNA agent of the invention targets nucleotides 200-290 of SEQ ID NO: 133, which is the target of the dsRNA agent.

[0275] In certain embodiments, the dsRNA agent targets nucleotides from any one of the nucleotide sequences of nucleotides 230-270, 233-262, 800-840, 800-830, 802-828, 1240-1290, 1240-1280, 1247-1288, 1590-1645, 1590-1620, and 1594-1642 of SEQ ID NO:121.

[0276] In certain embodiments, the dsRNA agent targets a nucleotide from any one of the nucleotide sequences 1594-1616, 802-824, 239-261, 1308-1330, 233-255, 1595-1617, 240-262, 1532-1554, 237-259, 3268-3290, 806-828, 1620-1642, 526-548, 1169-1191, 1266-1288, 1247-1269, 586-608, 1257-1279, and 400-422 of SEQ ID NO:121.

[0277] In one embodiment, the dsRNA of the present disclosure comprises at least two nucleotide sequences, i.e., sense strand and antisense strand.The sense strand sequence for C9orf72 can be selected from the group of sequences provided in any one of Tables 10A, 10B, 12-15, 19 or 20, and the corresponding nucleotides of the sense strand and the antisense strand can be selected from the group of sequences provided in any one of Tables 10A, 10B, 12-15, 19 or 20.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 mRNA generated during the expression of C9orf72 gene. Thus, in this embodiment, the dsRNA will comprise two oligonucleotides, one of which is described as the sense strand (passenger strand) in any one of Tables 10A, 10B, 12-15, 19, or 20, and the second oligonucleotide is described as the corresponding antisense strand (guide strand) to the sense strand in any one of Tables 10A, 10B, 12-15, 19, or 20.

[0278] 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.

[0279] Although the sequences in Tables 10A, 10B, 12-15, 19, or 20 are described as modified or conjugated sequences, it is understood that the RNA of the RNAi agent of the present disclosure, e.g., the dsRNA of the present disclosure, can include any one of the sequences described in any one of Tables 10A, 10B, 12-15, 19, or 20, unmodified, unconjugated, or modified or conjugated differently than described. For example, the sense strands of the agents of the present invention shown in Tables 13 and 15 are conjugated to GalNAc ligands, but these agents may also be conjugated to moieties that direct delivery to the CNS, e.g., C16 ligands, as described herein. Lipophilic ligands can be included at any of the positions provided herein.

[0280] 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. Thus, dsRNAs having a sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides derived from one of the sequences provided herein that differ in their ability to inhibit expression of the C9orf72 gene with no more than 10, 15, 20, 25, or 30% inhibition compared to dsRNAs containing the full-length sequence, e.g., using in vitro assays with Be(2)c cells and a 10 nM concentration of the RNA agent and PCR assays, as provided in the Examples herein, are expected to be within the scope of the present disclosure.

[0281] In addition, the RNA described herein specifies the site of C9orf72 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, which is coupled with additional nucleotide sequence taken from the region adjacent to the selected sequence in C9orf72 gene.

[0282] The dsRNA agents disclosed herein inhibit the expression of a C9orf72 target RNA containing a hexanucleotide repeat. Inhibiting expression includes any level of inhibition (e.g., partial inhibition of expression). For example, the dsRNA agent can inhibit the expression of a C9orf72 target RNA containing a hexanucleotide repeat by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% (or to the point where the C9orf72 target RNA is undetectable). For example, these levels of inhibition can occur within 24 to 48 hours after administration to cells expressing a C9orf72 target RNA containing a hexanucleotide repeat. The reduction can be, for example, compared to cells before treatment with the dsRNA agent or compared to control cells not treated with the dsRNA agent.

[0283] The dsRNA agent disclosed herein can also selectively inhibit the expression of C9orf72 target RNA that contains intronic hexanucleotide repeats, for example, compared with the expression of mature C9orf72 messenger RNA.In this situation, mature C9orf72 messenger RNA is the C9orf72 RNA transcript that has been spliced ​​and processed.Mature C9orf72 messenger RNA is composed exclusively of exons, and all introns have been removed.After administering dsRNA agent to the cell that expresses C9orf72 target RNA, if the relative decrease in the expression of C9orf72 target RNA is greater than the relative decrease in the expression of mature C9orf72 messenger RNA, then the dsRNA agent can selectively inhibit the expression of C9orf72 target RNA that contains intronic hexanucleotide repeats, compared with the expression of mature C9orf72 messenger RNA. For example, a dsRNA agent can inhibit expression of mature C9orf72 messenger RNA by less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, or less than about 5% (or, e.g., have no statistically significant or functionally significant effect on expression). For example, these levels of inhibition can be within 24 to 48 hours after administration to cells expressing mature C9orf72 messenger RNA.

[0284] The dsRNA agents disclosed herein can also, for example, reduce dipeptide repeat protein synthesis or dipeptide repeat protein levels in cells (e.g., within 24 to 48 hours after administration to the cells). For example, the dsRNA agent can reduce dipeptide repeat protein synthesis or dipeptide repeat protein levels by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%. The reduction can be, for example, compared to cells before treatment with the dsRNA agent or compared to control cells not treated with the dsRNA agent.

[0285] 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 include, 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 include 5, 4, 3, 2, or unmodified nucleotides.In yet other embodiments of the present disclosure, the RNAi agent of the present disclosure may include 5, 4, 3, 2, or 1 modified nucleotide.

[0286] 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.

[0287] 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 in 2'-5', and those with reverse polarity, where adjacent pairs of nucleoside units are linked in 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included. In some embodiments of the present invention, the dsRNA agent of the present invention is in free acid form. In other embodiments of the present invention, the dsRNA agent of the present invention is in salt form. In one embodiment, the dsRNA agent of the present invention is in sodium salt form. In certain embodiments, when the dsRNA agent of the present invention is in sodium salt form, sodium ion exists in the agent as the counterion for substantially all of the phosphodiester and / or phosphorothioate groups present in the agent.The agent in which substantially all of the phosphodiester and / or phosphorothioate linkages have sodium counterion comprises 5, 4, 3, 2 or 1 or less phosphodiester and / or phosphorothioate linkages that do not have sodium counterion.In some embodiments, when the dsRNA agent of the present invention is in sodium salt form, sodium ion exists as the counterion for all of the phosphodiester and / or phosphorothioate groups present in the agent.

[0288] 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.

[0289] 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.

[0290] 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.

[0291] In other embodiments, RNA mimetics suitable for use in RNAi agents are contemplated, in which both sugar and internucleoside linkage, i.e., the backbone of nucleotide unit, are replaced with novel groups. Base unit is maintained for hybridization with appropriate nucleic acid target compound. One such oligomeric compound, an RNA mimetic that has been found 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 part of the backbone. Representative US patents that teach the preparation of PNA compounds include, but are not limited to, US 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.

