Compositions and methods for improved gene silencing

Abstract

The present disclosure provides single-stranded or double-stranded interfering RNA molecules (e.g., siRNAs) that exhibit improved gene silencing. The present disclosure provides siRNA molecules having a fixed nucleobase region. For example, the antisense strand of the siRNA molecule may have a nucleobase region containing one or more mismatches relative to a target mRNA molecule. Alternatively or additionally, the antisense strand may contain a nucleobase region having a sequence independent of the target mRNA. An immobilization region may be included within a protruding end region of the antisense strand. The siRNA molecule may contain a specific pattern of nucleoside modification and a nucleoside-to-nucleoside linkage modification, as a pharmaceutical composition comprising the siRNA molecule. The siRNA molecule may be a branched siRNA molecule, such as a two-branched, three-branched or four-branched siRNA molecule. The disclosed siRNA molecules may further be characterized by a 5 '-phosphorus stabilizing moiety and / or a hydrophobic moiety. In addition, the present disclosure provides methods for delivering siRNA molecules of the present disclosure to a subject, such as the central nervous system of a subject identified as having a neurodegenerative disease.

Description

Technical Field

[0001] This disclosure relates to small interfering RNA (siRNA) molecules that exhibit improved gene silencing and compositions containing said siRNA molecules. This disclosure further describes methods for silencing target genes and treating diseases that may benefit from gene silencing, said methods being carried out by delivering siRNA molecules to target tissues of a subject in need. Background Technology

[0002] In many species, the introduction of double-stranded RNA (dsRNA) induces effective and specific gene silencing via RNA interference (RNAi). This phenomenon occurs in both plants and animals and plays a role in viral defense and transposon silencing mechanisms. Short interfering RNAs (siRNAs), typically much shorter than their target genes, have shown effectiveness in gene silencing and are therefore potential therapeutic agents for silencing genes, restoring genetic and biochemical pathway activity from disease states to normal, healthy states. However, there remains a need for siRNA constructs that exhibit improved gene silencing. Summary of the Invention

[0003] This disclosure provides compositions and methods for improving gene silencing. Therefore, this disclosure provides siRNA molecules having a fixed nucleotide region. For example, the antisense strand of the siRNA molecule may have a nucleotide region containing one or more mismatched nucleotides relative to the target mRNA molecule. Alternatively or additionally, the antisense strand may contain a nucleotide region having a sequence independent of the target mRNA. The fixed region may be contained within the overhanging region of the antisense strand.

[0004] The siRNA molecules disclosed herein can be delivered directly to subjects requiring gene silencing via, for example, intrathecal injection, intrastriatal injection, intraparenchymal injection, cerebellomedullary cistern injection (such as via catheter insertion), intravenous injection, subcutaneous injection, or intramuscular injection.

[0005] In one aspect, this disclosure provides a small interfering RNA (siRNA) molecule comprising an antisense strand and a sense strand complementary to a portion of said antisense strand; wherein: (i) The antisense strand comprises a first region of the linked nucleotide and a second region of the linked nucleotide in the 5' to 3' direction; (ii) The first region has complementarity sufficient to hybridize with a portion of the target mRNA transcript; (iii) The second region includes a protruding end extending beyond the semantic chain; and (iv) The second region has one or more nucleotide mismatches relative to the target mRNA transcript.

[0006] In some embodiments, the second region has 1 to 4 nucleotide mismatches (e.g., 1, 2, 3, or 4) relative to the target mRNA transcript. In some embodiments, the second region has 1 nucleotide mismatch relative to the target mRNA transcript. In some embodiments, the second region has 2 nucleotide mismatches relative to the target mRNA transcript. In some embodiments, the second region has 3 nucleotide mismatches relative to the target mRNA transcript. In some embodiments, the second region has 4 nucleotide mismatches relative to the target mRNA transcript. In some embodiments, the second region has 1 to 3 nucleotide mismatches relative to the target mRNA transcript. In some embodiments, the second region has 2 to 4 nucleotide mismatches relative to the target mRNA transcript. In some embodiments, the second region has 3 or 4 nucleotide mismatches relative to the target mRNA transcript. In some embodiments, the second region has 1 or 2 nucleotide mismatches relative to the target mRNA transcript. In some embodiments, the second region has 2 or 3 nucleotide mismatches relative to the target mRNA transcript.

[0007] In another aspect, this disclosure provides an siRNA molecule containing an antisense strand and a sense strand complementary to a portion of the antisense strand; wherein: (i) The antisense strand comprises a first region of the linked nucleotide and a second region of the linked nucleotide in the 5' to 3' direction; (ii) The first region has complementarity sufficient to hybridize with a portion of the target mRNA transcript; (iii) The second region includes a protruding end extending beyond the semantic chain; and (iv) The second region has a nucleobase sequence that is independent of the nucleobase sequence of the target mRNA transcript.

[0008] In some embodiments of any of the above aspects, the linked nucleotides are continuous nucleotides.

[0009] In some embodiments of the siRNA molecule described herein, the nucleotide sequence of the second region, relative to a corresponding antisense strand that is fully complementary to the target mRNA, confers an increased dissociation rate of the resulting mRNA cleavage product from the RISC complex upon cleavage of the target mRNA. In some embodiments, the nucleotide sequence of the second region confers increased binding affinity of the antisense strand to endogenous Argonaute (AGO) proteins, relative to a corresponding endogenous complex containing a corresponding antisense strand that is fully complementary to the target mRNA. In some embodiments, the nucleotide sequence of the second region improves the half-life of an endogenous complex containing both the antisense strand and the AGO protein, relative to a corresponding endogenous complex containing a corresponding endogenous complex containing a corresponding endogenous complex containing a corresponding endogenous complex containing a corresponding endogenous complex containing an antisense strand that is fully complementary to the target mRNA.

[0010] In some embodiments, the length of the second region is 1 to 10 nucleotides (e.g., 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 10, 3 to 10, 4 to 10, 5 to 10, 6 to 10, 7 to 10, 8 to 10, 9 to 10, 2 to 7, 3 to 7, 4 to 7, 5 to 7, 6 to 7, 2 to 6, 3 to 6, 4 to 6, 5 to 6, 2 to 5, 3 to 5, 4 to 5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some embodiments, the length of the second region is 1 nucleotide. In some embodiments, the length of the second region is 2 nucleotides. In some embodiments, the length of the second region is 3 nucleotides. In some embodiments, the length of the second region is 4 nucleotides. In some embodiments, the second region is 5 nucleotides long. In some embodiments, the second region is 6 nucleotides long. In some embodiments, the second region is 1 to 5 nucleotides long. In some embodiments, the second region is 1 to 4 nucleotides long. In some embodiments, the second region is 1 to 3 nucleotides long. In some embodiments, the second region is 2 to 4 nucleotides long. In some embodiments, the second region is 3 or 4 nucleotides long. In some embodiments, the second region is 1 or 2 nucleotides long. In some embodiments, the second region is 2 or 3 nucleotides long.

[0011] In some embodiments, the second region contains at least one uridine nucleotide. In some embodiments, the second region contains two uridine nucleotides.

[0012] In some embodiments, the second region contains at least one modified nucleoside interlink. In some embodiments of any fixed region described herein, the fixed region contains at least one (e.g., 1, 2, 3, 4, 5 or more) modified nucleoside interlink.

[0013] In some embodiments, at least one modified subunit inter-bonding has the formula E1: (E1); in: Each B is an independent base pairing part; W can be selected from the following groups: O, S, B, BR 2 , N, NR 2 OCH2, OCH, CH2 and CH, where W is selected from the group consisting of OCH2 and OCH; Each X is independently selected from the group consisting of: halogen (e.g., fluorine or chlorine), hydroxyl, and C.1-6 Alkoxy groups, optionally wherein each X is independently selected from the group consisting of: halogen groups (e.g., fluorine or chlorine) and C. 1-6 Alkyl groups (e.g., methoxy, ethoxy, 2-methoxyethoxy, n-propoxy, sec-propoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, or n-heptoxy); Y chooses from the following groups: O - OH, OR, NH - NH2, S - And SH, where Y can be freely selected from the following groups: O - OH and OR.

[0014] Z is selected from the following groups: O, S, BR 2 NR 2 and CH2; R is a protecting group; Each R 2 Independently H or optionally substituted C1-C6 alkyl; and It is an optional double bond.

[0015] In some implementations of E1, W is OCH2.

[0016] In some implementations of E1, W is OCH and It is a double bond.

[0017] In some implementations of E1, Z is O.

[0018] In some implementations of formula E1, Z is CH2.

[0019] In some implementations of E1, when Y is O - When Z or W is not O.

[0020] In some embodiments of formula E1, Z is CH2 and W is CH2. In some embodiments, the modified inter-subunit bonding of formula E1 is the same as the modified inter-subunit bonding of formula E2: (E2).

[0021] In some embodiments of formula E1, Z is CH2 and W is O. In some embodiments, the modified inter-subunit bonding of formula E1 is the modified inter-subunit bonding of formula E3: (E3).

[0022] In some embodiments of formula E1, Z is O and W is CH2. In some embodiments, the modified inter-subunit bonding of formula E1 is the modified inter-subunit bonding of formula E4: (E4).

[0023] In some embodiments of E1, Z is O and W is CH. In some embodiments, the modified inter-subunit bonding of E1 is the modified inter-subunit bonding of E5: (E5).

[0024] In some implementations, the modified inter-subunit bonding of formula E1 is the modified inter-subunit bonding of formula E6: (E6).

[0025] In some implementations of E6: Each B is an independent base pairing part; Each X is independently selected from the group consisting of: halogen, hydroxyl, and C. 1-6 Alkyl group; optionally, each X is independently selected from the group consisting of: halogen (e.g., fluorine) and C. 1-6 Alkyl groups (e.g., methoxy, ethoxy, n-propoxy, sec-propoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, or n-heptoxy); Y chooses from the following groups: O - OH, OR, NH - NH2, S - And SH, where Y can be freely selected from the following groups: O - OH and OR; Z is selected from the group consisting of O and CH2; and It is an optional double bond.

[0026] In some implementations of E6: Each X is independently selected from the group consisting of: fluorine, hydroxyl, and C. 1-6 Alkyl groups; optionally, each X is independently selected from the group consisting of fluorine and C. 1-6 Alkyl groups (e.g., methoxy, ethoxy, n-propoxy, sec-propoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, or n-heptoxy); Y chooses from the following groups: O - OH and OR; Z is selected from the group consisting of O and CH2; and It is an optional double bond.

[0027] In some implementations of E6: Each X is independently selected from the group consisting of: fluorine, hydroxyl, methoxy, ethoxy, n-propoxy, sec-propoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, and n-heptoxy; Y chooses from the following groups: O - OH and OR; Z is selected from the group consisting of O and CH2; and It is an optional double bond.

[0028] In some implementations of E6: Each X is independently selected from the group consisting of: fluorine, hydroxyl, methoxy, ethoxy, n-propoxy, sec-propoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, and n-heptoxy; Y chooses from the following groups: O - OH and OR; Z is O; and It is an optional double bond.

[0029] In some implementations of E6: Each X is independently selected from the group consisting of: fluorine, hydroxyl, methoxy, ethoxy, n-propoxy, sec-propoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, and n-heptoxy; Y chooses from the following groups: O - OH and OR; Z is CH2; and It is an optional double bond.

[0030] In some implementations of E6: Each X is independently selected from the group consisting of: fluorine, hydroxyl, and methoxy; Y chooses from the following groups: O - OH and OR; Z is O; and It is an optional double bond.

[0031] In some implementations of E6: Each X is independently selected from the group consisting of: fluorine, hydroxyl, and methoxy; Y chooses from the following groups: O - OH and OR; Z is CH2; and It is an optional double bond.

[0032] In some embodiments of formula E1, Z is O and W is OCH2. In some embodiments, the modified inter-subunit bonding of formula E1 is the modified inter-subunit bonding of formula E6a: (E6a).

[0033] In some embodiments of formula E1, Z is CH2 and W is CH. In some embodiments, the modified inter-subunit bonding of formula E1 is the modified inter-subunit bonding of formula E7: (E7)

[0034] In some embodiments of Formula E1, the base pairing portion B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0035] In some implementations, at least one modified subunit inter-bonding has the formula E8: (E8); in: D can be selected from the following groups: O, S, B, BR 2 , N, NR 2 OCH2, OCH, CH2 and CH, where D is selected from the group consisting of OCH2 and OCH; C chooses from the following groups: O - OH, OR 1 NH - NH2, S - And SH, where C can be chosen from the following groups: O - OH and OR 1 ; A chooses from the following groups: O, S, BR 2 NR 2 and CH2; R 1 It is a protecting group; Each R 2 Independently H or optionally substituted C1-C6 alkyl; It is an optional double bond; and Two optional modified nucleosides are bridged between subunits.

[0036] In some implementations, D is OCH2.

[0037] In some implementations, D is OCH and It is a double bond.

[0038] In some implementations, A is O.

[0039] In some implementations, A is CH2.

[0040] In some implementations, when C is O - When, A or D is not O.

[0041] In some embodiments, D is CH2. In another embodiment, the modified subunit inter-unit bond of formula E8 is the modified subunit inter-unit bond of formula E9: (E9).

[0042] In some embodiments, D is O. In another embodiment, the modified subunit inter-unit bonding of formula E8 is the modified subunit inter-unit bonding of formula E10: (E10).

[0043] In some embodiments, D is CH2. In another embodiment, the modified inter-subunit bond of formula E8 is the modified inter-subunit bond of formula E11: (E11).

[0044] In some embodiments, D is CH. In another embodiment, the modified subunit inter-unit bond of formula E8 is the modified subunit inter-unit bond of formula E12: .

[0045] (E12).

[0046] In some embodiments, D is OCH2. In another embodiment, the modified subunit inter-unit bond of formula E8 is the modified subunit inter-unit bond of formula E13: (E13).

[0047] In another embodiment, the modified subunit inter-bonding of formula E8 is the modified subunit inter-bonding of formula E14: (E14).

[0048] In another embodiment, the modified inter-subunit bonding of Formula E8 is the modified inter-subunit bonding of Formula E15: (E15).

[0049] In some implementations of modified siRNA-linked nucleotides, each optionally modified nucleotide is independently selected from the group consisting of adenosine, guanosine, cytidine, and uridine each time it appears.

[0050] In some implementations, at least one modified subunit inter-bonding has the formula E8: (E8); in: D can be any of the following groups: O, OCH2, OCH, CH2 and CH, where D can be any of the groups consisting of OCH2 and OCH; C chooses from the following groups: O - OH, OR 1 NH - NH2, S - And SH, where C can be chosen from the following groups: O - OH and OR 1 ; Choose the group consisting of O and CH2; R 1 The protecting group is selected from the group consisting of: dimethoxytriphenylmethyl (DMTr), succinate, tert-butyldimethylsilyl (TBDMS), benzoyl (Bz), benzyl (Bn), methoxyethoxymethyl ether (MOM), methoxybenzyl ether (PMB), methylthiomethyl ether, neopentanoyl (Piv), tetrahydropyranyl (THP), tetrahydrofuranyl (THF), triphenylmethyl (Trt), triisopropylsilyl (TIPS), tert-butyldiphenylsilyl (TBDPS), and acetate; It is an optional double bond; Furthermore, two optionally modified nucleosides are bridged between the subunits.

[0051] In some implementations, D is OCH2.

[0052] In some implementations, D is OCH and It is a double bond.

[0053] In some implementations, A is O.

[0054] In some implementations, A is CH2.

[0055] In some embodiments of any of the modified inter-subunit bonds described above, the base pairing portion B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0056] In some embodiments, R is a protecting group selected from the group consisting of: dimethoxytriphenylmethyl (DMTr), succinate, tert-butyldimethylsilyl (TBDMS), benzoyl (Bz), benzyl (Bn), methoxyethoxymethyl ether (MOM), methoxybenzyl ether (PMB), methylthiomethyl ether, neopentanoyl (Piv), tetrahydropyranyl (THP), tetrahydrofuranyl (THF), triphenylmethyl (Trt), triisopropylsilyl (TIPS), tert-butyldiphenylsilyl (TBDPS), and acetate; and It is an optional double bond.

[0057] In some implementations, the second region contains at least one thiophosphate nucleoside linker.

[0058] In some embodiments, the second region contains at least one nucleotide comprising a modified ribose. In some embodiments, the second region contains at least one 2'-methoxynucleotide. In some embodiments, the second region contains at least one 2'-fluoronucleotide.

[0059] In some implementations, the second region has any one of the following sequences in the 5' to 3' direction: -SASASA (Formula F1) -OASASA (Formula F2) -OAS-XA-S-XB (Formula F3) -SAS-XA-S-XB (Formula F4) -SAS-XA-S-XA (Formula F5) -SASASB (Formula F6) in Each S is an inter-nucleotide bond between thiophosphate esters; Each O represents a phosphodiester nucleoside linkage; Each A is a 2'-methoxyribonucleoside; Each B is a 2'-fluororibonucleoside; Each XA is a 2'-methoxynucleotide of formula E6a; and Each XB is a 2'-fluoronucleotide of formula E6a. In some embodiments, the second region has any one of the following sequences in the 5' to 3' orientation: -S-(mA)-S-(mA)-S-(mG) (Equation F7) -S-(mA)-S-(mU)-S-(mU) (Equation F8) -O-(mA)-S-(mU)-S-(mU) (Equation F9) -O-(mA)-S-(xU)-S-(yU) (Equation F10) -S-(mA)-S-(xU)-S-(yU) (Formula F11) -S-(mA)-S-(xU)-S-(xU) (Equation F12) -S-(mA)-S-(mU)-S-(fU) (Equation F13) in Each S is an inter-nucleotide bond between phosphate thioesters. Each O represents a phosphodiester nucleoside linkage. Each mA is 2'-methoxyadenosine. Each mG is 2'-methoxyguanidine. Each mU is 2'-methoxyuridine. Each xU is a 2'-methoxyuridine of formula E6a, and Each yU is a 2'-fluorouridine of formula E6a.

[0060] In some implementations, the antisense chain has a structure represented by Equation I, where Equation I is as follows in the 5' to 3' direction: AB-(A') j -CP 2 -DP 1 -(C'-P 1 ) k -C' Formula I; Where A is derived from CP 1 -DP 1 express; Each A' is derived from CP. 2 -DP 2 express; B is a type of CP 2 -DP 2 -DP 2 -DP 2 express; Each C is a 2'-O-methyl(2'-O-Me) ribonucleotide; Each C' is independently a 2'-O-Me ribonucleoside or a 2'-fluoro(2'-F) ribonucleoside; Each D is a 2'-F ribonucleotide; Each P 1 It is a phosphate ester nucleoside linkage; Each P 2 It is a phosphodiester nucleoside 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).

[0061] In some implementations, the antisense chain has a structure represented by formula A1, wherein formula A1 is in the 5' to 3' direction as follows: ASBSAOBOBOBOAOBOAOBOA-OBAOBOAOBSASASASBSA Formula A1; Where A represents 2'-O-Me ribonucleoside, B represents 2'-F ribonucleoside, O represents phosphate diester nucleoside linkage, and S represents thiophosphate nucleoside linkage.

[0062] In some implementations, the antisense chain has a structure represented by Formula II, wherein Formula II is in the 5' to 3' direction as follows: AB-(A') j -CP 2 -DP 1 -(CP 1 ) k -C' Formula II; Where A is derived from CP 1 -DP 1 express; Each A' is derived from CP. 2 -DP 2 express; B is a type of CP 2 -DP 2 -DP 2 -DP 2 express; Each C is a 2'-O-methyl(2'-O-Me) ribonucleotide; Each C' is independently a 2'-O-Me ribonucleoside or a 2'-fluoro(2'-F) ribonucleoside; Each D is a 2'-F ribonucleotide; Each P 1 It is a phosphate ester nucleoside linkage; Each P 2 It is a phosphodiester nucleoside 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).

[0063] In some implementations, the antisense chain has a structure represented by formula A2, wherein formula A2 is in the 5' to 3' direction as follows: ASBSAOBOBOBOAOBOAOBOA-OBAOBOAOBSASASASASA Formula A2; Where A represents 2'-O-Me ribonucleoside, B represents 2'-F ribonucleoside, O represents phosphate diester nucleoside linkage, and S represents thiophosphate nucleoside linkage.

[0064] In some implementations, the sense chain has a structure represented by Formula III, wherein Formula III is in the 5' to 3' direction as follows: E-(A') m -F Formula III; Where E is derived from formula (CP) 1 )2 indicates; F is derived from (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 indicates; A', C, D, P 1 and P 2 As defined in Equation II; and m is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7).

[0065] In some implementations, the sense chain has a structure represented by formula S1, wherein formula S1 is in the 5' to 3' direction as follows: ASASAOBOAOBOAOBOAOBOA-OAOAOBSASA Formula S1; Where A represents 2'-O-Me ribonucleoside, B represents 2'-F ribonucleoside, O represents phosphate diester nucleoside linkage, and S represents thiophosphate nucleoside linkage.

[0066] In some implementations, the sense chain has a structure represented by formula S2, wherein formula S2 is in the 5' to 3' direction as follows: ASASAOBOAOBOAOBOAOBOA-OAOAOBOAOA Formula S2; Where A represents 2'-O-Me ribonucleoside, B represents 2'-F ribonucleoside, O represents phosphate diester nucleoside linkage, and S represents thiophosphate nucleoside linkage.

[0067] In some implementations, the sense chain has a structure represented by formula S3, wherein formula S3 is in the 5' to 3' direction as follows: ASASAOBOAOBOAOBOAOBOA-OAOAOBSASB Formula S3; Where A represents 2'-O-Me ribonucleoside, B represents 2'-F ribonucleoside, O represents phosphate diester nucleoside linkage, and S represents thiophosphate nucleoside linkage.

[0068] In some implementations, the sense chain has a structure represented by formula S4, wherein formula S4 is in the 5' to 3' direction as follows: ASASAOBOAOBOAOBOAOBOA-OAOAOBOAOB Formula S4; Where A represents 2'-O-Me ribonucleoside, B represents 2'-F ribonucleoside, O represents phosphate diester nucleoside linkage, and S represents thiophosphate nucleoside linkage.

[0069] In some implementations, the antisense chain has a structure represented by formula IV, wherein formula IV is in the 5' to 3' direction as follows: A-(A') j -CP 2 -B-(CP 1 ) k -C' Formula IV; Where A is derived from CP 1 -DP 1 express; Each A' is derived from CP. 2 -DP 2 express; B is derived from DP 1 -CP1 -DP 1 express; Each C is a 2'-O-Me ribonucleotide; Each C' is independently either a 2'-O-Me ribonucleoside or a 2'-F ribonucleoside; Each D is a 2'-F ribonucleotide; Each P 1 It is a phosphate ester nucleoside linkage; Each P 2 It is a phosphodiester nucleoside 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).

[0070] In some implementations, the antisense chain has a structure represented by formula A3, wherein formula A3 is in the 5' to 3' direction as follows: ASBSAOBOAOBOAOBOAOBOA-OBAOBOAOBSASBSASASA Formula A3; Where A represents 2'-O-Me ribonucleoside, B represents 2'-F ribonucleoside, O represents phosphate diester nucleoside linkage, and S represents thiophosphate nucleoside linkage.

