Modified oligonucleotides with increased stability

Abstract

To provide a metabolically stable phosphorothioate-modified RNA.SOLUTION: The present invention provides a modified oligonucleotide, having a 5' end and a 3' end, and comprising a sense strand and an antisense strand complementary to a target, and at least one modified intersubunit linkage of Formula I, where at least one modified oligonucleotide is incorporated into the antisense strand or sense strand of an siRNA.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 824,136, filed March 26, 2019, U.S. Provisional Application No. 62 / 826,454, filed March 29, 2019, and U.S. Provisional Application No. 62 / 864,792, filed June 21, 2019, the entire disclosures of each of which are incorporated herein by reference.

[0002] STATEMENT REGARDING GOVERNMENT-SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under grant numbers NS104022 and OD020012 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] FIELD OF THE INVENTION The present invention relates to novel modified oligonucleotides and novel modified siRNAs. [Background technology]

[0004] background Currently, the most common metabolically stable backbone modification used in complex therapeutic RNAs is the phosphorothioate (PS) modification. Other available backbone modification options, such as peptide nucleic acids (PNAs) and phosphorodiamidate morpholino oligonucleotides (PMOs), perform well as sterically blocked antisense oligonucleotides but are not tolerated by many promising RNA-based therapeutic strategies. These strategies include siRNA, miRNA, RNase H-dependent antisense oligonucleotides, aptamer-based therapies, and CRISPR therapies. This poor tolerance is due to the inability of PNAs and PMOs to resist biological mechanisms, such as Argonaute proteins (siRNA / miRNA), Cas9 (CRISPR), and RNase H, which strictly recognize RNA structures when forming "functional" RNA-protein complexes. Summary of the Invention [Problem to be solved by the invention]

[0005] One of the most common RNA-based therapeutic strategies is the use of metabolically stable PS-modified RNA. However, one major drawback of this strategy is the toxicity of RNA due to nonspecific binding to a wide variety of proteins in vivo. Therefore, additional metabolically stable backbone modifications are urgently needed in the field of RNA therapeutics.

[0006] Provided herein are a variety of backbone modifications in which the bridging oxygen of the phosphodiester bond is replaced with various organic functional groups. The backbone modifications provided herein are not expected to significantly affect the structure of RNA, and therefore may provide compatibility with a wide variety of RNA binding biological mechanisms. Furthermore, these modifications are not expected to exhibit toxic nonspecific binding to proteins, and therefore may be incorporated into a wide range of therapeutic RNAs. [Means for solving the problem]

[0007] overview In one embodiment, the present invention provides a modified oligonucleotide, the oligonucleotide having a 5' end, a 3' end, and being complementary to a target, wherein the oligonucleotide comprises a sense strand and an antisense strand and at least one amino acid sequence of Formula I: [ka] [During the ceremony, each B is independently a base-pairing moiety; W is selected from the group consisting of O, OCH2, OCH, CH2, and CH, optionally wherein W is selected from the group consisting of OCH2 and OCH; Each X is independently selected from halo (e.g., fluoro or chloro), hydroxy, and C 1-6 alkoxy, optionally wherein each X is independently selected from the group consisting of halo (e.g., fluoro or chloro) and C 1-6alkoxy (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 - and SH, optionally wherein Y is O - , OH, and OR; Z is selected from the group consisting of O and CH2; R is a protecting group; and --- is an optional double bond. containing modified intersubunit bonds.

[0008] In some embodiments of formula I, W is OCH2.

[0009] In certain embodiments of formula I, W is OCH; --- is a double bond.

[0010] In some embodiments of formula I, Z is O.

[0011] In some embodiments of formula I, Z is CH2.

[0012] In certain embodiments of formula I, Y is O - When Z or W is not O.

[0013] In some embodiments of Formula I, Z is CH2 and W is CH2. In some embodiments, the modified intersubunit linkage of Formula I is [ka] The modified intersubunit bond of

[0014] In some embodiments of Formula I, Z is CH2 and W is O. In some embodiments, the modified intersubunit linkage of Formula I is [ka] The modified intersubunit bond of

[0015] In some embodiments of Formula I, Z is O and W is CH. In some embodiments, the modified intersubunit linkage of Formula I is [ka] The modified intersubunit bond of

[0016] In some embodiments of Formula I, Z is O and W is CH. In some embodiments, the modified intersubunit linkage of Formula I is represented by Formula V [ka] The modified intersubunit bond of

[0017] In certain embodiments, the modified intersubunit linkage of Formula I is represented by Formula VI [ka] The modified intersubunit bond of

[0018] In certain embodiments of Formula VI, each B is independently a base-pairing moiety; Each X is independently selected from halo, hydroxy, and C 1-6 alkoxy; optionally, wherein each X is independently selected from the group consisting of halo (e.g., fluoro) and C 1-6 alkoxy (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 - and SH, optionally wherein Y is O- selected from the group consisting of , OH and OR; Z is selected from the group consisting of O and CH; and --- is an optional double bond.

[0019] In certain embodiments of Formula VI, Each X is independently selected from fluoro, hydroxy and C 1-6 alkoxy; optionally wherein each X is independently selected from the group consisting of fluoro and C 1-6 alkoxy (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 CH; and --- is an optional double bond.

[0020] In certain embodiments of Formula VI, 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 CH; and --- is an optional double bond.

[0021] In certain embodiments of Formula VI, 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; and --- is an optional double bond.

[0022] In certain embodiments of Formula VI, 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; and --- is an optional double bond.

[0023] In certain embodiments of Formula VI, 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; and --- is an optional double bond.

[0024] In certain embodiments of Formula VI, 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; and --- is an optional double bond.

[0025] In some embodiments of Formula I, Z is O and W is OCH. In some embodiments, the modified intersubunit linkage of Formula I is represented by Formula VIa [ka] The modified intersubunit bond of

[0026] In some embodiments of Formula I, Z is CH2 and W is CH. In some embodiments, the modified intersubunit linkage of Formula I is [ka] The modified intersubunit bond of

[0027] In certain embodiments of Formula I, base-pairing moiety B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0028] In one embodiment, the modified oligonucleotide is incorporated into an siRNA, wherein the modified siRNA has a 5' end, a 3' end, and is complementary to a target, wherein the siRNA comprises a sense strand and an antisense strand and at least one nucleotide sequence of Formula I [ka] [During the ceremony, each B is independently a base-pairing moiety; W is selected from the group consisting of O, OCH2, OCH, CH2, and CH, optionally wherein W is selected from the group consisting of OCH2 and OCH; Each X is independently selected from halo (e.g., fluoro or chloro), hydroxy, and C 1-6 alkoxy, optionally wherein each X is independently selected from the group consisting of halo and C 1-6 alkoxy (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 - and SH, optionally wherein Y is O - selected from the group consisting of , OH and OR; Z is selected from the group consisting of O and CH2; R is a protecting group; and --- is an optional double bond. containing modified intersubunit bonds.

[0029] In certain embodiments of formula I, Y is O - When Z or W is not O. In some embodiments of formula I, Z is CH2 and W is CH2.

[0030] In certain embodiments, the modified intersubunit linkage of Formula I is represented by Formula II [ka] The modified intersubunit bond of

[0031] In some embodiments of Formula I, Z is CH2 and W is O. In some embodiments, the modified intersubunit linkage of Formula I is [ka] The modified intersubunit bond of

[0032] In some embodiments of Formula I, Z is O and W is CH. In some embodiments, the modified intersubunit linkage of Formula I is [ka] The modified intersubunit bond of

[0033] In some embodiments of Formula I, Z is O and W is CH. In some embodiments, the modified intersubunit linkage of Formula I is [ka] The modified intersubunit bond of

[0034] In an embodiment of Formula I, Z is CH=CH and W is CH2. In another embodiment, the modified intersubunit linkage of Formula I is represented by Formula VII [ka] The modified intersubunit bond of

[0035] In certain embodiments, the modified intersubunit linkage of Formula I is represented by Formula VI [ka] The modified intersubunit bond of

[0036] In certain embodiments of Formula VI, each B is independently a base-pairing moiety; Each X is independently selected from halo, hydroxy, and C 1-6 alkoxy; optionally, wherein each X is independently selected from the group consisting of halo (e.g., fluoro) and C 1-6 alkoxy (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 - and SH, optionally wherein Y is O - selected from the group consisting of , OH and OR; Z is selected from the group consisting of O and CH; and --- is an optional double bond.

[0037] In certain embodiments of Formula VI, Each X is independently selected from fluoro, hydroxy and C 1-6 alkoxy; optionally wherein each X is independently selected from the group consisting of fluoro and C 1-6 alkoxy (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 CH; and --- is any arbitrary double bond.

[0038] In certain embodiments of Formula VI, 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 CH; and --- is an optional double bond.

[0039] In certain embodiments of Formula VI, 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; and --- is an optional double bond.

[0040] In certain embodiments of Formula VI, 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; and --- is an optional double bond.

[0041] In certain embodiments of Formula VI, 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; and --- is an optional double bond.

[0042] In certain embodiments of Formula VI, 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; and --- is an optional double bond.

[0043] In some embodiments of Formula I, Z is O and W is OCH. In some embodiments, the modified intersubunit linkage of Formula I is represented by Formula VIa [ka] The modified intersubunit bond of

[0044] In some embodiments of Formula I, Z is CH2 and W is CH. In some embodiments, the modified intersubunit linkage of Formula I is [ka] The modified intersubunit bond of

[0045] In some embodiments, the base pairing moiety B is selected from the group consisting of adenine, guanine, cytosine, and uracil. In other embodiments, B is adenine. In yet other embodiments, B is guanine. In yet another embodiment, B is cytosine. In some embodiments, B is uracil.

[0046] In one embodiment, the modified oligonucleotide is incorporated into an siRNA, wherein the modified siRNA has a 5' end, a 3' end, and is complementary to a target, wherein the siRNA comprises a sense strand and an antisense strand and at least one nucleotide sequence of Formula I [ka] [During the ceremony, each B is independently a base-pairing moiety; W is selected from the group consisting of O, OCH2, OCH, CH2, and CH, optionally wherein W is selected from the group consisting of OCH2 and OCH; Each X is independently selected from halo (e.g., fluoro or chloro), hydroxy, and C 1-6 alkoxy, optionally wherein each X is independently selected from the group consisting of halo and C 1-6 alkoxy (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 - and SH, optionally wherein Y is O - selected from the group consisting of , OH and OR; Z is selected from the group consisting of O and CH2; R is a protecting group selected from the group consisting of dimethoxytrityl (DMTr), succinate, 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 acetate; and --- is an optional double bond. containing modified intersubunit bonds.

[0047] In some embodiments of formula I, W is OCH2.

[0048] In certain embodiments of formula I, W is OCH; --- is a double bond;

[0049] In some embodiments of formula I, Z is O.

[0050] In some embodiments of formula I, Z is CH2.

[0051] In certain embodiments, the modified oligonucleotide is incorporated into an siRNA, wherein the modified siRNA has a 5' end, a 3' end, is complementary to a target, and comprises a sense strand and an antisense strand, wherein the siRNA has the formula VIII [ka] [During the ceremony, D is selected from the group consisting of O, OCH2, OCH, CH2, and CH, optionally wherein D is selected from the group consisting of OCH2 and OCH; C is O - , OH, OR 1 , N.H. - , NH2, S - and SH, optionally wherein C is O - , OH and OR 1 selected from the group consisting of: A is selected from the group consisting of O and CH2; R 1 is a protecting group; --- is an optional double bond. wherein the intersubunit linkage bridges two optionally modified nucleosides.

[0052] In certain embodiments, D is OCH2.

[0053] In certain embodiments, D is OCH; --- is a double bond.

[0054] In certain embodiments, A is O.

[0055] In certain embodiments, A is CH2.

[0056] In some embodiments, C is O - Then A or D is not O.

[0057] In some embodiments, D is CH. In other embodiments, the modified intersubunit linkage of formula VIII is [ka] The modified intersubunit bond of

[0058] In certain embodiments, D is O. In other embodiments, the modified intersubunit linkage of formula VIII is [ka] The modified intersubunit bond of

[0059] In some embodiments, D is CH. In other embodiments, the modified intersubunit linkage of formula VIII is [ka] The modified intersubunit bond of

[0060] In certain embodiments, D is CH. In other embodiments, the modified intersubunit linkage of formula VIII is [ka] The modified intersubunit bond of

[0061] In other embodiments, the modified intersubunit linkage of formula VII is [ka] The modified intersubunit bond of

[0062] In some embodiments, D is OCH. In other embodiments, the modified intersubunit linkage of formula VII is [ka] The modified intersubunit bond of

[0063] In other embodiments, the modified intersubunit linkage of formula VII is [ka] The modified intersubunit bond of

[0064] In certain embodiments of modified siRNA conjugates, each optionally modified nucleoside is independently, at each occurrence, selected from the group consisting of adenosine, guanosine, cytidine, and uridine.

[0065] In certain embodiments, the modified oligonucleotide is incorporated into an siRNA, wherein the modified siRNA has a 5' end, a 3' end, is complementary to a target, and comprises a sense strand and an antisense strand, wherein the siRNA has the formula VIII [ka] [During the ceremony, D is selected from the group consisting of O, OCH2, OCH, CH2, and CH, optionally wherein D is selected from the group consisting of OCH2 and OCH; C is O - , OH, OR 1 , N.H. - , NH2, S - and SH, optionally wherein C is O -, OH and OR 1 selected from the group consisting of: A is selected from the group consisting of O and CH2; R 1 is a protecting group selected from the group consisting of dimethoxytrityl (DMTr), succinate, 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 acetate; --- is an optional double bond. wherein the intersubunit linkage bridges two optionally modified nucleosides.

[0066] In certain embodiments, D is OCH2.

[0067] In certain embodiments, D is OCH; --- is a double bond.

[0068] In certain embodiments, A is O.

[0069] In certain embodiments, A is CH2.

[0070] In certain embodiments, the present invention provides a method for treating or managing a neurodegenerative disease, comprising administering to a patient in need of such treatment or management a therapeutically effective amount of the above-described siRNA or modified oligonucleotide.

[0071] In certain embodiments, the siRNA or modified oligonucleotide is administered to the brain of the patient.

[0072] In certain embodiments, the siRNA or modified oligonucleotide is administered by intracerebroventricular (ICV) injection.

[0073] In some embodiments, there is provided a branched compound comprising two or more oligonucleotides, wherein (a) the oligonucleotides are linked to each other by one or more moieties selected from a linker, a spacer, and a branch point, and (b) at least one oligonucleotide has formula (I): [ka] [During the ceremony, each B is independently a base-pairing moiety; W is selected from the group consisting of O, OCH2, OCH, CH2, and CH, optionally wherein W is selected from the group consisting of OCH2 and OCH; Each X is independently selected from halo (e.g., fluoro or chloro), hydroxy, and C 1-6 alkoxy, optionally wherein each X is independently selected from the group consisting of halo and C 1-6 alkoxy (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 - and SH, optionally wherein Y is O - selected from the group consisting of , OH and OR; Z is selected from the group consisting of O and CH2; R is a protecting group; and --- is an optional double bond. is a modified oligonucleotide comprising at least one modified intersubunit linkage of

[0074] In certain embodiments, W is OCH2.

[0075] In certain embodiments, W is OCH; --- is a double bond.

[0076] In certain embodiments, Z is O.

[0077] In certain embodiments, Z is CH2.

[0078] In some embodiments, the branched compound comprises 2, 4, 6, or 8 oligonucleotides.

[0079] In certain embodiments, each oligonucleotide is double-stranded and comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand have respective 5' and 3' ends.

[0080] In certain embodiments, each double-stranded oligonucleotide is independently attached to a linker, spacer, or branch point at the 3' or 5' end of the sense or antisense strand.

[0081] In certain embodiments, each antisense strand independently comprises at least 16, at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides complementary to the target.

[0082] In some embodiments, each linker is independently selected from an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, and combinations thereof; wherein any of the carbon or oxygen atoms of the linker is optionally replaced with a nitrogen atom or has a hydroxyl substituent or an oxo substituent.

[0083] In some embodiments, Y is O - When either Z or W is not O.

[0084] In certain embodiments, Z is CH2 and W is CH2.

[0085] In certain embodiments, the modified intersubunit linkage of formula (I) is a linker of formula (II): [ka] The modified intersubunit bond of

[0086] In certain embodiments, Z is CH2 and W is O.

[0087] In certain embodiments, the modified intersubunit linkage of Formula (I) is represented by Formula (III): [ka] The modified intersubunit bond of

[0088] In certain embodiments, Z is O and W is CH2.

[0089] In certain embodiments, the modified intersubunit linkage of Formula (I) is represented by Formula (IV): [ka] The modified intersubunit bond of

[0090] In certain embodiments, Z is O and W is CH.

[0091] In certain embodiments, the modified intersubunit linkage of formula (I) is a linker of formula (V): [ka] The modified intersubunit bond of

[0092] In certain embodiments, Z is O and W is OCH2.

[0093] In certain embodiments, the modified intersubunit linkage of Formula I is represented by Formula VI [ka] The modified intersubunit bond of

[0094] In certain embodiments of Formula VI, each B is independently a base-pairing moiety; Each X is independently selected from halo, hydroxy, and C 1-6 alkoxy; optionally, wherein each X is independently selected from the group consisting of halo (e.g., fluoro) and C 1-6 alkoxy (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 - and SH, optionally wherein Y is O - selected from the group consisting of , OH and OR; Z is selected from the group consisting of O and CH; and --- is an optional double bond.

[0095] In certain embodiments of Formula VI, Each X is independently selected from fluoro, hydroxy and C 1-6 alkoxy; optionally wherein each X is independently selected from the group consisting of fluoro and C 1-6 alkoxy (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 CH; and --- is an optional double bond.

[0096] In certain embodiments of Formula VI, 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 CH; and --- is an optional double bond.

[0097] In certain embodiments of Formula VI, 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; and --- is an optional double bond.

[0098] In certain embodiments of Formula VI, 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; and --- is an optional double bond.

[0099] In certain embodiments of Formula VI, 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; and ---is an optional double bond.

[0100] In certain embodiments of Formula VI, 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; and --- is an optional double bond.

[0101] In certain embodiments, the modified intersubunit linkage of Formula (I) is represented by Formula (VIa): [ka] The modified intersubunit bond of

[0102] In certain embodiments, Z is CH2 and W is CH.

[0103] In certain embodiments, the modified intersubunit linkage of formula (I) is represented by formula (VII): [ka] The modified intersubunit bond of

[0104] In certain embodiments, base-pairing moiety B is selected from the group consisting of adenine, guanine, cytosine and uracil.

[0105] In one embodiment, the modified oligonucleotide is incorporated into a modified siRNA, which has a 5' end, a 3' end, and is complementary to a target, wherein the siRNA comprises a sense strand and an antisense strand and at least one modified intersubunit linkage of Formula (I).

[0106] In certain embodiments, R is a protecting group selected from the group consisting of dimethoxytrityl (DMTr), succinate, 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 acetate; and --- is an optional double bond.

[0107] In some embodiments, formula (1) [ka] wherein L is selected from an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, and combinations thereof, and wherein Formula (1) optionally includes one or more branch points Bp and one or more spacers, where Bp, at each occurrence, is independently a polyvalent organic species or a derivative thereof; and S, at each occurrence, is independently selected from an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, and combinations thereof; N is an RNA duplex comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand each independently comprise one or more chemical modifications; and n is 2, 3, 4, 5, 6, 7, or 8, wherein At least one N is a group of formula (I) [ka] [During the ceremony, each B is independently a base-pairing moiety; W is selected from the group consisting of O, OCH2, OCH, CH2, and CH, optionally wherein W is selected from the group consisting of OCH2 and OCH; Each X is independently selected from halo (e.g., fluoro or chloro), hydroxy, and C 1-6 alkoxy, optionally wherein each X is independently selected from the group consisting of halo and C 1-6 alkoxy (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 - and SH, optionally wherein Y is O - selected from the group consisting of , OH and OR; Z is selected from the group consisting of O and CH2; R is a protecting group; and --- is an optional double bond. containing modified intersubunit bonds.

[0108] In certain embodiments, W is OCH2.

[0109] In certain embodiments, W is OCH; --- is a double bond.

[0110] In certain embodiments, Z is O.

[0111] In certain embodiments, Z is CH2.

[0112] In some embodiments, the compound has a structure selected from formulas (1-1) through (1-9). [Table 1]

[0113] In one embodiment, the antisense strand is [ka] and a 5' terminal group R selected from the group consisting of:

[0114] In some embodiments, the compound has formula (2): [ka] [During the ceremony, X, for each occurrence, is independently selected from adenosine, guanosine, uridine, cytidine, and chemically modified derivatives thereof; Y, for each occurrence, is independently selected from adenosine, guanosine, uridine, cytidine, and chemically modified derivatives thereof; - is a phosphodiester internucleoside linkage; = is a phosphorothioate internucleoside linkage; and --- is, in each occurrence independently, a base-pairing interaction or a mismatch, provided that at least one of the - bonds or at least one of the = bonds in formula (2) is a modified subunit bond of formula (I). It has.

[0115] In some embodiments, the compound has formula (3): [ka] [During the ceremony, X is, for each occurrence independently, a nucleotide that includes a 2'-deoxy-2'-fluoro modification; X, for each occurrence, is independently a nucleotide containing a 2'-O-methyl modification; Y is, for each occurrence, independently a nucleotide that includes a 2'-deoxy-2'-fluoro modification; and Y, for each occurrence, is independently a nucleotide including a 2'-O-methyl modification; provided that at least one of the - bonds or at least one of the = bonds in formula (3) is a modified subunit bond of formula (I). It has the following structure.

[0116] In some embodiments, the compound has formula (4): [ka] wherein X, for each occurrence, is independently selected from adenosine, guanosine, uridine, cytidine, and chemically modified derivatives thereof; Y, for each occurrence, is independently selected from adenosine, guanosine, uridine, cytidine, and chemically modified derivatives thereof; - is a phosphodiester internucleoside linkage; = is a phosphorothioate internucleoside linkage; and --- is, in each occurrence independently, a base-pairing interaction or a mismatch, provided that at least one of the - bonds or at least one of the = bonds in formula (4) is a modified subunit bond of formula (I). It has the following structure.

[0117] In some embodiments, the compound has formula (5): [ka] [During the ceremony, X is, for each occurrence independently, a nucleotide that includes a 2'-deoxy-2'-fluoro modification; X, for each occurrence, is independently a nucleotide containing a 2'-O-methyl modification; Y is, for each occurrence, independently a nucleotide that includes a 2'-deoxy-2'-fluoro modification; and Y, for each occurrence, is independently a nucleotide containing a 2'-O-methyl modification. It has the following structure.

[0118] In some embodiments, the moiety L is L1 [ka] It has the following structure.

[0119] Part R is R 3 and n may be 2.

[0120] In some embodiments, L is L [ka] It has the following structure.

[0121] Part R is R 3 and n may be 2.

[0122] In some embodiments, Y is O - When Z or W is not O.

[0123] In certain embodiments, Z is CH2 and W is CH2.

[0124] In certain embodiments, the modified intersubunit linkage of formula (I) is a linker of formula (II): [ka] The modified intersubunit bond of

[0125] In certain embodiments, Z is CH2 and W is O.

[0126] In certain embodiments, the modified intersubunit linkage of Formula (I) is represented by Formula (III): [ka] The modified intersubunit bond of

[0127] In certain embodiments, Z is O and W is CH2.

[0128] In certain embodiments, the modified intersubunit linkage of Formula (I) is represented by Formula (IV): [ka] The modified intersubunit bond of

[0129] In certain embodiments, Z is O and W is CH.

[0130] In certain embodiments, the modified intersubunit linkage of formula (I) is a linker of formula (V): [ka] The modified intersubunit bond of

[0131] In certain embodiments, Z is O and W is OCH2.

[0132] In certain embodiments, the modified intersubunit linkage of Formula I is represented by Formula VI [ka] The modified intersubunit bond of

[0133] In certain embodiments of Formula VI, each B is independently a base-pairing moiety; Each X is independently selected from halo, hydroxy, and C 1-6 alkoxy; optionally, wherein each X is independently selected from the group consisting of halo (e.g., fluoro) and C 1-6 alkoxy (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 - and SH, optionally wherein Y is O - selected from the group consisting of , OH and OR; Z is selected from the group consisting of O and CH; and --- is an optional double bond.

[0134] In certain embodiments of Formula VI, Each X is independently selected from fluoro, hydroxy and C 1-6 alkoxy; optionally wherein each X is independently selected from the group consisting of fluoro and C 1-6 alkoxy (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 CH; and --- is an optional double bond.

[0135] In certain embodiments of Formula VI, 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 CH; and --- is an optional double bond.

[0136] In certain embodiments of Formula VI, 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; and --- is an optional double bond.

[0137] In certain embodiments of Formula VI, 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; and --- is an optional double bond.

[0138] In certain embodiments of Formula VI, 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; and --- is an optional double bond.

[0139] In certain embodiments of Formula VI, 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; and --- is an optional double bond.

[0140] In certain embodiments, the modified intersubunit linkage of Formula (I) is represented by Formula (VIa): [ka] The modified intersubunit bond of

[0141] In certain embodiments, Z is CH2 and W is CH.

[0142] In certain embodiments, the modified intersubunit linkage of formula (I) is represented by formula (VII): [ka] The modified intersubunit bond of

[0143] In certain embodiments, base-pairing moiety B is selected from the group consisting of adenine, guanine, cytosine and uracil.

[0144] In one embodiment, the present invention provides a compound of formula (6): [ka] wherein L is selected from an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, and a combination thereof, and wherein formula (6) optionally includes one or more branch points Bp and one or more spacers, where Bp, at each occurrence, is independently a polyvalent organic species or a derivative thereof; S is independently selected from an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, and a combination thereof; each cNA is independently a carrier nucleic acid including one or more chemical modifications; and n is 2, 3, 4, 5, 6, 7, or 8, wherein at least one chemical modification of at least one cNA is represented by formula (I): [ka] [During the ceremony, each B is independently a base-pairing moiety; W is selected from the group consisting of O, OCH2, OCH, CH2, and CH, optionally wherein W is selected from the group consisting of OCH2 and OCH; Each X is independently selected from halo (e.g., fluoro or chloro), hydroxy, and C 1-6alkoxy, optionally wherein each X is independently selected from the group consisting of halo and C 1-6 alkoxy (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 - and SH, optionally wherein Y is O - , OH and OR. Z is selected from the group consisting of O and CH2; R is a protecting group; and --- is an optional double bond. This is the intersubunit bond.

[0145] In certain embodiments, W is OCH2.

[0146] In certain embodiments, W is OCH; --- is a double bond.

[0147] In certain embodiments, Z is O.

[0148] In certain embodiments, Z is CH2.

[0149] In some embodiments, the delivery system has a structure selected from formulas (6-1) through (6-9). [Table 2]

[0150] In certain embodiments, each cNA independently comprises at least 15 contiguous nucleotides.

[0151] In certain embodiments, each cNA is composed of independently chemically modified nucleotides.

[0152] In some embodiments, the delivery system further comprises n therapeutic nucleic acids (NAs), wherein each NA hybridizes to at least one cNA.

[0153] In some embodiments, each NA independently comprises at least 16 contiguous nucleotides.

[0154] In some embodiments, each NA independently comprises 16 to 20 consecutive nucleotides.

[0155] In certain embodiments, each NA comprises an unpaired overhang of at least 2 nucleotides.

[0156] In certain embodiments, the nucleotides of the overhang are linked via phosphorothioate linkages.

[0157] In some embodiments, each NA is independently selected from the group consisting of DNA, siRNA, antagomiR, miRNA gapmer, mixmer, or guide RNA.

[0158] In some embodiments, each NA is identical.

[0159] In some embodiments, each NA is not identical.

[0160] In certain embodiments, the target of delivery is selected from the group consisting of brain, liver, skin, kidney, spleen, pancreas, colon, fat, lung, muscle, and thymus.

[0161] In some embodiments, Y is O - When Z or W is not O.

[0162] In certain embodiments, Z is CH2 and W is CH2.

[0163] In certain embodiments, the modified intersubunit linkage of formula (I) is a linker of formula (II): [ka] The modified intersubunit bond of

[0164] In certain embodiments, Z is CH2 and W is O.

[0165] In certain embodiments, the modified intersubunit linkage of Formula (I) is represented by Formula (III): [ka] The modified intersubunit bond of

[0166] In certain embodiments, Z is O and W is CH2.

[0167] In certain embodiments, the modified intersubunit linkage of Formula (I) is represented by Formula (IV): [ka] The modified intersubunit bond of

[0168] In certain embodiments, Z is O and W is CH.

[0169] In certain embodiments, the modified intersubunit linkage of formula (I) is a linker of formula (V): [ka] The modified intersubunit bond of

[0170] In certain embodiments, Z is O and W is OCH2.

[0171] In certain embodiments, the modified intersubunit linkage of Formula I is represented by Formula VI [ka] The modified intersubunit bond of

[0172] In certain embodiments of Formula VI, each B is independently a base-pairing moiety; Each X is independently selected from halo, hydroxy, and C 1-6 alkoxy; optionally, wherein each X is independently selected from the group consisting of halo (e.g., fluoro) and C 1-6 alkoxy (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 - and SH, optionally wherein Y is O - selected from the group consisting of , OH and OR; Z is selected from the group consisting of O and CH; and --- is an optional double bond.

[0173] In certain embodiments of Formula VI, Each X is independently selected from fluoro, hydroxy and C 1-6 alkoxy; optionally wherein each X is independently selected from the group consisting of fluoro and C 1-6 alkoxy (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 CH; and --- is an optional double bond.

[0174] In certain embodiments of Formula VI, 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 CH; and --- is an optional double bond.

[0175] In certain embodiments of Formula VI, 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; and --- is an optional double bond.

[0176] In certain embodiments of Formula VI, 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; and --- is an optional double bond.

[0177] In certain embodiments of Formula VI, 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; and --- is an optional double bond.

[0178] In certain embodiments of Formula VI, 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; and --- is an optional double bond.

[0179] In certain embodiments, the modified intersubunit linkage of Formula (I) is represented by Formula (VIa): [ka] The modified intersubunit bond of

[0180] In certain embodiments, Z is CH2 and W is CH.

[0181] In certain embodiments, the modified intersubunit linkage of formula (I) is represented by formula (VII): [ka] The modified intersubunit bond of

[0182] In certain embodiments, base-pairing moiety B is selected from the group consisting of adenine, guanine, cytosine and uracil.

[0183] In certain embodiments of any of the foregoing aspects of the invention, the RNA molecule (e.g., siRNA) is 8 nucleotides to 80 nucleotides in length (e.g., 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotides, 31 nucleotides, 32 nucleotides, 33 nucleotides, 34 nucleotides, 35 nucleotides, 36 nucleotides, 37 nucleotides, 38 nucleotides, 39 nucleotides, 40 nucleotides, 41 nucleotides). nucleotides, 42 nucleotides, 43 nucleotides, 44 nucleotides, 45 nucleotides, 46 nucleotides, 47 nucleotides, 48 ​​nucleotides, 49 nucleotides, 50 nucleotides, 51 nucleotides, 52 nucleotides, 53 nucleotides, 54 nucleotides, 55 nucleotides, 56 nucleotides, 57 nucleotides, 58 nucleotides, 59 nucleotides, 60 nucleotides, 61 nucleotides, 62 nucleotides, 63 nucleotides, 64 nucleotides, 65 nucleotides, 66 nucleotides, 67 nucleotides, 68 nucleotides, 69 nucleotides, 70 nucleotides, 71 nucleotides, 72 nucleotides, 73 nucleotides, 74 nucleotides, 75 nucleotides, 76 nucleotides, 77 nucleotides, 78 nucleotides, 79 nucleotides, or 80 nucleotides in length.

[0184] In certain embodiments, the RNA molecule is 10 to 50 nucleotides in length (e.g., 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotides, 31 nucleotides, 32 nucleotides, 33 nucleotides, 34 nucleotides, 35 nucleotides, 36 nucleotides, 37 nucleotides, 38 nucleotides, 39 nucleotides, 40 nucleotides, 41 nucleotides, 42 nucleotides, 43 nucleotides, 44 nucleotides, 45 nucleotides, 46 nucleotides, 47 nucleotides, 48 ​​nucleotides, 49 nucleotides, or 50 nucleotides in length).

[0185] In some embodiments, the RNA molecule comprises a length of about 15 nucleotides to about 25 nucleotides, hi some embodiments, the RNA molecule is 15 to 25 nucleotides in length (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length). [Brief explanation of the drawings]

[0186] The above and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings. This patent or application file contains at least one drawing filed in color. Copies of this patent or patent application file with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0187] [Figure 1] FIG. 1 summarizes the modified intersubunit linkers provided herein.

[0188] [Figure 2A]FIG. 2A shows a representative example of preparing a monomer of a modified phosphinate-containing oligonucleotide provided herein.

[0189] [Figure 2B] FIG. 2B shows a representative example of preparing monomers other than modified phosphinate-containing oligonucleotides provided herein.

[0190] [Figure 2C] FIG. 2C shows a representative example of preparing modified phosphinate-containing oligonucleotides provided herein.

[0191] [Figure 3A] FIG. 3A shows a representative example of preparing a monomer of a modified phosphonate-containing oligonucleotide provided herein.

[0192] [Figure 3B] FIG. 3B shows a representative example of preparing modified phosphonate-containing oligonucleotides provided herein.

[0193] [Figure 4] FIG. 4 provides an exemplary method for preparing the oligonucleotides provided herein on a solid support.

[0194] [Figure 5] FIG. 5 provides a method for producing vinyl phosphinate modified oligonucleotides.

[0195] [Figure 6] FIG. 6 shows a representative example of preparing monomers for synthesis of the oligonucleotides provided herein.

[0196] [Figure 7] FIG. 7 shows a representative example of preparing monomers for synthesizing modified oligonucleotides provided herein.

[0197] [Figure 8]FIG. 8 illustrates a method for producing modified oligonucleotides provided herein.

[0198] [Figure 9A] FIG. 9A shows the effect of internal -VP modifications on siRNA thermal stability.

[0199] [Figure 9B] FIG. 9B shows the effect of internal -VP modifications on duplex RNA thermal stability.

[0200] [Figure 10A] FIG. 10A shows RNA stability in a digestion test using SVPD (3' to 5' exonuclease) × 4 conditions for RNAs having PO, VP, and PS linkers.

[0201] [Figure 10B] FIG. 10B shows RNA stability in a digestion test using SVPDE (3' to 5' exonuclease) x 10 conditions for RNAs having PO, VP, and PS linkers.

[0202] [Figure 10C] FIG. 10C shows RNA stability in a digestion test using SVPDE (3' to 5' exonuclease) of a VP / PS mixed sequence.

[0203] [Figure 11] FIG. 11 shows the effect of adding mismatches in the siRNA sequence to improve allele discrimination without compromising silencing of the mutant allele.

