Double-stranded SIRNA with patterned chemical modifications

Abstract

The present disclosure provides single-stranded or double-stranded short interfering RNA (siRNA) molecules having a specific pattern of nucleoside and internucleoside linkage modifications, as well as pharmaceutical compositions comprising the same. The siRNA molecules may be branched siRNA molecules, such as bi-, tri-, or tetra-branched siRNA molecules. The disclosed siRNA molecules may further feature a 5' phosphorus stabilizing moiety and / or a hydrophobic moiety. Furthermore, the present disclosure provides a method for delivering the disclosed siRNA molecules to the central nervous system of a subject, such as a subject identified as having a disease.

Description

[Technical field]

[0001] The present disclosure relates to novel short interfering RNA (siRNA) molecules useful for RNA silencing, e.g., by RNA interference (RNAi), that contain patterns of chemically modified ribonucleotides, patterns of chemically modified internucleoside linkages, branched structures, hydrophobic moieties, and / or 5' phosphorus stabilizing moieties. [Background technology]

[0002] In many species, the introduction of double-stranded RNA (dsRNA) induces strong and specific gene silencing by RNA interference (RNAi). This phenomenon occurs in both plants and animals and has a role in viral defense and transposon silencing mechanisms. Short interfering RNA (siRNA), which is generally much shorter than the target gene, has been shown to be effective in gene silencing and is therefore useful as a therapeutic agent to silence genes and restore the activity of genetic and biochemical pathways from diseased states to normal, healthy states.

[0003] It is known that siRNA containing chemically modified ribonucleosides and / or chemically modified linkers exhibit increased nuclease resistance compared to corresponding unmodified siRNA while maintaining RNAi activity.There is still a need for siRNA molecules with improved nuclease resistance and gene silencing effect. Summary of the Invention

[0004] In a first aspect, the present disclosure provides a small interfering RNA (siRNA) molecule comprising an antisense strand and a sense strand having complementarity to the antisense strand, wherein the antisense strand comprises a structure represented by Formula I, which in the 5'-3' direction is: AB-(A') j -CP 2 -DP 1 -(C'-P 1 ) k -C' Formula I; where A is of the formula CP 1 -DP 1 each A' is independently 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 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; and k is an integer from 1 to 7.

[0005] In some embodiments, the antisense strand comprises a structure represented by Formula A1, which in the 5'-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.

[0006] In some embodiments, the antisense strand comprises a structure represented by Formula II, which in the 5'-3' direction is: AB-(A') j -CP 2 -DP 1 -(CP 1 ) k -C' Formula II; where A is of the formula CP 1 -DP 1each A' is independently 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 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 (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).

[0007] In some embodiments, the antisense strand comprises a structure represented by Formula A2, which, in the 5'-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.

[0008] In some embodiments, the sense strand comprises a structure represented by Formula III, which, in the 5'-3' direction, is: E-(A') m -F Formula III; In the formula, E is a compound represented by the formula (CP 1 )2; F is represented by the formula (CP 2 )3-DP 1 -CP 1 -C, (CP 2 )3-DP 2 -CP2 -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).

[0009] In some embodiments, j is 4 and k is 4. In some embodiments, m is 4.

[0010] In some embodiments, the sense strand comprises a structure represented by formula S1, which in the 5'-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.

[0011] In some embodiments, the sense strand comprises a structure represented by formula S2, which in the 5'-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.

[0012] In some embodiments, the sense strand comprises a structure represented by formula S3, which, in the 5'-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.

[0013] In some embodiments, the sense strand comprises a structure represented by formula S4, which, in the 5'-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.

[0014] In another aspect, the disclosure provides an siRNA molecule comprising an antisense strand and a sense strand having complementarity to the antisense strand, wherein the antisense strand comprises a structure represented by Formula IV, where Formula IV is in the 5'-3' direction: A-(A') j -CP 2 -B-(CP 1 ) k -C' Formula IV; where A is of the formula CP 1 -DP 1 each A' is independently represented by the formula CP 2 -DP 2 B is represented by the formula DP 1 -CP 1 -DP 1 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 1is independently a phosphorothioate internucleoside linkage; each P 2 is independently 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).

[0015] In some embodiments of the foregoing aspects, the antisense strand comprises a structure represented by Formula A3, which, in the 5'-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.

[0016] In some embodiments of the foregoing aspects, the sense strand comprises a structure represented by Formula V, where in the 5'-3' direction, Formula V is: E-(A') m -CP 2 -F formula V; In the formula, E is a compound represented by the formula (CP 1 ) 2; F is represented by the formula DP 1 -CP 1 -C, D.P. 2 -CP 2 -C, D.P. 1 -CP 1 -D, or DP 2 -CP 2 -D; A', C, D, PP 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).

[0017] In some embodiments of the foregoing aspects, j is 6 and k is 2. In some embodiments, m is 5.

[0018] In some embodiments of the foregoing aspects, the sense strand comprises a structure represented by formula S5, which, in the 5'-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.

[0019] In some embodiments of the foregoing aspects, the sense strand comprises a structure represented by formula S6, which, in the 5'-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.

[0020] In some embodiments of the foregoing aspects, the sense strand comprises a structure represented by formula S7, which, in the 5'-3' direction, is: ASASAOBOAOBOAOBOAOBOA-OBOAOBSASB Formula S7; wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0021] In some embodiments of the foregoing aspects, the sense strand comprises a structure represented by formula S8, which, in the 5'-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. 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 comprises a structure represented by Formula VI, where Formula VI is in the 5'-3' direction: A.B. j -EB k -EFG l -DP 1 -C' Formula VI; where A is of the formula CP 1 -DP 1 each B is independently represented by the formula CP 2 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 a group of the formula DP 2 -CP 2 F is represented by the formula DP 1 -CP 1 each G is independently represented by the formula CP 1 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 (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).

[0022] In some embodiments of the foregoing aspects, the antisense strand comprises a structure represented by formula A4, which, in the 5'-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.

[0023] In some embodiments of the foregoing aspects, the sense strand comprises a structure represented by formula VII, which, in the 5'-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 each H is independently represented by the formula (CP 1 each I is independently 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).

[0024] In some embodiments of the foregoing aspects, j is 3, k is 6, and l is 2. In some embodiments, m is 3, n is 3, and o is 3.

[0025] In some embodiments of the foregoing aspects, the sense strand comprises a structure represented by formula S9, which, in the 5'-3' direction, is: ASASAOAOAOBOBOBOAOBOA-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.

[0026] In some embodiments of any of the preceding aspects, the antisense strand further comprises a 5' phosphorus stabilization moiety at the 5' end of the antisense strand. In some embodiments of any of the preceding aspects, the sense strand further comprises a 5' phosphorus stabilization moiety at the 5' end of the antisense strand. In some embodiments, the 5' phosphorus stabilization moiety is represented by any one of Formulas VIII-XV: [ka] wherein Nuc represents a nucleobase and R represents an optionally substituted alkyl, an optionally substituted alkenyl, an optionally substituted alkynyl (e.g., an optionally substituted C1-C6 alkyl, an optionally substituted C2-C6 alkenyl, or an optionally substituted C2-C6 alkynyl), phenyl, benzyl, hydroxy, or hydrogen. In some embodiments, the nucleobase is adenine, uracil, guanine, thymine, or cytosine. In some embodiments, the 5' phosphorus stabilizing moiety is an (E)-vinyl phosphonate represented by formula X.

[0027] In some embodiments of any one of the preceding 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, and tocopherol.

[0028] In some embodiments of any one of the foregoing aspects, the length of the antisense strand is 10-30 (e.g., 12-28, 14-26, 16-24, or 18-22) nucleotides. In some embodiments, the length of the antisense strand is 15-25 (e.g., 16-24, 17-23, 18-22, or 19-21) 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.

[0029] In some embodiments of any one of the foregoing aspects, the sense strand is 12-20 nucleotides (e.g., 13-19, 14-18, or 15-17) nucleotides in length. 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.

[0030] In some embodiments, the antisense strand is 18 nucleotides in length and the sense strand is 14 nucleotides in length.

[0031] In some embodiments, the antisense strand is 18 nucleotides in length and the sense strand is 15 nucleotides in length.

[0032] In some embodiments, the antisense strand is 18 nucleotides in length and the sense strand is 16 nucleotides in length.

[0033] In some embodiments, the antisense strand is 18 nucleotides in length and the sense strand is 17 nucleotides in length.

[0034] In some embodiments, the antisense strand is 18 nucleotides in length and the sense strand is 18 nucleotides in length.

[0035] In some embodiments, the antisense strand is 19 nucleotides in length and the sense strand is 14 nucleotides in length.

[0036] In some embodiments, the antisense strand is 19 nucleotides in length and the sense strand is 15 nucleotides in length.

[0037] In some embodiments, the antisense strand is 19 nucleotides in length and the sense strand is 16 nucleotides in length.

[0038] In some embodiments, the antisense strand is 19 nucleotides in length and the sense strand is 17 nucleotides in length.

[0039] In some embodiments, the antisense strand is 19 nucleotides in length and the sense strand is 18 nucleotides in length.

[0040] In some embodiments, the antisense strand is 19 nucleotides in length and the sense strand is 19 nucleotides in length.

[0041] In some embodiments, the antisense strand is 20 nucleotides in length and the sense strand is 14 nucleotides in length.

[0042] In some embodiments, the antisense strand is 20 nucleotides in length and the sense strand is 15 nucleotides in length.

[0043] In some embodiments, the antisense strand is 20 nucleotides in length and the sense strand is 16 nucleotides in length.

[0044] In some embodiments, the antisense strand is 20 nucleotides in length and the sense strand is 17 nucleotides in length.

[0045] In some embodiments, the antisense strand is 20 nucleotides in length and the sense strand is 18 nucleotides in length.

[0046] In some embodiments, the antisense strand is 20 nucleotides in length and the sense strand is 19 nucleotides in length.

[0047] In some embodiments, the antisense strand is 20 nucleotides in length and the sense strand is 20 nucleotides in length.

[0048] In some embodiments, the antisense strand is 21 nucleotides in length and the sense strand is 14 nucleotides in length.

[0049] In some embodiments, the antisense strand is 21 nucleotides in length and the sense strand is 15 nucleotides in length.

[0050] In some embodiments, the antisense strand is 21 nucleotides in length and the sense strand is 16 nucleotides in length.

[0051] In some embodiments, the antisense strand is 21 nucleotides in length and the sense strand is 17 nucleotides in length.

[0052] In some embodiments, the antisense strand is 21 nucleotides in length and the sense strand is 18 nucleotides in length.

[0053] In some embodiments, the antisense strand is 21 nucleotides in length and the sense strand is 19 nucleotides in length.

[0054] In some embodiments, the antisense strand is 21 nucleotides in length and the sense strand is 20 nucleotides in length.

[0055] In some embodiments, the antisense strand is 21 nucleotides in length and the sense strand is 21 nucleotides in length.

[0056] In some embodiments, the antisense strand is 22 nucleotides in length and the sense strand is 14 nucleotides in length.

[0057] In some embodiments, the antisense strand is 22 nucleotides in length and the sense strand is 15 nucleotides in length.

[0058] In some embodiments, the antisense strand is 22 nucleotides in length and the sense strand is 16 nucleotides in length.

[0059] In some embodiments, the antisense strand is 22 nucleotides in length and the sense strand is 17 nucleotides in length.

[0060] In some embodiments, the antisense strand is 22 nucleotides in length and the sense strand is 18 nucleotides in length.

[0061] In some embodiments, the antisense strand is 22 nucleotides in length and the sense strand is 19 nucleotides in length.

[0062] In some embodiments, the antisense strand is 22 nucleotides in length and the sense strand is 20 nucleotides in length.

[0063] In some embodiments, the antisense strand is 22 nucleotides in length and the sense strand is 21 nucleotides in length.

[0064] In some embodiments, the antisense strand is 22 nucleotides in length and the sense strand is 22 nucleotides in length.

[0065] In some embodiments, the antisense strand is 23 nucleotides in length and the sense strand is 14 nucleotides in length.

[0066] In some embodiments, the antisense strand is 23 nucleotides in length and the sense strand is 15 nucleotides in length.

[0067] In some embodiments, the antisense strand is 23 nucleotides in length and the sense strand is 16 nucleotides in length.

[0068] In some embodiments, the antisense strand is 23 nucleotides in length and the sense strand is 17 nucleotides in length.

[0069] In some embodiments, the antisense strand is 23 nucleotides in length and the sense strand is 18 nucleotides in length.

[0070] In some embodiments, the antisense strand is 23 nucleotides in length and the sense strand is 19 nucleotides in length.

[0071] In some embodiments, the antisense strand is 23 nucleotides in length and the sense strand is 20 nucleotides in length.

[0072] In some embodiments, the antisense strand is 23 nucleotides in length and the sense strand is 21 nucleotides in length.

[0073] In some embodiments, the antisense strand is 23 nucleotides in length and the sense strand is 22 nucleotides in length.

[0074] In some embodiments, the antisense strand is 23 nucleotides in length and the sense strand is 23 nucleotides in length.

[0075] In some embodiments, the antisense strand is 24 nucleotides in length and the sense strand is 14 nucleotides in length.

[0076] In some embodiments, the antisense strand is 24 nucleotides in length and the sense strand is 15 nucleotides in length.

[0077] In some embodiments, the antisense strand is 24 nucleotides in length and the sense strand is 16 nucleotides in length.

[0078] In some embodiments, the antisense strand is 24 nucleotides in length and the sense strand is 17 nucleotides in length.

[0079] In some embodiments, the antisense strand is 24 nucleotides in length and the sense strand is 18 nucleotides in length.

[0080] In some embodiments, the antisense strand is 24 nucleotides in length and the sense strand is 19 nucleotides in length.

[0081] In some embodiments, the antisense strand is 24 nucleotides in length and the sense strand is 20 nucleotides in length.

[0082] In some embodiments, the antisense strand is 24 nucleotides in length and the sense strand is 21 nucleotides in length.

[0083] In some embodiments, the antisense strand is 24 nucleotides in length and the sense strand is 22 nucleotides in length.

[0084] In some embodiments, the antisense strand is 24 nucleotides in length and the sense strand is 23 nucleotides in length.

[0085] In some embodiments, the antisense strand is 24 nucleotides in length and the sense strand is 24 nucleotides in length.

[0086] In some embodiments, the antisense strand is 25 nucleotides in length and the sense strand is 14 nucleotides in length.

[0087] In some embodiments, the antisense strand is 25 nucleotides in length and the sense strand is 15 nucleotides in length.

[0088] In some embodiments, the antisense strand is 25 nucleotides in length and the sense strand is 16 nucleotides in length.

[0089] In some embodiments, the antisense strand is 25 nucleotides in length and the sense strand is 17 nucleotides in length.

[0090] In some embodiments, the antisense strand is 25 nucleotides in length and the sense strand is 18 nucleotides in length.

[0091] In some embodiments, the antisense strand is 25 nucleotides in length and the sense strand is 19 nucleotides in length.

[0092] In some embodiments, the antisense strand is 25 nucleotides in length and the sense strand is 20 nucleotides in length.

[0093] In some embodiments, the antisense strand is 25 nucleotides in length and the sense strand is 21 nucleotides in length.

[0094] In some embodiments, the antisense strand is 25 nucleotides in length and the sense strand is 22 nucleotides in length.

[0095] In some embodiments, the antisense strand is 25 nucleotides in length and the sense strand is 23 nucleotides in length.

[0096] In some embodiments, the antisense strand is 25 nucleotides in length and the sense strand is 24 nucleotides in length.

[0097] In some embodiments, the antisense strand is 25 nucleotides in length and the sense strand is 25 nucleotides in length.

[0098] In some embodiments, the antisense strand is 26 nucleotides in length and the sense strand is 14 nucleotides in length.

[0099] In some embodiments, the antisense strand is 26 nucleotides in length and the sense strand is 15 nucleotides in length.

[0100] In some embodiments, the antisense strand is 26 nucleotides in length and the sense strand is 16 nucleotides in length.

[0101] In some embodiments, the antisense strand is 26 nucleotides in length and the sense strand is 17 nucleotides in length.

[0102] In some embodiments, the antisense strand is 26 nucleotides in length and the sense strand is 18 nucleotides in length.

[0103] In some embodiments, the antisense strand is 26 nucleotides in length and the sense strand is 19 nucleotides in length.

[0104] In some embodiments, the antisense strand is 26 nucleotides in length and the sense strand is 20 nucleotides in length.

[0105] In some embodiments, the antisense strand is 26 nucleotides in length and the sense strand is 21 nucleotides in length.

[0106] In some embodiments, the antisense strand is 26 nucleotides in length and the sense strand is 22 nucleotides in length.

[0107] In some embodiments, the antisense strand is 26 nucleotides in length and the sense strand is 23 nucleotides in length.

[0108] In some embodiments, the antisense strand is 26 nucleotides in length and the sense strand is 24 nucleotides in length.

[0109] In some embodiments, the antisense strand is 26 nucleotides in length and the sense strand is 25 nucleotides in length.

[0110] In some embodiments, the antisense strand is 26 nucleotides in length and the sense strand is 26 nucleotides in length.

[0111] In some embodiments, the antisense strand is 27 nucleotides in length and the sense strand is 14 nucleotides in length.

[0112] In some embodiments, the antisense strand is 27 nucleotides in length and the sense strand is 15 nucleotides in length.

[0113] In some embodiments, the antisense strand is 27 nucleotides in length and the sense strand is 16 nucleotides in length.

[0114] In some embodiments, the antisense strand is 27 nucleotides in length and the sense strand is 17 nucleotides in length.

[0115] In some embodiments, the antisense strand is 27 nucleotides in length and the sense strand is 18 nucleotides in length.

[0116] In some embodiments, the antisense strand is 27 nucleotides in length and the sense strand is 19 nucleotides in length.

[0117] In some embodiments, the antisense strand is 27 nucleotides in length and the sense strand is 20 nucleotides in length.

[0118] In some embodiments, the antisense strand is 27 nucleotides in length and the sense strand is 21 nucleotides in length.

[0119] In some embodiments, the antisense strand is 27 nucleotides in length and the sense strand is 22 nucleotides in length.

[0120] In some embodiments, the antisense strand is 27 nucleotides in length and the sense strand is 23 nucleotides in length.

[0121] In some embodiments, the antisense strand is 27 nucleotides in length and the sense strand is 24 nucleotides in length.

[0122] In some embodiments, the antisense strand is 27 nucleotides in length and the sense strand is 25 nucleotides in length.

[0123] In some embodiments, the antisense strand is 27 nucleotides in length and the sense strand is 26 nucleotides in length.

[0124] In some embodiments, the antisense strand is 27 nucleotides in length and the sense strand is 27 nucleotides in length.

