Compositions and methods for treating Huntington's disease

Abstract

The present disclosure provides single- or double-stranded interfering RNA molecules (e.g., siRNAs) targeting the huntingtin (HTT) gene. Interfering RNA molecules, as well as pharmaceutical compositions containing them, may contain specific patterns of nucleoside and internucleoside linkage modifications. The siRNA molecules may be branched siRNA molecules, such as bi-, tri-, or tetra-antennary siRNA molecules. The disclosed siRNA molecules may further feature a 5' phosphorus-stabilizing moiety and / or a hydrophobic moiety. Furthermore, the present disclosure provides methods for delivering the disclosed siRNA molecules to the central nervous system of a subject, such as a subject identified as having Huntington's disease.

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 above XML copy was created on September 21, 2023, is titled "51436-035WO2_Sequence_Listing_9_21_23", and is 8,809 bytes in size.

[0002] The present disclosure relates to small interfering RNA (siRNA) molecules that target RNA transcripts (e.g., mRNA) of the huntingtin (HTT) gene and compositions containing the same. The present disclosure further describes methods for silencing HTT and treating diseases that may benefit from silencing HTT (e.g., Huntington's disease) by delivering HTT-targeting siRNA molecules to target tissues in subjects in need thereof. [Background technology]

[0003] HTT (huntingtin) encodes a protein involved in the development and progression of Huntington's disease. Studies have shown that individuals have a mutant form of HTT characterized by an abnormal trinucleotide repeat expansion. Currently, there are no treatments that can alter the course of Huntington's disease. Therefore, there is a need for therapeutic agents that can selectively reduce the activity of HTT in a manner that provides effective treatment for Huntington's disease or other HTT-related diseases or disorders. Summary of the Invention

[0004] The present disclosure provides compositions and methods for reducing huntingtin (HTT) expression through small interfering RNA (siRNA)-mediated silencing of HTT transcripts, which have the benefit of being highly selective for HTT over other genes.

[0005] The siRNA molecule of the present disclosure can be used to silence HTT gene, thereby preventing the translation of corresponding mRNA transcription product, and reducing the expression of HTT protein.By reducing HTT level in this way, the onset or progression of disease can be prevented.The siRNA molecule of the present disclosure can be directly delivered to the subject who needs to silence HTT, for example, by intrathecal injection, intracerebroventricular injection, intrastriatal injection, intraparenchymal injection, intracisternal injection by catheter insertion, intravenous injection, subcutaneous injection or intramuscular injection.

[0006] In a first aspect, the present disclosure provides an siRNA molecule comprising an antisense strand and a sense strand having complementarity to the antisense strand, wherein the antisense strand is 10 to 30 nucleotides in length (e.g., 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, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides in length). The antisense strand is 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 nucleotides in length, and is sufficiently complementary to hybridize to a region of equal length within a huntingtin (HTT) mRNA transcript having a nucleic acid sequence of any one of SEQ ID NOs: 1 to 3. The antisense strand comprises a structure represented by Formula I, which, in the 5' to 3' direction, is as follows: AB-(A') j -CP 2 -DP 1 -(C'-P 1 ) k -C' Formula I; where A is a compound of formula CP 1 -DP1 Represented by; Each A' is independently a group of formula CP 2 -DP 2 Represented by; B is the formula CP 2 -DP 2 -DP 2 -DP 2 Represented by; each C is independently 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 independently a 2'-F ribonucleoside; Each P 1 are independently a phosphorothioate internucleoside linkage; Each P 2 are independently 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).

[0007] 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.

[0008] In another aspect, the present disclosure provides an siRNA molecule comprising an antisense strand and a sense strand having complementarity to the antisense strand, wherein the antisense strand is 10 to 30 nucleotides in length (e.g., 10 to 29 nucleotides in length, 10 to 28 nucleotides in length, 10 to 27 nucleotides in length, 10 to 26 nucleotides in length, 10 to 25 nucleotides in length, 10 to 24 nucleotides in length, 10 to 23 nucleotides in length, 10 to 22 nucleotides in length, 10 to 21 nucleotides in length, or 10 to 20 nucleotides in length, 10 nucleotides in length, 11 nucleotides in length, 12 nucleotides in length, 13 nucleotides in length). The antisense strand is 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 nucleotides in length, and is sufficiently complementary to hybridize to a region of equal length within a huntingtin (HTT) mRNA transcript having a nucleic acid sequence of any one of SEQ ID NOs: 1-3. The antisense strand comprises 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 independently a group of formula CP 2 -DP 2 Represented by; B is the formula CP 2 -DP 2 -DP 2 -DP 2 Represented by; each C is independently 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 independently a 2'-F ribonucleoside; Each P 1 are independently a phosphorothioate internucleoside linkage; Each P 2 are independently 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).

[0009] 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.

[0010] In some embodiments, 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 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 (CP2 )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).

[0011] In some embodiments of any of the above aspects, j is 4 and k is 4. In some embodiments, m is 4.

[0012] 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.

[0013] In some embodiments, 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.

[0014] In some embodiments, 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.

[0015] In some embodiments, 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.

[0016] In yet another aspect, the present disclosure provides an siRNA molecule comprising an antisense strand and a sense strand having complementarity to the antisense strand, wherein the antisense strand is 10 to 30 nucleotides in length (e.g., 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, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides in length). The antisense strand is 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 nucleotides in length, and is sufficiently complementary to hybridize to a region of equal length within a huntingtin (HTT) mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-3. 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 independently a group of formula CP 2 -DP 2 Represented by; B is the formula DP 1 -CP 1 -DP 1 Represented by; each C is independently a 2'-O-Me ribonucleoside; each C' is independently a 2'-O-Me ribonucleoside or a 2'-F ribonucleoside; each D is independently a 2'-F ribonucleoside; Each P 1 are independently a phosphorothioate internucleoside linkage; Each P 2 are independently 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).

[0017] 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.

[0018] 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).

[0019] In some embodiments, j is 6 and k is 2. In some embodiments, m is 5.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] In another aspect, the present disclosure provides an siRNA molecule comprising an antisense strand and a sense strand having complementarity to the antisense strand, wherein the antisense strand is 10 to 30 nucleotides in length (e.g., 10 to 29 nucleotides in length, 10 to 28 nucleotides in length, 10 to 27 nucleotides in length, 10 to 26 nucleotides in length, 10 to 25 nucleotides in length, 10 to 24 nucleotides in length, 10 to 23 nucleotides in length, 10 to 22 nucleotides in length, 10 to 21 nucleotides in length, or 10 to 20 nucleotides in length, 10 nucleotides in length, 11 nucleotides in length, 12 nucleotides in length, 13 nucleotides in length). 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 independently has the formula CP 2 Represented by; each C is independently a 2'-O-Me ribonucleoside; each C' is independently a 2'-O-Me ribonucleoside or a 2'-F ribonucleoside; each D is independently a 2'-F ribonucleoside; Each E is independently of the formula DP 2 -CP 2 Represented by; F is the formula DP 1 -CP 1 Represented by; Each G independently has the formula CP 1 Represented by; Each P 1 are independently a phosphorothioate internucleoside linkage; Each P 2 are independently 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).

[0025] 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.

[0026] 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 independently represents a group 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 2 is as defined in formula VI; m is an integer from 1 to 7; n is an integer from 1 to 7; o is an integer from 1 to 7.

[0027] In some embodiments, j is 3, k is 6, and l is 2. In some embodiments, m is 3, n is 3, and o is 3.

[0028] 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.

[0029] In some embodiments of any of the above aspects, the regions of equal length within the HTT mRNA transcript have the nucleic acid sequence of SEQ ID NO: 1. In some embodiments of any of the above aspects, the regions of equal length within the HTT mRNA transcript have the nucleic acid sequence of SEQ ID NO: 2. In some embodiments of any of the above aspects, the regions of equal length within the HTT mRNA transcript have the nucleic acid sequence of SEQ ID NO: 3.

[0030] In some embodiments of any of the above aspects, the antisense strand has at least 70% (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%) complementarity to a region of 15 consecutive nucleobases within an HTT mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-3. In some embodiments, the antisense strand has at least 70% (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%) complementarity to a region of 16 consecutive nucleobases within an HTT mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-3. In some embodiments, the antisense strand has at least 70% (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%) complementarity to a region of 17 consecutive nucleobases within an HTT mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-3. In some embodiments, the antisense strand has at least 70% (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%) complementarity to a region of 18 consecutive nucleobases within an HTT mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-3. In some embodiments, the antisense strand has at least 70% (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%) complementarity to a region of 19 consecutive nucleobases within an HTT mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-3.In some embodiments, the antisense strand has at least 70% (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%) complementarity to a region of 20 consecutive nucleobases within an HTT mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-3. In some embodiments, the antisense strand has at least 70% (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%) complementarity to a region of 21 consecutive nucleobases within an HTT mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-3. In some embodiments, the antisense strand has at least 70% (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%) complementarity to a region of 22 consecutive nucleobases within an HTT mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-3. In some embodiments, the antisense strand has at least 70% (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%) complementarity to a region of 23 consecutive nucleobases within an HTT mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-3. In some embodiments, the antisense strand has at least 70% (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%) complementarity to a region of 24 consecutive nucleobases within an HTT mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-3.In some embodiments, the antisense strand has at least 70% (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%) complementarity to a region of 25 consecutive nucleobases within an HTT mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-3. In some embodiments, the antisense strand has at least 70% (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%) complementarity to a region of 26 consecutive nucleobases within an HTT mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-3. In some embodiments, the antisense strand has at least 70% (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%) complementarity to a region of 27 consecutive nucleobases within an HTT mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-3. In some embodiments, the antisense strand has at least 70% (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%) complementarity to a region of 28 consecutive nucleobases within an HTT mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-3. In some embodiments, the antisense strand has at least 70% (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%) complementarity to a region of 29 consecutive nucleobases within an HTT mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-3.In some embodiments, the antisense strand has at least 70% (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%) complementarity to a region of 30 consecutive nucleic acid bases within an HTT mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-3.

[0031] In some embodiments of any of the above aspects, the antisense strand has at least 70% (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%) complementarity to a region within an HTT mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-3. In some embodiments, the antisense strand has at least 75% (e.g., 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%) complementarity to a region within an HTT mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-3, and optionally, the antisense strand is complementary to an HTT mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-3. having at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementarity to a region within the mRNA transcript.

[0032] In some embodiments, 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 contiguous nucleotides that are completely complementary to a contiguous polynucleotide segment of equal length within a region of an HTT RNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-3.

[0033] In some embodiments, the antisense strand comprises 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 an HTT RNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-3. In some embodiments, the antisense strand comprises 12 to 30 contiguous nucleotides (e.g., 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 an HTT RNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-3. In some embodiments, the antisense strand comprises 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 an HTT RNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-3. In some embodiments, the antisense strand comprises 18 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 an HTT RNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-3. In some embodiments, the antisense strand comprises 15 to 21 contiguous nucleotides (e.g., 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides) that are perfectly complementary to a contiguous polynucleotide segment of equal length within a region of an HTT RNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-3.In some embodiments, the antisense strand comprises 15 contiguous nucleotides that are perfectly complementary to a contiguous polynucleotide segment of equal length within a region of an HTT RNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-3. In some embodiments, the antisense strand comprises 20 contiguous nucleotides that are perfectly complementary to a contiguous polynucleotide segment of equal length within a region of an HTT RNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-3. In some embodiments, the antisense strand comprises 21 contiguous nucleotides that are perfectly complementary to a contiguous polynucleotide segment of equal length within a region of an HTT RNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-3.

[0034] In some embodiments, the antisense strand has 9 or fewer nucleotide mismatches to a region within an HTT RNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-3, and 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 within an HTT RNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-3.

[0035] In some embodiments of the above aspects, the region of the HTT RNA transcript has the nucleic acid sequence of SEQ ID NO: 1. In some embodiments of the above aspects, the region of the HTT RNA transcript has the nucleic acid sequence of SEQ ID NO: 2. In some embodiments of the above aspects, the region of the HTT RNA transcript has the nucleic acid sequence of SEQ ID NO: 3.

[0036] In some embodiments of any of the above aspects, the antisense strand has a nucleic acid sequence that is at least 85% (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: 7-9. In some embodiments, 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: 7-9. In some embodiments, the antisense strand has a nucleic acid sequence that is at least 95% (e.g., 95%, 96%, 97%, 98%, 99%, or 100%) identical to the nucleic acid sequence of any one of SEQ ID NOs: 7-9, 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: 7-9. In some embodiments, the antisense strand has the nucleic acid sequence of any one of SEQ ID NOs: 7-9. In some embodiments, the nucleic acid sequence is SEQ ID NO: 7. In some embodiments, the nucleic acid sequence is SEQ ID NO: 8. In some embodiments, the nucleic acid sequence is SEQ ID NO: 9.

[0037] In some embodiments of any of the above aspects, the sense strand has a nucleic acid sequence that is at least 85% (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: 4-6. In some embodiments, the sense strand has a nucleic acid sequence that is at least 90% (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: 4-6. In some embodiments, the sense strand has a nucleic acid sequence that is at least 90% (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: 4-6, 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: 4-6. In some embodiments, the nucleic acid sequence is SEQ ID NO: 4. In some embodiments, the nucleic acid sequence is SEQ ID NO: 5. In some embodiments, the nucleic acid sequence is SEQ ID NO: 6.

[0038] In some embodiments of any of the aforementioned aspects or embodiments of the present disclosure, the antisense strand further comprises a 5' phosphorus stabilizing moiety at the 5' end of the antisense strand. In some embodiments of any of the aforementioned aspects or embodiments of the present disclosure, the sense strand further comprises a 5' phosphorus stabilizing moiety at the 5' end of the sense strand.

[0039] In some embodiments, each 5' phosphorus stabilizing moiety is independently represented by any one of Formulas IX-XVI: [ka] wherein Nuc represents a nucleobase 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. In some embodiments, the nucleobase is adenine, uracil, guanine, thymine, or cytosine.

[0040] In some embodiments, the 5' phosphorus stabilizing moiety is an (E)-vinyl phosphonate represented by formula XI.

[0041] In some embodiments of any of the above aspects, the siRNA molecule further comprises a hydrophobic moiety at the 5' or 3' end of the siRNA molecule, hi some embodiments, the hydrophobic moiety is selected from the group consisting of cholesterol, vitamin D, or tocopherol.

[0042] In some embodiments of any of the above aspects, the length of the sense strand is 12 to 30 nucleotides (e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides).

[0043] In some embodiments of any of the above aspects, the siRNA molecule is a branched siRNA molecule. In some embodiments, the branched siRNA molecule is biantennary, triantennary, or tetraantennary.

[0044] In some embodiments, the siRNA molecule is a biantennary siRNA molecule, optionally represented by any one of Formulas XVII-XIX: [ka] wherein each RNA is independently an siRNA molecule, L is a linker, and each X independently represents a branch point moiety.

[0045] 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.

[0046] In some embodiments, the siRNA molecule is a three-antennary siRNA molecule, optionally the three-antennary siRNA molecule is represented by any one of Formulas XX-XXIII: [ka] wherein each RNA is independently an siRNA molecule, L is a linker, and each X independently represents a branch point moiety.

[0047] 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.

[0048] In some embodiments, the siRNA molecule is a four-branched siRNA molecule, optionally the four-branched siRNA molecule is represented by any one of Formulas XXIV-XXVIII: [ka] wherein each RNA is independently an siRNA molecule, L is a linker, and each X independently represents a branch point moiety.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] In some embodiments, the ethylene glycol oligomer is PEG. In some embodiments, the PEG is TrEG. In some embodiments, the PEG is TEG.

[0053] 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).

[0054] In some embodiments, the linker connects one or more (eg, 1, 2, 3, 4, or more) siRNA molecules via a covalent bond-forming moiety.

[0055] 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.

[0056] In some embodiments, the linker comprises a structure of formula L1. [ka]

[0057] In some embodiments, the linker comprises a structure of formula L2. [ka]

[0058] In some embodiments, the linker comprises a structure of formula L3. [ka]

[0059] In some embodiments, the linker comprises a structure of formula L4. [ka]

[0060] In some embodiments, the linker comprises a structure of formula L5. [ka]

[0061] In some embodiments, the linker comprises a structure of formula L6. [ka]

[0062] In some embodiments, the linker comprises a structure of formula L7. [ka]

[0063] In some embodiments, the linker comprises a structure of formula L8. [ka]

[0064] In some embodiments, the linker comprises a structure of formula L9. [ka]

[0065] In some embodiments of any of the siRNA molecules described herein, the siRNA is formulated as a salt containing one or more divalent cations.The siRNA molecule may contain multiple cationic binding sites that are partially or completely saturated with one or more divalent cations.

[0066] In some embodiments of the divalent cation salt, the degree of saturation of the cationic binding sites with one or more divalent cations is about 10% to about 100% (e.g., about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, or about 90% to about 100%).

[0067] In some embodiments of the divalent cation salt, the cationic binding site is located within an internucleoside linkage, such as a phosphodiester and / or phosphorothioate linkage. For example, the cationic binding site can be an oxyanion moiety within a phosphodiester or phosphorothioate linkage.

[0068] In some embodiments of the divalent cation salt, one or more divalent cations are characterized as having an ionic radius in the range of about 30 picometers to about 150 picometers (e.g., about 30 picometers to about 140 picometers, about 40 picometers to about 130 picometers, about 50 picometers to about 120 picometers, about 60 picometers to about 110 picometers, about 60 picometers to about 100 picometers, or about 60 picometers to about 90 picometers).

[0069] In some embodiments of the divalent cation salt, one or more divalent cations comprise a hard Lewis acid. In some embodiments, one or more divalent cations comprise a hard Lewis acid. 2+ , Be 2+ , Ca 2+ , Cu 2+ , Mg 2+ , Mn 2+ , Ni 2+ , or Zn 2+ , or a combination thereof.

[0070] In some embodiments of the divalent cation salt, one or more divalent cations are Ba 2+ In some embodiments, the one or more divalent cations include Be 2+ In some embodiments, the one or more divalent cations include Ca 2+ In some embodiments, the one or more divalent cations include Cu. 2+ In some embodiments, the one or more divalent cations include Mg 2+ In some embodiments, the one or more divalent cations include Mn 2+ In some embodiments, the one or more divalent cations include Ni 2+ In some embodiments, the one or more divalent cations include Zn 2+ Includes.

