Human chromosome 9 open reading frame 72 (C9ORF72) iRNA agent compositions and methods of use thereof

Abstract

The present disclosure relates to double-stranded ribonucleic acid (dsRNAi) agents and compositions that target the human chromosome 9 open reading frame 72 (C9orf72) gene, and methods of inhibiting expression of the C9orf72 gene and treating subjects with a C9orf72-associated disease or disorder, such as C9orf72 amyotrophic lateral sclerosis, frontotemporal dementia, or Huntington-like syndrome resulting from C9orf72 expansions, using such dsRNAi agents and compositions.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 196,791, filed June 4, 2021, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] 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 astrocytes and neurons, and presynaptic terminals.

[0003] Differential use of alternative transcription start and termination sites results in the generation of three sense RNA transcripts from C9orf72 DNA. These encode two protein isoforms: a long, approximately 54 kDa isoform (isoform A) derived from variants 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). In addition to sense RNA transcripts from C9orf72 DNA, there are repeat-containing antisense RNA transcripts, which have been shown to be elevated in the brains of C9orf72 expansion-positive patients. Depending on the location of the transcription start site, repeat-free sense and antisense RNA transcripts also exist.

[0004] The two alternatively used first exons of the C9orf72 gene are exons 1a and 1b (see, e.g., Figure 1 in Barker et al., supra). A large GGGGCC (G4C2) hexanucleotide repeat expansion (from approximately 2-22 copies to 700-1600 copies) in the first intron of the C9orf72 gene between exons 1a and 1b has been shown to 1) disrupt transcription of repeat-free C9orf72 mRNA, thereby reducing C9orf72 mRNA and protein levels; 2) generate toxic dipeptide repeat proteins through RNA-initiated translation; and 3) generate nuclear and cytoplasmic RNA foci, both of which may be pathogenic and may result in several neurodegenerative diseases with distinct clinical features but common pathological features and genetic causes (Ling, et al. (2013) Neuron 79:416-438). Furthermore, repeat-containing antisense RNA transcripts have been shown to accumulate in nuclear and cytoplasmic RNA foci and contribute to the expression of antisense toxic dipeptide repeat proteins through RNA-initiated translation. In particular, the presence of a hexanucleotide repeat expansion in the C9orf72 gene is the most common genetic cause of familial and idiopathic amyotrophic lateral sclerosis (ALS), a severe degenerative disease of motor neurons in the brain and spinal cord. Indeed, C9orf72 mutant hexanucleotide repeat expansions are present in approximately 40% of familial ALS subjects and 8-10% of idiopathic ALS subjects. 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. This condition is characterized by behavioral and language impairments and manifests pathologically by neuronal atrophy in the frontal and anterior temporal lobes of the brain. Huntington's-like syndrome caused by C9orf72 expansion, characterized by movement disorders including dystonia, chorea, myoclonus, tremor and rigidity, cognitive and memory impairment, early psychosis, and behavioral problems, is also associated with hexanucleotide repeat expansions in the C9orf72 gene.

[0005] Although 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 endocytic trafficking. Additionally, multiple cellular pathways have been shown to be dysregulated in neurodegenerative diseases associated with C9orf72 hexanucleotide repeat expansions. For example, altered RNA processing has consistently emerged at the forefront of C9orf72 disease research. This includes bidirectional transcription of repeat sequences, accumulation of repeat RNAs in nuclear foci that sequester specific RNA-binding proteins (RBPs), and translation of RNA repeats into dipeptide repeat proteins (DPRs) via repeat-associated non-AUG (RAN)-initiated translation. Additionally, disruption of C9orf72 RNA release from RNA polymerase II, cytoplasmic translation, and degradation has been shown to be disrupted by C9orf72 hexanucleotide repeat expansions. Furthermore, several modifications have been identified in the processing of the C9orf72 RNA itself, with respect to its transcription, splicing, and localization (see, eg, Barker, et al., supra).

[0006] Regardless of the mechanism, several groups have identified the presence of sense and antisense C9orf72-containing foci, 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 either sense or antisense repeat-containing C9orf72 RNA in several cell types in 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 showed no disease, whereas mice with both C9orf72 alleles inactivated showed splenomegaly, lymphadenopathy, and mild impairment of social interaction, but no motor dysfunction. Additionally, mice expressing human C9orf72 RNA with up to 450 GGGGCC repeats showed age-, repeat-length-, and expression-level-dependent accumulation of sense- and antisense-RNA-containing foci and dipeptide repeat proteins synthesized by AUG-independent translation, accompanied by hippocampal neuron loss, increased anxiety, and cognitive impairment (Jiang, et al. (2016) Neuron 90:535-550).

[0007] 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., Huntington-like syndromes caused by C9orf72 expansions, Parkinsonism, olivopontocerebellar degeneration, corticobasal syndrome, or Alzheimer's disease; treatments are aimed only at alleviating symptoms and improving the patient's quality of life as the disease progresses.

[0008] Thus, there is a need in the art for agents that can selectively and efficiently inhibit the expression of the C9orf72 gene, e.g., hexanucleotide repeat-containing C9orf72 RNA, for example, for the treatment of subjects with C9orf72-associated disorders. Summary of the Invention

[0009] The present disclosure provides iRNA compositions that induce RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of C9orf72 genes, such as C9orf72 genes with expanded GGGGCC (G4C2) repeats. The C9orf72 RNA transcripts may be present in cells, for example, cells within a subject, such as a human. These iRNAs can be used to target and degrade one or more RNAs of corresponding genes (C9orf72 genes) in mammals.

[0010] The iRNAs of the present invention are designed to target C9orf72 gene transcripts, such as those containing an expanded GGGGCC hexanucleotide repeat in the gene's intron. The agents may target mature C9orf72 mRNA (mRNA from which the intron has been spliced ​​out) or sense or antisense C9orf72 RNA containing the hexanucleotide repeat (e.g., RNA containing C9orf72 intron 1A). The iRNAs described may have one or more nucleotide modifications or a combination of nucleotide modifications that increase the activity, delivery, and / or stability of the iRNA.

[0011] The agent may target the sense strand of mature C9orf72 mRNA (mRNA with introns spliced ​​out) or the sense or antisense strand of C9orf72 RNA containing hexanucleotide repeats (RNA containing C9orf72 intron 1A). In certain embodiments of the present invention, the RNAi agent of the present disclosure may target C9orf72 sense and / or antisense RNA transcripts containing hexanucleotide repeats (RNA containing C9orf72 intron 1A). Targeting C9orf72 sense and / or antisense strand RNAs containing hexanucleotide repeats can inhibit the expression or reduce the presence of 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 either the sense repeat-containing C9orf72 RNA or the antisense repeat-containing C9orf72 RNA in cells of the nervous system of a subject with a C9orf72-related disease. In some embodiments, a combination of an RNA agent targeting a C9orf72 sense strand RNA containing hexanucleotide repeats and an RNA agent targeting a C9orf72 antisense strand RNA containing hexanucleotide repeats is provided together.

[0012] iRNAs of the invention may reduce levels of C9orf72 mature mRNA to a lesser extent than they reduce levels of C9orf72 RNA containing hexanucleotide repeats. For example, iRNAs of the invention may reduce levels of C9orf72 mature mRNA by about 50% or less, or may reduce levels of sense- and antisense-containing C9orf72 RNA foci, reduce levels of one or more aberrant dipeptide repeat (DPR) proteins (poly(GA), poly(GR), poly(GP), poly(PA), and poly(PR)), and / or reduce levels of C9orf72 sense and / or antisense RNA containing hexanucleotide repeats by more than about 50%. Without intending to be bound by theory, it is believed that combinations or subcombinations of the aforementioned features with specific target sites or specific modifications in these iRNAs improve the efficacy, stability, potency, durability, and safety of iRNAs of the invention.

[0013] In one aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for knocking down a C9orf72 target RNA in a cell.

[0014] In one embodiment, the dsRNA agent targets a region of a C9orf72 target RNA that contains a hexanucleotide repeat, for example, multiple consecutive copies of a GGGGCC or CCCCGG hexanucleotide repeat. In some embodiments, the C9orf72 target RNA can be a sense C9orf72 RNA that contains a hexanucleotide repeat, an antisense C9orf72 target RNA that contains a hexanucleotide repeat, or a combination of a sense C9orf72 RNA that contains a hexanucleotide repeat and an antisense C9orf72 target RNA that contains a hexanucleotide repeat.

[0015] In one aspect, the invention provides 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, wherein the sense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO: 13 by no more than 3 nucleotides, e.g., 3, 2, 1, or 0 nucleotides, and the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ from the corresponding portion of the nucleotide sequence of SEQ ID NO: 14 by no more than 3 nucleotides, and wherein the sense strand, the antisense strand, or both the sense and antisense strands comprise at least one modified nucleotide.

[0016] In one aspect, the invention provides 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, wherein the sense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO: 17 by no more than 3 nucleotides, e.g., 3, 2, 1, or 0 nucleotides, and the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ from the corresponding portion of the nucleotide sequence of SEQ ID NO: 18 by no more than 3 nucleotides, and wherein the sense strand, the antisense strand, or both the sense and antisense strands comprise at least one modified nucleotide.

[0017] In one aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of C9orf72, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, wherein the sense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO: 19 by no more than 3 nucleotides, e.g., 3, 2, 1, or 0 nucleotides, and the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ from the corresponding portion of the nucleotide sequence of SEQ ID NO: 20 by no more than 3 nucleotides, and wherein the sense strand, the antisense strand, or both the sense and antisense strands comprise at least one modified nucleotide.

[0018] In one embodiment, an RNAi agent of the present disclosure targets a C9orf72 antisense RNA transcript in the region between the antisense RNA transcription start site and the 5' end of exon 1B. In another embodiment, an RNAi agent of the present disclosure targets a C9orf72 antisense RNA transcript in the region between the antisense RNA transcription start site and the hexanucleotide repeat. In another embodiment, an RNAi agent of the present disclosure targets a C9orf72 antisense RNA transcript in the region between the antisense RNA transcription start site and the 3' end of exon 1A. In another embodiment, an RNAi agent of the present disclosure targets a C9orf72 antisense RNA transcript in the region between the antisense RNA transcription start site and the 5' end of exon 1A. In another embodiment, an RNAi agent of the present disclosure targets a C9orf72 antisense RNA transcript in the region between the antisense RNA transcription start site and 500 bases upstream of the 5' end of exon 1A. In another embodiment, an RNAi agent of the present disclosure targets a C9orf72 antisense RNA transcript in the region between the antisense RNA transcription start site and 1000 bases upstream of the 5' end of exon 1A. In another embodiment, an RNAi agent of the present disclosure targets a C9orf72 antisense RNA transcript in the region between the antisense RNA transcription start site and 1500 bases upstream of the 5' end of exon 1A. In another embodiment, an RNAi agent of the present disclosure targets a C9orf72 antisense RNA transcript in the region between the antisense RNA transcription start site and 2000 bases upstream of the 5' end of exon 1A.

[0019] In another embodiment, an RNAi agent of the present disclosure targets a C9orf72 antisense RNA transcript in the region between the 5' end of exon 1B and the hexanucleotide repeat. In another embodiment, an RNAi agent of the present disclosure targets a C9orf72 antisense RNA transcript in the region between the 5' end of exon 1B and the 3' end of exon 1A. In another embodiment, an RNAi agent of the present disclosure targets a C9orf72 antisense RNA transcript in the region between the 5' end of exon 1B and the 5' end of exon 1A. In another embodiment, an RNAi agent of the present disclosure targets a C9orf72 antisense RNA transcript in the region between the 5' end of exon 1B and 500 bases upstream of the 5' end of exon 1A. In another embodiment, an RNAi agent of the present disclosure targets a C9orf72 antisense RNA transcript in the region between the 5' end of exon 1B and 1000 bases upstream of the 5' end of exon 1A. In another embodiment, an RNAi agent of the present disclosure targets a C9orf72 antisense RNA transcript in the region between the 5' end of exon 1B and 1500 bases upstream of the 5' end of exon 1 A. In another embodiment, an RNAi agent of the present disclosure targets a C9orf72 antisense RNA transcript in the region between the 5' end of exon 1B and 2000 bases upstream of the 5' end of exon 1A.

[0020] In another embodiment, an RNAi agent of the present disclosure targets a C9orf72 antisense RNA transcript in the region between the hexanucleotide repeat and the 3' end of exon 1A. In another embodiment, an RNAi agent of the present disclosure targets a C9orf72 antisense RNA transcript in the region between the hexanucleotide repeat and the 5' end of exon 1A. In another embodiment, an RNAi agent of the present disclosure targets a C9orf72 antisense RNA transcript in the region between the hexanucleotide repeat and 500 bases upstream of the 5' end of exon 1A. In another embodiment, an RNAi agent of the present disclosure targets a C9orf72 antisense RNA transcript in the region between the hexanucleotide repeat and 1000 bases upstream of the 5' end of exon 1A. In another embodiment, an RNAi agent of the present disclosure targets a C9orf72 antisense RNA transcript in the region between the hexanucleotide repeat and 1500 bases upstream of the 5' end of exon 1A. In another embodiment, an RNAi agent of the present disclosure targets the C9orf72 antisense RNA transcript in the region between the hexanucleotide repeat and 2000 bases upstream of the 5' end of exon 1A.

[0021] In one aspect, the invention provides 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 duplex region, wherein the sense strand comprises a nucleotide sequence comprising at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ by no more than 3 nucleotides from an mRNA target sequence in any one of Tables 4A-4G and 7A-7E, and the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ by no more than 3 nucleotides, e.g., 3, 2, 1, or 0 nucleotides, from the complement of the corresponding mRNA target sequence in any one of Tables 4A-4G and 7A-7E.

[0022] In certain embodiments, the sense strand, or the antisense strand, or both the sense and antisense strands are conjugated to one or more lipophilic moieties.

[0023] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a) 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, wherein the sense strand comprises a nucleotide sequence comprising at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ by no more than 3 nucleotides from an mRNA target sequence of any one of Tables 4A-4G, and the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ by no more than 3 nucleotides, e.g., 3, 2, 1, or 0 nucleotides, from the complement of the corresponding mRNA target sequence of any one of Tables 4A-4G; and b) 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, wherein the sense strand comprises a nucleotide sequence comprising at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ by no more than 3 nucleotides from an mRNA target sequence of any one of Tables 7A-7E, and the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ by no more than 3 nucleotides, e.g., 3, 2, 1, or 0 nucleotides, from the complement of the corresponding mRNA target sequence of any one of Tables 7A-7E.

[0024] In certain embodiments, the sense strand, or the antisense strand, or both the sense and antisense strands are conjugated to one or more lipophilic moieties.

[0025] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a) a dsRNA agent for inhibiting expression of a C9orf72 antisense strand transcript, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, the antisense strand comprising a nucleotide sequence comprising at least 15 contiguous nucleotides that differ from the complement of SEQ ID NO: 21 by no more than 3 nucleotides; b) a dsRNA agent for inhibiting expression of a C9orf72 sense strand transcript, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, wherein the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ by 3 or fewer, e.g., 3, 2, 1, or 0, nucleotides from the complement of any of the mRNA target sequences of any one of Tables 7A-7E.

[0026] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a) a dsRNA agent for inhibiting expression of a C9orf72 antisense strand transcript, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, the antisense strand comprising a nucleotide sequence comprising at least 15 contiguous nucleotides that differ from the complement of SEQ ID NO: 22 by no more than 3 nucleotides; b) a dsRNA agent for inhibiting expression of a C9orf72 sense strand transcript, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, wherein the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ by 3 or fewer, e.g., 3, 2, 1, or 0, nucleotides from the complement of any of the mRNA target sequences of any one of Tables 7A-7E.

[0027] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a) a dsRNA agent for inhibiting expression of a C9orf72 antisense strand transcript, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, the antisense strand comprising a nucleotide sequence comprising at least 15 contiguous nucleotides that differ from the complement of SEQ ID NO: 23 by no more than 3 nucleotides; b) a dsRNA agent for inhibiting expression of a C9orf72 sense strand transcript, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, wherein the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ by 3 or fewer, e.g., 3, 2, 1, or 0, nucleotides from the complement of any of the mRNA target sequences of any one of Tables 7A-7E.

[0028] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a) a dsRNA agent for inhibiting expression of a C9orf72 antisense strand transcript, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, the antisense strand comprising a nucleotide sequence comprising at least 15 contiguous nucleotides that differ from the complement of SEQ ID NO: 24 by no more than 3 nucleotides; b) a dsRNA agent for inhibiting expression of a C9orf72 sense strand transcript, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, wherein the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ by 3 or fewer, e.g., 3, 2, 1, or 0, nucleotides from the complement of any of the mRNA target sequences of any one of Tables 7A-7E.

[0029] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a) a dsRNA agent for inhibiting expression of a C9orf72 antisense strand transcript, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, the antisense strand comprising a nucleotide sequence comprising at least 15 contiguous nucleotides that differ from the complement of SEQ ID NO: 25 by no more than 3 nucleotides; b) a dsRNA agent for inhibiting expression of a C9orf72 sense strand transcript, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, wherein the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ by 3 or fewer, e.g., 3, 2, 1, or 0, nucleotides from the complement of any of the mRNA target sequences of any one of Tables 7A-7E.

[0030] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a) a dsRNA agent for inhibiting expression of a C9orf72 antisense strand transcript, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, the antisense strand comprising a nucleotide sequence comprising at least 15 contiguous nucleotides that differ from the complement of SEQ ID NO: 26 by no more than 3 nucleotides; b) a dsRNA agent for inhibiting expression of a C9orf72 sense strand transcript, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, wherein the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ by 3 or fewer, e.g., 3, 2, 1, or 0, nucleotides from the complement of any of the mRNA target sequences of any one of Tables 7A-7E.

[0031] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a) a dsRNA agent for inhibiting expression of a C9orf72 sense strand transcript, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, the antisense strand comprising a nucleotide sequence comprising at least 15 contiguous nucleotides that differ from the complement of SEQ ID NO: 51 by no more than 3 nucleotides; b) a dsRNA agent for inhibiting expression of a C9orf72 antisense strand transcript, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, wherein the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ by 3 or fewer, e.g., 3, 2, 1, or 0, nucleotides from the complement of any of the mRNA target sequences of any one of Tables 4A-4G.

[0032] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a) a dsRNA agent for inhibiting expression of a C9orf72 sense strand transcript, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, the antisense strand comprising a nucleotide sequence comprising at least 15 contiguous nucleotides that differ from the complement of SEQ ID NO: 52 by no more than 3 nucleotides; b) a dsRNA agent for inhibiting expression of a C9orf72 antisense strand transcript, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, wherein the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ by 3 or fewer, e.g., 3, 2, 1, or 0, nucleotides from the complement of any of the mRNA target sequences of any one of Tables 4A-4G.

[0033] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a) a dsRNA agent for inhibiting expression of a C9orf72 sense strand transcript, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, the antisense strand comprising a nucleotide sequence comprising at least 15 contiguous nucleotides that differ from the complement of SEQ ID NO: 53 by no more than 3 nucleotides; b) a dsRNA agent for inhibiting expression of a C9orf72 antisense strand transcript, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, wherein the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ by 3 or fewer, e.g., 3, 2, 1, or 0, nucleotides from the complement of any of the mRNA target sequences of any one of Tables 4A-4G.

[0034] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a) a dsRNA agent for inhibiting expression of a C9orf72 sense strand transcript, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, the antisense strand comprising a nucleotide sequence comprising at least 15 contiguous nucleotides that differ from the complement of SEQ ID NO: 54 by no more than 3 nucleotides; b) a dsRNA agent for inhibiting expression of a C9orf72 antisense strand transcript, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, wherein the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ by 3 or fewer, e.g., 3, 2, 1, or 0, nucleotides from the complement of any of the mRNA target sequences of any one of Tables 4A-4G.

[0035] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a) a dsRNA agent for inhibiting expression of a C9orf72 sense strand transcript, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, the antisense strand comprising a nucleotide sequence comprising at least 15 contiguous nucleotides that differ from the complement of SEQ ID NO: 55 by no more than 3 nucleotides; b) a dsRNA agent for inhibiting expression of a C9orf72 antisense strand transcript, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, wherein the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ by 3 or fewer, e.g., 3, 2, 1, or 0, nucleotides from the complement of any of the mRNA target sequences of any one of Tables 4a-4g.

[0036] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a) a dsRNA agent for inhibiting expression of a C9orf72 sense strand transcript, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, the antisense strand comprising a nucleotide sequence comprising at least 15 contiguous nucleotides that differ from the complement of SEQ ID NO: 56 by no more than 3 nucleotides; b) a dsRNA agent for inhibiting expression of a C9orf72 antisense strand transcript, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, wherein the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ by 3 or fewer, e.g., 3, 2, 1, or 0, nucleotides from the complement of any of the mRNA target sequences of any one of Tables 4A-4G.

[0037] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a) a dsRNA agent for inhibiting expression of a C9orf72 sense strand transcript, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, the antisense strand comprising a nucleotide sequence comprising at least 15 contiguous nucleotides that differ from the complement of SEQ ID NO: 57 by no more than 3 nucleotides; b) a dsRNA agent for inhibiting expression of a C9orf72 antisense strand transcript, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, wherein the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ by 3 or fewer, e.g., 3, 2, 1, or 0, nucleotides from the complement of any of the mRNA target sequences of any one of Tables 4A-4G.

[0038] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a) a dsRNA agent for inhibiting expression of a C9orf72 sense strand transcript, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, the antisense strand comprising a nucleotide sequence comprising at least 15 contiguous nucleotides that differ from the complement of SEQ ID NO: 58 by no more than 3 nucleotides; b) a dsRNA agent for inhibiting expression of a C9orf72 antisense strand transcript, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, wherein the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ by 3 or fewer, e.g., 3, 2, 1, or 0, nucleotides from the complement of any of the mRNA target sequences of any one of Tables 4A-4G.

[0039] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a) a dsRNA agent for inhibiting expression of a C9orf72 sense strand transcript, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, the antisense strand comprising a nucleotide sequence comprising at least 15 contiguous nucleotides that differ from the complement of SEQ ID NO: 59 by no more than 3 nucleotides; b) a dsRNA agent for inhibiting expression of a C9orf72 antisense strand transcript, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, wherein the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ by 3 or fewer, e.g., 3, 2, 1, or 0, nucleotides from the complement of any of the mRNA target sequences of any one of Tables 4A-4G.

[0040] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a) a dsRNA agent for inhibiting expression of a C9orf72 sense strand transcript, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, the antisense strand comprising a nucleotide sequence comprising at least 15 contiguous nucleotides that differ from the complement of SEQ ID NO: 60 by no more than 3 nucleotides; b) a dsRNA agent for inhibiting expression of a C9orf72 antisense strand transcript, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, wherein the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ by 3 or fewer, e.g., 3, 2, 1, or 0, nucleotides from the complement of any of the mRNA target sequences of any one of Tables 4A-4G.

[0041] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a) a dsRNA agent for inhibiting expression of a C9orf72 sense strand transcript, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, the antisense strand comprising a nucleotide sequence comprising at least 15 contiguous nucleotides that differ from the complement of SEQ ID NO: 61 by no more than 3 nucleotides; b) a dsRNA agent for inhibiting expression of a C9orf72 antisense strand transcript, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, wherein the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ by 3 or fewer, e.g., 3, 2, 1, or 0, nucleotides from the complement of any of the mRNA target sequences of any one of Tables 4A-4G.

[0042] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a) a dsRNA agent for inhibiting expression of a C9orf72 sense strand transcript, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, the antisense strand comprising a nucleotide sequence comprising at least 15 contiguous nucleotides that differ from the complement of SEQ ID NO: 62 by no more than 3 nucleotides; b) a dsRNA agent for inhibiting expression of a C9orf72 antisense strand transcript, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, wherein the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ by 3 or fewer, e.g., 3, 2, 1, or 0, nucleotides from the complement of any of the mRNA target sequences of any one of Tables 4A-4G.

[0043] In one aspect, the invention provides a combination of a first dsRNA agent that targets a C9orf72 antisense RNA transcript and a second dsRNA agent that targets a C9orf72 sense strand transcript, a) a first dsRNA agent comprises an antisense strand comprising a nucleotide sequence comprising at least 15 contiguous nucleotides that differ from the antisense sequence of AD-1446213 by no more than 3 nucleotides, and a second dsRNA agent comprises an antisense strand comprising a nucleotide sequence comprising at least 15 contiguous nucleotides that differ from the antisense sequence of AD-1285238 by no more than 3 nucleotides; b) a first dsRNA agent comprises an antisense strand comprising a nucleotide sequence comprising at least 15 contiguous nucleotides that differ from the antisense sequence of AD-1446213 by no more than 3 nucleotides, and a second dsRNA agent comprises an antisense strand comprising a nucleotide sequence comprising at least 15 contiguous nucleotides that differ from the antisense sequence of AD-1285234 by no more than 3 nucleotides; c) a first dsRNA agent comprises an antisense strand comprising a nucleotide sequence comprising at least 15 contiguous nucleotides that differ from the antisense sequence of AD-1446246 by no more than 3 nucleotides, and a second dsRNA agent comprises an antisense strand comprising a nucleotide sequence comprising at least 15 contiguous nucleotides that differ from the antisense sequence of AD-1285238 by no more than 3 nucleotides; d) a first dsRNA agent comprises an antisense strand comprising a nucleotide sequence comprising at least 15 contiguous nucleotides that differ from the antisense sequence of AD-1446246 by no more than 3 nucleotides, and a second dsRNA agent comprises an antisense strand comprising a nucleotide sequence comprising at least 15 contiguous nucleotides that differ from the antisense sequence of AD-1285234 by no more than 3 nucleotides; e) a first dsRNA agent comprises an antisense strand comprising a nucleotide sequence comprising at least 15 contiguous nucleotides that differ from the antisense sequence of AD-1446268 by no more than 3 nucleotides, and a second dsRNA agent comprises an antisense strand comprising a nucleotide sequence comprising at least 15 contiguous nucleotides that differ from the antisense sequence of AD-1285238 by no more than 3 nucleotides; f) a first dsRNA agent comprises an antisense strand comprising a nucleotide sequence comprising at least 15 contiguous nucleotides that differ from the antisense sequence of AD-1446268 by no more than 3 nucleotides, and a second dsRNA agent comprises an antisense strand comprising a nucleotide sequence comprising at least 15 contiguous nucleotides that differ from the antisense sequence of AD-1285234 by no more than 3 nucleotides.

[0044] In another aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of C9orf72, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, wherein the sense strand or the antisense strand is selected from the group consisting of any of the sense strands and antisense strands in any one of Tables 2, 3, 10A, 10C, 11, and 12, and wherein the sense strand, the antisense strand, or both the sense strand and the antisense strand comprise at least one modified nucleotide.

[0045] In another aspect, the invention provides 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, wherein the sense strand is selected from nucleotides 27573296 to 27573318, 27573314 to 27573336, 27573319 to 27573341, 27573562 to 27573584, 27573585 to 27573607, 27573592 to 27573614, 27573599 to 27573 73621, 27573608 to 27573630, 27573616 to 27573638, 27573619 to 27573641, 27573622 to 27573644, 27573633 to 27573655, 27573690 to 27573712, or 27573717 to 27573739, and the sense strand, the antisense strand, or both the sense strand and the antisense strand contain at least one modified nucleotide.

[0046] In one embodiment, the sense or antisense strand is a sense or antisense strand selected from the sense or antisense strand of a duplex selected from the group consisting of AD-1446213.1, AD-1446217.1, AD-1446222.1, AD-1446234.1, AD-1446243.1, AD-1446246.1, AD-1446252.1, AD-1446259.1, AD-1446265.1, AD-1446268.1, AD-1446271.1, AD-1446279.1, AD-1446289.1, and AD-1446294.1.

[0047] In one embodiment, the sense or antisense strand is a sense or antisense strand selected from the sense or antisense strand of a duplex selected from the group consisting of AD-1446213.1, AD-1446246.1, and AD-1446268.1.

[0048] In one aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of C9orf72, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, wherein the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ by 3 or fewer, e.g., 3, 2, 1, or 0 nucleotides, from any one of the antisense nucleotide sequences in any one of Tables 5, 6, 10B, and 10D, and wherein the sense strand, the antisense strand, or both the sense and antisense strands comprise at least one modified nucleotide.

[0049] In another aspect, the invention provides 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, wherein the sense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides that differ by no more than 3 nucleotides, e.g., 3, 2, 1, or 0 nucleotides, from any one of the nucleotide sequences of nucleotides 1-23, 15-37, 33-55, 37-59, 59-81, 62-84, or 69-91 of SEQ ID NO:1; and the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides from the corresponding nucleotide sequence of SEQ ID NO:5; and wherein the sense strand, the antisense strand, or both the sense and antisense strands comprise at least one modified nucleotide.

[0050] In one embodiment, the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ by 3 or fewer nucleotides, e.g., 3, 2, 1, or 0 nucleotides, from any one of the double-stranded antisense strand nucleotide sequences selected from the group consisting of AD-1446073.1, AD-1446075.1, AD-1285246.2, AD-1446084.1, AD-1446087.1, AD-1446090.1, and AD-1446095.1.

[0051] In one aspect, the invention provides 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 nucleotides 5197-5219, 5213-5235, 5223-5245, 5226-5248, 5227-5239, 5230-5249, 5231-5232, 5233-5234, 5235-5236, 5237-5238, 5239-5241, 5239-5242, 5243-5244, 5245-5246, 5247-5248, 5249-5250, 5251-5252 5249, 5228~5250, 5229~5251, 5230~5252, 5231~5253, 5233~5255, 5235~5256, 5241~5263, 5245~5267, 5233~5255, 5248~5270, 5539~5561, 5547~5569, 5917~5939, 5936~5958, 5954~ and the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides that differ by 3 or fewer nucleotides, e.g., 3, 2, 1, or 0 nucleotides, from any one of the nucleotide sequences of SEQ ID NO: 16, 5976, 6008-6030, 6021-6043, 6036-6058, 6043-6065, or 6048-6070; the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides from the corresponding nucleotide sequence of SEQ ID NO: 16; and the sense strand, the antisense strand, or both the sense strand and the antisense strand comprise at least one modified nucleotide.

[0052] In one embodiment, the antisense strand is selected from the group consisting of AD-1285231.1, AD-1285232.1, AD-1285233.1, AD-1285235.1, AD-1285237.1, AD-1285239.1, AD-1285240.1, AD-1285242.1, AD-1285244.1, AD-1285238.1, AD-1285243.1, AD-1285234.1, AD-1285241.1, AD-1285236.1, AD-1446111.1, AD-1446117.1, AD-1 and AD-1446205.1, AD-1446206.1, AD-1446207.1, AD-1446208.1, AD-1446209.1, AD-1446210.1, AD-1446211.1, AD-1446212.1, AD-1446213.1, AD-1446214.1, AD-1446215.1, AD-1446216.1, AD-1446217.1, AD-1446218.1, AD-14462196.1, AD-1446202.1, and AD-1446205.1, and which differ by 3 or less nucleotides, for example, 3, 2, 1, or 0 nucleotides, from any one of the double-stranded antisense strand nucleotide sequences selected from the group consisting of AD-1446206.1, AD-14462157.1, AD-1446168.1, AD-1446180.1, AD-1446189.1, AD-1446196.1, AD-1446202.1, and AD-1446205.1.