[0125] In some embodiments, the antisense strand is 28 nucleotides in length and the sense strand is 14 nucleotides in length.

[0126] In some embodiments, the antisense strand is 28 nucleotides in length and the sense strand is 15 nucleotides in length.

[0127] In some embodiments, the antisense strand is 28 nucleotides in length and the sense strand is 16 nucleotides in length.

[0128] In some embodiments, the antisense strand is 28 nucleotides in length and the sense strand is 17 nucleotides in length.

[0129] In some embodiments, the antisense strand is 28 nucleotides in length and the sense strand is 18 nucleotides in length.

[0130] In some embodiments, the antisense strand is 28 nucleotides in length and the sense strand is 19 nucleotides in length.

[0131] In some embodiments, the antisense strand is 28 nucleotides in length and the sense strand is 20 nucleotides in length.

[0132] In some embodiments, the antisense strand is 28 nucleotides in length and the sense strand is 21 nucleotides in length.

[0133] In some embodiments, the antisense strand is 28 nucleotides in length and the sense strand is 22 nucleotides in length.

[0134] In some embodiments, the antisense strand is 28 nucleotides in length and the sense strand is 23 nucleotides in length.

[0135] In some embodiments, the antisense strand is 28 nucleotides in length and the sense strand is 24 nucleotides in length.

[0136] In some embodiments, the antisense strand is 28 nucleotides in length and the sense strand is 25 nucleotides in length.

[0137] In some embodiments, the antisense strand is 28 nucleotides in length and the sense strand is 26 nucleotides in length.

[0138] In some embodiments, the antisense strand is 28 nucleotides in length and the sense strand is 27 nucleotides in length.

[0139] In some embodiments, the antisense strand is 28 nucleotides in length and the sense strand is 28 nucleotides in length.

[0140] In some embodiments, the antisense strand is 29 nucleotides in length and the sense strand is 14 nucleotides in length.

[0141] In some embodiments, the antisense strand is 29 nucleotides in length and the sense strand is 15 nucleotides in length.

[0142] In some embodiments, the antisense strand is 29 nucleotides in length and the sense strand is 16 nucleotides in length.

[0143] In some embodiments, the antisense strand is 29 nucleotides in length and the sense strand is 17 nucleotides in length.

[0144] In some embodiments, the antisense strand is 29 nucleotides in length and the sense strand is 18 nucleotides in length.

[0145] In some embodiments, the antisense strand is 29 nucleotides in length and the sense strand is 19 nucleotides in length.

[0146] In some embodiments, the antisense strand is 29 nucleotides in length and the sense strand is 20 nucleotides in length.

[0147] In some embodiments, the antisense strand is 29 nucleotides in length and the sense strand is 21 nucleotides in length.

[0148] In some embodiments, the antisense strand is 29 nucleotides in length and the sense strand is 22 nucleotides in length.

[0149] In some embodiments, the antisense strand is 29 nucleotides in length and the sense strand is 23 nucleotides in length.

[0150] In some embodiments, the antisense strand is 29 nucleotides in length and the sense strand is 24 nucleotides in length.

[0151] In some embodiments, the antisense strand is 29 nucleotides in length and the sense strand is 25 nucleotides in length.

[0152] In some embodiments, the antisense strand is 29 nucleotides in length and the sense strand is 26 nucleotides in length.

[0153] In some embodiments, the antisense strand is 29 nucleotides in length and the sense strand is 27 nucleotides in length.

[0154] In some embodiments, the antisense strand is 29 nucleotides in length and the sense strand is 28 nucleotides in length.

[0155] In some embodiments, the antisense strand is 29 nucleotides in length and the sense strand is 29 nucleotides in length.

[0156] In some embodiments, the antisense strand is 30 nucleotides in length and the sense strand is 14 nucleotides in length.

[0157] In some embodiments, the antisense strand is 30 nucleotides in length and the sense strand is 15 nucleotides in length.

[0158] In some embodiments, the antisense strand is 30 nucleotides in length and the sense strand is 16 nucleotides in length.

[0159] In some embodiments, the antisense strand is 30 nucleotides in length and the sense strand is 17 nucleotides in length.

[0160] In some embodiments, the antisense strand is 30 nucleotides in length and the sense strand is 18 nucleotides in length.

[0161] In some embodiments, the antisense strand is 30 nucleotides in length and the sense strand is 19 nucleotides in length.

[0162] In some embodiments, the antisense strand is 30 nucleotides in length and the sense strand is 20 nucleotides in length.

[0163] In some embodiments, the antisense strand is 30 nucleotides in length and the sense strand is 21 nucleotides in length.

[0164] In some embodiments, the antisense strand is 30 nucleotides in length and the sense strand is 22 nucleotides in length.

[0165] In some embodiments, the antisense strand is 30 nucleotides in length and the sense strand is 23 nucleotides in length.

[0166] In some embodiments, the antisense strand is 30 nucleotides in length and the sense strand is 24 nucleotides in length.

[0167] In some embodiments, the antisense strand is 30 nucleotides in length and the sense strand is 25 nucleotides in length.

[0168] In some embodiments, the antisense strand is 30 nucleotides in length and the sense strand is 26 nucleotides in length.

[0169] In some embodiments, the antisense strand is 30 nucleotides in length and the sense strand is 27 nucleotides in length.

[0170] In some embodiments, the antisense strand is 30 nucleotides in length and the sense strand is 28 nucleotides in length.

[0171] In some embodiments, the antisense strand is 30 nucleotides in length and the sense strand is 29 nucleotides in length.

[0172] In some embodiments, the antisense strand is 30 nucleotides in length and the sense strand is 30 nucleotides in length. In some embodiments of any one of the preceding aspects, 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 XVI-XVIII: [ka] wherein each RNA is independently an siRNA molecule, L is a linker, and each X independently represents a branch point moiety (e.g., a phosphoramidite, a tosylated solketal, 1,3-diaminopropanol, pentaerythritol, or any one of the branch point moieties described in U.S. Pat. No. 10,478,503). In some embodiments, the tri-branched siRNA molecule is represented by any one of formulas XIX-XXII: [ka] wherein each RNA is independently an siRNA molecule, L is a linker, and each X independently represents a branch point moiety. In some embodiments, the four-branched siRNA molecule is represented by any one of formulas XXIII-XXVII: [ka] wherein each RNA is independently a siRNA molecule, L is a linker, and each X independently represents a branch point moiety.

[0173] In some embodiments, the linker is selected from the group consisting of one or more consecutive subunits of ethylene glycol, alkyl, carbohydrate, block copolymer, peptide, RNA, and DNA, in some embodiments, the one or more consecutive subunits are 2-20 (e.g., 3-19, 4-18, 5-17, 6-16, 7-15, 8-14, 9-13, or 10-12) consecutive subunits.

[0174] In some embodiments, the linker is attached to one or more siRNA (e.g., one, two, or more) molecules of any of the above aspects and embodiments 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, phosphorothioate, and phosphoramidate.

[0175] In some embodiments, the antisense strand is selected from the group consisting of ABCA7, ABI3, ADAM10, APOC1, APOE, AXL, BIN1, C1QA, C3, C9ORF72, CASS4, CCL5, CD2AP, CD33, CD68, CLPTM1, CLU, CR1, CSF1, CST7, CTSB, CTSD, CTSL, CXCL10, CXCL13, DSG2, ECHDC3, EPHA1, FABP5, FERMT2, FTH1, GNAS, GRN, HBEGF, HLA-DRB1, HLA-DRB5, HTT, IFIT1, IFIT3, IFITM3, IFNAR1, IFNAR2, IGF 1, IL10RA, IL1A, IL1B, IL1RAP, INPP5D, ITGAM, ITGAX, KCNT1, LILRB4, LPL, MAPT, MEF2C, MMP12, MS4A4A, MS4A6A, MSH3, NLRP3, NME8, NOS2, PICALM, PILRA, PLCG2, PRNP, PTK2B, SCIMP, SCN9A, SLC24A4, SNCA, SORL1, SPI1, SPP1, SPPL2A, TBK1, TNF, TREM2, TREML2, TYROBP, and ZCWPW1.

[0176] In some embodiments, the antisense strand has sufficient complementarity to hybridize to a portion of a gene selected from HTT, MAPT, SNCA, C9ORF72, APOE, SCN9A, KCNT1, PRNP, and MSH3.

[0177] In some embodiments, the antisense strand has sufficient complementarity to hybridize with a portion of the HTT gene.

[0178] In another aspect, the present disclosure provides a pharmaceutical composition comprising the siRNA molecule of any one of the preceding aspects and embodiments, and a pharma- ceutically acceptable excipient, carrier, or diluent.

[0179] In another aspect, the present disclosure provides a method for delivering an siRNA molecule to the central nervous system (CNS) of a subject, comprising administering to the CNS of a subject an siRNA molecule of any one of the above aspects and embodiments, or a pharmaceutical composition of the above aspect. In some embodiments, the siRNA molecule or pharmaceutical composition is administered to the subject by intrastriatal, intracerebroventricular, or intrathecal injection.

[0180] In some embodiments of the above aspect, delivery of the siRNA molecule to the CNS of a subject results in gene silencing of the target gene in the subject. In some embodiments, the target gene is an overactive disease-driving gene. In some embodiments, the target gene is a negative regulator of a gene with reduced expression associated with a disease state in the subject. In some embodiments, the target gene is a positive regulator of a gene with increased expression associated with a disease state in the subject. In some embodiments, the target gene is a splice isoform of the target gene, and the splice isoform reduces the expression of the target gene. In some embodiments, gene silencing treats the disease state in the subject.

[0181] In some embodiments, the subject is a human.

[0182] In another aspect, the present disclosure provides a kit comprising an siRNA molecule of any one of the preceding aspects and embodiments, or a pharmaceutical composition of the preceding aspects, and a package insert, the package insert directing a user of the kit to carry out a method of the preceding aspects and embodiments. [Brief description of the drawings]