[0071] In some embodiments of the divalent cation salt, one or more divalent cations are Ca 2+ and Mg 2+ and optionally, Ca 2+ and Mg 2+ In some embodiments, the ratio of Ca to Ca is 1:100 to 100:1 (e.g., 1:75, 1:50, 1:25, 1:10, 1:5, 1:1, 5:1, 10:1, 25:1, 50:1, 75:1, or 100:1). 2+ and Mg 2+ are present in a 1:1 ratio.

[0072] In some embodiments of the divalent cation salt, the one or more divalent cations displace water from the cationic binding sites of the siRNA molecule.

[0073] In some embodiments of the divalent cation salt, the siRNA molecule comprises one or more atoms bearing a negative charge, and the divalent cation comprises two positive charges. In some embodiments, the ratio of negative to positive charges is between 0.75 and 7.5 (e.g., 0.76, 0.77, 0.78, 0.79, 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7 0.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5). In some embodiments, the ratio of negative to positive charges is between 1.0 and 2.0 (e.g., between 1.0 and 1.9, between 1.0 and 1.8, between 1.0 and 1.7, between 1.0 and 1.6, between 1.0 and 1.5, between 1.0 and 1.4, between 1.0 and 1.3, between 1.0 and 1.2, between 1.0 and 1.1, between 1.1 and 2.0, between 1.2 and 2.0, between 1.3 and 2.0, between 1.4 and 2.0, between 1.5 and 2.0, between 1.6 and 2.0, between 1.7 and 2.0, between 1.8 and 2.0, or between 1.9 and 2.0). In some embodiments, the ratio of negative to positive charges is 0.75 to 6.5 (e.g., 0.75 to 5.5, 0.75 to 4.5, 0.75 to 3.5, 0.75 to 2.5, 0.75 to 1.5, or 0.75 to 1). In some embodiments, the ratio of negative to positive charges is 1 to 7.5 (e.g., 1.5 to 7.5, 2.5 to 7.5, 3.5 to 7.5, 4.5 to 7.5, 5.5 to 7.5, or 6.5 to 7.5). In some embodiments, the molar ratio of siRNA molecules to divalent cations is 1:10 to 1:100 (e.g., 1:10 to 1:50, 1:18 to 1:38, 1:20 to 1:25, 1:25, or 1:20).In some embodiments, the concentration of one or more divalent cations is between 10 mM and 150 mM (e.g., between 20 mM and 150 mM, between 20 mM and 100 mM, between 25 mM and 150 mM, between 25 mM and 100 mM, between 30 mM and 90 mM, between 35 mM and 85 mM, between 35 mM and 75 mM, between 40 mM and 70 mM, between 40 mM and 65 mM, between 40 mM and 60 mM, or between 40 mM and 50 mM).

[0074] 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).

[0075] 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).

[0076] 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).

[0077] 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).

[0078] 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).

[0079] 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.

[0080] In some embodiments, 9 of the internucleoside linkages are phosphodiester or phosphorothioate linkages.

[0081] In some embodiments, the length of the antisense strand is 10 to 30 nucleotides (e.g., 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides), 15 to 25 nucleotides (e.g., 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, or 25 nucleotides), or 18 to 23 nucleotides (e.g., 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, or 23 nucleotides). In some embodiments, the 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.

[0082] 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.

[0083] In some embodiments, the sense strand is 12 to 30 nucleotides in length (e.g., 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides), or 14 to 18 nucleotides (e.g., 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, or 18 nucleotides). In some embodiments, the sense strand is 15 nucleotides in length. In some embodiments, the sense strand is 16 nucleotides in length. In some embodiments, the sense strand is 17 nucleotides in length. In some embodiments, the sense strand is 18 nucleotides in length. In some embodiments, the sense strand is 19 nucleotides in length. In some embodiments, the sense strand is 20 nucleotides in length. In some embodiments, the sense strand is 21 nucleotides in length. In some embodiments, the sense strand is 22 nucleotides in length. In some embodiments, the sense strand is 23 nucleotides in length. In some embodiments, the sense strand is 24 nucleotides in length. In some embodiments, the sense strand is 25 nucleotides in length. In some embodiments, the sense strand is 26 nucleotides in length. In some embodiments, the sense strand is 27 nucleotides in length. In some embodiments, the sense strand is 28 nucleotides in length. In some embodiments, the sense strand is 29 nucleotides in length. In some embodiments, the sense strand is 30 nucleotides in length.

[0084] In some embodiments, the four internucleoside linkages are phosphorothioate linkages.

[0085] 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 long and the sense strand is 18 nucleotides long. In some embodiments, the antisense strand is 26 nucleotides long and the sense strand is 19 nucleotides long. In some embodiments, the antisense strand is 26 nucleotides long and the sense strand is 20 nucleotides long. In some embodiments, the antisense strand is 26 nucleotides long and the sense strand is 21 nucleotides long. In some embodiments, the antisense strand is 26 nucleotides long and the sense strand is 22 nucleotides long. In some embodiments, the antisense strand is 26 nucleotides long and the sense strand is 23 nucleotides long. In some embodiments, the antisense strand is 26 nucleotides long and the sense strand is 24 nucleotides long. In some embodiments, the antisense strand is 26 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 26 nucleotides long. In some embodiments, the antisense strand is 27 nucleotides long and the sense strand is 14 nucleotides long. In some embodiments, the antisense strand is 27 nucleotides long and the sense strand is 15 nucleotides long. In some embodiments, the antisense strand is 27 nucleotides long and the sense strand is 16 nucleotides long. In some embodiments, the antisense strand is 27 nucleotides long and the sense strand is 17 nucleotides long. In some embodiments, the antisense strand is 27 nucleotides long and the sense strand is 18 nucleotides long. 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 in length and the sense strand is 23 nucleotides in length. In some embodiments, the antisense strand is 27 nucleotides in length and the sense strand is 24 nucleotides in length. In some embodiments, the antisense strand is 27 nucleotides in length and the sense strand is 25 nucleotides in length. In some embodiments, the antisense strand is 27 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 27 nucleotides in length. 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.

[0086] In another aspect, the present disclosure provides a pharmaceutical composition comprising an siRNA molecule of any of the above aspects or embodiments of the present disclosure and a pharmaceutically acceptable excipient, carrier, or diluent.

[0087] In yet another aspect, the present disclosure provides a method for delivering an siRNA molecule to a subject diagnosed with Huntington's disease, the method comprising administering to the subject a therapeutically effective amount of the siRNA molecule or pharmaceutical composition of any of the above aspects or embodiments of the present disclosure.

[0088] In yet another aspect, the present disclosure provides a method of treating Huntington's disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an siRNA molecule or pharmaceutical composition of any of the above aspects or embodiments of the present disclosure.

[0089] In a further aspect, the present disclosure provides a method for reducing HTT expression in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an siRNA molecule or pharmaceutical composition of any of the above aspects or embodiments of the present disclosure.

[0090] 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.

[0091] In some embodiments, the siRNA molecule is administered to the subject in the form of an aqueous solution or suspension. The siRNA molecule may be administered to the subject systemically or directly to the subject's central nervous system (CNS). For example, the siRNA molecule may be administered to the subject's cerebrospinal fluid (CSF), spinal cord, brain parenchyma, cortex, cerebellum, basal ganglia, caudate nucleus, putamen, thalamus, globus pallidus, substantia nigra, or another brain structure. In some embodiments, the siRNA molecule is administered intrathecally, intraventricularly, intrastriatally, or by intracisternal injection via catheter insertion. In some embodiments, the siRNA molecule is administered intrastriatally. In some embodiments, the siRNA molecule is administered intrathecally. In some embodiments, the siRNA molecule is administered intraventricularly.

[0092] In some embodiments of the methods described herein, the subject is a human.

[0093] 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 an accompanying instruction, the accompanying instruction instructing a user of the kit to perform a method of any of the above aspects or embodiments of the present disclosure. [Brief explanation of the drawings]