[0071] In some implementations, the sense chain has a structure represented by formula V, wherein formula V is in the 5' to 3' direction as follows: E-(A') m -CP 2 -F Formula V; Where E is derived from formula (CP) 1 )2 indicates; F is derived from DP 1 -CP 1 -C, DP 2 -CP 2 -C, DP 1 -CP 1 -D or DP 2 -CP 2 -D indicates; A', C, D, P 1 and P 2 As defined in Equation IV; and m is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7).

[0072] In some implementations, the sense chain has a structure represented by formula S5, wherein formula S5 is in the 5' to 3' direction: ASASAOBOAOBOAOBOAOBOA-OBOAOBSASA Formula S5; Where A represents 2'-O-Me ribonucleoside, B represents 2'-F ribonucleoside, O represents phosphate diester nucleoside linkage, and S represents thiophosphate nucleoside linkage.

[0073] In some implementations, the sense chain has a structure represented by formula S6, wherein formula S6 is in the 5' to 3' direction as follows: ASASAOBOAOBOAOBOAOBOA-OBOAOBOAOA Formula S6; Where A represents 2'-O-Me ribonucleoside, B represents 2'-F ribonucleoside, O represents phosphate diester nucleoside linkage, and S represents thiophosphate nucleoside linkage.

[0074] In some implementations, the sense chain has a structure represented by formula S7, wherein formula S7 is in the 5' to 3' direction as follows: ASASAOBOAOBOAOBOAOBOA-OBOAOBSASB Formula S7; Where A represents 2'-O-Me ribonucleoside, B represents 2'-F ribonucleoside, O represents phosphate diester nucleoside linkage, and S represents thiophosphate nucleoside linkage.

[0075] In some implementations, the sense chain has a structure represented by formula S8, wherein formula S8 is in the 5' to 3' direction as follows: ASASAOBOAOBOAOBOAOBOA-OBOAOBOAOB Formula S8; Where A represents 2'-O-Me ribonucleoside, B represents 2'-F ribonucleoside, O represents phosphate diester nucleoside linkage, and S represents thiophosphate nucleoside linkage.

[0076] In some implementations, the antisense chain has a structure represented by formula VI, wherein formula VI is in the 5' to 3' direction as follows: AB j -EB k -EFGl -DP 1 -C' Formula VI; Where A is derived from CP 1 -DP 1 express; Each B is derived from CP. 2 express; Each C is a 2'-O-Me ribonucleotide; Each C' is independently either a 2'-O-Me ribonucleoside or a 2'-F ribonucleoside; Each D is a 2'-F ribonucleotide; Each E is derived from DP. 2 -CP 2 express; F is derived from DP 1 -CP 1 express; Each G is a CP 1 express; Each P 1 It is a phosphate ester nucleoside linkage; Each P 2 It is a phosphodiester nucleoside 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).

[0077] In some implementations, the antisense chain has a structure represented by formula A4, wherein formula A4 is in the 5' to 3' direction as follows: ASBSAOAOAOBOAOAOAOAOA-OAOAOBOAOBSASASASBSA Formula A4; Where A represents 2'-O-Me ribonucleoside, B represents 2'-F ribonucleoside, O represents phosphate diester nucleoside linkage, and S represents thiophosphate nucleoside linkage.

[0078] In some implementations, the sense chain has a structure represented by Equation VII, wherein Equation VII is in the 5' to 3' direction: HB m -I n -A'-B o -HC Equation VII; Where A' is derived from CP2 -DP 2 express; Each H is derived from (CP) 1 )2 indicates; Each I is derived from equation (DP) 2 )express; B, C, D, P 1 and P 2 As defined in Formula VI; m is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7); n is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7); and o is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7).

[0079] In some implementations, the sense chain has a structure represented by formula S9, wherein formula S9 is in the 5' to 3' direction as follows: ASASAOAOAOBOBOBOAOBOA-OAOAOASASA Formula S9; Where A represents 2'-O-Me ribonucleoside, B represents 2'-F ribonucleoside, O represents phosphate diester nucleoside linkage, and S represents thiophosphate nucleoside linkage.

[0080] In some implementations, the antisense chain also has a 5' phosphorus-stabilizing portion at the 5' end of the antisense chain.

[0081] In some implementations, the sense chain also has a 5' phosphorus-stabilizing portion at the 5' end of the sense chain.

[0082] In some implementations, each 5'-phosphorus stabilizing moiety is independently represented by any one of formulas IX, XX, XI, XII, XIII, XIV, XV, or XVI: Wherein Nuc represents a nucleobase, optionally said nucleobase being selected from the group consisting of adenine, uracil, guanine, thymine, and cytosine, and R represents an optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, phenyl, benzyl, cation (e.g., monovalent cation), or hydrogen.

[0083] In some implementations, the nucleobases are adenine, uracil, guanine, thymine, or cytosine.

[0084] In some embodiments, the 5' phosphorus-stabilizing portion is (E)-vinylphosphonate represented by formula XI.

[0085] In some implementations, the siRNA molecule also has a hydrophobic portion at the 5' or 3' end of the siRNA molecule.

[0086] In some implementations, the hydrophobic portion is selected from the group consisting of cholesterol, vitamin D, and tocopherol.

[0087] In some implementations, the siRNA molecule is a branched siRNA molecule.

[0088] In some implementations, the branched siRNA molecule is dibranched, tribranched, or quadruple-branched.

[0089] In some embodiments, the siRNA molecule is bibranched, optionally wherein the bibranched siRNA molecule is represented by any one of formula XVII, XVIII, or XIX: Each RNA is an independent siRNA molecule, L is a linker, and each X independently represents a branch point portion.

[0090] In some embodiments, the bibranched siRNA molecule is represented by formula XVII. In some embodiments, the bibranched siRNA molecule is represented by formula XVIII. In some embodiments, the bibranched siRNA molecule is represented by formula XIX.

[0091] In some embodiments, the siRNA molecule is tribranched, optionally wherein the tribranched siRNA molecule is represented by any one of formulas XX, XXI, XXII, or XXIII: Each RNA is an independent siRNA molecule, L is a linker, and each X independently represents a branch point portion.

[0092] In some embodiments, the three-branched siRNA molecule is represented by formula XX. In some embodiments, the three-branched siRNA molecule is represented by formula XXI. In some embodiments, the three-branched siRNA molecule is represented by formula XXII. In some embodiments, the three-branched siRNA molecule is represented by formula XXIII.

[0093] In some embodiments, the siRNA molecule is four-branched, optionally wherein the four-branched siRNA molecule is represented by any one of formulas XXIV, XXV, XXVI, XXVII, or XXVIII: Each RNA is an independent siRNA molecule, L is a linker, and each X independently represents a branch point portion.

[0094] In some embodiments, a four-branched siRNA molecule is represented by formula XXIV. In some embodiments, a four-branched siRNA molecule is represented by formula XXV. In some embodiments, a four-branched siRNA molecule is represented by formula XXVI. In some embodiments, a four-branched siRNA molecule is represented by formula XXVII. In some embodiments, a four-branched siRNA molecule is represented by formula XXVIII.

[0095] In some implementations of branched siRNA, the adapter is selected from the group consisting of: ethylene glycol (e.g., polyethylene glycol (PEG), such as triethylene glycol (TrEG) or tetraethylene glycol (TEG)), alkyl groups, carbohydrates, block copolymers, peptides, one or more consecutive subunits of RNA and DNA.

[0096] In some embodiments, the linker is an ethylene glycol oligomer. In some embodiments, the linker is an alkyl oligomer. In some embodiments, the linker is a carbohydrate oligomer. In some embodiments, the linker is a block copolymer. In some embodiments, the linker is a peptide oligomer. In some embodiments, the linker is an RNA oligomer. In some embodiments, the linker is a DNA oligomer.

[0097] In some embodiments, the ethylene glycol oligomer is PEG. In some embodiments, PEG is TrEG. In some embodiments, PEG is TEG.

[0098] In some embodiments, the oligomer or copolymer contains 2 to 20 consecutive subunits (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 consecutive subunits).

[0099] In some implementations, the adapter attaches one or more (e.g., 1, 2, 3, 4 or more) siRNA molecules by forming a covalent bond.

[0100] In some embodiments, the covalent bond forming part is selected from the group consisting of: alkyl, ester, amide, carbamate, phosphonate, phosphate, thiophosphate, aminophosphate, triazole, urea, and methyl acetal.

[0101] In some implementations, the connector includes a structure of type L1: .

[0102] (Equation L1) In some implementations, the connector includes a structure of type L2: .

[0103] (Equation L2) In some implementations, the connector includes a structure of type L3: .

[0104] (Formula L3) In some implementations, the connector includes a structure of type L4: .

[0105] (Equation L4) In some implementations, the connector includes a structure of type L5: .

[0106] (Formula L5) In some implementations, the connector includes a structure of type L6: .

[0107] (Formula L6) In some implementations, the connector includes a structure of type L7: .

[0108] (Formula L7) In some implementations, the connector includes a structure of type L8: .

[0109] (Formula L8) In some implementations, the connector includes a structure of type L9: .

[0110] (Formula L9) In some embodiments of any siRNA molecule described herein, 50% or more of the ribonucleotides in the antisense strand are 2′-O-Me ribonucleotides (e.g., 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the ribonucleotides in the antisense strand may be 2′-O-Me ribonucleotides).

[0111] In some implementations, 60% or more of the ribonucleotides in the antisense strand are 2′-O-Me ribonucleotides (e.g., 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the ribonucleotides in the antisense strand may be 2′-O-Me ribonucleotides).

[0112] In some implementations, 70% or more of the ribonucleotides in the antisense strand are 2′-O-Me ribonucleotides (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the ribonucleotides in the antisense strand may be 2′-O-Me ribonucleotides).

[0113] In some implementations, 80% or more of the ribonucleotides in the antisense strand are 2′-O-Me ribonucleotides (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the ribonucleotides in the antisense strand may be 2′-O-Me ribonucleotides).

[0114] In some implementations, 90% or more of the ribonucleotides in the antisense strand are 2′-O-Me ribonucleotides (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the ribonucleotides in the antisense strand may be 2′-O-Me ribonucleotides).

[0115] In some embodiments, 10% or less of the inter-nucleoside bonds are phosphodiester bonds or thiophosphate bonds. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the inter-nucleoside bonds are phosphodiester bonds or thiophosphate bonds. In some embodiments, 100% of the inter-nucleoside bonds are phosphodiester bonds or thiophosphate bonds.

[0116] In some implementations, the nine nucleoside linkages are phosphodiester linkages or thiophosphate linkages.

[0117] In some embodiments, the antisense strand is 10 to 30 nucleotides long (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides), 15 to 25 nucleotides long (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides), or 18 to 23 nucleotides long (e.g., 18, 19, 20, 21, 22, or 23 nucleotides). In some embodiments, the antisense strand is 20 nucleotides long. In some embodiments, the antisense strand is 21 nucleotides long. In some embodiments, the antisense strand is 22 nucleotides long. In some embodiments, the antisense strand is 23 nucleotides long. In some embodiments, the antisense strand is 24 nucleotides long. In some embodiments, the antisense strand is 25 nucleotides long. In some embodiments, the antisense strand is 26 nucleotides long. In some embodiments, the antisense strand is 27 nucleotides long. In some embodiments, the antisense strand is 28 nucleotides long. In some embodiments, the antisense strand is 29 nucleotides long. In some embodiments, the antisense strand is 30 nucleotides long.

[0118] In some embodiments, the siRNA molecules of the branched compound are linked to each other via a linker (e.g., an ethylene glycol oligomer, such as tetraethylene glycol). In some embodiments, the siRNA molecules of the branched compound are linked to each other via a linker between the sense strand of one siRNA molecule and the sense strand of another siRNA molecule. In some embodiments, the siRNA molecules are linked to each other via a linker between the antisense strand of one siRNA molecule and the antisense strand of another siRNA molecule. In some embodiments, the siRNA molecules of the branched compound are linked to each other via a linker between the sense strand of one siRNA molecule and the antisense strand of another siRNA molecule.

[0119] In some embodiments, the sense strand is 12 to 30 nucleotides long (e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) or 14 to 18 nucleotides long (e.g., 14, 15, 16, 17, or 18 nucleotides). In some embodiments, the sense strand is 15 nucleotides long. In some embodiments, the sense strand is 16 nucleotides long. In some embodiments, the sense strand is 17 nucleotides long. In some embodiments, the sense strand is 18 nucleotides long. In some embodiments, the sense strand is 19 nucleotides long. In some embodiments, the sense strand is 20 nucleotides long. In some embodiments, the sense strand is 21 nucleotides long. In some embodiments, the sense strand is 22 nucleotides long. In some embodiments, the sense strand is 23 nucleotides long. In some embodiments, the sense strand is 24 nucleotides long. In some embodiments, the sense strand is 25 nucleotides long. In some embodiments, the sense strand is 26 nucleotides long. In some embodiments, the sense strand is 27 nucleotides long. In some embodiments, the sense strand is 28 nucleotides long. In some embodiments, the sense strand is 29 nucleotides long. In some embodiments, the sense strand is 30 nucleotides long.

[0120] In some implementations, the four nucleosides are linked by phosphate thioesters.

[0121] In some embodiments of the siRNA molecule described herein, the antisense strand is 18 nucleotides long and the sense strand is 14 nucleotides long. In some embodiments, the antisense strand is 18 nucleotides long and the sense strand is 15 nucleotides long. In some embodiments, the antisense strand is 18 nucleotides long and the sense strand is 16 nucleotides long. In some embodiments, the antisense strand is 18 nucleotides long and the sense strand is 17 nucleotides long. In some embodiments, the antisense strand is 18 nucleotides long and the sense strand is 18 nucleotides long. In some embodiments, the antisense strand is 19 nucleotides long and the sense strand is 14 nucleotides long. In some embodiments, the antisense strand is 19 nucleotides long and the sense strand is 15 nucleotides long. In some embodiments, the antisense strand is 19 nucleotides long and the sense strand is 16 nucleotides long. In some embodiments, the antisense strand is 19 nucleotides long and the sense strand is 17 nucleotides long. In some embodiments, the antisense strand is 19 nucleotides long and the sense strand is 18 nucleotides long. In some embodiments, the antisense strand is 19 nucleotides long and the sense strand is 19 nucleotides long. In some embodiments, the antisense strand is 20 nucleotides long and the sense strand is 14 nucleotides long. In some embodiments, the antisense strand is 20 nucleotides long and the sense strand is 15 nucleotides long. In some embodiments, the antisense strand is 20 nucleotides long and the sense strand is 16 nucleotides long. In some embodiments, the antisense strand is 20 nucleotides long and the sense strand is 17 nucleotides long. In some embodiments, the antisense strand is 20 nucleotides long and the sense strand is 18 nucleotides long. In some embodiments, the antisense strand is 20 nucleotides long and the sense strand is 19 nucleotides long. In some embodiments, the antisense strand is 20 nucleotides long and the sense strand is 20 nucleotides long. In some embodiments, the antisense strand is 21 nucleotides long and the sense strand is 14 nucleotides long. In some embodiments, the antisense strand is 21 nucleotides long and the sense strand is 15 nucleotides long. In some embodiments, the antisense strand is 21 nucleotides long and the sense strand is 16 nucleotides long. In some embodiments, the antisense strand is 21 nucleotides long and the sense strand is 17 nucleotides long. In some embodiments, the antisense strand is 21 nucleotides long and the sense strand is 18 nucleotides long. In some embodiments, the antisense strand is 21 nucleotides long and the sense strand is 19 nucleotides long. In some embodiments, the antisense strand is 21 nucleotides long and the sense strand is 20 nucleotides long. In some embodiments, the antisense strand is 21 nucleotides long and the sense strand is 21 nucleotides long.In some embodiments, the antisense strand is 22 nucleotides long and the sense strand is 14 nucleotides long. In some embodiments, the antisense strand is 22 nucleotides long and the sense strand is 15 nucleotides long. In some embodiments, the antisense strand is 22 nucleotides long and the sense strand is 16 nucleotides long. In some embodiments, the antisense strand is 22 nucleotides long and the sense strand is 17 nucleotides long. In some embodiments, the antisense strand is 22 nucleotides long and the sense strand is 18 nucleotides long. In some embodiments, the antisense strand is 22 nucleotides long and the sense strand is 19 nucleotides long. In some embodiments, the antisense strand is 22 nucleotides long and the sense strand is 20 nucleotides long. In some embodiments, the antisense strand is 22 nucleotides long and the sense strand is 21 nucleotides long. In some embodiments, the antisense strand is 22 nucleotides long and the sense strand is 22 nucleotides long. In some embodiments, the antisense strand is 23 nucleotides long and the sense strand is 14 nucleotides long. In some embodiments, the antisense strand is 23 nucleotides long and the sense strand is 15 nucleotides long. In some embodiments, the antisense strand is 23 nucleotides long and the sense strand is 16 nucleotides long. In some embodiments, the antisense strand is 23 nucleotides long and the sense strand is 17 nucleotides long. In some embodiments, the antisense strand is 23 nucleotides long and the sense strand is 18 nucleotides long. In some embodiments, the antisense strand is 23 nucleotides long and the sense strand is 19 nucleotides long. In some embodiments, the antisense strand is 23 nucleotides long and the sense strand is 20 nucleotides long. In some embodiments, the antisense strand is 23 nucleotides long and the sense strand is 21 nucleotides long. In some embodiments, the antisense strand is 23 nucleotides long and the sense strand is 22 nucleotides long. In some embodiments, the antisense strand is 23 nucleotides long and the sense strand is 23 nucleotides long. In some embodiments, the antisense strand is 24 nucleotides long and the sense strand is 14 nucleotides long. In some embodiments, the antisense strand is 24 nucleotides long and the sense strand is 15 nucleotides long. In some embodiments, the antisense strand is 24 nucleotides long and the sense strand is 16 nucleotides long. In some embodiments, the antisense strand is 24 nucleotides long and the sense strand is 17 nucleotides long. In some embodiments, the antisense strand is 24 nucleotides long and the sense strand is 18 nucleotides long. In some embodiments, the antisense strand is 24 nucleotides long and the sense strand is 19 nucleotides long. In some embodiments, the antisense strand is 24 nucleotides long and the sense strand is 20 nucleotides long.In some embodiments, the antisense strand is 24 nucleotides long and the sense strand is 21 nucleotides long. In some embodiments, the antisense strand is 24 nucleotides long and the sense strand is 22 nucleotides long. In some embodiments, the antisense strand is 24 nucleotides long and the sense strand is 23 nucleotides long. In some embodiments, the antisense strand is 24 nucleotides long and the sense strand is 24 nucleotides long. In some embodiments, the antisense strand is 25 nucleotides long and the sense strand is 14 nucleotides long. In some embodiments, the antisense strand is 25 nucleotides long and the sense strand is 15 nucleotides long. In some embodiments, the antisense strand is 25 nucleotides long and the sense strand is 16 nucleotides long. In some embodiments, the antisense strand is 25 nucleotides long and the sense strand is 17 nucleotides long. In some embodiments, the antisense strand is 25 nucleotides long and the sense strand is 18 nucleotides long. In some embodiments, the antisense strand is 25 nucleotides long and the sense strand is 19 nucleotides long. In some embodiments, the antisense strand is 25 nucleotides long and the sense strand is 20 nucleotides long. In some embodiments, the antisense strand is 25 nucleotides long and the sense strand is 21 nucleotides long. In some embodiments, the antisense strand is 25 nucleotides long and the sense strand is 22 nucleotides long. In some embodiments, the antisense strand is 25 nucleotides long and the sense strand is 23 nucleotides long. In some embodiments, the antisense strand is 25 nucleotides long and the sense strand is 24 nucleotides long. In some embodiments, the antisense strand is 25 nucleotides long and the sense strand is 25 nucleotides long. In some embodiments, the antisense strand is 26 nucleotides long and the sense strand is 14 nucleotides long. In some embodiments, the antisense strand is 26 nucleotides long and the sense strand is 15 nucleotides long. In some embodiments, the antisense strand is 26 nucleotides long and the sense strand is 16 nucleotides long. In some embodiments, the antisense strand is 26 nucleotides long and the sense strand is 17 nucleotides long. In some embodiments, the antisense strand is 26 nucleotides long and the sense strand is 18 nucleotides long. In some embodiments, the antisense strand is 26 nucleotides long and the sense strand is 19 nucleotides long. In some embodiments, the antisense strand is 26 nucleotides long and the sense strand is 20 nucleotides long. In some embodiments, the antisense strand is 26 nucleotides long and the sense strand is 21 nucleotides long. In some embodiments, the antisense strand is 26 nucleotides long and the sense strand is 22 nucleotides long. In some embodiments, the antisense strand is 26 nucleotides long and the sense strand is 23 nucleotides long.In some embodiments, the antisense strand is 26 nucleotides long and the sense strand is 24 nucleotides long. In some embodiments, the antisense strand is 26 nucleotides long and the sense strand is 25 nucleotides long. In some embodiments, the antisense strand is 26 nucleotides long and the sense strand is 26 nucleotides long. In some embodiments, the antisense strand is 27 nucleotides long and the sense strand is 14 nucleotides long. In some embodiments, the antisense strand is 27 nucleotides long and the sense strand is 15 nucleotides long. In some embodiments, the antisense strand is 27 nucleotides long and the sense strand is 16 nucleotides long. In some embodiments, the antisense strand is 27 nucleotides long and the sense strand is 17 nucleotides long. In some embodiments, the antisense strand is 27 nucleotides long and the sense strand is 18 nucleotides long. In some embodiments, the antisense strand is 27 nucleotides long and the sense strand is 19 nucleotides long. In some embodiments, the antisense strand is 27 nucleotides long and the sense strand is 20 nucleotides long. In some embodiments, the antisense strand is 27 nucleotides long and the sense strand is 21 nucleotides long. In some embodiments, the antisense strand is 27 nucleotides long and the sense strand is 22 nucleotides long. In some embodiments, the antisense strand is 27 nucleotides long and the sense strand is 23 nucleotides long. In some embodiments, the antisense strand is 27 nucleotides long and the sense strand is 24 nucleotides long. In some embodiments, the antisense strand is 27 nucleotides long and the sense strand is 25 nucleotides long. In some embodiments, the antisense strand is 27 nucleotides long and the sense strand is 26 nucleotides long. In some embodiments, the antisense strand is 27 nucleotides long and the sense strand is 27 nucleotides long. In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 14 nucleotides long. In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 15 nucleotides long. In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 16 nucleotides long. In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 17 nucleotides long. In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 18 nucleotides long. In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 19 nucleotides long. In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 20 nucleotides long. In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 21 nucleotides long. In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 22 nucleotides long.In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 23 nucleotides long. In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 24 nucleotides long. In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 25 nucleotides long. In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 26 nucleotides long. In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 27 nucleotides long. In some embodiments, the antisense strand is 28 nucleotides long and the sense strand is 28 nucleotides long. In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 14 nucleotides long. In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 15 nucleotides long. In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 16 nucleotides long. In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 17 nucleotides long. In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 18 nucleotides long. In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 19 nucleotides long. In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 20 nucleotides long. In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 21 nucleotides long. In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 22 nucleotides long. In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 23 nucleotides long. In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 24 nucleotides long. In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 25 nucleotides long. In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 26 nucleotides long. In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 27 nucleotides long. In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 28 nucleotides long. In some embodiments, the antisense strand is 29 nucleotides long and the sense strand is 29 nucleotides long. In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 14 nucleotides long. In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 15 nucleotides long. In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 16 nucleotides long. In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 17 nucleotides long.In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 18 nucleotides long. In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 19 nucleotides long. In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 20 nucleotides long. In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 21 nucleotides long. In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 22 nucleotides long. In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 23 nucleotides long. In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 24 nucleotides long. In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 25 nucleotides long. In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 26 nucleotides long. In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 27 nucleotides long. In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 28 nucleotides long. In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 29 nucleotides long. In some embodiments, the antisense strand is 30 nucleotides long and the sense strand is 30 nucleotides long.

