Compositions and methods for treating neurological disorders

Abstract

The present disclosure provides single-stranded or double-stranded interfering RNA molecules (e.g., siRNAs) that target the open reading frame 72 (C9orf72) gene of chromosome 9. The interfering RNA molecule may contain a specific pattern of nucleoside modification and inter-nucleoside linkage modification as a pharmaceutical composition comprising the interfering RNA molecule. The siRNA molecule may be a branched siRNA molecule, such as a two-branched, three-branched or four-branched siRNA molecule. The disclosed siRNA molecules may also be characterized by a 5 '-phosphorus stabilizing moiety and / or a hydrophobic moiety. In addition, the present disclosure provides methods for delivering siRNA molecules of the present disclosure to the central nervous system of a subject, such as a subject identified as having amyotrophic lateral sclerosis or frontotemporal dementia.

Description

[0001] Sequence Listing

[0002] This application contains a sequence listing that has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML copy was created on September 20, 2023, is named "51436-037WO2_Sequence_Listing_9_20_23" and is 1,011,545 bytes in size. Technical Field

[0003] The present disclosure relates to small interfering RNA (siRNA) molecules and compositions containing the siRNA molecules, the siRNA molecules targeting RNA transcripts (e.g., mRNA) of the chromosome 9 open reading frame 72 (C9orf72) gene. The present disclosure further describes methods for silencing C9orf72 and treating diseases that may benefit from silencing C9orf72 (e.g., frontotemporal dementia or amyotrophic lateral sclerosis), the methods being achieved by delivering siRNA molecules targeting C9orf72 to target tissues of subjects in need thereof. Background Art

[0004] C9orf72 (chromosome 9 open reading frame 72) encodes a protein that is involved in the pathology of diseases including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Studies have shown that repeat expansions within C9orf72 can cause ALS and FTD. There is a need for therapeutic approaches that can selectively reduce C9orf72 activity in a manner that provides effective treatment for ALS, FTD or other C9orf72-related diseases or conditions. Summary of the invention

[0005] The present disclosure provides compositions and methods for reducing the expression of chromosome 9 open reading frame 72 (C9orf72) by small interfering RNA (siRNA)-mediated silencing of C9orf72 transcripts. The advantage of the compositions and methods is that they exhibit high selectivity for C9orf72 relative to other genes.

[0006] The siRNA molecules of the present disclosure can be used to silence the C9orf72 gene, thereby preventing the translation of the corresponding mRNA transcript and reducing C9orf72 protein expression. Therefore, such a reduction in C9orf72 levels can prevent the onset or progression of the disease, because the hexanucleotide repeat expansion GGGGCC in the C9orf72 gene is a common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The siRNA molecules of the present disclosure can be delivered directly to a subject in need of C9orf72 silencing by, for example, intrathecal injection, intracerebroventricular injection, intrastriatal injection, intraparenchymal injection, intracerebellar medullary cisterna magna injection (such as via catheter insertion), intravenous injection, subcutaneous injection or intramuscular injection.

[0007] In one aspect, the present disclosure provides siRNA molecules comprising an antisense strand and a sense strand having complementarity to the antisense strand. The antisense strand has sufficient complementarity to hybridize to a region within a C9orf72 mRNA transcript having a nucleic acid sequence of any one of SEQ ID NOs: 1-384. In some embodiments, the antisense strand has sufficient complementarity to hybridize to a region within a C9orf72 mRNA transcript having a nucleic acid sequence of any one of SEQ ID NOs: 1-192. In some embodiments, the antisense strand has sufficient complementarity to hybridize to a region within a C9orf72 mRNA transcript having a nucleic acid sequence of any one of SEQ ID NOs: 193-384. The antisense strand can, for example, be 10 to 50 nucleotides in length (e.g., 10 to 45 nucleotides in length, 10 to 40 nucleotides in length, 10 to 35 nucleotides in length, 10 to 30 nucleotides in length, 10 to 29 nucleotides in length, 10 to 28 nucleotides in length, 10 to 27 nucleotides in length, 10 to 26 nucleotides in length, 10 to 25 nucleotides in length, 10 to 24 nucleotides in length, 10 to 23 nucleotides in length, 10 to 22 nucleotides in length, 10 to 21 nucleotides in length, or 10 to 20 nucleotides in length). In some embodiments, the antisense strand is 10 nucleotides in length, 11 nucleotides in length, 12 nucleotides in length, 13 nucleotides in length, 14 nucleotides in length, 15 nucleotides in length, 16 nucleotides in length, 17 nucleotides in length, 18 nucleotides in length, 19 nucleotides in length, 20 nucleotides in length, 21 nucleotides in length, 22 nucleotides in length, 23 nucleotides in length, 24 nucleotides in length, 25 nucleotides in length, 26 nucleotides in length, 27 nucleotides in length, 28 nucleotides in length, 29 nucleotides in length, 30 nucleotides in length, or longer.

[0008] In some embodiments of any of the preceding aspects, the antisense strand has at least 70% (e.g., at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) complementarity to a region of 15 consecutive nucleobases within a C9orf72 mRNA transcript having a nucleic acid sequence of any one of SEQ ID NOs: 1-384, SEQ ID NOs: 1-192, or SEQ ID NOs: 193-384. In some embodiments, the antisense strand has at least 70% (e.g., at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) complementarity to a region of 16 consecutive nucleobases within a C9orf72 mRNA transcript having a nucleic acid sequence of any one of SEQ ID NOs: 1-384. In some embodiments, the antisense strand has at least 70% (e.g., at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) complementarity to a region of 17 consecutive nucleobases within a C9orf72 mRNA transcript having a nucleic acid sequence of any one of SEQ ID NOs: 1-384, SEQ ID NOs: 1-192, or SEQ ID NOs: 193-384.In some embodiments, the antisense strand has at least 70% (e.g., at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) complementarity to a region of 18 consecutive nucleobases within a C9orf72 mRNA transcript having a nucleic acid sequence of any one of SEQ ID NOs: 1-384, SEQ ID NOs: 1-192, or SEQ ID NOs: 193-384. In some embodiments, the antisense strand has at least 70% (e.g., at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) complementarity to a region of 19 consecutive nucleobases within a C9orf72 mRNA transcript having a nucleic acid sequence of any one of SEQ ID NOs: 1-384, SEQ ID NOs: 1-192, or SEQ ID NOs: 193-384. In some embodiments, the antisense strand has at least 70% (e.g., at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) complementarity to a region of 20 consecutive nucleobases within a C9orf72 mRNA transcript having a nucleic acid sequence of any one of SEQ ID NOs: 1-384, SEQ ID NOs: 1-192, or SEQ ID NOs: 193-384.In some embodiments, the antisense strand has at least 70% (e.g., at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) complementarity to a region of 21 consecutive nucleobases within a C9orf72 mRNA transcript having a nucleic acid sequence of any one of SEQ ID NOs: 1-384, SEQ ID NOs: 1-192, or SEQ ID NOs: 193-384.

[0009] In some embodiments, the antisense strand has at least 70% (e.g., at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) complementarity to a region within a C9orf72 mRNA transcript having a nucleic acid sequence of any one of SEQ ID NOs: 1-384. In some embodiments, the region within the C9orf72 mRNA transcript has a nucleic acid sequence of any one of SEQ ID NOs: 1-192. In some embodiments, the region within the C9orf72 mRNA transcript has a nucleic acid sequence of any one of SEQ ID NOs: 193-384.

[0010] In some embodiments, the antisense strand has at least 75% complementarity to a region within a C9orf72 mRNA transcript having a nucleic acid sequence of any one of SEQ ID NOs: 1-384. For example, the antisense strand can have at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementarity to a region within a C9orf72 mRNA transcript having a nucleic acid sequence of any one of SEQ ID NOs: 1-384. In some embodiments, the region within the C9orf72 mRNA transcript has a nucleic acid sequence of any one of SEQ ID NOs: 1-192. In some embodiments, the region within the C9orf72 mRNA transcript has a nucleic acid sequence of any one of SEQ ID NOs: 193-384.

[0011] In some embodiments, the antisense strand has at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or 30 consecutive nucleotides that are fully complementary to a consecutive polynucleotide segment of equal length within the region of the C9orf72 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-384. In some embodiments, the region of the C9orf72 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 1-192. In some embodiments, the region of the C9orf72 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 193-384.

[0012] In some embodiments, the antisense strand has 10 to 30 consecutive nucleotides (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive nucleotides) that are fully complementary to a consecutive polynucleotide segment of equal length within the region of the C9orf72 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-384. In some embodiments, the region of the C9orf72 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 1-192. In some embodiments, the region of the C9orf72 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 193-384.

[0013] In some embodiments, the antisense strand has 12 to 30 consecutive nucleotides (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive nucleotides) that are fully complementary to a consecutive polynucleotide segment of equal length within the region of the C9orf72 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-384. In some embodiments, the region of the C9orf72 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 1-192. In some embodiments, the region of the C9orf72 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 193-384.

[0014] In some embodiments, the antisense strand has 15 to 30 consecutive nucleotides (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive nucleotides) that are fully complementary to a consecutive polynucleotide segment of equal length within the region of the C9orf72 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-384. In some embodiments, the region of the C9orf72 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 1-192. In some embodiments, the region of the C9orf72 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 193-384.

[0015] In some embodiments, the antisense strand has 18 to 30 consecutive nucleotides (e.g., 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive nucleotides) that are fully complementary to a consecutive polynucleotide segment of equal length within the region of the C9orf72 RNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-384. In some embodiments, the region of the C9orf72 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 1-192. In some embodiments, the region of the C9orf72 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 193-384.

[0016] In some embodiments, the antisense strand has 18 to 25 consecutive nucleotides (e.g., 18, 19, 20, 21, 22, 23, 24, or 25 consecutive nucleotides) that are fully complementary to a consecutive polynucleotide segment of equal length within the region of the C9orf72 mRNA transcript having a nucleic acid sequence of any one of SEQ ID NOs: 1-384. In some embodiments, the region of the C9orf72 mRNA transcript has a nucleic acid sequence of any one of SEQ ID NOs: 1-192. In some embodiments, the region of the C9orf72 mRNA transcript has a nucleic acid sequence of any one of SEQ ID NOs: 193-384.

[0017] In some embodiments, the antisense strand has 18 to 21 consecutive nucleotides (e.g., 18, 19, 20, or 21 consecutive nucleotides) that are fully complementary to a consecutive polynucleotide segment of equal length within the region of the C9orf72 mRNA transcript having a nucleic acid sequence of any one of SEQ ID NOs: 1-384. In some embodiments, the region of the C9orf72 mRNA transcript has a nucleic acid sequence of any one of SEQ ID NOs: 1-192. In some embodiments, the region of the C9orf72 mRNA transcript has a nucleic acid sequence of any one of SEQ ID NOs: 193-384.

[0018] In some embodiments, the antisense strand has 21 consecutive nucleotides that are fully complementary to a consecutive polynucleotide segment of equal length within the region of the C9orf72 mRNA transcript having a nucleic acid sequence of any one of SEQ ID NOs: 1-384. In some embodiments, the region of the C9orf72 mRNA transcript has a nucleic acid sequence of any one of SEQ ID NOs: 1-192. In some embodiments, the region of the C9orf72 mRNA transcript has a nucleic acid sequence of any one of SEQ ID NOs: 193-384.

[0019] In some embodiments, the antisense strand has 9 or fewer nucleotide mismatches relative to a region of 21 consecutive nucleobases of a C9orf72 RNA transcript having a nucleic acid sequence of any one of SEQ ID NOs: 1-384, optionally wherein the antisense strand contains 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or only 1 mismatch relative to the region of a C9orf72 mRNA transcript having a nucleic acid sequence of any one of SEQ ID NOs: 1-384. In some embodiments, the region of the C9orf72 mRNA transcript has a nucleic acid sequence of any one of SEQ ID NOs: 1-192. In some embodiments, the region of the C9orf72 mRNA transcript has a nucleic acid sequence of any one of SEQ ID NOs: 193-384.

[0020] In some embodiments, the antisense strand has a nucleic acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the nucleic acid sequence of any one of SEQ ID NOs: 769-1152. In some embodiments, the antisense strand has a nucleic acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the nucleic acid sequence of any one of SEQ ID NOs: 769-960. In some embodiments, the antisense strand has a nucleic acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the nucleic acid sequence of any one of SEQ ID NOs:961-1152.

[0021] In some embodiments, the antisense strand has a nucleic acid sequence that is at least 90% identical (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the nucleic acid sequence of any one of SEQ ID NOs: 769-1152. In some embodiments, the antisense strand has a nucleic acid sequence that is at least 90% identical (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the nucleic acid sequence of any one of SEQ ID NOs: 769-960. In some embodiments, the antisense strand has a nucleic acid sequence that is at least 90% identical (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the nucleic acid sequence of any one of SEQ ID NOs:961-1152.

[0022] In some embodiments, the antisense strand has a nucleic acid sequence that is at least 95% identical (e.g., 95%, 96%, 97%, 98%, 99% or 100% identical) to a nucleic acid sequence of any one of SEQ ID NOs: 769-1152, optionally wherein the antisense strand has a nucleic acid sequence that is at least 96%, 97%, 98% or 99% identical to a nucleic acid sequence of any one of SEQ ID NOs: 769-1152. In some embodiments, the antisense strand has a nucleic acid sequence that is at least 95% identical (e.g., 95%, 96%, 97%, 98%, 99% or 100% identical) to a nucleic acid sequence of any one of SEQ ID NOs: 769-960. In some embodiments, the antisense strand has a nucleic acid sequence that is at least 95% identical (e.g., 95%, 96%, 97%, 98%, 99% or 100% identical) to a nucleic acid sequence of any one of SEQ ID NOs: 961-1152.

[0023] In some embodiments, the antisense strand has a nucleic acid sequence of any one of SEQ ID NOs: 769-1152. In some embodiments, the antisense strand has a nucleic acid sequence of any one of SEQ ID NOs: 769-960. In some embodiments, the antisense strand has a nucleic acid sequence of any one of SEQ ID NOs: 961-1152.

[0024] In some embodiments, the sense strand has a nucleic acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the nucleic acid sequence of any one of SEQ ID NOs: 385-768. In some embodiments, the sense strand has a nucleic acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the nucleic acid sequence of any one of SEQ ID NOs: 385-576. In some embodiments, the sense strand has a nucleic acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the nucleic acid sequence of any one of SEQ ID NOs: 577-768.

[0025] In some embodiments, the sense strand has a nucleic acid sequence that is at least 90% identical (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the nucleic acid sequence of any one of SEQ ID NOs: 385-768. In some embodiments, the sense strand has a nucleic acid sequence that is at least 90% identical (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the nucleic acid sequence of any one of SEQ ID NOs: 385-576. In some embodiments, the sense strand has a nucleic acid sequence that is at least 90% identical (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the nucleic acid sequence of any one of SEQ ID NOs: 577-768.

[0026] In some embodiments, the sense strand has a nucleic acid sequence that is at least 95% identical (e.g., 95%, 96%, 97%, 98%, 99% or 100% identical) to a nucleic acid sequence of any one of SEQ ID NOs: 385-768, optionally wherein the sense strand has a nucleic acid sequence that is at least 96%, 97%, 98% or 99% identical to a nucleic acid sequence of any one of SEQ ID NOs: 385-768. In some embodiments, the sense strand has a nucleic acid sequence that is at least 95% identical (e.g., 95%, 96%, 97%, 98%, 99% or 100% identical) to a nucleic acid sequence of any one of SEQ ID NOs: 385-576. In some embodiments, the sense strand has a nucleic acid sequence that is at least 95% identical (e.g., 95%, 96%, 97%, 98%, 99% or 100% identical) to a nucleic acid sequence of any one of SEQ ID NOs: 577-768.

[0027] In some embodiments, the siRNA molecule has a sense strand having a nucleic acid sequence of any one of SEQ ID NOs: 385-768. In some embodiments, the sense strand has a nucleic acid sequence of any one of SEQ ID NOs: 385-576. In some embodiments, the sense strand has a nucleic acid sequence of any one of SEQ ID NOs: 577-768.

[0028] In some embodiments, the antisense strand has a structure represented by Formula I, wherein Formula I is, in the 5' to 3' direction:

[0029] AB-(A') j -CP 2 -DP 1 -(C'-P 1 ) k -C'

[0030] Formula I;

[0031] Where A is composed of the formula CP 1 -DP 1 express;

[0032] Each A' is represented by the formula CP 2 -DP 2 express;

[0033] B by CP 2 -DP 2 -DP 2 -DP 2 express;

[0034] Each C is a 2'-O-methyl (2'-O-Me) ribonucleoside;

[0035] Each C' is independently 2'-O-Me ribonucleoside or 2'-fluoro (2'-F) ribonucleoside;

[0036] Each D is a 2'-F ribonucleoside;

[0037] Each P 1 is a phosphorothioate internucleoside linkage;

[0038] Each P 2 It is a phosphodiester internucleoside linkage;

[0039] j is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6 or 7); and

[0040] k is an integer from 1 to 7 (eg, 1, 2, 3, 4, 5, 6, or 7).

[0041] In some embodiments, the antisense strand has a structure represented by Formula A1, wherein Formula A1 is:

[0042] ASBSAOBOBOBOAOBOAOBOA-OBAOBOAOBSASASASBSA

[0043] Formula A1;

[0044] wherein A represents a 2'-0-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0045] In some embodiments, the antisense strand has a structure represented by Formula II, wherein Formula II is, in the 5' to 3' direction:

[0046] AB-(A') j -CP 2 -DP 1 -(CP 1 ) k -C'

[0047] Formula II;

[0048] Where A is composed of the formula CP 1 -DP 1 express;

[0049] Each A' is represented by the formula CP 2 -DP 2 express;

[0050] B by CP 2 -DP 2 -DP 2 -DP2 express;

[0051] Each C is a 2'-O-methyl (2'-O-Me) ribonucleoside;

[0052] Each C' is independently 2'-O-Me ribonucleoside or 2'-fluoro (2'-F) ribonucleoside;

[0053] Each D is a 2'-F ribonucleoside;

[0054] Each P 1 is a phosphorothioate internucleoside linkage;

[0055] Each P 2 It is a phosphodiester internucleoside linkage;

[0056] j is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6 or 7); and

[0057] k is an integer from 1 to 7 (eg, 1, 2, 3, 4, 5, 6, or 7).

[0058] In some embodiments, the antisense strand has a structure represented by Formula A2, wherein Formula A2 is, in the 5' to 3' direction:

[0059] ASBSAOBOBOBOAOBOAOBOA-OBOOBOAOBSASASASASA

[0060] Formula A2;

[0061] wherein A represents a 2'-0-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0062] In some embodiments, the sense strand has a structure represented by Formula III, wherein Formula III is, in the 5' to 3' direction:

[0063] E-(A') m -F

[0064] Formula III;

[0065] Where E is given by the formula (CP 1 ) 2 express;

[0066] F is given by the formula (CP 2 ) 3 -DP 1 -CP 1 -C, (CP 2 ) 3-DP 2 -CP 2 -C, (CP 2 ) 3 -DP 1 -CP 1 -D or (CP 2 ) 3 -DP 2 -CP 2 -D means;

[0067] A', C, D, P 1 and P 2 As defined in Formula II; and

[0068] m is an integer from 1 to 7 (eg, 1, 2, 3, 4, 5, 6 or 7).