[0094] [Figure 1A] Shows the knockdown of HTT mRNA in different brain regions after 3 days in mice treated with the siRNA molecules of the present disclosure.The x-axis of each graph shows the pattern of antisense strand and sense strand modification and the amount of siRNA molecules administered.Three bars are shown for each condition tested, and from left to right, they represent the results in motor cortex, striatum and hippocampus, respectively. [Figure 1B] Figure 1 shows the accumulation of siRNA molecules in different brain regions after 3 days in mice treated with the siRNA molecules of the present disclosure.The x-axis of each graph shows the pattern of antisense strand and sense strand modification and the amount of siRNA molecules administered.Three bars are shown for each condition tested, and from left to right, they respectively represent the results in motor cortex, striatum and hippocampus. [Figure 2A] Shows the knockdown of HTT mRNA in different brain regions in mice treated with the siRNA molecules of the present disclosure after 7 days.The x-axis of each graph shows the pattern of antisense strand and sense strand modification and the amount of siRNA molecules administered.Three bars are shown for each condition tested, and from left to right, they represent the results in motor cortex, striatum and hippocampus, respectively. [Figure 2B] Figure 1 shows the accumulation of siRNA molecules in different brain regions in mice treated with the siRNA molecules of the present disclosure after 7 days.The x-axis of each graph shows the pattern of antisense strand and sense strand modification and the amount of siRNA molecules administered.Three bars are shown for each condition tested, and from left to right, they respectively represent the results in motor cortex, striatum and hippocampus. [Figure 3A]Illustrates the knockdown of HTT mRNA in different brain regions after 14 days in mice treated with the siRNA molecules of the present disclosure.The x-axis of each graph shows the pattern of antisense strand and sense strand modification and the amount of siRNA molecules administered.Four bars are shown for each condition tested, and from left to right, they represent the results in frontal cortex, motor cortex, striatum and hippocampus, respectively. [Figure 3B] Figure 1 shows the accumulation of siRNA molecules in different brain regions after 14 days in mice treated with the siRNA molecules of the present disclosure.The x-axis of each graph shows the pattern of antisense strand and sense strand modification and the amount of siRNA molecules administered.Four bars are shown for each condition tested, and from left to right, they respectively represent the results in frontal cortex, motor cortex, striatum and hippocampus. [Figure 4A] Illustrates the knockdown of HTT mRNA in different brain regions in mice treated with the siRNA molecules of the present disclosure after one month.The x-axis of each graph shows the pattern of antisense strand and sense strand modification and the amount of siRNA molecules administered.Four bars are shown for each condition tested, and from left to right, they represent the results in frontal cortex, motor cortex, striatum and hippocampus, respectively. [Figure 4B] Figure 1 shows the accumulation of siRNA molecules in different brain regions in mice treated with the siRNA molecules of the present disclosure after one month.The x-axis of each graph shows the pattern of antisense strand and sense strand modification and the amount of siRNA molecules administered.Four bars are shown for each condition tested, and from left to right, they respectively represent the results in frontal cortex, motor cortex, striatum and hippocampus. [Figure 5A] Shows the knockdown of HTT mRNA in different brain regions after 2 months in mice treated with the siRNA molecules of the present disclosure.The x-axis of each graph shows the pattern of antisense strand and sense strand modification and the amount of siRNA molecules administered.Four bars are shown for each condition tested, and from left to right, they represent the results in frontal cortex, motor cortex, striatum and hippocampus, respectively. [Figure 5B]Figure 1 shows the accumulation of siRNA molecules in different brain regions after 2 months in mice treated with the siRNA molecules of the present disclosure.The x-axis of each graph shows the pattern of antisense strand and sense strand modification and the amount of siRNA molecules administered.Four bars are shown for each condition tested, and from left to right, they respectively represent the results in frontal cortex, motor cortex, striatum and hippocampus. [Figure 6A] Illustrates the knockdown of HTT mRNA in different brain regions after 3 months in mice treated with the siRNA molecules of the present disclosure.The x-axis of each graph shows the pattern of antisense strand and sense strand modification and the amount of siRNA molecules administered.Four bars are shown for each condition tested, and from left to right, they represent the results in frontal cortex, motor cortex, striatum and hippocampus, respectively. [Figure 6B] Figure 1 shows the accumulation of siRNA molecules in different brain regions after 3 months in mice treated with the siRNA molecules of the present disclosure.The x-axis of each graph shows the pattern of antisense strand and sense strand modification and the amount of siRNA molecules administered.Four bars are shown for each condition tested, and from left to right, they respectively represent the results in frontal cortex, motor cortex, striatum and hippocampus. [Figure 7A] 7A shows the time course results in mice treated with siRNA molecules of the present disclosure. The figure shows HTT mRNA knockdown in mice treated with 2.5 nmol of siRNA molecules. The x-axis of the graph indicates the pattern of antisense and sense strand modification, as well as the brain region tested for each condition. In FIG. 7A, the data for the frontal cortex has four bars, from left to right, corresponding to the results after 14 days, 1 month, 2 months, and 3 months, respectively. For other brain regions, each bar, from left to right, corresponds to the results after 3 days, 7 days, 14 days, 1 month, 2 months, and 3 months, respectively. The data for the frontal cortex treated with antisense A3 / sense S6 has four bars, from left to right, corresponding to the results after 14 days, 1 month, 2 months, and 3 months, respectively. [Figure 7B]7B shows the time course results in mice treated with siRNA molecules of the present disclosure. The figure shows the accumulation of siRNA molecules in mice treated with 2.5 nmol of siRNA molecules. The x-axis of the graph indicates the pattern of antisense and sense strand modification, as well as the brain region tested for each condition. In FIG. 7B, the data for the frontal cortex has four bars, from left to right, corresponding to the results after 14 days, 1 month, 2 months, and 3 months, respectively. For other brain regions, each bar, from left to right, corresponds to the results after 3 days, 7 days, 14 days, 1 month, 2 months, and 3 months, respectively. The data for the frontal cortex treated with antisense A3 / sense S6 has four bars, from left to right, corresponding to the results after 14 days, 1 month, 2 months, and 3 months, respectively. [Figure 7C] 7C shows the time course results in mice treated with siRNA molecules of the present disclosure. The figure shows HTT mRNA knockdown in mice treated with 0.25 nmol of siRNA molecules. The x-axis of the graph indicates the pattern of antisense and sense strand modification, as well as the brain region tested for each condition. In FIG. 7C, the frontal cortex data has four bars, from left to right, corresponding to the results after 14 days, 1 month, 2 months, and 3 months, respectively. For other brain regions, each bar, from left to right, corresponds to the results after 3 days, 7 days, 14 days, 1 month, 2 months, and 3 months, respectively. The frontal cortex data for antisense A3 / sense S6 treatment has four bars, from left to right, corresponding to the results after 14 days, 1 month, 2 months, and 3 months, respectively. [Figure 7D]7D shows the time course results for mice treated with siRNA molecules of the present disclosure. The figure shows the accumulation of siRNA molecules in mice treated with 0.25 nmol of siRNA molecules. The x-axis of each graph indicates the pattern of antisense and sense strand modification and the brain region tested for each condition. In Figure 7D, the frontal cortex data for PBS and antisense A2 / sense S2 treatments have five bars, which correspond to the results after 7 days, 14 days, 1 month, 2 months, and 3 months, respectively, from left to right. The frontal cortex data for antisense A3 / sense S6 treatment have four bars, which correspond to the results after 14 days, 1 month, 2 months, and 3 months, respectively, from left to right. The remaining data in Figure 7D each have six bars, which correspond to the results after 3 days, 7 days, 14 days, 1 month, 2 months, and 3 months, respectively, from left to right. [Figure 8A] Illustrates the knockdown of HTT mRNA in different brain regions after 6 months in mice treated with the siRNA molecules of the present disclosure.The x-axis of each graph shows the pattern of antisense strand and sense strand modification and the amount of siRNA molecules administered.Four bars are shown for each condition tested, and from left to right, they represent the results in frontal cortex, motor cortex, striatum and hippocampus, respectively. [Figure 8B] Figure 1 shows the accumulation of siRNA molecules in different brain regions in mice treated with the siRNA molecules of the present disclosure after 6 months.The x-axis of each graph shows the pattern of antisense strand and sense strand modification and the amount of siRNA molecules administered.Four bars are shown for each condition tested, and from left to right, they respectively represent the results in frontal cortex, motor cortex, striatum and hippocampus. [Figure 9A] 1 shows the expression of HTT mRNA, HTT protein, and the amount of siRNA in mice treated with siRNA molecules of the present disclosure. The siRNA molecules had an antisense strand of formula A2 and a sense strand of formula S1. Results were analyzed in mice treated with 2.5 nmol of siRNA molecules in the motor cortex. [Figure 9B]1 shows the expression of HTT mRNA, HTT protein, and the amount of siRNA in mice treated with siRNA molecules of the present disclosure. The siRNA molecules had an antisense strand of formula A2 and a sense strand of formula S1. Results were analyzed in mice treated with 2.5 nmol of siRNA molecules in the hippocampus. [Figure 9C] 1 shows the expression of HTT mRNA, HTT protein, and the amount of siRNA in mice treated with siRNA molecules of the present disclosure. The siRNA molecules had an antisense strand of formula A2 and a sense strand of formula S1. Results were analyzed in mice treated with 2.5 nmol of siRNA molecules in the striatum. [Figure 9D] 1 shows the expression of HTT mRNA, HTT protein, and the amount of siRNA in mice treated with siRNA molecules of the present disclosure. The siRNA molecules had an antisense strand of formula A2 and a sense strand of formula S1. Results were analyzed in mice treated with 0.25 nmol of siRNA molecules in the motor cortex. [Figure 9E] 1 shows the expression of HTT mRNA, HTT protein, and the amount of siRNA in mice treated with siRNA molecules of the present disclosure. The siRNA molecules had an antisense strand of formula A2 and a sense strand of formula S1. Results were analyzed in mice treated with 0.25 nmol of siRNA molecules in the hippocampus. [Figure 9F] 1 shows the expression of HTT mRNA, HTT protein, and the amount of siRNA in mice treated with siRNA molecules of the present disclosure. The siRNA molecules had an antisense strand of formula A2 and a sense strand of formula S1. Results were analyzed in mice treated with 0.25 nmol of siRNA molecules in the striatum. [Figure 10A] 1 shows the expression of HTT mRNA over time in mice treated intrakidney with siRNA molecules of the present disclosure compared to PBS controls. The siRNA molecules had an antisense strand of formula A2 and a sense strand of formula S1. [Figure 10B]1 shows the expression of HTT mRNA over time in mice treated intrahepatically with siRNA molecules of the present disclosure compared to PBS controls. The siRNA molecules had an antisense strand of formula A2 and a sense strand of formula S1. [Figure 11] This figure shows the knockdown of HTT mRNA in different brain regions after 6 months in mice treated with 5 nmol of the siRNA molecule of the present disclosure.The x-axis of each graph shows the pattern of antisense strand and sense strand modification and the amount of siRNA molecule administered.Four bars are shown for each condition tested, and from left to right, they represent the results in frontal cortex, motor cortex, striatum and hippocampus, respectively. [Figure 12A] 1 shows knockdown of HTT mRNA in various brain regions of non-human primates treated with siRNA molecules of the present disclosure. The x-axis indicates the dose, route of administration (IT = intrathecal, ICV = intracerebroventricular), and brain region tested (fCtx = frontal cortex, mCtx = motor cortex, tCtx = temporal cortex, Hp = hippocampus, Put = putamen, Cd = caudate nucleus, SN = substantia nigra, pons, Med = medulla). [Figure 12B] 1 shows knockdown of HTT protein in various brain regions of non-human primates treated with siRNA molecules of the present disclosure. The x-axis indicates the dose, route of administration (IT = intrathecal, ICV = intracerebroventricular), and brain region tested (fCtx = frontal cortex, mCtx = motor cortex, tCtx = temporal cortex, Hp = hippocampus, Put = putamen, Cd = caudate nucleus, SN = substantia nigra, pons, Med = medulla). [Figure 12C] 1 shows the accumulation of siRNA molecules in various brain regions of non-human primates treated with the disclosed siRNA molecules. The x-axis indicates the dose, route of administration (IT = intrathecal, ICV = intracerebroventricular), and brain region tested (fCtx = frontal cortex, mCtx = motor cortex, tCtx = temporal cortex, Hp = hippocampus, Put = putamen, Cd = caudate nucleus, SN = substantia nigra, pons, Med = medulla). [Figure 13A]1 shows knockdown of HTT mRNA in the cerebellar cortex of non-human primates treated with siRNA molecules of the present disclosure. The x-axis indicates the dose and route of administration (IT = intrathecal, ICV = intracerebroventricular). "Ion conditioning" means that the siRNA molecules were formulated as salts. [Figure 13B] 1 shows knockdown of HTT protein in the cerebellar cortex of non-human primates treated with siRNA molecules of the present disclosure. The x-axis indicates the dose and route of administration (IT = intrathecal, ICV = intracerebroventricular). "Ion conditioning" means that the siRNA molecules were formulated as salts. [Figure 13C] 1 shows the accumulation of siRNA in the cerebellar cortex of non-human primates treated with siRNA molecules of the present disclosure. The x-axis indicates the dose and route of administration (IT = intrathecal, ICV = intracerebroventricular). "Ion conditioning" means that the siRNA molecules were formulated as salts. [Figure 13D] 1 shows knockdown of HTT mRNA in the deep cerebellar nuclei of non-human primates treated with siRNA molecules of the present disclosure. The x-axis indicates the dose and route of administration (IT = intrathecal, ICV = intracerebroventricular). "Ion conditioning" means that the siRNA molecules were formulated as salts. [Figure 13E] 1 shows knockdown of HTT protein in the deep cerebellar nuclei of non-human primates treated with siRNA molecules of the present disclosure. The x-axis indicates the dose and route of administration (IT = intrathecal, ICV = intracerebroventricular). "Ion conditioning" means that the siRNA molecules were formulated as salts. [Figure 13F] 1 shows the accumulation of siRNA in the deep cerebellar nuclei of non-human primates treated with siRNA molecules of the present disclosure. The x-axis indicates the dose and route of administration (IT = intrathecal, ICV = intracerebroventricular). "Ion conditioning" means that the siRNA molecules were formulated as salts. [Figure 14A]1 shows knockdown of HTT mRNA in the liver of non-human primates treated with siRNA molecules of the present disclosure. The x-axis indicates the dose and route of administration (IT = intrathecal, ICV = intracerebroventricular). "IC" means that the siRNA molecule was formulated as a salt. [Figure 14B] 1 shows knockdown of HTT protein in the liver of non-human primates treated with siRNA molecules of the present disclosure. The x-axis indicates the dose and route of administration (IT = intrathecal, ICV = intracerebroventricular). "IC" means that the siRNA molecule was formulated as a salt. [Figure 14C] 1 shows the accumulation of siRNA in the liver of non-human primates treated with siRNA molecules of the present disclosure. The x-axis indicates the dose and route of administration (IT = intrathecal, ICV = intracerebroventricular). "IC" means that the siRNA molecule was formulated as a salt. [Figure 15A] 1 shows knockdown of HTT mRNA in the kidney of non-human primates treated with siRNA molecules of the present disclosure. The x-axis indicates the dose and route of administration (IT = intrathecal, ICV = intracerebroventricular). "IC" means that the siRNA molecule was formulated as a salt. [Figure 15B] 1 shows the knockdown of HTT protein in the kidneys of non-human primates treated with siRNA molecules of the present disclosure. The x-axis indicates the dose and route of administration (IT = intrathecal, ICV = intracerebroventricular). "IC" means that the siRNA molecule was formulated as a salt. [Figure 15C] 1 shows the accumulation of siRNA in the kidneys of non-human primates treated with siRNA molecules of the present disclosure. The x-axis indicates the dose and route of administration (IT = intrathecal, ICV = intracerebroventricular). "IC" means that the siRNA molecule was formulated as a salt. [Figure 16A] 1 shows knockdown of HTT mRNA in the spinal cord of non-human primates treated with siRNA molecules of the present disclosure. The x-axis indicates the dose and route of administration (IT = intrathecal, ICV = intracerebroventricular). "Ion conditioning" means that the siRNA molecules were formulated as salts. [Figure 16B]1 shows knockdown of HTT protein in the spinal cord of non-human primates treated with siRNA molecules of the present disclosure. The x-axis indicates the dose and route of administration (IT = intrathecal, ICV = intracerebroventricular). "Ion conditioning" means that the siRNA molecules were formulated as salts. [Figure 16C] 1 shows the accumulation of siRNA in the spinal cord of non-human primates treated with siRNA molecules of the present disclosure. The x-axis indicates the dose and route of administration (IT = intrathecal, ICV = intracerebroventricular). "Ion conditioning" means that the siRNA molecules were formulated as salts. [Figure 17A] 1 shows the concentrations of siRNA molecules in the plasma of non-human primates treated with siRNA molecules of the present disclosure for animals in Group 2 as defined in Table 3 below. [Figure 17B] 1 shows the concentrations of siRNA molecules in the CSF of non-human primates treated with siRNA molecules of the present disclosure for animals in Group 2 as defined in Table 3 below. [Figure 17C] 1 shows the concentrations of siRNA molecules in the plasma of non-human primates treated with siRNA molecules of the present disclosure for animals in Group 3 as defined in Table 3 below. [Figure 17D] 1 shows the concentrations of siRNA molecules in the CSF of non-human primates treated with siRNA molecules of the present disclosure for animals in Group 3 as defined in Table 3 below. [Figure 17E] 1 shows the concentrations of siRNA molecules in the plasma of non-human primates treated with siRNA molecules of the present disclosure for animals in Group 4 as defined in Table 3 below. [Figure 17F] 1 shows the concentrations of siRNA molecules in the CSF of non-human primates treated with siRNA molecules of the present disclosure for animals in Group 4 as defined in Table 3 below. [Figure 17G] 1 shows the concentrations of siRNA molecules in the plasma of non-human primates treated with siRNA molecules of the present disclosure for animals in Group 6 as defined in Table 3 below. [Figure 17H]1 shows the concentrations of siRNA molecules in the CSF of non-human primates treated with siRNA molecules of the present disclosure for animals in Group 6 as defined in Table 3 below. [Figure 17I] 1 shows the concentrations of siRNA molecules in the plasma of non-human primates treated with siRNA molecules of the present disclosure for animals in Group 7 as defined in Table 3 below. [Figure 17J] 1 shows the concentrations of siRNA molecules in the CSF of non-human primates treated with siRNA molecules of the present disclosure for animals in Group 7 as defined in Table 3 below. [Figure 17K] 1 shows the concentrations of siRNA molecules in the plasma of non-human primates treated with siRNA molecules of the present disclosure for animals in Group 8 as defined in Table 3 below. [Figure 17L] 1 shows the concentrations of siRNA molecules in the CSF of non-human primates treated with siRNA molecules of the present disclosure for animals in Group 8 as defined in Table 3 below. [Figure 17M] 1 shows the concentrations of siRNA molecules in the plasma of non-human primates treated with siRNA molecules of the present disclosure for animals in Group 5 as defined in Table 3 below. [Figure 17N] 1 shows the concentrations of siRNA molecules in the CSF of non-human primates treated with siRNA molecules of the present disclosure for animals in Group 5 as defined in Table 3 below. [Figure 18A] 1 shows knockdown of HTT mRNA in the frontal cortex of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 4. The x-axis indicates dose and route of administration (IT = intrathecal, ICV = intracerebroventricular). [Figure 18B] 1 shows knockdown of HTT protein in the frontal cortex of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 4. The x-axis indicates dose and route of administration (IT = intrathecal, ICV = intracerebroventricular). [Figure 18C] Figure 1 shows siRNA accumulation in the frontal cortex of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 4. The x-axis indicates dose and route of administration (IT = intrathecal, ICV = intracerebroventricular). [Figure 19A] 1 shows knockdown of HTT mRNA in the motor cortex of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 4. The x-axis indicates dose and route of administration (IT = intrathecal, ICV = intracerebroventricular). [Figure 19B] 1 shows knockdown of HTT protein in the motor cortex of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 4. The x-axis indicates dose and route of administration (IT = intrathecal, ICV = intracerebroventricular). [Figure 19C] 1 shows siRNA accumulation in the motor cortex of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 4. The x-axis indicates dose and route of administration (IT = intrathecal, ICV = intracerebroventricular). [Figure 20A] 1 shows knockdown of HTT mRNA in the temporal cortex of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 4. The x-axis indicates dose and route of administration (IT = intrathecal, ICV = intracerebroventricular). [Figure 20B] 1 shows knockdown of HTT protein in the temporal cortex of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 4. The x-axis indicates dose and route of administration (IT = intrathecal, ICV = intracerebroventricular). [Figure 20C] Figure 1 shows siRNA accumulation in the temporal cortex of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 4. The x-axis indicates dose and route of administration (IT = intrathecal, ICV = intracerebroventricular). [Figure 21A] 1 shows knockdown of HTT mRNA in the hippocampus of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 4. The x-axis indicates dose and route of administration (IT = intrathecal, ICV = intracerebroventricular). [Figure 21B] 1 shows knockdown of HTT protein in the hippocampus of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 4. The x-axis indicates dose and route of administration (IT = intrathecal, ICV = intracerebroventricular). [Figure 21C] Figure 1 shows siRNA accumulation in the hippocampus of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 4. The x-axis indicates dose and route of administration (IT = intrathecal, ICV = intracerebroventricular). [Figure 22A] 1 shows knockdown of HTT mRNA in the putamen of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 4. The x-axis indicates dose and route of administration (IT = intrathecal, ICV = intracerebroventricular). [Figure 22B] 1 shows knockdown of HTT protein in the putamen of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 4. The x-axis indicates dose and route of administration (IT = intrathecal, ICV = intracerebroventricular). [Figure 22C] Figure 1 shows siRNA accumulation in the putamen of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 4. The x-axis indicates dose and route of administration (IT = intrathecal, ICV = intracerebroventricular). [Figure 23A] 1 shows knockdown of HTT mRNA in the caudate nucleus of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 4. The x-axis indicates dose and route of administration (IT = intrathecal, ICV = intracerebroventricular). [Figure 23B] 1 shows knockdown of HTT protein in the caudate nucleus of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 4. The x-axis indicates dose and route of administration (IT = intrathecal, ICV = intracerebroventricular). [Figure 23C] Figure 1 shows siRNA accumulation in the caudate nucleus of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 4. The x-axis indicates dose and route of administration (IT = intrathecal, ICV = intracerebroventricular). [Figure 24A] 1 shows knockdown of HTT mRNA in the substantia nigra of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 4. The x-axis indicates dose and route of administration (IT = intrathecal, ICV = intracerebroventricular). [Figure 24B]1 shows knockdown of HTT protein in the substantia nigra of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 4. The x-axis indicates dose and route of administration (IT = intrathecal, ICV = intracerebroventricular). [Figure 24C] Figure 1 shows siRNA accumulation in the substantia nigra of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 4. The x-axis indicates dose and route of administration (IT = intrathecal, ICV = intracerebroventricular). [Figure 25A] 1 shows knockdown of HTT mRNA in the pons of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 4. The x-axis indicates dose and route of administration (IT = intrathecal, ICV = intracerebroventricular). [Figure 25B] 1 shows knockdown of HTT protein in the pons of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 4. The x-axis indicates dose and route of administration (IT = intrathecal, ICV = intracerebroventricular). [Figure 25C] Figure 1 shows siRNA accumulation in the pons of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 4. The x-axis indicates dose and route of administration (IT = intrathecal, ICV = intracerebroventricular). [Figure 26A] 1 shows knockdown of HTT mRNA in the medulla of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 4. The x-axis indicates dose and route of administration (IT = intrathecal, ICV = intracerebroventricular). [Figure 26B] 1 shows knockdown of HTT protein in the medulla of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 4. The x-axis indicates dose and route of administration (IT = intrathecal, ICV = intracerebroventricular). [Figure 26C] Figure 1 shows the accumulation of siRNA in the medulla of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 4. The x-axis indicates the dose and route of administration (IT = intrathecal, ICV = intracerebroventricular). [Figure 27A]1 shows knockdown of HTT mRNA in the liver of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 4. The x-axis indicates dose and route of administration (IT = intrathecal, ICV = intracerebroventricular). [Figure 27B] 1 shows knockdown of HTT protein in the liver of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 4. The x-axis indicates dose and route of administration (IT = intrathecal, ICV = intracerebroventricular). [Figure 27C] Figure 1 shows siRNA accumulation in the liver of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 4. The x-axis indicates dose and route of administration (IT = intrathecal, ICV = intracerebroventricular). [Figure 28A] 1 shows knockdown of HTT mRNA in the kidney of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 4. The x-axis indicates dose and route of administration (IT = intrathecal, ICV = intracerebroventricular). [Figure 28B] 1 shows knockdown of HTT protein in the kidneys of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 4. The x-axis indicates the dose and route of administration (IT = intrathecal, ICV = intracerebroventricular). [Figure 28C] 1 shows siRNA accumulation in the kidneys of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 4. The x-axis indicates dose and route of administration (IT = intrathecal, ICV = intracerebroventricular). [Figure 29A]

[0033] Figure 1 shows knockdown of HTT mRNA in the spinal cord of non-human primates treated with siRNA molecules of the present disclosure, as described in Example 4 below. These values ​​were measured in the lumbar, thoracic, and cervical spinal cord. The x-axis indicates the dose and route of administration (IT = intrathecal, ICV = intracerebroventricular). [Figure 29B]

[0023] Figure 1 shows knockdown of HTT protein in the spinal cord of non-human primates treated with siRNA molecules of the present disclosure, as described in Example 4 below. These values ​​were measured in the lumbar, thoracic, and cervical spinal cord. The x-axis indicates the dose and route of administration (IT = intrathecal, ICV = intracerebroventricular). [Figure 30] 1 shows knockdown of HTT mRNA in the cerebellar cortex of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 4. The x-axis indicates dose and route of administration (IT = intrathecal, ICV = intracerebroventricular). [Figure 31] 1 shows knockdown of HTT mRNA in the deep nucleus of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 4. The x-axis indicates dose and route of administration (IT = intrathecal, ICV = intracerebroventricular). [Figure 32A]

[0033] Figure 1 shows the accumulation of siRNA molecules in the plasma and CSF of non-human primates administered siRNA molecules of the present disclosure, as described in Example 4 below. Results are shown for animals in Group 2 in Table 4 below. [Figure 32B]

[0033] Figure 1 shows the accumulation of siRNA molecules in the plasma and CSF of non-human primates administered siRNA molecules of the present disclosure, as described in Example 4 below. Results are shown for animals in Group 4 in Table 4 below. [Figure 32C] 1 shows the accumulation of siRNA molecules in the plasma and CSF of non-human primates administered siRNA molecules of the present disclosure, as described in Example 4 below. Results are shown for animals in Group 3 in Table 4 below. [Figure 32D] 1 shows the accumulation of siRNA molecules in the plasma and CSF of non-human primates administered siRNA molecules of the present disclosure, as described in Example 4 below. Results are shown for animals in Group 5 in Table 4 below. [Figure 33A] 1 shows knockdown of HTT mRNA in the frontal cortex of non-human primates treated with siRNA molecules of the present disclosure, as described in Example 5 below. This figure shows data from all animals, including those in which reduced CSF flow was observed upon administration. The x-axis indicates the amount administered by intrathecal injection. [Figure 33B] This figure shows the knockdown of HTT protein in the frontal cortex of non-human primates treated with siRNA molecules of the present disclosure, as described in Example 5 below. This figure shows data from all animals, including those in which reduced CSF flow was observed upon administration. The x-axis indicates the amount administered by intrathecal injection. [Figure 33C] 1 shows the accumulation of siRNA in the frontal cortex of non-human primates treated with siRNA molecules of the present disclosure, as described in Example 5 below. This figure shows data from all animals, including animals in which reduced CSF flow was observed upon administration. The x-axis shows the amount administered by intrathecal injection. The x-axis shows the amount administered by intrathecal injection. [Figure 34A] This figure shows the knockdown of HTT mRNA in the motor cortex of non-human primates treated with siRNA molecules of the present disclosure, as described in Example 5 below. This figure shows data from all animals, including those in which reduced CSF flow was observed upon administration. The x-axis indicates the amount administered by intrathecal injection. [Figure 34B] This figure shows the knockdown of HTT protein in the motor cortex of non-human primates treated with siRNA molecules of the present disclosure, as described in Example 5 below. This figure shows data from all animals, including those in which reduced CSF flow was observed upon administration. The x-axis indicates the amount administered by intrathecal injection. [Figure 34C]