[0204] [Figure 12] FIG. 12 shows the silencing efficacy of VP-modified siRNAs.

[0205] [Figure 13] FIG. 13 describes a method for preparing oligonucleotides having vinylphosphonate modified intersubunit linkages as described herein.

[0206] [Figure 14] Figure 14 lists the sequences of the synthesized vinylphosphonate-modified oligonucleotides. The antisense strand is written 5' to 3', with the SNP site in red and the mismatch in blue.

[0207] [Figure 15] FIG. 15 is a schematic representation of the hsiRNA antisense scaffold aligned to the HTT sequence surrounding the SNP site rs362273, with the green box indicating the location of the SNP site.

[0208] [Figure 16] FIG. 16 shows the change in mRNA expression when standard siRNA was used versus VP-modified siRNA.

[0209] [Figure 17] FIG. 17 shows on- or off-target HTT-mRNA knockdown with control and VP-modified siRNA.

[0210] [Figure 18] Figure 18 shows the structure of a di-hsiRNA. Black—2'-O-methyl; gray—2'-fluoro; red dotted line—phosphorothioate linkage; linker—tetraethylene glycol. Di-hsiRNA is two asymmetric siRNAs joined via a linker at the 3' end of the sense strand. Hybridization to the long antisense strand creates a protruding, single-stranded, fully phosphorothioated region necessary for tissue distribution, cellular uptake, and efficacy. The structure shown utilizes a four-monomer teg linker. The chemical identity of the linker can be modified without affecting efficacy. It can be adjusted for length, chemical composition (fully carbon), saturation, or by the addition of chemical targeting ligands.

[0211] [Figure 19] FIG. 19 shows the chemical synthesis, purification and quality control of biantennary siRNA.

[0212] [Figure 20] Figure 20 shows HPLC and quality control of compounds produced by the method described in Figure 19. Three major products were identified by mass spectrometry as the sense strand with a TEG (tetraethylene glycol) linker, a biantennary oligo, and a Vit-D (calciferol) conjugate. All products were independently purified by HPLC and tested in vivo. Only the biantennary oligo was characterized by unusual tissue distribution and efficacy, indicating that the branched structure is essential for tissue retention and distribution.

[0213] [Figure 21] Figure 21 shows mass spectrometry confirming the mass of the biantennary oligonucleotide. The observed mass of 11683 corresponds to the two sense strands joined via a TEG linker at the 3' end.

[0214] [Figure 22] FIG. 22 shows the synthesis of branched oligonucleotides using alternative chemical routes.

[0215] [Figure 23] FIG. 23 shows exemplary amidite linkers, spacers and branching moieties.

[0216] [Figure 24] Figure 24 shows oligonucleotide branching motifs. The double helix represents the oligonucleotide. Combinations of different linkers, spacers, and branch points allow for the production of a variety of branched hsiRNA structures.

[0217] [Figure 25] FIG. 25 shows structurally different branched oligonucleotides.

[0218] [Figure 26] FIG. 26 shows an asymmetric compound of the invention having four single-stranded phosphorothioate regions.

[0219] [Figure 27] Figures 27A-27C show in vitro efficacy data. (Figure 27A) HeLa cells were transfected (using RNAiMax) with the indicated concentrations of biantennary oligos for 72 hours. (Figure 27B) Primary cortical mouse neurons were treated with the indicated concentrations of biantennary oligos for one week. mRNA was measured using Affymetrix Quantigene 2.0. Data were normalized to a housekeeping gene (PPIB) and graphed as a percentage of the untreated control. (Figure 27C) HeLa cells were passively treated (without formulation) with the indicated concentrations of di-siRNA oligos for one week.

[0220] [Figure 28] Figures 28A-28B show the brain distribution of di-siRNA or TEG alone 48 hours after intrastriatal injection. Intrastriatal injection of 2 nmol (Figure 28A) biantennary oligo (4 nmol of the corresponding antisense strand) or (Figure 28B) TEG-oligo alone. N=2 mice / conjugate. Brains were collected 48 hours later and stained with Dapi (nuclei, blue). Red - oligo. The left side of the brain in (Figure 28A) is bright red, while the left side of the brain in (Figure 28B) is only faintly red.

[0221] [Figure 29] FIG. 29 shows that the single-injected di-siRNA was detected both ipsilaterally and contralaterally to the injection site.

[0222] [Figure 30] Figures 30A-30B show the broad distribution and efficacy of di-hsiRNA in the mouse brain. (Figure 30A) Robust Htt mRNA silencing in both the cortex and striatum 7 days after a single IS injection (25 μg), QuantiGene®. (Figure 30B) hsiRNA accumulation levels in tissues 7 days after injection (PNA assay).

[0223] [Figure 31]Figures 31A-31C show broad distribution and efficacy throughout the spinal cord after a bolus intrathecal injection of di-hsiRNA. Lumbar intrathecal injection of 3 nmol of biantennary oligo (6 nmol of the corresponding antisense HTT strand). (Figure 31A) Robust Htt mRNA silencing throughout the entire spinal cord, 7 days, n-6. Animals were sacrificed 7 days after injection. Tissue punches were taken from the cervical, thoracic, and lumbar regions of the spinal cord. mRNA was quantified using Affymetrix Quantigene 2.0 according to Coles et al. 2015. Data were normalized to the housekeeping gene, HPRT, and graphed as a percent of the aCSF control. aCSF = artificial CSF. (Figure 31B) Animals were injected in the lumbar IT with 75 μg of Cy3-Chol-hsiRNA, Cy-di-hsiRNA. Chol-hsiRNA exhibits a steep gradient of diffusion from the outside to the inside of the spinal cord. Di-hsiRNA shows broad distribution throughout the spinal cord (all regions). Leica 10x (20 mm bar). Image of biantennary oligo in the cervical region of the spinal cord 48 hours after intrathecal injection. Red = oligo, blue = Dapi. (Figure 31C) Image of biantennary oligo in the liver 48 hours after intrathecal injection. Red = oligo, blue = Dapi.

[0224] [Figure 32] Figures 32A-32C show branched oligonucleotides of the invention formed by annealing three oligonucleotides (Figure 32A). The long-chain oligonucleotide can contain a cleavable region in the form of unmodified RNA, DNA, or UNA; (Figure 32B) an asymmetrically branched oligonucleotide with 3' and 5' linkages to the previously described linker or spacer. This can be applied to the 3' and 5' ends of the sense or antisense strand, or a combination thereof; (Figure 32C) a branched oligonucleotide consisting of three different strands. Long dual-sense strands can be synthesized using 3' and 5' phosphoramidates to allow for 3'-3' adjacent ends or 5'-5' adjacent ends.

[0225] [Figure 33]FIG. 33 shows branched oligonucleotides of the invention conjugated with biologically active moieties.

[0226] [Figure 34] FIG. 34 shows the correlation between phosphorothioate content and stereoselectivity.

[0227] [Figure 35] FIG. 35 shows exemplary hydrophobic moieties.

[0228] [Figure 36] FIG. 36 shows exemplary internucleotide linkages.

[0229] [Figure 37] FIG. 37 shows exemplary internucleotide backbone linkages.

[0230] [Figure 38] Figure 38 shows exemplary sugar modifications.

[0231] [Figure 39] Figures 39A-39C show di-FM-hsiRNA. (Figure 39A) Chemical composition of four by-products produced by VitD-FM-hsiRNA synthesis and crude reverse-phase analytical HPLC of the original chemical synthesis. (Figure 39B) Efficacy of by-products in HeLa cells after lipid-mediated delivery of hsiRNA. Cells were treated for 72 hours. mRNA was measured using the QuantiGene 2.0 kit (Affymetrix). Data were normalized to the housekeeping gene HPRT and presented as a percentage of the untreated control. (Figure 39C) A single, unilateral intrastriatal injection (25 μg) of each hsiRNA by-product. Images were taken 48 hours after injection.

[0232] [Figure 40]Figures 40A-40B show that di-HTT-Cy3 does not efficiently induce silencing in the liver or kidney after intrastriatal injection. Figure 40A shows a scatter dot plot showing Htt mRNA expression in the liver 1 week after intrastriatal injection of di-HTT-Cy3 compared to a negative control (aCSF). Figure 40B shows a scatter dot plot showing Htt mRNA expression in the kidney 1 week after intrastriatal injection of di-HTT-Cy3 compared to a negative control (aCSF).

[0233] [Figure 41] Figures 41A-41B show that di-HTT efficiently silences HTT gene expression in both the striatum and cortex after intrastriatal injection, with di-HTT-Cy3 being slightly more effective than di-HTT (unlabeled). Figure 41A shows scatter dot plots depicting Htt mRNA expression in the striatum 1 week after intrastriatal injection of di-HTT, di-HTT-Cy3, or two negative controls (aCSF or di-NTC). Figure 41B shows scatter dot plots depicting Htt mRNA expression in the cortex 1 week after intrastriatal injection of di-HTT, di-HTT-Cy3, or two negative controls (aCSF or di-NTC).

[0234] [Figure 42] Figure 42 shows a scatter dot plot measuring di-HTT-Cy3 levels in the striatum and cortex. The plot shows that significant levels of di-HTT-Cy3 are still detectable two weeks after intrastriatal injection.

[0235] [Figure 43] Figures 43A-43B show that di-HTT-Cy3 effectively silences HTT mRNA and protein expression in both the striatum and cortex two weeks after intrastriatal injection. Figure 43A shows a scatter dot plot measuring Htt mRNA levels in the striatum and cortex two weeks after injection. Figure 43B shows a scatter dot plot measuring Htt protein levels in the striatum and cortex two weeks after injection.

[0236] [Figure 44] Figures 44A-44B show that high-dose di-HTT-Cy3 treatment does not cause significant toxicity in vivo two weeks after intrastriatal injection, but does result in substantial gliosis in vivo. Figure 44A shows scatter dot plots measuring DARPP32 signal in the striatum and cortex two weeks after di-HTT-Cy3 or aCSF injection. Figure 44B shows scatter dot plots measuring GFAP protein levels in the striatum and cortex two weeks after di-HTT-Cy3 or aCSF injection.

[0237] [Figure 45] FIG. 45 shows fluorescent imaging demonstrating that intrathecal injection of di-HTT-Cy3 results in robust and uniform distribution throughout the spinal cord.

[0238] [Figure 46] Figure 46 shows the merged fluorescence image of Figure 45 (magnified spinal cord). Blue - nuclei, red - di-HTT-Cy3.

[0239] [Figure 47] Figures 47A-47C show the widespread distribution of di-HTT-Cy3 48 hours after intracerebroventricular injection. Figure 47A shows fluorescent imaging of striatal, cortical, and cerebellar sections. Figure 47B shows bright-field images of whole brains injected with control (aCSF) or di-HTT-Cy3. Figure 47C shows fluorescent images of whole brain sections 48 hours after di-HTT-Cy3 injection.

[0240] [Figure 48] Figure 48 shows that di-HTT-Cy3 accumulates in multiple brain regions two weeks after intracerebroventricular injection. Scatter dot plots measure the levels of di-HTT-Cy3 in multiple brain regions.

[0241] [Figure 49]Figure 49A shows that di-HTT-Cy3 induces Htt gene silencing in multiple brain regions two weeks after intracerebroventricular injection, compared to a negative control injection (aCSF). Scatter dot plots measure Htt mRNA levels in multiple brain regions. Figure 49B shows that di-HTT-Cy3 induces Htt silencing in multiple brain regions two weeks after intracerebroventricular injection, compared to a negative control injection (aCSF). Scatter dot plots measure Htt protein levels in multiple brain regions.

[0242] [Figure 50] Figure 50 shows that intracerebroventricular injection of high dose di-HTT-Cy3 causes minimal toxicity in vivo. Scatter dot plots measure DARPP32 signal in multiple regions of the brain after di-HTT-Cy3 or aCSF injection.

[0243] [Figure 51] Figure 51 shows that intracerebroventricular injection of high dose di-HTT-Cy3 causes significant gliosis in vivo. Scatter dot plots measure DARPP32 signal in multiple regions of the brain after di-HTT-Cy3 or aCSF injection.

[0244] [Figure 52] Figure 52 shows that di-HTT-Cy3 distributes to multiple organs after intravenous injection. Fluorescent images show di-HTT-Cy3 levels in the heart, kidney, adrenal gland, and spleen after intravenous injection of di-HTT-Cy3 or negative control (PBS).

[0245] [Figure 53] Figure 53 shows that di-HTT-Cy3 accumulates in multiple organs after intravenous injection. Scatter dot plots measure the levels of di-HTT-Cy3 in multiple tissues.

[0246] [Figure 54]Figure 54 shows the structures of hsiRNA and fully metabolized (FM) hsiRNA.

[0247] [Figure 55] Figures 55A-55B show that complete metabolic stabilization of hsiRNA results in more effective gene silencing after intrastriatal injection of hsiRNA HTT or FM-hsiRNA HTT. Figure 55A shows a scatter dot plot measuring HTT mRNA levels up to 12 days after intrastriatal injection. Figure 55B shows a scatter dot plot measuring HTT mRNA levels up to 28 days after intrastriatal injection.

[0248] [Figure 56] Figure 56 shows the chemical diversity of single-stranded, fully modified oligonucleotides. The single-stranded oligonucleotides can consist of gapmers, mixmers, miRNA inhibitors, SSOs, PMOs, or PNAs.

[0249] [Figure 57] Figure 57 shows di-HTT with a TEG phosphoramidate linker.

[0250] [Figure 58] Figure 58 shows di-HTT with a TEG di-phosphate linker.

[0251] [Figure 59] Figure 59 shows variations of di-HTT with two oligonucleotide branches or four oligonucleotide branches.

[0252] [Figure 60] Figure 60 shows another variant of di-HTT in a structure with two oligonucleotide branches and R2 attached to a linker.

[0253] [Figure 61] Figure 61 shows a first strategy for incorporating hydrophobic moieties into branched oligonucleotide structures.

[0254] [Figure 62] Figure 62 shows a second strategy for incorporating hydrophobic moieties into branched oligonucleotide structures.

[0255] [Figure 63] Figure 63 shows a third strategy for incorporating hydrophobic moieties into branched oligonucleotide structures.

[0256] [Figure 64] Figure 64 shows the in vitro silencing efficacy of target mRNA with siRNA duplexes containing ex-NA intersubunit linkages at various positions.

[0257] [Figure 65] Figure 65 shows the ex-NA(2'O-methyl) phosphoramidate synthesis scheme.

[0258] [Figure 66] Figure 66 shows the synthesis scheme for ex-NA(2'-fluoro)phosphoramidates.

[0259] [Figure 67] Figure 67 shows the coupling of ex-NA phosphoramidates to a solid support. DETAILED DESCRIPTION OF THE INVENTION

[0260] DETAILED DESCRIPTION OF CERTAIN EXEMPLARY EMBODIMENTS Novel siRNAs are provided. Also provided are novel oligonucleotides.

[0261] Unless otherwise specified, the nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein is that which is well known and commonly used in the art. Unless otherwise specified, the methods and techniques provided herein are performed in accordance with conventional methods and as described in various general and more specific references well known in the art and cited and described elsewhere herein, unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclature used in connection with and laboratory methods and techniques of analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein are those which are well known and commonly used in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparations, formulation and delivery, and patient treatment.

[0262] Unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those of ordinary skill in the art. In the event of any ambiguity, the definitions provided herein take precedence over any dictionary or other definitions. Unless otherwise required, the singular includes the plural and the plural includes the singular. The use of "or" means "and / or" unless expressly stated otherwise. The terms "comprises" and other forms such as "including" and "including" are not limiting.

[0263] So that the present invention may be more readily understood, some terms are first defined.

[0264] The term "nucleoside" refers to a molecule having a purine or pyrimidine base covalently linked to a ribose or deoxyribose sugar. Examples of nucleosides include adenosine, guanosine, cytidine, uridine, and thymidine. Further examples of nucleosides include inosine, 1-methylinosine, pseudouridine, 5,6-dihydrouridine, ribothymidine, 2N-methylguanosine, and 2,2N,N-dimethylguanosine (also referred to as "rare" nucleosides). The term "nucleotide" refers to a nucleoside having one or more phosphate groups attached to the sugar moiety by an ester bond. Examples of nucleotides include nucleoside monophosphates, diphosphates, and triphosphates. The terms "polynucleotide" and "nucleic acid molecule" are used interchangeably herein and refer to a polymer of nucleotides linked by phosphodiester or phosphorothioate linkages between the 5' and 3' carbon atoms.

[0265] As used herein, "gene editing complex" refers to a biologically active molecule (e.g., a protein, one or more proteins, one nucleic acid, one or more nucleic acids, or any combination thereof) configured to add, destroy, or otherwise modify a genomic sequence (e.g., a gene sequence) by creating genetic lesions (e.g., double-strand breaks (DSBs) or single-strand breaks (SSBs)) in target DNA or other target nucleic acids, which can be loaded into the artificial exosomes of the present invention as cargo. Genetic lesions can be introduced by numerous methods known in the art. Examples of gene editing complexes include, but are not limited to, nucleases such as transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), engineered meganucleases, redesigned homing endonucleases, CRISPR / Cas systems, and meganucleases (e.g., meganuclease I-Scel). In some embodiments, the gene editing complex comprises proteins or molecules (e.g., components) associated with the CRISPR system, including, but not limited to, Cas9, Cas6, dCas9, CRISPR RNA (crRNA), transactivating crRNA (tracrRNA) and variants thereof. In some embodiments, the Cas protein is a Cpfl protein or a variant thereof.

[0266] As used herein, the terms "endonuclease" and "nuclease" refer to an enzyme that cleaves one or more phosphodiester bonds in a polynucleotide chain. Nucleases can be naturally occurring or engineered. Engineered nucleases are particularly useful for genome editing and are generally classified into four families: zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases (e.g., engineered meganucleases), and RNA-guided nucleases such as CRISPR-associated proteins (Cas nucleases).

[0267] The terms "RNA" or "RNA molecule" or "ribonucleic acid molecule" refer to a polymer of ribonucleotides (e.g., 2, 3, 4, 5, 10, 15, 20, 25, 30, or more ribonucleotides). The terms "DNA" or "DNA molecule" or "deoxyribonucleic acid molecule" refer to a polymer of deoxyribonucleotides. DNA and RNA can be synthesized naturally (e.g., by DNA replication or transcription of DNA, respectively). RNA can be post-transcriptionally modified. DNA and RNA can also be chemically synthesized. DNA and RNA can be single-stranded (i.e., ssRNA and ssDNA, respectively) or multi-stranded (e.g., double-stranded, i.e., dsRNA and dsDNA, respectively). "mRNA" or "messenger RNA" is a single-stranded RNA that specifies the amino acid sequence of one or more polypeptide chains. This information is translated when ribosomes bind to the mRNA during protein synthesis.

[0268] As used herein, the term "small interfering RNA" ("siRNA") (also referred to in the art as "short interfering RNA") refers to an RNA (or RNA analog) comprising about 10-50 nucleotides (or nucleotide analogs) that can direct or mediate RNA interference. Preferably, an siRNA comprises about 15-30 nucleotides or nucleotide analogs, more preferably about 16-25 nucleotides (or nucleotide analogs), even more preferably about 18-23 nucleotides (or nucleotide analogs), and even more preferably about 19-22 nucleotides (or nucleotide analogs) (e.g., 19, 20, 21, or 22 nucleotides or nucleotide analogs). The term "short" siRNA refers to an siRNA comprising about 21 nucleotides (or nucleotide analogs), e.g., 19, 20, 21, or 22 nucleotides. The term "long" siRNA refers to an siRNA comprising about 24-25 nucleotides, e.g., 23, 24, 25, or 26 nucleotides. In some cases, a short siRNA may contain fewer than 19 nucleotides, for example, 16, 17, or 18 nucleotides, as long as the short siRNA retains the ability to mediate RNAi. Similarly, in some cases, a long siRNA may contain more than 26 nucleotides, as long as the long siRNA retains the ability to mediate RNAi without further processing, for example, enzymatic processing, into a short siRNA.

[0269] The term "nucleotide analog" or "altered nucleotide" or "modified nucleotide" refers to a non-standard nucleotide, including non-naturally occurring ribonucleotides or deoxyribonucleotides. Exemplary nucleotide analogs are modified at any position so that certain chemical properties of the nucleotide are altered while the nucleotide analog still retains its ability to perform its intended function. Examples of nucleotide positions that can be derivatized include the 5-position, for example, 5-(2-amino)propyluridine, 5-bromouridine, 5-propyneuridine, 5-propenyluridine, etc.; the 6-position, for example, 6-(2-amino)propyluridine; the 8-position for adenosine and / or guanosine, for example, 8-bromoguanosine, 8-chloroguanosine, 8-fluoroguanosine, etc. Nucleotide analogs also include deazanucleotides, e.g., 7-deaza-adenosine; O- and N-modified (e.g., alkylated, e.g., N6-methyladenosine or as otherwise known in the art) nucleotides; and other heterocyclic-modified nucleotide analogs, such as those described in Herdewijn, Antisense Nucleic Acid Drug Dev., 2000 Aug. 10(4):297-310.

[0270] Nucleotide analogs can also contain modifications to the sugar portion of the nucleotide. For example, the 2'OH group can be substituted with a group selected from H, OR, R, F, Cl, Br, I, SH, SR, NH, NHR, NR, or COOR, where R is a substituted or unsubstituted C-C alkyl, alkenyl, alkynyl, aryl, etc. Other possible modifications include those described in U.S. Patents 5,858,988 and 6,291,438.

[0271] The phosphate group of nucleotide can also be modified, for example, by replacing one or more oxygens of the phosphate group with sulfur (for example, phosphorothioate), or by other substitutions that allow the nucleotide to perform its intended function, such as those described in Eckstein, Antisense Nucleic Acid Drug Dev. 2000 Apr. 10(2):117-21, Rusckowski et al. Antisense Nucleic Acid Drug Dev. 2000 Oct. 10(5):333-45, Stein, Antisense Nucleic Acid Drug Dev. 2001 Oct. 11(5):317-25, Vorobjev et al. Antisense Nucleic Acid Drug Dev. 2001 Apr. 11(2):77-85 and U.S. Patent 5,684,143. Some of the above-mentioned modifications (for example, phosphate group modification) preferably reduce the hydrolysis rate of the polynucleotide containing the analog, for example, in vivo or in vitro.

[0272] The term "oligonucleotide" refers to a short polymer of nucleotides and / or nucleotide analogs. The term "RNA analog" refers to a polynucleotide (e.g., a chemically synthesized polynucleotide) that has at least one modified or altered nucleotide compared to a corresponding unmodified or unmodified RNA, but retains the same or similar properties or functions as the corresponding unmodified or unmodified RNA. As described above, oligonucleotides can be linked with linkages that reduce the rate of hydrolysis of the RNA analog compared to RNA molecules with phosphodiester linkages. For example, analog nucleotides can contain methylenediol, ethylenediol, oxymethylthio, oxyethylthio, oxycarbonyloxy, phosphorodiamidate, phosphoroamidate, and / or phosphorothioate linkages. Preferred RNA analogs include sugar- and / or backbone-modified ribonucleotides and / or deoxyribonucleotides. Such modifications can further include the addition of non-nucleotide material, such as at the RNA termini or internally (to one or more nucleotides of the RNA). The RNA analog need only be sufficiently similar to natural RNA to have the ability to mediate RNA interference.

[0273] As used herein, the term "RNA interference" ("RNAi") refers to the selective intracellular degradation of RNA. RNAi occurs naturally in cells to remove foreign RNA (e.g., viral RNA). Natural RNAi proceeds through fragments cleaved from free dsRNA that direct the degradation mechanism to other similar RNA sequences. Alternatively, RNAi can be initiated manually, for example, to silence the expression of a target gene.

[0274] An RNAi agent, e.g., an RNA silencing agent, having a strand that is "sequence sufficiently complementary to a target mRNA sequence to direct target-specific RNA interference (RNAi)" means that the strand has a sequence sufficient to direct the destruction of the target mRNA by the RNAi machinery or process.

[0275] As used herein, the term "isolated RNA" (e.g., "isolated siRNA" or "isolated siRNA precursor") refers to an RNA molecule that is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

[0276] As used herein, the term "RNA silencing" refers to a group of sequence-specific regulatory mechanisms mediated by RNA molecules (e.g., RNA interference (RNAi), transcriptional gene silencing (TGS), post-transcriptional gene silencing (PTGS), repression, co-suppression, and translational repression) that result in the inhibition or "silencing" of the expression of corresponding protein-coding genes. RNA silencing has been observed in many types of organisms, including plants, animals, and fungi.

[0277] The term "differential RNA silencing" refers to the ability of an RNA molecule to substantially inhibit the expression of a "first" or "target" polynucleotide sequence, while not substantially inhibiting the expression of a "second" or "non-target" polynucleotide sequence, for example, when both polynucleotide sequences are present in the same cell.In some embodiments, the target polynucleotide sequence corresponds to a target gene, while the non-target polynucleotide sequence corresponds to a non-target gene.In other embodiments, the target polynucleotide sequence corresponds to a target allele, while the non-target polynucleotide sequence corresponds to a non-target allele.In some embodiments, the target polynucleotide sequence is a DNA sequence encoding the regulatory region (for example, promoter or enhancer element) of a target gene.In other embodiments, the target polynucleotide sequence is the target mRNA encoded by a target gene.

[0278] The term "in vitro" has its art-recognized meaning, e.g., involving purified reagents or extracts, e.g., cellular extracts. The term "in vivo" also has its art-recognized meaning, e.g., involving living cells, e.g., immortalized cells, primary cells, cell lines, and / or cells in an organism.

[0279] As used herein, the term "transgene" refers to any nucleic acid molecule that is strategically inserted into a cell and becomes part of the genome of the organism that develops from that cell. Such transgenes may include genes that are partially or entirely heterologous (i.e., foreign) to the transgenic organism or may represent genes that are homologous to endogenous genes of the organism. The term "transgene" also refers to a nucleic acid molecule comprising one or more selected nucleic acid sequences, e.g., DNA, encoding one or more engineered RNA precursors to be expressed in a transgenic organism, e.g., an animal, that are partially or entirely heterologous, i.e., foreign, to the transgenic animal, or homologous to an endogenous gene of the transgenic animal, but that are designed to be inserted into the animal's genome at a location different from the native gene. A transgene also includes one or more promoters and any other DNA, such as introns, necessary for expression of the selected nucleic acid sequence, all operably linked to the selected sequence, and may include enhancer sequences.

[0280] A gene "involved in" a disease or disorder includes a gene whose normal or abnormal expression or function affects or causes the disease or disorder or at least one symptom of the disease or disorder.

[0281] As used herein, the term "gain-of-function mutation" refers to any mutation in a gene in which the protein encoded by the gene (i.e., mutant protein) gains a function that causes or contributes to a disease or disorder not normally associated with the protein (i.e., wild-type protein). A gain-of-function mutation can be a deletion, addition, or substitution of one or more nucleotides in a gene that alters the function of the encoded protein. In certain embodiments, a gain-of-function mutation results in an altered function of the mutant protein or its interaction with other proteins. In other embodiments, a gain-of-function mutation results in a reduction or elimination of the normal wild-type protein, e.g., by altering, altering, or interacting the mutant protein with the normal wild-type protein.

[0282] As used herein, the term "target gene" refers to a gene whose expression is substantially inhibited or "silenced." This silencing is achieved, for example, by RNA silencing, such as by cleaving the mRNA of the target gene or by translational repression of the target gene. The term "non-target gene" refers to a gene whose expression is not substantially silenced. In some embodiments, the polynucleotide sequences of the target and non-target genes (e.g., mRNAs encoded by the target and non-target genes) may differ by one or more nucleotides. In other embodiments, the target and non-target genes may differ by one or more polymorphisms (e.g., single nucleotide polymorphisms or SNPs). In other embodiments, the target and non-target genes may share less than 100% sequence identity. In other embodiments, the non-target gene may be a homolog (e.g., an ortholog or paralog) of the target gene.

[0283] A "target allele" is an allele (e.g., an SNP allele) whose expression is selectively inhibited or "silenced." This silencing can be achieved by RNA silencing, for example, by cleavage of the target gene's mRNA or target allele by siRNA. The term "non-target allele" refers to an allele whose expression is not substantially silenced. In some embodiments, the target and non-target alleles can correspond to the same target gene. In other embodiments, the target allele corresponds to or is related to the target gene, and the non-target allele corresponds to or is related to a non-target gene. In some embodiments, the polynucleotide sequences of the target and non-target alleles can differ by one or more nucleotides. In other embodiments, the target and non-target alleles can differ by one or more allelic polymorphisms (e.g., one or more SNPs). In other embodiments, the target and non-target alleles can share less than 100% sequence identity.

[0284] As used herein, the term "polymorphism" refers to a difference (e.g., one or more deletions, insertions, or substitutions) in a gene sequence that is identified or detected when comparing the same gene sequences from different sources or subjects (but the same organism). For example, polymorphisms can be identified when comparing the same gene sequences from different subjects. Identifying such polymorphisms is routine in the art, and the method is similar to that used, for example, to detect breast cancer point mutations. Identification can be achieved, for example, by amplifying the polymorphic region from DNA extracted from the subject's lymphocytes using specific primers for the polymorphic region. Alternatively, polymorphisms can be identified when comparing two alleles of the same gene. In a specific embodiment, the polymorphism is a single nucleotide polymorphism (SNP).

[0285] A difference in sequence between two alleles of the same gene within an organism is referred to herein as an "allelic polymorphism." In certain embodiments, an allelic polymorphism corresponds to a SNP allele. For example, an allelic polymorphism can include a single nucleotide difference between two alleles of a SNP. A polymorphism can be in a nucleotide within a coding region, but due to the degeneracy of the genetic code, there is no change in the encoded amino acid sequence. Alternatively, a polymorphic sequence can encode a different amino acid at a specific position, but the amino acid change does not affect protein function. Polymorphic regions can also be found in non-coding regions of a gene. In exemplary embodiments, a polymorphism is found in the coding region of a gene or in the untranslated region of a gene (e.g., the 5'UTR or 3'UTR).

[0286] As used herein, the term "allelic frequency" refers to the relative frequency (e.g., proportion or percentage) of an allele (e.g., SNP allele) at a single locus in a population of individuals. For example, if a population of individuals carries n loci of a particular chromosomal locus (and the gene that occupies the locus) in each somatic cell, the allelic frequency of an allele is the fraction or percentage of loci that the allele occupies in the population. In a specific embodiment, the allelic frequency of an allele (e.g., SNP allele) is at least 10% (e.g., at least 15%, 20%, 25%, 30%, 35%, 40% or more) in a sample population.

[0287] As used herein, the term "sample population" refers to a group of individuals, which comprises a statistically significant number of individuals.For example, the sample population can comprise 50, 75, 100, 200, 500, 1000 or more individuals.In a specific embodiment, the sample population can comprise individuals who share at least one common disease phenotype (e.g., gain-of-function disorder) or mutation (e.g., gain-of-function mutation).

[0288] As used herein, the term "heterozygosity" refers to the proportion of individuals in a population who are heterozygous (e.g., contain two or more different alleles) at a particular locus (e.g., SNP). Heterozygosity can be calculated for a sample population using methods well known to those skilled in the art.

[0289] As used herein, the term "polyglutamine domain" refers to a protein segment or domain consisting of consecutive glutamine residues linked by peptide bonds. In some embodiments, the consecutive region contains at least five glutamine residues.

[0290] As used herein, the term "expanded polyglutamine domain" or "expanded polyglutamine segment" refers to a segment or domain of a protein that comprises at least 35 consecutive glutamine residues linked by peptide bonds. Such an expanded segment is found in subjects with a polyglutamine disorder, as described herein, whether or not the subject has overt symptoms.

[0291] As used herein, the term "trinucleotide repeat" or "trinucleotide repeat region" refers to a segment of a nucleic acid sequence consisting of consecutive repeats of a particular trinucleotide sequence. In some embodiments, the trinucleotide repeat comprises at least five consecutive trinucleotide sequences. Examples of trinucleotide sequences include, but are not limited to, CAG, CGG, GCC, GAA, CTG, and / or CGG.

[0292] As used herein, the term "trinucleotide repeat disease" refers to any disease or disorder characterized by an expanded trinucleotide repeat region located within a gene, where the expanded trinucleotide repeat region is the causative agent of the disease or disorder. Examples of trinucleotide repeat diseases include, but are not limited to, spinocerebellar ataxia type 12, spinocerebellar ataxia type 8, fragile X syndrome, fragile XE mental retardation, Friedreich's ataxia, and myotonic dystrophy. Examples of trinucleotide repeat diseases for treatment by the present invention are those characterized or caused by an expanded trinucleotide repeat region at the 5' end of the coding region of a gene encoding a mutant protein that causes or is the causative agent of the disease or disorder. Some trinucleotide diseases, such as fragile X syndrome, in which mutations are not associated with the coding region, may not be suitable for treatment by the methods of the present invention due to the lack of an appropriate mRNA to target by RNAi. In contrast, diseases such as Friedreich's ataxia may be amenable to treatment by the methods of the invention because the causative mutation is not within the coding region (i.e., within an intron), but the mutation may be, for example, within a pre-mRNA (e.g., a pre-splice pre-mRNA).

[0293] The term "examining the function of a gene in a cell or organism" refers to the examination or study of the expression, activity, function or phenotype resulting therefrom.

[0294] As used herein, the term "RNA silencing agent" refers to an RNA that can inhibit or "silence" the expression of a target gene. In some embodiments, the RNA silencing agent can prevent the complete processing (e.g., complete translation and / or expression) of an mRNA molecule through a post-transcriptional silencing mechanism. RNA silencing agents include small (<50 bp), non-coding RNA molecules, such as RNA duplexes containing paired strands, and precursor RNAs from which such small non-coding RNAs can be produced. Examples of RNA silencing agents include siRNA, miRNA, siRNA-like duplexes, antisense oligonucleotides, GAPMER molecules, and dual-function oligonucleotides, as well as their precursors. In some embodiments, the RNA silencing agent can induce RNA interference. In other embodiments, the RNA silencing agent can mediate translational repression.

[0295] As used herein, the term "rare nucleotide" refers to a naturally occurring nucleotide that occurs rarely, including a naturally occurring deoxyribonucleotide or ribonucleotide that is not guanosine, adenosine, cytosine, or uridine. Examples of rare nucleotides include, but are not limited to, inosine, 1-methylinosine, pseudouridine, 5,6-dihydrouridine, ribothymidine, 2N-methylguanosine, and 2,2N,N-dimethylguanosine.

[0296] The term "engineered," as in engineered RNA precursor or engineered nucleic acid molecule, indicates that the precursor or molecule is not found in nature, in that all or part of the nucleic acid sequence of the precursor or molecule is created or selected by humans. Once created or selected, the sequence can be replicated, translated, transcribed, or otherwise processed by machinery within the cell. Thus, an RNA precursor produced within a cell from a transgene containing an engineered nucleic acid molecule is an engineered RNA precursor.