[0292] 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--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.

[0293] Modified RNAs may also contain one or more substituted sugar moieties. 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 the alkyl, alkenyl, and alkynyl may be substituted or unsubstituted C1-C10 alkyl or C2-C10 alkenyl and alkynyl. Exemplary suitable modifications include O[(CH2)nO]mCH3, O(CH2).nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10. In other embodiments, the dsRNA comprises one of the following at the 2' position: C1-C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving group, reporter group, interfering substance, group for improving the pharmacokinetic properties of an RNAi agent or group for improving the pharmacodynamic properties of an RNAi agent, and other substituents with similar properties. In some embodiments, the modification comprises 2'-methoxyethoxy (2'-O--CH2CHOCH3, 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).

[0294] 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.

[0295] The RNAi agents 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.

[0296] 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.

[0297] 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].

[0298] 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.

[0299] 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.

[0300] 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).

[0301] 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."

[0302] 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.

[0303] 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.

[0304] In some embodiments, the RNAi agent of the present disclosure comprises one or more monomers that are UNA (unlocked nucleic acid) nucleotides. UNA is an unlocked acyclic nucleic acid, and any of the sugar bonds have been removed to form an unlocked "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].

[0305] 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.

[0306] 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.

[0307] 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.

[0308] 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.

[0309] Thus, the present disclosure provides double-stranded RNAi agents capable of inhibiting the expression of a target gene (i.e., the C9orf72 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.

[0310] 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.

[0311] 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.

[0312] 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.

[0313] 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.

[0314] 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.

[0315] 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.

[0316] In one embodiment, the RNAi agent is a 19-nucleotide double-ended bluntmer, where the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 7, 8, and 9 from the 5' end, and the antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0317] In another embodiment, the RNAi agent is a 20-nucleotide double-ended bluntmer, 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.

[0318] In yet another embodiment, the RNAi agent is a 21-nucleotide double-ended bluntmer, 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.

[0319] In one embodiment, the RNAi agent comprises a 21-nucleotide sense strand and a 23-nucleotide antisense strand, wherein the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 9, 10, and 11 from the 5' end, and the antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end, and one end of the RNAi agent is blunt, and the other end contains a two-nucleotide overhang. 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 the three nucleotides being overhanging nucleotides, and the third nucleotide being 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).

[0320] 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'-most 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'-most 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'-most nucleotide of the antisense strand is not paired with the sense strand, and up to six consecutive 3'-most 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 19 ribonucleotides of the sense strand, 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 of the antisense strand when the sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially double-stranded region between the sense and antisense strands, the antisense strand being sufficiently complementary to the target RNA along at least 19 ribonucleotides of the length of the antisense strand, and reducing target gene expression when the double-stranded nucleic acid is introduced into a mammalian cell, the sense strand containing at least one motif of three 2'-F modifications on three consecutive nucleotides, at least one of the motifs occurring at or near the cleavage site, and the antisense strand containing at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at or near the cleavage site.

[0321] 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 1-4 nucleotides longer at its 3' end than the first strand, the duplex region being at least 25 nucleotides in length, the second strand being sufficiently complementary to a target mRNA along at least 19 nucleotides of the second strand length, the RNAi agent reducing target gene expression when introduced into a mammalian cell, and Dicer cleavage of the RNAi agent preferentially yielding 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.

[0322] 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.

[0323] 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.

[0324] 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.

[0325] 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.

[0326] 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.

[0327] 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.

[0328] 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.

[0329] 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.

[0330] 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.

[0331] 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.

[0332] 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.

[0333] 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.

[0334] 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.

[0335] 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 the sense or antisense strand.

[0336] In one embodiment, the sense strand sequence has formula (I): 5'np-Na-(XXX)i-Nb-YYY-Nb-(ZZZ)j-Na-nq 3'(I) [In the formula, i and j each independently represent 0 or 1; p and q each independently represent 0 to 6; each Na independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides; each Nb independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides; each np and nq independently represents 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.

[0337] In one embodiment, Na or Nb comprises an alternating pattern of modifications.

[0338] 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.

[0339] 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' np-Na-YYY-Nb-ZZZ-Na-nq 3'(Ib)、 5' np-Na-XXX-Nb-YYY-Na-nq 3'(Ic), or 5' np-Na-XXX-Nb-YYY-Nb-ZZZ-Na-nq 3'(Id) It can be expressed as:

[0340] When the sense strand is represented by formula (Ib), Nb represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides.

[0341] Each Na can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0342] When the sense strand is represented by Formula (Ic), 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. Each Na may independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0343] When the sense strand is represented by formula (Id), each Nb independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. Preferably, Nb is 0, 1, 2, 3, 4, 5, or 6. Each Na may independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0344] Each of X, Y and Z may be the same as or different from one another.

[0345] In other embodiments, i is 0, j is 0, and the sense strand has the formula: 5' np-Na-YYY-Na-nq 3'(Ia) It can be expressed as:

[0346] When the sense strand is represented by Formula (Ia), each Na can independently comprise an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0347] In one embodiment, the antisense strand sequence of the RNAi has the formula (II): 5' nq'-Na'-(Z'Z'Z')k-Nb'-Y'Y'Y'-Nb'-(X'X'X')l-N'a-np' 3'(II) [In the formula, k and l each independently represent 0 or 1; p' and q' each independently represent 0 to 6; each Na' independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides; each Nb' independently represents an oligonucleotide sequence comprising 0 to 10 modified nucleotides; each np' and nq' independently represents an overhanging nucleotide; Nb' 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:

[0348] In one embodiment, Na' or Nb' comprises an alternating pattern of modifications.

[0349] 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.

[0350] In one embodiment, the Y'Y'Y' motif is all 2'-OMe modified nucleotides.

[0351] 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.

[0352] Thus, the antisense strand has the formula: 5' nq'-Na'-Z'Z'Z'-Nb'-Y'Y'Y'-Na'-np' 3'(IIb)、 5' nq'-Na'-Y'Y'Y'-Nb'-X'X'X'-np' 3'(IIc)、または 5' nq'-Na'-Z'Z'Z'-Nb'-Y'Y'Y'-Nb'-X'X'X'-Na'-np' 3'(IId) It can be expressed as:

[0353] When the antisense strand is represented by Formula (IIb), 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. Each Na' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0354] When the antisense strand is represented by Formula (IIc), 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. Each Na' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0355] When the antisense strand is represented by formula (IId), each Nb' independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. Each Na' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. Preferably, Nb is 0, 1, 2, 3, 4, 5, or 6.

[0356] In other embodiments, k is 0, l is 0, and the antisense strand has the formula: 5' np'-Na'-Y'Y'Y'-Na'-nq' 3'(Ia) It can be expressed as:

[0357] When the antisense strand is represented as formula (IIa), each Na' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0358] Each of X', Y' and Z' may be the same as or different from one another.