[0023] Figures 1A-1C show siRNA accumulation in the motor cortex of non-human primates treated with siRNA molecules of the present disclosure, as described in Example 5 below. These figures show data from all animals, including those in which reduced CSF flow was observed upon dosing. The x-axis shows the amount administered by intrathecal injection. [Figure 35A] 1 shows knockdown of HTT mRNA in the temporal cortex of non-human primates treated with siRNA molecules of the present disclosure, as described in Example 5 below. This figure shows data from all animals, including those in which reduced CSF flow was observed upon administration. The x-axis indicates the amount administered by intrathecal injection. [Figure 35B]This figure shows knockdown of HTT protein in the temporal cortex of non-human primates treated with siRNA molecules of the present disclosure, as described in Example 5 below. This figure shows data from all animals, including those in which reduced CSF flow was observed upon administration. The x-axis indicates the amount administered by intrathecal injection. [Figure 35C] This figure shows the accumulation of siRNA in the temporal cortex of non-human primates treated with siRNA molecules of the present disclosure, as described in Example 5 below. This figure shows data from all animals, including those in which reduced CSF flow was observed upon administration. The x-axis indicates the amount administered by intrathecal injection. [Figure 36A] 1 shows knockdown of HTT mRNA in the caudate nucleus of non-human primates treated with siRNA molecules of the present disclosure, as described in Example 5 below. This figure shows data from all animals, including those in which reduced CSF flow was observed upon administration. The x-axis indicates the amount administered by intrathecal injection. [Figure 36B] This figure shows the knockdown of HTT protein in the caudate nucleus of non-human primates treated with siRNA molecules of the present disclosure, as described in Example 5 below. This figure shows data from all animals, including those in which reduced CSF flow was observed upon administration. The x-axis indicates the amount administered by intrathecal injection. [Figure 36C] This figure shows the accumulation of siRNA in the caudate nucleus of non-human primates treated with siRNA molecules of the present disclosure, as described in Example 5 below. This figure shows data from all animals, including those in which reduced CSF flow was observed upon administration. The x-axis indicates the amount administered by intrathecal injection. [Figure 37A] 1 shows knockdown of HTT mRNA in the putamen of non-human primates treated with siRNA molecules of the present disclosure, as described in Example 5 below. This figure shows data from all animals, including those in which reduced CSF flow was observed upon administration. The x-axis indicates the amount administered by intrathecal injection. [Figure 37B]This figure shows knockdown of HTT protein in the putamen of non-human primates treated with siRNA molecules of the present disclosure, as described in Example 5 below. This figure shows data from all animals, including those in which reduced CSF flow was observed upon administration. The x-axis shows the amount administered by intrathecal injection. [Figure 37C] This figure shows the accumulation of siRNA in the putamen of non-human primates treated with siRNA molecules of the present disclosure, as described in Example 5 below. This figure shows data from all animals, including those in which reduced CSF flow was observed upon administration. The x-axis indicates the amount administered by intrathecal injection. [Figure 38A] This figure shows the knockdown of HTT mRNA in the hippocampus of non-human primates treated with siRNA molecules of the present disclosure, as described in Example 5 below. This figure shows data from all animals, including those in which reduced CSF flow was observed upon administration. The x-axis indicates the amount administered by intrathecal injection. [Figure 38B] This figure shows the knockdown of HTT protein in the hippocampus of non-human primates treated with siRNA molecules of the present disclosure, as described in Example 5 below. This figure shows data from all animals, including those in which reduced CSF flow was observed upon administration. The x-axis indicates the amount administered by intrathecal injection. [Figure 38C] This figure shows the accumulation of siRNA in the hippocampus of non-human primates treated with siRNA molecules of the present disclosure, as described in Example 5 below. This figure shows data from all animals, including those in which reduced CSF flow was observed upon administration. The x-axis indicates the amount administered by intrathecal injection. [Figure 39A] 1 shows knockdown of HTT mRNA in the cervical spinal cord of non-human primates treated with siRNA molecules of the present disclosure, as described in Example 5 below. The figure shows data from all animals, including those in which reduced CSF flow was observed upon dosing. The x-axis indicates the amount administered by intrathecal injection. [Figure 39B]

[0039] Figure 1 shows knockdown of HTT protein in the cervical spinal cord of non-human primates treated with siRNA molecules of the present disclosure, as described in Example 5 below. This figure shows data from all animals, including those in which reduced CSF flow was observed upon dosing. The x-axis indicates the amount administered by intrathecal injection. [Figure 39C] 1 shows the accumulation of siRNA in the cervical spinal cord of non-human primates treated with siRNA molecules of the present disclosure, as described in Example 5 below. The figure shows data from all animals, including those in which reduced CSF flow was observed upon dosing. The x-axis shows the amount administered by intrathecal injection. [Figure 40A] 1 shows knockdown of HTT mRNA in the thoracic spinal cord of non-human primates treated with siRNA molecules of the present disclosure, as described in Example 5 below. This figure shows data from all animals, including those in which reduced CSF flow was observed upon dosing. The x-axis indicates the amount administered by intrathecal injection. [Figure 40B] 1 shows knockdown of HTT protein in the thoracic spinal cord of non-human primates treated with siRNA molecules of the present disclosure, as described in Example 5 below. This figure shows data from all animals, including those in which reduced CSF flow was observed upon administration. The x-axis indicates the amount administered by intrathecal injection. [Figure 40C] This figure shows the accumulation of siRNA in the thoracic spinal cord of non-human primates treated with siRNA molecules of the present disclosure, as described in Example 5 below. This figure shows data from all animals, including those in which reduced CSF flow was observed upon administration. The x-axis indicates the amount administered by intrathecal injection. [Figure 41A] 1 shows knockdown of HTT mRNA in the lumbar spinal cord of non-human primates treated with siRNA molecules of the present disclosure, as described in Example 5 below. This figure shows data from all animals, including those in which reduced CSF flow was observed upon administration. The x-axis indicates the amount administered by intrathecal injection. [Figure 41B]This figure shows knockdown of HTT protein in the lumbar spinal cord of non-human primates treated with siRNA molecules of the present disclosure, as described in Example 5 below. This figure shows data from all animals, including those in which reduced CSF flow was observed upon administration. The x-axis indicates the amount administered by intrathecal injection. [Figure 41C] This figure shows the accumulation of siRNA in the lumbar spinal cord of non-human primates treated with siRNA molecules of the present disclosure, as described in Example 5 below. This figure shows data from all animals, including those in which reduced CSF flow was observed upon administration. The x-axis indicates the amount administered by intrathecal injection. [Figure 42A] This figure shows the knockdown of HTT mRNA in the liver of non-human primates treated with siRNA molecules of the present disclosure, as described in Example 5 below. This figure shows data from all animals, including those in which reduced CSF flow was observed upon dosing. The x-axis shows the amount administered by intrathecal injection. [Figure 42B] This figure shows the knockdown of HTT protein in the liver of non-human primates treated with siRNA molecules of the present disclosure, as described in Example 5 below. This figure shows data from all animals, including those in which reduced CSF flow was observed upon administration. The x-axis indicates the amount administered by intrathecal injection. [Figure 42C] This figure shows the accumulation of siRNA in the liver of non-human primates treated with siRNA molecules of the present disclosure, as described in Example 5 below. This figure shows data from all animals, including those in which reduced CSF flow was observed upon administration. The x-axis indicates the amount administered by intrathecal injection. [Figure 43A] 1 shows the knockdown of HTT mRNA in the kidneys of non-human primates treated with siRNA molecules of the present disclosure, as described in Example 5 below. This figure shows data from all animals, including those in which reduced CSF flow was observed upon dosing. The x-axis indicates the amount administered by intrathecal injection. [Figure 43B]1 shows the knockdown of HTT protein in the kidneys of non-human primates treated with siRNA molecules of the present disclosure, as described in Example 5 below. These figures show data from all animals, including those in which reduced CSF flow was observed upon administration. The x-axis shows the amount administered by intrathecal injection. [Figure 43C] Figures 1A and 1B show the accumulation of siRNA in the kidneys of non-human primates treated with siRNA molecules of the present disclosure, as described in Example 5 below. These figures show data from all animals, including those in which reduced CSF flow was observed upon administration. The x-axis shows the amount administered by intrathecal injection. [Figure 44A] 1 shows knockdown of HTT mRNA in the frontal cortex of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 5, excluding animals with occluded catheters. The x-axis indicates the amount administered by intrathecal injection. [Figure 44B] 1 shows knockdown of HTT protein in the frontal cortex of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 5, excluding animals with occluded catheters. The x-axis indicates the amount administered by intrathecal injection. [Figure 44C] Figure 1 shows siRNA accumulation in the frontal cortex of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 5, excluding animals with blocked catheters. The x-axis indicates the amount administered by intrathecal injection. [Figure 45A] 1 shows knockdown of HTT mRNA in the motor cortex of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 5, excluding animals with occluded catheters. The x-axis indicates the amount administered by intrathecal injection. [Figure 45B] 1 shows knockdown of HTT protein in the motor cortex of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 5, excluding animals with occluded catheters. The x-axis indicates the amount administered by intrathecal injection. [Figure 45C]Figure 1 shows siRNA accumulation in the motor cortex of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 5, excluding animals with occluded catheters. The x-axis indicates the amount administered by intrathecal injection. [Figure 46A] 1 shows knockdown of HTT mRNA in the temporal cortex of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 5, excluding animals with occluded catheters. The x-axis indicates the amount administered by intrathecal injection. [Figure 46B] 1 shows knockdown of HTT protein in the temporal cortex of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 5, excluding animals with occluded catheters. The x-axis indicates the amount administered by intrathecal injection. [Figure 46C] Figure 1 shows siRNA accumulation in the temporal cortex of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 5, excluding animals with occluded catheters. The x-axis indicates the amount administered by intrathecal injection. [Figure 47A] 1 shows knockdown of HTT mRNA in the caudate nucleus of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 5, excluding animals with occluded catheters. The x-axis indicates the amount administered by intrathecal injection. [Figure 47B] 1 shows knockdown of HTT protein in the caudate nucleus of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 5, excluding animals with occluded catheters. The x-axis indicates the amount administered by intrathecal injection. [Figure 47C] Figure 1 shows siRNA accumulation in the caudate nucleus of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 5, excluding animals with blocked catheters. The x-axis indicates the amount administered by intrathecal injection. [Figure 48A]1 shows knockdown of HTT mRNA in the putamen of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 5, excluding animals with occluded catheters. The x-axis indicates the amount administered by intrathecal injection. [Figure 48B] 1 shows knockdown of HTT protein in the putamen of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 5, excluding animals with occluded catheters. The x-axis indicates the amount administered by intrathecal injection. [Figure 48C] Figure 1 shows siRNA accumulation in the putamen of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 5, excluding animals with blocked catheters. The x-axis indicates the amount administered by intrathecal injection. [Figure 49A] 1 shows knockdown of HTT mRNA in the hippocampus of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 5, excluding animals with occluded catheters. The x-axis indicates the amount administered by intrathecal injection. [Figure 49B] 1 shows knockdown of HTT protein in the hippocampus of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 5, excluding animals with occluded catheters. The x-axis indicates the amount administered by intrathecal injection. [Figure 49C] Figure 1 shows siRNA accumulation in the hippocampus of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 5, excluding animals with occluded catheters. The x-axis indicates the amount administered by intrathecal injection. [Figure 50A] 1 shows knockdown of HTT mRNA in the cervical spinal cord of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 5, excluding animals with occluded catheters. The x-axis indicates the amount administered by intrathecal injection. [Figure 50B]1 shows knockdown of HTT protein in the cervical spinal cord of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 5, excluding animals with blocked catheters. The x-axis indicates the amount administered by intrathecal injection. [Figure 50C] Figure 1 shows siRNA accumulation in the cervical spinal cord of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 5, excluding animals with blocked catheters. The x-axis indicates the amount administered by intrathecal injection. [Figure 51A] 1 shows knockdown of HTT mRNA in the thoracic spinal cord of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 5, excluding animals with occluded catheters. The x-axis indicates the amount administered by intrathecal injection. [Figure 51B] 1 shows knockdown of HTT protein in the thoracic spinal cord of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 5, excluding animals with blocked catheters. The x-axis indicates the amount administered by intrathecal injection. [Figure 51C] Figure 1 shows siRNA accumulation in the thoracic spinal cord of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 5, excluding animals with blocked catheters. The x-axis indicates the amount administered by intrathecal injection. [Figure 52A] 1 shows knockdown of HTT mRNA in the lumbar spinal cord of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 5, excluding animals with occluded catheters. The x-axis indicates the amount administered by intrathecal injection. [Figure 52B] 1 shows knockdown of HTT protein in the lumbar spinal cord of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 5, excluding animals with occluded catheters. The x-axis indicates the amount administered by intrathecal injection. [Figure 52C]Figure 1 shows siRNA accumulation in the lumbar spinal cord of non-human primates treated with siRNA molecules of the present disclosure, as described below in Example 5, excluding animals with occluded catheters. The x-axis indicates the amount administered by intrathecal injection.

[0095] definition Unless otherwise defined herein, scientific and technical terms used herein have the meanings commonly understood by those of ordinary skill 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 "includes" and "included" is not limiting.

[0096] As used herein, the term "nucleic acid" refers to an RNA or DNA molecule composed of a chain of ribonucleotides or deoxyribonucleotides, respectively.

[0097] 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.

[0098] 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.

[0099] As used herein, the term "nucleoside" refers to a molecule consisting of a heterocyclic base and its sugar.

[0100] 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.

[0101] In the context of this disclosure, 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.

[0102] 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 single strands that optionally form a hairpin structure containing the double-stranded region.

[0103] 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.

[0104] As used herein, the term "sense strand" refers to the strand of an siRNA duplex that contains complementarity to the antisense strand.

[0105] As used herein, the term "divalent cation" refers to a positively charged ion (i.e., a cation) having a valence of 2+. Examples of divalent cations include Ba 2+ , Be 2+ , Ca 2+ , Cu 2+ , Mg 2+ , Mn 2+ , Ni 2+ , or Zn 2+ Due to their positive charge, divalent cations usually form ionic bonds with negatively charged atoms (e.g., oxyanions from phosphate or phosphorothioate groups, which have unit or partial negative charges).

[0106] As used herein, the terms "ionic radius" and "ionic radii" refer to the radius of one or more monoatomic ions (e.g., divalent cations) when measured in the form of an ionic crystal structure. Ionic radii are typically measured in picometers or angstroms.

[0107] As used herein, the term "salt" refers to any compound containing an ionic association between an anionic component (e.g., an oxyanion from a phosphate or phosphorothioate group bearing a unit or partial negative charge) and a cationic component (e.g., a divalent cation). Salts can have a variety of physical forms. For example, a salt may be a solid, crystalline, ionic compound, or may be in the form of a solution in which the salt is dissolved in a solvent and the salt's component ions are miscible with the solvent (e.g., water or another polar protic solvent). Salts may also be present in a suspension, such as a suspension formed by contacting (i) a homogeneous solution containing the salt of interest and a first solvent with (ii) a second solvent that is not completely miscible with the first solvent. An example of a suspension is one formed by contacting an aqueous solution containing the salt of interest with a solvent that is not completely miscible with water, such as an organic solvent containing one or more nonpolar functional groups. In the context of this disclosure, a "salt" includes a solution containing one or more divalent cations (e.g., Ba, 2+ , Be 2+ , Ca 2+ , Cu2+ , Mg 2+ , Mn 2+ , Ni 2+ , or Zn 2+ , or a combination thereof).

[0108] The term "cationic binding site" refers to a substituent in an siRNA molecule that carries either a partial or unit negative charge (e.g., an oxyanion of phosphate or phosphorothioate) and is capable of forming an ionic association with a cation (e.g., a divalent cation).

[0109] The term "saturation" refers to the relative proportion of cationic binding sites that are ionically bound by a particular cationic species (eg, a divalent cation).

[0110] The term "hard Lewis acid" refers to a chemical acid characterized by a low ionic radius, a high positive charge density, a strong ability to displace water, and a high-energy lowest unoccupied molecular orbital (LUMO).

[0111] 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.

[0112] As used herein, the terms "targeting," "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 such a manner as to reduce translation of the mRNA into a protein product.

[0113] 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 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., in the case of an enzyme, assessed using enzyme activity assays described herein or known in the art). As used herein, a cell is considered to "express" a gene or protein of interest if one or more, or all, of the above events are detectable within the cell or in 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 (i) the production by the cell, or population of cells, of a corresponding RNA transcript, such as an mRNA template (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) is detectable.

[0114] 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 to alter certain chemical properties of the nucleotide while retaining the ability of the nucleotide analog to perform its intended function.

[0115] 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.

[0116] 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.

[0117] As used herein, the terms "internucleoside" and "internucleotide" refer to the linkage between a nucleoside and a nucleotide, respectively.

[0118] As used herein, the term "antagomir" refers to a nucleic acid that can function as an inhibitor of miRNA activity.

[0119] 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.

[0120] As used herein, the term "mixmer" refers to a nucleic acid that contains a mixture of locked nucleic acid (LNA) and DNA.

[0121] 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.

[0122] 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 ", where 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 ", where 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 ", where this siRNA molecule comprises four siRNA molecules that are covalently linked to each other, for example, via a linker.

[0123] 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.

[0124] 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 preferably 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 can each independently contain one to three phosphate moieties that are unmodified (diphosphate or triphosphate) or modified.

[0125] 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 preferably 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 can each independently contain one to three phosphate moieties that are unmodified (diphosphate or triphosphate) or modified.

[0126] Certain internucleoside linkages provided herein, including, for example, phosphodiesters and phosphorothioates, contain a formal charge of −1 at physiological pH, which may be mediated by a cationic moiety, e.g., an alkali metal such as sodium or potassium, an alkaline earth metal such as calcium or magnesium, or an ammonium or guanidinium ion, or multiple divalent cations (e.g., Ba, 2+ , Be 2+ , Ca 2+ , Cu 2+ , Mg 2+ , Mn 2+ , Ni 2+ , Zn 2+ , or a combination thereof).