[0297] As used herein, the term "microRNA" ("miRNA"), also referred to as "small transient RNA" ("stRNA"), refers to small (10-50 nucleotide) RNAs that are genetically encoded (e.g., by viral, mammalian, or plant genomes) and can direct or mediate RNA silencing. "miRNA disorder" refers to a disease or disorder characterized by aberrant expression or activity of a miRNA.

[0298] As used herein, the term "bifunctional oligonucleotide" refers to an RNA silencing agent having the formula TL-μ, where T refers to the mRNA targeting portion, L refers to the binding portion, and μ refers to the miRNA recruitment portion. As used herein, the terms "mRNA targeting portion," "targeting portion," "mRNA targeting portion," or "targeting portion" refer to a domain, portion, or region of a bifunctional oligonucleotide that has sufficient size and complementarity to a portion or region of the mRNA selected or targeted for silencing (i.e., the portion has a sufficient sequence to capture the target mRNA). As used herein, the term "binding portion" or "binding portion" refers to a domain, portion, or region of an RNA silencing agent that covalently binds or links to mRNA.

[0299] As used herein, the term "antisense strand" of an RNA silencing agent, e.g., an siRNA or RNA silencing agent, refers to a strand that is substantially complementary to a segment of about 10-50 nucleotides, e.g., about 15-30, 16-25, 18-23, or 19-22 nucleotides, of the mRNA of a gene targeted for silencing. The antisense strand, or first strand, refers to a sequence that is sufficiently complementary to the desired target mRNA sequence for target-specific silencing, e.g., sufficiently complementary to induce destruction of the desired target mRNA by the RNAi machinery or process (RNAi interference) or sufficiently complementary to induce translational repression of the desired target mRNA.

[0300] The term "sense strand" or "second strand" of an RNA silencing agent, such as an siRNA or RNA silencing agent, refers to the strand complementary to the antisense strand or first strand. The antisense strand and the sense strand can also be referred to as the first strand or second strand, where the first strand or second strand is complementary to the target sequence, and the second strand or first strand is complementary to the first strand or second strand, respectively. The miRNA duplex intermediate or siRNA-like duplex is an miRNA strand that is sufficiently complementary to an approximately 10-50 nucleotide segment of the mRNA of the gene targeted for silencing, and an miRNA strand that is sufficiently complementary to form a duplex with the miRNA strand. * Contains chains.

[0301] As used herein, the term "guide strand" refers to the strand of an RNA silencing agent, e.g., the antisense strand of an siRNA duplex or siRNA sequence, that enters the RISC complex and directs cleavage of a target mRNA.

[0302] The term "asymmetry," as used herein, refers to the asymmetry of the duplex region of an RNA silencing agent (for example, the stem of an shRNA), and refers to the imbalance in the binding strength or base pairing strength between the ends of an RNA silencing agent (for example, between the terminal nucleotide of the first strand or stem portion and the terminal nucleotide of the opposite second strand or stem portion), such that the 5'-end of one strand of the duplex is more frequently transiently unpaired, for example, single-stranded, than the 5'-end of the complementary strand.This structural difference determines that one strand of the duplex is preferentially incorporated into the RISC complex.The strand whose 5'-end is less tightly paired with the complementary strand is preferentially incorporated into RISC and mediates RNAi.

[0303] As used herein, the term "binding strength" or "base pair strength" refers to the strength of the interaction between pairs of nucleotides (or nucleotide analogs) on opposing strands of an oligonucleotide duplex (e.g., an siRNA duplex), primarily due to H-bonding, van der Waals interactions, etc., between the nucleotides (or nucleotide analogs).

[0304] As used herein, the "5' end" of the antisense strand refers to the 5'-terminal nucleotide, e.g., 1 to about 5 nucleotides from the 5' end of the antisense strand. As used herein, the "3' end" of the sense strand refers to the region, e.g., 1 to about 5 nucleotides, that is complementary to the 5'-terminal nucleotide of the complementary antisense strand.

[0305] As used herein, the term "destabilizing nucleotide" refers to a first nucleotide or nucleotide analog that can base pair with a second nucleotide or nucleotide analog such that the base pair has a lower binding strength than conventional base pairs (i.e., Watson-Crick base pairs). In some embodiments, the destabilizing nucleotide can form a mismatch base pair with the second nucleotide. In other embodiments, the destabilizing nucleotide can form a wobble base pair with the second nucleotide. In yet other embodiments, the destabilizing nucleotide can form an ambiguous base pair with the second nucleotide.

[0306] As used herein, the term "base pair" refers to the interaction between a pair of nucleotides (or nucleotide analogs) on opposing strands of an oligonucleotide duplex (e.g., a duplex formed by a strand of an RNA silencing agent and a target mRNA sequence), primarily through H-bonding, van der Waals interactions, etc., between the nucleotides (or nucleotide analogs). As used herein, the terms "binding strength" or "base pair strength" refer to the strength of base pairing.

[0307] As used herein, the term "mismatched base pair" refers to a base pair that is non-complementary or non-Watson-Crick, e.g., a base pair that is not a normal, complementary G:C, A:T, or A:U base pair. As used herein, the term "ambiguous base pair" (also known as a non-discriminatory base pair) refers to a base pair formed by universal nucleotides.

[0308] As used herein, the term "universal nucleotide" (also known as "neutral nucleotide") refers to a nucleotide (e.g., a destabilizing nucleotide) that has a base (a "universal base" or "neutral base") that does not significantly discriminate against bases on a complementary polynucleotide when base-pairing. Universal nucleotides are hydrophobic molecules that can be efficiently packaged into antiparallel duplex nucleic acids (e.g., double-stranded DNA or RNA) primarily through stacking interactions. The base portion of a universal nucleotide generally contains a nitrogen-containing aromatic heterocyclic moiety.

[0309] As used herein, the term "sufficient complementarity" or "sufficient degree of complementarity" means that the RNA silencing agent has a sequence (e.g., in the antisense strand, mRNA targeting portion, or miRNA recruitment portion) sufficient to bind to the desired target RNA and induce RNA silencing of the target mRNA, respectively.

[0310] As used herein, the term "translational repression" refers to the selective inhibition of mRNA translation. Natural translational repression occurs through miRNA cleaved from shRNA precursors. Both RNAi and translational repression are mediated by RISC. Both RNAi and translational repression can occur naturally or can be initiated by human intervention, for example, to silence the expression of target genes.

[0311] As used herein, the term "alkoxy" refers to the group -O-alkyl, where alkyl is as defined herein. Alkoxy includes, by way of example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, t-butoxy, and the like. In some embodiments, C1-C6 alkoxy groups are provided herein.

[0312] The terms "halo" or "halogen," as used herein, alone or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom, preferably fluorine, chlorine, or bromine, and more preferably fluorine or chlorine.

[0313] The term "hydroxy," as used herein, alone or as part of another substituent, means, unless otherwise stated, an alcohol moiety having the formula --OH.

[0314] The preparation of linkers involves the protection and deprotection of various chemical groups. The need for protection and deprotection and the selection of appropriate protecting groups can be easily determined by those skilled in the art. The chemistry of protecting groups can be found, for example, in Greene, et al., Protective Groups in Organic Synthesis, 4th Ed., Wiley & Sons, 2007, which is incorporated herein by reference in its entirety. The suitability of the protecting groups and formation and cleavage methods described herein can be adjusted as necessary in light of each substituent.

[0315] Various methods of the present invention involve comparing a value, level, characteristic, feature, property, etc., to a "suitable control," which is used interchangeably herein with "suitable control." A "suitable control" or "suitable control" is any control or standard known to those of skill in the art to be useful for comparison purposes. In some embodiments, a "suitable control" or "suitable control" is a value, level, characteristic, feature, property, etc., determined prior to performing the RNAi methods described herein. For example, transcription rate, mRNA level, translation rate, protein level, biological activity, cellular feature or property, genotype, phenotype, etc., can be determined prior to introducing an RNA silencing agent of the present invention into a cell or organism. In other embodiments, a "suitable control" or "suitable control" is a value, level, characteristic, feature, property, etc., determined in a cell or organism, e.g., a control or normal cell or organism, exhibiting a normal trait. In yet other embodiments, a "suitable control" or "suitable control" is a predetermined value, level, characteristic, feature, property, etc.

[0316] Unless otherwise defined, all technical and scientific terms have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Furthermore, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0317] Various aspects of the invention are described in further detail in the following subsections.

[0318] I. Novel Modified Oligonucleotides In some embodiments, the present invention provides modified oligonucleotides, the oligonucleotides having a 5' end, a 3' end, and being complementary to a target, wherein the oligonucleotides comprise a sense strand and an antisense strand and at least one of Formula (I): [ka] [During the ceremony, each B is independently a base-pairing moiety; W is selected from the group consisting of O, OCH2, OCH2, CH2, and CH; Each X is independently selected from halo (e.g., fluoro or chloro), hydroxy, and C 1-6 selected from the group consisting of alkoxy; Y is O - , OH, OR, NH - , NH2, S - and SH; Z is selected from the group consisting of O and CH2; R is a protecting group; and --- is an optional double bond. containing modified intersubunit bonds.

[0319] In certain embodiments of Formula (I), Y is O- When Z or W is not O.

[0320] In certain embodiments of Formula (I), Z is CH2 and W is CH2. In other embodiments, the modified intersubunit linkage of Formula (I) is Formula (II): [ka] The modified intersubunit bond of

[0321] In certain embodiments of Formula (I), Z is CH and W is O. In other embodiments, the modified intersubunit linkage of Formula (I) is represented by Formula (III): [ka] The modified intersubunit bond of

[0322] In certain embodiments of Formula (I), Z is O and W is CH. In other embodiments, the modified intersubunit linkage of Formula (I) is represented by Formula (IV): [ka] The modified intersubunit bond of

[0323] In certain embodiments of Formula (I), Z is O and W is CH. In other embodiments, the modified intersubunit linkage of Formula (I) is represented by Formula V [ka] The modified intersubunit bond of

[0324] In certain embodiments, the modified intersubunit linkage of Formula I is represented by Formula VI [ka] The modified intersubunit bond of

[0325] In certain embodiments of Formula VI, each B is independently a base-pairing moiety; Each X is independently selected from halo, hydroxy, and C 1-6 alkoxy; optionally, wherein each X is independently selected from the group consisting of halo (e.g., fluoro) and C 1-6 alkoxy (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 - and SH, optionally wherein Y is O - selected from the group consisting of , OH and OR; Z is selected from the group consisting of O and CH; and --- is an optional double bond.

[0326] In certain embodiments of Formula VI, Each X is independently selected from fluoro, hydroxy and C 1-6 alkoxy; optionally wherein each X is independently selected from the group consisting of fluoro and C 1-6 alkoxy (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 CH; and --- is an optional double bond.

[0327] In certain embodiments of Formula VI, 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 CH; and --- is an optional double bond.

[0328] In certain embodiments of Formula VI, 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; and --- is an optional double bond.

[0329] In certain embodiments of Formula VI, 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; and --- is an optional double bond.

[0330] In certain embodiments of Formula VI, 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; and --- is an optional double bond.

[0331] In certain embodiments of Formula VI, 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; and --- is an optional double bond.

[0332] In certain embodiments of Formula (I), Z is O and W is OCH. In other embodiments, the modified intersubunit linkage of Formula (I) is represented by Formula VIa [ka] The modified intersubunit bond of

[0333] In some embodiments of Formula (I), Z is CH and W is CH. In other embodiments, the modified intersubunit linkage of Formula (I) is represented by Formula VII [ka] The modified intersubunit bond of

[0334] In certain embodiments of Formula (I), the base-pairing moiety B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0335] In certain embodiments, the modified oligonucleotide is incorporated into an siRNA, the modified siRNA having a 5' end, a 3' end, and being complementary to a target, wherein the siRNA comprises a sense strand and an antisense strand and at least one oligonucleotide of formula (I): [ka] [During the ceremony, each B is independently a base-pairing moiety; W is selected from the group consisting of O, OCH2, OCH2, CH2, and CH; Each X is independently selected from halo (e.g., fluoro or chloro), hydroxy, and C 1-6selected from the group consisting of alkoxy; Y is O - , OH, OR, NH - , NH2, S - and SH; Z is selected from the group consisting of O and CH2; R is a protecting group; and --- is an optional double bond. containing modified intersubunit bonds.

[0336] In certain embodiments of Formula (I), Y is O - When Z or W is not O. In other embodiments of Formula (I), Z is CH2 and W is CH2.

[0337] In yet other embodiments, the modified intersubunit linkage of formula (I) is [ka] The modified intersubunit bond of

[0338] In certain embodiments of Formula (I), Z is CH and W is O. In other embodiments, the modified intersubunit linkage of Formula (I) is represented by Formula (III): [ka] The modified intersubunit bond of

[0339] In certain embodiments of Formula (I), Z is O and W is CH. In other embodiments, the modified intersubunit linkage of Formula (I) is represented by Formula (IV): [ka] The modified intersubunit bond of

[0340] In certain embodiments of Formula (I), Z is O and W is CH. In other embodiments, the modified intersubunit linkage of Formula (I) is represented by Formula V [ka] The modified intersubunit bond of

[0341] In some embodiments of Formula (I), Z is CH=CH and W is CH. In other embodiments, the modified intersubunit linkage of Formula (I) is represented by Formula VII [ka] The modified intersubunit bond of

[0342] In certain embodiments, the modified intersubunit linkage of Formula I is represented by Formula VI [ka] The modified intersubunit bond of

[0343] In certain embodiments of Formula VI,

[0344] each B is independently a base-pairing moiety;

[0345] Each X is independently selected from halo, hydroxy, and C 1-6 alkoxy; optionally, wherein each X is independently selected from the group consisting of halo (e.g., fluoro) and C 1-6 alkoxy (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 - and SH, optionally wherein Y is O - selected from the group consisting of , OH and OR;

[0346] Z is selected from the group consisting of O and CH; and

[0347] --- is an optional double bond.

[0348] In certain embodiments of Formula VI,

[0349] Each X is independently selected from fluoro, hydroxy and C 1-6 alkoxy; optionally wherein each X is independently selected from the group consisting of fluoro and C 1-6 alkoxy (e.g., methoxy, ethoxy, n-propoxy, sec-propoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, or n-heptoxy);

[0350] Y is O - selected from the group consisting of , OH and OR;

[0351] Z is selected from the group consisting of O and CH; and

[0352] --- is an optional double bond.

[0353] In certain embodiments of Formula VI,

[0354] 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;

[0355] Y is O - selected from the group consisting of , OH and OR;

[0356] Z is selected from the group consisting of O and CH; and

[0357] --- is an optional double bond.

[0358] In certain embodiments of Formula VI,

[0359] 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;

[0360] Y is O - selected from the group consisting of , OH and OR;

[0361] Z is O; and

[0362] --- is an optional double bond.

[0363] In certain embodiments of Formula VI,

[0364] 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;

[0365] Y is O - selected from the group consisting of , OH and OR;

[0366] Z is CH2; and

[0367] --- is an optional double bond.

[0368] In certain embodiments of Formula VI,

[0369] each X is independently selected from the group consisting of fluoro, hydroxy, and methoxy;

[0370] Y is O - selected from the group consisting of , OH and OR;

[0371] Z is O; and

[0372] ---is an optional double bond.

[0373] In certain embodiments of Formula VI,

[0374] each X is independently selected from the group consisting of fluoro, hydroxy, and methoxy;

[0375] Y is O - selected from the group consisting of , OH and OR;

[0376] Z is CH2; and

[0377] --- is an optional double bond.

[0378] In certain embodiments of Formula (I), Z is O and W is OCH. In other embodiments, the modified intersubunit linkage of Formula (I) is represented by Formula VIa [ka] The modified intersubunit bond of

[0379] In some embodiments of Formula (I), Z is CH and W is CH. In other embodiments, the modified intersubunit linkage of Formula (I) is represented by Formula VII [ka] The modified intersubunit bond of

[0380] In some embodiments, the base pairing moiety B is selected from the group consisting of adenine, guanine, cytosine, and uracil. In other embodiments, B is adenine. In yet other embodiments, B is guanine. In yet another embodiment, B is cytosine. In some embodiments, B is uracil.

[0381] In certain embodiments, the modified oligonucleotide is incorporated into an siRNA, the modified siRNA having a 5' end, a 3' end, and being complementary to a target, wherein the siRNA comprises a sense strand and an antisense strand and at least one oligonucleotide of formula (I): [ka] [During the ceremony, each B is independently a base-pairing moiety; W is selected from the group consisting of O, OCH2, OCH2, CH2, and CH; Each X is independently selected from halo (e.g., fluoro or chloro), hydroxy, and C 1-6 selected from the group consisting of alkoxy; Y is O - , OH, OR, NH - , NH2, S - and SH; Z is selected from the group consisting of O and CH2; R is a protecting group selected from the group consisting of dimethoxytrityl (DMTr), succinate, 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 acetate; and --- is an optional double bond. containing modified intersubunit bonds.

[0382] In certain embodiments, the modified oligonucleotide is incorporated into an siRNA, wherein the modified siRNA has a 5' end, a 3' end, is complementary to a target, and comprises a sense strand and an antisense strand, wherein the siRNA has the formula VIII [ka] [During the ceremony, D is selected from the group consisting of O, OCH2, OCH2, CH2, and CH; C is O - , OH, OR 1 , N.H. - , NH2, S - and SH; A is selected from the group consisting of O and CH2; R 1 is a protecting group; --- is an optional double bond. wherein the subunit bridges two optionally modified nucleosides.

[0383] In some embodiments, C is O - Then A or D is not O.

[0384] In some embodiments, D is CH. In other embodiments, the modified intersubunit linkage of formula VIII is formula (IX): [ka] The modified intersubunit bond of

[0385] In certain embodiments, D is O. In other embodiments, the modified intersubunit linkage of formula VIII is represented by formula (X): [ka] The modified intersubunit bond of

[0386] In some embodiments, D is CH. In other embodiments, the modified intersubunit linkage of formula (VIII) is a linker of formula (XI): [ka] The modified intersubunit bond of

[0387] In some embodiments, D is CH. In other embodiments, the modified intersubunit linkage of formula VIII is represented by formula (XII): [ka] The modified intersubunit bond of

[0388] In other embodiments, the modified intersubunit linkage of formula (VII) is represented by formula (XIV): [ka] The modified intersubunit bond of

[0389] In some embodiments, D is OCH. In other embodiments, the modified intersubunit linkage of formula (VII) is a linker of formula (XIII): [ka] The modified intersubunit bond of

[0390] In other embodiments, the modified intersubunit linkage of formula (VII) is represented by formula (XXa): [ka] The modified intersubunit bond of

[0391] In certain embodiments of modified siRNA conjugates, each optionally modified nucleoside is independently, at each occurrence, selected from the group consisting of adenosine, guanosine, cytidine, and uridine.

[0392] In certain embodiments, the modified oligonucleotide is incorporated into an siRNA, wherein the modified siRNA has a 5' end, a 3' end, is complementary to a target, and comprises a sense strand and an antisense strand, wherein the siRNA has the formula (VIII): [ka] [During the ceremony, D is selected from the group consisting of O, OCH2, OCH2, CH2, and CH; C is O - , OH, OR 1 , N.H. - , NH2, S - and SH; A is selected from the group consisting of O and CH2; R 1 is a protecting group selected from the group consisting of dimethoxytrityl (DMTr), succinate, 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 acetate; --- is an optional double bond. wherein the intersubunit linkage bridges two optionally modified nucleosides.

[0393] In certain exemplary embodiments of Formula (I), W is O. In other embodiments, W is CH. In yet other embodiments, W is CH.

[0394] In certain exemplary embodiments of Formula (I), X is OH. In other embodiments, X is OCH. In yet other embodiments, X is halo.

[0395] In certain embodiments of Formula (I), the modified siRNA does not contain a 2'-fluoro substituent.

[0396] In certain embodiments of Formula (I), Y is O - In another embodiment, Y is OH. In yet another embodiment, Y is OR. In yet another embodiment, Y is NH -In some embodiments, Y is NH. In other embodiments, Y is S. - In yet another embodiment, Y is SH.

[0397] In certain embodiments of Formula (I), Z is O. In other embodiments, Z is CH2.

[0398] In some embodiments, the modified intersubunit bond is inserted at positions 1-2 of the antisense strand. In other embodiments, the modified intersubunit bond is inserted at positions 6-7 of the antisense strand. In yet other embodiments, the modified intersubunit bond is inserted at positions 10-11 of the antisense strand. In yet another embodiment, the modified intersubunit bond is inserted at positions 19-20 of the antisense strand. In some embodiments, the modified intersubunit bond is inserted at positions 5-6 and 18-19 of the antisense strand.

[0399] In some embodiments, R is DMTr. In other embodiments, R is succinate. In yet other embodiments, R is TBDPS. In yet still other embodiments, R is acetate.

[0400] In an exemplary embodiment of the modified siRNA linkage of formula (VIII), C is O - In other embodiments, C is OH. In yet other embodiments, C is OR 1 In still yet another embodiment, C is NH - In some embodiments, C is NH. In other embodiments, C is S. - In yet other embodiments, C is SH.

[0401] In exemplary embodiments of the modified siRNA conjugate of formula (VIII), A is O. In other embodiments, A is CH. In yet other embodiments, C is OR. 1 In still yet another embodiment, C is NH -In some embodiments, C is NH. In other embodiments, C is S. - In yet other embodiments, C is SH.

[0402] In some embodiments of the modified siRNA conjugate of formula (VIII), the optionally modified nucleoside is adenosine.In other embodiments of the modified siRNA conjugate of formula (VIII), the optionally modified nucleoside is guanosine.In other embodiments of the modified siRNA conjugate of formula (VIII), the optionally modified nucleoside is cytidine.In other embodiments of the modified siRNA conjugate of formula (VIII), the optionally modified nucleoside is uridine.

[0403] In some embodiments of modified siRNA conjugate, the conjugate is inserted at the 1-2 position of antisense strand.In other embodiments, the conjugate is inserted at the 6-7 position of antisense strand.In still other embodiments, the conjugate is inserted at the 10-11 position of antisense strand.In still yet another embodiment, the conjugate is inserted at the 19-20 position of antisense strand.In some embodiments, the conjugate is inserted at the 5-6 position and 18-19 position of antisense strand.

[0404] In some embodiments of Formula (I), base pairing moiety B is adenine. In some embodiments of Formula (I), base pairing moiety B is guanine. In some embodiments of Formula (I), base pairing moiety B is cytosine. In some embodiments of Formula (I), base pairing moiety B is uracil.

[0405] In certain embodiments of Formula (I), W is O. In certain embodiments of Formula (I), W is CH. In certain embodiments of Formula (I), W is CH.

[0406] In certain embodiments of Formula (I), X is OH. In certain embodiments of Formula (I), X is OCH. In certain embodiments of Formula (I), X is halo.

[0407] In an exemplary embodiment of Formula (I), the modified oligonucleotide does not include a 2'-fluoro substituent.

[0408] In certain embodiments of Formula (I), Y is O - In certain embodiments of Formula (I), Y is OH. In certain embodiments of Formula (I), Y is OR. In certain embodiments of Formula (I), Y is NH - In certain embodiments of Formula (I), Y is NH. In certain embodiments of Formula (I), Y is S - In certain embodiments of Formula (I), Y is SH.

[0409] In certain embodiments of Formula (I), Z is O. In certain embodiments of Formula (I), Z is CH2.

[0410] In some embodiments of Formula (I), the linkage is inserted at positions 1-2 of the antisense strand. In other embodiments of Formula (I), the linkage is inserted at positions 6-7 of the antisense strand. In yet other embodiments of Formula (I), the linkage is inserted at positions 10-11 of the antisense strand. In yet other embodiments of Formula (I), the linkage is inserted at positions 19-20 of the antisense strand. In some embodiments of Formula (I), the linkage is inserted at positions 5-6 and 18-19 of the antisense strand.

[0411] Provided herein is an embodiment of a method for producing an oligonucleotide of the invention, summarized in FIG.

[0412] In embodiments of the present invention, the oligonucleotides and siRNAs provided herein can be incorporated into a CRISPR / Cas system.

[0413] The genomic sequence of each target sequence can be found, for example, in publicly available databases maintained by NCBI.

[0414] II. siRNA design In one embodiment, siRNA is designed as follows: First, a portion of the target gene (e.g., a target gene of interest, such as the ApoE gene) is designed. Cleavage of the mRNA at these sites eliminates translation of the corresponding protein. A sense strand is designed based on the target sequence. Preferably, the portion (and the corresponding sense strand) contains approximately 19 to 25 nucleotides, e.g., 19, 20, 21, 22, 23, 24, or 25 nucleotides. More preferably, the portion (and the corresponding sense strand) contains 21, 22, or 23 nucleotides. However, those skilled in the art will recognize that siRNAs having a length of fewer than 19 nucleotides or more than 25 nucleotides can also function to mediate RNAi. Therefore, siRNAs of such lengths are within the scope of the present invention as long as they maintain the ability to mediate RNAi. Long RNAi agents have been shown to induce potentially undesirable interferon or PKR responses in certain mammalian cells. Preferably, the RNAi agents of the present invention do not induce a PKR response (i.e., are sufficiently short in length). However, long RNAi agents may be useful, for example, in cell types that cannot produce a PKR response or in situations where the PKR response is downregulated or attenuated by alternative means.

[0415] The sense strand sequence is designed so that the target sequence is essentially in the center of the strand. Shifting the target sequence to an off-center position can, in some cases, reduce the efficiency of cleavage by the siRNA. Such compositions, i.e., less efficient compositions, may be desirable for use if off-silencing of wild-type mRNA is detected.

[0416] The antisense strand is naturally the same length as the sense strand and contains complementary nucleotides. In some embodiments, the strands are completely complementary, i.e., the strands are blunt-ended when aligned or annealed. In other embodiments, the strands are aligned or annealed such that a 1, 2, 3, 4, 5, 6, or 7 nucleotide overhang is produced, i.e., the 3' end of the sense strand extends 1, 2, 3, 4, 5, 6, or 7 nucleotides beyond the 5' end of the antisense strand, and / or the 3' end of the antisense strand extends 1, 2, 3, 4, 5, 6, or 7 nucleotides beyond the 5' end of the sense strand. The overhang can comprise (or consist of) nucleotides corresponding to the target gene sequence (or its complement). Alternatively, the overhang can comprise (or consist of) deoxyribonucleotides, such as dTs, or nucleotide analogs or other suitable non-nucleotide materials.

[0417] To facilitate entry of the antisense strand into RISC (and thus increase or improve the efficiency of target cleavage and silencing), the base pairing strength between the 5' end of the sense strand and the 3' end of the antisense strand can be modified, e.g., reduced or decreased, as described in U.S. Patents 7,459,547, 7,772,203, and 7,732,593, entitled "Methods and Compositions for Controlling Efficacy of RNA Silencing," filed June 2, 2003, and U.S. Patents 8,309,704, 7,750,144, 8,304,530, 8,329,892, and 8,309,705, entitled "Methods and Compositions for Enhancing the Efficacy and Specificity of RNAi," filed June 2, 2003, the contents of which are incorporated herein by reference in their entireties. In certain embodiments of these aspects of the invention, the base pairing strength is weakened due to fewer G:C base pairs between the 5' end of the first or antisense strand and the 3' end of the first or antisense strand than between the 3' end of the first or antisense strand and the 5' end of the first or antisense strand. In other embodiments, the base pairing strength is weakened due to at least one mismatched base pair between the 5' end of the first or antisense strand and the 3' end of the first or antisense strand. In certain exemplary embodiments, the mismatched base pair is selected from the group consisting of G:A, C:A, C:U, G:G, A:A, C:C, and U:U. In other embodiments, the base pairing strength is weakened due to at least one wobble base pair, e.g., G:U, between the 5' end of the first or antisense strand and the 3' end of the first or antisense strand. In other embodiments, the base pairing strength is weakened due to at least one base pair containing a rare nucleotide, e.g., inosine (I). In certain exemplary embodiments, the base pair is selected from the group consisting of I:A, I:U, and I:C. In yet other embodiments, the base pair strength is weakened by at least one base pair comprising a modified nucleotide. In certain exemplary embodiments, the modified nucleotide is selected from the group consisting of 2-amino-G, 2-amino-A, 2,6-diamino-G, and 2,6-diamino-A.

[0418] The design of siRNA suitable for targeting the target sequence of interest is described in detail below. siRNA can be designed according to the above exemplary teachings for any other target sequence found in target gene. Furthermore, this technology can be applied to target any other target sequence, for example, target sequence that does not cause disease.

[0419] To verify the effectiveness of siRNA in disrupting mRNA (e.g., mRNA expressed from a target gene of interest), the siRNA can be incubated with cDNA (e.g., cDNA corresponding to the target gene of interest) in a Drosophila-based in vitro mRNA expression system. 32 Upon radiolabeling with P, newly synthesized mRNA (e.g., target mRNA) is detected by autoradiography on an agarose gel. The presence of cleaved mRNA indicates mRNA nuclease activity. Suitable controls include omitting siRNA. Alternatively, a control siRNA is selected that has the same nucleotide composition as the selected siRNA but lacks significant sequence complementarity with the appropriate target gene. Such a negative control can be designed by randomly mixing the nucleotide sequences of the selected siRNA; a homology search can be performed to confirm that the negative control lacks homology with any other genes in the appropriate genome. Additionally, a negative control siRNA can be designed by introducing one or more base mismatches into the sequence. The position of siRNA-mRNA complementarity that results in optimal mRNA specificity and maximal mRNA cleavage is selected.

[0420] III. RNAi Agents The present invention includes, for example, siRNA molecules, as described above, designed.The siRNA molecules of the present invention can be chemically synthesized or can be transcribed from DNA templates in vitro or in vivo, for example, by using recombinant human DICER enzyme to cleave shRNA or in vitro transcribed dsRNA templates into a pool of 20bp, 21bp or 23bp double-stranded RNA-mediated RNAi.The siRNA molecules can be designed using any method known in the art.

[0421] In some embodiments, instead of being interfering ribonucleic acid, such as siRNA or shRNA, as described above, RNAi agent can encode interfering ribonucleic acid, such as shRNA.In other words, RNAi agent can be the transcriptional template of interfering ribonucleic acid.Therefore, the RNAi agent of the present invention can also include small hairpin RNA (shRNA) and the expression construct that is engineered to express shRNA.The transcription of shRNA is thought to start at polymerase III (pol III) promoter and end at the 2nd position of 4-5-thymine transcription termination site. Upon expression, shRNAs are thought to fold into stem-loop structures with 3'UU-overhangs; the ends of these shRNAs are then processed, converting the shRNAs into siRNA-like molecules of approximately 21-23 nucleotides (Brummelkamp et al., 2002; Lee et al., 2002, supra; Miyagishi et al., 2002; Paddison et al., 2002, supra; Paul et al., 2002, supra; Sui et al., 2002 supra; Yu et al., 2002, supra. Further information regarding shRNA design and use can be found on the Internet at the following addresses: katandin.cshl.org:9331 / RNAi / docs / BseRI-BamHI_Strategy.pdf and katandin.cshl.org:9331 / RNAi / docs / Web_version_of_PCR_strategy1.pdf).

[0422] Expression constructs of the present invention include any construct suitable for use in an appropriate expression system, including, but not limited to, retroviral vectors, linear expression cassettes, plasmids, and viral or virus-derived vectors known in the art. Such expression constructs may contain one or more inducible promoters, RNA Pol III promoter systems such as the U6 snRNA promoter or the H1 RNA polymerase III promoter, or other promoters known in the art. Constructs may contain one or both strands of an siRNA. Expression constructs expressing both strands may also contain a loop structure connecting the two strands, or each strand may be transcribed separately from a separate promoter within the same construct. Each strand may also be transcribed from a separate expression construct (Tuschl, T., 2002, Supra).

[0423] Synthetic siRNAs can be delivered to cells by methods known in the art, including cationic liposome transfection and electroporation. To obtain long-term target gene suppression and to facilitate delivery under certain conditions, one or more siRNAs can be expressed intracellularly from recombinant DNA constructs. Such methods for expressing siRNA duplexes intracellularly from recombinant DNA constructs to enable long-term target gene suppression in cells are known in the art and include mammalian Pol III promoter systems (e.g., H1 or U6 / snRNA promoter systems capable of expressing functional double-stranded siRNAs (Tuschl, T., 2002, supra); (Bagella et al., 1998; Lee et al., 2002, supra; Miyagishi et al., 2002, supra; Paul et al., 2002, supra; Yu et al., 2002, supra; Sui et al., 2002, supra). RNA Pol III promoters can also be used. Transcription termination by T7 occurs at a run of four consecutive T residues in the DNA template, providing a mechanism for terminating siRNA transcripts at specific sequences. The siRNAs are complementary to the target gene sequence in the 5'-3' and 3'-5' orientations, and the two strands of the siRNA can be expressed in the same or separate constructs. Hairpin siRNAs expressed in cells driven by the H1 or U6 snRNA promoter can inhibit target gene expression (Bagella et al., 1998; Lee et al., 2002, supra; Miyagishi et al., 2002, supra; Paul et al., 2002, supra; Yu et al., 2002), supra; Sui et al., 2002, supra). Constructs containing siRNA sequences under the control of the T7 promoter also produce functional siRNAs when cotransfected with a vector expressing T7 RNA polymerase into cells (Jacque et al., 2002, supra).A single construct may contain multiple sequences encoding siRNAs, such as multiple regions of a target gene targeting the same gene or multiple genes, and may be driven, for example, by separate Pol III promoter sites.

[0424] Animal cells express a wide range of approximately 22-nucleotide noncoding RNAs called microRNAs (miRNAs), which can regulate gene expression at the transcriptional or post-translational level during animal development. One common feature of miRNAs is that they are all excised from an approximately 70-nucleotide precursor RNA stem-loop, presumably by Dicer, an RNase III enzyme, or its homologs. By replacing the stem sequence of a miRNA precursor with a sequence complementary to the target mRNA, vector constructs expressing the engineered precursor can be used to produce siRNAs to initiate RNAi against specific mRNA targets in mammalian cells (Zeng et al., 2002, supra). When expressed by a DNA vector containing a polymerase III promoter, microRNA-engineered hairpins can silence gene expression (McManus et al., 2002, supra). MicroRNAs targeting polymorphisms can also be useful for blocking translation of mutant proteins in the absence of siRNA-mediated gene silencing. Such applications may be useful, for example, in situations where the designed siRNA causes off-target silencing of a wild-type protein.