[0069] In some embodiments, the sense strand has a structure represented by Formula S1, wherein Formula S1 is, in the 5' to 3' direction:

[0070] ASASAOBOAOBOAOBOAOBOA-OAOAOBSASA

[0071] Formula S1;

[0072] wherein A represents a 2'-0-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0073] In some embodiments, the sense strand has a structure represented by Formula S2, wherein Formula S2 is, in the 5' to 3' direction:

[0074] ASASAOBOAOBOAOBOAOBOA-OAOAOBOAOA

[0075] Formula S2;

[0076] wherein A represents a 2'-0-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0077] In some embodiments, the sense strand has a structure represented by Formula S3, wherein Formula S3 is, in the 5' to 3' direction:

[0078] ASASAOBOAOBOAOBOAOBOA-OAOAOBSASB

[0079] Formula S3;

[0080] wherein A represents a 2'-0-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0081] In some embodiments, the sense strand has a structure represented by Formula S4, wherein Formula S4 is, in the 5' to 3' direction:

[0082] ASASAOBOAOBOAOBOAOBOA-OAOAOBOAOB

[0083] Formula S4;

[0084] wherein A represents a 2'-0-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0085] In some embodiments, the antisense strand has a structure represented by Formula IV, wherein Formula IV is, in the 5' to 3' direction:

[0086] A-(A') j -CP 2 -B-(CP 1 ) k -C'

[0087] Formula IV;

[0088] Where A is composed of the formula CP 1 -DP 1 express;

[0089] Each A' is represented by the formula CP 2 -DP 2 express;

[0090] B by DP 1 -CP 1 -DP 1 express;

[0091] Each C is a 2'-O-Me ribonucleoside;

[0092] Each C' is independently 2'-O-Me ribonucleoside or 2'-F ribonucleoside;

[0093] Each D is a 2'-F ribonucleoside;

[0094] Each P 1 is a phosphorothioate internucleoside linkage;

[0095] Each P2 It is a phosphodiester internucleoside linkage;

[0096] j is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6 or 7); and

[0097] k is an integer from 1 to 7 (eg, 1, 2, 3, 4, 5, 6, or 7).

[0098] In some embodiments, the antisense strand has a structure represented by Formula A3, wherein Formula A3 is:

[0099] ASBSAOBOAOBOAOBOAOBOA-OBAOBOAOBSASBSASASA

[0100] Formula A3;

[0101] wherein A represents a 2'-0-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0102] In some embodiments, the sense strand has a structure represented by Formula V, wherein Formula V is, in the 5' to 3' direction:

[0103] E-(A') m -CP 2 -F

[0104] Formula V;

[0105] Where E is given by the formula (CP 1 ) 2 express;

[0106] F by DP 1 -CP 1 -C, DP 2 -CP 2 -C, DP 1 -CP 1 -D or DP 2 -CP 2 -D means;

[0107] A', C, D, P 1 and P 2 As defined in Formula IV; and

[0108] m is an integer from 1 to 7 (eg, 1, 2, 3, 4, 5, 6 or 7).

[0109] In some embodiments, the sense strand has a structure represented by Formula S5, wherein Formula S5 is, in the 5' to 3' direction:

[0110] ASASAOBOAOBOAOBOAOBOA-OBOAOBSASA

[0111] Formula S5;

[0112] wherein A represents a 2'-0-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0113] In some embodiments, the sense strand has a structure represented by Formula S6, wherein Formula S6 is, in the 5' to 3' direction:

[0114] ASASAOBOAOBOAOBOAOBOA-OBOAOBOAOA

[0115] Formula S6;

[0116] wherein A represents a 2'-0-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0117] In some embodiments, the sense strand has a structure represented by Formula S7, wherein Formula S7 is, in the 5' to 3' direction:

[0118] ASASAOBOAOBOAOBOAOBOA-OBOAOBSASB

[0119] Formula S7;

[0120] wherein A represents a 2'-0-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0121] In some embodiments, the sense strand has a structure represented by Formula S8, wherein Formula S8 is, in the 5' to 3' direction:

[0122] ASASAOBOAOBOAOBOAOBOA-OBOAOBOAOB

[0123] Formula S8;

[0124] wherein A represents a 2'-0-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0125] In some embodiments, the antisense strand has a structure represented by Formula VI, wherein Formula VI is, in the 5' to 3' direction:

[0126] AB j -EB k -EFG l -DP 1 -C'

[0127] Formula VI;

[0128] Where A is composed of the formula CP 1 -DP 1 express;

[0129] Each B is represented by the formula CP 2 express;

[0130] Each C is a 2'-O-Me ribonucleoside;

[0131] Each C' is independently 2'-O-Me ribonucleoside or 2'-F ribonucleoside;

[0132] Each D is a 2'-F ribonucleoside;

[0133] Each E is represented by the formula DP 2 -CP 2 express;

[0134] F by DP 1 -CP 1 express;

[0135] Each G is represented by the formula CP 1 express;

[0136] Each P 1 is a phosphorothioate internucleoside linkage;

[0137] Each P 2 It is a phosphodiester internucleoside linkage;

[0138] j is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6 or 7);

[0139] k is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6 or 7); and

[0140] l is an integer from 1 to 7 (eg, 1, 2, 3, 4, 5, 6 or 7).

[0141] In some embodiments, the antisense strand has a structure represented by Formula A4, wherein Formula A4 is, in the 5' to 3' direction:

[0142] ASBSAOAOAOBOAOAOAOAOA-OAOAOBOAOBSASASASASBSA

[0143] Formula A4;

[0144] wherein A represents a 2'-0-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0145] In some embodiments, the sense strand has a structure represented by Formula VII, wherein Formula VII is, in the 5' to 3' direction:

[0146] HB m -I n -A'-B o -HC

[0147] Formula VII;

[0148] Where A' is composed of the formula CP 2 -DP 2 express;

[0149] Each H is represented by the formula (CP 1 ) 2 express;

[0150] Each I is represented by the formula (DP 2 )express;

[0151] B, C, D, P 1 and P 2 As defined in Formula VI;

[0152] m is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6 or 7);

[0153] n is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6 or 7); and

[0154] o is an integer from 1 to 7 (eg, 1, 2, 3, 4, 5, 6 or 7).

[0155] In some embodiments, the sense strand has a structure represented by Formula S9, wherein Formula S9 is, in the 5' to 3' direction:

[0156] ASASAOAOAOBOBOBOAOBOA-OAOAOASASA

[0157] Formula S9;

[0158] wherein A represents a 2'-0-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0159] In some embodiments, the antisense strand further has a 5' phosphorus stabilizing moiety at the 5' end of the antisense strand.

[0160] In some embodiments, the sense strand further has a 5' phosphorus stabilizing moiety at the 5' end of the sense strand.

[0161] In some embodiments, each 5'-phosphorus stabilizing moiety is independently represented by any of Formula IX, XX, XI, XII, XIII, XIV, XV, or XVI:

[0162]

[0163] wherein Nuc represents a nucleobase, optionally wherein the nucleobase is selected from the group consisting of adenine, uracil, guanine, thymine and cytosine, and R represents optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, phenyl, benzyl, a cation (e.g., a monovalent cation) or hydrogen.

[0164] In some embodiments, the nucleobase is adenine, uracil, guanine, thymine, or cytosine.

[0165] In some embodiments, the 5' phosphorus stabilizing moiety is an (E)-vinyl phosphonate represented by Formula XI.

[0166] In some embodiments, the siRNA molecule further has a hydrophobic portion at the 5' or 3' end of the siRNA molecule.

[0167] In some embodiments, the hydrophobic moiety is selected from cholesterol, vitamin D, or tocopherol.

[0168] In some embodiments, the siRNA molecule is a branched siRNA molecule.

[0169] In some embodiments, the branched siRNA molecule is bi-branched, tri-branched, or quad-branched.

[0170] In some embodiments, the siRNA molecule is bi-branched, optionally wherein the bi-branched siRNA molecule is represented by any one of Formula XVII, XVIII or XIX:

[0171]

[0172] Wherein each RNA is independently a siRNA molecule, L is a linker, and each X independently represents a branch point moiety.

[0173] In some embodiments, the bi-branched siRNA molecule is represented by Formula XVII. In some embodiments, the bi-branched siRNA molecule is represented by Formula XVIII. In some embodiments, the bi-branched siRNA molecule is represented by Formula XIX.

[0174] In some embodiments, the siRNA molecule is three-branched, optionally wherein the three-branched siRNA molecule is represented by any one of Formula XX, XXI, XXII, or XXIII:

[0175]

[0176] Wherein each RNA is independently a siRNA molecule, L is a linker, and each X independently represents a branch point moiety.

[0177] In some embodiments, the three-branched siRNA molecule is represented by formula XX. In some embodiments, the three-branched siRNA molecule is represented by formula XXI. In some embodiments, the three-branched siRNA molecule is represented by formula XXII. In some embodiments, the three-branched siRNA molecule is represented by formula XXIII.

[0178] In some embodiments, the siRNA molecule is four-branched, optionally wherein the four-branched siRNA molecule is represented by any one of Formula XXIV, XXV, XXVI, XXVII, or XXVIII:

[0179]

[0180] Wherein each RNA is independently a siRNA molecule, L is a linker, and each X independently represents a branch point moiety.

[0181] In some embodiments, the four-branched siRNA molecule is represented by formula XXIV. In some embodiments, the four-branched siRNA molecule is represented by formula XXV. In some embodiments, the four-branched siRNA molecule is represented by formula XXVI. In some embodiments, the four-branched siRNA molecule is represented by formula XXVII. In some embodiments, the four-branched siRNA molecule is represented by formula XXVIII.

[0182] In some embodiments of branched siRNA, the linker is selected from one or more consecutive subunits of ethylene glycol (e.g., polyethylene glycol (PEG), such as triethylene glycol (TrEG) or tetraethylene glycol (TEG)), alkyl, carbohydrate, block copolymer, peptide, RNA, and DNA.

[0183] In some embodiments, the linker is an ethylene glycol oligomer. In some embodiments, the linker is an alkyl oligomer. In some embodiments, the linker is a carbohydrate oligomer. In some embodiments, the linker is a block copolymer. In some embodiments, the linker is a peptide oligomer. In some embodiments, the linker is an RNA oligomer. In some embodiments, the linker is a DNA oligomer.

[0184] In some embodiments, the ethylene glycol oligomer is PEG. In some embodiments, the PEG is TrEG. In some embodiments, the PEG is TEG.

[0185] In some embodiments, the oligomer or copolymer contains 2 to 20 consecutive subunits (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive subunits).

[0186] In some embodiments, the linker is attached to one or more (eg, 1, 2, 3, 4, or more) siRNA molecules by way of a covalent bond forming part.

[0187] In some embodiments, the covalent bond forming moiety is selected from the group consisting of alkyl, ester, amide, carbamate, phosphonate, phosphate, phosphorothioate, phosphoramidate, triazole, urea, and methylal.

[0188] In some embodiments, the linker comprises a structure of Formula L1:

[0189]

[0190] In some embodiments, the linker comprises a structure of Formula L2:

[0191]

[0192]

[0193] In some embodiments, the linker comprises a structure of Formula L3:

[0194]

[0195] In some embodiments, the linker comprises a structure of Formula L4:

[0196]

[0197] In some embodiments, the linker comprises a structure of Formula L5:

[0198]

[0199] In some embodiments, the linker comprises a structure of Formula L6:

[0200]

[0201] In some embodiments, the linker comprises a structure of Formula L7:

[0202]

[0203]

[0204] In some embodiments, the linker comprises a structure of Formula L8:

[0205]

[0206] In some embodiments, the linker comprises a structure of Formula L9:

[0207]

[0208] In some embodiments of any of the siRNA molecules described herein, 50% or more of the ribonucleotides in the antisense strand are 2′-O-Me ribonucleotides (e.g., 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70% of the antisense strand). , 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% or 100% of the ribonucleotides can be 2'-O-Me ribonucleotides).

[0209] In some embodiments, 60% or more of the ribonucleotides in the antisense strand are 2'-O-Me ribonucleotides (e.g., 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the ribonucleotides in the antisense strand can be 2'-O-Me ribonucleotides).

[0210] In some embodiments, 70% or more of the ribonucleotides in the antisense strand are 2'-O-Me ribonucleotides (e.g., 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%, or 100% of the ribonucleotides in the antisense strand may be 2'-O-Me ribonucleotides).

[0211] In some embodiments, 80% or more of the ribonucleotides in the antisense strand are 2'-O-Me ribonucleotides (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the ribonucleotides in the antisense strand may be 2'-O-Me ribonucleotides).

[0212] In some embodiments, 90% or more of the ribonucleotides in the antisense strand are 2'-O-Me ribonucleotides (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the ribonucleotides in the antisense strand may be 2'-O-Me ribonucleotides).

[0213] In some embodiments, 10% or less of the internucleoside linkages are phosphodiester linkages or phosphorothioate linkages. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the internucleoside linkages are phosphodiester linkages or phosphorothioate linkages. In some embodiments, 100% of the internucleoside linkages are phosphodiester linkages or phosphorothioate linkages.

[0214] In some embodiments, the nine internucleoside linkages are phosphodiester linkages or phosphorothioate linkages.

[0215] In some embodiments, the antisense strand is 10 to 30 nucleotides in length (e.g., 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides), 15 to 25 nucleotides (e.g., 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, or 25 nucleotides), or 18 to 23 nucleotides (e.g., 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, or 23 nucleotides). In some embodiments, the antisense strand is 20 nucleotides in length. In some embodiments, the antisense strand is 21 nucleotides in length. In some embodiments, the antisense strand is 22 nucleotides in length. In some embodiments, the antisense strand is 23 nucleotides in length. In some embodiments, the antisense strand is 24 nucleotides in length. In some embodiments, the antisense strand is 25 nucleotides in length. In some embodiments, the antisense strand is 26 nucleotides in length. In some embodiments, the antisense strand is 27 nucleotides in length. In some embodiments, the antisense strand is 28 nucleotides in length. In some embodiments, the antisense strand is 29 nucleotides in length. In some embodiments, the antisense strand is 30 nucleotides in length.

[0216] In some embodiments, the siRNA molecules of the branched compounds are connected to each other by a joint (e.g., an ethylene glycol oligomer, such as tetraethylene glycol). In some embodiments, the siRNA molecules of the branched compounds are connected to each other by a joint between the sense strand of one siRNA molecule and the sense strand of another siRNA molecule. In some embodiments, the siRNA molecules are connected by a joint between the antisense strand of one siRNA molecule and the antisense strand of another siRNA molecule. In some embodiments, the siRNA molecules of the branched compounds are connected to each other by a joint between the sense strand of one siRNA molecule and the antisense strand of another siRNA molecule.

[0217] In some embodiments, the sense strand is 12 to 30 nucleotides in length (e.g., 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides) or 14 to 18 nucleotides (e.g., 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, or 18 nucleotides). In some embodiments, the sense strand is 15 nucleotides in length. In some embodiments, the sense strand is 16 nucleotides in length. In some embodiments, the sense strand is 17 nucleotides in length. In some embodiments, the sense strand is 18 nucleotides in length. In some embodiments, the sense strand is 19 nucleotides in length. In some embodiments, the sense strand is 20 nucleotides in length. In some embodiments, the sense strand is 21 nucleotides in length. In some embodiments, the sense strand is 22 nucleotides in length. In some embodiments, the sense strand is 23 nucleotides in length. In some embodiments, the sense strand is 24 nucleotides in length. In some embodiments, the sense strand is 25 nucleotides in length. In some embodiments, the sense strand is 26 nucleotides in length. In some embodiments, the sense strand is 27 nucleotides in length. In some embodiments, the sense strand is 28 nucleotides in length. In some embodiments, the sense strand is 29 nucleotides in length. In some embodiments, the sense strand is 30 nucleotides in length.

[0218] In some embodiments, the four internucleoside linkages are phosphorothioate linkages.

