Compositions and methods for improved gene silencing

Abstract

This disclosure provides single-stranded or double-stranded interfering RNA molecules (e.g., siRNA) that exhibit improved gene silencing. This disclosure provides siRNA molecules having fixed nucleic acid base regions. For example, the antisense strand of an siRNA molecule may have nucleic acid base regions containing one or more mismatches with respect to a target mRNA molecule. Alternatively, or in addition, the antisense strand may contain regions of nucleic acid bases having sequences irrelevant to the target mRNA. The fixed region may be contained within an overhang region of the antisense strand. The siRNA molecule may contain specific patterns of nucleoside modifications and internucleoside linkage modifications, such as in a pharmaceutical composition containing it. The siRNA molecule may be a branched siRNA molecule, e.g., a bibranched, tribranched, or tetrabranched siRNA molecule. The siRNA molecules of this disclosure may further feature a 5' phosphorus-stabilizing moiety and / or a hydrophobic moiety. In addition, this disclosure provides a method for delivering the siRNA molecules of this disclosure to the central nervous system of a target, e.g., a target identified as having a neurodegenerative disease.

Description

[Technical Field]

[0001] This disclosure relates to small interfering RNA (siRNA) molecules exhibiting improved gene silencing and compositions containing the same. This disclosure further describes methods for silencing target genes and treatments of diseases that may benefit from gene silencing by delivering siRNA molecules to target tissues requiring them. [Background technology]

[0002] In many species, the introduction of double-stranded RNA (dsRNA) induces potent 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. Generally, small interfering RNAs (siRNAs) that are significantly shorter than the target gene have been shown to be effective in gene silencing and are therefore useful as therapeutic agents to silence genes and restore the activity of genetic and biochemical pathways from diseased to normal healthy states. However, there is still a need for siRNA constructs that exhibit improved gene silencing. [Overview of the Initiative]

[0003] This disclosure provides compositions and methods for improved gene silencing. Accordingly, this disclosure provides siRNA molecules having fixed nucleic acid base regions. For example, the antisense strand of an siRNA molecule may have nucleic acid base regions containing one or more mismatches with respect to a target mRNA molecule. Alternatively, or in addition, the antisense strand may contain regions of nucleic acid bases having sequences irrelevant to the target mRNA. The fixed region may be contained within the overhang region of the antisense strand.

[0004] The siRNA molecules of this disclosure can be directly delivered to subjects requiring gene silencing by means of intrathecal injection, intraventricular injection, intrastriatal injection, intraparenchymal injection, intracisional injection, intracisional injection, intravenous injection, subcutaneous injection, or intramuscular injection, for example, by catheterization.

[0005] In one embodiment, the present disclosure provides a small interfering RNA (siRNA) molecule comprising an antisense strand and a sense strand complementary to a portion of the antisense strand, wherein (i) The antisense strand includes a first region of the linked nucleotide and a second region of the linked nucleotide in the 5'→3' direction, (ii) The first region has sufficient complementarity to hybridize with a portion of the target mRNA transcript, (iii) The second region includes an overhang that extends beyond the sense chain, (iv) The second region has one or more nucleotide mismatches with respect to the target mRNA transcript.

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

[0007] In another aspect, the present disclosure provides an siRNA molecule comprising an antisense chain and a sense chain complementary to a portion of the antisense chain, wherein, (i) The antisense strand includes a first region of the linked nucleotide and a second region of the linked nucleotide in the 5'→3' direction, (ii) The first region has sufficient complementarity to hybridize with a portion of the target mRNA transcript, (iii) The second region includes an overhang that extends beyond the sense chain, (iv) The second region has a nucleic acid sequence that is unrelated to the nucleic acid sequence of the target mRNA transcript.

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

[0009] In some embodiments of the siRNA molecules described herein, the nucleic acid sequence of the second region results in the formation of an mRNA degradation product with an increased rate of dissociation from the RISC complex after cleavage of the target mRNA, compared to a corresponding antisense strand that is fully complementary to the target mRNA. In some embodiments, the nucleic acid sequence of the second region results in an antisense strand with increased binding affinity to the endogenous Argonaut (AGO) protein compared to a corresponding antisense strand that is fully complementary to the target mRNA. In some embodiments, the nucleic acid sequence of the second region improves the half-life of the endogenous complex containing the antisense strand and AGO protein compared to a corresponding endogenous complex containing a corresponding antisense strand that is fully complementary to the target mRNA.

[0010] In some embodiments, the second region is 1 to 10 (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) nucleotides long. In some embodiments, the second region is 1 nucleotide long. In some embodiments, the second region is 2 nucleotides long. In some embodiments, the second region is 3 nucleotides long. In some embodiments, the second region is 4 nucleotides long. 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 bond. In some embodiments of the fixed region described herein, the fixed region contains at least one (e.g., one, two, three, four, five, or more) modified nucleoside bonds.

[0013] In some embodiments, at least one modified subunit bond is given by formula E1: [Chemical formula] and where each B is, independently, a base pair-forming moiety, W is O, S, B, BR 2 , N, NR 2 , OCH2, OCH, CH2, and CH, where optionally, W is selected from the group consisting of OCH2 and OCH, each X is, independently, halo (e.g., fluoro or chloro), hydroxy, and C 1~6 alkoxy, where optionally, each X is, independently, halo (e.g., fluoro or chloro), and C 1~6 alkoxy (e.g., methoxy, ethoxy, 2-methoxyethoxy, n-propoxy, sec-propoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, or n-heptoxy), Y is O - , OH, OR, NH -、 NH2, S - , and SH, where optionally, Y is selected from the group consisting of O - , OH, and OR, Z is O, S, BR 2 , NR 2 , and CH2, R is a protecting group, each R 2 is, independently, H or optionally substituted C1-C6 alkyl, [Chemical formula] is optionally a double bond.

[0014] In some embodiments of formula E1, W is OCH2.

[0015] In some embodiments of formula E1, W is OCH, [[ID=五十七]] [Chemical formula] It is a double bond.

[0016] In some embodiments of formula E1, Z is O.

[0017] In some embodiments of formula E1, Z is CH2.

[0018] In some embodiments of formula E1, Y is O - If that is the case, then either Z or W is not O.

[0019] In some embodiments of formula E1, Z is CH2 and W is CH2. In some embodiments, the modified subunit coupling of formula E1 is the modified subunit coupling of formula E2: [ka] That is the case.

[0020] In some embodiments of formula E1, Z is CH2 and W is O. In some embodiments, the modified subunit bond of formula E1 is the modified subunit bond of formula E3: [ka] That is the case.

[0021] In some embodiments of formula E1, Z is O and W is CH2. In some embodiments, the modified subunit bond of formula E1 is the modified subunit bond of formula E4: [ka] That is the case.

[0022] In some embodiments of formula E1, Z is O and W is CH. In some embodiments, the modified subunit bond of formula E1 is the modified subunit bond of formula E5: [ka] That is the case.

[0023] In some embodiments, the modified subunit coupling of formula E1 is the modified subunit coupling of formula E6: [ka] That is the case.

[0024] In some embodiments of formula E6, Each B is independently a base-pairing region. Each X is independently a halo, hydroxyl, and C 1~6 Selected from the group consisting of alkoxys, where optionally each X is independently a halo (e.g., fluoro) and a C. 1~6 Selected from the group consisting of alkoxys (e.g., methoxy, ethoxy, n-propoxy, sec-propoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, or n-heptoxy), Y is O - OH, OR, NH - NH2, S - Selected from the group consisting of , and SH, where Y is optionally O - Selected from the group consisting of OH and OR, Z is selected from the group consisting of O and CH2. [ka] The bond is arbitrarily a double bond.

[0025] In some embodiments of formula E6, Each X is independently fluoro, hydroxy, and C 1~6 Selected from the group consisting of alkoxys, where optionally each X is independently fluoro and C 1~6 Selected from the group consisting of alkoxys (e.g., methoxy, ethoxy, n-propoxy, sec-propoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, or n-heptoxy), Y is O -Selected from the group consisting of OH and OR, Z is selected from the group consisting of O and CH2. [ka] The bond is arbitrarily a double bond.

[0026] In some embodiments of formula E6, Each X is independently selected from the group consisting of fluoro, hydroxy, methoxy, ethoxy, n-propoxy, sec-propoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, and n-heptoxy. Y is O - Selected from the group consisting of OH and OR, Z is selected from the group consisting of O and CH2. [ka] The bond is arbitrarily a double bond.

[0027] In some embodiments of formula E6, Each X is independently selected from the group consisting of fluoro, hydroxy, methoxy, ethoxy, n-propoxy, sec-propoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, and n-heptoxy. Y is O - Selected from the group consisting of OH and OR, Z is O, [ka] The bond is arbitrarily a double bond.

[0028] In some embodiments of formula E6, Each X is independently selected from the group consisting of fluoro, hydroxy, methoxy, ethoxy, n-propoxy, sec-propoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, and n-heptoxy. Y is O -Selected from the group consisting of OH and OR, Z is CH2, [ka] The bond is arbitrarily a double bond.

[0029] In some embodiments of formula E6, Each X is independently selected from the group consisting of fluoro, hydroxy, and methoxy. Y is O - Selected from the group consisting of OH and OR, Z is O, [ka] The bond is arbitrarily a double bond.

[0030] In some embodiments of formula E6, Each X is independently selected from the group consisting of fluoro, hydroxy, and methoxy. Y is O - Selected from the group consisting of OH and OR, Z is CH2, [ka] The bond is arbitrarily a double bond.

[0031] In some embodiments of formula E1, Z is O and W is OCH2. In some embodiments, the modified subunit bond of formula E1 is the modified subunit bond of formula E6a: [ka] That is the case.

[0032] In some embodiments of formula E1, Z is CH2 and W is CH. In some embodiments, the modified subunit coupling of formula E1 is the modified subunit coupling of formula E7: [ka] That is the case.

[0033] In some embodiments of formula E1, the base-pairing moiety B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0034] In some embodiments, at least one modified subunit bond is given by formula E8: [ka] And, In the formula, D is O, S, B, BR 2 , N, NR 2 , selected from the group consisting of OCH2, OCH, CH2, and CH, where optionally, D is selected from the group consisting of OCH2 and OCH, C is O - , OH, OR 1 NH - NH2, S - Selected from the group consisting of , and SH, where C is optionally O - OH, and OR 1 Selected from the group consisting of, A is O, S, BR 2 , NR 2 Selected from the group consisting of , and CH2, R 1 It is a protecting group, Each R 2 These are independently H or optionally substituted C1-C6 alkyl groups. [ka] It is arbitrarily a double bond, The subunits are bridged by two optionally modified nucleosides.

[0035] In some embodiments, D is OCH2.

[0036] In some embodiments, D is OCH, [ka] It is a double bond.

[0037] In some embodiments, A is O.

[0038] In some embodiments, A is CH2.

[0039] In some embodiments, C is O - If that is the case, then either A or D is not O.

[0040] In some embodiments, D is CH2. In another embodiment, the modified subunit bond of formula E8 is the modified subunit bond of formula E9: [ka] That is the case.

[0041] In some embodiments, D is O. In another embodiment, the modified subunit bond of formula E8 is the modified subunit bond of formula E10: [ka] That is the case.

[0042] In some embodiments, D is CH2. In another embodiment, the modified subunit bond of formula E8 is the modified subunit bond of formula E11: [ka] That is the case.

[0043] In some embodiments, D is CH. In another embodiment, the modified subunit bond of formula E8 is the modified subunit bond of formula E12: [ka] That is the case.

[0044] In some embodiments, D is OCH2. In another embodiment, the modified subunit bond of formula E8 is the modified subunit bond of formula E13: [ka] That is the case.

[0045] In another embodiment, the modified subunit bond of formula E8 is the modified subunit bond of formula E14: [ka] That is the case.

[0046] In another embodiment, the modified subunit bond of formula E8 is the modified subunit bond of formula E15: [ka] That is the case.

[0047] In some embodiments of modified siRNA binding, each optionally modified nucleoside is independently selected in each instance from the group consisting of adenosine, guanosine, cytidine, and uridine.

[0048] In some embodiments, at least one modified subunit bond is given by formula E8: [ka] It is, In the formula, D is selected from the group consisting of O, OCH2, OCH, CH2, and CH, where optionally, D is selected from the group consisting of OCH2 and OCH. C is O - , OH, OR 1 NH - NH2, S - Selected from the group consisting of , and SH, where C is optionally O - OH, and OR 1 Selected from the group consisting of, A is selected from the group consisting of O and CH2. R 1 This is a protecting group selected from the group consisting of dimethoxytrityl (DMTr), succinic acid, tert-butyldimethylsilyl (TBDMS), benzoyl (Bz), benzyl (Bn), methoxyethoxymethyl ether (MOM), methoxybenzyl ether (PMB), methylthiomethyl ether, pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuranyl (THF), trityl (Trt), triisopropylsilyl (TIPS), tert-butyldiphenylsilyl (TBDPS), and acetic acid. [ka] It is arbitrarily a double bond, The subunits are bridged by two optionally modified nucleosides.

[0049] In some embodiments, D is OCH2.

[0050] In some embodiments, D is OCH, [ka] It is a double bond.

[0051] In some embodiments, A is O.

[0052] In some embodiments, A is CH2.

[0053] In some embodiments of the above-described modified subunit bonding, the base-pairing moiety B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0054] In some embodiments, R is a protecting group selected from the group consisting of dimethoxytrityl (DMTr), succinic acid, tert-butyldimethylsilyl (TBDMS), benzoyl (Bz), benzyl (Bn), methoxyethoxymethyl ether (MOM), methoxybenzyl ether (PMB), methylthiomethyl ether, pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuranyl (THF), trityl (Trt), triisopropylsilyl (TIPS), tert-butyldiphenylsilyl (TBDPS), and acetic acid. [ka] The bond is arbitrarily a double bond.

[0055] In some embodiments, the second region includes at least one internucleoside phosphorothioate bond.

[0056] In some embodiments, the second region contains at least one nucleotide comprising 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.

[0057] In some embodiments, the second region is arranged in one of the following ways in the 5'→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) It has, In the formula, each S is a nucleoside-phosphorothioate bond, Each oxygen atom is a phosphodiester bond between nucleosides. Each A is a 2'-methoxyribonucleoside, Each B is a 2'-fluororibonucleoside, Each XA is a 2'-methoxynucleotide of formula E6a, Each XB is a 2'-fluoronucleotide of formula E6a. In some embodiments, the second region is one of the following sequences in the 5'→3' direction: -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) (Formula F10) -S-(mA)-S-(xU)-S-(yU)(Equation F11) -S-(mA)-S-(xU)-S-(xU)(Formula F12) -S-(mA)-S-(mU)-S-(fU) (Formula F13) It has, In the formula, each S is a nucleoside-phosphorothioate bond, Each oxygen atom is a phosphodiester bond between nucleosides. Each mA is 2'-methoxyadenosine, Each mG is 2'-methoxyguanidine, Each mU is 2'-methoxyuridine, Each xU is 2'-methoxyuridine of formula E6a, Each yU is 2'-fluorouridine of formula E6a.

[0058] In some embodiments, the antisense chain has a structure represented by formula I, where formula I is in the 5'→3' direction. AB-(A') j -CP 2 -DP 1 -(C'-P 1 ) k -C' Equation I And, In the formula, A is in formula: CP 1 -DP 1 It is represented by, Each A' is given by formula: CP 2 -DP 2 It is represented by, B is the formula: CP 2 -DP 2 -DP 2 -DP 2 It is represented by, Each C is a 2'-O-methyl (2'-O-Me) ribonucleoside, Each C' is independently a 2'-O-Me ribonucleoside or a 2'-fluoro(2'-F) ribonucleoside. Each D is a 2'-F ribonucleoside, Each P 1 This is a nucleoside-phosphorothioate bond, Each P 2 This is a phosphodiester bond between nucleosides, j is an integer between 1 and 7 (for example, 1, 2, 3, 4, 5, 6, or 7), k is an integer between 1 and 7 (for example, 1, 2, 3, 4, 5, 6, or 7).

[0059] In some embodiments, the antisense chain has a structure represented by formula A1, where formula A1 is in the 5'→3' direction. ASBSAOBOBOBOAOBOAOBOA-OBOAOBOAOBSASASASBSA Formula A1 And, In the formula, A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents an internucleoside phosphodiester bond, and S represents an internucleoside phosphorothioate bond.

[0060] In some embodiments, the antisense chain has a structure represented by formula II, where formula II is in the 5'→3' direction. AB-(A') j -CP 2 -DP 1 -(CP 1 ) k-C' Formula II And, In the formula, A is in formula: CP 1 -DP 1 It is represented by, Each A' is given by formula: CP 2 -DP 2 It is represented by, B is the formula: CP 2 -DP 2 -DP 2 -DP 2 It is represented by, Each C is a 2'-O-methyl (2'-O-Me) ribonucleoside, Each C' is independently a 2'-O-Me ribonucleoside or a 2'-fluoro(2'-F) ribonucleoside. Each D is a 2'-F ribonucleoside, Each P 1 This is a nucleoside-phosphorothioate bond, Each P 2 This is a phosphodiester bond between nucleosides, j is an integer between 1 and 7 (for example, 1, 2, 3, 4, 5, 6, or 7), k is an integer between 1 and 7 (for example, 1, 2, 3, 4, 5, 6, or 7).

[0061] In some embodiments, the antisense chain has a structure represented by formula A2, where formula A2 is in the 5'→3' direction. ASBSAOBOBOBOAOBOAOBOA-OBOAAOBOAOBSASASASASA formula A2 And, In the formula, A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents an internucleoside phosphodiester bond, and S represents an internucleoside phosphorothioate bond.

[0062] In some embodiments, the sense strand has a structure represented by Formula III, where Formula III is in the 5’→3’ direction, E-(A’) m -F Formula III and is where E is represented by the formula: (C-P 1 )2, F is represented by the formula: (C-P 2 )3-D-P 1 -C-P 1 -C, (C-P 2 )3-D-P 2 -C-P 2 -C, (C-P 2 )3-D-P 1 -C-P 1 -D, or (C-P 2 )3-D-P 2 -C-P 2 -D, A’, C, D, P 1 , and P 2 are as defined in Formula II, and m is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7).

[0063] In some embodiments, the sense strand has a structure represented by Formula S1, where Formula S1 is in the 5’→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 and is where A represents a 2’-O-Me ribonucleoside, B represents a 2’-F ribonucleoside, O represents a internucleoside phosphodiester bond, and S represents an internucleoside phosphorothioate bond.

[0064] In some embodiments, the sense strand has a structure represented by Formula S2, where Formula S2 is in the 5’→;3’ direction, ASASAOBOAOBOAOBOAOBOA-OAOAOBOAOA formula S2 And, In the formula, A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents an internucleoside phosphodiester bond, and S represents an internucleoside phosphorothioate bond.

[0065] In some embodiments, the sense chain has a structure represented by formula S3, where formula S3 is in the 5'→3' direction. ASASAOBOAOBOAOBOAOBOA-OAOAOBSASB formula S3 And, In the formula, A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents an internucleoside phosphodiester bond, and S represents an internucleoside phosphorothioate bond.

[0066] In some embodiments, the sense chain has a structure represented by formula S4, where formula S4 is in the 5'→3' direction. ASASAOBOAOBOAOBOAOBOA-OAOAOBOAOB Formula S4 And, In the formula, A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents an internucleoside phosphodiester bond, and S represents an internucleoside phosphorothioate bond.

[0067] In some embodiments, the antisense chain has a structure represented by formula IV, where formula IV is in the 5'→3' direction. A-(A') j -CP 2 -B-(CP 1 ) k -C' Formula IV and wherein A is represented by the formula: C-P 1 -D-P 1 ; each A’ is represented by the formula: C-P -D-P 2 ; B is represented by the formula: D-P 2 -C-P -D-P 1 ; each C is a 2'-O-Me ribonucleoside, 1 each C’ is independently a 2'-O-Me ribonucleoside or a 2'-F ribonucleoside, 1 each D is a 2'-F ribonucleoside, each P is a phosphorothioate bond between nucleosides, each P is a phosphodiester bond between nucleosides, 1 j is an integer from 1 to 7 (for example, 1, 2, 3, 4, 5, 6, or 7), k is an integer from 1 to 7 (for example, 1, 2, 3, 4, 5, 6, or 7). 2 In some embodiments, the antisense strand has a structure represented by formula A3, where formula A3 is, in the 5'→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-S-A-S-B-S-A-S-A-S-A Formula A3

[0068] wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester bond between nucleosides, and S represents a phosphorothioate bond between nucleosides. 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-S-A-S-B-S-A-S-A-S-A Formula A3 and In some embodiments, the sense strand has a structure represented by formula V, where formula V is, in the 5'→3' direction,

[0069] E-(A’) -C-P m -F 2 - Formula V And, In the formula, E is given by the formula:(CP 1 ) is expressed by 2, F is given by equation: DP 1 -CP 1 -C, DP 2 -CP 2 -C, DP 1 -CP 1 -D, or DP 2 -CP 2 Represented by -D, A', C, D, P 1 , and P 2 This is defined in Equation IV, m is an integer between 1 and 7 (for example, 1, 2, 3, 4, 5, 6, or 7).