[0127] 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 other substitutions that allow the nucleotide to perform its intended function, such as those described in, for example, Eckstein, Antisense Nucleic Acid Drug Dev. 10:117-21, 2000; Rusckowski et al., Antisense Nucleic Acid Drug Dev. 10:333-45, 2000; Stein, Antisense Nucleic Acid Drug Dev. 11:317-25, 2001; Vorobjev et al., Antisense Nucleic Acid Drug Dev. 11:77-85, 2001; and US 5,684,143. Some of the above-referenced modifications (e.g., phosphate group modifications) preferably reduce the hydrolysis rate of polynucleotides containing the analogs in vivo or in vitro, for example.

[0128] 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.

[0129] "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 particular 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.

[0130] "Percent sequence identity (%)" with respect to a reference polynucleotide or 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 polypeptide sequence, after aligning the sequences to achieve the maximum percent sequence identity and introducing gaps as necessary.Alignment for determining percent nucleic acid or amino acid sequence identity 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. By way of 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 may alternatively be expressed 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 that program's alignment of A and B, and Y is the total number of nucleic acids in B. It will be recognized that 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 equal the percent sequence identity of B to A.

[0131] As used herein, the term "sufficiently complementary to hybridize" refers to a nucleic acid sequence or portion thereof that does not necessarily have 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 over the same portion of its length.

[0132] 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.

[0133] 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 well 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., NaCl concentration), e.g., 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).

[0134] The term "gene silencing" refers to the suppression of gene expression, for example, endogenous gene expression of HTT, which can be mediated through processes that affect transcription and / or processes that affect post-transcriptional mechanisms. In some embodiments, gene silencing occurs when an RNAi molecule initiates the inhibition or degradation of mRNA transcribed from a gene of interest in a sequence-specific manner by RNA interference, thereby preventing the translation of the gene product.

[0135] 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).

[0136] As used herein, the term "ethylene glycol chain" refers to a carbon chain having the formula ((CH2OH)2).

[0137] 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.

[0138] 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.

[0139] 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, iso-pentynyl, and tert-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.

[0140] 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.

[0141] 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.

[0142] As used herein, the term "amide" refers to an alkyl, alkenyl, alkynyl, or aromatic group attached to an aminocarbonyl functional group.

[0143] 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.

[0144] As used herein, the term "end group" refers to the group at which a carbon chain or nucleic acid ends.

[0145] 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.

[0146] 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).

[0147] 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).

[0148] 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.

[0149] As used herein, the term "between X and Y" includes values ​​of X and Y. For example, "between X and 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.

[0150] As used herein, the terms "subject" and "patient" are used interchangeably and refer to an organism, such as a mammal (e.g., a human), that experiences a neurodegenerative disease or disorder (e.g., Huntington's disease) and / or contains a gain-of-function HTT variant allele.

[0151] As used herein, the term "HTT" refers to the gene encoding huntingtin, including any native HTT gene from any source. The term encompasses "full-length," unprocessed HTT and any form of HTT resulting from processing in cells. The term also encompasses naturally occurring variants of HTT, such as splice variants or allelic variants. The nucleic acid sequence of an exemplary HTT gene is set forth in European Nucleotide Archive (ENA) Accession No. AB016794.1. The amino acid sequence of an exemplary protein encoded by the HTT gene is set forth in UNIPROT™ Accession No. P42858.

[0152] As used herein, the terms "microsatellite repeat expansion disorder," "microsatellite repeat expansion disease," "nucleotide repeat expansion disorder," and "nucleotide repeat expansion disease" are used interchangeably to refer to any disease or disorder caused by the instability and expansion of specific microsatellites. A "microsatellite" is a coding or non-coding DNA sequence containing a tandem repeat of base pairs. Exemplary microsatellite repeat expansion disorders include Fragile X syndrome, Fragile XE syndrome, Fragile X-associated tremor / ataxia syndrome, Fragile X primary ovarian failure, progressive myoclonic epilepsy type 1 / Unverricht-Lundborg disease, spinocerebellar ataxia (SCA) 12, neuronal intranuclear inclusion disease, glutaminase deficiency, Huntington's disease, SCA1, SCA2, SCA3, SCA6, SCA7, SCA17, dentatorubral-pallidoluysian atrophy, and spinal-bulbar muscular atrophy. Other microsatellite repeat expansion disorders include, but are not limited to, oculopharyngeal muscular dystrophy, Huntington's disease type 2, amyotrophic lateral sclerosis, myotonic dystrophy type 2 (DM2), Friedrich ataxia, Fuchs corneal endothelial dystrophy, SCA10, SCA31, SCA36, SCA37, cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS), benign adult familial myoclonic epilepsy, SCA8, and myotonic dystrophy type 1 (DM1). Other microsatellite repeat expansion disorders are described in Rodriguez et al., Neurobiology of Disease, 130:104515, 2019, the disclosure of which is incorporated herein by reference.

[0153] 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 remitting (whether partial or complete) the 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 the 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.

[0154] As used herein, the terms "benefit" and "response" are used interchangeably, for example, in the context of a subject receiving therapy for the treatment of Huntington's disease. For example, clinical benefit in the context of a subject with Huntington's disease administered an siRNA molecule or siRNA composition of the present disclosure includes, but is not limited to, a reduction in involuntary movements, memory loss, mood swings, or symptoms of anxiety and depression, and / or a reduction in wild-type HTT transcripts, mutant HTT transcripts, variant HTT transcripts, splice isoforms of HTT transcripts, and / or overexpressed HTT transcripts. DETAILED DESCRIPTION OF THE INVENTION

[0155] The present disclosure provides compositions of small interfering RNA (siRNA) molecules with sequence homology to the huntingtin (HTT) 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 include 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.

[0156] The siRNA molecules of the present disclosure may, for example, exhibit potent gene-specific suppression of HTT relative to other genes.

[0157] The siRNA molecules of the present disclosure can feature an antisense strand having a nucleic acid sequence that is complementary to a region within an HTT mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1 to 3. The degree of complementarity of the antisense strand to the region of the HTT mRNA transcript can be sufficient for the antisense strand to anneal over the entire length of the region of the HTT 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 an HTT mRNA transcript. In some embodiments, the region of an HTT RNA transcript has the sequence of SEQ ID NO: 1. In some embodiments, the region of an HTT RNA transcript has the sequence of SEQ ID NO: 2. In some embodiments, the region of the HTT RNA transcript has the sequence of SEQ ID NO:3.

[0158] In some embodiments, the siRNA molecules of the present disclosure are characterized by an antisense strand having a nucleic acid sequence at least 60% identical to, or at least 60% identical to, any one of SEQ ID NOs: 7-9. For example, the siRNA molecules of the present disclosure can be characterized by an antisense strand having a nucleic acid sequence 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: 7-9. In some embodiments, the nucleic acid sequence is SEQ ID NO: 7. In some embodiments, the nucleic acid sequence is SEQ ID NO: 8. In some embodiments, the nucleic acid sequence is SEQ ID NO: 9.

[0159] In some embodiments, the siRNA molecules of the present disclosure are characterized by a sense strand having a nucleic acid sequence at least 60% identical to, or at least 60% identical to, any one of SEQ ID NOs: 4-6. For example, the siRNA molecules of the present disclosure can be characterized by a sense strand having a nucleic acid sequence 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: 4-6. In some embodiments, the nucleic acid sequence is SEQ ID NO: 4. In some embodiments, the nucleic acid sequence is SEQ ID NO: 5. In some embodiments, the nucleic acid sequence is SEQ ID NO: 6.

[0160] 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 HTT mRNA transcript targeted by each antisense strand. [Table 1]

[0161] 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 having a 2' sugar modification.

[0162] 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.

[0163] 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.

[0164] 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.

[0165] The siRNA molecules described herein may employ modifications to the nucleobases, phosphate backbone, ribose core, 5' and 3' ends, and branching, where multiple strands of the siRNA can be covalently linked.

[0166] Length of the small interfering RNA molecule Within the scope of the present disclosure, 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 10 to 30 nucleotides (e.g., 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides), 15 to 25 nucleotides (e.g., 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, or 25 nucleotides), or 18 to 23 nucleotides (e.g., 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, or 23 nucleotides). In some embodiments, the antisense strand is 20 nucleotides. In some embodiments, the antisense strand is 21 nucleotides. In some embodiments, the antisense strand is 22 nucleotides. In some embodiments, the antisense strand is 23 nucleotides. In some embodiments, the antisense strand is 24 nucleotides. In some embodiments, the antisense strand is 25 nucleotides. In some embodiments, the antisense strand is 26 nucleotides. In some embodiments, the antisense strand is 27 nucleotides. In some embodiments, the antisense strand is 28 nucleotides. In some embodiments, the antisense strand is 29 nucleotides. In some embodiments, the antisense strand is 30 nucleotides.

[0167] In some embodiments, the sense strand of an siRNA molecule of the present disclosure is 12 to 30 nucleotides (e.g., 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides), or 14 to 23 nucleotides (e.g., 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, or 23 nucleotides). In some embodiments, the sense strand is 15 nucleotides in 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.

[0168] 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 with 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 comprises 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.

[0169] Nucleobase Modification The siRNA molecules of the present disclosure can 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 disclosure. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases, also referred to herein as heterocyclic base moieties, include other synthetic and natural nucleobases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl 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 7-deazaguanine and 3-deazaguanine. Nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone.Additional nucleobases include those disclosed in US3,687,808, 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 molecule of the present disclosure can also comprise polycyclic heterocyclic compounds instead of one or more heterocyclic base moieties.Many tricyclic heterocyclic compounds have been reported so far.These compounds are routinely used in antisense applications to increase the binding properties of modified strand to target strand.

[0170] Representative cytosine analogs that form three hydrogen bonds with guanosine in the second strand include 1,3-diazaphenoxazine-2-one (Kurchavov et al., Nucleosides and Nucleotides, 16:1837-46, 1997), 1,3-diazaphenothiazine-2-one (Lin et al., Am. Chem. Soc., 117:3873-4, 1995), and 6,7,8,9-tetrafluoro-1,3-diazaphenoxazine-2-one (Wang et al., Tetrahedron Lett., 39:8385-8, 1998). These base modifications have been shown to hybridize with complementary guanine when incorporated into oligonucleotides, and guanine has also been shown to hybridize with adenine, improving the thermal stability of the helix through extended stacking interactions (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).

[0171] Internucleoside bond modification Another variable in the design of the present disclosure 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 the present disclosure, but not limited to, is to protect part or the whole of siRNA molecules from hydrolysis.One example of the modification that reduces the hydrolysis rate is phosphorothioate.Any part or the whole of the backbone may contain phosphate substitution (for example, phosphorothioate). For example, the internucleoside linkages may be between 0 and 100% phosphorothioate, e.g., 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% 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.

[0172] 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, 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 having normal 3'-5' linkages, 2'-5' linked analogs thereof, and those having 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.

[0173] 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 with 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, 5,480,968, 5,490,970, 5,510,106, 5,520,108, 5,530,109, 5,540,110, 5,550,112, 5,561,114, 5,570,116, 5,580,118, 5,590,120, 5,591,122, 5,592,124, 5,593,126, 5,594,128, 5,595,129, 5,596,130, 5,597,140, ​​5,598,141, 5,599,150, 5,599,151, 5,599,152, 5,599,153, 5,599,160, 5,599,161, 5,599,162, 5,599,163, 5,599,170, 5,599,171, 5,599,182, 5,599,1 ,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.

[0174] 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.

[0175] 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 as follows: AB-(A')jC-P2-D-P1-(C'-P1)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.

[0176] 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.

[0177] 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 as follows: 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.

[0178] In some embodiments of the present disclosure, 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.

[0179] In some embodiments of the present disclosure, the antisense strand comprises a structure represented by Formula A2′, which, in the 5′ to 3′ direction, is as follows: ASBSAOBOBOBOAOBOAOBOA-OBOAOBSASBSASASASBSA 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.

[0180] 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.

[0181] 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 as follows: 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.

[0182] 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.

[0183] 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.

[0184] 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.

[0185] 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 as follows: 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 strand is complementary (e.g., fully or partially complementary) to a target nucleic acid.

[0186] In some embodiments of the present disclosure, 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.

[0187] 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' to 3' direction, as follows: 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.

[0188] 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.

[0189] 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' 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.

[0190] 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.

[0191] In some embodiments of the present disclosure, the siRNA may contain an antisense strand comprising a region represented by Formula VI, where Formula VI is, in the 5' to 3' direction, as follows: AB j -EB k -EFG l -DP 1 -C' Formula VI; where A is a compound of formula CP 1 -DP 1 Each B is represented by the formula CP 2 each C is a 2'-O-Me ribonucleoside; each C' is independently a 2'-O-Me ribonucleoside or a 2'-F ribonucleoside; each D is a 2'-F ribonucleoside; and each E is represented by the formula DP 2 -CP 2 F is represented by the formula DP 1 -CP 1 Each G is represented by the formula CP 1 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); 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.

[0192] In some embodiments of the present disclosure, 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.

[0193] 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 as follows: HB m -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.

[0194] In some embodiments of the present disclosure, the sense strand comprises 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.

[0195] 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).

[0196] siRNA synthesis method The siRNA molecules of the present disclosure can be synthesized by standard methods well 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.

[0197] 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.

[0198] Furthermore, it is contemplated that further optimization can be achieved for any siRNA agent disclosed herein 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).

[0199] The siRNA molecules of the present disclosure may contain one or more divalent cations (e.g., Ba 2+ , Be 2+ , Ca 2+ , Cu 2+ , Mg 2+ , Mn 2+ , Ni 2+ , or Zn 2+ , or a combination thereof). The composition may be prepared to contain multiple cationic binding sites saturated with divalent cations. For example, the composition may be prepared by hybridizing the therapeutic oligonucleotide molecule in the presence of divalent cations. Alternatively, the composition may be prepared by hybridizing the therapeutic oligonucleotide molecule without divalent cations and then adding divalent cations after hybridization. In the case of multiple divalent cations, the divalent cations may be added simultaneously or sequentially. For example, the therapeutic oligonucleotide molecule may be hybridized in the presence of two divalent cations. Alternatively, the therapeutic oligonucleotide molecule may be hybridized in the presence of one divalent cation, and then the second divalent cation is added after hybridization. As a further alternative, the therapeutic oligonucleotide molecule may be hybridized without divalent cations and then two divalent cations are added.

[0200] divalent cations The therapeutic oligonucleotides of the present disclosure are those in which the oxyanion moiety is Ba 2+ , Be 2+ , Ca 2+ , Cu 2+ , Mg 2+ , Mn 2+ , Ni 2+ , or Zn 2+ The nucleic acid may comprise one or more phosphodiester internucleoside linkages, such as phosphorothioate internucleoside linkages, electrostatically neutralized by ionic bonding with a divalent metal cation, such as methyltrimethylsilyl ...

[0201] The siRNA molecules of the present disclosure may contain one or more divalent cations (e.g., Ba 2+ , Be 2+ , Ca 2+ , Cu 2+ , Mg 2+ , Mn 2+ , Ni 2+ , or Zn 2+ , or combinations thereof). Due to their positive charge, divalent cations are typically reactive with negatively charged atoms (e.g., oxyanions from phosphate or phosphorothioate groups, which have unit or partial negative charges). The present disclosure provides novel evidence that saturating cationic binding sites on therapeutic oligonucleotide molecules with divalent cations significantly reduces toxicity when administered to the CNS of a subject.

[0202] The one or more divalent cations can have an ionic radius, when measured in the form of a crystal lattice, of about 30 picometers to about 150 picometers (e.g., about 30 picometers to about 140 picometers, about 40 picometers to about 130 picometers, about 50 picometers to about 120 picometers, about 60 picometers to about 110 picometers, about 60 picometers to about 100 picometers, or about 60 picometers to about 90 picometers). The calculated crystal radii of divalent cations disclosed by RD Shannon, Acta Crystallographica A. 32:751-767, 1976, are incorporated herein by reference.

[0203] The degree of saturation of the cationic binding sites of a therapeutic oligonucleotide molecule with one or more divalent cations can range from about 10% to about 100% (e.g., about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, or about 90% to about 100%).

[0204] In some embodiments, the antisense strand of a therapeutic oligonucleotide molecule may have a length of 10 to 30 nucleotides and may be ionically bound to a total of 10 to 30 divalent cations. For example, the molar ratio of antisense strand nucleotides to divalent cations in a therapeutic oligonucleotide molecule may be 1:3 to 3:1 (e.g., 1:3, 1.1:3, 1.2:3, 1.3:3, 1.4:3, 1.5:3, 1.6:3, 1.7:3, 1.8:3, 1.9:3, 2:3, 2.1:3, 2.2:3, 2.3:3, 2.4:3, 2.5:3, 2.6:3, 2.7:3, 2.8:3, 2.9:3, 3.0:3, 3.1:3, 3.2:3, 3.3:3, 3.4:3, 3.5:3, 3.6:3, 3.7:3, 3.8:3, 3.9 ... 3:1, 3:1.9, 3:1.8, 3:1.7, 3:1.6, 3:1.5, 3:1.4, 3:1.3, 3:1.2, 3:1.1, or 3:1).

[0205] In some embodiments, the sense strand of a therapeutic oligonucleotide molecule may have a length of 10-30 nucleotides and may be ionically bound to a total of 10-30 divalent cations. For example, the molar ratio of sense strand nucleotides to divalent cations in a therapeutic oligonucleotide molecule may be 1:3 to 3:1 (e.g., 1:3, 1.1:3, 1.2:3, 1.3:3, 1.4:3, 1.5:3, 1.6:3, 1.7:3, 1.8:3, 1.9:3, 2:3, 2.1:3, 2.2:3, 2.3:3, 2.4:3, 2.5:3, 2.6:3, 2.7:3, 2.8:3, 2.9:3, 3.0:3, 3.1:3, 3.2:3, 3.3:3, 3.4:3, 3.5:3, 3.6:3, 3.7:3, 3.8:3, 3.9:3, 3.10:3, 3.11:3, 3.12:3, 3.13:3, 3.14:3, 3.15:3, 3.16:3, 3.17:3, 3.18:3, 3.19:3, 3.20:3, 3.21:3, 3.22:3, 3.23:3, 3.24:3, 3.25:3, 3.26:3, 3.27:3, 3.28:3, 3.29:3, 3.30:3, 3.31:3, 3.32:3, 3.33:3, 3.34:3, 3.35:3, 3.36 3:1, 3:1.9, 3:1.8, 3:1.7, 3:1.6, 3:1.5, 3:1.4, 3:1.3, 3:1.2, 3:1.1, or 3:1).