[0219] In some embodiments of the siRNA molecules described herein, the length of the antisense strand is 18 nucleotides and the length of the sense strand is 14 nucleotides. In some embodiments, the length of the antisense strand is 18 nucleotides and the length of the sense strand is 15 nucleotides. In some embodiments, the length of the antisense strand is 18 nucleotides and the length of the sense strand is 16 nucleotides. In some embodiments, the length of the antisense strand is 18 nucleotides and the length of the sense strand is 17 nucleotides. In some embodiments, the length of the antisense strand is 18 nucleotides and the length of the sense strand is 18 nucleotides. In some embodiments, the length of the antisense strand is 19 nucleotides and the length of the sense strand is 14 nucleotides. In some embodiments, the length of the antisense strand is 19 nucleotides and the length of the sense strand is 15 nucleotides. In some embodiments, the length of the antisense strand is 19 nucleotides and the length of the sense strand is 16 nucleotides. In some embodiments, the length of the antisense strand is 19 nucleotides and the length of the sense strand is 17 nucleotides. In some embodiments, the length of the antisense strand is 19 nucleotides and the length of the sense strand is 18 nucleotides. In some embodiments, the length of the antisense strand is 19 nucleotides and the length of the sense strand is 19 nucleotides. In some embodiments, the length of the antisense strand is 20 nucleotides and the length of the sense strand is 14 nucleotides. In some embodiments, the length of the antisense strand is 20 nucleotides and the length of the sense strand is 15 nucleotides. In some embodiments, the length of the antisense strand is 20 nucleotides and the length of the sense strand is 16 nucleotides. In some embodiments, the length of the antisense strand is 20 nucleotides and the length of the sense strand is 17 nucleotides. In some embodiments, the length of the antisense strand is 20 nucleotides and the length of the sense strand is 18 nucleotides. In some embodiments, the length of the antisense strand is 20 nucleotides and the length of the sense strand is 19 nucleotides. In some embodiments, the length of the antisense strand is 20 nucleotides and the length of the sense strand is 20 nucleotides. In some embodiments, the length of the antisense strand is 21 nucleotides and the length of the sense strand is 14 nucleotides. In some embodiments, the length of the antisense strand is 21 nucleotides and the length of the sense strand is 15 nucleotides. In some embodiments, the antisense strand is 21 nucleotides in length and the sense strand is 16 nucleotides in length. In some embodiments, the antisense strand is 21 nucleotides in length and the sense strand is 17 nucleotides in length. In some embodiments, the antisense strand is 21 nucleotides in length and the sense strand is 18 nucleotides in length. In some embodiments, the antisense strand is 21 nucleotides in length and the sense strand is 19 nucleotides in length. In some embodiments, the antisense strand is 21 nucleotides in length and the sense strand is 20 nucleotides in length. In some embodiments, the antisense strand is 21 nucleotides in length and the sense strand is 21 nucleotides in length.In some embodiments, the length of the antisense strand is 22 nucleotides and the length of the sense strand is 14 nucleotides. In some embodiments, the length of the antisense strand is 22 nucleotides and the length of the sense strand is 15 nucleotides. In some embodiments, the length of the antisense strand is 22 nucleotides and the length of the sense strand is 16 nucleotides. In some embodiments, the length of the antisense strand is 22 nucleotides and the length of the sense strand is 17 nucleotides. In some embodiments, the length of the antisense strand is 22 nucleotides and the length of the sense strand is 18 nucleotides. In some embodiments, the length of the antisense strand is 22 nucleotides and the length of the sense strand is 19 nucleotides. In some embodiments, the length of the antisense strand is 22 nucleotides and the length of the sense strand is 20 nucleotides. In some embodiments, the length of the antisense strand is 22 nucleotides and the length of the sense strand is 21 nucleotides. In some embodiments, the length of the antisense strand is 22 nucleotides and the length of the sense strand is 22 nucleotides. In some embodiments, the length of the antisense strand is 23 nucleotides and the length of the sense strand is 14 nucleotides. In some embodiments, the length of the antisense strand is 23 nucleotides and the length of the sense strand is 15 nucleotides. In some embodiments, the length of the antisense strand is 23 nucleotides and the length of the sense strand is 16 nucleotides. In some embodiments, the length of the antisense strand is 23 nucleotides and the length of the sense strand is 17 nucleotides. In some embodiments, the length of the antisense strand is 23 nucleotides and the length of the sense strand is 18 nucleotides. In some embodiments, the length of the antisense strand is 23 nucleotides and the length of the sense strand is 19 nucleotides. In some embodiments, the length of the antisense strand is 23 nucleotides and the length of the sense strand is 20 nucleotides. In some embodiments, the length of the antisense strand is 23 nucleotides and the length of the sense strand is 21 nucleotides. In some embodiments, the length of the antisense strand is 23 nucleotides and the length of the sense strand is 22 nucleotides. In some embodiments, the length of the antisense strand is 23 nucleotides and the length of the sense strand is 23 nucleotides. In some embodiments, the length of the antisense strand is 24 nucleotides and the length of the sense strand is 14 nucleotides. In some embodiments, the antisense strand is 24 nucleotides in length and the sense strand is 15 nucleotides in length. In some embodiments, the antisense strand is 24 nucleotides in length and the sense strand is 16 nucleotides in length. In some embodiments, the antisense strand is 24 nucleotides in length and the sense strand is 17 nucleotides in length. In some embodiments, the antisense strand is 24 nucleotides in length and the sense strand is 18 nucleotides in length. In some embodiments, the antisense strand is 24 nucleotides in length and the sense strand is 19 nucleotides in length. In some embodiments, the antisense strand is 24 nucleotides in length and the sense strand is 20 nucleotides in length.In some embodiments, the length of the antisense strand is 24 nucleotides and the length of the sense strand is 21 nucleotides. In some embodiments, the length of the antisense strand is 24 nucleotides and the length of the sense strand is 22 nucleotides. In some embodiments, the length of the antisense strand is 24 nucleotides and the length of the sense strand is 23 nucleotides. In some embodiments, the length of the antisense strand is 24 nucleotides and the length of the sense strand is 24 nucleotides. In some embodiments, the length of the antisense strand is 25 nucleotides and the length of the sense strand is 14 nucleotides. In some embodiments, the length of the antisense strand is 25 nucleotides and the length of the sense strand is 15 nucleotides. In some embodiments, the length of the antisense strand is 25 nucleotides and the length of the sense strand is 16 nucleotides. In some embodiments, the length of the antisense strand is 25 nucleotides and the length of the sense strand is 17 nucleotides. In some embodiments, the length of the antisense strand is 25 nucleotides and the length of the sense strand is 18 nucleotides. In some embodiments, the length of the antisense strand is 25 nucleotides and the length of the sense strand is 19 nucleotides. In some embodiments, the length of the antisense strand is 25 nucleotides and the length of the sense strand is 20 nucleotides. In some embodiments, the length of the antisense strand is 25 nucleotides and the length of the sense strand is 21 nucleotides. In some embodiments, the length of the antisense strand is 25 nucleotides and the length of the sense strand is 22 nucleotides. In some embodiments, the length of the antisense strand is 25 nucleotides and the length of the sense strand is 23 nucleotides. In some embodiments, the length of the antisense strand is 25 nucleotides and the length of the sense strand is 24 nucleotides. In some embodiments, the length of the antisense strand is 25 nucleotides and the length of the sense strand is 25 nucleotides. In some embodiments, the length of the antisense strand is 26 nucleotides and the length of the sense strand is 14 nucleotides. In some embodiments, the length of the antisense strand is 26 nucleotides and the length of the sense strand is 15 nucleotides. In some embodiments, the length of the antisense strand is 26 nucleotides and the length of the sense strand is 16 nucleotides. In some embodiments, the length of the antisense strand is 26 nucleotides and the length of the sense strand is 17 nucleotides. In some embodiments, the length of the antisense strand is 26 nucleotides and the length of the sense strand is 18 nucleotides. In some embodiments, the length of the antisense strand is 26 nucleotides and the length of the sense strand is 19 nucleotides. In some embodiments, the length of the antisense strand is 26 nucleotides and the length of the sense strand is 20 nucleotides. In some embodiments, the length of the antisense strand is 26 nucleotides and the length of the sense strand is 21 nucleotides. In some embodiments, the length of the antisense strand is 26 nucleotides and the length of the sense strand is 22 nucleotides. In some embodiments, the length of the antisense strand is 26 nucleotides and the length of the sense strand is 23 nucleotides.In some embodiments, the length of the antisense strand is 26 nucleotides and the length of the sense strand is 24 nucleotides. In some embodiments, the length of the antisense strand is 26 nucleotides and the length of the sense strand is 25 nucleotides. In some embodiments, the length of the antisense strand is 26 nucleotides and the length of the sense strand is 26 nucleotides. In some embodiments, the length of the antisense strand is 27 nucleotides and the length of the sense strand is 14 nucleotides. In some embodiments, the length of the antisense strand is 27 nucleotides and the length of the sense strand is 15 nucleotides. In some embodiments, the length of the antisense strand is 27 nucleotides and the length of the sense strand is 16 nucleotides. In some embodiments, the length of the antisense strand is 27 nucleotides and the length of the sense strand is 17 nucleotides. In some embodiments, the length of the antisense strand is 27 nucleotides and the length of the sense strand is 18 nucleotides. In some embodiments, the length of the antisense strand is 27 nucleotides and the length of the sense strand is 19 nucleotides. In some embodiments, the length of the antisense strand is 27 nucleotides and the length of the sense strand is 20 nucleotides. In some embodiments, the length of the antisense strand is 27 nucleotides and the length of the sense strand is 21 nucleotides. In some embodiments, the length of the antisense strand is 27 nucleotides and the length of the sense strand is 22 nucleotides. In some embodiments, the length of the antisense strand is 27 nucleotides and the length of the sense strand is 23 nucleotides. In some embodiments, the length of the antisense strand is 27 nucleotides and the length of the sense strand is 24 nucleotides. In some embodiments, the length of the antisense strand is 27 nucleotides and the length of the sense strand is 25 nucleotides. In some embodiments, the length of the antisense strand is 27 nucleotides and the length of the sense strand is 26 nucleotides. In some embodiments, the length of the antisense strand is 27 nucleotides and the length of the sense strand is 27 nucleotides. In some embodiments, the length of the antisense strand is 28 nucleotides and the length of the sense strand is 14 nucleotides. In some embodiments, the length of the antisense strand is 28 nucleotides and the length of the sense strand is 15 nucleotides. In some embodiments, the length of the antisense strand is 28 nucleotides and the length of the sense strand is 16 nucleotides. In some embodiments, the antisense strand is 28 nucleotides in length and the sense strand is 17 nucleotides in length. In some embodiments, the antisense strand is 28 nucleotides in length and the sense strand is 18 nucleotides in length. In some embodiments, the antisense strand is 28 nucleotides in length and the sense strand is 19 nucleotides in length. In some embodiments, the antisense strand is 28 nucleotides in length and the sense strand is 20 nucleotides in length. In some embodiments, the antisense strand is 28 nucleotides in length and the sense strand is 21 nucleotides in length. In some embodiments, the antisense strand is 28 nucleotides in length and the sense strand is 22 nucleotides in length.In some embodiments, the length of the antisense strand is 28 nucleotides and the length of the sense strand is 23 nucleotides. In some embodiments, the length of the antisense strand is 28 nucleotides and the length of the sense strand is 24 nucleotides. In some embodiments, the length of the antisense strand is 28 nucleotides and the length of the sense strand is 25 nucleotides. In some embodiments, the length of the antisense strand is 28 nucleotides and the length of the sense strand is 26 nucleotides. In some embodiments, the length of the antisense strand is 28 nucleotides and the length of the sense strand is 27 nucleotides. In some embodiments, the length of the antisense strand is 28 nucleotides and the length of the sense strand is 28 nucleotides. In some embodiments, the length of the antisense strand is 29 nucleotides and the length of the sense strand is 14 nucleotides. In some embodiments, the length of the antisense strand is 29 nucleotides and the length of the sense strand is 15 nucleotides. In some embodiments, the length of the antisense strand is 29 nucleotides and the length of the sense strand is 16 nucleotides. In some embodiments, the length of the antisense strand is 29 nucleotides and the length of the sense strand is 17 nucleotides. In some embodiments, the length of the antisense strand is 29 nucleotides and the length of the sense strand is 18 nucleotides. In some embodiments, the length of the antisense strand is 29 nucleotides and the length of the sense strand is 19 nucleotides. In some embodiments, the length of the antisense strand is 29 nucleotides and the length of the sense strand is 20 nucleotides. In some embodiments, the length of the antisense strand is 29 nucleotides and the length of the sense strand is 21 nucleotides. In some embodiments, the length of the antisense strand is 29 nucleotides and the length of the sense strand is 22 nucleotides. In some embodiments, the length of the antisense strand is 29 nucleotides and the length of the sense strand is 23 nucleotides. In some embodiments, the length of the antisense strand is 29 nucleotides and the length of the sense strand is 24 nucleotides. In some embodiments, the length of the antisense strand is 29 nucleotides and the length of the sense strand is 25 nucleotides. In some embodiments, the length of the antisense strand is 29 nucleotides and the length of the sense strand is 26 nucleotides. In some embodiments, the length of the antisense strand is 29 nucleotides and the length of the sense strand is 27 nucleotides. In some embodiments, the antisense strand is 29 nucleotides in length and the sense strand is 28 nucleotides in length. In some embodiments, the antisense strand is 29 nucleotides in length and the sense strand is 29 nucleotides in length. In some embodiments, the antisense strand is 30 nucleotides in length and the sense strand is 14 nucleotides in length. In some embodiments, the antisense strand is 30 nucleotides in length and the sense strand is 15 nucleotides in length. In some embodiments, the antisense strand is 30 nucleotides in length and the sense strand is 16 nucleotides in length. In some embodiments, the antisense strand is 30 nucleotides in length and the sense strand is 17 nucleotides in length.In some embodiments, the length of the antisense strand is 30 nucleotides and the length of the sense strand is 18 nucleotides. In some embodiments, the length of the antisense strand is 30 nucleotides and the length of the sense strand is 19 nucleotides. In some embodiments, the length of the antisense strand is 30 nucleotides and the length of the sense strand is 20 nucleotides. In some embodiments, the length of the antisense strand is 30 nucleotides and the length of the sense strand is 21 nucleotides. In some embodiments, the length of the antisense strand is 30 nucleotides and the length of the sense strand is 22 nucleotides. In some embodiments, the length of the antisense strand is 30 nucleotides and the length of the sense strand is 23 nucleotides. In some embodiments, the length of the antisense strand is 30 nucleotides and the length of the sense strand is 24 nucleotides. In some embodiments, the length of the antisense strand is 30 nucleotides and the length of the sense strand is 25 nucleotides. In some embodiments, the length of the antisense strand is 30 nucleotides and the length of the sense strand is 26 nucleotides. In some embodiments, the length of the antisense strand is 30 nucleotides and the length of the sense strand is 27 nucleotides. In some embodiments, the antisense strand is 30 nucleotides in length and the sense strand is 28 nucleotides in length. In some embodiments, the antisense strand is 30 nucleotides in length and the sense strand is 29 nucleotides in length. In some embodiments, the antisense strand is 30 nucleotides in length and the sense strand is 30 nucleotides in length.

[0220] In another aspect, the present disclosure provides a pharmaceutical composition comprising the siRNA molecule of any one of the aforementioned aspects or embodiments of the present disclosure and a pharmaceutically acceptable excipient, carrier or diluent.

[0221] In another aspect, the present disclosure provides a method of delivering siRNA molecules to a subject diagnosed with ALS by administering to the subject a therapeutically effective amount of the siRNA molecule or pharmaceutical composition of any one of the foregoing aspects or embodiments of the present disclosure.

[0222] In another aspect, the present disclosure provides a method of delivering siRNA molecules to a subject diagnosed with FTD by administering to the subject a therapeutically effective amount of the siRNA molecule or pharmaceutical composition of any one of the foregoing aspects or embodiments of the present disclosure.

[0223] In another aspect, the present disclosure provides a method of treating ALS in a subject in need thereof, by administering to the subject a therapeutically effective amount of the siRNA molecule or pharmaceutical composition of any one of the foregoing aspects or embodiments of the present disclosure.

[0224] In another aspect, the present disclosure provides a method of treating FTD in a subject in need thereof by administering to the subject a therapeutically effective amount of the siRNA molecule or pharmaceutical composition of any one of the foregoing aspects or embodiments of the present disclosure.

[0225] In another aspect, the present disclosure provides a method of reducing C9orf72 expression in a subject in need thereof by administering a therapeutically effective amount of the siRNA or pharmaceutical composition of any one of the foregoing aspects or embodiments of the present disclosure.

[0226] In some embodiments, the siRNA molecule or the pharmaceutical composition is administered to the subject by intraventricular, intrastriatal, intraparenchymal or intrathecal injection. In some embodiments, the siRNA molecule or the pharmaceutical composition is administered to the subject by intravenous, intramuscular, or subcutaneous injection.

[0227] In some embodiments, the subject is a human.

[0228] In another aspect, the present disclosure provides a kit containing the siRNA molecule or pharmaceutical composition of any of the foregoing aspects or embodiments of the present disclosure, and a package insert instructing the user of the kit to perform the method of any of the foregoing aspects or embodiments of the present disclosure.

[0229] definition

[0230] Unless otherwise defined herein, the scientific and technical terms used herein have the meanings commonly understood by those of ordinary skill in the art. If there is any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. Unless the context otherwise requires, singular terms shall include plural terms and plural terms shall include singular terms. Unless otherwise indicated, the use of "or" means "and / or". The use of the term "including" and other forms such as "includes" and "included" is not restrictive.

[0231] As used herein, the term "nucleic acid" refers to an RNA molecule or a DNA molecule composed of a chain of ribonucleotides or deoxyribonucleotides, respectively.

[0232] As used herein, the term "therapeutic nucleic acid" refers to a nucleic acid molecule (eg, ribonucleic acid) that has partial or complete complementarity with a disease-associated target mRNA and interacts with and mediates silencing of mRNA expression.

[0233] As used herein, the term "carrier nucleic acid" refers to a nucleic acid molecule (e.g., ribonucleic acid) that has sequence complementarity with a therapeutic nucleic acid and hybridizes with the therapeutic nucleic acid. As used herein, the term "3' end" refers to a nucleic acid end containing an unmodified hydroxyl group at the 3' carbon of the ribose ring.

[0234] As used herein, the term "nucleoside" refers to a molecule consisting of a heterocyclic base and its sugar.

[0235] As used herein, the term "nucleotide" refers to a nucleoside having a phosphate group or variants thereof on its 3' or 5' sugar hydroxyl group. Examples of phosphate group variants include, but are not limited to, saturated alkyl phosphonates, unsaturated alkenyl phosphonates, phosphorothioates, and phosphoramidites.

[0236] In the context of the present disclosure, the term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or its mimetics. The term includes oligonucleotides composed of naturally occurring nucleobases, sugars, and covalent internucleoside (backbone) linkages, as well as oligonucleotides having non-naturally occurring (e.g., modified) portions of similar functions. Such modified or substituted oligonucleotides are often superior to the natural form because they have desirable properties, such as enhanced cellular uptake, enhanced affinity for nucleic acid targets, and increased stability in the presence of nucleases.

[0237] As used herein, the term "siRNA" refers to a small interfering RNA duplex that induces an RNA interference (RNAi) pathway. The length of the siRNA molecule may vary (usually between 10 and 30 base pairs) and may have varying degrees of complementarity with its target mRNA. The term "siRNA" includes a duplex of two separate strands, and optionally a single strand that forms a hairpin structure that includes the duplex region.

[0238] As used herein, the term "antisense strand" refers to the strand of a siRNA duplex that has a certain degree of complementarity with a target gene.

[0239] As used herein, the term "sense strand" refers to the strand of a siRNA duplex that has complementarity to the antisense strand.

[0240] The term "interfering RNA molecule" refers to an RNA molecule, such as small interfering RNA (siRNA), micro RNA (miRNA), short hairpin RNA (shRNA), or antisense oligonucleotide (ASO), that suppresses the endogenous function of a target RNA transcript.

[0241] As used herein, the term "expression" refers to one or more of the following events: (1) the generation of an RNA template from a DNA sequence (e.g., by transcription); (2) the processing of an RNA transcript (e.g., by splicing, editing, 5' cap formation and / or 3' end processing); and (3) the translation of RNA into a polypeptide or protein. In the context of a gene encoding a protein product, the terms "gene expression" and the like are used interchangeably with the terms "protein expression" and the like. The expression of a gene or protein of interest in a patient can be visualized, for example, by detecting in a sample obtained from the patient an increase in the amount or concentration of mRNA encoding the corresponding protein (e.g., assessed using an RNA detection procedure described herein or known in the art, such as quantitative polymerase chain reaction (qPCR) and RNAseq techniques), an increase in the amount or concentration of the corresponding protein (e.g., assessed using a protein detection method described herein or known in the art, such as an enzyme-linked immunosorbent assay (ELISA), etc.), and / or an increase in the activity of the corresponding protein (e.g., in the case of an enzyme, as assessed using an enzyme activity assay described herein or known in the art). As used herein, a cell is considered to "express" a gene or protein of interest if one or more or all of the above events can be detected in the cell or in the culture medium in which the cell resides. For example, a gene or protein of interest is considered to be "expressed" by a cell or cell population if the following events can be detected: (i) production of a corresponding RNA transcript, such as an mRNA template, by the cell or cell population (e.g., using an RNA detection procedure described herein); (ii) processing of the RNA transcript (e.g., splicing, editing, 5' cap formation and / or 3' end processing, such as using an RNA detection procedure described herein); (iii) translation of the RNA template into a protein product (e.g., using a protein detection procedure described herein); and / or (iv) post-translational modification of the protein product (e.g., using a protein detection procedure described herein).

[0242] As used herein, the terms "target," "targeting," and "targeted" in the context of siRNA design refer to generating the antisense strand so that it anneals to a region of interest within an mRNA transcript in a manner that results in reduced translation of the mRNA into a protein product.

[0243] As used herein, the terms "chemically modified nucleotides," "nucleotide analogs," "altered nucleotides," or "modified nucleotides" refer to non-standard nucleotides, including non-naturally occurring ribonucleotides or deoxyribonucleotides. Exemplary nucleotide analogs are modified at any position to alter certain chemical properties of the nucleotide, but retain the ability of the nucleotide analog to perform its intended function.