[0070] In some embodiments, the sense chain has a structure represented by formula S5, where formula S5 is in the 5'→3' direction. ASASAOBOAOBOAOBOAOBOA-OBOAOBSASA formula S5 And, In the formula, A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents an internucleoside phosphodiester bond, and S represents an internucleoside phosphorothioate bond.

[0071] In some embodiments, the sense chain has a structure represented by formula S6, where formula S6 is in the 5'→3' direction. ASASAOBOAOBOAOBOAOBOA-OBOAOBOAOA formula S6 And, In the formula, A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents an internucleoside phosphodiester bond, and S represents an internucleoside phosphorothioate bond.

[0072] In some embodiments, the sense chain has a structure represented by formula S7, where formula S7 is in the 5'→3' direction. ASASAOBOAOBOAOBOAOBOA-OBOAOBSASB formula S7 And, In the formula, A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents an internucleoside phosphodiester bond, and S represents an internucleoside phosphorothioate bond.

[0073] In some embodiments, the sense chain has a structure represented by formula S8, where formula S8 is in the 5'→3' direction, ASASAOBOAOBOAOBOAOBOA-OBOAOBOAOB formula S8 And, In the formula, A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents an internucleoside phosphodiester bond, and S represents an internucleoside phosphorothioate bond.

[0074] In some embodiments, the antisense chain has a structure represented by formula VI, where formula VI is in the 5'→3' direction. AB j -EB k -EFG l -DP 1 -C' Equation VI And, In the formula, A is in formula: CP 1 -DP 1 It is represented by, Each B is given by formula:CP 2 It is represented by, Each C is a 2'-O-Me ribonucleoside, Each C' is independently a 2'-O-Me ribonucleoside or a 2'-F ribonucleoside. Each D is a 2'-F ribonucleoside, each E is of the formula: D-P 2 -C-P 2 and is represented by F is of the formula: D-P 1 -C-P 1 and is represented by each G is of the formula: C-P 1 and is represented by each P 1 is a phosphorothioate internucleoside linkage, each P 2 is a phosphodiester internucleoside linkage, j is an integer from 1 to 7 (for example, 1, 2, 3, 4, 5, 6, or 7), k is an integer from 1 to 7 (for example, 1, 2, 3, 4, 5, 6, or 7), l is an integer from 1 to 7 (for example, 1, 2, 3, 4, 5, 6, or 7).

[0075] In some embodiments, the antisense strand has a structure represented by formula A4, where formula A4 is in the 5'→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 and in the formula, 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.

[0076] In some embodiments, the sense strand has a structure represented by formula VII, where formula VII is in the 5'→3' direction H-B m -I n -A'-B o -H-C Formula VII and in the formula, A' is of the formula: C-P 2 -D-P 2It is represented by, Each H is given by the formula:(CP 1 ) is expressed by 2, Each I is given by equation: (DP 2 ) is represented by, B, C, D, P 1 , and P 2 This is defined in Equation VI, m is an integer between 1 and 7 (for example, 1, 2, 3, 4, 5, 6, or 7), n is an integer between 1 and 7 (for example, 1, 2, 3, 4, 5, 6, or 7). o is an integer between 1 and 7 (for example, 1, 2, 3, 4, 5, 6, or 7).

[0077] In some embodiments, the sense chain has a structure represented by formula S9, where formula S9 is in the 5'→3' direction. ASASAOAOAOBOBOBOAOBOA-OAOAOAASASA formula S9 And, In the formula, A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents an internucleoside phosphodiester bond, and S represents an internucleoside phosphorothioate bond.

[0078] In some embodiments, the antisense chain also has a 5' phosphorus-stabilizing moiety at its 5' end.

[0079] In some embodiments, the sense chain also has a 5' phosphorus-stabilizing moiety at its 5' end.

[0080] In some embodiments, each 5' phosphorus-stabilized portion is independently one of formulas IX, XX, XI, XII, XIII, XIV, XV, or XVI: [ka] It is represented by, In the formula, Nuc represents a nucleobase, where optionally, the nucleobase is selected from the group consisting of adenine, uracil, guanine, thymine, and cytosine, and R represents optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, phenyl, benzyl, a cation (e.g., a monovalent cation), or hydrogen.

[0081] In some embodiments, the nucleobase is adenine, uracil, guanine, thymine, or cytosine.

[0082] In some embodiments, the 5'-phosphorus stabilizing moiety is an (E)-vinylphosphonate represented by formula XI.

[0083] In some embodiments, the siRNA molecule also has a hydrophobic moiety at the 5' or 3' end of the siRNA molecule.

[0084] In some embodiments, the hydrophobic moiety is selected from the group consisting of cholesterol, vitamin D, and tocopherol.

[0085] In some embodiments, the siRNA molecule is a branched siRNA molecule.

[0086] In some embodiments, the branched siRNA molecule is bifurcated, trifurcated, or quadrifurcated.

[0087] In some embodiments, the siRNA molecule is bifurcated, where optionally, the bifurcated siRNA molecule is any of formulas XVII, XVIII, or XIX:

Chemical formula

[0088] In some embodiments, the branched siRNA molecule is represented by formula XVII. In some embodiments, the branched siRNA molecule is represented by formula XVIII. In some embodiments, the branched siRNA molecule is represented by formula XV.

[0089] In some embodiments, the siRNA molecule is tribranched, where optionally the tribranched siRNA molecule is one of the formulas XX, XXI, XXII, or XXIII: [ka] It is represented by, In the formula, each RNA is independently an siRNA molecule, L is a linker, and each X is independently a branching point.

[0090] In some embodiments, the tribranched siRNA molecule is represented by formula XX. In some embodiments, the tribranched siRNA molecule is represented by formula XXI. In some embodiments, the tribranched siRNA molecule is represented by formula XXII. In some embodiments, the tribranched siRNA molecule is represented by formula XXIII.

[0091] In some embodiments, the siRNA molecule is tetrabranched, where optionally the tetrabranched siRNA molecule is one of the formulas XXIV, XXV, XXVI, XXVII, or XXVIII: [ka] It is represented by, In the formula, each RNA is independently an siRNA molecule, L is a linker, and each X is independently a branching point.

[0092] In some embodiments, the tetrabranched siRNA molecule is represented by formula XXIV. In some embodiments, the tetrabranched siRNA molecule is represented by formula XXV. In some embodiments, the tetrabranched siRNA molecule is represented by formula XXVI. In some embodiments, the tetrabranched siRNA molecule is represented by formula XXVII. In some embodiments, the tetrabranched siRNA molecule is represented by formula XXVIII.

[0093] In some embodiments of branched siRNA, the linker is selected from the group consisting of one or more consecutive ethylene glycols (e.g., polyethylene glycol (PEG), e.g., triethylene glycol (TrEG), or tetraethylene glycol (TEG)), alkyl groups, carbohydrates, block copolymers, peptides, RNA, and DNA subunits.

[0094] 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.

[0095] In some embodiments, the ethylene glycol oligomer is PEG. In some embodiments, PEG is TrEG. In some embodiments, PEG is TEG.

[0096] 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).

[0097] In some embodiments, the linker binds one or more (e.g., one, two, three, four, or more) siRNA molecules via a covalent bond-forming moiety.

[0098] In some embodiments, the covalent bond-forming moiety is selected from the group consisting of alkyl, ester, amide, carbamate, phosphonate, phosphate, phosphorothioate, phosphoramidate, triazole, urea, and formacetal.

[0099] In some embodiments, the linker is defined by formula L1: [ka] It includes the structure.

[0100] In some embodiments, the linker is defined by formula L2: [ka] It includes the structure.

[0101] In some embodiments, the linker is defined by formula L3: [ka] It includes the structure.

[0102] In some embodiments, the linker is defined by formula L4: [ka] It includes the structure.

[0103] In some embodiments, the linker is defined by formula L5: [ka] It includes the structure.

[0104] In some embodiments, the linker is defined by formula L6: [ka] It includes the structure.

[0105] In some embodiments, the linker is defined by formula L7: [ka] It includes the structure.

[0106] In some embodiments, the linker is defined by formula L8: [ka] It includes the structure.

[0107] In some embodiments, the linker is defined by formula L9: [ka] It includes the structure.

[0108] In some embodiments of any siRNA molecules described herein, more than 50% of the ribonucleotides of the antisense strand are 2'-O-Me ribonucleotides (for example, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65% of the ribonucleotides of the antisense strand). , 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% may be 2'-O-Me ribonucleotides.

[0109] In some embodiments, more than 60% of the ribonucleotides in the antisense strand are 2'-O-Me ribonucleotides (for example, 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).

[0110] In some embodiments, more than 70% of the ribonucleotides in the antisense strand are 2'-O-Me ribonucleotides (for example, 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 embodiments, more than 80% of the ribonucleotides in the antisense strand are 2'-O-Me ribonucleotides (for example, 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 embodiments, more than 90% of the ribonucleotides in the antisense strand are 2'-O-Me ribonucleotides (for example, 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 embodiments, less than 10% of the nucleoside bonds are phosphodiester or phosphorothioate bonds. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the nucleoside bonds are phosphodiester or phosphorothioate bonds. In some embodiments, 100% of the nucleoside bonds are phosphodiester or phosphorothioate bonds.

[0114] In some embodiments, the nine internucleoside bonds are phosphodiester bonds or phosphorothioate bonds.

[0115] In some embodiments, the length of the antisense chain is 10–30 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), 15–25 nucleotides (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides), or 18–23 nucleotides (e.g., 18, 19, 20, 21, 22, or 23 nucleotides). In some embodiments, the length of the antisense chain is 20 nucleotides. In some embodiments, the length of the antisense chain is 21 nucleotides. In some embodiments, the length of the antisense chain is 22 nucleotides. In some embodiments, the antisense chain length is 23 nucleotides. In some embodiments, the antisense chain length is 24 nucleotides. In some embodiments, the antisense chain length is 25 nucleotides. In some embodiments, the antisense chain length is 26 nucleotides. In some embodiments, the antisense chain length is 27 nucleotides. In some embodiments, the antisense chain length is 28 nucleotides. In some embodiments, the antisense chain length is 29 nucleotides. In some embodiments, the antisense chain length is 30 nucleotides.

[0116] In some embodiments, the siRNA molecules of the branched compound are linked to each other via a linker (e.g., an ethylene glycol oligomer, e.g., 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 the other 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 the other 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 the other siRNA molecule.

[0117] In some embodiments, the sense strand length is 12–30 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 14–18 nucleotides (e.g., 14, 15, 16, 17, or 18 nucleotides). In some embodiments, the sense strand length is 15 nucleotides. In some embodiments, the sense strand length is 16 nucleotides. In some embodiments, the sense strand length is 17 nucleotides. In some embodiments, the sense strand length is 18 nucleotides. In some embodiments, the sense strand length is 19 nucleotides. In some embodiments, the sense strand length is 20 nucleotides. In some embodiments, the sense strand length is 21 nucleotides. In some embodiments, the sense strand length is 22 nucleotides. In some embodiments, the sense chain length is 23 nucleotides. In some embodiments, the sense chain length is 24 nucleotides. In some embodiments, the sense chain length is 25 nucleotides. In some embodiments, the sense chain length is 26 nucleotides. In some embodiments, the sense chain length is 27 nucleotides. In some embodiments, the sense chain length is 28 nucleotides. In some embodiments, the sense chain length is 29 nucleotides. In some embodiments, the sense chain length is 30 nucleotides.

[0118] In some embodiments, the four nucleoside bonds are phosphorothioate bonds.

[0119] In some embodiments of the siRNA molecules 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 chain is 20 nucleotides long and the sense chain is 20 nucleotides long. In some embodiments, the antisense chain is 21 nucleotides long and the sense chain is 14 nucleotides long. In some embodiments, the antisense chain is 21 nucleotides long and the sense chain is 15 nucleotides long. In some embodiments, the antisense chain is 21 nucleotides long and the sense chain is 16 nucleotides long. In some embodiments, the antisense chain is 21 nucleotides long and the sense chain is 17 nucleotides long. In some embodiments, the antisense chain is 21 nucleotides long and the sense chain is 18 nucleotides long. In some embodiments, the antisense chain is 21 nucleotides long and the sense chain is 19 nucleotides long. In some embodiments, the antisense chain is 21 nucleotides long and the sense chain is 20 nucleotides long. In some embodiments, the antisense chain is 21 nucleotides long and the sense chain 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 chain is 24 nucleotides long and the sense chain is 20 nucleotides long. In some embodiments, the antisense chain is 24 nucleotides long and the sense chain is 21 nucleotides long. In some embodiments, the antisense chain is 24 nucleotides long and the sense chain is 22 nucleotides long. In some embodiments, the antisense chain is 24 nucleotides long and the sense chain is 23 nucleotides long. In some embodiments, the antisense chain is 24 nucleotides long and the sense chain is 24 nucleotides long. In some embodiments, the antisense chain is 25 nucleotides long and the sense chain is 14 nucleotides long. In some embodiments, the antisense chain is 25 nucleotides long and the sense chain is 15 nucleotides long. In some embodiments, the antisense chain is 25 nucleotides long and the sense chain is 16 nucleotides long. In some embodiments, the antisense chain is 25 nucleotides long and the sense chain 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.

[0120] In a further embodiment, the Disclosure provides a pharmaceutical composition comprising an siRNA molecule of any of the foregoing embodiments or models of the Disclosure, and a pharmaceutically acceptable excipient, carrier, or diluent.

[0121] In a further aspect, the Disclosure provides a method for delivering an siRNA molecule to the central nervous system (CNS) of a target by administering a composition of a polymerized oligonucleotide, an siRNA molecule, or a pharmaceutical composition of any of the foregoing aspects or embodiments of the Disclosure to the CNS of a target.

[0122] In some embodiments, the composition of a multimerized oligonucleotide, an siRNA molecule, or a pharmaceutical composition is administered to the subject by intrastriatal injection, intraventricular injection, or intrathecal injection.

[0123] In some embodiments, delivery of an siRNA molecule or pharmaceutical composition to the target CNS results in gene silencing of a target gene in the target.

[0124] 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 disease state in the subject. In some embodiments, the target gene is a positive regulator of a gene whose expression is enhanced in relation to the disease state in the subject. In some embodiments, the target gene is a splice isoform of the target gene, where the splice isoform reduces the expression of the target gene.

[0125] In some embodiments, gene silencing addresses a target disease condition.

[0126] In some embodiments of the methods described herein, the siRNA molecule or pharmaceutical composition is administered to the subject by intraventricular injection, intrastriatal injection, intraparenchymal injection, or intrathecal injection. In some embodiments, the siRNA molecule or pharmaceutical composition is administered to the subject by intravenous injection, intramuscular injection, or subcutaneous injection.

[0127] In some embodiments of any of the methods described herein, the subject is a human.

[0128] In another aspect, the Disclosure provides an siRNA molecule or pharmaceutical composition of any of the foregoing aspects or embodiments of the Disclosure, and a kit containing a document instructing a user of the kit to carry out any of the foregoing aspects or embodiments of the Disclosure. [Brief explanation of the drawing]

[0129] [Figure 1] This disclosure demonstrates in vitro knockdown of PRNPs by the siRNA molecule having a fixed nucleic acid base sequence at the 3' end of the antisense strand compared to a perfectly sequenced siRNA molecule. The siRNA molecule had the sequence shown in Table 3 below at the 3' end of the antisense strand. [Figure 2] Figures A, B, and C show graphs illustrating the ability of the siRNA molecules of this disclosure, which have a fixed nucleic acid base sequence at the 3' end of the antisense strand, to silence PRNPs in vivo. The siRNA molecules had a fixed region at the 3' end of the antisense strand, as shown in Table 4 below. Each siRNA was administered at doses of 5 nmol (Figure 2A), 1 nmol (Figure 2B), and 0.2 nmol (Figure 2C). [Figure 3-1] Figures A and B show graphs illustrating the ability of the siRNA molecules of this disclosure, which have a fixed nucleic acid base sequence at the 3' end of the antisense strand, to silence HPRT1 in vivo. The siRNA molecules had a fixed region at the 3' end of the antisense strand, as shown in Table 5 below. Each siRNA was administered at doses of 3 nmol (Figure 3A) and 1 nmol (Figure 3B). [Figure 3-2] Figures C-E show graphs illustrating the dose-dependent properties of HPRT1 knockdown by the siRNA molecules of this disclosure, which have a fixed nucleic acid base sequence at the 3' end of the antisense strand. The siRNA molecules had a fixed region at the 3' end of the antisense strand, as shown in Table 5 below. Figure 3C shows all the siRNA molecules in Table 5. Figure 3D shows only siRNA molecules 11 and 12 from Table 5. Figure 3D shows only siRNA molecules 11 and 15 from Table 5. [Figure 4] This graph shows the IC50 values ​​(pM), %mRNA expression, and goodness of fit (R2) for fixed nucleic acid sequences of all 64 possible three-terminal nucleic acid bases in the antisense strand of an siRNA molecule targeting HPRT1. [Modes for carrying out the invention]

[0130] definition Unless otherwise defined herein, scientific and technical terms used herein have the meanings generally understood by those skilled in the art. In the event of any potential ambiguity, the definitions provided herein shall prevail over any dictionary or external definitions. Unless otherwise required by context, singular terms encompass plural forms, and plural terms encompass singular forms. The use of “or” means “and / or” unless otherwise specified. The use of the term “including,” and other forms such as “includes” and “included,” is not limited to these.

[0131] As used herein, the term "nucleic acid" refers to RNA or DNA molecules, each consisting of a chain of ribonucleotides or deoxyribonucleotides.

[0132] As used herein, the term “therapeutic nucleic acid” refers to a nucleic acid molecule (e.g., ribonucleic acid) that has partial or complete complementarity to a disease-related target mRNA, interacts with it, and results in the silencing of mRNA expression.

[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 that contains an unmodified hydroxyl group at the 3' carbon of the ribose ring.

[0134] As used herein, the term "nucleoside" refers to a molecule composed of a heterocyclic base and a sugar.

[0135] As used herein, the term “nucleotide” refers to a nucleoside having a phosphate group or a variant thereof at the 3' or 5' sugar hydroxyl group. Examples of phosphate group variants include, but are not limited to, saturated alkyl phosphonates, unsaturated alkenyl phosphonates, phosphorothioates, and phosphoramidites.

[0136] When used in the context of a region of an siRNA molecule, a region of nucleic acid bases, a nucleic acid base sequence, a nucleotide region, or a nucleotide sequence, the term "fixed" refers to a sequence of nucleotides contained within an 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 be of any preferred length, but the silencing effect of the siRNA molecule is still maintained. Such a fixed sequence may contain one or more mismatches with the target mRNA transcript, but may also contain one or more nucleotides with matching sequences. That is, the fixed region has a default nucleotide sequence that can be inserted into any siRNA molecule regardless of the sequence of the target gene of interest. The fixed region may, but does not have to be, a region of consecutive or linked nucleotides. For example, the fixed region may be a region of 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) consecutive nucleotides with a sequence independent of the target mRNA transcript. Alternatively, there may be sequence-matching nucleotides (i.e., nucleotides complementary to the target mRNA transcript) interposed between nucleotides unrelated 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 permutation of matching and mismatched nucleotides.

[0137] As used herein, the term “continuous” means nucleotides linked to one another by direct covalent bonds.

[0138] As used herein, the term “linked” refers to both sequential nucleotides and nucleotides linked via linkers as defined herein. The linker may be, for example, an alkyl chain (e.g., methylene, ethylene, propylene, or any larger linear or branched alkylene group), 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 is sufficiently complementary to hybridize with the antisense strand of the siRNA molecule of this disclosure, thereby undergoing gene silencing.

[0140] "RISC" refers to RNA-induced silencing complexes that mediate RNA interference. As used herein, this term may refer to any RISC complex of any biological origin.