[0206] The therapeutic oligonucleotide molecules of the present disclosure can be combined with one or more divalent cations in a specific molar ratio, which can be related to the toxicity benefits provided by the divalent cations. For example, the molar ratio of therapeutic oligonucleotide molecules to divalent cations can range from 1:10 to 1:50 (e.g., 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40, 1:41, 1:42, 1:43, 1:44, 1:45, 1:46, 1:47, 1:48, 1:49, or 1:50). In some embodiments, the molar ratio of therapeutic oligonucleotide molecules to divalent cations can be in the range of 1:18 to 1:38 (e.g., 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, or 1:38). In some embodiments, the molar ratio of therapeutic oligonucleotide molecules to divalent cations can be in the range of 1:20 to 1:25 (e.g., 1:20, 1:21, 1:22, 1:23, 1:24, or 1:25). In some embodiments, the molar ratio of therapeutic oligonucleotide molecules to divalent cations can be 1:20. In some embodiments, the molar ratio of therapeutic oligonucleotide to divalent cation may be 1:25.

[0207] The therapeutic oligonucleotides of the present disclosure can be combined with one or more divalent cations, where the divalent cations are present at a particular concentration or concentration range, which can be related to the toxicity benefit provided by the divalent cations. For example, the concentration of divalent cations can range from 20 mM to 150 mM (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, , 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 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, or 150 mM). In some embodiments, the concentration of divalent cations is between 20 mM and 100 mM (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 mM).In some embodiments, the concentration of divalent cations can be between 35 mM and 75 mM (e.g., 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75 mM). In some embodiments, the concentration of divalent cations can be between 40 mM and 70 mM (e.g., 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 mM).

[0208] The therapeutic oligonucleotide may comprise one or more atoms having a negative charge, and the divalent cation may comprise a positive charge. In some embodiments, the therapeutic oligonucleotide and the divalent cation are present in an amount that results in a specific ratio of negative charge to positive charge present in the composition. Methods for determining the ratio of negative charge to positive charge are well known in the art, for example, in Furst et al., Electrophoresis., 37:2685-2691, 2016, the disclosure of which is incorporated herein by reference. In some embodiments, the ratio of negative to positive charges is between 0.75 and 7.5 (e.g., 0.76, 0.77, 0.78, 0.79, 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.8, 7.9, 8. 0.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5). In some embodiments, the ratio of negative to positive charges is between 1.0 and 2.0 (e.g., between 1.0 and 1.9, between 1.0 and 1.8, between 1.0 and 1.7, between 1.0 and 1.6, between 1.0 and 1.5, between 1.0 and 1.4, between 1.0 and 1.3, between 1.0 and 1.2, between 1.0 and 1.1, between 1.1 and 2.0, between 1.2 and 2.0, between 1.3 and 2.0, between 1.4 and 2.0, between 1.5 and 2.0, between 1.6 and 2.0, between 1.7 and 2.0, between 1.8 and 2.0, or between 1.9 and 2.0). In some embodiments, the ratio of negative to positive charges is between 0.75 and 6.5 (eg, between 0.75 and 5.5, between 0.75 and 4.5, between 0.75 and 3.5, between 0.75 and 2.5, between 0.75 and 1.5, or between 0.75 and 1).In some embodiments, the ratio of negative to positive charges is between 1 and 7.5 (eg, between 1.5 and 7.5, between 2.5 and 7.5, between 3.5 and 7.5, between 4.5 and 7.5, between 5.5 and 7.5, or between 6.5 and 7.5).

[0209] 5' phosphorus stabilizing moiety To further protect the siRNA molecules of the present disclosure from degradation, a 5'-phosphorus stabilizing moiety can be employed. The 5'-phosphorus stabilizing moiety replaces the 5'-phosphate, preventing hydrolysis of the phosphate. 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 strand of the siRNA molecule may independently and optionally employ any suitable 5'-phosphorus stabilizing moiety. [ka]

[0210] 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.

[0211] hydrophobic part The present disclosure also provides siRNA molecules with one or more hydrophobic moieties attached.The hydrophobic moiety can be covalently attached to the 5'-end or 3'-end of the siRNA molecule of the present disclosure.Non-limiting examples of the hydrophobic moiety suitable for use in the siRNA molecule of the present disclosure can include cholesterol, vitamin D, tocopherol, phosphatidylcholine (PC), docosahexaenoic acid, docosanoic acid, PC-docosanoic acid, eicosapentaenoic acid, lithocholic acid, or any combination of the aforementioned hydrophobic moiety and PC.

[0212] 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.

[0213] According to the present disclosure, the siRNA molecule disclosed herein may be a branched siRNA molecule. The siRNA molecule may be unbranched, or may be bi-, tri-, or tetra-branched, connected via a linker. Each main branch may be further branched to allow for 2, 3, 4, 5, 6, 7, or 8 separate RNA single- or double-stranded fragments. The branching points on the linker may originate from the same atom, or may originate from separate atoms along the linker. Some exemplary embodiments are listed in Table 2. [Table 2]

[0214] 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).

[0215] 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.

[0216] 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.

[0217] 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, any carbon or oxygen atom of the linker is optionally replaced with a nitrogen atom, has a hydroxyl substituent, or has an oxo substituent. 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 PEG, linear forked PEG, or branched forked PEG.

[0218] 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.

[0219] 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.

[0220] 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.).

[0221] In some embodiments, the linker has the structure of formula L1: [ka]

[0222] In some embodiments, the linker has the structure of formula L2: [ka]

[0223] In some embodiments, the linker has the structure of formula L3: [ka]

[0224] In some embodiments, the linker has the structure of formula L4: [ka]

[0225] In some embodiments, the linker has the structure of formula L5: [ka]

[0226] In some embodiments, the linker has the structure of formula L6: [ka]

[0227] In some embodiments, the linker has the structure of formula L7, as shown below: [ka]

[0228] In some embodiments, the linker has the structure of formula L8. [ka]

[0229] In some embodiments, the linker has the structure of formula L9: [ka]

[0230] 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.

[0231] 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.

[0232] Treatment method By delivering the HTT-targeting siRNA molecules of the present disclosure to a subject, Huntington's disease can be treated and / or phenotypes associated with Huntington's disease (e.g., movement disorders such as chorea and dystonia, cognitive impairment, and psychiatric disorders such as depression and anxiety) can be alleviated. Furthermore, Huntington's disease can also be treated by delivering the siRNA molecules of the present disclosure to a subject having a variant of the HTT gene, where siRNA-mediated gene silencing of the HTT variant gene reduces the expression level of the HTT transcript.

[0233] The present disclosure provides a method for treating a subject by silencing the HTT gene with one or more siRNA molecules described herein. This gene silencing can be achieved in a subject to silence wild-type HTT transcripts, mutant HTT transcripts, splice isoforms of HTT transcripts, and / or HTT transcripts that are overexpressed compared to healthy subjects. This method can include delivering a 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). 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 route of administration, the preferred dosage and / or the frequency of administration of an effective amount can vary depending on the subject's response. 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 individual subject.Administration can be carried out any suitable number of times per day for as long as necessary.Subjects can be adults or children, with or without co-morbidities.

[0234] Selecting a target Subjects that can be treated with the siRNA molecules disclosed herein include, for example, subjects in need of treatment for Huntington's disease and / or any other medical risk(s) associated with a pathogenic mutation in the HTT gene. Subjects that can be treated with the siRNA molecules disclosed herein include, for example, humans, monkeys, rats, mice, pigs, and other mammals that contain at least one orthologous copy of the HTT gene. Subjects can be adults or children with or without co-morbidities.

[0235] osmolality Administration of siRNA molecules of the present disclosure may affect the osmolality of a subject (e.g., of the cerebrospinal fluid (CSF)). The CSF osmolality of a subject being treated with an siRNA molecule of the present disclosure may be, for example, 250-450 mOsmol / kg. In some embodiments, the CSF osmolality is 250-350 mOsmol / kg. For siRNA molecules formulated as salts with one or more divalent cations, the subject's CSF osmolality may be affected by the concentration of the divalent cations. Persons overseeing a subject's treatment may monitor the subject's CSF osmolality and adjust the dosage accordingly. For example, a subject exhibiting a higher-than-normal osmolality may have their dosage reduced.

[0236] Alternatively, the concentration of sodium ion in the composition that contains siRNA molecule can be changed.For example, in the liquid formulation of siRNA molecule, the concentration of sodium can be adjusted to increase or decrease the osmolality that can be obtained, without adversely affecting the toxic benefits of divalent cations.By reducing the sodium level in formulation, it can be possible to maintain normal physiological osmolality level in the subject that is treated by the therapeutic oligonucleotide of the present disclosure.

[0237] Pharmaceutical Composition The siRNA molecule of the present disclosure can be formulated in pharmaceutical compositions for administering to subjects in biologically compatible forms 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).

[0238] Conventional 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 United States Pharmacopeial Convention, The National Formulary, United States Pharmacopeial, 2015, USP 38 NF 33).

[0239] Under ordinary conditions of storage and use, pharmaceutical compositions may contain preservatives, for example, to prevent the growth of microorganisms. Pharmaceutical compositions may include sterile aqueous solutions, sterile dispersions, or powders, for example, for the extemporaneous preparation of sterile solutions or dispersions. In all cases, the articles can be sterilized using techniques known in the art and can be fluidized to the extent that they can be easily administered to a subject in need of treatment.

[0240] The pharmaceutical compositions may be administered to a subject, e.g., a human subject, alone or in combination with a pharmaceutically acceptable carrier, the proportions of which may be determined by the solubility and / or chemical properties of the compound, the chosen route of administration, and standard pharmaceutical practice, as indicated herein.

[0241] Dosing regimen A physician with ordinary skill in the art can easily determine the effective amount of siRNA molecules to be administered to a mammalian subject (e.g., 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.In general, the suitable daily dose of one of the siRNA molecules of the present disclosure is the amount of the lowest dose of the siRNA molecule that is effective in producing a therapeutic effect.The ss-siRNA molecules or ds-siRNA molecules of the present disclosure can be administered by injection, for example, intrathecal, intracerebroventricular, intracisternal injection by catheter insertion, intraparenchymal, intravenous, subcutaneous, or intramuscular injection. The daily dosage of the therapeutic composition of the siRNA molecule of the present disclosure can be administered as a single dose, or as two, three, four, five, six or more doses that are administered separately at appropriate intervals throughout the day, week, month or year, optionally in unit dosage form.The siRNA molecule of the present disclosure can be administered alone, but can also be administered as a pharmaceutical formulation in combination with an excipient, a carrier, and optionally additional therapeutic agents.

[0242] Route of administration The methods of the present disclosure contemplate any route of administration tolerated by the therapeutic composition, some embodiments of which include intrathecal, intraventricular, intrastriatal, intraparenchymal, or intracisternal injection via catheterization.

[0243] 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.

[0244] 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.

[0245] 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.

[0246] 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.

[0247] 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.

[0248] 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.

[0249] 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.

[0250] 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.

[0251] 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]

[0252] The following examples are presented to provide one 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.

[0253] Example 1. Knockdown of HTT in mice with siRNA molecules of the present disclosure Introduction The purpose of these experiments was to test the ability of the siRNA molecules of the present disclosure to silence HTT. The siRNA molecules targeted a portion of the HTT mRNA transcript having the nucleobase sequence of any one of SEQ ID NOS: 1-3. The siRNA molecules had a sense strand having the nucleobase sequence of any one of SEQ ID NOS: 4-6 and an antisense strand having the nucleobase sequence of any one of SEQ ID NOS: 7-9. The siRNA molecules were branched (e.g., biantennary siRNA molecules having the structure of Formula XVII, Formula XVIII, or Formula XIX; triantennary siRNA molecules having the structure of Formula XX, Formula XXI, Formula XXII, or Formula XXIII; or tetraantennary siRNA molecules having the structure of Formula XXIV, Formula XXV, Formula XXVI, Formula XXVII, or Formula XXVIII). Each siRNA molecule was a ds-siRNA molecule having an antisense strand and a sense strand. The antisense strand had a modification pattern (e.g., the general structure of Formula I, Formula II, Formula IV, or Formula VI, or the specific structure of Formula A1, A2, A3, or A4). The sense strand also had a pattern of modifications (e.g., the general structure of Formula III, Formula V, or Formula VII, or the specific structures of Formula S1, S2, S3, S4, S5, S6, S7, S8, or S9).

[0254] Materials and Methods Mice were treated with either 0.25 or 2.5 nmol of a biantennary siRNA molecule having the structure of Formula XVII. The antisense strand had the nucleobase sequence of SEQ ID NO: 7, and the sense strand had the nucleobase sequence of SEQ ID NO: 4. The siRNA molecules were administered to FBV / NJ mice by bilateral intracerebroventricular injection. 5 μL was administered to each side of the bilateral injection at a flow rate of 0.5 μL / min. HTT mRNA knockdown was quantified by qRT-PCR. HTT protein knockdown was quantified by Western blot. siRNA accumulation was quantified by stem-loop PCR.

[0255] Results – Knockdown of HTT mRNA Mice were treated with siRNA molecules having the above nucleobase sequences and either (i) an antisense strand of Formula II (particularly, Formula A2) and a sense strand of Formula III (particularly, Formula S2), or (ii) an antisense strand of Formula IV (particularly, Formula A3) and a sense strand of Formula V (particularly, Formula S6). The mice were analyzed for HTT mRNA levels in various brain regions (frontal cortex, motor cortex, striatum, and hippocampus). The accumulation of siRNA molecules in the same brain regions was also analyzed. Data were collected after 3 days (Figures 1A and 1B), 7 days (Figures 2A and 2B), and 14 days (Figures 3A and 3B). These patterns indicated dose-dependent knockdown of HTT and accumulation of siRNA molecules, respectively.

[0256] One month later, mice treated with patterns (i) and (ii) above, in addition to the (iii) Formula II / Formula A2' antisense strand and Formula III / Formula S2 sense strand, were analyzed. Again, dose-dependent knockdown (Figure 4A) and accumulation (Figure 4B) were observed.

[0257] After 2 and 3 months, mice treated with patterns (i), (ii), and (iii) above, in addition to (iv) the antisense strand of Formula II (particularly Formula A2) and the sense strand of Formula III (particularly Formula S1), and (v) the antisense strand of Formula IV (particularly Formula A3) and the sense strand of Formula V (particularly Formula S8), were analyzed. Again, dose-dependent knockdown (Figures 5A and 6A) and accumulation (Figures 5B and 6B) were observed. These data show a correlation between knockdown and accumulation across the different patterns, with pattern (iv) showing the best knockdown among all compounds tested.

[0258] The above data for patterns (i) and (ii) were plotted together to show the time course of knockdown at the 2.5 nmol dose level (Figure 7A) and siRNA accumulation at the 2.5 nmol dose level (Figure 7B) in different brain regions. The same was plotted for the 0.25 nmol dose (Figures 7C and 7D). These data showed a time-dependent decrease in HTT transcripts across brain regions, with peak knockdown achieved at 1 month. Accumulation also decreased in a time-dependent manner.

[0259] Patterns (i), (ii), (iii), (iv), and (v) were tested over a 6-month period, and the results are shown in Figure 8A (mRNA knockdown) and Figure 8B (siRNA accumulation). These data indicate that pattern (iv), with an antisense strand of Formula A2 and a sense strand of Formula S1, exhibited superior properties. This pattern was plotted across several brain regions at doses of 2.5 and 0.25 nmol, respectively (Figures 9A-9F). HTT mRNA levels, HTT protein levels, and siRNA amounts were calculated. Potent knockdown was observed at 2.5 nmol, and the effect was more pronounced when total protein was measured. Protein and transcript amounts returned to baseline in approximately parallel fashion.

[0260] Finally, we measured HTT knockdown in the kidney (Figure 10A) and liver (Figure 10B). At all time points, less than 20% knockdown was observed in the liver and less than 10% knockdown was observed in the kidney.

[0261] Example 2. Knockdown of HTT in mice with siRNA molecules of the present disclosure Introduction The purpose of these experiments was to test the ability of different siRNA molecules of the present disclosure to silence HTT at doses different from those tested in Example 1. The siRNA molecules targeted a portion of the HTT mRNA transcript having the nucleobase sequence of any one of SEQ ID NOS: 1-3. The siRNA molecules had a sense strand having the nucleobase sequence of any one of SEQ ID NOS: 4-6 and an antisense strand having the nucleobase sequence of any one of SEQ ID NOS: 7-9. The siRNA molecules were branched (e.g., biantennary siRNA molecules having the structure of Formula XVII, Formula XVIII, or Formula XIX; triantennary siRNA molecules having the structure of Formula XX, Formula XXI, Formula XXII, or Formula XXIII; or tetraantennary siRNA molecules having the structure of Formula XXIV, Formula XXV, Formula XXVI, Formula XXVII, or Formula XXVIII). Each siRNA molecule was a ds-siRNA molecule having an antisense strand and a sense strand. The antisense strand had a modification pattern (e.g., the general structure of Formula I, Formula II, Formula IV, or Formula VI, or the specific structure of Formula A1, A2, A3, or A4). The sense strand also had a pattern of modifications (e.g., the general structure of Formula III, Formula V, or Formula VII, or the specific structures of Formula S1, S2, S3, S4, S5, S6, S7, S8, or S9).

[0262] Materials and Methods Mice were treated with either 0.2, 1.0, or 5.0 nmol of a biantennary siRNA molecule having the structure of Formula XVII. The antisense strand had the nucleobase sequence of SEQ ID NO: 7, and the sense strand had the nucleobase sequence of SEQ ID NO: 4. The siRNA molecules were administered to FBV / NJ mice by unilateral intracerebroventricular injection. 5 μL was administered at a flow rate of 0.5 μL / min. HTT mRNA knockdown was quantified by qRT-PCR. HTT protein knockdown was quantified by Western blot. siRNA accumulation was quantified by stem-loop PCR.