[0053] In one embodiment, the antisense strand is selected from the group consisting of AD-1285231.1, AD-1285232.1, AD-1285233.1, AD-1285234.1, AD-1285235.1, AD-1285236.1, AD-1285237.1, AD-1285239.1, AD-1285240.1, AD-1285241.1, AD-1285242.1, AD-1285243.1, AD-1285244.1, AD-1285245.1, AD-1285246.1, AD-1285247.1, AD-1285248.1, AD-1285249.1, AD-1285250.1, AD-1285251.1, AD-1285252.1, AD-1285253.1, AD-1285254.1, AD-1285255.1, AD-1285256.1, AD-1285257.1, AD-1285258.1, AD-1285259.1, AD-1285260.1, AD-1285261.1, AD-1285262.1, AD-1285263.1, AD-1285264.1, AD-1285265.1, AD-1285266.1, AD-1285267.1, AD-1285268.1, AD-1285269.1, AD-1285270.1, AD-1285271.1, AD-1285272.1, AD-12 and AD-1446090.1, and the antisense strand nucleotide sequence of the double-stranded ...

[0054] In one embodiment, the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides that differ by 3 or fewer nucleotides, e.g., 3, 2, 1, or 0 nucleotides, from any one of the double-stranded antisense strand nucleotide sequences selected from the group consisting of AD-1285238.1 and AD-1285234.1.

[0055] In one aspect, the invention provides 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 being nucleotides 5015-5052, 5017-5040, 5032-5059, 5032-5055, 5033-5055, 5035-5059, 5036-5059, 5058-5087, 5059-5087, 5059-5088 of SEQ ID NO: 15. 4, 5064~5087, 5197~5222, 5213~5267, 5223~5252, 5229~5252, 5233~5263, 5516~5570, 5539~5565, 5539~5562, 5545~5570, 5545~5569, 5593~5616, 5883~5950, 5917~5950, 5919~5950, 5923~5950, 5934~5977, 5934~5957, 5938~5977, 5938~5965, Any one of the nucleotide sequences of 5938 to 5961, 5947 to 5977, 5947 to 5973, 5972 to 6001, 5973 to 5997, 6006 to 6029, 6011 to 6070, 6011 to 6039, 6011 to 6038, 6015 to 6038, 6019 to 6045, 6019 to 6042, 6033 to 6070, 6035 to 6065, 6035 to 6059, and 6040 to 6063 has 3 or less nucleotides, for example, 3, 2, The antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides, which differ by 1 or 0, and the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides from the corresponding nucleotide sequence of SEQ ID NO: 16, and the sense strand, the antisense strand, or both the sense strand and the antisense strand comprise at least one modified nucleotide.

[0056] In one aspect, the invention provides 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, wherein the sense strand is 3 nucleotides or less, e.g., 3, 4, 5, 6, 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, 40, 41, 42, 43, 44, 45, 46, 47, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 11 The nucleotide sequence of the sense strand, the antisense strand, or both the sense strand and the antisense strand comprise at least one modified nucleotide.

[0057] In one aspect, the invention provides 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 being located at nucleotides 27573296-27573584, 27573296-27573575, 27573301-27573338, and 27573318-27573 of SEQ ID NO: 13. 342, 27573555~27573583, 27573581~27573607, 27573584~27573607, 27573588~27573671, 27573588~27573666, 27573588~27573624, 27573592~27573624, 27573592~27573617, 27573598~27573624, 2757359 and the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides that differ by 3 or fewer nucleotides, e.g., 3, 2, 1, or 0 nucleotides, from any one of the nucleotide sequences of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides from the corresponding nucleotide sequence of SEQ ID NO: 14, and the sense strand, the antisense strand, or both the sense strand and the antisense strand comprise at least one modified nucleotide.

[0058] In one aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of C9orf72, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, wherein the sense strand or the antisense strand is selected from the group consisting of any of the sense strands and antisense strands in any one of Tables 8 and 9, and wherein the sense strand, the antisense strand, or both the sense strand and the antisense strand comprise at least one modified nucleotide.

[0059] In one embodiment, the sense strand, the antisense strand, or both the sense and antisense strands are conjugated to one or more lipophilic moieties.

[0060] In one embodiment, the lipophilic moiety is conjugated to one or more internal positions within the double-stranded region of the dsRNA agent.

[0061] In one embodiment, the lipophilic moiety is conjugated via a linker or carrier.

[0062] In one embodiment, the lipophilicity of the lipophilic moiety is greater than 0, as measured by log Kow.

[0063] 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.

[0064] In one embodiment, the plasma protein binding assay is an electrophoretic mobility shift assay using human serum albumin protein.

[0065] In some embodiments, the dsRNA agent includes at least one modified nucleotide.

[0066] In one embodiment, no more than 5 of the nucleotides in the sense strand and no more than 5 of the nucleotides in the antisense strand are unmodified nucleotides.

[0067] In one embodiment, all of the nucleotides in the sense strand are modified nucleotides. In one embodiment, all of the nucleotides in the antisense strand are modified nucleotides. In one embodiment, all of the nucleotides in the sense strand and all of the nucleotides in the antisense strand are modified nucleotides.

[0068] 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 2'-O-hexadecyl nucleotide, a 2'-phosphate nucleotide, a 2'-5' linked ribonucleotide (3'-RNA), a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, an inverted abasic residue, a 2'-amino modified nucleotide, a 2'-O-allyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2'-hydroxy modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a 2',3'-seco modified nucleotide, a morpholino nucleotide, a phosphoramidate, or a non-natural base. nucleotides containing 5'-phosphate, tetrahydropyran-modified nucleotides, 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, glycol-modified nucleic acids (GNAs), nucleotides containing glycol nucleic acids (GNAs), nucleotides containing glycol nucleic acid S-isomers (S-GNAs), 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.

[0069] 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 deoxy-thymine nucleotides (dT), 2'-O-hexadecyl modified nucleotides, 2'-phosphate modified nucleotides, glycol modified nucleotides, locked nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, and nucleotides containing unnatural bases.

[0070] In one embodiment, the modified nucleotides include a short sequence of 3' terminal deoxy-thymine nucleotides (dT).

[0071] In one embodiment, the modified nucleotides are independently selected from the group consisting of 2'-O-methyl modified nucleotides, GNA modified nucleotides, and 2' fluoro modified nucleotides, 2'-phosphate modified nucleotides, 2'-O-hexadecyl modified nucleotides, and 2'-phosphate modified nucleotides.

[0072] In one embodiment, substantially all of the modified nucleotides in the sense strand are selected from the group consisting of 2'-O-methyl and 2'-fluoro modified nucleotides, hi some embodiments, substantially all of the modified nucleotides in the sense strand are selected from the group consisting of 2'-O-methyl and 2'-fluoro modified nucleotides.

[0073] In one embodiment, substantially all of the modified nucleotides in the antisense strand are selected from the group consisting of 2'-O-methyl modified nucleotides, 2'-phosphate modified nucleotides, glycol nucleic acid modified nucleotides, and 2'-fluoro modified nucleotides. In some embodiments, substantially all of the modified nucleotides in the antisense strand are selected from the group consisting of 2'-O-methyl modified nucleotides, 2'-phosphate modified nucleotides, glycol nucleic acid modified nucleotides, and 2'-fluoro modified nucleotides.

[0074] In one embodiment, substantially all of the modified nucleotides in the sense strand are selected from the group consisting of 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-O-hexadecyl modified nucleotides, and glycol nucleic acid (GNA) modified nucleotides. In some embodiments, substantially all of the modified nucleotides in the sense strand are selected from the group consisting of 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, '-O-hexadecyl modified nucleotides, and glycol nucleic acid (GNA) modified nucleotides.

[0075] In one embodiment, substantially all of the modified nucleotides in the antisense strand are selected from the group consisting of 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-phosphate modified nucleotides, and glycol nucleic acid (GNA) modified nucleotides. In some embodiments, all of the modified nucleotides in the antisense strand are selected from the group consisting of 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-phosphate modified nucleotides, and glycol nucleic acid (GNA) modified nucleotides.

[0076] In some embodiments, the dsRNA agent includes at least one phosphorothioate internucleotide linkage.

[0077] In one embodiment, the dsRNA agent includes 6 to 8 phosphorothioate internucleotide linkages.

[0078] 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.

[0079] In one embodiment, the sense strand comprises at least two phosphorothioate or methylphosphonate internucleotide linkages.

[0080] In one embodiment, the antisense strand comprises at least two, at least three, or at least four phosphorothioate or methylphosphonate internucleotide linkages.

[0081] In one embodiment, at least one phosphorothioate or methylphosphonate internucleotide linkage is at the 5'-terminus of one strand, the 3'-terminus of one strand, or both the 5'-terminus and the 3'-terminus of one strand.

[0082] 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. In some embodiments, the sense strand comprises one phosphorothioate internucleotide linkage at the 5'-end and one phosphorothioate internucleotide linkage at the 3'-end. In some embodiments, the sense strand comprises two phosphorothioate internucleotide linkages at the 5'-end and two phosphorothioate internucleotide linkages at the 3'-end.

[0083] In one embodiment, at least one phosphorothioate or methylphosphonate internucleotide linkage is present at both the 5'-end and the 3'-end 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. In some embodiments, the antisense strand comprises two phosphorothioate internucleotide linkages at the 5'-end and one phosphorothioate internucleotide linkage at the 3'-end. In some embodiments, the antisense strand comprises three phosphorothioate internucleotide linkages at the 5'-end and one phosphorothioate internucleotide linkage at the 3'-end. In some embodiments, the antisense strand comprises three phosphorothioate internucleotide linkages at the 5'-end and two phosphorothioate internucleotide linkages at the 3'-end.

[0084] In one embodiment, all of the modified nucleotides in the sense strand are selected from the group consisting of 2'-O-methyl modified nucleotides, 2'-O-hexadecyl modified nucleotides, and 2'-fluoro modified nucleotides; all of the modified nucleotides in the antisense strand are selected from the group consisting of 2'-O-methyl modified nucleotides, 2'-phosphate modified nucleotides, glycol nucleic acid modified nucleotides, and 2'-fluoro modified nucleotides; the sense strand comprises two phosphorothioate internucleotide linkages at the 5' end; and the antisense strand comprises two phosphorothioate internucleotide linkages at the 5' end and two phosphorothioate internucleotide linkages or vinyl-phosphonate at the 3' end.

[0085] 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.

[0086] 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.

[0087] The double-stranded region may be 15-30 nucleotide pairs in length, 17-23 nucleotide pairs in length, 17-25 nucleotide pairs in length, 23-27 nucleotide pairs in length, 19-21 nucleotide pairs in length, 21-23 nucleotide pairs in length, or 17, 18, 19, 20, 21, 22, or 23 nucleotide pairs in length. In some embodiments, the double-stranded region is 20 nucleotides in length. In some embodiments, the double-stranded region is 21 nucleotides in length. The double-stranded region may have 0, 1, 2, or 3 mismatches.

[0088] The sense strand and the antisense strand may each independently be 17-30 nucleotides, 17-25 nucleotides, 19-30 nucleotides, 19-25 nucleotides, 19-23 nucleotides, or 21-23 nucleotides in length, or 19, 20, 21, 22, or 23 nucleotides in length. In some embodiments, the sense strand is 20 nucleotides in length. In some embodiments, the antisense strand is 22 nucleotides in length. In some embodiments, the sense strand is 23 nucleotides in length. In some embodiments, the antisense strand is 21 nucleotides in length. In some embodiments, the sense strand is 23 nucleotides in length and the antisense strand is 21 nucleotides in length. In some embodiments, the sense strand is 23 nucleotides in length and comprises inverted abasic residues at the 3' and 5' terminal nucleotide positions.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] In some embodiments of the compositions and methods of the present invention, the RNAi agent further comprises one or more lipophilic moieties. As described herein, lipophilic moiety-conjugated RNAi agents are advantageous for in vivo delivery of nucleic acids and are also suitable compositions for in vivo therapeutic use. In one embodiment, one or more lipophilic moieties are conjugated to one or more internal positions on at least one strand. The lipophilic moieties can be conjugated to internal positions via a linker or carrier. In some embodiments, the lipophilic moiety facilitates or improves delivery of the RNAi agent to nerve cells or cells within nerve tissue.

[0093] In one embodiment, internal positions can include all positions except the two terminal positions from each end of at least one strand.

[0094] In another embodiment, internal positions can include all but the terminal three positions from each end of at least one strand.

[0095] In one embodiment, the internal position excludes the cleavage site region of the sense strand.

[0096] In one embodiment, internal positions can include all positions except positions 9 to 12 counting from the 5' end of the sense strand.

[0097] In another embodiment, internal positions can include all positions except positions 11-13 counting from the 3' end of the sense strand.

[0098] In one embodiment, the internal position excludes the cleavage site region of the antisense strand.

[0099] In one embodiment, internal positions can include all positions except positions 12 to 14 counting from the 5' end of the antisense strand.

[0100] In one embodiment, internal positions can be any position except positions 11-13 on the sense strand counting from the 3' end and positions 12-14 on the antisense strand counting from the 5' end.

[0101] 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 on the sense strand and positions 6-10 and 15-18 on the antisense strand, counting from the 5' end of each strand.

[0102] 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 on the sense strand and positions 15 and 17 on the antisense strand, counting from the 5' end of each strand.

[0103] In one embodiment, the internal position in the double-stranded region excludes the cleavage site region of the sense strand.

[0104] In one embodiment, the sense strand is 21 nucleotides in length, the antisense strand is 23 nucleotides in length, and the lipophilic moiety is conjugated to position 21, 20, 15, 1, 7, 6, or 2 of the sense strand or position 16 of the antisense strand, counting from the 5' end.

[0105] In one embodiment, the lipophilic moiety is conjugated to position 21, 20, 15, 1, or 7 of the sense strand.

[0106] In another embodiment, the lipophilic moiety is conjugated to position 21, 20, or 15 of the sense strand, counting from the 5' end.

[0107] In yet another embodiment, the lipophilic moiety is conjugated to position 20 or 15 of the sense strand, counting from the 5' end.

[0108] In one embodiment, the lipophilic moiety is conjugated to position 16 of the antisense strand, counting from the 5' end.

[0109] In one embodiment, the lipophilic moiety is an aliphatic compound, an alicyclic compound, or a polycyclic alicyclic compound.

[0110] 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 groups, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenoic acid, dimethoxytrityl, or phenoxazine.

[0111] In one embodiment, the lipophilic moiety comprises 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, alkenyl, and alkynyl. In one embodiment, the lipophilic moiety comprises a C6-C30 alkyl, a C6-C30 alkenyl, or a C6-C30 alkynyl.

[0112] In one embodiment, the lipophilic moiety comprises a saturated or unsaturated C6-C18 hydrocarbon chain. In one embodiment, the lipophilic moiety comprises a saturated or unsaturated C6, C7, C8, C9, C10, C11, C12, C13, C15, C15, C16, C17, or C18 hydrocarbon chain. The unsaturated C6-C18 can be monounsaturated or polyunsaturated C6-C18.

[0113] In one embodiment, the lipophilic moiety comprises a saturated or unsaturated C16 hydrocarbon chain. In one embodiment, the lipophilic moiety comprises a C16 alkyl, C16 alkenyl, or C16 alkynyl. The unsaturated C16 can be monounsaturated or polyunsaturated.

[0114] In one embodiment, the saturated or unsaturated C16 hydrocarbon chain is conjugated to the 6 position counting from the 5' end of the chain.

[0115] In one embodiment, the lipophilic moiety is conjugated via a carrier that substitutes for one or more nucleotides at an internal position or within the double-stranded region.

[0116] In one embodiment, the carrier is a cyclic group selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl, or is an acyclic portion of a serinol or diethanolamine backbone system.

[0117] In one embodiment, the lipophilic moiety is conjugated to the double-stranded iRNA agent via a linker that includes an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide linkage, the product of a click reaction, or a carbamate.

[0118] In one embodiment, the lipophilic moiety is conjugated to a nucleobase, a sugar moiety, or an internucleoside linkage.

[0119] In one embodiment, the lipophilic moiety or targeting ligand is conjugated via a biochemically cleavable linker selected from the group consisting of DNA, RNA, disulfides, amides, functionalized mono- or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.

[0120] 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.

[0121] In one embodiment, the dsRNA agent further comprises a targeting ligand that targets a neuronal cell, a cell in neuronal tissue, or a cell in central nervous system tissue.

[0122] In one embodiment, the dsRNA agent further comprises a targeting ligand that targets liver tissue.

[0123] In one embodiment, the targeting ligand is a GalNAc conjugate.

[0124] In one embodiment, the dsRNA agent further comprises a terminal chiral modification occurring at the first internucleotide linkage at the 3'-end of the antisense strand, where the linking phosphorus atom is in the Sp configuration; a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the antisense strand, where the linking phosphorus atom is in the Rp configuration; and a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the sense strand, where the linking phosphorus atom is in either the Rp or Sp configuration.

[0125] 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, where the linking phosphorus atom is in the Sp configuration; a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the antisense strand, where the linking phosphorus atom is in the Rp configuration; and a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the sense strand, where the linking phosphorus atom is in either the Rp or Sp configuration.

[0126] In yet another embodiment, the dsRNA agent further comprises a terminal chiral modification occurring at the first, second, and third internucleotide linkages at the 3'-end of the antisense strand, where the linking phosphorus atom is in the Sp configuration; a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the antisense strand, where the linking phosphorus atom is in the Rp configuration; and a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the sense strand, where the linking phosphorus atom is in either the Rp or Sp configuration.

[0127] 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, where the linking phosphorus atom is in the Sp configuration; a terminal chiral modification occurring at the third internucleotide linkage at the 3'-end of the antisense strand, where the linking phosphorus atom is in the Rp configuration; a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the antisense strand, where the linking phosphorus atom is in the Rp configuration; and a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the sense strand, where the linking phosphorus atom is in either the Rp or Sp configuration.

[0128] 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, where the linking phosphorus atom is in the Sp configuration; a terminal chiral modification occurring at the first and second internucleotide linkages at the 5'-end of the antisense strand, where the linking phosphorus atom is in the Rp configuration; and a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the sense strand, where the linking phosphorus atom is in either the Rp or Sp configuration.

[0129] In one embodiment, the dsRNA agent further comprises a phosphate or a phosphate mimetic at the 5'-end of the antisense strand.

[0130] In one embodiment, the phosphate mimetic is a 5'-vinylphosphonate (VP).

[0131] 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.

[0132] In one embodiment, the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

[0133] 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 a C9orf72 target RNA that includes a hexanucleotide repeat.

[0134] 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.

[0135] 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.

[0136] In one embodiment, the dsRNA agent reduces (poly(GA), poly(GR), poly(GP), poly(PA), and poly(PR) dipeptide repeat protein synthesis within 24 to 48 hours after administration to a cell expressing a C9orf72 target RNA that includes 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 to 48 hours after administration to the cell.

[0137] The present invention also provides cells and pharmaceutical compositions for inhibiting expression of the gene encoding C9orf72, including dsRNA agents of the invention.

[0138] In one embodiment, the dsRNA agent is in an unbuffered solution, such as saline or water.

[0139] In another embodiment, the dsRNA agent is in a buffer such as acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof, or phosphate buffered saline (PBS).

[0140] The invention further provides compositions comprising two or more, eg, two, three, or four, dsRNA agents for inhibiting expression of C9orf72.

[0141] In one embodiment, a composition includes a first dsRNA agent that targets the sense strand of C9orf72 (an exon or intron of C9orf72) and a second dsRNA agent that targets the antisense strand of C9orf72 (an exon or intron of C9orf72).

[0142] In some embodiments, suitable agents targeting the sense strand of C9orf72 for use in compositions of the invention comprising two or more dsRNA agents include a sense strand and an antisense strand that form a double-stranded region, a) a sense strand comprising at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 1 by no more than 3 nucleotides, and an antisense strand comprising a nucleotide sequence comprising at least 15 consecutive nucleotides that differ from the corresponding portion of the nucleotide sequence of SEQ ID NO: 5 by no more than 3 nucleotides; b) a sense strand comprising at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 15 by no more than 3 nucleotides, and an antisense strand comprising a nucleotide sequence comprising at least 15 consecutive nucleotides that differ from the corresponding portion of the nucleotide sequence of SEQ ID NO: 16 by no more than 3 nucleotides; c) an antisense strand comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the antisense nucleotide sequences in any one of Tables 5, 6, 10B, and 10D, wherein the sense strand, the antisense strand, or both the sense and antisense strands are conjugated to one or more lipophilic moieties; d) a sense strand comprising at least 15 consecutive nucleotides that differ by no more than three nucleotides from any one of the nucleotide sequences of nucleotides 1 to 23, 15 to 37, 33 to 55, 37 to 59, 59 to 81, 62 to 84, or 69 to 91 of SEQ ID NO: 1, and an antisense strand comprising at least 15 consecutive nucleotides from the corresponding nucleotide sequence of SEQ ID NO: 5; e) nucleotides 5197 to 5219, 5213 to 5235, 5223 to 5245, 5226 to 5248, 5227 to 5249, 5228 to 5250, 5229 to 5251, 5230 to 5252, 5231 to 5253, 5233 to 5255, 5235 to 5256, 5241 to 5263, 5245 to 5267, 5233 to 5255, 5248 to 5270, 5539 to 5561, 5547 to 5569, 5917 to 5950, 5951 to 5952, 5953 to 5954, 5955 to 5956, 5957 to 5958, 5959 to 5960, 5961 to 5962, 5963 to 5964, 5965 to 5966, 5967 to 5970, 5971 to 5972, 5973 to 5974, 5975 to 5976, 5976 to 5978, 5979 to 5980, 5981 to 5982, 5983 to 5984, 5985 to 5986, 5987 to 5988, 5989 to 5990, 5991 to 5992, 5993 to 5994, 5995 to 5996, 5996 to 5998, 5997 to 5999, 5 a sense strand comprising at least 15 consecutive nucleotides that differ by no more than three nucleotides from any one of the nucleotide sequences of SEQ ID NO: 16, 939, 5936-5958, 5954-5976, 6008-6030, 6021-6043, 6036-6058, 6043-6065, or 6048-6070, and an antisense strand comprising at least 15 consecutive nucleotides from the corresponding nucleotide sequence of SEQ ID NO: 16; f) nucleotides 5015 to 5052, 5017 to 5040, 5032 to 5059, 5032 to 5055, 5033 to 5055, 5035 to 5059, 5036 to 5059, 5058 to 5087, 5059 to 5087, 5059 to 5084, 5064 to 5087, 5197 to 5222, 5213 to 5267, 522 3~5252, 5229~5252, 5233~5263, 5516~5570, 5539~5565, 5539~5562, 5545~5570, 5545~5569, 5593~5616, 5883~5950, 5917~5950, 5919~5950, 5923~5950, 5934~5977, 5934~5957, 5 a sense strand comprising at least 15 consecutive nucleotides that differ by no more than three nucleotides from any one of the nucleotide sequences of SEQ ID NO: 938 to 5977, 5938 to 5965, 5938 to 5961, 5947 to 5977, 5947 to 5973, 5972 to 6001, 5973 to 5997, 6006 to 6029, 6011 to 6070, 6011 to 6039, 6011 to 6038, 6015 to 6038, 6019 to 6045, 6019 to 6042, 6033 to 6070, 6035 to 6065, 6035 to 6059, or 6040 to 6063, and an antisense strand comprising at least 15 consecutive nucleotides from the corresponding nucleotide sequence of SEQ ID NO: 16; g) a sense strand comprising at least 15 consecutive nucleotides that differ by no more than three nucleotides from any one of the nucleotide sequences of nucleotides 15-52, 17-40, 32-59, 32-55, 35-59, 36-59, 58-87, 59-87, 59-84, or 64-87 of SEQ ID NO: 1, and an antisense strand comprising at least 15 consecutive nucleotides from the corresponding nucleotide sequence of SEQ ID NO: 5; and h) an antisense strand comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the antisense nucleotide sequences in any one of Tables 8 and 9; The sense strand, the antisense strand, or both the sense and antisense strands comprise at least one modified nucleotide.

[0143] In certain embodiments, suitable agents targeting the sense strand of C9orf72, e.g., a C9orf72 exon or intron sense sequence, for use in compositions of the invention comprising two or more dsRNA agents, such as the dsRNA agents disclosed in PCT Publication No. WO2021 / 119226, the entire contents of which are incorporated herein by reference.

[0144] In certain embodiments, a suitable agent targeting the antisense strand of C9orf72 for use in a composition of the invention comprising two or more dsRNA agents comprises a sense strand and an antisense strand that form a double-stranded region, a) a sense strand comprising at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 13 by no more than 3 nucleotides, and an antisense strand comprising a nucleotide sequence comprising at least 15 consecutive nucleotides that differ from the corresponding portion of the nucleotide sequence of SEQ ID NO: 14 by no more than 3 nucleotides; b) a sense strand comprising at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 17 by no more than 3 nucleotides, and an antisense strand comprising a nucleotide sequence comprising at least 15 consecutive nucleotides that differ from the corresponding portion of the nucleotide sequence of SEQ ID NO: 18 by no more than 3 nucleotides; c) a sense strand comprising at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 19 by no more than 3 nucleotides, and an antisense strand comprising a nucleotide sequence comprising at least 15 consecutive nucleotides that differ from the corresponding portion of the nucleotide sequence of SEQ ID NO: 20 by no more than 3 nucleotides; d) an antisense comprising a nucleotide sequence selected from the group consisting of any of the antisense strand nucleotide sequences in any one of Tables 2, 3, 10A, 10C, 11, and 12; e) Nucleotides 27573296 to 27573318, 27573314 to 27573336, 27573319 to 27573341, 27573562 to 27573584, 27573585 to 27573607, 27573592 to 27573614, 27573599 to 27573621, 27573608 to 2757363 of SEQ ID NO: 13 a sense strand comprising at least 15 consecutive nucleotides that differ from 0, 27573616 to 27573638, 27573619 to 27573641, 27573622 to 27573644, 27573633 to 27573655, 27573690 to 27573712, or 27573717 to 27573739 by no more than 3 nucleotides; and f) nucleotides 27573296 to 27573584, 27573296 to 27573575, 27573301 to 27573338, 27573318 to 27573342, 27573555 to 27573583, 27573581 to 27573607, 27573584 to 27573607, 27573588 to 27573671, 27573588 to 27573666, 27573588 to 27573624, 27573592 to 27573624, 27573592 to 27573617, 27573598 to 275 a sense strand comprising at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from any one of the nucleotide sequences of SEQ ID NO: 14, 27573599 to 27573623, 27573606 to 27573655, 27573606 to 27573652, 27573606 to 27573647, 27573654 to 27573712, or 27573707 to 27573740, and an antisense strand comprising at least 15 consecutive nucleotides from the corresponding nucleotide sequence of SEQ ID NO: 14, The sense strand, the antisense strand, or both the sense and antisense strands comprise at least one modified nucleotide.

[0145] In one aspect, the invention provides a composition comprising two or more double-stranded ribonucleic acid (dsRNA) agents for inhibiting expression of C9orf72, each dsRNA agent independently comprises a sense strand and an antisense strand that form a double-stranded region; The first dsRNA agent targets the antisense strand of C9orf72: a) a dsRNA agent comprising: a sense strand comprising at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 13 by no more than 3 nucleotides; and an antisense strand comprising a nucleotide sequence comprising at least 15 consecutive nucleotides that differ from the corresponding portion of the nucleotide sequence of SEQ ID NO: 14 by no more than 3 nucleotides; b) a sense strand comprising at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 17 by no more than 3 nucleotides, and an antisense strand comprising a nucleotide sequence comprising at least 15 consecutive nucleotides that differ from the corresponding portion of the nucleotide sequence of SEQ ID NO: 18 by no more than 3 nucleotides; c) a sense strand comprising at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 19 by no more than 3 nucleotides, and an antisense strand comprising a nucleotide sequence comprising at least 15 consecutive nucleotides that differ from the corresponding portion of the nucleotide sequence of SEQ ID NO: 20 by no more than 3 nucleotides; d) a dsRNA agent comprising an antisense strand that comprises a nucleotide sequence selected from the group consisting of any of the antisense strand nucleotide sequences in any one of Tables 2, 3, 10A, 10C, 11, and 12; e) nucleotides 27573296 to 27573318, 27573314 to 27573336, 27573319 to 27573341, 27573562 to 27573584, 27573585 to 27573607, 27573592 to 27573614, 27573599 to 27573621, 27573608 to 27573630, 27573599 to 27573621, 27573630, 27573599 to 27573630, 27573599 to 27573641, 27573599 to 27573641, 27573599 to 27573650, 27573599 to 27573661, 27573599 to 27573671, 27573599 to 27573681, 27573599 to 27573691, 27573599 to 27573610, 27573599 to 27573621, 27573599 to 27573630, 27573599 to 27573641, 27573599 to 27573650, 27573599 to 275736610, 27573599 to 27573671, 27573599 to 2757 dsRNA agents comprising a sense strand that includes at least 15 contiguous nucleotides that differ from 73616-27573638, 27573619-27573641, 27573622-27573644, 27573633-27573655, 27573690-27573712, or 27573717-27573739 by no more than 3 nucleotides; f) nucleotides 27573296 to 27573584, 27573296 to 27573575, 27573301 to 27573338, 27573318 to 27573342, 27573555 to 27573583, 27573581 to 27573607, 27573584 to 27573607, 27573588 to 27573671, 27573588 to 27573666, 27573588 to 27573624, 27573592 to 27573624, 27573592 to 27573617, 27573598 to 27573624 of SEQ ID NO: 13 27573606-27573652, 27573606-27573647, 27573654-27573712, or 27573707-27573740; and an antisense strand comprising at least 15 contiguous nucleotides from the corresponding nucleotide sequence of SEQ ID NO: 14, A second dsRNA agent targeting the sense strand of C9orf72 a) a sense strand comprising at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 1 by no more than 3 nucleotides, and an antisense strand comprising a nucleotide sequence comprising at least 15 consecutive nucleotides that differ from the corresponding portion of the nucleotide sequence of SEQ ID NO: 5 by no more than 3 nucleotides; b) a sense strand comprising at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 15 by no more than 3 nucleotides, and an antisense strand comprising a nucleotide sequence comprising at least 15 consecutive nucleotides that differ from the corresponding portion of the nucleotide sequence of SEQ ID NO: 16 by no more than 3 nucleotides; c) a dsRNA agent comprising an antisense strand comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the antisense nucleotide sequences in any one of Tables 5, 6, 10B, and 10D; d) a dsRNA agent comprising a sense strand comprising at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from any one of the nucleotide sequences of nucleotides 1 to 23, 15 to 37, 33 to 55, 37 to 59, 59 to 81, 62 to 84, and 69 to 91 of SEQ ID NO: 1, and an antisense strand comprising at least 15 consecutive nucleotides from the corresponding nucleotide sequence of SEQ ID NO: 5; e) nucleotides 5197 to 5219, 5213 to 5235, 5223 to 5245, 5226 to 5248, 5227 to 5249, 5228 to 5250, 5229 to 5251, 5230 to 5252, 5231 to 5253, 5233 to 5255, 5235 to 5256, 5241 to 5263, 5245 to 5267, 5233 to 5255, 5248 to 5270, 5539 to 5561, 5547 to 5569, 5917 to 5939, 5 a sense strand comprising at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from any one of the nucleotide sequences of SEQ ID NO: 936-5958, 5954-5976, 6008-6030, 6021-6043, 6036-6058, 6043-6065, or 6048-6070, and an antisense strand comprising at least 15 consecutive nucleotides from the corresponding nucleotide sequence of SEQ ID NO: 16; f) nucleotides 5015 to 5052, 5017 to 5040, 5032 to 5059, 5032 to 5055, 5033 to 5055, 5035 to 5059, 5036 to 5059, 5058 to 5087, 5059 to 5087, 5059 to 5084, 5064 to 5087, 5197 to 5222, 5213 to 5267, 5223 to 5268, 5224 to 5269, 5225 to 5269, 5226 to 5269, 5227 to 5269, 5228 to 5269, 5229 to 5269, 5230 to 5230, 5231 to 5231, 5232 to 5232, 5233 to 5233, 5234 to 5234, 5235 to 5235, 5236 to 5236, 5237 to 5238, 5239 to 5240, 5241 to 5242, 5243 to 5244, 5245 to 5246, 5248 to 5249, 5249 to 5250, 5249 to 5251, 5249 to 5252, 5249 to 5253, 5249 to 5254, 5249 to 5255, 5249 to 5256, 5249 to 5257, 5249 to 5269, 5 5252, 5229~5252, 5233~5263, 5516~5570, 5539~5565, 5539~5562, 5545~5570, 5545~5569, 5593~5616, 5883~5950, 5917~5950, 5919~5950, 5923~5950, 5934~5977, 5934~5957, 5938~595 and an antisense strand comprising at least 15 consecutive nucleotides from the corresponding nucleotide sequence of SEQ ID NO: 16. g) a dsRNA agent comprising a sense strand comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the nucleotide sequences of nucleotides 15-52, 17-40, 32-59, 32-55, 35-59, 36-59, 58-87, 59-87, 59-84, or 64-87 of SEQ ID NO: 1, and an antisense strand comprising at least 15 contiguous nucleotides from the corresponding nucleotide sequence of SEQ ID NO: 5; and h) a dsRNA agent comprising an antisense strand comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the antisense nucleotide sequences in any one of Tables 8 and 9; The sense strand of the first dsRNA, the antisense strand of the first dsRNA, both the sense strand and the antisense strand of the first dsRNA, the sense strand of the second dsRNA, the antisense strand of the second dsRNA, and / or both the sense strand and the antisense strand of the second dsRNA comprise at least one modified nucleotide.