[0141] The term "degradation product" refers to mRNA that has been degraded after interacting with the RISC complex. After this interaction, the RISC complex can freely interact with other mRNA molecules.

[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 mimics. The term encompasses oligonucleotides composed of naturally occurring nucleic acid bases, sugars, and covalent nucleoside-to-backbone (skeletal) bonds, as well as oligonucleotides having similarly functioning non-natural (e.g., modified) moieties. Such modified or substituted oligonucleotides are often preferred over their natural forms for desirable properties such as enhanced cellular uptake, increased affinity for nucleic acid targets, and increased stability in the presence of nucleases.

[0143] As used herein, the term “siRNA” refers to a double-stranded, small interfering RNA that induces the RNA interference (RNAi) pathway. siRNA molecules may vary in length (generally 10–30 base pairs) and may have varying degrees of complementarity to their target mRNA. The term “siRNA” encompasses double-stranded molecules of two distinct strands, and single-stranded molecules that optionally form a hairpin structure containing the double-stranded region.

[0144] As used herein, the term “antisense strand” refers to a strand of siRNA double helix that has some degree of complementarity to the target gene.

[0145] As used herein, the term "sense strand" refers to a strand of siRNA double helix that is complementary to the antisense strand.

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

[0147] The term "interfering RNA molecule" refers to RNA molecules that suppress the endogenous function of target RNA transcripts, such as small interfering RNAs (siRNAs), microRNAs (miRNAs), short hairpin RNAs (shRNAs), or antisense oligonucleotides (ASOs).

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

[0149] As used herein, the terms “target,” “targeted,” and “targeted” in the context of siRNA design refer to the preparation of an antisense strand to be annealed to a region within an mRNA transcript of interest in order to reduce the translation of the mRNA into a protein product.

[0150] As used herein, the terms “chemically modified nucleotide,” “nucleotide analog,” “modified nucleotide,” and “modified nucleotide” refer to non-standard nucleotides, including non-natural ribonucleotides or deoxyribonucleotides. Exemplary nucleotide analogs are modified at arbitrary positions to alter certain chemical properties of a nucleotide, but to retain the ability of the nucleotide analog to perform its intended function.

[0151] As used herein, the term “metabolically stable” refers to an RNA molecule containing ribonucleotides that have been chemically modified to reduce the metabolic rate of the RNA molecule administered to a subject. Exemplary modifications include modifications from 2'-hydroxy to 2'-O-methoxy or 2'-fluoro, and modifications from phosphodiester to phosphorothioate.

[0152] As used herein, the term "phosphorothioate" refers to a phosphate group of a nucleotide that has been modified by substituting one or more of the oxygen atoms of the phosphate group with sulfur.

[0153] As used herein, terms such as "internucleoside" and "internucleotide" refer to the bonds between nucleosides in nucleic acid molecules.

[0154] As used herein, the term “antagomir” refers to nucleic acids that can function as inhibitors of miRNA activity.

[0155] As used herein, the term “gapmer” refers to a chimeric antisense nucleic acid containing a central block of deoxynucleotide monomers of sufficient length to induce RNase H cleavage. The deoxynucleotide block is sandwiched between ribonucleotide monomers or ribonucleotide monomers containing modifications.

[0156] As used herein, the term “mixmer” refers to nucleic acids containing a mixture of locked nucleic acids (LNA) and DNA.

[0157] As used herein, the term “guide RNA” refers to a nucleic acid that has sequence complementarity to a specific sequence in the genome immediately upstream or one base pair upstream of a protospacer adjacent motif (PAM) sequence used in the CRISPR / Cas9 gene editing system. Alternatively, “guide RNA” may refer to a nucleic acid (e.g., an antisense) that has sequence complementarity to a specific messenger RNA (mRNA) sequence. In this context, guide RNA may also have sequence complementarity to a “passenger RNA” sequence of equal or shorter length that is identical or substantially identical in sequence to the mRNA with which the guide RNA hybridizes.

[0158] As used herein, the term “branched siRNA” refers to a compound containing two or more double-stranded siRNA molecules covalently linked to one another. A branched siRNA molecule may be “di-branched,” also referred to herein as “disiRNA,” where this siRNA molecule comprises two siRNA molecules covalently linked to one another, for example, via a linker. A branched siRNA molecule may be “tri-branched,” also referred to herein as “tri-siRNA,” where this siRNA molecule comprises three siRNA molecules covalently linked to one another, for example, via a linker. A branched siRNA molecule may be “tetra-branched,” also referred to herein as “tetrasiRNA,” where this siRNA molecule comprises four siRNA molecules covalently linked to one another, for example, via a linker.

[0159] As used herein, the term “branching point” refers to a chemical portion of the branched siRNA structure of the Disclosure that may be covalently bonded to the 5' or 3' end of the antisense or sense strand of an siRNA molecule and may support the binding of additional single-stranded or double-stranded siRNA molecules. Non-limiting examples of branching points suitable for use with the methods and compositions of the Disclosure include, for example, phosphoramidites, tosylated solketals, 1,3-diaminopropanol, pentaerythritol, and any of the branching points described in US10,478,503.

[0160] As used herein, the term “phosphate moiety” refers to a terminal phosphate group encompassing phosphates and modified phosphates. The phosphate moiety may be located at any of the terminals, but is preferably located at the 5' terminal nucleoside. In one embodiment, the terminal phosphate has the formula:-OP(=O)(OH)OH and is unmodified. In another embodiment, the terminal phosphate is modified such that one or more of the O and OH groups are replaced with H, O, S, N(R'), or an alkyl group, where 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 each independently contain one to three phosphate moieties, which are either unmodified (diphosphate or triphosphate) or modified.

[0161] As used herein, the term “5' phosphate-stabilized moiety” refers to a terminal phosphate group encompassing phosphates and modified phosphates (e.g., phosphorothioates, phosphodiesters, phosphonates). The phosphate moiety may be located at any of the terminals, but is preferably located at the 5' terminal nucleoside. In one embodiment, the terminal phosphate has the formula:-OP(=O)(OH)OH and is unmodified. In another embodiment, the terminal phosphate is modified such that one or more of the O and OH groups are replaced with H, O, S, N(R'), or an alkyl group, where 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 each independently contain one to three phosphate moieties, which are either unmodified (diphosphate or triphosphate) or modified.

[0162] The phosphate group of a nucleotide may be modified, for example, by substituting one or more oxygen atoms of the phosphate group with sulfur (e.g., a phosphorothioate), or by making other substitutions that enable the nucleotide to perform its intended function, as described, for example, Eckstein, Antisense Nucleic Acid Drug Dev.10:117-21, 2000; Rusckowski et al., Antisense Nucleic Acid Drug Dev.10:333-45, 2000; Stein, Antisense Nucleic Acid Drug Dev.11:317-25, 2001; Vorobjev et al., Antisense Nucleic Acid Drug Dev.11:77-85, 2001; and US5,684,143. Some of the modifications described above (e.g., phosphate group modifications) preferably reduce the in vivo or in vitro hydrolysis rate of polynucleotides containing the above analogs.

[0163] As used herein, the term “complementary” refers to two nucleotides that form a regular Watson-Crick base pair. To avoid misunderstanding, in the context of this disclosure, Watson-Crick base pairs include adenine-thymine, adenine-uracil, and cytosine-guanine base pairs. In this context, a suitable Watson-Crick base pair is referred to as “matching,” while each nucleotide that does not form a pair, and each nucleotide that is improperly paired, is referred to as “mismatching.” Alignment for the purpose of determining nucleic acid sequence complementarity percentage can be achieved in various ways within the capabilities of those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software.

[0164] The “sequence complementarity percentage (%)” relative to a reference polynucleotide sequence is defined as the percentage of nucleic acids in a candidate sequence that is complementary to the nucleic acid in the reference polynucleotide sequence after the sequences have been aligned and gaps introduced as necessary to achieve the maximum possible sequence complementarity percentage. A given nucleotide is considered “complementary” to the reference nucleotide, as described herein, if two nucleotides form a regular Watson-Crick base pair. To avoid misunderstanding, in the context of this disclosure, Watson-Crick base pairs include adenine-thymine, adenine-uracil, and cytosine-guanine base pairs. In this context, a proper Watson-Crick base pair is referred to as a “match,” while each nucleotide that does not form a pair, and each nucleotide that improperly pairs, is referred to as a “mismatch.” Alignment for the purpose of determining the nucleic acid sequence complementarity percentage can be achieved in various ways within the capabilities of those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine suitable parameters for aligning sequences, including any algorithm required to achieve maximum complementarity over the entire length of the sequences being compared. Specifically, the sequence complementarity percentage of a given nucleic acid sequence A to a given nucleic acid sequence B (or, to be expressed as a given nucleic acid sequence A having a specific complementarity percentage to a given nucleic acid sequence B) is calculated as follows: 100 × (fraction X / Y), where X is the number of complementary base pairs in the alignment of A and B in a programmatic alignment (e.g., performed by computer software such as BLAST), and Y is the total number of nucleic acids in B. It will be understood that if the length of nucleic acid sequence A is not equal to the length of nucleic acid sequence B, the sequence complementarity percentage of A to B will not be equal to the sequence complementarity percentage of B to A. As used herein, a query nucleic acid sequence is considered "perfectly complementary" to a reference nucleic acid sequence if it has 100% sequence complementarity 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 gaps introduced as necessary to achieve the maximum possible sequence identity percentage. Alignment for the purpose of determining nucleic acid or amino acid sequence identity percentage can be achieved in various ways within the capabilities of a person skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. A person skilled in the art can determine suitable parameters for aligning sequences, including any algorithm required to achieve the maximum possible alignment over the entire length of the sequences being compared. For example, a sequence identity percentage value can be generated using the sequence comparison computer program BLAST. As a concrete example, the sequence identity percentage of a given nucleic acid or amino acid sequence A to, with, or against a given nucleic acid or amino acid sequence B (or, it can be expressed as a given nucleic acid or amino acid sequence A having a specific sequence identity percentage to, with, or against a given nucleic acid or amino acid sequence B) is calculated as follows: 100 × (fraction X / Y), where X is the number of nucleotides or amino acids that were evaluated as a perfect match by a sequence alignment program (e.g., BLAST) in the programmatic alignment of A and B, and Y is the total number of nucleic acids in B. It will 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 sequence identity percentage of A to B will not be equal to the sequence identity percentage of B to A.

[0166] As used herein, the term “sufficient complementarity for hybridization” means a nucleic acid sequence or part thereof that has one or more nucleotide mismatches with respect to the target region but can still hybridize to the target region under specified conditions, and does not need to be perfectly complementary (e.g., 100% complementary) to the target region or any part thereof. For example, a 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 can still form enough base pairs with the target to hybridize over its entire length.

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

[0168] The "stable double helix" formed during the annealing / hybridization of one nucleic acid to another is a double helix structure that does not denature with stringent washing. Exemplary stringent washing conditions are known in the art and include a temperature about 5°C lower than the melting temperature of the individual strands of the double helix, and a low concentration of monovalent salt, e.g., 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) of monovalent salt (e.g., NaCl concentration).

[0169] The term "gene silencing" refers to the suppression of gene expression, such as the endogenous gene expression of a target gene, which can be mediated by processes that affect transcription and / or post-transcriptional mechanisms. In some embodiments, gene silencing occurs when an RNAi molecule initiates inhibition or degradation of mRNA transcribed from a gene of interest in a sequence-specific manner through RNA interference, thereby preventing the translation of the gene product.

[0170] As used herein, the term “hyperactive disease driver gene” refers to a gene whose activity and / or expression is increased to contribute to or cause a disease condition in a subject (e.g., human). The disease condition may be caused or exacerbated directly or through an intermediate gene(s) by a hyperactive 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 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 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 if unsubstituted, they contain only C and H. When the name of an alkyl residue having a specific number of carbons is given, it is intended that all geometric isomers having that number of carbons are included and described. Thus, for example, “butyl” is intended to include n-butyl, sec-butyl, and isobutyl. Examples of alkyls include ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. In some embodiments, alkyls may be substituted. Preferred substituents that can be introduced to alkyl groups include, among others, hydroxy, alkoxy, amino, alkylamino, and halo.

[0175] As used herein, “alkenyl” refers to an acyclic or cyclic unsaturated hydrocarbon group having at least one olefinic unsaturated bond site (i.e., having at least one formula: C=C part). When an alkenyl group is unsubstituted, it contains only C and H. When the name of an alkenyl residue having a specific number of carbons is given, it is intended that all geometric isomers having that number of carbons are included and described. Thus, for example, “butenyl” is intended to include n-butenyl, sec-butenyl, and iso-butenyl. Examples of alkenyls include -CH=CH2, -CH2-CH=CH2, and -CH2-CH=CH-CH=CH2. In some embodiments, the alkenyl may be substituted. Preferred substituents that can be introduced to the alkenyl group include, among others, hydroxy, alkoxy, amino, alkylamino, and halo.

[0176] As used herein, “alkynyl” refers to an acyclic or cyclic unsaturated hydrocarbon group having at least one acetylene unsaturated bond site (i.e., having at least one part of the formula: C≡C). When an alkynyl group is unsubstituted, it contains only C and H. When the name of an alkynyl residue having a specific number of carbons is given, it is intended that all geometric isomers having that number of carbons are included and described. Thus, for example, “pentynyl” is intended to include n-pentynyl, sec-pentynyl, isopentynyl, and tert-pentynyl. Examples of alkynyls include -C≡CH and -C≡C-CH3. In some embodiments, the alkynyl may be substituted. Preferred substituents that can be introduced to the alkynyl group include, among others, hydroxy, alkoxy, amino, alkylamino, and halo.

[0177] As used herein, the term "phenyl" refers to a monocyclic arene obtained by removing one hydrogen atom from a carbon atom in the ring. The phenyl group may be unsubstituted or substituted with one or more suitable substituents, where the substituents replace the H of the phenyl group.

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

[0179] As used herein, the term "amide" refers to an alkyl, alkenyl, alkynyl, or aromatic group bonded to an aminocarbonyl functional group.

[0180] As used herein, the term “triazole” refers to a heterocyclic compound having the formula (C2H3N3) which has a five-membered ring of two carbon atoms and three nitrogen atoms, the positions of which can change to produce multiple isomers.

[0181] As used herein, the term “terminal group” refers to the group that terminates a carbon chain or nucleic acid.

[0182] As used herein, "amino acid" refers to a molecule containing amine and carboxyl functional groups as well as side chains specific to amino acids.

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

[0185] As used herein, the term “delivery target” refers to the organ or body part to which the branched oligonucleotide composition is desired to be delivered.

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

[0187] As used herein, the terms “subject” and “patient” are interchangeable and refer to an organism, such as a mammal (e.g., human), that is suffering from or at risk of suffering from a disease, disorder, or condition, as determined by a qualified professional (e.g., a physician or nurse) using, or without, clinical tests known in the art on, the subject(s) of the subject(s).

[0188] As used herein, the term “reference subject” refers to a healthy control subject that is the same as or similar to the subject treated with the compositions of this disclosure, for example, in terms of age, sex, geographical area, and / or educational level. A healthy reference subject is a reference subject that does not suffer from a disease associated with dysregulated gene expression or dysregulated gene pathway. Furthermore, a healthy reference subject is a reference subject that does not suffer from a disease associated with changes in gene expression and / or activity (e.g., increased or decreased).

[0189] As used herein, the terms “neuroinflammatory disease” and “neuroinflammatory disorder” are used interchangeably to refer to any pathological condition caused in any manner by neuroinflammation. “Neuroinflammation” refers to various immune responses in the central nervous system (e.g., microglia). Neuroinflammation may originate from the brain or may result from a systemic inflammatory response.

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

[0191] As used herein, the terms “to treat,” “to be treated,” and “to treat” mean both therapeutic treatment and preventive or deterrent measures, the purpose of which is to prevent, improve, or delay (reduce) an undesirable physiological condition, disorder, or disease, or to obtain a beneficial or desired clinical outcome. Beneficial or desired clinical outcomes include, but are not limited to, a reduction in the patient’s dependence on pharmacological treatment; relief of symptoms; a decrease in the severity of the condition, disorder, or disease; stabilization of the condition, disorder, or disease (i.e., no worsening); delay of onset or delay of progression of the condition, disorder, or disease; improvement or remission (whether partial or complete), whether detectable or undetectable, of the condition, disorder, or disease; improvement of at least one measurable physical, cognitive, or behavioral parameter, which is not necessarily identifiable by the patient; or improvement or reversal of the condition, disorder, or disease. Treatment encompasses inducing a clinically significant response without excessive levels of side effects. "Treatment" also includes extending the survival period compared to the survival period expected if no treatment is received.

[0192] As used herein, the terms “benefit” and “response” are interchangeable in the context of a subject receiving treatment for a disease. For example, clinical benefits in the context of a subject administered with an siRNA molecule or siRNA composition of this disclosure include, but are 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 wild-type transcripts, mutant transcripts, variant transcripts, or overexpressed transcripts, and / or splice isoforms of target gene transcripts.

[0193] The present invention provides small interfering RNA (siRNA) molecules, including single-stranded and double-stranded small interfering RNA (ds-siRNA), and methods for using them in the treatment of patients requiring gene silencing (e.g., patients with dysregulation of gene expression, such as Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, frontotemporal dementia, Huntington's disease, multiple sclerosis, or progressive supranuclear palsy). siRNA molecules can mediate RNA interference (RNAi) by degrading mRNA having a complementary nucleotide sequence, thereby reducing or completely preventing translation of a target gene.

[0194] The siRNA molecules of this disclosure may contain an immobilized sequence at the end of a sense strand or an antisense strand (e.g., 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 immobilized sequence may be contained within an overhang region of either strand (e.g., an overhang region of the sense strand extending beyond the antisense strand, or an overhang region of the antisense strand extending beyond the sense strand).

[0195] Various chemical modifications may be used on the siRNA molecules described herein. For example, the siRNA molecules described herein may include specific patterns of chemical modifications (e.g., 2'-ribose modification or nucleoside bond modification) to improve resistance to nuclease enzymes, toxicity profile, and physicochemical properties (e.g., thermal stability).

[0196] The siRNA molecules of this disclosure may be characterized by an antisense strand having a nucleic acid sequence complementary to the region of the mRNA transcript of a target gene. The degree of complementarity of the antisense strand to the region of the target mRNA transcript may be sufficient for the antisense strand to anneal over the entire length of the mRNA transcript region. For example, the antisense strand may have a nucleic acid sequence that is at least 60% complementary to the 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).

[0197] Fixed nucleic acid base region In some embodiments of the siRNA molecules of this disclosure, the antisense strand and / or sense strand contain a nucleotide region having an immobilized sequence of nucleic acid bases. In some embodiments, the immobilized nucleotide region is contained within the sense strand. In some embodiments, the immobilized nucleotide region is contained within the antisense strand. In some embodiments, the immobilized nucleotide region is part of the antisense strand and contains one or more mismatches with respect to the target mRNA transcript. In some embodiments, the immobilized nucleotide region has a sequence independent of the sequence of the target mRNA transcript.

[0198] While not bound by theory, certain fixed regions may result in the formation of mRNA degradation products with increased dissociation rates from the RISC complex after cleavage of target mRNA. Alternatively, or in addition, certain fixed regions may result in antisense strands with increased binding affinity to endogenous Argonaut (AGO) proteins compared to corresponding antisense strands that are fully complementary to the target mRNA.

[0199] This disclosure is independent of target mRNA transcripts and / or is at least partially based on the surprising discovery that the introduction of a fixed nucleic acid base region (e.g., to the 3' end of the antisense strand and / or to the antisense strand region protruding into the sense strand) having one or more (e.g., 1 to 4) nucleotide mismatches to an mRNA transcript brings surprising benefits to siRNA molecules. The fixed nucleic acid base regions of this disclosure achieve surprising results in a manner that depends on various factors, including, for example, the exact number of mismatches in the tail, as well as the nature and location of chemical modifications in the tail. Surprisingly, the length of the fixed nucleic acid base region has been found to be an important factor, and the benefits brought about by introducing the fixed nucleic acid base region may be affected by changes in the length of the fixed nucleic acid base region. The following sections describe these and other parameters in further detail.

[0200] In some embodiments, certain fixed nucleic acid sequences (e.g., the 3' end of the antisense strand and / or the 3' region of the antisense strand protruding into the sense strand) exhibit reduced neurotoxicity when administered to the CNS compared to the corresponding siRNA that is fully complementary to the target mRNA transcript. In some embodiments, certain fixed nucleic acid sequences (e.g., the 3' end of the antisense strand and / or the 3' region of the antisense strand protruding into the sense strand) do not exhibit acute neurotoxicity when administered to the CNS.