[0244] As used herein, the term "metabolic stable" refers to an RNA molecule containing ribonucleotides that have been chemically modified so as to reduce the metabolic rate of the RNA molecule administered to a subject. Exemplary modifications include modifications of 2'-hydroxy to 2'-O-methoxy or 2'-fluoro, and modifications of phosphodiester to phosphorothioate.

[0245] As used herein, the term "phosphorothioate" refers to a phosphate group of a nucleotide modified by replacing one or more oxygens of the phosphate group with sulfur.

[0246] As used herein, the terms "internucleoside" and "internucleotide" refer to the linkages between nucleosides and nucleotides, respectively.

[0247] As used herein, the term "antagomir" refers to a nucleic acid that can act as an inhibitor of miRNA activity.

[0248] As used herein, the term "gapmer" refers to a chimeric antisense nucleic acid containing a central block of deoxynucleotide monomers that is sufficiently long to induce cleavage by RNase H. The deoxynucleotide block is flanked by ribonucleotide monomers or ribonucleotide monomers containing modifications.

[0249] As used herein, the term "mixer" refers to a nucleic acid containing a mixture of locked nucleic acid (LNA) and DNA.

[0250] As used herein, the term "guide RNA" refers to a nucleic acid having sequence complementarity with a specific sequence in the genome, which is immediately upstream or 1 base pair of the protospacer adjacent motif (PAM) sequence, such as used in the CRISPR / Cas9 gene editing system. Alternatively, "guide RNA" can refer to a nucleic acid having sequence complementarity (e.g., antisense) with a specific messenger RNA (mRNA) sequence. In this regard, the guide RNA can also have sequence complementarity with a "passenger RNA" sequence of the same or shorter length, and the "passenger RNA" sequence is identical or substantially identical to the sequence of the mRNA to which the guide RNA hybridizes.

[0251] As used herein, the term "branched siRNA" refers to a compound containing two or more double-stranded siRNA molecules covalently bound to each other. The branched siRNA molecule can be "two-branched", also referred to herein as "two-siRNA", wherein the siRNA molecule includes, for example, 2 siRNA molecules covalently bound to each other through a linker. The branched siRNA molecule can be "three-branched", also referred to herein as "three-siRNA", wherein the siRNA molecule includes, for example, 3 siRNA molecules covalently bound to each other through a linker. The branched siRNA molecule can be "four-branched", also referred to herein as "four-siRNA", wherein the siRNA molecule includes, for example, 4 siRNA molecules covalently bound to each other through a linker.

[0252] As used herein, the term "branch point moiety" refers to a chemical moiety of the branched siRNA structure of the present disclosure that can be covalently linked to the 5' end or 3' end of the antisense strand or sense strand of the siRNA molecule and can support the attachment of additional single-stranded or double-stranded siRNA molecules. Non-limiting examples of branch point moieties suitable for use with the disclosed methods and compositions include, for example, phosphoramidites, glycerol tosylated acetonate, 1,3-diaminopropanol, pentaerythritol, and any of the branch point moieties described in US 10,478,503.

[0253] As used herein, the term "phosphate moiety" refers to a terminal phosphate group including phosphate and modified phosphate. The phosphate moiety may be located at either end, but is preferably located at the 5'-terminal nucleoside. In one aspect, the terminal phosphate is unmodified and has the formula -O-P(═O)(OH)OH. On the other hand, the terminal phosphate is modified so that one or more of the O and OH groups are replaced by H, O, S, N(R') or an alkyl group, wherein R' is H, an amino protecting group, or an unsubstituted or substituted alkyl group. In some embodiments, the 5' and or 3' terminal groups may include 1 to 3 phosphate moieties, each of which is independently unmodified (diphosphate or triphosphate) or modified.

[0254] As used herein, the term "5' phosphorus stabilizing moiety" refers to a terminal phosphate group including phosphates and modified phosphates (e.g., phosphorothioates, phosphodiesters, phosphonates). The phosphate moiety may be located at either end, but is preferably located at the 5'-terminal nucleoside. In one aspect, the terminal phosphate is unmodified and has the formula -O-P(=O)(OH)OH. On the other hand, the terminal phosphate is modified such that one or more of the O and OH groups are replaced by H, O, S, N(R') or an alkyl group, wherein R' is H, an amino protecting group, or an unsubstituted or substituted alkyl group. In some embodiments, the 5' and or 3' terminal groups may include 1 to 3 phosphate moieties, each of which is independently unmodified (diphosphate or triphosphate) or modified.

[0255] The phosphate group of the nucleotide can also be modified, for example, by replacing one or more oxygens of the phosphate group with sulfur (e.g., phosphorothioate), or by making other substitutions that allow the nucleotide to perform its intended function, such as in, for example, Eckstein, Antisense Nucleic Acid Drug Dev. 10: 117-21, 2000; Rusckowski et al., Antisense Nucleic Acid Drug Dev. 10: 333-45, 2000; Stein, Antisense Nucleic Acid Drug Dev. 11: 317-25, 2001; Vorobjev et al., Antisense Nucleic Acid Drug Dev. 11: 77-85, 2001; and US 5,684,143. Some of the above modifications (e.g., phosphate group modifications) preferably reduce the hydrolysis rate of the polynucleotides, for example, including the analogs, in vivo or in vitro.

[0256] As used herein, the term "complementary" refers to two nucleotides that form a standard Watson-Crick base pair. For the avoidance of doubt, the Watson-Crick base pair in the context of the present disclosure includes adenine-thymine, adenine-uracil and cytosine-guanine base pairs. In this article, the correct Watson-Crick base pair is referred to as "matching," and each unpaired nucleotide and each wrongly paired nucleotide are referred to as "mismatching." Comparisons for determining nucleic acid sequence identity percentages can be achieved in various ways within the capabilities of those skilled in the art, for example, using publicly available computer software, such as BLAST, BLAST-2 or Megalign software.

[0257] "Percent (%) sequence complementarity" relative to a reference polynucleotide sequence is defined as the percentage of nucleic acids in a candidate sequence that are complementary to nucleic acids in a reference polynucleotide sequence after alignment of the sequences and introduction of gaps, if necessary, to achieve maximum percentage sequence complementarity. A given nucleotide is considered to be "complementary" to a reference nucleotide as described herein if two nucleotides form a canonical Watson-Crick base pair. For the avoidance of doubt, Watson-Crick base pairs in the context of the present disclosure include adenine-thymine, adenine-uracil, and cytosine-guanine base pairs. In this article, a correct Watson-Crick base pair is referred to as a "match", while each unpaired nucleotide and each mispaired nucleotide are referred to as a "mismatch". Comparisons for determining nucleic acid sequence identity percentages can be implemented in various ways within the capabilities of those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required to achieve maximum complementarity over the full length of the compared sequence. For example, the sequence complementarity percentage between a given nucleic acid sequence A and a given nucleic acid sequence B (which can also be expressed as a given nucleic acid sequence A and a given nucleic acid sequence B having a certain complementarity percentage) is calculated as follows:

[0258] 100×(fraction X / Y)

[0259] Where X is the number of complementary base pairs in the alignment (e.g., performed by computer software such as BLAST) in a program alignment of A and B, and where Y is the total number of nucleic acids in B. It will be understood that when the length of nucleic acid sequence A is not equal to the length of nucleic acid sequence B, the percent sequence complementarity of A to B will not be equal to the percent sequence complementarity of B to A. As used herein, a query nucleic acid sequence is considered to be "fully complementary" to a reference nucleic acid sequence if they have 100% sequence complementarity.

[0260] " Percentage (%) of sequence identity " with respect to reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acid or amino acid consistent with the nucleic acid or amino acid in the reference polynucleotide or polypeptide sequence in the candidate sequence after the alignment sequence and the introduction of a space (if necessary) to achieve the maximum sequence identity percentage. The comparison for determining the nucleic acid or amino acid sequence identity percentage can be realized in various ways within the capabilities of those skilled in the art, for example, using publicly available computer software, such as BLAST, BLAST-2 or Megalign software. Those skilled in the art can determine the parameters applicable to the alignment sequence, including any algorithm required for the maximum comparison in the full length of the compared sequence. For example, the sequence comparison computer program BLAST can be used to generate a sequence identity percentage value. For example, the sequence identity percentage of a given nucleic acid or amino acid sequence A to, with or for a given nucleic acid or amino acid sequence B (it can also be alternatively described as a given nucleic acid or amino acid sequence A to, with or for a certain sequence identity percentage of a given nucleic acid or amino acid sequence B) is calculated as follows:

[0261] 100×(fraction X / Y)

[0262] wherein X is the number of nucleotides or amino acids scored as identical matches by a sequence alignment program (e.g., BLAST) in the program's alignment of A and B, and wherein Y is the total number of nucleic acids in B. It will be understood that where the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, the percent sequence identity of A to B will not be equal to the percent sequence identity of B to A.

[0263] As used herein, the term "complementarity sufficient for hybridization" refers to a nucleic acid sequence or portion thereof that is not necessarily completely complementary (e.g., 100% complementary) to a target region, or to a nucleic acid sequence or portion thereof that has one or more nucleotide mismatches relative to a target region but is still capable of hybridizing to a target region under specified conditions. For example, a nucleic acid can be, for example, 95% complementary, 90% complementary, 85% complementary, 80% complementary, 75% complementary, 70% complementary, 65% complementary, 60% complementary, 55% complementary, 50% complementary or less, but still forms enough base pairs with the target to hybridize over its length.

[0264] When one or more nucleoside residues in a polynucleotide are paired with one or more complementary nucleoside bases to form a stable duplex, "hybridization" or "annealing" of nucleic acid is achieved. Base pairing is usually driven by hydrogen bonding events. Hybridization includes Watson-Crick base pairs formed by natural and / or modified nucleobases. Hybridization can also include non-Watson-Crick base pairs, such as unstable base pairs (guanosine-uracil, hypoxanthine-uracil, hypoxanthine-adenine and hypoxanthine-cytosine), and Hoogsteen base pairs. Nucleic acids do not need 100% complementarity to hybridize. For example, a nucleic acid can be, for example, 95% complementary, 90% complementary, 85% complementary, 80% complementary, 75% complementary, 70% complementary, 65% complementary, 60% complementary, 55% complementary, 50% complementary or less relative to another nucleic acid, but two nucleic acids can still form enough base pairs with each other for hybridization.

[0265] A "stable duplex" formed after annealing / hybridization of a nucleic acid to another nucleic acid is a duplex structure that is not denatured by stringent washing. Exemplary stringent washing conditions are known in the art and include a temperature about 5°C lower than the melting temperature of a single strand of the duplex and a low monovalent salt concentration, such as a monovalent salt concentration (e.g., NaCl concentration) of less than 0.2 M (e.g., 0.2 M, 0.19 M, 0.18 M, 0.17 M, 0.16 M, 0.15 M, 0.14 M, 0.13 M, 0.12 M, 0.11 M, 0.1 M, 0.09 M, 0.08 M, 0.07 M, 0.06 M, 0.05 M, 0.04 M, 0.03 M, 0.02 M, 0.01 M or less).

[0266] The term "gene silencing" refers to the suppression of gene expression, such as the expression of the C9orf72 endogenous gene, which can be mediated by processes that affect transcription and / or by processes that affect post-transcriptional mechanisms. In some embodiments, gene silencing occurs when RNAi molecules initiate the inhibition or degradation of mRNA transcribed from a target gene in a sequence-specific manner through RNA interference, thereby preventing the translation of the gene product.

[0267] As used herein, the phrase "overactive disease driver gene" refers to a gene that has increased activity and / or expression that contributes to or causes a disease state in a subject (e.g., a human). A disease state can be caused or exacerbated by an overactive disease driver gene directly or through an intermediary gene.

[0268] As used herein, the term "ethylene glycol chain" refers to a glycol chain having the formula ((CH 2 OH) 2 )'s carbon chain.

[0269] As used herein, "alkyl" refers to a saturated hydrocarbon group. The alkyl group can be acyclic or cyclic and contains only C and H when unsubstituted. When naming an alkyl residue with a specific carbon number, it is intended to cover and describe all geometric isomers with that carbon number; therefore, for example, "butyl" is intended to include n-butyl, sec-butyl and isobutyl. Examples of alkyls include ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, etc. In some embodiments, the alkyl group can be substituted. Suitable substituents that can be introduced into the alkyl group include, for example, hydroxyl, alkoxy, amino, alkylamino and halo, etc.

[0270] As used herein, "alkenyl" refers to an acyclic or cyclic unsaturated hydrocarbon radical having at least one site of olefinic unsaturation (i.e., having at least one moiety of the formula C=C). When unsubstituted, an alkenyl group contains only C and H. When an alkenyl residue having a particular number of carbons is named, it is intended to encompass and describe all geometric isomers having that number of carbons; thus, for example, "butenyl" is intended to include n-butenyl, sec-butenyl, and isobutenyl. Examples of alkenyl groups include -CH=CH 2 , –CH 2 -CH=CH 2 and –CH 2 -CH=CH-CH=CH 2 In some embodiments, the alkenyl group may be substituted. Suitable substituents that may be introduced into the alkenyl group include, for example, hydroxy, alkoxy, amino, alkylamino, and halo.

[0271] As used herein, "alkynyl" refers to an acyclic or cyclic unsaturated hydrocarbon radical having at least one site of acetylenic unsaturation (i.e., having at least one moiety of the formula C≡C). When unsubstituted, an alkynyl group contains only C and H. When naming an alkynyl residue with a particular number of carbons, it is intended to encompass and describe all geometric isomers with that number of carbons; thus, for example, "pentynyl" is intended to include n-pentynyl, sec-pentynyl, isopentenyl, and tert-pentynyl. Examples of alkynyl groups include -C≡CH and -C≡C-CH 3 In some embodiments, the alkynyl group may be substituted. Suitable substituents that may be introduced into the alkynyl group include, for example, hydroxy, alkoxy, amino, alkylamino, and halo.

[0272] As used herein, the term "phenyl" refers to a monocyclic aromatic hydrocarbon in which one hydrogen atom on a carbon atom of the ring has been removed. The phenyl group may be unsubstituted or substituted with one or more suitable substituents, wherein the substituent replaces the H of the phenyl group.

[0273] As used herein, the term "benzyl" refers to a monovalent group obtained when a hydrogen atom attached to a methyl group of toluene is removed. The benzyl group typically has a phenyl -CH 2-. The benzyl group may be unsubstituted or substituted with one or more suitable substituents. For example, the substituents may replace the H and / or methylene (-CH 2 -) component H.

[0274] As used herein, the term "amide" refers to an alkyl, alkenyl, alkynyl, or aromatic group attached to an amino-carbonyl functional group.

[0275] As used herein, the term "triazole" refers to a triazole having the formula (C 2 H 3 N 3 ) is a heterocyclic compound having a five-membered ring of two carbons and three nitrogens, the positions of which can be changed to produce a variety of isomers.

[0276] As used herein, the term "terminal group" refers to a group at the end of a carbon chain or nucleic acid.

[0277] As used herein, "amino acid" refers to molecules containing amine and carboxyl functional groups and side chains characteristic of amino acids.

[0278] In some embodiments, the amino acid is selected from proteinogenic amino acids. In some embodiments, the amino acid is an L-amino acid or a D-amino acid. In some embodiments, the amino acid is a synthetic amino acid (e.g., a β-amino acid).

[0279] As used herein, the term "lipophilic amino acid" refers to an amino acid that includes a hydrophobic portion (eg, an alkyl chain or an aromatic ring).

[0280] As used herein, the term "delivery target" refers to an organ or part of the body to which delivery of the branched oligonucleotide composition is desired.

[0281] As used herein, the term "between X and Y" includes both X and Y values. For example, "between X and Y" refers to a range of values ​​between an X value and a Y value, as well as an X value and a Y value.

[0282] As used herein, the terms "subject" and "patient" are used interchangeably and refer to an organism, such as a mammal (e.g., a human) that experiences a neurodegenerative disease or disorder (e.g., amyotrophic lateral sclerosis with frontotemporal dementia) and / or contains a gain-of-function C9orf72 variant allele or a repeat expansion of the C9orf72 gene.

[0283] As used herein, the terms "neuroinflammatory disease" and "neuroinflammatory disorder" are used interchangeably to refer to any condition that is caused in some way by neuroinflammation. "Neuroinflammation" refers to a series of immune responses in the central nervous system (e.g., microglia). Neuroinflammation may be of brain origin or caused by a systemic inflammatory response.

[0284] As used herein, the terms "neurodegenerative disease" and "neurodegenerative disorder" are used interchangeably to refer to any condition that is caused in some way by loss of function or death of cells of the central nervous system or peripheral nervous system. Exemplary neurodegenerative diseases are Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, frontotemporal dementia, and spinocerebellar ataxia.

[0285] As used herein, the term "C9orf72" refers to a gene encoding a chromosome, including any native C9orf72 gene from any source. The term encompasses "full-length" unprocessed C9orf72 as well as any form of C9orf72 produced by intracellular processing. The term also encompasses naturally occurring variants of C9orf72, such as splice variants or allelic variants. The nucleic acid sequence of an exemplary C9orf72 gene is shown in the European Nucleotide Archive (ENA) Accession No. JN681271.1. The amino acid sequence of an exemplary protein encoded by the C9orf72 gene is shown in UNIPROT TM Accession number Q96LT7.

[0286] As used herein, the terms "treat," "treated," and "treating" mean therapeutic treatment and prophylactic or preventative measures, wherein the purpose is to prevent, ameliorate, or slow down (mitigate) an undesirable physiological condition, disorder, or disease, or to obtain a beneficial or desired clinical result. Beneficial or desired clinical results include, but are not limited to, reduced patient dependence on drug therapy; alleviation of symptoms; reduction in the extent of a condition, disorder, or disease; stabilization (i.e., not worsening) of the state of a condition, disorder, or disease; delay in the onset or slowing of the progression of a condition, disorder, or disease; improvement or remission (whether partial or complete), whether detectable or undetectable, of a condition, disorder, or disease state; improvement in at least one measurable physical, cognitive, or behavioral (such as depressive behavior or apathy) parameter, which improvement is not necessarily perceptible to the patient; or enhancement or amelioration of a condition, disorder, or disease. Treatment includes inducing a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival compared to expected survival if not receiving treatment.

[0287] As used herein, the terms "benefit" and "response" are used interchangeably in the context of a subject receiving a therapy for treating, for example, amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD). For example, clinical benefits in the context of a subject with ALS administered with an siRNA molecule or siRNA composition of the present disclosure include, but are not limited to, reduction in involuntary movements, muscle spasms, weakness, loss of motor control. As a further example, clinical benefits in the context of a subject with FTD administered with an siRNA molecule or siRNA composition of the present disclosure include, but are not limited to, reduction in memory problems, behavioral problems, and language problems. "Benefit" and "response" are also used interchangeably to refer to, for example, a reduction in wild-type C9orf72 transcripts, mutant C9orf72 transcripts, variant C9orf72 transcripts, splicing isoforms of C9orf72 transcripts, and / or overexpressed C9orf72 transcripts. DETAILED DESCRIPTION

[0288] The present disclosure provides compositions of small interfering RNA (siRNA) molecules having sequence homology to the chromosome 9 open reading frame 72 (C9orf72) gene, and methods for administering the siRNA molecules to a subject. In addition, the siRNA molecules described herein can be composed of branched siRNA structures, such as two-branched, three-branched, and four-branched siRNA structures, and can also include specific patterns of chemical modifications (e.g., 2' ribose modifications or internucleoside linkage modifications) to improve resistance to nucleases, toxicity profiles, and physicochemical properties (e.g., thermal stability). Small interfering RNA molecules are short double-stranded RNA molecules. They can mediate RNA interference (RNAi) by degrading mRNAs having complementary nucleotide sequences, thereby preventing the translation of target genes.