[0122] In another aspect, this disclosure provides a pharmaceutical composition comprising an siRNA molecule of any of the foregoing aspects or embodiments of this disclosure and a pharmaceutically acceptable excipient, carrier, or diluent.

[0123] In another aspect, this disclosure provides a method for delivering siRNA molecules to the CNS of a subject by administering a combination of multimeric oligonucleotides, siRNA molecules, or pharmaceutical compositions of any of the foregoing aspects or embodiments of this disclosure to the subject's central nervous system (CNS).

[0124] In some implementations, a combination of multimeric oligonucleotides, siRNA molecules, or pharmaceutical compositions is administered to the subject via intrastriatal, intraventricular, or intrathecal injection.

[0125] In some implementations, delivery of siRNA molecules or pharmaceutical compositions to a subject's CNS results in gene silencing of a target gene in the subject.

[0126] In some embodiments, the target gene is an overactive disease driver. In some embodiments, the target gene is a negative regulator of a gene whose expression is reduced in relation to the subject's disease state. In some embodiments, the target gene is a positive regulator of a gene whose expression is increased in relation to the subject's disease state. In some embodiments, the target gene is a splice isoform of the target gene, wherein the splice isoform reduces the expression of the target gene.

[0127] In some implementation schemes, the disease status of the subjects undergoing gene silencing therapy, In some embodiments of the methods described herein, the siRNA molecule or pharmaceutical composition is administered to the subject via intraventricular, striatal, intraparenchymal, or intrathecal injection. In some embodiments, the siRNA molecule or pharmaceutical composition is administered to the subject via intravenous, intramuscular, or subcutaneous injection.

[0128] In some implementations of any of the methods described herein, the subject is a human being.

[0129] In another aspect, this disclosure provides a pillbox containing an siRNA molecule or pharmaceutical composition of any of the foregoing aspects or embodiments of this disclosure, and a package insert instructing the user of the pillbox to perform any of the methods described in the foregoing aspects or embodiments of this disclosure.

[0130] definition Unless otherwise defined herein, the scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. In the event of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. Unless the context otherwise requires, singular terms shall include plural and plural terms shall include singular. Unless otherwise stated, the use of “or” means “and / or”. The use of the term “including” and other forms such as “includes” and “included” is not restrictive.

[0131] As used in this article, the term "nucleic acid" refers to RNA or DNA molecules composed of chains of ribonucleotides or deoxyribonucleotides, respectively.

[0132] As used herein, the term "therapeutic nucleic acid" refers to a nucleic acid molecule (e.g., ribonucleic acid) that is partially or completely complementary to disease-associated target mRNAs and interacts with them to mediate mRNA expression silencing.

[0133] As used herein, the term "carrier nucleic acid" refers to a nucleic acid molecule (e.g., ribonucleic acid) that is sequence complementary to and hybridizes with a therapeutic nucleic acid. As used herein, the term "3' end" refers to the end of a nucleic acid containing an unmodified hydroxyl group at the 3' carbon of the ribose ring.

[0134] As used in this article, the term "nucleoside" refers to a molecule composed of heterocyclic bases and their sugars.

[0135] As used herein, the term "nucleotide" refers to a nucleoside having a phosphate group or a variant thereof on its 3' or 5' sugar hydroxyl group. Examples of phosphate group variants include, but are not limited to, saturated alkyl phosphonates, unsaturated alkenyl phosphonates, thiophosphates, and phosphorous amides.

[0136] When used in the context of a region of an siRNA molecule, a region of nucleosides, a nucleobase sequence, a region of nucleotides, or a nucleotide sequence, the term "fixed" refers to a nucleotide sequence contained within the siRNA molecule (e.g., in the antisense strand of the siRNA molecule) that is independent of the sequence of the target mRNA transcript. The fixed region can have any suitable length while still maintaining the silencing effect of the siRNA molecule. Such fixed sequences may contain one or more mismatches relative to the target mRNA transcript, but may also contain one or more sequence-matching nucleotides. That is, the fixed region has a defined nucleotide sequence that can be inserted into any siRNA molecule, regardless of the sequence of the target gene. The fixed region can be a region of continuous or linked nucleotides, but it does not have to be. For example, a fixed region can be a region with 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) consecutive nucleotides independent of the sequence of the target mRNA transcript. Alternatively, there may be sequence-matching nucleotides inserted with nucleotides independent of the target mRNA transcript (i.e., nucleotides complementary to the target mRNA transcript). For example, there may be one or more mismatched nucleotides, followed by one or more sequence-matching nucleotides, followed by one or more mismatched nucleotides, or any other arrangement of matching and mismatched nucleotides.

[0137] As used in this article, the term "series" refers to nucleotides linked together by direct covalent bonds.

[0138] As used herein, the term “linked” refers to a continuous nucleotide as defined herein, as well as nucleotides linked by a linker. A linker may be, for example, an alkyl chain (e.g., methylene, ethylene, propylene, or any larger straight or branched alkylene chain), a polyethylene glycol chain (e.g., a TrEG or TEG linker), or any other linker described herein or known in the art.

[0139] "Target mRNA transcript" refers to an mRNA transcript that has complementarity sufficient to hybridize with the antisense strand of the siRNA molecule disclosed herein and thereby undergo gene silencing.

[0140] “RISC” refers to an RNA-induced silencing complex that mediates RNA interference. As used herein, the term may refer to any RISC complex from any organism.

[0141] The term "cleavage product" refers to mRNA that has been degraded after interacting with the RISC complex, after which the RISC complex freely interacts with another mRNA molecule.

[0142] In the context of this disclosure, the term "oligonucleotide" refers to oligomers or polymers of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or their analogues. This term includes oligonucleotides composed of naturally occurring nucleobases, sugars, and covalently linked nucleotides (backbone), as well as oligonucleotides having non-naturally occurring (e.g., modified) portions that serve a similar function. Such modified or substituted oligonucleotides are often superior to their natural forms because they possess desired properties, such as enhanced cellular uptake, enhanced affinity for nucleic acid targets, and increased stability in the presence of nucleases.

[0143] As used herein, the term "siRNA" refers to a small interfering RNA duplex that induces the RNA interference (RNAi) pathway. The length of siRNA molecules can vary (typically between 10 and 30 base pairs) and can have varying degrees of complementarity with their target mRNA. The term "siRNA" includes duplexes with two separate strands, as well as single strands optionally forming hairpin structures that include duplex regions.

[0144] As used in this article, the term "antisense strand" refers to the strand in the siRNA duplex that is complementary to the target gene to a certain extent.

[0145] As used in this article, the term "sense strand" refers to the strand of an siRNA duplex that is complementary to the antisense strand.

[0146] As used herein, the term “protruding end” refers to a single-stranded portion of a nucleic acid molecule located at one or both ends (i.e., at the 5' and / or 3' ends), which is otherwise a double-stranded nucleic acid molecule (e.g., double-stranded siRNA).

[0147] The term "interfering RNA molecule" refers to RNA molecules that inhibit the endogenous function of target RNA transcripts, such as small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), or antisense oligonucleotides (ASO).

[0148] As used herein, the terms “express” and “expression” refer to one or more of the following events: (1) the generation of an RNA template from a DNA sequence (e.g., by transcription); (2) the processing of RNA transcripts (e.g., by splicing, editing, 5' cap formation, and / or 3' end processing); and (3) the translation of RNA into a polypeptide or protein. In the context of a gene encoding a protein product, the terms “gene expression” and the like are used interchangeably with the terms “protein expression” and the like. The expression of a gene or protein of interest in a patient can be revealed, for example, by detecting the following in a sample obtained from the patient: an increase in the number or concentration of mRNA encoding the corresponding protein (e.g., assessed using RNA detection procedures described herein or known in the art, such as quantitative polymerase chain reaction (qPCR) and RNA seq techniques), an increase in the number or concentration of the corresponding protein (e.g., assessed using protein detection methods described herein or known in the art, such as enzyme-linked immunosorbent assay (ELISA), etc.), and / or an increase in the activity of the corresponding protein (e.g., in the case of an enzyme, assessed using enzyme activity assays described herein or known in the art). As used herein, a cell is considered to “express” the gene or protein of interest if one or more of the above events can be detected in the cell or the culture medium in which the cell resides. For example, a cell or cell population is considered to “express” the gene or protein of interest if the following events can be detected: (i) the cell or cell population produces a corresponding RNA transcript, such as an mRNA template (e.g., using the RNA detection procedure described herein); (ii) processing of the RNA transcript (e.g., splicing, editing, 5' cap formation, and / or 3' end processing, such as using the RNA detection procedure described herein); (iii) translation of the RNA template into a protein product (e.g., using the protein detection procedure described herein); and / or (iv) post-translational modifications of the protein product (e.g., using the protein detection procedure described herein).

[0149] As used in this article, the terms “target,” “targeted,” and “targeted” in the context of siRNA design refer to generating an antisense strand so that the antisense strand anneals with a region within the mRNA transcript of interest in a manner that results in a reduction in the translation of mRNA into a protein product.

[0150] As used herein, the terms “chemically modified nucleotide,” “nucleotide analog,” “altered nucleotide,” or “modified nucleotide” refer to non-standard nucleotides, including ribonucleotides or deoxyribonucleotides that are not naturally occurring. Exemplary nucleotide analogs are modified at any position to alter certain chemical properties of the nucleotide but retain the nucleotide analog’s ability to perform its intended function.

[0151] As used herein, the term "metabolic stable" refers to an RNA molecule containing chemically modified ribonucleotides to reduce the metabolic rate of the RNA molecule administered to the subject. Exemplary modifications include 2'-hydroxyl modification to 2'-O-methoxy or 2'-fluorine, and phosphodiester modification to thiophosphate.

[0152] As used herein, the term "thiophosphate" refers to the phosphate ester group of a nucleotide modified by replacing one or more oxygen atoms of the phosphate ester group with sulfur.

[0153] As used in this article, the terms "nucleoside linkage" and "nucleoside bond" refer to the bonds between nucleosides in nucleic acid molecules.

[0154] As used in this article, the term "antagomir" refers to a nucleic acid that can act as an inhibitor of miRNA activity.

[0155] As used herein, the term "gapmer" refers to a chimeric antisense nucleic acid containing a central block of deoxynucleotide monomers long enough to induce RNase H cleavage. The deoxynucleotide block is flanked by ribonucleotide monomers or modified ribonucleotide monomers.

[0156] As used in this article, the term "mixmer" refers to a nucleic acid containing a mixture of locked nucleic acid (LNA) and DNA.

[0157] As used herein, the term "guide RNA" refers to a nucleic acid that is sequence complementary to a specific sequence in the genome, said specific sequence being immediately upstream of or one base pair from a protospacer adjacent motif (PAM) sequence, as used in the CRISPR / Cas9 gene editing system. Alternatively, "guide RNA" may refer to a nucleic acid that is sequence complementary (e.g., antisense) to a specific messenger RNA (mRNA) sequence. In this regard, guide RNA may also be sequence complementary to a "passenger RNA" sequence of the same or shorter length, the "passenger RNA" sequence being identical or substantially identical to the sequence of the mRNA that the guide RNA hybridizes with.

[0158] As used herein, the term "branched siRNA" refers to a compound containing two or more double-stranded siRNA molecules covalently linked to each other. A branched siRNA molecule can be "dibranched," also referred to herein as "di-siRNA," wherein the siRNA molecule comprises, for example, two siRNA molecules covalently linked to each other via a linker. A branched siRNA molecule can be "tribranched," also referred to herein as "tri-siRNA," wherein the siRNA molecule comprises, for example, three siRNA molecules covalently linked to each other via a linker. A branched siRNA molecule can be "tetrabranched," also referred herein as "tetra-siRNA," wherein the siRNA molecule comprises, for example, four siRNA molecules covalently linked to each other via a linker.

[0159] As used herein, the term "branchpoint moiety" refers to a chemical portion of the branched siRNA structure of this disclosure that can be covalently linked to the 5' or 3' end of the antisense or sense strand of an siRNA molecule and can support the attachment of additional single-stranded or double-stranded siRNA molecules. Non-limiting examples of branchpoint moieties suitable for use in conjunction with the disclosed methods and compositions include, for example, phosphoramide, toluenesulfonyl acetone glycerol, 1,3-diaminopropanol, pentaerythritol, and any of the branchpoint moieties described in US 10,478,503.

[0160] As used herein, the term "phosphate moiety" refers to a terminal phosphate group comprising phosphoric acid and modified phosphoric acid. The phosphate moiety may be located at either end, but is preferably located at the 5′-terminal nucleoside. In one aspect, the terminal phosphate is unmodified and has the formula —O—P(═O)(OH)OH. In another aspect, the terminal phosphate is modified such that one or more of the O and OH groups are replaced by H, O, S, N (R'), or an alkyl group, wherein R' is H, an amino protecting group, or an unsubstituted or substituted alkyl group. In some embodiments, the 5' and / or 3' terminal groups may comprise one to three phosphate moieties, each of which is independently unmodified (diaphosphate or triphosphate) or modified.

[0161] As used herein, the term "5' phosphorus-stabilizing moiety" refers to a terminal phosphate group comprising phosphate esters and modified phosphate esters (e.g., thiophosphates, phosphate diesters, phosphonates). The phosphate moiety may be located at either end, but is preferably located at the 5'-terminal nucleoside. In one aspect, the terminal phosphate is unmodified and has the formula –O–P(=O)(OH)OH. In another aspect, the terminal phosphate is modified such that one or more of the O and OH groups are replaced by H, O, S, N (R'), or an alkyl group, wherein R' is H, an amino protecting group, or an unsubstituted or substituted alkyl group. In some embodiments, the 5' and / or 3' terminal groups may comprise one to three phosphate moieties, each of which is independently unmodified (diaphosphate or triphosphate) or modified.

[0162] The phosphate group of a nucleotide can also be modified, for example by replacing one or more oxygen atoms in the phosphate group with sulfur (e.g., thiophosphate), or by making other substitutions that allow the nucleotide to perform its intended function, such as in Eckstein, for example. 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. As described in 11:77-85, 2001; and US 5,684,143. Certain of the above modifications (e.g., phosphate group modifications) preferably reduce the rate of hydrolysis of polynucleotides, such as those comprising the like, in vivo or in vitro.

[0163] 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 this disclosure, Watson-Crick base pairs include adenine-thymine, adenine-uracil, and cytosine-guanine base pairs. In this document, a correct Watson-Crick base pair is referred to as a "match," and each unpaired nucleotide and each mispaired nucleotide is referred to as a "mismatch." Alignments used to determine the percentage of identity of nucleic acid sequences can be performed in various ways that are within the capabilities of those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software.

[0164] The "percentage (%) sequence complementarity" relative to a reference polynucleotide sequence is defined as the percentage of nucleic acids in the candidate sequence that are complementary to those in the reference polynucleotide sequence after sequence alignment and gap introduction, to achieve the maximum percentage of sequence complementarity if desired. A given nucleotide is considered "complementary" to the reference nucleotide described herein if two nucleotides form a canonical Watson-Crick base pair. For the avoidance of doubt, Watson-Crick base pairs in the context of this disclosure include adenine-thymine, adenine-uracil, and cytosine-guanine base pairs. In this document, a correct Watson-Crick base pair is referred to as a "match," and each unpaired nucleotide and each mispaired nucleotide is referred to as a "mismatch." Alignment used to determine the percentage of nucleic acid sequence identity can be performed in various ways within the capabilities of those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximum complementarity across the full length of the compared sequences. For example, the formula for calculating the percentage of sequence complementarity between a given nucleic acid sequence A and a given nucleic acid sequence B (which can also be expressed as a certain percentage of complementarity between a given nucleic acid sequence A and a given nucleic acid sequence B) is as follows: 100 × (fraction X / Y) Where X is the number of complementary base pairs in the alignment of A and B (e.g., performed by computer software such as BLAST), and Y is the total number of nucleic acids in B. It should be understood that when the length of nucleic acid sequence A is not equal to the length of nucleic acid sequence B, the percentage of sequence complementarity between A and B will not be equal to the percentage of sequence complementarity between B and A. As used herein, if the query nucleic acid sequence has 100% sequence complementarity with the reference nucleic acid sequence, then the query nucleic acid sequence is considered to be "perfectly complementary" to the reference nucleic acid sequence.

[0165] The "sequence identity percentage (%)" relative to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to those in the reference polynucleotide or polypeptide sequence after the sequences have been aligned and vacancies have been introduced (if necessary) to achieve the maximum sequence identity percentage. Alignments used to determine the nucleic acid or amino acid sequence identity percentage can be performed in various ways within the capabilities of those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine the parameters applicable to the aligned sequences, including any algorithms required to achieve the maximum alignment across the full length of the compared sequences. For example, the sequence identity percentage value can be generated using the sequence comparison computer program BLAST. For example, the sequence identity percentage for a given pair of nucleic acid or amino acid sequences A and / or for a given nucleic acid or amino acid sequence B (which can also be expressed alternatively as a certain sequence identity percentage possessed by a given pair of nucleic acid or amino acid sequences A and / or for a given nucleic acid or amino acid sequence B) is calculated as follows: 100 × (fraction X / Y) Where X is the number of nucleotides or amino acids that are scored as identical matches in the alignment of A and B by a sequence alignment program (e.g., BLAST), and Y is the total number of nucleic acids in B. It should be understood that if the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, the percentage of sequence identity of A with respect to B will not be equal to the percentage of sequence identity of B with respect to A.

[0166] As used herein, the term "sufficient complementarity for hybridization" refers to a nucleic acid sequence or portion thereof that does not need to be completely complementary to the target region (e.g., 100% complementary), or it refers to a nucleic acid sequence or portion thereof that has one or more nucleotide mismatches relative to the target region but is still capable of hybridizing with the target region under specified conditions. For example, the nucleic acid may be, for example, 95% complementary, 90% complementary, 85% complementary, 80% complementary, 75% complementary, 70% complementary, 65% complementary, 60% complementary, 55% complementary, 50% complementary, or less, but still forms sufficient base pairs with the target to hybridize in its length.

[0167] Nucleic acids undergo "hybridization" or "annealing" when one or more nucleoside residues within a polynucleotide pair with one or more complementary nucleoside bases to form a stable double strand. Base pairing is typically driven by hydrogen bonding events. Hybridization includes Watson-Crick base pairs formed from natural and / or modified nucleobases. Hybridization can also include non-Watson-Crick base pairs, such as unstable base pairs (guanosine-uracil, hypoxanthine-uracil, hypoxanthine-adenine, and hypoxanthine-cytosine), and Hoogsteen base pairs. Nucleic acids do not need to be 100% complementary to hybridize. For example, one nucleic acid may be 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, or less complementary to another nucleic acid, but the two nucleic acids can still form enough base pairs with each other for hybridization.

[0168] A “stable duplex” formed by annealing / hybridizing one nucleic acid with another is a duplex structure that will not denature due to rigorous washing. Exemplary rigorous washing conditions are those known in the art and include a temperature approximately 5°C lower than the melting temperature of a single strand of the duplex and a low monovalent salt concentration, such as a monovalent salt concentration (e.g., NaCl concentration) of less than 0.2 M (e.g., 0.2 M, 0.19 M, 0.18 M, 0.17 M, 0.16 M, 0.15 M, 0.14 M, 0.13 M, 0.12 M, 0.11 M, 0.1 M, 0.09 M, 0.08 M, 0.07 M, 0.06 M, 0.05 M, 0.04 M, 0.03 M, 0.02 M, 0.01 M or less).

[0169] The term "gene silencing" refers to the repression of gene expression, such as the expression of an endogenous gene of a target gene, which can be mediated by influencing the transcriptional process and / or by influencing post-transcriptional mechanisms. In some implementations, gene silencing occurs when RNAi molecules initiate the inhibition or degradation of mRNA transcribed from the target gene in a sequence-specific manner through RNA interference, thereby preventing the translation of the gene product.

[0170] As used herein, the phrase "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 can be caused or exacerbated directly by or through an intermediate gene of an overactive disease driver gene.

[0171] As used herein, the term "negative regulator" refers to a gene that negatively regulates (e.g., reduces or inhibits) the expression and / or activity of another gene or set of genes (e.g., a dysregulated gene or a dysregulated gene pathway).

[0172] As used herein, the term "positive regulator" refers to a gene that positively regulates (e.g., increases or saturates) the expression and / or activity of another gene or set of genes (e.g., a dysregulated gene or a dysregulated gene pathway).

[0173] As used herein, the term "ethylene glycol chain" refers to a carbon chain having the formula ((CH2OH)2).

[0174] As used herein, “alkyl” refers to a saturated hydrocarbon group. Alkyl groups can be acyclic or cyclic and, when unsubstituted, contain only C and H. When naming alkyl residues with a specific number of carbon atoms, it is intended to encompass and describe all geometric isomers having that number of carbon atoms; thus, for example, “butyl” is intended to include n-butyl, sec-butyl, and isobutyl. Examples of alkyl groups include ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, etc. In some embodiments, the alkyl group may be substituted. Suitable substituents that can introduce alkyl groups include, for example, hydroxyl, alkoxy, amino, alkylamino, and halogen groups.

[0175] As used herein, "alkenyl" refers to an acyclic or cyclic unsaturated hydrocarbon group having at least one olefinic unsaturated site (i.e., having at least one C=C portion). When unsubstituted, the alkenyl group contains only C and H. When naming alkenyl residues with a specific number of carbons, it is intended to encompass and describe all geometric isomers having that number of carbons; thus, for example, "butenyl" is intended to include n-butenyl, sec-butenyl, and isobutenyl. Examples of alkenyl groups include –CH=CH2, –CH2-CH=CH2, and –CH2-CH=CH-CH=CH2. In some embodiments, the alkenyl group may be substituted. Suitable substituents that can be introduced into the alkenyl group include, for example, hydroxyl, alkoxy, amino, alkylamino, and halogen groups.

[0176] As used herein, “alkynyl” refers to an acyclic or cyclic unsaturated hydrocarbon group having at least one alkynyl unsaturated site (i.e., having at least one C≡C portion). When unsubstituted, the alkynyl group contains only C and H. When naming alkynyl residues with a specific number of carbons, it is intended to encompass and describe all geometric isomers having that number of carbons; thus, for example, “pentynyl” is intended to include n-pentynyl, sec-pentynyl, isopentenynyl, and tertiary-pentynyl. Examples of alkynyl groups include –C≡CH and –C≡C-CH3. In some embodiments, the alkynyl group may be substituted. Suitable substituents that can be introduced into the alkynyl group include, for example, hydroxyl, alkoxy, amino, alkylamino, and halogen groups.

[0177] As used herein, the term "phenyl" refers to a monocyclic aromatic hydrocarbon from which one hydrogen atom has been removed from the carbon atom of the ring. The phenyl group may be unsubstituted or substituted with one or more suitable substituents, wherein the substituents replace the H atom of the phenyl group.