[0201] In some embodiments, the immobilized nucleic acid base region has 1 to 4 (e.g., 1, 2, 3, or 4) nucleotide mismatches with respect to the target mRNA transcript. In some embodiments, the immobilized nucleic acid base region has 1 nucleotide mismatch with respect to the target mRNA transcript. In some embodiments, the immobilized nucleic acid base region has 2 nucleotide mismatches with respect to the target mRNA transcript. In some embodiments, the immobilized nucleic acid base region has 3 nucleotide mismatches with respect to the target mRNA transcript. In some embodiments, the immobilized nucleic acid base region has 4 nucleotide mismatches with respect to the target mRNA transcript. In some embodiments, the immobilized nucleic acid base region has 1 to 3 nucleotide mismatches with respect to the target mRNA transcript. In some embodiments, the immobilized nucleic acid base region has 2 to 4 nucleotide mismatches with respect to the target mRNA transcript. In some embodiments, the immobilized nucleic acid base region has 3 or 4 nucleotide mismatches with respect to the target mRNA transcript. In some embodiments, the immobilized nucleic acid base region has 1 or 2 nucleotide mismatches with respect to the target mRNA transcript. In some embodiments, the immobilized nucleic acid base region has two or three nucleotide mismatches with respect to the target mRNA transcript.

[0202] Since perfect complementarity between the antisense strand loaded into RISC and the target mRNA can destabilize the RISC complex and / or induce ubiquitin ligase-mediated degradation of the RISC complex, a 3' end mismatch of the antisense strand can improve RISC-mediated target silencing.

[0203] In some embodiments, the 3' end of the antisense strand has 1 to 4 (e.g., 1, 2, 3, or 4) nucleotide mismatches with respect to the target mRNA transcript. In some embodiments, the 3' end of the antisense strand has 1 nucleotide mismatch with respect to the target mRNA transcript. In some embodiments, the 3' end of the antisense strand has 2 nucleotide mismatches with respect to the target mRNA transcript. In some embodiments, the 3' end of the antisense strand has 3 nucleotide mismatches with respect to the target mRNA transcript. In some embodiments, the 3' end of the antisense strand has 4 nucleotide mismatches with respect to the target mRNA transcript. In some embodiments, the 3' end of the antisense strand has 1 to 3 nucleotide mismatches with respect to the target mRNA transcript. In some embodiments, the 3' end of the antisense strand has 2 to 4 nucleotide mismatches with respect to the target mRNA transcript. In some embodiments, the 3' end of the antisense strand has 3 or 4 nucleotide mismatches with respect to the target mRNA transcript. In some embodiments, the 3' end of the antisense strand has one or two nucleotide mismatches with respect to the target mRNA transcript. In some embodiments, the 3' end of the antisense strand has two or three nucleotide mismatches with respect to the target mRNA transcript.

[0204] In some embodiments, the siRNA molecule may contain one or more of the above-described regions at the 5' end of the sense strand. In some embodiments, the siRNA molecule may contain one or more of the above-described regions at the 5' end of the antisense strand. In some embodiments, the siRNA molecule may contain one or more of the above-described regions at the 3' end of the sense strand. In some embodiments, the siRNA molecule may contain one or more of the above-described regions at the 3' end of the antisense strand. In some embodiments, the fixed region is contained within a region of the sense strand that protrudes into the antisense strand. In some embodiments, the fixed region is contained within a region of the antisense strand that protrudes into the sense strand.

[0205] In some embodiments, the fixed region is 1 nucleotide long. In some embodiments, the fixed region is 2 nucleotide long. In some embodiments, the fixed region is 3 nucleotide long. In some embodiments, the fixed region is 4 nucleotide long. In some embodiments, the fixed region is 5 nucleotide long. In some embodiments, the fixed region is 6 nucleotide long. In some embodiments, the fixed region is 7 nucleotide long. In some embodiments, the fixed region is 8 nucleotide long. In some embodiments, the fixed region is 9 nucleotide long. In some embodiments, the fixed region is 10 nucleotide long.

[0206] The fixed region may, but does not have to be, a region of consecutive or linked nucleotides. For example, the fixed region may be a region of 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) consecutive nucleotides having a sequence unrelated to the target mRNA transcript. Alternatively, there may be a sequence-matching nucleotide (i.e., a nucleotide complementary to the target mRNA transcript) interposed between nucleotides unrelated to the target mRNA transcript. For example, one or more mismatched nucleotides, followed by one or more sequence-matching nucleotides, followed by one or more mismatched nucleotides, or any other permutation of matching and mismatched nucleotides may be present without affecting the ability of the siRNA molecule to achieve gene silencing.

[0207] In some embodiments, the fixed region is 3 nucleotides long and is located at the 3' end of the antisense strand region protruding from the sense strand. In some embodiments, the fixed region has a nucleotide sequence selected from the sequences in Table 1a below. [Table 1]

[0208] In some embodiments, the fixed region is 4 nucleotides long and is located at the 3' end of the antisense strand region protruding from the sense strand. In some embodiments, the fixed region has a nucleotide sequence selected from the sequences in Table 1b below. [Table 2]

[0209] In some embodiments, the fixed regions described herein may have any combination of modifications (e.g., 2' sugar modifications, nucleoside bond modifications, modified nucleic acid bases, and / or modified subunit bonds of formula E1 and its subformations).

[0210] In some embodiments of the fixed regions described herein, the fixed region includes at least one (e.g., one, two, three, four, five, or more) modified nucleoside bonds.

[0211] In some embodiments, at least one modified subunit bond is given by formula E1: [ka] And, In the formula, each B is independently a base-pairing region. W is O, S, B, BR 2 , N, NR 2 , selected from the group consisting of OCH2, OCH, CH2, and CH, where optionally W is selected from the group consisting of OCH2 and OCH, Each X is independently a halo (e.g., fluoro or chloro), hydroxy, and C 1~6 Selected from the group consisting of alkoxys, where optionally each X is independently a halo (e.g., fluoro or chloro) and C 1~6Selected from the group consisting of alkoxys (e.g., methoxy, ethoxy, 2-methoxyethoxy, n-propoxy, sec-propoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, or n-heptoxy), Y is O - OH, OR, NH -、 NH2, S - Selected from the group consisting of , and SH, where Y is optionally O - Selected from the group consisting of OH and OR, Z is O, S, BR 2 , NR 2 Selected from the group consisting of , and CH2, R is a protecting group, Each R 2 These are independently H or optionally substituted C1-C6 alkyl groups. [ka] The bond is arbitrarily a double bond.

[0212] In some embodiments of formula E1, W is OCH2.

[0213] In some embodiments of formula E1, W is OCH, [ka] It is a double bond.

[0214] In some embodiments of formula E1, Z is O.

[0215] In some embodiments of formula E1, Z is CH2.

[0216] In some embodiments of formula E1, Y is O - If that is the case, then either Z or W is not O.

[0217] In some embodiments of formula E1, Z is CH2 and W is CH2. In some embodiments, the modified subunit coupling of formula E1 is the modified subunit coupling of formula E2: [ka] That is the case.

[0218] In some embodiments of formula E1, Z is CH2 and W is O. In some embodiments, the modified subunit bond of formula E1 is the modified subunit bond of formula E3: [ka] That is the case.

[0219] In some embodiments of formula E1, Z is O and W is CH2. In some embodiments, the modified subunit bond of formula E1 is the modified subunit bond of formula E4: [ka] That is the case.

[0220] In some embodiments of formula E1, Z is O and W is CH. In some embodiments, the modified subunit bond of formula E1 is the modified subunit bond of formula E5: [ka] That is the case.

[0221] In some embodiments, the modified subunit coupling of formula E1 is the modified subunit coupling of formula E6: [ka] That is the case.

[0222] In some embodiments of formula E6, Each B is independently a base-pairing region. Each X is independently a halo, hydroxyl, and C 1~6Selected from the group consisting of alkoxys, where optionally each X is independently a halo (e.g., fluoro) and a C. 1~6 Selected from the group consisting of alkoxys (e.g., methoxy, ethoxy, n-propoxy, sec-propoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, or n-heptoxy), Y is O - OH, OR, NH - NH2, S - Selected from the group consisting of , and SH, where Y is optionally O - Selected from the group consisting of OH and OR, Z is selected from the group consisting of O and CH2. [ka] The bond is arbitrarily a double bond.

[0223] In some embodiments of formula E6, Each X is independently fluoro, hydroxy, and C 1~6 Selected from the group consisting of alkoxys, where optionally each X is independently fluoro and C 1~6 Selected from the group consisting of alkoxys (e.g., methoxy, ethoxy, n-propoxy, sec-propoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, or n-heptoxy), Y is O - Selected from the group consisting of OH and OR, Z is selected from the group consisting of O and CH2. [ka] The bond is arbitrarily a double bond.

[0224] In some embodiments of formula E6, Each X is independently selected from the group consisting of fluoro, hydroxy, methoxy, ethoxy, n-propoxy, sec-propoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, and n-heptoxy. Y is O - Selected from the group consisting of OH and OR, Z is selected from the group consisting of O and CH2. [ka] The bond is arbitrarily a double bond.

[0225] In some embodiments of formula E6, Each X is independently selected from the group consisting of fluoro, hydroxy, methoxy, ethoxy, n-propoxy, sec-propoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, and n-heptoxy. Y is O - Selected from the group consisting of OH and OR, Z is O, [ka] The bond is arbitrarily a double bond.

[0226] In some embodiments of formula E6, Each X is independently selected from the group consisting of fluoro, hydroxy, methoxy, ethoxy, n-propoxy, sec-propoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, and n-heptoxy. Y is O - Selected from the group consisting of OH and OR, Z is CH2, [ka] The bond is arbitrarily a double bond.

[0227] In some embodiments of formula E6, Each X is independently selected from the group consisting of fluoro, hydroxy, and methoxy. Y is O - Selected from the group consisting of OH and OR, Z is O, [ka] The bond is arbitrarily a double bond.

[0228] In some embodiments of formula E6, Each X is independently selected from the group consisting of fluoro, hydroxy, and methoxy. Y is O - Selected from the group consisting of OH and OR, Z is CH2, [ka] The bond is arbitrarily a double bond.

[0229] In some embodiments of formula E1, Z is O and W is OCH2. In some embodiments, the modified subunit bond of formula E1 is the modified subunit bond of formula E6a: [ka] That is the case.

[0230] In some embodiments of formula E1, Z is CH2 and W is CH. In some embodiments, the modified subunit coupling of formula E1 is the modified subunit coupling of formula E7: [ka] That is the case.

[0231] In some embodiments of formula E1, the base-pairing moiety B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0232] In some embodiments, at least one modified subunit bond is given by formula E8: [ka] And, In the formula, D is O, S, B, BR 2 , N, NR2 , selected from the group consisting of OCH2, OCH, CH2, and CH, where optionally, D is selected from the group consisting of OCH2 and OCH, C is O - , OH, OR 1 NH - NH2, S - Selected from the group consisting of , and SH, where C is optionally O - OH, and OR 1 Selected from the group consisting of, A is O, S, BR 2 , NR 2 Selected from the group consisting of , and CH2, R 1 It is a protecting group, Each R 2 These are independently H or optionally substituted C1-C6 alkyl groups. [ka] It is arbitrarily a double bond, The subunits are bridged by two optionally modified nucleosides.

[0233] In some embodiments, D is OCH2.

[0234] In some embodiments, D is OCH, [ka] It is a double bond.

[0235] In some embodiments, A is O.

[0236] In some embodiments, A is CH2.

[0237] In some embodiments, C is O - If that is the case, then either A or D is not O.

[0238] In some embodiments, D is CH2. In another embodiment, the modified subunit bond of formula E8 is the modified subunit bond of formula E9: [ka] That is the case.

[0239] In some embodiments, D is O. In another embodiment, the modified subunit bond of formula E8 is the modified subunit bond of formula E10: [ka] That is the case.

[0240] In some embodiments, D is CH2. In another embodiment, the modified subunit bond of formula E8 is the modified subunit bond of formula E11: [ka] That is the case.

[0241] In some embodiments, D is CH. In another embodiment, the modified subunit bond of formula E8 is the modified subunit bond of formula E12: [ka] That is the case.

[0242] In some embodiments, D is OCH2. In another embodiment, the modified subunit bond of formula E8 is the modified subunit bond of formula E13: [ka] That is the case.

[0243] In another embodiment, the modified subunit bond of formula E8 is the modified subunit bond of formula E14: [ka] That is the case.

[0244] In another embodiment, the modified subunit bond of formula E8 is the modified subunit bond of formula E15: [ka] That is the case.

[0245] In some embodiments of modified siRNA binding, each optionally modified nucleoside is independently selected in each instance from the group consisting of adenosine, guanosine, cytidine, and uridine.

[0246] In some embodiments, at least one modified subunit bond is given by formula E8: [ka] And, In the formula, D is selected from the group consisting of O, OCH2, OCH, CH2, and CH, where optionally, D is selected from the group consisting of OCH2 and OCH. C is O - , OH, OR 1 NH - NH2, S - Selected from the group consisting of , and SH, where C is optionally O - OH, and OR 1 Selected from the group consisting of, A is selected from the group consisting of O and CH2. R 1 This is a protecting group selected from the group consisting of dimethoxytrityl (DMTr), succinic acid, tert-butyldimethylsilyl (TBDMS), benzoyl (Bz), benzyl (Bn), methoxyethoxymethyl ether (MOM), methoxybenzyl ether (PMB), methylthiomethyl ether, pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuranyl (THF), trityl (Trt), triisopropylsilyl (TIPS), tert-butyldiphenylsilyl (TBDPS), and acetic acid. [ka] is optionally a double bond, Between the subunits, two optionally modified nucleosides are crosslinked.

[0247] In some embodiments, D is OCH2.

[0248] In some embodiments, D is OCH,

Chemical formula

[0249] In some embodiments, A is O.

[0250] In some embodiments, A is CH2.

[0251] In some embodiments of any of the above modified subunit - subunit linkages, the base - pairing moiety B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0252] In some embodiments, R is a protecting group selected from the group consisting of dimethoxytrityl (DMTr), succinic acid, tert - butyldimethylsilyl (TBDMS), benzoyl (Bz), benzyl (Bn), methoxyethoxymethyl ether (MOM), methoxybenzyl ether (PMB), methylthiomethyl ether, pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuranyl (THF), trityl (Trt), triisopropylsilyl (TIPS), tert - butyldiphenylsilyl (TBDPS), and acetic acid,

Chemical formula

[0253] In some embodiments, the fixed region contains at least one (e.g., 1, 2, 3, 4, 5, or more) phosphorothioate bond between nucleosides.

[0254] In some embodiments, the fixed region contains at least one (e.g., 1, 2, 3, 4, 5, or more) nucleotides containing modified ribose. In some embodiments, the fixed region contains at least one (e.g., 1, 2, 3, 4, 5, or more) 2'-methoxynucleotides. In some embodiments, the fixed region contains at least one (e.g., 1, 2, 3, 4, 5, or more) 2'-fluoronucleotides.

[0255] In some embodiments, the fixed region is 3 nucleotides long at the 3' end of the antisense strand, where optionally the fixed region is located within a portion of the antisense strand protruding into the sense strand. In some embodiments, the fixed region is one of the 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) It has, In the formula, S is an internucleoside phosphorothioate bond, O is an internucleoside phosphodiester bond, A is a 2'-methoxynucleotide, B is a 2'-fluoronucleotide, XA is the 2'-methoxynucleotide of formula E6a, and XB is the 2'-fluoronucleotide of formula E6a.

[0256] In some embodiments, the fixed area is one 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) (Formula F10) -S-(mA)-S-(xU)-S-(yU)(Equation F11) -S-(mA)-S-(xU)-S-(xU)(Formula F12) -S-(mA)-S-(mU)-S-(fU) (Formula F13) It has, In the formula, S is an internucleoside phosphorothioate bond, O is an internucleoside phosphodiester bond, 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.

[0257] Structure of siRNA The siRNA molecules of this disclosure may be in the form of a single-stranded (ss) or double-stranded (ds) oligonucleotide structure. In some embodiments, the siRNA molecules may be bibranched, tribranched, or tetrabranched molecules. Furthermore, the siRNA molecules of this disclosure may contain one or more nucleoside-to-phosphodiester bonds and / or analogs thereof, such as nucleoside-to-phosphorothioate bonds. The siRNA molecules of this disclosure may further contain chemically modified nucleosides having 2' sugar modifications.

[0258] The simplest siRNA consists of ribonucleic acid containing an ss- or ds-structure formed by a first strand (i.e., an antisense strand) and, in the case of ds-siRNA, a second strand (i.e., a sense strand). The first strand contains a sequence of nucleotides that is at least partially complementary to the target nucleic acid. The second strand also contains a sequence of nucleotides, which is at least partially identical to the target nucleic acid. The first strand and the second strand can hybridize to form a double-stranded structure. Hybridization typically occurs by Watson-Crick base pairing.

[0259] Depending on the sequences of the first and second strands, hybridization or base pairing may not be complete or precise, meaning that the first and second strands are not 100% base-paired due to mismatches. One or more mismatches may be present within the double helix without necessarily affecting the RNAi activity of the siRNA.

[0260] The first strand contains a sequence of nucleotides that is essentially complementary to the target nucleic acid. Typically, the target nucleic acid sequence is ss-RNA, preferably mRNA, according to the mode of action of the interfering ribonucleic acid. Such hybridization is most likely, but not limited to, Watson-Crick base pairing. The degree to which the first strand has a sequence of nucleotides complementary to the target nucleic acid sequence can be 80% to 100%, and may be, for example, 80%, 85%, 90%, 95%, or 100% complementary.

[0261] The siRNA molecules described herein may employ nucleic acid bases, phosphate backbones, ribose cores, 5' and 3' terminal modifications, and branching, where multiple siRNA chains may be covalently linked.

[0262] Length of small interfering RNA molecules It is within the scope of this disclosure that any length known in the art and any length previously unknown may be used in the present invention. As described herein, the possible lengths of the antisense strand of the siRNA molecule of this disclosure 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 chain is 20 nucleotides. In some embodiments, the antisense chain is 21 nucleotides. In some embodiments, the antisense chain is 22 nucleotides. In some embodiments, the antisense chain is 23 nucleotides. In some embodiments, the antisense chain is 24 nucleotides. In some embodiments, the antisense chain is 25 nucleotides. In some embodiments, the antisense chain is 26 nucleotides. In some embodiments, the antisense chain is 27 nucleotides. In some embodiments, the antisense chain is 28 nucleotides. In some embodiments, the antisense chain is 29 nucleotides. In some embodiments, the antisense chain is 30 nucleotides.

[0263] In some embodiments, the sense strand of the siRNA molecule of this disclosure 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.

[0264] 2' sugar modification This disclosure may encompass ss-siRNA molecular compositions and ds-siRNA molecular compositions comprising at least one (e.g., at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, or more) nucleosides having 2' sugar modifications. Possible 2' modifications include all possible orientations of OH;F;O-, S-, or N-alkyl;O-, S-, or N-alkenyl;O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl may be substituted or unsubstituted C1-C10 alkyl or C2-C10 alkenyl and alkynyl. In some embodiments, modifications include 2'-O-methyl (2'-O-Me) modifications. Other possible sugar substituents include C1-C10 lower alkyl groups, substituted lower alkyl groups, alkenyl groups, alkynyl groups, alkali groups, aralkyl groups, O-alkaryl groups or O-aralkyl groups, SH groups, SCH3 groups, OCN groups, Cl groups, Br groups, CN groups, CF3 groups, OCF3 groups, SOCH3 groups, SO2CH3 groups, ONO2 groups, NO2 groups, N3 groups, NH2 groups, heterocycloalkyl groups, heterocycloalkaryl 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 having similar properties. In some embodiments, modifications include 2'-methoxyethoxy (2'-O-(2-methoxyethyl) or 2'-MOE, also known as 2'-O-CH2CH2OCH3). In some embodiments, modifications include 2'-dimethylaminooxyethoxy, also known as 2'-DMAOE, i.e., the O(CH2)2ON(CH3)2 group, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O-CH2OCH2N(CH3)2. Other possible sugar substituents include, for example, aminopropoxy (-OCH2CH2CH2NH2), allyl (-CH2-CH=CH2), -O-allyl (-O-CH2-CH=CH2), and fluoro (F). The 2' sugar substituent may be located at the arabino position (upper position) or the ribo position (lower position).In some embodiments, the 2'-arabino modification is 2'-F. Similar modifications may be made at other positions on the siRNA molecule, particularly at the 3' position of the sugar in the 3'-terminal nucleoside or 2'-5'-linked oligonucleotide, and at the 5' position of the 5'-terminal nucleotide. The oligonucleotide may also have sugar mimetic molecules such as the cyclobutyl moiety instead of the pentofuranosyl sugar.