[0359] 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.

[0360] 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.

[0361] 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.

[0362] The sense strand represented by any one of the above formulas (Ia), (Ib), (Ic) and (Id) forms a duplex with the antisense strand represented by any one of the formulas (IIa), (IIb), (IIc) and (IId), respectively.

[0363] Thus, an RNAi agent for use in the methods of the disclosure can include a sense strand and an antisense strand, each strand having 14-30 nucleotides, and the RNAi duplex can have the formula (III): Sense: 5' np-Na-(XXX)i-Nb-YYY-Nb-(ZZZ)j-Na-nq 3' Antisense: 3' np'-Na'-(X'X'X')k-Nb'-Y'Y'Y'-Nb'-(Z'Z'Z')l-Na'-nq' 5' (III) [In the formula, i, j, k, and l each independently represent 0 or 1; p, p', q and q' each independently represent 0 to 6; each Na and Na' independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides; each Nb and Nb' independently represents an oligonucleotide sequence comprising 0 to 10 modified nucleotides; each np', np, nq', and nq independently represents an overhanging nucleotide, 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

[0364] In one embodiment, i is 0 and j is 0, or i is 1 and j is 0, or i is 0 and j is 1, or i and j are both 0, or i and j are both 1. In another embodiment, k is 0 and l is 0, or k is 1 and l is 0, or k is 0 and l is 1, or k and l are both 0, or k and l are both 1.

[0365] An exemplary combination of sense and antisense strands that form an RNAi duplex has the following formula: 5' np-Na-YYY-Na-nq 3' 3' np'-Na'-Y'Y'Y'-Na'nq' 5' (IIIa) 5' np-Na-YYY-Nb-ZZZ-Na-nq 3' 3' np'-Na'-Y'Y'Y'-Nb'-Z'Z'Z'-Na'nq'5' (IIIb) 5' np-Na-XXX-Nb-YYY-Na-nq 3' 3' np'-Na'-X'X'X'-Nb'-Y'Y'Y'-Na'-nq' 5' (IIIc) 5' np-Na-XXX-Nb-YYY-Nb-ZZZ-Na-nq 3' 3' np'-Na'-X'X'X'-Nb'-Y'Y'Y'-Nb'-Z'Z'Z'-Na-nq' 5' (IIId) Includes.

[0366] When the RNAi agent is represented by Formula (IIIa), each Na independently represents an oligonucleotide sequence comprising from 2 to 20, from 2 to 15, or from 2 to 10 modified nucleotides.

[0367] When an RNAi agent is represented by Formula (IIIb), each Nb independently represents an oligonucleotide sequence containing 1 to 10, 1 to 7, 1 to 5, or 1 to 4 modified nucleotides. Each Na independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0368] When an RNAi agent is represented as Formula (IIIc), each Nb, Nb' independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. Each Na independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0369] When an RNAi agent is represented as Formula (IIId), each Nb, Nb' independently represents an oligonucleotide sequence comprising 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. Each Na, Na' independently represents an oligonucleotide sequence comprising 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. Each of Na, Na', Nb, and Nb' independently comprises an alternating pattern of modifications.

[0370] In one embodiment, when the RNAi agent is represented by formula (IIId), the Na modification is a 2'-O-methyl or 2'-fluoro modification. In another embodiment, when the RNAi agent is represented by formula (IIId), the Na modification is a 2'-O-methyl or 2'-fluoro modification, np'>0, and at least one np' is linked to an adjacent nucleotide via a phosphorothioate linkage. In yet another embodiment, when the RNAi agent is represented by formula (IIId), the Na modification is a 2'-O-methyl or 2'-fluoro modification, np'>0, and at least one np' is linked to an adjacent nucleotide via a phosphorothioate linkage, and the sense strand is conjugated to one or more C16 (or related) moieties attached by a bivalent or trivalent branched linker (described below). In another embodiment, when the RNAi agent is represented by formula (IIId), the Na modification is a 2'-O-methyl or 2'-fluoro modification, np'>0, and at least one np' is linked to an adjacent nucleotide via a phosphorothioate linkage, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more lipophilic, e.g., C16 (or related) moieties, which may be attached by a divalent or trivalent branched linker.

[0371] In one embodiment, when the RNAi agent is represented by formula (IIIa), the Na modification is a 2'-O-methyl or 2'-fluoro modification, np'>0, and at least one np' is linked to an adjacent nucleotide via a phosphorothioate linkage, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more lipophilic, e.g., C16 (or related) moieties attached by a divalent or trivalent branched linker.

[0372] In one embodiment, the RNAi agent is a multimer that contains at least two double strands represented by formula (III), (IIIa), (IIIb), (IIIc) and (IIId), and the double strands are connected by a linker. The linker may be cleavable or non-cleavable. 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.

[0373] In one embodiment, the RNAi agent is a multimer that contains 3, 4, 5, 6 or more double strands represented by formula (III), (IIIa), (IIIb), (IIIc) and (IIId), and the double strands are connected by a linker. The linker may be cleavable or non-cleavable. 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.

[0374] In one embodiment, two RNAi agents represented by formula (III), (IIIa), (IIIb), (IIIc) and (IIId) 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.

[0375] 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.

[0376] In certain embodiments, the compositions and methods of the present disclosure include vinyl phosphonate (VP) modifications of RNAi agents as described herein. In an exemplary embodiment, the vinyl phosphonate of the present disclosure has the following structure:

Chemical formula

[0377] 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.

[0378] Vinyl phosphate modifications are also contemplated for the compositions and methods of the present disclosure. Exemplary vinyl phosphate structures include:

Chemical formula

[0379] 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.

[0380] Thermally destabilizing modifications can include, but are not limited to, abasic modifications, mismatches with the opposing nucleotide in the opposing strand, and sugar modifications, such as 2'-deoxy modifications or acyclic nucleotides, such as unlocked nucleic acids (UNAs) or glycol nucleic acids (GNAs).

[0381] Exemplary abasic modifications include, but are not limited to, the following: [ka] wherein R=H, Me, Et, or OMe; R′=H, Me, Et, or OMe; and R″=H, Me, Et, or OMe. [ka] wherein B is a modified or unmodified nucleobase. Examples include:

[0382] Exemplary sugar modifications include, but are not limited to, the following: [ka] [ka] wherein B is a modified or unmodified nucleobase. Examples include:

[0383] In some embodiments, the thermally destabilizing modification of the duplex is one of the following: [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:

[0384] 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 [ka] [wherein B is a modified or unmodified nucleobase, R1 and R2 are independently H, halogen, OR3, or alkyl; and R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar]. The term "UNA" refers to an unlocked acyclic nucleic acid in which one of the sugar bonds has been removed to form an unlocked "sugar" residue. In one example, UNA also encompasses a monomer in which the bond between C1'-C4' has been removed (i.e., a covalent carbon-oxygen-carbon bond between the C1' and C4' carbons). In another example, the C2'-C3' bond of the sugar (i.e., the covalent carbon-carbon bond between the C2' and C3' carbons) is removed (see Mikhailov et al., Tetrahedron Letters, 26(17): 2059 (1985) and Fluiter et al., Mol. Biosyst., 10: 1039 (2009), the entire contents of which are incorporated herein by reference). Acyclic derivatives provide greater backbone flexibility without affecting Watson-Crick pairing. Acyclic nucleotides can be linked via 2'-5' or 3'-5' linkages.