[0178] As used herein, the term "benzyl" refers to a monovalent group obtained when a hydrogen atom is removed from a methyl group attached to toluene. The benzyl group typically has the formula phenyl-CH2-. The benzyl group can be unsubstituted or substituted with one or more suitable substituents. For example, the substituents may replace the hydrogen atom of the phenyl component and / or the hydrogen atom of the methylene (-CH2-) component.

[0179] As used herein, the term "amide" refers to an alkyl, alkenyl, alkynyl, or aromatic group attached to an amino-carbonyl functional group.

[0180] As used herein, the term “triazole” refers to a heterocyclic compound having the formula (C2H3N3) and having a five-membered ring with two carbons and three nitrogens, the positions of which can be changed to produce a variety of isomers.

[0181] As used in this article, the term "terminal group" refers to a group at the end of a carbon chain or nucleic acid.

[0182] As used in this article, "amino acid" refers to a molecule containing amine and carboxyl functional groups, as well as the side chains characteristic of amino acids.

[0183] In some embodiments, the amino acid is selected from the group consisting of protein amino acids. In some embodiments, the amino acid is an L-amino acid or a D-amino acid. In some embodiments, the amino acid is a synthetic amino acid (e.g., a β-amino acid).

[0184] As used herein, the term "lipophilic amino acid" refers to an amino acid that includes a hydrophobic moiety (e.g., an alkyl chain or an aromatic ring).

[0185] As used herein, the term "delivery target" refers to a body organ or part to which a branched oligonucleotide composition needs to be delivered.

[0186] As used in this article, the term "between X and Y" includes both X and Y values. For example, "between X and Y" refers to the range of values ​​between X and Y values, as well as both X and Y values.

[0187] As used herein, the terms “subject” and “patient” are used interchangeably and refer to an organism, such as a mammal (e.g., a human), which is determined by a qualified professional (e.g., a physician or nursing practitioner) to have or be at risk of having a disease, condition or disorder, with or without having undergone one or more laboratory tests known in the art on one or more samples from the subject.

[0188] As used herein, the term "reference subject" refers to a healthy control subject who is the same as or similar to a subject treated with the compositions of this disclosure, for example, in terms of age, sex, geographic region, and / or educational level. A healthy reference subject is a subject who does not have a disease associated with the expression of an dysregulated gene or a dysregulated gene pathway. Furthermore, a healthy reference subject is a subject who does not have a disease associated with alterations in gene expression and / or activity (e.g., increase or decrease).

[0189] As used herein, the terms “neuritis disorder” and “neuritis syndrome” are used interchangeably to refer to any condition caused by inflammation of the nerves in some way. “Neuritis” refers to a series of immune responses in the central nervous system, such as microglia. Neuroinflammation can be of brain origin or caused by a systemic inflammatory response.

[0190] As used herein, the terms “neurodegenerative disease” and “neurodegenerative disorder” are used interchangeably to refer to any condition caused in some way by the loss or death of function of cells in the central or peripheral nervous system. Exemplary neurodegenerative diseases include Alzheimer’s disease, Huntington’s disease, amyotrophic lateral sclerosis (ALS), frontotemporal dementia, and spinocerebellar ataxia.

[0191] As used herein, the terms “treat,” “treated,” and “treating” refer to therapeutic and prophylactic (or preventative) measures aimed at preventing, improving, or mitigating (alleviating) an undesirable physical ailment, condition, or disease, or achieving a beneficial or desired clinical outcome. Beneficial or desired clinical outcomes include, but are not limited to, a reduction in patient dependence on medication; relief of symptoms; a decrease in the severity of the ailment, condition, or disease; stabilization (i.e., non-deterioration) of the state of the ailment, condition, or disease; a delay in the onset or slowing of the progression of the ailment, condition, or disease; improvement or remission of the ailment, condition, or disease state (whether partial or complete), whether detectable or undetectable; improvement in at least one measurable physical, cognitive, or behavioral parameter, which may not necessarily be identifiable to the patient; or enhancement or improvement of the ailment, condition, or disease. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival compared to expected survival without treatment.

[0192] As used herein, the terms “benefit” and “response” are used interchangeably in the context of a subject receiving a therapy for treating a disease. For example, a clinical benefit in the context of a subject receiving the siRNA molecule or siRNA composition of this disclosure includes, but is not limited to, a reduction in the duration and / or frequency of disease symptoms experienced by the subject, and / or a reduction in disease-related phenotypes, and / or a reduction in splice isotypes of wild-type transcripts, mutant transcripts, variant transcripts, or overexpressed transcripts and / or transcripts of target genes. Attached Figure Description

[0193] Figure 1 The diagram illustrates the in vitro knockdown of PRNPs using the siRNA molecule of this disclosure, which contains a fixed nucleobase sequence at the 3' end of the antisense strand, compared to a fully sequence-matched siRNA molecule. The siRNA molecule has the sequence listed in Table 3 below at the 3' end of the antisense strand.

[0194] Figure 2A , 2B Figures 2 and 2C are diagrams illustrating the ability of the disclosed siRNA molecules, containing a fixed nucleotide sequence at the 3' end of the antisense strand, to silence PRNPs in vivo. The siRNA molecules have the fixed region at the 3' end of the antisense strand listed in Table 4 below. Each siRNA is packaged at 5 nmol (…). Figure 2A ), 1 nmol ( Figure 2B ) and 0.2 nmol ( Figure 2C Dosage application.

[0195] Figure 3A and 3B This diagram illustrates the ability of the disclosed siRNA molecules, containing a fixed nucleotide sequence at the 3' end of the antisense strand, to silence HPRT1 in vivo. The siRNA molecules have the fixed region at the 3' end of the antisense strand as listed in Table 5 below. Each siRNA is packaged at 3 nmol (… Figure 3A ) and 1 nmol ( Figure 3B Dosage application.

[0196] Figures 3C-3E This is a graph illustrating the dose-dependent HPRT1 knockdown performed using the siRNA molecule of this disclosure, which contains a fixed nucleobase sequence at the 3' end of the antisense strand. The siRNA molecule has the fixed region at the 3' end of the antisense strand as listed in Table 5 below. Figure 3C All siRNA molecules are shown in Table 5. Figure 3D Only siRNA molecules 11 and 12 from Table 5 are shown. Figure 3D Only siRNA molecules 11 and 15 from Table 5 are shown.

[0197] Figure 4This displays the IC50 value (pM), mRNA expression % and goodness-of-fit (R) of all 64 possible fixed nucleobase sequences at the three terminal nucleosides of the antisense strand of the siRNA molecule targeting HPRT1. 2 (The image is missing.) Detailed Implementation

[0198] This invention provides short interfering RNA (siRNA) molecules, including single-stranded and double-stranded short interfering RNA (ds-siRNA), and methods for treating patients requiring gene silencing (e.g., patients with dysregulated gene expression, such as those suffering from Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, frontotemporal dementia, Huntington's disease, multiple sclerosis, or progressive supranuclear palsy). The siRNA molecules are capable of mediating RNA interference (RNAi) by degrading mRNA with complementary nucleotide sequences, thereby reducing or completely blocking the translation of target genes.

[0199] The siRNA molecule disclosed herein may contain a fixed sequence at the end of either the sense strand or the antisense strand (e.g., at the 5' end of the sense strand, the 5' end of the antisense strand, the 3' end of the sense strand, or the 3' end of the antisense strand). The fixed sequence may be contained within a protruding portion of either strand (e.g., extending beyond the protruding region of the sense strand beyond the antisense strand or extending beyond the protruding region of the antisense strand beyond the sense strand).

[0200] The siRNA molecules described herein can be chemically modified in various ways. Furthermore, the siRNA molecules described herein may include specific patterns of chemical modification (e.g., 2' ribose modification or internucleotide linkage modification) to improve resistance to nucleases, toxicity profile, and physicochemical properties (e.g., thermal stability).

[0201] The siRNA molecule disclosed herein may be characterized by having an antisense strand with a nucleic acid sequence complementary to a region of the mRNA transcript in the target gene. The complementarity of the antisense strand to the region of the target mRNA transcript may be sufficient to cause the antisense strand to anneal along the full length of the region of the mRNA transcript. For example, the antisense strand may have a nucleic acid sequence that is at least 60% complementary to a region of the target mRNA transcript (e.g., 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary).

[0202] Fixed nucleobase region In some embodiments of the siRNA molecule disclosed herein, the antisense strand and / or sense strand contains a region of nucleotides having a fixed nucleobase sequence. In some embodiments, the region of the fixed nucleotide is contained within the sense strand. In some embodiments, the region of the fixed nucleotide is contained within the antisense strand. In some embodiments, the region of the fixed nucleotide is part of the antisense strand and contains one or more mismatches relative to the target mRNA transcript. In some embodiments, the region of the fixed nucleotide has a sequence independent of the sequence of the target mRNA transcript.

[0203] Unbound by theory, certain fixed regions can confer an increased dissociation rate of the resulting mRNA cleavage products from the RISC complex after target mRNA cleavage. Alternatively, or furthermore, certain fixed regions can confer an increased binding affinity of the antisense strand to endogenous Argonaute (AGO) proteins relative to the corresponding antisense strand that is fully complementary to the target mRNA.

[0204] This disclosure is based, at least in part, on the unexpected discovery that installing a fixed nucleobase region (e.g., at the 3' end of the antisense strand and / or in a region of the antisense strand that protrudes from the sense strand) independent of the target mRNA transcript and / or relative to the mRNA transcript, imparts unexpected benefits to siRNA molecules. The fixed nucleobase region of this disclosure achieves unexpected results in a manner dependent on a variety of factors, including, for example, the precise number of mismatches in the tail and the nature and location of chemical modifications in the tail. It has been unexpectedly found that the length of the fixed nucleobase region is an important factor and that the benefits imparted by installing the fixed nucleobase region can be influenced by changing the length of the fixed nucleobase region. These and other parameters are described in further detail in the following sections.

[0205] In some embodiments, certain fixed nucleobase sequences (e.g., at the 3' end of the antisense strand and / or in a 3' region of the antisense strand that protrudes beyond the sense strand) exhibit reduced neurotoxicity upon administration to the CNS compared to the corresponding siRNA that is fully complementary to the target mRNA transcript. In some embodiments, no acute neurotoxicity was observed after administration of certain fixed nucleobase sequences (e.g., at the 3' end of the antisense strand and / or in a 3' region of the antisense strand that protrudes beyond the sense strand) to the CNS.

[0206] In some embodiments, the fixed nucleotide region has 1 to 4 (e.g., 1, 2, 3, or 4) nucleotide mismatches relative to the target mRNA transcript. In some embodiments, the fixed nucleotide region has 1 nucleotide mismatch relative to the target mRNA transcript. In some embodiments, the fixed nucleotide region has 2 nucleotide mismatches relative to the target mRNA transcript. In some embodiments, the fixed nucleotide region has 3 nucleotide mismatches relative to the target mRNA transcript. In some embodiments, the fixed nucleotide region has 4 nucleotide mismatches relative to the target mRNA transcript. In some embodiments, the fixed nucleotide region has 1 to 3 nucleotide mismatches relative to the target mRNA transcript. In some embodiments, the fixed nucleotide region has 2 to 4 nucleotide mismatches relative to the target mRNA transcript. In some embodiments, the fixed nucleotide region has 3 or 4 nucleotide mismatches relative to the target mRNA transcript. In some embodiments, the fixed nucleotide region has 1 or 2 nucleotide mismatches relative to the target mRNA transcript. In some embodiments, the fixed nucleotide region has 2 or 3 nucleotide mismatches relative to the target mRNA transcript.

[0207] Mismatch at the 3' end of the antisense strand can improve RISC-mediated target silencing because the perfect complementarity between the antisense strand of the RISC load and the target mRNA can destabilize the RISC complex and / or trigger ubiquitin ligase-mediated degradation of the RISC complex.

[0208] In some embodiments, the 3' end of the antisense strand has 1 to 4 (e.g., 1, 2, 3, or 4) nucleotide mismatches relative to the target mRNA transcript. In some embodiments, the 3' end of the antisense strand has 1 nucleotide mismatch relative to the target mRNA transcript. In some embodiments, the 3' end of the antisense strand has 2 nucleotide mismatches relative to the target mRNA transcript. In some embodiments, the 3' end of the antisense strand has 3 nucleotide mismatches relative to the target mRNA transcript. In some embodiments, the 3' end of the antisense strand has 4 nucleotide mismatches relative to the target mRNA transcript. In some embodiments, the 3' end of the antisense strand has 1 to 3 nucleotide mismatches relative to the target mRNA transcript. In some embodiments, the 3' end of the antisense strand has 2 to 4 nucleotide mismatches relative to the target mRNA transcript. In some embodiments, the 3' end of the antisense strand has 3 or 4 nucleotide mismatches relative to the target mRNA transcript. In some embodiments, the 3' end of the antisense strand has 1 or 2 nucleotide mismatches relative to the target mRNA transcript. In some implementations, the 3' end of the antisense strand has a 2 or 3 nucleotide mismatch relative to the target mRNA transcript.

[0209] In some embodiments, the siRNA molecule may contain one or more regions as described above at the 5' end of the sense strand. In some embodiments, the siRNA molecule may contain one or more regions as described above at the 5' end of the antisense strand. In some embodiments, the siRNA molecule may contain one or more regions as described above at the 3' end of the sense strand. In some embodiments, the siRNA molecule may contain one or more regions as described above at the 3' end of the antisense strand. In some embodiments, the fixation region is contained within a region of the sense strand that protrudes from the antisense strand. In some embodiments, the fixation region is contained within a region of the antisense strand that protrudes from the sense strand.

[0210] In some embodiments, the fixed region is 1 nucleotide long. In some embodiments, the fixed region is 2 nucleotides long. In some embodiments, the fixed region is 3 nucleotides long. In some embodiments, the fixed region is 4 nucleotides long. In some embodiments, the fixed region is 5 nucleotides long. In some embodiments, the fixed region is 6 nucleotides long. In some embodiments, the fixed region is 7 nucleotides long. In some embodiments, the fixed region is 8 nucleotides long. In some embodiments, the fixed region is 9 nucleotides long. In some embodiments, the fixed region is 10 nucleotides long.

[0211] The fixed region can be a region of continuous or linked nucleotides, but it does not have to be. For example, the fixed region can be a region of 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) consecutive nucleotides independent of the sequence of the target mRNA transcript. Alternatively, there may be sequence-matching nucleotides inserted with nucleotides independent of the target mRNA transcript (i.e., nucleotides complementary to the target mRNA transcript). For example, there may be one or more mismatched nucleotides, followed by one or more sequence-matching nucleotides, followed by one or more mismatched nucleotides, or any other arrangement of matching and mismatched nucleotides, without affecting the ability of the siRNA molecule to perform gene silencing.

[0212] In some embodiments, the fixed region is 3 nucleotides in length and is located at the 3' end of the antisense strand, protruding from the sense strand. In some embodiments, the fixed region has a nucleotide sequence selected from the sequences in Table 1a below.

[0213] Table 1a. Exemplary fixed nucleobase sequences

[0214] In some embodiments, the fixed region is 4 nucleotides in length and is located at the 3' end of the antisense strand, protruding from the sense strand. In some embodiments, the fixed region has a nucleotide sequence selected from the sequences in Table 1b below.

[0215] Table 1b. Exemplary fixed nucleobase sequences

[0216] In some embodiments, the fixed region described herein may have any combination of modifications (e.g., any of 2' sugar modification, nucleoside inter-linking modification, modified nucleobases and / or modified subunit inter-linking of E1 and its derivatives).

[0217] In some embodiments of any of the fixed regions described herein, the fixed region comprises at least one (e.g., 1, 2, 3, 4, 5 or more) modified nucleoside-to-nucleotide linkages.

[0218] In some embodiments, at least one modified subunit inter-bonding has the formula E1: (E1); in: Each B is an independent base pairing part; W can be selected from the following groups: O, S, B, BR 2 , N, NR 2 OCH2, OCH, CH2 and CH, where W is selected from the group consisting of OCH2 and OCH; Each X is independently selected from the group consisting of: halogen (e.g., fluorine or chlorine), hydroxyl, and C. 1-6 Alkoxy groups, optionally wherein each X is independently selected from the group consisting of: halogen groups (e.g., fluorine or chlorine) and C. 1-6 Alkyl groups (e.g., methoxy, ethoxy, 2-methoxyethoxy, n-propoxy, sec-propoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, or n-heptoxy); Y chooses from the following groups: O - OH, OR, NH - NH2, S - And SH, where Y can be freely selected from the following groups: O - OH and OR.

[0219] Z is selected from the following groups: O, S, BR 2 NR 2 and CH2; R is a protecting group; Each R 2Independently H or optionally substituted C1-C6 alkyl; and It is an optional double bond.

[0220] In some implementations of E1, W is OCH2.

[0221] In some implementations of E1, W is OCH and It is a double bond.

[0222] In some implementations of E1, Z is O.

[0223] In some implementations of formula E1, Z is CH2.

[0224] In some implementations of E1, when Y is O - When Z or W is not O.

[0225] In some embodiments of formula E1, Z is CH2 and W is CH2. In some embodiments, the modified inter-subunit bonding of formula E1 is the same as the modified inter-subunit bonding of formula E2: (E2).

[0226] In some embodiments of formula E1, Z is CH2 and W is O. In some embodiments, the modified inter-subunit bonding of formula E1 is the modified inter-subunit bonding of formula E3: (E3).

[0227] In some embodiments of formula E1, Z is O and W is CH2. In some embodiments, the modified inter-subunit bonding of formula E1 is the modified inter-subunit bonding of formula E4: (E4).

[0228] In some embodiments of E1, Z is O and W is CH. In some embodiments, the modified inter-subunit bonding of E1 is the modified inter-subunit bonding of E5: (E5).

[0229] In some implementations, the modified inter-subunit bonding of formula E1 is the modified inter-subunit bonding of formula E6: (E6).

[0230] In some implementations of E6: Each B is an independent base pairing part; Each X is independently selected from the group consisting of: halogen, hydroxyl, and C. 1-6 Alkyl group; optionally, each X is independently selected from the group consisting of: halogen (e.g., fluorine) and C. 1-6 Alkyl groups (e.g., methoxy, ethoxy, n-propoxy, sec-propoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, or n-heptoxy); Y chooses from the following groups: O - OH, OR, NH - NH2, S - And SH, where Y can be freely selected from the following groups: O - OH and OR; Z is selected from the group consisting of O and CH2; and It is an optional double bond.

[0231] In some implementations of E6: Each X is independently selected from the group consisting of: fluorine, hydroxyl, and C. 1-6 Alkyl groups; optionally, each X is independently selected from the group consisting of fluorine and C. 1-6 Alkyl groups (e.g., methoxy, ethoxy, n-propoxy, sec-propoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, or n-heptoxy); Y chooses from the following groups: O - OH and OR; Z is selected from the group consisting of O and CH2; and It is an optional double bond.

[0232] In some implementations of E6: Each X is independently selected from the group consisting of: fluorine, hydroxyl, methoxy, ethoxy, n-propoxy, sec-propoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, and n-heptoxy; Y chooses from the following groups: O - OH and OR; Z is selected from the group consisting of O and CH2; and It is an optional double bond.

[0233] In some implementations of E6: Each X is independently selected from the group consisting of: fluorine, hydroxyl, methoxy, ethoxy, n-propoxy, sec-propoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, and n-heptoxy; Y chooses from the following groups: O- OH and OR; Z is O; and It is an optional double bond.

[0234] In some implementations of E6: Each X is independently selected from the group consisting of: fluorine, hydroxyl, methoxy, ethoxy, n-propoxy, sec-propoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, and n-heptoxy; Y chooses from the following groups: O - OH and OR; Z is CH2; and It is an optional double bond.

[0235] In some implementations of E6: Each X is independently selected from the group consisting of: fluorine, hydroxyl, and methoxy; Y chooses from the following groups: O - OH and OR; Z is O; and It is an optional double bond.

[0236] In some implementations of E6: Each X is independently selected from the group consisting of: fluorine, hydroxyl, and methoxy; Y chooses from the following groups: O - OH and OR; Z is CH2; and It is an optional double bond.

[0237] In some embodiments of formula E1, Z is O and W is OCH2. In some embodiments, the modified inter-subunit bonding of formula E1 is the modified inter-subunit bonding of formula E6a: (E6a).

[0238] In some embodiments of formula E1, Z is CH2 and W is CH. In some embodiments, the modified inter-subunit bonding of formula E1 is the modified inter-subunit bonding of formula E7: (E7)

[0239] In some embodiments of Formula E1, the base pairing portion B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0240] In some implementations, at least one modified subunit inter-bonding has the formula E8: (E8); in: D can be selected from the following groups: O, S, B, BR 2 , N, NR 2 OCH2, OCH, CH2 and CH, where D is selected from the group consisting of OCH2 and OCH; C chooses from the following groups: O - OH, OR 1 NH - NH2, S - And SH, where C can be chosen from the following groups: O - OH and OR 1 ; A chooses from the following groups: O, S, BR 2 NR 2 and CH2; R 1 It is a protecting group; Each R 2 Independently H or optionally substituted C1-C6 alkyl; It is an optional double bond; and Two optional modified nucleosides are bridged between subunits.

[0241] In some implementations, D is OCH2.

[0242] In some implementations, D is OCH and It is a double bond.

[0243] In some implementations, A is O.

[0244] In some implementations, A is CH2.

[0245] In some implementations, when C is O - When, A or D is not O.

[0246] In some embodiments, D is CH2. In another embodiment, the modified subunit inter-unit bond of formula E8 is the modified subunit inter-unit bond of formula E9: (E9).

[0247] In some embodiments, D is O. In another embodiment, the modified subunit inter-unit bonding of formula E8 is the modified subunit inter-unit bonding of formula E10: (E10).

[0248] In some embodiments, D is CH2. In another embodiment, the modified inter-subunit bond of formula E8 is the modified inter-subunit bond of formula E11: (E11).

[0249] In some embodiments, D is CH. In another embodiment, the modified subunit inter-unit bond of formula E8 is the modified subunit inter-unit bond of formula E12: (E12).

[0250] In some embodiments, D is OCH2. In another embodiment, the modified subunit inter-unit bond of formula E8 is the modified subunit inter-unit bond of formula E13: (E13).

[0251] In another embodiment, the modified subunit inter-bonding of formula E8 is the modified subunit inter-bonding of formula E14: (E14).

[0252] In another embodiment, the modified inter-subunit bonding of Formula E8 is the modified inter-subunit bonding of Formula E15: (E15).

[0253] In some implementations of modified siRNA-linked nucleotides, each optionally modified nucleotide is independently selected from the group consisting of adenosine, guanosine, cytidine, and uridine each time it appears.

[0254] In some implementations, at least one modified subunit inter-bonding has the formula E8: (E8); in: D can be any of the following groups: O, OCH2, OCH, CH2 and CH, where D can be any of the groups consisting of OCH2 and OCH; C chooses from the following groups: O - OH, OR 1 NH - NH2, S -And SH, where C can be chosen from the following groups: O - OH and OR 1 ; Choose the group consisting of O and CH2; R 1 The protecting group is selected from the group consisting of: dimethoxytriphenylmethyl (DMTr), succinate, tert-butyldimethylsilyl (TBDMS), benzoyl (Bz), benzyl (Bn), methoxyethoxymethyl ether (MOM), methoxybenzyl ether (PMB), methylthiomethyl ether, neopentanoyl (Piv), tetrahydropyranyl (THP), tetrahydrofuranyl (THF), triphenylmethyl (Trt), triisopropylsilyl (TIPS), tert-butyldiphenylsilyl (TBDPS), and acetate; It is an optional double bond; Furthermore, two optionally modified nucleosides are bridged between the subunits.