[0265] Nucleic acid base modification The siRNA molecules of this disclosure may also include nucleosides or other substitutes or monomer subunit mimics containing nucleic acid bases (often simply referred to in the art as “bases” or “heterocyclic base moieties”). Nucleic acid bases are other moieties that are extensively modified or substituted, and such modified and / or substituted nucleic acid bases are suitable for this disclosure. As used herein, “unmodified” or “natural” nucleic acid bases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). In this specification, modified nucleic acid bases, also referred to as heterocyclic base moieties, include other synthetic and natural nucleic acid bases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, adenine and guanine, as well as other alkyl derivatives of adenine and guanine, 6-methyl derivatives and other alkyl derivatives of adenine and guanine, 2-propyl derivatives and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl(-C=C-CH3)uracil and cytosine. Other alkynyl derivatives of pyrimidine bases include 6-azouracil, cytosine, and thymine, 5-uracil (pseudracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, and other 8-substituted adenines and guanines, 5-halo, in particular 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaguanine, and 3-deazaguanine and 3-deazaguanine. Nucleic acid bases may include those in which a purine or pyrimidine base is replaced by another heterocycle, such as 7-deazaadenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone.Further nucleic acid bases include those disclosed in US3,687,808, Kroschwitz, JI, ed. The Concise Encyclopedia of Polymer Science and Engineering, New York, John Wiley & Sons, 1990, pp.858-859, Englisch et al., Angewandte Chemie, International Edition 30:613, 1991, and Sanghvi, YS, Chapter 16, Antisense Research and Applications, CRC Press, Gait, MJ ed., 1993, pp.289-302. The siRNA molecules of this disclosure may include polycyclic heterocyclic compounds instead of one or more heterocyclic base moieties. Numerous tricyclic heterocyclic compounds have been previously reported. These compounds are routinely used in antisense applications to enhance the binding properties of the modified chain to the target chain.

[0266] Representative cytosine analogs that form three hydrogen bonds with guanosine in the second chain include 1,3-diazafenoxazine-2-one (Kurchavov et al., Nucleosides and Nucleotides, 16:1837-46, 1997), 1,3-diazafenothiazine-2-one (Lin et al., Am. Chem. Soc., 117:3873-4, 1995), and 6,7,8,9-tetrafluoro-1,3-diazafenoxazine-2-one (Wang et al., Tetrahedron Lett., 39:8385-8, 1998). These base modifications have been shown to hybridize with complementary guanine upon incorporation into oligonucleotides, the latter of which has been shown to hybridize with adenine, improving helical thermal stability through extended stacking interactions (see also US10 / 155,920 and US10 / 013,295 (both of which are incorporated herein by reference in their entirety)). Further helical stabilization properties have been observed when cytosine analogs / alternatives have an aminoethoxy moiety bound to a rigid 1,3-diazaphenoxazine-2-one scaffold (Lin et al., Am. Chem. Soc., 120:8531-2, 1998).

[0267] Nucleoside bond modification Another variable in the design of this disclosure is the nucleoside-to-nucleoside bonds that constitute the phosphate backbone of the siRNA molecule. While the natural RNA phosphate backbone may be employed herein, derivatives thereof may be used to enhance the desired characteristics of the siRNA molecule. Of particular importance in this disclosure, though not limited to, is the protection of part or all of the siRNA molecule from hydrolysis. An example of a modification that reduces the rate of hydrolysis is a phosphorothioate. Any part or all of the backbone may contain phosphate substitutions (e.g., phosphorothioates). For example, 0-100% of nucleoside bonds can be phosphorothioates, and for instance, 0-100%, 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, 90-100%, 0-90%, 0-80%, 0-70%, 0-60%, 0-50%, 0-40%, 0-30%, 0-20%, 0-10%, 10-90%, 20-80%, 30-70%, 40-60%, 10-40%, 20-50%, 30-60%, 40-70%, 50-80%, or 60-90% can be phosphorothioate bonds. Similarly, 0-100% of the nucleoside bonds can be phosphodiester bonds, for example, 0-100%, 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, 90-100%, 0-90%, 0-80%, 0-70%, 0-60%, 0-50%, 0-40%, 0-30%, 0-20%, 0-10%, 10-90%, 20-80%, 30-70%, 40-60%, 10-40%, 20-50%, 30-60%, 40-70%, 50-80%, or 60-90% can be phosphodiester bonds.

[0268] Specific examples of useful siRNA molecule candidates in the present invention include oligonucleotides containing modified nucleoside bonds, such as unnatural nucleoside bonds. As defined herein, oligonucleotides having modified nucleoside bonds include nucleoside bonds that retain a phosphorus atom and nucleoside bonds that do not contain a phosphorus atom. For the purposes of this specification and as sometimes referred to in the art, modified oligonucleotides that do not have a phosphorus atom in the nucleoside skeleton may also be considered oligonucleosides. A preferred phosphorus-containing modified nucleoside bond is a nucleoside phosphorothioate bond. In some embodiments, modified oligonucleotide skeletons containing a phosphorus atom include, for example, methylphosphonates and other alkylphosphonates, phosphinates, 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphories, selenophosphates, boranophosphates having a normal 3'-5' bond, their 2'-5' bond analogues, and those having inverted polarity with one or more internucleotide bonds being 3'-3', 5'-5', or 2'-2' bonds.Illustrative U.S. patents describing the preparation of phosphorus-containing bonds include, but are not limited to, U.S. Patent Nos. 3,687,808, 4,469,863, 4,476,301, 5,023,243, 5,177,195, 5,188,897, 5,264,423, 5,276,019, 5,278,302, and 5,286,717. No. 5,321,131, No. 5,399,676, No. 5,405,939, No. 5,453,496, No. 5,455,233, No. 5,466,677, No. 5,476 ,925, No. 5,519,126, No. 5,536,821, No. 5,541,316, No. 5,550,111, No. 5,563,253, No. 5,571,799, No. 5 , 587,361, 5,625,050, 6,028,188, 6,124,445, 6,160,109, 6,169,170, 6,172,209 No. 6,239,265, No. 6,277,603, No. 6,326,199, No. 6,346,614, No. 6,444,423, No. 6,531,590, No. 6,534 Examples include U.S. Patent Nos. 639, 6,608,035, 6,683,167, 6,858,715, 6,867,294, 6,878,805, 7,015,315, 7,041,816, 7,273,933, 7,321,029, and U.S. Patent No. RE 39464 (the full contents of each of these are incorporated herein by reference).

[0269] In some embodiments, the phosphorus atom-free modified oligonucleotide skeletons have skeletons formed by short-chain alkyl or cycloalkyl nucleoside bonds, mixed heteroatoms and alkyl or cycloalkyl nucleoside bonds, or one or more short-chain heteroatoms or heterocyclic nucleoside bonds. These include morpholino bonds (partially formed from the sugar moiety of the nucleoside); siloxane skeletons; sulfide, sulfoxide, and sulfone skeletons; formacetyl and thioformacetyl skeletons; methyleneformacetyl and thioformacetyl skeletons; riboacetyl skeletons; alkene-containing skeletons; sulfamate skeletons; methyleneimino and methylenehydrazino skeletons; sulfonate and sulfonamide skeletons; amide skeletons; and other skeletons having mixed N, O, S, and CH2 component moieties. Non-limiting examples of U.S. patents teaching the preparation of non-phosphorus skeletons include, but are not limited to, U.S. Patents No. 5,034,506, No. 5,166,315, No. 5,185,444, No. 5,214,134, No. 5,216,141, No. 5,235,033, No. 5,64,562, No. 5,264,564, No. 5,405,938, No. 5,434,257, No. 5,466,677, No. 5,470,967, and No. 5 Examples include Nos. 489,677, 5,541,307, 5,561,225, 5,596,086, 5,602,240, 5,608,046, 5,610,289, 5,618,704, 5,623,070, 5,663,312, 5,633,360, 5,677,437, and 5,677,439 (the entire contents of each of these are incorporated herein by reference).

[0270] Inter-subunit modification Alternatively or in addition to the modifications described herein, this section discloses additional intersubunit modifications that may be included in the siRNA molecules of this disclosure. An 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) intersubunit binding modifications as disclosed herein. These modifications may be located in fixed or overhanging regions of the antisense strand. In some embodiments, one, two, three, four, five, or more modifications as described in this section may be located in a second region of the antisense strand protruding from the sense strand.

[0271] In some embodiments, at least one modified subunit bond is given by formula E1: [ka] And, In the formula, each B is independently a base-pairing region. W is O, S, B, BR 2 , N, NR 2 , selected from the group consisting of OCH2, OCH, CH2, and CH, where optionally W is selected from the group consisting of OCH2 and OCH, Each X is independently a halo (e.g., fluoro or chloro), hydroxy, and C 1~6 Selected from the group consisting of alkoxys, where optionally each X is independently a halo (e.g., fluoro or chloro) and C 1~6 Selected from the group consisting of alkoxys (e.g., methoxy, ethoxy, 2-methoxyethoxy, n-propoxy, sec-propoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, or n-heptoxy), Y is O - OH, OR, NH - NH2, S - Selected from the group consisting of , and SH, where Y is optionally O - Selected from the group consisting of OH and OR, Z is O, S, BR2 , NR 2 Selected from the group consisting of , and CH2, R is a protecting group, Each R 2 These are independently H or optionally substituted C1-C6 alkyl groups. [ka] The bond is arbitrarily a double bond.

[0272] In some embodiments of formula E1, W is OCH2.

[0273] In some embodiments of formula E1, W is OCH, [ka] It is a double bond.

[0274] In some embodiments of formula E1, Z is O.

[0275] In some embodiments of formula E1, Z is CH2.

[0276] In some embodiments of formula E1, Y is O - If that is the case, then either Z or W is not O.

[0277] In some embodiments of formula E1, Z is CH2 and W is CH2. In some embodiments, the modified subunit coupling of formula E1 is the modified subunit coupling of formula E2: [ka] That is the case.

[0278] In some embodiments of formula E1, Z is CH2 and W is O. In some embodiments, the modified subunit bond of formula E1 is the modified subunit bond of formula E3: [ka] That is the case.

[0279] In some embodiments of formula E1, Z is O and W is CH2. In some embodiments, the modified subunit bond of formula E1 is the modified subunit bond of formula E4: [ka] That is the case.

[0280] In some embodiments of formula E1, Z is O and W is CH. In some embodiments, the modified subunit bond of formula E1 is the modified subunit bond of formula E5: [ka] That is the case.

[0281] In some embodiments, the modified subunit coupling of formula E1 is the modified subunit coupling of formula E6: [ka] That is the case.

[0282] In some embodiments of formula E6, Each B is independently a base-pairing region. Each X is independently a halo, hydroxyl, and C 1~6 Selected from the group consisting of alkoxys, where optionally each X is independently a halo (e.g., fluoro) and a C. 1~6 Selected from the group consisting of alkoxys (e.g., methoxy, ethoxy, n-propoxy, sec-propoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, or n-heptoxy), Y is O - OH, OR, NH - NH2, S - Selected from the group consisting of , and SH, where Y is optionally O - Selected from the group consisting of OH and OR, Z is selected from the group consisting of O and CH2. [ka] The bond is arbitrarily a double bond.

[0283] In some embodiments of formula E6, Each X is independently fluoro, hydroxy, and C 1~6 Selected from the group consisting of alkoxys, where optionally each X is independently fluoro and C 1~6 Selected from the group consisting of alkoxys (e.g., methoxy, ethoxy, n-propoxy, sec-propoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, or n-heptoxy), Y is O - Selected from the group consisting of OH and OR, Z is selected from the group consisting of O and CH2. [ka] The bond is arbitrarily a double bond.

[0284] In some embodiments of formula E6, Each X is independently selected from the group consisting of fluoro, hydroxy, methoxy, ethoxy, n-propoxy, sec-propoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, and n-heptoxy. Y is O - Selected from the group consisting of OH and OR, Z is selected from the group consisting of O and CH2. [ka] The bond is arbitrarily a double bond.

[0285] In some embodiments of formula E6, Each X is independently selected from the group consisting of fluoro, hydroxy, methoxy, ethoxy, n-propoxy, sec-propoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, and n-heptoxy. Y is O - Selected from the group consisting of OH and OR, Z is O, [ka] The bond is arbitrarily a double bond.

[0286] In some embodiments of formula E6, Each X is independently selected from the group consisting of fluoro, hydroxy, methoxy, ethoxy, n-propoxy, sec-propoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, and n-heptoxy. Y is O - Selected from the group consisting of OH and OR, Z is CH2, [ka] The bond is arbitrarily a double bond.

[0287] In some embodiments of formula E6, Each X is independently selected from the group consisting of fluoro, hydroxy, and methoxy. Y is O - Selected from the group consisting of OH and OR, Z is O, [ka] The bond is arbitrarily a double bond.

[0288] In some embodiments of formula E6, Each X is independently selected from the group consisting of fluoro, hydroxy, and methoxy. Y is O - Selected from the group consisting of OH and OR, Z is CH2, [ka] The bond is arbitrarily a double bond.

[0289] In some embodiments of formula E1, Z is O and W is OCH2. In some embodiments, the modified subunit bond of formula E1 is the modified subunit bond of formula E6a: [ka] That is the case.

[0290] In some embodiments of formula E1, Z is CH2 and W is CH. In some embodiments, the modified subunit coupling of formula E1 is the modified subunit coupling of formula E7: [ka] That is the case.

[0291] In some embodiments of formula E1, the base-pairing moiety B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0292] In some embodiments, at least one modified subunit bond is given by formula E8: [ka] And, In the formula, D is O, S, B, BR 2 , N, NR 2 , selected from the group consisting of OCH2, OCH, CH2, and CH, where optionally, D is selected from the group consisting of OCH2 and OCH, C is O - , OH, OR 1 NH - NH2, S - Selected from the group consisting of , and SH, where C is optionally O - OH, and OR 1Selected from the group consisting of, A is O, S, BR 2 , NR 2 Selected from the group consisting of , and CH2, R 1 It is a protecting group, Each R 2 These are independently H or optionally substituted C1-C6 alkyl groups. [ka] It is arbitrarily a double bond, The subunits are bridged by two optionally modified nucleosides.

[0293] In some embodiments, D is OCH2.

[0294] In some embodiments, D is OCH, [ka] It is a double bond.

[0295] In some embodiments, A is O.

[0296] In some embodiments, A is CH2.

[0297] In some embodiments, C is O - If that is the case, then either A or D is not O.

[0298] In some embodiments, D is CH2. In another embodiment, the modified subunit bond of formula E8 is the modified subunit bond of formula E9: [ka] That is the case.

[0299] In some embodiments, D is O. In another embodiment, the modified subunit bond of formula E8 is the modified subunit bond of formula E10: [ka] That is the case.

[0300] In some embodiments, D is CH2. In another embodiment, the modified subunit bond of formula E8 is the modified subunit bond of formula E11: [ka] That is the case.

[0301] In some embodiments, D is CH. In another embodiment, the modified subunit bond of formula E8 is the modified subunit bond of formula E12: [ka] That is the case.

[0302] In some embodiments, D is OCH2. In another embodiment, the modified subunit bond of formula E8 is the modified subunit bond of formula E13: [ka] That is the case.

[0303] In another embodiment, the modified subunit bond of formula E8 is the modified subunit bond of formula E14: [ka] That is the case.

[0304] In another embodiment, the modified subunit bond of formula E8 is the modified subunit bond of formula E15: [ka] That is the case.

[0305] In some embodiments of modified siRNA binding, each optionally modified nucleoside is independently selected in each instance from the group consisting of adenosine, guanosine, cytidine, and uridine.

[0306] In some embodiments, at least one modified subunit bond is given by formula E8: [ka] And, In the formula, D is selected from the group consisting of O, OCH2, OCH, CH2, and CH, where optionally, D is selected from the group consisting of OCH2 and OCH. C is O - , OH, OR 1 NH - NH2, S - Selected from the group consisting of , and SH, where C is optionally O - OH, and OR 1 Selected from the group consisting of, A is selected from the group consisting of O and CH2. R 1 This is a protecting group selected from the group consisting of dimethoxytrityl (DMTr), succinic acid, tert-butyldimethylsilyl (TBDMS), benzoyl (Bz), benzyl (Bn), methoxyethoxymethyl ether (MOM), methoxybenzyl ether (PMB), methylthiomethyl ether, pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuranyl (THF), trityl (Trt), triisopropylsilyl (TIPS), tert-butyldiphenylsilyl (TBDPS), and acetic acid. [ka] It is arbitrarily a double bond, The subunits are bridged by two optionally modified nucleosides.

[0307] In some embodiments, D is OCH2.

[0308] In some embodiments, D is OCH, [ka] It is a double bond.

[0309] In some embodiments, A is O.

[0310] In some embodiments, A is CH2.

[0311] In some embodiments of the above-described modified subunit bonding, the base-pairing moiety B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0312] In some embodiments, R is a protecting group selected from the group consisting of dimethoxytrityl (DMTr), succinic acid, tert-butyldimethylsilyl (TBDMS), benzoyl (Bz), benzyl (Bn), methoxyethoxymethyl ether (MOM), methoxybenzyl ether (PMB), methylthiomethyl ether, pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuranyl (THF), trityl (Trt), triisopropylsilyl (TIPS), tert-butyldiphenylsilyl (TBDPS), and acetic acid. [ka] The bond is arbitrarily a double bond.

[0313] Modification patterns of siRNA molecules The following sections provide a set of exemplary scaffolds in which the siRNA molecules of this disclosure may be incorporated.

[0314] In some embodiments of this disclosure, the siRNA may contain an antisense chain comprising a region represented by formula I, where formula I is in the 5'→3' direction. AB-(A') j -CP 2 -DP 1 -(C'-P 1) k -C' Equation I And, In the formula, A is in formula: CP 1 -DP 1 It is expressed by, and each A' is given by equation:CP 2 -DP 2 It is expressed by the formula: CP 2 -DP 2 -DP 2 -DP 2 Represented by, each C is a 2'-O-methyl(2'-O-Me) ribonucleoside, each C' is independently a 2'-O-Me ribonucleoside or a 2'-fluoro(2'-F) ribonucleoside, each D is a 2'-F ribonucleoside, and each P 1 This is a nucleoside-phosphorothioate bond, and each P 2 is a nucleoside-internucleoside phosphodiester bond, j is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7), and k is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7). In some embodiments, j is 4. In some embodiments, k is 4. In some embodiments, j is 4 and k is 4. The antisense is complementary (e.g., completely or partially complementary) to the target nucleic acid sequence.

[0315] In some embodiments, the antisense chain includes a structure represented by formula A1, where formula A1 is in the 5'→3' direction. ASBSAOBOBOBOAOBOAOBOA-OBOAOBOAOBSASASASBSA Formula A1 And, In the formula, A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents an internucleoside phosphodiester bond, and S represents an internucleoside phosphorothioate bond.

[0316] In some embodiments of the present disclosure, the siRNA may contain an antisense strand comprising a region represented by formula II, where formula II is in the 5'→3' direction. AB-(A') j -CP 2 -DP 1 -(CP 1 ) k -C' Formula II And, In the formula, A is in formula: CP 1 -DP 1 It is expressed by, and each A' is given by equation:CP 2 -DP 2 It is expressed by the formula: CP 2 -DP 2 -DP 2 -DP 2 Represented by, each C is a 2'-O-methyl(2'-O-Me) ribonucleoside, each C' is independently a 2'-O-Me ribonucleoside or a 2'-fluoro(2'-F) ribonucleoside, each D is a 2'-F ribonucleoside, and each P 1 This is a nucleoside-phosphorothioate bond, and each P 2 is a nucleoside-internucleoside phosphodiester bond, j is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7), and k is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7). In some embodiments, j is 4. In some embodiments, k is 4. In some embodiments, j is 4 and k is 4. The antisense is complementary (e.g., completely or partially complementary) to the target nucleic acid sequence.

[0317] In some embodiments of the present disclosure, the antisense chain includes a structure represented by formula A2, where formula A2 is in the 5'→3' direction. ASBSAOBOBOBOAOBOAOBOA-OBOAAOBOAOBSASASASASA formula A2 And, In the formula, A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents an internucleoside phosphodiester bond, and S represents an internucleoside phosphorothioate bond.