[0385] 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: [ka]

[0386] 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 invention.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.

[0387] 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 the complementary base on the target mRNA, such as: [ka] Includes.

[0388] 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.

[0389] Thermally destabilizing modifications can also include universal base and phosphate modifications that have reduced or eliminated ability to form hydrogen bonds with opposing bases.

[0390] 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: [ka] There is.

[0391] 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: [ka] wherein R is H, OH, OCH, F, NH, NHMe, NMe, or O-alkyl. Includes:

[0392] Exemplary phosphate modifications known to reduce the thermal stability of dsRNA duplexes compared to native phosphodiester linkages include: [ka] There is.

[0393] 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.

[0394] 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 that can be used and generally exist 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.

[0395] 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.

[0396] 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.

[0397] 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.

[0398] 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.

[0399] 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.

[0400] 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.

[0401] Exemplary thermally stabilizing modifications include, but are not limited to, 2'-fluoro modifications. Other thermally stabilizing modifications include, but are not limited to, LNA.

[0402] 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.

[0403] 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.

[0404] 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 the position -1 and +1 from the position of destabilizing modification.

[0405] 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.

[0406] 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.

[0407] 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.

[0408] 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 comprise: (i) the antisense strand comprises 2, 3, 4, 5, or 6 2'-fluoro modifications, (ii) the antisense strand comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages, (iii) the sense strand is conjugated with a ligand, (iv) the sense strand comprises 2, 3, 4, or 5 2'-fluoro modifications, (v) the sense strand comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages, (vi) the dsRNA comprises at least four 2'-fluoro modifications, and (vii) the dsRNA comprises a blunt end at the 5' end of the antisense strand. Preferably, a 2-nt overhang is at the 3' end of the antisense strand.

[0409] 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. the antisense strand comprises a nucleotide sequence of at least 19 ribonucleotides along its length, thereby forming a single-stranded 5' overhang of 10 to 30 nucleotides, 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 along its length, thereby reducing target gene expression when the double-stranded nucleic acid is introduced into a mammalian cell, and the antisense strand contains at least one thermally destabilizing nucleotide, the at least one thermally destabilizing nucleotide being in the seed region of the antisense strand (i.e., at positions 2 to 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.

[0410] 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 comprises at least one The dsRNA contains thermally destabilized nucleotides, with at least one thermally destabilized nucleotide in the seed region of the antisense strand (i.e., positions 2-9 at the 5' end of the antisense strand), and 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; (i) 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.

[0411] 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.

[0412] 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.

[0413] 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 part 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 modified deoxyribonucleotides, 2'-deoxy-2'-fluoro (2'-F) or 2'-O-methyl, instead of the ribosugar of the nucleic acid base, and modifications at the phosphate group, for example, phosphorothioate modifications.The overhang does not need to be homologous to the target sequence.

[0414] In some embodiments, each residue of sense strand and antisense strand is independently modified with LNA, HNA, 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 sense strand and 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 double strand that exists in the antisense strand.

[0415] 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 nucleotides with two different modifications selected from 2'-O-methyl or 2'-deoxy. In some embodiments, each residue in the sense strand and the antisense strand is independently modified with 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.

[0416] In some embodiments, the dsRNA molecules of the present disclosure comprise alternating patterns of 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 may refer to one every other nucleotide or one every third nucleotide, or similar patterns. For example, if A, B, and C each represent one type of modification to a nucleotide, the alternating motif may be "ABABABABABAB...", "AABBAABBAABB...", "AABAABAABAAB...", "AAABAAABAAAB...", "AABBBAAABBB...", or "ABCABCABCABC...", etc.

[0417] 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...".

[0418] 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 begin with "ABABAB" from the 5'-3' end of the strand, and the alternating motif in the antisense strand may begin 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 begin with "AABBAABB" from the 5'-3' end of the strand, and the alternating motif in the antisense strand may begin 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.

[0419] 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.

[0420] 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 nucleotide that is adjacent to the overhang nucleotide.For example, there can be at least two phosphorothioate internucleotide linkages between the terminal three nucleotides, two of which are overhang nucleotides, and the third is the nucleotide that is adjacent to the overhang nucleotide that is adjacent to the overhang nucleotide.Preferably, these terminal three nucleotides can be the 3' end of antisense strand.

[0421] 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.

[0422] 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.

[0423] 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.

[0424] 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.

[0425] 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, methylphosphonate, or phosphate linkages.

[0426] 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.

[0427] 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.

[0428] 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.

[0429] 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, methylphosphonate, or phosphate linkages.

[0430] 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.

[0431] 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 ends.

[0432] 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).

[0433] 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 positions 1 and 2 and two phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) of the antisense strand.

[0434] 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 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 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 positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) of the antisense strand.

[0436] 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 positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 (counting from the 5' end) of the antisense strand.

[0437] 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.

[0438] 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.

[0439] 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 of the antisense strand (counting from the 5' end).

[0440] 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 positions 1 and 2 and two phosphorothioate internucleotide linkage modifications (counting from the 5' end) within positions 18-23 of the antisense strand.

[0441] 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.

[0442] 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.

[0443] 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 positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) of the antisense strand.

[0444] 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 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.

[0445] 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.

[0446] 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.

[0447] 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.

[0448] 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.

[0449] 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.

[0450] 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 no more than 8 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 eight or fewer non-chiral 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 noncontiguous. In some embodiments, the internucleotide linkages in the Rp configuration may be contiguous or noncontiguous. In some embodiments, the nonchiral internucleotide linkages may be contiguous or noncontiguous.

[0451] In some embodiments, compounds of the present disclosure include blocks that are stereochemical blocks. In some embodiments, the blocks are Rp blocks, in that each internucleotide linkage of the block is Rp. In some embodiments, the 5'-block is an Rp block. In some embodiments, the 3'-block is an Rp block. In some embodiments, the blocks are Sp blocks, in that each internucleotide linkage of the block is Sp. In some embodiments, the 5'-block is an Sp block. In some embodiments, the 3'-block is an Sp block. In some embodiments, provided oligonucleotides include both Rp and Sp blocks. In some embodiments, provided oligonucleotides include one or more Rp blocks but 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.

[0452] 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.

[0453] 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, e.g., 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.