[0183] [Figure 1A]1 shows the antisense strand of an exemplary "first generation (F1)" double-stranded (ds-) short interfering (si) RNA (ds-siRNA) molecule (ds-siRNA A_V1) used to compare knockdown efficacy to one or more of the "second generation (F2)" ds-siRNA molecules of the present disclosure. The antisense strand exhibits an alternating pattern (i.e., motif) of 2'-O-methyl (2'-O-Me) and 2'-fluoro (2'-F) modified ribonucleosides. The ds-sRNA molecule also features a central block of approximately 11-13 modified ribonucleosides linked by phosphodiester internucleoside linkages flanked at the 5' and 3' ends, or at the 5' end only, by blocks of 2-5 modified ribonucleosides linked by phosphorothioate internucleoside linkages. [Figure 1B] 1 shows the sense strand of an exemplary "first generation (F1)" double-stranded (ds-) short interfering (si) RNA (ds-siRNA) molecule (ds-siRNA A_V1) used to compare knockdown efficacy to one or more of the "second generation (F2)" ds-siRNA molecules of the present disclosure. The sense strand exhibits an alternating pattern (i.e., motif) of 2'-O-methyl (2'-O-Me) and 2'-fluoro (2'-F) modified ribonucleosides. The ds-sRNA molecule also features a central block of approximately 11-13 modified ribonucleosides linked by phosphodiester internucleoside linkages flanked at the 5' and 3' ends, or at the 5' end only, by blocks of 2-5 modified ribonucleosides linked by phosphorothioate internucleoside linkages. [Figure 2A]1 shows the antisense strand of another exemplary F1 ds-siRNA molecule (ds-siRNA A_V2) used to compare knockdown efficacy to one or more F2 ds-siRNA molecules of the present disclosure. The antisense strand exhibits an alternating pattern (i.e., motif) of 2'-O-Me and 2'-F modified ribonucleosides. The ds-sRNA molecule also features a central block of approximately 11-13 modified ribonucleosides linked by phosphodiester internucleoside linkages flanked at the 5' and 3' ends, or only at the 5' end, by blocks of 2-7 modified ribonucleosides linked by phosphorothioate internucleoside linkages. [Figure 2B] 1 shows the sense strand of another exemplary F1 ds-siRNA molecule (ds-siRNA A_V2) used to compare knockdown efficacy to one or more F2 ds-siRNA molecules of the present disclosure. The sense strand exhibits an alternating pattern (i.e., motif) of 2'-O-Me and 2'-F modified ribonucleosides. The ds-sRNA molecule also features a central block of approximately 11-13 modified ribonucleosides linked by phosphodiester internucleoside linkages flanked at the 5' and 3' ends, or only at the 5' end, by blocks of 2-7 modified ribonucleosides linked by phosphorothioate internucleoside linkages. [Figure 3A] 1 shows the antisense strand of an exemplary F2 double-stranded (ds-) short interfering (si)RNA (ds-siRNA) molecule (ds-siRNA A_V3) of the present disclosure. The antisense strand exhibits an alternating pattern (i.e., motif) of 2'-O-Me and 2'-F modified ribonucleosides. The ds-sRNA molecule also features a central block of approximately 11-13 modified ribonucleosides linked by phosphodiester internucleoside linkages flanked at the 5' and 3' ends, or at the 5' end only, by blocks of 2-5 modified ribonucleosides linked by phosphorothioate internucleoside linkages. [Figure 3B]1 shows the sense strand of an exemplary F2 double-stranded (ds-) short interfering (si)RNA (ds-siRNA) molecule (ds-siRNA A_V3) of the present disclosure. The sense strand exhibits an alternating pattern (i.e., motif) of 2'-O-Me and 2'-F modified ribonucleosides. The ds-sRNA molecule also features a central block of approximately 11-13 modified ribonucleosides linked by phosphodiester internucleoside linkages flanked at the 5' and 3' ends, or at the 5' end only, by blocks of 2-5 modified ribonucleosides linked by phosphorothioate internucleoside linkages. [Figure 4A] The antisense strand of an exemplary F2 ds-siRNA molecule (ds-siRNA A_V4) of the present disclosure is shown. The antisense strand exhibits an alternating pattern (i.e., motif) of 2'-O-Me and 2'-F modified ribonucleosides. The ds-sRNA molecule also features a central block of approximately 11-13 modified ribonucleosides linked by phosphodiester internucleoside linkages flanked at the 5' and 3' ends, or only at the 5' end, by blocks of 2-5 modified ribonucleosides linked by phosphorothioate internucleoside linkages. [Figure 4B] The sense strand of an exemplary F2 ds-siRNA molecule (ds-siRNA A_V4) of the present disclosure (FIG. 4B) is shown. The sense strand exhibits an alternating pattern (i.e., motif) of 2'-O-Me and 2'-F modified ribonucleosides. The ds-sRNA molecule also features a central block of approximately 11-13 modified ribonucleosides linked by phosphodiester internucleoside linkages flanked at the 5' and 3' ends, or at the 5' end only, by blocks of 2-5 modified ribonucleosides linked by phosphorothioate internucleoside linkages. [Figure 5A]1 shows the antisense strand of an exemplary F2 ds-siRNA molecule (ds-siRNA A_V5) of the present disclosure. The antisense strand exhibits an alternating pattern (i.e., motif) of 2'-O-Me and 2'-F modified ribonucleosides. The ds-sRNA molecule also features a central block of approximately 11-13 modified ribonucleosides linked by phosphodiester internucleoside linkages flanked at the 5' and 3' ends, or only at the 5' end, by blocks of 2-5 modified ribonucleosides linked by phosphorothioate internucleoside linkages. [Figure 5B] 1 shows the sense strand of an exemplary F2 ds-siRNA molecule (ds-siRNA A_V5) of the present disclosure. The sense strand exhibits an alternating pattern (i.e., motif) of 2'-O-Me and 2'-F modified ribonucleosides. The ds-sRNA molecule also features a central block of approximately 11-13 modified ribonucleosides linked by phosphodiester internucleoside linkages flanked at the 5' and 3' ends, or only at the 5' end, by blocks of 2-5 modified ribonucleosides linked by phosphorothioate internucleoside linkages. [Figure 6A] The antisense strand of an exemplary F2 ds-siRNA molecule (ds-siRNA A_V6) of the present disclosure is shown. The antisense strand exhibits an alternating pattern (i.e., motif) of 2'-O-Me and 2'-F modified ribonucleosides. The ds-sRNA molecule also features a central block of approximately 11-13 modified ribonucleosides linked by phosphodiester internucleoside linkages flanked at the 5' and 3' ends, or only at the 5' end, by blocks of 2-5 modified ribonucleosides linked by phosphorothioate internucleoside linkages. [Figure 6B]1 shows the sense strand of an exemplary F2 ds-siRNA molecule (ds-siRNA A_V6) of the present disclosure. The sense strand exhibits an alternating pattern (i.e., motif) of 2'-O-Me and 2'-F modified ribonucleosides. The ds-sRNA molecule also features a central block of approximately 11-13 modified ribonucleosides linked by phosphodiester internucleoside linkages flanked at the 5' and 3' ends, or only at the 5' end, by blocks of 2-5 modified ribonucleosides linked by phosphorothioate internucleoside linkages. [Figure 7A] 1 shows the antisense strand of another exemplary F1 ds-siRNA molecule (ds-siRNA B_V1) used to compare knockdown efficacy to one or more F2 ds-siRNA molecules of the present disclosure. The antisense strand exhibits an alternating pattern (i.e., motif) of 2'-O-Me and 2'-F modified ribonucleosides. The ds-sRNA molecule also features a central block of approximately 11-13 modified ribonucleosides linked by phosphodiester internucleoside linkages flanked at the 5' and 3' ends, or only at the 5' end, by blocks of 2-5 modified ribonucleosides linked by phosphorothioate internucleoside linkages. [Figure 7B] 1 shows the sense strand of another exemplary F1 ds-siRNA molecule (ds-siRNA B_V1) used to compare knockdown efficacy to one or more F2 ds-siRNA molecules of the present disclosure. The sense strand exhibits an alternating pattern (i.e., motif) of 2'-O-Me and 2'-F modified ribonucleosides. The ds-sRNA molecule also features a central block of approximately 11-13 modified ribonucleosides linked by phosphodiester internucleoside linkages flanked at the 5' and 3' ends, or only at the 5' end, by blocks of 2-5 modified ribonucleosides linked by phosphorothioate internucleoside linkages. [Figure 8A]1 shows the antisense strand of another exemplary F1 ds-siRNA molecule (ds-siRNA B_V2) used to compare knockdown efficacy to one or more F2 ds-siRNA molecules of the present disclosure. The antisense strand exhibits an alternating pattern (i.e., motif) of 2'-O-Me and 2'-F modified ribonucleosides. The ds-sRNA molecule also features a central block of approximately 11-13 modified ribonucleosides linked by phosphodiester internucleoside linkages flanked at the 5' and 3' ends, or only at the 5' end, by blocks of 2-7 modified ribonucleosides linked by phosphorothioate internucleoside linkages. [Figure 8B] 1 shows the sense strand of another exemplary F1 ds-siRNA molecule (ds-siRNA B_V2) used to compare knockdown efficacy to one or more F2 ds-siRNA molecules of the present disclosure. The sense strand exhibits an alternating pattern (i.e., motif) of 2'-O-Me and 2'-F modified ribonucleosides. The ds-sRNA molecule also features a central block of approximately 11-13 modified ribonucleosides linked by phosphodiester internucleoside linkages flanked at the 5' and 3' ends, or only at the 5' end, by blocks of 2-7 modified ribonucleosides linked by phosphorothioate internucleoside linkages. [Figure 9A] 1 shows the antisense strand of an exemplary F2 ds-siRNA molecule (ds-siRNA B_V3) of the present disclosure. The antisense strand exhibits an alternating pattern (i.e., motif) of 2'-O-Me and 2'-F modified ribonucleosides. The ds-sRNA molecule also features a central block of approximately 11-13 modified ribonucleosides linked by phosphodiester internucleoside linkages flanked at the 5' and 3' ends, or only at the 5' end, by blocks of 2-5 modified ribonucleosides linked by phosphorothioate internucleoside linkages. [Figure 9B]1 shows the sense strand of an exemplary F2 ds-siRNA molecule (ds-siRNA B_V3) of the present disclosure. The sense strand exhibits an alternating pattern (i.e., motif) of 2'-O-Me and 2'-F modified ribonucleosides. The ds-sRNA molecule also features a central block of approximately 11-13 modified ribonucleosides linked by phosphodiester internucleoside linkages flanked at the 5' and 3' ends, or only at the 5' end, by blocks of 2-5 modified ribonucleosides linked by phosphorothioate internucleoside linkages. [Figure 10A] 1 shows the antisense strand of an exemplary F2 ds-siRNA molecule (ds-siRNA B_V4) of the present disclosure. The antisense strand exhibits an alternating pattern (i.e., motif) of 2'-O-Me and 2'-F modified ribonucleosides. The ds-sRNA molecule also features a central block of approximately 11-13 modified ribonucleosides linked by phosphodiester internucleoside linkages flanked at the 5' and 3' ends, or only at the 5' end, by blocks of 2-5 modified ribonucleosides linked by phosphorothioate internucleoside linkages. [Figure 10B] 1 shows the sense strand of an exemplary F2 ds-siRNA molecule (ds-siRNA B_V4) of the present disclosure. The sense strand exhibits an alternating pattern (i.e., motif) of 2'-O-Me and 2'-F modified ribonucleosides. The ds-sRNA molecule also features a central block of approximately 11-13 modified ribonucleosides linked by phosphodiester internucleoside linkages flanked at the 5' and 3' ends, or only at the 5' end, by blocks of 2-5 modified ribonucleosides linked by phosphorothioate internucleoside linkages. [Figure 11A]1 shows the antisense strand of an exemplary F2 ds-siRNA molecule (ds-siRNA B_V5) of the present disclosure. The antisense strand exhibits an alternating pattern (i.e., motif) of 2'-O-Me and 2'-F modified ribonucleosides. The ds-sRNA molecule also features a central block of approximately 11-13 modified ribonucleosides linked by phosphodiester internucleoside linkages flanked at the 5' and 3' ends, or only at the 5' end, by blocks of 2-5 modified ribonucleosides linked by phosphorothioate internucleoside linkages. [Figure 11B] 1 shows the sense strand of an exemplary F2 ds-siRNA molecule (ds-siRNA B_V5) of the present disclosure. The sense strand exhibits an alternating pattern (i.e., motif) of 2'-O-Me and 2'-F modified ribonucleosides. The ds-sRNA molecule also features a central block of approximately 11-13 modified ribonucleosides linked by phosphodiester internucleoside linkages flanked at the 5' and 3' ends, or only at the 5' end, by blocks of 2-5 modified ribonucleosides linked by phosphorothioate internucleoside linkages. [Figure 12A] 1 shows the antisense strand of an exemplary F2 ds-siRNA molecule (ds-siRNA B_V6) of the present disclosure. The antisense strand exhibits an alternating pattern (i.e., motif) of 2'-O-Me and 2'-F modified ribonucleosides. The ds-sRNA molecule also features a central block of approximately 11-13 modified ribonucleosides linked by phosphodiester internucleoside linkages flanked at the 5' and 3' ends, or only at the 5' end, by blocks of 2-5 modified ribonucleosides linked by phosphorothioate internucleoside linkages. [Figure 12B]1 shows the sense strand of an exemplary F2 ds-siRNA molecule (ds-siRNA B_V6) of the present disclosure. The sense strand exhibits an alternating pattern (i.e., motif) of 2'-O-Me and 2'-F modified ribonucleosides. The ds-sRNA molecule also features a central block of approximately 11-13 modified ribonucleosides linked by phosphodiester internucleoside linkages flanked at the 5' and 3' ends, or only at the 5' end, by blocks of 2-5 modified ribonucleosides linked by phosphorothioate internucleoside linkages. [Figure 13A] 1 shows the antisense strand of an exemplary F2 ds-siRNA molecule (ds-siRNA C_V1) of the present disclosure. The antisense strand exhibits an alternating pattern (i.e., motif) of 2'-O-Me and 2'-F modified ribonucleosides. The ds-sRNA molecule also features a central block of approximately 11-13 modified ribonucleosides linked by phosphodiester internucleoside linkages flanked at the 5' and 3' ends, or only at the 5' end, by blocks of 2-5 modified ribonucleosides linked by phosphorothioate internucleoside linkages. [Figure 13B] 1 shows the sense strand of an exemplary F2 ds-siRNA molecule (ds-siRNA C_V1) of the present disclosure. The sense strand exhibits an alternating pattern (i.e., motif) of 2'-O-Me and 2'-F modified ribonucleosides. The ds-sRNA molecule also features a central block of approximately 11-13 modified ribonucleosides linked by phosphodiester internucleoside linkages flanked at the 5' and 3' ends, or only at the 5' end, by blocks of 2-5 modified ribonucleosides linked by phosphorothioate internucleoside linkages. [Figure 14A]FIG. 1 is a series of scatter plots showing expression levels of huntingtin (HTT) mRNA levels normalized to the housekeeping gene ATP5b in different brain regions (i.e., hippocampus, cortex, and striatum) of FVB / NJ female mice treated with various doses of exemplary di-siRNAs of the present disclosure (i.e., 0.2 nmol, 1.0 nmol, or 5.0 nmol), including F1 molecule ds-siRNA A_V1, or vehicle control (PBS), administered by intracerebroventricular (ICV) injection. [Figure 14B] FIG. 1 is a series of scatter plots showing expression levels of huntingtin (HTT) mRNA levels normalized to the housekeeping gene ATP5b in different brain regions (i.e., hippocampus, cortex, and striatum) of FVB / NJ female mice treated with various doses of exemplary di-siRNAs of the present disclosure (i.e., 0.2 nmol, 1.0 nmol, or 5.0 nmol), including F1 molecule ds-siRNA A_V2, or vehicle control (PBS), administered by intracerebroventricular (ICV) injection. [Figure 14C] FIG. 1 is a series of scatter plots showing expression levels of huntingtin (HTT) mRNA levels normalized to the housekeeping gene ATP5b in different brain regions (i.e., hippocampus, cortex, and striatum) of FVB / NJ female mice treated with various doses of exemplary di-siRNAs of the present disclosure (i.e., 0.2 nmol, 1.0 nmol, or 5.0 nmol), including F2 molecule ds-siRNA A_V3, or vehicle control (PBS), administered by intracerebroventricular (ICV) injection. [Figure 14D] FIG. 1 is a series of scatter plots showing expression levels of huntingtin (HTT) mRNA levels normalized to the housekeeping gene ATP5b in different brain regions (i.e., hippocampus, cortex, and striatum) of FVB / NJ female mice treated with various doses of exemplary di-siRNAs of the present disclosure (i.e., 0.2 nmol, 1.0 nmol, or 5.0 nmol), including F2 molecule ds-siRNA A_V4, or vehicle control (PBS), administered by intracerebroventricular (ICV) injection. [Figure 14E]FIG. 1 is a series of scatter plots showing expression levels of huntingtin (HTT) mRNA levels normalized to the housekeeping gene ATP5b in different brain regions (i.e., hippocampus, cortex, and striatum) of FVB / NJ female mice treated with various doses of exemplary di-siRNAs of the present disclosure (i.e., 0.2 nmol, 1.0 nmol, or 5.0 nmol), including F2 molecule ds-siRNA A_V5, or vehicle control (PBS), administered by intracerebroventricular (ICV) injection. [Figure 14F] FIG. 1 is a series of scatter plots showing expression levels of huntingtin (HTT) mRNA levels normalized to the housekeeping gene ATP5b in different brain regions (i.e., hippocampus, cortex, and striatum) of FVB / NJ female mice treated with various doses of exemplary di-siRNAs of the present disclosure (i.e., 0.2 nmol, 1.0 nmol, or 5.0 nmol), including F2 molecule ds-siRNA A_V6, or vehicle control (PBS), administered by intracerebroventricular (ICV) injection. [Figure 15A] FIG. 13 is a series of scatter plots showing expression levels of HTT mRNA levels normalized to the housekeeping gene ATP5b in different brain regions (i.e., hippocampus, cortex, and striatum) of FVB / NJ female mice treated with various doses of exemplary di-siRNAs of the present disclosure (i.e., 0.2 nmol, 1.0 nmol, or 5.0 nmol), including F1 molecule ds-siRNA B_V1, or vehicle control (PBS) administered by ICV injection. [Figure 15B] FIG. 13 is a series of scatter plots showing expression levels of HTT mRNA levels normalized to the housekeeping gene ATP5b in different brain regions (i.e., hippocampus, cortex, and striatum) of FVB / NJ female mice treated with various doses of exemplary di-siRNAs of the present disclosure (i.e., 0.2 nmol, 1.0 nmol, or 5.0 nmol), including F1 molecule ds-siRNA B_V2, or vehicle control (PBS) administered by ICV injection. [Figure 15C]FIG. 13 is a series of scatter plots showing expression levels of HTT mRNA levels normalized to the housekeeping gene ATP5b in different brain regions (i.e., hippocampus, cortex, and striatum) of FVB / NJ female mice treated with various doses of exemplary di-siRNAs of the present disclosure (i.e., 0.2 nmol, 1.0 nmol, or 5.0 nmol), including F2 molecule ds-siRNA B_V3, or vehicle control (PBS) administered by ICV injection. [Figure 15D] FIG. 13 is a series of scatter plots showing expression levels of HTT mRNA levels normalized to the housekeeping gene ATP5b in different brain regions (i.e., hippocampus, cortex, and striatum) of FVB / NJ female mice treated with various doses of exemplary di-siRNAs of the present disclosure (i.e., 0.2 nmol, 1.0 nmol, or 5.0 nmol), including F2 molecule ds-siRNA B_V4, or vehicle control (PBS) administered by ICV injection. [Figure 15E] FIG. 13 is a series of scatter plots showing expression levels of HTT mRNA levels normalized to the housekeeping gene ATP5b in different brain regions (i.e., hippocampus, cortex, and striatum) of FVB / NJ female mice treated with various doses of exemplary di-siRNAs of the present disclosure (i.e., 0.2 nmol, 1.0 nmol, or 5.0 nmol), including F2 molecule ds-siRNA B_V5, or vehicle control (PBS) administered by ICV injection. [Figure 15F] FIG. 13 is a series of scatter plots showing expression levels of HTT mRNA levels normalized to the housekeeping gene ATP5b in different brain regions (i.e., hippocampus, cortex, and striatum) of FVB / NJ female mice treated with various doses of exemplary di-siRNAs of the present disclosure (i.e., 0.2 nmol, 1.0 nmol, or 5.0 nmol), including F2 molecule ds-siRNA B_V6, or vehicle control (PBS) administered by ICV injection. [Figure 16]FIG. 1 is a scatter plot showing expression levels of HTT mRNA levels normalized to the housekeeping gene ATP5b in different brain regions (i.e., hippocampus, cortex, and striatum) of FVB / NJ female mice treated with various doses (i.e., 0.2 nmol, 1.0 nmol, or 5.0 nmol) of an exemplary di-siRNA of the present disclosure, i.e., ds-siRNA C_V1, administered by ICV injection, or vehicle control (PBS). [Figure 17A] FIG. 13 is a graph showing expression levels of HTT mRNA levels normalized to the housekeeping gene ATP5b in different brain regions (i.e., hippocampus, cortex, and striatum) of FVB / NJ female mice treated with a dose of 0.5 nmol of exemplary di-siRNAs of the disclosure, including F1 molecules ds-siRNA A_V1, ds-siRNA A_V2, ds-siRNA B_V1, and ds-siRNA B_V2, and F2 molecules ds-siRNA A_V3, ds-siRNA A_V4, ds-siRNA A_V5, ds-siRNA A_V6 ds-siRNA B_V3, ds-siRNA B_V4, ds-siRNA B_V5, ds-siRNA B_V6, and ds-siRNA C_V1, or vehicle control (PBS) administered by ICV injection. [Figure 17B] FIG. 13 is a graph showing expression levels of HTT mRNA levels normalized to the housekeeping gene ATP5b in different brain regions (i.e., hippocampus, cortex, and striatum) of FVB / NJ female mice treated with a dose of 2.5 nmol of exemplary di-siRNAs of the disclosure, including F1 molecules ds-siRNA A_V1, ds-siRNA A_V2, ds-siRNA B_V1, and ds-siRNA B_V2, and F2 molecules ds-siRNA A_V3, ds-siRNA A_V4, ds-siRNA A_V5, ds-siRNA A_V6 ds-siRNA B_V3, ds-siRNA B_V4, ds-siRNA B_V5, ds-siRNA B_V6, and ds-siRNA C_V1, or vehicle control (PBS) administered by ICV injection. [Figure 18]Scatter plot showing the toxicity profile of exemplary di-siRNAs of the present disclosure, including F1 molecule ds-siRNA A_V1 and F2 molecules ds-siRNA A_V3, ds-siRNA A_V4, ds-siRNA B_V3, ds-siRNA B_V4, ds-siRNA B_V6, and ds-siRNA C_V1, as quantified by EvADINT scoring assay. Higher scores indicate higher toxicity levels.

[0184] definition Unless otherwise defined herein, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or extrinsic definitions. Unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. The use of "or" means "and / or" unless otherwise specified. The use of the term "including" as well as other forms such as "includes" and "included" is not limiting.

[0185] The term "nucleic acid" as used herein refers to an RNA or DNA molecule that is composed of a chain of ribonucleotides or deoxyribonucleotides, respectively.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 disease-related target mRNA, interacts with disease-related target mRNA, and mediates the silencing of mRNA expression.

[0186] 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 at the 3' carbon of the ribose ring.

[0187] As used herein, the term "nucleoside" refers to a molecule that consists of a heterocyclic base and its sugar.

[0188] As used herein, the term "nucleotide" refers to a nucleoside having a phosphate group on its 3' or 5' sugar hydroxyl group.

[0189] As used herein, the term "siRNA" refers to small interfering RNA duplexes that induce the RNA interference (RNAi) pathway. siRNA molecules may be of various lengths (generally 18-30 base pairs) and may 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 a double-stranded region.

[0190] 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.

[0191] As used herein, the term "sense strand" refers to the siRNA duplex that contains complementarity to the antisense strand.

[0192] As used herein, the term "chemically modified nucleotide" or "nucleotide analog" or "altered nucleotide" or "modified nucleotide" refers to a non-standard nucleotide, 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.

[0193] As used herein, the term "metabolically stabilized" refers to an RNA molecule that contains ribonucleotides that have been chemically modified from a 2'-hydroxyl group to a 2'-O-methyl group.

[0194] 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.

[0195] As used herein, the term "ethylene glycol chain" refers to a carbon chain having the formula ((CH2OH)2).

[0196] As used herein, "alkyl" refers to a saturated hydrocarbon group. Alkyl groups can be acyclic or cyclic and, if unsubstituted, contain only C and H. When naming an alkyl residue with a specific number of carbons, all geometric isomers with that number of carbons 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.

[0197] 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). Alkenyl groups, when unsubstituted, contain 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=CH2, -CH2-CH=CH2, and -CH2-CH=CH-CH=CH2. 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.

[0198] 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). Alkynyl groups contain only C and H when unsubstituted. When an alkynyl residue having a specific number of carbons is named, all geometric isomers having that number of carbons are intended to be encompassed and described. Thus, for example, "pentynyl" is meant to include n-pentynyl, sec-pentynyl, and iso-pentynyl. Examples of alkynyl include -C≡CH and -C≡C-CH3. In some embodiments, alkynyl can be substituted. Suitable substituents that can be introduced into alkynyl groups include, for example, hydroxy, alkoxy, amino, alkylamino, and halo, among others.

[0199] As used herein, the term "phenyl" refers to a monocyclic arene having one hydrogen atom removed from a carbon atom of the ring. A phenyl group can be unsubstituted or substituted with one or more suitable substituents, which substitute for the H of the phenyl group.

[0200] 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. Benzyl 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.

[0201] As used herein, the term "amide" refers to an alkyl, alkenyl, alkynyl, or aromatic group bound to an aminocarbonyl functional group.

[0202] As used herein, the terms "internucleoside" and "internucleotide" refer to the linkage between a nucleoside and a nucleotide, respectively.

[0203] 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.

[0204] As used herein, the term "end group" refers to the group at which a carbon chain or nucleic acid ends.

[0205] 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).

[0206] As used herein, the term "antagomir" refers to a nucleic acid that can function as an inhibitor of miRNA activity.

[0207] As used herein, the term "gapmer" refers to a chimeric antisense nucleic acid that contains 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.

[0208] As used herein, the term "mixmer" refers to a nucleic acid composed of a mixture of locked nucleic acid (LNA) and DNA.

[0209] As used herein, the term "guide RNA" refers to a nucleic acid that has sequence complementarity to a specific sequence in a genome immediately upstream or one base pair upstream of a 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.

[0210] 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.

[0211] 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 molecules can be "bi-branched", 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 molecules can be "tri-branched", 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 molecules 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.

[0212] 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 attached to the 5'-end or 3'-end of the antisense or sense strand of siRNA molecule, and can support the attachment of additional single-stranded or double-stranded siRNA molecules.Non-limiting examples of branch point moieties suitable for use with the disclosed methods and compositions include, for example, phosphoramidite, tosylated solketal, 1,3-diaminopropanol, pentaerythritol, and any one of the branch point moieties described in US Patent No. 10,478,503.