[0318] In some embodiments of the present disclosure, the sense chain includes a structure represented by formula III, where formula III is in the 5'→3' direction. E-(A') m -F Formula III And, In the formula, E is given by the formula:(CP 1 ) is expressed by 2, and F is given by equation: (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 is represented by A', C, D, P 1 , and P 2 m is defined in Equation I, where m is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7). In some embodiments, m is 4. The sense chain is complementary to the antisense chain (e.g., fully or partially complementary).

[0319] In some embodiments of the present disclosure, the sense chain includes a structure represented by formula S1, where S1 is in the 5'→3' direction. ASASAOBOAOBOAOBOAOBOA-OAOAOBBSASA Formula S1 And, In the formula, A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents an internucleoside phosphodiester bond, and S represents an internucleoside phosphorothioate bond.

[0320] In some embodiments of the present disclosure, the sense chain includes a structure represented by formula S2, where S2 is in the 5'→3' direction. ASASAOBOAOBOAOBOAOBOA-OAOAOBOAOA formula S2 And, In the formula, A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents an internucleoside phosphodiester bond, and S represents an internucleoside phosphorothioate bond.

[0321] In some embodiments of the present disclosure, the sense chain includes a structure represented by formula S3, where S3 is in the 5'→3' direction. ASASAOBOAOBOAOBOAOBOA-OAOAOBSASB formula S3 And, In the formula, A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents an internucleoside phosphodiester bond, and S represents an internucleoside phosphorothioate bond.

[0322] In some embodiments of the present disclosure, the sense chain includes a structure represented by formula S4, where S4 is in the 5'→3' direction. ASASAOBOAOBOAOBOAOBOA-OAOAOBOAOB Formula S4 And, In the formula, A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents an internucleoside phosphodiester bond, and S represents an internucleoside phosphorothioate bond.

[0323] In some embodiments of the present disclosure, the siRNA may contain an antisense strand comprising a region represented by formula IV, where formula IV is in the 5'→3' direction. A-(A') j -CP 2 -B-(CP 1 ) k -C' Formula IV And, In the formula, A is in formula: CP 1 -DP 1 It is expressed by, and each A' is given by equation:CP 2 -DP 2 It is expressed by the equation: DP 1 -CP 1 -DP 1 Represented by, each C is a 2'-O-Me ribonucleoside, each C' is independently a 2'-O-Me ribonucleoside or a 2'-F ribonucleoside, each D is a 2'-F ribonucleoside, and each P 1 This is a nucleoside-phosphorothioate bond, and each P 2 is a nucleoside-internucleoside phosphodiester bond, where 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., completely or partially complementary).

[0324] In some embodiments of the present disclosure, the antisense chain includes a structure represented by formula A3, where formula A3 is in the 5'→3' direction. ASBSAOBOAOBOAOBOAOBOA-OBOAOBOAOBSASASASA formula A3 And, In the formula, A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents an internucleoside phosphodiester bond, and S represents an internucleoside phosphorothioate bond.

[0325] In some embodiments of the present disclosure, the siRNA of the present disclosure may have a sense strand represented by formula V, where formula V is in the 5'→3' direction. E-(A') m -CP 2 -F Formula V And, In the formula, E is given by the formula:(CP 1 ) is expressed by 2, and F is given by equation: DP 1 -CP 1 -C, DP 2 -CP 2 -C, DP 1 -CP 1 -D, or DP 2 -CP 2 -D is represented by A', C, D, P 1 , and P 2 m is defined in Equation IV, where m is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7). In some embodiments, m is 5. The sense chain is complementary to the antisense chain (e.g., fully or partially complementary).

[0326] In some embodiments of the present disclosure, the sense chain includes a structure represented by formula S5, where S5 is in the 5'→3' direction. ASASAOBOAOBOAOBOAOBOA-OBOAOBSASA formula S5 And, In the formula, A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents an internucleoside phosphodiester bond, and S represents an internucleoside phosphorothioate bond.

[0327] In some embodiments of the present disclosure, the sense chain includes a structure represented by formula S6, where S6 is in the 5'→3' direction. ASASAOBOAOBOAOBOAOBOA-OBOAOBOAOA formula S6 And, In the formula, A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents an internucleoside phosphodiester bond, and S represents an internucleoside phosphorothioate bond. In some embodiments of the present disclosure, the sense chain includes a structure represented by formula S7, where S7 is in the 5'→3' direction. ASASAOBOAOBOAOBOAOBOA-OBOAOBSASB formula S7 And, In the formula, A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents an internucleoside phosphodiester bond, and S represents an internucleoside phosphorothioate bond.

[0328] In some embodiments of the present disclosure, the sense chain includes a structure represented by formula S8, where S8 is in the 5'→3' direction. ASASAOBOAOBOAOBOAOBOA-OBOAOBOAOB formula S8 And, In the formula, A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents an internucleoside phosphodiester bond, and S represents an internucleoside phosphorothioate bond.

[0329] In some embodiments of this disclosure, the siRNA may contain an antisense strand comprising a region represented by formula VI, where formula VI is in the 5'→3' direction. AB j -EB k-EFG l -DP 1 -C' Equation VI And, In the formula, A is in formula: CP 1 -DP 1 It is expressed by the formula:CP 2 Represented by equation: DP, where each C is a 2'-O-Me ribonucleoside, each C' is independently a 2'-O-Me ribonucleoside or a 2'-F ribonucleoside, each D is a 2'-F ribonucleoside, and each E is a DP. 2 -CP 2 It is expressed by the equation: DP 1 -CP 1 It is expressed by the formula:CP 1 Represented by, each P 1 This is a nucleoside-phosphorothioate bond, and each P 2 is a nucleoside-internucleoside phosphodiester bond, where 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., completely or partially complementary).

[0330] In some embodiments of the present disclosure, the antisense chain comprises a structure represented by formula A4, where formula A4 is in the 5'→3' direction. ASBSAOAOAOBOAOAOAOAOA-OAOAOBOAOBSASASASBSA formula A4 And, In the formula, A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents an internucleoside phosphodiester bond, and S represents an internucleoside phosphorothioate bond.

[0331] In some embodiments of this disclosure, the siRNA may contain a sense strand comprising a region represented by formula VII, where formula VII is in the 5'→3' direction. HB m -I n -A'-B o -HC Formula VII And, In the formula, A' is in formula: CP 2 -DP 2 It is expressed by the formula: (CP 1 ) is expressed by 2, and each I is given by equation: (DP 2 ) is represented by B, C, D, P 1 , and P 2 The sense chain is defined in formula VI, where m is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7), n is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7), and o is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7). In some embodiments, m is 3. In some embodiments, n is 3. In some embodiments, o is 3. In some embodiments, m is 3, n is 3, and o is 3. The sense chain is complementary to the antisense chain (e.g., fully or partially complementary).

[0332] In some embodiments of the present disclosure, the sense chain includes a structure represented by formula S9, where S9 is in the 5'→3' direction. ASASAOAOAOBOBOBOAOBOA-OAOAOAASASA formula S9 And, In the formula, A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents an internucleoside phosphodiester bond, and S represents an internucleoside phosphorothioate bond.

[0333] siRNA is given by formula VIII: Z-((AP-) n (BP-) m ) q Formula VIII It may contain an antisense chain that includes a region represented by In the formula, Z is the 5' phosphorus-stabilizing moiety, each A is a 2'-O-methyl(2'-O-Me) ribonucleoside, each B is a 2'-fluororibonucleoside, each P is an internucleoside bond independently selected from phosphodiester bonds and phosphorothioate bonds, 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 from 1 to 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).

[0334] siRNA synthesis method The siRNA molecules of this disclosure can be synthesized by standard methods known in the art, for example, by using automated DNA synthesizers commercially available from Biosearch, Inc. and Applied Biosystems, Inc., as will be further discussed below.

[0335] siRNA agents may be prepared using liquid-phase organic synthesis, solid-phase organic synthesis, or both. Organic synthesis offers the advantage of readily preparing oligonucleotides, including non-natural or modified nucleotides. The siRNA molecules of this disclosure may be prepared using liquid-phase organic synthesis, solid-phase organic synthesis, or both.

[0336] Furthermore, it is intended that any siRNA agent disclosed herein may be further optimized by systematically adding or removing linked nucleosides to create longer or shorter sequences. Such optimized sequences may be further refined by introducing modified nucleosides and / or modified nucleoside bindings, such as those described herein or known in the art, including alternative nucleosides, alternative sugar moieties, and / or alternative nucleoside bindings, as known and / or discussed herein, to further optimize the molecule (e.g., increase serum stability or circulating half-life, increase thermal stability, enhance transmembrane delivery, and / or targeting to specific locations or cell types).

[0337] 5' Phosphorus-stabilized portion To further protect the siRNA molecules of this disclosure from degradation, a 5'-phosphorus stabilizing moiety may be employed. The 5'-phosphorus stabilizing moiety replaces the 5'-phosphate, preventing its hydrolysis. Hydrolysis of the 5'-phosphate prevents binding to RISC, a necessary step in gene silencing. Any phosphate substitution that does not prevent binding to RISC is intended in this disclosure. In some embodiments, the 5'-phosphate substitution is also stable against hydrolysis in vivo. Each chain of the siRNA molecule may independently and optionally employ any preferred 5'-phosphorus stabilizing moiety. [ka]

[0338] Several exemplary end caps are shown in formulas IX to XVI. In formulas IX to XVI, Nuc represents a nucleic acid base or a nucleic acid base derivative or substitute as described herein. In formulas IX to XVI, X represents a 2'-modification as described herein. Some embodiments employ a hydroxyl group as in formula IX, a phosphate group as in formula X, a vinyl phosphonate group as in formulas XI and XIV, a 5'-methyl-substituted phosphate group as in formulas XII, XIII, and XVI, a methylene phosphonate group as in formula XV, or a vinyl 5'-vinyl phosphonate as a 5'-phosphorus-stabilizing moiety as shown in formula XI.

[0339] hydrophobic part This disclosure further provides siRNA molecules to which one or more hydrophobic moieties are attached. The hydrophobic moieties may be covalently attached to the 5' or 3' end of the siRNA molecules of this disclosure. Non-limiting examples of hydrophobic moieties suitable for use with the siRNA molecules of this disclosure include cholesterol, vitamin D, tocopherol, phosphatidylcholine (PC), docosahexaenoic acid, docosanic acid, PC-docosanoic acid, eicosapentaenoic acid, lithocholic acid, or any combination of the aforementioned hydrophobic moieties and PC.

[0340] siRNA branching The siRNA molecules of this disclosure may be branched. For example, the siRNA molecules of this disclosure may have one of several branching patterns, such as those described herein.

[0341] According to this disclosure, the siRNA molecules disclosed herein may be branched siRNA molecules. The siRNA molecules may be unbranched, or they may be bifurcated, trifurcated, or tetrafurcated via linkers. Each main branch may be further branched to allow for 2, 3, 4, 5, 6, 7, or 8 distinct single-stranded or double-stranded RNAs. The branching points on the linker may originate from the same atom, or from different atoms along the linker. Several exemplary embodiments are listed in Table 2. [Table 3]

[0342] In some embodiments, the siRNA molecule is a branched siRNA molecule. In some embodiments, the branched siRNA molecule is bifid, trifid, or tetrafid. In some embodiments, a bifid siRNA molecule is represented by one of the formulas XVII to XIX, where each RNA is independently an siRNA molecule, L is a linker, and each X is independently a branching point (e.g., any of the branching point parts described in US10,478,503, such as phosphoramidite, tosylated solketal, 1,3-diaminopropanol, pentaerythritol, or US10,478,503).

[0343] In some embodiments, a tribranched siRNA molecule is represented by one of the formulas XX to XXIII, where each RNA is independently an siRNA molecule, L is a linker, and each X independently represents a branching point.

[0344] In some embodiments, a tetrabranched siRNA molecule is represented by one of the formulas XXIV to XXVIII, where each RNA is independently an siRNA molecule, L is a linker, and each X independently represents a branching point.

[0345] Linker Multiple strands of siRNA described herein may be covalently linked via a linker. This branching effect, in particular, improves cell permeability, allowing for better access to cells in the CNS (e.g., nerve cells or glial cells). Any linking portion that is not incompatible with the siRNA of the present invention may be employed. Examples of linkers include 2 to 10 subunits (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 subunits) of ethylene glycol chains, alkyl chains, carbohydrate chains, block copolymers, peptides, RNA, DNA, etc. In some embodiments, any carbon or oxygen atom of the linker may optionally be replaced with a nitrogen atom and have a hydroxyl substituent or an oxo substituent. In some embodiments, the linker is a polyethylene glycol (PEG) linker. Suitable PEG linkers for use with the compositions and methods of this disclosure include linear or non-linear PEG linkers. Examples of non-linear PEG linkers include branched PEG, linear fork PEG, or branched fork PEG.

[0346] PEG linkers of various weights may be used with the compositions and methods of this disclosure. For example, the PEG linker may have a weight of 5 to 500 daltons. In some embodiments, a PEG linker having a weight of 500 to 1,000 daltons may be used. In some embodiments, a PEG linker having a weight of 1,000 to 10,000 daltons may be used. In some embodiments, a PEG linker having a weight of 200 to 20,000 daltons may be used. In some embodiments, the linker is covalently bonded to the sense strand of the siRNA. In some embodiments, the linker is covalently bonded to the antisense strand of the siRNA. In some embodiments, the PEG linker is a triethylene glycol (TrEG) linker. In some embodiments, the PEG linker is a tetraethylene glycol (TEG) linker.

[0347] In some embodiments, the linker is an alkyl chain linker. In some embodiments, the linker is a peptide linker. In some embodiments, the linker is an RNA linker. In some embodiments, the linker is a DNA linker.

[0348] The linker may covalently link two, three, four, or five independent siRNA chains. The linker may be covalently bonded to any portion of the siRNA oligomer. In some embodiments, the linker is bonded to the 3' end of the nucleoside of each siRNA chain. In some embodiments, the linker is bonded to the 5' end of the nucleoside of each siRNA chain. In some embodiments, the linker is bonded to the nucleoside of the siRNA chain (e.g., sense or antisense chain) via a covalent bonding moiety. In some embodiments, the covalent bonding moiety is selected from the group consisting of alkyl, ester, amide, carbonate, carbamate, triazole, urea, formacetal, phosphonate, phosphate, and phosphate derivatives (e.g., phosphorothioate, phosphoramidate, etc.).

[0349] In some embodiments, the linker has the structure of formula L1: [ka] It has.

[0350] In some embodiments, the linker has the structure of formula L2: [ka] It includes the structure.

[0351] In some embodiments, the linker has the structure of formula L3: [ka] It has.

[0352] In some embodiments, the linker has the structure of formula L4: [ka] It includes the structure.

[0353] In some embodiments, the linker has the structure of formula L5: [ka] It includes the structure.

[0354] In some embodiments, the linker has the structure of formula L6: [ka] It includes the structure.

[0355] In some embodiments, the linker has the structure of formula L7 as shown below: [ka] It includes the structure.

[0356] In some embodiments, the linker has the structure of formula L8: [ka] It has.

[0357] In some embodiments, the linker has the structure of formula L9: [ka] It has.

[0358] In some embodiments, the selection of a linker for use with one or more branched siRNA molecules disclosed herein may be based on the hydrophobicity of the linker, for example, so that a desired hydrophobicity is achieved for one or more branched siRNA molecules of this disclosure. For example, a linker containing an alkyl chain may be used to increase the hydrophobicity of the branched siRNA molecule compared to a branched siRNA molecule having a less hydrophobic or hydrophilic linker.

[0359] The siRNA agents disclosed herein may be synthesized and / or modified by methods established in the art, for example, those described by reference herein in Beaucage, SL et al. (edrs.), Current Protocols in Nucleic Acid Chemistry, John Wiley & Sons, Inc., New York, NY, 2000.

[0360] Treatment method This disclosure provides a method for treating subjects requiring gene silencing. Gene silencing may be performed, insofar as the objective is to restore genetic and biochemical pathway activity from a diseased state to a healthy state, in particular to silence defective or hyperactive genes, to silence negative regulators of genes with reduced expression, to silence wild-type genes that play a role in activation in pathways that increase the activity of disease driver genes, or to silence splice isoforms of such genes that, when selectively knocked down, can increase the total expression of such genes. The method may include delivering the siRNA molecule of this disclosure or a pharmaceutical composition containing it to the CNS of a subject (e.g., human) by any suitable route of administration (e.g., intrastriatal, intraventricular, intrathecal, intrastriatal, intracavitary, intracisional, intraparenchymal, intraintravenous, subcutaneous, or intramuscular). The active compound may be administered in any preferred dose. The actual dose of the composition of this disclosure administered to a patient may be determined by physical and physiological factors such as body weight, severity of condition, past or concomitant therapeutic interventions, the patient's idiopathic disease, and route of administration. Depending on the dose and route of administration, the preferred dose and / or effective dose frequency may vary depending on the subject's response. The administering physician will, in any case, determine the concentration of the active ingredient(s) in the composition and the appropriate dose(s) for each individual subject. Administration may be given any preferred number of times per day and for the required duration. Subjects may be adults or children with or without comorbidities.

[0361] Selection of target Subjects that can be treated with the siRNA molecules disclosed herein are, for example, subjects requiring treatment for any medical risk(s) associated with gain-of-function mutations in the target gene. Subjects that can be treated with the siRNA molecules disclosed herein include, for example, humans, monkeys, rats, mice, pigs, and other mammals having at least one orthologascopy of the target gene. Subjects may be adults or children with or without comorbidities.

[0362] 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. Accordingly, this disclosure provides pharmaceutical compositions containing the siRNA molecules of this disclosure mixed with a suitable diluent, carrier, or excipient. The siRNA molecules can be administered directly to the CNS or affected tissue of a subject, for example, by intrastriatal, intraventricular, intrathecal injection, intracisional injection via catheter, intraparenchymal injection, intravenous injection, subcutaneous injection, or intramuscular injection.

[0363] Conventional procedures and components for selecting and preparing suitable formulations are, for example, Remington, JP. The Science and Practice of Pharmacy, Easton, PA. Mack Publishers, 2012, 22 nd This is described in the ed., and in The United States Pharmacopeial Convention, The National Formulary, United States Pharmacopeial, 2015, USP 38 NF 33.

[0364] Under normal storage and use conditions, the pharmaceutical composition may contain, for example, preservatives to prevent the growth of microorganisms. The pharmaceutical composition may include a sterile aqueous solution, a sterile dispersion, or a powder for immediate preparation of a sterile solution or sterile dispersion, for example. In all cases, the form may be sterilized using techniques known in the art and may be fluidized to the extent that it can be easily administered to an object requiring treatment.

[0365] The pharmaceutical composition may be administered to a subject, for example, a human subject, alone or in combination with a pharmaceutically acceptable carrier, and the ratio may be determined by the solubility and / or chemical properties of the compound, the selected route of administration, and standard pharmaceutical techniques, as shown herein.

[0366] Administration regimen A physician with ordinary skill in the art can easily determine an effective dose of an siRNA molecule for administration to a mammalian subject (e.g., human) requiring administration. For example, a physician can start by prescribing a dose of one of the siRNA molecules of the Disclosure at a level lower than the level required to achieve the desired therapeutic effect, and gradually increase the dose until the desired effect is achieved. Alternatively, a physician can initiate a treatment regimen by administering a high dose of one of the siRNA molecules of the Disclosure, and then gradually increase the dose until the minimum dose at which the therapeutic effect (e.g., reduction in the expression of a target gene sequence) is reached. Generally, a preferred daily dose of one of the siRNA molecules of the Disclosure is the amount of siRNA molecule that is the minimum dose effective to produce a therapeutic effect. The ss-siRNA molecules or ds-siRNA molecules of the Disclosure may be administered by injection, for example, intrathecally, intravenously, intraventricularly, intracerebrally, intracisionally, intracisionally, intramuscularly, intracisionally, intracerebral The daily dose of the therapeutic composition of the siRNA molecule disclosed herein may be administered as a single dose, or as two, three, four, five, six, or more doses administered separately at appropriate intervals throughout the day, week, month, or year, in unit dosage forms as optional. The siRNA molecule disclosed herein may be administered alone, but may also be administered as a pharmaceutical formulation in combination with excipients, carriers, and optionally additional therapeutic agents.

[0367] Route of administration The methods described herein involve any route of administration permitted by the therapeutic composition. Some embodiments of the methods include intrathecal injection, intraventricular injection, intrastriatal injection, intraparenchymal injection, or intracisional injection via catheter.