[0454] 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; (ii) the antisense strand comprises two, three, four, five, or six 2'-fluoro modifications; (iii) the sense strand is conjugated to a ligand; (iv) the sense strand comprises two, three, four, or five 2'-fluoro modifications; (v) the sense strand comprises one, two, three, four, or five phosphorothioate internucleotide linkages; (vi) the dsRNA comprises at least four 2'-fluoro modifications; (vii) the dsRNA comprises a 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.

[0455] 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 comprises at least two, three, four, five, or six 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 between 12 and 40 nucleotide pairs in length; (vii) the dsRNA comprises a duplex region between 12 and 40 nucleotide pairs in length; and (viii) the dsRNA has a blunt end at the 5' end of the antisense strand.

[0456] 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., 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; (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.

[0457] 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, 2): (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.

[0458] In some embodiments, the dsRNA molecule of the present disclosure comprises a mismatch (or mismatches) or combinations thereof within the double strand with the target. Mismatches can occur in the overhang region or the double-stranded region. 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.

[0459] 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.

[0460] 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.

[0461] It has been found that the introduction of a 4'- or 5'-modified nucleotide at the 3' end of a dinucleotide phosphodiester (PO), phosphorothioate (PS) or phosphorodithioate (PS2) linkage at any position in a single- or double-stranded oligonucleotide can exert a steric effect on the internucleotide linkage, thus protecting it from nucleases and stabilizing it.

[0462] 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.

[0463] 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.

[0464] 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.

[0465] 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.

[0466] 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.

[0467] 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 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.

[0468] 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.

[0469] 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.

[0470] 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.

[0471] Ligands can be attached to polynucleotides via carriers. The 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, e.g., a hydroxyl group, or generally a bond available and suitable for incorporation of the carrier into a backbone, e.g., a phosphate or modified phosphate, e.g., sulfur-containing backbone, of a ribonucleic acid. "Tethering attachment point" (TAP) refers, in some embodiments, to a constituent ring atom, e.g., a carbon atom or heteroatom (separate from the atom providing the backbone attachment point), of a cyclic carrier that connects the selected moiety. The moiety can be, for example, a carbohydrate, e.g., a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide. The selected moiety may be connected to the cyclic carrier by an intervening tether. Thus, cyclic carriers often contain a functional group, e.g., an amino group, or generally provide a bond suitable for incorporation or tethering another chemical entity, e.g., a ligand, to the constituent ring.

[0472] 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.

[0473] In certain specific embodiments, an RNAi agent for use in the methods of the disclosure is an agent selected from the group of agents listed in any one of Tables 10A, 10B, 12-15, 19, or 20.

[0474] IV. Ligand-Conjugated iRNA Another modification of the iRNA of the invention 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).

[0475] In certain embodiments, the ligand alters the distribution, targeting, or life span of the iRNA agent into which it is incorporated. In some embodiments, the ligand provides enhanced affinity for a selected target, for example, a molecule, a cell or cell type, a compartment, for example, a cellular or organ compartment, a tissue, an organ, or a 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.

[0476] 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.

[0477] 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.

[0478] 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.

[0479] 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.

[0480] 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.

[0481] In some embodiments, the ligand attached to the iRNA described herein acts as a pharmacokinetic modulator (PK modulator). PK modulators include lipophilic substances, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, and the like. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, and the like. Oligonucleotides containing several phosphorothioate linkages 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 invention. 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.

[0482] Ligand-conjugated iRNAs of the invention 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.

[0483] The oligonucleotides used in the conjugates of the invention may be conveniently and routinely made through the known technique of solid phase synthesis. Equipment for such synthesis is available, for example, from Applied Biosystems (登録商標) These oligonucleotides are sold by several vendors, including Sigma-Aldrich (Foster City, CA). Any other means for such synthesis known in the art may additionally or alternatively be used. It is also known to use similar techniques to prepare other oligonucleotides, such as phosphorothioates and alkylated derivatives.

[0484] For the ligand-conjugated oligonucleotides and molecules having ligand-sequence-specific linked nucleosides of the present invention, 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.

[0485] When using a nucleotide-conjugate precursor that already has a linking moiety, the synthesis of the sequence-specific linked nucleoside is usually completed, and then a ligand molecule is reacted with the linking moiety to form a ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or linked nucleosides of the present invention 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.

[0486] 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.

[0487] 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.

[0488] 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.

[0489] 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.

[0490] 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).

[0491] 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.

[0492] 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.

[0493] 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: ). An RFGF analog containing a hydrophobic MTS (e.g., the amino acid sequence AALLPVLLAAP (SEQ ID NO: )) 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: )) and the sequence derived from the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: )) 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.

[0494] The RGD peptide for use in the compositions and methods of the present invention can be linear or cyclic, and can be modified, for example, glycosylated or methylated, to facilitate targeting to specific 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.

[0495] 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 deliver iRNA agents to tumor cells expressing αVβ3 (Haubner et al., Jour. Nucl. Med., 42:326-336, 2001).

[0496] 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...

Claims

1. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of C9orf72, wherein the dsRNA agent comprises a sense strand and an antisense strand that form a double-stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the antisense nucleotide sequences set forth in any one of Tables 10A, 10B, 12-15, 19, 20, or 21.

2. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of C9orf72, the dsRNA agent comprising a sense strand and an antisense strand forming a double-stranded region, the sense strand comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from nucleotides 200-290 of SEQ ID NO: 133, and the antisense strand comprising at least 15 contiguous nucleotides from the corresponding nucleotide sequence of SEQ ID NO: 134; A dsRNA agent wherein the sense strand, the antisense strand, or both the sense and antisense strands are conjugated to one or more lipophilic moieties.

3. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of C9orf72, wherein the dsRNA agent comprises a sense strand and an antisense strand that form a double-stranded region, and the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 125; A dsRNA agent wherein the sense strand, the antisense strand, or both the sense and antisense strands are conjugated to one or more lipophilic moieties.

4. 3. The dsRNA agent of claim 1 or 2, wherein the nucleotide sequences of the sense and antisense strands comprise any one of the nucleotide sequences of the sense and antisense strands listed in any one of Tables 14, 15, 19, or 20.

5. The antisense strand is AD-348904.1, AD-348136.1, AD-347612.1, AD-348639.1, AD-347606.1, AD-348905.1, AD-347613.1, AD-348842.1, AD-347610.1, AD-350329.1, AD-348140.1, AD-348930.1, AD-347863.1, AD-3485 5. The dsRNA agent of any one of claims 1, 2, and 4, comprising 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-348597.1, AD-348578.1, AD-347923.1, AD-348588.1, and AD-347773.

1.

6. 1. A double-stranded ribonucleic acid (dsRNA) agent for selectively inhibiting a C9orf72 target RNA comprising a hexanucleotide repeat comprising multiple consecutive copies of SEQ ID NO:1, The dsRNA agent comprises a sense strand and an antisense strand that form a double-stranded region, wherein the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the antisense nucleotide sequences set forth in any one of Tables 14, 15, and 21.

