Compositions and methods for treating neurodegenerative diseases

Abstract

The present invention provides single- or double-stranded interfering RNA molecules (e.g., siRNAs) targeting the TAR DNA-binding protein (TARDBP) gene. The interfering RNA molecules, as well as pharmaceutical compositions comprising them, may contain specific patterns of nucleoside and internucleoside linkage modifications. The siRNA molecules may be branched siRNA molecules, such as biantennary, triantennary, or tetraantennary siRNA molecules. The disclosed siRNA molecules may further feature a 5' phosphorus-stabilizing moiety and / or a hydrophobic moiety. Furthermore, the present invention provides methods for delivering the disclosed siRNA molecules to the central nervous system of a subject (e.g., a subject identified as having a neurodegenerative disease (e.g., amyotrophic lateral sclerosis or frontotemporal dementia)).

Description

[Technical Field]

[0001] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format and is incorporated herein by reference in its entirety. The XML copy, created on September 19, 2023, is named "51436-039WO2_Sequence_Listing_9_19_23" and is 505,859 bytes in size.

[0002] Technical Field This disclosure relates to small interfering RNA (siRNA) molecules that target RNA transcripts (e.g., mRNA) of the transactive response DNA-binding protein 43 (TARDBP) gene, and compositions containing the same. This disclosure further describes methods for silencing TARDBP by delivering TARDBP-targeting siRNA molecules to target tissues in subjects in need thereof, and the treatment of diseases that may benefit from silencing TARDBP (e.g., neurodegenerative diseases, such as amyotrophic lateral sclerosis and frontotemporal dementia). [Background technology]

[0003] TARDBP (transactive response DNA-binding protein 43) encodes a protein that is involved in the pathology of diseases, including neurodegenerative diseases (e.g., amyotrophic lateral sclerosis and frontotemporal dementia). Thus, there is a need for therapies that can selectively reduce TARDBP activity in a manner that provides effective treatment for neurodegenerative diseases (e.g., amyotrophic lateral sclerosis and frontotemporal dementia) or other TARDBP-associated diseases or disorders. Summary of the Invention

[0004] The present disclosure provides compositions and methods for reducing expression of transactive response DNA-binding protein 43 (TARDBP) by small interfering RNA (siRNA)-mediated silencing of the TARDBP transcript, which offer the advantage of being highly selective for TARDBP over other genes.

[0005] The siRNA molecules of the present invention can be used to silence the TARDBP gene, thereby preventing translation of the corresponding mRNA transcript and reducing the expression of transactive response DNA-binding protein 43 (TDP-43) protein. Therefore, since mislocalization and aggregation of TDP-43 are the cause of neurodegenerative diseases (e.g., amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD)), reducing the TDP-43 level prevents the onset or progression of the disease. The siRNA molecules of the present disclosure can be directly delivered to a subject requiring TARDBP silencing, for example, by intrathecal injection, intracerebroventricular injection, intrastriatal injection, intraparenchymal injection, intracisternal injection (e.g., catheterization), intravenous injection, subcutaneous injection, or intramuscular injection.

[0006] In one aspect, the present disclosure provides an siRNA molecule comprising an antisense strand and a sense strand complementary to the antisense strand. The antisense strand has sufficient complementarity to hybridize to a region within a TARDBP mRNA transcript having a nucleic acid sequence set forth in any one of SEQ ID NOS: 1-192. The antisense strand may be, for example, 10 to 50 nucleotides in length (e.g., 10 to 45 nucleotides, 10 to 40 nucleotides, 10 to 35 nucleotides, 10 to 30 nucleotides, 10 to 29 nucleotides, 10 to 28 nucleotides, 10 to 27 nucleotides, 10 to 26 nucleotides, 10 to 25 nucleotides, 10 to 24 nucleotides, 10 to 23 nucleotides, 10 to 22 nucleotides, 10 to 21 nucleotides, or 10 to 20 nucleotides in length). In some embodiments, the antisense strand is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more nucleotides in length.

[0007] In some embodiments of any of the above-described 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 TARDBP mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-192. 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 TARDBP mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-192.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 TARDBP mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-192. 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 TARDBP mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-192.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 TARDBP mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-192. 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 TARDBP mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-192.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 TARDBP mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-192.

[0008] 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 TARDBP mRNA transcript having a nucleic acid sequence of any one of SEQ ID NOs: 1-192.

[0009] In some embodiments, the antisense strand has at least 75% complementarity to a region within a TARDBP mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1 to 192. 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 of a TARDBP mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1 to 192.

[0010] 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 completely complementary to a contiguous polynucleotide segment of equal length within a region of a TARDBP mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-192.

[0011] In some embodiments, the antisense strand has 10 to 30 contiguous 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 contiguous nucleotides) that are completely complementary to a contiguous polynucleotide segment of equal length within a region of a TARDBP mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-192.

[0012] In some embodiments, the antisense strand has 12 to 30 contiguous 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 contiguous nucleotides) that are completely complementary to a contiguous polynucleotide segment of equal length within a region of a TARDBP mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-192.

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

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

[0015] In some embodiments, the antisense strand has 18 to 25 contiguous nucleotides (e.g., 18, 19, 20, 21, 22, 23, 24, or 25 contiguous nucleotides) that are completely complementary to a contiguous polynucleotide segment of equal length within a region of a TARDBP mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-192.

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

[0017] In some embodiments, the antisense strand has 21 contiguous nucleotides that are perfectly complementary to a contiguous polynucleotide segment of equal length within a region of a TARDBP mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-192.

[0018] In some embodiments, the antisense strand has 9 or fewer nucleotide mismatches to a region of 21 consecutive nucleic acid bases of a TARDBP RNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-192; optionally, 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 to a region of a TARDBP mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-192.

[0019] 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: 385-576.

[0020] 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: 385-576.

[0021] 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 the nucleic acid sequence of any one of SEQ ID NOs: 385-576, and optionally, 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: 385-576.

[0022] In some embodiments, the antisense strand has the nucleic acid sequence of any one of SEQ ID NOs: 385-576.

[0023] 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: 193-384.

[0024] 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: 193-384.

[0025] 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 the nucleic acid sequence of any one of SEQ ID NOs: 193-384, and optionally, 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: 193-384. In some embodiments, the siRNA molecule has a sense strand having the nucleic acid sequence of any one of SEQ ID NOs: 193-384.

[0026] In some embodiments, the antisense strand has a structure represented by Formula I, which, in the 5' to 3' direction, is: AB-(A') j-CP 2 -DP 1 -(C'-P 1 ) k -C' Formula I; where A is a compound of formula CP 1 -DP 1 Represented by; Each A' is of the formula CP 2 -DP 2 Represented by; B is the formula CP 2 -DP 2 -DP 2 -DP 2 Represented by; 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); k is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7).

[0027] In some embodiments, the antisense strand has a structure represented by Formula A1, which, in the 5' to 3' direction, is:

[0028] ASBSAOBOBOBOAOBOAOBOA-OBOAOBOAOBSASASASBSA Formula A1; wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0029] In some embodiments, the antisense strand has a structure represented by Formula II, which, in the 5' to 3' direction, is: AB-(A') j -CP 2 -DP 1 -(CP 1 ) k -C' Formula II; where A is a compound of formula CP 1 -DP 1 Represented by; Each A' is of the formula CP 2 -DP 2 Represented by; B is the formula CP 2 -DP 2 -DP 2 -DP 2 Represented by; 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); k is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7).

[0030] In some embodiments, the antisense strand has a structure represented by Formula A2, which, in the 5' to 3' direction, is: ASBSAOBOBOBOAOBOAOBOA-OBOAAOBOAOBSASASASASA Formula A2; wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0031] In some embodiments, the sense strand has a structure represented by Formula III, which, in the 5' to 3' direction, is: E-(A') m -F Formula III; where E is a group represented by the formula (CP 1 )2; F is 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 - represented by D; A', C, D, P 1 , and P 2 is as defined in formula II; m is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7).

[0032] In some embodiments, the sense strand has a structure represented by formula S1, which, in the 5' to 3' direction, is: ASASAOBOAOBOAOBOAOBOA-OAOAOBBSASA Formula S1; wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0033] In some embodiments, the sense strand has a structure represented by Formula S2, which, in the 5' to 3' direction, is: ASASAOBOAOBOAOBOAOBOA-OAOAOBOAOA Formula S2; wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0034] In some embodiments, the sense strand has a structure represented by Formula S3, which, in the 5' to 3' direction, is: ASASAOBOAOBOAOBOAOBOA-OAOAOBSASB formula S3; wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0035] In some embodiments, the sense strand has a structure represented by Formula S4, which, in the 5' to 3' direction, is: ASASAOBOAOBOAOBOAOBOA-OAOAOBOAOB Equation S4; wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0036] In some embodiments, the antisense strand has a structure represented by Formula IV, which, in the 5' to 3' direction, is: A-(A') j -CP 2 -B-(CP 1 ) k-C' Formula IV; where A is a compound of formula CP 1 -DP 1 Represented by; Each A' is of the formula CP 2 -DP 2 Represented by; B is the formula DP 1 -CP 1 -DP 1 Represented by; 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 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); k is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7).

[0037] In some embodiments, the antisense strand has a structure represented by Formula A3, which, in the 5' to 3' direction, is: ASBSAOBOAOBOAOBOAOBOA-OBOAOBOAOBSASASASA formula A3; wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0038] In some embodiments, the sense strand has a structure represented by Formula V, where Formula V is, in the 5' to 3' direction: E-(A') m -CP 2 -F formula V; where E is a group represented by the formula (CP 1 )2; F is the formula DP 1 -CP 1 -C, DP 2 -CP 2 -C, DP 1 -CP 1 -D, or DP 2 -CP 2 - represented by D; A', C, D, P 1 , and P 2 is as defined in formula IV; m is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7).

[0039] In some embodiments, the sense strand has a structure represented by Formula S5, which, in the 5' to 3' direction, is: ASASAOBOAOBOAOBOAOBOA-OBOAOBSASA Formula S5; wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0040] In some embodiments, the sense strand has a structure represented by Formula S6, which, in the 5' to 3' direction, is: ASASAOBOAOBOAOBOAOBOA-OBOAOBOAOA Formula S6; wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0041] In some embodiments, the sense strand has a structure represented by Formula S7, which, in the 5' to 3' direction, is: ASASAOBOAOBOAOBOAOBOA-OBOAOBSASB formula S7; wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0042] In some embodiments, the sense strand has a structure represented by Formula S8, which, in the 5' to 3' direction, is: ASASAOBOAOBOAOBOAOBOA-OBOAOBOAOB formula S8; wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0043] In some embodiments, the antisense strand has a structure represented by Formula VI, which, in the 5' to 3' direction, is: AB j -EB k -EFG l -DP 1 -C' Formula VI; where A is a compound of formula CP 1 -DP 1 Represented by; Each B is a function of the formula CP 2 Represented by; 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 the formula DP 2 -CP 2 Represented by; F is the formula DP 1 -CP1 Represented by; Each G is a function of the formula CP 1 Represented by; 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); k is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7); l is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7).

[0044] In some embodiments, the antisense strand has a structure represented by Formula A4, which, in the 5' to 3' direction, is: ASBSAOAOAOBOAOAOAOAOA-OAOAOBOAOBSASASASBSA formula A4; wherein A represents a 2'-O-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 sense strand has a structure represented by Formula VII, which, in the 5' to 3' direction, is: HB m -I n -A'-B o -HC Formula VII; where A' is a compound of the formula CP 2 -DP 2 Represented by; Each H is a function of the formula (CP 1 )2; Each I independently represents a group of the formula (DP 2 ) is represented by; B, C, D, P 1 , and P 2is 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); o is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7).

[0046] In some embodiments, the sense strand has a structure represented by Formula S9, which, in the 5' to 3' direction, is: ASASAOAOAOBOBOBOBOAOBOA-OAOAOAASASA Formula S9; wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0047] In some embodiments, the antisense strand also has a 5' phosphorus stabilizing moiety at the 5' end of the antisense strand.

[0048] In some embodiments, the sense strand also has a 5' phosphorus stabilizing moiety at the 5' end of the sense strand.

[0049] In some embodiments, each 5' phosphorus stabilizing moiety is independently represented by any one of formulas IX, XX, XI, XII, XIII, XIV, XV, or XVI: [ka] wherein Nuc represents a nucleobase, optionally the nucleobase is selected from the group consisting of adenine, uracil, guanine, thymine, and cytosine, and R represents an optionally substituted alkyl, an optionally substituted alkenyl, an optionally substituted alkynyl, phenyl, benzyl, a cation (e.g., a monovalent cation), or hydrogen.

[0050] In some embodiments, the nucleobase is adenine, uracil, guanine, thymine, or cytosine.

[0051] In some embodiments, the 5' phosphorus stabilizing moiety is an (E)-vinyl phosphonate represented by formula XI.

[0052] In some embodiments, the siRNA molecule also has a hydrophobic portion at the 5' or 3' end of the siRNA molecule.

[0053] In some embodiments, the hydrophobic moiety is selected from the group consisting of cholesterol, vitamin D, or tocopherol.

[0054] In some embodiments, the siRNA molecule is a branched siRNA molecule.

[0055] In some embodiments, the branched siRNA molecule is biantennary, triantennary, or tetraantennary.

[0056] In some embodiments, the siRNA molecule is biantennary, and optionally, the biantennary siRNA molecule is represented by any one of Formulas XVII, XVIII, or XIX: [ka] wherein each RNA is independently an siRNA molecule, L is a linker, and each X independently represents a branch point moiety.

[0057] In some embodiments, the biantennary siRNA molecule is represented by Formula XVII. In some embodiments, the biantennary siRNA molecule is represented by Formula XVIII. In some embodiments, the biantennary siRNA molecule is represented by Formula XIX.

[0058] In some embodiments, the siRNA molecule is tri-antennary, and optionally, the tri-antennary siRNA molecule is represented by any one of Formulas XX, XXI, XXII, or XXIII: [ka] wherein each RNA is independently an siRNA molecule, L is a linker, and each X independently represents a branch point moiety.

[0059] In some embodiments, the three-antennary siRNA molecule is represented by formula XX. In some embodiments, the three-antennary siRNA molecule is represented by formula XXI. In some embodiments, the three-antennary siRNA molecule is represented by formula XXII. In some embodiments, the three-antennary siRNA molecule is represented by formula XXIII.