[0425] Viral delivery mechanisms can also be used to induce specific silencing of target genes through the expression of siRNA, for example, by producing recombinant adenoviruses carrying siRNA under the transcriptional control of an RNA Pol II promoter (Xia et al., 2002, supra). Infection of HeLa cells with these recombinant adenoviruses allows for the reduction of endogenous target gene expression. Injection of recombinant adenoviral vectors into transgenic mice expressing the siRNA target gene results in the reduction of target gene expression in vivo. In animal models, whole embryo electroporation can efficiently deliver synthetic siRNA to postimplantation mouse embryos (Calegari et al., 2002). In adult mice, efficient delivery of siRNA can be achieved by "high pressure" delivery techniques, rapid injection (within 5 seconds) of large volumes of siRNA-containing solution into animals via the tail vein (Liu et al., 1999, supra; McCaffrey et al., 2002, supra; Lewis et al., 2002). Nanoparticles and liposomes can also be used to deliver siRNA to animals. In certain exemplary embodiments, recombinant adeno-associated viruses (rAAV) and related vectors can be used to deliver one or more siRNAs to cells, such as neuronal cells (e.g., brain cells) (U.S. Patent Applications 2014 / 0296486, 2010 / 0186103, 2008 / 0269149, 2006 / 0078542, and 2005 / 0220766).

[0426] The nucleic acid composition of the present invention can comprise both unmodified siRNA and modified siRNA, as known in the art, such as crosslinked siRNA derivatives or derivatives with non-nucleotide moieties, for example, linked to the 3' or 5' end.Modifying siRNA derivatives in this way can improve the cellular uptake or enhance the cell targeting activity of the obtained siRNA derivatives compared with corresponding siRNAs, which is useful for tracking siRNA derivatives in cells, or improve the stability of siRNA derivatives compared with corresponding siRNAs.

[0427] As described herein, engineered RNA precursors introduced into cells or whole organisms can produce desired siRNA molecules.Then, these siRNA molecules are associated with the endogenous protein components of the RNAi pathway, and bind and target specific mRNA sequences for cleavage and destruction.In this way, the mRNA targeted by the siRNA produced from engineered RNA precursors is depleted from cells or organisms, leading to the reduction of the concentration of the protein coded by this mRNA in cells or organisms.RNA precursors are generally nucleic acid molecules that individually code one strand of dsRNA or code the entire nucleotide sequence of RNA hairpin loop structure.

[0428] The nucleic acid compositions of the invention can be unconjugated or can be conjugated to other moieties, such as nanoparticles, to enhance the properties of the composition, e.g., pharmacokinetic parameters such as absorption, efficacy, bioavailability and / or half-life. Conjugation can be achieved by methods known in the art, for example, using Lambert et al., Drug Deliv. Rev.: 47(1), 99-112 (2001) (describing nucleic acids loaded onto polyalkylcyanoacrylate (PACA) nanoparticles); Fattal et al., J. Control Release 53(1-3):137-43 (1998) (describing nucleic acids bound to nanoparticles); Schwab et al., Ann. Oncol. 5 Suppl. 4:55-8 (1994) (describing nucleic acids bound to intercalating agents, hydrophobic groups, polycations, or PACA nanoparticles); and Godard et al., Eur. J. Biochem. 232(2):404-10 (1995) (describing nucleic acids bound to nanoparticles).

[0429] The nucleic acid molecules of the present invention can also be labeled using any method known in the art. For example, the nucleic acid molecules can be labeled with a fluorophore, such as Cy3, fluorescein, or rhodamine. Labeling can be accomplished using kits such as SILENCER TMThis can be achieved using an siRNA labeling kit (Ambion). Furthermore, siRNA can be, for example, 3 H, 32 It may be radiolabeled using P or other suitable isotopes.

[0430] Furthermore, because RNAi is believed to proceed via at least one single-stranded RNA intermediate, those skilled in the art will recognize that ss-siRNAs (e.g., the antisense strand of a ds-siRNA) can also be designed (e.g., for chemical synthesis), produced (e.g., enzymatically produced), or expressed (e.g., from a vector or plasmid) as described herein and utilized by the methods of the present invention. Furthermore, in invertebrates, RNAi can be efficiently induced by long dsRNAs (e.g., dsRNAs about 100 to 1,000 nucleotides in length, preferably about 200 to 500, e.g., about 250, 300, 350, 400, or 450 nucleotides in length) that act as effectors of RNAi (Brondani et al., Proc Natl Acad Sci USA. 2001 Dec. 4; 98(25):14428-33. Epub 2001 Nov. 27.).

[0431] IV. RNA Silencing Agents In some embodiments, the present invention provides novel RNA silencing agents (such as siRNA and shRNA), methods for producing the RNA silencing agents, and methods (such as research and / or therapeutic methods) for using the improved RNA silencing agents (or parts thereof), for example, for the RNA silencing of ApoE, C9ORF72 or Htt protein.The RNA silencing agent comprises antisense strand (or parts thereof), wherein the antisense strand has sufficient complementarity with heterozygous single nucleotide polymorphism for the mediation of RNA-mediated silencing mechanism (such as RNAi).

[0432] In certain embodiments, siRNA compounds are provided that have one or any combination of the following properties: (1) fully chemically stabilized (i.e., no unmodified 2'-OH residues); (2) asymmetric; (3) 11-16 base pair duplexes; (4) alternating patterns of chemically modified nucleotides (e.g., 2'-fluoro and 2'-methoxy modifications); and (5) a single-stranded, fully phosphorothioated tail of 5-8 bases. The number of phosphorothioate modifications varies from 6 to 17 total in various embodiments.

[0433] In certain embodiments, the siRNA compounds described herein can be conjugated to a wide variety of targeting agents, including, but not limited to, cholesterol, DHA, phenyltropane, cortisol, vitamin A, vitamin D, GalNac, and gangliosides. Cholesterol modifications have demonstrated 5-10 fold improved efficacy in vitro over previously used chemical stabilization patterns (e.g., all purines modified, rather than only purimidines) in a wide range of cell types (e.g., HeLa, neurons, hepatocytes, trophoblasts).

[0434] The compound of the present invention having the structural properties described herein can be called "hsiRNA-ASP" (hydrophobic modification characterized by highly stable pattern, small interfering RNA).Furthermore, this hsiRNA-ASP pattern shows dramatic improvement in distribution through the brain, spinal cord, liver, placenta, kidney, spleen and some other tissues, and can be used for therapeutic intervention.

[0435] In the liver, hsiRNA-ASP was specifically delivered to endothelial and Kupffer cells, but not to hepatocytes, making this chemical modification pattern a complementary, rather than competitive, technology to GalNac conjugates.

[0436] The compounds of the present invention can be described in the following aspects and embodiments.

[0437] In a first embodiment, provided is an oligonucleotide of at least 16 contiguous nucleotides, the oligonucleotide having a 5' end, a 3' end and complementarity to a target, wherein (1) the oligonucleotide comprises alternating 2'-methoxy-ribonucleotides and 2'-fluoro-ribonucleotides; (2) the nucleotides at positions 2 and 14 from the 5' end are not 2'-methoxy-ribonucleotides; and (3) the nucleotides are connected by modified linkages as shown in Figure 1.

[0438] a) Design of siRNA molecules The siRNA molecules of the present invention are duplexes consisting of a sense strand and a complementary antisense strand. Preferably, the siRNA molecules are about 10-50 or more nucleotides long, i.e., each strand contains 10-50 nucleotides (or nucleotide analogs). More preferably, the siRNA molecules have each strand about 15-30, e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides long, where one of the strands is fully complementary to the target region. Preferably, the strands are aligned such that there are at least 1, 2, or 3 bases at the ends of the strands that are not aligned (i.e., no complementary bases on the opposite strand), resulting in an overhang of 1, 2, or 3 residues at one or both ends of the duplex when the strands are annealed. Preferably, the siRNA molecules are about 10-50 or more nucleotides long, i.e., each strand contains 10-50 nucleotides (or nucleotide analogs). More preferably, the siRNA molecule comprises each strand about 15 to 30, e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length, wherein one of the strands is substantially complementary to the target sequence and the other strand is identical or substantially identical to the first strand.

[0439] In certain embodiments, the antisense strand is 20 nucleotides in length and the sense strand is 15 or 16 nucleotides in length.

[0440] In certain embodiments, the antisense strand is 21 nucleotides in length and the sense strand is 15 or 16 nucleotides in length.

[0441] In certain embodiments, the antisense strand is 20 or 21 nucleotides in length and the sense strand is 15 nucleotides in length.

[0442] In certain embodiments, the antisense strand is 20 or 21 nucleotides in length and the sense strand is 16 nucleotides in length.

[0443] In certain embodiments, the antisense strand is 20 nucleotides in length and the sense strand is 15 nucleotides in length.

[0444] In one embodiment, the antisense strand is 21 nucleotides in length and the sense strand is 16 nucleotides in length.

[0445] Generally, siRNAs can be designed using any method known in the art, for example, using the following protocol.

[0446] 2. The sense strand of the siRNA is designed based on the sequence of the selected target site. Preferably, the sense strand contains approximately 19 to 25 nucleotides, e.g., 19, 20, 21, 22, 23, 24, or 25 nucleotides. More preferably, the sense strand contains 21, 22, or 23 nucleotides. However, those skilled in the art will recognize that siRNAs with lengths fewer than 19 nucleotides or greater than 25 nucleotides can also function to mediate RNAi. Thus, siRNAs of such lengths are within the scope of the present invention, so long as they retain the ability to mediate RNAi. Long RNA silencing agents have been shown to induce potentially undesirable interferon or protein kinase R (PKR) responses in certain mammalian cells. Preferably, the RNA silencing agents of the present invention do not induce the PKR response (i.e., are sufficiently short in length). However, long RNA silencing agents may be useful, for example, in cell types that cannot generate a PKR response or in situations where the PKR response is downregulated or attenuated by alternative means.

[0447] The siRNA molecules of the present invention have sufficient complementarity with the target sequence so that the siRNA can mediate RNAi. Generally, siRNAs containing a nucleotide sequence sufficiently identical to a portion of the target sequence of a target gene are preferred to achieve RISC-mediated cleavage of the target gene. Thus, in a preferred embodiment, the sense strand of the siRNA is designed to have a sequence sufficiently identical to a portion of the target. For example, the sense strand may have 100% identity to the target site. However, 100% identity is not required. Greater than 80% identity between the sense strand and the target RNA sequence is preferred, for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identity. The present invention has the advantage of tolerating some sequence variation, which can improve the efficiency and specificity of RNAi. In some embodiments, the sense strand has 4, 3, 2, 1 or 0 mismatched nucleotides with the target region, such as the target region that is different by at least one base pair between wild type and mutant allele, that is, the target region that contains gain-of-function mutation, and the other strand is identical or substantially identical to the first strand.In addition, the siRNA sequence that has small insertion or deletion of 1 or 2 nucleotides can also be effective for mediating RNAi.Alternatively, the siRNA sequence that has nucleotide analog substitution or insertion can be effective for inhibition.

[0448] Sequence identity can be determined by sequence comparison and alignment algorithms known in the art. To determine the percent identity of two nucleic acid sequences (or two amino acid sequences), the sequences are aligned for optimal comparison (e.g., gaps can be introduced into the first or second sequence for optimal alignment). The nucleotides (or amino acid residues) at corresponding nucleotide (or amino acid) positions are then compared. If a position in the first sequence is occupied by the same residue as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = number of identical positions / total number of positions x 100), and optionally penalizes the score for the number of gaps introduced and / or the length of the gaps introduced.

[0449] The sequence comparison and percent identity determination of two sequences can be achieved using a mathematical algorithm.In some embodiments, the alignment is made by aligning the portion of the sequence that has sufficient identity, rather than the portion with a lower degree of identity (i.e., local alignment).A preferred, non-limiting example of the local alignment algorithm used for sequence comparison is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified by Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77.Such an algorithm is incorporated into the BLAST program (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10.

[0450] In another embodiment, the alignment is optimized by introducing appropriate gaps, and the percent identity is determined over the entire length of the aligned sequences (i.e., gapped alignment). To obtain a gapped alignment for comparison purposes, Gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. In another embodiment, the alignment is optimized by introducing appropriate gaps, and the percent identity is determined over the entire length of the aligned sequences (i.e., global alignment). A preferred, non-limiting example of a mathematical algorithm used for global comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When using the ALIGN program for comparing amino acid sequences, a PAM120 weighted residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

[0451] 3. The antisense or guide strand of an siRNA is usually the same length as the sense strand and contains complementary nucleotides. In some embodiments, the guide strand and the sense strand are fully complementary, i.e., the strands are blunt-ended when aligned or annealed. In other embodiments, the strands of an siRNA can be paired in such a way as to have a 3' overhang of 1 to 7 (e.g., 2, 3, 4, 5, 6, or 7) or 1 to 4, e.g., 2, 3, or 4, nucleotides. The overhang can comprise (or consist of) nucleotides corresponding to the target gene sequence (or its complement). Alternatively, the overhang can comprise (or consist of) deoxyribonucleotides, such as dTs, or nucleotide analogs or other suitable non-nucleotide materials. Thus, in other embodiments, the nucleic acid molecule can have a 3' overhang of two nucleotides, such as TT. The overhanging nucleotides can be RNA or DNA. As described above, it is desirable to select a target region in which the mutant:wild-type mismatch is a purine:purine mismatch.

[0452] 4. Compare potential targets to an appropriate genome database (human, mouse, rat, etc.) using any method known in the art, and filter out any target sequences that are significantly homologous to other coding sequences. One such method for such sequence homology searching is known as BLAST, available at the National Center for Biotechnology Information website.

[0453] 5. Selection of one or more sequences that meet the evaluation criteria.

[0454] Further information regarding the design and use of siRNAs can be found in "The siRNA User Guide," available at the Max-Plank-Institut für Biophysikalische Chemie website.

[0455] Alternatively, siRNA may be functionally defined as a nucleotide sequence (or oligonucleotide sequence) capable of hybridizing with a target sequence (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 12-16 hours, 50°C or 70°C hybridization; subsequent washing). Further preferred hybridization conditions include hybridization at 1xSSC at 70°C or 1xSSC, 50% formamide at 50°C, followed by washing with 0.3xSSC at 70°C, or hybridization at 4xSSC at 70°C or 4xSSC, 50% formamide at 50°C, followed by washing with 1xSSC at 67°C. The hybridization temperature for hybrids predicted to be less than 50 base pairs in length is 5-10°C lower than the melting temperature (Tm) of the hybrid, where Tm is determined according to the following formula: For hybrids shorter than 18 base pairs in length, Tm(°C) = 2(A+T bases) + 4(G+C bases). For hybrids 18-49 base pairs in length, Tm(°C) = 81.5 + 16.6(log 10[Na +]) + 0.41(%G+C) - (600 / N), where N is the number of bases in the hybrid and [Na + ] is the sodium ion concentration in the hybridization buffer ([Na + ]=0.165M). Further examples of stringency conditions for polynucleotide hybridization are provided in Sambrook, J., E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, chapters 9 and 11, and Current Protocols in Molecular Biology, 1995, F. M. Ausubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4, which are incorporated herein by reference.

[0456] Negative control siRNA has the same nucleotide composition as selected siRNA, except that it does not have significant sequence complementarity with the appropriate genome.Such negative control can be designed by randomly scrambling the nucleotide sequence of selected siRNA.Homology search can be carried out to confirm that negative control lacks homology with any other gene in the appropriate genome.In addition, negative control siRNA can be designed by introducing one or more base mismatches into the sequence.

[0457] 6. To verify the effectiveness of each siRNA in disrupting a target mRNA (e.g., wild-type or mutant mRNA), the siRNA can be incubated with the target cDNA in a Drosophila-based in vitro mRNA expression system. 32Using radiolabeling with P, newly synthesized target mRNA is detected by autoradiography on an agarose gel. The presence of cleaved target mRNA indicates mRNA nuclease activity. Suitable controls include omitting the siRNA and using non-targeting cDNA. Alternatively, a control siRNA is selected that has the same nucleotide composition as the selected siRNA but lacks significant sequence complementarity with the appropriate target gene. Such a negative control can be designed by randomly scrambling the nucleotide sequence of the selected siRNA. A homology search can be performed to confirm that the negative control lacks homology with any other genes in the appropriate genome. Additionally, a negative control siRNA can be designed by introducing one or more base mismatches into the sequence.

[0458] siRNA can be designed to target any of the above-mentioned target sequences.The siRNA is the antisense strand that is sufficiently complementary to the target sequence to mediate the silencing of the target sequence.In some embodiments, the RNA silencing agent is siRNA.

[0459] In certain embodiments, the siRNA is a sense strand containing the bond shown in FIG. 1 or an antisense strand containing the bond shown in FIG.

[0460] The position of siRNA-mRNA complementarity that results in optimal mRNA specificity and maximal mRNA cleavage is selected.

[0461] b) siRNA-like molecules The siRNA-like molecules of the present invention have a sequence that is "sufficiently complementary" to the target sequence of mRNA (i.e., have a strand with a sequence) to direct gene silencing by RNAi or translational repression. siRNA-like molecules are designed in the same manner as siRNA molecules, but the degree of sequence identity between the sense strand and the target RNA is similar to that observed between miRNA and its target. Generally, as the degree of sequence identity between the miRNA sequence and the corresponding target gene sequence decreases, the tendency to mediate post-transcriptional gene silencing by translational repression rather than RNAi increases. Therefore, in another embodiment, when post-transcriptional gene silencing by translational repression of the target gene is desired, the miRNA sequence has partial complementarity with the target gene sequence. In some embodiments, the miRNA sequence has partial complementarity (complementary site) with one or more short sequences dispersed within the target mRNA (e.g., within the 3'-UTR of the target mRNA) (Hutvagner and Zamore, Science, 2002; Zeng et al., Mol. Cell, 2002; Zeng et al., RNA, 2003; Doench et al., Genes & Dev., 2003). Because the mechanism of translational repression is cooperative, in some embodiments, multiple complementary sites (e.g., 2, 3, 4, 5, or 6) can be targeted.

[0462] The ability of an siRNA-like duplex to mediate RNAi or translational repression can be predicted by the distribution of non-identical nucleotides between the target gene sequence and the silencing agent nucleotide sequence at the complementary site. In some embodiments, when gene silencing by translational repression is desired, at least one non-identical nucleotide is present in the central portion of the complementary site so that the duplex formed by the miRNA guide strand and the target mRNA contains a central "bulge" (Doench JG et al., Genes & Dev., 2003). In other embodiments, 2, 3, 4, 5, or 6 consecutive or non-consecutive non-identical nucleotides are introduced. The non-identical nucleotides can be selected to form wobble base pairs (e.g., G:U) or mismatch base pairs (G:A, C:A, C:U, G:G, A:A, C:C, U:U). In a more preferred embodiment, the "bulge" is centered at nucleotide positions 12 and 13 from the 5' end of the miRNA molecule.

[0463] c) short hairpin RNA (shRNA) molecules In certain embodiments, the present invention provides shRNA that can mediate target sequence RNA silencing with enhanced selectivity.In contrast to SiRNA, shRNA mimics the natural precursor of microRNA (miRNA) and enters at the top of gene silencing pathway.For this reason, shRNA is thought to mediate gene silencing more efficiently by being delivered throughout the natural gene silencing pathway.

[0464] miRNAs are non-coding RNAs of approximately 22 nucleotides that can regulate gene expression at the transcriptional or post-translational level during plant and animal development. One common characteristic of miRNAs is that they are all presumably excised from an approximately 70-nucleotide precursor RNA stem-loop called a pre-miRNA by Dicer, an RNase III enzyme, or its homologs. Naturally occurring miRNA precursors (pre-miRNAs) generally contain a single strand that forms a double-stranded stem containing two complementary portions and a loop connecting the two portions of the stem. In a typical pre-miRNA, the stem contains one or more bulges, e.g., external nucleotides that create a one-nucleotide "loop" in one portion of the stem, and / or one or more unpaired nucleotides that create a gap in the hybridization of the two portions of the stem to each other. The short hairpin RNAs or engineered RNA precursors of the present invention are artificial constructs based on these naturally occurring pre-miRNAs, but are engineered to deliver a desired RNA silencing agent (e.g., an siRNA of the present invention). By replacing the stem sequence of a pre-miRNA with a sequence complementary to a target mRNA, an shRNA is formed. shRNAs are processed throughout the cell's gene silencing pathways, thereby efficiently mediating RNAi.

[0465] An essential requirement of an shRNA molecule is that it contains a first and a second portion that are sufficiently complementary to anneal or hybridize to form a duplex or double-stranded stem portion. The two portions do not need to be fully or completely complementary. The first and second "stem" portions are connected by a portion whose sequence has insufficient sequence complementarity to anneal or hybridize with the other portion of the shRNA. This latter portion is referred to as the "loop" portion of the shRNA molecule. The shRNA molecule is processed to produce siRNA. The shRNA may also contain one or more bulges, i.e., extra nucleotides that create small nucleotide "loops" in the stem portion, e.g., loops of one, two, or three nucleotides. The stem portions may be the same length, or one portion may contain an overhang of, e.g., one to five nucleotides. The overhanging nucleotides may include, for example, uracils (Us), e.g., all Us. Such Us are typically encoded by thymidines (Ts) in the shRNA-encoding DNA, which signal transcription termination.

[0466] In the shRNA (or engineered precursor RNA) of the present invention, one portion of the double-stranded stem is a nucleic acid sequence that is complementary (or antisense) to the target sequence. Preferably, one strand of the stem portion of the shRNA is sufficiently complementary (e.g., antisense) to the target RNA (e.g., mRNA) sequence to mediate degradation or cleavage of the target RNA via RNA interference (RNAi). Thus, the engineered RNA precursor comprises a two-part double-stranded stem and a loop connecting the two parts. The antisense portion can be at the 5' or 3' end of the stem. The stem portion of the shRNA is preferably about 15 to about 50 nucleotides in length. Preferably, the two stem portions are about 18 or 19 to about 21, 22, 23, 24, 25, 30, 35, 37, 38, 39, or 40 or more nucleotides in length. In a preferred embodiment, the length of the stem portion must be 21 nucleotides or more. When used in mammalian cells, the length of the stem portion should be less than about 30 nucleotides to avoid inducing non-specific responses such as the interferon pathway. In non-mammalian cells, the stem may be longer than 30 nucleotides. In fact, the stem may contain a much larger segment complementary to the target mRNA (up to and including the entire mRNA). In fact, the stem portion may contain a much larger segment complementary to the target mRNA (up to and including the entire mRNA).

[0467] The two portions of the double-stranded stem must be sufficiently complementary to hybridize to form the double-stranded stem. Thus, the two portions may, but need not, be fully or completely complementary. Furthermore, the two stem portions may be the same length, or one portion may include an overhang of 1, 2, 3, or 4 nucleotides. The overhanging nucleotides may, for example, include uracils (Us), e.g., all Us. The loop in an shRNA or engineered RNA precursor may differ from the native pre-miRNA sequence by modifying the loop sequence to increase or decrease the number of paired nucleotides, or by replacing all or part of the loop sequence with a tetraloop or other loop sequence. Thus, the loop in an shRNA or engineered RNA precursor may be 2, 3, 4, 5, 6, 7, 8, 9, or more nucleotides long, e.g., 15 or 20 or more nucleotides.

[0468] The loop in an shRNA or engineered RNA precursor can differ from the native pre-miRNA sequence by modifying the loop sequence to increase or decrease the number of paired nucleotides, or by replacing all or part of the loop sequence with a tetraloop or other loop sequence. Thus, the loop portion of an shRNA can be about 2 to about 20 nucleotides in length, i.e., about 2, 3, 4, 5, 6, 7, 8, 9, or more nucleotides, e.g., 15 or 20 or more nucleotides in length. Preferred loops consist of or include a "tetraloop" sequence. Examples of tetraloop sequences include, but are not limited to, the sequences GNRA (where N is any nucleotide and R is a purine nucleotide), GGGG, and UUUU.

[0469] In some embodiments, the shRNA of the present invention comprises the sequence of the desired siRNA molecule described above. In other embodiments, the sequence of the antisense portion of the shRNA can be designed essentially as described above or generally from within the target RNA, for example, by selecting a sequence of 18, 19, 20, 21, or more nucleotides from a region 100 to 200 or 300 nucleotides upstream or downstream of the translation initiation. Generally, the sequence can be selected from any part of the target RNA (e.g., mRNA), including the 5'UTR (untranslated region), coding sequence, or 3'UTR. This sequence may optionally immediately follow a region of the target gene containing two adjacent AA nucleotides. The last two nucleotides of the nucleotide sequence may be selected to be UU. This approximately 21-nucleotide sequence is used to create one portion of the double-stranded stem of the shRNA. This sequence can replace the stem portion of the wild-type pre-miRNA sequence, for example, enzymatically, or be included in the complete sequence synthesized. For example, DNA oligonucleotides encoding the entire stem-loop engineered RNA precursor or encoding only the portion to be inserted into the double-stranded stem of the precursor can be synthesized and restriction enzymes used to form the engineered RNA precursor construct, e.g., from the wild-type pre-miRNA.

[0470] The engineered RNA precursor contains, in its duplex stem, approximately 21–22 nucleotides of the desired siRNA or siRNA-like duplex to be produced in vivo. Thus, the stem portion of the engineered RNA precursor contains at least 18 or 19 nucleotide pairs corresponding to the sequence of an exon of the gene whose expression is to be reduced or inhibited. The two 3′ nucleotides flanking this region of the stem are selected to maximize siRNA production from the engineered RNA precursor in vivo and in vitro and maximize the effectiveness of the resulting siRNA in targeting the corresponding mRNA for translational repression or disruption by RNAi.

[0471] In some embodiments, the shRNA of the present invention comprises an miRNA sequence, optionally a terminally modified miRNA sequence, to increase insertion into RISC. The miRNA sequence can be similar to or identical to any naturally occurring miRNA (see, for example, The miRNA Registry; Griffiths-Jones S, Nuc. Acids Res., 2004). More than 1000 naturally occurring miRNAs have been identified to date, and together they are thought to account for approximately 1% of all predicted genes in the genome. Many natural miRNAs cluster together in the introns of pre-mRNAs and can be identified computationally using homology-based searches (Pasquinelli et al., 2000; Lagos-Quintana et al., 2001; Lau et al., 2001; Lee and Ambros, 2001) or computer algorithms (e.g., MiRScan, MiRSeeker) that predict the ability of candidate miRNA genes to form stem-loop structures in pre-mRNAs (Grad et al., Mol. Cell., 2003; Lim et al., Genes Dev., 2003; Lim et al., Science, 2003; Lai EC et al., Genome Bio., 2003). Online registries provide searchable databases of all published miRNA sequences (The miRNA Registry at the Sanger Institute website; Griffiths-Jones S, Nuc. Acids Res., 2004). By way of example, naturally occurring miRNAs include lin-4, let-7, miR-10, miR-15, miR-16, miR-168, miR-175, miR-196 and their homologs, as well as other naturally occurring miRNAs from humans and certain model organisms, including Drosophila melanogaster, Caenorhabditis elegans, zebrafish, Arabidopsis thaliana, House Mouse, and Rattus norvegicus, as described in International PCT Publication WO 03 / 029459.

[0472] Naturally occurring miRNAs are expressed by endogenous genes in vivo and processed by Dicer or other RNAi from hairpin or stem-loop precursors (pre-miRNA or pri-miRNA) (Lagos-Quintana et al., Science, 2001; Lau et al., Science, 2001; Lee and Ambros, Science, 2001; Lagos-Quintana et al., Curr. Biol., 2002; Mourelatos et al., Genes Dev., 2002; Reinhart et al., Science, 2002; Ambros et al., Curr. Biol., 2003; Brennecke et al., 2003; Lagos-Quintana et al., RNA, 2003; Lim et al., Genes Dev., 2003; Lim et al., Science, 2003). Although miRNAs can temporarily exist as double-stranded duplexes in vivo, only one strand is incorporated into the RISC complex and directs gene silencing. Some miRNAs, for example, plant miRNAs, have perfect or near-perfect complementarity with target mRNAs, thus directing the cleavage of target mRNAs. Other miRNAs have less than perfect complementarity with target mRNAs, thus directing translational repression of target mRNAs. The degree of complementarity between a miRNA and its target mRNA is thought to determine its mechanism of action. For example, perfect or near-perfect complementarity between a miRNA and its target mRNA is a predictor of the cleavage mechanism (Yekta et al., Science, 2004), while less than perfect complementarity is a predictor of the translational repression mechanism. In a specific embodiment, the miRNA sequence is a naturally occurring miRNA sequence, and its abnormal expression or activity is associated with a miRNA disorder.

[0473] d) Bifunctional Oligonucleotide Tethers In other embodiments, the RNA silencing agent of the present invention comprises a bifunctional oligonucleotide tether useful for intracellular mobilization of miRNA. Animal cells express a wide range of miRNAs, non-coding RNAs of approximately 22 nucleotides that can regulate gene expression at the transcriptional or post-translational level. By binding RISC-bound miRNAs and recruiting them to target mRNAs, bifunctional oligonucleotide tethers can suppress the expression of genes involved in, for example, the atherosclerotic process. The use of oligonucleotide tethers offers several advantages over existing techniques for suppressing the expression of specific genes. First, the methods described herein mediate RNA silencing through endogenous molecules, often abundant, such as miRNAs. Thus, the methods described herein eliminate the need to introduce exogenous molecules (e.g., siRNAs) to mediate RNA silencing. Second, RNA silencing agents, and in particular, binding moieties (e.g., oligonucleotides such as 2'-O-methyl oligonucleotides), can be made stable and resistant to nuclease activity. As a result, the tethers of the present invention can be designed for direct delivery, eliminating the need for indirect delivery (e.g., viruses) of precursor molecules or plasmids designed to produce the desired gene within cells. Third, the tether and its individual portions can be designed to accommodate specific mRNA sites and specific miRNAs. Designs can be cell- and gene-product-specific. Fourth, the methods disclosed herein leave mRNA intact, allowing those skilled in the art to shut off protein synthesis with short pulses using the cell's own machinery. As a result, these methods of RNA silencing are highly controllable.

[0474] The bifunctional oligonucleotide tethers ("tethers") of the present invention are designed to recruit miRNAs (e.g., endogenous cellular miRNAs) to target mRNAs to induce regulation of a gene of interest. In a preferred embodiment, the tether has the formula TL-μ, where T is the mRNA targeting moiety, L is the binding moiety, and μ is the miRNA recruitment moiety. Any one or more moieties can be double-stranded. Preferably, however, each moiety is single-stranded.

[0475] The moieties within the tether can be positioned or linked (5' to 3' direction) as described by the formula TL-μ (i.e., the 3' end of the targeting moiety is attached to the 5' end of the binding moiety, which is attached to the 5' end of the miRNA recruitment moiety). Alternatively, the moieties can be positioned or linked in the tether as follows: μ-TL (i.e., the 3' end of the miRNA recruitment moiety is attached to the 5' end of the binding moiety, which is attached to the 5' end of the targeting moiety).

[0476] As described above, the mRNA targeting moiety is capable of capturing a specific target mRNA. According to the present invention, expression of the target mRNA is undesirable, and therefore translational repression of the mRNA is desirable. The mRNA targeting moiety must be of a size sufficient to efficiently bind to the target mRNA. The length of the targeting moiety will depend, in part, on the length of the target mRNA and the degree of complementarity between the target mRNA and the targeting moiety. In various embodiments, the targeting moiety is less than about 200, 100, 50, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 nucleotides in length. In specific embodiments, the targeting moiety is about 15 to about 25 nucleotides in length.

[0477] As described above, the miRNA recruitment portion can bind to miRNA. According to the present invention, the miRNA can be any miRNA that can suppress target mRNA. It has been reported that mammals have more than 250 endogenous miRNAs (Lagos-Quintana et al. (2002) Current Biol. 12:735-739; Lagos-Quintana et al. (2001) Science 294:858-862; and Lim et al. (2003) Science 299:1540). In various embodiments, the miRNA can be any miRNA recognized in the art.

[0478] The binding moiety can be any factor that can bind the targeting moiety so that the activity of the targeting moiety is maintained.The binding moiety is preferably an oligonucleotide moiety that contains a sufficient number of nucleotides so that the targeting agent can fully interact with each of its targets.The binding moiety has little or no sequence homology with cellular mRNA or miRNA sequence.Examples of binding moieties include one or more 2'-O-methyl nucleotides, such as 2'-β-methyl adenosine, 2'-O-methyl thymidine, 2'-O-methyl guanosine or 2'-O-methyl uridine.

[0479] e) Gene Silencing Oligonucleotides In some exemplary embodiments, gene expression (for example, target gene expression) can be regulated by using oligonucleotide-based compounds, comprising two or more single-stranded antisense oligonucleotides linked via their 5'-ends, allowing two or more accessible 3'-ends to exist, so as to effectively inhibit or reduce target gene expression.Such linked oligonucleotides are also known as gene silencing oligonucleotides (GSO) (for example, US8,431,544, assigned to Idera Pharmaceuticals, Inc., which is incorporated herein by reference in its entirety for all purposes).Provided herein is a novel and improved GSO and its embodiment, comprising the intersubunit bond of formula (I).

[0480] The linkage at the 5' end of the GSO is independent of other oligonucleotide linkages and can be direct via the 5', 3', or 2' hydroxyl group, or indirect via a non-nucleotidic linker or nucleoside, utilizing the 2' or 3' hydroxyl position of the nucleoside. Linkage can also utilize a functionalized sugar or nucleobase of the 5'-terminal nucleotide.

[0481] GSOs can contain two identical or different sequences conjugated to the 5'-5' end via phosphodiester, phosphorothioate, or non-nucleoside linkers. Such compounds contain 15-27 nucleotides complementary to specific portions of mRNA targets of interest for antisense downregulation of gene products. GSOs containing identical sequences can bind to specific mRNAs through Watson-Crick hydrogen bonding interactions and inhibit protein expression. GSOs containing different sequences can bind to two or more distinct regions of one or more mRNA targets and inhibit protein expression. Such compounds contain heteronucleotide sequences complementary to the target mRNA and form stable duplex structures through Watson-Crick hydrogen bonding. Under certain conditions, GSOs containing two free 3' ends (5'-5 linked antisense) are more potent inhibitors of gene expression than those containing a single free 3' end or no free 3' end.

[0482] In certain embodiments, the non-nucleotidic linker has the formula HO—(CH) o --CH(OH)--(CH2) p glycerol or glycerol homolog of the formula HO--OH, where o and p are independently integers from 1 to about 6, from 1 to about 4, or from 1 to about 3. In certain other embodiments, the non-nucleotidic linker is a derivative of 1,3-diamino-2-hydroxypropane. Some such derivatives have the formula HO--(CH2) m --C(O)NH--CH2--CH(OH)--CH2--NHC(O)--(CH2) m --OH, where m is an integer from 0 to about 10, 0 to about 6, 2 to about 6, or 2 to about 4.