[0255] In some implementations, D is OCH2.

[0256] In some implementations, D is OCH and It is a double bond.

[0257] In some implementations, A is O.

[0258] In some implementations, A is CH2.

[0259] In some embodiments of any of the modified inter-subunit bonds described above, the base pairing portion B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0260] In some embodiments, R is a protecting group selected from the group consisting of: dimethoxytriphenylmethyl (DMTr), succinate, tert-butyldimethylsilyl (TBDMS), benzoyl (Bz), benzyl (Bn), methoxyethoxymethyl ether (MOM), methoxybenzyl ether (PMB), methylthiomethyl ether, neopentanoyl (Piv), tetrahydropyranyl (THP), tetrahydrofuranyl (THF), triphenylmethyl (Trt), triisopropylsilyl (TIPS), tert-butyldiphenylsilyl (TBDPS), and acetate; and It is an optional double bond.

[0261] In some implementations, the fixed region contains at least one (e.g., 1, 2, 3, 4, 5 or more) inter-nucleotide bonds of thiophosphate.

[0262] In some embodiments, the fixed region contains at least one (e.g., 1, 2, 3, 4, 5 or more) nucleotide containing a modified ribose. In some embodiments, the fixed region contains at least one (e.g., 1, 2, 3, 4, 5 or more) 2'-methoxynucleotide. In some embodiments, the fixed region contains at least one (e.g., 1, 2, 3, 4, 5 or more) 2'-fluoronucleotide.

[0263] In some embodiments, the fixation region at the 3' end of the antisense strand is three nucleotides in length, optionally wherein the fixation region is located within the portion of the antisense strand that protrudes from the sense strand. In some embodiments, the fixation region has any one of formulas F1-F6: -SASASA (Formula F1) -OASASA (Formula F2) -OAS-XA-S-XB (Formula F3) -SAS-XA-S-XB (Formula F4) -SAS-XA-S-XA (Formula F5) -SASASB (Formula F6) Where S is a phosphate thioester nucleoside link, O is a phosphate diester nucleoside link, A is a 2'-methoxynucleotide, B is a 2'-fluoronucleotide, XA is a 2'-methoxynucleotide of formula E6a, and XB is a 2'-fluoronucleotide of formula E6a.

[0264] In some implementations, the fixed area has any of the following patterns: -S-(mA)-S-(mA)-S-(mG) (Equation F7) -S-(mA)-S-(mU)-S-(mU) (Equation F8) -O-(mA)-S-(mU)-S-(mU) (Equation F9) -O-(mA)-S-(xU)-S-(yU) (Equation F10) -S-(mA)-S-(xU)-S-(yU) (Formula F11) -S-(mA)-S-(xU)-S-(xU) (Equation F12) -S-(mA)-S-(mU)-S-(fU) (Equation F13) Where S is a thiophosphate nucleoside linkage, O is a phosphodiester nucleoside linkage, mA is 2'-methoxyadenosine, mG is 2'-methoxyguanidine, mU is 2'-methoxyuridine, xU is 2'-methoxyuridine of formula E6a, and yU is 2'-fluorouridine of formula E6a.

[0265] siRNA structure The siRNA molecules disclosed herein can be in the form of single-stranded (ss) or double-stranded (ds) oligonucleotide structures. In some embodiments, the siRNA molecules can be dibranched, tribranched, or tetrabranched molecules. Furthermore, the siRNA molecules disclosed herein may contain one or more phosphodiester nucleoside links and / or their analogues, such as thiophosphate nucleoside links. The siRNA molecules disclosed herein may also contain chemically modified nucleosides with 2' sugar modifications.

[0266] The simplest siRNA consists of ribonucleic acid containing an ss- or ds- structure, formed by a first strand (i.e., the antisense strand), and in the case of ds-siRNA, a second strand (i.e., the sense strand). The first strand comprises a continuous nucleotide chain that is at least partially complementary to the target nucleic acid. The second strand also comprises a continuous nucleotide chain, wherein the second strand is at least partially identical to the target nucleic acid. The first and second strands can hybridize with each other to form a double-stranded structure. Hybridization typically occurs via Watson-Crick base pairing.

[0267] Depending on the sequences of the first and second strands, hybridization or base pairing is not necessarily perfect or complete. This means that the first and second strands may have mismatches rather than 100% base pairing. One or more mismatches may also exist within the double strand, but this does not necessarily affect siRNA RNAi activity.

[0268] The first strand contains a continuous nucleotide chain that is substantially complementary to the target nucleic acid. Typically, based on the mode of interference with RNA, the target nucleic acid sequence is ss-RNA, preferably mRNA. Such hybridization is most likely to occur via Watson-Crick base pairing, but is not necessarily limited to this. The degree to which the first strand has a continuous nucleotide chain complementary to the target nucleic acid sequence can be between 80% and 100%, for example, 80%, 85%, 90%, 95%, or 100% complementarity.

[0269] The siRNA molecules described in this article can be modified by altering the nucleobases, phosphate backbone, ribose core, 5'-end and 3'-end, as well as branches, wherein multiple strands of the siRNA can be covalently linked.

[0270] The length of small interfering RNA molecules Within the scope of this disclosure, any length known in the art and previously unknown may be used in this invention. As described herein, the possible lengths of the antisense strand of the siRNA molecule disclosed herein are 10 to 30 nucleotides (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides), 15 to 25 nucleotides (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides), or 18 to 23 nucleotides (e.g., 18, 19, 20, 21, 22, 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.

[0271] In some embodiments, the sense strand of the siRNA molecule disclosed herein is 12 to 30 nucleotides (e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) or 14 to 23 nucleotides (e.g., 14, 15, 16, 17, 18, 19, 20, 21, 22, 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.

[0272] 2′ Sugar Modification This disclosure may include ss-siRNA and ds-siRNA molecular compositions comprising at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or more) nucleosides with 2' sugar modifications. Possible 2'-modifications include OH; F; O-alkyl, S-alkyl or N-alkyl; O-alkenyl, S-alkenyl or N-alkenyl; O-ynyl, S-ynyl or N-ynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl, and ynyl groups may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and ynyl groups. In some embodiments, the modification includes a 2'-O-methyl (2'-O-Me) modification. Other possible sugar substituents include: C1 to C10 lower alkyl groups, substituted lower alkyl groups, alkenyl groups, alkynyl groups, aryl groups, O-alkaneyl or O-aryl groups, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocyclic alkyl groups, heterocyclic alkaneyl groups, aminoalkylamino groups, polyalkylamino groups, substituted silyl groups, groups for improving the pharmacokinetic properties of oligonucleotides or groups for improving the pharmacodynamic properties of oligonucleotides, and other substituents with similar properties. In some embodiments, the modification includes 2'-methoxyethoxy (2′-O-CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2′-MOE). In some embodiments, the modification includes a 2'-dimethylaminoethoxy group, i.e., the O(CH2)2ON(CH3)2 group, also known as 2′-DMAOE; and a 2'-dimethylaminoethoxyethoxy group (also known in the art as 2'-O-dimethylamino-ethoxy-ethyl or 2′-DMAEOE), i.e., 2′-O-CH2OCH2N(CH3)2. Other possible sugar substituents include, for example, aminopropoxy (-OCH2CH2CH2NH2), allyl (-CH2-CH=CH2), -O-allyyl (-O-CH2-CH=CH2), and fluorine (F). The 2'-sugar substituent can be located at the arabinose (top) position or the ribose (bottom) position. In some embodiments, the 2'-arabinose modification is 2'-F. Similar modifications can also be made at other positions on the siRNA molecule, specifically at the 3' terminal nucleotide or at the 3' position of the sugar and the 5' position of the 5' terminal nucleotide in 2'-5' linked oligonucleotides. Oligonucleotides can also have sugar analogs such as cyclobutyl moiety to replace pentofuranosyl sugar.

[0273] Nucleobase modification The siRNA molecules disclosed herein may also include nucleosides or other alternative or analogous monomeric subunits, which include nucleobases (generally referred to in the art simply as "bases" or "heterocyclic base moieties"). Nucleobases are another part that has been extensively modified or substituted, and such modified and / or substituted nucleobases are applicable to this disclosure. As used herein, "unmodified" or "natural" nucleobases include purine bases adenine (A) and guanine (G), and 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, adenine, and guanine 6-methyl derivatives and other alkyl derivatives, adenine and guanine 2-propyl derivatives and other alkyl derivatives, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C=C-CH3)uracil and cytosine, and other alkynyl derivatives of pyrimidine bases. 6-Azouracil, cytosine, and thymine; 5-uracil (pseudouracil); 4-thiouracil; 8-halogenated, 8-amino, 8-mercapto, 8-thioalkyl, 8-hydroxy, and other 8-substituted adenine and guanine; 5-halogenated (specifically 5-bromo), 5-trifluoromethyl, and other 5-substituted uracil and cytosine; 7-methylguanine and 7-methyladenine; 2-F-adenine; 2-amino-adenine; 8-azaguanine and 8-azaadenine; 7-deadenine and 7-deadenine; and 3-deadenine and 3-deadenine. The nucleobases may also include those in which the purine or pyrimidine base is replaced by other heterocycles such as 7-deadenine, 7-deadenine, 2-aminopyridine, and 2-pyridone. Other nucleobases include those disclosed in US 3,687,808 and those disclosed in Kroschwitz, JI. The Concise Encyclopedia of Polymer Science and Engineering, New York, John Wiley & Sons, 1990, those on pages 858-859; by Englisch et al. Angewandte Chemie, The international edition, 30:613, those published in 1991; and by Sanghvi, YS, Chapter 16, Antisense Research and Applications, Those disclosed in CRC Press, Gait, MJ (ed.), 1993, pp. 289-302. The siRNA molecules disclosed herein may also include polycyclic heterocyclic compounds to replace one or more heterocyclic base moieties. Many tricyclic heterocyclic compounds have been previously reported. These compounds are often used in antisense applications to enhance the binding properties of the modified strand to the target strand.

[0274] Representative cytosine analogues that form three hydrogen bonds with guanosine in the second chain include 1,3-diazaphenoxazine-2-one (Kurchavov et al.) ,Nucleosides and Nucleotides, 16:1837-46, 1997), 1,3-diazaphenthiazin-2-one (Lin et al.) Am. Chem. Soc., 117:3873-4, 1995) and 6,7,8,9-tetrafluoro-1,3-diazaphenoxazine-2-one (Wang et al., Tetrahedron Lett., 39:8385-8, 1998). These base modifications incorporated into oligonucleotides have been shown to hybridize with complementary guanine, and the latter has also been shown to hybridize with adenine and enhance helical thermal stability through extended stacking interactions (see also US 10 / 155,920 and US 10 / 013,295, both of which are incorporated herein by reference in their entirety). Further helical stability has been observed when cytosine analogs / substitutes have an aminoethoxy moiety attached to a rigid 1,3-diazaphenoxazine-2-one scaffold (Lin et al., 1998). Am. Chem. Soc., 120:8531-2, 1998).

[0275] Nucleoside inter-linking modification Another variable in this design is the internucleotide bonds of the phosphate backbone constituting the siRNA molecule. While a native RNA phosphate backbone may be used, derivatives thereof that enhance the desired characteristics of the siRNA molecule may be used. While not limiting, it is particularly important in this disclosure to protect portions or the entire siRNA molecule from hydrolysis. One example of a modification that reduces the rate of hydrolysis is a thiophosphate. Any portion or the entire backbone may contain phosphate ester substituents (e.g., thiophosphate, etc.). For example, the nucleoside linkage can be a thiophosphate with a concentration between 0% and 100%, such as between 0% and 100%, between 10% and 100%, between 20% and 100%, between 30% and 100%, between 40% and 100%, between 50% and 100%, between 60% and 100%, between 70% and 100%, between 80% and 100%, between 90% and 100%, between 0% and 90%, between 0% and 80%, between 0% and 70%, etc. Thiophosphate ester bonds between 0% and 60%, between 0% and 50%, between 0% and 40%, between 0% and 30%, between 0% and 20%, between 0% and 10%, between 10% and 90%, between 20% and 80%, between 30% and 70%, between 40% and 60%, between 10% and 40%, between 20% and 50%, between 30% and 60%, between 40% and 70%, between 50% and 80%, or between 60% and 90%. Similarly, internucleotide linkages can be phosphodiester linkages between 0% and 100%, such as between 0% and 100%, between 10% and 100%, between 20% and 100%, between 30% and 100%, between 40% and 100%, between 50% and 100%, between 60% and 100%, between 70% and 100%, between 80% and 100%, between 90% and 100%, between 0% and 90%, between 0% and 80%, between 0% and 70%. Phosphodiester bonding between 0% and 60%, between 0% and 50%, between 0% and 40%, between 0% and 30%, between 0% and 20%, between 0% and 10%, between 10% and 90%, between 20% and 80%, between 30% and 70%, between 40% and 60%, between 10% and 40%, between 20% and 50%, between 30% and 60%, between 40% and 70%, between 50% and 80%, or between 60% and 90%.

[0276] Specific examples of possible siRNA molecules that can be used in this invention include oligonucleotides containing modified (e.g., non-naturally occurring) internucleotide links. As defined herein, oligonucleotides having modified internucleotide links include internucleotide links that retain a phosphorus atom and those that do not. For the purposes of this specification, and as sometimes mentioned in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleotide backbone may also be considered oligonucleotides. Preferred phosphorus-containing modified internucleotide links are phosphate thioester internucleotide links. In some embodiments, the modified oligonucleotide backbone containing a phosphorus atom includes, for example, thiophosphates, dithiophosphates, phosphate triesters, aminoalkyl phosphate triesters, methyl and other alkylphosphonates (including 3'-alkylphosphonates, 5'-alkylphosphonates), phosphonates, aminophosphates (including 3'-aminoaminophosphates and aminoalkylaminophosphates), thioaminophosphates, thioalkylphosphonates, thioalkyl phosphate triesters, selenophosphates and borophosphates having normal 3'-5' linkages, analogs of these with 2'-5' linkages, and those with antipolarity, wherein the linkage between one or more nucleotides is 3' to 3', 5' to 5', or 2' to 2' linkage. Exemplary U.S. patents describing the preparation of phosphorus-containing bonds include, but are not limited to, U.S. patent numbers 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,3 21,131;5,399,676;5,405,939;5,453,496;5,455,233;5,466,677;5,476,925;5,519,126;5,536,821;5,541,316;5,550,111;5,563,253;5,571,799;5,587 361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534 U.S. Patent RE39464, the entire contents of each of these patents are hereby incorporated herein by reference.

[0277] In some embodiments, the modified oligonucleotide backbone, which does not include a phosphorus atom, has a backbone formed by inter-linking of short-chain alkyl or cycloalkyl nucleosides, inter-linking of mixed heteroatoms and alkyl or cycloalkyl nucleosides, or inter-linking of one or more short-chain heteroatomic nucleosides or heterocyclic nucleosides. These include backbones having: morpholino linkages (partially formed from the sugar moiety of the nucleoside); siloxane backbones; sulfide, sulfoxide, and sulfone backbones; formylacetyl and thioformylacetyl backbones; methyleneformylacetyl and thioformylacetyl backbones; riboacetyl backbones; olefinic backbones; aminosulfonate backbones; methyleneimine and methylenehydrazine backbones; sulfonate and sulfonamide backbones; amide backbones; and other backbones having mixed N, O, S, and CH2 components. Non-limiting examples of U.S. patents teaching the preparation of non-phosphorus skeletons include, but are not limited to, U.S. patent numbers 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5, 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 these patents are hereby incorporated herein by reference.

[0278] Inter-subunit modification Alternatively, or in addition to the modifications discussed above, this section discloses other inter-subunit modifications that may be included within the siRNA molecules disclosed herein. The siRNA molecule may contain at least one (e.g., at least two, at least three, at least four, at least five, at least six, or more) inter-subunit linking modification as disclosed herein. These modifications may be present in the fixed region or the overhanging region of the antisense strand. In some embodiments, one, two, three, four, five, or more modifications as described in this section may be present in a second region of the antisense strand overhanging the sense strand.

[0279] In some embodiments, at least one modified subunit inter-bonding has the formula E1: (E1); in: Each B is an independent base pairing part; W can be selected from the following groups: O, S, B, BR 2 , N, NR2 OCH2, OCH, CH2 and CH, where W is selected from the group consisting of OCH2 and OCH; Each X is independently selected from the group consisting of: halogen (e.g., fluorine or chlorine), hydroxyl, and C. 1-6 Alkoxy groups, optionally wherein each X is independently selected from the group consisting of: halogen groups (e.g., fluorine or chlorine) and C. 1-6 Alkyl groups (e.g., methoxy, ethoxy, 2-methoxyethoxy, n-propoxy, sec-propoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, or n-heptoxy); Y chooses from the following groups: O - OH, OR, NH - NH2, S - And SH, where Y can be freely selected from the following groups: O - OH and OR.

[0280] Z is selected from the following groups: O, S, BR 2 NR 2 and CH2; R is a protecting group; Each R 2 Independently H or optionally substituted C1-C6 alkyl; and It is an optional double bond.

[0281] In some implementations of E1, W is OCH2.

[0282] In some implementations of E1, W is OCH and It is a double bond.

[0283] In some implementations of E1, Z is O.

[0284] In some implementations of formula E1, Z is CH2.

[0285] In some implementations of E1, when Y is O - When Z or W is not O.

[0286] In some embodiments of formula E1, Z is CH2 and W is CH2. In some embodiments, the modified inter-subunit bonding of formula E1 is the same as the modified inter-subunit bonding of formula E2: (E2).

[0287] In some embodiments of formula E1, Z is CH2 and W is O. In some embodiments, the modified inter-subunit bonding of formula E1 is the modified inter-subunit bonding of formula E3: (E3).

[0288] In some embodiments of formula E1, Z is O and W is CH2. In some embodiments, the modified inter-subunit bonding of formula E1 is the modified inter-subunit bonding of formula E4: (E4).

[0289] In some embodiments of E1, Z is O and W is CH. In some embodiments, the modified inter-subunit bonding of E1 is the modified inter-subunit bonding of E5: (E5).

[0290] In some implementations, the modified inter-subunit bonding of formula E1 is the modified inter-subunit bonding of formula E6: (E6).

[0291] In some implementations of E6: Each B is an independent base pairing part; Each X is independently selected from the group consisting of: halogen, hydroxyl, and C. 1-6 Alkyl group; optionally, each X is independently selected from the group consisting of: halogen (e.g., fluorine) and C. 1-6 Alkyl groups (e.g., methoxy, ethoxy, n-propoxy, sec-propoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, or n-heptoxy); Y chooses from the following groups: O - OH, OR, NH - NH2, S - And SH, where Y can be freely selected from the following groups: O - OH and OR; Z is selected from the group consisting of O and CH2; and It is an optional double bond.

[0292] In some implementations of E6: Each X is independently selected from the group consisting of: fluorine, hydroxyl, and C. 1-6 Alkyl groups; optionally, each X is independently selected from the group consisting of fluorine and C. 1-6 Alkyl groups (e.g., methoxy, ethoxy, n-propoxy, sec-propoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, or n-heptoxy); Y chooses from the following groups: O - OH and OR; Z is selected from the group consisting of O and CH2; and It is an optional double bond.

[0293] In some implementations of E6: Each X is independently selected from the group consisting of: fluorine, hydroxyl, methoxy, ethoxy, n-propoxy, sec-propoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, and n-heptoxy; Y chooses from the following groups: O - OH and OR; Z is selected from the group consisting of O and CH2; and It is an optional double bond.

[0294] In some implementations of E6: Each X is independently selected from the group consisting of: fluorine, hydroxyl, methoxy, ethoxy, n-propoxy, sec-propoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, and n-heptoxy; Y chooses from the following groups: O - OH and OR; Z is O; and It is an optional double bond.

[0295] In some implementations of E6: Each X is independently selected from the group consisting of: fluorine, hydroxyl, methoxy, ethoxy, n-propoxy, sec-propoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, and n-heptoxy; Y chooses from the following groups: O - OH and OR; Z is CH2; and It is an optional double bond.

[0296] In some implementations of E6: Each X is independently selected from the group consisting of: fluorine, hydroxyl, and methoxy; Y chooses from the following groups: O - OH and OR; Z is O; and It is an optional double bond.

[0297] In some implementations of E6: Each X is independently selected from the group consisting of: fluorine, hydroxyl, and methoxy; Y chooses from the following groups: O - OH and OR; Z is CH2; and It is an optional double bond.

[0298] In some embodiments of formula E1, Z is O and W is OCH2. In some embodiments, the modified inter-subunit bonding of formula E1 is the modified inter-subunit bonding of formula E6a: (E6a).

[0299] In some embodiments of formula E1, Z is CH2 and W is CH. In some embodiments, the modified inter-subunit bonding of formula E1 is the modified inter-subunit bonding of formula E7: (E7)

[0300] In some embodiments of Formula E1, the base pairing portion B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0301] In some implementations, at least one modified subunit inter-bonding has the formula E8: (E8); in: D can be selected from the following groups: O, S, B, BR 2 , N, NR 2 OCH2, OCH, CH2 and CH, where D is selected from the group consisting of OCH2 and OCH; C chooses from the following groups: O - OH, OR 1 NH - NH2, S - And SH, where C can be chosen from the following groups: O - OH and OR 1 ; A chooses from the following groups: O, S, BR 2 NR 2 and CH2; R 1 It is a protecting group; Each R 2 Independently H or optionally substituted C1-C6 alkyl; It is an optional double bond; and Two optional modified nucleosides are bridged between subunits.

[0302] In some implementations, D is OCH2.

[0303] In some implementations, D is OCH and It is a double bond.

[0304] In some implementations, A is O.

[0305] In some implementations, A is CH2.

[0306] In some implementations, when C is O - When, A or D is not O.

[0307] In some embodiments, D is CH2. In another embodiment, the modified subunit inter-unit bond of formula E8 is the modified subunit inter-unit bond of formula E9: (E9).

[0308] In some embodiments, D is O. In another embodiment, the modified subunit inter-unit bonding of formula E8 is the modified subunit inter-unit bonding of formula E10: (E10).

[0309] In some embodiments, D is CH2. In another embodiment, the modified inter-subunit bond of formula E8 is the modified inter-subunit bond of formula E11: (E11).

[0310] In some embodiments, D is CH. In another embodiment, the modified subunit inter-unit bond of formula E8 is the modified subunit inter-unit bond of formula E12: (E12).

[0311] In some embodiments, D is OCH2. In another embodiment, the modified subunit inter-unit bond of formula E8 is the modified subunit inter-unit bond of formula E13: (E13).

[0312] In another embodiment, the modified subunit inter-bonding of formula E8 is the modified subunit inter-bonding of formula E14: (E14).

[0313] In another embodiment, the modified inter-subunit bonding of Formula E8 is the modified inter-subunit bonding of Formula E15: (E15).

[0314] In some implementations of modified siRNA-linked nucleotides, each optionally modified nucleotide is independently selected from the group consisting of adenosine, guanosine, cytidine, and uridine each time it appears.