[0146] In one embodiment, the sense or antisense strand is a sense or antisense strand selected from the sense or antisense strand of a duplex selected from the group consisting of AD-1446213.1, AD-1446217.1, AD-1446222.1, AD-1446234.1, AD-1446243.1, AD-1446246.1, AD-1446252.1, AD-1446259.1, AD-1446265.1, AD-1446268.1, AD-1446271.1, AD-1446279.1, AD-1446289.1, and AD-1446294.1.

[0147] In one embodiment, the sense or antisense strand is a sense or antisense strand selected from the sense or antisense strand of a duplex selected from the group consisting of AD-1446213.1, AD-1446246.1, and AD-1446268.1.

[0148] In one embodiment, the antisense strand comprises at least 15 consecutive nucleotides that differ by no more than 3, 2, or 1 nucleotide from any one of the antisense strand nucleotide sequences and / or sense strand nucleotide sequences of the double strand selected from the group consisting of AD-1446073.1, AD-1446075.1, AD-1285246.2, AD-1446084.1, AD-1446087.1, AD-1446090.1, and AD1446095.1.

[0149] In one embodiment, the antisense strand is selected from the group consisting of AD-1285231.1, AD-1285232.1, AD-1285233.1, AD-1285235.1, AD-1285237.1, AD-1285239.1, AD-1285240.1, AD-1285242.1, AD-1285244.1, AD-1285238.1, AD-1285234.1, AD-1285243.1, AD-1285241.1, AD-1285236.1, AD-1446111.1, AD-1 and AD-1446205.1, AD-1446202.1, and AD-1446205.1.

[0150] In one embodiment, the antisense strand comprises at least 15 consecutive nucleotides that differ by 3, 2, or 1 or less nucleotides from any one of the antisense strand nucleotide sequences and / or sense strand nucleotide sequences of a double strand selected from the group consisting of AD-1285238.1 and AD-1285234.1.

[0151] In one embodiment, the antisense strand of a first dsRNA agent comprises at least 15 contiguous nucleotides that differ by no more than 3, 2, or 1 nucleotide from any one of the antisense strand nucleotide sequences of a double strand selected from the group consisting of AD-1446213.1, AD-1446246.1, and AD-1446268.1, and the antisense strand of a second dsRNA agent comprises at least 15 contiguous nucleotides that differ by no more than 3, 2, or 1 nucleotide from any one of the antisense strand nucleotide sequences of a double strand selected from the group consisting of AD-1285238.1 and AD-1285234.1.

[0152] In one embodiment, a) a first dsRNA agent comprises an antisense strand comprising a nucleotide sequence comprising at least 15 contiguous nucleotides that differ from the antisense sequence of AD-1446213 by no more than 3 nucleotides, and a second dsRNA agent comprises an antisense strand comprising a nucleotide sequence comprising at least 15 contiguous nucleotides that differ from the antisense sequence of AD-1285238 by no more than 3 nucleotides; b) a first dsRNA agent comprises an antisense strand comprising a nucleotide sequence comprising at least 15 contiguous nucleotides that differ from the antisense sequence of AD-1446213 by no more than 3 nucleotides, and a second dsRNA agent comprises an antisense strand comprising a nucleotide sequence comprising at least 15 contiguous nucleotides that differ from the antisense sequence of AD-1285234 by no more than 3 nucleotides; c) a first dsRNA agent comprises an antisense strand comprising a nucleotide sequence comprising at least 15 contiguous nucleotides that differ from the antisense sequence of AD-1446246 by no more than 3 nucleotides, and a second dsRNA agent comprises an antisense strand comprising a nucleotide sequence comprising at least 15 contiguous nucleotides that differ from the antisense sequence of AD-1285238 by no more than 3 nucleotides; d) a first dsRNA agent comprises an antisense strand comprising a nucleotide sequence comprising at least 15 contiguous nucleotides that differ from the antisense sequence of AD-1446246 by no more than 3 nucleotides, and a second dsRNA agent comprises an antisense strand comprising a nucleotide sequence comprising at least 15 contiguous nucleotides that differ from the antisense sequence of AD-1285234 by no more than 3 nucleotides; e) a first dsRNA agent comprises an antisense strand comprising a nucleotide sequence comprising at least 15 contiguous nucleotides that differ from the antisense sequence of AD-1446268 by no more than 3 nucleotides, and a second dsRNA agent comprises an antisense strand comprising a nucleotide sequence comprising at least 15 contiguous nucleotides that differ from the antisense sequence of AD-1285238 by no more than 3 nucleotides; f) a first dsRNA agent comprises an antisense strand comprising a nucleotide sequence comprising at least 15 contiguous nucleotides that differ from the antisense sequence of AD-1446268 by no more than 3 nucleotides, and a second dsRNA agent comprises an antisense strand comprising a nucleotide sequence comprising at least 15 contiguous nucleotides that differ from the antisense sequence of AD-1285234 by no more than 3 nucleotides.

[0153] In another embodiment, a) a first dsRNA agent comprises the antisense and / or sense strand of AD-1446213 and a second dsRNA agent comprises the antisense and / or sense strand of AD-1285238; or b) the first dsRNA agent comprises the antisense and / or sense strand of AD-1446213 and the second dsRNA agent comprises the antisense and / or sense strand of AD-1285234; or c) the first dsRNA agent comprises the antisense and / or sense strand of AD-1446246 and the second dsRNA agent comprises the antisense and / or sense strand of AD-1285238; or d) the first dsRNA agent comprises the antisense and / or sense strand of AD-1446246 and the second dsRNA agent comprises the antisense and / or sense strand of AD-1285234; or e) the first dsRNA agent comprises the antisense and / or sense strand of AD-1446268 and the second dsRNA agent comprises the antisense and / or sense strand of AD-1285238; or f) The first dsRNA agent comprises the antisense and / or sense strand of AD-1446268 and the second dsRNA agent comprises the antisense and / or sense strand of AD-1285234.

[0154] In one embodiment, the sense strand, the antisense strand, or both the sense and antisense strands are conjugated to one or more lipophilic moieties.

[0155] In one embodiment, the lipophilic moiety is conjugated to one or more internal positions within the double-stranded region of the first dsRNA agent, the second dsRNA agent, or both the first and second dsRNA agents.

[0156] In one embodiment, the lipophilic moiety is conjugated to the first dsRNA agent, the second dsRNA agent, or both the first and second dsRNA agents via a linker or carrier.

[0157] In one embodiment, the lipophilicity of the lipophilic moiety conjugated to the first dsRNA agent, the second dsRNA agent, or both the first and second dsRNA agents, as measured by log Kow, is greater than 0.

[0158] In one embodiment, the hydrophobicity of the first dsRNA agent, the second dsRNA agent, or both the first and second dsRNA agents, is greater than 0.2 as measured by the unbound fraction in a plasma protein binding assay of the dsRNA agents.

[0159] In one embodiment, the plasma protein binding assay is an electrophoretic mobility shift assay using human serum albumin protein.

[0160] In one embodiment, the first dsRNA agent, the second dsRNA agent, or both the first and second dsRNA agents comprise at least one modified nucleotide.

[0161] In one embodiment, no more than five of the sense strand nucleotides and no more than five of the antisense strand nucleotides of the first dsRNA agent, the second dsRNA agent, or both the first and second dsRNA agents, are unmodified nucleotides.

[0162] In one embodiment, all of the nucleotides in the sense strand and all of the nucleotides in the antisense strand of the first dsRNA agent, the second dsRNA agent, or both the first and second dsRNA agents, are modified nucleotides.

[0163] 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 2'-O-hexadecyl nucleotide, a 2'-phosphate nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, an inverted abasic residue, a 2'-amino modified nucleotide, a 2'-O-allyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2'-hydroxy modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a 2',3'-seco modified nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide containing a non-natural base, a tetrahydropyridine, a methyl nucleotide, a methyl ester ... The nucleotides are selected from the group consisting of methyl-, 2' ...

[0164] 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 deoxy-thymine nucleotides (dT), locked nucleotides, 2'-O-hexadecyl nucleotides, 2'-phosphate nucleotides, glycol nucleotides, vinyl-phosphonate nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, and nucleotides containing unnatural bases.

[0165] In one embodiment, the modified nucleotides include a short sequence of 3' terminal deoxy-thymine nucleotides (dT).

[0166] In one embodiment, the modified nucleotides are independently selected from the group consisting of 2'-O-methyl modified nucleotides, GNA modified nucleotides, 2'-O-hexadecyl modified nucleotides, 2'-phosphate modified nucleotides, vinyl-phosphonate modified nucleotides, and 2' fluoro modified nucleotides.

[0167] In one embodiment, the first dsRNA agent, the second dsRNA agent, or both the first and second dsRNA agents include at least one phosphorothioate internucleotide linkage.

[0168] In one embodiment, the first dsRNA agent, the second dsRNA agent, or both the first and second dsRNA agents include 6-8 phosphorothioate internucleotide linkages.

[0169] In one embodiment, each strand of the first dsRNA agent, the second dsRNA agent, or both the first and second dsRNA agents is 30 nucleotides or less in length.

[0170] In one embodiment, at least one strand of the first dsRNA agent, the second dsRNA agent, or both the first and second dsRNA agents includes a 3' overhang of at least one nucleotide.

[0171] In one embodiment, at least one strand of the first dsRNA agent, the second dsRNA agent, or both the first and second dsRNA agents includes a 3' overhang of at least two nucleotides.

[0172] In one embodiment, the double-stranded region of the first dsRNA agent, the second dsRNA agent, or both the first and second dsRNA agents is 15-30 nucleotide pairs in length.

[0173] In one embodiment, the double-stranded region of the first dsRNA agent, the second dsRNA agent, or both the first and second dsRNA agents is 17-23 nucleotide pairs in length.

[0174] In one embodiment, the double-stranded region of the first dsRNA agent, the second dsRNA agent, or both the first and second dsRNA agents is 17-25 nucleotide pairs in length.

[0175] In one embodiment, the double-stranded region of the first dsRNA agent, the second dsRNA agent, or both the first and second dsRNA agents is 23-27 nucleotide pairs in length.

[0176] In one embodiment, the double-stranded region of the first dsRNA agent, the second dsRNA agent, or both the first and second dsRNA agents is 19-21 nucleotide pairs in length.

[0177] In one embodiment, the double-stranded region of the first dsRNA agent, the second dsRNA agent, or both the first and second dsRNA agents is 21-23 nucleotide pairs in length.

[0178] In one embodiment, each strand of the first dsRNA agent, the second dsRNA agent, or both the first and second dsRNA agents is 19-30 nucleotides in length.

[0179] In one embodiment, each strand of the first dsRNA agent, the second dsRNA agent, or both the first and second dsRNA agents is 19-23 nucleotides in length.

[0180] In one embodiment, each strand of the first dsRNA agent, the second dsRNA agent, or both the first and second dsRNA agents is 21-23 nucleotides in length.

[0181] In one embodiment, the one or more lipophilic moieties are conjugated via a linker or carrier to one or more internal positions on at least one strand of the first dsRNA agent, the second dsRNA agent, or both the first and second dsRNA agents.

[0182] In one embodiment, an internal position in a first dsRNA agent, a second dsRNA agent, or both a first dsRNA agent and a second dsRNA agent includes any position except the two terminal positions from each end of at least one strand.

[0183] In one embodiment, an internal position in a first dsRNA agent, a second dsRNA agent, or both a first dsRNA agent and a second dsRNA agent includes every position except the terminal three positions from each end of at least one strand.

[0184] In one embodiment, the internal position of the first dsRNA agent, the second dsRNA agent, or both the first and second dsRNA agents excludes the cleavage site region of the sense strand.

[0185] In one embodiment, the internal position in the first dsRNA agent, the second dsRNA agent, or both the first and second dsRNA agents includes any position except positions 9-12 counting from the 5' end of the sense strand.

[0186] In one embodiment, the internal position in the first dsRNA agent, the second dsRNA agent, or both the first and second dsRNA agents includes any position except positions 11-13 from the 3' end of the sense strand.

[0187] In one embodiment, the internal position of the first dsRNA agent, the second dsRNA agent, or both the first and second dsRNA agents excludes the cleavage site region of the antisense strand.

[0188] In one embodiment, the internal position in the first dsRNA agent, the second dsRNA agent, or both the first and second dsRNA agents includes any position except positions 12-14 from the 5' end of the antisense strand.

[0189] In one embodiment, the internal position in the first dsRNA agent, the second dsRNA agent, or both the first and second dsRNA agents includes any position except positions 11-13 on the sense strand, counting from the 3' end, and positions 12-14 on the antisense strand, counting from the 5' end.

[0190] In one embodiment, the one or more lipophilic moieties are conjugated to one or more internal positions of the first dsRNA agent, the second dsRNA agent, or both the first and second dsRNA agents, selected from the group consisting of positions 4-8 and 13-18 on the sense strand and positions 6-10 and 15-18 on the antisense strand, counting from the 5' end of each strand.

[0191] In one embodiment, the one or more lipophilic moieties are conjugated to one or more internal positions on the first dsRNA agent, the second dsRNA agent, or both the first and second dsRNA agents, selected from the group consisting of positions 5, 6, 7, 15, 17 on the sense strand and positions 15 and 17 on the antisense strand, counting from the 5' end of each strand.

[0192] In one embodiment, the internal position within the double-stranded region of the first dsRNA agent, the second dsRNA agent, or both the first and second dsRNA agents excludes the cleavage site region of the sense strand.

[0193] In one embodiment, the sense strand is 21 nucleotides in length, the antisense strand is 23 nucleotides in length, and the lipophilic moiety is conjugated to position 21, 20, 15, 1, 7, 6, or 2 of the sense strand or position 16 of the antisense strand, counting from the 5' end.

[0194] In one embodiment, the lipophilic moiety is conjugated to the first dsRNA agent, the second dsRNA agent, or both the first and second dsRNA agents at position 21, 20, 15, 1, or 7 of the sense strand, counting from the 5' end.

[0195] In one embodiment, the lipophilic moiety is conjugated to the first dsRNA agent, the second dsRNA agent, or both the first and second dsRNA agents at position 21, 20, or 15 of the sense strand, counting from the 5' end.

[0196] In one embodiment, the lipophilic moiety is conjugated to the first dsRNA agent, the second dsRNA agent, or both the first and second dsRNA agents at position 20 or 15 of the sense strand, counting from the 5' end.

[0197] In one embodiment, the lipophilic moiety is conjugated to the first dsRNA agent, the second dsRNA agent, or both the first and second dsRNA agents at position 16 of the antisense strand, counting from the 5' end.

[0198] In one embodiment, the lipophilic moiety conjugated to the first dsRNA agent, the second dsRNA agent, or both the first and second dsRNA agents is an aliphatic, alicyclic, or polycyclic alicyclic compound.

[0199] 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 groups, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenoic acid, dimethoxytrityl, or phenoxazine.

[0200] In one embodiment, the lipophilic moiety comprises 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.

[0201] In one embodiment, the lipophilic moiety comprises a saturated or unsaturated C6 to C18 hydrocarbon chain.

[0202] In one embodiment, the lipophilic moiety comprises a saturated or unsaturated C16 hydrocarbon chain.

[0203] In one embodiment, the saturated or unsaturated C16 hydrocarbon chain is conjugated to the 6 position counting from the 5' end of the chain.

[0204] In one embodiment, the lipophilic moiety is conjugated to the first dsRNA agent, the second dsRNA agent, or both the first and second dsRNA agents via a carrier that substitutes one or more nucleotides at an internal position or within the double-stranded region.

[0205] In one embodiment, the carrier is a cyclic group selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl, or is an acyclic portion of a serinol or diethanolamine backbone system.

[0206] In one embodiment, the lipophilic moiety is conjugated to the double-stranded iRNA agent, the first dsRNA agent, the second dsRNA agent, or both the first and second dsRNA agents via a linker that includes 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.

[0207] In one embodiment, the lipophilic moiety is conjugated to a nucleobase, sugar moiety, or internucleoside linkage of the first dsRNA agent, the second dsRNA agent, or both the first and second dsRNA agents.

[0208] In one embodiment, the lipophilic moiety or targeting ligand is conjugated to the first dsRNA agent, the second dsRNA agent, or both the first and second dsRNA agents via a biochemically cleavable linker selected from the group consisting of DNA, RNA, disulfides, amides, functionalized mono- or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.

[0209] In one embodiment, the 3'-end of the sense strand of the first dsRNA agent, the second dsRNA agent, or both the first and second dsRNA agents 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.

[0210] In one embodiment, the first dsRNA agent, the second dsRNA agent, or both the first and second dsRNA agents further comprise a targeting ligand that targets a neuron, a cell in neuronal tissue, or a cell in central nervous system tissue, or liver tissue.

[0211] In one embodiment, the targeting ligand is a GalNAc conjugate.

[0212] In one embodiment, the first dsRNA agent, the second dsRNA agent, or both the first and second dsRNA agents are a terminal chiral modification occurring at the first internucleotide linkage at the 3' end of the antisense strand, wherein the linking phosphorus atom is in the Sp configuration; a terminal chiral modification occurring at the first internucleotide linkage at the 5' end of the antisense strand, wherein the linking phosphorus atom is in the Rp configuration; and It further comprises a terminal chiral modification occurring at the first internucleotide linkage at the 5' end of the sense strand, where the linking phosphorus atom is in either the Rp or Sp configuration.

[0213] In one embodiment, the first dsRNA agent, the second dsRNA agent, or both the first and second dsRNA agents are a terminal chiral modification occurring at the first and second internucleotide linkages at the 3' end of the antisense strand, wherein the linking phosphorus atom is in the Sp configuration; a terminal chiral modification occurring at the first internucleotide linkage at the 5' end of the antisense strand, wherein the linking phosphorus atom is in the Rp configuration; and It further comprises a terminal chiral modification occurring at the first internucleotide linkage at the 5' end of the sense strand, where the linking phosphorus atom is in either the Rp or Sp configuration.

[0214] In one embodiment, the first dsRNA agent, the second dsRNA agent, or both the first and second dsRNA agents are terminal chiral modifications occurring at the first, second, and third internucleotide linkages at the 3' end of the antisense strand, wherein the linking phosphorus atom is in the Sp configuration; a terminal chiral modification occurring at the first internucleotide linkage at the 5' end of the antisense strand, wherein the linking phosphorus atom is in the Rp configuration; and It further comprises a terminal chiral modification occurring at the first internucleotide linkage at the 5' end of the sense strand, where the linking phosphorus atom is in either the Rp or Sp configuration.

[0215] In one embodiment, the first dsRNA agent, the second dsRNA agent, or both the first and second dsRNA agents are a terminal chiral modification occurring at the first and second internucleotide linkages at the 3' end of the antisense strand, wherein the linking phosphorus atom is in the Sp configuration; a terminal chiral modification occurring at the third internucleotide linkage at the 3' end of the antisense strand, wherein the linking phosphorus atom is in the Rp configuration; a terminal chiral modification occurring at the first internucleotide linkage at the 5' end of the antisense strand, wherein the linking phosphorus atom is in the Rp configuration; and It further comprises a terminal chiral modification occurring at the first internucleotide linkage at the 5' end of the sense strand, where the linking phosphorus atom is in either the Rp or Sp configuration.

[0216] In one embodiment, the first dsRNA agent, the second dsRNA agent, or both the first and second dsRNA agents are a terminal chiral modification occurring at the first and second internucleotide linkages at the 3' end of the antisense strand, wherein the linking phosphorus atom is in the Sp configuration; a terminal chiral modification occurring at the first and second internucleotide linkages at the 5' end of the antisense strand, wherein the linking phosphorus atom is in the Rp configuration; and It further comprises a terminal chiral modification occurring at the first internucleotide linkage at the 5' end of the sense strand, where the linking phosphorus atom is in either the Rp or Sp configuration.

[0217] In one embodiment, the first dsRNA agent, the second dsRNA agent, or both the first and second dsRNA agents further comprise a phosphate or a phosphate mimetic at the 5'-end of the antisense strand.

[0218] In one embodiment, the phosphate mimetic is a 5'-vinylphosphonate (VP).

[0219] In one embodiment, the base pair at one position at the 5' end of the antisense strand of the first dsRNA agent, the second dsRNA agent, or both the first and second dsRNA agents, is an AU base pair.

[0220] In one embodiment, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

[0221] The present invention also provides a cell comprising the composition of the present invention.

[0222] In some embodiments, the compositions of the present invention are pharmaceutical compositions, which in some embodiments comprise a lipid formulation.

[0223] In one aspect, the invention provides a method of reducing the level of one or more C9orf72 RNA transcripts, such as a C9orf72 RNA comprising hexanucleotide repeats, such as a C9orf72 gene comprising multiple consecutive copies of the hexanucleotide repeat, in a cell, e.g., a neuron, such as a motor neuron, the method including contacting the cell with a dsRNA agent of the invention, two or more, e.g., two, three, or four, dsRNA agents of the invention, two or more, e.g., two, three, or four, dsRNA agents for inhibiting expression of one or more C9orf72 RNA transcripts, e.g., a composition comprising a first dsRNA agent that targets a C9orf72 sense transcript (an exon or intron of C9orf72) and a second dsRNA agent that targets a C9orf72 antisense transcript (an exon or intron of C9orf72), as described herein, or a pharmaceutical composition of the invention, thereby inhibiting expression of the C9orf72 gene in the cell.

[0224] In another aspect, the present invention provides a method for reducing dipeptide repeat protein synthesis or dipeptide repeat protein aggregates in a cell, the method comprising introducing into a cell a dsRNA agent of the invention, two or more, e.g., two, three, or four, dsRNA agents of the invention, two or more, e.g., two, three, or four, dsRNA agents for inhibiting expression of one or more C9orf72 RNA transcripts, e.g., a first dsRNA agent that targets a C9orf72 sense transcript (an exon or intron of C9orf72) and a second dsRNA agent that targets a C9orf72 antisense transcript (an exon or intron of C9orf72), as described herein, or a pharmaceutical composition of the invention, thereby reducing dipeptide repeat protein synthesis or dipeptide repeat protein aggregates in the cell.

[0225] In another aspect, the invention provides a method for reducing accumulation or aggregation of poly(glycine-alanine) peptide, poly(glycine-proline) peptide, poly(glycine-arginine) peptide, poly(alanine-proline) peptide, or poly(proline-arginine) peptide in a cell. The method includes introducing into a cell a dsRNA agent of the invention, two or more, e.g., two, three, or four, dsRNA agents of the invention, two or more, e.g., two, three, or four, dsRNA agents for inhibiting expression of C9orf72, e.g., a first dsRNA agent that targets a C9orf72 sense transcript (an exon or intron of C9orf72) and a second dsRNA agent that targets a C9orf72 antisense transcript (an exon or intron of C9orf72), as described herein, or a pharmaceutical composition of the invention, thereby reducing accumulation or aggregation of poly(glycine-alanine) peptide, poly(glycine-proline) peptide, poly(glycine-arginine) peptide, poly(alanine-proline) peptide, or poly(proline-arginine) peptide in the cell.

[0226] In another aspect, the invention provides a method for reducing repeat-length-dependent formation of C9orf72 RNA foci in a cell, the method including introducing into a cell a dsRNA agent of the invention, two or more, e.g., two, three, or four, dsRNA agents of the invention, two or more, e.g., two, three, or four, dsRNA agents for inhibiting expression of C9orf72, e.g., a first dsRNA agent that targets a C9orf72 sense transcript (an exon or intron of C9orf72) and a second dsRNA agent that targets a C9orf72 antisense transcript (an exon or intron of C9orf72), as described herein, or a pharmaceutical composition of the invention, thereby reducing repeat-length-dependent formation of C9orf72 RNA foci in the cell.

[0227] In another aspect, the invention provides a method for reducing nuclear and / or cytoplasmic sense and / or antisense C9orf72 RNA foci in a cell, the method including introducing into a cell a dsRNA agent of the invention, two or more, e.g., two, three, or four, dsRNA agents of the invention, two or more, e.g., two, three, or four, dsRNA agents for inhibiting expression of C9orf72, e.g., a first dsRNA agent that targets a C9orf72 sense transcript (an exon or intron of C9orf72) and a second dsRNA agent that targets a C9orf72 antisense transcript (an exon or intron of C9orf72), as described herein, or a pharmaceutical composition of the invention, thereby reducing nuclear and / or cytoplasmic sense and / or antisense C9orf72 RNA foci in the cell.

[0228] In one embodiment, the cell is in a subject.

[0229] In one embodiment, the subject is a human.

[0230] In one embodiment, the subject has or is at risk of developing a C9orf72-associated disorder, such as a C9orf72 hexanucleotide repeat expansion-associated disease, condition, or disorder.

[0231] In one embodiment, the C9orf72-associated disorder is selected from the group consisting of C9orf72 amyotrophic lateral sclerosis, frontotemporal dementia, Huntington's disease, Huntington-like syndrome due to C9orf72 hexanucleotide repeat expansion, Parkinsonism, olivopontocerebellar degeneration, corticobasal syndrome, and Alzheimer's disease.

[0232] In one embodiment, contacting a cell with a dsRNA agent inhibits the level of sense and / or antisense hexanucleotide repeat-containing C9orf72 RNA transcripts by at least 50%, 60%, 70%, 80%, 90%, or 95%.

[0233] In one embodiment, inhibiting the levels of sense and / or antisense hexanucleotide repeat-containing C9orf72 RNA transcripts reduces the levels of one or more aberrant dipeptide repeat (DPR) proteins selected from the group consisting of poly(glycine-alanine), poly(glycine-arginine), poly(glycine-proline), poly(proline-alanine), and poly(proline-arginine) by at least 50%, 60%, 70%, 80%, 90%, or 95%.

[0234] In one embodiment, contacting a cell with a dsRNA agent inhibits expression of C9orf72 mRNA by no more than 50%, 40%, 30%, 20%, 10%, or 5%.

[0235] In one embodiment, the dsRNA agent inhibits expression of a C9orf72 target mRNA 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 a C9orf72 target RNA that includes a hexanucleotide repeat.

[0236] In some embodiments, 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. In other embodiments, the dsRNA agent inhibits expression of the 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 that express the mature C9orf72 messenger RNA.

[0237] In some embodiments, the dsRNA agent reduces dipeptide repeat (poly(GA), poly(GR), poly(GP), poly(PA), and / or poly(PR)) protein synthesis or dipeptide repeat (poly(GA), poly(GR), poly(GP), poly(PA), and / or poly(PR)) protein aggregates in a cell.

[0238] In some embodiments, the dsRNA agent reduces nuclear and / or cytoplasmic sense and / or antisense C9orf72 RNA foci in the cell.

[0239] In one embodiment, inhibiting expression of C9orf72 reduces C9orf72 protein levels in the subject's serum by no more than 50%, 40%, 30%, 20%, 10%, or 5%.

[0240] In some embodiments, the dsRNA agent reduces dipeptide repeat (poly(GA), poly(GR), poly(GP), poly(PA), and / or poly(PR)) protein synthesis or dipeptide repeat (poly(GA), poly(GR), poly(GP), poly(PA), and / or poly(PR)) protein aggregates 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.

[0241] 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, such as a C9orf72 hexanucleotide repeat expansion-associated disease, condition, or disorder, the method comprising administering to the subject a therapeutically effective amount of a dsRNA agent of the invention, two or more, e.g., two, three, or four, dsRNA agents of the invention, two or more, e.g., two, three, or four or more dsRNA agents for inhibiting expression of one or more C9orf72 RNAs, e.g., a first dsRNA agent that targets a C9orf72 sense strand transcript (an exon or intron of C9orf72) and a second dsRNA agent that targets a C9orf72 antisense strand transcript (an exon or intron of C9orf72), as described herein, or a pharmaceutical composition of the invention, thereby treating the subject having a disorder that would benefit from reduced C9orf72 expression.

[0242] In another aspect, the invention provides a method of preventing at least one symptom in a subject having a disorder that would benefit from reduced expression of a C9orf72 RNA comprising a hexanucleotide repeat expansion, such as a C9orf72 hexanucleotide repeat expansion-associated disease, condition, or disorder, the method comprising administering to the subject a prophylactically effective amount of a dsRNA agent of the invention, two or more, e.g., two, three, or four, dsRNA agents of the invention, two or more, e.g., two, three, or four or more dsRNA agents for inhibiting expression of C9orf72, e.g., a first dsRNA agent that targets a C9orf72 sense strand transcript (an exon or intron of C9orf72) and a second dsRNA agent that targets a C9orf72 antisense strand transcript (an exon or intron of C9orf72), as described herein, or a pharmaceutical composition of the invention, thereby preventing at least one symptom in the subject having a disorder that would benefit from reduced C9orf72 expression.

[0243] In one embodiment, the method includes administering a first dsRNA agent that targets the sense strand of C9orf72 (an exon or intron of C9orf72) and a second dsRNA agent that targets the antisense strand of C9orf72 (an exon or intron of C9orf72).