[0060] In some embodiments, the siRNA molecule is four-antennary, and optionally, the four-antennary siRNA molecule is represented by any one of Formulas XXIV, XXV, XXVI, XXVII, or XXVIII: [ka] wherein each RNA is independently an siRNA molecule, L is a linker, and each X independently represents a branch point moiety.

[0061] 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.

[0062] In some embodiments of the branched siRNA, the linker is selected from the group consisting of one or more consecutive subunits of ethylene glycol (e.g., polyethylene glycol (PEG), e.g., triethylene glycol (TrEG) or tetraethylene glycol (TEG)), alkyl, carbohydrate, block copolymer, peptide, RNA, and DNA.

[0063] 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.

[0064] In some embodiments, the ethylene glycol oligomer is PEG. In some embodiments, the PEG is TrEG. In some embodiments, the PEG is TEG.

[0065] 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).

[0066] In some embodiments, the linker connects one or more (eg, 1, 2, 3, 4, or more) siRNA molecules via a covalent bond-forming moiety.

[0067] 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 formacetal.

[0068] In some embodiments, the linker comprises a structure of formula L1: [ka]

[0069] In some embodiments, the linker comprises a structure of formula L2: [ka]

[0070] In some embodiments, the linker comprises a structure of formula L3: [ka]

[0071] In some embodiments, the linker comprises a structure of formula L4: [ka]

[0072] In some embodiments, the linker comprises a structure of formula L5: [ka]

[0073] In some embodiments, the linker comprises a structure of formula L6: [ka]

[0074] In some embodiments, the linker comprises a structure of formula L7: [ka]

[0075] In some embodiments, the linker comprises a structure of formula L8: [ka]

[0076] In some embodiments, the linker comprises a structure of formula L9: [ka]

[0077] 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%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151 %, 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% can be 2'-O-Me ribonucleotides).

[0078] 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).

[0079] 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 can be 2'-O-Me ribonucleotides).

[0080] 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 can be 2'-O-Me ribonucleotides).

[0081] 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 can be 2'-O-Me ribonucleotides).

[0082] In some embodiments, 10% or less of the internucleoside linkages are phosphodiester or phosphorothioate linkages. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the internucleoside linkages are phosphodiester or phosphorothioate linkages. In some embodiments, 100% of the internucleoside linkages are phosphodiester or phosphorothioate linkages.

[0083] In some embodiments, 9 of the internucleoside linkages are phosphodiester or phosphorothioate linkages.

[0084] In some embodiments, the length of the antisense strand is between 10 and 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), between 15 and 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 between 18 and 23 nucleotides (e.g., 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, or 23 nucleotides). In some embodiments, the length of the antisense strand is 20 nucleotides. In some embodiments, the length of the antisense strand is 21 nucleotides. In some embodiments, the length of the antisense strand is 22 nucleotides. In some embodiments, the length of the antisense strand is 23 nucleotides. In some embodiments, the length of the antisense strand is 24 nucleotides. In some embodiments, the length of the antisense strand is 25 nucleotides. In some embodiments, the length of the antisense strand is 26 nucleotides. In some embodiments, the length of the antisense strand is 27 nucleotides. In some embodiments, the length of the antisense strand is 28 nucleotides. In some embodiments, the length of the antisense strand is 29 nucleotides. In some embodiments, the length of the antisense strand is 30 nucleotides.

[0085] In some embodiments, the siRNA molecules of the branched compound are linked to each other by a linker (e.g., an ethylene glycol oligomer such as tetraethylene glycol). In some embodiments, the siRNA molecules of the branched compound are linked to each other via a linker between the sense strand of one siRNA molecule and the sense strand of another siRNA molecule. In some embodiments, the siRNA molecules are linked to each other via a linker 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 compound are linked to each other via a linker between the sense strand of one siRNA molecule and the antisense strand of another siRNA molecule.

[0086] In some embodiments, the length of the sense strand is between 12 and 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 between 14 and 18 nucleotides (e.g., 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, or 18 nucleotides). In some embodiments, the length of the sense strand is 15 nucleotides. In some embodiments, the length of the sense strand is 16 nucleotides. In some embodiments, the length of the sense strand is 17 nucleotides. In some embodiments, the length of the sense strand is 18 nucleotides. In some embodiments, the length of the sense strand is 19 nucleotides. In some embodiments, the length of the sense strand is 20 nucleotides. In some embodiments, the length of the sense strand is 21 nucleotides. In some embodiments, the length of the sense strand is 22 nucleotides. 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.

[0087] In some embodiments, the four internucleoside linkages are phosphorothioate linkages.

[0088] In some embodiments of the siRNA molecules described herein, the antisense strand is 18 nucleotides in length and the sense strand is 14 nucleotides in length. In some embodiments, the antisense strand is 18 nucleotides in length and the sense strand is 15 nucleotides in length. In some embodiments, the antisense strand is 18 nucleotides in length and the sense strand is 16 nucleotides in length. In some embodiments, the antisense strand is 18 nucleotides in length and the sense strand is 17 nucleotides in length. In some embodiments, the antisense strand is 18 nucleotides in length and the sense strand is 18 nucleotides in length. In some embodiments, the antisense strand is 19 nucleotides in length and the sense strand is 14 nucleotides in length. In some embodiments, the antisense strand is 19 nucleotides in length and the sense strand is 15 nucleotides in length. In some embodiments, the antisense strand is 19 nucleotides in length and the sense strand is 16 nucleotides in length. In some embodiments, the antisense strand is 19 nucleotides in length and the sense strand is 17 nucleotides in length. In some embodiments, the antisense strand is 19 nucleotides in length and the sense strand is 18 nucleotides in length. In some embodiments, the antisense strand is 19 nucleotides long and the sense strand is 19 nucleotides long. In some embodiments, the antisense strand is 20 nucleotides long and the sense strand is 14 nucleotides long. In some embodiments, the antisense strand is 20 nucleotides long and the sense strand is 15 nucleotides long. In some embodiments, the antisense strand is 20 nucleotides long and the sense strand is 16 nucleotides long. In some embodiments, the antisense strand is 20 nucleotides long and the sense strand is 17 nucleotides long. In some embodiments, the antisense strand is 20 nucleotides long and the sense strand is 18 nucleotides long. In some embodiments, the antisense strand is 20 nucleotides long and the sense strand is 19 nucleotides long. In some embodiments, the antisense strand is 20 nucleotides long and the sense strand is 20 nucleotides long.In some embodiments, the antisense strand is 21 nucleotides long and the sense strand is 14 nucleotides long. In some embodiments, the antisense strand is 21 nucleotides long and the sense strand is 15 nucleotides long. In some embodiments, the antisense strand is 21 nucleotides long and the sense strand is 16 nucleotides long. In some embodiments, the antisense strand is 21 nucleotides long and the sense strand is 17 nucleotides long. In some embodiments, the antisense strand is 21 nucleotides long and the sense strand is 18 nucleotides long. In some embodiments, the antisense strand is 21 nucleotides long and the sense strand is 19 nucleotides long. In some embodiments, the antisense strand is 21 nucleotides long and the sense strand is 20 nucleotides long. In some embodiments, the antisense strand is 21 nucleotides long and the sense strand is 21 nucleotides long. In some embodiments, the antisense strand is 22 nucleotides long and the sense strand is 14 nucleotides long. In some embodiments, the antisense strand is 22 nucleotides long and the sense strand is 15 nucleotides long. In some embodiments, the antisense strand is 22 nucleotides long and the sense strand is 16 nucleotides long. In some embodiments, the antisense strand is 22 nucleotides long and the sense strand is 17 nucleotides long. In some embodiments, the antisense strand is 22 nucleotides long and the sense strand is 18 nucleotides long. In some embodiments, the antisense strand is 22 nucleotides long and the sense strand is 19 nucleotides long. In some embodiments, the antisense strand is 22 nucleotides long and the sense strand is 20 nucleotides long. In some embodiments, the antisense strand is 22 nucleotides long and the sense strand is 21 nucleotides long. In some embodiments, the antisense strand is 22 nucleotides long and the sense strand is 22 nucleotides long. In some embodiments, the antisense strand is 23 nucleotides long and the sense strand is 14 nucleotides long.In some embodiments, the antisense strand is 23 nucleotides long and the sense strand is 15 nucleotides long. In some embodiments, the antisense strand is 23 nucleotides long and the sense strand is 16 nucleotides long. In some embodiments, the antisense strand is 23 nucleotides long and the sense strand is 17 nucleotides long. In some embodiments, the antisense strand is 23 nucleotides long and the sense strand is 18 nucleotides long. In some embodiments, the antisense strand is 23 nucleotides long and the sense strand is 19 nucleotides long. In some embodiments, the antisense strand is 23 nucleotides long and the sense strand is 20 nucleotides long. In some embodiments, the antisense strand is 23 nucleotides long and the sense strand is 21 nucleotides long. In some embodiments, the antisense strand is 23 nucleotides long and the sense strand is 22 nucleotides long. In some embodiments, the antisense strand is 23 nucleotides long and the sense strand is 23 nucleotides long. In some embodiments, the antisense strand is 24 nucleotides long and the sense strand is 14 nucleotides long. In some embodiments, the antisense strand is 24 nucleotides long and the sense strand is 15 nucleotides long. In some embodiments, the antisense strand is 24 nucleotides long and the sense strand is 16 nucleotides long. In some embodiments, the antisense strand is 24 nucleotides long and the sense strand is 17 nucleotides long. In some embodiments, the antisense strand is 24 nucleotides long and the sense strand is 18 nucleotides long. In some embodiments, the antisense strand is 24 nucleotides long and the sense strand is 19 nucleotides long. In some embodiments, the antisense strand is 24 nucleotides long and the sense strand is 20 nucleotides long. In some embodiments, the antisense strand is 24 nucleotides long and the sense strand is 21 nucleotides long. In some embodiments, the antisense strand is 24 nucleotides long and the sense strand is 22 nucleotides long.In some embodiments, the antisense strand is 24 nucleotides long and the sense strand is 23 nucleotides long. In some embodiments, the antisense strand is 24 nucleotides long and the sense strand is 24 nucleotides long. In some embodiments, the antisense strand is 25 nucleotides long and the sense strand is 14 nucleotides long. In some embodiments, the antisense strand is 25 nucleotides long and the sense strand is 15 nucleotides long. In some embodiments, the antisense strand is 25 nucleotides long and the sense strand is 16 nucleotides long. In some embodiments, the antisense strand is 25 nucleotides long and the sense strand is 17 nucleotides long. In some embodiments, the antisense strand is 25 nucleotides long and the sense strand is 18 nucleotides long. In some embodiments, the antisense strand is 25 nucleotides long and the sense strand is 19 nucleotides long. In some embodiments, the antisense strand is 25 nucleotides long and the sense strand is 20 nucleotides long. In some embodiments, the antisense strand is 25 nucleotides long and the sense strand is 21 nucleotides long. In some embodiments, the antisense strand is 25 nucleotides long and the sense strand is 22 nucleotides long. In some embodiments, the antisense strand is 25 nucleotides long and the sense strand is 23 nucleotides long. In some embodiments, the antisense strand is 25 nucleotides long and the sense strand is 24 nucleotides long. In some embodiments, the antisense strand is 25 nucleotides long and the sense strand is 25 nucleotides long. In some embodiments, the antisense strand is 26 nucleotides long and the sense strand is 14 nucleotides long. In some embodiments, the antisense strand is 26 nucleotides long and the sense strand is 15 nucleotides long. In some embodiments, the antisense strand is 26 nucleotides long and the sense strand is 16 nucleotides long. In some embodiments, the antisense strand is 26 nucleotides long and the sense strand is 17 nucleotides long.In some embodiments, the antisense strand is 26 nucleotides in length and the sense strand is 18 nucleotides in length. In some embodiments, the antisense strand is 26 nucleotides in length and the sense strand is 19 nucleotides in length. In some embodiments, the antisense strand is 26 nucleotides in length and the sense strand is 20 nucleotides in length. In some embodiments, the antisense strand is 26 nucleotides in length and the sense strand is 21 nucleotides in length. In some embodiments, the antisense strand is 26 nucleotides in length. The sense strand is 22 nucleotides in length. In some embodiments, the antisense strand is 26 nucleotides in length and the sense strand is 23 nucleotides in length. In some embodiments, the antisense strand is 26 nucleotides in length and the sense strand is 24 nucleotides in length. In some embodiments, the antisense strand is 26 nucleotides in length and the sense strand is 25 nucleotides in length. In some embodiments, the antisense strand is 26 nucleotides in length and the sense strand is 26 nucleotides in length. In some embodiments, the antisense strand is 27 nucleotides in length and the sense strand is 14 nucleotides in length. In some embodiments, the antisense strand is 27 nucleotides in length and the sense strand is 15 nucleotides in length. In some embodiments, the antisense strand is 27 nucleotides in length and the sense strand is 16 nucleotides in length. In some embodiments, the antisense strand is 27 nucleotides in length and the sense strand is 17 nucleotides in length. In some embodiments, the antisense strand is 27 nucleotides in length and the sense strand is 18 nucleotides in length. In some embodiments, the antisense strand is 27 nucleotides long and the sense strand is 19 nucleotides long. In some embodiments, the antisense strand is 27 nucleotides long and the sense strand is 20 nucleotides long. In some embodiments, the antisense strand is 27 nucleotides long and the sense strand is 21 nucleotides long. In some embodiments, the antisense strand is 27 nucleotides long and the sense strand is 22 nucleotides long. In some embodiments, the antisense strand is 27 nucleotides long and the sense strand is 23 nucleotides long. In some embodiments, the antisense strand is 27 nucleotides long and the sense strand is 24 nucleotides long. In some embodiments, the antisense strand is 27 nucleotides long and the sense strand is 25 nucleotides long. In some embodiments, the antisense strand is 27 nucleotides long and the sense strand is 26 nucleotides long. In some embodiments, the antisense strand is 27 nucleotides long and the sense strand is 27 nucleotides long.In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 14 nucleotides long. In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 15 nucleotides long. In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 16 nucleotides long. In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 17 nucleotides long. In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 18 nucleotides long. In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 19 nucleotides long. In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 20 nucleotides long. In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 21 nucleotides long. In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 22 nucleotides long. In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 23 nucleotides long. In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 24 nucleotides long. In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 25 nucleotides long. In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 26 nucleotides long. In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 27 nucleotides long. In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 28 nucleotides long. In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 14 nucleotides long. In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 15 nucleotides long. In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 16 nucleotides long.In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 17 nucleotides long. In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 18 nucleotides long. In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 19 nucleotides long. In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 20 nucleotides long. In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 21 nucleotides long. In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 22 nucleotides long. In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 23 nucleotides long. In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 24 nucleotides long. In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 25 nucleotides long. In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 26 nucleotides long. In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 27 nucleotides long. In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 28 nucleotides long. In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 29 nucleotides long. In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 14 nucleotides long. In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 15 nucleotides long. In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 16 nucleotides long. In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 17 nucleotides long. In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 18 nucleotides long.In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 19 nucleotides long. In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 20 nucleotides long. In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 21 nucleotides long. In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 22 nucleotides long. In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 23 nucleotides long. In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 24 nucleotides long. In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 25 nucleotides long. In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 26 nucleotides long. In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 27 nucleotides long. In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 28 nucleotides long. 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.