[0289] The siRNA molecules of the present disclosure can exhibit robust gene-specific repression of, for example, C9orf72 relative to other human genes.

[0290] The siRNA molecules of the present disclosure may be characterized by an antisense strand having a nucleic acid sequence complementary to a region of a C9orf72 mRNA transcript having a nucleic acid sequence of any one of SEQ ID NOs: 1 to 384. The degree of complementarity of the antisense strand to the region of the C9orf72 mRNA transcript may be sufficient to allow the antisense strand to anneal over the entire length of the region of the C9orf72 mRNA transcript. For example, the antisense strand can have a nucleic acid sequence that is at least 60% complementary (e.g., 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary) to a region of a C9orf72 mRNA transcript. In some embodiments, the region of the C9orf72 RNA transcript has the sequence of any one of SEQ ID NOs: 1-192. In some embodiments, the region of the C9orf72 RNA transcript has the sequence of any one of SEQ ID NOs: 193-384.

[0291] In some embodiments, the siRNA molecules of the present disclosure are characterized by having an antisense strand of a nucleic acid sequence of any one of SEQ ID NOs: 769-1152, or a nucleic acid sequence at least 60% identical thereto. For example, the siRNA molecules of the present disclosure can be characterized by having an antisense strand having a nucleic acid sequence that is at least 60% identical (e.g., 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the nucleic acid sequence of any one of SEQ ID NOs: 769-1152. In some embodiments, the nucleic acid sequence is any one of SEQ ID NOs: 769-960. In some embodiments, the nucleic acid sequence is any one of SEQ ID NOs:961-1152.

[0292] In some embodiments, the siRNA molecules of the present disclosure feature a sense strand having a nucleic acid sequence of any one of SEQ ID NOs: 385-768, or a nucleic acid sequence at least 60% identical thereto. For example, the siRNA molecules of the present disclosure can be characterized by having a sense strand having a nucleic acid sequence that is at least 60% identical (e.g., 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the nucleic acid sequence of any one of SEQ ID NOs: 385-768. In some embodiments, the nucleic acid sequence is any one of SEQ ID NOs: 385-576. In some embodiments, the nucleic acid sequence is any one of SEQ ID NOs: 577-768.

[0293] Exemplary siRNA molecules of the present disclosure are those shown below in Table 1. Table 1 summarizes the antisense strand, the sense strand, and the corresponding regions of the C9orf72 mRNA transcript targeted by each antisense strand.

[0294] Table 1. Nucleotide sequences of gene-specific siRNA targeting C9orf72

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309]

[0310]

[0311]

[0312]

[0313]

[0314]

[0315]

[0316]

[0317]

[0318]

[0319]

[0320]

[0321]

[0322] siRNA structure

[0323] The siRNA molecules of the present disclosure can be in the form of single-stranded (ss) or double-stranded (ds) oligonucleotide structures. In some embodiments, the siRNA molecules can be bi-branched, tri-branched or quad-branched molecules. In addition, the siRNA molecules of the present disclosure can contain one or more phosphodiester internucleoside linkages and / or their analogs, such as thiophosphate internucleoside linkages. The siRNA molecules of the present disclosure can also contain chemically modified nucleosides with 2' sugar modifications.

[0324] The simplest siRNA is composed of a ribonucleic acid comprising an ss- or ds-structure, which is formed by a first strand (i.e., an antisense strand), and in the case of a ds-siRNA, there is also a second strand (i.e., a sense strand). The first strand includes a continuous nucleotide segment that is at least partially complementary to the target nucleic acid. The second strand also includes a continuous nucleotide segment, wherein the second segment is at least partially identical to the target nucleic acid. The first strand and the second strand can hybridize to each other to form a double-stranded structure. Hybridization usually occurs through Watson-Crick base pairing.

[0325] Depending on the sequence of the first and second strands, hybridization or base pairing may not be complete or perfect, meaning that the first and second strands are not 100% base paired due to mismatches. One or more mismatches may also exist within the duplex, but may not necessarily affect siRNA RNAi activity.

[0326] The first strand contains a continuous nucleotide segment that is substantially complementary to the target nucleic acid. Typically, according to the mode of action of the interfering RNA, the target nucleic acid sequence is ss-RNA, preferably mRNA. Such hybridization is most likely to occur through Watson-Crick base pairing, but is not necessarily limited to this. The degree to which the first strand has a continuous nucleotide segment that is complementary to the target nucleic acid sequence can be between 80% and 100%, for example 80%, 85%, 90%, 95% or 100% complementary.

[0327] The siRNA molecules described herein may employ modifications to the nucleobases, phosphate backbone, ribose core, 5'- and 3'-ends, and branching, wherein multiple strands of the siRNA may be covalently linked.

[0328] Length of small interfering RNA molecules

[0329] Any length known in the art and previously unknown within the scope of the present disclosure may be used in the present invention. As described herein, the antisense strand of the siRNA molecules of the present disclosure may be 10 to 30 nucleotides (e.g., 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides), 15 to 25 nucleotides (e.g., 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, or 25 nucleotides), or 18 to 23 nucleotides (e.g., 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, or 23 nucleotides). In some embodiments, the antisense strand is 20 nucleotides. In some embodiments, the antisense strand is 21 nucleotides. In some embodiments, the antisense strand is 22 nucleotides. In some embodiments, the antisense strand is 23 nucleotides. In some embodiments, the antisense strand is 24 nucleotides. In some embodiments, the antisense strand is 25 nucleotides. In some embodiments, the antisense strand is 26 nucleotides. In some embodiments, the antisense strand is 27 nucleotides. In some embodiments, the antisense strand is 28 nucleotides. In some embodiments, the antisense strand is 29 nucleotides. In some embodiments, the antisense strand is 30 nucleotides.

[0330] In some embodiments, the sense strand of the siRNA molecules of the present disclosure is 12 to 30 nucleotides (e.g., 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides) or 14 to 23 nucleotides (e.g., 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, or 23 nucleotides). In some embodiments, the sense strand is 15 nucleotides. In some embodiments, the sense strand is 16 nucleotides. In some embodiments, the sense strand is 17 nucleotides. In some embodiments, the sense strand is 18 nucleotides. In some embodiments, the sense strand is 19 nucleotides. In some embodiments, the sense strand is 20 nucleotides. In some embodiments, the sense strand is 21 nucleotides. In some embodiments, the sense strand is 22 nucleotides. In some embodiments, the sense strand is 23 nucleotides. In some embodiments, the sense strand is 24 nucleotides. In some embodiments, the sense strand is 25 nucleotides. In some embodiments, the sense strand is 26 nucleotides. In some embodiments, the sense strand is 27 nucleotides. In some embodiments, the sense strand is 28 nucleotides. In some embodiments, the sense strand is 29 nucleotides. In some embodiments, the sense strand is 30 nucleotides.

[0331] 2′ sugar modification

[0332] The present disclosure may include ss-siRNA and ds-siRNA molecular compositions comprising at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or more) nucleoside with a 2' sugar modification. Possible 2'-modifications include OH in all possible orientations; F; O-alkyl, S-alkyl, or N-alkyl; O-alkenyl, S-alkenyl, or N-alkenyl; O-alkynyl, S-alkynyl, or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl, and alkynyl groups may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. In some embodiments, the modification includes a 2'-O-methyl (2'-O-Me) modification. Other possible sugar substituent groups include: C1 to C10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl, or O-aralkyl, SH, SCH 3 、OCN、Cl、Br、CN、CF 3 、OCF 3 、SOCH3 、SO 2 CH 3 、ONO 2 、NO 2 、N 3 NH 2 , heterocycloalkyl, heterocycloalkylaryl, aminoalkylamino, polyalkylamino, substituted silyl, groups for improving the pharmacokinetic properties of oligonucleotides or groups for improving the pharmacodynamic properties of oligonucleotides, and other substituents with similar properties. In some embodiments, the modification includes 2'methoxyethoxy (2'-O-CH 2 CH 2 OCH 3 , also known as 2'-O-(2-methoxyethyl) or 2'-MOE). In some embodiments, the modification includes 2'-dimethylaminooxyethoxy, ie, O(CH 2 ) 2 ON(CH 3 ) 2 group, also known as 2′-DMAOE; and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylamino-ethoxy-ethyl or 2′-DMAEOE), i.e., 2′-O-CH 2 OCH 2 N(CH 3 ) 2 Other possible sugar substituent groups include, for example, aminopropoxy (-OCH 2 CH 2 CH 2 NH 2 ), allyl (-CH 2 -CH=CH 2 ), -O-allyl (-O-CH 2 -CH=CH 2 ) and fluorine (F). The 2'-sugar substituent can be located at the arabinose (upper) position or the ribose (lower) position. In some embodiments, the 2'-arabinose modification is 2'-F. Similar modifications can also be made at other positions on the siRNA molecule, specifically at the 3' position of the sugar on the 3' terminal nucleoside or in the 2'-5' linked oligonucleotide and the 5' position of the 5' terminal nucleotide. Oligonucleotides can also have sugar mimetics such as cyclobutyl moieties instead of pentofuranosyl sugars.

[0333] Nucleobase modification

[0334] The siRNA molecules of the present disclosure may also include nucleosides or other substituted or mimetic monomeric subunits, which include nucleobases (often referred to in the art as "bases" or "heterocyclic base moieties"). Nucleobases are another moiety that has been extensively modified or substituted and such modified and or substituted nucleobases are suitable for use in the present disclosure. 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 (also referred to herein as heterocyclic base moieties) include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl derivatives and other alkyl derivatives of adenine and guanine, 2-propyl derivatives and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C=C-CH3) uracil and cytosine, and other alkynyl derivatives of pyrimidine bases. Nucleobases include 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-sulfhydryl, 8-sulfanyl, 8-hydroxyl and other 8-substituted adenine and guanine, 5-halo (particularly 5-bromo), 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Nucleobases may also include those in which purine or pyrimidine bases are replaced by other heterocycles such as 7-deaza-adenine, 7-deazaguanine, 2-aminopyridine and 2-pyridone. Additional nucleobases include those disclosed in US 3,687,808, those disclosed in Kroschwitz, JI, ed. The Concise Encyclopedia of Polymer Science and Engineering, New York, John Wiley & Sons, 1990, pp. 858-859; those disclosed by Englisch et al., Angewandte Chemie, International Edition, 30:613, 1991; and those disclosed by Sanghvi, YS, Chapter 16, Antisense Research and Applications, CRC Press, Gait, MJ, ed., 1993, pp. 289-302. The siRNA molecules disclosed herein may also include polycyclic heterocyclic compounds to replace one or more heterocyclic base moieties. Many tricyclic heterocyclic compounds have been previously reported.These compounds are often used in antisense applications to increase the binding properties of the modified strand to the target strand.

[0335] Representative cytosine analogs that generate three hydrogen bonds with guanosine in the second strand include 1,3-diazaphenoxazine-2-one (Kurchavov et al., Nucleosides and Nucleotides, 16: 1837-46, 1997), 1,3-diazaphenoxazine-2-one (Lin et al. Am. Chem. Soc., 117: 3873-4, 1995) and 6,7,8,9-tetrafluoro-1,3-diazaphenoxazine-2-one (Wang et al., Tetrahedron Lett., 39: 8385-8, 1998). These base modifications incorporated into oligonucleotides have been shown to hybridize with complementary guanines, and the latter have been shown to hybridize with adenines and enhance helical thermal stability through extended stacking interactions (see also US 10 / 155,920 and 10 / 013,295, both of which are incorporated herein by reference in their entirety). Further helix-stabilizing properties have been observed when cytosine analogs / substitutes have an aminoethoxy moiety attached to a rigid 1,3-diazaphenoxazin-2-one scaffold (Lin et al., Am. Chem. Soc., 120:8531-2, 1998).

[0336] Internucleoside linkage modification

[0337] Another variable in the disclosed design is the internucleoside linkage of the phosphate backbone that constitutes the siRNA molecule. Although the natural RNA phosphate backbone can be used here, its derivatives that enhance the desired characteristics of the siRNA molecule can be used. Although not restrictive, it is particularly important in the disclosure to protect the part or the entirety of the siRNA molecule from hydrolysis. An example of the modification that reduces the hydrolysis rate is thiophosphate. Any part or the entirety of the backbone can contain a phosphate substituent (e.g., thiophosphate, etc.). For example, the internucleoside linkages can be 0 to 100% phosphorothioate, e.g., 0 to 100%, 10 to 100%, 20 to 100%, 30 to 100%, 40 to 100%, 50 to 100%, 60 to 100%, 70 to 100%, 80 to 100%, 90 to 100%, 0 to 90%, 0 to 80%, 0 to 70%, 0 to 60%, 0 to 50%, 0 to 40%, 0 to 30%, 0 to 20%, 0 to 10%, 10 to 90%, 20 to 80%, 30 to 70%, 40 to 60%, 10 to 40%, 20 to 50%, 30 to 60%, 40 to 70%, 50 to 80%, or 60 to 90% phosphorothioate linkages. Similarly, the internucleoside linkages can be 0 to 100% phosphodiester linkages, e.g., 0 to 100%, 10 to 100%, 20 to 100%, 30 to 100%, 40 to 100%, 50 to 100%, 60 to 100%, 70 to 100%, 80 to 100%, 90 to 100%, 0 to 90%, 0 to 80%, 0 to 70%, 0 to 60%, 0 to 50%, 0 to 40%, 0 to 30%, 0 to 20%, 0 to 10%, 10 to 90%, 20 to 80%, 30 to 70%, 40 to 60%, 10 to 40%, 20 to 50%, 30 to 60%, 40 to 70%, 50 to 80%, or 60 to 90% phosphodiester linkages.

[0338] The specific examples of some possible siRNA molecules that can be used for the present invention include oligonucleotides containing modified (e.g., non-naturally occurring) internucleoside linkages. As defined in this specification, oligonucleotides with modified internucleoside linkages include internucleoside linkages retaining phosphorus atoms and internucleoside linkages without phosphorus atoms. For the purposes of this specification, and as sometimes mentioned in this area, modified oligonucleotides without phosphorus atoms in their internucleoside backbones can also be considered as oligonucleosides. Preferably, the modified internucleoside linkages containing phosphorus are phosphorothioate internucleoside linkages. In some embodiments, modified oligonucleotide backbones containing phosphorus atoms include, for example, phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates (including 3'-alkylene phosphonates, 5'-alkylene phosphonates), phosphinates, phosphoramidates (including 3'-amino phosphoramidate and aminoalkyl phosphoramidates), thiophosphoramidates, thioalkylphosphonates, thioalkylphosphotriesters, selenophosphates, and borophosphates with normal 3'-5' linkages, 2'-5' linked analogs of these, and those with reversed polarity, wherein one or more internucleotide linkages are 3' to 3', 5' to 5', or 2' to 2' linkages. Exemplary U.S. patents describing the preparation of 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; 5,286,717; 5,3 21,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587 ,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534, 639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat. No. RE39464, the entire contents of each of which are hereby incorporated herein by reference.

[0339] In some embodiments, the modified oligonucleotide backbone that does not include a phosphorus atom has a backbone formed by short-chain alkyl or cycloalkyl nucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl nucleoside linkages, or one or more short-chain heteroatoms or heterocyclic nucleoside linkages. These include those with: morpholino linkages (partially formed by the sugar portion of the nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formyl acetyl and thioformyl acetyl backbones; methylene formyl acetyl and thioformyl acetyl backbones; riboacetyl backbones; olefin-containing backbones; aminosulfonate backbones; methylene imino and methylene hydrazine backbones; sulfonate and sulfonamide backbones; amide backbones; and those with mixed N, O, S and CH 2 Non-limiting examples of U.S. patents that teach the preparation of non-phosphorus frameworks include, but are not limited to, U.S. Patent Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5, 5,677,437; and 5,677,439, the entire contents of each of which are hereby incorporated by reference herein.

[0340] Modification patterns of siRNA molecules

[0341] The following section provides a set of exemplary scaffolds into which the siRNA molecules of the present disclosure can be incorporated.

[0342] In some embodiments of the present disclosure, the siRNA may contain an antisense strand comprising a region represented by Formula I, wherein Formula I is

[0343] AB-(A') j -CP 2 -DP 1 -(C'-P 1 ) k -C'

[0344] Formula I;

[0345] Where A is composed of the formula CP 1 -DP 1 Indicates; each A' is represented by the formula CP 2 -DP2 Indicated by; B is represented by the formula CP 2 -DP 2 -DP 2 -DP 2 represents; each C is a 2'-O-methyl (2'-O-Me) ribonucleoside; each C' is independently a 2'-O-Me ribonucleoside or a 2'-fluoro (2'-F) ribonucleoside; each D is a 2'-F ribonucleoside; each P 1 is a phosphorothioate internucleoside linkage; each P 2 is a phosphodiester internucleoside linkage; j is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7); and k is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7). In some embodiments, j is 4. In some embodiments, k is 4. In some embodiments, j is 4 and k is 4. The antisense is complementary (e.g., fully or partially complementary) to the target nucleic acid sequence.

[0346] In some embodiments, the antisense strand comprises a structure represented by Formula A1, wherein Formula A1 is:

[0347] ASBSAOBOBOBOAOBOAOBOA-OBAOBOAOBSASASASBSA

[0348] Formula A1;

[0349] wherein A represents a 2'-0-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0350] In some embodiments of the present disclosure, the siRNA may contain an antisense strand comprising a region represented by Formula II, wherein Formula II is, in the 5' to 3' direction:

[0351] AB-(A') j -CP 2 -DP 1 -(CP 1 ) k -C'

[0352] Formula II;

[0353] Where A is composed of the formula CP 1 -DP 1 Indicates; each A' is represented by the formula CP 2 -DP 2 Indicated by; B is represented by the formula CP 2 -DP 2 -DP2 -DP 2 represents; each C is a 2'-O-methyl (2'-O-Me) ribonucleoside; each C' is independently a 2'-O-Me ribonucleoside or a 2'-fluoro (2'-F) ribonucleoside; each D is a 2'-F ribonucleoside; each P 1 is a phosphorothioate internucleoside linkage; each P 2 is a phosphodiester internucleoside linkage; j is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7); and k is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7). In some embodiments, j is 4. In some embodiments, k is 4. In some embodiments, j is 4 and k is 4. The antisense is complementary (e.g., fully or partially complementary) to the target nucleic acid sequence.