[0244] In some embodiments, suitable agents targeting the sense strand of C9orf72 for use in methods of the invention that include two or more dsRNA agents include a sense strand and an antisense strand that form a double-stranded region, wherein the strands are: a) a sense strand comprising at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 1 by no more than 3 nucleotides, and an antisense strand comprising a nucleotide sequence comprising at least 15 consecutive nucleotides that differ from the corresponding portion of the nucleotide sequence of SEQ ID NO: 5 by no more than 3 nucleotides; b) a sense strand comprising at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 15 by no more than 3 nucleotides, and an antisense strand comprising a nucleotide sequence comprising at least 15 consecutive nucleotides that differ from the corresponding portion of the nucleotide sequence of SEQ ID NO: 16 by no more than 3 nucleotides; c) an antisense strand comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the antisense nucleotide sequences in any one of Tables 5, 6, 10B, and 10D; d) a sense strand comprising at least 15 consecutive nucleotides that differ by no more than three nucleotides from any one of the nucleotide sequences of nucleotides 1 to 23, 15 to 37, 33 to 55, 37 to 59, 59 to 81, 62 to 84, or 69 to 91 of SEQ ID NO: 1, and an antisense strand comprising at least 15 consecutive nucleotides from the corresponding nucleotide sequence of SEQ ID NO: 5; e) nucleotides 5197 to 5219, 5213 to 5235, 5223 to 5245, 5226 to 5248, 5227 to 5249, 5228 to 5250, 5229 to 5251, 5230 to 5252, 5231 to 5253, 5235 to 5256, 5241 to 5263, 5245 to 5267, 5233 to 5255, 5248 to 5270, 5539 to 5561, 5547 to 5569, 5917 to 5939, 5 a sense strand comprising at least 15 consecutive nucleotides that differ by no more than three nucleotides from any one of the nucleotide sequences of SEQ ID NO: 936 to 5958, 5954 to 5976, 6008 to 6030, 6021 to 6043, 6036 to 6058, 6043 to 6065, or 6048 to 6070, and an antisense strand comprising at least 15 consecutive nucleotides from the corresponding nucleotide sequence of SEQ ID NO: 16; f) nucleotides 5015 to 5052, 5017 to 5040, 5032 to 5059, 5032 to 5055, 5033 to 5055, 5035 to 5059, 5036 to 5059, 5058 to 5087, 5059 to 5087, 5059 to 5084, 5064 to 5087, 5197 to 5222, 5213 to 5267, 522 3~5252, 5229~5252, 5233~5263, 5516~5570, 5539~5565, 5539~5562, 5545~5570, 5545~5569, 5593~5616, 5883~5950, 5917~5950, 5919~5950, 5923~5950, 5934~5977, 5934~5957, 5 a sense strand comprising at least 15 consecutive nucleotides that differ by no more than three nucleotides from any one of the nucleotide sequences of SEQ ID NO: 938 to 5977, 5938 to 5965, 5938 to 5961, 5947 to 5977, 5947 to 5973, 5972 to 6001, 5973 to 5997, 6006 to 6029, 6011 to 6070, 6011 to 6039, 6011 to 6038, 6015 to 6038, 6019 to 6045, 6019 to 6042, 6033 to 6070, 6035 to 6065, 6035 to 6059, or 6040 to 6063, and an antisense strand comprising at least 15 consecutive nucleotides from the corresponding nucleotide sequence of SEQ ID NO: 16; g) a sense strand comprising at least 15 consecutive nucleotides that differ by no more than three nucleotides from any one of the nucleotide sequences of nucleotides 15-52, 17-40, 32-59, 32-55, 35-59, 36-59, 58-87, 59-87, 59-84, or 64-87 of SEQ ID NO: 1, and an antisense strand comprising at least 15 consecutive nucleotides from the corresponding nucleotide sequence of SEQ ID NO: 5; and h) an antisense strand comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the antisense nucleotide sequences in any one of Tables 8 and 9; The sense strand, the antisense strand, or both the sense and antisense strands comprise at least one modified nucleotide.

[0245] In certain embodiments, suitable agents targeting C9orf72, e.g., the sense strand of a C9orf72 exon or intron sense sequence, for use in methods of the invention comprising two or more dsRNA agents are dsRNA agents disclosed in PCT Publication No. WO2021 / 119226, the entire contents of which are incorporated herein by reference.

[0246] In certain embodiments, suitable agents targeting the antisense strand of C9orf72 for use in methods of the invention comprising two or more dsRNA agents include a sense strand and an antisense strand that form a double-stranded region, wherein the strands are: a) a sense strand comprising at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 13 by no more than 3 nucleotides, and an antisense strand comprising a nucleotide sequence comprising at least 15 consecutive nucleotides that differ from the corresponding portion of the nucleotide sequence of SEQ ID NO: 14 by no more than 3 nucleotides; b) a sense strand comprising at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 17 by no more than 3 nucleotides, and an antisense strand comprising a nucleotide sequence comprising at least 15 consecutive nucleotides that differ from the corresponding portion of the nucleotide sequence of SEQ ID NO: 18 by no more than 3 nucleotides; c) a sense strand comprising at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 19 by no more than 3 nucleotides, and an antisense strand comprising a nucleotide sequence comprising at least 15 consecutive nucleotides that differ from the corresponding portion of the nucleotide sequence of SEQ ID NO: 20 by no more than 3 nucleotides; d) an antisense comprising a nucleotide sequence selected from the group consisting of any of the antisense strand nucleotide sequences in any one of Tables 2, 3, 10C, 10B, 11, and 12; e) Nucleotides 27573296 to 27573318, 27573314 to 27573336, 27573319 to 27573341, 27573562 to 27573584, 27573585 to 27573607, 27573592 to 27573614, 27573599 to 27573621, 27573608 to 2757363 of SEQ ID NO: 13 a sense strand comprising at least 15 consecutive nucleotides that differ from 0, 27573616 to 27573638, 27573619 to 27573641, 27573622 to 27573644, 27573633 to 27573655, 27573690 to 27573712, or 27573717 to 27573739 by no more than 3 nucleotides; and f) nucleotides 27573296 to 27573584, 27573296 to 27573575, 27573301 to 27573338, 27573318 to 27573342, 27573555 to 27573583, 27573581 to 27573607, 27573584 to 27573607, 27573588 to 27573671, 27573588 to 27573666, 27573588 to 27573624, 27573592 to 27573624, 27573592 to 27573617, 27573598 to 275 a sense strand comprising at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from any one of the nucleotide sequences of SEQ ID NO: 14, 27573599 to 27573623, 27573606 to 27573655, 27573606 to 27573652, 27573606 to 27573647, 27573654 to 27573712, or 27573707 to 27573740, and an antisense strand comprising at least 15 consecutive nucleotides from the corresponding nucleotide sequence of SEQ ID NO: 14, The sense strand, the antisense strand, or both the sense and antisense strands comprise at least one modified nucleotide.

[0247] In one embodiment, the sense strand, the antisense strand, or both the sense and antisense strands are conjugated to one or more lipophilic moieties.

[0248] In one embodiment, the disorder is a C9orf72-associated disorder.

[0249] In one embodiment, the C9orf723-associated disorder is selected from the group consisting of C9orf72 amyotrophic lateral sclerosis, frontotemporal dementia, Huntington's disease, Huntington-like syndrome due to C9orf72 expansion, Parkinsonism, olivopontocerebellar degeneration, corticobasal syndrome, and Alzheimer's disease.

[0250] In one embodiment, the subject is a human.

[0251] In one embodiment, administration of the agent to a subject results in a decrease in C9orf72 protein accumulation.

[0252] In some embodiments, the method reduces dipeptide repeat protein synthesis or reduces dipeptide repeat protein aggregation in the subject. In some embodiments, the method reduces expression of a C9orf72 target RNA that includes a hexanucleotide repeat comprising multiple consecutive copies of SEQ ID NO:1 in the subject.

[0253] In one embodiment, administration of the agent to a subject results in a decrease in the level of one or more dipeptide repeat (DPR) proteins selected from the group consisting of poly(glycine-alanine), poly(glycine-arginine), poly(glycine-proline), poly(proline-alanine), and poly(proline-arginine).

[0254] In one embodiment, the level of one or more abnormal dipeptide repeat (DPR) proteins is reduced by more than 50%, 60%, 70%, 80%, 90%, or 95%.

[0255] In one embodiment, the level of poly(glycine-alanine) and / or poly(glycine-proline) is reduced by more than 50%, 60%, 70%, 80%, 90%, or 95%.

[0256] 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.

[0257] In one embodiment, the dsRNA agent is administered to the subject subcutaneously.

[0258] In another embodiment, the dsRNA agent is administered to the subject intrathecally.

[0259] In yet another embodiment, the dsRNA agent is administered to the subject intracerebroventricularly.

[0260] In one embodiment, the method of the invention further comprises determining the level of C9orf72 in a sample from the subject.

[0261] In one embodiment, the level of C9orf72 in the subject sample is the C9orf72 protein level in a blood, serum, or cerebrospinal fluid sample.

[0262] In one embodiment, the methods of the invention further comprise administering to the subject an additional therapeutic agent.

[0263] In one aspect, the invention provides a kit including any one or more of a dsRNA agent of the invention, a composition of the invention, or a pharmaceutical composition of the invention.

[0264] In another aspect, the invention provides a vial containing any one or more of a dsRNA agent of the invention, a composition of the invention, or a pharmaceutical composition of the invention.

[0265] In yet another aspect, the invention provides a syringe comprising any one or more of a dsRNA agent of the invention, a composition of the invention, or a pharmaceutical composition of the invention.

[0266] In one embodiment, an RNAi agent is a pharmaceutically acceptable salt thereof. The "pharmaceutically acceptable salt" of each RNAi agent herein includes, but is not limited to, sodium, calcium, lithium, potassium, ammonium, magnesium salts, and mixtures thereof. Those skilled in the art will appreciate that when an RNAi agent is provided as a polycationic salt, it will have one cation per free acid group of the optionally modified phosophodiester backbone and / or any other acidic modifications (e.g., a phosphonate group at the 5' end). For example, an oligonucleotide "n" nucleotides in length will contain n-1 optionally modified phosophodiesters, such that an oligonucleotide 21 nt in length can be provided as a salt with up to 20 cations (e.g., 20 sodium cations). Similarly, an RNAi agent having a 21 nt sense strand and a 23 nt antisense strand can be provided as a salt with up to 42 cations (e.g., 42 sodium cations). In the preceding examples, if the RNAi agent also includes a 5'-terminal phosphate or a 5'-terminal vinylphosphonate group, the RNAi agent can be provided as a salt with up to 44 cations (e.g., 44 sodium cations). [Brief explanation of the drawings]

[0267] [Figure 1] Graph showing the results of a single-dose screen in Cos-7 cells of the indicated agents at final concentrations of 10 nM, 1 nM, or 0.1 nM. [Figure 2] FIG. 2 is a graph showing the results of a subset of drugs from FIG. 1 selected for further analysis based on single-dose screening in Cos-7 cells at final concentrations of 10 nM, 1 nM, or 0.1 nM. [Figure 3] Graph showing the results of a single-dose screen in Cos-7 cells of the indicated agents at final concentrations of 10 nM, 1 nM, or 0.1 nM. [Figure 4]FIG. 4 is a graph showing the results of a subset of drugs from FIG. 3 selected for further analysis based on single-dose screening in Cos-7 cells at final concentrations of 10 nM, 1 nM, or 0.1 nM. [Figure 5A-5B] This graph shows the effect of target duplexes on C9orf72 RNA accumulation. Embryonic stem cells harboring an approximately 300× G4C2 repeat expansion were electroporated with 1 μM of two different dsRNA agents targeting sense RNA (solid dark bars), two different dsRNA agents targeting antisense RNA transcribed from the region of the C9orf72 gene between exon 1A and the repeat expansion (white bars), or a combination of sense RNA (AD1285238.1) targeting siRNA-1 and one of each antisense siRNA (hatched bars). Knockdown of transcripts containing sequences derived from the region of the C9orf72 gene between exon 1A and the repeat expansion (Figure 5A) was assayed by RT-qPCR using an assay that detects sequences from this region. Note that this assay primarily detects sense RNA, as antisense RNA levels are eight-fold lower than sense RNA. C9orf72 spliced ​​mRNA (Figure 5B) was assayed by RT-qPCR using an assay that recognizes RNA containing sequences spanning the junction between exon 2 and exon 3. Data were normalized to the mean of two control samples (black bars) treated with vehicle, artificial cerebrospinal fluid (aCSF). [Figures 6A-6C]Western slot blot (Figure 6A) and blot quantification graphs (Figures 6B and 6C) show the effect of a duplex of interest on the levels of dipeptide repeat proteins. Embryonic stem cells harboring an approximately 300× G4C2 repeat expansion were electroporated with 1 μM of two different dsRNA agents targeting sense RNA (solid dark bars, Figures 6B-6C), antisense RNA (white bars, Figures 6B-6C), or a combination as in Figure 5 (hatched bars, Figures 6B-6C). Dipeptide repeat protein levels after knockdown were assayed using antibodies against poly(GlyAla) (right panel, Figure 6A) and poly(GlyPro) (left panel, Figure 6A). Relative protein levels of poly(GlyPro) (Figure 6B) and poly(GlyAla) (Figure 6C) after siRNA treatment were quantified and normalized to aCSF-treated samples. [Figure 7] Graph showing the percentage of C9orf72 mRNA remaining after intrathecal administration of a single 3 mg / kg dose of the indicated duplex or PBS. [Figure 8] 1 is a graph showing the use of nanostring probes for mapping transcription start sites in C9orf72 antisense RNA. DETAILED DESCRIPTION OF THE INVENTION

[0268] The present disclosure provides an RNAi composition that causes the RNA transcript of C9orf72 gene, such as the C9orf72 gene with an expanded GGGGCC (G4C2) repeat, to be cleaved by RNA-induced silencing complex (RISC).The C9orf72 gene can be in a cell, for example, a cell in a subject, such as a human.The use of these iRNAs allows the targeted degradation of the RNA of corresponding gene (C9orf72 gene) in mammals.

[0269] The iRNA of the present invention is designed to target C9orf72 target RNA, for example, C9orf72 target RNA with an expanded GGGGCC hexanucleotide repeat in the intron of the gene. The agent may target mature C9orf72 mRNA (mRNA with the intron spliced ​​out) or C9orf7 mRNA precursor (mRNA containing an intron). In certain embodiments of the present invention, the RNAi agent of the present disclosure may target C9orf72 sense and / or antisense RNA transcripts containing hexanucleotide repeats (RNA containing C9orf72 intron 1A). Targeting C9orf72 sense and / or antisense strand RNAs containing hexanucleotide repeats can inhibit the expression or reduce the presence of 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 either the sense repeat-containing C9orf72 RNA or the antisense repeat-containing C9orf72 RNA in cells of the nervous system of a subject with a C9orf72-related disease. In some embodiments, a combination of an RNA agent targeting a C9orf72 sense strand RNA containing hexanucleotide repeats and an RNA agent targeting a C9orf72 antisense strand RNA containing hexanucleotide repeats is provided together.

[0270] The described iRNAs can have one or more nucleotide modifications or a combination of nucleotide modifications that increase the activity, delivery, and / or stability of the iRNA.

[0271] In some embodiments, iRNAs of the invention inhibit expression of the C9orf72 gene (e.g., mature mRNA) by about 50% or less, reduce the levels of sense- and antisense-containing C9orf72 RNA foci by more than about 50%, reduce the levels of one or more aberrant dipeptide repeat (DPR) proteins (poly(GA), poly(GR), poly(GP), poly(PA), and poly(PR)), and / or reduce the expression of C9orf72 sense and / or antisense RNAs containing hexanucleotide repeats. Without intending to be bound by theory, it is believed that combinations or subcombinations of the foregoing features with specific target sites or specific modifications in these iRNAs improve the efficacy, stability, potency, durability, and safety of iRNAs of the invention.

[0272] Accordingly, the present disclosure also provides methods of using the RNAi compositions of the present disclosure, including compositions comprising one or more, e.g., two, three, or four, dsRNA agents of the invention, to knock down or inhibit expression of one or more C9orf72 RNAs, or to treat a subject having a disorder that would benefit from knocking down or inhibiting expression of one or more C9orf72 RNAs, e.g., a C9orf72-related disease, e.g., C9orf72 amyotrophic lateral sclerosis, frontotemporal dementia, or Huntington's disease, e.g., a disease associated with an expanded GGGGCC hexanucleotide repeat in an intron of the C9orf72 gene, such as Huntington-like syndrome caused by C9orf72 expansion, parkinsonism, olivopontocerebellar degeneration, corticobasal syndrome, or Alzheimer's disease.

[0273] RNAi agents of the present disclosure can be about 30 nucleotides in length or less, e.g., 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-25, 19-26, 19-25, 19-24, 19-23 ...6, 19-25, 19-24, 19-23, 1 The RNA strand (antisense strand) has a region that is 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 a C9orf72 gene, e.g., 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, which region is substantially complementary to at least a portion of a target RNA transcript of a C9orf72 gene, e.g., a C9orf72 intron.

[0274] The presence of sense and antisense C9orf72-containing foci, as well as aberrant dipeptide repeat (DPR) proteins (poly(GA), poly(GR), poly(GP), poly(PA), and poly(PR)) produced from all reading frames of C9orf72 RNA containing either sense or antisense repeats through repeat-associated non-AUG-dependent (RAN) translation, have been identified in several cell types in 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. al. (2016), in certain embodiments of the present invention, the RNAi agents of the present disclosure are about 30 nucleotides in length or less, e.g., 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-2 8, 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, 2 The RNAi agent of the present disclosure comprises an RNA strand (antisense strand) having a region that is 0-24, 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 is substantially complementary to at least a portion of a target RNA transcript of a C9orf72 gene, e.g., a C9orf72 intron. In certain embodiments, the RNAi agent of the present disclosure comprises an RNA strand (antisense strand) having a region that is approximately 21-23 nucleotides in length, which is substantially complementary to at least a portion of a target RNA transcript of a C9orf72 gene, e.g., a C9orf72 intron.

[0275] In certain embodiments, RNAi agents of the present disclosure include an RNA strand (antisense strand) that can be longer in length, e.g., up to 66 nucleotides, e.g., 36-66, 26-36, 25-36, 31-60, 22-43, 27-53 nucleotides in length, where a region of at least 19 contiguous nucleotides is substantially complementary to at least a portion of an mRNA transcript of the C9orf72 gene. RNAi agents with longer antisense strands preferably include a second RNA strand (sense strand) that is 20-60 nucleotides in length, where the sense and antisense strands form a duplex of 18-30 contiguous nucleotides.

[0276] These RNAi agents allow for targeted degradation of target RNAs of the C9orf72 gene in mammals. Thus, methods and compositions comprising these RNAi agents are useful for treating subjects who would benefit from knocking down target C9orf72 RNA, reducing normal C9orf72 protein, and / or reducing pathogenic dipeptide repeat proteins generated from pathogenic hexanucleotide repeat expansions, such as subjects with C9orf72-related diseases, such as C9orf72 amyotrophic lateral sclerosis, frontotemporal dementia, Huntington's disease, e.g., Huntington-like syndromes caused by C9orf72 expansions, parkinsonism, olivopontocerebellar degeneration, corticobasal syndrome, or Alzheimer's disease.

[0277] The following detailed description discloses methods for making and using compositions comprising RNAi agents that inhibit expression of the C9orf72 gene, as well as compositions and methods for treating subjects with diseases and disorders that may benefit from inhibiting or reducing expression of the gene.

[0278] I. Definition So 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.

[0279] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. For example, "an element" means one element or to more than one element, e.g., a plurality of elements.

[0280] 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.

[0281] The term "about" is used herein to mean within a typical tolerance in the art. For example, "about" can be understood as about 2 standard deviations from the mean. In certain embodiments, about means ±10%. In certain embodiments, about means ±5%. When about is before a series of numbers or ranges, it is understood that "about" can modify each of the consecutive numbers or ranges.

[0282] The term "at least" preceding a number or series of numbers, when clear from the context, is understood to include the number adjacent to the term "at least," and all subsequent numbers or integers that may logically be 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 at least precedes a series of numbers or ranges, it is understood that "at least" can modify each of the consecutive numbers or ranges.

[0283] As used herein, "less than or equal to" or "less than" is understood to refer to the value adjacent to the term and any logically smaller value or integer than that value, up to zero, where logical from the context. For example, a duplex having an overhang of "two or fewer nucleotides" has an overhang of 2, 1, or 0 nucleotides. When a series of numbers or ranges are preceded by "less than," it is understood that "less than or equal to" can modify each of the consecutive numbers or ranges.

[0284] As used herein, a method of detection may include determining that the amount of analyte present is below the detection level of the method.

[0285] In the event of a conflict between a shown target site and the nucleotide sequence for either the sense or antisense strand, the shown sequence controls.

[0286] In the event of a conflict between a chemical structure and a chemical name, the chemical structure shall prevail.

[0287] A composition or method "comprising" or "including" one or more recited elements may include other elements not specifically recited. For example, a composition "comprising" or "including" a protein may include the protein alone or in combination with other components. The transitional phrase "consisting essentially of" means that the claim should be construed to include the specific elements recited in the claim, as well as elements that do not materially affect the basic and novel characteristics of the claimed invention. Thus, when used in the claims of the present invention, the term "consisting essentially of" is not intended to be interpreted as equivalent to "comprising."

[0288] "Optional" or "optionally" means that the event or circumstance described below may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where the event or circumstance does not occur.

[0289] The term "C9orf72" gene refers to the gene C9orf72, also known as "C9orf72-SMCR8 complex subunit," "guanine nucleotide exchange C9orf72," "chromosome 9 open reading frame 72," "protein C9orf72," "DENNL72," "FTDALS1," "ALSFTD," and "FTDALS," which encodes a well-known protein involved in regulating endosomal trafficking. The C9orf72 protein has been shown to interact with Rab proteins involved in autophagy and endocytic trafficking. Expansion of GGGGCC repeats from about 2 to about 22 copies to about 700 to about 1600 copies in the intronic sequence between alternative 5' exons in transcripts from this gene has been associated with C9orf72 amyotrophic lateral sclerosis, frontotemporal dementia, Huntington's disease, e.g., Huntington-like syndrome due to C9orf72 expansion, parkinsonism, olivopontocerebellar degeneration, corticobasal syndrome, or Alzheimer's disease. Alternative splicing results in multiple transcript variants encoding different isoforms.

[0290] Exemplary nucleotide and amino acid sequences for C9orf72 can be found, for example, in GenBank Accession No. NM_001256054.2 (Homo sapiens C9orf72, SEQ ID NO:1, reverse complement SEQ ID NO:5); GenBank Accession No. XM_005581570.2 (Macaca fascicularis C9orf72, SEQ ID NO:2, reverse complement SEQ ID NO:6); GenBank Accession No. NM_001081343.2 (Mus musculus C9orf72, SEQ ID NO:3, reverse complement SEQ ID NO:7), and GenBank Accession No. NM_001007702.1 (Rattus norvegicus C9orf72, SEQ ID NO:4, reverse complement SEQ ID NO:8).

[0291] 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: 9, reverse complement SEQ ID NO: 10), and NM_018325.5, transcript variant 2 (SEQ ID NO: 11, reverse complement SEQ ID NO: 12).

[0292] The nucleotide sequence of the genomic region of human chromosome 9 harboring the C9orf72 gene can be found, for example, in Genome Reference Consortium Human Build 38 (also referred to as Human Genome build 38 or GRCh38), available at GenBank. The nucleotide sequence of the genomic region of human chromosome 9 harboring the C9orf72 gene can also be found, for example, in GenBank accession number NC_000009.12 (SEQ ID NO: 13 provides nucleotides 27546546..27573866 of the chromosome 9 assembly; reverse complement SEQ ID NO: 14). The nucleotide sequence of the human C9orf72 gene can be found, for example, in GenBank accession number NG_031977.1 (SEQ ID NO: 15; reverse complement SEQ ID NO: 16).

[0293] SEQ ID NO: 13 provides nucleotides 27546546..27573866 (NC_000009.12) of the assembly of chromosome 9. When a range of a target sequence within SEQ ID NO: 13 is provided, it will be understood that the nucleotide position range corresponds to the nucleotide position in the assembly of chromosome 9, e.g., nucleotides 27573086-27573106 of SEQ ID NO: 13 refers to the nucleotide position in the assembly of human chromosome 9 to which SEQ ID NO: 13 provides the nucleotide at position 27546546..27573866.

[0294] Further examples of C9orf72 sequences can be found in publicly available databases such as GenBank, OMIM, and UniProt.

[0295] Additional information regarding C9orf72 can be found, for example, at www.ncbi.nlm.nih.gov / gene / 203228. As used herein, the term C9orf72 also refers to variations in the C9orf72 gene, including variants provided in the Clinical Variant Database, for example, at www.ncbi.nlm.nih.gov / clinvar / ?term=NM_001256054.2.

[0296] 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.

[0297] As used herein, "target sequence" refers to a contiguous portion of the nucleotide sequence of an RNA molecule formed during transcription of the C9orf72 gene, such as a sense or antisense C9orf72 RNA molecule, including mRNA, which is a product of RNA processing of a primary transcript. In one embodiment, the target portion of the sequence will be at least long enough to serve as a substrate for RNAi-directed cleavage at or near a portion of the nucleotide sequence of an mRNA molecule formed during transcription of the C9orf72 gene. In one embodiment, the target sequence is within the protein-coding region of the C9orf72 gene. In another embodiment, the target sequence is within an intron of the C9orf72 gene, for example, the intron between exons 1A and 1B. In one embodiment, the target sequence is a sense C9orf72 RNA molecule. In another embodiment, the target sequence is an antisense C9orf72 RNA molecule. In one embodiment, the target sequence includes a transcription start site, e.g., a transcription start site of an antisense C9orf72 RNA molecule, e.g., about 171 bp downstream of the 3' end of exon 1B encoding DNA, or approximately 270 bp downstream of the GGGGCC hexanucleotide repeat expansion, e.g., nucleotide 5607 of NG_031977 (SEQ ID NO: 15). In some embodiments, the target sequence includes the region between the transcription start site and exon 1A, e.g., nucleotides 5001-5607, 5026-5607, 5127-5607, or 5130-5607 of NG_031977 (SEQ ID NO: 15). Exons 1A and 1B correspond to positions 5001-5158 and 5386-5436 of NG_031977. In some embodiments, the target sequence includes a region starting at the transcription start site, extending through the hexanucleotide repeat expansion region, and extending at least about 200 bp, 500 bp, 900 bp, 1200 bp, 1500 bp, or 2000 bp into the 5' flanking sequence of the C9orf72 gene. When the nucleotide sequence of the target sequence is provided, for example, as a cDNA or genomic sequence, or the reverse complement of a cDNA or genomic sequence, e.g., SEQ ID NOs: 1-20, it is understood that "Ts" is "Us" in the corresponding mRNA sequence.

[0298] C9orf72 mRNA (target C9orf72 RNA) is RNA transcribed from the C9orf72 gene, either the sense or antisense strand of the transcription message. C9orf72 RNA includes C9orf72 mature mRNA, C9orf72 precursor RNA, or any portion thereof (e.g., spliced-out intron regions 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 RNA is C9orf72 RNA from which at least one intron, particularly the first intron (intron 1), has not been removed.

[0299] C9orf72 proteins include any protein expressed from C9orf72 RNA, including proteins expressed from C9orf72 mature RNA, as well as dipeptide repeat proteins resulting from repeat-associated non-AUG (AUG) translation from C9orf72 RNA containing hexanucleotide repeats (e.g., poly(glycine-alanine), poly(glycine-proline), poly(glycine-arginine), poly(alanine-proline), and poly(proline-arginine)).

[0300] The C9orf72 target RNA may include a C9orf72 RNA having a hexanucleotide repeat expansion. The hexanucleotide repeat expansion may include, but is not limited to, multiple consecutive copies of SEQ ID NO: 1 or a sequence having at least 90% identity to multiple consecutive copies of SEQ ID NO: 1. The C9orf72 target RNA may include, but is not limited to, C9orf72 sense and antisense RNA transcripts having a hexanucleotide repeat expansion. The C9orf72 target RNA may, for example, have 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).

[0301] The target sequence can be about 15 to 30 nucleotides in length. For example, the target sequence can be about 15 to 30 nucleotides, 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to 29, 19 The target sequence may be 19-23, 21-22, 21-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. In certain embodiments, the target sequence is 19-23 nucleotides in length, and optionally 21-23 nucleotides in length. Ranges and lengths between the ranges and lengths listed above are also contemplated as part of this disclosure.

[0302] As used herein, the term "strand comprising a sequence" refers to an oligonucleotide comprising a chain of nucleotides described by a sequence referenced using standard nucleotide nomenclature.

[0303] "G", "C", "A", "T", and "U" generally refer to nucleotides containing guanine, cytosine, adenine, thymidine, and uracil as bases, respectively, in the context of modified or unmodified nucleotides. However, it will be understood that the term "ribonucleotide" or "nucleotide" can also refer to modified nucleotides, as described in more detail below, or alternative replacement moieties (see, for example, Table 1). Those skilled in the art will appreciate that guanine, cytosine, adenine, thymidine, and uracil can be substituted with other moieties without substantially altering the base pairing properties of oligonucleotides containing nucleotides bearing such replacement moieties. For example, but not limited to, a nucleotide containing inosine as a base can base pair with a nucleotide containing adenine, cytosine, or uracil. Thus, a nucleotide containing uracil, guanine, or adenine can be substituted with a nucleotide containing inosine, for example, in the nucleotide sequence of a dsRNA featured in the present disclosure. In another example, adenine and cytosine anywhere within an oligonucleotide can be substituted with guanine and uracil, respectively, to form a GU wobble that base pairs with the target mRNA. Sequences containing such substitutions are suitable for the compositions and methods featured in this disclosure.

[0304] As used interchangeably herein, the terms "iRNA," "RNAi agent," "iRNA agent," and "RNA interference agent" refer to an agent that comprises an RNA, as that term is defined herein, and mediates targeted cleavage of an RNA transcript via the RNA-induced silencing complex (RISC) pathway. RNA interference (RNAi) is a process that governs the sequence-specific degradation of mRNA. RNAi knocks down (i.e., reduces the amount of) or regulates (i.e., inhibits) the expression of C9orf72, a C9orf72-associated transcript, or a C9orf72-associated peptide (e.g., a dipeptide repeat) in a cell, e.g., a cell within a subject, such as a mammalian subject.

[0305] In one embodiment, the RNAi agent of the present disclosure comprises a single-stranded RNAi that interacts with a target RNA sequence, e.g., a C9orf72 target mRNA sequence (either a sense or antisense RNA transcript sequence), to mediate cleavage of the target RNA. Without wishing to be bound by theory, it is believed that long double-stranded RNAs introduced into cells are degraded into double-stranded small interfering RNAs (siRNAs) comprising a sense strand and an antisense strand by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, an RNase III-like enzyme, processes these dsRNAs into 19-23 base pair small interfering RNAs with characteristic two-base 3' overhangs (Bernstein, et al., (2001) Nature 409:363). These siRNAs are then incorporated into RNA-induced silencing complex (RISC), where one or more helicases can unwind the siRNA duplex, thereby inducing target recognition for complementary antisense strands (Nykanen, et al., (2001) Cell 107:309). When bound to appropriate target mRNA, one or more endonucleases in RISC cleave the target and induce silencing (Elbashir, et al., (2001) Genes Dev.15:188). Therefore, in one aspect, the present disclosure relates to single-stranded RNA (ssRNA) (the antisense strand of the siRNA duplex) that is produced in cells and promotes the formation of RISC complex to silence target gene, i.e., C9orf72 gene. Therefore, the term "siRNA" is used herein to also mean the RNAi described above.

[0306] In another embodiment, an RNAi agent may be a single-stranded RNA introduced into a cell or organism to inhibit a target mRNA. The single-stranded RNAi agent binds to the RISC endonuclease Argonaute 2, which then cleaves the target mRNA. Single-stranded siRNAs are generally 15-30 nucleotides long and chemically modified. The design and testing of single-stranded RNAs are described in U.S. Patent No. 8,101,348 and Lima et al., (2012) Cell 150:883-894, the entire contents of each of which are incorporated herein by reference. Any of the antisense nucleotide sequences described herein may be used as single-stranded siRNAs described herein or as single-stranded siRNAs chemically modified by the methods described in Lima et al., (2012) Cell 150:883-894.