7. 7. The dsRNA agent of any one of claims 1, 2, 4, 5, and 6, wherein 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-463863.1, AD-463862.1, AD-463869.1, AD-463873.1, AD-463872.1, and AD-463860.

1.

8. 8. The dsRNA of claim 7, wherein the antisense strand comprises at least 15 consecutive nucleotides from any one of the antisense strand nucleotide sequences of the duplex selected from the group consisting of AD-463863.1, AD-463862.1, AD-463869.1, AD-463873.1, AD-463872.1, and AD-463860.

1.

9. 8. The dsRNA agent of claim 7, wherein the antisense strand comprises a nucleotide sequence that differs 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-463863.1, AD-463862.1, AD-463869.1, AD-463873.1, AD-463872.1, and AD-463860.

1.

10. 8. The dsRNA agent of claim 7, wherein the antisense strand comprises the nucleotide sequence of any one of the antisense strand nucleotide sequences of the duplex selected from the group consisting of AD-463863.1, AD-463862.1, AD-463869.1, AD-463873.1, AD-463872.1, and AD-463860.

1.

11. 8. The dsRNA agent of claim 7, wherein the antisense strand consists of the nucleotide sequence of any one of the antisense strand nucleotide sequences of the duplex selected from the group consisting of AD-463863.1, AD-463862.1, AD-463869.1, AD-463873.1, AD-463872.1, and AD-463860.

1.

12. 12. The dsRNA agent of any one of claims 7-11, wherein the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the sense strand nucleotide sequences of the duplex selected from the group consisting of AD-463863.1, AD-463862.1, AD-463869.1, AD-463873.1, AD-463872.1, and AD-463860.

1.

13. 13. The dsRNA agent of any one of claims 7-12, wherein the sense strand comprises at least 15 contiguous nucleotides from any one of the duplexed sense strand nucleotide sequences selected from the group consisting of AD-463863.1, AD-463862.1, AD-463869.1, AD-463873.1, AD-463872.1, and AD-463860.

1.

14. 14. The dsRNA agent of any one of claims 7-13, wherein the sense strand comprises a nucleotide sequence that differs by no more than 3 nucleotides from any one of the sense strand nucleotide sequences of the duplex selected from the group consisting of AD-463863.1, AD-463862.1, AD-463869.1, AD-463873.1, AD-463872.1, and AD-463860.

1.

15. 15. The dsRNA agent of any one of claims 7-14, wherein the sense strand comprises the nucleotide sequence of any one of the sense strand nucleotide sequences of the duplex selected from the group consisting of AD-463863.1, AD-463862.1, AD-463869.1, AD-463873.1, AD-463872.1, and AD-463860.

1.

16. 16. The dsRNA agent of any one of claims 7 to 15, wherein the sense strand consists of the nucleotide sequence of any one of the duplex sense strand nucleotide sequences selected from the group consisting of AD-463863.1, AD-463862.1, AD-463869.1, AD-463873.1, AD-463872.1, and AD-463860.

1.

17. 4. The dsRNA agent of claim 1 or 3, wherein the nucleotide sequences of the sense and antisense strands comprise any one of the nucleotide sequences of the sense and antisense strands listed in any one of Tables 12 or 13.

18. 18. The dsRNA agent of any one of claims 1, 3, and 17, 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 230-270, 233-262, 800-840, 800-830, 802-828, 1240-1290, 1240-1280, 1247-1288, 1590-1645, 1590-1620, and 1594-1642 of SEQ ID NO: 121, and the antisense strand comprises at least 15 contiguous nucleotides from the corresponding nucleotide sequence of SEQ ID NO:

125.

19. the sense strand is selected from the group consisting of nucleotides 1594-1616, 802-824, 239-261, 1308-1330, 233-255, 1595-1617, 240-262, 1532-1554, 237-259, 3268-3290, 806-828, 1620-1642, 526-548, 1169-1191, 1266-1288, 1247-1269, 58 of SEQ ID NO:121 19. The dsRNA agent of any one of claims 1, 3, 17, and 18, wherein 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: 6-608, 1257-1279, and 400-422, and wherein the antisense strand comprises at least 15 contiguous nucleotides from the corresponding nucleotide sequence of SEQ ID NO:

125.

20. 20. The dsRNA agent of any one of claims 1 and 4-19, wherein the sense strand, the antisense strand, or both the sense and antisense strands are conjugated to one or more lipophilic moieties.

21. 21. The dsRNA agent of claim 20, wherein the lipophilic moiety is conjugated to one or more internal positions in the double-stranded region of the dsRNA agent.

22. 22. The dsRNA agent of claim 20 or 21, wherein the lipophilic moiety is conjugated via a linker or carrier.

23. 23. The dsRNA agent of any one of claims 20-22, wherein the lipophilicity of the lipophilic moiety is greater than 0 as measured by log Kow.

24. 24. The dsRNA agent of any one of claims 1-23, wherein the hydrophobicity of the double-stranded RNAi agent is greater than 0.2 as measured by the unbound fraction in a plasma protein binding assay of the double-stranded RNAi agent.

25. 25. The dsRNA agent of claim 24, wherein the plasma protein binding assay is an electrophoretic mobility shift assay using human serum albumin protein.

26. 26. The dsRNA agent of any one of claims 1-25, wherein the dsRNA agent comprises at least one modified nucleotide.

27. 27. The dsRNA agent of claim 26, wherein no more than five of the nucleotides in the sense strand and no more than five of the nucleotides in the antisense strand are unmodified nucleotides.

28. 27. The dsRNA agent of claim 26, wherein all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides.

29. At least one of the modified nucleotides is a deoxynucleotide, a 3'-terminal deoxythymine (dT) nucleotide, a 2'O-methyl modified nucleotide, a 2'fluoro modified nucleotide, a 2'deoxy modified nucleotide, a locked nucleotide, a non-locked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino modified nucleotide, a 2'-O-allyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2'-hydroxyl modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'O-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide containing a non-natural base, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a 2'-amino modified nucleotide, a 2'-O-allyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2'-hydroxyl modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'- ...

29. The dsRNA agent of any one of claims 26-28, selected from the group of nucleotides, cyclohexenyl-modified nucleotides, nucleotides comprising a 5' phosphorothioate group, nucleotides comprising a 5' methylphosphonate group, nucleotides comprising a 5' phosphate or a 5' phosphate mimic, nucleotides comprising a vinylphosphonate, nucleotides comprising adenosine-glycol nucleic acid (GNA), nucleotides comprising thymidine-glycol nucleic acid (GNA) S-isomers, nucleotides comprising 2-hydroxymethyl-tetrahydrofuran-5-phosphate, nucleotides comprising 2'-deoxythymidine-3' phosphate, nucleotides comprising 2'-deoxyguanosine-3'-phosphate, and terminal nucleotides linked to a cholesteryl derivative and a dodecanoic acid bisdecylamide group; and combinations thereof.