[0263] result The mice were cultured with: (i) an antisense strand of Formula II (particularly Formula A2) and a sense strand of Formula III (particularly Formula S2); (ii) an antisense strand of Formula IV (particularly Formula A3) and a sense strand of Formula V (particularly Formula S6); (iv) an antisense strand of Formula II (particularly Formula A2) and a sense strand of Formula III (particularly Formula S1); (v) an antisense strand of Formula III (particularly Formula A3) and a sense strand of Formula V (particularly Formula S8); (vi) an antisense strand of Formula IV (particularly Formula A3) and a sense strand of Formula V (particularly, Formula S5); (vii) an antisense strand of Formula IV (particularly, Formula A3) and a sense strand of Formula V (particularly, Formula S7); (viii) an antisense strand of Formula II (particularly, Formula A2) and a sense strand of Formula III (particularly, Formula S4); and (ix) an antisense strand of Formula VI (particularly, Formula A4) and a sense strand of Formula VII (particularly, Formula S9). The results are shown in Figure 11. Each of these patterns resulted in knockdown of HTT in various brain regions after 6 months, with the effect being most pronounced in the hippocampus.

[0264] Example 3. HTT knockdown, pharmacokinetics, and pharmacodynamics of siRNA molecules of the present disclosure in non-human primates Introduction The purpose of these experiments was to test the ability of different siRNA molecules of the present disclosure to silence HTT. Pharmacokinetic (PK) and pharmacodynamic (PD) properties were also investigated. The siRNA molecules targeted a portion of the HTT mRNA transcript having the nucleobase sequence of any one of SEQ ID NOS: 1-3. The siRNA molecules had a sense strand having the nucleobase sequence of any one of SEQ ID NOS: 4-6 and an antisense strand having the nucleobase sequence of any one of SEQ ID NOS: 7-9. The siRNA molecules were branched (e.g., biantennary siRNA molecules having the structure of Formula XVII, Formula XVIII, or Formula XIX; triantennary siRNA molecules having the structure of Formula XX, Formula XXI, Formula XXII, or Formula XXIII; or tetraantennary siRNA molecules having the structure of Formula XXIV, Formula XXV, Formula XXVI, Formula XXVII, or Formula XXVIII). Each siRNA molecule was a ds-siRNA molecule having an antisense strand and a sense strand. The antisense strand had a modification pattern (e.g., the general structure of Formula I, Formula II, Formula IV, or Formula VI, or the specific structure of Formula A1, A2, A3, or A4). The sense strand also had a modification pattern (e.g., the general structure of Formula III, Formula V, or Formula VII, or the specific structure of Formula S1, S2, S3, S4, S5, S6, S7, S8, or S9).

[0265] Materials and Methods Animals were treated with various doses of biantennary siRNA molecules having the structure of Formula XVII. The antisense strand had the nucleobase sequence of SEQ ID NO: 7, and the sense strand had the nucleobase sequence of SEQ ID NO: 4. The siRNA molecules were administered either intrathecally or intracerebroventricularly. The antisense strand had the structure of Formula II (particularly Formula A2), and the sense strand had the structure of Formula III (particularly Formula S1). In some experiments, the siRNA molecules were formulated as salts. In these experiments, the cation was Mg 2+ and di-siRNA Mg 2+ The ratio to Mg is 1:25. 2+ The concentration of was 33 mM. The conditions tested are shown in Table 3 below. [Table 3]

[0266] result Various brain regions were analyzed for percent HTT mRNA expression compared to the PBS control, percent HTT protein expression compared to the PBS control, and siRNA content. Regions examined included the frontal cortex, motor cortex, temporal cortex, hippocampus, caudate nucleus, putamen, substantia nigra, pons, and medulla (Figures 12A-12C), as well as the cerebellar cortex (Figures 13A-13C) and deep cerebellar nuclei (Figures 13D-13F). Other tissues analyzed included the liver (Figures 14A-14C), kidney (Figures 15A-15C), and spinal cord (Figures 16A-16C). The amount of siRNA in cerebrospinal fluid (CSF) and plasma was also determined for all treatment groups (Figures 17A-17N).

[0267] Collectively, these data demonstrate that HTT was silenced at all doses tested. Furthermore, formulating the siRNA molecules as salts allowed for dosing at 100 mg without acute neurological symptoms. When administered as salts, the siRNA molecules demonstrated durable gene silencing throughout the brain and spinal cord, even within deep brain regions, while also exhibiting limited pharmacodynamic effects in the liver and kidney. Intrathecal administration performed comparably to intracerebroventricular administration. Furthermore, animals with robust target silencing had more siRNA molecules found in the CSF and fewer in the plasma. High concentrations of siRNA molecules in the CSF were observed and maintained for 1 week.

[0268] Example 4. Knockdown of HTT using siRNA molecules of the present disclosure in a one-month study in non-human primates Introduction The purpose of these experiments was to test the ability of different siRNA molecules of the present disclosure to silence HTT. Pharmacokinetic (PK) and pharmacodynamic (PD) properties were also investigated. The siRNA molecules targeted a portion of the HTT mRNA transcript having the nucleobase sequence of any one of SEQ ID NOS: 1-3. The siRNA molecules had a sense strand having the nucleobase sequence of any one of SEQ ID NOS: 4-6 and an antisense strand having the nucleobase sequence of any one of SEQ ID NOS: 7-9. The siRNA molecules were branched (e.g., biantennary siRNA molecules having the structure of Formula XVII, Formula XVIII, or Formula XIX; triantennary siRNA molecules having the structure of Formula XX, Formula XXI, Formula XXII, or Formula XXIII; or tetraantennary siRNA molecules having the structure of Formula XXIV, Formula XXV, Formula XXVI, Formula XXVII, or Formula XXVIII). Each siRNA molecule was a ds-siRNA molecule having an antisense strand and a sense strand. The antisense strand had a modification pattern (e.g., the general structure of Formula I, Formula II, Formula IV, or Formula VI, or the specific structure of Formula A1, A2, A3, or A4). The sense strand also had a modification pattern (e.g., the general structure of Formula III, Formula V, or Formula VII, or the specific structure of Formula S1, S2, S3, S4, S5, S6, S7, S8, or S9).

[0269] Materials and Methods Naive animals were treated with various doses of biantennary siRNA molecules having the structure of Formula XVII. The antisense strand had the nucleobase sequence of SEQ ID NO: 7, and the sense strand had the nucleobase sequence of SEQ ID NO: 4. The siRNA molecules were administered by either intrathecal (IT) or intracerebroventricular (ICV) injection. The antisense strand had the structure of Formula II (particularly, Formula A2), and the sense strand had the structure of Formula III (particularly, Formula S1). In some experiments, the siRNA molecules were formulated as salts. In these experiments, the cation was Mg 2+ and di-siRNA and Mg 2+ The ratio of Mg to Mg was 1:25. 2+The concentrations of were 16.7 mM in animals dosed with 50 mg, 8.3 mM in animals dosed with 25 mg, and 4.2 mM in animals dosed with 12.5 mg. The conditions tested are shown in Table 4 below. [Table 4]

[0270] result Various brain regions were analyzed for percent HTT mRNA expression compared to the PBS control, percent HTT protein expression compared to the PBS control, and siRNA quantity. The regions examined were the frontal cortex (Figures 18A-18C), motor cortex (Figures 19A-19C), temporal cortex (Figures 20A-20C), hippocampus (Figures 21A-21C), putamen (Figures 22A-22C), caudate nucleus (Figures 23A-23C), substantia nigra (Figures 24A-24C), pons (Figures 25A-25C), and medulla (Figures 26A-26C). Other tissues analyzed were the liver (Figures 27A-27C) and kidney (Figures 28A-28C), which did not show substantial silencing. HTT mRNA and protein expression were also measured in the spinal cord (Figures 29A and 29B). Finally, HTT mRNA expression was measured in the cerebellar cortex (Figure 30) and deep nuclei (Figure 31). Furthermore, the accumulation and clearance of di-siRNA molecules in plasma and CSF were examined in each of the four treatment groups (Groups 2-5 in Table 4 above, Figures 32A-32B).

[0271] Taken together, these data demonstrate that HTT was silenced at all doses tested. Consistent with the data presented in Example 3, 50-75% protein knockdown was observed in all cortical regions and the hippocampus. When siRNA was administered via intravenous delivery, 25-50% protein knockdown was observed in the caudate nucleus, putamen, substantia nigra, pons, and medulla, whereas approximately 50% knockdown was observed when siRNA was administered via intracerebroventricular delivery. Furthermore, low levels of knockdown were observed in the liver and kidney, and siRNA molecules were cleared from plasma within hours, whereas they persisted in the CSF for approximately two weeks.

[0272] Example 5. Evaluation of siRNA molecules at various time points in non-human primates Introduction The purpose of these experiments was to evaluate the tissue distribution and pharmacokinetics (PK) of siRNA molecules of the present disclosure for silencing HTT. The siRNA molecules targeted a portion of the HTT mRNA transcript having the nucleobase sequence of any one of SEQ ID NOS: 1-3. The siRNA molecules had a sense strand having the nucleobase sequence of any one of SEQ ID NOS: 4-6 and an antisense strand having the nucleobase sequence of any one of SEQ ID NOS: 7-9. The siRNA molecules were branched (e.g., biantennary siRNA molecules having the structure of Formula XVII, Formula XVIII, or Formula XIX; triantennary siRNA molecules having the structure of Formula XX, Formula XXI, Formula XXII, or Formula XXIII; or tetraantennary siRNA molecules having the structure of Formula XXIV, Formula XXV, Formula XXVI, Formula XXVII, or Formula XXVIII). Each siRNA molecule was a ds-siRNA molecule having an antisense strand and a sense strand. The antisense strand had a modification pattern (e.g., the general structure of Formula I, Formula II, Formula IV, or Formula VI, or the specific structure of Formula A1, A2, A3, or A4). The sense strand also had a pattern of modifications (e.g., the general structure of Formula III, Formula V, or Formula VII, or the specific structures of Formula S1, S2, S3, S4, S5, S6, S7, S8, or S9).

[0273] Materials and Methods Naive cynomolgus monkeys were treated with various doses of biantennary siRNA molecules having the structure of Formula XVII, where the antisense strand had the nucleobase sequence of SEQ ID NO:7 and the sense strand had the nucleobase sequence of SEQ ID NO:4.

[0274] The siRNA molecules were administered either as a single 50 mg dose of the siRNA molecule (Groups 2-5 below) or as three separate 50 mg doses of the siRNA molecule (the second and third doses were administered 3 and 6 months after the first dose, respectively). The doses were administered as an intrathecal bolus injection. The antisense strand had the structure of Formula II (specifically, Formula A2), and the sense strand had the structure of Formula III (specifically, Formula S1). The conditions tested are shown in Table 5 below. [Table 5]

[0275] The animals dosed in this study were males aged 3 to 7 years. Animals were dosed as shown in Table 5 above via a surgically implanted CSF (cerebrospinal fluid) catheter and a port system with the catheter tip placed in the mid-thoracic region. One animal in Group 3 and two animals in Group 4 exhibited catheter blockage during dosing. These animals were previously identified as being at risk for intrathecally administered drug loss. Upon study completion, little to no di-siRNA exposure was determined in the CNS, including all brain regions and lumbar spinal cord evaluated. An alternative analysis of CNS tissue is also provided, in which these animals (referred to as "outliers") were removed from the dataset.

[0276] HTT mRNA expression was measured by quantitative real-time PCR (qRT-PCR). Data were normalized to a housekeeping gene (PPIA) and % expression was calculated based on PBS-treated animals. HTT protein expression was measured by enzyme-linked immunosorbent assay (ELISA). Data were normalized to total protein content using a BCA assay and % expression was calculated based on PBS-treated animals. siRNA was quantified using a locked nucleic acid-based hybridization assay using Meso-Scale Discovery for detection.

[0277] result Various brain regions were analyzed for percent HTT mRNA expression compared to the PBS control, percent HTT protein expression compared to the PBS control, and siRNA quantity. Regions examined included the frontal cortex (Figures 33A-33C), motor cortex (Figures 34A-34C), temporal cortex (Figures 35A-35C), caudate nucleus (Figures 36A-36C), putamen (Figures 37A-37C), hippocampus (Figures 38A-38C), cervical spinal cord (Figures 39A-39C), thoracic spinal cord (Figures 40A-40C), and lumbar spinal cord (Figures 41A-41C). Liver (Figures 42A-42C) and kidney (Figures 43A-43C) were also measured. Further analysis was performed excluding outlier animals, and results are shown for the frontal cortex (Figures 44A-44C), motor cortex (Figures 45A-45C), temporal cortex (Figures 46A-46C), caudate nucleus (Figures 47A-47C), putamen (Figures 48A-48C), hippocampus (Figures 49A-49C), cervical spinal cord (Figures 50A-50C), thoracic spinal cord (Figures 51A-51C), and lumbar spinal cord (Figures 52A-52C).

[0278] Taken together, these data demonstrate that HTT was silenced at all doses tested, and furthermore, silencing persisted even 6 months after treatment.

[0279] Example 6. Generation of HTT-targeting siRNA molecules The siRNA molecules of the present disclosure can be synthesized by standard methods well 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.

[0280] 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 are shown in Table 1 above, along with the nucleotide sequences of the sense and antisense strands and the target sequence of huntingtin (HTT) mRNA. Those skilled in the art will recognize that antisense (AS) strands can be annealed to corresponding sense (S) strands to obtain ds-siRNA molecules. Alternatively, those skilled in the art can use only antisense strands to derive ss-siRNA molecules.

[0281] Example 7. Optimization of HTT-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.

[0282] 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 7-deazaadenine, and / or 3-deazaguanine and 3-deazaadenine. siRNA molecules can also include nucleic acid bases in which the purine or pyrimidine base is replaced with other heterocycles, such as 7-deazaadenine, 7-deazaguanosine, 2-aminopyridine, and / or 2-pyridone. Further optimization of the siRNA molecules of the present disclosure may also 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.

[0283] Optimizing siRNA through 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 can be 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.

[0284] 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.

[0285] Optimization of siRNA using hydrophobic moieties The optimization of the siRNA molecule of the present disclosure can include covalently linked hydrophobic moieties at 5'-end or 3'-end.Non-limiting examples of the hydrophobic moieties suitable for use in the siRNA molecule of the present disclosure can include cholesterol, vitamin D, tocopherol, phosphatidylcholine (PC), docosahexaenoic acid, docosanoic acid, PC-docosanoic acid, eicosapentaenoic acid, lithocholic acid, or any combination of the aforementioned hydrophobic moieties and PC.

[0286] Optimization of siRNA using stabilizing moieties The 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.

[0287] 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 siRNAs connected via linkers.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 different atoms along the linker.Some exemplary embodiments are listed in Table 2 above.

[0288] 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, tosylated solketal, 1,3-diaminopropanol, pentaerythritol, or any one of the branch point moieties described in U.S. Pat. No. 10,478,503).

[0289] Example 8. Preparation and administration of HTT-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 diluent, carrier or excipient, and can further comprise preservatives, for example, to prevent microbial growth.The conventional procedures and ingredients for selecting and preparing suitable formulations can be found in, for example, Remington, JP The Science and Practice of Pharmacy, Easton, PA.Mack Publishers, 2012, 22 nd ed, and the United States Pharmacopeial Convention, The National Formulary, United States Pharmacopeial, 2015, USP 38 NF 33).

[0290] 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.

[0291] Example 9. Methods for treating Huntington's disease using HTT-targeting siRNA molecules A subject who needs to be treated for Huntington's disease is treated with a predetermined dose of the siRNA molecule or siRNA composition of the present disclosure formulated as a salt, at a frequency determined by a physician.A physician with ordinary skill in the art can easily determine the effective amount of siRNA molecule to be administered to a mammalian subject (e.g., human) who needs administration.For example, a physician can start by prescribing a dose of one of the siRNA molecules of the present disclosure at a level lower than the level required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved.Alternatively, a physician can start a treatment regimen by administering one of the siRNA molecules of the present disclosure at a high dose, and then administer gradually decreasing doses until the minimum dose that produces a therapeutic effect (e.g., reducing the expression of HTT mRNA or suitable biomarker) is reached.In general, the suitable daily dose of one of the siRNA molecules of the present disclosure is the minimum dose amount that is effective for producing a therapeutic effect. The ss-siRNA molecule or ds-siRNA molecule of the present disclosure can be administered by injection, for example, intrathecal, intracerebroventricular, intrastriatal, intraparenchymal, intravenous, intramuscular, subcutaneous, or intracisternal injection by catheter insertion.The daily dose of one of the therapeutic compositions 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 that are administered separately at appropriate intervals throughout the day, week, month, or year, optionally in unit dosage form.Any of the siRNA molecules of the present disclosure can be administered alone, or can be administered as a pharmaceutical preparation in combination with excipients, carriers, and optionally additional therapeutic agents.The dosage and frequency are determined based on the height, weight, age, sex, and other disorders of the subject.

[0292] 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.

[0293] 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, subcutaneous, 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.

[0294] 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, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0295] While the invention has been described in conjunction with specific embodiments thereof, it will be understood that further modifications are possible, and this application is intended to cover any variations, uses, or applications of the invention which generally follow the principles of the invention and are within known or customary practice in the art to which the invention pertains and which are applicable to the essential features described above, including departures from the invention that fall within the scope of the appended claims.

[0296] Other embodiments are within the scope of the following claims.

Claims

1. A small interfering RNA (siRNA) molecule comprising an antisense strand and a sense strand having complementarity to the antisense strand, wherein the antisense strand has sufficient complementarity to hybridize to a region of equal length within a huntingtin (HTT) mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-3, wherein the antisense strand comprises a structure represented by Formula I, which, in the 5' to 3' direction, is as follows: 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 It is represented by; Each A' is independently a group of the formula C-P ２ -D-P ２ It is represented by; B is a compound of the formula C-P ２ -D-P ２ -D-P ２ -D-P ２ It is represented by; each C is independently 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 independently a 2'-F ribonucleoside; Each P １ is independently a phosphorothioate internucleoside linkage; Each P ２ is independently a phosphodiester internucleoside linkage; j is an integer from 1 to 7; The siRNA molecule, wherein k is an integer of 1 to 7.

2. The antisense strand comprises a structure represented by formula A1, which, in the 5' to 3' direction, is as follows: 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; 2. The siRNA molecule of claim 1, 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.