[0307] In another embodiment, an "RNAi agent" for use in the compositions and methods of the present disclosure is double-stranded RNA, and is also referred to herein as a "double-stranded RNAi agent," "double-stranded RNA (dsRNA) molecule," "dsRNA agent," or "dsRNA." The term "dsRNA" refers to a complex of ribonucleic acid molecules having a double-stranded structure comprising two antiparallel, substantially complementary nucleic acid strands, referred to as having a "sense" or "antisense" orientation with respect to a target RNA, i.e., the sense strand of the C9orf72 gene or the antisense strand of the C9orf72 gene. In some embodiments of the present disclosure, the double-stranded RNA (dsRNA) induces degradation of the target RNA, e.g., mRNA, via a post-transcriptional gene silencing mechanism referred to herein as RNA interference or RNAi.

[0308] The dsRNA agents described herein can be distinct from (ie, do not include) antisense oligonucleotides (ASOs) or gapmer antisense oligonucleotides (ASOs).

[0309] In some embodiments, any of the antisense oligonucleotide sequences disclosed herein can be used alone as an ASO or ribozyme. The ASO can comprise 16-20 contiguous nucleotides from any of the described antisense oligonucleotide sequences. In some embodiments, the ASO targets the same target RNA region as any of the described dsRNAs. The ASO can downregulate the target by inducing RNase H endonucleolytic cleavage of the target RNA, sterically hindering ribosomal activity, inhibiting 5' cap formation, or altering splicing. The ASO can be a gapmer or morpholino. A "gapmer" is an oligonucleotide containing an internal region with multiple nucleosides that support RNase H cleavage, positioned between external regions with one or more nucleosides, where the nucleosides comprising the internal region are chemically distinct from the nucleoside(s) comprising the external regions. The internal region may be referred to as the "gap," and the external regions may be referred to as "wings." A gapmer can have 5' and 3' wings, each having 2 to 6 nucleotides, and a gap having 7 to 12 nucleotides. Gapmers can have a 3-10-3 or 5-10-5 configuration. All of the nucleotides of a gapmer have phosphorothioate linkages, optionally with one or more chiral mesyl-phosphoramidate- or methylphosphonate-linked nucleotides. The wing nucleotides can be, but are not limited to, 2'-O-methoxyethyl (2'-MOE)-modified nucleotides, LNA-modified nucleotides, cET-modified nucleotides, or combinations thereof. The gap nucleotide can be a deoxyribonucleotide. Any cytosine nucleotide in the ASO can be methyl-cytosine.

[0310] Generally, dsRNA molecules can contain ribonucleotides, but as described in detail herein, each or both strands can also contain one or more non-ribonucleotides, such as deoxyribonucleotides, modified nucleotides.In addition, as used herein, "RNAi agent" can include ribonucleotides with chemical modifications, and RNAi agents can contain substantial modifications in multiple nucleotides.As used herein, the term "modified nucleotide" refers to a nucleotide that independently has a modified sugar moiety, a modified internucleotide linkage, or a modified nucleobase.Thus, the term modified nucleotide encompasses the substitution, addition, or removal of, for example, functional groups or atoms, to the internucleoside linkage, sugar moiety, or nucleobase.Modifications suitable for use in the agents of the present disclosure include 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 purposes of this specification and claims.

[0311] In certain embodiments of the present disclosure, the inclusion of deoxy-nucleotides, if present within an RNAi agent, can be considered to constitute modified nucleotides.

[0312] The double-stranded region may be of any length that allows for specific degradation of the desired target RNA via the RISC pathway, and may be 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 22, 18 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 24, 18 to 25, 18 to 26, 18 to 27, 18 to 28, 18 to 29, 18 to 30, 18 to 29, 18 to 31, 18 to 21, 18 to 22, 18 to 23, 18 to 24, 18 to 25, 18 to 26, 18 to 29, 18 to 26, 18 to 26, 18 to 27, The length may be in the range of 8, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 30, 20 to 29, 20 to 28, 20 to 27, 20 to 26, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, or 21 to 22 base pairs. In certain embodiments, the double-stranded region is 19-21 base pairs in length, e.g., 21 base pairs in length. Ranges and lengths between the ranges and lengths listed above are also intended to be part of this disclosure.

[0313] The two strands forming the double-stranded structure may be different portions of a single larger RNA molecule, or they may be separate RNA molecules. When the two strands are part of a single larger molecule and are therefore connected by an uninterrupted chain of nucleotides between the 3' end of one strand and the corresponding 5' end of the other strand forming the double-stranded structure, the connected RNA strands are referred to as a "hairpin loop." A hairpin loop may contain at least one unpaired nucleotide. In some embodiments, a hairpin loop may contain at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23, or more unpaired nucleotides or nucleotides not directed toward the target site of the dsRNA. In some embodiments, a hairpin loop may be 10 or fewer nucleotides. In some embodiments, a hairpin loop may be 8 or fewer unpaired nucleotides. In some embodiments, a hairpin loop may be 4 to 10 unpaired nucleotides. In some embodiments, a hairpin loop may be 4 to 8 nucleotides.

[0314] When the two substantially complementary strands of dsRNA are composed of separate RNA molecules, these molecules can, but do not necessarily, be covalently connected. In certain embodiments, the two strands are covalently linked by means other than an uninterrupted chain of nucleotides between the 3'-end of one strand and the corresponding 5'-end of the other strand that form a double-stranded structure, this connecting structure is referred to as a "linker" (note, however, that certain other structures as defined elsewhere herein can also be referred to as "linkers"). The RNA strands may have the same number of nucleotides or different numbers of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of dsRNA minus any overhangs present in the double strand. In addition to the double-stranded structure, RNAi may also include one or more nucleotide overhangs. In one embodiment of an RNAi agent, at least one strand includes a 3'-overhang of at least one nucleotide. In another embodiment, at least one strand comprises a 3' overhang of at least two nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments, at least one strand of the RNAi agent 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, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In yet other embodiments, both the 3' end and the 5' end of one strand of the RNAi agent comprise an overhang of at least one nucleotide.

[0315] In one embodiment, an RNAi agent of the 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, and directs cleavage of the target RNA.

[0316] In some embodiments, the iRNA of the invention is a 24-30 nucleotide dsRNA that interacts with a target RNA sequence, for example, a C9orf72 target mRNA sequence, and mediates cleavage of the target RNA.

[0317] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide protruding from the double-stranded structure of an RNAi agent, such as a dsRNA. For example, a nucleotide overhang exists when the 3'-end of one strand of a dsRNA extends beyond the 5'-end of the other strand, or vice versa. A dsRNA can include an overhang of at least one nucleotide, or the overhang can include at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more. A nucleotide overhang can comprise or consist of a nucleotide / nucleoside analog, such as a deoxynucleotide / nucleoside. An overhang can be on the sense strand, the antisense strand, or any combination thereof. Furthermore, a given overhanging nucleotide can be present on the 5'-end, the 3'-end, or both ends of either the antisense strand or the sense strand of a dsRNA.

[0318] In one embodiment, the antisense strand of the dsRNA has an overhang of 1 to 10 nucleotides at the 3' or 5' end, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In one embodiment, the sense strand of the dsRNA has an overhang of 1 to 10 nucleotides at the 3' or 5' end, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In another embodiment, one or more of the nucleotides in the overhang are replaced with a nucleoside thiophosphate.

[0319] In certain embodiments, the antisense strand of the dsRNA has an overhang of 1 to 10 nucleotides at the 3' or 5' end, for example, 0 to 3, 1 to 3, 2 to 4, 2 to 5, 4 to 10, 5 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In one embodiment, the sense strand of the dsRNA has an overhang of 1 to 10 nucleotides at the 3' or 5' end, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In another embodiment, one or more of the nucleotides in the overhang are substituted with a nucleoside thiophosphate.

[0320] In certain embodiments, the overhang on the sense strand or the antisense strand can comprise an extended length of more than 10 nucleotides, for example, a length of 1 to 30 nucleotides, 2 to 30 nucleotides, 10 to 30 nucleotides, or 10 to 15 nucleotides. In certain embodiments, the extended overhang is on the sense strand of the duplex. In certain embodiments, the extended overhang is on the 3'-end of the sense strand of the duplex. In certain embodiments, the extended overhang is on the 5'-end of the sense strand of the duplex. In certain embodiments, the extended overhang is on the antisense strand of the duplex. In certain embodiments, the extended overhang is on the 3'-end of the antisense strand of the duplex. In certain embodiments, the extended overhang is on the 5'-end of the antisense strand of the duplex. In certain embodiments, one or more of the nucleotides in the overhang are substituted with a nucleoside thiophosphate. In certain embodiments, the overhang comprises a self-complementary portion that enables the overhang to form a stable hairpin structure under physiological conditions.

[0321] In certain embodiments, at least one end of at least one strand extends beyond the double-stranded targeting region, including structures in which one of the strands contains a thermodynamically stable tetraloop structure (see, e.g., U.S. Pat. Nos. 8,513,207 and 8,927,705 and WO 2010 / 033225, the contents of each of which are incorporated by reference in their entirety). Such structures may include single-stranded extensions (on one or both sides of the molecule) and double-stranded extensions.

[0322] 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 comprises a tetraloop sequence. In certain embodiments, the sense strand is 25 to 35 nucleotides in length.

[0323] The tetraloop may comprise ribonucleotides, deoxyribonucleotides, modified nucleotides, and combinations thereof. Typically, a tetraloop has 4-5 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'-fhioro-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 preferred embodiments, 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. See, for example, PCT Publication No. WO 2020 / 206350, the entire contents of which are incorporated herein by reference.

[0324] Exemplary 2' adem-modified nucleotides are shown below. [ka]

[0325] The term "blunt" or "blunt-ended" as used herein with respect to dsRNA means that there are no unpaired nucleotides or nucleotide analogs at a given end of the dsRNA, i.e., there are no nucleotide overhangs. One or both ends of the dsRNA can be blunt. If 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 molecules will be double-stranded throughout their entire length.

[0326] The term "antisense strand" or "guide strand" of an RNAi agent refers to the strand of an RNAi agent, e.g., dsRNA, that includes a region that is substantially complementary to a target sequence, e.g., C9orf72 mRNA.

[0327] As used herein, the term "region of complementarity," as defined herein, refers to a region on the antisense strand that is substantially complementary to a sequence, e.g., a target sequence, e.g., a C9orf72 nucleotide sequence. If the region of complementarity is not perfectly complementary to the target sequence, the mismatch can be within an internal region or a terminal region of the molecule. Generally, the most tolerable mismatch is within the terminal region, e.g., within 5, 4, 3, or 2 nucleotides of the 5' or 3' end of the RNAi agent. In some embodiments, a double-stranded RNA agent of the invention contains nucleotide mismatches within the antisense strand. In some embodiments, the antisense strand of a double-stranded RNA agent of the invention contains four or fewer mismatches with the target mRNA, e.g., the antisense strand contains four, three, two, one, or zero mismatches with the target mRNA. In some embodiments, the antisense strand of a double-stranded RNA agent of the 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 4, 3, 2, 1, or 0 mismatches with the antisense strand. In some embodiments, the nucleotide mismatch is within, e.g., 5, 4, or 3 nucleotides from the 3' end of the iRNA. In another embodiment, the nucleotide mismatch is within, e.g., the 3' terminal nucleotide of the iRNA agent. In some embodiments, the mismatch is not in the seed region.

[0328] Thus, the RNAi agents described herein may contain one or more mismatches to the target sequence. In one embodiment, the RNAi agents described herein contain three or fewer mismatches (i.e., three, two, one, or zero mismatches). In one embodiment, the RNAi agents described herein contain two or fewer mismatches. In one embodiment, the RNAi agents described herein contain one or fewer mismatches. In one embodiment, the RNAi agents described herein contain zero mismatches. In certain embodiments, when the antisense strand of an RNAi agent contains a mismatch to the target sequence, this mismatch can optionally be limited to be within the last five nucleotides from either the 5'-end or the 3'-end of the complementary region. For example, in such an embodiment, in the case of a 23-nucleotide RNAi agent, the strand complementary to a region of the C9orf72 gene generally does not contain any mismatches within the central 13 nucleotides. Using the methods described herein or known in the art, it is possible to determine whether an RNAi agent containing a mismatch to a target sequence is effective in inhibiting expression of the C9orf72 gene. It is important to consider the effectiveness of an RNAi agent with a mismatch in inhibiting expression of the C9orf72 gene, particularly when a particular complementary region in the C9orf72 gene is known to have polymorphic sequence variation within the population. In some embodiments, the RNAi agent contains a single nucleotide mismatch with the target sequence, and the mismatch occurs at the 3' or 5' end of the RNAi agent. The mismatch can be in the antisense strand, the sense strand, or both the sense and antisense strands. For RNAi agents with 3' or 5' terminal mismatches with the target RNA on both the sense and antisense strands, the terminal nucleotides of the sense and antisense strands can be base-paired. Therefore, for any of the antisense or sense sequences described herein, the 5' or 3' nucleotide can be a replacement for the nucleotide that forms a mismatch with the target RNA.

[0329] As used herein, "substantially all of the nucleotides are modified" means extensively but not entirely modified and may include no more than 5, 4, 3, 2, or 1 unmodified nucleotides.

[0330] The term "sense strand" or "passenger strand" of an RNAi agent refers to the strand of an RNAi agent that includes a region that is substantially complementary to a region of the antisense strand, as that term is defined herein.

[0331] As used herein, the term "cleavage region" refers to a region located immediately adjacent to the cleavage site. The cleavage site is the site on the target where cleavage occurs. In some embodiments, the cleavage region comprises three bases immediately adjacent to either end of the cleavage site. In some embodiments, the cleavage region comprises two bases immediately adjacent to either end of the cleavage site. In some embodiments, the cleavage site specifically occurs at the site bounded by nucleotides 10 and 11 of the antisense strand, and the cleavage region comprises nucleotides 11, 12, and 13.

[0332] As used herein, unless otherwise indicated, the term "complementary," when used to describe a first nucleotide sequence in the context of a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising a first nucleotide sequence to hybridize to form a double-stranded structure with an oligonucleotide or polynucleotide comprising the second nucleotide sequence under specified conditions, as would be understood by one of skill in the art. Such conditions can 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 a wash (see, e.g., "Molecular Cloning: A Laboratory Manual," Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). Other conditions, such as physiologically relevant conditions that may be encountered in an organism, can be applied. Those skilled in the art can determine the most appropriate set of conditions for testing the complementarity of two sequences depending on the ultimate use of the hybridized nucleotides.

[0333] A complementary sequence in an RNAi agent, such as in the dsRNA described herein, comprises base pairing of an oligonucleotide or polynucleotide comprising a first nucleotide sequence with an oligonucleotide or polynucleotide comprising a second nucleotide sequence throughout the entire length of one or both nucleotide sequences. Such sequences may be referred to herein as "fully complementary" to each other. However, when a first sequence is referred to herein as "substantially complementary" to a second sequence, the two sequences may be fully complementary, or they may form one or more, but generally no more than 5, 4, 3, or 2 mismatched base pairs during hybridization of a duplex of up to 30 base pairs, while maintaining the ability to hybridize under conditions most relevant to its final application, such as inhibiting gene expression via the RISC pathway. However, if two oligonucleotides are designed to form one or more single-stranded overhangs during hybridization, these overhangs shall not be considered mismatches when determining complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, where the longer oligonucleotide comprises a 21-nucleotide sequence that is perfectly complementary to the shorter oligonucleotide, may still be referred to as "fully complementary" for purposes described herein.

[0334] "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 they satisfy the above requirements regarding their ability to hybridize, including, but not limited to, G:U wobble or Hoogsteen base pairing.

[0335] The terms "complementary," "fully complementary," and "substantially complementary" herein may be used 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 will be understood from the context of their use.

[0336] As used herein, a polynucleotide that is "substantially complementary to at least a portion of" an RNA transcript refers to a polynucleotide that is substantially complementary to a contiguous portion of an RNA transcript of interest (e.g., a C9orf72 RNA, either the sense strand or the antisense strand). For example, a polynucleotide is complementary to at least a portion of a C9orf72 RNA if the sequence is substantially complementary to an uninterrupted portion of the RNA.

[0337] 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% 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 the equivalent region of the nucleotide sequence of any one of SEQ ID NOs: 1-4, 9, 11, 13, 15, 17, and 19, over its entire length.

[0338] As described above, the pathogenic large GGGGCC (G4C2) hexanucleotide repeat expansion in the first intron of the C9orf72 gene between exons 1a and 1b can be bidirectionally transcribed. Thus, in some embodiments, antisense strand polynucleotides complementary to either strand of the C9orf72 gene are disclosed herein. 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% 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 over its entire length to the equivalent region of the nucleotide sequence of any one of SEQ ID NOs: 5-8, 10, 12, 14, 16, 18, or 20.

[0339] In some embodiments, the antisense polynucleotides disclosed herein are substantially complementary to a fragment of a target C9orf72 sequence, including nucleotides 27573296-27573318, 27573314-27573336, 27573319-27573341, 27573562-27573584, 27573585-27573607, 27573592-27573614, 27573599-27573621, 27573608-27573630, 27573599-27573641, 27573599-27573650, 27573599-27573661, 27573599-27573671, 27573599-27573681, 27573599-27573692, 27573599-27573603, 27573599-27573614, 27573599-27573621, 27573608-27573630, 27573599-27573641, 27573599-27573652, 27573599-27573662, 27573599-27573674, 27573599-27573685, 27573599-27573696, 27573599-27573610, 27 The fragment of SEQ ID NO: 13 selected from the group consisting of 3616 to 27573638, 27573619 to 27573641, 27573622 to 27573644, 27573633 to 27573655, 27573690 to 27573712, and 27573717 to 27573739 comprises 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 over its entire length to a fragment of SEQ ID NO: 13 selected from the group consisting of 3616 to 27573638, 27573619 to 27573641, 27573622 to 27573644, 27573633 to 27573655, 27573690 to 27573712, and 27573717 to 27573739.

[0340] In some embodiments, the antisense polynucleotides disclosed herein are substantially complementary to a fragment of a target C9orf72 sequence and comprise 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 over its entire length to a fragment of SEQ ID NO: 1, such as nucleotides 1-23, 15-37, 33-55, 37-59, 62-84, or 69-91 of SEQ ID NO: 1. Ranges intermediate to the above-listed ranges are also contemplated as part of the present disclosure.

[0341] In some embodiments, the antisense polynucleotides disclosed herein are substantially complementary to a fragment of a target C9orf72 sequence, including nucleotides 5197-5219, 5223-5245, 5226-5248, 5227-5249, 5233-5255, 5248-5270, 5539-5561, 5547-5569, 5917-5939, 5936-5958, 5940-5949, 5950-5951, 5952-5953, 5954-5955, 5956-5958, 5958-5959, 5960-5961, 5962-5963, 5964-5965, 5966-5967, 5968-5969, 5970-5971, 5972-5972, 5974-5975, 5976-5976, 5978-5979, 5979-5980, 5981-5981, 5982-5982, 5983-5983, 5984-5985, 5986-5986, 5988-5989, 5990-5991, 5992-5993, 5994-5995, 5996-5996, 5998-6000, 5999-6001, 5999-6002, 6003-6004, 6005-6006, 6007-6008, 6009 , 5954-5976, 6008-6030, 6021-6043, 6036-6058, 6043-6065, and 6048-6070. Ranges intermediate to the above-listed ranges are also contemplated as part of this disclosure.

[0342] In some embodiments, the antisense polynucleotides disclosed herein are substantially complementary to a fragment of a target C9orf72 sequence, such as nucleotides 5015-5052, 5017-5040, 5032-5059, 5032-5055, 5033-5055, 5035-5059, 5036-5059, 5058-5087, 5059 of SEQ ID NO: 15. 9~5087, 5059~5084, 5064~5087, 5197~5222, 5213~5267, 5223~5252, 5229~5252, 5233~5263, 5516~5570, 5539~5565, 5539~5562, 5545~5570, 5545~5569, 5593~5616, 5883~5950, 5917~5950, 5919~5950 , 5923~5950, 5934~5977, 5934~5957, 5938~5977, 5938~5965, 5938~5961, 5947~5977, 5947~5973, 5972~6001, 5973~5997, 6006~6029, 6011~6070, 6011~6039, 6011~6038, 6015~6038, 6019~6045, 6019~ 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 over its entire length to a fragment of SEQ ID NO: 15, such as 6042, 6033-6070, 6035-6065, 6035-6059, or 6040-6063. Ranges intermediate to the above-listed ranges are also contemplated as part of this disclosure.

[0343] In some embodiments, the antisense polynucleotides disclosed herein are substantially complementary to a fragment of a target C9orf72 sequence and comprise 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 over its entire length to a fragment of SEQ ID NO: 1, such as nucleotides 15-52, 17-40, 32-59, 32-55, 35-59, 36-59, 58-87, 59-87, 59-84, or 64-87 of SEQ ID NO: 1. Ranges intermediate to the above-listed ranges are also contemplated as part of the present disclosure.

[0344] In some embodiments, the antisense polynucleotides disclosed herein are substantially complementary to a fragment of a target C9orf72 sequence, including nucleotides 27573296-27573584, 27573296-27573575, 27573301-27573338, 27573318-27573342, 27573555-27573583, 27573581-27573607, 27573584-27573607, 27573588-27573671, 27573588-27573666, 27573588-27573624, 27573592-27573593 of SEQ ID NO: 13. 27573606 to 27573652, 27573606 to 27573647, 27573654 to 27573712, or 27573707 to 27573740. Ranges intermediate to the above-listed ranges are also contemplated as part of this disclosure.

[0345] In other embodiments, the sense polynucleotides disclosed herein are substantially complementary to a target C9orf72 sequence and comprise a contiguous nucleotide sequence that is at least about 80% complementary, 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 Tables 2, 3, 10A, 10C, 11, or 12, or to a fragment of any one of the sense strand nucleotide sequences in any one of Tables 2, 3, 10A, 10C, 11, or 12.

[0346] 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 about 80% complementary, 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 Tables 5, 6, 10B, or 10D, or to a fragment of any one of the sense strand nucleotide sequences in any one of Tables 5, 6, 10B, or 10D.

[0347] In certain embodiments, the sense strand and the antisense strand are selected from any one of the duplexes AD-1446213.1, AD-1446217.1, AD-1446222.1, AD-1446234.1, AD-1446243.1, AD-1446246.1, AD-1446252.1, AD-1446259.1, AD-1446265.1, AD-1446268.1, AD-1446271.1, AD-1446279.1, AD-1446289.1, and AD-1446294.1.

[0348] In certain embodiments, the sense strand and the antisense strand are selected from any one of the duplexes AD-1446213.1, AD-1446246.1, and AD-1446268.1.

[0349] In certain embodiments, the sense strand and the antisense strand are selected from any one of the duplexes AD-1446073.1, AD-1446075.1, AD-1285246.2, AD-1446084.1, AD-1446087.1, AD-1446090.1, and AD-1446095.1.

[0350] In certain embodiments, the sense and antisense strands are selected from any one of the duplexes AD-1446087.1 and AD-1446090.1.

[0351] In certain embodiments, the sense and antisense strands are selected from any one of the duplexes AD-1285238.1 and AD-1285234.1.

[0352] In certain embodiments, the sense strand and the antisense strand are the duplex AD-1285231.1, AD-1285232.1, AD-1285233.1, AD-1285235.1, AD-1285237.1, AD-1285239.1, AD-1285240.1, AD-1285242.1, AD-1285244.1, AD-1285243.1, AD-128 5241.1, AD-1285236.1, AD-1446111.1, AD-1446117.1, AD-1446147.1, AD-1446157.1, AD-1446168.1, AD-1446180.1, AD-1446189.1, AD-1446196.1, AD-1446202.1, AD-1446205.1.

[0353] In certain embodiments, the sense strand and the antisense strand are selected from any one of the duplexes AD-1285231.1, AD-1285232.1, AD-1285233.1, AD-1285234.1, AD-1285235.1, AD-1285236.1, AD-1285237.1, AD-1285239.1, AD-1285240.1, AD-1285241.1, AD-1285242.1, and AD-1285243.1.

[0354] 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 about 80% complementary, e.g., about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% complementary to any one of the sense strand nucleotide sequences in any one of Tables 8 or 9, or a fragment of any one of the sense strand nucleotide sequences in any one of Tables 8 or 9, over its entire length.

[0355] As used herein, the phrase "inhibiting C9orf72 expression" includes inhibiting expression of mature C9orf72 mRNA, knocking down or inhibiting expression or reducing the level of C9orf72 RNA containing hexanucleotide repeats in introns, and knocking down or inhibiting expression or reducing the level of the antisense strand of C9orf72 RNA containing hexanucleotide repeats. Knocking down or inhibiting expression or reducing the level of C9orf72 RNA containing hexanucleotide repeats includes inhibiting the production of sense and antisense C9orf72-containing foci and / or inhibiting the production of aberrant dipeptide repeat (DPR) proteins (e.g., poly(glycine-alanine) or poly(GA) peptides, poly(glycine-proline) or poly(GP) peptides, poly(glycine-arginine) or poly(GR) peptides, poly(alanine-proline) or poly(PA) peptides, or poly(proline-arginine) or poly(PR) peptides). In some embodiments, the repeat length-dependent formation of RNA foci, the fractionation of specific RNA-binding proteins, or the 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 is inhibited or reduced by more than 50%, e.g., more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, or more than 95%, and expression of C9orf72 mature RNA is inhibited or reduced by less than 50%, e.g., less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, or less than 5%.

[0356] In one embodiment, at least partial suppression of expression of the C9orf72 gene is assessed by a reduction in the amount of C9orf72 RNA, e.g., sense RNA transcripts, antisense RNA transcripts, total C9orf72 RNA transcripts, sense C9orf72 repeat-containing RNA transcripts, and / or antisense C9orf72 repeat-containing RNA transcripts, which can be isolated from or detected in a first cell or group of cells in which the C9orf72 gene is transcribed and which have been treated to inhibit expression of the C9orf72 gene. The degree of inhibition is assessed by a decrease in the amount of RNA transcripts, sense C9orf72 repeat-containing RNA transcripts, and / or antisense C9orf72 repeat-containing RNA transcripts compared to a second cell or group of cells that is substantially identical to the first cell or group of cells but has not been treated like the first cell or group of cells (control cells). The degree of inhibition can be expressed as:

number

[0357] As used herein, the phrase "contacting cells with RNAi agent" such as dsRNA includes contacting cells by any possible means.Contacting cells with RNAi agent includes contacting cells with RNAi agent in vitro or contacting cells with RNAi agent in vivo.Contacting can be carried out directly or indirectly.Therefore, for example, RNAi agent can be physically contacted with cells by separately carrying out a method, or RNAi agent can be placed in a situation that can allow or cause it to contact cells afterwards.

[0358] Contacting cells in vitro may be performed, for example, by incubating the cells with an RNAi agent. Contacting cells in vivo may be performed, for example, by injecting the RNAi agent into or near the tissue in which the cells are located, or by injecting the RNAi agent into another region, such as the central nervous system (CNS), optionally by intrathecal, intravitreal, or other injection, or by injecting the RNAi agent into the bloodstream or subcutaneous space so that the agent subsequently reaches the tissue in which the cells to be contacted are located. For example, the RNAi agent may include or be coupled to a ligand that directs or otherwise stabilizes the RNAi agent to the desired site, for example, in the CNS, such as a lipophilic moiety, as described below and further detailed in, for example, PCT / US2019 / 031170, which is incorporated herein by reference. A combination of in vitro and in vivo contacting methods is also possible. For example, cells may be contacted with an RNAi agent in vitro and then transferred to a subject.

[0359] In one embodiment, contacting a cell with an RNAi agent includes "introducing" or "delivering an RNAi agent into a cell" by promoting or effecting uptake or absorption into the cell. Absorption or uptake of the RNAi agent can occur by spontaneous diffusive or active cellular processes, or by auxiliary agents or devices. Introduction of an RNAi agent into a cell can be in vitro or in vivo. For example, for in vivo introduction, the RNAi agent can be injected into a tissue site or administered systemically. In vitro introduction into a cell can include methods known in the art, such as electroporation and lipofection. Additional approaches are described herein below or known in the art.

[0360] The terms "lipid-soluble" or "lipophilic moiety" refer broadly to any compound or chemical moiety that has an affinity for lipids. One way to characterize the lipophilicity of a lipophilic moiety is by the octanol-water partition coefficient, log K ow In this case, K ow is the ratio of the concentration of a chemical in the octanol phase to the concentration of the chemical in the aqueous phase of a two-phase system at equilibrium. The octanol-water partition coefficient is a laboratory-measured property of a substance. However, it can also be predicted by using coefficients attributable to the structural components of a chemical calculated using first-principles or empirical methods (see, e.g., Tetko et al., J. Chem. Inf. Comput. Sci. 41:1407-21 (2001), incorporated herein by reference in its entirety). This provides a thermodynamic measure of a substance's tendency to prefer a non-aqueous or oily environment rather than water (i.e., its hydrophilic / lipophilic balance). In principle, a chemical can be expressed as its logK ow is greater than 0, the lipophilic moiety is lipophilic in nature. Typically, the lipophilic moiety has a logK ow is 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 K ow For example, the log K of cholesteryl N-(hexan-6-ol)carbamate is predicted to be approximately 0.7. ow is predicted to be 10.7.

[0361] The lipophilicity of a molecule can be changed depending on the functional groups it contains. For example, adding a hydroxyl or amine group to the end of the lipophilic moiety can increase the partition coefficient (e.g., logK ow ) may increase or decrease in value.

[0362] 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, if the unbound fraction of the plasma protein binding assay of double-stranded RNAi agent is determined to be positively correlated with the relative hydrophobicity of double-stranded RNAi agent, then it will be positively correlated with the silencing activity of double-stranded RNAi agent.

[0363] In one embodiment, the plasma protein binding assay to be determined is an electrophoretic mobility shift assay (EMSA) using human serum albumin protein. An exemplary protocol for this binding assay is described in detail, for example, in PCT / US2019 / 031170. The hydrophobicity of the double-stranded RNAi agent, as measured by the unbound fraction of siRNA in the binding assay, is greater than 0.15, greater than 0.2, greater than 0.25, greater than 0.3, greater than 0.35, greater than 0.4, greater than 0.45, or greater than 0.5, in the case of enhanced in vivo delivery of siRNA.

[0364] Thus, conjugating a lipophilic moiety to an internal position of a double-stranded RNAi agent provides optimal hydrophobicity for enhanced in vivo delivery in siRNA. In some embodiments, the lipophilic moiety facilitates or improves delivery of the RNAi agent to neural cells, or cells in neural tissue, or cells in central nervous system tissue.

[0365] The term "lipid nanoparticle" or "LNP" refers to a vesicle comprising a lipid layer that encapsulates a pharmaceutically active molecule, such as a nucleic acid molecule, e.g., an RNAi agent or a plasmid from which an RNAi agent is transcribed. LNPs are described, for example, in U.S. Patent Nos. 6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents of which are incorporated herein by reference.

[0366] As used herein, a "subject" refers to an animal, such as a mammal, including a primate (human, non-human primate, e.g., monkey and chimpanzee) or a non-primate (e.g., rat or mouse). In preferred embodiments, the subject is a human, e.g., a human being treated or evaluated for a disease, disorder, or condition that would benefit from reduced levels of target C9orf72 RNA as described herein; a human at risk for a disease, disorder, or condition that would benefit from reduced levels of target C9orf72 RNA; a human having a disease, disorder, or condition that would benefit from reduced C9orf72 expression; or a human being treated for a disease, disorder, or condition that would benefit from reduced C9orf72 expression. In some embodiments, the subject is a female human. In other embodiments, the subject is a male human. In one embodiment, the subject is an adult subject. In one embodiment, the subject is a child subject. In another embodiment, the subject is a juvenile subject, i.e., a subject under the age of 20.