[0089] In a further aspect, the present disclosure provides a pharmaceutical composition comprising an siRNA molecule of any of the preceding aspects or embodiments of the present disclosure and a pharmaceutically acceptable excipient, carrier, or diluent.

[0090] In a further aspect, the present disclosure provides a method of delivering an siRNA molecule to a subject diagnosed with a neurodegenerative disease by administering to the subject a therapeutically effective amount of the siRNA molecule or a pharmaceutical composition of any of the above-mentioned aspects or embodiments of the present disclosure.

[0091] In a further aspect, the present disclosure provides a method of treating a neurodegenerative disease in a subject in need thereof by administering to the subject a therapeutically effective amount of an siRNA molecule or a pharmaceutical composition of any of the above-mentioned aspects or embodiments of the present disclosure.

[0092] In some embodiments of the methods described herein, the neurodegenerative disease is amyotrophic lateral sclerosis (ALS). In some embodiments of the methods described herein, the neurodegenerative disease is frontotemporal dementia (FTD).

[0093] In another aspect, the present disclosure provides a method of reducing TARDBP expression in a subject in need thereof by administering a therapeutically effective amount of an siRNA or pharmaceutical composition of any of the above-mentioned aspects or embodiments of the present disclosure.

[0094] In some embodiments of the methods described herein, siRNA molecule or pharmaceutical composition is administered to subject by intracerebroventricular, intrastriatal, intraparenchymal or intrathecal injection.In some embodiments, siRNA molecule or pharmaceutical composition is administered to subject by intravenous, intramuscular or subcutaneous injection. In some embodiments of any of the methods described herein, the subject is a human.

[0095] In another aspect, the present disclosure provides a kit comprising an siRNA molecule or pharmaceutical composition of any of the above aspects or embodiments of the present disclosure and a package insert instructing a user of the kit to perform a method of any of the above aspects or embodiments of the present disclosure.

[0096] definition Unless otherwise defined herein, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or extrinsic definitions. Unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. The use of "or" means "and / or" unless otherwise stated. The use of the term "including" and other forms such as "include" and "included" is not limiting.

[0097] As used herein, the term "nucleic acid" refers to an RNA or DNA molecule composed of a chain of ribonucleotides or deoxyribonucleotides, respectively.

[0098] As used herein, the term "therapeutic nucleic acid" refers to a nucleic acid molecule (e.g., ribonucleic acid) that has partial or complete complementarity with, interacts with, and mediates silencing of expression of a disease-associated target mRNA.

[0099] As used herein, the term "carrier nucleic acid" refers to a nucleic acid molecule (e.g., a ribonucleic acid) that has sequence complementarity with and hybridizes to a therapeutic nucleic acid. As used herein, the term "3' end" refers to the end of a nucleic acid that contains an unmodified hydroxyl group on the 3' carbon of the ribose ring.

[0100] As used herein, the term "nucleoside" refers to a molecule consisting of a heterocyclic base and its sugar.

[0101] As used herein, the term "nucleotide" refers to a nucleoside having a phosphate group or a variant thereof at 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.

[0102] In the context of this invention, the term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This 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 that function similarly. Such modified or substituted oligonucleotides are often preferred over native forms due to desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid targets, and increased stability in the presence of nucleases.

[0103] As used herein, the term "siRNA" refers to a small interfering RNA duplex that induces the RNA interference (RNAi) pathway. siRNA molecules can be of various lengths (generally 10-30 base pairs) and contain various degrees of complementarity to their target mRNA. The term "siRNA" includes duplexes of two separate strands, and, optionally, single strands that form a hairpin structure containing the double-stranded region.

[0104] As used herein, the term "antisense strand" refers to the strand of the siRNA duplex that contains a degree of complementarity to a target gene.

[0105] As used herein, the term "sense strand" refers to the siRNA duplex that contains complementarity to the antisense strand.

[0106] The term "interfering RNA molecule" refers to an RNA molecule, such as a small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), or antisense oligonucleotide (ASO), that suppresses the endogenous function of a target RNA transcript.

[0107] As used herein, the terms "express" and "expression" refer 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 the RNA transcript (e.g., by splicing, editing, 5' capping, and / or 3' end processing); and (3) the translation of the RNA into a polypeptide or protein. In the context of a gene encoding a protein product, terms such as "gene expression" are used interchangeably with terms such as "protein expression." Expression of a gene or protein of interest in a patient can be determined, 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 RNA detection procedures described herein or known in the art, such as quantitative polymerase chain reaction (qPCR) and RNA-seq techniques), an increase in the amount or concentration of the corresponding protein (e.g., assessed using protein detection methods described herein or known in the art, such as enzyme-linked immunosorbent assay (ELISA), among others), and / or an increase in the activity of the corresponding protein (e.g., assessed using enzyme activity assays described herein or known in the art, in the case of an enzyme). 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 are detectable within the cell or the medium in which the cell resides. For example, a gene or protein of interest is considered to be "expressed" by a cell, or population of cells, if it is possible to detect (i) the production of a corresponding RNA transcript, such as an mRNA template, by the cell, or population of cells (e.g., using the RNA detection procedures described herein); (ii) processing of the RNA transcript (e.g., splicing, editing, 5' capping, and / or 3' end processing, using the RNA detection procedures described herein); (iii) translation of the RNA template into a protein product (e.g., using the protein detection procedures described herein); and / or (iv) post-translational modification of the protein product (e.g., using the protein detection procedures described herein).

[0108] As used herein, the terms "target," "targeting," and "targeted" in the context of siRNA design refer to generating an antisense strand such that it anneals within a region within an mRNA transcript of interest in a manner that reduces translation of the mRNA into a protein product.

[0109] As used herein, the terms "chemically modified nucleotide," "nucleotide analog," "altered nucleotide," and "modified nucleotide" refer to non-standard nucleotides, including non-naturally occurring ribonucleotides or deoxyribonucleotides. Exemplary nucleotide analogs are modified at any position such that certain chemical properties of the nucleotide are altered while retaining the ability of the nucleotide analog to perform its intended function.

[0110] As used herein, the term "metabolically stabilized" refers to an RNA molecule containing chemically modified ribonucleotides to reduce the metabolic rate of the RNA molecule administered to a subject. Exemplary modifications include 2'-hydroxy to 2'-O-methoxy or 2'-fluoro, and phosphodiester to phosphorothioate.

[0111] As used herein, the term "phosphorothioate" refers to a phosphate group of a nucleotide that is modified by replacing one or more of the oxygens of the phosphate group with sulfur.

[0112] As used herein, the terms "internucleoside" and "internucleotide" refer to the linkage between a nucleoside and a nucleotide, respectively.

[0113] As used herein, the term "antagomir" refers to a nucleic acid that can function as an inhibitor of miRNA activity.

[0114] As used herein, the term "gapmer" refers to a chimeric antisense nucleic acid containing a central block of deoxynucleotide monomers of sufficient length to induce RNase H cleavage. The deoxynucleotide block is flanked by ribonucleotide monomers or ribonucleotide monomers containing modifications.

[0115] As used herein, the term "mixmer" refers to a nucleic acid composed of a mixture of locked nucleic acid (LNA) and DNA.

[0116] As used herein, the term "guide RNA" refers to a nucleic acid that has sequence complementarity to a specific sequence in the genome immediately upstream or one base pair upstream of the protospacer adjacent motif (PAM) sequence used in the CRISPR / Cas9 gene editing system. Alternatively, "guide RNA" may refer to a nucleic acid that has sequence complementarity (e.g., is antisense) to a specific messenger RNA (mRNA) sequence. In this context, a guide RNA may also have sequence complementarity to a "passenger RNA" sequence of equal or shorter length that is identical or substantially identical to the sequence of the mRNA to which the guide RNA hybridizes.

[0117] As used herein, the term " branched siRNA " refers to a compound that contains two or more double-stranded siRNA molecules that are covalently linked to each other.Branched siRNA molecule can be " bibranched ", also referred to herein as " di-siRNA ", and this siRNA molecule comprises two siRNA molecules that are covalently linked to each other, for example, via a linker.Branched siRNA molecule can be " tribranched ", also referred to herein as " tri-siRNA ", and this siRNA molecule comprises three siRNA molecules that are covalently linked to each other, for example, via a linker.Branched siRNA molecule can be " four-branched ", also referred to herein as " tetra-siRNA ", and this siRNA molecule comprises four siRNA molecules that are covalently linked to each other, for example, via a linker.

[0118] As used herein, the term "branch point moiety" refers to the chemical moiety of the branched siRNA structure of the present disclosure, which can be covalently linked to the 5'-end or 3'-end of the antisense or sense strand of siRNA molecule, and can support the binding of additional single-stranded or double-stranded siRNA molecules.Non-limiting examples of branch point moieties suitable for use in conjunction with the disclosed methods and compositions include, for example, phosphoramidite, tosylated solketal, 1,3-diaminopropanol, pentaerythritol, and any one of the branch point moieties described in US Patent No. 10,478,503.

[0119] As used herein, the term "phosphate moiety" refers to a terminal phosphate group, including phosphate and modified phosphates. The phosphate moiety can be located at either terminus, but is preferred at the 5'-terminal nucleoside. In one aspect, the terminal phosphate has the formula -OP(=O)(OH)OH and is unmodified. In another aspect, the terminal phosphate is modified such that one or more of the O and OH groups are replaced with H, O, S, N(R'), or an alkyl where R' is H, an amino protecting group, or an unsubstituted or substituted alkyl. In some embodiments, the 5'- and / or 3'-terminal groups may each independently contain one to three phosphate moieties that are unmodified (diphosphate or triphosphate) or modified.

[0120] As used herein, the term "5' phosphorus stabilizing moiety" refers to a terminal phosphate group, including phosphate and modified phosphates (e.g., phosphorothioates, phosphodiesters, phosphonates). The phosphate moiety can be located at either terminus, but is preferred at the 5'-terminal nucleoside. In one aspect, the terminal phosphate has the formula -OP(=O)(OH)OH and is unmodified. In another aspect, the terminal phosphate is modified such that one or more of the O and OH groups are replaced with H, O, S, N(R'), or an alkyl where R' is H, an amino protecting group, or an unsubstituted or substituted alkyl. In some embodiments, the 5' and / or 3' terminal groups may each independently contain one to three phosphate moieties that are unmodified (diphosphate or triphosphate) or modified.

[0121] The phosphate group of a nucleotide may also be modified, for example, by substituting one or more oxygen atoms of the phosphate group with sulfur (e.g., phosphorothioate) or by creating other substitutions that allow the nucleotide to perform its intended function, as described, for example, in 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-referenced modifications (e.g., phosphate group modifications) preferably reduce the hydrolysis rate of polynucleotides containing the analogs in vivo or in vitro, for example.

[0122] As used herein, the term "complementary" refers to two nucleotides that form a canonical Watson-Crick base pair. For the avoidance of doubt, in the context of the present disclosure, Watson-Crick base pairs include adenine-thymine, adenine-uracil, and cytosine-guanine base pairs. In this context, proper Watson-Crick base pairs are referred to as "matches," while unpaired and improperly paired nucleotides are referred to as "mismatches." Alignment for purposes of determining percent nucleic acid sequence complementarity can be achieved in a variety of 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.

[0123] "Percent sequence complementarity (%)" to a reference polynucleotide sequence is defined as the percentage of nucleic acids in a candidate sequence that are complementary to nucleic acids in the reference polynucleotide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence complementarity. A given nucleotide is considered "complementary" to a reference nucleotide, as described herein, if the two nucleotides form a standard Watson-Crick base pair. For the avoidance of doubt, in the context of the present disclosure, Watson-Crick base pairs include adenine-thymine, adenine-uracil, and cytosine-guanine base pairs. In this context, proper Watson-Crick base pairs are referred to as "matches," while unpaired and improperly paired nucleotides are referred to as "mismatches." Alignment for purposes of determining percent nucleic acid sequence complementarity can be achieved in a variety of 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 algorithms needed to achieve maximal complementarity over the entire length of the sequences being compared. By way of illustration, the percent sequence complementarity of a given nucleic acid sequence A to a given nucleic acid sequence B (which can alternatively be referred to as a given nucleic acid sequence A having a certain percent complementarity to a given nucleic acid sequence B) is calculated as follows: 100×(fraction X / Y) where X is the number of complementary base pairs in a programmed alignment of A and B (e.g., as performed by computer software such as BLAST), and Y is the total number of nucleic acids in B. It is understood that if the length of nucleic acid sequence A is not equal to the length of nucleic acid sequence B, then the percent sequence complementarity 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 the query nucleic acid sequence has 100% sequence complementarity to the reference nucleic acid sequence.

[0124] "Percent sequence identity (%)" with respect to a reference polynucleotide or reference polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to the nucleic acids or amino acids in the reference polynucleotide or reference polypeptide sequence, after aligning the sequences to achieve the maximum percent sequence identity and introducing gaps as necessary. Alignment for determining percent sequence identity of nucleic acids or amino acids can be achieved in a variety of 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 appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment across the entire length of the sequences being compared. For example, percent sequence identity values ​​can be generated using the sequence comparison computer program BLAST. As an illustration, the percent sequence identity of a given nucleic acid or amino acid sequence A to, with, or against a given nucleic acid or amino acid sequence B (which can alternatively be referred to as a given nucleic acid or amino acid sequence A having a certain percent sequence identity to, with, or against a given nucleic acid or amino acid sequence B) is calculated as follows: 100×(fraction X / Y) where X is the number of nucleotides or amino acids scored as identical matches by a sequence alignment program (e.g., BLAST) in a programmatic alignment of A and B, and Y is the total number of nucleic acids in B. If the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, then the percent sequence identity of A to B will not be considered equal to the percent sequence identity of B to A.