[0354] In some embodiments of the present disclosure, the antisense strand comprises a structure represented by Formula A2, wherein Formula A2 is:

[0355] ASBSAOBOBOBOAOBOAOBOA-OBOOBOAOBSASASASASA

[0356] Formula A2;

[0357] wherein A represents a 2'-0-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0358] In some embodiments of the present disclosure, the sense strand comprises a structure represented by Formula III, wherein Formula III is:

[0359] E-(A') m -F

[0360] Formula III;

[0361] Where E is given by the formula (CP 1 ) 2 Represented by; F is represented by the formula (CP 2 ) 3 -DP 1 -CP 1 -C, (CP 2 ) 3 -DP 2 -CP 2 -C, (CP 2 ) 3 -DP 1 -CP 1 -D or (CP2 ) 3 -DP 2 -CP 2 -D means; A', C, D, P 1 and P 2 As defined in Formula I; and m is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7). In some embodiments, m is 4. In some embodiments, the sense strand is complementary to the antisense strand (e.g., fully or partially complementary).

[0362] In some embodiments of the present disclosure, the sense strand comprises a structure represented by Formula S1, wherein Formula S1 is:

[0363] ASASAOBOAOBOAOBOAOBOA-OAOAOBSASA

[0364] Formula S1;

[0365] wherein A represents a 2'-0-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0366] In some embodiments of the present disclosure, the sense strand comprises a structure represented by Formula S2, wherein Formula S2 is, in the 5' to 3' direction:

[0367] ASASAOBOAOBOAOBOAOBOA-OAOAOBOAOA

[0368] Formula S2;

[0369] wherein A represents a 2'-0-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0370] In some embodiments of the present disclosure, the sense strand comprises a structure represented by Formula S3, wherein Formula S3 is:

[0371] ASASAOBOAOBOAOBOAOBOA-OAOAOBSASB

[0372] Formula S3;

[0373] wherein A represents a 2'-0-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0374] In some embodiments of the present disclosure, the sense strand comprises a structure represented by Formula S4, wherein Formula S4 is, in the 5' to 3' direction:

[0375] ASASAOBOAOBOAOBOAOBOA-OAOAOBOAOB

[0376] Formula S4;

[0377] wherein A represents a 2'-0-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0378] In some embodiments of the present disclosure, the siRNA may contain an antisense strand comprising a region represented by Formula IV, wherein Formula IV is, in the 5' to 3' direction:

[0379] A-(A') j -CP 2 -B-(CP 1 ) k -C'

[0380] Formula IV;

[0381] Where A is composed of the formula CP 1 -DP 1 Indicates; each A' is represented by the formula CP 2 -DP 2 Indicated by; B is represented by the formula DP 1 -CP 1 -DP 1 represents; each C is 2'-O-Me ribonucleoside; each C' is independently 2'-O-Me ribonucleoside or 2'-F ribonucleoside; each D is 2'-F ribonucleoside; each P 1 is a phosphorothioate internucleoside linkage; each P 2 is a phosphodiester internucleoside linkage; j is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7); and k is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7). In some embodiments, j is 6. In some embodiments, k is 4. In some embodiments, j is 6 and k is 4. The antisense strand is complementary (e.g., fully or partially complementary) to the target nucleic acid.

[0382] In some embodiments of the present disclosure, the antisense strand comprises a structure represented by Formula A3, wherein Formula A3 is:

[0383] ASBSAOBOAOBOAOBOAOBOA-OBAOBOAOBSASBSASASA

[0384] Formula A3;

[0385] wherein A represents a 2'-0-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0386] In some embodiments of the present disclosure, the siRNA of the present disclosure may have a sense strand represented by Formula V, wherein Formula V is, in the 5' to 3' direction:

[0387] E-(A') m -CP 2 -F

[0388] Formula V;

[0389] Where E is given by the formula (CP 1 ) 2 Indicated by: F by DP 1 -CP 1 -C, DP 2 -CP 2 -C, DP 1 -CP 1 -D or DP 2 -CP 2 -D means; A', C, D, P 1 and P 2 As defined in Formula IV; and m is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7). In some embodiments, m is 5. In some embodiments, the sense strand is complementary to the antisense strand (e.g., fully or partially complementary).

[0390] In some embodiments of the present disclosure, the sense strand comprises a structure represented by Formula S5, wherein Formula S5 is, in the 5' to 3' direction:

[0391] ASASAOBOAOBOAOBOAOBOA-OBOAOBSASA

[0392] Formula S5;

[0393] wherein A represents a 2'-0-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0394] In some embodiments of the present disclosure, the sense strand comprises a structure represented by Formula S6, wherein Formula S6 is, in the 5' to 3' direction:

[0395] ASASAOBOAOBOAOBOAOBOA-OBOAOBOAOA

[0396] Formula S6;

[0397] wherein A represents a 2'-0-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0398] In some embodiments of the present disclosure, the sense strand comprises a structure represented by Formula S7, wherein Formula S7 is, in the 5' to 3' direction:

[0399] ASASAOBOAOBOAOBOAOBOA-OBOAOBSASB

[0400] Formula S7;

[0401] wherein A represents a 2'-0-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0402] In some embodiments of the present disclosure, the sense strand comprises a structure represented by Formula S8, wherein Formula S8 is, in the 5' to 3' direction:

[0403] ASASAOBOAOBOAOBOAOBOA-OBOAOBOAOB

[0404] Formula S8;

[0405] wherein A represents a 2'-0-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0406] In some embodiments of the present disclosure, the siRNA may contain an antisense strand comprising a region represented by Formula VI, wherein Formula VI is, in the 5' to 3' direction:

[0407] AB j -EB k -EFG l -DP 1 -C'

[0408] Formula VI;

[0409] Where A is composed of the formula CP 1 -DP 1 Indicates; each B is represented by the formula CP 2 Each C is a 2'-O-Me ribonucleoside; each C' is independently a 2'-O-Me ribonucleoside or a 2'-F ribonucleoside; each D is a 2'-F ribonucleoside; each E is of the formula DP 2 -CP 2 Indicated by: F by DP 1 -CP 1 Indicated; each G is represented by the formula CP 1 Indicates that each P 1 is a phosphorothioate internucleoside linkage; each P 2 is a phosphodiester linkage; j is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7); k is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7); and l is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7). In some embodiments, j is 3. In some embodiments, k is 6. In some embodiments, l is 2. In some embodiments, j is 3, k is 6, and l is 2. The antisense strand is complementary (e.g., fully or partially complementary) to the target nucleic acid.

[0410] In some embodiments of the present disclosure, the antisense strand comprises a structure represented by Formula A4, wherein Formula A4 is:

[0411] ASBSAOAOAOBOAOAOAOAOA-OAOAOBOAOBSASASASASBSA

[0412] Formula A4;

[0413] wherein A represents a 2'-0-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0414] In some embodiments of the present disclosure, the siRNA may contain a sense strand comprising a region represented by Formula VII, wherein Formula VII is, in the 5' to 3' direction:

[0415] HB m -I n -A'-B o -HC

[0416] Formula VII;

[0417] Where A' is composed of the formula CP 2 -DP 2Represented; each H is represented by the formula (CP 1 ) 2 Represented; each I is represented by the formula (DP 2 ) indicates; B, C, D, P 1 and P 2 As defined in Formula VI; m is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7); n is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7); and o is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7). In some embodiments, m is 3. In some embodiments, n is 3. In some embodiments, o is 3. In some embodiments, m is 3, n is 3, and o is 3. The antisense strand is complementary (e.g., fully or partially complementary) to the sense strand.

[0418] In some embodiments of the present disclosure, the sense strand comprises a structure represented by Formula S9, wherein Formula S9 is, in the 5' to 3' direction:

[0419] ASASAOAOAOBOBOBOAOBOA-OAOAOASASA

[0420] Formula S9;

[0421] wherein A represents a 2'-0-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0422] In some embodiments of the present disclosure, the siRNA may contain an antisense strand comprising a region represented by Formula VIII:

[0423] Z-((AP-) n (BP-) m ) q ;

[0424] Formula VIII

[0425] wherein Z is a 5' phosphorus stabilizing moiety; each A is a 2'-O-methyl (2'-O-Me) ribonucleoside; each B is a 2'-fluoro-ribonucleoside; each P is independently an internucleoside linkage selected from a phosphodiester linkage and a phosphorothioate linkage; n is an integer from 1 to 5 (e.g., 1, 2, 3, 4, or 5); m is an integer from 1 to 5 (e.g., 1, 2, 3, 4, or 5); and q is an integer between 1 and 30 (1, 2, 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, or 30).

[0426] siRNA Synthesis Method

[0427] The siRNA molecules of the present disclosure can be synthesized by standard methods known in the art, as discussed further below, for example, by using an automated DNA synthesizer, such as commercially available from, for example, Biosearch, Applied Biosystems, Inc.

[0428] siRNA agents can be prepared using solution phase or solid phase organic synthesis or both. Organic synthesis provides the advantage that oligonucleotides including non-natural or modified nucleotides can be easily prepared. siRNA molecules of the present disclosure can be prepared using solution phase or solid phase organic synthesis or both.

[0429] In addition, it is considered that for any siRNA reagent disclosed herein, further optimization can be achieved by systematically adding or removing the nucleosides connected to produce longer or shorter sequences. In addition, such optimized sequences can be adjusted by, for example, introducing modified nucleosides described herein or known in the art and / or modified internucleoside linkages, including alternative nucleosides, alternative sugar moieties and / or alternative internucleoside linkages as known in the art and / or discussed herein, to further optimize molecules (e.g., increase serum stability or circulation half-life, increase thermal stability, enhance transmembrane delivery and / or target specific locations or cell types).

[0430] 5′ phosphorus stabilizing moiety

[0431] In order to further protect the siRNA molecules of the present disclosure from degradation, a 5'-phosphorus stabilizing portion can be used. The 5'-phosphorus stabilizing portion replaces the 5'-phosphate to prevent phosphate hydrolysis. The hydrolysis of the 5'-phosphate prevents binding to RISC, which is a necessary step for gene silencing. Any phosphate replacement that does not hinder binding to RISC is considered in the present disclosure. In some embodiments, the substitute of the 5'-phosphate is also stable to hydrolysis in vivo. Each chain of the siRNA molecule can independently and optionally adopt any suitable 5'-phosphorus stabilizing portion.

[0432]

[0433] Some exemplary end caps are shown in Formulas IX-XVI. Nuc in Formulas IX-XVI represents a nucleobase or a nucleobase derivative or substitution as described herein. X in Formulas IX-XVI represents a 2'-modification as described herein. Some embodiments employ a hydroxyl as in Formula IX, a phosphate as in Formula X, a vinyl phosphonate as in Formulas XI and XIV, a 5'-methyl substituted phosphate as in Formulas XII, XIII, and XVI, a methylene phosphonate as in Formula XV, or a vinyl 5'-vinyl phosphonate as shown in Formula XI as a 5'-phosphorus stabilizing moiety.

[0434] Hydrophobic part

[0435] The present disclosure also provides siRNA molecules having one or more hydrophobic moieties attached thereto. The hydrophobic moiety can be covalently attached to the 5' end or the 3' end of the siRNA molecules of the present disclosure. Non-limiting examples of hydrophobic moieties suitable for use with the siRNA molecules of the present disclosure can include cholesterol, vitamin D, tocopherol, phosphatidylcholine (PC), docosahexaenoic acid, docosanoic acid, PC-docosanoic acid, eicosapentaenoic acid, lithocholic acid, or any combination of the above hydrophobic moieties and PC.

[0436] siRNA branch

[0437] The siRNA molecules of the present disclosure can be branched. For example, the siRNA molecules of the present disclosure can have one of several branching patterns, as described herein.

[0438] According to the present disclosure, the siRNA molecules disclosed herein can be branched siRNA molecules. The siRNA molecules may not be branched, or may be two-branched, three-branched or four-branched, connected by a joint. Each main branch may further branch to allow 2, 3, 4, 5, 6, 7 or 8 independent RNA single strands or double strands. The branching points on the joint may be derived from the same atom, or along the individual atoms of the joint. Some exemplary embodiments are listed in Table 2.

[0439] Table 2. Branched siRNA structures

[0440]

[0441] In some embodiments, the siRNA molecule is a branched siRNA molecule. In some embodiments, the branched siRNA molecule is bi-branched, tri-branched or quad-branched. In some embodiments, the bi-branched siRNA molecule is represented by any one of Formulas XVII-XIX, wherein each RNA is independently a siRNA molecule, L is a linker, and each X independently represents a branch point portion (e.g., phosphoramidite, tosylated acetone glycerol, 1,3-diaminopropanol, pentaerythritol, or any one of the branch point portions described in US10,478,503).

[0442] In some embodiments, the three-branched siRNA molecule is represented by any of Formulae XX-XXIII, wherein each RNA is independently a siRNA molecule, L is a linker, and each X independently represents a branch point moiety.

[0443] In some embodiments, a four-branched siRNA molecule is represented by any one of Formulae XXIV-XXVIII, wherein each RNA is independently a siRNA molecule, L is a linker, and each X independently represents a branch point moiety.

[0444] Connectors

[0445] The multiple chains of siRNA described herein can be covalently attached by a joint. The effect of this branching especially improves cell permeability, thereby allowing better access to cells (e.g., neurons or microglia) in the CNS. Any connecting part incompatible with the siRNA of the present invention can be adopted. The joint includes ethylene glycol chains, alkyl chains, carbohydrate chains, block copolymers, peptides, RNA, DNA, etc. of 2 to 10 subunits (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10 subunits). In some embodiments, any carbon or oxygen atom of the joint is optionally replaced by a nitrogen atom, with a hydroxyl substituent, or with an oxo substituent. In some embodiments, the joint is a polyethylene glycol (PEG) joint. The PEG joint suitable for use with the disclosed compositions and methods includes a linear or nonlinear PEG joint. The example of a nonlinear PEG joint includes a branched PEG, a linear forked PEG or a branched forked PEG.

[0446] PEG joints of various weights can be used with the disclosed compositions and methods. For example, a PEG joint can have a weight between 5 and 500 daltons. In some embodiments, a PEG joint having a weight between 500 and 1,000 daltons can be used. In some embodiments, a PEG joint having a weight between 1,000 and 10,000 daltons can be used. In some embodiments, a PEG joint having a weight between 200 and 20,000 daltons can be used. In some embodiments, the joint is covalently attached to the sense strand of the siRNA. In some embodiments, the joint is covalently attached to the antisense strand of the siRNA. In some embodiments, the PEG joint is a triethylene glycol (TrEG) joint. In some embodiments, the PEG joint is a tetraethylene glycol (TEG) joint.

[0447] In some embodiments, the linker is an alkyl linker. In some embodiments, the linker is a peptide linker. In some embodiments, the linker is an RNA linker. In some embodiments, the linker is a DNA linker.

[0448] The joint can be covalently connected to 2,3,4 or 5 unique siRNA chains. The joint can be covalently bonded to any part of the siRNA oligomer. In some embodiments, the joint is attached to the 3' end of the nucleoside of each siRNA chain. In some embodiments, the joint is attached to the 5' end of the nucleoside of each siRNA chain. In some embodiments, the joint is attached to the nucleoside of the siRNA chain (such as sense strand or antisense strand) by covalent bond formation. In some embodiments, the covalent bond formation part is selected from the group consisting of alkyl, ester, amide, carbonate, carbamate, triazole, urea, methylal, phosphonate, phosphate and phosphate derivatives (such as, phosphorothioate, phosphoramidate etc.).

[0449] In some embodiments, the linker has the structure of Formula L1:

[0450]

[0451] In some embodiments, the linker has the structure of Formula L2:

[0452]

[0453] In some embodiments, the linker has the structure of Formula L3:

[0454]

[0455] In some embodiments, the linker has the structure of Formula L4:

[0456]

[0457] In some embodiments, the linker has the structure of Formula L5:

[0458]

[0459] In some embodiments, the linker has the structure of Formula L6:

[0460]

[0461] In some embodiments, the linker has the structure of Formula L7, as shown below:

[0462]

[0463] In some embodiments, the linker has the structure of Formula L8:

[0464]

[0465] In some embodiments, the linker has the structure of Formula L9:

[0466]

[0467] In some embodiments, the choice of linker used with one or more branched siRNA molecules disclosed herein can be based on the hydrophobicity of the linker such that, for example, a desired hydrophobicity is achieved for one or more branched siRNA molecules of the present disclosure. For example, compared to branched siRNA molecules with lower hydrophobicity linkers or hydrophilic linkers, linkers containing alkyl chains can be used to increase the hydrophobicity of the branched siRNA molecules.

[0468] The siRNA reagents disclosed herein can be synthesized and / or modified by methods well established in the art, such as those described in Beaucage, S.L. et al. (eds.), Current Protocols in Nucleic Acid Chemistry, John Wiley & Sons, Inc., New York, N.Y., 2000, which is hereby incorporated by reference herein.

[0469] Therapeutic methods

[0470] The siRNA molecules targeting C9orf72 of the present disclosure can be delivered to a subject to treat amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and / or mitigate phenotypes associated with the disorder. For example, the siRNA molecules can be delivered to a subject to treat ALS and / or mitigate ALS disease-related phenotypes (e.g., involuntary movements, muscle spasms, weakness, loss of motor control). Alternatively, the siRNA molecules targeting C9orf72 of the present disclosure can be delivered to a subject to treat FTD and / or mitigate FTD-related phenotypes (e.g., memory problems, behavioral problems, and language problems). In addition, the siRNA molecules of the present disclosure can also be delivered to a subject having a C9orf72 gene variant, for which siRNA-mediated gene silencing of the C9orf72 variant gene reduces the expression level of the C9orf72 transcript, thereby treating ALS, FTD, or other C9orf72-related diseases or disorders.

[0471] The present disclosure provides a method for treating a subject by silencing the C9orf72 gene with one or more siRNA molecules described herein. Relative to healthy subjects, gene silencing can be performed in a subject to silence wild-type C9orf72 transcripts, mutant C9orf72 transcripts, splicing isoforms of C9orf72 transcripts, and / or overexpressed C9orf72 transcripts thereof. The method may include delivering the siRNA molecules of the present disclosure or a pharmaceutical composition containing the same to the CNS or affected tissue of a subject (e.g., a human) by any appropriate route of administration (e.g., intraventricular, intrathecal, intrastriatal, via catheterization into the cerebellomedullary cisterna, intraparenchymal, intravenous, subcutaneous, or intramuscular). The active compound may be administered at any suitable dose. The actual dose of the composition of the present disclosure administered to a patient may be determined by physical and physiological factors, such as body weight, severity of the condition, previous or concurrent therapeutic interventions, idiopathic diseases of the patient, and route of administration. Depending on the dose and route of administration, the preferred dose and / or number of administrations of the effective amount may vary depending on the subject's response. In any case, the practitioner responsible for administration will determine the concentration of active ingredients in the composition and the appropriate dosage for the individual subject. Administration may be performed any suitable number of times per day and for as long as necessary. The subject may be an adult or child with or without comorbidities.

[0472] Subject selection

[0473] Subjects that can be treated with the siRNA molecules disclosed herein are subjects who need treatment for, for example, ALS, FTD, and / or any one or more other medical risks associated with C9orf72 gene repeat expansion or gain-of-function mutations. Subjects that can be treated with the siRNA molecules disclosed herein can include, for example, humans, monkeys, rats, mice, pigs, and other mammals containing at least one orthologous copy of the C9orf72 gene. Subjects can be adults or children with or without coexisting diseases.