[0213] As used herein, the term "5' phosphorus stabilizing moiety" refers to a terminal phosphate group, including phosphate as well as modified phosphates (e.g., phosphorothioates, phosphodiesters, phosphonates). The phosphate moiety may be located at either terminus, but is preferred at the 5' terminal nucleoside. In one aspect, the terminal phosphate has the formula -OP(=O)(OH)OH and is unmodified. In another aspect, the terminal phosphate is modified such that one or more of the O and OH groups are replaced with H, O, S, N(R'), or an alkyl where R' is H, an amino protecting group, or an unsubstituted or substituted alkyl. In some embodiments, the 5' and or 3' terminal groups can each independently comprise one to three phosphate moieties that are unmodified (diphosphate or triphosphate) or modified.

[0214] As used herein, the term "between X and Y" is inclusive of values ​​of X and Y. For example, "between X and Y" refers to a range of values ​​between a value of X and a value of Y, as well as a value of X and a value of Y.

[0215] As used herein, "amino acid" refers to a molecule that contains an amine and a carboxyl functional group as well as a side chain characteristic of an amino acid.

[0216] 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).

[0217] For example, certain internucleotide linkages provided herein, including phosphodiesters and phosphorothioates, contain a formal charge of −1 at physiological pH, which is understood to be balanced 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.

[0218] The phosphate group of the nucleotide may also be modified, for example, by replacing one or more oxygens of the phosphate group with sulfur (e.g., phosphorothioate), or by making other substitutions that allow the nucleotide to perform its intended function, as described, for example, in Eckstein, Antisense Nucleic Acid Drug Dev. 10:117-21, 2000; Rusckowski et al., Antisense Nucleic Acid Drug Dev. 10:333-45, 2000; Stein, Antisense Nucleic Acid Drug Dev. 11:317-25, 2001; Vorobjev et al., Antisense Nucleic Acid Drug Dev. 11:77-85, 2001; and US5,684,143. Certain modifications (e.g., phosphate group modifications) of the above-referenced modifications preferably reduce the hydrolysis rate of, for example, the polynucleotides containing the above-mentioned analogs in vivo or in vitro.

[0219] 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 the respective unpaired and improperly paired nucleotides are referred to as "mismatches." Alignment for purposes of measuring nucleic acid sequence complementarity percentages can be accomplished in a variety of ways within the capabilities of one of skill in the art, using publicly available computer software, such as, for example, BLAST, BLAST-2, or Megalign software.

[0220] "Percent sequence complementarity" to a reference polynucleotide sequence is defined as the percent 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 to be "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, a proper Watson-Crick base pair is referred to as a "match," while each unpaired nucleotide and an improperly paired nucleotide are referred to as a "mismatch." Alignment for purposes of measuring percent nucleic acid sequence complementarity can be accomplished in a variety of ways within the capabilities of one of skill in the art, using publicly available computer software, such as, for example, BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum complementarity over the entire length of the sequences being compared. By way of illustration, the percent sequence complementarity of a given nucleic acid sequence A to a given nucleic acid sequence B (which can alternatively be referred to as a given nucleic acid sequence A having a certain percent complementarity to a given nucleic acid sequence B) is calculated as follows: 100×(fraction X / Y) where X is the number of complementary base pairs in a program 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 is not 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.

[0221] The term "sufficiently complementary to hybridize" as used herein refers to a nucleic acid sequence or a portion thereof that does not have to be fully complementary (e.g., 100% complementary) to a target region or a nucleic acid sequence or a portion thereof, which 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 its entire length.

[0222] Nucleic acid "hybridization" or "annealing" is achieved when one or more nucleoside residues in a polynucleotide base pair with one or more complementary nucleosides to form a stable duplex. Base pairing is usually caused by hydrogen bonding phenomena. Hybridization includes Watson-Crick base pairs formed from natural and / or modified nucleobases. Hybridization may also include 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.

[0223] 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 harsh washing conditions are known in the art and include temperatures about 5°C below the melting temperatures of the individual strands of the duplex and low concentrations of monovalent salt, such as monovalent salt concentrations (e.g., NaCl concentrations) of less than 0.2M (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).

[0224] The term "gene silencing" refers to the suppression of gene expression, for example, transgene, heterologous gene and / or endogenous gene expression, which can be mediated through the process of affecting transcription and / or the process of affecting post-transcriptional mechanisms.In some embodiments, gene silencing occurs when RNAi molecules initiate the inhibition or degradation of the mRNA transcribed from a gene of interest in a sequence-specific manner through RNA interference, thereby preventing the translation of the gene's product.

[0225] 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 by an intermediary gene(s).

[0226] The term "negative regulator," as used herein, refers to a gene that negatively regulates (eg, reduces or inhibits) the expression and / or activity of another gene or set of genes.

[0227] The term "positive regulator," as used herein, refers to a gene that positively regulates (eg, increases or saturates) the expression and / or activity of another gene or set of genes.

[0228] The term "phosphate moiety" as used herein refers to a terminal phosphate group, including phosphate and modified phosphates. The phosphate moiety may be located at either terminus, but is preferred at the 5'-terminal nucleoside. In one aspect, the terminal phosphate has the formula -OP(=O)(OH)OH and is unmodified. In another aspect, the terminal phosphate is modified such that one or more of the O and OH groups are replaced with H, O, S, N(R'), or an alkyl where R' is H, an amino protecting group, or an unsubstituted or substituted alkyl. In some embodiments, the 5' and or 3' terminal groups can each independently comprise 1-3 phosphate moieties that are unmodified (diphosphate or triphosphate) or modified.

[0229] In the context of the present invention, the term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term includes oligonucleotides composed of naturally occurring nucleobases, sugars and covalent internucleoside (backbone) linkages, as well as oligonucleotides having non-naturally occurring (e.g., modified) portions that function similarly. Such modified or substituted oligonucleotides are often preferred over natural 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.

[0230] As used herein, the terms "treat", "treated" or "treating" refer to both therapeutic treatment and prophylactic or preventative measures, the purpose of which is to prevent or slow (alleviate) an undesirable physiological condition, disorder, or disease, or to obtain a beneficial or desired clinical outcome. Beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms; reduction in the extent of the condition, disorder, or disease; a stabilized (i.e., non-worsening) state of the condition, disorder, or disease; a delay in the onset or progression of the condition, disorder, or disease; an improvement or remission (whether partial or total) of the condition, disorder, or disease, whether detectable or undetectable; an improvement in at least one measurable physical parameter, not necessarily discernible by the patient; or an enhancement or amelioration of the condition, disorder, or disease. Treatment includes eliciting a clinically significant response without excessive levels of side effects. "Treatment" also includes prolonging survival compared to the expected survival if not receiving treatment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0231] The present invention provides new forms of siRNA, such as single-stranded (ss-) or double-stranded short interfering RNA (ds-siRNA) molecules with 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). The siRNA compositions of the present disclosure can employ a variety of modifications previously known and / or unknown in the art. Additionally, the present disclosure features branched siRNA structures, such as biantennary, triantennary, and tetraantennary ds-siRNA structures.

[0232] The siRNA of the disclosure may contain an antisense strand comprising a region represented by formula I, AB-(A') j -CP 2 -DP 1-(C'-P 1 ) k -C' Formula I; where A is of the formula CP 1 -DP 1 each A' is independently 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 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; and k is an integer from 1 to 7.

[0233] In some embodiments, the antisense strand comprises a structure represented by formula A1, where in the 5'-3' direction, formula A1 is:

[0234] The siRNA of the disclosure may contain an antisense strand that includes a region represented by Formula II, AB-(A') j -CP 2 -DP 1 -(CP 1 ) k -C' Formula II; where A is of the formula CP 1 -DP 1 each A' is independently represented by the formula CP 2 -DP 2 B is represented by the formula CP 2 -DP 2 -DP 2 -DP 2each 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 are independently 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.

[0235] In some embodiments, the siRNA of the disclosure may have a sense strand represented by Formula III: E-(A') m -F Formula III; In the formula, E is a compound represented by 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 II; 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.

[0236] Alternatively, the siRNA of the disclosure may contain an antisense strand that includes a region represented by formula IV: A-(A') j -CP 2 -B-(CP 1 ) k -C' Formula IV; where A is of the formula CP 1 -DP 1 each A' is independently represented by the formula CP 2 -DP 2 B is represented by the formula DP 1 -CP 1 -DP 1 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 are independently 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.

[0237] In some embodiments, the siRNA of the disclosure may have a sense strand represented by Formula V: E-(A') m -CP 2 -F formula V; In the formula, E is a compound represented by the formula (CP 1 ) 2; F is represented by the formula DP 1 -CP 1 -C, D.P. 2 -CP 2 -C, D.P. 1 -CP 1 -D, or DP 2 -CP2 -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.

[0238] Alternatively, the siRNA of the disclosure may contain an antisense strand that includes a region represented by Formula VI: A.B. j -EB k -EFG l -DP 1 -C' Formula VI; where A is of the formula CP 1 -DP 1 each B is independently represented by the formula CP 2 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 a group of the formula DP 2 -CP 2 F is represented by the formula DP 1 -CP 1 each G is independently represented by the formula CP 1 Each P 1 is 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); 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.

[0239] In some embodiments, the siRNA of the disclosure may have a sense strand represented by Formula VII: 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 independently represented by the formula (CP 1 each I is independently 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); 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.

[0240] The siRNA molecules of the present disclosure can be synthesized by standard methods known in the art, for example, by use of an automated DNA synthesizer such as those commercially available from Biosearch, Applied Biosystems, Inc., as discussed further below.

[0241] siRNA agent can be prepared by using liquid phase organic synthesis or solid phase organic synthesis or both.Organic synthesis has the advantage that it is easy to prepare the oligonucleotide that contains non-natural nucleotide or modified nucleotide.The siRNA molecule of the present disclosure can be prepared by using liquid phase organic synthesis or solid phase organic synthesis or both.

[0242] Furthermore, it is contemplated that any siRNA agent disclosed herein can be further optimized 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 modified 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., increase serum stability or circulating half-life, increase thermal stability, enhance transmembrane delivery, and / or target to specific locations or cell types).

[0243] siRNA structure The simplest siRNA consists of a ribonucleic acid containing a ss- or ds-structure, formed by a first strand (i.e., antisense strand) and, in the case of ds-siRNA, by a second strand (i.e., sense strand). The first strand comprises a series of consecutive nucleotides that are at least partially complementary to the target nucleic acid. The second strand also comprises a stretch of consecutive nucleotides, the second stretch being at least partially identical to the target nucleic acid. The first strand and said second strand can hybridize with each other to form a double-stranded structure. Hybridization typically occurs by Watson-Crick base pairing.

[0244] Depending on the sequence of the first and second strand, hybridization or base pairing is not necessarily complete or complete, which means that the first and second strands are not 100% base paired due to mismatch.One or more mismatches can also exist within the double strand, without necessarily affecting the RNA interference (RNAi) activity of siRNA.

[0245] The first strand comprises a stretch of contiguous nucleotides essentially complementary to the target nucleic acid. Typically, the target nucleic acid sequence is ss-RNA, preferably mRNA, according to the mode of action of the interfering ribonucleic acid. Such hybridization is most likely through Watson-Crick base pairing, but is not necessarily limited thereto. The extent to which the first strand has a complementary stretch of contiguous nucleotides to the target nucleic acid sequence can be 80% to 100%, for example, 80%, 85%, 90%, 95%, or 100% complementary.

[0246] The siRNAs described herein may employ modifications to the nucleobases, phosphate backbone, ribose core, 5' and 3' ends, and branching, where multiple strands of the siRNA may be covalently linked.

[0247] Length of siRNA molecule Within the scope of the present invention, any length known in the art and previously unknown can be employed for the present invention. As described herein, the potential length of the antisense strand of the branched siRNA of the present invention is between 10 and 30 nucleotides (e.g., 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides), between 15 and 25 nucleotides (e.g., 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, or 25 nucleotides), or between 18 and 23 nucleotides (e.g., 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, or 23 nucleotides). In some embodiments, the antisense strand is 20 nucleotides. In some embodiments, the antisense strand is 21 nucleotides. In some embodiments, the antisense strand is 22 nucleotides. In some embodiments, the antisense strand is 23 nucleotides. In some embodiments, the antisense strand is 24 nucleotides. In some embodiments, the antisense strand is 25 nucleotides. In some embodiments, the antisense strand is 26 nucleotides. In some embodiments, the antisense strand is 27 nucleotides. In some embodiments, the antisense strand is 28 nucleotides. In some embodiments, the antisense strand is 29 nucleotides. In some embodiments, the antisense strand is 30 nucleotides.

[0248] In some embodiments, the sense strand of the branched siRNA of the invention is between 12-30 nucleotides (e.g., 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides), or between 14-23 nucleotides (e.g., 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, or 23 nucleotides). In some embodiments, the sense strand is 15 nucleotides. In some embodiments, the sense strand is 16 nucleotides. In some embodiments, the sense strand is 17 nucleotides. In some embodiments, the sense strand is 18 nucleotides. In some embodiments, the sense strand is 19 nucleotides. In some embodiments, the sense strand is 20 nucleotides. In some embodiments, the sense strand is 21 nucleotides. In some embodiments, the sense strand is 22 nucleotides. In some embodiments, the sense strand is 23 nucleotides. In some embodiments, the sense strand is 24 nucleotides. In some embodiments, the sense strand is 25 nucleotides. In some embodiments, the sense strand is 26 nucleotides. In some embodiments, the sense strand is 27 nucleotides. In some embodiments, the sense strand is 28 nucleotides. In some embodiments, the sense strand is 29 nucleotides. In some embodiments, the sense strand is 30 nucleotides.

[0249] 2' sugar modification The present invention includes ss-siRNA and ds-siRNA compositions that include 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 all possible orientations of OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl 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. Some embodiments include a 2'-O-methyl (2'-O-Me) modification, such as O[(CH2) n O] m CH3, O(CH2) n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2 and O(CH2) n ON [(CH2) nCH3]2 is used, with n and m ranging from 1 to about 10. 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 pharmacokinetic properties of oligonucleotides, and other substituents with similar properties. In some embodiments, the modification includes 2'-methoxyethoxy (also known as 2'-O-CH2CHOCH3, 2'-O-(2-methoxyethyl) or 2'-MOE). In some embodiments, modifications include the group 2'-dimethylaminooxyethoxy, i.e., O(CH2)2ON(CH3)2, also known as 2'-DMAOE, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylamino-ethoxy-ethyl or 2'-DMAEOE), i.e., 2'-O-CHOCH2N(CH3). 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 be made at other positions in oligomeric compounds, 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.

[0250] Nucleobase Modifications Oligomeric compounds may also include nucleosides or other surrogate or mimetic monomeric subunits that contain a nucleobase (often referred to in the art simply as a "base" or a "heterocyclic base moiety"). Nucleobases are other moieties that can be extensively modified or substituted, and such modified and / or substituted nucleobases can be amenable to the present invention. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases, also referred to herein as heterocyclic base moieties, include other synthetic and natural nucleobases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C=C-CH3) uracil and cytosine, and other alkynyl derivatives of pyrimidine bases. , 6-azo-uracil, 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 by other heterocycles, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone.Further 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 oligomeric compounds of the present invention can also contain polycyclic heterocyclic compounds in place of one or more heterocyclic base moieties. Many tricyclic heterocyclic compounds have been previously reported. These compounds are routinely used in antisense applications to increase the binding properties of the modified strand to the target strand.

[0251] Representative cytosine analogs that make three hydrogen bonds with a guanosine in the second strand include 1,3-diazaphenoxazin-2-one (Kurchavov et al., Nucleosides and Nucleotides, 16:1837-46, 1997), 1,3-diazaphenothiazin-2-one (Lin et al. Am. Chem. Soc., 117:3873-4, 1995), and 6,7,8,9-tetrafluoro-1,3-diazaphenoxazin-2-one (Wang et al., Tetrahedron Lett., 39:8385-8, 1998). These base modifications incorporated into oligonucleotides have been shown to hybridize with complementary guanines, which in turn have been shown to hybridize with adenines through extended stacking interactions, improving the thermal stability of the helix (see also US10 / 155,920 and US10 / 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).

[0252] Internucleoside bond modification Another variable in the design of the present invention is the internucleoside bond that constitutes the phosphate backbone.Natural RNA phosphate backbone may be employed herein, but its derivatives, known and yet unknown in the art, may be used to improve the desired properties of siRNA.Of particular importance in the present invention, but not limited to, is to protect part or all of siRNA from hydrolysis.One example of the modification that reduces the hydrolysis rate is phosphorothioate.Any part or all of the backbone may contain phosphate substitution (e.g., phosphorothioate, phosphodiester, etc.). For example, the internucleoside bond 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 bond. Similarly, the internucleoside bond 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.

[0253] Specific examples of some potential oligomeric compounds useful in the present invention include oligonucleotides that contain modified, e.g., non-naturally occurring, internucleoside linkages.As defined herein, oligonucleotides with modified internucleoside linkages include internucleoside linkages that retain phosphorus atoms and internucleoside linkages that do not have phosphorus atoms.For the purposes of this specification, and as sometimes referred to in the art, modified oligonucleotides that do not have phosphorus atoms in their internucleoside backbone can also be considered to be oligonucleosides.Preferred phosphorus-containing modified internucleoside linkages are phosphorothioate internucleoside linkages. In some embodiments, modified oligonucleotide backbones comprising a phosphorus atom therein include, for example, phosphorothioates, phosphodioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidates and aminoalkyl phosphoramidates, thinophosphoramidates, thinoalkyl phosphonates, thinoalkyl phosphotriesters, selenophosphates, boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs thereof, as well as those having inverted 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, Nos. 6,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Patent No. RE39464, the entire contents of each of which are incorporated herein by reference.

[0254] In some embodiments, modified oligonucleotide backbones that do not contain phosphorus atoms therein have backbones formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatom or heterocyclic internucleoside linkages. These include morpholino linkages (formed in part from the sugar portion of the nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methyleneformacetyl and thioformacetyl backbones; 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 CH2 moieties. Non-limiting examples of U.S. patents that teach the preparation of non-phosphorus scaffolds include, but are not limited to, U.S. Pat. Nos. 5,034,506, 5,166,315, 5,185,444, 5,214,134, 5,216,141, 5,235,033, 5,264,562, 5,264,564, 5,405,938, 5,434,257, 5,466,677, 5,470,967, 5,520,970, and 5,530,740. Nos. 5,489,677, 5,541,307, 5,561,225, 5,596,086, 5,602,240, 5,608,046, 5,610,289, 5,618,704, 5,623,070, 5,663,312, 5,633,360, 5,677,437, and 5,677,439, the entire contents of each of which are incorporated herein by reference.