[0125] As used herein, the term "sufficiently complementary to hybridize" refers to a nucleic acid sequence or portion thereof that does not need to be perfectly complementary (e.g., 100% complementary) to a target region or nucleic acid sequence or portion thereof, that has one or more nucleotide mismatches with the target region but can still hybridize to the 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 form sufficient base pairs with the target to hybridize along its entire length.

[0126] Nucleic acid "hybridization" or "annealing" occurs when one or more nucleoside residues within a polynucleotide base pair with one or more complementary nucleosides to form a stable duplex. Base pairing is typically mediated by hydrogen bonding events. Hybridization can include Watson-Crick base pairs formed from natural and / or modified nucleobases. Hybridization can also involve non-Watson-Crick base pairs, such as wobble base pairs (guanosine-uracil, hypoxanthine-uracil, hypoxanthine-adenine, and hypoxanthine-cytosine) and Hoogsteen base pairs. Nucleic acids do not need to be 100% complementary to undergo hybridization. For example, one nucleic acid can be, e.g., 95% complementary, 90% complementary, 85% complementary, 80% complementary, 75% complementary, 70% complementary, 65% complementary, 60% complementary, 55% complementary, 50% complementary, or less, to another nucleic acid, yet the two nucleic acids can form sufficient base pairs with each other to hybridize.

[0127] A "stable duplex" formed upon annealing / hybridization of one nucleic acid to another is a duplex structure that is not denatured by harsh washing. Exemplary stringent washing conditions are known in the art and include a temperature about 5°C below the melting temperature of each strand of the duplex and a low monovalent salt concentration, e.g., a monovalent salt concentration (e.g., NaCl concentration) of less than 0.2M (e.g., 0.2M, 0.19M, 0.18M, ​​0.17M, 0.16M, 0.15M, 0.14M, 0.13M, 0.12M, 0.11M, 0.1M, 0.09M, 0.08M, 0.07M, 0.06M, 0.05M, 0.04M, 0.03M, 0.02M, 0.01M, or lower).

[0128] The term "gene silencing" refers to the suppression of gene expression (e.g., endogenous gene expression of TARDBP), which can be mediated through processes that affect transcription and / or 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 gene of interest in a sequence-specific manner by RNA interference, thereby preventing translation of the gene's product.

[0129] As used herein, the term "overactive disease driver gene" refers to a gene with increased activity and / or expression that contributes to or causes a disease state in a subject (e.g., a human). The disease state may be caused or exacerbated by the overactive disease driver gene directly or through an intermediary gene(s).

[0130] As used herein, the term "ethylene glycol chain" refers to a carbon chain having the formula ((CH2OH)2).

[0131] As used herein, "alkyl" refers to a saturated hydrocarbon group. An alkyl group can be acyclic or cyclic and, if unsubstituted, contains only C and H. When an alkyl residue having a specific number of carbon atoms is named, all geometric isomers having that number of carbon atoms are intended to be encompassed and described. Thus, for example, "butyl" is meant to include n-butyl, sec-butyl, and iso-butyl. Examples of alkyl include ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like. In some embodiments, alkyl can be substituted. Suitable substituents that can be introduced into alkyl groups include, for example, hydroxy, alkoxy, amino, alkylamino, and halo, among others.

[0132] As used herein, "alkenyl" refers to an acyclic or cyclic unsaturated hydrocarbon group 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 specific number of carbons is named, all geometric isomers having that number of carbons are intended to be encompassed and described. Thus, for example, "butenyl" is meant to include n-butenyl, sec-butenyl, and iso-butenyl. Examples of alkenyl include -CH=CH, -CH-CH=CH, and -CH-CH=CH-CH=CH. In some embodiments, alkenyl can be substituted. Suitable substituents that can be introduced into alkenyl groups include, for example, hydroxy, alkoxy, amino, alkylamino, and halo, among others.

[0133] As used herein, "alkynyl" refers to an acyclic or cyclic unsaturated hydrocarbon group 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 an alkynyl residue having a specific number of carbons is named, all geometric isomers having that number of carbons are intended to be encompassed and described. Thus, for example, "pentynyl" is meant to include n-pentynyl, sec-pentynyl, and iso-pentynyl. Examples of alkynyl include -C≡CH and -C≡C-CH3. In some embodiments, alkynyl can be substituted. Suitable substituents that can be introduced into alkynyl groups include, for example, hydroxy, alkoxy, amino, alkylamino, and halo, among others.

[0134] As used herein, the term "phenyl" refers to a monocyclic arene in which one hydrogen atom has been removed from a carbon atom of the ring. Phenyl groups can be unsubstituted or substituted with one or more suitable substituents, which replace the H of the phenyl group.

[0135] As used herein, the term "benzyl" refers to the monovalent radical obtained when the hydrogen atom attached to the methyl group of toluene is removed. A benzyl group generally has the formula phenyl-CH2-. A benzyl group can be unsubstituted or substituted with one or more suitable substituents. For example, a substituent may replace an H in the phenyl moiety and / or an H in the methylene (-CH2-) moiety.

[0136] As used herein, the term "amide" refers to an alkyl, alkenyl, alkynyl, or aromatic group attached to an aminocarbonyl functional group.

[0137] As used herein, the term "triazole" refers to a heterocyclic compound having the formula (C2H3N3) that has a five-membered ring of two carbons and three nitrogens, the positions of which can vary to give rise to multiple isomers.

[0138] As used herein, the term "end group" refers to the group at which a carbon chain or nucleic acid ends.

[0139] As used herein, "amino acid" refers to a molecule containing an amine and a carboxyl functional group as well as a side chain characteristic of an amino acid.

[0140] In some embodiments, the amino acid is selected from the group of 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 beta-amino acid).

[0141] As used herein, the term "lipophilic amino acid" refers to an amino acid that contains a hydrophobic moiety (eg, an alkyl chain or an aromatic ring).

[0142] As used herein, the term "delivery target" refers to an organ or part of the body to which it is desired to deliver a branched oligonucleotide composition.

[0143] As used herein, the term "X to Y" is inclusive of values ​​of X and Y. For example, "X to Y" refers to a range of values ​​between the value of X and the value of Y, as well as the value of X and the value of Y.

[0144] As used herein, the terms "subject" and "patient" are used interchangeably and refer to an organism (e.g., a mammal (e.g., a human)) experiencing a neurodegenerative disease or disorder (e.g., amyotrophic lateral sclerosis with frontotemporal dementia) and / or containing a gain-of-function TARDBP variant allele.

[0145] As used herein, the term "TARDBP" refers to a gene encoding transactive response DNA-binding protein 43 kDa (TDP-43), including any native TARDBP gene from any source. The term encompasses "full-length" unprocessed TARDBP and any form of TARDBP resulting from processing within a cell. The term also encompasses natural variants of TARDBP (e.g., splice variants or allelic variants). The nucleic acid sequence of an exemplary TARDBP gene is set forth in European Nucleotide Archive (ENA) Accession No. U23731.1. The amino acid sequence of an exemplary protein encoded by a TARDBP gene is set forth in UNIPROT™ Accession No. Q13148.

[0146] As used herein, the terms "neuroinflammatory disease" and "neuroinflammatory disorder" are used interchangeably to refer to any condition caused in any way by neuroinflammation. "Neuroinflammation" refers to various immune responses in the central nervous system (e.g., microglia). Neuroinflammation can originate in the brain or be the result of a systemic inflammatory response.

[0147] As used herein, the terms "neurodegenerative disease" and "neurodegenerative disorder" are used interchangeably to refer to any condition caused in any way by the loss of function or death of cells in the central or peripheral nervous system. Exemplary neurodegenerative diseases are Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, frontotemporal dementia, and spinocerebellar ataxia.

[0148] As used herein, the terms "treat," "treated," and "treating" refer to both therapeutic treatment and prophylactic or preventative measures, the purpose of which is to prevent, ameliorate, or slow (alleviate) 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, reducing a patient's dependence on drug treatment; alleviating symptoms; reducing the severity of a condition, disorder, or disease; stabilizing (i.e., not worsening) a condition, disorder, or disease, delaying or slowing the progression of a condition, disorder, or disease; improving or remission (whether partial or complete) of a condition, disorder, or disease, whether detectable or undetectable; improving at least one measurable physical, cognitive, or behavioral (e.g., depressive behavior or apathy) parameter, not necessarily recognized by the patient; or improving or ameliorating a condition, disorder, or disease. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment.

[0149] As used herein, the terms "benefit" and "response" are used interchangeably in the context of a subject undergoing therapy for the treatment of a neurodegenerative disease (e.g., amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD)). For example, clinical benefits associated with an ALS subject administered an siRNA molecule or siRNA composition of the present disclosure include, but are not limited to, a reduction in involuntary movements, muscle spasms, weakness, and loss of motor control. As a further example, clinical benefits associated with a subject with FTD administered an siRNA molecule or siRNA composition of the present disclosure include, but are not limited to, a reduction in memory impairment, behavioral impairment, and language impairment. "Benefit" and "response" are used interchangeably to refer, for example, to a reduction in wild-type TARDBP transcripts, mutant TARDBP transcripts, variant TARDBP transcripts, splice isoforms of TARDBP transcripts, and / or overexpressed TARDBP transcripts. DETAILED DESCRIPTION OF THE INVENTION

[0150] The present invention provides compositions of small interfering RNA (siRNA) molecules having sequence homology with the TAR DNA-binding protein-43 (TARDBP) gene, and methods for administering the siRNA molecules to subjects. Furthermore, the siRNA molecules described herein may be configured as branched siRNA structures, such as biantennary, triantennary, and tetraantennary siRNA structures, and may further contain specific patterns of chemical modifications (e.g., 2' ribose modifications or internucleoside linkage modifications) to improve resistance to nuclease enzymes, toxicity profiles, and physicochemical properties (e.g., thermal stability). Small interfering RNA molecules are short double-stranded RNA molecules. They mediate RNA interference (RNAi) by degrading mRNAs with complementary nucleotide sequences, thereby preventing the translation of target genes.

[0151] The siRNA molecules of the present invention may exhibit potent gene-specific suppression of TARDBP, for example, relative to other human genes.

[0152] The siRNA molecules of the present disclosure may feature an antisense strand having a nucleic acid sequence complementary to a region of a TARDBP mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1 to 192. The degree of complementarity of the antisense strand with the region of the TARDBP mRNA transcript may be sufficient for the antisense strand to anneal over the entire length of the region of the TARDBP 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 TARDBP mRNA transcript.

[0153] In some embodiments, the siRNA molecules of the present disclosure feature an antisense strand having the nucleic acid sequence of any one of SEQ ID NOs: 385-576, or a nucleic acid sequence at least 60% identical thereto. For example, siRNA molecules of the present disclosure can be characterized by 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: 385-576.

[0154] In some embodiments, the siRNA molecules of the present disclosure are characterized by a sense strand having the nucleic acid sequence of any one of SEQ ID NOs: 193-384, or a nucleic acid sequence at least 60% identical thereto. For example, siRNA molecules of the present disclosure are characterized by 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: 193-384.

[0155] Exemplary siRNA molecules of the present disclosure are shown below in Table 1. Table 1 summarizes the antisense strand, the sense strand, and the corresponding region of the TARDBP mRNA transcript targeted by each antisense strand. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12]

[0156] siRNA structure The siRNA molecules of the present disclosure may be in the form of a single-stranded (ss) or double-stranded (ds) oligonucleotide structure. In some embodiments, the siRNA molecules may be biantennary, triantennary, or tetraantennary molecules. Furthermore, the siRNA molecules of the present disclosure may contain one or more phosphodiester internucleoside linkages and / or their analogs, such as phosphorothioate internucleoside linkages. The siRNA molecules of the present disclosure may further contain chemically modified nucleosides with 2' sugar modifications.

[0157] The simplest siRNAs consist of ribonucleic acids containing either a single strand or double strand structure, formed by a first strand (i.e., antisense strand) and, in the case of ds-siRNA, a second strand (i.e., sense strand). The first strand contains a stretch of consecutive nucleotides that is at least partially complementary to the target nucleic acid. The second strand also contains a stretch of consecutive nucleotides, and the second stretch is at least partially identical to the target nucleic acid. The first and second strands can hybridize to each other to form a double-stranded structure. Hybridization typically occurs via Watson-Crick base pairing.

[0158] Depending on the sequences of the first and second strands, hybridization or base pairing is not necessarily perfect or complete, which means that the first and second strands are not 100% base-paired due to mismatches.One or more mismatches can also exist within the double strand, without necessarily affecting the RNAi activity of siRNA.

[0159] The first strand contains a stretch of contiguous nucleotides that is essentially complementary to the target nucleic acid. Typically, the target nucleic acid sequence is ss-RNA, preferably mRNA, in accordance with the mode of action of the interfering ribonucleic acid. Such hybridization most likely occurs via Watson-Crick base pairing, but is not necessarily limited to this. The degree to which the first strand has a stretch of contiguous nucleotides complementary to the target nucleic acid sequence can be 80% to 100%, for example, 80%, 85%, 90%, 95%, or 100% complementary.

[0160] The siRNA molecules described herein may employ modifications to the nucleobases, phosphate backbone, ribose core, 5' and 3' ends, and branching, and multiple strands of the siRNA can be covalently linked.

[0161] Length of the small interfering RNA molecule Within the scope of the present invention, any length known in the art and previously unknown can be employed for the present invention. As described herein, the potential length of the antisense strand of the siRNA molecule of the present disclosure is between 10 and 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), between 15 and 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 between 18 and 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.

[0162] In some embodiments, the sense strand of an siRNA molecule of the present disclosure is between 12 and 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 between 14 and 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 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.

[0163] 2' sugar modification The present disclosure may include ss- and ds-siRNA molecular compositions containing at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or more) nucleoside having a 2' sugar modification. Possible 2'-modifications include OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or all possible orientations of O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl can be substituted or unsubstituted C1-C10 alkyl or C2-C10 alkenyl and alkynyl. In some embodiments, the modification includes a 2'-O-methyl (2'-O-Me) modification. Other potential sugar substituents include C1-C10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, 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-CH2CHOCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE). In some embodiments, modifications include the group O(CH)ON(CH), also known as 2'-dimethylaminooxyethoxy, i.e., 2'-DMAOE, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylamino-ethoxy-ethyl or 2'-DMAEOE), i.e., 2'-O-CHOCHN(CH). Other potential sugar substituents include, for example, aminopropoxy (-OCHCHCHNH), allyl (-CH-CH=CH), -O-allyl (-O-CH-CH=CH), and fluoro (F). The 2'-sugar substituent can be in the arabino (up) or ribo (down) position. In some embodiments, the 2'-arabino modification is 2'-F.Similar modifications may also be made elsewhere in the siRNA molecule, particularly the 3' position of the sugar in the 3'-terminal nucleoside or 2'-5'-linked oligonucleotides and the 5' position of the 5'-terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.