[0315] In some implementations, at least one modified subunit inter-bonding has the formula E8: (E8); in: D can be any of the following groups: O, OCH2, OCH, CH2 and CH, where D can be any of the groups consisting of OCH2 and OCH; C chooses from the following groups: O - OH, OR 1 NH - NH2, S - And SH, where C can be chosen from the following groups: O - OH and OR 1 ; Choose the group consisting of O and CH2; R 1 The protecting group is selected from the group consisting of: dimethoxytriphenylmethyl (DMTr), succinate, tert-butyldimethylsilyl (TBDMS), benzoyl (Bz), benzyl (Bn), methoxyethoxymethyl ether (MOM), methoxybenzyl ether (PMB), methylthiomethyl ether, neopentanoyl (Piv), tetrahydropyranyl (THP), tetrahydrofuranyl (THF), triphenylmethyl (Trt), triisopropylsilyl (TIPS), tert-butyldiphenylsilyl (TBDPS), and acetate; It is an optional double bond; Furthermore, two optionally modified nucleosides are bridged between the subunits.

[0316] In some implementations, D is OCH2.

[0317] In some implementations, D is OCH and It is a double bond.

[0318] In some implementations, A is O.

[0319] In some implementations, A is CH2.

[0320] In some embodiments of any of the modified inter-subunit bonds described above, the base pairing portion B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0321] In some embodiments, R is a protecting group selected from the group consisting of: dimethoxytriphenylmethyl (DMTr), succinate, tert-butyldimethylsilyl (TBDMS), benzoyl (Bz), benzyl (Bn), methoxyethoxymethyl ether (MOM), methoxybenzyl ether (PMB), methylthiomethyl ether, neopentanoyl (Piv), tetrahydropyranyl (THP), tetrahydrofuranyl (THF), triphenylmethyl (Trt), triisopropylsilyl (TIPS), tert-butyldiphenylsilyl (TBDPS), and acetate; and It is an optional double bond.

[0322] Modification patterns of siRNA molecules The following sections provide a set of exemplary scaffolds into which the siRNA molecules of this disclosure can be incorporated.

[0323] In some embodiments of this disclosure, the siRNA may contain an antisense strand including a region represented by Formula I, wherein Formula I is oriented from 5' to 3'. AB-(A') j -CP 2 -DP 1 -(C'-P 1 ) k -C' Formula I; Where A is derived from CP 1 -DP 1 Indicates; each A' is represented by formula CP 2 -DP 2 Indicates; B is derived from CP 2 -DP 2 -DP 2 -DP 2 Indicates that each C is a 2'-O-methyl (2'-O-Me) ribonucleoside; each C' is independently a 2'-O-Me ribonucleoside or a 2'-fluoro (2'-F) ribonucleoside; each D is a 2'-F ribonucleoside; each P 1 It is an internucleotide linking of thiophosphate esters; each P 2It is a phosphodiester nucleoside link; 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 to the target nucleic acid sequence (e.g., fully or partially complementary).

[0324] In some implementations, the antisense chain comprises a structure represented by formula A1, wherein formula A1 is in the 5' to 3' direction as follows: ASBSAOBOBOBOAOBOAOBOA-OBAOBOAOBSASASASBSA Formula A1; Where A represents 2'-O-Me ribonucleoside, B represents 2'-F ribonucleoside, O represents phosphate diester nucleoside linkage, and S represents thiophosphate nucleoside linkage.

[0325] In some embodiments of this disclosure, the siRNA may contain an antisense strand including a region represented by Formula II, wherein Formula II is oriented from 5' to 3'. AB-(A') j -CP 2 -DP 1 -(CP 1 ) k -C' Formula II; Where A is derived from CP 1 -DP 1 Indicates; each A' is represented by formula CP 2 -DP 2 Indicates; B is derived from CP 2 -DP 2 -DP 2 -DP 2 Indicates that each C is a 2'-O-methyl (2'-O-Me) ribonucleoside; each C' is independently a 2'-O-Me ribonucleoside or a 2'-fluoro (2'-F) ribonucleoside; each D is a 2'-F ribonucleoside; each P 1 It is an internucleotide linking of thiophosphate esters; each P 2 It is a phosphodiester nucleoside link; 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 to the target nucleic acid sequence (e.g., fully or partially complementary).

[0326] In some embodiments of this disclosure, the antisense chain includes a structure represented by formula A2, wherein formula A2 is in the 5' to 3' direction as follows: ASBSAOBOBOBOAOBOAOBOA-OBAOBOAOBSASASASASA Formula A2; Where A represents 2'-O-Me ribonucleoside, B represents 2'-F ribonucleoside, O represents phosphate diester nucleoside linkage, and S represents thiophosphate nucleoside linkage.

[0327] In some embodiments of this disclosure, the semantic chain includes a structure represented by Formula III, wherein Formula III is in the 5' to 3' direction as follows: E-(A') m -F Formula III; Where E is derived from formula (CP) 1 )2 represents; F is derived from 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 indicates: A', C, D, P 1 and P 2 As defined in Equation I; and m is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7). In some embodiments, m is 4. In some embodiments, the sense chain and the antisense chain are complementary (e.g., fully or partially complementary).

[0328] In some embodiments of this disclosure, the semantic chain includes a structure represented by formula S1, wherein formula S1 is in the 5' to 3' direction as follows: ASASAOBOAOBOAOBOAOBOA-OAOAOBSASA Formula S1; Where A represents 2'-O-Me ribonucleoside, B represents 2'-F ribonucleoside, O represents phosphate diester nucleoside linkage, and S represents thiophosphate nucleoside linkage.

[0329] In some embodiments of this disclosure, the semantic chain includes a structure represented by formula S2, wherein formula S2 is in the 5' to 3' direction as follows: ASASAOBOAOBOAOBOAOBOA-OAOAOBOAOA Formula S2; Where A represents 2'-O-Me ribonucleoside, B represents 2'-F ribonucleoside, O represents phosphate diester nucleoside linkage, and S represents thiophosphate nucleoside linkage.

[0330] In some embodiments of this disclosure, the semantic chain includes a structure represented by formula S3, wherein formula S3 is in the 5' to 3' direction as follows: ASASAOBOAOBOAOBOAOBOA-OAOAOBSASB Formula S3; Where A represents 2'-O-Me ribonucleoside, B represents 2'-F ribonucleoside, O represents phosphate diester nucleoside linkage, and S represents thiophosphate nucleoside linkage.

[0331] In some embodiments of this disclosure, the semantic chain includes a structure represented by formula S4, wherein formula S4 is in the 5' to 3' direction as follows: ASASAOBOAOBOAOBOAOBOA-OAOAOBOAOB Formula S4; Where A represents 2'-O-Me ribonucleoside, B represents 2'-F ribonucleoside, O represents phosphate diester nucleoside linkage, and S represents thiophosphate nucleoside linkage.

[0332] In some embodiments of this disclosure, the siRNA may contain an antisense strand including a region represented by Formula IV, wherein Formula IV is oriented from 5' to 3'. A-(A') j -CP 2 -B-(CP 1 ) k -C' Formula IV; Where A is derived from CP 1 -DP 1 Indicates; each A' is represented by formula CP 2 -DP 2 Indicates; B is derived from formula DP 1 -CP 1 -DP1 Indicates that each C is a 2'-O-Me ribonucleotide; each C' is independently a 2'-O-Me ribonucleotide or a 2'-F ribonucleotide; each D is a 2'-F ribonucleotide; each P 1 It is an internucleotide linking of thiophosphate esters; each P 2 It is a phosphodiester nucleoside linker; 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 to the target nucleic acid (e.g., fully or partially complementary).

[0333] In some embodiments of this disclosure, the antisense chain includes a structure represented by formula A3, wherein formula A3 is in the 5' to 3' direction as follows: ASBSAOBOAOBOAOBOAOBOA-OBAOBOAOBSASBSASASA Formula A3; Where A represents 2'-O-Me ribonucleoside, B represents 2'-F ribonucleoside, O represents phosphate diester nucleoside linkage, and S represents thiophosphate nucleoside linkage.

[0334] In some embodiments of this disclosure, the siRNA of this disclosure may have a sense strand represented by formula V, wherein formula V is in the 5' to 3' orientation as follows: E-(A') m -CP 2 -F Formula V; Where E is derived from formula (CP) 1 )2 represents; F is derived from formula DP 1 -CP 1 -C, DP 2 -CP 2 -C, DP 1 -CP 1 -D or DP 2 -CP 2 -D indicates: A', C, D, P 1 and P 2 As defined in Equation IV; and m is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7). In some embodiments, m is 5. In some embodiments, the sense chain and the antisense chain are complementary (e.g., fully or partially complementary).

[0335] In some embodiments of this disclosure, the semantic chain includes a structure represented by formula S5, wherein formula S5 is in the 5' to 3' direction: ASASAOBOAOBOAOBOAOBOA-OBOAOBSASA Formula S5; Where A represents 2'-O-Me ribonucleoside, B represents 2'-F ribonucleoside, O represents phosphate diester nucleoside linkage, and S represents thiophosphate nucleoside linkage.

[0336] In some embodiments of this disclosure, the semantic chain includes a structure represented by formula S6, wherein formula S6 is in the 5' to 3' direction as follows: ASASAOBOAOBOAOBOAOBOA-OBOAOBOAOA Formula S6; Wherein A represents 2'-O-Me ribonucleoside, B represents 2'-F ribonucleoside, O represents phosphodiester nucleoside linkage, and S represents thiophosphate nucleoside linkage. In some embodiments of this disclosure, the sense chain includes a structure represented by formula S7, wherein formula S7 is in the 5' to 3' orientation as follows: ASASAOBOAOBOAOBOAOBOA-OBOAOBSASB Formula S7; Where A represents 2'-O-Me ribonucleoside, B represents 2'-F ribonucleoside, O represents phosphate diester nucleoside linkage, and S represents thiophosphate nucleoside linkage.

[0337] In some embodiments of this disclosure, the semantic chain includes a structure represented by formula S8, wherein formula S8 is in the 5' to 3' direction as follows: ASASAOBOAOBOAOBOAOBOA-OBOAOBOAOB Formula S8; Where A represents 2'-O-Me ribonucleoside, B represents 2'-F ribonucleoside, O represents phosphate diester nucleoside linkage, and S represents thiophosphate nucleoside linkage.

[0338] In some embodiments of this disclosure, the siRNA may contain an antisense strand including a region represented by Formula VI, wherein Formula VI is oriented from 5' to 3'. AB j -EB k -EFGl -DP 1 -C' Formula VI; Where A is derived from CP 1 -DP 1 Indicates; each B is represented by CP. 2 Indicates that each C is a 2'-O-Me ribonucleoside; each C' is independently a 2'-O-Me ribonucleoside or a 2'-F ribonucleoside; each D is a 2'-F ribonucleoside; each E is derived from formula DP. 2 -CP 2 It is represented by F from formula DP. 1 -CP 1 Indicates; each G is represented by CP. 1 Indicates; each P 1 It is an internucleotide linking of thiophosphate esters; each P 2 The linkage is a phosphodiester bond; 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 to the target nucleic acid (e.g., fully or partially complementary).

[0339] In some embodiments of this disclosure, the antisense chain includes a structure represented by formula A4, wherein formula A4 is in the 5' to 3' direction as follows: ASBSAOAOAOBOAOAOAOAOA-OAOAOBOAOBSASASASBSA Formula A4; Where A represents 2'-O-Me ribonucleoside, B represents 2'-F ribonucleoside, O represents phosphate diester nucleoside linkage, and S represents thiophosphate nucleoside linkage.

[0340] In some embodiments of this disclosure, the siRNA may contain a sense strand including a region represented by Formula VII, wherein Formula VII is in the 5' to 3' orientation: HB m -I n -A'-B o -HC Equation VII; Where A' is derived from CP 2 -DP 2 Indicates; each H is represented by formula (CP) 1)2 represents; each I is represented by equation (DP) 2 ) represents; B, C, D, P 1 and P 2 As defined in Equation VI; m is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7); n is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7); and o is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7). In some embodiments, m is 3. In some embodiments, n is 3. In some embodiments, o is 3. In some embodiments, m is 3, n is 3, and o is 3. The antisense chain is complementary to the sense chain (e.g., fully or partially complementary).

[0341] In some embodiments of this disclosure, the semantic chain includes a structure represented by formula S9, wherein formula S9 is in the 5' to 3' direction: ASASAOAOAOBOBOBOAOBOA-OAOAOASASA Formula S9; Where A represents 2'-O-Me ribonucleoside, B represents 2'-F ribonucleoside, O represents phosphate diester nucleoside linkage, and S represents thiophosphate nucleoside linkage.

[0342] In some embodiments of this disclosure, the siRNA may contain an antisense strand including the region represented by Formula VIII: Z-((AP-) n (BP-) m ) q ; Formula VIII Where Z is the 5' phosphorus-stabilized moiety; each A is a 2'-O-methyl (2'-O-Me) ribonucleoside; each B is a 2'-fluoro-ribonucleoside; each P is independently an inter-nucleoside linker selected from phosphodiester-linked and thiophosphate-linked links; n is an integer from 1 to 5 (e.g., 1, 2, 3, 4, or 5); m is an integer from 1 to 5 (e.g., 1, 2, 3, 4, or 5); and q is an integer between 1 and 30 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30).

[0343] siRNA synthesis method The siRNA molecules disclosed herein can be synthesized using standard methods known in the art, as discussed further below, for example, by using an automated DNA synthesizer, such as those commercially available from Biosearch, Applied Biosystems, Inc.

[0344] siRNA reagents can be prepared using solution-phase or solid-phase organic synthesis, or both. Organic synthesis offers the advantage of readily preparing oligonucleotides comprising non-natural or modified nucleotides. The siRNA molecules disclosed herein can be prepared using solution-phase or solid-phase organic synthesis, or both.

[0345] Furthermore, given that any siRNA reagent disclosed herein can be further optimized by systematically adding or removing linked nucleosides to produce longer or shorter sequences, such optimized sequences can be further tailored by, for example, the introduction of modified nucleosides and / or modified nucleoside linkages as described herein or known in the art, including alternative nucleosides, alternative sugar moieties, and / or alternative nucleoside linkages as known in the art and / or discussed herein, to further optimize the molecule (e.g., increasing serum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, and / or targeting specific sites or cell types).

[0346] 5′ Phosphorus stable part To further protect the siRNA molecules of this disclosure from degradation, a 5'-phospho-stabilizing moiety may be employed. The 5'-phospho-stabilizing moiety replaces the 5'-phosphate ester to prevent phosphate ester hydrolysis. Hydrolysis of the 5'-phosphate ester prevents binding to RISC, a necessary step in gene silencing. Any phosphate ester substitution that does not impede binding to RISC is considered in this disclosure. In some embodiments, the 5'-phosphate ester substitute is also stable against in vivo hydrolysis. Each strand of the siRNA molecule may independently and optionally employ any suitable 5'-phospho-stabilizing moiety.

[0347] Some exemplary end caps are shown in Formulas IX-XVI. In Formulas IX-XVI, Nuc represents a nucleobase or nucleobase derivative or substitution as described herein. In Formulas IX-XVI, X represents a 2'-modification as described herein. Some embodiments employ a hydroxyl group as in Formula IX, a phosphate ester as in Formula X, a vinyl phosphonate as in Formulas XI and XIV, a 5'-methyl-substituted phosphate ester as in Formulas XII, XIII, and XVI, a methylene phosphonate as in Formula XV, or a vinyl 5'-vinyl phosphonate as shown in Formula XI as the 5'-phosphorus stabilizing moiety.

[0348] Hydrophobic part This disclosure also provides siRNA molecules having one or more hydrophobic portions thereto. The hydrophobic portions may be covalently attached to the 5' or 3' end of the siRNA molecule of this disclosure. Non-limiting examples of hydrophobic portions suitable for use with the siRNA molecule of this disclosure may include cholesterol, vitamin D, tocopherol, phosphatidylcholine (PC), docosahexaenoic acid, docosanoic acid, PC-docosaanoic acid, eicosapentaenoic acid, lithocholic acid, or any combination of the above hydrophobic portions with PC.

[0349] siRNA branch The siRNA molecules disclosed herein can be branched. For example, the siRNA molecules disclosed herein can have one of several branching patterns, as described herein.

[0350] According to this disclosure, the siRNA molecules disclosed herein can be branched siRNA molecules. The siRNA molecule may not be branched, or it may be dibranched, tribranched, or tetrabranched, connected by a linker. Each main branch may further branch to allow 2, 3, 4, 5, 6, 7, or 8 separate single-stranded or double-stranded RNA strands. Branching points at the linker may originate from the same atom or along separate atoms of the linker. Some exemplary embodiments are listed in Table 2.

[0351] Table 2. Structure of branched siRNAs

[0352] In some embodiments, the siRNA molecule is a branched siRNA molecule. In some embodiments, the branched siRNA molecule is dibranched, tribranched, or tetrabranched. In some embodiments, a dibranched siRNA molecule is represented by any one of formulas XVII-XIX, wherein each RNA is independently an siRNA molecule, L is a linker, and each X independently represents a branch point portion (e.g., phosphorous acid, toluenesulfonated acetone glycerol, 1,3-diaminopropanol, pentaerythritol, or any one of the branch point portions described in US 10,478,503).

[0353] In some implementations, the three-branched siRNA molecule is represented by any one of formulas XX-XXIII, where each RNA is an siRNA molecule independently, L is a linker, and each X independently represents a branch point portion.

[0354] In some implementations, the four-branched siRNA molecule is represented by any one of formulas XXIV-XXVIII, wherein each RNA is an siRNA molecule independently, L is a linker, and each X independently represents a branch point portion.

[0355] connector Multiple strands of the siRNA described herein can be covalently attached via a linker. This branching action particularly improves cell permeability, thereby allowing better entry into cells in the CNS (e.g., neurons or microglia). Any linker portion incompatible with the siRNA of this invention may be employed. Linkers comprise 2 to 10 subunits (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 subunits) of glycol chains, alkyl chains, carbohydrate chains, block copolymers, peptides, RNA, DNA, etc. In some embodiments, any carbon or oxygen atom of the linker is optionally replaced by a nitrogen atom, has a hydroxyl substituent, or has an oxygen substituent. In some embodiments, the linker is a polyethylene glycol (PEG) linker. PEG linkers suitable for use with 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.

[0356] Various weights of PEG adapters can be used with the disclosed compositions and methods. For example, PEG adapters can have a weight between 5 and 500 Daltons. In some embodiments, PEG adapters with a weight between 500 and 1,000 Daltons can be used. In some embodiments, PEG adapters with a weight between 1,000 and 10,000 Daltons can be used. In some embodiments, PEG adapters with a weight between 200 and 20,000 Daltons can be used. In some embodiments, the adapter is covalently attached to the sense strand of the siRNA. In some embodiments, the adapter is covalently attached to the antisense strand of the siRNA. In some embodiments, the PEG adapter is a triethylene glycol (TrEG) adapter. In some embodiments, the PEG adapter is a tetraethylene glycol (TEG) adapter.

[0357] In some embodiments, the adapter is an alkyl linker. In some embodiments, the adapter is a peptide adapter. In some embodiments, the adapter is an RNA adapter. In some embodiments, the adapter is a DNA adapter.

[0358] The adapter can covalently link 2, 3, 4, or 5 distinct siRNA strands. The adapter can covalently bind to any portion of the siRNA oligomer. In some embodiments, the adapter attaches to the 3' end of the nucleoside of each siRNA strand. In some embodiments, the adapter attaches to the 5' end of the nucleoside of each siRNA strand. In some embodiments, the adapter is covalently formed to a nucleoside of the siRNA strand (e.g., sense or antisense strand). In some embodiments, the covalently formed portion is selected from the group consisting of alkyl, ester, amide, carbonate, carbamate, triazole, urea, methyl acetal, phosphonate, phosphate ester, and phosphate ester derivatives (e.g., thiophosphate, aminophosphate, etc.).

[0359] In some implementations, the connector has a structure of type L1: .

[0360] (Equation L1) In some implementations, the connector has a structure of type L2: .

[0361] (Equation L2) In some implementations, the connector has the structure of type L3: .

[0362] (Formula L3) In some implementations, the connector has the structure of type L4: .

[0363] (Equation L4) In some implementations, the connector has the structure of type L5: .

[0364] (Formula L5) In some implementations, the connector has a structure of type L6: .

[0365] (Formula L6) In some implementations, the connector has a structure of type L7, as shown below: .

[0366] (Formula L7) In some implementations, the connector has a structure of type L8: .

[0367] (Formula L8) In some implementations, the connector has a structure of type L9: .

[0368] (Formula L9) In some embodiments, the selection of a adapter used with one or more branched siRNA molecules disclosed herein can be based on the hydrophobicity of the adapter, such that, for example, a desired hydrophobicity is achieved for one or more branched siRNA molecules disclosed herein. For example, an adapter containing an alkyl chain can be used to increase the hydrophobicity of the branched siRNA molecule compared to branched siRNA molecules with a less hydrophobic adapter or a hydrophilic adapter.

[0369] The siRNA agents disclosed herein can be synthesized and / or modified using methods well-established in the art, such as those described by Beaucage, SL, et al. (editors). Current Protocols in Nucleic Acid Chemistry The methods described in John Wiley & Sons, Inc., New York, NY, 2000 are hereby incorporated herein by reference.

[0370] Treatment This disclosure provides a method for treating a subject requiring gene silencing. Gene silencing can be performed, among other reasons, to silence defective or overactive genes, to silence negative regulators of genes with reduced expression, to silence wild-type genes that have an activating effect in one or more pathways that increase the activity of disease-driving genes, or to silence splice isotypes of one or more genes that, upon selective knockdown, can increase the total expression of one or more genes, provided the goal is to restore the activity of genetic and biochemical pathways from a disease state to a healthy state. The method may include delivering the siRNA molecule of this disclosure or a pharmaceutical composition comprising it to the CNS of a subject (e.g., a human) via any suitable route of administration (e.g., intrastriatal, intraventricular, intrathecal, intrastriatal injection, intracerebellomedullary cistern via catheter insertion, intraparenchymal injection, intravenous injection, subcutaneous injection, or intramuscular injection). The active compound may be administered at any suitable dose. The actual dose of the composition of this disclosure administered to the patient can be determined by physical and physiological factors such as weight, disease severity, prior or concurrent treatment interventions, the patient's idiopathic condition, and the route of administration. Depending on the dosage and route of administration, the preferred dosage and / or the frequency of administration of the effective amount may vary based on the subject's response. In any case, the practitioner responsible for administration will determine the concentration of one or more active ingredients in the composition and one or more appropriate doses for the individual subject. Administration may be performed any suitable number of times per day and for as long as necessary. Subjects may be adults or children with or without comorbidities.

[0371] Subject selection Subjects who can be treated with the siRNA molecules disclosed herein are those who require treatment for any one or more medical risks associated with gain-of-function mutations in the target gene. Subjects who can be treated with the siRNA molecules disclosed herein may include, for example, humans, monkeys, rats, mice, pigs, and other mammals containing at least one orthologous copy of the target gene. Subjects may be adults or children with or without comorbid diseases.

[0372] Pharmaceutical Composition The siRNA molecules of this disclosure can be formulated into pharmaceutical compositions for administration to a subject in a biocompatible form suitable for in vivo administration. Therefore, this disclosure provides a pharmaceutical composition comprising the siRNA molecules of this disclosure mixed with a suitable diluent, carrier, or excipient. The siRNA molecules can be administered, for example, directly to the CNS or affected tissues of a subject (e.g., via intraventricular, intrastriatal, intrathecal, intracerebrospinal, intraparenchymal, intravenous, subcutaneous, or intramuscular injection).