[0483] Some non-nucleotide linkers allow for the attachment of more than two GSO moieties. For example, the non-nucleotide linker glycerol has three hydroxyl groups to which GSO moieties can be covalently attached. Some oligonucleotide-based compounds of the present invention therefore contain two or more oligonucleotides attached to nucleotide or non-nucleotide linkers. Such oligonucleotides according to the present invention are referred to as "branched".

[0484] In some embodiments, the GSO is at least 14 nucleotides in length. In some exemplary embodiments, the GSO is 15-40 nucleotides in length or 20-30 nucleotides in length. Thus, the component oligonucleotides of the GSO can independently be 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length.

[0485] These oligonucleotides can be prepared by art-recognized methods, such as phosphoramidate or H-phosphonate chemistry, which can be performed manually or by automated synthesizer. These oligonucleotides can also be modified in a number of ways without impairing their ability to hybridize to mRNA. Such modifications can include at least one internucleotide linkage of the oligonucleotide that is alkyl phosphonate, phosphorothioate, phosphorodithioate, methyl phosphonate, phosphate ester, alkyl phosphonothioate, phosphoramidate, carbamate, carbonate, phosphate hydroxyl, acetamidate, or carboxymethyl ester, or a combination thereof, and other internucleotide linkages between the 5' end of one nucleotide and the 3' end of the other nucleotide, in which the 5' nucleotide phosphodiester bond is replaced with any number of chemical groups.

[0486] V. Modified RNA Silencing Agents In some embodiments of the present invention, the above-mentioned oligonucleotides, siRNAs and RNA silencing agents of the present invention (or any part thereof) can be modified to further improve the activity of the agent.For example, the RNA silencing agents described in Section II above can be modified with any of the following modifications.Modifications can, in part, further enhance target discrimination, enhance drug stability (e.g., prevent degradation), promote cellular uptake, enhance targeting efficiency, improve binding (e.g., to target), improve patient tolerance of the drug and / or reduce toxicity.

[0487] 1) Modifications to enhance target discrimination In some embodiments, the oligonucleotide, siRNA and RNA silencing agent of the present invention can be substituted with destabilizing nucleotide to enhance single-nucleotide target discrimination (see U.S. Application No. 11 / 698,689 filed on January 25, 2007 and U.S. Provisional Application No. 60 / 762,225 filed on January 25, 2006, both of which are incorporated herein by reference).This modification can be sufficient to eliminate the specificity of the RNA silencing agent for non-target mRNA (for example, wild-type mRNA) without appreciably affecting the specificity of the RNA silencing agent for target mRNA (for example, gain-of-function mutant mRNA).

[0488] In a preferred embodiment, the RNA silencing agent of the present invention is modified by introducing at least one universal nucleotide into its antisense strand. A universal nucleotide contains a base moiety that can indiscriminately base pair with any of the four common nucleotide bases (e.g., A, G, C, and U). Universal nucleotides are preferred because they have relatively little effect on the stability of the RNA duplex or the duplex formed by the guide strand of the RNA silencing agent and the target mRNA. Examples of universal nucleotides include those having an inosine base moiety or an inosine analog base moiety selected from the group consisting of deoxyinosine (e.g., 2'-deoxyinosine), 7-deaza-2'-deoxyinosine, 2'-aza-2'-deoxyinosine, PNA-inosine, morpholino-inosine, LNA-inosine, phosphoramidate inosine, 2'-O-methoxyethyl-inosine, and 2'-OMe-inosine. In a particularly preferred embodiment, the universal nucleotide is an inosine residue or a naturally occurring analog thereof.

[0489] In some embodiments, the RNA silencing agent of the present invention is modified by introducing at least one destabilizing nucleotide within 5 nucleotides from the specificity-determining nucleotide (i.e., the nucleotide that recognizes the disease-related polymorphism).For example, the destabilizing nucleotide can be introduced at a position within 5, 4, 3, 2, or 1 nucleotide from the specificity-determining nucleotide.In exemplary embodiments, the destabilizing nucleotide is introduced at a position that is 3 nucleotides from the specificity-determining nucleotide (i.e., there are two stabilizing nucleotides between the destabilizing nucleotide and the specificity-determining nucleotide).In RNA silencing agents (such as siRNA and shRNA) that have two strands or strand portions, the destabilizing nucleotide can be introduced into the strand or strand portion that does not contain the specificity-determining nucleotide.In a preferred embodiment, the destabilizing nucleotide is introduced into the same strand or strand portion that contains the specificity-determining nucleotide.

[0490] 2) Modifications to enhance efficacy and specificity In some embodiments, the oligonucleotide, siRNA and RNA silencing agent of the present invention can be modified according to asymmetric design rules to promote the effectiveness and specificity of RNAi mediation (see U.S. Patent 8,309,704, 7,750,144, 8,304,530, 8,329,892 and 8,309,705).This modification preferentially promotes the antisense strand of siRNA (for example, the siRNA produced by the siRNA or shRNA designed by the method of the present invention) to enter RISC, so that the antisense strand preferentially guides the cleavage or translational suppression of target mRNA, thereby increasing or improving the efficiency of target cleavage and silencing. Preferably, the asymmetry of the RNA silencing agent is enhanced by reducing the base pair strength between the 5' end of the antisense strand (AS 5') and the 3' end of the sense strand (S 3') of the RNA silencing agent compared to the base pair strength between the 3' end of the antisense strand (AS 3') and the 5' end of the sense strand (S '5') of the RNA silencing agent.

[0491] In some embodiments, the asymmetry of the RNA silencing agents of the present invention can be increased so that there are fewer G:C base pairs between the 5'-end of the first or antisense strand and the 3'-end of the sense strand than between the 3'-end of the first or antisense strand and the 5'-end of the sense strand. In other embodiments, the asymmetry of the RNA silencing agents of the present invention can be increased so that there is at least one mismatched base pair between the 5'-end of the first or antisense strand and the 3'-end of the sense strand. Preferably, the mismatched base pair is selected from the group consisting of G:A, C:A, C:U, G:G, A:A, C:C, and U:U. In other embodiments, the asymmetry of the RNA silencing agents of the present invention can be increased so that there is at least one wobble base pair, e.g., G:U, between the 5'-end of the first or antisense strand and the 3'-end of the sense strand. In other embodiments, the asymmetry of the RNA silencing agents of the present invention can be increased so that there is at least one base pair containing at least one rare nucleotide, e.g., inosine (I). Preferably, the base pair is selected from the group consisting of I:A, I:U and I:C. In yet another embodiment, the asymmetry of the RNA silencing agent of the present invention can be enhanced by the presence of a base pair comprising at least one modified nucleotide. In a preferred embodiment, the modified nucleotide is selected from the group consisting of 2-amino-G, 2-amino-A, 2,6-diamino-G and 2,6-diamino-A.

[0492] 3) RNA silencing agents with enhanced stability The RNA silencing agent of the present invention can be modified to improve its stability in serum or cell culture medium.To enhance stability, the 3' residue can be stabilized against degradation, and can be selected to be composed of, for example, purine nucleotides, particularly adenosine or guanosine nucleotides.Alternatively, the substitution of pyrimidine nucleotides with modified analogs, for example, the substitution of uridine with 2'-deoxythymidine, is tolerated and does not affect the efficiency of RNA interference.

[0493] In one embodiment, the present invention relates to an RNA silencing agent comprising a first strand and a second strand, wherein the second strand and / or the first strand is modified by substituting an internal nucleotide with a modified nucleotide so as to enhance in vivo stability compared to the corresponding unmodified RNA silencing agent. As defined herein, an "internal" nucleotide is one located at any position other than the 5' or 3' end of a nucleic acid molecule, polynucleotide, or oligonucleotide. The internal nucleotide can be within a single-stranded molecule or within a strand of a double-stranded or duplex molecule. In one embodiment, the sense strand and / or antisense strand is modified by substituting at least one internal nucleotide. In another embodiment, the sense strand and / or antisense strand is modified by substituting at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more internal nucleotides. In other embodiments, the sense and / or antisense strands are modified by substitution of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of the internal nucleotides. In yet other embodiments, the sense and / or antisense strands are modified by substitution of all the internal nucleotides.

[0494] In certain embodiments, the present invention relates to RNA silencing agents that are at least 80% chemically modified. In preferred embodiments of the present invention, the RNA silencing agent can be fully chemically modified, i.e., 100% of the nucleotides are chemically modified.

[0495] In a preferred embodiment of the present invention, RNA silencing agent can comprise at least one modified nucleotide analogue.Nucleotide analogue can be present, for example, in the 5'-end and / or 3'-end region of siRNA molecule, at the position where target-specific silencing activity, for example, RNAi-mediated activity or translational repression activity, is not substantially affected.In particular, the end can be stabilized by incorporating modified nucleotide analogue.

[0496] Examples of nucleotide analogs include sugar- and / or backbone-modified ribonucleotides (i.e., phosphate-sugar backbone modifications). For example, the phosphodiester bond of natural RNA can be modified to include at least one nitrogen or sulfur heteroatom. In exemplary backbone-modified ribonucleotides, the phosphoester group connected to adjacent ribonucleotides is replaced with a modified group, such as a phosphothioate group. In exemplary sugar-modified ribonucleotides, the 2'OH group is replaced with a group selected from H, OR, R, halo, SH, SR, NH, NHR, NR, or ON, where R is C-C alkyl, alkenyl, or alkynyl, and halo is F, Cl, Br, or I.

[0497] In a specific embodiment, the modification is 2'-fluoro, 2'-amino and / or 2'-thio modification. Particularly preferred modifications include 2'-fluoro-cytidine, 2'-fluoro-uridine, 2'-fluoro-adenosine, 2'-fluoro-guanosine, 2'-amino-cytidine, 2'-amino-uridine, 2'-amino-adenosine, 2'-amino-guanosine, 2,6-diaminopurine, 4-thio-uridine and / or 5-amino-allyl-uridine. In a specific embodiment, the 2'-fluoro ribonucleotide is all uridine and cytidine. Further exemplary modifications include 5-bromo-uridine, 5-iodo-uridine, 5-methyl-cytidine, ribo-thymidine, 2-aminopurine, 2'-amino-butyryl-pyrene-uridine, 5-fluoro-cytidine and 5-fluoro-uridine. 2'-deoxy-nucleotide and 2'-Ome nucleotide can also be used in the modified RNA silencing agent portion of the present invention.Additional modified residues include deoxy-abasic, inosine, N3-methyl-uridine, N6,N6-dimethyl-adenosine, pseudouridine, purine ribonucleoside and ribavirin.In a particularly preferred embodiment, the 2' portion is a methyl group, so that the binding portion is a 2'-O-methyl oligonucleotide.

[0498] In exemplary embodiments, the RNA silencing agent of the present invention comprises locked nucleic acid (LNA). LNA is a sugar-modified nucleotide that is resistant to nuclease activity (highly stable) and has single-nucleotide discrimination ability against mRNA (Elmen et al., Nucleic Acids Res., (2005), 33(1): 439-447; Braasch et al. (2003) Biochemistry 42:7967-7975, Petersen et al. (2003) Trends Biotechnol 21:74-81). These molecules have 2'-O,4'-C-ethylene-bridged nucleic acids, with possible modifications such as 2'-deoxy-2"-fluorouridine. Furthermore, LNA constrains the sugar moiety in a 3'-endo conformation, thereby pre-organizing the nucleotide for base pairing and increasing the melting temperature of the oligonucleotide by approximately 10°C per base, thereby increasing the specificity of the oligonucleotide.

[0499] In another exemplary embodiment, the RNA silencing agent of the present invention comprises a peptide nucleic acid (PNA). PNA comprises a modified nucleotide in which the sugar-phosphate moiety of the nucleotide is replaced with a neutral 2-aminoethylglycine moiety, which can form a polyamide backbone that is highly resistant to nuclease digestion and provides the molecule with improved binding specificity (Nielsen, et al., Science, (2001), 254: 1497-1500).

[0500] Also preferred are nucleobase-modified ribonucleotides, i.e., ribonucleotides that contain at least one non-naturally occurring nucleobase instead of a naturally occurring nucleobase.The base can be modified to block the activity of adenosine deaminase.Examples of modified nucleobases include, but are not limited to, uridine and / or cytidine modified at the 5th position, such as 5-(2-amino)propyluridine, 5-bromouridine; adenosine and / or guanosine modified at the 8th position, such as 8-bromoguanosine; deazanucleotides, such as 7-deaza-adenosine; O- and N-alkylated nucleotides, such as N6-methyladenosine.It should be noted that the above modifications can be combined.

[0501] In other embodiments, crosslinking can be used to modify the pharmacokinetics of RNA silencing agents, for example, to extend their half-life in the body.Therefore, the present invention includes RNA silencing agents that have two complementary strands of nucleic acid, where the two strands are crosslinked.The present invention also includes RNA silencing agents that are conjugated or not (for example, at their 3' end) with other moieties (for example, non-nucleic acid moieties such as peptides), organic compounds (for example, dyes), etc.).The modification of siRNA derivatives in this way can improve the cellular uptake or enhance the cell targeting activity of the obtained siRNA derivatives compared with corresponding siRNAs, which is useful for tracking siRNA derivatives in cells, or improve the stability of siRNA derivatives compared with corresponding siRNAs.

[0502] Other exemplary modifications include: (a) 2' modifications, such as providing a 2'OMe moiety at the sense or antisense strand, particularly U in the sense strand, or a 3' overhang, such as providing a 2'OMe moiety at the 3' end (the 3' end refers to the 3' atom or the 3'-most portion of the molecule, e.g., the 3'-most P or 2' position, as the context dictates); (b) modifications of the backbone, such as by substituting O with S in a phosphate backbone, such as providing a phosphorothioate modification of U or A or both, particularly in the antisense strand; e.g., substituting O with S; (c) substituting U with a C5 amino linker; (d) substituting A with G (preferably, the sequence change is located in the sense strand, not the antisense strand); and (d) modifications at the 2', 6', 7', or 8' positions. Exemplary embodiments are those in which one or more of these modifications are present in the sense strand but not in the antisense strand, or in which the antisense strand has fewer such modifications. Further exemplary modifications include a 3' overhang, e.g., the use of a methylated P at the 3' end; a combination of 2' modifications, e.g., providing a 2'OMe moiety and modifying the backbone, e.g., by substituting S for O, e.g., providing a phosphorothioate modification, or using a 3' overhang, e.g., a methylated P at the 3' end; modification with a 3' alkyl; modification of a 3' overhang, e.g., with an abasic pyrrolidone at the 3' end; modification with naproxen, ibuprofen, or other moieties that prevent degradation at the 3' end.

[0503] 4) Modifications to enhance cellular uptake In other embodiments, the RNA silencing agent may be modified with a chemical moiety, for example, to enhance cellular uptake by target cells (e.g., neuronal cells). Thus, the present invention includes RNA silencing agents that are conjugated or unconjugated (e.g., at their 3' ends) to other moieties (e.g., non-nucleic acid moieties such as peptides), organic compounds (e.g., dyes), etc. Conjugation can be achieved by methods known in the art, for example, using the methods of Lambert et al., Drug Deliv. Rev.: 47(1), 99-112 (2001) (describing nucleic acids loaded onto polyalkylcyanoacrylate (PACA) nanoparticles); Fattal et al., J. Control Release 53(1-3):137-43 (1998) (describing nucleic acids bound to nanoparticles); Schwab et al., Ann. Oncol. 5 Suppl. 4:55-8 (1994) (describing nucleic acids bound to intercalating agents, hydrophobic groups, polycations, or PACA nanoparticles); and Godard et al., Eur. J. Biochem. 232(2):404-10 (1995) (describing nucleic acids bound to nanoparticles).

[0504] In a specific embodiment, the RNA silencing agent of the present invention is conjugated to a lipophilic moiety. In some embodiments, the lipophilic moiety is a ligand containing a cationic group. In other embodiments, the lipophilic moiety is bound to one or both strands of siRNA. In an exemplary embodiment, the lipophilic moiety is bound to one end of the sense strand of siRNA. In other exemplary embodiments, the lipophilic moiety is bound to the 3' end of the sense strand. In some embodiments, the lipophilic moiety is selected from the group consisting of cholesterol, vitamins, vitamin K, vitamin A, folic acid, or cationic dyes (e.g., Cy3). In an exemplary embodiment, the lipophilic moiety is cholesterol. Other lipophilic moieties include cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.

[0505] 5) Tethered Ligand Other entities can be tethered to the RNA silencing agents of the present invention. For example, ligands can be tethered to RNA silencing agents to improve stability, hybridization thermodynamics with the target nucleic acid, targeting to specific tissues or cell types, or cell permeability, for example, via endocytosis-dependent or -independent mechanisms. Ligands and related modifications can also increase sequence specificity, resulting in reduced off-site targeting. Tethered ligands can contain one or more modified bases or sugars that can function as intercalators. These can preferably be located in internal regions, such as the bulge, of the RNA silencing agent / target duplex. Intercalators can be aromatic, such as polycyclic aromatic or heterocyclic aromatic compounds. Polycyclic intercalators can have stacking capabilities and can include systems with two, three, or four fused rings. Universal bases as described herein can be included in the ligand. In certain embodiments, the ligand can contain a leaving group that contributes to target gene inhibition by cleaving the target nucleic acid. The leaving group can be, for example, bleomycin (e.g., bleomycin-A5, bleomycin-A2, or bleomycin-B2), pyrene, phenanthroline (e.g., O-phenanthroline), polyamine, tripeptide (e.g., lys-tyr-lys tripeptide), or metal ion chelating group. The metal ion chelating group can include, for example, Lu(III) or EU(III) macrocyclic complex, Zn(II) 2,9-dimethylphenanthroline derivative, Cu(II) terpyridine, or acridine, which can promote the selective cleavage of target RNA at the bulge region of free metal ions such as Lu(III). In some embodiments, peptide ligands can be tethered to RNA silencing agents to promote cleavage at the bulge region of target RNA, for example. For example, 1,8-dimethyl-1,3,6,8,10,13-hexaazacyclotetradecane (cyclam) can be conjugated to a peptide (e.g., via an amino acid derivative) to promote target RNA cleavage. The tethered ligand can be an aminoglycoside ligand, which can improve the hybridization properties or sequence specificity of the RNA silencing agent.Examples of aminoglycosides include glycosylated polylysine, galactosylated polylysine, neomycin B, tobramycin, kanamycin A, and acridine conjugates of aminoglycosides, such as Neo-N-acridine, Neo-S-acridine, Neo-C-acridine, Tobra-N-acridine, and KanaA-N-acridine. The use of acridine analogs increases sequence specificity. For example, neomycin B has a higher affinity for RNA than DNA, but lower sequence specificity. The acridine analog, neo-5-acridine, has increased affinity for the HIV Rev response element (RRE). In some embodiments, a guanidine analog (guanidinoglycoside) of the aminoglycoside ligand is tethered to the RNA silencing agent. In guanidinoglycosides, the amine group of the amino acid is replaced with a guanidine group. The binding of a guanidine analog can enhance the cell permeability of the RNA silencing agent. The tethered ligand can be a poly-arginine peptide, peptoid, or peptidomimetic, which can enhance cellular uptake of an oligonucleotide agent.

[0506] Exemplary ligand is directly or indirectly linked to ligand-conjugated carrier via intervening tether, preferably by covalent bond.In exemplary embodiments, ligand is linked to carrier via intervening tether.In exemplary embodiments, ligand changes the distribution, targeting or life span of incorporated RNA silencing agent.In exemplary embodiments, ligand provides enhanced affinity to selected target, for example, molecule, cell or cell type, compartment, for example, cell or organ compartment, tissue, organ or body region, for example, compared with species without such ligand.

[0507] Exemplary ligands can improve transport, hybridization, and specificity properties, and can also improve the nuclease resistance of the resulting natural or modified RNA silencing agent or polymer molecule, which contains any combination of monomers and / or natural or modified ribonucleotides described herein. Ligands generally can include, for example, therapeutic modifiers to enhance uptake; diagnostic compounds or reporter groups, for example, to monitor distribution; cross-linking agents; nuclease-resistance-conferring moieties; and natural or unconventional nucleobases. Common examples include lipid-soluble substances, lipids, steroids (e.g., uvaol, hecogenin, diosgenin), terpenes (e.g., triterpenes, e.g., sarsasapogenin, friedelin, epifriedelinol-derivatized lithocholic acid), vitamins (e.g., folic acid, vitamin A, biotin, pyridoxal), carbohydrates, proteins, protein-binding agents, integrin-targeting molecules, polycations, peptides, polyamines, and peptidomimetics. Ligands may include naturally occurring substances (e.g., human serum albumin (HSA), low-density lipoprotein (LDL) or globulins); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); amino acids or lipids. Ligands may be recombinant or synthetic molecules such as synthetic polymers, e.g., synthetic polyamino acids. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer or polyphosphazine.Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimeric polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or alpha helical peptides.

[0508] Ligands can also include targeting groups, e.g., cell or tissue targeting agents, e.g., lectins, glycoproteins, lipids, or proteins, e.g., antibodies that bind to specific cell types such as kidney cells. The targeting group can be thyroid stimulating hormone, melanocyte hormone, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyvalent mannose, polyvalent fucose, glycosylated polyamino acids, polyvalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, lipid, cholesterol, steroid, bile acid, folic acid, vitamin B12, biotin, or an RGD peptide or RGD peptidomimetic. Other examples of ligands include dyes, intercalating agents (e.g., acridine and substituted acridine), crosslinkers (e.g., psoralens, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine, phenanthroline, pyrene), lys-tyr-lys tripeptides, aminoglycosides, guanidinium aminoglycosides, artificial endocrine disrupters (e.g., EDTA), lipophilic molecules, e.g., cholesterol (and its thio analogs), cholic acid, cholanic acid, lithocholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, glycerol (e.g., esters (e.g., mono-, bis-, or tris-fatty acid esters, e.g., C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 or C20 fatty acids) and their ethers, e.g., C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 or C 20 alkyl; e.g., 1,3-bis-O(hexadecyl)glycerol, 1,3-bis-O(octadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, stearic acid (e.g., glyceryl distearate), oleic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenoic acid, dimethoxytrityl or phenoxazine) and peptide conjugates (e.g., ante napedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, naproxen, vitamins, folic acid), synthetic ribonucleases (e.g., imidazole, bis-imidazole, histamine, imidazole clusters, acridine-imidazole conjugates, tetraazamacrocycles of Eu 3+ complex), dinitrophenyl, HRP or AP.

[0509] Ligands can be proteins, e.g., glycoproteins or peptides, e.g., molecules with specific affinity for co-ligands, or antibodies, e.g., antibodies that bind to specific cell types, such as cancer cells, endothelial cells, or bone cells. Ligands can also include hormones and hormone receptors. They can also include lipids, lectins, carbohydrates, vitamins, cofactors, non-peptidic species, such as multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, multivalent mannose, or multivalent fucose. Ligands can be, for example, lipopolysaccharides, activators of p38 MAP kinase, or activators of NF-κB.

[0510] The ligand can be a substance, such as a drug, that can increase the uptake of the RNA silencing agent into cells, for example, by disrupting the cytoskeleton of the cell, for example, by disrupting the microtubules, microfilaments, and / or intermediate filaments of the cell. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, jasplakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin. The ligand can increase the uptake of the RNA silencing agent into cells, for example, by activating an inflammatory response. Examples of ligands with such an effect include tumor necrosis factor alpha (TNFα), interleukin-1 beta, or gamma interferon. In some embodiments, the ligand is a lipid or lipid-based molecule. Such lipid or lipid-based molecule preferably binds to a serum protein, for example, human serum albumin (HSA). The HSA-binding ligand allows the conjugate to be distributed to target tissues, for example, non-renal target tissues of the body. For example, the target tissue can be the liver, including liver parenchymal cells. Other molecules capable of binding HSA can also be used as ligands. For example, neproxin or aspirin can be used. Lipids or lipid-based ligands can be used to (a) increase the resistance of the conjugate to degradation, (b) increase targeting or transport to target cells or cell membranes, and / or (c) regulate binding to serum proteins, such as HSA. Lipid-based ligands can be used to regulate, for example, control, the binding of the conjugate to target tissues. For example, lipids or lipid-based ligands that bind more strongly to HSA are less likely to be targeted to the kidney and therefore less likely to be eliminated from the body. Lipids or lipid-based ligands that bind less strongly to HSA can be used to target the conjugate to the kidney. In a preferred embodiment, the lipid-based ligand binds to HSA. The lipid-based ligand can bind to HSA with sufficient affinity so that the conjugate is preferably distributed in non-renal tissues. However, it is preferred that the affinity is not so strong that HSA-ligand binding cannot be reversed.In other preferred embodiments, the lipid-based ligand binds weakly or not at all to HSA such that the conjugate preferably distributes to the kidney. Other moieties that target kidney cells can also be used in place of or in addition to the lipid-based ligand.

[0511] In another embodiment, the ligand is a moiety, such as a vitamin, that is taken up by target cells, for example, proliferating cells. These are particularly useful for treating disorders characterized by unwanted cell proliferation, for example, malignant or non-malignant, for example, cancer cells. Examples of vitamins include vitamins A, E, and K. Examples of other vitamins include B vitamins, such as folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients taken up by cancer cells. Also included are HSA and low-density lipoprotein (LDL).

[0512] In another embodiment, the ligand is a cell-penetrating agent, preferably a helical cell-penetrating agent. Preferably, the agent is amphipathic. Examples of agents are peptides such as tat or antennapedia. If the agent is a peptide, it can be modified, including peptidyl mimetics, invertomers, non-peptide or pseudo-peptide bonds, and the use of D-amino acids. The helical agent preferably has a lipophilic and lipophobic phase, and is preferably an alpha-helical agent.

[0513] The ligand can be a peptide or peptidomimetic. Peptidomimetics (also referred to herein as oligopeptidomimetics) are molecules that can fold into defined three-dimensional structures similar to natural peptides. Attachment of peptides and peptidomimetics to oligonucleotide agents can affect the pharmacokinetic distribution of RNA silencing agents, such as by enhancing cellular recognition and uptake. The peptide or peptidomimetic moiety can be about 5 to 50 amino acids in length, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length. The peptide or peptidomimetic can be, for example, a cell-penetrating peptide, a cationic peptide, an amphipathic peptide, or a hydrophobic peptide (e.g., composed primarily of Tyr, Trp, or Phe). The peptide moiety can be a dendrimeric peptide, a constrained peptide, or a cross-linked peptide. The peptide moiety can be an L-peptide or a D-peptide. Alternatively, the peptide moiety can include a hydrophobic membrane translocating sequence (MTS). The peptide or peptidomimetic can be encoded by a random sequence of DNA, such as peptides identified from a phage display library or a one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature 354:82-84, 1991). In an exemplary embodiment, the peptide or peptidomimetic tethered to the RNA silencing agent via an incorporated monomer unit is a cell-targeting peptide, such as an arginine-glycine-aspartic acid (RGD)-peptide or RGD mimic. The peptide portion can range in length from about 5 amino acids to about 40 amino acids. The peptide portion can have structural modifications, such as for increased stability or to direct conformational properties. Any of the following structural modifications can be utilized:

[0514] 6) Branched oligonucleotides Two or more oligonucleotides, at least one of which comprises an intersubunit bond according to the embodiment of formula (I), can be connected to each other by one or more moieties independently selected from linker, spacer and branch point to form a branched compound.For example, a branched compound can comprise two or more RNA silencing agents of the above-mentioned type, resulting in a new type of RNA silencing agent with branched structure.In a typical embodiment, each oligonucleotide comprises an antisense strand (or a part thereof), wherein the antisense strand has sufficient complementarity to heterozygous single nucleotide polymorphism for the mediation of RNA-mediated silencing mechanism (such as RNAi).

[0515] In exemplary embodiments, the branched compound may have 2 to 8 RNA silencing agents attached via a linker. The linker may be hydrophobic. In typical embodiments, the branched oligonucleotide of the present application has 2 to 3 oligonucleotides. In certain embodiments, the oligonucleotides are independently substantially chemically stabilized (e.g., at least 40% of the component bases are chemically modified). In specific embodiments, the oligonucleotides are fully chemically stabilized (i.e., all component bases are chemically modified). In certain embodiments, the branched oligonucleotides comprise one or more single-stranded phosphorothioated tails, each tail independently having 2 to 20 nucleotides. In non-limiting embodiments, each single-stranded tail has 8 to 10 nucleotides.

[0516] In some embodiments, branched compounds are characterized by three properties: (1) branched structure, (2) complete metabolic stabilization, and (3) the presence of a single-stranded tail containing a phosphorothioate linker. In exemplary embodiments, branched oligonucleotides have two or three branches. Increasing the overall size of the branched structure promotes increased uptake. Also, without being bound by a particular theory of activity, it is believed that multiple adjacent branches (e.g., two or three) allow each branch to act cooperatively, thereby dramatically enhancing the rate of internalization, transport, and excretion.

[0517] Branched compounds are provided in a variety of structurally distinct embodiments. For example, as shown in Figure 24, in some embodiments, the nucleic acid attached at the branch point is single-stranded and includes an miRNA inhibitor gapmer, mixmer, SSO, PMO, or PNA. These single strands can be attached at their 3' or 5' ends. Combinations of siRNA and single-stranded oligonucleotides are also used for dual functions. In other embodiments, short nucleic acids complementary to gapmers, mixmers, miRNA inhibitors, SSOs, PMOs, and PNAs are used to deliver and enhance the distribution and cellular internalization of these active single-stranded nucleic acids. The short double-stranded region has a low melting temperature (T ) for fast dissociation upon internalization of the branched structure into cells. m approximately 37°C).

[0518] As shown in Figure 33, "di-siRNA" compounds, i.e., branched oligonucleotides having two siRNAs and a linker, can contain chemically diverse conjugates. Conjugated bioactive ligands can be used to enhance cell specificity and promote membrane binding, internalization, and serum protein binding. Examples of bioactive moieties used for conjugation include DHAg2, DHA, GalNAc, and cholesterol. These moieties can be attached to the di-siRNA via a connecting linker or spacer or added via an additional linker or spacer attached to other free siRNA ends.

[0519] Without being bound by any specific theory, it has been found that the presence of branched structure improves the tissue retention level in the brain by more than 100 times compared with the unbranched compound of the same chemical composition, suggesting a new mechanism of cell retention and distribution.Branched oligonucleotides have unexpectedly uniform distribution throughout the spinal cord and brain.In addition, branched oligonucleotides show unexpectedly efficient systemic delivery to a wide variety of tissues and extremely high levels of tissue accumulation.

[0520] Branched oligonucleotides can include a variety of therapeutic nucleic acids, including ASO, miRNA, miRNA inhibitor, splice switching, PMO, PNA. In some embodiments, branched oligonucleotides further comprise conjugated hydrophobic moieties and show exceptional silencing and efficacy in vitro and in vivo.

[0521] Non-limiting embodiments of branched oligonucleotide configurations are disclosed in Figures 18, 24-26, 32-34, and 57-62. Non-limiting examples of linkers, spacers, and branch points are disclosed in Figure 24.

[0522] Linker In some embodiments of the branched oligonucleotide compound, each linker is independently selected from an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, and combinations thereof; wherein either the carbon or oxygen atom of the linker optionally has a nitrogen atom, a hydroxyl substituent, or an oxo substituent. In some embodiments, each linker is an ethylene glycol chain. In other embodiments, each linker is an alkyl chain. In other embodiments, each linker is a peptide. In other embodiments, each linker is RNA. In other embodiments, each linker is DNA. In other embodiments, each linker is a phosphate. In other embodiments, each linker is a phosphonate. In other embodiments, each linker is a phosphoramidate. In other embodiments, each linker is an ester. In other embodiments, each linker is an amide. In other embodiments, each linker is a triazole. In other embodiments, each linker has a structure selected from the formula of Figure 23.

[0523] VI. Compound of formula (1) In other embodiments, provided herein are compounds of formula (1): [ka] wherein L is selected from an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, and a combination thereof, and wherein Formula (1) optionally further comprises one or more branch points Bp and one or more spacers; wherein Bp, at each occurrence, is independently a polyvalent organic species or a derivative thereof; S, at each occurrence, is independently selected from an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, and a combination thereof; N is an RNA duplex comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand each independently comprise one or more chemical modifications; and n is 2, 3, 4, 5, 6, 7, or 8. In certain embodiments, at least one N comprises a modified intersubunit linkage of Formula (I). In some embodiments, the compound of formula (1) has a structure selected from formulas (1-1) to (1-9) in Table 1. [Table 3]

[0524] In some embodiments, the compound of Formula (1) is Formula (1-1). In other embodiments, the compound of Formula (1) is Formula (1-2). In other embodiments, the compound of Formula (1) is Formula (1-3). In other embodiments, the compound of Formula (1) is Formula (1-4). In other embodiments, the compound of Formula (1) is Formula (1-5). In other embodiments, the compound of Formula (1) is Formula (1-6). In other embodiments, the compound of Formula (1) is Formula (1-7). In other embodiments, the compound of Formula (1) is Formula (1-8). In other embodiments, the compound of Formula (1) is Formula (1-9).

[0525] In some embodiments of the compound of Formula (1), each linker is independently selected from an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, and combinations thereof; wherein either the carbon or oxygen atom of the linker has a hydroxyl substituent, optionally replaced with a nitrogen atom, or has an oxo substituent. In some embodiments of the compound of Formula (1), each linker is an ethylene glycol chain. In other embodiments, each linker is an alkyl chain. In other embodiments of the compound of Formula (1), each linker is a peptide. In other embodiments of the compound of Formula (1), each linker is RNA. In other embodiments of the compound of Formula (1), each linker is DNA. In other embodiments of the compound of Formula (1), each linker is a phosphate. In other embodiments, each linker is a phosphonate. In other embodiments of the compound of Formula (1), each linker is a phosphoramidate. In other embodiments of the compound of Formula (1), each linker is an ester. In other embodiments of the compound of Formula (1), each linker is an amide. In other embodiments of the compound of Formula (1), each linker is a triazole. In other embodiments of the compound of Formula (1), each linker is a structure selected from the formulas of Figure 23.

[0526] In certain embodiments of the compound of Formula (1), Bp is a polyvalent organic species. In other embodiments of the compound of Formula (1), Bp is a derivative of a polyvalent organic species. In certain embodiments of the compound of Formula (1), Bp is a triol or tetraol derivative. In other embodiments, Bp is a tri- or tetra-carboxylic acid derivative. In other embodiments, Bp is an amine derivative. In other embodiments, Bp is a tri- or tetra-amine derivative. In other embodiments, Bp is an amino acid derivative. In other embodiments of the compound of Formula (1), Bp is selected from the formulae of Figure 23.