[0164] Nucleobase Modification The siRNA molecules of the present disclosure may also comprise nucleosides or other surrogate or mimic monomer subunits that contain nucleobases (often referred to in the art simply as "bases" or "heterocyclic base moieties").Nucleobases are other moieties that can be extensively modified or substituted, and such modified and / or substituted nucleobases can be in accordance with the present invention.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 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 cytosine, and other alkynyl derivatives of pyrimidine bases. Derivatives include 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 3-deazaguanine. Nucleobases may also include those in which the purine or pyrimidine base is replaced by other heterocycles, such as 7-deaza-adenine, 7-deazaguanosine, 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 in Englisch et al., Angewandte Chemie, International Edition 30:613, 1991; and those disclosed in Sanghvi, YS, Chapter 16, Antisense Research and Applications, CRC Press, Gait, MJ ed., 1993, pp. 289-302. The siRNA molecules of the present disclosure can also contain polycyclic heterocyclic compounds instead of one or more heterocyclic base moieties. Many tricyclic heterocyclic compounds have been reported. These compounds are routinely used in antisense applications to increase the binding properties of modified strands to target strands.

[0165] Representative cytosine analogs that make three hydrogen bonds with guanosine in the second strand include 1,3-diazaphenoxazin-2-one (Kurchavov et al., Nucleosides and Nucleotides, 16:1837-46, 1997), 1,3-diazaphenothiazin-2-one (Lin et al. Am. Chem. Soc., 117:3873-4, 1995), and 6,7,8,9-tetrafluoro-1,3-diazaphenoxazin-2-one (Wang et al., Tetrahedron Lett., 39:8385-8, 1998). These base modifications incorporated into oligonucleotides have been shown to hybridize with complementary guanine, which in turn hybridizes with adenine through extended stacking interactions, improving the thermal stability of the helix (see also US 10 / 155,920 and US 10 / 013,295, both of which are incorporated herein by reference in their entireties). Further helix-stabilizing properties have been observed when cytosine analogs / substitutes bear aminoethoxy moieties attached to a rigid 1,3-diazaphenoxazin-2-one scaffold (Lin et al., Am. Chem. Soc., 120:8531-2, 1998).

[0166] Internucleoside bond modification Another variable in the design of the present invention is the internucleoside bond that constitutes the phosphate backbone of siRNA molecules.Natural RNA phosphate backbone may be used herein, but its derivative may be used to improve the desirable properties of siRNA molecules.Of particular importance in this disclosure, but not limited to, is to protect part or the whole of siRNA molecules from hydrolysis.One example of the modification that reduces hydrolysis rate is phosphorothioate.Any part or the whole of backbone may contain phosphate substitution (for example, phosphorothioate). For example, the internucleoside linkages may 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 may be between 0 and 100% phosphodiester bonds, for example, between 0 and 100%, 10 and 100%, 20 and 100%, 30 and 100%, 40 and 100%, 50 and 100%, 60 and 100%, 70 and 100%, 80 and 100%, 90 and 100%, 0 and 90%, 0 and 80%, 0 and 70%, 0 and 60%, 0 and 50%, 0 and 40%, 0 and 30%, 0 and 20%, 0 and 10%, 10 and 90%, 20 and 80%, 30 and 70%, 40% and 60%, 10 and 40%, 20 and 50%, 30 and 60%, 40 and 70%, 50 and 80%, or 60 and 90% phosphodiester bonds.

[0167] Specific examples of some potential siRNA molecules useful in the present invention include the oligonucleotide that contains modified, for example, non-naturally occurring internucleoside linkages.As defined herein, the oligonucleotide that has modified internucleoside linkages includes the internucleoside linkages that retain phosphorus atom and the internucleoside linkages that do not have phosphorus atom.For the purpose of this specification and as sometimes referred to in the art, the modified oligonucleotide that does not have phosphorus atom in internucleoside backbone can also be considered as oligonucleoside.Preferred phosphorus-containing modified internucleoside linkage is phosphorothioate internucleoside linkage. In some embodiments, modified oligonucleotide backbones comprising a phosphorus atom therein include, for example, phosphorothioates, phosphodioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkyl phosphoramidates, thinophosphoramidates, thinoalkylphosphonates, thinoalkylphosphotriesters, selenophosphates, boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs thereof, and those with reversed polarity where one or more internucleotide linkages are 3'-3', 5'-5', or 2'-2' linkages.Exemplary United States 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; Same No. 5,321,131; Same No. 5,399,676; Same No. 5,405,939; Same No. 5,453,496; Same No. 5,455,233; Same No. 5,466,677; Same No. 5,476, No. 925; No. 5,519,126; No. 5,536,821; No. 5,541,316; No. 5,550,111; No. 5,563,253; No. 5,571,799; No. 5, No. 587,361; No. 5,625,050; No. 6,028,188; No. 6,124,445; No. 6,160,109; No. 6,169,170; No. 6,172,209 ; Same No. 6,239,265; Same No. 6,277,603; Same No. 6,326,199; Same No. 6,346,614; Same No. 6,444,423; Same No. 6,531,590; Same No. 6,534, 6,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Patent No. RE39464, the entire contents of each of which are incorporated herein by reference.

[0168] In some embodiments, modified oligonucleotide backbones that do not contain a phosphorus atom have backbones formed by short-chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short-chain heteroatom or heterocyclic internucleoside linkages, including morpholino linkages (formed in part from the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide, and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, riboacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and other backbones containing mixed N, O, S, and CH moieties. Non-limiting examples of U.S. patents that teach the preparation of non-phosphorus scaffolds 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, Nos. 489,677, 5,541,307, 5,561,225, 5,596,086, 5,602,240, 5,608,046, 5,610,289, 5,618,704, 5,623,070, 5,663,312, 5,633,360, 5,677,437, and 5,677,439, the entire contents of each of which are incorporated herein by reference.

[0169] Modification patterns of siRNA molecules The following section provides a set of exemplary scaffolds into which the siRNA molecules of the present disclosure can be incorporated.

[0170] In some embodiments of the present disclosure, the siRNA may contain an antisense strand comprising a region represented by Formula I, which, in the 5' to 3' direction, is: AB-(A') j -CP 2 -DP 1 -(C'-P 1 ) k -C' Formula I; where A is a compound of formula CP 1 -DP 1 Each A' is represented by the formula CP 2 -DP 2 B is represented by the formula CP 2 -DP 2 -DP 2 -DP 2 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; and 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 a target nucleic acid sequence.

[0171] In some embodiments, the antisense strand comprises a structure represented by Formula A1, which, in the 5' to 3' direction, is: ASBSAOBOBOBOAOBOAOBOA-OBOAOBOAOBSASASASBSA Formula A1; wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0172] In some embodiments of the present disclosure, the siRNA may contain an antisense strand comprising a region represented by Formula II, which, in the 5' to 3' direction, is: AB-(A') j -CP 2 -DP 1 -(CP 1 ) k -C' Formula II; where A is a compound of formula CP 1 -DP 1 Each A' is represented by the formula CP 2 -DP 2 B is represented by the formula CP 2 -DP 2 -DP 2 -DP 2 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; and 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 a target nucleic acid sequence.

[0173] In some embodiments, the antisense strand comprises a structure represented by Formula A2, which, in the 5' to 3' direction, is: ASBSAOBOBOBOAOBOAOBOA-OBOAAOBOAOBSASASASASA Formula A2; wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0174] In some embodiments of the present disclosure, the sense strand comprises a structure represented by Formula III, which, in the 5' to 3' direction, is: E-(A') m -F Formula III; where E is the formula (CP 1 )2; 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 (CP 2 )3-DP 2 -CP 2 -D; A', C, D, P 1 , and P 2 is 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. The sense strand is complementary (e.g., fully or partially complementary) to the antisense strand.

[0175] In some embodiments of the present disclosure, the sense strand comprises a structure represented by formula S1, which, in the 5' to 3' direction, is: ASASAOBOAOBOAOBOAOBOA-OAOAOBBSASA Formula S1; wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0176] In some embodiments of the present disclosure, the sense strand comprises a structure represented by formula S2, which, in the 5' to 3' direction, is: ASASAOBOAOBOAOBOAOBOA-OAOAOBOAOA Formula S2; wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0177] In some embodiments of the present disclosure, the sense strand comprises a structure represented by formula S3, which, in the 5' to 3' direction, is: ASASAOBOAOBOAOBOAOBOA-OAOAOBSASB formula S3; wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0178] In some embodiments of the present disclosure, the sense strand comprises a structure represented by formula S4, which, in the 5' to 3' direction, is: ASASAOBOAOBOAOBOAOBOA-OAOAOBOAOB Equation S4; wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0179] In some embodiments of the present disclosure, the siRNA may contain an antisense strand comprising a region represented by Formula IV, which, in the 5' to 3' direction, is: A-(A') j -CP 2 -B-(CP 1 ) k -C' Formula IV; where A is a compound of formula CP 1 -DP 1 Each A' is represented by the formula CP 2 -DP 2 B is represented by the formula DP 1 -CP 1 -DP 1 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; and 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 is complementary (e.g., fully or partially complementary) to a target nucleic acid.

[0180] In some embodiments, the antisense strand comprises a structure represented by Formula A3, which, in the 5' to 3' direction, is: ASBSAOBOAOBOAOBOAOBOA-OBOAOBOAOBSASASASA formula A3; wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0181] In some embodiments of the present disclosure, the siRNA of the present disclosure can have a sense strand represented by Formula V, where Formula V is, in the 5'→3' direction, E-(A') m -CP 2 -F formula V; where E is the formula (CP 1 )2; F is represented by the formula DP 1 -CP 1 -C, DP 2 -CP 2 -C, DP 1 -CP 1 -D, or DP 2 -CP 2 -D; A', C, D, P 1 , and P 2 is 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. The sense strand is complementary (e.g., fully or partially complementary) to the antisense strand.

[0182] In some embodiments of the present disclosure, the sense strand comprises a structure represented by formula S5, which, in the 5' to 3' direction, is: ASASAOBOAOBOAOBOAOBOA-OBOAOBSASA Formula S5; wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0183] In some embodiments of the present disclosure, the sense strand comprises a structure represented by formula S6, which, in the 5' to 3' direction, is: ASASAOBOAOBOAOBOAOBOA-OBOAOBOAOA Formula S6; wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage. In some embodiments of the present disclosure, the sense strand comprises a structure represented by formula S7, which, in the 5'→3' direction, is: ASASAOBOAOBOAOBOAOBOA-OBOAOBSASB formula S7; wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0184] In some embodiments of the present disclosure, the sense strand comprises a structure represented by formula S8, which, in the 5' to 3' direction, is: ASASAOBOAOBOAOBOAOBOA-OBOAOBOAOB formula S8; wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0185] In some embodiments of the present disclosure, the siRNA may contain an antisense strand comprising a region represented by Formula VI, which, in the 5' to 3' direction, is: AB j -EB k -EFG l -DP 1 -C' Formula VI; where A is a compound of formula CP 1 -DP1 Each B is represented by the formula CP 2 wherein 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, and each E is a nucleotide of the formula DP 2 -CP 2 and F is expressed by the formula DP 1 -CP 1 Each G is represented by the formula CP 1 Each P 1 is a phosphorothioate internucleoside linkage, and 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), 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 a target nucleic acid.

[0186] In some embodiments, the antisense strand comprises a structure represented by Formula A4, which, in the 5' to 3' direction, is: ASBSAOAOAOBOAOAOAOAOA-OAOAOBOAOBSASASASBSA formula A4; wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0187] In some embodiments of the present disclosure, the siRNA may contain a sense strand comprising a region represented by Formula VII, which, in the 5' to 3' direction, is: HBm -I n -A'-B o -HC Formula VII; where A' is a compound of the formula CP 2 -DP 2 Each H is represented by the formula (CP 1 )2; each I is represented by the formula (DP 2 ) are represented by B, C, D, P 1 , and P 2 is 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 sense strand is complementary (e.g., fully or partially complementary) to the antisense strand.

[0188] In some embodiments of the present disclosure, the sense strand comprises a structure represented by formula S9, which, in the 5'→3' direction, is: ASASAOAOAOBOBOBOBOAOBOA-OAOAOAASASA Formula S9; wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0189] In some embodiments of the present disclosure, the siRNA may contain an antisense strand comprising a region represented by Formula VIII: Z-((AP-) n (BP-) m ) q ; Formula VIII 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).

[0190] siRNA synthesis method The siRNA molecules of the present disclosure can be synthesized by standard methods known in the art, for example, by use of an automated DNA synthesizer such as those commercially available from Biosearch, Applied Biosystems, Inc., as discussed further below.

[0191] siRNA agent can be prepared by liquid phase organic synthesis or solid phase organic synthesis or both.Organic synthesis has the advantage that it can easily prepare the oligonucleotide that contains non-natural nucleotide or modified nucleotide.The siRNA molecule of the present disclosure can be prepared by liquid phase organic synthesis or solid phase organic synthesis or both.

[0192] Furthermore, it is contemplated that for any siRNA agent disclosed herein, further optimization can be achieved by systematically adding or removing linked nucleosides to generate longer or shorter sequences. Furthermore, such optimized sequences can be adjusted by introducing modified nucleosides and / or internucleoside linkages, as described herein or known in the art, including, for example, alternative nucleosides, alternative sugar moieties, and / or alternative internucleoside linkages, as known in the art and / or discussed herein, to further optimize the molecule (e.g., increasing serum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, and / or targeting to specific locations or cell types).

[0193] 5' phosphorus stabilizing moiety To further protect the siRNA molecules of the present disclosure from degradation, 5'-phosphorus stabilizing moiety can be employed.The 5'-phosphorus stabilizing moiety replaces 5'-phosphate to prevent phosphate hydrolysis.The hydrolysis of 5'-phosphate prevents binding to RISC, which is a necessary step in gene silencing.Any phosphate substitution that does not prevent binding to RISC is contemplated in the present disclosure.In some embodiments, the 5'-phosphate substitution is also stable against in vivo hydrolysis.Each strand of siRNA molecule can independently and optionally employ any suitable 5'-phosphorus stabilizing moiety. [ka]

[0194] Some exemplary end caps are illustrated in Formulas IX-XVI. Nuc in Formulas IX-XVI represents a nucleobase or nucleobase derivative or substitute as described herein. X in Formulas IX-XVI represents a 2'-modification as described herein. Some embodiments employ a hydroxy 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 the 5'-phosphorus stabilizing moiety as shown in Formula XI.