[0373] Routine procedures and ingredients for selecting and preparing suitable formulations, for example, at Remington, JP The Science and Practice of Pharmacy , Easton, PA. Mack Publishers, 2012, 22nd Edition and The United States Pharmacopeial Convention, The National Formulary It is described in United States Pharmacopeial, 2015, USP 38 NF 33.

[0374] Under normal storage and use conditions, the pharmaceutical composition may contain preservatives, for example, to prevent microbial growth. The pharmaceutical composition may comprise a sterile aqueous solution, a dispersion, or, for example, a powder for the ad hoc preparation of a sterile solution or dispersion. In all cases, the form may be sterilized using techniques known in the art and fluidized to a degree readily applicable to a subject requiring treatment.

[0375] The pharmaceutical composition may be administered to a subject, for example, a human subject, alone or in combination with a pharmaceutically acceptable carrier as described herein, in proportions determined by the solubility and / or chemical properties of the compound, the chosen route of administration, and standard pharmaceutical practice.

[0376] Dosing regimen Physicians with ordinary skill in the art can readily determine the effective amount of siRNA molecules to be administered to a mammalian subject (e.g., a human) in need. For example, a physician may begin a prescribed dose of one of the disclosed siRNA molecules at a level below that required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. Alternatively, a physician may begin a treatment regimen by administering a high dose of one of the disclosed siRNA molecules and then gradually administer lower doses until the lowest dose is reached to achieve a therapeutic effect (e.g., a reduction in the expression of the target gene sequence). Generally, a suitable daily dose of one of the disclosed siRNA molecules will be the amount of the lowest dose of siRNA molecules that effectively produces a therapeutic effect. The disclosed ss- or ds-siRNA molecules may be administered by injection, for example, intrathecal, intraventricular, via catheter insertion into the cerebellomedullary cistern, intraparenchymal, intravenous, subcutaneous, or intramuscular administration. The daily dose of the therapeutic composition of the disclosed siRNA molecules may be administered as a single dose or as two, three, four, five, six, or more doses administered individually at appropriate intervals over a day, week, month, or year, optionally in unit dosage form. Although the siRNA molecule disclosed herein can be administered alone, it can also be administered as a pharmaceutical formulation in combination with excipients, carriers, and optionally additional therapeutic agents.

[0377] Application route The methods disclosed herein take into account any route of administration to which the therapeutic composition is tolerated. Some embodiments of the methods include intrathecal injection, intraventricular injection, striatal injection, intraparenchymal injection, or injection via catheter insertion into the cerebellomedullary cistern.

[0378] Intrathecal injection involves direct injection into the spinal cord or subarachnoid space. By direct injection into the spinal CSF, the siRNA molecules disclosed herein can directly enter cells within the spinal cord (e.g., neurons and microglia) and have pathways to bypass the blood-brain barrier and enter cells in the brain.

[0379] Intraventricular ventricle (ICV) injection is a method of direct injection into the cerebrospinal fluid (CSF) of the brain. Similar to intrathecal injection, ICV is an injection method that bypasses the blood-brain barrier. The advantage of using ICV is that it can enter cells in the brain and spinal cord without the risk of therapeutic agent degradation in the bloodstream.

[0380] Intrastriatal injection involves direct injection into the striatum, a region of the basal ganglia beneath the brain cortex. Injection into the striatum bypasses the blood-brain barrier and the pharmacokinetic challenges of blood flow, allowing direct access to brain cells.

[0381] Intraplasmic administration involves direct injection into the parenchyma (e.g., brain parenchyma). Injection into the brain parenchyma allows direct administration to brain regions affected by disease or condition, while bypassing the blood-brain barrier.

[0382] Injection via catheter insertion into the cerebellomedullary cistern involves direct injection into the cistern, a brain region located between the dorsal surface of the cerebellum and medulla oblongata. Injection into the cistern induces more direct delivery of cells to the cerebellum, brainstem, and spinal cord.

[0383] In some embodiments of the methods described herein, the therapeutic composition may be delivered to the subject via systemic administration (e.g., intravenous, intramuscular, or subcutaneous).

[0384] Intravenous (IV) administration is a method of direct injection into the bloodstream of a subject. IV administration can be in the form of a bolus dose or by continuous infusion, or any other method of treatment for tolerance of the therapeutic composition.

[0385] Intramuscular (IM) injection is administered into a muscle of the recipient, such as the deltoid or gluteal muscles. IM allows for rapid absorption of the therapeutic composition.

[0386] Subcutaneous injection involves injecting the medication into the subcutaneous tissue. The absorption of subcutaneously delivered compositions may be slower than with IV or IM injections, which can be advantageous for compositions requiring continuous absorption.

[0387] Example The following embodiments are provided to provide a description of how the compositions and methods described herein can be used, prepared, and evaluated by those skilled in the art, and the embodiments are intended only as examples of this disclosure and not to limit the scope of the disclosure as believed by the inventors.

[0388] Example 1. Effect of fixed nucleobase sequence at the 3' end of the antisense strand of ds-siRNA molecule This embodiment demonstrates the ability of the disclosed siRNA molecule to target PRNPs with a fixed nucleotide sequence at the 3' end of the antisense strand. An siRNA molecule with two uridines at the 3' end of the antisense strand was compared with a matching sequence.

[0389] PRNP-targeting siRNA molecules were delivered to HeLa cells via lipid-mediated cellular uptake (RNAiMax). HeLa cells were seeded using RNAiMax and simultaneously transfected with different concentrations of siRNA molecules. PRNP mRNA expression was measured 24 hours post-transfection. Figure 1 Table 3 demonstrates that each siRNA molecule effectively silences PRNP.

[0390] Table 3

[0391] In Table 3, O represents phosphate diester nucleoside linkage, S represents thiophosphate nucleoside linkage, mG represents 2'-methoxyguanosine, mC is 2'-methoxycytosine, mA is 2'-methoxyadenosine, mU is 2'-methoxyuridine, and fU is 2'-fluorouridine.

[0392] Example 2. Effect of including modified nucleoside-to-nucleotide linkages or modified ribose moieties in the fixed nucleotide sequence at the 3' end of the antisense strand of the ds-siRNA molecule. This embodiment demonstrates the advantages of including modified internucleotide links in the fixed nucleobase region at the 3' end of the antisense strand of the siRNA molecule. Modified internucleotide links include modified internucleotide links of any one of formulas E1-E15 (e.g., modified internucleotide links of formula E6a) or phosphate thioester internucleotide links. Furthermore, this embodiment investigates the effects of including modified ribose moieties (e.g., 2'-methoxynucleoside and / or 2'-fluoronucleoside).

[0393] PRNP Tg26378 mice aged 7 to 8 weeks were injected bilaterally into the intracerebroventricular tract using either PBS control or targeted PBS. PRNP siRNA molecules from mRNA transcripts were injected at a rate of 2.0 µL / min, 10 µL / animal (5 µL / ventilator). Tissues were collected from the frontal cortex (fCtx), motor cortex (mCTx), striatum (Cpu), and hippocampus (HP). For each test condition, N=8. The concentrations of siRNA in tissues relative to the PBS control were analyzed. PRNP The amount of mRNA expressed was measured. Each condition was tested using injections of 0.2 nmol, 1 nmol, and 5 nmol of siRNA molecules. The siRNA molecules tested are described in Table 4 below.

[0394] Table 4.

[0395] The results of the experiment are shown in Figure 2A In 2B (5 nmol injection), 2C (1 nmol injection), and 2D (0.2 nmol injection), the results were reported as measured relative to control mice treated with PBS. PRNP % of mRNA. These results demonstrate that, including fixing the nucleobase sequence, effectively silences mRNA in vivo. PRNP mRNA.

[0396] Example 3. siRNA molecules with fixed nucleobase regions This embodiment further demonstrates the advantages of including modified internucleotide links in the fixed nucleobase region at the 3' end of the antisense strand of the siRNA molecule. Modified internucleotide links include modified internucleotide links of any one of formulas E1-E15 (e.g., modified internucleotide links of formula E6a) or phosphate thioester internucleotide links. Furthermore, this embodiment investigates the effects of including modified ribose moieties (e.g., 2'-methoxynucleoside and / or 2'-fluoronucleoside).

[0397] As described in Example 2, PBS control or targeted PBS was administered to 8- to 9-week-old FVB / N female mice via bilateral intraventricular injection. HPRT1 siRNA molecules from mRNA transcripts. Tissues were collected from the frontal cortex (fCtx), motor cortex (mCTx), striatum (Cpu), and hippocampus (HP). For each assay condition, N = 8–10. After 28 days, tissues were analyzed relative to a PBS control. HPRT1 mRNA expression was tested under each condition using either 1 nmol or 3 nmol of siRNA molecules. The siRNA molecules tested are described in Table 5 below.

[0398] Table 5.

[0399] The result is Figure 3A (3 nmol) and Figure 3B These results are shown in (1 nmol). These results demonstrate that, including the effective silencing of fixed nucleobase sequences in vivo, [the following is a description of the results]. HPRT1 mRNA.

[0400] In separate experiments, the siRNA molecules described in Table 5 were tested in vitro at various doses using HeLa cells and a 10pt series: 100 nM and 10-fold decreased to 0.00000001 nM and simulated treatment. Cells were reverse stained with 0.1% RNAiMAX, 0.5% FBS, and 0.5% penicillin-streptomycin, and their IC50 values ​​were calculated. Results are as follows... Figure 3C (Comparison of all siRNA molecules), 3D (comparison of formula F1 containing a terminal 2'-methoxy nucleoside with formula F6 containing a terminal 2'-fluoro nucleoside), and 3E (comparison of formula F1 with formula F5, wherein a modified nucleoside linker of formula E6a has been added). IC50 values ​​are also reported in Table 6 below.

[0401] Table 6

[0402] Example 4. Evaluation of all possible fixed nucleobase sequences at the 3' end of the antisense strand of the ds-siRNA molecule. In this experiment, the effects of all possible trinucleotide sequences (i.e., the 3' sequences in Table 1a) were examined. This embodiment used a targeting system with a 21-mer antisense strand and a 16-mer sense strand. HPRT1 The ds-siRNA molecule. The three terminal nucleobases of the 3' end of the antisense strand were fixed to the sequences in Table 1a, independent of the target mRNA transcript. These fixed nucleobase regions were chemically modified as shown in Formula F1.

[0403] HeLa cells were transfected with siRNA molecules at doses ranging from 0.00000001 nM to 100 nM. The IC50 values ​​for each assay condition were then calculated, and the results are shown in [the table / data]. Figure 4 The lowest IC50 value (1.9 pM) was observed for the GGU sequence, while the highest IC50 value (165 pM) was observed for the GUC sequence. The IC50 values ​​are also reported in Table 7 below.

[0404] Table 7. This example demonstrates that the identity of the 3' nucleobase sequence of the antisense strand affects the silencing activity of the siRNA molecule.

[0405] Example 5. Method for delivering ds-siRNA molecules to the central nervous system of a patient. Subjects diagnosed with a disease (such as human subjects) are treated by administration of the siRNA molecule of this disclosure or a pharmaceutical composition containing said siRNA molecule at a dose and frequency determined by the practitioner (e.g., three times daily, twice daily, once daily, once weekly, once monthly). The dose and frequency are determined based on the subject's height, weight, age, sex, and other conditions.

[0406] siRNA molecules with the chemical modification patterns disclosed herein (e.g., branched siRNA molecules) are selected by practitioners based on compatibility with the disease and the patient. Single-stranded or double-stranded branched siRNAs are available. The selected siRNA has an antisense strand, and in the case of double-stranded siRNA, a sense strand, the sequence and RNA modifications of which (e.g., natural and non-natural nucleoside linkages, modified sugars, and 5'-phosphorus stabilizing moieties) are best suited to the targeted patient and disease. For example, the antisense strand may have any of the antisense strand modification patterns disclosed herein, such as, for example, antisense pattern 1: ASBSAOBOBOBOAOBOAOBOA-OBOAOBOAOBSASASASASA (Formula A1); antisense pattern 2: ASBSAOBOAOBOAOBOAOBOA-OBOAOBOAOBSASBSASASA (Formula A2); or antisense pattern 3: ASBSAOAOAOBOAOAOAOAOA-OAOAOBOAOBSASASASBSA (Formula A3). In the case of ds-siRNA, antisense mode 1 may have a fully or partially complementary sense strand, which has any of the following chemical modification modes: sense mode 1: ASASAOBOAOBOAOBOAOBOA-OAOAOBSASA (formula S1); sense mode 2: ASASAOBOAOBOAOBOAOBOA-OAOAOBOAOA (formula S2); sense mode 3: ASASAOBOAOBOAOBOAOBOA-OAOAOBSASB (formula S3); or sense mode 4: ASASAOBOAOBOAOBOAOBOA-OAOAOBOAOB (formula S4).In the case of ds-siRNA with antisense pattern 2, the sense strand may have any 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 ds-siRNA with antisense pattern 3, the sense strand may have a modified pattern with sense pattern 9: ASASAOAOAOBOBOBOAOBOA-OAOAOASASA (Formula S9); where A and B are different nucleosides (e.g., A is 2-O-methylribonucleoside; B is 2'-fluororibonucleoside), T is thiophosphate, P is phosphodiester, and PSM is the 5'-phosphorus stabilizing moiety (e.g., 5'-vinylphosphonate).

[0407] siRNA is delivered at a rate tolerable to the patient via the route most suitable for the patient and disease (e.g., intrathecal, intraventricular, or striatum) until the subject reaches the maximum tolerated dose or until the symptoms of the disease are satisfactorily improved.

[0408] Example 6. Optimization of siRNA molecules For any small interfering RNA (siRNA) agent disclosed herein, modifications to the siRNA are expected to further optimize the molecule's efficacy or biophysical properties (e.g., increased serum stability or circulating half-life, increased thermal stability, enhanced transmembrane delivery, and / or targeting of specific sites or cell types). Such optimization can be achieved by systematically adding or removing linked nucleosides to generate longer or shorter sequences. Further siRNA optimization may include, for example, incorporating one or more alternative nucleosides, replacing the 2' sugar moiety, and / or replacing inter-nucleoside linkages. Furthermore, such optimized siRNA molecules may include the introduction of hydrophobic and / or stable moieties at the 5' and / or 3' ends.

[0409] siRNA optimization using alternative nucleosides The optimization of the siRNA molecule disclosed herein may include one or more of the following nucleoside modifications: 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymidine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C=C-CH3)uracil and other alkynyl derivatives of cytosine and pyrimidine bases, 6-azo Uracil, cytosine, and thymine; 5-uracil (pseudouracil); 4-thiouracil; 8-halogenated, 8-amino, 8-mercapto, 8-thioalkyl, 8-hydroxy, and other 8-substituted adenine and guanine; 5-halogenated, especially 5-bromo, 5-trifluoromethyl, and other 5-substituted uracil and cytosine; 7-methylguanine and 7-methyladenine; 2-F-adenine; 2-amino-adenine; 8-azaguanine and 8-azaadenine; 7-deadenine and 7-deadenine; and / or 3-deadenine and 3-deadenine. siRNA molecules may also include nucleobases in which a purine or pyrimidine base is replaced by another heterocycle such as 7-deadenine, 7-deadenine, 2-aminopyridine, and / or 2-pyridone. Further optimization of the siRNA molecule disclosed herein may include the nucleobases disclosed in the following literature: US 3,687,808; Kroschwitz, JI (edited); The Concise Encyclopedia of Polymer Science and Engineering, New York, John Wiley & Sons, 1990, pp. 858-859; Englisch et al. Angewandte Chemie, International Edition, 30:613, 1991; and Sanghvi, YS, Chapter 16. Antisense Research and Applications, CRC Press, Gait, MJ (eds.), 1993, pp. 289-302.

[0410] siRNA optimization using alternative sugar modifications The optimization of the siRNA molecule disclosed herein may include one or more of the following 2' sugar modifications: 2'-O-methyl (2'-O-Me); 2'-methoxyethoxy (2′-O-CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE); 2'-dimethylaminooxyethoxy, i.e., the O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE; and / or 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylamino-ethoxy-ethyl or 2'-DMAEOE), i.e., 2′-O-CH2OCH2N(CH3)2. Other possible 2′-modifications that can optimize the siRNA molecule of this disclosure include OH, F, O-alkyl, S-alkyl, or N-alkyl; O-alkenyl, S-alkenyl, or N-alkenyl; O-ynyl, S-ynyl, or N-ynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl, and ynyl groups can be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and ynyl groups. Other possible sugar substituents include, for example, aminopropoxy (-OCH2CH2CH2NH2), allyl (-CH2-CH=CH2), -O-allyl (-O-CH2-CH=CH2), and fluorine (F). The 2′-sugar substituent can be located at the arabinose (top) position or the ribose (bottom) position. In some embodiments, the 2′-arabinose modification is 2′-F. Similar modifications can be made at other positions on the siRNA molecule, specifically at the 3' terminal nucleotide or at the 3' position of the sugar and the 5' position of the 5' terminal nucleotide in oligonucleotides linked at 2'-5'. Oligonucleotides can also have sugar mimics such as a cyclobutyl moiety to replace the pentofuranosyl sugar.

[0411] siRNA optimization using alternative nucleoside-to-nucleotide linkages The optimization of the siRNA molecules disclosed herein may include one or more of the following internucleotide modifications: thiophosphate, dithiophosphate, phosphate triester, aminoalkyl phosphate triester, methyl and other alkylphosphonates (including 3'-alkylphosphonene esters, 5'-alkylphosphonene esters), phosphonates, aminophosphates (including 3'-aminoaminophosphate and aminoalkylaminophosphate), thioaminophosphate, thioalkylphosphonate, thioalkyl phosphate triester, selenophosphate and borophosphates having normal 3'-5' linkages, analogs of these with 2'-5' linkages, and those with antipolarity, wherein one or more internucleotide linkages are 3' to 3', 5' to 5', or 2' to 2' linkages.

[0412] siRNA optimization using the hydrophobic portion Optimization of the siRNA molecules disclosed herein may include covalently attached hydrophobic portions to the 5' or 3' ends. Non-limiting examples of hydrophobic portions suitable for use with the siRNA molecules disclosed herein may include cholesterol, vitamin D, tocopherol, phosphatidylcholine (PC), docosahexaenoic acid, docosanoic acid, PC-docosaanoic acid, eicosapentaenoic acid, lithocholic acid, or any combination of the above hydrophobic portions with PC.

[0413] siRNA optimization using the stable portion Optimization of the siRNA molecules disclosed herein may include a 5'-phosphate-stabilizing moiety that protects the siRNA molecule from degradation. The 5'-phosphate-stabilizing moiety replaces the 5'-phosphate ester to prevent phosphate ester hydrolysis. Hydrolysis of the 5'-phosphate ester prevents binding to RISC, a necessary step in gene silencing. Any phosphate ester replacement that does not impede binding to RISC is considered in this disclosure. In some embodiments, the 5'-phosphate ester substitute is also stable against in vivo hydrolysis. Each siRNA strand may independently and optionally employ any suitable 5'-phosphate-stabilizing moiety. Non-limiting examples of 5'-stabilizing moieties suitable for use with the siRNA molecules of this disclosure may include those shown in Formulas IX-XVI above.

[0414] siRNA optimization using branched siRNA Optimization of the siRNA molecules disclosed herein may include incorporating branching patterns, such as two-, three-, or four-branched siRNAs linked by a linker. Each main branch may further branch to allow 2, 3, 4, 5, 6, 7, or 8 separate single- or double-stranded RNAs. Branching points at the linker may originate from the same atom or from separate atoms along the linker. Some exemplary embodiments are listed in Table 2 above.

[0415] The siRNA compositions disclosed herein can be optimized to the following forms: bibranched siRNA molecules, as represented by any one of formulas XVII-XIX; tribranched siRNA molecules, as represented by any one of formulas XX-XXIII; and / or tetrabranched siRNA molecules, as represented by any one of formulas XXIV-XXVIII, wherein each RNA is independently an siRNA molecule, L is a linker, and each X independently represents a branch point portion (e.g., phosphoramide, toluenesulfonated acetone glycerol, 1,3-diaminopropanol, pentaerythritol, or any one of the branch point portions described in US 10,478,503).

[0416] Example 7. Preparation and administration of siRNA molecules The siRNA molecules of this disclosure can be formulated into pharmaceutical compositions for administration to subjects in a biocompatible form suitable for in vivo administration. For example, the siRNA molecules of this disclosure can be administered in suitable diluents, carriers, or excipients, and may also contain preservatives, for example, to prevent microbial growth. Conventional procedures and ingredients for selecting and preparing suitable formulations are available, for example, at Remington, JP. The Science and Practice of Pharmacy , Easton, PA.Mack Publishers, 2012, 22nd Edition and The United States Pharmacopeial Convention, The National Formulary It is described in United States Pharmacopeial, 2015, USP 38 NF 33.

[0417] The methods of this disclosure consider any route of administration to the CNS of a subject that is tolerable for the siRNA compositions of this disclosure. Non-limiting examples of siRNA injection into the CNS include intrathecal injection, intraventricular injection, or injection into the cerebellomedullary cistern via catheter insertion. A physician with ordinary skill in the art can readily determine an effective route of administration.

[0418] Example 8. Method for treating subjects requiring gene silencing Subjects requiring gene silencing will be treated with a dose of the disclosed siRNA molecule or siRNA composition (prepared as a salt) at a frequency determined by the practitioner. A physician with ordinary skill in the art can readily determine the effective amount of siRNA molecule to administer to a mammalian subject (e.g., a human) in need. For example, a physician may begin a prescribed dose of one of the disclosed siRNA molecules at a level below that required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. Alternatively, a physician may begin a treatment regimen by administering a high dose of one of the disclosed siRNA molecules and then gradually administer lower doses until the minimum dose required to produce a therapeutic effect (e.g., a reduction in the expression of the target mRNA or a suitable biomarker) is reached. Generally, an appropriate daily dose of one of the disclosed siRNA molecules will be the minimum dose required to effectively produce a therapeutic effect. The disclosed ss- or ds-siRNA molecules can be administered by injection, for example, intrathecal, intraventricular, or via catheter insertion into the cerebellomedullary cistern. The daily dose of a therapeutic composition of one of the siRNA molecules disclosed herein can be administered as a single dose, or as two, three, four, five, six or more doses administered alone at appropriate intervals throughout a day, week, month or year, optionally in unit dosage form. While one of the siRNA molecules disclosed herein can be administered alone, it can also be administered as a pharmaceutical formulation in combination with excipients, carriers and optionally additional therapeutic agents. Dosage and frequency are determined based on the subject's height, weight, age, sex and other conditions.

[0419] One or more siRNA molecules disclosed herein are selected by the practitioner based on compatibility with the subject. Single-stranded or double-stranded siRNA molecules (e.g., unbranched siRNA, bibranched siRNA, tribranched siRNA, tetrabranched siRNA, covalently linked siRNA) are available. The selected siRNA molecule has an antisense strand and may also have a sense strand, the sequence and RNA modifications of which (e.g., natural and non-natural nucleoside linkages, modified sugars, 5'-phospho-stabilizing moieties, hydrophobic moieties, and / or branching structures) being most suitable for the patient.