[0368] Intrathecal injection is a direct injection into the spinal column or subarachnoid space. By direct injection into the CSF of the spinal column, the siRNA molecules of this disclosure reach cells in the spinal column (e.g., nerve cells and glial cells) directly and take a pathway to reach cells in the brain by bypassing the blood-brain barrier.

[0369] Intracerebroventricular (ICV) injection is a method of direct injection into the cerebrospinal fluid (CSF) of the ventricles. Similar to intrathecal injection, ICV is an injection method that bypasses the blood-brain barrier. Using ICV offers the advantage of allowing therapeutic drugs to reach brain and spinal column cells without the risk of degradation in the bloodstream.

[0370] Intrastriatal injection is a direct injection into the striatum or corpus striatum. The striatum is a region of the subcortical basal ganglia in the brain. Injection into the striatum bypasses the blood-brain barrier, avoiding the pharmacokinetic challenges of injection into the bloodstream and allowing direct delivery to brain cells.

[0371] Intraparenchymal administration involves direct injection into the parenchyma (e.g., the brain parenchyma). Injection into the brain parenchyma allows for direct injection into the affected brain region while bypassing the blood-brain barrier.

[0372] Catheter-assisted intracisional injection is a direct injection into the cisterna magna. The cisterna magna is a region of the brain located between the cerebellum and the dorsal surface of the medulla oblongata. Injection into the cisterna magna results in more direct delivery to cells of the cerebellum, brainstem, and spinal cord.

[0373] In some embodiments of the methods described herein, the therapeutic composition may be delivered to the subject by systemic administration, for example, intravenous, intramuscular, or subcutaneous administration.

[0374] Intravenous (IV) injection is a method of direct injection into the target bloodstream. IV administration may be in the form of a bolus, a continuous infusion, or any other method permitted by the therapeutic composition.

[0375] Intramuscular (IM) injection is an injection into the target muscle, such as the deltoid or gluteal muscle. IM can allow for rapid absorption of the therapeutic composition.

[0376] Subcutaneous injection is an injection into the subcutaneous tissue. The absorption of compositions delivered subcutaneously may be slower than with IV or IM injections, which can be beneficial for compositions requiring sustained absorption. [Examples]

[0377] The following examples are provided to those skilled in the art to explain how the compositions and methods described herein may be used, prepared, and evaluated, and are intended merely to illustrate the disclosure and not to limit the scope of what the inventors consider to be the disclosure.

[0378] Example 1. Effect of fixed nucleic acid base sequence at the 3' end of the antisense strand of a ds-siRNA molecule This example demonstrates the ability of the PRNP-targeting siRNA molecule of this disclosure to have a nucleic acid base sequence immobilized at the 3' end of the antisense strand. An siRNA molecule having two uridines at the 3' end of the antisense strand was compared with a sequence-matched one.

[0379] PRNP-targeting siRNA molecules were delivered to HeLa cells via lipid-mediated cell uptake (RNAiMax). HeLa cells were seeded and simultaneously translocated using RNAiMax with different concentrations of siRNA molecules. PRNP mRNA expression was measured 24 hours after translocation. Figure 1 and Table 3 demonstrate that each siRNA molecule effectively silences PRNP. [Table 4]

[0380] In Table 3, O represents an internucleoside phosphodiester bond, S represents an internucleoside phosphorothioate bond, mG represents 2'-methoxyguanosine, mC represents 2'-methoxycytosine, mA represents 2'-methoxyadenosine, mU represents 2'-methoxyuridine, and fU represents 2'-fluorouridine.

[0381] Example 2. Effects of including a modified nucleoside bond or modified ribose moiety in the fixed nucleic acid base sequence at the 3' end of the antisense strand of a ds-siRNA molecule. This example demonstrates the advantages of including a modified nucleoside linkage in the fixed nucleic acid base region at the 3' end of the antisense strand of an siRNA molecule. Examples of modified nucleoside links include any of the modified nucleoside links of formulas E1-E15 (e.g., the modified nucleoside linkage of formula E6a) or nucleoside phosphorothioate links. In addition, this example investigates the effect of including a modified ribose moiety (e.g., a 2'-methoxynucleoside and / or a 2'-fluoronucleoside).

[0382] PRNP Tg26378 mice aged 7-8 weeks were administered either a PBS control or an siRNA molecule targeting PRNP mRNA transcript by bilateral intracerebroventricular injection. 10 μL (5 μL / pore) was injected per animal at a rate of 2.0 μL / min. Tissue samples were collected from the frontal cortex (fCtx), motor cortex (mCTx), striatum (Cpu), and hippocampus (HP). N=8 mice were tested for each condition. PRNP mRNA expression levels were analyzed by comparing the tissue samples to the PBS control. Each condition was tested separately with injections of 0.2 nmol, 1 nmol, and 5 nmol of siRNA molecules. The tested siRNA molecules are listed in Table 4 below. [Table 5]

[0383] The results of this experiment are shown in Figures 2A (5 nmol injection), 2B (1 nmol injection), and 2C (0.2 nmol injection), and the results are reported as the percentage of PRNP mRNA measured compared to mice administered PBS. These results demonstrate that PRNP mRNA is effectively silenced in vivo by including a fixed nucleic acid base sequence.

[0384] Example 3. siRNA molecule having an immobilized nucleic acid base region This example further demonstrates the advantages of including a modified nucleoside linkage in the fixed nucleic acid base region at the 3' end of the antisense strand of an siRNA molecule. Examples of modified nucleoside links include any of the modified nucleoside links of formulas E1-E15 (e.g., the modified nucleoside linkage of formula E6a) or nucleoside phosphorothioate links. In addition, this example investigates the effect of including a modified ribose moiety (e.g., a 2'-methoxynucleoside and / or a 2'-fluoronucleoside).

[0385] 8-9 week old FVB / N female mice were administered siRNA molecules targeting either PBS control or HPRT1 mRNA transcript by bilateral intraventricular injection as described in Example 2. Tissues were collected from the frontal cortex (fCtx), motor cortex (mCTx), striatum (Cpu), and hippocampus (HP). N=8-10 mice were tested for each condition. After 28 days, HPRT1 mRNA expression was analyzed in the tissues compared to the PBS control. Each condition was tested separately by injection of 1 nmol or 3 nmol of siRNA molecules. The tested siRNA molecules are listed in Table 5 below. [Table 6]

[0386] The results are shown in Figures 3A (3 nmol) and 3B (1 nmol). These results demonstrate that HPRT1 mRNA is effectively silenced in vivo by including a fixed nucleic acid base sequence.

[0387] In independent experiments, the siRNA molecules listed in Table 5 were tested in vitro at various doses using HeLa cells and 10 series of 10-fold dilutions from 100 nM to 0.00000001 nM, as well as mock preparations. Cells were reverse transfected with 0.1% RNAiMAX, 0.5% FBS, and 0.5% penicillin-streptomycin, and their IC50 values ​​were calculated. The results are shown in Figure 3C (comparing all siRNA molecules), 3D (comparing formula F1 containing a terminal 2'-methoxynucleoside to formula F6 containing a terminal 2'-fluoronucleoside), and 3E (comparing formulas F1 to F5 with the addition of modified nucleoside binding of formula E6a). The IC50 values ​​are also reported in Table 6 below. [Table 7]

[0388] Example 4. Evaluation of all possible fixed trinucleotide 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 tested. A ds-siRNA molecule targeting HPRT1, having a 21-mer antisense strand and a 16-mer sense strand, was used in this example. The 3-terminal nucleic acid bases at the 3' end of the antisense strand were immobilized as the sequences in Table 1a, independently of the target mRNA transcript. These immobilized nucleic acid base regions were chemically modified as shown in formula F1.

[0389] HeLa cells were transfused using siRNA molecules at doses ranging from 0.00000001 nM to 100 nM. The IC50 values ​​were then calculated for each tested condition. The results are shown in Figure 4. The lowest IC50 value was observed for the GGU sequence (1.9 pM), while the highest IC50 value was observed for the GUC sequence (165 pM). The IC50 values ​​are also reported in Table 7 below. [Table 8-1] [Table 8-2]

[0390] This example demonstrates that the identity of the nucleic acid base sequence at the 3' end of the antisense strand affects the silencing activity of the siRNA molecule.

[0391] Example 5. Method for delivering ds-siRNA molecules to the central nervous system of a patient. Subjects, such as human subjects diagnosed with a disease, are treated by administering the siRNA molecule or a pharmaceutical composition containing it at a dose and frequency determined by the attending physician (e.g., three times daily, twice daily, once daily, once a week, or once a month). The dose and frequency are determined based on the subject's height, weight, age, sex, and other disorders.

[0392] For suitability to the disease and target, siRNA molecules (e.g., branched siRNA molecules) having the chemical modification patterns disclosed herein are selected by the physician. Single-stranded or double-stranded branched siRNAs can be selected. The selected siRNA has an antisense strand, and in the case of double-stranded siRNA, the sense strand having sequence and RNA modifications (e.g., natural and unnatural nucleoside binding, modified sugars, and 5' phosphorus stabilization moieties) is best suited to the patient and the targeted disease. For example, the antisense strand may have any of the antisense strand modification patterns disclosed herein, e.g., 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 pattern 1 may have a fully or partially complementary sense strand having one of the following chemical modification patterns: sense pattern 1: ASASAOBOAOBOAOBOAOBOA-OAOAOBSASA (formula S1); sense pattern 2: ASASAOBOAOBOAOBOAOBOA-OAOAOBOAOA (formula S2); sense pattern 3: ASASAOBOAOBOAOBOAOBOA-OAOAOBSASB (formula S3); or sense pattern 4: ASASAOBOAOBOAOBOAOBOA-OAOAOBOAOB (formula S4).In the case of ds-siRNA having antisense pattern 2, the sense strand may have one of the following chemical modification patterns: sense pattern 5: ASASAOBOAOBOAOBOAOBOA-OBOAOBSASA (formula S5); sense pattern 6: ASASAOBOAOBOAOBOAOBOA-OBOAOBOAOA (formula S6); sense pattern 7: ASASAOBOAOBOAOBOAOBOA-OBOAOBSASB (formula S7); or sense pattern 8: ASASAOBOAOBOAOBOAOBOA-OBOAOBOAOB (formula S8). In the case of ds-siRNA having antisense pattern 3, the sense strand may have a sense strand having the modified pattern of sense pattern 9: ASASAOAOAOBOBOBOAOBOA-OAOAOASASA (formula S9), where A and B are different nucleosides (e.g., A is a 2-O-methylribonucleoside and B is a 2'-fluororibonucleoside), T is a phosphorothioate, P is a phosphodiester, and PSM is a 5'-phosphorus stabilizing moiety (e.g., 5'-vinylphosphonate).

[0393] siRNA is delivered via a route best suited to the patient and their condition (e.g., intrathecal, intraventricular, or striatal) at a rate acceptable to the patient until the target reaches the maximum tolerated dose or until the symptoms of the disease are sufficiently improved.

[0394] Example 6. Optimization of siRNA molecules For any small interfering RNA (siRNA) agent disclosed herein, modification of the siRNA is expected to further optimize the efficacy or biophysical properties of the molecule (e.g., increased serum stability or circulating half-life, increased thermal stability, enhanced transmembrane delivery, and / or targeting to specific locations or cell types). Such optimization may be achieved by systematically adding or removing linked nucleosides to create longer or shorter sequences. Further optimization of the siRNA may include, for example, the incorporation of one or more alternative nucleosides, alternative 2' sugar moieties, and / or alternative nucleoside bonds. Furthermore, such optimization of the siRNA molecule may include the introduction of hydrophobic and / or stabilizing moieties to the 5' and / or 3' ends.

[0395] Optimization of siRNA with alternative nucleosides Optimization of the siRNA molecules of this disclosure may include one or more of the following nucleoside modifications: 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl derivatives and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C=C-CH3)uracil and cytosine, and other alkynyl derivatives of pyrimidine bases, 6 -Azouracil, cytosine, and thymine, 5-uracil (pseudracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, and other 8-substituted adenines and guanines, 5-halo, in particular 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and / or 3-deazaguanine and 3-deazaadenine. siRNA molecules may also include nucleic acid bases in which a purine or pyrimidine base is replaced by another heterocycle, such as 7-deazaadenine, 7-deazaguanosine, 2-aminopyridine, and / or 2-pyridone. Further optimizations of the siRNA molecules disclosed herein may include the nucleic acid bases disclosed in US3,687,808, Kroschwitz, JI, ed. The Concise Encyclopedia of Polymer Science and Engineering, New York, John Wiley & Sons, 1990, pp.858-859, Englisch et al., Angewandte Chemie, International Edition 30:613, 1991, and Sanghvi, YS, Chapter 16, Antisense Research and Applications, CRC Press, Gait, MJ ed., 1993, pp.289-302.

[0396] Optimization of siRNA by alternative sugar modifications Optimization of the siRNA molecule of this disclosure 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-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O-CH2OCH2N(CH3)2. Other possible 2' modifications that may optimize the siRNA molecules of this disclosure include OH;F;O-, S-, or N-alkyl;O-, S-, or N-alkenyl;O-, S-, or N-alkynyl; or all possible orientations of O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl can be substituted or unsubstituted C1-C10 alkyl or C2-C10 alkenyl and alkynyl. Other possible sugar substituents include, for example, aminopropoxy (-OCH2CH2CH2NH2), allyl (-CH2-CH=CH2), -O-allyl (-O-CH2-CH=CH2), and fluoro (F). The 2' sugar substituent may be located at the arabino position (upper position) or the ribo position (lower position). In some embodiments, the 2'-arabino modification is 2'-F. Similar modifications can occur at other positions on the siRNA molecule, particularly at the 3' position of the sugar in the 3'-terminal nucleoside or 2'-5' linked oligonucleotide, and at the 5' position of the 5'-terminal nucleotide. Oligonucleotides may also have sugar mimetic forms, such as the cyclobutyl moiety, instead of the pentofuranosyl sugar.

[0397] Optimization of siRNA through alternative nucleoside binding. Optimization of the siRNA molecules of this disclosure may include one or more of the following internucleoside modifications: methylphosphonates and other alkylphosphonates, phosphinates, 3'-alkylenephosphonates, 5'-alkylenephosphonates, phospholamidates, 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, boranophosphates having a normal 3'-5' bond, their 2'-5' bond analogues, and those having inverted polarity with one or more internucleotide bonds being 3'-3', 5'-5', or 2'-2' bonds.

[0398] Optimization of siRNA by hydrophobic moiety Optimization of the siRNA molecules of this disclosure may involve a hydrophobic moiety covalently attached to the 5' or 3' end. Non-limiting examples of hydrophobic moieties suitable for use with the siRNA molecules of this disclosure include cholesterol, vitamin D, tocopherol, phosphatidylcholine (PC), docosahexaenoic acid, docosanic acid, PC-docosanoic acid, eicosapentaenoic acid, lithocholic acid, or any combination of the aforementioned hydrophobic moieties and PC.

[0399] Optimization of siRNA by stabilization portion Optimization of the siRNA molecules of this disclosure may include a 5'-phosphorus stabilization moiety that protects the siRNA molecule from degradation. The 5'-phosphorus stabilization moiety replaces the 5'-phosphate and prevents its hydrolysis. Hydrolysis of the 5'-phosphate prevents binding to RISC, a necessary step in gene silencing. Any phosphate substitution that does not prevent binding to RISC is intended in this disclosure. In some embodiments, the 5'-phosphate substitution is also stable against hydrolysis in vivo. Each siRNA chain may independently and optionally employ any suitable 5'-phosphorus stabilization moiety. Non-limiting examples of suitable 5'-stabilization moieties for use with the siRNA molecules of this disclosure may be those represented by formulas IX to XVI above.

[0400] Optimization of siRNA using branched siRNA Optimization of the siRNA molecules of this disclosure may include, for example, the incorporation of branching patterns such as bifurcated, trifurcated, or tetrafurcated siRNAs linked via linkers. Each main branch may be further branched to allow for 2, 3, 4, 5, 6, 7, or 8 distinct single-stranded or double-stranded RNAs. The branching points on the linker may originate from the same atom or from different atoms along the linker. Several exemplary embodiments are listed in Table 2 above.

[0401] The siRNA compositions of this disclosure may be optimized to take the form of a bibranched siRNA molecule represented by any of formulas XVII to XIX; a tribranched siRNA molecule represented by any of formulas XX to XXIII; and / or a tetrabranched siRNA molecule represented by any of formulas XXIV to XXVIII, where each RNA is independently an siRNA molecule, L is a linker, and each X independently represents a branching point (e.g., any of the branching points described in US10,478,503).

[0402] Example 7. Preparation and administration of siRNA molecules The siRNA molecules of this disclosure can be formulated into pharmaceutical compositions for administration to a target in a biocompatible form suitable for in vivo administration. For example, the siRNA molecules of this disclosure may be administered with a suitable diluent, carrier, or excipient, and may further contain, for example, preservatives to prevent microbial growth. Conventional procedures and components for selecting and preparing a suitable formulation are, for example, Remington, JP. The Science and Practice of Pharmacy, Easton, PA. Mack Publishers, 2012, 22. nd This is described in the ed., and in The United States Pharmacopeial Convention, The National Formulary, United States Pharmacopeial, 2015, USP 38 NF 33.

[0403] The methods described herein assume any route of administration to the target CNS that is permissible with the siRNA compositions of this disclosure. Non-limiting examples of siRNA injection into the CNS include intrathecal, intraventricular, or cisterna magna injection via catheter. A physician with the usual skills in the art can easily determine an effective route of administration.

[0404] Example 8. Treatment method for subjects requiring gene silencing. Subjects requiring gene silencing are treated by administering the siRNA molecules or siRNA compositions of the Disclosure, formulated as salts, at a frequency determined by the physician. A physician with ordinary skill in the art can easily determine an effective dose of the siRNA molecules to administer to a mammalian subject (e.g., human) requiring treatment. For example, a physician may begin by prescribing a dose of one of the siRNA molecules of the Disclosure at a level lower than the level required to achieve the desired therapeutic effect, and gradually increase the dose until the desired effect is achieved. Alternatively, a physician may initiate a treatment regimen by administering a high dose of one of the siRNA molecules of the Disclosure, and then gradually increase the dose until the minimum dose that produces a therapeutic effect (e.g., a reduction in the expression of target mRNA or a preferred biomarker) is achieved. Generally, a preferred daily dose of one of the siRNA molecules of the Disclosure is the minimum dose that is effective in producing a therapeutic effect. The ss-siRNA molecules or ds-siRNA molecules of the Disclosure may be administered by injection, for example, intrathecal, intraventricular, or intracisional injection via catheter. The daily dose of one therapeutic composition of the siRNA molecules disclosed herein may be administered as a single dose, or as two, three, four, five, six, or more doses administered separately at appropriate intervals throughout the day, week, month, or year, in unit dosage forms as optional. Any of the siRNA molecules disclosed herein may be administered alone, but may also be administered as a pharmaceutical formulation in combination with excipients, carriers, and optionally additional therapeutic agents. The dose and frequency should be determined based on the subject's height, weight, age, sex, and other disorders.

[0405] For suitability to the patient, the siRNA molecule(s) of this disclosure are selected by the physician. Single-stranded or double-stranded siRNA (e.g., unbranched siRNA, bibranched siRNA, tribranched siRNA, tetrabranched siRNA, covalently linked siRNA) may be selected. The selected siRNA molecule may have an antisense strand and a sense strand with the most suitable sequence and RNA modifications for the patient (e.g., natural and unnatural nucleoside binding, modified sugars, 5' phosphate stabilization moieties, hydrophobic moieties, and / or branched structures).

[0406] The siRNA molecule is delivered at a rate acceptable to the patient, via a route best suited to the patient and their condition (e.g., intrathecal, intraventricular, or intracisional injection via catheter), until the target reaches the maximum tolerated dose or until symptoms are sufficiently improved.

[0407] Other Embodiments All publications, patents, and patent applications described herein are incorporated herein by reference to the extent that each individual publication or patent application is specifically and individually indicated as being incorporated by reference.

[0408] Although the present invention has been described in relation to its specific embodiments, it can be further modified, and this application will be understood to encompass any variations, uses, or modifications in general that conform to the principles of the present invention, including departures from the invention that are known or customary practices within the scope of the art to which the invention belongs and that may apply to the basic features described above, and that are subject to the scope of the claims.

[0409] Other embodiments are within the scope of the claims.