[0195] hydrophobic part The present disclosure further provides siRNA molecules that are linked with one or more hydrophobic moieties.The hydrophobic moiety can be covalently linked to the 5'-end or 3'-end of the siRNA molecule of the present disclosure.Non-limiting examples of the hydrophobic moiety that is suitable for use with the siRNA molecule of the present disclosure can include cholesterol, vitamin D, tocopherol, phosphatidylcholine (PC), docohexaenoic acid, docosanoic acid, PC-docosanoic acid, eicosapentaenoic acid, lithocholic acid, or any combination of the aforementioned hydrophobic moieties and PC.

[0196] siRNA branching The siRNA molecules of the present disclosure may be branched. For example, the siRNA molecules of the present disclosure may have one of several branching patterns, as described herein.

[0197] According to the present disclosure, the siRNA molecule disclosed herein can be a branched siRNA molecule.The siRNA molecule can be unbranched, or can be bi-, tri-, or tetra-branched, connected via a linker.Each main branch can be further branched to allow 2, 3, 4, 5, 6, 7, or 8 separate RNA single-strands or double-strands.The branching points on the linker can be from the same atom, or can be from separate atoms along the linker.Some exemplary embodiments are listed in Table 2. [Table 2]

[0198] In some embodiments, the siRNA molecule is a branched siRNA molecule. In some embodiments, the branched siRNA molecule is biantennary, triantennary, or tetraantennary. In some embodiments, the biantennary siRNA molecule is represented by any one of Formulas XVII to XIX, wherein each RNA is independently an siRNA molecule, L is a linker, and each X independently represents a branch point moiety (e.g., phosphoramidite, tosylated solketal, 1,3-diaminopropanol, pentaerythritol, or any one of the branch point moieties described in US 10,478,503).

[0199] In some embodiments, the three-branched siRNA molecule is represented by any one of Formulas XX-XXIII, wherein each RNA is independently an siRNA molecule, L is a linker, and each X independently represents a branch point moiety.

[0200] In some embodiments, the four-branched siRNA molecule is represented by any one of Formulas XXIV-XXVIII, wherein each RNA is independently an siRNA molecule, L is a linker, and each X independently represents a branch point moiety.

[0201] Linker The multiple strands of the siRNAs described herein may be covalently linked by a linker. This branching effect, among other things, improves cell permeability, allowing for better access to cells within the CNS (e.g., neurons or glial cells). Any linker moiety that is not incompatible with the siRNAs of the present invention can be employed. Linkers include ethylene glycol chains of 2 to 10 subunits (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 subunits), alkyl chains, carbohydrate chains, block copolymers, peptides, RNA, DNA, and others. In some embodiments, the carbon or oxygen atoms of the linker are optionally replaced with nitrogen atoms, have hydroxyl substituents, or have oxo substituents. In some embodiments, the linker is a polyethylene glycol (PEG) linker. PEG linkers suitable for use in the disclosed compositions and methods include linear or non-linear PEG linkers. Examples of non-linear PEG linkers include branched PEGs, linear forked PEGs, or branched-forked PEGs.

[0202] PEG linkers of various weights may be used in the disclosed compositions and methods. For example, the PEG linker may have a weight between 5 and 500 daltons. In some embodiments, a PEG linker having a weight between 500 and 1,000 daltons may be used. In some embodiments, a PEG linker having a weight between 1,000 and 10,000 daltons may be used. In some embodiments, a PEG linker having a weight between 200 and 20,000 daltons may be used. In some embodiments, the linker is covalently attached to the sense strand of the siRNA. In some embodiments, the linker is covalently attached to the antisense strand of the siRNA. In some embodiments, the PEG linker is a triethylene glycol (TrEG) linker. In some embodiments, the PEG linker is a tetraethylene glycol (TEG) linker.

[0203] In some embodiments, the linker is an alkyl chain 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.

[0204] The linker may covalently link two, three, four, or five unique siRNA strands. The linker may be covalently attached to any portion of the siRNA oligomer. In some embodiments, the linker is attached to the 3'-end of a nucleoside in each siRNA strand. In some embodiments, the linker is attached to the 5'-end of a nucleoside in each siRNA strand. In some embodiments, the linker is attached to a nucleoside in an siRNA strand (e.g., a sense strand or an antisense strand) via a covalent bond-forming moiety. In some embodiments, the covalent bond-forming moiety is selected from the group consisting of alkyl, ester, amide, carbonate, carbamate, triazole, urea, formacetal, phosphonate, phosphate, and phosphate derivatives (e.g., phosphorothioate, phosphoramidate, etc.).

[0205] In some embodiments, the linker has the structure of formula L1: [ka]

[0206] In some embodiments, the linker has the structure of formula L2: [ka]

[0207] In some embodiments, the linker has the structure of formula L3: [ka]

[0208] In some embodiments, the linker has the structure of formula L4: [ka]

[0209] In some embodiments, the linker has the structure of formula L5: [ka]

[0210] In some embodiments, the linker has the structure of formula L6: [ka]

[0211] In some embodiments, the linker has a structure of formula L7, as shown below: [ka]

[0212] In some embodiments, the linker has the structure of formula L8: [ka]

[0213] In some embodiments, the linker has the structure of formula L9: [ka]

[0214] In some embodiments, the selection of a linker for use in one or more of the branched siRNA molecules disclosed herein may be based on the hydrophobicity of the linker, e.g., to achieve a desired hydrophobicity for one or more of the branched siRNA molecules of the present disclosure. For example, a linker containing an alkyl chain may be used to increase the hydrophobicity of the branched siRNA molecule compared to a branched siRNA molecule having a less hydrophobic or hydrophilic linker.

[0215] The siRNA agents disclosed herein can be synthesized and / or modified by methods well established in the art, such as those described in Beaucage, SL et al. (eds.), Current Protocols in Nucleic Acid Chemistry, John Wiley & Sons, Inc., New York, NY, 2000, which is incorporated herein by reference.

[0216] Treatment method The TARDBP-targeting siRNA molecules of the present disclosure may be delivered to a subject to treat a neurodegenerative disease (e.g., amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD)) and / or alleviate a phenotype associated with the disorder. For example, the siRNA molecules may be delivered to a subject to treat ALS and / or alleviate an ALS disease-associated phenotype (e.g., involuntary movements, muscle spasms, weakness, loss of motor control). Alternatively, the TARDBP-targeting siRNA molecules of the present disclosure may be delivered to a subject to treat FTD and / or alleviate an FTD-associated phenotype (e.g., memory impairment, behavioral impairment, and language impairment). Furthermore, the siRNA molecules of the present disclosure can also be delivered to subjects having a variant of the TARDBP gene, and siRNA-mediated gene silencing of the TARDBP variant gene reduces the expression level of the TARDBP transcript, thereby treating a neurodegenerative disease (e.g., ALS or FTD) or other TARDBP-related disease or disorder.

[0217] The present disclosure provides methods for treating a subject by TARDBP gene silencing using one or more of the siRNA molecules described herein. Gene silencing can be performed in a subject to silence wild-type TARDBP transcripts, mutant TARDBP transcripts, splice isoforms of TARDBP transcripts, and / or overexpressed TARDBP transcripts compared to healthy subjects. The method can include delivering an siRNA molecule 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 administration route (e.g., intracerebroventricular, intrathecal, intrastriatal, intracisternal via catheter insertion, intraparenchymal, intravenous, subcutaneous, or intramuscular injection). The active compound can be administered at any suitable dose. The actual dosage of the composition of the present disclosure administered to a patient can be determined by physical and physiological factors, such as body weight, severity of the condition, prior or concurrent therapeutic interventions, the patient's idiopathic disease, and the route of administration. Depending on the dosage and administration route, the preferred dosage and / or the administration frequency of the effective amount can vary depending on the response of the subject.The practitioner in charge of administration will in any case determine the concentration of active ingredient(s) in the composition and the appropriate dose(s) for each subject.Administration can be carried out any suitable number of times per day for the required period.The subject can be an adult or a pediatric human, with or without coexisting diseases.

[0218] Selecting a target Subjects that can be treated with the siRNA molecules disclosed herein are, for example, subjects in need of treatment for a neurodegenerative disease (e.g., ALS or FTD) and / or other medical risk(s) associated with a gain-of-function mutation in the TARDBP gene. Subjects that can be treated with the siRNA molecules disclosed herein may include, for example, humans, monkeys, rats, mice, pigs, and other mammals containing at least one homologous copy of the TARDBP gene. The subject may be an adult or pediatric human, with or without co-morbidities.

[0219] Pharmaceutical Composition The siRNA molecule of the present disclosure can be formulated in pharmaceutical compositions for administering to subjects in a biologically compatible form suitable for in vivo administration.Therefore, the present disclosure provides pharmaceutical compositions that contain the siRNA molecule of the present disclosure mixed with suitable diluent, carrier or excipient.For example, siRNA molecule can be directly administered to subject CNS or affected tissue (for example, by intracerebroventricular, intrastriatal, intrathecal injection, intracisternal injection by catheter insertion, intraparenchymal injection, intravenous injection, subcutaneous injection or intramuscular injection).

[0220] Procedures and ingredients for selecting and preparing suitable formulations are described, for example, in Remington, J.P. The Science and Practice of Pharmacy, Easton, PA. Mack Publishers, 2012, 22 nd ed. and The National Formulary, United States Pharmacopeial, 2015, USP 38 NF 33).

[0221] Under ordinary conditions of storage and use, pharmaceutical compositions may contain preservatives, for example, to prevent the growth of microorganisms. Pharmaceutical compositions may comprise sterile aqueous solutions, sterile dispersions, or, for example, powders for the extemporaneous preparation of sterile solutions or dispersions. In all cases, the form can be sterilized using techniques known in the art and can be fluidized to the extent that it can be easily administered to a subject in need of treatment.

[0222] Pharmaceutical compositions may be administered to a subject, e.g., a human subject, alone or in combination with pharmaceutically acceptable carriers, as provided herein, the proportions of which may be determined by the solubility and / or chemical properties of the compound, the selected route of administration, and standard pharmaceutical practice.

[0223] Dosing regimen 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 of administration. For example, a physician can begin by prescribing a dose of one of the siRNA molecules of the present disclosure at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. Alternatively, a physician can begin a treatment regimen by administering one of the siRNA molecules of the present disclosure at a high dose, and then gradually decrease the dosage until the minimum dosage that achieves the therapeutic effect (e.g., reduction in the expression of the target gene sequence) is reached. Generally, the suitable daily dose of one of the siRNA molecules of the present disclosure will be the amount of the lowest dose of the siRNA molecule that is effective in producing a therapeutic effect. The ss- or ds-siRNA molecules of the present disclosure can be administered by injection, for example, intrathecal, intracerebroventricular, intracisternal injection via catheter insertion, intraparenchymal, intravenous, subcutaneous, or intramuscular injection. The daily dosage of a 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 throughout the day, week, month, or year, optionally in a unit dosage form. While the siRNA molecules of the present disclosure can be administered alone, they can also be administered as pharmaceutical formulations in combination with excipients, carriers, and, optionally, additional therapeutic agents.

[0224] Route of administration The disclosed methods contemplate any route of administration tolerated by the therapeutic composition, some embodiments of which include intrathecal, intracerebroventricular, intrastriatal, intraparenchymal, or intracisternal infusion via catheterization.

[0225] Intrathecal injection is a direct injection into the spinal column or subarachnoid space. By injecting directly into the CSF of the spinal column, the siRNA molecules of the present disclosure have direct access to cells in the spinal column (e.g., neurons and glial cells) and have a route to bypass the blood-brain barrier and access cells in the brain.

[0226] Intracerebroventricular (ICV) injection is a method of injection directly into the CSF in the ventricles of the brain. Like intrathecal injection, ICV is an injection method that bypasses the blood-brain barrier. ICV has the advantage of allowing therapeutic agents to access cells in the brain and spinal column without the risk of degradation in the blood.

[0227] Intrastriatal injection is a direct injection into the striatum (or corpus striatum), a subcortical basal ganglia region of the brain. Injection into the striatum bypasses the blood-brain barrier, avoiding the pharmacokinetic challenges of injection into the bloodstream and allowing direct access to brain cells.

[0228] Intraparenchymal administration is direct injection into the parenchyma (e.g., brain parenchyma). Injection into the brain parenchyma allows for direct injection into the brain region affected by the disease or disorder, bypassing the blood-brain barrier.

[0229] Intracisternal injection via catheterization is a direct injection into the cisterna magna, a region of the brain located between the cerebellum and the dorsal aspect of the medulla oblongata. Injection into the cisterna magna provides more direct delivery to cells in the cerebellum, brainstem, and spinal cord.

[0230] In some embodiments of the methods described herein, the therapeutic composition can be delivered to the subject by systemic administration, for example, intravenous, intramuscular, or subcutaneous administration.

[0231] Intravenous (IV) injection is a method of injection directly into a subject's bloodstream. IV administration may be in the form of a bolus dose, by continuous infusion, or any other method acceptable to the therapeutic composition.

[0232] Intramuscular (IM) injections are injections into a muscle of interest, such as the deltoid or gluteal muscles. IM may allow for rapid absorption of the therapeutic composition.

[0233] Subcutaneous injections are injections into the subcutaneous tissue. Absorption of compositions delivered subcutaneously may be slower than IV or IM injections, which may be beneficial for compositions that require continuous absorption. [Example]

[0234] The following examples are presented to provide those of ordinary skill in the art with an illustration of how the compositions and methods described herein can be used, made, and evaluated, and are intended to be purely exemplary of the disclosure and are not intended to limit the scope of what the inventors regard as their disclosure.

[0235] Example 1. TARDBP knockdown the purpose This example describes the results of a series of experiments conducted to investigate the ability of siRNA molecules complementary to specific regions within the human TARDBP mRNA transcript to achieve a reduction in expression of the TARDBP gene.

[0236] Materials and Methods HeLa cells were actively transfected with TARDBP siRNA at either 20 nM or 0.5 nM concentrations. After 24 hours, cells were lysed, and mRNA levels of TARDBP and a housekeeping gene (ATP5B) were assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) using standard reagents and an Applied Biosystems TaqMan assay. Results are presented as the percentage of remaining TARDBP mRNA compared to untreated control cells in the same assay.