30. 30. The dsRNA agent of claim 29, wherein the modified nucleotide is selected from the group consisting of 2' deoxy-2'-fluoro modified nucleotides, 2' deoxy modified nucleotides, 3' terminal deoxythymine nucleotides (dT), locked nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, and nucleotides containing unnatural bases.

31. 30. The dsRNA agent of claim 29, wherein the modified nucleotides comprise a short sequence of 3' terminal deoxythymine nucleotides (dT).

32. 30. The dsRNA agent of claim 29, wherein the modifications to the nucleotide are 2'-O-methyl, GNA, and 2' fluoro modifications.

33. The dsRNA agent of any one of claims 1 to 32, further comprising at least one phosphorothioate internucleotide linkage.

34. 34. The dsRNA agent of claim 33, comprising 6 to 8 phosphorothioate internucleotide linkages.

35. 35. The dsRNA agent of any one of claims 1-34, wherein each strand is 30 or fewer nucleotides in length.

36. The dsRNA agent of any one of claims 1 to 35, wherein at least one strand comprises a 3' overhang of at least one nucleotide.

37. The dsRNA agent of any one of claims 1 to 36, wherein at least one strand comprises a 3' overhang of at least 2 nucleotides.

38. 38. The dsRNA agent of any one of claims 1 to 37, wherein the double-stranded region is 15 to 30 nucleotide pairs in length.

39. 39. The dsRNA agent of claim 38, wherein the double-stranded region is 17 to 23 nucleotide pairs in length.

40. 39. The dsRNA agent of claim 38, wherein the double-stranded region is 17 to 25 nucleotide pairs in length.

41. 39. The dsRNA agent of claim 38, wherein the double-stranded region is 23 to 27 nucleotide pairs in length.

42. 39. The dsRNA agent of claim 38, wherein the double-stranded region is 19 to 21 nucleotide pairs in length.

43. 39. The dsRNA agent of claim 38, wherein the double-stranded region is 21 to 23 nucleotide pairs in length.

44. 44. The dsRNA agent of any one of claims 1-43, wherein each strand is 19-30 nucleotides in length.

45. 44. The dsRNA agent of any one of claims 1-43, wherein each strand is 19-23 nucleotides in length.

46. 44. The dsRNA agent of any one of claims 1-43, wherein each strand is 21-23 nucleotides in length.

47. 47. The dsRNA agent of any one of claims 21-46, wherein the one or more lipophilic moieties are conjugated to one or more internal positions in at least one strand.

48. 48. The dsRNA agent of claim 47, 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.

49. 49. The dsRNA agent of claim 48, wherein the internal positions include all positions except the two terminal positions from each end of at least one strand.

50. 49. The dsRNA agent of claim 48, wherein the internal positions include all but the three terminal positions from each end of at least one strand.

51. 51. The dsRNA agent of any one of claims 48-50, wherein the internal position excludes the cleavage site region of the sense strand.

52. 52. The dsRNA agent of claim 51, wherein the internal positions include all positions except positions 9-12, counting from the 5' end of the sense strand.

53. 52. The dsRNA agent of claim 51, wherein the internal positions include all positions except positions 11-13, counting from the 3' end of the sense strand.

54. 51. The dsRNA agent of any one of claims 48-50, wherein the internal position excludes the cleavage site region of the antisense strand.

55. 55. The dsRNA agent of claim 54, wherein the internal positions include all positions except positions 12-14, counting from the 5' end of the antisense strand.

56. The dsRNA agent of any one of claims 48-50, wherein the internal positions include all positions except positions 11-13 counting from the 3' end and positions 12-14 counting from the 5' end.

57. 57. The dsRNA agent of any one of claims 21-56, 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.

58. 58. The dsRNA agent of claim 57, wherein 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.

59. 22. The dsRNA agent of claim 21, wherein the internal position in the double-stranded region excludes the cleavage site region of the sense strand.

60. 60. The dsRNA agent of any one of claims 20-59, wherein the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length, and wherein the lipophilic moiety is conjugated to position 21, 20, 15, 1, 7, 6, or 2 of the sense strand or position 16 of the antisense strand.

61. 61. The dsRNA agent of claim 60, wherein the lipophilic moiety is conjugated to position 21, 20, 15, 1, or 7 of the sense strand.

62. 61. The dsRNA agent of claim 60, wherein the lipophilic moiety is conjugated to position 21, 20, or 15 of the sense strand.

63. 61. The dsRNA agent of claim 60, wherein the lipophilic moiety is conjugated to position 20 or 15 of the sense strand.

64. 61. The dsRNA agent of claim 60, wherein the lipophilic moiety is conjugated to position 16 of the antisense strand.

65. 65. The dsRNA agent of any one of claims 20-64, wherein the lipophilic moiety is an aliphatic, alicyclic, or polyalicyclic compound.

66. 66. The dsRNA agent of claim 65, wherein 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.

67. 66. The dsRNA agent of claim 65, wherein 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.

68. 68. The dsRNA agent of claim 67, wherein the lipophilic moiety comprises a saturated or unsaturated C6 to C18 hydrocarbon chain.

69. 68. The dsRNA agent of claim 67, wherein the lipophilic moiety contains a saturated or unsaturated C16 hydrocarbon chain.

70. 70. The dsRNA agent of claim 69, wherein the saturated or unsaturated C16 hydrocarbon chain is conjugated to position 6, counting from the 5' end of the chain.

71. 71. The dsRNA agent of any one of claims 20-70, wherein the lipophilic moiety is conjugated via a carrier that replaces one or more nucleotides at an internal position or in the double-stranded region.

72. 72. The dsRNA agent of claim 71, wherein 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 backbone or a diethanolamine backbone.

73. 71. The dsRNA agent of any one of claims 20-70, wherein the lipophilic moiety is conjugated to the double-stranded iRNA agent via a linker that contains an ether, a thioether, a urea, a carbonate, an amine, an amide, a maleimide-thioether, a disulfide, a phosphodiester, a sulfonamide linkage, a product of a click reaction, or a carbamate.

74. 74. The double-stranded iRNA agent of any one of claims 20-73, wherein the lipophilic moiety is conjugated to a nucleobase, a sugar moiety, or an internucleoside linkage.

75. 75. The dsRNA agent of any one of claims 20-74, wherein the lipophilic moiety or targeting ligand is conjugated via a biocleavable linker selected from the group consisting of DNA, RNA, disulfide, amide, galactosamine, glucosamine, glucose, galactose, mannose functionalized mono- or oligosaccharides, and combinations thereof.