Claims

1. A small interfering RNA (siRNA) molecule comprising an antisense strand and a sense strand complementary to a portion of the antisense strand, (i) The antisense strand includes a first region of linked nucleotides and a second region of linked nucleotides in the 5'→3' direction, (ii) The first region has sufficient complementarity to hybridize with a portion of the target mRNA transcript, (iii) The second region includes an overhang that extends beyond the sense chain, (iv) The small interfering RNA (siRNA) molecule wherein the second region has one or more nucleotide mismatches with respect 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, (i) The antisense strand includes a first region of linked nucleotides and a second region of linked nucleotides in the 5'→3' direction, (ii) The first region has sufficient complementarity to hybridize with a portion of the target mRNA transcript, (iii) The second region includes an overhang that extends beyond the sense chain, (iv) The siRNA molecule wherein the second region has a fixed nucleic acid base sequence independent of the nucleic acid base sequence of the target mRNA transcript.

3. The siRNA molecule according to claim 1 or 2, wherein the siRNA molecule is introduced into an RNA-induced silencing complex (RISC), and when the RISC is exposed to the target mRNA transcript, the target mRNA transcript is cleaved, thereby forming a degradation product, the degradation product dissociates from the RISC at an increased dissociation rate compared to a RISC formed from a corresponding siRNA containing an antisense strand completely complementary to the target mRNA transcript.

4. The siRNA molecule according to any one of claims 1 to 3, wherein the nucleic acid base sequence of the second region results in an antisense strand having increased binding affinity to an endogenous Argonaut (AGO) protein compared to a corresponding antisense strand that is completely complementary to the target mRNA.

5. The siRNA molecule according to any one of claims 1 to 4, wherein the nucleic acid base sequence of the second region improves the half-life of the endogenous complex comprising the antisense strand and the AGO protein compared to the corresponding endogenous complex comprising a corresponding antisense strand that is completely complementary to the target mRNA.

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

7. The siRNA molecule according to claim 6, wherein the second region is one nucleotide in length.

8. The siRNA molecule according to claim 6, wherein the second region is 2 nucleotides long.

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

10. The siRNA molecule according to any one of claims 1 to 9, wherein the second region comprises at least one uridine nucleotide.

11. The siRNA molecule according to claim 10, wherein the second region comprises two uridine nucleotides.

12. The siRNA molecule according to any one of claims 1 to 11, wherein the second region comprises at least one modified nucleoside bond.

13. The second region is given by equation E1: 【Chemistry 1】 It includes at least one modified nucleoside bond, In the formula, each B is independently a base-pairing region. W is O, S, B, BR ２ , N, NR ２ , OCH ２ , OCH, CH ２ Selected from the group consisting of , and CH, Each X independently comprises a halo, hydroxyl, and C. １～６ Selected from the group consisting of alkoxys, Y is O － , OH, OR, NH － , NH ２ , S － , and is selected from the group consisting of SH Z is O, S, BR ２ , NR ２ , and CH ２ Selected from the group consisting of, R is a protecting group, Each R ２ However, independently, H or any substituted C １ ~C ６ It is alkyl, 【Chemistry 2】 The siRNA molecule according to claim 12, wherein the bond is optionally a double bond.

14. Y is O － In this case, the siRNA molecule according to claim 13, wherein either Z or W is not O.

15. Z is CH ２ And W is CH ２ The siRNA molecule according to claim 13.

16. The modified nucleoside bond in formula E1 is, formula E2: 【Transformation 3】 The siRNA molecule according to claim 12, wherein the modified nucleoside bond is a modified nucleoside bond.

17. Z is CH ２ The siRNA molecule according to claim 13, wherein W is O.

18. The modified nucleoside bond in formula E1 is in formula E3: 【Chemistry 4】 The siRNA molecule according to claim 12, wherein the modified nucleoside bond is a modified nucleoside bond.

19. Z is O, and W is CH ２ The siRNA molecule according to claim 13.

20. The modified nucleoside bond in formula E1 is in formula E4: 【Transformation 5】 The siRNA molecule according to claim 12, wherein the modified nucleoside bond is a modified nucleoside bond.

21. The siRNA molecule according to claim 13, wherein Z is CH and W is O.

22. The modified nucleoside bond in formula E1 is, formula E5: 【Transformation 6】 The siRNA molecule according to claim 12, wherein the modified nucleoside bond is a modified nucleoside bond.

23. Z is O, and W is OCH ２ The siRNA molecule according to claim 13.

24. The modified nucleoside bond in formula E1 is in formula E6: 【Transformation 7】 It is a modified nucleoside bond, In the formula, each X is independently fluoro, hydroxy, and C. １～６ Selected from the group consisting of alkoxys, Y is O － OH, OR, S － Selected from the group consisting of SH and SR, Z is O and CH ２ Selected from the group consisting of, 【Transformation 8】 The siRNA molecule according to claim 12, wherein the bond is optionally a double bond.

25. The modified nucleoside bond in formula E1 is formula E6a: 【Chemistry 9】 The siRNA molecule according to claim 24, wherein the modified nucleoside bond is a modified nucleoside bond.

26. Z is CH ２ The siRNA molecule according to claim 13, wherein W is CH.

27. The modified nucleoside bond in formula E1 is in formula E7: 【Chemistry 10】 The siRNA molecule according to claim 12, wherein the modified nucleoside bond is a modified nucleoside bond.

28. The siRNA molecule according to any one of claims 1 to 27, wherein the second region comprises at least one internucleoside phosphorothioate bond.

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

30. The siRNA molecule according to any claim 29, wherein the second region comprises at least one 2'-methoxynucleotide.

31. The siRNA molecule according to claim 29 or 30, wherein the second region comprises at least one 2'-fluoronucleotide.

32. The second region described above has one of the following sequences in the 5'→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-SB (Formula F6) It has, In the formula, each S is a nucleoside-phosphorothioate bond, Each oxygen atom is a phosphodiester bond between nucleosides. Each A is a 2'-methoxyribonucleoside, Each B is a 2'-fluororibonucleoside, Each XA is a 2'-methoxynucleotide of formula E6a, An siRNA molecule according to any one of claims 1 to 31, wherein each XB is a 2'-fluoronucleotide of formula E6a.

33. The second region described above has one of the following sequences in the 5'→3' direction: -S-(mA)-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) (Equation F11) -S-(mA)-S-(xU)-S-(xU) (Formula F12) -S-(mA)-S-(mU)-S-(fU) (Formula F13) It has, In the formula, each S is a nucleoside-phosphorothioate bond, Each oxygen atom is a phosphodiester bond between nucleosides. Each mA is 2'-methoxyadenosine, Each mG is 2'-methoxyguanidine, Each mU is 2'-methoxyuridine, Each xU is 2'-methoxyuridine of formula E6a, The siRNA molecule according to claim 32, wherein each yU is 2'-fluorouridine of formula E6a.

34. The antisense chain includes a structure represented by formula I, wherein formula I is in the 5'→3' direction. A-B-(A’) ｊ -C-P ２ -D-P １ -(C’-P １ ) ｋ -C’ Equation I And, In the equation, A is equal to the equation: C - P １ -D-P １ It is represented by, Each A' is given by equation: C - P ２ -D-P ２ It is represented by, B is given by equation: C - P ２ -D-P ２ -D-P ２ -D-P ２ It is represented by, Each C is a 2'-O-methyl (2'-O-Me) ribonucleoside, Each C' is independently a 2'-O-Me ribonucleoside or a 2'-fluoro(2'-F) ribonucleoside. Each D is a 2'-F ribonucleoside, Each P １ However, this is a nucleoside-phosphorothioate bond, Each P ２ However, it is a phosphodiester bond between nucleosides, j is an integer from 1 to 7, The siRNA molecule according to any one of claims 1 to 33, wherein k is an integer from 1 to 7.

35. The antisense chain includes a structure represented by formula A1, wherein formula A1 is in the 5'→3' direction. A-S-B-S-A-O-B-O-B-O-BO-A-O-BO-A-O-BO-A-O-BO-O-A-O-B-O-A-O-B-S-A-S-A-S-A-S-B-S Formula A1 And, The siRNA molecule according to claim 34, wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents an internucleoside phosphodiester bond, and S represents an internucleoside phosphorothioate bond.

36. The antisense chain includes a structure represented by formula I, wherein formula I is in the 5'→3' direction. A-B-(A’) ｊ -C-P ２ -D-P １ -(C-P １ ) ｋ -C’ Formula II And, In the equation, A is equal to the equation: C - P １ -D-P １ It is represented by, Each A' is given by equation: C - P ２ -D-P ２ It is represented by, B is given by equation: C - P ２ -D-P ２ -D-P ２ -D-P ２ It is represented by, Each C is a 2'-O-methyl (2'-O-Me) ribonucleoside, Each C' is independently a 2'-O-Me ribonucleoside or a 2'-fluoro(2'-F) ribonucleoside. Each D is a 2'-F ribonucleoside, Each P １ However, this is a nucleoside-phosphorothioate bond, Each P ２ However, it is a phosphodiester bond between nucleosides, j is an integer from 1 to 7, The siRNA molecule according to any one of claims 1 to 33, wherein k is an integer from 1 to 7.

37. The antisense chain includes a structure represented by formula A2, wherein formula A2 is in the 5'→3' direction. A-S-B-SA Formula A2 And, The siRNA molecule according to claim 36, wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents an internucleoside phosphodiester bond, and S represents an internucleoside phosphorothioate bond.

38. The sense chain includes a structure represented by formula III, where formula III is in the 5'→3' direction. E-(A') ｍ -F Formula III And, In the equation, E is given by (C - P １ ) ２ It is represented by, F is given by the formula: (C - P ２ ) ３ -D-P １ -C-P １ -C, (C-P ２ ) ３ -D-P ２ -C-P ２ -C, (C-P ２ ) ３ -D-P １ -C-P １ -D, or (C-P ２ ) ３ -D-P ２ -C-P ２ - Represented by D, A', C, D, P １ , and P ２ However, as defined in Equation II, The siRNA molecule according to any one of claims 1 to 37, wherein m is an integer from 1 to 7.

39. The sense chain includes a structure represented by formula S1, wherein formula S1 is in the 5'→3' direction, A-S-A-S-A-O-B-O-A-O-BO-A-O-BO-A-O-BO-O-A-O-A-O-A-O-B-S-A-S-A Formula S1 And, The siRNA molecule according to claim 38, wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents an internucleoside phosphodiester bond, and S represents an internucleoside phosphorothioate bond.

40. The sense chain includes a structure represented by formula S2, wherein formula S2 is in the 5'→3' direction, A-S-A-S-A-O-BO-O-A-O-BO-A-O-BO-A-O-BO-O-A-O-A-O-A-O-BO-O-A-O-A Formula S2 And, The siRNA molecule according to claim 38, wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents an internucleoside phosphodiester bond, and S represents an internucleoside phosphorothioate bond.

41. The sense chain includes a structure represented by formula S3, wherein formula S3 is in the 5'→3' direction, A-S-A-S-A-O-B-O-A-O-BO-A-O-BO-A-O-BO-O-A-O-A-O-A-O-B-S-A-S-B Formula S3 And, The siRNA molecule according to claim 38, wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents an internucleoside phosphodiester bond, and S represents an internucleoside phosphorothioate bond.

42. The sense chain includes a structure represented by formula S4, wherein formula S4 is in the 5'→3' direction, A-S-A-S-A-O-BO-O-A-O-BO-A-O-BO-A-O-BO-O-A-O-A-O-A-O-BO-O-A-O-B Formula S4 And, The siRNA molecule according to claim 38, wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents an internucleoside phosphodiester bond, and S represents an internucleoside phosphorothioate bond.

43. The antisense chain includes a structure represented by formula IV, wherein formula IV is in the 5'→3' direction. A-(A’) ｊ -C-P ２ -B-(C-P １ ) ｋ -C’ Formula IV And, In the equation, A is equal to the equation: C - P １ -D-P １ It is represented by, Each A' is given by equation: C - P ２ -D-P ２ It is represented by, B is given by equation: D - P １ -C-P １ -D-P １ It is represented by, Each C is a 2'-O-Me ribonucleoside, Each C' is independently a 2'-O-Me ribonucleoside or a 2'-F ribonucleoside. Each D is a 2'-F ribonucleoside, Each P １ However, this is a nucleoside-phosphorothioate bond, Each P ２ is a phosphodiester bond between nucleosides, j is an integer from 1 to 7, The siRNA molecule according to any one of claims 1 to 33 and 38 to 42, wherein k is an integer from 1 to 7.

44. The antisense chain includes a structure represented by formula A3, wherein formula A3 is in the 5'→3' direction. A-S-B-S-A-O-B-O-A-O-B-O-A-O-BO-A-O-B-O-A-O-BO-O-A-O-B-O-A-O-B-S-A-S-B-S-A-S-A Formula A3 And, The siRNA molecule according to claim 43, wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents an internucleoside phosphodiester bond, and S represents an internucleoside phosphorothioate bond.

45. The sense chain includes a structure represented by formula V, where formula V is in the 5'→3' direction. E-(A’) ｍ -C-P ２ -F Formula V And, In the equation, E is given by (C - P １ ) ２ It is represented by, F is represented by the formula: D - P １ - C - P １ - C, D - P ２ - C - P ２ - C, D - P １ - C - P １ - D, or D - P ２ - C - P ２ - D, A', C, D, P １ , and P ２ However, as defined in formula IV, An siRNA molecule according to any one of claims 1 to 37, 43, and 44, wherein m is an integer from 1 to 7.

46. The sense chain includes a structure represented by formula S5, where formula S5 is in the 5'→3' direction, A-S-A-S-A-O-B-O-A-O-BO-A-O-BO-A-O-BO-O-A-O-B-O-A-O-B-S-A-S-A Formula S5 And, The siRNA molecule according to claim 45, wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents an internucleoside phosphodiester bond, and S represents an internucleoside phosphorothioate bond.

47. The sense chain includes a structure represented by formula S6, wherein formula S6 is in the 5'→3' direction, A-S-A-S-A-O-BO-O-A-O-BO-A-O-BO-A-O-BO-O-A-O-BO-O-A-O-B-O-A-O-A Formula S6 And, The siRNA molecule according to claim 45, wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents an internucleoside phosphodiester bond, and S represents an internucleoside phosphorothioate bond.

48. The sense chain includes a structure represented by formula S7, wherein formula S7 is in the 5'→3' direction, A-S-A-S-A-O-B-O-A-O-BO-A-O-BO-A-O-BO-O-A-O-B-O-A-O-B-S-A-S-B Formula S7 And, The siRNA molecule according to claim 45, wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents an internucleoside phosphodiester bond, and S represents an internucleoside phosphorothioate bond.

49. The sense chain includes a structure represented by formula S8, wherein formula S8 is in the 5'→3' direction, A-S-A-S-A-O-B-O-A-O-BO-A-O-BO-A-O-BO-O-A-O-B-O-A-O-B-O-A-O-B Formula S8 And, The siRNA molecule according to claim 45, wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents an internucleoside phosphodiester bond, and S represents an internucleoside phosphorothioate bond.

50. The antisense chain includes a structure represented by formula VI, wherein formula VI is in the 5'→3' direction. A-B ｊ -E-B ｋ -E-F-G ｌ -D-P １ -C’ Formula VI And, In the equation, A is equal to the equation: C - P １ -D-P １ It is represented by, Each B is given by the formula: C - P ２ It is represented by, Each C is a 2'-O-Me ribonucleoside, Each C' is independently a 2'-O-Me ribonucleoside or a 2'-F ribonucleoside. Each D is a 2'-F ribonucleoside, Each E is given by equation: D - P ２ -C-P ２ It is represented by, F is given by the formula: D - P １ -C-P １ It is represented by, Each G is given by the formula: C - P １ It is represented by, Each P １ However, this is a nucleoside-phosphorothioate bond, Each P ２ However, it is a phosphodiester bond between nucleosides, j is an integer from 1 to 7, k is an integer from 1 to 7, The siRNA molecule according to any one of claims 1 to 33, 38 to 42, and 45 to 49, wherein l is an integer from 1 to 7.

51. The antisense chain includes a structure represented by formula A4, wherein formula A4 is in the 5'→3' direction. A-S-B-S-A-O-A-O-A-O-BO-A-O-A-O-A-O-A-O-A-O-A-O-A-O-B-O-A-O-B-S-A-S-A-S-A-S-B-S-A Formula A4 And, The siRNA molecule according to claim 50, wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents an internucleoside phosphodiester bond, and S represents an internucleoside phosphorothioate bond.

52. The sense chain includes a structure represented by formula VII, wherein formula VII is in the 5'→3' direction. H-B ｍ -I ｎ -A’-B ｏ -H-C Formula VII And, In the equation, A' is equal to equation: C - P ２ -D-P ２ It is represented by, Each H is given by the formula: (C - P １ ) ２ It is represented by, Each I is given by equation: (D - P ２ ) is represented by, B, C, D, P １ , and P ２ However, as defined in formula VI, m is an integer from 1 to 7, n is an integer from 1 to 7, An siRNA molecule according to any one of claims 1 to 37, 43, 44, 50, and 51, wherein o is an integer from 1 to 7.

53. The sense chain includes a structure represented by formula S9, wherein formula S9 is in the 5'→3' direction, A-S-A-S-A-O-A-O-A-O-BO-BO-O-BO-A-O-BO-O-A-O-A-O-A-O-A-S-A-S-A Formula S9 And, The siRNA molecule according to claim 52, wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents an internucleoside phosphodiester bond, and S represents an internucleoside phosphorothioate bond.

54. The siRNA molecule according to any one of claims 1 to 53, wherein the antisense strand further comprises a 5' phosphorus-stabilizing moiety at the 5' end of the antisense strand.

55. The siRNA molecule according to any one of claims 1 to 54, wherein the sense strand further comprises a 5' phosphorus-stabilizing moiety at the 5' end of the sense strand.

56. Each 5' phosphorus-stabilizing moiety independently corresponds to one of the formulas IX to XVI: 【Chemistry 11】 It is represented by, The siRNA molecule according to claim 54 or 55, wherein Nuc represents a nucleic acid base 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, hydroxy, or hydrogen.

57. The siRNA molecule according to claim 56, wherein the nucleic acid base is adenine, uracil, guanine, thymine, or cytosine.

58. The siRNA molecule according to any one of claims 54 to 57, wherein the 5' phosphorus-stabilizing moiety is an (E)-vinylphosphonate represented by formula XI.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

79. The siRNA molecule according to claim 78, wherein the branched siRNA molecule is bifurcated, trifurcated, or tetrafurcated.

80. The siRNA molecule is a bifurcated siRNA molecule, and optionally the bifurcated siRNA molecule is one of the formulas XVII to XIX: 【Chemistry 12】 It is represented by, The siRNA molecule according to claim 79, wherein each RNA is independently an siRNA molecule, L is a linker, and each X independently represents a branching point.

81. The siRNA molecule is a tribranched siRNA molecule, and optionally the tribranched siRNA molecule is one of the formulas XX to XXIII: 【Chemistry 13】 It is represented by, The siRNA molecule according to claim 79, wherein each RNA is independently an siRNA molecule, L is a linker, and each X independently represents a branching point.

82. The siRNA molecule is a tetrabranched siRNA molecule, and optionally the tetrabranched siRNA molecule is one of the formulas XXIV to XXVIII: 【Chemistry 14】 It is represented by, The siRNA molecule according to claim 79, wherein each RNA is independently an siRNA molecule, L is a linker, and each X independently represents a branching point.

83. The siRNA molecule according to any one of claims 80 to 82, wherein the linker is selected from the group consisting of one or more consecutive ethylene glycols, alkyls, carbohydrates, block copolymers, peptides, RNA, and DNA subunits.

84. The siRNA molecule according to claim 83, wherein the one or more consecutive subunits are two to twenty consecutive subunits.

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

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

87. A method for reducing the expression of a target gene in a subject in whom it is necessary to reduce the expression of the target gene, comprising administering a therapeutically effective amount of an siRNA molecule according to any one of claims 1 to 84 or a pharmaceutical composition according to claim 85 to the CNS of the subject.

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

89. The method according to claim 87 or 88, wherein the target gene is an overactive disease driver.

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

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

92. The method according to claim 87 or 88, wherein the target gene is a splice isoform of the target gene, and the splice isoform reduces the expression of the target gene.

93. The method according to any one of claims 87 to 92, wherein the reduction of gene expression treats the target disease condition.

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

95. A kit comprising a polymerized oligonucleotide according to any one of claims 1 to 84 or a pharmaceutical composition according to claim 85, and a package insert, wherein the package insert instructs the user of the kit to carry out the method according to any one of claims 86 to 94.

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