[0237] result 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 a percentage of the residual mRNA expression of untreated at 20 nM and 0.5 nM. The knockdown results are reported in Table 3 (20 nM) and Table 4 (0.5 nM). [Table 3-1] [Table 3-2] [Table 3-3] [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4]

[0238] Example 2. Generation of TARDBP-targeting siRNA molecules The siRNA molecules of the present disclosure can be synthesized by standard methods known in the art, for example, by use of an automated DNA synthesizer such as those commercially available from Biosearch, Applied Biosystems, Inc., as discussed further below.

[0239] siRNA agents can be prepared using liquid phase organic synthesis or solid phase organic synthesis, or both. Organic synthesis has the advantage that oligonucleotides containing unnatural or modified nucleotides can be easily prepared. Specific examples of siRNA molecules containing the nucleotide sequences of sense and antisense strands and TARDBP mRNA target sequences are shown in Table 1 above. Those skilled in the art will understand that antisense (AS) strands can be annealed to corresponding sense (S) strands to generate ds-siRNA molecules. Alternatively, those skilled in the art can use only antisense strands to induce ss-siRNA molecules.

[0240] Example 3. Optimization of TARDBP-targeting siRNA molecules It is contemplated that any small interfering RNA (siRNA) agent disclosed herein can be further optimized for its efficacy or biophysical properties (e.g., increased serum stability or circulating half-life, increased thermal stability, enhanced transmembrane delivery, and / or targeting to specific locations or cell types) by modifying the siRNA. Such optimization can be achieved by systematically adding or removing linked nucleosides to generate longer or shorter sequences. Further optimization of the siRNA can include, for example, the incorporation of one or more alternative nucleosides, alternative 2' sugar moieties, and / or alternative internucleoside linkages. Furthermore, such optimized siRNA molecules can include the introduction of hydrophobic and / or stabilizing moieties at the 5' and / or 3' ends.

[0241] Optimizing siRNA with alternative nucleosides 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 cytosine, and other alkynyl derivatives of pyrimidine bases. , 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine, and / or 7-deazaguanine and 3-deazaguanine. siRNA molecules may also contain nucleobases in which the purine or pyrimidine base is replaced with other heterocycles, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and / or 2-pyridone. Further optimization of the siRNA molecules of the present disclosure may include nucleobases disclosed in US 3,687,808; Kroschwitz, JI, ed. The Concise Encyclopedia 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.

[0242] Optimizing siRNA with alternative sugar modifications 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-CH2CHOCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE), 2'-dimethylaminooxyethoxy, i.e., an O(CH2)2ON(CH3)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-CHOCH2N(CH3)2. Other possible 2' modifications that can optimize the siRNA molecules of the present disclosure include OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or all possible orientations of O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl include all possible orientations of substituted or unsubstituted C1-C10 alkyl or C2-C10 alkenyl and alkynyl. Other potential sugar substituents include, for example, aminopropoxy (-OCH2CH2CH2NH2), allyl (-CH2-CH=CH2), -O-allyl (-O-CH2-CH=CH2), and fluoro (F). The 2'-sugar substituent can be in the arabino (up) or ribo (down) position. In some embodiments, the 2'-arabino modification is 2'-F. Similar modifications may also be made elsewhere in the siRNA molecule, particularly the 3' position of the sugar in the 3'-terminal nucleoside or 2'-5'-linked oligonucleotides and the 5' position of the 5'-terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.

[0243] Optimizing siRNA with alternative internucleoside linkages Optimization of the siRNA molecules of the present disclosure may include one or more of the following internucleoside modifications: 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, thinophosphoramidates, thinoalkylphosphonates, thinoalkylphosphotriesters, selenophosphates, boranophosphates with normal 3'-5' linkages, 2'-5' linked analogs of these, and those with reversed polarity where one or more internucleotide linkages are 3'-3', 5'-5', or 2'-2' linkages.

[0244] Optimization of siRNA using hydrophobic moieties The optimization of the siRNA molecule of the present disclosure can comprise the hydrophobic moiety covalently linked to the 5'-end or 3'-end.The non-limiting example of the hydrophobic moiety suitable for use with the siRNA molecule of the present disclosure can include cholesterol, vitamin D, tocopherol, phosphatidylcholine (PC), docohexaenoic acid, docosanoic acid, PC-docosanoic acid, eicosapentaenoic acid, lithocholic acid, or any combination of the aforementioned hydrophobic moiety and PC.

[0245] Optimization of siRNA using stabilizing molecules Optimization of the siRNA molecules of the present disclosure may include a 5'-phosphorus stabilizing moiety that protects the siRNA molecule from degradation. The 5'-phosphorus stabilizing moiety replaces the 5'-phosphate to prevent phosphate hydrolysis. Hydrolysis of the 5'-phosphate prevents binding to RISC, a necessary step in gene silencing. Any phosphate substitution that does not prevent binding to RISC is contemplated in the present disclosure. In some embodiments, the 5'-phosphate substitution is also stable against in vivo hydrolysis. Each siRNA strand may independently and optionally employ any suitable 5'-phosphorus stabilizing moiety. Non-limiting examples of 5'-stabilizing moieties suitable for use in the siRNA molecules of the present disclosure may include those shown in Formulas IX to XVI above.

[0246] Optimization of siRNA using branched siRNA The optimization of the siRNA molecule of the present disclosure can include incorporating branching patterns, such as bi-branched, tri-branched or tetra-branched siRNA, which are connected via linker.Each main branch can be further branched to allow 2, 3, 4, 5, 6, 7 or 8 separate RNA single strands or double strands.The branching points on linker can be from the same atom, or can be from different atoms along the linker.Some exemplary embodiments are listed in Table 2 above.

[0247] The siRNA compositions of the present disclosure can be optimized to be in the form of a biantennary siRNA molecule represented by any one of Formulas XVII to XIX; a triantennary siRNA molecule represented by any one of Formulas XX to XXIII; and / or a tetraantennary siRNA molecule represented by any one of Formulas XXIV to XXVIII, wherein each RNA is independently an siRNA molecule, L is a linker, and each X independently represents a branch point moiety (e.g., a phosphoramidite, a tosylated solketal, 1,3-diaminopropanol, pentaerythritol, or any one of the branch point moieties described in U.S. Pat. No. 10,478,503).

[0248] Example 4. Preparation and administration of TARDBP-targeting siRNA molecules The siRNA molecule of the present disclosure can be formulated in pharmaceutical compositions for administration to subjects in a biologically compatible form suitable for in vivo administration.For example, the siRNA molecule of the present disclosure can be administered with suitable diluents, carriers or excipients, and can further comprise preservatives, for example, to prevent microbial growth.The procedures and ingredients for selecting and preparing suitable formulations can be found, for example, in Remington, JP, The Science and Practice of Pharmacy, Easton, PA.Mack Publishers, 2012, 22 nd ed. and The National Formulary, United States Pharmacopeial, 2015, USP 38 NF 33).

[0249] The method of the present disclosure contemplates any administration route that can be tolerated by the siRNA composition of the present disclosure to subjects.Non-limiting examples of siRNA injection into CNS include intrathecal, intracerebroventricular, intrastriatal, intraparenchymal, or intracisternal injection by catheter insertion.Examples of systemic administration include intravenous, intramuscular, and subcutaneous injection.A doctor with ordinary skill in the art can easily determine effective administration route.

[0250] Example 5. Methods for the treatment of neurodegenerative diseases using TARDBP-targeting siRNA molecules A subject in need of treatment for a neurodegenerative disease (e.g., amyotrophic lateral sclerosis type 3 (ALS) or frontotemporal dementia (FTD)) is treated with a dosage of the siRNA molecule or siRNA composition of the present invention formulated as a salt at a frequency determined by the treating physician. A physician of ordinary skill in the art can readily determine an effective amount of the siRNA molecule to administer to a mammalian subject (e.g., a human) in need thereof. For example, a physician can begin by prescribing a dose of one of the siRNA molecules of the present disclosure at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. Alternatively, a physician can begin a treatment regimen by administering one of the siRNA molecules of the present disclosure at a high dose, and then gradually decrease the dose until the minimum dose that produces a therapeutic effect (e.g., a reduction in the expression of TARDBP mRNA or a suitable biomarker, or a reduction in an ALS- or FTD-associated phenotype) is achieved. Generally, a suitable daily dose of one of the siRNA molecules of the present disclosure is the minimum dose amount effective to produce a therapeutic effect. The ss- or ds-siRNA molecules of the present disclosure can be administered by injection, for example, intrathecally, intracerebroventricularly, intrastriatally, intraparenchyma, intravenously, intramuscularly, or by catheterization into the cisterna magna.The daily dose of one 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 throughout the day, week, month, or year, optionally in a unit dosage form.Any of the siRNA molecules of the present disclosure can be administered alone, or can be administered as a pharmaceutical formulation in combination with an excipient, a carrier, and optionally, an additional therapeutic agent.The dosage and frequency are determined based on the subject's height, weight, age, sex, and other disorders.

[0251] The siRNA molecule of the present disclosure is selected by a physician taking into consideration its compatibility with the subject. Single-stranded or double-stranded siRNA molecules (e.g., unbranched siRNA, bi-branched siRNA, tri-branched siRNA, tetra-branched siRNA) can be selected. The selected siRNA molecule can have an antisense strand and a sense strand with the most suitable sequence and RNA modification (e.g., natural and non-natural internucleoside linkages, modified sugars, 5' phosphorus stabilizing moieties, hydrophobic moieties, and / or branched structures) for the patient.

[0252] The siRNA molecules are delivered by the route most appropriate for the patient and condition (e.g., injection, e.g., intrathecal, intracerebroventricular, intrastriatal, intraparenchymal, intravenous, intramuscular, or intracisternal injection via catheterization) at a rate tolerable by the patient until the subject reaches the maximum tolerated dose or until symptoms are sufficiently alleviated.

[0253] Other embodiments All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0254] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications, and that this application is generally intended to cover any modifications, uses, and applications of the invention in accordance with the principles of the invention, and includes such departures from the invention as come within known or customary practice in the art to which the invention pertains and which may be applied to the essential features described above and which fall within the scope of the appended claims.

[0255] Other embodiments are within the scope of the following claims.

Claims

1. 1. A small interfering RNA (siRNA) molecule comprising an antisense strand and a sense strand that is complementary to the antisense strand, wherein the antisense strand has sufficient complementarity to hybridize to a region within a transactive response DNA binding protein 43 (TARDBP) mRNA transcript having a nucleic acid sequence of any one of SEQ ID NOs: 1-192.

2. 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 TARDBP mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-192, and optionally, the antisense strand has at least 70% complementarity to the TARDBP mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-192.

3. 3. The siRNA molecule of claim 2, wherein the antisense strand has at least 75% complementarity to a region of 21 contiguous nucleobases within the TARDBP mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-192, and optionally, 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 TARDBP mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-192.

4. The siRNA molecule of any one of claims 1 to 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 completely complementary to a contiguous polynucleotide segment of equal length within the region of the TARDBP RNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1 to 192.

5. The siRNA molecule of claim 4, wherein the antisense strand comprises 10 to 30 contiguous nucleotides that are completely complementary to a contiguous polynucleotide segment of equal length within the region of the TARDBP RNA transcript having a nucleic acid sequence of any one of SEQ ID NOs: 1 to 192.

6. The siRNA molecule of claim 5, wherein the antisense strand comprises 12 to 30 consecutive nucleotides that are completely complementary to a contiguous polynucleotide segment of equal length within the region of the TARDBP RNA transcript having a nucleic acid sequence of any one of SEQ ID NOs: 1 to 192.

7. The siRNA molecule of claim 6, wherein the antisense strand comprises 15 to 30 contiguous nucleotides that are completely complementary to a contiguous polynucleotide segment of equal length within the region of the TARDBP RNA transcript having a nucleic acid sequence of any one of SEQ ID NOs: 1 to 192.

8. The siRNA molecule of claim 7, wherein the antisense strand comprises 18 to 30 contiguous nucleotides that are completely complementary to a contiguous polynucleotide segment of equal length within the region of the TARDBP RNA transcript having a nucleic acid sequence of any one of SEQ ID NOs: 1 to 192.

9. The siRNA molecule of claim 8, wherein the antisense strand comprises 18 to 25 contiguous nucleotides that are completely complementary to a contiguous polynucleotide segment of equal length within the region of the TARDBP RNA transcript having a nucleic acid sequence of any one of SEQ ID NOs: 1 to 192.

10. The siRNA molecule of claim 9, wherein the antisense strand comprises 18 to 21 contiguous nucleotides that are completely complementary to a contiguous polynucleotide segment of equal length within the region of the TARDBP RNA transcript having a nucleic acid sequence of any one of SEQ ID NOs: 1 to 192.

11. The siRNA molecule of claim 10, wherein the antisense strand comprises 21 contiguous nucleotides that are perfectly complementary to a contiguous polynucleotide segment of equal length within the region of the TARDBP RNA transcript having a nucleic acid sequence of any one of SEQ ID NOs: 1 to 192.

12. 12. The siRNA molecule of any one of claims 1 to 11, wherein the antisense strand contains no more than 9 nucleotide mismatches to a region of 21 consecutive nucleobases of the TARDBP RNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1 to 192, and optionally, the antisense strand contains no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or only 1 mismatch to said region of the TARDBP RNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1 to 192.

13. The siRNA molecule of any one of claims 1 to 12, 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: 385 to 576.

14. The siRNA molecule of claim 13, 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: 385 to 576.

15. The siRNA molecule of claim 14, wherein the antisense strand has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NOs: 385-576, and optionally, 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: 385-576.

16. The siRNA molecule of claim 15, wherein the antisense strand has a nucleic acid sequence of any one of SEQ ID NOs: 385 to 576.

17. The siRNA molecule of any one of claims 1 to 16, 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: 193 to 384.

18. The siRNA molecule of claim 17, 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: 193 to 384.

19. 19. The siRNA molecule of claim 18, wherein the sense strand has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NOs: 193-384, and optionally, 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: 193-384.

20. The siRNA molecule of claim 19, wherein the sense strand has a nucleic acid sequence of any one of SEQ ID NOs: 193 to 384.