[0255] siRNA patterning Nucleosides used in the present invention are characterized by various modifications in nucleobase and sugar. A complete ss-siRNA or ds-siRNA may have one, two, three, four, five or more different nucleosides, each of which appears one or more times in the siRNA strand. Nucleosides may appear in a repeating pattern (e.g., alternating between two modified nucleosides), or may be a chain of one type of nucleoside with a substitution of a second type of nucleoside. Similarly, internucleoside linkages may be one or more types appearing in a single-stranded or double-stranded siRNA in a repeating pattern (e.g., alternating between two internucleoside linkages), or may be a chain of one type of internucleoside linkage with a substitution of a second type of internucleoside linkage. Although the siRNAs of the present disclosure tolerate a variety of substitution patterns, below are exemplified some preferred patterns of siRNA modifications in the antisense strand of a single-stranded or ds-siRNA molecule, where A and B represent two types of nucleosides and S and O represent two types of internucleoside linkages. Antisense pattern 1: ASBSAOBOBOBOAOBOAOBOA-OBOAOBOAOBSASASASBSA (Formula A1) Antisense pattern 2: ASBSAOBOBOBOAOBOAOBOA-OBOAAOBOAOBSASASASASA (Formula A2) Antisense pattern 3: ASBSAOBOAOBOAOBOAOBOA-OBOAOBOAOBSASASASA (Formula A3) Antisense pattern 4: ASBSAOAOAOBOAOAOAOAOA-OAOAOBOAOBSASASASBSA (Formula A4)

[0256] Non-limiting examples of siRNA modification patterns in the sense strand of a ds-siRNA molecule are shown below. Sense patterns compatible with antisense patterns 1 or 2: Sense Pattern 1: ASASAOBOAOBOAOBOAOBOA-OAOAOBBSASA (Formula S1) Sense Pattern 2: ASASAOBOAOBOAOBOAOBOA-OAOAOBOAOA (Formula S2) Sense Pattern 3: ASASAOBOAOBOAOBOAOBOA-OAOAOBSASB (Formula S3) Sense Pattern 4: ASASAOBOAOBOAOBOAOBOA-OAOAOBOAOB (Equation S4) Sense pattern compatible with antisense pattern 3: Sense Pattern 5: ASASAOBOAOBOAOBOAOBOA-OBOAOBSASA (Formula S5) Sense Pattern 6: ASASAOBOAOBOAOBOAOBOA-OBOAOBOAOA (Formula S6) Sense Pattern 7: ASASAOBOAOBOAOBOAOBOA-OBOAOBSASB (Formula S7) Sense Pattern 8: ASASAOBOAOBOAOBOAOBOA-OBOAOBOAOB (Formula S8) Sense patterns compatible with antisense pattern 4: Sense Pattern 9: ASASAOAOAOBOBOBOBOAOBOA-OAOAOAASASA (Formula S9)

[0257] In some embodiments, A represents a 2'-O-Me nucleoside. In some embodiments, B represents a 2'-F nucleoside. In some embodiments, O represents a phosphodiester internucleoside linkage. In some embodiments, S represents a phosphorothioate internucleoside linkage.

[0258] 5' phosphorus stabilizing moiety To further protect siRNA from degradation, 5'-phosphorus stabilizing moiety can be adopted. 5'-phosphorus stabilizing moiety replaces 5'-phosphate to prevent hydrolysis of phosphate. Hydrolysis of 5'-phosphate prevents binding to RISC, which is a necessary step of gene silencing. Any substitution of phosphate that does not prevent binding to RISC is contemplated in the present disclosure. In some embodiments, the substitution of 5'-phosphate is also stable to hydrolysis in vivo. Each siRNA strand may independently adopt any suitable 5'-phosphorus stabilizing moiety, optionally. [ka]

[0259] Some exemplary end caps are demonstrated in formulas VIII-XV. Nuc in formulas VIII-XV represents a nucleobase or nucleobase derivative or substitute as described herein. X in formulas VIII-XV represents a 2'-modification as described herein. Some embodiments employ hydroxy as in formula VIII, sulfonate as in formula IX, vinyl phosphonate as in formulas X and XIII, 5'-methyl substituted sulfonate as in formulas XI, XIII and XV, or methylene phosphonate as in formula XIV. Vinyl 5'-vinyl phosphonate as the 5'-phosphorus stabilizing moiety as shown in formula X.

[0260] hydrophobic part The present disclosure further provides siRNA molecules with one or more hydrophobic moieties attached.Hydrophobic moieties can be covalently attached to the 5'-end or 3'-end of the siRNA molecules of the present disclosure.Non-limiting examples of the hydrophobic moieties suitable for use with the siRNA of the present disclosure include cholesterol, vitamin D, tocopherol, phosphatidylcholine (PC), docohexaenoic acid, docosanoic acid, PC-docosanoic acid, eicosapentaenoic acid, lithocholic acid, or any combination of the above hydrophobic moieties and PC.

[0261] siRNA branching According to the present disclosure, the siRNA molecule disclosed herein may be a branched siRNA molecule. The siRNA molecule may be unbranched or bi-, tri- or tetra-branched, connected via a linker. Each main branch may be further branched to allow 2, 3, 4, 5, 6, 7 or 8 separate RNA single or double strands. 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 1. [Table 1]

[0262] 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 XVI-XVIII, where 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 US10,478,503).

[0263] In some embodiments, a three-branched siRNA molecule represented by any one of formulas XIX-XXII, wherein each RNA is independently an siRNA molecule, L is a linker, and each X independently represents a branch point moiety.

[0264] In some embodiments, in a four-branched siRNA molecule represented by any one of Formulas XXIII-XXVIII, each RNA is independently an siRNA molecule, L is a linker, and each X independently represents a branch point moiety.

[0265] Linker The multiple strands of the siRNA described herein may be covalently linked by a linker. This branching effect, among other things, improves cell permeability, allowing better access to cells (e.g., neurons or glial cells) in the CNS. Any linker moiety that is not incompatible with the siRNA of the present invention may be employed. Linkers include ethylene glycol chains of 2-10 subunits (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 subunits), alkyl chains, carbohydrate chains, block copolymers, peptides, RNA, DNA, and others. In some embodiments, the carbon or oxygen atoms of the linker are optionally replaced with nitrogen atoms, have hydroxyl substituents, or have oxo substituents. In some embodiments, the linker is a polyethylene glycol (PEG) linker. PEG linkers suitable for use in the disclosed compositions and methods include linear or non-linear PEG linkers. Examples of non-linear PEG linkers include branched PEG, linear forked PEG, or branched forked PEG.

[0266] 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 linker (TEG).

[0267] 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.

[0268] The linker may covalently link two, three, four, or five unique siRNA strands. The linker may be covalently linked to any portion of the siRNA oligomer. In some embodiments, the linker is attached to the 3' end of the nucleoside of each siRNA strand. In some embodiments, the linker is attached to the 5' end of the nucleoside of each siRNA strand. In some embodiments, the linker is attached to the nucleoside of the siRNA strand (e.g., the sense strand or the 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.).

[0269] In some embodiments, the linker has a structure of formula L1, as shown below: [ka]

[0270] In some embodiments, the linker has a structure of formula L2, as shown below: [ka]

[0271] In some embodiments, the linker has a structure of formula L3, as shown below: [ka]

[0272] In some embodiments, the linker has the structure of formula L4, as shown below. [ka]

[0273] In some embodiments, the linker has the structure of formula L5, as shown below. [ka]

[0274] In some embodiments, the linker has the structure of formula L6, as shown below. [ka]

[0275] In some embodiments, the linker has the structure of formula L7, as shown below. [ka]

[0276] In some embodiments, the linker has the structure of formula L8, as shown below. [ka]

[0277] In some embodiments, the linker has the structure of formula L9, as shown below. [ka]

[0278] 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, for example, 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.

[0279] 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.

[0280] gene The siRNA molecules of the present disclosure may comprise an antisense strand and a sense strand having complementarity to the antisense strand, wherein the antisense strand is 10-30 nucleotides in length and is complementary to one or more of the following genes: ABCA7, ABI3, ADAM10, APOC1, APOE, AXL, BIN1, C1QA, C3, C9ORF72, CASS4, CCL5, CD2AP, CD33, CD68, CLPTM1, CLU, CR1, CSF1, CST7, CTSB, CTSD, CTSL, CXCL10, CXCL13, DSG2, ECHDC3, EPHA1, FABP5, FERMT2, FTH1, GNAS, GRN, HBEGF, HLA-DRB1, HLA-DRB5, HTT, I and has sufficient complementarity to hybridize to any region of FIT1, IFIT3, IFITM3, IFNAR1, IFNAR2, IGF1, IL10RA, IL1A, IL1B, IL1RAP, INPP5D, ITGAM, ITGAX, KCNT1, LILRB4, LPL, MAPT, MEF2C, MMP12, MS4A4A, MS4A6A, MSH3, NLRP3, NME8, NOS2, PICALM, PILRA, PLCG2, PRNP, PTK2B, SCIMP, SCN9A, SLC24A4, SNCA, SORL1, SPI1, SPP1, SPPL2A, TBK1, TNF, TREM2, TREML2, TYROBP, and ZCWPW1. In some embodiments, the antisense strand has sufficient complementarity to hybridize to a region of any of the following genes: APOE, BIN1, C1QA, C3, C9ORF72, CCL5, CD33, CLU / APOJ, CR1, CXCL10, CXCL13, IFIT1, IFIT3, IFITM3, IFNAR1, IFNAR2, IL10RA, IL1A, IL1B, IL1RAP, INPP5D, ITGAM, MEF2C, MMP12, NLRP3, NOS2, PILRA, PLCG2, PTK2B, SLC24A4, TBK1, and TNF. In some embodiments, the antisense strand has sufficient complementarity to hybridize to a region of any of the following genes: HTT, MAPT, SNCA, C9ORF72, APOE, SCN9A, KCNT1, PRNP, and MSH3.In some embodiments, the siRNA molecule has sufficient complementarity to hybridize to a region of the HTT gene.

[0281] Treatment methods The present invention provides a method of treating a subject in need of gene silencing. Gene silencing may be performed to silence defective or overactive genes, to silence negative regulators of under-expressed genes, to silence wild-type genes that have an activating role in pathway(s) that increase the activity of disease-driving genes, to silence gene(s) splice isoforms that may increase the total expression of gene(s) when selectively knocked down, among others, as long as the goal is to restore genetic and biochemical pathway activity from a disease state toward a healthy state. The method may include delivering the siRNA molecule of the present disclosure or a pharmaceutical composition containing it to the CNS of a subject (e.g., a human) by any suitable route of administration (e.g., intrastriatal, intracerebroventricular, or intrathecal injection). The active compound may be administered at any suitable dose. The actual dosage of the composition of the present invention administered to a patient may be determined by physical and physiological factors such as body weight, severity of the condition, previous or concurrent therapeutic interventions, idiopathic disease of the patient, and route of administration. Depending on the dosage and route of administration, the preferred dosage and / or the number of times of administration of the effective amount may vary depending on the subject's response.The practitioner responsible for 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 performed any suitable number of times per day for the period required.The subject may be an adult or pediatric human, with or without co-morbidities.

[0282] disease The subject in need of gene silencing may need silencing of a gene found in the CNS (e.g., microglial cells). The gene may be associated with a particular disease or disorder. For example, the gene may be associated with Huntington's disease, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), dementia with Lewy bodies (DLB), pure autonomic failure, Lewy body dysphagia, incidental Lewy body disease (ILBD), hereditary Lewy body disease, olivopontocerebellar atrophy (OPCA), striatonigral degeneration, Shy-Drager syndrome, epilepsy or epilepsy disorder, prion disease, or pain or pain disorder.

[0283] gene The gene silencing methods described herein may be performed to silence defective or overactive genes, to silence negative regulators of under-expressed genes, to silence wild-type genes that have an activating role in pathway(s) that increase the activity of disease-driving genes, to silence gene(s) splice isoforms that, when selectively knocked down, may increase the total expression of the gene(s), among others, so long as the goal is to restore genetic and biochemical pathway activity from a disease state towards a healthy state.

[0284] The disease or disorder may be associated with a gene mutation in one or more of the following genes: ABCA7, ABI3, ADAM10, APOC1, APOE, AXL, BIN1, C1QA, C3, C9ORF72, CASS4, CCL5, CD2AP, CD33, CD68, CLPTM1, CLU, CR1, CSF1, CST7, CTSB, CTSD, CTSL, CXCL10, CXCL13, DSG2, ECHDC3, EPHA1, FABP5, FERMT2, FTH1, GNAS, GRN, HBEGF, HLA-DRB1, HLA-DRB5, HTT, IFIT1, IFIT3, IFITM3, IFNAR1, IFN-γ ... It may be associated with any of the following: AR2, IGF1, IL10RA, IL1A, IL1B, IL1RAP, INPP5D, ITGAM, ITGAX, KCNT1, LILRB4, LPL, MAPT, MEF2C, MMP12, MS4A4A, MS4A6A, MSH3, NLRP3, NME8, NOS2, PICALM, PILRA, PLCG2, PRNP, PTK2B, SCIMP, SCN9A, SLC24A4, SNCA, SORL1, SPI1, SPP1, SPPL2A, TBK1, TNF, TREM2, TREML2, TYROBP, and ZCWPW1. In some embodiments, the disease or disorder is associated with any of the following genes: APOE, BIN1, C1QA, C3, C9ORF72, CCL5, CD33, CLU / APOJ, CR1, CXCL10, CXCL13, IFIT1, IFIT3, IFITM3, IFNAR1, IFNAR2, IL10RA, IL1A, IL1B, IL1RAP, INPP5D, ITGAM, MEF2C, MMP12, NLRP3, NOS2, PILRA, PLCG2, PTK2B, SLC24A4, TBK1, and TNF. In some embodiments, the disease or disorder is associated with any of the following genes: HTT, MAPT, SNCA, C9ORF72, APOE, SCN9A, KCNT1, PRNP, and MSH3. In some embodiments, the disease or disorder is associated with the HTT gene.

[0285] Pharmaceutical Compositions The branched siRNA molecule of the present invention can be formulated into pharmaceutical compositions for administering to subjects in a biologically compatible form suitable for in vivo administration.Therefore, the present disclosure provides pharmaceutical compositions comprising the siRNA of the present disclosure mixed with suitable diluent, carrier or excipient.The siRNA can be, for example, directly administered to the CNS of subjects (for example, by intrastriatal, intracerebroventricular or intrathecal injection).

[0286] Procedures and ingredients for selecting and preparing suitable formulations are described, for example, in P. The Science and Practice of Pharmacy, Easton, PA. Mack Publishers, 2012, 22 nd ed. and the National Formulary, United States Pharmacopeial, 2015, USP 38 NF 33).

[0287] Under normal storage and use conditions, the pharmaceutical composition may contain a preservative, for example, to prevent the growth of microorganisms.The pharmaceutical composition includes a sterile aqueous solution, a sterile dispersion, or a powder for extemporaneous preparation of a sterile solution or dispersion.In all cases, the form can be sterilized using techniques known in the art and can be fluidized to the extent that it can be easily administered to the subject in need of treatment.

[0288] Pharmaceutical compositions may be administered to a subject, e.g., a human subject, alone or in combination with pharma- ceutically acceptable carriers, as provided herein, 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.

[0289] Regimen A physician of ordinary skill in the art can easily determine the effective amount of siRNA to be administered to a mammalian subject (e.g., human) in need of siRNA. For example, a physician can begin prescribing the dosage of the siRNA 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 the siRNA at a high dose, and then administer progressively lower doses until a therapeutic effect (e.g., reduction in the expression of a target gene sequence) is achieved. In general, a suitable daily dosage of the siRNA of the present disclosure is the amount of the lowest dosage of siRNA effective to produce a therapeutic effect. Single-stranded or double-stranded siRNA molecules of the present disclosure can be administered, for example, intrathecally, intracerebroventricularly, or intrastriatally, by injection (e.g., injection into the caudate nucleus or putamen). The daily dosage of the siRNA therapeutic composition of the present disclosure may be administered as a single dose or as two, three, four, five, six or more doses administered separately at appropriate intervals throughout the day, week, month or year, optionally in unit dosage form.The siRNA of the present disclosure can be administered alone, but can also be administered in pharmaceutical formulations in combination with excipients, carriers and, optionally, additional therapeutic agents.

[0290] Route of administration The methods of the present disclosure contemplate any route of administration permitted by the therapeutic composition. Some embodiments of the methods include intrathecal, intracerebroventricular, or intrastriatal injection.

[0291] 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 (e.g., neurons and glial cells) in the spinal column and have a route to bypass the blood-brain barrier and access cells in the brain.

[0292] 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. The use of ICV offers the advantage of accessing cells in the brain and spinal column without the risk of the therapeutic agent being degraded in the blood.

[0293] Intrastriatal injection is a direct injection into the striatum (or corpus striatum). The striatum is a subcortical basal ganglia region of the brain. Injection into the striatum bypasses the blood-brain barrier and the pharmacokinetic challenges of injection into the bloodstream, allowing direct access to the cells of the brain. EXAMPLES

[0294] 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 illustrative of the disclosure and are not intended to limit the scope of what the inventors regard as their disclosure.

[0295] Example 1: Method for preparing double-stranded short interfering RNA molecules with patterned ribonucleoside and internucleoside linkage modifications (I) The double-stranded (ds-) short interfering (si) RNA (ds-siRNA) molecules with patterned ribonucleoside modifications and internucleoside linkage modifications of the present disclosure are prepared according to methods known in the art, such as the methods disclosed herein. The ds-siRNA molecule is a double-stranded oligoribonucleotide from which the sense strand originates and has complete (i.e., 100%) or partial (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to the mRNA sequence of the target gene. The nucleic acid sequence of the antisense strand is completely (i.e., 100%) complementary or partially (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) complementary to the nucleic acid sequence of the sense strand / mRNA of the target gene. The antisense strand and the sense strand of the ds-siRNA agent are each synthesized according to established methods (e.g., synthesis and ligation or tandem synthesis) and contain alternating patterns (motifs) of modified ribonucleosides, such as 2'-O-methyl (2'-O-Me) ribonucleosides and 2'-fluoro (2'-F) ribonucleosides, and modified internucleoside linkages, such as phosphorothioate linkages. The antisense strand is generated to be of a desired length so that functional advantages (e.g., RNA interference, thermostability, and / or resistance to nucleases) are achieved. Exemplary antisense strands can be, for example, 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 in length. The sense strand is generated to be of a desired length so that a functional advantage is achieved (eg, efficient RISC loading, thermostability, and / or resistance to nucleases).Exemplary sense strands can be, for example, 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 in length.Depending on the difference in length between antisense strand and sense strand, ds-siRNA double-stranded structure includes 5' overhang, 3' overhang, or both.Exemplary antisense strands can have the following patterns: Antisense pattern 2 (Formula A2): ASBSAOBOBOBOAOBOAOBOA-OBOAOBOAOBSASASASASA (Figures 3A, 4A, 5A, and 6A). Exemplary sense strands can have any one of the following patterns: Sense pattern 1 (equation S1): ASASAOBOAOBOAOBOAOBOA-OAOAOBSASA (Fig. 3B); Sense pattern 2 (equation S2): ASASAOBOAOBOAOBOAOBOA-OAOAOBOAOAOA (Figure 4B) Sense pattern 3 (equation S3): ASASAOBOAOBOAOBOAOBOA-OAOAOBSASB ( Figure 5B ); Sense pattern 4 (equation S4): ASASAOBOAOBOAOBOAOBOA-OAOAOBOAOB (Figure 6B) 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.