[0474] Pharmaceutical composition

[0475] The siRNA molecules in the present disclosure can be formulated into pharmaceutical compositions and applied to subjects in a biocompatible form suitable for in vivo administration. Therefore, the present disclosure provides a pharmaceutical composition comprising siRNA molecules of the present disclosure mixed with a suitable diluent, carrier or excipient. The siRNA molecules can, for example, be directly administered to the CNS of the subject or to the affected tissue (e.g., by intraventricular, intrastriatal, intrathecal injection, via catheter insertion in the cerebellomedullary cisterna, intraparenchymal injection, intravenous injection, subcutaneous injection or intramuscular injection).

[0476] Conventional procedures and ingredients for selecting and preparing suitable formulations are described, for example, in Remington, JP The Science and Practice of Pharmacy, Easton, PA. Mack Publishers, 2012, 22nd edition and The United States Pharmacopeial Convention, The National Formulary, United States Pharmacopeial, 2015, USP 38 NF 33).

[0477] Under ordinary conditions of storage and use, the pharmaceutical composition may contain a preservative, for example to prevent the growth of microorganisms. The pharmaceutical composition may comprise a sterile aqueous solution, dispersion, or, for example, a powder for the extemporaneous preparation of a sterile solution or dispersion. In all cases, the form may be sterilized using techniques known in the art and may be fluidized to the extent that it can be easily administered to a subject in need of treatment.

[0478] The pharmaceutical composition can be administered to a subject, e.g., a human subject, alone or in combination with a pharmaceutically acceptable carrier as described herein, the proportion of which can be determined by the solubility and / or chemical properties of the compound, the chosen route of administration, and standard pharmaceutical practice.

[0479] Dosage regimen

[0480] Physicians of ordinary skill in the art can easily determine the effective amount of siRNA molecules to be administered to mammalian subjects (e.g., people) in need. For example, the physician can start the prescribed dose of one of the disclosed siRNA molecules at a level lower than that required to obtain the desired therapeutic effect, and gradually increase the dose until the desired effect is achieved. Alternatively, the physician can start the treatment regimen by administering one of the disclosed siRNA molecules at a high dose, and then gradually administer a lower dose until the lowest dose for achieving the therapeutic effect (e.g., the expression of the target gene sequence is reduced) is reached. In general, the suitable daily dose of one of the disclosed siRNA molecules will be the amount of the siRNA molecules at the lowest dose that effectively produces the therapeutic effect. The disclosed ss-siRNA or ds-siRNA molecules can be administered by injection, such as intrathecal injection, intraventricular injection, injection in the cerebellomedullary cisterna magna via catheter insertion, intraparenchymal injection, intravenous injection, subcutaneous or intramuscular administration. The daily dose of the therapeutic composition of the siRNA molecules of the present disclosure can be administered as a single dose, or as two, three, four, five, six or more doses administered separately at appropriate intervals in a day, a week, a month or a year, optionally, in unit dosage form. Although the siRNA molecules of the present disclosure can be administered alone, it can also be administered as a pharmaceutical formulation in combination with an excipient, a carrier and optionally an additional therapeutic agent.

[0481] Route of administration

[0482] The methods of the present disclosure contemplate any route of administration tolerated by the therapeutic composition.Some embodiments of the methods include intrathecal injection, intracerebroventricular injection, intrastriatal injection, intraparenchymal injection, or injection into the cisterna magna by catheterization.

[0483] Intrathecal injection is injection directly into the spine or subarachnoid space. By injecting directly into the CSF of the spine, the siRNA molecules of the present disclosure can directly enter cells (e.g., neurons and microglia) in the spine and have a pathway that can bypass the blood-brain barrier and enter cells in the brain.

[0484] Intraventricular (ICV) injection is a method of injecting directly into the CSF of the ventricles of the brain. Similar to intrathecal injection, ICV is an injection method that bypasses the blood-brain barrier. The advantage of using ICV is that cells in the brain and spine can be accessed without the risk of the therapeutic agent being degraded in the blood.

[0485] Intrastriatal injections are injections directly into the striatum or corpus striatum. The striatum is a region of the basal ganglia beneath the cortex of the brain. Injection into the striatum bypasses the blood-brain barrier and the pharmacokinetic challenges of injection into the bloodstream and allows direct access to brain cells.

[0486] Intraparenchymal administration is injection directly into a parenchyma (e.g., brain parenchyma). Injection into the brain parenchyma allows for direct injection into the area of ​​the brain affected by the disease or disorder while bypassing the blood-brain barrier.

[0487] Intra-cisternal injection via catheterization is an injection directly into the cisterna magna. The cisterna magna is an area of ​​the brain located between the cerebellum and the dorsal surface of the medulla oblongata. Injection into the cisterna magna results in more direct delivery of cells to the cerebellum, brainstem, and spinal cord.

[0488] In some embodiments of the methods described herein, the therapeutic composition can be delivered to the subject by systemic administration (eg, intravenously, intramuscularly, or subcutaneously).

[0489] Intravenous (IV) injection is a method of direct injection into the subject's bloodstream. IV administration can be in the form of a bolus dose or by continuous infusion, or any other method tolerated by the therapeutic composition.

[0490] Intramuscular (IM) injection is injection into a muscle of a subject, such as the deltoid or gluteal muscle. IM can allow for rapid absorption of the therapeutic composition.

[0491] Subcutaneous injection is injection into the tissue beneath the skin. Absorption of compositions delivered subcutaneously may be slower than IV or IM injection, which may be beneficial for compositions that require continuous absorption.

[0492] Example

[0493] The following examples are put forth so as to provide one of ordinary skill in the art with a description of how the compositions and methods described herein may be used, prepared, and evaluated and are intended solely as exemplary of the disclosure and are not intended to limit the scope of what the inventors regard as their disclosure.

[0494] Example 1. C9orf72 knockdown

[0495] Target

[0496] This example describes the results of a series of experiments performed to investigate the ability of siRNA molecules complementary to specific regions within the human C9orf72 mRNA transcript to achieve reduction in C9orf72 gene expression.

[0497] Materials and methods

[0498] SK-Mel-28 cells were actively transfected with C9orf72 siRNA at 20 nM or 0.5 nM concentrations. After 72 hours, cells were lysed and mRNA levels of C9orf72 and housekeeping genes (ATP5b) were assessed via reverse transcription quantitative polymerase chain reaction (RT-qPCR) using standard reagents and Applied Biosystems TaqMan assays. Results are presented as the percentage of C9orf72 mRNA remaining relative to untreated control cells in the same assay (untreated C9orf72 mRNA %).

[0499] result

[0500] Cells were treated with siRNA molecules of the present disclosure having antisense and sense strands as shown in Tables 3 and 4 below. The knockdown efficiency of the siRNA molecules was measured as the percentage of residual mRNA expression at 20 nM and 0.5 nM relative to the untreated. The knockdown results are reported in Table 3 (20 nM) and Table 4 (0.5 nM).

[0501] Table 3. Knockdown of C9orf72 using siRNA molecules of the present disclosure at 20 nM

[0502]

[0503]

[0504]

[0505]

[0506] Table 4. Knockdown of C9orf72 with siRNA molecules of the present disclosure at 0.5 nM

[0507]

[0508]

[0509]

[0510]

[0511] Example 2. Generation of siRNA molecules targeting C9orf72

[0512] The siRNA molecules of the present disclosure can be synthesized by standard methods known in the art, as discussed further below, for example, by using an automated DNA synthesizer, such as commercially available from, for example, Biosearch, Applied Biosystems, Inc.

[0513] siRNA agents can be prepared using solution phase or solid phase organic synthesis or both. Organic synthesis provides the advantage that oligonucleotides including non-natural or modified nucleotides can be easily prepared. Specific examples of siRNA molecules and the nucleotide sequences of the sense and antisense strands, as well as the C9orf72 mRNA target sequence are shown in Table 1 above. It is understood that one skilled in the art can anneal the antisense (AS) strand to the corresponding sense (S) strand to produce a ds-siRNA molecule. Alternatively, one skilled in the art can use only the antisense strand to obtain a ss-siRNA molecule.

[0514] Example 3. Optimization of siRNA molecules targeting C9orf72

[0515] For any small interfering RNA (siRNA) reagent disclosed herein, it is expected that modifications to the siRNA can further optimize the efficacy or biophysical properties of the molecule (e.g., increasing serum stability or circulation half-life, increasing thermal stability, enhancing transmembrane delivery and / or targeting a specific location or cell type). Such optimization can be achieved by systematically adding or removing connected nucleosides to generate longer or shorter sequences. Further siRNA optimization can include incorporating, for example, one or more alternative nucleosides, alternative 2' sugar moieties and / or alternative internucleoside linkages. In addition, such optimized siRNA molecules can include introducing hydrophobic moieties and / or stabilizing moieties at the 5' and / or 3' ends.

[0516] siRNA Optimization with Alternative Nucleosides

[0517] Optimization of the siRNA molecules of the present disclosure may include one or more of the following nucleoside modifications: 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-propynyl (-C=C-CH3) uracil and other alkynyl derivatives of cytosine and pyrimidine bases, 6-azo Uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-sulfanyl, 8-hydroxyl and other 8-substituted adenine and guanine, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and / or 3-deazaguanine and 3-deazaadenine. siRNA molecules can also include nucleobases in which purine or pyrimidine bases are replaced by other heterocycles such as 7-deaza-adenine, 7-deazaguanine, 2-aminopyridine and / or 2-pyridone. Further optimization of the siRNA molecules of the present disclosure may include the nucleobases disclosed in the following documents: US 3,687,808; Kroschwitz, JI, ed.; The Concise Encyclop edia of Polymer Science and Engineering, New York, John Wiley & Sons, 1990, pp. 858-859; Englisch et al., Angewandte Chemie, International Edition, 30:613, 1991; and Sanghvi, YS, Chapter 16, Antisense Research and Applications, CRC Press, Gait, MJ, ed., 1993, pp. 289-302.

[0518] siRNA optimization using alternative sugar modifications

[0519] Optimization of the siRNA molecules of the present disclosure may include one or more of the following 2' sugar modifications: 2'-O-methyl (2'-O-Me); 2'-methoxyethoxy (2'-O-CH 2 CH 2 OCH 3 , also known as 2'-O-(2-methoxyethyl) or 2'-MOE); 2'-dimethylaminooxyethoxy, also known as O(CH 2 ) 2ON(CH 3 ) 2 group, also known as 2'-DMAOE; and / or 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylamino-ethoxy-ethyl or 2'-DMAEOE), i.e., 2'-O-CH 2 OCH 2 N(CH 3 ) 2 Other possible 2′-modifications that can optimize the siRNA molecules of the present disclosure include OH in all possible orientations; F; O-alkyl, S-alkyl, or N-alkyl; O-alkenyl, S-alkenyl, or N-alkenyl; O-alkynyl, S-alkynyl, or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl, and alkynyl groups can be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl groups. Other possible sugar substituent groups include, for example, aminopropoxy (—OCH 2 CH 2 CH 2 NH 2 ), allyl (-CH 2 -CH=CH 2 ), -O-allyl (-O-CH 2 -CH=CH 2 ) and fluorine (F). The 2'-sugar substituent can be located at the arabinose (upper) position or the ribose (lower) position. In some embodiments, the 2'-arabinose modification is 2'-F. Similar modifications can also be made at other positions on the siRNA molecule, specifically at the 3' position of the sugar on the 3' terminal nucleoside or in the 2'-5' linked oligonucleotide and the 5' position of the 5' terminal nucleotide. Oligonucleotides can also have sugar mimetics such as cyclobutyl moieties instead of pentofuranosyl sugars.

[0520] siRNA Optimization Using Alternative Internucleoside Linkages

[0521] Optimization of the siRNA molecules of the present disclosure can include one or more of the following internucleoside modifications: phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates (including 3'-alkylenephosphonates, 5'-alkylenephosphonates), phosphinates, phosphoramidates (including 3'-aminophosphoramidate and aminoalkylphosphoramidates), thiophosphoramidates, thioalkylphosphonates, thioalkylphosphotriesters, selenophosphates, and borophosphates with normal 3'-5' linkages, 2'-5' linked analogs of these, and those with reversed polarity, wherein one or more of the internucleotide linkages are 3' to 3', 5' to 5', or 2' to 2' linkages.

[0522] siRNA optimization using hydrophobic moieties

[0523] Optimization of the siRNA molecules of the present disclosure may include a hydrophobic moiety covalently attached to the 5' end or the 3' end. Non-limiting examples of hydrophobic moieties suitable for use with the siRNA molecules of the present disclosure may include cholesterol, vitamin D, tocopherol, phosphatidylcholine (PC), docosahexaenoic acid, docosanoic acid, PC-docosanoic acid, eicosapentaenoic acid, lithocholic acid, or any combination of the above hydrophobic moieties and PC.

[0524] siRNA optimization using stabilizing fractions

[0525] The optimization of siRNA molecules of the present disclosure can include a 5'-phosphorus stabilizing portion that protects the siRNA molecules from degradation. The 5'-phosphorus stabilizing portion replaces the 5'-phosphate to prevent phosphate hydrolysis. The hydrolysis of the 5'-phosphate prevents the combination with RISC, which is a necessary step for gene silencing. Any phosphate replacement that does not hinder the combination with RISC is considered in the present disclosure. In some embodiments, the substitute of the 5'-phosphate is also stable to hydrolysis in vivo. Each siRNA chain can independently and optionally adopt any suitable 5'-phosphorus stabilizing portion. Non-limiting examples of 5' stabilizing portions suitable for use with siRNA molecules of the present disclosure can include those shown in Formulas IX-XVI above.

[0526] siRNA Optimization Using Branched siRNA

[0527] Optimization of siRNA molecules disclosed herein can include incorporation of branching patterns, such as two-branch, three-branch or four-branch siRNAs connected by joints. Each main branch can be further branched to allow 2, 3, 4, 5, 6, 7 or 8 independent RNA single strands or double strands. The branching points on the joint can be derived from the same atom, or along the individual atoms of the joint. Some exemplary embodiments are listed in Table 2 above.

[0528] The siRNA compositions disclosed herein can be optimized into the following forms: a two-branched siRNA molecule, as represented by any one of Formulas XVII-XIX; a three-branched siRNA molecule, as represented by any one of Formulas XX-XXIII; and / or a four-branched siRNA molecule, as represented by any one of Formulas XXIV-XXVIII, wherein each RNA is independently a siRNA molecule, L is a linker, and each X independently represents a branch point moiety (e.g., phosphoramidite, glycerol tosylated acetonate, 1,3-diaminopropanol, pentaerythritol, or any of the branch point moieties described in US 10,478,503).

[0529] Example 4. Preparation and administration of siRNA molecules targeting C9orf72

[0530] The siRNA molecules in the present disclosure can be formulated into pharmaceutical compositions and applied to subjects in a biocompatible form suitable for in vivo administration. For example, the siRNA molecules of the present disclosure can be applied in a suitable diluent, carrier or excipient, and can also contain a preservative, for example, to prevent the growth of microorganisms. Conventional procedures and ingredients for selecting and preparing suitable preparations are described, for example, in Remington, JP The Science and Practice of Pharmacy, Easton, PA. Mack Publishers, 2012, the 22nd edition and The United States Pharmacopeial Convention, The National Formulary, United States Pharmacopeial, 2015, USP 38 NF 33).

[0531] The method of the present disclosure encompasses any route of administration to a subject that is tolerated by the siRNA compositions of the present disclosure. Non-limiting examples of siRNA injections to the CNS include intrathecal injections, intraventricular injections, intrastriatal injections, intraparenchymal injections, or injections in the cerebellomedullary cistern via catheter insertion. Examples of systemic administration include intravenous injections, intramuscular injections, and subcutaneous injections. A physician with ordinary skill in the art can easily determine an effective route of administration.

[0532] Example 5. Method for treating amyotrophic lateral sclerosis or frontotemporal dementia using siRNA molecules targeting C9orf72

[0533] Subjects in need of treatment for amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD) are treated with a dose of the siRNA molecules or siRNA compositions of the present disclosure (formulated as salts) at a frequency determined by the practitioner. A physician of ordinary skill in the art can easily determine the effective amount of siRNA molecules to be administered to a mammalian subject (e.g., a human) in need thereof. For example, a physician may start a prescribed dose of one of the siRNA molecules of the present disclosure at a level lower than that required to obtain the desired therapeutic effect, and gradually increase the dose until the desired effect is achieved. Alternatively, a physician may initiate a treatment regimen by administering a high dose of one of the siRNA molecules of the present disclosure, and subsequently gradually administer lower doses until a minimum dose is reached that produces a therapeutic effect (e.g., a reduction in the expression of C9orf72 mRNA or a suitable biomarker, or a reduction in an ALS- or FTD-related phenotype). In general, a suitable daily dose of one of the siRNA molecules of the present disclosure will be the amount of the lowest dose that is effective to produce a therapeutic effect. The ss- or ds-siRNA molecules disclosed herein can be administered by injection, such as intrathecal injection, intraventricular injection, intrastriatal injection, intraparenchymal injection, intravenous injection, intramuscular injection, subcutaneous injection, or by injection in the cerebellomedullary cisterna via catheter insertion. The daily dose of the therapeutic composition of one of the siRNA molecules disclosed herein can be administered as a single dose, or as two, three, four, five, six or more doses administered separately at appropriate intervals in a day, a week, a month or a year, optionally, in a unit dosage form. Although one of the siRNA molecules disclosed herein can be administered alone, it can also be administered as a pharmaceutical preparation combined with an excipient, a carrier and optionally another therapeutic agent. Dosage and frequency are determined according to the height, weight, age, sex and other conditions of the subject.

[0534] The siRNA molecules disclosed herein are selected by the practitioner based on compatibility with the subject. Single-stranded or double-stranded siRNA molecules (e.g., non-branched siRNA, two-branched siRNA, three-branched siRNA, four-branched siRNA) are available for selection. The selected siRNA molecules have an antisense strand and may have a sense strand with a sequence and RNA modification (e.g., natural and non-natural internucleoside linkages, modified sugars, 5'-phospho-stabilizing moieties, hydrophobic indistinction and / or branched structures) that is most suitable for the patient.

[0535] The siRNA molecules are delivered by the route most appropriate for the patient (e.g., intrathecal, intraventricular, intrastriatal, intraparenchymal, intravenous, intramuscular, subcutaneous, or by injection into the cisterna magna via catheterization) and condition at a rate tolerated by the patient until the subject reaches the maximum tolerated dose, or until a satisfactory improvement in symptoms is achieved.

[0536] Other Implementations

[0537] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.

[0538] Although the present invention has been described in conjunction with its specific embodiments, it should be understood that it is capable of further modifications and this application is intended to cover any changes, uses or adaptations of the present invention based on the principles of the present invention, including changes that are not part of the present invention but are known or customary implementation methods in the field to which the present invention belongs and that belong to the essential features described above and that are within the scope of the claims.

[0539] Other embodiments are within the claims.

Claims

1. A small interfering RNA (siRNA) molecule comprising an antisense strand and a sense strand having complementarity to the antisense strand, wherein the antisense strand has sufficient complementarity to hybridize to a region within a chromosome 9 open reading frame 72 (C9orf72) mRNA transcript having a nucleic acid sequence of any one of SEQ ID NOs: 1-384.

2. The siRNA molecule of claim 1, wherein the antisense strand has at least 70% complementarity to a region of 19, 20, 21 or more consecutive nucleobases within the C9orf72 mRNA transcript having a nucleic acid sequence of any one of SEQ ID NOs: 1-384, optionally wherein the antisense strand has at least 70% complementarity to the C9orf72 mRNA transcript having a nucleic acid sequence of any one of SEQ ID NOs: 1-384.