[0367] As used herein, the terms "treating" or "treatment" refer to a beneficial or desired outcome, for example, but not limited to, the alleviation or amelioration of one or more signs or symptoms associated with a C9orf72 hexanucleotide repeat expansion transcript or its dipeptide repeat product, e.g., a C9orf72-associated disease, such as a C9orf72-associated disease. "Treatment" can also mean increasing survival time compared to expected survival time in the absence of treatment.

[0368] The term "lower" in the context of C9orf72 levels or disease markers or symptoms in a subject refers to a statistically significant decrease in such levels. The decrease can be, for example, at least 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more. In certain embodiments, the decrease is at least 20%. In certain embodiments, the decrease is at least 50%, e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more, in the level of a disease marker, e.g., sense- or antisense-containing foci and / or the level of an aberrant dipeptide repeat protein. In some embodiments, the decrease is 50% or less, e.g., 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less, for C9orf72 protein and / or C9orf72 mRNA levels. "Reducing" in the context of C9orf72 levels in a subject preferably refers to reducing to a level that is accepted as within the normal range for individuals without such a disorder. In certain embodiments, "reducing" refers to a reduction in the difference between the level of a marker or symptom in a subject suffering from a disease and a level that an individual would consider within the normal range, e.g., a reduction in weight between an obese individual and an individual whose weight is considered within the normal range.

[0369] As used herein, "prevention" or "preventing," when used in reference to a disease, disorder, or condition that would benefit from reduced expression of a C9orf72 hexanucleotide repeat expansion or its dipeptide product, refers to a reduced likelihood that a subject will develop symptoms associated with such disease, disorder, or condition, e.g., symptoms of a C9orf72-associated disease. A failure to develop the disease, disorder, or condition, or a reduction in the onset of symptoms associated with such disease, disorder, or condition (e.g., a reduction of at least about 10% of a clinically acceptable magnitude for the disease or disorder), or a delay in the onset of symptoms (e.g., a delay of days, weeks, months, or years) is considered effective prevention.

[0370] As used herein, the term "C9orf72-associated disease" or "C9orf72-associated disorder" includes any disease or disorder that may benefit from reduced expression and / or activity of a C9orf72 hexanucleotide repeat expansion transcript. Exemplary C9orf72-associated diseases include diseases in which a subject carries a hexanucleotide repeat (GGGCC) expansion in the intron between exons 1a and 1b of the C9orf72 gene, such as amyotrophic lateral sclerosis, frontotemporal dementia, Huntington's disease, e.g., Huntington-like syndromes caused by C9orf72 expansions, Parkinsonism, olivopontocerebellar degeneration, corticobasal syndrome, or Alzheimer's disease.

[0371] Normal G4C2 repeats are approximately 25 units or less, while highly penetrant disease alleles are typically larger than approximately 60 repeat units, ranging up to over 4,000 units. Rarely, 47-60 repeats segregate with the disease within families. Repeat-primed PCR assays are typically used to detect smaller expansions (less than 80), but accurate sizing of larger repeats requires other techniques (e.g., Southern blot hybridization) that provide length estimates.

[0372] Because subjects with a GGGGCC (or G4C2) hexanucleotide expansion in an intron of the C9orf72 gene can manifest as amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD), even within the same family, the neurodegeneration associated with this expansion is referred to herein as "C9orf72 amyotrophic lateral sclerosis / frontotemporal dementia" or "C9orf72 ALS / 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 of clinical ALS. It is also a common cause of FTD, accounting for approximately one-quarter of familial FTD cases. The age at onset of symptoms ranges from 30 to 70 years, with the average onset occurring in the late 50s. C9orf72-mediated ALS often resembles typical ALS, can have bulbar or limb onset, can (but does not necessarily) progress rapidly, and can be associated with later cognitive symptoms. For this reason, C9orf72-mediated ALS is evaluated and treated similarly to any 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 executive dysfunction. Less commonly, C9orf72-mediated FTD presents with semantic variant primary progressive aphasia (PPA) or non-fluent variant PPA, and in very rare cases, it can resemble corticobasal syndrome, progressive supranuclear palsy, or HD-like syndrome. Parkinsonian features may also be present in C9orf72-mediated ALS or FTD.

[0373] Subjects may present with frontotemporal lobar degeneration (FTLD), characterized by progressive changes in behavior, executive dysfunction, and / or language impairment. Of the three FTLD clinical syndromes, behavioral variant FTD (bvFTD) is the most frequent, but not exclusive, entity. It is characterized by progressive behavioral disturbances and decline in executive function accompanied by predominant frontal lobe atrophy on brain MRI. Motor neuron disease, including upper motor neuron dysfunction or lower motor neuron dysfunction (or both), may also be present, which may or may not meet criteria for the full ALS phenotype. Many individuals with C9orf72-associated bvFTD have some degree of parkinsonism, typically rigidity without tremor and unresponsive to levodopa.

[0374] Huntington's disease-like syndromes (HD-like syndromes, or HDL syndromes) are a family of inherited neurodegenerative disorders that closely resemble Huntington's disease (HD) in that they typically produce a combination of chorea, cognitive decline or dementia, and behavioral or psychiatric disturbances.

[0375] Subjects with Huntington's disease-like syndrome caused by C9orf72 expansion are characterized by movement disorders including dystonia, chorea, myoclonus, tremor, and rigidity. Related features also include cognitive and memory impairment, early psychiatric disturbances, and behavioral disturbances. The average age at onset is approximately 43 years (range 8-60). Early mental and behavioral disturbances (including depression, apathy, obsessive-compulsive behavior, and psychosis) are common. Cognitive symptoms manifest as executive dysfunction. Movement disorders are prominent. Parkinsonian features and pyramidal features may also be present. As used herein, a "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, the method of administration of the agent, the disease and its severity and medical history, age, weight, family history, genetic makeup, type of prior or concurrent treatment, if any, and other personal characteristics of the subject to be treated.

[0376] As used herein, a "prophylactically effective amount" is intended to include an amount of an RNAi agent that, when administered to a subject with a C9orf72-associated disorder, is sufficient to prevent or ameliorate the disease or one or more symptoms of the disease. Ameliorating the disease includes slowing the course of the disease or reducing the severity of any future disease. A "prophylactically effective amount" can vary depending on the RNAi agent, the method of administration of the agent, the degree of risk of the disease, and the patient's medical history, age, weight, family history, genetic makeup, type of prior or concurrent treatment, if any, and other personal characteristics of the patient to be treated.

[0377] A "therapeutically effective amount" or a "prophylactically effective amount" also encompasses an 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 agents used in the methods of the present disclosure can be administered in amounts sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0378] The term "pharmaceutically acceptable" is used herein to refer to those compounds, materials (including salts), compositions or dosage forms that are suitable for use in contact with the tissues of human and animal subjects without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio, within the scope of sound medical judgment.

[0379] The phrase "pharmaceutically acceptable carrier," as used herein, means 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, that is involved in the transport or transportation of a subject compound from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the subject being treated. Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants, such as magnesium state, sodium lauryl sulfate, and talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; and (13) agar. (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide, (15) alginic acid, (16) pyrogen-free water, (17) isotonic saline, (18) Ringer's solution, (19) ethyl alcohol, (20) pH buffer solutions, (21) polyesters, polycarbonates, or polyanhydrides, (22) bulking agents, such as polypeptides and amino acids, (23) serum components, such as serum albumin, HDL, and LDL, and (22) other non-toxic, compatible substances employed in pharmaceutical formulations.

[0380] The term "sample," as used herein, encompasses similar bodily fluids, cells, or tissues isolated from a subject, as well as collections of bodily fluids, cells, or tissues present within a subject. Examples of biological fluids include blood, serum, and serous fluid, plasma, cerebrospinal fluid, ocular fluid, lymphatic fluid, urine, saliva, and the like. Tissue samples may include samples from tissues, organs, or localized regions. For example, samples may be derived from specific organs, parts of organs, or bodily fluids or cells within those organs. In certain embodiments, samples may be obtained from the brain (e.g., the whole brain or specific segments of the brain, such as the striatum, or specific types of cells within the brain, such as neurons and glial cells (astrocytes, oligodendrocytes, microglia)). In some embodiments, a "sample obtained from a subject" refers to blood obtained from a subject or plasma or serum obtained therefrom. In further embodiments, a "sample obtained from a subject" refers to brain tissue (or a subcomponent thereof) or retinal tissue (or a subcomponent thereof) obtained from a subject.

[0381] II. RNAi Agents of the Disclosure As described elsewhere herein, mutations in C9orf72 are associated with familial frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). These mutations result in the expansion of a G4C2 (SEQ ID NO: 1) hexanucleotide repeat located within the intron between exons 1A and 1B of the C9orf72 gene. The hexanucleotide repeat may be translated through a mechanism other than non-AUG priming. Accumulation of the repeat expansion-containing RNA (target RNA) or translation of the repeat sequence may cause or contribute to FTD and / or ALS, or disease symptoms associated with FTD and / or ALS.

[0382] Thus, the present invention provides dsRNA agents that selectively and efficiently reduce the expression of C9orf72-associated expression products, RNAs, and / or translated polypeptides associated with hexanucleotide repeat expansions. In some embodiments, the dsRNA agents target (e.g., selectively target) hexanucleotide repeat-containing RNAs (target RNAs) and knock down the target RNAs and polypeptides expressed from the hexanucleotide repeat-containing RNAs. The dsRNA agents may be used in methods for the therapeutic treatment and / or prevention of signs or symptoms associated with FTD and / or ALS, including, but not limited to, the repeat-length-dependent formation of RNA foci resulting from repeat-associated non-AUG (AUG) translation in neurons, the compartmentalization 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)). The dsRNA agent may be used in a method for the therapeutic treatment and / or prevention of signs or symptoms associated with FTD and / or ALS, including, but not limited to, signs and symptoms of motor neuron disease and signs and symptoms of dementia. Signs and symptoms of motor neuron disease may include, for example, stumbling, dropping things, unusual fatigue in the arms and / or legs, slurred speech, muscle spasms and twitching, periods of uncontrollable laughing or crying, and difficulty breathing. Signs and symptoms of dementia may include, for example, behavioral changes, personality changes, speech and language disorders, and movement-related disorders. Such methods include administering one or more dsRNA agents described herein to a subject (e.g., a human or animal subject).

[0383] The dsRNA agents described herein can stop or reduce the accumulation of repeat-containing C9orf72 RNA (eg, assayed as RNA foci), thereby preventing RNA translation from synthesizing dipeptide repeat proteins.

[0384] In some embodiments, a dsRNA agent of the invention targets mature C9orf72 mRNA (i.e., mRNA with an intron spliced ​​out). In other embodiments, a dsRNA agent of the invention targets C9orf72 RNA that includes an intron, such as intron 1A (i.e., sense or antisense RNA with the intron not spliced ​​out, an RNA region spliced ​​out from a precursor mRNA, or an alternatively spliced ​​RNA).

[0385] dsRNA comprises two RNA strands, which are complementary and hybridize to form a double-stranded structure under the conditions in which the dsRNA is used. In some embodiments, one strand of the dsRNA (the antisense strand) comprises a complementary region that is substantially complementary, and generally completely complementary, to the target sequence. The target sequence can be obtained from the sequence of the RNA formed during the expression of the C9orf72 gene. The other strand (the sense strand) comprises a region that is complementary to the antisense strand, so that these two strands hybridize to form a double-stranded structure when combined under suitable conditions. In some embodiments, one strand of the dsRNA (the sense strand) comprises a complementary region that is substantially complementary, and generally completely complementary, to the target sequence obtained from the antisense sequence of the RNA formed during the expression of the C9orf72 gene. The other strand (antisense strand) comprises the region that is complementary to sense strand, so that when these two strands are combined under suitable conditions, they hybridize to form a double-stranded structure.As described elsewhere herein and known in the art, the complementary sequence of dsRNA can also be comprised as the self-complementary region of a single nucleic acid molecule, so that it is opposite on separate oligonucleotides.

[0386] Generally, the double-stranded structure is 15-30 base pairs in length, e.g., 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-22, 19-23, 19-24, 19-25, 19-26, 19-27, 19-28, 19-29, 19-30, 19-31, 19-32, 19-33, 19-34, 19-35, 19-36, 19-37, 19-38, 19-39, 19-40, 19-41, 19-42, 19-43, 19-44, 19-45, 19-46, 19-47, 19-48, 19-49, 19-50, 19-51, 19-52, 19-53, 19-54, 19-55, 19-56, 19-57, 19-58, 19-59, 19-60, 19-61, 19-62, 19-63, 19-64, 19-65, 1 The length is 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 certain preferred embodiments, the double-stranded structure is 18 to 25 base pairs in length, e.g., 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 25, 21 to 24, 21 to 23, 21 to 22, 22 to 25, 22 to 24, 22 to 23, 23 to 25, 23 to 24, or 24 to 25 base pairs in length, e.g., 19 to 21 base pairs in length. Ranges and lengths between the ranges and lengths listed above are also intended to be part of this disclosure.

[0387] Similarly, the region of complementarity to the target sequence may be 15-30 nucleotides in length, e.g., 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 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 that lie between the ranges and lengths listed above are also intended to be part of this disclosure.

[0388] In some embodiments, the double-stranded structure is 19-30 base pairs in length. Similarly, the region of complementarity to the target sequence is 19-30 nucleotides in length.

[0389] In some embodiments, the dsRNA is 15-23 nucleotides long, 19-23 nucleotides long, or 25-30 nucleotides long. Generally, the dsRNA is long enough to serve as a substrate for the Dicer enzyme. For example, it is well known in the art that dsRNAs longer than about 21-23 nucleotides can function as substrates for Dicer. As those skilled in the art will recognize, the region of an RNA targeted for cleavage will most often be a portion of a longer RNA molecule, often an mRNA molecule. Where relevant, a "portion" of an mRNA target is a contiguous sequence of the mRNA target long enough to allow it to serve as a substrate for RNAi-dependent cleavage (i.e., cleavage via the RISC pathway).

[0390] Those skilled in the art will appreciate that the double-stranded region is a primary functional portion of a dsRNA, e.g., about 15-36 base pairs, e.g., 15-36, 15-35, 15-34, 15-33, 15-32, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, It will also recognize a double-stranded region of 18 to 20, 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 30, 20 to 29, 20 to 28, 20 to 27, 20 to 26, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, or 21 to 22 base pairs, for example, 19 to 21 base pairs. That is, in one embodiment, an RNA molecule or complex of RNA molecules having a double-stranded region of more than 30 base pairs is a dsRNA, as long as it is processed into a functional duplex of, for example, 15-30 base pairs that targets the desired RNA for cleavage. Thus, those skilled in the art will recognize that, in one embodiment, an miRNA is a dsRNA. In another embodiment, the dsRNA is not a naturally occurring miRNA. In another embodiment, an RNAi agent useful for targeting C9orf72 expression is not generated in a target cell by cleavage of a larger dsRNA.

[0391] The dsRNA described herein can further comprise one or more single-stranded nucleotide overhangs, for example, 1, 2, 3, or 4 nucleotides. The nucleotide overhangs can comprise or consist of nucleotide / nucleoside analogs, such as deoxynucleotides / nucleosides. The overhangs can be on the sense strand, the antisense strand, or any combination thereof. Furthermore, a given overhang nucleotide can be present on the 5'-end, 3'-end, or both ends of either the antisense strand or the sense strand of the dsRNA.

[0392] 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.These component strands are then 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 containing 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.

[0393] Regardless of synthesis method, siRNA preparation can be prepared in a solution (for example, aqueous solution or organic solution) suitable for formulation.For example, siRNA preparation can be precipitated, redissolved in pure double distilled water, and lyophilized.Then, dried siRNA can be resuspended in a solution suitable for the intended formulation process.

[0394] In certain embodiments, a dsRNA agent of the invention targets a C9orf72 target RNA that contains a hexanucleotide repeat that includes multiple consecutive copies, e.g., a C9orf72 target RNA with 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).

[0395] In one embodiment, the dsRNA of the present disclosure comprises at least two nucleotide sequences, a sense strand and an antisense strand. The sense strand sequence for C9orf72 may be selected from the group of sequences provided in any one of Tables 2, 3, 5, 6, 8, 9, 10A, 10B, 10C, 10D, 11, and 12, and the corresponding nucleotide sequence of the antisense strand of this sense strand may be selected from the group of sequences provided in any one of Tables 2, 3, 5, 6, 8, 9, 10A, 10B, 10C, 10D, 11, and 12. In this embodiment, one of the two sequences is complementary to the other of the two sequences, and one of the sequences is substantially complementary to the sequence of the RNA generated upon expression of the C9orf72 locus. Thus, in this embodiment, the dsRNA will comprise two oligonucleotides, one oligonucleotide described as the sense strand (passenger strand) in any one of Tables 2, 3, 5, 6, 8, 9, 10A, 10B, 10C, 10D, 11, and 12, and the second oligonucleotide described as the corresponding antisense strand (guide strand) of the sense strand in any one of Tables 2, 3, 5, 6, 8, 9, 10A, 10B, 10C, 10D, 11, and 12.

[0396] In one embodiment, the substantially complementary sequences of the dsRNA are contained on separate oligonucleotides, hi another embodiment, the substantially complementary sequences of the dsRNA are contained on a single oligonucleotide.

[0397] Although the sequences in Tables 2 and 5 are described as modified or conjugated sequences, it is understood that an RNA of an RNAi agent of the disclosure, e.g., a dsRNA of the disclosure, can comprise any one of the sequences shown in any one of Tables 2, 3, 5, 6, 8, 9, 10A, 10B, 10C, 10D, 11, and 12, unmodified, unconjugated, or modified or conjugated differently than described herein.

[0398] Those skilled in the art are well aware that dsRNAs having a duplex structure of approximately 20-23 base pairs, e.g., 21 base pairs, have been hailed as particularly effective in inducing RNA interference (Elbashir et al., (2001) EMBO J., 20:6877-6888). However, others have discovered that shorter or longer RNA duplex structures can also be effective (Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226). In the above-described embodiments, due to the nature of the oligonucleotide sequences provided herein, the dsRNAs described herein can comprise at least one strand that is a minimum of 21 nucleotides in length. It can be reasonably predicted that shorter duplexes, minus only a few nucleotides at one or both ends, can be similarly effective compared to the dsRNAs described above. Thus, dsRNAs having a sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides derived from one of the sequences provided herein, and which differ in their ability to inhibit expression of the C9orf72 gene by no more than 10, 15, 20, 25, or 30% inhibition from dsRNAs containing the entire sequence, e.g., using in vitro assays with Be(2)c cells and an RNA agent at a 10 nM concentration and the PCR assays provided in the Examples herein, are intended to be within the scope of this disclosure.

[0399] In addition, the RNA described herein identifies the site in C9orf72 transcript that is susceptible to RISC-mediated cleavage.Therefore, the present disclosure also features the RNAi agent that targets within this site.As used herein, if the RNAi agent promotes the cleavage of the transcript at any of the specific sites, the RNAi agent is said to target within the specific site of the RNA transcript.This RNAi agent will generally comprise at least about 15 consecutive nucleotides, preferably at least 19 nucleotides, from one of the sequences provided herein, which is combined with additional nucleotide sequences taken from the region adjacent to the selected sequence in C9orf72 gene.

[0400] 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, a dsRNA agent may inhibit 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 may occur within 24 to 48 hours after administration to cells expressing a C9orf72 target RNA containing a hexanucleotide repeat. The reduction may be, for example, compared to cells prior to treatment with the dsRNA agent or compared to control cells not treated with the dsRNA agent.

[0401] The dsRNA agents disclosed herein selectively reduce the level of or inhibit the expression of a C9orf72 target RNA containing an intronic hexanucleotide repeat relative to the expression of a mature C9orf72 messenger RNA. In this context, the mature C9orf72 messenger RNA is a spliced ​​and processed C9orf72 RNA transcript. The mature C9orf72 messenger RNA consists of only exons, with all introns removed. A dsRNA agent can selectively inhibit the expression of a C9orf72 target RNA containing an intronic hexanucleotide repeat relative to the expression of a mature C9orf72 messenger RNA if the relative decrease in expression of the C9orf72 target RNA is greater than the relative decrease in expression of the mature C9orf72 messenger RNA after administration of the dsRNA agent to cells expressing the C9orf72 target RNA. For example, a dsRNA agent may 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 may be within 24 to 48 hours after administration to cells expressing mature C9orf72 messenger RNA.

[0402] The dsRNA agents disclosed herein can also, for example, reduce dipeptide repeat protein synthesis or dipeptide repeat protein levels in a cell (e.g., within 24-48 hours after administration to the cell). For example, the dsRNA agent may 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 the cells before treatment with the dsRNA agent, or compared to control cells that were not treated with the dsRNA agent.

[0403] According to certain aspects of the invention, an iRNA agent may be designed to target a hotspot region of any of the target RNAs described herein, including any specified portion of the target RNA (e.g., a particular exon). As used herein, a hotspot region may refer to a region of about 19-200, 19-150, 19-100, 19-75, 19-50, 21-200, 21-150, 21-100, 21-75, 21-50, 50-200, 50-150, 50-100, 50-75, 75-200, 75-150, 75-100, 100-200, or 100-150 nucleotides of a target RNA sequence where targeting with an RNAi agent provides a significantly higher likelihood of effective silencing compared to targeting other regions of the same target RNA. According to certain embodiments of the present invention, a hotspot region may comprise a limited region of a target RNA, or in some cases, a substantially limited region of the target, for example, comprising less than half the length of the target RNA, for example, about 5%, 10%, 15%, 20%, 25%, or 30% of the length of the target RNA. Conversely, other regions to which a hotspot is compared may cumulatively comprise at least half the length of the target RNA. For example, the other regions may cumulatively comprise at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 95% of the length of the target RNA.

[0404] Comparative regions of the target RNA may be empirically evaluated to identify hotspots using efficacy data obtained from in vitro or in vivo screening assays. For example, RNAi agents targeting various regions spanning the target RNA may be compared for the frequency of effective iRNA agents binding to each region (e.g., the amount of inhibition of target gene expression, as measured by mRNA expression or protein expression). Generally, hotspots may be recognized by observing the clustering of multiple effective RNAi agents binding to a defined region of the RNA target. A hotspot may be fully characterized by observing the efficacy of iRNA agents that cumulatively span at least about 60% of the target region identified as a hotspot, e.g., about 70%, about 80%, about 90%, or about 95% or more of the length of the region, including both ends of the region (i.e., at least about 60%, 70%, 80%, 90%, or 95% or more of the nucleotides within the region, including the nucleotides at each end of the region, are targeted by the iRNA agent). According to some embodiments of the invention, an iRNA agent may be identified as effective that exhibits at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% inhibition across the region (e.g., about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or less of mRNA remaining).

[0405] The suitability of RNA regions for targeting may also be assessed using a quantitative comparison of inhibition measurements across different regions of defined size (e.g., 25, 30, 40, 50, 60, 70, 80, 90, or 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nt). For example, an average level of inhibition may be determined for each region, and the averages for each region may be compared. The average level of inhibition within a hotspot region may be substantially higher than the average of all regions evaluated. According to some embodiments, the average level of inhibition in a hotspot region may be at least about 10%, 20%, 30%, 40%, or 50% higher. According to some embodiments, the average level of inhibition in a hotspot region may be at least about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 standard deviations higher than the mean of the average. The average level of inhibition may be higher by a statistically significant amount (e.g., p<0.05). According to some embodiments, each inhibition measurement within a hotspot region may exceed a threshold amount (e.g., equal to or less than a threshold amount of residual mRNA). According to some embodiments, each inhibition measurement within a region may be substantially higher than the average of all inhibition measurements across all measured regions. For example, each inhibition measurement in a hotspot region may be at least about 10%, 20%, 30%, 40%, or 50% higher than the average of all inhibition measurements. According to some embodiments, each inhibition measurement may be at least about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 standard deviations higher than the average of all inhibition measurements. Each inhibition measurement may be higher than the average of all inhibition measurements by a statistically significant amount (e.g., p<0.05). Criteria for assessing hotspots may include various combinations of the above standards being met (e.g., an average level of inhibition of at least about a first amount, with no inhibition measurements below a threshold level of a second amount less than the first amount).

[0406] Therefore, it is expressly contemplated that any iRNA agent, including certain exemplary iRNA agents described herein, that targets a hotspot region of a target RNA can be selected to induce RNA interference of a target mRNA, preferably because targeting such a hotspot region is more likely to exhibit a robust inhibitory response than targeting a region that is not a hotspot region. RNAi agents that target target sequences that substantially overlap (e.g., at least about 70%, 75%, 80%, 85%, 90%, 95% of the target sequence length), or preferably reside entirely within a hotspot region, can be considered to target a hotspot region. Hotspot regions of RNA targets of the present invention may include any region that the data disclosed herein show a higher frequency of targeting by an effective RNAi agent that includes any of the criteria described elsewhere herein, regardless of whether the scope of such a hotspot region is explicitly specified.

[0407] In various embodiments, the dsRNA agents of the invention target hotspot regions. In one embodiment, the hotspot regions comprise any one of the nucleotide sequences selected from SEQ ID NOs: 21-47 and 51-93. In another embodiment, the hotspot regions comprise nucleotides 220-256, 220-266, and 200-290 of SEQ ID NO: 13.

[0408] III. Modified RNAi Agents of the Present Disclosure In one embodiment, the nucleotides of an RNAi agent of the present disclosure, e.g., dsRNA, are unmodified and do not contain, for example, chemical modifications or conjugations known in the art and described herein. In a preferred embodiment, the nucleotides of an RNAi agent of the present disclosure, e.g., dsRNA, are chemically modified to enhance stability or other beneficial characteristics. In certain embodiments of the present disclosure, substantially all of the nucleotides of an RNAi agent of the present disclosure are modified. In other embodiments of the present disclosure, all of the nucleotides of an RNAi agent of the present disclosure are modified. An RNAi agent of the present disclosure in which "substantially all of the nucleotides are modified" may be widely, but not entirely, modified and may contain 5, 4, 3, 2 or fewer, or unmodified nucleotides. In yet other embodiments of the present disclosure, an RNAi agent of the present disclosure may contain 5, 4, 3, 2, or fewer than 1 modified nucleotide.

[0409] 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. Lett. et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference. Modifications include, for example, terminal modifications, such as 5'-end modifications (phosphorylation, conjugation, inverted linkage) or 3'-end modifications (conjugation, DNA nucleotides, inverted linkages, etc.), base modifications, such as substitution with stabilizing bases, destabilizing bases, or bases that base-pair with partners in an extended repertoire, base removal (abasic nucleotides) or conjugated bases, sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions, or backbone modifications, including modification or substitution of phosphodiester bonds. 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. The RNA with modified backbone includes, among others, that does not have phosphorus atom in backbone.For the purpose of this specification, and as sometimes referred to in the art, the modified RNA that does not have phosphorus atom in their internucleoside backbone can also be considered as oligonucleoside.In some embodiments, modified RNAi agent will have phosphorus atom in its internucleoside backbone.

[0410] Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates, including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates with normal 3'-5' linkages, their 2'-5' linked analogs, and those with reversed polarity, where adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included. In some embodiments of the present invention, the dsRNA agents of the present invention are in free acid form. In other embodiments of the present invention, the dsRNA agents of the present invention are in salt form. In one embodiment, the dsRNA 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 ions exist in the agent as counterions to 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 counterions comprises 5, 4, 3, 2, or 1 or less phosphodiester and / or phosphorothioate linkages that do not have sodium counterions.In some embodiments, when the dsRNA agent of the present invention is in sodium salt form, sodium ions exist in the agent as counterions to all of the phosphodiester and / or phosphorothioate groups present in the agent.

[0411] Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Patent Nos. 3,687,808, 4,469,863, 4,476,301, 5,023,243, 5,177,195, 5,188,897, 5,264,423, 5,276,019, 5,278,302, 5,286,711, and the like. No. 7, No. 5,321,131, No. 5,399,676, No. 5,405,939, No. 5,453,496, No. 5,455,233, No. 5,466,677, No. 5,47 No. 6,925, No. 5,519,126, No. 5,536,821, No. 5,541,316, No. 5,550,111, No. 5,563,253, No. 5,571,799, No. 5 , 587,361, 5,625,050, 6,028,188, 6,124,445, 6,160,109, 6,169,170, 6,172,209 No. 6,239,265, No. 6,277,603, No. 6,326,199, No. 6,346,614, No. 6,444,423, No. 6,531,590, No. 6,534, Nos. 6,639, 6,608,035, 6,683,167, 6,858,715, 6,867,294, 6,878,805, 7,015,315, 7,041,816, 7,273,933, 7,321,029, and U.S. Patent No. RE39464, the entire contents of each of which are incorporated herein by reference.

[0412] Modified RNA backbones that do not contain a phosphorus atom in the backbone have backbones formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatom or heterocyclic internucleoside linkages, including those with morpholino linkages (formed in part from the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and others with mixed N, O, S, and CH2 constituent moieties.

[0413] Representative U.S. 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, and 5,470,967. , 5,489,677, 5,541,307, 5,561,225, 5,596,086, 5,602,240, 5,608,046, 5,610,289, 5,618,704, 5,623,070, 5,663,312, 5,633,360, 5,677,437, and 5,677,439, the entire contents of each of which are incorporated herein by reference.

[0414] In another embodiment, RNA mimics suitable for use in RNAi agents are contemplated, in which both the sugar and internucleoside linkages, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with appropriate nucleic acid target compounds. One such oligomeric compound, an RNA mimic known to have excellent hybridization properties, is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleobases are retained and are directly or indirectly linked to the aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents teaching the preparation of PNA compounds include, but are not limited to, U.S. Patent Nos. 5,539,082, 5,714,331, and 5,719,262, the entire contents of each of which are incorporated herein by reference. 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.

[0415] Some embodiments featured in this disclosure include RNA 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-- (representing the natural phosphodiester backbone as --O--P--O--CH2--) of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNA featured herein has the morpholino backbone structure of the above-referenced US Pat. No. 5,034,506.

[0416] Modified RNAs can also contain one or more substituted sugar moieties. RNAi agents, e.g., dsRNAs, featured herein can contain one of the following at the 2' position: OH, F, O-, S-, or N-alkyl, O-, S-, or N-alkenyl, O-, S-, or N-alkynyl, or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C6. 10 Alkyl or C2-C 10 (It can be alkenyl or alkynyl). Exemplary suitable modifications include O[(CH) n O] m CH3, O(CH2). n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2 and O(CH2) n ON[(CH2) n CH3)]2, where n and m are from 1 to about 10. In other embodiments, the dsRNA includes one of the following at the 2' position: C1 to C 10lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH, OCN, Cl, Br, CN, CF, OCF, SOCH, SOCH, ONO, NO, N, NH, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving groups, reporter groups, interfering substances, groups for improving the pharmacokinetic properties of RNAi agents or groups for improving the pharmacodynamic properties of RNAi agents, and other substituents with similar properties. In some embodiments, the modification includes 2'-methoxyethoxy (2'-O-CHCHOCH, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., an alkoxy-alkoxy group. Another exemplary modification is the 2'-dimethylaminooxyethoxy, i.e., O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, as described herein below in the Examples, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O-CH2-O-CH2-N(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 within these three families), 2'-alkoxyalkyl, and 2'-NMA (N-methylacetamide).