3. 1. An siRNA molecule comprising an antisense strand and a sense strand complementary to the antisense strand, wherein the antisense strand has sufficient complementarity to hybridize to a region of equal length within a huntingtin (HTT) mRNA transcript having a nucleic acid sequence of any one of SEQ ID NOs: 1-3, wherein the antisense strand comprises a structure represented by Formula II, which, in the 5' to 3' direction, is: 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 It is represented by; Each A' is independently a group of the formula C-P ２ -D-P ２ It is represented by; B is a compound of the formula C-P ２ -D-P ２ -D-P ２ -D-P ２ It is represented by; each C is independently 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 independently a 2'-F ribonucleoside; Each P １ is independently a phosphorothioate internucleoside linkage; Each P ２ is independently a phosphodiester internucleoside linkage; j is an integer from 1 to 7; The siRNA molecule, wherein k is an integer of 1 to 7.

4. The antisense strand comprises a structure represented by Formula A2, which, in the 5' to 3' direction, is as follows: A-S-B-SA Formula A2; 4. The siRNA molecule of claim 3, 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.

5. the sense strand comprises a structure represented by Formula III, which, in the 5' to 3' direction, is: E-(A') ｍ -F Formula III; In the formula, E is a group represented by the formula (C-P １ ) ２ It is represented by; 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 4, wherein m is an integer of 1 to 7.

6. The siRNA molecule of any one of claims 1 to 5, wherein j is 4 and k is 4.

7. The siRNA molecule of claim 5 or claim 6, wherein m is 4.

8. The sense strand comprises a structure represented by formula S1, which, in the 5' to 3' direction, is as follows: 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; 6. The siRNA molecule of claim 5, 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.

9. The sense strand comprises a structure represented by formula S2, which, in the 5' to 3' direction, is as follows: 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; 6. The siRNA molecule of claim 5, 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.

10. The sense strand comprises a structure represented by formula S3, which, in the 5' to 3' direction, is as follows: 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; 6. The siRNA molecule of claim 5, 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.

11. The sense strand comprises a structure represented by formula S4, which, in the 5' to 3' direction, is as follows: 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; 6. The siRNA molecule of claim 5, 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.

12. 1. An siRNA molecule comprising an antisense strand and a sense strand having complementarity to the antisense strand, wherein the antisense strand has sufficient complementarity to hybridize to a region of equal length within a huntingtin (HTT) mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-3, wherein the antisense strand comprises a structure represented by Formula IV, which, in the 5' to 3' direction, is: 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 It is represented by; Each A' is independently a group of the formula C-P ２ -D-P ２ It is represented by; B is a compound of the formula D-P １ -C-P １ -D-P １ It is represented by; each C is independently a 2'-O-Me ribonucleoside; each C' is independently a 2'-O-Me ribonucleoside or a 2'-F ribonucleoside; each D is independently a 2'-F ribonucleoside; Each P １ is independently a phosphorothioate internucleoside linkage; Each P ２ is independently a phosphodiester internucleoside linkage; j is an integer from 1 to 7; The siRNA molecule, wherein k is an integer of 1 to 7.

13. The antisense strand comprises a structure represented by Formula A3, which, in the 5' to 3' direction, is 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; 13. The siRNA molecule of claim 12, 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.

14. the sense strand comprises a structure represented by Formula V, which, in the 5' to 3' direction, is: E-(A’) ｍ -C-P ２ -F Formula V; In the formula, E is a group represented by the formula (C-P １ ) ２ It is represented by; 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; A', C, D, P １ , and P ２ is as defined in formula IV; The siRNA molecule of claim 12 or 13, wherein m is an integer of 1 to 7.

15. The siRNA molecule of claim 14, wherein j is 6 and k is 2.

16. 16. The siRNA molecule of claim 14 or claim 15, wherein m is 5.

17. The sense strand comprises a structure represented by formula S5, which, in the 5' to 3' direction, is as follows: 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; 15. The siRNA molecule of claim 14, 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.

18. The sense strand comprises a structure represented by formula S6, which, in the 5' to 3' direction, is as follows: 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; 15. The siRNA molecule of claim 14, 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.

19. The sense strand comprises a structure represented by formula S7, which, in the 5' to 3' direction, is as follows: 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; 15. The siRNA molecule of claim 14, 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.

20. The sense strand comprises a structure represented by formula S8, which, in the 5' to 3' direction, is as follows: 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; 15. The siRNA molecule of claim 14, 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.

21. 1. An siRNA molecule comprising an antisense strand and a sense strand having complementarity to the antisense strand, wherein the antisense strand has sufficient complementarity to hybridize to a region of equal length within a huntingtin (HTT) mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-3, wherein the antisense strand comprises a structure represented by Formula VI, which, in the 5' to 3' direction, is: 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 It is represented by; Each B independently represents a group of the formula C-P ２ It is represented by; each C is independently a 2'-O-Me ribonucleoside; each C' is independently a 2'-O-Me ribonucleoside or a 2'-F ribonucleoside; each D is independently a 2'-F ribonucleoside; Each E independently represents a group of the formula D-P ２ -C-P ２ It is represented by; F is a compound of the formula D-P １ -C-P １ It is represented by; Each G independently represents a group of the formula C-P １ It is represented by; Each P １ is independently a phosphorothioate internucleoside linkage; Each P ２ is independently a phosphodiester internucleoside linkage; j is an integer from 1 to 7; k is an integer from 1 to 7; The siRNA molecule, wherein 1 is an integer of 1 to 7.

22. The antisense strand comprises a structure represented by formula A4, which, in the 5' to 3' direction, is 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; 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 sense strand comprises Formula VII, which, in the 5' to 3' direction, is: 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 It is represented by; Each H independently represents a group of the formula (C-P １ ) ２ It is represented by; Each I independently represents a group of the formula (D-P ２ ) is represented by; 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; The siRNA molecule of claim 21 or 22, wherein o is an integer from 1 to 7.

24. The siRNA molecule of claim 23, wherein j is 3, k is 6, and l is 2.

25. 25. The siRNA molecule of claim 23 or claim 24, wherein m is 3, n is 3, and o is 3.

26. The sense strand comprises a structure represented by formula S9, which, in the 5' to 3' direction, is as follows: 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; 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.

27. The siRNA molecule of any one of claims 1 to 26, wherein the equal length regions within the HTT mRNA transcript have the nucleic acid sequence of SEQ ID NO:

1.

28. The siRNA molecule of any one of claims 1 to 26, wherein the equal length regions within the HTT mRNA transcript have the nucleic acid sequence of SEQ ID NO:

2.

29. The siRNA molecule of any one of claims 1 to 26, wherein the equal length regions within the HTT mRNA transcript have the nucleic acid sequence of SEQ ID NO:

3.

30. 30. The siRNA molecule of any one of claims 1 to 29, wherein the antisense strand has at least 70% complementarity to a region of 19, 20, 21, or more contiguous nucleobases within the HTT mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1 to 3, and optionally, the antisense strand has at least 70% complementarity to the HTT mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1 to 3.

31. 31. The siRNA molecule of claim 30, wherein the antisense strand has at least 75% complementarity to a region within the HTT mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-3, 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 a region within the HTT mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-3.

32. 32. The siRNA molecule of any one of claims 1 to 31, 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 perfectly complementary to a contiguous polynucleotide segment of equal length within the region of the HTT RNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1 to 3.

33. 33. The siRNA molecule of claim 32, wherein the antisense strand comprises 10 to 30 contiguous nucleotides that are perfectly complementary to a contiguous polynucleotide segment of equal length within the region of the HTT RNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1 to 3.

34. The siRNA molecule of claim 33, wherein the antisense strand comprises 12 to 30 contiguous nucleotides that are perfectly complementary to a contiguous polynucleotide segment of equal length within the region of the HTT RNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1 to 3.

35. 35. The siRNA molecule of claim 34, wherein the antisense strand comprises 15 to 30 contiguous nucleotides that are perfectly complementary to a contiguous polynucleotide segment of equal length within the region of the HTT RNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1 to 3.

36. 36. The siRNA molecule of claim 35, 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 HTT RNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1 to 3.

37. 37. The siRNA molecule of claim 36, 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 HTT RNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1 to 3.

38. 36. The siRNA molecule of claim 35, wherein the antisense strand comprises 15 consecutive nucleotides that are perfectly complementary to a contiguous polynucleotide segment of equal length within the region of the HTT RNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1 to 3.

39. 38. The siRNA molecule of claim 37, wherein the antisense strand comprises 20 consecutive nucleotides that are perfectly complementary to a contiguous polynucleotide segment of equal length within the region of the HTT RNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1 to 3.

40. 38. The siRNA molecule of claim 37, 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 HTT RNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1 to 3.

41. 41. The siRNA molecule of any one of claims 1 to 40, wherein the antisense strand contains no more than 9 nucleotide mismatches to a region within the HTT RNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1 to 3, 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 the region within the HTT RNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1 to 3.

42. 42. The siRNA molecule of any one of claims 30 to 41, wherein said region of said HTT RNA transcript has the nucleic acid sequence of SEQ ID NO:

1.

43. 42. The siRNA molecule of any one of claims 30 to 41, wherein said region of said HTT RNA transcript has the nucleic acid sequence of SEQ ID NO:

2.

44. 42. The siRNA molecule of any one of claims 30 to 41, wherein said region of said HTT RNA transcript has the nucleic acid sequence of SEQ ID NO:

3.

45. The siRNA molecule of any one of claims 1 to 44, 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: 7 to 9.

46. The siRNA molecule of claim 45, 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: 7 to 9.

47. The siRNA molecule of claim 46, wherein the antisense strand has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of any one of SEQ ID NOs: 7-9, 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: 7-9.

48. The siRNA molecule of claim 47, wherein the antisense strand has a nucleic acid sequence of any one of SEQ ID NOs: 7 to 9.

49. The siRNA molecule of any one of claims 45 to 48, wherein the nucleic acid sequence is SEQ ID NO:

7.

50. The siRNA molecule of any one of claims 45 to 48, wherein the nucleic acid sequence is SEQ ID NO:

8.

51. The siRNA molecule of any one of claims 45 to 48, wherein the nucleic acid sequence is SEQ ID NO:

9.

52. The siRNA molecule of any one of claims 1 to 51, 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: 4 to 6.

53. The siRNA molecule of claim 52, 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: 4 to 6.

54. 54. The siRNA molecule of claim 53, 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: 4-6, 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: 4-6.

55. The siRNA molecule of claim 54, wherein the sense strand has the nucleic acid sequence of any one of SEQ ID NOs: 4 to 6.

56. The siRNA molecule of any one of claims 52 to 55, wherein the nucleic acid sequence is SEQ ID NO:

4.

57. 56. The siRNA molecule of any one of claims 52 to 55, wherein the nucleic acid sequence is SEQ ID NO:

5.

58. The siRNA molecule of any one of claims 52 to 55, wherein the nucleic acid sequence is SEQ ID NO:

6.

59. The siRNA molecule of any one of claims 1 to 58, wherein the antisense strand further comprises a 5' phosphorus stabilizing moiety at the 5' end of the antisense strand.

60. The siRNA molecule of any one of claims 1 to 59, wherein the sense strand further comprises a 5' phosphorus stabilizing moiety at the 5' end of the sense strand.

61. each 5' phosphorus stabilizing moiety is independently represented by any one of formulas IX-XVI: 【Chemical 1】 61. The siRNA molecule of claim 59 or 60, 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.

62. 62. The siRNA molecule of claim 61, wherein the nucleobase is adenine, uracil, guanine, thymine, or cytosine.

63. 63. The siRNA molecule of any one of claims 59 to 62, wherein the 5' phosphorus stabilizing moiety is an (E)-vinyl phosphonate of formula XI.

64. The siRNA molecule of any one of claims 1 to 63, further comprising a hydrophobic moiety at the 5'-end or 3'-end.

65. 65. The siRNA molecule of claim 64, wherein the hydrophobic moiety is selected from the group consisting of cholesterol, vitamin D, or tocopherol.

66. 66. The siRNA molecule of any one of claims 1 to 65, wherein the sense strand has a length of 10 to 30 nucleotides.

67. 67. The siRNA molecule of claim 66, wherein the sense strand is 10 to 25 nucleotides in length.

68. 68. The siRNA molecule of claim 67, wherein the sense strand is 12 to 25 nucleotides in length.

69. 69. The siRNA molecule of claim 68, wherein the sense strand is 12 to 20 nucleotides in length.

70. 70. The siRNA molecule of claim 69, wherein the sense strand is 12 to 19 nucleotides in length.

71. 71. The siRNA molecule of claim 70, wherein the sense strand is 15 nucleotides in length.

72. 71. The siRNA molecule of claim 70, wherein the sense strand is 16 nucleotides in length.

73. 71. The siRNA molecule of claim 70, wherein the sense strand is 18 nucleotides in length.

74. The siRNA molecule of any one of claims 1 to 73, wherein the length of the antisense strand is 10 to 30 nucleotides.

75. 75. The siRNA molecule of claim 74, wherein the length of the antisense strand is 12 to 30 nucleotides.

76. 76. The siRNA molecule of claim 75, wherein the length of the antisense strand is 15 to 30 nucleotides.

77. 77. The siRNA molecule of claim 76, wherein the length of the antisense strand is 18 to 30 nucleotides.

78. 78. The siRNA molecule of claim 77, wherein the length of the antisense strand is 18 to 25 nucleotides.

79. 79. The siRNA molecule of claim 78, wherein the length of the antisense strand is 18 to 21 nucleotides.

80. 80. The siRNA molecule of claim 79, wherein the antisense strand is 18 nucleotides in length.

81. 80. The siRNA molecule of claim 79, wherein the antisense strand is 20 nucleotides in length.

82. 80. The siRNA molecule of claim 79, wherein the antisense strand is 21 nucleotides in length.

83. The siRNA molecule of any one of claims 1 to 82, which is a branched siRNA molecule.

84. 84. The siRNA molecule of claim 83, wherein the branched siRNA molecule is biantennary, triantennary, or tetraantennary.

85. the siRNA molecule is a biantennary siRNA molecule, optionally the biantennary siRNA molecule is represented by any one of Formulas XVII to XIX: 【Chemistry 2】 85. The siRNA molecule of claim 84, wherein each RNA is independently an siRNA molecule, L is a linker, and each X independently represents a branch point moiety.

86. the siRNA molecule is a tri-antennary siRNA molecule, optionally the tri-antennary siRNA molecule is represented by any one of formulas XX-XXIII: 【Chemistry 3】 85. The siRNA molecule of claim 84, wherein each RNA is independently an siRNA molecule, L is a linker, and each X independently represents a branch point moiety.

87. the siRNA molecule is a four-antennary siRNA molecule, optionally the four-antennary siRNA molecule is represented by any one of Formulas XXIV to XXVIII: 【Chemistry 4】 85. The siRNA molecule of claim 84, wherein each RNA is independently an siRNA molecule, L is a linker, and each X independently represents a branch point moiety.

88. 88. The siRNA molecule of any one of claims 85 to 87, 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.

89. 89. The siRNA molecule of claim 88, wherein the one or more consecutive subunits is between 2 and 20 consecutive subunits.

90. 90. The siRNA molecule of any one of claims 1 to 89, wherein the siRNA molecule is formulated as a salt comprising one or more divalent cations.

91. 91. The siRNA molecule of claim 90, wherein the siRNA molecule comprises multiple cationic binding sites that are partially or fully saturated with the one or more divalent cations.

92. The one or more divalent cations are Ba ２＋ , Be ２＋ , Ca ２＋ , Cu ２＋ , Mg ２＋ , Mn ２＋ , Ni ２＋ , or Zn ２＋ 92. The siRNA molecule of claim 90 or 91, comprising:

93. The one or more divalent cations are Mg ２＋ 93. The siRNA molecule of claim 92, comprising:

94. 94. The siRNA molecule of any one of claims 90 to 93, wherein the siRNA molecule comprises one or more atoms having a negative charge, the divalent cation comprises two positive charges, and the ratio of negative to positive charges is between 0.75 and 7.5, and optionally, the ratio of negative to positive charges is between 1.0 and 2.

0.

95. a) a ratio of negative to positive charges of 0.75 to 6.5, optionally a ratio of negative to positive charges of 0.75 to 5.5, 0.75 to 4.5, 0.75 to 3.5, 0.75 to 2.5, 0.75 to 1.5, or 0.75 to 1; or b) The siRNA molecule of claim 94, wherein the ratio of negative to positive charges is between 1 and 7.5, between 1.5 and 7.5, between 2.5 and 7.5, between 3.5 and 7.5, between 4.5 and 7.5, between 5.5 and 7.5, or between 6.5 and 7.

5.

96. 96. The siRNA molecule of any one of claims 90 to 95, wherein the molar ratio of the siRNA molecule to the one or more divalent cations is 1:10 to 1:

100.

97. 97. The siRNA molecule of claim 96, wherein the molar ratio of the siRNA molecules to the one or more divalent cations is between 1:10 and 1:50, optionally the molar ratio of the siRNA molecules to the one or more divalent cations is between 1:18 and 1:38, optionally the molar ratio of the siRNA molecules to the one or more divalent cations is between 1:20 and 1:25, optionally the molar ratio of the siRNA molecules to the one or more divalent cations is about 1:20, optionally the molar ratio of the siRNA molecules to the one or more divalent cations is about 1:

25.

98. A pharmaceutical composition comprising the siRNA molecule of any one of claims 1 to 97 and a pharmaceutically acceptable excipient, carrier, or diluent.

99. 100. A method of delivering an siRNA molecule to a subject diagnosed with Huntington's disease, comprising administering to the subject a therapeutically effective amount of the siRNA molecule of any one of claims 1 to 97 or the pharmaceutical composition of claim 98.

100. 100. A method of treating Huntington's disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an siRNA molecule of any one of claims 1 to 97 or a pharmaceutical composition of claim 98.

101. 99. A method of reducing HTT expression in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of an siRNA molecule of any one of claims 1 to 97 or a pharmaceutical composition of claim 98.

102. The method of any one of claims 99 to 101, wherein the siRNA molecule or the pharmaceutical composition is administered to the subject by intracerebroventricular, intrastriatal, intraparenchymal, or intrathecal injection.

103. The method of any one of claims 99 to 101, wherein the siRNA molecule or the pharmaceutical composition is administered to the subject by intravenous, intramuscular, or subcutaneous injection.

104. The method of any one of claims 99 to 103, wherein the subject is a human.

105. A kit comprising an siRNA molecule according to any one of claims 1 to 97, or a pharmaceutical composition according to claim 98, and a package insert, the package insert instructing a user of the kit to carry out the method according to any one of claims 99 to 104.

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