[0420] siRNA molecules are delivered at a rate tolerable by the most suitable route for the patient (e.g., intrathecal, intraventricular, or via catheter insertion into the cerebellomedullary cistern) and the disease, until the subject reaches the maximum tolerated dose or until symptoms are satisfactorily improved.

[0421] Other implementation plans All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication or patent application is specifically and individually identified and incorporated by reference.

[0422] Although the invention has been described in conjunction with its specific embodiments, it should be understood that further modifications can be made to it, and this application is intended to cover any variations, uses, or adaptations of the invention based on the principles of the invention, including known or conventional practices that, while not part of the invention, fall within the scope of the invention and changes that fall within the essential features described above and are within the scope of the claims.

[0423] Other embodiments are defined in the claims.

Claims

1. A small interfering RNA (siRNA) molecule comprising an antisense strand and a sense strand having complementarity to a portion of the antisense strand; wherein: (i) the antisense strand comprises, in the 5' to 3' direction, a first region of linked nucleotides and a second region of linked nucleotides; (ii) the first region has complementarity sufficient to hybridize to a portion of a target mRNA transcript; (iii) the second region comprises an overhang extending beyond the sense strand; and (iv) the second region has one or more nucleotide mismatches relative to the target mRNA transcript.

2. A siRNA molecule comprising an antisense strand and a sense strand having complementarity to a portion of the antisense strand; wherein: (i) the antisense strand comprises, in the 5' to 3' direction, a first region of linked nucleotides and a second region of linked nucleotides; (ii) the first region has complementarity sufficient to hybridize to a portion of a target mRNA transcript; (iii) the second region comprises an overhang extending beyond the sense strand; and (iv) the second region has a fixed nucleobase sequence independent of the nucleobase sequence of the target mRNA transcript.

3. The siRNA molecule of claim 1 or 2, wherein upon introduction of the siRNA molecule into an RNA-induced silencing complex (RISC) and exposure of the RISC to the target mRNA transcript, the target mRNA transcript is cleaved, thereby forming a cleavage product, and the cleavage product dissociates from the RISC at an increased off-rate relative to a RISC formed from a corresponding siRNA comprising an antisense strand that is perfectly complementary to the target mRNA transcript.

4. The siRNA molecule of any one of claims 1-3, wherein the nucleobase sequence of the second region confers increased binding affinity of the antisense strand to an endogenous Argonaute (AGO) protein relative to a corresponding antisense strand that is perfectly complementary to the target mRNA.

5. The siRNA molecule of any one of claims 1-4, wherein the nucleobase sequence of the second region improves the half-life of an endogenous complex comprising the antisense strand and an AGO protein relative to a corresponding endogenous complex comprising a corresponding antisense strand that is perfectly complementary to the target mRNA.

6. The siRNA molecule of any one of claims 1-5, wherein the second region is 1 to 10 nucleotides in length, optionally wherein the second region is 1 to 6 nucleotides in length.

7. The siRNA molecule of claim 6, wherein the second region is 1 nucleotide in length.

8. The siRNA molecule of claim 6, wherein the second region is 2 nucleotides in length.

9. The siRNA molecule of claim 6, wherein the second region is 3 or 4 nucleotides in length.

10. The siRNA molecule of any one of claims 1-9, wherein the second region comprises at least one uridine nucleotide. ​ ​ ​ ​ ​ ​ ​ ​ 11. The siRNA molecule of claim 10, wherein the second region comprises two uridine nucleotides.

12. The siRNA molecule of any one of claims 1-11, wherein the second region comprises at least one modified internucleoside linkage.

13. The siRNA molecule of claim 12, wherein the second region comprises at least one modified internucleoside linkage of Formula El: (E1)； wherein: each B is independently a base pairing moiety; W is selected from the group consisting of O, S, B, BR 2 , N, NR 2 , OCH2, OCH, CH2, and CH; each X is independently selected from the group consisting of halo, hydroxyl, and C 1-6 alkoxy; Y is selected from the group consisting of: O - , OH, OR, NH - , NH2, S - , and SH; Z is selected from the group consisting of O, S, BR 2 , NR 2 , and CH2; R is a protecting group; Each R 2 Independently H or optionally substituted C1-C6 alkyl; and is an optional double bond.

14. The siRNA molecule of claim 13, wherein when Y is O - Z or W is not O.

15. The siRNA molecule of claim 13, wherein Z is CH2and W is CH2.

16. The siRNA molecule of claim 12, wherein the modified internucleoside linkage of Formula El is a modified internucleoside linkage of Formula E2: (E2)。 17. The siRNA molecule of claim 13, wherein Z is CH2and W is O.

18. The siRNA molecule of claim 12, wherein the modified internucleoside linkage of Formula El is a modified internucleoside linkage of Formula E3: (E3)。 19. The siRNA molecule of claim 13, wherein Z is O and W is CH2.

20. The siRNA molecule of claim 12, wherein the modified internucleoside linkage of Formula El is a modified internucleoside linkage of Formula E4: (E4)。 21. The siRNA molecule of claim 13, wherein Z is O and W is CH.

22. The siRNA molecule of claim 12, wherein the modified internucleoside linkage of Formula El is a modified internucleoside linkage of Formula E5: (V)。 23. The siRNA molecule of claim 13, wherein Z is O and W is OCH2.

24. The siRNA molecule of claim 12, wherein the modified internucleoside linkage of Formula El is a modified internucleoside linkage of Formula E6: (E6) wherein each X is independently selected from the group consisting of fluorine, hydroxyl, and C 1-6 alkoxy; Y is selected from the group consisting of O - , OH, OR, S - , SH, and SR; Z is selected from the group consisting of O and CH2; and is an optional double bond.

25. The siRNA molecule of claim 24, wherein the modified internucleoside linkage of Formula El is a modified internucleoside linkage of Formula E6a: (E6a).

26. The siRNA molecule of claim 13, wherein Z is CH2and W is CH.

27. The siRNA molecule of claim 12, wherein the modified internucleoside linkage of Formula El is a modified internucleoside linkage of Formula E7: (E7)。 28. The siRNA molecule of any one of claims 1-27, wherein the second region comprises at least one phosphorothioate internucleoside linkage.

29. The siRNA molecule of any one of claims 1-28, wherein the second region comprises at least one nucleotide comprising a modified ribose.

30. The siRNA molecule of claim 29, wherein the second region comprises at least one 2’- methoxy nucleotide.

31. The siRNA molecule of claim 29 or 30, wherein the second region comprises at least one 2’- fluoro nucleotide.

32. The siRNA molecule of any one of claims 1-31, wherein the second region has any one of the following sequences in the 5' to 3' direction: -S-A-S-A-S-A (Formula F1) -O-A-S-A-S-A (Formula F2) -O-A-S-XA-S-XB (Formula F3) -S-A-S-XA-S-XB (Formula F4) -S-A-S-XA-S-XA (Formula F5) -S-A-S-A-S-B (Formula F6) wherein each S is a phosphorothioate internucleoside linkage; each O is a phosphodiester internucleoside linkage; each A is a 2'-methoxyribose nucleoside; each B is a 2'-fluoro ribose nucleoside; each XA is a 2'-methoxy nucleotide of Formula E6a; and each XB is a 2'-fluoro nucleotide of Formula E6a.

33. The siRNA molecule of claim 32, wherein the second region has any one of the following sequences in the 5' to 3' direction: -S-(mA)-S-(mG) (Formula F7) -S-(mA)-S-(mU)-S-(mU) (Formula F8) -O-(mA)-S-(mU)-S-(mU) (Formula F9) -O-(mA)-S-(xU)-S-(yU) (Formula F10) -S-(mA)-S-(xU)-S-(yU) (Formula F11) -S-(mA)-S-(xU)-S-(xU) (Formula F12) -S-(mA)-S-(mU)-S-(fU) (Formula F13) wherein each S is a phosphorothioate internucleoside linkage, each O is a phosphodiester internucleoside linkage, each mA is a 2'-methoxyadenosine, each mG is a 2'-methoxy guanine, each mU is a 2'-methoxy uridine, each xU is a 2'methoxy uridine of Formula E6a, and each yU is a 2'-fluoro uridine of Formula E6a.

34. The siRNA molecule of any one of claims 1-33, wherein the antisense strand comprises a structure represented by Formula I, wherein Formula I is in the 5' to 3' direction: Formula I; each C is a 2’-O-methyl (2’-O-Me) ribose nucleoside; A-B-(A') j -C-P 2 -D-P 1 -(C'-P 1 ) k -C' each C’ is independently a 2’-O-Me ribose nucleoside or a 2’-fluoro (2’-F) ribose nucleoside; wherein A is represented by formula C-P 1 -D-P 1 ; each A' is represented by formula C-P 2 -D-P 2 ; B is represented by the formula C-P 2 -D-P 2 -D-P 2 -D-P 2 ; each D is a 2’-F ribose nucleoside; j is an integer from 1 to 7; and k is an integer from 1 to 7. each P 1 is a phosphorothioate internucleosidic linkage; each P 2 is a phosphodiester internucleosidic linkage; 35. The siRNA molecule of claim 34, wherein the antisense strand comprises a structure represented by Formula A1, wherein Formula A1 is in the 5' to 3' direction: A-S-B-S-A-O-B-O-B-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-S-A-S-A-S-A-S-B-S-A Formula A1; wherein A represents a 2’-O-Me ribose nucleoside, B represents a 2’-F ribose nucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage. ​ ​ ​ 36. The siRNA molecule of any one of claims 1-33, wherein the antisense strand comprises a structure represented by Formula II, wherein Formula II is in the 5' to 3' direction: A-B-(A') j -C-P 2 -D-P 1 -(C-P 1 ) k -C' Formula II; wherein A is represented by formula C-P 1 -D-P 1 ; each A' is represented by formula C-P 2 -D-P 2 ; B is represented by the formula C-P 2 -D-P 2 -D-P 2 -D-P 2 ; each C is a 2'-0-methyl (2'-0-Me) ribose nucleoside; each C' is independently a 2'-0-Me ribose nucleoside or a 2'-fluoro (2'-F) ribose nucleoside; each D is a 2'-F ribose nucleoside; each P 1 is a phosphorothioate internucleosidic linkage; each P 2 is a phosphodiester internucleosidic linkage; j is an integer from 1 to 7; and k is an integer from 1 to 7.

37. The siRNA molecule of claim 36, wherein the antisense strand comprises a structure represented by Formula A2, wherein Formula A2 is in the 5' to 3' direction: A-S-B-S-A-O-B-O-B-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-S-A-S-A-S-A-S-A-S-A Formula A2; wherein A represents a 2'-0-Me ribose nucleoside, B represents a 2'-F ribose nucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

38. The siRNA molecule of any one of claims 1-37, wherein the sense strand comprises a structure represented by Formula III, wherein Formula III is in the 5' to 3' direction: E-(A') m -F Formula III; wherein E is represented by formula (C-P 1 )2; F from the formula (C-P 2 )3-D-P 1 -C-P 1 -C, (C-P 2 )3-D-P 2 -C-P 2 -C, (C-P 2 )3-D-P 1 -C-P 1 -D or (C-P 2 )3-D-P 2 -C-P 2 -D means; A', C, D, P 1 and P 2 as defined in formula II; and m is an integer from 1 to 7.

39. The siRNA molecule of claim 38, wherein the sense strand comprises a structure represented by Formula S1, wherein Formula S1 is in the 5' to 3' direction: A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-A-O-A-O-B-S-A-S-A Formula S1; wherein A represents a 2'-0-Me ribose nucleoside, B represents a 2'-F ribose nucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

40. The siRNA molecule of claim 38, wherein the sense strand comprises a structure represented by Formula S2, wherein Formula S2 is in the 5' to 3' direction: A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-A-O-A-O-B-O-A-O-A Formula S2; wherein A represents a 2'-0-Me ribose nucleoside, B represents a 2'-F ribose nucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

41. The siRNA molecule of claim 38, wherein the sense strand comprises a structure represented by Formula S3, wherein Formula S3 is in the 5' to 3' direction: A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-A-O-A-O-B-S-A-S-B Formula S3; wherein A represents a 2'-0-Me ribose nucleoside, B represents a 2'-F ribose nucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

42. The siRNA molecule of claim 38, wherein the sense strand comprises a structure represented by Formula S4, wherein Formula S4 is in the 5' to 3' direction: A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-A-O-A-O-B-O-A-O-B Formula S4; wherein A represents a 2'-O-Me ribose nucleoside, B represents a 2'-F ribose nucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

43. The siRNA molecule of any one of claims 1-33 and 38-42, wherein the antisense strand comprises a structure represented by Formula IV, wherein Formula IV is in the 5' to 3' direction: A-(A') j -C-P 2 -B-(C-P 1 ) k -C' Formula IV; wherein A is represented by formula C-P 1 -D-P 1 ; each A' is represented by formula C-P 2 -D-P 2 ; B is represented by formula D-P 1 -C-P 1 -D-P 1 ; each C is a 2'-O-Me ribose nucleoside; each C' is independently a 2'-O-Me ribose nucleoside or a 2'-F ribose nucleoside; each D is a 2'-F ribose nucleoside; each P 1 is a phosphorothioate internucleosidic linkage; each P 2 is a phosphodiester internucleosidic linkage; j is an integer from 1 to 7; and k is an integer from 1 to 7.

44. The siRNA molecule of claim 43, wherein the antisense strand comprises a structure represented by Formula A3, wherein Formula A3 is in the 5' to 3' direction: A-S-B-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-S-A-S-B-S-A-S-A-S-A Formula A3; wherein A represents a 2'-O-Me ribose nucleoside, B represents a 2'-F ribose nucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

45. The siRNA molecule of any one of claims 1-37, 43, and 44, wherein the sense strand comprises a structure represented by Formula V, wherein Formula V is in the 5' to 3' direction: E-(A') m -C-P 2 -F Formula V; wherein E is represented by formula (C-P 1 )2; F from formula D-P 1 -C-P 1 -C, D-P 2 -C-P 2 -C, D-P 1 -C-P 1 -D or D-P 2 -C-P 2 -D means; A', C, D, P 1 and P 2 as defined in formula IV; and m is an integer from 1 to 7.

46. The siRNA molecule of claim 45, wherein the sense strand comprises a structure represented by Formula S5, wherein Formula S5 is in the 5' to 3' direction: A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-S-A-S-A Formula S5; wherein A represents a 2'-O-Me ribose nucleoside, B represents a 2'-F ribose nucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

47. The siRNA molecule of claim 45, wherein the sense strand comprises a structure represented by Formula S6, wherein Formula S6 is in the 5' to 3' direction: A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-A Formula S6; wherein A represents a 2'-O-Me ribose nucleoside, B represents a 2'-F ribose nucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

48. The siRNA molecule of claim 45, wherein the sense strand comprises a structure represented by Formula S7, wherein Formula S7 is in the 5' to 3' direction: A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-S-A-S-B Formula S7; wherein A represents a 2'-0-Me ribose nucleoside, B represents a 2'-F ribose nucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

49. The siRNA molecule of claim 45, wherein the sense strand comprises a structure represented by Formula S8, wherein Formula S8 is in the 5' to 3' direction: A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B Formula S8; wherein A represents a 2'-0-Me ribose nucleoside, B represents a 2'-F ribose nucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

50. The siRNA molecule of any one of claims 1-33, 38-42, and 45-49, wherein the antisense strand comprises a structure represented by Formula VI, wherein Formula VI is in the 5' to 3' direction: A-B j -E-B k -E-F-G l -D-P 1 -C’ Formula VI; wherein A is represented by formula C-P 1 -D-P 1 ; each B is represented by the formula C-P 2 ; each C is a 2'-0-Me ribose nucleoside; each C' is independently a 2'-0-Me ribose nucleoside or a 2'-F ribose nucleoside; each D is a 2'-F ribose nucleoside; each E is represented by formula D-P 2 -C-P 2 ; F is of the formula D-P 1 -C-P 1 represents; each G is represented by formula C-P 1 ; each P 1 is a phosphorothioate internucleosidic linkage; each P 2 is a phosphodiester internucleosidic 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.

51. The siRNA molecule of claim 50, wherein the antisense strand comprises a structure represented by Formula A4, wherein Formula A4 is in the 5' to 3' direction: A-S-B-S-A-O-A-O-A-O-B-O-A-O-A-O-A-O-A-O-A-O-A-O-A-O-B-O-A-O-B-S-A-S-A-S-A-S-B-S-A Formula A4; wherein A represents a 2'-0-Me ribose nucleoside, B represents a 2'-F ribose nucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

52. The siRNA molecule of any one of claims 1-37, 43, 44, 50, and 51, wherein the sense strand comprises a structure represented by Formula VII, wherein Formula VII is in the 5' to 3' direction: H-B m -I n -A'-B o -H-C Formula VII; wherein A' is represented by the formula C-P 2 -D-P 2 ; each H is represented by formula (C-P 1 )2; each I is represented by formula (D-P 2 ) ; B, C, D, P 1 and P 2 as defined in formula VI; m is an integer from 1 to 7; n is an integer from 1 to 7; and o is an integer from 1 to 7.

53. The siRNA molecule of claim 52, wherein the sense strand comprises a structure represented by Formula S9, wherein Formula S9 is in the 5' to 3' direction: A-S-A-S-A-O-A-O-A-O-B-O-B-O-B-O-A-O-B-O-A-O-A-O-A-O-A-S-A-S-A 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.

54. The siRNA molecule of any one of claims 1-53, wherein the antisense strand further comprises a 5’ phosphoramidate moiety at the 5’ end of the antisense strand.

55. The siRNA molecule of any one of claims 1-54, wherein the sense strand further comprises a 5’ phosphoramidate moiety at the 5’ end of the sense strand.

56. The siRNA molecule of claim 54 or 55, wherein each 5’ phosphoramidate moiety is independently represented by any one of formulae IX-XVI: wherein Nuc represents a nucleobase selected from the group consisting of adenine, uracil, guanine, thymine, and cytosine, and R represents an optionally substituted alkyl group, an optionally substituted alkenyl group, an optionally substituted alkynyl group, a phenyl group, a benzyl group, a hydroxyl group, or hydrogen.

57. The siRNA molecule of claim 56, wherein the nucleobase is adenine, uracil, guanine, thymine, or cytosine.

58. The siRNA molecule of any one of claims 54-57, wherein the 5’ phosphoramidate moiety is an (E)-vinyl phosphonate represented by formula XI.

59. The siRNA molecule of any one of claims 1-58, wherein the siRNA molecule further comprises a hydrophobic moiety at the 5’ end or 3’ end of the siRNA molecule.

60. The siRNA molecule of claim 59, wherein the hydrophobic moiety is selected from the group consisting of cholesterol, vitamin D, or tocopherol.

61. The siRNA molecule of any one of claims 1-60, wherein the length of the sense strand is 10 to 30 nucleotides.

62. The siRNA molecule of claim 61, wherein the length of the sense strand is 10 to 25 nucleotides.

63. The siRNA molecule of claim 62, wherein the length of the sense strand is 12 to 25 nucleotides.

64. The siRNA molecule of claim 63, wherein the length of the sense strand is 12 to 20 nucleotides.

65. The siRNA molecule of claim 64, wherein the length of the sense strand is 12 to 19 nucleotides.

66. The siRNA molecule of claim 65, wherein the length of the sense strand is 15 nucleotides.

67. The siRNA molecule of claim 65, wherein the length of the sense strand is 16 nucleotides.

68. The siRNA molecule of claim 65, wherein the length of the sense strand is 18 nucleotides.

69. The siRNA molecule of any one of claims 1-68, wherein the length of the antisense strand is 10 to 30 nucleotides.

70. The siRNA molecule of claim 69, wherein the length of the antisense strand is 12 to 30 nucleotides.

71. The siRNA molecule of claim 70, wherein the length of the antisense strand is 15 to 30 nucleotides.

72. The siRNA molecule of claim 71, wherein the antisense strand is 18 to 30 nucleotides in length.

73. The siRNA molecule of claim 72, wherein the antisense strand is 18 to 25 nucleotides in length.

74. The siRNA molecule of claim 73, wherein the antisense strand is 18 to 21 nucleotides in length.

75. The siRNA molecule of claim 74, wherein the antisense strand is 18 nucleotides in length.

76. The siRNA molecule of claim 74, wherein the antisense strand is 20 nucleotides in length.

77. The siRNA molecule of claim 74, wherein the antisense strand is 21 nucleotides in length.

78. The siRNA molecule of any one of claims 1-77, wherein the siRNA molecule is a branched siRNA molecule.

79. The siRNA molecule of claim 78, wherein the branched siRNA molecule is bi- branched, tri-branched, or tetra-branched.

80. The siRNA molecule of claim 79, wherein the siRNA molecule is a bi-branched siRNA molecule, optionally wherein the bi-branched siRNA molecule is represented by any one of formulae XVII-XIX: wherein each RNA is independently a siRNA molecule, L is a linker, and each X independently represents a branch point moiety.

81. The siRNA molecule of claim 79, wherein the siRNA molecule is a tri- branched siRNA molecule, optionally wherein the tri-branched siRNA molecule is represented by any one of formulae XX-XXIII: wherein each RNA is independently a siRNA molecule, L is a linker, and each X independently represents a branch point moiety.

82. The siRNA molecule of claim 79, wherein the siRNA molecule is a tetra- branched siRNA molecule, optionally wherein the tetra-branched siRNA molecule is represented by any one of formulae XXIV-XXVIII: wherein each RNA is independently a siRNA molecule, L is a linker, and each X independently represents a branch point moiety.

83. The siRNA molecule of any one of claims 80-82, wherein the linker is selected from the group consisting of an ethylene glycol, an alkyl, a carbohydrate, a block copolymer, a peptide, one or more contiguous subunits of RNA, and one or more contiguous subunits of DNA.

84. The siRNA molecule of claim 83, wherein the one or more contiguous subunits is 2 to 20 contiguous subunits.

85. A pharmaceutical composition comprising the siRNA molecule of any one of claims 1-84 and a pharmaceutically acceptable excipient, carrier, or diluent.

86. A method of delivering a siRNA molecule to the central nervous system (CNS) of a subject, the method comprising administering to the subject a therapeutically effective amount of the siRNA molecule of any one of claims 1-84 or the pharmaceutical composition of claim 85.

87. A method of reducing expression of a target gene in a subject in need thereof, the method comprising administering to the CNS of the subject a therapeutically effective amount of the siRNA molecule of any one of claims 1-84 or the pharmaceutical composition of claim 85.

88. The method of claim 86 or 87, wherein the siRNA molecule or the pharmaceutical composition is administered to the subject by intrastriatal, intracerebroventricular, or intrathecal injection.

89. The method of claim 87 or 88, wherein the target gene is a disease-driving gene that is overactive.

90. The method of claim 87 or 88, wherein the target gene is a negative regulator of a gene whose expression is decreased in relation to a disease state of the subject.

91. The method of claim 87 or 88, wherein the target gene is a negative regulator of a gene whose expression is increased in relation to a disease state of the subject.

92. The method of claim 87 or 88, wherein the target gene is a splice isoform of the target gene, wherein the splice isoform decreases expression of the target gene.

93. The method of any one of claims 87-92, wherein the decrease in gene expression treats a disease state of the subject.

94. The method of any one of claims 86-93, wherein the subject is a human.

95. A kit comprising the multimeric oligonucleotide of any one of claims 1-84 or the pharmaceutical composition of claim 85 and a package insert, wherein the package insert directs a user of the kit to perform the method of any one of claims 86-94.

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