[0417] Other modifications include 2'-methoxy (2'-OCH), 2'-aminopropoxy (2'-OCHCHCHNH), 2'-O-hexadecyl, and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA of an RNAi agent, particularly the 3' position of the sugar on the 3'-terminal nucleotide or in a 2'-5'-linked dsRNA and the 5' position of the 5'-terminal nucleotide. RNAi agents can also have sugar mimetics, such as a cyclobutyl moiety in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Patent Application Nos. 4,981,957, 5,118,800, 5,319,080, 5,359,044, 5,393,878, 5,446,137, 5,466,786, 5,514,785, 5,519,134, 5,519,134, 5,519,136, 5,519,138, 5,519,139, 5,519,140, ​​5,519,141, 5,519,142, 5,519,143, 5,519,144, 5,519,145, 5,519,146, 5,519,147, 5,519,148, 5,519,149, 5,519,150, 5,519,151, 5,519,152, 5,519,153, 5,519,154, 5,519,155, 5,519,156, 5,519,157, 5,519,158, 5,519,159 ... Nos. 5,567,811, 5,576,427, 5,591,722, 5,597,909, 5,610,300, 5,627,053, 5,639,873, 5,646,265, 5,658,873, 5,670,633, and 5,700,920, some of which are commonly owned with the present application. The entire contents of each of the foregoing are incorporated herein by reference.

[0418] The RNAi agents of the present disclosure may also include modifications or substitutions of nucleobases (often simply referred to in the art as "bases"). As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include other synthetic and natural nucleobases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, These include 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine, and 3-deazaguanine and 3-deazaadenine.Additional nucleobases include those disclosed in 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, pp. 858-859, Kroschwitz, JL, 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, pp. 289-302, Crooke, ST and Lebleu, B., Ed., CRC Press, 1993. Some of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the present disclosure. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and 0-6 substituted purines, 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, Y.S., Crooke, S.T., and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), and are exemplary base substitutions, especially when combined with 2'-O-methoxyethyl sugar modifications.

[0419] Representative United States patents that teach the preparation of certain of the above modified nucleobases, as well as other modified nucleobases, include, but are not limited to, the above-referenced U.S. Patent Nos. 3,687,808, 4,845,205, 5,130,30, 5,134,066, 5,175,273, 5,367,066, 5,432,272, 5,457,187, 5,459,255, 5,484,908, 5,502,177, 5,525,711, 5,552,540, 5,587,469, Nos. 5,594,121, 5,596,091, 5,614,617, 5,681,941, 5,750,692, 6,015,886, 6,147,200, 6,166,197, 6,222,025, 6,235,887, 6,380,368, 6,528,640, 6,639,062, 6,617,438, 7,045,610, 7,427,672, and 7,495,088, the entire contents of each of which are incorporated herein by reference.

[0420] The RNAi agents of the present disclosure can also be modified to include one or more locked nucleic acids (LNAs). Locked nucleic acids are nucleotides with modified ribose moieties, in which the ribose moiety contains an additional bridge connecting the 2' and 4' carbons. 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 Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).

[0421] RNAi agents of the present disclosure can also be modified to include one or more bicyclic sugar moieties. A "bicyclic sugar" is a furanose 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 system. In certain embodiments, the bridge connects the 4'-carbon and 2'-carbon of the sugar ring. Thus, in some embodiments, agents of the present disclosure may 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 in a 3'-endo conformation. The addition of a locking nucleic acid to siRNA has been shown to increase siRNA stability in serum and reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, OR. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). Examples of bicyclic nucleosides for use in the polynucleotides of the present disclosure include, but are not limited to, nucleosides comprising a bridge between the 4'-ribosyl ring atom and the 2'-ribosyl ring atom. 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 referred to as "constrained ethyl" or "cEt") and 4'-CH(CHOCH3)-O-2' (and analogs thereof, see e.g., U.S. Pat. No. 7,399,845), 4'-C(CH3)(CH3)-O-2' (and analogs thereof, see e.g., U.S. Pat. No. 7,399,845). No. 8,278,283), 4'-CH2-N(OCH3)-2' (and analogs thereof, see, e.g., U.S. Pat. No. 8,278,425), 4'-CH2-ON(CH3)-2' (see, e.g., U.S. Patent Publication No. 2004 / 0171570), 4'-CH2-N(R)-O-2', where 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 the foregoing are incorporated herein by reference.

[0422] 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, 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.

[0423] Any of the foregoing bicyclic nucleosides can be prepared with one or more stereochemical sugar configurations, including, for example, α-L-ribofuranose and β-D-ribofuranose (see WO 99 / 14226).

[0424] The RNAi agents of the present disclosure can also be modified to include one or more constrained ethyl nucleotides. As used herein, a "constrained ethyl nucleotide" or "cEt" is a locked nucleic acid containing a bicyclic sugar moiety containing a 4'-CH(CH3)-O-2' bridge. In one embodiment, the constrained ethyl nucleotide is in the S conformation and is referred to herein as an "S-cEt."

[0425] 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, increasing hybridization affinity to mRNA. The linker is of sufficient length to position the oxygen in an optimal position for stability and affinity, thereby reducing puckering of the ribose ring.

[0426] 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.

[0427] In some embodiments, the RNAi agent of the present disclosure includes one or more monomers that are UNA (unlocked nucleic acid) nucleotides. UNAs are unlocked acyclic nucleic acids in which any of their sugar linkages have been removed to form an unlocked "sugar" residue. In one example, UNAs also encompass monomers in which the C1'-C4' bond has been removed (i.e., a covalent carbon-oxygen-carbon bond between the C1' and C4' carbons). In another example, the C2'-C3' bond of the sugar has been removed (i.e., a covalent carbon-carbon bond between the C2' and C3' carbons) (see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039, which are incorporated herein by reference).

[0428] Representative U.S. publications that teach the preparation of UNAs include, but are not limited to, U.S. Pat. No. 8,314,227, and U.S. Patent Application Publication Nos. 2013 / 0096289, 2013 / 0011922, and 2011 / 0313020, the entire contents of each of which are incorporated herein by reference.

[0429] Potential stabilizing modifications to the ends of RNA molecules can include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3'-phosphate, inverted 2'-deoxy-modified ribonucleotides such as inverted dT (idT) and inverted dA (idA), and inverted abasic 2'-deoxyribonucleotides (iAb), and others. Disclosure of these modifications can be found in WO2011 / 005861.

[0430] In one example, the 3' or 5' end of the oligonucleotide is linked to an inverted 2'-deoxy modified ribonucleotide, such as an inverted dT (idT), an inverted dA (idA), or an inverted abasic 2'-deoxyribonucleotide (iAb). In one particular example, the inverted 2'-deoxy modified ribonucleotide is linked to the 3' end of the oligonucleotide, such as the 3' end of the sense strand described herein, where the linkage is via a 3'-3' phosphodiester linkage or a 3'-3' phosphorothioate linkage.

[0431] In another example, the 3' end of the sense strand is linked to an inverted abasic ribonucleotide (iAb) via a 3'-3' phosphorothioate linkage. In another example, the 3' end of the sense strand is linked to an inverted dA (idA) via a 3'-3' phosphorothioate linkage.

[0432] In another example, the 5' end of the sense strand is linked to an inverted abasic ribonucleotide (iAb) via a 3'-3' phosphorothioate linkage. In another example, the 5' end of the sense strand is linked to an inverted dA (idA) via a 3'-3' phosphorothioate linkage.

[0433] In another example, the 3' and 5' ends of the sense strand are linked to an inverted abasic ribonucleotide (iAb) via a 3'-3' phosphorothioate linkage. In another example, the 3' and 5' ends of the sense strand are linked to an inverted dA (idA) via a 3'-3' phosphorothioate linkage.

[0434] In one particular example, the inverted 2'-deoxy modified ribonucleotide is linked to the 3' end of the oligonucleotide, such as the 3' end of the sense strand described herein, where the linkage is via a 3'-3' phosphodiester linkage or a 3'-3' phosphorothioate linkage.

[0435] In another example, the 3' terminal nucleotide of the sense strand is an inverted dA (idA) and is joined to the preceding nucleotide via a 3'-3' linkage (eg, a 3'-3' phosphorothioate linkage).

[0436] Other modifications of the RNAi agent of the present disclosure include 5' phosphate or 5' phosphate mimics, such as 5' terminal phosphate or phosphate mimics on the antisense strand of the RNAi agent. Suitable phosphate mimics are disclosed, for example, in US2012 / 0157511, the entire contents of which are incorporated herein by reference.

[0437] A. Modified RNAi Agents Comprising Motifs of the Present Disclosure In certain aspects of the present disclosure, the double-stranded RNAi agents of the present disclosure include agents having chemical modifications, such as those 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, particularly at or near the cleavage site. In some embodiments, the sense and antisense strands of the RNAi agent may be otherwise completely modified. The introduction of these motifs interrupts the modification pattern of the sense or antisense strand, if present. The RNAi agent may optionally be conjugated with a lipophilic ligand, for example, a C16 ligand, for example, on the sense strand. The RNAi agent may optionally be modified with an (S)-glycol nucleic acid (GNA) modification, for example, at one or more residues of the antisense strand. The resulting RNAi agent exhibits excellent gene silencing activity.

[0438] Thus, the present disclosure provides a double-stranded RNAi agent capable of inhibiting 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 may be 15-30 nucleotides in length. For example, each strand may be 16-30 nucleotides in length, 17-30 nucleotides in length, 25-30 nucleotides in length, 27-30 nucleotides in length, 17-23 nucleotides in length, 17-21 nucleotides in length, 17-19 nucleotides in length, 19-25 nucleotides in length, 19-23 nucleotides in length, 19-21 nucleotides in length, 21-25 nucleotides in length, or 21-23 nucleotides in length. In certain embodiments, each strand is 19-23 nucleotides in length.

[0439] The sense strand and antisense strand typically form a duplex double-stranded RNA ("dsRNA"), also referred to herein as an "RNAi agent." The double-stranded region of an RNAi agent may be 15-30 nucleotide pairs in length. For example, the double-stranded region may be 16-30 nucleotide pairs in length, 17-30 nucleotide pairs in length, 27-30 nucleotide pairs in length, 17-23 nucleotide pairs in length, 17-21 nucleotide pairs in length, 17-19 nucleotide pairs in length, 19-25 nucleotide pairs in length, 19-23 nucleotide pairs in length, 19-21 nucleotide pairs in length, 21-25 nucleotide pairs in length, or 21-23 nucleotide pairs in length. In another example, the double-stranded region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides in length. In a preferred embodiment, the double-stranded region is 19 to 21 nucleotide pairs in length.

[0440] In one embodiment, the RNAi agent may include one or more overhang regions or capping groups at the 3'-end, 5'-end, or both ends of one or both strands. The overhangs can be 1 to 6 nucleotides in length, e.g., 2 to 6 nucleotides in length, 1 to 5 nucleotides in length, 2 to 5 nucleotides in length, 1 to 4 nucleotides in length, 2 to 4 nucleotides in length, 1 to 3 nucleotides in length, 2 to 3 nucleotides in length, or 1 to 2 nucleotides in length. In a preferred embodiment, the nucleotide overhang region is 2 nucleotides in length. The overhang can be the result of one strand being longer than the other, or the result of two strands of the same length being twisted. The overhang can form a mismatch with the target mRNA, be complementary to the targeted gene sequence, or be a different sequence. The first and second strands can also be linked by additional bases, for example, to form a hairpin, or by other non-basic linkers.

[0441] In one embodiment, the nucleotides in the overhang region of an RNAi agent can each independently be modified or unmodified nucleotides, including, but not limited to, 2'-sugar modifications, such as 2-F, 2'-O-methyl, thymidine (T), and any combination thereof.

[0442] For example, TT can be an overhang sequence for either end on either strand, which can form a mismatch with the target mRNA, or can be complementary to the targeted gene sequence, or can be another sequence.

[0443] The 5'- or 3'-overhang of the sense strand, the antisense strand, or both strands of the RNAi agent may be phosphorylated. In some embodiments, the overhang region comprises two nucleotides with a phosphorothioate between them, and these two nucleotides may be the same or different. In one embodiment, the overhang is present at the 3'-end of the sense strand, the 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.

[0444] RNAi agent may only have 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 may 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 asymmetry between the blunt end at the 5'-end of the antisense strand and the 3'-end overhang of the antisense strand is favorable for the insertion of the guide strand into the RISC process.

[0445] In one embodiment, the RNAi agent is a double-ended bluntmer 19 nucleotides in length, the sense strand of which 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 of which 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.

[0446] In another embodiment, the RNAi agent is a double-ended bluntmer 20 nucleotides in length, the sense strand containing at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 8, 9, and 10 from the 5' end, and the antisense strand containing at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0447] In yet another embodiment, the RNAi agent is a double-ended bluntmer 21 nucleotides in length, the sense strand containing 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 containing at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0448] In one embodiment, the RNAi agent comprises a 21-nucleotide sense strand and a 23-nucleotide antisense strand, wherein the sense strand comprises at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 9, 10, and 11 from the 5' end, and the antisense strand comprises at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end, and one end of the RNAi agent is blunt and the other end comprises a two-nucleotide overhang. Preferably, the two-nucleotide overhang is at the 3' end of the antisense strand. When the two-nucleotide overhang is at the 3' end of the antisense strand, there may be two phosphorothioate internucleotide linkages between the three terminal nucleotides, two of which are overhanging nucleotides, and the third nucleotide is a paired nucleotide adjacent to the overhanging nucleotide. In one embodiment, the RNAi agent further comprises two phosphorothioate internucleotide linkages between the terminal three nucleotides at both the 5'-end of the sense strand and the 5'-end of the antisense strand. In one embodiment, every nucleotide in the sense and antisense strands of the RNAi agent, including a nucleotide that is part of a motif, is a modified nucleotide. In one embodiment, each residue, independently, for example, within an alternating motif, is modified with 2'-O-methyl or 3'-fluoro. Optionally, the RNAi agent further comprises a ligand (e.g., a lipophilic ligand, optionally a C16 ligand).

[0449] In one embodiment, the RNAi agent comprises a sense strand and an antisense strand, wherein the sense strand is 25 to 30 nucleotide residues in length and, starting from the 5'-most nucleotide (position 1), comprises at least 8 ribonucleotides at positions 1 to 23 of the first strand; and the antisense strand is 36 to 66 nucleotide residues in length and, starting from the 3'-most nucleotide, comprises at least 8 ribonucleotides at positions 1 to 23 of the sense strand to form a duplex; wherein at least the 3'-most nucleotide of the antisense strand is unpaired with the sense strand, and up to 6 consecutive 3'-most nucleotides are unpaired with the sense strand, thereby forming a 3' single-stranded overhang of 1 to 6 nucleotides; and wherein the 5'-end of the antisense strand is 10 to 30 nucleotides that are not paired with the sense strand. the sense strand comprises three consecutive nucleotides, thereby forming a single-stranded 5' overhang of 10 to 30 nucleotides, at least the 5'- and 3'-terminal nucleotides of the sense strand base-pair with nucleotides of the antisense strand when the sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially double-stranded region between the sense and antisense strands, and the antisense strand is sufficiently complementary to the target RNA along at least 19 ribonucleotides of the antisense strand length, such that the double-stranded nucleic acid reduces target gene expression when introduced into a mammalian cell, and the sense strand comprises at least one motif of three 2'-F modifications on three consecutive nucleotides, at least one of the motifs occurring at or near the cleavage site. The antisense strand comprises at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at or near the cleavage site.

[0450] In one embodiment, the RNAi agent comprises a sense strand and an antisense strand, the RNAi agent comprising a first strand having a length of at least 25 nucleotides and at most 29 nucleotides, and a second strand having a length of at most 30 nucleotides with at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end, the 3' end of the first strand and the 5' end of the second strand form blunt ends, the second strand is 1 to 4 nucleotides longer at its 3' end than the first strand, the double-stranded region is at least 25 nucleotides in length, and the second strand is sufficiently complementary to a target mRNA along at least 19 nucleotides of the length of the second strand, the RNAi agent reduces target gene expression when introduced into a mammalian cell, and dicer cleavage of the RNAi agent preferentially results in an siRNA comprising the 3' end of the second strand, thereby reducing target gene expression in the mammalian cell. Optionally, the RNAi agent further comprises a ligand.

[0451] In one embodiment, the sense strand of the RNAi agent contains at least one motif of three identical modifications on three consecutive nucleotides, one of which motifs occurs at the cleavage site in the sense strand.

[0452] In one embodiment, the antisense strand of the RNAi agent can also contain at least one motif of three identical modifications on three consecutive nucleotides, one of which motifs occurs at or near the cleavage site in the antisense strand.

[0453] For RNAi agents having a double-stranded region 17 to 23 nucleotides in length, the cleavage sites of the antisense strand are typically approximately 10, 11, and 12 positions from the 5' end. Thus, three identical modification motifs may occur at positions 9, 10, 11, 10, 11, 12, 11, 12, 13, 12, 13, 14, or 13, 14, 15 of the antisense strand, starting from the first nucleotide from the 5' end of the antisense strand, or starting from the first paired nucleotide within the double-stranded region from the 5' end of the antisense strand. The cleavage site within the antisense strand may also vary depending on the length of the double-stranded region of the RNAi from the 5' end.

[0454] The sense strand of RNAi agent can comprise at least one motif of three identical modifications on three consecutive nucleotides at the cleavage site of its strand, and the antisense strand can have at least one motif of three identical modifications on three consecutive nucleotides at or near the cleavage site of its strand.When sense strand and antisense strand form dsRNA duplex, sense strand and antisense strand can be aligned so that one motif of three nucleotides on sense strand and one motif of three nucleotides on antisense strand have at least one nucleotide overlap, that is, at least one of the three nucleotides of the motif in sense strand and at least one of the three nucleotides of the motif in antisense strand form base pairing.Alternatively, at least two nucleotides can overlap, or all three nucleotides can overlap.

[0455] 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 motif may be a wing modification. As used herein, the term "wing modification" refers to a motif that occurs in another portion of the strand separated from a motif at or near the cleavage site of the same strand. The wing modification may be adjacent to the first motif or separated by at least one or more nucleotides. When the motifs are immediately adjacent to each other, the chemical nature of the motifs is distinct from each other, and when the motifs are separated by one or more nucleotides, the chemical nature can 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.

[0456] Like the sense strand, the antisense strand of an RNAi agent may contain two or more motifs of three identical modifications on three consecutive nucleotides, at least one of which occurs at or near the cleavage site of that strand. The antisense strand may also contain one or more wing modifications in the same alignment as the wing modifications that may be present on the sense strand.

[0457] 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.

[0458] In another embodiment, wing modifications on the sense or antisense strand of an RNAi agent typically do not include the first one or two paired nucleotides in the double-stranded region at the 3' end, 5' end, or both ends of the strand.

[0459] When the sense and antisense strands of an RNAi agent each contain at least one wing modification, the wing modifications may be at the same end of the double-stranded region, with an overlap of 1, 2, or 3 nucleotides.

[0460] When the sense and antisense strands of an RNAi agent each contain at least two wing modifications, the sense and antisense strands can be aligned such that the two modifications from each single strand are at one end of a double-stranded region with 1, 2, or 3 nucleotide overlap, the two modifications from each single strand are at the other end of the double-stranded region with 1, 2, or 3 nucleotide overlap, and the two modifications from the single strand are on either side of a lead motif with 1, 2, or 3 nucleotide overlap within the double-stranded region.

[0461] In one embodiment, the RNAi agent contains mismatches or combinations thereof within the duplex with the target. Mismatches may occur within the overhang region or within the duplex region. Base pairs can be ranked based on their propensity to promote dissociation or melting (e.g., based on the free energy of association or dissociation of a particular pairing; the simplest approach is to examine each pair individually, but side-by-side or similar analyses 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 or non-canonical pairings (described elsewhere herein), are preferred over canonical (A:T, A:U, G:C) pairings, and pairings involving universal bases are preferred over canonical pairings.

[0462] In one embodiment, the RNAi agent comprises the first 1, 2, 3, 4, or 5 base pairs within the double-stranded region from the 5' end of the antisense strand independently selected from the group of A:U, G:U, I:C, and at least one mismatch pair, e.g., a non-canonical pairing or a non-canonical pairing or a pairing containing a universal base, to promote dissociation of the antisense strand at the 5' end of the duplex.

[0463] 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.

[0464] 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 on the 3' end of the sense strand or antisense strand.

[0465] In one embodiment, the sense strand sequence is represented by formula (I): 5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3'(I) During the ceremony, i and j are each independently 0 or 1; p and q each independently represent 0 to 6; each N a independently represent oligonucleotide sequences containing 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides; each N b independently represent an oligonucleotide sequence comprising 0 to 10 modified nucleotides; each n p and n q independently represent an overhanging nucleotide, wherein Nb and Y do not have the same modification; XXX, YYY, and ZZZ each independently represent one motif of three identical modifications on three consecutive nucleotides. Preferably, YYY are all 2'-F modified nucleotides.

[0466] In one embodiment, N a or N b includes alternating pattern modifications.

[0467] 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 double-stranded region of 17 to 23 nucleotides in length, the YYY motif can occur at or near the cleavage site of the sense strand (e.g., at positions 6, 7, 8, 7, 8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12, or 11, 12, 13), starting from the first nucleotide from the 5' end, or optionally starting from the first paired nucleotide in the double-stranded region from the 5' end.

[0468] In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 1. Thus, the sense strand has the following formula: 5'n p -N a -YYY-N b -ZZZ-N a -n q 3'(Ib), 5'n p -N a -XXX-N b -YYY-N a -n q 3'(Ic), or 5'n p -Na -XXX-N b -YYY-N b -ZZZ-N a -n q 3'(Id) is expressed by

[0469] When the sense strand is represented by formula (Ib), N b represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides.

[0470] each N a can independently represent an oligonucleotide sequence that includes 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0471] When the sense strand is represented by formula (Ic), N b represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a can independently represent an oligonucleotide sequence that includes 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0472] When the sense strand is represented by formula (Id), each N b independently represent an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. b is 0, 1, 2, 3, 4, 5, or 6. Each N a can independently represent an oligonucleotide sequence that includes 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0473] Each of X, Y, and Z may be the same or different from one another.

[0474] In other embodiments, i is 0, j is 0, and the sense strand has the formula: 5'n p -N a -YYY-N a -nq 3'(Ia) It can be represented by:

[0475] When the sense strand is represented by formula (Ia), each N a can independently represent an oligonucleotide sequence that includes 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0476] In one embodiment, the antisense strand sequence of the RNAi has the formula (II): 5'n q’ -N a '-(Z'Z'Z') k -N b '-Y'Y'Y'-N b '-(X'X'X') l -N' a -n p '3'(II) can be represented by During the ceremony, k and l are each independently 0 or 1; p' and q' each independently represent 0 to 6; each N a ' independently represent oligonucleotide sequences containing 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides; each N b ' independently represents an oligonucleotide sequence comprising 0 to 10 modified nucleotides; each n p ' and n q ' independently represents an overhanging nucleotide, N b ' and Y' do not have the same modification, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides.

[0477] In one embodiment, N a ' or N b ' includes alternating pattern modifications.

[0478] The Y'Y'Y' motif occurs at or near the cleavage site of the antisense strand.For example, when the RNAi agent has a double-stranded region of 17-23 nucleotides in length, the Y'Y'Y' motif can occur at positions 9, 10, 11, 10, 11, 12, 11, 12, 13, 12, 13, 14, or 13, 14, 15 of the antisense strand, where the numbers start from the first nucleotide from the 5' end, or optionally, the numbers start from the first paired nucleotide in the double-stranded region from the 5' end.Preferably, the Y'Y'Y' motif occurs at positions 11, 12, 13.

[0479] In one embodiment, the Y'Y'Y' motif is all 2'-OMe modified nucleotides.

[0480] In one embodiment, k is 1 and l is 0, or k is 0 and l is 1, or both k and l are 1.

[0481] Thus, the antisense strand has the following formula: 5'n q’ -N a '-Z'Z'Z'-N b '-Y'Y'Y'-N a '-n p’ 3'(IIb), 5'n q’ -N a '-Y'Y'Y'-N b '-X'X'X'-n p’ 3'(IIc), or 5'n q’ -N a '-Z'Z'Z'-N b '-Y'Y'Y'-N b '-X'X'X'-N a '-n p’ 3'(IId) is expressed by

[0482] When the antisense strand is represented by formula (IIb), N b ’represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a ' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0483] When the antisense strand is represented by formula (IIc), N b ' represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a ' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0484] When the antisense strand is represented by formula (IId), each N b Each N' independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a ' independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. b is 0, 1, 2, 3, 4, 5, or 6.

[0485] In other embodiments, k is 0, l is 0, and the antisense strand has the formula: 5'n p’ -N a’ -Y'Y'Y'-N a’ -n q’ 3'(Ia) is expressed by

[0486] When the antisense strand is represented by formula (IIa), each N a ' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0487] Each of X', Y', and Z' may be the same as or different from one another.

[0488] Each nucleotide in the sense strand and the antisense strand may be independently modified with LNA, 1,5-anhydrohexitol (HNA), cyclohexenyl (CeNA), 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-hydroxyl, or 2'-fluoro. For example, each nucleotide in the sense strand and the antisense strand may be independently modified with 2'-O-methyl or 2'-fluoro. Each X, Y, Z, X', Y', and Z' may specifically represent a 2'-O-methyl modification or a 2'-fluoro modification.

[0489] In one embodiment, the sense strand of the RNAi agent may include a YYY motif occurring at positions 9, 10, and 11 of the strand when the double-stranded region is 21 nt, where the numbers start from the first nucleotide from the 5' end, or optionally, the numbers start from the 5' end with the first paired nucleotide in the double-stranded region, and Y represents a 2'-F modification. The sense strand may additionally include a XXX motif or a ZZZ motif as a wing modification at the opposite end of the double-stranded region, where XXX and ZZZ each independently represent a 2'-OMe modification or a 2'-F modification.

[0490] In one embodiment, the antisense strand may include a Y'Y'Y' motif occurring at positions 11, 12, or 13 of the strand, where the numbers start from the first nucleotide from the 5' end, or optionally, the numbers start from the first paired nucleotide in the double-stranded region from the 5' end, and Y' represents a 2'-O-methyl modification. The antisense strand may additionally include an X'X'X' motif or a Z'Z'Z' motif as a wing modification at the opposite end of the double-stranded region, where X'X'X' and Z'Z'Z' each independently represent a 2'-OMe modification or a 2'-F modification.

[0491] The sense strand represented by any one of the above formulas (Ia), (Ib), (Ic), and (Id) forms a duplex with the antisense strand represented by any one of the above formulas (IIa), (IIb), (IIc), and (IId), respectively.

[0492] Thus, the RNAi agent used in the methods of the present disclosure may include a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, and the RNAi duplex is represented by formula (III): [Table 1] During the ceremony, i, j, k, and l are each independently 0 or 1; p, p', q, and q' each independently represent 0 to 6; each N a and N a ’ independently represent oligonucleotide sequences containing 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides; each N b and N b ’ independently represent an oligonucleotide sequence comprising 0 to 10 modified nucleotides; During the ceremony, each n p ',n p , n q ', and n q each of which may or may not be present independently represents an overhanging nucleotide; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides.

[0493] In one embodiment, i is 0 and j is 0, or i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 0, or both i and j are 1. In another embodiment, k is 0 and l is 0, or k is 1 and l is 0, k is 0 and l is 1, or both k and l are 0, or both k and l are 1.

[0494] Exemplary combinations of sense and antisense strands that form RNAi duplexes include the following formulas: [Table 2]

[0495] When the RNAi agent is represented by formula (IIIa), each N a represents an oligonucleotide sequence that independently contains 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0496] When the RNAi agent is represented by formula (IIIb), each N b represents an oligonucleotide sequence containing, independently, 1 to 10, 1 to 7, 1 to 5, or 1 to 4 modified nucleotides. a represents an oligonucleotide sequence that independently contains 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0497] When the RNAi agent is represented by formula (IIIc), each N b , N b Each N' independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a represents an oligonucleotide sequence that independently contains 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0498] When the RNAi agent is represented by formula (IIId), each N b , N b Each N' independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a , N a ’ represents an oligonucleotide sequence containing, independently, 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. a , N a ', N b , and N b’ Each of independently comprises an alternating pattern of modifications.

[0499] In one embodiment, when the RNAi agent has formula (IIId), N a The modification is a 2'-O-methyl modification or a 2'-fluoro modification. In another embodiment, when the RNAi agent has formula (IIId), N a The modification is a 2'-O-methyl or 2'-fluoro modification, p '>0 and at least one n p In yet another embodiment, when the RNAi agent has formula (IIId), N' is linked to the adjacent nucleotide via a phosphorothioate linkage. a The modification is a 2'-O-methyl or 2'-fluoro modification, p '>0 and at least one n p In another embodiment, when the RNAi agent has formula (IIId), N' is linked to adjacent nucleotides via phosphorothioate linkages, and the sense strand is conjugated to one or more C16 (or related thereto) moieties attached via a divalent or trivalent branched linker (described below). a The modification is a 2'-O-methyl or 2'-fluoro modification, p '>0 and at least one n p ' are linked to adjacent nucleotides via phosphorothioate linkages, and the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more lipophilic moieties, e.g., C16 (or related) moieties, optionally attached via a bivalent or trivalent branched linker.

[0500] In one embodiment, when the RNAi agent has formula (IIIa), N a The modification is a 2'-O-methyl or 2'-fluoro modification, p '>0 and at least one n p' are linked to adjacent nucleotides via phosphorothioate linkages, and the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more li...