[0296] The antisense and sense strands of the ds-siRNA molecule are generated such that the resulting double-stranded structure has 0-5 nucleotide mismatches (e.g., 0, 1, 2, 3, 4, or 5) between the sense strand and the antisense and / or the antisense strand and the target mRNA sequence, respectively. The ds-siRNA molecule may be further modified to incorporate 5' phosphorus stabilizing moieties (e.g., 5'-vinyl phosphonate) and / or hydrophobic moieties (e.g., cholesterol, vitamin D, or tocopherol) on the antisense strand, the sense strand, or both. Additionally, the ds-siRNA molecule may contain branched structures as disclosed herein, such as biantennary structures, triantennary structures, or tetraantennary structures as disclosed herein. The ds-siRNA agent may further be incorporated into a pharmaceutical composition containing a pharma-ceutically acceptable excipient, carrier, or diluent.

[0297] Example 2: Method for preparing ds-siRNA molecules with patterned ribonucleoside and internucleoside linkage modifications (II) The ds-siRNA molecules with patterned ribonucleoside modifications and internucleoside linkage modifications of the present disclosure are prepared according to methods known in the art, such as those disclosed herein. The ds-siRNA molecule is a double-stranded oligoribonucleotide from which the sense strand originates and has complete (i.e., 100%) or partial (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to the mRNA sequence of the target gene. The nucleic acid sequence of the antisense strand is completely (i.e., 100%) complementary or partially (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) complementary to the nucleic acid sequence of the sense strand / mRNA of the target gene. The antisense strand and the sense strand of the ds-siRNA agent are each synthesized according to well-known methods (e.g., synthesis and ligation or tandem synthesis) and include an alternating pattern (motif) of modified ribonucleosides, such as 2'-O-Me ribonucleosides and 2'-F ribonucleosides, and modified internucleoside linkages, such as phosphorothioate linkages. The antisense strand is generated to a desired length so that functional advantages (e.g., RNA interference, thermostability, and / or resistance to nucleases) are achieved. Exemplary antisense strands can be, for example, 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 in length. The sense strand is generated to a desired length so that functional advantages (e.g., efficient RISC loading, thermostability, and / or resistance to nucleases) are achieved. Exemplary sense strands can be, for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.Depending on the difference in length between the antisense and sense strands, the ds-siRNA double-stranded structure contains a 5' overhang, a 3' overhang, or both. Exemplary antisense strands can have the following patterns: Antisense pattern 3 (Formula A3): ASBSAOBOAOBOAOBOAOBOA-OBOAOBOAOBSASBSASASA (Figs. 9A, 10A, 11A, and 12A); Exemplary sense strands can have any one of the following patterns: Sense pattern 5 (equation S5): ASASAOBOAOBOAOBOAOBOA-OBOAOBSASA (Fig. 9B); Sense pattern 6 (equation S6): ASASAOBOAOBOAOBOAOBOA-OBOAOBOAOAOA (Figure 10B) Sense pattern 7 (equation S7): ASASAOBOAOBOAOBOAOBOA-OBOAOBSASB ( Figure 11B ); Sense pattern 8 (equation S8): ASASAOBOAOBOAOBOAOBOA-OBOAOBOAOB (Figure 12B) 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.

[0298] The antisense and sense strands of the ds-siRNA molecule are generated such that the resulting double-stranded structure has 0-5 nucleotide mismatches (e.g., 0, 1, 2, 3, 4, or 5) between the sense strand and the antisense and / or the antisense strand and the target mRNA sequence, respectively. The ds-siRNA molecule may be further modified to incorporate 5' phosphorus stabilizing moieties (e.g., 5'-vinyl phosphonate) and / or hydrophobic moieties (e.g., cholesterol, vitamin D, or tocopherol) on the antisense strand, the sense strand, or both. Additionally, the ds-siRNA molecule may contain branched structures as disclosed herein, such as biantennary structures, triantennary structures, or tetraantennary structures as disclosed herein. The ds-siRNA agent may further be incorporated into a pharmaceutical composition containing a pharma-ceutically acceptable excipient, carrier, or diluent.

[0299] Example 3: Method for preparing ds-siRNA molecules with patterned ribonucleoside and internucleoside linkage modifications (III) The ds-siRNA molecules with patterned ribonucleoside modifications and internucleoside linkage modifications of the present disclosure are prepared according to methods known in the art, such as those disclosed herein. The ds-siRNA molecule is a double-stranded oligoribonucleotide from which the sense strand originates and has complete (i.e., 100%) or partial (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to the mRNA sequence of the target gene. The nucleic acid sequence of the antisense strand is completely (i.e., 100%) complementary or partially (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) complementary to the nucleic acid sequence of the sense strand / mRNA of the target gene. The antisense strand and the sense strand of the ds-siRNA agent are each synthesized according to well-known methods (e.g., synthesis and ligation or tandem synthesis) and include an alternating pattern (motif) of modified ribonucleosides, such as 2'-O-Me ribonucleosides and 2'-F ribonucleosides, and modified internucleoside linkages, such as phosphorothioate linkages. The antisense strand is generated to a desired length so that functional advantages (e.g., RNA interference, thermostability, and / or resistance to nucleases) are achieved. Exemplary antisense strands can be, for example, 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 in length. The sense strand is generated to a desired length so that functional advantages (e.g., efficient RISC loading, thermostability, and / or resistance to nucleases) are achieved. Exemplary sense strands can be, for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.Depending on the difference in length between the antisense and sense strands, the ds-siRNA double-stranded structure contains a 5' overhang, a 3' overhang, or both. Exemplary antisense strands can have the following patterns: Antisense pattern 4 (Formula A4): ASBSAOAOAOBOAOAOAOAOA-OAOAOAOBOAOBSASASASBSA (Figure 13A); An exemplary sense strand can have the following pattern: Sense pattern 9 (equation S9): ASASAOAOAOBOBOAOBOA-OAOAOASASA (Figure 13B); 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.

[0300] The antisense and sense strands of the ds-siRNA molecule are generated such that the resulting double-stranded structure has 0-5 nucleotide mismatches (e.g., 0, 1, 2, 3, 4, or 5) between the sense strand and the antisense and / or the antisense strand and the target mRNA sequence, respectively. The ds-siRNA molecule may be further modified to incorporate 5' phosphorus stabilizing moieties (e.g., 5'-vinyl phosphonate) and / or hydrophobic moieties (e.g., cholesterol, vitamin D, or tocopherol) on the antisense strand, the sense strand, or both. Additionally, the ds-siRNA molecule may contain branched structures as disclosed herein, such as biantennary structures, triantennary structures, or tetraantennary structures as disclosed herein. The ds-siRNA agent may further be incorporated into a pharmaceutical composition containing a pharma-ceutically acceptable excipient, carrier, or diluent.

[0301] Example 4: Methods for delivering ds-siRNA molecules to the central nervous system of a patient A subject, e.g., a human subject, diagnosed with a disease is treated by administering the disclosed siRNA molecules of a pharmaceutical composition containing the siRNA molecules at a dosage and frequency determined by a treating physician (e.g., three times a day, twice a day, once a day, once a week, once a month). The dosage and frequency are determined based on the subject's height, weight, age, sex, and other disorders.

[0302] The siRNA molecules (e.g., branched siRNA molecules) having the chemical modification patterns disclosed herein are selected by the practitioner for compatibility with the disease and subject. Single-stranded or double-stranded branched siRNAs are available for selection. The selected siRNA has an antisense strand, and in the case of double-stranded siRNAs, the sense strand has the sequence and RNA modification (e.g., natural and non-natural internucleoside linkages, modified sugars, and 5'-phosphorus stabilizing moieties) that is most suitable for the patient and the disease being targeted. For example, the antisense strand may have any one of the antisense strand modification patterns disclosed herein, for example, antisense pattern 2: ASBSAOBOBOBOAOBOAOBOA-OBOAOBOAOBSASASASASA (Formula A2); antisense pattern 3: ASBSAOBOAOBOAOBOAOBOA-OBOAOBOAOBSASBSASASA (Formula A3); or antisense pattern 4: ASBSAOAOAOBOAOAOAOAOAOA-OAOAOBOAOBSASASASBSA (Formula A4). In the case of ds-siRNA, antisense pattern 1 may have a fully or partially complementary sense strand with any one of the following patterns of chemical modification: sense pattern 1: ASASAOBOAOBOAOBOAOBOA-OAOAOBSASA (Formula S1); sense pattern 2: ASASAOBOAOBOAOBOAOBOA-OAOAOBOAOA (Formula S2); sense pattern 3: ASASAOBOAOBOAOBOAOBOA-OAOAOBSASB (Formula S3); or sense pattern 4: ASASAOBOAOBOAOBOAOBOA-OAOAOBOAOB (Formula S4).In the case of a ds-siRNA having antisense pattern 2, the sense strand may have any one of the following chemical modification patterns: sense pattern 5: ASASAOBOAOBOAOBOAOBOA-OBOAOBSASA (Formula S5); sense pattern 6: ASASAOBOAOBOAOBOAOBOA-OBOAOBOAOA (Formula S6); sense pattern 7: ASASAOBOAOBOAOBOAOBOA-OBOAOBSASB (Formula S7); or sense pattern 8: ASASAOBOAOBOAOBOAOBOA-OBOAOBOAOB (Formula S8). For a ds-siRNA with antisense pattern 3, the sense strand may have a modification pattern of sense pattern 9: ASASAOAOAOBOBOAOBOA-OAOAOASASA (Formula S9); where A and B are different nucleosides (e.g., A is a 2-O-methyl ribonucleoside; B is a 2'-fluoro ribonucleoside), T is a phosphorothioate, P is a phosphodiester, and PSM is a 5'-phosphorus stabilizing moiety (e.g., 5'-vinyl phosphonate).

[0303] The siRNA is delivered by the route most suitable for the patient and condition (e.g., intrathecal, intracerebroventricular, or intrastriatal) at a rate tolerated by the patient until the subject reaches the maximum tolerated dose or until the symptoms of the disease are sufficiently ameliorated.

[0304] Example 5: In vivo gene silencing using di-siRNA pattern variants in a mouse model To determine the efficacy of gene silencing using the di-siRNA constructs described herein, gene silencing was performed against the huntingtin (HTT) gene using ds-siRNA pattern variants in FVB / NJ female mice in vivo.

[0305] method In vivo administration of di-siRNA. Thirteen di-siRNA scaffolds with a consensus sequence targeting HTT were designed and tested in vivo by intracerebroventricular (ICV) administration to FVB / NJ female mice. Among these ds-siRNA scaffolds, four scaffolds, namely ds-siRNA_A_V1, ds-siRNA_A_V2, ds-siRNA_B_V1, and ds-siRNA_B_V2, were first generation (F1) ds-siRNA molecules used to compare knockdown efficacy against the remaining second generation (F2) ds-siRNA scaffolds. Animals were divided into 14 groups (13 di-siRNA treatments and 1 PBS vehicle) with 10 animals per group and were intracerebroventricularly injected with the test articles shown in Table 2. On day 1, stereotaxic injections were performed in FVB / NJ female mice, where a single unilateral ICV injection (10 μL) was performed at 0.5 μL / min into the right side of the brain after needle insertion at the following coordinates from bregma: -0.45 mm AP, +1 mm medial-lateral, and -2.5 mm dorsoventral. Three dose levels (0.1, 0.5, and 2.5 nmol total compound) were tested for each scaffold. One month after injection, animals were perfused with cold 1×PBS and brains were harvested and sliced. Tissue punches of consistent diameter and thickness were taken from different brain regions (motor cortex, hippocampus, and striatum) and flash frozen on dry ice. [Table 2]

[0306] RNA Expression Analysis. To assess huntingtin mRNA expression levels, total RNA was extracted from mouse brain tissue punches using phenol:chloroform extraction. This was performed by first disrupting tissue samples in TRIzol reagent (Invitrogen) using a TissueLyserII (Qiagen) and adding chloroform to the homogenized samples at a ratio of 5:1 TRIzol:chloroform. The tubes were then vigorously shaken and spun at 12,000×g for 15 min, and the resulting upper aqueous phase containing total RNA was carefully removed and added to a clean tube. An equal volume of 70% ethanol was added to each sample and mixed gently. Samples were further purified using Qiagen RNeasy column purification according to the standard kit protocol. Samples were eluted in 40 μL of RNase-free water. After elution, RNA was analyzed on a TapeStation 4200 Bioanalyzer (Agilent) to assess concentration and quality. All samples were normalized for total RNA. cDNA synthesis was carried out using the Applied Biosystems High-Capacity cDNA Reverse Transcription kit in a response volume of 20 μL. After RT-PCR, nuclease-free water was added to the cDNA to bring the final sample volume to 50 μL.

[0307] For HTT gene expression analysis, qPCR was performed using TaqMan reagents on a QuantStudio7 real-time PCR instrument (Life Technologies). Samples were probed for total mouse HTT expression (assay ID Mm01213820_m1) and normalized to the housekeeping gene reference ATP5b (assay ID Mm00443967_g1) in the wells. Comparative analysis of relative HTT quantification (DeltaDeltaCt) to samples taken from PBS-treated mice was performed. Results showing HTT expression after administration of different doses of di-siRNA are shown in Tables 3-5. Mean% = mean % of HTT mRNA expression; SD = standard deviation; n = number of animals per group. [Table 3] [Table 4] [Table 5]

[0308] Each ds-siRNA construct tested caused a dose-dependent reduction in HTT mRNA levels in each of the brain regions tested, with the HTT mRNA levels after knockdown. Exemplary scatter plots demonstrating the dose-dependence of HTT knockdown in mice using the ds-siRNA constructs of the present disclosure are provided in Figures 14A-14F, 15A-15F, and 16, respectively, for ds-siRNA A, ds-siRNA B, and ds-siRNA C constructs, respectively.

[0309] Additional studies were performed to compare the relative patterns of HTT mRNA level reduction at dose levels of 0.5 nmol and 2.5 nmol, as shown in Figures 18A and 18B, respectively. The mRNA levels were examined after one month. Figure 18B shows that some of the F2 patterns have increased potency over the comparative F1 patterns.

[0310] Example 6: Reducing the toxic effects of interfering RNA delivery to the central nervous system. introduction In many species, the introduction of double-stranded RNA induces strong and specific gene silencing by RNA interference (RNAi). This phenomenon occurs in both plants and animals and has a role in viral defense and transposon silencing mechanisms. For example, short interfering RNA (siRNA), which is generally much shorter than the target gene, has been shown to be effective in gene silencing and is therefore useful as a therapeutic agent for silencing genes to restore the activity of genetic and biochemical pathways from disease states to normal, healthy states. However, delivery of interfering RNA molecules such as short interfering RNA (siRNA) to a subject, particularly to the central nervous system of a subject, carries the risk of toxic side effects including, among others, seizures, tremors, and hyperactive motor behavior. There remains a need for interfering RNA molecules that, when administered to a subject in need thereof, result in reduced toxicity.

[0311] result The severity of acute CNS toxicity of the siRNA molecules of the present disclosure was quantified by using the EvADINT scoring assay (Table 6). The higher the score, the more toxic the experimental condition was considered to be. [Table 6]

[0312] Various motifs of exemplary siRNA molecules of the present disclosure were evaluated for toxicity benefit using the EvADINT scoring assay, and the average across multiple tests is shown in Table 7 below. The data is graphically represented as a scatter plot in Figure 19. As shown in the data, some F2 molecules (e.g., ds-siRNA A_V3, ds-siRNA A_V4, ds-siRNA B_V6, ds-siRNA C_V2, and ds-siRNA B_V3) show improved toxicity benefit when compared to F1 molecules (e.g., ds-siRNA A_V1). [Table 7]

[0313] Specific embodiments Some specific embodiments are listed below. The embodiments listed below should not be construed as limiting the scope of the present invention, but rather the following are presented as some examples of the utility of the present invention.

[0314] E1. A small interfering RNA (siRNA) molecule, comprising an antisense strand and a sense strand having complementarity to the antisense strand, wherein the antisense strand comprises a structure represented by Formula I, which in the 5'-3' direction is: AB-(A') j -CP 2 -DP 1 -(C'-P 1 ) k -C' Formula I where A is of the formula CP 1 -DP 1 Represented by; Each A' is independently a group of the formula CP 2 -DP 2 Represented by; B is the formula CP 2 -DP 2 -DP 2 -DP 2 Represented by; each C is 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; and k is an integer from 1 to 7, a small interfering RNA (siRNA) molecule.

[0315] E2. The antisense strand has a structure represented by formula A1, which in the 5'-3' direction is: ASBSAOBOBOBOAOBOAOBOA-OBOAOBOAOBSASASASBSA Formula A1; An siRNA molecule described in E1, 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.

[0316] E3. A small interfering RNA (siRNA) molecule, comprising an antisense strand and a sense strand having complementarity to the antisense strand, wherein the antisense strand comprises a structure represented by Formula II, which in the 5'-3' direction is: AB-(A') j -CP 2 -DP 1 -(CP 1 ) k -C' Formula II; where A is of the formula CP 1 -DP 1 Represented by; Each A' is independently a group of the formula CP 2 -DP 2 Represented by; B is the formula CP 2 -DP 2 -DP 2 -DP 2 Represented by; each C is 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; and k is an integer from 1 to 7, a small interfering RNA (siRNA) molecule.

[0317] The siRNA molecule according to E1 or E3, wherein E4.j is 1 to 6.

[0318] E5. The siRNA molecule according to E1 or E3, wherein j is 1 to 5.

[0319] The siRNA molecule according to E1 or E3, wherein E6.j is 1 to 4.

[0320] The siRNA molecule according to E1 or E3, wherein E7.j is 1 to 3.

[0321] The siRNA molecule according to E1 or E3, wherein E8.j is 1 to 2.

[0322] The siRNA molecule of E1 or E3, wherein E9.j is 1.

[0323] E10.j is 2, the siRNA molecule of E1 or E3.

[0324] E11.j is 3, the siRNA molecule of E1 or E3.

[0325] E12.j is 4, the siRNA molecule of E1 or E3.

[0326] E13.j is 5, the siRNA molecule of E1 or E3.