[0527] Polyvalent organic species are moieties that contain carbon and three or more valencies (i.e., points of attachment to a moiety such as S, L, or N, as defined above). Non-limiting examples of polyvalent organic species include triols (e.g., glycerol, phloroglucinol, etc.), tetraols (e.g., ribose, pentaerythritol, 1,2,3,5-tetrahydroxybenzene, etc.), tri-carboxylic acids (e.g., citric acid, 1,3,5-cyclohexanetricarboxylic acid, trimesic acid, etc.), tetra-carboxylic acids (e.g., ethylenediaminetetraacetic acid, pyromellitic acid, etc.), tertiary amines (e.g., tripropargylamine, triethanolamine, etc.), triamines (e.g., diethylenetriamine, etc.), tetramines, and species that contain combinations of hydroxyl, thiol, amino, and / or carboxyl moieties (e.g., amino acids such as lysine, serine, cysteine, etc.).

[0528] In some embodiments of the compound of Formula (1), each nucleic acid comprises one or more chemically modified nucleotides. In some embodiments of the compound of Formula (1), each nucleic acid consists of chemically modified nucleotides. In some embodiments of the compound of Formula (1), >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55%, or >50% of each nucleic acid comprises chemically modified nucleotides.

[0529] In certain embodiments, each antisense strand comprises a 5'-terminal group R independently selected from the groups in Table 2. [Table 4]

[0530] In some embodiments, R is R1. In other embodiments, R is R2. In other embodiments, R is R3. In other embodiments, R is R4. In other embodiments, R is R5. In other embodiments, R is R6. In other embodiments, R is R7. In other embodiments, R is R8.

[0531] Structure of formula (2) In certain embodiments, the compound of Formula (1) is represented by Formula (2): [ka] wherein X, at each occurrence, is independently selected from adenosine, guanosine, uridine, cytidine, and chemically modified derivatives thereof; Y, at each occurrence, is independently selected from adenosine, guanosine, uridine, cytidine, and chemically modified derivatives thereof; - is a phosphodiester internucleoside linkage; = is a phosphorothioate internucleoside linkage; and ---, at each occurrence, is independently a base pairing interaction or a mismatch. Furthermore, at least one of the internucleoside linkages may be replaced with a modified intersubunit linkage of formula (I).

[0532] In some embodiments, the structure of formula (2) does not contain any mismatches. In some embodiments, the structure of formula (2) contains one mismatch. In other embodiments, the compound of formula (2) contains two mismatches. In other embodiments, the compound of formula (2) contains three mismatches. In other embodiments, the compound of formula (2) contains four mismatches. In some embodiments, each nucleic acid is composed of chemically modified nucleotides.

[0533] In some embodiments, >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55%, or >50% of the X in the structure of Formula (2) are chemically modified nucleotides. In other embodiments, >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55%, or >50% of the X in the structure of Formula (2) are chemically modified nucleotides.

[0534] Structure of formula (3) In certain embodiments, the compound of Formula (1) is represented by Formula (3): [ka] wherein: Xis, for each occurrence, independently a nucleotide that includes a 2'-deoxy-2'-fluoro modification; X is, for each occurrence, independently a nucleotide that includes a 2'-O-methyl modification; Y is, for each occurrence, independently a nucleotide that includes a 2'-deoxy-2'-fluoro modification; and Y is, for each occurrence, independently a nucleotide that includes a 2'-O-methyl modification.

[0535] In some embodiments, X is selected from the group consisting of 2'-deoxy-2'-fluoro modified adenosine, guanosine, uridine, or cytidine. In some embodiments, X is selected from the group consisting of 2'-O-methyl modified adenosine, guanosine, uridine, or cytidine. In some embodiments, Y is selected from the group consisting of 2'-deoxy-2'-fluoro modified adenosine, guanosine, uridine, or cytidine. In some embodiments, Y is selected from the group consisting of 2'-O-methyl modified adenosine, guanosine, uridine, or cytidine.

[0536] In some embodiments, the structure of Formula (3) does not contain any mismatches. In some embodiments, the structure of Formula (3) contains one mismatch. In other embodiments, the compound of Formula (3) contains two mismatches. In other embodiments, the compound of Formula (3) contains three mismatches. In other embodiments, the compound of Formula (3) contains four mismatches.

[0537] Structure of formula (4) In certain embodiments, the compound of Formula (1) is represented by Formula (4): [ka] wherein X, at each occurrence, is independently selected from adenosine, guanosine, uridine, cytidine, and chemically modified derivatives thereof; Y, at each occurrence, is independently selected from adenosine, guanosine, uridine, cytidine, and chemically modified derivatives thereof; - is a phosphodiester internucleoside linkage; = is a phosphorothioate internucleoside linkage; and ---, at each occurrence, is independently a base pairing interaction or a mismatch. Also, at least one of the internucleoside linkages may be replaced with a modified intersubunit linkage of formula (I).

[0538] In some embodiments, the structure of formula (4) does not contain any mismatches. In some embodiments, the structure of formula (4) contains one mismatch. In other embodiments, the compound of formula (4) contains two mismatches. In other embodiments, the compound of formula (4) contains three mismatches. In other embodiments, the compound of formula (4) contains four mismatches. In some embodiments, each nucleic acid is composed of chemically modified nucleotides.

[0539] In some embodiments, >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55%, or >50% of the X in the structure of Formula (2) are chemically modified nucleotides. In other embodiments, >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55%, or >50% of the X in the structure of Formula (2) are chemically modified nucleotides.

[0540] Structure of formula (5) In certain embodiments, the compound of Formula (1) is represented by Formula (5): [ka] wherein: X is, for each occurrence, independently a nucleotide that includes a 2'-deoxy-2'-fluoro modification; X is, for each occurrence, independently a nucleotide that includes a 2'-O-methyl modification; Yis, for each occurrence, independently a nucleotide that includes a 2'-deoxy-2'-fluoro modification; and Y is, for each occurrence, independently a nucleotide that includes a 2'-O-methyl modification.

[0541] In some embodiments, X is selected from the group consisting of 2'-deoxy-2'-fluoro modified adenosine, guanosine, uridine, or cytidine. In some embodiments, X is selected from the group consisting of 2'-O-methyl modified adenosine, guanosine, uridine, or cytidine. In some embodiments, Y is selected from the group consisting of 2'-deoxy-2'-fluoro modified adenosine, guanosine, uridine, or cytidine. In some embodiments, Y is selected from the group consisting of 2'-O-methyl modified adenosine, guanosine, uridine, or cytidine.

[0542] In some embodiments, the structure of formula (5) contains no mismatches. In some embodiments, the structure of formula (6) contains one mismatch. In other embodiments, the compound of formula (5) contains two mismatches. In other embodiments, the compound of formula (5) contains three mismatches. In other embodiments, the compound of formula (V) contains four mismatches.

[0543] Flexible Linker In certain embodiments of compounds of Formula (1), L is L1 [ka] It has the following structure. In some embodiments of L1, R is R 3 and n is 2.

[0544] In some embodiments of the structure of Formula (II), L has the structure of L1. In some embodiments of the structure of Formula (III), L has the structure of L1. In some embodiments of the structure of Formula (IV), L has the structure of L1. In some embodiments of the structure of Formula (V), L has the structure of L1. In some embodiments of the structure of Formula (VI), L has the structure of L1. In some embodiments of the structure of Formula (VII), L has the structure of L1.

[0545] In certain embodiments of compounds of Formula (1), L is L2 [ka] It has the following structure. In some embodiments of L2, R is R 3 and n is 2. In some embodiments of the structure of Formula (2), L has the structure of L2. In some embodiments of the structure of Formula (3), L has the structure of L2. In some embodiments of the structure of Formula (4), L has the structure of L2. In some embodiments of the structure of Formula (5), L has the structure of L2.

[0546] delivery system In other embodiments, provided is a compound of formula (6): [ka] wherein L is selected from an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, and a combination thereof, and wherein formula (6) optionally further comprises one or more branch points Bp and one or more spacers; wherein Bp, at each occurrence, is independently a polyvalent organic species or a derivative thereof; S, at each occurrence, is independently selected from an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, and a combination thereof; each cNA is independently a carrier nucleic acid comprising one or more chemical modifications; and n is 2, 3, 4, 5, 6, 7, or 8. In some embodiments, at least one cNA comprises a modified intersubunit linkage of formula (I).

[0547] In some embodiments of the delivery system, L is an ethylene glycol chain. In other embodiments of the delivery system, L is an alkyl chain. In other embodiments of the delivery system, L is a peptide. In other embodiments of the delivery system, L is RNA. In other embodiments of the delivery system, L is DNA. In other embodiments of the delivery system, L is a phosphate. In other embodiments of the delivery system, L is a phosphonate. In other embodiments of the delivery system, L is a phosphoramidate. In other embodiments of the delivery system, L is an ester. In other embodiments of the delivery system, L is an amide. In other embodiments of the delivery system, L is a triazole.

[0548] In some embodiments of the delivery system, S is an ethylene glycol chain. In other embodiments, S is an alkyl chain. In other embodiments of the delivery system, S is a peptide. In other embodiments, S is RNA. In other embodiments of the delivery system, S is DNA. In other embodiments of the delivery system, S is a phosphate. In other embodiments of the delivery system, S is a phosphonate. In other embodiments of the delivery system, S is a phosphoramidate. In other embodiments of the delivery system, S is an ester. In other embodiments, S is an amide. In other embodiments, S is a triazole.

[0549] In some embodiments of the delivery system, n is 2. In other embodiments of the delivery system, n is 3. In other embodiments of the delivery system, n is 4. In other embodiments of the delivery system, n is 5. In other embodiments of the delivery system, n is 6. In other embodiments of the delivery system, n is 7. In other embodiments of the delivery system, n is 8.

[0550] In some embodiments, each cN comprises >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55% or >50% chemically modified nucleotides.

[0551] In certain embodiments, the compound of formula (6) has a structure selected from formulas (6-1) to (6-9) in Table 3. [Table 5]

[0552] In certain embodiments, the compound of formula (6) has the structure of formula (6-1). In certain embodiments, the compound of formula (6) has the structure of formula (6-2). In certain embodiments, the compound of formula (6) has the structure of formula (6-3). In certain embodiments, the compound of formula (6) has the structure of formula (6-4). In certain embodiments, the compound of formula (6) has the structure of formula (6-5). In certain embodiments, the compound of formula (6) has the structure of formula (6-6). In certain embodiments, the compound of formula (6) has the structure of formula (6-7). In certain embodiments, the compound of formula (6) has the structure of formula (6-8). In certain embodiments, the compound of formula (6) has the structure of formula (6-9).

[0553] In some embodiments of a compound of formula (6) (e.g., one of formulas (6-1) to (6-9)), each cNA independently comprises at least 15 consecutive nucleotides. In some embodiments, each cNA independently comprises chemically modified nucleotides.

[0554] In some embodiments, each NA hybridizes to at least one cNA. In some embodiments, at least one NA comprises a modified intersubunit linkage of formula (I). In some embodiments, the compound of the present invention is characterized by the following properties: (1) two or more branched oligonucleotides, for example, with unequal numbers of 3'-ends and 5'-ends; (2) substantially chemically stabilized (for example, free of RNA and optionally free of DNA), for example, with more than 40%, optionally 100% of the oligonucleotide being chemically modified; and (3) phosphorothioated single oligonucleotides, comprising at least three, optionally 5-20 phosphorothioated linkages.

[0555] VII. Nucleic Acids, Vector Host Cells, and Methods of Introducing Branched Oligonucleotide Compounds The RNA silencing agent of the present invention can be directly introduced into cells (e.g., neurons) (i.e., intracellularly); or can be introduced extracellularly into cavities, interstitial spaces, into the circulation of organisms, orally, or by immersing cells or organisms in a solution containing nucleic acid. Vascular or extravascular circulation, blood or lymphatic system, and cerebrospinal fluid are the sites where nucleic acid can be introduced.

[0556] The RNA silencing agent of the present invention can be introduced using nucleic acid delivery methods known in the art, including injection of nucleic acid-containing solution, irradiation of particles coated with nucleic acid, immersion of cells or organisms in nucleic acid solution, or electroporation of cell membrane in the presence of nucleic acid.Other methods known in the art for introducing nucleic acid into cells can be used, such as lipid-mediated carrier transport, chemically mediated transport, and cationic liposome transfection such as calcium phosphate.Nucleic acid can be introduced with other components that perform one or more of the following activities: enhance nucleic acid uptake by cells or increase the inhibition of target genes in other ways.

[0557] Physical methods for introducing nucleic acids include injection of RNA-containing solutions, irradiation of RNA-coated particles, immersion of cells or organisms in RNA solutions, or electroporation of cell membranes in the presence of RNA. Viral constructs packaged in viral particles achieve both efficient introduction of expression constructs into cells and transcription of the RNA encoded by the expression construct. Other methods known in the art for introducing nucleic acids into cells, such as lipid-mediated carrier transport and chemically mediated transport using calcium phosphate or other methods, can also be used. Thus, RNA can be introduced with components that perform one or more of the following activities: enhancing RNA uptake by cells, preventing single-strand annealing, stabilizing single strands, or otherwise increasing inhibition of target genes.

[0558] RNA can be introduced directly into cells (i.e., intracellularly); or extracellularly into cavities, interstitial spaces, into the circulation of an organism, orally, or by bathing a cell or organism in a solution containing RNA. Vascular or extravascular circulation, the blood or lymphatic system, and cerebrospinal fluid are sites where RNA can be introduced.

[0559] The cells bearing the target gene may be derived from germline or somatic, totipotent or pluripotent, dividing or non-dividing, parenchymal or epithelial, immortalized or transformed, etc. The cells may be stem cells or differentiated cells. Differentiated cell types include adipocytes, fibroblasts, myocytes, cardiomyocytes, endothelium, neurons, glia, blood cells, megakaryocytes, lymphocytes, macrophages, neutrophils, eosinophils, basophils, mast cells, leukocytes, granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts, hepatocytes, and cells of endocrine or exocrine glands.

[0560] Depending on the specific target gene and the dose of double-stranded RNA material delivered, this process can result in partial or complete loss of target gene function. Examples include a reduction or loss of gene expression in at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% or more of the target cells. Inhibition of gene expression refers to the absence (or observable decrease) in the levels of protein and / or mRNA product from the target gene. Specificity refers to the ability to inhibit the target gene without affecting other genes in the cell. Inhibition results can be confirmed by tests of the surface properties of the cell or organism (as shown below in the Examples) or by biochemical techniques such as RNA solution hybridization, nuclease protection, Northern hybridization, reverse transcription, gene expression monitoring using microarrays, antibody binding, enzyme-linked immunosorbent assay (ELISA), Western blotting, radioimmunoassay (RIA), other immunoassays, and fluorescence-activated cell sorting (FACS).

[0561] For RNA-mediated inhibition in cell lines or whole organisms, gene expression is conveniently assayed by the use of reporter or drug resistance genes whose protein products can be easily assayed. Such reporter genes include acetohydroxyacid synthase (AHAS), alkaline phosphatase (AP), beta-galactosidase (LacZ), beta-glucuronidase (GUS), chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase (NOS), octopine synthase (OCS), and their derivatives. Several selectable markers are available that confer resistance to ampicillin, bleomycin, chloramphenicol, gentamicin, hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin, and tetracycline. By quantification of gene expression levels, assays can determine the degree of inhibition, such as greater than 10%, 33%, 50%, 90%, 95%, or 99%, compared to cells not treated with the present invention. A small dose of RNAi agent administered over a long period of time can result in inhibition of a small proportion of cells (e.g., at least 10%, 20%, 50%, 75%, 90%, or 95% of target cells). Quantification of gene expression in cells can show similar amounts of inhibition at the level of target mRNA accumulation or target protein translation. For example, inhibition efficiency can be determined by assessing the amount of gene product in cells; mRNA can be detected using a hybridization probe with a nucleotide sequence outside the region used for the inhibitory double-stranded RNA, or translated polypeptides can be detected with antibodies raised against the polypeptide sequence of that region.

[0562] RNA can be introduced in an amount that allows delivery of at least one copy per cell. Higher doses of material (e.g., at least 5, 10, 100, 500, or 1000 copies / cell) result in more effective inhibition; lower doses may also be useful for certain applications.

[0563] In an exemplary embodiment, the effectiveness of the RNAi agent of the present invention (e.g., siRNA targeting a target sequence of interest) is tested for its ability to specifically degrade mutant mRNA (e.g., target mRNA and / or target protein production) in cells, particularly neurons (e.g., striatal or cortical neuronal clonal cell lines and / or primary neurons). Other easily transfectable cells, such as HeLa cells or COS cells, are also suitable for cell-based validation assays. Cells are transfected with human wild-type or mutant cDNA (e.g., human wild-type or mutant target cDNA). Standard siRNA, modified siRNA, or vectors that can produce siRNA from U-loop mRNA are co-transfected. The selective reduction of target mRNA and / or target protein is measured. The reduction of target mRNA or protein can be compared with the target mRNA or protein level in the absence of an RNAi agent or in the presence of an RNAi agent that does not target the target mRNA. Exogenously introduced mRNA or protein (or endogenous mRNA or protein) can be assayed for comparison purposes. When utilizing neural cells, which are known to be somewhat resistant to standard transfection techniques, it may be desirable to introduce the RNAi agent (eg, siRNA) by passive uptake.

[0564] Recombinant adeno-associated viruses and vectors In certain exemplary embodiments, recombinant adeno-associated viruses (rAAVs) and related vectors can be used to deliver one or more siRNAs to cells, such as neural cells (e.g., brain cells). AAVs can infect many different cell types, but infection efficiency varies depending on the serotype, which is determined by the sequence of the capsid protein. Several natural AAV serotypes have been identified, with serotypes 1 to 9 being the most commonly used recombinant AAVs. AAV-2 is the most well-studied and published serotype. The AAV-DJ system includes serotypes AAV-DJ and AAV-DJ / 8. These serotypes were created through DNA mixing of multiple AAV serotypes to produce AAVs with hybrid capsids, which improve transduction efficiency in vitro (AAV-DJ) and in vivo (AAV-DJ / 8) in a wide variety of cells and tissues.

[0565] In a specific embodiment, broad central nervous system (CNS) delivery can be achieved by intravascular delivery of recombinant adeno-associated virus 7 (rAAV7), RAAV9, and rAAV10, or other suitable rAAV (Zhang et al. (2011) Mol. Ther. 19(8):1440-8. doi: 10.1038 / mt.2011.98. Epub 2011 May 24). rAAV and related vectors are well known in the art and are described in U.S. Patent Applications 2014 / 0296486, 2010 / 0186103, 2008 / 0269149, 2006 / 0078542, and 2005 / 0220766, each of which is incorporated herein by reference in its entirety for all purposes.

[0566] rAAV can be delivered to a subject in a composition by any suitable method known in the art. rAAV can be suspended in a physiologically compatible carrier (i.e., composition) and administered to a subject, i.e., a host animal such as a human, mouse, rat, cat, dog, sheep, rabbit, horse, cow, goat, pig, guinea pig, hamster, chicken, turkey, or non-human primate (e.g., macaque). In some embodiments, the host animal is a non-human host animal.

[0567] Delivery of one or more rAAVs to a mammalian subject can be performed, for example, by intramuscular injection or administration into the mammalian subject's bloodstream. Administration into the bloodstream can be by injection into a vein, artery, or any other vascular conduit. In some embodiments, one or more rAAVs are administered into the bloodstream by isolated limb perfusion, a technique well known in the surgical field that essentially allows a technician to isolate one limb from the systemic circulation prior to administration of the rAAV virions. A modification of the isolated limb perfusion technique described in U.S. Patent 6,177,403 can also be used by those skilled in the art to administer virions to the vasculature of an isolated limb, potentially enhancing transduction of muscle cells or tissues. Furthermore, in some situations, it may be desirable to deliver virions to the subject's central nervous system (CNS). "CNS" refers to all cells and tissues of the vertebrate brain and spinal cord. Thus, the term includes, but is not limited to, neurons, glial cells, astrocytes, cerebrospinal fluid (CSF), interstitial spaces, bone, cartilage, and the like. Recombinant AAV can be delivered directly to the CNS or brain using neurosurgical techniques known in the art, such as by stereotactic injection, using a needle, catheter, or related device to inject, for example, into the ventricular region, as well as the striatum (e.g., the caudate nucleus or putamen of the striatum), the spinal cord, and neuromuscular junction or cerebellar lobules (see, e.g., Stein et al., J Virol 73:3424-3429, 1999; Davidson et al., PNAS 97:3428-3432, 2000; Davidson et al., Nat. Genet. 3:219-223, 1993; and Alisky and Davidson, Hum. Gene Ther. 11:2315-2329, 2000).

[0568] Compositions of the invention can include rAAV alone or in combination with one or more other viruses (e.g., a second rAAV carrying one or more different encoding transgenes). In certain embodiments, a composition includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different rAAVs, each carrying one or more different transgenes.

[0569] The effective amount of rAAV is sufficient for the target infection of animals to target desired tissue.In some embodiments, the effective amount of rAAV is sufficient for producing stable somatic transgenic animal models.Effective amount mainly depends on factors such as the species, age, weight, health and target tissue of the subject, and therefore may vary between animals and tissues.For example, the effective amount of one or more rAAVs is generally about 10 9 ~10 16 The volume of the solution containing the genome copies ranges from about 1 ml to about 100 ml. 11 ~10 12 A dose of 10 rAAV genome copies is appropriate. 12 rAAV genome copies are effectively targeted to heart, liver, and pancreatic tissues. In some cases, stable transgenic animals are produced by multiple administrations of rAAV.

[0570] In certain embodiments, the rAAV composition is particularly useful when the rAAV is present at high concentrations (e.g., about 10 13 The composition is formulated to reduce aggregation of AAV particles (genome copies / mL or higher). Methods for reducing rAAV aggregation are well known in the art and include, for example, adding detergents, adjusting pH, adjusting salt concentration, etc. (See, e.g., Wright et al. (2005) Molecular Therapy 12:171-178, the contents of which are incorporated herein by reference).

[0571] A "recombinant AAV (rAAV) vector" minimally comprises a transgene and its regulatory sequences, as well as 5' and 3' AAV inverted terminal repeats (ITRs). It is this recombinant AAV vector that is packaged into capsid proteins and delivered to selected target cells. In some embodiments, the transgene encoding a polypeptide, protein, functional RNA molecule (e.g., siRNA) or other gene product of interest is a nucleic acid sequence heterologous to the vector sequence. The nucleic acid coding sequence is operably linked to regulatory elements in a manner that allows transgene transcription, translation, and / or expression in cells of the target tissue.

[0572] The AAV sequence of the vector generally includes cis-acting 5' and 3' inverted terminal repeat (ITR) sequences (see, e.g., BJ Carter, in "Handbook of Parvoviruses", ed., P. Tijsser, CRC Press, pp. 155-168 (1990)). The ITR sequences are usually about 145 base pairs in length. In some embodiments, substantially the entire ITR-encoding sequence is used in the molecule, although some minor modifications of these sequences are tolerated. The ability to modify these ITR sequences is within the skill of one in the art (see, e.g., textbooks such as Sambrook et al., "Molecular Cloning. A Laboratory Manual", 2nd ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., J. Virol., 70:520-532 (1996)). An example of such a molecule for use in the present invention is a "cis-acting" plasmid containing a transgene in which the transgene sequence of choice and associated regulatory elements are flanked by 5' and 3' AAV ITR sequences, which may be obtained from any known AAV, including the mammalian AAV types described further herein.

[0573] VIII. Treatment Methods As used herein, "treatment" or "treating" is defined as the application or administration of a therapeutic agent (e.g., an RNA agent or a vector or transgene encoding same) to a patient, or the application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a disease or disorder, a symptom of a disease or disorder, or a predisposition to a disease or disorder, for the purpose of curing, repairing, alleviating, mitigating, altering, relieving, ameliorating, or affecting the disease or disorder, the symptom of a disease or disorder, or the predisposition to a disease.

[0574] In certain embodiments, the present invention provides methods of preventing a disease or disorder in a subject by administering to the subject a therapeutic agent (e.g., an RNAi agent or a vector or transgene encoding same). Subjects at risk for a disease can be identified, for example, by any or a combination of the diagnostic or prognostic assays described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the disease or disorder, such that the disease or disorder is prevented or, alternatively, its progression is delayed.

[0575] Another aspect of the invention relates to a method of therapeutically treating a subject, ie, altering the onset of symptoms of a disease or disorder.

[0576] With respect to both prophylactic and therapeutic treatment methods, such treatments may be specifically tailored or modified based on knowledge gained from the field of pharmacogenomics. As used herein, "pharmacogenomics" refers to the application of genomic technologies, such as gene sequencing, statistical genetics, and gene expression analysis, to drugs in clinical development and on the market. More specifically, the term refers to the study of how a patient's genes determine their response to drugs (e.g., the patient's "drug response phenotype" or "drug response genotype"). Thus, another aspect of the present invention provides methods for tailoring an individual's prophylactic or therapeutic treatment according to that individual's drug response genotype using the target gene molecules or target gene modulators of the present invention. Pharmacogenomics allows clinicians or physicians to target prophylactic or therapeutic treatments to patients who will most benefit from the treatment and avoid treating patients who will experience toxic drug-related side effects.

[0577] Therapeutic agents can be tested in suitable animal models.For example, the RNAi agent described herein (or the vector or transgene that encodes it for expression) can be used in animal models to determine the efficacy, toxicity or side effects of treatment with the agent.Alternatively, therapeutic agents can be used in animal models to determine the mechanism of action of such agents.For example, agents can be used in animal models to determine the efficacy, toxicity or side effects of treatment with the agent.Alternatively, agents can be used in animal models to determine the mechanism of action of such agents.

[0578] The pharmaceutical composition comprising the RNA silencing agent of the present invention can be administered to any patient who is diagnosed as having or at risk of developing neurodegenerative disease.In some embodiments, the patient is diagnosed as having neuropathy, and the patient's other overall health is good.For example, the patient is not terminally ill, and the patient may survive at least 2, 3, 5 or more years after diagnosis.The patient can be treated immediately after diagnosis, or treatment can be delayed until the patient experiences more debilitating symptoms, such as motor fluctuations and abnormal movements in Parkinson's disease patients.In other embodiments, the patient does not reach the advanced stage of the disease.

[0579] RNA silencing agents modified for enhanced neuronal uptake may be administered at less than about 1.4 mg / kg body weight or less than 10 mg, 5 mg, 2 mg, 1 mg, 0.5 mg, 0.1 mg, 0.05 mg, 0.01 mg, 0.005 mg, 0.001 mg, 0.0005 mg, 0.0001 mg, 0.00005 mg, or 0.00001 mg / kg body weight and less than 200 nmoles of RNA agent (e.g., about 4.4 x 10 16The unit dose of the RNA silencing agent can be administered at a dose of less than 1500nmole, 750nmole, 300nmole, 150nmole, 75nmole, 15nmole, 7.5nmole, 1.5nmole, 0.75nmole, 0.15nmole, 0.075nmole, 0.015nmole, 0.0075nmole, 0.0015nmole, 0.00075nmole, 0.00015nmole per kg of body weight.For example, the unit dose can be administered by injection (for example, intravenous or intramuscular, intrathecal or directly into the brain), inhalation or topical application.Particularly preferred dosage is less than 2mg, 1mg or 0.1mg per kg of body weight.

[0580] Delivery of an RNA silencing agent directly to an organ (e.g., directly to the brain) can be at a dose of about 0.00001 mg to about 3 mg / organ, or preferably about 0.0001 to 0.001 mg / organ, about 0.03 to 3.0 mg / organ, about 0.1 to 3.0 mg / eye, or about 0.3 to 3.0 mg / organ. The dose can be an amount effective for treating or preventing a neurodegenerative disease or disorder, such as AD or ALS. In some embodiments, the unit dose is often administered less than once a day, for example, less than every 2, 4, 8, or 30 days. In other embodiments, the unit dose is not administered at a fixed frequency (e.g., not at a fixed frequency). For example, the unit dose can be administered all at once. In some embodiments, an effective dose is administered in conjunction with other traditional therapeutic modalities.

[0581] In some embodiments, a subject is administered an initial dose of an RNA silencing agent and one or more maintenance doses. The one or more maintenance doses are generally lower than the initial dose, for example, half the initial dose. The maintenance regimen can include treating the subject with one or more doses ranging from 0.01 μg to 1.4 mg / kg body weight / day, for example, 10, 1, 0.1, 0.01, 0.001, or 0.00001 mg / kg body weight / day. The maintenance dose is preferably administered no more than once every 5, 10, or 30 days. Furthermore, the treatment regimen can be continued for a period that varies depending on the nature of the specific disease, its severity, and the patient's overall condition. In a preferred embodiment, the dosage can be delivered no more than once a day, for example, no more than once every 24, 36, 48, or more hours, for example, no more than once every 5 or 8 days. After treatment, the patient can be monitored for changes in condition and relief of symptoms of the disease state. The dosage of the compound can be increased if the patient does not respond significantly to the current dosage level, or the dosage can be decreased if a reduction in the symptoms of the disease state is observed, the disease state is eliminated, or unwanted side effects are observed.

[0582] An effective dose can be administered in a single dose or in two or more doses as needed or as deemed appropriate in a particular situation. If repeated or frequent infusion is desired, implantation of a delivery device, such as a pump, a semi-permanent stent (e.g., intravenous, intraperitoneal, intracisternal or intracapsular) or a reservoir is preferred. In some embodiments, the pharmaceutical composition comprises multiple RNA silencing agent species. In other embodiments, the RNA silencing agent species comprises sequences that are non-overlapping and non-adjacent with other species with respect to naturally occurring target sequences. In other embodiments, the multiple RNA silencing agent species are specific for different naturally occurring target genes. In other embodiments, the RNA silencing agent is allele-specific. In other embodiments, the multiple RNA silencing agent species target two or more target sequences (e.g., 2, 3, 4, 5, 6 or more target sequences).

[0583] After successful treatment, it may be desirable for the patient to undergo maintenance therapy to prevent the recurrence of the disease state, wherein the compounds of the invention are administered at a maintenance dose ranging from 0.01 μg to 100 g / kg body weight (see U.S. Patent 6,107,094).

[0584] The concentration of the RNA silencing agent composition is sufficient to be effective in treating or preventing a disorder or regulating a physiological condition in humans. The concentration or amount of RNA silencing agent administered depends on the parameters determined for the agent and the method of administration, e.g., nasal, buccal, or pulmonary. For example, nasal formulations tend to require much lower concentrations of some components to avoid irritation or injury to the nasal passages. It may be desirable to dilute oral formulations up to 10-100 times to provide a suitable nasal formulation.

[0585] Certain factors, including but not limited to the severity of the disease or disorder, previous treatment, general physical condition, and / or age of the subject, and other existing diseases, may affect the dosage required to effectively treat the subject. Furthermore, treatment of a subject with a therapeutically effective amount of an RNA silencing agent may include a single treatment or, preferably, a series of treatments. It is also recognized that the effective dosage of an RNA silencing agent for treatment may increase or decrease during the course of a particular treatment. Changes in dosage may be evident due to the results of the diagnostic assays described herein. For example, the subject may be monitored after administration of the RNA silencing agent composition. Based on the monitored information, an additional amount of the RNA silencing agent composition may be administered.

[0586] Administration depends on the severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal administration schedules can be calculated by measuring drug accumulation in the patient's body. Those skilled in the art can easily determine optimal dosages, administration methods, and repetition rates. Optimal dosages may vary depending on the relative potency of individual compounds and are generally determined based on the EC50 / EC60 / EC80 / EC9 ... 50It can be estimated based on. In some embodiments, the animal model comprises a transgenic animal that expresses a human gene, for example, a gene that produces target RNA, for example, an RNA that is expressed in neural cells. Transgenic animals can lack corresponding endogenous RNA. In other embodiments, the test composition comprises an RNA silencing agent that is complementary to the sequence that is conserved between the target RNA in the animal model and the target RNA in humans, at least in the internal region.

[0587] IX. Pharmaceutical Compositions and Methods of Administration The present invention relates to the use of the above-mentioned agents for preventive and / or therapeutic treatment, as described below. Therefore, the modulators of the present invention (e.g., branched oligonucleotides containing RNA silencing agents) can be incorporated into pharmaceutical compositions suitable for administration. Such compositions generally comprise nucleic acid molecules, proteins, antibodies, or branched oligonucleotide compounds and pharmaceutically acceptable carriers. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., that are compatible with pharmaceutical administration. Such media and agents for pharmaceutically active substances are well known in the art. As long as conventional media or agents are not incompatible with the active compounds, their use in the composition is contemplated. Supplementary active compounds can also be incorporated into the composition.

[0588] The pharmaceutical composition of the present invention is formulated to be compatible with its intended administration route.Examples of administration routes include parenteral, for example, intravenous, intradermal, subcutaneous, intraperitoneal, intramuscular, oral (for example, inhalation), transdermal (topical) and transmucosal administration.In some exemplary embodiments, the pharmaceutical composition of the present invention is delivered to cerebrospinal fluid (CSF) by administration routes including, but not limited to, intrastriatal (IS) administration, intracerebroventricular (ICV) administration and intrathecal (IT) administration (for example, via pump, infusion, etc.). Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may contain the following components: a sterile diluent, such as water for injection, saline solution, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; an antibacterial agent, such as benzyl alcohol or methylparaben; an antioxidant, such as ascorbic acid or sodium bisulfite; a chelating agent, such as ethylenediaminetetraacetic acid; a buffer, such as acetate, citrate, or phosphate, and a tonicity adjusting agent, such as sodium chloride or dextrose. The pH may be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be enclosed in glass or plastic ampoules, disposable syringes, or multiple-dose vials.

[0589] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous, IS, ICV and / or IT administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL TM(BASF, Parsippany, NJ) or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. The prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols, such as mannitol, sorbitol, sodium chloride, and the like, in the compositions. Prolonged absorption of the injectable compositions can be brought about by including the composition in an agent that delays absorption, for example, aluminum monostearate and gelatin.

[0590] Sterile injectable solution can be prepared by incorporating the active compound in the required amount into a suitable solvent with one or a combination of the above-mentioned components as needed, and then sterilizing by filtration.Generally, dispersion can be prepared by incorporating the active compound into a sterile medium that contains a basic dispersion medium and other necessary components from above.For the preparation of sterile injectable solution, the preferred method of preparing sterile powder is vacuum drying and freeze-drying, which produces a powder of the active compound and any additional desired components from the solution that has been previously sterile-filtered.

[0591] Oral compositions generally contain an inert diluent or an edible carrier. They can be placed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound is incorporated with additives and used in the form of tablets, lozenges or capsules. Oral compositions can also be prepared using a fluid carrier used as a mouthwash, where the compound in the fluid carrier is orally applied, swished, expectorated or swallowed. Pharmaceutically compatible binding agents and / or adjuvants can be incorporated as part of the composition. The tablets, pills, capsules, troches and the like may contain the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an additive such as starch or lactose, a disintegrating agent such as alginic acid, primogel or corn starch; a lubricant such as magnesium stearate or sterols; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring such as peppermint, methyl salicylate or orange flavoring.

[0592] For administration by inhalation, the compounds are delivered in the form of an aerosol spray or nebulizer from pressured container or dispenser which contains a suitable propellant, eg, a gas such as carbon dioxide.