76. 76. The dsRNA agent of any one of claims 20-75, wherein the 3' end of the sense strand is protected via an end cap that is a cyclic group having an amine, the cyclic group being selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl.

77. The dsRNA agent of any one of claims 1 to 19, further comprising a targeting ligand that targets liver tissue.

78. 78. The dsRNA agent of claim 77, wherein the targeting ligand is a GalNAc conjugate.

79. 79. The dsRNA agent of any one of claims 1-78, further comprising: a terminal chiral modification at the first internucleotide linkage at the 3'-end of the antisense strand having a linking phosphorus atom in the Sp configuration; a terminal chiral modification at the first internucleotide linkage at the 5'-end of the antisense strand having a linking phosphorus atom in the Rp configuration; and a terminal chiral modification at the first internucleotide linkage at the 5'-end of the sense strand having a linking phosphorus atom in either the Rp or Sp configuration.

80. terminal chiral modifications 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 a 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 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.

79. The dsRNA agent of any one of claims 1-78, further comprising:

81. Terminal chiral modifications 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 a 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 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.

79. The dsRNA agent of any one of claims 1-78, further comprising:

82. terminal chiral modifications 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 occurs at the third internucleotide linkage at the 3' end of the antisense strand, with the linking phosphorus atom in the Rp configuration. a terminal chiral modification occurring at a 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 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.

79. The dsRNA agent of any one of claims 1-78, further comprising:

83. terminal chiral modifications 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 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.

79. The dsRNA agent of any one of claims 1-78, further comprising:

84. The dsRNA agent of any one of claims 1 to 83, further comprising a phosphate or phosphate mimetic at the 5'-end of the antisense strand.

85. 85. The dsRNA agent of claim 84, wherein the phosphate mimetic is a 5'-vinylphosphonate (VP).

86. The dsRNA agent of any one of claims 1 to 83, wherein the base pair at one position on the 5' end of the antisense strand of the duplex is an AU base pair.

87. 84. The dsRNA agent of any one of claims 1 to 83, wherein the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

88. 88. A cell containing the dsRNA agent of any one of claims 1-87.

89. 88. A pharmaceutical composition for inhibiting the expression of C9orf72, comprising the dsRNA agent of any one of claims 1 to 87.

90. 88. A pharmaceutical composition comprising the dsRNA agent of any one of claims 1-87 and a lipid formulation.

91. 91. The pharmaceutical composition of claim 89 or 90, wherein the dsRNA agent is in an unbuffered solution.

92. 89. The pharmaceutical composition of claim 88, wherein the unbuffered solution is saline or water.

93. 91. The pharmaceutical composition of claim 89 or 90, wherein the dsRNA agent is in a buffered solution.

94. 94. The pharmaceutical composition of claim 93, wherein the buffer solution comprises acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof.

95. 94. The pharmaceutical composition of claim 93, wherein the buffer solution is phosphate buffered saline (PBS).

96. 96. A method of inhibiting expression of C9orf72 in a cell, comprising contacting the cell with the dsRNA agent of any one of claims 1-87 or the pharmaceutical composition of any one of claims 89-95, thereby inhibiting expression of C9orf72 in the cell.

97. 97. The method of claim 96, wherein the cell is in a subject.

98. 98. The method of claim 97, wherein the subject is a human.

99. 99. The method of claim 98, wherein the subject has a C9orf72-associated disorder.

100. 100. The method of claim 99, wherein the C9orf72-associated disorder is selected from the group consisting of C9orf72 amyotrophic lateral sclerosis / frontotemporal dementia or Huntington-like syndrome due to C9orf72 expansion.

101. 101. The method of any one of claims 96-100, wherein contacting the cell with the dsRNA agent inhibits expression of C9orf72 by 50% or less.

102. 102. The method of any one of claims 96-101, wherein inhibiting expression of C9orf72 reduces C9orf72 protein levels in the subject's serum by 50% or less.

103. 96. A method of treating a subject having a disorder that would benefit from reduced C9orf72 expression, comprising administering to the subject a therapeutically effective amount of the dsRNA agent of any one of claims 1-87 or the pharmaceutical composition of any one of claims 89-95, thereby treating the subject having a disorder that would benefit from reduced C9orf72 expression.

104. 96. A method of preventing at least one symptom in a subject having a disorder that would benefit from reduced C9orf72 expression, comprising administering to the subject a prophylactically effective amount of the dsRNA agent of any one of claims 1-87 or the pharmaceutical composition of any one of claims 89-95, thereby preventing at least one symptom in a subject having a disorder that would benefit from reduced C9orf72 expression.

105. 105. The method of claim 103 or 104, wherein the disorder is a C9orf72-associated disorder.

106. 106. The method of claim 105, wherein the C9orf723-associated disorder is selected from the group consisting of C9orf72 amyotrophic lateral sclerosis / frontotemporal dementia and Huntington-like syndrome caused by C9orf72 expansion.

107. 107. The method of claim 106, wherein the subject is a human.

108. 108. The method of claim 106 or 107, wherein administration of the agent to a subject results in a decrease in repeat length-dependent formation of RNA aggregates, capture of specific RNA-binding proteins, or accumulation or aggregation of poly(glycine-alanine) peptides, poly(glycine-proline) peptides, poly(glycine-arginine) peptides, poly(alanine-proline) peptides, or poly(proline-arginine) peptides.

109. 109. The method of claim 108, wherein repeat length-dependent formation of RNA aggregates, trapping of specific RNA-binding proteins, or accumulation or aggregation of poly(glycine-alanine), poly(glycine-proline), poly(glycine-arginine), poly(alanine-proline), or poly(proline-arginine) peptides is reduced by more than 50%, and expression of C9orf72 mature RNA is inhibited by less than 50%.

110. 110. The method of any one of claims 103-109, wherein the dsRNA agent is administered to the subject at a dose of from about 0.01 mg / kg to about 50 mg / kg.

111. 111. The method of any one of claims 103-110, wherein the dsRNA agent is administered subcutaneously to the subject.

112. 111. The method of any one of claims 93-110, wherein the dsRNA agent is administered to the subject intrathecally.

113. 113. The method of any one of claims 93 to 112, further comprising determining the level of C9orf72 in a sample from the subject.

114. The method of claim 113, wherein the level of C9orf72 in the subject sample is the C9orf72 protein level in a blood, serum, or cerebrospinal fluid sample.

115. 115. The method of any one of claims 103 to 114, further comprising administering to the subject an additional therapeutic agent.

116. 96. A kit comprising the dsRNA agent of any one of claims 1 to 87 or the pharmaceutical composition of any one of claims 89 to 95.

117. A vial comprising the dsRNA agent of any one of claims 1-87 or the pharmaceutical composition of any one of claims 89-95.

118. 96. A syringe comprising the dsRNA agent of any one of claims 1-87 or the pharmaceutical composition of any one of claims 89-95.

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