[0327] E14.j is 6, the siRNA molecule of E1 or E3.

[0328] E15.j is 7, the siRNA molecule of E1 or E3.

[0329] E16. The siRNA molecule according to any one of E1 to E15, wherein k is 1 to 6.

[0330] E17. The siRNA molecule according to E16, wherein k is 1 to 5.

[0331] E18. The siRNA molecule according to E16, wherein k is 1 to 4.

[0332] E19. The siRNA molecule according to E16, wherein k is 1 to 3.

[0333] E20. The siRNA molecule according to E16, wherein k is 1 to 2.

[0334] E21. The siRNA molecule of E16, wherein k is 1.

[0335] E22. The siRNA molecule of E16, wherein k is 2.

[0336] E23. The siRNA molecule of E16, wherein k is 3.

[0337] E24. The siRNA molecule of E16, wherein k is 4.

[0338] E25. The siRNA molecule of E16, wherein k is 5.

[0339] E26. The siRNA molecule of E16, wherein k is 6.

[0340] E27.The siRNA molecule of E16, wherein k is 7.

[0341] E28. The siRNA molecule of E1 or E3, wherein j is 4 and k is 4.

[0342] E29. The antisense strand has a structure represented by formula A2, which in the 5'-3' direction is: ASBSAOBOBOBOAOBOAOBOA-OBOAOBOAOBSASASASASA Formula A2; An siRNA molecule described in E3, 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.

[0343] E30. The sense strand comprises a structure represented by formula III, which in the 5'-3' direction is: E-(A') m -F Formula III; In the formula, E is a compound represented by the formula (CP 1 )2; F is the formula (CP 2 )3-DP 1 -CP 1 -C, (CP 2 )3-DP 2 -CP 2 -C, (CP 2 )3-DP 1 -CP 1 -D, or (CP 2 )3-DP 2 -CP 2 - represented by D; A', C, D, P 1 , and P 2 is as defined in formula II; The siRNA molecule of any one of E1 to E29, wherein m is an integer of 1 to 7.

[0344] The siRNA molecule of E30, wherein E31.m is 1 to 6.

[0345] The siRNA molecule of E30, wherein E32.m is 1 to 5.

[0346] The siRNA molecule of E30, wherein E33.m is 1 to 4.

[0347] The siRNA molecule of E30, wherein E34.m is 1 to 3.

[0348] The siRNA molecule of E30, wherein E35.m is 1 to 2.

[0349] The siRNA molecule of E30, wherein E36.m is 1.

[0350] The siRNA molecule of E30, wherein E37.m is 2.

[0351] The siRNA molecule of E30, wherein E38.m is 3.

[0352] The siRNA molecule of E30, wherein E39.m is 4.

[0353] The siRNA molecule of E30, wherein E40.m is 5.

[0354] The siRNA molecule of E30, wherein E41.m is 6.

[0355] The siRNA molecule of E30, wherein E42.m is 7.

[0356] E43. The sense strand has a structure represented by formula S1, which in the 5'-3' direction is: ASASAOBOAOBOAOBOAOBOA-OAOAOBBSASA Formula S1; The siRNA molecule of E30, 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.

[0357] E44. The sense strand has a structure represented by formula S2, which in the 5'-3' direction is: ASASAOBOAOBOAOBOAOBOA-OAOAOBOAOA Formula S2; The siRNA molecule of E30, 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.

[0358] E45. The sense strand has a structure represented by formula S3, which in the 5'-3' direction is: ASASAOBOAOBOAOBOAOBOA-OAOAOBSASB formula S3; The siRNA molecule of E30, 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.

[0359] E46. The sense strand has a structure represented by formula S4, which in the 5'-3' direction is: ASASAOBOAOBOAOBOAOBOA-OAOAOBOAOB Equation S4; The siRNA molecule of E30, 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.

[0360] E47. An siRNA molecule comprising an antisense strand and a sense strand having complementarity to the antisense strand, wherein the antisense strand comprises a structure represented by formula IV, which in the 5'-3' direction is: A-(A') j -CP 2 -B-(CP 1 ) k -C' Formula IV; where A is of the formula CP 1 -DP 1 Represented by; Each A' is independently a group of the 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 is independently a phosphorothioate internucleoside linkage; Each P 2 is, independently, a phosphodiester internucleoside linkage; j is an integer from 1 to 7; and k is an integer from 1 to 7.

[0361] The siRNA molecule according to E47, wherein E48.j is 1 to 6.

[0362] The siRNA molecule according to E47, wherein E49.j is 1 to 5.

[0363] The siRNA molecule according to E47, wherein E50.j is 1 to 4.

[0364] The siRNA molecule described in E47, wherein E51.j is 1 to 3.

[0365] The siRNA molecule according to E47, wherein E52.j is 1 to 2.

[0366] The siRNA molecule according to any one of E47 to E52, wherein E53.j is 1.

[0367] The siRNA molecule according to any one of E47 to E52, wherein E54.j is 2.

[0368] The siRNA molecule according to any one of E47 to E52, wherein E55.j is 3.

[0369] The siRNA molecule according to any one of E47 to E52, wherein E56.j is 4.

[0370] The siRNA molecule according to any one of E47 to E52, wherein E57.j is 5.

[0371] The siRNA molecule of E47 or E48, wherein E58.j is 6.

[0372] The siRNA molecule of E47, wherein E59.j is 7.

[0373] E60. The siRNA molecule according to any one of E47 to E59, wherein k is 1 to 6.

[0374] E61. The siRNA molecule of E60, wherein k is 1 to 5.

[0375] The siRNA molecule of E60, wherein k is 1 to 4.

[0376] E63. The siRNA molecule according to E60, wherein k is 1 to 3.

[0377] The siRNA molecule according to E60, wherein k is 1 to 2.

[0378] The siRNA molecule of E60, wherein k is 1.

[0379] The siRNA molecule of E60, wherein k is 2.

[0380] The siRNA molecule of E60, wherein k is 3.

[0381] The siRNA molecule of E60, wherein k is 4.

[0382] The siRNA molecule of E60, wherein k is 5.

[0383] E70. The siRNA molecule of E60, wherein k is 6.

[0384] The siRNA molecule of E60, wherein E71.k is 7.

[0385] E72. The siRNA molecule of E47, wherein j is 6 and k is 2.

[0386] E73. The antisense strand has a structure represented by formula A3, which in the 5'-3' direction is: ASBSAOBOAOBOAOBOAOBOA-OBOAOBOAOBSASASASA formula A3; The siRNA molecule of E47, 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.

[0387] E74. The sense strand comprises a structure represented by formula V, which in the 5'-3' direction is: E-(A') m -CP 2 -F formula V; In the formula, E is a compound represented by the formula (CP 1 )2; F is for DP 1 -CP 1 -C, D.P. 2 -CP 2 -C, D.P. 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; The siRNA molecule according to any one of E47 to E73, wherein m is an integer of 1 to 7.

[0388] The siRNA molecule described in E74, wherein E75.m is 1 to 6.

[0389] The siRNA molecule described in E74, wherein E76.m is 1 to 5.

[0390] The siRNA molecule described in E74, wherein E77.m is 1 to 4.

[0391] The siRNA molecule described in E74, wherein E78.m is 1 to 3.

[0392] The siRNA molecule described in E74, wherein E79.m is 1 to 2.

[0393] The siRNA molecule of E74, wherein E80.m is 1.

[0394] The siRNA molecule of E74, wherein E81.m is 2.

[0395] The siRNA molecule of E74, wherein E82.m is 3.

[0396] The siRNA molecule of E74, wherein E83.m is 4.

[0397] The siRNA molecule of E74, wherein E84.m is 5.

[0398] The siRNA molecule of E74, wherein E85.m is 6.

[0399] The siRNA molecule of E74, wherein E86.m is 7.

[0400] E87. The sense strand has a structure represented by formula S5, which in the 5'-3' direction is: ASASAOBOAOBOAOBOAOBOA-OBOAOBSASA Formula S5; The siRNA molecule of E74, 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.

[0401] E88. The sense strand has a structure represented by formula S6, which in the 5'-3' direction is: ASASAOBOAOBOAOBOAOBOA-OBOAOBOAOA Formula S6; The siRNA molecule of E74, 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.

[0402] E89. The sense strand has a structure represented by formula S7, which in the 5'-3' direction is: ASASAOBOAOBOAOBOAOBOA-OBOAOBSASB Formula S7; The siRNA molecule of E74, 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.

[0403] E90. The sense strand has a structure represented by formula S8, which in the 5'-3' direction is: ASASAOBOAOBOAOBOAOBOA-OBOAOBOAOB formula S8; The siRNA molecule of E74, 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.

[0404] E91. An siRNA molecule comprising an antisense strand and a sense strand having complementarity to the antisense strand, wherein the antisense strand comprises a structure represented by formula VI, where formula VI is in the 5'-3' direction: A.B. j -EB k -EFG l -DP 1 -C' Formula VI; where A is of the formula CP 1 -DP 1 Represented by; Each B is independently a group of 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 is independently of the formula CP 1 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; and l is an integer from 1 to 7.

[0405] The siRNA molecule according to E91, wherein E92.j is 1 to 6.

[0406] The siRNA molecule according to E91, wherein E93.j is 1 to 5.

[0407] The siRNA molecule according to E91, wherein E94.j is 1 to 4.

[0408] The siRNA molecule according to E91, wherein E95.j is 1 to 3.

[0409] The siRNA molecule according to E91, wherein E96.j is 1 to 2.

[0410] The siRNA molecule of E91, wherein E97.j is 1.

[0411] The siRNA molecule of E91, wherein E98.j is 2.

[0412] The siRNA molecule of E91, wherein E99.j is 3.

[0413] The siRNA molecule of E91, wherein E100.j is 4.

[0414] The siRNA molecule of E91, wherein E101.j is 5.

[0415] The siRNA molecule of E91, wherein E102.j is 6.

[0416] The siRNA molecule of E91, wherein E103.j is 7.

[0417] E104. The siRNA molecule according to any one of E91 to E103, wherein k is 1 to 6.

[0418] E105. The siRNA molecule according to E104, wherein k is 1 to 5.

[0419] The siRNA molecule according to E104, wherein k is 1 to 4.

[0420] E107. The siRNA molecule according to E104, wherein k is 1 to 3.

[0421] E108. The siRNA molecule according to E104, wherein k is 1 to 2.

[0422] The siRNA molecule according to E104, wherein k is 1.

[0423] The siRNA molecule of E104, wherein k is 2.

[0424] The siRNA molecule of E104, wherein E111.k is 3.

[0425] The siRNA molecule of E104, wherein k is 4.

[0426] The siRNA molecule of E104, wherein k is 5.

[0427] The siRNA molecule of E104, wherein k is 6.

[0428] The siRNA molecule of E104, wherein k is 7.

[0429] The siRNA molecule according to any one of E91 to E115, wherein E116.l is 1 to 6.

[0430] The siRNA molecule according to E116, wherein E117.l is 1 to 5.

[0431] The siRNA molecule according to E116, wherein E118.l is 1 to 4.

[0432] The siRNA molecule according to E116, wherein E119.l is 1 to 3.

[0433] The siRNA molecule according to E116, wherein E120.l is 1 to 2.

[0434] The siRNA molecule of E116, wherein E121.l is 1.

[0435] The siRNA molecule of E116, wherein E122.l is 2.

[0436] The siRNA molecule of E116, wherein E123.l is 3.

[0437] The siRNA molecule of E116, wherein E124.l is 4.

[0438] The siRNA molecule of E116, wherein E125.l is 5.

[0439] The siRNA molecule of E116, wherein E126.l is 6.

[0440] The siRNA molecule of E116, wherein E127.l is 7.

[0441] E128. The siRNA molecule of E91, wherein j is 3, k is 6, and l is 2.

[0442] E129. The antisense strand has a structure represented by formula A4, which in the 5'-3' direction is: ASBSAOAOAOBOAOAOAOAOA-OAOAOBOAOBSASASASBSA formula A4; The siRNA molecule of E91, 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.

[0443] E130. The sense strand comprises a structure represented by formula VII, which in the 5'-3' direction is: HB m -I n -A'-B o -HC Formula VII; where A' is a compound of the formula CP 2 -DP 2 Represented by; Each H is independently a group of the formula (CP 1 )2; Each I is independently represented by the formula (DP 2 ) is represented as; 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 any one of E91 to E129, wherein o is an integer of 1 to 7.

[0444] The siRNA molecule of E130, wherein E131.m is 1 to 6.

[0445] The siRNA molecule according to E130, wherein E132.m is 1 to 5.

[0446] The siRNA molecule according to E130, wherein E133.m is 1 to 4.

[0447] The siRNA molecule according to E130, wherein E134.m is 1 to 3.

[0448] The siRNA molecule of E130, wherein E135.m is 1 or 2.

[0449] The siRNA molecule of E130, wherein E136.m is 1.

[0450] The siRNA molecule of E130, wherein E137.m is 2.

[0451] The siRNA molecule of E130, wherein E138.m is 3.

[0452] The siRNA molecule of E130, wherein E139.m is 4.

[0453] The siRNA molecule of E130, wherein E140.m is 5.

[0454] The siRNA molecule of E130, wherein E141.m is 6.

[0455] The siRNA molecule of E130, wherein E142.m is 7.

[0456] E143. The siRNA molecule according to E130, wherein n is 1 to 6.

[0457] The siRNA molecule according to E130, wherein n is 1 to 5.

[0458] The siRNA molecule according to E130, wherein E145.n is 1 to 4.

[0459] The siRNA molecule according to E130, wherein E146.n is 1 to 3.

[0460] The siRNA molecule according to E130, wherein E147.n is 1 to 2.

[0461] The siRNA molecule of E130, wherein E148.n is 1.

[0462] The siRNA molecule of E130, wherein E149.n is 2.

[0463] The siRNA molecule of E130, wherein E150.n is 3.

[0464] The siRNA molecule of E130, wherein E151.n is 4.

[0465] The siRNA molecule of E130, wherein E152.n is 5.

[0466] The siRNA molecule of E130, wherein E153.n is 6.

[0467] The siRNA molecule of E130, wherein E154.n is 7.

[0468] The siRNA molecule according to any one of E130 to E154, wherein E155.o is 1 to 6.

[0469] The siRNA molecule according to E155, wherein E156.o is 1 to 5.

[0470] The siRNA molecule according to E155, wherein E157.o is 1 to 4.

[0471] The siRNA molecule according to E155, wherein E158.o is 1 to 3.

[0472] The siRNA molecule according to E155, wherein E159.o is 1 to 2.

[0473] The siRNA molecule of E155, wherein E160.o is 1.

[0474] The siRNA molecule of E155, wherein E161.o is 2.

[0475] The siRNA molecule of E155, wherein E162.o is 3.

[0476] The siRNA molecule of E155, wherein E163.o is 4.

[0477] The siRNA molecule of E155, wherein E164.o is 5.

[0478] The siRNA molecule according to E155, wherein E165.o is 6.

[0479] The siRNA molecule of E155, wherein E166.o is 7.

[0480] E167. The siRNA molecule of E130, wherein m is 3, n is 3, and o is 3.

[0481] E168. The sense strand has a structure represented by formula S9, which in the 5'-3' direction is: ASASAOAOAOBOBOBOBOAOBOA-OAOAOAASASA Formula S9; The siRNA molecule of E130, 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.

[0482] E169.P 1 The siRNA molecule according to any one of E1 to E168, wherein is a phosphorothioate bond.

[0483] E170.P 2 The siRNA molecule of any one of E1 to E169, wherein is a phosphodiester bond.

[0484] E171. The siRNA molecule of any one of E1 to E170, wherein the antisense strand further comprises a 5' phosphorus stabilizing moiety at the 5' end of the antisense strand.

[0485] E172. The siRNA molecule of any one of E1-E171, wherein the sense strand further comprises a 5' phosphorus stabilizing moiety at the 5' end of the sense strand.

[0486] E173. The siRNA molecule of any one of E1-E172, wherein at least 10% of the ribonucleosides are 2'-O-Me ribonucleosides.

[0487] E174. The siRNA molecule of E173, wherein at least 20% of the ribonucleosides are 2'-O-Me ribonucleosides.

[0488] E175. The siRNA molecule of E174, wherein at least 30% of the ribonucleosides are 2'-O-Me ribonucleosides.

[0489] E176. The siRNA molecule of E175, wherein at least 40% of the ribonucleosides are 2'-O-Me ribonucleosides.

[0490] E177. The siRNA molecule of E176, wherein at least 50% of the ribonucleosides are 2'-O-Me ribonucleosides.

[0491] E178. The siRNA molecule of E177, wherein at least 60% of the ribonucleosides are 2'-O-Me ribonucleosides.

[0492] E179. The siRNA molecule of E178, wherein at least 70% of the ribonucleosides are 2'-O-Me ribonucleosides.

[0493] E180. The siRNA molecule of E179, wherein at least 80% of the ribonucleosides are 2'-O-Me ribonucleosides.

[0494] E181. The siRNA molecule of E180, wherein at least 90% of the ribonucleosides are 2'-O-Me ribonucleosides.

[0495] E182. The siRNA molecule of any one of E1-E181, wherein at least 10% of the ribonucleosides are 2'-F ribonucleosides.

[0496] E183. The siRNA molecule of E182, wherein at least 20% of the ribonucleosides are 2'-F ribonucleosides.

[0497] E184. The siRNA molecule of E183, wherein at least 30% of the ribonucleosides are 2'-F ribonucleosides.

[0498] E185. The siRNA molecule of E184, wherein at least 40% of the ribonucleosides are 2'-F ribonucleosides.

[0499] E186. The siRNA molecule of E185, wherein at least 50% of the ribonucleosides are 2'-F ribonucleosides.

[0500] E187. The siRNA molecule of E186, wherein at least 60% of the ribonucleosides are 2'-F ribonucleosides.

[0501] E188. The siRNA molecule of E187, wherein at least 70% of the ribonucleosides are 2'-F ribonucleosides.

[0502] E189. The siRNA molecule of E188, wherein at least 80% of the ribonucleosides are 2'-F ribonucleosides.

[0503] E190. The siRNA molecule of E189, wherein at least 90% of the ribonucleosides are 2'-F ribonucleosides.

[0504] E191. The siRNA molecule of any one of E1 to E190, wherein the antisense strand has 12 2'-O-Me ribonucleosides.

[0505] E192. The siRNA molecule of any one of E1 to E191, wherein the antisense strand has nine 2'-F ribonucleosides.

[0506] E193. The siRNA molecule of E191 or E192, wherein the sense strand has 11 2'-O-Me ribonucleosides.

[0507] E194. The siRNA molecule of E191 or E192, wherein the sense strand has 10 2'-O-Me ribonucleosides.