[0593] Systemic administration can also be via transmucosal or transdermal means.For transmucosal or transdermal administration, a penetrant suitable for the barrier to be permeated is used in the formulation.Such penetrants are generally known in the art, and include, for example, for transmucosal administration, surfactants, bile salts and fusidic acid derivatives.Transmucosal administration can be achieved through the use of nasal sprays or suppositories.For transdermal administration, the active compound is formulated into ointments, salves, gels or creams as is generally known in the art.

[0594] The compounds can also be formulated in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0595] RNA silencing agents can also be administered by transfection or infection using methods including, but not limited to, those described in McCaffrey et al. (2002), Nature, 418(6893), 38-9 (hydrodynamic transfection); Xia et al. (2002), Nature Biotechnol., 20(10), 1006-10 (viral-mediated delivery); or Putnam (1996), Am. J. Health Syst. Pharm. 53(2), 151-160, erratum at Am. J. Health Syst. Pharm. 53(3), 325 (1996).

[0596] RNA silencing agents can also be administered by any method suitable for administering nucleic acid agents such as DNA vaccines. These methods include gene guns, bio-syringes, and skin patches, as well as needle-free methods such as the microparticle DNA vaccine technology disclosed in US Patent 6,194,389 and the mammalian transdermal needle-free vaccination of powder-form vaccines disclosed in US Patent 6,168,587. In addition, intranasal delivery is possible, as described, inter alia, in Hamajima et al. (1998), Clin. Immunol. Immunopathol., 88(2), 205-10. Liposomes (e.g., as described in US Patent 6,472,375) and microencapsulation can also be used. Biodegradable targetable microparticle delivery systems can also be used (e.g., as described in US Patent 6,471,996).

[0597] In some embodiments, the active compounds can be prepared with carriers that protect the compounds from rapid elimination from the body, such as controlled-release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art. Materials can also be purchased from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies against viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared by methods known to those skilled in the art, for example, as described in U.S. Patent 4,522,811.

[0598] For ease of administration and uniformity of dosage, it is particularly advantageous to prepare oral or parenteral compositions in dosage units.Dosage unit form as used herein refers to a physically separate unit suitable as a unitary dose for treatment subject; each unit contains a predetermined amount of active compound calculated to produce desired therapeutic effect together with necessary pharmaceutical carrier.The specification of dosage unit form of the present invention is supported and directly depends on the specific characteristics of active compound and the specific therapeutic effect to be achieved and the inherent limitations in the field of preparation of this active compound for individual treatment.

[0599] The toxicity and therapeutic efficacy of such compounds can be determined, for example, by LD 50 (50% of the population) and ED 50 The dose ratio between toxic and therapeutic effects is the therapeutic index, and the LD 50 / ED 50 The therapeutic index may be expressed as: Compounds that exhibit large therapeutic indices are preferred. Compounds that exhibit toxic side effects may also be used, although care should be taken to design delivery systems that target such compounds to the site of affected tissues in order to minimize the potential for damage to uninfected cells, thereby reducing side effects.

[0600] The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds is preferably administered with little or no toxicity and with an ED 50 The therapeutically effective dose of any compound used in the method of the invention can be estimated initially from cell culture assays. A fixed dose can be formulated in animal models to achieve a circulating concentration of 100 mg / kg / day, based on the EC 200 / 2000 / 2000 determined in cell culture. The dosage can vary within this range depending on the dosage used and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. 50 (i.e., the concentration of the test compound which achieves a half-maximal response) can be used to more accurately determine useful doses in humans. Plasma levels can be measured, for example, by high performance liquid chromatography.

[0601] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration, if desired.

[0602] The therapeutically effective amount (i.e., effective dosage) of an RNA silencing agent as defined herein will depend on the RNA silencing agent selected. For example, once a plasmid encoding an shRNA is selected, a single dose ranging from about 1 μg to 1000 mg is administered; in some embodiments, 10 μg, 30 μg, 100 μg, or 1000 μg may be administered. In some embodiments, 1 to 5 g of the composition may be administered. The composition may be administered from once or more times daily to once or more times weekly, including once every other day. Those skilled in the art will recognize that certain factors, including the severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and other diseases present, may affect the dosage and timing required to effectively treat a subject. Furthermore, treatment of a subject with a therapeutically effective amount of a protein, polypeptide, or antibody may include a single treatment or, preferably, a series of treatments.

[0603] The nucleic acid molecules of the present invention can be introduced into expression constructs, such as viral vectors, retroviral vectors, expression cassettes, or plasmid viral vectors, using methods known in the art, including, but not limited to, those described in Xia et al. (2002), Supra. The expression constructs can be delivered to a subject, for example, by inhalation, oral administration, intravenous infusion, topical administration (see U.S. Patent No. 5,328,470), or stereotactic injection (see, for example, Chen et al. (1994), Proc. Natl. Acad. Sci. USA, 91, 3054-3057). A pharmaceutical preparation of the delivery vector can contain the vector in an acceptable diluent or a slow-release matrix in which the delivery vehicle is embedded. Alternatively, if the complete delivery vector can be produced intact from recombinant cells, such as retroviral vectors, the pharmaceutical preparation can include one or more cells that produce the gene delivery system.

[0604] The nucleic acid molecules of the present invention also include small hairpin RNAs (shRNAs) and expression constructs engineered to express shRNAs. Transcription of shRNAs initiates at the polymerase III (pol III) promoter and terminates at position 2 of the 4-5-thymine transcription termination site. Upon expression, shRNAs are thought to fold into stem-loop structures with 3'UU-overhangs; the ends of these shRNAs are then processed, converting them into siRNA-like molecules of approximately 21 nucleotides. Brummelkamp et al. (2002), Science, 296, 550-553; Lee et al. (2002). supra; Miyagishi and Taira (2002), Nature Biotechnol., 20, 497-500; Paddison et al. (2002), supra; Paul (2002), supra; Sui (2002), supra; Yu et al. (2002), supra.

[0605] The expression construct can be any construct suitable for use in an appropriate expression system, including, but not limited to, retroviral vectors, linear expression cassettes, plasmids, and viral or virus-derived vectors, as known in the art. Such expression constructs can include one or more inducible promoters, RNA Pol III promoter systems such as the U6 snRNA promoter or the H1 RNA polymerase III promoter, or other promoters known in the art. The construct can contain one or both strands of the siRNA. Expression constructs that express both strands can also include a loop structure connecting the two strands, or each strand can be transcribed separately from a separate promoter within the same construct. Each strand can also be transcribed from a separate expression construct; see Tuschl (2002), Supra.

[0606] In some exemplary embodiments, the composition comprising the RNA silencing agent of the present invention can be delivered to the nervous system of a subject by various routes.Examples of routes include intrathecal, parenchymal (for example, brain), nasal and ocular delivery.The composition can also be delivered systemically, for example, by intravenous, subcutaneous or intramuscular injection, which is particularly useful for delivering RNA silencing agents to peripheral neurons.Preferred delivery route is directly to the brain, for example, the ventricle or hypothalamus, or the lateral or dorsal part of the brain.The RNA silencing agent for neuronal delivery can be incorporated into pharmaceutical compositions suitable for administration.

[0607] For example, the composition may comprise one or more RNA silencing agent species and a pharmaceutically acceptable carrier. The pharmaceutical composition of the present invention can be administered in a variety of ways, depending on whether local or systemic treatment is desired and the treatment area. Administration can be topical (including ophthalmic, intranasal, transdermal), oral, or parenteral. Parenteral administration includes intravenous infusion, subcutaneous, intraperitoneal, or intramuscular injection, intrathecal, or intravenous (e.g., intracerebroventricular) administration. In some exemplary embodiments, the RNA silencing agent of the present invention is delivered across the blood-brain-barrier (BBB) ​​using a variety of suitable compositions and methods described herein.

[0608] The delivery route depends on the patient's disorder.For example, for a subject diagnosed with neurodegenerative disease, the RNA silencing agent of the present invention can be directly administered to the brain (for example, near the globus pallidus or the striatum and the medium spiny neurons of the striatum of the basal ganglia).In addition to the RNA silencing agent of the present invention, the patient can be administered a second treatment, for example, a symptomatic treatment and / or a disease-specific treatment.The second treatment can be, for example, symptomatic (for example, to reduce symptoms), neuroprotective (for example, to slow or stop disease progression) or restorative (for example, to reverse disease process).Other treatments can be psychotherapy, physical therapy, speech therapy, communication and memory aids, social support services and dietary advice.

[0609] The RNA silencing agent can be delivered to neurons in the brain. A delivery method that does not require the composition to cross the blood-brain barrier can be used. For example, a pharmaceutical composition containing an RNA silencing agent can be delivered to a patient by directly injecting it into an area containing disease-affected cells. For example, the pharmaceutical composition can be delivered by direct injection into the brain. The injection can be by stereotaxic injection into a specific area of ​​the brain (e.g., the substantia nigra, cortex, hippocampus, striatum, or globus pallidus). The RNA silencing agent can be delivered to multiple areas of the central nervous system (e.g., multiple areas of the brain and / or the spinal cord). The RNA silencing agent can be delivered to a generalized area of ​​the brain (e.g., generalized delivery to the cortex of the brain).

[0610] In some embodiments, RNA silencing agents can be delivered by using a cannula or other delivery device, one end of which is implanted into tissue, for example, the brain, for example, the substantia nigra, cortex, hippocampus, striatum or globus pallidus of the brain.The cannula can be connected to a reservoir of RNA silencing agents.The flow or delivery can be mediated by a pump, for example, an osmotic pump or a minipump, such as an Alzet pump (Durect, Cupertino, CA).In some embodiments, the pump and reservoir are implanted in a region distal to the tissue, for example, the abdomen, and delivery is carried out by a conduit from the pump or reservoir to the release site.Devices for delivery to the brain are described, for example, in US Patents 6,093,180 and 5,814,014.

[0611] The RNA silencing agent of the present invention can be further modified so that it can pass through the blood-brain barrier.For example, the RNA silencing agent can be conjugated with the molecule that allows the agent to pass through the barrier.This modified RNA silencing agent can be administered by any desired method, for example, by intracerebroventricular or intramuscular injection or pulmonary delivery.

[0612] In certain embodiments, exosomes are used to deliver the RNA silencing agents of the present invention. Exosomes can cross the BBB and deliver siRNA, antisense oligonucleotides, chemotherapeutic agents, and proteins, specifically to neurons, after systemic injection (Alvarez-Erviti L, Seow Y, Yin H, Betts C, Lakhal S, Wood MJ. (2011). Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nat Biotechnol. 2011 Apr;29(4):341-5. doi: 10.1038 / nbt.1807; El-Andaloussi S, Lee Y, Lakhal-Littleton S, Li J, Seow Y, Gardiner C, Alvarez-Erviti L, Sargent IL, Wood MJ. (2011). Exosome-mediated delivery of siRNA in vitro and in vivo. Nat Protoc. 2012 Dec;7(12):2112-26. doi: 10.1038 / nprot.2012.131; EL Andaloussi S, Maeger I, Breakefield XO, Wood MJ. (2013). Extracellular vesicles: biology and emerging therapeutic opportunities. Nat Rev Drug Discov. 2013 May;12(5):347-57. doi: 10.1038 / nrd3978; El Andaloussi S, Lakhal S, Maeger I, Wood MJ. (2013). Exosomes for targeted siRNA delivery across biological barriers. Adv Drug Deliv Rev. 2013 Mar;65(3):391-7. doi: 10.1016 / j.addr.2012.08.008).

[0613] In some embodiments, one or more lipophilic molecules are used to enable the delivery of the RNA silencing agent of the present invention across the BBB (Alvarez-Ervit (2011)). The RNA silencing agent is then activated, for example, by enzymatic degradation of the lipophilic mimicking compound to release the drug in its active form.

[0614] In some embodiments, one or more receptors that mediate compound permeabilization can be used to increase the permeability of the BBB, enabling delivery of the RNA silencing agents of the present invention. These drugs increase the permeability of the BBB by temporarily increasing the osmotic pressure in the blood, which loosens the tight junctions between endothelial cells (El-Andaloussi (2012)). By loosening the tight junctions, conventional intravenous injection of the RNA silencing agent can be performed.

[0615] In certain embodiments, a nanoparticle-based delivery system is used to deliver the RNA silencing agents of the present invention across the BBB. As used herein, "nanoparticle" refers to polymeric nanoparticles, which are generally solid, biodegradable, colloidal systems that have been widely studied as drug or gene carriers (S.P. Egusquiaguirre, M. Igartua, R.M. Hernandez, and J.L. Pedraz, "Nanoparticle delivery systems for cancer therapy: advances in clinical and preclinical research," Clinical and Translational Oncology, vol. 14, no. 2, pp. 83-93, 2012). Polymeric nanoparticles are classified into two broad categories: natural polymers and synthetic polymers. Natural polymers for siRNA delivery include, but are not limited to, cyclodextrin, chitosan, and atelocollagen (Y. Wang, Z. Li, Y. Han, L.H. Liang, and A. Ji, "Nanoparticle-based delivery system for application of siRNA in vivo," Current Drug Metabolism, vol. 11, no. 2, pp. 182-196, 2010).Synthetic polymers have been intensively studied, including, but not limited to, polyethyleneimine (PEI), poly(dl-lactide-co-glycolide) (PLGA), and dendrimers (X. Yuan, S. Naguib, and Z. Wu, “Recent advances of siRNA delivery by nanoparticles,” Expert Opinion on Drug Delivery, vol. 8, no. 4, pp. 521-536, 2011). For a review of nanoparticles and other suitable delivery systems, see Jong-Min Lee, Tae-Jong Yoon, and Young-Seok Cho, “Recent Developments in Nanoparticle-Based siRNA Delivery for Cancer Therapy,” BioMed Research International, vol. 2013, Article ID 782041, 10 pages, 2013. doi:10.1155 / 2013 / 782041 (incorporated by reference in its entirety)).

[0616] The RNA silencing agent of the present invention can be administered to the eye, for example, for the treatment of retinal disorders, such as retinopathy. For example, the pharmaceutical composition can be applied to the ocular surface or adjacent tissues, for example, into the eyelid. It can be applied topically, for example, as a spray, eye drops, eye wash, or ointment. Ointments or droppable liquids can be delivered by ocular delivery systems known in the art, such as applicators or eyedroppers. Such compositions can include mucosal mimetics, such as hyaluronic acid, chondroitin sulfate, hydroxypropylmethylcellulose, or poly(vinyl alcohol), preservatives, such as sorbic acid, EDTA, or benzylcuronium chloride, and a conventional amount of diluent and / or carrier. Pharmaceutical compositions can also be administered intraocularly, and can be introduced by a needle or other delivery device that can be introduced into a selected area or structure. Compositions containing RNA silencing agents can also be administered via an eye patch.

[0617] Generally, the RNA silencing agent of the present invention can be administered by any suitable method. As used herein, local delivery refers to the direct application of the RNA silencing agent to any surface of the body, including the eye, mucous membrane, the surface of a body cavity, or any internal surface. Topical administration preparations can include transdermal patches, ointments, lotions, creams, gels, drops, sprays, and liquids. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners, etc. may be necessary or desirable. Topical administration can also be used as a means of selectively delivering the RNA silencing agent to the epidermis or dermis of a subject, or specific layers or underlying tissues thereof.

[0618] Compositions for intrathecal or intraventricular (e.g., intracerebroventricular) administration can include sterile aqueous solutions that can also include buffers, diluents, and other suitable additives. Compositions for intrathecal or intraventricular administration preferably do not include any additional lipophilic moieties other than the transfection reagent or, for example, the lipophilic moiety attached to the RNA silencing agent.

[0619] Formulations for parenteral administration include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives. Intraventricular infusion may be facilitated, for example, by an intraventricular catheter attached to a reservoir. For intravenous use, the total concentration of solutes should be controlled to render the preparation isotonic.

[0620] The RNA silencing agent of the present invention can be administered to a subject by pulmonary delivery.Pulmonary delivery composition can be delivered by inhalation of dispersion, so that the composition in dispersion reaches the lung, and can be easily absorbed directly into blood circulation through alveolar region.Pulmonary delivery can be effective for both systemic delivery and localized delivery for treating pulmonary disease.In some embodiments, the RNA silencing agent administered by pulmonary delivery is modified so that it can pass through the blood-brain barrier.

[0621] Pulmonary delivery can be achieved by various approaches, including nebulization, aerosolization, micellar, and dry powder-based formulations. Delivery can be achieved with liquid nebulizers, aerosol-based inhalers, and dry powder dispersion devices. Metered-dose devices are preferred. One advantage of using an atomizer or inhaler is that the device is self-contained, minimizing the possibility of contamination. Dry powder dispersion devices deliver drugs that can be easily formulated as dry powders, for example. RNA silencing agent compositions can be stably stored as lyophilized or spray-dried powders, either by themselves or in combination with a suitable powder carrier. Delivery of the inhalable composition can be mediated by a dosing timing element, which, when incorporated into the device, can include a timer, dose counter, timing device, or time indicator that allows dose tracking, compliance monitoring, and / or patient dosing trigger during aerosol pharmaceutical administration.

[0622] Types of pharmaceutical excipients useful as carriers include stabilizers, such as human serum albumin (HSA), bulking agents, such as carbohydrates, amino acids, and polypeptides; pH regulators or buffers; salts, such as sodium chloride, etc. These carriers may be in crystalline or amorphous form, or may be a mixture of the two.

[0623] Particularly valuable bulking agents include compatible carbohydrates, polypeptides, amino acids, or combinations thereof. Suitable carbohydrates include monosaccharides such as galactose, D-mannose, sorbose, and the like; disaccharides such as lactose, trehalose, and the like; cyclodextrins such as 2-hydroxypropyl-beta-cyclodextrin; and polysaccharides such as raffinose, maltodextrin, dextran, and the like; alditols such as mannitol, xylitol, and the like. Preferred carbohydrate groups include lactose, trehalose, raffinose maltodextrin, and mannitol. Suitable polypeptides include aspartame. Amino acids include alanine and glycine, with glycine being preferred.

[0624] Suitable pH regulators or buffers include organic salts prepared from organic acids and bases, such as sodium citrate, sodium ascorbate, and the like; sodium citrate is preferred.

[0625] The RNA silencing agent of the present invention can be administered by oral and nasal delivery.For example, the drug administered through these membranes has a rapid onset of action, provides therapeutic plasma levels, avoids the first-pass liver metabolism, and avoids the drug exposure to the adverse gastrointestinal (GI) environment.Another advantage includes the easy accessibility of the membrane site, so that the drug can be easily applied, localized and removed.In some embodiments, the RNA silencing agent administered by oral or nasal delivery is modified so that it can pass through the blood-brain barrier.

[0626] In some embodiments, the composition of unit dose or fixed amount comprising RNA silencing agent is delivered by implantable device.Device can comprise the sensor for monitoring the parameter in subject.For example, device can comprise pump such as osmotic pump and optionally related electronic device.

[0627] The RNA silencing agent may be packaged into the virus's natural capsid or into an artificial capsid produced chemically or enzymatically or into structures derived therefrom.

[0628] X. Kit In some other embodiments, the present invention provides a suitable container containing a modified siRNA, the modified siRNA having a 5' end and a 3' end and complementary to a target, wherein the siRNA comprises a sense strand and an antisense strand and at least one modified intersubunit linkage of formula (I) as described above. The kit can include a pharmaceutical formulation of an RNA silencing agent, for example, a double-stranded RNA silencing agent or sRNA agent (e.g., a precursor that can be processed into an RNA silencing agent, for example, an sRNA agent, such as a DNA encoding a large RNA silencing agent or a double-stranded RNA silencing agent or sRNA agent or its precursor). In some embodiments, the individual components of the pharmaceutical formulation can be provided in a single container. Alternatively, it may be desirable to provide two or more components of the pharmaceutical formulation, for example, one container for the RNA silencing agent formulation and at least one other container for the carrier compound. The kit can be packaged in a variety of configurations, such as one or more containers in a single box. The various components can be combined, for example, according to instructions provided with the kit. The ingredients can be combined, for example, by the methods described herein to prepare and administer a pharmaceutical composition. The kit can also include a delivery device.

[0629] It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein may be made, using appropriate equivalents, without departing from the scope of the embodiments disclosed herein. While certain embodiments have been described in detail, the same will be more clearly understood by reference to the following examples, which are included for purposes of illustration only and are not intended to be limiting. [Example]

[0630] Example 1. Synthesis of phosphinate-modified intersubunit linkages The method for preparing the phosphinate-modified intersubunit linkages of the present invention is summarized in Figures 2A-2C. The method involves Jones oxidation of the free alcohol to the corresponding ketone, followed by Wittig olefination to achieve the exomethylene moiety shown in intermediate compound 3. Amide protection with BOM followed by hydroboration / oxidation affords the free alcohol intermediate 5. Mesylation followed by a modified Finkelstein reaction affords the iodinated intermediate 7, which is then subjected to further functionalization to lead to the methylphosphinate monomer 9.

[0631] Various protection and deprotection steps are performed to achieve intermediate 13. IBX oxidation gives the corresponding ketone, followed by Wittig olefination to access the methylene. Again, hydroboration / oxidation followed by mesylation and Finkelstein reaction gives monomer 18.

[0632] Combination of monomers 9 and 18 under basic conditions affords the phosphinate-linked dimer 19. Acid-mediated and Pearlman-catalyzed deprotection, followed by further phosphanamine functionalization, affords dimer 22.

[0633] Example 2. Synthesis of phosphonate-modified intersubunit linkages The method for preparing the phosphonate-modified intersubunit linkages of the present invention is summarized in Figures 3A and 3B.

[0634] Amide protection with BOM followed by methoxyphosphanamine functionalization gives intermediate 24. Reduction using tetrazole and water gave monomer 25.

[0635] Monomers 18 and 25 were combined under basic conditions to produce phosphonate dimer 26. Acid-mediated and Pearlman-catalyzed deprotection followed by further phosphanamine functionalization affords dimer 29.

[0636] Example 3. Solid-supported oligonucleotide extension Methods for assembling modified oligonucleotides having phosphinate and phosphonate intersubunit linkages are summarized in Figure 4. The oligonucleotides provided herein can be assembled using solid support mechanisms, where modified intersubunit linkages can be selectively inserted into the oligonucleotide sequence.

[0637] Example 4. Synthesis of vinylphosphonate-modified intersubunit linkages The synthetic approach for the preparation of the vinylphosphonate-modified intersubunit linkages of the present invention is summarized in FIG.

[0638] Synthesis of compound 2a TBDPS protection of the secondary alcohol of compound 1a was carried out under standard conditions well known in the art of organic synthesis to produce compound 2a.

[0639] Synthesis of compound 3a A dry solution of compound 2a (16.6 g, 20.8 mmol) in pyridine (100 mL) was added to anhydrous DIPEA (6.5 mL, 37.4 mmol) and benzoyl chloride (3.6 mL, 31.2 mmol). After stirring the mixture for 4 hours at room temperature, excess pyridine was evaporated and the mixture was diluted with CHCl. ​​The organic solution was washed with saturated aqueous NaHCO, filtered, and evaporated. The organic layer was collected, dried over MgSO, filtered, and evaporated. The crude material was purified by silica gel column chromatography (hexane-ethyl acetate, 4:1 to 1:1) to give compound 3a as a slightly yellow foam (14.5 g, 78%). 1 H NMR (500 MHz, CDCl3) δ 7.88-7.87 (m, 2H), 7.84 (d, 1H, J=8.3 Hz), 7.67-7.58 (m, 5H), 7.48-7.45 (m, 4H), 7.39-7.32 (m, 4H), 7.25-7.23 (m, 3H), 7.18-7.17 (m, 2H), 7.12-7.07 (m, 4H), 6.80-6.75 (m, 4H), 6.08 (dd, 1H, J HH =1.5 Hz, J HF =15.2 Hz), 5.14, (d, 1H, JHH =8.3 Hz), 4.59 (ddd, 1H, J HH =3.7, 1.5 Hz, J HF =51.9 Hz), 4.43 (ddd, 1H, J HH =7.4, 4.0 Hz, J HF =19.1 Hz), 4.24-4.23 (m, 1H), 3.79 (s, 6H), 3.62 (dd, 1H, J HH =11.2, 2.0 Hz), 3.35 (dd, 1H, J HH =11.1, 2.0 Hz), 1.00 (s, 9H); 13 C NMR (126 Hz, CDCl3) δ 168.4, 161.8, 158.72, 158.66, 148.9, 143.9, 139.4, 135.71. 135.70, 135.1, 134.8, 134.7, 132.3, 132.2, 131.3, 130.4, 130.2, 130.1, 129.1, 128.2, 128.0,127.91, 127.89, 127.2, 113.19, 113.16, 102.2, 92.5 (d, JCF=194.4 Hz), 87.7 (d, J CF =34.5 Hz), 87.2, 82.4, 70.0 (d, J CF =15.4 Hz), 60.7, 60.4, 55.2, 26.6

[0640] Synthesis of compound 4a Compound 3a (14.5 g, 16.3 mmol) was dissolved in 3% trichloroacetic acid / CHCl solution (200 mL) containing triethylsilane (8.0 mL, 50.1 mmol) and stirred for 1 h at rt. After washing the solution three times with saturated aqueous NaHCO, the combined organic layer was dried over MgSO, filtered, and evaporated. The crude material was purified by silica gel column chromatography (hexane / ethyl acetate, 4:1 to 3:7) to give compound 4a as a white foam (8.67 g, 91%). 1H NMR (500 MHz, CDCl3) δ 7.89-7.88 (m, 2H), 7.68-7.64 (6H, m), 7.51-7.45 (m, 4H), 7.42-7.38 (4H, m), 5.93 (dd, 1H, J HH =2.9 Hz, J HF =15.1 Hz), 5.73 (d, 1H, J HH =8.2 Hz), 4.74 (ddd, 1H, J HH =4.1, 3.2 Hz, J HF =52.2 Hz), 4.31 (ddd, 1H, J HH =5.8, 4.7, J HF =15.4 Hz), 4.11-4.09 (m, 1H), 3.82-3.79 (m, 1H), 3.39 (ddd, 1H, J HH =12.1, 5.6, 1.5 Hz), 1.64 (br, 1H), 1.11 (s, 9H); 13 C NMR (126 Hz, CDCl3) δ 168.3, 161.8, 149.0, 140.5, 135.7, 135.2, 132.8, 132.3, 131.3, 130.5, 130.4, 130.3, 129.2, 128.02, 127.96, 102.4, 91.8 (d, J CF =91.8 Hz), 89.5 (d, J CF =33.6 Hz), 69.5 (d, J CF =69.5 Hz), 60.3, 26.8

[0641] Synthesis of compound 6a An anhydrous solution of compound 4a (6.5 g, 11.0 mmol) was added to IBX (7.7 g, 27.6 mmol) and stirred at 85 °C for 2 h. After cooling the mixture in an ice bath, the precipitate was filtered off through Celite. The combined eluent was evaporated and co-evaporated three times with anhydrous CH3CN under an argon atmosphere. Compound 5a was obtained as a white foam and used without further purification. In a separate flask, PPh3 (11.6 g, 44.2 mmol) was added to a solution of CBr4 (7.3 g, 22.1 mmol) in anhydrous CHCl2 (25 mL) at 0 °C and stirred for 0.5 h at 0 °C. To this solution, a solution of compound 5a in anhydrous CHCl2 (25 mL) was added dropwise at 0 °C (10 min) and stirred for 2 h at 0 °C. After dilution with CHCl, the organic solution was washed with saturated aqueous NHCl, dried over MgSO, filtered, and evaporated. The resulting material was dissolved in a minimum amount of diethyl ether and added dropwise to an excess of diethyl ether solution with vigorous stirring at 0°C. The precipitate in the solution was filtered off through Celite, and the eluent was evaporated. The crude material was purified by silica gel column chromatography (hexane / ethyl acetate, 9:1 to 1:1) to give compound 6a as a white foam (4.3 g, 52%). 1 H NMR (500 MHz, CDCl3) δ 7.68-7.84 (m, 2H), 7.70-7.65 (m, 3H), 7.60-7.58 (m, 2H), 7.52-7.49 (m, 2H), 7.42-7.36 (m,4H), 7.31-7.28 (m, 2H), 7.09 (d, 1H, J=8.2 Hz), 6.25 (d, 1H, J=8.9 Hz), 5.75 (dd, 1H, J HF =8.24 Hz), 5.49 (dd, 1H, J HF =21.4 Hz), 4.77 (t, 1H, J HH =8.5 Hz, J HF =8.5 Hz), 4.38 (dd, 1H, J HH =4.1 Hz, J HF =52.1 Hz), 4.25 (ddd, 1H, J HH =8.1, 4.9 Hz, J HF =19.4 Hz), 1.10 (s, 9H);13 C NMR (126 Hz, CDCl3) δ 167.9, 161.6, 148.3, 141.4, 135.8, 134.7 (d, J C-Br =139.0 Hz), 132.5, 132.2, 131.1, 130.5, 130.3, 130.2, 129.2, 127.9, 102.7, 97.3, 93.3 (d, J CF =39.1 Hz), 91.5 (d, J CF =190.7 Hz), 82.4, 73.9 (d, J CF =16.4 Hz), 26.7

[0642] Synthesis of compounds 7a-E and 7a-Z A dry solution of compound 6a (4.2 g, 5.66 mmol) in DMF (25 mL) was added to dimethyl phosphite (2.09 mL, 22.6 mmol) and triethylamine (1.58 mL, 11.3 mmol) at 0 °C, followed by stirring overnight at room temperature. After diluting the solution with ethyl acetate, the organic solution was washed with saturated aqueous NH4Cl and brine. The organic solution was then dried over MgSO4, filtered, and evaporated. The resulting crude material was repeatedly purified by silica gel column chromatography (hexane / ethyl acetate, 9:1 to 1:1) until all the pure isomeric compounds were collected, yielding compound 7a-E (1.95 g, 52%). 1 H NMR (500 MHz, CDCl3) δ 7.87-7.85 (m, 2H), 7.89-7.85 (m, 3H), 7.61-7.59 (m, 2H), 7.52-7.48 (m, 2H), 7.45-7.32 (m, 6H), 7.08 (d, 1H, J HH =8.2), 6.49 (d, 1H, J HH =13.7), 5.99 (dd, 1H, J HH =13.7 Hz, 8.1 Hz), 5.75 (d, 1H, J HH =8.2), 5.63 (d, 1H, J HF =19.8 Hz), 4.43 (dd, 1H, J HF =52.6 Hz, JHH =4.3 Hz), 4.42 (t, 1H, J HH =8.0 Hz), 4.07 (ddd, J HH =7.8, 4.7 Hz, J HF =19.5 Hz), 1.08 (s, 9H); 13 C NMR (126 Hz, CDCl3) δ 148.4, 140.4, 135.8, 135.7, 135.3, 133.3, 132.3, 132.4, 132.1, 131.1, 130.5, 130.4, 130.3, 129.2, 127.95, 127.93, 112.4, 102.7, 91.7 (d, J CF =36.3 Hz), 91.6 (d, J CF =191.6 Hz), 82.8, 73.9 (d, J CF =16.4 Hz), 26.7, 19.1;および7a-Z (0.58g, 15%); 1 H NMR (500 MHz, CDCl3) δ 7.87-7.85 (m, 2H), 7.68-7.65 (m, 3H), 7.61-7.59 (m, 2H), 7.52-7.48 (m, 2H), 7.42-7.39 (m, 2H), 7.34-7.29 (m, 4H), 7.12 (d, 1H, J HH =8.2 Hz), 6.51 (d, 1H, J HH =7.4 Hz), 5.96 (dd, 1H, J HH =8.4 Hz, 7.4 Hz), 5.75 (d, 1H, J HH =8.2 Hz), 5.57 (dd, 1H, J HH =1.2 Hz, J HF =20.6 Hz), 5.04 (dd, 1H, J HH =8.2 Hz), 4.48 (J HH =3.5 Hz, J HF =53.1 Hz), 4.24 (ddd, 1H, J HH =7.8, 4.9 Hz, J HF =18.6 Hz), 1.09 (s, 9H); 13C NMR (126 Hz, CDCl3) δ 168.0, 161.7, 148.4, 141.4, 135.9, 135.8, 135.2, 132.6, 132.5, 131.2, 130.6, 130.5, 130.2, 130.1, 129.2, 127.8, 127.7, 114.5, 102.6, 93.0 (d, J CF =37.2 Hz), 91.6 (d, J CF =191.6 Hz), 80.3, 74.3 (d, J CF =16.4 Hz), 26.7, 19.1

[0643] Synthesis of compound 9a Anhydrous compound 7a-E (1.95 g, 2.94 mmol), Pd(OAc) (125 mg, 0.59 mmol), and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (652 mg, 1.18 mmol) were purged with argon and then dissolved in anhydrous THF (50 mL). Propylene oxide (2.06 mL, 29.4 mmol) was added, followed by compound 8a (2.07 g, 3.24 mmol) in one portion. The mixture was stirred at 70 °C for 4 h. After removing the solvent under reduced pressure, the crude mixture was purified by silica gel column chromatography (hexane / ethyl acetate, 50:50 to 0:100). The resulting fraction containing compound 9a was further purified by silica gel column chromatography (CHCl-MeOH, 0% to 5%) to give compound 9a as a diastereoisomeric mixture (2.04 g, 57%). 31 P NMR (202 MHz, CDCl3) δ 18.3

[0644] Synthesis of compound 10a A solution of compound 9a (2.0 g, 1.64 mmol) in anhydrous THF (22.5 mL) was added to 1.0 M TBAF-THF (2.5 mL, 2.5 mmol) and stirred at ambient temperature for 30 minutes. After dilution with CHCl (120 mL), the organic layer was washed with brine, dried over MgSO, filtered, and evaporated. The resulting crude material was purified by silica gel column chromatography (1% TEA-CHCl / MeOH, 0% to 6%) to give compound 10a (1.52 g, 94%). 31 P NMR (202 MHz, CDCl3) δ 19.0, 18.7

[0645] Synthesis of compound 11a Compound 10a (589.7 mg, 0.6 mmol) was anhydrous by repeated coevaporation with anhydrous CHCN and then dissolved in anhydrous CHCl (6.0 mL). To this solution, N,N-diisopropylethylamine (0.31 mL, 1.8 mmol) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidate (0.16 mL, 0.7...