Claims

1. A composition for inhibiting the expression of C9orf72, comprising: (i) a first double-stranded ribonucleic acid (dsRNA) agent or a pharmaceutically acceptable salt thereof for reducing the level of C9orf72 sense RNA transcript and a second dsRNA agent or a pharmaceutically acceptable salt thereof for reducing the level of C9orf72 antisense RNA transcript, wherein the first dsRNA agent or a pharmaceutically acceptable salt thereof and the second dsRNA agent or a pharmaceutically acceptable salt thereof each independently comprise a sense strand and an antisense strand forming a double-stranded region, the antisense strand of the first dsRNA agent comprises at least 15 consecutive nucleotides from the complement of nucleotides 5230 to 5256 of SEQ ID NO: 13, and the antisense strand of the second dsRNA agent comprises at least 15 consecutive nucleotides from nucleotides 5155 to 5275 of SEQ ID NO: 13, all nucleotides of the sense strand and antisense strand of the first dsRNA agent and all nucleotides of the sense strand and antisense strand of the second dsRNA agent each independently comprise a nucleotide modification, wherein the sense strand, the antisense strand, or both the sense strand and the antisense strand of the first dsRNA agent, the second dsRNA agent, or both the first dsRNA agent and the second dsRNA agent are each independently conjugated to one or more lipophilic moieties, a composition, and (ii) a first dsRNA agent or a pharmaceutically acceptable salt thereof for inhibiting the expression of C9orf72 and a second dsRNA agent or a pharmaceutically acceptable salt thereof for inhibiting the expression of C9orf72, wherein the first dsRNA agent or a pharmaceutically acceptable salt thereof targeting the antisense strand of C9orf72 comprises a) a sense strand comprising at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 17 by 3 or fewer nucleotides and an antisense strand comprising a nucleotide sequence comprising at least 15 consecutive nucleotides that differ from the corresponding portion of the nucleotide sequence of SEQ ID NO: 18 by 3 or fewer nucleotides, a dsRNA agent or a pharmaceutically acceptable salt thereof. b) A dsRNA agent or a pharmaceutically acceptable salt thereof, comprising a sense strand containing at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 19 by 3 or fewer nucleotides, and an antisense strand containing a nucleotide sequence that contains at least 15 consecutive nucleotides that differ from the corresponding portion of the nucleotide sequence of SEQ ID NO: 20 by 3 or fewer nucleotides. c) A dsRNA agent or a pharmaceutically acceptable salt thereof, comprising an antisense containing a nucleotide sequence selected from the group consisting of any of the antisense strand nucleotide sequences in any one of Tables 1 to 6 below. 【Table 1-1】 【Table 1-2】 【Table 1-3】 【Table 1-4】 【Table 1-5】 【Table 1-6】 【Table 2-1】 【Table 2-2】 【Table 2-3】 【Table 2-4】 【Table 2-5】 【Table 2-6】 【Table 3】 【Table 4】 【Table 5】 【Table 6】 d) A dsRNA agent or a pharmaceutically acceptable salt thereof, comprising a sense strand containing at least 15 consecutive nucleotides that differ from nucleotides 27573296 to 27573318, 27573314 to 27573336, 27573319 to 27573341, 27573562 to 27573584, 27573585 to 27573607, 27573592 to 27573614, 27573599 to 27573621, 27573608 to 27573630, 27573616 to 27573638, 27573619 to 27573641, 27573622 to 27573644, 27573633 to 27573655, 27573690 to 27573712, or 27573717 to 27573739 of SEQ ID NO: 13 by 3 or fewer nucleotides, and ​ e) A sense strand comprising at least 15 consecutive nucleotides that differ by 3 or fewer nucleotides from any one of the nucleotide sequences of nucleotides 27573296 to 27573584, 27573296 to 27573575, 27573301 to 27573338, 27573318 to 27573342, 27573555 to 27573583, 27573581 to 27573607, 27573584 to 27573607, 27573588 to 27573671, 27573588 to 27573666, 27573588 to 27573624, 27573592 to 27573624, 27573592 to 27573617, 27573598 to 27573624, 27573599 to 27573623, 27573606 to 27573655, 27573606 to 27573652, 27573606 to 27573647, 27573654 to 27573712, or 27573707 to 27573740 of SEQ ID NO: 13, and an antisense strand comprising at least 15 consecutive nucleotides from the corresponding nucleotide sequence of SEQ ID NO: 14, a dsRNA agent or a pharmaceutically acceptable salt thereof, selected from the group consisting of, and a second dsRNA agent that targets the sense strand of C9orf72 or a pharmaceutically acceptable salt thereof, a) A sense strand comprising at least 15 consecutive nucleotides that differ by 3 or fewer nucleotides from the nucleotide sequence of SEQ ID NO: 1, and an antisense strand comprising a nucleotide sequence comprising at least 15 consecutive nucleotides that differ by 3 or fewer nucleotides from the corresponding portion of the nucleotide sequence of SEQ ID NO: 5, a dsRNA agent or a pharmaceutically acceptable salt thereof, b) A sense strand comprising at least 15 consecutive nucleotides that differ by 3 or fewer nucleotides from the nucleotide sequence of SEQ ID NO: 15, and an antisense strand comprising a nucleotide sequence comprising at least 15 consecutive nucleotides that differ by 3 or fewer nucleotides from the corresponding portion of the nucleotide sequence of SEQ ID NO: 16, a dsRNA agent or a pharmaceutically acceptable salt thereof, c) Tables 7 to 10 below 【Table 7-1】 【Table 7-2】 【Table 7-3】 【Table 7-4】 【Table 7-5】 【Table 7-6】 【Table 7-7】 【Table 7-8】 【Table 8-1】 【Table 8-2】 【Table 8-3】 【Table 8-4】 【Table 8-5】 【Table 8-6】 【Table 8-7】 【Table 8-8】 【Table 9】 【Table 10】 The dsRNA agent or a pharmaceutically acceptable salt thereof, comprising an antisense strand containing at least 15 consecutive nucleotides that differ by 3 or fewer nucleotides from any one of the antisense nucleotide sequences in any one of them. d) The dsRNA agent or a pharmaceutically acceptable salt thereof, comprising a sense strand containing at least 15 consecutive nucleotides that differ by 3 or fewer nucleotides from any one of the nucleotide sequences of nucleotides 1 to 23, 15 to 37, 33 to 55, 37 to 59, 59 to 81, 62 to 84, or 69 to 91 of SEQ ID NO: 1, and an antisense strand containing at least 15 consecutive nucleotides from the corresponding nucleotide sequence of SEQ ID NO:

5. e) The dsRNA agent or a pharmaceutically acceptable salt thereof, comprising a sense strand containing at least 15 consecutive nucleotides that differ by 3 or fewer nucleotides from any one of the nucleotide sequences of nucleotides 5197 to 5219, 5213 to 5235, 5223 to 5245, 5226 to 5248, 5227 to 5249, 5228 to 5250, 5229 to 5251, 5230 to 5252, 5231 to 5253, 5233 to 5255, 5235 to 5256, 5241 to 5263, 5245 to 5267, 5233 to 5255, 5248 to 5270, 5539 to 5561, 5547 to 5569, 5917 to 5939, 5936 to 5958, 5954 to 5976, 6008 to 6030, 6021 to 6043, 6036 to 6058, 6043 to 6065, or 6048 to 6070 of SEQ ID NO: 15, and an antisense strand containing at least 15 consecutive nucleotides from the corresponding nucleotide sequence of SEQ ID NO:

16. f) A sense strand containing at least 15 consecutive nucleotides that differ from any one of the nucleotide sequences of nucleotides 5015-5052, 5017-5040, 5032-5059, 5032-5055, 5033-5055, 5035-5059, 5036-5059, 5058-5087, 5059-5087, 5059-5084, 5064-5087, 5197-5222, 5213-5267, 5223-5252, 5229-5252, 5233-5263, 5516-5570, 5539-5565, 5539-5562, 5545-5570, 5545-5569, 5593-5616, 5883-5950, 5917-5950, 5919-5950, 5923-5950, 5934-5977, 5934-5957, 5938-5977, 5938-5965, 5938-5961, 5947-5977, 5947-5973, 5972-6001, 5973-5997, 6006-6029, 6011-6070, 6011-6039, 6011-6038, 6015-6038, 6019-6045, 6019-6042, 6033-6070, 6035-6065, 6035-6059, or 6040-6063 by 3 or fewer nucleotides, and an antisense strand containing at least 15 consecutive nucleotides from the corresponding nucleotide sequence of SEQ ID NO: 16, a dsRNA agent or a pharmaceutically acceptable salt thereof, g) A sense strand containing at least 15 consecutive nucleotides that differ from any one of the nucleotide sequences of nucleotides 15-52, 17-40, 32-59, 32-55, 35-59, 36-59, 58-87, 59-87, 59-84, or 64-87 of SEQ ID NO: 1 by 3 or fewer nucleotides, and an antisense strand containing at least 15 consecutive nucleotides from the corresponding nucleotide sequence of SEQ ID NO: 5, a dsRNA agent or a pharmaceutically acceptable salt thereof, and h) An antisense strand containing at least 15 consecutive nucleotides that differ from any one of the antisense nucleotide sequences in any one of Tables 11-12 below by 3 or fewer nucleotides, a dsRNA agent or a pharmaceutically acceptable salt thereof, 【Table 11】 【Table 12-1】 【Table 12-2】 selected from the group consisting of a composition selected from the group consisting of a composition. ​ **Claim 2**: The composition according to claim 1, wherein in (i), the antisense strand of the first dsRNA agent comprises at least 15 consecutive nucleotides from the nucleotide sequence 5'-UUUAAUCUUAUCA GGU CUUUUUC-3' or 5'-UCUGGUUAAUCUUAUCA GGU CU-3'. **Claim 3**: The composition according to claim 1, wherein in (i), the antisense strand of the second dsRNA agent is 5'-UCA A GAAA A GAC CUG AUA AAG AU-3'; 5'-UAUUAA CCA GAA GAAA ACA AGGA-3'; 5'-UCUG AUA AAG AUUAA CCA GAA GA-3'; 5'-UAGA A GAAA ACA AGGA GGG AAA C-3'; and 5'-UGAG GUG UGU GUG UUUUU GUG UUUUC-3' The composition according to claim 1, comprising at least 15 consecutive nucleotides from any one of the antisense strand nucleotide sequences selected from the group consisting of. **Claim 4**: The composition according to claim 1, wherein in (ii), the first dsRNA agent or a pharmaceutically acceptable salt thereof, the second dsRNA agent or a pharmaceutically acceptable salt thereof, or both the first dsRNA agent or a pharmaceutically acceptable salt thereof and the second dsRNA agent or a pharmaceutically acceptable salt thereof are independently conjugated to one or more lipophilic moieties. **Claim 5**: The composition according to claim 1, wherein the lipophilic moiety is conjugated to one or more internal positions in the double-stranded region of the first dsRNA agent or a pharmaceutically acceptable salt thereof, the second dsRNA agent or a pharmaceutically acceptable salt thereof, or both the first dsRNA agent or a pharmaceutically acceptable salt thereof and the second dsRNA agent or a pharmaceutically acceptable salt thereof. **Claim 6**: The composition according to claim 1, wherein the lipophilic moiety is conjugated to the first dsRNA agent or a pharmaceutically acceptable salt thereof, the second dsRNA agent or a pharmaceutically acceptable salt thereof, or both the first dsRNA agent or a pharmaceutically acceptable salt thereof and the second dsRNA agent or a pharmaceutically acceptable salt thereof via a linker or a carrier. **Claim 7** The composition according to claim 1, wherein in (ii), all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand of the first dsRNA agent or a pharmaceutically acceptable salt thereof, the second dsRNA agent or a pharmaceutically acceptable salt thereof, or both the first dsRNA agent or a pharmaceutically acceptable salt thereof and the second dsRNA agent or a pharmaceutically acceptable salt thereof contain nucleotide modifications. Item 8: The composition according to Item 1, wherein in (i), at least one of the nucleotide modifications is selected from the group consisting of deoxy-nucleotide modification, 3'-terminal deoxy-thymine (dT) nucleotide modification, 2'-O-methyl nucleotide modification, 2'-fluoro nucleotide modification, 2'-deoxy nucleotide modification, 2'-O-hexadecyl nucleotide modification, 2'-phosphate nucleotide modification, locked nucleotide modification, unlocked nucleotide modification, conformationally restricted nucleotide modification, constrained ethyl nucleotide modification, abasic nucleotide modification, reverse abasic residue modification, 2'-amino nucleotide modification, 2'-O-allyl nucleotide modification, 2'-C-alkyl nucleotide modification, 2'-hydroxy nucleotide modification, 2'-methoxyethyl nucleotide modification, 2'-O-alkyl nucleotide modification, 2',3'-seco nucleotide modification, morpholino nucleotide modification, phosphoramidate modification, nucleotide modification containing unnatural base, tetrahydropyran nucleotide modification, 1,5-anhydrohexitol nucleotide modification, cyclohexenyl nucleotide modification, nucleotide modification containing 5'-phosphorothioate group, nucleotide modification containing 5'-methylphosphonate group, nucleotide modification containing 5' phosphate or 5' phosphate mimetic, nucleotide modification containing vinylphosphonate, nucleotide modification containing adenosine-glycol nucleic acid (GNA), nucleotide modification containing thymidine-glycol nucleic acid (GNA) S isomer, nucleotide modification containing 2-hydroxymethyl-tetrahydrofuran-5-phosphate, nucleotide modification containing 2'-deoxythymidine-3' phosphate, nucleotide modification containing 2'-deoxyguanosine-3'-phosphate, and terminal nucleotide modification linked to cholesteryl derivative and bisdecylamide group of dodecanoic acid, and combinations thereof. Item 9 The composition according to Item 1, wherein the first dsRNA agent or a pharmaceutically acceptable salt thereof, the second dsRNA agent or a pharmaceutically acceptable salt thereof, or both the first dsRNA agent or a pharmaceutically acceptable salt thereof and the second dsRNA agent or a pharmaceutically acceptable salt thereof contain at least one phosphorothioate internucleotide linkage. Item 10 The composition according to claim 9, wherein the first dsRNA agent or a pharmaceutically acceptable salt thereof, the second dsRNA agent or a pharmaceutically acceptable salt thereof, or both the first dsRNA agent or a pharmaceutically acceptable salt thereof and the second dsRNA agent or a pharmaceutically acceptable salt thereof contain phosphorothioate internucleotide linkages between 6 and 8 nucleotides.

11. The composition according to claim 1, wherein at least one strand of the first dsRNA agent, the second dsRNA agent, or both the first dsRNA agent and the second dsRNA agent contains a 3'-overhang of at least one nucleotide.

12. The composition according to claim 1, wherein the double-stranded region of the first dsRNA agent, the second dsRNA agent, or both the first dsRNA agent and the second dsRNA agent is 15 to 30 nucleotide pairs in length.

13. The composition according to claim 1, wherein each strand of the first dsRNA agent, the second dsRNA agent, or both the first dsRNA agent and the second dsRNA agent is 19 to 30 nucleotides in length.

14. The composition according to claim 5, wherein the one or more lipophilic moieties are conjugated via a linker or a carrier to one or more internal positions on at least one strand of the first dsRNA agent, the second dsRNA agent, or both the first dsRNA agent and the second dsRNA agent.

15. The composition according to claim 5, wherein the one or more lipophilic moieties are conjugated to one or more of the internal positions of the first dsRNA agent, the second dsRNA agent, or both the first dsRNA agent and the second dsRNA agent, selected from the group consisting of positions 4 to 8 and 13 to 18 on the sense strand and positions 6 to 10 and 15 to 18 on the antisense strand, counted from the 5'-end of each strand.

16. The composition according to claim 5, wherein the lipophilic moiety conjugated to the first dsRNA agent, the second dsRNA agent, or both the first dsRNA agent and the second dsRNA agent is an aliphatic, alicyclic, or polycyclic alicyclic compound.

17. The composition according to claim 16, wherein the lipophilic moiety comprises 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.

18. The composition according to claim 16, wherein the lipophilic moiety comprises a saturated or unsaturated C6-C18 hydrocarbon chain.

19. The composition according to claim 16, wherein the lipophilic moiety comprises a saturated or unsaturated C16 hydrocarbon chain.

20. The composition according to claim 19, wherein the saturated or unsaturated C16 hydrocarbon chain is conjugated at the 6th position counted from the 5'-end of the chain.

21. The composition according to claim 5, wherein the lipophilic moiety is conjugated to the first dsRNA agent, the second dsRNA agent, or both the first dsRNA agent and the second dsRNA agent via a carrier that replaces one or more nucleotides within the internal position or the double-stranded region.

22. The composition according to claim 5, wherein the lipophilic moiety is conjugated to the first dsRNA agent, the second dsRNA agent, or both the first dsRNA agent and the second dsRNA agent of the double-stranded iRNA agent via a linker comprising ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide linkage, a product of a click reaction, or carbamate.

23. The composition according to claim 5, wherein the lipophilic moiety is conjugated to the nucleobase, sugar moiety, or internucleoside linkage of the first dsRNA agent, the second dsRNA agent, or both the first dsRNA agent and the second dsRNA agent.

24. The composition according to claim 5, wherein the lipophilic moiety or the targeting ligand is conjugated to the first dsRNA agent, the second dsRNA agent, or both the first dsRNA agent and the second dsRNA agent via a biochemically cleavable linker selected from the group consisting of DNA, RNA, disulfide, amide, a functionalized monosaccharide or oligosaccharide, and combinations thereof, the functionalized monosaccharide or oligosaccharide being selected from galactosamine, glucosamine, glucose, galactose, mannose.

25. The 3'-end of the sense strand of the first dsRNA agent, the second dsRNA agent, or both the first dsRNA agent and the second dsRNA agent is protected via a terminal cap which is a cyclic group having an amine, and the cyclic group is selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl, the composition according to claim 5.

26. The composition according to claim 1, wherein the first dsRNA agent, the second dsRNA agent, or both the first dsRNA agent and the second dsRNA agent further comprise a phosphate or a phosphate mimetic at the 5'-end of the antisense strand.

27. A cell comprising the composition according to claim 1.

28. The composition according to claim 1, which is a pharmaceutical composition for inhibiting the expression of C9orf72.

29. A method of reducing the level of one or more C9orf72 RNA transcripts in a cell, the method comprising contacting the cell with the composition according to claim 1, thereby inhibiting the expression of C9orf72 in the cell.

30. The composition according to claim 1, for use in a method for treating a subject having a disorder that may benefit from a reduction in C9orf72 expression, the method comprising administering to the subject a therapeutically effective amount of the composition, thereby treating the subject having a disorder that may benefit from a reduction in C9orf72 expression.

31. The composition according to claim 1, for use in a method for preventing at least one symptom in a subject having a disorder that may benefit from a reduction in C9orf72 expression, the method comprising administering to the subject a prophylactically effective amount of the composition, thereby preventing at least one symptom in the subject having a disorder that may benefit from a reduction in C9orf72 expression.

32. The composition according to claim 30 or 31, wherein the disorder is a C9orf72-related disorder.

33. The composition according to claim 32, wherein the C9orf72-related disorder is selected from the group consisting of C9orf72 amyotrophic lateral sclerosis, frontotemporal dementia, Huntington's-like syndrome caused by C9orf72 expansion, parkinsonism, olivopontocerebellar degeneration, corticobasal syndrome, and Alzheimer's disease.

34. The composition according to claim 30 or 31, wherein the subject is a human.

35. The composition according to claim 30 or 31, wherein the dsRNA agent is administered intrathecally or intraventricularly to the subject.

36. The composition according to claim 30 or 31, wherein an additional therapeutic agent is administered to the subject.

37. A kit, vial or syringe comprising the composition according to claim 1.

38. A double-stranded ribonucleic acid (dsRNA) agent or a pharmaceutically acceptable salt thereof for inhibiting the expression of C9orf72, wherein the dsRNA agent or a pharmaceutically acceptable salt thereof targets the antisense strand of C9orf72 and comprises a sense strand and an antisense strand that form a double-stranded region, (a) the sense strand comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 17 by 3 or fewer nucleotides, and the antisense strand comprises a nucleotide sequence comprising at least 15 consecutive nucleotides that differ from the corresponding portion of the nucleotide sequence of SEQ ID NO: 18 by 3 or fewer nucleotides, and the sense strand, the antisense strand, or both the sense strand and the antisense strand comprise at least one nucleotide modification; or (b) the sense strand comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 19 by 3 or fewer nucleotides, and the antisense strand comprises a nucleotide sequence comprising at least 15 consecutive nucleotides that differ from the corresponding portion of the nucleotide sequence of SEQ ID NO: 20 by 3 or fewer nucleotides, and the sense strand, the antisense strand, or both the sense strand and the antisense strand comprise at least one nucleotide modification; or (c) the sense strand or the antisense strand is a sense strand or an antisense strand selected from the group consisting of any one of the sense strands and antisense strands in Tables 13-18 below 【Table 13-1】 【Table 13-2】 【Table 13-3】 【Table 13-4】 【Table 13-5】 【Table 13-6】 【Table 14-1】 【Table 14-2】 【Table 14-3】 【Table 14-4】 【Table 14-5】 【Table 14-6】 【Table 15】 【Table 16】 【Table 17】 【Table 18】 and the sense strand, the antisense strand, or both the sense strand and the antisense strand comprise at least one nucleotide modification. The sense strand, the antisense strand, or both the sense strand and the antisense strand contain at least one nucleotide modification; or (d) the sense strand contains at least 15 consecutive nucleotides that differ from nucleotides 27573296 to 27573318, 27573314 to 27573336, 27573319 to 27573341, 27573562 to 27573584, 27573585 to 27573607, 27573592 to 27573614, 27573599 to 27573621, 27573608 to 27573630, 27573616 to 27573638, 27573619 to 27573641, 27573622 to 27573644, 27573633 to 27573655, 27573690 to 27573712, or 27573717 to 27573739 of SEQ ID NO: 13 by 3 or fewer nucleotides, and The sense strand, the antisense strand, or both the sense strand and the antisense strand contain at least one nucleotide modification; or (e) the sense strand contains at least 15 consecutive nucleotides that differ from any one of the nucleotide sequences of nucleotides 27573296 to 27573584, 27573296 to 27573575, 27573301 to 27573338, 27573318 to 27573342, 27573555 to 27573583, 27573581 to 27573607, 27573584 to 27573607, 27573588 to 27573671, 27573588 to 27573666, 27573588 to 27573624, 27573592 to 27573624, 27573592 to 27573617, 27573598 to 27573624, 27573599 to 27573623, 27573606 to 27573655, 27573606 to 27573652, 27573606 to 27573647, 27573654 to 27573712, or 27573707 to 27573740 of SEQ ID NO: 13 by 3 or fewer nucleotides, and the antisense strand contains at least 15 consecutive nucleotides from the corresponding nucleotide sequence of SEQ ID NO: 14, and The sense strand, the antisense strand, or both the sense strand and the antisense strand contain at least one nucleotide modification. A dsRNA agent or a pharmaceutically acceptable salt thereof.

39. A double-stranded ribonucleic acid (dsRNA) agent or a pharmaceutically acceptable salt thereof for inhibiting the expression of C9orf72, wherein the dsRNA agent or a pharmaceutically acceptable salt thereof targets the sense strand of C9orf72 and comprises a sense strand and an antisense strand that form a double-stranded region, wherein (a) the antisense strand comprises at least 15 consecutive nucleotides that differ by 3 or fewer nucleotides from any one of the antisense nucleotide sequences in any one of Tables 19 to 22 below, and 【Table 19-1】 【Table 19-2】 【Table 19-3】 【Table 19-4】 【Table 19-5】 【Table 19-6】 【Table 19-7】 【Table 19-8】 【Table 20-1】 【Table 20-2】 【Table 20-3】 【Table 20-4】 【Table 20-5】 【Table 20-6】 【Table 20-7】 【Table 20-8】 【Table 21】 【Table 22】 the sense strand, the antisense strand, or both the sense strand and the antisense strand comprise at least one nucleotide modification; or wherein (b) the sense strand comprises at least 15 consecutive nucleotides that differ by 3 or fewer nucleotides from any one of the nucleotide sequences of nucleotides 1 to 23, 15 to 37, 33 to 55, 37 to 59, 59 to 81, 62 to 84, or 62 to 91 of SEQ ID NO: 1, the antisense strand comprises at least 15 consecutive nucleotides from the corresponding nucleotide sequence of SEQ ID NO: 5, and the sense strand, the antisense strand, or both the sense strand and the antisense strand comprise at least one nucleotide modification; or wherein (c) the sense strand comprises at least 15 consecutive nucleotides that differ by 3 or fewer nucleotides from any one of the nucleotide sequences of nucleotides 5197 to 5219, 5213 to 5235, 5223 to 5245, 5226 to 5248, 5227 to 5249, 5228 to 5250, 5229 to 5251, 5230 to 5252, 5231 to 5253, 5233 to 5255, 5235 to 5256, 5241 to 5263, 5245 to 5267, 5248 to 5270, 5539 to 5561, 5547 to 5569, 5917 to 5939, 5936 to 5958, 5954 to 5976, 6008 to 6030, 6021 to 6043, 6036 to 6058, 6043 to 6065, or 6048 to 6070 of SEQ ID NO: 15, and the antisense strand comprises at least 15 consecutive nucleotides from the corresponding nucleotide sequence of SEQ ID NO: 16, and the sense strand, the antisense strand, or both the sense strand and the antisense strand comprise at least one nucleotide modification; or The sense strand, the antisense strand, or both the sense strand and the antisense strand contain at least one nucleotide modification; or (d) the sense strand contains at least 15 consecutive nucleotides that differ from any one of the nucleotide sequences of nucleotides 5015 - 5052, 5017 - 5040, 5032 - 5059, 5032 - 5055, 5033 - 5055, 5035 - 5059, 5036 - 5059, 5058 - 5087, 5059 - 5087, 5059 - 5084, 5064 - 5087, 5197 - 5222, 5213 - 5267, 5223 - 5252, 5229 - 5252, 5233 - 5263, 5516 - 5570, 5539 - 5565, 5539 - 5562, 5545 - 5570, 5545 - 5569, 5593 - 5616, 5883 - 5950, 5917 - 5950, 5919 - 5950, 5923 - 5950, 5934 - 5977, 5934 - 5957, 5938 - 5977, 5938 - 5965, 5938 - 5961, 5947 - 5977, 5947 - 5973, 5972 - 6001, 5973 - 5997, 6006 - 6029, 6011 - 6070, 6011 - 6039, 6011 - 6038, 6015 - 6038, 6019 - 6045, 6019 - 6042, 6033 - 6070, 6035 - 6065, 6035 - 6059, or 6040 - 6063 of SEQ ID NO: 15 by 3 or fewer nucleotides, and the antisense strand contains at least 15 consecutive nucleotides from the corresponding nucleotide sequence of SEQ ID NO: 16, and the sense strand, the antisense strand, or both the sense strand and the antisense strand contain at least one nucleotide modification; or (e) the sense strand contains at least 15 consecutive nucleotides that differ from any one of the nucleotide sequences of nucleotides 15 - 52, 17 - 40, 32 - 59, 32 - 55, 35 - 59, 36 - 59, 58 - 87, 59 - 87, 59 - 84, or 64 - 87 of SEQ ID NO: 1 by 3 or fewer nucleotides, and the antisense strand contains at least 15 consecutive nucleotides from the corresponding nucleotide sequence of SEQ ID NO: 5, and the sense strand, the antisense strand, or both the sense strand and the antisense strand contain at least one nucleotide modification; or (f) The antisense strand is any one of the antisense nucleotide sequences in any one of Tables 23 to 24 below 【Table 23】 【Table 24-1】 【Table 24-2】 and contains at least 15 consecutive nucleotides that differ from any one of them by 3 or fewer nucleotides, and the sense strand, the antisense strand, or both the sense strand and the antisense strand contain at least one nucleotide modification, a dsRNA agent or a pharmaceutically acceptable salt thereof.

40. The dsRNA agent or a pharmaceutically acceptable salt thereof according to Claim 38 or 39, wherein the sense strand, the antisense strand, or both the sense strand and the antisense strand are conjugated to one or more lipophilic moieties.

41. The dsRNA agent or a pharmaceutically acceptable salt thereof according to Claim 38 or 39, wherein the lipophilic moiety is conjugated to one or more internal positions in the double-stranded region of the dsRNA agent.

42. The dsRNA agent or a pharmaceutically acceptable salt thereof according to Claim 40, wherein the lipophilic moiety is conjugated via a linker or a carrier.

43. The dsRNA agent or a pharmaceutically acceptable salt thereof according to Claim 38 or 39, wherein all the nucleotides of the sense strand and all the nucleotides of the antisense strand contain nucleotide modifications.

44. At least one of the nucleotide modifications is selected from the group consisting of deoxy-nucleotide modification, 3'-terminal deoxy-thymine (dT) nucleotide modification, 2'-O-methyl nucleotide modification, 2'-fluoro nucleotide modification, 2'-deoxy nucleotide modification, 2'-O-hexadecyl nucleotide modification, 2'-phosphate nucleotide modification, 2'-5' linked ribonucleotide (3'-RNA) modification, locked nucleotide modification, unlocked nucleotide modification, conformationally restricted nucleotide modification, constrained ethyl nucleotide modification, abasic nucleotide modification, reverse abasic residue modification, 2'-amino nucleotide modification, 2'-O-allyl nucleotide modification, 2'-C-alkyl nucleotide modification, 2'-hydroxy nucleotide modification, 2'-methoxyethyl nucleotide modification, 2'-O-alkyl nucleotide modification, 2',3'-seco nucleotide modification, morpholino nucleotide modification, phosphoramidate modification, nucleotide modification containing unnatural bases, tetrahydropyran nucleotide modification, 1,5-anhydrohexitol nucleotide modification, cyclohexenyl nucleotide modification, nucleotide modification containing 5'-phosphorothioate group, nucleotide modification containing 5'-methylphosphonate group, nucleotide modification containing 5'-phosphate or 5'-phosphate mimetic, nucleotide modification containing vinylphosphonate, nucleotide modification containing glycol nucleic acid (GNA), nucleotide modification containing glycol nucleic acid S isomer (S-GNA), nucleotide modification containing 2-hydroxymethyl-tetrahydrofuran-5-phosphate, nucleotide modification containing 2'-deoxythymidine-3'-phosphate, nucleotide modification containing 2'-deoxyguanosine-3'-phosphate, and terminal nucleotide modification linked to cholesteryl derivative and bisdecylamide group of dodecanoic acid, and combinations thereof, the dsRNA agent according to claim 38 or 39, or a pharmaceutically acceptable salt thereof.

45. The dsRNA agent according to claim 38 or 39, or a pharmaceutically acceptable salt thereof, comprising at least one phosphorothioate nucleotide internucleoside linkage.

46. The dsRNA agent or a pharmaceutically acceptable salt thereof according to claim 38 or 39, wherein at least one strand comprises a 3' overhang of at least one nucleotide.

47. The dsRNA agent or a pharmaceutically acceptable salt thereof according to claim 38 or 39, wherein the double-stranded region has a length of 15 to 30 nucleotide pairs.

48. The dsRNA agent or a pharmaceutically acceptable salt thereof according to claim 38 or 39, wherein each strand has a length of 19 to 30 nucleotides.

49. The dsRNA agent or a pharmaceutically acceptable salt thereof according to claim 40, wherein one or more of the lipophilic moieties are conjugated to one or more internal positions selected from the group consisting of positions 4 to 8 and 13 to 18 on the sense strand and positions 6 to 10 and 15 to 18 on the antisense strand, counted from the 5' end of each strand.

50. The dsRNA agent or a pharmaceutically acceptable salt thereof according to claim 40, wherein the lipophilic moiety is an aliphatic, alicyclic, or polycyclic alicyclic compound.

51. The lipophilic moiety is conjugated via a carrier that replaces one or more nucleotides at the internal position or within the double-stranded region, or The lipophilic moiety is conjugated to the double-stranded iRNA agent via a linker comprising an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide linkage, a product of a click reaction, or a carbamate, or The lipophilic moiety is conjugated to a nucleobase, sugar moiety, or internucleoside linkage, or The lipophilic moiety or targeting ligand is a DNA, RNA, disulfide, amide, functionalized monosaccharide or oligosaccharide, and is conjugated via a biochemically cleavable linker selected from the group consisting of functionalized monosaccharides or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof. The dsRNA agent or a pharmaceutically acceptable salt thereof according to claim 40.

52. The 3'-end of the sense strand is protected via a terminal cap which is a cyclic group having an amine, and the cyclic group is selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl, the dsRNA agent according to claim 40 or a pharmaceutically acceptable salt thereof.

53. The dsRNA agent according to claim 38 or 39 or a pharmaceutically acceptable salt thereof, further comprising a phosphate or a phosphate mimetic at the 5'-end of the antisense strand.

54. A cell comprising the dsRNA agent according to claim 38 or 39 or a pharmaceutically acceptable salt thereof.

55. A pharmaceutical composition for inhibiting the expression of C9orf72, comprising the dsRNA agent according to claim 38 or 39 or a pharmaceutically acceptable salt thereof.

56. A method for reducing the level of one or more C9orf72 RNA transcripts in a cell, the method comprising contacting the cell with the dsRNA agent according to claim 38 or 39 or a pharmaceutically acceptable salt thereof, thereby inhibiting the expression of C9orf72 in the cell.