3. The siRNA molecule of claim 2, wherein the antisense strand has at least 75% complementarity to a region of 21 consecutive nucleobases within the C9orf72 mRNA transcript having a nucleic acid sequence of any one of SEQ ID NOs: 1-384, optionally wherein the antisense strand has at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementarity to the region within the C9orf72 mRNA transcript having a nucleic acid sequence of any one of SEQ ID NOs: 1-384.

4. The siRNA molecule of any one of claims 1-3, wherein the antisense strand comprises at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29 or 30 consecutive nucleotides that are fully complementary to a consecutive polynucleotide segment of equal length within the region of the C9orf72 RNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-384.

5. The siRNA molecule of claim 4, wherein the antisense strand comprises 10 to 30 consecutive nucleotides that are fully complementary to a consecutive polynucleotide segment of equal length within the region of the C9orf72 RNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-384.

6. The siRNA molecule of claim 5, wherein the antisense strand comprises 12 to 30 consecutive nucleotides that are fully complementary to a consecutive polynucleotide segment of equal length within the region of the C9orf72 RNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-384.

7. The siRNA molecule of claim 6, wherein the antisense strand comprises 15 to 30 consecutive nucleotides that are fully complementary to a consecutive polynucleotide segment of equal length within the region of the C9orf72 RNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-384.

8. The siRNA molecule of claim 7, wherein the antisense strand comprises 18 to 30 consecutive nucleotides that are fully complementary to a consecutive polynucleotide segment of equal length within the region of the C9orf72 RNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-384.

9. The siRNA molecule of claim 8, wherein the antisense strand comprises 18 to 25 consecutive nucleotides that are fully complementary to a consecutive polynucleotide segment of equal length within the region of the C9orf72 RNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-384.

10. The siRNA molecule of any one of claims 9, wherein the antisense strand comprises 18 to 21 consecutive nucleotides that are fully complementary to a consecutive polynucleotide segment of equal length within the region of the C9orf72 RNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-384.

11. The siRNA molecule of claim 10, wherein the antisense strand comprises 21 consecutive nucleotides that are fully complementary to a consecutive polynucleotide segment of equal length within the region of the C9orf72 RNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-384.

12. The siRNA molecule of any one of claims 1-11, wherein the antisense strand comprises 9 or fewer nucleotide mismatches relative to a region of 21 consecutive nucleobases of the C9orf72 RNA transcript having a nucleic acid sequence of any one of SEQ ID NOs: 1-384, optionally wherein the antisense strand comprises 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or only 1 mismatch relative to the region of the C9orf72 RNA transcript having a nucleic acid sequence of any one of SEQ ID NOs: 1-384.

13. The siRNA molecule of any one of claims 1-12, wherein the region of the C9orf72 RNA transcript has a nucleic acid sequence of any one of SEQ ID NOs: 1-192.

14. The siRNA molecule of any one of claims 1-12, wherein the region of the C9orf72 RNA transcript has a nucleic acid sequence of any one of SEQ ID NOs: 193-384.

15. The siRNA molecule of any one of claims 1-14, wherein the antisense strand has a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence of any one of SEQ ID NOs: 769-1152.

16. The siRNA molecule of claim 15, wherein the antisense strand has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of any one of SEQ ID NOs: 769-1152.

17. The siRNA molecule of claim 16, wherein the antisense strand has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NOs: 769-1152, optionally wherein the antisense strand has a nucleic acid sequence that is at least 96%, 97%, 98% or 99% identical to the nucleic acid sequence of any one of SEQ ID NOs: 769-1152.

18. The siRNA molecule of claim 17, wherein the antisense strand has a nucleic acid sequence of any one of SEQ ID NOs: 769-1152.

19. The siRNA molecule of any one of claims 15-18, wherein the nucleic acid sequence is any one of SEQ ID NOs: 769-960.

20. The siRNA molecule of any one of claims 15-18, wherein the nucleic acid sequence is any one of SEQ ID NOs: 961-1152.

21. The siRNA molecule of any one of claims 1-20, wherein the sense strand has a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence of any one of SEQ ID NOs: 385-768.

22. The siRNA molecule of claim 21, wherein the sense strand has a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of any one of SEQ ID NOs: 385-768.

23. The siRNA molecule of claim 22, wherein the sense strand has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NOs: 385-768, optionally wherein the sense strand has a nucleic acid sequence that is at least 96%, 97%, 98% or 99% identical to the nucleic acid sequence of any one of SEQ ID NOs: 385-768.

24. The siRNA molecule of claim 23, wherein the sense strand has a nucleic acid sequence of any one of SEQ ID NOs: 385-768.

25. The siRNA molecule of any one of claims 21-24, wherein the nucleic acid sequence is any one of SEQ ID NOs: 385-576.

26. The siRNA molecule of any one of claims 21-24, wherein the nucleic acid sequence is any one of SEQ ID NOs: 577-768.

27. The siRNA molecule of any one of claims 1-26, wherein the antisense strand comprises a structure represented by Formula I, wherein Formula I in the 5' to 3' direction is: A-B-(A’) j -C-P 2 -D-P 1 -(C’-P 1 ) k -C’ Formula I; Where A is composed of the formula CP 1 -DP 1 express; Each A' is represented by the formula CP 2 -DP 2 express; B by CP 2 -DP 2 -DP 2 -DP 2 express; Each C is a 2'-O-methyl (2'-O-Me) ribonucleoside; Each C' is independently 2'-O-Me ribonucleoside or 2'-fluoro (2'-F) ribonucleoside; Each D is a 2'-F ribonucleoside; Each P 1 is a phosphorothioate internucleoside linkage; Each P 2 It is a phosphodiester internucleoside linkage; j is an integer from 1 to 7; and k is an integer from 1 to 7.

28. The siRNA molecule of claim 27, wherein the antisense strand comprises a structure represented by Formula A1, wherein Formula A1 is: ASBSAOBOBOBOAOBOAOBOA-OBAOBOAOBSASASASBSA Formula A1; wherein A represents a 2'-0-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

29. The siRNA molecule of any one of claims 1-26, wherein the antisense strand comprises a structure represented by Formula II, wherein Formula II is: A-B-(A’) j -C-P 2 -D-P 1 -(C-P 1 ) k -C’ Formula II; Where A is composed of the formula CP 1 -DP 1 express; Each A' is represented by the formula CP 2 -DP 2 express; B by CP 2 -DP 2 -DP 2 -DP 2 express; Each C is a 2'-O-methyl (2'-O-Me) ribonucleoside; Each C' is independently 2'-O-Me ribonucleoside or 2'-fluoro (2'-F) ribonucleoside; Each D is a 2'-F ribonucleoside; Each P 1 is a phosphorothioate internucleoside linkage; Each P 2 It is a phosphodiester internucleoside linkage; j is an integer from 1 to 7; and k is an integer from 1 to 7.

30. The siRNA molecule of claim 29, wherein the antisense strand comprises a structure represented by Formula A2, wherein Formula A2 is: ASBSAOBOBOBOAOBOAOBOA-OBOOBOAOBSASASASASA Formula A2; wherein A represents a 2'-0-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

31. The siRNA molecule of any one of claims 1-30, wherein the sense strand comprises a structure represented by Formula III, wherein Formula III is: E-(A') m -F Formula III; Where E is given by the formula (CP 1 ) 2 express; F is given by the formula (CP 2 ) 3 -DP 1 -CP 1 -C, (CP 2 ) 3 -DP 2 -CP 2 -C, (CP 2 ) 3 -DP 1 -CP 1 -D or (CP 2 ) 3 -DP 2 -CP 2 -D means; A', C, D, P 1 and P 2 As defined in Formula II; and m is an integer of 1-7.

32. The siRNA molecule of claim 31, wherein the sense strand comprises a structure represented by Formula S1, wherein Formula S1 is: ASASAOBOAOBOAOBOAOBOA-OAOAOBSASA Formula S1; wherein A represents a 2'-0-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

33. The siRNA molecule of claim 31, wherein the sense strand comprises a structure represented by Formula S2, wherein Formula S2 is: ASASAOBOAOBOAOBOAOBOA-OAOAOBOAOA Formula S2; wherein A represents a 2'-0-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

34. The siRNA molecule of claim 31, wherein the sense strand comprises a structure represented by Formula S3, wherein Formula S3 is: ASASAOBOAOBOAOBOAOBOA-OAOAOBSASB Formula S3; wherein A represents a 2'-0-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

35. The siRNA molecule of claim 31, wherein the sense strand comprises a structure represented by Formula S4, wherein Formula S4 is: ASASAOBOAOBOAOBOAOBOA-OAOAOBOAOB Formula S4; wherein A represents a 2'-0-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

36. The siRNA molecule of any one of claims 1-26 and 31-35, wherein the antisense strand comprises a structure represented by Formula IV, wherein Formula IV in the 5' to 3' direction is: A-(A’) j -C-P 2 -B-(C-P 1 ) k -C’ Formula IV; Where A is composed of the formula CP 1 -DP 1 express; Each A' is represented by the formula CP 2 -DP 2 express; B by DP 1 -CP 1 -DP 1 express; Each C is a 2'-O-Me ribonucleoside; Each C' is independently 2'-O-Me ribonucleoside or 2'-F ribonucleoside; Each D is a 2'-F ribonucleoside; Each P 1 is a phosphorothioate internucleoside linkage; Each P 2 It is a phosphodiester internucleoside linkage; j is an integer from 1 to 7; and k is an integer from 1 to 7.

37. The siRNA molecule of claim 36, wherein the antisense strand comprises a structure represented by Formula A3, wherein Formula A3 is: ASBSAOBOAOBOAOBOAOBOA-OBAOBOAOBSASBSASASA Formula A3; wherein A represents a 2'-0-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

38. The siRNA molecule of any one of claims 1-30, 36 and 37, wherein the sense strand comprises a structure represented by Formula V, wherein Formula V in the 5' to 3' direction is: E-(A’) m -C-P 2 -F Formula V; Where E is given by the formula (CP 1 ) 2 express; F by DP 1 -CP 1 -C, DP 2 -CP 2 -C, DP 1 -CP 1 -D or DP 2 -CP 2 -D means; A', C, D, P 1 and P 2 As defined in Formula IV; and m is an integer of 1-7.

39. The siRNA molecule of claim 38, wherein the sense strand comprises a structure represented by Formula S5, wherein Formula S5 is: ASASAOBOAOBOAOBOAOBOA-OBOAOBSASA Formula S5; wherein A represents a 2'-0-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

40. The siRNA molecule of claim 38, wherein the sense strand comprises a structure represented by Formula S6, wherein Formula S6 is: ASASAOBOAOBOAOBOAOBOA-OBOAOBOAOA Formula S6; wherein A represents a 2'-0-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

41. The siRNA molecule of claim 38, wherein the sense strand comprises a structure represented by Formula S7, wherein Formula S7 is: ASASAOBOAOBOAOBOAOBOA-OBOAOBSASB Formula S7; wherein A represents a 2'-0-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

42. The siRNA molecule of claim 38, wherein the sense strand comprises a structure represented by Formula S8, wherein Formula S8 is: ASASAOBOAOBOAOBOAOBOA-OBOAOBOAOB Formula S8; wherein A represents a 2'-0-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

43. The siRNA molecule of any one of claims 1-26, 31-35, and 38-42, wherein the antisense strand comprises a structure represented by Formula VI, wherein Formula VI is, in the 5' to 3' direction: A-B j -E-B k -E-F-G l -D-P 1 -C’ Formula VI; Where A is composed of the formula CP 1 -DP 1 express; Each B is represented by the formula CP 2 express; Each C is a 2'-O-Me ribonucleoside; Each C' is independently 2'-O-Me ribonucleoside or 2'-F ribonucleoside; Each D is a 2'-F ribonucleoside; Each E is represented by the formula DP 2 -CP 2 express; F by DP 1 -CP 1 express; Each G is represented by the formula CP 1 express; Each P 1 is a phosphorothioate internucleoside linkage; Each P 2 It is a phosphodiester internucleoside linkage; j is an integer from 1 to 7; k is an integer from 1 to 7; and l is an integer from 1 to 7.

44. The siRNA molecule of claim 43, wherein the antisense strand comprises a structure represented by Formula A4, wherein Formula A4 is: ASBSAOAOAOBOAOAOAOAOA-OAOAOBOAOBSASASASASBSA Formula A4; wherein A represents a 2'-0-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

45. The siRNA molecule of any one of claims 1-30, 36, 37, 43 and 44, wherein the sense strand comprises a structure represented by Formula VII, wherein Formula VII is: H-B m -I n -A'-B o -H-C Formula VII; Where A' is composed of the formula CP 2 -DP 2 express; Each H is represented by the formula (CP 1 ) 2 express; Each I is represented by the formula (DP 2 )express; B, C, D, P 1 and P 2 As defined in Formula VI; m is an integer from 1 to 7; n is an integer from 1 to 7; and o is an integer from 1 to 7.

46. ​​The siRNA molecule of claim 45, wherein the sense strand comprises a structure represented by Formula S9, wherein Formula S9 is: ASASAOAOAOBOBOBOAOBOA-OAOAOASASA Formula S9; wherein A represents a 2'-0-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

47. The siRNA molecule of any one of claims 1-46, wherein the antisense strand further comprises a 5' phospho-stabilizing moiety at the 5' end of the antisense strand.

48. The siRNA molecule of any one of claims 1-47, wherein the sense strand further comprises a 5' phosphorus stabilizing moiety at the 5' end of the sense strand.

49. The siRNA molecule of claim 47 or 48, wherein each 5' phosphorus stabilizing moiety is independently represented by any one of Formulas IX-XVI: wherein Nuc represents a nucleobase, optionally wherein the nucleobase is selected from the group consisting of adenine, uracil, guanine, thymine and cytosine, and R represents optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, phenyl, benzyl, a cation or hydrogen.

50. The siRNA molecule of claim 49, wherein the nucleobase is adenine, uracil, guanine, thymine or cytosine.

51. The siRNA molecule of any one of claims 47-50, wherein the 5' phosphorus stabilizing moiety is an (E)-vinyl phosphonate represented by Formula XI.

52. The siRNA molecule of any one of claims 1-51, wherein the siRNA molecule further comprises a hydrophobic portion at the 5' end or the 3' end of the siRNA molecule.

53. The siRNA molecule of claim 52, wherein the hydrophobic moiety is selected from cholesterol, vitamin D or tocopherol.

54. The siRNA molecule of any one of claims 1-53, wherein the sense strand is 10 to 30 nucleotides in length.

55. The siRNA molecule of claim 54, wherein the sense strand is 10 to 25 nucleotides in length.

56. The siRNA molecule of claim 55, wherein the sense strand is 12 to 25 nucleotides in length.

57. The siRNA molecule of claim 56, wherein the sense strand is 12 to 20 nucleotides in length.

58. The siRNA molecule of claim 57, wherein the sense strand is 12 to 19 nucleotides in length.

59. The siRNA molecule of claim 58, wherein the sense strand is 15 nucleotides in length.

60. The siRNA molecule of claim 58, wherein the sense strand is 16 nucleotides in length.

61. The siRNA molecule of claim 58, wherein the sense strand is 18 nucleotides in length.

62. The siRNA molecule of any one of claims 1-61, wherein the antisense strand is 10 to 30 nucleotides in length.

63. The siRNA molecule of claim 62, wherein the antisense strand is 12 to 30 nucleotides in length.

64. The siRNA molecule of claim 63, wherein the antisense strand is 15 to 30 nucleotides in length.

65. The siRNA molecule of claim 64, wherein the antisense strand is 18 to 30 nucleotides in length.

66. The siRNA molecule of claim 65, wherein the antisense strand is 18 to 25 nucleotides in length.

67. The siRNA molecule of claim 66, wherein the antisense strand is 18 to 21 nucleotides in length.

68. The siRNA molecule of claim 67, wherein the antisense strand is 18 nucleotides in length.

69. The siRNA molecule of claim 67, wherein the antisense strand is 20 nucleotides in length.

70. The siRNA molecule of claim 67, wherein the antisense strand is 21 nucleotides in length.

71. The siRNA molecule of any one of claims 1-70, wherein the siRNA molecule is a branched siRNA molecule.

72. The siRNA molecule of claim 71, wherein the branched siRNA molecule is bi-branched, tri-branched or quad-branched.

73. The siRNA molecule of claim 72, wherein the siRNA molecule is a bi-branched siRNA molecule, optionally wherein the bi-branched siRNA molecule is represented by any one of Formulas XVII-XIX: Wherein each RNA is independently a siRNA molecule, L is a linker, and each X independently represents a branch point moiety.

74. The siRNA molecule of claim 72, wherein the siRNA molecule is a three-branched siRNA molecule, optionally wherein the three-branched siRNA molecule is represented by any one of Formulae XX-XXIII: Wherein each RNA is independently a siRNA molecule, L is a linker, and each X independently represents a branch point moiety.

75. The siRNA molecule of claim 72, wherein the siRNA molecule is a four-branched siRNA molecule, optionally wherein the four-branched siRNA molecule is represented by any one of Formulae XXIV-XXVIII: Wherein each RNA is independently a siRNA molecule, L is a linker, and each X independently represents a branch point moiety.

76. The siRNA molecule of any one of claims 73-75, wherein the linker is selected from one or more consecutive subunits of ethylene glycol, an alkyl, a carbohydrate, a block copolymer, a peptide, RNA, and DNA.

77. The siRNA molecule of claim 76, wherein the one or more consecutive subunits are 2 to 20 consecutive subunits.

78. A pharmaceutical composition comprising the siRNA molecule of any one of claims 1-77 and a pharmaceutically acceptable excipient, carrier or diluent.

79. A method of delivering an siRNA molecule to a subject diagnosed with amyotrophic lateral sclerosis (ALS), the method comprising administering to the subject a therapeutically effective amount of the siRNA molecule of any one of claims 1-77 or the pharmaceutical composition of claim 78.

80. A method of delivering an siRNA molecule to a subject diagnosed with frontotemporal dementia (FTD), the method comprising administering to the subject a therapeutically effective amount of the siRNA molecule of any one of claims 1-77 or the pharmaceutical composition of claim 78.

81. A method of treating ALS in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the siRNA molecule of any one of claims 1-77 or the pharmaceutical composition of claim 78.

82. A method of treating FTD in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the siRNA molecule of any one of claims 1-77 or the pharmaceutical composition of claim 78.

83. A method of reducing C9orf72 expression in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the siRNA molecule of any one of claims 1-77 or the pharmaceutical composition of claim 78.

84. The method of any one of claims 79-83, wherein the siRNA molecule or the pharmaceutical composition is administered to the subject by intracerebroventricular, intrastriatal, intraparenchymal or intrathecal injection.

85. The method of any one of claims 79-83, wherein the siRNA molecule or the pharmaceutical composition is administered to the subject by intravenous, intramuscular, or subcutaneous injection.

86. The method of any one of claims 79-85, wherein the subject is a human.

87. A kit comprising the siRNA molecule of any one of claims 1-77, or the pharmaceutical composition of claim 78, and a package insert, wherein the package insert instructs a user of the kit to perform the method of any one of claims 79-86.

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