[0508] E195. The siRNA molecule of any one of E191 to E194, wherein the sense strand has five 2'-F ribonucleosides.

[0509] E196. The siRNA molecule of any one of E191 to E194, wherein the sense strand has six 2'-F ribonucleosides.

[0510] E197. The siRNA molecule of any one of E1-E190, wherein the antisense strand has 17 2'-O-Me ribonucleosides.

[0511] E198. The siRNA molecule of any one of E190 or E197, wherein the antisense strand has five 2'-F ribonucleosides.

[0512] E199. The siRNA molecule of any one of E197-E198, wherein the sense strand has 12 2'-O-Me ribonucleosides.

[0513] E200. The siRNA molecule of any one of E197 to E199, wherein the sense strand has four 2'-F ribonucleosides.

[0514] E201. The siRNA molecule of any one of E1-E200, wherein at least 10% of the internucleoside linkages are phosphodiester or phosphorothioate linkages.

[0515] E202. The siRNA molecule of E201, wherein at least 20% of the internucleoside linkages are phosphodiester or phosphorothioate linkages.

[0516] E203. The siRNA molecule of E202, wherein at least 30% of the internucleoside linkages are phosphodiester or phosphorothioate linkages.

[0517] E204. The siRNA molecule of E203, wherein at least 40% of the internucleoside linkages are phosphodiester or phosphorothioate linkages.

[0518] E205. The siRNA molecule of E204, wherein at least 50% of the internucleoside linkages are phosphodiester or phosphorothioate linkages.

[0519] E206. The siRNA molecule of E205, wherein at least 60% of the internucleoside linkages are phosphodiester or phosphorothioate linkages.

[0520] E207. The siRNA molecule of E206, wherein at least 70% of the internucleoside linkages are phosphodiester or phosphorothioate linkages.

[0521] E208. The siRNA molecule of E207, wherein at least 80% of the internucleoside linkages are phosphodiester or phosphorothioate linkages.

[0522] E209. The siRNA molecule of E208, wherein at least 90% of the internucleoside linkages are phosphodiester or phosphorothioate linkages.

[0523] E210. The siRNA molecule of any one of E1-E209, wherein the antisense strand has seven phosphorothioate linkages.

[0524] E211. The siRNA molecule of any one of E1-E210, wherein the antisense strand has 13 phosphodiester bonds.

[0525] E212. An siRNA molecule of E210 or E211, wherein the sense strand has four phosphorothioate linkages.

[0526] E213. The siRNA molecule of any one of E210 to E212, wherein the sense strand has 11 phosphodiester bonds.

[0527] E214. The siRNA molecule of any one of E210 to E212, wherein the sense strand has 13 phosphodiester bonds.

[0528] E215.5' a phosphorus stabilizing moiety represented by any one of formulas VIII-XV; [ka] The siRNA molecule of any one of E171 to E214, wherein Nuc represents a nucleobase 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, hydroxy, or hydrogen.

[0529] E216. The siRNA molecule of E215, wherein the nucleobase is adenine, uracil, guanine, thymine, or cytosine.

[0530] E217. The siRNA molecule of E215 or E216, wherein the 5' phosphorus stabilizing moiety is an (E)-vinyl phosphonate represented by formula X.

[0531] E218. The siRNA molecule of any one of E1-E217, wherein the siRNA molecule further comprises a hydrophobic moiety at the 5' end or 3' end of the siRNA molecule.

[0532] E219. The siRNA molecule of E218, wherein the hydrophobic portion is at the 5' end of the siRNA molecule.

[0533] E220. The siRNA molecule of E218 or E219, wherein the hydrophobic portion is at the 3' end of the siRNA molecule.

[0534] E221. The siRNA molecule of any one of E218-E220, wherein the hydrophobic moiety is selected from the group consisting of cholesterol, vitamin D, or tocopherol.

[0535] E222. The siRNA molecule of E221, wherein the hydrophobic moiety is cholesterol.

[0536] E223. The siRNA molecule of E221, wherein the hydrophobic moiety is Vitamin D.

[0537] E224. The siRNA molecule of E221, wherein the hydrophobic moiety is tocopherol.

[0538] E225. The siRNA molecule of any one of E1 to E224, wherein the length of the antisense strand is between 10 and 30 nucleotides (e.g., 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 nucleotides).

[0539] E226. The siRNA molecule of E225, wherein the length of the antisense strand is between 15 and 30 nucleotides (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 nucleotides).

[0540] E227. The siRNA molecule of E226, wherein the length of the antisense strand is between 18 and 23 nucleotides (eg, 19, 20, 21, or 22 nucleotides).

[0541] E228. The siRNA molecule of E227, wherein the length of the antisense strand is 20 nucleotides.

[0542] E229. The siRNA molecule of E227, wherein the length of the antisense strand is 21 nucleotides.

[0543] E230. The siRNA molecule of E227, wherein the length of the antisense strand is 22 nucleotides.

[0544] E231. The siRNA molecule of E227, wherein the length of the antisense strand is 23 nucleotides.

[0545] E232. The siRNA molecule of E226, wherein the length of the antisense strand is 24 nucleotides.

[0546] E233. The siRNA molecule of E226, wherein the length of the antisense strand is 25 nucleotides.

[0547] E234. The siRNA molecule of E226, wherein the length of the antisense strand is 26 nucleotides.

[0548] E235. The siRNA molecule of E226, wherein the length of the antisense strand is 27 nucleotides.

[0549] E236. The siRNA molecule of E226, wherein the length of the antisense strand is 28 nucleotides.

[0550] E237. The siRNA molecule of E226, wherein the length of the antisense strand is 29 nucleotides.

[0551] E238. The siRNA molecule of E226, wherein the length of the antisense strand is 30 nucleotides.

[0552] E239. The siRNA molecule of any one of E1-E238, wherein the length of the sense strand is between 12-30 nucleotides.

[0553] E240. The siRNA molecule of E239, wherein the length of the sense strand is between 14 and 24 nucleotides (e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides).

[0554] E241. The siRNA molecule of E240, wherein the length of the sense strand is 15 nucleotides.

[0555] E242. The siRNA molecule of E240, wherein the length of the sense strand is 16 nucleotides.

[0556] E243. The siRNA molecule of E240, wherein the length of the sense strand is 17 nucleotides.

[0557] E244. The siRNA molecule of E240, wherein the length of the sense strand is 18 nucleotides.

[0558] E245. The siRNA molecule of E240, wherein the length of the sense strand is 19 nucleotides.

[0559] E246. The siRNA molecule of E240, wherein the length of the sense strand is 20 nucleotides.

[0560] E247. The siRNA molecule of E240, wherein the length of the sense strand is 21 nucleotides.

[0561] E248. The siRNA molecule of E240, wherein the length of the sense strand is 22 nucleotides.

[0562] E249. The siRNA molecule of E240, wherein the length of the sense strand is 23 nucleotides.

[0563] E250. The siRNA molecule of E240, wherein the length of the sense strand is 24 nucleotides.

[0564] E251. The siRNA molecule of E239, wherein the length of the sense strand is 25 nucleotides.

[0565] E252. The siRNA molecule of E239, wherein the length of the sense strand is 26 nucleotides.

[0566] E253. The siRNA molecule of E239, wherein the length of the sense strand is 27 nucleotides.

[0567] E254. The siRNA molecule of E239, wherein the length of the sense strand is 28 nucleotides.

[0568] E255. The siRNA molecule of E239, wherein the length of the sense strand is 29 nucleotides.

[0569] E256. The siRNA molecule of E239, wherein the length of the sense strand is 30 nucleotides.

[0570] E257. The siRNA molecule according to any one of E1 to E256, wherein the siRNA molecule is a branched siRNA molecule.

[0571] E258. The siRNA molecule of E257, wherein the branched siRNA molecule is biantennary, triantennary, or tetraantennary.

[0572] E259. The siRNA molecule of E258, wherein the siRNA molecule is biantennary.

[0573] E260. The siRNA molecule of E258, wherein the siRNA molecule is triantennary.

[0574] E261. The siRNA molecule of E258, wherein the siRNA molecule is tetraantennary.

[0575] E262 The biantennary siRNA molecule is represented by any one of formulas XVI to XVIII, [ka] The siRNA molecule of E258 or E259, wherein each RNA is independently an siRNA molecule, L is a linker, and each X independently represents a branch point moiety.

[0576] E263 The siRNA molecule according to E262, wherein the bisecting siRNA molecule is represented by formula XVI.

[0577] E264 The siRNA molecule according to E262, wherein the bisecting siRNA molecule is represented by formula XVII.

[0578] E265 The siRNA molecule of E262, wherein the bisecting siRNA molecule is represented by formula XVIII.

[0579] E266. The three-branched siRNA molecule is represented by any one of formulas XIX-XXII, [ka] The siRNA molecule of E258 or E260, wherein each RNA is independently an siRNA molecule, L is a linker, and each X independently represents a branch point moiety.

[0580] E267. The siRNA molecule according to E266, wherein the three-branched siRNA molecule is represented by formula XIX.

[0581] E268. The siRNA molecule according to E266, wherein the three-branched siRNA molecule is represented by formula XX.

[0582] E269. The siRNA molecule according to E266, wherein the three-branched siRNA molecule is represented by formula XXI.

[0583] E270. The siRNA molecule according to E266, wherein the three-branched siRNA molecule is represented by formula XXII.

[0584] E271. The four-branched siRNA molecule is represented by any one of formulas XXIII-XXVII, [ka] The siRNA molecule of E258 or E261, wherein each RNA is independently an siRNA molecule, L is a linker, and each X independently represents a branch point moiety.

[0585] E272. The siRNA molecule of E271, wherein the four-branched siRNA molecule is represented by formula XXIII.

[0586] E273. The siRNA molecule of E271, wherein the four-branched siRNA molecule is represented by formula XXIV.

[0587] E274. The siRNA molecule of E271, wherein the four-branched siRNA molecule is represented by formula XXV.

[0588] E275. The siRNA molecule of E271, wherein the four-branched siRNA molecule is represented by formula XXVI.

[0589] E276. The siRNA molecule of E271, wherein the four-branched siRNA molecule is represented by formula XXVII.

[0590] E277. The siRNA molecule of any one of E262 to E276, wherein the linker is selected from the group consisting of one or more consecutive subunits of ethylene glycol (e.g., polyethylene glycol (PEG), such as triethylene glycol (TrEG) or tetraethylene glycol (TEG)), alkyl, carbohydrate, block copolymer, peptide, RNA, and DNA.

[0591] E278. The siRNA molecule of E277, wherein the linker is an ethylene glycol oligomer.

[0592] E279. The siRNA molecule of E278, wherein the ethylene glycol oligomer is PEG.

[0593] The siRNA molecule of E279, wherein the PEG is TrEG.

[0594] The siRNA molecule of E279, wherein the PEG is TEG.

[0595] E282. The siRNA molecule of E277, wherein the linker is an alkyl oligomer.

[0596] E283. The siRNA molecule of E277, wherein the linker is a carbohydrate oligomer.

[0597] E284. The siRNA molecule of E277, wherein the linker is a block copolymer.

[0598] E285. The siRNA molecule of E277, wherein the linker is a peptide oligomer.

[0599] E286. The siRNA molecule of E277, wherein the linker is an RNA oligomer.

[0600] E287. The siRNA molecule of E277, wherein the linker is a DNA oligomer.

[0601] E288. The siRNA molecule of any one of E277-E287, wherein the oligomer or copolymer contains 2-20 consecutive subunits.

[0602] E289. The siRNA molecule of E288, wherein the oligomer or copolymer contains 4 to 18 consecutive subunits.

[0603] E290. The siRNA molecule of E289, wherein the oligomer or copolymer contains 6-16 consecutive subunits.

[0604] E291. The siRNA molecule of E230, wherein the oligomer or copolymer contains 8-14 consecutive subunits.

[0605] E292. The siRNA molecule of E231, wherein the oligomer or copolymer contains 10-12 consecutive subunits.

[0606] E293. The siRNA molecule of E277, wherein the linker connects one or more (eg, one, two or more) siRNA molecules via a covalent bond forming moiety.

[0607] E294. The siRNA molecule of E293, wherein the covalent bond forming moiety is selected from the group consisting of alkyl, ester, amide, carbamate, phosphonate, phosphate, phosphorothioate, phosphoramidate, triazole, urea, and formacetal.

[0608] E295. The linker comprises a structure of formula L1, wherein L1 is [ka] The siRNA molecule of E277,

[0609] E296. The linker comprises a structure of formula L2, [ka] The siRNA molecule of E277,

[0610] E297. The linker comprises a structure of formula L3, [ka] The siRNA molecule of E277,

[0611] E298. The linker comprises a structure of formula L4, [ka] The siRNA molecule of E277,

[0612] E299. The linker comprises a structure of formula L5, [ka] The siRNA molecule of E277,

[0613] E300. The linker comprises a structure of formula L6, wherein formula L6 is [ka] The siRNA molecule of E277,

[0614] E301. The linker comprises a structure of formula L7, [ka] The siRNA molecule of E277,

[0615] E302. The linker comprises a structure of formula L8, [ka] The siRNA molecule of E277,

[0616] E303. The linker comprises a structure of formula L9, [ka] The siRNA molecule of E277,

[0617] E304.The antisense strand is ABCA7, ABI3, ADAM10, APOC1, APOE, AXL, BIN1, C1QA, C3, C9ORF72, CASS4, CCL5, CD2AP, CD33, CD68, CLPTM1, CLU, CR1, CSF1, CST7, CTSB, CTSD, CTSL, CX CL10, CXCL13, DSG2, ECHDC3, EPHA1, FABP5, FERMT2, FTH1, GNAS, GRN, HBEGF, HLA-DRB1, HLA-DRB5, HTT, IFIT1, IFIT3, IFITM3, IFNAR1, IFNAR2, IGF1, IL10RA, IL1A, IL 1B, IL1RAP, INPP5D, ITGAM, ITGAX, KCNT1, LILRB4, LPL, MAPT, MEF2C, MMP12, MS4A4A, MS4A6A, MSH3, NLRP3, NME8, NOS2, PICALM, PILRA, PLCG2, PRNP, PTK2B, SCIMP, SCN9A, SLC24A4, SNCA, SORL1, SPI1, SPP1, SPPL2A, TBK1, TNF, TREM2, TREML2, TYROBP, and ZCWPW1.

[0618] E305. The siRNA molecule of E304, wherein the antisense strand has sufficient complementarity to hybridize to a portion of a gene selected from APOE, BIN1, C1QA, C3, C9ORF72, CCL5, CD33, CLU / APOJ, CR1, CXCL10, CXCL13, IFIT1, IFIT3, IFITM3, IFNAR1, IFNAR2, IL10RA, IL1A, IL1B, IL1RAP, INPP5D, ITGAM, MEF2C, MMP12, NLRP3, NOS2, PILRA, PLCG2, PTK2B, SLC24A4, TBK1, and TNF.

[0619] E306. The siRNA molecule of E304, wherein the antisense strand has sufficient complementarity to hybridize with a portion of a gene selected from HTT, MAPT, SNCA, C9ORF72, APOE, SCN9A, KCNT1, PRNP, and MSH3.

[0620] E307. The siRNA molecule of E306, wherein the antisense strand has sufficient complementarity to hybridize with a portion of the HTT gene.

[0621] E308. The siRNA molecule of E306, wherein the antisense strand has sufficient complementarity to hybridize with a portion of the MAPT gene.

[0622] E309. The siRNA molecule of E306, wherein the antisense strand has sufficient complementarity to hybridize with a portion of the SNCA gene.

[0623] E310. The siRNA molecule of E306, wherein the antisense strand has sufficient complementarity to hybridize with a portion of the C9ORF72 gene.

[0624] E311. The siRNA molecule of E306, wherein the antisense strand has sufficient complementarity to hybridize with a portion of the APOE gene.

[0625] E312. The siRNA molecule of E306, wherein the antisense strand has sufficient complementarity to hybridize with a portion of the SCN9A gene.

[0626] E313. The siRNA molecule of E306, wherein the antisense strand has sufficient complementarity to hybridize with a portion of the KCNT1 gene.

[0627] E314. The siRNA molecule of E306, wherein the antisense strand has sufficient complementarity to hybridize with a portion of the PRNP gene.

[0628] E315. The siRNA molecule of E306, wherein the antisense strand has sufficient complementarity to hybridize with a portion of the MSH3 gene.

[0629] E316. A pharmaceutical composition comprising the siRNA molecule according to any one of E1 to E315, and a pharma- ceutically acceptable excipient, carrier, or diluent.

[0630] E317. A method for delivering an siRNA molecule to the central nervous system (CNS) of a subject, comprising administering to the CNS of the subject an siRNA molecule described in any one of E1-E315 or a pharmaceutical composition described in E302.

[0631] E318. The method of E317, wherein the siRNA molecule or pharmaceutical composition is administered to the subject by intrastriatal, intracerebroventricular, or intrathecal injection.

[0632] E319. The method of E317 or E318, wherein delivery of the siRNA molecule or pharmaceutical composition to the CNS of a subject results in gene silencing of a target gene in the subject.

[0633] E320. The method of E319, wherein the target gene is an overactive disease driver gene.

[0634] E321. The method of E319, wherein the target gene is a negative regulator of a gene with decreased expression associated with a disease state in the subject.

[0635] E322. The method of E319, wherein the target gene is a positive regulator of a gene with increased expression associated with a disease state in the subject.

[0636] E323. The method of E319, wherein the target gene is a splice isoform of the target gene, and the splice isoform reduces expression of the target gene.

[0637] E324. The method of any one of E319-E323, wherein a disease condition in a subject is treated by gene silencing.

[0638] E325. The method according to any one of E317 to E324, wherein the siRNA molecule or pharmaceutical composition is administered to the subject by intrastriatal injection.

[0639] E326. The method according to any one of E317 to E324, wherein the siRNA molecule or pharmaceutical composition is administered to the subject by intracerebroventricular injection.

[0640] E327. The method according to any one of E317 to E324, wherein the siRNA molecule or pharmaceutical composition is administered to the subject by intrathecal injection.

[0641] E328. The method of any one of E317 to E327, wherein the subject is a human.

[0642] E329. A kit comprising an siRNA molecule described in any one of E1 to E315, or the pharmaceutical composition described in E316, and an accompanying instruction, the accompanying instruction instructing a user of the kit to carry out a method described in any one of E317 to E328.

[0643] Other embodiments Various modifications and variations of the disclosed content described will be apparent to those skilled in the art without departing from the scope and spirit of the disclosure. Although the disclosure has been described in combination with specific embodiments, it should be understood that the disclosure of the claims should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the disclosure that are obvious to those skilled in the art are intended to be within the scope of the disclosure. Other embodiments are within the scope of the claims.

Claims

[Claim 1] The invention described in the specification and drawings.

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