Claims

1. A modified oligonucleotide, the oligonucleotide having a 5' end and a 3' end and complementary to a target, wherein the oligonucleotide comprises a sense strand and an antisense strand and at least one of Formula I 【Chemistry 1】 [During the ceremony, each B is independently a base pairing moiety; W is O, OCH ２ , O.C.H., C.H. ２ and CH; Each X is independently selected from halo, hydroxy and C １－６ alkoxy; Y is O － , OH, OR, NH － , N.H. ２ , S － and SH; Z is O and CH ２ selected from the group consisting of: R is a protecting group; and --- is an optional double bond. A modified oligonucleotide comprising a modified intersubunit linkage of

2. Y is O － 2. The modified oligonucleotide of claim 1, wherein Z or W is not O when:

3. Z is CH ２ and W is CH ２ The modified oligonucleotide of claim 1,

4. The modified intersubunit linkage of formula I is represented by formula II 【Chemistry 2】 The modified oligonucleotide of claim 3, wherein the modified intersubunit linkage is:

5. Z is CH ２ and W is O.

6. The modified intersubunit linkage of formula I is represented by formula III 【Chemistry 3】 The modified oligonucleotide of claim 5, wherein the modified intersubunit linkage is:

7. Z is O and W is CH ２ The modified oligonucleotide of claim 1,

8. The modified intersubunit linkage of formula I is represented by formula IV 【Chemistry 4】 The modified oligonucleotide of claim 7, wherein the modified intersubunit linkage is:

9. The modified oligonucleotide of claim 1, wherein Z is O and W is CH.

10. The modified intersubunit linkage of formula I is represented by formula V 【Chemistry 5】 The modified oligonucleotide of claim 9, wherein the modified intersubunit linkage is:

11. Z is O and W is OCH ２ The modified oligonucleotide of claim 1,

12. The modified intersubunit linkage of formula I is represented by formula VI 【Chemistry 6】 wherein each X is independently fluoro, hydroxy, or C １－６ alkoxy; Y is O － , OH, and OR; Z is O and CH ２ and --- is an optional double bond. or the modified intersubunit linkage of formula I is of formula VIa 【Chemistry 7】 The modified oligonucleotide of claim 11, wherein the modified intersubunit linkage is:

13. Z is CH ２ and W is CH.

14. The modified intersubunit linkage of formula I is represented by formula VII 【Chemistry 8】 The modified oligonucleotide of claim 13, wherein the modified intersubunit linkage is:

15. 2. The modified oligonucleotide of claim 1, wherein each base pairing moiety B is independently selected from the group consisting of adenine, guanine, cytosine and uracil.

16. 16. The modified oligonucleotide of claim 15, wherein one or both of B's ​​is adenine.

17. 16. The modified oligonucleotide of claim 15, wherein one or both of B's ​​is guanine.

18. 16. The modified oligonucleotide of claim 15, wherein one or both of B's ​​is cytosine.

19. 16. The modified oligonucleotide of claim 15, wherein one or both of B's ​​is uracil.

20. The modified oligonucleotide of claim 1 , wherein W is O.

21. W is CH ２ The modified oligonucleotide of claim 1,

22. The modified oligonucleotide of claim 1, wherein W is CH.

23. 2. The modified oligonucleotide of claim 1, wherein one or both of X is OH.

24. One or both of X's are OCH ３ The modified oligonucleotide of claim 1,

25. 2. The modified oligonucleotide of claim 1, wherein one or both X's are halo.

26. 2. The modified oligonucleotide of claim 1, wherein the modified oligonucleotide does not include a 2'-fluoro substituent.

27. Y is O － The modified oligonucleotide of claim 1,

28. The modified oligonucleotide of claim 1, wherein Y is OH.

29. The modified oligonucleotide of claim 1, wherein Y is OR.

30. Y is NH － The modified oligonucleotide of claim 1,

31. Y is NH ２ The modified oligonucleotide of claim 1,

32. Y is S － The modified oligonucleotide of claim 1,

33. The modified oligonucleotide of claim 1, wherein Y is SH.

34. The modified oligonucleotide of claim 1 , wherein Z is O.

35. Z is CH ２ The modified oligonucleotide of claim 1,

36. 2. The modified oligonucleotide of claim 1, wherein the modified intersubunit linkage is inserted at positions 1-2 of the antisense strand.

37. 2. The modified oligonucleotide of claim 1, wherein the modified intersubunit linkage is inserted at positions 6-7 of the antisense strand.

38. 2. The modified oligonucleotide of claim 1, wherein the modified intersubunit linkage is inserted at positions 10-11 of the antisense strand.

39. 2. The modified oligonucleotide of claim 1, wherein the modified intersubunit linkage is inserted at positions 19-20 of the antisense strand.

40. 2. The modified oligonucleotide of claim 1, wherein modified intersubunit linkages are inserted at positions 5-6 and 18-19 of the antisense strand.

41. The modified oligonucleotide is incorporated into an siRNA, the modified siRNA having a 5' end, a 3' end, and complementary to a target, wherein the siRNA comprises a sense strand and an antisense strand and at least one nucleotide sequence of Formula I. 【Chemistry 9】 [During the ceremony, each B is independently a base pairing moiety; W is O, OCH ２ , O.C.H., C.H. ２ and CH; Each X is independently halo, hydroxy, and C １－６ alkoxy; Y is O － , OH, OR, NH － , N.H. ２ , S － and SH; Z is O and CH ２ selected from the group consisting of: --- is an optional double bond. The modified oligonucleotide of claim 1 , comprising a modified intersubunit linkage of

42. Y is O － The modified siRNA of claim 41, wherein Z or W is not O when

43. Z is CH ２ and W is CH ２ The modified siRNA of claim 41,

44. The modified intersubunit linkage of formula I is represented by formula II 【Chemistry 10】 The modified siRNA of claim 43, wherein the modified intersubunit linkage is:

45. Z is CH ２ and W is O.

46. The modified intersubunit linkage of formula I is represented by formula III 【Chemistry 11】 The modified siRNA of claim 45, wherein the modified intersubunit linkage is:

47. Z is O and W is CH ２ The modified siRNA of claim 41,

48. The modified intersubunit linkage of formula I is represented by formula IV 【Chemistry 12】 The modified siRNA of claim 47, wherein the modified intersubunit linkage is:

49. The modified siRNA of claim 41, wherein Z is O and W is CH.

50. The modified intersubunit linkage of formula I is represented by formula V 【Chemistry 13】 The modified siRNA of claim 49, wherein the modified intersubunit linkage is:

51. Z is O and W is OCH ２ The modified siRNA of claim 41,

52. The modified intersubunit linkage of formula I is represented by formula VI 【Chemistry 14】 wherein each X is independently fluoro, hydroxy, or C １－６ alkoxy; Y is O － , OH, and OR; Z is O and CH ２ and --- is an optional double bond. or the modified intersubunit linkage of formula I is of formula VIa 【Chemistry 15】 The modified siRNA of claim 51, wherein the modified intersubunit linkage is:

53. Z is CH ２ and W is CH.

54. The modified intersubunit linkage of formula I is represented by formula VII 【Chemistry 16】 The modified siRNA of claim 53, wherein the modified intersubunit linkage is:

55. The modified siRNA of claim 41, wherein the base-pairing moiety B is selected from the group consisting of adenine, guanine, cytosine and uracil.

56. The modified siRNA of claim 55, wherein one or both of B's ​​is adenine.

57. The modified siRNA of claim 55, wherein one or both of B's ​​is guanine.

58. The modified siRNA of claim 55, wherein one or both of B's ​​is cytosine.

59. The modified siRNA of claim 55, wherein one or both of B's ​​is uracil.

60. The modified siRNA of claim 41, wherein W is O.

61. W is CH ２ The modified siRNA of claim 41,

62. The modified siRNA of claim 41, wherein W is CH.

63. The modified siRNA of claim 41, wherein one or both of X is OH.

64. One or both of X's are OCH ３ The modified siRNA of claim 41,

65. The modified siRNA of claim 41, wherein one or both of X is halo.

66. 42. The modified siRNA of claim 41, wherein the modified siRNA does not contain a 2'-fluoro substituent.

67. Y is O － The modified siRNA of claim 41,

68. The modified siRNA of claim 41, wherein Y is OH.

69. The modified siRNA of claim 41, wherein Y is OR.

70. Y is NH － The modified siRNA of claim 41,

71. Y is NH ２ The modified siRNA of claim 41,

72. Y is S － The modified siRNA of claim 41,

73. The modified siRNA of claim 41, wherein Y is SH.

74. The modified siRNA of claim 41, wherein Z is O.

75. Z is CH ２ The modified siRNA of claim 41,

76. The modified siRNA of claim 41, wherein the modified intersubunit linkage is inserted at positions 1-2 of the antisense strand.

77. The modified siRNA of claim 41, wherein the modified intersubunit linkage is inserted at positions 6-7 of the antisense strand.

78. The modified siRNA of claim 41, wherein the modified intersubunit linkage is inserted at positions 10-11 of the antisense strand.

79. The modified siRNA of claim 41, wherein the modified intersubunit linkage is inserted at positions 19-20 of the antisense strand.

80. The modified siRNA of claim 41, wherein modified intersubunit linkages are inserted at positions 5-6 and 18-19 of the antisense strand.

81. The modified oligonucleotide is incorporated into an siRNA, the modified siRNA having a 5' end, a 3' end, and complementary to a target, wherein the siRNA comprises a sense strand and an antisense strand and at least one nucleotide sequence of Formula I. 【Chemistry 17】 [During the ceremony, each B is independently a base pairing moiety; W is O, OCH ２ , O.C.H., C.H. ２ and CH; Each X is independently halo, hydroxy, and C １－６ alkoxy; Y is O － , OH, OR, NH － , N.H. ２ , S － and SH; Z is O and CH ２ selected from the group consisting of: R is a protecting group selected from the group consisting of dimethoxytrityl (DMTr), succinate, 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 acetate; and --- is an optional double bond. The modified oligonucleotide of claim 1 , comprising a modified intersubunit linkage of

82. Y is O － The modified siRNA of claim 81, wherein Z or W is not O.

83. Z is CH ２ and W is CH ２ The modified siRNA of claim 81,

84. The modified intersubunit linkage of formula I is represented by formula II 【Chemistry 18】 The modified siRNA of claim 83, wherein the modified intersubunit linkage is:

85. Z is CH ２ and W is O.

86. The modified intersubunit linkage of formula I is 【Chemistry 19】 The modified siRNA of claim 85, wherein the modified intersubunit linkage is:

87. Z is O and W is CH ２ The modified siRNA of claim 81,

88. The modified intersubunit linkage of formula I is represented by formula IV 【Chemistry 20】 The modified siRNA of claim 87, wherein the modified intersubunit linkage is:

89. The modified siRNA of claim 81, wherein Z is O and W is CH.

90. The modified intersubunit linkage of formula I is represented by formula V 【Chemistry 21】 The modified siRNA of claim 89, wherein the modified intersubunit linkage is:

91. Z is O and W is OCH ２ The modified siRNA of claim 82,

92. The modified intersubunit linkage of formula I is represented by formula VI 【Chemical 22】 wherein each X is independently fluoro, hydroxy, or C １－６ alkoxy; Y is O － , OH, and OR; Z is O and CH ２ and --- is an optional double bond. or the modified intersubunit linkage of formula I is of formula VIa 【Chemistry 23】 The modified siRNA of claim 91, wherein the modified intersubunit linkage is:

93. Z is CH ２ and W is CH.

94. The modified intersubunit linkage of formula I is represented by formula VII 【Chemistry 24】 The modified siRNA of claim 93, wherein the modified intersubunit linkage is:

95. The modified siRNA of claim 81, wherein each base-pairing moiety B is independently selected from the group consisting of adenine, guanine, cytosine and uracil.

96. The modified siRNA of claim 95, wherein one or both of B's ​​is adenine.

97. The modified siRNA of claim 95, wherein one or both of B's ​​is guanine.

98. The modified siRNA of claim 95, wherein one or both of B's ​​is cytosine.

99. The modified siRNA of claim 95, wherein one or both of B's ​​is uracil.

100. The modified siRNA of claim 81, wherein W is O.

101. W is CH ２ The modified siRNA of claim 81,

102. The modified siRNA of claim 81, wherein W is CH.

103. The modified siRNA of claim 81, wherein one or both of X is OH.

104. One or both of X's are OCH ３ The modified siRNA of claim 81,

105. The modified siRNA of claim 81, wherein one or both of X is halo.

106. The modified siRNA of claim 81, wherein the modified siRNA does not contain a 2'-fluoro substituent.

107. Y is O － The modified siRNA of claim 81,

108. The modified siRNA of claim 81, wherein Y is OH.

109. The modified siRNA of claim 81, wherein Y is OR.

110. Y is NH － The modified siRNA of claim 81,

111. Y is NH ２ The modified siRNA of claim 81,

112. Y is S － The modified siRNA of claim 81,

113. The modified siRNA of claim 81, wherein Y is SH.

114. The modified siRNA of claim 81, wherein Z is O.

115. Z is CH ２ The modified siRNA of claim 81,

116. The modified siRNA of claim 81, wherein R is DMTr.

117. The modified siRNA of claim 81, wherein R is succinate.

118. The modified siRNA of claim 81, wherein R is TBDPS.

119. The modified siRNA of claim 81, wherein R is acetate.

120. The modified siRNA of claim 81, wherein the modified intersubunit linkage is inserted at positions 1-2 of the antisense strand.

121. The modified siRNA of claim 81, wherein the modified intersubunit linkage is inserted at positions 6-7 of the antisense strand.

122. The modified siRNA of claim 81, wherein the modified intersubunit linkage is inserted at positions 10-11 of the antisense strand.

123. The modified siRNA of claim 81, wherein the modified intersubunit linkage is inserted at positions 19-20 of the antisense strand.

124. The modified siRNA of claim 81, wherein modified intersubunit linkages are inserted at positions 5-6 and 18-19 of the antisense strand.

125. The modified oligonucleotide is incorporated into an siRNA, the modified siRNA having a 5' end, a 3' end, complementary to a target and comprising a sense strand and an antisense strand, wherein the siRNA is represented by Formula VIII 【Chemistry 25】 [During the ceremony, D is O, OCH ２ , O.C.H., C.H. ２ and CH; C is O － , O.H., O.R. １ , N.H. － , N.H. ２ , S － and SH; A is O and CH ２ selected from the group consisting of: R １ is a protecting group; --- is an optional double bond.

2. The modified oligonucleotide of claim 1, comprising at least one modified intersubunit linkage of the formula:

126. C is O － When A or D is not O, the modified siRNA bond of claim 125 is

127. D is CH ２ The modified siRNA bond of claim 125,

128. The modified intersubunit linkage of formula VIII is represented by formula IX: 【Chemistry 26】 The modified siRNA linkage of claim 127, which is a modified intersubunit linkage of:

129. The modified siRNA bond of claim 125, wherein D is O.

130. The modified intersubunit linkage of formula VIII is represented by formula X 【Chemical 27】 The modified siRNA linkage of claim 129, which is a modified intersubunit linkage of

131. D is CH ２ The modified siRNA bond of claim 125,

132. The modified intersubunit linkage of formula VIII is represented by formula XI 【Chemistry 28】 The modified intersubunit linkage of claim 131 , the modified siRNA linkage of claim 131 .

133. The modified siRNA bond of claim 125, wherein D is CH.

134. The modified intersubunit linkage of formula VIII is represented by formula XII ​ The modified siRNA linkage of claim 133, which is a modified intersubunit linkage of

135. The modified intersubunit linkage of formula VIII is represented by formula XIV 【Chemistry 30】 The modified siRNA linkage of claim 125, which is a modified intersubunit linkage of

136. D is OCH ２ The modified siRNA bond of claim 125,

137. The modified intersubunit linkage of formula VII is represented by formula XIII 【Chemistry 31】 The modified siRNA linkage of claim 136, which is a modified intersubunit linkage of

138. The modified siRNA bond of claim 125, wherein A is O.

139. The modified siRNA conjugate of claim 125, wherein each optionally modified nucleoside is independently selected at each occurrence from the group consisting of adenosine, guanosine, cytidine and uridine.

140. 140. The modified siRNA conjugate of claim 139, wherein the optionally modified nucleoside is adenosine.

141. 140. The modified siRNA conjugate of claim 139, wherein the optionally modified nucleoside is guanosine.

142. 140. The modified siRNA conjugate of claim 139, wherein the optionally modified nucleoside is a cytidine.

143. 140. The modified siRNA conjugate of claim 139, wherein the optionally modified nucleoside is a uridine.

144. 126. The modified siRNA conjugate of claim 125, wherein the modified nucleoside does not comprise a 2'-fluoro substituent.

145. C is O － The modified siRNA bond of claim 125,

146. The modified siRNA bond of claim 125, wherein C is OH.

147. C is OR １ The modified siRNA bond of claim 125,

148. C is NH － The modified siRNA bond of claim 125,

149. C is NH ２ The modified siRNA bond of claim 125,

150. C is S － The modified siRNA bond of claim 125,

151. The modified siRNA bond of claim 125, wherein C is SH.

152. The modified siRNA bond of claim 125, wherein A is O.

153. A is CH ２ The modified siRNA bond of claim 125,

154. The modified siRNA linkage of claim 125, wherein the linkage is inserted at positions 1-2 of the antisense strand.

155. The modified siRNA linkage of claim 125, wherein the linkage is inserted at position 6-7 of the antisense strand.

156. The modified siRNA linkage of claim 125, wherein the linkage is inserted at positions 10-11 of the antisense strand.

157. The modified siRNA linkage of claim 125, wherein the linkage is inserted at positions 19-20 of the antisense strand.

158. The modified siRNA linkage of claim 125, wherein the linkages are inserted at positions 5-6 and 18-19 of the antisense strand.

159. The modified oligonucleotide is incorporated into an siRNA, the modified siRNA having a 5' end, a 3' end, complementary to a target and comprising a sense strand and an antisense strand, wherein the siRNA is represented by Formula VIII 【Chemistry 32】 [During the ceremony, D is O, OCH ２ , O.C.H., C.H. ２ and CH; C is O － , O.H., O.R. １ , N.H. － , N.H. ２ , S － and SH; A is O and CH ２ selected from the group consisting of: R １ is a protecting group selected from the group consisting of dimethoxytrityl (DMTr), succinate, 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 acetate; --- is an optional double bond.

2. The modified oligonucleotide of claim 1, comprising at least one modified intersubunit linkage of the formula:

160. C is O － When A or D is not O, the modified siRNA bond of claim 159 is

161. D is CH ２ The modified siRNA bond of claim 159,

162. The modified intersubunit linkage of formula VIII is represented by formula IX: 【Chemical 33】 The modified siRNA linkage of claim 161, which is a modified intersubunit linkage of

163. The modified siRNA bond of claim 159, wherein D is O.

164. The modified intersubunit linkage of formula VIII is represented by formula X 【Chemical 34】 The modified siRNA linkage of claim 163, which is a modified intersubunit linkage of

165. D is CH ２ The modified siRNA bond of claim 159,

166. The modified intersubunit linkage of formula VIII is represented by formula XI 【Chemistry 35】 The modified siRNA linkage of claim 165, which is a modified intersubunit linkage of

167. The modified siRNA bond of claim 159, wherein D is CH.

168. The modified intersubunit linkage of formula VIII is represented by formula XII 【Chemical Formula 36】 The modified siRNA linkage of claim 167, which is a modified intersubunit linkage of:

169. D is OCH ２ The modified siRNA bond of claim 159,

170. The modified intersubunit linkage of formula VIII is represented by formula XIII 【Chemical 37】 The modified siRNA linkage of claim 169, which is a modified intersubunit linkage of

171. The modified siRNA bond of claim 159, wherein C is OH.

172. The modified intersubunit linkage of formula VIII is represented by formula XIV 【Chemical Formula 38】 The modified siRNA linkage of claim 167, which is a modified intersubunit linkage of:

173. 160. The modified siRNA conjugate of claim 159, wherein each optionally modified nucleoside is independently, at each occurrence, selected from the group consisting of adenosine, guanosine, cytidine and uridine.

174. The modified siRNA conjugate of claim 173, wherein the optionally modified nucleoside is adenosine.

175. The modified siRNA conjugate of claim 173, wherein the optionally modified nucleoside is guanosine.

176. The modified siRNA conjugate of claim 173, wherein the optionally modified nucleoside is a cytidine.

177. The modified siRNA conjugate of claim 173, wherein the optionally modified nucleoside is a uridine.

178. 160. The modified siRNA conjugate of claim 159, wherein the modified nucleoside does not comprise a 2'-fluoro substituent.

179. C is O － The modified siRNA bond of claim 159,

180. The modified siRNA bond of claim 159, wherein C is OH.

181. C is OR １ The modified siRNA bond of claim 159,

182. C is NH － The modified siRNA bond of claim 159,

183. C is NH ２ The modified siRNA bond of claim 159,

184. C is S － The modified siRNA bond of claim 159,

185. The modified siRNA bond of claim 159, wherein C is SH.

186. The modified siRNA bond of claim 159, wherein A is O.

187. A is CH ２ The modified siRNA bond of claim 159,

188. R １ The modified siRNA conjugate of claim 159, wherein is DMTr.

189. R １ The modified siRNA linkage of claim 159, wherein is a succinate.

190. R １ The modified siRNA conjugate of claim 159, wherein is TBDPS.

191. R １ The modified siRNA linkage of claim 159, wherein is acetate.

192. 160. The modified siRNA linkage of claim 159, wherein the linkage is inserted at positions 1-2 of the antisense strand.

193. 160. The modified siRNA linkage of claim 159, wherein the linkage is inserted at position 6-7 of the antisense strand.

194. 160. The modified siRNA linkage of claim 159, wherein the linkage is inserted at positions 10-11 of the antisense strand.

195. 160. The modified siRNA linkage of claim 159, wherein the linkage is inserted at positions 19-20 of the antisense strand.

196. 160. The modified siRNA linkage of claim 159, wherein the modified intersubunit linkages are inserted at positions 5-6 and 18-19 of the antisense strand.

197. 1. A branched compound comprising two or more oligonucleotides, (a) oligonucleotides are connected to each other by one or more moieties selected from a linker, a spacer, and a branch point; and (b) at least one oligonucleotide has at least one formula (I) 【Chemical Formula 39】 [During the ceremony, each B is independently a base pairing moiety; W is O, OCH ２ , O.C.H., C.H. ２ and CH; Each X is independently halo, hydroxy, and C １－６ alkoxy; Y is O － , OH, OR, NH － , N.H. ２ , S － and SH; Z is O and CH ２ selected from the group consisting of: R is a protecting group; and --- is an optional double bond. A branched compound, which is a modified intersubunit bond of.

198. 200. The branched compound of claim 197, comprising 2, 4, 6 or 8 oligonucleotides.

199. 200. The branched compound of claim 197, wherein each oligonucleotide is double-stranded and comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand have respective 5' and 3' ends.

200. 200. The branched compound of claim 199, wherein each double-stranded oligonucleotide is independently connected to a linker, spacer or branch point at the 3' or 5' end of the sense or antisense strand.

201. 201. The branched compound of any of claims 199-200, wherein each antisense strand independently comprises at least 16, at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides and has complementarity to a target.

202. each linker is independently selected from an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, and combinations thereof; 202. The branched compound of any of claims 197-201, wherein any carbon or oxygen atom of the linker is optionally replaced with a nitrogen atom, has a hydroxyl substituent or has an oxo substituent.

203. Y is O － 200. The branched compound of claim 197, wherein Z or W is not O when

204. Z is CH ２ and W is CH ２ 200. The branched compound of claim 197,

205. The modified intersubunit linkage of formula (I) is 【Chemistry 40】 The branched compound of claim 204, wherein the modified intersubunit bond is:

206. Z is CH ２ and W is O.

207. The modified intersubunit linkage of formula (I) is represented by formula (III) 【Chemistry 41】 The branched compound of claim 206, wherein the modified intersubunit bond is:

208. Z is O and W is CH ２ 200. The branched compound of claim 197,

209. The modified intersubunit linkage of formula (I) is represented by formula (IV) 【Chemistry 42】 The branched compound of claim 208, wherein the modified intersubunit bond is:

210. 200. The branched compound of claim 197, wherein Z is O and W is CH.

211. The modified intersubunit linkage of formula (I) is represented by formula (V) 【Chemistry 43】 211. The branched compound of claim 210, wherein the modified intersubunit bond is:

212. Z is O and W is OCH ２ 200. The branched compound of claim 197,

213. The modified intersubunit linkage of formula I is represented by formula VI 【Chemistry 44】 wherein each X is independently fluoro, hydroxy, or C １－６ alkoxy; Y is O － , OH, and OR; Z is O and CH ２ and --- is an optional double bond. or the modified intersubunit bond of formula (I) is of formula (VIa) 【Chemistry 45】 The branched compound of claim 212, wherein the modified intersubunit bond is:

214. Z is CH ２ and W is CH.

215. The modified intersubunit linkage of formula (I) is represented by formula (VII) 【Chemistry 46】 215. The branched compound of claim 214, wherein the modified intersubunit bond is:

216. 200. The branched compound of claim 197, wherein the base pairing moiety B is selected from the group consisting of adenine, guanine, cytosine and uracil.

217. The branched compound of claim 197, wherein the modified oligonucleotide is incorporated into a modified siRNA, the modified siRNA having a 5' end, a 3' end and being complementary to a target, wherein the siRNA comprises a sense strand and an antisense strand and at least one modified intersubunit linkage of formula (I).

218. R is a protecting group selected from the group consisting of dimethoxytrityl (DMTr), succinate, 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 acetate; and The branched compound of claim 197, wherein -- is an optional double bond.

219. Formula (1) 【Chemistry 47】 A compound of the formula: wherein L is selected from an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, and combinations thereof, wherein formula (1) optionally further comprises one or more branch points Bp and one or more spacers, and wherein Bp is independently at each occurrence a polyvalent organic species or derivative thereof; S is independently selected from each occurrence of an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, phosphates, phosphonates, phosphoramidates, esters, amides, triazoles, and combinations thereof; N is an RNA duplex comprising a sense strand and an antisense strand, where the sense strand and the antisense strand each independently comprise one or more chemical modifications; and n is 2, 3, 4, 5, 6, 7 or 8, At least one N is of formula (I) 【Chemistry 48】 [During the ceremony, each B is independently a base pairing moiety; W is O, OCH ２ , O.C.H., C.H. ２ and CH; Each X is independently halo, hydroxy, and C １－６ alkoxy; Y is O － , OH, OR, NH － , N.H. ２ , S － and SH; Z is O and CH ２ selected from the group consisting of: R is a protecting group; and --- is an optional double bond. The compound comprises a modified intersubunit bond of

220. The compound of claim 219 having a structure selected from formulas (1-1) to (1-9): 【Table 1】

221. The antisense strand is 【Chemistry 49】 220. The compound of claim 219, comprising a 5' terminal group R selected from:

222. Formula (2) 【Chemistry 50】 [During the ceremony, X, for each occurrence, is independently selected from adenosine, guanosine, uridine, cytidine, and chemically modified derivatives thereof; Y, for each occurrence, is independently selected from adenosine, guanosine, uridine, cytidine, and chemically modified derivatives thereof; - is a phosphodiester internucleoside linkage; = is a phosphorothioate internucleoside linkage; and --- is, independently in each occurrence, a base pairing interaction or a mismatch, with the proviso that at least one of the - bonds or at least one of the = bonds in formula (2) is a modified subunit bond of formula (I).

220. The compound of claim 219 having the structure:

223. Formula (3) 【Chemistry 51】 [During the ceremony, X is, for each occurrence, independently, a nucleotide containing a 2'-deoxy-2'-fluoro modification; X is, independently for each occurrence, a nucleotide containing a 2'-O-methyl modification; Y, for each occurrence, is independently a nucleotide containing a 2'-deoxy-2'-fluoro modification; and Y is, for each occurrence, independently, a nucleotide containing a 2'-O-methyl modification; and with the proviso that at least one of the - bonds or at least one of the = bonds in formula (3) is a modified subunit bond of formula (I).

223. The compound of claim 222 having the structure:

224. Formula (4) 【Chemistry 52】 wherein X, for each occurrence, is independently selected from adenosine, guanosine, uridine, cytidine, and chemically modified derivatives thereof; Y, for each occurrence, is independently selected from adenosine, guanosine, uridine, cytidine, and chemically modified derivatives thereof; - is a phosphodiester internucleoside linkage; = is a phosphorothioate internucleoside linkage; and --- is, independently in each occurrence, a base pairing interaction or a mismatch, with the proviso that at least one of the - bonds or at least one of the = bonds in formula (4) is a modified subunit bond of formula (I).

220. The compound of claim 219 having the structure:

225. Formula (5) 【Chemistry 53】 [During the ceremony, X is, for each occurrence, independently, a nucleotide containing a 2'-deoxy-2'-fluoro modification; X is, independently for each occurrence, a nucleotide containing a 2'-O-methyl modification; Y, for each occurrence, is independently a nucleotide containing a 2'-deoxy-2'-fluoro modification; and Y, for each occurrence, is independently a nucleotide containing a 2'-O-methyl modification.

225. The compound of claim 224 having the structure:

226. L is L1 【Chemical 54】 The compound of any of claims 219 to 225, having the structure:

227. R is R ３ and n is 2.

228. L is L2 【Chemistry 55】 The compound of any of claims 219 to 225, having the structure:

229. R is R ３ and n is 2.

230. Y is O － When Z or W is not O, the compound of claim 219.

231. Z is CH ２ and W is CH ２ 220. The compound of claim 219,

232. The modified intersubunit linkage of formula (I) is 【Chemistry 56】 The branched compound of claim 231, wherein the modified intersubunit bond is:

233. Z is CH ２ and W is O.

234. The modified intersubunit linkage of formula (I) is represented by formula (III) 【Chemistry 57】 The compound of claim 233, wherein the modified intersubunit bond is:

235. Z is O and W is CH ２ 235. The compound of claim 234,

236. The modified intersubunit linkage of formula (I) is represented by formula (IV) 【Chemistry 58】 The compound of claim 235, wherein the modified intersubunit bond is:

237. 220. The compound of claim 219, wherein Z is O and W is CH.

238. The modified intersubunit linkage of formula (I) is represented by formula (V) 【Chemistry 59】 The compound of claim 237, wherein the modified intersubunit bond is:

239. Z is O and W is OCH ２ 220. The branched compound of claim 219, wherein:

240. The modified intersubunit linkage of formula I is represented by formula VI 【Chemistry 60】 wherein each X is independently fluoro, hydroxy, or C １－６ alkoxy; Y is O － , OH, and OR; Z is O and CH ２ and --- is an optional double bond. or the modified intersubunit bond of formula (I) is of formula (VIa) 【Chemistry 61】 The branched compound of claim 239, wherein the modified intersubunit bond is:

241. Z is CH ２ and W is CH.

242. The modified intersubunit linkage of formula (I) is represented by formula (VII) 【Chemistry 62】 The branched compound of claim 241, wherein the modified intersubunit bond is:

243. 220. The branched compound of claim 219, wherein the base pairing moiety B is selected from the group consisting of adenine, guanine, cytosine and uracil.

244. Formula (6) 【Chemistry 63】 A delivery system for a therapeutic nucleic acid having the structure Where: L is selected from an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, and combinations thereof, wherein formula (6) optionally further comprises one or more branch points Bp and one or more spacers, wherein: Bp is independently at each occurrence a polyvalent organic species or derivative thereof; S is independently selected from each occurrence of an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, phosphates, phosphonates, phosphoramidates, esters, amides, triazoles, and combinations thereof; each cNA is independently a carrier nucleic acid that contains one or more chemical modifications; and n is 2, 3, 4, 5, 6, 7 or 8; wherein at least one chemical modification of at least one cNA is represented by formula (I): 【Chemistry 64】 [During the ceremony, each B is independently a base pairing moiety; W is O, OCH ２ , O.C.H., C.H. ２ and CH; Each X is independently halo, hydroxy, and C １－６ alkoxy; Y is O － , OH, OR, NH － , N.H. ２ , S － and SH; Z is O and CH ２ selected from the group consisting of: R is a protecting group; and --- is an optional double bond. A delivery system comprising an intersubunit bond of

245. The delivery system of claim 244, having a structure selected from formulas (6-1) to (6-9): 【Table 2】

246. The delivery system of claim 244, wherein each cNA independently comprises at least 15 consecutive nucleotides.

247. The delivery system of claim 244, wherein each cNA is independently comprised of a chemically modified nucleotide.

248. The delivery system of claim 244, further comprising n therapeutic nucleic acids (NAs), wherein each NA hybridizes to at least one cNA.

249. The delivery system of claim 248, wherein each NA independently comprises at least 16 consecutive nucleotides.

250. The delivery system of claim 248, wherein each NA independently comprises 16 to 20 contiguous nucleotides.

251. The delivery system of claim 248, wherein each NA comprises an unpaired overhang of at least two nucleotides.

252. The delivery system of claim 251, wherein the nucleotides of the overhang are joined via phosphorothioate bonds.

253. The delivery system of claim 248, wherein each NA is independently selected from the group consisting of DNA, siRNA, antagomiR, miRNA gapmer, mixmer or guide RNA.

254. The delivery system of claim 248, wherein each NA is identical.

255. The delivery system of claim 248, wherein each NA is not identical.

256. The delivery system of claim 244 having the structure of any one of claims 23 to 47.

257. 245. The delivery system of claim 244, wherein the target of delivery is selected from the group consisting of brain, liver, skin, kidney, spleen, pancreas, colon, fat, lung, muscle and thymus.

258. Y is O － When Z or W is not O, the delivery system of claim 244.

259. Z is CH ２ and W is CH ２ The delivery system of claim 244,

260. The modified intersubunit linkage of formula (I) is 【Chemistry 65】 The delivery system of claim 259, wherein the modified intersubunit bond is:

261. Z is CH ２ and W is O.

262. The modified intersubunit linkage of formula (I) is represented by formula (III) 【Chemistry 66】 The compound of claim 261, wherein the modified intersubunit bond is:

263. Z is O and W is CH ２ 245. The compound of claim 244,

264. The modified intersubunit linkage of formula (I) is represented by formula (IV) 【Chemistry 67】 The compound of claim 263, wherein the modified intersubunit bond is:

265. 50. The compound of claim 48, wherein Z is O and W is CH.

266. The modified intersubunit linkage of formula (I) is represented by formula (V) 【Chemistry 68】 The compound of claim 265, wherein the modified intersubunit bond is:

267. Z is O and W is OCH ２ 245. The branched compound of claim 244, wherein:

268. The modified intersubunit linkage of formula I is represented by formula VI 【Chemistry 69】 wherein each X is independently fluoro, hydroxy, or C １－６ alkoxy; Y is O － , OH, and OR; Z is O and CH ２ and --- is an optional double bond. or the modified intersubunit bond of formula (I) is of formula (VIa) 【Chemistry 70】 The branched compound of claim 267, wherein the modified intersubunit bond is:

269. Z is CH ２ and W is CH.

270. The modified intersubunit linkage of formula (I) is represented by formula (VII) 【Chemistry 71】 270. The branched compound of claim 269, wherein the modified intersubunit bond is:

271. 245. The branched compound of claim 244, wherein the base pairing moiety B is selected from the group consisting of adenine, guanine, cytosine and uracil.

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