21. The antisense strand comprises a structure represented by Formula I, wherein Formula I is in the 5' to 3' direction: A-B-(A’) ｊ -C-P ２ -D-P １ -(C’-P １ ) ｋ -C’ Formula I, (Wherein A is a compound of the formula C-P １ -D-P １ is expressed as Each A' is of the formula C-P ２ -D-P ２ is expressed as B is a compound of the formula C-P ２ -D-P ２ -D-P ２ -D-P ２ is expressed as 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, The siRNA molecule of any one of claims 1 to 20, wherein k is an integer from 1 to 7.

22. The antisense strand comprises a structure represented by formula A1, wherein formula A1 is in the 5'→3' direction: A-S-B-S-A-O-B-O-B-O-BO-A-O-BO-A-O-BO-A-O-BO-O-A-O-B-O-A-O-B-S-A-S-A-S-A-S-B-S Formula A1; 22. The siRNA molecule of claim 21, wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

23. the antisense strand comprises a structure represented by Formula II, wherein Formula II is in the 5' to 3' direction: A-B-(A’) ｊ -C-P ２ -D-P １ -(C-P １ ) ｋ -C’ Formula II; (Wherein A is a compound of the formula C-P １ -D-P １ is expressed as Each A' is of the formula C-P ２ -D-P ２ is expressed as B is a compound of the formula C-P ２ -D-P ２ -D-P ２ -D-P ２ is expressed as 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, The siRNA molecule of any one of claims 1 to 20, wherein k is an integer from 1 to 7.

24. The antisense strand comprises a structure represented by formula A2, and formula A2 is in the 5'→3' direction: A-S-B-SA Formula A2; 24. The siRNA molecule of claim 23, wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

25. the sense strand comprises a structure represented by Formula III, wherein Formula III is in the 5' to 3' direction: E-(A') ｍ -F Formula III; (Wherein E is a group represented by the formula (C-P １ ) ２ is expressed as F is represented by the formula (C-P ２ ), ３ -D-P １ -C-P １ -C, (C-P ２ ), ３ -D-P ２ -C-P ２ -C, (C-P ２ ), ３ -D-P １ -C-P １ -D, or (C-P ２ ), ３ -D-P ２ -C-P ２ -D, A', C, D, P １ , and P ２ is as defined in Formula II, The siRNA molecule of any one of claims 1 to 24, wherein m is an integer from 1 to 7.

26. The sense strand comprises a structure represented by formula S1, wherein formula S1 is in the 5'→3' direction: A-S-A-S-A-O-B-O-A-O-BO-A-O-BO-A-O-BO-O-A-O-A-O-A-O-B-S-A-S-A Formula S1; 26. The siRNA molecule of claim 25, wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

27. The sense strand comprises a structure represented by formula S2, and formula S2 is in the 5'→3' direction: A-S-A-S-A-O-BO-O-A-O-BO-A-O-BO-A-O-BO-O-A-O-A-O-A-O-BO-O-A-O-A Formula S2; 26. The siRNA molecule of claim 25, wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

28. The sense strand comprises a structure represented by formula S3, and formula S3 is in the 5'→3' direction: A-S-A-S-A-O-B-O-A-O-BO-A-O-BO-A-O-BO-O-A-O-A-O-A-O-B-S-A-S-B Formula S3; 26. The siRNA molecule of claim 25, wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

29. The sense strand comprises a structure represented by formula S4, and formula S4 is in the 5'→3' direction: A-S-A-S-A-O-BO-O-A-O-BO-A-O-BO-A-O-BO-O-A-O-A-O-A-O-BO-O-A-O-B Formula S4; 26. The siRNA molecule of claim 25, wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

30. The antisense strand comprises a structure represented by Formula IV, wherein Formula IV is in the 5' to 3' direction: A-(A’) ｊ -C-P ２ -B-(C-P １ ) ｋ -C’ Formula IV; (Wherein A is a compound of the formula C-P １ -D-P １ is expressed as Each A' is of the formula C-P ２ -D-P ２ is expressed as B is a compound of the formula D-P １ -C-P １ -D-P １ is expressed as 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 P １ is a phosphorothioate internucleoside linkage, Each P ２ is a phosphodiester internucleoside linkage, j is an integer from 1 to 7, The siRNA molecule of any one of claims 1 to 20 and 25 to 29, wherein k is an integer from 1 to 7.

31. The antisense strand comprises a structure represented by formula A3, and formula A3 is arranged in the 5' to 3' direction as follows: A-S-B-S-A-O-B-O-A-O-B-O-A-O-BO-A-O-B-O-A-O-BO-O-A-O-B-O-A-O-B-S-A-S-B-S-A-S-A Formula A3; 31. The siRNA molecule of claim 30, wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

32. The sense strand comprises a structure represented by Formula V, wherein Formula V is in the 5' to 3' direction: E-(A’) ｍ -C-P ２ -F Formula V; (Wherein E is a group represented by the formula (C-P １ ) ２ is expressed as F is represented by the formula D - P １ - C - P １ - C, D - P ２ - C - P ２ - C, D - P １ - C - P １ - D, or D - P ２ - C - P ２ - D, and is represented by A', C, D, P １ , and P ２ is as defined in formula IV, The siRNA molecule of any one of claims 1 to 24, 30, and 31, wherein m is an integer from 1 to 7.

33. The sense strand comprises a structure represented by formula S5, and formula S5 is in the 5'→3' direction: A-S-A-S-A-O-B-O-A-O-BO-A-O-BO-A-O-BO-O-A-O-B-O-A-O-B-S-A-S-A Formula S5; 33. The siRNA molecule of claim 32, wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

34. The sense strand comprises a structure represented by formula S6, and formula S6 is in the 5'→3' direction: A-S-A-S-A-O-BO-O-A-O-BO-A-O-BO-A-O-BO-O-A-O-BO-O-A-O-B-O-A-O-A Formula S6; 33. The siRNA molecule of claim 32, wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

35. The sense strand comprises a structure represented by formula S7, wherein formula S7 is in the 5'→3' direction: A-S-A-S-A-O-B-O-A-O-BO-A-O-BO-A-O-BO-O-A-O-B-O-A-O-B-S-A-S-B Formula S7; 33. The siRNA molecule of claim 32, wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

36. The sense strand comprises a structure represented by formula S8, and formula S8 is in the 5'→3' direction: A-S-A-S-A-O-B-O-A-O-B-O-A-O-BO-A-O-BO-O-A-O-B-O-A-O-B-O-A-O-B Formula S8; 33. The siRNA molecule of claim 32, wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

37. the antisense strand has 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; (Wherein A is a compound of the formula C-P １ -D-P １ is expressed as Each B is of the formula C-P ２ is expressed as 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 D-P ２ -C-P ２ is expressed as F is a compound of the formula D-P １ -C-P １ is expressed as Each G is of the formula C-P １ is expressed as Each P １ is a phosphorothioate internucleoside linkage, Each P ２ is a phosphodiester internucleoside linkage, j is an integer from 1 to 7, k is an integer from 1 to 7, 1 is an integer from 1 to 7. The siRNA molecule of any one of claims 1 to 20, 25 to 29, and 32 to 36, wherein 1 is an integer from 1 to 7.

38. The antisense strand comprises a structure represented by formula A4, and formula A4 is arranged in the 5' to 3' direction as follows: A-S-B-S-A-O-A-O-A-O-BO-A-O-A-O-A-O-A-O-A-O-A-O-A-O-B-O-A-O-B-S-A-S-A-S-A-S-B-S-A Formula A4; 38. The siRNA molecule of claim 37, wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

39. the sense strand comprises a structure represented by Formula VII, wherein Formula VII is in the 5' to 3' direction: H-B ｍ -I ｎ -A’-B ｏ -H-C Formula VII; (Wherein A' is a compound of the formula C-P ２ -D-P ２ is expressed as Each H is a member of the formula (C-P １ ) ２ is expressed as Each I independently represents a group of the formula (D-P ２ ) and B, C, D, P １ , and P ２ is as defined in formula VI, m is an integer from 1 to 7, n is an integer from 1 to 7, 39. The siRNA molecule of any one of claims 1 to 24, 30, 31, 37, and 38, wherein o is an integer from 1 to 7.

40. The sense strand comprises a structure represented by formula S9, wherein formula S9 is in the 5'→3' direction: A-S-A-S-A-O-A-O-A-O-B-O-B-O-BO-A-O-BO-O-A-O-A-O-A-O-A-S-A-S-A Formula S9; 40. The siRNA molecule of claim 39, wherein A represents a 2'-O-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 any one of claims 1 to 40, wherein the antisense strand further comprises a 5' phosphorus stabilizing moiety at the 5' end of the antisense strand.

42. The siRNA molecule of any one of claims 1 to 41, wherein the sense strand further comprises a 5' phosphorus stabilizing moiety at the 5' end of the sense strand.

43. each 5' phosphorus stabilizing moiety is independently represented by any one of formulas IX-XVI: 【Chemical 1】 43. The siRNA molecule of claim 41 or 42, 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.

44. 44. The siRNA molecule of claim 43, wherein the nucleobase is adenine, uracil, guanine, thymine, or cytosine.

45. The siRNA molecule of any one of claims 41 to 44, wherein the 5' phosphorus stabilizing moiety is an (E)-vinyl phosphonate of formula XI.

46. The siRNA molecule of any one of claims 1 to 45, wherein the siRNA molecule further comprises a hydrophobic moiety at the 5' end or the 3' end of the siRNA molecule.

47. 47. The siRNA molecule of claim 46, wherein the hydrophobic moiety is selected from the group consisting of cholesterol, vitamin D, or tocopherol.

48. The siRNA molecule of any one of claims 1 to 47, wherein the sense strand has a length of 10 to 30 nucleotides.

49. 49. The siRNA molecule of claim 48, wherein the sense strand is 10 to 25 nucleotides in length.

50. 50. The siRNA molecule of claim 49, wherein the sense strand is 12 to 25 nucleotides in length.

51. 51. The siRNA molecule of claim 50, wherein the sense strand is 12 to 20 nucleotides in length.

52. 52. The siRNA molecule of claim 51, wherein the sense strand is 12 to 19 nucleotides in length.

53. 53. The siRNA molecule of claim 52, wherein the sense strand is 15 nucleotides in length.

54. 53. The siRNA molecule of claim 52, wherein the sense strand is 16 nucleotides in length.

55. 53. The siRNA molecule of claim 52, wherein the sense strand is 18 nucleotides in length.

56. 56. The siRNA molecule of any one of claims 1 to 55, wherein the length of the antisense strand is 10 to 30 nucleotides.

57. 57. The siRNA molecule of claim 56, wherein the length of the antisense strand is 12 to 30 nucleotides.

58. 58. The siRNA molecule of claim 57, wherein the length of the antisense strand is 15 to 30 nucleotides.

59. 59. The siRNA molecule of claim 58, wherein the length of the antisense strand is 18 to 30 nucleotides.

60. 60. The siRNA molecule of claim 59, wherein the antisense strand is 18 to 25 nucleotides in length.

61. 61. The siRNA molecule of claim 60, wherein the length of the antisense strand is 18 to 21 nucleotides.

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

63. 62. The siRNA molecule of claim 61, wherein the antisense strand is 20 nucleotides in length.

64. 62. The siRNA molecule of claim 61, wherein the antisense strand is 21 nucleotides in length.

65. The siRNA molecule of any one of claims 1 to 64, wherein the siRNA molecule is a branched siRNA molecule.

66. 66. The siRNA molecule of claim 65, wherein the branched siRNA molecule is biantennary, triantennary, or tetraantennary.

67. The siRNA molecule is a biantennary siRNA molecule, and optionally, the biantennary siRNA molecule is represented by any one of Formulas XVII to XIX: 【Chemistry 2】 67. The siRNA molecule of claim 66, wherein each RNA is independently an siRNA molecule, L is a linker, and each X independently represents a branch point moiety.

68. The siRNA molecule is a tri-antennary siRNA molecule, and optionally the tri-antennary siRNA molecule is represented by any one of Formulas XX to XXIII: 【Chemistry 3】 67. The siRNA molecule of claim 66, wherein each RNA is independently an siRNA molecule, L is a linker, and each X independently represents a branch point moiety.

69. The siRNA is a four-antennary siRNA molecule, and optionally, the four-antennary siRNA molecule is represented by any one of Formulas XXIV to XXVIII: 【Chemistry 4】 67. The siRNA molecule of claim 66, wherein each RNA is independently an siRNA molecule, L is a linker, and each X independently represents a branch point moiety.

70. 70. The siRNA molecule of any one of claims 67 to 69, wherein the linker is selected from the group consisting of one or more consecutive subunits of ethylene glycol, alkyl, carbohydrate, block copolymer, peptide, RNA, and DNA.

71. The siRNA molecule of claim 70, wherein the one or more consecutive subunits are 2 to 20 consecutive subunits.

72. A pharmaceutical composition comprising the siRNA molecule of any one of claims 1 to 71 and a pharmaceutically acceptable excipient, carrier, or diluent.

73. 73. A method of delivering an siRNA molecule to a subject diagnosed with a neurodegenerative disease, said method comprising administering to said subject a therapeutically effective amount of the siRNA molecule of any one of claims 1 to 71 or the pharmaceutical composition of claim 72.

74. 73. A method of treating a neurodegenerative disease in a subject in need thereof, said method comprising administering to said subject a therapeutically effective amount of the siRNA molecule of any one of claims 1 to 71 or the pharmaceutical composition of claim 72.

75. 75. The method of claim 73 or 74, wherein the neurodegenerative disease is amyotrophic lateral sclerosis (ALS).

76. 75. The method of claim 73 or 74, wherein the neurodegenerative disease is frontotemporal dementia (FTD).

77. 73. A method of reducing TARDBP expression in a subject in need thereof, said method comprising administering to said subject a therapeutically effective amount of an siRNA molecule of any one of claims 1 to 71 or a pharmaceutical composition of claim 72.

78. 78. The method of any one of claims 73 to 77, wherein the siRNA molecule or pharmaceutical composition is administered to the subject by intracerebroventricular, intrastriatal, intraparenchymal, or intrathecal injection.

79. The method of any one of claims 73 to 77, wherein the siRNA molecule or pharmaceutical composition is administered to the subject by intravenous, intramuscular, or subcutaneous injection.

80. 80. The method of any one of claims 73 to 79, wherein the subject is a human.

81. A kit comprising the siRNA molecule of any one of claims 1 to 71 or the pharmaceutical composition of claim 72 and a package insert, the package insert instructing a user of the kit to carry out the method of any one of claims 73 to 80.

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